JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 15
antibiotics isolation, separation and purification
JOURNAL OF CHROMATOGRAPHY LIBRARY Volume 1 Chromatography of Antibiotics by G.H. Wagman and M J . Weinstein Volume 2
Extraction Chromatography edited by T. Braun and G. Ghersini
Volume 3 Liquid Column Chromatography. A Survey of Modern Techniques and Applications edited by Z. Deyl, K. Mac& and J. J a d k Volume 4
Detectors in Gas Chromatography by J. Sev&
Volume 5
Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods by N.A. Parris
Volume 6 Isotachophoresis. Theory, Instrumentation and Applications by F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen Volume 7 Chemical Derivatization in Liquid Chromatography by J.F. Lawrence and R.W. Frei Volume 8 Chromatography of Steroids by E. Heftmann Volume 9 HPTLC - High Performance Thin-Layer Chromatography edited by A. Zlatkis and R.E. Kaiser Volume 10 Gas Chromatography of Polymers by V.G. Berezkin, V.R. Alishoyev and LB. Nemirovskaya Volume 11 Liquid Chromatography Detectors by R.P.W. Scott Volume 12 Affinity Chromatography by J. Turkovi Volume 13 Instrumentation for High-Performance Liquid Chromatography edited by J.F.K. Huber Volume 14 Radiochromatography. The Chromatography and Electrophoresis of Radiolabelled Compounds by T.R. Roberts Volume 15 Antibiotics. Isolation, Separation and Purification edited by M.J. Weinstein and G.H. Wagman Volume 16 Porous Silica. Its Properties and Use as Support in Column Liquid Chromatography by K.K. Unger
JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 15
antibiotics isolation, separation and purification
edited by Marvin J. Weinstein Microbiological Sciences, Schering-Plough Corporation, Bloomfield, N.J.
Gerald H. Wagman Microbiological Sciences, Schering-Plough Corporation, Kenilworth, N.J.
ELSEVIER SCIENTIFIC PUBLISHING COMPANY Amsterdam - Oxford - New York 1978
ELSEVIER SCIENTIFIC PUBLISHING COMPANY 336 Jan van Galenstraat P.O. Box 211, 1000 AE Amsterdam, The Netherlands Distributors for the United States and Canada: ELSEVIER NORTH-HOLLAND INC. 52,Vanderbilt Avenue New York, N.Y. 10017
ISBN 0-444-41727-3 (Vol. 16) ISBN 0-444-41616-1 (Series) @ Elsevier Scientific Publishing Company, 1975 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, P.O. Box 330, 1000 AH Amsterdam, The Netherlands
Printed in The Netherlands
The most recent developments in antibiotic chemotherapy have been accomplished through the creation of new effective agents resulting from chemical manipulations. Active substances, as well as inactive molecular fragments, have been used as substrates to accomplish dramatic changes in biological activity. New chemical reactions, developed in recent years, have enabled the research laboratory to provide a steady flow of interesting semi-synthetic antibiotics. In the aminocyclitol family, several very promising antibiotics have reached the clinic either with improved activity against resistant bacteria or with reduced toxicity. Certain derivatives have shown improvement in both of these parameters. Among the beta-lactam antibiotics, the flow of new agents is better characterized as a flood. Modification of the C-aminopenicillanic acid, 7-aminocephalosporanic acid and 7-methoxyS 7-aminocephalosporanic acid nucleus has resulted in a plethora of agents that could alter the ego of any chemist! These new derivatives are t o o numerous to name, and such an effort would likely omit one destined to be the major bullet. More recently the thienamycin, nocardicin and clavulanic acid class of beta-lactams have become the chemist's playground. We know this is not the beginning--nor is the end in sight. In earlier days, chemists tried their hands at doctoring the now-ancient warriors such as streptomycin, neomycin, the tetracyclines, chloramphenicol and erythromycin, always changing, but rarely improving the patient, literally and figuratively. The latest breed of chemist armed with new technology, new reactions and novel substrates has made remarkable strides in new, useful drug development,..and the better is yet to come! It is with these thoughts in mind that we have asked the most competent scientists in the field of antibiotic isolation to contribute to this latest vade m e c u m . We have chosen key chemical families to be represented, and in each chapter the authors write from hard personal experience. This volume anticipates a continuation of this renaissance in the field of antibiotic discovery. Old substrates will be worked again for nuggets of new activity and the only source for many of these starting materials is the fermentation vessel. For those who would venture forth in this fermentation broth, this text will present the latest techniques for transferring the hopeful substrates from the fermentor to the retort. We are convinced that a worthwhile contribution to the antibiotic armamentarium lies somewhere in the molecular manipulation of the various chemical types reviewed in this publication and will feel a certain satisfaction in having eased the way for that happening to occur.
Bloomfield, N.J., May 1978
USA
Marvin J. Weinstein Gerald H. Wagman
This Page Intentionally Left Blank
vi i
CONTENTS L i s t of C o n t r i b u t o r s
Actinomycins
....................................
vii
............................................
1
Anthony Maugeh and Edwatd K a t z
Ansamycins
..............................................
39
.............................
69
A n h i t Ganguty
Cephalosporin A n t i b i o r i c s . .
H a m i t t and L o u i n e W . C h a n d a t t
Robeht L.
Coumarin - C l y c o s i d e An t i b i o t i cs
..........................
1 01
J u l i u n Z3ehgea and Andhew V . B a t c h o
...............
2-Deoxys t r e p t a m i n e - Con t a i n i n g An t i b i o t i cs 159 J o s e p h A . Mahquez and A l l a n Kehbhnch Griseofulvins.
..........................................
215
........................
231
G e t a l d H . Wagman and M a h u i n J . W e i n n t e i n
Lincomycin R e l a t e d A n t i b i o t i c s . .
Thoman E . E b l e
Macrolide A n t i b i o t i c s
...................................
273
J a h o s t a v P. M a j e h
M a r i n e - Deri v e d A n t i b i o t i cs
..............................
L o i n S . S h i e l d and K e n n e t h L . R i n e h a h t ,
30 9 Jh.
................... 387
P e n i c i l l i n s and R e l a t e d A n t i b i o t i c s . . B a h i d 8 . Mukhehjee and Bong K .
Lee
-
Peptide Antibiotics Plant-Derived
.....................................
415
.............................
46 3
Antibiotics..
Eh h ahd Gh o n n
Lenteh A.
Mitbcheh
.............. 479 ......................................... 5 2 1 Hubeht Maeht
Polyether Antibiotics.....................
R o b e t t L. HamiPP. and L o u i n e W . C h a n d a t l
Siderochromes..
......................
S t r e p t a m i n e - Con t a i n i n g A n t i b i o t i cs 587 V a u i d Pettman and Y a d u a k i Ogawa
S t r e p t o t h r i c i n s and R e l a t e d A n t i b i o t i c s . .
............... 6 1 7 A . S . Kh o k h t o v
Tetracyclines
...........................................
S a u t Neidteman
S u b j e c t I n d e x ..............,............................761
715
This Page Intentionally Left Blank
ix
LIST OF CONTRIBUTORS Andrew D. Batcho, Chemical Rcbeahch Depahtment, Hoddmann-La
Roche, l n c . , N u t l e y , New Jehbey J u l i u s B e r g e r , Depahtment 0 6 MichobioLogy, H066mann-LaRoche, I n c . , N u t t e y , New Jehbey. Louise W .
C r a n d a l l , Fehmentax%on Phoductb Rebeahch, ELL L i U y
and Co.
, l n d i a n a p o t i b , 'Indiana.
Thomas E b l e , The Upjohn Company, KaLamazoo, Michigan. A s h i t Ganguly, ChemicaL Rebeahch, Schehing C O h p O h U t i O M ,
Bloom6ietd, New Jehbey. Erhard Gross, S e c t i o n on MoLecuLah S t h u c t u h e , N a t i o n a l
Znbtitute
06
HeaLth, Bethebda, Mahyland.
Fehmentation Phoductb Rebeahch, ELL L i L L y lndianapotib, Indiana.
Robert L . H a m i l l ,
and C o . ,
Edward Katz, SchooL
0 6 Medicine and v e n t i d t h y , Geohgetown U n i v e h b i t y , Wabhington, V . C .
A l l a n Kershner, A n t i b i o t i c b Red e a h c h , Schehing Cohpohation,
BLoom6ieLd, New J e h d e y .
A. S . Khokhlov, USSR Academy 06
0 6 S c i e n c e b , Shemyakin ' I n b t i t U t e Bio-ohganic Chemibthy, Mobcow, USSR.
Lee, A n t i b i o t i c b Rebeahch, Schehing Cohpohation, 860o m d i e l d , New 3ehb e y .
Bong K .
Hubert Maehr, Depahtment
0 6 Michobiotogy, Haddmann- La Roche, Tnc., N u t l e y , New Jehbey.
06 BLochemibthy, S c h o o l 06 Medicine and D e n f i b t h y , Nohthwebtehn U n i V e h b i t y , Chicago, ILlinoib
J a r o s l a v P . Majer, Depahtment
.
Joseph A. Marquez, A n t i b i o t i c b Rebeahch, Schehing Cohpohation,
gLoom6ieLd, New Jehbey. AntKony Mauger, Rebeahch Foundation a d t h e Wabhington H o b p i t a t
Cenzeh, Wadhington, V . C . L e s t e r A. M i t s c h e r , Depahdment
School
06
0 6 Natuhal Phoductb Chemibthy, Phahmacy, U n i v e h b i t y 0 6 Kanbah, Lawhence, Kanbab.
B a r i d B . Mukherjee, Univehbidad Autonoma de Ciudad Juahez
EbcueLa de M e d i d n a , J u a h e z , Chihuahua, Mexico. Saul Neidleman, CetUb Cohpoaation, BehkeLey, CaLi6ohn.h. Yasuaki Ogawa, C e n t h a l Rebeahch Labohatohied, M e i j i Seika
Kaidha L t d . ,
Yokohama, Japan.
X
David P e r l m a n , D e p a h t m e n t 06 P h a a m a c e u t i c a L B i o c h e m i b t h y , S c h o o L 06 P h a a m a c y , U n i v e h b i t y 06 W i b c o n b i n , M a d i d o n , Wid condin. Kenneth R i n e h a r t , Jr., D e p a h t m e n t 0 6 C h e m i b t h y , U n i v e h b i t y 06 I L L i n o i d , Uhbana, I t e i n o i b .
Lois S . S h i e l d , D e p a h t m e n t 0 6 C h e m i b t h y , U n i v e h d i t y TLLinoi6, Uabana, I L L i n o i 6 . G e r a l d H. Wagman, M i c h o b i o C o g i c a L S c i e n c e 4 C o a p o h a t i o n , B L o o m 6 i e L d , New J e h b e y .
06
, Schehing
Marvin J. W e i n s t e i n , M i c h o b i o L o g i c a L S c i e n c e b , S c h e a i n g C o a p o h a t i o n , B L o o m d i e l d , New J e h b e y .
1 Actinomycins Anthony B. Mauger and Edward Katz Research Foundation of the Washington Hospital Center, Washington, D. C. and Department of Microbiology, Georgetown University Schools of Medicine and Dentistry, Washington, D. C.
1.
Techniques for separation of actinomycin mixtures Introduction Biological activity 1 ;3 Recovery of actinomycin mixtures. 1.4 Countercurrent distribution 1.5 Paper chromatography 1.6 Column chromatography 1.6.1 Cellulose powder 1.6.2 Sephadex. 1.6.3 Alumina. 1.6.4 Silicic acid 1.7 Thin-layer chromatography 1.8 High-pressure liquid chromatography -
1.1
-
2.
................................ ......................... ........... ................. ........................ ....................... .......................... ................................. .................................. .............................. ................... .........
Methods for identification of amino acids from actinomycins 2.1 Introduction., 2,2 Paper chromatography.. 2.3 High-voltage paper electrophoresis 2.4 Thin-layer chromatography 2.5 Ion-exchange chromatography., 2.6 Gas-liquid chromatography Stereochemistry of the amino acids from 2.7 actinornycins 2.8 Amino acid sequence in actinomycins -
.............................. ...................... .......... ................... ............... ................... ................................ .........
3,
4. 5.
6.
The structure of the actinomycin chromophore..
....
.............. Abbreviations.. ................................... Literature cited .................................. Nuclear magnetic resonance studies..
2 8 8 9
9
12 12 14 14 15 15 16
17 17 20 22 22 25
26 27 29
32 33
34
2
1. Techniques for Separation of Actinomycin Mixtures 1.1 Introduction -
The actinomycins are a series of chromopeptide lactone antibiotics with a common structural format differing only in certain amino acid residues (Fig. 1; Table 1, following pagell,
MeVal
MeVal
‘1
Sar Pro
I
D-Val
I
Thr
F i g u r e 1.
co
co
CH3
CH3
S t r u c t u r e o f actinomycin D
The chromophore, actinocin, is Z-amino-4,6-dimethylphenoxazinone (3)-1,9-dicarboxylic acid. Examination of the structures in the table reveals the extent of the variations in amino acid composition of the naturally occurring actinomycins. Thus, in site 1 of the peptide chain, L-threonine may be replaced by a amino-$,y-dihydroxybutyric acid (hydroxythreonine); in site 2 there may be present D-valine and/or D-alloisoleucine. L-Proline, in site 3 , may be substituted f o r b y 4 - h y d r o x y - L - p r o l i n e , 4-keto-L-proline, sarcosine, 4-keto-5-methylproline, 5-methylproline or 3-hydroxy-5-methylproline. Site 4 is always occupied
TABLE 1 Amino A c i d Sequences o f Actinomycins S i t e o f Actinomycin Peptides Actinomycins produced by S-tkep.tomyceb species 1, A I ’ 61’ XOB
‘la
c2€l
1 -
2 -
3 -
4 -
-5
Thr Thr
Val Val
Pro HyPro
Sar Sar
MeVal MeVal
Thr Thr
Val Val
-aHyPro
Pro
Sar Sar
MeVal MeVal
Thr Thr
Val Val
Sar Sar
Sar Sar
MeVal MeVal
Thr Thr
Val Val
Pro Sar
Sar Sar
MeVal MeVal
Thr Thr
Val Val
Pro Pro
Sar Sar
MeVal MeVal
Thr Thr
Val Val
Sar KetoPro
Sa r Sar
MeVal MeVal
Thr Thr
Val Val
Pro KetoPro
Sar Sar
MeVal MeVal
Thr Thr
Va 1
Pro Pro
Sar
aIleu -
Sar
MeVal MeVal
a1l e u -
Pro
Sar Sar
MeVal MeVal
Thr Thr
Val
Pro
w
TABLE 1 (Continued)
Actinomycins produced by Sa2e.pZomycu species
S i t e o f Actinomycin Peptides
1 -
2 -
3 -
4 -
Thr Th r
aIleu aIleu -
Pro Pro
Sar Sar
MeVal MeVal
Thr Hy Th r
Val Val
4-Keto-5-MePro 3-Hy-5-MePro
Sar Sar
MeVal MeAla
Thr
Val Val
4-Keto-5-MePro 5-MePro
Sar
HyThr
Sa r
MeVal MeAla
Schering l a
Thr Thr
Val Val
4-Keto-5-MePro 5-MePro
Sar Sa r
MeVal MeVal
Schering l b
Thr Thr
Val Val
4-Keto-5-MePro 5-MePro
Sar
Sar
MeV a1 MeAla
Thr Thr
Val Val
4-Keto-5-MePro 3-Hy-5-MePro
Sa r
MeVal MeVal
Thr Thr
Val
V a1
4-Keto-54ePro 3-Hy-5-MePro
Sar Sar
MeVal MeAla
Th r HyThr
Val Val
4-Keto-5-MePro 5-MePro
Sar Sar
MeVal MeAla
Thr Thr
V a1
Sar Sa r
MeVal MeVal
VII,
c3
z1
5 -
Actinomycins produced by
Mktomonohpom & C o ~ d ~ m ~
Shering l c
S h e r i n g 6a
Schering 10
Sar
D i r e c t e d b i o s y n t h e s i s with pipecolic acid Pip 2
Val
Pip Pip
TABLE 1 (Continued) Directed biosynthesis with pipecolic acid
S i t e o f Actinomycin Peptides
1 -
2 -
-3
-4
P i p 18
Thr Thr
Val Val
Pip Pro
Sar Sar
MeVal MeVal
Pip l a
Thr Thr
Val Val
4-Ke t oPip Pip
Sar Sar
MeVal MeV a1
P i p 16
Thr Thr
Val Val
4 -Ke t oPip Pro
Sar Sar
MeVal MeVal
Th r Th r
Val Val
4-HyPip Pip
S ar
MeVal MeVal
Th r Thr
Val Val
4-HyPip Pro
S ar Sar
MeVal MeVal
Thr Thr
a1 l e u -
Sar Sar
Sar Sar
MeV61 MeVal
Thr Thr
a1l e u Val
Sar Pro
Sa r Sar
MeVal MeVal
Thr Th r
aIleu aIleu
Sar
Sar
Sar Sar
MeVal MeVal
Thr Thr
aIleu aIleu
Sar Pro
Sar S ar
MeVal MeVal
P i p ly P i p 1~
5ar
5 -
Directed biosynthesis w i t h sarcosine F1
F2
F3
F4
Val
TABLE 1 (Continued)
Directed b i o s y n t h e s i s with sarcosine
S i t e o f Actinomycin P e p t i c k s
-1
-2
-3
-4
-5
See actinomycin I1
Fa
See actinomycin 111 Directed b i o s y n t h e s i s with isoleucine stereoisomers*
El (S.
ChZgbOItIaeeub)
Pro
Sar
MeVal
Thr
{a1l e u 1 C Val 1 C Ileu3
Pro
Sar
MeaIleu
Th r Thr
-a II ll ee uu
Pro Prcl
Sar Sar
MeaI l e u MeaIleu
Thr Thr
Ileu
Ileu
Pro Pro
Sar Sar
MegI l e u
Thr Thr
Val Val
Sar Sar
MeVal MeVal
Thr Thr
Val Val
Sar Sar
MeVal MeVal
Thr Thr
Val Val
Sar Sar
MeVal MeVal
Thr Thr
V a1
Sar Sar
MeVal
Thr
MeEIleu
D i r e c t e d b i o s y n t h e s i s with o r .OLUYI&-4-methylproline
d.A
KlC
Vel
MeVal
TABLE 1 (Continued)
Directed biosynthesis with azetidine-2-carboxylic a c i d
S i t e o f Actinomycin Peptides
1
2
3
4
Thr Thr
Val Val
Pro Aze t
Sar Sa r
MeVal MeVal
Th r Thr
Val Val
Azet Aze t
Sar Sar
MeVal MeVal
oid-chloro A
Thr Thr
Val Val
Ci6-4-clPro d-4-ClPro
Sa r Sar
MeVal MeVal
oid-chloro B
Thr Thr
Val Val
Pro Ci6-4-ClPro
Sar Sar
MeVal MeVal
ZtW-chloro A
Th r Thr
Val Val
Ztunb-4-ClPro .ikzn~-4-C1Pro
Sar Sar
MeVal MeVal
Ztthavls-chloro B
Th r Thr
Val Val
Pro ahan.h-4-ClPro
Sar Sar
MeVal MeVal
Azetomycin
I, AzB
Azetomycin 11, Aza
5
Directed biosynthesis with
Ci6- o r ~ n ~ - 4 - c h l o r o p r o l i n e
.*Recent i n v e s t i g a t i o n s w i t h i s o l e u c i n e stereoisomersbb do n o t c o n f i r m an e a r l i e r report3'.
8
by s a r c o s i n e ; f i n a l l y , t h e amino a c i d a t s i t e 5 , N-methyl-Lv a l i n e , can be r e p l a c e d by N - m e t h y l a l a n i n e i n some a c t i n o m y c i n s o f t h e Z s e r i e s . Amino a c i d s u b s t i t u t i o n s i n t h e p e p t i d e c h a i n s a r e responsible f o r t h e q u a n t i t a t i v e d i f f e r e n c e s observed i n t h e b i o l o g i c a l a c t i v i t i e s of n a t u r a l a c t i n o m y c i n s a n d t h o s e formed via directed biosynthesis. With t h e e x c e p t i o n o f a c t i n o m y c i n D (Am D), t h e n a t u r a l l y o c c u r r i n g a c t i n o m y c i n p r e p a r a t i o n s were shown by t h e t e c h n i q u e s d e s c r i b e d h e r e t o b e m i x t u r e s o f a t l e a s t t h r e e components. I n i t i a l l y , s u c h complexes were d e s i g n a t e d as A , B y C , D , X , e t c . a c c o r d i n g t o t h e p r o d u c i n g o r g a n i s m . However, A , B a n d X cont a i n t h e same components (I-V) and d i f f e r o n l y i n t h e i r r e l a t i v e a b u n d a n c e , which c a n a l s o v a r y w i t h c u l t u r e c o n d i t i o n s . The s o l e component of Am D ( = I V ) i s p r e s e n t i n a l l t h e abovementioned complexes. The 2 complex, i n c o n t r a s t , h a s no compon e n t s i n common w i t h t h e above m i x t u r e s . In addition t o the n a t u r a l l y p r o d u c e d a c t i n o m y c i n complexes, m i x t u r e s c o n t a i n i n g n o v e l components have b e e n produced by d i r e c t e d b i o s y n t h e s i s , i n which an added amino a c i d o r amino a c i d a n a l o g u e competes f o r incorporation i n t o s p e c i f i c s i t e s i n the peptide moieties'. S e p a r a t i o n of m i x t u r e s of s u c h c l o s e l y - r e l a t e d compounds r e p r e s e n t s a f o r m i d a b l e t a s k . P a r t i t i o n chromatography h a s i n g e n e r a l p r o v e d more u s e f u l t h a n a d s o r p t i o n c h r o m a t o g r a p h y , p a r t i c u l a r l y i n t h e s e p a r a t i o n of components d i f f e r i n g o n l y i n a l k y l s i d e c h a i n s ( f o r example, D - a U o i s o l e u c i n e i n p l a c e o f Dv a l i n e ) . H i s t o r i c a l l y , t h e f i r s t s e p a r a t i o n s (1951) were a c c o m p l i s h e d by c o u n t e r c u r r e n t d i s t r i b u t i o n , w i t h p a r t i t i o n chromatography on p a p e r o r on columns of c e l l u l o s e o r Sephadex d e v e l o p e d soon t h e r e a f t e r . More r e c e n t d e v e l o p m e n t s i n c l u d e t h e u s e of t h i n - l a y e r and h i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h y . A b b r e v i a t i o n s u s e d i n t h i s c h a p t e r w i l l be f o u n d i n S e c t i o n 5.
1 . 2 Biological
activity
I n v e s t i g a t i o n s c o n c e r n i n g t h e mode o f a c t i o n of Am D h a v e r e v e a l e d t h a t t h e a n t i b i o t i c i s a p o t e n t i n h i b i t o r o f deoxyConsequently, t h e r i b o n u c l e i c a c i d - d e p e n d e n t RNA s y n t h e s i s . a n t i b i o t i c h a s become a n e x t r e m e l y u s e f u l p r o b e f o r i n v e s t i g a t i o n s r e l a t i n g t o t h e s y n t h e s i s o f m e s s e n g e r RNA and p r o t e i n as w e l l as v i r u s r e p l i c a t i o n . C l i n i c a l l y , Am D i s o f c o n s i d e r a b l e i m p o r t a n c e i n t h e t r e a t m e n t o f W i l m ' s tumor, g e s t a t i o n a l c h o r i o c a r c i n o m a a n d mixed metastatic c a r c i n o m a o f t h e t e s t e s .
1 . 3 Recovery
of a c t i n o m y c i n m i x t u r e s
The r e c o v e r y of a c t i n o m y c i n s from c u l t u r e f i l t r a t e s a f t e r f e r m e n t a t i o n s h a s u s u a l l y b e e n e f f e c t e d by e x t r a c t i o n w i t h o r g a n i c s o l v e n t s s u c h as e t h y l a c e t a t e o r b u t y l a c e t a t e . The mycelium which g e n e r a l l y c o n t a i n s r e l a t i v e l y s m a l l amounts o f t h e a n t i b i o t i c s may b e e x t r a c t e d w i t h z - b u t a n o l o v e r n i g h t . A f t e r evaporation under reduced p r e s s u r e , t h e crude actinomycin m i x t u r e s s o o b t a i n e d are t h e n s u b j e c t e d t o t h e p r o c e d u r e s d e s c r i b e d below,
9
1.4 Countercurrent -
distribution
The f i r s t d e m o n s t r a t i o n of t h e m u l t i p l i c i t y o f a n a c t i n o mycin p r e p a r a t i o n was made w i t h t h e C complex ( C 1 + C p + C 3 ) using c o u n t e r c u r r e n t d i s t r i b u t i o n with t h e s o l v e n t system, ether/5.6% hydrochloric acid (system.5, Table ? I 3 . A s t h e r e s o l u t i o n o f t h i s complex was r e l a t i v e l y p o o r a n d t h e r e was e x t e n s i v e d e g r a d a t i o n o f t h e a c t i n o m y c i n s , more e f f e c t i v e s y s t e m s ( T a b l e 2 , n e x t p a g e ) , u s u a l l y h a v i n g a sodium a r y l s u l f o n a t e as s o l u b i l i z i n g a g e n t i n t h e a q u e o u s p h a s e , were i n t r o duced. This methodology r e s u l t e d i n u s e f u l p r o c e d u r e s f o r t h e p r e p a r a t i v e i s o l a t i o n o f t h e i n d i v i d u a l components o f t h e C a n d X c o m p l e x e s 4 y 5 . The C complex h a s b e e n s e p a r a t e d w i t h a l l t h e s o l v e n t s y s t e m s l i s t e d ( e x c e p t j ) 3 - 8 . The X complex was r e s o l ~ e d ~ w i, t~h s y s t e m a n d a p a r t i a l s e p a r a t i o n of t h e 2 comp l e x g was o b t a i n e d w i t h s y s t e m
i.
1 . 5 Paper
c h r o m a t o g r a p h y (PC)
All t h e s u c c e s s f u l PC p r o c e d u r e s f o r t h e s e p a r a t i o n o f actinomycin m i x t u r e s have involved p a r t i t i o n systems h a v i n g a s o l u b i l i z i n g a g e n t i n t h e a q u e o u s p h a s e , Such a g e n t s h a v e i n c l u d e d v a r i o u s a r o m a t i c s u l f o n i c a c i d s a n d t h e i r sodium s a l t s , a n d sodium 0- or m - c r e s o t i n a t e ( s e e T a b l e 3 ? p a g e f o l l o w i n g Table 2), The t e c h n i q u e r e q u i r e s i m p r e g n a t i o n of t h e p a p e r w j t h The a q u e o u s p h a s e p r i o r t o a p p l i c a t i o n o f t h e a c t i n o m y c i n m i x t u r e , u s u a l l y i n a c e t o n e s o l u t i o n . Most o f t h e m o b i l e p h a s e s u s e d ( T a b l e 3 ) h a v e c o n s i s t e d of b i n a r y m i x t u r e s of a l e s s p o l a r ( u s u a l l y n - d i b u t y l e t h e r ) a n d more p o l a r o r g a n i c s o l v e n t , t h e c o m p o s i i i o n o f which c a n b e v a r i e d a c c o r d i n g t o t h e p a r t i c u l a r m i x t u r e u n d e r s t u d y . For e x a m p l e , Rf v a l u e s c a n b e i n c r e a s e d by a r e d u c t i o n i n t h e n - d i b u t y l e t h e r c o n t e n t o f t h e m o b i l e p h a s e . The b e s t a n a l y t i c a l s e p a r a t i o n s h a v e b e e n o b t a i n e d by t h e c i r c u l a r c h r o m a t o g r a p h i c t e c h n i q u e , w i t h t h e d e s c e n d i n g p r o c e d u r e p r e f e r r e d f o r p r e p a r a t i v e work. The n a r r o w z o n e s o b t a i n e d on c i r c u l a r p a p e r c h r o m a t o g r a m s f a c i l i t a t e comp a r i s o n s of d i f f e r e n t m i x t u r e s and t h e i r s e p a r a t e d components. The e a r l i e s t PC s t u d i e s o f a c t i n o m y c i n s w e r e a p p l i e d t o t h e C complex7310 w i t h s o l v e n t systems a and affording separ a t i o n i n t o t h r e e components ((21, C CT). Subsequently, a f o u r t h component ( C z a ) was d e t e c t e d ” u s 1 n g s y s t e m s h a n d k Comparisons of t h e A , B, C, D and X complexes were ? i ~ a d e ~ ~ - ~ ~ w i t h s o l v e n t systems b and T h e s e i n v e s t i g a t i o n s demons t r a t e d t h e r e l a t i o n s F i p s b e t w e e n t h e v a r i o u s a c t i n o m y c i n comp l e x e s a n d i n t r o d u c e d t h e more g e n e r a l l y u s e f u l a q u e o u s p h a s e o f sodium n - c r e s o t i n a t e . The Z complex was r e s o l v e d i n t o s i x c o m p o n e n t s g w i t h s y s t e m d. C h e m i c a l c o n v e r s i o n s o f a c t i n o m y c i n X 2 i n t o X I , X o B and X o 6 w e r e i n v e s t i g a t e d 1 6 u s i n g s y s t e m 1 a n d
c,
s.
0. -
The f i r s t s t u d i e s o f d i r e c t e d b i o s y n t h e s i s w e r e c o n d u c t e d w i t h i s o l e u c i n e u s i n g Stheptomyced chhydoma~tud (system c ) a n d w i t h s a r c o s i n e a n d t h e o r g a n i s m s S. c h h y d o m a ~ L ~ 4 ~ ~ ~ ~ ~ ( s y s t e m m ) a n d S . a n t i b i o t i c u ~ ’ ~? ’s y~s ~t e m i ) . I n t h e l a t t e r case, in-which o n e or b o t h p r o l i n e s a r e r e p l a c e d by s a r c o s i n e ( a c t i n o m y c i n s I11 a n d 11, r e s p e c t i v e l y ) , i t was p o s s i b l e t o
P 0
TABLE 2 Solvent Systems f o r Countercurrent D i s t r i b u t i o n Organic Phase
Aqueous Phase
diethyl ether
5.6% h y d r o c h l o r i c a c i d
methyl b u t y l e t h e r / n - d i b u t y l e t h e r (7:3)
30% urea
C -
methyl b u t y l e t h e r / n - d i b u t y l e t h e r (3:2)
5% Na p x y l o l s u l f o n a t e
-d
methyl b u t y l e t h e r
1.5 or 1.75% Na naphthalene-Bs u l f onate
e -
cy clohexane / m y 1 ace t ate (1:l or 4:6)
5% Na naphthalene-6-sulfonate
cyclohexane/benzene
Methanol/water (7:3)
1
(1:l)
Ref. 3 697
i s o a n y l acetate
5% Na naphthalene-8-sulfonate
carbon t e t r a c h l o r i d e
Methanol/water
carbon t e t r a c h l o r i d e
2.5 or 4.5% Na naphtha1ene-Bs u l f onate
diisopropyl ether/ethyl acetate ( 4 : l )
2 % Na naphthalene-6 -sulFonate
6
495,7 8
(4: 1)
9
TABLE 3
Solvent Systems f o r Paper Chromatography Ref. -
S t a t i o n a r y (aqueous) Phase
Mobile ( o r g a n i c ) Phase n-dibutyl ether
10% Na naphthalene-116-disulfonate
10
n - d i b u t y l e f h e r / e t h y l acetate ( 3 : l )
2% naphthalene-B-sulfonic
11
n - d i b u t y l ether/n-butanol
(3: 2)
acid
10% Na p c r e s o t i n a t e
7,17
n - d i b u t y l e t h e r / b u t y l acetate (1:3)
10L Na p c r e s o t i n a t e
n - d i b u t y l e t h e r / e t h y l acetate (1:3)
10% Na g - c r e s o t i n a t e
71 12 23
n - d i b u t y l e t h e r / e t h y l acetate (1:l)
2% p t o l u e n e s u l f o n i c a c i d
n - d i b u t y l e t h e r / e t h y l acetate (1:3)
2% 1-hydroxynaphthalene-2-sulfonic
13 acid
13
n - d i b u t y l e t h e r / n - b u t a n o l (3:2 o r 7 : l )
10% Na g - c r e s o t i n a t e
14,22
n - d i b u t y l ether/s-tetrachloroethane (2: 1-5: 1)
10X Na p c r e s o t i n a t e
15,19 ,201
n - d i b u t y l ether/n-butanol
(5:l)
10% t h i o u r e a
8
n - d i b u t y l ether/n-butanol
(5: 1)
2% Na naphthalene-B-sulfonate
8
isoamyl acetate
5% Na naphthalene-B-sulfonate
8
n-dibutyl etherjn-dipropyl ether/ n-butanol (7:1:2)
7% Na p c r e s o t i n a t e
amyl acetate
8.5% Na m-cresotinate
b u t y l acetate b u t y l acetate/n-dibutyl amyl a c e t a t e / n - d i b u t y l
ether (3:l) ether (3:l)
amyl acetate d i i s c p r o p y l ether/chloroform
(3:2)
21,24
16,17,18 18
10% Na p c r e s o t i n a t e
16
10% Na o - c r e s o t i n a t e
22
10% Na 2 - c r e s o t i n a t e
22
5% Na 2 - c r e s o t i n a t e
24
10% Na 2 - c r e s o t i n a t e
24
o b s e r v e n o t o n l y a s e p a r a t i o n of components I - V , b u t , an o c c a s i o n , a r e s o l u t i o n o f 111 i n t o i t s two isomers. I s o l a t i o n o f a c t i n o m y c i n s I1 a n d I11 f r o m t h e l a t t e r m i x t u r e was e f f e c t e d by p r e p a r a t i v e PCpl ( s y s t e m i ) . S u b s e q u e n t l y , a z e t i d i n e - 2 - c a r b o x y l i c a c i d and p i p e c o l i c a c i d were a l s o shown ( s y s t e m i ) t o be i n c o r p o r a t e d by S . a n t i b i o t i c u d w i t h t h e f o r m a t i o n of-new a c t i n o m y c i n s Z 0 . The p r e p a r a t i v e s e a r a t i o n o f t h e p i p e c o l i c a c i d - s t i m u l a t e d a c t i n o m y c i n complexg2 r e q u i r e d a c o m b i n a t i o n of Recent column a n d PC w i t h s e v e r a l s o l v e n t s y s t e m s ( h , $1. s t u d i e s i n c l u d e t h e u s e o f p r e p a r a t i v e PC f o r t e i s o l a t i o n o f a c t i n o m y c i n s p r o d u c e d by i n c o r p o r a t i o n o f a z e t i d i n e - 2 - c a r b o x y l i c a c i d z 3 ( s y s t e m e ) and ~ i A - 4 - m e t h y l p r o l i n e ~( s~y s t e m r ) , C i r c u l a r PC a n a l y s i s - o f m i x t u r e s produced by s. palrvutub w i t h b o t h ciA- and t l r a n A - 4 - m e t h y l p r o l i n e ~ ~h a~v e u t i l i z e d s y s t e m s a n d 2.
8,
i,
PC h a s been employed i n t h e s e p a r a t i o n of r a d i o l a b e l e d a c t i n o m y c i n s r e s u l t i n g from b i o s y n t h e t i c e x p e r i m e n t s . A f t e r i n c o r p o r a t i o n o f 1 4 C - L - p r o l i n e o r 3H-L-hydroxyproline b y S . a n t L b i o t i c u A a c t i n o m y c i n s I , I V and V were s e p a r a t e d by PC (system i)25:26S . i m i l a r l y , various 14C-labeled i s o l e u c i n e i s o m e r s were i n c o r p o r a t e d by s. chhyAomaLLuA a n d t h e r a d i o a c t i v e a c t i n o m y c i n s ( C l + C2 + C J + E l + E p ) were r e s o l v e d u s i n g s y s t e m
s27.
1 . 6 Column 1.6.1
chromatography C e l l u l o s e powder
The t e c h n i q u e s used f o r p a r t i t i o n c h r o m a t o g r a p h i c s e p a r a t i o n s o f a c t i n o m y c i n m i x t u r e s on c e l l u l o s e powdeb columns f o l low t h e same p r i n c i p l e s a s t h o s e a p p l i e d t o PC. T h u s , t h e c o l umn is f i r s t t r e a t e d w i t h t h e a q u e o u s p h a s e , which u s u a l l y cont a i n s sodium m- o r o - c r e s o t i n a t e , and i s t h e n e q u i l i b r a t e d w i t h t h e mobile phzse. s b l e 4 ( n e x t page) l i s t s t h e s o l v e n t systems which have b e e n employed. I n d i v i d u a l actinomycins have u s u a l l y been r e c o v e r e d by e x t r u s i o n and e x t r a c t i o n from a p p r o p r i a t e zones of t h e c e l l u l o s e column a f t e r t h e s o l v e n t h a s been removed by a f l o w o f a i r . Se a r a t i o n o f t h e Am C complex i n t o t h r e e components was ( s y s t e m s d, 2, d ) a n d a more c a r e f u l s t u d y z 9 p e r m i t t e d i s o l a t i o n of a f o u r t 5 component, C p a ( s y s t e m a ) . Ext e n s i v e work w i t h b o t h c e l l u l o s e ( s y s t e m s 5 a n d 5) a n d s i l i c i c a c i d (see b e l o w ) columns was n e c e s s a r y t o s e p a r a t e t h e major ( X O B , X , X 2 ) a n d minor X g a , Xla, X J , XI,) components of t h e X c o m p l e ~ ~ ~ Chemical 1 ~ ~ ~c o n~v e~r s i o~n s~ of~ X 2~ to ~ X )O B ,, X ' J ~ a n d X 1 a l s o i n v o l v e d t h e u s e of c e l l u l o s e columns f o r s e p a r a t i o n and p u r i f i c a t i o n of the products16 (system e l . Separation and c h a r a c t e r i z a t i o n o f t h e m a j o r components (17 I V , V ) o f t h e A and B complexes, which resemble t h e X complex, was a l s o a c h i e v e d v i a c e l l u l o s e powder column c h r o m a t ~ g r a p h y ' (~s y s t e m b ) . A p a r t i a l s e p a r a t i o n of t h e Z complex ( s y s t e m 2) p e r m i t T e d i s o l a t i o n o f Am 21, a n d , a f t e r a c e t y l a t i o n , i t s mono- a n d d i a c e t a t e were s e p a r a t e d on a c e l l u l o s e column32 ( s y s t e m g).
TABLE 4 Solvent Systems f o r P a r t i t i o n Chromatography on C e l l u l o s e o r Sephadex" Columns Mobile (organic) Phase
-a
S t a t i o n a r y (aqueous) Phase
n - d i b u t y l ether/n-butanol (3:2 o r 4 : l )
10% Na m-cresotinate
b
n-dibutyl ether/s-tetrachloroethane ( 4 : l o r 5:l)
lo?;
C
n - d i b u t y l ether/n-butanol
d
Ref. -
7,28,29, 30,34
(5:l)
Na o - c r e s o t i n a t e
15,19
2% Na naphthalene-8-sulfonate
8
isoamyl acetate
5% Na n a p h t h a l e n e - B s u l f o n a t e
8
e
b u t y l acetate
10L Na m-cresotinate
f
b u t y l acetate/n-dibutyl (3:l)
ether
10% Na p c r e s o t i n a t e
9
b u t y l acetateh-dibutyl ether (3:l)
10% Na 2 - c r e s o t i n a t e
"Systems
2
and
9 have
16,28,31,32
28,32
22
been used w i t h Sephadex.
P W
14 C e l l u l o s e columns have a l s o been used t o s e p a r a t e a c t i n o mycins g e n e r a t e d by d i r e c t e d b i o s y n t h e s i s . By t h i s means, a c t i n o m y c i n s I 1 and I11 were i s o l a t e d ( s y s t e m b ) from a m i x t u r e produced by s a r c o s i n e - s t i m u l a t i o n of S . anaXbiZ.ticub 1 9 . Addit i o n of L - l e u c i n e t o A c t i n o m y c e d 6Luoked t e n d c u l t u r e s r e s u l t e d i n a m i x t u r e o f a c t i n o m y c i n s o f which some components c o n t a i n e d D - l e u c i n e i n p l a c e o f D-aLLoisoleucine; t h i s m i x t u r e was r e s o l v e d on a c e l l u l o s e ~ o l u m n ~ ~ d( en toa i l s g i v e n ) . 1.6.2
Sephadex
Sephadex G 2 5 has been u t i l i z e d i n t h e same manner a s c e l l u l o s e a s a s u p p o r t f o r t h e aqueous p h a s e i n p a r t i t i o n column c h r o m a t o g r a p h i c s e p a r a t i o n s of a c t i n o m y c i n s . T h i s c o n c e p t was s u c c e s s f u l l y i n v e s t i g a t e d i n a s e p a r a t i o n of t h e C ( s y s t e m a ) . P a r t i a l s e p a r a t i o n s o f t h e h i g h l y complex a c t i n o mycin mixture s y n t h e s i z e d by p i e c o l i c a c i d s t i m u l a t i o n of S . a n t i b i o t i e u d were a l s o r e p o r t e d g 2 w i t h Sephadex 6 2 5 columns ( s y s t e m g ) . F o r t h e s e p a r a t i o n of t h e s i m p l e r m i x t u r e ( I V + P i p 18 +-Pip 2 ) produced by S . pahvuLud w i t h p i p e c o l i c a c i d i n t h e medium, p a r t i t i o n chromatography ( s y s t e m g ) on a c e l i t e column was e f f e c t i v e 3 5 . 1.6.3
Alumina
Chromatography on a l u m i n a columns h a s been employed f o r ( i ) p u r i f i c a t i o n of c r u d e a c t i n o m y c i n m i x t u r e s a f t e r t h e i r ext r a c t i o n from t h e c u l t u r e b r o t h o r mycelium, ( i i ) p u r i f i c a t i o n of i n d i v i d u a l components a f t e r s e p a r a t i o n by p a r t i t i o n t e c h n i q u e s , ( i i i ) s e p a r a t i o n of a c t i n o m y c i n m i x t u r e s , The f i r s t a c t i n o m y c i n p r e p a r a t i o n , d e s i g n a t e d a c t i n o m y c i n A from S. ( A c t i n o m y c c b ) a n t i b i o t i c u b , was p u r i f i e d by a d s o r p t i o n on a column o f a l u m i n a i n benzene, f o l l o w e d by e l u t i o n w i t h i n c r e a s i n g c o n c e n t r a t i o n s (15-30%) of a c e t o n e i n b e n z e n e 3 6 . Simil a r l y , t h e Am C complex was p u r i f i e d on a column of alumina (Brockmann a c t i v i t y 11) u s i n g benzene f o l l o w e d by e l u t i o n w i r h e t h y l a c e t a t e 3 . The same g r o c e d u r e was u t i l i z e d t o p u r i f y i n d i v i d u a l components of t h e C ,?, X7, and I7 complexes f o l l o w i n g t h e i r s e p a r a t i o n by c o u n t e r c u r r e n t d i s t r i b u t i o n . More r e c e n t l y , alumina (Brockmann a c t i v i t y I V ) o r "acid-washed alumina" 2 1 7 2 4 9 3 7 h a s been p r e f e r r e d f o r p u r i f i c a t i o n of a c t i n o m y c i n s . The e f f i c a c y of a l u m i n a columns f o r s e p a r a t i o n of a c t i n o mycins depends upon t h e t y p e o f m i x t u r e under s t u d y . I f t h e components d i f f e r o n l y i n t h e a l k y l s i d e c h a i n s of amino a c i d r e s i d u e s , as i n t h e C complex (D-aLLoisoleucine v d , D - v a l i n e ) , t h i s method i s i n e f f e c t i v e and p a r t i t i o n chromatography i s p r e f e r r e d . Where t h e r e a r e f u n c t i o n a l g r o u p d i f f e r e n c e s , a s i n t h e X o r B complexes ( h y d r o x y p r o l i n e , p r o l i n e o r 4 - k e t o p r o l i n e ) , t h e u s e of a d s o r p t i o n chromatography on s i l i c i c a c i d ( s e e below) o r alumina h a s been s u c c e s s f u l , A p a r t i a l s e p a r a t i o n of t h e X complex was o b t a i n e d on alumina 11; a f t e r e l u t i o n of X 1 and X 2 w i t h e t h y l a c e t a t e , t h e X o f r a c t i o n was e l u t e d w i t h a c e t o n e and s e p a r a t e d f u r t h e r on c e l l u l o s e c 0 1 u m n s ~ 3 1 y~ ~ 38. I s o l a t i o n of minor components s u c h a s Xla a l s o depended on a combination o f c e l l u l o s e powder and alumina chromatographyz8. S e p a r a t i o n of
15
actinomycins X I and X 6 resulting from catalytic hydrogenation (platinum/acetic acid? of X2 was accomplished on alumina I1 by elution first with ethyl acetate (XI) and then acetone (Xqg)16. The B complex, which is similar to X (and the similar S comp l e ~ ) ~was ~ , separated on a column o f acid-washed alumina with c h l o r o f o r m / b e n ~ e n e ~ ~In . two separate studies of the 2 complex, one with methanol ( 2 - 5 % ) in chloroformq and the other with ethyl acetate on a column of alumina IV3*, three fractions were obtained: Zo, 2 1 and 22-25. 1.6.4
Silicic acid
The general observations made above concerning adsorption chromatography apply also to the use of silicic acid, which has been employed both for purification and, in some cases, for separation of actinomycin mixtures. Am D was purified on a column of silicic acid with benzene/ethyl acetate (1:4) followed by elution with ethyl acetate12. Individual components of the A complex, resulting from cellulose column separation, were similarly purified on a silicic acid c01umn'~. The same mixture could also be separated into individual components on a silicic acid column with ethyl acetateI4, as could the S complex3q. A partial separation of the mixture from a sarcosine-stimulated S. a n A i b i o t i c u d complex was effected on silicic acid with increasing concentrations of methanol (0-2%) in ethyl acetate", Silicic acid polumn chromatography also proved successful to separate mixtures of actinomycins produced by s . pahuueud in the presence of c i d - or Xhand-4-chloro-L-proline. The latter two complexes qenerally contain small amounts of IV plus two new components 5.
1.7 Thin-layer chromatography
(w
Paper chromatography (partition) is generally superior to TLC (mainly adsorption) for separations of actinomycin mixtures. Nevertheless, the more rapid and convenient TLC technique has been employed with increasing frequency in recent years. As with adsorption chromatography on columns, i t is most effective where structural differences between components include the presence of a functional group such as hydroxyl. Separation of the C complex was unsatisfactory on silica gel, but was achieved on a l ~ m i n a ~ O -(solvents ~~, 5 , 2 , Table 5, next page). The same system also separated rhe components, F l and F3 (from the C plus F mixtures), produced by S. e h h y b u m a ~ ~ u in n the presence of added sarcosine, whereas silica gel only separated the C group from the F group41943 (solvents d, e, However, silica gel was more effective in separaTing actinomycins F 8 and Fq (solvent g ) from the same mixture40. The solvent systems (Table 5 ) used in the above studies were designed to function as partition systems, but adsorption effects probably predominate. A tritiated A complex (I + IV + V) was separated preparatively on silica gel TLC (solvent h)44. Preparative TLC was also employed in the separation of-the three components (IV + K1, + K2 in the mixture produced by S. pahuuLud in the pres~ . this case, Gelman ITLC ence of ~ h a ~ d - 4 - m e t h y l - L - p r o l i n e ~ ~In silicic acid-impregnated glass fiber sheets were employed with ethyl acetate. This system did not separate the corresponding actinomycin mixture containing cid-4-methyl-L-proline. Similar
c,
r).
16 TABLE 5 Solvent S y s t e m f o r Thin-layer Chromatography
Ref -
Solvents
-a
e t h y l acetate/a-tetrachloroethane/water (3:1:3) (bottom l a y e r )
-b
41,42,43
e t h y l a c e t a t e / n - d i b u t y l ether/water (2:1:2 o r 3:1:3) ( t o p l a y e r )
43
-C
benzene/e t h y 1 acetate /methanol (20:5:2 or 6:4:1)
43
-d
n-butanol/m thanol/water ( 6 :1:3)
43
n - b u t e n o l / a c e t i c acid/water (10:1:3)
43
f
e t h y l acetate/isopropanol/water (5:2:1)
43
9
e t h y l acetate/methenol/water (top layer)
40
e
-
-h
.
e t h y l acetate/acetone
(20:1:20)
(2:l)
44
TLC systems have proved effective in the separation of the Z complex and related actinomycin preparations 35.
1.8 High-pressure liquid chromatography (HPLC) The use of HPLC for analytical separation of two actinomycin mixtures (the C complex and a mixture produced by S. p a t v u l u b in the presence of cib-4-chloroproline) has been des~ r i b e d ~ A~ .Bondapak Cle/Corasil column was employed with a UV detector and acetonitrile/water (1:l) as solvent at 6000 p.s.i. The separations obtained compare with those observed on paper or thin-layer chromatography, respectively, but the method offers the advantage of simpler and more accurate quantitation.
17
2. Methods for identification of amino acids from actinomycins 2.1 Introduction In the earliest reported investigation46y47 of the amino acid content of an actinomycin preparation, D-valine, L-proline, L-threonine, sarcosine and N-methyl-L-valine were identified in hydrolysates of the A and B complexes. The amino acids (from the B complex) were separated preparatively by a combination of ion-exchange and cellulose powder column Chromatography. They were characterized by melting point, elementary analysis and derivative formation, and compared with the authentic amino acids by PC in several solvent systems, Identification of the three secondary, or ”imino” acids, was aided by deamination of hydrolysates with nitrous fumes prior to PC. Soon thereafter, the amino acid content of the C complex was r e p ~ r t e d ~ which ’ ~ ~ consists of the same amino acids in addition to D-allaisoleucine. In this investigation all separations, including preparative isolation, were done by PC. Microbiological methods were employed in the identification of Lthreonine and the D-configuration of valine and alloisoleucine. PC has been used in several phases of the study of actinomycin hydrolysates: identification,.quanritation (colorimetric, with ninhydrin), preparative isolation and radioisotope assay (biosynthetic studies). High-voltage paper electrophoresis has proven to be a valuable complementary technique, which can be enhanced by two-dimensional combination with PC. Ion-exchange chromatography has been utilized analytically, in the form of the automatic amino acid analyzer, and preparatively, including the isolation of radioactive amino acids resulting from isotope incorporation experiments. Gas-liquid chromatography of derivatized amino acids from actinomycin hydrolysates has been a useful identification technique, particularly when combined with mass spectrometry. The latter procedure can also be utilized to measure stable isotopes in amino acids incorporated biosynthetically. The use of high-voltage paper electrophoresis, as well as ion-exchange and gas chromatographic techniques has largely displaced classical methods for the investigation of the stereochemistry of amino acids from ac tinomyc ins. A chromatographic procedure for the confirmation of amino acid sequence in actinomycins consists of thermal degradation to diketopiperazines representing contiguous amino acid pairs (excluding threonine). The diketopiperazines can be identified by gas-liquid chromatography, if necessary combined with mass spectr~metry~~-~~.
The methods outlined above are surveyed in greater detail below.
2.2 Paper chromatography The various solvent systems which have been utilized for
18
PC with actinomycin hydrolysates are shown in Table 6, and the diagnostic spray reagents in Table 7 (following page). TABLE 6 Solvents f o r Paper Chromatography o f Amino Acids Ref. -
Solvents
a
n - b u t a n o l / a c e t i c acid/water (4:1:x)*
b c d e
s-collidine/water phenol/water
3,48
(variable r a t i o )
n-butanol/water
(upper phase)
o-cresol/water benzyl alcohol/water (upper Phase)
g
sec.-butanol/water
-i
3,9,21,22,48 46,47 3,7,46,48
-f
sec.-butanol/3%
3,4,6,7,9,21, 46,47,40,49
3,48
(upper phase)
aqueous ammonia (7:Z)
3,48 15,21
t-b ut an ol/e t h an01/NH 4 M/H 20 ( 4 :12 :1 :3)
41
J
meth anol/e thanol/w ate r ( 9 :9 :2 )
k
n-butanol/phenol/acetic (2:2:1:5 o r 3:1:1:5)
1
n-butanol/phenol/acetic a c i d / c i t r a t e b u f f e r , pH 6.4 (20:16:1:20)
55
t-butanol/methyl e t h y l ketone/formic acid/water (8: 6: 3 :3)
22
80% aqueous phenol/ethanol/ammonia
52
-m n
acid/water
41 21,22
(15:4:1)
o
77% e t h a n o l
p
t-butanol/formic
acid/water (5:l: 1)
52
9
n-butanol/formi c acid/wate r ( 10 :1:2 )
56,67
r s t
n-butanol/ethano1/3% NHkOH (7:7:6)
52
23
(1:l:l)
23
me thanol/wate r / p y r i d i n e ( 20 :5 :1)
24
n-butanol/pyridine/water
*x denotes v a r i a n t s o l v e n t ; c o n s u l t o r i g i n a l paper for details.
For the early work on the amino acids from the actinomycin A and B complexes, solvent systems a-d were employed46y47. In the investigation of the C complex hydroiysate, solvents 2 , b, 5, f were used, with a and e serving for preparative separation; 3%t 8. Quantirative-amino-acid analyses of the separared components C1, C 2 , C J , 11, 12, X1 and X2 were effected with two-dimensional PC (solvents d and 5 ) by colorimetry after ninhydrin t r e a t r ~ e n t ~ ~ ~Quantitative a~. determinations of amino
e,
19 TABLE 7 Spray Reagents f o r Paper Chromatography Reagent
Specificity
Ninhydrin
General, except 4-Ketopr0, 4-Keto-5-MePro
Isatin
Pro, Hypro, 5-MePro, Hydroxy-5-MePro
Ehr l ich
Hypro, 4-Chloropro
Triphenyltetrazolium chloride
4-Ketop ro
p-Nitrobenzoyl c h l o r i d e / p y r i d i n e
N-methylamino acids
not 3-
acids from the A, B and D preparations were carried out by densitometry on paper chromatograms (solvent The amino acid content of actinomycins I1 and I11 was investigated by two-dimensional PC (solvents c and a), by the descending method (solvent & ) and by the circular technique (solvent k); the latter procedure was utilized in conjunction with densitometry for quantitation21.
PC was used extensively for the identification of the amino acids present in various components of the X complex 1 6 3 2 8 , 3 1 , such as the hydroxyproline of X o b and the 4-ketopro4-Ketoproline was identified through forline of X2 and XI,. mation of a red spot on paper chromatograms treated with the triphenyltetrazolium chloride reagent, and also by reduction of the actinomycin prior to hydrolysis. In particular, reduction with aluminum isopropoxide produced actinomycins X O and X o 6 , which were shown to contain hydroxyproline and aLlaRydroxyproline, respective1yl6. The latter imino acids were detectable with isatin o r Ehrlich’s reagent. A similar conclusion resulted from examination of a hydrolysate of the actinomycins produced by S. antibioticus with 14C-L-proline as precursors2. Reduction with sodium borohydride of the radiolabeled 4-ketoproline (isolated by ion-exchange chromatography) afforded 4-hydroxyproline, identified by paper chromatographic comparisons (solvents k , fl,
0, E). The amino acid content of the 2 complex actinomycins was initially investigated by two-dimensional PC (solvents 2 and aI9. The p-nitrobenzoyl chloride/pyridine reagent53 was used to detect the N-methylamino acids (sarcosine, N-methylalanine, N-methylvaline) in the 2 components. The absence of proline was a noteworthy feature of these actinomycins. In a later study32,54y55 4-keto-5-methylproline was identified as a component of Am 2 1 via reduction with aluminum isopropoxide prior to hydrolysis. The formation 0-f two isomers of 4-hydroxy-5methylproline was demonstrated by paper chromatographic comparisons with the synthetic compounds using solvent a. 4-Keto-5methylproline was similarly identified in actinoiiiycin 25 hydro-
20
by PC with solvent lysates, together with 5-methylpr0line~~, which was said to distinguish 3-,4- and 5-methylproline.
I,
In studies of directed biosynthesis, hydrolysates of actinomycins Pip lu, Pip 18 and Pip 2, which were produced by S. a n t i b i a t i c u b with pipecolic acid, were identified by descending (solvent k) and two-dimensional (solvents m and c) PCZ2. Pipecolic acid was present in all three actinomycins, an3 Pip la contained 4-ketopipecolic acid. The latter gave a yellow ninhydrin color, no isatin color, and was Ehrlich reagent positive. Incorporation of 4-methylproline into actinomycins syn~ ~ PC with s o l thesized by S. a n t i b i a t i c u b was d e m ~ n s t r a t e dby vents d, lc, g . In investigations of the incorporation of isoleucine isomers into actinomycins by S. a n f i b i o t i c u b reparative PC (solvent a) was em loyed to isolate D-isoleucineP7 and N - r n e t h y l a L L ~ i s o l e u c i n e ~5g~ ’ from hydrolysates.
o,
PC has been effective for the isolation of radioisotopically labeled amino acids from actinomycin hydrolysates obtained during biosynthetic studies. Areas containing radioactive amino acids are generally cut out, eluted from the paper with water and a s s a ed by liquid scintillation counting. Thus, incorporation of y4C-labeled L-valine, L-proline, glycine, sarcosine and L-methionine into actinomycins by S. a n t i b i a f i c u s was measured by the pa er chromatographic technique (solvent a)59. Incorporation of P4C-labeled L-isoleucine (as D-aLlaisolFucine) and threonine into actinomycins by S. a n t i b i o t i c u b was examined by PC (solvent a)42, and in studies with 14C-labeled sarcosine, glycine and methionine, solvents d, i, j were employed41. Incorporation of tritiated thanb-3-methylproline (but not the c i b isomer) into actinomycin by S. a n t i b i a t i c u b was demonstrated by descending (solvents 2, k) and two-dimensional (solvents m and c) PCfi0. In experiments involving incorporation of l5N-L=valine into actinomycins by S. a n t i b i a t i c u b , PC with solvent 5 was employed for isolation of labeled N-methyl-valine6 l.
T
2.3 High-voltage
paper electrophoresis (HVE)
HVE separations of amino acids in actinomycin hydrolysates have been obtained with formate or formate/acetate buffers of pH 1.9 and at 2200-4600 volts. In some cases adequate separations were observed with HVE alone. In other situations, twodimensional combination with PC has been a valuable technique, with PC (solvent a or t) as the second dimension. For example, see Fig. 2 , next page.HVE was employed to identify the amino acids in hydrolywhich include sates of actinomycins X O B , Xo6, Xoq and X hydroxyproline and aLLohydroxyproline16 6?’ In two independent studies of actinomycin Z5, HVE alone and in combination with PC6*, revealed the presence of 5-methylproline. The presence of 4-keto-5-methylproline in actinomycins Z 1 5 4 and Z 5 5 5 was demonstrated by identification of two isomers of 4-hydroxy-5-methylproline by HVE after reduction of the actinomycins with aluminum isopropoxide. Two additional novel amino acids were discovered as componenxs of 54y55,
21
Sar
0
Q
r
va10 0
N-MeAla Thr
0H-Thr
N-MeVal
0
0
OH-Thr
0
unknown
Q-MeVal
3-OH-5-MePro
PC S e p a r a t i o n o f amino a c i d s i n h y d r o l y s a t e s o f a c t i n o T c i n Z 1 by t h e two-dimensional p a p e r e l e c t r o p h o r e t i c - C h r o m a t o g r a p h i c p r o c e d u r e 4. The unknown n i n h y d r i n - p o s i t i v e component may r e p r e s e n t a d e g r a d a t i o n p r o d u c t der i v e d from h y d r o x y t h r e o n i m o r 4-keto-5-methylproline; the l a t t e r compound i s r e p o r t e d t o be c o m p l e t e l y d e s t r o y e d d u r i n g a c i d h y d r o l y s i s 3 2 9 54. Rep r i n t e d w i t h t h e p e r m i s s i o n of Academic Press, I n c .
FIGURE 2.
22 actinomycin Z1 with the aid of two-dimensional HVE-PC: a-aminoB,y-dihydroxybut ric acid (11hydroxythreonine11)63 and 3-hydroxy5-methylproline6x. In the latter case, HVE distinguished the four synthetic diastereoisomers, of which the 2;3-thanb-2,5cib isomer corresponded with that in the Z1 hydroly~ate~'. 5 Methylproline, unlike 3-hydroxy-5-methylpro1iney gave a blue color with isatin; both gave yellow ninhydrin colors. HVE alone and in combination with PC was used in studies on the incorporation of various isoleucine isomers into actinomycin by S. antibioticub and S. ~ h h y b o r n a ~ ~ u These b ~ ~ ~ ~ ~ procedures established the presence o# D-isoleucine and N-methylL-aetoisoleucine in these actinomycins. HVE alone and in conjunction with PC was employed to identify pipecolic, thanb-4hydroxypipecolic and 4-ketopipecolic acids in the new actinomycins formed by S. anabioticub in the presence of pipecolic acidz2, and to demonstrate incorporation of 4-methylproline into actinomycins by the same organism56y67. HVE-PC was also used to characterize the amino acid content of four new actinomycins produced by directed biosynthesis (S. pahvueud with cib- and than&-4-methylprolines In biosynthetic studies, HVE has been effective for studying the incorporation of radioisotopically labeled amino acid precursors, both preparatively for liquid scintillation counting and by autoradiographyS9. The HVE technique was used in studies of the incorporation of I5N-labeled6l and 12,3-3H21labeled68 L-valine, and of tritiated thanb-3-methylpr0line~~ into actinomycins.
2.4 Thin-layer chromatography (TLC) Two-dimensional TLC was used to identify the amino acids, including azetidine-2-carboxylic acid, in alkaline hydrolysates of the new actinomycins produced when the latter imino acid was . powder TLC added to cultures of S . a n t i b i o L L c u ~ ~ ~Cellulose plates were employed with solvents and 5.
A two-dimensional combination of thin-layer electrophoresis and TLC on cellulose powder has been described6', which is essentially a miniturization of the HVE-PC system described above. A formate/acetate buffer (pH 1.9) was used for electrophoresis at Y5 volts/cm, and solvent 5 for chromatography in the second dimension. Fluorescarnine was employed for visualization of the amino acids. Separations were described for hydrolysates of actinomycins D, C3, X 2 and some preparations related to the Z complex.
2.5 Ion-exchange chromatography In the original preparative isolation of the amino acids from the actinomycin rlBrl complex47, a sulfonic acid (cation exchange) resin was employed with aqueous ammonia ("displacement method"). This procedure gave only a partial separation but the later application of the "elution method" with dilute hydrochloric acid or buffers in the pH range 2.91-5.26 (Table 8, next page) produced far superior separations.
23 TABLE 8
Ion-Exchange Chromatography B u f f e r s Buffer
Ref. -
a
0.37 M c i t r a t e / s o d i u m c i t r a t e , pH 2.91
52
b
0.2 M p y r i d i n e / a c e t i c a c i d , pH 3.5
58
c
0.2 M p y r i d i n e / a c e t i c a c i d , pH 4.25
58
d
0 . 2 M c i t r a t e / s o d i u m c i t r a t e , pH 3.05
27,40,58,60, 62-65,70,71
e
0.2 M c i t r a t e / s o d i u m c i t r a t e , pH 4.25
27,40,58,60, 62-65,70,71
f
0 . 2 M c i t r a t e / s o d i u m c i t r a t e , pH 5.26
57
Ion-exchange chromatography (with dilute HCl) was used to purify D-valine and N-methylvaline (after elution from paper chromatograms) from actinomycin hydrolysates after biosynthetic incorporation of 1SN-L-valine61. Ion-exchange chromatography was also employed for the isolation o f radioactive proline, hydroxyproline and 4-ketoproline from actinomycin hydro1 sates after utilization of 14C-L-proline (elution with 1 N H C L ) Y 2 Reduction of the labeled 4-ketoproline with sodium borohydride produced a mixture of hydroxyproline and aeeohydroxyproline (1:3 ratio) which was also separated by ion-exchange chromatography with buffer a. Ion-exchange chromatography was utilized in the prepararive isolation of N-methyl-alLoisoleucine (buffer b), and D-isoleucine (buffers b and clfrom hydrolysates of actinomycins produced by S. a n ? i b i o t ~ c u s 8.
.
e)
The automatic amino acid analyzer ( A M ) (buffers d and has become an important analytical method for actinomycin example, see Fig. 3 , next page. The h y d r o l y ~ a t e s ~ ~For . following are further examples of its application. It was used to confirm the presence of D-isoleucine and N-methylaLLoisoleucine in actinomycins after utilization of isoleucine isomers”, and for quantitative amino acid analyses of the various fractions produced under these conditions by S. a n t i b i a t i c u s , S. pahvubus and 3 . c h y b omaLLus Ci6 - 5-methylproline was identified in hydrolysates of Am Z562; this technique distinguishes 3-,4- and 5-methylproline and their diastereoisomers except for those of 4-methylpr0line~~. a-Amino-B,y-dihydroxybutyric acid (”hydr~xythreonine”)~and 2,3-thUttb-2,5-ci6-3h y d r o x y - 5 - m e t h y l p r 0 1 i n e ~ ~were ’ ~ ~ identified as components of Am Z1. The AAA was used for analysis of the alkaline hydrolysates of actinomycins produced by directed biosynthesis with azetidine-2-carboxylic acid23. In the case of directed biosynthesis with pipecolic acid, analysis confirmed the presence of pipecolic, thans-4-hydroxypipecolic and 4-ketopipecolic acid in various components35. The use of fluorescarnine in place of ninhydrin for amino acid analysis of actinomycin hydrolysates
‘.
2.0
Sarcosine
Alloisoleucint
0.8
soleucine
Proline
L 100
125
150
175
200
Effluent (ml)
FIGURE 3. S e p a r e t i m of an amino a c i d hydrolysate obtained from actinomycin E2 synthesized by S. chJybomaeeuS i n the presence o f L - d t o i s o l e u c i n e ( o r D-isoleucine)L6. N-Methylvaline i s absent and there are equimolar amounts o f D-&oisoleucine and D-isoleucine present. Reprinted w i t h the permission of the American Society f o r Microbiology.
25 was reported7I. The primary amino acids gave products which were both fluorescent and UV-absorbing, whereas the secondary imino acids gave only UV-absorbing products.
2.6 Gas-liquid
chromatography (GLC)
GLC separation of several amino acids found in actinomycins (sarcosine, N-methylvaline, valine, aLLaisoleucine, proline, threonine, hydroxyproline, alLohydroxyproline) was reported72. The amino acids were derivatized to N-acetyl n-amyl esters and chromatographed on column 5 (Table 9 ) . TABLE 9 Column Packings f o r Gas-Liquid Chromatography Ref. -
Column Packing,
-a
0.2% Versemid + 0.2% Carbowax 1540 NGS on Chromosorb W
-b -
1.8% o r 3% EGSP-Z on Gas Chrom Q
52,62
C
5X EGS on Gas Chrom Q
35,52
-d
0.5% EGA on Chromosorb W
24,62
-e -f
3% O V 1 7 on Gas Chrom Q 3% OV225 on Gas Chrom Q
+
0.2%
72
63,64,65 5 1,63,64,65
4-Ketoproline was identified in hydrolysates of Am V by derivatization to the N-trifluoroacetyl methyl ester and GLC comparison with the synthetic compound (columns and Cib - 5-meth lproline6 and 2,3-tmMb - 2 , s - cib -3 -hydroxy-5m e t h y l p r ~ l i n ewere ~ ~ ~identified ~~ in hydrolysates of actinomycins Z 5 and Z 1 respectively, by GLC, after trifluoroacetylation of their methyl esters. For 5-methylproline, GLC columns b and d , which both resolve the diastereoisomers, were employed. for 3-~ydroxy-5-methylproline, columns g and f, which distinguigh all four diastereoisomers, were used. Tn both cases identity was confirmed by combined gas chromatography-mass spectrometry (GC-MS) of the same derivatives. For identification of a-amino-B,y-dihydroxybutyric acid ("hydroxythreoninett) as a component of actinomycin Z1, trimethylsilylation was employed (columns e and f) and identity was confirmed by comparison with the synyhetic-substance by combined GC-MS63. For the determination of the deuterium content of sarcosine in actinomycins 11, I11 and IV after incorporation of {N-C2H33-sarcosine by S. a n t i b i a t i c u d , combined GC-MS was employed with the N-formyl amino acid methyl esters on column f51. c i b - and thanb-4-methylprolines from hydrolysates of actinomycins Klc, KPC and Kit, K2t, respectively, were distinguished as their trifluoroacetyl methyl esters24 on GLC column &. Pipecolic, 4-hydroxypipecolic and 4-ketopipecolic acids from actino-
26 mycins were identified by GLC (column c) and combined GC-MS as their trifluoroacetylated methyl esters35.
2.7 Stereochemistry
of the amino acids from actinomycins
In the early studies relating to the structure of the actinomycins, the enantiomeric configurations of the various crystalline amino acids were established by (i) optical rotation3943 (in water and hydrochloric acid) and (ii) enzymatic and microbiological methods3 (for D-valine and D-aeloisoleucine). The absolute configuration of the latter amino acid at the % carbon was determined by ninhydrin oxidation to 2-methylbutyraldehyde which was characterized as its 2,4-dinitrophenylhydrazone. Confirmation of the stereochemistry of D-aLtoisoleucine was obtained by comparisons of several derivatives with the authentic isomers3. The L-configuration of hydroxyproline and 4-ketoproline present in actinomycins X o g and X,, respectively, was deduced from their interconversions with X 1 which contains L-prolinel6, More recently, optical rotatory dispersion was used to establish the L-configuration of pipecolic, 4-ketopipecolic and thanb-4-hydroxypipecolic acids from actinomycins formed by S . antibiodicub (directed biosynthesis with DL-pipecolic acid). The t h a n b stereochemis try of the 4-hydroxypipecolic acid was demonstrated by HVE and GLC comparisons with the authentic diastereoi~omers~~. In studies of the actinomycins synThesized by S. a n t i b i o and S. chhybomaLLub in the presence of various isoleucine isomers, optical rotatory d i s p e r s i ~ n ~ and ~ ’L~ ~and D-amino acid o x i d a ~ e s ~ were ~ , employed ~ ~ in establishing the stereochemistry of D-valine, D-isoleucine and D-alloisoleucine. The presence of D-isoleucine in certain of these actinomycins was demonstrated by formation of the L-leucyl dipeptides and subsequent ion-exchange chromatographys7 (system f, Table 8 ) . ticub
Hydroxyproline and altohydroxyproline, from actinomycins X o a and X o B , respectively, were distinguished by HVE16’28.
This separation can also be effected by GLC72. The latter technique was employed to distinguish c i b - and thanb-4-methylproline in the actinomycins Klc, KzC and Kit, KZt, respectively+ The two diastereoisomers of 4 - h y d r o x y - 5 - m e t h y l p r o l i n e resulting from aluminum isopropoxide reduction of 4-keto-5methylproline in intact actinomycins Z 1 and 25 were distinguished b high-voltage paper electrophoresis, but were not identified 5zy73. Recent, independent studies35 indicate c i b , c i b stereochemistry for the 4 - h y d r o x y - 5 - m e t h y l p r o l i n e resulting from sodium borohydride reduction of 4-keto-S-methylproline, and hence c i b stereochemistry for this imino acid from actinomycin Z1. This is expected, since the 5-methylproline in actinomycin 2 5 was shown to be ciA by ion-exchange and gas-liquid chromatography6’. Also, the 3-hydroxy-5-methylprolineb4 from actinomycin 21 was shown to have 2,3-thanb-2,5-cib s t e r e ~ c h e m i s t r y ~ ~ . In the latter case, all four synthetic diastereoisomers were
27
distinguishable by HVE, AAA and GLC, and compared by these methods with the natural imino acid. The stereochemistry of the synthetic isomers was established by epimerization studies and high-field proton magnetic resonance6’. Two generalizations emerge from the investigations of cyclic imino acids from actinomycins: (i) In the naturally occurring or ”biosynthetically directed” hydroxy imino acids (hydroxyproline, 4-hydroxypipecolic acid, 3-hydroxy-5-methylproline), the hydroxyl group is always t h a n 6 to the carboxyl. (ii) In the proline series having a 5-methyl group (5-methylproline, 4-keto-5-methylproline, 3-hydroxy-5-methylproline), the methyl group is always C i 6 to the carboxyl.
2.8 Amino
acid sequence in actinomyeins
The amino acid sequence74 of actinomycin C3 (Table 1 ) was established by the following methods: (i) DesaminoactinocylL-threonine7’, actinocyl-bi6-L-threonine76 and actinocyl-bibL-threonyl-D-aLLoisoleucine76 were identified as partial acid hydrolysis products, in the latter two cases by comparisons with the synthetic compounds. (ii) Actinomycinic acid C, (obtained by opening the lactone rings via mild alkaline hydrolysis) was subjected to reaction with hot acetic anhydride in i s known78 to modify the C-terminal amino ~ y r i d i n e ~which ~, acids of peptides. After acid hydrolysis, the absence of Nmethylvaline identified this as the C-terminal amino acid. (iii) Begradation of actinomycin C2 or C3 in hydrazine hydrate at 140 produced cyclo(sarcosy1-N-methylvalyl), indicating that these two amino acids are contiguous in the actinomycin peptide moieties; a similar degradation of actinomycin I1 ( = C1) produced cyclo(D-valyl-L-prolyl) 79. The analogous amino acid sequence was deduced for actinomycin D, with D-aLLoisoleucine replaced by D-valineso. A novel feature of this investigation was the oxidative cleavages1 of the actinomycin chromophore, permitting separate sequence determination of the two peptide chains, (Fig. 4, next page). Partial acid hydrolysis of each peptide was monitored by paper chromatography; in each case N-methylvaline was released first, followed by sarcosine and proline; threonine was released slowest of all the amino acids. N-Methylvaline was also identified as C-terminal by the pyridine/acetic anhydride degradation procedureomentioned above. Thermal degradation of each peptide at 180 produced cyclo(sarcosy1-N-methylvalyl), which was purified by vacuum sublimation. Chemical conversions of actinomycins X2 to X1 ( = D) and in which a 4-ketoproline residue was reduced to proline “,8 hydroxyproline, respectively16, served to establish the amino acid sequences of X2 and X O B as analogous to that of actinomycin D. Oxidative cleavage of the chromophore permitted separate amino acid analysis of the u and B peptide chains in these actinomycins revealing that the hydroxyproline (in X O B ) or ketoproline (in X p ) is located in the $-peptideE2. Likewise in actinornycins C2 and Cza, D-valine was found in the a and B chains, respectively, D-alloisoleucine occupying the analogous
,
28
a
6
5 COOH
COOH
I
I
peptide
I
FIGURE 4.
peptide
I
+
Oxidative cleavage o f the actinomycin chromophore
co
I
COOH
..
site in the other ~ e p t i d e ~ ~ ' ~ ~ . A gas chromatographic study of the cyclodipeptides (diketopiperazines) formed during thermal degradation of actinomycins forms the basis of a microanalytical method for obtaining sequence information. De radationocan be performef in dimethylformamide s o l ~ t i o nat~ 200 ~ ~ ~ or ~ dry at 4 0 0 as a pyrolysis-gas chromatographic pro~edure'~. In addition to cyclo(D-valyl-L-prolyl) and cyclo(sarcosy1-N-methylvalyl), the earlier isolation of which is mentioned above, cyclo(proly1sarcosyl) was identified as a degradation product of actinomycin D. This information, together with the observation that cyclo (sarcosyl-N-methylvalyl) was the first diketopiperazine released 5 0 confirms the amino acid sequence of actinomycin D. Similar sequences were demonstrated for actinomycins C 3 , 11, 111, P i p 2 and A z u , confirming that direcred biosynrhesis with proline analogues does not alrer the amino acid sequence of the resulting actinomycin variants49,
29 3. The structure of the actinomycin chromophore
The structure of the chromophore common to all the naturally occurring actinomycins is based upon the following evidence :
(i) Mild acid hydrolysis of actinomycin C 3 (I, Fig. 5 , next page) gave deaminoactinomycin C 3 (11) and ammonia, while more vigorous hydrolysis produced actinocinin (111) and actinocinyl-L-threonine (IV)75. The structure of actinocinin followed from its molecular formula, the presence of two acidic groups (one a carboxyl) and one weakly basic group, the presence of two C-methyls and of a carbonyl group which was quinonoidal in character (reversibly reducible with stannous chloride). Actino cinin was synthesized by condensation of 3-hydroxy-4-methylanthranilic acid with 2 , 5 - d i h y d r o ~ y t o l u q u i n o n e ~ ~ . (ii) Prolonged hydrolysis of I with hydrochloric acid gave two additional colored degradation products, 2-hydroxy-4,6dimethylphenoxazone (v) and 2,5-dihydroxytoluquinone ( ~ 1 ) ~ ~ . Condensation of VI with 2-amino-6-methylphenol gave V. (iii) Reductive acetylation experiments demonstrated that the actinomycin chromophore has an aminoquinone, as distinct from hydroxyiminoquinone, structure77.
(iv) Actinocyl-bib-glycine methyl ester (VII) was synthesized by ferricyanide oxidation (Fig. 6, page following Fig. 5 ) of 3-hydroxy-4-methylanthraniloylglycine methyl ester (VIII)86. The ultraviolet and visible absorption spectrum of VII and I were virtually identical (maxima at 238 and 443, shoulder at 4 2 5 nm). (v) Treatment of 1 (Fig. 5 ) with hot barium hydroxide solution caused extensive rearran ement of the chromophore with formation of llactinomycinolll (IX)fo’86. The same compound was also produced from actinomycins B (complex) and D, confirming that these actinomycins all have a common chromophore.
30
t 0
0
0
0
I 0
0
I
v
I
o
I 0
M
O’M
31
Gly-OMe
Gly-OMe
I
I
co 2
NH2
Gly-OMe
I
IIII+c
OH
CH3
m FIGURE 6 .
Synthesis of actinocyl-b&-glycine
CHJ
CH3
PII methyl e s t e r .
32
4. Nuclear magnetic resonance (NMR)
Proton NMR studies of actinomycin D in organic solvents and in aqueous solution88392 have been reported. The 13C NMR spectrum of actinomycin D has also been describedg3 and some of the original assignments corrected in independent studies94y95. Proton NMR studies of actinomycins I1 and IIIg6, and of those containing c i b - or Zhans-4-methylproline (KJt, K2t, Klc, K2c)24y35 have also been undertaken. Conformational investigations of various actinomycins by NMR and other techniques have been reviewedg4. It was concluded that all the natural actinomycins possess a common conformation with only slight variations, and that this conformation is similar to that revealed by X-ray crystallo raphic studies of the deoxyguanosineactinomycin D complex97Sgf. 87-91
Acknowledgement We acknowledge financial support from the National Cancer Institute, U.S. Public Health Service, through a contract “01-CM-43732) and research grants (CA-11627 to A.B.M.) and (CA-09626 to E . K . ) .
33 5 . Abbreviations Am C complex Actinomycin C complex Am D = Actinomycin D Am Z 1 = Actinomycin Z l Am 25 Actinomycin Z 5 Am V Actinomycin V
PC = Paper chromatography TLC = Thin-layer chromatography HPLC = High-pressure liquid chromatography HVE High-voltage paper electrophoresis GLC = Gas-liquid chromatography Gas chromatography-mass spectrometry GC-MS NMR = Nuclear magnetic resonance Thr = L-Threonine Hydroxythreonine (u-amino-B,y-dihydroxybutyric acid) HyThr Val D-Valine aIleu = D-aLLoisoleucine Ileu = D-Isoleucine L-Proline Pro HyPro thand-4-Hydroxy-L-proline aHyPro = aLLo ( c i d 1-4-Hydroxy-L-proline KetoPro 4-Keto-L-proline 4-Keto-5-MePro 4-Keto-cid-5-methylproline 3-Hy-5 -MePro t h a n d - 3 -hydroxy-c i d - 5-methy lprol ine 5-MePro = cid-5-Methylproline Sar = Sarcosine Pip L-Pipecolic acid 4-KetoPip 4-Keto-L-pipecolic acid 4-HyPip = thand-4-Hydroxy-L-pipecolic acid c i d -4-MePro = cid-4-Methylproline thand-4-MePro = thand-4-Methylproline Azetidine-2-carboxylic acid Azet ci6-4-C1Pro cid -4-Chloroproline than6-4-C1PrO thand-4-Chloroproline MeVal = N-Methyl-L-valine MeAla N-Methylalanine MeaIleu N-Methyl-L-aLLoisoleucine
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39
Ansamycins
. .
A K Ganguly Chemical Research Department Schering Corporation. Bloomfield. N.J.
. 2.
1
Ansamycins:
General introduction
.................
Rifamycins 2.1 Introduction Structural determination 2.3 Biological activity 2;4 Structures 2.5 Rifamycin B n.l Producing organism 2.5.2 Physical properties 2.5.3 Isolation and purification 2.6 Rifamycin 0 m.1 Producing organism 2.6.2 Physical properties 2.6.3 Isolation and purification 2.7 Rifamycin S 2.7.1 Producing organism 2.7.2 Physical properties 2.7.3 Structure.. 2.8 Rifamycin SV 2.8.1 Producing organism 2.8.2 Physical properties 2.8.3 Isolation and purification 2.9 Rifamycin L 2.9.1 Producing organism 2.9.2 Physical properties 2.9.3 Isolation and purification., 2.10 Rifamycin W 2.10.1 Producing organism 2.10.2 Physical properties 2.10.3 Isolation and purification 2.10.4 Structure 2 . 1 1 Rifamycin Y
................................ .................... ......................... .................................. ................................. ........................ ....................... ................ ................................. ........................ ....................... ................ ................................. ........................ ....................... ............................... ................................ ........................ ....................... ................ ................................. ........................ ....................... .............. ................................. ........................ ....................... ................ ................................. ................................. 3. Halomicins 3.1 Introduction................................ 3 . 1 Chemistry. .................................. 3.3 Biological activity ......................... Physical properties ......................... 3.4.1 Halomicin A. .............................. 3.4.2 Halomicin B ............................... 3.4.3 Halomicin C ............................... 3.4.4 Halomicin D ............................... 3.5 Isolation and purification .................. c
_
41 43 43 44 46 46 46 46 47 47 48 48 48 48 48 48 49 49 49 49 50 50 50 50 50 50 50 51 51 51 51 53 53 54 54 54 54 56 56 56
40
4
.
Streptovaricins 4.1 Introduction 4.2 Structures 4.3 Chemistry Biological activity y,5 Chemical and physical properties 4.3.1 Streptovaricin A . 4.5.2 Streptovaricin B 4.5.3 Streptovaricin C 4.5.4 Streptovaricin D 4.5.5 Streptovaricin E 4.5.6 Streptovaricin F 4.5.7 Streptovaricin G 4.5.8 Streptovaricin J 4.5 Isolation and purification of streptovaricins
-
5
5
. .
Tolypomycin Y 5.1 Producing organism Physical properties Biological activity Isolation and purification 375 Structure
-
8
.
............................ ........................... ........................... .................... .....................................
Naphthomycin 5.1 Producing organism i;TI Physical properties 6.3 Chemistry 6.4 Structure Isolation and purification
7.
.................................. .................................... ..................................... ........................... .............. ........................... ............................ ............................ ............................ ............................ ............................ ............................ ............................ .
............................ ........................... ..................................... ..................................... ....................
Geldanamycin 7.1 Producing organism Chemical and physical properties ';f;3 Biological activity Isolation and purification 7.5 - Structure..
............................ .............. ........................... .................... ...................................
Maytansine 8.1 Producing organism 8.2 Structure 8.3 - Biological activity
............................ ..................................... ........................... Acknowledgements.................................... 9. Literature Cited ....................................
57 57 57 59 59 59 59 59 59 59 59
60 60 60 61 61 61
62 62 63 63 63 63 63 64 64 65
65 65
66 66 66 67 67
1. Ansamycins: General Introduction The name ansamycin was given by Prelogl to a new class of antibiotics in which an aliphatic chain (ansa bridge) connected two nonadjacent positions of an aromatic nucleus. Ansamycins can be divided into two sub-groups, those in which the ansa bridge is attached to a substituted naphthalene or naphthoquinone moiety and the others in which the ansa bridge is attached to a substituted benzene ring. Rifamycins, halomicins, streptovaricins, tolypomycins and naphthomycins belong to the first category whereas geldanamycin and maytansinoids belong to the second group. To show the differences between the two subclasses the structures of rifamycin B (1) and geldanamycin ( 2 ) are shown below.
Me
Me
Me
Me
OMe
0
Me
(1 1
The members belonging to the first category of ansamycins i.e. rifamycins, halomicins, streptovaricins, tolypomycins, contain 17-skeletal carbons in their ansa bridges whereas in geldanamycin and maytansinoids there are 15-skeletal carbon atoms. Naphthomycin contains a much larger 23-skeletal carbon in its ansa bridge. Biosynthetic studies of rifamycins, streptovaricins and geldanamycins indicate that there is a common biosynthetic pathway for all these antibiotics. Based on studies using I 3 C precursors i t is concluded that a seven-carbon amino compound containing a six membered ring initiates a single polyketide chain composed of two acetate and eight propionate units in the “amide-head” direction. The naphthalenic chromophore is formed by closure of a second ring including the first propionate and acetate unit of the polyketide chain. For details of biosyn-
42
thetic studies the reader is referred to the excellent work by Lancini', Rinehart3 and their co-workers. Ansamycins,are highly active against gram-positive bacteria and Mycobactehium t u b e k c u L o b i d . Some of them also possess activity against gram-negative bacteria. The most successful member of this class of antibiotics is rifampicin (3) which is
Me
Me
widely used clinically for curing tuberculosis and other infections caused by gram-positive bacteria. Ansamycins inhibit bacterial RNA synthesis in a highly specific manner by forming a complex with bacterial DNA-dependent RNA polymerase. Some ansamycins also show antiviral activity. Rifampicin inhibits replication of Vaccinia pox virus and N-demethyl rifampicin shows greater activity against Vaccinia virus and Herpes simplex. The mode of action of rifampicin in the inhibition of bacterial and viral replications are quite different. Recently it has been claimed that several rifamycin derivatives possess antitumor activity by inhibiting the RNA-dependent DNA polymerase activity of many RNA tumor viruses. Streptovaricin complex is highly active in the above test.
43 2.
Rifamycins
2.1 Introduction Rifamycins are produced4by Nocasdia m e d i t e w a n e i (initially considered to belong to the genus S t t e p t o m q c e b f . Fermentation of N o c a t d i a m e d i t e h t a n e i yields a mixture designated rifamycin A-E on the basis of their chromatographic behavior. These components show antibacterial activity. From the above mixture rifamycin B was easily isolated because of its strongly acidic character. Subsequently, it was discovered5 that rifamycin B could be exclusively produced by the addition of sodium diethyl barbiturate to the fermentation broth. Rifamycin B is essentially inactive against bacteria. However, when allowed to stand in aqueous solution in the presence of air, it yields rifamycin 0 which is converted under acidic pH to a highly active antibacterial called rifamycin S. On reduction using ascorbic acid rifamycin S yields another active antibacterial, rifamycin SV. The conversion of rifamycin B to rifamycin 0 is more conveniently achieved either by treatment with nitrous acid or by electrochemical oxidation6. To the best knowledge of the reviewer, rifamycin A, C, D and E have not been isolated in pure state and their structures determined. Rifamycin S when incubated with washed mycelium of Nocahdia m e d i t e h h a n e i (strain ME/291) yields rifamycin B, L and G. Rifamycin G has no antibacterial activity. Rifamycin L possesses antibacterial activity and unlike rifamycin B it does not undergo spontaneous oxidation and hydrolysis. In recent years extensive investigations of mutant strains of Nocaadia m e d i t e h t a n e i have lead to the production and isolation of rifamycin SV, 27-Desmethoxy-27-hydroxy rifamycin SV and its 25-desacetyl derivative. Very recently a mutant of Nocahdia m e d i t e 4 k a n e i has been claimed to produce rifamycin P, Q and R. Although the structures of rifamycin P and Q are unknown, they are claimed to belong to the group of ansamycins and possess an atom of sulphur and an extra nitrogen atom in their molecules. l n withe and i n w i v o activity of rifamycin P equals those of rifampicin, the presently used compound in humans. Rifamycin Q is less active than rifamycin P and rifamycin R is similar in biological activity to rifamycin S. From a morphological variant of Nocahdia m c d i t e t m n e i rifamycin W has been isolated. Although rifamycin W is devoid of antibacterial activity, its structural resemblance to streptovaricins is of biogenetic significance. This confirms the suggestion that all naphthalenic ansamycins are derived from a common biogenetic precursor having basically similar carbon skeleton as streptovaricins. 2.2 Structural -
determination
Elucidation of the structure of rifamycin S by Prelog7 using chemical degradation and spectroscopic methods opened a new chapter in the chemistry of antibiotics, It was recognized that rifamycins belong to a new group of antibiotic in which a long aliphatic chain bridged an aromatic ring system. Such compounds were named by Prelog as ansamycins. The proposed structures of rifamycin B and Y were confirmed by X-ray analysis of their p-iodoanilides.
44
For the details of chemistry of rifamycins the reader is referred to the ~ l e v i e w s ~on ’ ~this subject. Rifamycin S on methylation using methyl iodide and silver oxide yields 8-0methyl ether of rifamycin S ( 4 ) and the imino methyl ether of the amide (5). Methanolysis of ( 5 ) yields a naphthoquinone derivative ( 6 ) and an aliphatic dimethyl acetal methyl ester (7). The structure of ( 6 ) is based on further chemical degradation and spectroscopic evidence. The location of the nitrogen atom in ( 5 ) is deduced as follows. Catalytic hydrogenation of the diazo product ( 8 ) (prepared by reacting rifamycin 0 with p-toluene sulphonylhydrazine) yields the dihydroxy naphthalene derivative (9) in which the phenolic hydroxyl groups are in peri-relationship and the compound also shows the presence of two ortho aromatic protons. The structure of the aliphatic dimethyl acetal ( 7 ) follows from further chemical degradations. As rifamycin S is a dienamide, one point of linkage of ( 6 ) to ( 7 ) is easily assigned. The other point of attachment of the aliphatic chain to the aromatic chromophore is established as follows. Although rifamycin S has one phenolic hydroxyl and two aliphatic hydroxyl groups , the aliphatic dimethyl acetal ( 7 ) has only one aliphatic hydroxyl group. The second aliphatic hydroxyl group is cyclized in ( 7 ) to the tetrahydropyran ring. The presence of a vinyl ether (0-CH=CH-) in rifamycin S is shown by comparison of the nmr spectra of rifamycin S and its hexahydro derivative. As the methyoxyl group in rifamycin S corresponds to a saturated ether, it followed that the vinyl ether is attached to the aromatic nucleus and as this methoxyl group is easily lost by cyclization in (7) its position is assigned to be allylic. Based on all these observations Prelog proposed structure (10) for rifamycin S.
2.3 Biological
activity
Rifamycins are active against gram-positive bacteria and particularly against Mycobactehium t u b e h c u L o d i d , They also possess low activity against gram-negative bacteria. Rifamycin B, the first naturally occurring antibiotic of this group to be isolated pure, shows very little antibacterial activity. In aqueous solution rifamycin B is converted into a much more active antibacterial named rifamycin S. Although rifamycin S has very high antibacterial activity, it has never been used clinically because of its high acute toxicity and poor oral absorption properties. Rifamycin S is easily reduced with ascorbic acid to rifamycin SV. As the sodium salt of rifamycin SV has high antibacterial activity, low toxicity and favorable distribution pattern in various tissues, it has been tried with success in the clinic to verify its usefulness in the treatment of diseases caused by gram-positive infections and particularly against tuberculosis. During the past several years extensive chemical modification of rifamycin B, S and SV have been carried out to obtain compounds with improved potency against gram-positive bacteria, gram-negative bacteria and Mycobactehium t u b e h c u k o d i d , low toxicity and better absorption properties. It is not the purpose of this chapter to list all the compounds prepared by chemical modification in this group of antibiotics. Excellent reviews438
45
Me
M e M e
Me
Me
Me
(5)
OAc
46
have been written on this subject. Much of the success in chemical modification is derived by manipulation at C 3 and C 4 . Chemical modifications in other centers of rifamycins seem to result in compounds with low or insignificant antibacterial activity
.
Conversion of the carboxyl group of rifamycin B to amides, hydrazides and esters result in increased activity. Rifamide, the N,N-diethylamide of rifamycin B , has been used clinically. Rifamycin 0 on treatment9 with result in substitution with nitrogen tionlo of halomicin B from rifamycin this process. All these derivatives activity.
primary or secondary amines function at C 4 . Prepara0 is an illustration of possess high antibacterial
Michael type of addition at C 3 in rifamycin S has been achieved with primary and secondary amines to yield C3-substituted derivative of rifamycin SV. These compounds show significant antibacterial activity. Similarly conjugated addition of mercaptans, carbanions and enamines have been achieved but these derivatives do not possess useful antibacterial activity. The most numerous and clinically most successful rifamycins are represented by the general structure, 3-amino methyl derivative of rifamycin SV. These compounds are easily made by Mannich reaction of rifamycin S with secondary amines in the presence of formaldehyde. Rifampicin, a rifamycin SV derivative, with a (Q-methyl-piperazinyl)-imino methyl group at C 3 is the most successful (clinically and commercially) ansamycin known. It has a wide antibacterial spectrum, high activity against gram-positive bacteria and Mycobactehium t u b e ~ c u ~ o ~ i ~ , and is less active against gram-negative bacteria. It is orally active. Rifampicin is now being used very effectively in the clinic against tuberculosis and also against other infectious diseases. 2 .Q Structures -
Structures of some of the rifamycins to be described are shown on the following page.
2 . 5 Rifamycin 2.5.1
B
Producing organism
The fermentation broth of N o c a h d i a m e d i t e h h a n e i contains several substances displaying antibiotic activity. The main component of this complex is rifamycin B 4 . 2.5.2
Physical properties
A. Rifamycin B crystallizes from benzene as brilliant yellow prismatic needles.
-
i t has no definite melting point. B. Melting point decomposes at 1 6 O - 1 6 U 0 C and does not melt until 300 C.
It
47
Me
Me
Me RlMMYClN 0: X * -0CH2COORlFAMYClN S: X = 0
C. It is a dibasic acid; electrometric t i t r a t i o n i n a q u e o u s m e t h a n o l shows t w o a c i d i c g r o u p s , pH4 = 2.8; pH% = 6 . 7 . 1
D.
Molecular formula C3gH49NOl,.
E.
Optical activity:
CulZo
589
= -11'
2
( C = 1%i n m e t h a n o l ) .
F. U . V . and v i s i b l e s p e c t r u m : i n a b u f f e r s o l u t i o n pH 7 . 3 r i f a m y c i n B shows a b s o r p t i o n a t 223 nm ( E l = 5 5 5 1 , 304 nm 1 (E: = 2 7 5 1 , 425 nm ( E l = 2 2 0 ) . 1
G. P a p e r c h r o m a t o g r a p h y : t h e Rf v a l u e s i n s e v e r a l solv e n t s y s t e m s are: water c o n t a i n i n g 3% N H b C l , 1% ascorbic acid: 0.25; butanol s a t u r a t e d with water: 0.87; butanol, a c e t i c a c i d , w a t e r ( 4 : l : S ) : 0 . 9 5 ; b u t a n o l , a c e t i c a c i d , e t h a n o l , water ( 2 5 : 3 : 2 5 : 4 7 ) : 0 . 8 5 ; a c e t o n e , w a t e r (1:1):0.88; chloroform, c y c l o h e x a n e , water ( 8 : 1:2 1 : 0 . 9 2 .
2.5.3 I s o l a t i o n and p u r i f i c a t i o n The f e r m e n t a t i o n b r o t h i s a d j u s t e d t o pH 7 t o 8 a n d f i l tered. The f i l t r a t e i s a c i d i f i e d t o pH 2 . 0 a n d e x t r a c t e d w i t h e t h y l a c e t a t e . R i f a m y c i n B i s t h e n e x t r a c t e d i n t o a q u e o u s pH 6 . 5 t o 7 . 5 , r e a c i d i f i e d t o pH 2 and e x t r a c t e d b a c k i n t o e t h y l a c e t a t e . The o r g a n i c l a y e r is w a s h e d , d r i e d a n d c o n c e n t r a t e d t o y i e l d c r y s t a l l i n e rifamycin B.
2 . 6 Rifamycin
0
R i f a m y c i n O 4 was f i r s t o b t a i n e d by a e r i a l o r c h e m i c a l o x i R e c e n t l y 6 i t h a s b e e n shown t h a t e l e c d a t i o n of r i f a m y c i n B . trochemical oxidation of rifamycin B y i e l d s rifamycin 0 quanti-
48
tat ively
.
2.6.1
Producing organism
Rifamycin 0 has been isolated as the main active component from a culture beer of StheptOmyCeteb which was isolated from a soil sample collected in Miyagi-ken, Japan and is designated as strain No. 4107 Az. 2.6.2 A.
Physical properties
From methanol it is obtained as pale yellow crystals.,
B. It has no definite melting point: rifamycin 0 decomposes at 160'C and does not melt until 300'C. C.
Specific rotation: Icrlko = +71.5'.
D. U.V. absorption: in methanol solution containing 5% of acetate buffer solution (pH 4.62) it shows absorption maxima at 226 nm (El = 3651, 273 nm (El 4 4 0 ) and 370 nm (El = 60). 1 I 1 It has no absorption in the visible region of the spectrum. E. Infrared abSOrDtiOn: unlike all other rifamvcins. rifamycin 0 shows a very-characteristic strong absorp;ion a t 1822 cm-l.
F. Molecular formula: C39H47N014. 2.6.3
Isolation and purification
The fermentation broth is adjusted to acidic pH and then extracted with ethyl acetate. The organic layer is washed, dried and evaporated to dryness to yield a solid which is chromatographed on silica gel to yield pure rifamycin 0 .
2.7 Rifamycin
S
Rifamycin 0 ' on acidic hydrolysis yields rifamycin S4,l. 2.7.1
Producing organism
The blocked mutant (ATCC 21271) of N o c a h d h meditehhanei besides producing rifamycin SV also produces rifamycin S. 2.7.2
Physical properties
A. Rifamycin S is an orange yellow compound. lizes from methanol.
B
Melting point: 180-182'C.
C
Specific rotation:
= +476'
It crystal-
(C = 0.1% in meth-
anal).
D U.V. and visible absorption spectrum: in phosphate buffer pH 7.3) rifamycin S shows absorption maxima at 317 nm
49 ( E l = 426) and 525 nm; in 0.1N hydrochloric acid it shows absobption.maxima at 279 nm (El = 3291, 340 nm ( E i 117) and 4 0 1 nm (E: 84).
E.
Molecular formula: C37H4,NOI2.
2.7.3
Structure
Me
Me
AcO Me
RIFAMYON S (RED)
In a recent publication it has been shown that rifamycin S could exist in two tautomeric forms (red and yellow in color) which are interconvertible. Both the tautomers show identical biological activities.
2.8 Rifamycin SV Rifamycin SV4” was initially obtained by chemical reduction of rifamycin S which was in turn prepared by acidic cleavage of rifamycin 0, a product of oxidation of rifamycin B. 2.8.1
Producing organism
It is produced in the fermentation of a blocked mutant (ATCC 21271) of Nocahdia m e d i t e h h a n e i . Recently1’ i t has been reported that rifamycin SV is produced by Michomono6poha e.k?.k?Lpho6 doha.
2.8.2
Physical properties
A.
It is obtained from benzene as yellow-orange crystals.
B.
Melting point:
it
has no definite melting point, de-
50
composes a t 1 4 O o C and d o e s n o t m e l t u n t i l 300OC. C.
I t i s a s t r o n g monobasic a c i d (pH
+
2.7).
D. U.V. and v i s i b l e s p e c t r u m : i n p h o s p h a t e b u f f e r s o l u t i o n (pH 7.3) r i f a m y c i n SV shows a b s o r p t i o n a t 223 nm (E: = 586 314 nm ( E i 322) and 445 nm (Ei = 204). E.
Specific rotation: {a);*
F.
Molecular formual:
2.8.3
-4'
(C = 1 . 0 % i n methanol)
C37H47N012.
I s o l a t i o n and p u r i f i c a t i o n
The f e r m e n t a t i o n b r o t h i s t r e a t e d w i t h a s o l u t i o n of a s c o r b i c a c i d ( 5 g / l i t r e ) , a d j u s t e d t o pH 7 and f i l t e r e d . The f i l t r a t e is a c i d i f i e d t o pH 3 and e x t r a c t e d w i t h e t h y l a c e t a t e . The o r g a n i c l a y e r i s washed, d r i e d and e v a p o r a t e d t o d r y n e s s t o y i e l d a s o l i d which i s chromatographed o v e r s i l i c a g e l t o y i e l d p u r e r i f a m y c i n SV.
2.9 Rifamycin L 2.9.1
Producing organism
I n c u b a t i o n of r i f a m y c i n S w i t h washed mycelium of Nocakdia mediteaaanei ( s t r a i n ME/291) p r o d u c e s r i f a m y c i n s B, Y and LIZ. 2.9.2
Physical properties
A. Rifamycin L c r y s t a l l i z e s from a m i x t u r e of a c e t o n e and benzene.
B.
M e l t i n g p o i n t : i t decomposes a t 152-153OC.
C. U . V . and v i s i b l e a b s o r p t i o n : i n b u f f e r e d s o l u t i o n (pH 7.38) i t shows a b s o r p t i o n a t 298 nm ( E , 19800), 412 nm (17500).
D. NMR s p e c t r u m i s similar t o r i f a m y c i n B e x c e p t i t shows a s i g n a l a t 64.58 (2H) f o r t h e g l y c o l i c methylene g r o u p . E.
Molecular formula:
2.9.3
C39H49N014.
I s o l a t i o n and p u r i f i c a t i o n
The f i l t e r e d b r o t h is a c i d i f i e d t o pH 2 and e x t r a c t e d w i t h e t h y l a c e t a t e . The combined o r g a n i c l a y e r i s c o n c e n t r a t e d and The aqueous r i f a m y c i n s e x t r a c t e d i n t o p h o s p h a t e b u f f e r pH 7.38. s o l u t i o n a f t e r a c i d i f i c a t i o n i s re-extracted w i t h e t h y l acetate. E v a p o r a t i o n o f t h e s o l v e n t and p r e c i p i t a t i o n i n t o p e t r o l e u m e t h e r y i e l d s a crude m i x t u r e .of r i f a m y c i n s which a r e f u r t h e r s e p a r a t e d u s i n g c o u n t e r c u r r e n t d i s t r i b u t i o n and column chromatography
-
' -
2 . 1 0 Rifamycin W 2.10.1
Producing organism
Y
51
R i f a m y c i n W I 3 i s p r o d u c e d by f e r m e n t a t i o n o f a m u t a n t s t r a i n ( m u t a n t 1 2 6 ) o f Nocahdia meditehhanei. 2.10.2
Physical properties
A.
I t c r y s t a l l i z e s f r o m e t h y l a c e t a t e as y e l l o w n e e d l e s .
B.
I t h a s no d e f i n i t e m e l t i n g p o i n t .
C. U.V. and v i s i b l e spectrum: i t s spectrum is d i f f e r e n t from t h a t of r i f a m y c i n S . I n 0.1M s o d i u m h y d r o x i d e s o l u t i o n i t shows a b s o r p t i o n a t 239 nm ( l o g E = 4 . 5 6 1 , 350 nm ( l o g E Y.O9), 5 4 0 nm ( l o g E = 3 . 7 3 ) .
D. pKa
Y.9
a n d 11.1.
E. I n f r a r e d spectrum: I t l a c k s t h e a b s o r p t i o n due t o a c e t o x y l ( 1 7 0 0 em-') a n d t h e d i h y d r o f u r a n o n e c a r b o n y l ( 1 7 2 5 cm-') w h i c h a r e c h a r a c t e r i s t i c o f o t h e r r i f a m y c i n s . As in r i f a m y c i n S i t shows b a n d s a t 1 6 9 0 a n d 1 E 3 0 cm-l. F.
Molecular formula:
2.10.3
C35H45011N.
I s o l a t i o n and p u r i f i c a t i o n
The f e r m e n t a t i o n b r o t h is f i l t e r e d , a d j u s t e d t o pH 2 a n d e x t r a c t e d w i t h e t h y l a c e t a t e . The o r g a n i c l a y e r i s e x t r a c t e d w i t h a n a q u e o u s s o l u t i o n o f sodium p h o s p h a t e b u f f e r (pff 7 . 3 8 ) . A f t e r washing t h e aqueous l a y e r w i t h chloroform it i s r e a d j u s t e d t o pH 2 a n d r i f a m y c i n W e x t r a c t e d i n t o e t h y l a c e t a t e . R i f a m y c i n W i s p u r i f i e d by c o u n t e r c u r r e n t d i s t r i b u t i o n u s i n g e t h y l a c e t a t e and sodium p h o s p h a t e b u f f e r pH 6 . 5 (1:l) a s t h e s o l v e n t system. 2.10.4
Structure
The s t r u c t u r e o f r i f a m y c i n W i s shown on t h e f o l l o w i n g Page *
2 . 1 1 Rifamycin
Y
R i f a m y c i n Y i s p r o d u c e d 5 as a b y - p r o d u c t when t h e f e r m e n t a t i o n o f Nocahdia meditchhanei i s c a r r i e d o u t i n t h e p r e s e n c e of sodium d i e t h y l b a r b i t u r a t e . The s t r u c t u r e o f r i f a m y c i n Y h a s b e e n e s t a b l i s h e d i n d e p e n d e n t l y by c h e m i c a l d e g r a d a t i 0 1 - 1 ~ ~ a n d s p e c t r o s c o p i c method a n d by X-ray a n a l y s i s 1 4 a o f t h e p - i o d o a n i l i d e of r i f a m y c i n Y . A l t h o u g h t h e s t r u c t u r e o f r i f a m y c i n Y d i f f e r s from r i f a m y c i n B o n l y a t C ~ and O C21, it is devoid of any a n t i b a c t e r i a l a c t i v i t y . ( S t r u c t u r e on f o l l o w i n g p a g e ) .
52
CH3
RIFAMYCIN W
Me
Me
Ma
Me RIFAMYCN Y
53
3. Halomicins 3 .IIntroduction -
Halomicin complex, consisting of a mixture of halomicins A, B, C and D, is produced15 by M i c f i o m o n o d p o h a h a l o p h y t i c a (NRRL 2 9 9 8 ) . These compounds have been isolated in pure state and the structures of halomicin A 1 6 , B I O and C 1 6 have been determined. Me
HALOMICINS
A 8 C
Me
R2 - R1
H H
OH
OH H H
3.2 Chemistry -
Halomicins are closely related to rifamycins. They contain a substituted pyrrolidine ring at the C 4 position of rifamycins. Halomicins differ from each other in their hydroxylation pattern in the ansa ring or in the pyrrolidine moiety. Halomicin B is the major constituent of the halomicin complex. The structural elucidation of halomicins by Ganguly10’16 e t a t . is based on chemical degradation and spectroscopic evidence. Due to th’eir paramagnetic nature halomicins do not give good nmr spectra. However,, the nmr spectra of the methyl ethers of halomicins are very informative. Using decoupling experiments, chemical shifts and coupling constants of all the protons of halomicins A , B and C have been assigned. The presence of an
54
abnormally high-field methyl doublet at 6 -0.61 in the nmr spectrum of halomicin B di-0-methyl ether (11) suggested that halomicins belong to the ansamycin group of antibiotics and mass spectral fragmentation pattern of (11) parallels the fragmentation of rifamycin SV tri-0-methyl ether. High resolution mass spectra established the molecular formula of halomicin B to be C43H58N2012. Acetylation experiments of (11) established the presence of three alcoholic hydroxyl groups in the molecule. From these and other observations it was clear that halomicin B and rifamycin B have the same structure of the ansa rings and also that in halomicin B a C6H12NO unit is attached to the aromatic portion of the molecule. The basic nature of the nitrogen atom (in the side chain) was shown by non-aqueous titration of halomicin B di-0-methyl ether ( A HNP = 7.6; A HNP represents effective basicity of the compounds and is represented by the differences in their half neutralization potentials from that of 8-hydroxyquinoline) which compared very closely with the corresponding value for p-methoxy aniline ( A HNP = 7.1). Halomicin B on treatment with nitrous acid or on electrochemical oxidation yields rifamycin S and a basic compound (121, C6H13N0. The nmr and mass spectra of (12) established its gross structure. The relative stereochemistry of (12) was proven by its conversion to (13) as follows. Compound (12) on treatment with t-butyl bromoacetate yielded (14) which on refluxing in benzene containing p-toluenesulphonic acid yielded (13). Comparison of the nmr spectra of (13) and a related compound (15) prepared from L-prolinol established the relative stereochemistry and conformation of (13). Compound (12) was converted in two steps to (161, I @ 1 2 2 e + 7122 and similarly compound (171, I 8 1 2 2 8 - 5333 was prepared from L-prolinol. Thus the stereochemistry of (12) was established to be (2R, 3s). Finally, halomicin B was reconstituted by heating rifamycin 0 with compound (12) thus establishing the structure and absolute stereochemistry of halomicin B. The structures16 of halomicin A and C have been determined using the above chemical degradation sequence and by the application of spectroscopic methods.
3.3 Biological activity The halomicin complex and also the pure components are active against gram-positive bacteria and M y c o b a c t e t i a t u b e t c u t o h i h . They have no activity against gram-negative bacteria. Methyl ethers and acetates of halomicins were essentially inactive as antibacterials.
3.4 Physical 3.4.1
properties
Halomicin
A
Halomicin A is a yellow crystalline solid (acetone solvate), C43H58N2013, map. 192-194 d, + 100.5°(chloroform), A max 234 (48000), 302 (16700) and 419 Rm (14160). 3 -4.2 Halomicin B Halomicin B crystallizes from acetone-benzene as a yellow
55
Me
(13)
Me
56
crystalline solid, C43H58N2012, m.p. 178-182', h max 238 (421611, 298 (14187) and 415 nm (20341). 3.4.3
+
73.1',
Halomicin C
Halomicin C is a yellow amorphous solid, C43H58N2013, h max 2 3 7 (383001, 301 ( 1 0 9 0 0 ) , 4 2 0 nm (164CO).
{ a f g 6 + 153',
3.4.4
Halomicin D
Halomicin D :i a yellow cryszalline solid (acetone-benzene), m.p. 177-182 I o J D + 142.1 , A max 234 (48268), 328 ( 1 4 9 4 6 ) and 440 nm ( 6 3 1 9 ) .
3.5 Isolation and purification Halomicins are producedl by M i c h o m o n o d p o h a h a t o p h y ~ c a (NRRL 2998) and M i c a o m o n o b p o h a h a l o p h y f i c a var. n . n i g h a ( M I R L 3097). The crude halomicin complex is extracted with ethyl acetate from.the fermentation broth at pH 7.5 and then separated into its constiruents using column chromatography on silica gel or paper chromatography.
57
4.
Streptovaricins 4.1 Introduction -
Streptovaricin complex, consisting of a mixture of streptovaricins A , B, C, D, E , G, and J , is produced17 by S t h e p t o myceb 6 p e c t a b i i % . These components have been obtained pure and their structures determined. Like rifamycins, streptovaricins are also highly active against gram-positive bacteria and Mycobactehium t u b e h c u l o 6 i 6 . 4.2 Structures -
0
II
H\
0
I
STREPTOVARICINS
y43
H
U
V
W
X
Y
Z
A
W O H H
AcH
OH
B
H
AcH
OH
C
H OHH H
H
OH
D
H
OHH
H
H
H
E
H
0 0 H
H
H
OH
G
OH OH H
H
H
OH
J
H O & H H H O H
OHH
R
4.3 Chemistry Extensive chemical degradation1' and the application of spectroscopic methods established the structural relationship
58
between various streptovaricins. The final verification of the structures came from X-ray analysislg of the cyclic p-bromophenylboronate of streptovaricin C triacetate. For details of chemical degradation which lead to the structural elucidation of streptovaricin, the reader is referred to the publications” of Rinehart and his colleagues. The presence of a common chromophore in streptovaricins is indicated by the similarity of their electronic spectra. On degradation with osmium tetraoxide-sodium periodate all the streptovaricins except streptovaricin D gave streptovarone (18).
ke M e
High resolution mass spectra of the streptovaricins (except streptovaricin D) contain all the prominent peaks present in the mass spectra of prostreptovarone (19). The chemical shifts and coupling constants values of all the protons present in the aliphatic side chain of streptovaricin have been summarized17. It is clear from these data that streptovaricins differ from each other in the degree of oxygenation, oxidation and acetylation.
As the coupling constants and chemical shifts of the streptovaricins are similar to streptovaricin C (the structure of which has been rigidly established by X-ray) it is assumed that the stereochemistry of a l l the streptovaricins are the same. It is to be notedlg that the relative configuration at every comparable chiral center of streptovaricin C is identical with that in rifamycin B and Y and tolypomycin excepting the geometry of the dienamide unit (trans-cis in streptovaricin and cis-trans in rifamycins).
59
4.4 Biological
activity
Streptovaricins20 are active against gram-positive and gram-negative bacteria and especially against Myco6actchium tu6ehcuLodid. Individual components of the streptovaricin complex vary in their activity. Based on the number of strains of microorganisms inhibited and the degree of activity the results could be summarized as follows: streptovaricins A,G > B,C > D?J > E > > F. Like naturally occurring rifamycins and rifampicin, streptovaricins inhibit the growth of E. cot% by inhibition of DNA-dependent RNA polymerase of E. coli. Streptovaricins are active in inhibiting reverse transcriptase of Rauscher leukemia virus. Streptovaricin D is the most active component and streptovaricin F is essentially inactive and others have activity in the intermediary range. Streptovaricin C and streptovaricin G acetates are considerably more active than the parent compounds. It should be noted that the acetates of streptovaricins are essentially devoid of antibiotic activity.
4.5 Chemical
and physical properties
Note: Rf values refer to paper chromatography using benzene:methanol :water ( 2: 1:1). 4.5.1
Streptovaricin A
Streptovaricin A, C42H53N016, precipitated from l-chlorobutane had m.p. 200-201° (m.p. 233-243; after drying for 25 hr. over P205 in high vacuum. + 610 (C 0.118, CHC13), molecular weight 827.3364 (HRMS), f 0.37. 4.5.2
Streptovaricin B
Streptovaricin B, C~+~H53N015, when precipitated from 1chlorobutane had m.p. 187-188OC, + 5760,Rf 0.67. 4.5.3
S treptovaricin
C
Streptovaricin C, C40H51N014, precipitated from l-chloro+ 602, Rf 0.79, molecular butane had m.p. 189-19l0C, weight 769.3253 (HRMS). 4.5.4
Streptovaricin D
Streptovaricin D, C40Ha1N013, yrecipitaied from chloro+ 590.3 , molecular form-hexane had m.p. 172-175 C, {a}; weight 753.3374 (HRMS), Rf 0.96. ,4.5.5 Streptovaricin E Streptovaricin E, C40H gN011+,crystallized from l-chlorobutane, m.p. 198-200°C, { a } i $ + 412.3O,Rf 1.00. 4.5.6
Streptovaricin F
Streptovaricin F, C39H47N0l4, crystallized from ethyl
60
-
acetate -methanol, m. p. 22 2 2 24OC. 4.5.7
Streptovaricin G
Streptovaricin G precipitated from 1-chlorobutane, + 473', Rf 0 . 6 5 . C1+0H51N015,m a p . 190-192'C, 4.5.8
Streptovaricin J
Streptovaricin J, purified by countercurrent distribution followed by preparative'tlc, precipitated from ether, solid C42H53N015,m.p. 177-180 , { a ] , + 326'.
4.6 Isolation
and purification of streptovaricins
To a solution of streptovaricin in dioxan, n-hexane is added. On allowing the mixture to stand at room temperature overnight, an orange yellow precipitate (Fraction I) separates out consisting mainly of streptovaricin A . Further addition of hexane to the mother liquor yields a mixture consisting of all the streptovaricins (Fraction 11). Fraction I containing mainly streptovaricin A is further purified by chromatography on silica gel; elution with 3 % methanol in chloroform yields pure streptovaricin A and B. Column chromatography of Fraction I1 on silica gel (using 2% methanol in chloroform as eluent) and following the fractionation on tlc (silica gel; 4% methanol in chloroform) purer fractions dd all the components are collected, These fractions are further purified by chromatography on silica gel or using preparative tlc.
61 5.
Tolypomycin Y
5.1 Producing
organism
Tolypomycin Y is an ansamycin belonging to the rifamycin It is produced' by S t h e p t o m y c e A t o t y p o p h o h u d Cof amily produced in the same fermentation are rifamycins B and 0.
.
.
5 . 2 Physical
properties
A. Tolypomycin Y crystallizes from ethyl acetate as yellow needles.
B. Melting point: yellow needles lose their anisotropy at 1 2 0 - 1 2 5 O C but do not show a definite melting point below 30OoC.
C.
Molecular formula:
CL,~H~I+N~O~L,.
D. Specific rotation: ( a ) 2 2 + 3 2 6 ' ( C 1, ethanol); + 376O ( C 0 . 5 , acetone); {a?:' + 3 2 5 ' ( C 0 . 3 5 , chloroform). E. Rf values: using Whatman filter paper No. 1, Rf values of tolypomycin Y are as follows: n-hexane-benzene-ethanol-water ( 1 : 3 : 1 : 3 ) ,
Rf 0 . 7 8 .
n-hexane-benzene-acetone-water ( 3 0 : 1 0 : 1 8 : 2 2 ) ;
Rf 0 . 6 8 .
n-hexane-ether-acetone-water (15: 5 :8:12) ; Rf 0 . 2 7 .
Rf values obtained by thin layer chromatography on silica gel containing 2 % of oxalic acid are as follows: ethyl acetate and acetone (1:l) containing 1% oxalic acid; Rf 0.05 acetone containing 1% oxalic acid; Rf 0 . 2 .
F. U . V . and visible spectra: in ethanol solution tolypomycin Y shows absorption maxima at 2 3 2 nm ( 2 9 0 0 0 1 , 2 9 0 nm ( 2 3 8 0 0 1 , 3 3 7 nm ( 1 2 7 0 0 ) and 3 7 0 to 4 3 0 nm (shoulder). In pH 7 . 2 buffered solution it shows maxima at 2 3 4 . 5 ( 2 7 9 0 0 1 , 3 1 9 (25530),
387 (30001,
465
(33001,
485 nm ( 3 2 0 0 ) .
G. Infrared spectrum shows absorption at 3 5 6 5 , 3 5 0 0 , (-OH, NH), 1 7 1 5 (ester), 1684, 1 6 6 5 (carbonyl), 1620 cm-1 (amidel.
3400
5.3 Biological
activity
Tolypomycin Y is highly active against gram-positive bacteria and also has some activity against gram-negative and acid-fast bacteria. It is active against experimental infection in mice against gram-positive bacteria when administered subcutaneously, intraperitoneally and orally.
62
5.b Isolation and
purification
The culture filtrate of S t h e p t o m y c e b t o l y p o p h o h u b is adjusted to pH 8 and then extracted with ethyl acetate. After concentration of the organic layer, the antibiotic is precipitated into ether-n-hexane (1:lO). The crude antibiotic mixture is then purified on a silica gel column ( o r using preparative tlc). 5.5 Structure -
Mo
Me
Me
The structurezz of tolypomyc-.?one was deduced us,.?g chemical degradation and spectroscopic methods and confirmed by Xray analysis with 8,21, 23-tri-Pbromobenzoyl tolypomycinone. It should be noted that tolypomycinone is very closely related to rifamycin S with a methyl and an adjacent double bond replaced by a cyclopropane ring and a carbonyl group.
63 6 . Naphthomycin
6 . 1 Producing
organism
Naphthomycin was f i r s t 2 3 i s o l a t e d from t h e c u l t u r e s of s t r a i n Tu 1 0 5 of S t J t e p t o m g c e b c o L L i n u b L i n d e r b e i n and l a t e r 2 4 from t h e c u l t u r e o f s t r e p t o m y c e s s p . X-12384.
6.2 P h y s i c a l
properties
A. Naphthomycin c r y s t a l l i z e s from a c e t o n e - h e x a n e as d e e p yellow n e e d l e s .
ing
.
B.
Molecular formula: C 4 0 H 4 6 C l N O g .
C.
Melting p o i n t :
D.
Specific rotation:
E.
For U.V.
and I . R .
i t decomposes a t 2 0 0 ° C w i t h o u t m e l t {a],
+ 432'
C , 0.5
i n chloroform).
curves s e e r e f . 23.
6 . 3 Chemistry The s t r u c t u r e of naphthomycin i s b a s e d on s p e c t r o s c o p i c e v i d e n c e ; i n p a r t i c u l a r nmr s p e c t r u m u s i n g l a n t h a n i d e s h i f t rea g e n t h a s been found t o be most u s e f u l i n a s s i g n i n g t h e c h e m i c a l s h i f t and c o u p l i n g c o n s t a n t v a l u e s . Naphthomycin r e s e m b l e s m a y t a n s i n e which p o s s e s s e s a c h l o r i n e o r t h o t o t h e a i d e n i t r o g e n , c o n t a i n s a n e x t e n d e d c o n j u g a t e d s e c o n d a r y amide f u n c t i o n l i k e geldanamycin and r i f a m y c i n s b u t u n l i k e t h e s e a n t i b i o t i c s , naphthomycin d o e s n o t c o n t a i n an o x y g e n a t e d f u n c t i o n a t C g . The e i g h t c a r b o n f r a g m e n t s a t t a c h e d a t C 5 h a s t h e same c o n s t i t u t i o n as i n s t r e p t o v a r i c i n s . Naphthomycin c o n t a i n s 2 3 - s k e l e t a l c a r b o n atoms compared t o 1 5 found i n m a y t a n k i n e and 1 7 found i n r i f amycins. 6.4 Structure -
The s t r u c t u r e o f naphthomycin i s on t h e f o l l o w i n g page.
6.5 Isolation
and p u r i f i c a t i o n
The f e r m e n t a t i o n b r o t h i s e x t r a c t e d w i t h e t h y l a c e t a t e and t h e o r g a n i c l a y e r t h e n washed w i t h aqueous sodium c a r b o n a t e s o l u t i o n , The b l u e g r e e n a l k a l i n e e x t r a c t i s a c i d i f i e d w i t h concentrated hydrochloric acid. The a n t i b i o t i c is t h e n e x t r a c t e d i n t o e t h y l a c e t a t e , washed w i t h water, d r i e d o v e r sodium s u l p h a t e and e v a p o r a t e d t o d r y n e s s t o y i e l d a y e l l o w c r y s t a l l i n e solid. On t l c ( s i l i c a g e l ; M e r c k F-254; e t h y l acetate:water: f o r m i c acid:100:30:2.5) t h e c r u d e m i x t u r e showed t h e p r e s e n c e of t h e m a j o r component, naphthomycin ( R f 0 . 7 5 ) and two m i n o r i m p u r i t i e s ( R f 0 . 6 3 and 0 . 3 3 ) . The c r u d e a n t i b i o t i c s complex i s p u r i f i e d by column chromatography ( s i l i c a g e l ) u s i n g c h l o r o form:methanol (19:l) as t h e e l u t i n g s o l v e n t .
64..
/
/
4
Mb \\
CH
\
CH-
I
CH
CH
/CH
1
NAPHTHO MY C IN
7. ’
Geldanamycin 7.1 Producing organism
Geldanamycin is produced2 var. geLdanu6 var. n o v a .
7.2 Chemical
by S t h e p t O m y C e d h y g h o 6 copicud
and physical properties
A. Geldanamycin is a yellow crystalline solid (chloroform-ether).
B.
Melting point: 252-255OC.
C.
Specific rotation:
101;5
+ 55’.
65
D. U.V. spectrum: i n methanol s o l u t i o n geldanamycin shows maxima a t 2 5 5 (163501, 304 (19300) and a broad weak s h o u l d e r a t $ 4 0 0 nm (980). E. I n f r a r e d spectrum: i t shows a b s o r p t i o n a t 3 5 1 0 , 3445, 3350, 3315, 1734, 1700, 1676, 1655, 1635, 1608 cm-’.
see r e f . 2 5 .
F.
N m r spectrum:
G.
Molecular formula:
C29H1,0NzOg(m/e
560.273).
Using e x t e n s i v e c h e m i c a l d e g r a d a t i o n z 6 , h i g h r e s o l u t i o n mass s p e c t r a and nmr s p e c t r a ? geldanamycin h a s been a s s i g n e d s t r u c t u r a l formula ( 2 ) . Unlike r i f a m y c i n s , s t r e p t o v a r i c i n s , e t c . , geldanarnycin c o n t a i n s a benzoquinone n u c l e u s .
7.3 B i o l o g i c a l
activity
Geldanarnycin i s m o d e r a t e l y a c t i v e i n v i f k o a g a i n s t p r o t o zoa, b a c t e r i a and f u n g i .
7.4 I s o l a t i o n
and p u r i f i c a t i o n
Geldanarnycin i s produced by S t k e p t o m y c e h h y g k o h c o p i c u d v a r . getdanuh v a r . n o v a . The c u l t u r e d medium is f i l t e r e d and the f i l t r a t e extracted with n-butanol. The n - b u t a n o l e x t r a c t i s c o n c e n t r a t e d and t h e c r u d e s o l i d i s f i l t e r e d . Geldanamycin i s c r y s t a l l i z e d from a m i x t u r e of c h l o r o f o r m and e t h e r . 7.5 Structure -
See f o r m u l a ( 2 ) .
66
8. Maytansine 8.1 Producing organism
Maytansinoids are obtained from plant sources and possess "ansa macrolidel' structures. MaytansineZ7 is obtained from an alcoholic extract of Maytenud ohatus Loes whereas maytanprine' a and maytanbutineZa are isolated from Maytenud buchananii Loeb. 8.2 Structure -
The structure of maytansine has been established by X-ray analysis of (3-bromopropy)-maytansine. N m r and mass spectra of maytansine, maytanpurine and maytanbutine established the interrelationship between the three compounds.
Ma
0
Me0
Me
MAYTANSINE
OMe
; R = Me
MAYTANPRINE ; R = CH2Me MAYTANBUTINE; -CHMt2
8.3 Biological activity Maytansinoids show significant inhibitory activity i n v i t h o against cells derived from human carcinoma of the nasopharynx (KB) and against sarcoma 180, Lewis lung carcinoma and L-1210 and P-388 leukemias in the mouse and Walker 256 intramuscular carcinomsarcoma in the rat.
67 Acknowledgements The a u t h o r w i s h e s t o t h a n k D r . G . C . L a n c i n i f o r s u p p l y i n g a c o m p l e t e l i s t o f all p u b l i s h e d work o n r i f a m y c i n a n d P r o f e s s o r K . L. R i n e h a r t f o r s e n d i n g a c o p y o f t h e p r e - p r i n t of h i s a r t i c l e on c h e m i s t r y of t h e a n s a m y c i n a n t i b i o t i c s p u b l i s h e d i n F o r t s c h r i t t e d e r Chemie O r g a n i s c h e r N a t u r s t o f f e . 9.
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R . J. W h i t e , E . M a r t i n e l l i a n d G . L a n c i n i , P r o c . N a t . Acad. S c i . 7 1 ( 1 9 7 4 1 , 3260 a n d r e f e r e n c e s c i t e d t h e r e i n .
3.
R . D . J o h n s o n , A. H a b e r a n d K . L. R i n e h a r t , J. h e r . Chem. SOC. 9 6 ( 1 9 7 4 1 , 3316 a n d r e f e r e n c e s c i t e d t h e r e i n .
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11.
M. J . W e i n s t e i n , G . H . Wagman, M . G . P a t e l a n d J . A. M a r q u e z , U.S. P a t e n t 3 , 9 0 1 , 7 6 4 ; A u g u s t 2 6 , 1 9 7 5 .
12.
G. C. L a n c i n i , G . G. G a l l o , G . S a r t o r i and P. S e n s i , J. A n t i b i o t i c s , 22 ( 1 9 6 9 1 , 369.
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E . M a r t i n e l l i , G . G . Gallo, P . A n t o n i n i a n d R . J. W h i t e , T e t r a h e d r o n 30 ( 1 9 7 4 1 , 3 0 8 7 .
14.
J. L e i t i c h , V . 505.
P r e l o g and P. S e n s i , E x p e r e n t i a , 2 3 (19671,
1 4 a . M . B r u f a n i , W . F e d e d i , 6. G i a c o m e l l o a n d A . V a c i a g o , E x p e r e n t i a , 23 (19671, 508. 15'.
J . W e i n s t e i n , G . M . Luedemann, E . M. Oden a n d G . H . Wagman, A n t i m i c r o b i a l A g e n t s a n d C h e m o t h e r a p y ( 1 9 6 7 1 , 1 4 3 5 .
M.
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A. K . Ganguly, Y . T . L i u , 0 . Z . S a r r e a n d S . Szmulewicz, J . A n t i b i o t i c s 30 (19771 625.
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p . S i m i n o f f , R . M . S m i t h , W . T . S o k o l s k i and G . M . S a v a g e , Amer. Rev. Tuberc. Pulm. D i s . , 7 5 (19571, 576.
18.
K. L. R i n e h a r t , J r . , M . L. Mahashwari, F. J . Antosz, H . H . Mathur, K . S a s a k i and R . J . S c h a c h t , J . A m e r . Chem. SOC. 9 3 (19711, 6273 a n d r e f e r e n c e s c i t e d t h e r e i n .
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A. H. J . Wang, I . C . P a u l , K . L. R i n e h a r t , Jr., and F . J . Antosz, J . Amer. Chem. S O C . 9 3 (19711, 6275.
20.
K . L. R i n e h a r t , J r . , F. J . A n t o s z , K. S a s a k i , P. K . M a r t i n , M . L. Mahashwari, F. Reusser, L . H . L i , D . Moran and P . F. Wiley, B i o c h e m i s t r y 1 3 (19741, 861.
21.
M.
S h i b a t a , T. Hasegawa and E. H i g a s h i d e , J . A n t i b i o t i c s T. Hasegawa, E . H i g a s h i d e and M . S h i b a t a , J . A n t i b i o t i c s 24 (19711, 817.
XXIV (19711, 810; 22.
T. K i s h i , M. Asai, M. Muroi, S. Harada, E . M i z u t t a , S . T e r e o , T. Muki and K . Mizuno, T e t r a h e d r o n L e t t e r s (1969), 91; K . Kamiya, T. S u g i n o , Y . Wada, M . Nishikawa and T. K i s h i , E x p e r e n t i a , 25 (19691, 901.
23.
M . B a l e r n a , W . K e l l e r - S c h i e r l e i n , C . M a r t i n s , H. Wolf a n d H . Zahner, Arch. M i k r o b i o l . 65 (19691, 3 0 3 .
24.
T. H. Williams, J . A n t i b i o t i c s 28 (19751, 85.
25.
C . DeBoer, P. A. Meulman R . J . Wnuk and D. H . P e t e r s o n , J. A n t i b i o t i c s , 23 (19701, 4 4 2 .
26.
K. S a s a k i , K . L. R i n e h a r t , J r . , G . Slomp, M . F. G r o s t r e and E. C. Olson, J . Amer. Chem. SOC. 9 2 (19701, 7591.
27.
S . M. Kupchan, Y. Komoda, W. A. C o u r t , G . J . Thomas, R . M . S m i t h , A. K a r i n , C . J. Gilmore, R . C . H a l t i w a n g e r and R . F. Bryan, J. Amer. Chem. SOC, 9 4 (19721, 1354.
28.
S . M . Kupchan, Y . Komoda, G . Chem. Comm. (19721, 1065.
J . Thomas and H . P. J . H i n t z ,
69
Cephalosporin A n t i b i o t i c s
.
.
R o b e r t L H a m i l l and L o u i s e W C r a n d a l l M i c r o b i o l o g i c a l and Fermentation P r o d u c t s Research L i l l y Research L a b o r a t o r i e s . I n d i a n a p o l i s . I n d i a n a
.
1 2
.
-
Cephalosporin C Introduction Producing organism 2.1.2 P h y s i c a l and c h e m i c a l p r o p e r t i e s 2.1.3 S t r u c t u r a l formula 2.2 Summary of b i o l o g i c a l a c t i v i t i e s and t h e r a p e u t i c uses 2.3 I s o l a t i o n and p u r i f i c a t i o n methods 2.3.1 I s o l a t i o n b a s e d on t h e method r e p o r t e d by Abraham 2.3.2 Use of c a r b o n column chromatography 2.3.3 P r e p a r a t i o n and i s o l a t i o n of t h e q u i n o l i n e salt of N-chloroacetyl cephalosporin C 2.3.4 P r e p a r a t i o n of c e p h a l o s p o r i n C-zinc complex 2.3.5 D e t e c t i o n and a s s a y 2.4 Literature Cited -
2.1 2.1.1
-
-
3
.
-
.
................................. ......................... ........... ......................... ............................. ........... .................................... ........ ..... .................................... ........................ .............................
7-Amii n o c e p h a l o s p o r a n i c Acid Introduction Producing organism P h y s i c a l and c h e m i c a l p r o p e r t i e s 3.1.2 S t r u c t u r a l formula 3.1.3 Summary of b i o l o g i c a l a c t i v i t i e s 3.2 P r e p a r a t i o n of 7-aminocephalosporanic a c i d 3.3 (7-ACA) P r e p a r a t i o n of 7-ACA from c e p h a l o s p o r i n C 3.3.1 using n i t r o s y l chloride P r e p a r a t i o n of 7-ACA from t h e q u i n o l i n e 3.3.2 s a l t of N - c h l o r o a c e t y l c e p h a l o s p o r i n C D e t e c t i o n and a s s a y 3.3.3 Literature Cited 3.4
3.1 3.1.1
-
4
............................... .............................
Introductory Section 1.1 Literature Cited
................................. ......................... ........... ......................... .............
...................................... .................... ..... ........................ .............................
M e t h o x y c e p h a l o s p o r i n s (Cephamycins A and B ; C-2801X) Introduction Producing organisms 4.1.2 P h y s i c a l and c h e m i c a l p r o p e r t i e s 4.1.3 S t r u c t u r a l formulas 4.2 Summary o f b i o l o g i c a l a c t i v i t i e s 4.3 I s o l a t i o n and p u r i f i c a t i o n methods 4.3.1 I s o l a t i o n of cephamycins A and B 4.3.2 S e p a r a t i o n of cephamycins A and B 4.3.3 S e p a r a t i o n and p u r i f i c a t i o n of C - 2 8 0 1 X 4.3.4 Assay and d e t e c t i o n 4.4 Literature Cited
4.1 h.1.1
-
-
-
................................. ........................ ........... ........................ ............. ........... ........... .......... ..... ........................
.............................
71 71 78 78 78 78
78 78 78 79 80
81 81 82 84
84 84 84 84 84 84 85 85 86 87
87 87 88
88 89 89 89 89 90 90
70
5
.
A 1 6 8 8 4 (7-Methoxycephalosporin C )
.............................. ...................... ........ 5.1.2 ...................... 5.1.3 5.2 . ........ 5.3.1 ............... 5.3.2 ......................... 5.3.3 ..................... 5.4 .......................... 6. A 1 6 8 8 6 A and A16886B (Cephamycin C ) 6.1 I n t r o d u c t i o n .............................. 6.1.1 Producing organisms ..................... 6.1.2 P h y s i c a l and chemical p r o p e r t i e s ........ 6.1.3 S t r u c t u r a l formula ...................... 6.2 Summary of b i o l o g i c a l a c t i v i t i e s .......... 5.1 5.1.1
Introduction Producing organism Chemical and p h y s i c a l p r o p e r t i e s S t r u c t u r a l formula Summary of b i o l o g i c a l a c t i v i t i e s and u s e s I s o l a t i o n and p u r i f i c a t i o n methods I s o l a t i o n of crude A 1 6 8 8 4 S e p a r a t i o n of A 1 6 8 8 4 and deacetoxycephalosporin C D e t e c t i o n and a s s a y Literature Cited
........ A 1 6 8 8 6 B ................................. 6.3.2 I s o l a t i o n of cephamycin C ............... 6.3.3 D e t e c t i o n and a s s a y ..................... 6.4 L i t e r a t u r e C i t e d .......................... 6.3
6.3.1
I s o l a t i o n and p u r i f i c a t i o n methods I s o l a t i o n and s e p a r a t i o n of A 1 6 8 8 6 A and
92 92 92 92 92 92 92 93 94 94 95 95 95 95 96 96 96 97 98 99
71
1. Introductory Section At present, 13 different compounds containing the cephalosporin nucleus have been isolated from fermentations of various strains of streptomycetes and fungi. These are listed in Table 1. While these compounds do not have the properties necessary for therapeutic efficacy, their discovery has made possible extensive microbiological and chemical modifications of the cephalosporin nucleus. These studies are well-documented. The monograph edited by Flynn in 1972l includes reviews concerned with both chemistry and biology of cephalosporins and penicillins. More recently, Webber and Ott2 reviewed structureactivity relationships of the cephalosporin antibiotics. From the introduction of cephalothin in 1964, the cephalosporin antibiotics have proven to be important therapeutic agents in the treatment of infections caused by both gram-positive and gram negative organisms. The semi-synthetic cephalosporin antibiotics currently marketed in the United States are listed in Table 2. Moellering and Swartz3 have recently reviewed the newer cephalosporins, including some not yet released for clinical use. The cephalosporin nucleus, 7-aminocephalosporanic acid, was a necessary prerequisite for the preparation of the semisynthetic cephalosporins. Although 7-aminocephalosporanic acid is not a naturally occurring fermentation product, we have included chemical methods of preparation in this chapter. Isolation methods for cephalosporin C; methoxycephalosporins (cephamycins A and B, C-2801x1; A16884 (7-methoxycephalosporin C); A16886A and A16886B (cephamycin C) are given in separate sections. The references in Table 1 give isolation methods for the other cephalosporin antibiotics.
1.1 Literature Cited 1.
E. H. Flynn (editor), Cephalosporins and Penicillins: Chemistry and Biology, Academic Press, New York, 1972.
2.
J. A. Webber and J. L. Ott, Advances in Applied Microbiology (in press).
3.
R. C. Moellering and M. N. Swartz, New England J. Med. 294 (1976) 24-48.
4.
G. G. F. Newton and E. P. Abraham, Nature 175 (1955) 548.
5.
R. L. Hamill and R, Nagarajan, U.S. Patent 3,862,008; January 21, 1975.
6.
T. Kanzaki, Y. Fuj,isawa, H. Shirafugi, K. Nara and M. Yoneda, U.S. Patent 3,926,729; December 16, 1975.
7.
R. Nagarajan, L. D. Boeck, M. Gorman, R. L. Hamill, C. E. Higgens, M. M. Hoehn, W. M. Stark and J. G. Whitney, J. Amer. Chem. SOC. 93 (1971) 2308-2310.
72 8.
E . P . S t a p l e y , M . J a c k s o n , S. H e r n a n d e z , S . B . Zirnmerman, S . A. C u r r i e , S . M o c h a l e s , J . M . Mata, H . B. W o o d r u f f and D . H e n d l i n , A n t i m i c r o b . Ag. a n d C h e m o t h e r . 2 ( 1 9 7 2 ) 122-131.
9.
H. F u k a s e , T. Hasegawa, K. Hatano, H . I w a s a k i a n d M . Y o n e d a , J. A n t i b i o t i c s 29 ( 1 9 7 6 ) 1 1 3 - 1 2 0 .
10.
P. T r a x l e r , H . J . T r e i c h l e r a n d J . N u e s c h , J . A n t i b i o t i c s 2E ( 1 9 7 5 ) 605-606.
11.
T. K a n z a k i , T. F u k i t a , H . S h i r a f u g i a n d Y . F u j i s a w a , J. A n t i b i o t i c s 27 ( 1 9 7 4 ) 361-362.
12.
T. S h o r n u r a , H . W a t a n a b e , Y . Ogawa, K . O h b a , Y . Kondo, M. Kojima, S . I n o u e a n d T . N i i d a , U.S. P a t e n t 3 , 9 7 4 , 0 3 5 ; August 1 0 , 1976.
13.
H . I m a n a k a , J . H o s o d a , K . Jomon, I . U e d a , D. M o r i n o a n d H . S a k a i , German P a t e n t 2 , 3 3 2 , 0 6 5 ; J a n u a r y 1 7 , 1 9 7 4 .
&
0
DZ
Y I
x x "-5.'
m E m
L
CT
u
u
m
5 0
u 0
n
E?
u)
n
0
I I
CT
C
u
a
.-I k 0
E?
m
5
m
5 8 0 I
I1
I
0 I1
-a r -k
73
Table 1 (continued)
Na m 7-Methoxy-3-deacety1-3-0carbamylcephalospbrin C (A168B68, cephamycin C)
Producing Organism
saLep.tOnyces C&UUUgpnub
s&ep.tonyces
eactamdunavLs
Cephamycin A
Structure
R = -H
R' =
-0CH3
R"
-0CONH2
~l
-H
R =
Reference 798
8
R ' = -OM-13
R" = Cephamycin
B
C2081X
R = - H
R'
=
-0CHq
R"
=
-OCOC=CH t OCH 3
R R'
N-acetyldeacetoxycephalosporin C
-OCOC=CH I OCH 3
-H ~l
8
9
-OCH?
10
Table 1 (continued)
Name 3-Deace toxy-3-thiome t h y l cephalosporin C
Structure
Producing Organism
Cephdo~poaiwn
achernonium
R = -H R'
R"
3-De acet y 1-3-0 -carb amoy 1cephalosporin C (A16886A)
7-Methoxy &ace t y l cephalosporin C
7-Me thoxy &ace toxy cephalosporin C ( WS-3442-D)
Sfitrrepbmycw ChVUUQenUA
11
31
=
-S-CH3
R=-H R'
-H
R"
-OCONH,
R=-H z
R"
= -OH
-0CH3
Rz-H -0CH3
R' 2
7
12
R'
R"
Reference
-H
13
Table 2 Cephalosporin Antibiotics Marketed i n the United States
COOH
Generic Name Cephalothin
Cephaloridine
Cephalogly cin
Route o f Administration
Structure
Intravenous Intramua cular
Intravenous Intramuscular
Oral
2-
R =
UCH 2-
II
=
II
-= II
a
C
0
.r( 4
m
L
8
II
a
II
a
II
a
II
a
8
r
u
II
a
m
>
m
4J L
L
r(
77
.,
d m
d m
n o
$!
d
78 2 . Cephalosporin C 2 . 1 Introduction As t h e p a r e n t s u b s t a n c e of group of c l i n i c a l l y important a n t i b i o t i c s , cephalosporin C occupies a unique p l a c e i n t h e h i s t o r y of a n t i b i o t i c d e v e l o p m e n t . Abraham and Loder' h a v e reviewed t h e work which l e d t o t h e i s o l a t i o n and s t r u c t u r e of cephalosporin C.
2.1.1
Producing organism1
C e p h a t o A p onium acaemonium 2.1.2
.
P h y s i c a l and c h e m i c a l p r o p e r t i e s 2
C 1 6 H 2 1 N 3 0 8 S mol. w t . 4 1 5 . 4 4 1% 2 0 8 ) Xmax 2 6 0 nm (Elcm pKa's 3 . 9 , 5 . 3 , 1 0 . 5 ( 6 6 % d i m e t h y l f o r m a m i d e ) 2.1.3
S t r u c t u r a l formula
COOH
2 . 2 Summary
of b i o l o g i c a l a c t i v i t i e s and t h e r a p e u t i c uses
C e p h a l o s p o r i n C i s m o d e r a t e l y a c t i v e a g a i n s t gram p o s i t i v e and gram n e g a t i v e b a c t e r i a b o t h i n v i t k o a n d i n v i v o 3 , b u t t h e a c t i v i t y i s n o t s u f f i c i e n t f o r t h e r a p e u t i c u s e s . The d e r i v a t i v e s of c e p h a l o s p o r i n C have p r o v e n t o b e of g r e a t c l i n i c a l significance.
2.3 I s o l a t i o n
and p u r i f i c a t i o n methods
I t i s d i f f i c u l t t o c h o o s e r e p r e s e n t a t i v e i s o l a t i o n methods from t h e v a s t number of p u b l i c a t i o n s on c e p h a l o s p o r i n C. We h a v e s e l e c t e d two methods which i l l u s t r a t e t h e i s o l a t i o n of c e p h a l o s p o r i n C i t s e l f a n d two methods which i l l u s t r a t e t h e i s o l a t i o n of c e p h a l o s p o r i n C d e r i v a t i v e s , These d e r i v a t i v e s can s u b s e q u e n t l y be used as s t a r t i n g material f o r c l e a v a g e r e a c t i o n s t o produce 7-aminocephalosporanic a c i d .
2.3.1
I s o l a t i o n b a s e d on t h e method r e p o r t e d b y Abrahamgy
The f e r m e n t a t i o n b r o t h ( 2 0 0 m l f is f i l t e r e d w i t h f i l t e r a i d , and t h e f i l t e r cake i s washed w i t h water. The f i l t r a t e
79
and washed a r e combined and r u n o v e r a column o f Dowex 50 x 8 ( H t ) t o a d j u s t t h e pH t o 2 . 8 . The e l u a t e i s k e p t a t 37OC f o r 3 h r t o destroy p e n i c i l l i n N. The s o l u t i o n i s r e f i l t e r e d t o c l a r i f y i t and t h e f i l t r a t e i s p a s s e d o v e r a 1 x 3 . 1 c m column of A m b e r l i t e 4 0 0 , a c e t a t e form, 1 0 0 - 2 0 0 mesh (Rohm and Haas, Philadelphia, PA).* The s i z e of t h e column i s c o n t r o l l e d t o a b s o r b C 1 - and o t h e r i n o r g a n i c a n i o n s b u t n o t t o a b s o r b t h e cephalosporin C. The e f f l u e n t , c o n t a i n i n g c e p h a l o s p o r i n C , i s f r e e z e - d r i e d t o y i e l d a brown powder. The powder i s d i s s o l v e d i n 0 . 2 M ammonium a c e t a t e b u f f e r , pH 5 . 0 , and t h e s o l u t i o n i s a p p l i e d t o a 1 x 50 c m column of A m b e r l i t e XE-58 ( a f i n e l y d i v i d e d form of A m b e r l i t e I R - 4 B , 1 0 0 - 1 5 0 mesh, a c e t a t e form) i n e q u i l i b r i u m w i t h t h e ammonium a c e t a t e b u f f e r . E l u t i o n i s carr i e d o u t w i t h t h e same b u f f e r a t a f l o w r a t e o f 2 m l p e r 5 min. The 2 m l f r a c t i o n s a r e a s s a y e d by m e a s u r i n g t h e a b s o r b e n c y a t 2 6 0 nm a n d t h e n i c o t i n a m i d e a s s a y . Fractions containing the h i g h e s t amount of c e p h a l o s p o r i n C a r e combined and f r e e z e - d r i e d t o remove most of t h e ammonium a c e t a t e . The d r i e d powder i s d i s s o l v e d i n 2 m l of w a t e r , and t h e s o l u t i o n i s a p p l i e d t o a 1 x 5 cm column of Dowex 5 0 x 8 ( H t ) ( 2 0 0 - 4 0 0 mesh) (Dow Chemical Co. Midland, M I ) . The column i s washed q u i c k l y w i t h 1 3 m l of water. The r e s i n i s e x t r u d e d , s u s p e n d e d i n 1 0 m l of water, and t h e s u s p e n s i o n i s t i t r a t e d t o pH 6 . 0 w i t h 1 N NaOH. The m i x t u r e i s f i l t e r e d , washed w i t h water, and t h e f i l t r a t e i s f r e e z e - d r i e d t o y i e l d a powder of c e p h a l o s p o r i n C sodium s a l t . The s a l t can be c r y s t a l l i z e d from a minimum q u a n t i t y of w a t e r , b u t t h e p r o duct i s usually not pure. The c r u d e c e p h a l o s p o r i n C (Na s a l t ) i s c o n v e r t e d t o t h e f r e e a c i d by d i s s o l v i n g t h e powder i n w a t e r ( 2 8 mg/ml) and addi n g Dowex 5 0 x 8 (H') t o t h e s t i r r e d s o l u t i o n u n t i l t h e pH The r e s i n is f i l t e r e d o f f and t h e f i l t r a t e i s f a l l s t o 2.6. freeze-dried. The r e s u l t i n g d r y powder i s d i s s o l v e d i n 0 . 2 m l of water, and t h e s o l u t i o n i s a p p l i e d t o a column ( 0 . 5 x 2 5 c m ) of Dowex 1 x 8 ( a c e t a t e c y c l e , 1 0 0 - 2 0 0 m e s h ) . E l u t i o n i s c a r r i e d o u t w i t h 0.5 N a c e t i c a c i d , c o l l e c t i n g 1 m l f r a c t i o n s p e r 5 min. The e l u t i o n i s m o n i t o r e d by r e a d i n g t h e e x t i n c t i o n a t 2 6 0 nm o r by n i n h y d r i n c o l o r d e n s i t y . The f r a c t i o n s c o n t a i n i n g t h e h i g h e s t amount o f c e p h a l o s p o r i n C a r e p o o l e d and f r e e z e d r i e d t o y i e l d t h e f r e e a c i d of c e p h a l o s p o r i n C . The. sodium s a l t i s o b t a i n e d by d i s s o l v i n g t h e powder i n 2 m l of w a t e r , t i t r a t i n g t o pH 6 . 0 w i t h 0 . 0 2 N N a O H , a n d f r e e z e - d r y i n g . The sodium s a l t of c e p h a l o s p o r i n C i s c r y s t a l l i z e d as t h e d i h y d r a t e from water ( 1 5 0 mg/ml) w i t h c a r e f u l a d d i t i o n of e t h a n o l . 2.3.2
Use of c a r b o n column chromatography6
Whole b r o t h c o n t a i n i n g a p p r o x i m a t e l y 2 mg c e p h a l o s p o r i n C / m l and 1 mg p e n i c i l l i n N / m l i s f i l t e r e d w i t h H y f l o S u p e r c e l (Johns M a n v i l l e Co., N e w Y o r k ) . Two t h o u s a n d m l o f t h e f i l t r a t e "Higher y i e l d i n g fermentation b r o t h s (e.g., 2 mg/ml o r above) can be s e l e c t i v e l y adsorbed on anion exchange r e s i n column u s i n g l e a d and t r a i l columns as described i n Ref. 5. The trail Amberlite IRA-68 ( a c e t a t e form) column can be e l u t e d w i t h 0.85M sodium acetate (pH 5.8) and t h e cephalos p o r i n C r i c h f r a c t i o n s are pooled and concentrated u n t i l c r y s t a l l i z a t i o n o f cephalosporin C sodium s a l t is i n i t i a t e d , and t h e mixture i s c h i l l e d a t
inor
fnr m m n l a t p rrvstalli7ation.
80
is p a s s e d o v e r a column ( 3 . 7 x 6 1 cm) c o n t a i n i n g 550 m l o f c h r o m a t o g r a p h i c c a r b o n ( 4 0 mesh), a n d t h e column i s washed w i t h 1 0 0 0 m l o f water. The e f f l u e n t a c i d w a s h i n g s c o n t a i n p e n i c i l l i n N b u t l i t t l e o r n o c e p h a l o s p o r i n C. The column is e l u t e d w i t h a s o l v e n t m i x t u r e (pH 1 2 . 0 ) o f 2 0 0 0 m l o f a 7 % ( b y volume) a q u e o u s s o l u t i o n of n - b u t a n o l a n d 2 0 0 m l o f a 0 . 1 N a q u e o u s s o l u t i o n of Na OH . F r a c t i o n s of 50 m l a r e c o l l e c t e d and t h e e l u t i o n s a r e m o n i t o r e d by s p e c t r o p h o t o m e t r y a t 260 nm f o r c e p h a l o s p o r i n C a n d a t 420 nm f o r p i g m e n t s . The o p t i c a l d e n s i t y v a l u e s a t 260 nm s t a r t t o i n c r e a s e a t a b o u t f r a c t i o n 1 5 , p e a k a t f r a c t i o n 2 0 and f a l l t o n e a r zero a t about f r a c t i o n 30, while t h e o p t i c a l d e n s i t y v a l u e s a t 4 2 0 nm s t a r t t o i n c r e a s e a t a b o u t f r a c t i o n 23, peak a t f r a c t i o n 2 7 a n d f a l l t o n e a r z e r o a t f r a c t i o n 30. F r a c t i o n 2 0 a s s a y s a t a b o u t 1 2 mg c e p h a l o s p o r i n C / m l . Fractions 1 0 t h r o u g h 1 8 c o n t a i n p e n i c i l l i n N as i n d i c a t e d by TLC on c e l l u l o s e p l a t e s using a s o l v e n t system o f n-butano1:glacial acetic a c i d : w a t e r (3:l:l) a n d n i n h y d r i n s p r a y . F r a c t i o n s 1 9 t h r o u g h 2 6 are p o o l e d a n d a d j u s t e d t o pH 6 . 5 w i t h 1 N N a O H a n d a r e conc e n t r a t e d i n vacu0 a t a n e x t e r n a l t e m p e r a t u r e o f no h i g h e r t h a n 3OoC, The r e s u l t a n t c r y s t a l s are f i l t e r e d o f f a n d d r i e d . F u r t h e r c r o p s are o b t a i n e d by c o n c e n t r a t i o n of t h e f i l t r a t e t o g i v e a t o t a l o f a b o u t 2 . 2 g of c e p h a l o s p o r i n C sodium ( 9 0 . 6 % p u r i t y , y i e l d of a b o u t 5 0 % ) . The c r y s t a l s a r e r e c r y s t a l l i z e d from 10 m l of w a t e r : e t h a n o l (1:l) t o y i e l d 1 . 6 8 g o f p u r e c e p h a l o s p o r i n C sodium s a l t . 2.3.3
P r e p a r a t i o n and i s o l a t i o n o f t h e q u i n o l i n e s a l t of N-chloroacetyl cephalosporin C7
S u f f i c i e n t whole b r o t h t o c o n t a i n 25.8 g o f c e p h a l o s p o r i n C i s t r e a t e d w i t h 1 3 g aluminum s u l f a t e o c t a d e c a h y d r a t e a n d 1 0 m l of 6 . 6 % p o l y e t h y l e n e i m i n e p e r l i t e r o f b r o t h and f i l t e r e d . The f i l t r a t e i s mixed w i t h 2 volumes of m e t h a n o l , and t h e p r e c i p i t a t e which forms i s s e p a r a t e d a n d d i s c a r d e d . The f i l t r a t e is C o n c e n t r a t e d t o 530 m l i n vacuo. The c o n c e n t r a t e d f i l t r a t e i s c h i l l e d t o 1 0 ° C a n d a c y l a t e d w i t h 105 m l of c h l o r o a c e t i c propionic anhydride s o l u t i o n (an estimated 5 equivalents of The a c e t y l a t i n g mixa n h y d r i d e / e q u i v a l e n t of c e p h a l o s p o r i n C ) . t u r e i s made by r e a c t i n g sodium p r o p i o n a t e a n d c h l o r o a c e t y l The pH of t h e c h l o r i d e i n e t h y l a c e t a t e and i s u s e d as s u c h . m i x t u r e is m a i n t a i n e d a t 9 . 0 w i t h 145 m l o f b o r a t e b u f f e r . A f t e r t h e a c y l a t i o n i s c o m p l e t e d , 39 m l of q u i n o l i n e i s a d d e d , a n d t h e pH i s a d j u s t e d t o 2 . 9 w i t h 1 0 5 m l o f 3 0 % H2SOk. The a c i d i f i e d m i x t u r e i s s t i r r e d f o r 30 min. w h i l e c r y s t a l l i z a t i o n of t h e N-acyl c e p h a l o s p o r i n C q u i n o l i n e s a l t monohydrate o c c u r s . The m i x t u r e i s c h i l l e d t o 15OC o v e r n i g h t , f i l t e r e d , a n d t h e p r e c i p i t a t e i s washed w i t h w a t e r . The c r y s t a l s a r e d r i e d i n vaCu0 o v e r n i g h t a t 4OoC. Y i e l d s of a b o u t 32.5 g of t h e Nc h l o r o a c e t y l cephalosporin C q u i n o l i n e s a l t (95.4% p u r i t y ) a r e obtained. The a c y l a t i o n r e a c t i o n can be r u n on t h e a n i o n r e s i n e l u a t e s c o n t a i n i n g cephalosporin C as o b t a i n e d i n s e c t i o n 2.3.1 and 2.3.4 except t h a t only 2 e q u i v a l e n t s of t h e anhydride/equiv a l e n t of cephalosporin C a r e used.
81 2.3.4
P r e p a r a t i o n o f c e p h a l o s p o r i n C - z i n c complex8
The f e r m e n t a t i o n b r o t h 8 c o n t a i n i n g a b o u t 2 . 1 mg c e p h a l o s p o r i n C / m l , i s c o o l e d t o 1 5 C a n d a d j u s t e d t o pH 2 . 8 - 3 . 0 w i t h 50% w/v s u l f u r i c a c i d o r o x a l i c a c i d . The a c i d i f i e d b r o t h i s f i l t e r e d w i t h f i l t e r a i d , and 30 R of t h e f i l t r a t e i s p a s s e d o v e r a column ( i n t e r i o r d i a m e t e r o f 1 5 cm) o f A m b e r l i t e XAD-5 ( 3 0 R ) i n water a t a f l o w r a t e o f 500 ml/min. The column i s washed w i t h 1 5 .8 of w a t e r and t h e c e p h a l o s p o r i n C i s e l u t e d w i t h 6 0 .8 o f 1 0 % a q u e o u s i s o p r o p a n o l , a t a f l o w r a t e o f 1 R/min. c o l l e c t i n g 1 .8 f r a c t i o n s . " The e l u t i o n i s m o n i t o r e d by UV s p e c t r o p h o t o m e t r y a t 2 6 0 nm o r by b i o a s s a y . The f i r s t few e l u a t e f r a c t i o n s u s u a l l y c o n t a i n o n l y a trace o f c e p h a l o s p o r i n C. About 9 5 % o f t h e c e p h a l o s p o r i n C p r e s e n t i n t h e o r i g i n a l f i l t r a t e as w e l l as 20-25% of t h e o r i g i n a l i m p u r i t i e s a r e p r e s e n t i n 45 R of e l u a t e . A t t h i s p o i n t c r u d e c e p h a l o s p o r i n C can b e o b t a i n e d b y c o n c e n t r a t i n g t h e e l u a t e a n d p r e c i p i t a t i n g t h e c e p h a l o s p o r i n C w i t h a c e t o n e . A l t e r n a t i v e l y , t h e 4.5 R act i v e e l u a t e i s p a s s e d o v e r a column ( 5 cm i n t e r n a l d i a m e t e r ) c o n t a i n i n g 1 .8 o f A m b e r l i t e I R A - 6 8 s u s p e n d e d i n 1 0 % a q u e o u s i s o propanol. The f l o w r a t e i s 1 0 . 8 / h r . The column i s washed w i t h 1 R o f water, a n d t h e c e p h a l o s p o r i n C i s e l u t e d w i t h p y r i d i n e a c e t a t e ( s o d i u m a c e t a t e , 0.85M, may a l s o b e u s e d f o r e l u t i o n ) b u f f e r , pH 5 . 5 , a t a f l o w r a t e o f 1 R / h r . The b u f f e r i s 0 . 4 4 M p y r i d i n e a n d 0.2M a c e t i c a c i d . Z i n c a c e t a t e ( 1 8 5 g) i s a d d e d t o 3 I o f e l u a t e c o n t a i n i n g t h e m a j o r amount o f c e p h a l o s p o r i n C , and w i t h i n 2 0 min. 3 I o f i s o p r o p a n o l a r e s t i r r e d i n t o t h e c l e a r s o l u t i o n . Toward t h e end o f t h e a d d i t i o n c e p h a l o s p o r i n C - z i n c complex w i l l b e g i n t o c r y s t a l l i z e o u t . The m i x t u r e i s cooled t o 2OC. The p r e c i p i t a t e i s f i l t e r e d o f f i n v a c u o , washed twice w i t h 300 m l o f w a t e r , once w i t h 300 m l a c e t o n e a n d d r i e d i n vacuo a t 4 0 ° C t o y i e l d a b o u t 3 6 . 6 g of w h i t e , c r y s t a l l i n e c e p h a l o s p o r i n C - z i n c complex. A l t e r n a t i v e p r o c e d u r e s f o r p r e p a r i n g t h e c e p h a l o s p o r i n Cz i n c complex a s w e l l as o t h e r m e t a l complexes h a v e b e e n r e p o r t ed9. 2.3.5
D e t e c t i o n and a s s a y
A n a l y t i c a l p r o c e d u r e s for c e p h a l o s p o r i n C a n d i t s d e r i v a t i v e s h a v e b e e n t h o r o u g h l y r e v i e w e d b y M a r r e l l i ' O , b u t a few of t h e most u s e f u l methods w i l l be d i s c u s s e d i n t h i s s e c t i o n . The d i s c - p l a t e a g a r d i f f u s i o n a s s a y u s i n g Saemonctea gaLeinaJdum as t h e t e s t o r g a n i s m i s t h e method o f c h o i c e f o r t h e m i c r o b i o l o g i c a l a s s a y of ce h a l o s p o r i n C . d i s c u s s e d i n d e t a i l by Kavanagh?'.
T h i s method i s
S e v e r a l r a p i d and accurate c h e m i c a l methods h a v e b e e n d e v e l o p e d f o r r o u t i n e a n a l y t i c a l u s e . An i o d o m e t r i c t i t r a t i o n of t h e a l k a l i n e h y d r o l y s i s p r o d u c t o f c e p h a l o s p o r i n C 1 2 i s a *The XAD-2 r e s i n can be regenerated with 30 R o f methano1:ZN NaOH (1:l) and thorough washing o r successively w i t h 15 I 1N NaOH, 10 R 0.2N H,SO, w i t h water.
82
r a p i d method which compares i n a c c u r a c y t o t h e m i c r o b i o l o g i c a l assay. The h y d r o x y l a m i n e method13 can be used f o r d e t e r m i n a t i o n of b o t h c e p h a l o s p o r i n C and p e n i c i l l i n N . An a u t o m a t e d p r o c e d u r e h a s been d e v e l o p e d f o r t h e s i m u l t a n e o u s d e t e r m i n a t i o n of t h e s e a n t i b i o t i c s i n f e r m e n t a t i o n b r o t h s 1 4 . The n i c o t i n a m i d e a s s a y 1 5 i s a p p l i c a b l e t o c e p h a l o s p o r i n C and d e r i v a t i v e s which have r e t a i n e d t h e a c e t y l m o i e t y i n t h e 3 p o s i t i o n f u n c t i o n . P a p e r c h r o m a t o g r a p h i c s e p a r a t i o n o f c e p h a l o s p o r i n C from p e n i c i l l i n N , d e a c e t y l c e p h a l o s p o r i n C , and c e p h a l o s p o r i n C l a c t o n e can b e a c h i e v e d u s i n g Whatman No. 1 o r No. 4 p a p e r w i t h s y s t e m s s u c h as: n - b u t a n o 1 : a c e t i c a c i d : w a t e r ( 4 : l : h ) ; n - b u t a n o l : a c e t i c a c i d : water ( 4: 1:5 1 ; m e t h a n o l : p r o p a n o l : water ( 6 : 2 :1); nb u t a n o 1 : a c e t i c a c i d : w a t e r ( 3 : l : l ) ; isopropano1:water:pyridine (65:30:5). Our p r e f e r r e d s y s t e m i s methano1:n-propano1:water ( 6 : 2 : 1 ) u s i n g Whatman No. 1 p a p e r b u f f e r e d w i t h 0.75M KHzPO,, The o r d e r o f movement from t h e f a s t e s t t o slowest p H 4, 2 0 h r . i s p e n i c i l l i n N, c e p h a l o s p o r i n C , a n d d e a c e t y l c e p h a l o s p o r i n C . C e p h a l o s p o r i n C l a c t o n e g e n e r a l l y moves f a s t e r t h a n e n i c i l l i n A number o f s y s t e m s h a v e b e e n r e v i e w e d by B e t i n a y s . BioN. a u t o g r a p h y i s t h e most s e n s i t i v e d e t e c t i o n s y s t e m and t h e p r e f e r r e d o r g a n i s m s a r e Salmonella gallinanurn ( L i l l y R e s e a r c h Labo r a t o r i e s , X-142) and Pacudomonaa aolanaccaJtum ( L i l l y R e s e a r c h L a b o r a t o r i e s , X-185). Eacillua 4 u b t i l i d ATCC 6 6 3 3 i s a l s o v e r y u s e f u l b u t i s less s e n s i t i v e . N i n h y d r i n and s t a r c h - i o d i n e s p r a y s can be used f o r d e t e c t i o n , and UV l i g h t w i l l d e t e c t t h e cephalosporins but not p e n i c i l l i n N. Thin l a y e r chromatography h a s n o t p r o v e n t o b e v e r y s u c c e s s f u l f o r s e p a r a t i o n of p e n i c i l l i n N a n d c e p h a l o s p o r i n C . C e l l u l o s e p l a t e s a p p e a r t o work b e t t e r t h a n s i l i c a g e l p l a t e s , b u t p a p e r chromatography is s t i l l t h e p r e f e r r e d p r o c e d u r e . R e c e n t l y , h i g h p e r f o r m a n c e l i q u i d chromatography h a s been a p p l i e d t o s e p a r a t i o n problems i n t h e c e p h a l o s p o r i n s e r i e s o f antibiotics. Both i o n - e x c h a n g e 1 7 a n d r e v e r s e p h a s e l a HPLC h a v e been r e p o r t e d . A m i x t u r e of 1 0 cephalosporin d e r i v a t i v e s has been r e s o l v e d by u s e of microbonded p r o p y l a m i n e s i l i c a (Microbondapak N H 2 , Waters A S S O C . , M i l f o r d , MA) a n d a s o l v e n t s y s t e m The o f a c e t i c acid:methanol:acetonitrile:H20 ( 2 : 4 : 7 . 5 : 8 6 . 5 ) . equipment a n d c o n d i t i o n s u s e d were: Waters M-6000 pump; U6K s e p t u m l e s s i n j e c t o r ; 254 nm UV d e t e c t i o n ( W a t e r s A s s o c . ) w i t h F i s c h e r O m n i s c r i b e r e c o r d e r ; 4 x 3 0 0 mm column o f p NH, bonded p h a s e p r o p y l a m i n e on 1 0 1.1 s i l i c a g e l ( W a t e r s Assoc.); 3,400 p s i ; 3 ml/min flow r a t e 1 9 , T h i s method i s a p p l i c a b l e t o t h e d e t e c t i o n and s e p a r a t i o n of c e p h a l o s p o r i n C and d e a c e t y l c e p h a l o s p o r i n C i n fermentation broths.
2. 4 Literature
Cited
1.
E . P . Abraham a n d P. B . Loder, i n E . H . F l y n n ( E d i t o r ) , Cephalosporins and P e n i c i l l i n s : Chemistry and Biology, Academic P r e s s , N e w York, 1972, p p . 2 - 2 6 .
2.
R. Nagarajan, i b i d . ,
3.
E . F. Abraham, P h a r m a c o l . Rev. 1 4 ( 1 9 6 2 ) 473-498.
pp. 643, 6 5 3 .
83
-4. P. W . Trown, E. P. Abra ha m , G . G . F . N e w t o n , C . W . Hale a n d G. A. M i l l e r , B ioc he m . J. 8 4 ( 1 9 6 2 ) 1 5 7 - 1 6 6 . 5.
E. P . Abraha m , G . G . F . Newton a n d C . W . Hale, U.S. 3 , 1 8 4 , 4 5 4 ; May 1 8 , 1 9 6 5 .
6.
K . Nara, K . O h t a , K . Katamoto, N . Mizokami a n d H . F u k u d a , U . S . P a t e n t 3 , 9 2 6 , 9 7 3 ; De c e m be r 1 6 , 1 9 7 5 .
7.
G . M . W i l d , U.S.
8.
W.
9.
H . B i c k e l , R . B o s s h a r d t , B . F e c h t i g , W . Voser, J . M u e l l e r a n d H . P e t e r , U.S. P a t e n t 3 , 6 6 1 , 9 0 1 ; May 9 , 1 9 7 2 .
Voser, U.S.
P a t e n t 3,835,129;
P a t e n t 3,725,400;
Patent
September 1 0 , 1974.
A p r i l 3, 1973.
10.
L, P. M a r r e l l i , i n E. H . F l y n n ( E d i t o r ) , C e p h a l o s p o r i n s a n d Penicillins: Chemistry and B i o l o g y , Academic P r e s s , New Y o r k , 1 9 7 2 , p p . 609-635.
11.
F . K a v a n a g h , i n F. Ka va na gh ( E d i t o r ) , A n a l y t i c a l M i c r o b i o l o g y , Vol. I , Academic P r e s s , N e w Y o r k , 1 9 6 3 , p p . 2 6 5 - 2 7 0 .
12.
J . F . A l i c i n o , A n a l . Chem.
13.
E. H. F l y n n , i n E. H. Flynn ( E d i t o r ) , C e p h a l o s p o r i n s and Penicillins: C h e m i s t r y and B i o l o g y , Academic P r e s s , New York, 1 9 7 2 , Appendix, p. 680.
14.
H. E . R o u d e b u s h , P r o c . 1 9 6 9 T e c h n i c o n I n t e r n . Cong. Automated A n a l y s e s , Chicago, 1969.
15.
E . H . F l y n n , i n E. H . F l y n n ( E d i t o r ) , C e p h a l o s p o r i n s a n d Penicillins: Chemistry and B i o l o g y , Academic P r e s s , New York, 1972, Appendix, pp. 679-680.
16.
V . B e t t i n a , i n M. L e d e r e r ( E d i t o r ) , C h r o m a t o g r a p h i c R e v i e w s , Vol. 7 , E l s e v i e r , N e w Y o r k , 1 9 6 5 , p p . 1 1 9 - 1 6 6 .
17.
J. Konecny, E . F e l b e r a n d J. G r u n e r , J. A n t i b i o t i c s 26 1973) 135-141.
18.
E . R . W h i t e , M. A . C a r r o l l , J . E . Zaremba a n d D. J . B e n d e r , J. A n t i b i o t i c s 2 8 ( 1 9 7 5 ) 2 0 5 - 2 1 4 .
19.
R . D. Miller a n d N . N e u s s , J. A n t i b i o t i c s 2 9 ( 1 9 7 6 ) 9 0 2 - 9 0 6 .
33 ( 1 9 6 1 ) 648-649.
84 3. 7-Aminocephalosporanic Acid
3.1 Introduction 7-Aminocephalosporanic a c i d (7-ACA) i s n o t a n a t u r a l l y o c c u r r i n g f e r m e n t a t i o n p r o d u c t . We h a v e i n c l u d e d methods of p r e p a r a t i o n b e c a u s e of i t s i m p o r t a n c e as a n i n t e r m e d i a t e i n t h e s y n t h e s i s of t h e c l i n i c a l l y u s e f u l s e m i - s y n t h e t i c c e p h a l o s p o r i n s . 3.1.1
Producing organism
None. 3.1.2
P h y s i c a l and c h e m i c a l p r o p e r t i e s ’
C10H12N205S mol. w t . 272.3 Xmax 2 6 5 nm ( E i E m 3 1 0 ) 3.1.3
S t r u c t u r a l formula’
3 . 2 Summary
of b i o l o g i c a l a c t i v i t i e s
7-ACA h a s v e r y weak a n t i b a c t e r i a l a c t i v i t y 2 . S i n c e 7-ACA h a s been shown t o i n h i b i t D - a l a n i n e c a r b o x y p e p t i d a s e from E . c o l i , i t i s assumed t h a t 7-ACA h a s t h e same mechanism o f a c t i o n as t h e more a c t i v e d e r i v a t i v e s 3.
3.3 Preparation
of 7 - a m i n o c e p h a l o s p o r a n i c a c i d ( 7 - A C A )
Huber e t have c o m p r e h e n s i v e l y r e v i e w e d t h e methods of p r e p a r a t i o n o f 7-ACA u s i n g as s t a r t i n g m a t e r i a l c e p h a l o s p o r i n C d e r i v a t i v e s as w e l l as c e p h a l o s p o r i n C i t s e l f . Two methods are g i v e n h e r e . The f i r s t method uses n i t r o s y l c h l o r i d e t o form a n i m i n o - l a c t o n e which i s s u b s e q u e n t l y h y d r o l y z e d t o 7-ACA. The s e c o n d method uses p h o s p h o r u s p e n t a c h l o r i d e as t h e c l e a v i n g a g e n t a n d N-chloroacetylcephalosporin C as t h e s t a r t i n g mater i a l . T h i s method h a s t h e a d v a n t a g e of u s i n g a d e r i v a t i v e as a starting material. T h i s d e r i v a t i v e can be p r e p a r e d from c r u d e c e p h a l o s p o r i n C ( s e e method 2.3.21, thus bypassing the laborious p u r i f i c a t i o n of cephalosporin C. 3.3.1
P r e p a r a t i o n o f 7-ACA from c e p h a l o s p o r i n C u s i n g nitrosyl chloride3
Twenty grams o f c e p h a l o s p o r i n C , sodium s a l t i s d i s s o l v e d i n 79 m l 98-100% f o r m i c a c i d . The r e s u l t i n g s o l u t i o n i s s t i r r e d and c o o l e d i n a n i c e b a t h . A c o l d , f r e s h l y p r e p a r e d s o l u t i o n of
85
5 . 5 g n i t r o s y l c h l o r i d e i n f o r m i c a c i d i s added i n one p o r t i o n . A f t e r a 5 min. r e a c t i o n t i m e , t h e m i x t u r e i s c o n c e n t r a t e d i n vacuo a n d t h e r e s i d u e i s d i s s o l v e d i n 75 m l w a t e r . The r e s u l t i n g s o l u t i o n i s s t i r r e d and c o o l e d i n an i c e b a t h . The pH o f t h e s o l u t i o n i s a d j u s t e d t o 3 . 5 , t h e i s o e l e c t r i c p o i n t of 7-ACA, w i t h 1N MaOH. The p r e c i p i t a t e d 7-ACA i s f i l t e r e d , washed w i t h water, a n d d r i e d . T h i s r e a c t i o n r e s u l t s i n a 30% y i e l d o f 9 0 % p u r e 'I-ACA. 3.3.2
P r e p a r a t i o n of 7-ACA from t h e q u i n o l i n e s a l t of N-chloroacetyl cephalosporin C6
N - c h l o r o a c e t y l c e p h a l o s p o r i n C q u i n o l i n e s a l t ( 1 5 . 3 g ) is d i s s o l v e d i n 5 2 m l c h l o r o f o r m and 8 . 8 m l d i m e t h y l a c e t a m i d e i s added t o t h e r e s u l t i n g s o l u t i o n . To t h i s s o l u t i o n i s a d d e d , w i t h s t i r r i n g , 1 0 . 1 m l of a c e t y l c h l o r i d e . S t i r r i n g i s c o n t i n ued f o r 5 min. and t h e n t h e t e m p e r a t u r e of t h e m i x t u r e i s lowT w e n t y - s i x m l of d i e t h y l a n i l i n e i s a d d e d , f o l ered to -15OC. lowed by a s o l u t i o n of 9 . 8 g p h o s p h o r o u s p e n t a c h l o r i d e i n 1 0 0 m l of c h l o r o f o r m . The r e s u l t i n g s o l u t i o n i s s t i r r e d f o r 30 min, 5 0 m l o f c o l d m e t h a n o l i s a d d e d , and s t i r r i n g i s c o n t i n u e d f o r 30 min. The r e a c t i o n m i x t u r e i s d i l u t e d w i t h 1 0 0 m l water. The aqueous p h a s e i s s e p a r a t e d and i s washed w i t h 1 0 0 m l c h l o r o form. The pH o f t h e a q u e o u s p h a s e i s a d j u s t e d t o 3 . 5 w i t h ammonium h y d r o x i d e . The r e s u l t i n g s o l u t i o n i s a l l o w e d t o s t i r f o r 30 min. d u r i n g which t i m e 7-ACA p r e c i p i t a t e s . The p r e c i p i t a t e i s f i l t e r e d , washed w i t h w a t e r and a l c o h o l , a n d d r i e d , y i e l d i n g 4.38 g of 7-ACA. Cephalosporin C, z i n c s a l t , prepared according t o S e c t i o n 2 . 3 . 4 can be u s e d as t h e s t a r t i n g m a t e r i a l i n p l a c e o f t h e Nchloroacetyl derivative. 3.3.3
D e t e c t i o n and a s s a y
M a r r e l l i 7 h a s d e s c r i b e d v a r i o u s chromatography s y s t e m s t h a t have been used f o r 7-ACA. P a p e r chromatography h a s been more u s e f u l t h a n TLC. The p r e f e r r e d s o l v e n t s y s t e m s a r e watera c e t o n e m i x t u r e s o f v a r i o u s c o n c e n t r a t i o n s . UV a b s o r b a n c e a n d n i n h y d r i n s p r a y are commonly used d e t e c t i n g a g e n t s . 3ecause 7ACA h a s a low o r d e r o f a n t i m i c r o b i a l a c t i v i t y , b i o a u t o g r a p h y i s n o t a method of c h o i c e . However, Baci&!ud d u b t i e i d can b e u s e d as a d e t e c t i n g o r g a n i s m i f s u f f i c i e n t l y h i g h l e v e l s o f 7-ACA have been c h r o m a t o g r a p h e d . Another bioautography technique involves t h e chemical c o n v e r s i o n o f 7-ACA t o i t s N - p h e n y l a c e t y l d e r i v a t i v e by a p p r o p r i a t e l y t r e a t i n g t h e f i l t e r p a p e r a f t e r chromatography of t h e 7-ACA8. This d e r i v a t i v e i s highly a c t i v e a g a i n s t gram-positive organisms.
An a u t o m a t i c method u s i n g t h e Moore-Stein p r o c e d u r e f o r amino a c i d a n a l y s i s h a s been d e s c r i b e d g . T h i s method i s p a r t i c u l a r l y a p p l i c a b l e t o q u a n t i t a t i n g t h e amounts o f 7-ACA i n t h e p r e s e n c e of o t h e r d e r i v a t i v e s . The HPLC method r e p o r t e d by Miller and Neuss'O w i l l s e p a r a t e 7-ACA from o t h e r c e p h a l o s p o r i n s . Details o f t h i s method a r e o u t l i n e d i n S e c t i o n 2 . 3 . 5 .
86
3.4 L i t e r a t u r e
Cited
1.
E. H. F l y n n ( E d i t o r ) , C e p h a l o s p o r i n s a n d P e n i c i l l i n s : C h e m i s t r y a n d B i o l o g y , Academic P r e s s , N e w York, 1 9 7 2 , pp. 371, 631.
2.
M. Gorman a n d C. W . Ryan, i b i d . ,
3.
K . I s a k i a n d J. L. S t r o m i n g e r , J . B i o l . Chem. 243 ( 1 9 6 8 ) 319 3-3201.
4.
F. M. H u b e r , R . R . C h a u v e t t e a n d B . G . J a c k s o n , i n E . H . Flynn ( E d i t o r ) , Cephalosporins and P e n i c i l l i n s : Chemistry a n d B i o l o g y , Academic P r e s s , N e w York, 1 9 7 2 , pp. 27-73.
5.
R . B. M o r i n , B . G . J a c k s o n , E . H . F l y n n , R . W . Roeske a n d S. L. Andrews, J. h e r . Chem. SOC. 9 1 ( 1 9 6 9 ) 1396-1400.
6.
H. B. H a y e s , U.S.
7.
L. P. Marrelli, i n E . H . F l y n n ( E d i t o r ) , C e p h a l o s p o r i n s a n d P e n i c i l l i n s : C h e m i s t r y a n d B i o l o g y , Academic Press, N e w York, 1 9 7 2 , p p . 610-635.
8.
B . L o d e r , G . G . F. Newton a n d E . P. Abraham, Biochem. J. 79 ( 1 9 6 1 ) 408-416.
9.
E. H. F l y n n , i n E. H . F l y n n ( E d i t o r ) , C e p h a l o s p o r i n s a n d P e n i c i l l i n s : C h e m i s t r y and B i o l o g y , Academic P r e s s , N e w York, 1 9 7 2 , Appendix, pp. 680-683.
10.
pp. 533-582.
P a t e n t 3 , 8 1 3 , 3 8 9 ; May 28, 1 9 7 4 .
R . D. Miller a n d N . N e u s s , J . A n t i b i o t i c s 2 9 ( 1 9 7 6 ) 9 0 2 - 9 0 6 .
87 4 . Methoxycephalosporins (Cephamycins
A and B; C-2801x1
4.1 Introduction Cephamycins A and B were included in the group of 7methoxycephalosporin antibiotics first reported in 1971' and 19722. In addition to cephamycins A and B, this group included A16886B (identical with cephamycin C) and A16884 (7-methoxycephalosporin C). These publications were the first reports of formation of cephalosporin compounds by fermentation of strepto. mycetes. More recently, C-2801X3 was isolated from a fermentation beer which also contained cephamycins A and B. The structure of C-2801X is identical to the structure of cephamycin B except for an additional hydroxyl on the phenyl moiety. These compounds possess activity against gram-positive and gram-negative bacteria and are much more resistant than cephalosporin C to enzymatic degradation by microorganisms known to produce cephalosporinases. 4.1.1
Producinp organisms
gAideud NRRL 38512; Sheptomyced hetenorno&S&eptomyced phud 3 ; Sheptomyced pandyendid 3.
4.1.2
Physical and chemical properties
Cephamycin A4 C25H29N3014S2 mol. wt. 659.6 1% 432) hmax 287 nm, 0.1N H C 1 (Elcm 1% 280 nm, 0.1N NaOH (Elcm 432)
Cephamycin A, sodium Salt3: +141.4'
(c-0.5, H,O)
Cephamycin B C25H29N3011S mol. wt. 579.56 1% 524) hmax 305 nm, 0.1N HC1 (Elcm 328 nm, 0.1N NaOH
Cephamycin B, sodium salt3 +137'
(C.0.5,
H2O)
C-2 801X C25H2gN3012S
mol. Wt. 595.56
C-2801X, sodium salt
564)
88
1% Xmax (H20) 2 3 4 nm (Elcm 2 0 8 ) 1% 2 9 5 nm (Elcm 255) 1% 2 7 4 )
3 1 8 nm (Elcm { a } $ 5 +124.4'
(c=0.5,
H,O)
S o l u b i l i t y - The sodium s a l t s a r e s o l u b l e i n water, methanol and e t h a n o l , b u t are i n s o l u b l e i n o t h e r o r g a n i c solvents
.
+
Stabilit above p
-
The compounds a r e u n s t a b l e below pH 2 and
Structural formulas3~4
4.1.3
The methoxy g r o u p a t t h e 7 p o s i t i o n h a s t h e a - c o n f i g u r a tion.
NH 2 H HOOC-CHI (CH2 ) 3 -CON
@-' OCH 3
fH 0 0
I
II I
CHz-O-C-C=CHR CO ZH
Cephamycin A:
R = O O S O z O H
Cephamycin B:
C-2801X:
R = -@OH
OH
4 . 2 Summary
of b i o l o g i c a l a c t i ~ i t i e s ~ ' ~
Cephamycins A a n d B a n d C-2801X a r e f a i r l y a c t i v e ifl v i a 2 0 a g a i n s t g r a m - p o s i t i v e and g r a m - n e g a t i v e b a c t e r i a i n c l u d i n g S t a p h y t o c o c c u d audeuo, B a C i U U A A u b t i t i A , PkOteuA V u t g u h i b , Pnoteud m o d g a f l i i , E d c h e d i c h i a C O I L , KtebdieLta pneumoniae, S a t m o f l e t t a typhimudium, a n d A t c a t i g e n e d duecatin b u t n o t v i r t u a l l y i n a c t i v e a g a i n s t Poeudomonad a e d u g i f l o d a , S e d d U t i b mM c e d c e f l b , and S&eptoCOccuA d a e c a t i b . The compounds a r e v e r y r e s i s t a n t t o e n z y m a t i c d e g r a d a t i o n by c e p h a l o s p o r i n a s e s from AJkaligefleA duecatid a n d Aedobacten c l o a c a e .
-
The ED50 v a l u e s a g a i n s t E . C o t i i n f e c t i o n s i n mice a r e 30 mg/kg f o r C-2801X a n d '100 mg/kg f o r cephamycins A and B .
89 These compounds a r e r e l a t i v e l y n o n - t o x i c , a c h a r a c t e r i s t i c of compounds of t h e c e p h a l o s p o r i n g r o u p ( e . g . , L D 5 0 >400 mg/kg, mice, I V ) .
4.3 I s o l a t i o n 4.3.1
and P u r i f i c a t i o n Methods
I s o l a t i o n o f cephamycins A a n d B 4
The f e r m e n t a t i o n b r o t h i s a c i d i f i e d a n d t h e n f i l t e r e d . Four l i t e r s of f i l t r a t e (80 ug cephamycin/ml) i s p a s s e d o v e r a column c o n t a i n i n g 380 R o f A m b e r l i t e XAD-2 (Rohm and Haas Co., P h i l a d e l p h i a , PA). A f t e r a water wash t h e column i s e l u t e d w i t h 60% aq. methanol. The e l u a t e c o n t a i n i n g t h e cephamycins ( a b o u t 800 R ) i s c o n c e n t r a t e d t o a b o u t 1 / 4 volume, a n d t h e pH i s a d j u s t e d t o 3 . 5 w i t h a q . ammonia. One h a l f of t h e r e s u l t i n g conc e n t r a t e i s d i l u t e d w i t h a h a l f volume o f water a n d i s p a s s e d o v e r a column o f 2 2 . 5 R A m b e r l i t e I R A - 6 8 ( C 1 - 1 (Rohm a n d Haas Co., P h i l a d e l p h i a , PA). The column is e l u t e d w i t h 2 0 0 .t of a s o l u t i o n o f 1M sodium n i t r a t e and 0.1M sodium a c e t a t e a t pH 7 . 5 . T h i s e l u a t e i s a d j u s t e d t o pH 3 and i s a d s o r b e d on a 4 5 .t bed o f A m b e r l i t e XAD-2 (Rohm and Haas Co., P h i l a d e l p h i a , PA). A f t e r a water wash, t h e column i s e l u t e d w i t h 3 2 0 .t of 25% a q . a c e tone. T h i s e l u a t e i s c o n c e n t r a t e d t o a b o u t 5 % of t h e o r i g i n a l volume u n d e r r e d u c e d p r e s s u r e , a d j u s t e d t o pH 4 . 0 w i t h a q . ammon i a and f r e e z e - d r i e d . 4.3.2
S e p a r a t i o n of cephamycins A a n d B 4
Ten grams o f a c r u d e p r e p a r a t i o n from t h e p r e c e d i n g p r o c e d u r e a r e d i s s o l v e d i n 1 8 m l o f a 0.5M NH4Br-0.05M a c e t i c a c i d buffer. T h i s s o l u t i o n i s c h r o m a t o g r a p h e d on a column ( 2 . 5 x 1 0 0 cm) o f DEAE Sephadex A - 2 5 ( P h a r m a c i a F i n e C h e m i c a l s , I n c . , P i s c a t a w a y , NJ) a n d d e v e l o p e d w i t h t h e same b u f f e r a t a r a t e o f 80 m l / h r . The column e l u t i o n i s m o n i t o r e d w i t h a r e f r a c t o m e t e r and bioassay. Cephamycin B is r e c o v e r e d i n f r a c t i o n s 80-133 and cephamycin A i s r e c o v e r e d i n f r a c t i o n s 170-230. The s o l u t i o n s are d e s a l t e d by a d s o r p t i o n on a 1 0 0 m l column o f A m b e r l i t e XAD-2 (Rohm a n d Haas Co., P h i l a d e l p h i a , PA), and s u b s e q u e n t e l u t i o n with 300 m l of 9 0 % aq. methanol. The m e t h a n o l i s removed u n d e r r e d u c e d p r e s s u r e and t h e a q . c o n c e n t r a t e i s f r e e z e - d r i e d t o y i e l d p r e p a r a t i o n s o f cephamycins A and B of a p p r o x i m a t e l y 70% p u r i t y . . P u r i f i c a t i o n o f cephamycin A and B i s a c h i e v e d 3 by r e chromatography o v e r A m b e r l i t e XAD-2 u s i n g 3 % a q . m e t h a n o l as developer f o r t h e former and 7% aq. e t h a n o l f o r t h e l a t t e r . 4.3.3
S e p a r a t i o n a n d p u r i f i c a t i o n o f C-2801X3
C - 2 8 0 1 X coproduced w i t h cephamycin A a n d B can b e s e p a r a t e d from t h e l a t t e r compounds by A m b e r l i t e XAD-2 column chromat o g r a p h y u s i n g a s t e p w i s e g r a d i e n t o f 9 : 1 , 4:1, a n d 1:l m i x t u r e s of 1 / 1 5 M p h o s p h a t e b u f f e r (pH 6 . 5 ) a n d m e t h a n o l . The o r d e r of e l u t i o n i s cephamycin A , C - 2 8 0 1 X , and cephamycin 9 . The f r a c t i o n s r i c h i n C - 2 8 0 1 X a r e d i l u t e d w i t h p h o s p h a t e b u f f e r CpH 5 . 6 ) a n d a r e a p p l i e d t o an XAD-2 column. Complete s e p a r a t i o n i s a c h i e v e d by e l u t i o n w i t h w a t e r a n d t h e n w i t h 1 0 % a q . e t h a n o l .
90
P u r i f i c a t i o n of C-2801X i s a c h i e v e d by Dowex SOW x 4 “ a + ) (Dow Chemical Company, Midland, M I ) chromatography a n d e l u t i o n w i t h 0.1M a c e t a t e b u f f e r , pH 4 . 4 : e t h a n o l ( 9 : 1 ) , f o l l o w e d by d e s a l t i n g The 1 0 % e t h a n o l e l u a t e c o n t a i n i n g C - 2 8 0 1 X on a n XAD-2 column. i s c o n c e n t r a t e d and f r e e z e - d r i e d t o y i e l d c r u d e C-2801X-Na ( 7 5 % ) . T h i s p r e p a r a t i o n is p u r i f i e d on a d i e t h y l a m i n o e t h y l ( D E A E I - c e l l u l o s e ( S c h l e i c h e r and S c h e u l l I n c . , Keene, NH) column u s i n g 0.1M a c e t a t e b u f f e r (pH 4 . 4 c o n t a i n i n g 0.1M N a C 1 ) as a d e v e l o p e r o r on a c e l l u l o s e column u s i n g t h e upper phase of n - b u t a n o l : a c e t i c a c i d : w a t e r ( 4 : 1 : 5 ) as a d e v e l o p e r . The f r a c t i o n s cont a i n i n g C-2801X are d e s a l t e d on an XAD-2 column a n d f r e e z e - d r i e d t o y i e l d a c o l o r l e s s powder o f p u r e C - 2 8 0 1 X - N a s a l t . 4.3.4
Assay and d e t e c t i o n z p 3
The a n t i b i o t i c p o t e n c y of b r o t h o r p u r i f i e d samples i s d e t e r m i n e d by d i s c - p l a t e d i f f u s i o n a s s a y s u s i n g P k o t e u d V u l g U i A MB-838 or V i b h i o pedCO&ZnA M B - 1 2 7 2 . The r a n g e o f c o n c e n t r a t i o n s i n a s s a y s performed w i t h P . v u l g a n i d i s from 4 t o 500 ug/rnl, w h i l e t h e r a n g e o f c o n c e n t r a t i o n i n a s s a y s performed w i t h V . pencoland i s from < 1 t o 2 0 pg/ml. P a p e r s t r i p chromatography and e l e c t r o p h o r e s i s a r e u s e f u l A t o d i f f e r e n t i a t e between cephamycin A and B and C-2801X. p a p e r c h r o m a t o g r a p h i c s y s t e m of i s o p r o p a n o 1 : w a t e r ( 7 ) :3 0 ) u s i n g Whatman No. 1 p a p e r g i v e s a p p r o x i m a t e R f v a l u e s of 0 . 2 9 f o r cephamycin A, 0 . 4 2 f o r cephamycin B , a n d 0.33 f o r C-2801X as d e t e r m i n e d by b i o a u t o g r a p h y . P a p e r - s t r i p e l e c t r o p h o r e s i s is performed i n a r e f r i g e r a t e d u n i t o p e r a t e d f o r 2.5 h r a t 6 0 0 v w i t h 0.165M p h o s p h a t e b u f f e r a t pH 7 . 0 on s t r i p s of S c h l e i c h e r and S c h e u l l SS-598 f i l t e r p a p e r 5 2 cm i n l e n g t h and t h e c h a r g e measured i n cm i s -5.9 f o r cephamycin A and - 2 . 0 f o r cephamycin B as d e t e r m i n e d by b i o a u t o g r a m s .
A r e f r a c t o m e t e r can b e used t o m o n i t o r column e l u t i o n and can be used j o i n t l y w i t h b i o a s s a y s . The compounds react w i t h n i n h y d r i n t o p r p d u c e c o l o r which can b e measured s p e c t r o p h o t o m e t r i c a l l y t o f o l l o w column e l u t i o n s . Ninhydrin s p r a y can b e u t i l i z e d f o r p a p e r chromatography o r TLC d e t e c t i o n . FeC13-K3Fe ( C N 1 6 r e a g e n t can b e used f o r d e t e c t i o n of cepharnycin B and C - 2 8 0 1 X due t o t h e i r p h e n o l i c h y d r o x y l g r o u p s . 4.4 Literature -
1.
2.
Cited
R . N a g a r a j a n , L. D. Boeck, M . Gorman, R . L . H a m i l l , C . E . Higgens, M. M. Hoehn, W . M . S t a r k and J. G . Whitney, J. A m e r . Chem. SOC. 9 3 ( 1 9 7 1 ) 2308-2310. E . 0. S t a p l e y , M.
Jackson,
S . Hernandez, S . B .
Zimmerrnan,
S. A. C u r r i e , S. Mochales, J . M. Mata, H . B . Woodruff a n d D. H e n d l i n , A n t i m i c r o b . Agents and Chemother.
2 (1973) 1 2 2 -
131.
3.
H. Fukase, T. Hasegawa, K . Hatano, H . Iwasaki and M . Yoneda, J. A n t i b i o t i c s 29 (1976) 1 1 3 - 1 2 0 .
91
4.
T . W . M i l l e r , R . T . Goegelman, R . G . Weston, I . P u t t e r and F. J. Wolf, A n t i m i c r o b . Agents Chemother. 2 ( 1 9 7 2 ) 132-135.
92 5. A16884 (7-Methoxycephalosporin C)1’2
5.1 Introduction 7-Methoxycephalosporin C (A16884) was one of the group of cephalosporins isolated from fermentations of streptomycetes’. Penicillin N and deacetoxycephalosporin C were also produced in this fermentation. 5.1.1 Producing organism StAepZomyced t i p m a n i i NRRL 3584
5.1.2
Chemical and physical properties’
C17H23N309S mol. wt. 445.43 1% 155) Amax (H20) 265 nm (Elcm 242 nm (E;Zm
130)
pKa’s 3.9, 5.3, 10.5 (66% dimethylformamide). 5.1.3
Structural formula’
The methoxy group has the a-configuration.
I
HOOCCH (CHz 1 5 CON CHzOCOCH 3
5.2 Summary of biological activities and
uses
A16884 exhibits i n v i a h o and i n V i v O activity against gram-positive and gram-negative bacteria. In comparison with cephalosporin C, A16884 is more active against gram-negative bacteria and is less active against gram-positive bacteria3. A16884 is active against infections of Pdcudomonad dolanacearrum in tomato plants*.
5.3 Isolation and 5.3.1
purification methods
Isolation of crude A16884
The fermentation broth ( 6 0 a ) is adjusted to pH 2.5 and held at room temperature for 30 min. to destroy most of the penicillin N present. The acid broth is filtered with Hyflo Supercel (Johns-Manville Co., New Y o r k ) and the filtrate is adjusted to pH 4.5 and passed over a 9.6 x 150 cm column of carbon (Type CAL 12 x 40, Pittsburgh Activated Carbon Co., Pittsburgh, PA?. The column is washed with water until the effluent
93
i s c o l o r l e s s , and t h e a c t i v i t y i s e l u t e d with 5 0 % aqueous acetone. F r a c t i o n s e q u i v a l e n t t o a column volume a r e c o l l e c t e d a n d a s s a y e d . Most o f t h e a c t i v i t y i s g e n e r a l l y c o n t a i n e d i n f r a c t i o n s 2 t h r o u g h 4 . These f r a c t i o n s a r e combined a n d can be e i t h e r c o n c e n t r a t e d i n vacuo t o remove t h e a c e t o n e b e f o r e t h e a n i o n r e s i n s t e p o r a p p l i e d d i r e c t l y t o a 5 . 9 x 104 c m column o f IRA-68 ( a c e t a t e o r f o r m a t e c y c l e , Rohm a n d Haas Co., P h i l a d e l p h i a , PA) r e s i n . The column i s washed w i t h 5 column volumes of w a t e r o r u n t i l t h e e f f l u e n t i s c o l o r l e s s and c l e a r . The a c t i v i t y i s t h e n e l u t e d w i t h 0.15M ammonium a c e t a t e o r ammonium f o r m a t e d e p e n d i n g on t h e r e s i n c y c l e . Sodium a c e t a t e can be u s e d i n p l a c e of t h e ammonium s a l t s a n d t h e f i n a l p r o d u c t w i l l be A16884, sodium s a l t . I s o l a t i o n o f t h e sodium s a l t may b e a d v a n t a g e o u s b e c a u s e of e a s e i n p r e c i p i t a t i n g , f i l t e r i n g , and drying t h e product. Column volume f r a c t i o n s a r e c o l l e c t e d a n d a s s a y e d , and t h e most a c t i v e f r a c t i o n s a r e combined a n d p a s s e d o v e r a 4.3 x 7 2 cm column o f c a r b o n ( P i t t s b u r g h Type CAL, 1 2 x 40). The column i s washed w i t h s i x column volumes o f water a n d the a c t i v i t y i s eluted with water:acetonitrile (7:3). Column volume f r a c t i o n s a r e c o l l e c t e d a n d a s s a y e d . A c t i v e f r a c t i o n s a r e combined, c o n c e n t r a t e d i n v a c u o t o remove t h e a c e t o n i t r i l e , and f r e e z e - d r i e d t o y i e l d 2 5 - 3 0 g o f c r u d e A16884 c o n t a i n i n g some d e a c e t o x y c e p h a l o s p o r i n C . T h i s f r e e z e - d r i e d p r e p a r a t i o n i s d i s s o l v e d i n a minimum o f w a t e r and a p p l i e d t o a 7 . 2 x 6 0 c m column o f m i c r o c r y s t a l l i n e c e l l u l o s e * ( A v i c e l PH101, FMC Corp., P h i l a d e l p h i a , PA) packed i n a c e t o n i t r i 1 e : H Z O ( 7 : 3 ) , a n d washed w i t h a c e t o n i t r i l e b e f o r e a p p l i c a t i o n of t h e s a m p l e . A f t e r a p p l y i n g t h e s a m p l e , t h e c o l umn i s washed w i t h one column volume of a c e t o n i t r i l e and t h e a c t i v i t y i s e l u t e d w i t h m e t h a n o l . Column volume f r a c t i o n s a r e c o l l e c t e d and a s s a y e d . The a c t i v e f r a c t i o n s a r e combined, conc e n t r a t e d i n vacuo t o 2 0 0 m l and t h e a c t i v i t y i s p r e c i p i t a t e d by t h e a d d i t i o n o f 2 L! o f a c e t o n e . The p r e c i p i t a t e i s f i l t e r e d o f f , washed w i t h a c e t o n e , and d r i e d i n Vacuo t o y i e l d 9-12 g o f semi-purified product. 5.3.2
S e p a r a t i o n of A 1 6 8 8 4 a n d d e a c e t o x y c e p h a l o s p o r i n C
Twenty grams o f t h e above p r e p a r a t i o n i s d i s s o l v e d i n a minimum amount o f water a n d a p p l i e d t o a column o f s i l i c a g e l , grade 9 5 0 . P r i o r t o u s e , t h e s i l i c a g e l 950 s h o u l d b e washed w i t h 0.1M sodium a c e t a t e , f o l l o w e d by a water wash, and d r i e d . The s i l i c a g e l i s t h e n washed w i t h water, m e t h a n o l , a n d s u s p e n d e d i n 7 0 % a c e t o n i t r i l e f o r p a c k i n g t h e column. A f t e r a p p l i c a t i o n of t h e s a m p l e , t h e column i s washed w i t h a c e t o n i t r i l e , a n d the a c t i v i t y is e l u t e d with acetonitrile:H20 ( 7 : 3 ) . Fractions o f 2 5 0 m l a r e c o l l e c t e d , and t h e e l u t i o n i s m o n i t o r e d by b i o a s s a y and p a p e r c h r o m a t o g r a p h i c b i o a u t o g r a p h y . A16884 i s %Alternatively, the dissolved sample can be mixed w i t h a small amount o f s i l i c a gel grade 950 (W.R. Grace, Davison Chemical Co., Baltimore, MD) and d r i e d under vacuum a t room temperature. The s i l i c a g e l containing t h e a c t i v i t y is a p p l i ed d i r e c t l y on top o f a cellulose column packed i n methanol and drained t o t h e top o f t h e s u r f a c e , The e l u t i o n is c a r r i e d out w i t h methanol and t h e a c t i v i t y is p r e c i p i t a t e d a5 described above.
94
e l u t e d f i r s t , f o l l o w e d by d e a c e t o x y c e p h a l o s p o r i n C . I f any p e n i c i l l i n N i s p r e s e n t , i t i s e l u t e d between A16884 and d e a c e toxycephalosporin C. F r a c t i o n s r i c h e s t i n A16884 a r e combined, c o n c e n t r a t e d t o d r y n e s s i n v a c u o , and d i s s o l v e d i n methanol. Ten volumes of a c e t o n e a r e added t o p r e c i p i t a t e a b o u t 8 g of p u r i f i e d A16884 ammonium s a l t . F i n a l p u r i f i c a t i o n i s a c h i e v e d by d i s s o l v i n g 1 g of t h e above produce i n 4 m l of water and a p p l y i n g t h e s o l u t i o n t o a 2 x 6 0 cm column packed w i t h s i l i c a g e l ( g r a d e 950) i n a c e t o n i t r i l e and e l u t i n g w i t h a c e t o n i t r i 1 e : w a t e r ( 4 : l ) . The e l u t i o n i s f o l l o w e d by b i o a s s a y and p a p e r c h r o m a t o g r a p h i c b i o a u t o g r a p h y The f r a c t i o n s c o n t a i n i n g o n l y A16884 are combined a n d concent r a t e d t o d r y n e s s . The r e s i d u e i s d i s s o l v e d i n a small volume of d i m e t h y l s u l f o x i d e , a few m l of e t h a n o l i s a d d e d , and t h e a c t i v i t y i s p r e c i p i t a t e d w i t h a d d i t i o n of e x c e s s e t h e r . The p r e c i p i t a t e is f i l t e r e d o r c e n t r i f u g e d o f f and d r i e d i n vacuo t o y i e l d 9 1 mg o f A 1 6 8 8 4 monoammonium s a l t a s a n amorphous powder. To d a t e , A16884 h a s n o t been c r y s t a l l i z e d e i t h e r as t h e f r e e a c i d o r as a s a l t .
Less p u r i f i e d p r e p a r a t i o n s as d e t e r m i n e d b y b i o a s s a y a n d TLC can be p u r i f i e d by r e c y c l i n g t h r o u g h t h e c e l l u l o s e and s i l i c a g e l column c h r o m a t o g r a p h i c s t e p s . 5.3.3
D e t e c t i o n and a s s a y
S e c t i o n 6 , A16886A and A 1 6 8 8 6 B (cephamycin C ) i n c l u d e s methods f o r d e t e c t i o n and a s s a y of 7-methoxycephalosporin C .
5.4 Literature
Cited
1.
R . N a g a r a j a n , L. D. Boeck, M. Gorman, R . L. H a m i l l , C . E . H i g g e n s , M. M. Hoehn, W . M . S t a r k and J. G . Whitney, J . Amer. Chem. SOC. 93 ( 1 9 7 1 ) 2 3 0 8 - 2 3 1 0 .
2.
R . L . H a m i l l , C . E . Higgens and M . M. Hoehn, U . S . 3 , 7 1 9 , 5 6 3 ; March 6 , 1973.
3.
J . A . Webber and J. L. O t t , Advances i n A p p l i e d M i c r o b i o l o g y i n press.
Patent
95 6. A16886A and A16886B (Cephamycin C ) 6.1 Introduction A16886A (deacetyl-3-0-carbamoyl-cephalosporinC) and A16886B (deacetyl-3-0-carbamoyl-7-methoxycepahlospor~nC ) are produced by fermentation of Stneptomyced ck?avu.k?igebudl. This culture also produced penicillin N and deacetoxycephalos orin C. A16886B was isolated independently by Stapley e t at.’ and designated cephamycin C by that group. The producing organisms, S . tactamdunand and S . a t b o g n i d e o t u d , also produce the unrelated antibiotics efrotomycin3 and toyocamycin4, respectively. 6.1.1
Producing organisms
S & ~ p t O m y C e A clauu.k?igenud NRRL 3585I; S.tneptomyce.4 h c t a mdunand NRRL 38022; S.tneptomyce.4 a t b o g A i d e o l u . 4 NRRL 57354.
6.1.2
Physical and chemical properties
A168 86A’ C15H2,-,N408S
mol. wt. 416.3
Xmax ( H 2 0 ) 261 nm (E:Zm
161)
pKa’s 4.0, 5.3, 10.5 (66% dimethylformamide) A16886B (cephamycin C)1’5 Cl6HZ2N4O9S mol. wt. 446.37 Xmax ( H 2 0 ) 242 (E:Em
124)
264 (Eitm 149) pKa’s 4.2, 516, 10.4 (66% dimethylformamide) 6.1.3
Structural formula’
The methoxy group of A16886B has the a-configuration.
p&
R O W - CNH I H 2(CH2)jCON H R
N /
CH2OCONH2
COOH A16886A:
R = H
A16886B (Cephamycin C):
R = OCHr
96
6 . 2 Summary
of b i o l o g i c a l a c t i v i t i e s
A16886A h a s e s s e n t i a l l y t h e same i n v i t a 0 a n t i m i c r o b i a l s p e c t r u m as c e p h a l o s p o r i n C 6 . A 1 6 8 8 6 B shows as i n c r e a s e i n a c t i v i t y a g a i n s t gram-negative organisms along with decreased a c t i v i t y a g a i n s t gram-positive organisms5y6. This spectrum i s c o n s i s t e n t w i t h t h e a c t i v i t i e s r e p o r t e d f o r o t h e r 7-methoxycephalosporin d e r i v a t i v e s . A16886B is a l s o q u i t e r e s i s t a n t t o h y d r o l y s i s by c e p h a l o s p o r i n a s e s 5 . C e f o x i t i n , a s e m i - s y n t h e t i c d e r i v a t i v e o f deacetyl-3-0-carbamoyl-7-methoxycephalospor~n C i s now u n d e r g o i n g c l i n i c a l t r i a l s 7 .
6.3 Isolation 6.3.1
and p u r i f i c a t i o n methods
I s o l a t i o n and s e p a r a t i o n of A16886A a n d A16886B8
The f e r m e n t a t i o n b e e r of Staeptomyce4 ck!avuk!ige4uA cont a i n s small amounts of p e n i c i l l i n N a n d d e a c e t o x y c e p h a l o s p o r i n C which can i n t e r f e r e w i t h t h e i s o l a t i o n o f t h e major f a c t o r s . A f t e r f i l t r a t i o n o f t h e b r o t h (75 R ) w i t h f i l t e r a i d , t h e pH o f The a c i d i f i e d b r o t h i s t h e f i l t r a t e i s a d j u s t e d t o pH 2 . 5 . a l l o w e d t o s t a n d f o r 30 min. i n o r d e r t o d e s t r o y most o f t h e p e n i c i l l i n N p r e s e n t , The pH of t h e f i l t r a t e i s t h e n a d j u s t e d t o 4.5. T h i s f i l t r a t e is a p p l i e d t o a column c o n t a i n i n g 8 R of P i t t s b u r g h Carbon (Type CAL, 1 2 x 4 0 , P i t t s b u r g h A c t i v a t e d Carbon Co., P i t t s b u r g h , PA) a t a f l o w r a t e o f 6 0 ml/min. After t h e column is washed w i t h 1 0 R water, t h e a c t i v i t y is e l u t e d w i t h 5 0 % aqueous a c e t o n e . The e l u t i o n is m o n i t o r e d by b i o a s s a y , a n d t h e a c t i v e f r a c t i o n s a r e combined and a p p l i e d t o a 9 . 5 x 1 4 0 cm column o f A m b e r l i t e IRA-68, a c e t a t e c y c l e r e s i n (Rohm and Haas Co., P h i l a d e l p h i a , PA) a t a 40 ml/min flow r a t e . The column i s washed w i t h 1 0 I. o f water, and t h e a c t i v i t y i s removed w i t h O.15M sodium a c e t a t e , c o l l e c t i n g f i f t e e n 2 R f r a c t i o n s a t 6 0 ml/min flow r a t e . The e l u t i o n i s a g a i n m o n i t o r e d by b i o a s s a y . The e i g h t most a c t i v e f r a c t i o n s a r e combined and d e s a l t e d by p a s s i n g o v e r a 9 . 5 x 1 0 0 c m c a r b o n column ( P i t t s b u r g h Type CAL, 1 2 x 40). The column i s washed w i t h 8 R of water a n d t h e a c t i v i t y is e l u t e d with 4 : l water:acetone s o l u t i o n . The a c t i v e f r a c t i o n s a r e combined, c o n c e n t r a t e d i n v a c u o t o remove t h e a c e t o n e , and f r e e z e - d r i e d (20-30 g ) . F i v e grams o f t h e f r e e z e - d r i e d p r e p a r a t i o n i s d i s s o l v e d i n a b o u t 1 2 m l o f water a n d a p p l i e d t o a 3 x 9 5 cm column o f m i c r o c r y s t a l l i n e c e l l u l o s e ( A v i c e l PH 1 0 1 , FMC C o r p . , P h i l a d e l p h i a , PA) packed i n acetonitri1e:n-propano1:water (1:1:0.5) a n d t h e column i s d e v e l o p e d w i t h t h e same s o l v e n t s y s t e m a t a f l o w r a t e of 3 ml/min, c o l l e c t i n g 5 0 m l f r a c t i o n s . The e l u t i o n i s m o n i t o r e d by b i o a s s a y and p a p e r c h r o m a t o g r a p h i c b i o a u t o g r a p h y . The f o l l o w i n g s i n g l e a n t i b i o t i c s are e l u t e d i n t h i s o r d e r : p e n i c i l l i n N, i f p r e s e n t ( f r a c t i o n s 2 0 - 2 8 ) ; deacetoxycephalos p o r i n C ( f r a c t i o n s 3 4 - 3 7 ) ; A 1 6 8 8 6 B ( f r a c t i o n s 5 6 - 6 2 ) ; A16886A (74-78). The in-between f r a c t i o n s c o n t a i n m i x t u r e s of t h e two f a c t o r s and can be r e c y c l e d f o r f u r t h e r f r a c t i o n a t i o n . The f r a c t i o n s c o n t a i n i n g A16886B a r e combined and c o n c e n t r a t e d i n V a c u o t o a low volume, f i v e volumes o f m e t h a n o l a r e a d d e d , t h e n 2 0 volumes o f a c e t o n e t o p r e c i p i t a t e t h e A 1 6 8 8 6 B sodium s a l t .
97
The a d d i t i o n o f m e t h a n o l a l o n e w i l l o f t e n p r e c i p i t a t e p a r t of the activity. The p r e c i p i t a t e i s f i l t e r e d , washed w i t h a c e t o n e , and d r i e d t o y i e l d a b o u t 4 0 0 mg ( a b o u t 7 5 % p u r e ) . The A 1 6 8 8 6 B i s r e c y c l e d o v e r a c e l l u l o s e column i n t h e a c e t o n i t r i 1 e : n - p r o pano1:water ( 1 : 1 : 0 . 5 ) s y s t e m and t h e most a c t i v e f r a c t i o n s are combined, c o n c e n t r a t e d , and p r e c i p i t a t e d w i t h t h e w a t e r : m e t h a n o 1 : a c e t o n e c o m b i n a t i o n t o y i e l d p u r e A 1 6 8 8 6 B (cephamycin C) sodium s a l t as a n amorphous powder. A16886A, sodium s a l t , can be p u r i f i e d i n a s i m i l a r manner. 6.3.2
I s o l a t i o n o f cephamycin C 5
B r o t h from a f e r m e n t a t i o n o f Sfieptorngced lactamdu,tand i s f i l t e r e d , a n d 8 0 0 9. of f i l t r a t e c o n t a i n i n g 3 . 7 y g / a n t i b i o t i c i s c h r o m a t o g r a p h e d on a column of 2 7 L o f Dowex 1 x 2 , c h l o r i d e The s p e n t c y c l e , r e s i n (Dow Chemical Company, Midland, M I ) . e f f l u e n t , which c o n t a i n s a b o u t 2 0 % of t h e a c t i v i t y , i s d i s c a r d e d . A 5 % sodium c h l o r i d e s o l u t i o n i s u s e d t o e l u t e t h e a c t i v i t y . N i n e t e e n 1 L f r a c t i o n s a r e c o l l e c t e d . F r a c t i o n s 2 a n d 3 , which c o n t a i n a b o u t 5 6 % of t h e c h a r g e d a c t i v i t y , a r e combined. The pH i s a d j u s t e d t o pH 2 w i t h H C 1 a n d t h e r e s u l t i n g s o l u t i o n i s chromatographed o v e r 2 7 9. of Dowex 50 x 2 , hydrogen c y c l e , res i n . A f t e r a w a t e r wash, t h e a c t i v i t y i s e l u t e d w i t h 2 % p y r i d i n e i n water. E i g h t e e n 4 a f r a c t i o n s a r e c o l l e c t e d a n d by a s s a y c o n t a i n a b o u t 43% of t h e a c t i v i t y . F r a c t i o n s 4 t h r o u g h 8 c o n t a i n i n g 2 4 % of t h e b r o t h a c t i v i t y are combined, n e u t r a l i z e d This w i t h d i l u t e N a O H , a n d c o n c e n t r a t e d i n vaCuO t o 100 m l . c o n c e n t r a t e i s chromatographed on a column c o n t a i n i n g 2 . 3 9. o f Dowex 1 x 2 ( c h l o r i d e c y c l e , 2 0 0 - 4 0 0 mesh) r e s i n . The column, e q u i l i b r a t e d beforehand with p y r i d i n e hydrochloride b u f f e r (0.1M pH 5 . 0 1 , i s d e v e l o p e d w i t h t h i s b u f f e r a t 4 ml/min. The column e l u a t e i s m o n i t o r e d w i t h a r e f r a c t o m e t e r . A f t e r 4,260 m l of eluate, 2 0 m l f r a c t i o n s a r e c o l l e c t e d and assayed. F r a c t i o n s 1 5 0 t o 2 1 0 , d e t e r m i n e d t o be t h e b e s t by comparison of b i o a s s a y and r e f r a c t o m e t e r r e a d i n g s , c o n t a i n 5 6 % of t h e a c t i v i t y c h a r g e . These f r a c t i o n s a r e p o o l e d , a d j u s t e d t o pH 8 , and c o n c e n t r a t e d t o d r y n e s s t o remove t h e p y r i d i n e . The sample i s d e s a l t e d by d i s s o l v i n g i t i n 50 m l o f w a t e r , a d j u s t e d t o pH 7, and a p p l i e d t o column c o n t a i n i n g 2 , 2 8 0 m l of Bio-Gel P-2 (200-400 mesh) (BioRad L a b o r a t o r i e s , Richmond, C A I . The column i s washed w i t h water a t 1 0 ml/min, c o l l e c t i n g 2 0 m l f r a c t i o n s and m o n i t o r i n g F r a c t i o n s a r e b i o a s s a y e d and compared with a refractometer. w i t h t h e r e f r a c t o m e t e r c u r v e . The peak a c t i v i t y i s i n f r a c t i o n 7 7 and f r a c t i o n s 7 5 t o 7 9 a r e combined a n d f r e e z e - d r i e d t o y i e l d 828 mg of cephamycin C ( 4 0 % p u r e ) . ~
The c r u d e cephamycin C can b e p u r i f i e d by d i s s o l v i n g t h e p r e p a r a t i o n i n 2 0 m l o f 1% a q u e o u s n - b u t a n o l and c h r o m a t o g r a p h i n g t h e s o l u t i o n on a column c o n t a i n i n g 2,530 m l of Sephadex G - 1 0 ( P h a r m a c i a F i n e C h e m i c a l s , P i s c a t a w a y , NJ). The column i s d e v e l o p e d w i t h 1%aqueous n - b u t a n o l . F r a c t i o n s of 10.5 m l are c o l l e c t e d , a n d t h e e l u t i o n i s m o n i t o r e d w i t h a refractometer and b i o a s s a y . The b i o a c t i v i t y a p p e a r s i n f r a c t i o n s of a b o u t 9 0 through 1 2 2 . The h i g h e s t a c t i v i t y f r a c t i o n s a r e p o o l e d a n d c o n c e n t r a t e d t o d r y n e s s i n vacuo o r f r e e z e - d r i e d t o y i e l d h i g h l y p u r i f i e d cephamycin C (A16886B). T h i s 7 - m e t h o x y c e p h a l o s p o r i n d e r i v a t i v e has r e s i s t e d a l l attempts a t c r y s t a l l i z a t i o n .
98
6.3.3
D e t e c t i o n and a s s a y
D e t e c t i o n and a s s a y methods f o r A16884 (7-methoxycephalos p o r i n C ) a r e i n c l u d e d i n t h i s s e c t i o n , s i n c e t h e methods a r e similar t o t h o s e used f o r A16886B (cephamycin C ) . These compounds have s u f f i c i e n t a n t i m i c r o b i a l a c t i v i t y f o r e f f e c t i v e use of p a p e r d i s c a g a r p l a t e a s s a y s . S t a p l e y et a t . 2 used V i b k i o pekco&znA MG-1272 as t h e a s s a y organism f o r cephamycin C . Howe v e r , t h i s organism i s a l s o s e n s i t i v e t o t h e coproduced a n t i b i o t i c , e f r o t o m y c i n 3 . SatmoneLta gaCLinaJxum ( L i l l y R e s e a r c h L a b o r a t o r i e s c u l t u r e X - 1 4 2 ) has been used f o r b o t h A16884 and A16886 f a c t o r s . T h i s s t r a i n of S. gattinakum i s a l s o i n h i b i t e d by p e n i c i l l i n N . P e n i c i l l i n a s e can be i n c o r p o r a t e d i n t o t h e a g a r p l a t e medium i n o r d e r t o i n a c t i v a t e t h e p e n i c i l l i n N , t h u s permitting a d i f f e r e n t i a l assay. P a p e r chromatography i s more u s e f u l t h a n TLC i n d e t e c t i n g t h e i n d i v i d u a l f a c t o r s . The s y s t e m g i v e n as f o l l o w s w i l l s e p a r a t e t h e cephamycin f a c t o r s 2 . Whatman 3MM p a p e r Developing s o l v e n t : a p p r o x i m a t e l y 3.5 h r . D e t e c t i n g organism:
1sopropanol:water ( 7 : 3 ) f o r V i b k i o p e k C O t a n A MG-1272
A n t i b i o ti c
Rf -
Cephamycin A
0.64
Cephamycin B
0.73
Cephamycin C
0.44
Penicillin N
0.54
Cephalosporin C
0.56
Two s y s t e m s f o r s e p a r a t i n g t h e a n t i b i o t i c s produced by t h e S t 4 e p t O m y c s A L i p m a n i i a n d t h e SLteptomyceA ctavutigekuA fermentations a r e given.
Whatman No. 1 p a p e r n - p r o p a n o l : p y r i d i n e : a c e t i c a c i d : ace t o n i t r i l e : S o l v e n t 1: water (45:30:9:40:36) Solvent 2: a c e t o n i t r i 1 e : w a t e r (4:l) w i t h 400 m l of s o l v e n t 1 i n t h e bottom of t h e d e v e l o p i n g t a n k . D e t e c t i n g organism: PAeudomonaA Research L a b o r a t o r i e s c u l t u r e X-185).
A
Ohtaceakum ( L i l l y
Chromatograms are d e v e l o p e d f o r 2 2 h r .
99 Rf>k
An t i b i o t i c
Solvent 1
Solvent 2
A168811
0.66
0.75
Penicillin N
0.61
0.61
0.51
0.55
A16886B
0.40
0.55
A16886A
0.32
0.43
Deace t o x y c e p h a l o s p o r i n C
"Rf v a l u e s c a l c u l a t e d from p o i n t of o r i g i n t o e n d of p a p e r , as t h e s o l v e n t r u n s o f f t h e e n d of t h e p a p e r during t h e development. S i l i c a g e l a n d c e l l u l o s e TLC h a v e b e e n u s e d f o r s e p a r a t i o n of p e n i c i l l i n N a n d A 1 6 8 8 4 . U s i n g a c e t o n i t r i 1 e : w a t e r ( 7 : 3 ) as t h e d e v e l o p i n g s o l v e n t , t h e f o l l o w i n g Rf v a l u e s a r e o b s e r v e d :
Rf S i l i c a G e l Cellulose A16884
0.46
0.51
Penicillin N
0.39
0.39
T h e c o m p o u n d s are d e t e c t e d b y e i t h e r n i n h y d r i n s p r a y r e a g e n t or b i o a u t o g r a p h y u s i n g P . A o e a n a c e a A u m . Some r e s o l u t i o n of t h e A16886 f a c t o r s c a n be o b t a i n e d u s i n g c e l l u l o s e TLC w i t h e t h a n o 1 : l M ammonium a c e t a t e (pH 5 . 2 w i t h H C 1 ) ( 6 : 4 ) as t h e d e v e l o p i n g s o l v e n t . R f v a l u e s a r e : A16886A, 0 . 2 9 ; A16886B, 0 . 3 5 ; d e a c e t o x y c e p h a l o s p o r i n C , 0 . 3 9 ; a n d p e n i c i l l i n N, 0.48. P . AO$UflUCf2UhUm i s u s e d as t h e d e t e c t i n g o r g a n i sm
.
6.4 L i t e r a t u r e -
Cited
1.
R . N a g a r a j a n , L. D . B o e c k , M . Gorman, R . L. H a m i l l , C . E . H i g g e n s , M . M . H o e h n , W . M . S t a r k a n d J . G . W h i t n e y , J. h e r . Chem. SOC. 9 3 ( 1 9 7 1 ) 2308-2310.
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E . 0. S t a p l e y , M . J a c k s o n , S . H e r n a n d e z , S . B . Zimmerman, S . A. C u r r i e , S . M o c h a l e s , J. M . Mata, H . B . W o o d r u f f a n d D . H e n d l i n , A n t i m i c r o b . Ag. C h e m o t h e r . 2 ( 1 9 7 2 ) 1 2 2 - 1 3 1 .
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R . Wax, W. Maiese, R . W e s t o n a n d J . B i r n b a u m , J. A n t i b i o t i c s 29 ( 1 9 7 6 ) 6 7 0 - 6 7 3 .
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E . 0. S t a p l e y a n d J . M . b e r 2 1 , 1975.
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T. W . M i l l e r , R . T. G o e g e l m a n , R . G . W e s t o n , I . P u t t e r and F. J . Wolf, A n t i m i c r o b . A g . C h e m o t h e r . 2 ( 1 9 7 2 ) 1 3 2 - 1 3 5 .
Mata, U.S.
P a t e n t 3,914,158;
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100 6.
M. Gorman a n d C . W . R y a n , i n E. H . F l y n n ( E d i t o r ) , Cephalosporins and P e n i c i l l i n s : Chemistry and Biology, Academic P r e s s , N e w Y o r k , 1 9 7 2 , p p . 5 3 2 - 5 8 2 .
7.
R . C. M o e l l e r i n g a n d M. ( 1 9 7 6 ) 24-28.
N. S w a r t z , N e w E n g l a n d J . Med. 2 9 4
8.
M . Gorman, C . E . H i g g e n s a n d R . N a g a r a j a n , U.S. P a t e n t 3,801,464; A p r i l 2 , 1974.
9.
R . T. Goegelman a n d T . W . M i l l e r , U . S . P a t e n t 3 , 7 0 9 , 8 8 0 ; January 9, 1973.
Acknowledgment T h e a u t h o r s are g r a t e f u l t o Mrs. P h y l i s Hager f o r h e r c a p a b l e a s s i s t a n c e i n p r e p a r a t i o n of t h e m a n u s c r i p t .
101 C o u m a r i n .G l y c o s i d e A n t i b i o t i c s
.
J u l i u s B e r g e r a n d Andrew D B a t c h 0 Chemical Research Department H o f f m a n n - L a Roche I n c Nutley. New J e r s e y
.
.
1
Novobiocin Introduction Producing organisms 1.1.2 Brief chemical description 1.1.3 S t r u c t u r a l formula 1.2 Summary o f t h e r a p e u t i c u s e 1.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n 1.3.1 Detection 1.3.2 I s o l a t i o n of novobiocin 1.4 Literature cited
1.1 1.1.1
2
.
-
-
.
.
106 108 108
110 121
126 126
126 126 126 126 126
126 127
Novobiocin analogs from Ring A and B Precursors Introduction 4.1.1 Producing organisms 4.1.2 Brief c h e m i c a l d e s c r i p t i o n 4.1.3 S t r u c t u r a l formula 4.2 Summary of t h e r a p e u t i c u s e 4.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n 4.4 Literature cited
131 131 131 132 132 132 132
Clorobiocin Introduction Producing organisms 5.1.2 B r i e f chemical d e s c r i p t i o n 5.1.3 S t r u c t u r a l formula 5.2 Summary o f t h e r a p e u t i c u s e
134 134 134 134 135
4.1
5
104 106
128 128 128 128 129 129 129 129 129
-
.
.................................. ......................... .................. .......................... .................... ....... ................................... ................................... ..............................
103 103
Methoxynovobiocin Introduction 3.1.1 Producing organisms Brief c h e m i c a l d e s c r i p t i o n 3.1.2 3.1.3 S t r u c t u r a l formula 3.2 Summary of t h e r a p e u t i c u s e 3.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n 3.3.1 Detection 3.3.2 sol at ion... 3.4 Literature cited
3.1 -
4
.................................. ......................... .................. .......................... .................... ....... ................................... ..................... ..............................
Dihydronovobiocin Introduction Producing organisms 2.1.2 B r i e f chemical d e s c r i p t i o n 2.1.3 S t r u c t u r a l formula 2.2 Summary of t h e r a p e u t i c u s e 2.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n 2.3.1 Detection 2.3.2 Isolation 2.4 Literature cited
2.1 2.1.1
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3
.
5.1 5.1.1
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.................................. ......................... .................. .......................... .................... ....... ................................... ................................ .............................. .................................. ......................... ..................
.......................... .................... ....... ..............................
.................................. ......................... .................. .......................... ....................
102
. Clorobiocin ( c o n t i n u e d ) 5.3 E x t r a c t i o n . s e p a r a t i o n a n d p u r i f i c a t i o n ....... 1 3 7 137 5.3.1 D e t e c t i o n ................................... 5.3.2 I s o l a t i o n ................................... 1 3 8 141 5.4 L i t e r a t u r e c i t e d .............................. 6 . Coumermycin 6.1 I n t r o d u c t i o n .................................. 143 6.1.1 P r o d u c i n g organisms ......................... 143 6.1.2 Brief c h e m i c a l d e s c r i p t i o n .................. 1 4 3 6.1.3 S t r u c t u r a l f o r m u l a .......................... 144 6.2 Summary o f t h e r a p e u t i c u s e .................... 145 6.3 E x t r a c t i o n . s e p a r a t i o n a n d p u r i f i c a t i o n ....... 1 4 8
5
6.3.1 6.3.2 6.4
-
................................... ................................... 148 153 .............................. 157
Detection Isolation Literature cited
103
One of t h e c o n s t i t u e n t s o c c u r r i n g most f r e q u e n t l y i n almost a l l a n t i b i o t i c f a m i l i e s is a s u g a r moiety. T h i s p r o p e r t y h a s b e e n u t i l i z e d by Y o n e h a r a ' i n h i s p r o p o s a l o f a t e n t a t i v e c l a s s i f i c a t i o n o f a n t i b i o t i c s by c h e m i c a l s t r u c t u r e i n t o t e n famil i e s , w h e r e i n two f a m i l i e s a r e a s s i g n e d t o s a c c h a r i d e s a n d g l y c o s i d e s . However, e v e n t h o u g h t h e p r e s e n c e i n a n a n t i b i o t i c o f t h e c a r b o h y d r a t e m o i e t y may b e e s s e n t i a l f o r i t s a n t i m i c r o b i a l a c t i v i t y , a f a m i l y - c l a s s i f i c a t i o n of g l y c o s i d e a n t i b i o t i c s i s n o t t o o u s e f u l , b e c a u s e of t h e e x t r e m e c h e m i c a l d i v e r s i t y of t h e many members t h a t would f a l l i n t o t h e f a m i l y 2 . I n view o f t h i s , and t h e f a c t t h a t s e v e r a l subgroups of c a r b o h y d r a t e - c o n t a i n i n g antibiotics a r e being treated separately in other chapters , t h i s chapter w i l l restrict i t s e l f t o t h e i s o l a t i o n , separation and p u r i f i c a t i o n of t h e coumarin-glycoside a n t i b i o t i c s . T h i s g r o u p i n c l u d e s n o v o b i o c i n , d i h y d r o n o v o b i o c i n , methoxyn o v o b i o c i n , c l o r o b i o c i n a n d coumermycins ( s u g o r d o r n y c i n s ) . 1. N o v o b i o c i n
1.1 I n t r o d u c t i o n 1.1.1 P r o d u c i n g o r g a n i s m s
The name n o v o b i o c i n (1)was a c c e p t e d i n 195€i3 f o r t h e a n t i b i o t i c i n d e p e n d e n t l y d i s c o v e r e d by a t l e a s t f i v e l a b o r a t o r i e s . I t was f i r s t r e p o r t e d t o h a v e b e e n i s o l a t e d i n 1 9 5 1 by Waga a n d T a k a h a s h i 4 , b u t t h e o r i g i n a l d e s c r i p t i o n i n 1 9 5 3 u n d e r t h e name g r i s e o f l a v i n 5 o m i t t e d c e r t a i n e s s e n t i a l p h y s i c o - c h e m i cal d a t a ( I . R . , U.V. ) a n d c o n t a i n e d s e v e r a l d i s c r e p a n c i e s . Kuroya e t ax.6 a g a i n i s o l a t e d n o v o b i o c i n from Waga's c u l t u r e , a n d by d i r e c t comparison w i t h a novobiocin sample (Upjohn) demonstrated i d e n t i t y of the a n t i b i o t i c s . They a l s o f o u n d by c o m p a r i s o n o f t h e i r c u l t u r e S t A e p t o m y c e A g A i A e O ~ & % V u A No. 1 6 0 w i t h l i t e r a t u r e d e s c r i p t i o n s of S . n i v e u d 7 , S . A p h t 4 0 i d e A 8 a n d t h e c a r d e l m y c i n p r o d u c i n g s t r a i n of S. g A i A e u A S P . ~t h a t t h e s e s t r a i n s are a l l s i m i l a r t o e a c h o t h e r t a x o n o m i c a l l y . R o l l a n d e t ae:lo d e s c r i b e d t h e p r o d u c t i o n o f n o v o b i o c i n by a "new" s p e c i e s S t A e p t O m g C e A S . 800 b u t g a v e o n l y a s k e t c h y t a x o n o m i c d e s c r i p t i o n . Kanzaki e t ae.ll r e p o r t e d t h a t t h e i r S t A e p t o m g c e A s t r a i n No. 58383 p r o d u c e d n o v o b i o c i n b u t d i f f e r e d f r o m known p r o d u c e r s i n c a r b o n a s s i m i l a t i o n p a t t e r n and i n n i t r i t e and hydrogen s u l f i d e - p r o d u c ib i l i t y
.
Synonyms employed f o r n o v o b i o c i n i n c l u d e : s t r e p t o n i v i c i n , a n t i b i o t i c 6 6 a , Albamycin ( U p j o h n ) ; c a t h o m y c i n , C a t h o c i n , Spheromycin ( M e r c k , S h a r p E Dohme) ; c a r d e l m y c i n , c r y s t a l l i n i c a c i d , PA 9 3 ( P f i z e r L a b s ) ; g r i s e o f l a v i n (Waga); v u l c a m y c i n , a n t i b i o t i c S . 800 (Le P e t i t S . P . A . ) ; i n a m y c i n ( H o e c h s t ) ; b i o t e x i n ( G l a x o ) ; r o b i o c i n a ( P r a d e l , M a d r i d ) . Welch a n d W r i g h t 1 2 r e p o r t e d on t h e common i d e n t i t y o f c a t h o m y c i n , s t r e p t o n i v i c i n and c a r d e l m y c i n b a s e d on U . V . a n d I . R . a b s o r p t i o n s p e c t r a , o p t i c a l r o t a t i o n s , a n t i b a c t e r i a l s p e c t r a , e f f e c t of s e r u m a n d b a c terial cross-resistance. An e x c e l l e n t r e v i e w of t h e n o v o b i o c i n d e v e l o p m e n t was p r e -
104 s e n t e d by Hoeksema a n d S m i t h i n 1 9 6 1 1 3 . S t r e p t o m y c e s c u l t u r e s for n o v o b i o c i n p r o d u c t i o n s u c h as SthtneptOmyCCZA d p h c A o i d e 4 (MA-319 ) NRRL 2 4 4 9 a n d StheptOmgce.4 n i U e U A NRRL 2466, are a v a i l a b l e from p u b l i c c u l t u r e d e p o s i t agencies. 1.1.2
Brief chemical description
N o v o b i o c i n ( & I , i s o l a t e d i n many l a b o r a t o r i e ~ a~n d~ ' ~ ~ ~ ~ w e l l c h a r a c t e r i z e d by w o r k e r s from t h e Upjohn a n d Merck l a b o r a t o r i e s 7 y 1 4 - 1 8 i, s a d i b a s i c a c i d , p a l e y e l l o w t o w h i t e c r y s t a l s , e x i s t i n f i n Lwo p o l y m o r p h i c f o r m s which show e q u a l o p t i c a l a c t i v i t y { a ) $ -63 (C=l.O, 9 5 % E t O H ) a n d i d e n t i c a l U . V . a b s o r p t i o n b u t w i t h d i f f e r e n t I . R . s p e c t r a . They are i n t e r c o n v e r t i b l e t h r o u g h t h e amorphous f o r m made by s o l u t i o n i n a c e t o n e , f o l lowed by r a p i d p r e c i p i t a t i o n w i t h p e t r o l e u m e t h e r : I ( m o s t common) from a c e t o n e - w a t e r , e t h a n o l or e t h y l a c e t a t e , m.p. 152-156' d e c . ; and 11, t h e r a r e r m o d i f i c a t i o n from a c e t o n e - h e x a n e , m:p. 174-178' d e c . ; s o l u b l e i n w a t e r a t pH > 7 . 5 a n d p r a c t i c a l l y i n s o l u b l e a t l o w e r pH. The a c i d i c form i s s o l u b l e i n p o l a r s o l v e n t s s u c h as a c e t o n e , e t h y l a n d amyl a c e t a t e s , e t h a n o l , metha n o l , p ~ r i d i n e ' ~ .I t i s s t a b l e i n t h e d a r k b u t s l i g h t l y l i g h t s e n s i t i v e z 0 . The e m p i r i c a l f o r m u l a i s C31H36011N2; mol. w t . 6 1 2 . 6 5 ; pKal 4 . 3 f o r t h e ' e n o l a n d pKa2 9 . 1 f o r t h e p h e n o l i c g r o u p ( w a t e r ) ; {cr164 -63 (c=l.O, 9 5 % E t O H ) , -27' ( c . l . 0 , 1N N a O H ) , -44' (c=l.O, p y r i d i n e ) . A b s o r p t i o n maxima a r e 307 nm, E %m 6 0 0 i n 0 . 1 N N a O H ; 324 nm, E$E 390 i n 0 . 1 N m e t h a n o l i c H ?; 304 nm, E i g m 350 i n pH 7 phospffate b u f f e r . The I . R . s p e c t r a are g i v e n 6 ~l o Y ~ ~ .
E
The monosodium s a l t f o r m s c r y s t a l s w i t h m.p. 210-215' decal8; -34' (c.1.081, w a t e r ) ; i t i s f r e e l y s o l u b l e i n w a t e r , methanol and e t h a n o l . Other s o l u b i l i t i e s a r e givenlq. Disodium n o v o b i o c i n and c a l c i u m a c i d n o v o b i o c i n h a v e been p r e p a r e d a n d d e s c r i b e d 1 8 . The monosodium and t h e c a l c i u m a c i d s a l t s were u s e d most c o m m e r c i a l l y .
{cr)b5
PC s t u d i e s 3 1 showed t h a t n o v o b i o c i n can s o l v a t e r e a d i l y w i t h m e t h a n o l and e t h a n o l , a n d a l s o form a h y d r a t e , t h u s g i v i n g r i s e t o two s p o t s on c h r o m a t o g r a p h i c d e v e l o p m e n t w i t h a c e t o n e or b u t a n o n e . S o l i d a d d u c t s o f s o d i u m and p o t a s s i u m s a l t s o f novobiocin w i t h methanol and e t h a n o l have been r e p o r t e d and u s e d f o r p u r i f i c a t i o n o f t h e a n t i b i o t i c 3 z . The sodium s a l t m e t h a n o l a d d u c t f o r t s n e e d l e s , m.p. 2 2 5 ' +5' ( d e c . ) w i t h loss o f b i r e f r i n g e n c e 1 0 b e f o r e m e l t i n g ; i t i s l e s s s o l u b l e i n metha n o l t h a n s o d i u m n o v o b i o c i n . The s o d i u m s a l t e t h a n o l a d d u c t i s s t r o n g l y b i r e f r i n g e n t , m.p. 2 1 7 - 2 2 2 ' (dec.). Amine s a l t s i n c l u d i n g t h o s e w i t h b a s i c a n t i b i o t i c s 2 l Y z 2 have been r e p o r t e d . The o r g a n i c b a s e s a r e g e n e r a l l y t o o w e a k t o p e r m i t t h e f o r m a t i o n of n e u t r a l s a l t s . Acid s a l t s of primary, s e c o n d a r y and t e r t i a r y amines are e a s i l y madez0, Mono-, d i - a n d t r i e s t e r s as w e l l as e t h e r s of n o v o b i o c i n h a v e been r e p o r t e d z 3 ; t h e m o n o e s t e r i s s a i d t o form on t h e s u g a r and i n t h e d i e s t e r t h e p h e n o l i c hydroxyl i s a l s o blocked. The
1 05
t r i e s t e r includes t h e e n o l i c hydroxyl and forms only w i t h d i f f i c u l t y . All o f t h e s e d e r i v a t i v e s were r e p o r t e d t o b e a c t i v e b u t q u i t e weakly, both in v i t h o and i n V i V O . N o v o b i o c i n d r y p o w d e r s i n a c i d or s a l t f o r m a r e q u i t e stable. I n a q u e o u s s o l u t i o n , pH 3-6 c a u s e s l i t t l e d e c o m p o s i t i o n , b u t a t pH 1 0 t h e r e i s a r a p i d d r o p i n a c t i v i t y t o a b o u t 65-70% i n 2 h o u r s , wh er e an e q u i l i b r i u m i s e s t a b l i s h e d b e tw e e n n o v o b i o c i n a n d i n a c t i v e i s o n o v o b i o c i n ( 2 I z 4 . The l a t t e r i s f o r m e d as a r e s u l t o f a m i g r a t i o n o f t h e c a r b a m y l g r o u p t o t h e a d j a c e n t s u g a r h y d r o x y l Z 5 . P u r e i s o n o v o b i o c i n , on e q u i l i b r a t i o n i n base, g i v e s 5 5 4 0 % conversion back to novobiocin. F u r t h e r a l k a l i n e t r e a t m e n t o f 1. l e a d s t o i n a c t i v e d e s c a r b a m y l n o v o b i o c i n ( 3 ) . Novobiocin can be d e t e r m i n e d i n t h e p r e s e n c e of i s o n o v o b i o c i n by a c h e m i c a l method b a s e d on t h e p e r i o d a t e o x i d a t i o n o f 3-0-carbamylnoviose , p r o d u c e d from n o v o b i o c i n by a c i d cleavage of t h e g l y c o s i d i c bond25. Acid degradation of novobiocin under various c o n d i t i o n s y i e l d s : 290’
(L) i n
m e t h a n o l or e t h a n o l
A. C y c l o n o v o b i o c i c a c i d ; p a l e y e l l o w c r y s t a l s , m.p. 2 8 8 ( d e c . ) ; pKal 6 . 3 ( i n 75% DMF); o p t i c a l l y i n a c t i ~ e ~ ~ ’ ~ ~ ’ ~
B. N o v o b i o c i c a c i d (i),m.p. 217.-232’, t h e a g l y c o n e o f has a l s o b e e n p r o d u c e d e n n o v o b i ~ c i n ~ ~ ’ T~h~i s ’ compound ~ ~ . z y m a t i c a l l y from t h e s u b s t i t u t e d b e n z o i c a c i d moiety o f novobioc i n ( r i n g A) and t h e coumarin moiety ( r i n g B ) w i t h a d e n o s i n e t r i p h o s p h a t e and a novobiocic a c i d s y n t h e t a s e p r e s e n t i n cellf r e e e x t r a c t s of S t h e p t O m y C e A n i V e U A Z 8 . C.
The m e t h y l g l y c o s i d e of 3 - O - c a r b a m y l n o v i o s e ,
D.
The e t h y l g l y c o s i d e o f 3 - 0 - c a r b a m y l n o v i o s e ,
192’18,29,
175
’ *.
30.
m.p. m .p.
191173-
The a m i d e b o n d i n n o v o b i o c i n h a s b e e n h y d r o l y z e d b y a microbial e n z y m e 6 4 . A b a c t e r i u m (NRRL B-3652 A h t h h o b a c t e h Ap. N-18) i s o l a t e d f r o m sewage a n d c e l l - f r e e e nz ym e s o l u t i o n s p r e p a r e d f r o m t h e b a c t e r i u m were f o u n d t o h y d r o l y z e n o v o b i o c i n i n t o t h e two c o m p o n e n t s o f t h e a n t i b i o t i c , w h i c h were i s o l a t e d a n d i d e n t i f i e d as n o v e n a m i n e (6-1 a n d 3-isopentenyl-4-hydroxybenzoic a c i d . N o v e n a m i ne was i n t u r n u s e d f o r t h e c h e m i c a l p r e p a r a t i o n o f n o v e l s e m i s y n t h e t i c n o v o b i o c i n a c y l a t e s 6 5 . The a v a i l a b i l i t y o f novenamine h a s a l l o w e d t h e p r e p a r a t i o n o f a v a r i e t y o f a n a l o g s n o t r e a d i l y a c c e s s i b l e b y t h e b i o s y n t h e t i c means e m p l o y e d by Walton e t
F o u r s yn t h e t i c n o v o b i o c i n d e r i v a t i ve s , 8 -de sme t h y l n o v o b i oc i n , 8-desmethyldihydronovobiocin, 8-desme t h y l c y c l o n o v o b i o c i n were a n d 7-~3-carbamylnov~osyl~-3-acetam~no-4-hydroxycoumar~n, p r e p a r e d by u s e o f an i n t e r m e d i a t e d e r i v e d from n a t u r a l n o v i o s e . They were r e p o r t e d 6 6 t o b e “ u s e f u l a n t i m i c r o b i a l a g e n t s . . e f f e c t i v e i n t h e t r e a t m e n t a n d c o n t r o l o f p l a n t d i s e a s e s . ” Howe v e r , n o a n t i m i c r o b i a l d a t a were p r e s e n t e d .
. .. . . . .
When t h e c y c l i c p r e c u r s o r 2 ,2-dimethyl-6-carboxy-3-chroman
106 was a d d e d t o a n o v o b i o c i n f e r m e n t a t i o n , a new d e r i v a t i v e was o b t a i n e d which h a d o n l y 4 % o f t h e a n t i b a c t e r i a l a c t i v i t y o f n o v o b i ~ c i n ~ The ~ . same compound, s y n t h e s i z e d a t F . Hoffmann-La Roche & Co.A.G., Basle, as Ro 4-5151, was f o u n d t o h a v e l e s s t h a n 2 % o f t h e a c t i v i t y o f n o v o b i o c i n . The N-methyl (Ro 4-5311) a n d t h e N,N-dimethyl d e r i v a t i v e s on t h e c a r b a m a t e n i t r o g e n o f d i h y d r o n o v o b i o c i n were a l s o d e v o i d of a c t i v i t y . O k ~ m u r ap ~r e~p a r e d a s e r i e s o f 8 4 o r more d e r i v a t i v e s o f n o v o b i o c i n a n d r e l a t e d compounds. Among t h e s e 3 - a c y l a m i n o - 4 h y d r o x y c o u m a r i n d e r i v a t i v e s , some "were e f f e c t i v e " i n i n v i 2 J L o a n t i m i c r o b i a l tests a g a i n s t n i n e t e e n b a c t e r i a and f u n g i , b u t t h e p o t e n c i e s w e r e g e n e r a l l y q u i t e low. I n a s e r i e s o f p a p e r s , w o r k e r s a t F . Hoffmann-La Roche E C O . A.G. , Basle, d e s c r i b e d t h e s y n t h e s i s o f many n o v o b i o c i n i n t e r m e d i a t e s i n c l u d i n g n o v i o s e , c u l m i n a t i n g i n t h e t o t a l s y n t h e s i s o f i s o n o v o b i o c i n a n d novob i o c i n 6 8 . The i d e n t i t y o f t h e s y n t h e t i c a n d n a t u r a l n o v o b i o c i n was e s t a b l i s h e d from c o m p a r i s o n s o f I . R . ,r o t a t o r y d i s p e r s i o n c u r v e s and m i c r o b i o l o g i c a l a c t i v i t y a g a i n s t S t a p h y l o c o c c u d auaeud P 6 5 3 8 . 1.1.3
S t r u c t u r a l formula
S t r u c t u r a l s t u d i e s c a r r i e d o u t i n d e p e n d e n t l y by t h e Merck and U*.. ~ i o h n grouDs -~ .~ e~s t a~b l i s~h e d- t h~ e ~ ~ u n i q u e s t r u c t u r e o f n o v o b i o c i n as 7-(3,0-carbamoyl-5,5-dimethyl4- 0 -me t h y l - a - l - l y x o s y 1) - 4 -hydroxy - 3 - 4-hydroxy - 3 - ( 3 - m e t h y l b u t - 2 e n y l ) ben zami d o I- 8 - m e t h y l c o u m a r i n (1) 14r27,29r33y34
.
A s may b e s e e n i n F i g u r e 1, t h e a n t i b i o t i c c o n s i s t s of a s u b s t i t u t e d b e n z o i c a c i d m o i e t y ( A r i n g ) l i n k e d by a n arnide bond t o an aminohydroxycoumarin moiety ( B r i n g ) , which i n t u r n i s j o i n e d t o an u n i q u e s u g a r , n o v i o s e , w i t h one h y d r o x y l c a r r y i n g a carbamyl group ( C r i n g ) . C o n f o r m a t i o n a l s t u d i e s on n o v i o s e g l y c o s i d e s a n d on n o v o b i o c i n l e d t o c o m p l e t e a s s i g n m e n t o f i t s s t e r e o c h e m i ~ t r y ~ ~I t. s o r t h o r h o m b i c c r y s t a l s t r u c t u r e was r e c e n t l y s o l v e d 3 6 and t h e assignments i n t h e 1 3 C N.M.R. spectrum h a v e been made 7.
1 . 2 Summary
of Therapeutic use
The a n t i m i c r o b i a l a c t i v i t y o f n o v o b i o c i n h a s b e e n e v a l u a t e d by many i n v e s t i g a t o r s . I n V ~ ~ R Oi t, i s a c t i v e a t 0 . 1 t o 1 mtg/ml a g a i n s t some of t h e g r a m - p o s i t i v e cocci, s u c h as S t a p h y .!OCOCCuO auReuA or p y o g e n e d ( i n c l u d i n g p e n i c i l l i n - r e s i s t a n t s t r a i n s ) a n d t h e pneumococci. S t h c p t o c o c c u d pyogencA i s l e s s s e n s i t i v e ( 0 . 5 t o 4 mcg/ml), StJLep. vilridand s t r a i n s vary i n t h e i r s e n s i t i v i t y a n d S t h e p . daecalid i s u s u a l l y r e s i s t a n t 3 8 . N o v o b i o c i n - s e n s i t i v e g r a m - p o s i t i v e b a c i l l i i n c l u d e Baci.!IuA a n t h n a c i d ( 2 mcg/ml), C l o d t L d i u m p e k 6 h i n g e n A ( 1 mcg/rnl) a n d C O A y n e b a c t e ~ ~ i u dmi p h t h c n i a c Some gram-nega t i v e b a c t e r i a 3 9 s u c h as Hacmophilud i n d l u e n z a e (1 m c g / m l ) , NeiAAeAia d p . (1-4 mcg/ml) a n d c e r t a i n s t r a i n s o f PROteuA vulgaJLi.4 a r e s e n s i t i v e , b u t o t h e r P n o t c u d s p . are c o m p l e t e l y r e s i s t a n t . The d r u g was r a r e l y u s e d a g a i n s t t h e s e o r g a n i s m s i n c l i n i c a l p r a c t i c e . Many b a c t e r i a o r i g i n a l l y s e n s i t i v e t o novobiocin r e a d i l y acquire r e s i s t a n c e i n v ~ ~ Aby o c u l t i v a t i o n i n t h e p r e s e n c e o f d r u g .
.
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RING C
RING A #
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NOVOBIOCIC ACID, 4 NOVENAMINE, 6
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-9
2, 4,
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-9
H Novobiocin CONH2 Isonovobiocin H Descarbamylnovobiocin Novobiocic a c i d ( l a c k s Ring C) Oihydronovobiocin ( s a t u r a t e d H s i d e chain i n Ring A ) Novenamine ( l a c k s Ring A ) H
!L% CONH2
H H
CH3 CH3 CH3
CONH2
CH3
CONH,
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FIGURE 1
I n a n e a r l y i n v e s t i g a t i o n , n o v o b i o c i n was a d m i n i s t e r e d t o mice, r a t s , g u i n e a p i g s , c a t s , r a b b i t s and dogs i n a c u t e , s u b a c u t e a n d c h r o n i c t o x i c i t y s t u d i e s 4 0 . I n mice, a c u t e L D s o , i n mg/kg, was 262-300 i . p . , 407 i . v . , 962->1000 p . 0 . Guinea p i g s were much more s u s c e p t i b l e , LDs0 1 1 . 5 i . p . , 2 7 . 8 S . C . Long term o r a l t o x i c i t y s t u d i e s i n r a t s and dogs d i d n o t d i s c l o s e any s i g n i f i c a n t p a t t e r n o f t o x i c i t y . S i m i l a r mouse t o x i c i t y d a t a were r e p o r t e d l o , L D 5 0 4 2 4 mg/kg i . v . and 1 1 2 0 mg/kg p . 0 . Nov’obiocin a d m i n i s t e r e d t o h e a l t h y men i n s i n g l e d o s e s of 250 mg gave good s e r u m l e v e l s a f t e r 3 h r by e i t h e r i . m . ( 4 . 2 mcg/ml) or i . v . (11 mcg/ml) r o u t e . High l e v e l s (11 mcg/ml) were found 2 h r a f t e r i n i t i a l o r a l a d m i n i s t r a t i o n i n f o u r divided d o s e s ( 2 5 0 mg e a c h ) when t h e i n i t i a l d o s e was t a k e n on a n empty s t o m a c h 4 1 . Recovery o f n o v o b i o c i n from t h e u r i n e r a n g e d from 0 . 3 t o 15%. N o v o b i o c i n , c a l c i u m o r monosodium, was w i d e l y u s e d f o r some t i m e i n human m e d i c i n e , e i t h e r a l o n e (Albamycin or Cathomycin) o r i n c o m b i n a t i o n ( 1 : 2 ) w i t h t e t r a c y c l i n e ( P a n a l b a ) . I n 1 9 6 1 t o t a l a n n u a l p r o d u c t i o n of t h e d r u g i n t h e U . S . A . a l o n e was e s t i m a t e d t o . b e a b o u t 1 5 0 0 0 k g 1 3 . The s u g g e s t e d o r a l a d u l t d o s a g e i s 1 g i n i t i a l l y , f o l i o w e d by 0 . 2 5 g e v e r y 6 h r or 0 . 5 g
108 every 1 2 h r . I n c h i l d r e n , t h e s u g g e s t e d d o s e i s 1 5 mg/kg body w e i g h t p e r d a y , i n d i v i d e d d o s e s . The d r u g h a d i t s m a j o r use i n s t a p h y l o c o c c a l i n f e c t i o n s 4 2 b u t evidence accumulated f o r many a n d f r e q u e n t a d v e r s e e f f e c t s . They i n c l u d e u r t i c a r i a a n d o t h e r a l l e r g i c symptoms ( i n some s e r i e s o c c u r r i n g i n 30 t o 53% of p a t i e n t s ) , h e p a t o t o x i c i t y d u e t o i n h i b i t i o n o f t h e c o n j u g a t i n g l i v e r enzyme g l u c u r o n y l t r a n s f e r a s e , h y p e r b i l i r u b i n e m i a , h a e m a t o l o g i c a l d i s o r d e r s and g a s t r o i n t e s t i n a l i n t o l e r a n c e . A s a r e s u l t o f t h e a b o v e p l u s t h e a v a i l a b i l i t y of new p e n i c i l l i n a s e - r e s is t a n t p e n i c i l l i n s and o t h e r p o t e n t a n t i -s t a p h y l o c o c c a l a g e n t s , t h e d r u g i s no l o n g e r u s e d f o r t h e s e i n f e c t i o n s 4 3 a n d h a s become more o r l e s s o b s o l e t e . C o m b i n a t i o n s o f n o v o b i o c i n w i t h o t h e r a n t i b i o t i c s s u c h as t e t r a c y c l i n e , s o d i u m f u s i d a t e a n d r i f a m p i c i n were u s e d t o p r e v e n t emergence o f d r u g r e s i s t a n c e , b u t t h e evidence t h a t they a c t s y n e r g i s t i c a l l y h a s been seriously q ~ e s t i o n e d ~ A ~ l.t h o u g h i t i s e f f e c t i v e f o r t h e t r e a t ment o f p n e u m o c o c c a l pneumonia, w h e r e p e n i c i l l i n i s t h e d r u g of c h o i c e , i t d o e s n o t a p p e a r t o h a v e any o t h e r r o l e i n c u r r e n t t h e r a p e u t i c s 38. I n t h e a n i m a l h e a l t h a n d v e t e r i n a r y f i e l d , n o v o b i o c i n was a p p r o v e d f o r u s e i n f e e d s o f young a n i m a l s as a g r o w t h p r o m o t a n t , i n b o v i n e m a s t i t i s f o r m u l a t i o n s ( B i o t e x i n , G l a x o ) a n d f o r cert a i n i n d i c a t i o n s i n c a t s a n d d o g s , e s p e c i a l l y Staphylococcud auheud i n f e c t i o n s when p r e s e n t i n e i t h e r p r i m a r y i n f e c t i o n s ( s e p t i c e m i a ) or secondary complications (as i n distemper o r f e l i n e p a n l e u k o p e n i a ) . Dose i s 10-30 mg/kg i . m . H a r r i e s 4 5 r e p o r t e d a r a t h e r unique t o x i c i t y of novobiocin a g a i n s t t h e f e m a l e c o d l i n g moth, Ca,tpocapda pornoneela (L. 1, a t c o n c e n t r a t i o n s as low as 37.5-75 p . p . m . , as i n d i c a t e d by i n h i b i t i o n of e g g - l a y i n g and m o r t a l i t y . The d r u g was much more t o x i c t h a n a z i n p h o s m e t h y l , one o f t h e most e f f e c t i v e i n s e c t i c i d e s u s e d ( i n 1 9 6 7 ) a g a i n s t t h e c o d l i n g moth.
1.3 Extraction, 1.3.1
s e p a r a t i o n and p u r i f i c a t i o n
Detection
B e f o r e and, a f t e r c r y s t a l l i n e n o v o b i o c i n was o b t a i n e d , fermentation b r o t h p o t e n c i e s , e x t r a c t i o n procedures and sample p u r i t i e s w e r e f o l l o w e d by o n e o r more a s s a y s i n v o l v i n g cupp l a t e a g a r d i f f u s i o n b i o a s s a y , b i o a u t o g r a p h y on s e e d e d a g a r p l a t e s o f p a p e r chromatograms or t h i n - l a y e r p l a t e s , U . V . a b s o r p t i o n and c o u n t e r c u r r e n t d i s t r i b u t i o n a n a l y s i s . The employment of most o f t h e s e p r o c e d u r e s i s b r i e f l y r e f e r r e d t o w h e r e t h e y are u s e d i n p a r t i c u l a r i s o l a t i o n s . The o f f i c i a l l y a c c e p t e d a s s a y method (as o f 1 9 7 3 1 , by t h e Food and Drug A d m i n i s t r a t i o n , f o r the potency determination of novobiocin is the microbiologi c a l c y l i n d e r cup a g a r - d i f f u s i o n a s s a y u s i n g S t a p h y l o c o c c w epide,tmidin ATCC 1 2 2 2 8 as t h e t e s t o r g a n i s m . However, t h i s method i s n o t as p r e c i s e q u a n t i t a t i v e l y as d e s i r a b l e , n o r c a n i t d e t e c t t h e p r e s e n c e o f m i n o r i m p u r i t i e s . A d e s c r i p t i o n of some of t h e o t h e r m e t h o d s , as u s e d b y KominekZ8, i s g i v e n b e l o w . A r a p i d s p e c t r o p h o t o m e t r i c U.V. a s s a y t o d e t e r m i n e novob i o c i n i n f e r m e n t a t i o n b r o t h s i s b a s e d on t h e U . V . a b s o r b a n c e o f t h e a n t i b i o t i c i n n - b u t y l a c e t a t e a t 338 nm46. The a s s a y i s
109 s e n s i t i v e , simple and r a p i d b u t n o t completely s p e c i f i c s i n c e n o v o b i o c i n - r e l a t e d compounds s u c h a s d e c a r b a m y l n o v o b i o c i n i s o n o v o b i o c i n (21, e t c . a l s o r e s p o n d . I f specificity is req u i r e d , b i o a s s a y s or p a p e r chromatography a r e u s e d .
(z),
Novobiocin, n o v o b i o c i n p r e c u r s o r s a n d n o v o b i o c i c a c i d (5) c o n t a i n i n g s a m p l e s f o r a n a l y s i s by PC a r e e x t r a c t e d i n t o b u t y l a c e t a t e a t pH 6 . 5 , t h e s o l v e n t i s e v a p o r a t e d , and t h e r e s i d u e r e d i s s o l v e d i n acetone. Whatman No. 4 or 20 p a p e r d i p p e d i n e t h y l e n e g l y c o l c o n t a i n i n g 2 % of 8 5 % l a c t i c a c i d i s t h e s t a t i o n a r y phase. The m o b i l e p h a s e c o n s i s t s of i s o p r o p y l e t h e r s a t u r a ted with ethylene glycol. The s a m p l e , d i s s o l v e d i n a c e t o n e , i s s p o t t e d s o t h a t e a c h s p o t c o n t a i n s a b o u t 7 5 mcg of n o v o b i o c i n or related material. The chromatograms are d e v e l o p e d by d e s c e n d i n g C o n t a c t p r i n t s are made method f o r ca. 1 6 h r a t 2 8 ' and d r i e d . t o i d e n t i f y t h e U . V . a b s o r b i n g components. The a b s o r p t i v i t i e s a t 3 2 4 nm a n d t h e o r d e r o f p o l a r i t y of t h e s e compounds from more p o l a r t o l e s s p o l a r i s as f o l l o w s : 0 - d e m e t h y l n o v o b i o c i n ( 4 0 . 0 ) , 0-demethyldecarbamylnovobiocin ( 4 2 . 6 ) , n o v o b i o c i n ( 3 9 . 9 ) , i s o novobiocin ( 3 9 . 9 ) , decarbamylnovobiocin ( 4 2 . 5 ) , and novobiocic a c i d ( 5 1 . 9 ) . Whatman N o . 4 p a p e r i s u s e d t o d e v e l o p n o v o b i o c i n and t h e more p o l a r compounds, and No. 2 0 p a p e r i s u s e d t o s e p a r a t e i s o n o v o b i o c i n a n d o t h e r l e s s p o l a r compounds. A f t e r c h r o m a t o g r a p h i c s e p a r a t i o n h a s been a c h i e v e d , t h e p r o p e r U . V . a b s o r b i n g s p o t s a r e c u t from t h e p a p e r and e l u t e d w i t h 10 r n l of a c i d i c methanol ( 0 . 0 0 2 N H 2 S 0 4 ) . The a b s o r b a n c e o f t h e e l u a t e i s r e a d a t 3 2 4 nm w i t h t h e e l u a t e from a b l a n k c h a n n e l of p a p e r as r e f e r e n c e . The c o n c e n t r a t i o n of t h e compound measured i s d e t e r m i n e d from t h e a b s o r b a n c e r e a d i n g a t 3 2 4 nm a n d i t s a b s o r p tivity. Walton e t 0 . 1 . u~s e~d d e s c e n d i n g P C 4 8 t o e v a l u a t e t h e 1 9 new n o v o b i o c i n d e r i v a t i v e s which t h e y p r o d u c e d . U s i n g Whatman No. 1 p a p e r , 10-50 p 1 o f b r o t h i s s p o t t e d on t h e p a p e r s t r i p s along with a r e f e r e n c e s t a n d a r d , and developed with a system c o n t a i n i n g c a p r y l a l c o h o l as t h e s t a t i o n a r y p h a s e and 0 . 1 M y pH 8 . 2 phosphate b u f f e r s o l u t i o n e q u i l i b r a t e d with c a p r y l a l c o h o l for t h e m o b i l e p h a s e . A f t e r d e v e l o p m e n t , t h e p a p e r s t r i p s were a i r - d r i e d a t room t e m p e r a t u r e t o e x h a u s t a l l s o l v e n t . The d r y s t r i p s were t h e n s c a n n e d u n d e r U . V . and p l a c e d on agar. s e e d e d w i t h Baci11uo - b u b . t i C i A . A f t e r t h e c u l t u r e h a d grown i n t h e a g a r , t h e p o s i t i o n s of t h e zones o f i n h i b i t i o n i n r e l a t i o n t o s t a n d a r d c r y s t a l l i n e n o v o b i o c i n were d e t e r m i n e d . An HPLC method f o r t h e a n a l y s i s of n o v o b i o c i n h a s been d e s c r i b e d by T s u j i and R o b e r t s o n 4 9 . The method u s e s a 1 m-long Zipax HCP column w i t h a mobile p h a s e of 1 5 % m e t h a n o l i n 0 . 0 2 M p h o s p h a t e b u f f e r , pH 7 . 0 , a t a f l o w - r a t e o f 0 . 8 5 ml/min ( 6 8 a t m ) . Novobiocin i s o n o v o b i o c i n (2),d i h y d r o n o v o b i o c i n decarbamylnovob i o c i n (3-1 desmethyldescarbamylnovobiocin ( 8 ), novob i o c i c a c i d (41, a n d novenamine ( S ) , a r e s e p a r a t e d i n a b o u t 3 0 min. The r e l a t i v e s t a n d a r d d e v i a t i o n f o r t h e a n a l y s i s of novob i o c i n i s l e s s t h a n 1%. These same a u t h o r s a l s o mention t h e a v a i l a b i l i t y o f a G C method b a s e d on t h e a c e t y l a t i o n of n o v o b i o c i n a n d chromatography u s i n g a n O V - 1 7 column, b u t r e s u l t s a r e unpublished. I t has e x c e l l e n t p r e c i s i o n ( r e l a t i v e s t a n d a r d dev i a t i o n 0 . 3 4 % ) b u t t h e method c l e a v e s g l y c o s i d i c or amide bonds
(L),
110
a n d i s i n c a p a b l e o f d i f f e r e n t i a t i n g i s o n o v o b i o c i n from n o v o b i o c i n . Nishijima e t ~ 1 have . r~e p o ~ r t e d on t h e G C a n a l y s i s of n o v o b i o c i n a n d o t h e r a n t i b i o t i c s which were c o n v e r t e d t o t h e i r t r i m e t h y l s i l y l e t h e r s o r e s t e r s . Each o f t h e s e was d e v e l o p e d on a column f i l l e d w i t h 0 . 7 5 % s i l i c o n e O V - 1 onogaschrom Q o r 0 . 5 % s i l i c o n e O V - 1 7 on Chromosorb W a t 1 9 2 - 2 2 0 A flame-ioniz a t i o n d e t e c t o r was used.
.
Chemical a s s a y methods d e s c r i b e d by Ikekawa e t ~ 1 and. F o r i s t e.t a t . 2 5 are c a p a b l e o f d i f f e r e n t i a t i n g a n d q u a n t i t a t i n g n o v o b i o c i n i n t h e p r e s e n c e of i s o m e r s a n d d e g r a d a t i o n p r o d u c t s , I n t h e i r s t u d i e s on t h e b i o s y n t h e s i s of n o v o b i o c i n , C a l v e r t e Z mention s i x c h r o m a t o g r a p h i c s y s t e m s t h a t t h e y u s e d f o r n o v o b i o c i n p u r i f i c a t i o n , i n c l u d i n g TLC w i t h c h l o r o f o r m - f o r m i c a c i d - w a t e r - m e t h a n o l (100:O.e: 8 : 1 2 . 5 ) , w i t h c h l o r o f o r m - a c e t i c a c i d ( 9 : l ) and with the system of K ~ r a c h a g i n ~ ~ . 1.3.2
I s o l a t i o n o f n o v o b i o c i n (1)
Methods employed t o i s o l a t e n o v o b i o c i n from f e r m e n t a t i o n b r o t h e x p l o i t t h e a c i d c h a r a c t e r o f t h e a n t i b i o t i c . Three gene r a l p r o c e d u r e s have i n c l u d e d t h e t r a d i t i o n a l s t e p s o f f i l t r a t i o n t o remove m y c e l i a a n d i n s o l u b l e i m p u r i t i e s , and s u b s e q u e n t c o n c e n t r a t i o n a n d p u r i f i c a t i o n s t e p s s u c h as: 1. P r e c i p i t a t i o n o f a c r u d e m a t e r i a l by a c i d o r a s u i t able reactant. 2.
Solvent extraction.
3.
A d s o r p t i o n a n d e l u t i o n , as on c a r b o n , o t h e r a d s o r b a n t
or i o n - e x c h a n g e r e s i n s , E l i m i n a t i o n o f t h e cost o f f i l t r a t i o n i s a f e a t u r e of one commercial w h o l e - b e e r p r o c e s s employing a c o u n t e r c u r r e n t f l o w i n columns c h a r g e d w i t h a n i o n exchange r e s i n 5 4 . I t s h o u l d be p o i n t e d o u t t h a t most o f t h e e x t r a c t i o n a n d i s o l a t i o n p r o c e d u r e s d e s c r i b e d below a r e more s u i t e d f o r small-scale l a b o r a t o r y work. Although m o d i f i c a t i o n s o f some o f them may have been employed ,n l a r g e scale c o m m e r c i a l l y f o r a t i m e ? p r o d u c t i o n p r o c e d u r e s g e n e r a l l y s h i f t e d t o l e s s l a b o r - c o n s u m i n g a n d d e c r e a s e d number of s t e p s which c o u l d b e l a r g e l y a u t o m a t e d . A . Y . Wagas f i r s t i s o l a t e d n o v o b i o c i n ( u n d e r t h e name g r i s e o f l a v i n ) by t h e f o l l o w i n g scheme: C u l t u r e f i l t r a t e ( a g e 5 0 - 6 0 h o u r s , pH 8 . 2 ) was a d j u s t e d t o pH 2 . 0 w i t h d i l u t e h y d r o c h l o r i c a c i d , and then adsorbed f o r 1 / 2 hour with a c i d c l a y u s e d a t 1%l e v e l , w i t h a g i t a t i o n , a t room t e m p e r a t u r e . Thg a d s o r b a t e was e l u t e d 3 times w i t h 80% a c e t o n e (pH 7 . 0 ) a t 6 0 , u s i n g 1 / 2 0 volume o f t h e o r i g i n a l f i l t r a t e e a c h time. The comb i n e d e l u a t e s were a d j u s t e d t o pH 5 . 2 and f l a s h e v a p o r a t e d t o a c o n c e n t r a t e , which was e x t r a c t e d 2-3 times w i t h e q u a l volumes o f ethyl acetate. The combined e t h y l a c e t a t e e x t r a c t was washed w i t h w a t e r a t pH 5.2 a n d t h e n a t pH 7 . 0 . The washed e t h y l acet a t e e x t r a c t was t h e n e v a p o r a t e d i n v a c u a and d r i e d t o a brown powder. Y i e l d o f b i o a c t i v i t y , measured by S t a p h y ~ o c o c c u b auheud a s s a y , was 80-90%. The powder was d i s s o l v e d i n d r y m e t h a n o l ,
~
~
111 i n a c t i v e i n s o l u b l e s were r e m o v e d , a n d t h e m e t h a n o l c o n c e n t r a t e was c h r o m a t o g r a p h e d on a l u m i n a ( B L 6 , J a p a n Aluminum Co., L t d . ) using dry methanol f o r development. The e l u a t e s were t a k e n i n The f i r s t c o l a b o u t 10 m l p o r t i o n s a c c o r d i n g t o t h e i r c o l o r . o r l e s s a n d t h e s e c o n d b r o w n i s h f r a c t i o n s were i n a c t i v e , b u t t h e t h i r d colorless f r a c t i o n c o n t a i n e d almost a l l t h e a c t i v i t y . A c t i v e p o r t i o n s were c o m b i n e d a n d c o n c e n t r a t e d t o d r y n e s s i n v a c u o . The r e s u l t i n g f a i n t y e l l o w s u b s t a n c e was r e c r y s t a l l i z e d from d r y e t h a n o l t o g i v e a c t i v e , colorless n e e d l e s . Papergrams o f h i s novobiocin ( g r i s e o f l a v i n ) w i t h 1 3 s o l v e n t s y s t e m s were d e s c r i b e d , w h i c h t o g e t h e r w i t h s a l t i n g - o u t m e t h o d o l o g y , a n t i b a c t e r i a l s p e c t r a , m . p . , a few c h e m i c a l p r o p e r t i e s and c r o s s - i n h i b i t i o n s with o t h e r a n t a g o n i s t i c s t r e p t o myces were u s e d t o d i s t i n g u i s h t h e new a n t i b i o t i c f r o m known a n t i b i o t i cs
.
B. K a c z k a e t a1.14 r e p o r t e d i n r a t h e r g e n e r a l o u t l i n e t h e f i r s t i s o l a t i o n of c r y s t a l l i n e n o v o b i o c i n (cathomycin) from f e r m e n t a t i o n o f S t A e p t o m y c e d A p h e J t o i d e A , NRRL 2 4 4 9 , u t i l i z i n g as a f i r s t s t e p t h e p r e c i p i t a t i o n o f a c r u d e m a t e r i a l b y a c i d i f i c a t i o n o f a s o l u t i o n o f e v a p o r a t e d b r o t h f i l t r a t e t o a b o u t pH 2. The d r i e d p r e c i p i t a t e was t r i t u r a t e d w i t h a c e t o n e a n d t h e i n s o l u b l e m a t e r i a l was r e m o v e d . The a c e t o n e s o l u t i o n was e v a p o r a t e d i n v a c u o a n d t h e r e s i d u e was t r i t u r a t e d w i t h m e t h a n o l . The i n s o l u b l e m a t e r i a l was r emo v ed a n d t h e m e t h a n o l f i l t r a t e was e v a p o r a t e d i n v a c u o . The m e t h a n o l - s o l u b l e r e s i d u e was t r i t u r a t e d w i t h p e t r o l e u m e t h e r w h i c h d i s s o l v e d most of t h e d a r k colored substances. The r e m a i n i n g r e s i d u e was d i s s o l v e d i n d i l u t e s o d i u m h y d r o x i d e a n d t h e n h y d r o c h l o r i c a c i d was a d d e d t o cause p r e c i p i t a t i o n . The d r i e d p r e c i p i t a t e was t r i t u r a t e d r e p e a t e d l y w i t h e t h e r a n d t h e e t h e r e x t r a c t was e v a p o r a t e d . The a m o r p h o u s r e s i d u e c r y s t a l l i z e d f r o m a q u e o u s a c e t o n e or e t h a n o l or m i x t u r e s o f p e t r o l e u m e t h e r a n d a c e t o n e o r e t h a n o l . A m odif i c a t i o n of t h i s p r o c e s s , p r e c i p i t a t i o n o f t h e a n t i b i o t i c d i r e c t l y from t h e f i l t e r e d b r o t h , e l i m i n a t e d t h e e v a p o r a t i o n s t e p which is n o t v e r y p r a c t i c a l commercially55.
An i s o l a t i o n p r o c e d u r e r e s e m b l i n g t h e o n e u s e d f o r u r i f i c a t i o n o f p e n i c i l l i n was e m p l o y e d b y Hoeksema e Z ~ 1 . ~ ~B ' r o t~h a~d j.u s t e d t o pH 8 . 0 w i t h s o d i u m h y d r o x i d e , t o e n s u r e maximum s o l u t i o n o f t h e a c i d i c a n t i b i o t i c , was f i l t e r e d w i t h t h e a i d o f d i a t o m a c e o u s e a r t h . The a c t i v e f i l t r a t e was a c i d i f i e d t o pH 6 . 0 a n d e x t r a c t e d w i t h a m y l a c e t a t e (1/5 v o l u m e ) . A n t i b i o t i c a c t i v i t y was t h e n e x t r a c t e d i n t o a n a q u e o u s b u f f e r a t pH 1 0 a n d t h e a q u e o u s e x t r a c t a c i d i f i e d f o r a s e c o n d t r a n s f e r i n t o a m y l a c e t a t e (1/5 v o l u m e ) . When a c e t o n e a n d p e t r o l e u m e t h e r were a d d e d t o t h e c o n c e n t r a t e d amyl a c e t a t e , c r y s t a l l i z a t i o n of t h e novobiocin ( s t r e p t o n i v i c i n ) occurred. The a n t i b i o t i c was f u r t h e r p u r i f i e d b y r e c r y s t a l l i z a t i o n f r o m a c e t o n e , A 627-tube acetone-water, and acetone-hexane combinations. Craig c o u n t e r c u r r e n t d i s t r i b u t i o n a n a l y s i s ( s o l v e n t system n o t g i v e n ) was u s e d t o e s t a b l i s h t h e d e g r e e o f p u r i t y o f t h e c r y s t a l l i n e material o b t a i n e d above. R e s u l t s of p a p e r c h r o m a t o g r a p h y i n f i v e s o l v e n t s y s t e m s were p r e s e n t e d as shown i n F i g u r e 2 . C.
early
D. R o l l a n d e t al.l0 i s o l a t e d n o v o b i o c i n ( a n t i b i o t i c S . 8 0 0 ) f r o m t h e i r StAeptomyceA S . 8 0 0 c u l t u r e b r o t h i n a v e r y s i m i l a r
112
SOLVENT SYSTEM 0
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0.2 1
VALUES 0.4 I
0.6
0.8
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e
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FIGURE 2. Paper chromatography o f novobiocin. System I: 81% n-butanol, 19% water p l u s 0.25% p - t o l u e n e s u l f o n i c acid. Syatem 11: 50% butanol, 25% water and 25% a c e t i c acid. System 111: 78.4% n-butanol, 18.6% water p l u s 2% p i p e r i d i n e . System I V ; 96% water and 4% n-butanol. System V: 94% water, 41 n-butanol p l u s 2% p - t o l u e n e s u l f o n i c acid.
manner. A c t i v e f i l t r a t e was a c i d i f i e d t o pH 6 . 0 w i t h s u l f u r i c a c i d , and t h e a n t i b i o t i c e x t r a c t e d w i t h 1 / 1 0 volume o f amyl a c e t a t e . T h i s was e x t r a c t e d w i t h water a t pH 9 . 5 , from which t h e a n t i b i o t i c was p r e c i p i t a t e d by a c i d i f i c a t i o n . I t c o u l d be c r y s t a l l i z e d from aqueous a l c o h o l o r aqueous a c e t o n e . E. Kuroya e t L Z ~ c. o~n f i r m e d t h e i s o l a t i o n o f n o v o b i o c i n from Waga’s c u l t u r e , employing b o t h w a t e r - s o l v e n t d i s t r i b u t i o n s a n d a l u m i n a c h r o m a t o g r a p h y , as o u t l i n e d i n t h e f o l l o w i n g p r o c e d u r e : Whole b r o t h c o n t a i n i n g 3-50 m g / & was h a r v e s t e d a t 120 h r a t pH 8 . 4 t o 8 . 6 . The b r o t h was a d j u s t e d t o pH 8 . 0 , f i l t e r e d , t h e f i l t r a t e a c i d i f i e d t o pH 4 . 0 and e x t r a c t e d w i t h e t h y l acetate. The s o l v e n t e x t r a c t was washed w i t h w a t e r a n d a r e d p i g ment was removed. The a n t i b i o t i c was t r a n s f e r r e d from t h e s o l v e n t i n t o w a t e r a t pH 8 . 0 - 8 . 4 a n d , a f t e r a c i d i f i c a t i o n , was a g a i n transferred t o ethyl acetate. The c o n c e n t r a t e d e x t r a c t was d r i e d w i t h sodium s u l f a t e and c h r o m a t o g r a p h e d on an a l u m i n a column. The column was d e v e l o p e d and e l u t e d w i t h m e t h a n o l . The a c t i v e p i g m e n t l e s s e l u a t e was d r i e d i n uacuo, t h e r e s i d u e was d i s s o l v e d i n 0 . 0 1 N NaOH a n d t h e n 0.1 N H C 1 was added t o c a u s e p r e c i p i t a tion. The d r i e d p r e c i p i t a t e was r e c r y s t a l l i z e d from a c e t o n e w a t e r , y i e l d i n g f a i n t l y y e l l o w i s h n e e d l e s of t h e f r e e a c i d .
F . Celmer and Koe32 r e f e r r e d t o a c o m m e r c i a l l y i m p r a c t i cal p u r i f i c a t i o n procedure involving a series of e x t r a c t i o n and c o n c e n t r a t i o n s t e p s i n which f i l t e r e d b r o t h was a d j u s t e d t o a b o u t pH 2 by t h e a d d i t i o n o f a m i n e r a l a c i d a n d was t h e n ext r a c t e d w i t h a s o l v e n t s u c h as m e t h y l i s o b u t y l k e t o n e . This ext r a c t was washed w i t h d i l u t e sodium b i c a r b o n a t e t o remove impur-
113
i t i e s a n d e x t r a c t e d w i t h a q u e o u s ammonium h y d r o x i d e t o p r o d u c e t h e ammonium s a l t . A f t e r a d j u s t m e n t o f t h e a q u e o u s s o l u t i o n t o low pH t o o b t a i n t h e f r e e a c i d , t h e a n t i b i o t i c was f u r t h e r e x tracted with e t h e r , etc. I n p l a c e of t h i s p r o c e d u r e , t h e a u t h o r s p r o p o s e d a p u r i f i c a t i o n method b a s e d on t h e f i n d i n g t h a t sodium a n d p o t a s s i u m s a l t s o f n o v o b i o c i n form a d d u c t s w i t h metha n o l a n d e t h a n o l , w h i c h c a n b e u s e d a s i n t e r m e d i a t e s from w h i c h t o r e g e n e r a t e s u b s t a n t i a l l y pure a n t i b i o t i c by simple h e a t i n g , e.g., air-drying. O t h e r a l c o h o l s s u c h as n - p r o p a n o l , a n d 2 p r o p a n o l a n d n - b u t a n o l do n o t form a d d u c t s . The a d d u c t s a r e p r e p a r e d f r o m a m e t h a n o l or e t h a n o l s o l u t i o n of a c r u d e sodium o r p o t a s s i u m s a l t o f n o v o b i o c i n a f t e r c o n c e n t r a t i o n t o form a s u p e r s a t u r a t e d s o l u t i o n of n o v o b i o c i n s a l t i n a l c o h o l a n d r e c o v e r i n g t h e r e s u l t i n g p r e c i p i t a t e d c r y s t a l l i n e a l c o h o l a d d u c t of t h e n o v o b i o c i n s a l t . A s u p e r s a t u r a t e d s o l u t i o n may a l s o b e a c c o m p l i s h e d by a d d i t i o n o f f u r t h e r c r u d e n o v o b i o c i n s a l t . C r y s t a l l i z a t i o n may b e a i d e d by s e e d i n g w i t h n o v o b i o c i n s a l t c r y s t a l s a n d / o r by c o o l i n g . Scheme 1 i l l u s t r a t e s t h e p r o c e d u r e . SCHEME 1
337 g crude novobiocin i n 3 -!methanol; a g i t a t e , and add 135 g d e c o l o r i z i n g carbon ( N o r i t A). S t i r 15 min, f i l t e r
n
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Carbon cake, wash with 370 rnl methanol
F i I tr a t e
[----L---TI
Carbon cake, discarded
Combine
I
A g i t a t e , add 202 r n l of 2.54 N s o h . o f NaOH i n methanol Then, evaporate h vacua t o ca. 2.4 -!. t o give pH 8.25. Cool t o 5' with s t i r r i n g . F i l t e r .
A
Methanol adduct o sodium novobiocin c r y s t a l s . Wash s e v e r a l times w i t h c o l d methanol, then a i r - d r y a t 25'. Y i e l d 175 g.
[I
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Conc. t o 8.5 Cool t o 5
2nd crop c r y s t a l s 52 g a i r - d r y
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3 r d crop c r y s t a l s 62 g a i r - d r y
114 G. K a c ~ k ar e~p o~r t e d i n g r e a t e r d e t a i l on h i s i s o l a t i o n a n d p u r i f i c a t i o n methods, t h i s time i n c l u d i n g a l u m i n a c h r o m a t o graphy a l o n g with water-solvent d i s t r i b u t i o n s . The d e t a i l s a r e g i v e n i n Schemes 2 a n d 3 . The s t a r t i n g m a t e r i a l a f t e r t h e a l u m i n a c h r o m a t o g r a p h y must b e a t l e a s t 7 % p u r e or n o a c c e p t a b l e c r y s t a l l i n e p r o d u c t i s o b t a i n a b l e 5 8 . W a l l i ~ kr e~p o~ r t e d t h e same i s o l a t i o n p r o c e d u r e s as Kaczka.
SCHEME 2 Staqe I, Extraction.
-
Whole b r o t h a d j . t o pH 9.0 ( c o n t g . antibiotic/,!) and f i l t e r e d
80-90 mg p u r e
F i l t r a t e + 2.28 k g Hy’ilo S u p e r c e l p e r 380 1; s l o w l y a c i d i f y t o pH 2.0 w i t h H C I . A g i t a t e 10 rnin, f i l t e r , wash c a k e w i t h water
Cells d i k c a r d e d
H
Washed f i l t e r c a k e (0.2-0.3% p u r e , e x c l u d i n g f i l t e r - a i d )
I
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I E x t r a c t 2X w i t h 85% aq. %OH,
S p e n t cake
1/10 v o l . e a c h time; a t pH 9.0
Methanol e x t r a c t (80% r e c o v . o f b r o t h b i o a c t i v i t y ; 1-1.57; p u r e s o l i d s ) C o n c e n t r a t e t o 1/10 v o l . A d j u s t t o pH 9.0 E x t r a c t 2X w i t h e q u a l v o l s . o f n - b u t a n o l ( A p p a r e n t d i s t r i b . r a t i o is CQ.40:l)
S p e n t aqueous C o n c e n t r a t e t o 1/10 vol. Add t o 15 v o l s . of H20, pH 9.0 end f i l t e r a i d a t ca. 0.5 9/3.8 ,! o r i g i n a l b r o t h v o l . A d j u s t s l o w l y t o pH 2.0 w i t h HC1; f i l t e r F i l t e r cake (10-127: s o l i d s p u r i t y , e x c l u d i n g f i l t e r - a i d )
Aq. f i l t r a t e d i s c a r d e d
D r i e d i n vacua a t 4OoC Milled and t r i t u r a t e d with petroleum e t h e r till c o l o r l e s s
1
F i l t e r cake P e t . e t h e r washes d i s c a r d (12-15% s o l i d s p u r i t y , e x c l u d i n g f i l t e r a i d )
1
E x t r a c t w i t h d r y e t h a n o l u n t i l e x t r a c t s a r e v. It. y e l l o w Combine e x t r a c t s and c o n c e n t r a t e t o s o h . o f 15-20?; s o l i d s
Conc. e t h a n o l s o l u t i o n (20-30:; s o l i d s p u r i t y )
115
SCHEME 3 S t a q e 11, P u r i f i c a t i o n .
C o n c e n t r a t e d e t h a n o l s o l u t i o n (15-20:; o f 20-30:; p u r i t y )
solids
Chromatograph on a c i d - w a s h e d alumina (50:l r a t i o t o s o l i d s )
I m p u r i t i e s remain on A1203 ColUm
A c t i v e e l u a t e s i n 2.5-3 column void-volumes (95% of t h e a c t i v i t y )
I , Concentrate t o CU. 5%s o l i d s Add s l i g h t l y more t h a n e q u a l v o l . of H20 and a l l o w t o crystallize
C r y s t a l h e novobiocin (50-609; p u r e )
I
F i l t r a t e s t i l l h a s up t o 15%of o r i g . a c t i v i t y
D i s s o l v e i n dry a c e t o n e t o 30% s o h . T r e a t w i t h 2X w e i g h t o f Darco G-60; f i l t e r
Darco a d s o r b a t e washed repeatedly with acetone
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J n
Combined f i l t r a t e and washes c o n c e n t r a t e d t o 5% s o l i d s Add p e t . e t h e r t o t u r b i d i t y and allow t o c r y s t a l l i z e
C r y s t a l l i n e novobiocin (90-95:; r e c o v e r y o f b i o a c t i v i t y , 90-100% p u r i t y )
I
Spent f i l t r a t e
H. J . G i 1 l i 1 - 1 ~ d~ e s c r i b e d a n improvement i n p r e p a r a t i o n of p u r e c r y s t a l l i n e monosodium n o v o b i o c i n which i n v o l v e d d i s s o l v i n g n o v o b i o c i n f r e e a c i d o f 8 0 - 9 0 % p u r i t y i n a mixed o r g a n i c s o l v e n t c o n t a i n i n g 2 p a r t s by volume o f an a l c o h o l s u c h as metha n o l t o 1 p a r t by volume o f an a r o m a t i c s o l v e n t s u c h as b e n z e n e , and a d d i n g s o d i u m m e t h o x i d e t o pH 7 . 2 t o d i r e c t l y p r e c i p i t a t e c r y s t a l l i n e monosodium n o v o b i o c i n , i n e x c e s s of 9 0 % y i e l d , w i t h good c o l o r a n d h i g h p u r i t y . D e t a i l s o f o n e e x a m p l e are g i v e n i n Scheme 4. I. C h a i e t 5 B d e s c r i b e d some m o d i f i c a t i o n s i n t h e Merck p u r i f i c a t i o n and c r y s t a l l i z a t i o n of novobiocin, t h e p r o g r e s s of which was m o n i t o r e d by t h e s t a n d a r d c u p - p l a t e d i f f u s i o n a s s a y ,
116 SCHEME 4
1175 g novobiocin f r e e a c i d , p u r i t y 88.5X D i s s o l v e i n mixt. of 2350 m l T t h a n o l + 1175 m l benzene, w i t h a g i t a t i o n , a t 30 F i l t e r on Buchner f u n n e l through f i l t e r p a p e r c o a t e d with 15 g Supercel f i l t e r - a i d Rinse f i l t e r pad w i t h 450 rnl of 2 : l rnethanol-benzene
_ I _
D i s c a r d f i l t e r cake
I
F i 1t r a te p l u s f r&h l y -p r e p a r e d , f i l t e r e d sodium methoxide (120 g) i n methanol (1200 m l ) , added over 1/2 h r , w i t h s g i t a t i o n , t o pH 7.2 t o 7.3 C r y s t a l l i z a t i o n occurs. A g i t a t e !he s l u r r y f o r 1 h r st 25-30 , t h e n add 8800 m l benzene o v e r 10 min, and a g i t a t e f o r 2nd h r a t 25-30'
n
C r y s t . monosodium s a l t , washed 4X 500 m l benzene; dry a t 50' in VIUIUO. Yield, 983 g ( 9 1 . 4 % of t h e o r y ) o f 99% p u r i t y
F i l t e r through p a p e r on a Lapp p o r c e l a i n f i l t e r Spent f i l t r a t e
u s i n g 8aciLlua mcgathefiium ATCC 9885 as t h e t e s t o r g a n i s m . The p u r e a n t i b i o t i c a r b i t r a r i l y h a s a m i c r o b i o l o g i c a l a c t i v i t y of 5000 u n i t s p e r mg. I n comparison w i t h t h e p r o c e d u r e s d e s c r i b e d e a r l i e r by K a ~ z k a ~t h~e , m o d i f i c a t i o n s of C h a i e t were more d i r e c t , r e q u i r e d l e s s h a n d l i n g of a c t i v e m a t e r i a l , took', less t i m e t o c a r r y t h r o u g h , and p e r m i t t e d o b t a i n i n g s u b s t a n t i a l l y p u r e c r y s t a l l i n e n o v o b i o c i n even from c r u d e c o n c e n t r a t e s from which n o c r y s t a l l i n e p r o d u c t c o u l d p r e v i o u s l y be o b t a i n e d . They f e a t u r e d t h e u s e of a c o u n t e r c u r r e n t e x t r a c t i o n a p p a r a t u s t o h a n d l e d i s t r i b u t i o n s between aqueous s o l u t i o n s a n d amyl a c e t a t e , f i r s t a t pH 6 . 5 , t h e n a t pH 1 0 , f o l l o w e d by c r y s t a l l i z a t i o n from a c a r e f u l l y s e l e c t e d m i x t u r e of m e t h a n o l - w a t e r - a c e t i c a c i d (10:4: 1 v/v). Details of one example are g i v e n i n Scheme 5 . I n a n o t h e r example , Scheme 6 , p u r i f i c a t i o n of 2 5 % c r u d e n o v o b i o c i n was a c h i e v e d by a d s o r p t i o n and e l u t i o n from an A m b e r l i t e XE 98 column, r e m o v a l of i m p u r i t i e s on an a c i d - w a s h e d a l u m i n a column a n d c r y s t a l l i z a t i o n from e t h a n o l - w a t e r - a c e t i c a c i d . F . J. d e s c r i b e s e s s e n t i a l l y t h e same m a j o r i s o l a t i o n p r o c e d u r e as C h a i e t , w i t h t h e a d d i t i o n a l claims t h a t i n p l a c e of amyl a c e t a t e as e x t r a c t a n t (which i s p r e f e r r e d ) , one can a l s o u s e m e t h y l i s o b u t y l ketone, e t h y l a c e t a t e , isoamyl a l c o h o l o r n-butyl a c e t a t e ; a l s o , i n p l a c e o f 5 % m e t h a n o l i n t h e aqueous b u f f e r s o l u t i o n one
117 SCHEME 5 Whole fermentation b r o t h , pH 7.0-8.0 Add diatomaceous, e a r t h , f i l t e r
11
F i l t e r cake, wash w i t h warm water, b r o t h volume; f i l t e r
f
Spent f i l t e r cake
11
Filtrate
1/5 o f o r i g .
'
4
Was h ings
Combined aq. s o l n . i s e x t d . a t pH 6.5 i n a countercurrent e x t r a c t i o n apparatus w i t h 2 X 1/10 v o l . o f amyl acetate
I
Amy1 acetate e x t r a c t s are extd. i n a countercurrent apparatus w i t h 2 X 1/5 vol. o f a l k a l i n e b u f f e r soln., pH 9.5-10 (10 g sodium carbonate -c 8 g sodium bicarbonate p e r 1 ?.! of 5% aqueous methanol or NHkOH i n 5% methanol)
I1
Aqueous b u f f e r e x t r a c t s Conc. by evapn., a d j u s t t o pH 2.3-2.6 w i t h H C 1
I
n '
I
Spent organic s o l v e n t
Filter
Ppt. o f novobiocin acid; weigh. Dissolve i n methanol t o give 10% s o l i d s , then add water and a c e t i c a c i d s o t h a t f i n a l p r o p o r t i o n s by v o l . o f methanol, water, a c e t i c a c i d are 10:4:1.
n '
Spent aqueous
1
Spent f i l t r a t e d i s c a r d
Filter
Pure c r y s t a l s ; wash w i t h methanol-water
1
Spent f i l t r a t e
( 1 0 : 4 v/v) Dry in uucuo below 45'
can a l s o u s e N,N-dimethylformamide
o r ethylene glycol.
J . When n o v o b i o c i n i s e x p o s e d t o a l k a l i , s u c h as a t pH during a recovery process, inactive isonovobiocin (with c a r b a m y l g r o u p i n t h e 2 - i n s t e a d o f t h e 3- p o s i t i o n ) i s f o r m e d De Vries60 r e p o r t e d t h a t a n a d m i x t u r e i n s u b s t a n t i a l amounts. of n c v o b i c c i n a n d i s c n o v o b i o c i n c o u l d b e u p g r a d e d b y t r e a t m e n t with acetone-water ( 1 . 5 : l v/v). The a m o u n t of isomer p r e s e n t 10,
118 SCHEME 6
1 1 e t h a n o l s o l u t i o n o f novobiocin (25% p u r i t y ) 2 1 water, a d j u s t e d t o pH 2.0 w i t h H C 1 15 g diatomaceous earth; f i l t e r
1
n
F i l t e r cake S l u r r y w i t h NH,OH
I
Spent f i l t r a t e soh.,
a t pH 10, f i l t e r I
600 m l f l l t r a t e . About 400 ml o f t h i s passed through column contg. 150 m l Amberlite XE 9 8 r e s i n (Cl-); r e s i n i s washed w i t h 85% methanol
Spent f i l t e r cake
I
II
Spent f i l t r a t e and washes
Resin adsorbate E l u t e w i t h methanol-conc. HC1 (85:16 v/v)
E l u a t e contg. s o l i d s o f 74% p u r i t y 2 vols. o f water t o p p t .
Spent r e s i n
+
I
Filter Precipitate i s dissolved i n ethanol, and chromatographed on 20 g o f acid-washed alumina
I
Spent f i l t r a t e
I
1
Alumina, contg. i m p u r i t i e s g l a c i a l a c e t i c acid, and slow a d d i t i o n o f water t o c r y s t a l l i zation. F i l t e r
E
C r y s t a l s o f pure novobiocin
Spent f i l t r a t e
can be d e t e r m i n e d by C r a i g d i s t r i b u t i o n a n a l y s i s o r by t h e d i f f e r e n c e between t h e U.V. a s s a y a n d t h e b i o a s s a y u s i n g Sakcina 1 u t e a as t h e t e s t o r g a n i s m . The f o l l o w i n g e x a m p l e , summarized i n Scheme 7 , was p r e s e n t e d . K. Wolf e t r e p o r t e d t h a t a t t e m p t s t o e l u t e novob i o c i n from q u a t e r n a r y amine a n i o n - e x c h a n g e r e s i n s (upon which i t h a s been a b s o r b e d ) have r e s u l t e d i n c o m m e r c i a l l y u n s a t i s f a c t o r y r e c o v e r i e s of t h e a n t i b i o t i c . Elution with aqueous a l k a l i r e s u l t s i n c o m p l e t e i n a c t i v a t i o n of t h e a n t i b i o t i c , as d o e s e ? l i t i o n w i t h a c i d s , though t o a somewhat l e s s d e g r e e .
119
SCHEME 7 Fermentation beer (13 runs) E x t r a c t w i t h b u t y l acetate
II
B u t y l acetat! e x t r a c t p l u s aq. Na2C03 s o h . t o pH 10
Spent aqueous
D
I
Spent s o l v e n t
Aqueous e x t r a c t adjusted t o pH 5.5 Acetone added f o r c r y s t a l l i z a t i o n
I Filter
n
C r y s t a l s contg. 358.2 k g novobiocin and 39.9 k g i s o n o v o b i o c i n ( r a t i o 9:1), p l u s other impurities
I
Spent f i 1t r a t e
Wash with 65% aqueoua acetone (1017 a ) , then w i t h 60% aqueous acetone ( 7 6 4 a )
I
I
408.6 k g washed c r y s t a l s , 86.3% novobiocin (novobiocin t o isonovobiocin 1 3 . 5 : l )
I
Washes, c o n t a i n 6.2 k g novobiocin and 13.9 k g i s o n o v o b i o c i n ( 0 . 4 4 6 : 1)
R e c r y s t a l l i z e t o remove o t h e r impurities, with or without f u r t h e r washes with 60X acetone
However, e l u t i o n w i t h t h e i r w a t e r - m i s c i b l e o r g a n i c s o l v e n t - w a t e r s a l t e l u a n t gave high y i e l d s and no i n a c t i v a t i o n of t h e a n t i biotic. R e s i n r e g e n e r a t i o n was g e n e r a l l y u n n e c e s s a r y s i n c e a k i n d of a u t o r e g e n e r a t i o n of t h e r e s i n o c c u r s w i t h t h e i r e l u a n t s . One e x a m p l e i s g i v e n b e l o w i n T a b l e 1. Two a of Dowex 1-X2 r e s i n , i n t h e c h l o r i d e c y c l e , was added t o a m i x t u r e c o n t a i n i n g sodium novobiocin and t h e m i x t u r e stirred for 3 hr. The r e s i n was s e p a r a t e d b y f i l t r a t i o n a n d w a s h e d w i t h water. 4 0 m l a l i q u o t s of t h e r e s i n were t h e n t a k e n , p l a c e d i n c h r o m a t o g r a p h i c c o l u m n s a n d e l u t e d . The p e r c e n t a g e o f n o v o b i o c i n e l u t e d w i t h t h e v a r i o u s e l u a n t s i s shown i n T a b l e 1. The e l u a n t s were made u p b y d i s s o l v i n g t h e i n d i c a t e d quant i t y of s a l t i n water, d i l u t i n g t o 6 0 m l a n d t h e n d i l u t i n g t h i s a q u e o u s s a l t s o l u t i ' o n t o 200 m l w i t h t h e i n d i c a t e d o r g a n i c s o l vent
.
I n a n o t h e r e x a m p l e , t o 2 II o f n o v o b i o c i n was a d d e d 2 0 0 m l of Dowex mixture s t i r r e d for 1 h r . The r e s i n w i t h 800 m l water. Samples o f LO m l
a m i x t u r e c o n t a i n i n g sodium 1-2X r e s i n (Cl-), a n d t h e was f i l t e r e d o f f a n d w a s h e d of t h i s r e s i n were t a k e n
120 TABLE 1 E l u t i o n o f Novobiocin f r o m Oowex 1-X2 Resin Novobiocin eluted ( pe r cen t
Volume (ml)
-
Eluant Methanol/ammonium c h l o r i d e (10.7 g) Isopropanol/ammonium c h l o r i d e (10.7 g) O i met h y 1 s u lf o x i de /ammoni um ch 1o r ide (10.7 g) Ethanol/ammonium c h l o r i d e (10.7 g) Acetone/potassium i o d i d e (33.2 g) Ethylene glycol/ammonium c h l o r i d e (10 g) Oioxane/ammonium c h l o r i d e (10 g) Methanol/calcium c h l o r i d e (22.2 g) Methanol/dirnethylamine h y d r o c h l o r i d e (16.3 9) Methanol/lithium c h l o r i d e (0.2 g)
140 150
99.8 99.3
150 140 140 120 116 120
98.7 99.0 85.0 39.7 66.3 84.9
120 2,000
89.7 85 .O
a n d e a c h e l u t e d column-wise w i t h 4 5 m l o f t h e p a r t i c u l a r e l u a n t , as shown i n T a b l e 2 . TABLE 2 E l u t i o n o f Novobiocin f r o m Oowex 1 4 2 Resin Novobiocin e l u t e d (percent)
Eluant
70% methanol 5% ammonium c h l o r i d e (aq. ) Aq. s o l n . contg. 70% methanol by vol., 5% (w/v) ammonium c h l o r i d e Aq. s o h . contg. 90% m t h a n o l by vol., 3% (w/v) ammonium c h l o r i d e Aq. s o l n . contg. 98% methanol by vol., 5% (w/v) l i t h i u m c h l o r i d e
0 0
and
85.4 and 100 and 96.2
1. P u b l i c a t i o n s on s c a l e - u p of a column a d s o r p t i o n p r o c e s s by computer s i m u l a t i o n and on computer s i m u l a t i o n o f p l a n t - s c a l e mu 1t i column a d s o r p t i o n p r o c e s s e s u n d e r p e r i o d i c countercurrent d e s c r i b e t h e m a t h e m a t i c s and groundwork of t h e n e x t n o v o b i o c i n i s o l a t i o n p r o c e s s . To e l i m i n a t e t h e c o s t o f r n y c e l i a l f i l t r a t i o n and t h e i n s t a b i l i t y of t h e a n t i b i o t i c i n t h e f i l t e r c a k e , B e l t e r e t al.54 d e v e l o p e d a p e r i o d i c c o u n t e r c u r r e n t w h o l e - b e e r p r o c e s s f o r r e c o v e r y of novobiocin. T h i s p r o c e d u r e employed some of t h e t e c h n o l o g y d e v e l o p e d i n t h e l a t e 1 9 5 0 ' s f o r t h e a d s o r p t i o n of t h e b a s i c a n t i b i o t i c s t r e p t o m y c i n an a c a t i o n i c ion-exchange r e s i n from a whole, u n f i l t e r e d b e e r , t h u s e l i m i n a t i n g l o s s e s and c o s t s o f the f i l t r a t i o n step. To i s o l a t e t h e a c i d i c n o v o b i o c i n , whole b e e r from t h e f e r m e n t o r was p a s s e d o v e r a h i g h c a p a c i t y v i b r a t i n g s c r e e n t o remove h u l l s , c h a f f a n d o t h e r i n s o l u b l e m a t e r i a l s t h a t might f o u l t h e r e s i n - b e d . The s c r e e n e d b e e r was t h e n con-
1 21 t a c t e d w i t h t h e a n i o n i c - e x c h a n g e r e s i n - - D o w e x 21K--in a s e r i e s o f t h r e e s p e c i a l l y d e s i g n e d , w e l l - m i x e d c o l u m n s . Whole b e e r was i n t r o d u c e d a t t h e b o t t o m o f t h e f i r s t column, and e x i t e d from t h e t o p t o t h e b o t t o m o f t h e s e c o n d column, e t c . Each o f t h e c o l u m n s was e q u i p p e d w i t h a s p e c i a l h e a d s c r e e n s i z e d t o p e r m i t passage of mycelia and o t h e r i n s o l u b l e s b u t t o r e t a i n t h e r e s i n particles. R e s i n i s u n i f o r m l y d i s p e r s e d i n t h e column by v i g orous a g i t a t i o n a b o v e a c e r t a i n d e g r e e o f m i x i n g . The f i r s t or l e a d column i s removed f r o m t h e s e r i e s p e r i o d i c a l l y , w a s h e d f r e e o f i n s o l u b l e s a n d e l u t e d i n t h e f i x e d b e d made w i t h a m e t h a n o l i c ammonium c h l o r i d e s o l u t i o n . This elutes t h e adsorbed novobiocin a n d s i m u l t a n e o u s l y r e g e n e r a t e s t h e r e s i n t o t h e c h l o r i d e form. The r e s u l t i n g e l u a t e s a r e s u b s e q u e n t l y p r o c e s s e d t o c r y s t a l l i n e a n t i b i o t i c by c o n v e n t i o n a l p u r i f i c a t i o n methods. Each o f t h e u n e l u t e d columns i s advanced o n e p o s i t i o n a n d t h e f r e s h l y e l u t e d column i s p l a c e d i n t h e t r a i l p o s i t i o n o f t h e t r a i n s o t h a t 3 columns are c o n s t a n t l y i n t h e a d s o r p t i o n c y c l e w h i l e o n e column i s b e i n g e l u t e d . The b e e r f e e d c y c l e i s t h e n r e p e a t e d . E f f l u e n t f r o m t h e t h i r d c o l u m n , s u b s t a n t i a l l y d e v o i d of n o v o b i o c i n , i s d i s c a r d e d . A s s a y s f o r n o v o b i o c i n were d o n e b y m e a s u r i n g U.V. a b s o r p t i o n of b u t y l a c e t a t e e x t r a c t s a t 3 3 8 nm. The U . V . a s s a y was more p r e c i s e a n d s i m p l e r t o c o n d u c t t h a n b i o a s s a y s or p a p e r grams. D i s t r i b u t i o n o f r e s i d e n c e times w i t h i n t h e c o l u m n s , s p a c e v e l o c i t y a n d v o l u m e r a t i o were c a r e f u l l y a d j u s t e d t o g i v e maximum e x t r a c t i o n e f f i c i e n c y . An o v e r a l l y i e l d i n c r e a s e o f 5 7 % was o b t a i n e d a t f u l l scale o p e r a t i o n o f t h i s t y p e of p r o c e s s i n g o v e r t h e c o n v e n t i o n a l f i 1t r a t i o n m e t h o d . 1.4 Literature -
Cited
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1 4*
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28.
L. A .
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C . H . S h u n k , C . H . S t a m m e r , E . A . K a c z k a , E . W a l t o n , C . F. S p e n c e r , A . N . W i l s o n , J . W . R i c h t e r , F. W . H o l l y a n d K . F o l k e r s , J . A m e r . Chem. S o c . 78 (1956) 1770-1771.
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C. H . Stammer, E. Walton, A. N . Wilson, R . W . Walker, N . R . T r e n n e r , F . W . H o l l y a n d K . F o l k e r s , J . A m e r . Chem. S O C . 80 (1958) 137-140.
31.
W . T. S o k o l s k i , N . J . E i l e r s a n d J . W . A n n u a l , (1956-57) 1031-1034.
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W . D. Celmer a n d B . 1 6 , 1959.
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E . W a l t o n , J. 0. R o d i n , C. H . Stammer, F. W . H o l l y a n d K . F o l k e r s , J. A m e r . Chem. S O C . 78 (1956) 5454-5455.
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Kominek, A n t i m i c r o b i a l A g e n t s a n d Chemo. 1 (1972) 123-124. A m e r . SOC. f o r M i c r o b i o l .
K.
Koe, U.S.
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E. A. K a c z k a , C . H. S h u n k , J . W . R i c h t e r , F . J . Wolf, M . Gasser a n d K . F o l k e r s , J . A m e r . 4125-4127.
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A. K u c e r s a n d N . M . B e n n e t t , The Use o f A n t i b i o t i c s , 2 n d Ed. (1975) 325-331. J . B. L i p p i n c o t t Company, P h i l a d e l p h i a
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L. D . S a b a t h , D . A . G e r s t e i n a n d M . F i n l a n d , A n t i m i c r o b i a l A g e n t s a n d C h e m o t h e r a p y (1968), A m e r . SOC. for M i c r o b i o l . (1969) 398-404.
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(1956-57)
J . M a r t i n , F. R . H e i l m a n , D . R . N i c h o l s , W . E . Wellman and J . E. Geraci, J.A.M.A. 162 (1956) 1150-1153.
L. Masson,
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1 24 46.
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R . B. W a l t o n , L. E. M c D a n i e l a n d H. B . W o o f r u f f , Developm e n t s i n I n d u s t r i a l M i c r o b i o l o g y 3 ( 1 9 6 2 ) 370-375. Plenum P r e s s , New York.
48.
F. J. Wolf a n d R. N e s c o t , A n t i b i o t i c s A n n u a l , ( 1 9 5 6 - 5 7 ) 1035-1039.
49.
K . T s u j i a n d J . H . R o b e r t s o n , J. C h r o m a t o g r . 9 4 ( 1 9 7 4 ) 245-253.
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0 . N i s h i j i m a , 0. Matano a n d T . K a s h i w a , Noyaku K e n s a s h a Hokoku, 11 ( 1 9 7 1 ) 1 3 3 - 1 3 5 .
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T. I k e k a w a , F . Iwai, E . A k i t a a n d H . Umezawa, J . A n t i b i o tics 1 6 ( 1 9 6 3 ) 56-57.
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P . A . B e l t e r , F . L. C u n n i n g h a m a n d J . W . a n d B i o e n g . 1 5 ( 1 9 7 3 ) 533-549.
55.
E. A . K a c z k a , U.S.
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H. W a l l i c k , C a n a d i a n P a t e n t 6 4 4 , 3 9 2 ; J u l y 1 0 , 1 9 6 2 .
57.
J. G i l l i n , U.S.
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October 1 3 , 1959.
58.
L. C h a i e t , U.S.
P a t e n t 2,923,705;
February 2 , 1960.
59 *
F. J. W o l f , U.S.
60.
W. H.
61.
,
P a t e n t 2,895,952;
P a t e n t 3,125,566;
D e Vries, U.S.
Chem.
Chen, B i o t e c h n .
J u l y 21, 1959.
March 1 7 , 1 9 6 4 .
P a t e n t 2,931,796;
A p r i l 5, 1960.
F . J . Wolf, I . P u t t e r , G . V . Downing J r . a n d J . G i l l i n , November 3 0 , 1 9 6 5 .
U.S. P a t e n t 3 , 2 2 1 , 0 0 8 ;
C h e n , J . A. B u e g e , F. L. C u n n i n g h a m a n d J . I . N o r t h a m , I . E EC. P r o c e s s D e s i g n a n d D e v e l o p . 7 ( 1 9 6 8 ) 2 6 - 3 1 .
62.
J. W .
63.
J . W . C h e n , F. L. C u n n i n g h a m a n d J . A . Buege, I . E EC. P r o c e s s D e s i g n a n d D e v e l o p . 11 ( 1 9 7 2 ) 4 3 0 - 4 3 4 .
64.
0. K . S e b e k a n d H . H o e k s e m a , J . A n t i b i o t i c s 2 5 ( 1 9 7 2 ) 4344 3 6 ; German P a t e n t 2 , 0 2 6 , 6 8 7 ; D e c e m b e r 1 0 , 1 9 7 0 .
65.
L. D o l a k , 0. K . S e b e k . C . L e w i s a n d H . H o e k s e m a , 1 2 t h I n t e r s c i e n c e Conf. Antimicrob. Agents and Chemother. (1972) A b s t . No. 5 3 .
1 25 66.
C. F. S p e n c e r a n d E. W a l t o n , A u s t r a l i a n P a t e n t 2 2 1 , 8 8 2 ( a p p l i c a t i o n No. 2 6 , 8 9 0 ) ; J u n e 3 , 1 9 5 9 .
67.
K . Okumura, J . P h a r m a c e u t i c a l S O C . J a p a n 80 ( 1 9 6 0 ) 5 2 5 - 5 3 2 . A l s o , 8 1 ( 1 9 6 1 ) 308, 312, 316, 323, 453 a n d 1482.
68.
B . P . V a t e r l a u s , K . Doebel, J . Kiss, A. I . R a c h l i n a n d H . S p i e g e l b e r g , H e l v . C h i m . A c t a 47 ( 1 9 6 4 ) 3 9 0 - 3 9 8 .
126
2. Dihydronovobiocin ( 5 ) 2.1 I n t r o d u c t i o n 2.1.1
Producing organism
D i h y d r o n o v o b i o c i n was p r o d u c e d b i osyn;the t i c a l l y b y t h e a d d i t i o n o f 4-hydroxy-3-(3-methylbutyl)benzo~c a c i d t o t h e f e r m e n t a t i o n medium w i t h Sthtp.tOmyCeA ~ p h t h o i d t h ~ . Brief c h e m i c a l d e s c r i p t i o n
2.1.2
D i h y d r o n o v o b i o c i n ( 5 ) was f i r s t p r o d u c e d b y t h e c a t a l y t i c h y d r o g e n a t i o n o f n o v o b i o c ' i n , w i t h t h e u p t a k e of o n e mole o f hydrogen and the s a t u r a t i o n of t h e side-chain i n t h e A I t has m.p. 1 6 3 - 1 6 5 ', w i t h p o t e n t i o m e t r i c t i t r a t i o n a n d o p t i c a l a c t i v i t y v a l u e s s i m i l a r t o t h o s e of n o v o b i o c i n . The U.V. a b s o r p t i o n max. i s 3 2 8 nm ( i n 0 . 0 1 N H2S0 , 7 0 % e t h a n o l ) . Its t o t a l c h e m i c a l s y n t h e s i s was d e s c r i b e d l h . S t r u c t u r a l formula
2.1.3
T h i s i s shown i n F i g u r e 1, i n t h e n o v o b i o c i n s e c t i o n .
2 . 2 Summary
o f t h e r a p e u t i c use
D i h y d r o n o v o b i o c i n h a s LDso v a l u e s a n d a n t i b a c t e r i a l p r o p e r t i e s in with0 a n d in viva n e a r l y i d e n t i c a l t o t h o s e o f n o v o biocin. I t s t o x i c i t y i n mice was f o u n d 8 t o b e 325 mg/kg i . v . a n d a b o u t 1 1 0 0 rng/kg p . 0 . I t is s t a b l e i n aqueous v e h i c l e s and is r e a d i l y a d s o r b a b l e and b i o l o g i c a l l y a c t i v e . This c o n t r a s t s with the i n s t a b i l i t y of novobiocin i n s o l u t i o n and unreadilya b s o r b e d s t a b l e c r y s t a l f o r m o f n o v o b i o c i n 3.
2.3 E x t r a c t i o n , 2.3.1
s e p a r a t i o n and p u r i f i c a t i o n
,Detection
A PC m e t h o d h a s b e e n d e s c r i b e d 5 f o r d i s t i n g u i s h i n g d i h y d r o n o v o b i o c i n ( R f 0 . 6 5 ) f r o m n o v o b i o c i n ( R 1.0) a n d f r o m descarbamylnovobiocin ( 3 ) (Rf 1.13). N o v o g i o c i c a c i d (5)h a s a n Rf s i m i l a r t o t h a t o f d i h y d r o n o v o b i o c i n ( 5 ) . D e s c a r b a m y l d i h y d r o n o v o b i o c i n , w h i c h is p r o d u c e d f r o m d i h y d r o n o v o b i o c i n b y a l k a l i n e degradation a l s o has s l i g h t l y g r e a t e r mobility than i t s p a r e n t compound, w h i l e d i h y d r o n o v o b i o c i c a c i d h a s a c o r r e s p o n d i n g l y lower m o b i l i t y
.
S e n s i tt ~ 1 d . esc~ r i b e d t h e a n a l y s i s o f a m i x t u r e of n o v o b i o c i n and dihydronovobiocin, b u t i t d i d not d i f f e r e n t i a t e i s o novobiocin. 2.3.2
I s o l a t i o n of dihydronovobiocin
D i h y d r o n o v o b i o c i n was i s o l a t e d f r o m f e r m e n t a t i o n b r o t h s as m e n t i o n e d a b o v e b y7. S u c c e s s f u l p u r i f i c a t i o n of s o d i u m d i h y dronovobiocin by an ion-exchange procedure h a s been r e p o r t e d g . To a m i x t u r e c o n t a i n i n g t h e a n t i b i o t i c was a d d e d 5 0 m l o f Dowex
1 27 1 - X 2 r e s i n ( C l - ) y a n d t h e m i x t u r e was s t i r r e d f o r 3 h r . The r e s i n was t h e n r e m o v e d b y f i l t r a t i o n a n d w a s h e d w i t h water. A 20 m l a l i q u o t of r e s i n was e l u t e d c o l u m n - w i s e w i t h f i v e 20 r n l p o r t i o n s of a n a q u e o u s s o l u t i o n c o n t a i n i n g 7 0 % b y v o l u m e m e t h a n o l a n d 5 % ( w / v ) ammonium c h l o r i d e . A t o t a l of 9 7 . 4 % of t h e a n t i b i o t i c was e l u t e d .
2.4 Literature 1.
Cited
H . H o e k s e m a , J . L. J o h n s o n a n d J. W . H i n m a n , J. Amer. C h e m . SOC. 77 ( 1 9 5 5 ) 6710-6711.
2.
J . W . Hinman, E. L. C a r o n a n d H . H o e k s e m a , J. Amer. Chem. SOC. 79 ( 1 9 5 7 ) 3 7 8 9 - 3 8 0 0 .
3.
J . D. M u l l i n s a n d T. J. Macek, E d . ) 49 ( 1 9 6 0 ) 2 4 5 - 2 4 8 .
4.
R . B . Walton, L. E. M c D a n i e l a n d H . B . W o o d r u f f , D e v e l o p m e n t s
J. Amer.
Pharm. Assoc.
i n I n d u s t r i a l M i c r o b i o l o g y 3 ( 1 9 6 2 ) 370-375. New York. 5.
(Sci.
Plenum P r e s s ,
F . J. Wolf a n d R . Nescot, A n t i b i o t i c s A n n u a l , ( 1 9 5 6 - 5 7 ) 1035-1039. P. S e n s i , G. 1611-1613.
7.
K. F o l k e r s and H. B. Woodruff, 11, 1 9 6 3 .
8.
G . R o l l a n d , P . S e n s i , G . A. De F e r r a r i , G. M a f f i i , M. T . T i m b a l a n d L. G . S i l v e s t r i , 11 F a r m a c o ( E d . S c i . ) 11 ( 1 9 5 6 ) 549-561.
9.
F. J . W o l f , I . P u t t e r , G . V . Downing Jr. a n d J . G i l l i n , U.S. P a t e n t 3 , 2 2 1 , 0 0 8 ; November 3 0 , 1 9 6 5 .
LO.
G.
G a l l o a n d L.
6.
C h i e s a , A n a l . Chem. U.S.
29 ( 1 9 5 7 )
P a t e n t 3,093,549;
June
B . P . V a t e r l a u s , K . D o e b e 1 , J . Kiss, A. I . R a c h l i n a n d H . S p i e g e l b e r g , H e l v . Chim. Acta 4 7 ( 1 9 6 4 ) 3 9 0 - 3 9 8 .
1 28
3 . Me thoxynovobi o c i n ( 7 1 3 . 1 Introduction -
3.1.1
Producing organism
was f i r s t r e p o r t e d t o be p r o d u c e d Methoxynovobiocin (1) b i o s y n t h e t i c a l l y when 4-me thoxy- 3-0-methyl-2 - b u t e n y l ) b e n z o i c a c i d was added a s a g r e c u r s o r t o a f e r m e n t a t i o n b r o t h o f SfAeptomyced d p h e k o i d e d l ’ The y i e l d of a n t i b a c t e r i a l a c t i v i t y was d o u b l e d , w i t h a p p r o x i m a t e l y e q u a l amounts o f methoxynovobiocin and novobiocin being p r e s e n t i n t h e b r o t h .
.
3.1.2
Brief chemical d e s c r i p t i o n
Methoxynovobiocin i s s o l u b l e i n m e t h a n o l , e t h a n o l , acet o n e and e t h y l a c e t a t e . A s o l u t i o n o f t h e a n t i b i o t i c had t h e following c h a r a c t e r i s t i c peaks2: nm 304 254 240 230 225
(max.) (rnax.) (sh.) (min.) (end. abs.)
3U5 308 269 246 287
No d i f f e r e n c e s i n U . V . s p e c t r u m between measurements made i n n e u t r a l and a l k a l i n e s o l u t i o n s were o b s e r v e d as would be e x p e c t e d , i n c o n t r a s t t o n o v o b i o c i n and o t h e r a n a l o g s w i t h t h e f r e e a c i d i c 4-hydroxyl g r o u p , which e x h i b i t a c h a r a c t e r i s t i c shift. The I . R . s p e c t r u m o f methoxynovobiocin was similar t o t h a t o f n o v o b i o c i n , e x c e p t i t showed no 3 . 1 micron phenolic-OH band, a n d some d i f f e r e n c e s i n l o n g e r w a v e l e n g t h s 2 . 3.1.3
S t r u c t u r a l formula
-1 Novobiocin, R = H -7 Methoxynovobiocin, R = CH3
129
3 . 2 Summary
o f t h e r a p e u t i c use
M e t h o x y n o v o b i o c i n was r e p o r t e d t o b e " a c t i v e a g a i n s t
S t a p h y t o c o c c u d s p . , B a c i l t u d a u b t i t i d , Paeudomonad s p . , E . c o l i , Pkoteud v u t g a n i d , S t ~ ~ e p t o c o c c udecatia, d Satmonella typhoda and t h e l i k e " , a n d t o d i f f e r somehow f r o m n o v o b i o c i n i n s p e c i f i c i t y against individual bacteria. However, n o q u a n t i t a t i v e a c t i v i t y M e t h o x y n o v o b i o c i n was s y n t h e s i z e d c h e m i d a t a were p r e s e n t e d . c a l l y a t F . H o f f m a n n - L a Roche E Co.,A.G.,Basle,as Ro 4 - 5 3 1 3 , w h e r e i t was f o u n d t o h a v e a b o u t 1 / 3 t h e a n t i b a c t e r i a l a c t i v i t y of n o v o b i o c i n ( u n p u b l i s h e d ) .
3.3 Extraction, 3.3.1
s e p a r a t i o n and p u r i f i c a t i o n
Detection
Methoxynovobiocin had a r e l a t i v e m o b i l i t y of 0 . 8 0 t o 0 . 8 4 compared t o 1 . 0 0 f o r n o v o b i o c i n and 0 . 6 0 f o r d i h y d r o n o v o b i o c i n , as d e t e r m i n e d b y d e s c e n d i n g P C 3 , b i o a u t o g r a m s w i t h B a c i t L u d a u b t i l i d a n d U.V. r e c o r d i n g s o f t h e p a p e r s t r i p c h r o m a t o g r a m s . 3.3.2
Isolation
M e t h o x y n o v o b i o c i n was i s o l a t e d a n d p u r i f i e d 2 as s u m m a r i z e d i n Scheme 8 .
3.4 L i t e r a t u r e
Cited
1. R . B . W a l t o n , L . E . M c D a n i e l a n d H . B . W o o d r u f f , D e v e l o p m e n t s i n I n d u s t r i a l M i c r o b i o l o g y 3 ( 1 9 6 2 ) 370-375. Plenum P r e s s , New York.
2.
K . F o l k e r s a n d H . B . W o o d r u f f , U.S. P a t e n t 3 , 0 9 3 , 5 4 9 ; 11, 1 9 6 3 .
3.
F . J . Wolf a n d R . Nescot, A n t i b i o t i c s A n n u a l ( 1 9 5 6 - 5 7 ) 1035-1039.
June
130 SCHEME 8
1.5 .! b r o t h contg. methoxynovobiocin
1
IF i l t e r with
(z)
filter-aid
I
TI
, I ,
F il t e r-cake E x t r a c t w i t h 1 .! and 0.5
ll
1 EtOAc
I
I
E x t r a c t 5X w i t h t o t a l o f 910 ml EtOAc
Spent ceke discarded
E t h y l acetate extract
E t h y l acetate e x t r a c t s Wash 3X w i t h 150 m l pet. ether II
I Conc. t o y e l l . o i l and a s o l i d residue. E x t r a c t with 3X 500 m l H20 and o f p e t . e t h e r
T----llResidue,
Water and pet. ether washes
,
F i l t r a t e contg. 173 mg
1
Spent aqueous
1
Ethyl ajetate extract; Spent p e t . Concentrate, diss. r e s i d u e e t h e r washes i n 100 ml EtOH, pl 7 diss.
i n 100 m l EtOH, cool, f i l t e r o f f white s o l i d White' s o l i d discarded
I
EtOH f i l t r a t e combine
L
concentrate, diss. r e s i d u e i n 25 m l warm E t W , cool, f i l t e r F i l t r a t e evap. t o dryness under NS. Diss. r e s i d . i n ca. 3 m l 0.5 M phosphate b u f f e r , pH 8.5
White' s o l i d discarded
I
Chromatograph on c h l o r o s i l o r k - t r e a t e d s i l i c i c a c i d i n column 12 mm X 60 cm. Develop w i t h phosphate b y f f e r
IT
Remainder ldiscarckd
F r a c t i o n s ( 8 ml each) 51-65 combined (125 m l ) Wash 2 X 25 m l pet. e t h e r
n
Aqueous e x t r a c t ; Plus 2 X 25 ml EtOAc
I Pet. e t h e r washes
Filter
Me thoxyibvob i o c i n precipitate
I
F i l t r a t e discard
131
4. Novobiocin a n a l o g s from Ring A and B p r e c u r s o r s
4.1 I n t r o d u c t i o n 4.1.1
Producing organisms
Both Sltneptomyceo h p h e / r o i d e A a n d S . n i u e u o have been used i n f e r m e n t a t i o n e x p e r i m e n t s t o p r o d u c e n o v o b i o c i n a n a l o g s from s u i t a b l e precursor^^'^' l l . 4.1.2
Brief chemical d e s c r i p t i o n
I n a s y n t h e t i c f e r m e n t a t i o n medium i n o c u l a t e d w i t h S .
A p h U o i d e A , Miller a n d W a l t o n l f o u n d t h a t a d d i t i o n o f p o t e n t i a l p r e c u r s o r s , s u c h as n o v o b i o c i c a c i d , p - a m i n o s a l i c y l i c a c i d and 4-hydroxy-3-(3-methyl-2-butenyl)benz0~~ a c i d , s t i m u l a t e d novobiocin yield. F u r t h e r m o r e , by a d d i n g o t h e r s u i t a b l e b e n z o i c a c i d d e r i v a t i v e s t o t h e medium', i t was p o s s i b l e t o b i o - s y n t h e s i z e 1 9 d i f f e r e n t analogs of novobiocin d i f f e r i n g i n t h e a c y l Ring A. F o l k e r s a n d Woodruff3 p r e s e n t e d a l i s t o f a c y l p r e c u r s o r s which t h e y f o u n d t o s u p p o r t p r o d u c t i o n of t h e c o r r e s p o n d i n g n o v o b i o c i n - l i k e compounds. They w e r e : 3-allyl-4-hydroxybenzoic a c i d , 3-(2 - b u t e n y l ) -4-hydroxybenzoic a c i d , 3-( a-me t h y l a l l y l ) -4hy droxyben z o i c a c i d , 3 - ( 3 -me t h y 1 2 -b u t e n y 1) 4 -e t h oxybenzo i c a c i d , 3-(3-phenylpropyl)-4-hydroxybenzoic a c i d , 3 - n - b u t y l - 4 h y d r o x y b e n z o i c a c i d , 3,4-dichloro-4-hydroxybenzoic a c i d , 3 - ( a methylallyl)-4-hydroxybenzoic a c i d , 3-ethyl-4-hydroxybenzoic a c i d , 3-amyl-4-hydroxybenzoic a c i d , 3-isobutyl-4-hydroxybenzoic a c i d , 3 - h e x y l b e n z o i c a c i d , 3-(2 ,3-dichloropropyl)-4-methoxyb e n z o i c a c i d . The n o v o b i o c i n a n a l o g s r e s u l t i n g from t h e above p r e c u r s o r s h a d c o m p a t i b l e s i m i l a r i t i e s w i t h n o v o b i o c i n on U . V . a n d I . R . measurements b u t d e t a i l e d c h a r a c t e r i z a t i o n was n o t given.
-
-
B i r c h 4 f o u n d t h a t w i t h c e r t a i n S . niueuo m u t a n t s , p r e c u r s o r s c o n s i s t i n g o f a l m o s t any s u b s t i t u t e d b e n z o i c a c i d , when added t o t h e f e r m e n t a t i o n b r o t h , were c o n v e r t e d t o n o v o b i o c i n No chema n a l o g s , b u t t h e p r o d u c t s were mixed w i t h n o v o b i o c i n . i c a l c h a r a c t e r i z a t i o n s were g i v e n . By s e l e c t i n g S. n i V Q U b mut a n t s which c o u l d n o t g e n e r a t e t h e coumarin p o r t i o n , b u t . which c o u l d s y n t h e s i z e n o v i o s e a n d c o n v e r t added a c y l a m i n o c o u m a r i n d e r i v a t i v e s i n t o a n a l o g s o f n o v o b i o c i n , h e was a b l e t o p r e p a r e chloronovobiocin (chlorodesme thylnovobiocin,z). Desme t h y l coumarin p r e c u r s o r f e e d i n g y i e l d e d some d e s m e t h y l n o v o b i o c i n
cg,.
KominekS r e p o r t e d t h a t a m u t a n t o f s . n i v e u d (FD-1161) o b t a i n e d from t h e B o o t ' s P u r e Drug Co., L t d . i n England d i d n o t produce novobiocin b u t could convert descarbamylnovobiocin ( 3 ) and 0-desme thyldescarbamylnovobiocin ( 8) t o n o v o b i o c i n . A n o t h e r S. n i W e U A m u t a n t (Upjohn BC-384) l i k e w i s e c o u l d n o t s y n t h e s i z e n o v o b i o c i n b u t d i d p r o d u c e d e s c a r b a m y l n o v o b i o c i n and O-desmethyld e s c a r b a m y l n o v o b i o c i n i n g r e a t a b u n d a n c e , These o b s e r v a t i o n s were u s e d i n Korninek's p r o p o s a l s f o r p o s s i b l e b i o s y n t h e t i c r o u t e s i n novobiocin production6. I n d e e d , Kominek a n d S e b e k 7 r e p o r t e d
132 t h a t of t h e t o t a l p r o d u c t i o n ( m e a s u r e d by U.V. a s s a y ) i n a t y p i c a l S . n i u s u d f e r m e n t a t i o n ( 7 d a y s ) , n o v o b i o c i n (1)a c c o u n t e d f o r 4 5 % i s o n o v o b i o c i n (2)f o r 2 1 %, O-desmethyldescarbamylnovob i o c i n (4) f o r l o % , d e s c a r b a m y l n o v o b i o c i n ( 3 ) f o r 1 4 % a n d O-desm e t h y l n o v o b i o c i n (lo)f o r 2%.
-
4.1.3
S t r u c t u r a l formula
The s t r u c t u r e s of a l l o f t h e b e n z o i c a c i d s u b s t i t u t e d a n a l o g s d e s c r i b e d above can b e deduced r e a d i l y by t h e r e p l a c e ment o f Ring A i n n o v o b i o c i n (1)w i t h t h e c o r r e s p o n d i n g a c y l m o i e t y . S i m i l a r l y , i n Ring B , t h e 8-methyl s u b s t i t u e n t may be c o m p l e t e l y a b s e n t ( d e s m e t h y l ) o r i t may b e r e p l a c e d by c h l o r i n e (chloronovobiocin). I n Ring C, t h e c a r b a m y l may b e r e p l a c e d by hydrogen ( d e s c a r b a m y l n o v o b i o c i n ) o r 8-methyl a n d carbamyl may b o t h be a b s e n t (desmethyldescarbamylnovobiocin).
RING 0
RING C 10, -9,
Desmethylnovobiocin Chloronovobiocin -3, Descerbarnylnovobiocin -8, Desmet h y ldescarbarny l n o v o b i o c i n
4 . 2 Summary
RING A
Rl -
R2
H
c1
CONH2 CONH2
CH3
H
H
H
of t h e r a p e u t i c u s e
T h e new d e r i v a t i v e s d e s c r i b e d by F o l k e r s a n d Woodruff3 v a r i e d i n t h e i r r e l a t i v e a c t i v i t i e s a g a i n s t various t e s t bact e r i a from n o v o b i o c i n i n an u n p r e d i c t a b l e manner. Certain anal o g s h a d l o w e r a c t i v i t y f o r one o r g a n i s m , e . g . StaphyLococcud auRCud; h i g h e r a c t i v i t y f o r o t h e r o r g a n i s m s , e . g . B a c i U u A d u b t i t i & , P R o t e u d V d g a n i d ; and were unchanged f o r o t h e r s , s u c h as E d c h c R i c h i a c0t.L. B i r c h 4 s t a t e d t h a t a l t e r a t i o n o f t h e a c y l g r o u p ( R i n g A ) was, i n a l l c a s e s t e s t e d , u n f a v o r a b l e . Chloron o v o b i o c i n was h i g h l y a c t i v e , w i t h a s p e c t r u m o f a c t i v i t y s i m i l a r t o b u t n o t i d e n t i c a l w i t h t h a t o f n o v o b i o c i n (compare w i t h clorobiocin). None o f t h e s e a n a l o g s h a s been r e p o r t e d t o h a v e been t e s t e d i n humans.
4.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
The a n a l o g s l i s t e d by Folkers a n d Woodruff3 were i s o l a t e d i n a manner similar t o t h a t d e s c r i b e d f o r methoxynovobiocin ( s e e Scheme 8).
4.4 Literature 1.
Cited
I . M. M i l l e r and R . B . Walton, P a t i b i o t i c s Annual, 1040-1045.
(1956-57)
133
. . . ,
R . 8. W a l t o n , L. E . M c D a n i e l a n d H . B . W o o d r u f f , D e v e l o p ments i n I n d u s t r i a l M i c r o b i o l o g y 3 ( 1 9 6 2 ) 370-375. Plenum P r e s s , N e w York.
K. F o l k e r s a n d H . B . W o o d r u f f , U.S. P a t e n t 3 , 0 9 3 , 5 4 9 ; J u n e 11, 1 9 6 3 .
A. J. B i r c h , A d v a n c . A n t i m i c r o b . A n t i n e o p l a s t i c C h e m o t h e r . P r o c . I n s t . Congr. Chemother., 7 t h 1 9 7 1 (Pu b . 1 9 7 2 ) 1 ( P t 2 1 , 1023-1024. E d i t o r , M. H e j z l a r ; U n i v e r s i t y P a r k P r e s s , Baltimore.
L . A. Kominek, A n t i b i o t i c s 2 ( 1 9 6 7 ) 2 3 1 - 2 3 9 . E d i t o r s , D. G o t t l i e b a n d P . D. Shaw; S p r i n g e r - V e r l a g N e w York I n c . , N e w
York.
. .
,
L. A. Kominek, A n t i m i c r o b i a l A g e n t s a n d Chemo. 1 ( 1 9 7 2 ) 123-134. A m e r . S O C . for M i c r o b i o l . L . A. Kominek a n d 0 . K . S e b e k , D e v e l o p m e n t s i n I n d u s t r i a l M i c r o b i o l o g y , 1 5 ( 1 9 7 4 ) 60-69. SOC. I n d . M i c r o b i o l .
134 5. C l o r o b i o c i n
5.1 Introduction 5.1.1
Producing organisms
C l o r o b i o c i n h a s been i s o l a t e d from c u l t u r e b r o t h s of s e v e r a l s t r a i n s of streptomyces belonging t o c l e a r l y d i f f e r e n t S&eptornyced h y g a a d c o p i c u d DS 9 ,7 5 1 (Union o f S. species A f r i c a ) , NRRL 3418; S . hoAeochkornogeneA v a r . OACitanA DS 1 2 , 9 7 6 ( I n d i a ) , NRRL 3504; and S. a l b o c i n e A e A c e n A DS 2 1 , 6 4 7 ( F r a n c e ) , NRRL 3 9 1 4 . F o r example, S. A o A ~ o c h A o m o g ~ n e grown A 3 days a t 30' i n a e r a t e d a g i t a t e d medium p r o d u c e d a b o u t 250 mg/k of a n t i biotic'. 5.1.2
Chemical d e s c r i p t i o n
The a n t i b i o t i c was i s o l a t e d by w o r k e r s of Rhone-Poulenc F r a n c e , i n 1 9 6 8 a n d c o m p l e t e p a t e n t s p e c i f i c a t i o n s were p u b l i s h e d i n 1 9 7 0 2 , 1 9 7 2 3 and 1 9 7 4 ' + . A b r i e f c h e m i c a l and o v e r a l l d e s c r i p t i o n was g i v e n by N i n e t e t at.1. S.A.,
C l o r o b i o c i n ( f i r s t c a l l e d a n t i b i o i i c 1 8 , 6 3 1 R.P.) i s a w h i t e m i c r o c r y s t a l l i n e powder, m.p. 2 0 6 , e a s i l y s o l u b l e i n dimethylsulfoxide , dioxane o r acetone; s o l u b l e i n d i l u t e s t r o n g b a s e s , m e t h a n o l , e t h a n o l , c h l o r o f o r m , dimethylformamide a n d e t h y l a c e t a t e ; s p a r i n g l y s o l u b l e o r i n s o l u b l e i n water, a q u e o u s s o l u t i o n o f sodium b i c a r b o n a t e , d i l u t e s t r o n g a c i d s , c a r b o n t e t r a c h l o r i d e , a c e t o n i t r i l e a n d h e x a n e . The e m p i r i c a l f o r m u l a i s C 5H37011N2Cl; mol. w t . 696.5; { a I f i o - 6 8 ' ( c . 1 , e t h a n o l ) ; { a 1 5 q - 8 0 0 ( ~ ~ 0 . a6c ,e t o n e ) . e U . V . a b s o r p t i o n max. ( C H C 1 s o l u t i o n , 1 0 m / a ) 275 nm, 444; s h o u l d e r a t 3 0 7 nm, 2 0 1 ; 337 nm, EfEm 434. The i n T r a r e d s p e c t r u m i s g i v e n by Mancy e t ~ 1 . ~ . C l o r o b i o c i n i s a weak a c i d i n aqueous s o l u t i o n , forming c r y s t a l l i n e s a l t s with c e r t a i n metallic o r organic bases. The d i e t h a n o l a m i n e s a l t i s v e r y s o l u b l e i n w a t e r , t h e sodium s a l t is l i t t l e soluble.
Eir
5.1.3
El&,
S t r u c t u r a l formula
Examination o f U.V., I . R . , N . M . R . and mass s p e c t r a p e r m i t t e d N i n e t et a t . 1 t o p r e s e n t a c o r r e c t s t r u c t u r a l f o r m u l a , a l t h o u g h i n s u f f i c i e n t e x p e r i m e n t a l o r s p e c t r a l d e t a i l s were g i v e n . An e a r l i e r s t r u c t u r e p r o p o s a l by Mancy e t u - ! . ~ l e f t u n d e t e r m i n e d t h e p o s i t i o n s of t h e c h l o r i n e atom a n d a m e t h y l g r o u p on t h e p y r r o l e m o i e t y . D o l a k 5 p r e s e n t e d e v i d e n c e from d e g r a d a t i v e and N.M.R. techniques f o r t h e following s t r u c t u r e .
I t i s c l e a r t h a t t h e s t r u c t u r e of c l o r o b i o c i n i s very similar t o t h a t of n o v o b i o c i n , d i f f e r i n g o n l y by t h e s u b s t i t u t i o n o f c h l o r i n e f o r m e t h y l i n t h e coumarin Ring B , and of m e t h y l p y r r o y l f o r c a r b a m y l on t h e n o v i o s e s u g a r Ring C . In this l a t t e r r e s p e c t , i t h a s a f e a t u r e i n common w i t h coumermycin. Although t h e f e r m e n t a t i o n medium f o r c l o r o b i o c i n c o n t a i n s c o b a l t s a l t s , p r e s u m a b l y t o f a c i l i t a t e t h e s y n t h e s i s o f t h e methylpyr r o y l moiety i n analogy with the cobalt-stimulated b i o s y n t h e s i s of p y r r o l e - m e t h y l a t e d coumermycin ( A l l , n o e v i d e n c e i s o f f e r e d 4
135
RING C
novobiocin R1 = CH3; R2 =
RING B
RING A
H~N-CO-
clorobiocin R1 = C1; R2
=
mco-
CH3
N
H
t h a t , with a d e f i c i e n c y of c o b a l t , a non-methylated p y r r o y l a n a l o g o f c l o r o b i o c i n may o c c u r . No e v i d e n c e h a s been o f f e r e d e i t h e r as t o t h e e x i s t e n c e o f a n i s o c l o r o b i o c i n , i n a n a l o g y w i t h isonovobiocin6.
A summary of t h e i n v i t h o a n t i b a c t e r i a l a c t i v i t i e s o f c l o r o b i o c i n , measured by b r o t h d i l u t i o n t e s t s 4 i s g i v e n i n Table 3. TABLE 3
I n v i t 4 0 A c t i v i t y o f Clorobiocin Test Organism
S . a ~ h e u ,209P, ATCC 6538P S. awLeuA, 209P, Smith Sancina .&.tea, ATCC 9341 St4ep~OcOccrcd ~ W C & A , ATCC 9790 S t ~ p . t o c o c cpyogenes ~ hemof.y&icw D.ip-fococcw pneumovciae
N e h e n h ca.tahrthaU~
NebAuLa men.&giZkf&
Nebiadenia gonomhoeae
Lac&badUu c a e i ATCC 7469
B a c i t h A u b Z X i A ATCC 6633 Mycoba&iwn species ATCC 607 EAchenichia coi5 ATCC 9637 Pho.teuA v u t g a n b K l e b A i e U a pnewnonia ATCC 10,031 Pbeudomonab aenug-iuzona Bhucek'la 6honchhep;tica P ~ . I i u t e U am u t t o c i d a Reim 'A ,?%?ponema
Min. b a c t e r i o s t a t i c concn. i n mcg/ml 0.005 0.003
0.02 0.04 0.05
0.03 0.005 0.05 0.4 1 0.6 10 10 13 1 4 0.3 7 12
An a d d i t i o n a l comparison of t h e r e l a t i v e i n v i L 4 0 a c t i v i t i e s o f c l o r o b i o c i n , n o v o b i o c i n and coumermycin A l from unpubl i s h e d d a t a of t h e M i c r o b i o l o g y Department a t Hoffmann-La Roche
136 I n c . , Nutley, is given i n Table 4. TABLE 4 Comparative In v h 2 0 A c t i v i t i e s o f C l o r o b i o c i n , Novobiocin, Methylpyrroyldescarbamylnovobiocin (Ro 7-9760) and Coumermycin A,
X A c t i v i t y Relative t o Coumermycin = 100* Test Organism
Ehchehichia cofi ATCC 27856 Pdeudomonas amuginoha ATCC 8709 KlebdieLLa pneumonia& ATCC 27858 AcinetobacWt c d c o a c e C h ~ATCC 10 15 3
S m d a mmVrcencen6
ATCC 27857
Sun/ra~%sp. ATCC 93 BadLLw s p . E ATCC 27859 B a ~ U u d4 u b ~ X . hNRRL 558
SXteptomyce~ceLLuloda&
C l o r o b i ocin
Novobiocin
RO 7-9760
400
20 25 400
100 500 100
4000 150 150 75 100
300 25 15 400 500
1000 16 200 20 30
150 100 150 20 -200
20 8-20 30
100 120 100 25 500
150
1000
ATCC
3313
SmVrdna llLtea ATCC 9341 Staphylococcud aWrew ATCC 6538P BdUuA megczteAium ATCC 8011 MycobaWium phlei ATCC 355
-
*Calculations were made from the diameters o f i n h i b i t i o n zones read on n u t r i e n t agar d i f f u s i o n cup-assays; each o f t h e t h r e e a n t i b i o t i c s was t e s t e d a t a s e r i e s of concentrations from 1, 10, 100 and 1000 mcg/ml
.
I t may b e s e e n f r o m t h e d a t a i n T a b l e s 3 and 4 t h a t c l o r o b i o c i n is i n d e e d g e n e r a l l y more a c t i v e i n w i t 4 0 t h a n novob i o c i n , as c l a i m e d by N i n e t e t al.l. I t has high a c t i v i t y a g a i n s t c e r t a i n g r a m - p o s i t i v e b a c t e r i a , and a more r e s t r i c t e d a c t i v i t y a g a i n s t gram-negative b a c t e r i a , although i t s t i l l e x e r t s v e r y h i g h a c t i v i t y a g a i n s t c e r t a i n NeihAe/ria. I n l i q u i d media, t h e min i m u m b a c t e r i o s t a t i c c o n c e n t r a t i o n s f o r S t a p hy L o coccud aukeud , S t 4 e p t o c o c c u 4 pyogened and N e i d d U i a m e n i n g i t i d i d were a b o u t 0 . 0 5 mcg/ml, w h i l e M . I . C . v a l u e s f o r Edchehichia C a l i P t o t e u h u u l g a a i h , Pbeudomonad ae/ruginoda and Padteu4elLa m u l t o c i d a were 5 - 1 0 mcg/ml. N i n e t e t a 1 . l c o n c l u d e d t h a t t h e p r e s e n c e of t h e c h l o r i n e atom on t h e n u c l e u s i n t h e n o v o b i o c i n f a m i l y increased the a n t i b i o t i c a c t i v i t y without notably modifying t h e t o x i c i t y ; however, t h e p o s s i b i l i t y d o e s n o t seem t o h a v e been c o n s i d e r e d t h a t i t may b e t h e m e t h y l p y r r o y l f u n c t i o n which is responsible f o r the increased i n witno a n t i b a c t e r i a l a c t i v i t y of c l o r o b i o c i n o v e r n o v o b i o c i n . A d i r e c t c o m p a r i s o n of t h e b i o a c t i v i t i e s of c l o r o b i o c i n , novobiocin, chloronovobiocin7 and r n e t h y l p y r r o y l d e s c a r b a m y l n o v o b i o c i n r e s o l v e d t h i s q u e s t i o n . As shown i n T a b l e 4 , w i t h two e x c e p t i o n s , s y n t h e t i c 5-methyl-2pyrroyldescarbamylnovobdocin does indeed have e s s e n t i a l l y t h e same improved q u a l i t a t i v e a n d q u a n t i t a t i v e a c t i v i t i e s as d o e s clorobiocin over novobiocin. Additional d a t a kindly s u p p l i e d b y t h e Upjohn CompanyB c l e a r l y s u p p o r t t h e same c o n c l u s i o n t h a t
,
137 the chlorine s u b s t i t u t i o n is not responsible f o r t h e increased potency. T h u s , i t may b e s e e n from t h e i r d a t a i n T a b l e 5 t h a t i n a d i r e c t comparison, chloronovobiocin (8-chloro-8-desmethyln o v o b i o c i n ) i s j u s t a b o u t e q u a l i n a n t i b a c t e r i a l p o t e n c y t o novob i o c i n , a n d i s c o n s i d e r a b l y weaker t h a n c l o r o b i o c i n . TABLE 5
In w i . 0 ~A n t i b a c t e r i a l S p e c t r a o f Novobiocin, Clorobiocin and Chloronovobiocin*
M.I.C. Organism
S . aUnelL6 UC 76 S . aUnelL6 UC 607 s. @xa.U uc 3235
P. mul-tocida
uc
264
S . hemO&ZklLs UC 203 E. cofi UC 5 1 K . pneumonia UC 57 9 . vuLgm.in uc 93 P. vulgcCCrh UC 189
P. auuginooa
S.
uc
95 ochoMmtefleni UC 263
i n mcg/ml a f t e r 20 h r a t 37'
Novobiocin*
Clorobiocin
<0.09 200 6.3 0.38 0.38
>zoo
1.5 6.3 >200 >200
>zoo
1.5 1.5 <0.09
8-chloro-8desmethylnovobiocin 0.19 200 25 0.75 0.75 50 0.75 3.1
>zoo
>200
>zoo
*The compounds were d i s s o l v e d i n 1 m l dimethylforrnamide a n d d i l u t e d s e r i a l l y i n b r a i n h e a r t i n f u s i o n b r o t h . The maximum c o n c e n t r a t i o n u s e d was 200 mcg/ml.
I n mice, c l o r o b i o c i n h a s a n L D 5 0 o f 2 . 2 g / k g p . 0 . a n d 1 . 7 g / k g s . c . ~ ,b e i n g s l i g h t l y i n f e r i o r t o n o v o b i o c i n . C l o r o b i o c i n i s a c t i v e a g a i n s t e x p e r i m e n t a l s t r e p t o c o c c u s , pneumococcus a n d s t a p h y l o c o c c u s i n f e c t i o n s i n mice when a d m i n i s t e r e d o r a l l y o r s u b c u t a n e o u s l y . T h u s , 5-10 mg/kg p . 0 . or S . C . were r e p o r t e d t o cure s t a p h y l o c o c c u s a n d m e n i n g o c o c c a l i n f e c t i o n s ' . Unpublished r e s u l t s from t h e Hoffmann-La Roche C h e m o t h e r a p y D e p a r t m e n t , N u t l e y , on material k i n d l y s u p p l i e d b y Rh o n e -Po u le n c , i n d i c a t e C D 5 0 ' s i n mice w i t h s. auneua i n f e c t i o n s o f 5 . 6 mg/kg S . C . a n d 1 9 mg/kg p . 0 . The a n t i b i o t i c was i n a c t i v e a g a i n s t s y s t e m i c i n f e c t i o n s o f K . pneumowae > 5 0 mg/kg s . c . ; Pnoteuo VUlgaAiA > 2 5 0 mg/kg S . C . a n d > 5 0 0 mg/kg P.o., a n d i n a c t i v e a g a i n s t l o c a l Tnichomonao v a g i n a t i o i n f e c t i o n s a t 250 mg/kg p . 0 . To d a t e , n o r e s u l t s h a v e a p p e a r e d on t h e use o f c l o r o b i o c i n i n humans. Ho wev er , Mancy e t at.4 s u g g e s t e d t h a t f o r t h e treatment of i n f e c t i o n s by gram-positive microorganisms i n a human a d u l t , t h e d o s e i s g e n e r a l l y b e t w e e n 1 a n d 3 g p e r d a y a d m i n i s t e r e d o r a l l y or r e c t a l l y a n d b e t w e e n 0 . 2 a n d 2 g p e r d a y administered parenterally.
5.3 Extraction, 5.3.1
s e p a r a t i o n and p u r i f i c a t i o n techniques
Detection
I n a d d i t i o n t o t h e m i c r o b i o l o g i c a l a s s a y , c l o r o b i o c i n may
138
be d e t e c t e d by i t s c h r o m a t o g r a p h i c m o b i l i t y , u s i n g p l a t e s o f n u t r i e n t a g a r s e e d e d w i t h Staphylococcuh aLbu4 f o r b i o a u t o g r a p h y . The d a t a i n T a b l e 6 summarize t h e Rf v a l u e s o b t a i n e d w i t h a number o f s o l v e n t s y s t e m s on p a p e r , a l u m i n a , a n d s i l i c a g e l chromatography4.
TABLE 6
Chromatographic Behavior of Clorobiocin4 System (composition by volume)
Support Non-buffered Non-buf f e r e d Non -bu f f e red Non-buffered
paper* paper* paper* paper*
Non-buffered paper* Paper * imp re gn a t e d w i t h a phosphate b u f f e r pH 7, M/3 Alumina ( t h i n l a y e r ) Kieselgel G ( s i l i c a g e l ) , t h i n layer Kieselgel G ( s i l i c a gel), t h i n layer
Rf -
Butanol, s a t d . with water Benzene-methanol ( 4 : l ) N H , + C l , 30 q / L i n water B utanol-ace t i c a c i d-w a t e r (4:1:5 upper phase) E t h y 1 ace take -cyclohexane (1:l) s a t d . w i t h water Chloroform
0.95 0.95 0.05
Me thanol-water
0.27
(95:5)
B u t anol-ace ti c acid-water (4:1:5,
1.oo
0.50 0.50
1 .oo
upper phase)
Carbon t e t r a c h l o r i d e - e t h a n o l a c e t i c a c i d (90:6:6)
0.50
*Arches 302 5.3.2
Isolation
C l o r o b i o c i n can b e i s o l a t e d from f e r m e n t a t i o n b r o t h s by v a r i o u s m e t h ~ d s l ’ ~ .A c t i v i t y i s d e t e r m i n e d by t h e a g a r d i f f u s i o n a s s a y , u s i n g M i c ~ o c o c c u hpyogened aLbud as t h e s e n s i t i v e t e s t o r g a n i s m , and p u r e c l o r o b i o c i n as a r e f e r e n c e s t a n d a r d . I f b r o t h i s f i l t e r e d a t pH 7 or g r e a t e r , a c t i v i t y i s p r e s e n t i n b o t h f i l t r a t e and mycelium. From t h e f i l t r a t e , a c t i v i t y may b e e x t r a c t e d w i t h an i m m i s c i b l e s o l v e n t s u c h as b u t a n o l or a n y 1 a l c o h o l , or w i t h c h l o r o f o r m , m e t h y l e n e c h l o r i d e or e t h y l acet a t e . To a v o i d t h e n e e d of s e p a r a t e e x t r a c t i o n s of f i l t r a t e and mycelium, t h e whole b r o t h i s f i l t e r e d a t pH 5 , where a l l t h e a n t i b i o t i c r e m a i n s i n t h e f i l t e r cake, from which i t c a n b e e x t r a c t e d w i t h 6 0 % aqueous m e t h a n o l (or w i t h e t h a n o l or p r o p a n 01). The e x t r a c t i s e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e and t h e a n t i b i o t i c i s e x t r a c t e d from t h e c o n c e n t r a t e d s o l u t i o n a t pH 3 w i t h one of t h e w a t e r - i m m i s c i b l e s o l v e n t s , s u c h as n - b u t a n o l . The b u t a n o l e x t r a c t i s c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e , and a f t e r n e u t r a l i z a t i o n w i t h sodium m e t h y l a t e , a d d i t i o n o f h e x a n e p r o d u c e s a p r e c i p i t a t e o f amorphous a n t i b i o t i c of a b o u t 4 0 % purity. F u r t h e r p u r i f i c a t i o n i n v o l v e s a) c h r o m a t o g r a p h y of an aqueous s o l u t i o n of t h e p r o d u c t on Dowex 1 - X 2 (Cl-) i o n - e x c h a n g e r e s i n ; t h e a c t i v e f r a c t i o n i s e l u t e d w i t h an a q u e o u s m e t h a n o l s o l u t i o n c o n t a i n i n g 8 0 % m e t h a n o l and 3 % ammonium c h l o r i d e ; b ) e x t r a c t i o n of t h e above e l u a t e w i t h e t h y l a c e t a t e ; t h e o r g a n i c p h a s e i s d e c o l o r i z e d by p a s s i n g t h r o u g h an a l u m i n a column, t h e n
139 c o n c e n t r a t e d by e v a p o r a t i o n under r e d u c e d p r e s s u r e t o b r i n g about t h e c r y s t a l l i z a t i o n of t h e c l o r o b i o c i n ; c ) a r e c r y s t a l l i z a t i o n o f t h e p r o d u c t i s e f f e c t e d b y d i s s o l v i n g i n a m i x t u r e of a c e t o n e a n d d i o x a n e or a c e t o n i t r i l e , a n d b y a d d i t i o n o f water. A d e t a i l e d e x a m p l e i s o u t l i n e d as follows4: 990 d b r o t h , contg. 288 mg/d ( t o t a l 285 g a n t i b i o t i c )
STAGE 1
adj. t o H 5 w.10 d 5 N H E 1 with s t i r r i n g , A f t e r 1/% h r , add 50 k g f i l t e r a i d and f i l t e r on f i l t e r press.
I
11
F i l t e r cake washed w. 200
F i 1t r a t e discarded
HO ,
I Wash aiscarded
F i l t e r t a k e (199 kg) susp. i n 750 d o f m i x t . o f water-kOH contg. 600 d MeOH. Adjust apparent pH t o 7 by addn. o f 1 d 5 N NaOH. A g i t a t e f o r 1 h r , then f i l t e r
n I
F i l t e r cake w!shed w. 100 d o f 60% (by v o l . ) MeOH-H20 mixture. F i l t e r Filtiate-
F i l t e r (cake discarded
I
n
Lower phase e x t r a c t w. 30 d BuOH Spent mother l i q . discarded
Upper l a y e r
98
d corrbined e x t r . washed
I
Water wash discarded
Crude Na s a l t , wh a!
Yield:
Combined vo1.= 840 d contg. 239.4 g a n t i b i o t i c (84% recov.) Concentrate a t 35', 35 mm Hg, t o 100 k?. Add 50 k BuoH, s t i r , adj. t o pH 3 w.5 N HC1. S t i r 30 min, decant, separate
II
Upper phase
L-
F i I tr a t e
W.
10 d H2O
ll
BuOH e x t r a c t concd. a t 20 mm Hg and 37' t o 3 k?; n e u t r . t o pH 7 by addn. o f 25X (by vol.) s o h . of Na methylate i n BuOH. Add 30 d hexane t o p p t . a n t i b i o t i c ; f i l t e r w. hexane, dry a t 40°,
5 mm Hg
477 g o f 43.5% p u r i t y ( o r 207.5 g "pure"
=
D is t a r d f i I tr a t e
73% recov.)
140 P u r i f i c a t i o n o f Crude A n t i b i o t i c
STAGE 11
1345 q crude a n t i b i o t i c (33% pure) d i a s . - i n 30 t f meth anol-wat e r ( 1 : l v/v)
add t o upper p a r t o f column (12 cm I.D.) contg. Dowex 1-X2 r e s i n (30 1) i n C1-; f l o w - r a t e 3 t / h r
n I Wash r e s i n successively, a t flow-rate
30 t i n i t i a l soln. through column
Wash s o l u t i o n s discarded
of 25 L/hr with: 20 1 kOH-HzO (1:l V/V) ,120 f MeOH-HzO (3:2 V / V ) contg. 15 g / t o f NH4C1,60 L MeOH-H20 (7:3 v/v) contg. 15 g / t o f NH,Cl
E l u t e column w i t h 6 X 20 t ( t o t a l 120 1) MeOH420 ( 4 : l V / V ) contg. 30 g/e NHkC1
f Fraction 1 discarded (20 1)
Combine the e x t r a c t s , wash
w. 5 1 H20, dry over Na2S04 and conc. t o 10 1 under reduced press. and <40° t o y i e l d " P u r i f i e d EtOAc Extract"
F r a c t i o n s 5 t o 6 of t h e Dowex 1 - X 2 chromatogram s i m i l a r l y y i e l d 35 g c r y s t a l l i n e p r o d u c t , f r e e a c i d , 7 0 2 mcg/mg. Furthermore, 1 2 4 g of p r o d u c t f o r r e c y c l i n g ( 2 5 0 mcg/mg p o t e n c y ) can b e r e c o v e r e d by c o n c e n t r a t i n g t h e c r y s t a l l i z a t i o n mother l i q u o r s and p r e c i p i t a t i o n w i t h hexane.
141
STAGE I11
P u r i f i e d E t h y l Acetate E x t r a c t from Stage I 1 (10
e)
Percolate through washed alumina (pH 4) column ( I . D . 5 cm, 500 9). I Wash column w. 2
Percolate
e
EtOAc
Combine e f f l u e n t s and washings, conc. a t reduc. press. t o 1/10 v o l . t o cause c r y s t a l l i z a t i o n . F i l t e r a f t e r 2 h r a t 0'
: Mother l i q u o r s
Crystals,owash w. c o l d EtOAc (200 m l ) and <5 mm Hg f o r 24 h r . dry a t 40 Y i e l d : 170 g f r e e acid, 85.5% pure (Equiv. t o 33% recov. from crude, o r 24L o v e r a l l recovery from b r o t h )
-
,
(recovered s e p a r a t e l y )
For r e c r y s t a l l i z a t i o n , 2 7 6 g of material s u c h a s o i s d e s c r i b e d a b o v e ( 8 5 5 mcg/mg p o t e n c y ) i s d i s s o l v e d a t 30 i n 2 . 4 p, o f a m i x t u r e of a c e t o n e - d i o x a n e (5:l v / v ) . The s o l u t i o n i s c l a r i f i e d ; 2 . 5 p, o f water a r e a d d e d o v e r 3 h r a t a m b i e n t t e m p e r a t u r e with slow s t i r r i n g . A f t e r s t a n d i n g o v e r n i g h t , t h e c r y s t a l s a r e f i i t e r e d o f f , washed w i t h 1 p, o f w a t e r a n d d r i e d for 4 8 h r a t 6 0 , 1 mm Hg p r e s s u r e . Clorobiocin f r e e acid, 231 g ( 8 4 % r e c o v e r y on r e c r y s t a l l i z a t i o n , or 2 0 % o v e r a l l r e c o v e r y from 9 9 0 p, whole b r o t h , by c a l c u l a t i o n ) , i s o b t a i n e d as w h i t e For f i n a l clean-up, 1 6 4 g of c r y s t a l s , w i t h 9 9 0 mcg/ml p o t e n c y . t h e above c r y s t a l l i n e f r e e a c i d i s d i s s o l v e d i n 5 0 0 ml a c e t o n e ; t h e s o l u t i o n i s c l a r i f i e d by f i l t e r i n g t h r o u g h a b e d o f C l a r c e l D I C , and 4 . 5 o f a m i x t u r e o f a c e t o n i t r i l e - w a t e r (1:l v / v ) i s a d d e d w i t h slow s t i r r i n g o v e r 1 - 1 / 2 h r . A f t e r s t a n d i n g o v e r n i g h t , t h e r e s u l t i n g c r y s t a l s are f i l t e r e d o f f , washed w i t h 1 o f a c e t o n e - w a t e r (1:l v / v ) a n d t h e n w i t h 1 fi w a t e r , a n d d r i e d The d r i e d w h i t e c r y s t a l s 4 8 h r a t 5 0 ' u n d e r l e s s t h a n 5 m m Hg. ( 1 2 4 g ) a s s a y i n g 1,000 mcg/mg, a r e o b t a i n e d i n 7 6 % y i e l d (or 1 5 % o v e r a l l y i e l d from b r o t h ) .
5.4 Literature 1.
Cited
L . N i n e t , F. B e n a z e t , Y . D . Mancy, J. Preud'Homrne, D . E . W r i g h t , A . Abraham, Godard a n d J. T h e i l l e u x ,
C h a r p e n t i e , M . Dubost, J . F l o r e n t , T . L. T h r e l f a l l , B . V u i l l e m i n , M . C a r t i e r , N . De C h e z e l l e s , C . C . R . Acad. S c . P a r i s 2 7 5 ( 1 9 7 2 )
455-458. 2.
D. Mancy, L. N i n e t a n d J . Preud'Homme, 1,208,842; October 14, 1 9 7 0 .
British Patent
3.
D. Mancy, L . N i n e t a n d J . Preud'Homme, August 8, 1 9 7 2 .
U.S. P a t e n t 3 , 6 8 2 , 8 8 6 ;
142 4.
D. Mancy, L . N i n e t a n d J. Preud’Homme, U.S. P a t e n t 3 , 7 9 3 , 1 h 7 ; February 19, 1974.
5.
L . D o l a k , J . A n t i b i o t i c s 26 ( 1 9 7 3 ) 1 2 1 - 1 2 5 .
6.
A. A . F o r i s t , S . T h e a l a n d W . A. S t r u c k , A n a l . Chem. 3 1 (1959) 100-102.
7.
A . J. B i r c h , A d v a n c . A n t i m i c r o b . A n t i n e o p l a s t i c C h e m o t h e r . P r o c . I n t . Congr. C h e m o t h e r . , 7 t h 1 9 7 1 ( P u b . 1 9 7 2 ) , 1 ( P t 21, 1 0 2 3 - 1 0 2 4 . E d i t o r , M. H e j z l a r , U n i v . P a r k P r e s s , Baltimore, Maryland.
8.
P e r s o n a l communication, 1976.
,
G. B . Whitfield, Jr., September 1 3 ,
143
6 . Coumermycin
.6,1 Introduction Producing organisms
6.1.1
The name coumermycin ( B r i s t o l - B a n y u R e s e a r c h I n s t i t u t e ) f i r s t a p p e a r e d i n 1 9 6 4 l ' * f o r t h e a n t i b i o t i c p r o d u c e d by S & e p t O m y C e A R i A h i R i e n A i A ATCC 1 4 8 1 2 w h i c h was i s o l a t e d f r o m a s o i l s a m p l e c o l l e c t e d on R i s h i r i I s l a n d , H o k k a i d o , J a p a n . O r i g i n a l l y t h e a n t i b i o t i c h a d b e e n r e f e r r e d t o as Bu-260 a n d as n o t o m y c i n 3 :
E a r l i e r , w o r k e r s a t Hoffmann-La Roche I n c . , N u t l e y , New J e r s e y , h a d i s o l a t e d a n a n t i b i o t i c complex c a l l e d s u g o r d o m y c i n from t h e i r c u l t u r e , S t A e p t o m y c e A h a z e l i e n A i A v a r . h a z e l i e n n i d NRRL 2938, which was o b t a i n e d f r o m a s o i l s a m p l e c o l l e c t e d on t h e Gaspe P e n i n s u l a , C a n a d a . S i n c e t h e B r i s t o l - B a n y u p u b l i c a t i o n a p p e a r e d f i r s t , t h e name s u g o r d o m y c i n was w i t h d r a w n . A U n i t e d S t a t e s p a t e n t c o v e r i n g t h e s u b s t a n c e was f i r s t awarded t o Kawaguchi e t a l . , B r i s t o l - B a n y u R e s e a r c h I n s t i t u t e , L t d . 4, b u t s u b s e q u e n t l y t h e a s s i g n m e n t was r e v e r s e d i n f a v o r o f B a t c h o e t . a t . , Hoffmann-La Roche I n c q 5 . The p r o c e s s p a t e n t was i s s u e d t o J. Berger6. Two a d d i t i o n a l o r g a n i s m s , S. A p i n i c h J t o m o g e n e A a n d S . A p i n i C O U m l x R e n A i A , h a v e been f o u n d t o b e coumermycin p r o d u c e r s ? . Submerged a e r o b i c f e r m e n t a t i o n s o f S . A i n h i t i e n n i a i n s h a k e f l a s k s i n a medium c o n t a i n i n g 6 % s t a r c h , 3 % b r e w e r s y e a s t a n d 1%c a l c i u m c a r b o n a t e p r o d u c e d 300-400 mg/R o f coumermycin4. B r i e f chemical d e s c r i p t i o n
6.1.2
The s t r u c t u r e s of t h e coumermycins are shown i n F i g u r e 3. Coumermycin A l ( s u g o r d o m y c i n D-la, 1) i s a weak d i b a s i c a c i d o f pKa 7 . 7 6 i n 75% a q . d i o x a n e 8 h a v i n g t h e e m p i r i c a l f o r m u l a C55H5,N502, w i t h mol. w t . 1 1 1 0 a n d m.p. 258-260' d e c . 8 ; f o r m s c r y s t a l s , m. p . 2 7 5 d e c . f rom t e t r a h y d r o f u r a n - w a t e r - a c e t o n i t r i l e ( u n p u b l i s h e d d a t a from Hoffmann-La Roche, I n c . ) . I t is s o l u b l e i n dioxane, t e t r a h y d r o f u r a n , dimethylformamide, a c e t o n e , methyl i s o b u t y l ketoneb methyl e t h y l ketone, e t h y l a c e t a t e , and e t h a n - 1 4 1 ( c . 1 , 7 5 % a q . a c e t o n e ) a n d h a s U . V . max. a t 2 8 0 nm 018' E "5 !;' a n d 336 nm E#Bm 380 i n e t h a n o l 8 ; a l s o a U . V . max. a t 21;gmnrn i n 6 0 % a q . met m o l y . The I . R . s p e c t r u m i s g i v e n 8 ; i t i s u n s t a b l e i n s o l u t i o n s o f low or h i g h pH.
'
The c r y s t a l l i n e monosodium s a l t 8 (m.p. 2 4 5 ' d e c . , n e e d l e s from e t h a n o l ) was u s e f u l i n t h e i s o l a t i o n a n d p u r i f i c a t i o n . The I . R . s p e c t r u m i s g i v e n . The d i s o d i u m s a l t , s e v e r a l amine s a l t s a n d a d m i x t u r e s o f coumermycin A 1 a n d a m i n e s , p a r t i c u l a r l y w i t h N-mgthylglucamine, h a v e b e e n f o u n d t o be t h e r a p e u t i c a l l y usefu110-i2
.
Coumermycin A2 ( s u g o r d o m y c i n D - l d , 2 ) i s a weak d i b a s i c a c i d having t h e e m p i r i c a l formula C 5 3 H 5 5 N 5 0 2 ~ , mol. w t . 1 0 8 2 a n d s o l u b i l i t y similar t o coumermycin A , . The I . R . s p e c t r u m i s g i v e n 1 3 ; t h e U . V . max. are a t 2 6 7 nm a n d 3 4 0 nm i n e t h a n o l 1 3
144 a n d a t 2 7 0 nm i n 6 0 % a q . m e t h a n o l g . The c r y s t a l l i n e monosodium s a l t was f o u n d t o be u s e f u l i n t h e p u r i f i c a t i o n . The I . R . i s g i v e n 1 3. Coumermycin D - l b , c (sugordomycin D - l b , c ; and/or is a weak d i b a s i c a c i d w i t h t h e e m p i r i c a l f o r m u l a C54H57N5020 mol. w t . 1 0 9 6 , a n d h a s s o l u b i l i t y similar t o coumermycin A1 w i t h U . V . max. 2 7 4 nm i n 6 0 % aq.methano1. I t has n o t been e s t a b l i s h e d w h e t h e r coumermycin D-lb o r D-lc o r a m i x t u r e of t h e two i s p r e s e n t g .
(s)
Coumermycin B (sugordomycin 0 - 2 , ‘a a n d / o r 4B) i s a w e a k d i b a s i c a c i d w i t h t h e e m p i r i c a l f o r m u l a C I + ~ H ~ $ N ~ Omol. Z , wt. 1 0 0 3 , and h a s s o l u b i l i t y similar t o coumermycin A 1 w i t h U . V . max. 2 8 4 nm i n 6 0 % a q . m e t h a n o l . I t h a s n o t b e e n e s t a b l i s h e d w h e t h e r coumermycin B i s % o r ‘(b o r a m i x t u r e o f % and 4b9. and/or i s a weak Coumermycin C (sugordomycin D - 3 , d i b a s i c a c i d w i t h t h e e m p i r i c a l f o r m u l a C 4 8 H 5 2 N 4 0 1 9 , mol. w t . 9 8 9 , and h a s s o l u b i l i t y similar t o coumermycin A 1 w i t h U . V . max. 2 7 2 i n 60% a q . m e t h a n o l . I t h a s n o t been e s t a b l i s h e d w h e t h e r coumermycin C i s or o r a mixture of t h e twog. Coumermycin D (sugordomycin D - 4 , 6 ) i s a weak d i b a s i c a c i d h a v i n g t h e e m p i r i c a l f o r m u l a C 4 3 H 4 9 N 3 0 1 8 , mol. w t . 908, a n d s o l u b i l i t y similar t o coumermycin A The U . V . s p e c t r u m h a s no maximum i n t h e 270-282 nm r e g i o n d : 6.1.3
S t r u c t u r a l formula
The Hoffmann-La Rocheg a n d B r i s t o l - B a n y ~ ~l ~4 Yteams i n d e p e n d e n t l y e l u c i d a t e d t h e s t r u c t u r e s of coumermycin A 1 (1)a n d A2 coumermycin A 1 a n d s u ordomycin D - l a were shown t o be i d e n t i c a l by TLC a n d b i o a s s a y Hoffmann-La Roche ( B a s l e ) chemists, t a k i n g advantage of t h e i r experience i n t h e s y n t h e s i s of n o v ~ b i o c i n ~soon ~ , a c h i e v e d a t o t a l s y n t h e s i s 9 ’ 1 6 o f coumermycin A 1 .
(z?;
8.
The s t r u c t u r e s o f t h e o t h e r components, a l t h o u g h n o t r i g i d l y e s t a b l i s h e d , have been deduced from p h y s i c o - c h e m i c a l d a t a and l i m i t e d d e g r a d a t i o n s t u d i e s There e x i s t s , however, t h e u n c e r t a i n t y as t o w h e t h e r t h e p y r r o l e m o i e t y ( m o i e t i e s i s a t t a c h e d s p e c i f i c a l l y o r i n d i s c r i m i n a t e l y ( p r o d u c i n g mixtures).
’.
The s i m i l a r i t y o f t h e coumermycins t o n o v o b i o c i n a n d c l o r o b i o c i n is r e a d i l y apparent ( c f . S e c t i o n s I and V ) . I f the pyrroyl (pyrrolecarbonyl) function migrates t o the a d j a c e n t h y d r o x y l g r o u p i n t h e coumermycins, as d o e s t h e carbamyl g r o u p i n n o v o b i o c i n ( t o i s o n o v o b i o c i n ) , t h e c o m p l e x i t y o f p o s s i b l e i s o m e r s would b e g r e a t l y i n c r e a s e d . S i n c e t h e molec u l e i s n o t s y m m e t r i c a l , t h e number of p o s s i b l e i s o m e r s m u l t i p l i e s even more. Thus p r o l o n g e d c o u n t e r - c u r r e n t d i s t r i b u t i o n s and m u l t i p l e manipulations might i n c r e a s e t h e r i s k of production o f isocoumermycins.
145
The b i o s y n t h e s i s of coumermycin A 1 h a s b e e n s t u d i e d by S c a n n e l l a n d Kong18, who c o n c l u d e d t h a t L - p r o l i n e was u t i l i z e d f o r pyrrole production.
Slruc turr
Rl
1
C H , QHC O -
2
FIGURE 3.
ChJpCO-
pcopco-
30
QH c o -
3b
Qnc o -
40
cn, a cH o -
4b
H
so
R2
QHc o -
Sb
n
6
H
H
tl
p c o H
Structures o f the coumermycins.
6 . 2 Summary
of t h e r a p e u t i c use
l n v b h o , t h e coumermycins a r e a c t i v e p r e d o m i n a n t l y a g a i n s t g r a m - p o s i t i v e b a c t e r i a , w i t h weaker a c t i v i t y a g a i n s t gram-ne a t i v e o r g a n i s m s . Kawaguchi e t u - ! ? . a~n d G r u n b e r g a n d B e n n e t t y g r e p o r t e d t h a t 8 s t r a i n s o f Staphy-!?ococcu4 uuReu.4 w i t h v a r y i n g r e s i s t a n c e p a t t e r n s t o o t h e r a n t i b i o t i c s were s e n s i t i v e t o coumermycin A, a t M . I . C . v a l u e s of 0 . 0 0 0 3 t o 0 . 0 0 2 5 mcg/ml.
146
T h e r e was some c r o s s - o v e r r e s i s t a n c e o f a n o v o b i o c i n - r e s i s t a n t
S. auheud c u l t u r e , w h i c h r e q u i r e d 0 . 1 5 6 mcg/ml o f coumermycin Al f o r i n h i b i t i o n . Other s e n s i t i v e gram-positive organisms i n c l u d e d S a n c i n a l u t e a ( 0 . 0 0 5 mcg/ml), Conynebacte~tiumxenodid ( 0 . 0 0 0 6 mcg/ml), B a c i l l u d d u b t i l i d ( 0 . 3 1 2 m c g / m l ) , B a c i l l u d ceneud ( 0 . 1 5 6 mcg/ml), S t h e p t o c o c c u a pyogenea ( 0 . 0 9 t o 0 . 7 8 mcg/ml), U i p ~ o c o c c u d pneumoniae ( 0 . 0 9 t o 0 . 7 8 m c g / m l ) , Lactob a c i l l u d cadei (>lo mcg/ml) a n d Mycobactenium s p . ( 0 . 7 8 t o 1 2 . 5 mcg/ml). G r a m - n e g a t i v e b a c t e r i a , i n c l u d i n g Eachenichia c o l i ,
Klebdiella pneumoniae, S a l m o n e l l a t y p h i , S h i g e l l a d y d e n t e n i a e , Neioaenia s p . a n d Pdeudomonaa aehuginoaa, were s e n s i t i v e t o 0 . 7 8 t o 1 2 . 5 mcg/ml.
Yeasts and f u n g i were i n s e n s i t i v e ( > l o 0 mcg/ml).
The r e l a t i v e a c t i v i t i e s g o f t h e coumermycins are p r e s e n t e d Coumermycin D i s v i r t u a l l y i n a c t i v e a n d a l t h o u g h i t i n Table 7. was n o t d e t e c t e d on PC b i o a u t o g r a p h y i n 9 , i t was d e t e c t e d i n 8 because of i t s presence i n high concentration. TABLE 7 Relative
ln
v i t 4 0 A c t i v i t i e s o f Coumermycins
Antibacterial* a c t i v i t y
Cornponent A1
100 25 3.6 20 1.6 0.01
,
D-lb c A2
B(D-2) C(D-3) D(D-4)
YBy cup-plate agar d i f f u s i o n assay, using S4kphylococcud W e u d ATCC 6538P.
A c o m p a r i s o n o f b r o t h d i l u t i o n a c t i v i t i e s of coumermycins i s p r e s e n t e d i n T a b l e 8 ( u n p u b l i s h e d , Hoffmann-La Roche I n c . )
.
TABLE 8 B r o t h D i l u t i o n A c t i v i t i e s o f Coumermycins and C l i n i c a l l y used A n t i b i o t i c s Antibiotic Coumermycin A, Coumermycin A, Coumermycin D-lb, c Coumermycin B ( 0-2) Coumermycin C(D-3) Tetracycline Strep tomy c i n s u l f a t e Chloramphenicol
M I C * (rncg/ml)
0.005-0.01 2.5-5.0 0.25 0.12-0.5 5.0 0.6-1.2 1.2-2.5 5 -10
Re l a t i v e Potency 10,000 25 200 200-500 10
100 50 10
*Determined i n peptone-nutrient broth, u s i n g an inoculum o f lo5 S4kphylococc~d~ e c e ~l l s per d m l . Lower inoculum concentration would show g r e a t e r s e n s i t i v i t i e s . Observations were recorded f o r complete growth i n h i b i t i o n a f t e r 1 8 h r , using t w o - f o l d d i l u t i o n s .
147 lfl v i u o , t o x i c i t y ( L D 5 0 ) i n mice was r e p o r t e d * t o b e 3 8 0 mg/kg S . C . a n d > 2 0 0 0 mg/kg p . 0 . In chronic toxicity trials w h e r e r a t s r e c e i v e d 2 5 m g / k g / d a y i . m . or 1 0 0 m g / k g / d a y p . 0 . , w e i g h t g a i n was n o r m a l a n d n o a d v e r s e e f f e c t was n o t e d o v e r 6 0 d a y s ( 6 d a y / w e c k b a s i s ) . I n S . auReuA i n f e c t i o n s o f m i c e , C D 5 0 was 0 . 1 3 mg/kg S . C . a n d q . 3 mg / k g p . 0 . I n comparative tests, C D 5 , v a l u e s for n o v o b i o c i n were 3.0 mg/kg a n d 5 . 6 m g/kg, r e s p e c t i v e l y 8.
G r u n b e r g a n d B e n n e t t l g r e p o r t e d L D 5 0 of c oum e rm yc in A 1 i n mice, i n m g / k g : >lo00 P . o . , 250 s . c . , 1 5 9 i . p . , a n d 25 i . v . In a s e r i e s o f b a c t e r i a l i n f e c t i o n s i n mice t h e y r e p o r t e d t h e following d a t a i n Table 9. TABLE 9
Activity o f Cournermycin A 1 Against B a c t e r i a l I n f e c t i o n s i n Mice CD5o
Orqanism
S.C.
S ~ h y e o c o c c u nauReW Kfeb~e&?apflewnofliae S ~ e p t o c o c c wpyogeflu u i p t o c o c c ~pfleumovLiae
(rng/kg) p.0.
0.3
3
11.0
229 >500 >500
17.0 28
Edchehichia c o t i , Pdeudomonan aehUgiflOAa, Pnoteun v u f g n h i n , S a t m a n e t f a t y p h i , S a f m o n e t t a o c h o t t m u e t f e h i , Mycobactenium t u b e n c u t o A i A , a n d Mycobactenium eephaemuhium i n f e c t i o n s were r e s i s t a n t a t d o s e s of > 5 0 mg/kg
S.C.
a n d > 5 0 0 mg/kg p . 0 .
A d d i t i o n a l b a c t e r i a l s t u d i e s i n mice, r e p o r t e d b y G r u n b e r g
e t af.zo, r e v e a l e d a n e x c e l l e n t a c t i v i t y a g a i n s t “LiAAehia mefli n g i t i d i d i n f e c t i o n s w i t h C D 5 0 v a l u e s o f 2 rng/kg S . C . a n d 2 2 mgfkg p . 0 . Coumermycin A 1 g l v e s a s t r i k i n g r e s p o n s e , C D 5 o 1 4 mg/kg s . c . , on mice i n f e c t e d w i t h a m e n i n g o p n e u m o n i t i s a g e n t (psitticosis-lymphogranuloma-trachoma g r o u p ) . B e c a u s e o f i t s v e r y g o o d s p e c t r u m of a c t i v i t y v e r s u s gra m p o s i t i v e organisms , p a r t i c u l a r l y a g a i n s t staphylococcus, i n a d d i t i o n t o a p p r e c i a b l e a c t i v i t y a g a i n s t a v a r i e t y of gramn e g a t i v e b a c t e r i a , co u mer my ci n A 1 was r e c o g n i z e d as a p o t e n t i a l l y u s e f u l a n t i b i o t i c . Ho wev er , i n s p i t e o f i t s e x c e l l e n t ifl u i t n o a c t i v i t y , t h e i n s o l u b l e n a t u r e o f c oum e rm yc in A 1 made p a r e n t e r a l a d m i n i s t r a t i o n u n s a t i s f a c t o r y . Poor o r a l a b s o r p t i o n was a l s o o b s e r v e d . The l a t t e r was p a r t l y o v e r c o m e b y t h e u s e o f a water s o l u b l e N-methylglucamine a d m i x t u r e l o . I n 2 2 of 4 9 (45%) human cases ( d o s e d y r e s u m a b l y w i t h 1 . 0 g o f c o u m e r m y c i n A 1 P . o . ) , Nakazawa e t reported side eff e c t s o f a g a s t r o i n t e s t i n a l n a t u r e v i z . a n o r e x i a , n a u s e a , emesis, a b d o m i n a l p a i n , d i a r r h e a , e t c . S i d e e f f e c t s were more p r o n e t o develop with l a r g e r s i n g l e doses and l a r g e r t o t a l doses. Since b l o o d l e v e l c o n c e n t r a t i o n s were low a f t e r o r a l a d m i n i s t r a t i o n , i t would a p p e a r t h a t t h e g a s t r o i n t e s t i n a l e f f e c t s are due m a i n l y t o local r e a c t i o n s i n t h e g u t .
148 D a i l y 2 . 0 g d o s e s o f t h e d i s o d i u m s a l t of coumermycin A 1 were found t o g i v e b e t t e r c l i n i c a l r e s p o n s e t h a n d a i l y 1 . 0 g d o s e s 2 * . T r e a t m e n t of 64 p a t i e n t s w i t h l y m p h a d e n i t i s , m a s t i t i s , b r o n c h i t i s , t o n s i l l i t i s , phlegmon, f u r u n c l e , e t c . gave s a t i s f a c t o r y r e s u l t s i n 49 ( 7 7 % ) c a s e s w i t h f a i l u r e t o cure i n 1 2 (19%) cases. S a t i s f a c t o r y c l i n i c a l r e s u l t s were o b t a i n e d i n 1 8 of 2 2 ( 8 2 % ) S . au)LeuA i n f e c t i o n s w i t h f a i l u r e i n 4 cases. I n f e c t i o n s due t o a- and 8 - s t r e p t o c o c c i , c o r y n e b a c t e r i u m , and mixed o r g a n i s m s were almost a l l s a t i s f a c t o r y . C l i n i c a l s t u d i e s c a r r i e d o u t w i t h a n N-methylglucamine a d m i x t u r e o f coumermycin A 1 u n d e r Hoffmann-La Roche s p o n s o r s h i p i n i t i a l l y i n d i c a t e d e n c o u r a g i n g r e s u l t s . The a n t i b i o t i c seemed t o b e l a r g e l y f r e e from s i d e e f f e c t s . Of a t o t a l of 1 1 3 cases of v a r i o u s b a c t e r i a l i n f e c t i o n s , 75 ( 6 6 % ) appeared t o respond. O f t h e 5 7 s t a p h y l o c o c c a l i n f e c t i o n s , 36 ( 6 3 % ) seemed t o r e s p o n d . F u r t h e r t e s t i n g gave l e s s e n c o u r a g i n g r e s u l t s , s o t h e a n t i b i o t i c i n ve s t i g a t i on s we re t e r m i n a t e d
.
A n o t h e r a p p r o a c h t o overcoming t h e s h o r t c o m i n g s of s o l u b i l i t y a n d a b s o r p t i o n o f coumermycin A 1 was t a k e n by B r i s t o l L a b o r a t o r i e s , who p r e p a r e d a s e r i e s of c h e m i c a l l y m o d i f i e d cour n e r r n y ~ l n s ~ ~The . most p r o m i s i n g o f t h e s e , BL-e43, was t e s t e d Studies u t i l i z i n g i n humans a n d f o u n d to b e v e r y w e l l a b s o r b e d .
BL-C43 t h e mono- and d i s o d i u m s a l t s of coumermycin A 1 ( w i t h o u t Nm e t h y l g l u c a m i n e ) c o n s i s t e n t l y gave b l o o d l e v e l s o f l e s s t h a n 0 . 1 mcg/ml a f t e r an o r a l d o s e of 5 0 0 mg, w h e r e a s BL-C43 r e a c h e d a peak b l o o d l e v e l o f o v e r 1 5 mcg/ml i n 3 h o u r s a n d was s t i l l h i g h a f t e r 10 h o u r s ( 8 mcg/ml). However, i n s p i t e o f t h e e x t e n s i v e a n i m a l s t u d i e s i n d i c a t i n g t h e i n n o c u o u s n a t u r e of BL-C43, Phase I c l i n i c a l s t u d i e s r e v e a l e d an u n f a v o r a b l e t o x i c i t y a f t e r 10 d a y s o f d o s i n g a t 1 0 t o 2 0 % o f t h e h i g h e s t d o s e u s e d i n t h e dog t o x i c o l o g y s t u d i e s . F u r t h e r s t u d i e s were t h e r e f o r e t e r m i n a t e d .
6.3 Extraction, 6.3.1
s e p a r a t i o n and p u r i f i c a t i o n
Detection
E a r l y p a p e r chromatography a n d b i o a u t o g r a p h y ( a g a i n s t S . s t u d i e s a t B r i s t o l - B a n y u showed t h e p r e s e n c e of 4 s e p a r a t e a n t i b i o t i c m o i e t i e s (coumermycins A , B , C and D) i n t h e UU)LeUA)
149 f e r m e n t a t i o n b r ~ t h ~ ’ ~ ’ ~ At ’ ~Hoffmann-La ~ ) . Roche, o n l y 3 a n t i b i o t i c components ( a l s o a g a i n s t s . a u 4 e u d ) , sugordomycins D - 1 , D-2, and D-3, were d e t e c t e d ( F i g u r e 3 ) . H o w e v e r , a f o u r t h comp o n e n t , sugordomycin D-4, which h a d v i r t u a l l y n o a c t i v i t y ( v e r s u s S. auRCub), was d i s c o v e r e d g . F u r t h e r c o m p l e x i t y was d e m o n s t r a t e d by a 2 0 0 - t r a n s f e r C r a i g c o u n t e r c u r r e n t d i s t r i b u t i o n a t pH 8.2 ( F i g u r e 4 ) which r e v e a l e d t h a t sugordomycin D - 1 was i n f a c t a m i x t u r e o f a n t i b i o t i c components named i n o r d e r o f t h e i r a p p e a r a n c e i n t h e a p p a r a t u s : sugordomycin D - l a , D-lb, D - l c , a n d D-ld.
D-la
160
I40
I20
0-2, 0-3 A
I\
I
b
---
100
U.V. 80
=O
’ I
20
-
10
-
I I
60
40
20 0.
0
’ 20
I
0
~~
40
80
eo
100
120
140
160
180
200
TUBE N O
FIGURE 4: Craig countercurrent d i s t r i b u t i o n (200 tubes) o f coumermycin complex. Solvent system: d l o r o f o r m - i s o p r o p a n o l ~ . 0 6 7Mphosphate buffer a t pH 8.2 (1:1:2).
An e f f e c t i v e r e s o l u t i o n of sugordomycin D-2 and D-3 was a c h i e v e d a t pH 7 a f t e r 4 0 0 t r a n s f e r s ( F i g u r e 59).
However, a sample o f coumermycin o b t a i n e d from B r i s t o l Laboratories , subjected t o a Craig countercurrent d i s t r i b u t i o n , was f o u n d t o c o n t a i n t h e same components, a l b e i t i n d i f f e r e n t p r o p o r t i o n s , as i n t h e Hoffmann-La Roche c r u d e i s o l a t e s (unpubl i s h e d d a t a , Hoffmann-La Roche I n c . 1. Thus , a l l a c c u m u l a t e d e v i d e n c e l e a d s t o t h e c o n c l u s i o n t h a t sugordomycin D-2 i s i d e n -
150
60
4s 40
3s 30 26 20 16
10
6 0 0
40
80
(20
100
200
TUBE NO.
FIGURE 5 : Craig countercurrent d i s t r i b u t i o n (400 tubes) o f 0-2 and 0-3 f r a c t i o n (from F i g u r e 4). Solvent system: chloroform-isopropanol-0.067 M phosphate b u f f e r a t pH 7.0 (1:l:Z).
t i c a l w i t h coumermycin B , sugordomycin D - 3 i s i d e n t i c a l w i t h coumermycin C , and sugordomycin D - 4 ( w h i c h h a s e x t r e m e l y w e a k a c t i v i t y ) i s i d e n t i c a l w i t h coumermycin D . A s d e s c r i b e d e a r l i e r , t h e i d e n t i t y of sugordomycins D - l a and D-ld w i t h coumermycins A 1 and A P , r e s p e c t i v e l y , h a s b e e n e s t a b l i s h e d a n d t h e name sugordomycin h a s been w i t h d r a w n . E v i dence f o r t h e p r e s e n c e of a d d i t i o n a l D - l components, D-lb a n d D - l c , was g l e a n e d from t h e C r a i g d i s t r i b u t i o n ( F i g . 4 ) and s u p p o r t e d by t h e s p e c t r a l c h a r a c t e r i s t i c s ( I . R . , U.V. ,N.M.R.)of t h e c o r r e s p o n d i n g f r a c t i o n s ( t u b e s 5 0 - 8 0 ) . Whether t h e p e a k s ( D - l b a n d D - l c ) were c a u s e d by one or by two components was n e v e r s e c u r e l y e s t a b l i s h e d , as n o o t h e r d i f f e r e n c e among t h e f r a c t i o n s i n t h i s r e g i o n was d i s c e r n i b l e . Thus t h e d e n o t a t i o n D - l b , was u s e d t o imply t h a t e i t h e r D - l b (&I or D-lc or a m i x t u r e of t h e t w o was p r e s e n t . I t is also reasonable to expect t h a t B ( D - 2 ) and C (D-3) may e a c h be e i t h e r o f 2 i s o m e r s ('a o r 2, 5a or 2 )or m i x t u r e s o f b o t h 8 . N o d e t a i l e d a n a l y s e s o f t h e C r a z distributions carried out at Bristol-Banyu R e s e a r c h I n s t i t u t e have a p p e a r e d .
(s)
A.
Bioassay
Coumermycins a r e d e t e c t e d by t h e s t a n d a r d c u p - p l a t e a g a r d i f f u s i o n a s s a y , w i t h S . auRLW as t h e t e s t o r g a n i s m .
A s o l v e n t s y s t e m u s e d f o r n o v ~ b i o c i nwas ~ ~ adapted a t Roche as f o l l o w s for coumermycin. Samples (0.5-10 mcg) were a p p l i e d t o Whatman No. 1 p a p e r which, a f t e r e v a p o r a t i o n o f t h e
1 51 s o l v e n t , was wet w i t h t h e s t a t i o n a r y p h a s e ( m e t h a n o l - 1 - o c t a n o l , 5 : l ) t o a p o i n t j u s t below t h e sample s p o t . A f t e r b l o t t i n g , t h e p a p e r s t r i p was a l l o w e d t o e q u i l i b r a t e a t room t e m p e r a t u r e i n a c o v e r e d chromatography jar c o n t a i n i n g some s t a t i o n a r y p h a s e . Development was c a r r i e d o u t by d e s c e n t of t h e mobile p h a s e , 0 . 1 M p o t a s s i u m hydrogen p h o s p h a t e a d j u s t e d t o pH 8 . 2 and s a t u r a t e d with 1-octanol. Development t i m e was 1 5 - 2 0 h r . V i s u a l i z a t i o n was most r e l i a b l y c a r r i e d o u t by b i o a u t o g r a p h y v e r s u s S. a U R C U . 4 . A t y p i c a l PC of c r u d e a n t i b i o t i c s o l i d s i s s k e t c h e d i n F i g u r e 69. A c w a 2 0 - 2 5 m O...I.~I.~ TI-*
DLVCNT
FRONT
'DP\
nf
Whatman No. 1 paper was wet w i t h methanol-1-octanol ( 5 : l ) t o a p o i n t j u s t below t h e sample spot. A f t e r e q u i l i b r a t i o n , descending development was c a r r i e d o u t w i t h 0.1 M K,HPO, a t pH 8.2 s a t u r a t e d with 1-octanol (15-20 h r ) Assay organism was S@ky~ococcr~AaWeu.6.
.
The Rf of coumermycin (presumably s t i l l a m i x t u r e ) i n a number of s o l v e n t s y s t e m s i s d e s c r i b e d 3 3 4 . R e s o l u t i o n i n t o coumermycins A, B, C and D was e f f e c t e d by d e s c e n d i n g PC f o r 18 h r a t 22-23' u s i n g a c e t o n e and 0 . 1 M t r i e t h a n o l a m i n e a d j u s t e d t o pH 7 w i t h a c e t i c a c i d ( 2 : 3 ) ; b i o a u t o g r a p h y was c a r r i e d o u t w i t h S. auRCu.4. Q u a n t i t a t i v e a n a l y s i s a t 31tO nm was e f f e c t e d by s c a n n i n g w i t h a P h o t o v o l t D e n s i c o r d Z 4 .
c.
TLC
R e s o l u t i o n of t h e coumermycin components was a c c o m p l i s h e d on s i l i c a g e l G w i t h a c e t i c a c i d - w a t e r - m e t h a n o l - c a r b o n t e t r a chloride (2:0.7:15:100) a s shown i n F i g u r e 79. V i s u a l i z a t i o n was e f f e c t e d by s p r a y i n g w i t h a 1 0 % f e r r i c c h l o r i d e and p o t a s s i um f e r r i c y a n i d e s o l u t i o n ( 1 : l ) .
152
FIGURE 7: Thin-layer chromatographic separation o f coumermycin complex on s i l i c a g e l G : a c e t i c acid-water-methanol-carbon tetrachloride (2:0.7:15: 100). Visualization by spraying w i t h 10%f e r r i c chloride-potassium f e r r i cyanide (1:l).
D.
Craig countercurrent analysis
C r a i g c o u n t e r c u r r e n t a n a l y s i s was u t i l i z e d f o r b o t h p r e p a r a t i v e and a n a l y t i c a l p u r p o s e s . I t was p a r t i c u l a r l y u s e f u l f o r analyzing t h e performance of the f r a c t i o n a l l i q u i d e x t r a c t i o n column, and i t w i l l be t r e a t e d u n d e r t h a t t o p i c .
E.
Degradative chemical a n a l y s i s
The p u r i f i c a t i o n o f m i x t u r e s o f c e r t a i n coumermycins can b e f o l l o w e d by c a r r y i n g o u t a q u a n t i t a t i v e d e t e r m i n a t i o n of t h e carried out a r a t i o of t h e p y r r o l e m o i e t i e s . Kawaguchi e t 1x1.~ t r a n s e s t e r i f i c a t i o n with methanol and analyzed t h e r e s u l t i n g Claridge e t hydrolyzed with barium p y r r o l e e s t e r s by G C . h y d r o x i d e and employed a PC-UV a n a l y s i s f o r 5 - m e t h y l p y r r o l e - 2 c a r b o x y l i c a c i d and p y r r o l e - 2 - c a r b o x y l i c a c i d . S c a n n e l l ’ u t i l i z e d d i r e c t p y r o l y s i s - G C t o q u a n t i t a t i v e l y a n a l y z e f o r 2-methylp y r r o l e and p y r r o l e .
1 53 6.3.2
Isolation
The r a t i o of coumermycins i n t h e f e r m e n t a t i o n b r o t h s was f o u n d t o v a r y c o n s i d e r a b l y d e p e n d i n g on b o t h medium c o m p o s i t i o n and c u l t u r e s t r a i n , w i t h t h e r e l a t i v e amount of coumermycin A l r a n g i n g from 4 0 t o 7 5 % 2 4 . The a d d i t i o n o f c o b a l t s a l t s t o t h e f e r m e n t a t i o n medium was r e p o r t e d t o l e a d t o a l m o s t e x c l u s i v e p r o d u c t i o n o f coumermycin A 1 ( i n e x c e s s of 9 3 % ) making i t s i s o l a t i o n r e l a t i v e l y easy. C l a r i d g e e t ~ 2 1 a.l s~o ~r e p o r t e d t h e i s o l a t i o n of a UV m u t a n t which p r o d u c e d e s s e n t i a l l y o n l y coumermycin A components. The i s o l a t i o n of p u r e coumermycin A , from t h e coumermycin complex i n a p r a c t i c a l manner would have been u n t e n a b l e were i t not f o r t h e f r a c t i o n a l l i q u i d e x t r a c t i o n column26. D i s t r i b u t i o n d a t a from t h e C r a i g a p p a r a t u s were a p p l i e d d i r e c t l y t o column t e c h n o l o g y making p o s s i b l e t h e i s o l a t i o n o f p u r e coumermycin A , on a r e l a t i v e l y s m a l l s c a l e ( l a b o r a t o r y ) t o l a r g e s c a l e ( p r o d u c tion) basis. The d i s c o v e r y o f f e r m e n t a t i o n c o n d i t i o n s which r e s u l t e d i n a l m o s t e x c l u s i v e p r o d u c t i o n o f coumermycin A, o b v i a t e d t h e need f o r t h i s s e p a r a t i o n . Nevertheless, the i s o l a t i o n of p u r e coumermycin A 1 which was c a r r i e d o u t a t Hoffmann-La Roche i n two s t a g e s i s a l s o p r e s e n t e d . The i s o l a t i o n o f t h e o t h e r a n t i b i o t i c components h a s been c a r r i e d o u t by d i s t r i b u t i o n and r e - d i s t r i b u t i o n i n t h e C r a i g a p p a r a t u s , making o n l y l i m i t e d amounts o f t h e s e s u b s t a n c e s F i r s t p r i o r i t y was t o o b t a i n t h e most a c t i v e a n t i available. b i o t i c coumermycin A, f o r b i o l o g i c a l e v a l u a t i o n . The i s o l a t i o n o f coumermycin A2 i s p r e s e n t e d l a t e r as a t y p i c a l example.
An i s o l a t i o n p r o c e d u r e 3 f o r a coumermycin A1 r i c h ferment a t i o n i s d e s c r i b e d below: The whole b r o t h ( 2 6 5 0 a ) was a d j u s t e d t o pH 6 w i t h s u l f u r i c a c i d a n d e x t r a c t e d w i t h a n e q u a l volume o f m e t h y l i s o b u t y l C o n c e n t r a t i o n i n uacuo p r e c i p i t a t e d t h e c r u d e ketone (MIBK). a n t i b i o t i c which was s l u r r i e d s e v e r a l times w i t h M I B K and metha n o l t o remove c o n t a m i n a t i n g l i n s e e d o i l . The p r o d u c t ( 3 6 7 . 3 g ) was t h e n d i s s o l v e d i n 2 0 % a q . a c e t o n e w i t h t h e a d d i t i o n o f s u f f i c i e n t ammonium h y d r o x i d e t o b r i n g t h e s o l u t i o n t o pH 1 0 , dec o l o r i z e d w i t h c h a r c o a l , and r e p r e c i p i t a t e d by a d j u s t i n g t o pH 6. T h e f i l t e r e d p r e c i p i t a t e ( 3 0 0 g ) was d i s s o l v e d i n 1 5 0 0 m l of dimethylformamide a n d f i l t e r e d . To t h e f i l t r a t e was added 1 0 0 m l o f a 4 2 % s o l u t i o n o f sodium 2 - e t h y l h e x a n o a t e i n d r y nbutanol. Spontaneous c r y s t a l l i z a t i o n occurred a f f o r d i n g 2 7 2 . 5 g of t h e monosodium s a l t o f coumermycin A1 of b e t t e r t h a n 9 9 % p u r i t y by G C a s s a y ( f o r p y r r o l e e s t e r s ) . Kawaguchi e t described the following i s o l a t i o n proc e d u r e f o r coumermycin ( p r e s u m a b l y t h e c o m p l e x ) . The f e r m e n t a t i o n b r o t h ( 5 a ) was f i l t e r e d a t pH 8 . 0 . The f i l t r a t e was a d j u s t e d t o pH 6 . 0 a n d was e x t r a c t e d w i t h 2 R o f M I B K . T h e mycel i a l cake was e x t r a c t e d w i t h 5 0 0 m l o f a c e t o n e . The e x t r a c t was c o n c e n t r a t e d in uacuo a n d t h e n e x t r a c t e d a t pH 6 w i t h 2 0 0 m l of M I B K . The two MIBK e x t r a c t s ( f r o m c e l l s and f i l t r a t e ) were combined, washed w i t h 5 0 0 m l of w a t e r , a n d mixed w i t h 1 a o f
154
c o l d water f o l l o w e d by sodium h y d r o x i d e t o pH 1 0 . 0 . The a q u e o u s p h a s e was s e p a r a t e d , a d j u s t e d t o pH 6 . 0 , a n d e x t r a c t e d w i t h 3 0 0 m l o f e t h y l a c e t a t e . A f t e r c o n c e n t r a t i o n i n U Q C U O t o 30 m l , 1 5 0 m l of p e t . e t h e r was a d d e d . The l i g h t brown p r e c i p i t a t e was washed with' m e t h a n o l t o remove some c o l o r e d i m p u r i t i e s a n d d r i e d t o g i v e 7 5 0 mg o f coumermycin. The coumermycin ( 4 0 0 mg) was d i s s o l v e d i n 1 0 0 m l of e t h y l a c e t a t e a n d a d s o r b e d on a column o f 2 0 g of a l u m i n a p r e v i o u s l y t r e a t e d w i t h s u l f u r i c a c i d . A 2 0 0 ml wash w i t h e t h y l a c e t a t e was f o l l o w e d by e l u t i o n w i t h m e t h a n o l c o l l e c t e d i n 1 0 m l f r a c tions. Combination o f t h e a c t i v e f r a c t i o n s and e v a p o r a t i o n i n vacu0 gave a p a l e y e l l o w s o l i d which was r e c r y s t a l l i z e d ;wice from methanol t o g i v e 7 0 mg of coumermycin, m.p. 2 1 8 - 2 2 0
.
F u r t h e r p u r i f i c a t i o n was a c h i e v e d by a C r a i g c o u n t e r c u r r e n t d i s t r i b u t i o n u s i n g c a r b o n tetrachloride-chloroformmethanol-water (5:1:5:1 v / v ) . A f t e r 50 t r a n s f e r s t h e bioassay, U.V.,andweight p e a k s were f o u n d i n t u b e 2 6 . Lyophilization of t e n t u b e s a r o u n d t h i s peak gave " p u r e " coumermycin. P u r i f i c a t i o n by a l u m i n a c h ~ i o m a t o g r a p h ywas ~ e f f e c t e d by d i s s o l v i n g 3 9 . 7 mg ( 5 0 % coumermycin A l ) or c r u d e coumermycin i n e t h y l a c e t a t e and a b s o r b i n g i t on 2 g o f s u l f u r i c a c i d - t r e a t e d a l u m i n a (pH 4 . 7 ) . A wash w i t h e t h y l a c e t a t e f o l l o w e d by e l u t i o n w i t h methanol and a c i d i c methanol a f f o r d e d , on combining t h e a c t i v e f r a c t i o n s , 1 2 . 4 mg of p a l e y e l l o w i s h powder ( p r e s u m a b l y 7 0 % coumermycin A l l . The i s o l a t i o n of p u r e coumermycin A 1 a t Hoffmann-La Roche l9 and Stage 2 2 7 .
was e f f e c t e d i n two s t a g e s - - S t a g e
S t a g e 1. Crude a n t i b i o t i c s o l i d s ( f o r use i n S t a g e 2 ) were i s o l a t e d as f o l l o w s . Whole b r o t h ( 2 3 , 0 0 0 L c o n t a i n i n g 1 3 0 mg/ L) was a d j u s t e d t o pH 4 . 5 and f i l t e r e d . The f i l t r a t e was d i s c a r d e d . The f i l t e r - c a k e ( c e l l s ) was washed w i t h 7 6 0 0 II of n - b u t a n o l , f i l t e r e d , a n d t h e b u t a n o l e x t r a c t was a d j u s t e d t o pH 5 . 5 and c o n c e n t r a t e d i n UQCUO t o 1 / 4 0 volume. A d d i t i o n of 5 volumes o f p e t . e t h e r and f i l t r a t i o n gave 1 6 kg of c r u d e sodium s a l t ( c a . 1 0 % p u r e ) which was d i g e s t e d w i t h 380 L o f HC1-acetone. The i n s o l u b l e p o r t i o n ( 6 - 9 k g ) was removed by f i l t r a t i o n and discarded. The a c e t o n e s o l u t i o n was c o n c e n t r a t e d i n U Q C U O t o 8 - 1 0 II , 5 7 L o f w a t e r was added a n d t h e p r e c i p i t a t e f i l t e r e d , washed w i t h 1 9 I I of b e n z e n e and t h e n w i t h 2 0 & o f m e t h a n o l t o g i v e 1 . 3 kg o f c r u d e a n t i b i o t i c s o l i d s . A c o u n t e r - c u r r e n t d i s t r i b u t i o n of t h e c r u d e a n t i b i o t i c s o l i d s i s shown i n F i g u r e 4 . The a p p a r a t u s was o v e r l o a d e d s o maximal amounts o f p u r e a n t i b i o t i c s c o u l d be o b t a i n e d a f t e r r e c y c l i n g some o f t h e r e s u l t i n g fractions
-
.
Stage 2 . F r a c t i o n a l l i q u i d e x t r a c t i o n was c a r r i e d o u t as f o l l o w s : A p r e l i m i n a r y p u r i f i c a t i o n was c a r r i e d o u t by d i s s o l v i n g 1 kg b a t c h e s ( t o t a l o f 3.85 k g ) o f c r u d e a n t i b i o t i c s o l i d s ( c a . 4 0 % coumermycin A l ) i n 1 7 0 L of a s o l v e n t m i x t u r e (SO) c o n t a i n i n g ( b y w e i g h t ) 2 3 % i s o p r o p a n o l , 75% c h l o r o f o r m , a n d 2 % water. A f t e r f i l t r a t i g n o f i n s o l u b l e m a t e r i a l , t h e f i l t r a t e was c o n c e n t r a t e d a t 25 i n U Q C U O ( 5 0 mm Hg) s o as t o remove
155 v i r t u a l l y a l l o f t h e c h l o r o f o r m , a d j u s t i n g t h e volume w i t h i s o p r o p a n o l as r e q u i r e d t o m a i n t a i n a volume o f 5 0 a . The r e s u l t i n g s u s p e n s i o n was f i l t e r e d , a n d t h e f i l t e r - c a k e was washed thoroughly with isopropanol a f f o r d i n g a t o t a l of 3.0 kg (on a d r y b a s i s ) of wet s o l i d c o n t a i n i n g 9 1 % of t h e a n t i b i o t i c a c t i v ity. The f i l t r a t e ( 0 . 8 6 k g , 2 2 % o f t h e t o t a l w e i g h t ) c a n b e reworked f o r a second c r o p of crude a n t i b i o t i c . The 3 . 0 kg ( d r y b a s i s ) o f w e t s o l i d , d i s s o l v e d i n b a t c h e s o f 7 5 0 g i n a m i x t u r e o f 1 . 2 3 a o f i s o p r o p a n o l and 5 . 6 7 k of f r e s h l y d i s t i l l e d t e t r a h y d r o f u r a n , was pumped a t t h e r a t e o f 4 0 0 m l (40 g ) p e r h o u r i n t o t h e c e n t e r o f a p r e - e q u i l i b r a t e d 75 mm x 7 m r e c i p r o c a t i n g p l 2 t e f r a c t i o n a l l i q u i d e x t r a c t i o n c o l umn26 m a i n t a i n e d a t 25-27 , a m p l i t u d e 0 . 9 4 cm, a g i t a t i o n 2 0 0 s t r o k e s p e r min. I n t o t h e t o p o f t h e column was f e d 1 2 0 ml/min o f s o l v e n t m i x t u r e SL a n d i n t o t h e b o t t o m was f e d 4 0 0 ml/min SA (pH 8 . 5 - 8 . 6 ) . SL was t h e l o w e r p h a s e a n d S was t h e u p p e r p h a s e disodium phosphate, o b t a i n e d by m i x i n g a s o l u t i o n o f 2 2 . 1 1 k g 9.6 o f 2 % p h o s p h o r i c a c i d , a n d 2,430 2 of d e i o n i z e d water w i t h 42.0 kg of c h l o r o f o r m and 421 kg of i s o p r o p a n o l f o l l o w e d by 1 5 2 5 k of S O . A s SA emerged from t h e column, t h e pH was a d j u s t e d t o 6.2-6.4 w i t h a s t r e a m o f 1 0 % p h o s p h o r i c a c i d ( c a . 1 6 m l / m i n ) . The o r g a n i c p h a s e S L was c o n d u c t e d i n t o an a g i t a t e d r e c e i v e r c o n t a i n i n g some SA a d j u s t e d t o a n d m a i n t a i n e d a t pH 5 . 5 - 5 . 7 . A f t e r t h e f e e d s u p p l y was e x h a u s t e d (100 h r ) , t h e s o l v e n t s (SL and SA) were pumped f o r an a d d i t i o n a l 3 h r b e f o r e t h e e q u i p m e n t was s h u t down.
04
Coumermycin A 1 ( 1 . 5 8 kg or 4 1 % o f t h e o r i g i n a l w e i g h t ) o f b e t t e r t h a n 9 7 % p u r i t y was r e c o v e r e d f r o m t h e p H - a d j u s t e d o r g a n i c l a y e r (SL) a f t e r s e p a r a t i o n o f :he p h a s e s , w a s h i n g w i t h w a t e r a n d c o n c e n t r a t i o n t o 6 0 9, a t 2 5 i n vacua (as d e s c r i b e d i n t h e p r e l i m i n a r y p u r i f i c a t i o n ) , f i l t r a t i o n , and washing with isopropanol. The r e m a i n i n g coumermycin a n t i b i o t i c s ( 1 . 0 4 k g , 2 7 % of t h e o r i g i n a l w e i g h t ) w e r e r e c o v e r e d f r o m t h e a d j u s t e d (pH 5 . 5 5 . 7 ) a q u e o u s p h a s e (SA) By e x t r a c t i o n w i t h 3 X 1 5 2! o f S O , conc e n t r a t i o n t o 10 k a t 2 5 in v a c u o ( a s w i t h coumermycin A l l , f i l t r a t i o n , a n d washing w i t h i s o p r o p a n o l .
T o e s t a b l i s h t h e p u r i t y , a s a m p l e ( 1 . 0 0 g ) o f coumermycin
A l o b t a i n e d from t h e f r a c t i o n a l l i q u i d e x t r a c t i o n column was d i s s o l v e d i n 40 m l of e q u i l i b r a t e d o r g a n i c ( l o w e r ) phase and placed i n tube 0 i n a Craig a l l - g l a s s apparatus c o n s i s t i n g of 2 0 0 t u b e s , each having a c a p a c i t y of 1 0 0 m l . The s o l v e n t s y s tem was p r e p a r e d by m i x i n g 5 k o f c h l o r o f o r m , 5 k o f i s o p r o p a n o l , and 10 2 o f 0 . 1 M d i p o t a s s i u m h y d r o g e n p h o s p h a t e a d j u s t e d t o pH 8.4 with potassium hydroxide. I n t o e a c h t u b e was a d d e d 4 0 m l o f e q u i l i b r a t e d lower ( s t a t i o n a r y ) p h a s e . The u p p e r ( m o b i l e ) p h a s e was f e d a u t o m a t i c a l l y a t 2 5 - 2 7 ' f o r 2 0 0 c y c l e s w i t h a r e s t t i m e o f 1 2 m i n u t e s p e r c y c l e . A f t e r t h e d i s t r i b u t i o n was comp l e t e ( 2 6 h r ) , a 0 . 1 m l a l i q u o t o f e a c h p h a s e was w i t h d r a w n from e v e r y f i f t h t u b e a n d d i l u t e d t o 2 m l w i t h 6 0 % a q u e o u s metha n o l f o r u l t r a v i o l e t a n a l y s i s . The a b s o r b a n c e a t t h e maximum i n t h e 260-285 nm r e g i o n ( t h e e x a c t p o s i t i o n v a r i e d f o r e a c h component) was d e t e r m i n e d a n d a p l o t o f t h e t o t a l a b s o r b a n c e f o r
1 56
t h e sampled t u b e shown i n F i g u r e 8 , was u s e d as a g u i d e i n comb i n i n g f r a c t i o n s . A n a l y s i s c o u l d a l s o be c a r r i e d o u t by b i o a s s a y , TLC, o r PC b i o a u t o g r a p h y . The f r a c t i o n s were combined as i n d i c a r e d below and a d j u s t e d t o pH 4 w i t h p h o s p h o r i c a c i d . A f t e r s e p a r a t i n g t h e p h a s e s , t h e u p p e r aqueous l a y e r was e x t r a c t e d twice w i t h a n e q u a l volume of e t h y l a c e t a t e . The combined o r g a n i c p h a s e s were d r i e d (Na2S04), f i l t e r e d , and e v a p o r a t e d . Tubes 0-35 3 6-70 71-90 91-200
Weight (discarded
Coumermycin Component
-
1
8 0 0 mg 5 0 mg
A1 m i x t u r e o f A 1 and D - l b , c
(discarded)
-
Pur'e coumermycin A1 ( 8 0 0 mg) a p p e a r e d . i n t u b e s 35-70. Tubes 7 1 - 9 0 c o n t a i n e d a m i x t u r e ( 5 0 mg) e s t i m a t e d t o c o n t a i n a maximum o f 2 5 mg ( 3 % of t h e t o t a l 8 5 % r e c o v e r y ) o f D - l b , c .
TUBE No. FIGURE 8. C r a i g counter-current d i s t r i b u t i o n (200 tubes) o f coumermycin A 1 i s o l a t e d by f r a c t i o n a l l i q u i d e x t r a c t i o n . Solvent ayetem: Chloroformisopropanol-0.067M phosphate b u f f e r a t pH 8.2 (1:1:2).
Coumermycin A2 was o b t a i n e d from a f e r m e n t a t i o n which p r o duced a r e l a t i v e l y h i g h p r o p o r t i o n o f t h i s component4. Crude coumermycin ( 3 0 0 g ) , i s o l a t e d by M I B K e x t r a c t i o n as p r e v i o u s l y describedpwas s t i r r e d with 3 of t e t r a h y d r o f u r a n a n d , a f t e r removal o f 5 1 g of u n d i s s o l v e d m a t e r i a l , was c o n v e r t e d t o t h e sodium s a l t by d r o p w i s e a d d i t i o n o f 0 . 1 N N a O H u n t i l pH 6.5 was r e a c h e d . A d d i t i o n o f 1 5 a o f e t h a n o l f o l l o w e d by c o n c e n t r a t i o n i n vacuo t o a small volume gave t h e sodium s a l t which was r e c r y s t a l l i z e d from 1:l m e t h y l e n e c h l o r i d e - m e t h a n o l (1 g / 1 5 r n l ) by
157 h e a t i n g t o b o i l i n g , f i l t e r i n g , b o i l i n g o f f some m e t h y l e n e c h l o r i d e , a d d i n g m e t h a n o l t o t h e h a z e p o i n t , a n d a l l o w i n g t o cool slowly. T h e p r o d u c t was t h e n p u r i f i e d b y C r a i g c o u n t e r - c u r r e n t d i s t r i b u t i o n ( 2 0 0 t u b e s ) u s i n g 40 m l of a q u e o u s p h a s e a n d 20 m l of s o l v e n t ( u p p e r ) p h a s e . T h e s o l v e n t s y s t e m was p r e p a r e d b y e q u i l i b r a t i n g 1 5 g of 0 . 2 5 M aq. t r i e t h a n ~ l a m i n e l i~n water m i x e d w i t h 30 a o f m e t h y l e t h y l k e t o n e a n d a d j u s t e d t o pH 8 . 0 w i t h acetic a c i d . A s o l u t i o n of 2 0 g o f material was p l a c e d i n t h e f i r s t 1 6 t u b e s a n d 700 t r a n s f e r s were made. The coumermycin A Z , i s o l a t e d from t u b e s 1 0 5 - 1 2 9 from two r u n s ( 9 . 0 g t o t a l ) , was d i s s o l v e d a n d p l a c e d i n t h e f i r s t 1 6 t u b e s a n d , a f t e r 700 t r a n s f e r s , was c o l l e c t e d ( 2 . 5 g ) i n t u b e s 1 1 5 - 1 3 4 . A f t e r conversion t o t h e f r e e a c i d a n d r e c o n v e r s i o n t o t h e s o d i u m s a l t , 1 . 1 4 g of c r y s t a l l i n e sodium s a l t was o b t a i n e d . A n a l y t i c a l d a t a were given.
6.4 L i t e r a t u r e
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General Introduction
Gentamicin Introduction Producing organism 2.1.2 Chemical d e s c r i p t i o n and s t r u c t u r e 2.1.3 Summary of t h e r a p e u t i c u s e 2.2 E x t r a c t i o n of t h e g e n t a m i c i n c o m p l e x 2.2.1 E x t r a c t i o n of t h e c o m p l e x ( M e t h o d 1) 2.2.2 E x t r a c t i o n of t h e complex (Method 2 ) 2.3 S e p a r a t i o n o f the g e n t a m i c i n components 2.3.1 S e p a r a t i o n by p a p e r a n d t h i n l a y e r chromatography 2.3.2 S e p a r a t i o n o f t h e g e n t a r n i c i n c o m p l e x by column c h r o m a t o g r a p h y 2.4 S e p a r a t i o n of some m i n o r c o m p o n e n t s of t h e g e n t a m i c i n complex 2.4.1 S e p a r a t i o n a n d i s o l a t i o n of g e n t a m i c i n A 2.4.2 S e p a r a t i o n of m i n o r c o m p o n e n t s by p a p e r chromatography 2.4.3 Column c h r o m a t o g r a p h i c s e p a r a t i o n o f t h e minor g e n t a m i c i n components 2.4.4 S e p a r a t i o n of C z a 2.4.5 S e p a r a t i o n of C2b 2.5 Q u a n t i t a t i v e d e t e r m i n a t i o n of t h e g e n t a m i c i n components 2.6 Literature cited
2.1 2.1.1
-
-
3
.
................................
.................................. .......................... .......... .................. .......... ........ ........ ....... .............................. ....................... ............................ ....
.............................. ................. ........................... ........................... .................................... ..............................
Sisomicin Introduction Producing organism 3.1.2 Chemical d e s c r i p t i o n and s t r u c t u r e 3.1.3 Structure 3.1.4 Summary o f t h e r a p e u t i c u s e 3.2 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n 3.2.1 E x t r a c t i o n of c r u d e a n t i b i o t i c 3.2.2 P u r i f i c a t i o n of s i s o m i c i n 3.2.3 A l t e r n a t e p u r i f i c a t i o n of s i s o m i c i n 3.3 Microbiological assay 3.4 Minor components 3.4.1 S e p a r a t i o n of minor components 3.4.2 Chemistry and s t r u c t u r e 3.5 Literature cited
3.1 3.1.1
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-
.................................. .......................... .......... ................................... .................. ....... .............. ................... ......... ......................... .............................. .............. ..................... ..............................
162 163 163 163 163 164 164 165 165 166 167 168 168 169 170 171 172 172 173 176 176 176 176 177 177 177 177 178 179 179 179 179 179
160 4.
K a n amy c i n 4.1 Introduction 4.1.1 Producing organism 4.1.2 Chemical d e s c r i p t i o n a n d s t r u c t u r e 4.1.3 Summary o f t h e r a p e u t i c u s e 4.2 E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n 4.2.1 S e p a r a t i o n of k a n a m y c i n A 4.2.2 S e p a r a t i o n of kanamycin B 4.2.3 S e p a r a t i o n of k a n a m y c i n C 4.3 Literature cited
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5
.
................................... 181 ........................... 181 ........... 1 8 1 ................... 181 ........ 1 8 2 .................... .................... 11 8823 .................... 183 ............................... 1 8 4
Tobramycin Introduction 5 1. 1 Producing organism 5.1.2 Chemical d e s c r i p t i o n a n d s t r u c t u r e 5.1.3 Summary o f t h e r a p e u t i c u s e . , 5.2 E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n 5.2.1 E x t r a c t i o n of t h e c o m p l e x 5.2.2 S e p a r a t i o n of t h e n e b r a m y c i n c o m p o n e n t s 5.2.3 P r o d u c t i o n of t o b r a m y c i n b y h y d r o l y s i s 5.3 Literature cited -
5.1 -
.
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................................... ........................... ........... ................. ........ .................... ......
....... ...............................
6
.
186
186 186 186 187 187 187 188 189
V e r d a m i c i n a n d A n t i b i o t i c 6-52 Introduction Producing organism 6.1.2 Chemical d e s c r i p t i o n a n d s t r u c t u r e 6 . 1 . 2 . 1 Verdamicin 6 . 1 . 2 . 2 A n t i b i o t i c G.52 6.1.3 Summary of b i o l o g i c a l a c t i v i t y o f v e r d a m i c i n a n d 6.52 6 . 1 . 3 . 1 Verdamicin 6 . 1 . 3 . 2 A n t i b i o t i c 6.52 6.2 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n 6.3 Literature cited -
................................... ........................... 11 99 00 ........... 1190 90 ................................... .............................. 191 ..................................... 1 9 1 ................................... 191 .............................. 192 ........ 1 9 2 ............................... 193 7 . N eomy c i n s 7.1 I n t r o d u c t i o n ................................... 1 9 4 7.1.1 P r o d u c i n g o r g a n i s m s .......................... 194 7.1.2 Chemical d e s c r i p t i o n a n d s t r u c t u r e ........... 1 9 4 7.2 T h e r a p e u t i c u s a g e .............................. 1 9 5 7.3 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n ........ 1 9 6 7.3.1 E x t r a c t i o n of c r u d e a n t i b i o t i c ............... 1 9 6 7.3.2 D e t e c t i o n .................................... 197 6.1 6.1.1
7.3.3 7.3.3.1
.....
P u r i f i c a t i o n and c h r o m a t o g r a p h i c methods S e p a r a t i o n u s i n g anion-exchange chromatography 7 . 3 . 3 . 2 S e p a r a t i o n u s i n g c a t i o n e x c h a n g e chromatography 7.3.3.3 Gas-liquid chromatographic a n a l y s i s 7.4 Literature cited
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198
....................................... 1 9 8 ....................................... 198 .......... 1 9 9 ............................... 1 9 9
161
8
.
Paromomycins Introduction 9.1.1 Producing organisms 8.1.2 Chemical d e s c r i p t i o n and s t r u c t u r e 8.2 Therapeutic usage 8.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n 8.3.1 E x t r a c t i o n of c r u d e a n t i b i o t i c 8.3.2 Detection 8.3.3 P u r i f i c a t i o n and c h r o m a t o g r a p h i c methods 8 . 3 . 3 . 1 R e s o l u t i o n of N - a c e t y l p a r o m o m y c i n s by c e l l u l o s e p a r t i t i o n chromatography 8 . 3 . 3 . 2 R e s o l u t i o n of paromomycins by a n i o n exchange chromatography 8 . 3 . 3 . 3 R e s o l u t i o n o f p a r o m o m y c i n s by g a s - l i q u i d chromatography 8.4 Literature cited 8.1 -
................................. ........................ ......... ............................ ...... ............. .................................. ... ......... ...................... ............................. .............................
Lividomycins Introduction 9.1.1 Producing organism 9.1.2 Chemical d e s c r i p t i o n and s t r u c t u r e 9.2 Therapeutic usage 9.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n E x t r a c t i o n of c r u d e a n t i b i o t i c 9.3.1 Detection 9.3.2 9.3.3 Purification 9.4 Literature cited 9.1 -
-
10
.
................................. ......................... ......... ............................ ...... ............. .................................. ............................... .............................
Butirosins 10.1 Introduction 10.1.1 P r o d u c i n g o r g a n i s m 10.1.2 Chemical d e s c r i p t i o n and s t r u c t u r e 1 0 . 2 Therapeutic usage 10.3 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n 10.3.1 E x t r a c t i o n of c r u d e a n t i b i o t i c 10.3.2 Detection 10.3.3 P u r i f i c a t i o n 10.4 Literature cited
................................. ......................... ......... ............................ ...... ............. .................................. ............................... .............................
202 202 202 202 203 203 204 204
205 205 205 206
208 208 208 208 208 208 208 210 210 211
211 211 212 212 212 212 212 213
162
i.
General Introduction
The a m i n o g l y c o s i d e s a r e , as a c l a s s , one o f t h e most pot e n t a n d w e l l s t u d i e d g r o u p s of a n t i b i o t i c s . Common t o aminog l y c o s i d e s i s an a m i n o c y c l i t o l r i n g w h i c h may b e e i t h e r s t r e p t amine ( I ) or 2 - d e o x y s t r e p t a m i n e ( I I ) , t h u s a l l o w i n g a f u r t h e r s u b c l a s s i f i c a t i o n ( F i g . 1). The s t r e p t a m i n e - c o n t a i n i n g a n t i b i o t i c s w i l l b e d e a l t w i t h e l s e w h e r e i n t h i s volume w h e r e a s t h i s c h a p t e r w i l l be concerned e x c l u s i v e l y w i t h t h e l a t t e r group.
M
I
n
H
FIGURE 1 .
Streptemine
(I) and Z-deoxystreptemine( 11).
The 2 -deoxys t r e p t a m i n e component i s r e a d i l y i s o l a t e d by a c i d h y d r o l y s i s o f a m i n o c y c l i t o l s . I t was f i r s t d e s c r i b e d as a d e g r a d a t i o n p r o d u c t o f neomycin A (nearnine or 1 , 3 - d i a m i n o - 4 , 5 , 6 t r i h y d r o x y c y c l o h e x a n e ) and was s u b s e q u e n t l y shown t o be c l o s e l y r e l a t e d t o t h e s t r e p t a m i n e m o i e t y of s t r e p t o m y c i n t h u s e r m i t t i n g t h e u s e of t h e more common name 2 - d e o x y s t r e p ~ a m i n e ~ ’ ~ . The a b s o l u t e c o n f i g u r a t i o n was d e t e r m i n e d by p r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y a n d was c o n s i s t e n t w i t h an a l l - t r a n s s t e r e o c h e m i s t r y f o r t h e s u b s t i t u e n t g r o u p s on t h e p a r e n t cyclohexane ring3. While p o s s e s s i n g n o b i o l o g i c a l a c t i v i t y i t s e l f , t h e subs e q u e n t w i d e s p r e a d o c c u r r e n c e o f 2 - d e o x y s t r e p t a m i n e as a m o i e t y became t h e b a s i s f o r f u r t h e r a n t i b i o t i c c l a s s i f i c a t i o n d e p e n d e n t on. t h e p l a c e m e n t of s u b s t i t u e n r g r o u p s on t h e r i n g . R e p r e s e n t a t i v e o f compounds h a v i n g 4 , 6 - s u b s r i t u t i o n a r e t h e kanamycins , g e n t a m i c i n s and s i s o m i c i n s w h i l e t h o s e h a v i n g 4 , 5 - s u b s t i t u t i o n on 2 - d e o x y s t r e p t a m i n e a r e t h e n e o m y c i n s , l i v i d o m y c i n s and paromomycins
.
1.1 L i t e r a t u r e C i t e d 1. F. A. K u e h l , Jr., M. N . B i s h o p a n d K. SOC. 7 3 ( 1 9 5 1 ) 8 8 1 - 8 8 2 . _c
F o l k e r s , J . Am. Chem.
2.
H. E . C a r t e r , J. R . D y e r , P. D. Shaw, K. L. R i n e h a r t , Jr., and M. H i c h e n s , J. Am. Chem. SOC. 8 3 (1961) 3723-2734.
3.
R. U. Lemieux and R . 858-862.
J . C u s h l y , Canad. J . Chem. 4 1 ( 1 9 6 3 )
1 63
2.
Gentamicin
2 . 1 Introduction Producing organism
2.1.1
I n 1963 W e i n s t e i n and co-workers’ d e s c r i b e d g e n t a m i c i n , a b r o a d s p e c t r um , bas i c , w a t e r-so l u b l e a n t i b i o t i c m i x t u r e i sol a t e d f r o m two p r e v i o u s l y u n d e s c r i b e d s p e c i e s o f m i c r o o r g a n i s m s b e l o n g i n g t o t h e g e n u s Michomononpona, s p e c i f i c a l l y M. p u h p u n e a s p n . ( N R R L 2 9 5 3 ) a n d M. eChinOApOkU s p n . ( N R R L 2 9 8 5 ) 2 . The g e n u s Michomononpohu h a d n o t p r e v i o u s l y b e e n e x t e n s i v e l y exami n e d as a s o u r c e o f n o v e l a n t i b i o t i c s u b s t a n c e . S i n c e 1 9 6 3 s e v e r a l a d d i t i o n a l aminoglycoside a n t i b i o t i c s h a v e been isol a t e d f r o m t h i s g e n u s , as w i l l b e d i s c u s s e d b e l o w . Chemical d e s c r i p t i o n and s t r u c t u r e
2 .l.2
I s o l a t i o n and preliminary chemical s t u d i e s 3 demonstrated t h a t g e n t a m i c i n i s a complex o f a m i n o g l y c o s i d e a n t i b i o t i c s cont a i n i n g t h e a m i n o c y c l i t o l 2 - d e o x y s t r e p t a m i n e a n d tw o a d d i t i o n a l a m i n o s u g a r s . The c o m p l e x i s i s o l a t e d as a n o p t i c a l l y a c t i v e amorphous powder. I t is r e a d i l y s o l u b l e i n water, p y r i d i n e and d i m e t h y l f o r m a m i d e ; m o d e r a t e l y s o l u b l e i n m e t h a n o l , a c e t o n e and e t h a n o l ; i n s o l u b l e i n e t h e r , b e n z e n e a n d h a l o g e n a t e d h y d ro carbons. Gentamicin g i v e s p o s i t i v e Elson-Morgan and n i n h y d r i n tests b u t n e g a t i v e m a l t o l , f u r f u r a l and Sakaguchi r e a c t i o n s . C h r o m a t o g r a p h i c s e p a r a t i o n of t h e g e n t a m i c i n c o m p l e x show i t t o c o n s i s t o f 3 major c o m p o n e n t s d e s i g n a t e d as C , , C, a n d Mass s p e c t r a l a n a l y s i s a n d m i c r o a n a l y s e s of t h e comCla4y5. p o n e n t s s u g g e s t m o l e c u l a r w e i g h t s 4 7 7 , 463 and 449 c o r r e s p o n d i n g t o m o l e c u l a r f o r m u l a e C21H43N507, C2,H,,N507 a n d ClgH39N507 respectively
.
The e h c i d a t i o n o f t h e s t r u c t u r e a n d s t e r e o c h e m i s t r y o f t h e g e n r r a m i c i n C c o m p o n e n t s are d e s c r i b e d i n p u b l i c a t i o n s b y Cooper e t U C . ~ - ~ O a n d D a n i e l s e t aC. l l . Nucl ear m a g n e t i c r e s o n a n c e m e a s u r e m e n t s o f t h e C c o m p o n e n t s show t h a t g e n t a r n i c i n C 1 c o n t a i n s 2 N-CH3 g r o u p s , o n e s e c o n d a r y C-CH3 group and o n e t e r t i a r y C-CH3 g r o u p . Gentamicin C2 c o n t a i n s one less N - C H 3 g r o u p w h i l e g e n t a m i c i n C1, d i f f e r s from C 2 by t h e a b s e n c e o f t h e s e c o n d a r y C-CH3 g r o u p . M e t h a n o l y s i s of e a c h o f t h e g e n t a m i c i n c o m p o n e n ts g i v e s rise t o a monosaccharide methylglycoside c a l l e d methylgarosaminide and a gentamine. The t h r e e d i f f e r e n t g e n t a m i c i n s a r e p s e u d o d i s a c c h a r i d e s c o n s i s t i n g of a p y r a n o s e s u g a r c a l l e d purpurosamine a t t a c h e d v i a a g l y c o s i d i c l i n k a g e t o 2-deoxystreptamine. The s t r u c t u r e of t h e g e n t a m i c i n C components i s shown i n f i g u r e 2, ( n e x t p a g e ) . 2.1.3
Summary o f t h e r a p e u t i c u s e
G e n t a m i c i n i s e f f e c t i v e a g a i n s t a w i d e v a r i e t y of p a t h o The f a c t t h a t g e n i c gram-negative and gram-positive b a c t e r i a . i t i s a c t i v e a g a i n s t Pneudamanad a s w e l l as P h O t e U A s p e c i e s i s
164
2'
10 GENTAMICIN C1 R s R ' s C H 3 l b GENTAMICIN C2 R s CH3; R ' * H IC GENTAlUlClN Cla R s R' 8 H
FIGURE 2.
Gentamicins
one of t h e most i m p o r t a n t a t t r i b u t e s of g e n t a r n i c i n as i s i t s a c t i v i t y a g a i n s t bac t e r i a r e s i s t a n t t o o t h e r a m i n o g l y c o s i d e antibiotics. Gentamicin i s c u r r e n t l y a v a i l a b l e f o r t o p i c a l use and p a r e n t e r a l i n j e c t i o n . The d r u g i s p o o r l y a b s o r b e d o r a l l y e x c e p t d u r i n g a c u t e s t a g e s of b a c i l l a r y d y s e n t e r y 1 2 . T o p i c a l l y , g e n t a m i c i n i s used i n a 0 . 1 % o i n t m e n t or a 0 . 1 % cream and i s v e r y e f f e c t i v e a g a i n s t p r i m a r y and s e c o n d a r y b a c t e r i a l i n f e c tions. P a r e n t e r a l l y , t h e d r u g i s used a g a i n s t s u s c e p t i b l e gram-negative i n f e c t i o n s . It is effective in infected surgical wounds , s e v e r e s o f t t i s s u e , r e s p i r a t o r y t r a c t and u r i n a r y t r a c t infections. L i k e o t h e r aminoglycosides, gentamicin i n j e c t a b l e i s p o t e n t i a l l y n e p h r o t o x i c and t h e r e f o r e p a t i e n t s t r e a t e d s h o u l d be u n d e r c l o s e c l i n i c a l o b ~ e r v a t i o n ~ ~ - ' ~ .
2.2 Extraction
of t h e g e n t a m i c i n complex
We h a v e e l e c t e d t o r e p o r t two e x t r a c t i o n p r o c e d u r e s f o r t h e g e n t a m i c i n complex and s e v e r a l p r o c e d u r e s f o r t h e s e p a r a t i o n and d e t e r m i n a t i o n of i t s components. 2.2.1
E x t r a c t i o n of t h e complex (Method 1)
I n 1 9 6 3 Rosselet3 and co-workers r e p o r t e d on t h e i s o l a t i o n of t h e g e n t a m i c i n complex by an ion-exchange p r o c e d u r e . The pH of t h e whole b r o t h was a d j u s t e d t o 2 . 0 w i t h s t r o n g m i n e r a l a c i d t o r e l e a s e t h e major p a r t of t h e a n t i b i o t i c from t h e mycelium. A f t e r f i l t r a t i o n of t h e b r o t h , o x a l i c a c i d was added t o t h e c l a r i f i e d b r o t h t o p r e c i p i t a t e c a l c i u m i o n s and
16 5 t h e s o l u t i o n was n e u t r a l i z e d w i t h s t r o n g a l k a l i . The p r e c i p i t a t e d c a l c i u m o x a l a t e was s e p a r a t e d by f i l t r a t i o n a n d t h e f i l t r a t e was p a s s e d t h r o u g h a c o l u m n c o n t a i n i n g A m b e r l i t e I R C - 5 0 “a+; 2 0 t o 5 0 mes h ) i o n - e x c h a n g e r e s i n , a n d t h e s p e n t b r o t h was d i s c a r d e d . The a n t i b i o t i c m i x t u r e , c o n t a i n i n g g e n t a m i c i n a n d cop r o d u c e d a n t i b i o t i c s , was e l u t e d f r o m t h e r e s i n w i t h s t r o n g mineral acid. The n e u t r a l i z e d e l u a t e was t h e n t i t r a t e d w i t h s o d i um d o d e c y l b e n z e n e s u l f o n a t e ( J o h n s on a n d H a r d c a s t 1e , 1 9 6 1) t o a p r e d e t e r m i n e d e n d p o i n t , t h u s p r e c i p i t a t i n g m o s t of t h e g e n t a m i c i n a n d l e a v i n g t h e c o - p r o d u c e d a n t i b i o t i c s a n d some gentamicin i n s o l u t i o n . Th e e n d p o i n t was c a l c u l a t e d as 7 0 % o f t h e a m o u n t of d o d e c y l b e n z e n e s u l f o n a t e n e c e s s a r y t o c a u s e comp l e t e p r e c i p i t a t i o n of t h e a c t i v i t y . ( T h e a c t i v i t y was m e a s u r e d by b i o l o g i c a l a s s a y as d e s c r i b e d by Oden e d at. 17). Gentarnicin d o d e c y l b e n z e n e s u l f o n a t e was s e p a r a t e d b y f i l t r a t i o n , w a she d w i t h water, p a r t i a l l y d r i e d , a n d d i s s o l v e d i n m e t h y l a l c o h o l . T h e m e t h a n o l i c s o l u t i o n was p a s s e d t h r o u g h a c o l u m n c o n t a i n i n g A m b e r l i t e IRA-401s ( O H - ) i o n - e x c h a n g e r e s i n t o r e g e n e r a t e g e n t a micin from i t s s a l t form. T h e r e s i n e f f l u e n t was t h e n c o n c e n t r a t e d a n d a c i d i f i e d ( t o pH 4 . 5 ) w i t h d i l u t e s u l f u r i c a c i d . The g e n t a m i c i n s u l f a t e t h u s p r o d u c e d was p r e c i p i t a t e d w i t h m e t h a n o l a n d s e p a r a t e d by f i l t r a t i o n . Approximately 55% of t h e total a c t i v i t y o r i g i n a l l y present in the b r o t h could be isol a t e d i n t h i s way. G e n t a m i c i n was p r e p a r e d by p a s s i n g a n a q u e o u s s o l u t i o n of i t s s u l f a t e s a l t t h r o u g h a co l u mn c o n t a i n i n g A m b e r l i t e IRA-400 (OH-) f o l l o w e d by l y o p h i l i z a t i o n o f t h e e f f l u e n t . 2.2.2
E x t r a c t i o n of t h e complex (Method 2 )
I n 1 9 7 4 Leela a n d c o - w o r k e r s r e p o r t e d a v a r i a t i o n i n t h e i s o l a t i o n o f g e n t a m i c i n . The pH o f t h e w h o l e b r o t h was a d j u s t e d t o 2 w i t h s u l f u r i c a c i d and t h e a c i d i f i e d b r o t h s t i r r e d a n d f i l t e r e d by g r a v i t y . The pH o f t h e f i l t e r e d b r o t h was a d j u s t e d t o 7 w i t h c o n c e n t r a t e d ammonium h y d r o x i d e a n d t h e n e u t r a l i z e d f i l t r a t e p a s s e d t h r o u g h a colum n o f IRC-50 r e s i n (NHI, + f o r m ) . T h e r e s i n was w a s h e d w i t h w a t e r a n d e l u t e d w i t h 2 N NH,+OH a n d t h e e l u a t e c o n c e n t r a t e d a n d a i r d r i e d . The r e s u l t i n g d a r k brown c r u d e was p a s s e d t h r o u g h a c olum n o f IRA-401s r e s i n (OH- form) and e l u t e d w i t h water. E l u a t e s c o l l e c t e d The d e c o l o r b e t w e e n pH 8 a n d 1 2 were c o m b i n e d a n d a i r d r i e d . i z e d c r u d e was a d s o r b e d on a co l u mn p a c k e d w i t h Dowex 1 x 2 res i n (OH- f o r m ) a n d t h e g e n t a m i c i n c o m p l e x e l u t e d w i t h water.
2.3 Separation
o f t h e gentamicin components
S e p a r a t i o n of t h e g e n t a m i c i n c o m p o n e n t s 1 9 ’ 2 o i s b e s t a c c o m p l i s h e d w i t h t h i n l a y e r or p a p e r c h r o m a t o g r a p h y u s i n g t h e l o w e r p h a s e o f a s o l v e n t s y s t e m cmrnposed o f c h 1 o r o f o r m : m e t h a n o l : 1 7 % ammonium h y d r o x i d e i n a r a t i o of 2 : l : l . This system is a Table m o d i f i c a t i o n o f t h e m e t h o d r e p o r t e d by I k e k a w a e t uL. ’ l . 1 s h o w s a c o m p a r i s o n o f Rf v a l u e s o f t h e g e n t a m i c i n c o m p o n e n t s C1, C2 and C l a i n v a r i o u s systems.
1 66 TABLE 1 Conparative
Rf Values o f t h e Gentamicin Components Gent ami c i n Conponents
zu
Ll
L2
BOX methanol plus 3% sodium c h l o r i d e ( w t / v o l ) (l:l), descendinga
0.57
0.56
0.48
Propanol :py r idine :a c e t i c a c i d :water (6:4:1:3), ascending (v/v)
0.34
0.30
0.22
80% phenol ascending( v/v)
0.45
0.45
0 -45
Benzene:mthanol descending
0.0
0.0
0 .o
N-bu t a n o l :water :a c e t i c a c i d ( 4 :5 :1) upper phase used ascending
0.0
0.0
0 .o
[ h l o r o f orm: methano&: 17?6 ammonium hydroxide (2: 1: 1)
0.67b
0.40b
0.21
Paper Chromatoqrqhic System
(v/v)
(9:1),
b u f f e r e d with 0.95
‘Paper
M
Na2S04 and 0.05
b
M NaHS04.
b R t = distance o f zone from o r i g i n / d i s t a n c e from o r i g i n t o end o f paper a t 2 C
=
6 h.
Lower phase.
2.3.1
S e p a r a t i o n by p a p e r and t h i n l a y e r chromatography
T h i n - l a y e r chromatography of t h e g e n t a m i c i n complex was c a r r i e d o u t on p l a t e s c o a t e d w i t h s i l i c a g e l G u s i n g t h e chloroform-methanol-ammonia s o l v e n t s y s t e m . P l a t e s were d e v e l o p e d f o r 16-18 hours; t h e p r e s e n t time is 4 h o u r s . D e t e c t i o n o f spots was acconplished by two methods: 1) by t h e use o f reagents; and 2) by a m i c r o b i o l o g i c a l procedure. The f i r s t method was c a r r i e d out by t h e usual technique o f spraying the p l a t e s w i t h 0.25% n i n h y d r i n i n pyridine-acetone and consequent h e a t i n g a t 105 C f o r s e v e r a l minutes. The zones appear as p u r p l e o r b l u e spots against a w h i t e background. Also, starch-potassium i o d i d e reagent spray can be used, w i t h which t h e zones shciw up as dark b l u e spots a g a i n s t a white background. M i c r o b i o l o g i c a l d e t e c t i o n o f t h e a n t i b i o t i c zones was c a r r i e d out by l a y i n g t h e p l a t e face-down on a sheet o f Whatman No. 1 paper i n contact with agar seeded with S&ZphyLacaccub ~Wteub ATCC 6538P. A f t e r 10 minutes t h e paper and p l a t e were removed and a f t e r a s u i t a b l e i n c u b a t i o n p e r i o d t h e zones o f i n h i b i t i o n , r e p r e s e n t i n g t h e gentamicin components, were observed.
1 67
The method of s e p a r a t i o n on t h e t h i n - l a y e r p l a t e s was o r i g i n a l l y s l o w compared t o most t h i n - l a y e r t e c h n i q u e s . Inc r e a s e d s p e e d of s e p a r a t i o n of t h e components was r e a l i z e d by use of paper chromatography u t i l i z i n g t h e chloroform-methanolammonia s y s t e m . Upper p h a s e was p l a c e d i n t h e b o t t o m of t h e c h r o m a t o g r a p h i c jars s e v e r a l h o u r s p r i o r t o u s e , and lower p h a s e was u s e d t o d e v e l o p t h e p a p e r g r a m s which were r u n d e s c e n d i n g on Whatman No. 1 p a p e r i n i n s u l a t e d c h a m b e r s , f o r 6 hours at 25OC. The s o l v e n t was a l l o w e d t o d r i p o f f t h e p a p e r s d u r i n g development. D e t e c t i o n o f z o n e s was a c c o m p l i s h e d by t h e p r e v i o u s l y d e s c r i b e d n i n h y d r i n t e c h n i q u e or by t h e b i o a u t o g r a p h i c method. I n t h e l a t t e r , t h e p a p e r was p l a t e d d i r e c t l y on t h e agar:, p r e p a r e d as p r e v i o u s l y d e s c r i b e d , f o r a 1 0 - m i n u t e p e r i o d p r i o r t o incubation. The complex s e p a r a t e d i n t o t h r e e components and t h e s e components c o r r e s p o n d e d t o t h o s e o b s e r v e d u s i n g t h e t h i n - l a y e r chromatographic technique except t h a t t h e paper chromatographic The zone o f i n h i b i t i o n method was more s e n s i t i v e a n d r a p i d . n e a r e s t t o t h e f r o n t i s i d e n t i f i e d as C 1 , t h e z o n e f o l l o w i n g d e s i g n a t e d as C2 and t h a t c l o s e s t t o t h e o r i g i n , C1,. 2.3.2
S e p a r a t i o n o f t h e g e n t a m i c i n complex by column chromatography
Whatman No. 1 c e l l u l o s e powder was p a c k e d i n a column h a v i n g a n i n n e r d i a m e t e r of 2 . 4 c m t o a h e i g h t of 30 c m , and washed w i t h t h e u p p e r p h a s e o f t h e c h l o r o f o r m - m e t h a n o l - 1 7 % ammonia s y s t e m ( 2 : l : l ) v / v . 2 0 0 mg of t h e g e n t a m i c i n s u l f a t e was mixed w i t h some c e l l u l o s e powder p a c k e d a t o p t h e c e l l u l o s e i n t h e column and e l u t e d w i t h t h e l o w e r p h a s e of t h e s o l v e n t s y s tern. The column was r u n a t t h e r a t e o f 2 ml/min and 1 6 m l f r a c t i o n s were c o l l e c t e d . The column was m o n i t o r e d by m i c r o b i o l o g i c a l l y d i s c t e s t i n g e a c h f r a c t i o n and a l s o t r e a t i n g w i t h n i n h y d r i n r e a g e n t t o d e t e r m i n e t h e a b s e n c e or p r e s e n c e of antibiotic. The p o s i t i v e f r a c t i o n s were t h e n c h r o m a t o g r a p h e d i n t h e paper system previously described. L i k e f r a c t i o n s were p o o l e d and c o n c e n t r a t e d t o d r y n e s s . T h i s method g a v e a 9 4 % r e c o v e r y of s t a r t i n g m a t e r i a l . P a p e r c h r o m a t o g r a p h y of t h e s e p a r a t e d components and b i o a u t o g r a p h y of t h e p a p e r g r a m s a g a i n s t S ~ a p h ~ - ! O C o C C u bau/Leub showed t h a t t h e C 1 and C 2 components were f r e e of i m p u r i t i e s and t h a t C1, c o n t a i n e d a p p r o x i m a t e l y 5% of C 2 . Rechromatog r a p h y of t h e C1, f r a c t i o n u s i n g t h e d e s c r i b e d column r e s u l t e d i n i s o l a t i o n o f t h i s compound f r e e of t h e C2 componenr. A m a j o r p r o b l e m i n s e p a r a t i o n o f q u a n t i t i e s of t h e g e n t a m i c i n complex i n t o i t s t h r e e components i s s c a l e - u p of e x i s t i n g procedures. Major d i s a d v a n t a g e s o f t h e a b o v e methods a r e t h a t t h i n l a y e r o r p a p e r c h r o m a t o g r a p h y can b e u s e d o n l y f o r d e t e c t i o n or p r e p a r a t i o n of m i n u t e q u a n t i t i e s of material and c e l l u l o s e powder c h r o m a t o g r a p h y i s time-consuming b e c a u s e of t h e
168
c a r e t h a t m u s t be t a k e n i n p a c k i n g t h e column. An improved t e c h n i q u e was d e v i s e d u t i l i z i n g t h e s o l v e n t s y s t e m p r e v i o u s l y d e s c r i b e d and Chromosorb W R Y an i n e r t adsorbent. A l t h o u g h t h e mechanics of t h e s e p a r a t i o n are e s s e n t i a l l y s i m i l a r t o t h o s e d e s c r i b e d , t h i s p r o c e d u r e i s more e a s i l y c a r r i e d o u t , d o e s n o t r e q u i r e hand p a c k i n g , can b e s c a l e d up r e a d i l y a n d , because i t u s e s a low r a t i o of a d s o r b e n t t o g e n t a m i c i n complex, can be used t o s e p a r a t e l a r g e q u a n t i t i e s of mat e r i a1
.
Chromosorb W ( 6 0 t o 1 0 0 mesh) m a n u f a c t u r e d by JohnsM a n v i l l e , N.Y.) was s l u r r i e d w i t h t h e upper p h a s e of c h l o r o f o r m methanol-17% ammonia (2:l:l v / v ) and f i l t e r e d . The Chromosorb was packed i n t o a column h a v i n g an I . D . of 3 cm. t o a h e i g h t of 50 c m . , i n 5 c m segments ( a p p r o x i m a t e l y 150 gm as d r y Chromos o r b ) and 1 l i t e r of lower p h a s e was r u n t h r o u g h t h e column. A 3 gm p o r t i o n of g e n t a m i c i n base was d i s s o l v e d i n 1 0 m l o f m e t h a n o l , a d s o r b e d t o t h e s m a l l e s t p o s s i b l e q u a n t i t y of Chromosorb and d r i e d u n d e r vacuum u s i n g a Rinco f i l m e v a p o r a t o r . T h i s m i x t u r e was packed a t o p t h e column and wet w i t h a s m a l l q u a n t i t y of lower p h a s e . The column was e l u t e d w i t h lower p h a s e a t t h e r a t e of 1 m l / m i n u t e , c o l l e c t i n g 5 f r a c t i o n s / h r . for a t o t a l of 2 3 1 f r a c t i o n s . F r a c t i o n s were t e s t e d as d e s c r i b e d p r e v i o u s l y and t h e 3 major component p e a k s were l o c a t e d , p o o l e d , d e c o l o r i z e d on IRA 401s ( O H - ) r e s i n and c o n c e n t r a t e d t o d r y n e s s . P a p e r chromatography of t h e b a s e s and b i o a u t o g r a p h y of t h e papergrams i n d i c a t e d t h a t a l l components were f r e e o f m i c r o b i o l o g i c a l l y a c t i v e i m p u r i t i e s . The g e n t a m i c i n s C 1 , C 2 and C1, were r e c o v e r e d i n good y i e l d s .
2 . 4 Separation
of some minor components of t h e g e n t a m i c i n
complex
Weins t e i n e t al.l r e p o r t e d t h a t two minor components , g e n t a m i c i n A and B were co-produced w i t h t h e g e n t a m i c i n comp l e x ( g e n t a m i c i n C 1 , C 2 and C l g ) . Since t h e r e l e a s e of t h i s i n i t i a l r e p o r t , s e v e r a l i n v e s t i g a t o r s have r e p o r t e d o t h e r minor components t h a t a r e produced w i t h g e n t a m i c i n . These o t h e r minor components a r e g e n t a m i c i n B I , X , Cp, and C 2 b . 2.4.1
S e p a r a t i o n a n d i s o l a t i o n of g e n t a m i c i n A
Maehr a n d S c h a f f n e r 2 2 used Dowex 1 x 2 r e s i n (OH- form) i n a g l a s s column w i t h a i n n e r d i a m e t e r of 75 mm packed t o a h e i g h t of 6 7 0 mm t o s e p a r a t e 3 5 g o f a g e n t a m i c i n complex. The column was m o n i t o r e d w i t h a Model R C - 1 6 B c o n d u c t i v i t y b r i d g e o p e r a t i n g a t a f r e q u e n c y o f 1 0 0 0 C . P . S . which was modified with a voltage d i v i d e r i n t h e output c i r c u i t t o provide f o r recorder readout. A g l a s s flow t h r o u g h c e l l , w i t h p l a t i n u m e l e c t r o d e s , h a v i n g a c e l l c o n s t a n t of 0 . 2 c m - l was used w i t h o u t s p e c i a l t e m p e r a t u r e c o n t r o l . A Leeds and N o r t h r u p Spedomax H was used t o r e c o r d t h e 0-10 m V r a n g e . Thin l a y e r chromatography was p e r f o r m e d on n o n - a c t i v a t e d l a y e r s of Brinkman S i l i c a Gel G , 0.75 mm t h i c k n e s s . They w e r e d e v e l o p e d with t h e following s o l v e n t systems: 1) chloroform-methanol-2 8%
169 ammonium h y d r o x i d e - w a t e r ( 1 : 4 : 2 : 1 v / v ) ; 2 ) c h l o r o f o r m - m e t h a n o l 2 8 % ammonium h y d r o x i d e ( 2 : l : l ) ; 3 ) c h l o r o f o r m - m e t h a n o l - 2 8 % ammonium h y d r o x i d e - w a t e r ( 1 0 : 5 : 3 : 2 v / v ) . Nin h y d r i n s p r a y was u s e d as t h e d e t e c t o r ( 1 2 5 mg n i n h y d r i n d i s s o l v e d i n 25 m l b u t a n o l and 2 5 m l a c e t o n e ) . The c o l u m n c h a r g e d w i t h t h e g e n t a m i c i n w a s e l u t e d w i t h water a n d t h e c o n d u c t i v i t y o f t h e e f f l u e n t measured. Ten f r a c t i o n s were c o l l e c t e d ; t h e s e c o n d p e a k 1 9 5 0 m l - 2670 m l c o n t a i n e d t h e g e n t a m i c i n C , , C, a n d C1, m i x t u r e . The n e x t s i x f r a c t i o n s ( 2 6 7 0 m l t o 5549 m l ) c o n t a i n e d c o m p l e x m i x t u r e s and f i n a l l y f r a c t i o n 9 ( 5 5 5 0 m l 9430 m l ) c o n t a i n e d g e n t a m i c i n A as t h e m a j o r c o n s t i t u e n t .
-
2.4.2
S e p a r a t i o n o f m i n o r c o m p o n e n t s by p a p e r c h r o m a t o graphy
G e n t a m i c i n s A , B , B 1 a n d X were s e p a r a t e d by Wagman e-t a e . 2 3 i n t h e f o l l o w i n g t h r e e s y s t e m s . T h e i r Rf v a l u e s i n t h e s e s y s t e m s a r e summarized i n T a b l e 2 . 1. S y s t e m No. 1. A s o l v e n t m i x t u r e c o n s i s t i n g of c h l o r o f o r m - m e t h a n o l - l 7 % ammonium h y d r o x i d e ( 2 : l : l ) was u s e d t o s e p a r a t e t h e components o f t h e a n t i b i o t i c complex. The q u a n t i t y s p o t t e d on t h e p a p e r s f o r g e n t a m i c i n s A , B y X a n d B 1 was 5 mcg. The u p p e r p h a s e was p l a c e d i n t h e b o t t o m o f t h e c h r o m a t o g r a p h i c jars s e v e r a l h o u r s p r i o r t o u s e a n d lower p h a s e was u s e d t o d e v e l o p t h e p a p e r c h r o m a t o g r a m s w h i c h were r u n downward on Whatman No. 1 p a p e r i n i n s u l a t e d c h a m b e r s , f o r 1 6 h r . a t 25'. The s o l v e n t was a l l o w e d t o d r i p o f f t h e p a p e r d u r i n g d e v e l o p ment.
M i c r o b i a l d e t e c t i o n s o f t h e a n t i b i o t i c s was c a r r i e d o u t as d e s c r i b e d p r e v i o u s l y . Very l i t t l e movement of t h e m i n o r c o m p o n e n t s was s e e n i n t h i s s y s t e m as d e s c r i b e d i n T a b l e 2 . 2. S y s t e m No. 2 . A s o l v e n t s y s t e m was p r e p a r e d c o n s i s t i n g o f 2 - b u t a n o n e - t e r t . - b u t a n o l - m e t h a n o l - 2 8 % ammonium T h i s i s a m o d i f i c a t i q n of a s o l v e n t hydroxide (16:3:1:6). m i x t u r e d e s c r i b e d by Majumdar a n d Majumdar The c h r o m a t o g r a p h i c j a r s were e q u i l i b r a t e d f o r s e v e r a l h o u r s p r i o r t o u s e . T h e a n t i b i o t i c c o m p l e x was s p o t t e d on Whatman No. 1 p a p e r a n d run descending f o r 16 h o u r s . The s o l v e n t was a l l o w e d t o d r i p o f f t h e paper d u r i n g development. M i c r o b i o l o g i c a l d e t e c t i o n of t h e a n t i b i o t i c c o m p o n e n t s was c a r r i e d o u t as p r e v i o u s l y d e s cribed.
.
Rf v a l u e s r e l a t i v e t o g e n t a m i c i n B 1 , t h e most m o b i l e component, are d e s c r i b e d i n Table 2 . 3. S y s t e m No. 3 . The l o w e r p h a s e o f a c h l o r o f o r m m e t h a n o l - 2 8 % ammonium h y d r o x i d e ( 1 : l : l ) s y s t e m was u s e d t o s e p a r a t e t h e m i n o r c o m p o n e n t s o n ChromAR s h e e t 5 0 0 ( s i l i c i c a c i d - g l a s s f i b e r s h e e t ) . The c h r o m a t o g r a m s were d e v e l o p e d i n e q u i l i b r a t e d i n s u l a t e d c h a m b e r s , a s c e n d i n g f o r 5 0 m in. Microb i o l o g i c a l d e t e c t i o n was c a r r i e d o u t as d e s c r i b e d p r e v i o u s l y . Rf v a l u e s are d e t a i l e d i n T a b l e 2 , ( n e x t p a g e ) .
170 TABLE 2
Chromatoqraphy of Minor Gentamicin Components System 1 (mm f r o m o r i qin )
System 2 Rf B 1 *
System 3 Rf
Gentamicin A1**
0 .o
0.36
0.10
Gentamicin A
0 .o
0.41
0.16
Gentamicin B
9.0
0.52
0.22
Gentamicin X
13.5
0.55
0.28
Unknown component
27 .O
0.72
0.32
Gentamicin 81
27 .O
1.00
0.40
*Rf B 1 = d i s t a n c e c o n p m e n t from o r i q i n d i s t a n c e component B 1 f r o m o r i g i n **Present i n component A a s a t r a c e . 2.4.3
Column c h r o m a t o g r a p h i c s e p a r a t i o n o f t h e m i n o r g e n t a m i c i n components
A t t h e e n d of t h e g e n t a m i c i n f e r m e n t a t i o n , t h e a n t i b i o t i c s p r o d u c e d a r e e x t r a c t e d from t h e b r o t h by a d s o r b i n g t h e t o t a l a n t i b i o t i c complex o n t o a c a t i o n - e x c h a n g e r e s i n t y p e s u c h as A m b e r l i t e I R C - 5 0 i n t h e ammonium c y c l e , and t h e c r u d e a n t i b i o t i c m i x t u r e e l u t e d w i t h d i l u t e ammonium h y d r o x i d e . The e l u a t e i s d e c o l o r i z e d by p a s s a g e t h r o u g h an a n i o n - e x c h a n g e column c o n t a i n i n g A m b e r l i t e IRA-QOlS r e s i n i n t h e h y d r o x y l form. A f t e r d e c o l o r i z a t i o n , t h e e l u a t e i s c o n c e n t r a t e d i n uacuo t o a s o l u t i o n c o n t a i n i n g from a b o u t 3 0 % t o 6 0 % s o l i d s .
T h i s s o l u t i o n c o n t a i n s a l l of t h e a n t i b i o t i c s p r o d u c e d i n t h e fermentation. Removal of t h e g e n t a m i c i n C components i s a c c o m p l i s h e d by a d s o r b i n g t h e c r u d e c o n c e n t r a t e on a column of Dowex 1 x 2 and s e l e c t i v e l y e l u t i n g t h e g e n t a m i c i n C components as a g r o u p w i t h d e i o n i z e d water. The c o - p r o d u c e d minor compone n t s a r e e l u t e d from t h e Dowex 1 x 2 column s u b s e q u e n t t o t h e g e n t a m i c i n C components and t h e a q u e o u s e l u a t e c o n c e n t r a t e d i n v a c u a t o a s u i t a b l e volume and l y o p h i l i z e d . A 2 kg p o r t i o n of f e r m e n t a t i o n p r o d u c t f r o m w h i c h most of t h e g e n t a m i c i n C h a d been removed was d i s s o l v e d i n 4 . 5 l i t e r s of hot methanol. The s o l u t i o n was c l a r i f i e d by f i l t r a t i o n , c o o l e d t o room t e m p e r a t u r e w i t h e x p o s u r e t o a i r and s e e d e d w i t h g e n t a m i c i n A. Gentamicin A c r y s t a l l i z e d as i t s c o l o r l e s s carb o n a t e s a l t whigh was f i l t e r e d , washed s p a r i n g l y w i t h m e t h a n o l and d r i e d a t 8 0 it? v a c u o a f f o r d i n g 1 2 0 g o f t h e p r o d u c t .
The m o t h e r l i q u o r and w a s h i n g s were combined and c o n c e n t r a t e d i n v a c u o . Based on p a p e r c h r o m a t o g r a p h y t h i s m a t e r i a l c o n t a i n e d r e s i d u a l A and C components t o g e t h e r w i t h B y B 1 and X. T h i s complex was c h r o m a t o g r a p h e d as f o l l o w s : two c h r o m a t o g r a p h i c columns ( 1 0 cm I.D. x 150 cm l o n g ) were c o n n e c t e d i n
171
series and f i l l e d w i t h s i l i c a g e l (Baker) which had p r e v i o u s l y b e e n c o n d i t i o n e d by c o n t a c t w i t h t h e lower p h a s e o f a m i x t u r e o f c h l o r o f o r m - m e t h a n o l - 2 8 % ammonium h y d r o x i d e (1:l:l). A s o l u t i o n o f 7 5 0 g o f t h e r e s i d u e was p r e p a r e d i n a minimum volume ( a p p r o x i m a t e l y 2 l i t e r s ) o f t h e l o w e r l a y e r o f t h e same s o l v e n t mixture. T h e a n t i b i o t i c s o l u t i o n was pumped o n t o t h e f i r s t column w i t h a m e t e r i n g pump a t t a c h e d t o a p r e s s u r e v a l v e s e t at 5 p.s.i. The column was e l u t e d by pum ping s o l v e n t t o t h e t o p a t a f l o w r a t e o f a b o u t 58 m l / m i n . a n d c o l l e c t i n g f r a c t i o n s o f a p p r o x i m a t e l y 2000 m l each. The e f f l u e n t was m o n i t o r e d by t h i n - l a y e r a n d p a p e r c h r o m a t o g r a p h y as d e s c r i b e d e a r l i e r . App r o p r i a t e f r a c t i o n s were c o m b i n e d a n d e v a p o r a t e d t o d r y n e s s . R e s i d u a l g e n t a m i c i n C was e l u t e d f i r s t , f o l l o w e d by a n unknown component, B , , X and B . When e l u t i o n o f g e n t a m i c i n B was a l m o s t c o m p l e t e t h e s o l v e n t was c h a n g e d t o a m i x t u r e c o m p r i s i n g e q u a l v o l u m e s o f m e t h a n o l a n d c o n c . ammonium h y d r o x i d e t o e l u t e g e n t a m i c i n A. The p o o l e d f r a c t i o n s c o n t a i n i n g e i t h e r g e n t a m i c i n B or g e n t a m i c i n B 1 were d r i e d , d i s s o l v e d i n e t h a n o l a n d c o n c e n trated to a residue. R e p e t i t i o n of t h e f o r e g o i n g s t e p r e s u l t e d i n c r y s t a l l i n e s u s p e n s i o n s of t h e e t h a n o l s o l v a t e s o f g e n t a m i c i n B and g e n t a m i c i n B , . A summary o f t h e f r a c t i o n a t i o n a n d y i e l d s o f t h e g e n t a m i c i n c o m p o n e n t s a r e shown i n T a b l e 3 . TABLE 3 Column Chromatographic Separation o f Minor Gent ami c i n Conponents F r a c t i o n No.
Component
Weight* ( 9 )
11-12
Fare-run
13-21
Gentamicin C
22-26
Unknown Component gentamicin B 1
27-29
Gentamicin 81
42 .O
30-34
Gentamicins B1 + X
67.8
35
Gentamicin X
12.3 74.9
36-40
Gentamicins S + B
41-50
Gentamicin 6
51-80
Gentamicins B + A
4.8
110
+
196.8
85.8 165 .O
*Not c o r r e c t e d f o r m o i s t u r e .
2.4.4
S e p a r a t i o n o f C2a
D a n i e l s and Marquez25 a l o n g w i t h K e r s h n e r 2 6 and Byrne 2 7 have r e p o r t e d t h e i s o l a t i o n of a n o t h e r minor component, Except, g e n t a m i c i n C p , which i s co-produced w i t h gentamicin. f o r m i n o r v a r i a t i o n s , a l l of t h e r e p o r t e d m e t h o d s f o r t h e i s o l a t i o n of g e n t a m i c i n C Z a~r e v i r t u a l l y i d e n r i c a l . Daniels and M a r q u e z z s i s o l a t e d C 2 a as f o l l o w s : Gentamicin C base ( 9 6 grams)
e-t aL.
172 p r e p a r e d as d e s c r i b e d i n t h e e x t r a c t i o n s e c t i o n , was d i s s o l v e d i n 4 0 0 m l of t h e u p p e r p h a s e o f t h e c h l o r o f o r m - m e t h a n o l 1 7 % ammonium h y d r o x i d e m i x t u r e ( 2 : l : l ) . F o r t y m l of t h e s o l u t i o n was a d d e d t o e a c h of t h e f i r s t 1 0 t u b e s i n a 5 0 0 x 80 m l tube countercurrent extraction. A l l of t h e 5 0 0 t u b e s a r e f i l l e d w i t h t h e l o w e r p h a s e of t h e above d e s c r i b e d m i x t u r e . The a p p a r a t u s was s e t t o d e l i v e r 40 m l o f t h e u p p e r p h a s e t o t u b e o n e f o r e a c h t r a n s f e r and t h e e x t r a c t o r s e t f o r 5 0 0 t r a n s f e r s . A f t e r c o m p l e t i o n o f t h e t r a n s f e r s e v e r y 8 t h t u b e was sampled f o r c h r o m a t o g r a p h y on S c h l e i c h e r and S c h u e l l p a p e r No. 589 u s i n g t h e l o w e r p h a s e of t h e a b o v e - d e s c r i b e d s o l v e n t mixture. The p a p e r i s d e v e l o p e d f o r a b o u t 1 6 h r s , d r i e d and p l a t e d on a n a g a r p l a t e s e e d e d w i t h StaphyLococcuA au4cuA ( A T C C 6 5 3 8 P ) and a d u p l i c a t e p a p e r s p r a y e d w i t h n i n h y d r i n and heated. The a g a r p l a t e i s i n c u b a t e d a t 3 7 O C o v e r n i g h t . The m a t e r i a l t h a t m i g r a t e s a s C , ( i . e . t u b e s 290-360) were combined. Tubes 2 9 0 - 3 6 0 were r e p l a c e d w i t h f r e s h t u b e s c o n t a i n i n g 40 m l of u p p e r p h a s e and 40 m l o f l o w e r p h a s e . The a p p a r a t u s was r e - s e t f o r 2800 t r a n s f e r s and t h e c h r o m a t o g r a p h i c p r o c e d u r e s p e r f o r m e d above r e p e a t e d . Chromatographically g e n t a m i c i n CZa m i g r a t e s a l i t t l e f a s t e r t h a n C 2 and was f o u n d i n t u b e s 1 - 1 6 . The f r a c t i o n s a r e combined and c o n c e n t r a t e d and 1 . 3 g of g e n t a m i c i n C Z a i s o l a t e d . 2.4.5
S e p a r a t i o n of c2b
G e n t a m i c i n c 2 b i s a n o t h e r m i n o r component co-produced i n t h e standard gentamicin fermentation. KershnerZ6 f i r s t desc r i b e d i t s p r e s e n c e i n t r a c e amounts i n g e n t a m i c i n p r e p a r a t i o n s . Byrne e x ae. 2 7 a l s o r e p o r t e d i t f r o m g e n t a m i c i n f e r m e n t a t i o n s . D a n i e l s and co-workers 2 8 r e p o r t e d i t s i s o l a t i o n , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n from f e r m e n t a t i o n b r o t h of a Michomonobpoha puapuhea m u t a n t w h i c h p r o d u c e d g e n t a m i c i n C1, as a ma:or p r o d u c t and g e n t a m i c i n C2b as a minor p r o d u c t . B o t h K e r s h n e r a 6 a n d Byrne and c o - w o r k e r s 2 ? i s o l a t e d g e n t a m i c i n C2b v i a t h e same p r o c e s s as t h e i s o l a t i o n of g e n t a m i c i n C z a p r e v i o u s l y m e n t i o n e d , Craig countercurrent d i s t r i b u t i o n . The i n v i t h o s p e c t r u m of b o t h g e n t a m i c i n C z a and C2b i s s u b s t a n t i a l l y t h e same as g e n t a m i c i n c1, C 2 and Cia. Byrne'? claims t h a t g e n t a m i c i n C z a and g e n t a m i c i n c2b r e p r e s e n t o n l y 4 % of t h e t o t a l C complex m i x t u r e .
2.5 Quantitative
d e t e r m i n a t i o n of t h e g e n t a m i c i n components
S e v e r a l p r o c e d u r e s have b e e n r e p o r t e d f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n of t h e g e n t a m i c i n complex. Wagman ex at. 2 9 r e p o r t e d on a d i f f e r e n t i a l p a p e r chromat o g r a p h i c a s s a y f o r t h e components of t h e g e n t a m i c i n complex ( C l , C 2 , Cia). The g e n t a m i c i n s a r e s e p a r a t e d on p a p e r s t r i p s w i t h t h e l o w e r p h a s e o f t h e c h l o r o f o r m - m e t h a n o l - 1 7 % ammonium hydroxide ( 2 : l : l ) s o l v e n t system. After separation the paper s t r i p s a r e p l a t e d a g a i n s t SRaphyLococcub auheub a n d t h e z o n e s of a c t i v i t y a r e q u a n t i f i e d a g a i n s t known s t a n d a r d s . Wagman e.2 a t . 3 0 a l s o r e p o r t e d on a d i f f e r e n t i a l n i n h y d r i n c h r o m a t o g r a p h i c a s s a y f o r t h e m a j o r g e n t a m i c i n components. The same s o l v e n t s y s t e m i s u s e d as d e s c r i b e d a b o v e . A f t e r d e v e l o p -
173 ment, the s t r i p s c o n t a i n i n g t h e a n t i b i o t i c are t r e a t e d w i t h n i n h y d r i n r e a g e n t , c o l o r d e v e l o p e d by h e a t i n g a n d c o l o r i n t e n s i t i e s of t h e z o n e s are r e a d on a n i n t e g r a t i n g s c a n n e r f r o m w h i c h component p r o p o r t i o n s c a n b e d e t e r m i n e d . Results are i n e x c e l l e n t a g r e e m e n t w i t h t h e m i c r o b i o l o g i c a l m e t h o d . A n h a l t 3 1 r e p o r t e d on a n a s s a y f o r g e n t a m i c i n i n s e r u m b y high pressure l i q u i d chromatography. He e x t r a c t e d t h e g e n t a m i c i n f r o m s e r u m by u s i n g a CM-Sephadex c o l u m n a n d a n a l y s i s b y r e v e r s e - p h a s e i o n - p a i r Chromatography. S i n c e t h e g e n t a m i c i n c o m p o n e n t s h a v e n o u l t r a v i o l e t a b s o r p t i o n , a d e r i v a t i v e was made w i t h 0 - p h t h a l a d e h y d e t o g i v e a f l u o r e s c e n t p r o d u c t t h a t could be used f o r d e t e c t i o n . K a b a s a k a l i a n 3 2 r e p o r t e d on t h e d e t e r m i n a t i o n o f t h e g e n t a m i c i n c o m p o n e n t s i n f e r m e n t a t i o n b r o t h by i n - s i t u f l u o r e m e t r i c m e a s u r e m e n t s o f 4 - c h l o r o - 7 - n i t r o b e n z o - 2 -oxo-1 , J - d i a z o l e d e r i v a tives. The n e e d f o r a r a p i d , s e n s i t i v e m e t h o d f o r d e t e r m i n i n g t h e g e n t a m i c i n c o m p o n e n t s d u r i n g f e r m e n t a t i o n , l e d ro t h e i n v e s t i g a t i o n of fluorescence procedures. A f t e r t h i n - l a y e r chromat o g r a p h y t h e a b o v e d e r i v a t i v e s were made a n d r e c h r o m a t o g r a p h e d . The p l a t e s a r e t h e n s c a n n e d u s i n g e x c i t a t i o n a n d i m m i s i o n wave l e n g t h o f 420 a n d 5 3 0 nm r e s p e c t i v e l y . The o f f i c i a l Food a n d D r u g s A d m i n i s t r a t i o n me-chod f o r t h e d e t e r m i n a t i o n of t h e g e n t a m i c i n C c o m p o n e n t s 3 3 i s a p a p e r chromatographic t e c h n i q u e similar t o t h o s e p r e v i o u s l y d e s c r i b e d . The a c t i v i t i e s o f g e n t a m i c i n C 1 , C 2 a n d C1, are c a l c u l a t e d r e l a t i v e t o t h e t o t a l gentamicin a c t i v i t y of the gentamicin master s t a n d a r d .
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176 3. S i s o m i c i n
3.1 Introduction 3.1.1
Producing organism
I n 1970 W e i n s t e i n and c o - w o r k e r s r e p o r t e d on an aminoglycoside a n t i b i o t i c , s i s o m i c i n ' , i s o l a t e d from t h e fermentaThe t i o n b r o t h o f a new s p e c i e s of t h e g e n u s Michomonoopoka. o r g a n i s m was i s o l a t e d from t h e s o i l of t h e I n y o N a t i o n a l F o r e s t i n C a l i f o r n i a and was named Michomonoopoha i n y o e n b i b (NRRL 3 9 2 9 ) . 3.1.2
Chemical d e s c r i p t i o n and s t r u c t u r e
S i s o m i c i n i s c r y s t a l l i z e d as n e e d l e s f r o m e t h a n o l w i t h a m e l t i n g p o i n t of 198-201' and { a } 2 6 + 188.9 (H20). L i k e g e n t a m i c i n , s i s o m i c i n is b a s i c , h i g h y p o l a r and water s o l u b l e . I t i s s p a r i n g l y s o l u b l e i n methanol, s l i g h t l y s o l u b l e i n c h l o r o f o r m , i n s o l u b l e i n a c e t o n e , e t h e r and b e n z e n e . 3.1.3
Structure
Chemical a n d p h y s i c a l s t u d i e s have e s t a b l i s h e d t h e s t r u c t u r e f o r s i s o m i c i n 2 t h a t i s shown i n f i g u r e 3.
FIGURE 3 .
Sisomicin.
Mass s p e c t r o m e t r y s t u d i e s i n d i c a t e d t h a t t h e m o l e c u l a r c o m p o s i t i o n o f s i s o m i c i n i s C!gH37N501. A c a l c u l a t e d m o l e c u l a r w e i g h t of 447 and m i c r o a n a l y s i s ' w e r e i n a g r e e m e n t w i t h a monoThe m o l e c u l a r w e i g h t a n d h y d r a t e of t h e a b v e c o m p o s i t i o n . c o m p o s i t i o n of s i s o m i c i n s u g g e s t e d t h a t i t i s a d e h y d r o - a n a l o g of gentamicin The i n f r a r e d s p e c t r u m e x h i b i t e d an abs o r p t i o n band a t 1 6 9 0 c m - I a t t r i b u t a b l e t o a v i n y l i c e t h e r function i n t h e molecule. C o n c l u s i v e e v i d e n c e of t h e p r e s e n c e a n d s p e c i f i c l o c a t i o n for r h e s i t e of u n s a t u r a t i o n was o b t a i n e d from a d e t a i l e d s t u d y o f t h e mass
1 77
3.1.4
Summary o f t h e r a p e u t i c u s e
S i s o m i c i n , l i k e g e n t a m i c i n , is f o u n d t o b e a c t i v e a g a i n s t most s p e c i e s o f b o t h g r a m - n e g a t i v e a n d g r a m - p o s i t i v e b a c t e r i a i n c l u d i n g some S t a p h y e o c o c c u b s t r a i n s r e s i s t a n t t o p e n i c i l l i n I t i s a l s o a c t i v e a g a i n s t some c l i n i c a l i s o and m e t h i c i l l i n . l a t e s o f b a c t e r i a r e s i s t a n t t o o t h e r r e l a t e d d r u g s s u c h as k a n a m y c in a n d n e o m y c i n . S i s o m i c i n h a s t h e p o t e n t i a l t o x i c p a t t e r n s of t h e o t h e r a m i n o g l y c o s i d e g r o u p of a n t i b i o t i c s , t h e r e f o r e t r e a t m e n t i s g i v e n under close c l i n i c a l o b s e r v a t i o n s .
3.2 E x t r a c t i o n , 3.2.1
s e p a r a t i o n and p u r i f i c a t i o n
Extraction of crude a n t i b i o t i c
The e x t r a c t i o n p r o c e s s f o r s i s o m i c i n p a r a l l e l s t h e e x t r a c t i o n p r o c e d u r e d e v i s e d f o r g e n t a m i c i n . The f e r m e n t a t i o n b r o t h was a c i d i f i e d t o pH 2 w i t h 6 N s u l f u r i c a c i d a n d f i l t e r e d . The pH was a d j u s t e d t o 7 w i t h 6N ammonium h y d r o x i d e a n d s u f f i c i e n t o x a l i c a c i d added t o p r e c i p i t a t e t h e calcium p r e s e n t a n d filtered. The f i l t r a t e i s r e n e u t r a l i z e d w i t h ammonium h y d r o x i d e a n d c h a r g e d o n t o a c a t i o n i c e x c h a n g e a d s o r p t i o n c olum n c o n t a i n i n g 1 5 0 0 - 2 0 0 0 g o f IRC-50 A m b e r l i t e i n i t s ammonium f o r m . The e l u a t e was d i s c a r d e d , t h e r e s i n w a s h e d w i t h water a n d e l u t e d w i t h 2 N ammonium h y d r o x i d e . 4 0 0 m l f r a c t i o n s were c o l l e c t e d a n d m o n i t o r e d by d i s c t e s t i n g w i t h S t a p h y C o c a c c u b a U 4 e U b ATCC 6 5 3 8 P . The a c t i v e f r a c t i o n s were c o m b i n e d a n d e v a p o r a t e d t o d r y n e s s u n d e r vacuum y i e l d i n g 2 8 g of c r u d e s i s o m i c i n . Material p r o d u c e d i n t h i s m a n n e r h a d a p o t e n c y o f a b o u t 500 mcg/mg a c c o r d i n g t o t h e m i c r o b i o l o g i c a l a s s a y a n d i s c om pose d of e s s e n t i a l l y s i s o m i c i n ( a s t h e b a s e ) w i t h s m a l l q u a n t i t i e s of m i n o r components. 3.2.2
P u r i f i c a t i o n of s i s o m i c i n
P u r i f i c a t i o n o f t h e a n t i b i o t i c was c a r r i e d o u t by d i s s o l v i n g 2 8 g o f c r u d e s i s o m i c i n i n 1 0 0 ml o f d i s t i l l e d w a t e r a n d c h a r g i n g i t o n a n a n i o n e x c h a n g e a d s o r p t i o n c olum n (Dowex 1 x 2 ) i n t h e h y d r o x y l form. 2000 g of t h e r e s i n was s l u r r i e d i n water i n t o a co l u mn 2 % i n c h e s i n d i a m e t e r t o a h e i g h t o f 36 inches. The c o l umn was e l u t e d w i t h d i s t i l l e d water a t a r a t e o f a b o u t 2 3 m l / m i n . a n d 1 0 0 ml f r a c t i o n s c o l l e c t e d a n d m o n i t o r e d w i t h a c o n d u c t i v i t y meter a n d by d i s c t e s t i n g a g a i n s t Staphyeacoccub auneub. T o i n s u r e t h a t t h e f r a c t i o n s were p r o p e r l y comb i n e d , a p o r t i o n o f e a c h f r a c t i o n was c h r o m a t o g r a p h e d on Whatman No. 1 p a p e r u s i n g t h e l o w e r p h a s e o f a c h l o r o f o r m - m e t h a n o l - l 7 % E a c h p a p e r was s p r a y e d w i t h ammonium h y d r o x i d e s y s t e m 2 : l : l . n i n h y d r i n a n d t h e e l u a t e s c o n t a i n i n g l i k e m a t e r i a l s were comb i n e d a n d l y o p h i l i z e d y i e l d i n g a b o u t 5 . 7 g of s i s o m i c i n a s s a y i n g a b o u t 9 0 0 mcg/mg. T h i s s o l v e n t s y s t e m was a l s o u s e d t o h e l p d i f f e r e n t i a t e s i s o m i c i n from o t h e r a n t i b i o t i c s . T a b l e 4 shows a c o m p a r i s o n o f t h e Rf of s i s o m i c i n i n v a r i o u s s y s t e m s .
178 TABLE 4 Rf
Values o f Sisomicin
Paper Chromatographic System
€! !
80% methanol p l u s 3% sodium c h l o r i d e (w/v) 1:1, descending*
0.49
Prop an0 1:py r idine :ace t ic a c i d : water ( 6 : 4 : 1 : 3 ) , ascending
0.29
(v/v) 80% phe no1 ascending ( v / v )
0.45
Benzene:methanol ( v / v ) descending
0 .o
9:l
0 .o
n-bu t a n o l :water :a c e t i c a c i d '140:50:100) (upper phase used) ascending
0.51 R t f o r 26 hrs**
2X p - t o l u e n e s b l f u r i c a c i d i n water s a t u r a t e d n-butanol
C h1or0 f orm :met h an o 1:17% ammoni um hydroxide 2:l:l *Paper b u f f e r e d with 0.95 molar NazS04
**Rz 3.2.3
=
0.21 R t f o r 6 hrs
+ 0.05 molar NaHS04.
distance o f zone from o r i q i n distance f r o m o r i g i n t o end o f paper
Alternate purification of sisomicin
200 g o f Whatman No. 1 c e l l u l o s e pow de r was m i x e d w i t h 20 ml o f t h e t o p p h a s e o f a s o l v e n t s y s t e m composed of c h l o r o f o r m : m e t h a n o l : l 7 % ammonium h y d r o x i d e ( 2 : l : l ) a n d p a c k e d i n small s e g m e n t s i n a c o l u m n h a v i n g a n i n n e r d i a m e t e r o f 0 . 2 5 i n c h a n d a h e i g h t o f 20 i n c h e s . The lower p h a s e o f t h e s o l v e n t s y s t e m was r u n t h r o u g h t h e column u n t i l a y e l l o w b a n d of i m p u r i t i e s e m e r g e d . Two g r a m s o f s i s o m i c i n s u l f a t e ( p r e p a r e d by d i s s o l v i n g t h e b a s e i s o l a t e d p r e v i o u s l y and t r e a t i n g w i t h s u l f u r i c a c i d ) was d i s s o l v e d i n a b o u t 3 m l o f t h e u p p e r s o l v e n t p h a s e , m i x e d w i t h some c e l l u l o s e p o w d e r , d r i e d u n d e r vacuum a n d packed a t o p t h e c e l l u l o s e column. Lower p h a s e was a l l o w e d t o r u n t h r o u g h t h e co l u mn a t t h e r a t e o f 1 m l / m i n . c o l l e c t i n g 5 m l f r a c t i o n s every 1 5 minutes. A l i q u o t s o f e a c h f r a c t i o n were s p o t t e d on f i l t e r p a p e r a n d t e s t e d w i t h n i n h y d r i n r e a g e n t t o d e t e r m i n e t h e p r e s e n c e or a b s e n c e of a n t i b i o t i c . P a p e r c h r o m a t o g r a p h y o f t h e a n t i b i o t i c c o n t a i n i n g f r a c t i o n s e s t a b l i s h e d t h a t t h e d e s i r e d m a t e r i a l was l o c a t e d between f r a c t i o n s 1 2 1 t o 1 9 0 . F r a c t i o n s 1 2 1 t o 190 a r e combined, e v a p o r a t e d t o d r y n e s s , r e d i s s o l v e d i n w a t e r , a n d p a s s e d t h r o u g h IRA 401s ( a n a n i o n exchange r e s i n ) i n t h e hydroxyl c y c l e . The pH o f t h e e l u a t e was a d j u s t e d t o 4 . 5 w i t h s u l f u r i c a c i d a n d t r e a t e d w i t h c h a r c o a l , f i l t e r e d a n d c o n c e n t r a t e d t o a s m a l l e r v o l u m e . The c o n c e n t r a t e was a d d e d t o a n e x c e s s amo u n t o f m e t h a n o l a n d t h e
179 w h i t e p r e c i p i t a t e t h a t was f o r m e d was s e p a r a t e d by f i l t r a t i o n . The p r e c i p i t a t e was d i s s o l v e d i n water a n d p a s s e d t h r o u g h a n I R A 401s r e s i n column i n t h e h y d r o x y l f o r m . The e f f l u e n t was c o l l e c t e d , c o n c e n t r a t e d a n d l y o p h i l i z e d , y i e l d i n g a b o u t 300 mg of s i s o m i c i n b a s e h a v i n g a n a s s a y of a b o u t 1 0 0 0 mcg/mg.
3.3 Microbiological
assay
The i n v i t h a a s s a y i s a c y l i n d e r c u p a s s a y u s i n g S t a p h y t a c v c c u h auheuh ATCC 6 5 3 8 P as t h e t e s t o r g a n i s m . The c o n d i t i o n s of t h e a s s a y a r e s i m i l a r t o t h e c y l i n d e r p l a t e a s s a y d e s c r i b e d A u n i t o f s i s o m i c i n a c t i v i t y i s t h e amount o f f o r gentamicin. material which produces a z o n a l r e s p o n s e of 1 6 . 3 0 . 9 mm u n d e r t h e c o n d i t i o n s o f t h i s a s s a y a n d h a s b e e n d e f i n e d as o n e m i c r o gram. +_
3.4 Minor 3.4.1
components S e p a r a t i o n of m i n o r c o m p o n e n t s
Minor components produced i n t h e f e r m e n t a t i o n i n c l u d e a n t i b i o t i c s 6 6 - 4 0 B a n d 66-40D8. The r e s i d u a l a n t i b i o t i c c o m p l e x , a f t e r r e m o v a l o f s i s o m i c i n by c r y s t a l l i z a t i o n a n d c h r o m a t o g r a p h y o n s i l i c a g e l c o l u m n w i t h c h l o r o f o r m - r n e t h a n o l - l 5 % ammonium h y d r o x i d e ( 3 : 5 : 3 ) as e l u a n t , c o n t a i n e d t h e m i n o r c o m p o n e n t s a n t i b i o t i c 66-40B a n d 66-40D. T h e c r u d e m i x t u r e o f f r a c t i o n s r i c h i n t h e s e compounds was c h r o m a t o g r a p h e d r e p e a t e d l y on a s i l i c a g e l c o l u m n ( 1 7 0 x 7 . 5 ) w i t h t h e same e l u e n t t o g i v e ( i n o r d e r of e l u t i o n ) 1 . 6 g o f 6 6 - 4 0 B a n d 0 . 8 g o f 66-4OD. 3.4.2
Chemistry and s t r u c t u r e
B o t h a n t i b i o t i c s 66-40B a n d 6 6 - 4 0 D e x h i b i t b r o a d s p e c t r u m a c t i v i t y . M i c r o a n a l y s i s a n d h i g h r e s o l u t i o n mass s p e c t r a were i n a g r e e m e n t w i t h a c o m p o s i t i o n o f C18H35N507 f o r b o t h . The I R s p e c t r a s h o w e d a b s o r p t i o n for 66-40B a t 1 6 9 0 cm-l a n d 1 6 8 0 c m - I for 66-40D w h i c h i s c o n s i s t e n t for t h e p r e s e n c e o f a v i n y l e t h e r group. More d e t a i l e d s t u d i e s i n d i c a t e d a d o u b l e b o n d a t C (4')-C(5). The s t r u c t u r e f o r 66-40B a n d 66-40D i s shown i n f i g u r e 4, ( f o l l o w i n g p a g e ) . 3.5 L i t e r a t u r e -
cited
J. W e i n s t e i n , E. 23 ( 1 9 7 0 ) 551.
Oden a n d J . A.
W a i t z , J. A n t i b i o t i c s
1.
M.
2.
D. C o o p e r , R . S . J a r e t a n d H. R e i m a n n , Chem. Commun. ( 1 9 7 1 ) 2 8 5 ; H . R e i m a n n , R. S . J a r e t a n d D . J . C o o p e r , i b i d (1971) 9 2 4 ; M. K u g e l m a n , A. K . Mallams a n d H . F . V e r n a y , J. A n t i b i o t i c s 2 6 ( 1 9 7 3 ) 394.
3.
D. J . C o o p e r , H . M. M a r i g l i a n o , M. J . I n f e c . D i s . 1 1 9 ( 1 9 6 9 ) 342.
M.
D.
Y u d i s a n d T. T r a u b e l ,
180
4.' D. J . C o o p e r , M. D. Y u d i s , R . D. G u t h r i e a n d A. M. P r i o r , J . Chem. S O C . C ( 1 9 7 1 ) 9 6 0 . 5.
D. J. C o o p e r , M. D. Y u d i s , H . M. J . Chem. S O C . C ( 1 9 7 1 ) 2876.
6.
D. J . C o o p e r , P . J. L . D a n i e l s , M . D. Y u d i s , H . M. M a r i g l i a n o , R. D. G u t h r i e a n d S . T . K . B u k h a r i , J . Chem. S OC. C ( 1 9 7 1 ) 3126.
7.
H . R e i m a n n , D. J . C o o p e r , A. K . Mallams, R. S. J a r e t , A. Y e h a s k e l , M. K u g e l m a n , H . F. V e r n a y , D. S c h u m a c h e r , J . O r g . Chem. 37 ( 1 9 7 4 ) 1 4 5 1 - 1 4 5 7 .
8.
D. H . D a v i e s , D. G r e e v e s , A. K . Mallams, J. B . Morton a n d R. W . T k a c h , J . Chem. S O C . ( 1 9 7 5 ) 8 1 4 .
M a r i g l i a n o a n d T. T r a u b e l ,
2' "
F I G U E 4.
66-408 and 66-40D.
'
Z
3' A
4
'
181 Kanamycin
4.
4.1 Introduction 4.1.1
Producing organism
Kanamycin i s a b a s i c water s o l u b l e a n t i b i o t i c f i r s t d e s c r i b e d by Umezawa e t a t . ’ i n 1 9 5 7 . I t is a c t i v e against Mycobacteria, gram-posi r i v e and gram-negative organisms and i s p r o d u c e d by a s p e c i e s o f S A h t p t o m y c e b , S . k a n a m y c e t i c u b 2 .
L1.1.2
Chemical d e s c r i p t i o n and s t r u c t u r e
Kanamycin i s p r o d u c e d as a c o m p l e x c o n s i s t i n g o f 3 a n t i b i o t i c s : kanamycin A’, t h e m a j o r component a n d k a n a m y c i n s B and C 3 ’ 4 , t h e 2 minor components. l e d t o t h e e l u c i d a t i o n of t h e S t u d i e s by C r o n e-t s t r u c t u r e o f kanamycin A ( F i 5 ) w h i l e p a r a l l e l work by Umezawa e t at.9-16, Ogawa a t . 1 7 - 2 F ; a n d I n o n e a n d Ogawa22 c o n f i r m e d i t . Kanamycin A h a s t h e m o l e c u l a r f o r m u l a C 1 8 H 3 6 N 4 0 1 1 a n d t h e Ia154 + 1 4 6 ( c = l N/10 H 2 S O 4 ) ; n o c h a r following properties: a c t 2 r i s t i c u 1t r a v i ole t a bs o r p t i on ; hy d r o c h l or i d e s a 1t r e a d i l y s o l u b l e i n water a n d m e t h a n o l , s l i g h t l y s o l u b l e i n e t h a n o l b u t i n s o l u b l e i n n o n - p o l a r s o l v e n t s ; p o s i t i v e ninhydrin and Molisch t e s t s b u t n e g a t i v e T o l l i n s , F e h l i n g and S a k a g u c h i t e s t s . Ito Kanamycin B h a s t h e m o l e c u l a r f o r m u l a C 1 8 H 3 7 N 5 0 1 1 . h a v e d e s c r i b e d t h e s t r u c t u r e as shown i n F l g u r e 5 , (next page). 11 h a s s i m i l a r p r o p e r t i e s as t h o s e d e s c r i b e d for k a n a m y c i n A. Kanamycin B d e c o m p o s e s a t 1 7 O o C , +114 ( c = 0 . 9 8 % i n water).
et
Kanamycin C h a s t h e m o l e c u l a r f o r m u l a C18H36N,011 a n d h a s t h e s t r u c t u r e shown i n F i g u r e 5 , as e l u c i d a t e d by Murare i n 1 9 6 l Z 4 . I t s c h e m i c a l p r o p e r t i e s are s i m i l a r t o t h o s e o f k a n a m y c i n A a n d B w i t ; { a ) $ 1 + 1 2 6 ( ~ 1 i n%w a t e r ) a n d a decomp o s i t i o n p o i n t a t 270 C. 4.1.3
Summary o f t h e r a p e u t i c use
Kanamycin i s a c t i v e a g a i n s t S t a p h y L a c o c c u h a u h e u b , S t a p h y L o e o c e u s a L b u b , E b c h e h i c h i a c o l i a n d many o t h e r c o l i f o r m s , A . a e h o g e n e b , S h i g e L L a , S a L m o n e L l a , KLebnieLPa pneumoniae a n d many s t r a i n s of PhOteUb. Kanamycin i s u s e d i n u r i n a r y t r a c t i n f e c t i o n s , t o r y i n f e c t i o n s , s k i n , s o f t tissue and p o s t - s u r g i c a l ti ons
.
respirainfec-
The m a j o r t o x i c e f f e c t of p a r e n t e r a l l y a d m i n i s t e r e d k a n a m i c i n i s i t s a c t i o n on t h e a u d i t o r y p o r t i o n of t h e e i g h t h n e r v e . High f r e q u e n c y d e a f n e s s u s u a l l y o c c u r s f i r s t . T i n n i t u s or v e r t i g o may o c c u r a n d a r e e v i d e n c e o f v e s t i b u l a r i n j u r y a n d irreversible deafness.
182
R * OH ; R1 * NH2 A . R1* NH2
R.* N k ; R1* OH
FIGURE 5.
4.2 E x t r a c t i o n ,
KANAMYCIN A KANAMYCIN B KANAMYCINC
Kanarnycins.
s e p a r a t i o n and p u r i f i c a t i o n
T h e r e a r e s e v e r a l ways t h a t k an amyc in c a n b e re m ove d f r o m t h e f e r m e n t a t i o n b r o t h . One m e t h o d i s b y c o n c e n t r a t i n g t h e b r o t h by vacuum or by s p r a y d r y i n g . The k a n a m y c i n c a n t h e n be e x t r a c t e d f r o m t h e r e s i d u e w i t h w a t e r , m e t h a n o l , e t h a n o l or a c e t o n e . A c i d i f i c a t i o n of t h e r e s i d u e i m p r o v e s t h e s o l u b i l i t y . A n o t h e r m e t h o d i s b y a d s o r b i n g t h e k an am yc in i n t h e f i l t e r e d b r o t h on a c t i v a t e d c h a r c o a l a n d e l u t i n g w i t h a q u e o u s a c e t o n e o r a l c o h o l a d j u s t e d t o pH 2 . The m e t h o d of c h o i c e i s a n i o n exchange p r o c e d u r e . 4.2.1
S e p a r a t i o n of k a n a m y c i n A
One h u n d r e d a n d s i x t y l i t e r s o f r h e w h o l e b r o t h were f i l t e r e d ( c o n t a i n i n g 250 mcg/ml o f k a n a m y c i n ) a t pH 2 a n d p a s s e d t h r o u g h a column o f IRC-50 r e s i n ( N a ' f o r m ) a t t h e r a t e of 1 6 k / h r . The v o l u me of t h e co l u mn was 6 II a n d t h e d i a m e t e r was 1 5 cm. A f t e r t h e co l u mn was w a s h e d w i t h water t h e ka na m yc in was e l u t e d w i t h 1N H C 1 a t t h e r a t e of 0 . 8 I I / h r . a n d 2 II f r a c tions collected. The e f f l u e n t i n t h e s e v e n t h t o t e n t h c u t contained the active substance. T h e f r a c t i o n s were c o m b i n e d a n d t h e pH a d j u s t e d t o 6 e v a p o r a t e d a n d l y o p h i l i z e d . 500 g of p o w d e r t h u s o b t a i n e d c o n t a i n e d 8 g of k ana rnyc in. The p o w d e r was d i s s o l v e d i n 2 II of m e t h a n o l , f i l t e r e d a n d 20 II of a c e t o n e was a d d e d , y i e l d i n g 8 g of a p r e c i p i t a t e c o n t a i n i n g 8 gm o f kanamycin. Kanamycin A s u l f a t e was i s o l a t e d by r e p e a t i n g t h e r e s i n a d s o r p t i o n o n IRC-50 ( N H 4 ' f o r m ) . Th e c olum n was e l u t e d w i t h
1 83
0 . 2 N ammonium h y d r o x i d e a n d t h e e l u a t e c o n c e n t r a t e d t o 5 0 - 1 0 0 mg/ml o f k a n a m y ci n a c t i v i t y , d i l u t e d w i t h 0 . 8 II volum e o f m e t h a n o l a n d a d j u s t e d t o pH 8 w i t h s u l f u r i c a c i d . Repeated c r y s t a l l i z a t i o n w i t h m e t h a n o l a t pH 8 y i e l d e d p u r e k a n a m y c i n A.
4.2.2
S e p a r a t i o n of k an amy ci n B
Kanamycin i s a m i x t u r e o f k a n a m y c i n A as a major c om pone nt and t h e 2 minor components kanamycin B and C . Kanamycin B was s e p a r a t e d f r o m k a n a m y c i n c o m p l e x by p a p e r s t r i p c h r o m a t o g r a p h y i n P e t e r s o n ' s n - b u t a n o l :water : 2 % p - t o l u e n e s u l f o n i c a c i d s y s t e m on S c h l e i c h e r a n d S c h u e l l 589 B l u e r i b b o n o r Whatman No. 1 p a p e r s . I n t h i s s y s t e m k a n a m y c i n A h a s a n Rf o f 0 . 3 5 a n d k a n a m y c i n B h a s an Rf a b o u t 0 . 6 . Kanamycin B was i s o l a t e d i n a s i m i l a r way t o ka na m yc in A e x c e p t t h a t a f t e r t h e IRC-50 r e s i n ( N H 4 ' f o r m ) h a s b e e n e l u t e d w i t h ammonium h y d r o x i d e , t h e e l u a t e c o n t a i n i n g k a n a m y c i n s A a n d B was a d j u s t e d t o a pH o f 6 . The e l u a t e was c o n c e n t r a t e d t o a b o u t 5 0 0 0 mcg/ml b y p l a t e a s s a y a n d 7 . 0 g o f s o d i u m d o d e c y l b e n z e n e s u l f o n a t e d i s s o l v e d i n 70 m l was a d d e d . Kanamycin B d o d e c y l b e n z e n e s u l f o n a t e p r e c i p i t a t e s a n d was c o l l e c t e d by f i l t r a t i o n ( f i l t e r a i d Dicolite is used). Kanamycin B s a l t was d i s s o l v e d by s l u r r y i n g t h e c a k e i n 1 0 0 m l o f m e t h a n o l for a b o u t one h o u r and f i l t e r i n g . To t h e c l e a r f i l t r a t e c o n t a i n i n g t h e k a n a m y c i n B s a l t , d i l u t e s u l f u r i c a c i d was a d d e d u n t i l n o more p r e c i p i t a t e was f o r m e d . The p u r i f i e d k ana m yc in was f i l t e r e d a n d f o u n d t o c o n t a i n a much lower p e r c e n t a g e of k a n a m y c i n A t h a n was f o u n d i n t h e r e s i n e l u a t e . Kanamycin B i s p u r i f i e d by d i s s o l v i n g t h e p r e c i p i t a t e i n w a t e r a t 5000 mcg/ml a n d a d j u s t i n g t o pH 6 . T h i s s o l u t i o n was a d s o r b e d i n IRC-50 r e s i n ( N H 4 + f o r m ) The a c t i v e e l u a t e a n d e l u t e d w i t h 1 . 0 N ammonium h y d r o x i d e . was c o n c e n t r a t e d t o a b o u t 3 0 0 0 0 0 mcg/ml a n d a d d e d t o 7 v o l u m e s o f m e t h a n o l w h i c h r emo v es a p r e c i p i t a t e r i c h i n k a n a m y c i n A . T h e f i l t r a t e was a d d e d t o f i v e v o l u m e s o f a c e t o n e y i e l d i n g a p r e c i p i t a t e c o n t a i n i n g p u r i f i e d kanamycin B b a s e . P u r i f i c a t i o n o f k a n a m y c i n B was a c h i e v e d by r e p e a t i n g t h e d o d e c y l b e n z e n e s u l f o n a t e s t e p , f i l t e r i n g , e x t r a c t i n g t h e cake w i t h methanol and a d d i n g s u l f u r i c a c i d t o t h e methanol. This p r o c e d u r e y i e l d e d k an amy ci n B f r e e o f k a n a m y c i n A. 4.2.3
S e p a r a t i o n o f k an amy ci n C
Kanamycin C i s s e p a r a t e d f r o m k a n a m y c i n A a n d B by p a p e r c h r o m a t o g r a p h y u s i n g Toyo f i l t e r p a p e r No. 52 a n d water-saturated n-butanol containing 1.0% p-toluenesulfonic acid. In this c h r o m a t o g r a p h y t h e s p o t of k a n a m y c i n C a p p e a r e d b e t w e e n s p o t s T h e s e s p o t s were d e t e c t e d by a c h r o m a t o g r a p h i c o f A a n d B. m e t h o d d e s c r i b e d by R o t h r o c k e t aL.
'.
The m o t h e r l i q u o r o f k a n a m y c i n A m o n o s u l f a t e c r y s t a l l i z a t i o n was d i l u t e d t o 2 0 % o f t h e s o l i d c o n t e n t ( 3 2 0 0 0 mcg/ml as kanamycin A) and t r e a t e d w i t h d i t h i z o n e a n d a c t i v a t e d c a r b o n . The f i l P r a t e was d i l u t e d t o a b o u t 1%of t h e s o l i d c o n t e n t ( 1 8 0 0 mcg/ml as k a n a m y c i n A ) , a p p l i e d t o a c olum n o f IRC-50
184
(NH,' f o r m ) a n d t h e a n t i b i o t i c s e l u t e d w i t h 1 . 0 N NH40H. The e l u a t e was c o n c e n t r a t e d t o 2 5 % o f t h e s o l i d c o n t e n t ( 3 8 0 0 0 mcg/ml as k a n a m y c i n b a s e ) a n d f e d t o a column of Dowex 1 x 2 r e s i n a n d e l u t e d w i t h d i s t i l l e d water. The r a t e was a b o u t 1 m l / m i n . a n d 1 0 m l f r a c t i o n s were c o l l e c t e d . T h e f r a c t i o n s were e x a m i n e d for w e i g h t a n d c h e c k e d f o r b i o l o g i c a l a c t i v i t y b y d i s c t e s t i n g a g a i n s t S. auteun and chromatographed i n t h e above d e s c r i b e d s y s t e m . The k a n a m y c i n c o m p l e x s e p a r a t e d as f o l l o w s : kanamycin B i n f r a c t i o n s 1 3 - 1 6 ; m i x t u r e o f kanamycin B a n d C i n f r a c t i o n s 27-33, kanamycin C i n f r a c r i o n s 34-50; kanamycin A i n a f t e r f r a c t i o n 6 2 . T h e k a n a m y c i n C f r a c t i o n s were c o m b i n e d a n d One gram o f t h i s p o w d e r was d i s s o l v e d evaporated t o dryness. i n 1 ml o f d i s t i l i e d water a n d a d d e d t o t h r e e v o l u m e s o f m e t h a n o l warmed t o 40 C a n d e t h a n o l was a d d e d d r o p w i s e u n t i l a white p r e c i p i t a t e a p p e a r e d . Kanamycin C was i s o l a t e d as w h i t e crystals.
4.3 Literature
cited
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H . Umezawa, M. U e d a , K . Maeda, K . Y a g i s h i t a , S . Kondo, Y . Okami, R . U t a h a r a , Y. O r a t o , K . N i t t a a n d T . T a k e u c h i , J. A n t i b i o t i c s , J a p a n S e r . A. 10 ( 1 9 5 7 ) 1 8 1 .
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T. T a k e u c h i , T. H i k i j a , K . N i t t a , S . Y a m a z a k i , S . A b e , H . Takayama a n d H . Umezawa, J . A n t i b i o t i c s , J a p a n S e r . A. 10 ( 1 9 5 7 ) 1 0 7 .
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H . U . S c h m i t z , B . F a r d i g , F. A. O ' H e r r o n , M . A. R o u s c h e a n d I . R . H o o p e r , J . Am. Chem. S O C . 80 ( 1 9 5 8 ) 2 9 1 1 .
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J . W. R o r h r o c k , R. T . Goegelman a n d F. J . W o l f , A n t i b i o t . Ann. 1 9 5 8 / 5 9 , ( 1 9 5 9 ) 796-803.
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M. J . C r o n , D. L. E v a n s , F. M. I . R . H o o p e r , P. Chu a n d U. R. 80 ( 1 9 5 8 ) 4741.
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M . J . C r o n , 0. B . F a r d i g , D. J . J o h n s o n , H. S c h m i t z , D . F. W h i t e h e a d , I . R . H o o p e r a n d R . U. L e m i e u x , J . Am. Chem. SOC. 80 ( 1 9 5 8 ) 2342.
7.
M. J . C r o n , 0 . B . F a r d i g , D . L. J o h n s o n , D. F. W h i t e h e a d , I . R. H o o p e r a n d R. U. L e m i e u x , J . Am. Chem. S O C . 80 (1958) 4115.
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M. J. C r o n , D. L. J o h n s o n , F. M. P a l e r m i t i , Y . P e r r o n , H . D. T a y l o r , D. F. W h i t e h e a d a n d I . R . H o o p e r , J . Am. Chem. S O C . 80 ( 1 9 5 8 ) 752.
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P a l e r m i t i , D. F. W h i t e h e a d , L e m i e u x , J. Am. Chem. S O C .
I t o , J. A n t i b i o t i c s , 1 3 (1960) 358.
S. Umezawa, Y . I t o a n d S . F u k a t s u , J . A n t i b i o t i c s , 11 ( 1 9 5 8 ) 120.
11.
S. Umezawa, Y . I t o a n d S . F u k a t s u , J . A n t i b i o t i c s , 11 (1958) 1 6 2 .
185 12.
S . Umezawa, Y . I t o a n d S . F u k a t s u , B u l l . J a p a n 32 (1959) 81.
13.
S. Urnezawa, Y . I t o , S . F u k a t s u a n d H . Urnezawa, J.
Chern. S O C .
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14.
H. Urnezawa, K . Maeda a n d M. U e d a , U.S. P a t e n r 2 , 9 3 1 , 7 9 8 , April 5, 1960.
Umezawa a n d T. T s u c h i y a , J . A n t i b i o t i c s ,
15 (1962) 51.
15.
S.
16.
H. Umezawa, M . U e d a , K . M a e d a , K . Y a g i s h i t a , S . K o n d o , Y . Okarni, R . U t a h a r a , Y . O s a t o , K . N i t t a a n d T. T a k e u c h i , J. A n t i b i o t i c s , 1 0 ( 1 9 5 7 ) 1 8 1 .
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H. Ogawa a n d T . I t o , J . A n t i b i o t i c s , 10 ( 1 9 5 7 ) 2 6 7 .
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H. Ogawa, T . I t o , S . I n o u e a n d S . K o n d o , J . A n r i b i o t i c s , 11 ( 1 9 5 8 ) 7 0 .
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H . Ogawa, T . I t o , S . I n o u e a n d S . K o n d o , J . A n t i b i o t i c s , 11 ( 1 9 5 8 ) 7 2 .
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H. Ogawa, T. I t o , S . I n o u e a n d S . K o n d o , J. A n t i b i o t i c s , 11 ( 1 9 5 8 ) 1 6 6 .
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H. O g a w a , T. I t o , S . Kondo a n d S . I n o u e , J . A n t i b i o t i c s , 11 ( 1 9 5 8 ) 1 6 9 .
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S. I n o u e a n d H . Ogawa, Chern. Pharrn. B u l l . 8 ( 1 9 6 0 ) 7 9 .
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T. I t o , M. N i s h i o a n d H. S e r . A. 1 7 ( 1 9 6 4 ) 1 8 9 .
24.
M.
25.
M. M u r a s e , T . W a k a z a w a , M. Abe a n d I . Kawaji, J .
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1 4 (1961) 156.
1 4 (1961) 367.
186 .5, Tobramycin
5.1 Introduction 5 .l.1 P r o d u c i n g o r g a n i s m T o b r a m y c i n , f o r m e r e l y known as n e b r a m y c i n f a c t o r 6 , was f i r s t d e s c r i b e d i n 1 9 6 7 l as b e i n g p r o d u c e d by a new s p e c i e s of S t u p t o m y c u , namely S. t e n e b a a f i i u b . Two c u l t u r e s o f t h e p r o d u c i n g o r g a n i s m s w e r e i s o l a t e d from t h e s o i l and d e s i g n a t e d a s ATCC 1 7 9 2 0 a n d ATCC 1 7 9 2 1 ' . Tobramycin ( n e b r a m y c i n f a c t o r 6 ) was i n i t i a l l y r e p o r t e d as a b i o l o g i c a l l y a c t i v e component t h a t was co-produced i n t h e n e b r a m y c i n complex a l o n g w i t h 6 o t h e r I n 1 9 7 3 Koch3 r e p o r t e d t h a t t o b r a m y c i n was p r o d u c e d components. as a r e s u l t o f a b a s e c a t a l y z e d h y d r o l y s i s of n e b r a m y c i n f a c t o r 5 which occurs d u r i n g i s o l a t i o n . 5.1.2
Chemical d e s c r i p t i o n a n d s t r u c t u r e
Tobramycin i s a w h i t e c r y s t a l l i n e b a s i c s u b s t a n c e t h a t , l i k e o t h e r a m i n o g l y c o s i d e s , forms s a l t s w i t h b o t h o r g a n i c a n d i n o r g a n i c a c i d s . I t i s s o l u b l e i n w a t e r and d i m e t h y l s u l f o x i d e , s l o w l y s o l u b l e i n m e t h a n o l and i n s o l u b l e i n most o r g a n i c s o l v e n t s . Tobramycin g i v e s a p o s i t i v e Elson-Morgan , n i n h y d r i n and A n t h r o n e t e s t b u t d o e s n o t react t o t h e b i u r e t or S a k a g u c h i test. E l e c t r o m e t r i c t i t r a t i o n of t o b r a m y c i n shows t h e p r e s e n c e o f 5 t i t r a t a b l e g r o u p s h a v i n g pKa v a l u e s of 5 . 8 , 6 . 8 , 7 . 1 , 7 . 9 and 9.3 Analysis:
C a l c u l a t e d for C 1 e H 3 7 N 5 0 9 H 2 0
Specific rotation:
+ 1 2 8 (c-1% i n water).
The a b o v e d a t a s u g g e s t s a s t r u c t u r a l s i m i l a r i t y t o t h e kanamycins. The i s o l a t i o n o f d e r i v a t i v e s of 2 - d e o x y s t r e p t a m i n e and 2 s u g a r s , 3-amino-3-deoxy-D-glucose and n e b r o s a m i n e s u p p o r t e d t h i s assumption. F i n a l l y e v a l u a t i o n of t h e mass s p e c t r u m and cmr s p e c t r u m , t o b r a m y c i n e s t a b l i s h e d i t s s t r u c t u r e t o b e a g l y c o s i d i c l i n k a g e s of 3 -amino-3 -deoxy-D-gl u c o s e and n e b r o s a m i n e t o 2 - d e o x y s t r e p t a m i n e as shown i n F i g . 6 4 ( n e x t p a g e 1. 5.1.3
Summary of t h e r a p e u t i c u s e
Tobramycin e x h i b i t s t h e same k i n d o f a c t i v i t y as d o e s g e n t a m i c i n a n d s i s o m i c i n . I t h a s a b r o a d s p e c t r u m of a c t i v i t y a g a i n s t both gram-negative and gram-positive b a c t e r i a b u t i s n o t e f f e c t i v e a g a i n s t f u n g a l compounds. Like gentamicin and sisomicin i t i s rapidly absorbed a f t e r intramuscular i n j e c t i o n w i t h r e s u l t a n t p e a k s e r u m l e v e l s o c c u r r i n g between 30 and 9 0 minutes. Tobramycin i s i n d i c a t e d f o r t h e t r e a t m e n t of s e p t i c e m i a ; central-nervous system i n f e c t i o n s , including meningitis , s e r i o u s lower r e s p i r a t o r y i n f e c t i o n s , g a s t r o i n t e s t i n a l i n f e c t i o n s and
187
-1
FIGURE 6 .
Tobramycin.
s o f t t i s s u e infections including burns o r g a n i sm s
.
5.2 E x t r a c t i o n ,
,
c a u s e d by s u s c e p t i b l e
s e p a r a t i o n and p u r i f i c a t i o n
T o b r a m y c i n was f i r s t d e s c r i b e d as i s o l a t e d as p a r t o f a c o m p l e x by a n i o n e x c h a n g e p r o c e d u r e 5 . 5.2.1
E x t r a c t i o n of t h e complex
880 l i t e r s o f w h o l e b r o t h were a d j u s t e d t o pH 2 w i t h s u l f u r i c a c i d and f i l t e r e d . The f i l t r a t e was a d j u s t e d t o pH 5 . 5 w i t h s o d i u m h y d r o x i d e a n d p a s s e d t h r o u g h a column c o n The n e b r a m y c i n comt a i n i n g 9 II o f I R C - 5 0 r e s i n ( N H 4 ' f o r m ) . p l e x was e l u t e d f r o m t h e r e s i n w i t h 0 . 1 N s u l f u r i c a c i d . The e l u a t e ( 9 . 4 & ) was c o n c e n t r a t e d t o 6 . 8 II u n d e r vacuum. The pH o f t h e c o n c e n t r a t e d e l u a t e was a d j u s t e d t o 11 w i t h s o d i u m The i n a c t i v e h y d r o x i d e a n d 6 v o l u m e s o f a c e t o n e were a d d e d . p r e c i p i t a t e w h i c h f o r m e d was f i l t e r e d , d i s c a r d e d , a n d t h e pH o f the f i l t r a t e adjusted to 3.5 with s u l f u r i c a c i d i n order t o p r e c i p i t a t e t h e n e b r a m y c i n s u l f a t e s a l t . The n e b r a m y c i n s u l f a t e c o m p l e x was f i l t e r e d a n d r e d i s s o l v e d i n a s m a l l a m o u n t o f d e i o n i z e d w a t e r a n d p a s s e d t h r o u g h a c o l u m n c o n t a i n i n g 1 8 II o f Dowex 1 x 1 r e s i n (OH- form). T h e a c t i v e e l u a n t s were p o o l e d , c o n c e n t r a t e d a n d d r i e d t o y i e l d 425 g o f n e b r a m y c i n b a s e a s s a y i n g 622 u n i t s / m g . 5.2.2
S e p a r a t i o n of t h e n e b r a m y c i n c o m p o n e n t s
The n e b r a m y c i n c o m p l e x ( a s s a y 690 mcg/mg) was d i s s o l v e d i n d e i o n i z e d water. The pH a d j u s t e d t o 4 . 5 w i t h s u l f u r i c a c i d and d e c o l o r i z e d w i t h c h a r c o a l . T h e m i x t u r e was f i l t e r e d a n d f o r m ) column t h e f i l t r a t e p a s s e d t h r o u g h a I R C - 5 0 r e s i n (NH,' (dimensions 9.2 x 10.5 c m ) . The column was w a s h e d w i t h water a n d e l u t e d w i t h 0.1N NH40H a t a f l o w r a t e o f 20 m l / m i n . a n d
188
900 m l fractions collected.
N e b r a m y c i n f a c t o r s 1 - 5 were e l u t e d a t t h i s c o n c e n t r a t i o n b u t n e b r a m y c i n f a c t o r 6 ( t o b r a m y c i n ) was e l u t e d w i t h 0 . 3 N NH40H. E l u a t e s were p o o l e d on t h e bases o f c h r o m a t o g r a p h i c p u r i t y and a s s a y . The p o o l e d e l u a t e s were c o n c e n t r a t e d a n d d r i e d i n vacuo t o y i e l d t h e amo r p h o u s p ow de r of t h e r e s p e c t i v e components. C h r o m a t o g r a p h y was p e r f o r m e d b y r u n n i n g Whatman No. 1 p a p e r i n a d e s c e n d i n g man n er i n a s o l v e n t s y s t e m c o n s i s t i n g o f water-saturated n-butyl alcohol containing 2% p-toluenesulfonic a c i d . The a c t i v e c o m p o n e n t s were l o c a t e d by b i o a u t o g r a p h y o f t h e d e v e l o p e d p a p e r s on a g a r p l a t e s s e e d e d w i t h BaciLLub b u b t i l i b (ATCC 6633). T h e i s o l a t e d c o m p o n e n t s were f u r t h e r p u r i f i e d by d e c o l o r i z i n g w i t h carbon and p a s s a g e o f t h e carbon t r e a t e d s o l u t i o n s t h r o u g h a Bio-Red (AG l l A 8 ) r e s i n c o l u m n . The d e s i r e d f r a c t i o n s were p o o l e d a n d l y o p h i l i z e d . C h r o m a t o g r a p h i c a l l y p u r e n e b r a m y c i n s 2 , 4 , 5 a n d 6 were o b t a i n e d b u t o n l y e n r i c h e d n e b r a m y c i n s 1, 1' a n d 3 w e r e o b t a i n e d i n t h i s m a n n e r . 5.2.3
P r o d u c t i o n of t o b r a m y c i n by h y d r o l y s i s
As m e n t i o n e d b e f o r e , Ko ch 3 r e p o r t e d t h a t t o b r a m y c i n i s n o t p r o d u c e d by f e r m e n t a t i o n b u t i n s t e a d i t i s p r o d u c e d b y a b a s e c a t a l y z e d h y d r o l y s i s of nebramycin f a c t o r 5 ' . Koch a l s o r e p o r t e d t h a t nebramycin f a c t o r 5 (kanamycin B ) i s a l s o p r o d u c e d by h y d r o l y s i s ( h y d r o l y s i s o f f a c t o r 4 ) a n d n o t by f e r m e n tation. The i s o l a t i o n o f t h e n e b r a m y c i n c o m p l e x was a c c o m p l i s h e d by p a s s i n g t h e f i l t e r e d b r o t h t h r o u g h a c olum n o f CG-50 ( N H L + + form) r e s i n . The c o l u m n was w a s h e d w i t h w a t e r a n d t h e n e b r a m y c i n f a c t o r s were e l u t e d w i t h a g r a d i e n t p r e p a r e d by m i x i n g 0.2N ammonium h y d r o x i d e w i t h 0.05N ammonium h y d r o x i d e . Nebram y c i n f a c t o r s 2 a n d 4 a n d a new compound, f a c t o r 5 ' , were eluted i n t h a t order. Un d er t h e s e c o n d i t i o n s of i s o l a t i o n a n d s e p a r a t i o n , n e b r a m y c i n f a c t o r 5 ( k a n a m y c i n B) a n d t o b r a m y c i n were n o t d e t e c t e d i n t h e f e r m e n t a t i o n b r o t h . D e t e c t i o n o f t h e n e b r a m y c i n f a c t o r s was d o n e on t h i n l a y e r chromatography p l a t e s . F a c t o r s 2 , 4 a n d 5 ' were s e p a r a t e d o n Merck s i l i c a g e l G p l a t e s u s i n g a s o l v e n t s y s t e m composed o f c h l o r o f orm-met h a n o l - 2 8 % ammonium h y d r o x i d e ( 1: 3 :2 ) The comp o u n d s were d e t e c t e d by n i n h y d r i n . A m o d i f i c a t i o n of t h e Pa nD u t c h e r 6 r e a g e n t i n w h i c h s t a r c h i o d i d e was r e p l a c e d by 1 0 0 mg b e n z i d i n e , 1 0 0 mg K I a n d 2 m l o f g l a c i a l a c e t i c a c i d i n 1 0 0 m l o f water.
.
N e b r a m y c i n f a c t o r 5 a n d t o b r a m y c i n c a n be i s o l a t e d f r o m t h e a b o v e f e r m e n t a t i o n by f o l l o w i n g t h e o r i g i n a l i s o l a t i o n p r o c e d u r e d e s c r i b e d by Thompson5. The d i f f e r e n c e i n t h e p r o cess is t h a t T h o m p s o n ' s p r o c e d u r e t h e c o m p l e x was e x p o s e d t o s t r o n g l y b a s i c c o n d i t i o n s . The pH o f t h e IRC-50 e l u a t e was a d j u s t e d t o 11 a n d i n a c t i v e s o l i d s were p r e c i p i t a t e d w i t h
189
acetone.
The p r e c i p i t a t e a t t h i s pH s t o o d o v e r n i g h t . Later t h e s u l f a t e d f o r m o f t h e c o m p l e x was c o n v e r t e d t o t h e f r e e b a s e by p a s s a g e t h r o u g h a Dowex 1 x 1 ( O H - f o r m ) r e s i n . Since e i t h e r of t h e s e 2 s t e p m i g h t c a u s e a b a s e c a t a l y z e d h y d r o l y s i s , K o c h 3 e x a m i n e d t h e e f f e c t of a q u e o u s b a s e as f a c t o r s 4 and 5 ' . F a c t o r 4 was c o n v e r t e d t o f a c t o r 5 when h e a t e d a t 0 . 1 N N a O H a t 99'C, a n d u n d e r s i m i l a r c o n d i t i o n s f a c t o r 5 ' was c o n v e r t e d t o t o b r a m y c i n as shown b e l o w : C19H38N2011
' H20
OH-
Factor 4 C19H38N6010
Kanamycin B ( F a c t o r 4 )
' H20
OH-
C18H37N50g
+
CO2
' NH3
To bramy c i n
Factor 5 5.3 L i t e r a t u r e -
C18H37N5010 + C O 2 t N H 3
cited
1.
W . M . S t a r k , M. M. Itoehn a n d N . G . Knox, A n t i m i c r o b i a l A g e n t s a n d Chemotherapy ( 1 9 6 7 ) 314-323.
2.
C. E . I t i g g i n s a n d R. E. K a s t n e r , A n t i m i c r o b i a l A g e n t s a n d Chemotherapy ( 1 9 6 7 ) 324-331.
3.
K . F. Koch, F. A. D a v i s a n d J . A . R h o a d e s , J . A n t i b i o t i c s 26 ( 1 9 7 3 ) 7 4 5 - 7 5 1 .
4.
K . F. Koch a n d J . A. R h o a d e s , A n t i m i c r o b i a l A g e n t s and Chemotherapy ( 1 9 6 7 ) 309-313.
5.
R . Q . Thompson a n d E . A. P r e s t i , A n t i b i o t i c s a n d C h e m o t h e r a p y ( 1 9 6 7 ) 332-340.
6.
S.
C.
P a n a n d J , D.
D u t c h e r , A n a l . Chem. 28 ( 1 9 5 6 ) 8 3 6 - 8 3 8 .
190 6.
V e r d a m i c i n a n d A n t i b i o t i c G-52
6 . 1 Introduction 6.1.1
Producing organism
Two s p e c i e s o f Michomonobpoha, n a m e l y M. puhpuhea a n d have previously been described t o produce, respect i v e l y , t h e aminoglycoside a n t i b i o t i c s , gentamicinl and sisomicin Two a d d i t i o n a l a m i n o g l y c o s i d e a n t i b i o t i c s , v e r d a m i c i n 3 a n d G-5Z4 p r o d u c e d by two o t h e r s p e c i e s o f Michomoflobpoha, n a m e l y M . g f i i b e a and M . z i o n e n n i b , r e s p e c t i v e l y , w i l l b e d i s cussed i n t h i s section.
M.
iflyOCnhiA
.
V e r d a m i c i n , r e p o r t e d by W e i n s t e i n e t ab. i n 1 9 7 0 d e r i v e d i t s name f r o m t h e g r a y - g r e e n c o l o n y c o l o r t h a t i s t y p i c a l o f t h e p r o d u c i n g o r g a n i s m o n a v a r i e t y o f c u l t u r e medium.
G-52 , a more r e c e n t l y d i s c o v e r e d b r o a d s p e c t r u m a n t i b i o t i c , was r e p o r t e d by Marquez e t ab. i n 1 9 7 6 . 6.1.2 6.1.2.1
Chemical d e s c r i p t i o n and s t r u c t u r e Verdami c i n
D a n i e l s and Yehaske15 d e s c r i b e d t h e s t r u c t u r e of verdam i c i n i n a p r e s e n t a t i o n a t t h e 1 3 t h I n t e r s c i e n c e C o n f e r e n c e on A n t i m i c r o b i a l Agents and Chemotherapy i n 1973. A c o m p a r i s o n o f t h e pmr s p e c t r a o f v e r d a m i c i n a n d s i s o m i c i n s h o w t h e mar k ed s i m i l a r i t y of t h e t w o c om pounds. B u t t h e d i f f e r e n c e s n o t e d , s u g g e s t t h a t verdamicin is a 6'-C-methylsisomicin. T h i s i s s u p p o r t e d by e v a l u a t i o n o f t h e mass s p e c F i g . 7 shows t h e v e r d a t r u m a n d cmr s p e c t r u m o f v e r d a m i c i n . micin s t r u c t u r e .
FIGURE 7.
Verdamicin.
19 1 Verdamicin r e a d i l y forms salts w i t h o r g a n i c and i n o r g a n i c acids. I t i s e x t r e m e l y water s o l u b l e a n d h a s some s o l u b i l i t y V e r d a m i c i n e x h i b i t s no a b s o r p i n most p o l a r o r g a n i c s o l v e n t s . t i o n i n t h e u l t r a v i o l e r r a n g e of 220-400. Further, i t is s t a b l e t o b o i l i n g f o r a t l e a s t 30 m i n u t e s i n t h e pH range of 2-10. I t i s o p t i c a l l y a c t i v e Ia166 + 1 5 7 . 9 (c.0.38 a n d h a s a n e q u i v a l e n t w e i g h t o f 1 0 6 . 8 a n d a pKa
6.1. 2. 2
i n water) 8.1.
A n t i b i o t i c 6-52
The s t r u c t u r e o f 6-52 was e l u c i d a t e d by D a n i e l s 1 9 7 6 a n d i s shown i n F i g . 8.
FIGURE 8.
et
A n t i b i o t i c G-52.
E v a l u a t i o n of i t s pmr a n d mass s p e c r r u m c o u p l e d w i t h t h e a n a l y s i s of i t s p r o t o n d e c o u p l e d cmr s p e c r r a l e d t o t h e a s s i g n ment of 6 ' - N - m e t h y l s i s o m i c i n as t h e s t r u c t u r e o f 6 - 5 2 . This a s s i g n m e n t was c o n f i r m e d b y t h e s y n t h e t i c c o n v e r s i o n of s i s o m i c i n t o 6-52. S i s o m i c i n was r e a c t e d w i t h t - b u t o x y c a r b o n y l o x y phthalimide t o a f f o r d t h e 6'-N-t-butoxycarbonyl d e r i v a t i v e w h i c h was r e d u c e d w i t h l i t h i u m a l u m i n u m h y d r i d e t o a n t i b i o t i c 6-52. 6-52 i s a w h i t e s o l i d w i t h t h e same c h e m i c a l p r o p e r t i e s as v e r d a m i c i n . Its o p t i c a l r o t a t i o n is + 156.4 ( ~ 0 . 3 % i n water) a n d h a s a pKa o f 8 . 2 .
6.1.3 6.1.3.1
Summary of b i o l o g i c a l a c t i v i t y of v e r d a m i c i n a n d 6-52 Verdami c i n
Verdamicin h a s b r o a d s p e c r r u m a c t i v i t y i n v i t h o b u t i t is n o t a c ~ i v ea g a i n s t Candida or S a c c h a n o m y c e s . I t shows a h i g h
19 2 d e g r e e of a c t i v i t y a g a i n s t k a n a m y c i n - r e s i s t a n t s t r a i n s of K . pneumoniae b u t a p p e a r s t o be c r o s s - r e s i s t a n t w i t h g e n t a -
micin. I n mouse p r o t e c t i o n t e s t s i t shows s i m i l a r a c t i v i t y t o g e n t a m i c i n w i t h a s u g g e s t i o n of improved a c t i v i t y a g a i n s t StheptOCOCCUb.
6 .l. 3 . 2
A n t i b i o t i c 6-52
6-52 has b r o a d s p e c t r u m a c t i v i t y b u t a p p e a r s t o be l e s s a c t i v e than genramicin. I t i s a c t i v e a g a i n s t kanamycin-resist a n t K L e b d i d L a s t r a i n s b u t not a g a i n s t gentamicin-resistant
PA eudomo n a d
.
R e s u l t s of l i m i t e d i n v i v a t e s t s i n mice s u g g e s t t h a t G-52 i s s l i g h t l y l e s s a c t i v e t h a n g e n t a m i c i n or s i s o m i c i n .
I t s a c u t e t o x i c i t y e x p r e s s e d as LD,, i s c o m p a r a b l e to g e n t a m i c i n v i a t h e i n t r a v e n o u s and s u b c u t a n e o u s r o u t e s b u t is more t o x i c v i a t h e i n t r a p e r i t o n e a l r o u t e .
6 . 2 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
Verdamicin i s co-produced w i t h s i s o m i c i n and t h e y are i s o l a t e d as a complex from a f e r m e n t a t i o n b r o t h by an i o n exchange p r o c e d u r e . O x a l i c a c i d was added t o t h e whole b r o t h t o p r e c i p i t a t e calcium i o n s , and t h e pH of t h e f e r m e n t a t i o n f u r t h e r adjusted t o 2.0 with s t r o n g mineral a c i d t o r e l e a s e t h e major p a r t of t h e a n t i b i o t i c from t h e mycelium. After f i l t r a t i o n t h e c l a r i f i e d b r o t h was n e u t r a l i z e d w i t h ammonium h y d r o x i d e and t h e a n t i b i o t i c a d s o r b e d t o I R C 5 O r e s i n (NHL,+ form) and t h e s p e n t b r o t h d i s c a r d e d . The a n t i b i o t i c complex was e l u t e d from t h e r e s i n w i t h 2 N ammonium h y d r o x i d e and t h e e l u a t e c o n c e n t r a t e d and e v a p o r a t e d t o dryness. The complex c o n s i s t s o f v e r d a m i c i n , s i s o m i c i n and some minor components and has a p o t e n c y of a b o u t 1 3 6 mcg/mg (as gen t a m i c i n ) 7. S e p a r a t i o n of v e r d a m i c i n was a c h i e v e d by s i l i c a g e l chromatography. It was e l u t e d w i t h t h e lower p h a s e of a s o l v e n t m i x t u r e c o n s i s t i n g of chloroform-isopropanol-concentrated ammonium h y d r o x i d e ( 2 : 1:1). D e t e c t i o n of t h e a n t i b i o t i c s and d e t e r m i n a t i o n of homog e n i c i t y o f f r a c t i o n s was c a r r i e d o u t by p a p e r chromatography and was a c c o m p l i s h e d by t h e two methods p r e v i o u s l y d e s c r i b e d f o r gentamicin. The i s o l a t e d v e r d a m i c i n h a d a p o t e n c y of 8 1 3 mcg/mg i n the assay previously described. F u r t h e r p u r i f i c a t i o n by conv e r s i o n t o t h e s u l f a t e d e r i v a t i v e and r e c o n v e r s i o n to i t s b a s e r e s u l t e d i n a component which a s s a y e d 1 0 0 0 mcg/mg. 6 - 5 2 i s a l s o produced as a m i x t u r e w i t h s i s o m i c i n . Its i s o l a t i o n p r o c e d u r e p a r a l l e l s t h a t of v e r d a m i c i n . A f t e r e l u t i o n
193
from t h e r e s i n i t h a s a p o t e n c y of a b o u t 3 0 0 mcg/mg ( a s g e n t a micin). The s e p a r a t i o n a n d p u r i f i c a r : on p r o c e d u r e i s t h e same as t h a t used f o r verdamicin. F i n a l p u r i f i c a t i o n y i e l d s G-52 b a s e w i t h a p o t e n c y of 8 6 5 mcg/mg as c o m p a r e d w i t h g e n t a m i c i n .
6.3 Literature
cited
1.
M. J. W e i n s t e i n , G . M . Luedemann, E . M . Oden a n d G . H . Wagman, A n t i m i c r o b i a l A g e n t s a n d C h e m o t h e r a p y ( 1 9 6 3 ) 1 - 7 .
2.
M. J . W e i n s t e i n , J . A. M a r q u e z , R . T. Testa, G . H. Wagman, E . M. Oden a n d J . A . W a i t z , J . A n t i b i o t i c s , 2 3 ( 1 9 7 0 ) 5 5 1 .
3.
M. J . W e i n s t e i n , G. H . Wagman, J. A. M a r q u e z , R. T. Testa a n d J . A. W a i t z , A n t i m i c r o b i a l A g e n t s a n d C h e m o t h e r a p y (1975) 246-249.
4.
J . A. M a r q u e z , G . H . Wagman, R . T. T e s t a , J . A. W a i t z a n d M. J . W e i n s t e i n , J . A n t i b i o t i c s , 2 9 ( 1 9 7 6 ) 4 8 3 - 4 8 7 .
5.
P. J . L. C a n i e l s and A. S . Y e h a s k e l , p r e s e n t e d a t t h e 1 3 t h I n t e r s c i e n c e Conference on A n t i m i c r o b i a l Agents and Chemotherapy , 1 97 3.
6.
P . J . L.
7.
E.
D a n i e l s , R. S . J a r e t , T . L . N a ga bhusha n a n d W . N . T u r n e r , J. A n t i b i o t i c s , 2 9 ( 1 9 7 6 ) 4 8 8 - 4 9 1 .
M. Oden, H . S t a n d e r a n d M . J . W e i n s t e i n , A n t i m i c r o b i a l Agents and Chemotherapy ( 1 9 6 3 ) 8 .
1 9 II
7 . Neomycins
7.1 Introduction 7 . 1 . 1 Producing organisms
Neomycin was f i r s t d e s c r i b e d i n 1 9 4 9 i n t h e now c l a s s i c a l p a p e r of Waksman a n d L e C h e v a l i e r as b e i n g p r o d u c e d f r o m S t h e p Xomqceh d h a d i a e a n o r g a n i s m o r i g i n a l l y s t u d i e d by Waksman a n d C u r t i s i n 1916"*. S e v e r a l o t h e r s p e c i e s o f Stkeptomyced h a v e a l s o b e e n n o t e d w h i c h p r o d u c e n e o m y c i n , most n o t a b l y S . aLbog h i d e o l u d 3 . A n t i b i o t i c p r o d u c t i o n was a l s o f o u n d i n two s t r a i n s o f Stheptomyced h a v i n g b l u e a e r i a l m y c e l i u m , a n d i n two cases f r o m d i f f e r e n t g e n e r a , Elyptoopahangium bkaAiLLende4 a n d M i c h o mono6poha A p . 6 9 - 6 8 3 5 . 7.1.2
Chemical d e s c r i p t i o n and s t r u c t u r e
L i k e most a n t i b i o t i c s t h e n e o m y c i n s are g e n e r a l l y p r o d u c e d as a c o m p l e x o f c l o s e l y r e l a t e d compounds i n v a r y i n g r a t i o s d e p e n d i n g upon t h e f e r m e n t a t i o n c o n d i t i o n s . T h e major c o m p o n e n t s were d e s i g n a t e d o n t h e b a s i s o f e a r l i e r c h r o m a t o g r a p h i c s e p a r a t i o n s as n e o m y c i n s A , B a n d C 6 . Physico-chemical s t u d i e s p e r f o r m e d o n t h e n eo my ci n c o m p o n e n t s g a v e t h e i n f o r m a t i o n shown i n T a b l e 5 . TABLE 5
-A Formula
Neomycin
-B
C -
C23H46N6013
c2 3H46N60 1 3
Melting Point
2 5 6 O C dec.
N.A.
N.A.
S p e c i f i c r o t at i o n "
+
+
+
Absorption
End
12H26N4O6
123'
Absorption
83O**
End
Absorption
121O
End Absorption
The n e o m y c i n s a r e b a s i c , h i g h l y p o l a r a n d w a t e r s o l u b l e . They p o s s e s s d e c r e a s i n g s o l u b i l i t y i n m e t h a n o l a n d e t h a n o l r e s p e c t i v e l y and are v i r t u a l l y i n s o l u b l e i n l e s s p o l a r o r g a n i c solvents. I n a d d i t i o n , t h e y e x h i b i t h i g h v o l a t i l i t y a nd a n a b s e n c e of clear m e l t i n g p o i n t s . The c h e m i s t r y of t h e n e o m y c i n s h a s b e e n e x t e n s i v e l y r e v i e w e d by R i n e h a r t 7 w h i c h l e d t o t h e p r o p o s a l o f t h e s t r u c t u r e s shown i n F i g u r e 9 ( n e x t p a g e ) . T h e m i n o r c o m p o n e n t s LP, a n d LPb, s o named b e c a u s e of t h e i r low p o t e n c y a nd f o r m a l l y known as LP-I a n d LP-11, were n o t e d as b e i n g p r o d u c e d i n c e r t a i n n e o m y c i n c u l t u r e s a n d are t h u s r e p r e s e n t e d h e r e . Neomycin A ( n e a r n i n e ) i s a c o n s t i t u e n t of i n t a c t n e o m y c i n s B a n d C a n d i s b o t h a fermentation and a degradation product8. N e om yc ins B a n d C a r e e a c h composed o f a n ear n i n e m o i e t y l i n k e d v i a g l y c o -
19 5 s i d i c bonds t o a n e o b i o s a m i n e m o i e t y . The d i f f e r e n c e b e t w e e n t h e t w o r e s t s i n t h e s t e r e o c h e m i s t r y o f t h e C - 6 p o s i t i o n of n e o s a m i n e s B a n d C b e i n g 2,5-diamino-2,6-dideoxy-L-idose f o r t h e f o r m e r a n d 2,5-diamino-2,6-dideoxy-D-glucose f o r t h e l a t t e r 9 > 1 0 . T h i s same s u b t l e d i f f e r e n c e i s a l s o s e e n i n t h e paromomycins and w i l l b e d e s c r i b e d i n a l a t e r s e c t i o n .
1: R'*H. R z * C W , R3.H. R4*NH2 I R3m &,H* NH2 2: R 1 * W , R2mH, G * H . R4*NH2 5: #mAc,R4*NH2 4:
(MOMVCIN B ) (NEOMYON LP-B) (NU)MVUNC) (NEOMYCIN LP-C)
H NH2
no H
FIGURE 9 .
7.2 T h e r a p e u t i c
Neornycins
.
u s age
The d i s c o v e r y o f n e o m y c i n a n d i t s s u b s e q u e n t b r o a d s p e c t r u m of a c t i v i t y h e r a l d e d a n e x p l o s i v e e f f o r t i n a n t i b i o t i c research. The a n t i b i o t i c h a s a b r o a d s p e c t r u m of a c t i v i t y a g a i n s t b o t h g r a m - p o s i t i v e and gram-negative b a c t e r i a , l i t t l e a n t i p r o t o z o a l a c t i v i t y a n d i s i n e f f e c t i v e as an a n t i v i r a l o r a n t i f u n g a l compound. The l a c k o f a n t i p r o t o z o a l a c t i v i t y o f n e o m y c i n i s i n c o n t r a s t t o t h a t of p ar o m om yc in w h i c h d i f f e r s by t h e r e p l a c e m e n t o f a n a m i n o g r o u p b y a h y d r o x y g r o u p l l . S u b s e q u e n t s t u d y h a s shown n e o m y c i n B t o b e b e t w e e n 3 a n d 1 0 times more a c t i v e Than e i t h e r n e o m y c i n C or n e a m i n e
19 6 a l t h o u g h e v e n more p r o n o u n c e d d i f f e r e n c e s h a v e b e e n n o t e d ” . A w i d e r a n g e of a p p l i c a t i o n s f o r n e o m y c i n h a v e b e e n described13. Administered p a r e n t e r a l l y , it is used i n t h e t r e a t m e n t of s y s t e m i c g r a m - p o s i t i v e and gram-negative i n f e c t i o n s a n d as a p r o p h y l a c t i c p r e v e n t i o n o f p e r i t o n i t i s due t o i n t e s t i n a l contamination during abdominal surgery. I t has been i n s t i l l e d i n t o t h e b l a d d e r f o r u r i n a r y t r a c t i n f e c t i o n s . G i v e n o r a l l y , n e o m y c i n i s u s e d as a p r e - o p e r a t i v e bow e l s t e r i l i z a t i o n a g e n t , i n t h e t r e a t m e n t o f d i a r r h e a s c a u s e d by c e r t a i n e n t e r e o - p a t h o g e n i c E . c o e i s t r a i n s a n d as t h e a g e n t o f c h o i c e f o r t h e p r e v e n t i o n o f h e p a t i c p r e c o m a or coma. Neomycin i s , h o w e v e r , by f a r most w i d e l y u s e d as a t o p i c a l a n t i b i o t i c a n d i s f o r m u l a t e d i n a v a r i e t y o f creams, o i n t m e n t s , d r o p s a n d l o t i o n s w i t h c o r t i c o s t e r o i d s or a n t i f u n g a l a g e n t s or by i t s e l f f o r a l a r g e number o f de r m a t o l o g i c a l , o p t h a m o l o g i c a l or o t o l o g i c a l infections. L i k e most o t h e r a m i n o g l y c o s i d e s , n e o m y c i n e x h i b i t s c h r o n i c e i g h t h c r a n i a l nerve and r e n a l t o x i c i t i e s . Acute t o x i c i t i e s i n mice (LDS0) r a n g e f r o m 1 2 0-265 mg/kg s u b c u t a n e o u s l y , 1 1 6 - 1 3 3 mg/kg i n t r a p e r i t o n e a l l y , 1 5 -370 mg/kg i n t r a v e n o u s l y a n d 2800 mg/kg o r a l l y .
7.3 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n
7 . 3 . 1 E x t r a c t i o n of c r u d e a n t i b i o t i c I n i t i a l l y , e x t r a c t i o n o f t h e n e o m y c i n s was p e r f o r m e d u s i n g a v a r i a n t o f t h e p r o c e d u r e d e v i s e d e a r l i e r for t h e i s o l a t i o n of ~ t r e p t o m y c i n l ~ .The f e r m e n t a t i o n b r o t h i s a c i d i f i e d t o pH 2 . 7 w i t h s u l f u r i c acid and f i l t e r e d u s i n g a diatomaceous e a r t h - t y p e f i l t e r aid. The pH o f t h e r e s u l t a n t f i l t r a t e i s t h e n a d j u s t e d t o 8.0 w i t h 5 0 % s o d i u m h y d r o x i d e . To t h e b a s i c s o l u t i o n , a c t i v a t e d c h a r c o a l i s a d d e d a n d t h e s u s p e n s i o n i s a g i t a t e d for 1 hour and f i l t e r e d . C r u d e a n t i b i o t i c i s e l u t e d from t h e charc o a l c a k e w i t h f o u r a l i q u o t s o f 1 0 % a c e t o n e (pH 2 . 0 ) w h i c h are t h e n c o m b i n e d f o l l o w e d by a d d i t i o n o f n e a t a c e t o n e y i e l d i n g a p r e c i p i t a t e w h i c h i s t h e n r e d i s s o l v e d i n water a n d a d j u s t e d t o pH 6 . 2 . A t t h i s p o i n t t h e a n t i b i o t i c could be l y o p h i l i z e d or f u r t h e r p u r i f i e d u s i n g a p i c r a t e p r e c i p i t a t i o n m e t h o d 1 5 . Neom y c i n p i c r a t e s a r e d i s s o l v e d i n m i n i m a l a m o u n t s of a c e t o n e t o w h i c h s u l f u r i c a c i d i s a d d e d y i e l d i n g n e o m y c i n s u l f a t e . The p r e c i p i t a t e i s t h e n washed w i t h a c e t o n e , r e d i s s o l v e d i n water, a d j u s t e d t o pH 4 . 7 w i t h b a s e a n d l y o p h i l i z e d . The p r e c e d i n g m e t h o d , h o w e v e r , g a v e p o o r y i e l d s o f a n t i b i o t i c with varying potencies generally unsatisfactory f o r large scale separation. The met h o d o f c h o i c e f o r n e o m y c i n as w e l l as most o t h e r a m i n o g l y c o s i d e s i s t h e i o n - e x c h a n g e p r o c e d u r e 1 6 - 1 8 . S e v e r a l v a r i a t i o n s o f t h i s p r o c e d u r e h a v e be e n r e p o r t e d w i t h y i e l d s i n t h e 7 0 % or g r e a t e r r a n g e . A t y p i c a l procedure is t h a t used f o r t h e e x t r a c t i o n o f neomycin from Michomonodpoha A?. 69-68?”. Th e pH o f t h e w h o l e b r o t h i s a c i d i f i e d w i t h mineral a c i d and f i l t e r e d . O x a l i c a c i d i s t h e n added t o t h e n e u t r a l f i l t r a t e a n d t h e r e s u l t a n t p r e c i p i t a t e i s removed by filtration. T h e f i l t r a t e i s t h e n n e u t r a l i z e d a n d a d s o r b e d on
1 97
a n A m b e r l i t e IRC-50 c a t i o n e x c h a n g e r e s i n i n t h e ( N H k t f o r m ) . The r e s i n i s t h e n w a s h e d w i t h s e v e r a l v o l u m e s o f water a n d t h e w a s h i n g s and i n a c t i v e f i l t r a t e are d i s c a r d e d . The a n t i b i o t i c m i x t u r e i s t h e n e l u t e d w i t h 2M ammonium h y d r o x i d e . The a m m o n i a c a l e l u a t e i s t h e n c o n c e n t r a r e d , a c i d i f i e d t o pH 4 . 5 with s u l f u r i c a c i d and t r e a t e d w i t h a c t i v a t e d carbon. The neomcyin m i x t u r e i s t h e n ad d ed t o m e t h a n o l t h u s p r e c i p i t a t i n g i t as t h e s u l f a t e s a l t . I t s h o u l d b e n o t e d t h a t t h i s t e c h n i q u e is similar t o s e v e r a l o t h e r p r o c e d u r e s w e l l documented i n t h e neomycin l i t e r a t u r e . E x c e p t f o r minor v a r i a t i o n s , a l l o f t h e r e p o r t e d methods are v i r t u a l l y i d e n t i c a l . I n a d d i t i o n , as a p r o c e d u r a l n o t e , a d s o r b t i o n t o t h e c a t i o n exchange r e s i n h a s been r e p o r t e d u s i n g e i t h e r t h e b a t c h method w i t h i t s a t t e n d e n t e q u i l i b r a t i o n problems, or t h e c o l u m n m e t h o d a n d i t s i n h e r e n t s l o w n e s s .
7.3.2 D e t e c t i o n I n o r d e r t o d i f f e r e n t i a t e t h e neomycin complex, s e v e r a l paper and t h i n l a y e r chromatographic systems have been proposed over the years. T h i s was d o n e f o r t h e p u r p o s e o f b o t h s u p p l e menting a non-resolving bioassay procedure, to v i s u a l i z e t h e c o m p o n e n r s i n t h e a n t i b i o t i c m i x t u r e a n d as a m e t h o d t o f o l l o w t h e c o u r s e of f e r m e n t a t i o n , e x t r a c t i o n and p u r i f i c a t i o n . The f o l l o w i n g s o l v e n t systems f o r p a p e r chromatography of i n t a c t neomycins h a v e been u t i l i z e d w i t h v a r y i n g d e g r e e s o f s u c c e s s : A.
n-propano1:glacial
des cending
a c e t i c a c i d : w a t e r (9:l:lO v / v ) ,
5.
B. 2-butanone:t-butanol:methanol:6.5N (16:3:1:6 v / v ) , d e s c e n d i n g z 1 .
ammonium h y d r o x i d e
V i s u a l i z a t i o n i s u s u a l l y c a r r i e d o u t by e i t h e r t h e u s e of bioautography a g a i n s r a s u s c e p t i b l e organism (e.g. B a c i U u b 6 u b . C L f i b ) or a p r i m a r y a m i n o g r o u p r e a g e n t s u c h as n i n h y d r i n o r f l u o r e s cami n e
.
Two t h i n l a y e r c h r o m a t o g r a p h i c s y s t e m s h a v e a l s o b e e n d e v e l o p e d f o r r e s o l u t i o n of i n t a c t n e o m y c i n m i x t u r e s w h i c h y i e l d f a i r l y good r e s u l t s :
A.
' B. (10:3.5:3:7
0.5N s u l f u r i c a c i d u t i l i z i n g carbon p l a t e s z 2 .
M e t h y l e t h y l k e t o n e : m e t h a n o l : i s o p r o p a n o l : 7.9N NHbOH v / v ) on c e l l u l o s e p l a t e s w i t h m u l t i p l e d e v e l o p m e n t Z 3 .
E x c e l l e n t r e s o l u t i o n of N - a c e t y l a t e d d e r i v a t i v e s o f n e o m y c i n s were r e p o r t e d f o r d i f f e r e n t i a l a s s a y a n d c o m p o s i t i o n o f the a n t i b i o r i c mixture. The o n l y d r a w b a c k i s t h e i n a b i l i t y t o r e g e n e r a t e d e a c e t y l a t e d s t a r t i n g p r o d u c t s once s e p a r a t i o n had occurred and hence bioautography cannot be used to v i s u a l i z e separation:
A.
n-butano1:pyridine:water ( 6 : 4 : 3 v / v ) d e s c e n d i n g
papergramz".
198
n-butano1:water:pyridine B: p a p e r g r a m 25 .
(
84:16:2
C. isoamyl alcoho1:water:pyridine a s c e n d i n g p a p e r g r a m 26 . D.
v/v) descending
( 1 : O . 8:l v / v )
1-butano1:pyridine:water ( 3:2:2 v / v ) d e s c e n d i n g 2 ' .
Using t h e N - a c e t y l a t e d d e r i v a t i v e s , v i s u a l i z a t i o n c a n be achieved e i t h e r w i t h t h e starch-potassium i o d i d e spray (PanD u t c h e r r e a g e n t ) or as r e p o r t e d i n case B ( a b o v e ) u s i n g "+C-Ha c e t y l d e r i v a t i v e s and d e t e c t i o n w i t h a r a d i o c h r o m a t o g r a m scanner
.
7.3.3
P u r i f i c a t i s o n and c h r o m a t o g r a p h i c methods
Using t h e c a t i o n exchange r e s i n e x t r a c t i o n procedure p r e v i o u s l y m e n t i o n e d and t h e a t t e n d a n t f o r m a t i o n o f t h e s u l f a t e s a l t , h i g h p o t e n c i e s are u s u a l l y a c h i e v e d . The neomycin s u l f a t e complex can b e f u r t h e r p u r i f i e d by r e d i s s o l v i n g i n water, a d d i n g d e c o l o r i z i n g c h a r c o a l , h e a t i n g and f i l t r a t i o n . L y o p h i l i z a t i o n of t h e f i l t r a t e y i e l d s a w h i t e powder w i t h somewhat higher assay values than t h e s t a r t i n g s u l f a t e . 7.3.3.1
S e p a r a t i o n u s i n g anion-exchange chromatography
R e s o l u t i o n of t h e neomycin complex i n t o i t s c o n s t i t u e n t com o n e n t s can b e r e a d i l y a c h i e v e d u s i n g a n i o n - e x c h a n g e r e s i n 28yq9. More c o r r e c t l y , t h i s i s a n i o n - e x c l u s i o n p r o c e d u r e where s e p a r a t i o n occurs p r i m a r i l y as a r e s u l t o f t h e s t r e n g t h of London f o r c e i n t e r a c t i o n s between t h e s o l u t e and r e s i n . The u s u a l t e c h n i q u e i s t o p r e p a r e a column u s i n g a s t r o n g l y b a s i c ( q u a t e r n a r y amine t y p e ) a n i o n - e x c h a n g e r e s i n s u c h as Dowex 1 x 2 o r A m b e r l i t e 4 0 1 w i t h p a r t i c l e s i z e s of 50-100 mesh i n t h e h y d r o x y l form. A p p r o x i m a t e l y 25% s o l u t i o n s of t h e a n t i b i o t i c s u l f a t e are p r e p a r e d and washed o n t o t h e t o p of t h e r e s i n bed w i t h d i s t i l l e d water. D i s t i l l e d water i s u s e d as t h e eluant. D e t e r m i n a t i o n of t h e e l u t e d components can be made by n i n h y d r i n a s s a y , b i o l o g i c a l a s s a y , o p t i c a l r o t a t i o n or q u a l i tat i v e m o n i t o r i n g u s i n g t h i n l a y e r o r p a p e r chromatograms of selected fractions. 7.3.3.2
S e p a r a t i o n u s i n g c a t i o n exchange chromatography
The more c l a s s i c a l form of i o n e x c h a n g e c h r o m a t o g r a p h y u t i l i z i n g c a t i o n exchange by v a r y i n g s o l v e n t s t r e n g t h has a l s o been r e p o r t e d 30. Aqueous neomycin s u l f a t e s o l u t i o n i s t r e a t e d w i t h ammonium h y d r o x i d e t o a d j u s t t h e pH t o 9 . This s o l u t i o n is t h e n i n s t i l l e d i n t o t h e bed of a c a r b o x y l i c a c i d i o n - e x c h a n g e r e s i n ( A m b e r l i t e I R C - 5 0 ) i n t h e ( N H 4 + ) form. The column i s t h e n s u c c e s s i v e l y washed w i t h d i s t i l l e d water and 0 . 2 N ammonium hydroxide. I n c r e a s i n g t h e i o n i c s t r e n g t h t o 0 . 2 5 N ammonium h y d r o x i d e s e l e c t i v e l y e l u t e s neomycin C. A further increase t o 1N ammonium h y d r o x i d e t h e n e l u t e s neomycin B. F r a c t i o n s can b e m o n i t o r e d as p r e v i o u s l y s t a t e d f o r u s i n g t h e a n i o n e x c h a n g e procedure.
199 7 -3.3.3
Gas-liquid
chromatographic a n a l y s i s
The g a s - l i q u i d c h r o m a t o g r a p h i c m e t h o d for t h e d e t e r m i n a t i o n o f n e o m y c i n cmmponents p r o v i d e s s i m u l t a n e o u s q u a l i t a t i v e and q u a n t i t i v e a n a l y s e s w i t h a h i g h d e g r e e o f s e n s i t i v i t y 31. Neomycin s a m p l e s a r e a s s a y e d as t h e i r p e r t r i m e t h y l s i l y l d e r i v a t i z e s . T y p i c a l l y , l y o p h i l i z e d neomycin s u l f a t e i s h e a t e d a t 75 C f o r 40 m i n u t e s i n a m i x t u r e o f t r i m e t h y l s i l y l i m i d a z o l e i n trimethylsilyld r y p y r i d i n e (TRI-SIL Z ; P i e r c e C h e m i c a l Co.) d e t h y l a m i n e a n d t r i l a u r i n as an i n t e r n a l s t a n d a r d . R e s o l u t i o n o f t h e s i l y l d e r i v a t i v e s of n e o m y c i n c o m p o n e n t s i s a c h i e v e d using the following parameters:
,
"Column: Packing:
g l a s s , 3 mm x 1 8 3 6 ( 6 f t . ) 0 . 7 5 % OV-1
Temperatures:
Carrier g a s :
on Gas Chrom Q ( 1 0 0 - 1 2 0 m e s h )
1.
oven*"
2.
d e t e c t o r ; 31OoC
3.
i n j e c t o r ; 29OoC
2 9 O o C isothermal 15O-31O0C a t 1 0 ° C / m i n progra m m i ng
H e l i u m 40 m l s / m i n
"The p a c k e d c o l u m n i s n o n - f l o w c o n d i t i o n e d a t 33OoC f o l l o w e d by i n j e c t i o n o f S i l y l - 8 ( P i e r c e Chemical Co.) t o f u l l y s i l y l a t e any remaining a c t i v e s i t e s . f c f : E i t h e r i s o t h e r m a l or t e m p e r a t u r e p r o g r a m m i n g can b e used. A l t e r n a t i v e l y s h o r t e r c o l u m n s c a n be u s e d by i n c r e a s i n g t h e l i q u i d p h a s e l o a d i n g t o 3% OV-1 on Gas Chrom Q , 1 0 0 - 1 2 0 mesh 3 2 . S l i g h t l y d i f f e r e n t d e r i v a t i z a t i on c o n d i t i o n s h a v e b e e n r e p o r t e d as w e l l as a p r e c o n d i t i o n i n g p r o c e d u r e for t h e g l a s s c o l u m n s . A s these t e c h n i q u e s r e q u i r e c a r e f u l a r t e n t i o n t o d e t a i l t o i n s u r e success, t h e r e a d e r i s r e f e r r e d t o t h e l i t e r a t u r e c i t e d ( a l s o 33, 3b) f o r a r i g o r o u s d e s c r i p t i o n o f experimental procedures.
7.4 L i t e r a t u r e
Cited
1.
S . A. Waksman a n d H. 305-30 7 .
A.
L e C h e v a l i e r , S c i e n c e 109 ( 1 9 4 9 )
2.
S . A. Waksman a n d R.
E.
Curtis, Soil Sci.
(1916) 99.
200
3.
R. G. B e n e d i c t , 0. L. S h u t w e l l , T. G . P r i d h a m , L . A. L i n d e n f e l s e r a n d W . C. Haynes , A n t i m i c r o b . a n d C h e m o t h e r . 4 (1954) 653-656.
4.
H . A. L e c h e v a l i e r , CRC C r i t i c a l R e v i e w s i n M i c r o b i o l o g y ( 1 9 7 5 ) 359-397.
5.
G . H . Wagman, J. A. M a r q u e z , P . D. W a t k i n s , J . V. B a i l e y , F . G e n t i l e a n d M. J . W e i n s t e i n , J . A n t i b i o t i c s , 26 ( 1 9 7 3 ) 732-736. J . D. D u t c h e r , N. H o s a n s k y , M . N . D o n i n a n d 0 . W i n t e r s t e i n e r , J. Am. Chem. S O C . 7 3 ( 1 9 5 1 ) 1 3 8 4 .
K.
L. R i n e h a r t , Jr. ; T h e N e o m y c i n s a n d R e l a t e d A n t i b i o t i c s (1964). J o h n W i l e y a n d Dons, I n c . , N e w York.
B . E . L e a c h a n d C. M . 2794-2 7 9 7 .
T e e t e r s , J. Am.
Chem.
SOC. 7 3 ( 1 9 5 1 )
K . L. R i n e h a r t , Jr. , A. D. A r g o u d e l i s , W . A. G O S S , A. S c h l e r a n d C. P . S c h a f f n e r , J . Am. Chem. S O C . 82 ( 1 9 6 0 ) 3938-3946.
10.
K.
L. R i n e h a r t , Jr. and P. W. 83 ( 1 9 6 1 ) 643-648.
K.
11.
D. L o e b e n b e r g ; A n a l y s i s a n d M e c h a n i s m of t h e D i f f e r e n t i a l A n t i a m o e b i c A c t i v i t y of Some A m i n o g l y c o s i d e A n t i b i o t i c s I n c l u d i n g t h e G e n t a m i c i n s , a N e w Group of A n t i b i o t i c s ( 1 9 7 5 ) Ph.D. T h e s i s , New York U n i v e r s i t y , New York.
12.
0. K .
13.
S . A. Waksman; N e o m y c i n . Its N a t u r e and P r a c t i c a l Applic a t i o n (1958). T h e Williams a n d W i l k i n s Co. , B a l t i m o r e , Md
Moo, J . Am. Chem.
SOC.
S e b e k , J. B a c t e r i o l . 75 ( 1 9 5 8 ) 1 9 9 .
.
14.
B . E . L e a c h , W . H . DeVries, H . A. N e l s o n , W . G. J a c k s o n a n d S . J. E v a n s , J. Am. Chem. S O C . 7 3 ( 1 9 5 1 ) 2 7 9 7 - 2 8 0 0 .
15.
E . A. S w a r t , D. H u t c h i n s o n a n d S . A. Waksman, A r c h . Biochem. 2 4 ( 1 9 4 9 ) 92-103.
16.
S . A. Waksman; N e o m y c i n s ; n a t u r e , f o r m a t i o n , i s o l a t i o n and p r a c t i c a l a p p l i c a t i o n (1953). Rutgers University P r e s s , New Brunswick, N . J .
17.
U.
18.
T. W .
1 9*
G. H . Wagman, J. A. M a r q u e z , P. D. W a t k i n s , J. V . B a i l e y , F. G e n t i l e a n d M. J. W e i n s t e i n , J. A n t i b i o t i c s 26 ( 1 9 7 3 ) 732-736.
20.
op. c i t . K .
F. Nager, U.S. P a t e n t 2 , 6 6 7 , 4 4 1 ;
M i l l e r , U.S. P a t e n t 3 , 0 0 5 , 8 1 5 ;
L.
R i n e h a r t , Jr.
, A.
J a n u a r y 26, 1954. October 2 4 ,
1961.
D. A r g o u d e l i s e t at.
201
21.
M. K. M a j u m d a r a n d S . K . M a j u m d a r , A p p l . M i c r o b i o l . ( 1 9 6 9 ) 763-764.
22.
T. F. B r o d a s k y , A n a l .
23.
N.
24.
S . C. 838.
25.
D.
26.
M. K . Majumdar a n d S . K . M a j u m d a r , A n a l . 215-217.
27.
H. Maehr a n d C. P . S c h a f f n e r , Anal. Chem. 36 ( 1 9 6 4 ) 1011108.
28.
i b i d . H.
29 30
. .
31. 32 33 34
. . .
R.
Chem.
35 ( 1 9 6 3 ) 3 4 3 - 3 4 5 .
C h a t t e r j e e , I n d . J . Chem. P a n a n d J . D.
G. Kaiser, A n a l .
17
1 3 ( 1 9 7 5 ) 1282-1284.
D u t c h e r , Anal. Chem. 2 8 ' ( 1 9 5 6 ) 8 3 6 Chem.
35 ( 1 9 6 3 ) 5 5 2 - 5 5 4 . Chem.
39 ( 1 9 6 7 )
Maehr and C. P . S c h a f f n e r .
S . H . P i n e s , U.S.
P a t e n t 3,329,566;
G. Nomine a n d L. P e n a s s e , U.S. 6 , 1962. K . T s u j i a n d J. H. 1335.
P a t e n t 3,062,807;
Robertson, Anal.
B. V a n G i e s s e n a n d K . 1068-1070.
J u l y 4, 1967. November
Chem. 4 1 ( 1 9 6 9 ) 1 3 3 2 -
T s u j i , J. P h a r m . S c i . ,
60 ( 1 9 7 1 )
K. T s u j i i n Methods i n E n z y m o l o g y X L I I I A n t i b i o t i c s . E d i t o r , J. H . H a s h , A c a d e m i c P r e s s , 2 2 0 - 2 2 8 , ( 1 9 7 5 ) . K . T s u j i , J . H. R o b e r t s o n , R . Baas a n d D. J . M c I n n i s , App. M i c r o . 1 8 ( 1 9 6 9 ) 3 9 6 - 3 9 8 .
202 8 . Paromomycins
8.1 Introduction 8.1.1
Producing organisms
Due t o t h e d i f f i c u l t i e s i n i d e n t i f y i n g a n t i b i o t i c s i n t h e e a r l y y e a r s o f a n t i b i o t i c w o r k , t h e paromomycins were " d i s c o v e r e d " n o less t h a n s i x times u n t i l t h e y were a l l shown t o be identical. They were f i r s t d e s c r i b e d i n 1952 as c a t e n u l i n ' b e i n g p r o d u c e d by StheptOmyCeb c a t e n u l a e , a g a i n i n 1 9 5 6 as paromomycin2 and i n 1958 as h y d r o x y m y c i n 3 b o t h b e i n g e l i c i t e d f r o m StheptOmyCeb h . i m O b U b dohma pahomomycinub. The complex i s a l s o known as c r e s t o m y c i n a n d f a r m i g l u c i n p r o d u c e d by S t h e p t o myceb Che4tOmyC&tiCUb a n d StheptOtnyCeb 6hadiae v a r . i t a l i c u s r e s p e c t i v e l y 4 and a m i n o s i d i n 5 . The paromomycins h a v e a l s o b e e n known as zygomycins A, a n d A Z 6 which a r e p r o d u c e d by S t h e p t a m y C e b pulvahaceub and neomycins D , E a n d F p r o d u c e d by S t h e p t o myceb 6hadiae7. A n o t h e r synonomous compound, monomycin i s p r o d u c e d f r o m a d i f f e r e n t g e n u s , Actinamyceh CiJLcUt?atub Vah.
mono m y c i n i 8. 8.1.2
Chemical d e s c r i p t i o n a n d s t r u c t u r e
The paromornycins e x h i b i t much o f t h e same t y p e o f chemic a l b e h a v i o r n o t e d i n t h e n e o m y c i n s . They a r e p o l a r , b a s i c , water s o l u b l e a n t i b i o t i c s h a v i n g t h e c h a r a c t e r i s t i c a b s e n c e of clear cut melting points. Structural studies led t o the elucid a t i o n o f a compound d i f f e r i n g from neomycin by t h e s u b s t i t u t i o n o f a h y d r o x y l g r o u p f o r an amino g r o u p as shown i n F i g u r e 109 (next page). S i m i l a r s t u d i e s p e r f o r m e d a t a l a t e r d a t e on zygornycin and neomycin l e d t o t h e d i s c o v e r y o f two m a j o r components w i t h i n t h e com l e x d e n o t e d as paromomycins I a n d I1 ( z y g o m y c i n s A1 a n d A 2 ) 1 0 y 1 P . A s can be s e e n t h e d i f f e r e n c e s i n t h e two components are e p i m e r i c a b o u t t h e C-6 p o s i t i o n o f n e o s a m i n e a s i t u a t i o n c l e a r l y a n a l a g o u s t o t h a t of neomycin. Finally, c a r e f u l l y c o n d u c t e d s t u d i e s on t h e i n t a c t a n d d e g r a d a t i o n d u c t s o f c a t e n u l i n , p a r o m y c i n , hydroxymycin a n d a m i n o s i d i n K O, a n d t h e e l u c i d a t i o n of t h e zygornycin A1 and A 2 s t r u c t u r e s 1 3 demonstrated t h e i r i d e n t i t y .
-
8.2 Therapeutic
usage
The paromomycins e x h i b i t t h e same t y p e o f b r o a d s p e c t r u m a c t i v i t y s e e n w i t h t h e n e o m y c i n s . However, u n l i k e t h e neomycins a n d most o t h e r a r n i n o c y c l i t o l a n t i b i o t i c s , t h e y a l s o show s t r o n g a n t i - p r o t o z o a l a c t i v i t y as w e l l i 4 . I n V i t h o paromomycin i s a m e b i c i d a l a t 3.9 mg/ml a g a i n s t Entamoeba h i h t o l y t i c a . A g a i n s t i n t e s t i n a l a m e b i a s i s i n r a t s , t h e C D 5 o was f o u n d t o b e b e t w e e n 22-35 mg/kg/day. A d o s e o f 5 - 2 2 mg/kg/day, f i v e d a y s a w e e k f o r t m w e e k s was s u f f i c i e n t t o e r a d i c a t e i n t e s t i n a l t r i c h o monads i n 7 5 % of t h e d o g s t r e a t e d i n one p a r t i c u l a r s t u d y . Acute t o x i c i t y s t u d i e s i n r a t s g a v e LD5o v a l u e s of g r e a t e r t h a n 1 6 2 5 mg/kg o r a l l y , 650 mg/kg s u b c u t a n e o u s l y a n d 156 mg/kg i n t r a v e n o u s l y . C h r o n i c t o x i c i t y s t u d i e s showed e v i d e n c e o f k i d n e y f a t d e p o s i t s o n l y when t h e d r u g i s g i v e n
203
FIGURE 10.
Paromomycins
parenterally. A t low d o s e s t h e s e c h a n g e s were r e v e r s i b l e a f t e r t h e d r u g was w i t h d r a ~ n ' ~ .
8.3 Extraction , separation 8.3.1
and p u r i f i c a t i o n
E x t r a c t i o n of c r u d e a n t i b i o t i c
A s i n t h e c a s e of t h e neomycin a n t i b i o t i c s , t h e paromomycins are b e s t e x t r a c t e d u s i n g a c a t i o n e x c h a n g e r e s i n p r o c e d u r e s u c h as t h e f o i l o w i n g 1 6 :
1. The f e r m e n t a t i o n b r o t h i s a d j u s t e d P O pH 3 , f i l t e r e d and t h e f i l t r a t e and w a s h i n g s a r e combined. 2. The pH i s a d j u s t e d t o 7 w i t h s o d i u m h y d r o x i d e and t h e s o l u t i o n i s t h e n p a s s e d t h r o u g h a column of A m b e r i i t e I R C - 5 0 ( N H ~ + form).
3. The column i s washed t h o r o u g h l y w i t h water; c r u d e a n t i b i o t i c i s t h e n e l u t e d w i t h 0.5N a q u e o u s ammonia.
204 '4. The i n a c t i v e f r a c t i o n s are d i s c a r d e d a n d t h e a c t i v e f r a c t i o n s are c o n c e n t r a t e d in vacuo.
5. The pH of t h e c o n c e n t r a t e i s a d j u s t e d t o I , f i l t e r e d and t h e f i l t r a t e i s p a s s e d o n t o a column o f A m b e r l i t e CG-50 (NH3- form). 6. The column i s t h e n washed s u c c e s s i v e l y w i t h t h r e e volumes of water and 1 2 volumes of 0 . 0 5 N aqueous ammonia.
I. The column i s t h e n washed w i t h 0.1N aqueous ammonia t o remove a d d i t i o n a l b y - p r o d u c t s and i s f i n a l l y t r e a t e d w i t h 0 . 3 N aqueous ammonia t o e l u t e paromornycin. 8.
The paromomycin e l u a t e i s t h e n e v a p o r a t e d t o d r y n e s s
i n vacuo t o o b t a i n paromomycin. A t t h i s s t a g e , t h e a n t i b i o t i c b a s e may b e r e d i s s o l v e d i n water a n d l y o p h i l i z e d o r c o n v e r t e d t o t h e s u l f a t e by t i t r a t i o n w i t h s u l f u r i c a c i d and s u b j e c t e d t o a d d i t i o n a l p u r i f i c a t i o n procedures i f necessary.
8.3.2
Detection
Because o f t h e i r c l o s e s i m i l a r i t y t o t h e neomycins, t h e paromomycins can be d e t e c t e d u s i n g t h e same s y s t e m s a l r e a d y d e s c r i b e d . The i n i t i a l s t u d i e s on t h e paromomycins u t i l i z e d the a c e t y l a t e d d e r i v a t i v e s t o achieve separation. As i n t h e case o f t h e n e o m y c i n s , t h e s o l v e n t s y s t e m of c h o i c e f o r p a p e r chromatography o f p e r - N - a c e t y l a t e d paromomycin i s composed of n-butano1:pyridine:water ( 3 : 2 : 2 v / v ) and v i s u a l i z e d by t h e Pan - D u t c h e r r e a c t i o n . The r e s o l u t i o n of zygomycin components h a s been r e p o r t e d by p a p e r chromatography u s i n g t h r e e d i f f e r e n t s o l v e n t s y s t e m s 1 7 :
, descending.
A.
5 0 % aqueous p h e n o l
B.
1-butano1:acetic acid:water (2:1:2
v/v)
, descending.
C.
1-butano1:acetic
v/v)
, descending.
acid:water
(4:1:5
A mixed bed t h i n l a y e r c h r o m a t o g r a p h i c s y s t e m h a s a l s o b e e n s p e c i f i e d f o r paromomycinls. A m i x t u r e of s i l i c a g e l G ( K i e s e l g e l GI and a l u m i n a i s d e p o s i t e d a s a t h i n l a y e r on glass plates. S e p a r a t i o n o f paromomycin i s a c h i e v e d w i t h a s o l v e n t s y s t e m composed o f n - p r o p a n o 1 : e t h y l a c e t a t e : w a t e r : ammonium h y d r o x i d e ( 5 0 : 10 : 30 :1 0 v / v ) . I n a l l cases, v i s u a l i z a t i o n can b e a c h i e v e d w i t h n i n h y d r i n , i o d i n e v a p o r , E l s o n Morgan r e a g e n t ( o r any o t h e r r e a g e n t s p e c i f i c f o r p r i m a r y amino g r o u p s ) or b i o a u t o g r a p h y a g a i n s t a s u s c e p t i b l e o r g a n i s m .
8.3.3 P u r i f i c a t i o n and c h r o m a t o g r a p h i c methods S e p a r a t i o n of paromomycin I from paromomycin I1 h a s been a c h i e v e d by two d i f f e r e n t methods. The o l d e r , more c l a s s i c a l a p p r o a c h i n v o l v e s c e l l u l o s e column chromatography of N-
205
a c e t y l a t e d d e r i v a t i v e s b a s e d on s i m i l a r p r o c e d u r e s d e r i v e d f o r The . s e c o n d p r o c e d u r e i s by r e s o l u t i o n of N - a c e t y l n e o m y c i n ~ ~ ~ i o n - e x c l u s i o n c h r o m a t o g r a p h y on an i o n - e x c h a n g e r e s i n a l s o b a s e d on s e p a r a t i o n s d e r i v e d f o r i n t a c t , u n d e r i v a t i z e d n e o m y c i n s 2 0 . 8.3.3.1
R e s o l u t i o n of N - a c e t y l paromomycins by c e l l u l o s e
p a r t i t i o n chromatopraphy T y p i c a l l y , a column i s p r e p a r e d by m e c h a n i c a l l y t a m p i n g small a l i q u o t s of powdered c e l l u l o s e i n t o a s u i t a b l e s i z e t u b e u n t i l i t i s f u l l . N - a c e t y l paromomycin i s a p p l i e d t o t h e b e d a n d e l u t e d w i t h 1-butano1:pyridine:water ( 6 0 : 4 0 : 4 0 or 6 0 : 4 0 : 3 0 v/v). Under t h e s e c o n d i t i o n s , paromomycin I i s e l u t e d b e f o r e paromomycin 11. 8.3.3.2
R e s o l u t i o n o f paromomycins by a n i o n e x c h a n g e
chromatoRraphy
A s w i t h n e o m y c i n , p a r t i a l r e s o l u t i o n o f paromomycin ( r e p o r t e d as c a t e n u l i n ) h a s been n o t e d u s i n g a n i o n exchange resins. I n u s u a l p r o c e d u r e s e i t h e r Dowex 1 x 2 , 5 0 - 1 0 0 mesh or AG 1 - X 2 , 5 0 - 1 0 0 mesh a n a l y t i c a l g r a d e s a r e u s e d a f t e r c o n v e r s i o n t o t h e h y d r o x y l f o r m by t r e a t m e n t w i t h 1 N a q u e o u s sodium hydroxide. The r e s i n i s t h e n t h o r o u g h l y washed w i t h water t o remove e x c e s s b a s e a n d t h e n s l u r r i e d i n t o a g l a s s column. Approximately 2 5 % aqueous s o l u t i o n s of t h e a n t i b i o t i c s u l f a t e i s p r e p a r e d and i n s t i l l e d o n t o t h e column bed. Elution i s a c h i e v e d w i t h d i s t i l l e d water at a l i n e a r f l o w r a t e o f 0 . 4 - 0 . 6 cm/min. D e t e c t i o n i s a c h i e v e d q u a l i t a t i v e l y by u s e o f b i o l o g i c a l a c t i v i t y m o n i t o r i n g o r , q u a n t i t a t i v e l y by b i o a s s a y o f f r a c t i o n s , n i n h y d r i n a s s a y of Moore a n d S t e i n 2 I , or o p t i c a l rotation. 8.3.3.3
R e s o l u t i o n o f paromomycins by g a s - l i q u i d chroma t o g r a ph y
L i k e t h e n e o m y c i n s , paromomycins I and I1 can be r e s o l v e d f r o m e a c h o t h e r by u s i n g g a s c h r o m a t o g r a p h i c p r o c e d u r e s 2 2 . O f p a r t i c u l a r i n t e r e s t a r e t h e c a s e s i n w h i c h paromomycin i s cop r o d u c e d w i t h neomycin. I n s u c h i n s t a n c e s y s e p a r a t i o n and q u a n t i t a t i o n o f a l l t h e a n t i b i o t i c s p r e s e n t becomes e x c e p t i o n a l l y d i f f i c u l t u n l e s s h i g h r e s o l u t i o n c a n be a c h i e v e d . F o r a n a l y t i c a l purposes, t h e gas chromatographic procedure o f f e r s t h e combined a d v a n t a g e s o f h i g h s p e e d , q u a n t i t a t i v e d e t e c t i o n and high s e n s i t i v i t y . A 1 0 mg/ml a q u e o u s s a m p l e of paromomycin s u l f a t e i s p r e p a r e d by p l a c i n g a 1 . 0 m l a l i q u o t i n t o 2 m l s e r u m v i a l s a n d freeze drying. The v i a l s a r e t h e n c a p p e d a n d s e a l e d a n d 1 . 0 ml o f an i n t e r n a l s i a n d a r d s i l y l a t i o n r e a g e n t " i s added f o l l o w e d by h e a t i n g a t 75 C i n an o i l b a t h f o r 4 5 m i n u t e s . The s a m p l e s are then i n j e c t e d u s i n g t h e f o l l o w i n g parameters:
-
*A s o l u t i o n c o n t a i n i n g 3 mg o f t r i l a u r i n and 70 rnls of N - t r i r n e t h y l s i l y l d i e t h y l a m i n e per ml. of T r i - S i l 2 ( P i e r c e Chemical Corrpany) (1.5 mcg/rnl trirnethylsilyirnidazole i n d r y p y r i d i n e )
.
206
x 1 8 3 0 mm ( 6 f t . ) g l a s s
Column :
3 mm i . d .
Packing":
0 . 7 5 % O V - 1 on Gas Chrom Q , 1 0 0 - 1 2 0 mesh
Column T e m p e r a t u r e :
Detector:
2 9 0 ° C isothermal
Flame i o n i z a t i o n
*Columns a r e p a c k e d a n d n o n - f l o w c o n d i t i o n e d a t 33OoC f o r 1 h o u r , f o l l o w e d by a n i n j e c t i g n of 50 m l s S i l y l - 8 ( P i e r c e C h e m i c a l Company) w i t h fl o w a t 2 2 0 a n d 25OoC.
8.4 L i t e r a t u r e
cited
1.
J. W . D a v i s s o n , I . A. S o l o m o n s a n d T. M. a n d Chemotherapy, 2 ( 1 9 5 2 ) 460-461.
2.
R. P . F r o h a r d t , T. H . H a s k e l l , J . E h r l i c h a n d M. P. K n u d s e n , B e l g i a n P a t e n t 5 4 7 , 9 7 6 ; October 1 2 , 1 9 5 6 .
3.
G. H a g e m a n n , G. Nomine a n d L. P e n a s s e , Am. P h a r m . F r a n c . 1 6 ( 1 9 5 8 ) 596-600.
4.
Y. Okami i n Handbook o f M i c r o b i o l o g y , Vol. I11 M i c r o b i a l Products. E d i t o r s , A. I . L a s k i n a n d H . A. L e c h e v a l i e r , CRC P r e s s 7 2 8 ( 1 9 7 3 ) .
5.
F. A r m a c o n e , C. B e r t a z o l l i , M. G h i o n e a n d T. S c o t t i , G i o r n . Microbiol. 7 ( 1 9 5 9 ) 251-272.
6.
H . H i t o m i , S . H o r i i , T. Y a m a g u c h i , M . I m a n i s h i a n d A. M i k a y e , J. A n t i b i o t i c s , 1 4 ( 1 9 6 1 ) 6 3 - 6 7 .
7.
E . J. H e s s l e r , H . K . J a n h k e , J. H. R o b e r t s o n , K . T s u j i , K . R i n e h a r t a n d W . T. S h i e r , J . A n t i b i o t i c s , 2 3 (1570) 464-466.
8.
G. F. G a u z e , T. P . P r e o b r a z h e n s k a y a , L. P . I v a n i t s k a y a a n d V. K . K o v a l e n k o v a , A n t i b i o t i k i 4 ( 1 9 6 0 ) 3 .
9.
T. H . H a s k e l l , J. C. F r e n c h a n d Q. R. B a r t z , J. Am. Chem. SOC. 8 1 ( 1 9 5 9 ) 3 4 8 2 - 3 4 8 3 .
Lees, A n t i b i o t i c s
,
H o r i i , J. A n t i b i o t i c s , S e r . A, 1 4 ( 1 9 6 1 ) 2 4 9 - 2 5 0 .
10.
S.
11.
K . L. R i n e h a r t , J r . , M. H i c h e n s , A. D. A r g o u d e l i s , W . S. C h i l t o n , H . E . C a r t e r , M. P. G e o r g i a d i s , C. P . S c h a f f n e r a n d R. T. S h i l l i n g s , J. Am. Chem. S O C . 8 4 ( 1 9 6 2 ) 3 2 1 8 - 3 2 2 0 .
12.
R. T . S c h i l l i n g s and C . P . S c h a f f n e r , A n t i b i o t i c s a n d C h e m o t h e r a p y , ( 1 9 6 1) 2 74-2 85
13.
S . H o r i i , J. A n t i b i o t i c s , S e r . A.
14.
P. E. T h o m p s o n , A. B a y l e s , S . F. Herbst, 8 . O l s z e w s k i a n d J. E. M e i s e n h e l d e r , A n t i b i o t . and C h e m o t h e r . 9 ( 1 9 5 9 ) 6 1 8 626.
.
1 5 ( 1 9 6 2 ) 187-188.
207 15.
G. L. C o f f e y , L. E . A n d e r s o n , M. W . F i s h e r , M. M. G a l b r a i t h , A. 8 . H i l l e g a s , D. L. K o h b e r g e r , P . E. T h o m p s o n , K . S . Weston a n d J. E h r l i c h , A n t i b i o t . a n d Chemother. 9 ( 1 9 5 9 ) 7 3 0 - 7 3 8 .
16.
Japanese P a t e n t 7,036,158;
17.
K . N a k a z a w a , M. S h i b a t a , E. H i g a s h i d e , T . K a n z a k i , H . Yamamoto, A. M i y a k e , H. H i t o m i , S . H o r i i , T. Y a m a g u c h i , T. A r a k i , K . T s u c h i y a , Y . Oka a n d A. A m a i , U.S. P a t e n t 3,089,827;
November 1 7 , 1 9 7 0 .
May 1 4 , 1 9 6 3 .
18.
V . R. H u t t e n r a u c h a n d I . S c h u l z e , P h a r m . Z e n t r a l b l a t t , 1 0 4 ( 1 9 6 5 ) 85-87.
19.
op. c i t . S . H o r i i p . 2 4 9 .
20.
H . Maehr a n d C . P. S c h a f f n e r , Anal. Chem. 36 ( 1 9 6 4 ) 1 0 4 - 1 0 8 .
21.
S . Moore a n d W . 388.
22.
K . T s u j i a n d J. H . 1661-1663.
H . S t e i n , J. B i o l . Chem., Robertson, Anal.
Chem.,
176 ( 1 9 4 8 ) 36742 ( 1 9 7 0 )
208 9 . Lividomycins
9 . 1 Introduction 9.1.1
Producing organism
The l i v i d o m y c i n s , o r i g i n a l l y known as q u i n t o m y c i n o r a n t i b i o t i c 2 2 3 0 , was f i r s t r e p o r r e d i n 1 9 7 1 as h a v i n g b e e n p r o d u c e d by S t h c p t o m y c e b l i v i d u a n o v . s p . ] . While t h e l i v i d o mycins t h e m s e l v e s are n o v e l compounds , t h e o r g a n i s m was a l s o shown t o p r o d u c e mannosyl paromomycin and paromomycin I w h i c h w e r e p r e v i o u s l y d e n o t e d as No. 2230-C and No. 2230-D, r e s p e c tively. 9.1.2
Chemical d e s c r i p t i o n and s t r u c t u r e
S t r u c t u r a l e l u c i d a t i o n s t u d i e s p e r f o r m e d on l i v i d o m y c i n r e v e a l e d a compound s i m i l a r to paromomycin h a v i n g b o t h a 2 amino-2 ,3-dideoxy-D-gluco-pyranose and 4-0-a-D-mannopyranosyl m o i e t i e s t h e l a t t e r as a s u b s t i t u e n t on t h e n e o s a m i n e r i n g ( F i g . 1 1 2 * 3 , n e x t p a g e ) . L i k e t h e paromomycins and n e o m y c i n s , t h e l i v i d o m y c i n s a r e p o l a r b a s i c , water s o l u b l e a n t i b i o t i c s . T h e i r s o l u b i l i t y i n a l c o h o l s r a p i d l y d e c r e a s e s as t h e c h a i n length is increased. The compounds show s t a b i l i t y a t n e u t r a l and b a s i c pH and t e n d t o decompose i n a c i d 4 .
9.2 T h e r a p e u t i c
usage
Although f a r l e s s d a t a r e g a r d i n g t h e b i o l o g i c a l a c t i v i t y of t h e l i v i d o m y c i n s h a s b e e n r e p o r t e d as compared w i t h t h e body of knowledge now s u r r o u n d i n g t h e neomycins and paromomycins t h o s e s t u d i e s which have been p e r f o r m e d show them t o b e b r o a d s p e c t r u m a n t i b i o t i c s 5 . Lividomycin A showed good a c t i v i t y a g a i n s t b o t h g r a m - p o s i t i v e and g r a m - n e g a t i v e o r g a n i s m s i n c l u d i n g Myco bacte/rium t u b e h c u l o d i a and P a eudomonab aehuginod a i n b o t h i n v i t h o and i n v i v a s t u d i e s .
9.3 E x t r a c t i o n , 9.3.1
s e p a r a t i o n and p u r i f i c a t i o n
E x t r a c t i o n o f crude a n t i b i o t i c
The w h o l e b r o t h o f a l i v i d o m y c i n f e r m e n t a t i o n i s f i l t e r e d w i t h H y f l o S u p e r c e l and t h e r e s u l t a n t f i l t r a t e i s t h e n a d j u s t e d t o pH 7 . 0 and a d s o r b e d o n t o t h e bed o f an A m b e r l i t e IRC-84 The column i s t h e n c a t i o n exchange r e s i n i n t h e NH4+ phase. washed w i t h d e i o n i z e d water f o l l o w e d by 1 N NH40H. The a c t i v e e l u a t e i s c o n c e n t r a t e d , a d j u s t e d t o pH 7.0 and l y o p h i l i z e d t o y i e l d a powder o f c r u d e l i v i d o m y c i n complex. 9.3.2
Detection
A s w i t h t h e neomycins, p r i m a r y d i f f e r e n t i a t i o n and d e t e c t i o n of t h e l i v i d o m y c i n complex i s b e s r a c h i e v e d u s i n g p a p e r or t h i n l a y e r c h r o m a t o g r a p h y f o l l o w e d by d e t e c t i o n u s i n g b i o a u t o graphy o r ninhydrin. For p a p e r Chromatography t h e f o l l o w i n g s y s t e m has been u s e d 6 : n - b u t a n o l ( s a t u r a t e d w i t h water): pt o l u e n e s u l f o n i c a c i d : t - b u t a n o l ( 8 8 : 2 : 1 0 v/v). In addition, t h i n l a y e r chromatography using e i t h e r s i l i c a g e l o r alumina
209
Rl R2 LlVlDOMYClN A H H LlVlDOMYClN 0 PAROMOMYCIN OH MANNOSYL PAROMOMYClN OH
FIGURE 11.
MANNOSYL
OH OH MA"0SYL
Lividomycins
p l a t e s can b e used w i t h t h e f o l l o w i n g s y s t e m s : 1. c h l o r o f o r m : m e t h a n o l : 1 7 % ammonium h y d r o x i d e ( 2 :1:1 v/v) upper phase (alumina and s i l i c a g e l ) . a c i d : w a t e r (1:l:l v / v ) ( s i l i c a g e l
2.
n-butano1:acetic
3.
m e t h a n o l : l O % ammonium a c e t a t e (1:l v / v ) ( s i l i c a g e l
only). only). D e t e c t i o n c a n be a c h i e v e d e i t h e r by b i o a u t o g r a p h y or w i t h ninhydrin spray reagent. The N - a c e t a t e d e r i v a t i v e s of t h e l i v i d o m y c i n s g i v e good s e p a r a t i o n of t h e i n d i v i d u a l components on s i l i c a g e l d e v e l o p e d i n c h 1 o r o f o r m : m e t h a n o l (I: 1 ) . V i s u a l i z a t i o n of t h e s e p a r a t e d components is a c h i e v e d by s p r a y i n g w i t h a n i s a l d e h y d e - H 2 S 0 , reagent. High v o l t a g e p a p e r e l e c t r o p h o r e s i s can also be u s e d i n r e s o l v i n g t h e l i v i d o m y c i n complex. The c o n d i t i o n s for t h e
2 10 s e p a r a t i o n are: Support: Buffer:
Toyo No. 5 1 p a p e r . Formic a c i d : a c e t i c a c i . d : w a t e r pH 1. 8 ( 2 2 : 7 5 : 9 0 0 v/v).
Conditions: D e t e c t i on : 9.3.3
3000 V ( 2 0 mA/10 c m ) . b i oau t o g r a p h y and n i n hy d r i n
.
Purification
P r e p a r a t i v e s e p a r a t i o n of e a c h of t h e l i v i d o m y c i n compon e n t s has been achieved u s i n g g r a d i e n t e l u t i o n c a t i o n exchange c h r o m a t o g r a p h y . An a q u e o u s s o l u t i o n of t h e c r u d e powder i s a d s o r b e d on t h e bed of a 10 x 4 0 0 mm ( i . d < x l e n g t h ) CMS e p h a d e x C-25 column i n t h e (NH4') phase. A f t e r washing w i t h water, a c o n c a v e g r a d i e n t was g e n e r a t e d w i t h 0 . 1 2 N NH40H t o 0 . 3 5 N NH40H t o o b t a i n r e s o l u t i o n of t h e l i v i d o m y c i n complex. Large s c a l e r e s o l u t i o n of t h e l i v i d o m y c i n s can a l s o b e o b t a i n e d by a d s o r b i n g t h e c r u d e powder on an A m b e r l i t e CG-50 t y p e I r e s i n column i n t h e (NH4') phase. Following washing w i t h water and 0 . 0 8 N N H h O H , e a c h o f t h e l i v i d o m y c i n components was e l u t e d s t e p w i s e w i t h 0.1N NH,OH, 0 . 1 2 N N H Q O H , 0 . 1 5 N NH40H a n d 0.17N NH40H r e s p e c t i v e l y . Each o f t h e p o o l e d e l u a t e s was c o n c e n t r a t e d , a d s o r b e d on a Dowex 1 x 2 column i n t h e ( O H - ) p h a s e , e l u t e d w i t h water, c o n c e n t r a t e d a g a i n a n d l y o p h i l i z e d yielding t h e pure f r e e bases.
9.4 Literature
cited
1.
T . Oda, T. M o r i , H. I t o , T. Kunieda and K. A n t i b i o t i c s , 2 4 ( 1 9 7 1 ) 333-338.
2.
T. Oda, T. Mori and Y . K y o t a n i , J . A n t i b i o t i c s , 2 4 ( 1 9 7 1 ) 503-510.
3.
T . Oda, T. Mori, Y. 24 ( 1 9 7 1 ) 511-519.
4.
T. Mori, T . I c h i y a n a d i , H . Kondo, K . Tukunaga, and T. Oda, J. A n t i b i o t i c s , 24 ( 1 9 7 1 ) 339-346.
5.
F. K o b a y a s h i , T. Nagoya, Y . Yoshimura, K. Kaneko, S . Ogata and S . G o t o , J. A n t i b i o t i c s , 25 ( 1 9 7 2 1 128-136.
6.
T. Mori, Y . K y o t a n i , I . Watanabe and T.Oda, J. A n t i b i o t i c s 2 5 ( 1 9 7 2 ) 149-150.
K y o t a n i a n d M.
Munakata, J.
Nakayama, J . A n t i b i o t i c s ,
2 11
10. Butirosins
10.1 Introduction 10.1.1
Producing organism
The b u t i r o s i n s , a l s o known as a m b u t y r o s i n e , a r e u n i q u e among t h e a m i n o g l y c o s i de -aminocy c l i t o 1 a n t i b i o t i c s i n t h a t t h e y are p r o d u c e d by a b a c t e r i u m r a t h e r t h a n an a c t i n o m y c e t e . The p r o d u c i n g o r g a n i s m was named BaciLLud ciacuLann 10.1.2
Chemical d e s c r i p t i o n a n d s t r u c t u r e
S t r u c t u r a l s t u d i e s p e r f o r m e d on t h e b u t i r o s i n s demons t r a t e d t h e e x i s t e n c e of a n u n u s u a l e n t i t y t h a t o f a (S)-(-)4-amino-2-hydroxybutyric a c i d s i d e c h a i n 2 I . The c o m p l e t e s t r u c t u r e is shown below. The b u t i r o s i n s e x h i b i t no s h a r p m e l t i n g p o i n t , are water s o l u b l e a n d s l i g h t l y s o l u b l e i n l o w e r a l c o h o l s as t h e i r f r e e b a s e s .
FIGURE 12.
Butirosin
212 10.2 Therapeutic usage E x p e r i m e n t a l l y , b u t i r o s i n h a s b e e n shown t o p o s s e s s s i g n i f i c a n t b r o a d s p e c t r u m i n h i b i t o r y a n t i b a c t e r i a1 a c t i v i t y i n b o t h i n u i t h o and i n v i u o s y s t e m s 4 . Although t h e range o f o r g a n i s m s t e s t e d was n o t r e m a r k a b l e i t i s o f s p e c i a l n o t e s i n c e i t d e m o n s t r a t e s a c t i v i t y a g a i n s t some s t r a i n s o f g e n t a m i c i n r e s i s t a n t Pneudomonas a e h u g i n o s a i n i n u i u o s t u d i e s . 10.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n 10.3.1
E x t r a c t i o n of crude a n t i b i o t i c
E x t r a c t i o n of b u t i r o s i n f r o m c u l t u r e f i l t r a t e s i s a c h i e v e d by b a t c h w i s e a d s o r p t i o n a t b e t w e e n pH 6 a n d pH 7 o n t o a n IRC-50 c a t i o n e x c h a n g e r e s i n i n t h e ( N H 4 + ) f o r m . T h e r e s i n i s t h e n p a c k e d i n t o a co l u mn a n d w a s h e d w i t h 0.1M ammonium h y d r o x i d e ; t h e a c t i v e e l u a t e i s t h e n r e a d s o r b e d o n t o t h e same t y p e o f r e s i n c o l u mn a n d w a s h e d w i t h 3 v o l u m e s of 0.1M ammonium h y d r o x i d e a n d t h e a n t i b i o t i c c o m p l e x i s e l u t e d w i t h 1.OM a m m o n i u m h y d r o x i d e , c o n c e n t r a t e d i n uacuo a n d l y o p h i l i z e d 5 . 10.3.2
Detection
Three p a p e r Chromatography s o l v e n t s y s t e m s f o r t h e c h a r a c t e r i z a t i o n of b u t i r o s i n have been r e p o r t e d a l t h o u g h t h e s e d o n o t r e s o l v e t h e c o m p l e x i n t o i t s a c t i v e c o m p o n e n t s . They are u s e f u l , h o w e v e r , f o r d i f f e r e n t i a t i n g t h e b u t i r o s i n s f r o m o t h e r a m i n o g l y c o s i d e s a n d are g i v e n b e l o w : 1. m e t h a n o 1 : w a t e r : a c e t i c a c i d : 2 5 % a q u e o u s N a C L ( 6 0 0 : 2 1 2 : 7 5 : 2 0 . 8 v / v ) , (Whatman No. 1 p a p e r b u f f e r e d a t pH 3 ) .
2. m e t h a n o l : 5 % a q u e o u s N a C l ( 2 : l v / v ) (Whatman No. p a p e r b u f f e r e d a t pH 3 ) . 3. ethano1:water:acetic 500:38:7.5 v / v ) .
1
acid:25% aqueous N a C l (250:
V i s u a l i z a t i o n i s o b t a i n e d by e i t h e r n i n h y d r i n s p r a y
or b i o a u t o g r a p h y . R e s o l u t i o n o f t h e t e t r a N-acetates o f t h e b u t i r o s i n c o m p l e x i n t o b u t i r o s i n s A a n d B c a n be a c h i e v e d u s i n g t h e following s o l v e n t system f o r paper chromatography: 1-butanol: pyridine:5% boric acid (6:4:3 v/v). D e t e c t i o n o f t h e r e s o l v e d c o m p o n e n t s i s o b t a i n e d by a s l i g h t m o d i f i c a t i o n o f the Pan-Dutcher p r o c e d u r e c i t e d e a r l i e r i n t h e s e c t i o n on n e o m y c i n s . Using t h i s technique t h e r e l a t i v e r a t i o s of b u t i r o s i n A t o b u t i r o s i n B i s a p p r o x i m a t e l y 8 :2 . 10.3.3
Purification
R e s o l u t i o n o f t h e u n d e r i v a t i z e d b u t i r o s i n complex c a n be a c h i e v e d u s i n g ion-exchange chromatography. B u t i r o s i n B i s a d s o r b e d o n t o t h e b ed o f a Dowex 1 x 1 or 1 x 2 column i n t h e
213
b o r a t e f o r m . S e p a r a t i o n o f A f r o m B o c c u r s by d e v e l o p m e n t of t h e column w i t h water. B u t i r o s i n B can t h e n be e l u t e d by s u b s e q u e n t d e v e l o p m e n t w i t h b o r i c a c i d up t o 5 % . F u r t h e r p u r i f i c a t i o n of e a c h o f t h e i s o l a t e d f r e e b a s e s o c c u r s by t r e a t m e n t w i t h A m b e r l i t e IRC-50 i n t h e ( N H 3 ' ) form. 10.4 Literature cited
1. J . D. H o w e l l s , L. E . A n d e r s o n , G . L. C o f f e y , G. D. S e n o s , M. A. U n d e r h i l l , D. L. V o g l e r and J . E h r l i c h , A n t i m i c r o b . A g e n t s and C h e m o t h e r . , 2 ( 1 9 7 2 ) 7 9 - 8 3 . 2.
P . W.
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K . Woo, H . W . Dion a n d Q . L e t t . 2 8 (1971) 2625-2628.
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K . Woo and R . D. W e s t h a n d , Carbohyd. Res.,
3 1 (19731
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C. L. H e i f e t z , M. W . F i s h e r , J. A. Chodubski a n d M. 0 . DeCarlo, A n t i m i c r o . A g e n t s and Chemother., 2 ( 1 9 7 2 ) 8 9 - 9 4 .
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H . W . D i o n , P . W . K . Woo, N . E . Willmer, D. L. K e r n , J . Onaga a n d S. A. F u s a r i , A n t i m i c r o . A g e n t s and Chemother., 2 ( 1 9 7 2 ) 84-88.
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T. Nara, S . T a k a s a w a , R. O k a c h i , I . Kawamoto, M. Yamamoto, August 2 4 , 1 9 7 6 . S. S a t o a n d T . S a t o , U.S. P a t e n t 3 , 9 7 6 , 7 6 8 ;
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2 15 Griseofulvins
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.
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G e r a l d H Wagman a n d Marvin J W e i n s t e i n M i c r o b i o l o g i c a l S c i e n c e s S c h e r i n g C o r p o r a t i on Bloomf i e l d . N e w J e r s e y
.
1
Griseofulvin
1.1 I n t r o d u c t i o n ................................ 1.1.1 P r o d u c i n g o r g a n i s m s ....................... B r i e f c h e m i c a l d e s c r i p t i o n ................ 1.1.2 1.1.3 S t r u c t u r a l f o r m u l a ........................ T h e r a p e u t i c u s e a n d p h a r m a c o l o g y ............ 1.2 1.3 A n a l y t i c a l methods .......................... 1.4 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n ..... 1.4.1 I n t r o d u c t i o n .............................. E x t r a c t i o n a n d p u r i f i c a t i o n methods ....... 1.4.2 1.5 L i t e r a t u r e C i t e d ............................ 2. (+)-l-thio-Griseofulvin 2.1 I n t r o d u c t i o n ................................ 2.1.1 P r o d u c i n g o r g a n i s m ........................ 2.1.2 B r i e f c h e m i c a l d e s c r i p t i o n ................ 2.1.3 S t r u c t u r a l f o r m u l a ........................ 2.2 T h e r a p e u t i c u s e ............................. A n a l y t i c a l methods .......................... 2.3 2.4 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n ..... 2.5 L i t e r a t u r e C i t e d ............................ 3 . (+)-5'-Hydroxy-l-thiogriseofulvin 3.1 I n t r o d u c t i o n ................................ 3.1.1 P r o d u c i n g o r g a n i s m ........................ 3.1.2 B r i e f c h e m i c a l d e s c r i p t i o n ................ 3.1.3 S t r u c t u r a l f o r m u l a ........................ 3.2 T h e r a p e u t i c u s e ............................. A n a l y t i c a l methods .......................... 3.3 3.4 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n ..... L i t e r a t u r e C i t e d ............................ 3.5 4 . (+)-5'-Hydroxy-griseofulvin Ir.1 I n t r o d u c t i o n ................................ 4.1.1 P r o d u c i n g o r g a n i s m ........................ 4.1.2 B r i e f c h e m i c a l d e s c r i p t i o n ................ S t r u c t u r a l f o r m u l a ........................ 4.1.3 T h e r a p e u t i c u s e ............................. 4.2 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n ..... 4.3 4.4 L i t e r a t u r e C i t e d ............................ -
216 216 216 216 216 217 218 218 218 220 223 223 223 223 223 224 224 225 226 226 226 226 226 227 227 227 228 228 228 228 228 229 230
216
1. G r i s e o f u l v i n
1.1 I n t r o d u c t i o n 1.1.1 P r o d u c i n g o r g a n i s m s
G r i s e o f u l v i n was f i r s t i s o l a t e d i n 1939 by Oxford, R a i s t r i c k and S i m o n a r t ’ from c u l t u r e s of P e n i c i l l i u m g n i d e o 6ulvum. I n 1946 B r i a n e t ~ 1 d . i s c o~v e r e d a f a c t o r i n c u l t u r e s of P . j a n c z e w a k i i p r o d u c i n g m o r p h o l o g i c a l changes i n f u n g i which t h e y named ” c u r l i n g f a c t o r ” , a n d which was f o u n d , i n t h e f o l l o w i n g y e a r by Grove and McGowan3, t o be i d e n t i c a l w i t h g r i s eof u l v i n
.
The f o l l o w i n g a d d i t i o n a l s p e c i e s of t h e genus P e n i c i l k i u m p r o d u c e g r i s e o f u l v i n ; P . gnideo6ut?vum D i e r c k x l : P . u n t i c a e B a i n i e r 4 , P . patulurn B a i n i e r l Y 5 which was c l a s s i f i e d by Raper and Thorn6 i n t o t h e s p e c i e s P . u 4 t i c a e . A l s o , p r o d u c t i o n h a s been found i n o t h e r s p e c i e s o f t h e genus Penicillkurn i d e n t i f i e d as P . j a n c e z e w d k i i Z a l e ~ k i ’ ~P ~. ,n a i d Z h i c h i i Smith’, P . v i n i d i c y c l o p i u m Abea, P . n i g n i c a n d , P . albidurn Supp., P . k a c i b o n d k i Zal. and P . m e l i n i i Thorng. More r e c e n t l y l O g r i s e o f u l v i n h a s been produced by Khudkia o h y z a e , Nigbodpona ~ R y z a e ,N . d p h a e a i c a , N . d a c c h a a i and N . mudae. 1.1.2
Brief chemical d e s c r i p t i o n 1 1 y 1 2
The e m p i r i c a l f o r m u l a f o r g r i s e o f u l v i n i s C 1 7 H 1 7 O 6 C l p and h a s t h e c h e m i c a l name 7-chloro-2’,4,6-trimethoxy-6~~-methylspiro { b e n z o f u r a n - 2 ( 3 H ) , 1 ’ I 2 1 - ~ y c l o h e x e n e ~ - 3 , 4 ~ - d i o n e .I t c r y s t a l l i z e s i n o c t a h e d r a l o r rhombic form f r o ? benzene w i t h a m e l t i n g In p o i n t of 2 2 O o C and r o t a t i o n { a ) b 7 + 370 ( s a t ’ d C H C 1 , ) . m e t h a n o l , g r i s e o f u l v i n e x h i b i t s an u l t r a v i o l e t s p e c t r u m w i t h maxima a t 3 2 4 , 2 9 1 and 236 nm. I t is p r a c t i c a l l y i n s o l u b l e i n water and p e t r o l e u m e t h e r ; s l i g h t l y s o l u b l e i n e t h a n o l , methan o l , a c e t o n e , b e n z e n e , c h l o r o f o r m , e t h y l a c e t a t e and a c e t i c a c i d . The s o l u b i l i t y i n N,N-dimethylformamide i s 12-14 g/100 m l a t 25’C. 1.1.3
S t r u c t u r a l formula
The d e t e r m i n a t i o n o f t h e s t r u c t u r e of g r i s e o f u l v i n was r e v i e w e d by G r o v e l 3 and t h e s t e r e o c h e m i s t r y c o n f i r m e d by x - r a y The m o l e c u l e c o n t a i n s t h r e e r i n g s ; an a r o c r y s t a l l o g r a p h y 4. matic benzene r i n g , a five-membered o x y g e n - c o n t a i n i n g h e t e r o c y c l i c r i n g and a h y d r o a r o m a t i c s i x membered r i n g ( n e x t p a g e ) .
1.2 T h e r a p e u t i c
use and pharmacology
Griseofulvin e x h i b i t s mycostatic a c t i v i t y a g a i n s t a v a r i e t y of f u n g i 1 5 i n c l u d i n g ZygomyCetQd, Adcomyceted ( e x c e p t Sacchanomyced c e & e v i d i a e ) Badidiomyceted ( e x c l u d i n g Coniophona cenebella) and many Fungi i m p e n d e c t i . Actinomyceted and Oomyceted are n o t I t is p a r t i c u l a r l y a c t i v e a g a i n s t sensitive t o griseofulvin. numerous s p e c i e s of T n i c h op h y t o n , E p i d enmop h y t o n and Micn odp on urn and is u s e d i n t h e t r e a t m e n t of human mycoses. I t i s a l s o act i v e a g a i n s t s e v e r a l p l a n t p a t h ~ g e n s l ~ and ” ~ dermatomycosis i n
217
1
CI
c a t s l a , dogs19, p o u l t r y 2 0 and o t h e r s p e c i e s . The LD50 d o s e f o r g r i s e o f u l v i n i n mice by t h e i n t r a v e n o u s r o u t e i s 280 mg/kg; by i n t r a p e r i t o n e a l i n j e c t i o n i t i s 1 , 1 5 0 mg/kg; by s u b c u t a n e o u s r o u t e i t i s > 1 2 , 0 0 0 mg/kg a n d 5 0 , 0 0 0 mg/kg on o r a l a d m i n i s t r a t i o n z 1 . G r i s e o f u l v i n i s a b s o r b e d from t h e a l i m e n t a r y t r a c t ; i n humans, s i n g l e d o s e s of 0 . 2 5 , 1 . 0 and 2 . 0 g a v e b l o o d l e v e l s of 0 . 7 , 1 . 5 a n d 2 . 5 mcg/ml, r e s p e c t i v e l y z z y 2 , A b s o r p t i o n v a r i e s c o n s i d e r a b l y among i n d i v i d u a l s m a i n l y due t o i n s o l u b i l i t y i n aqueous media o f t h e upper G . I . t r a c t z 4 , The peak serum l e v e l f o u n d i n f a s t i n g a d u l t s w i t h a 0 . 5 g oral d o s e o c c u r s a t a b o u t 4 h r . and r a n g e s between 0 . 5 t o 2 . 0 m ~ g / m l ~ G ~ r. i s e o f u l v i n is d e p o s i t e d i n t h e k e r a t i n p r e c u r s o r c e l l s a n d h a s a greater a f f i n i t y f o r diseased tissue. I t i s t i g h t l y bound t o t h e new k e r a t i n which becomes h i g h l y r e s i s t a n t t o f u n g a l i n v a s i0ns24.
s
1.3 Analytical
methods
G r i s e o f u l v i n can b e d e t e r m i n e d by a v a r i e t y of a n a l y t i c a l methods. M i c r o b i o l o g i c a l and s p e c t r o p h o t o m e t r i c t e c h n i q u e s a r e d e s c r i b e d by Oden e t O t h e r methods o f a s s a y f o r t h e p u r e d r u g a r e g i v e n i n t h e U . S . P h a r m a c o p e i a Z 6 , t h e B r i t i s h Pharmac o p e i a Z 7 and t h e European P h a r m a c o p e i a Z 8 . A s i m p l e u l t r a v i o l e t s p e c t r o p h o t o m e t r i c method f o r t h e d e t e r m i n a t i o n of t h e g r i s e o f u l v i n m e t a b o l i t e , 6-dimethyl-griseofulvin, w i t h a l i m i t of d e t e c t i o n o f 1 mg/ml was d e s c r i b e d by Roland and R i e g e l m a n Z 9 . The g a s c h r o m a t o g r a p h i c a n a l y s i s o f g r i s e o f u l v i n was r e p o r t e d 3 0 ; g r i s e o f u l v i n and d e c h l o r o - g r i s e o f u l v i n were d e t e r m i n e d i n c r u d e e x t r a c t s o f P e n i c i l l i u r n u A Z i c a e by t h i n l a y e r and g a s - l i q u i d ~ h r o m a t o g r a p h y ~ ~Using . t h i n l a y e r chromatography-fluorometrica s s a y m e t h o d o l ~ g y ~c~o ,n c e n t r a t i o n s of 0.25 t o 2 rng/ml of g r i s e o f u l v i n and g r i s e o f u l V i n - 4 ' - 0 1 have been d e t e c t e d . G r i s e o f u l v i n h a s also been d e t e r m i n e d by t i m e - r e s o l v e d p h ~ s p h o r i m e t r ywhich ~~ was employed f o r c o n c e n t r a t i o n s i n t h e r a n g e 5 x lo-'+ t o 1 x molar.
218
1.4 1.4.1
Introduction
Most of t h e d a t a f o r p u r i f i c a t i o n of g r i s e o f u l v i n i s i n t h e p a t e n t l i t e r a t u r e ; v e r y l i t t l e i s t o be found i n s c i e n t i f i c publications. Numerous p a t e n t s have been i s s u e d and examples from some o f t h e s e a r e p r e s e n t e d i n t h e f o l l o w i n g p a g e s . I n g e n e r a l , t h e medium a n d / o r mycelium a r e e x t r a c t e d w i t h a s u i t a b l e s o l v e n t and t h e c r u d e g r i s e o f u l v i n t h u s o b t a i n e d p u r i f i e d by r e c r s t a l l i z a t i o n . I n one s t u d y of s o l v e n t e f f i c i e n c y r e p o r t e d 3 { g r i s e o f u l v i n was e x t r a c t e d from t h e b r o t h u s i n g a v a r i e t y o f s o l v e n t s and t h e h i g h e s t y i e l d ( 9 5 % ) was obt a i n e d w i t h methylene c h l o r i d e . The y i e l d was lower when b u t y l a c e t a t e , t r i c h l o r o e t h y l e n e , aqueous a c e t o n e and o t h e r s o l v e n t s were used. I t was concluded t h a t methylene c h l o r i d e was s i m p l e r and l e s s e x p e n s i v e t o u s e . However, i n t h e examples t h a t f o l lows, a number of s o l v e n t s and p u r i f i c a t i o n t e c h n i q u e s a r e d e s c r i b e d , a l l of which r e s u l t e d i n p u r i f i e d g r i s e o f u l v i n . 1.4.2
E x t r a c t i o n and p u r i f i c a t i o n methods
-
A p r o c e s s f o r p r o d u c t i o n and i s o l a t i o n of g r i s e o EXAMPLE 1 f u l v i n under c o n d i t i o n s of submerged a e r o b i c f e r m e n t a t i o n on a l a r g e s c a l e i s d e s c r i b e d 3 5. 310 II of whole broth c6ntairIlin.g an e s t i m a t e d 244 g of g r i s e o f u l v i n were f i l t e r e d on a r o t a r y s t r i n g d i s c h a r g e f i l t e r t o g i v e 47 kg of m y c e l i a l s o l i d . The s o l i d was s t i r r e d f o r 1 5 min. w i t h 45 11 of b u t y l a c e t a t e and t h e n t h e s o l v e n t was s e p a r a t e d by d e c a n t a t i o n , y i e l d i n g 4 0 II of b u t y l a c e t a t e a s s a y The s o l i d was t h e n s t i r r e d f o r a f u r t h e r i n g a t 2 , 5 1 0 mcg/ml. 1 5 mins. w i t h 40 II of f r e s h b u t y l a c e t a t e and t h e s o l v e n t s e p a r a t e d by d e c a n t a t i o n , y i e l d i n g 40 L o f b u t y l a c e t a t e a s s a y i n g a t 1 , 1 7 0 mcg/ml. The s o l i d was mixed w i t h s o l v e n t f o r a t h i r d time, 40 L of b u t y l a c e t a t e a s s a y i n g a t 355 mcg/ml b e i n g obtained.
The b u t y l a c e t a t e e x t r a c t s were c l a r i f i e d by a d d i n g 0 . 3 % k i e s e l g u h r and f i l t e r i n g . The c l a r i f i e d s o l v e n t was t h e n conc e n t r a t e d i n W U C U O a t a maximum t e m p e r a t u r e of 5 O o C t o 500 mls, On c o o l i n g t o room t e m p e r a t u r e t h e c r u d e g r i s e o f u l v i n s e p a r a t e d as a brown powder. This c r u d e m a t e r i a l was washed w i t h 200 m l s of b u t y l a c e t a t e , a c o n s i d e r a b l e amount of c o l o r b e i n g removed, The s o l i d r e m a i n i n g was d r i e d o v e r phosphorous p e n t o x i d e under vacuum t o g i v e 1 8 0 g of c r u d e g r i s e o f u l v i n ( 7 9 . 5 % p u r e ) . This washed c r u d e m a t e r i a l was d i s s o l v e d i n 5 1 of a c e t o n e , f i l t e r e d and 8 0 II of w a t e r added. The c r y s t a l l i n e p r o d u c t which immediately s e p a r a t e d was f i l t e r e d , washed w i t h 1 0 0 m l of b u t y l a c e t a t e and t h e n d r i e d i n vacua o v e r phosphorous p e n t o x i d e t o g i v e 1 1 0 g of p u r e g r i s e o f u l v i n , m.p. 220'~'
-
EXAMPLE 2 3 5 4 2 5 II of whole b r o t h c o n t a i n i n g an e s t i m a t e d 3 0 1 g o f g r i s e o f u l v i n were f i l t e r e d on a r o t a r y s t r i n g d i s c h a r g e f i l -
219
The s o l i d was s t i r r e d t e r t o g i v e 7 0 kg o f m o i s t m y c e l i a l s o l i d . f o r 1 5 min. w i t h 55 R o f b u t y l a c e t a t e and t h e n t h e s o l v e n t w a s s e p a r a t e d by d e c a n t a t i o n , y i e l d i n g 45 II of b u t y l a c e t a t e a s s a y i n g a t 3,080 mcg/ml. The s o l i d was t h e n s t i r r e d f o r a f u r t h e r 1 5 min. w i t h 5 0 R o f b u t y l a c e t a t e a n d t h e s o l v e n t s e p a r a t e d by d e c a n t a t i o n , y i e l d i n g 48 R of b u t y l a c e t a t e assaying a t , 1 , 3 3 5 mcg/ml. T h i s was r e p e a t e d a t h i r d t i m e , 40 II o f b u t y l acetate a s s a y i n g a t 5 5 4 mcg/ml b e i n g r e c o v e r e d . The b u t y l a c e t a t e e x t r a c t s were c l a r i f i e d by c e n t r i f u g a t i o n a n d c o n c e n t r a t e d i n vacuo a t a maximum t e m p e r a t u r e of 5OoC On c o o l i n g t o room t e m p e r a t u r e t h e c r u d e g r i s e o f u l v i n t o 1 R. This crude s e p a r a t e d as a n o i l y , p a l e brown mass o f c r y s t a l s . material was s t i r r e d a n d f i l t e r e d w i t h 4 0 0 m l s o f c h l o r o f o r m t o remove t h e o i l a d h e r i n g t o t h e c r y s t a l s . The s o l i d s o o b t a i n e d was d r i e d o v e r phosphorous p e n t o x i d e u n d e r vacuum t o g i v e 340 g of a s o l i d . T h i s washed c r u d e material was s t i r r e d w i t h 5 of a c e t o n e t o g i v e a s a t u r a t e d s o l u t i o n , f i l t e r e d a n d 2 5 II o f water a d d e d . The c r y s t a l l i n e material which i m m e d i a t e l y s e p a r a t e d , was f i l t e r e d and washed w i t h 100 m l s o f b u t y l a c e t a t e . Y i e l d 175 g , m.p.,
220'~.
I n a n o t h e r e x p e r i m e n t , 0 . 8 g o f c r u d e g r i s e o f u l v i n from an e v a p o r a t e d b u t y l a c e t a t e e x t r a c t of mycelium was d i s s o l v e d i n 1 0 m l s of d i m e t h y l f o r m a m i d e and f i l t e r e d . To t h e f i l t r a t e 200 m l s o f water were added and t h e s o l i d which i m m e d i a t e l y c r y s t a l l i z e d was f i l t e r e d t o g i v e 0 . 5 g of p u r e p r o d u c t , m.p. 220
.
-
EXAMPLE 3 I n t h i s method36 t h e mycelium was s e p a r a t e d from 21.3 kg of m o i s t t h e h a r v e s t e d b r o t h and t r e a t e d as f o l l o w s . mycelium w i t h an a c t i v i t y o f 4.8 m c g / m l were o b t a i n e d from 300 R o f n u t r i e n t medium. T h i s was e x t r a c t e d t h r e e times w i t h 4 2 . 6 , 2 1 . 3 a n d 2 1 . 3 R of m e t h y l e n e c h l o r i d e r e s p e c t i v e l y . The comb i n e d e x t r a c t was e v a p o r a t e d t o 1 / 1 0 o f t h e i n i t i a l volume a n d cooled t o + 4 O C . The p r e c i p i t a t e d f l o c c u l e n t b r o w n i s h irnpuri t i e s were s e p a r a t e d on a p r e s s u r e f i l t e r . The f i l t r a t e was c l a r i f i e d w i t h 0 . 5 % a c i d a c t i v a t e d c h a r c o a l w h i l e warming t o 3SoC, u s i n g a r e f l u x c o n d e n s e r ; t h e s p e n t c h a r c o a l was s e p a r a t e d on t h e p r e s s u r e f i l t e r a n d washed w i t h m e t h y l e n e c h l o r i d e t a k e n i n t h e amount o f 2.5% o f t h e f i l t r a t e . The c l a r i f i e d f i l t r a t e was e v a p o r a t e d t o 1 / 1 5 of t h e i n i t i a l volume a t a t e m p e r a t u r e n o t e x c e e d i n g 5OoC and t h e n c o o l e d t o O°C. The c r y s t a l l i z e d c r u d e g r i s e o f u l v i n was removed i n a c e n t r i f u g e , washed w i t h c o l d a c e t o n e t a k e n i n t h e amount o f 8 0 % o f t h e f i l t r a t e by volume, a n d d r i e d i n a vacuum d r i e d a t 50 a n d a p r e s s u r e o f 2 5 mm. Hg. 1 0 2 g o f c r u d e g r i s e o f u l v i n was obt a i n e d , i . e . , a y i e l d o f 9 5 % & Assay 95% ( s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n ) ; m.p. 216-218 C .
-
EXAMPLE 4 G r i s e o f u l v i n was o b t a i n e d from t h e c u l t u r e l i q u i d and mycelium by e x t r a c t i o n w i t h m e t h y l e n e c h l o r i d e 3 7 . The f i l t e r e d e x t r a c t was e v a p o r a t e d t o 1 / 3 of i t s o r i g i n a l volume, p u r i f i e d w i t h a c t i v e c a r b o n and c o n c e n t r a t e d t o 1 / 1 5 volume. The c o n c e n t r a t e was c o o l e d t o - 8 O C a n d c r u d e g r i s e o f u l v i n crystallized. I t was p u r i f i e d by r e c r y s t a l l i z a t i o n from a c e t o n e .
220
EXAMPLE 5 - G r i s e o f u l v i n p r o d u c e d by K h u d k i a oayzae was ext r a c t e d by t h e f o l l o w i n g p r o c e d ~ r e ~ A~ .o n e p. p o r t i o n o f h a r v e s t b r o t h was f i l t e r e d t o s e p a r a t e t h e mycelium from t h e b r o t h . The f e r m e n t a t i o n medium was e x t r a c t e d o n c e w i t h 2 0 0 m l o f c h l o r o f o r m a n d t w i c e w i t h 1 0 0 m l of c h l o r o f o r m , The e x t r a c t s were combined, washed w i t h s a t u r a t e d sodium c h l o r i d e s o l u t i o n , d r i e d and e v a p o r a t e d . The r e s i d u e was r e c r y s t a l l i z e d from m e t h a n o l t o g i v e 130 mg of p u r e g r i s e o f u l v i n , m.p. 2 1 8 - 2 2 1 ° C . , which showed no d e p r e s s i o n on a d m i x t u r e w i t h a n a u t h e n t i c samp l e o f g r i s e o f u l v i n and whose I . R . s p e c t r u m was i d e n t i c a l w i t h t h a t o f an a u t h e n t i c sample of g r i s e o f u l v i n . The mycelium, r e t a i n e d a b o v e , was d r i e d i n a vacuum oven a t 4OoC, c r u s h e d and t h e n e x t r a c t e d c o n t i n u o u s l y w i t h 500 m l of c h l o r o f o r m f o r 2 h r s . The e x t r a c t was e v a p o r a t e d t o g i v e a r e d s o l i d ( 3 2 0 mg) which was shown by U.V. e s t i m a t i o n t o contain g r i s e o f u l v i n t o t h e extent of 90%.
-
EXAMPLE 6 A method i s d e s c r i b e d f o r o b t a i n i n g v e r y h i g h l y p u r i f i e d g r i s e o f u l v i n from a m e t h y l e n e c h l o r i d e f e r m e n t a t i o n A s o l u t i o a of c r u d e g r i s e o f u l v i n i n m e t h y l e n e c h l o r i d e was c o o l e d t o 4 C and t r e a t e d w i t h a c t i v a t e d c a r b o n a n d c r y s t a l l i z a t i o n a t O°C. Y e l l o w i s h c r y s t a l s of 95% p u r i t y were o b t a i n e d . Four kg of c r u d e g r i s e o f u l v i n was s t i r r e d w i t h 6 p. of c a r b o n t e t r a c h l o r i d e f o r 30 min, and t h e e x t r a c t s e p a r a t e d from t h e r e s i d u e by f i l t r a t i o n . The c r u d e p r o d u c t was washed w i t h 1 c a r b o n t e t r a c h l o r i d e r e f l u x e d w i t h 4 0 p. of m e t h y l e n e c h l o r i d e a t 25'C. To t h e s o l u t i o n was added 200 g o f a c t i v a t e d c a r b o n , s t i r r e d f o r o n e h o u r a t 35OC a n d t h e mass f i l t e r e d on a p r e s s u r e f i l t e r . The m e t h y l e n e c h l o r i d e was conc e n t r a t e d a t 38-4O'C t o a t h i c k consistency and allowed t o c r y s t a l l i z e a t 15OC, f i l t e r e d , a n d d r i e d a t 25OC i n vacuo ( 1 5 mm) f o r 2 4 h o u r s . The y i e l d of g r i s e o f u l v i n was 3.53 kg ( 9 3 % of t h e i n i t i a l q u a n t i t y o f c r u d e g r o d u c t ) . The p u r i t y o f t h e 341.8-347'. s u b s t a n c e was 9 8 . 5 % ; m.p. 2 1 8 - 2 2 2 C; { a ) 1.5 Literature Cited
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22 3 :2
(+)-l-thio-Griseofulvin
2.1 I n t r o d u c t i o n 2.1.1
P r o d u c i n g organism132
The f e r m e n t a t i o n of s e l e c t e d s u b s t r a t e s under c o n t r o l l e d c o n d i t i o n s w i t h a s t r a i n o f S&eptomyceA cineJteocaocatu-5 N.S. ( L e d e r l e AC-209) l e a d s t o t h e f o r m a t i o n of ( + I - 1 - t h i o - g r i s e o This s e c t i o n f u l v i n and (+)-5‘-hydroxy-l-thio-griseofulvin. o n l y d e a l s w i t h t h e f o r m e r compound. 2.1.2
B r i e f chemical d e s c r i p t i o n 2
A sample o f ( + I - 1 - t h i o g r i s e o f u l v i n , p r e p a r e d by c r y s t a l l i z a t i o n from a m i x t u r e of a c e t o n e and hexane, f o l l o w e d by d r y i n g i n vacuo w i t h P 2 0 5 o v e r n i g h t , has t h e f o l l o w i n g c h a r a c t e r i s t i c s 6 m e l t i n g p o i n t , 192-194’C; o p t i c a l r o t a t i o n = +321° ( 5 4.5 i C = 0.670 i n CHC13). The compound h a s u l t r a v i o l e t maxima i n methanol a t 232 nm (35.3501, 246 nm (36,800),306 nm (15,800), and 345 nm (3,900). ~e mass s p e c t r u m showed a molec u l a r i o n a t 368. The nmr s p e c t r u m (60 m c , C D C 1 3 ) showed peaks a t 66 c p s , (132 c p s . , 3-HI; 150 c p s . , m u l t i p l e t (3-HI; 219 c p s . s i n g l e t (3-HI; 239 c p s . , s i n g l e t (3-HI; 242 c p s . , s i n g l e t (3-H) 335 c p s . , s i n g l e t (1-HI; and 377 c p s . , s i n g l e t (1-HI; r e l a t i v e t o TMS.
2.1.3
S t r u c t u r a l formulal”
The s t r u c t u r a l f o r m u l a of ( + I - 1 - t h i o g r i s e o f u l v i n i s i l l u s t r a t e d as f o l l o w s .
2.2 T h e r a p e u t i c
usel
The a n t i f u n g a l compound ( + ) - l - t h i o g r i s e o f u l v i n h a s a s p e c t r u m of a c t i v i t y as shown i n T a b l e 1. The d a t a were d e v e l I n t h i s procedure g r e a t e r oped u s i n g t h e a g a r d i l u t i o n method. amounts of t e s t compounds were i n c o r p o r a t e d i n f l u i d n u t r i e n t a g a r and t h e m i x t u r e s were p l a c e d i n p e t r i d i s h e s . D i l u t e s u s p e n s i o n s of t h e v a r i o u s f u n g a l c u l t u r e s were a p p l i e d t o t h e hardened a g a r s u r f a c e s . A f t e r a p p r o p r i a t e i n c u b a t i o n p e r i o d s
22 4
t h e s e were examined t o d e t e r m i n e t h e l o w e s t c o n c e n t r a t i o n l e v e l s a t which growth o f t h e f u n g u s was i n h i b i t e d . Table 1 Antifungal A c t i v i t y of (+) -1-thiogriaeofulvin Minimal Inhibitory Concentration ( mcg/ml) 2.5 5.0 5.0 5.0
12.5 50.0 50.0 50 .O
2.3 A n a l y t i c a l
methods
Thin l a y e r c h r o m a t o g r a p h y 2 i s u s e d t o f o l l o w t h e f e r m e n t a t i o n and s e p a r a t i o n o f ( + ) - t h i o g r i s e o f u l v i n . Eastman c e l l u l o s e w i t h f l u o r e s c e n t indicator-MN polygram c e l l 300/UV-, i m p r e g n a t e d w i t h t h e l o w e r p h a s e o f a s o l v e n t s y s t e m composed o f h e x a n e , e t h y l a c e t a t e , m e t h a n o l a n d water i n t h e r a t i o 70:30:15:6 i s suitable. S p o t s are d e t e c t e d by u l t r a v i o l e t l i g h t . (+)-l-thiog r i s e o f u l v i n h a s a n Rf = 0 . 9 2 i n t h i s s y s t e m .
2.4 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n
A f t e r t h e f e r m e n t a t i o n i s completed’, t h e f e r m e n t e d mixt u r e c o n t a i n i n g t h e n o v e l compounds i s f i l t e r e d p r e f e r a b l y a t pH 6 - 7 t o remove t h e mycelium. Diatomaceous e a r t h o r any o t h e r c o n v e n t i o n a l f i l t r a t i o n a i d may b e u s e d t o a s s i s t i n t h e f i l t r a t i o n . Normally t h e m y c e l i a l c a k e i s washed w i t h water a n d t h e wash i s p o o l e d w i t h t h e f i l t r a t e . The combined f i l t r a t e a n d wash i s e x t r a c t e d w i t h 2 p o r t i o n s o f c h l o r o f o r m ( e a c h p o r t i o n b e i n g a b o u t 1 / 3 t h e volume of t h e f i l t r a t e and wash) which a r e combined and c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e t o a r e s i d u e . I n p l a c e of c h l o r o f o r m , o t h e r s o l v e n t s s u c h as e t h y l - a c e t a t e , butanol, methylene c h l o r i d e , etc., can be used. This r e s i d u e is t h e n s u b j e c t e d t o p a r t i t i o n chromatography on a d i a t o m a c e o u s e a r t h column u s i n g a s o l v e n t s y s t e m composed o f : h e x a n e , e t h y l a c e t a t e , m e t h a n o l , water ( 3 8 0 : 1 2 0 : 7 5 : 3 0 ) . The column e f f l u e n t , c o n t i n u o u s l y m o n i t o r e d u s i n g p e r c e n t t r a n s m i s s i o n a t 245 nm, i s c o l l e c t e d i n s e p a r a t e f r a c t i o n s i n s u i t a b l e volumes. The compounds p r o d u c e d i n t h e f e r m e n t a t i o n s a r e e l u t e d a p p r o x i m a t e l y between t h e s e c o n d t h r o u g h t h e f o u r t h and s i x t h t h r o u g h n i n t h hold-back volumes. F r a c t i o n s o f e f f l u e n t c o n t a i n i n g t h e n o v e l compounds of t h i s i n v e n t i o n a r e p o o l e d and t h e compounds recovered therefrom using standard techniques. An example of t h e i s o l a t i o n o f ( + I - 1 - t h i o g r i s e o f u l v i n i s as f o l l o w s 2 . A f i v e l i t e r p o r t i o n o f f e r m e n t a t i o n b r o t h i s c l a r i f i e d by f i l t r a t i o n t h r o u g h H y f l o R and t h e f i l t r a t e ext r a c t e d w i t h 1 / 2 volume o f c h l o r o f o r m . The c h l o r o f o r m e x t r a c t
225
i s d r i e d u s i n g a n h y d r o u s sodium s u l f a t e , which i s removed by f i l t r a t i o n , and c o n c e n t r a t e d t o a r e s i d u e ( 5 3 5 mg) u n d e r reduced p r e s s u r e . The r e s i d u e i s p u r i f i e d by means of a d s o r p t i o n column chromatography on s i l i c a g e l ( 4 0 gms). The column i s p r e p a r e d by s l u r r y i n g t h e a d s o r b a n t i n an a p p r o p r i a t e volume of c h l o r o f o r m a n d p u r i f y i n g t h e s l u r r y i n t o a g l a s s column. The r e s i d u e , d i s s o l v e d i n a minimal amount of c h l o r o f o r m , i s c h a r g e d o n t o t h e column. The column i s d e v e l o p e d w i t h a d d i t i o n a l c h l o roform. The e l u a t e c o n t a i n s b o t h ( + I - 1 - t h i o g r i s e o f u l v i n a n d (+)-5’-hydroxy-l-thiogriseofulvin and i s c o n c e n t r a t e d t o a r e s i d u e ( 2 7 mg) u n d e r r e d u c e d p r e s s u r e . P u r i f i c a t i o n o f t h e l a t t e r compound w i l l be d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n . The r e s i d u e o b t a i n e d as d e s c r i b e d i s f u r t h e r p u r i f i e d by means o f p a r t i t i o n chromatography on d i a t o m a c e o u s e a r t h . The column s u p p o r t i s p r e p a r e d by mixing 0.50 m l o f t h e l o w e r p h a s e from t h e s y s t e m N-hexane, e t h y l a c e t a t e , m e t h a n o l a n d water (380:120:75:30) w i t h e a c h 1 g of d i a t o m a c e o u s e a r t h . The w e t s u p p o r t i s u s e d t o pack a g l a s s column. The r e s i d u e , d i s s o l v e d i n a small amount of lower p h a s e , i s c h a r g e d o n t o t h e d i a t o maceous e a r t h . The column i s t h e n e l u t e d w i t h t h e u p p e r p h a s e of t h e described s o l v e n t system c o l l e c t i n g e f f l u e n t i n s e p a r a t e f r a c t i o n s of s u i t a b l e volumes. The column development i s f o l lowed by m o n i t o r i n g t h e e f f l u e n t w i t h o p t i c a l d e n s i t y r e a d i n g s a t 245 nm. ( + I - 1 - t h i o g r i s e o f u l v i n i s e l u t e d i n t h e second t h r o u g h f o u r t h h o l d - b a c k volumes. Appropriate e f f l u e n t f r a c t i o n s a r e combined a n d c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e t o . d r y n e s s . The r e s i d u e s are r e c r y s t a l l i z e d i n a m i x t u r e of a c e t o n e and hexane. Approximately 1 2 mg of ( * ) - l - t h i o g r i s e o f u l v i n , m.p. 188-189°C was o b t a i n e d . 2.5 Literature -
Cited
1.
H. Newman, W . W . A n d r e s , U.S. 1970.
2.
H. Newman and P . Shu, U.S. P a t e n t 3 , 6 1 6 , 2 3 7 ; 1971.
P a t e n t 3,532,714;
October 6 ,
October 2 6 ,
226
-3.
(+)-5'-hydroxy-l-thiogriseofulvin
3.1 Introduction 3.1.1
Producing organisrnly2
The antifungal compound (+)-51-hydroxy-l-thiogriseofulvin is co-produced with (+I-1-thiogriseofulvin as described in the previous section using selected substrates under controlled conditions with a strain of S t n e p t o m y c e d cLne4eocAocatu.4 n. s. (Lederle AC-209). 3.1.2
Brief chemical description'
The properties of (+)-51-hydroxy-l-thiogriseofulvin are as follows. A sample of this compound was prepared by crystallization from a mixture of acetone and hexane, followed by drying i n vacua with P205 overnight. It has a mglting goint of 206-208'C. The optical rotation { a ) 2 5 = +325 (k7.3 ; C 0.410 CHC1). Ultraviolet maxima occur in #ethanol; 232 nm (30,600), 246 nm (30,500), 308 nm (13,4501, 350 nm (3,450). The mass spectrum showed a molecular ion at 384. The nmr spectrum ( 6 0 mc, CDC13) showed peaks at 74 cps., 149 cps., (3-HI; 150 cps., multiplet (1-HI; 219 CPS., singlet (3-HI; 238 cps., singlet (3-H); 240 cps., singlet (3-HI; 284 cps., doublet (1-HI; 336 cps., singlet (1-H) and 375 c p s . , singlet (1-H) relative to
TMS
.
3.1.3
Structural formula "
The structural formula of (+)-5'-hydroxy-l-thiogriseofulvin is represented as follows.
3.2 Therapeutic use The antifungal activity of (+)-5'-hydroxy-l-thiogriseofulvin was determined as for (+I-1-thiogriseofulvin in Section 2.2. A table indicating the antifungal activity of this compound follows.
227 Table 2 A n t i f u n g a l A c t i v i t y o f (+) -5'-hydroxy-l-thiogriaeofulvin Minimal I n h i b i t o r y Concentration (mcq/ml)
Organisms
Tnichophytan .tov~bunavld TnLchophytan mnZagmphy.ted T h i c h o p h y t a n nubnum UiCnoApoAum can& UiCnOApohum gypACwn PhiaRophthaa gepnsetmei Candida dbicand clryptacoccud neodonmavLb
12.5 12.5 12 .o 55 .O 12 .o 500 .O 50.0 55.0
I t can be s e e n t h a t t h e a c t i v i t y o f ( + ) - 5 ' - h y d r o x y - l - t h i o g r i s e o f u l v i n is considerably less than t h a t f o r ( + ) - l - t h i o griseofulvin.
3.3 A n a l y t i c a l
methods
Thin l a y e r c h r o m a t o g r a p h y l i s u s e d t o f o l l o w t h e p r o d u c t i o n and i s i d e n and e x t r a c t i o n o f (+)-5'-hydroxy:l-thiogriseofulvin tical t o t h a t described in Section 2.3 f o r (+)-l-thiogriseofulvin. However, t h e Rf i n t h i s s y s t e m f o r t h e f o r m e r compound is 0.65.
3.4 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
The i s o l a t i o n of (+)-5'-hydroxy-l-thiogriseofulvin i s e s s e n t i a l l y i d e n t i c a l t o t h a t f o r ( + I - 1 - t h i o g r i s e o f u l v i n as described in Section 2.4. The p r e l i m i n a r y i s o l a t i o n c o n t a i n s a m i x t u r e o f b o t h components as p r e v i o u s l y n o t e d . However, t h e p a r t i t i o n chromatography on d i a t o m a c e o u s e a r t h e n a b l e s one t o s e p a r a t e t h e two a n t i f u n g a l compounds'. A s n o t e d p r e v i o u s l y , ( + ) - 1 - t h i o g r i s e o f u l v i n is e l u t e d i n t h e second through t h e f o u r t h h o l d - b a c k volumes b u t (+)-5'-hydroxy-l-thiogriseofulvin i s e l u t e d from t h e s i x t h t o t h e n i n t h h o l d - b a c k volumes. Approp r i a t e e f f l u e n t f r a c t i o n s a r e combined and c o n c e n t r a t e d u n d e r reduced p r e s s u r e t o dryness. The r e s i d u e s a r e r e c r y s t a l l i z e d from a m i x t u r e of a c e t o n e and h e x a n e . S i x mg o f ( + ) - 5 ' - h y d r o x y 1 - t h i o g r i s e o f u l v i n , m.p. 202-203OC was o b t a i n e d .
3.5 L i t e r a t u r e
Cited
1.
H . Newman a n d W . W . A n d r e s , U . S . 6 , 1970.
2.
H . Newman a n d P . Shu, U.S. 1 971.
P a t e n t 3,532,714;
P a t e n t 3,616,237;
October
October 2 7 ,
228
-4.
- 5 ' -hy droxy - g r i s eo f ul v i n 4 . 1 Introduction (+
4.1.1
P r o d u c i n g organism'
The compound (+)-5'-hydroxy-griseofulvin i s produced by f e r m e n t a t i o n from d e h y d r o g r i s e o f u l v i n o r ( + ) - g r i s e o f u l v i n u s i n g t h e microorganism S2%cp.tomyccA CincnocnocatuA ( L e d e r l e AC-209 1 , NRRL 3443.
4.1.2
B r i e f chemical d e s c r i p t i o n '
The a n t i f u n g a l compound (+I-5'-hydroxy-griseofulvin, p r e p a r e d by c r y s t a l l i z a t i o n from a c e t o n e / h e x a n e , f o l l o w e d by d r y i n g i n vacuo w i t h P20s o v e r n i g h t . I t has a mglting p o i n t of 222-224OC. The o p t i c a l r o t a t i o n { a l k 5 = *292 (+3 C = 1 . 0 0 i n CHC13). U l t r a v i o l e t maxima measured i n nanameters o c c u r i n methanol a t 2 1 1 (21,500), 217 (21,150), 235 (20,940), 292 The mass s p e c t r u m showed a m o l e c u l a r 21,500) and 325 (4,790). i o n a t 368. The nmr s p e c t r u m ( 6 0 mc, CDC13) showed peaks a t ( r e l a t i v e t o TMSI 6 6 c p s . , d o u b l e t (3-HI; 150 c p s . , m u l t i p l e t (1-H); 219 c p s . , s i n g l e t (3-HI; 239 c p s . , s i n g l e t (3-HI; 242 c p s . , s i n g l e t (3-HI; 281 c p s . , d o u b l e t (1-HI; 336 c p s . , s i n g l e t ( 1 - H I ; 368 c p s . , s i n g l e t (1-HI.
4.1.3
S t r u c t u r a l formula'
The s t r u c t u r a l f o r m u l a f o r (+)-5'-hydroxy-griseofulvin i s i l l u s t r a t e d as f o l l o w s .
4.2 T h e r a p e u t i c
usel
( + 1-5 ' - h y d r o x y - g r i s e o f u l v i n shows s i g n i f i c a n t a n t i f u n g a l a c t i v i t y a g a i n s t a v a r i e t y of f u n g i when t e s t e d u s i n g t h e a g a r d i l u t i o n method. T h i s t e c h n i q u e i s t h e same as i l l u s t r a t e d i n S e c t i o n 2.2. Table 3 which f o l l o w s , summarized r e s u l t s o b s e r v e d w i t h t h i s a n t i f u n g a l compound.
229 Table 3 A n t i f u n g a l A c t i v i t y of (+)-5 Organisms
-hydroxy-griseofulvin Minimal I n h i b i t o r y Concentration ( mcg/rnl)
Can&& dbicavts 300 (E83) &Yp&COCCu6 fleO~OJtmavLs SP (E138) Epidumiphytin dCoc0~wnATCC 10227 (E129) h!hAodpOJtWn d o & ATCC 14057 (E140) bkhOdpOJtWn d ATCC 10214 (E55) &CnOdpo/rWn @JpdeWn ATCC 14683 (E130) Pkidophtho/ra g e a n A e h i N I H 8274 (E16) ThichophyZon mn.tagJtophy.tea ( E l l ) T ~ c h o p h y t o nn u k m (E97) TJtichophy&ton ton6Urtan6 N I H 662 (E10)
64 64 8 8 10 4 64 8 4 4
(+)-5'-hydroxy-griseofulvin h a s been f o u n d t o have s i g n i f i c a n t a c t i v i t y a g a i n s t Mic&oApo&um c a n i d when t e s t e d as a t o p i c a l a n t i f u n g a l agent i n guinea pigs. The t e s t compound was p r e p a r e d i n a 2 % o i n t m e n t i n a Carbowax b a s e .
4.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
S e v e r a l examples have b e e n p u b l i s h e d ' f o r t h e i s o l a t i o n o f (+)-5'-hydroxy-griseofulvin; t h e s e a r e i l l u s t r a t e d as f o l lows.
-
T w e n t y - e i g h t k of f e r m e n t a t i o n b r o t h which was EXAMPLE 1 p r e p a r e d by a d d i n g ( + ) - g r i s e o f u l v i n t o a f e r m e n t a t i o n o f S . cineJteocnocatuA and a l l o w e d t o f e r m e n t f o r an a d d i t i o n a l 6 6 h o u r s , and h a v i n g a pH o f 6.9, was c l a r i f i e d by f i l t r a t i o n t h r o u g h H y f l o R a n d t h e f i l t r a t e e x t r a c t e d w i t h t w o - e i g h t .9 p o r t i o n s of chloroform. The combined c h l o r o f o r m e x t r a c t was conc e n t r a t e d u n d e r r e d u c e d p r e s s u r e t o a r e s i d u e (2.2 gms). The r e s i d u e was p u r i f i e d by p a r t i t i o n chromatography on a column ( 5 0 0 gms) of d i a t o m a c e o u s e a r t h . The column s u p p o r t was p r e p a r e d by m i x i n g 0.5 m l o f t h e l o w e r p h a s e from t h e s o l v e n t s y s t e m composed of h e x a n e : e t h y l a c e t a t e : m e t h a n o l : w a t e r ( 7 0 : 3 0 : 1 5 : 6 ) w i t h e a c h gram o f d i a t o m a c e o u s e a r t h . The r e s i d u e , d i s s o l v e d i n a smaller amount o f lower p h a s e , was mixed w i t h a small amount o f d i a t o m a c e o u s e a r t h and c h a r g e d t o t h e column. The upper p h a s e o f t h e above s o l v e n t s y s t e m was u s e d t o e l u t e t h e column. The column e f f l u e n t was m o n i t o r e d a t 245 nm: t h e p e a k , l o c a t e d a p p r o x i m a t e l y between t h e s i x t h a n d s e v e n t h h o l d back volumes, c o r r e s p o n d e d t o (+)-5'-hydroxy-griseofulvin. A p p r o p r i a t e f r a c t i o n s of e f f l u e n t c o n t a i n i n g t h e d e s i r e d p r o d u c t were combined and c o n c e n t r a t e d t o a r e s i d u e u n d e r r e d u c e d p r e s s u r e . The r e s i d u e was d i s s o l v e d i n a c e t o n e and t h e s o l u t i o n decolorized with activated charcoal. Hexane was added t o t h e d e c o l o r i z e d s o l u t i o n and c o n c e n t r a t i o n of t h i s s o l u t i o n p r e c i p i t a t e d (+)-5'-hydroxy-griseofulvin. The c r y s t a l s were s e p a r a t e d by f i l t r a t i o n ( 1 7 0 mg) h a v i n g a m e l t i n g p o i n t o f 222224OC. EXAMPLE 2
-
Two h u n d r e d k o f f e r m e n t a t i o n b r o t h (pH 6 . 9 ) were
2 30
c l a r i f i e d by f i l t r a t i o n t h r o u g h HyfloR and t h e f i l t r a t e e x t r a c t e d w i t h two 1 / 3 volumes o f c h l o r o f o r m . The combined c h l o r o f o r m e x t r a c t was c o n c e n t r a t e d t o a r e s i d u e (15.4 gms) u n d e r reduced p r e s s u r e . The r e s i d u e was p u r i f i e d by means of p a r t i t i o n chromatography on 1 . 7 kg o f d i a t o m a c e o u s e a r t h i n a manner e s s e n t i a l l y as d e s c r i b e d i n Example 1. C r y s t a l l i z a t i o n of t h e c r u d e p r o d u c t o b t a i n e d from t h e a c e t o n e - h e x a n e y i e l d e d 2 . 4 gms o f p u r i f i e d (+)-5'-hydroxy-griseofulvin.
-
A 300 m l p o r t i o n of f e r m e n t a t i o n b r o t h was f i l t e r e d t h r o u g h RyfloR and t h e f i l t r a t e e x t r a c t e d w i t h 150 m l of c h l o roform. The c h l o r o f o r m e x t r a c t was d r i e d u s i n g anhydrous sodium s u l f a t e and t h e d r y e x t r a c t c o n c e n t r a t e d t o a r e s i d u e u n d e r reduced p r e s s u r e . The r e s i d u e was p u r i f i e d by means of p a r t i t i o n chromatography on 50 gms of d i a t o m a c e o u s e a r t h i n a manner e s s e n t i a l l y as d e s c r i b e d i n S e c t i o n 4 . 3 . 1 . The ( + ) - g r i s e o f u l v i n i s e l u t e d i n t h e t h i r d t h r o u g h t h e f i f t h hold-back volumes a n d (+)-5'-hydroxy-griseofulvin from t h e s i x t h and s e v e n t h h o l d - b a c k volumes. A c o m b i n a t i o n of m o n i t o r i n g t h e column e f f l u e n t w i t h u l t r a v i o l e t l i g h t and t h i n l a y e r chromatography was used t o d e t e r m i n e i n which of t h e s e v e r a l f r a c t i o n s e a c h of t h e compounds was l o c a t e d . A p p r o p r i a t e f r a c t i o n s were combined and concent r a t e d s e p a r a t e l y t o d r y n e s s by r e d u c e d p r e s s u r e . The r e s i d u e s were r e c r y s t a l l i z e d i n methanol r e s u l t i n g i n 8 mg of ( + ) - g r i s e o f u l v i n and 4 mg o f (+)-5'-hydroxy-griseofulvin. EXAMPLE 3
4.4 L i t e r a t u r e 1.
Cited
Andres and M. P . Kuntsmann, U.S. October 2 6 , 1 9 7 1 .
W . W.
P a t e n t 3,616,238;
23 1
Lincomcyin Related Antibiotics
.
Thomas E Eble The Upjohn Company. Kalamazoo. Michigan
1
.
Lincomycin
1.1 Introduction................................... 1.1.1 Producing organisms ..........................
................... ........................... ............... ........ S . t i n c o t n e n b i b .............................. 1.3.1.1 Detection .................................... 1.3.2 Purification................................. 1.3.3 Crystallization .............................. 1.3.3.1 Partition chromatography ..................... Countercurrent distribution .................. 1.3.3.2 1.3.4 Isolation from S . .................. 2 . 4'-Depropyl-4'-ethyllincomycin 2.1 Introduction................................... 2.1.1 Producing organism ........................... 2.1.2 Brief chemical description ................... Strcutural formula........................... 2.1.3 2.2 Extraction. separation and purification ........ 2.2.1 Detection.................................... 2.2.2 Isolation of 11; separation from lincomycin.. Purification................................. 2.2.3 2.2.3.1 Countercurrent distribution .................. Florisil chromatography ...................... 2.2.3.2 2.2.4 Analysis for I1 .............................. 2.2.4.1 Thin-layer chromatography .................... 2.2.4.2 Pyrolysis-gas chromatography................. Gas-liquid chromatography .................... 2.2.4.3 3 . 1-Demethylthio-1-ethylthiolincomycin 3.1 Introduction................................... 3.1.1 Brief chemical description ................... 3.1.2 Structural formula........................... Extraction. separation and purification ........ 3.2 3.2.1 Isolation of 111 from S . u m b k i n u b ............ 3.2.2 Isolation of I11 from S . t i n c o t n e n b i o ........ 3 . 2 . 2 . 1 Countercurrent distribution.,................ 3.2.2.2 Crystallization of I11 hydrochloride. Isolation of two crystalline forms........... 4 . 1'-Demethyllincomycin 4.1 Introduction................................... 4.1.1 Brief chemical description ................... 4.1.2 Structural formula........................... 1.1.2 1.1.3 1.2 1.3 1.3.1
Brief chemical description Structural formula Therapeutic use and pharmacology Extraction. separation and purification Isolation of crude lincomycin from
ebp+MObub
234 234 234 234 235 235 235 236 236 238 238 238 239 240 240 240 240 240 240 242 242 242 242 243 243 244 244 244 245 245 245 245 246 246 246 248 248 248
2 32 4
.
1'-Demethyllincomycin (continued) 4.2 Extraction. separation and purification Recovery from the 4.2.1 Isolation of IV fermentation broth after addition of MTL Isolation of 4.2.2 Countercurrent distribution crystalline IV hydrochloride 4.2.3 Isolation after addition of methylene inhibitors
.
.......
.... . ................ ..................................
5
.
248 248 249 2 49
l-Demethylthio-l-ethylthio-l'-demethyl-lte thyllincomycin
.................................. .................. .......................... ....... .................. .. .............................. 1-Demethylthio-1-ethylthio-1'-demethyllincomycin 6. 6.1 Introduction.................................. 6.1.1 Brief chemical description .................. 6.1.2 Structural formula.......................... 6.2 Extraction, separation and purification ....... 6.2.1 Isolation................................... 6.2.2 Countercurrent distribution - crystallization of the hydrochloride................... 7. Lincomycin sulfoxide and l-demethylthio-lhydroxylincomycin 7.1 Introduction, structures and isolation ........ Celestice tins 8. 8.1 Introduction .................................. 8.1.1 Producing organisms ......................... 8.1.2 Brief chemical description .................. 8.1.3 Structural formula.......................... 8.2 Extraction, separation and purification., ..... 8.2.1 Isolation of the free base .................. 8.2.2 Isolation of crystalline celesticetin hydrochloride............................... Desalicetin and celesticetins B, C and D 9. 9.1 Introduction.................................. 9.1.1 Brief chemical description .................. 9.1.1.1 Desalicetin................................. 9.1.1.2 Celesticetin B .............................. 9.1.1.3 Celesticetin C .............................. 9.1.1.4 Celesticetin D .............................. 9.1.2 Structural formulae......................... 9.2 Extraction. separation and purification ....... 9.2.1 Isolation of crude mixture .................. 9.2.2 Isolation of desalicetin (X) ................ 5.1 5.1.1 5.1.2 5.2
Introduction Brief chemical description Structural formula Extraction, separation and purification 5.2.1 Isolation of crude mixture 5.2.2 Separation of (I) and (11) from (111) and (V) by counter double current distribution 5.2.3 Separation of V from I11 by silica gel chromatography
9.2.3
Isolation of celesticetin B (XI).
...........
251 251 251 252 252 252 252
252 254 254 254 254 255
255 256 256 256 256 257 257 257 257 257 257 257 259 259 259 259 259 260 261
233 9
.
Desalicetin and celesticetins B. C and D (continued) 9.2.3.1 Counter double current distribution 9.2.3.2 Countercurrent distribution 9.2.4 Isolation of celesticetin C (XI11 9.2.5 Isolation of celesticetin D (XIII) 9.2.5.1 Counter double current distribution 9.2.5.2 Countercurrent distribution
........ ................ .......... ......... ........ ................ Desalicetin 2"- (4-aminosalicylate 10 . 10.1 Introduction .................................. 10.1.1 Brief chemical description ................. 10.1.2 Structural formula ......................... 10.2 Extraction. separation and purification ....... 10.2.1 Isolation .................................. 10.2.1.1 Counter double current distribution ........ 10.2.1.2 Separation of XIV from celesticetin ........ 7-0-Demethylcelesticetin 11. 11.1 Introduction .................................. 11.1.1 Brief chemical description ................. 11.1.2 Structural formula ......................... 11.2 Extraction. separation and purification ....... 11.2.1 Isolation .................................. 11.2.1.1 Filtration and adsorption on Amberlite XAD-2 ...................................... 11.2.1.2 Silica gel chromatography. Isolation of XV hydrochloride ........................... 12
.
264 264 264 265 265 265 265
267
Other lincosaminides and celestosaminides Introduction and structures Extraction. separation and purification 13.2.1 Isolation and characterization
267 268 268
-
.................................. ................. ....... .................... ........................ ....... .................................. ......................................
.
.
262 263 263 263 263 263 264
l'-Demethyl-7-0-demethylcelesticetin and 1'demethylcelesticetin 12.1 Introduction 12.1.1 Brief chemical description 12.1.1.1 l'-Demethyl-7-0-demethylcelesticetin 12.1.1.2 1'-Demethylcelesticetin 12.1.2 Structural formulae 12.2 Extraction. separation and purification 12.2.1 Isolation 12.2.1.1 Filtration and adsorption on Amberlite XAD-2 12.2.1.2 Purification by counter double current Isolation of XVII distribution Counter12.2.1.3 Separation of XVI from XVII current distribution Silica 12.2.1.4 Isolation of XVI hydrochloride gel chromatography
-
13
261 261 261 262 262 262
........... ....................... . ......................... .
................... ....... ............. 14. Literature Cited .................................... 13.1 13.2
265 265 265 266 266 266 266 266 266 267
269
234
1. Lincomycin't
1.1 I n t r o d u c t i o n 1.1.1 P r o d u c i n g o r g a n i s m s
The f e r m e n t a t i o n c o n d i t i o n s a n d t h e a n t i b a c t e r i a l a c t i v i t y of l i n c o m y c i n were d e s c r i b e d by Mason e t i n 1962. The p r o d u c i n g a c t i n o m y c e t e was named Stncptomyced t i n c o l n e n n i n v a r . t i n c o l n e n d i d , NRRL 2936. B e s i d e s S. t i n c o t h e n d i d , l i n c o m y c i n can b e p r o d u c e d by StJteptomyccd umbhinud v a r . n o v a . ; by Stheptomyced k?dpinOdUd D i e t z , s p . n o v a . r 4 ; by St4cptomyck?d pdeudoghideolud chemouah C i n m y c c t i c u d D i e t z v a r . n o v a . 5 ; by S2&k?ptomyccd v a n i a b o l i b chemovah. l i n i a b i t i a D i e t z v a r . nova.6; by s t r a i n #1146 Actinomyced h o d e o ~ u A 7and by Sthcptomyced v e t t o d u d s p . nova.8.
1.1.2
Brief chemical d e s c r i p t i o n
The a n t i b i o t i c , i s o l a t e d o r i g i n a l l y by Herr and Bergy9 was a c o l o r l e s s c r y s t a l l i n e h y d r o c h l o r i d e s a l t , h a v i n g an H20. I t was q u i t e e m p i r i c a l f o r m u l a of C18H34N,O,S.HC1.1/2 s o l u b l e i n water, m e t h a n o l and e t h a n o l b u t r e l a t i v e l y i n s o l u b l e i n less p o l a r o r g a n i c s o l v e n t s . P o t e n t i o m e t r i c t i t r a t i o n s of t h e h y d r o c h l o r i d e showed l i n c o m y c i n t o be monobasic, w i t h a p k a ' o f 7 . 6 a n d an e q u i v a l e n t w e i g h t of 455. O p t i c a l r o t a t o r y d i s p e r s i o n measurements showed l i n c o m y c i n h y d r g c h l o r i d e t o be d e x t r o r o t a t o r y w i t h n o maximum and { a 1 6 5 = +137 ( c . 1 , water). Lincomycin showed n o c h a r a c t e r i s t i c a b s o r p t i o n from 2 2 0 t o 4 0 0 nm. I t was found t o be s t a b l e as t h e d r y h y d r o c h l o r i d e f o r 6 months a t 7 O o C . The l i n c o m y c i n h y d r o c h l o r i d e d e s c r i b e d by Herr a n d Bergy was a h e m i h y d r a t e and c o n s i s t e d o f l i g h t , f l u f f y , n e e d l e - l i k e c r y s t a l s . A s e c o n d form o f c r y s t a l s , i s o l a t e d by V a n O v e r l o o p l o , e x i s t e d as small, d e n s e c u b e s . T h i s form e x i s t e d as a monohydrate. I t was shown t h a t t h e two were p o l y m o r p h i c by d i f f e r e n c e s i n t h e i r X-ray d i f f r a c t i o n p a t t e r n s . P o l y m o r p h i c f o r m I1 ( t h e monohydrate) i s t h e c o m m e r c i a l l y a v a i l a b l e m a t e r i a l . The two p o l y m o r p h i c forms r e s u l t from d i f f e r i n g c o n d i t i o n s of c r y s t a l l i z a t i o n . Form I .is o b t a i n e d when a c e t o n e i s added q u i c k l y t o a n a q u e o u s s o l u t i o n of l i n c o m y c i n h y d r o c h l o r i d e h e l d a t 5-10°C. Form I1 r e s u l t s when t h e a c e t o n e i s added s l o w l y and t h e s o l u t i o n i s h e l d a t t e m p e r a t u r e s above 25OC. 1.1.3
Structural formula
o w e d S t r u c t u r a l ~ t u d i e s ~ ~ ~ ~ ~ t~h a~ t ~l i n~ c o~m~y csi n h (I) c o n s i s t s of a c a r b o h y d r a t e p o r t i o n m e t h y l 6-amino-6,a-dideoxy1-thio-D-enythAo-a-D-gatacto-octopyranoside** bound by amide " L i n c o c i n i s t h e t r a d e m a r k of The Upjohn Company f o r l i n c o m y c i n h y d r o c h l o r i d e , monohydrate.
2 35
linkage to 1-methy1-2kanA- Y-propyl-L-pyrrolidine-2- carboxylic acid. *
1.2 Therapeutic use
and pharmacology
Lincomycin has found usage in a variety of disease states as an antibacterial agent active against susceptible strains of streptococci, pneumococci and staphylococci (including penicillinase producing strains). Because lincomycin reaches effective concentrations in bone, it has been used with good results in the treatment of chronic osteomyelitis caused by lincomycinsensitive staphylococci. The absorption and excretion of lincomycin have been studied by many investigators. The initial findings of Vavra e t at.15 concerning serum levels and urinary excretion in humans have been verified by others. These authors found that after oral administration to normal adult volunteers, lincomycin attained peak serum levels at 4 hr. At an oral dosage of 5 0 0 mg every 6 hr, antibiotic serum levels were maintained between 2.4 and 3.9 pg/ml. An average of 3 to 5% of the oral dose was detected in the urine in the 24 hr period after dosing. Peak serum levels were obtained within 1 hr after intramuscular administration. Single intramuscular doses of 100, 200 and 6 0 0 mg resulted in the following respective average peak serum levels: 2.7, 3.8 and 1 1 . 6 ug/ml. After intramuscular administration, 27 to 47% was detected in the urine in the 24 hr period after dosing. Intravenous administration of lincomycin (2 hr infusion period) resulted in peak serum levels slightly higher than those seen after a comparable intramuscular dose. Intravenous infusion of 600 mg every 6 hr maintained serum levels between 6.3 and 19.8 ug/ml, and a dosage of 300 mg every 12 hr maintained levels between 1.3 and 9.8 pg/ml. Urine excretion of lincomycin after intravenous dosage averaged 12.7% in the 24 hr period after dasing.
1.3 Extraction, separation and 1.3.1
purification
Isolation of crude lincomycin from S. C i n c o t n e n A i . 4
Lincomycin was isolated from fermentations of S t 4 e p t o he trivial name meth lthiolincosaminide.(MTL) h b en given ?:.the aTino sugar poryion and propylhygric acid @HAY to the amino acid portion of the molecule.
2 36 rnycsd t i n c o l n e n d i d v a r . b i n c o b n e n d i d , NRRL 2936 (Herr and Bergyg and Bergy e t al.16) by a d j u s t i n g t h e whole b e e r from a h a r v e s t pH of 7 . 9 t o pH 6 . 7 w i t h c o n c e n t r a t e d s u l f u r i c a c i d and f i l t e r i n g u s i n g a b o u t 4% f i l t e r - a i d .
The c l e a r beer was a d j u s t e d t o pH 1 0 w i t h 5 0 % aqueous sodium h y d r o x i d e and e x t r a c t e d twice w i t h 1/3 volume o f 1b u t a n o l . The combined e x t r a c t s were mixed w i t h 1 / 2 volume of water and a d j u s t e d under c o n s t a n t a g i t a t i o n t o pH 2 w i t h conc e n t r a t e d s u l f u r i c a c i d . The aqueous phase was r e a d j u s t e d t o pH 10 and a g a i n e x t r a c t e d twice w i t h 1/3 volume of 1 - b u t a n o l . The combined b u t a n o l e x t r a c t s were washed w i t h 0 . 1 volume of water and c o n c e n t r a t e d i n v a c u o t o d r y n e s s . The e x t r a c t i o n was f o l l o w e d by s t a n d a r d b i o - a s s a y p r o c e d u r e s a g a i n s t S a n c i n a h t e a on a g a r t r a y s b u f f e r e d t o pH 6.8 w i t h 0 . 1 M pH 7 . 0 p h o s p h a t e b u f f e r 1 7 . The c r u d e p r e p a r a t i o n was o b t a i n e d i n a b o u t 5 0 % y i e l d and was a p p r o x i m a t e l y 5 0 % p u r e . 1.3.1.1
Detect i o n
U n t i l c r y s t a l l i n e l i n c o m y c i n h y d r o c h l o r i d e was o b t a i n e d i t was deemed n e c e s s a r y t o f o l l o w t h e e x t r a c t i o n p r o c e d u r e n o t o n l y by b i o a s s a y b u t a l s o by some means which c o u l d assure t h a t t h e p r o c e d u r e was i n d e e d c a p a b l e o f i s o l a t i n g t h e d e s i r e d a n t i b i o t i c . The method employed was p a p e r chromatography f o l l o w e d by b i o - a u t o g r a p h y on a g a r t r a y s s e e d e d w i t h Sancina k . t t e a , The s o l v e n t s y s t e m s were as f o l l o w s :
I. 11.
111.
IV.
V. VI.
1-Bu t a n o l - w a t e r
( 84 :1 6
v/v)
, 17
hrs.
1-Butanol-water (84:16 v / v ) p l u s 0.25% p-toluenesulfonic acid (w/v), 1 6 hrs. 1-Butanol-acetic acid-water ( 2 : l : l 16 hrs.
v/v),
1 - B u t a n o l - w a t e r (84:16 v / v ) p l u s 2% p i p e r t d i n e (v/v), 16 hrs. 1-Butanol-water
(4:96 v/v), 5 hrs.
1 - B u t a n o l - w a t e r ( 4 : 9 6 v / v ) p l u s 0.25% ptoluenesulfonic acid (w/v), 5 hrs.
F i g u r e I shows t h e bioautogram of l i n c o m y c i n on S. l u t e a . The s t r e a k i n g on system V i s c h a r a c t e r i s t i c of p u r i f i e d p r e p a r a t i o n s . Such b e h a v i o r of l i n c o m y c i n i n f e r m e n t a t i o n b r o t h s i s rare.
1.3.2
Purification
Crude l i n c o m y c i n was p u r i f i e d by r e d i s s o l v i n g t h e l i n e p r o d u c t i n water ( c a 2 0 0 mg/ml) and a d j u s t i n g t h e pH t o 2 w i t h c o n c e n t r a t e d s u l f u r i c a c i d . A brown p r e c i p i t a t e was removed by
237
Rf-
System
0.2
0.4
0.6
0.8
I
I1 111
IV V VI
FIGURE 1 P a p e r chromatography of l i n c o m y c i n . The a n t i b i o t i c was d e t e c t e d by b i o a u t o g r a p h y on S a Q C i n U &~.tQaseeded agar.
238
f i l t r a t i o n a n d t h e c l e a r f i l t r a t e was e x t r a c t e d w i t h an e q u a l volume o f m e t h y l e n e c h l o r i d e t o remove i m p u r i t i e s . The a q u e o u s l a y e r was a d j u s t e d t o pH 5.0 w i t h 50% sodium h y d r o x i d e and a g a i n e x t r a c t e d w i t h an e q u a l volume of m e t h y l e n e c h l o r i d e t o remove more i m p u r i t i e s . F i n a l l y t h e a q u e o u s l a y e r was a d j u s t e d t o pH 1 0 w i t h 50% sodium h y d r o x i d e a n d e x t r a c t e d e x h a u s t i v e l y w i t h m e t h y l e n e chloride. A small volume of water was a d d e d t o t h e combined e x t r a c t s and t h e m e t h y l e n e c h l o r i d e was removed i n v a c u o . The r e s u l t i n g a q u e o u s s o l u t i o n was f r e e z e d r i e d t o a f f o r d material a p p r o x i m a t e l y 80% p u r e a n d i n y i e l d s of 9 0 t o 95% from t h e crude s t a r t i n g material. 1.3.3
Crystallization
S e v e r a l p r o c e d u r e s were u s e d f o r t h e f i n a l p u r i f i c a t i o n and c r y s t a l l i z a t i o n of l i n c o m y c i n h y d r o c h l o r i d e . 1.3.3.1
P a r t i t i o n chromatography
A s o l v e n t system c o n s i s t i n g of cyclohexane, methyl e t h y l k e t o n e , and pH 1 0 b u f f e r (70:30:20 v / v ) was e q u i l i b r a t e d . (The b u f f e r was made by a d d i n g s u f f i c i e n t NaHC03 t o a 0 . 2 M N a 2 C 0 3 s o l u t i o n t o a d j u s t t h e pH t o 1 0 . 0 ) . One h u n d r e d grams of f i l t e r a i d was s l u r r i e d i n t h e u p p e r p h a s e of t h i s s y s t e m ; f o r t y m l of lower p h a s e was added and t h e whole s l u r r y was homogeni z e d . T h i s s o l v e n t - f i l t e r a i d m i x t u r e was p o u r e d i n t o a g l a s s column (32 mm I . D . ) a n d packed t o a c o n s t a n t h e i g h t u s i n g . 1 8 k g / s q . cm. o f a i r p r e s s u r e . The f e e d f o r t h e column, 2 . 5 g o f c a . 9 0 % p u r e l i n c o m y c i n , was d i s s o l v e d i n 2 m l of l o w e r p h a s e a n d t h e n homogenized w i t h u p p e r p h a s e and p l a c e d o n t o t h e t o p o f t h e column b e d . The column was d e v e l o p e d w i t h u p p e r p h a s e a t a rate o f a p p r o x i m a t e l y 2 m l / m i n u t e . Two h u n d r e d 2 0 m l f r a c t i o n s were c o l l e c t e d . F r a c t i o n s 1 t h r o u g h 85 were i n a c t i v e ; f r a c t i o n s 9 0 t h r o u g h 200 a l l p o s s e s s e d a b o u t t h e same a c t i v i t y . F r a c t i o n s 1 0 0 t h r o u g h 200 were p o o l e d a n d c o n c e n t r a t e d
i n vucuo ( l e s s t h a n 5OoC) t o d r y n e s s . The r e s i d u e was d i s s o l v e d i n 20 m l o f water a n d t h e pH was a d j u s t e d t o 2.0 u s i n g c o n c e n t r a t e d h y d r o c h l o r i c a c i d . The aqueous s o l u t i o n was e x t r a c t e d 2 times w i t h 1 0 m l of 1 - b u t a n o l a n d t h e aqueous was d i s t i l l e d i n vacuo a t l e s s t h a n 5OoC t o 10 m l t o remove t h e dissolved 1-butanol. Acetone (50-60 m l ) was added s l o w l y t o t h e aqueous c o n c e n t r a t e and c r y s t a l l i z a t i o n s t a r t e d . A f t e r 30 m i n u t e s a t room t e m p e r a t u r e t h e c r y s t a l s were f i l t e r e d o f f and vacuum d r i e d t o a c o n s t a n t w e i g h t o f 388 mg o f c r y s t a l l i n e lincomycin hydrochloride. 1.3.3.2
Countercurrent d i s t r i b u t i o n
A s o l u t i o n of 32 g o f a p p r o x i m a t e l y 50% p u r e l i n c o m y c i n i n water (pH 9 . 1 ) was a d j u s t e d t o pH 2 . 3 w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d . The a q u e o u s s o l u t i o n was e x t r a c t e d t w i c e w i t h e q u a l volumes o f m e t h y l e t h y l k e t o n e (MEK). The MEK e x t r a c t s removed 4 7 1 mg o f i m p u r i t i e s . The a q u e o u s p h a s e was c o n c e n t r a t e d t o a volume o f g0 m l a n d mixed w i t h 600 m l of acetone. The s o l u t i o n was c h i l l e d t o - 2 O O C a n d t h e c r y s t a l s
2 39
which s e p a r a t e d were r e c o v e r e d by f i l t e r i n g , washed w i t h acet o n e , and vacuum d r i e d t o c o n s t a n t w e i g h t o f 7 . 4 g o f c r y s t a l l i n e lincomycin hydrochloride. T h i s material was p o o l e d w i t h o t h e r comparable p r e p a r a A two gram a l i q u o t was d i s s o l v e d i n 1 0 0 m l o f water a n d 100 m l of 2 - b u t a n o l . The s t a r t i n g material was p l a c e d i n t u b e s 0 - 1 9 o f a CCD a p p a r a t u s a n d d i s t r i b u t e d t h r o u g h 500 t r a n s f e r s . The d i s t r i b u t i o n was a n a l y z e d by s o l i d s d e t e r m i n a t i o n . An e x c e l l e n t a g r e e m e n t w i t h t h e t h e o r e t i c a l c u r v e was o b t a i n e d . tions.
The p o o l from t u b e s 1 0 0 - 1 4 0 was c o n c e n t r a t e d t o a volume o f 10 m l , f i l t e r e d a n d c o n c e n t r a t e d f u r t h e r t o 5 m l . Fifty m l of r e a g e n t g r a d e a c e t o n e was added and t h e m i x t u r e was c h i l l e d t o - 2 0 C , A f t e r s e v e r a l h o u r s t h e c r y s t a l s were c o l l e c t e d , washed w i t h a c e t o n e , and vacuum d r i e d t o a c o n s t a n t w e i g h t . The y i e l d was 1 . 4 2 grams of l i n c o m y c i n h y d r o c h l o r i d e c r y s t a l s . 1.3.4
I s o l a t i o n from S. e b p h 0 b u A
The i s o l a t i o n ' from h a r v e s t b e e r s o f S. eApinOdUd i n v o l v e d f i l t e r i n g 9 11 of whole b e e r a t h a r v e s t pH u s i n g f i l t e r aid. The f i l t e r cake was washed w i t h 1 .t o f w a t e r and t h e wash combined w i t h t h e c l e a r b e e r . The c l e a r beer-wash ( 7 . 2 a ) was a d j u s t e d t o pH 8.8 a n d e x t r a c t e d 3 times w i t h m e t h y l e n e c h l o r i d e u s i n g o n e - t h i r d o f t h e c l e a r b e e r volume e a c h t i m e . The methy l e n e c h l o r i d e e x t r a c t s were combined a n d t h e n c o n c e n t r a t e d t o d r y n e s s t o g i v e an o i l y m a t e r i a l . T h i s m a t e r i a l was d i s s o l v e d i n 5 0 0 m l of e t h e r and t h e s o l u t i o n mixed w i t h 5 m l of 1 N m e t h a n o l i c hydrogen c h l o r i d e from which l i n c o m y c i n h y d r o c h l o r i d e p r e c i p i t a t e d as a c o l o r l e s s amorphous m a t e r i a l ; y i e l d 450 mg. T h i s p r e p a r a t i o n was c h a r a c t e r i z e d by t h i n l a y e r chromatography u s i n g s i l i c a g e l G (Merck A . G . , D a r m s t a d t ) as s u p p o r t and m e t h y l e t h y l k e t o n e - a c e t o n e - w a t e r ( 1 5 0 : 5 0 : 2 0 v / v ) as t h e s o l vent (Figure 2 ) .
I t was l a t e r f o u n d 4 t h a t S. enpinooud when grown a t e l e v a t e d t e m p e r a t u r e s (44-48'C) produced h i g h e r t i t e r s of l i n comycin t h a n when grown a t u s u a l t e m p e r a t u r e s . The l i n c o m y c i n p r o d u c e d was e x t r a c t e d by t h e f o l l o w i n g p r o c e d u r e . Whole f e r m e n t a t i o n b r o t h ( a p p r o x i m a t e l y 48 1 was f i l t e r e d using f i l t e r a i d . The f i l t e r cake was washed w i t h 1 a o f water and t h e aqueous wash was combined w i t h t h e f i l t r a t e . The c l e a r b e e r was p a s s e d o v e r a column c o n t a i n i n g 2 5 0 m l of A m b e r l i t e XAD-2 a t a f l o w r a t e o f 2 5 m l p e r m i n u t e . The column was washed w i t h 500 m l o f water and was t h e n e l u t e d w i t h m e t h a n o l - w a t e r (95:5 v / v ) . F r a c t i o n s of 2 0 m l were c o l l e c t e d , and a s s a y e d a g a i n s t S. lutes. The a c t i v e f r a c t i o n s were combined a n d c o n c e n t r a t e d t o d r y n e s s t o g i v e 3.5 gms of a l i n c o m y c i n p r e p a r a t i o n a s s a y i n g 3 1 0 mcg o f lincomycin/mg. T h i s m a t e r i a l was t r i t u r a t e d w i t h m e t h y l e n e c h l o r i d e , t h e m e t h y l e n e c h l o r i d e e x t r a c t was concent r a t e d t o d r y n e s s and t h e r e s i d u e was t r i t u r a t e d w i t h a c e t o n e . The f i l t r a t e was mixed w i t h e t h e r t o g i v e a p r e c i p i t a t e which was removed. The r e m a i n i n g f i l t r a t e was mixed w i t h m e t h a n o l i c hydrogen c h l o r i d e (1 N ) t o p r e c i p i t a t e c o l o r l e s s l i n c o m y c i n
2 40
h y d r o c h l o r i d e which was i s o l a t e d by f i l t r a t i o n and c r y s t a l l i z e d from w a t e r - a c e t o n e t o g i v e c r y s t a l l i n e l i n c o m y c i n h y d r o c h l o r i d e . 2.
4'-Depropyl-4'-ethyllincomycin 2 . 1 Introduction -
A r g o u d e l i s Qta l . 1 8 ' 1 9 3 2 0 r e p o r t e d t h e i s o l a t i o n and s t r u c t u r e o f a n a n t i b i o t i c co-produced w i t h l i n c o m y c i n i n f e r m e n t a t i o n s o f S . linco.!!nenbib. T h i s a n t i b i o t i c i s t h e 4 ' d e p r o p y l - 4 ' - e t h y l homolog of lincomycin (11). 2.1.1
Producing organism
4'-Depropyl-4~-ethyllincomycin and a l l s u b s e q u e n t l y d e s c r i b e d l i n c o s a m i n i d e s were i s o l a t e d from S2Aeptontyceb l i n c o l ncndid var. lincolnenbib
.
2.1.2
B r i e f chemical d e s c r i p t i o n
4'-Depropyl-4 I - e t h y l l i n c o m y c i n i s o l a t e d as t h e f r e e b a s e h a s t h e f o r m u l a C17H32N206S.1/2 H20 and as t h e c r y s t a l l i n e h y d r o c h l o r i d e C17H32N206S.HC1.1/2 H20. I t s s o l u b i l i t y p r o p e r t i e s were similar t o t h o s e of l i n c o m y c i n . P o t e n t i o m e t r i c aqueous t i t r a t i o n s showed a pKa' of 7 . 6 0 . 25
The h y d r o c h l o r i d e showed a s p e c i f i c r o t a t i o n { a l D =+147.5' (c=l, w a t e r ) , and no a b s o r p t i o n i n t h e UV and v i s i b l e r a n g e . 2.1.3
S t r u c t u r a l formula
4'-Depropyl-4'-ethyllincomycin
2 . 2 Extraction, 2.2.1
has t h e s t r u c t u r a l formula
s e p a r a t i o n and p u p i f i c a t i o n
Detection
The p a p e r c h r o m a t o g r a p h i c b e h a v i o r of I1 i s s o similar t o t h a t of l i n c o m y c i n t h a t i t was n o t d e t e c t e d i n t h e e a r l y s t a g e s of lincomycin development. I t was d e t e c t e d i n c r u d e l i n c o m y c i n p r e p a r a t i o n s by TLC. A t y p i c a l t h i n - l a y e r chromatogram is shown i n Figure 2 .
241
Rf 0.2
0.4
0.6
0.8
FIGURE 2 T h i n - l a y e r chromatography of I1 h y d r o c h l o r i d e . Upper I1 h y d r o c h l o r i d e ; middle I1 h y d r o c h l o r i d e and l i n c o mycin h y d r o c h l o r i d e ; l o w e r , l i n c o m y c i n h y d r o c h l o r i d e . T h i n - l a y e r p l a t e s were p r e p a r e d from S i l i c a G e l G (Merck, D a r m s t a d t ) . T h i c k n e s s o f t h e f i l m was 0 . 4 mm. The s o l v e n t s y s t e m c o n s i s t e d o f m e t h y l e t h y l k e t o n e , a c e t o n e , a n d water ( 1 5 0 : 5 0 : 2 0 v / v ) . Detection s y s t e m s u s e d were p e r i o d a t e - p e r r n a n g a n a t e s p r a y and b i o a u t o g r a p h y on S a h c i n a Rutea s e e d e d a g a r .
2 42 2.2.2
I s o l a t i o n of II; s e p a r a t i o n from l i n c o m y c i n
A c u l t u r e b r o t h o f l i n c o m y c i n f e r m e n t a t i o n ( 2 4 k % ) was a d j u s t e d t o pH 3 . 0 w i t h c o n c e n t r a t e d s u l f u r i c a c i d , a n d f i l t e r e d using f i l t e r aid. The f i l t e r e d b e e r was a d j u s t e d t o pH 8 . 0 w i t h 50% aqueous sodium h y d r o x i d e . The a l k a l i n e c l e a r b e e r was t h e n p a s s e d t h r o u g h columns c o n t a i n i n g P i t t s b u r g h Type CAL, 1 2 - 4 0 mesh g r a n u l a r c a r b o n (109 k g ) . The c a r b o n columns were washed w i t h water a t 4 O o C .
Lincomycin and I1 were e l u t e d from t h e c a r b o n u s i n g a n a c e t o n e - w a t e r m i x t u r e (4:l v / v ) a t 5OoC. The a c e t o n e e l u a t e s ( 8 . 7 k L) were c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e t o an a q u e o u s c o n c e n t r a t e o f 0 . 3 kL. The aqueous c o n c e n t r a t e was a d j u s t e d t o pH 1 0 . 6 w i t h 5 0 % sodium h y d r o x i d e s o l u t i o n and e x t r a c t e d w i t h The m e t h y l e n e m e t h y l e n e c h l o r i d e ( t o t a l volume of 0.4 kk). c h l o r i d e e x t r a c t s were mixed w i t h water and t h e m i x t u r e was c o n c e n t r a t e d a z e o t r o p i c a l l y t o an a q u e o u s s o l u t i o n ( 7 2 a ) , which was a d j u s t e d t o pH 1 . 0 w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d . Acetone ( 0 . 7 kk) was added t o t h e a c i d i c aqueous concent r a t e and t h e m i x t u r e was a l l o w e d t o s t a n d o v e r n i g h t . Crude c r y s t a l s o f l i n c o m y c i n a n d I1 h y d r o c h l o r i d e s were i s o l a t e d by f i l t r a t i o n and d r i e d ( 3 0 . 7 5 kg)
.
T h i s m a t e r i a l was d i s s o l v e d i n water (32 k) a n d mixed with 0.3 k L of acetone. Lincomycin h y d r o c h l o r i d e s t a r t e d p r e c i p i t a t i n g a l m o s t i m m e d i a t e l y . The c r y s t a l s of l i n c o m y c i n h y d r o c h l o r i d e were i s o l a t e d by f i l t r a t i o n a n d d r i e d ( 2 4 . 3 k g ) . 2.2.3
Purification
The m o t h e r l i q u o r s c o n t a i n i n g I1 h y d r o c h l o r i d e a n d r e s i d u a l l i n c o m y c i n h y d r o c h l o r i d e were c o n c e n t r a t e d t o an a q u e o u s s o l u t i o n ( 2 1 a ) . One R o f t h i s s o l u t i o n was a d j u s t e d t o pH 9.5 by u s i n g 2 N a q u e o u s sodium h y d r o x i d e s o l u t i o n . The a l k a l i n e s o l u t i o n was t h e n e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e . The methy l e n e c h l o r i d e e x t r a c t was c o n c e n t r a t e d t o d r y n e s s t o g i v e 85.0 g o f c r u d e I1 f r e e b a s e which was u s e d as t h e s t a r t i n g m a t e r i a l f o r countercurrent distribution. 2.2.3.1
Countercurrent d i s t r i b u t i o n
Ten grams o f t h i s c r u d e I1 f r e e b a s e was d i s s o l v e d i n 75 m l of t h e l o w e r p h a s e o f t h e s o l v e n t s y s t e m 1 - b u t a n o l a n d water (1:l v / v ) . The pH was a d j u s t e d t o 4.2 by a d d i t i o n o f 1 N aqueo u s h y d r o c h l o r i c a c i d . T h i s s o l u t i o n was t h e n mixed w i t h an e q u a l volume o f u p p e r p h a s e o f t h e same s o l v e n t s y s t e m and t h e m i x t u r e was t r a n s f e r r e d i n an a l l - g l a s s CCD a p p a r a t u s (10 ml/ phase). The d i s t r i b u t i o n was s t o p p e d when 1 0 0 0 t r a n s f e r s h a d been c o m p l e t e d and a n a l y z e d by d e t e r m i n a t i o n o f s o l i d s and t h i n l a y e r chromatography. Two p e a k s w i t h K v a l u e s o f 0 . 0 9 and 0 . 1 5 were found. T h i n - l a y e r c h r o m a t o g r a p h y showed t h a t t u b e s 60-90 c o n t a i n e d I1 as t h e o n l y b i o a c t i v e m a t e r i a l . However, t h e s e f r a c t i o n s were f o u n d t o c o n t a i n i m p u r i t i e s which were removed by F l o r i s t 1 chromatography. 2.2.3.2 F l o r i s i l chromatography F r a c t i o n s 60-90 f r o m t h e CCD were combined a n d concen-
243
t r a t e d t o an a q u e o u s s o l u t i o n . The pH was a d j u s t e d t o 10.5 a n d t h e s o l u t i o n was f r e e z e - d r i e d t o g i v e 900 mg o f c o l o r l e s s amorphous m a t e r i a l . T h i s material ( 8 0 0 mg) was d i s s o l v e d i n 2 0 m l o f a c e t o n e and t h e s o l u t i o n was added t o t h e t o p o f a 2 5 mm (1.D.) column c o n t a i n i n g 4 0 g o f F l o r i s i l packed i n S k e l l y s o l v e B. The column was t h e n e l u t e d w i t h S k e l l y s o l v e - a c e t o n e m i x t u r e s of i n c r e a s i n g acetone content. I1 was e l u t e d from t h e column w i t h a m i x t u r e o f S k e l l y s o l v e - a c e t o n e i n t h e volume r a t i o of 10:90. The e l u a t e s c o n c e n t r a t e d t o d r y n e s s gave 400 mg o f I1 f r e e base. The f r e e b a s e (100 mg) was d i s s o l v e d i n 0 . 7 m l o f 1 N aqueous h y d r o c h l o r i c a c i d . The s o l u t i o n was t h e n mixed w i t h 30 m l o f a c e t o n e and 40 m l o f e t h e r , and t h e m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 1 5 h o u r s . The c r y s t a l l i n e c o l o r l e s s I1 h y d r o c h l o r i d e was i s o l a t e d by f i l t r a t i o n a n d d r i e d ( 6 0 mg). 2.2.4
A n a l y s i s f o r I1
S i n c e I1 i s t h e o n l y known a n a l o g o f l i n c o m y c i n f o u n d t o be co-produced i n normal S. f i n c o f n c n n i n f e r m e n t a t i o n s , i t was n e c e s s a r y t o d e v e l o p p r o c e d u r e s for i t s q u a n t i t a t i o n i n commerc i a l l i n c o m y c i n . A l l o w a b l e l i m i t s f o r c o n c e n t r a t i o n o f I1 i n l i n c o m y c i n h a d t o be s e t . Control s p e c i f i c a t i o n s permit no more t h a n 5 % . S e v e r a l t e c h n i q u e s have been d e v e l o p e d f o r t h e d e t e c t i o n and q u a n t i t a t i o n of 11. 2.2.4.1
T h i n - l a y e r chromatography
Brodasky and Lummis i n 1 9 6 4 2 1 d e s c r i b e d a p r o c e d u r e which combined t h i n - l a y e r c h r o m a t o g r a p h y and d i s c - p l a t e a s s a y t e c h n i ques. Chromatography o f l i n c o m y c i n and 11, as h y d r o c h l o r i d e s , was c a r r i e d o u t on s i l i c a - g e l p l a t e s w i t h e i t h e r o f two m o b i l e p h a s e s , one composed of m e t h y l e t h y l k e t o n e - a c e t o n e - w a t e r ( 9 . 3 : 2 . 6 : l v / v ) a n d t h e o t h e r composed o f m e t h y l p r o p y l k e t o n e methyl e t h y l ketone-water-methanol ( 2 : Z : l : l v / v ) . The t h i n l a y e r p l a t e s were p r e p a r e d by a p p l y i n g a s l u r r y o f s i l i c a g e l G ( M e r c k , D a r m s t a d t ) c o n t a i n i n g 3 0 g o f s i l i c a g e l and 6 0 m l o f d i s t i l l e d water t o a c e t o n e - w a s h e d g l a s s p l a t e s (10 x 2 0 cm). The s l u r r y was a p p l i e d w i t h a Camag a p p a r a t u s s e t f o r a f i l m thickness of 4 x cm. The p l a t e s were a i r - d r i e d and s t o r e d without f u r t h e r treatment. Development of t h e p l a t e s was c a r r i e d o u t i n e i t h e r o f t h e two m o b i l e p h a s e s m e n t i o n e d u n t i l t h e s o l v e n t f r o n t h a d t r a v e l e d a b o u t 1 0 cm. T h i s r e q u i r e d 45 t o 60 minutes f o r e i t h e r mobile phase. The a n t i b i o t i c s were d e t e c t e d by e x p o s i n g t h e p l a t e s t o iodine vapors. A f t e r removal o f t h e i o d i n e t h e s i l i c a g e l i n t h e area marked was m e c h a n i c a l l y r e d u c e d t o a powder a n d removed w i t h a d e v i c e d e s c r i b e d i n t h e p u b l i c a t i o n . The a n t i b i o t i c s were removed from t h e s i l i c a g e l by e x t r a c t i o n w i t h w a t e r f o r l i n c o m y c i n and m e t h a n o l f o r 11. A 1 0 0 - u l a l i q u o t was a p p l i e d t o 6 . 5 mm p a p e r d i s c s .
The
2 44 d i s c s were t h e n a p p l i e d t o a g a r t r a y s s e e d e d w i t h Saacina i u t e a . The t r a y s were i n c u b a t e d f o r 1 6 h r a t 3 O o C and t h e z o n e s o f i n h i b i t i o n were compared t o t h o s e o f s t a n d a r d s on t h e same t r a y . The a s s a y was shown t o b e e x p e r i m e n t a l l y f e a s i b l e i n a n a l y z i n g fermentations. 2.2.4.2
Pyrolysis-gas
chromatography
I n 1 9 6 7 BrodaskyZ2 r e p o r t e d on a low t e m p e r a t u r e p y r o l y s i s g a s c h r o m a t o g r a p h i c s e p a r a t i o n o f d e g r a d a t i o n p r o d u c t s of v a r i o u s analogs of lincomycin. The low t e m p e r a t u r e (375’C) pyrogram o f l i n c o m y c i n showed many f a s t moving p e a k s a n d a b r o a d peak a t a r e t e n t i o n time o f 24.6 min. Samples of t h i s material were c o l l e c t e d and a n a l y z e d I t was shown t o be a s s o c i a t e d w i t h t h e by mass s p e c t r o s c o p y . d e g r a d a t i o n of t h e PHA p o r t i o n of t h e m o l e c u l e and was i d e n t i f i e d as t h e 1 - m e t h y l - 3 - p r o p y l p y r r o l e . Under t h e same p y r o l y t i c c o n d i t i o n s , I1 showed a peak w i t h T h i s was shown t o b e t h e 1-methyl-3-ethyl p y r r o l e .
a s h i f t i n r e t e n t i o n time t o 1 5 . 8 min. 2.2.4.3
G a s - l i q u i d chromatography
The g a s - l i q u i d chromatography o f t h e t r i m e t h y l s i l y l e t h e r s of l i n c o m y c i n and some o f i t s a n a l o g s was r e p o r t e d i n 1 9 6 8 by Houtman ct a t . 2 3 . Under t h e s t a n d a r d i z e d c o n d i t i o n s d e s c r i b e d i n t h i s work, t h e y f o u n d a u s e a b l e d i f f e r e n c e i n t h e r e t e n t i o n times of s i l y l a t e d l i n c o m y c i n and 11. The r e s p o n s e f a c t o r s f o r l i n c o m y c i n a n d I1 were d e t e r m i n e d b y s i l y l a t i n g and c h r o m a t o g r a p h i n g a 5 0 - 5 0 m i x t u r e of t h e two compounds. The d i f f e r e n c e i n r e s p o n s e p e r mg f o r t h e two m a t e r i a l s was l e s s t h a n 1%. T h i s small d i f f e r e n c e was i n s i g n i f i c a n t i n t h e d e t e r m i n a t i o n of small amounts of I1 i n l i n c o m y c i n . The r e s p o n s e r e l a t i o n s h i p was f u r t h e r e s t a b l i s h e d by a r e c o v e r y s t u d y . E i g h t p r e p a r e d m i x t u r e s r a n g i n g from 0-10% I1 i n l i n c o m y c i n by w e i g h t were a s s a y e d . S t a t i s t i c a l a n a l y s i s of t h e d a t a i n d i c a t e d a s l o p e of 0 . 9 9 . Twenty r e p l i c a t e a s s a y s , e a c h on d i f f e r e n t d a y s , of a sample o f l i n c o m y c i n c o n t a i n i n g 2 . 3 3 % I1 gave a s t a n d a r d d e v i a t i o n o f 0 . 1 0 8 % f o r 11. T h i s r e p r e s e n t s a c o e f f i c i e n t of v a r i a t i o n of 4.8% f o r t h e minor c o n s t i t u e n t . T h i s p r o c e d u r e was m o d i f i e d by Margosis i n 1 9 6 E Z 4 a n d a g a i n as s l i g h t l y m o d i f i e d was p u b l i s h e d as t h e o f f i c i a l method f o r t h e d e t e r m i n a t i o n of I1 i n l i n c o m y c i n Z 5 . 3. 1-Demethylthio-1-ethylthiolincomycin
3.1 Introduction P a t t e r s o n & 0.1.~a n d A r g o u d e l i s e x a t . 1 8 ’ 2 6 r 2 showed 7 t h a t when Stncptomyced umbninud v a r . cyanconigcn v a r . nova or
2 45 S . d i n c o d n e n d i d was grown i n t h e p r e s e n c e o f e t h i o n i n e , a new homolog o f l i n c o m y c i n was p r o d u c e d . I n t e r p r e t a t i o n of t h e NMR s p e c t r a o f t h e new a n t i b i o t i c i n r e l a t i o n s h i p t o t h a t of lincomycin, coupled with t h e d i f f e r ences i n elemental a n a l y s i s , e s p e c i a l l y f u n c t i o n a l group analys i s a n d , i n t h e case o f t h a t a n t i b i o t i c p r o d u c e d by 3 . t i n c o d n e n d i d , degradation s t u d i e s 1 8 , allowed these authors t o conclude t h a t t h e new a n t i b i o t i c was 1-demethylthio-1-ethylthiolincomycin (111). 3.1.1
Brief chemical d e s c r i p t i o n
1-Demethylthio-1-ethylthiolincomycinh y d r o c h l o r i d e h a s t h e e m p i r i c a l formula C19H36N206S.HC1.H20. I t s s o l u b i l i t y p r o p e r t i e s were similar t o t h o s e o f l i n c o m y c i n . P o t e n t i o m e t r i c aqueous t i t r a t i o g s showed a pk,' o f 7.73. I t s s p e c i f i c r o t a t i o n was +143 (c=O.62, w a t e r ) . 3.1.2
S t r u c t u r a l formula
1-Demethylthio-1-ethylthiolincomcyin f o r m u l a 111. y43 CH3
has the structural
I
F:H$
Ho-fH
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"0 SCHzCH3
I11
3.2 E x t r a c t i o n , 3.2.1
OH
s e p a r a t i o n and p u r i f i c a t i o n
I s o l a t i o n o f 111 from S. u m b a i n u ~
I11 was formed when S. umbhinuh was grown u n d e r c o n d i t i o n s c o n d u c i v e t o t h e p r o d u c t i o n of l i n c o m y c i n b u t t o which 0 . 0 0 6 % D L - e t h i o n i n e w a s added a f t e r 118 h r s o f g r o w t h . I t was d e t e c t e d a t h a r v e s t t i m e i n t h e c u l t u r e b r o t h by p a p e r b i o a u t o g r a p h y a g a i n s t C o h y n e b a c t e n i u m x e & o d i d on pH 7.9 n u t r i e n t a g a r medium. By d e v e l o p i n g on No. 1 Whatman p a p e r i n 1 - b u t a n o l - w a t e r - i s o a m y l a l c o h o l - d i c h l o r o a c e t i c a c i d ( 1 0 0 : 7 5 : 5 0 : 1 v / v ) , t h e Rf v a l u e s of l i n c o m y c i n and t h e new compound were 0 . 5 0 a n d 0.65, r e s p e c t i v e l y .
The a n t i b i o t i c s were a d s o r b e d from t h e c u l t u r e f i l t r a t e on a c t i v a t e d c h a r c o a l ( 1 . 5 p e r c e n t w/v) a n d e l u t e d w i t h a mixt u r e o f a c e t o n e a n d water ( 9 : l v / v ) . The e l u a t e was c o n c e n t r a t e d t o an a q u e o u s p h a s e and a d j u s t e d t o pH 1 0 . 5 . T h i s was e x t r a c t e d w i t h two e q u a l volumes o f 1 - b u t a n o l . The combined e x t r a c t s were e x t r a c t e d twice w i t h o n e - f i f t h volume o f water a d j u s t e d t o pH 2.5 w i t h h y d r o c h l o r i c a c i d . The a q u e o u s p h a s e was l y o p h i l i z e d . The r e s i d u a l s o l i d was a b o u t 9 0 p e r c e n t p u r e a n t i b i o t i c , c o n t a i n i n g l i n c o m c y i n and t h e new compound i n t h e r a t i o
246 o f a b o u t 1O:l. Lincomycin and I11 were r e s o l v e d by p a r t i t i o n chromatog r a p h y on d i a t o m a c e o u s e a r t h , i n e t h y l a c e t a t e 0.5 M p h o s p h a t e b u f f e r , pH 6.2. The d i s t r i b u t i o n c o e f f i c i e n t s were 0.05 a n d 0 . 0 9 f o r l i n c o m y c i n a n d I11 r e s p e c t i v e l y . Most of t h e 111, which c o n t a i n e d l e s s t h a n 2 p e r c e n t l i n c o m y c i n as j u d g e d by p a p e r c h r o m a t o g r a p h y , was e l u t e d w i t h a n amount o f d e v e l o p i n g f l u i d e q u a l t o 3 t o 5.5 times t h e v o i d volume of t h e column. T h i s p o r t i o n o f t h e e l u a t e was e x t r a c t e d w i t h water which h a d been a d j u s t e d t o pH 2 . 5 w i t h h y d r o c h l o r i c a c i d . The e x t r a c t was n e u t r a l i z e d t o pH 4.5 w i t h A m b e r l i t e IR-45 a n d l y o p h i l i z e d . The r e s i d u e was d i s s o l v e d i n m e t h a n o l (200 mg/ml), and f i v e volumes o f a c e t o n e were a d d e d . A f t e r s t o r a g e o v e r n i g h t a t 4OC, t h e c r y s t a l l i n e h y d r o c h l o r i d e s a l t o f I11 was c o l l e c t e d a n d r e c r y s t a l l i z e d twice i n t h e same way.
-
3.2.2
I s o l a t i o n o f I11 from S. l i n c o l n e n h i o
I11 was formed on t h e a d d i t i o n of 2 g l a o f D L - e t h i o n i n e a t 48 h o u r s of growth o f S. k?incolncnAiA u n d e r o t h e r w i s e u s u a l f e r m e n t a t i o n c o n d i t i o n s 2 8 . I t was d e t e c t e d by t h i n - l a y e r c h r o matography ( F i g u r e 3 ) .
F e r m e n t a t i o n b e e r ( 2 3 5 1 ) was f i l t e r e d a t h a r v e s t pH u s i n g f i l t e r a i d . The m y c e l i a l c a k e was washed w i t h water and t h e combined f i l t e r e d b e e r a n d w a t e r wash ( 2 7 5 a ) was s t i r r e d f o r 45 m i n u t e s w i t h 1 2 . 5 kg of a c t i v a t e d c a r b o n and 2 . 5 kg of d i a t o maceous e a r t h . The m i x t u r e was f i l t e r e d and t h e f i l t r a t e was discarded. The c a k e was t h e n e l u t e d w i t h a m i x t u r e of water and a c e t o n e ( 9 : l v / v ) . T h i s e l u a t e was c o n c e n t r a t e d t o an aqueous s o l u t i o n which was a d j u s t e d t o pH 1 0 . 0 w i t h 5 0 % a q u e o u s sodium h y d r o x i d e , and t h e n e x t r a c t e d t h r e e times w i t h 20-1 p o r t i o n s o f m e t h y l e n e c h l o r i d e . The m e t h y l e n e c h l o r i d e e x t r a c t s were c o n c e n t r a t e d t o d r y n e s s t o g i v e a m i x t u r e o f e q u a l amounts of I11 a n d l i n c o m y c i n i n t h e f r e e - b a s e form ( 7 . 1 4 g ) . T h i s mat e r i a l was t h e n d i s s o l v e d i n 100 m l o f 1 N m e t h a n o l i c h y d r o g e n c h l o r i d e and t h e s o l u t i o n mixed w i t h 3 . 2 $ o f e t h y l e t h e r w i t h s t i r r i n g . The p r e c i p i t a t e d c o l o r l e s s amorphous m a t e r i a l was i s o l a t e d by f i l t r a t i o n a n d d r i e d . 3.2.2.1
Countercurrent d i s t r i b u t i o n
Crude I11 h y d r o c h l o r i d e ( 7 g ) was d i s s o l v e d i n 20 ml of water and 2 0 m l o f 1 - b u t a n o l . The pH was a d j u s t e d t o 4 . 2 w i t h 1 N aqueous h y d r o c h l o r i c a c i d , and t h e m i x t u r e was t r a n s f e r r e d i n a CCD a p p a r a t u s (10 m l / p h a s e ) . The d i s t r i b u t i o n was i n t e r r u p t e d when 1 0 0 0 t r a n s f e r s h a d been c o m p l e t e d a n d a n a l y z e d by d e t e r m i n a t i o n o f s o l i d s a n d by t h i n - l a y e r chromatography. Two p e a k s w i t h K v a l u e s o f 0 . 1 4 and 0.20 were f o u n d . Tubes 135-190, which c o n t a i n e d I11 o n l y , were combined, and t h e s o l u t i o n was c o n c e n t r a t e d t o d r y n e s s i n vacuo t o g i v e 2.44 g o f c o l o r l e s s amorphous I11 h y d r o c h l o r i d e . 3.2.2.2
C r y s t a l l i z a t i o n of I11 h y d r o c h l o r i d e . o f two c r y s t a l l i n e forms
Isolation
2 47
Rf
-
FIGURE 3 T h i n - l a y e r chromatography of I11 h y d r o c h l o r i d e . Upper I11 h y d r o c h l o r i d e ; m i d d l e , I11 h y d r o c h l o r i d e and l i n c o m y c i n h y d r o c h l o r i d e ; l o w e r , lincomycin hydrochloride. T h i n - l a y e r p l a t e s were p r e p a r e d from S i l i c a G e l G (Merck, D a r m s t a d t ) . T h i c k n e s s of t h e The s o l v e n t s y s t e m c o n s i s t e d o f f i l m was 0 . 4 mm. m e t h y l e t h y l k e t o n e , a c e t o n e , and water ( 1 5 0 : 5 0 : 2 0 D e t e c t i o n s y s t e m s used were p e r i o d a t e v/v). permanganate s p r a y and b i o a u t o g r a p h y on Slvlcina lutes s e e d e d a g a r .
2 48
I11 h y d r o c h l o r i d e ( 5 0 0 mg) was d i s s o l v e d i n 2 m l o f water, 1 m l of methanol and 100 m l of a c e t o n e . T h i s s o l u t i o n was mixed w i t h e t h e r u n t i l t h e f i r s t c r y s t a l s a p p e a r e d . The m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 1 h o u r . The p r e c i p i t a t e d c r y s t a l s h a v i n g t h e a p p e a r a n c e of c u b e s (Form 11) were
s e p a r a t e d from t h e s u p e r n a t a n t by d e c a n t a t i o n and r e c r y s t a l l i z e d from w a t e r - m e t h a n o l - a c e t o n e - e t h e r t o g i v e 2 5 0 mg o f I11 h y d r g c h l o r i d e (Form 11). The s u p e r n a t a n t was a l l o w e d t o s t a n d a t 5 C f o r 4 h o u r s . The c r y s t a l l i n e I11 h y d r o c h l o r i d e ( n e e d l e s , Form I > p r e c i p i t a t e d , was i s o l a t e d by f i l t r a t i o n , and was d r i e d (150 mg). These polymorphic forms c o r r e s p o n d e d t o Forms I1 and I of lincomycin r e s p e c t i v e l y . 4 . 1'-Demethyllincomycin 4.1 Introduction --
During t h e c o u r s e o f e a r l y b i o s y n t h e t i c s t u d i e s , A r g o u d e l i s e t at.29 d e c i d e d t o d e t e r m i n e what e f f e c t t h e a d d i t i o n of t h e two h a l v e s of t h e l i n c o m y c i n molecule ( i . e . PHA and MTL) would have on t h e f e r m e n t a t i o n of S . t i n c o l n e n A i A . A l though no i n c r e a s e i n l i n c o m y c i n p r o d u c t i o n was o b s e r v e d a f t e r a d d i n g e i t h e r m o i e t y , t h e a d d i t i o n of MTL r e s u l t e d i n t h e p r o d u c t i o n o f a new a n t i b i o t i c , 1 ' - d e m e t h y l l i n c o m y c i n ( I V ) . 4.1.1
B r i e f chemical d e s c r i p t i o n
1'-Demethyllincomycin h y d r o c h l o r i d e h a s t h e e m p i r i c a l I t was more p o l a r t h a n l i n c o f o r m u l a : C17H,2N206S.HC1.H20. mycin. P o t e n t i o m e t r i c aqueous t i t r a t i o n s gave a pKa' of 7 . 5 8 . The o p t i c a l r o t a t i o n { a 1 $ 5 was +149 ( c = 0 . 9 , water). 4.1.2
S t r u c t u r a l formula
&
1'-Demethyllincomycin h a s t h e s t r u c t u r a l f o r m u l a I V . H
CH 3
I
HO-CH I
CONH-CH
IV
4.2 Extraction, 4.2.1
H'QscH3
s e p a r a t i o n and p u r i f i c a t i o n
I s o l a t i o n o f I V . Recovery from t h e f e r m e n t a t i o n b r o t h a f t e r a d d i t i o n of MTL
Best y i e l d s were o b t a i n e d when 2 g / a o f MTL was added t o normal S. e i n c o k h c n A i A f e r m e n t a t i o n s a f t e r 4 8 h o u r s of growth. A t h a r v e s t t i m e , b o t h lincomycin and I V were p r e s e n t . I n t h i s case, t h e p o l a r i t y d i f f e r e n c e s between t h e two a n t i b i o t i c s was
2 49
s u c h t h a t I V c o u l d be d e t e c t e d n o t o n l y by t h i n - l a y e r chromatog r a p h y ( F i g u r e 4 ) b u t slso by p a p e r chromatography ( F i g u r e 5 ) . F e r m e n t a t i o n b r o t h ( 3 5 a ) was f i l t e r e d a t h a r v e s t pH u s i n g f i l t e r a i d . The f i l t r a t e was s t i r r e d f o r 30 m i n u t e s w i t h a c t i v a t e d c a r b o n ( 5 % , w/v) a n d f i l t e r e d . The c a r b o n c a k e was washed s u c c e s s i v e l y w i t h water and s u c c e s s i v e w a t e r - a c e t o n e m i x t u r e s (1:4; 7 : 3 and 9 : l v / v ) . The c a r b o n cake was t h e n ’ s l u r r i e d w i t h 1 - b u t a n o l - a c e t o n e - w a t e r (3:3:2 v / v ) . The m i x t u r e was f i l t e r e d a n d t h e f i l t r a t e was c o n c e n t r a t e d t o an a q u e o u s s o l u t i o n which was f r e e z e - d r i e d t o g i v e 9 . 0 g o f c o l o r l e s s material. T h i s was d i s s o l v e d i n 4 0 0 m l o f water, a n d a d j u s t e d t o pH 10 w i t h a q u e o u s sodium h y d r o x i d e a n d t h e n e x t r a c t e d s u c c e s s i v e l y , o n c e w i t h 5 0 0 ml o f S k e l l y s o l v e B , and f o u r times w i t h 250 ml p o r t i o n s of methylene c h l o r i d e . The S k e l l y s o l v e e x t r a c t a n d t h e m e t h y l e n e c h l o r i d e e x t r a c t s (which c o n t a i n e d l i n c o m y c i n ) were d i s c a r d e d . The a q u e o u s s o l u t i o n was t h e n e x t r a c t e d f i v e times w i t h 250 m l p o r t i o n s o f 1 - b u t a n o l . The combined b u t a n o l e x t r a c t was c o n c e n t r a t e d t o d r y n e s s . The r e s i due was d i s s o l v e d i n 5 0 m l o f 0 . 5 N m e t h a n o l i c hydrogen c h l o r i d e a n d t h i s s o l u t i o n was mixed w i t h 5 0 0 m l o f e t h y l e t h e r . The p r e c i p i t a t e d material was i s o l a t e d by f i l t r a t i o n . T h i s material c o n t a i n i n g I V and l i n c o m c y i n as t h e h y d r o c h l o r i d e s a l t s was u s e d as t h e s t a r t i n g material f o r c o u n t e r c u r r e n t d i s t r i b u t i o n . 4.2.2
Countercurrent d i s t r i b u t i o n . c r y s t a l l i n e I V hydrochloride
I s o l a t i o n of
The c r u d e m i x t u r e o f l i n c o m y c i n a n d I V h y d r o c h l o r i d e s was d i s s o l v e d i n 1 0 0 m l o f t h e lower p h a s e o f e q u a l volumes o f 1b u t a n o l and water. T h i s s o l u t i o n was mixed w i t h an e q u a l v o l ume o f t h e u p p e r p h a s e and t r a n s f e r r e d t o a CCD a p p a r a t u s . A f t e r 930 t r a n s f e r s , t h e d i s t r i b u t i o n was a n a l y z e d by s o l i d s d e t e r m i n a t i o n a n d t h i n - l a y e r c h r o m a t o g r a p h y . Tubes 1 0 4 - 1 7 0 , which c o n t a i n e d m a i n l y I V h y d r o c h l o r i d e , were combined ( 6 6 0 ml) and c o n c e n t r a t e d t o a volume o f 8 0 m l . C r y s t a l l i n e I V hydroc h l o r i d e f o r m e d , was i s o l a t e d by f i l t r a t i o n a n d d r i e d ( 2 0 0 mg). An a d d i t i o n a l 2 0 0 mg was o b t a i n e d by f u r t h e r c o n c e n t r a t i o n o f t h e f i l t r a t e . These two p r e p a r a t i o n s were combined a n d d i s s o l v e d i n water (17 ml). Addition of 60 ml of acetone r e s u l t e d i n t h e c r y s t a l l i z a t i o n o f I V h y d r o c h l o r i d e i n t h e form of c o l o r l e s s , l o n g , f e a t h e r y c r y s t a l s , which were i s o l a t e d by f i l t r a t i o n and d r i e d ; y i e l d 1 7 0 mg. 4.2.3
I s o l a t i o n a f t e r a d d i t i o n of methylation i n h i b i t o r s
I n a s t u d y of t h e e f f e c t o f m e t h y l a t i o n i n h i b i t o r s on S. described the following e x t r a c t i o n process f o r I V and lincomycin.
e i n c o e n e n o i d f e r m e n t a t i o n s A r g o u d e l i s e t ae. 3 0
’
H a r v e s t f e r m e n t a t i o n s t o which v a r i o u s m e t h y l a t i o n i n h i b i t o r s h a d been a d d e d were f i l t e r e d w i t h t h e a i d of f i l t e r a i d . The f i l t e r cake was washed w i t h water a n d t h e wash was combined with the f i l t r a t e . The combined c l e a r b e e r and wash ( 4 e ) was
250
Af
0.2
-
0.4
‘0.6
0.8
FIGURE 4 T h i n - l a y e r chromatography of I V . Upper, I V ; m i d d l e , I V , I1 and l i n c o m y c i n ; l o w e r , l i n c o mycin. T h i n - l a y e r p l a t e s were p r e p a r e d from S i l i c a G e l G (Merck, D a r m s t a d t ) . T h i c k n e s s of t h e f i l m was 0 . 4 mm. The s o l v e n t - s y s t e m c o n s i s t e d of methyl e t h y l k e t o n e , a c e t o n e water (150:50:20 v / v ) . D e t e c t i o n systems used were p e r i o d a t e - p e r m a n g a n a t e s p r a y and b i o a u t o g r a p h y on Sakcina Lutes s e e d e d a g a r .
Rf
System
0.2
-
0.4
0.6
0.8
1.0
FIGURE 5 P a p e r chromatography o f I V . The a n t i b i o t i c was d e t e c t e d by b i o a u t o g r a p h y on Sakcina lu&a seeded agar.
251 adjusted to pH 6 . 0 and extracted once with 400 ml of a 9% solution of sodium dinonylnaphthalene sulfonate (NaDNNS) in Skellysolve B. The spent beer was then extracted once with 400 ml of Skellysolve B. The NaDNNS and the Skellysolve B extracts were combined. The combined extract was washed with 200 m l of water. The aqueous wash was discarded and the NaDNNS-Skellysolve B phase was mixed with 160 ml of a 25% solution of Aliquat-336 (industrial grade tricaproyl methyl ammonium chloride produced by General Mills, Chemical Division, Kankakee, Illinois) in Skellysolve B and 200 ml of water. The mixture was shaken well and then the two phases were allowed to separate. The aqueous phase was removed and the organic phase was extracted twice more with 200 ml of water each time. The aqueous extracts were combined and washed twice with 200 ml of Skellysolve B each time. The aqueous solution was then adjusted to pH 10 and extracted three times with 350 ml portions of methylene chloride each time to remove the lincomycin. The aqueous solution was then extracted with 1-butanol (four 350 ml portions). The butanol extracts were combined and concentrated to dryness. The residue was triturated with 95% ethanol and 140 mg of insoluble crystalline material was isolated by filtration. This preparation was found by paper chromatography to contain mainly IV with small amounts of related lincomycin compounds. Recrystallization of this preparation by dissolving in 4 ml of water and adding 25 ml of acetone, afforded 50 mg of crystalline IV hydrochloride. 5. l-Demethylth~o-l-ethylth~o-l'-demethyl-l'-ethyll~ncomyc~n
5.1 Introduction Argoudelis c t a l ? . 3 2 s 3 3reported that the addition of ethionine to S . k?incok?nenAiA growing in synthetic media results not only in the production of l-demethylthio-l-ethylthiolincomycin but also another antibiotic which proved to be l-demethylthio-1-ethylthio-1'-demethyl-1 '-ethyllincomycin (V)
.
5.1.1
Brief chemical description
1-Demethylthio -1-ethy1thio-1 -demethyl-1 -ethyllincomycin Its solubilhydrochloride has the formula C2oH3,N,06S.HCl.H,?. ities were sizilar to lincomycin. It had an optical rotation +144.5 (c=0.38, water).
5.1.2
Structural formula
The structural formula of l-demethylthio-l-ethylthio-1'demethyl-1'-ethyllincomycin is V.
252
5.2 E x t r a c t i o n , 5.2.1
s e p a r a t i o n and p u r i f i c a t i o n
I s o l a t i o n of c r u d e m i x t u r e
For t h e p r o d u c t i o n o f V , 5 0 0 mcg/ml o f DL-ethionine was added t o f e r m e n t a t i o n s o f S. LincolnenAiA growing i n a medium i n which c i t r a t e and g l u c o s e were t h e s o l e c a r b o n s o u r c e s . The e t h i o n i n e was added 72 h r s a f t e r i n o c u l a t i o n and t h e b e e r s were h a r v e s t e d a t 1 4 4 h r s . The a n t i b i o t i c s produced were e x t r a c t e d by t h e f o l l o w i n g p r o c e d u r e . F e r m e n t a t i o n b e e r ( 2 5 0 a ) was f i l t e r e d a t h a r v e s t pH u s i n g f i l t e r a i d . The m y c e l i a l cake was washed w i t h water and t h e cake was t h e n d i s c a r d e d . The comb i n e d f i l t e r e d b e e r and water wash (240 a ) was t h e n a d j u s t e d t o pH 1 0 ( w i t h aqueous sodium h y d r o x i d e ) and e x t r a c t e d t h r e e times w i t h 6 0 a of methylene c h l o r i d e e a c h time. The methylene c h l o r i d e e x t r a c t s were combined and t h e s o l u t i o n was c o n c e n t r a t e d t o an o i l y m a t e r i a l ( 7 4 . 8 g ) . T h i n - l a y e r chromatography showed t h e p r e s e n c e o f l i n c o m y c i n ( I ) , 4'-depropyl-4'-ethyllincomycin (111, 1-demethylthio-1-ethylthio-lincomycin (111) and t h e new a c t i v i t y A t y p i c a l t h i n - l a y e r chromatogram i s p r e s e n t e d i n F i g u r e 6 . V.
5.2.2
S e p a r a t i o n o f (I) and (11) from (111) and ( V ) b y c o u n t e r double c u r r e n t d i s t r i b u t i o n
F o r t y grams of t h e o i l y material was d i s s o l v e d i n 240 ml of a b s o l u t e methanol. T h i s s o l u t i o n was c l a r i f i e d by f i l t r a t i o n and mixed w i t h 8 a o f e t h y l e t h e r . M e t h a n o l i c hydrogen c h l o r i d e (1 N ; 1 6 0 ml) was t h e n added under c o n t i n u o u s s t i r r i n g . The p r e c i p i t a t e d h y d r o c h l o r i d e s were f i l t e r e d and d r i e d ; y i e l d 1 9 . 5 g. T h i s material was t h e n d i s t r i b u t e d i n a c o u n t e r d o u b l e c u r r e n t d i s t r i b u t i o n (CDCD) a p p a r a t u s ( 2 5 m l p e r p h a s e ) u s i n g e q u a l volumes o f 1 - b u t a n o l and w a t e r . The d i s t r i b u t i o n was a n a l y z e d a f t e r 130 t r a n s f e r s by t h i n - l a y e r chromatography. (11) (K=0.10) and l i n c o m y c i n (K=0.14) were s e p a r a t e d from 111 (K=0.20) and V (K=0.25). F r a c t i o n s c o n t a i n i n g I11 and V were combined and c o n c e n t r a t e d t o d r y n e s s ; y i e l d 4.33 g . 5.2.3
S e p a r a t i o n of V from I11 by s i l i c a g e l chromatography
T h r e e g r a m s of t h e m i x t u r e of I11 and V was chromatographed o v e r 9 0 0 g o f s i l i c a g e l (Merck, Darrnstadt No. 7734) u s i n g methyl e t h y l k e t o n e - a c e t o n e - w a t e r (100:30:5 v / v ) . TLC showed t h a t e a r l y f r a c t i o n s c o n t a i n e d V . Concentration of t h e e l u a t e s c o n t a i n i n g V a f f o r d e d 2 7 0 mg of c r y s t a l l i n e V hydrochloride. 6.
l-Demethvlthio-l-ethylthio-l'-dernethyllincomyc~n
6 . 1 Introduction When 1-demethylthio-1-ethylthiolincomycin (111) i s t r e a t e d w i t h h y d r a z i n e a r e a c t i o n t a k e s p l a c e similar t o t h a t w i t h l i n comycin. The h y d r a z i d e o f PHA i s formed and t h e s u g a r p o r t i o n e t h y l t h i o l i n c o s a m i n i d e (ETL) i s o b t a i n e d .
253
Rf
0.2
0.4
-
0.6
0.8
FIGURE 6 A: 4'-Depropyl-4'-ethyllincomycin
(11) B: Lincomycin (I) C: 1 - D e m e t h y l t h i o - 1 - e t h y l t h i o l i n c o m y c i n (111) D: 1-Demethylthio-1-ethylthio-1'-demethyl-1'ethyllincomycin (V) Thin-layer plates were prepared from Silica Gel G (Merck, Darmstadt). The solvent system consisted of methyl ethyl ketone, acetone, and water (140:40:22 v/v). Detection systems used were periodate-permanganate spray and bioautography on Sancina tu-tea seeded agar.
254 A r g o u d e l i s L t ( ~ 1 . l f~o u'n d~ t~h a t t h e a d d i t i o n of ETL t o n o r m a l f e r m e n t a t i o n s o f S. .&tco1nenAh r e s u l t e d i n t h e product i o n of t h e a n t i b i o t i c 1-demethylthio-1-ethylthio-1'-demethyll i n c o m y c i n (VI 1. 6.1.1
B r i e f chemical d e s c r i p t i o n
l-Demethylthio-l-ethylthio-lt-demethyllincornycin hydrot ~was c h l o r i d e h a s t h e m o l e c u l a r f o r m u l a C , & I ~ I + N ~ ~ ~ S . H C ~ .IH O. s l i g h t l y more p o l a r t h a n l i n c o m y c i n a n d l e s s p o l a r t h a n 1'demethyllincomycin. I t showed a pKa' o f 8 . 0 by p o t e n t i o m e p i c t i t r a t i o n i n water and an o p t i c a l r o t a t i o n 0 f I a ) i 5 = +157 ( c = 0 . 5 7 , w a t e r ) . 6.1.2
S t r u c t u r a l formula
1-Demethylthio-1-ethylthio-1'-demethyllincomycin s t r u c t u r a l formula V I . CH 3
49
has t h e
I
CONH-CH "O-Y
VI
6.2 E x t r a c t i o n , 6.2.1
HQ
SC2H5
OH s e p a r a t i o n and p u r i f i c a t i o n
Isolation
V I was r e c o v e r e d u s i n g t h e f o l l o w i n g p r o c e d u r e : Two a n d one h a l f a o f b e e r was f i l t e r e d a n d t h e cake was washed w i t h water. The combined b e e r a n d wash (3.12 a ) was a d j u s t e d t o pH 6 . 0 w i t h 1 N a q u e o u s h y d r o c h l o r i c a c i d a n d e x t r a c t e d w i t h 1 . 5 a. o f S k e l l y s o l v e B . The aqueous s o l u t i o n was t h e n e x t r a c t e d w i t h two 400 m l p a r t i o n s o f a 9 % s o l u t i o n o f NaDNNS i n S k e l l y s o l v e B. The NaDNNS e x t r a c t was washed w i t h 2 0 0 ml of water, which was d i s c a r d e d . The NaDNNS e x t r a c t was t h e n mixed w i t h 3 5 0 m l of water a n d 9 0 m l o f A l i q u a t 336. The aqueous p h a s e was s e p a r a t e d and t h e s p e n t NaDNNS e x t r a c t was e x t r a c t e d two a d d i t i o n a l times w i t h t h e same p o r t i o n s as above of A l i q u a t 336 a n d water. The s p e n t NaDNNS was d i s c a r d e d . The a q u e o u s e x t r a c t s were comb i n e d , and t h e combined s o l u t i o n was washed w i t h two 2 0 0 m l p o r t i o n s o f S k e l l y s o l v e B , t h e n a d j u s t e d t o pH 1 0 . 0 w i t h 1 N aqueous sodium h y d r o x i d e and e x t r a c t e d w i t h t h r e e 500 ml p o r t i o n s o f m e t h y l e n e c h l o r i d e t o remove t h e l i n c o m y c i n . The a q u e o u s was t h e n e x t r a c t e d f i v e t i m e s w i t h 4 0 0 m l p o r t i o n s o f 1-butanol. The b u t a n o l i c e x t r a c t s were combined, mixed w i t h 200 ml o f water, a d j u s t e d t o pH 1 . 0 b y u s i n g 1 N aqueous h y d r o c h l o r i c a c i d a n d c o n c e n t r a t e d t o a volume o f 4 0 ml. Four hund r e d m l of a c e t o n e was added and t h e p r e c i p i t a t e d m a t e r i a l was s e p a r a t e d by f i l t r a t i o n and d r i e d , y i e l d 4 . 0 g . T h i s m a t e r i a l was d i s s o l v e d i n 60 m l o f w a t e r and mixed w i t h 8 0 0 ml o f ace-
255 tone. The c r y s t a l l i n e V I h y d r o c h l o r i d e a n d a small amount o f i m p u r i t y was s e p a r a t e d by f i l t r a t i o n (420 mg). An a d d i t i o n a l 2 0 0 mg o f V I h y d r o c h l o r i d e and a small amount o f i m p u r i t y was o b t a i n e d from t h e f i l t r a t e . These p r e p a r a t i o n s were combined ( 6 2 0 mg) and d i s s o l v e d i n 1 8 m l of w a t e r . Acetone ( 1 8 0 ml) was added a n d c o l o r l e s s c r y s t a l s were f i l t e r e d o f f and d r i e d ( 3 6 0 mg). An a d d i t i o n a l 1 4 0 mg o f c r y s t a l s were o b t a i n e d from t h e filtrate. These p r e p a r a t i o n s ( 3 6 0 mg a n d 1 4 0 mg) were f o u n d by TLC t o c o n t a i n a s e c o n d b i o a c t i v e component i n a d d i t i o n t o V I . S e p a r a t i o n o f t h e s e compounds was a c h i e v e d by CCD. 6.2.2
Countercurrent d i s t r i b u t i o n the hydrochloride
-
cry,stalliza.tion of
The s t a r t i n g material (360 mg and 1 4 0 mg combined) was d i s s o l v e d i n t h e l o w e r p h a s e ( 8 0 m l ) o f 1 - b u t a n o l - w a t e r (1:l v/v). The s o l u t i o n was mixed w i t h e q u a l amounts o f u p p e r p h a s e and t h e s y s t e m was e q u i l i b r a t e d . The p h a s e s were added t o a 2 m l / p h a s e C C D a p p a r a t u s and t h e d i s t r i b u t i o n s t a r t e d a n d completed after 1000 transfers. Thin l a y e r chromatography showed The s o l u t i o n i n t h e s e t u b e s t h a t tubes 140-210 contained V I . was c o n c e n t r a t e d t o d r y n e s s and t h e r e s i d u e was d i s s o l v e d i n 1 2 m l of w a t e r . A d d i t i o n o f s u c c e s s i v e amounts of a c e t o n e (i.L. 7 0 m l , 3 0 m l , 1 0 0 m l ) gave c r y s t a l l i n e p r e p a r a t i o n s o f 4 4 mg, 2 3 6 mg, a n d 54 mg. These p r e p a r a t i o n s were combined a n d r e c r y s t a l l i z e d from 3 2 m l o f water and 3 9 2 m l o f a c e t o n e t o g i v e 2 7 0 mg of V I h y d r o c h l o r i d e c r y s t a l s . 7.
Lincomycin s u l f o x i d e and 1-deme thylthio-1-hydroxylincomycin
7.1 Introduction,
s t r u c t u r e s and i s o l a t i o n
E x t e n s i o n o f t h e nobmal f e r m e n t a t i o n of S . lincolnenAi4 from t h e u s u a l s i x d a y s t o t w e l v e d a y s r e s u l t e d i n t h e format i o n o f two a d d i t i o n a l a n t i b i o t i c s b e s i d e s l i n c o m y c i n and 4 ' d e p r o p y l 4 ' -e t h y l l i n comyci n ( I I )
-
.
These were i s o l a t e d by A r g o u d e l i s and Mason35 a n d shown t o be l i n c o m y c i n s u l f o x i d e ( V I I ) a n d l-demethylthio-l-hydroxylincomycin ( V I I I ) .
H OH '
OH
VIII Lincomycin, 11, V I I and VIII were e x t r a c t e d from f i l t e r e d b r o t h by l i q u i d i o n e x c h a n g e r s as d e s c r i b e d a b o v e . The two new compounds were s e p a r a t e d from l i n c o m y c i n by
256 CDCD were same tic
u s i n g 1 - b u t a n o l - w a t e r (1:l v / v ) . The two new a c t i v i t i e s s e p a r a t e d from e a c h o t h e r by c o n v e n t i o n a l CCD u s i n g t h e s o l v e n t s y s t e m and were i d e n t i f i e d by comparison t o a u t h e n samples p r e p a r e d c h e m i c a l l y from l i n c o m y c i n .
8. C e l e s t i c e t i n s
8.1 I n t r o d u c t i o n 8.1.1
Producing organisms
Hoeksema e t a t . 3 6 r e p o r t e d t h e i s o l a t i o n o f c e l e s t i c e t i n i n 1954 from c u l t u r e b r o t h s of Stncptomyccd c a e k A L b s p . nova NRRL 241837, Although c e l e s t i c e t i n was known t o p o s s e s s a c t i v i t y a g a i n s t gram p o s i t i v e o r g a n i s m s b o t h i n v i t n o a n d i n it was deemed l a c k i n g i n c l i n i c a l p o t e n t i a l and was n o t d e v e l o p e d past laboratory studies. 8.1.2
B r i e f chemical d e s c r i p t i o n
C e l e s t i c e t i n was i s o l a t e d as t h e f r e e b a s e , a n d t h e oxal a t e a n d s a l i c y l a t e s a l t s . A n a l y t i c a l d a t a on a l l forms a g r e e d The f r e e b a s e was w i t h an e m p i r i c a l f o r m u l a C24H36,40OgN2S. soluble in acidic or strongly basic solutions but insoluble i n t h e r a n g e pH 7 t o 10. The s a l t s were w a t e r s o l u b l e .
Electrometric t i t r a t i o n s i n w a t e r a n d i n 6 7 % d i m e t h y l formamide showed a b a s i c f u n c t i o n o f pKal 7.7 a n d an a c i d i c g r o u p o f pKa' 9 . 8 . O p t i c a l r o t a t i o n s C ~ ) ~ ~ ~ wof e r oex :a l a t e = t106.6' ( c - 0 . 5 , ( c - 0 . 5 , water); a n d o f f r e e b a s e = +126.6 (cm0.5, chloroform).
water18 o f s a l i c y l a t e = t 9 0 . 2
C r y s t a l l i n e c e l e s t i c e t i n h y d r o c h l o r i d e was c h a r a c t e r i z e d = 6 7 a t 305 nm; by
by UV maxima o f ,1% = 188 a t 2 4 0 nm a n d ,1% L L 1cm 1cm
an o p t i c a l r o t a t i o n of 8.1.3
= t96.7'
( c - 0 . 5 , water).
S t r u c t u r a l formula
The p a r t i a l s t r u c t u r e was p r e s e n t e d i n 1 9 5 6 3 8 a n d puba disl i s h e d i n 196439. The f i n a l s t r ~ c t u r e (1x1, ~ ~ * shows ~ ~ t i n c t i v e r e l a t i o n s h i p between l i n c o m y c i n a n d c e l e s t i c e t i n .
257
8.2 E x t r a c t i o n , 8.2.1
s e p a r a t i o n and p u r i f i c a t i o n
I s o l a t i o n of t h e f r e e b a s e
The p r o d u c t i o n o f c e l e s t i c e t i n was f o l l o w e d by t h e p a p e r d i s c method42 u s i n g BacieeuA d u b t i t i 4 as t h e t e s t organism and was c h a r a c t e r i z e d i n f e r m e n t a t i o n b e e r s by p a p e r chromatography (Figure 7 ) .
A s d e s c r i b e d by Hoeksema e t and DeBoet and Hoeksema was h a r v e s t e d ( 6 7 h r s ) ; pH 7.8 and f i l t e r e d . A 1 5 0 0 m l a l i q u o t was e x t r a c t e d w i t h two 5 0 0 m l p o r t i o n s o f methylene c h l o r i d e . The e x t r a c t was c o n c e n t r a t e d t o two m l under r e d u c e d p r e s s u r e and t h e n added t o t e n m l of S k e l l y s o l v e B. A floccul e n t p r e c i p i t a t e was o b t a i n e d . A f t e r washing w i t h f i f t y m l o f S k e l l y s o l v e B and d r y i n g i n v a c u o , 38 mg (43% y i e l d ) o f c e l e s t i c e t i n was o b t a i n e d . T h i s f r e e b a s e was g r e a t e r t h a n 9 0 % p u r e by UV measurements. 43, b e e r
8.2.2
I s o l a t i o n of c r y s t a l l i n e c e l e s t i c e t i n h y d r o c h l o r i d e
A methylene c h l o r i d e s o l u t i o n c o n t a i n i n g 7 5 0 mg of c e l e s t i c e t i n f r e e b a s e was t r e a t e d w i t h d r y hydrogen c h l o r i d e . On e v a p o r a t i o n , a gummy r e s i d u e was o b t a i n e d . A f t e r t r i t u r a t i o n of t h i s r e s i d u e w i t h anhydrous e t h e r , 4 3 5 mg of a w h i t e , microc r y s t a l l i n e powder was o b t a i n e d which was i d e n t i f i e d as c e l e s t i c e t i n hydrochloride.
The o x a l a t e and s a l i c y l a t e s a l t s were a l s o made from t h e extracted f r e e base. 9 . D e s a l i c e t i n and c e l e s t i c e t i n s B , C and D
9 . 1 Introduction Hinman e t showed t h a t c e l e s t i c e t i n i s c o n v e r t e d t o d e s a l i c e t i n ( X I by b a s i c h y d r o 1 s i s o f t h e s a l i c y o l y l g r o u p . I n 1 9 7 2 A r g o u d e l i s and BrodaskyX5 r e p o r t e d t h a t , i n a d d i t i o n t o c e l e s t i c e t i n and d e s a l i c e t i n , s e v e r a l new c e l e s t i c e t i n s were produced i n f e r m e n t a t i o n s of S . c a e & A t i A . The new c e l e s t i c e t i n s were named c e l e s t i c e t i n B , C and D , ( X I ) , ( X I I ) , and (XIII). 9.1.1 9.1.1.1
B r i e f chemical d e s c r i p t i o n
Desali ce t i n
C r y s t a l l i n e d e s a l i c e t i n hydrochloride has t h e formula C17H32N207S.HC1. P o t e n t i o m e t r i c t i t r a t i o n i n water showed t h e p r e s e n c e o f a b a g i c g r o u p , pK I t showed o p t i c a l r o t a 7.5. tion = + 1 5 0 ( c = 1,waterT. The f r e e b a s e h a s a m o l e c u l a r w e i g h t ~~t.1'408. I t showed n o maxima between 2 2 0 and 4 0 0 nm. 9.1.1.2
Celesticetin B
C r y s t a l l i n e c e l e s t i c e t i n B hydrochloride has t h e formula
258
Rf System
0.2
-
0.4
0.6
0.8
I I1 I11 IV
V VI
FIGURE 7 Paper chromatography of c e l e s t i c e t i n . The a n t i b i o t i c was d e t e c t e d by bioautography on BaciCCuQ d u b t i C i d seeded a g a r .
259
C21H38N208S.HC1. P o t e n t i o m e t r i c aqueous t i t r a t i o n s stowed i t t c Its o p t i c a l r o t a t i o n = + 1 4 6 (c=l, have a pKa' of 7 . 4 . water). C e l e s t i c e t i n B had a m o l e c u l a r weigh? = Mt 4 7 8 . It showed no maxima between 2 2 0 and 4 0 0 nm. 9.1.1.3
Celesticetin C
Crystalline c e l e s t i c e t i n C hydrochloride, C ~ L , H ~ ~ N ~ O ~ S . H C ~ showed t h e p r e s e n c e o f a b a s i c g r o u p . P o t e n t i o m e t r i c aqueous I t s o p t i c a l r o t a t i o n Calk5 =+ t i t r a t i o n s showed a pKa' o f 7.6. +123' (c.1, water). C e l e s t i c e t i n C had a m o l e c u l a r w e i g h t = M 527. I t showed U V maxima a t 2 4 3 nm ( a = 1 1 . 6 ) and 328 nm (a=6.3) i n water. 9.1.1.4
Celesticetin D
S i n c e o n l y small amounts of c e l e s t i c e t i n D were i s o l a t e d i t s , p r o p e r t i e s were n o t d e t e r m i n e d . I t had a m o l e c u l a r w e i g h t = M 450 ( c a l c . f o r ClgH23N208S, 4 5 0 ) . 9.1.2
S t r u c t u r a l formulae
The s t r u c t u r a l f o r m u l a e o f d e s a l i c e t i n a n d c e l e s t i e e t i n s B , C and D are g i v e n below.
CH2CH20R
.
.
OH
Desalicetin ( X I
R=H
Celesticetin B (XI)
R-C-CH,
.CH 3
*O
CH3
Celesticetin C (XII) R=-C NH2
Celesticetin 0 (XIII) R=-C\;-CH3
9.2 E x t r a c t i o n , 9.2.1
0 s e p a r a t i o n and p u r i f i c a t i o n
I s o l a t i o n of c r u d e m i x t u r e
These a n t i b i o t i c s were i n i t i a l l y d e t e c t e d i n mother l i q u o r s , a f t e r c e l e s t i c e t i n s a l i c y l a t e c r y s t a l l i z a t i o n , by TLC on s i l i c a g e l G a n d b i o a u t o g r a p h y on S. L u t e a . T a b l e 1 shows t h e
260
TABLE 1 Rf Values o f X, X I , X I I , X I 1 1 and Celesticetin
0.70
R f s i n t h e t h r e e s o l v e n t s y s t e m s u s e d : System A. m e t h y l e t h y l k e t o n e - a c e t o n e - w a t e r ( 1 8 6 : 5 2 : 2 0 v / v ) ; System B . m e t h y l propyl k e t o n e - m e t h y l e t h y l k e t o n e - m e t h a n o l - w a t e r ( 2 : 2 : 1 : 1 v / v ) ; System C. chloroform-methanol ( 6 : l v/v). The a n t i b i o t i c s were i s o l a t e d by t h e f o l l o w i n g p r o c e d u r e S. c a e e e d t i d was a d j u s t e d t o pH 2 . 5 - 3 w i t h c o n c e n t r a t e d s u l f u r i c a c i d and f i l t e r e d u s i n g f i l t e r a i d . The f i l t e r e d b e e r was a d j u s t e d t o pH 7 . 8 - 8 w i t h 5 0 % sodium h y d r o x i d e a n d e x t r a c t e d w i t h 0 . 2 volume o f m e t h y l e n e c h l o r i d e . 46.
A 1 0 0 k l f e r m e n t a t i o n of
The m e t h y l e n e c h l o r i d e was c o n c e n t r a t e d t o 2 0 0 a a n d e x The aqueous l a y e r was t r a c t e d w i t h 1 5 0 a o f water a t pH 4 . 5 . reextracted with 100 o f m e t h y l e n e c h l o r i d e a t pH 7 . 0 . The m e t h y l e n e c h l o r i d e e x t r a c t was d e c o l o r i z e d w i t h c a r b o n , t h e c a r b o n was removed, and t h e m e t h y l e n e c h l o r i d e was c o n c e n t r a t e d by d i s t i l l a t i o n . Twenty a o f e t h y l a c e t a t e was a d d e d t o 10 and t h e d i s t i l l a t i o n c o n t i n u e d u n t i l a l l o f t h e m e t h y l e n e c h l o r i d e was removed. A c a l c u l a t e d amount o f s a l i c y l i c a c i d d i s s o l v e d i n h o t e t h y l a c e t a t e was added s l o w l y w i t h s t i r r i n g t Q t h e e t h y l acet a t e c o n c e n t r a t e . The s o l u t i o n was a l l o w e d t o cool t o 2OC and t h e n h e l d o v e r n i g h t a t -2OOC.
The c r y s t a l l i n e c e l e s t i c e t i n s a l i c y l a t e was removed by filtration. The combined e t h y l a c e t a t e m o t h e r l i q u o r s were c o n c e n t r a t e d t o d r y n e s s and s e r v e d as s t a r t i n g m a t e r i a l . 9.2.2
I s o l a t i o n o f d e s a l i c e t i n (XI
One kg o f t h e m o t h e r l i q u o r c o n c e n t r a t e was d i s t r i b u t e d between 2 a of t h e u p p e r p h a s e and 2 a o f t h e lower p h a s e of e q u a l p a r t s o f 1 - b u t a n o l a n d water a n d 2 0 0 m l of 6 N a q u e o u s h y d r o c h l o r i c a c i d . The two p h a s e s were s e p a r a t e d . Each p h a s e was t h e n r e e x t r a c t e d w i t h 2 a o f t h e c o r r e s p o n d i n g f r e s h u p p e r and lower phase. The combined l o w e r p h a s e s r e s u l t i n g from t h e s e c o n d d i s t r i b u t i o n were f r e e z e d r i e d t o g i v e 2 6 9 g o f material. Ten transfers
8e tofw e et hn i 1s - bmaterial was d i s t r i b u t e d u t a n o l - w a t e r (1:l v / v ) .
i n a CCD f o r 1 0 0 0 The d i s t r i b u t i o n
261
was a n a l y z e d by s o l i d s d e t e r m i n a t i o n , b i o a c t i v i t y (wd. S. L u t e a ) and UV s p e c t r a . The b i o a c t i v e material i n t u b e s 80-120, h a v i n g n o maximum i n t h e U V , were p o o l e d and c o n c e n t r a t e d t o d r y n e s s . The r e s i due was d i s s o l v e d i n m e t h a n o l and p r e c i p i t a t e d w i t h e t h e r . A f t e r d r y i n g , t h i s material ( 1 . 6 8 g ) was shown t o be, t h e h y d r o c h l o r i d e o f X by comparison w i t h an a u t h e n t i c sample p r e p a r e d by c h e m i c a l d e g r a d a t i o n of c e l e s t i c e t i n . 9.2.3 9.2.3.1
I s o l a t i o n of c e l e s t i c e t i n B ( X I ) Counter double c u r r e n t d i s t r i b u t i o n
F o r t y grams o f t h e m o t h e r l i q u o r c o n c e n t r a t e was d i s s o l v e d i n 1 0 0 m l o f u p p e r p h a s e and 1 0 m l o f l o w e r p h a s e . The pH o f t h e mixed p h a s e s was a d j u s t e d t o 4.0 w i t h h y d r o c h l o r i c a c i d and p l a c e d i n t h e 4 c e n t e r t u b e s of a 2 5 m l / p h a s e 1 0 0 t u b e CDCD a p p a r a t u s . The d i s t r i b u t i o n was r u n f o r 50 t r a n s f e r s w i t h o u t c o l l e c t i n g f r a c t i o n s , t h e n an a d d i t i o n a l 5 0 t r a n s f e r s c o l l e c t i n g b o t h u p p e r a n d lower p h a s e s .
Pools Two a d d i t i o n a l i d e n t i c a l d i s t r i b u t i o n s were r u n . from a l l t h r e e d i s t r i b u t i o n s of t u b e s 12-50 i n t h e l o w e r p h a s e c o l l e c t o r were combined a n d c o n c e n t r a t e d t o d r y n e s s t o g i v e 6 . 6 g of m a t e r i a l r i c h i n X I . 9.2.3.2
Countercurrent d i s t r i b u t i o n
T h i s material was t h e n r e d i s s o l v e d i n b o t h p h a s e s o f t h e 1 - b u t a n o l - w a t e r s y s t e m and d i s t r i b u t e d i n . a c o n v e n t i o n a l count e r c u r r e n t d i s t r i b u t i o n apparatus f o r 800 transfers. Fractions 110-150 were f o u n d by TLC t o c o n t a i n X I . They were p o o l e d a n d c o n c e n t r a t e d t o d r y n e s s , a f f o r d i n g 2.75 g o f m i c r o - c r y s t a l l i n e X I hydrochloride. 9.2.4
I s o l a t i o n of c e l e s t i c e t i n C ( X I I )
The p r o c e d u r e d e s c r i b e d f o r t h e i s o l a t i o n of d e s a l i c e t i n A f t e r 1000 t r a n s f e r s f r a c t i o n s 220-270 h a v i n g maxima a t 242 and 325 nm, were p o o l e d and e v a p o r a t e d t o g i v e 1.36 g of material. TLC showed t h i s t o be m o s t l y X I I .
( X I was u s e d .
A column was p r e p a r e d from 3 0 0 g o f s i l i c a g e l (Merck) packed i n c h l o r o f o r m - m e t h a n o l (6:l v / v ) . The 1.36 g o f c r u d e X I 1 was d i s s o l v e d i n 20 m l o f t h i s s o l v e n t , mixed w i t h 2 0 g of s i l i c a g e l and t h e mixture evaporated t o dryness. I t was p l a c e d on t o p o f t h e column which was t h e n d e v e l o p e d w i t h t h e c h l o r o form-methanol s y s t e m . F r a c t i o n s o f 2 0 m l were c o l l e c t e d and a n a l y z e d by b i o a c t i v i t y ( v o . S. L u t e a ) a n d TLC. F r a c t i o n s 35 t o 65 were f o u n d t o c o n t a i n X I I . They were combined a n d c o n c e n t r a t e d t o d r y n e s s . T h i s r e s i d u e was r e d i s s o l v e d i n 1 0 m l o f m e t h a n o l . E t h e r was added f o l l o w e d by 1 N m e t h a n o l i c hydrogen c h l o r i d e p r e c i p i t a t i n g X I 1 as t h e m i c r o c r y s t a l l i n e hydrochloride.
262
9.2.5
I s o l a t i o n o f c e l e s t i c e t i n D (XI111
One kg o f t h e m o t h e r l i q u o r c o n c e n t r a t e was d i s t r i b u t e d as d e s c r i b e d i n t h e i s o l a t i o n o f d e s a l i c e t i n ( X I . I n t h i s case, however, t h e u p p e r p h a s e s a f t e r t h e s e c o n d e x t r a c t i o n were combined, and concentrated t o an aqueous s o l u t i o n and f r e e z e d r i e d . T h i s f r e e z e d r i e d m a t e r i a l was d i s t r i b u t e d between 2 a e a c h of u p p e r and l o w e r p h a s e s o f c y c l o h e x a n e - e t h y l a c e t a t e 95% e t h a n o l - w a t e r (1:l:l:l v / v ) . The l o w e r p h a s e was t h e n ree x t r a c t e d t h r e e times w i t h 2 a p o r t i o n s o f u p p e r p h a s e . The u p p e r p h a s e s were combined and e x t r a c t e d once w i t h 2 a o f l o w e r phase. The two lower p h a s e s were combined a n d c o n c e n t r a t e d t o d r y n e s s t o g i v e 320 g which by TLC was r i c h i n X I I I . 9.2.5.1
Counter double c u r r e n t d i s t r i b u t i o n
The 320 g o f material was d i s s o l v e d i n 1250 m l e a c h o f t h e u p p e r a n d l o w e r p h a s e s o f t h e s y s t e m 1 - b u t a n o l - w a t e r (1:l v / v ) . 250 m l o f e a c h p h a s e was i n t r o d u c e d i n t u b e s 25-35 ( l o w e r p h a s e s i d e of a CDCD a p p a r a t u s ( 2 5 m l / p h a s e ) . The d i s t r i b u t i o n ( u s i n g t h e 1 - b u t a n o l - w a t e r s o l v e n t s y s t e m ) was r u n a c c o r d i n g t o the following schedule. 1) S i x t e e n t r a n s f e r s were r u n w i t h o u t c o l l e c t i n g f r a c t i o n s . 2 ) F i f t y - n i n e t r a n s f e r s were r u n c o l l e c t i n g upper p h a s e o n l y . 3 ) F i n a l l y , f i f t y t r a n s f e r s were r u n c o l l e c t i n g both phases. The d i s t r i b u t i o n when a n a l y z e d by s o l i d s ' d e t e r m i n a t i o n , b i o a c t i v i t y a n d TLC, showed X I 1 1 t o b e i n t h e lower p h a s e c o l l e c t o r . F r a c t i o n s 5-15 from t h i s c o l l e c t o r were p o o l e d a n d conc e n t r a t e d t o dryness. T h i s whole p r o c e d u r e was r e p e a t e d f o u r a d d i t i o n a l times. The f i n a l p o o l a f f o r d e d 4 g o f material. 9.2.5.2
Countercurrent d i s t r i b u t i o n
The 4 g was d i s t r i b u t e d between t h e 1 - b u t a n o l - w a t e r s y s t e m i n a c o n v e n t i o n a l CCD a p p a r a t u s f o r 2 0 0 0 t r a n s f e r s . Tubes 95-130 were p o o l e d , c o n c e n t r a t e d t o d r y n e s s , r e d i s s o l v e d i n a b s o l u t e m e t h a n o l and p r e c i p i t a t e d as s e m i c r y s t a l l i n e X I 1 1 hydroc h l o r i d e ( 7 5 mg) w i t h t h e a d d i t i o n o f e t h e r . 10. Desalicetin 2"-(4-aminosalicylate)
10.1 Introduction I n 1974 A r g o u d e l i s a n d C o a t s 4 7 showed t h a t r a d i o a c t i v e s a l i c y l i c a c i d a d d e d t o f e r m e n t a t i o n s of S. CaQeQAtiA was i n c o r porated i n t o c e l e s t i e e t i n without randomization. S i n c e the e n z y m a t i c s y s t e m r e s p o n s i b l e f o r e s t e r format i o n a t t h e C-2" h y d r o x y l o f d e s a l i c e t i n was n o t s p e c i f i c f o r s a l i c y l i c a c i d (as shown b y t h e i s o l a t i o n o f X I , X I 1 a n 9 X I I I ) , A r g o u d e l i s e t a l . 4 8 added d i f f e r e n t a c i d s t o f e r m e n t a t i o n s o f
263
S. c a e k k ~ t i ~ . Amongst t h e s e a c i d s was 4 - a m i n o s a l i c y l i c a c i d which i n duced t h e f o r m a t i o n of a new a n t i b i o t i c d e s a l i c e t i n 2"-(4-aminosalicylate) (XIV). 10.1.1
B r i e f chemical d e s c r i p t i o n
C r y s t a l l i n e d e s a l i c e i i n 2"-( 4-arninosalicylate) hydrochlor i d e showed a n +111 (c=l, water). The UV s p e c t r u m showed maxima a t 288, 288 and 306 nm i n water. I t h a s a molec u l a r w e i g h t = Mt 543.2 (Calc. f o r C24H37N309S, 5 4 3 . 2 ) . 10.1.2
S t r u c t u r a l formula
Desalice t i n 2"- ( 4 - a m i n o s a l i c y l a t e ) formula X I V .
Cr+
has the s t r u c t u r a l
CH 3 CH3OCH I
-I
CONH CH I
XIV 10.2 Extraction, -
10.2.1
s e p a r a t i o n and p u r i f i c a t i o n
Isolation
X I V was d e t e c t e d by TLC on Eastman 6061 s h e e t s u s i n g t h e s o l v e n t s y s t e m c h l o r o f o r m - m e t h a n o l (6:l v / v ) . B i o a u t o g r a m s on S. ku&a showed i t t o have a n Rf ( 0 . 7 ) s l i g h t l y s l o w e r t h a n t h e coproduced c e l e s t i c e t i n . F e r m e n t a t i o n b r o t h ( c a 1 0 a ) , c o n t a i n i n g X I V , was f i l The f i l t e r c a k e was t e r e d w i t h t h e a i d of d i a t o m a c e o u s e a r t h . washed w i t h 2 a o f water a n d t h e a q u e o u s wash was combined w i t h t h e c l e a r f i l t r a t e . T h i s s o l u t i o n was p a s s e d o v e r a column cont a i n i n g 5 0 0 m l of A m b e r l i t e XAD-2 packed i n water. The s p e n t The column was washed b e e r was b i o i n a c t i v e and was d i s c a r d e d . w i t h 2 P o f water and t h e n e l u t e d w i t h 3 II o f 9 5 % a q u e o u s m e t h a n o l . The m e t h a n o l i c e l u a t e was c o n c e n t r a t e d t o d r y n e s s t o g i v e 1 1 . 7 g of material c o n t a i n i n g a l l t h e b i o a c t i v i t i e s p r e s e n t in the fermentation broth. 10.2.1.1
Counter double c u r r e n t d i s t r i b u t i o n
The m a t e r i a l ( 1 1 . 7 g ) o b t a i n e d by A m b e r l i t e XAD-2 c h r o matography was d i s s o l v e d i n 1 0 0 m l of e a c h p h a s e o f e q u a l v o l umes of 1 - b u t a n o l - w a t e r . The s o l u t i o n was a d j u s t e d t o pH 3 . 5 u s i n g 2 N a q u e o u s h y d r o c h l o r i c a c i d and t h e n added i n f o u r cent e r t u b e s of a CDCD a p p a r a t u s ( 1 0 0 t u b e s , 2 5 m l / p h a s e ) . The
264
d i s t r i b u t i o n was a n a l y z e d a f t e r 178 t r a n s f e r s by TLC and b i o a c t i v i t y d e t e r m i n a t i o n u s i n g S. lutes a s t h e a s s a y organism. Tubes c o n t a i n i n g X I V a l o n e o r i n m i x t u r e w i t h c e l e s t i c e t i n were combined and t h e s o l u t i o n was c o n c e n t r a t e d t o d r y n e s s t o g i v e 1 . 5 g of h i g h l y b i o a c t i v e material c o n t a i n i n g b o t h X I V and c e l e s t i c e t i n hydrochlorides. 10.2.1.2
S e p a r a t i o n of X I V from c e l e s t i c e t i n
A column was p r e p a r e d from 500 g of s i l i c a g e l ( M e r c k , D a r m s t a d t , 7734) packed i n chloroform-methanol (6:l v / v ) . The m i x t u r e o f t h e a n t i b i o t i c s o b t a i n e d by CDCD, ca. 1 . 5 g , was d i s s o l v e d i n 5 0 m l of t h i s s o l v e n t s y s t e m , mixed w i t h 30 g o f s i l i c a g e l and t h e m i x t u r e was c o n c e n t r a t e d t o d r y n e s s . The powder o b t a i n e d was added t o t h e t o p of t h e column and t h e c o l umn was t h e n e l u t e d w i t h t h e chloroform-methanol s o l v e n t s y s t e m . B i o a c t i v e f r a c t i o n s were a n a l y z e d by TLC. C e l e s t i c e t i n was e l u t e d f i r s t , f o l l o w e d by X I V . F r a c t i o n s c o n t a i n i n g X I V were combined and c o n c e n t r a t e d t o d r y n e s s . The r e s i d u e was d i s s o l v e d i n 10 m l o f m e t h a n o l , 10 m l of c h l o r o f o r m and 500 m l of e t h e r . M e t h a n o l i c hydrogen c h l o r i d e (1 N , 5 m l ) added t o t h e s o l u t i o n r e s u l t e d i n t h e p r e c i p i t a t i o n of c o l o r l e s s X I V h y d r o c h l o r i d e , which was i s o l a t e d by f i l t r a t i o n ; y i e l d 2 5 0 mg.
11. 7 - 0 -Deme t h y l c e l e s ti c e t i n 11.1 I n t r o d u c t i o n -
I n 1 9 7 2 Argoudelis e t r e p o r t e d on t h e i s o l a t i o n of 7 - 0 d e m e t h y l c e l e s t i c e t i n XV from a m u t a n t of S. C a e k A t i A d e s i g nated s t r a i n 22227a. 11.1.1 B r i e f c h e m i c a l d e s c r i p t i o n
7-O-Demethylce&esticetin h y d r o c h l o r i d e had an o p t i c a l = + 1 1 5 ( c = 0.85, w a t e r ) . I t s s o l u b i l i t y c h a r rotation a c t e r i s t i c s @ere similar t o t h o s e o f c e l e s t i c e t i n . P o t e n t i o metric t i t r a t i o n i n water showed a b a s i c g r o u p , pKa' 7 . 5 , and a weak a c i d i c g r o u p , pKa' 9 . 6 . Mass s p e c t r a l d a t a a g r e e d w i t h t h e f o r m u l a C23H34N209S (MW 514) f o r t h e f r e e b a s e . The UV s p e c t r u m i n methanol showed maxima a t 238 ( a = 1 8 ) and 304 ( a z 7 . 3 )
nm
.
11.1.2
S t r u c t u r a l formula
7-0 -Deme t h y l c e l e s t i c e t i n h a s t h e s t r u c t u r e XV.
xv
265
11.2 Extraction, 11.2.1
s e p a r a t i o n and p u r i f i c a t i o n
Isolation
S. cae&4t& s t r a i n 2 2 2 2 7 a produced o n l y two d e t e c t a b l e a n t i b i o t i c s XV and what was t h o u g h t t o be 7 - 0 - d e m e t h y l d e s a l i c e t i n . No c e l e s t i c e t i n was found. XV was d e t e c t e d and f o l l o w e d by b i o a c t i v i t y v 4 . S. l u t e a and by TLC on s i l i c a g e l G i n t h e chloroform-methanol ( 6 : l v / v ) s y s t e m where i t had an Rf o f 0 . 4 0 ( c e l e s t i c e t i n had an R f o f 0.70 i n t h i s s y s t e m ) .
,
11.2.1.1
F i l t r a t i o n and a d s o r p t i o n on A m b e r l i t e XAD-2
F e r m e n t a t i o n b r o t h ( c a . 1 2 . 5 & ) was f i l t e r e d u s i n g f i l t e r a i d . The f i l t e r cake was washed w i t h 2 l i t e r s o f water and t h e aqueous wash was combined w i t h t h e c l e a r f i l t r a t e . T h i s s o l u t i o n was p a s s e d o v e r a column p r e p a r e d from 500 ml o f A m b e r l i t e XAD-2 packed i n water. The s p e n t b e e r was found b i o i n a c t i v e and was d i s c a r d e d . The column was washed w i t h water and t h e n The methane l u t e d w i t h 3 l i t e r s o f methanol-water ( 9 5 : 5 v / v ) . o l i c e l u a t e was c o n c e n t r a t e d t o d r y n e s s t o g i v e 3.3 g o f materi a l c o n t a i n i n g e s s e n t i a l l y a l l of t h e b i o a c t i v i t y p r e s e n t i n the fermentation broth. 11.2.1.2
S i l i c a g e l chromatography. hydrochloride
I s o l a t i o n of XV
A column was p r e p a r e d from 6 0 0 g o f s i l i c a g e l ( M e r c k , D a r m s t a d t , 7734) packed i n chloroform-methanol (6:l v / v ) . Crude XV (ca. 3.0 g > , was d i s s o l v e d i n 1 0 0 m l o f methanol and 1 0 0 m l of t h e s o l v e n t system. T h i s s o l u t i o n was mixed w i t h 5 0 g of s i l i c a g e l , c o n c e n t r a t e d t o d r y n e s s and added t o t h e t o p of t h e column which was t h e n e l u t e d w i t h t h e chloroform-methanol s o l v e n t system. F r a c t i o n s ( 2 0 m l e a c h ) were a n a l y z e d f o r b i o a c t i v i t y and by UV s p e c t r a and TLC. F r a c t i o n s 98-149 c o n t a i n i n g XV were combined and t h e s o l u t i o n was c o n c e n t r a t e d t o d r y n e s s . The r e s i d u e was d i s s o l v e d i n a m i x t u r e of 1 0 m l of m e t h a n o l and 5 ml o f 1 N m e t h a n o l i c hydrogen c h l o r i d e and 500 m l o f e t h e r was added. The p r e c i p i t a t e d , c o l o r l e s s X V h y d r o c h l o r i d e was i s o l a t e d by f i l t r a t i o n and d r i e d ; y i e l d 3 3 0 mg. 1 2 . l'-Demethyl-7-0-demethylcelesticetin celes t i c e t i n
and 1 ' - d e m e t h y l -
1 2 . 1 Introduction An S. c a e & a t h mutant s t r a i n 22218a was shown t o p r o duce two new a n t i b i o t i c s s 0 , 1 ' - d e m e t h y l c e l e s t i c e t i n ( X V I ) and l'-demethyl-7-0-demethylcelesticetin ( X V I I ) .
12.1.1 12.1.1.1
Brief c h e m i c a l d e s c r i p t i o n
l'-Demethyl-7-O-demethylcelesticetin
C r y s t a l l i n e l'-demethyl-7-0-demethylcelesticetin hydroc h l o g i d e , C22H32N209S.HCl.H20 had an o p t i c a l r o t a t i o n { a } 2 s = + 1 2 2 (c=l, w a t e r ) . The UV s p e c t r u m was i d e n t i c a l t o t h a ? of
266 c e l e s t i c e t i n showing maxima a t 238 ( a = 1 7 . 6 6 ) a n d 303 ( a = 7 . 2 0 ) nm i n m e t h a n o l . 1 2 . l .1 . 2
1 ' -Deme t h y l c e l e s t i ce t i n
1 ' - D e m e t h y l c e l e s t i c e t i n h y d r o c k l o r i d e , C23H3~N209S.HC1 h a d an o p t i c a l r o t a t i o n =+112.5 (c=l, water). The UV s p e c t r u m showed maxima at 238 ( a z 1 5 . 5 ) a n d 3 0 4 ( a c 7 . 2 ) nm i n m e t h a n o l . 12.1.2
S t r u c t u r a l formulae
1'- D e m e t h y l c e l e s t i c e t i n and 1' -deme t h y l - 7-0-demethylc e l e s t i c e t i n h a v e s t r u c t u r e s X V I and X V I I r e s p e c t i v e l y .
H O r " ,
OH
XVI XVII 1 2 . 2 Extraction, separation andgurification 12.2.1
Lsolation
X V I and X V I I a l t h o u g h d e t e c t e d a n d d i f f e r e n t i a t e d f r o m c e l e s t i c e t i n by t h e i r TLC b e h a v i o r were n o t d i s t i n g u i s h a b l e from e a c h o t h e r by t h i s means. The two were d i s t i n g u i s h e d by t h e i r d i f f e r e n t b e h a v i o r i n t h e i s o l a t i o n p r o c e s s a n d were c h a r a c t e r i z e d b t h e i r GC-MS s p e c t r a as d e s c r i b e d by Brodasky and A r g o u d e l i s s Y .
12.2.1.1
F i l t r a t i o n and a d s o r p t i o n on A m b e r l i t e XAD-2
Fermentation broth ( 4 , 4 0 0 1 was f i l t e r e d u s i n g f i l t e r aid. The f i l t e r e d b e e r was p a s s e d o v e r a column p r e p a r e d from 500 l i t e r s of A m b e r l i t e XAD-2 packed i n water. The s p e n t b e e r was b i o - i n a c t i v e and was d i s c a r d e d . The column was washed w i t h 7 5 R of water a n d t h e n w i t h 1 , 1 0 0 of 9 5 % aqueous methanol. The m e t h a n o l i c e l u a t e was c o n c e n t r a t e d t o d r y n e s s t o g i v e 1 , 2 7 5 kg of material c o n t a i n i n g p r a c t i c a l l y a l l o f t h e b i o a c t i v i t i e s present in the fermentation broth.
12.2.1.2
P u r i f i c a t i o n by c o u n t e r d o u b l e c u r r e n t distribution. I s o l a t i o n of X V I I .
F i f t y grams o f t h e m a t e r i a l o b t a i n e d as d e s c r i b e d above was d i s s o l v e d i n 2 0 0 m l of e a c h p h a s e o f e q u a l volumes of 1butanol-water. The s o l u t i o n was a d j u s t e d t o pH 3.5 u s i n g 2 N aqueous h y d r o c h l o r i c a c i d . T h i s s o l u t i o n was t h e n added i n
267
e i g h t c e n t e r t u b e s of a CDCD a p p a r a t u s ( 1 0 0 t u b e s , 2 5 m l p e r p h a s e ) . The d i s t r i b u t i o n was a n a l y z e d a f t e r 1 1 2 t r a n s f e r s by d e t e r m i n a t i o n of b i o a c t i v i t y of s e l e c t e d f r a c t i o n s a g a i n s t S. e u t e a and by TLC. F r a c t i o n s c o n t a i n i n g b i o a c t i v e materials were combined and c o n c e n t r a t e d t o d r y n e s s t o y i e l d 2 . 1 3 g of a mixt u r e o f X V I and X V I I h y d r o c h l o r i d e s . F o l l o w i n g t h e above p r o c e d u r e f i v e a d d i t i o n a l CDCD r u n s were c a r r i e d o u t e a c h time u s i n g 5 0 g of t h e same s t a r t i n g mat e r i a l . The p u r i f i e d m i x t u r e s o f t h e a n t i b i o t i c s o b t a i n e d i n t h e s e d i s t r i b u t i o n s were t h e n combined a n d t r i t u r a t e d w i t h 1 5 0 m l o f a b s o l u t e m e t h a n o l . C r y s t a l l i n e material i n s o l u b l e i n m e t h a n o l was s e p a r a t e d by f i l t r a t i o n . R e c r y s t a l l i z a t i o n from w a t e r - a c e t o n e y i e l d e d 7 0 0 mg of c o l o r l e s s n e e d l e s i d e n t i f i e d as X V I I hydrochloride. The f i l t r a t e from t h e t r i t u r a t i o n w i t h m e t h a n o l was mixed w i t h 2 l i t e r s o f e t h e r . The r e s u l t i n g p r e c i p i t a t e ( 1 2 . 7 g ) c o n t a i n e d b o t h X V I a n d X V I I as t h e i r h y d r o c h l o r i d e s a l t s . S e p a r a t i o n o f t h e two a n t i b i o t i c s was o b t a i n e d by CCD as d e s c r i b e d below. 12.2.1.3
S e p a r a t i o n o f X V I from X V I I . d i s t r i bu t i on
Countercurrent
The s o l v e n t s y s t e m u s e d c o n s i s t e d o f e q u a l volumes of 1-butanol-water. The s t a r t i n g m a t e r i a l , 1 2 . 7 g , o b t a i n e d a s d e s c r i b e d a b o v e , was d i s s o l v e d i n 50 m l of e a c h p h a s e and a d d e d i n 5 t u b e s of a CCD (500 t u b e s , 1 0 m l / p h a s e ) . The d i s t r i b u t i o n was a n a l y z e d a f t e r 1 , 5 0 0 t r a n s f e r s by t e s t i n g f o r a n t i b i o t i c a c t i v i t y a n d by TLC. F r a c t i o n s 280-330 y i e l d e d 2 . 2 g of X V I I by c o n c e n t r a t i o n t o d r y n e s s and c r y s t a l l i z a t i o n from m e t h a n o l . F r a c t i o n s 3 4 0 - 4 0 0 c o n t a i n e d X V I , small amounts o f X V I I and b i o i n a c t i v e m a t e r i a l ( s ) . These f r a c t i o n s were t h e n combined and t h e s o l u t i o n was c o n c e n t r a t e d t o d r y n e s s ; y i e l d 1.5 g . T h i s material was f u r t h e r p u r i f i e d by s i l i c a g e l c h r o m a t o g r a p h y . 12.2.1.4
I s o l a t i o n of X V I h y d r o c h l o r i d e . chromatography
Silica gel
A column was p r e p a r e d from 2 5 0 g o f s i l i c a g e l (Merck, D a r m s t a d t , 7734) p a c k e d i n c h l o r o f o r m - m e t h a n o l (6:l v / v ) . The s t a r t i n g m a t e r i a l , 1 . 3 g , o b t a i n e d by t h e c o u n t e r c u r r e n t d i s t r i b u t i o n d i s c u s s e d a b o v e , was d i s s o l v e d i n 20 m l o f t h e s o l v e n t s y s t e m , mixed w i t h 2 0 g of s i l i c a g e l , and t h e m i x t u r e c o n c e n t r a t e d t o d r y n e s s . T h i s powder was a d d e d t o t h e t o p of t h e column and t h e column was e l u t e d w i t h t h e above s o l v e n t s y s t e m . F r a c t i o n s ( 2 0 ml e a c h ) were a n a l y z e d by UV a n d b i o a c t i v i t y determinations. Fractions 160-240 , containing XVI , were combined and t h e s o l u t i o n was c o n c e n t r a t e d t o d r y n e s s . The r e s i d u e was d i s s o l v e d i n 1 0 m l o f 1 N m e t h a n o l i c h y d r o g e n c h l o r i d e a n d t h i s s o l u t i o n was mixed w i t h e t h e r . X V I h y d r o c h l o r i d e , which p r e c i p i t a t e d as a c o l o r l e s s amorphous m a t e r i a l , was i s o l a t e d by f i l t r a t i o n and d r i e d ; y i e l d 1 3 0 mg. 1 3 . O t h e r l i n c o s a m i n i d e s and c e l e s t o s a m i n i d e s
1 3 . 1 I n t r o d u c t i o n and s t r u c t u r e s
268 A r g o u d e l i s and BrodaskyS2 d e s c r i b e d t h e s e p a r a t i o n and i d e n t i f i c a t i o n of f o u r p r e v i o u s l y unknown a n t i b i o t i c s produced d u r i n g t h e f e r m e n t a t i o n o f S . C a e t e A t i A . They were t h e d e s a l i ce t in-2 "-0-bu t y r a t e ( X V I I I ) ; d e s a l i c et in-2 'I-0-pen t a n o a t e ( X I X ) ; 7-'O-deme t h y l d e s a l i c e t i n - 2 'I-0-bu t y r a t e (XX) and 7-0-deme t h y l d e s a l i c e t i n - 2 "-0-an t h r a n i l a t e ( X X I 1.
s
XVII I R=CH3 R ' P - C - C H ~ C H ~ C H ~
13.2 E x t r a c t i o n , 13.2.1
XX
R=H
8
R1=-C-C3H7
s e p a r a t i o n and p u r i f i c a t i o n
I s o l a t i o n and c h a r a c t e r i z a t i o n
The b i o a c t i v e materials were e x t r a c t e d from t h e c l e a r f i l t r a t e w i t h m e t h y l e n e c h l o r i d e a t a l k a l i n e pH ( C a . 8.0). C e l e s t i c e t i n was removed by c r y s t a l l i z a t i o n as t h e s a l i c y l a t e s a l t and t h e mother l i q u o r s were c o n c e n t r a t e d t o a n o i l y r e s i due. Examination o f t h i s c r u d e material by TLC r e v e a l e d t h e p r e s e n c e o f s e v e r a l compounds which i n h i b i t e d t h e growth of Salccina t u t e a . D i s t r i b u t i o n of t h e o i l y r e s i d u e between 1b u t a n o l - w a t e r (1:l v / v , a d j u s t e d w i t h aqueous H C 1 t o pH 2 . 0 1 , f o l l o w e d by c o n c e n t r a t i o n of t h e aqueous p h a s e t o d r y n e s s , y i e l d e d material c o n t a i n i n g several a n t i b i o t i c s . C o u n t e r c u r r e n t d i s t r i b u t i o n { l - b u t a n o l - w a t e r (1:l v / v ) ) s e p a r a t e d c e l e s t i c e t i n (1x1, d e s a l i c e t i n ( X ) and c e l e s t i c e t i n B ( X I ) from a m i x t u r e of c e l e s t i c e t i n C (XI11 and s e v e r a l unknown a n t i b i o t i c s . S i l i c a g e l chromatography of t h e m i x t u r e u s i n g c h l o r o form-methanol (6:l v / v ) removed t h e c e l e s t i c e t i n C (XI11 from a m i x t u r e of c l o s e l y r e l a t e d a n t i b i o t i c s . These a n t i b i o t i c s were t r a n s f o r m e d t o t h e i r t r i m e t h y l s i l y l (TMS) e t h e r d e r i v a t i v e s and t h i s m i x t u r e was a n a I y z e d by GC-MS. The compounds were c h a r a c t e r i z e d by a n a l y s i s of t h e i r f r a g m e n t a t i o n p a t t e r n s i n r e l a t i o n s h i p t o p a t t e r n s of p r e v i o u s l y known l i n e a s a m i n i d e s and c e l e s t o s a m i n i d e s and were c h a r a c t e r i z e d as X V I I I , X I X , XX and X X I .
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J . M a s o n , J. A n t i b i o t i c
34. A. D. A r g o u d e l i s a n d D. J . Mason, U.S. December 1 0 , 1 9 6 8 .
P a t e n t 3,415,811;
35. A. D. A r g o u d e l i s a n d D. J. M a s o n , J . A n t i b i o t i c s 22 ( 1 9 6 9 ) 289-291. 3 6 . H. H o e k s e m a , G. F. Crum a n d W . H . Annual, (1954-55) 837-841.
DeVries, A n t i b i o t i c s
37. C. DeBoet, A. D i e t z , J . R. W i l k i n s , C . L e w i s a n d G. Savage, Antibiotics Annual (1954-55) 831-836.
Md
2 71 3 8 . H.
H o e k s e m a a n d J . W . H i n m a n , 1 2 9 t h N a t i m a l M e e t i n g of t h e American C h e m i c a l S o c i e t y , Dallas, T e x a s ( 1 9 5 6 ) .
3 9 . H. H o e k s e m a a n d J . W. 4979-4982.
H i n m a n , J. Amer. Chem. SOC. 86 ( 1 9 6 4 )
40. H . H o e k s e m a , J . Amer. Chem. S O C . 8 6 ( 1 9 6 4 ) 4 2 2 4 . 4 1 . H. H o e k s e m a , J . Amer.
Chem. SOC. 9 0 ( 1 9 6 8 ) 7 5 5 - 7 5 7 .
4 2 . Y. H. Loo, P . S. S k e l l , H . H . T h o r n b e r r y , J. E h r l i c h , J. M. M c G u i r e , G . M. S a v a g e a n d J . C. S y l v e s t e r , J . Bact. 50 ( 1 9 4 5 ) 701-709. 43.
C. DeBoet a n d H. H o e k s e m a , U.S. 1960.
P a t e n t 2,928,844;
44. J. W . Hinman', H . H o e k s e m a a n d W . 2,851,463; September 9 , 1958.
G.
March 1 5 ,
Jackson, U.S.Patent
4 5 . A. D. A r g o u d e l i s and T. F. B r o d a s k y , J . A n t i b i o t i c s 2 5 (1972) 194-196. 46. A. D. A r g o u d e l i s , P e r s o n a l C o m m u n i c a t i o n . 47. A. D. A r g o u d e l i s a n d J . H . 674-676.
I
Coats, J . A n t i b i o t i c s 2 7 ( 1 9 7 4 )
4 8 . A. D. A r g o u d e l i s , J. H . Coats a n d L. E . J o h n s o n , J. A n t i b i o t i c s 2 7 ( 1 9 7 4 ) 738-743. 4 9 . A. D. A r g o u d e l i s , J . H. C o a t s , P . G . Lemaux a n d 0 . K . J . A n t i b i o t i c s 25 ( 1 9 7 2 ) 4 4 5 - 4 5 5 .
Sebek,
G . Lemaux a n d 0 . K .
Sebek,
5 0 . A , D. A r g o u d e l i s , J. H. C o a t s , P . J. A n t i b i o t i c s 26 ( 1 9 7 3 ) 7 - 1 4 .
51. T. F . B r o d a s k y a n d A. D. A r g o u d e l i s , J . A n t i b i o t i c s 2 6 (1973) 131-134. 5 2 . A. D. A r g o u d e l i s a n d T. F. B r o d a s k y , J . A n t i b i o t i c s 27 ( 1 9 7 4 ) 642-645.
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273
Macrolide Antibiotics Jaroslav Majer Department of Biochemisrry. Northwestern University Medical and Dental Schools. Chicago. Illinois
1
.
Erythromycin 1.1 Producing organisms 1I-I Physical and chemical properties Structural formulae 1.4 Therapeutic use 1.3 Isolation and purification m . l Extraction 1.5.2 Countercurrent distribution 1.5.3 Paper chromatography 1.5.4 Thin layer chromatography 1.5.5 Column chromatography 1.5.6 Silica gel chromatography 1.5.7 Cellulose column chromatography 1.5.8 Sephadex LH-20 column chromatography
.......................... ............. .......................... .............................. ................... ................................. ................ ....................... .................. ...................... .................. ............ ....... 2 . Oleandomycin 2.1 Producing organism ........................... Physical and chemical properties ............. 2.3 Structure .................................... - Therapeutic use .............................. 2.5 Isolation and purification ................... n . l Extraction ................................. 2.5.2 Countercurrent distribution ................ 2.5.3 Thin layer chromatography .................. 3
.
Tylosin 3.1 Producing organisms 3.1 Physical and chemical properties 3.3 Structure Therapeutic use 3.5 Isolation 3.5.1 General procedure 3.5.2 Laboratory procedure 3.5.3 Column chromatography 3.5.4 Extraction and separation from biological materials and feeds 3.5.5 Countercurrent distribution 3.5.6 Thin layer chromatography 3.5.7 Paper chromatography
.......................... ............. .................................... .............................. .................................... .......................... ....................... ...................... ........................ ................ .................. ....................... 4 . Rosamicin 4.1 Producing organism ........................... Physical and chemical properties ............. 4,3 Structure .................................... 4.4 Therapeutic activity ......................... K-5 Isolation.................................... 4.5.1 Extraction ................................. 4.5.2 Column chromatography...................... 4.5.3 Thin layer chromatography ..................
275 275 275 275 276 276 277 277 278 280 280 280 281 282 282 282 283 283 283 283 283 285 285 285 285 286 286 287 287 287 288 288 289 290 290 290 290 290 290 291
291
2 74
.
5
Leucomycin 5.1 Producing organisms 3-72 - Structure 5.3 Therapeutic activity 5.4 Isolation 5.4.1 Extraction 5.4.2 Column chromatography 5.4.3 Thin layer chromatography 5.Q.Q Paper chromatography 5.4.5 High pressure liquid chromatography..
........................... 293 ..................................... 293 .......................... 293 ..................................... 294 .................................. 294 ....................... 295 ................... 295 ........................ ....... 295 295 6 . Maridomycin 297 6.1 Producing organism ............................ 6.2 Physical and chemical properties .............. 297 6.3 Structure ..................................... 297 6.4 Therapeutic activity .......................... 297 6,5 Isolation and purification .................... 297 6.5.1 Extraction .................................. 297 6.5.2 Column chromatography ....................... 298 6.5.3 Thin layer chromatography ................... 299 7 . Midecamycin (SF-837) Producing organism ............................ 300 7.1 7.2 Physical and chemical properties .............. 300 Structure ............................ 300 7.3 Therapeutic activity .......................... 300 7.4 Isolation and purification .................... 301 7.5 301 Extraction .................................. 7.5.1 Countercurrent distribution ................. 301 7.5.2 301 Column chromatography ....................... 7.5.3 Thin layer chromatography ................... 301 .......I..
7.5.4
8
.
Literature cited
....................................
304
2 75 1. Erythromycin
1.1 Producing organisms Industrial production of erythromycin employs derivatives of Stheptomyceb ehythheub originally isolated from a soil sample from the Island of Panay, city of 110-110, in the Philippines Archipelago in 1952l. Another erythromycin producing species e.g. S t h e p t o m y c e b ghibeopeanub has been reported more recently2.
1.2 Physical
and chemical properties
The structure elucidation of erythromycin was mainly due to the effort of the Eli Lilly research team during the midf if tie^^-^. The 14 membered lactone aglycone was called erythronolide, the dimethylaminodeoxy sugar was named desosamine and the neutral sugar cladinose. The other two erythromycins B and C - differ from erythromycin A in the absence of the 0methyl group in the cladinose moiety: erythromycin C 8 , or by the absence of the tertiary hydroxyl group (C-12) in the lactone portion: erythromycin B9-l
-
.
Recently, two new erythromycin congeners were described. Erythromycin D is the most primitive diglycoside of the erythromycins, having mycarose (des-!-methyl cladinose) and desosamine attached to the simpler B form of the lactone12. Erythromycin E contains an ortho ester grouping and re resents a later stage of the erythromycin biosynthetic pathway' P . Detailed knowledge exists re arding the stereochemistry and conformation of erythromycins1'-'6. Of the erythromycins only erythromycin A is certified by the FDA. It exists in two allotropic modifications melting at 135-140 and 190-193OC respectively. Empirical formula C37H67N013 corresponds to molecular weight 733,4772. Erythromycin is basic (pK4 8 . 6 1 , has a specific optical rotation - 78* (c 1.9 ethanol) and a UV hmax at 278 nm (E 50). 1.3 Structural formulae The structural formulae are on the following page. 1.4 Therapeutic use The principal use of erythromycin is in group A betahemolytic streptococcal, staphylococcal, and pneumococcal infections. It is also active against many atypical mycobacteria. Otherwise, erythromycin preparations are most useful as penicillin substitutes in individuals with streptococcal or pneumococcal infections who are hypersensitive to penicillin. Erythromycin is available as the base and as salts and esters of the base. All of its forms have the same spectrum of activity and indications for use. Erythromycin (E-Mycin, Erythrocin, Ilotycin), erythromycin stearate (Erythrocin Stearate), and erythromycin estolate (Ilosone) are used orally; erythromycin gluceptate (Ilotycin Gluceptate) and erythromycin lacto-
276
Erythromycin A
B C D
R,
R,
OH H
Erythromycin E
Ma
M
I
OH
H
H
H
bionate (Erythrocin Lactobionate 1 are recommended primarily for intravenous use; and erythromycin ethylsuccinate (Erythromycin Ethyl Succinate, Pediamycin) is given both orally and intramuscularly. The base also i s used rectally and topically. Usual dosage expressed in terms of erythromycin base is 250-500 mg every 6 hours for adults, children 30 mg/kg body weight daily in divided doses. In severe cases the doses may be doubled. Erythromycin has a low acute and chronic toxicity. Oral and subcutaneous LD50 values of rhe free base in the mouse are about 3 0 0 0 and 2500 mg/kg respectively. Corresponding figures for the hydrochloride are 2900 mg/kg (oral), 1849 mg/kg (subcutaneous) 452 mg/kg (intravenous) and 490 mg/kg (intraperitoneal). Clinical experience with humans has substantiated the generally low toxicity of erythromycin. Adverse reactions e.g. nausea, vomiting and diarrhea may occasionally accompany oral administration. Erythromycin estolate can produce acute cholestatic hepatitis with fever and jaundice or a subclinically impaired liver function. Up to 15% of patients receiving this prepararion for more than two weeks may have abnormal liver function tests. Some of these abnormal tests (elevated SGOT) may be false positives, but others indicate impairment of liver functionl7.
1.5 Isolation 1.5.1
and purification
Extraction
Eight liters of crude broth containing a mixture of
2 77 e r y t h r o m y c i n A, B a n d C was f i l t e r e d w i t h t h e a i d of 5 % D i c o l i t e . The f i l t r a t e was a d j u s t e d t o pH 9 . 7 5 u s i n g a 4 0 % sodium h y d r o x i d e s o l u t i o n a n d e x t r a c t e d w i t h f o u r two l i t e r p o r t i o n s of c h l o r o f o r m . A f t e r c o n c e n t r a t i o n and c h i l l i n g o f t h e e x t r a c t t h e c r y s t a l l i n e m a t e r i a l contained erythromycin A ( 9 0 % ) and e r y t h r o mycin B (10%)8. 1.5.2
Countercurrent d i s t r i b u t i o n
Mother l i q u o r s a f t e r c r y s t a l l i z a t i o n o f e r y t h r o m y c i n A a n d B w i t h a n a c t i v i t y c o r r e s p o n d i n g t o 5 , 6 0 0 mg o f e r y t h r o m y c i n were d i s s o l v e d i n 1 0 0 m l of t h e u p p e r p h a s e of a n e q u i l i b r a t e d m e t h y l i s o b u t y l k e t o n e - s o d i u m p h o s p h a t e b u f f e r ( 0 . 1 N, pH 6.5)acetone system (20:20:1). After s i x t y t r a n s f e r s tubes 10-20 w e r e shown t o c o n t a i n e r y t h r o m y c i n C , t u b e s 2 1 - 2 6 a m i x t u r e o f The e r y t h r o m y c i n C a n d A a n d t u b e s 27-38 o n l y e r y t h r o m y c i n A . c o n t e n t s o f t u b e s 1 0 - 2 0 were combined a n d c o n c e n t r a t e d u n d e r reduced p r e s s u r e a l l o w i n g t h e r e s u l t i n g aqueous phase t o b e a d j u s t e d t o pH 9 . 7 5 a n d e x t r a c t e d w i t h c h l o r o f o r m . The c h l o r o f o r m e x t r a c t s were d r i e d o v e r a n h y d r o u s sodium s u l f a t e a n d r e d u c e d t o a 1 0 m l volume. E r y t h r o m y c i n C c r y s t a l l i z e d from t h e c o n c e n t r a t e d s o l u t i o n y i e l d i n g 1 g as a f i r s t c r o p 8 . The a b o v e s o l v e n t s y s t e m was u s e d f o r s e p a r a t i o n o f e r y t h romycin A a n d B (590 mg) i n a 1 0 0 - t u b e c o u n t e r c u r r e n t d i s t r i b u t i o n a p p a r a t u s 1 8 w i t h a 1 0 m l tube c a p a c i t y of each phase. A s shown by p a p e r c h r o m a t o g r a p h y t u b e s 3 0 t o 5 0 c o n t a i n e d e r y t h r o mycin A w h i l e t u b e s 60-80 c o n t a i n e d e r y t h r o m y c i n B. The l a t t e r f r a c t i o n s w e r e combined, t h e i r volume r e d u c e d t o 4 0 m l , a d j u s t e d t o pH 9 . 6 and e x t r a c t e d w i t h c h l o r o f o r m . The r e s i d u e a f t e r r e m o v a l o f t h e c h l o r o f o r m was c r y s t a l l i z e d from 6 m l of a c e t o n e . The f i r s t c r o p weighed 9 5 mg9. 1.5.3
Paper chromatography
For a n a l y t i c a l p u r p o s e s d u r i n g t h e e a r l y p e r i o d o f s t u d i e s on e r y t h r o m y c i n p a p e r c h r o m a t o g r a p h y was f r e q u e n t l y employed f o r s e p a r a t i o n of r a d i o l a b e l l e d e r y t h r o m y c i n s a n d , i n p a r t i c u l a r , f o r b i o a u t o g r a p h y . W i l e y and h i s c o - w o r k e r s u s e d a s o l v e n t system o f m e t h a n o l - a c e t o n e - w a t e r ( 1 9 : 6 : 75) i n which e r y t h r o m y c i n C was t h e f a s t e s t moving, f o l l o w e d by e r y t h r o m y c i n s A ( R f 0 . 7 ) and B ( R f 0 . 6 ) ' . A more d e t a i l e d p r o c e d u r e was d e s c r i b e d by Friedrnan e t Whatman No. 1 or 3MM p a p e r was d i p p e d i n t h e lower p h a s e
of t h e f o l l o w i n g m i x t u r e : Methylcyclohexane-tert. buranol-ethyl isobutyl ketone-0.1 M p o t a s s i u m p h o s p h a t e b u f f e r (pH 7 . 0 ) ( 7 5 : 1 5 : 1 0 : 2 5 ) a n d t h e e x c e s s i v e s o l v e n t s w e r e removed by b l o t t i n g b e t w e e n s h e e t s o f f i l t e r p a p e r a n d by a e r a t i o n f o r a b o u t f i v e m i n u t e s . A f t e r a p p l i c a t i o n of t h e e r y t h r o m y c i n s a m p l e ( e x t r a c t ) a n d r e f e r e n c e s a m p l e s , t h e p a p e r was l e f t t o d r y f o r a n o t h e r f i f t e e n m i n u t e s a n d d e v e l o p e d o n c e i n t h e d e s c e n d i n g u p p e r p h a s e of t h e a b o v e s y s t e 3 . D e t e c t i o n w i t h v a n i l l i n ( 0 . 5 % ) i n p e r c h l o r i c a c i d (1M) a t 75 C r e v e a l e d e r y t h r o m y c i n A as a b l u e or b l u e - g r a y s p o t w e l l s e p a r a t e d from r e l a t e d compounds.
2 78 An a s c e n d i n g p a p e r chromatography s y s t e m u s i n g EatonDikeman No. 613 p a p e r g a v e s a t i s f a c t o r y s e p a r a t i o n s of e r y t h r o mycin A a n d B. The s o l v e n t s y s t e m was t h e aqueous p h a s e of a m i x t u r e o f 9 0 0 m l 0.01N ammonium h x d r o x i d e and 1 0 0 m l m e t h y l i s o b u t y l k e t o n e e q u i l i b r a t e d a t 28 C . Q u a n t i t a t i o n was a c h i e v e d b y m e a s u r i n g zone s i z e on b i o a u t o g r a p h i c p l a t e s u s i n g BaciCLub d u b t i l i b as t h e t e s t o r g a n i s m z 0 . E x c e l l e n t s e p a r a t i o n of t h e known e r y t h r o m y c i n s was a l s o o b t a i n e d w i t h a n a s c e n d i n g s y s t e m on Whatman No. 3MM p a e r w i t h 2 % aqueous d i p o t a s s i u m p h o s p h a t e as t h e s o l v e n t system' g
.
A t w o - s t e p p a p e r c h r o m a t o g r a p h i c t e c h n i q u e was successf u l l y employed f o r s e p a r a t i o n o f e r y t h r o m y c i n a n d p r o p i o n y l e r y t h r o m y c i n from b i o l o g i c a l s a m p l e s , e . g . whole b l o o d , serum, plasma, u r i n e and s a l i v a . Samples a n d s e r i e s of s t a n d a r d s were a p p l i e d t o Whatman No. 1 p a p e r and d e v e l o p e d by d e s c e n d i n g chromatography i n a b s o l u t e methanol ( s o l v e n t s y s t e m No. 1). A f t e r a p p r o x i m a t e l y o n e h o u r t h e chromatograms were removed and a i r d r i e d . The u p p e r p o r t i o n of t h e chromatogram was c u t o f f 5 cm below t h e s t a r t i n g l i n e . The l a n e numbers were t r a n s p o s e d t o a p o i n t 6 . 3 5 c m from t h e u p p e r e n d o f t h e c u t chromatogram. The s e c o n d s t e p o f development of t h e chromatogram was c a r r i e d o u t a g a i n i n d e s c e n d i n g a r r a n g e m e n t w i t h a s o l v e n t s y s t e m No. 2 p r e p a r e d by d i s s o l v i n g 1 2 . 5 g of ammonium c h l o r i d e and 3 5 g of sodium c h l o r i d e i n 1 0 0 m l o f d i s t i l l e d water a n d by a d d i n g 2 5 m l of d i o x a n e a n d 1 2 . 5 m l o f m e t h y l e t h y l k e t o n e . The volume was made t o one l i t e r w i t h d i s t i l l e d w a t e r and t h e pH was a d j u s t e d t o 5 . 7 w i t h 1 N ammonium h y d r o x i d e . The chromatography was s t o p p e d when t h e s o l v e n t f r o n t n e a r e d t h e lower end of t h e t a p e . A f t e r d r y i n g i n t h e a i r t h e chromatograms were q u a n t i t a t e d b b i o a u t o g r a p h y on p l a t e s s e e d e d w i t h S a h c i n a L u t e a ATCC 9 3412y, 1.5.4
Thin l a y e r chromatography
Thin l a y e r chromatography h a s been found i n v a l u a b l e i n s t u d i e s done w i t h e r y t h r o m y c i n and i t s d e r i v a t i v e s . A number o f s o l v e n t s y t e m s and d e t e c t i o n r e a g e n t s h a v e b e e n d e s c r i b e d i n the literature. Anderson u s e d f o r K i e s e l g e l G l a y e r s a s y s t e m composed of m e t h y l e n e chloride-methanol-benzene-formamide ( 8 0 : 2 0 : 2 0 : 2 - 5 ) . T h i s method r e s o l v e d e r y t h r o m y c i n A, B , a n h y d r o e r y t h r o m y c i n and a c e t y l e r y t h r o m y c i n s . However, s h o r t c o m i n g s i n v o l v e d an u n s a t i s f a c t o r y r e p r o d u c i b i l i t y and a f o r m a t i o n o f two s o l v e n t fronts", Buffered s i l i c a g e l and a mobile phase c o n s i s t i n g of 2butanol-nitromethane-ethyl a c e t a t e - w a t e r ( 6 0 : 3 0 : 2 0 : 2 0 ) s e p a r a t e d e r y t h r o m y c i n A from a n h y d r o e r y t h r o m c i n y e t f a i l e d t o r e s o l v e erythromycin A from, erythromycin B Z 3 . An improvement was a c h i e v e d w i t h p l a t e s p r e p a r e d from a m i x t u r e of K i e s e l g e l G a n d K i e s e l g u h r G (1:l) i m p r e g n a t e d w i t h formamide ( 1 5 % i n a c e t o n e ) . A f t e r few m i n u t e s a t room t e m p e r a t u r e t h e p l a t e s a r e d e v e l o p e d i n a t e r n a r y o r q u a r t e r n a r y solv e n t s y s t e m , e . g . n-hexane o r b e n z e n e ( 4 0 - 6 0 1 , c h l o r o f o r m o r
279 methylene chloride (55-35) and methanol or ethanol ( 5 ) ; n-hexane or benzene or carbon tetrachloride (15-451, chloroform or methylene chloride (40-201, ethyl acetate ( 4 0 - 3 0 1 , and methanol (5). According to the authors chloroform must be phosgene and hydrogen chloride free. After developing for 30-50 minutes the plates were kept at llO°C for 10 minutes to remove the formamide. Detection was then done by any conventional method such as with cerium sulfate (l%), molybdic acid (2.5%) in 10% sulfuric acid. Blue spots developed after a few minutes at llO°C and stayed unchanged for about 12 hours. The ternary system satisfactorily resolved the erythromycins A, B and C, whereas anhydroerythromycin C can cochromatograph with erythromycin A as does anhydroerythromycin A with erythromycin B. In this regard the quarternary solvent system was shown superior.24 Another system devised for erythromycins employs a mixture of ethyl acetate--isopropanol--ammonium acetate (15% aqueous, adjusted to pH 9.6) 9:7:8 (upper phase)". This system together with an anisaldehyde spray were i n t e h aeia instrumental in the isolation of erythromycin D from mother liquors after crystallization of erythromycin A. The Rf values relative to erythromycin A (RER A- 1.0) were 1.05 for erythromycin B, 0.92 for erythromycin g,-and 0 . 8 7 for erythromycin C. The anisaldehyde spray reagent (ethanol--anisaldehyde--conc. sulfuric acid, 9:l:l) was shown to be "lactone specific" since i t produced, after a slow heating to BOOC, a different coloration of compounds possessing the A- or B-type lactone portions12. Differentiation of erythromycin and its esters has been achieved on sodium acetate-buffered silica gel plates. Plates (20 x 20 cm 0.25 mm thickness) were spread using a slurry (1:2 w/v) silica gel G (Merck) in sodium acetate (0,02N, aqueous) and dried overnight. For development, a solvent system of methanol--O.O2N sodium acetate (12:3) was used. Optimal resolution was obtained when the solvent front had travelledo 15 cm. Spraying with a derection reagent and heating at 150 C for five minutes produced blue-gray coloration with erythromycin and its esters. The spray reagent consisting of glucose--phosphoric acid--water--ethanol--n-butanol (2:10:40:30:30) had to be freshly prepared daily. The spots were quantitated by direct densitometry. Erythromycin base and its salts e.g. stearate, lactobionate and gluceptate displayed identical Rf values of 0.28; anhydroerythromycin had an Rf of 0.33. The ester salt of erythromycin (estolate) was slightly slower--Rf = 0.65 than the esters of erythromycin, e.g. succinate or ethyl carbonate, which both migrated with an Rf o f 0.6726. Erythromycin A was resolved from erythromycin C on silica gel plates with a solvent system of meth lene chloride -methanol (95%) -conc. ammonium hydroxide 90:10:12x,38. Spots were visualized by7spraying with the original arsenomolybdate reagent of Nelson (Dissolve ammonium molybdate ( 2 5 g) in 450 ml of distilled water; add 21 ml of conc. sulfuric acid and mix with a solution of 3 g of sodium arsenate (Na2HAs04.7 H20) in 25 ml of water, Incubate at 37OC for 24-48 hours in a brown bottle). After heating at 100°C for 10 minutes, quantitation was carried out immediately by direct densitometry along with appropriate standardsz0.
.
2 80 1.5.5
Column chromatography
A m b e r l i t e I R C - 5 0 r e s i n column chromatography was used f o r r e c o v e r y and s e p a r a t i o n of l a b e l e d e r y t h r o m y c i n . The myceliumf r e e f e r m e n t a t i o n b e e r was f i r s t d i l u t e d ( t w o - f o l d ) w i t h water and p a s s e d t h r o u g h a column ( 2 x 1 0 cm) of A m b e r l i t e IRC-50 r e s i n which had been e q u i l i b r a t e d w i t h p o t a s s i u m p h o s p h a t e b u f f e r (lmM, pH 7 . 0 ) c o n t a i n i n g EDTA (ImM). The column was washed w i t h a p p r o x i m a t e l y 2 5 0 m l of t h e same b u f f e r and e l u t i o n was accomplished w i t h 1M b u f f e r . The b u l k of r a d i o a c t i v i t y and a l l t h e a n t i b a c t e r i a l a c t i v i t y a p p e a r e d between 1 2 0 t o 150 ml of t h e e f f l u e n t . T h i s f r a c t i o n was a d j u s t e d t o pH 9.5 and e x t r a c t e d w i t h 4 x 3 0 m l o f m e t h y l c y c l o h e x a n e . The combined e x t r a c t s were washed 10 times w i t h 0 . 5 ml of w a t e r , d u r i n g which p r o c e d u r e i n c r e a s i n g amounts o f e r y t h r o m y c i n were d e t e c t a b l e i n t h e aqueous p h a s e . The l a t t e r was e v e n t u a l l y a d ' u s t e d t o pH 7 and r e d u c e d t o a volume of 0 . 5 m l under n i t r o g e n 2 (See n e x t p a r a g ra p h ) ,
d.
1.5.6
S i l i c a g e l chromatography
S i l i c a g e l SG 34 Whatman (85 g ) was mixed t h o r o u g h l y w i t h 8 5 m l of t h e lower p h a s e of n-heptane--benzene--acetone--isop r o p a n o l and 10 mM p o t a s s i u m p h o s p h a t e pH 7 . 0 ( 2 5 : 5 0 : 1 5 : 1 0 : 2 5 ) and added s l o w l y w i t h tamping t o a column ( 2 0 mm d i a m e t e r ) c o n t a i n i n g t h e upper p h a s e o f t h e s o l v e n t s y s t e m which f i n a l l y gave an e f f i c i e n t t o t a l volume 2 4 0 ml. The sample a p p l i e d i n t h e lower p h a s e was chromatographed w i t h t h e u p p e r p h a s e a t a flow r a t e 0 . 1 5 ml/cm2/min. The peak of t h e b i o l o g i c a l a c t i v i t y a p p e a r e d a t a r e l a t i v e e l u t i o n volume" o f 1 . 5 5 . This m a t e r i a l was homogeneous and v i r t u a l l y i d e n t i c a l w i t h e r y t h r o m y c i n A 2 8 . 1.5.7
C e l l u l - o s e column chromatography
A c r u d e c o n c e n t r a t e c o n t a i n i n g e r y t h r o m y c i n A and B ( 1 7 6 mg) o b t a i n e d by e x t r a c t i o n of t h e f e r m e n t a t i o n b r o t h w i t h c h l o r o f o r m o r amyl a c e t a t e was mixed w i t h 3 g of c e l l u l o s e powder ( S o l k a - F l o c , a p u r i f i e d wood c e l l u l o s e ) . The c e l l u l o s e w i t h t h e a d s o r b e d e r y t h r o m y c i n s was t h o r o u g h l y d r i e d , packed i n t o t h e t o p of a column p r e v i o u s l y packed w i t h d r y c e l l u l o s e and t h e column was developed by g r a v i t y w i t h 0.01N ammonium h y d r o x i d e s a t u r a t e d w i t h m e t h y l i s o b u t y l k e t o n e . F r a c t i o n s of 1 5 m l were c o l l e c t e d and m o n i t o r e d by p a p e r chromatographys. F r a c t i o n s 6 t o 1 4 c o n t a i n e d erythromycin A, f r a c t i o n s 1 5 - 2 0 c o n t a i n e d a m i x t u r e of A and B , and f r a c t i o n s 21-48 c o n t a i n e d o n l y e r y t h r o m y c i n B. The r e c o v e r y from t h e column was a l m o s t quantitative. The e r y t h r o m y c i n B f r a c t i o n s were combined, adj u s t e d t o pH 1 0 . 5 w i t h sodium h y d r o x i d e (40%), and e x t r a c t e d w i t h two 1 0 0 m l p o r t i o n s of c h l o r o f o r m . A f t e r d r y i n g o v e r anhydrous sodium s u l f a t e and e v a p o r a t i o n of t h e s o l v e n t , t h e r e s i d u e was e x t r a c t e d w i t h t h r e e 2 5 ml p o r t i o n s of e t h e r , which were combined and d r i e d . The r e s i d u e a f t e r e v a p o r a t i o n o f t h e e t h e r was d i s s o l v e d i n 5 m l of w a r m a c e t o n e . C o o l i n g d e p o s i t e d c r y s t a l s of e r y t h r o m y c i n B 9 , q e l a t i v e e l u t i o n volume (REV1 is thus expressed as a r a t i o o f the e l u t i o n volume, Ve over t h e t o t a l ( e f f e c t i v e volume) V t o f the column,
281 1.5.8
Sephadex LH-20 column chromatography
Sephadex LH-20 f u l f i l l s a l m o s t i d e a l l y t h e r e q u i r e m e n t s f o r t h e c h r o m a t o g r a p h i c s e p a r a t i o n of a m p h i p a t h i c compounds l i k e macrolides. As well as separating substances according t o s i z e , Sephadex LH-20 e x h i b i t s a number of g e l - s o l u t e i n t e r a c t i o n e f f e c t s of p a r t i t i o n chromatography. Sephadex LH-20 was used by t h e a u t h o r i n a f o l l o w i n g a r r a n g e m e n t 1 2 . A column (2.0 x 1 2 0 c m , bed volume 3 0 0 ml) o f Sephadex LH-20 i n chloroform-hexane ( l : l , v / v ) was l o a d e d w i t h a p p r o x i m a t e l y 2 5 0 mg of a mother l i q u o r c o n c e n t r a t e c o n t a i n i n g e r y t h r o mycin A, B , C and I?, t h e s p i r o k e t a l s (6+ 9 ; 1 2 +9) of e r y t h r o mycin A and C, and a number of o t h e r e r y t h r o m y c i n r e l a t e d comp o n e n t s . F r a c t i o n s o f 5 m l were c o l l e c t e d a t a flow r a t e o f 0 . 1 ml/cm2/min. Erythromycin B was e l u t e d f i r s t ( R E V 0 . 8 )
f o l l o w e d by e r y t h r o m y c i n D ( 0 . 8 5 1 , A ( 0 . 9 ) and C ( 0 . 9 5 ) r e s p e c t i v e l y . The o v e r l a p p i n g of e r y t h r o m y c i n s D and A was encount e r e d , and, i n o r d e r t o i s o l a t e t h e s u b s t a n c e s of h i g h e r p u r i t y , p r e p a r a t i v e TLC and a r e p e a t e d column chromatography were n e c e s s a r y . S p i r o k e t a l s of e r y t h r o m y c i n A and C e l u t e d w i t h t h e r e s p e c t i v e e r y t h r o m y c i n s , which was a m a j o r s h o r t c o m i n g of t h e o t h e r w i s e e f f i c i e n t method.
282
2 . Oleandomyc i n "
2 . 1 Producing
organism
Oleandomycin i s e l a b o r a t e d by a s t r a i n o f S t h e p t o m y c e b a n t i b i o t i c u d (ATCC 1 1 8 9 1 ) 2 9 - 3 2 when grown i n a v a r i e t y of o r g a n i c media u n d e r submerged a e r o b i c c o n d i t i o n s .
2.2 Physical
and c h e m i c a l p r o p e r t i e s
Oleandomycin i s a c r y s t a l l i n e , c o l o r l e s s , b a s i c compound (pKA 8 . 5 1 , m o l e c u l a r w e i g h t 6 8 7 . I t i s s l i g h t l y s o l u b l e i n water and l i g r o i n , b u t d i s s o l v e s r e a d i l y i n most of t h e o t h e r The compound c r y s t a l l i z e s grom aqueous common o r g a n i c s o l v e n t s . methanol or aqueous a c e t o n e s o l u t i o n s m e l t i n g a t 1 1 0 C . With c h l o r i n a t e d h y d r o c a r b o n s r h e a n t i b i o t i c forms s o l v a t e d c r y s t a l s . The c h l o r o f o r m s o l v a t e , m.p. 1 2 0 - 1 2 1 ° C , c o n t a i n s one e q u i v a l e n r : of t e n a c i o u s l y bound s o l v e n t . Oleandomycin i s r e a d i l y s o l u b l e i n a c i d i c media and forms c r y s t a l l i n e s a l t s w i t h m i n e r a l and organic acids. The g p e c i f i c o p t i c a l r o t a t i o n of t h e f r e e b a s e i n m e t h a n o l 1 ; i n t h e U V r e g i o n i t a b s o r b s weakly n e a r 2 9 0 nm ( E 5 0 ) . The I R s p e c t r u m ( C H C 1 3 ) d l s p l a y s a b s o r p t i o n b a n d s a t 3 5 0 9 cm-l ( - C H 2 - ) , 1 7 1 8 and 1 7 1 2 cm-1 ( > C = O ) among o t h e r s . P a r t i a l and c o m p l e t e a c y l a t i o n h a s been a c h i e v e d u n d e r c o n t r o l l e d c o n d i t i o n s 3 3 . T r i a c e t y l o l e a n d o m y c i n ( T A O ) h a s become t h e r a p e u t i c a l l y important.
is -65
*
2.3 Structure -
D e g r a d a t i o n s t u d i e s r e s u l t e d i n t h e e l u c i d a t i o n of t h e Two s u g a r s a r e a t t a c h e d a t The p o s i t i o n s C-3 a n d C - 5 t o t h e f o u r t e e n membered l a c t o n e r i n g c a l l e d o l e a n d o l i d e . The n e u t r a l s u g a r i s L - o l e a n d r o s e and t h e b a s i c s u g a r i s D-desosamine. A b s o l u t e c o n f i g u r a t i o n of oleandomycin has b e e n a s s i g n e d by C e l ~ n e r ~ ~ .
0
Oleandomycin
-riginally
r e f e r r e d t o as PA 1 U > ; P f i z e r ' s t r a d e name i s Matromycin.
283
2.4 Therapeutic use Oleandomycin and TAO have the same i n u i t h o antimicrobial spectrum which involves numerous gram-positive bacteria, exerting the strongest effect against streptococci, pneumococci and staphylococci.
A few gram-negative bacteria, e.g. some species of N e i d s e h i a , BhuceLLa and HemophiLu6 also fall within the in u i t h o antimicrobial spectrum. Erythromycin-resistant staphylo-
cocci freshly isolated from patients sometimes display complete cross-resistance to oleandomycin. On the other hand, some staphylococcal infections resistant to erythromycin may yield to troleandomycin37. Oleandomycin preparations may cause adverse reactions like other macrolide antibiotics, e.g. nausea, anorexia, diarrhea and vomiting. Prolonged administration may result in symptoms of hepatotoxicity recognized only from abnormal results of laboratory tests, or worse, manifested as clinical cholestatic jaundice. The oral dose of TAO for acute infections in adults is 1000 to 2000 mg daily,equally divided in four doses, Children can tolerate doses of 125 to 250 mg every 6 hours.
2.5 Isolation and purification 2.5.1
Extraction
The general method for recovery and purification of oleandomycin involves filtration, extraction of the filtrate with methyl isobutyl ketone, distribution into dilute aqueous acid, adjustment of the aqueous phase to pH 9 and distribution into methyl ethyl ketone. Addition of an equivalent amount of hydrochloric acid to the methyl ethyl ketone solution produces a crystalline material which is the solvated oleandomycin hydrochloridez9. 2.5.2
Countercurrent distribution
For countercurrent distribution analysis a modified system originally used for erythromycin' is suitable: methyl isobutyl ketone--acetone--phosphate-citrate buffer pH 7 (20:1:20). Another solvent system consists of benzene--cyclohexane--ethanol--water ( 5 : 5:8:2I3l. In a recent work on biotransformation of erythronolide A oxime by an oleandomycin-producing strain of Stht?ptOmyCe6 a n t i b i a t i c u d ATCC 11891, the separation of the semi-synthetic product from the endogenous oleandomycin was accomplished in a system of n-buranol--acetic acid--water (10:l:l) with the aqueous phase adjusted to pH 2.95. After carrying out 100 transfers with 4 0 ml of each phase per tube, oleandomycin was found in tubes 6 0 - 6 7 3 8 . 2.5.3
Thin layer chromatography
Brinkmann or Merck silica gel plates were developed twice in a solvent system of methylene chloride--methanol--water--
284
ammonium hydroxide ( 9 0 : 9 : 5:0.5:1). Detection was done by spraying with phosphomolybdic acid (5% in ethanol) or xanthydrol (0.15%)in a mixture of conc. hydrochloric and acetic acid (12:l) and heating at 100°C20*3e,
2 85 3 . Tylosin
3 . 1 Producing
organisms
Two t y l o s i n p r o d u c i n g s t r a i n s were i s o l a t e d from s o i l s a m p l e s c o l l e c t e d i n T h a i l a n d i n 1 9 5 5 . They were a s s i g n e d l a b o r a t o r y numbers M48-E2724 a n d M48-E2728. Taxonomic s t u d i e s i n d i c a t e d t h a t i n many r e s p e c t s t h e y were similar t o o r i d e n t i c a l w i t h S t h e p t o m y c e b dhadiae. Growth c o n d i t i o n s , e . g . s p o r u l a t i o n and v e g e t a t i v e media as w e l l as f e r m e n t a t i o n s were d e s c r i b e d i n d e t a i l 3 9 . F o r t h a t work t h e c u l t u r e M48-E2724 was used. T y l o s i n i s also p r o d u c e d by S t h e p t o m ced h y g h o b c o p i c u d i n m a ~ r o c i na n~d~ minor q u a n t i t i e s 4 0 . G r a d u a l l y , desmycosin", l a ~ t e n o c i nwere ~ ~ d e s c r i b e d as companion p r o d u c t s i n f e r m e n t a t i o n s of S t a e p t o m y c e d dhadiae. T h e i r r o l e s i n b i o s n t h e s i s of t y l o s i n were r e v e a l e d r e c e n t l y i n a n e l e g a n t s t u d y 4 $ . M a c r o c i n a n d d e s m y c o s i n a r e d i r e c t p r e c u r s o r s of t y l o s i n , w h i l e l a c t e n o c i n i s a key i n t e r m e d i a t e of t h e f o r m e r two. Tylosin is f u r t h e r transformed t o relomycin and an u n i d e n t i f i e d p r o d u c t .
3.2 P h y s i c a l
and c h e m i c a l p r o p e r t i e s
SpeThe c r y s t a l l i n e form o f t y s o s i n m e l t s a t 128-132OC. c i f i c o p t i c a l r o t a t i o n {a)$5 - 4 6 ( c = 2 , i n m e t h a n o l ) . Electrometric t i t r a t i o n i n 66% d i m e t h y l f o r m a m i d e g a v e a n e u t r a l i z a t i o n e q u i v a l e n t of 9 0 4 , pK4 7.1. E m p i r i c a l f o r m u l a C46H77NO17 c o r r e s p o n d s to a m o l e c u l a r w e i g h t 915. T e UV a b s o r p t i o n s p e c t r u m i n m e t h a n o l h a s a Xmax a t 282 nm, = 2 4 5 . The I R s p e c t r u m showed a b s o r p t i o n maxima a t 350 cm-' i n d i c a t i v e o f t h e p r e s e n c e of h d r o x y l s , 1724 cm-l d u e to t h e l a c t o n e c a r b o n y l , 1685 a n d 1 5 9 5 r e v e a l t h e p r e s e n c e of a d i e n i c s y s t e m conjugated with a carbonyl function. The 1 1 6 0 c m - ' band i s a s s i g n e d t o C-0-C s t r e t c h i n g a b s o r p t i o n . The t y l o s i n b a s e is s o l u b l e i n most o r g a n i c s o l v e n t s e . g . l o w e r a l c o h o l s , l o w e r k e t o n e s , c h l o r i n a t e d h y d r o c a r b o n s , and I t s s o l u b i l i t y i n water v a r i e s i n v e r s e l y w i t h t e m p e r a benzene. S a l t s of t y l o s i n w i t h a c i d s t u r e ; a t 25OC i t e q u a l s 5 mg/ml. were p r e p a r e d and c h a r a c t e r i z e d a s were i t s e s t e r s w i t h a c e t i c and p r o p i o n i c a c i d . 3 . 3 Structure -
The s t r u c t u r a l f o r m u l a h a s been d e d u c e d from d e g r a d a t i o n c o m p a r i s o n w i t h r e l a t e d m a ~ r o l i d e s ~a n~ d, c o n f i r m e d r e c e n t l y as c o r r e c t b y c a r b o n - 1 3 N . M . R . a n a l y s i s 4 6 . The a t t a c h ment o f t h e mycaminosylmycarose d i s a c c h a r i d e i s t h u s a t C - 5 o f t h e a g l y c o n e , and t h e s t r u c t u r e o f t y l o s i n is a s shown on t h e n e x t page.
3.4 Therapeutic
use
The o r i g i n a l s t u d i e s showed a p r o n o u n c e d a c t i v i t y i n v i t h o o f t y l o s i n a g a i n s t gram-posi t i v e b a c t e r i a , c e r t a l n gram-
286
Ty lor in n e g a t i v e b a c t e r i a , and m y c o b a c t e r i a .
Of p a r t i c u l a r i n t e r e s t
was i t s h i g h l e v e l o f a c t i v i t y a g a i n s t mycoplasma i s o l a t e d from domestic animals. The t o x i c i t y o f t y l o s i n h a s b e e n low: t h e i n t r a v e n o u s L D 5 0 f o r mice was a b o u t 4 0 0 mg/kg; d o s e s u p t o 5 0 0 0 mg/kg a d m i n i s t e r e d o r a l l y t o r a t s were w i t h o u t h a r m f u l effects. Dogs t o l e r a t e d a d a i l y d o s e of 1 0 0 mg/kg f o r more t h a n a y e a r . Minimal s i d e e f f e c t s may be o b s e r v e d w i t h h i g h e r d o s e s , however. P a r t i a l c r o s s r e s i s t a n c e with erythromycin has been recorded. T y l o s i n h a s found a wide p r a c t i c a l u s e as a n a n i m a l h e a l t h a n d growth a g e n t , e s p e c i a l l y i n p o u l t r y and s w i n e . S i n c e 1 9 6 2 , i t h a s b e e n m a r k e t e d by E l i L i l l y u n d e r t h e name T y l a n ; t h e h i g h e s t recommended l e v e l f o r t y l o s i n i n a c o m p l e t e swine f e e d h a s been 1 0 0 g / t o n . 3.5 Isolation 3.5.1
General procedure
Large scale i s o l a t i o n procedure u s u a l l y involves f i l t r a t i o n o f t h e whole b r o t h w i t h t h e a i d o f a n i n e r t d i a t o m a c e o u s t y p e f i l t e r a g e n t . Then, t y l o s i n a c t i v i t y from t h e f i l t r a t e i s t r a n s f e r r e d t o an o r g a n i c s o l v e n t a t pH 9 , u s u a l l y i n a continuous two-stage l i q u i d - l i q u i d e x t r a c t i o n . To t h i s e f f e c t s e v e r a l intermediate molecular weight e s t e r s can be used such as b u t y l or amyl a c e t a t e . I n t h i s manner a n a d e q u a t e p u r i f i c a t i o n and c o n c e n t r a t i o n u s u a l l y i s a c h i e v e d . The b a s i c p r o d u c t i s converted t o a water s o l u b l e s a l t , e:g. phosphate o r tartrate, by r e - e x t r a c t i o n i n t o t h e aqueous s o l u t i o n s o f t h e r e s p e c t i v e a c i d s . These c o n c e n t r a t e s or t h e d r i e d s a l t s are a l s o u t i l i z e d i n t h e f i n a l f o r m u l a t i o n as s t a b i l i z e d f e e d mixes. (Any p r o c e d u r e s h o u l d b e m a i n t a i n e d w i t h i n a pH r a n g e o f 4 t o 9 i n o r d e r t o minimize p o s s i b l e d e g r a d a t i o n ) . A l t e r n a t i v e l y , t h e t y l o s i n b a s e , which i s r e l a t i v e l y i n s o l u b l e i n water, can be c r y s t a l l i z e d from s o l v e n t f r e e , c o n c e n t r a t e d aqueous e x t r a c t s by c a r e f u l l y c o n t r o l l e d pH a d j u s t m e n t 4 7 .
3.5.2
Laboratory procedure
A l a b o r a t o r y s c a l e method f o r i s o l a t i o n a n d p u r i f i c a t i o n o f l a b e l e d t y l o s i n and t y l o s i n - l i k e a n t i b i o t i c s h a s a l s o been described4 : Four 9 0 m l c u l t u r e s were h a r v e s t e d by t h e a d d i t i o n of t h r e e volumes o f a c e t o n e . The whole b r o t h - a c e t o n e m i x t u r e was f i l t e r e d t h r o u g h Whatman No. 4 2 p a p e r . The f i l t e r c a k e was washed twice w i t h 7 0 % a q u e o u s a c e t o n e , a n d t h e wash was comb i n e d w i t h t h e f i l t r a t e . A f t e r e v a p o r a t i o n o f a c e t o n e under r e d u c e d p r e s s u r e , t h e aqueous s o l u t i o n was a d j u s t e d t o pH 9 . 3 a n d e x t r a c t e d w i t h t h r e e volumes of c h l o r o f o r m . The e x t r a c t s were e v a p o r a t e d t o d r y n e s s a n d t h e r e s i d u e was d i s s o l v e d i n 1 0 0 m l of amyl a c e t a t e . The b a s e s were t r a n s f e r r e d from t h e amyl a c e t a t e i n t o a p h o s p h a t e b u f f e r ( 0 . 5 M , pH 4 . 5 ) and t h e a q u e o u s p h a s e r e - e x t r a c t e d w i t h c h l o r o f o r m a t pH o f 8 . 5 . 3.5.3
Column chromatography
The c h l o r o f o r m s o l u t i o n c o n t a i n i n g t h e c r u d e material was a p p l i e d t o a s i l i c a g e l column (Merck 7 7 3 4 ) p r e p a r e d i n c h l o r o form and d e v e l o p e d w i t h a m i x t u r e o f e t h y l a c e t a t e - - a c e t o n e (1:l). T y l o s i n was e l u t e d from t h e column i n t h e e a r l y f r a c t i o n s , f o l l o w e d by a p a r t i a l l y r e s o l v e d m i x t u r e of m a c r o c i n and relomycin. The f r a c t i o n s c o n t a i n i n g t y l o s i n were p o o l e d and e ~ a p o r a t e d ~ ~ , F r a c t i o n s c o n t a i n i n g m a c r o c i n a n d r e l o m y c i n were p o o l e d , e v a p o r a t e d a n d t h e r e s i d u e was d i s s o l v e d i n a small volume o f e t h y l acetate. Chromatography on a column o f a c t i v a t e d aluminum o x i d e (Woelrn, a c t i v i t y g r a d e 1) p r e p a r e d i n e t h y l a c e t a t e a n d developed with a mixture of e t h y l a c e t a t e - - e t h a n o l (80:20) s e p a r a t e d m a c r o c i n from r e l o m y c i n a n d from t h e r e s i d u a l t y l o sin43. 3.5.4
E x t r a c t i o n a n d s e p a r a t i o n from b i o l o g i c a l materials a n d f e e d s
Because o f t h e f o r m a t i o n o f e m u l s i o n s t h e o p t i m l r e c o v e r y o f t y l o s i n from materials l i k e l i v e r ( h o m o g e n a t e s ) , b l o o d ( p l a s m a ) , m i l k e t c . r e q u i r e s a t h o r o u g h d e p r o t e i n i z a t i o n and a suitable solvent. The p r o c e d u r e t o t h i s e f f e c t was d e s c r i b e d a s f o l l o w s : S a t u r a t e d ammonium s u l f a t e s o l u t i o n ( 3 m l ) , 5 % z i n c s u l f a t e ( 2 5 m l ) a n d s a t u r a t e d b a r i u m h y d r o x i d e ( 2 5 ml) were added c o n s e c u t i v e l y t o 25 ml of b l o o d plasma w i t h v i g o r o u s s h a k i n g a f t e r each a d d i t i o n . A f t e r c e n t r i f u g a t i o n f o r 1 5 minutes a t 1500 rpm, t h e s u p e r n a t a n t was t r a n s f e r r e d i n t o a 100-ml g l a s s s t o p p e r e d e x t r a c t i o n t u b e , a d j u s t e d t o pH 9 and e x t r a c t e d t h r e e times w i t h 2 0 m l p o r t i o n s of a c h l o r o f o r m - e t h y l a c e t a t e m i x t u r e (2:l). The combined e x t r a c t s were d r i e d o v e r a n h y d r o u s sodium s u l f a t e , f i l t e r e d and e v a p o r a t e d . The r e s i d u e s d i s s o l v e d i n a p p r o p r i a t e volumes o f m e t h a n o l were a s s a y e d s e m i - q u a n t i t a t i v e l y a l o n g w i t h t y l o s i n s t a n d a r d s by t h i n l a y e r c h r o m a t o g r a p h y as The y i e l d s o f r e c o v e r y v a r i e d between 6 0 - 7 0 % 4 8 , d e s c r i b e d below.
288
I n some cases, t h e t y l o s i n s p o t c o u l d b e o v e r l a p p e d w i t h some i n t e r f e r i n g s u b s t a n c e s . I f t h i s was t h e case, t h e r e s i d u e was d i s s o l v e d i n s e v e r a l m i l l i l i t e r s o f d i l u t e d h y d r o c h l o r i c a c i d (pH 5-61 a n d e x t r a c t e d t h r e e times w i t h e t h e r . The e t h e r e a l e x t r a c t s were d i s c a r d e d and t h e a q u e o u s l a y e r make a l k a l i n e , e x t r a c t e d , d r i e d a n d e v a p o r a t e d as a b o v e 4 8 . 3.5.5
Countercurrent d i s t r i b u t i o n
E t h y l acetate e q u i l i b r a t e d w i t h 0 . 1 M p h o s p h a t e b u f f e r (pH 6 . 0 ) was u s e d t o r e s o l v e desmycosin o r m a c r o c i n from t y l o s i n . Using 6 0 t r a n s f e r s between e t h y l a c e t a t e - - 0 . 1 M p h o s p h a t e b u f f e r (pH 6.01, desmycosin was f o u n d i n t u b e s No. 4 - 1 7 , macroc i n between 16-32 and t y l o s i n i n 3 5 - 5 5 r e s p e c t i ~ e l y ~ ~ . 3.5.6
Thin l a y e r chromatography
S i l i c a g e l 2 5 4 (Merck) p l a t e s were u s e d i n a s t u d y d e a l ing with d e t e c t i o n of t y l o s i n i n b i o l o g i c a l m a t e r i a l s e.g. b l o o d p l a s m a , t i s s u e homogenates, u r i n e and a n i m a l f e e d . S o l v e n t s y s t e m s o f c h l o r o f o r m - - a c e t o n e (60:40) and e t h y l a c e t a t e - methanol (85:15) were employed f o r t h e f i r s t and s e c o n d d i r e c t i o n r e s p e c t i v e l y i n a two-dimentional arrangement. I n t h e f i r s t s o l v e n t s y s t e m , t y l o s i n had a n Rf v a l u e of a b o u t 0.12 and i n t h e s e c o n d s o l v e n t s y s t e m a b o u t 0.60. A f t e r l o c a t i o n o f s p o t s u n d e r UV l i g h t t h e p l a t e s were v i s u a l i z e d with t h r e e s p r a y r e a g e n t s i n t h e following sequence:
(i) I o d o p l a t i n a t e . PtC14.2HC1.6HzO (1 gm) and K1 ( 2 0 gm) were d i s s o l v e d i n c o n c . H C 1 ( 8 ml) a n d made up t o a volume of 4 0 0 ml w i t h d i s t i l l e d water. A t t h i s s t a g e t y l o s i n a p p e a r s as a brown s p o t on a p i n k background. ( i i ) D r a g e n d o r f f ' s r e a g e n t . S o l u t i o n A: b a s i c bismuth n i t r a t e (0.85 gm), a c e t i c a c i d (10 ml), d i s t i l l e d water ( 4 0 ml). S o l u t i o n B : K 1 ( 2 0 gm) d i s s o l v e d i n water (50 m l ) . Both s o l u t i o n s were mixed a n d s t o r e d i n a brown b o t t l e . For s p r a y i n g 5 m l of t h e s t o c k s o l u t i o n were mixed w i t h 3 5 ml of water and 10 m l of a c e t i c a c i d . This s p r a y r e a g e n t i n t e n s i f i e s t h e c o l o r a t i o n of t y l o s i n .
( i i i ) S a t u r a t e d s o l u t i o n of s i l v e r s u l f a t e i n 1 0 % s u l f u r i c a c i d . After spraying w i t h t h i s reagent t h e t y l o s i n s p o t s c h a n g e t h e c o l o r t o orange-brown on a d a r k b a ~ k g r o u n d " ~ . The s e n s i t i v i t y of t h e method i s between 2-4 mcg a n d allows f o r a s e m i - q u a n t i t a t i v e a n a l y s i s .
Microliter s a m p l e s of f e r m e n t a t i o n b r o t h o f S t k e p t o m y c e b dhadiae were a s s a y e d q u a n t i t a t i v e l y on s i l i c a g e l p l a t e s (EM L a b o r a t o r i e s , 0.25 mm t h i c k n e s s ) d e v e l o p e d i n s y s t e m s c o n t a i n i n g
e t h y l a c e t a t e a n d d i e t h y l a m i n e ( 9 5 5 ) . The d e v e l o p e d p l a t e s were a n a l y z e d d i r e c t l y on a t h i n l a y e r p l a t e s c a n n e r ( S c h o e f f e l SD 3000) a t 2 8 3 nm. The Rf v a l u e s of t y l o s i n a n d i t s c o n g e n e r s i n t h e above s y s t e m were as f o l l o w s : t y l o s i n : 0.81; d e s m y c o s i n : 0.63; r e l o m y c i n :0.52; m a c r o c i n : 0.36; l a c t e n o c i n : 0.27"'.
2 89 Those compounds c o n t a i n i n g t h e m y c a r o s y l m o i e t y e . g . m a c r o c i n , t y l o s i n a n d r e l o m y c i n c o u l d be v i s u a l i z e d w i t h a x a n t h y d r o l s p r a y . All e x c e p t r e l o m y c i n r e a c t e d w i t h 4-amino-3-hydrazinoS-nercapto-1,Z , 4 - t r i a z o l e ( P u r p a l d , A l d r i c h Chemical Co. ) 4 7 . 3.5. 7
P a p e r chromatography
S e v e r a l s o l v e n t s y s t e m s were employed f o r s e p a r a t i o n a n d s u b s e q u e n t b i o a u t o g r a p h y of t y l o s i n a n d companion p r o d u c t s . T a b l e 1 l i s t s t h e Rf v a l u e s and t h e c o m p o s i t i o n of t h e s o l v e n t systems 42
.
TABLE 1 Solvent System
Macrocin
Lactenocin
Tylosin
Oeamycosin
Methyl e t h y l ketone, pH 4 b u f f e r e d p q e r
0.45
0.20
0.45
0
Methyl e t h y l ketone
0.66
0.38
0.81
0.63
n - B u t m o l s a t u r a t e d with water, pH 4 b u f f e r e d paper
0.92
0.75
0.90
0.90
n d u t a n o l saturated w i t h water
0.80
0.60
0.84
0.74
0.74
0.86
0.57 0.91
0.76
E t h y l acetate s a t u r a t e d with water, pH 4 b u f f e r e d paper
0.39
0.05
0.89
0.11
MgSO~+--tmthyl e t h y l ketone-water (7:2.5:90)
---
---
0.33
0.65
---
---
0.48
---
NaC1--methyl e t h y l ke tone--water ( 7: 2.5 :90.5)
.a
Oiisq,ropy1 ether--me t h y 1 i s o b u t y l ketone--O.2% ammonium carbonate (2:l:Z)
290 4.
Rosamicin"
4.1 P r o d u c i n g
organism
The p r o d u c e r o f r o s a r n i c i n was i s o l a t e d from a Texas s o i l sample c o l l e c t e d i n San J a c i n t o County. I t was a s s i g n e d NRRL No. 3718 and shown t o be a new s p e c i e s of t h e g e n u s Mickomonob p o n a e . g . h o b ~ h i a ~The ~ . m a j o r component from t h e a n t i b i o t i c complex produced by ~ i c h o m o n o b p o h ahobeopuhpuhea i s c o n s i d e r e d t o b e i d e n t i c a l w i t h ~ o s a m i c i n ~as~ ,i s j u v e n i m y c i n A 3 5 1 , 5 2 . 4.2 P h y s i c a l a n d c h e m i c a l p r o p e r t i e s Rosamicin i s a n example of a 16-membered m a c r o l i d e monog l y c o s i d e . I t has a m e l t i n g g o i n t a t 1 1 9 - 1 2 2 ' C ( c r y s t a l l i z e d from c h l o r o f o r m ) , I a 3 h 0 -35 ( e t h a n o l ) , UV Ama 238) and a pKA 8.7. Rosamicin i s r e a d i l y s o l u ~ l f 4 ! n n ~ e ~ E h ~ ~ 8 ! , a c e t o n e , c h l o r o f o r m and benzene , s p a r i n g l y s o l u b l e i n d i e t h y l e t h e r and s l i g h t l y s o l u b l e i n w a t e r . 4.3 S t r u c t u r e The s t r u c t u r e o f r o s a m i c i n as shown was e l u c i d a t e d by Reimann a n d J a r e t 3 ,
Roramici n
4.4 T h e r a p e u t i c
activity
Based on b i o l o g i c a l d a t a , r o s a m i c i n p o s s e s s e s i n v i t h o and i n v i v o p r o p e r t i e s common t o o t h e r m a c r o l i d e s , w i t h some b e t t e r a c t i v i t i e s a g a i n s t g r a m - n e g a t i v e o r g a n i s m s as w e l l as showing s u p e r i o r s t a b i l i t y . I t i s a l s o a c t i v e a g a i n s t M y c a plasma s p e c i e s 5 4 . P r o t e c t i v e a c t i v i t y a g a i n s t l e t h a l i n f e c t i o n s i n mice i s comparable w i t h e r y t h r o m y c i n . The a c u t e t o x i c i t y i n terms o f LD50 (mg/kg) was 1 0 0 0 o r a l l y , 625 s u b c u t a n e o u s l y , 350 i n t r a p e r i t o n e a l l y a n d 1 5 5 i n t r a v e n o u s l y .
4.5 Isolation 4.5.1
and p u r i f i c a t i o n
Extraction
The f e r m e n t a t i o n f l u i d ( 6 0 a ) was a d j u s t e d t o a pH of 9 . 5 w i t h sodium h y d r o x i d e and e x t r a c t e d t w i c e w i t h 2 volumes o f * a t first named rosaramicin.
291
e t h y l a c e t a t e . The s o l v e n t p h a s e was t h e n s e p a r a t e d a n d c o n c e n t r a t e d t o a v o l u me o f 1 . 5 l i t e r s . The b a s e s from t h e c o n c e n t r a t e were t a k e n i n t o a d i l u t e d s o l u t i o n of s u l f u r i c a c i d (0.1N) by t h r e e 6 0 0 m l e x t r a c t i o n s . The c o m b i n e d a q u e o u s p h a s e s were a d j u s t e d t o a pH o f 9 . 5 w i t h s o d i u m h y d r o x i d e a n d e x t r a c t e d two t i m e s i n small p o r t i o n s w i t h e q u a l v o l u m e s o f e t h y l a c e t a t e . The s o l v e n t p h a s e was s e p a r a t e d a n d c o n c e n t r a t e d t o a volume of a b o u t 7 0 m l . T h i s c o n c e n t r a t e was s l o w l y a d d e d t o a mixture of ether--hexane (3:2) w i t h s t i r r i n g . The r e s u l t i n g p r e c i p i t a t e was f i l t e r e d , d i s c a r d e d a n d t h e m o t h e r l i q u o r s evaporated to dryness. The r e s i d u e was r e d i s s o l v e d i n 1 0 0 m l o f e t h y l a c e t a t e a n d w a s h e d t h r e e t i m e s w i t h 25 m l o f water. The s o l v e n t p h a s e was s e p a r a t e d , d r i e d o v e r s o d i u m s u l f a t e , f i l t e r e d and concentrated t o dryness. T h i s m a t e r i a l was t h e n d i s s o l v e d i n 2 0 m l o f e t h y l e t h e r a n d i n s o l u b l e s i f a n y , were f i l t e r e d o f f a n d d i s c a r d e d . The e t h e r s o l u t i o n was a d d e d t o a b o u t 1 0 0 m l of p e t r o l e u m e t h e r ( b . p . 3O-6O0C) a n d t h e r e s u l t i n g b u f f - c o l o r e d p r e c i p i t a t e f i l t e r e d a n d d r i e d i n v a c u o . The m o t h e r l i q u o r s a f t e r c o n c e n t r a t i o n a f f o r d e d a d d i t i o n a l p r e c i p i t a t e w h i c h was a l s o f i l t e r e d , d r i e d and combined w i t h t h e i n i t i a l p r e c i p i t a t e . The w e i g h t o f t h e c o m b i n e d p r e c i p i t a t e s was a b o u t 1 . 5 g 4 9 . (Materi a 1 p r o d u c e d i n t h i s way was a s s i g n e d a w o r k i n g p o t e n c y o f a b o u t 7 0 0 mcg/mg o f r o s a m i c i n c o m p l e x a c c o r d i n g t o t h e b i o a s s a y procedure). 4.5.2
Column c h r o m a t o g r a p h y
S e p a r a t i o n o f t h e major c o m p o n e n t was a c h i e v e d b y a p p l y i n g t h e r o s a m i c i n c o m p l e x i n a m i x t u r e of c h l o r o f o r m - - m e t h a n o l ( 9 : l ) a t t h e t o p of a s i l i c i c a c i d c o l u m n . The c olum n was developed with chloroform--methanol ( 4 : l ) and e l u a t e s c o l l e c t e d . The i n d i v i d u a l f r a c t i o n s were m o n i t o r e d b y d i s c t e s t i n g e a c h f r a c t i o n a g a i n s t S t a p h y L o c o c c u 6 auheun a n d t h i n l a y e r c h r o m a t o g r a p h y . By t h i s p r o c e d u r e , r o s a m i c i n was s e p a r a t e d a n d i s o l a t e d a f t e r p o o l i n g h o mo g en eo u s f r a c t i o n s , d r y i n g , d i s s o l v i n g i n acetone and p r e c i p i t a t i n g with e t h y l erher. The mother l i q u o r s were e v a p o r a t e d t o d r y n e s s y i e l d i n g t h e a n t i b i o t i c as a w h i t e powder. A t t h i s s t a g e t h e p o t e n c y o f r o s a m i c i n was d e f i n e d as 1000 m ~ g / m g ~ ~ . 4.5.3
Thin l a y e r chromatography
Two s o l v e n t s y s t e m s were u s e d t o d i f f e r e n t i a t e r o s a m i c i n f r o m o t h e r m a c r o l i d e s as s h o wn i n T a b l e 2 , ( n e x t p a g e ) .
292
TABLE 2 Comparative Thin-layer Chromatography o f Roearnicin with some Macrolide A n t i b , i ~ t i c a ~ ~
-
System Chloroform-methanol17% ammonia, 2 : l : l
Eutanol-acetic acidwater, 3 : l : l
Antibiotic
Rf and Color by H ~ S O I +Spray*
Roaamicin Megalomicin Oleandomycin Erythromycin Spiramycin Carbomycin
0.82 0.97 0.78 0.94 0.95 0.96
Roaarnicin
0.31 0.44 0.21 0.38 0.16 0.35 0.14 0.58
Megalomicin Erythromycin Spirarnycin Oleandomycin Carbornycin
0.98 0.95
Tan Black Black Brown Red-B rown Purple
0.37 %
0.45 0.31
0.45 0.36
Tan Purple Brown Red-Brown Black Purple
*Plate heated a t 100°C and sprayed w i t h H2SO4 i n methanol ( 1 : l ) .
293 5. Leucomycin*
5.1 Producing
organisms
The leucomycin complex of antibiotics is synthesized by The original strains of Stkeptomyceb k i t a d a t o e n b i d producer was isolated in 1953. Another strain, designated A 204-P2 and classified as a new variety of S t h e p t o m y c e d nahbonenA i d var. j o 6 a m y c e f i c u b var. noua was reported in 1964 to produce a new macrolide antibiotic called j o ~ a m y c i n ~ ~Josamycin . was shown eventually to be identical with one of the leucomycin components e.g. leucomycin A 3 5 7 . The major product in S t h e p t o mycin h i t u b a t o e n d i d is leucomycin A 1 whereas the other producer favors the A 3 form. 5.2 Structure At present the leucomycin complex comprises ten congeneric 16-membered macrolides Characterized by a mycaminosylmycarose disaccharide attached at the C-5 of the chromophoric lactone. Different acyl functions may be present or absent at the C-3 of the leuconolide and at the C-4 of mycarose whereas in common they have an a , 6, A , (I diene to the secondary alcohol at C - 9 and a formylmethyl group at C-6. Leucomycins are weakly basic -pKA lipophilic and form water soluble salts.
2,
6.75 in 50% ethanol,
Structural studies were initiated by Watanabe and his co-workers5 8 9 59. Afterwards bmura and his associates made numerous contributions as to structure, stereochemistry of l e u c c m y ~ i n s ~ ~their ’ ~ ~ structural relationship and microbiological activities7d as well as their b i o ~ y n t h e s i s ~ ~The . structures of the leucomycins are shown on the next page.
5.3 Therapeutic activity The antimicrobial acrivity of leucomycins was shown to be similar to most of the other macrolide antibiotics. Some properties have been found to be superior, e.g. acid stability. They also have been reported as resistance non-inducing macrolides 72. Practical use of leucomycin started in 1955 and many clinical data have thus been published. Similarly, extensive studies were conducted with josamycin (leucomycin A,) which resulted in the most com lete documentation of various clinical and non-clinical aspectsP2, Clinical studies confirmed leucomycin to be as effective as erythromycin in the treatment of staphylococcal and gram-.positive infections in patients. Excellent responses were observed in acute respiratory infections, streptococcal infections and purulent infections caused by staphylococci. *Named Kitasamycin by the International Center o f Information on Antibiotics, Liege, Belgium, 1969.
294
Laucomycin
H
A,
A4
dIf
A5
H
A3
0
I f
0 A7
H
A8
0
A9
H
U
0
V
I f
I f
H
H
E f f e c t i v e d o s e s s t a r t w i t h 6 0 0 mg p e r day t o o u t p a t i e n t s w i t h m i l d c a s e s . Good t h e r a p e u t i c r e s u l t s were o b t a i n e d w i t h 8 0 0 - 1 2 0 0 mg p e r day d i v i d e d i n t h r e e o r f o u r p o r t i o n s . In c h i l d r e n , 2 0 - 4 0 mg/kg p e r day o r a l l y a d m i n i s t e r e d gave s a t i s f a c t o r y r e s u l t s . The a n t i b i o t i c showed low i n c i d e n c e of s i d e e f f e c t s such a s a n o r e x i a , e p i g a s t r i c d i s t r e s s o r n a u s e a 7 3 . 5.4 Isolation 5.4.1
Extraction
According t o t h e o r i g i n a l p r o c e d u r e t h e f e r m e n t a t i o n b e e r of S t h e p t o m y c e d k i t a d a t o e n d i d was a d j u s t e d t o pH 4 and f i l t e r e d . A l k a l i n i z a t i o n of t h e f i l t r a t e t o a pH of 8 . 0 was f o l l o w e d by e x t r a c t i o n w i t h b u t y l a c e t a t e , The b u t y l a c e t a t e l a y e r was ext r a c t e d t h r e e times w i t h d i l u t e d h y d r o c h l o r i c a c i d . From t h e
295
combined a c i d e x t r a c t s t h e b a s e s p r e c i p i t a t e d a t pH 8 . 0 . Six d e f i n e d components d e s i g n a t e d A , and A and B l - B , were o b t a i n e d d countercurrent disa f t e r Amberli t e IRC-50 c h r ~ m a t o g r a p h ya~n ~ t r i b u t i o n 5. 5.4.2
Column c h r o m a t o g r a p h y
Improvement i n p r o d u c t i v i t y of t h e p r o d u c i n g o r g a n i s m l e d t o i s o l a t i o n o f e i g h t major components which were shown t o b e n o v e l , e x c e p t A l , when compared w i t h t h e e a r l i e r m i x t u r e . The f l o w d i a g r a m d e p i c t s t h e i s o l a t i o n p r o c e d u r e . The new l e u c o mycins h a v e b e e n r e f e r r e d t o from A 3 up t o A g . Interestingly enough t h e A 2 and t h e B-group w e r e n o t d e t e c t e d among t h e metab o l i t e s o f t h e improved s t r a i n 7 3 . Leucomycin Complex
column chromatography on alumina
ethyl acetate--benzene
( 3 : l ) e luate
on s i l i c a g e l benzene--acetone (5: 1-72) A3
A4
5.4.3
A6
e t h y l acetate eluate
I
c o l . chrom. on s i l i c a gel
A8
Thin l a y e r c h r o m a t o g r a p h y
A s u i t a b l e t h i n l a y e r chromatography system c o n s i s t i n g Leuo f b e n z e n e - - a c e t o n e (2:l) was u s e d on K i e s e l g u h r p l a t e s . comycin A 3 had an Rf 0 . 6 9 i n t h i s s y s t e m . F o r d e t e c t i o n 2 0 % s u l f u r i c a c i d was u s e d 5 7 .
5.4.4
Paper chromatography
Paper chromatography is o f h i s t o r i c a l s i g n i f i c a n c e s i n c e i t was u s e d i n m o n i t o r i n g t h e c o m p o s i t i o n of f r a c t i o n s f r o m c o u n t e r c u r r e n t d i s t r i b u t i o n procedure of a mixture c o n t a i n i n g mainly t h e leucomycins of t h e B type. L a t e r , w i t h t h e improvement of t h e p r o d u c t i v i t y of t h e s t r a i n t h e s e compounds were n o t d e t e c t e d 7 3 . The chromatograms were d e v e l o p e d i n b u t y l a c e t a t e s a t u r a t e d w i t h p h o s p h a t e b u f f e r pH 6 or 8 . 5.4.5
High p r e s s u r e l i q u i d c h r o m a t o g r a p h y
HPLC h a s b e e n shown s u p e r i o r t o a n y p r e v i o u s a n a l y t i c a l p r o c e d u r e d e a l i n g w i t h d e t e c t i o n and s e p a r a t i o n of t h e l e u c o -
296 'mycin complex. The eight components of the complex ( A l and A 3 - A 9 ) produced by S t J L e p t o m y c e b k i t a b a t o e n b i d No. 23-1 including leucomycin U and V were resolved satisfactorily on a reverse phase column with a mobile phase composed of methanol--O.O6M acetate buffer (pH 4,9)--acetonitrile (35:60:5, v/v/v). The composition of the eluent mixture was critical, as was the pH of the buffer, since it influenced retention time and resolution. The detector was preset at a wave length which agreed with the Amax of the leucomycin chromophor thus ensuring the highest sensitivity. The retention times of the known leucomycins are listed below
: Leucomycin
Retention time (min)
A1
17.5
A3
25.8
A5
8.0
A4
11.5
A7
4.0
A6
6.0
A9
2.5
A8
3.5
U
2 .a
V
1.5
Instrument: JASCO FLC-150. Detector: JASCO UVIDEC-2 (232.5 nm) Column : JASC OP ACK S V-02-500. Column temperaspe c t rophot omete r ture: anbient. Column pressure: 65 kg/cm2. Flow r a t e : 1 ml/min.
.
297 6. Maridomycin
6.1 Producing organism The producer strain of maridomycin(s) was isolated from a soil sample collected in Chichibu City, Saitama Prefecture, Taxonomical Japan, and designated Stheptomyced sp. No. B - 5 0 5 0 . characteristics allowed the classification of No- B - 5 0 5 0 as a strain of StheptomgceA hgghodcopicud (Jensen, 1931) Waksman et Henrici, 194877.
6.2 Physical and
chemical properties
The maridomycin complex com rises six closely related 16membered macrolide bis-glycosides78-80. They are weak bases soluble in sethanol, ethanol, acetone, ethyl acetate, and chloroform; slightly soluble in ethyl ether and benzene; sparingly soluble in hexane, petroleum ether and water. Their physico-chemical properties are listed in Table 3. TABLE 3 I* -
Maridomycin
II* -
Ill* -
I V -
V -
VI -
('c)
129-32
134-6
135-8
143-6
144-9
149-54
( E l , EtOH)
-72.3'
-71.9'
-76.0'
-76.2'
-73.2'
-77.7'
Mol. weight M+
857
843
8 29
815
815
801
PKL
6.8
6.8
7.1
6.9
7.1
7.1
m.p.
* I d e n t i c a l w i t h YL-704C3, YL-704Cz and YL-704C 1
respectivelye1
6.3 Structure_ The structure of maridomycin(s) is on the following page.
6.4 Therapeutic activity Maridomycin I, the most potent of the maridomycin complex exerts a pronounced antibacterial effect against certain grampositive bacteria, N e i d b e h i a gonohkhoeae and V i b h i o c h o L e h a e e 2 . It has also been shown to be active against clinical strains of staphylococci of the group B and C as classified by Mitsuhashie3 In experimental infections (mice) caused by StaphyLococcu6 aukeu4 3 0 8 A - I , StheptocOccuA pgogened E - 1 4 and DipLococcuA pneumoniae t y p e 7 , maridomycin was as effective as leucomycin. The acute toxicity after intraperitoneal administration was more than 1 g/kg in mice.
6.5 Isolation 6.5.1
and purification
Extraction
The broth filtrate (4,000 liters) was adjusted to pH 8
298
OH
Rl
Maridomycin
I
n m
0
zp
0
Y XI
'
f
1
0
l
P
d
0
and e x t r a c t e d w i t h one t h i r d of e t h y l a c e t a t e . A f t e r volume r e d u c t i o n t o 100 l i t e r s i n v a c u o and washing w i t h water, t h e c o n c e n t r a t e was e x t r a c t e d t h r e e times w i t h 5 0 l i t e r s o f 0.33M K H ~ P O I , s o l u t i o n a d j u s t e d t o pH 3 w i t h p h o s p h o r i c a c i d . The combined aqueous e x t r a c t s were made a l k a l i n e (pH 9 ) and ext r a c t e d a g a i n w i t h e t h y l a c e t a t e . The e x t r a c t was washed w i t h w a t e r and c o n c e n t r a t e d t o a volume of 1 . 5 l i t e r s . A d d i t i o n of n-hexane (30 l i t e r s ) t o t h e c o n c e n t r a t e p r e c i p i t a t e d a t o t a l of 5 2 2 gm of c r u d e m a t e r i a l , which gave 2 7 2 gm o f maridomycin complex upon c r y s t a l l i z a t i o n from benzene a s c o l o r l e s s n e e d l e s , m.p. 137-141OC. Next, 4 0 gm o f t h e maridomycin complex i n 4 l i t e r s of e t h y l a c e t a t e were r o u g h l y f r a c t i o n a t e d by p a r t i t i o n w i t h 0.1M c i t r a t e b u f f e r pH 4 . 1 6 ( t w i c e w i t h 2 l i t e r s ) . The o r g a n i c phase a f t e r washing w i t h w a t e r and e v a p o r a t i o n y i e l d e d c r y s t a l s from benzene ( 9 . 4 gm) c o n s i s t i n g o f maridomycin I and I1 ( f r a c t i o n 1). The aqueous p h a s e s were combined, a d j u s t e d t o pH 8 and e x t r a c t e d w i t h e t h y l a c e t a t e . The r e s i d u e a f t e r evap o r a t i o n of t h e o r g a n i c s o l v e n t ( 2 6 . 8 gm) c o n t a i n e d maridomycin 111, I V , V and VI ( f r a c t i o n 2 ) . 6.5.2
Column chromatography
Chromatography of f r a c t i o n 1 ( 2 gm) was c a r r i e d o u t on a s i l i c a g e l column (Merck,O.OS-0.2 mm, 2 5 0 gm) c o l l e c t i n g f r a c t i o n s of 2 0 m l . The column was f i r s t d e v e l o p e d w i t h an e t h y l
299
a c e t a t e - - b e n z e n e m i x t u r e ( l : l , 2 0 0 m l ) f o l l o w e d by a n 3 : 2 r a t i o of t h e same p a i r o f s o l v e n t s . The c o m p o s i t i o n o f t h e i n d i v i d u a l f r a c t i o n s was m o n i t o r e d by t h i n l a y e r c h r o m a t o g r a p h y u s i n g a s o l v e n t system o f benzene--acetone ( 3 : 2 ) . Chromarographic a l l y homogenous f r a c t i o n s were combined and e v a p o r a t e d . I n t h i s manner 435 mg o f maridomycin I a n d 1 8 5 mg of maridomycin I1 were o b t a i n e d in p u r e , c r y s t a l l i n e form. For c h r o m a t o g r a p h y o f f r a c t i o n 2 ( 5 gm) a column was p r e p a r e d from 500 gm of s i l i c a g e l s a t u r a t e d w i t h t h e u p p e r l a y e r r e s u l t i n g from t h e mixing of n - h e x a n e - - e t h y l a c e t a t e - - e t h a n o l - water (1:1:1:2). The column was d e v e l o p e d w i t h t h e same s o l vent mixture. F r a c t i o n s o f 2 0 m l were c o l l e c t e d and m o n i t o r e d by t h i n l a y e r c h r o m a t o g r a p h y as a b o v e . F r a c t i o n s which g a v e a s i n g l e s p o t were p o o l e d and e v a p o r a t e d t o d r y n e s s . C r y s t a l l i z a t i o n a f f o r d e d maridomycin I 1 1 ( 1 . 6 4 gm), I V ( 4 5 0 mg), V ( 5 0 0 mg) and V I ( 2 2 0 mg). An a l t e r n a t e s e p a r a t i o n of t h e maridomycin complex was a c c o m p l i s h e d on a smaller s c a l e on a C e l i t e 535 column ( J o h n s M a n v i l l e , 5 gm) by e m p l o y i n g t h e l o w e r p h a s e of a s y s t e m of n - h e x a n e - - e t h y l e n e dichloride--methanol--water ( 1 2 0 : 9 0 : 3 0 : 6 ) a n d c h l o r o p h e n o l r e d as an i n d i c a t o r . The i n d i c a t o r was t i t r a t e d w i t h hydrogen c h l o r i d e t i l l t h e a p p e a r a n c e of t h e y e l l o w c o l o r p e r s i s t e d . Maridomyein complex ( 2 5 mg) d i s s o l v e d i n t h e u p p e r l a y e r ( 0 . 4 m l ) was a p p l i e d a t t h e t o p of t h e column w h i c h was s l o w l y d e v e l o p e d w i r h t h e u p p e r l a y e r . P u r p l e b a n d s r e v e a l e d t h e s e p a r a t i o n of t h e components w h i c h were e l u t e d i n t h e s e q u e n c e maridomycin I, 11, 111, IV, V a n d VI. U s u a l l y y i e l d i n g 4 , 3 , 5 mg of t h e f i r s t t h r e e compounds, a m i x t u r e of IV and V (4 mg), and 2 mg of V I e 4 . 6.5.3
Thin l a y e r c h r o m a t o g r a p h y
Rf v a l u e s of maridomycins a r e shown i n T a b l e 4 . TABLE 4
S o l v e n t System
I I1 I11 IV V VI
* Spotfilm, *+ Kieselgel
***
0.48 0.42 0.37 0.32 0.30 0.27
0.68 0.63 0.57 0.53 0.50 0.43
0.71 0.66 0.61 0.55
0.52 0.48
Tokyo-kasi G Merck
Kieselgel G Merck d e v e l o p e d t h r e e times. F o r d e t e c t i o n the f o l l o w i n g r e a g e n t s were u s e d : i ) c o n c . s u l f u r i c a c i d , i i ) 5% i o d i n e i n c h l o r o f o r m , i i i ) 5% phosphomolybdic a c i d i n e t h a n o l , i v ) 5% c e r i c s u l f a t e i n 1 N s u l f u r i c a c i d a 4 .
300 7 . Midecamycin ( S F - 8 3 7 ) 7 . 1 Produc ing -
organism
Stheptomyceb mycano daciens Shomura and Niida sp. nova elaborates an antibiotic complex which comprises four closely interrelated antibiotic substances originall designated S F - 8 3 7 , S F - 8 3 7 A 2 , S F - 8 3 7 A 3 and SF-837Ak r e s p e c t i ~ e l y ~ ~ , ~ ~ .
7 . 2 Physical and
chemical properties
As a white powder the major component melts at 1 2 2 - 1 2 4 ' C , when crystallized $rom benzene-gexane it forms colorless needles me ting at 1 5 5 - 1 5 6 C . { a I z 2 - 6 7 (c = 1, ethanol), Amax 2 3 2 nm El cm 3 2 5 , (ethanol), pKA i . 9 ( 5 0 % ethanol). S F - 8 3 7 has been skown to be identical with YL-704 B1e7.
B
7 . 3 Structure -
The structures of the midecamycins differ at C-9 by having a secondary hydroxyl or an 0x0-function, and propionyl or butyryl ester functions at C-4" of mycarose as shown by the structural formulae88-40. X
0
SF- 837A,
0
SF- 8 3 7 4
0
SF- 837A,
0
7 . 4 Therapeutic
H, OH
I f \
SF- 837
I
F
H,
0
0
F
0
OH
activity
Midecamycin is primarily active against gram-positive bacteria, less active against gram-negative bacteria, yeast and molds. It does not show cross-resistance with erythromycin and its toxicity is low: 6 0 0 0 mg/kg in micee5.
30 1
7.5 I s o l a t i o n 7.5.1
and p u r i f i c a t i o n
Extraction
F i l t e r e d b r o t h ( 1 5 0 l i t e r s ) h a s b e e n e x t r a c t e d a t pH 8 w i t h f i f t y l i t e r s o f e t h y l a c e t a t e . The b a s i c m a t e r i a l from t h e e t h y l a c e t a t e l a y e r was t a k e n i n t o t w e n t y l i t e r s o f a d i l u t e d a c i d s o l u t i o n (pH 2 ) a n d , a f t e r a d j u s t i n g t h e a q u e o u s p h a s e t o pH 8 , e x t r a c t e d a g a i n w i t h e t h y l a c e t a t e (10 l i t e r s ) . Dec o l o r i z a t i o n o f t h e f i n a l e t h y l a c e t a t e e x t r a c t w i t h a c t i v e car bon a n d e v a p o r a t i o n l e f t a r e s i d u e which was d i s s o l v e d i n 300 m l o f b e n z e n e . E v a p o r a t i o n of b e n z e n e a f f o r d e d 6 2 gm of c r u d e m a t e r i a l w i t h an a c t i v i t y o f 720 mcg/mg. 7.5.2
Countercurrent d i s t r i b u t i o n
T h i s material h a s b e e n s u b j e c t e d t o a c o u n t e r c u r r e n t d i s t r i b u t i o n procedure using a s o l v e n t system c o n s i s t i n g of b e n z e n e a n d 0.3M p h o s p h a t e b u f f e r pH 4 . 1 . Two l i t e r p h a s e s h a v e b e e n u s e d d u r i n g t h e s e v e n t r a n s f e r s , The b u l k of S F - 8 3 7 was d i s t r i b u t e d i n t o t u b e s n o s . 2 ~ 4 ,w h e r e a s t u b e n o . 1 s t i l l c o n t a i n e d a p o r t i o n o f SF-837 a l o n g w i t h t h e minor components A 2 , A 3 , a n d AL,. Evaporation of t h e no. 1 f r a c t i o n gave a w h i t e powder ( 8 . 3 gm) from w h i c h t h e i n d i v i d u a l components were i s o l a t e d by s i l i c a g e l column Chromatography ( 7 0 0 m l ) i n benzene-a c e t o n e ( 4 : 1 ) , c o l l e c t i n g f r a c t i o n s o f 50 m l . SF-837Ab was e l u t e d f i r s t i n f r a c t i o n s n o s , 2 5 ~ 2 8 , a f f o r d i n g 180 mg of t h e p u r e a n t i b i o t i c , A2 a n d A 3 e l u t e d n e x t i n f r a c t i o n s no. 36-52 Fractions nos. w i t h an a d m i x t u r e o f SF-837, t o t a l l i n g 2 . 1 gm. 64-86 c o n t a i n e d SF-837 ( 2 - 3 gm). More o f SF-837 ( 2 4 gm) was r e c o v e r e d ( f r o m t u b e s n o s . 2-4 o f c o u n t e r c u r r e n t d i s t r i b u t i o n ) a f t e r column c h r o m a t o g r a p h y on s i l i c a g e l . 7.5.3
Column c h r o m a t o g r a p h y
The m i x t u r e o f SE-837A2 a n d A 3 ( 2 gm) i n b e n z e n e (10 m l ) was r e c h r o m a t o g r a p h e d on an a l u m i n a column ( 2 0 0 ml) d e v e l o p e d w i t h e t h y l a c e t a t e - - b e n z e n e (1:l) m i x t u r e . 2 0 m l f r a c t i o n s were c o l l e c t e d of which n o s , 52-76 c o n t a i n e d p u r e SF-837A3 ( 2 8 0 mg). S t a r t i n g w i t h f r a c t i o n n o . 9 0 , when t h e e l u e n t s o l v e n t r a t i o u s e d was c h a n g e d t o e t h y l a c e t a t e - - b e n z e n e ( 2 : 1 ) , SF-837 A 2 was e l u t e d i n f r a c t i o n s n o s . 115-132. The whole p r o c e d u r e is summarized i n t h e f l o w c h a r t s g , ( n e x t p a g e ) . 7.5.4
Rf v a l u e s of t h e f o u r midecamycins i n two s o l v e n t s y s t e m s on s i l i c a g e l a n d a l u m i n a r h i n l a y e r c h r o m a t o g r a p h y a r e shown i n Table 5 (page following flow c h a r t ) .
30 2
countercurrent d i a t r ib u t i o n ( 7 t r a n s f e r s i n benzene--0.3M phosphate b u f f e r pH 4.1)
1 i l i c a gel c o l u m ~enzene--acetone (4:1)
s i l i c a gel chromat. benzene-acetone (4:l)
SF-a37 (24gm)
J
evaporation
J
evaporation
SF-837Ak (180 IMJ)
alumina c o l . chrom. E t0Ac--benzene
SF-837 (2.6 cjin)
(l:l, 2:l)
evaporation
j evaporation
SF-837A3 (280 mg)
SF-sj7A2 (210 mg)
J
30 3
TABLE 5 _L_
SF-837
TLC* S i l i c a gel
2 !
benzene--acetone
3!
&t
SF-837**
0.51
0.50
0.55
0.45
0.41
0.45
0.52
0.32
(2:U Alumina
Et OAc--benzene
(1:l)
*
**
10% s u l f u r i c a c i d served as a d e t e c t i o n reagent. Rf i n n-butanol--acetic 0.82 and acetone--water
acid--water ( 3 : l : l ) (49:l) O.9Zs5.
was 0.67,
i n methanol
Acknowledgement The a u t h o r g r a t e f u l l y a c k n o w l e d g e s t h e c o o p e r a t i o n of Dr. S . Omura, Kirasato U n i v e r s i t y , T o k y o ; D r . W . D . Celmer, P f i z e r I n c . ; a n d Drs. R . L . H a m i l l , H . M . H i g g i n s a n d E . T. S e n o , E l i L i l l y a n d Co.
30 4 8. L i , t e r a t u r e c i t e d
,
1.
J . M . McGuire, R . L. Bunch, R . C . A n d e r s o n , H. E. Boaz, E . H . F l y n n , H . P o w e l l a n d J. E . S m i t h , A n t i b i o t . a n d Chemoth. 2 (1952) 281-283.
2.
R . M . Thompson a n d F . M . S t r o n g , Biochem. B i o p h y s . Res. Commun. 43 (1971) 213-216.
3.
R . K . C l a r k , J r . , A n t i b i o t . a n d Chemoth. 3 (1953) 663-671.
4.
E. H. F l y n n , M . V . S i g a l , J r . , P . F. W i l e y a n d K . G e r z o n ,
J. Am. Chem. SOC. 76 (1954) 3121-3131.
'
5.
P . F. W i l e y , K . G e r z o n , E . H . F l y n n , M . V . S i g a l , J r . , a n d V . C . Q u a r c k , J. Am. Chem. SOC. 77 (1955) 3676-3677.
6.
M . V . S i g a l , J r . , P. F. W i l e y , K . G e r z o n , E. H. F l y n n , V . C . Q u a r c k and 0 . W eaver, J . Am. Chem. S o c . 78 (1956) 388-395.
7.
P. F. W i l e y , K . G e r z o n , E. H . F l y n n , M . V . S i g a 1 , J r . , 0 . Weaver, V . C . Q u a r c k , R . R . C h a u v e t t e a n d R . Monahan, J. Am. C 5 e m . SOC. 79 (1957) 6062-6070.
8.
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J . A n t i b i o t . 24 (1971) 460-475.
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309 Marine -Derived An tib iot ics Lois S
. Shield
.
and Kenneth L Rinehart. Jr Department of Chemistry University of Illinois. Urbana. Illinois
1
.
2.Cyano.4, 5.dibromopyrrole 1.1 Introduction 1.1.1 Source organism 1.1.2 Chemical description 1.1.3 Structural formula 1.2 Bioactivity 1.3 Extraction. separation and purification Literature cited
321 321 321 321 321 321 321
4-Bromopyrrole-2-carbonylguanidine 2.1 Introduction 2.1.1 Source organism Chemical description 2.1.2 Structural formula 2.1.3 2.2 Bioactivity Extraction, separation and purification 2.3 2.4 Literature cited -
322 322 322 322 322 322 322
1.acetamide Introduction Source organism 3.1.2 Chemical description 3.1.3 Structural formula 3.2 Bioactivity 3.3 Extraction, separation and purification 3.4 Literature cited -
322 322 322 323 323 323 323
3,5.Dibromo.l.hydroxy.4.oxo.2~5.cyclohexad~en e. 1-acetamide 4.1 Introduction 4.1.1 Source organisms 4.1.2 Chemical description 4.1.3 Structural formula 4.2 Bioactivity 4.3 Extraction, separation and purification 4.1, Literature cited -
323 323 323 324 324 324 324
2,4-Dibromo-3, 6-dihydroxybenzeneacetamide 5.1 Introduction 5.1.1 Source organism 5.1.2 Chemical description 5.1.3 Structural formula 5.2 Bioactivity 5.3 Extraction, separation and purification 5.4 - Literature cited
325 325 325 325 325 325 326
1.4 2
3
4
.
.
.
.
............................... .......................... ..................... ....................... .................................... ...........................
............................... .......................... ..................... ....................... .................................... ...........................
3,5.Dibromo.4.hydroxybenzene.
3.1 3.1.1
............................... .......................... ..................... ....................... .................................... ...........................
............................... ......................... ..................... ....................... .................................... ...........................
5
.
............................... .......................... ..................... ....................... ....................................
...........................
310 6
.
(1~,6~).(.).3,5.Dibromo.l,6.dihydroxy.4.methox y. 2 ,4. cy clohexad iene-1-aceton itri le I(-)-Aeroplysinin-ll 6.1 Introduction 6.T.1 Source organism 6.1.2 Chemical des.cription 6.1.3 Structural formula 6.2 Bioactivity 6.5 Extraction. separation and purification 6.4 Literature cited -
326 326 326 326 326 326 327
(1~,6~).(+).3,5.Dibromo.l,6.dihydroxy.4.methox y. 2,4.cyclohexadiene. 1.acetonitrile C(+)-Aeroplysinin-l? 7.1 Introduction Source organisms 7.1.1 Chemical description 7.1.2 Structural formula 7.1.3 7.2 Bioactivity Extraction. separation and purification 7.3 Literature cited 7.4 -
............................... ......................... ..................... ....................... .................................... ...........................
327 327 327 327 328 328 328
l.(3.$.D.Arabofuranosyl)uracil (Spongouridine) Introduction Source organism 8.1.2 Chemical description 8.1.3 Structural formula 8.2 Bioactivity 8.3 Extraction. separation and purification 8.4 Literature cited -
. ............................... .......................... ..................... ....................... .................................... ...........................
328 328 329 329 329 329 330
5,6.Dibromo.lH.indole. 3.ethanamine 9.1 Introdu-dtion 9.1.1 Source organism 9.1.2 Chemical description 9.1.3 Structural formula 9.2 Bioactivity 9.3 - Extraction, separation and purification 9.4 Literature cited -
330 330 330 330 330 330 330
3,5.Dibromo.l.hydroxy.4,4.dimethoxy.2,5.cycl 0 . hexadiene-1-acetami.de 10.1 Introduction TKI.1 Source organisms 10.1.2 Chemical description 10.1.3 Structural formula 10.2 Bioactivity 10.3 Extraction. separation and purification 1 0.4 Literature cited -
331 331 331 331 331 331 331
............................... ........................... ..................... ....................... ................................ .... ...........................
7
.
8
9
10
.
.
.
8.1 8.1.1
............................... .......................... ...................... ....................... .................................... ........................... ............................... ......................... ..................... ....................... .................................... ...........................
311
11
.
12*
13
14
.
.
. ................................ ........................... ...................... ........................ ................................. ................................. Dibromophakellin 12.1 Introduction................................ m.1 Source organism ........................... 12.1.2 Chemical description ...................... 12.1.3 Structural formula ........................ 12.2 Bioactivity ................................. 12.3 Extraction. separation and purification ..... 12.4 Literature cited ............................ (Spongothymidine) Introduction 11.1.1 Source organism 11.1.2 Chemical description 11.1.3 Structural formula 11.2 Bioactivity 11.3 Extraction. separation and purification 11.4 Literature cited 1.(3.B.D.Arabofuranosyl)thymine
11.1
5,6.Dibromo.lH.indole.3.( N.methylethanamine1 13.1 Introduction 13.1.1 Source organism 13.1.2 Chemical description 13.1.3 Structural formula 13.2 Bioactivity 13.3 Extraction, separation and purification Literature cited
................................ ........................... ...................... ........................ ................................. .................................
3,5-Dibromo.4.ethoxy-l-hydroxy-4.methoxy-2,
15
.
333 333 333 333 333 333 333
333 334 334 334 334 334 334
5.
cyclohexadiene-1-acetamide 14.1 Introduction m.1 Source organism 14.1.2 Chemical description 14.1.3 Structural formula 14.2 Bioactivity 14.3 Extraction, separation and purification 14.4 Literature cited -
-
332 332 332 332 332 332 332
................................ ........................... ...................... ........................ ................................. .................................
334 334 334 335 335 335 335
3-Bromo-2-(4-bromophenoxy)-phenol
................................ ........................... ...................... ........................ ...................................... ............................ 1 6 . 3.4.5-Tribromo-2-(2.4-dibromophenoxy )-phenol 16.1 Introduction................................ TKi.1 Source organism ........................... 16.1.2 Chemical description ...................... 16.1.3 Structural formula ........................ 16.2 Bioactivity ................................. Extraction. separation and purification ..... 16.4 Literature cited ............................ 15.1 Introduction TS.l.1 Source organism 15.1.2 Chemical description 15.1.3 Structural formula 15.2 Bioactivity 15.3 Extraction. separation and purification Literature cited
335 335 335 335 336 336 336 336 336 336 337 337 337 337
312
17
.
{l~.(la.2~.4a~.8a.8a.)}.Decahydro.l.isocyano.4a. dimethyl-2-(l-methylethenyl)-naphthalene ( Acan th el 1in-1) 17.1 Introduction 17.1.1 Source organism 17.1.2 Chemical description 17.1.3 Structural formula 17.2 Bioactivity
17.4 18
.
. 8
337 ............................... 337 .......................... 337 ..................... 337 ....................... 337 ................................ Extraction. separation and purification .... 337 338 Literature cited ...........................
(Z,E).(R).(.).5.{5.(3.Furyl).2.methyl.l.pentenyl }. 3~{5-~3=furyl)propylidene}-dihydro-2(3~)-furanone
(Nitenin)
............................... .......................... ..................... ....................... ................................ Extraction, separation and purification .... Literature cited ...........................
18.1 Introduction m.1 Source organism 18.1.2 Chemical description 18.1.3 Structural formula 18.2 Bioactivity
18.3 18.4 19
.
.
E S - { R* S*- ( E) 1 1 -a-{ 5- ( 3 .Furanyl).2-methyl-2. pFn tenyTI . y =me thy1. 3. f ur anhexano1 (Furospongin-1) 19.1 Introduction 19.1.1 Source organisms 19.1.2 Chemical description 19.1.3 Structural formula 19.2 Bioactivity 19.3 Extraction. separation and purification Literature cited
............................... ......................... ..................... ....................... .................................... ...........................
20
.
338 338 338 338 338 339 339
339 339 339 339 339 340 340
5.I9.C5.(3.Furanylmethyl).3.furanyl}.2,6.dimethy 1. 6-nonenylidene}-4-hydroxy-3-methyl-2(5~)-furanone (Ircinin-1) and 5-{9-{5-(3-Furanylrnethyl)-3furanyl~.2,6.dimethyl.5.nonenylidene~.4.hydr0xy. 3.
methyl-2(5E)-furanone (Ircinin-2) 20.1 Introduction 20.1.1 Source organism 20.1.2 Chemical description 20.1.3 Structural formulas 20.2 Bioactivity 20.3 Extraction, separation and purification Literature cited
-
21
.
............................... 340 .......................... 340 ..................... 340 ...................... 340 ................................ 341 .... 341 ........................... 341
5.~13.(3.F~ranyl).2,6~lO.trimethyl.6,10.tr i. decadienylideneI-4-hydroxy-3-methyl-2(5H).
furanone (Variabilin) Introduction Source organisms 21.1.2 Chemical description 21.1.3 Structural formula
21.1 21.1.1
............................... ......................... ..................... .......................
341 341 342
342
313
21
.
5.{13.(3.Fhranyl).2.6.10.trimethyl.6.lO.tr i. decadienylidene~-4-hydroxy-3-1nethyl-2(5~)-
furanone (Variabilin) (continued) 21.2 Bioactivity Extraction. separation and purification 21.4 Literature cited
................................ .... ...........................
22
.
3aB.4.5a.6.7.8.11.12.13.14.15aci.Dodecahydro.6 hydroxy.6.10.14B.trimethyl.3.methylene.5.15.epox cyclotetradeca{blfuran-2(3H)-one (Eunicin) 22.1 Introduction
22.1.1
a.
y.
............................... .......................... ..................... ....................... ................................ ...............................
Source organism 22.1.2 Chemical description 22.1.3 Structural formula 22.2 Bioactivity 22.3 Extraction. separation and purification 22.4 Literature cited 23
.
(Asperdiol) Introduction 23.1.1 Source organisms 23.1.2 Chemical description 23.1.3 Structural formula 23.2 Bioactivity 23.3 Extraction. separation and purification 23.4 Literature cited 23.1
-
.
............................... ......................... ..................... ....................... ................................ .... ........................... ............................... ......................... ..................... ....................... .................................... ...........................
.
343 343 344 344 344 344 344
(1~.2~.12~.13S).2.12.Dihydroxy.4.8.12.trimethy 1. 16-methylene-l4-oxabicyclo~ll.3.13heptadeca-4,8-
dien-15-one (Crassin acetate) 24.1 Introduction T T i . 1 Source organisms 24.1.2 Chemical description 24.1.3 Structural formula 24.2 Bioactivity 24.3 Extraction. separation and purification 24.4 Literature cited 25
342 342 343 343 343 343 343
(1R.2E.6R.7S.10E.14R).6.7.€poxy.3.hydroxymethy 1. 14~is~pr~pefiyl.7.11-dimethyl.cyclotetradeca.2.1 0.
dienol
24
342 342 342
a.(1,2.Dihydroxypropyl).2,5.dihydro.2.methyl. 0x0-2-furantetradecanoic acid 25.1 Introduction 233.1 Source organism 25.1.2 Chemical description 25.1.3 Structural formula 25.2 Bioactivity
-
344 344 345 345 345 345 346
. 5
............................... .......................... ..................... ....................... ................................ Extraction, separation and purification .... Literature cited ...........................
346 346 346 347 347 347 347
3 14
26
.
6.Bromo.3.7.7.trimethyl.2.13.dioxat~icycl (9 .3.0.03~eltetradec.10.en.1 2.one
0 .
(Aplysistatin)
............................... .......................... ..................... ....................... ................................ .... ...........................
26.1 Introduction 16.1.1 Source organism 26.1.2 Chemical description 26.1.3 Structural formula 26.2 Bioactivity Extraction. separation and purification 16.4 Literature cited 27
.
Holotoxin A 27.1 Introduction Source organism 27.1.2 Chemical description 27.1.3 Structural formula 27.2 Bioactivity 17.3 Extraction. separation and purification Literature cited
m.l
28
.
.
30
.
............................... 348 .......................... 348 ..................... 348 ....................... 348 ................................ 350 .... 350 ........................... 350
Holotoxin B 28.1 Introduction 28.1.1 Source organism 28.1.2 Chemical description 28.1.3 Structural formula 28.2 Bioactivity 28.3 Extraction. separation and purification 28.4 Literature cited
29
347 347 347 348 348 348 348
............................... .......................... ..................... ....................... .................................... ...........................
351 351 351
351
351 351 351
Holotoxin C 29.1 Introduction 19.1.1 Source organism 29.1.2 Chemical description 29.1.3 Structural formula 29.2 Bioactivity 29.3 Extraction. separation and purification Literature Cited
............................... 351 .......................... 351 ..................... 351 ....................... 352 352 ................................ ............................... 352 352
1.hydroquinone (Geranylhydroquinone) 30.1 Introduction 3D.1.1 Source organism 30.1.2 Chemical description 30.1.3 Structural formula 30.2 Bioactivity 30.3 Extraction. separation and purification -3 Literature cited 2.(2E.3,7.Dimethyl.2,6.octadienyl
-
............................... 352 .......................... 352 ..................... 352 352 ....................... .................................... 352 352 ........................... 353
315
31
.
13-Hydroxy-9-~l-hydroxyethyl~-3-{4-~3-hydroxyphenyl~4.methoxy.l.methylbutyl)-4,14,16,16.tetramethyl.2.6. 10,17-tetraoxatr~cyclo~ll.3.l.l1~5~octadecane-7,lldione (Debromoaplysiatoxin) 31.1 Introduction 353 35 3 31.1.1 Source organisms 31.1.2 Chemical description 35 3 31.1.3 Structural formula 353 31.2 Bioactivity 35 3 31.3 Extraction. separation and purification 354 354 Literature Cited
................................ .......................... ...................... ........................ ................................. ..... ............................
32
33
.
.
Acrylic acid 32.1 Introduction 32.1.1 Source organism 32.1.2 Chemical description 32.1.3 Structural formula 32.2 Bioactivity of sodium acrylate 32.3 Extraction, separation and purification 32.4 Literature cited
................................ ........................... ...................... ........................ .............. .................................
{l~.(la.4aB.8aa)~.2.I(Decahydro.5.5.8a.tr~methy 1 . 2.methylene.l.naphthalenyl~.rnethyl~.ly 4.benzenediol ( Zonarol) 33.1 Introduction 33.1.1 Source organism 33.1.2 Chemical description 33.1.3 Structural formula 33.2 Bioactivity 33.3 Extraction, separation and purification 33.4 - Literature cited
................................ ........................... ...................... ........................ ................................. .................................
34
.
~l~.(la.4a~.8aa)~.2.~1.4.4a.5.6.7.8.8a.0ctahydr 0. 2.5.5.8a.tetramethyl.l.naphthalenyl)methyl~.l. 4. benzenediol (Isozonarol) 34.1 Introduction 34.1.1 Source organism 34.1.2 Chemical description 34.1.3 Structural formula 34.2 Bioactivity 34.3 Extraction. separation and purification 34.4 Literature cited
35
.
................................ ........................... ...................... ........................ ................................. ..... ............................
1.2.3.5. 6.Pentathiepane (Lenthioninel Introduction 35.1 35.1.1 Source organisms 35.1.2 Chemical description Structural formula 35.1.3 35.2 Bioactivity 3 5.3 Extraction. separation and purification 35.4 Literature cited -
................................ .......................... ...................... ........................ ................................. .................................
354 354 354 354 354 355 355
355 355 355 355 356 356 356
356 356 356 356 357 357 357
357 357 357 357 358 358 358
316 36.42
43
44
.
.
. Polyhaloketones 1. Introduction.............................. 1.1 Source organisms ........................ 1.2 Chemical description .................... 1.3 Structural formulas..................... 2. Bioactivity ............................... 5 . Extraction. separation and purification ... -6 . Literature cited ..........................
359 359 359 359 359 359 360
1.2.4. 6.Tetrathiepane Introduction Source organisms 43.1.2 Chemical description 43.1.3 Structural formula..... 43.2 Bioactivity 43.3 Extraction. separation and purification Literature cited
360 360 360 360 360 360 360
4.Dioxo.1.2.4. 6.tetrathiepane 44.1 Introduction 44.1.1 Source organism 44.1.2 Chemical description Structural formula 44.1.3 44.2 Bioactivity 44.3 Extraction. separation and purification 44.4 Literature cited
360 360 361 361 361 361 361
43.1 43.1.1
............................... .......................... ..................... ....................... .................................... ...........................
45-48
.
Polyhalo-3-buten-2-ones Introduction Source organisms 1.2 Chemical description 1.3 Structural formulas 2 Bioactivity 3 Extraction. separation and purification - Literature cited
361 361 361 362 362 362 362
Bromophenols Introduction Source organisms 1.2 Chemical description 1.3 Structural formulas 2 Bioactivity -5 - Extraction. separation and purification Literature cited.. -4
362 362 363 363 363 364 364
. -11.1 .. .
49.56
.
. -11.1
.. .
57
.
............................... ......................... ..................... .................. ................................ ........................... ....
.............................. ........................ .................... ..................... ............................... ... ..........................
.............................. ........................ .................... ..................... ............................... ........................ ...
2-Hydroxybenzaldehyde 57.1 Introduction m.1 Source organisms Chemical description 57.1.2 57.1.3 Structural formula 57.2 Bioactivity 57.3 - Extraction. separation and purification 57.4 Literature cited
............................... ......................... ..................... ....................... .................................... ...........................
365 365 365 365 365 365 365
317
58
59
.
.
1.1.3.3.Tetrabromo. 2.heptanone 58.1 Introduction m.1 Source organism 58.1.2 Chemical description 58.1.3 Structural formula 58.2 Bioactivity 5-8;3 Extraction. separation and purification Literature cited
366 366 366 366 366 366 366
2S_,3~,4~.2.Carboxy.4.isopropenyl.3.pyrrolidin e. acetic acid (a-Kainic acid) 59.1 Introduction m.1 Source organism 59.1.2 Chemical description 59.1.3 Structural formula 59.2 Bioactivity Extraction. separation and purification Literature cited
367 367 367 367 367 367 367
toxyfimbrolide s . Ace 1. Introduction............................... 1.1 Source organism .......................... 1.2 Chemical descriptions .................... 1.3 Structural formulas...................... 2 . Bioactivity ................................ 3 -. Extraction. separation and purification .... 4. Literature cited ........................... -
368 368 368 368 368 368 369
{3&.(2E.3R*.7E*.ER*. (Z))l-2-(l-Bromopropylidene)7.chloro.3.6.7.8.tetrahydro.8.(2.penten.4.ynyl 1. 2H-oxocin-3-01 (Chondriol) 66.1 Introduction 66.1,1 Source organism 66.1.2 Chemical description 66.1.3 Structural formula 66.2 Bioactivity F6.3 Extraction. separation and purification - Literature cited
369 369 369 369 370 370 370
............................... .......................... ..................... ....................... ................................ .... ........................... ............................... .......................... ..................... ....................... ................................ .... ...........................
60.65
66
.
............................... .......................... ..................... ....................... ................................ ........................... ....
67
.
4-Bromo-6-~1S,2R-1,2-d~methylb~cycloI3.1.O~hex-2-
yl)-m-cresol-(L~urinterol) 67.1 Introduction m.1 Source organisms 67.1.2 Chemical description 67.1.3 Structural formula 67.2 Bioactivity Extraction. separation and purification 67.4 Literature cited
............................... ......................... ..................... ....................... ................................ ...............................
370 370 370 370 311 371 311
318
68
.
6-(1~,2~-1,2-Dirnethylb~cyclo~3.l.O~hex-2-y1~-~cresol ( Debromolaurinterol) 372 68.1 Introduction 372 68.1.1 Source organisms Chemical description 372 68.1.2 Structural formula 373 68.1.3 373 68.2 Bioactivity 373 68.3 Extraction, separation and purification 373 68.4 Literature cited
-
'-
69
.
............................... ......................... ..................... ....................... ................................ .... ...........................
41.5.D~bromo.4.chloro.1',3'.31. 4.tetramethylspiro {cyclohexane-1,2'-{7~-oxabicyclo{4.l.O)hept~4~en}-
2-01 69.1
(Prepacifenol) Introduction m.1 Source organisms 69.1.2 Chemical description 69.1.3 Structural formula 69.2 Bioactivity Extraction. separation and purification Literature cited -
374 375 375
2S.3S.4R.2.Carbo~y.4.(1'~.3~~.5~~.l'-methy1.5 '. c~rb~xy?l.,3-hexadienyl)-3-pyrrolidineacetic acid (Domoic acid) 70.1 Introduction 773.1 Source organism 70.1.2 Chemical description 70.1.3 Structural formula 70.2 Bioactivity 70.3 Extraction. separation and purification 70.1, Literature cited
375 375 375 375 376 376 376
{3~.(3a.3aa.46.76.7aB)1.7.Bromooctahydro.4.7 a. dimethyl-3-(2-methyl-l-propenyl)-l~-inden-4-01 (Oppositol) 71.1 Introduction 71.1.1 Source organism 71.1.2 Chemical description 71.1.3 Structural formula 71.2 Bioactivity Extraction. separation and purification - Literature cited
376 376 376 376 376 376 377
Decahydro.a.a.4a.trimethy l.cyclopropaId}naphthalene -7-methanol (Cycloeudesmol) 72.1 Introduction T T . 1 Source organism 72.1.2 Chemical description 72.1.3 Structural formula 72.2 Bioactivity 72.3 Extraction. separation and purification 72.14 Literature cited
377 377 377 377 378 378 378
............................... 374 ......................... 374 ..................... 374 ....................... 374
.................................... ...........................
70
.
-
71
.
-
72
.
-
............................... .......................... ..................... ....................... .................................... ...........................
............................... .......................... ..................... ....................... .................................... ........................... ............................... .......................... ..................... ....................... .................................... ...........................
319
73
74
.
.
2.3.4.5.Tetrabromo.l 5.pyrrole Introduction 73.1 Source organism 73.1.1 Chemical description 73.1.2 Structural formula 73.1.3 Bioactivity 73.2 73.3 Extraction. separation and purification 73.4 Literature cited
............................... .......................... ..................... ....................... ................................ .... ...........................
74.1.1 74.1.2
74.1.3 74.2
7 4.4 75
76
.
.
)-phenol ............................... Source organisms......................... Chemical description..................... Structural formula....................... Bioactivity ................................ Extraction. separation and purification .... Literature cited ............................
2.4.Dibrom0.6.(3.4.5.tribromopyrrol.2.y1 74.1 Introduction
2-;-Pentyl-4-quinolone Introduction Source organism 75.1.2 Chemical description 75.1.3 Structural formula 75.2 Bioactivity Extraction. separation and purification Literature cited
75.1 75.1.1
............................... .......................... ..................... ....................... ................................ .... ...........................
2-n_.-Heptyl-4-quinolone 76.1 Introduction
............................... Chemical description..................... Structural formula....................... Bioactivity ................................ Extraction. separation and purification .... Literature cited ...........................
76.1.1 Source organism.,........................ 76.1.2 76.1.3 76.2
76.3 77
.
4a.12b-Dihydr0-4a.8.12b.trihydroxy-4-methylben (a)anthracene-2.7.12(lH- 1-trione (SS-228 Y) 71.1 Introduction
‘77.1.1
379 379 379 380 380 380 381
381 381 381 381 381 381 382
382 382 382 382 382 383 383
Z-
............................... ....................... ..................... ..................... ................................ .... ...........................
Producing organism 77.1.2 Chemical description 77.1.3 Structural formula.. 77.2 Bioactivity Extraction. separation and purification 1 7.4 Literature cited -
378 378 378 378 378 379 379
383 383 383 383 383 383 3 84
32 0
The p r e s e n t c h a p t e r d i f f e r s i n several ways from o t h e r c h a p t e r s i n t h i s book and i t s w r i t i n g posed some r a t h e r s p e c i a l d i f f i c u l t i e s . The most immediate was t h e q u e s t i o n of which compounds t o i n c l u d e . Although t h e y are o f t e n r e f e r r e d t o a s " a n t i b i o t i c s " by t h e a u t h o r s i n v o l v e d (who, f o r t h e most p a r t , have n o t been i n v o l v e d i n t r a d i t i o n a l a n t i b i o t i c s r e s e a r c h ) , v e r y few of t h e compounds l i s t e d h e r e a r e a n t i b i o t i c s i n t h e s t r i c t (Waksman) s e n s e o f (la c h e m i c a l s u b s t a n c e produced by microorganisms which i n v e r y low c o n c e n t r a t i o n has t h e c a p a c i t y t o i n h i b i t t h e growth and even t o d e s t r o y b a c t e r i a and o t h e r microorganisms." F i r s t , o n l y t h e compaunds a t t h e end of t h e c h a p t e r a r e produced by microorganisms--pseudomonads and a n a c t i n o m y c e t e . (We have s p e c i f i c a l l y and r e g r e t f u l l y o m i t t e d c e p h a l o s p o r i n , s i n c e i t i s covered i n a s e p a r a t e c h a p t e r ) . The r e m a i n d e r are produced by marine p l a n t s and a n i m a l s , mainly sponges and r e d a l g a e ( s e a w e e d s ) , Second ( p r o b a b l y due t o t h e a u t h o r s ' l a c k of e x p e r i e n c e w i t h t r a d i t i o n a l a n t i b i o t i c s ) , many of t h e compounds' b i o a c t i v i t y p r o p e r t i e s have o n l y been d e s c r i b e d i n t h e v a g u e s t terms as l t a n t i b a c t e r i a l t f , "an a n t i b i o t i c " , e t c . We have i n c l u d e d t h e compounds i n t h e c h a p t e r b u t have a l s o i n c l u d e d t h e q u o t e s f o r t h e s e a l l u s i o n s t o a c t i v i t y . I n t h e same v e i n , many of t h e compounds whose a c t i v i t i e s are q u a n t i f i e d would s i m p l y n o t p a s s m u s t e r i n a p h a r m a c e u t i c a l a n t i m i c r o b i a l s c r e e n ; some of t h e i r a c r i v i t i e s c o n s i s t of an 1 8 mm zone of i n h i b i t i o n from a 3 mg sample (compound 3 ) o r a 1 - 6 nun zone from 5 mg o f sample (compound 57). mese m a r g i n a l a n t i b a c t e r i a l a g e n t s a r e a l s o i n c l u d e d . Having l o w e r e d t h e b a r , we have l o w e r e d i t a b i t more by i n c l u d i n g m a r i n e - d e r i v e d compounds whose s o l e r e p o r t e d a c t i v i t i e s a r e as a n t i t u m o r , , a n t i v i r a l o r a n t i h e l m i n t h i c agents, Even w i t h t h e s e c o n c e s s i o n s , t h e l i s t of marine " a n t i m i c r o b i a l " a g e n t s i s n o t y e t e x c e e d i n g l y l o n g ; p e r h a p s we can be more s e l e c t i v e i n a r e v i s i o n . F i n a l l y , t h e r e i s f o r t h e compounds d e s c r i b e d v e r y l i t t l e i n t h e way o f c h r o m a t o g r a p h i c d a t a . Almost no p a p e r o r t h i n l a y e r c h r o m a t o g r a p h i c Rf v a l u e s have been r e c o r d e d . Conseq u e n t l y , we have d e v o t e d c o n s i d e r a b l e a t t e n t i o n t o d e t a i l s of i s o l a t i o n , which u s u a l l y i n c l u d e d c h r o m a t o g r a p h i c s e p a r a t i o n s , as an i n d i c a t o r of t h e compounrls' c h r o m a t o g r a p h i c b e h a v i o r . We have a r r a n g e d t h e compounds, f i r s t , a c c o r d i n g t o b i o l o g i c a l s o u r c e a n d , s e c o n d , a c c o r d i n g t o f o r m u l a . Compounds from a n i m a l s - - P o r i f e r a ( s p o n g e s ) , C n i d a r i a ( g o r g o n i a n s ) , Mollusca ( a s e a h a r e ) , Echinodermata ( a sea cucumber), C h o r d a t a ( a n a s c i d i a n l - - c o m e f i r s t , i n t h a t o r d e r , f o l l o w e d by compounds from plants--Cyanophyta, Chrysophyta, Phaeophyta, Rhodophyta ( b l u e - g r e e n , g o l d e n , brown, r e d a l g a e ) - - i n t h a t o r d e r . F i n a l l y come t h e few compounds from microorganisms. W i t h i n e a c h Phylum ( e . g . P o r i f e r a ) , compounds are a r r a n g e d i n o r d e r of i n c r e a s i n g c a r b o n c u m b e r s . Both common and s y s t e m a t i c names a r e us ed, as a p p r o p r i a t e . The s y s t e m a t i c names are t h o s e g i v e n by Chemical A b s t r a c t s , where a b s t r a c t s i n c l u d e s y s t e m a t i c names,
Within t h e d e s c r i p t i o n f o r each compound t h e s e c t i o n s a r e
32 1 s e l f - e x p l a n a t o r y , w i t h t h e p o s s i b l e e x c e p t i o n of mass s p e c t r a l d a t a which show o n l y t h e i o n s f o r t h e most a b u n d a n t i s o t o p e (1Hyi2C, 14N, l60, C1, 7 9 B r , e t c . ) . Thus, t h e m o l e c u l a r i o n f o r c h l o r o f o r m would be g i v e n a s m/e 118 ( MI , t h a t of bromoform a s m/e 250 ( M I . The l i s t o f " a n t i m i c r o b i a l " a g e n t s s h o u l d be r e a s o n a b l y c o m p l e t e t h r o u g h t h e l i t e r a t u r e o f A p r i l , 1977.
1. 2-Cyano-4,5-dibromopyrrole
1.1 I n t r o d u c t i o n 1 1.1.1 S o u r c e organism
AgelaA onoideA ( s p o n g e ) . 1.1.2
Chemical d e s c r i p t i o n m.p. 248 (MI.
C5H2Br2N2;
MS
-m/e
1.1.3
172-173';
I R (CHC13)umaX 2220
cm-l;
S t r u c t u r a l formula
Br
QCN
Br
I
1.2 Bioactivd " A c t i v e a g a i n s t S 2 2 e p t o COccuA, U~pk?OcoCCuA,Candida
ak?bicanA, T h i c h o p h y t o n . 1.3 Extraction, -
s e p a r a t i o n and - purification'
The f r e s h sponge was e x t r a c t e d w i t h m e t h a n o l , s o l v e n t was removed, a n d t h e aqueous r e s i d u e was e x t r a c t e d w i t h e t h e r , The e t h e r s o l u b l e material y i e l d e d 1 ( 0 . 0 2 % d r y w e i g h t ) . 1.4 Literature -
cited
1.
S . F o r e n z a , L . M i n a l e , A. R i c c i o a n d E . F a t t o r u s s o , Chem. Commun. ( 1 9 7 1 ) 1129-1130.
2.
L . Minale, G . Cimino, S. De S t e f a n 0 and G . Sodano, F o r t s c h r . Chem. Org. N a t u r s t . 33 ( 1 9 7 6 ) 1 - 7 2 .
322 2. 4-B romopyrrole- 2- carbonylguanidin e
2.1 Introduction 2.1.1
Source organism
A g e l a d sp. (sponge).
2.1.2
Chemical description
C6H7BrN4O ; tan solid. 2.1.3
Gructural formula
Br NH CONHCNH,
I
2.2 Bioactivity -2
is the active component of the antibiotic extract.
2.3 Extraction , separation and purification -
The dried sponge was extracted exhaustively with methanol. The concentrated extracts were triturated with benzene, dissolved in water, then extracted with ethyl acetate followed by n-butanol. Chromatography on columns of Polyamide-6 and Sephadex gave 2. 2.4 Literature -
cited
1. M. F. Stempien, Jr., R . F. Nigrelli and J. S. Chib, 164th ACS Meeting, Abstracts (1972) MEDI 21. 3 . 3,5-Dibromo-4-hydroxybenzene-l-acetamide
3.1 Introduction 3.1.1
Source organism
Oelrongia aacheni (sponge, hard and soft varieties). 3.1.2
Chemical description
C8H7Br2NO2; white crystals, ?.p. 190-191'; IR, UV, NMR in agreement with data for an authentic synthetic sample,
32 3 3.1.3
S t r u c t u r a l formula
OH
3.3 E x t r a c t i o n , -
s e p a r a t i o n and p u r i f i c a t i o n
The d r i e d , ground sponge was e x t r a c t e d e x h a u s t i v e l y w i t h methanol. The e x t r a c t was c o n c e n t r a t e d t o a gum a n d t h e n t r i t u r a t e d r e p e a t e d l y w i t h e t h y l a c e t a t e and d r i e d t o a gum. Ext r a c t i o n w i t h d i e t h y l e t h e r and a c e t o n i t r i l e was f o l l o w e d by chromatography o f t h e a c e t o n i t r i l e - s o l u b l e material on Polyamide-6, e l u t i n g w i t h c h l o r o f o r m t o g i v e 2.
3.4 L i t e r a t u r e 1.
4.
cited
M . F. S t e m p i e n , J r . , J. S. Chib, R . F . N i g r e l l i and R . A . Mierzwa i n Food-Drugs from t h e S e a P r o c e e d i n g s 1972. E d i t o r , L . R . Worthen, Mar. T e c h n o l . SOC., Washington, D.C. 105-110, 1973.
3,5-Dibromo-l-hydroxy-4-oxo-2,5-cyclohexadiene-l-acetam~de 4.1 Introduction -
4.1.1
Source organisms
Ueaongia ( = A p . t y d i n Q ) a e h o p h o b a l , 0. c a u C i d o a m i b 2 , U. 6 i ~ t u l a ~ i ~ 3 ' 4U., t h i o n a 5 , u n i d e n t i f i e d V e a o n g i a s p . ( s p o n g e s ) $ 4.1.2
Chemical d e s c r i p t i o n 2
3 95' d e c . ; TLC Rf 0.5 C8H7Br2N03; c r y s t a l s , m.p. ( s i l i c a g e l , e t h 1 a c e t a t e ) . UV AEH36R nm ( c ) 257 ( 8 , 0 0 0 ) ; I R ( N u j o l ) v cm-y 3,445, 3,420, 3,y%, 1,700, 1,675, 1 , 6 6 0 , 1,650, 1,590; NMR 6 ppm 2.75 (2 H , s ) , 2.97 ( 3 H, s ) , 7.59 ( 2 H , s ) ; MS m/e 323 ( M I , 308, 280, 2 4 6 , 244.
*This compomd may be an a r t i f a c t o f the e x t r a c t i o n p r o ~ e d u r e ~ ' ~
32 4 4.1.3
S t r u c t u r a l formula
0
HOW CH2CONH2
4.2 B i o a c t i v i t y ' Test Organism
I n h i b i t i o n zone r a d i u s (mn) 200 pg/ml 40 ug/ml 3.0 3.0
0.5 0.5 0 0 0.5 1.0 1.0 2.5 1.0
1 .o
1.o 2 .o 2.5 3 .O 5.5 3.0
4.3 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n
The wet sponge was ground twice i n a b l e n d e r w i t h metha n o l . The s o l v e n t was removed i n vacuo and by f r e e z e - d r y i n g t o a d a r k - g r e e n r e s i d u e , which was t h e n e x t r a c t e d w i t h e t h y l acet a t e , d r i e d and e v a p o r a t e d . Chromatography on a s i l i c a g e l column (75 x 4 . 8 cm) e l u t i n g w i t h e t h y l a c e t a t e gave c r y s t a l l i n e 3,5-d~bromo-l-hydroxy-4,4-dimethoxy-2,5-cyclohexad~ene-l-acet a m i d e (lo), m.p. 191' ( v i d e i n 6 a a ) . Rechromatography o v e r s i l i c a g e l e l u t i n g w i t h e t h e r gave 2 2 . T r e a t m e n t of ( - ) - a e r o p l y s i n i n - l (6) w i t h t r i f l u o r o a c e t i c a c i d i n a c e t o n e , f o l l o w e d by TLC gave 47.
4 . 4 L i t e r a t u r e cited 1.
E . F a t t o r u s s o , L . Minale and G . Sodano, J.C.S. ( 1 9 7 2 ) 16-18.
2.
G. M. Sharma and P. R .
Perkin I, 1
B u r k h o l d e r , J . A n t i b i o t i c s , S e r . A.
20 (1967) 200-203. 3.
G. M. Sharma, B . Vig a n d P . R . B u r k h o l d e r i n Drugs from t h e Sea. E d i t o r , H . D . F r e u d e n t h a l , J. Ocean Tech. Mar. Technol. Soc.,
Washington, D . C . ,
119-126
(1968).
32 5 B u r k h o l d e r , J. Org. Chem. 3 5
4.
G . M . S h a r m a , B . Vig a n d P . R . (1970) 2823-2826.
5.
R . J. A n d e r s e n a n d D. J . F a u l k n e r , T e t r a h e d r o n L e t t . 1175-1178.
6.
R . J . A n d e r s e n and D. J . F a u l k n e r i n F o o d - D r u g s from t h e S e a P r o c e e d i n g s 1 9 7 2 . E d i t o r , L. R . W o r t h e n , Mar. T e c h n o l . S o c . , W a s h i n g t o n , D.C. , 1 1 1 - 1 1 5 , 1 9 7 3 .
7.
W . F u l m o r , G . E . Van L e a r , G . 0 . Morton a n d R . D. Mills, T e t r a h e d r o n L e t t . ( 1 9 7 0 ) 4551-4552.
5.
(1973)
2,4-Dibromo-3,6-dihydroxybenzeneacetamide 5.1 Introduction 5.1.1
Source organism
Venongia aunea H y a t t , 1 8 7 5 ( s p o n g e ) . 5.1.2
uv
Chemical d e s c r i p t i o n
170-172' d e c . ; o'1'8w'''zN~~i~)c o2l 5o0r l e( 4s,s0 0c0 )r,y s t 2a 8l 0s , ( m.p. 2 , 9 0 0 ) , 368 (7,200);
%ax-
1 , 6 5 8 , 1 , 6 2 6 ; HRMS m/e 3 2 4 . 8 7 7 1 ( M ) , 3 0 7 . 8 5 0 6 ( M NH3), 279.8556 (M NH, - C O ) , 2 5 1 . 8 6 0 9 ( M - .NH, -2CO), 2 2 1 (M NH3 3CO H), 1 9 8 . 9 3 9 5 ( M NH, CO - B r ) , 1 7 0 . 9 4 3 1 ( M - NH3 2CO - B r ) , 1 4 2 . 9 4 9 7 ( M NH, 3CO B r ) , 92.0140 (M NH, - 2CO - 2 B r ) .
(KBr)cm'l
-
-
-
5.1.3
-
-
-
-
-
IR
-
-
S t r u c t u r a l formula
OH
5.2 B i o a c t i v i t y
OH
5 i n h i b i t s t h e g r o w t h of B a c i l f u ~o u b L L f i A , E d c h e n i c h i a
cofi an3
P e n i c i f e i u m a&avenetum.
5.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
T h e s p o n g e was g r o u n d i n a b l e n d e r w i t h i s o p r o p a n o l . T h e m i x t u r e was c o n c e n t r a t e d , r e d i s s o l v e d p a r t i a l l y i n e t h e r , t h e n r e c o n c e n t r a t e d t o give c r y s t a l s . R e c r y s t a l l i z a t i o n from e t h e r - m e t h a n o l ( 1 9 : 1) gave S.
326
5.4 Literature
cited
1. G. E. Krejcarek, R. H. White, L. P. Hager, W. 0. McClure, R, D. Johnson, K. L. Rinehart,.Jr., J. A . McMillan, I. C. Paul, P. D. Shaw and R. C. Brusca, Tetrahedron Lett. (1975) 507-510,
6. ~1R,6S~-~-~-3,5-Dibromo-1,6-dihydroxy-4-methoxy-2,4-cyc1oero sinin-
1 6.1 Introduction 6.1.1
Source organism1"
P&zudoce&atina claA.ba (formerly 1ankhe.tta 6.1.2
Mdid,
sponge).
Chemical description] 0
cHtofi.:i
C9H9Br2N03; crystals, m.p. 112-116'; {ctlD-198 (CH3)2CO); CD (CH30H) I 0 1 2 8 2 ~ 4 9 0 0 ,A ~ 2 8 2-15; UV A ( E ) 284 (5,000); IR (KBr) em- 3,390, 2,262; NMR (CDC13/DMSO-d6) 6 ppm 6.34 (1 H, d , J = 1.3 Hz), 5.5 (2 HI, 4.27 ( 1 H, m), 3.74 ( 3 H), 2.80 (2 HI; MS g / e 336.896 (MI. 6.1.3
Structural formulalY3
OCH, I
CN
6.2 B i ~ a c t i v i t y ~ ' ~
Results
Test system
Inhibition a t 20-100 ug/ml
Gram-poaitivs, gramnegative organisms Fungi
i n vL&o agar dilution essay Not active in v h o
Four standard b a c t e r i a l infections i n mice L1210
B ub cutaneous )
6.3 Extraction, -
No p r o t e c t i w (128 mg/kg, Active
separation and purifi,cation
No information available.
327
6.4 Literature 1.
cited
W. Fulmor, G. E. Van Lear, G. 0. Morton and R. D. Mills, Tetrahedron Lett. (1970) 4551-4552.
2.
R. J. Andersen and D. J. Faulkner i n Food-Drugs from the Sea Proceedings 1974. Editors, H. H. Webber and G. D. Ruggieri, Mar. Technol. SOC., Washington, D.C., 263-267, 1976.
3.
D. B. Cosulich and F. M. Lovell, Chem. Commun. (1971) 397-398.
4.
R. J. Andersen and D. J. Faulkner, J. Am. Chem. S O C . 97 (1975) 936-937.
7.
(1S,6~)-(+~-3,5-Dibromo-l,6-dihydroxy-4-methoxy-2,4-cyclohexadiene-1-acetoni trile {(+)-Aeroplysinin-l}
.
7.1 Introduction 7.1.1
Source organisms
Veaongia ( = A p l y d i n a ) aeaophoba, 0. a a c h e a i , Z a n t h e l l a sp.
(sponges). 7.1.2
Chemical description
C9H9Br2N03; crystals, m.p. 120-&W$ { a l p +186' ( c 0.5, CH,OH), +182O Ic 0.5, (CH312CO]; UV Amax nm E ) 231 (3,2201, 284 (4,910); IR (Nujol) cm-1 3,380, 2,265, 1,635, 1,585; NMR (CD3CN) 6 ppm 2.28 (1 H, s ) , 2.74 (2 H, s ) , 3.70 (3H, s ) , 4.10 (2 H,bm), 6.34 ( 1 H, s ) ; MS m/e 341 (MI, 323, 240. 7.1.3
Structural formula
OCH,
CN
32 8
7.2 B i o a c t i v i t y
-5
Reeults
Test system
s.taphy~ococcu~ dbuA
Active
8acLttU &.tit.& Bacil& cueun
Active Active
Fungi
Not a c t i v e & vifitnc
Four standard b a c t e r i a l i n f e c t i o n s i n mice
No p r o t e c t i o n (128 mg/kg, sub cutaneous)
Gram-positive, . gramnegative organisms
I n h i b i t i o n a t 20-100 pg/ml i n v i t h o , agar d i l u t i o n assay
3 mg-37 mm zone Active
7.3 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n 2
The f r e s h s p o n g e was e x t r a c t e d f o u r times w i t h a c e t o n e a t room t e m p e r a t u r e f o r t h r e e d a y s , t h e n c o n c e n t r a t e d a n d e x t r a c t e d f o u r times w i t h e t h e r . A f t e r d r x i n g , t h e gummy'mass was t r e a t e d w i t h l i g h t p e t r o l e u m ( b . p . 40-70 ) f o l l o w e d by e t h e r , The e t h e r s o l u b l e m a t e r i a l was c h r o m a t o g r a p h e d o n s i l i c a g e l ( M e r c k ) e l u t e d w i t h c h l o r o f o r m - e t h e r (1:l) t o g i v e c r y s t a l s , w h i c h were rec r y s t a l l i z e d f r o m c h l o r o f o r m t o g i v e ?.
7.4 L i t e r a t u r e
cited
1. E. F a t t o r u s s o , L. M i n a l e a n d G . S o d a n o , Chem. Commun. ( 1 9 7 0 ) 751-752. 2.
E . F a t t o r u s s o , L . M i n a l e a n d G . S o d a n o , J . C . S. P e r k i n I , 1 (1972) 16-18.
3.
W. F u l m o r , G . E . Van L e a r , G . 0 . Morton a n d R . D. Mills, T e t r a h e d r o n L e t t . (1970) 4551-4552.
4.
L.
Mazzarella a n d R. P u l i t i , Gazz. Chim. I t . 1 0 2 ( 1 9 7 2 )
391- 39 4 ,
5.
R . J . A n d e r s e n a n d D. J. F a u l k n e r i n Food-Drugs from t h e S e a P r o c e e d i n g s 1 9 7 4 . E d i t o r s , H . H . Webber a n d G . D . R u g g i e r i , Mar. T e c h n o l . S o c . , W a s h i n g t o n , D . C . , 2 6 3 - 2 6 7 , 1976.
8. l-(3-8-D-Arabofuranosyl)uracil.
8.1 I n t r o d u c t i o n ' 8.1.1
Source organism
C q p t o t e t h y a c&ypta ( s p o n g e ) .
(Spongouridine)
329
8.1.2
Chemical description
C9H12N206; white, almost cubic crystals, m.p. 226-2028'. PC, Rf 0.13 1-butanog:ammonia:water (sat.:l:4); {a), + 9 7 ( d 0.6, 8% NaOH), + 126 (C 1.0, H20).
8.1.3
Structural formula
0
OH
8 . 2 Bioactivity2 Although no antibacterial activity was reported for 8 or for the thymine nucleoside, spongothymidine (GI, also isoiated from C. c n y p t a , they are included here because they served as a model for synthesis of the active compounds described below. D e r i v a t i v e o r Analog o f 8
Activity
1-B-D-arabinofursnosylcytosine (cytosine arabinoside, ara C )
I n h i b i t s growth o f Sarcoma 180, E h r l i c h carcinoma and L1210 leukemia i n n i e e . I n h i b i t s . development o f f r o g and sea urchin embryos. E f f e c t i v e Vd. k e r a t i t i s due t o HUpU d h p & x v i r u s .
3 ' -Azido-3' -deoxyspongouridine
An t i v i r a 1
3~-Deoxy-3'-halogenoapongouridines
A n t i v i r a l , antitumor
8.3 Extraction,
, ant i t umor
separation and purification'
The sponge was preserved in a 5 % formalin-sea water solution, then dried in a vacuum oven at 6OoC, ground and extracted with acetone in a modified Soxhlet apparatus to give crystals. The nucleoside mixture was applied to a column of Dowex-1 resin (OH- form). Ammonium hydroxide-ammonium formate buffers were used to separate 8 at pH 8 . 3 . The aqueous solution of 8 was applied to a Dowex-1 resin (OH- form) column, eluted wit5 0.05 N formic acid, then evaporated and recrystallized from methanol.
330
8.4 Literature -
Cited
1. W. Bergmann and D. C. Burke, J. Org. Chem. 20 (1955) 15011507. 2.
M. H. Baslow; Maarine Phaamacotogy (1969). Williams and Wilkins Co., Baltimore, pp. 97-99 (and references therein).
9. 5,6-Dibromo-lH-indole-3-ethanamine
9.1 Introduction 9.1.1
Source organism
Poly&LbaoApongia maynaadii Hyatt (sponge). 9.1.2
Chemical description
nm ( E ) 232 (29,200), C l 0 H I 0 B r 2 N 2 ;m.p. 110-120°; UV 297 (3,700), 307.(3,300); NMR (CDC13/DMSO-d6) 6 ppm 2.15 (1 H, s l br s ) , 2.98 (4H, b r s), 3.3 ( b r m), 7.73 (1 H , s ) , 7.78 (1 H, s ) ; MS m/g 315.9209 (M), 286, 207, 128, 30. 9.1.3
Structural formula
Br
CH2CH2NH,
Br
I
H
9.2 Bioactivity Test System
Results
Gram-positive, . gramnegative organisms
I n h i b i t i o n a t 25-250 pg/ml, agar d i l u t i o n assay
Four standard bacte r ie l i n f e c t i o n s i n mice
No p r o t e c t i o n t o 64 mg/kg, subcutaneous o r o r a l
9.3 Extraction,
separation and purification
The sponge was extracted with aqueous ethanol. 9.4 Literature -
Cited
1. G. E. Van Leap, G. 0. Morton and W. Fulmor, Tetrahedron Lett. (1973) 299-300.
3 31
10. 3,5-Dibromo-l-hydroxy-4,4-dimethoxy-2,5-cyclohexad~ene-lacetamide 10.1 IntroductionlY2 10.1.1
Source organisms
Uellongia cauLi6oami4, U. di4tu-!ahiA (sponges)." 10.1.2
Chemical description
C10H13Br2N04;crystals, m.p. 191'; TLC Rf 0.5 (silica gel, ethyl acetate); IR (Nujol) v cm-1 3,420, 3,375, 3,315, 1,698, 1,640; NMR 6 ppm 2.6 (3 H), 2.8 (2 H, br), 3.1 (3 HI, 3.15 (4 HI, 6.82 (3 HI; MS m/e 369 (MI, 338, 320, 311. 10.1.3
Structural formula
*YQLr HO
CH,CONH,
10.2 Bioa,ctivi,tylY2 Very slightly active c h i a coli.
VA.
BaciLLuA A u b t i l i b and Eachehi-
10.3 Extraction, separation and purification See Section 4.3.1. 10.4 Literature cited
1. G. M. Sharma and P. R. Burkholder, J. Antibiot., Ser. A 20 (1967) 200-203. 2.
G. M. Sharma, B. Vig and P. R. Burkholder, J. Org. Chem. 35 (1970) 2823-2826.
3.
R. J. Andersen and D. J. Faulkner, Tetrahedron Lett. (1973) 1175-1178.
T h i s compound may be
M
a r t i f a c t o f the e x t r a c t i o n procedure3.
332
(Spongothymidine)
11. 1-(3-8-D-Arabofuranosyl)thypine
11.1 I n t r o d u c t i o n 11.1.1
Source organism
C ~ y p t o t e t h y ac ~ y p t a( s p o n g e ) , 11.1.2
Chemical d e s c r i p t i o n
C 1 0 H 1 ~ N 2 0 6 ;c l e a r prisms, m.p. 2 4 6 - 2 4 7 ' Rf 0 . 2 8 11butano1:ammonia:water ( s a t . 1:4)); +80.0°'(C 1.1, 8% NaOH); UV Aaq., n e u t - nm ( € 1 269 ( 9 , 2 5 8 ) .
11.1.3
S t r u c t u r a l formula
0
OH
11.2 Bioactivity See S e c t i o n 8 . 2 , 1 1 . 3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
The sponge was p r e s e r v e d i? 5 % f o r m a l i n - s e a w a t e r s o l u t i o n , d r i e d i n a vacuum-oven a t 6 0 , ground and e x t r a c t e d w i t h a c e t o n e i n a modified S o x h l e t a p p a r a t u s t o g i v e c r y s t a l s , A f t e r two days t h e s e were c o l l e c t e d and t r i t u r a t e d w i t h b o i l i n g e t h a n o l ; i n s o l u b l e m a t e r i a l was d i s s o l v e d i n h o t w a t e r . The s o l u t i o n was t r e a t e d w i t h N o r i t , f i l t e r e d and cooled slowly t o g i v e c l u s t e r s of c l e a r prisms of 11. F u r t h e r crops of c r y s t a l s were o b t a i n e d by c o o l i n g of t h e a E o h o l i c e x t r a c t of t h e f i r s t c r y s t a l l i n e m a t e r i a l and then by slow e v a p o r a t i o n of t h e mother l i q u o r . These were r e c r y s t a l l i z e d t h r e e times from 5 0 % e t h a n o l , then from w a t e r .
11.4 Literature cited 1.
W . Bergmann and R . J . Feeney, J . Org. Chem. 1 6 (1951) 981987.
333
12. Dibromophakellin
12.1 Introduction 12.1.1
Source organism
P h a h e l l i a dlabettata (sponge). 12.1.2
Chemical description
C l 1 H l l B r 2 N ~ Om.; 237;245' dec Ka ca. 7.7 (20% methyl cellosolve); {u)bp'-203 ; UV iCHibHPnm ( € 1 233 (8,877), 281 (8,813); IR (KBr) v cm-1 3,400 bry?,975, 2,950, 1,675, 1,640, 1,587, 1,550, 1,490, 1,435, 972, 721; MS m/g 370, 345. 12.1.3
Structural formula
8 r.
H,N 1 , 2 Bioact-fity -
12 has
"broad spectrum antimicrobial activity".
12.3 Extraction, separation and -
purification
Not reported. 6-Bromophakellin, C11H12BrN50, with the same bioactivity, was also obtained (see numbering system above). 12.4 Literature -
1.
cited
G. M. Sharrna and P. R. Burkholder, Chem. Commun. (1971) 151-152.
2.
G. M. Sharrna, B. Vig and P. R. Burkholder i n Food-Drugs from the Sea Proceedings 1969. Editor, H. W. Youngken, Jr., Mar. Technol. SOC., Washington, D.C., 307-310, 1970,
13. 5,6-Dibromo-l~-indole-3-(N-methylethanam~ne)
13.1 Introduction -
334 13.1.1
Source organism
PoLy d i b n o d p o n g i a m a y n a h d i i Hyatt (sponge 1 13.1.2
.
Chemical d e s c r i p t i o n
C l l H 1 2 B r 2 N 2 ; m.p. 1 3 2 - 1 3 4 ' ; UV XCH30H nrn ( € 1 2 3 2 NMR ( a c e t o n e - d 6 ) 6 ppm 2 . 3 8 (29,200), 297 (3,700), 3 0 7 (3,300); ( 3 H, s ) , 2 . 6 8 ( 4 H, s ) , 7 . 2 3 (1 H , sl b r s), 7 . 7 7 (1 H , s ) , 7 . 9 3 (1 H , s ) ; MS m/e 3 2 9 . 9 3 7 2 ( M I , 2 8 7 , 2 0 8 , 1 2 8 , 4 4 . 13.1.3
S t r u c t u r a l formula
I
H 13.2 Bioactivity
See S e c t i o n 9 . 2 .
13.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i , o n
See S e c t i o n 9 . 3 . 13.4 Literature -
cited
See S e c t i o n 9 . 4 . ohex ad ie n e -
14. 1-ace tam1 de 1 4 . 1 Introduction -
14.1.1
Source organism
V e n o n g i a sp. 1 (sponge)." 14.1.2
Chemical d e s c r i p t i o n 1
C l l H l 5 B r 2 N O 4 ; I R ( C H 2 C 1 2 ) cm'' 3,525, 3,400, 3,220, 1 , 1 0 0 ; NMR ( C D C 1 3 ) 6 ppm 1 . 2 5 ( 3 H , t , 5 = 7 Hz), 1,680, 1,600, 2.57 ( 2 H, s ) , 3.15 ( 3 H, s ) , 3.38 ( 2 H, q, J = 7 Hz), 5.33 (1 H , bs), 6 . 3 8 (1 H , bs), 6 . 7 7 ( 2 H, 6 ) ; MS m/e 3 5 2 , 3 3 8 , 2 7 9 , 2 6 5 , 53. e-
an a r t i f a c t o f the extraction procedure2.
3 35 14.1.3 S t r u c t u r a l formula
CH,O
OC,H,
BfQOr HO
CH,CONH,
14.2 B i o a c t i v i t y
1 4 is
new a n t i b i o t i c " ' .
14.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n ' T h e s p o n g e was e x t r a c t e d w i t h e t h a n o l . was b y F l o r i s i l c h r o m a t o g r a p h y .
Purification
14.4 L i t e r a t u r e . Cited _ I
1. ,R. J . A n d e r s e n a n d D. J. F a u l k n e r i n F o o d - D r u g s from t h e S e a P r o c e e d i n g s 1 9 7 2 . E d i t o r , L. R . W o r t h e n , Mar. T e c h n o l . S o c . , Washington, D.C. 1 1 1 - 1 1 5 , 1 9 7 3 . 2.
R . J . Andersen a n d D. J. F a u l k n e r , T e t r a h e d r o n L e t t . ( 1 9 7 3 ) 1175-1178.
-
1 5 . 3 -Bromo- 2 ( 4 - b r o m o p h e n o x y - p h e n o l
15.1 Introduction1 15.1.1 Source organism
U y o i d e a h e ~ b a c e a( s p o n g y ) . I
15.1.2
Chemical d e s c r i p ion
nm 2 7 8 , 2 8 5 ; I R C,,H,Br,O,; m.p. 9 5 - 9 8 ' h ( K B r ) v em-' 3 , 5 0 0 - 3 , 4 0 0 , 1 , 5 8 7 1 , 5 7 0 , 1 , 4 8 0 , 1 , 4 5 5 , 1 , 2 4 2 , 9 2 0 , 8 3 0 , 7 4 5 , 7 0 0 ; NMR ( 2 2 0 MH&, DMSO-d,) 6 ppm 6 . 8 4 ( 2 H , d , J = 9 H z ) , 7 . 0 9 (1 H , d of d , J = 8 , 1 . 9 H z ) , 7 . 1 6 (1 H , t , J 8 H Z ) , 7 . 2 5 (1 H , d O f d , J 8, 1 . 9 H Z ) , 7 . 5 6 ( 2 H , d , J = 9 H z ) , I4S m/e 3 4 2 ( M I .
-
1 5 . 1 . 3 S t r u c t u r a l formula S e e n e x t page.
3 36
Br
HO
15.2 B i o a c t i v i t y I y 2 A c t i v e vA. g r a m - p o s i t i v e and gram-negative o r g a n i s m s .
-15.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n l *
The sponge was e x t r a c t e d w i t h benzene, t h e n chromatog r a p h e d on F l o r i s i l . A c t i v e f r a c t i o n s were c o l l e c t e d and rep e a t e d c r y s t a l l i z a t i o n from benzene gave 1 6 ( c d . f o l l o w i n g ) . The mother l i q u o r s r e m a i n i n g a f t e r c r y s t a n i z a t i o n of 16 were combined and s o l v e n t was e v a p o r a t e d . Steam d i s t i l l a t i o n of t h e r e s i d u e gave 15.
15.4 L i t e r a t u r e
cited
1.
G . M.
Sharrna and B . Vig, T e t r a h e d r o n L e t t . (1972) 1715-1718.
2.
G . M. Sharma, B . Vig and P . R . B u r k h o l d e r i n Food-Drugs from t h e Sea P r o c e e d i n g s 1969. E d i t o r , H. W . Youngken, J r . , Mar. Technol. S O C . , Washington, D . C . , 307-310, 1970.
16. 3,4,5-Tribromo-2-(2,4-dibromophenoxy)
phenol
16.1 I n t r o d u c t i o n 16.1.1
Source organism
U y d i d e a hehbacea ( s p o n g e ) . 16.1.2 Chemical d e s c r i p t i o n CI2H5Br502;
A C H ~ O H Y O H -303; I R max 1,470, 1,4VO, 890, (1 H , d , J 9 Hz), (1 H , d , J = 3 Hz);
*
m.p. 185-186'; UV max nm ( 0 1 288 (b,OOO), ( K B r ) u cm-l 3,500, 3 , 4 1 0 , 1,580, 1,555, 875, 862, 841, 820; NMR 6 pprn 3 . 1 , 6.56 7.38 (1 H , d of d , J = 9 , J = 3 Hz), 7.5, 7.8 MS m/e - 580 ( M I .
2 mdl6,
The following compoulds, with b i o a c t i v i t i e s s i m i l a r t o a l s o obtained from &idea kunbacea2: 3,4,5- Tri romo-2 -( 2 I ,4 -dib romophenoxy )-phenol 4,6-Dibromo-2-(2 ' ,4 I -dib romophenoxy ) -phenol 3,5-Dibromo-2-( 4'-bromophenoxy) -phenol
?
were
337 16.1.3 S t r u c t u r a l formula
16.2 Bioactivity
A c t i v e vh. gram-posi t i v e and gram-negative organisms.
16.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
See S e c t i o n 15.3. 16.4 L i t e r a t u r e 1.
Cited
G . M . Sharma a n d B . V i g , T e t r a h e d r o n L e t t . ( 1 9 7 2 ) 1 7 1 5 - 1 7 1 8 .
1 7 . (1~-~lcr,26,4a6,8a,8aa)3-Decahydro-l-isocyano-4a,8-dimethyl2 - ( 1 - m e t h y l e t h e n y l ) -naph t h a l e n e (Acant h e l l i n - 1 1
17.1 Introduction' 17.1.1 Source organism A c a n t h e t t a acuza ( s p o n g e ) . 17.1.2
Chemical d e s c r i p t i o n
C 1 6 H 2 5 N ; o i l ; I a l D -41.2' (C 5 , C H C 1 3 ) ; I R ( C C 1 , ) v cm-l 2 , 9 7 0 , 2 , 1 4 0 , 1 , 6 4 0 , 8 8 5 ; NMR ( 1 0 0 MHz, C C 1 4 ) 6 ppm 0 . 8 4 ( 3 H , s), 1 . 2 2 ( 3 H, d, J 6 H z ) , 1 . 7 5 ( 3 H , d , J = 1 H z ) , 2.15 (1 H, m ) , 3.27 (1 H , J = 11 H z ) , 4.85 ( 2 H , bs); MS m/e ( r e l . i n t e n s i t y , % ) 231 ( M , 2 6 1 , 2 1 6 ( 1 8 1 , 2 0 4 (101, 189 ( i 3 T , 1 8 8 ( 1 3 1 , 1 2 3 ( 5 7 1 , 109 ( 1 0 0 1 , 1 5 ( 5 8 ) .
17.1.3 S t r u c t u r a l formula See n e x t page. 17.2 B i o a c t i v i t y " A c t i v e a g a i n s t Mycobacte-tium s p .
17.3 E x t r a c t i o n ,
'I2
s e p a r a t i o n and p u r i f i c a t i o n '
The f r e s h s p o n g e was e x t r a c t e d t h r e e times w i t h c o l d a c e t o n e f o r 2 4 h r s , t h e n c o n c e n t r a t e d . The a q u e o u s r e s i d u e was t r e a t e d t h r e e times w i t h e t h e r a n d t a k e n t o d r y n e s s , The o i l y
338
r e s i d u e was c h r o m a t o g r a p h e d o v e r s i l i c a g e l ( 0 . 0 5 - 0 . 2 mm M e r c k ) , e l u t e d with l i g h t petroleum-benzene t o give three f r a c t i o n s - l i g h t petroleum-benzene (8:2) gave acanthellin-2; l i g h t p e t r o l e u m - b e n z e n e ( 7 : 3 ) g a v e a m i x t u r e of a t l e a s t t h r e e s u b s t a n c e s followed by
17.
17.4 L i t e r a t u r e cited
1.
L. M i n a l e , R . R i c c i o a n d G . S o d a n o , T e t r a h e d r o n 30 ( 1 9 7 4 ) 1341-1343.
2.
L. M i n a l e , G . Cimino, J. D e S t e f a n o a n d G . S o d a n o , F o r t s c h r . Chem. Org. N a t u r s t . 3 3 ( 1 9 7 6 ) 1 - 7 2 .
18. ( z , E ) - ( R ) - ( - ) - 5 - { 5 - ( 3 - F u r 1)-2-methyl-l-penteny1}-3-{3-(3~ u r y l ) p r o p y l i d e n e l - d i h y d r ~ - 2 ~ 3 ~ ) - f u r a n o n e( N i t e n i n 1 18.1 Introduction'
18.1.1 Source organism
S p o n g i a n i t e n a ( s p o n g e 1. 18.1.2
Chemical d e s c r i p t i o n
C21H2401,; C O h r l e S S o i l ; Rf 0 . 4 {C6H6-40-7O0 l i g h t p e t r o l e u m ( 9 t l d on s i l i c a g e l F254 ( M e r c k ) } ; { a } , -4 . 4 O ( c 2 , CHC13); U V h 6 1 2 nm ( € 1 2 2 1 ( 1 4 , 0 0 0 ) ; I R ( f i l m ) u cm-1 1 , 7 4 5 , max 1 , 6 7 0 , 1 , 1 7 0 ; NMR (100 MHz, C C 1 4 ) 6 ppm 7 . 2 6 ( 2 H, t , J = 2 H z ) , 7 . 1 6 (1 H I , 7 . 1 2 (1 HI, 6 . 2 3 ( 1 H I , 6 . 1 7 (1 H I , 6 . 0 4 ; MS m/e ( r e l . i n t e n s i t y , % ) 340 (10, M I , 3 2 5 ( 5 ) , 9 5 ( 2 0 1 , 81 (lO7fLT)T 67 (11); HRMS 3 4 0 . 1 6 3 8 ( M I . 18.1.3 S t r u c t u r a l formula See n e x t page. 18.2 B i o a c t i v i t y
" A c t i v e a g a i n s t Mycobacteaium s p .
339
1 8 . 3 E x t r a c t i o n , separation a n d p u r i f i c a t i o n ] T h e f r e s h s p o n g e was e x t r a c t e d t h r e e t i m e s w i t h m e t h a n o l f o r t h r e e d a y s a t room t e m p e r a t u r e . The combined m e t h a n o l i c e x t r a c t s were c o n c e n t r a t e d a t r e d u c e d p r e s s u r e a n d e x t r a c t e d w i t h 3 p o r t i o n s of e t h e r . S o l v e n t was r e m o v e d a n d t h e o i l y material was c h r o m a t o g r a p h e d on s i l i c a g e l ( M e r c k ) , e l u t e d w i t h b e n z e n e followed b y b e n z e n e - e t h y l e t h e r (9:l) t o y i e l d a m i x t u r e of n i t e n i n and dihydronitenin. 1 8 was i s o l a t e d b y p r e p TLC u s i n g benzene-40-70'
l i g h t p e t r o l e u m ( 9 : l ) (0.43% d r y sponge).
18.4 L i t e r a t u r e -
cited
1.
E . F a t t o r u s s o , L. M i n a l e , G . S o d a n o a n d E. T r i v e l l o n e , T e t r a h e d r o n 27 ( 1 9 7 1 ) 3909-3917.
2.
L. M i n a l e , G . C i m i n o , S . D e S t e f a n o a n d G . Chem. Org. N a t u r s t . 33 ( 1 9 7 6 ) 1 - 7 2 .
19.
Sodano, F o r t s c h r .
{z-{&*,S*(E) ]-cY,-{5- ( 3 - F u r a n y l ) - 2 - m e t h y l - 2 - p e n t e n y l methyl-3-furanhexanol (Furospongin-1)
}-'y-
19.1 Introduction 19.1.1 Source organisms
H i p p o o p o n g i a communio, S p o n g i a oddicinalio ( s p o n g e s ) . 1 9 . 1 . 2 Chemical d e s c r i p t i o n C2,H3003; m.p. 35' ( f r o m 80-100' t8.8 ( C 1, C H C 1 3 ) ; U V ( c y c l o h e x a n e ) Xmax
{a}, l i g h t petroleum) nm ( € 1 2 2 0 ( 9 , 1 0 0 ) ;
I R ( f i l m ) w cm-l 3 , 4 0 0 ( b ) , 3 , 1 4 0 , 1 , 5 7 0 , 1 , 5 1 0 , 1 , 4 5 0 , 1 , 3 8 0 , 1 , 1 7 0 , 1 , 0 7 0 , 1 , 0 2 0 , 8 7 5 , 780; NMR ( C D C l , , 1 0 0 MHz) 6 ppm 2 . 4 0 ( 4 H , J = 6 H z ) , 3 . 7 5 (1 H , m), 6 . 1 6 (2 H I , 7 . 1 6 (2 H ) , 7 . 2 8 (2 H ) MS m/e ( r e l . i n t e n s i t y , % ) 330 ( 1 0 , M I , 312 (21, 1 8 1 ( 1 5 ) 1 5 0 ( 5 0 1 , 1 3 3 (201, 1 0 9 ( 5 1 , 9 5 ( 1 5 1 , 9 4 (201, 8 1 (1001, 6 7 (10). 19.1.3 S t r u c t u r a l formula S e e n e x t page. 19. 2 B i o a c t i v it y
"Has some a n t i b a c t e r i a l a c t i v i t y ~ D ~ p ~ o c o c c uS&epZod,
coccuo ) I t .
340
19.3 Extraction,
separation and purification
The fresh sponge was extracted three times with methanol for three days and the combined extracts were concentrated i n vacuo. The aqueous solution was extracted with three portions of ether and the solvent was evaporated to give a dark brown oil. Chromatography on silica gel with benzene was monitored by TLC to collect fractions yielding l.9, a colorless oil, crystallizing on standing. 19.4 Literature cited 1. G. Cimino, S . DeStefano, L. Minale and E. Fattorusso, Tetrahedron 27 (1971) 4673-4679.
l}-2 6-dimeth 1-5(dfur :none I?cin in-2
and 5-{9-{5-(3-Furanylmethy1)-3-furan nonenylidene1 -4-hydroxy-3 -methyl-2
(
341
I r c i n in-1
I r c i n in-2 20.2 C 0 CCUA
.
Bioactivity " A c t i v e a g a i n s t S . auRCuA, StheptOCOccu.4, and P i p l o -
20.3 Extraction
,
s e p a r a t i o n and p u r i f i c a t i o n '
The f r e s h s p o n g e was e x t r a c t e d t h r e e times w i t h m e t h a n o l f o r t h r e e d a y s a n d t h e combined e x t r a c t s were c o n c e n t r a t e d i n vacuo. The r e m a i n i n g a q u e o u s s o l u t i o n was e x t r a c t e d w i t h t h r e e p o r t i o n s of e t h e r a n d s o l v e n t was e v a p o r a t e d t o g i v e a brown o i l , which was chromatographed on s i l i c a g e l . E l u t i o n w i t h b e n z e n e , f o l l o w e d by b e n z e n e - e t h e r ( 9 : 1 ) , gave a m i x t u r e of i r c i n i n s 1 and 2 . The m i x t u r e was r e s i s t a n t t o s e p a r a t i o n ( T L C on S i 0 2 a n d A 1 2 0 3 w i t h and w i t h o u t AgNO,). 20.4 Literature cited
1.
G . Cimino, S . D e S t e f a n o , L . M i n a l e a n d E . F a t t o r u s s o , T e t r a h e d r o n 2 8 ( 1 9 7 2 ) 333-341.
2.
L . M i n a l e , G . Cimino, S . D e S t e f a n o and G . Sodano, F o r t s c h r . Chem. Org. N a t u r s t . 33 ( 1 9 7 6 ) 1 - 7 2 .
2 1 . 5-{13-(3-Furanyl)-2,6,10-trimethyl-E 4-hydroxy- 3 -methyl- 2 ( 5m
10-tridecadienylideneli lf in
2 1 . 1 Introduction 21.1.1
Source organisms
F a d c i o d p o n g i a doveal,
lacinia A a o b i t i n a 2 , and 1 .
342 ( s p o n g e s 1.
vaniabilid Schmidt 21.1.2
Chemical d e s c r i p t i o n 3
~ i t i ~ ~
CH30Hy0H- 249, 3 0 9 ; I R u nm 2 5 5 , C25H3404; uv cm-1 1 , 7 3 0 , 1 , 6 3 0 , 1 , 0 3 0 , 8 8 0 , 7 6 5 ; NMR 6 ppm 1 . 6 2 , 1 . 6 7 , 1 . 8 1 8 Hz), 6.25 (1 H , b r ( 3 H , s ) , 5.08 ( 2 H , m ) , 5.41 (1 H , d, J s ) , 7 . 1 8 (1 H, b r s ) , 7 . 3 1 (1 H , b r s ) .
21.1.3
S t r u c t u r a l formula3"
21.2 B i o a c t i v i t y 3
2 1 is
a c t i v e vd. S t a p h y l o c o c c u d auReud.
21.3 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n 2 '
T h e s p o n g e was e x t r a c t e d w i t h e t h e r , t h e n chromatog r a p h e d on s i l i c a g e l , e m p l o y i n g h e x a n e - d i o x a n e ( 6 5 : 3 5 ) t o g i v e 2 1 i n 0 . 2 % y i e l d from I . v a 4 i a b i l i d .
-
21.4 L i t e r a t u r e -
cited
1.
R . K a z l a u s k a s , P . T. Murphy, R . J. Q u i n n a n d R . J . W e l l s , . T e t r a h e d r o n L e t t . ( 1 9 7 6 ) 2635-2636.
2.
I. R o t h b e r g a n d P . S h u b i a k , T e t r a h e d r o n L e t t . ( 1 9 7 5 ) 7 6 9 772.
3.
D. J . F a u l k n e r , T e t r a h e d r o n L e t t . ( 1 9 7 3 ) 3 8 2 1 - 3 8 2 2 .
2 2 . 3a~,4,5n,6,7,8,11,12,13,14,15u,15aa-Dodecahydro-6a-hydrox -6 1 0 148-trimethyl-3-rnethylene-5,l~- e p o x y eye l o t e trade cat b -3HI-one (Eunicin) 22.1 Introduction 22.1.1 Source organism
Eunicea mamrnoda L a m o u r o u x ( g o r g o n i a n ) . " O c c u r s a t least i n p a r t w i t h i n t h e u n i c e l l u l a r algal symbionts (zooxanthellae) associated with the invertebrate hostff1. *Stereochemistry o f o l e f i n s unknown.
-
343
22.1.2
Chemical d e s c r i p t i o n 1
~ 2 0 ~ 3 0 0 4c;o l o r l e s s
I R u cm-'
(CHC13);
22.1.3
3,623,
c r y s t a l s , m.p. 1,765, 1 , 6 6 4 .
155';
(a1k5 -89'
S t r u c t u r a l formula2
22.2 Bioactivityl
.
A c t i v e ud c i l i a t e d p r o t o z o a n s , and C l o d t a i d i u m dedeni.
22.3 E x t r a c t i o n ,
Staphylococcud
Uuheud
s e p a r a t i o n and p u r i f i c a t i o n 3
The a n i m a l s were d r i e d o u t d o o r s i n t h e s h a d e a n d t h e n The c o r t e x was s t r i p p e d a n d c r u s h e d w i t h a meat g r i n d e r o r m e c h a n i c a l b l e n d e r , t h e n e x t r a c t e d ( S o x h l e t ) w i t h n - p e n t a n e or e t h e r . The i n s o l u b l e m a t e r i a l y i e l d e d c r y s t a l l i n e (22).
i n an oven a t 6 O o C .
22.4 Literature -
cited
1.
A , J. Weinheimer, R . E. M i d d l e b r o o k , J. 0. B l e d s o e , Jr., W . E . M a r s i c o a n d T . K . B . K a r n s , Chem. Commun. ( 1 9 6 8 ) 384-385.
2.
M . B. H o s s a i n , A . F. N i c h o l a s a n d D. Commun. ( 1 9 6 8 ) 385-386.
3.
L. S . C i e r e s z k o , D . H . S i f f o r d a n d A . J. Weinheimer, Ann. N . Y . Acad. S c i . 9 0 ( 1 9 6 0 ) 917-919.
23.
van d e r H e l m , Chem.
(1R,2E,6R,7S,10E,14B~-6,7-Epoxy-3-hydroxymethyl-l4-~sopro( A s pe r d To1 )
)2,lO-dienol 2 3 .1 Introduction 23.1.1 Source organisms
Eunicea a d p e a u t a , E . t o u h n e d o h t i ( g o r g o n i a n s )
.
344 23.1.2
Chemical d e s c r i p t i o n
-87' ( C H C 1 3 ) ; C 2 0 H 3 2 I l 3 ; c r y s t a l s , m.p. 109-110°C; I R ( K B r ) v cm' 3 , 4 5 0 , 1 , 6 4 5 ; NMR ( C D C 1 3 ) 6 ppm 1 . 2 0 (s), 1 . 6 2 (bs), 1.77 ( b s ) , 2.70 (dd, J 4 , 6 Hz), 4.05 ( 2 H, b s ) , 4 . 5 0 (1 H , d d , J = 5 , 8 Hz), 4 . 7 5 ( b s ) , 4.94 ( b s ) , 5 . 1 4 ( b t , J 7 Hz), 5 . 4 5 ( b d , J = 8 Hz); MS 320 ( M ) .
m/e
23.1.3
S t r u c t u r a l formula
' " 3
23.2 B i o a c t i v i t y E f f e c t i v e d o s e s f o r 50% i n h i b i t i o n of t h e i n v i i 2 0 P-388 l y m p h o c y t i c l e u k e m i a , KB a n d LE c e l l l i n e s ( c y t o t o x i c i t y ) a r e 24, 6 a n d 6 u g / m l , r e s p e c t i v e l y . 2 3 . 3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n The a n i m a l s were e x t r a c t e d w i t h w a t e r - c h l o r o f o r m a n d b i o a c t i v i t y was c o n c e n t r a t e d i n t h e c h l o r o f o r m p h a s e u s i n g t h e i n v i t a 0 P-388 a n d KB b i o a s s a y s as g u i d e s . T h r e e s u c c e s s i v e p a r t i t i o n s u s i n g hexane, carbon t e t r a c h l o r i d e and chloroform v d . 1 0 , 2 5 and 3 5 % water i n m e t h a n o l , r e s p e c t i v e l y , w i t h b i o a c t i v i t y a g a i n l o c a l i z e d i n t h e c h l o r o f o r m p h a s e , f o l l o w e d by c h r o m a t o g r a p h y t h r o u g h Sephadex LH-20 w i t h m e t h a n o l a n d t h r o u g h s i l i c a g e l w i t h a c e t o n e - h e x a n e m i x t u r e s g a v e c r y s t a l l i n e 23. Slow e v a p o r a t i o n f r o m b e n z e n e a f f o r d e d c l e a r p r i s m a t i c n e e d l e s .
2 3.4 L i t e r a t u r e c i t e d 1.
24.
A . J . Weinheimer, J. A . Matson, D. van d e r H e l m a n d M . P o l i n g , T e r r a h e d r o n L e t t . ( 1 9 7 7 ) 1295-1298.
(lS,ZS,12B, 1 3 s ) -2,12-Dihydroxy-4 ,8,12- t r i m e t h y l - 1 6 - m e t h y l e n e 1 4 - o x a b i c y c l o Ill,3.l)heptadeca-4,8-di,e,n-15-one . ( C r a s s i n acetate) 24.1 Introduction -
24.1.1 S o u r c e o r g a n i s m s
Pdeudoptexauna c auc idl, P . dLageCLoda H o u t t u n 2 , P. pOhOda ( = c a a . u a )
H~uttuyn~'~ P., wagenaaai S t i a s n y 2 ' 1 ,
345 (gorgonians). 24.1.2
Chemical d e s c r i p t i o n 3
C22H3,05; w h i t e n e e d l e s , m.p. 144-145.5'; TLC Rf 0 . 4 9 ( s i l i c a g e l , 2 0 % a c e t o n e i n b e n z e n e ) , Rf 0.59 (aluminum o x i d e , 2 0 % d i o x a n e i n b e n z e n e ) , Rf 0 . 7 5 {aluminum o x i d e , a c e t o n e - b e n zene ( 1 : 4 ) } ; t70.7' ( c 4.0, C 2 H 5 0 H ) . 24.1.3 S t r u c t u r a l formula6
H,C
24.2 Bioactivi t y l
9 33
OH
4y
Toxic t o p a r r o t f i s h . I n h i b i t s development of f e r t i l i z e d e g g s of t h e sea u r c h i n L y t e c h i n u A v a h i e g a h A a t 10 ppm. C i d a l i n v i & o ub. Endamoeba h i ~ t o l y t i c aa t 1 / 5 0 , 0 0 0 . Toxic a t v e r y low c o n c e n t r a t i o n t o j u v e n i l e forms of b a r n a c l e s . Active i n PS t e s t ( i n v i v o b i o a s s a y a g a i n s t P-388 l y m p h o c y t i c l e u k e mia). A c t i v e i n KB t e s t ( i n V ~ & O b i o a s s a y a g a i n s t a human carcinoma of t h e n a s o p h a r y n x ; T / C 1 3 0 a t 5 0 mg/kg; ED50 %2pg/ml) A c t i v e v b . Tethahymena p y h i $ o h m i A (0.106, 0.133 mM c a u s e d d e c r e a s e d m o t i l i t y and c e l l d e a t h ; 0 . 0 2 7 , 0.053, and 0.08mM a l t e r e d t h e growth r a t e and f i n a l p o p u l a t i o n d e n s i t y ) .
24.3 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n
1. From P. cha4ba a c c o r d i n g t o r e f . 4 : The expanded p o l y p s were s t r i p p e d from f r e s h l y c u t a n i m a l s w i t h a " l i o n jaw" bone h o l d e r , s t i r r e d w i t h s e a water, t h e n a l l o w e d t o s e t t l e . The s u p e r n a t a n t was d i s c a r d e d and t h e r e s i d u e m a c e r a t e d i n a Waring B l e n d o r w i t h sea water, s t r a i n e d t h r o u g h g l a s s wool and c e n t r i f u g e d , t h e n washed r e p e a t e d l y w i t h s e a water, d i l u t e d s e a water and f i n a l l y w i t h f r e s h water, t h e n c e n t r i f u g e d a g a i n . A f t e r two e x t r a c t i o n s w i t h a c e t o n e ( t h e s e c o n d t i m e i n a S o x h l e t a p p a r a t u s ) , t h e d a r k g r e e n i s h - b r o w n e x t r a c t s were combined a n d c o n c e n t r a t e d i n vacuo, t h e n e x t r a c t e d w i t h isohexane ( S o x h l e t ) t o g i v e a s o l u b l e o i l and an i n s o l u b l e d a r k s o l i d , which was d i s s o l v e d i n benzene t o a brown s o l u t i o n and chromatographed on a F l o r i s i l column. The c o l o r l e s s f r a c t i o n s were c o n c e n t r a t e d and r e c r y s t a l l i z e d from benzene-hexane t o g i v e 2. (Zooxant h e l l a e of P . po/roba y i e l d e d 8 % , t h e c o r t e x as much a s 1.5%, d r y w e i g h t ; P . w a g e n u m i and P . dlagek?k!oba y i e l d e d 1%;o n l y a t r a c e of 24 was found i n P. C h u C i A ) .
346 2. From P . p o A O A a a c c o r d i n g t o r e f . 1: The a n i m a l was chopped i n t o 1 - 3 c m l o n g p i e c e s a n d s t o r e d i n 9 5 % i s o p r o p a n o l , which was l a t e r d e c a n t e d a n d removed by f l a s h e v a p o r a t i o n a t 35-40'C. The r e s i d u a l a q u e o u s m i x t u r e was l y o p h i l i z e d a n d t h e f o l l o w i n g f r a c t i o n a t i o n was f o l l o w e d by i n v&to cytotoxic activ i t y i n t h e KB t e s t . The a c t i v e r e s i d u e was p a r t i t i o n e d b e tween water a n d m e t h y l e n e c h l o r i d e , t h e n b e t w e e n 1 - b u t a n o l a n d water. The m e t h y l e n e c h l o r i d e p h a s e was d r i e d w i t h magnesium s u l f a t e , f i l t e r e d and e v a p o r a t e d t o g i v e a d a r k r e d d i s h brown v i s c o u s s y r u p w h i c h was d i s s o l v e d i n m e t h a n o l a n d e x t r a c t e d r e p e a t e d l y w i t h h e x a n e , t h e n c o n c e n t r a t e d by f l a s h e v a p o r a t i o n a t r e d u c e d p r e s s u r e . B i o l o g i c a l a c t i v i t y was f o u n d t o b e c o n c e n t r a t e d i n t h e h e x a n e r e s i d u e , w h i c h was c h r o m a t o g r a p h e d o v e r a s i l i c a g e l (Woelm) d r y column w i t h e t h e r . S e c t i o n s of t h e column were e a c h e l u t e d b a t c h w i s e w i t h e t h e r , f i l t e r e d a n d e v a p o r a t e d a n d t h e a c t i v e r e s i d u e was i d e n t i f i e d . The s l i g h t l y y e l l o w c r y s t a l l i n e mass was t r e a t e d w i t h a c t i v a t e d c a r b o n i n b e n z e n e a n d r e c r y s t a l l i z e d from b e n z e n e - h e x a n e t o g i v e w h i t e n e e d l e s of 2. 24.4 L i t e r a t u r e c i t e d
1.
A . J . Weinheimer a n d J . A . M a t s o n , L l o y d i a 38 ( 1 9 7 5 ) 378382,
2.
A . J . Weinheimer, F. J . S c h m i t z a n d L . S . C i e r e s z k o i n Drugs from t h e S e a . E d i t o r , H. D. F r e u d e n t h a l , J . Ocean Tech. Mar. T e c h n o l . S O C . , W a s h i n g t o n , D . C . , 1 3 5 - 1 4 0 , 1 9 6 8 .
3.
L. S. C i e r e s z k o , D. H . S i f f o r d a n d A . J . W e i n h e i m e r , Ann. N . Y . Acad. S c i . 9 0 ( 1 9 6 0 ) 9 1 7 - 9 1 9 .
4.
L. S . C i e r e s z k o , T r a n s , N . Y . Acad. S c i . 2 4 ( 1 9 6 2 ) 502-503.
5.
J . R . Rice, C . P a p a s t e p h a n o u and D. G . A n d e r s o n , B i o l . B u l l . 1 3 8 ( 1 9 7 0 ) 334-343.
6.
M . B . H o s s a i n a n d D . van d e r H e l m , Rec. T r a v . Chim. Pays-Bas 88 ( 1 9 6 9 ) 1 4 1 3 - 1 4 2 3 .
7.
D. L. P e r k i n s and L. S . C i e r e s z k o , H y d r o b i o l o g i a 42 ( 1 9 7 3 ) 77-84.
25. a - ~ 1 , 2 - D ~ h y d r o x y p r o p y l ~ - 2 , 5 - d ~ h y d r o - 2 - m e t h y l - 5 - 0 ~ 0 - 2 - f u r a n tetradecanoic acid 25.1 Introduction 25.1.1 Source organism
P t Q A o g o A g i a g u a d a t h p Q n A i A Duchassaing and Michelin (gorgonian). 25.1.2
Chemical d e s c r i p t i o n
C26H4z08;
w h i t e s o l i d , m.p.
81.1-82.9';
{a],
-8.3'
347 0 . 4 7 , C H C 1 3 ) ; UV X g 5 % C 2 H 5 0 H nm ( € 1 2 0 4 ( 1 7 , 4 3 6 ) ; I R (CHC13) max cm-l 3 , 5 0 0 ( w , b r ) , 1,7QO ( s , b r , w i d t h a t h a l f i n t e n s i t y , 1,700-1,770), 1 , 2 1 5 ( s , b r ) ; NMR (CHC13) 6 ppm 1 . 2 8 , 1 . 4 0 ( d , J = 7 H z ) , 2 . 0 8 , 4 . 7 8 - 5 . 4 2 ( c o m p l e x m ) , 7 . 0 2 ( 9 , J = 1 . 5 Hz). (C
w
25.1.3
S t r u c t u r a l formula
25.2 B i o a c t i v i t y M i l d a c t i v i t y v d , StaphyLococcud auReud and Mycobactelrium d m e g m a t i d . 25.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
T h e d r i e d , c o a r s e l y g r o u n d a n i m a l was e x t r a c t e d i n a continuous percolator-extractor s e q u e n t i a l l y according to: h e x a n e , 1 8 h + 4 8 h + 9 6 h ; b e n z e n e , 2 8 h + 72 h ; m e t h a n o l , 4 8 h + 7 2 . h t o g i v e a p r e c i p i t a t e of 25 from t h e t h i r d h e x a n e ext r a c t . T h i s was c h r o m a t o g r a p h e d o n s i l i c i c a c i d , e l u t i n g w i t h b e n z e n e followed b y b e n z e n e - e t h y l acetate m i x t u r e s w i t h e t h y l a c e t a t e c o n c e n t r a t i o n i n c r e a s i n g t o lo%, t h e n r e c r y s t a l l i z e d from a q u e o u s i s o p r o p a n o l . 25.4 L i t e r a t u r e c i t e d
1.
F. J. S c h m i t z a n d E . D. L o r a n c e , J. Org. Chem. 3 6 ( 1 9 7 1 ) 719 - 72 1.
26. 6 - B r o m o - 3 , 7 , 7 - t r ~ m e t h y l - 2 , 1 3 - d ~ o x a t r ~ c y c l 0 { 9 . 3 . O . O . ~ , ~ ~ ~ tetradec-10-en-12-one (Aplysistatin) 26.1 Introduction 26.1.1
Source organism
ApLydia a n g u i ( s e a h a r e ) . 2 6.1.2
Chemical d e s c r i p t i o n
C15H21Br03; m.p. 173-175OC; ORD ( C H 3 0 H ) {al?j70 + 1 7 , 5 0 0 ; CD ( C H 3 O H )
+21,500,
Ial:&
-375', +8,580 ( 2 5 9 ) ;
I R ( K B r ) cm-1 1 , 7 6 5 , 1 , 6 7 6 , 1 , 2 3 0 , 1 , 2 0 5 , 1 , 0 1 0 , 1 , 0 0 0 , 6 2 8 , 5 9 0 ; l H NMR ( C D C l , ) 6 ppm 0 . 9 6 ( 3 H , s ) , 1 . 1 6 ( 3 H , s ) , 1 . 2 8 ( 3 H, s ) , 1 . 6 - 2 . 4 ( 5 H , m ) , 2 . 5 8 ( 2 H , m), 3 . 9 ( 2 H , m), 4 . 5 2 (1 H , t , J = 8 . 5 H z ) , 5 . 1 7 ( 1 H , m ) , 7 . 0 0 (1 H, m); MS m/e - 330
348
26.1.3 s r u c t u r a l formula
26.2 B i o a c t i v i t y C y t o t o x i c vA. N a t i o n a l Cancer I n s t i t u t e ' s murine lymphocytic l e u k e m i a P-388 ( E D 5 0 2.7 pg/ml) and KB ( E D 5 0 2.4 pgAml).
26.3 E x t r a c t i o n , s e p a r a t i o n an0 p u r i f i c a t i o n
_L_
The a n i m a l was e x t r a c t e d w i t h i s o p r o p a n o l , t h e n w i t h c h l o r o f o r m . The c h l o r o f o r m - s o l u b l e f r a c t i o n was a p p l i e d t o a prepacked s i l i c a g e l column, e l u t i n g w i t h l i g r o i n - e t h y l a c e t a t e (9:l). A c t i v i t y was f o l l o w e d w i t h t h e P-388 i n v i & o c e l l l i n e . S i n g l e c r y s t a l s were p r e p a r e d from a c e t o n e - h e x a n e .
26.4 L i t e r a t u r e c i t e d 1.
G. R . P e t t i t , C . L. H e r a l d , M . S . A l l e n , R . B . Von D r e e l e , L. D. V a n e l l , J . P . Y . Kao, W. B l a k e , J. Am. Chem. SOC. 9 9 (1977) 262-263.
27. Holotoxin A 27.1 I n t r o d u c t i o n 27.1.1 Source organism
S f i c h o p u A j a p o n i c u a S e l e n k a ( s e a cucumber). 27.1.2 Chemical d e s c r i p t i o n :59H~,4027; c o l o r l e s s n e e d l e s ( d i h y d r a t e ) , m.p. 248-252'; (cr3h4-53 ( c 0.3, C 5 H 5 N ) ; CD ( c 1.24 x lo-", C H J O H ) 0, (0)3a5 -9,900 ( n e g . m a x . ) , (Q)263 -550 ( n e g . m i n . ) , (0)233 0 (8)205+22,000 ( p o s . max.); -13,700 ( n e g . max.), uv max t r a n s p a r e n t above 210 nm; I R ( K B r ) u cm-1 3,400 b r , 1,750 b r , 1 , 0 7 0 b r .
27.1.3 S t r u c t u r a l f o r m u l a See n e x t p a g e ,
a
349
350
27.2 B i o a c t i v i t y M I C (trg/rnl) Test organism
Thichophyton hubhum Thichophyton m e n h g h o p h y t u Micnodpohum gypdeum Candida d b i c a n a Candida u.ti.(?ib
Tohula
d.(?ib
A a p u g i l l u b ohyzae Penici.Uium ch.yAogenwn
Thichomonab wagin& Supe r f ic i e l de rmatophy tos i s ( c l i n i c a l tests)
Holotoxins
B 28 -
C 29 -
1.56
0.78 1.56
6 -25 12.5
3.12 6.25 3.12
1.56
12.5
6.25 3.12
25.0
A 27 -
0.78
3.12 6.25 3.12 3.12
3.12 12.5 6.25
12.5 12.5 25.0 12.5
1.56
3.12 Some improvement in 88.5X o f cases, w i t h almost no side effects.
27.3 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n
The w h o l e , f r e s h a n i m a l was e x t r a c t e d w i t h m e t h a n o l under r e f l u x t h r e e times , c o n c e n t r a t e d a t reduced p r e s s u r e , t h e n t r e a t e d w i t h m e t h a n o l a t room t e m p e r a t u r e . S o l v e n t was removed from t h e m e t h a n o l s o l u b l e p o r t i o n a n d t h e b e n z e n e s o l u b l e p o r t i o n was removed. The b e n z e n e i n s o l u b l e p o r t i o n was t r e a t e d t w i c e w i t h 9 5 % a l c o h o l u n d e r r e f l u x f o r 30 min. t h e n c o n c e n t r a t e d a n d c h r o m a t o g r a p h e d on s i l i c a g e l ( M e r c k , 0 . 0 5 - 0 . 2 nun), e l u t i n g w i t h c h l o r o f o r m - m e t h a n o l - w a t e r ( 7 : 3 : 1 , lower l a y e r ) . The f r a c t i o n c o n t a i n i n g 2 7 was r e c h r o m a t o g r a p h e d on s i l i c a g e l t o g i v e c r u d e 2 7 , 3 s p z s on TLC { s i l i c a g e l D-5 Camag, s p r a y 1%C e ( S 0 , ) 2 i n H2S04 w i t h h e a t 3 u s i n g c h l o r o f o r m m e t h a n o l - w a t e r ( 7 : 3 : 1 , l o w e r l a y e r ) . Crude 2 7 was s u b j e c t e d t o d r o p l e t c o u n t e r e u r r e n t c h r o m a t o g r a p h y u s i n g -diloroform-methanolwater ( 5 : 6 : 4 ) m i x t u r e ( u p p e r l a y e r - m o v i n g p h a s e , l o w e r l a y e r s t a t i o n a r y p h a s e ) , 6 ml/h, t o g i v e f r a c t i o n s c o n t a i n i n g holot o x i n B ( 2 1 , h o l o T o x i n C ( 2 9 , t r a c e ) a n d 2 7 , w h i c h was rec r y s t a l l i z e d from chloroform-methanol-water.
lm
27.4 L i t e r a t u r e
ci,ted
1.
I . K i t a g a w a , T . Sugawara, I . Y o s i o k a a n d K . Pharm. B u l l , 24 ( 1 9 7 6 ) 266-274.
2.
I . K i t a g a w a , T . Sugawara a n d I . Y o s i o k a , Chem. Pharm. B u l l . 2 4 ( 1 9 7 6 ) 274-204.
3.
S . Shimada, S c i e n c e 1 6 3 ( 1 9 6 9 ) 1 4 6 2 .
4.
W. L . Tan, C . D j e r a s s i , J. Fayos a n d J . C l a r d y , J . Org. Chem. 40 ( 1 9 7 5 ) 466-470.
Kuriyama, Chem.
351 5.
I . K i t a g a w a , T. Sugawara a n d I . Y o s i o k a , T e t r a h e d r o n L e t t . ( 1 9 7 4 ) 4111-4114.
6.
I . K i t a g a w a , T. S u g a w a r a , I . Y o s i o k a a n d K . Kuriyama, T e t r a h e d r o n L e t t . ( 1 9 7 5 ) 963-966.
28. H o l o t o x i n B 28.1 Introduction 28.1.1 Source organism
Stichopud japonicud S e l e n k a ( s e a cucumber). 28.1.2
Chemical d e s c r i p t i o n
{ct}29-520 ( c 0 . 4 , C 6 5 H 1 0 4 0 3 3 ( d i h y d r a t e ) ; m.p. 236-239'. I R ( K B r ) v c m - l 3,360 ( b r ) , 1 , 7 5 0 ( b r j , 1 , 8 7 0 ( b r ) ; uv CH30H t r a n s p a r e n t a b o v e 2 1 0 nm; CD ( e 5.9 x lo-'+, CH30H) Xmax ( O } ~ , + ~ O , { 0 3 ~ ~ ~ . ~ - 1 2 , 6( n0 e0g . m a x . ) , CO1262-1,100 ( n e g . m i n . ) ,
C5H5N);
{ 0 ) ~ ~ ~ - 1 0 , 3 (0n 0e g . max. I . ,
( 012150,
{O1205+14,200 (POS. max. 1.
28.1.3 S t r u c t u r a l formula S e e f o r m u l a f o r h o l o t o x i n A ( 2 1 , where R o r R ' o r R" = B-D-glucopyranosyl f o r h o l o t o x i n B. 2 8.2 B i o a c t i v it y
See S e c t i o n 27.2. 28.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n See S e c t i o n 27.3. 28.4 L i t e r a t u r e c i t e d See S e c t i o n 2 7 . 4 . 29. H o l o t o x i n C 29.1 Introduction -
29.1.1 Source organism
Stichopud japonicud S e l e n k a (sea cucumber). 29.1.2.Chemical d e s c r i p t i o n Amorphous; CD (CH30H) nm { Q } 3 0 5 ( n e g ; m a x . ) , I 0 1 ( n e g . m i n . ) , {8}232 ( n e g . m a x . ) , i O 1 2 0 0 ( p o s . ) ; UV Xmax CH30fi6O t r a n s p a r e n t a b o v e 2 1 0 nm; I R ( K B r ) u cm-l 3,380 ( b r ) , 1 , 7 5 5
352 (br), 1,070 (br). 29.1.3 Structural formula Not known; isolated in trace amount only.
29.2 Bioactivity See Section 27.2.
29.3 Extraction, separation and purification See Section 27.3. 29.4 Literature -
cited
See Section 27.4. 30. 2-(2~-3,7-Dimethyl-2,6-octadienyl)-hydroquinone (Geranylhydroquinone) 30.1 Introduction' 30.1.1 Source organism A p t i d i u m s p . (colonial tunicate).
30.1.2 Chemical description C16H2202; viscous oil; NMR (CCl,,) 6 ppm 1.59, 1.69, 1.75 (3 s), 2.10 ( 4 H, br), 3.28 ( 2 H, m), 5.25 (1 H, s), 5.50 ( 1 H, s ) , 6.50 (3 H, m); MS m/e 246 (M, 32%), 123. 30.1.3 Structural formula
OH 3 0.2 Bioact ivi ty
*
Advance administration of geranylhydroquinone protects test animals against some forms of leukemia, Rous sarcoma and mammary carcinoma, 30.3 Extraction, separation and purification' The tunicate was extracted with chloroform and fractions
353 were separated by silica gel column chromatography. Diethyl ether in benzene ( 5 % ) eluted 30 (7% dry weight). 30.4 Literature cited
1. W. Fenical i n Food-Drugs from the Sea Proceedings 1974. Editors, H. H. Webber and G. D. Ruggieri, Mar. Technol. SOC. , Washington, D.C. , 388-394 (1976).
P. Baranger, French Patent No. M2694; August 31, 1964;
2.
Chem. Abstr. 61 (1964) 1594Oe. 31. t e t r a o x a t r i c y c l o l l l . 3 . 1 , l ~ ~ ~ ~ o c t a d e c a n e - 7-dione 3 1.1 (Debromoaplysiatoxin)
31.1 Introduction
31.1.1 Source organisms
L y n g b y a g h a c i l i d (blue-green alga) l ; O b c i l l a t o h i a n i g R o vinidid Gomont and S c h i z o t h h i x c a l c i c o l a (inseparable mixture S t y l o c h e i l u d l o n g i c a u d a (Quoy and of two blue-green algae)' Gaimard, 1824) (sea hareIi. 31.1.2 Chemical description
C32H48010; colorless needles, m.p. 105.5-107.0°; UV nm ( E ) 283 (1,9501, X CH30H,O*1!. NaQH 290 (3,000); MS
-m/e-
592 (MI, 574. 31.1.3 Structural formula
OH 31.2 Bioactivity LD50
Ca.
1.5 ug/mouse; T/C 167 at LD50 for P-388
354
lymphocytic leukemia ( i n t r a p e r i t o n e a l ) 31.3 E x t r a c t i o n ,
.
s e p a r a t i o n and p u r i f i c a t i o n
The f r o z e n a l g a was homogenized a n d e x t r a c t e d w i t h a m i x t u r e of c h l o r o f o r m and methanol (1:2). The c h l o r o f o r m l a y e r was washed r e p e a t e d l y w i t h water, d r i e d o v e r anhydrous sodium s u l f a t e and e v a p o r a t e d , t h e n chromatographed on F l o r i s i l , e l u t i n g w i t h c h l o r o f o r m and methanol (9:l). Gel p e r m e a t i o n c h r o matography on Sephadex LH-20 u s i n g c h l o r o f o r m a n d methanol ( l ; l ) ,f o l l o w e d by column chromatography ( t h i n - l a y e r chromatography g r a d e s i l i c a g e l ) e l u t i n g w i t h chloroform-methanol ( 9 : l ) g a v e 3 1 ( 0 . 0 1 3 % y i e l d ) , which was c r y s t a l l i z e d as a microc r y s t x l i n e powder from d i e t h y l e t h e r and p e n t a n e , t h e n r e c r y s t a l l i z e d from aqueous methanol. 31.4 L i t e r a t u r e c i t e d
1.
J. S . Mynderse, R . E . Moore, M . Kashiwagi and T . R . N o r t o n , S c i e n c e 196 ( 1 9 7 7 ) 5 3 8 - 5 4 0 .
2.
Y . Kato and P . J. S c h e u e r , J . Am, Chem. SOC. 9 6 ( 1 9 7 4 ) 2245-2246.
32. Acrylic a c i d
32.1 I n t r o d u c t i o n ’ 3 2 . 1 . 1 Source organism
Phaeocybtib p o u c h e t i i ( c o l o n i a l g o l d e n a l g a ) . 32.1.2
Chemical d e s c r i p t i o n
C 3 H b O z ; I R , X-ray a n a l y s i s , f i l t e r p a p e r chromatograms i d e n t i c a l t o t h o s e of commercial m a t e r i a l .
32.1.3
S t r u c t u r a l formula
CHz=CH-COOH
32.2 B i o a c t i v i t y of sodium a c r y l a t e l ” MI C Test organism
(mg/rnl, f i l t e r paper disk assay on PH 6 . 5 h e a r t i n f u s i o n agar, Difco) 0.030 0.21 0.35 1.3 1.6 2.5 9 .D 5.0 5.0
38.0
355 3 2 . 3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n l Y 2 T h e s u p e r n a t a n t f l u i d of t h e t h a w e d a l g a l c e l l s (pH 5 . 5 ) was a d j u s t e d t o pH 3 . 0 w i t h h y d r o c h l o r i c o r s u l f u r i c a c i d , t h e n was v o l a t i l i z e d u n d e r r e d u c e d p r e s s u r e ( R i n c o r o t a t i n g vacuumt y p e e v a p o r a t o r ) t o g i v e a c o n d e n s a t e (pH 3 . 7 ) f r e e of m i n e r a l T h e pH was a d j u s t e d t o t h e e q u i v a l e n c e p o i n t ? pH 7 . 5 , acids. w i t h s o d i u m h y d r o x i d e ; e v a p o r a t i o n i n V ~ C U Ogave s o d i u m a c r y l a t e ( y i e l d of 3 2 , 7 . 4 % d r y w e i g h t ) . 32.4 L i t e r a t u r e cited
1.
J. McN. S i e b u r t h , S c i e n c e 1 3 2 ( 1 9 6 0 ) 676-677.
2.
M . H . Baslow, " M a r i n e P h a r m a c o l o g y " . Williams a n d W i l k i n s Co., B a l t i m o r e , 5 0 - 5 2 , 1 9 6 9 a n d r e f e r e n c e s t h e r e i n .
3 3 . tLS-(la,4a8,8aa)}-2-I(Decahydro-5,5,8a-tr.i.meth~l-2-methylene-
l-naphthalenyl)methyl~-l~~-benzenediol ( Z o n a r o l l 33.1 3 3 . 1 . 1 S o u r c e orga-
V i c t y o p t & t i A z o n a z i o i d e d ( = u n d u l a . t a ) ( P a c i f i c Ocean brown a l g a ) . 33.1.2
Chemical d e s c r i p t i o n
~ 2 1 ~ 3 0 0 2n ;o n c r y s t a l l i n e
(8,400),
295 ( 3 , 1 5 0 ) ;
I R 13::v:
gum;
uv
~
' ~ 3 nm ' ~( € 1 2 1 1 max 3,400, 2,960,
cm-l 3 , 5 5 0 ,
1 , 6 5 0 1 , 4 9 0 , 1 , 4 4 0 , 1 , 1 7 0 , 9 1 0 ; NMR ( 6 0 MHz) 6 ppm 3 . 3 ( 3 ~ , m ) , 4 . 7 (1 H, s ) , 4 . 7 5 (1 H , s ) , 4 . 6 4 (1 H , s ) , 2 . 6 0 ( 2 H , m), 2 . 4 1 . 0 ( 1 2 E, m), 0 . 8 5 ( 9 H, "1; MS m/e ( r e l . i n t e n s i t y ) 4 1 ( 2 5 1 , 4 3 (111, 5 5 ( 2 7 1 , 5 7 ( 8 1 , 6 7 ( 1 8 1 5 6 9 ( 3 2 ) , 7 7 ( 1 2 1 , 79 ( 1 4 1 , 8 1 ( 2 8 1 , 8 3 ( l o ) , 9 1 (151, 9 3 ( 1 5 1 , 9 5 ( 4 9 1 , 9 7 ( 1 1 1 , 1 0 5 ( 1 2 1 , 107 (241, 109 (431, 119 ( 1 2 ) , 1 2 1 ( 2 9 ) , 122 (101, 123 (901, 124 (201, 135 ( 2 1 1 , 136 (161, 137 (241, 147 (81, 149 (141, 1 6 1 (291, 162 (8), 163 (181, 175 (181, 176 (101, 1 7 7 (161, 178 (211, 189 ( 1 4 1 , 1 9 0 ( 1 6 1 , 1 9 1 ( l o o ) , 1 9 2 ( 1 8 1 , 299 ( 6 1 , 314 (521, 315 ( l o ) , 316 ( 2 ) . 33.1.3
S t r u c t u r a l formula
356
33.2 B i o a c t i v i t y ' M o d e r a t e l y f u n g i t o x i c V A . P h y t o p h t h o - t a cinnamomi, Rhizoctonia Aolani, Sctekotina Acleaotiozum, Sclehotium L o l & i i . 33.3 Extraction,separation
and p u r i f i c a t i o n '
The a i r - d r i e d a l g a was g r o u n d t o 1 mm i n a W i l e y m i l l , t h e n e x t r a c t e d w i t h m e t h a n o l ( S o x h l e t , 48 h r ) . S o l v e n t was removed i n vacuo t o g i v e a t a r w h i c h was t a k e n up i n excess d i e t h y l e t h e r , d r i e d , and c o n c e n t r a t e d t o a d a r k t a r . T h i s was t a k e n up i n p e t r o l e u m e t h e r ( b . p . 30-60') a n d c h r o m a t o g r a p h e d o v e r s i l i c a g e l (Davison grade 21, e l u t i n g with petroleum e t h e r , b e n z e n e a n d d i e t h y l e t h e r - b e n z e n e ; 30% d i e t h y l e t h e r i n b e n z e n e g a v e 3 3 , w h i c h was d i s s o l v e d i n b e n z e n e a n d p r e c i p i t a t e d w i t h petroleum e t h e r , 33.4 L i t e r a t u r e -
cited
1. W. F e n i c a l , J . J. S i m s , D. S q u a t r i t o , R . M . Wing a n d P. R a d l i c k , J . Org. Chem. 3 8 ( 1 9 7 3 ) 2383-2386. 2.
G . C i m i n o , S . D e S t e f a n o , W . F e n i c a l , L . M i n a l e a n d J . J. Sims, E x p e r i e n t i a 3 1 ( 1 9 7 5 ) 1250-1251.
3 4 , ElS.-(la,4aB, 8 a a ) I-2-{lA4.4a,5 ,6 7 8 8 a - O c t a h y d r o - 2 , 5 , 5 *tetramethyl-l-naphthalenyJ)methy%-benzenedi.ol (Isozonarol)
,8 a -
34.1 I n t roduction 34.1.1 Source organism D i c t y o p t e - t i ~ zona-tioideA ( = u n d u t a t a ) ( G u l f of C a l i f o r n i a brown a l g a ) .
34.1.2 - d e s c r i p t i o n
C21H3002; n o n c r y s t a l l i n e gum; UV nm ( € 1 209 (8,210), 296 ( 2 , 7 0 0 ) ; I R cm-1 3 , 5 7 0 , 3 , 3 4 0 , 2 , 9 2 0 , 1 , 6 0 5 , 1 , 5 0 0 , 1 , 4 5 0 , 1 , 3 7 7 , 1 , 2 9 0 , 1 , 1 7 0 , 1 , 0 9 5 , 9 5 5 , 8 7 2 ; NMR ( C H C 1 3 1 6 ppm 6 . 8 0 (1 H , s), 6.60 ( 2 H , s ) , 5.45 (1 H, m ) , 4.90 ( 2 H , m ) , 2.60 ( 2 H, m ) , 1 . 0 - 2 . 5 ( 1 2 H, m ) , 0.90 (9 H, m); MS ( r e l . i n t e n s i t y ) 39 (171, 4 1 ( 7 6 1 , 42 ( 7 1 , 4 3 ( 3 9 1 , 53 (151, 55 ( 5 5 1 , 57 (131, 65 ( 1 1 1 , 67 ( 4 1 1 , 69 (431, 77 ( 2 2 1 , 79 ( 2 3 1 , 81 ( 2 3 1 , 8 3 (101, 9 1 ( 2 9 1 , 9 3 ( 2 1 1 , 9 5 ( 5 7 1 , 97 (111, 1 0 5 ( 1 9 1 , 1 0 7 ( 2 9 1 , 1 0 9 ( 7 2 1 , 1 1 9 ( 1 5 1 , 1 2 1 ( 3 2 1 , 1 2 3 ( 7 4 ) , 124 (13), 1 3 5 ( 2 9 1 , 1 4 7 ( 9 1 , 149 (101, 1 6 1 ( 2 4 1 , 1 6 3 (111, 1 7 3 (111, 1 7 5 ( 3 2 1 , 1 9 0 (131, 1 9 1 (1001, 1 9 2 (131, 314 (171, 315 ( 5 1 , 316 ( 1 ) .
YE:^^
34.1.3 S t r u c t u r a l f o r m u l a See next page.
357
34.2 Bioactivi ty Same as zonarol
(33).
34.3 Extraction, separation and purification See Section 33.3. From the Pacific Ocean alga obtained, from the Gulf of California alga, 2.
33
is
34.4 Literature cited See Section 33.4. 35. lY2,3,5,6-Pentathiepane
(Lenthioninel
35.1 Introduction 35.1.1 Source organisms
C h o n d h i a c a l i d o h n i c a (red alga)', S h i i t a k e LQntinuA e d o d e 4 (Berk.) Sing. (terrestrial mushroom)*. 35.1.2 Chemical description C2HbS5; white crystals, m.p. 56-5701, 6O-6lo2 Rf 0.3 MS (silica gel, hexane); NMR (CDC1,) 6 ppm 4.33 (4 H, m/e (rel. intensity) 188 (31, MI, 174 (21, 156 (501, 142 (711, f24 $411, 110 (331, 9 6 (621, 78 ( l o o ) , 64 (341, 46 (631, 45 (99) ; HRMS 187.8890 (MI
.
35.1.3 Structural formula
358
35.2 B i o a c t i v i t y The c r u d e a l g a l e x t r a c t was a c t i v e vA. V i b J l i o a n g u i l T h r e e d i s t i n c t b a n d s o f a n t i m i c r o b i a l a c t i v i t y were o b t a i n e d ; from o n e o f them were i s o l a t e d t h e a n t i b a c t e r i a l comp o n e n t s 1 , 2 , 3 ,5 , 6 - p e n t a t h i e p a n e a n d 1 , 2 $ 4 6 - t e t r a t h i e p a n e l. .htUm.
(s)
L e n t h i o n i n e i s o l a t e d from t h e mushrooms showed a n t i microbial a c t i v i t i e s : Test
strain
8a~CCw.&tb.tieib
S&zphy.tococcu~w e u d
Ebchuichia c o U Pitohud vdgaAi.6
P-ihicuhia onyzae GComeUa c h g u l a t a TAichophyZon nrentagJtophytu
M I C (us/ml)
50 250 250 50 12.5 12.5 3.12
CandLda atbicarcs
6.25
Sacchanonyces cuevAiae Cltyptococc~~b neo doitman.4
6.25 6.25
Taichophyton ~ubitum
3.12
35.3 E x t r a c t i o n
s e p a r a t i o n and puri-f-ication]
The f r o z e n a l g a was thawed, g r o u n d u n d e r e t h y l a c e t a t e i n a Waring B l e n d o r , t h e n f i l t e r e d a n d e x t r a c t e d w i t h c h l o r o form ( S o x h l e t ) . The e t h y l a c e t a t e f i l t r a t e was s e p a r a t e d from t h e aqueous p h a s e and d r i e d o v e r sodium s u l f a t e . S o l v e n t s w e r e removed from t h e combined o r g a n i c e x t r a c t s and t h e r e s u l t i n g v i s c o u s g r e e n o i l was a p p l i e d t o a 100-200 mesh F l o r i s i l column which was e l u t e d w i t h s o l v e n t s of i n c r e a s i n g p o l a r i t y f r o m hexane to m e t h a n o l . F r a c t i o n s were combined i n t o t h r e e b i o l o g i c a l l y a c t i v e bands. The s e m i - s o l i d m a t e r i a l from t h e f i r s t band was r e c h r o m a t o g r a p h e d on p r e p s i l i c a g e l GF p l a t e s , e l u t i n g w i t h h e x a n e t o o b t a i n components a t Rf 0 . 4 and a t Rf 0.3. The l a t t e r was e x t r a c t e d a n d c r y s t a l l i z e d from 1 0 % d i c h l o r o m e t h a n e i n d i e t h y l e t h e r t o o b t a i n 35 ( 0 . 9 % e x t r a c t a b l e o i l ) . 35.4 L i t e r a t u r e c i t e d 1.
S . J . W r a t t e n a n d D. J. F a u l k n e r , J . Org. Chem. 4 1 ( 1 9 7 6 ) 2465-2467.
2.
K . M o r i t a a n d S . K o b a y a s h i , T e t r a h e d r o n L e t t . ( 1 9 6 6 ) 573577.
3.
K . M o r i t a a n d S . K o b a y a s h i , Chem. Pharm. B u l l . 1 5 ( 1 9 6 7 ) 988-993.
359 36-42. Polyhaloketones 1. Qtroduction
1.1 Source organisms
taxidoamid (Delile) Trev. (36-42l) and Adp~nag0pd.i~
A. aamata (36-411) (red algae). 1.2 Chemical
36 37 38 39 40 41 -
C3H2Br3C10 C3H2Br40 C3H3Br2C10 C3H3Br2C10 C3H3Br30 C3H4BrC10 C3H4Br20
42
1,3,3-tribromo-l-chloroacetone 1,1,3,3-tetrabromoacetone 3,3-dibromo-l-chloroacetone 1,3-dibromo-l-chloroacetone
1,lY3-tribromoacetone 1-bromo-3-chloroacetone 1,3-dibromoacetone
IR v cm-l 1,710-1,760; NMR and M S were superimposable on spectra of synthetic products. 1.3 Structural
h!?.zh!!t
-
R -nc R1
/" \ cn-
I
I
R2
Rq
Br
H
Br
Br
C1
Br
H
Br
37 38
39 40 -
42 41
2.
Br
C1
C1
-
R3
Br
Br
Br Br
36
Br
Br
H
Br
Br
Br
H
H
Br
H
C1 Br
H
Bioactivity
Compounds 36-42 "show strong antimicrobial activity against a variety of microorganisms (Staphylococcud, Fu.4aaLum, Vi bai 0 1
.
3. Extraction,. se~aration and purification 1. From A. tax : The partially air-dried let extracted with chloroalga was ground in a form, concentrated in wacud and chromatographed twice on silica gel, eluting with benzene to give twelve fractions which were analyzed by GC-MS (3% O V - 1 7 column operating isothermally at 120'). Moderate separation of eleven major compounds was observed. Seven isomers of polyhaloacetone (36-42) were identified as well as four polyhalo-3-buten-2-one z r z a t i v e s (see Section 45-48 1.
36 0 2. From A.-amata (ref. 2): The lightly-dried alga was ground in a i l e y m i r and Soxhlet extracted with methylene chloride then chromatographed over silica gel, eluting with 10% benzene in petrol and then 100% benzene to give 36-41.
4. Literature cited 1.
W. Fenical, Tetrahedron Lett. (1974) 4463-4466.
2.
0. McConnell and W. Fenical, Phytochem. 16 (1977) 367-374.
43. 1,2,4,6-Tetrathiepane 43.1 Introduction -
43.1.1 Source organisms C h o n d n i a c a t i d o n n i c a (re d alga) ( 3 5.4.1) edoded (terrestrial mushroom) (35.4.2).
, Lentinud
43.1.2 Chemical description C3H6S4; long, colorless needles, m.p. 78-79'; Rf 0.4, (silica gel, hexane); NMR (CDC131 6 ppm 4.22 ( 2 H, s ) , 4.26 (4 H, a ) (35.4.1); MS m/e 124, 7 8 , 60, Li5 (35.4.3). 43.1.3 Structural formula
43.2 Bioactivity See Section 35.2. 43.3 Extraction, separation and purification The minor component (Rf 0 . 4 ) obtained in 35.3 was sublimed or recrystallized to yieid 4" (0.06% extractable oil). 43.4 Literature Cited
.__
See Section 35.4. 44. 4-Dioxo-1,2,kY6-tetrathiepane
44.1 Introduction 44.1.1 Source organism C h o n d r t i a c a l i d o n n i c a (red alga).
36 1
44.1.2 Chemical description C3H602S4; prisms, m.p. 154-155'; IR (CHC13) cm-' 1,330, 1,125, 1,120; 'H NMR (CDC13) 6 ppm 4.18 ( 2 H, s ) , 4.43 (2 H, s ) , 4.56 (2 H, s ) ; I3C NMR (DMs0-d~)ppm 43.8, 54.1, 63.3; MS m/e (rel. intensity) 202 ( 3 3 1 , 138 (161, 124 (81, 110 (91, 92 T2n), 64 (301, 46 (971, 45 (100); HRMS 201.9257 (MI, 109.9319 (CHzS3'). 44.1.3 Structural formula
44.2 Bioactiviry "Is particularly responsible for the antibiotic activity a~~ of C . c a L i ~ a ~ n i c(35.4.1).
44.3 Extraction,
separation and purification
The second antibiotic band obtained in 35.3 was triturated under diethyl ether to give a white powder, which was crystallized from chloroform to yield 43 (17.5% extractable oil). 44.4 Literature cited See Section 35.4.1. 45-48.
Polyhalo-3-buten-2-ones
1. Introduction 1.1 Source organisms A A p a h a g o p A i A t a x i b o n m i . 4 (Delile) Trev. (45-48,l ) , A . t a x i o h m i d (Delile) Collins and Hervey 48,2), A . anmata (46, 1.
(5,
4
1.2 Chemical description
45 46 47 48
C4H2Br3C10 C4H2Br40 C4H3Br2C10 C4H3Br30
Tribromochloro-3-buten-2-one Tetrabromo-3-buten-2-one Dibromochloro-3-buten-2-one Tribromo-3-buten-2-one
IR v cm-l 1,670-1,680; NMR 6 ppm 7.40-7.65; MS m/g 382 (MI, 211; 48 304 (MI, 211.
36 2 1.3 S t r u c t u r a l formulas
0
p4 \
,c,
R3, R4, R5
45 -
Br
.Ez c1
46
Br
Br
48 -
H
Br
El
11
I -RI
= H, B r , B r
2. Bioactivit y
Same as compounds 36-42 ( S e e s e c t i o n 3 6 - 4 1 . 2 ) . 3. E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
1. From A . t a x i d o a m i b . ( D e l i l e ) T r e v . ( r e f . 1): S e e S e c t i o n 36-42. 3 ) . 2. From A . t a x i d o a m i d ( D e l i l e ) C o l l i n s a n d H e r v e y ( r e f . V o l a t i l e m a t e r i a l was c o l l e c t e d f r o m t h e wet a l g a w i t h water i n vacua on a d r y i c e - c o o l e d c o n d e n s e r f i n g e r , t h e n was t a k e n u p i n m e t h y l e n e c h l o r i d e , d r i e d o v e r magnesium s u l f a t e a n d e v a p o r a t e d t o t h e e s s e n t i a l o i l . The h i g h - b o i l i n g f r a c t i o n g a v e 46 b y p r e p TLC o n s i l i c a g e l HF w i t h b e n z e n e . 48 was ident-ied b y GC-MS i n t r a c e a m o u n t s .
2):
3. From A . aamata ( r e f . 3 ) : S e e S e c t i o n 36-42.3. t i o n w i t h 7 0 0 % b e n z e n e a l s o g a v e E. 4.
Elu-
Literature c i s
1.
W.
2.
8 . J. B u r r e s o n ,
3.
0. M c C o n n e l l a n d W .
Fenical, Tetrahedron L e t t .
( 1 9 7 4 ) 4463-4466.
R . E . Moore a n d P . R o l l e r , T e t r a h e d r o n L e t t . ( 1 9 7 5 ) 473-476.
49-56.
F e n i c a l , P h y t o c h e m . 1 6 ( 1 9 7 7 ) 367-37Q.
Bromophenols"
1. I n t r o d u c t i o n
1.1 S o u r c e o r g a n i s m s
Antithamnion pLumuLa ( 5 5 1 , Baongniantck'La byAAoide.4 ( 5 5 ) , CaLothaix bRevibbima (51-53),CeRamium Aubaum ( 5 3 ) , CoaaLdina oddicinaLi.4 (53), Cyb.tTZLZ72um puapuaeum ( 2,)=cub V ~ A ~ C U ~ O 3 I t has been suggested'
t h a t bromophenols from red a lga e , such as 49 and a r e a r t i f a c t s of t h e i s o l e t i e n procedures w i n g h o t aqueous a c z o r methmol-acid e x t r a c t i o n , a r i s i n g from sqwous hydrolysis of the a n tim i c r o b i a l agent dipotasaium 5,6-dibromo-3,4-dihydroxybenzyl a lc ohol 1' , 4 - d i s u l f a t e . In o t h er work6, t h i s compound has been reported i n a c t i v e .
53,
A U A
363
1.2 Chemical description:*
"9
50 52 53 54 55 56
C7H4Br203
2,3-Dibromo-4,5-dihydroxybenzaldehyde(5,6-Dibromoprotocatechualdehyde 1 C7H5Br03 3-Bromo-4,5-dihydroxybenzaldehyde 2,3,6-Tribromo-4,5-dihydroxybenzyl C7H5Br303 alcohol C7H6Br202 3,5-Dibromo-4-hydroxybenzyl a l c o h o l C7H6Br203 2,3-Dibromo-4,5-dihydroxybenzyl alcohol (Lanosol) C7H7Br02 3-Bromo-4-hydroxybenzyl alcohol C7H7Br03 3-Bromo-4,5-dihydroxybenzyl alcohol C,10H12Br203 2,3-Dibromo-4,5-dihydroxybenzyl propyl ether
1.3 Structural formulas
lil CH20H
Br 4 OH 49-53,55,56
Br
49 50 51 52 53 -
OH
E2
E3
Br
CH 0
H
H
CH0
H
Br
CH20H
Br
H
CH2oH
H
Br
CH,OH
H
55 -
H
CH20H
H
56
Br CH2OCH2CH2CH3 H
-
54 -
2. Bioactivity
49-56 - "had
antibiotic activityff6.
*Other chemical md physical properties were i d e n t i c a l t o those o f s y ntheti c samples.
36 4 3 . E x t r a c t , i onnoi,t -s
1. 52 and 53 from 0. dentata a n d R. c o n d e h ~ o i d e b ~ The : b l o t t e d alga was e x t r a c t e d twice w i t h b o i l i n g 8 0 % e t h a n o l , conr e d u c e d p r e s s u r e a n d a d j u s t e d t o 1 N with h y d r o centrated c h l o r i c a c i d . A f t e r b o i l i n g f o r 1 0 min. and c o n F i n u o u s e x t r a c t i o n i n t o d i e t h y l e t h e r f o r 24 h r , c h r o m a t o g r a p h y of t h e e t h e r s o l u b l e c o n c e n t r a t e on p o l y a m i d e t h i n l a y e r s u s i n g m e t h a n o l water ( 9 : l ) a l l o w e d l o c a t i o n o f p h e n o l i c components by color r e a c t i o n s w i t h UV l i g h t , FeC13 s p r a y r e a g e n t a n d f a s t Bordeaux s a l t BD. The m a j o r p h e n o l , 5 3 , was e l u t e d w i t h e t h y l a c e t a t e , a c e t y l a t e d w i t h a c e t i c a n h y d z d e i n p y r i d i n e and c r y s t a l l i z e d fro! m e t h a n o l as c o l o r l e s s p r i s m s o f i t s t r i a c e t a t e , m.p. 1 0 5 1 0 6 , i d e n t i c a l t o an a u t h e n t i c s a m p l e . P h e n o l 52 was o b t a i n e d by t r e a t m e n t of t h e e x t r a c t w i t h c h a r c o a l and p r e p TLC on a c i d methanol i n chloroform washed s i l i c a g e l G d e v e l o p e d w i t h 1% f o l l o w e d b y e l u t i o n w i t h e t h y l a c e t a t e and r e c r y s t a l l i z a t i o n t h r e e times from b e n z e n e { y i e l d 0.024% from 0. d e n t a t a , 0 . 0 0 3 % from R. c o n d e h v o i d e b ( f r e s h w e i g h t ) ) .
at
2 . 49, 50, 52, 53 a n d 55 f r o m s e v e r a l r e d a l g a e 7 : Ovend r i e d o r d e e p - f r o z e n a l g a was e x t r a c t e d w i t h 90% m e t h a n o l u n d e r r e f l u x f o r 3 - h r , twice w i t h 8 0 % m e t h a n o l , t h e n f i l t e r e d a n d r o t a r y e v a p o r a t e d . The a q u e o u s s o l u t i o n was f i l t e r e d t h r o u g h Hyflow c e l i t e , a d j u s t e d t o 0.05 M h y d r o c h l o r i c acid a n d h e a t e d f o r 1 0 min, t h e n e x t r a c t e d w i t h e t h y l a c e t a t e and f r o z e n . An a p p r o x i m a t e l y e q u a l volume o f p e t r o l ( b . p . 40-60') was a d d e d and r e s u l t a n t c r y s t a l s were d i s s o l v e d i n a m i n i m a l amount o f water. F r e e z e d r i e d s a m p l e s were e i t h e r s i l y l a t e d i n acetonit r i l e w i t h BSTFA f o r a n a l y s i s by GC-MS or c h r o m a t o g r a p h e d o v e r s i l i c a g e l or p o l y a m i d e , s p r a y i n g w i t h FeC13 a n d Na*MoOt,. S i m i l a r p r o c e d u r e s y i e l d e d 49-56 from o t h e r red a l g a e . 4.
Literature cited
1.
B . W e i n s t e i n , T . L. R o l d , C . E . H a r r e l l , J r . , M . W . B u r n s I11 a n d J. R . Waaland, Phytochem. 1 4 ( 1 9 7 5 ) 2667-2670.
2.
K. K u r a t a , T. Amiya a n d N . Nakano, Chem. L e t t . ( 1 9 7 6 ) 821-822.
3.
P. S a e n g e r , M . P e d e r s e n a n d K . S . Rowan, Phytochem. 1 5 ( 1 9 7 6 ) 1957-1958.
4.
T. Higa and P. J. S c h e u e r , J. Am. Chem. SOC. 9 6 ( 1 9 7 4 ) 2246-2248.
5.
J. S. C r a i g i e a n d D . E . G r u e n i g , S c i e n c e 1 5 7 ( 1 9 6 7 ) 10581059
6.
K. W. G l o m b i t z a , H. S t o f f e l e n , U. Murawski, J. B i e l a c z e k a n d H. Egge, P l a n t a Med. 25 ( 1 9 7 4 ) 105-114. 81 ( 1 9 7 4 ) 1 0 1 8 0 8 ~ .
7.
Chem. A b s t r .
M . P e d e r s e n , P. S a e n g e r and L. F r i e s , Phytochem. 1 3 ( 1 9 7 4 ) 2273-2279.
365 57. p-Hydroxybenzaldehyde 57.1 Introduction 57.1.1 Source organisms V e d y a p e d i c e t t a t a var. ~ t a n d o a d i a n a(red alga)I, Chaomobactedium sp.2 and Paeudomonad 102-33 (marine bacteria).
57.1.2 CJemical
description
C7H602; m.p. 114-116'. IR, NMR, MS identical to spectra of a commercial sample; NMR (6DC13) 6 7.02 (2 H, d, J = 8 Hz), 7.80 ( 2 H, d, J = 8 Hz), 9.00 (1 H, b), 9.10 (1 H, s). 57.1.3 Structural formula
CHO
57.2 Bioactivi ty llAppearsto be the active component of the CH2C12 extract, which shows moderate antimicrobial activity against O i b a i o a n g u i l l a k u m , C a n d i d a a t b i c a n . 6 , and S t a p h y l o c o c c u A ~ u R ~ u A In ~ ~ a~ disk . assay at 5 mg/disk, zones of inhibition v A . S . auaCu.6, C . a t b i c a n d and V . h a v e y i were 1-6 mm293. It also inhibited marine bacteria P h o t o b a c t e n i u m i i d h e l r i , P. mandapamendid, Benechea h a a v e y i , and Chaomobactea I-L-33 (1-6 mm zones of inhibition) and P . pho4phoaeum (>6 mm zone of inhibition).
57.3 Extraction, separati.on and pu.rifi.c.ation 1. The air dried alga was extracted with methylene chloride and chromatographed over silica gel (Davison grade 62), eluted with benzene-eth 1 ether (1:l) to give 58 followed by p-hydroxybenzyl alcohol7
.
2. pyrrole
(75)3 .
From Chaomobactekium sp., see 2,3,4,5-tetrabromo-lH-
( E ) ~ .
3.
From Pdeudomonad 102-3, see 2-n-pentyl-4-quinolone
57.4 Literature cited -
1. W. Fenical and 0. McConnell, Phytochem. 15 (1976) 435-436.
366 2.
R . J. Andersen, M. S. Wolfe and D. J. Faulkner, Mar. Biol. 27 (1974) 281-285.
3.
S. J. Wratten, M. S. Wolfe, R. J. Andersen and D. J. Faulkner, Antimicrob. Ag. Chemother. 11 (1977) 411-414.
58. 1,1,3,3-Tetrabromo-2-heptanone 58.1 Introduction 58.1.1 Source organism
Bonnemaibonia humi@VLa ( r e d alga). 58.1.2
Chemical description
C7H10Br40; IR (CC14) cm-I 1,738; NMR (60 MHz, CCl4) 6 ppm 1.00 (t), 1.2-1.8 (m), 2.4-2.7 ( m ) , 6.83 (s); GC-MS analysis m/e 426 (MI, 373.6795 (M C4He), 256.8998 ( C I , H ~ C ~ ~ B ~ ~ ~ B ~ C E ~ ) 228.9045 (CbHgC79Br81Br), 200.8372 (6ECCH79Bra1Br) 172.8424 (CH79Br81Br), 188.9916 (C7HloBrO1, 148.9789 (C5H881Br).
-
-
58.1.3 Structural formula 0
1
CH CH2CH2CH2CBr2 CCHB r2 58.2 Bioactivity
7
Test organism
MIC, u g h 1
100
100 500 500 1,000 1,000 1,000 1,000
58.3 Extraction, separation .and purification The alga was ground under ethanol, filtered, concentrated in a rotary evaporator, then redissolved in petroleum ether and chromatographed over silicic acid. 58.4 Literature cited I _
1.
J. F. Siuda, G. R. van Blaricom, P. D. Shaw, R. D. Johnson, R. H. White, L. P. Hager and K. L. Rinehart, Jr., J. Am.
Chem. Soc. 91 (1975) 937-938.
36 7 4&-2-Carboxy-4-isopropenyl-3-pyrrolidineacetic (a-Kainic acid)
acid
59.1 Introduction 59 ,I,1 Source organism
Uigenea A i m p t e x (Wulf 1 Aq. (red alga). 59.1.2 Chemical description CIoH1,NO,; colorless needles or prismatic crystals, m.p. dec.; soluble in water; { ~ r l i l- 1 4 . 8 k 0 . 5 ' ( c 1.01, H20); IR v cm-' 1,653, 892.
251'
59.1.3 Structural formula
H 59.2 Bioactivity Antihelminthic action of c a . 70% in 10-2G mg dose. 59.3 Extraction,
separation and purification
The alga was extracted with water and ethanol was added to form a precipitate. After centrifuging, the soluble phase was applied to a column of activated alumina, eluting with dilute ethanol (to remove inorganic chlorides followed by sodium mannosidoglycerate). Elution with distilled water and evaporation gave a pale-yellowish brown powder, which was purified with lead diacetate to give
so.
59.4 Literature cited
1. M. H. Baslow, "M.arine Pharmacology," Williams and Wilkins Co., Baltimore, 1969, 69-71, and references given therein.
36 8 60-65. Acetoxyf imbrolides
1, Introduction'
y2
1.1 Source organism V e t i d e a dimbziata ( = p u t c h n a ) (lamour.) Mont. (red alga).
1.2 Chemical descriptions
60
61 62
C11HllBr304, 11R-3-(l~-Acetoxybutylf-4-bromo-5dibromomethylene=3-butenolide, MS m/g 450 (MI. CllH12BrC104,1'R,5Z-3-(lt-Acetoxybutyl)-4-bromo-5chloromethylene-3-bztenolide , MS m/e 326 (MI. C 1 1H 12Brl04, 1IR, 5E-3- (l'-Acetoxybutyl)-4-bromo-5iodomethylene-3=bufenolide
, MS m/e
416 (MI.
63 CllH12Br104,1'R,5Z-3-(lt-Acetox buty&)-4-bromo-5iodomethylene-3=bufenolide, { a 1 5 x +26 m/e 416 (MI. MS -
64
1.4, CHC13);
CllH12Br204,lfI?,5_E-3-(lt-Acetoxybutyl)-4;bromo-5bromgmethylene-3-butenolide, m . g . 80-80.5 ; { a 1 a 5 -4.1 ( C 1.3, CHC13); ( ( ~ 1 6+47 ~ ( C 0.24, CHC13); MS m/e 370 (MI.
-65
(C
-
, 1 )g3,-:5 ( 1 ' -Ace toxybutyl I -4-bromo- 5 ylene-3-butenolideY +29' ( C 5.8, CHC13) b r o m 8 W ~ b R nm ( € 1 291 (8,900); IR (CHClB) u cm-1 1,792, ~~7h:x1,645, 1,613; lH NMR (100 MHz, CC14) 6 ppm 0.97 (t), 1.39 (m), 1.86 (m), 2.01 (s), 5.44 (dd, J = 7.4, 6.3 Hz), 6.24 (s); 13C NMR (CFT-PO, CDC13) ppm 14 (q), 19 (t), 21 (q), 3 3 (t), 6 8 (d), 92, 130, 131, 149, 162 (s), 170 ( 6 ) ; MS m/e 370 (MI. C
H 2Br2 0
60-65 - mixture:
nm ( € 1 293; IR v cm-l 1,780, 1,740; NMR 6 ppm 0.94 (3 H, t, J = 7 Hz), 1.34 ( 2 H, m), 1.86 (2 H, m), 2.09 ( 3 H, 8 1 , 5.52 (1 H, t), 6.70 and 6.38 (1 H combined, ratio 1: 3).
UV hmax CH30H
1.3 Structural formulas See next page, 2. B i o a c t i a
60-65 - "have
antimicrobial activity"'.
3 . Extraction, separation an.d purification
The air dried alga was extracted with methylene chloride, then subjected to open column silica gel chromatography followed by HPLC to give 60-65.
369
60 61 62 63 64 65 -
! l
%
Br
Br
H
c1
I
H
H
I H Br
Br H
4. Literature cited
1.
R. Kazlauskas, P. T. Murphy, R. J. Quinn and R. J. Wells, Tetrahedron Lett. (1977) 37-40.
2.
J. A. Pettus, Jr., R. M. Wing and J. J. Sims, Tetrahedron Lett. (1977) 41-44.
66. {3B-(2E,3B*, 7&*, 8&*, (Z))l-2-(l-Bromopropylidene)-7chloro-3,6,7,8-tetrahydro-8-~2-penten-4-ynylJ-2H-oxocin-3-01 (Chondriol) 66.1 Introduction 66.1.1 Source organism1” L a u n e n c i a sp. (originally thought to be C h o n d n i a o p p ~ d i t i c l a d a )(red alga). 66.1.2 Chemical description3 C15H18BrC102; colorless oil; UV nm ( € 1 221 max (9,800); IR vmax m/e cm” 3,400, 3,290; MS - 344 ( M I . 66.1.3 Structural formula3
370 66.2 B i ~ a c t i v i t y ~ ' ~ ug/ml f o r complete inhibition, 48 h.
Test organism
10-100
10-100 Chondriol is also a n t i v i r a l . 6 6 . 3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n ' T h e a l g a was e x t r a c t e d w i t h h e x a n e , t h e n c h r o m a t o g r a p h e d o v e r silica gel t o give c h o n d r i o l (0.1% d r y w e i g h t ) . 66.4 L i t e r a t u r e Cited F e n i c a l a n d J. N.
N o r r i s , J . P h y c o l o g y 11 ( 1 9 7 5 ) 1 0 4 - 1 0 8 .
1.
W.
2.
W . F e n i c a l , J. J. S i m s a n d P. R a d l i c k , T e t r a h e d r o n L e t t . ( 1 9 7 3 ) 313-316.
3.
W. F e n i c a l , K. B. G i f k i n s a n d J. C l a r d y , T e t r a h e d r o n L e t t . (1974) 1507-1510.
4.
J . J. S i m s , M . S . D o n n e l l , J. V . L e a r y a n d G . H. L a c y , A n t i m i c r o b . Ag. C h e m o t h e r . 7 ( 1 9 7 5 1 3 2 0 - 3 2 1 .
67. 4 - B r o m o - 6 - ( 1 ~ . 2 ~ - 1 , 2 - d i m e t h y l b i c y c l o ~ 3 . 1 . 0 ~ h e x - 2 - y l ~ - m - e r e s o ~ (Laurinterol) 67.1 Introduction 67.1.1 Source organisms
C o h a l l i n a c h i l e n d i h l, L a u h e n c i a d e c i d u a 2 , L , i n t e h m e d i a Yamada3, 1. , j o h n n t o n i i l , L . n i d i d i c a 4 , 1. n i p p o n i c a Yamada5, 1. obamuhai Yamada6, 1. pacidica7' 0 1 Rhodymenia cati{ohnical, ( r e d a l g a e ) , an u n i d e n t i f i e d B r y o z o a n ( E c t o p r o c t a ) l , Ophiodehma v a / l i e atum ( b r i t t l e s t a r l l , A p L y ~ i acalidohnica Cooper ( s e a hare)
B.
6 7.
I.2 C h e m i c a l d e s c r i p t i o n ' D
(c.1.88,
C15H19Br0; c o l o r l e s s c r y s t a l s , m.p. 54-55'; {c~}~~+13.3' CHC13); w nm ( E ) 2 2 5 ( 7 , 1 0 0 1 , 2 8 3 ( 2 , 2 0 0 1 ,
289 (2,100);
I R vmax CHC1'aEm-l
3,600,
3,450,
3,060,
1,610,
1,495,
1 , 1 5 2 , 1 , 0 8 0 , 1 , 0 2 5 , 9 0 0 , 8 6 5 , 8 5 0 ; NMR ( 6 0 MHz, C H C 1 3 ) 6 ppm 1.1 (1 H , s ) , 6 . 4 2 (1 H, s ) , 7 . 4 7 (1 H , s ) ; MS m/e ( r e l . a b u n d . 1 CH3, 1001, 265 (321, 251 (321,737 ( 6 5 1 , 226 294 ( M I , 279 ( M Br, 3 5 ) , 2 0 1 ( 7 2 ) , 200 ( 6 5 1 , 1 9 9 ( 5 5 1 , 1 7 6 (671, ( 7 5 1 , 215 ( M 1 5 9 ( 6 0 1 , 1 4 8 ( 4 0 1 , 1 4 4 ( 5 8 ) , 1 2 1 (18), 1 1 5 ( 5 2 1 , 9 1 ( 4 5 1 , 8 3 ( 5 2 1 , 77 ( 3 0 1 , 7 4 ( 7 2 1 , 5 9 ( 8 2 ) , 4 5 ( 7 8 1 .
-
-
371 6 7 . 1 . 3 S t r u c t u r a l formula11’12
67.2 Bioactivity4’13 Test orqanism
ug/ml for complete i n h i b i t i o n , 48 h . 1-5
1-5 10-100 67.3 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n l a The m e t h a n o l e x t r a c t o f t h e a i r - d r i e d a l g a was c o n c e n The t r a t e d i n vacuo a n d t h e r e s i d u e was p e r c o l a t e d w i t h e t h e r . e t h e r s o l u t i o n was s h a k e n w i t h 5 % a q u e o u s p o t a s s i u m h y d r o x i d e , S o l v e n t was removed t o g i v e a then with 1 N hydrochloric acid. n e u t r a l , brown oil which was c h r o m a t o g r a p h e d o v e r a l u m i n a , e l u Repeated rechromatography t i n g w i t h n-hexane-benzene ( 5 : l ) . o v e r s i l i c a g e l g a v e l a u r i n t e r o l a n d d e b r o m o l a u r i n t e r o l (E). More c o n v e n i e n t l y , t h e n - h e x a n e - b e n z e n e ( 5 : l ) e l u e n t was a c e t y l a t e d with acetic anhydride and p y r i d i n e , then chromatographed o v e r s i l i c a g e l , e l u t i n g w i t h n - h e x a n e - b e n z e n e (3:l). S o l v e n t was removed a n d r e c r y s t a l l i z a t i o n from m e t h a n o l o a f f o r d e d l a u r i n t e r o l a c e t a t e , c o l o r l e s s c r y s t a l s , m.p. 93-93.5 Hydrolysis wirh methanol-potassium hydroxide gave l a u r i n t e r o l ( c a . 0.22% from L . LnteArnedLa).
.
67.4 L i t e r a t u r e c i t e d 1.
K . L. R i n e h a r t , J r . , R . D. Johnson, J. F. S i u d a , G. E . K r e j c a r e k , P. D. Shaw, J . A . McMillan, a n d I . C . P a u l , in The N a t u r e o f S e a w a t e r . E d i t o r , E . D . G o l d b e r g , P h y s i c a l a n d C h e m i c a l S c i e n c e s R e s e a r c h R e p o r t 1, Abakon V e r l a g s g e s e l l s c h a f t , B e r l i n , 1 9 7 5 , p p . 651-665.
2.
S.
3.
T. I r i e , M . S u z u k i , E. Kurosawa a n d T. Masamune, T e t r a h e d r o n L e t t , ( 1 9 6 6 ) 1837-1840.
4.
S. M . Waraszkiewicz and K . ( 1 9 7 4 ) 2003-2006.
Caccamese and K . tion.
L . R i n e h a r t , Jr., m a n u s c r i p t i n p r e p a r a -
L. E r i c k s o n , T e t r a h e d r o n L e t t .
372 5.
T. S u z u k i , M . Suzuki and E. Kurosawa, T e t r a h e d r o n L e t t . ( 1 9 7 5 ) 3 0 5 7- 3 0 5 8 .
6.
T. I r i e , M . Suzuki and Y . Hayakawa, B u l l . Chem. Soc. J a p . 42 ( 1 9 6 9 ) 8 4 3 - 8 4 4 .
7.
K . L . R i n e h a r t , J r . , R . D . Johnson, I . C . P a u l , J . A. McMillan, J. F. S i u d a and G . E. K r e j c a r e k i n Food-Drugs From t h e S e a P r o c e e d i n g s 1 9 7 4 . E d i t o r s , H. H. Webber and G. D. R u g g i e r i , Mar. Technol. Soc., Washington, D . C . , 4 3 4 442 ( 1 9 7 6 ) .
8.
J. J. Sims, W . F e n i c a l , R . M . Wing and P. R a d l i c k , J. Am. Chem. Soc. 9 3 ( 1 9 7 1 ) 3 7 7 4 - 3 7 7 5 .
9.
M . 0. S t a l l a r d and D . J . F a u l k n e r , Comp. Biochem. P h y s i o l . 49B ( 1 9 7 4 ) 2 5 - 3 5 .
10.
T. I r i e , M . S u z u k i , E. Kurosawa and T. Masamune, Tetrahedron 2 6 ( 1 9 7 0 ) 3 2 7 1 - 3 2 7 7 .
11.
A . F. Cameron, G . Ferguson and J . M .
R o b e r t s o n , Chem.
Commun. ( 1 9 6 7 ) 2 7 1 - 2 7 2 . 12.
A . F. Cameron, G . Ferguson and J. M . R o b e r t s o n , J. Chem. SOC. (3) ( 1 9 6 9 ) 6 9 2 - 6 9 7 .
13.
J . J. Sims, M , S . D o n n e l l , J . V . Leary and G . H. Lacy, A n t i m i c r o b . Ag. Chemother. 7 ( 1 9 7 5 ) 3 2 0 - 3 2 1 .
68.
6-~1S,2B-1,2-D~methylb~cycloE3.l.0)hex-2-yl~-m-cresol (Debromolaurinterol)
68.1 1ntroducti.on 6 8 . 1 . 1 S o u r c e orkanisms
Launencia i n t e n m e d i a Yamada 1, L , j o h n b t o n i i 2 , L , obamunai Yamada3, L . p a c i 6 i c a 4 , Plocamium pacidicum2 ( r e d a l g a e ) , Laxodubeniteb s p . (sponge12, A p l y a i a c a e i d o a n i c a Cooper ( s e a hareI5. 6 8 . 1 . 2 Chemical d e s c r i p t i o n 6 C15H20O;
colorless o i l ; (alko -12.2'
(C
1.80,
CHC13);
nm ( E ) 2 7 6 ( 3 , 5 0 0 1 , 2 8 2 ( 3 , 4 0 0 ) ; I R vmax cm-l 3 , 6 8 0 , 3,060, 1,620, 1,580, 1,520, 1,420, 1,392, 1,378, 1,295, 1,230, 1 , 1 8 5 , 1 , 1 3 0 , 1 , 1 0 0 , 1 , 0 6 0 , 1 0 2 0 , 9 5 5 , 8 5 5 , 8 1 5 ; NMR 6 ppm c a . 0 . 5 ( 2 H , m ) , c a . 1.1 (1 H , m j , 1 . 2 9 ( 3 H , s), 1 . 3 5 ( 3 H , s), 2 . 2 0 ( 3 H , s ) , 4 . 9 6 (1 H , s), 6 . 3 0 (1 H , b r s), 6 . 4 9 (1 H , b r d , J 7 . 5 Hz); MS ( r e l . a b u n d . ) 2 1 6 (MI, 2 0 1 ( M CH3, l o o ) , 1 8 5 ( 5 1 1 , 1 1 5 (4r6), 9 1 ( 4 4 1 , 7 7 ( 4 0 1 , 74 ( 5 2 1 , 59 ( 5 7 1 , 45 ( 5 3 ) .
UV ACZHsoH max
m/e
-
373 6 8.1.3
S t r u c t u r a l formula7'
6 8.2 Bioact i v i t y ug/ml f o r conplete
Teat organism
S.tophglococcus w e u d !&eobactehium m t g n n z t h candida diw
--i n h i b i t i o n ,
46 h.
10-30 10-50 10-50
68.3 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n 6 D e b r o m o l a u r i n t e r o l was o b t a i n e d w i t h l a u r i n t e r o l (67) f r o m 1 . i n t e k m e d i a by r e p e a t e d c h r o m a t o g r a p h y of t h e n - h e x z e A g a i n more c o n v e n i e n t l y , t h e b e n z e n e ( 5 : l ) e l u e n t (see 67.3). a c e t y l a t e d c - h e x a n e - b e n z e n e (5:l) e l u e n t , c h r o m a t o g r a p h e d o v e r s i l i c a g e l e l u t e d w i t h n - h e x a n e - b e n z e n e (1:l) a n d t h e n r e c h r o m a t o g r a p h e d o v e r s i l i c a - g e l a f f o r d e d d e b r o m o l a u r i n t e r o l aceHydrolysis t a t e , a c o l o r l e s s o i l , {alD-28.9'(c 2.04, C H C 1 3 ) . f o l l o w e d b y c h r o m a t o g r a p h y o v e r s i l i c a g e l gave d e b r o m o l a u r i n terol.
68.4 L i t e r a t u r e -
Cited
1.
T. I r i e , M . S u z u k i , E. Kurosawa a n d T. Masamune, T e t r a h e d r o n L e t t . (1966) 1837-1840.
2.
K . L . R i n e h a r t , J r . , R . D. J o h n s o n , J. F. S i u d a , G . E. Krejcarek, P. D. Shaw, J. A . McMillan, a n d I . C . P a u l , i n The N a t u r e o f S e a w a t e r . E d i t o r , E . D. G o l d b e r g , P h y s i c a l a n d C h e m i c a l S c i e n c e s R e s e a r c h R e p o r t 1, Abakon V e r l a g s g e s e l l s c h a f t , B e r l i n , 1975, p p . 651-665.
3.
T. I r i e , M. S u z u k i , a n d Y. Hayakawa, B u l l . Chern. SOC. Japan 42 (1969) 843-844.
4.
K . L. R i n e h a r t , J r . , R . D. J o h n s o n , I . C. P a u l , J. A . McMillan, J. F. S i u d a a n d G . E . Krejcarek i n Food-Drugs from t h e S e a P r o c e e d i n g s 1974. E d i t o r s , H. H. Webber a n d G . D. R u g g i e r i , Mar. T e c h n o l . SOC., W a s h i n g t o n , D.C. , 4 3 4 442 (1976).
5.
M. 0. S t a l l a r d a n d D. J . F a u l k n e r , Comp. Biochem. P h y s i o l . 49B (1974) 25-35.
374 6.
T. I r i e , M. S u z u k i , E . Kurosawa and T . Masamune, T e t r a h e d r o n 26 (1970) 3271-3277.
7.
A. F. Cameron, G . Ferguson and J. M . R o b e r t s o n , Chem. Commun. (1967) 271-272.
8.
A. F. Cameron, G . Ferguson a n d J. M . R o b e r t s o n , J. Chem. SOC. ( B ) (1969) 692-697.
9.
J . J. Sims, M . S . D o n n e l l , J. V . Leary and G . H . Macy, A n t i m i c r o b . Ag. Chemother. 7 (1975) 320-321.
69. 4' 5-Dibromo-4-chloro-l',3',3' , 4 - t e t r a r n e t h y l s p i r o { c y c l o - - o x a b i c y c l o { 4 . 1 .O I h e p t I 4 l e n I - Z - o l (Prepacif e n o l ) 69.1 I n t r o d u c t i o n 1 69.1.1
Source
organism
Laulrencia dilidonmi41, Laulrencia p a c i d i c a K y l i n 2 . 69.1.2
Chemical d e s c r i p t i o n
C 1 5 H 2 1 B r 2 C 1 0 2 ; c r y s t a l s , m.p. 109-126' s o l i d i f y , then r e m e l t a t 1470; MS m/e 426 (MI, 409.9477 (M - k 2 0 ) ; NMR (100 MHz, C D C 1 , ) 6 ppm 1T2q ( s ) , 1.42 (s), 1.50 ( d , J 5 H z ) , 1.63 (s), 1 . 8 8 ( s ) , 2.4 (m), 3.02 ( d , J 3 H z ) , 4.41, 4.72, 6.25 ( d , J = 3 Hz).
69.1.3 S t r u c t u r a l f o r m u l a 3
Br
69.2 B i o a c t i v i t y 2 Test organiem
SAaphylococcu w e w hkjcobactuhm bmegmath
vg/ml for complete inhibitj.bn, 48 h.
10-100 10-100
375
69.5 Extraction,
s e p a r a t i o n and p u r i r i c a t i o n '
The a l g a was e x t r a c t e d w i t h hexane and s o l v e n t was removed. The p a r t l y c r y s t a l l i n e r e s i d u e was r e c r y s t a l l i z e d from hexane t o g i v e p r e p a c i f e n o l ( 0 . 3 % of d r y p l a n t ) . 69.4 Literature cited
1.
J . J . S i m s , W . F e n i c a l , R . M . Wing and P . R a d l i c k , J . Am. Chem. SOC. 9 5 ( 1 9 7 3 ) 9 7 2 .
2.
W.
3.
J . J . S i m s , W . F e n i c a l , R . M. Wing and P . R a d l i c k , J. Am. Chem. SOC. 9 3 ( 1 9 7 1 ) 3 7 7 4 - 3 7 7 5 .
4.
J. J. Sims, M . S . D o n n e l l , J. V . Leary and G . H. Lacy, A n t i m i c r o b . Ag. Chemother. 7 ( 1 9 7 5 ) 3 2 0 - 3 2 1 .
F e n i c a l , Phytochem. 1 5 ( 1 9 7 6 ) 5 1 1 - 5 1 2 .
7 0 . 2 S , 3 S 4R-2-Carboxy-4-(ltE,3'E,5'S-l'-met h e x a d f e n y l ) 3-py r r , o l i d i n e a c et i c a c i d
-
7 0 . 1 Introduction 7 0 . 1 . 1 S o u r c e organism ChondfiLa akma.ta ( K i t z i n g ) Okamura ( r e d a l g a ) . 7 0 . 1 . 2 Chemical d e s c r i p t i o n 2
C15H21N06; c o l o r l e s s n e e d l e s ( d i h y d r a t e ) , m.p. 217' d e c . ; s o l u b l e i n water, a c e t i c a c i d , m i n e r a l a c i d s and a l k a l i ; s p a r i n g l y s o l u b l e i n methanol and e t h a n o l ; i n s o l u b l e i n c h l o r o form e t h e r a benzene and a c e t o n e ; pKa 2 . 1 0 , 3 . 7 2 , 4 . 9 7 , 9 . 8 2 ; ( H 2 0 ) ; UV A:", nm ( l o g E ) 2 4 2 ( 4 . 2 4 ) ; I R ( K B r ) v {a1b5-109.6
cm-1 9 7 0 ; NMR (CF3COOH) 6 ppm 1 . 4 2 ( d , J = 6 . 6 Hz), 1 . 9 4 ( b r s ) . 7 0 . 1 . 3 S t r u c t u r a l formula3
COOH
I CH,--C-C=C-C=C, I I I H
H
H
CH3
I
C , H,C 00 H
I
H QCOOH
I
H
3 76
70.2 B i o a c t i v i z l
"Oral a d m i n i s t r a t i o n o f 2 0 mg o f was f o u n d t o be m a r k e d l y e f f e c t i v e i n e x p e l l i n g ascaris and pinworm, w i t h o u t any o b s e r v a b l e s i d e e f f e c t t t 1 . 70.3 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n 2
1. The a l g a was e x t r a c t e d w i t h water f o l l o w e d by m e t h a n o l , t h e n a d s o r b e d on aluminum o x i d e , e l u t i n g w i t h water t o g i v e 2 ( 2 - 3 mg/g d r y w e i g h t ) . 2. The a l g a was e x t r a c t e d w i t h water t h e n a d s o r b e d on a c t i v a t e d c h a r c o a l e l u t i n g with methanol t o g i v e
2.
70.4 L i t e r a t u r e c i t e d
_L
1. K . D a i g o , Yakugaku Z a s s h i 79 ( 1 9 5 9 ) 350-358; Chem. A b s t r . 53 ( 1 9 5 9 ) 14218. 2.
T. Takemoto and K . D a i g o , Arch. Pharm. 293 ( 1 9 6 0 ) 627-633; Chem. A b s t r . 54 ( 1 9 6 0 ) 23187b.
3.
T. Takemoto, K. D a i g o , Y . Kondo and K. Kondo, Yakugaku Z a s s h i 86 (1966) 874-877; Chem. A b s t r . 66 ( 1 9 6 7 ) 28604m.
71. {3S-~30,,3a~,48,78,7a~~~-7-Bromooctahydr~o-4,7a-d~methy1-3n - m e t h y l - 1 - p r o p e n y l ) - I ? T ~ X G Z C ~( o ~p p o s i t o l ) 71.1 Introduction -
7 1 . 1 . 1 Source organism
Lauhencia A u b o p p o a i t a S e t c h e l l ( r e d a l g a ) . 71.1.2
Chemical d e s c r i p t i o n
C15H 5BrO; c o l o r l e s s { a y z 3 -232' (c 0.19, 54- 5 5 H , 81, 1 . 1 5 ( 3 H , s ) , 1.05 (1 H.. m), 3 . 8 3 (1 H , d o f d , Hz); MS m/g 300 ( M I .
'(b
o i l , c r y s t a l l i z e s s l o w l y , m.p. C2H50H); NMR (220 MHz, C C l t , ) 6 ppm 1 . 6 2 ( 6 H, s ) , 2 . 2 8 (1 H , m), 2.99 J = 1 2 . 4 H z ) , 5.02 ( 1 H , d , J = 9.5
71.1.3 S t r u c t u r a l formula See n e x t page.
--
71.2 Bioactlvify IlModerately a c t i v e "
VA.
S t a p h y l o c o c c u s auReuA.
7 1 . 3 E x t r a c t i o n , s e g a r a t i o ? and p u r i f i c g t i o n The a i r - d r i e d a l g a was e x t r a c t e d w i t h h e x a n e . chromatography y i e l d e d o p p o s i t o l ( 0 . 1 % d r y w e i g h t ) .
Repeated
377
Br
Q?
HO
CH3
-C
71.4 L i t e r a t u r e c i t e d
1.
S. S . H a l l , D. J . F a u l k n e r , J . F a y o s a n d J . C l a r d y , J. Am. Chem. SOC. 9 5 ( 1 9 7 3 ) 7 1 8 7 - 7 1 8 9 .
7 2 . D e c a h y d r o - a , a , 4a-trimethyl-cyclopropa{d ) n a p h t h a l e n e - 7 methanol ( Cy c l o e u d e s m o l ) 72.1 Introduction1 72.1.1
Source organism
Chondnia o p p o d i t i c l a d a Dawson ( r e d a l g a ) . 72.1.2
Chemical d e s c r i p t i o n
C 1 5 H 2 6 O ; c o l o r l e s s n e e d l e s , m.p. 94-95'; ( c 0 . 3 2 , C H 3 0 H ) ; IR v cm-1 3 , 6 8 0 , 3 , 5 5 0 , 3 , 0 9 5 ; NMR 2 2 0Irl MHz, I * C C 1 4 ) 6 ppm 0 . 3 5 ( d , J = 5 H z ) , 0 . 4 7 ( d , J = 5 Hz), 1.00 ( 3 H , s ) , 1 . 2 5 ( 3 H, s ) , 1 . 3 3 ( 3 H, s ) , 1 . 0 - 2 . 3 ( m ) ; MS m/e 2 2 2 (MI, M 18, intense.
-
-
72.1.3
S t r u c t u r a l formula
C H3
378 72.2 Bioactivity2 pg/ml f o r complete inhibition, 48 h.
Teat organism
SAaphylococcLLs w e u d
10-50 50-100
S&oneUa choluaed.iLLa Uycobactuium Arnegmetid Ccvrdida d b i c w
10-50
10-50
72.3 Extraction, separation and purification’ The alga was extracted with methylene chloride, chromatographed repeatedly and then crystallized from hexane to yield cycloeudesmol (0.021% dry weight). 72.4 Literature -
1.
cited
W. Fenical and J. J. Sims, Tetrahedron Lett. (1974) 11371140.
2.
J. J. Sims, M. S. Donnell, J. V. Leary and G. H. Lacy, Anrimicrob. Ag. Chemother. 7 (1975) 320-321.
73. 2,3,4,5-Tetrabromo-lH-pyrrole 73.1 Introduction 73.1.1
Source organism
Chnomobactekium sp. (Sneath, 1956) (marine bacterium). 73.1.2
Chemical description
C4HBr4N; MS (70 eV) m/e 379 (MI, 300 (M HCNBr), 2 2 1 (M - Br2), 194 - HCNBr2), 142 (M HCNBr3), 6 3 (CkHN), 62 (C4N).
(x
73.1.3
-
Br), 273 (M Br3), 115 (M -
Structural formula
B)ii-JBr
Br
Br
I
H
73.2 Bioactivity In disk-assay screens using 5 mg/disk, 73 inhibited EAchenichia CO-!~, PAeudomonaA aenuginoha and C a a i d a albicanh (1-6 mm zones of inhibition) and StaphylOCoccuh auReuA ( > 6 mm zone of inhibition). It also inhibited the growth of the marine
3 79
b a c t e r i a Photobacteaium i i ~ h e a ia n d P . mandapamendid ( > 6 mm z o n e s o f i n h i b i t i o n ) a n d P . phoAphoaeum, Beneckea haaueyi a n d ChaomobacteA I-L-33 ( 1 - 6 mm z o n e s o f i n h i b i t i o n ) . 73.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t - i o n The m a r i n e b a c t e r i u m I-L-33 was m a s s - c u l t u r e d by o n e o f two methods:
1. F i l t e r e d seawater, B a c t o - P e p t o n e ( 5 g / i ) , y e a s t e x t r a c t (1 g / t ) , and N a B r (500 mg/a) were a u t o c l a v e d i n P y r e x c u l t u r e b o t t l e s f i t t e d w i t h f r i t t e d g l a s s b u b b l e r s , 500-ml i n o c u l a o f a 2 4 - h r seawater b r o t h c u l t u r e of I-L-$3 were a d d e d and t h e c u l t u r e was i n c u b a t e d w i t h a e r a t i o n a t 25 f o r 2 w e e k s . E t h y l a c e t a t e ( 3 a ) a n d sodium c h l o r i d e ( 2 , 4 0 0 g ) were a d d e d , t h e m i x t u r e was s t i r r e d f o r s e v e r a l h o u r s , a n d t h e e t h y l acet a t e l a y e r was removed a n d c o n c e n t r a t e d . 2 . Heavy l a w n s o f b a c t e r i a were grown on t r a y s ( 2 x 30 x 60 cm) c o n t a i n i n g B a c t o - T r y p t o n e (10 g / t ) , y e a s t e x t r a c t (1 g / a ) and N a B r ( 5 0 0 m g / a ) . Twelve-hour b r o t h i n o c u l a were s p r e a d u n i f o r m l y o v e r t h e a g a r s u r f a c g s , w h i c h were t h e n i n c u b a t e d u n d e r h e a v y aluminum f o i l a t 25 f o r 4 d a y s . A d a r k p u r p l e p a s t e of w e t c e l l s was s c r a p e d from t h e a g a r s u r f a c e , f r e e z e - d r i e d , homogenized i n m e t h a n o l , e x t r a c t e d w i t h e t h y l acetate, and c o n c e n t r a t e d . The c r u d e e x t r a c t o b t a i n e d a c c o r d i n g t o 7 3 . 3 . 1 o r 73. 3.2 was t r i t u r a t e d w i t h b e n z e n e (100 ml/g c r u d e e x t r a c t ) , f i l tered and c o n c e n t r a t e d i n vacuo. Chromatography on F l o r i s i l e l u t i n g w i t h hexane, benzene, and e t h y l acetate g r a d i e n t s a f f o r d e d 73, p - h y d r o x y b e n z a l d e h y d e (571, 2 , 4 - d i b r o m o - 6 - ( 3 , 4 , 5 tribromopyrrol-2-yl)-phenol ( 2 1 , hexabromo-2,2'-bipyrrole and n - p r o p y l 4-hydroxybenzoate . 73.4 L i t e r a t u r e c i t e d
R . J . A n d e r s e n , M. 27 ( 1 9 7 4 ) 281-285.
1.
S . Wolfe a n d D.
J . F a u l k n e r , Mar. B i o l .
74. 2 , 4 - D i b r o m o - 6 - ( 3 , 4 , 5 - t r i b r o m o p y r r o l - 2 - y l ~ - p h e n o l 74.1 I n t r o d u c t i o n 74.1.1 Source organisms
Chhomobactea I - L - 3 3 l , PAQudomonaA baomoutilid from ThaZaAdia ( t u r t l e g r a s s I 2 ( m a r i n e b a c t e r i a ) . 74.1.2
~k;"
C1 H4B
200';
uv
Chemical d e c . 135-155', tar NQoH nm 284, 293, 355;
sNO; f a i n t g r e e n c r y s t a l s nm 286, 308;UV
NMR (CDC13) 6 ppm 6.07 (1 H, s ) , 7.60 (1 H, d, J = 2.5 Hz), 8 . 1 3 ( 1 H , d , J = 2.5 Hz), 9.5 (1 H , b r ) ; MS ( 7 0 eV) m/e 548
3 80
-
-
-
-
(MI, 4 7 0 (M B r ) , 4 4 2 (M H C N B r ) , 391 (M B r p ) , 364 (M H C N B r 3 1 , 80 ( H B r , b a s e p e a k ) . B r ) , 285 (M H C N B r 2 ) , 3 1 2 (M
-
-
74.1.3 S t r u c t u r a l f o r m u l a
Y
H
Br
74.2 B
i o a c t i ~ i t y ~ ~ ~ ~ ~ Activity
Test organism
Conplete inhibition a t 0.0063 s ~ h y . e o c o c c u bauReud pg/ml (Trypticase Soy Broth) D.ip.-!ococcud pnewnodaa. S.tnep.tdcoccu.4 pyo@nU kjcobac&&un.tubUcdoA.id Inhibition a t 0 . 2 pg/ml i n H37R
Gram-negati ve organisms
synthetic medium Inacti ve
Candida aebicans Mouse t o x i c i t y 25 mg/kg ( s i n g l e dose, intravenous ) 50 mg/kg (single dose, intravenous) 250 mg/kg ( s i n g l e dose, subcutaneous ) 200 mg/kg ( s i n g l e dose, subcutaneous)
Tolerated Lethal Tolerated Lethal No protection vd. UC-76 i n f e c t i o n
-
S.
auheud
74.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
1. From Chnomobacteh I-L-33 t e t r a b r o m o - 1 H - p y r r o l e (73).
( r e f . 1).
See 2,3,4,5-
-
2. From Pdeudomona..6 bhomoutilLd (ref.. 2 ) . The growth medium u s e d was seawater (1 & I , N-Z Case (2 g ) , S o y t o n e ( 2 g), y e a s t e x t r a c t (1 g ) , d e x t r o s e ( 2 g ) , v i t a m i n B I Z !1 g ) , D i f c o a g a r ( 2 0 g ) . An a c t i v e l y growing ( 2 4 h r ) s u s p e n s i o n of c e l l s (1 ml of 1 0 0 g of t u r t l e g r a s s s h a k e n w i t h 9 9 m l s t e r i l e o s e a water) was s t r e a k e d o v e r t h e s u r f a c e and i n c u b a t e d a t 30 f o r
381 The b a c t e r i a l s l u d g e was b l e n d e d f o r 5 min. i n a Waring 3 days. B l e n d o r w i t h 5 v o l . o f m e t h a n o l , t h e n c e n t r i f u g e d a t 2,000 x g f o r 1 0 min. The s u p e r n a t a n t l i q u i d was d e c a n t e d a n d t h e comb i n e d m e t h a n o l i c e x t r a c t s were c o n c e n t r a t e d i n Vacuo t o a d a r k o i l y r e s i d u e w h i c h was s t i r r e d r e p e a t e d l y w i t h e t h y l e t h e r . The e t h e r was removed, c h l o r o f o r m was a d d e d a n d c h r o m a t o g r a p h y on s i l i c a g e l (No. 3405, B a k e r ) , e l u t i n g w i t h 1 0 0 % c h l o r o f o r m , g a v e a c t i v e f r a c t i o n s ( b y 8 . d u b f i l i a a s s a y ) which were t a k e n t o d r y n e s s . S g l u t i o n i n a m i n i m a l amount of c h l o r o f o r m and c o o l i n g t o - 1 0 g a v e l o n g n e e d l e s which were r e c r y s t a l l i z e d by s l o w c o o l i n g from c h l o r o f o r m t o g i v e l a r g e m o n o c l i n i c c r y s t a l s . 74.4 L i t e r a t u r e -
1. 2.
cited
R. J . A n d e r s e n , M. S . Wolfe a n d D . J . F a u l k n e r , Mar. B i o l . 27 ( 1 9 7 4 ) 281-285. P . R . B u r k h o l d e r , R . M . P f i s t e r a n d F. H . L e i t z , Appl.
Microbial. 1 4 ( 1 9 6 6 ) 649-653.
3.
F. M . L o v e l l , J . Am. Chem. SOC. 88 ( 1 9 6 6 ) 4510-4511.
4.
S . H a n e s s i a n a n d J. S . K a l t e n b r o n n , J. Am. ( 1 9 6 6 ) 4509-4510.
Chem. S o c . 88
75. 2 - g - P e n t y l - 4 - q u i n o l o n e 75.1 I n t r o d u c t i o n 75.1.1 Source organism
Pdcudomonad 102-3 ( m a r i n e b a c t e r i u m ) , 7 5 . 1 . 2 Chemical d e s c r i p t i o n C1bH17NO;
I R (CHC13) 0.80 ( 3 H, 6.26 (1 H , 8.38 (1 H , 159 (loo),
m.p.
141-142';
UV
max
nm 235, 3 1 5 , 3 2 6 ;
u cm-1 3 , 3 0 0 , 1 , 6 4 5 , 1 , 5 9 0 , 1 , 5 5 0 ; NMR ( C D C 1 3 ) 6 ppm t ) , 1 . 2 3 (4 H, m), 1 . 7 2 ( 2 H , m ) , 2.70 (1 H , t ) , 7.34 (1 H , t ) , 7.59 (1 H , t ) , 7.85 (1 H , d ) , d ) ; MS m / e ( r e l . i n t e n s i t y , % ) 215 (211, 172 ( 2 3 1 , 130 (337,-77 ( 2 5 ) ; HRMS 215.1309 ( M I . s),
75.1.3
S t r u c t u r a l formula
See n e x t page. 75.2 B i o a c t i v i t y I n a g a r d i f f u s i o n t e s t i n g a t 50 p g / d i s k ,
2
inhibited
Staphylococcua autreua ( > 6 m m zone of i n h i b i t i o n ) a n d V i b t r i o hatrueyi a n d V . a n g u i l l a a u m ( 1 - 6 mm z o n e s o f i n h i b i t i o n ) .
75.3 E x t r a c t i o n ,
s e p a r s t i o n and p u r i f i c a t i o n
pacudomonad 1 0 2 - 3 was c u l t u r e d o n a t r a y (60 x 30 x 2 cm) of p e p t o n e ( 5 g/k), y e a s t e x t r a c t ( 1 g / k ) and a g a r (10 g / k )
382
0
i n f i l t e r e d seawater for 4 d a y s a t room t e m p e r a t u r e . C e l l s were s c r a p e d g e n t l y from t h e a g a r s u r f a c e a n d homogenized i n e t h y l acetate ( 1 0 0 m l / g of wet c e l l s ) . The s o l u t i o n was f i l tered, d r i e d o v e r a n h y d r o u s sodium s u l f a t e ( t h r e e times), a n d c o n c e n t r a t e d i n vacuo t o a v i s c o u s o i l . The c r u d e e x t r a c t was c h r o m a t o g r a p h e d o v e r F l o r i s i l , e l u t i n g w i t h s o l v e n t s o f i n c r e a s i n g p o l a r i t y from benzene t o 20% m e t h a n o l i n e t h y l a c e t a t e . The f r a c t i o n e l u t e d w i t h b e n zene g a v e 2 - h y d r o x y b e n z a l d e h y d e ( 5 7 ) . F r a c t i o n s e l u t e d w i t h e t h e r were c h r o m a t o g r a p h e d on s i l E a g e l p l a t e s u s i n g 1 5 % acet o n i t r i l e i n c h l o r o f o r m t o s e p a r a t e t h e major component 75, which was c r y s t a l l i z e d f r o m benzene-hexane ( l : l ) ,from tE minor component 2 - n - h e p t y l - 4 - q u i n o l o n e (El. 75.4 L i t e r a t u r e c * S . J . W r a t t e n , M. S . Wolfe, R . J . Andersen a n d D. J. F a u l k n e r , A n t i m i c r o b . Ag. Chemother. 11 ( 1 9 7 7 ) 411-414.
1.
76, 2 - n - H e p t y l - 4 - q g i n o l o n e 76.1 I n t r o d u c t i o n 76.1.1 Source organism
PAeudomonad 1 0 2 - 3 l ( m a r i n e b a c t e r i u m ) , PAeudomonu.4 aehugLnoAa2 ( t e r r e s t r i a l b a c t e r i u m ) . 76.1.2
Chemical d e s c r i p t i o n
C 1 $-I2 1 N O ; m.p.
i d e n t i c a l t o a u t h e n t i c materi-
146-147';
a13.
76.1.3
S t r u c t u r a l formula
S e e s t r u c t u r e o f 75; for
E,
n = 6.
76.2 Bioactivity I n a d i s k a s s a y a t 50 u g / d i s k , StaphyRococcuA auheud
383 (>6 mm zone of inhibition) and V i b A i o h u v e y i (1-6 mm zone of inhibition) were inhibited. 76.3 Extraction, separation and purification See Section 75.3. 76.4 Literature Cited
J. Wratten, M. S. Wolfe, R. J. Andersen and D. J. Faulkner, Antimicrob. Ag. Chemother. 11 (1977) 411-414.
1.
S.
2.
E. E. Hays, I. C. Wells, P. A. Katzman, C. K. Cain, F. A. Jacobs, S. A. Thayer, E. A. Doisy, W. L. Gaby, E. C. Roberts, R. D. Muir, C. J. Carroll, L. R. Jones and N. J. Wade, J. Biol. Chem. 159 (1945) 725-750.
3.
I. C. Wells, J. Biol. Chem. 196 (1952) 331-340.
77. 4a,12b-Dihydro-4a,8,12b-trihydroxy-4-methylbenz(a~anthracene(SS-228 Y) 2 ,7 ,1773X)- trione
77.1 Introduction 77.1.1 Producing organism
Chainia punpuaogena SS-228 (actinomycete). 77.1.2 Chemical description Cl9H1406; orange powder, m.p. 256-266' dec. ; {0}~-85' ~ N 80% CH30H nm ( € 1 218 (22.000). I c 1.0. (CH2)9CO): - - , uv A ~ *HC1, 228 sh (21,800), 415 sh (4,000), 440-460 (4,100), Aoa2E NaoHy CH30H 269 (17,900), 408 (6,4001, 552 (4,400); IR (KBr) u cm-1 1,680, 1,662, 1,630; NMR (db-methanol) 6 ppm 1.93 (3 H, s ) , 2.68 (2 H, br s), 6.12 (1 H, m), 6.47 (1 H, d , J 10.0 Hz), 6.96 (1 H, d, J = 10.0 Hz), 7.24 ( 1 H, dd, J = 7.5, 2.0 H z ) , 7.45-7.85 (2 H, m); MS I J / ~ 320 (M - 18). 77.1.3 Structural formula See next page,
77.2 Bioactivity See next page. 77.3 Extraction, segaration and purification Straig SS-228 was cultivated on an agar slant of GG medium, at 27 for 2-3 weeks, then inoculated (125 ml in 500 ml Sakaguchi flask) into a culture medium of Kobu-Cha (powdered tangle seaweed, Laminahia) (l.O%),.glycerol (1.0%), pH 7.4, and maintained on a reciprocating shaking machine of 8 cm amplitude,
384
0
Test organism
Mycoba&.aLum AmgmatiS kkjcobacaWdutn p h W
M I C (pg/ml) ATCC 607
B&LU AUbZLU.6 NRRL 8-558 8 a C i l . h dubtiU.6 P C 1 219 8dUud dn.thn&
Conynebac&mbunbow& 1810 u i r n o c o c c ~~ f h . u A FDA 16 Sancina &&a PC1.1001 S ~ h y l o c a c c u 4auheud FDA 209 P
50 25 3.12 1.56 1.56 12.5 0.78 1.56 3.12 1.56 100 50
50
50
Sa&one& q p h o d a T-63 Ski@& dlexneni 4b JA 11811 Candida &icand 3147
100 50 50
77 prolonged t h e s u r v i v a l o f mice i n o c u l a t e d w i t h Ehrlieh a s c i t e a tumor X e n dose waa >1.56 vg/mouse/day, 10 da. Acute t o x i c i t y i n mice: LD50 1.56-6.25 r n g h g , i n t r a p e r i t o n e a l . 77 i n h i b i t e d dopemine B-hydroxylase (65.2% i n h i b i t i o n a t 0.1 ug/ml).
130 s t r o k e s / m i n a t 27'' f o r 72 h o u r s . A p o r t i o n of t h e broth ( 2 0 ml) was t r a n s f e r r e d t o 1.250 m l of t h e c u l t u r e medium and s h a k e - c u l t u r e d as above f o r 1 2 0 h o u r s . S . awLeuA FDA 2 0 9 P was used f o r t h e m i c r o b i o l o g i c a l a s s a y by t h e c y l i n d e r p l a t e method, The b r o t h was a d j u s t e d to pH 5 . 0 w i t h d i l u t e hydro-
3 85
c h l o r i c a c i d a n d f i l t e r e d , t h e n e x t r g c t e d twice w i t h e t h y l acet a t e and c o n c e n t r a t e d i n vacuo a t 4 0 t o g i v e a r e d d i s h brown powder which was c h r o m a t o g r a p h e d twice o v e r s i l i c i c a c i d ( A . R . , 1 0 0 mesh M a l l i n c k r o d t ) , e l u t i n g once w i t h c h l o r o f o r m a n d t h e n w i t h w a t e r - s a t u r a t e d b u t y l a c e t a t e t o g i v e SS-228 Y ( 4 0 % y i e l d ) . 77. 4 L i t e r a t u r e c i t e d
1.
T. Okazaki, T. K i t a h a r a a n d Y . (1975)
2.
Okami, J. A n t i b i o t i c s 28
176-184.
T. K i t a h a r a , H. Naganawa, T . O k a z a k i , Y . Okami a n d H . Umezawa, J. A n t i b i o t i c s 2 8 (1975) 2 8 0 - 2 8 5 .
This Page Intentionally Left Blank
387
Penicillins and Related Antibiotics
.
. .
Barid B Mukherjee and 8 K Lee Universidad Autonoma de Ciudad Juarez Escuela de Medicina. Juarez. Chihuahua. Mexico and Schering Corporation. Bloomf ield. New Jersey 1.
Natural penicillins with 6-APA nucleus 1.1 Introduction 1.1.1 Producing organisms 1.1.2 Structural formulae 1.1.3 Physical and chemical properties 1.1.3.1 Penicillin G 1.1.3.2 Penicillin F 1.1.3.3 Penicillin K 1.1.3.4 Penicillin X 1.1.3.5 Penicillin N 1.2 Therapeutic use and pharmacology of penicillins 1.3 Methods for assaying penicillins 1.4 Isolation of penicillins from fermentation broths 1.4.1 Solvent extraction method 1.4.2 Charcoal adsorption method 1.4.3 Chromatographic methods of purifying penicillins 1.4.4 Precipitation Of penicillins with organic base or ammonia 1.4.5 Isolation of penicillin N and isopenicillin N
.................................... ........................... ........................... .............. .................................. .................................. .................................. .................................. ..................................
................. .......................................... ..................... .................... ................................... ...............................
2
.
6-Aminopenicillanic acid (6-APA) Introduction Producing organisms 2.1.2 Physical and chemical properties 2.2 Antibacterial activity of 6-aminopenicillanic acid 2.3 Assay methods for 6-aminopenicillanic acid 2.3.1 Cup plate assay 2.3.2 Hydroxylamine assay 2.3.3 Phenylacetylation method of assay 2.4 Isolation and purification of 6-aminopenicillanic acid 2.4.1 Preparation of a crude concentrate (I) 2.4.2 Purification of the crude concentrate (11) 2.4.3 Purification of 6-APA by Dowex 1 Resin 2.4.4 Recrystallization of 6-APA Formation of 6-APA from penicillin by enzymic 2.5 hydrolysis 2.5.1 Purification and general properties of penicillin amidase 2.5.2 Isolation of 6-APA from acylase treated penicillins 2.5.3 Isolation of 6-APA from phenoxymethyl penicillin using spores of fusaria 2.5.4 Preparation of 6-APA from benzyl penicillin using immobilized penicillin amidase 2.1 2.1.1
389 389 389 391 39 1 391 39 1 39 1 39 1 392 392 39 3 39 4 39 4 394
.
.................................... ........................... .............. ............................................
395 395 397 39 8 39 8
39 8 ...... 39 8 ............................... 398 ........................... 39 8 ............. 400 ...................................... 40 0 ........ 400 .... 401 ........ .................... 402 402 ...................................... 402 ............................ 40 3 404 ................................... ............ 405 .......... 405
3aa
. 6-Aminopenicillanic acid (6-APA)(continued) 2.6 Nonenzymatic conversion of penicillins to 6.APA ......................................... 406 3 . Other naturally occurring $-lactam antibiotics Nocardicin .................................... 407 3.1 Discovery and brief chemical description .... 4 0 7 3.1.1 Producing organism.......................... 4.07 3.1.2 Physical and chemical properties............ 4 0 7 3.1.3 Fermentation and isolation procedures....... 4 0 7 3.1.4 Clavulanic acid ............................... 408 3.2 Discovery and chemical description. ......... 408 3.2.1 Producing organism .......................... 408 3.2.2 Fermentation and isolation .................. 4 0 9 3.2.3 Thienamycin ................................... 410 3.3 Discovery and chemical description .......... 410 3.3.1 Producing organism .......................... 410 3.3.2 Biological activity ......................... 410 3.3.3 Physical and chemical properties ............ 411 3.3.4 Fermentation and isolation .................. 4 1 1 3.3.5 4. Literature Cited .................................... 411
2
3 89 1. N a t u r a l p e n i c i l l i n s w i t h 6-APA n u c l e u s
1.1 I n t r o d u c t i o n P e n i c i l l i n G a n d p e n i c i l l i n F were t h e f i r s t o f t h i s g r o u p t o b e d i s c o v e r e d f r o m t h e c u l t u r e f i l t r a t e o f P . chnybogenum. P e n i c i l l i n G , i s o l a t e d i n t h e U n i t e d S t a t e s , p r o v e d t o b e b e n z y l p e n i c i l l i n , a n d p e n i c i l l i n F , d i s c o v e r e d by t h e SubB r i t i s h workers, proved t o be A2-pentenyl p e n i c i l l i n ’ . s e q u e n t work showed t h a t t h e p e n i c i l l i n p r o d u c e d by P. chnyaogenum was n o t a s i n g l e compound, a n d b o t h t h e q u a n t i t y a n d q u a l i t y of t h e p e n i c i l l i n s s y n t h e s i z e d d e p e n d e d l a r g e l y on t h e i n g r e d i e n t s o f t h e f e r m e n t a t i o n medium. A d d i t i o n of p h e n y l acetic a c i d , n o t o n l y s t i m u l a t e d t h e f o r m a t i o n of b e n z y l p e n i c i l l i n over other penicillins, but a l s o increased the yield of t h e a n t i b i o t i c 2 - ‘ + . By a d d i n g a l a r g e v a r i e t y of s i d e c h a i n p r e c u r s o r s t o t h e f e r m e n t a t i o n medium, a l a r g e v a r i e t y of p e n i c i l l i n s were I n t h e absence of exogenously added p r e c u r s o r s , s e v e r a l p e n i c i l l i n s w e r e f o r m e d by P . chnydogenwn, e . g . , p e n i c i l l i n s K , F, d i h y d r o F 5 . A s p e c i e s o f Cephalobponium was f o u n d t o p r o d u c e a n a n t i b a c t e r i a l s u b s t a n c e c a l l e d c e p h a l o This s p o r i n N o r p e n i c i l l i n N , which was a new p e n i c i l l i n was shown t o c o n t a i n D-4-amino-b-carboxybutyl o r Da - a m i n o a d i p y l m o i e t y as t h e s i d e c h a i n . I s o p e n i c i l l i n N ( L - a a m i n o a d i p y l p e n i c i l l i n ) from P . chnydogcnum was r e p o r t e d by Flynn and co-workers O . 1.1.1 P r o d u c i n g o r g a n i s m s A l l the naturally occurring p e n i c i l l i n s containing the 6-APA n u c l e u s are p r o d u c e d e i t h e r by t r u e f u n g i ( e u k a r y o t e s ) o r Stneptomyced ( p r o k a r y o t e s 1. Among f u n g i , t h e y are p r o d u c e d b y many s p e c i e s o f t h e g e n e r a P e n i c i l l i u m , AApehgi&?ud a n d some 1 - ~ The m o s t t h o r o u g h l y s t u d i e d s p e c i e s o f C e p h a t o ~ p o n i ~ m 13. m i c r o o r g a n i s m f o r t h e commercial p r o d u c t i o n of p e n i c i l l i n s i s P . c h n y ~ o g e n u m l ~ .O t h e r s p e c i e s o f p e n i c i l l i n - p r o d u c i n g m i c r o o r g a n i s m s a r e two d e r m a t o p h y t e s Tnichophyton mentagnophyted a n d Epide&mophyton dloccoaum’ 3’15 , E m e h i c e t l o p d i d s p e c i e s 1 6 - 1 9 , Cephalodponium specie^^^-^ 3 , a n d PaQCilOmyCed pehdicinu.4 s p e c i e ~ ~I n~ a .d d i t i o n , p e n i c i l l i n N i s p r o d u c e d by S L t Q p t o -
myCQ412’ 2 5
1.1.2
m u r a l formulae
The n a t u r a l l y o c c u r r i n g p e n i c i l l i n s c o n t a i n i n g t h e 6-APA n u c l e u s are r e p r e s e n t e d by t h e f o l l o w i n g s t r u c t u r e s ( s e e n e x t page).
390
Generic Name Penicillin
G
Structure ( R)
-
@CH2
Penicillin F
CH3CH= CHCH2CH2 -
Dihyd ropenicillin F
CH3CCH2)4-
Penicillin K
CH3C CH2)6
Penicillin X
HO@CH~-
Penicillin N
HOOC-CH-(CH2)3
-
AH2
lsopenicillin N
H2N- CH-( CH2)3I
COOH
-
391 1.1.3
P h y s i c a l and chemical p r o p e r t i e s
1.1.3.1
Formula :
P e n i c i l l i n G1 1 6H1 8O 4N2S
pK = 2.76 a t 25OC i n water 264.4 nm a n d 258.6 nm -1 Major IR c a r b o n y l bands: 1790-1770 cm 'max
( 8 - l a c t a m c a r b o n y l ) 1 6 9 0 - 1 6 8 0 cm-1 ( s e c o n d a r y amide c a r b o n y l ) 1750 cm'' (ester carbonyl). (a)$5
m.p. 1.1.3.2 Formula :
for s o d i u m s a l t + 305 (water). ( s o d i u m s a l t ) = 215OC ( d e c . ) .
P e n i c i l l i n F1 c I 4H2004N2S
pK = 2.84 a t 25OC i n water
No c h a r a c t e r i s t i c a b s o r p t i o n i n t h e U.V. only e n d a b s o r p t i o n below 260 nm. = + 276-316. m.p. ( s o d i u m s a l t ) = 204-5OC ( d e c . ) . 1.1.3.3 Formula:
range;
P e n i c i l l i n K1 16H26O 4N2S
No c h a r a c t e r i s t i c a b s o r p t i o n i n t h e U.V.range; o n l y e n d a b s o r p t i o n below 260 nm. = +258. 1.1.3.4
Formu l a :
Penicillin XI c1g H 1 8' 5N2S
pK = 2 . 6 2 a t 2 3 O C i n water 'max 278 nm ( t h e p e a k is more i n t e n s e t h a n t h a t of o t h e r p e n i c i l l i n s b e c a u s e of t h e p r e s e n c e of p-hydroxy g r o u p i n t h e a r o m a t i c r i n g ) . ( E l c m = 42.51, ( e x h i b i t s t h e c h a r a c t e r i s t i c b e h a v i o u r of t h e p h e n o l i c g r o u p i n t h e U . V . range, showing i n c r e a s e d e x t i n c t i o n i n a l k a l i n e s o l u t i o n due t o i o n i z a t i o n of t h e p h e n o l i c hydroxyl group). of s o d i u m s a l t +267. m.p. ( s o d i u m s a l t ) 228-35OC ( d e c . ) . 1.1.3.5 Formula:
P e n i c i l l i n N2 c l 4H2 1 06N3S
-
No c h a r a c t e r i s t i c a b s o r p t i o n i n t h e U . V . only end absorption.
range;
392
1.2 T h e r a p e u t i c
use and pharmacology o f p e n i c i l l i n s
The n a t u r a l p e n i c i l l i n s v a r y c o n s i d e r a b l y i n t h e i r i n t r i n s i c a n t i b a c t e r i a l a c t i v i t y . Although p e n i c i l l i n K h a s t h e h i g h e s t a c t i v i t y , it has a high p r o t e i n binding property. P e n i c il l i n e
Uni ta/rnq
F
K
1550 1667 2300
X
900
G
The h i g h a c t i v i t y o f p e n i c i l l i n G a g a i n s t p y o g e n i c c o c c i h a s made i t a d r u g o f c h o i c e . H e a t l e y a n d F l o r e y r e p o r t e d t h a t i n p e n i c i l l i n N, t h e p r e s e n c e o f t h e a-amino s i d e c h a i n , i n s t e a d o f t h e b e n z y l s i d e c h a i n , was a s s o c i a t e d w i t h a h u n d r e d - f o l d loss o f a c t i v i t y a g a i n s t S . a u ~ e u d , b u t w i t h a h i g h e r a c t i v i t y against coliform bacilli27-29. In considering t h e r e l a t i v e e f f i c a c y of t h e s e d i f f e r e n t n a t u r a l l y occurring p e n i c i l l i n s i n V ~ V O , t h e i r pharmacological as w e l l as t h e i r a n t i b a c t e r i a l p r o p e r t i e s s h o u l d b e t a k e n i n t o a c c o u n t . The b l o o d l e v e l s a c h i e v e d by t h e s e p e n i c i l l i n s a r e i n an i n c r e a s i n g o r d e r o f K
S . pyogened
S. pneumoniae
T. paeeidum
(mg/ks)
(mdkg)
(mdkg)
260 100 50
160 100 83
100 8
9
19
14
11.5
I n g e n e r a l i t i s c l e a r t h a t p e n i c i l l i n K i s t h e most e f f e c t i v e of t h e four n a t u r a l p e n i c i l l i n s a g a i n s t s t r e p t o c o c c a l and pneumococcal i n f e c t i o n s , a n d p e n i c i l l i n G a g a i n s t s p i r o c h a e t a l i n f e c t i o n s . A l l c l i n i c a l r e p o r t s show t h a t p e n i c i l l i n G given intramuscularly i n t h e r a p e u t i c e f f e c t i v e doses produces no t o x i c e f f e c t s , 1 . 3 Methods -
f o r assaying p e n i c i l l i n s
Analysis of the d i f f e r e n t p e n i c i l l i n s i n a fermentation b r o t h can be e a s i l y a c c o m p l i s h e d by means o f p a p e r chromatog r a p h y u s i n a n h y d r o u s e t h e r o r p h o s p h a t e b u f f e r as a d e v e l o p i n g s o l v e n t 3 0 y 3 g . A b e t t e r r e s o l u t i o n o f p e n i c i l l i n s G , F and V was r e p o r t e d by Watanabe a n d c o - w o r k e r s , u s i n g a p a r a f f i n t r e a t e d f i l t e r p a p e r s a t u r a t e d w i t h b u t y l a c e t a t e as a s t a t i c p h a s e a n d p h o s p h a t e b u f f e r as a m o b i l e p h a s e . A c c o r d i n g t o t h i s method, t h e f i l t e r p a p e r i s immersed i n 2 % l i q u i d p a r a f f i n i n e t h e r a n d d r i e d i n a i r , t h e n t h e s a m p l e s are a p p l i e d ( a b o u t 0 . 0 1 m l o f 500 u n i t s / m l i n s t e r i l e water) a n d e q u i l i b r a t e d i n b u t y l The chromatogram i s d e v e l o p e d f o r a c e t a t e v a p o r s f o r two h r . 1 6 h r a t room t e m p e r a t u r e w i t h 2 7 % d i h y d r o g e n p o t a s s i u m p h o s p h a t e a t pH 6 . 0 . Rf v a l u e s o b t a i n e d from t h e chromatography
39 3
are p r e s e n t e d i n t h e f o l l o w i n g t a b l e . Rf -
Penicillins
0.48 0.22 0.14 0.36 0.59
Another p a p e r c h r o m a t o g r a p h i c method i s t o d e v e l o p a Whatman No. 1 p a p e r i n a s o l v e n t s y s t e m of t h e upper phase of n - b u t a n o l : e t h a n o l : w a t e r (V:l: 5 by volume) , o r n - b u t a n o 1 : p y r i d i n e : w a t e r o ( l : l : l by volume). The chromatograms are r u n f o r 1 7 h r a t 5 C. D e t e c t i o n of t h e p e n i c i l l i n s i s made by b i o a u t o g r a p h y u s i n g 8 . h u b t i l i d as a t e s t o r g a n i s m . The Rf v a l u e s of t h e p e n i c i l l i n s i n t h e s e s y s t e m s are as f o l l o w s : Penicillins
G F
Dihydro F K X N 6-APA
Butenol :e t h enol :w ate r (4:1:5)
0.68 0.78 0.78 0.78 0.47 0.05 0.20
But enol: r i dine :water v py
0.75 0.73 0.73 0.73 0.72 0.23 0.48
The y i e l d s o f p e n i c i l l i n s i n a c u l t u r e f i l t r a t e may be a s s a y e d i n many d i f f e r e n t ways. I n g e n e r a l two a s s a y methods a r e commonly used: ( a ) b i o l o g i c a l a s s a y method, u s i n g 8 . Aubas a t e s t organism, measures c o m p a r a t i v e a c t i v i t y o f t h e b r o t h sample t o a n a u t h e n t i c p e n i c i l l i n G as a s t a n d a r d , and ( b ) c h e m i c a l a s s a y method, u s i n g hydroxylamine, measures t h e t o t a l p e n i c i l l i n nucleus i n the broth.
1.4 Isolation
o f p e n i c i l l i n s from f e r m e n t a t i o n b r o t h s
The most i m p o r t a n t fungus f o r p e n i c i l l i n p r o d u c t i o n i s P. chayeogenum, and i m p r e s s i v e improvements i n y i e l d s have been a c c o m p l i s h e d o v e r t h e l a s t t h i r t y y e a r s by s e l e c t i n g h i g h y i e l d i n g m u t a n t s . A s t r a i n of P . chayaogenum, d e s i g n a t e d as NRRL 1 9 5 1 was found t o be most s u i t a b l e f o r t h e p e n i c i l l i n f e r m e n t a t i 0 n 3 ~ 3 ~ ~ 3 A3 f,t e r c o m p l e t i o n of a f e r m e n t a t i o n by t h i s s t r a i n , f o r example, t h e b r o t h i s s e p a r a t e d from t h e mycelium by f i l t r a t i o n , and p e n i c i l l i n s are removed from t h e c u l t u r e b r o t h by e x t r a c t i o n w i t h o r g a n i c s o l v e n t s o r by a d s o r p t i o n o n t o c h a r c o a l . Most of t h e m a n u f a c t u r i n g methods today , a r e b a s e d on t h e s o l v e n t - e x t r a c t i o n method. P e n i c i l l i n s i n t h e form of f r e e a c i d s can be e x t r a c t e d from aqueous f e r m e n t a t i o n b r o t h s w i t h a v a r i e t y of o r g a n i c s o l v e n t s such as e t h e r , i s o p r o p y l a c e t a t e , n - b u t y l a c e t a t e , amyl a c e t a t e and c h l o r o f o r m , and can b e r e - e x t r a c t e d from t h e r e s u l t i n g o r g a n i c phase t o t h e aqueous phase as a s a l t under n e u t r a l o r s l i g h t l y a l k a l i n e c o n d i t i o n s . By r e p e a t i n g these s o l v e n t e x t r a c t i o n p r o c e s s e s , f u r t h e r p u r i f i c a t i o n s and
39 4 concentrations a r e achieved. In other i n d u s t r i a l processes , p e n i c i l l i n s a r e removed from t h e c u l t u r e b r o t h s by a c h a r c o a l a d s o r p t i o n t e c h n i q u e . P e n i c i l l i n s are a d s o r b e d on c h a r c o a l and e l u t e d w i t h aqueous a c e t o n e . I n i t i a l l y t h e c h a r c o a l a d s o r p t i o n method was t h e method of c h o i c e , b e c a u s e i t gave a b e t t e r q u a l i t y p r o d u c t and was c h e a p e r i n comparison t o t h e e x p e n s i v e s o l v e n t e x t r a c t i o n p r o c e s s e s . With t h e d i s c o v e r y of h i g h y i e l d i n g s t r a i n s t o d a y , t h e c h a r c o a l a d s o r p t i o n method has b e e n almost c o m p l e t e l y r e p l a c e d by t h e s o l v e n t e x t r a c t i o n t e c h n i q u e . 1.4.1
S o l v e n t e x t r a c t i o n method
The s o l v e n t e x t r a c t i o n method i n most i n d u s t r i a l p r o c e s s e s c o n s i s t s of f o u r d i f f e r e n t s t e p s : (1) e x t r a c t i o n of p e n i c i l l i n s from t h e c u l t u r e b r o t h , ( 2 ) t r a n s f e r from s o l v e n t t o b u f f e r , (3) e x t r a c t i o n from b u f f e r t o s o l v e n t and ( 4 ) t r a n s f e r from s o l v e n t t o water. The f i r s t s t e p i s u s u a l l y carried o u t a t a pH range of 2.0 t o 4.0. The e x a c t pH d e p e n d s on t h e n a t u r e o f t h e s o l v e n t u s e d . Amy1 a c e t a t e or b u t y l a c e t a t e i s commonly used f o r S t e p 1. I n some commercial p r o c e s s e s a d i f f e r e n t s o l v e n t s u c h as c h l o r o f o r m i s used f o r S t e p 3 , whereas i n o t h e r p r o c e s s e s t h e same s o l v e n t is used f o r b o t h e x t r a c t i o n S t e p s 1 aad 3. The f i r s t e x t r a c t i o n s t e p i s g e n e r a l l y c a r r i e d o u t a t 0-5 C. b e c a u s e o f t h e r e l a t i v e i n s t a b i l i t y of p e n i c i l l i n s a t a c i d i c pH v a l u e s . A f t e r t h e p e n i c i l l i n s are s u f f i c i e n t l y c o n c e n t r a t e d i n aqueous s o l u t i o n s f o l l o w i n g one o r two e x t r a c t i o n - c o n c e n t r a t i o n s t e p s , c r u d e c r y s t a l l i n e p r e p a r a t i o n s are p r e p a r e d by v a r i o u s methods; e . g . , d i r e c t p r e c i p i t a t i o n o f p o t a s s i u m s a l t s , p r o c a i n e s a l t s or ammonium s a l t s of p e n i c i l l i n s from t h e concent r a t e d aqueous s o l u t i o n s . R e c r y s t a l l i z a t i o n of p o t a s s i u m o r sodium s a l t s of p e n i c i l l i n s can be a c h i e v e d by d e h y d r a t i o n of s o l v e n t s from s o l u t i o n s of p e n i c i l l i n s a l t s i n water, or by a d d i n g s e v e r a l volumes o f anhydrous s o l v e n t s , e . g , a c e t o n e , t o t h e c o n c e n t r a t e d aqueous s o l u t i o n s o f t h e s a l t s . 1.4.2
C h a r c o a l a d s o r p t i o n method’
P e n i c i l l i n s from t h e c u l t u r e b r o t h can b e r e a d i l y a d s o r b e d o n t o c h a r c o a l a t n e u t r a l pH a t room t e m p e r a t u r e . About 2-3% of c h a r c o a l by w e i g h t i s added t o t h e b r o t h w i t h s t i r r i n g , The c h a r c o a l and a d s o r p t i o n i s c a r r i e d o u t f o r 1 5 t o 30 min. s.uspension i s f i l t e r e d a n d washed w i t h d i s t i l l e d water. P e n i c i l l i n s are e l u t e d f r o m t h e c h a r c o a l w i t h a h a l f t o o n e volume of 8 0 % a c e t o n e . Acetone i s removed from t h e e l u a t e s e i t h e r bg d i s t i l l a t i o n u n d e r r e d u c e d p r e s s u r e a t a t e m p e r a t u r e below 25 C , o r by a d d i t i o n of an e q u a l volume o f a wateri m m i s c i b l e o r g a n i c s o l v e n t . The c o n c e n t r a t e d aqueous s o l u t i o n o f p e n i c i l l i n s i s a d j u s t e d t o n e u t r a l i t y , and s u b j e c t e d t o f u r t h e r c o n c e n t r a t i o n s and p u r i f i c a t i o n s . 1.4.3
C h r o m a t o g r a p h i c methods of p u r i f y i n g p e n i c i l l i n s ’
I n g e n e r a l , s i l i c a g e l o r a l u m i n a i s used f o r t h e chromat o g r a p h i c p u r i f i c a t i o n o f p e n i c i l l i n s . When a l u m i n a i s u s e d , i t has t o b e n e u t r a l i z e d b e f o r e use t o a v o i d l o s s e s of p e n i c i l l i n s v i a i n a c t i v a t i o n by t h e a l k a l i n e a l u m i n a . The n e u t r a l i z a t i o n i s done by a d d i n g H2S04 o r H C 1 t o a s u s p e n s i o n o f a l u m i n a
395 i n water u n t i l t h e pH o f t h e s u s g e n s i o n r e m a i n s a t 5.0 o r 6 . 0 . The a l u m i n a i s t h e n d r i e d a t 1 1 0 C f o r 2 d a y s a f t e r which a s o l u t i o n of a p e n i c i l l i n (A*-pentenyl p e n i c i l l i n o r benzyl p e n i c i 1 l i n ) i s a p p l i e d t o t h e column. T h i s r e s u l t s i n t h e format i o n o f t h r e e b a n d s : a d a r k brown u p p e r band c o n t a i n i n g l i t t l e o r no p e n i c i l l i n s , a l i g h t y e l l o w m i d d l e band c o n t a i n i n g t h e b u l k o f p u r i f i e d p e n i c i l l i n s , and a d a r k y e l l o w b o t t o m band c o n t a i n i n g some impure p e n i c i l l i n s . T h e s e b a n d s a r e d i s c e r n i b l e u n d e r u l t r a v i o l e t l i g h t , a n d e a c h band i s e l u t e d w i t h p h o s p h a t e b u f f e r . The d e g r e e o f p u r i f i c a t i o n a c h i e v e d by a l u m i n a chromatography is i n t h e o r d e r of 50-100%. S i l i c a g e l h a s been u s e d s u c c e s s f u l l y as a medium o f p a r t i t i o n i n g sodium s a l t s o f t h e p e n i c i l l i n s between water a n d o r g a n i c s o l v e n t s s u c h as e t h e r a n d c h l o r o f o r m . The s i l i c a p h o s p h a t e g e l i s p r e p a r e d by m i x i n g s i l i c a g e l w i t h o n e - h a l f volume o f a p h o s p h a t e b u f f e r . The r e s u l t i n g m i x t u r e i s s t i r r e d w i t h t h e s o l v e n t t o b e u s e d f o r d e v e l o p i n g t h e p a r t i t i o n chromatogram. S o l u t i o n s o f p e n i c i l l i n s i n t h e form o f f r e e a c i d s i n e t h e r o r c h l o r o f o r m , o r of t h e sodium s a l t s i n water, a r e c h a r g e d o n t o t h e column, P e n i c i l l i n s a r e e l u t e d w i t h e t h e r . n - H e p t y l p e n i c i l l i n , A 2 - p e n t e n y l p e n i c i l l i n , b e n z y l p e n i c i l l i n a n d phydroxy b e n z y l p e n i c i l l i n a r e e l u t e d i n s e q u e n t i a l o r d e r . 1.4.4
P r e c i p i t a t i o n Of p e n i c i l l i n s w i t h o r g a n i c b a s e o r ammonia'
A p u r e c r y s t a l l i n e s a l t can b e o b t a i n e d i n t h e f o r m of a t r i e t h y l a m i n e s a l t b y a d d i t i o n of t r i e t h y l a m i n e t o a n e t h e r s o l u t i o n of benzyl p e n i c i l l i n . I t i s u s u a l l y c a r r i e d o u t by a d d i n g a 1 0 % s o l u t i o n o f t r i e t h y l a m i n e i n e t h e r t o a cold d r y s o l u t i o n of b e n z y l p e n i c i l l i n i n e t h e r , c o n t a i n i n g 5,000-10,000 units/ml. The t r i e t h y l a m i n e s a l t c a n b e r e c r y s t a l l i z e d by d i s s o l v i n g i n c h l o r o f o r m a n d p r e c i p i t a t i n g w i t h a c e t o n e , o r by d i s s o l v i n g i n methylene c h l o r i d e and p r e c i p i t a t i n g with methyl e t h y l k e t o n e . Ammonium s a l t s of p e n i c i l l i n s c a n b e o b t a i n e d by g r a d u a l a d d i t i o n of a f r e s h l y p r e p a r e d s o l u t i o n of l i q u i d ammonia i n e t h e r (1:5) t o a n e t h e r e a l s o l u t i o n o f t h e p e n i c i l lins. 1.4.5
I s o l a t i o n of p e n i c i l l i n N and i s o p e n i c i l l i n N
P e n i c i l l i n N , which was l a t e r f o u n d t o b e i d e n t i c a l t o c e p h a l o s p o r i n N , was i s o l a t e d from a C e p h a l o d p o a i u m bp. 27-29, I n t e r e s t i n t h i s new n a t u r a l p e n i c i l l i n was v e r y h i g h , b e c a u s e i t h a d a c t i v i t y a g a i n s t some g r a m - n e g a t i v e b a c t e r i a . A p e n i c i l l i n N - l i k e a n t i b i o t i c was d i s c o v e r e d f r o m CephaloApoJtium A a L m o A y n n e m a t u m 2 1 y 2 2 . T h i s a n t i b i o t i c , c a l l e d synemmatin B , was c h a r a c t e r i z e d by Abraham and c o - w o r k e r s , a n d f o u n d t o b e Besides t h e CephaLochemically i d e n t i c a l t o p e n i c i l l i n N34. A p o A L u m s p e c i e s , o t h e r s ecies were s u b s e q u e n t l y r e c o g n i z e d t o produce p e n i c i l l i n N16-19324. P e n i c i l l i n N-producing s t r a i n s h a v e n o t been f o u n d s o f a r t o p r o d u c e a n y o t h e r p e n i c i l l i n s , and are i n s e n s i t i v e t o i n c o r p o r a t i o n o f s i d e c h a i n p r e c u r s o r s i n t o t h e a n t i b i o t i c . A n o t h e r n a t u r a l p e n i c i l l i n h a s been i s o l a t e d f r o m t h e c u l t u r e medium o f P e n i c i L L i u m c h a y b o g e n u m grown i n a s i m p l e medium i n t h e a b s e n c e of a d d e d p r e c u r s o r s 5 y 3 5 .
396 Under t h i s c o n d i t i o n small amounts o f s o l v e n t e x t r a c t a b l e c i l l i n s , 6-APA, and a n o t h e r B-lactam compound w i t h no t e r i a l a c t i v i t y were produced. T h i s new compound was i s o l a t e d by a d s o r p t i o n on a c t i v a t e d carbon, e l u t i o n w i t h aqueous acet o n e , and chromatography on p o t a t o s t a r c h i n aqueous t e r t i a r y b u t a n o l . The p u r i f i e d p r o d u c t was i n d i s t i n g u i s h a b l e from p e n i c i l l i n N by a l l c h r o m a t o g r a p h i c comparisons. I t was s e n s i t i v e t o p e n i c i l l i n a s e , and i t s a n t i b a c t e r i a l a c t i v i t y a g a i n s t S, auaCuA 2 0 9 P was lower t h a n t h a t of p e n i c i l l i n G, b u t was e q u a l t o t h a t of p e n i c i l l i n N. Hydrolysis w i t h 1 . 0 N H C 1 yielded L a - a m i n o a d i p i c a c i d , i n s t e a d of D-a-aminoadipic a c i d . The i d e n t i f i c a t i o n o f L-a-aminoadipic a c i d was b a s e d on i n f r a r e d and X-ray d i f f r a c t i o n p a t t e r n s , which were i d e n t i c a l t o t h o s e o f a n a u t h e n t i c sample of L- a-aminoadipic a c i d . However, t h e o p t i c a l r o t a t o r y d i s p e r s i o n c u r v e was e q u a l i n magnitude, b u t o p p o s i t e i n s i g n t o t h a t g i v e n by t h e a u t h e n t i c sample. On t h e b a s i s of t h i s d a t a , t h e name i s o p e n i c i l l i n N was proposed f o r t h i s a n t i b i o t i c e . Another p e n i c i l l i n , i n i t i a l l y c a l l e d p e n i c i l l i n M , was i s o l a t e d from t h e mycelium of P. chhyAogenum grown i n a c o r n s t e e p l a c t o s e medium35. The mycelium was washed exhaust i v e l y i n t o water, and t h e c o n c e n t r a t e e x t r a c t e d w i t h 7 0 % aqueous a c e t o n e ( 4 . 0 g wet mycelium t o 10 m l a c e t o n e ) . The acet o n e e x t r a c t showed t h e p r e s e n c e of 6-APA, p e n i c i l l i n s X , G , F , dihydro-F, K , methyl p e n i c i l l i n , and p e n i c i l l i n M . I n t h e b r o t h p e n i c i l l i n s X , G , F and dihydro-F and K were predominant. In t h e m y c e l i a l e x t r a c t s , however, t h e s e p e n i c i l l i n s were minor components, and t h e major p r o d u c t s were 6-APA, p e n i c i l l i n M and methyl p e n i c i l l i n . P e n i c i l l i n M was i s o l a t e d from t h e a c e t o n e e x t r a c t s of t h e mycelium. The a c e t o n e e x t r a c t s were concent r a t e d under r e d u c e d p r e s s u r e , and i n a c t i v e o i l was removed by e x t r a c t i o n w i t h e t h e r . The r e m a i n i n g material was a d s o r b e d on c h a r c o a l and e l u t e d w i t h a c e t o n e . The p o o l e d e l u a t e s were e v a p o r a t e d t o d r y n e s s , and a n aqueous s o l u t i o n o f t h e r e s i d u e was a d s o r b e d o n t o "De A c i d i t e FF" r e s i n i n c h l o r i d e c y c l e , and e l u t e d w i t h .05 N HC1. For f u r t h e r p u r i f i c a t i o n c e l l u l o s e chromatography, u s i n g a m i x t u r e of n-butano1:pyridine:water f o r e l u t i o n , and a s u b s e q u e n t p r e p a r a t i v e e l e c t r o p h o r e s i s a t pH 7 . 0 , u s i n g a c e t i c a c i d b u f f e r , were a p p l i e d . P e n i c i l l i n M was l a t e r found t o b e i d e n t i c a l t o p e n i c i l l i n N i n many r e s p e c t s . The a c t i v i t y o f p e n i c i l l i n M a g a i n s t 8. A u b t i l i A was similar to t h a t o f p e n i c i l l i n N, b u t t h e a c t i v i t y a g a i n s t S . typhimuaium was s i g n i f i c a n t l y less. Since t h e a n t i b a c t e r i a l a c t i v i t y of penic i l l i n s was known t o be i n f l u e n c e d by t h e c o n f i g u r a t i o n o f t h e s i d e c h a i n 3 6 , i t was s u g g e s t e d t h a t t h e d i f f e r e n c e i n a n t i b a c t e r i a l a c t i v i t y was due t o t h e f a c t t h a t p e n i c i l l i n M might have a s i d e c h a i n w i t h t h e L - c o n f i g u r a t i o n i n s t e a d o f D-configu r a t i o n . E v e n t u a l l y , i t was e s t a b l i s h e d t h a t p e n i c i l l i n M was i d e n t i c a l t o i s o p e n i c i l l i n N d e s c r i b e d by Flynn and co-workers7. Both p e n i c i l l i n M and i s o p e n i c i l l i n N were shown t o b e i d e n t i c a l t o p e n i c i l l i n W d e t e c t e d by Hale, M i l l e r and K e l l y 3 7 .
antigizi-
39 7 2.
6-Aminopenicillanic acid (6-APA)
2.1 Introduction During the early days, two methods of assay were used to measure penicillin concentrations in fermentation broths. One method was the biological cup plate assay of Heatley, in which antimicrobial activity of the fermentation broths was measured by comparison with a standard penicillin solution38. The second method was a chemical assay, in which penicillins were converted to hydroxylamine derivatives by treating with hydroxylamine, and measured colorimetrically after adding a ferric salt to the h y d r o ~ a m a t e s ~ ~In . penicillin fermentations carried out with added side chain precursors, the two assay methods were in fairly good agreement with one another. However, Ballio and co-workers observed, during the fermentative synthesis of paminobenzyl penicillin, that there were discrepancies between the biological and the chemical assays when a precursor was omitted from the fermentation40. The chemical assay always showed higher values than the biological assay. They argued that the discrepancies between the two assay methods were in part due to the presence of a penicillin molecule which was devoid of the side chain. Such a molecule would have a much lower antimicrobial activity than a penicillin, but would respond to the hydroxylamine reaction. The presence of such a compound was confirmed by two findings: (a) when the fermentation broth was treated with an excess of phenylacetylchloride following removal of natural penicillins by solvent extraction at a low pH, an antibiotic identical to penicillin G was formed, and (b) the 6-aminopenicillanic acid was isolated from the fermentation broth4'. In penicillin fermentations carried out in the absence of side chain precursors, Kato observed that a compound, which was biologically inactive but sensitive to penicillinase, was present in addition to natural penicillins42y43. However, his attempts to isolate the compound from the fermentation broth was unsuccessful. The 6-aminopenicillanic acid is represented by the following structure.
6-Aminopenicil lank acid
39 8
2.1.1
P r o d u c i n g organisms
6 - A m i n o p e n i c i l l a n i c a c i d is a n a t u r a l l y o c c u r r i n g p r o d u c t o f p e n i c i l l i n - p r o d u c i n g s t r a i n s o f P e n i c i t l i u m chaydogenum, p a r t i c u l a r l y when t h e f e r m e n t a t i o n is c a r r i e d o u t i n t h e a b s e n c e of s i d e chain p r e c u r s o ~ s ~ B ~ e. s i d e s P . chaydogenum 6-APA a l s o a p p e a r s t o be a p r o d u c t o f c e p h a l o s p o r i n - p r o d u c i n g s t r a i n s l i k e CephatoApoaium Aa.tmoAynemmatum and Emeaiee.ttopAiA minimal7. I n a d d i t i o n , some dermatophytes a l s o roduce 6-APA i n t h e i r fermentation broths. They are E p i d e a m o p g y t o n i n t e a d i g i t a l e and
Taichophyton g ~ j p A e u m ~ ~ . 2.1.2
P h y s i c a l and chemical p r o p e r t i e s
Formula: CBH1203N2S m.p.
2
208-209OC ( d e c . )
6-APA i s d e s t r o y e d by p e n i c i l l i n a s e ( B . ceReu.4 b r o t h f i l t r a t e ) , b u t a t a much s l o w e r r a t e t h a n b e n z y l p e n i c i l l i n . L i k e t h e p e n i c i l l i n s i t i s r a p i d l y decomposed by s t r o n g a l k a l i , b u t is r e l a t i v e l y s t a b l e t o a c i d s . I t is a s t r o n g a c i d , t h e isoe l e c t r i c point being about 4.3. E l e c t r o m e t r i c t i t r a t i o n showed an a c i d group o f pK 2.3 a n d a s u r p r i s i n g l y weak amino group of O p t i c a l r o t a t i o n was dependent on pH, p a r t i c u l a r l y pK 5 . 1 . below pH 6 . 0 . Between pH 7 . 0 and 1 0 . 5 t h e s p e c i f i c r o t a t i o n was c o n s t a n t at a v a l u e of { a l s o +337. Below pH 6 . 0 . t h e rot a t i o n showed a marked d e c r e a s g g i v i n g a v a l u e a; pH 2:O of = + 284.
2.2 Antibacterial
a c t i v i t y of 6-Aminopenicillanic a c i d
The m . i . c. v a l u e s o f 6 - a m i n o p e n i c i l l a n i c a c i d i n comparison t o t h o s e of b e n z y l p e n i c i l l i n a r e g i v e n i n t h e f o l l o w i n g t a b l e ( n e x t p a g e ) . I n g e n e r a l 6-APA shows much less a c t i v i t y t h a n p e n i c i l l i n G a g a i n s t g r a m - p o s i t i v e b a c t e r i a . A g a i n s t gramn e g a t i v e organisms t h e d i f f e r e n c e i n a c t i v i t y was r e l a t i v e l y slight.
2.3 Assay 2.3.1
methods f o r 6 - A m i n o p e n i c i l l a n i c a c i d Cup p l a t e a s s a y
T h i s a s s a y was d e v e l o p e d by H e a t l e y and c o - w o r k e r ~ ~ ~ . B a c i k ! t u ~A u b f i l i A is used as t h e t e s t organism, and b e n z y l peni c i l l i n as t h e s t a n d a r d a n t i b i o t i c . 2.3.2
_ I _
Hydroxylamine a s s a y
This method is a m o d i f i c a t i o n of t h e p r o c e d u r e d e s c r i b e d by Ford, and h a s been r e p o r t e d by B a t c h e l o r s and 0 t h e r s 3 ~ " + ~ , and can be used f o r t h e a s s a y o f p e n i c i l l i n s or 6-APA. When i t i s used f o r t h e a s s a y of p e n i c i l l i n s , b e n z y l p e n i c i l l i n i s used as t h e s t a n d a r d . I n t h e case o f a s s a y i n g for t h e 6-APA, t h e same a c i d is u s e d as t h e s t a n d a r d . The 6-APA a s s a y is c a r r i e d o u t by a l l o w i n g t h e sample t o s t a y a t pH 2 . 0 f o r 2 h r s a t room t e m p e r a t u r e . Under t h e s e c o n d i t i o n s p e n i c i l l i n s a r e
399 M i n i m u m Concentrations (mcg/ml) Required t o I n h i b i t Growth o f Bacteria. S e r i a l D i l u t i o n s i n Agar. I n c h a t i o n f o r 24 H r a t 37OC. 6-Aminop e n i c i l l m i c acid
Benzyl p e n ic i1l i n
62.5 250 250 50 50 50 50 >250 >250 50 125 62.5 50 50 >250 1.25 1.25 6.25 1.25 50 125 125 50 50 125 50 >250 >250 50 50 50 50 50 50 50 125 125
0.02 >250 >250 125 125 0 .001 0.02 0.5 0.5 0.01 0.05 0.005 0.012 5 .O 250 0.01 0.001 0.5 0.06 0.6 125 5 50 5 5 25 >250 >250 2.5 5.0 5.0 1.o 5 .O 50 125 >250 0.5
Staph. W e u d (Oxford) S-h. pyogeneA BRL. 1083" Staph. pqogenu BRL. 1093* BRL. 1234** BRL. 1235**
srvrcina lu&a
S&Qp. V M d W BRL. 1479 Sf2ep. vLtidun.4 BRL. 1480 S&ep. u h i d w BRL. 1247 Satnep. pyogeneA 6 haemolytic BRL. 1231 S&ep. pyogened ¶, haemolytic BRL. 1275 Uip&coccUA pneumonh BRL. 1221 ~ ~ O C O C C UpnemoLae A BRL. 1222 B<w Aub&&d 8deeud C U Q U Cownebuctuium ho&nmni BRL. 1218 Conynebac&tLum h06nmn.i BRL. 1219 Conynebacteniwn b e h e BRL. 1318 Co/ynebac.tuiwn beBRL. 1451 BondetetXa bbonchdqxXca Aaobaoten mowed A l d g Q n & A daecaeid EAchUnickia coU K.febAi&a pnewnoniae PnolIzLL6 minabiLiA P,to&UA vdgaAL4 Pnotm.6 rno4gad.l PAeudomonaa pyoyanea SdmoneUa %phi Sdmonelkh pahztgpki A Salmonella pczrtatyphi B SaLmoneUa @phi-munium S h i g e l l a Ahigae SkigeUa donnei C h t o m o b a W u m psodigio&m
Vibhio
P~~O&&A
Myco6ac.tuiu.m p h l e i
*Penicillinase-producing p e n i c i l l i n - r e s i s t a n t stephylococci, **BRL. 1234 i s o l a t e d from c u l t u r e o f Oxford Sa%phytacaccu,5 i n medium c o n t a i n i n g an i n h i b i t o r y c o n c e n t r a t i o n o f b e n z y l p e n i c i l l i n . BRL. 1235 sim i 1a r l y o b t a i n ed u s i n g 6 amin ope n i c i 11a nic a c i d
.
-
destroyed, b u t n o t 6-APA. Reagents:
The assay procedures a r e as follows:
Hydroxylamine h y d r o c h l o r i d e solution Alkaline b u f f e r solution F e r r i c ammonium s u l f a t e solution
347.0 g/L 173.0 g NaCH and 20.6 g Na acetate/L 200.0 g F e r r i c ammonium sulf a t e and 94.3 g conc.H$Q/L
400 The hydroxylamine r e a g e n t i s p r e p a r e d by mixing t h e hydroxylamine s o l u t i o n , t h e a l k a l i n e b u f f e r s o l u t i o n and e t h a n o l i n a r a t i o of 1:1:4 ( b y volume). The pH of t h e m i x t u r e s h o u l d be 7 . 0 . I f not, it is adjusted t o n e u t r a l i t y . Two 2 . 0 m l samp l e s are t a k e n i n s e p a r a t e t u b e s . To t h e f i r s t t u b e ( b l a n k ) i s added 0 . 2 5 m l of 5 . 0 N NaOH. The b l a n k t u b e is a l l o w e d t o s t a n d f o r 1 0 min and n e u t r a l i z e d by a d d i n g 0 . 2 5 m l of 5 N H2S04. To t h e second t u b e i s added 0 . 5 m l o f H20. To b o t h t u b e s 6 m l p o r t i o n s o f hydroxylamino r e a g e n t s are a d d e d , a n d t h e t u b e s a r e a l l o w e d t o s t a n d a t room t e m p e r a t u r e for t e n min. Two m l o f f e r r i c ammonium s u l f a t e s o l u t i o n i s added t o e a c h t u b e . The c o n t e n t s of t h e t u b e s a r e t h o r o u g h l y mixed. C o l o r i s developed from t h e r e a c t i o n , and i t i s measured by a c o l o r i m e t e r a t 623 m u . 2.3.3
P h e n y l a c e t y l a t i o n method of a s s a y
P e n i c i l l i n s a r e f i r s t d e s t r o y e d by e x p o s i n g t h e sample a t pH 2.0 for 2 h r , o r by e x t r a c t i n g t h e sample w i t h b u t y l acetate a t pH 2 . 0 . A f t e r n e u t r a l i z a t i o n , t h e samples are s p o t t e d i n d u p l i c a t e on Whatman No. 1 p a p e r . One s e r i e s o f t h e p a p e r s t r i p s i s d i r e c t l y p l a c e d on a g a r p l a t e s s e e d e d w i t h 8 . A u b Z i t i A , w h i l e t h e o t h e r s e r i e s i s used f o r t h e p h e n y l a c e t y l a t i o n of 6APA t o b e n z y l p e n i c i l l i n . The p h e n y l a c e t y l a t i o n i s c a r r i e d o u t by s p r a y i n g t h e p a p e r s t r i p s s u c c e s s i v e l y w i t h 5 % NaHC03 s o l u t i o n , 5 % p h e n y l a c e t y l c h l o r i d e i n d r y a c e t o n e , and 5 % NaHC03 s o l u t i o n . Under t h e s e c o n d i t i o n s t h e c o n v e r s i o n o f 6-APA t o b e n z y l p e n i c i l l i n i s r a p i d and goes t o c o m p l e t i o n . Vapious conc e n t r a t i o n s (10-100 u n i t / m l ) o f a s t a n d a r d b e n z y l p e n i c i l l i n s o l u t i o n a r e s p o t t e d on a s t r i p . The c o n t r o l and t h e p h e n y l a c e t y l a t e d s t r i p s a r e p l a c e d on a s e e d e d a g a r p l a t e . The pot e n c i e s of t h e p h e n y l a c e t y l a t e d s a m p l e s are d e t e r m i n e d by comp a r i n g g r o w t h - i n h i b i t i o n zone s i z e s of t h e samples t o t h o s e o f t h e b e n z y l p e n i c i l l i n s t a n d a r d . One mg of 6-APA a f t e r phenyla c e t y l a t i o n , i s e q u i v a l e n t t o 2 , 7 5 2 u n i t s of b e n z y l p e n i c i l l i n .
2.4 I s o l a t i o n
and p u r i f i c a t i o n of 6 - A m i n o p e n i c i l l a n i c a c i d
Unlike t h e p e n i c i l l i n s which a r e a l s o p r e s e n t i n t h e b r o t h , 6-APA c a n n o t be e x t r a c t e d i n t o common o r g a n i c s o l v e n t s s u c h as b u t a n o l o r b u t y l a c e t a t e . T h i s p r o p e r t y i s t a k e n i n t o a c c o u n t f o r i t s i s o l a t i o n . 6 - A m i n o p e n i c i l l a n i c a c i d is i s o l a t e d u s i n g any one o r a combination of t h e f o l l o w i n g methods, 2.4.1
P r e p a r a t i o n of a c r u d e c o n c e n t r a t e ( I ) 4 6
The c u l t u r e b r o t h is a d j u s t e d t o pH 7 . 5 , and c o n c e n t r a t e d t o one t e n t h o f t h e o r i g i n a l volume i n vacuo a t 2 5 t o 3OoC. The pH of t h e c o n c e n t r a t e i s r e - a d j u s t e d t o 3 . 0 w i t h 5 0 % H3P04 t o y i e l d a b i o i n a c t i v e p r e c i p i t a t e . The p r e c i p i t a t e i s removed by c e n t r i f u g a t i o n , and t h e c l e a n s u p e r n a t a n t i s e x t r a c t e d w i t h a h a l f volume of n - b u t y l a c e t a t e t o remove p e n i c i l l i n s . The pH of t h e aqueous l a y e r i s r e - a d j u s t e d t o 7 . 5 w i t h N a O H , and t o t h e l i q u i d i s added t h r e e volumes of a c e t o n e , T h i s res u l t s i n t h e f o r m a t i o n o f a s e m i - s o l i d p h a s e i n t h e bottom. The upper p h a s e i s c l a r i f i e d by c e n t r i f u g a t i o n , and c o n c e n t r a t e d t o one t w e n t i e t h of t h e o r i g i n a l volume. R e s u l t s of t h e d i f -
40 1 f e r e n t s t e p s i n t h e p r e p a r a t i o n o f t h e crude c o n c e n t r a t e are shown i n t h e f o l l o w i n g t a b l e . P r e p a r a t i o n o f t h e c r u d e concent r a t e r e s u l t s i n t h e l o s s o f 30-60% of 6-APA, b u t t h i s l e a d s t o an o v e r a l l p u r i f i c a t i o n of seven-fold. Recovery Data f o r Preparation o f 6-AminoP e n i c i l l e n i c A c i d Concentrate Total Solida
% 6-AminoPeni c i l l a n i c Acid
13
100
100
01
VOl.
(1.) -
Stege
Units/
ml
Units/ mg
Culture f i l t r a t e
40
345
Concentrete t o one t e n t h volume
4
3,300
12.5
100
95
Adjusted t o pH 3.0 and p r e c i p i t a t e removed
3.3
3,400
12.0
98
90
3.3
2,800
11
96
81
10.9
865
80
11.5
70
72
11.5
68
After extraction with b u t y l acetate Acetone supernatant F i n a l concentrate
2.4.2
2
4,600
P u r i f i c a t i o n o f t h e c r u d e c o n c e n t r a t e (IIIh6
A column ( 2 x 35 i n . ) , c o n t a i n i n g 7 0 0 g of d e a c i d i t e r e s i n , i s c h a r g e d w i t h 8.0 L o f t h e c r u d e c o n c e n t r a t e a t a flow r a t e of 400 m l / h r . The column i s washed w i t h 2 . 0 L o f water. The combined p e r c o l a t e a n d w a t e r wash r e s u l t i n a l o s s o f 1 1 . 5 % o f t h e t o t a l 6-APA a p p l i e d t o t h e column. The column i s n e x t Active e l u t e d with 6% acetic a c i d a t a flow r a t e of 800 ml/hr. f r a c t i o n s are c o l l e c t e d ( 3 . 0 L); t h e s e a c c o u n t f o r a 47% of t h e t o t a l 6-APA a p p l i e d t o t h e column. T h i s material i s o n l y 1 0 % p u r e (252 u n i t s / m g I . F u r t h e r p u r i f i c a t i o n o f t h e material i s a c h i e v e d by s u b s e q u e n t c e l l u l o s e column chromatography. The a c t i v e e l u a t e from t h e d e a c i d i t e r e s i n column i s e v a p o r a t e d t o d r y n e s s i n vacuo t o remove a c e t i c a c i d . T h i s s t e p r e s u l t s i n a loss o f 6-30% o f 6-APA, d e p e n d i n g on t h e volume h a n d l e d a n d t h e t i m e i n v o l v e d . The d r i e d r e s i d u e i s d i s t i l l e d i n a m i x t u r e o f acetone-isopropanol-water ( 2 : 2 : 1 I , and a p p l i e d t o a c e l l u l o s e column. I n a t y p i c a l e x p e r i m e n t a s a m p l e of 55 m l , h a v i n g a s p e c i f i c a c t i v i t y of 2 0 8 u n i t s / m g a n d a t o t a l a c t i v i t y o f 1 . 3 6 x l o 6 u n i t s of G-APA, i s a p p l i e d t o a column of 2 x 1 0 i n . and e l u t e d w i t h a m i x t u r e of a c e t o n e - i s o p r o analwater. The f i r s t 12 m l o f t h e e l u a t e i s d i s c a r d e d , an$ 25 m l f r a c t i o n s are c o l l e c t e d . F r a c t i o n s 2 0 - 2 2 c o n t a i n e d 9 0 % o f t h e t o t a l 6-APA a p p l i e d t o t h e column. T h i s a c c o u n t s f o r 1 5 % o f t h e t o t a l w e i g h t o f material a p p l i e d t o t h e column, t h u s achieving a p u r i t y of about s i x - f o l d . This material (45% p u r e ) has a s p e c i f i c a c t i v i t y of 1,250 units/mg. The e l u a t e s from t h e c e l l u l o s e column are e v a p o r a t e d t o d r y n e s s i n vacuo, a n d t h e r e s i d u e d i s s o l v e d i n water t o a c o n c e n t r a t i o n o f a b o u t 1 0 0 mg/ml. The pH of t h i s material i s a d j u s t e d t o 4.3 u s i n g 2 0 % H C 1 , a n d t h e m i x t u r e i s c o o l e d i n i c e b a t h . C r y s t a l s o f 6-APA, which
40 2
a p p e a r on c o o l i n g t h e m i x t u r e , are removed by f i l t r a r i o n , washed w i t h water and a c e t o n e , and d r i e d . 2.4.3
P u r i f i c a t i o n of 6-APA by Dowex 1 R e s i n 4 6
S i n c e 6-APA i s s t r o n g l y a c i d i c , Dowex 1 r e s i n i n H+ c y c l e a p p e a r s to b e a good medium f o r a s e l e c t i v e a d s o r p t i o n of t h e material. Once a d s o r b e d , i t can b e e l u t e d w i t h O.1N H C 1 . I f t h i s chromatography i s c a r r i e d o u t a t room t e m p e r a t u r e , 70-80% recovery i s u s u a l i y a c h i e v e d , whereas, i f t h e experiment i s c a r r i e d o u t a t 1 0 C o r a l o w e r t e m p e r a t u r e u s i n g j a c k e t e d columns, r e c o v e r i e s as h i g h as 95% a r e n o r m a l l y a c h i e v e d . I n a t y p i c a l e x p e r i m e n t , 2 kg of c h a r g e d Dowex 1 r e s i n i s poured i n t o a 2 i n c h d i a m e t e r column, and c h a r g e d w i t h 1 0 . 8 o f 6-APA c o n c e n t r a t e c o n t a i n i n g 3 2 . 5 x lo6 u n i t s a t a p u r i t y of 40.7 units/ml. P e r c o l a t e i s c o l l e c t e d a t a flow r a t e of 40 ml/min a t room t e m p e r a t u r e . The column i s washed w i t h 2 . 0 of water. The p e r c o l a t e a n d t h e water-wash c o n t a i n a b o u t 1 1 . 2 % of 6-APA o r i g i n a l l y a p p l i e d t o t h e column. The column i s c h a r g e d w i t h 0 . 1 N H C 1 , and e l u a t e s are c o l l e c t e d i n 4 0 0 m l f r a c t i o n s . F r a c t i o n s 62-70 c o n t a i n 2 3 . 2 x lo6 u n i t s o f 6-APA w i t h a s p e c i f i c a c t i v i t y o f 954 u n i t s / m g and a c c o u n t f o r a b o u t a 2 3 - f o l d p u r i f i c a t i o n of t h e s t a r t i n g material. The a c t i v e f r a c t i o n s a r e combined, and a d j u s t e d t o a pH of 7 . 0 w i t h 4 . 0 N NH4OH. The n e u t r a l i z e d material i s r e d u c e d t o a volume of 1 0 0 m l by c o n c e n t r a t i o n i n vacuo, and 6-APA i s c r y s t a l l i z e d from t h i s c o n c e n t r a t e by slow a d d i t i o n o f 2 0 % H C 1 u n t i l t h e f i n a l pH becomes 4 . 3 . The c r y s t a l s , which a p p e a r on c h i l l i n g , a r e f i l t e r e d , washed w i t h water a n d c o l d a c e t o n e . O v e r a l l y i e l d is 69.5%. 2.4.4
R e c r y s t a l l i z a t i o n of 6-APA46
R e c r y s t a l l i z a t i o n is c a r r i e d o u t by s l o w l y a d d i n g 1 0 % H C 1 t o an aqueous s o l u t i o n o f t h e 6-APA a t a c o n c e n t r a t i o n of 1 0 0 mg/ml, u n t i l t h e f i n a l pH o f t h e m i x t u r e r e a c h e s 4.3. The rec r y s t a l l i z e d material i s washed w i t h water and c o l d a c e t o n e , The r e c r y s t a l l i z e d 6-APA h a s a s p e c i f i c a c t i v i t y of 2,740 u n i t s / m g , a m e l t i n g p o i n t o f 208-209°C ( w i t h d e c . ) , a n d an i s o e l e c t r i c p o i n t a t pH 4.3. O p r i c a l r o t a t i o n o f t h e 6-APA i s d e p e n d e n t on pH v a l u e s , p a r t i c u l a r l y a t v a l u e s below 6 . 0 . The s p e c i f i c r o t a t i o n i s +337 a t between pH v a l u e s of 7 . 0 and 10.5.
2.5 Formation
of 6-APA from p e n i c i l l i n s b y e n z y m i c h y d r o l y s i s
Many m i c r o o r g a n i s m s , i n c l u d i n g b o t h b a c t e r i a and f u n g i , p r o d u c e enzymes which h y d r o l y z e t h e amide bond i n t h e 6 p o s i t i o n of p e n i c i l l i n s . The enzymes are g e n e r a l l y known as p e n i c i l l i n a c y l a s e s o r amidases4 7. The a c y l a s e - p r o d u c i n g f u n g i are
Alteanania, Adpeagiltud, B o t a y Z L d , Cephalospoaium, C n y p t o c o c c w , EmelciceleOpbib,Epicoccum, Epidemnophyton, Fudaaium, M U C O ~ , P e n i c i l l i u m , Phoma, Tnichodeama, Taichophyton a n d Taichodpoaon. The a c y l a s e - p r o d u c i n g b a c t e r i a a r e Aenobactea, ALcaLigeneA, 8 olcdotella, CeLluLomona4, Colcyne bacteaium, Eawinia, Edchenichia, Flavobacteaium, MicRoco~cud, Nocaadia, Pnoteu4, Pbeudomonad,
40 3
SaLmoneLLa, Sahcina and Xanthomonab. Of t h e s e t h e most w i d e l y s t u d i e d i s Ebchekichia coLi (ATCC 9 6 3 7 ) . 2.5.1
Purification-and acylas-
g e n e r a l p r o p e r t i e s of p e n i c i l l i n
There are two d i s t i n c t t y p e s o f p e n i c i l l i n a c y l a s e s a c y l a s e s o b t a i n e d from f u n g i and s t r e p t o m y c e s , h y d r o l y z e phenoxy methyl p e n i c i l l i n more r a p i d l y t h a n b e n z y l p e n i c i l l i n , and t h e enzymes i s o l a t e d from b a c t e r i a l s p e c i e s h y d r o l y z e b e n z y l p e n i c i l l i n more r a p i d l y t h a n phenoxymethyl p e n i c i l l i n . Among b a c t e r i a l a m i d a s e s , t h e E. C O I Land ALcaLigeneb enzymes a r e most i m p o r t a n t . The most t h o r o u g h l y s t u d i e d enzyme is t h a t from E. C O Up, a r t i c u l a r l y from t h e s t r a i n ATCC 9637. The E . c o e i enzyme i s u s u a l l y i n t r a c e l l u l a r , whereas some o t h e r b a c t e r i a l enzymes a r e e x t r a c e l l u l a r . The f o r m a t i o n of p e n i c i l l i n a c y l a s e i n E. coLi i s i n d u c e d by p h e n y l a c e t i c a c i d and o t h e r s t r u c t u r a l l y r e l a t e d compounds, and r e p r e s s e d by h i g h concent r a t i o n s o f c a r b o h y d r a t e i n t h e media. Seed c u l t u r e s (4 a ) of E . coLi ATCC 9637 grown on p h e n y l a c e t i c a c i d and g l u t a m a t e were p r e p a r e d by s e r i a l t r a n s f e r s , and u s e d t o i n o c u l a t e 1 0 0 medium of t h e f o l l o w i n g c o m p o s i t i o n : mZp04
3.0 g
KH2HPO4
7.0 g
( NHI+) 2soi+
1.0 g
&s04.7H20
0.2 g
Phenylacetic acid
2.0 g
Sodium glutamate
5.0 g
Water
1.0 L
The c u l t u r e was i n c u b a t e d a t 3OoC. The i n t r a c e l l u l a r l e v e l of p e n i c i l l i n amidase r e a c h e d a maximum a t 8 - 1 0 h r s , a n d t h e c u l t u r e h a r v e s t e d and c e n t r i f u g e d . The h a r v e s t e d b a c t e r i a (250-300 g w e t w e i g h t ) were k e p t f r o z e n u n t i l r e q u i r e d . The b a c t e r i a were r e s u s p e n d e d i n 0 . 0 1 M p h o s p h a t e b u f f e r (pH 7 1 , and d i s r u p t e d by r e c y c l i n g f o r 5 m i n . t h r o u g h a Morton-Garlin homogenizer. The t e m p e r a t u r e of t h e l i q u i d was m a i n t a i n e d below 5 C , t h e d e b r i s removed by c e n t r i f u g a t i o n . The s u p e r n a t a n t was t r e a t e d w i t h 1 7 . 0 g of s t r e p t o m y c i n s u l p h a t e t o p r e c i p i t a t e n u c l e i c a c i d s , which were removed by c e n t r i f u g a tion. The acylase enzyme was p r e c i p i t a t e d from t h e s u p e r n a t a n t by a d d i t i o n of s u f f i c i e n t ammonium s u l f a t e t o g i v e a f i n a l c o n c e n t r a t i o n of 6 0 % . A f t e r o v e r n i g h t s t a n d i n g t h e p r e c i p i t a t e d enzyme was c o l l e c t e d by c e n t r i f u g a t i o n , r e d i s s o l v e d i n 0 . 0 1 M p h o s p h a t e b u f f e r (pH 7 . 0 1 , and r e f r a c t i o n a t e d with polyethylene glycol. The p o l y e t h y l e n e g l y c o l f r a c t i o n ( 1 0 - 2 0 % ) was r e d i s s o l v e d i n 0 . 0 1 M p h o s p h a t e b u f f e r (pH 7.01, and p a s s e d t h r o u g h a DEAE c e l l u l o s e column ( 2 0 x 2 . 5 cm diame t e r ) . The f i r s t few f r a c t i o n s ( e a c h 10 m l ) were c o l l e c t e d , and s t o r e d i n t h e d e e p f r e e z e u n t i l r e q u i r e d .
40 4 The activity of the acylase enzyme is optimum at pH 9.0. The enzyme is optimally stable at a pH 10.0, with a slight loss of activity at pH 9.0 or 11.0. However, the enzyme is completely deactivated at pH 3.0. 2.5.2
Isolation of 6-APA from acylase treated penicil-
linsgy -
Fifty !t of clarified fermentation broth was obtained from a fermentation of an acylase producing organism, carried out in a 90 II fermenter. A suitable penicillin was added to give a concentration of 10 mg/ml. The pH was maintained at 8.0 by automatic addition of 10% sodium hgdroxide and the temperature of the reaction mixture held at 45 C. Formation of 6-aminopenicillanic acid was followed by assay at intervals of time. Results of a typical hydrolysis are shown in the following table. Hydrolysis to 6-aminopenicillanic acid proceeded rapidly at the beginning of the reaction but became progressively slower a s 6-aminopenicillanic acid accumulated. After 5 hrs, the conversion to 6-aminopenicillanic acid was 56.4%. The remaining penicillin was destroyed as a result of penicillinase activity and the instability of the penicillin at that temperature and pH. Crystalline 6-aminopenicillanic acid was isolated from this reaction mixture using the ion-exchange resin Dowex 1 and eluting with hydrochloric acid. Before percolating the reaction mixture through the resin column, however, the phenoxyacetic acid was removed by extracting with one half volume of butylacetate at pH 2.0. The extracted aqueous phase was then adjusted to pH 7.0 with sodium hydroxide. H y d r o l y s i s o f Phenoxymethyl P e n i c i l l i n by P e n i c i l l i n Acylase C e l l - f r e e f e r m e n t a t i o n b r o t h o f Stktptomyces .&venduRue BRL. 198 w a s i n c u b a t e d with p h e n o x y r n e t h y l p e n i c i l l i n a t a c o n c e n t r a t i o n of 1%. Terrperature m a i n t a i n e d a t 4 5 O C End pH m a i n t a i n e d a t 8.0.
6-arninopeni c i l l a n i c a c i d formed
x
Time ( h r )
(d m l )
Conversion
1 2 3 4 5
1770 2470
28.7 40 .O 47 .o 52.5 56.4
2930 3230 3470
The recovery of crystalline 6-aminopenicillanic acid from the enzyme treated broth was 50%. Recrystallization of this 6aminopenicillanic acid gave a preparation which assayed at 97% pure by the method of phenylacetylation to convert to benzylpenicillin and the subsequent microbiological assay of the latter.
40 5
The s p o r e s o f FuAaAium m o l i n i ~ o & m ch y d r o l y z e phenoxym e t h y l p e n i c i l l i n , b u t n o t b e n z y l p e n i c i l l i n t o 6-APA. The spore a c y l a s e , l i k e t h e mycelial a c y l a s e s of actinomycetes and f i l a m e n t o u s f u n g i , h y d r o l y z e phenoxymethyl p e n i c i l l i n i n p r e f erence t o benzyl p e n i c i l l i n . S p o r e s were o b t a i n e d by g r o w i n g t h e o r g a n i s m on moist " p o t barley". To 8 0 m l o f t a p w a t e r i n 2 . 8 R F e r n b a c h f l a s k was added 200 g o f b a r l e y , The f l a s k s were s t e r i l i z e d f o r 1 h r a t 1 2 l o C , c o o l e d t o 25OC, i n o c u l a t e d w i t h a s p o r e s u s p e n s i o n o f F . mon.Ll.Ldo&me,and i n c u b a t e d f o r 8-10 d a y s a t 2 5 O C i n an atmos p h e r e o f 6 0 % r e l a t i v e h u m i d i t y . Average y i e l d o f t h e s p o r e s were 1011 s p o r e s / f l a s k . S p o r e s w e r e h a r v e s t e d , washed, and s u s pended i n 0 . 1 M p h o s p h a t e b u f f e r (pH 8.0) t o g i v e a c o n c e n t r a t i o n o f 2 x 108 t o 5 x 108 s p o r e s / m l . To 50 m l o f t h e s p o r e s u s p e n s i o n i n a 2 5 0 ml B l e n m e y e r f l a s k w a s a d d e d 1 0 0 mg o f phenoxymethyl p e n i c i l l i n , a n d t h e f l a s k s w e r e i n c u b a t e d on a r o t a r y s h a k e r a t 28OC f o r 24 t o 48 h r . T h i s gave a 70-80% c o n v e r s i o n y i e l d o f phenoxymethyl p e n i c i l l i n t o 6-APA. 2.5.4
P r e p a r a t i o n o f 6-APA from b e n z y l p e n i c i l l i n u s i n g immobilized p e n i c i l l i n acylase3
An a g a r o s e s o l u t i o n ( S e p h a r o s e 4 B , 4 % , 2 . 5 m l ) was f i l t e r e d , and t h e wet r e s i d u a l s o l i d was s t i r r e d f o r 8 m i n . a t pH 1 1 . 5 - 1 2 . 0 i n 2 . 0 m l o f a 5% s o l u t i o n o f i c e c o l d aqueous cyanogen b r o m i d e . The s o l i d g e l was removed by f i l t r a t i o n , t h o r o u g h l y washed w i t h i c e water a n d a n i c e c o l d s o l u t i o n o f 0 . 1 M borax. I t was t h e n s u s p e n d e d i n 4 . 0 m l o f 0 . 1 M b o r a x , and s t i r r e d w i t h E . c o l i a c y l a s e ( 0 . 1 0 6 g , a c t i v i t y 159,000 p/g) f o r 2 4 h r a t 4OC. The m i x t u r e was f i l t e r e d a n d t h e s o l i d res i d u e was washed t h o r o u g h l y w i t h water t o g i v e 1 . 2 g o f wet polymer w i t h a s p e c i f i c a c t i v i t y o f 679 p / g .
To an i m m o b i l i z e d enzyme s o l u t i o n ( 2 8 3 g , a c t i v i t y 4 2 p / g ) was added a s o l u t i o n of b e n z y l p e n i c i l l i n ( 2 0 g ) as p o t a s s i u m The m i x t u r e was i n c u b a t e d a t 37OC, s a l t i n water (317 m l ) . and t h e pH k e p t a t 7 . 8 by p e r i o d i c a d d i t i o n o f 2 . 5 N N a O H . The r e a c t i o n m i x t u r e was shown t o c o n t a i n 1 0 . 7 g o f 6 - a m i n o p e n i c i l l a n i c a c i d ( 9 2 % ) a f t e r 6 h r i n c u b a t i o n , a n d i t was f i l t e r e d . The pH of t h e s o l u t i o n was a d j u s t e d t o 3 . 0 w i t h 5 N H C 1 , a n d e x t r a c t e d w i t h o n e - h a l f volume of m e t h y l i s o b u t y l k e t o n e t o remove t h e r e s i d u a l b e n z y l p e n i c i l l i n and t h e p h e n y l a c e t i c a c i d s i d e c h a i n . A f t e r t h e e x t r a c t i o n t h e water s o l u t i o n was s e p a r a t e d from t h e o r g a n i c p h a s e , The pH o f t h e aqueous p h a s e was a d j u s t e d t o 7 . 5 , a n d t h e volume r e d u c e 2 t o 1 2 0 m l i n vaCu0. The c o n c e n t r a t e d s o l u t i o n was c o o l e d t o 5 C , a n d f i l t e r e d . When t h e f i l t e r e d s o l u t i o n was a c i d i f i e d t o pH 4 . 3 w i t h 5 N HC1,the c r y s t a l l i n e 6-APA p r e c i p i t a t e d . The c r y s t a l s were washed w i t h a small amount o f c o l d water a n d d r y a c e t o n e , a n d d r i e d i n vacuo t o g i v e 8 . 8 g o f 6-APA. The p u r i t y o f t h e p r o d u c t was 95% and t h e t o t a l y i e l d c a l c u l a t e d on t h e b a s i s of b e n z y l p e n i c i l l i n added was 7 2 % .
406 2 . 6 Nonenzymatic -
c o n v e r s i o n of p e n i c i l l i n s t o 6-APAS2
6-APA can be produced i n commercial q u a n t i t i e s e i t h e r by d i r e c t f e r m e n t a r i o n i n a p r e c u r s o r - f r e e p e n i c i l l i n b r o t h or by enzymatic c l e a v a g e of n a t u r a l p e n i c i l l i n s . A t h i r d and v e r y e f f i c i e n t nonenzymatic route t o 6-APA from p e n i c i l l i n s i s through a s e r i e s of c h e m i c a l r e a c t i o n s i n v o l v i n g (1) s i l y l a t i o n , ( 2 ) t r e a t m e n t o f t h e s i l y l ester w i t h PCl5 and p y r i d i n e t o form an imino c h l o r i d e i n t e r m e d i a t e , ( 3 ) a d d i t i o n of an a l c o h o l t o c o n v e r t t h e i n t e r m e d i a t e t o an imino e t h e r , and ( 4 ) a d d i t i o n o f water t o e f f e c t h y d r o l y s i s t o 6-APA. The r e a c t i o n s a r e i l l u s t r a t e d i n t h e f o l l o w i n g scheme:
0
R-i-%x 0
COOH
0
1, PC15, pyridine 2. R'OH, pyridine
OR'
0 H ,%O, mild
COOH
R' = R-CO =
olkyl, hydroxyalkyl, or phenylalkyl acyl radical
COOH
40 7
3 . O t h e r n a t u r a l l y o c c u r r i n g B-lactam a n t i b i o t i c s
3 . 1 Nocardicin5 3.1.1
Discovery and b r i e f chemical d e s c r i p t i o n
T h i s a n t i b i o t i c was d i s c o v e r e d i n t h e r e s e a r c h l a b o r a t o r i e s of F u j i s a w a P h a r m a c e u t i c a l Company i n Japan. I t i's r e p r e s e n t e d by t h e f o l l o w i n g s t r u c t u r e .
NOH- C&
- CO-NH
E'pm
0
II
COOH O-C&-C&-CH-
C m
I
Nocord icin 3.1.2
Prqducing organism
The a n t i b i o t i c is p r o d u c e d by a s t r a i n of Nocandia, i s o l a t e d from a s o i l i n Tsuyama C i t y , J a p a n . The p r o d u c i n g s t r a i n h a s b e e n d e s i g n a t e d as Nocandia unidonmid v a r . t s u y a m a n e n s i s ATCC 2 1 8 0 6 . 3.1.3
P h y s i c a l and c h e m i c a l p r o p e r t i e s
Nocardicin is s o l u b l e i n a l k a l i n e s o l u t i o n s , e . g . , i n aqueous ammonia, a q u e o u s p y r i d i n e , e t c . I t i s a l s o s o l u b l e i n dimethyl s u l f o x i d e and methanol, b u t i n s o l u b l e i n chloroform, e t h y l a c e t a t e o r d i e t h y l e t h e r . The a n t i b i o t i c becomes brown a t 1 8 7 O C , a n d decomposes a t 2 1 4 - 2 1 6 O C . The U.V. s p e c t r u m shows a peak a t 2 2 0 nm a n d a maxima a t 2 7 2 nm (Ei:m 310). 3 . l .4
F e r m e n t a t i o n and i s o l a t i o n p r o c e d u r e s
The a n t i b i o t i c i s p r o d u c e d when a s t r a i n o f Nocandia u n i donmi4 i s grown u n d e r submerged ( a e r o b i c ) c o n d i t i o n s i n a n u t r i e n t medium c o n t a i n i n g c a r b o n a n d n i t r o g e n s o u r c e s . '$he ferment a t i o n i s u s u a l l y c a r r i e d o u t a t a t e m p e r a t u r e of 30 C o v e r a p e r i o d of 3 0 t o 5 0 h r . A t t h e end of f e r m e n t a t i o n , t h e c e l l s aTe removed from t h e b r o t h . b y e i t h e r f i l t r a t i o n or c e n t r i f u g a t i o n . The c l e a r f i l t r a t e I S e x t r a c t e d w i t h an o r g a n i c s o l v e n t
40 8 s u c h as p y r i d i n e o r b u t a n o l . A l t e r n a t i v e l y , t h e a n t i b i o t i c is adsorbed onto charcoal, a c t i v a t e d alumina, s i l i c a g e l o r cellul o s e powder. The a d s o r b e d a n t i b i o t i c i s e l u t e d by means of a p o l a r organic s o l v e n t . For f u r t h e r p u r i f i c a t i o n of t h i s a n t i b i o t i c , i t i s a d s o r b e d o n t o A m b e r l i t e XAD-4 from t h e c u l t u r e b r o t h , a n d e l u t e d w i t h 5 % m e t h a n o l , F o r example, t o 2,400 a of t h e c l e a r f i l t r a t e o f a f e r m e n t a t i o n b r o t h was added 2 4 kg of a c t i v a t e d c h a r c o a l , and t h e m i x t u r e s t i r r e d f o r 1 5 min. The c h a r c o a l s u s p e n s i o n was f i l t e r e d , a n d t h e c a k e washed o n c e w i t h 500 a o f water and e x t r a c t e d t w i c e w i t h 6 0 0 a o f a m i x t u r e Tha e x t r a c t s o f a c e t o n e : w a t e r : 2 5 % a q u e o u s ammonia ( 1 O O : l O O : l ) . were combined a n d c o n c e n t r a t e d t o a volume of 100 a . The conc e n t r a t e was t r e a t e d w i t h D u o l i t e C - 2 0 , a n d t h e pH a d j u s t e d t o 3.0. T h i s material was f i l t e r e d t o remove any p r e c i p i t a t e s . The clear f i l t r a t e was d i l u t e d w i t h 5 0 0 .9 o f w a t e r , a n d t h e d i l u t e d material p a s s e d t h r o u g h a column f i l l e d w i t h 8 0 1 o f The column was s u c c e s s i v e l y washed w i t h 8 0 k of D u o l i t e A-6. w a t e r , 240 k o f 0 . 5 N a c e t i c a c i d , 240 a water a n d f i n a l l y e l u t e d w i t h 320 a o f a m i x t u r e o f w a t e r : p y r i d i n e : a c e t i c a c i d (1OO:lOO:l). The e l u a t e s were c o n c e n t r a t e d t o 2 0 a , a n d d i l u t e d w i t h one volume o f a m i x t u r e of e t h y l a c e t a t e : n - b u t a n o l (1:l). The m i x t u r e was s t i r r e d f o r 1 5 min, and t h e a q u e o u s p h a s e was s e p a r a t e d a n d removed. The aqueous l a y e r was n e u t r a l i z e d w i t h 4 . 0 N aqueous N a O H , a n d c o n c e n t r a t e d t o a b o u t 6.0 a , a n d t h e pH a d j u s t e d t o 2.5. A t t h i s p o i n t a p a l e brown p r e c i p i t a t e was obtained. T h i s p r e c i p i t a t e was washed w i t h a c e t o n e and w a t e r , a n d d r i e d t o y i e l d 1 5 2 gm o f a p a l e b r o w n i s h powder, The powder was s u s p e n d e d i n 2 water, and t h e pH b r o u g h t t o 7 . 5 w i t h 4 N The s o l u t i o n was p a s s e d t h r o u g h a column packed w i t h 1 k NaOH. DEAE Sephadex A - 2 5 . The e l u t i o n was done w i t h 3 k of a 5% aqueous ammonia, a n d t h e e l u a t e b r o u g h t t o pH 2 . 5 w i t h a q u e o u s H C 1 t o p r o d u c e 1 2 6 gm of a p a l e b r o w n i s h a n t i b i o t i c c r u d e . The c r u d e a n t i b i o t i c was s u s p e n d e d i n 9 0 0 m l water, a n d t h e s u s p e n s i o n S i x t y gms o f a c t i v a t e d b r o u g h t t o pH 7 . 5 w i t h aqueous N a O H . c a r b o n was a d d e d , a n d s t i r r e d f o r 1 5 min. The a c t i v a t e d c a r b o n was removed by f i l t r a t i o n , 9 0 0 m l o f m e t h a n o l added t o t h e rem a i n i n g f i l t r a t e , a n d t h e pH b r o u g h t t o 2 . 5 w i t h 4 . 0 N H C 1 . T h i s r e s u l t e d i n t h e f o r m a t i o n of 10 gm of n o c a r d i c i n i n t h e form of w h i t e n e e d l e s h a p e d c r y s t a l s .
3.2 C l a v u l a n i c 3.2.1
acid54
Discovery and chemical d e s c r i p t i o n
C l a v u l a n i c a c i d was d i s c o v e r e d by Beecham g r o u p i n England. I t i s a n a t u r a l product having 8-lactamase-inhibiting a c t i v i t y . I t i s c a p a b l e of i n h i b i t i n g t h e growth of S. aU)LLuA s t r a i n s , a n d shows a s y n e r g i s t i c a c t i v i t y w i t h a m p i c i l l i n a g a i n s t 0 - l a c t a m a s e - p r o d u c i n g s t r a i n s o f E . c ~ l i ,K l e b h i e L t a aeaOgene.4 and Staphylococcu.4 aulleu.4, and w i t h c e p h a l o r i d i n e a g a i n s t 8 - l a c t a m a s e - p r o d u c i n g s t r a i n s of P A o t e u A m i n a b i t i d a n d Staphytococcud auaeud, C l a v u l a n i c a c i d i s r e p r e s e n t e d by t h e s t r u c t u r e on t h e n e x t p a g e . 3.2.2
Producing organism
C l a v u l a n i c a c i d i s p r o d u c e d by t h e s t r a i n StAeptomyceA
409
COOH
Clovulanic acid
cLauulagenud ATCC 2 7 0 6 4 . 3.2.3
F e r m e n t a t i o n and i s o l a t i o n
The f e r m e n t a t i o n of t h i s a n t i b i o t i c i s c a r r i e d o u t on an a e r o b i c s u r f a c e o r i n a submerged c u l t u r e . A n u t r i e n t medium, c o n t a i n i n g 0.1-10% o f a complex o r g a n i c n i t r o g e n s o u r c e s u c h as y e a s t e x t r a c t , maize s t e e p l i q u o r , v e g e t a b l e p r o t e i n , p e p t o n e o r c a s e i n h y d r o l y s a t e s , a r e u s e d . A t t h e end o f f e r m e n t a t i o n t h e c u l t u r e f i l t r a t e i s a c i d i f i e d t o pH 2 . 3 , and e x t r a c t e d w i t h an o r g a n i c s o l v e n t such as n - b u t y l a c e t a t e , methyl i s o b u t y l ketone, n-butanol o r e t h y l a c e t a t e . The B - l a c t a m a s e - i n h i b i t i n g s u b s t a n c e i s r e - e x t r a c t e d from t h e o r g a n i c s o l v e n t i n aqueous b i c a r b o n a t e s o l u t i o n . The aqueous e x t r a c t i s c o n c e n t r a t e d u n d e r a vacuum a n d f r e e z e - d r i e d t o y i e l d a c r u d e p r o d u c t , T h i s c r u d e p r o d u c t i s s t a b l e when s t o r e d i n t h e d r y s t a t e a t - 2 O O C . C l a v u l a n i c a c i d i s a l s o s e p a r a t e d from t h e c u l t u r e f i l t r a t e by means of a n i o n exchange r e s i n chromatography. The c e l l f r e e f i l t r a t e i s p e r c o l a t e d a t an a p p r o x i m a t e l y n e u t r a l o r s l i g h t l y a c i d i c pH ( 6 - 7 ) i n a column packed w i t h a weakly o r s t r o n g l y b a s i c a n i o n exchange r e s i n s u c h as A m b e r l i t e I R 4 8 o r Z e o l i t e FFII'. The column i s washed w i t h water and e l u t e d w i t h aqueous sodium c h l o r i d e s o l u t i o n . A s an example, 2 0 L o f a c u l t u r e f i l t r a t e i s c o n c e n t r a t e d t o 5 . 0 L and l y o p h i l i z e d . The m a t e r i a l i s s u s p e n d e d i n 9 0 0 m l of anhydrous d i m e t h y l formamide, and d i s p l a c e d w i t h 150 m l b e n z y l bromide. The m i x t u r e i s s t i r r e d 2 h r a t room t e m p e r a t u r e a n d d i l u t e d w i t h 1 . 0 L of e t h y l a c e t a t e . The r e a c t i o n m i x t u r e i s f i l t e r e d , and c o n c e n t r a t e d t o t h e s m a l l e s t p o s s i b l e volume. The o i l y r e s i d u e i s e x t r a c t e d w i t h a n o t h e r L o f e t h y l a c e t a t e . A f t e r f i l t e r i n g the e x t r a c t , i t i s again concentrated t o an o i l y r e s i d u e , which is a p p l i e d t o a 7 . 6 x 35.6 c m s i l i c a g e l column. The column i s e l u t e d w i t h c y c l o h e x a n e t o remove b e n z y l bromide,
410
and t h e e l u a t e e x t r a c t e d w i t h e t h y l a c e t a t e . Twenty m l f r a c t i o n s are c o l l e c t e d , and examined by TLC. TLC p l a t e s are s p r a y e d w i t h 2,3,5-triphenyltetrazolium c h l o r i d e r e a g e n t t o d e t e r m i n e t h e p r e s e n c e of t h e b e n z y l e s t e r of c l a v u l a n i c a c i d . The f r a c t i o n s showing t h e p r e s e n c e of t h e e s t e r are comb i n e d and c o n c e n t r a t e d t o 1 5 ml. The c o n c e n t r a t e i s c h a r g e d t o a 3.8 x 40 cm s i l i c a g e l column and e l u t e d w i t h c h l o r o f o r m : ethyl acetate (8:2). F i f t e e n ml f r a c t i o n s a r e c o l l e c t e d , and t e s t e d f o r t h e b e n z y l e s t e r i n t h e a f o r e m e n t i o n e d manner, The b e n z y l e s t e r - p o s i t i v e f r a c t i o n s a r e p o o l e d , and c o n c e n t r a t e d t o 8.0 m l . The c o n c e n t r a t e i s charged t o a 2 . 5 x 4 0 cm s i l i c a g e l column, and e l u t e d w i t h e t h y l a c e t a t e : c y c l o h e x a n e m i x t u r e ( 8 : 2 ) The b e n z y l e s t e r - p o s i t i v e f r a c t i o n s are combined and c o n c e n t r a t e d under r e d u c e d p r e s s u r e . T h i s g i v e s 1 6 0 m l of p u r e b e n z y l e s t e r as an oil'.
3 . 3 Thienamycinss 3.3.1
Discovery and c h e m i c a l d e s c r i p t i o n
The a n t i b i o t i c was d i s c o v e r e d by Merck s c i e n t i s t s i n 1 9 7 6 , and i s r e p r e s e n t e d by t h e f o l l o w i n g s t r u c t u r e .
m-
CH3-CH (OH 1
S-Cb- C e N H 2
0
COOH
Thienamycin 3.3.2
P r o d u c i n g organism
Thienamycin i s produced by StaeptomyceA c a t t l e y a , i s o l a t e d from a s o i l sample. I t i s a new a c t i n o m y c e t e , and h a s been d e s i g n a t e d a s MA 4 2 9 7 . 3.3.3
Biological a c t i v i t y
Thienamycin i s a c t i v e a g a i n s t b o t h g r a m - p o s i t i v e and gramn e g a t i v e b a c t e r i a , i n c l u d i n g Pdeudomonaa aeRuginoaa, S. ae&ueal P . m i k a b i l i b , E. coli, K . pneumoniac, and Entenobacten c l o a c a e .
411 3.3.4
P h y s i c a l and chemical p r o p e r t i e s
T h i e n a m y c i n i s a w h i t e s o l i d , s o l u b l e i n water, a n d h a s a l i m i t e d s o l u b i l i t y i n methanol. The e m p e r i c a l f o r m u l a o f t h i e n a m y c i n i s C l l H 1 6 N 2 0 4 S as d e d u c e d from h i g h r e s o l u t i o n mass spectroscopy. Thienamycin h a s a s p e c i f i c o p t i c a l rotation of f u I z 7 = + 82.7 ( C = O . l % , w / v i n a 1 0 mM a q u e o u s p h o s p h a t e b u f f e r , pH g . 9 ) . The u l t r a v i o l e t a b s o r p t i o n s p e c t r u m o f t h e i n a m y c i n t a k e n i n a q u e o u s s o l u t i o n i n t h e pH r a n g e o f 4 t o 8 h a s a p e a k a t 2 9 6 . 5 nm ( E l % 2 9 0 ) a n d a t r o u g h a t 242 nm ( E % = 8 8 ) . S p e c t r a t a k e n f h % e d i a t e l y a f t e r making a s o l u t i o n h"pH 2.0 show a r e d s h i f t i n t h e a b s o r p t i o n maxima t o 309 nm, a n d s p e c t r a t a k e n f o l l o w i n g a d j u s t m e n t t o pH 1 2 show a r e d s h i f t of t h e a b s o r p t i o n maxima t o 3 0 0 . 5 nm. Thienamycin i s a i n t e r n a l l y n e u t r a l i z e d a m p h o t e r i c s u b s t a n c e whose a c i d i c f u n c t i o n h a s a pKa = 3 . 0 8 , when m e a s u r e d i n a p h o s p h a t e b u f f e r ( 3 0 mM). 3.3.5
Fermentation and i s o l a t i o n
A f t e r 53 h r of f e r m e n t a t i o n , t h e b r o t h f r o m Sa%Lp~OmyCeA caZ.t.te u ( 5 0 0 m l ) i s f i l t e r e d , a d j u s t e d t o pH 7 . 0 , a n d f r e e z e d r i e d t o y i e l d 1 0 . 7 g o f s o l i d . A 1 . 5 gm p o r t i o n of t h e s o l i d i s t a k e n up i n 25 m l of n - b u t y l a l c o h o 1 : w a t e r ( 1 : 9 9 ) . This s o l u t i o n (pH 7 . 0 ) i s a p p l i e d t o a 5 x 1 1 8 cm c o l u m n of B i o g e l P-2 ( 2 0 0 - 4 0 0 m e s h ) , w h i c h h a s p r e v i o u s l y b e e n e q u i l i b r a t e d w i t h n - b u t y l a l c o h o l : w a t e r , a n d e l u t e d w i t h t h e same s o l u t i o n a t a f l o w r a t e of 1 0 m l / m i n , c o l l e c t i n g a 650 m l f o r e r u n f o l l o w e d by 7 5 f r a c t i o n s o f 20 m l e a c h . The e f f l u e n t stream is m o n i t o r e d w i t h a r e c o r d i n g d i f f e r e n t i a l refractometer. E a c h f r a c t i o n is assayed for a n t i b a c t e r i a l a c t i v i t y . Thienamycin is found i n f r a c t i o n s 3 4 t h r o u g h 4 0 w i t h a maximum i n f r a c t i o n 3 7 . Ten ml o f f r a c t i o n 37 a r e f r e e z e - d r i e d t o y i e l d 20 mg o f s o l i d s . The s o l i d s o b t a i n e d a r e t a k e n u p i n 5 . 0 m l o f water f o r a s s a y . A s s a y p l a t e s are i n c u b a t e d o v e r n i g h t a t 28OC a n d , t h e r e s u l t s a r e shown b e l o w : Concentration
Zone size i n mm, against S. aUteu.4 ATCC 6538P
80 g/ml 40 g/ml 20 g/ml 10 g/ml
30 mm 25 mm
21 mm 17 mm
The a s s a y i s c a r r i e d o u t b y d i s c d i f f u s i o n a s s a y u s i n g S . au)Leud ATCC 6538P as t h e t e s t o r g a n i s m . 4. Literature c i t e d
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P e p t i d e A n t i b i o t i cs E r h a r d Gross S e c t i o n on M o l e c u l a r S t r u c t u r e En d o c r i n o l o g y and Rep r od u c t i on R e s e a r c h B r a n c h N a t i o n a l I n s t i t u t e o f C h i l d H e a l t h and Human Development N a t i o n a l I n s t i t u t e s of H e a l t h B e t h e s d a . Maryland
.
1
P e p t i d e s w i t h a . B - u n s a t u r a t e d a n d r e l a t e d amino a c i d s Introduction Nisin Producing organism 1.2.2 Chemistry 1.2.3 Biology 1.2. 3.1 Antibiotic properties., 1.2.3.2 E f f e c t on n e o n a t a l t i s s u e 1.2.3.3 E f f e c t on n e o p l a s t i c t i s s u e 1.2.4 E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n 1.3 Monodinitrophenylnisin 1.3.1 Preparative procedure 1.3.2 Chemistry 1.3.3 Biology 1.3.4 E x t r a c t i o n s e p a r a t i o n and p u r i f i c a t i o n . , 1.4 Subtilin 1.4.1 Producing s t r a i n 1.4.2 Chemistry 1.4.3 Biology 1.4.4 E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n 1.5 Cinnamycin Producing organism 1.5.1 1.5.2 Chemistry 1.5.3 Biology 1.5.4 E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n 1.6 Duramycin 1.6.1 Producing organism 1.6.2 Chemistry 1.6.3 Biology 1.6.4 E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n 1.7 Summary
1.1 1.2 1.2.1
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2
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.................................. 4 1 7 ......................................... 420 .......................... ................................... 44202 0 ..................................... ..................... 44 22 11 ................... 421 ................. ..... 4421 21 ........................ 426 ....................... 4 2 6 ................................... 4 2 6 ..................................... 426 ... 426 ...................................... 27 ............................ 4429 ................................... 4 2 9 .......................................... 430 430 .................................... 430 .......................... 430 ................................... 431 ..................................... 431 ..... 4431 ..................................... 31 .......................... 432 ................................... 432 ..................................... 433 ..... 433
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P e p t i d e s w i t h a @ - u n s a t u r a t e d amino a c i d u n r e l a t e d t o o t h e r r e s i d u e s i n t h e molecule 2.1 Telomycin 2.1.1 Producing organism 2.1.2 Chemistry 2.1.3 Biology E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n 2.1.4 Stendomycin 2.2 Producing organism 2.2.1 2.2.2 Chemistry 2.2.3 Biology 2.2.4 E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n
-
433
..................................... 434 .......................... 1r34 ................................... 434 ..................................... 434 ..... 434 ................................... 436 .......................... 436 ................................... 437 .......................................... 437 439
416 2
.
P e p t i d e s w i t h a . 8.unsaturated amino a c i d u n r e l a t e d t o o t h e r r e s i d u e s i n t h e molecule (continued) 2.3 Tuberactinomycins (Capreomycin; viomycin) 2.3.1 Producing organism 2.3.2 Chemistry 2.3.3 Biology 2.3.4 E x t r a c t i o n s e p a r a t i o n and p u r i f i c a t i o n
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3
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P e p t i d e s w i t h s e q u e n c e s of a l t e r n a t i n g D- a n d Lamino a c i d s 3.1 Introduction 3.2 G r a m i c i d i n A . B. a n d C 3.2.1 Producing organism 3.2.2 Chemistry 3.2.3 Biology 3.2.3.1 Antibiotic properties 3.2.3.2 E f f e c t on n e o n a t a l t i s s u e 3.2.3.3 E f f e c t on n e o p l a s t i c t i s s u e 3.2.3.4 E f f e c t on m i t o c h o n d r i a 3.2.3.5 E f f e c t on s p o r u l a t i o n 3.2.3.6 B i o s y n t h e s i s of t h e l i n e a r g r a m i c i d i n s 3.2.4 E x t r a c t i o n . s e p a r a t i o n and p u r i f i c a t i o n
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................................. ....................... ......................... .................................. .................................... ...................... .................. ................ ..................... ........................... ......
4
5
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S y n t h e t i c v a l i n e - g r a m i c i d i n s A. B. and C Introduction Chemistry Biology E f f e c t of L i p i d B i l a y e r s E x t r a c t i o n s e p a r a t i o n and p u r i f i c a t i o n Summary
4.1 4.2 4.3 4.4 4.5 4.6 -
Literature
439
440 440 441
441
442 442 442 444 444 444
446 446 446 446
447 447
................................. 450 450 .................................... ...................................... 451 ..................... 451 ............................................ 44 55 33 C i t e d ....................................457
.
417
1. P e p t i d e s w i t h a , 6 - U n s a t u r a t e d a n d R e l a t e d Amino A c i d s
1.1 I n t r o d u c t i o n The i s o l a t i o n i n 1 9 2 8 of a s u b s t a n c e f r o m S ~ U p ~ o c O c C u A $actidl w i t h i n h i b i t o r y p r o p e r t i e s on t h e g r o w t h o f s t r e p t o c o c c i and LactobacilluA bu4!gahicuA2 a n t e d a t e d t h a t o f p e n i cillin3 belongs name o f in, the
by one y e a r . The p r o d u c i n g s t r a i n of t h e m i c r o o r g a n i s m t o t h e L a n c e f i e l d Group N 4 , a f a c t r e f l e c t e d i n t h e t h e a n t i b i o t i c (NISIN = Group N I n h i b i t o r y S u b s t a n c e + t e r m i n a t i n g l e t t e r s i n names g i v e n a n t i b i o t i c s ) .
Thus f a r , n i s i n d i d n o t s h a r e t h e p r o m i n e n c e of p e n i c i l l i n as a n t i m i c r o b i a l a g e n t of c l i n i c a l s i g n i f i c a n c e . H i s t o r i c a l l y , t h i s may h a v e b e e n , i n p a r t , t h e r e s u l t of t h e r e p o r t e d p o o r s o l u b i l i t y o f t h e p e p t i d e . N i s i n d o e s , however, r e s e r v e f o r i t s e l f a d i s t i n c t r o l e as u n i q u e f o o d p r e s e r v a t i v e 5 i n European c o u n t r i e s and i n o t h e r p a r t s of t h e w o r l d , b u t n o t i n t h e United S t a t e s . P e n i c i l l i n w i l l be d i s c u s s e d e l s e w h e r e i n t h i s t r e a t i s e . NISIN - f a l l i n g i n t o a n o v e l class of p e p t i d e a n t i b i o t i c s w i t h unique s t r u c t u r a l f e a t u r e s w i l l be d e a l t w i t h h e r e .
-
N i s i n 6 i s t h e member of a g r o u p of p e p t i d e s c o n t a i n i n g t h e a , 6 - u n s a t u r a t e d amino a c i d s DEHYDROALANINE ( F i g u r e 1) a n d DEHYDROBUTYRINE ( F i g u r e 1) a n d / o r d e r i v a t i v e s o f t h e l a t t e r two i n t h e form of ( a ) t h e t h i o e t h e r amino a c i d s LANTHIONINE and
....
fIGUREL1.
,
. ..
The structure o f a,6-unsaturated amino acid8 ( R alanine; R = CH3: dehydrobutyrine).
H: dehydro-
6-METHYLLANTHIONINE ( F i g u r e 2 , n e x t p a g e ) , and ( b ) LYSINOThe amino a c i d c o m p o s i t i o n s ALANINE ( F i g u r e 3 , n e x t p a g e ) . g i v e n i n T a b l e 1 show t h e d i s t r i b u t i o n of t h e s e r e s i d u e s i n n i s i n , SUBTILIN’, C I N N A M Y C I N B and DURAMYCIN9. C i nn amy c i n a n d d u r amy c i n c o n t a i n a l s o 6 -HY DROXY AS PART I C A C I D ( T a b l e 1). A r e l a t i o n s h i p o f t h i s r e s i d u e w i t h i t s a,Bu n s a t u r a t e d c o u n t e r p a r t is n o t r e a d i l y t o b e d e n i e d .
I
HOOC~CH-BCH~S-BCH-"CH-COOH I I NH2
NH2
FIGURE 2 . The s t r u c t u r e o f t h e t h i o e t h e r amino acids l a n t h i o n i n e and b-methyllanthionine( R = C H 3 ) .
(R = H)
H
FIGURE 3.
Lysinolanine, formed by the a d d i t i o n o f t h e €-amino group o f l y s i n e across the double bond o f dehydroelanine, e.g. i n model e x p e r i m n t s with n i s i n end n i s i n fragments and p o s t u l a t e d f o r t h e b i o s y n t h e s i s i n cinnsmycin and duramycin.
The p r e s e n c e of a , B - u n s a t u r a t e d amino a c i d s i s n o t r e s t r i c t e d t o t h e p e p t i d e s a l r e a d y accounted f o r . I t is n o t r e s t r i c t e d e i t h e r t o t h e s e p e p t i d e s - and o t h e r s l i k e l y t o be found - as a c l a s s . The s i n g u l a r f e a t u r e of r e p r e s e n t a t i v e s of t h e group i s t h e p r e s e n c e of amino a c i d s l a n t h i o n i n e , Bmethyllanthionine lysinolanine t h a t are c h e m i c a l l y r e l a t e d t o a, B - u n s a t u r a t e d p r e c u r s o r s . T h i s r e l a t i o n s h i p e x t e n d s t o f o r t h e time b e i n g a t l e a s t as a p o s t u l a t e * : biosynthesis t h e l a n t h i o n i n e s and l y s i n o a l a n i n e are t h e p r o d u c t s of t h e a d d i t i o n of n u c l e o p h i l e s , s u c h as t h e s u l f h y d r y l group of c y s t e i n e ( F i g u r e 4, n e x t p a g e ) and t h e €-amino group of l y s i n e ( F i g u r e 3) across the d o u b l e bond of d e h y d r o a l a n i n e o r dehydrobutyrine.
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-
-
Nature produces also o t h e r p e p t i d e s w i t h t h e s e a,B-unsatu r a t e d amino a c i d s as w e l l as p e p t i d e s w i t h a , b - u n s a t u r a t e d
TABLE 1 The Amino A c i d Composition o f Peptides w i t h a,BUnsaturated and Related Amino Acid Nunber o f Residues Amino Acid Hydroxyaspartic A c i d Aspartic Acid Serine 6-Methyllant hioninea Lanthioninea Glutamic A c i d Proline Gly cine Alanine Valine Me t h i o n i n e Isoleucine Leucine Pheny 1a l m i n e Lysinoalaninea Lysine Hystidine Ammonia Arginine Tryptophan Dehy droalanine Dehydrobutyrine Total
Nisin
_ I _
Subtil i n
-
-
1
1
1
-
4 1
4 1 3 1
-
1 3 2 1 2 3 2
-
2
1 1
-
1 4 1
-
-
3 2 (4)
-
-
2
1
29
Cinnamycin
3
(6)
-
1 2 1 27
aLanthionine, 6 - b t h y l l a n t h i o n i n e , and l y s i n o a l a n i n e are counted as one r e s i d u e each. I n s t r u c t u r a l p r e s e n t a t i m s t h e c o n t r i b u t i n g amino acids are counted i n d i v i d u a l l y ( c f . for i n s t a n c e Figure 5 ) .
420
LANTHIONINE
Q P
P
Q
HOOC-CH-CH2- S - C h - C H - C O O H
I
I
NH I
Q P - SH +
HOOC-CH -C+ I NH*
P
a
HzC= C I
- COOH
NH I AC
CYSTEINE
DEHYDROALANINE
The addition o f cysteine t o dehydroalanine w i t h t h e formation o f lanthionine. Ac = a c y l or amino a c y l group; dehydroalenine with the unprotected amino g r o w w i l l decorpose i n the presence o f water t o give ammonia and p y r u v i c acid.
amino a c i d s o t h e r t h a n d e h y d r o a l a n i n e and d e h y d r o b u t y r i n e . These p e p t i d e s d i f f e r from t h e former group i n t h a t t h e r e i s no o b v i o u s r e l a t i o n s h i p between t h e a , B - u n s a t u r a t e d amino a c i d and o t h e r amino a c i d r e s i d u e s p r e s e n t . A s e l e c t a c c o u n t w i l l be g i v e n of r e p r e s e n t a t i v e s of t h i s t y p e of p e p t i d e a n r i b i o t i c . 1 . 2 Nisin -1 . 2 . 1 P r o d u c i n g organism
S t 4 ~ p t o c o c c u 4 l a c t i h , Lancef i e l d , Group N 1 * 4. 1.2.2
Chemistry
N i s i n i s a h e t e r o d e t i c p e n t a c y c l i c p e p t i d e 6 ( F i g u r e 5 ) of t h i r t y f o u r amino a c i d r e s i d u e s . D e t a i l s of t h e amino a c i d comp o s i t i o n are g i v e n i n T a b l e 1. Unique t o n i s i n i s t h e p r e s e n c e
42 1
o f n o f e w e r t h a n t h r e e r e s i d u e s of a , b - u n s a t u r a t e d a m i n o a c i d s , two o f them d e h y d r o a l a n i n e l O , a n d one d e h y d r o b u t y r i n e ’ l . The h e t e r o d e t i c r i n g s y s t e m s are b e l i e v e d t o r e s u l t f r o m t h e a d d i t i o n of s u l f h y d r y l groups a c r o s s t h e a , g - u n s a t u r a t i o n s i n dehydroalanine and dehydrobutyrine8. T h i s a d d i t i o n r e a c t i o n i s l i k e l y t o b e u n d e r t h e s t e r e o c h e m i c a l c o n t r o l o f an enzyme or a s y s t e m o f enzymes. The a - c a r b o n atoms of r e s i d u e s 3, 8 , 1 3 , 2 3 , a n d 2 5 ( F i g u r e 5 , n e x t p a g e ; l e f t hand h a l f of l a n t h i o n i n e and B - m e t h y l l a n t h i o n i n e s ; d e h y d r o a l a n i n e o r d e h y d r o b u t y r i n e i n t h e p r e c u r s o r f o r m ) are of t h e D - c o n f i g u r a t i o n . 1.2.3 Biology 1.2.3.1
Antibiotic Properties
Nisin is primarily e f f e c t i v e against gram-positive o r g a n i s m s , i n c l u s i v e of BacieeuA t u b Q & c u l o n i o . Great v a l u e h a s been a t t r i b u t e d t o i t s b a c t e r i c i d a l n a t u r e . S i n g l e i n j e c t i o n s were f o u n d t o be r e m a r k a b l y e f f e c t i v e a g a i n s t c e r t a i n s t r e p t o c o c c a l and c l o s t r i d i a l i n f e c t i o n s i n t h e mouse1’. In a field t r i a l a g a i n s t b o v i n e s t r e p t o c o c c a l and s t a p h y l o c o c c a l m a s t i t i s t h e p r e c e n t a g e of c u r e s was h i g h w i t h s i n g l e i n f u s i o n s of n i s i n in 0i113.
I t i s i m p o r t a n t t o b e a r i n mind t h e l e v e l o f p u r i t y o f n i s i n w i t h which t h e s t u d i e s r e c o r d e d a b o v e w e r e c o n d u c t e d . With t h e s t r u c t u r e o f t h e a n t i b i o t i c e s t a b l i s h e d a n d w i t h access t o a d v a n c e d t e c h n i q u e s o f p u r i f i c a t i o n , a more b r o a d l y based r e - e v a l u a t i o n of t h e a n t i m i c r o b i a l p r o p e r t i e s ought t o b e r e w a r d i n g . From s u c h s t u d i e s one o u g h t t o a l s o a r r i v e a t applicable toxicity data. 1.2.3.2
E f f e c t on N e o n a t a l T i s s u e
B a s e d on t h e c o n c e p t o f t h e i n t e r c e p t i o n o f , f o r i n s t a n c e , e s s e n t i a l s u l f h y d r y l groups , n i s i n and n o n e n z y m a t i c a l l y d e r i v e d f r a g m e n t s of n i s i n were t e s t e d f o r t h e i r e f f e c t on i n t r a u t e r i n e tissue. The p a r e n t m o l e c u l e a n d t h e H z N - t e r m i n a l f r a g m e n t s 6 ¶ 1 4 F i g u r e 5 ; r e s i d u e s 1 - 2 1 o b t a i n e d by c l e a v a g e w i t h cyanogen b r o m i d e 1 5; r e s i d u e 1 8 : g l y c i n e or h o m o s e r i n e ( l a c t o n e ) ; r e s i d u e 2 1 : homoserine ( l a c t o n e ) i n both f r a g m e n t s ) induce f e t a l r e s o r p t i o n i n r a b b i t s and r a t s a t v a r i o u s s t a g e s of g e s t a t i o n I 6 . I n h i b i t o r y e f f e c t s on i m p l a n t a t i o n were a l s o o b s e r v e d . The COOH-terminal f r a g m e n t d i d n o t p r o d u c e t h e same e f f e c t s , p o s s i b l y f o r r e a s o n s of f a s t p r o t e o l y t i c breakdown. 1.2.3.3
E f f e c t on N e o p l a s t i c T i s s u e
When t h e e f f e c t s o f n i s i n a n d n i s i n f r a g m e n t s on n e o n a t a l t i s s u e were o b s e r v e d ( v i d e s u p r a ) , t h e p a r e n t m o l e c u l e was a l s o t e s t e d on n e o p l a s t i c t i s s u e . N i s i n shows l i m i t e d g r o w t h i n h i b i t i o n on l e u c e m i a c e l l s i n mice. 1.2.4
E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
An improved method f o r t h e p r e p a r a t i o n o f n i s i n 1 7 r e s o r t s t o e x t r a c t i o n o f t h e a c i d i f i e d ( h y d r o c h l o r i c a c i d ; pH 1 . 8 - 2 . 0 ) g r o w t h medium a l m o s t s a t u r a t e d w i t h s o d i u m c h l o r i d e w i t h
F N
Po
COOH FIGURE 5 . The structure of n i s i n (top) End subt'ilin (bottom). ABA = aminobutyric a c i d ; M A dehydroalanine; DHB = dehydrabutyrine (6-mthyldehydroalanine); ALA-S-ALA = lanthionine; ABA-S-ALA = B-methyllanthionine. Centers of c h i r a l i t y a t a-carbon atom are o f the L-configuration with the exception o f those o f residues 3, 8 , 13, 23, and 25. The a-carbon atoms o f t h e s e residues show the D-configuration. The a-aminobutyric a c i d moieties of the B-methyllanthionine residues are o f t h e threo-form.
42 3 of broth). o r g a n i c s o l v e n t ( e . g . n - p r o p a n o l , 100 m l p e r 1 N i s i n i s s a l t e d o u t from t h e o r g a n i c l a y e r by t r e a t m e n t w i t h 0 . 1 N h y d r o c h l o r i c a c i d and e x c e s s s o d i u m c h l o r i d e . The material t o b e s u b j e c t e d t o c o u n t e r c u r r e n t d i s t r i b u t i o n ( v i d e i n f r a ) was o b t a i n e d from A p l i n E B a r r e t t , Y e o v i l , England. I t had been p r e p a r e d from u n s t a n d a r d i z e d N i s a p l i n ( p r o t e i n a c e o u s commercial p r o d u c t i n which n i s i n i s e n r i c h e d ) i n t h e f o l l o w i n g way. N i s a p l i n was s u s p e n d e d i n m e t h y l a l c o h o l and t h e pH a d j u s t e d to 2 . 0 by t h e a d d i t i o n of h y d r o c h l o r i c acid. A f t e r s t a n d i n g , t h e s o l i d s were c e n t r i f u g e d and d i s c a r d e d . F o l l o w i n g c o o l i n g and r e c e n t r i f u g i n g , an e q u a l volume o f c o o l e d a c e t o n e was added t o t h e c o o l e d s o l u t i o n . The p r e c i p i t a t e was s p u n down i n a c e n t r i f u g e c o o l e d t o - 1 5 O C . The i s o l a t e d mater i a l was d r i e d and g r o u n d . P r e p a r a t i o n s s o o b t a i n e d may b e s u b j e c t e d d i r e c t l y t o c o u n t e r c u r r e n t d i s t r i b u t i o n , P r i o r p a s s a g e o v e r S e p h a d e x 6-25 i n 0.2N a c e t i c a c i d removes a d d i t i o n a l p r o t e i n a c e o u s material of h i g h m o l e c u l a r w e i g h t . P r e p u r i f i c a t i o n on S e p h a d e x 6-25 o f t h e commercial p r o d u c t was i m p o r t a n t a t one time in-as-much as i t r e v e a l e d i m m e d i a t e l y t h e p r e s e n c e o f PYRUVYLLYSINE, t h e p r o d u c t t h a t r e s u l t s from t h e c l e a v a g e o f t h e amino a c i d r e s i d u e i n t h e penultimate p o s i t i o n of n i s i n . Chemical i n v e s t i g a t i o n s e s t a b l i s h e d t h a t r e s i d u e t o b e t h e f i r s t o n e l o of two d e h y d r o a l a n i n e s t o be s e e n i n n i s i n ' l . C o u n t e r c u r r e n t d i s t r i b u t i o n o f commercial p r o d u c t ( a f t e r p a s s a g e o v e r Sephadex 6 - 2 5 ] i n t h e s o l v e n t s y s t e m n - b u t a n o l : water: a c e t i c a c i d = 3 : 4 : 1 gave t h e d i s t r i b u t i o n p a t t e r n shown i n Figure 6 , n e x t page. The p r e s e n c e o f a t l e a s t f o u r compone n t s is i n d i c a t e d . The l e f t - h a n d b r a n c h of t h e c u r v e f o r t h e major component d i f f e r s from t h a t t o t h e r i g h t , i n d i c a t i n g t h e p r e s e n c e o f more t h a n one component. Keeping i n mind t h e p r e s e n c e of a , B - u n s a t u r a t e d amino a c i d s , t h e p r e s e n c e of cons t a n t l y s h i f t i n g e q u i l i b r a m u s t n o t be excluded. The a,@u n s a t u r a t e d amino a c i d s are c a p a b l e o f a d d i n g water across t h e double bond, a r e a c t i o n t h a t i s a c i d c a t a l y z e d . The s o l v e n t i s a c i d i c o u t of n e c e s s i t y s i n c e n i s i n i s n o t s o l u b l e a t pHv a l u e s c l o s e t o n e u t r a l and u n d e r a l k a l i n e c o n d i t i o n s . The t e t r a h e d r a l i n t e r m e d i a t e r e s u l t i n g from t h e a d d i t i o n o f water t o t h e a , B - u n s a t u r a t i o n i s u n s t a b l e a n d b r e a k s down w i t h t h e f o r m a t i o n o f amide and k e t o a c i d ( v i d e s u p r a t h e o c c u r r e n c e o f PYRUVYLCYSTINE i n commercial p r e p a r a t i o n of n i s i n ) . C o n s i d e r i n g t h e r e v e r s e r e a c t i o n , namely t h e a d d i t i o n of t h e amide f u n c t i o n a c r o s s t h e k e t o g r o u p i n a - k e t o c a r b o x y l i c a c i d s , e q u i l i b r i a a r e c o n c e i v a b l e t h a t o s c i l l a t e between a,@u n s a t u r a t e d amino a c i d s on t h e one hand and a m i d e s and k e t o a c i d s on t h e o t h e r . When m a t e r i a l r e p r e s e n t e d by t h e c r o s s - h a t c h e d area o f F i g u r e 6 was r e d i s t r i b u t e d i n t h e same s o l v e n t s y s t e m , t h e d i s t r i b u t i o n c u r v e of t h e l e f t - h a n d p a n e l o f F i g u r e 7 was o b t a i n e d ( n e x t p a g e ) . The t h e o r e t i c a l c u r v e ( d a s h e s ) d o e s n o t
424
TUBE NO.
FIGURE 6. Countercurrent d i s t r i b u t i o n o f n i s i n i n the solvent system The m a t e r i a l represented by the area butmo1:water:acetic a c i d = 3:4:1. o f crosaed l i n e s w a s r e d i s t r i b u t e d i n the sane sovlent system ( c f . Figure 7, next page).
match t h e e x p e r i m e n t a l c u r v e d u c t lacks homogeneity.
-
s t i l l i n d i c a t i n g t h a t t h e pro-
When a r e l a t i v e l y small q u a n t i t y of t h e p r o d u c t r e p r e s e n t e d by t h e d i s t r i b u t i o n c u r v e i n t h e l e f t - h a n d p a n e l of F i g u r e 7 was newly d i s t r i b u t e d i n t h e s o l v e n t s y s t e m n - b u t a n o l : w a t e r : a c e t i c a c i d = 3:4:1, t h e c u r v e i n t h e r i g h t - h a n d p a n e l of
Y25
PURIFICATION OF NlSlN 1.5
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CCDNISIN I (113-124) I I
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CCDNISIN (113-124) I 49-59
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FIGURE 7. Countercurrent d i s t r i b u t i o n o f t h e major component o f n i s i n . L e f t panel: R e d i s t r i b u t i o n o f t h e m a t e r i a l represented by t h e crosshatched area i n F i g u r e 6 . R i g h t panel: R e d i s t r i b u t i o n o f m a t e r i a l represented by t h e p a t t e r n i n t h e l e f t panel. Solvent system, butano1:water: a c e t i c a c i d = 3:4:1. Experimental curve: s o l i d l i n e ; t h e o r e t i c a l curve; dashed l i n e .
F i g u r e 7 was o b t a i n e d . While t h e o r e t i c a l a n d e x p e r i m e n t a l curve are now i n good a g r e e m e n t , t h i s i s l i k e l y t o m e r e l y b e t h e r e s u l t of i n a d e q u a c i e s i n a n a l y s i s n o l o n g e r c a p a b l e of d e t e c t i n g t h e p r e s e n c e of more t h a n one component.
42 6
The o b s e r v a t i o n s made r a i s e q u e s t i o n s a b o u t t h e u t i l i t y of t h e c r i t e r i a of c o u n t e r c u r r e n t d i s t r i b u t i o n t o e s t a b l i s h t h e HOMOGENEITY of s u b s t a n c e s . A r e t h e s e c r i t e r i a no l o n g e r v a l i d ? The a n s w e r i s u n e q u i v o c a l . The c r i t e r i a f o r homogeneity as d e r i v e d from c o u n t e r c u r r e n t d i s t r i b u t i o n d a t a , r e m a i n v a l i d and a p p l i c a b l e as b e f o r e . The c o n f r o n t a t i o n a r i s e s w i t h n o v e l s t r u c t u r a l f e a t u r e s , s u c h as t h o s e s e e n i n n i s i n . The m o l e c u l e may w e l l be p u r i f i e d t o a h i g h l e v e l o f h o m o g e n e i t y . However, i t s c h e m i c a l dynamics ( c f , t h e r e a c t i o n s and o s c i l l a t i n g e q u i l i b r i a d i s c u s s e d a b o v e ) are s u c h , t h a t o t h e r m o l e c u l a r e n t i t i e s w i t h d i f f e r e n t p h y s i c a l p r o p e r t i e s a r e c o n s t a n t l y being formed. I t i s h e r e , where one m u s t s e e k t h e r e a s o n s f o r t h e d i s c r e p a n c i e s s e e n i n t h e p a t t e r n s of c o u n t e r c u r v e n t d i s t r i b u t i o n . Adaptation t o t h i s s i t u a t i o n new a n d d i f f e r e n t as i t may be m u s t n o t be made d i f f i c u l t . These m o l e c u l e s w i t h p a r t i c u l a r f e a t u r e s of c h e m i c a l dynamics are a l s o u n i q u e i n t h e i r b i o l o g ical properties,
-
-
1 . 3 Monodinitrophenylnisin Monodini t r o p h e n y l a t e d n i s i n was g r e p a r e d t o e s t a b l i s h unequivocally the molecular of t h e a n t i b i o t i c by t h e p a r t i a l s u b s t i t u t i o n t e c h n i q u e 2 O. 1.3.1 Preparative procedure Monodini t r o p h e n y l n i s i n was s y n t h e s i z e d a c c o r d i n g t o t h e p r o c e d u r e o f B a t t e r s b y and C r a i g z 0 . A l i m i t e d amount of d i n i t r o p h e n y l a t i n g a g e n t ( f l u o r o d i n i t r o b e n z e n e ) was employed i n o r d e r to.secure t h e - l a r g e s t p o s s i b l e q u a n t i t y of p a r t i a l l y s u b s t i t u t e d product. 1. 3.2 C h e m i s t r y The d i n i t r o p h e n y l g r o u p i n monodini t r o p h e n y l n i s i n i s a t t a c h e d t o t h e €-amino g r o u p of t h e l y s i n e r e s i d u e i n p o s i t i o n 22 (Figure 5). T h i s a s s i g n m e n t i s b a s e d on t h e n o n p r o t e o l y t i c f r a g m e n t a t i o n o f m o n o d i n i t r o p h e n y l n i s i n w i t h cyanogen bromide 5, s e p a r a t i o n of t h e r e s u l t i n g f r a ments by c o u n t e r c u r r e n t d i s t r i b u t i o n , a n d amino a c i d a n a l y s i s f 6 . 1 . 3 . 3 Biology A n t i b i o t i c a c t i v i t i e s f o r m o n o d i n i t r o p h e n y l n i s i n are shown i n T a b l e 11, n e x t p a g e . Minimum i n h i b i t o r y c o n c e n t r a t i o n s compare f a v o r a b l y w i t h t h o s e r e p o r t e d f o r t h e p a r e n t m o l e c u l e of n i s i n . The i n c o r p o r a t i o n of t h e h y d r o p h o b i c d i n i t r o p h e n y l g r o u p a c t u a l l y t e n d s t o improve a n t i b i o t i c properties. T h i s i s p e r h a p s due t o f a c i l i t a t e d p e n e t r a t i o n o f t h e h y d r o p h o b i c e n v i r o n m e n t of t h e membranes of m i c r o b i a l organisms. 1 . 3 . 4 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
Monodini t r o p h e n y l n i s i n a n d b y - p r o d u c t s of t h e d i n i t r o p h e n y l a t i o n were i s o l a t e d from t h e r e a c t i o n m i x t u r e by p a s s i n g t h e same o v e r a Sephadex 6-25 column o p e r a t e d w i t h 0.2N a c e t i c
42 7
Table 2 A n t i b i o t i c A c t i v i t i e s o f Monodinitrcphenylnisin Minimum I n h i b i t o r y C m c e n t r a t i ons (mcg/ml)
Orqmism
Baci.!%A d u b ~ f i ATCC 6633
0.015
SancLna .&&a
0.0075
ATCC 9341
Stclphylococcub m e u b ATCC 116312 S.taphylococcu m e u ~DA 209P Stclphg,fococcuA u u ~ e u ~DA 2001
0.12
0.015 3.0
l D i n i t r o p h e n y l a t i o n a t t h e l y s i n e residue i n p o s i t i m 2 2 . 2 P e n i c i l l i n resistant s t r a i n . k d i u m : yeast beef b r o t h , Minimum I n h i b i t o r y pH 6.8; inoculum s i z e = l o 4 c e l l s / m l . Concentrations were g r e a t e r than 50 mcg/ml f o r S&tneptOCOCcub ~ DA 11, Edchenichia co& pyogenu C, S ~ e p t o c o c c upyogenu ATCC 10536, Pdeudomonad aeAug&ona ATCC 8689, Pneudomonad aenug&oda ATCC 8709, and K&b&eUa p n e u m o n h DA 20.
acid. Countercurrent d i s t r i b u t i o n ( F i g u r e 8 , next page) s e p a r a t e d m o n o d i n i t r o p h e n y l n i s i n from u n r e a c t e d n i s i n and p r o d u c t s of h i g h e r d e g r e e s of d i n i t r o p h e n y l a t i o n . A h i g h l e v e l of homogeneity i s i n d i c a t e d f o r m o n o d i n i t r o p h e n y l n i s i n ( c f . t h e a g r e e m e n t between e x p e r i m e n t a l and t h e o r e t i c a l c u r v e i n t h e l e f t panel of Figure 8 ) . With t h e s t r u c t u r e of n i s i n e s t a b l i s h e d and w i t h t e c h n i q u e s a v a i l a b l e t o f r a g m e n t t h e m o l e c u l e , i t i s now r e a s o n a b l e t o attempt molecular m o d i f i c a t i o n w i t h t h e i n t e n t i o n t o improve antibiotic activity Monodini t r o p h e n y l a t i on ( v i d e s u p r a ) and t h e r e s u l t a n t good a n t i b i o t i c c h a r a c t e r i s t i c s of t h e d e r i v a t i v e are an a l m o s t a c c i d e n t a l f i r s t step in t h i s direction.
.
-
-
The t o t a l s y n t h e s i s of n i s i n w i l l be f o r m i d a b l e . The approach chosen calls f o r t h e assembly of t h e i n d i v i d u a l r i n g s y s t e m s of t h e m o l e c u l e ( c f . F i g u r e 5 ) and t h e i r s u b s e q u e n t condensation. The s y n t h e s i s of Rin A of n i s i n ( c f . F i g u r e 5 ; r e s i d u e s 3 - 8 ) i s p r e s e n t l y underwayf1.
1.4 Subtilin and B - m e t h y l l a n t h i onine The de t e c t i on of l a n t h i o n i n e i n s u b t i l i n prompted an i n v e s t i g a t i o n aimed a t e s t a b l i s h i n g t h e p r e s e n c e o r a b s e n c e o f a , B - u n s a t u r a t e d amino a c i d s f r o m t h e The q u e s t i o n was a s k e d : Do p e p t i d e s o t h e r t h a n n i s i n i n w h i c h l a n t h i o n i n e and B - m e t h y l l a n t h i o n i n e are f o u n d a l s o c o n t a i n t h e a , g - u n s a t u r a t e d amino a c i d s d e h y d r o a l a n i n e and dehydrobutyrine
.
F h)
W
OD 250 mp
DINITROPHENYLATION OF NlSlN
OD 160mp
DINITROPHENYLATION OF NISI N
1.500
NlSlN
.oo(
I
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mono-DNP-NIS IN
am
FIGURE 8. The s e p a r a t i o n and p u r i f i c a t i o n of monodinitrophenylnisin by c o u n t e r c u r r e n t d i s t r i b u t i m . S o l v e n t Monodinitrophenylnisin is s e p a r a t e d from u n r e a c t e d n i s i n and system: n-butano1:water:acetic a c i d = 3:4:1. p r o d u c t s o f h i g h e r degrees o f d i n i t r c p h e n y l a t i m . Left p m e l : a n a l y s i s a t 250 nm a b s o r p t i o n due t o t h e presence of u,B-unsaturated amino acids a d t o a lesser e x t e n t t h e t h i o e t h e r b r i d g e s o f l m t h i o n i n e s ; right panel: a n a l y s i s a t 360 nm ( a b s o r p t i m o f t h e d i n i t r o p h e n y l group).
429 1 . 4 . 1 Producing s t r a i n
Baci&?un A u b t i t i A Z 5 . S u b t i l i n was f i r s t p r o d u c e d and named i n 1 9 4 4 2 5 when an e f f o r t was made t o d i s t i n q u i s h p e p t i d e l i k e p r o d u c t s f r o m B a c i t l u a h u b t i l i d from t h e t y r o t h r i c i n ( v i d e i n f r a ) of 8 a c i t t u A b h e u i A Z 6 . 1.4.2
Chemistry
The p r e s e n c e o f d e h y d r o a l a n i n e i n ~ u b t i l i nwas ~ ~e s t a b l i s h e d by ( a ) t h e r e l e a s e o f p y r u v y l l y s i n e upon t r e a t m e n t . o f t h e p e p t i d e w i t h hydrochloric a c i d i n g l a c i a l acetic a c i d , ( b ) t h e d e t e c t i o n of S - c a r b o x y m e t h y l c y s t e i n e i n h y d r o l y s a t e s of s u b t i l i n t h a t had been t r e a t e d w i t h a 6 0 0 - f o l d e x c e s s o f m e t h y l m e r c a p t o a c e t a t e f o r t h i r t e e n d a y s a t room t e m p e r a t u r e . The r e l e a s e of p y r u v y l l y s i n e from s u b t i l i n r e v e a l e d i d e n t i t y f o r The t h e l a s t two amino a c i d r e s i d u e s i n n i s i n and s u b t i l i n . p e n u l t i m a t e and COOH-terminal p o s i t i o n a r e o c c u p i e d by DEHYDROALANINE a n d l y s i n e , r e s p e c t i v e l y . The p r e s e n c e o f 1 . 7 r e s i d u e s o f S - c a r b o x y m e t h y l c y s t e i n e i n t h e h y d r o l y s a t e of s u b t i l i n t o which m e t h y l m e r c a p t o a c e t a t e had been a d d e d , g a v e a f i r s t i n d i c a t i o n of t h e p r e s e n c e of more t h a n one r e s i d u e o f de hy d r o a l a n i n e i n s ub t i l i n
.
T h a t t h e r e a r e , i n d e e d , two r e s i d u e s o f d e h y d r o a l a n i n e p r e s e n t i n s u b t i l i n was e s t a b l i s h e d by a d d i t i o n o f b e n z y l m e r c a p t a n t o t h e a n t i b i o t i c f o l l o w e d by h y d r o l y s i s and amino a c i d analysis2’. The same s t u d y r e v e a l e d a l s o t h e p r e s e n c e of DEHYDROBUTYRINE. The b e n z y l m e r c a p t a n a d d i t i o n p r o d u c t s of dehydroalanine and dehydrobutyrine as w e l l as t h e d i a s t e r e o i s o m e r s of t h e l a t t e r are r e a d i l y s e p a r a t e d on t h e i o n - e x change columns of t h e amino a c i d a n a l y z e r . A t t h i s s t a g e o f t h e i n v e s t i g a t i o n i d e n t i c a l number of d e h y d r o a l a n i n e ( t w o r e s i d u e s ) and d e h y d r o b u t y r i n e ( o n e r e s i d u e ) were shown t o b e p r e s e n t i n n i s i n and s u b t i l i n Z 7 .
-
-
The amino a c i d c o m p o s i t i o n of s u b t i l i n Z 8 ( c f . T a b l e 1) provided t h e information necessary t o select approaches f o r t h e f r a g m e n t a t i o n of t h e m o l e c u l e . C l e a v a g e with t r y p ~ i n( c~f .~ F i g u r e 5 , t h e p e t i d e bonds between r e s i d u e s 2 - 3 and 2 9 - 3 0 ) , and t h e r m o l y s i n Z 9 ( c f . F i g u r e 5 , t h e p e p t i d e bonds between r e s i d u e s 1 9 - 2 0 , 1 5 - 1 6 , and 1 1 - 1 2 ) g a v e f r a g m e n t s t h a t were s u i t a b l e f o r s e q u e n c e d e t e r m i n a t i o n and t h e a s s i g n m e n t of t h e s u l f i de b r i d g e s 30 , l . The s t r u c t u r e o f SUBTILIN ( F i g u r e 5 ) shows f e a t u r e s r e m a r k a b l y r e m i n i s c e n t of t h o s e of n i s i n . The s i z e s of Rings A t h r o u g h E ( l e f t t o r i g h t ) are i d e n t i c a l . Rings B ( r e s i d u e s 8 - 1 1 ) are i d e n t i c a l i n amino a c i d c o m p o s i t i o n . The d e h y d r o a l a n i n e r e s i d u e s occupy i d e n t i c a l p o s i t i o n s i n b o t h m o l e c u l e s . DEHYDROBUTYRINE, l o c a t e d i n p o s i t i o n 2 i n n i s i n i s f o u n d i n t h e endo-cyclic p o s i t i o n 1 8 i n s u b t i l i n . The a - c a r b o n atoms o f r e s i d u e s 3, 8 , 1 3 , 2 3 , a n d 25 a r e of t h e D - c o n f i g u r a t i o n . This u n i q u e r e g u l a r i t y i n c h i r a l e x p r e s s i o n was e a r l i e r c o r r e l a t e d w i t h a s p e c t s of t h e b i o s y n t h e s i s of n i s i n and s u b t i l i n ( v i d e s u p r a ; c f . r e f . 8).
430 1.4.3
Biology
I n m i c r o b i o l o g i c a l a s s a y s f o r ~ u b t i l i n ~t h~e , p e p t i d e was found t o be a c t i v e a g a i n s t MichOCoccua conglomehatua, S t & t p t o coccuo d a e c a l i a and S t a p h y l o c o c c u a auhCua. S u b t i l i n d i d n o t i n h i b i t t h e growth of E . t y ~ h i ~I n~ a. c o m p a r a t i v e s t u d y w i t h t y r o t h r i c i n ( v i d e i n f r a ) , whole c u l t u r e s of B a c i Q l u a a u b t i l i a showed e x c e p t i o n a l l y h i g h a c t i v i t y a g a i n s t Phytomonaa m i c h i g anenaia 3 3 . 1.4.Q
E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
S u b t i l i n may be i s o l a t e d from whole c u l t u r e by e x t r a c t i o n
From the l a t t e r w i t h e i t h e r aqueous e t h a n o l 3 4 o r n - b ~ t a n o l ~ ~ . s o l v e n t , t h e a n t i b i o t i c is e x t r a c t e d i n t o 1%aqueous a c e t i c a c i d f o l l o w i n g t h e a d d i t i o n of o r g a n i c s o l v e n t , f o r i n s t a n c e , p e t r o l e u m e t h e r , t o t h e aqueous n - b u r a n o l e x t r a c t . Up t o 8 5 - 9 0 % of a n t i b i o t i c a c t i v i t y i n t h e n - b u t a n o l l a y e r is removed i n t h i s way. S u b t i l i n i s then p r e c i p i t a t e d o u t of t h e 1% aqueous a c e t i c a c i d e x t r a c t by t h e a d d i t i o n of sodium c h l o r i d e t o a c o n c e n t r a t i o n o f 1 0 % . Direct p r e c i p i t a t i o n of s u b t i l i n by s a t u r a t i n g t h e n - b u t a n o l l a y e r ( a d j u s t e d t o pH 5 ) w i t h sodium c h l o r i d e h a s a l s o been r e p o r t e d 3 6 . For a d e t a i l e d s t u d y t o f u r t h e r c h a r a c t e r i z e s u b t i l i n , t h e a n t i b i o t i c was e x t e n s i v e l y p u r i f i e d by c o u n t e r c u r r e n t d i s t r i b ~ t i o n ~ With ~ . samples of t h e same h i s t o r y comparable r e s u l t s were o b t a i n e d i n two s o l v e n t s y s t e m s : ( a ) 2 0 % a c e t i c a c i d : n - b u t a n o l = 5:4, ( b ) 0 . 1 % a c e t i c a c i d : n - b u t a n o l : 33:18:6. A sample t h a t had been s t o r e d f o r s e v e r a l pyridine y e a r s gave a complex d i s t r i b u t i o n p a t t e r n . I n r e t r o s p e c t , t h i s i s n o t s u r p r i s i n g i n view of t h e r e a c t i v e s t r u c t u r a l f e a t u r e s now e s t a b l i s h e d f o r s u b t i l i n ( v i d e s u p r a ) . Samples o f s u b t i l i n f o r s t r u c t u r a l s t u d i e s were p u r i f i e d by c o u n t e r c u r r e n t d i s t r i b u t i o n i n t h e s o l v e n t s y s t e m , b u t a n o l : w a t e r : a c e t i c a c i d = 3:4:116.
1 . 5 Cinnamycin Cinnamycin was d i s c o v e r e d i n t h e c o u r s e of a s e a r c h f o r a n t i b i o t i c s w i t h a c t i v i t i e s d i r e c t e d a g a i n s t C l o a t a i d i u m bo-tu~. was t h e tinurn and Mycobactekium t ~ b e h c u l o a i a ~ Cinnamycin f i r s t a c t i n o m y c e t e p o l y p e p t i d e a n t i b i o t i c found t o c o n t a i n s u l f i d e amino a c i d s . S i n c e t h e l a t t e r had been i d e n t i f i e d t o b e l a n t h i o n i n e and t h e C7Hl,+N204S amino a c i d of s u b t i l i n ( v i d e s u p r a ; s u b s e q u e n t l y shown t o be B - m e t h y l l a n r h i o n i n e ) , i t was of i n t e r e s t t o i n v e s t i g a t e cinnamycin f o r t h e p o s s i b l e p r e s e n c e of a , b - u n s a t u r a t e d amino a c i d s 1 6 . 1.5.1
P r o d u c i n g organism
StheptomyceA cinnamoneua 3 8 , The StheptomyceA c u l F u r e s employed i n e a r l y s t u d i e s of cinnamycin rese.mbled S . gk.ueoCahneua t o a d e g r e e , b u t were r e a d i l y d i f f e r e n t i a t e d from t h e l a t t e r . The name g i v e n t h e p r o d u c i n g s t r a i n , r e f l e c t s t h e change of t h e w h i t e a e r i a l mycelium o f young c u l t u r e s t o a l i g h t cinnamon c o l o r .
431 1.5.2
Chemistry
T e s t s f o r t h e p r e s e n c e of a , b - u n s a t u r a t e d amino a c i d s were n e g a t i v e when c i n n a m y c i n was e x p o s e d t o 0 . 1 N h y d r o c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d and when t h e a d d i t i o n o f m e r c a p t a n was a t t e m p t e d ( c f . t h e p r o d u c t s f r o m t h e s e r e a c t i o n s when c a r r i e d o u t w i t h n i s i n a n d s u b t i l i n ) . The amino a c i d s a n a l y s i s revealed t h e presence of residues previously not i d e n t i f i e d i n cinnamycin a n d / o r h e r e t o f o r e n o t observed t o o c c u r n a t u r a l l y ( c f . T a b l e 1). T h e r e are p r e s e n t i n c i n n a m y c i n one r e s i d u e of l a n t h i o n i n e a n d two r e s i d u e s of 5 - m e t h y l l a n t h i o n i n e . The d i a s t e r e o i s o m e r s of h y d r o x y a s p a r t i c a c i d are b o t h f o u n d i n t h e h y d r o l y s a t e of cinnamycin, t h e threo-form b e l i e v e d t o a r i s e from changes i n c h i r a l i t y d u r i n g h y d r o l y s i s . LYSINOALANINE ( F i g u r e 3) i s t h e amino a c i d s e e n i n c i n n a m y c i n f o r t h e f i r s t time t o o c c u r n a t u r a l l y 6. Cinnamycin and duramycin ( v i d e i n f r a ) are c l o s e l y related. One d i f f e r e n c e a p p a r e n t a t t h i s time i s t h e p r e s e n c e of a r g i n i n e i n c i n n a m y c i n ,where t h e r e i s l y s i n e i n d u r a m y c i n . The s t r u c t u r a l e l u c i d a t i o n o f b o t h p e p t i d e s i s s t i l l i n p r o g r e s s 1 6 . A d d i t i o n a l d a t a on t h e i r c h e m i c a l c h a r a c t e r i z a t i o n w i l l be d i s c u s s e d i n t h e s e c t i o n c o v e r i n g d u r a m y c i n . 1.5.3
Biology
Cinnamycin i n h i b i t e d t h e g r o w t h o f C l o h t a i d i u m a t c o n c e n t r a t i o n s o f 0 . 0 8 5 m ~ g / m l ~ The ~ . inhibitory t i o n f o r Mycobact'LAium tubeAcuCoAiA was 5 . 2 mcg/ml. c o n c e n t r a t i o n s i n h i b i t e d t h e growth of g r a m - p o s i t i v e
botulinum
concentraComparable r o d s , e .g. % a C i e l ? U h C ' L h ' L U 4 , 8aCieeuh CihCUfAnA, BaciCluh mQgaeOAiUm, a n d B a c i U u ~h u b t . L l i d . The a n t i b i o t i c i s l e s s e f f e c t i v e a g a i n s t g r a m - n e g a t i v e r o d s , a c i d - f a s t r o d s , g r a m - p o s i t i v e c o c c i , and yeasts
.
1.5.4
E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
C u l t u r e ' s of StAeptomyc'Lh CinnUmOneUA were c e n t r i f u g e d u s i n g c e l i t e as f i l t e r a i d . The s u p e r n a t a n t was p a s s e d t h r o u g h columns p a c k e d w i t h I R C - 5 0 c a t i o n e x c h a n g e r e s i n (Rohm and Haas) and t h e a n t i b i o t i c a l l y a c t i v e m a t e r i a l e l u t e d w i t h 0 . 1 N h y d r o c h l o r i c a c i d . P a s s a g e o v e r IR-48 r e s i n s (Rohn a n d Haas) was f o l l o w e d by c h r o m a t o g r a p h y on a l u m i n a (Harshaw 1 e l u t i n g w i t h 8 0 % a q u e o u s m e t h a n o l 3 8 . Cinnamycin h y d r o c h l o r i d e f r o m c h r o m a t o g r a p h y on a l u m i n a was f u r t h e r p u r i f i e d by c o u n t e r c u r r e n t d i s t r i b u t i o n 3 9 . The s o l v e n t s y s t e m was n - b u t a n o l w i t h a v o l a t i l e 0 . 2 N ammonium a c e t a t e b u f f e r o f pH 5 . 4 . Two m a j o r comp o n e n t s were s e p a r a t e d . One of them ( F a c t o r 1) r e m a i n e d i n t h e s t a r t i n g r u b e s of t h e d i s t r i b u t i o n , showed a s p e c t r u m of a n t i b i o t i c a c t i v i t y similar t o t h a t of cinnamycin b u t n o e f f e c t on C io Ath i di um bot ul i num . Cinnamycin f rom c o u n t e r c u r r e n t d i s t r i b u t i o n i s however, e f f e c t i v e a g a i n s t t h i s microo r g a n i s m a t c o n c e n t r a t i o n s of 0 . 5 - 1 . 0 mcglml.
1 . 6 Duramycin The i s o l a t i o n of c i n n a m y c i n was s o o n f o l l o w e d by t h a t o f a c l o s e l y r e l a t e d p o l y p e p t i d e w i t h promising a n t i b i o t i c prop-
432
e r t i e s . The new p r o d u c t was named DURAMYCIN f o r r e a s o n s of i t s s t a b i l ity40. 1.6.1
P r o d u c i n g organism
Duramycin was i s o l a t e d from Stneptomyced cinnamaneud forma a z a c o l u t a , an o r anism known t o produce f a c t o r s a c t i v e a g a i n s t p l a n t d i s e a s e s f 1 . S t u d i e s conducted w i t h c u l t u r e f i l t r a t e s i n d i c a t e d s t a b i l i t y of t h e a c t i v e p r i n c i p l e t o h e a t i n t h e pH-range from 3 t o 9 . 1.6.2
Chemistry
P e p t i d e s s u c h as duramycin and cinnamycin are r a t h e r r e s i s t a n t t o p r o t e o l y t i c d i g e s t i o n . The p r e s e n c e o f l y s i n e and p h e n y l a l a n i n e i n b o t h p e p t i d e s s u g g e s t e d t h e u s e of t r y p s i n and chymotrypsin f o r f r a g m e n t a t i o n p u r p o s e s . N e i t h e r of t h e p r o teases a t t a c k s cinnamycin o r duramycin, n o t even under f o r c e d c o n d i t i o n s t h a t leave t h e p r o t e o l y t i c a c t i v i t i e s i n t a c t . Thermoiysin , t h e p r o t e a s e s t a b l e a t e l e v a t e d t e m p e r a t u r e s ( 6 0 - 8 0 C) gave f r a g m e n t s t h a t a r e u s e f u l f o r s t r u c t u r a l i n v e s t i g a t i on. I n v i e w of t h e a b s e n c e of e f f e c t i v e means t o degrade cinnamycin o r duramycin p r o t e o l y t i c a l l y , nonenzymatic methods of p e p t i d e bond c l e a v a g e had t o be employed. The p r e s e n c e of a s p a r t i c acid offered the opportunity f o r treatment with d i l u t e a c i d a t e l e v a t e d t e m p e r a t u r e and p o t e n t i a l l y t h e c l e a v a g e of b o t h p e p t i d e bonds of t h i s amino a c i d 4 2 . H y d r o x y a s p a r t i c b e i n g a l s o p r e s e n t , t h e q u e s t i o n a r o s e whether i t s p e p t i d e bonds w i l l a l s o be c l e a v e d ? When duramycin, f o r i n s t a n c e , was t r e a t e d w i t h 0.03N h y d r o c h l o r i c a c i d a t l l O ° C f o r 5 4 h o u r s , t h i s o b s e r v a t i o n was m a d e 9 y 1 6 ; a s p a r t i c a c i d , h y d r o x y a s p a r t i c a c i d , and g l y c i n e were l i b e r a t e d i n e q u i m o l a r q u a n t i t i e s as shown by amino a c i d a n a l y s i s . Under i d e n t i c a l c o n d i t i o n s t h e same amino a c i d s were a l s o r e l e a s e d i n e q u a l amounts from cinnamycin16. T h i s answered t h e q u e s t i o n posed b e f o r e i n t h e a f f i r m a t i v e and t h e d a t a may be i n t e r p r e t e d i n t h i s way: s i n c e a s p a r t i c a c i d and h y d r o x y a s p a r t i c a c i d a r e b o t h r e l e a s e d , g l y c i n e had t o be l o c a t e d between t h e two d i c a r b o x y l i c monamino a c i d s i n t h e sequence of t h e p e p t i d e c h a i n . The f o l l o w i n g o r d e r of t h e t h r e e amino a c i d s i s i n agreement w i t h a d d i t i o n a l d a t a 1 6 from sequence d e t e r m i n a t i o n s : -OH-ASP-GLY-ASP-.
The s t r u c t u r a l e l u c i d a t i o n of duramycin i s s t i l l i n p r o g r e s s . A t t h i s s t a g e of t h e i n v e s t i g a t i o n t h e o v e r a l l s t r u c t u r e of t h e molecule p r e s e n t i t s e l f a s t h a t of a h e t e r o d e t i c p e n t a c r y l i c p e p t i d e : t h r e e r i n g s t r u c t u r e r e s u l t from t h e f o r m a t i o n of t h e t h i o e t h e r b r i d g e s of l a n t h i o n i n e and t h e two r e s i d u e s of B - m e t h y l l a n t h i o n i n e ; two a d d i t i o n a l r i n g s y s t e m s are formed !hen t h e l y s i n o a l a n i n e b r i d g e a r i s e s from t h e a d d i t i o n of t h e NH2-group of t h e COOH-terminal l y s i n e r e s i d u e a c r o s s t h e d o u b l e bond of a r e s i d u e of d e h y d r o a l a n i n e i n a p o s i t i o n i n t h e p e p t i d e c h a i n t h a t i s n o t t h e p e n u l t i m a t e one. What h a s been s a i d a b o u t s t r u c t u r a l f e a t u r e s of duramycin , a p p l i e s t o cinnamycin i n l a r g e measure 6.
433
1.6.3
Biology
Duramycin was i n i t i a l l y f o u n d t o be a c t i v e a g a i n s t gramp o s i t i v e r o d s , c e r t a i n y e a s t s , and f u n g i ' + O . A more e x t e n s i v e s t u d y i n c l u d e d t h i r t y - f i v e p h y t o p a t h o g e n s , e l e v e n o f which were i n h i b i t e d by c o n c e n t r a t i o n s of duramycin o f l e s s t h a n 1 0 0 mcg/ml 43. Duramycin was b a c t e r i c i d a l f o r BaciICun o u b t i l i a a t conc e n t r a t i o n s of 0 . 2 mcg/ml. The a n t i b i o t i c d i s p l a y s marked a c t i v i t y a g a i n s t t h e a n a e r o b e C l o a O i d i u r n botulinurn, a concent r a t i o n of 0 . 0 5 mcg/ml r e s u l t i n g i n c o m p l e t e i n h i b i t i o n . 1.6.4
E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
Duramycin was e x t r a c t e d w i t h 1 - b u t a n o l from t h e f i l t r a t e of t h e f e r m e n t a t i o n b r o t h . A d d i t i o n of h e p t a n e t o t h e b u t a n o l e x t r a c t c a u s e d t h e s e p a r a t i o n of a water l a y e r c o n t a i n i n g most of t h e a n t i b i o t i c a c t i v i t y . Crude duramycin i s o l a t e d f r o m t h e a q u e o u s l a y e r a f t e r e v a p o r a t i o n in v a c u o a n d l y o p h i l i z a t i o n was s u b j e c t e d t o c h r o m a t o g r a p h y on a l u m i n a e l u t i n g w i t h 80% a q u e o u s methanol. C o u n t e r c u r r e n t d i s t r i b u t i o n s were c a r r i e d o u t i n s e p a r a t o r y f u n n e l s w i t h t h e s o l v e n t s y s t e m 2 - b u t a n o l a n d 0.01N ammonium h y d r o x i d e o r i n t h e a u t o m a t e d e q u i p m e n t of C r a i g and P o s t 4 ' + u s i n g a s o l v e n t s y s t e m c o n s i s t i n g of 1 - b u t a n o l and S o l u t e d i s t r i b u t i o n followed ammonium a c e t a t e (0.2M; pH 5 . 3 5 ) . g r a v i m e t r i c a l l y a n d a n t i b i o t i c a c t i v i t y were i n good a g r e e m e n t .
1 . 7 Summary N i s i n , s u b t i l i n , c i n n a m y c i n a n d d u r a m y c i n are p e p t i d e a n t i b i o t i c s w i t h u n i q u e n o v e l s t r u c t u r a l f e a t u r e s . The f o u r p e p t i d e s have i n common t h e p r e s e n c e of a , B - u n s a t u r a t e d a n d / o r r e l a t e d amino a c i d s ( l a n t h i o n i n e , B - m e t h y l l a n t h i o n i n e , l y s i n o a l a n i n e ) . The c h e m i s t r y o f t h e s e amino a c i d s i s now well e s t a b l i s h e d and molecular m o d i f i c a t i o n a n d s y n t h e s i s are f e a s i b l e . Beyond t h e a n t i m i c r o b i a l p o t e n c i e s of t h e f o u r p e p t i d e s , c e r t a i n o t h e r b i o l o g i c a l a c t i v i t i e s have a l r e a d y been demons t r a t e d f o r n i s i n and p a r t i a l s t r u c t u r e s of n i s i n ( v i d e s u p r a ) . These o b s e r v a t i o n s w a r r a n t f u r t h e r e x p l o r a t i o n a n d e x t e n s i o n of t h e s e i n v e s t i g a t i o n s t o s u b t i l i n , c i n n a m y c i n , a n d duramycin as w e l l as t o f r a g m e n t s of t h e s e p e p t i d e s . S t u d i e s on t h e r e l a t i o n s h i p between s t r u c t u r e and f u n c t i o n w i l l c o n t r i b u t e t o a b e t t e r understanding of t h e p r o p e r t i e s of t h e s e molecules i n t h e p h y s i o l o g i c a l environment. There is r e a s o n t o l o o k f o r w a r d t o a r o l e f o r t h e s e p e p t i d e s more s i g n i f i c a n t a t t h e l e v e l of t h e r a p y and i n t h e c l i n i c t h a n i t Was The p o s s i b i l i t y o f l i n k i n g t h e s t r u c t u r a l f e a in the past. tures considered here t o h i g h l y hydrophobic p e p t i d e s f o r ease of p e n e t r a t i o n of t h e l i p i d b i l a y e r o f t h e biomembrane - w l l l b e f u r t h e r d i s c u s s e d i n s e c t i o n 4.
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434 2 . P e p t i d e s w i t h a , @ - u n s a t u r a t e d Amino A c i d s U n r e l a t e d t o O t h e r Residues i n t h e Molecule N i s i n and s u b t i l i n c o n t a i n DEHYDROALANINE and DEHYDROBUTYRINE as w e l l as LANTHIONINE and 6-METHYLLANTHIONINE r e l a t e d t o t h e f o r m e r t w o by t h e a d d i t i o n of t h e s u l f h y d r y l g r o u p o f c y s t e i n e a c r o s s t h e d o u b l e bond i n t h e a , b - u n s a t u r a t e d amino acid. I n c i n n a m y c i n and duramycin one f i n d s o n l y t h e amino a c i d s r e l a t e d t o a,B-unsaturated precursor residues: Lanthionine @ - m e t h y l l a n t h i o n i n e , and LYSINOLANINE h e r e s e e n f o r t h e f i r s t time t o occur n a t u r a l l y . I n cinnamycin a n d duramycin t h e p r e c u r s o r r e s i d u e s - d e h y d r o a l a n i n e and dehydrobutyrine all participated i n addition reactions.
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There a r e o t h e r p o l y p e p t i d e a n t i b i o t i c s w i t h a , @ - u n s a t u r a t e d amino a c i d s t h a t are d i s t i n c t l y d i f f e r e n t from t h e p e p t i d e s a c c o u n t e d f o r above. I n t h e s e p e p t i d e s t h e r e a r e n o o t h e r amino a c i d r e s i d u e s w i t h an o b v i o u s r e l a t i o n s h i p s u c h as, f o r i n s t a n c e , t h e f o r m a t i o n of l a n t h i o n i n e or @ - m e t h y l l a n t h i o n i n e t o a , B - u n s a t u r a t e d amino a c i d s . One of t h e f i r s t r e p r e s e n t a t i v e s t o b e s t u d i e d w i t h i n t h i s c l a s s of p o l y p e p t i d e a n t i b i o t i c s was TELOMYCIN4 5.
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2 . 1 Telomycin The a , @ - u n s a t u r a t e d amino a c i d p r e s e n t i n t e l o m y c i n i s DEHYDROTRYPTOPHAN46. 2.1.1
Producing organism
S&tep2krnyceA s t r a i n t h a t r e m a i n s t o b e 2.1.2
Chemistry
Telomycin i s a h e t e r o d e t i c m o n o c y c l i c p e p t i d e ( F i g u r e 9 , n e x t p a g e ) . By d e f i n i t i o n , t h e a n t i b i o t i c may b e c o n s i d e r e d a d e p s i p e p t i d e , in-as-much as a r e s i d u e of c i s - 3 - h y d r o x y p r o l i n e i s l i n k e d t o t h e h y d r o x y l g r o u p of a t h r e o n i n e . Considering t e l o m y c i n i n l i n e a r form w i t h c i s - 3 - h y d r o x y p r o l i n e o c c u p y i n g t h e COOH-terminal p o s i t i o n , DEHYDROTRYPTOPHAN i s s e e n i n t h e penultimate position. B e s i d e s t h e a , @ - u n s a t u r a t e d amino a c i d ( d e h y d r o t r y p t o p h a n ) t h e s e rare amino a c i d s are f o u n d i n t e l o mycin: t r a n s - 3 - h y d r o x y p r o l i n e , 3 - h y d r o x y l e u c i n e , B - m e t h y l t r y p t o phan , c i s - 3 - h y d r o x y p r o l i n e ( v i d e s u p r a ; i n t h e COOH-terminal p o s i t i o n of t h e l i n e a r f o r m of t h e a n t i b i o t i c ) .
,
2 . l .3
Biology
Telomycin i s a c t i v e a g a i n s t g r a m - p o s i t i v e o r g a n i s m s , i n c l u s i v e of s t r a i n s of Michococcun pyogeneo v a r . Q U h e U A , t h a t are r e s i s t a n t t o o t h e r a n t i b i o t i c s 4 7 . The a n t i b i o t i c h a s l i t t l e or n o e f f e c t on g r a m - n e g a t i v e b a c t e r i a 4 ? . 2.1.4
E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
The p u r i t y o f t e l o m y c i n i s o l a t e d from c u l t u r e b r o t h of t h e p r o d u c i n g s t r a i n may be e s t a b l i s h e d by p a p e r e l e c t r o p h o r e s i s (pH 1 . 9 and 6 . 4 ) and t h i n l a y e r c h r o m a t o g r a p h y ( i s o p r o p y l alcohol 3% ammonia on s i l i c a g e l G ) . Telomycin u s e d i n s t r u c t u r a l s t u d i e s was p u r i f i e d by c o u n t e r c u r r e n t d i s t r i b u t i o n between an u p p e r p h a s e o f t e r t - b u t y l a l c o h o l a n d a l o w e r p h a s e of w a t e r c o n t a i n i n g 4 % sodium c h l o r i d e 4 6y 4 8 .
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FIGURE 9 . The s t r u c t u r e o f Telomycin. The a,b-unsaturated amino a c i d dehydrotryptophan i s i n th e p en ultimate p o s i t i o n o f tte l i n e a r f o r m of the a n t i b i o t i c . Here shown is t h e d i p s i p e p t i d e s t r u c t u r e 0-acyl bond between the carboxyl te r minal r e s i d u e of cis-3-hydroxyproline and threonine i n p o s i t i o n 3.
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436 Worthy of mention i n c o n n e c t i o n w i t h t e l o m y c i n a r e E C H I N U L I N 4 9 a n d t h e NEOECHINULINS A t h r o u g h ESD. These p e p t i d e s c o n t a i n a l s o d e h y d r o t r y p t o p h a n , t h e p r e s e n c e of which was f i r s t s u s p e c t e d when a c o m p a r i s o n was made between t h e UV-spectra of n e o e c h i n u l i n s and t e l o m y c i n S 0 . A r e p r e s e n t a t i v e s t r u c t u r e i s shown i n F i g u r e 1 0 f o r N e o e c h i n u l i n B S 1 which c o n r a i n s a l s o DEHYDROALANINE i n d e k e t o p i p e r a z i n e s t r u c t u r e w i t h t h e d e h y d r o tryptophan d e r i v a t i v e
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.Figure 10. The s t r u c t u r e of Neoechinulin 8. R=H. I n t h e 2 p o s i t i o n o f t h e i n d o l e nucleus a hydrogon is r e p l a c e d by t h e 1.1-dimethyl-prop-2-enyl gfoup. Dehydrotryptophan so d e r i v a t i z e d is i n d i k e t o p i p e r a z i n e s t r u c t u r e w i t h a r e s i d u e of dehydroalanine.
The f u n g a l mat o f A A p e R g i l l u d a m t e t o d a m i , grown on molasses b e e t c u l t u r e 8 was c o n t i n u o u s l y e x t r a c t e d w i t h l i g h t p e t r o l e u m (b.p. 6 0 - 8 0 C), d i e t h y l e t h e r , and c h l o r o f o r m . Ext r a c t s o b t a i n e d w i t h t h e l a t t e r two s o l v e n t s c o n t a i n e d , b e s i d e s l a r g e amounts o f e c h i n u l i n and o t h e r d e h y d r o t r y p t o p h a n comp o u n d ~ ~minor ~ , q u a n t i t i e s of t h e n e o e c h i n u l i n s A-E and n e o e c h i n u l i n . The d i k e t o p i p e r a z i n e s w e r e s e p a r a t e d by column c h r o m a t o g r a p h y on s i l i c a g e l u s i n g h e x a n e - e t h y l a c e t a t e as e l u a n t . P r i o r t o c r y s t a l l i z a t i o n t h e p e p t i d e s were f u r t h e r p u r i f i e d by p r e p a r a t i v e t h i n l a y e r c h r ~ m a t o g r a p h y ~ ~ . F o r r e a s o n s of i t s s t r u c t u r e ALBONOURSIN52 d e s e r v e s t o b e m e n t i o n e d h e r e . P r o d u c e d by two a c t i n o m y c e t e s , StRep.tomyceA a l b u d v a r . dungatun a n d SheptornyceA n o u A A e i , t h e a n t i b i o t i c c o n s i s t s of t h e two a , @ - u n s a t u r a t e d amino a c i d s d e h y d r o l e u c i n e and dehydrophenylalanine l i n k e d t o a d i k e t o p i p e r a z i n e structureS2.
2 . 2 Stendomycin This h e t e r o d e t i c monocyclic p e p t i d e c o n t a i n s t h e a,Bu n s a t u r a t e d amino a c i d d e h y d r o b u t y r i n e 53. 2.2.1
Producing organism
StJLeptomyced enduA5'+ a s t r e p t o m y c e t e i s o l a t e d f r o m s o i l . The i d e n t i t y of t h e p r o d u c i n g s t r a i n was a s c e r t a i n e d by comparison w i t h other streptomycetes.
437 2.2.2
Chemistry
Stendomycin d i s p l a y s t h e s t r u c t u r e ( F i g u r e 11, n e x t p a g e ) of a h e t e r o d e t i c m o n o c y c l i c p e p t i d e . Ring f o r m a t i o n o c c u r s v i a l a c t o n i z a t i o n between t h e c a r b o n x y l t e r m i n u s and a r e s i d u e of D-allo-threonine i n p o s i t i o n 9 of t h e molecule. Besides DEHYDROBUTYRINE ( p o s i t i o n 8 ) , t h e s e r a r e amino a c i d s are f o u n d i n stendomycin: N-methyl-L-threonine , D - a l l o - i s o l e u c i n e ( 2 r e s i d u e s ; p o s i t i o n s 5 and 131, D-allo-threonine ( 2 r e s i d u e s ; p o s i t i o n s 8 and 1 1 1 , and STENDOMYCIDINE55y56. T h e r e i s a t o t a l of s e v e n D-amino a c i d s i n t h e m o l e c u l e , t h r e e o f them i n s u c c e s s i v e p o s i t i o n s and s e p a r a t e d by d e h y d r o b u t y r i n e from two a d d i t i o n a l ones i n a d j a c e n t p o s i t i o n . Two of t h e D - r e s i d u e s a l t e r n a t e w i t h amino a c i d s o f t h e L - c o n f i g u r a t i o n ( F i g u r e 11). The H2N-terminus ( L - p r o l i n e ) i s a c y l a t e d by a f a t t y a c i d isomyristic acid. The t e r m i n a l COOH-group as i n d i c a t e d before i s i n l a c t o n e bond w i t h t h r e o n i n e i n p o s i t i o n 8 .
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STENDOMYCIDINE o c c u p i e s t h e COOH-terminal p o s i t i o n ( c f F i g u r e 11 f o r t h e s t r u c t u r e of t h i s amino a c i d ) . T h i s amino a c i d may b e c o n s i d e r e d a d e r i v a t i v e of a r g i n i n e w i t h t h e amino and w-ni t r o g e n of t h e g u m i d 0 g r o u p m e t h y l a t e d . Biosynthetically t h e 6-membered r i n g may be t h e r e s u l t of t h e a d d i t i o n of t h e w-ni t r o g e n f u n c t i o n a c r o s s t h e d o u b l e bond o f DEHYDROARGININE, a p o s t u l a t e s t a t i n g t h a t a s e c o n d a , g - u n s a t u r a t e d amino a c i d was p r e s e n t i n t h e m o l e c u l e a t one time. A t t h e same time t h e p o s t u l a t e s a y s a l s o t h a t s t e n d o m y c i d i n e i s r e l a t e d t o an a , b - u n s a t u r a t e d amino a c i d , h e r e , s o t o s a y , by way of an "intramolecular" r e a c t i o n , i .e. t h e nucleophile t h e w-nitrogen f u n c t i o n of d e h y d r o a r g i n i n e a d d i n g a c r o s s t h e d o u b l e bond was p r e s e n t i n t h e a , B - u n s a t u r a t e d amino a c i d i t s e l f . The e a r l i e r c l a s s i f i c a t i o n o f a , b - u n s a t u r a t e d and r e l a t e d amino a c i d s s p e c i f i e s t h e n u c l e o p h i l e t o be c o n t r i b u t e d by an amino a c i d o t h e r t h a n t h a t b e g r i n g t h e a , B - u n s a t u r a t i o n ( e . g . SH-groups of c y s t e i n e or NH2-group of l y s i n e ) .
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S i m i . l a r o b s e r v a t i o n s on c l o s e l y r e l a t e d s t r u c t u r e s were made w i t h t h e t u b e r a c t i n o m y c i n s ( v i d e i n f r a ) and p e p t i d e s r e l a t e d t o them.
A n t i m i c r o b i a l a c t i v i t i e s of s t e n d o m y c i n have been d e t e r mined on a s i z a b l e number of m i c r o o r g a n i s m s ( c f . for i n s t a n c e , Ref. 5 4 ) . S e l e c t e d lowest minimum i n h i b i t o r y c o n c e n t r a t i o n s mcg/ml a r e : b a c t e r i a , Mycobactesium avium: 6 . 2 5 ; f u n g i (dermat o p h y t e s ) ; EpideAmophyton ~ t o c c o o u m :0 . 7 8 ; f u n g i ( m i s c e l l a n e o u s ) ; PolypoJtuo c i n n a b a n i n u o : 0 . 7 8 . T e s t e d i n v i v o a g a i n s t human p a t h o g e n i c f u n g i 5 7 , s t e n d o m y c i n p r o d u c e d 5 0 % i n h i b i t i o n of t h e s e o r g a n i s m s a t t h e c o n c e n t r a t i o n s (mcg/ml) g i v e n : B . k A t O m y C e A d c n m a t i t i d i o , 0 . 4 6 ; Candida a l b i c a n o , 3.80 ; Cnypto C O C C U A neodonmano, 1.00; f f i o t o p t a o m a CapAU$atUm, 0 . 4 5 ; Spanothichum o c h e n e k i i , 0 . 3 2 . A t t w o - f o l d s e r i a l d i l u t i o n s from 0 . 2 1 0 0 mcg/ml s t e n d o m y c i n was f u n g i c i d a l t o C e n a t o o t o m e l l a u t m i , G l o m e n e l l a c i n g u t a , and PulJhlaAia A p . a t c o n c e n t r a t i o n s g r e a t e r
t h a n 3 . 1 3 mcg/ml.
FIGURE 11. The s t r u c t u r e o f Stendomycin. The sequence o f amino acids shown here i s t h a t o f the peptide m o s t ablndant ammg others o f the stendomycin family. I n other analogs o f the group i s o m y r i s t i c a c i d i s replaced by lower homologs and allo-isoleucine by v a l i n e or leucine. FA = f a t t y acid; L-Ste = Stmdomycidine.
439 2 0 2 * 4 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
A n t i f u n g a l a c t i v i t y i s o n l y found i n minute amounts i n f i l t e r e d b r o t h . F i l t e r cakes ( H y f l o Super-Cel used as f i l t e r a i d ) were d r i e d u n d e r reduced p r e s s u r e a t t e m p e r a t u r e s below 4 0 ° C and e x t r a c t e d w i t h benzene: methanol = 9:l. Following e v a p o r a t i o n of t h e s o l v e n t s , t h e v i s c o u s r e s i d u e was e x t r a c t e d w i t h methanol. The m e t h a n o l - i n s o l u b l e p o r t i o n was d i s c a r d e d . The methanol e x t r a c t was t r e a t e d r e p e a t e d l y w i t h N o r i t e and t h e carbon removed by f i l t r a t i o n . Stendomycin was r e c o v e r e d from t h e f i l t r a t e as a g l a s s y , cream c o l o r e d s o l i d by removal of t h e s o l v e n t under reduced p r e s s u r e . F u r t h e r p u r i f i c a t i o n of s t e n d o mycin i s p o s s i b l e by p r e c i p i t a t i o n w i t h sodium s u l f a t e from aqueous s o l u t i o n s . C o u n t e r c u r r e n t d i s t r i b u t i o n between methyl i s o b u t y l ketone and p h o s p h a t e b u f f e r ( 0 . 0 5 M ; pH 6 ) s e p a r a t e d two comp o n e n t s t h a t were named stendomycin A and B . The r a t i o o f component A t o B may v a r y from f e r m e n t a t i o n t o f e r m e n t a t i o n . The name stendomycin a p p l i e s t o t h e m i x t u r e of two c l o s e l y related antifungal antibiotics. 2 . 3 T u b e r a c t i n o m y c i n s ( Capreomycin ; v i omycin 1 The t u b e r a c t i n o m y ~ i n s(~F~i g u r e 12) c o n t a i n t h e a,Bu n s a t u r a t e d amino a c i d 3-ureidodehydroalanineb0. I n aqueous
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OH OH H
H
FIGURE 12. The s t r u c t u r e o f t h e tuberactinomycins, s u b s t i t u e n t s R, and R2 as i n d i c a t e d i n the t a b l e . S u b s t i t u e n t R 1 a t the y-carbon atom o f 8-lysine: the guanidine H o r a-l. S u b s t i t u e n t R2 i n the 4 p o s i t i o n o f t u b e r a c t i d i n e amino acid: H or OH. Tuberactinomycin B = viomycin; capreomycin: R, = H; R2 = OH. Serine n e x t t o 3-ureidodehydroalanine replaced by alanine. Desylsylviomycin (LL-BM547a) and t h e p e p t i d e (LL-BM547B) i n w h i c h 8 - l y s i n e i s replaced by BN-CH3-8-arginine have also been described5*.
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440
s o l u t i o n t h i s amino a c i d may u n d e r g o t h e c h a n g e s i n d i c a t e d i n F i g u r e 13. C a p r e o m y c i n 6 1 , viomycin ( = t u b e r a c t i n o m y c i n B * 6 2 - 6 4 , a n d t h e a n t i b i o t i c s LL-BM 5 4 7 B(I) a n d LL-BM 5 4 7 2 (11)58 a r e c l o s e l y r e l a t e d t o t h e t u b e r a c t i n o m y c i n s . The d i f f e r e n c e s are s e e n i n the e x c h a n g e of s u b s t i t u e n t i n amino a c i d s i d e c h a i n s or t h a t o f one amino a c i d r e s i d u e a t most ( c f . F i g u r e 1 2 ) . 2.3.1
Producing organism
T u b e r a c t i n o m y c i n was i s o l a t e d f r o m t h e f e r m e n t a t i o n b r o t h o f a s t r e p t o m y c e s named StAeptomycea gRideOveJcticik?tcLtuA v a r . t u b ~ t a c t i c u ~The ~ ~ same , a n t i b i o t i c was a l s o r e p o r t e d u n d e r t h e name t u b e r a c t i n 6 6 . The g e n e r i c name, t u b e r a c t i n o m y c i n , i s i n a c c o r d a n c e w i t h r u l e s e s t a b l i s h e d i n J a p a n for names of nonproprietary drugs.
2.3.2
Chemistry
The t u b e r a c t i n o m y c i n s ( F i g u r e 1 2 1 are h o m o d e t i c m o n o c y c l i c p l a c e d i n an e x o c y c l i c p e p t i d e s w i t h one amino a c i d - 8 - l y s i n e position. T h i s r e s i d u e of B - l y s i n e i s a t t a c h e d t o t h e a-amino g r o u p of a , d - d i a m i n o p r o p i o n i c a c i d . The y - c a r b o n atom of B l y s i n e may b e h y d r o x y l a t e d - 2 - h y d r o x y - B - l y ~ i n e ~ ~ - ~The ~ . cons tit u e n t a , B - u n s a t u r a t e d a m i n o a c i d 3-ureidodehydroalanine60 h a s b e e n t h e s u b j e c t of d e t a i l e d c h e m i c a l i n v e s t i g a t i o n . I n aqueous medium t h i s amino a c i d i s s u b j e c t t o h y d r o l y s i s w i t h t h e l o s s of t h e u r e i d o group and f o r m a t i o n of C-formylglycine ( c f . F i g u r e 1 3 1 , a r e a c t i v e amino a c i d d e r i v a t i v e i t s e l f 6 ' + .
-
I FIGURE 13. medium. I
P
m
Structural changes o f 3-ureidodehydroalanine in aqueous ureido form; I1 ureido- "imino" - form; I11 = C-formylglycine.
T u b e r a c t i d i n e 7 0 y 7 1 ( V i o m y c i n d i n e i n ~ i o m y c i n ~is~ )t h e g u a n i d i n o amino a c i d i n t h e t u b e r a c t i n o m y c i n s A a n d B ( F i g u r e 12). Capreomycidine i s p r e s e n t i n t h e t u b e r a c t i n o m y c i n s N and 0 (Figure 1 2 ) . The c h e m i s t r y of t h e s e d e h y d r o a r g i n i n e d e r i v e d compounds i s w e l l e s t a b l i s h e d 6 3 y 7 0y 7 1 ; v i d e s u p r a t h e r e l a t i o n s h i p t o stendomycidin and t h e d i s c u s s i o n t h e r e ) . The c y c l i c p o r t i o n s o f t h e t u b e r a c t i n o m y c i n s A a n d B ( B - l y s i n e m o i e t i e s removed) a r e known as T u b e r a c t i n a n i n e A , t h o s e of t h e t u b e r a c t i n o m y c i n s N and 0 as t u b e r a c t i n a n i n e N .
441
C o n v e r s i o n o f t u b e r a c t i n a m i n e N t o t h e t u b e r a c t i n o m y c i n s N and 0 h a s been a c c o m p l i s h e d 7 2 . The r e m o v a l o f t h e 8 - l y s i n e r e s i d u e s and r e a c y l a t i o n o f f e r a b r o a d r a n g e of s t r u c t u r a l m o d i f i c a t i o n s f o r t h e i n v e s t i g a t i o n of t h e b i o l o g i c a l c h a r a c t e r i s t i c s of t h e t u b e r ac t i n omy c i n s
.
T u b e r a c t i n o m y c i n 0 h a s been o b t a i n e d by t o t a l s y n t h e s i s 5 9 . 2.3.3
Biology
The t u b e r a c t i n o m y c i n s a r e a c t i v e a g a i n s t gram-pos i t i ve as w e l l as g r a m - n e g a t i v e b a c t e r i a . The g r o w t h of f u n g i and y e a s t s was n o t i n h i b i t e d . P a r t i c u l a r l y good e f f e c t s were s e e n i n s t u d i e s on t h e g r o w t h i n h i b i t i o n of m y c o b a c t e r i a a n d p a t h o genic tubercular b a c i l l i including s t r a i n s r e s i s t a n t to s t r e p t o mycin a n d kanamycin6 5. T u b e r a c t i n o m y c i n s were a l s o e f f e c t i v e a g a i n s t f r e s h l y i s o l a t e d s t r a i n s t h a t were h i g h l y r e s i s t a n t t o kanamycin. I n experimental t u b e r c u l o s i s i n guinea p i g s , tubera c t i n o m y c i n s were e f f e c t i v e when g i v e n s u b c u t a n e o u s l y i n d o s e s of 40 mg d a i l y f o r f i v e weeks. D a i l y s u b c u t a n e o u s d o s e s of 4 mg i n h i b i t e d t h e m u l t i p l i c a t i o n o f t u b e r c a b a c i l l i and f o r m a t i o n o f pulmonary l e s i o n s i n mice66. 2.3.4
E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
Tuberactinomycine are r e c o v e r e d from t h e f e r m e n t a t i o n b r o t h by a d s o r p t i o n t o an i o n e x c h a n g e r e s i n . The c u l t u r e f i l t r a t e i s a l l o w e d t o p a s s t h r o u g h a column of A m b e r l i t e IRC-50 i n t h e Na+-form. The r e s i n i s washed w i t h water p r i o r t o t h e a p p l i c a t i o n of 1N h y d r o c h l o r i d e a c i d . Column e f f l u e n t s a r e n e u t r a l i z e d w i t h NaOH and c o n c e n t r a t e d under reduced p r e s s u r e . The c o n c e n t r a t e s are t r e a t e d w i t h a c t i v e c a r b o n t o remove c o l o r b e f o r e b e i n g p o u r e d i n t o e i g h t volumes o f m e t h a n o l . The p r e c i p i t a t i o n may b e r e p e a t e d . Crude t u b e r a c t i n o m y c i n h y d r o c h l o r i d e i s f u r t h e r p u r i f i e d by i o n e x c h a n g e c h r o m a t o g r a p h y on CMSephadex C - 2 5 o r A m b e r l i t e CG-50 e q u i l i b r a t i n g and e l u t i n g w i t h ammonium a c e t a t e b u f f e r ( 0 . 6 M ; pH 8 . 6 ) . Repeating t h e chromatog r a p h y on I R C - S O “a+) w i t h t h e p o o l e d m a j o r f r a c t i o n and t h e p r e c i p i t a t i o n w i t h m e t h a n o l p r o d u c t s a r e o b t a i n e d t h a t a r e homog e n e o u s by t h i n l a y e r c h r o m a t o g r a p h y .
442 3. P e p t i d e s w i t h Sequences of A l t e r n a t i n g D- and L-Amino Acids 3.1 I n t r o d u c t i o n Although t h e p r a c t i c a l v a l u e o f a n t i b i o t i c s had been c o n s i d e r e d by most i n v e s t i g a t o r s i n t h e f i e l d as e a r l y as p r i o r t o t h e t u r n of t h e c e n t u r y , i t remained f o r D ~ b o ts o~ i ~ solate i n 1 9 3 9 a " p u r i f i e d " p r o d u c t from BacilLuA b / r e v i o t h a t became of t h e r a p e u t i c s i g n i f i c a n c e . The a u t h o r chose t o r e f e r t o t h e a c t i v e p r i n c i p l e as TYROTHRICIN. One y e a r l a t e r Dubos i s o l a t e d t o g e t h e r w i t h Hotchkiss t h r e e " p r o t e i n - f ree" f r a c t i o n s from t h i s a c t i v e p r i n c i p l e which were named G R A M I C I D I N , g r a m i n i c a c i d , and g r a m i c i d i n i c a c i d 7 4 . A s t h i s s e c t i o n of t h e r e v i e w w i l l p l a c e emphasis on t h e a n t i b i o t i c p r i n c i p l e r e f e r r e d t o as G R A M I C I D I N by Dubos and H o t c h k i s s , i t i s i m p o r t a n t t o r e c o r d t h a t t h e same a u t h o r s e s t a b l i s h e d - a s e a r l y a s 1 9 4 0 t h a t one h a l f of t h e a-amino a c i d s i n g r a m i c i d i n are of t h e D - c o n E u r a tion'>. I t was n o t b e f o r e a n o t h e r t w e n t y - f i v e y e a r s had p a s s e d t h a t i t was shown c o n c l u s i v e l y t h a t t h e amino a c i d s of t h e Dc o n f i g u r a t i o n are i n t e r s p a c e d - i n a r e g u l a r p a t t e r n between Lamino a c i d s 7 6 . T h i s a l t e r n a t i n g p a t t e r n of L- and D- c o n f i g u r a t i o n i n t h e s e q u e n c e of t h e g r a m i c i d i n s i s of n o minor conseI t i m p a r t s upon quence f o r t h e c o n f o r m a t i o n of t h e s e p e p t i d e s . t h e s e m o l e c u l e s a unique h e l i c a l f e a t u r e 7 7 t h a t e n a b l e s them t o embed i n l i p i d b i l a y e r s and l i k e l y i n biomembranes t o b r i n g a b o u t i o n t r a n s p o r t a c r o s s t h e s e b a r r i e r s between aqueous comp a r t m e n t s . T h i s p r o p e r t y i s due t o t h e p r e s e n c e o f h y d r o p h o b i c groups i n t h e s i d e c h a i n s of t h e c o n s t i t u e n t amino a c i d s .
-
-
3.2 Gramicidin
A , B , and C
The g r a m i c i d i n s A , B , and C a r e a group of s t r u c t u r a l l y unique p e n t a d e c a p e p t i d e s . The p r i m a r y amino group of t h e H z N t e r m i n u s and t h e c a r b o x y l group of t h e COOH-terminus a r e blocked. C o n f i g u r a t i o n a l l y , t h e r e is t h e s t r i c t l y o b s e r v e d p a t t e r n of L- and D-amino a c i d s . The l i n e a r g r a m i c i d n s are h y d r o p h o b i c , t h u s c a p a b l e of embedding r e a d i l y i n l i p i d bil a y e r s and l i k e l y i n biomembranes. The d e s c r i p t i v e term " l i n e a r g r a m i c i d i n s " w i l 1 be used t o make r e f e r e n c e t o t h e g r a m i c i d i n s A, B , and C , as t h e s e m o l e c u l e s c o n s i s t of p e p t j d e c h a i n s of \ f i f t e e n amino a c i d r e s i d u e s e a c h . I n c o n t r a - d i s t i n c t i o n ? g r a m i c i d i n S , a l s o i s o l a t e d from a s t r a i n of B U C i L h A bRev L4 , i s a c y c l i c p e p t i d e composed o f two p e n t a p e p t i d e s of i d e n t i c a l sequence77. By way of a s p e c i f i c t y p e of h e l i x 7 % ( e a r l i e r r e f e r r e d t o as I I ~ , D - h e l i x , now known as 8 9 . 3 - h e l i x ) 7 9 t h e g r a m i c i d i n s mediate t h e t r a n s p o r t of u n i v a l e n t c a t i o n s across l i p i d bilayers.
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3.2.1
Producing organism
The g r a m i c i d i n s a r e produced by B U C i l l U A b R Q v i A , a member of t h e T y r o t h r i x group of b a c t e r i a . Other p e p t i d e s i s o l a t e d from BaciQLuA b R e v i 4 are shown i n t h e flow s h e e t of F i g u r e 1 4 (next page).
Tyrocidine A Tyrocidine
Tyrocidine B Tyrocidine C Valine
-
Gramicidin A
Gramicidin A
Bacillus brevis--pTyrothricin
Isoleucine
-c -c
Valine
Gramicidin
Gramicidin B
Gramicidin B
Isoleucine
Valine
Gramicidin C
Gramicidin A
Gramicidin B
Gramicidin C
Isoleucine
Gramicidin C
FIGURE 14. Peptide a n t i b i o t i c s i s o l a t e d f r o m c u l t u r e s o f B d l l l M b ~ l ? u L 5 . Other p e p t i d e s produced by B a c L L f h ~
m
u
t n o t st-uwn h e r e ) are: gramicidin S7’,
b r e v ~ l i n ~ * b’ r~e ~ v i,n l o O a d ~ ? d e i n e ~ O ~ - ~ ~ ‘ + .
F F
w
444
3.2.2
Chemistry
The g r a m i c i d i n s A, B , and C are l i n e a r p e n t a d e c a p e p t i d e s ( F i g u r e 1 5 , n e x t p a g e ) . With t h e e x c e p t i o n of g l y c i n e , a l l amino a c i d r e s i d u e s c a r r y h y d r o p h o b i c s i d e c h a i n s . The p r i m a r y amino groups a t amino t e r m i n i a r e b l o c k e d by f o r m y l a t i o n . The c a r b o x y l t e r m i n a l r e s i d u e s are c o n v e r t e d t o amides t h a t a r e hydroxyethylated (ethanolamine ; s e r i n e decarboxylated) The HzN-terminal p o s i t i o n i s o c c u p i e d by e i t h e r v a l i n e (dominant a n a l o g ) o r i s o l e u c i n e . A s p o s i t i o n 11 i s taken by TRYPTOPHAN i n g r a m i c i d i n A , PHENYLALANINE i n g r a m i c i d i n 8 , and TYROSINE i n g r a m i c i d i n C , t h e r e are t h r e e p a i r s of a n a l o g s , d i f f e r i n g w i t h i n t h e p a i r i n t h e occupant o f p o s i t i o n 1 ( f r o m p a i r t o p a i r i n t h a t found i n p o s i t i o n 11).
.
A most unique f e a t u r e of the l i n e a r g r a m i c i d i n s i s t h e s t r i c t l y o b s e r v e d p a t t e r n of a l t e r n a t i n g L- and D-configurat i o n s . G l y c i n e , t h e n o n - c h i r a l amino a c i d , i s found i n a p o s i t i o n t h a t would be o c c u p i e d by a D-amino a c i d i n t h e placement of r e g u l a r l y changing c h i r a l i t i e s . T h i s r e g u l a r i t y i n p l a c i n g L- and D-amino a c i d s i n t h e p e p t i d e c h a i n i s of profound consequence f o r t h e s t r u c t u r e ( c o n f o r m a t i o n ) of t h e g r a m i c i d i n s A , 8 , and C . The l i n e a r g r a m i c i d i n s are c a p a b l e of forming t h e n o v e l t y p e of 8 $ , 3 - h e l i c e ~ ~ ~t hye ~s u~p;e r s c r i p t 6 i n d i c a t e s t h e number of amino a c i d r e s i d u e s p e r t u r n of t h e h e l i x ; t h e subscripts 3.3 s t a t e the f a c t that three C 0 g r o u p s e a c h of t h e s i x p e p t i d e bonds a r e p o i n t e d i n o p p o s i t e d i r e c t i o n s w i t h i n a t u r n of t h e h e l i x . The h e l i x i s s t a b i l i z e d by hydrogen bonds between NH-groups and oxygens i n t h e capbonyl f u n c t i o n s of t h e p e p t i d e bonds. The d i r e c t i o n of t h e p e p t i d e C = 0 groups a l t e r n a t e s r e g u l a r l y w i t h i n t h e t u r n s of t h e h e l i x . The FORMYL-group a t t h e H2N-terminus p a r t i c i p a t e s i n t h e hydrogen bonding scheme. Thus, i t i s p o s s i b l e t o form head-to-head d i m e r s E 0 t h a t are s u f f i c i e n t l y l o n g t o s p a n ’ t h e t h i c k n e s s of l i p i d b i l a y e r s and b r i n g a b o u t t h e t r a n s p o r t of u n i v a l e n t c a t i o n s t h r o u g h t h e i n t e r i o r of t h e h e l i x (TRANSMEMBRANE CHANNELS; c f . Ref. 8 1 ) . The hydrophobic s i d e c h a i n s of t h e amino a c i d s i n t h e l i n e a r g r a m i c i d i n s are l i n i n g t h e c i r c u m f e r e n c e of t h e h e l i x , a f a c t t h a t g r e a t l y f a c i l i t a t e s t h e embedding of t h e s e m o l e c u l e s i n t h e h y d r o p h o b i c environment of t h e l i p i d b i l a y e r .
3.2.3
Biology
The b i o l o g i c a l p r o p e r t i e s of t h e g r a m i c i d i n s a r e no l o n g e r r e s t r i c t e d t o t h e i r a c t i o n a s a n t i b i o t i c s . Other b i o l o g i c a l a c t i v i t i e s have been s e e n r e c e n t l y . The unique p r o p e r t y of t h e l i n e a r g r a m i c i d i n s t o embed ( a n d t o p e n e t r a t e ) l i p i d b i l a y e r s make ( a ) s t i l l o t h e r b i o l o g i c a l p o t e n t i a l s a l i k e l i hood, ( b ) p o i n t t o t h e i r u t i l i t y a s v e h i c l e s f o r o t h e r molec u l e s t o b e b r o u g h t i n t o t h e l i p i d b i l a y e r of t h e biomembrane. 3.2.3.1
Antibiotic Properties
Mixtures of t h e l i n e a r g r a m i c i d i n s were found t o be e f f e c t i v e a g a i n s t many b u t n o t a l l g r a m - p o s i t i v e o r g a n i s m s . This action is r a t h e r b a c t e r i o s t a t i c not necessarily bacteric i d a l . C o n c e n t r a t i o n s of 1 t o 30 mcg/ml t o 10-5M) p r o v e d
FIGURE 15. The s t r u c t u r e s o f the gramicidins A, 8 , and C. Amino t e r m i n i are formylated, carboxyl t e r m i n i are hydroxyethylamidated. C o n f i g u r a t i m s a l t e r n a t e r e g u l a r l y between the L- and D-form. The n o n - h i r a l amino a c i d glycine occupies a p o s i t i o n where an amino a c i d o f the D-configuration would be found i n t h e regular p a t t e r n o f alternating chiralities.
446
u s e f u l i n many cases. The g r o w t h of a number o f g r a m - n e g a t i v e cocci was a l s o i n h i b i t e d . I t h a s b e e n c l a i m e d t h a t g r a m i d d i n must be g i v e n p a r e n t e r a l l y to p r o d u c e s y s t e m i c e f f e c t s . S y s t e m i c t o x i c i t y , however, i s s o h i g h t h a t c l i n i c a l u s e i s l i m i t e d t o local a p p l i c a t i o n . 3.2.3.2
E f f e c t on N e o n a t a l T i s s u e
I n a s t u d y on t h e e f f e c t of v a r i o u s p e p t i d e s on n e o n a t a l t i s s u e , g r a m i d d i n A was g i v e n t o r a t s o r a l l y . A t d o s e s of 3 t o 30 mg i n t h e form of s u s p e n s i o n s i n w a t e r a n d a d m i n i s t e r e d d u r i n g t h e l a s t trimester o f g e s t a t i o n c o m p l e t e r e s o r p t i o n o f a l l n e o n a t a l t i s s u e was o b s e r v e d 6 . 3.2.3.3
E f f e c t on N e o p l a s t i c T i s s u e
G r a m i c i d i n A and B are c a p a b l e of r e d u c i n g t h e i n v i v a g r o w t h o f leucemic c e l l s i n rnicel6. I n c e l l c u l t u r e s t h e r e i s g r o w t h r e t a r d a t i o n of leucemia c e l l s t o t h e e x t e n t of 80-85%16. 3.2.3.4
E f f e c t on M i t o c h o n d r i a
The l i n e a r g r a m i c i d i n s c a u s e i o n - d e p e n d e n t c h a n g e s i n t h e volume o f m i t o c h o n d r i a t h a t are e n e r g i z e d by a d e n o s i n e 5 ' - t r i p h o ~ p h a t e ~( c~f .- ~F i~g u r e 1 8 f o r a c o m p a r i s o n of t h e s e c h a n g e s b r o u g h t a b o u t by n a t u r a l a n d s y n t h e t i c g r a m i c i d i n A ) . In a r e c e n t s t u d y i t was d e m o n s t r a t e d t h a t a l s o 2 , 2 - d i m e t h y l s u c c i n i c a n h v d r i d e i n d u c e s an immediate and r a p i d i n c r e a s e i n o r g a n e l l a r volbme i n s u s p e n s i o n s of m i t o c h o n d r i a - f o r t i f i e d w i t h g r a m i c i d i n and p o t a s s i u m i o n B 5 . 3.2.3.5
E f f e c t on S p o r u l a t i o n
Most i n t e r e s t i n g o b s e r v a t i o n s on t h e b i o l o g i c a l f u n c t i o n of t h e l i n e a r g r a m i c i d i n s ( i n t h e i r n a t i v e e n v i r o n m e n t ) h a v e been made i n s t u d i e s on g r a m i c i d i n - n e g a t i v e m u t a n t s B 6 . Such s t r a i n s of BaciLtuA bnfivih are n o t c a p a b l e of f o r m i n g n o r m a l spores unless provided with the peptide(s1. The l i n e a r g r a m i c i d i n s i n h i b i t RNA s y n t h e s i s by p u r i f i e d RNA p o l y m e r a s e (nucleosidetriphosphate: RNA n u c l e o t i d y l t r a n s f e r a s e , EC 2 . 7 . 7 . 6 ) by i n t e r f e r i n g w i t h t h e b i n d i n g of RNA p o l y The e f f e c t o b s e r v e d i s m e r a s e to DNAB7 ( c f . a l s o 8 8 and 8 9 ) . i n t e r p r e t e d as d e s t a b i l i z a t i o n of t h e RNA polymerase-DNA comp l e x , a mode o f a c t i o n t h a t i s c o n s i s t e n t w i t h t h e c o n t r o l of p r o m o t e r s e l e c t i o n . G r a m i c i d i n - n e g a t i ve m u t a n t s of B a c i t l u ~ bJtfivi.4 showed g r e a t l y r e d u c e d l e v e l s o f s e r i n e p r o t e a s e i n e a r l y s t a g e s o f s p o r u l a t i o n . The s p o r e s were low i n d i p i c o l i n i c a c i d and h i g h l y s e n s i t i v e t o h e a t . I t was shown t h a t t h e gramicidin-dependent s y n t h e s i s of s e r i n e p r o t e a s e r e q u i r e d transcription. The i n h i b i t i o n of t r a n s c r i p t i o n was r e s t r i c t e d t o c e r t a i n r e g i o n s of t h e DNA-template b u t n o t t o o t h e r s . I n as-much as t h e e x p e r i m e n t s were c o n d u c t e d i n a h i g h l y p u r i f i e d s y s t e m d e v o i d o f membranes t h e e f f e c t s o b s e r v e d a r e d i s t i n c t f r o m t h e i o n o p h o r e t i c p r o p e r t i e s ( v i d e s u p r a ) of t h e l i n e a r g r a m i c i d i n s . R a t h e r , a l l i n d i c a t i o n s are i n f a v o r of a d i r e c t r e l a t i o n s h i p b e t w e e n t h i s g r o u p of p e p t i d e a n t i b i o t i c s and
447
b a c t e r i a l s p o r u l a t i o n . In a l l l i k e l i h o o d , t h e p e p t i d e s act i n t h e i r n a t i v e environment i n a way t h a t d i f f e r s from t h e i r a n t i b a ct e r i a 1 a c t i on. 3.2.3.6
B i o s y n t h e s i s of t h e l i n e a r g r a m i c i d i n s
The b i o s y n t h e s i s o f t h e l i n e a r g r a m i c i d i n s (as w e l l as t h a t o f o t h e r p e p t i d e s produced by BaciLLu.5 b h e v i b ) i s mediated by multi-enzyme complexes through c o n s e c u t i v e c y c l e s of p a n t o thenate-assisted condensations v i a t h i o l ester activation. F. Lipmann has reviewed t h i s non-ribosomal t y p e o f p o l y p e p t i d e s y n t h e s i s on polyenzyme t e m p l a t e s g 0 . 3.2.4
E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
Adjustment of t h e f i l t r a t e of a c u l t u r e of BaciCLua b l t e v i ~ t o pH 4.5-4.8 by t h e a d d i t i o n o f h y d r o c h l o r i c a c i d p r e c i p i t a t e s a m i x t u r e of a n t i b i o t i c s . The d r i e d s o l i d material i s e x t r a c t e d w i t h a l c o h o l . A d d i t i o n of s a l i n e t o t h e a l c o h o l i c e x t r a c t newly p r e c i p i t a t e s a m i x t u r e of a n t i b i o t i c s t h a t is known as T Y R O T H R I C I N 7 3 ( c f . F i g u r e 15). E x t r a c t i o n of t y r o t h r i c i n w i t h an a c e t o n e - e t h e r m i x t u r e s e c u r e s a s o l u b l e n e u t r a l f r a c t i o n , t h e l i n e a r g r a m i c i d i n s l e a v i n g on i n s o l u b l e b a s i c ~ ~t y~r o~c i d~ i n~e s' ~ are~ . p o r t i o n , t h e t y r o c i d i n e ~ ~ ~The c y c l i c d e c a p e p t i d e s . F i v e of t h e amino a c i d r e s i d u e s a r e i n a sequence t h a t i s i d e n t i c a l w i t h t h o s e s e e n i n g r a m i c i d i n S 7 7 . C o u n t e r c u r r e n t d i s t r i b u t i o n of t h e n e u t r a l f r a c t i o n from t y r o t h r i c i n i n t h e s o l v e n t s y s t e m benzene:chloroform:methanol: water 15:15:23:7 ( v / v ) s e p a r a t e s t h r e e component^^^^^^. The major component i s g r a m i c i d i n A ( F i g u r e 1 6 , n e x t p a g e ) . Gramicidin B and C are p r e s e n t i n s m a l l e r amounts. F r a c t i o n D ( F i g u r e 1 6 ) i s a minor component c o n s i s t i n g p r i m a r i l y of mater i a l t h a t i s more h y d r o p h i l i c t h a n t h e g r a m i c i d i n s and p o s s i b l y a l s o of h i g h e r m o l e c u l a r w e i g h t . Components A, B y and C cont a i n t h e g r a m i c i d i n a n a l o g s t h a t d i f f e r i n t h e r e s i d u e occupying p o s i t i o n 1 ( v a l i n e - and i s o l e u c i n e g r a m i c i d i n A , B , and C ) . The t r a n s f e r number of 9 9 9 was n o t s u f f i c i e n t l y l a r g e t o s e p a r a t e these a n a l o g s . R e d i s t r i b u t i o n of r e p r e s e n t a t i v e f r a c t i o n s of t h e mixt u r e s of t h e v a l i n e and i s o l e u c i n e g r a m i c i d i n s A, B , and C gave t h e p a t t e r n s shown i n F i g u r e 1 7 , ( f o l l o w i n g F i g u r e 1 6 ) . The d i s c r e p a n c i e s i n t h e r i g h t - h a n d p o r t i o n of t h e e x p e r i m e n t a l and t h e o r e t i c a l c u r v e s are due t o t h e p r e s e n c e of t h e i s o l e u c i n e a n a l o g s (minor components). More e x t e n s i v e d i s t r i b u t i o n s ( i n c r e a s e d t r a n s f e r numbers) and t h e i s o l a t i o n of s o l u t e from i n d i v i d u a l t u b e s o f t h e d i s t r i b u t i o n t r a i n ( a t r e l a t i v e l y low 200 t o 4 0 0 ) p r o v i d e p u r e g r a m i c i d i n a n a l o g s 1 6 . t r a n s f e r numbers
-
D r o p l e t c o u n t e r c u r r e n t chromatographyg6 and h i g h - p e r f ormance l i q u i d chromatographyg have been a p p l i e d t o t h e s e p a r a t i o n of t h e a n a l o g s of t h e l i n e a r g r a m i c i d i n s . Under a p p r o p r i a t e c o n d i t i o n s s u c c e s s f u l s e p a r a t i o n i s p o s s i b l e . However, n e i t h e r of t h e two t e c h n i q u e s l e n d s i t s e l f a t t h i s time t o t h e s e p a r a t i o n of q u a n t i t i e s beyond t h e m i l l i g r a m s c a l e .
OO/IO
OO/I 280mp
280 mp
F
c OD
IO.0Oi-
TNO.o o Solvent system benzene:chloroFIGURE 16. Countercurrent d i s t r i b u t i o n o f a mixture o f the l i n e a r gramicidins. forn:methanol:water = 15:15:23:7 (v/v); 40 m l l a v e r phase, 40 ml upper phase; 5 0 0 - t ~ b e unit. Gramicidin (60 9) dissolved i n the lower phase volums o f tubes 0-30; forerm, tubes 31-79 lower + upper phase; tubes 80-129, two A: volums o f lower phase; lower phase analyzed;...., cpper phase analyzed;t h e o r e t i c a l curve. Gramicidin A; K ( d i s t r i b u t i o n coefficient: conc. i n upper phase/conc. i n lower phase) = 0.56; 6: gramicidin 8; K = 0.30; C: gramicidin C; K = 1.56; D: gramicidin 0; K = 16.65.
-,
,
50
TUBE NO.
100
FIGURE 17.
Solvent systems benzene: R e d i s t r i b u t i o n o f gramicidin A, 6, C (frm d i s t r i b u t i o n shown i n Figure 16). ch1oroform:methanol:water = 15:15:23:7 (v/v); upper phase 10 ml, lower phase 10 ml, 200 tube unit.. A-A-A, experimental curve; 0-0-0, t h e o r e t i c a l ' c u r v e . ( A ) gramicidin A f r o m pooled f r a c t i o n s 340-380 i n Figure 16; (B) gramic i d i n B f r o m pooled f r a c t i o n 222-242 i n Figure 16; ( C ) gramicidin C from pooled f r a c t i o n s 800-840 i n Figure 16;
F
discrepancies between experimental and t h e o r e t i c a l curves are the r e f l e c t i o n o f the presence o f the i s o l e u c i n e analogs (minor components).
W
c
450 4. S y n t h e t i c V a l i n e - G r a m i c i d i n s A, B , and C
4.1 Introduction The v a l i n e a n a l o g s of g r a m i c i d i n gramicidin B106, and g r a m i c i d i n C 1 0 6 have b e e n made a v a i l a b l e s y n t h e t i c a l l y v i a s o l i d p h a s e t e c h n i q u e s 7 . C o n s i d e r i n g t h e h i g h l e v e l of h y d r o p h o b i c i t y of t h e l i n e a r g r a m i c i d i n s , it c a n n o t be d e n i e d t h a t a d v a n t a g e s are a s s o c i a t e d w i t h t h e e x e c u t i o n of t h e s y n t h e s i s i n t h e heterogeneous environment of t h e s o l i d phase supp o r t e d p e p t i d e . The s o l u b i l i t i e s of p r e c u r s o r s and i n t e r mediates do n o t p o s e p r o b l e m s . P u r i f i c a t i o n i s p o s t p o n e d a l m o s t a l w a y s u n t i l t h e l e v e l of t h e f i n a l p r o d u c t h a s been r e a c h e d . T h e r e i t h a s t o be f a c e d w i t h a l l i t s i m p a c t . The e x p e r i e n c e gained w i t h t h e p u r i f i c a t i o n of t h e n a t u r a l l y o c c u r r i n g l i n e a r g r a m i c i d i n s g 4 9 5 h e l d a l l t h e answers t o p r o v i d i n g s y n t h e t i c p r o d u c t s o f h i g h l e v e l s o f p u r i t y w i t h t h e f u l l complement of biological properties.
The h y d r o p h o b i c i t y o f t h e l i n e a r g r a m i c i d i n s d o e s n o t p r e c l u d e t h e i r s y n t h e s i s v i a s o l u t i o n methods. S y n t h e s e s o f v a l i n e a n d i s o l e u c i n e g r a m i c i d i n A i n s o l u t i o n are r e c o r d e d i n t h e l i t e r a t u r e 1 0 8 . The o c t a p e p t i d e s e q u e n c e of r e s i d u e s 3 t h r o u g h 10 - common t o a l l l i n e a r g r a m i c i d i n h a s been s n t h e s i z e d i n s o l u t i o n using a c t i v e esters i n a c t i v a t i o n s t e p s y 6 . Analogs of t h e COOH-terminal s e q u e n c e o f r e s i d u e s 11-15 h a v e b e e n p r o v i d e d v i a t h e same a p p r o a c h 1 6 . Varying w i t h t h e t y p e o f g r o u p a t t a c h e d t o t h e HZN-terminus, t h e s e p e p t i d e s d i s p l a y a c t i v i t i e s t h a t are e i t h e r a g o n i s t i c o r a n t a g o n i s t i c i n l e u c o c y t e c h e m o t a ~ i s ~ The ~ ~ . a n t i b i o t i c p r o p e r t i e s o f t h e s e pept i d e s a r e ’ p r e s e n t l y t h e s u b j e c t of an i n v e s t i g a t i o n .
-
4.2 C h e m i s t r y Under t h e g r o w t h c o n d i t i o n s e m p l o y e d 7 4 y 7 5 , BacLLLud p r o d u c e d t h e v a l i n e a n a l o g s of t h e l i n e a r g r a m i c i d i n s i n g r e a t e r amounts t h a n t h e i s o l e u c i n e a n a l o g s . O f t h e s i x molec u l a r f o r m s , v a l i n e g r a m i c i d i n A ( F i g u r e 14) is t h e one i s o l a t e d i n l a r g e s t q u a n t i t y . V a l i n e - g r a m i c i d i n A 1 0 5 was s y n t h e s i z e d f i r s t , f o l l o w e d by g r a m i c i d i n B l o b and g r a m i c i d i n C 1 0 6 . Analogs of t h e g r a m i c i d i n s h a v e also b e e n p r e p a r e d 1 6 . bheuid
’
The s y n t h e s e s were c a r r i e d o u t on p o l y s t y r e n e r e s i n s , t h e t r y p t o p h a n r e s i d u e of p o s i t i o n 1 5 o f t h e g r a m i c i d i n s b e i n g l i n k e d t o t h e s u p p o r t i n b e n z y l e s t e r bond. The t e r t . - b u t y l o x y c a r b o n y l g r o u p s e r v e d t o p r o t e c t a-amino g r o u p s . D i c y c l o h e x y l c a r b o d i i m i d e was employed i n a c t i v a t i o n s t e p s , The s y n t h e s e s were p e r f o r m e d i n a u t o m a t e d i n s t r u m e n t s b u i l t e i t h e r according t o t h e published design of M e r r i f i e l d ’ l o o r a v a i l a b l e c o m m e r c i a l l y (Beckman S y n t h e s i z e r Model 9 9 0 ; Beckman I n s t r u m e n t s , P a l o A l t o , C a l i f o r n i a ) . A g i t a t i o n of t h e resin i n t h e f i r s t i n s t r u m e n t was b y s h a k i n g t h e r e a c t i o n v e s s e l ( r o c k i n g m o t i o n s ) , i n t h e s e c o n d by m e c h a n i c a l s t i r r i n g . The p e p t i d e s were d i s p l a c e d from t h e r e s i n by t r e a t m e n t w i t h ethanolamine i n organic s o l v e n t s o r s o l v e n t mixtures, thus providing d i r e c t l y the d e s i r e d pentadecapeptide ethanolamides.
451
F o r m y l a t i o n i s t h e l a s t s t e p of n e c e s s a r y o p e r a t i o n s i n t h e s y n t h e s i s . I t was accomplished by a c t i v a t i n g f o r m i c a c i d v i a e i t h e r an e s t e r ( e . g . w i t h d i n i t r o p h e n o l ) o r a s a n h y d r i d e (formic acid - a c e t i c anhydride). The f i n a l p r o d u c t s were p u r i f i e d by c o u n t e r c u r r e n t d i s t r i b u t i o n ( c f . t h e s e c t i o n on e x t r a c t i o n , s e p a r a t i o n and p u r i fication). 4 . 3 Biology -
The s y n t h e t i c p r o d u c t s c h a r a c t e r i z e d a s and found i d e n t i c a l by a number of chemical and p h y s i c a l c r i t e r i a w i t h t h e v a l i n e a n a l o g s of g r a m i c i d i n A, B , and C , showed t h e b i o l o g i c a l a c t i v i t i e s of t h e i r n a t u r a l c o u n t e r p a r t s . Examples of t h e s e a c t i v i t i e s a r e , f o r i n s t a n c e , t h e growth i n h i b i t i o n of microorganism. For t h e t h r e e s y n t h e t i c g r a m i c i d i n s minimum i n h i b i t o r y c o n c e n t r a t i o n s compared most f a v o r a b l y w i t h t h o s e of t h e n a t u r a l p r o d u c t s ( c f . T a b l e 3 ) where t h e d a t a are shown f o r n a t u r a l and s y n t h e t i c g r a m i c i d i n A. TABLE 3 h t i b i o t i c A c t i v i t y o f N a t u r a l and Synthetic Valine-Cramicidin A* Minimum I n h i b i t o r y Concentration, mcq/ml Organism
8 a d . t h aubatieis sancina .&Ltea S&lphylococw weU.4 S.tneptococw pyogeneb
E b c W c k i a cokX Paerrdomom aenu&oaa
Nat u r a1 Gramicidin A
Synthetic Gramicidin A
2.5
2.5
0.25
0.25
2.5
2.5
0.075
0.05
50.0
50 .O
25 .O
25 .O
*Minimum i n h i b i t o r y concentr&ions were determined by t h e tube d i l u t i o n method. Mdium:
Yeast beef broth, pH 6.8.
E x c e l l e n t agreement was a l s o s e e n € o r t h e e f f e c t of t h e g r a m i c i d i n s on m i t o c h o n d i r a . F i g u r e 1 8 , n e x t p a g e , compares t h e volume changes measured a s d e c r e a s e i n t h e a b s o r b a n c e a t 5 2 0 nm) i n d u c e d i n th’e o r g a n e l l e by n a t u r a l and s y n t h e t i c valine-gramicidin B.
4.4 Effect
on L i p i d B i l a y e r s
Due t o t h e i r u n i q u e c o n f o r m a t i o n ( B q . 3 - h e l i ~ ’ ~ )and t h e i r a b i l i t y ( b a s e d on h y d r o p h o b i c i t y ) t o embed i n l i p i d b i l a y e r s , the l i n e a r gramicidins transport univalent cations across t h i s
‘Tp
-1
I
4TP
Decrease 0.1 Absorbance
1
I
Decrease 0.1 Absorbance
1
2mM K+
1
2mM K+
I
2mM Malate
I
2mM Malate
\
2mM K+ 2mM Malate
2mM K+ 2mM Malate
pH 74
pH 7.4
Synthetic GB
Natural GB
II 6
12
1
I
I
I
18 24 6 12 MINUTES AFTER ADDITION OF MITOCHONDRIA
I
18
I
24
FIGURE 18. Mitochondria1 volume changes induced by gramicidin El. L e f t hand panel: synthetic gramicidin 8; right h m d panel: n a t u r a l gramicidin 8. Volume chmge measured as decrease i n o p t i c a l density a t 520 nm.
45 3
’’’
b a r r i e r between a q u e o u s c o m p a r t m e n t s . I n s i n g l e c h a n n e l cond u c t a n c e measurements ( c f . 81) t h e s y n t h e t i c v a l i n e - g r a m i c i d i n s B and C g a v e t h e same r e s u l t s as t h e n a t u r a l a n a l o g s . I n F i g u r e 1 9 on t h e f o l l o w i n g p a g e , i t i s shown f o r n a t u r a l and s y n t h e t i c v a l i n e - g r a m i c i d i n C how l o n g s i n g l e c h a n n e l s a r e open f o r t h e p a s s a g e o f i o n s and t h e f l o w of c u r r e n t a c r o s s t h e l i p i d bilayer. I t must be b o r n e i n mind t h a t e a c h e v e n t , s o r e c o r d e d , i s b r o u g h t a b o u t by two molecules o f g r a m i c i d i n t h a t j o i n i n a head-to-head dimer a p p r o p r i a t e l y o r i e n t e d i n the l i p i d b i l a y e r t o f u n c t i o n as TRANSMEMBRANE CHANNEL.
4.5 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
In s o l i d phase p e p t i d e s y n t h e s i s p u r i f i c a t i o n i s p o s t poned t o t h e l e v e l o f t h e f i n a l p r o d u c t . The e x p e r i e n c e g a i n e d w i t h t h e s e p a r a t i o n and p u r i f i c a t i o n of t h e n a t u r a l p r o d u c t s , was of g r e a t v a l u e when a f t e r t h e s y n t h e s i s b y p r o d u c t s h a d t o b e s e p a r a t e d from t h e d e s i r e d g r a m i c i d i n s and t h e l a t t e r had t o be p u r i f i e d . U s i n g t h e s o l v e n t s y s t e m (benzene:chloroform:methanol: water 1 5 : 1 5 : 2 3 : 7 ) from t h e p u r i f i c a t i o n o f t h e n a t u r a l g r a m i c i d i n s , t h e s y n t h e t i c a n a l o g s were r e a d i l y p u r i f i e d . F i g u r e 2 0 , on t h e f o l l o w i n g p a g e , shows t h e p a t t e r n o f t h e f i r s t d i s t r i b u t i o n . When t h e m a j o r component was r e d i s t r i b u t e d i n t h e same s o l v e n t s y s t e m , t h e s o l u t e was r e p r e s e n t e d by a s i n g l e symmet r i c a l peak ( F i g u r e 2 1 ; u p p e r p a n e l ) . W i t h p u r i f i e d n a t u r a l v a l i n e - g r a m i c i d i n A on h a n d , e q u a l q u a n t i t i e s of t h e n a t u r a l and s y n t h e t i c a n a l o g were mixed f o r c o u n t e r c u r r e n t d i s t r i b u t i o n . The p a t t e r n was a g a i n t h a t of a s i n g l e symmetrical curve. S o l i d phase p e p t i d e s y n t h e s i s , t h u s , p r o v i d e s a p r o d u c t t h e d i s t r i b u t i o n c r i t e r i a o f which are i n f u l l agreement w i t h t h o s e of t h e n a t u r a l p r o d u c t .
4 . 6 Summary With t h e accomplished s y n t h e s i s ( d i s c u s s e d h e r e ) of t h e v a l i n e a n a l o g s of t h e g r a m i c i d i n s A , B , and C , i t may b e assumed t h a t t h e i s o l e u c i n e analogs w i l l also be a c c e s s i b l e v i a s y n t h e t i c routes. A number of a n a l o g s ( e x c h a n g e of amino a c i d r e s i d u e s ) of t h e l i n e a r g r a m i c i d i n s h a v e a l r e a d y been p r o v i d e d l 6 ) . T h i s s y n t h e t i c p o t e n t i a l w i l l b e o f i m p o r t a n c e i n more t h a n one way. On t h e one h a n d , i t w i l l make i t p o s s i b l e t o u t i l i z e t h e g r a m i c i d i n s as p o s s i b l e c a r r i e r s f o r o t h e r molec u l a r s p e c i e s t o b e p l a c e d i n t h e h y d r o p h o b i c e n v i r o n m e n t of t h e l i p i d b i l a y e r of t h e biomembrane. O t h e r p r o s p e c t s are numerous c o n c e i v a b l e s t r u c t u r a l m o d i f i c a t i o n s w i t h t h e o b j e c t i v e t o improve s p e c i f i c a c t i v i t i e s ( v i d e s u p r a ) of t h e b r o a d s p e c t r u m o f b i o l o g i . c a 1 p r o p e r t i e s t h a t h a s been u n f o l d e d a l r e a d y . Last, b u t n o t l e a s t , t h e r e i s e v e r y r e a s o n t o a n t i c i p a t e improvements from s t r u c t u r a l v a r i a t i o n w i t h r e g a r d t o u n d e s i r a b l e s i d e e f f e c t s , and h o p e f u l l y , t h e r e t u r n of t h i s c l a s s of p o l y p e p t i d e s , n o t o n l y as a n t i b i o t i c s , t o a n , a g a i n , more p r o m i n e n t p l a c e i n c l i n i c a l medicine.
F
Ln
F
GRAMlClDlN C d
nat
Eyn.
FIGURE 19. S i n g l e c h m n e l conductance measurements with grarnicidin C. Syn: s y n t h e t i c gramicidin C; nat: natural gramicidin C. S i n g l e channels vary i n the length o f time they are open (conducting). For the s y n t h e t i c preparation of gramicidin C two c h m n e l s are open simultaneously (second increase i n conductance) f o r longer periods o f time than i s the case f o r t h e natural analog.
I .2
Solvent System ( v/v 1 benzene 15 chloroform I 5 methanol 23 water 7
3E 0 (lo
cu 0.8 >-
0
z a
m U
0
0
0.
m
a
FIGURE 20.
Countercurrent d i s t r i b u t i o n o f synthetic gramicidin A.
be redistributed ( c f . Figure 21).
Material represented by the major peek w i l l
F u1 u1
456
Solvent System (v/v) 15 benzene chloroform I 5 methanol 23
I .6
A
I .2
0.8 3. E
8 0.4 cu
t Y2 o
I
a m U
8 0.4 m a
0.2
C TUBE NUMBER
FIGURE 21. R e d i s t r i b u t i o n of the major component from the d i s t r i b u t i o n o f the crude product o f s y n t h e t i c valine-gramicidin A (upper p a n e l ) . The lower panel shows the d i s t r i b u t i o n curve o f s mixture o f equal amounts o f n a t u r a l and s y n t h e t i c valine gramicidin A.
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46 3
P l a n t - D e r i v e d A n t i b i o t i cs
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Lester A M i t s c h e r The U n i v e r s i t y of Kansas. Lawrence. Kansas
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1
5
6
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. 8. 9. 10. 11. 7
................................ S p e c i f i c I n t r o d u c t i o n ............................... Q u a t e r n a r y A l k a l o i d s . B e r b e r i n e ....................
General Introduction
Tertiary Alkaloids
.
.
Liriodenine
....................
464 464 467 469
.
Phenols C h r y s o p h a n i c Acid-9-Anthrone a n d 5.7. 2 4 -Te t r a h y d r o x y - 6 - ( 3. 3 - D i m e t h y l a l l y l ) Isoflavone
......................................... A c i d s . Wyerone A c i d ................................ N e u t r a l S u b s t a n c e s . P i p e r i n e ....................... G l y c o s i d e s . T u l i p o s i d e s A and B .................... C o n c l u s i o n .......................................... L i t e r a t u r e C i t e d .................................... A d d i t i o n a l B i b l i o g r a p h y .............................
470 471 472 472 473 473 475
46 4
1. G e n e r a l I n t r o d u c t i o n
C l e a r l y t h e f i r s t a n t i b i o t i c s u s e d by man were t h o s e f o u n d amongst t h e m e t a b o l i c p r o d u c t s o f t h e h i g h e r p l a n t s . T h e i r most s u c c e s s f u l a p p l i c a t i o n a p p e a r s t o h a v e been as p r e s e r v a t i v e s and a n t i s e p t i c s a u s e which c o n t i n u e s t o d a y . D e s p i t e t h e i r l o n g h i s t o r y of use, n o a g e n t d e r i v e d from t h e s e s o u r c e s r e m a i n s i n s y s t e m i c use i n modern times. With t h e a d v e n t o f t h e s u l f a d r u g s ? p e n i c i l l i n and then t h e streptomycete products, i n t e r e s t i n a n t i m i c r o b i a l a g e n t s from h i g h e r p l a n t s r e s u r g e d b r i e f l y and t h e n waned a g a i n . I n t e r e s t i n t h e s e materials h a s o n c e a g a i n reawakened now t h a t t h e p o i n t o f d i m i n i s h i n g r e t u r n s h a s been r e a c h e d i n s e a r c h i n g f o r new a n t i b i o t i c s from t r a d i t i o n a l f e r m e n t a t i o n s o u r c e s . P r e s e n t r e s e a r c h on h i g h e r p l a n t e x t r a c t s i n v o l v e s i s o l a t i o n o f i n d i v i d u a l a g e n t s i n p u r e form a n d e v a l u a t i o n of t h e i r p o t e n t i a l u s i n g modern t e c h n i q u e s a n d , i n p a r t i c u l a r , i n d i c a t o r m i c r o o r g a n i s m s o f known p e d i g r e e ’ y 2 . T h i s f a c t o r h a s seldom been g i v e n p r o p e r a t t e n t i o n i n t h e p a s t s o t h a t much of t h e l i t e r a t u r e p r i o r t o a b o u t 19613 i s h a r d t o evaluate.
-
The p u r p o s e of t h i s c h a p t e r i s t o p r e s e n t s e l e c t e d works i n v o l v i n g c a r e f u l s t u d y o f i n d i v i d u a l a n t i m i c r o b i a l a g e n t s from h i g h e r p l a n t s whose c h e m i c a l n a t u r e b e l o n g t o s e v e r a l d i f f e r e n t c h e m i c a l c l a s s e s i n o r d e r t o g u i d e t h o s e who a r e i n t e r e s t e d i n t a k i n g up t h e c h a l l e n g e t h a t t h e s e s u b s t a n c e s r e p r e s e n t . I n t h i s s e n s e , i t d i f f e r s from t h e p r e c e e d i n g c h a p t e r s f o r i t i s n o t p o s s i b l e a t p r e s e n t t o s e l e c t d i s c r e t e examples i n v o l v i n g a g e n t s p r e s e n t l y i n c l i n i c a l use f o r t h e s y s t e m i c t r e a t m e n t o f human d i s e a s e . The v e r y l a r g e number o f r e p o r t s of a n t i m i c r o b i a l a c t i v i t y i n h i g h e r p l a n t e x t r a c t s and t h e r e l a t i v e l y few r e p o r t s o f c o m p l e t e and s y s t e m a t i c s t u d y o f t h e s e compounds l e a d s t o t h e o p t i m i s t i c , and one h o p e s u l t i m a t e l y j u s t i f i a b l e , hope t h a t c o n t i n u e d work w i l l l e a d t o c l i n i c a l s u c c e s s . 2. Specific Introduction
-
I n any we 11-con ce i ve d , b i o a s s a y d i r e c t e d f r a c t i o n a t i o n a n d i s o l a t i o n scheme, w h e t h e r i t be d i r e c t e d toward m i c r o b i a l p r o d u c t s o r t o t h o s e from h i g h e r p l a n t s , i t i s e s s e n t i a l t h a t a s y s t e m a t i c f r a c t i o n a t i o n scheme be f o l l o w e d i n t h e e a r l y s t a g e s . The s t e p s i n c o r p o r a t e d s h o u l d be t h o s e which can be p e r f o r m e d e f f i c i e n t l y w i t h l a r g e q u a n t i t i e s of m a t e r i a l s o t h a t t h e more time consuming a n d e x p e n s i v e c h r o m a t o g r a p h i c s t e p s can be c a r r i e d o u t l a t e r when t h e amount of m a t e r i a l i s s m a l l and t h e amount o f column a b s o r b a n t and s o l v e n t r e q u i r e d i s a l s o s m a l l . A g e n e r a l scheme h a s been d e v e l o p e d f o r s u c h work w i t h h i g h e r p l a n t e x t r a c t s 3 . I t i s r a t h e r s i m i l a r i n some r e s p e c t s t o t h a t u s e d e f f e c t i v e l y by Kupchan and h i s co-workers i n f r a c t i o n a t i n g higher p l a n t s f o r antitumor agents4-?. I f t h e p l a n t material c o n s i s t s of s e e d s or o t h e r tissues r i c h i n l i p i d s , i t i s a d v i s a b l e t o e x t r a c t t h e material a t room t e m p e r a t u r e by p e r c o l a t i o n o r m a c e r a t i o n w i t h h e x a n e o r p e t r o leum e t h e r t o g i v e e x t r a c t l a b e f o r e p r o c e e d i n g ( F i g u r e l ) . T h i s c o n c e p t i s s i m i l a r t o t h a t u s e d i n i s o l a t i n g myceliumbound s t r e p t o m y c e t e a n t i b i o t i c s . O t h e r w i s e , one may p r o c e e d
465 Powdered P l a n t M a t e r i a l
I Hexme*
Neutrals
Aci ds
Water Solubles,
Alkaloida
I
9OXbOH/ Pet. Ether Extract 6 (MeOH) TerpenesSterols
Extract 7 (P.E.) WaxesFats
I
Extract 8 (CHf) A c i da
I Extr!ct 11 (CHf) Tertiary Alkaloids
a-l-
E x t r a c t 12' (OH-)
I NHhCl/CHf E x t r a c t 13 ( C H f ) Phenolic A l k a l o i d a
*This step i s o p t i o n a l . FIGURE 1. Generalized Scheme f o r I s o l a t i o n o f A n t i m i c r o b i a l Agents from Higher P l a n t s .
d i r e c t l y t o t h e n e x t s t e p which i s p e r c o l a t i o n o r m a c e r a t i o n o f t h e a i r - d r i e d ground p l a n t material w i t h 9 5 % e t h a n o l u n t i l t h e material i s e x h a u s t e d . E v a p o r a t i o n p r o d u c e s e x t r a c t 1. E x t r a c t l a a n d / o r 1 a r e t e s t e d f o r a n t i m i c r o b i a l s p e c t r a by t h e c l a s s i c a g a r - d i l u t i o n s t r e a k method i n t h e u s u a l way. F u r t h e r p u r i f i c a t i o n o f e x t r a c t 1 i s a c h i e v e d by p a r t i t i o n i n g between 5 % h y d r o c h l o r i c a c i d and c h l o r o f o r m t o p r o d u c e e x t r a c t 2 ( t h e c h l o r o form l a y e r ; c o n t a i n s n e u t r a l s u b s t a n c e s and a c i d s ) and e x t r a c t 3 ( t h e aqueous p h a s e ; c o n t a i n s a l k a l o i d s and water s o l u b l e s u b s t a n c e s ) . E x t r a c t 2 i s f u r t h e r p u r i f i e d by e x t r a c t i o n w i t h 5 % sodium h y d r o x i d e t o p r o d u c e e x t r a c t 4 ( t h e c h l o r o f o r m l a y e r ;
46 6
c o n t a i n s n e u t r a l m a t e r i a l s ) and e x t r a c t 5 ( t h e a l k a l i n e p h a s e ; c o n t a i n s t h e a c i d s ) . E x t r a c t 4 i s t a k e n t o a r e s i d u e and p a r t i t i o n e d between 9 0 % methanol and p e t r o l e u m e t h e r . These s o l v e n t s form an i m m i s c i b l e m i x t u r e and r e s u l t i n e x t r a c t 6 ( t h e aqueous methanol p h a s e ; c o n t a i n s t h e more p o l a r n e u t r a l materia l s , p a r t i c u l a r l y t h e t e r p e n e s and s t e r o l s ) and e x t r a c t 7 ( t h e p e t r o l e u m e t h e r ; c o n t a i n s f a t s and w a x e s ) . E x t r a c t s 6 and 7 are e v a p o r a t e d t o r e s i d u e s a t l e s s t h a n 40’ i n uacuo and t e s t e d f o r b i o a c t i v i t y . E x t r a c t 5 i s a c i d i f i e d s t r o n g l y w i t h hydroc h l o r i c a c i d and e x t r a c t e d w i t h c h l o r o f o r m t o produce e x t r a c t 8 ( t h e c h l o r o f o r m l a y e r ; c o n t a i n s t h e a c i d s ) which i s c a r e f u l l y e v a p o r a t e d and t e s t e d f o r a c t i v i t y . E x t r a c t 3 i s made a l k a l i n e t o a t l e a s t pH 1 0 w i t h ammonia and e x t r a c t e d w i t h c h l o r o f o r m t o produce e x t r a c t 9 ( t h e aqueous p h a s e ; c o n t a i n s t h e water s o l u b l e material, i n c l u d i n g any q u a t e r n a r y a l k a l o i d s p r e s e n t 1 and e x t r a c t 1 0 ( t h e c h l o r o f o r m l a y e r ; c o n t a i n s t h e a l k a l o i d s ) . Ext r a c t 1 0 i s e x t r a c t e d w i t h 5 % sodium h y d r o x i d e s o l u t i o n t o p r o duce e x t r a c t 11 ( t h e c h l o r o f o r m p h a s e ; c o n t a i n s t h e t e r t i a r y a l k a l o i d s ) and e x t r a c t 1 2 ( t h e aqueous p h a s e ; c o n t a i n s t h e p h e n o l i c a l k a l o i d s and o t h e r a m p h o t e r i c m a t e r i a l s ) . E x t r a c t 1 2 i s s a t u r a t e d w i t h ammonium c h l o r i d e and e x t r a c t e d w i t h c h l o r o form t o produce e x t r a c t 1 3 ( t h e c h l o r o f o r m l a y e r ; c o n t a i n s t h e p h e n o l i c a l k a l o i d s ) . E x t r a c t s 11 and 1 3 are d r i e d w i t h anhyd r o u s sodium s u l f a t e and e v a p o r a t e d t o r e s i d u e s which are t e s t e d f o r a n t i m i c r o b i a l a c t i v i t y . E x t r a c t 9 i s n e u t r a l i z e d and t h e n a p o r t i o n i s a c i d i f i e d w i t h g l a c i a l a c e t i c a c i d and t h e n a s a t u r a t e d aqueous s o l u t i o n o f ammonium r e i n e c k a t e i s added u n t i l p r e c i p i t a t i o n of t h e q u a t e r n a r y r e i n e c k e s a l t s ceases. T h i s p r e c i p i t a t e i s suspended i n 5 0 % a c e t o n e and p a s s e d o v e r an a n i o n exchange r e s i n column ( I R A - 4 1 0 , Cl’form, 300 g/60 g of r e i n e c k e s a l t ) . The e f f l u e n t c o n t a i n s t h e q u a t e r n a r y c h l o r i d e s a l t s and t h e s e are o b t a i n e d by l y o p h y l i z a t i o n a f t e r e v a p o r a t i o n of t h e a c e t o n e . The c h l o r i d e s a l t s are d i s s o l v e d i n water f o r a n t i m i c r o b i a l t e s t i n g . The r e m a i n i n g n e u t r a l aqueous phase i s l y o p h y l i z e d and t e s t e d f o r a n t i b i o t i c a c t i v i t y . Through t h i s s y s t e m a t i c p r o c e s s , o n l y u n s t a b l e o r trace s u b s t a n c e s can be missed. I f one works o n l y on b i o a c t i v e f r a c t i o n s , much o f t h i s p r o c e s s can be o m i t t e d once t h e p a t h of t h e a c t i v e material i s uncovered. F i n a l p u r i f i c a t i o n t o c r y s t a l l i n e o r o t h e r w i s e p u r e material depends upon t h e c h e m i c a l n a t u r e and number o f c o n s t i t u e n t s s o t h a t i t i s n o t p o s s i b l e t o g e n e r a l i z e beyond t h i s p o i n t e x c e p t t o i n d i c a t e t h a t i n most cases t h e e x t r a c t s can be b e s t f u r t h e r p u r i f i e d a t t h i s s t a g e by a b s o r p t i o n chromatography on s i l i c a g e l u s i n g t h e u s u a l e l u o t r o p i c s e r i e s of benzene t o e t h e r t o c h l o r o f o r m t o methanol. The acids and t h e q u a t e r n a r y bases may r e q u i r e i o n exchange chromatography and t h e l a t t e r may r e q u i r e c h a r c o a l chromatography as w e l l . U s u a l l y , however, s i l i c i c a c i d w i l l s u f f i c e . I n many i n s t a n c e s , a f t e r p r e l i m i n a r y s e p a r a t i o n by s i l i c a g e l chromatography, ? r e p a r a t i v e t h i n - l a y e r chromatography a n d / o r chromatography u s i n g TLC g r a d e s i l i c a g e l o r Sephadex LH-20 i s advantageous i n p r o ducing p u r e materials. P a r t i c u l a r d e t a i l s are b e s t h a n d l e d by t h e s p e c i f i c examples which f o l l o w . A s e r i e s o f p a p e r s can be c o n s u l t e d f o r an examp l e of a v e r y thorough u s e of t h i s s y s t e m t o i s o l a t e n e a r l y two
Q67
dozen p r o d u c t s from an a c t i v e e x t r a c t o f Thalictltum R u g o A ~ r n ~ ’ ~ . 3 . Quaternary Alkaloids.
Berberine.
While many q u a t e r n a r y a l k a l o i d s p o s s e s s a t l e a s t weak a n t i m i c r o b i a l a c t i v i t y , b e r b e r i n e d e s e r v e s t o be s i n g l e d o u t as t h e p r o t o t y p e on s e v e r a l g r o u n d s . Berberine-containing p l a n t s a r e v e r y w i d e s p r e a d o c c u r r e n c e b e i n g f o u n d i n many s p e c i e s o f t h e
BeltbeAidaceae, R a n u n c u l a c e a e , h o n a c e a e , Mcnibpeltmaceae, Papaweltaceae a n d R u t a c e a e and h a v e been u s e d f o r c e n t u r i e s i n ethnobotanical medicine. Recent s c i e n t i f i c e x a m i n a t i o n i n a number o f l a b o r a t o r i e s h a s c o n f i r m e d b e r b e r i n e ’ s r e l a t i v e l y weak b u t q u i t e b r o a d s p e c t r u m a c t i v i t y toward a number of p a t h o g e n i c m i c r o o r g a n i s m s l O . B e r b e r i n e seems n o t t o h a v e been s t u d i e d very thoroughly i n w i w o f o r t h i s purpose although t h e use o f p l a n t s r i c h i n b e r b e r i n e , s u c h as Phellodsndnon amuREnAell a n d P. w i l A O n i i l 2 by i n d i g e n o u s p o p u l a t i o n s i n t h e F a r East i s w e l l e s t a b l i s h e d and a t t r i b u t e d t o t h e i r b e r b e r i n e c o n t e n t . T h e r e are s i m i l a r r e p o r t s t h a t b e r b e r i n e - r i c h e x t r a c t s o f BenbeAih a a i A t a t a h a s been u s e d as a c h l o r a m p h e n i c o l s u b s t i t u t e i n t r e a t m e n t o f c h o l e r a 1 3 ’ 1 4 . Much o f t h i s work h a s r e c e n t l y been r e v i e w e d and n e e d n o t b e r e p e a t e d h e r e 1 ° ’ 1 5 . B e r b e r i n e , C20H100~N, i s a yellow, non-basic, quaternary ammonium s u b s t a n c e f o r m i n g well c h a r a c t e r i z e d , c r y s t a l l i n e , water-insoluble salts w i t h a v a r i e t y of companion the iodide is om3 it oy np si c oa fl . which The c h l o r i d e , on t h e o t h e r hand, p o s s e s s e s omJ s u f f i c i e n t water s o l u b i l i t y f o r most p u r p o s e s , b u t i s 1 i n s o l u b l e i n a l c o h o l o r common o r g a n i c s o l v e n t s . Melti n g p o i n t s are v a r i a b l e a n d , therefore, e s s e n t i a l l y useless i n t h i s s e r i e s . Berberine is s t a b l e i n a c i d i c and n e u t r a l s o l u t i o n s , b u t is converted t o a number o f p r o d u c t s , some o f which p o s s e s s s i g n i f i c a n t a n t i m i c r o b i a l a c t i v i t y t h e m s e l v e s when s t o r e d i n a l k a l i n e s o l u t i o n s 1 6 . These r e a c t i o n s are q u i t e g e n e r a l f o r t h e c l a s s as a c o n s e q u e n c e of t h e iminium f u n c t i o n a l i t y a n d are i l l u s t r a t e d i n t h e f o l l o w i n g f o r m u l a e (1-7). A d d i t i o n o f h y d r o x i d e i o n p r o d u c e s p s e u d o b a s e 2 which p o s s e s s e s more o r l e s s a l d e h y d i c p r o p e r t i e s i n t a u t o m e r 7 c form 2. T h i s s u b s t a n c e can u n d e r g o an a p p a r e n t C a n n i z z a r o r e a c t i o n t o p r o d u c e i n t e r m e d i a t e s 2 a n d 2. Cyclod e h y d r a t i o n p r o d u c e s r e s p e c t i v e l y l a c t a m 6 and d i h y d r o b a s e 1. A l k a l o i d 1 i s r e g a r d e d as an a r t i f a c t a n d r e c e n t work h a s shown t h a t i t r e t a i n s some i n W ~ Z J LaOn t i m i c r o b i a l a c t i v i t y 1 2 . A more amusing example was r e c e n t l y u n c o v e r e d when f r a c t i o n a t i n g b e r b e r i n e - r i c h p l a n t s by p a r t i t i o n i n g ammoniacal s o l u t i o n s w i t h c h l o r o f o r m 1 2 . Under t h e s e c o n d i t i o n s a d d u c t 8 i s i s o l a t e d a n d was found t o be more p o t e n t t h a n b e r b e r i n e i t s e l f . I t conceiva b l y i s a p r o d r u g a b l e t o p e n e t r a t e c e l l walls more successf u l l y than b e r b e r i n e i t s e l f because i t i s a t e r t i a r y base. U n t i l f u l l y understood, these p e c u l i a r i t i e s of berberine l e d t o
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When b e r b e r i n e i s t h e p r i n c i p a l a l k a l o i d of t h e p l a n t , i t can o f t e n b e c r y s t a l l i z e d d i r e c t l y 1 7 . The e t h a n o l e x t r a c t o f t h e powdered p l a n t i s e v a p o r a t e d a n d t h e r e s i d u e d i s s o l v e d as w e l l as p o s s i b l e i n h o t water. R e s i n s s e p a r a t e and t h e h o t s o l u t i o n i s f i l t e r e d . E x c e s s h y d r o c h l o r i c a c i d i s added and c h i l l i n g p r o d u c e s c r y s t a l s of b e r b e r i n e c h l o r i d e . T h i s i s p u r i f i e d by d i s s o l v i n g i n e t h a n o l a n d p r e c i p i t a t i n g w i t h e t h e r . N e x t , t h e c h l o r i d e i s d i s s o l v e d i n h o t w a t e r , t h i s i s made a l k a l i n e w i t h a few d r o p s of 1 0 % N a O H . Acetone i s added and d i l u t i o n w i t h an e q u a l amount of water p r e c i p i t a t e s b e r b e r i n e - a c e t o n e (2). A f t e r c o o l i n g o v e r n i g h t , t h i s i s f i l t e r e d o f f , washed w i t h c o l d water and d r i e d . B e r b e r i n e i s r e g e n e r a t e d i n a h i g h s t a t e of p u r i t y by d i s s o l v i n g i n e t h a n o l - c h l o r o f o r m ( 1 0 : 1) a n d boiling. On c o o l i n g , b e r b e r i n e i s o b t a i n e d . T h i s method i s q u i t e convenient b u t r a t h e r s p e c i f i c . A more g e n e r a l method which i s a d a p t e d also t o t h e i s o l a t i o n of o t h e r q u a t e r n a r y b a s e s when a m i x t u r e i s p r e s e n t i n v o l v e s c h r o m a t o g r a p h y l s . The m i x t u r e of q u a t e r n a r y c h l o r i d e s r e g e n e r a t e d from t h e r e i n e c k e s a l t s by i o n e x c h a n g e , as d e s c r i b e d i n t h e g e n e r a l s e c t i o n , i s d i s s o l v e d i n t h e minimum amount o f 1%metha n o l i n c h l o r o f o r m and i s chromatographed o v e r a s i l i c i c a c i d column ( a p p r o x i m a t e l y 30 g o f a b s o r b e n t p e r g o f m i x t u r e ) u s i n g a p p r o x i m a t e l y 5 column volumes o f 1%MeOH i n CHf, 5 o f 5% MeOH i n CHf, 15 o f 1 0 % MeOH i n CHf, 1 0 o f 2 0 % MeOH i n CHf, 5 o f 4 0 % MeOH i n CHf a n d 10 o f MeOH. Berberine u s u a l l y e l u t e s with 5% MeOH i n c h l o r o f o r m and i s e a s i l y d e t e c t e d b e c a u s e o f i t s c o l o r . 4. T e r t i a r y A l k a l o i d s .
Liriodee.
A g r e a t many t e r t i a r y a l k a l o i d s show a n t i m i c r o b i a l a c t i v i t y . L i r i o d e n i n e (lo),C , , H , N O 3 , m.p. 28O-28l0C, h a s been chosen t o t y p i f y t h i s g r o u p b e c a u s e of t h e c l e a r way i n which i t s i s o l a t i o n i s described and because i t is q u i t e p o t e n t i n v i t h o l g . The a i r - d r i e d ground h e a r t w o o d ( 2 . 2 k g ) o f L i t i o d e n d h o n t u l i p i d Q A a L. ( t u l i p t r e e o r y e l l o w p o p l a r ) was p e r c o l a t e d w i t h e t h a n o l and e v a p o r a t e d t o g i v e 7 1 g o f a r e s i d u e which was a n t i m i c r o b i a l l y a c t i v e i n an a g a r - w e l l a s s a y a g a i n s t s e v e r a l b a c t e r i a . A 35 g sample was p a r t i t i o n e d between 125 m l e a c h o f e t h e r a n d 2 % c i t r i c a c i d . The e t h e r l a y e r was e x t r a c t e d twice more w i t h 1 2 5 m l p o r t i o n s of a c i d a n d f i l t e r e d t o remove an inert interfacial precipitate. A l l t h e a c t i v i t y was i n t h e a c i d
470
l a y e r s so t h e s e were a d j u s t e d t o pH 9-10 w i t h ammonia a n d t h e n e x t r a c t e d t h r e e times w i t h 1 - L p o r t i o n s o f c h l o r o f o r m . A f t e r d r y i n g w i t h a n h . sodium s u l f a t e , 6.2 g o f an a c t i v e a l k a l o i d r e s i d u e was o b t a i n e d , T h i s was s e p a r a t e d i n t o t e r t i a r y a n d t e r t i a r y p h e n o l i c a l k a l o i d s by d i s s o l v i n g t h e r e s i d u e i n 250 m l o f c h l o r o f o r m a n d e x t r a c t i n g w i t h 3 X 250 m l p o r t i o n s of 5 % NaOH s o l u t i o n . A f t e r d r y i n g a n d e v a p o r a t i n g t h e c h l o r o f o r m l a y e r , t h e t e r t i a r y n o n - p h e n o l i c a l k a l o i d s weighed 4 . 7 g a n d c o n t a i n e d a l l t h e a c t i v i t y . A 2 g p o r t i o n was d i s s o l v e d i n c h l o r o f o r m and chromatographed o v e r 200 g o f aluminum o x i d e (Woelm, n e u t r a l , g r a d e 111). L i r i o d e n i n e was t h e most p o t e n t c o n s t i t u e n t a n d e l u t e d w i t h 1%m e t h a n o l i n c h l o r o f o r m i n p u r e enough c o n d i t i o n t o c r y s t a l l i z e .
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11 10 5 . P h e n o l s . C h r y s o p h a n i c Acid-9-Anthrone and 5 , 7 , 2 ' , 4 ' Te t r a h y d r o x y - 6 - ( 3 ,3 - D i m e t h y l a l l y l ) -1sof l a v o n e
P h e n o l s h a v e l o n g been known t o h a v e a n t i s e p t i c a c t i v i t y , b u t t h e s i m p l e p h e n o l s a r e membrane t o x i n s and h a v e i n s u f f i c i e n t s a f e t y f o r i n t e r n a l u s e . A s might b e e x p e c t e d , s i n c e p l a n t s are
rich i n phenolic e x t r a c t i v e s ? phenolic p l a n t products have f r e q u e n t l y been f o u n d t o be a n t i m i c r o b i a l a g e n t s . I n o r d e r t o a v o i d n o n s e l e c t i v i t y as f a r as p o s s i b l e , modern work h a s a v o i d e d t h e v o l a t i l e o r " e s s e n t i a l " o i l s and h a s c o n c e n t r a t e d on more complex m a t e r i a l s which p r o m i s e t o b e more s e l e c t i v e . One o f t h e more p o t e n t p l a n t p h e n o l i c s t o b e d e s c r i b e d i n r e c e n t y e a r s i s chrysophanic acid-9-anthrone. T h i s m a t e r i a l comes from Cdddia t o k a Z 0 and v a r i o u s o t h e r p l a n t s , s u c h as a l o e s , which h a v e been u s e d f o r l a x a t i v e s s i n c e p r e h i s t o r i c times. A p a s t e made o f t h e g r o u n d s e e d s o f t h i s p l a n t h a s been u s e d i n r u r a l p a r t s o f I n d i a f o r t r e a t m e n t o f s k i n d i s e a s e s . Dry m a t u r e s e e d s of Caddis t a k a were ground and t h e f i n e y e l l o w s e e d powd e r was d e f a t t e d by S o x h l e t e x t r a c t i o n w i t h p e t r o l e u m e t h e r f o r 1 2 hours. The d e f a t t e d s e e d powder (250 g ) was made i n t o a p a s t e w i t h 625 m l o f water and t h e p a s t e was e x t r a c t e d by s h a k i n g w i t h 1 . 6 1 o f b e n z e n e . E v a p o r a t i o n o f t h e benzene p r o d u c e d a r e d d i s h y e l l o w powder (1 g ) which was a c t i v e i n u i h o a g a i n s t T f i i c h o p h y t o n fiubkum. The r e s i d u e was d i s s o l v e d i n 40 m l o f h o t c h l o r o f o r m a n d chromatographed on a 50 g column o f s i l i c a g e l G. The f a s t e s t m o v i n g o c o l o r e d band p r o v e d t o be t h e a n t h r o n e 11, C 1 ~ H 1 ~ 0 3m.p. , 200 which showed ronounced a c t i v i t y i n 7 v.ict/ro ( 3 t o 10 mcg/ml) a g a i n s t T a i c h o p y t o n fiubaum, T . m e n t a g f i o p h y t e d , Michodponum C a n i d , M . g y p d e u m a n d G e o t f i i c h u m c a n d i -
,
5:
durn. Another i n t e r e s t i n g compound is 5 , 7 , 2 ' 4 ' - t e t r a h y d r o x y - 6 -
471
12 (3,3-dimethylallyl)-isoflavone (GIz1. The b i o a c t i v e immature f r u i t s o f LupinuA & ~ i k u A (60 k g ) were minced i n m e t h a n o l a n d t h e s o l v e n t was c o n c e n t r a t e d c a r e f u l l y i n vacu0 t o a n a q u e o u s p h a s e . T h i s c o n c e n t r a t e was a d j u s t e d t o pH 3 w i t h ( 6 N ) H C 1 and e x t r a c t e d w i t h b e n z e n e . E v a p o r a t i o n of t h e b e n z e n e l a y e r g a v e 120 g of a b i o a c t i v e c o n c e n t r a t e which was c h r o m a t o r a p h e d on a 1 : 2 m i x t u r e o f s i l i c i c a c i d and CeliteR ( t h e C e l i t e f a c i l i t a t e s t h e f l o w r a t e ) . E l u t i o n began w i t h b e n z e n e and when 30% e t h y l a c e t a t e was p r e s e n t i n t h e m i x t u r e , t h e b i o a c t i v i t y e l u t e d . E v a p o r a t i o n gave 33 g o f material which was p u r i f i e d by chrometography o v e r F l o r i s i l R . The d e s i r e d p r o d u c t , C Z 0 H l 8 O 6 , m.p. 225-227, 0.83 g , e l u t e d w i t h 5 % e t h y l acetate i n b e n z e n e . T h i s p r o d u c t i s a c t i v e a g a i n s t b a c t e r i a a t 2 5 t o 1 0 0 mcg/ml a n d a g a i n s t s e l e c t e d f u n g i a t a b o u t 10 mcg/ml.
fi
6 . Acids.
Wyerone A c i d .
Many f a t t y a c i d d e r i v a t i v e s h a v e some a n t i f u n g a l a c t i v i t y s o i t i s n o t s u r p r i s i n g t h a t some a n t i m i c r o b i a l a g e n t s from h i g h e r p l a n t s b e l o n g t o t h i s c h e m i c a l c l a s s as w e l l . One o f some r e c e n t i n t e r e s t b e c a u s e of t h e b i o l o g i c a l i n t e r a c t i o n s i n v o l v e d i s a p h y t o a l e x i n i s o l a t e d from t h e b r o a d b e a n ( U i c i a $aba L . ) a f t e r i n f e c t i o n w i t h t h e p l a n t p a t h o g e n i c f u n g u s BotaytLtid dabae22. F r o z e n , i n f e c t e d l e a v e s ( 3 5 0 g ) were macera t e d w i t h 80% e t h a n o l ( 1 . 5 l), f i l t e r e d a n d t h e f i l t r a t e conc e n t r a t e d t o an a q u e o u s s o l u t i o n ( 5 0 m l ) .
13 The c o n c e n t r a t e was d i l u t e d w i t h 50 m l o f 5% s o d i u m c a r b o n ate a n d e x t r a c t e d w i t h f o u r 500 m l p o r t i o n s of e t h e r . The aqueous p h a s e was a d j u s t e d t o pH 4 w i t h p h o s p h o r i c a c i d a n d e x t r a c t e d w i t h f o u r 500 m l p o r t i o n s o f e t h e r . The e t h e r l a y e r s were c o n c e n t r a t e d t o a r e s i d u e which p a r t i a l l y d i s s o l v e d i n m e t h y l e n e c h l o r i d e . The C H 2 C l 2 s o l u t i o n was s u b j e c t e d t o p r e p a t i v e TLC on s i l i c a g e l u s i n g M e O H / C H 2 C l Z 1 : 5 f o r d e v e l o p m e n t . The a c t i v e b l u e f l u o r e s c e n t b a n d was e x t r a c t e d w i t h w a r m e t h a n o l a n d e v a p o r a t e d t o g i v e wyerone a c i d , C I 4 H 1 , O 4 , ( 1 3 ) . The p r o d u c t is moderately a c t i v e a g a i n s t t h e i n f e c t i n g E a y a A s t r a i n . Wyerone i t s e l f i s t h e c o r r e s p o n d i n g m e t h y l e s t e r a n d has b e e n i s o l a t e d f r o m t h e n o n - i n f e c t e d p l a n t z 3 . I t is a n t i f u n g a l a l s o , b u t i s n o t as p o t e n t as t h e a c i d . P r e s u m a b l y , t h e i n f e c t i n g
472
s t r a i n h y d r o l y z e s c o n s t i t u i t i v e wyerone ' i n r e s i s t a n t s p e c i e s a n d t h e r e b y limits i t s own p a t h o g e n i c i t y . - Wyerone i s i s o l a t e d by u s i n g low t e m p e r a t u r e s a n d w r a p p i n g s i l i c a g e l columns w i t h b l a c k c l o t h . T h i s i s a w o r t h w h i l e t e c h n i q u e when working w i t h p h o t o s e n s i t i v e acetylenes of t h i s kind. 7 . Neutral S u b s t a n c e s .
Piperine.
A n t i m i c r o b i a l a c t i v i t y used i n t h e e t h n o m e d i c a l s y s t e m of West A f r i c a was r e p o r t e d f o r t h e A s h a n t i p e p p e r p l a n t , Pipeh The d r i e d f r u i t (500 g ) was gUinAenAe Schum. and
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14 m a c e r a t e d w i t h a c e t o n e to g i v e 1 7 . 9 4 g of e x t r a c t i v e . This was p a r t i t i o n e d between 500 ml e a c h of e t h e r a n d water. The b i o a c t i v e e t h e r l a y e r was washed s u c c e s s i v e l y w i t h t h r e e 500 m l p o r t i o n s of 1%H C 1 and t h r e e 500 m l p o r t i o n s of 2 % NaHC03. Evap o r a t i o n o f t h e e t h e r l a y e r s gave 1 6 . 8 3 g of an o i l which was c h r o m a t o g r a p h e d on 500 g of s i l i c i c a c i d . P e t r o l e u m e t h e r / as t h e main a c t i v e c h l o r o f o r m 3:7 gave 1 . 3 3 g of p i p e r i n e (2) component. I t s a c t i v i t y , however, was r e s t r i c t e d t o a p p r o x i m a t e l y 1 0 0 mcg/ml a g a i n s t Mycobactehium dmegmaZiA a n d C a n d i d a
atbicanA ,
8. G l y c o s i d e s .
Tuliposides A and B.
T s c h e s c h e ' s l a b o r a t o r y h a s i n v e s t i g a t e d t h e p r o p e r t i e s of numerous a n t i m i c r o b i a l l y a c t i v e g l y c o s i d e s o f p l a n t o r i ' The The t u l i p o s i d e s are i l l u s t r a t i v e and of r e c e n t i n t e r e s t stems a n d l e a v e s ( 3 5 k g ) of TuLipa gednehiana L. were minced w i t h 30 1 of 80% MeOH t h r e e times aifd t h e f i l t r a t e s were conc e n t r a t e d t o an aqueous p h a s e a t 40 T h i s was e x t r a c t e d s u c c e s s i v e l y w i t h c y c l o h e x a n e a n d t h e n c h l o r o f o r m . The b i o a c t i v e aqueous p h a s e was c o n c e n t r a t e d t o 1 . 9 kg o f a s y r u p . Of t h i s , 7 g were c h r o m a t o g r a p h e d on 6 0 0 g o f c e l l u l o s e powder ( M u n k t a l l no. 4 0 0 ) e l u t i n g w i t h e t h y l a c e t a t e / p y r i d i n e / w a t e r 3.6:1:1.15. R a p i d r u n n i n g (1 L / h r ) o f a g h o r t , f a t , column gave 7 2 0 mg of t u l i p o s i d e A , ( 1 5 ) { ~ 3+ ~6 4~ (c=l.O, H20) which i s a c t i v e a g a i n s t B . d u b t m A ae 1 0 0 mcg/ml. The p r o d u c t i s n o t s t a b l e , l o s i n g 75% of i t s a c t i v i t y i n f o u r weeks a t - 2 0 ' whereupon t h e p o s i t i o n a l isomer (19)i s i s o l a b l e . I t is a c t i v e only at about 1 2 0 0 mcg/ml. H y d r o l y s i s w i t h d i l u t e a c i d or s n a k e venom g i v e s Further a-methylene l a c t o n e 2 which i s a c t i v e a t 200 mcg/ml. e l u t i o n o f t h e c z l l u l o s e column gave 2 6 0 mg of t u l i p o s i d e B ( 2 1 , IaIiO + 56 ( c = l . O , H20) which i s a c t i v e a t 40 mcg/ml a g a i n s t 8 . d u b a t i A and can b e h y d r o l y z e d t o l a c t o n e (2) which i s a c t i v e a t 1 0 0 mcg/ml. I n e a c h i n s t a n c e h y d r o l y s i s o r rea r r a n g e m e n t l e d t o l e s s a c t i v e m a t e r i a l s . The h y d r o l y s i s p r o d u c t s 16 a n d 18 are s i m p l e members of t h e a - m e t h y l e n e l a c t o n e s , a g r o u p f r e q u e n t l y s t u d i e d of l a t e b e c a u s e o f t h e i r a n t i t u m o r
s5?
.
473
15
16
CH20H
OH
"O
0
CHCHZOH I
OH
17
18
19 activity. 9. C o n c l u s i o n T h e s e r e l a t i v e l y f e w e x a m p l e s i l l u s t r a t e t h e r a n g e of t e c h n i q u e s i n u s e a n d t h e d i v e r s i t y of t h e s t r u c t u r e s i n v o l v e d . A d d i t i o n a l s p e c i f i c e x a m p l e s can b e f o u n d b y c o n s u l t i n g t h e most recent r e v i e w s i n a n d r e f e r e n c e s 26-66.
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1.
2.
............................ 481 ................................ Q82 ............................. 483 ......................... 483 .......................... 4 8 4 Monensin 2.1 Introduction....,...... ..................... 4 9 2 2.1.1 P r o d u c i n g o r g a n i s m . . ...................... h92 Chemical and p h y s i c a l p r o p e r t i e s .......... lrg2 2.1.2 2.1.3 S t r u c t u r a l f o r m u l a . . ...................... 492 2.2 Summary of b i o l o g i c a l a c t i v i t i e s and u s e s . . . 4 9 2 2.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n . . ... 4 9 3 General I n t r o d u c t i o n . . Extraction.. Fractionation.. D e t e c t i o n and a s s a y Literature Cited..
1.1 1.2 1.3 1.4 -
2.3.1 2.3.2 2.3.3 2.3.4 2.4
3.
I s o l a t i o n method No. l.................... I s o l a t i o n method No. 2 . . . . . . . . . . . . . . . . . . . . S e p a r a t i o n of i n d i v i d u a l f a c t o r s Methods f o r d e t e c t i o n and a s s a y L i t e r a t u r e Cited..
49 3 49 4 495 lrg5 496
.......... ........... .......................... L a s a l o c i d Complex ( X - 5 3 7 A Y R 0 - 2 - 2 9 8 5 ) 3.1 Introduction............ .................... 44 99 88 3.1.1 P r o d u c i n g o r g a n i s m . . ...................... 3.1.2 Chemical and p h y s i c a l p r o p e r t i e s . . ........ 4 9 8 3.1.3 S t r u c t u r a l f o r m u l a . . ...................... 498 Summary of b i o l o g i c a l a c t i v i t i e s and uses... 4 9 9 3.2 3.3 E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n . . ... 4 9 9 3.3.1 L a s a l o c i d i s o l a t i o n . . ..................... 499 3.3.2 I s o l a t i o n of i d o - l a s a l o c i d A and l a s a l o c i d 500 f a c t o r s B , C, D, and E .................... 3.3.3 D e t e c t i o n and a s s a y . ...................... 501 3.4 L i t e r a t u r e C i t e d . . .......................... -
4.
Narasin ( A 2 8 0 8 6 A ) Introduction.. Producing organism.. Chemical and p h y s i c a l p r o p e r t i e s . . 4.1.2 4.1.3 Structural formula........................ 4.2 Summary of b i o l o g i c a l a c t i v i t i e s and u s e s . . . S e p a r a t i o n and p u r i f i c a t i o n m e t h o d s . . . . . . . . . 4.3 I s o l a t i o n of c r u d e n a r a s i n complex.. 4.3.1 P u r i f i c a t i o n of n a r a s i n complex.... 4.3.2 b.3.3 S e p a r a t i o n of n a r a s i n A, B , and D . . . . . . . . . 4.3.4 P r e p a r a t i o n of t h e sodium s a l t of n a r a s i n . 4.3.5 ' D e t e c t i o n and a s s a y . . . . . . . . . . . . . . . . . . . . . . . 4.4 L i t e r a t u r e Cited..
4.1 4.1.1
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.............................. 503 ...................... ........ 55 0033
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50 3
503 50 4 504 504
505 505 506 507
480
5
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Lonomycin 5.1 Introduction Producing organisms 5.1.1 Chemical a n d p h y s i c a l p r o p e r t i e s 5.1.2 5.1.3 S t r u c t u r a l formula 5.2 Summary of b i o l o g i c a l a c t i v i t i e s a n d u s e s Extraction. s e p a r a t i o n and p u r i f i c a t i o n 5.3 5.3.1 I s o l a t i o n m e t h o d f o r DE-3936 5.3.2 Detection and assay 5.4 L i t e r a t u r e Cited
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6.1 6.1.1 6.1.2 6.1.3 6.2 6.3 6.3.1 6.3.2 6.3.3
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6.3.4 6.3.5
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A23187
7.1 7.1.1
7.1.2 7.1.3 7.2 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.4
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................................. 508 ........................ 508 ........... ............................. 555 000 888 ...... 509 ............... 509 ........................ ............................. 509 509 I n t r o d u c t i o n ................................. 5 1 1 P r o d u c i n g o r g a n i s m ......................... 511 C h e m i c a l d e s c r i p t i o n ....................... 511 S t r u c t u r a l f o r m u l a ......................... 511 Summary of b i o l o g i c a l a c t i v i t i e s a n d u s e s .... 5 1 1 S e p a r a t i o n a n d p u r i f i c a t i o n m e t h o d s .......... 512 I s o l a t i o n of A204 c o m p l e x .................. 512 S e p a r a t i o n o f A204 f a c t o r s ................. 5 1 3 P r e p a r a t i o n of A204A m e t h y l e t h e r d e r i v a t i v e from A204A s o d i u m s a l t ................ 5 1 3 P r e p a r a t i o n of A204A m e t h y l e t h e r d e r i v a t i v e f r o m A204A ............................ 513 514 D e t e c t i o n a n d a s s a y ........................ L i t e r a t u r e C i t e d ............................. 514 ................................. 516 ......................... 516 ........... 516 ......................... .... 516 ...... 55 11 77 .. ..................... 517 ..................... 518 ..... 518 ........................ .............................519 519
Introduction Producing organism Chemical and p h y s i c a l p r o p e r t i e s S t r u c t u r a l formula Summary o f b i o l o g i c a l a c t i v i t i e s and u s e s Separation. e x t r a c t i o n and p u r i f i c a t i o n I s o l a t i o n m e t h o d No 1 I s o l a t i o n m e t h o d No 2 P r e p a r a t i o n of t h e a c i d form of A23187 Detection and assay L i t e r a t u r e Cited
481 1.. G e n e r a l I n t r o d u c t i o n
I n 1 9 6 7 , Agtarap e t a1.l r e p o r t e d t h e d e t e r m i n a t i o n of t h e s t r u c t u r e of t h e newly i s o l a t e d a n t i b i o t i c , monensin. T h i s p u b l i c a t i o n marked t h e r e c o g n i t i o n of a new c l a s s of a n t i b i o t i c s c h a r a c t e r i z e d by a s i n g l e c a r b o x y l i c a c i d g r o u p , a number of c y c l i c e t h e r s , a few h y d r o x y l s , and a l a r g e number of C - a l k y l g r o u p s . I n 1 9 5 1 , Harned e t a.!.2 had r e p o r t e d t h e i s o l a t i o n of had reported n i g e r i c i n , and, i n t h e same y e a r , B e r g e r e t t h e i s o l a t i o n of t h r e e a n t i b i o t i c s d e s i g n a t e d X-537A (now known as l a s a l o c i d ) , X - 2 0 6 , and X - 4 6 4 ( l a t e r shown t o b e t h e same as n i g e r i c i n ) . However, t h e s t r u c t u r e s of t h e s e a n t i b i o t i c s were n o t d e t e r m i n e d u n t i l 1968 and l a t e r . A s of mid-1976, t h e p o l y e t h e r c l a s s c o n s i s t e d of 1 9 a n t i b i o t i c s a n d , i n a d d i t i o n , t h e i r coproduced a n a l o g s . These a r e l i s t e d i n Table 1. The l i t e r a t u r e c i t e d includes r e f e r e n c e s t o t h e i s o l a t i o n procedure and, where a p p l i c a b l e , t h e s t r u c t u r e . The a n t i b i o t i c A 2 3 1 8 7 i s a t y p i c a l of t h i s c l a s s b e c a u s e i t c o n t a i n s n i t r o g e n ; however, i t i s i n c l u d e d i n t h i s c h a p t e r b e c a u s e i t does s h a r e many of t h e unique f e a t u r e s o f t h e p o l y e t h e r s . I n t e r e s t was s t i m u l a t e d i n t h e s e a n t i b i o t i c s when monensin was found t o be e f f e c t i v e i n c o n t r o l l i n g c o c c i d i o s i s i n p o u l t r y and o t h e r a n i m a l s 4 . More r e c e n t l y , monensin has been r e p o r t e d t o improve f e e d e f f i c i e n c y i n r u m i n a n t s 5 . Thus, t h e f i r s t p o l y e t h e r a n t i b i o t i c s , r e p o r t e d i n 1951, which were i n i t i a l l y t h o u g h t t o have l i t t l e o r no p r a c t i c a l u s e d u e t o t h e i r r e l a t i v e l y h i g h p a r e n t e r a l t o x i c i t y and l a c k of good i n v i v a a n t i m i c r o b i a l a c t i v i t y , now have commercial p o s s i b i l i t i e s . A n t i o c c c i d i a l and f e e d - e f f i c i e n c y - i m p r o v i n g p r o p e r t i e s have been r e p o r t e d f o r many of t h e p o l y e t h e r a n t i b i o t i c s . Another g e n e r a l p r o p e r t y p o s s e s s e d by t h e p o l y e t h e r a n t i b i o t i c s i s t h e i r a b i l i t y t o t r a n s p o r t c a t i o n s a c r o s s membranes a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . These compounds, t h e r e f o r e , b e l o n g t o a g e n e r a l c l a s s d e s i g n a t e d as i o n o p h o r e s ( i o n - b e a r e r s ) . S e e Pressman f o r a r e c e n t review. Although i o n o p h o r e s were i n i t i a l l y i n t e r e s t i n g a s biochemical t o o l s i n experimental biol o g i c a l s y s t e m s , r e c e n t l y , t h e p o s s i b l e u s e of t h e s e compounds i n man as p o s i t i v e i n o t r o p i c a e n t s ( s t i m u l a t i n g c a r d i a c cont r a c t i l i t y ) h a s been s u g g e s t e d , The d i f f e r e n c e s i n c a t i o n s p e c i f i c i t y among t h e p o l y e t h e r a n t i b i o t i c s have s u g g e s t e d nonb i o l o g i c a l uses s u c h as s p e c i f i c - i o n e l e c t r o d e s e and r e c o v e r y of m e t a l s t h r o u g h s e m i - s o l i d membranesg.
9
The p o l y e t h e r s have a number of common p r o p e r t i e s . A s mentioned above, A23187 i s n o t t y p i c a l of t h i s group and t h e g e n e r a l i z a t i o n s do n o t a p p l y . Each p o l y e t h e r h a s an a p p r o x i m a t e ranges i n molecular weight e m p i r i c a l f o r m u l a of ( C 3 - 4 H 5 - 7 0 ) n , from 5 0 0 t o 1 0 0 0 , and i s a monocarboxylic a c i d . The h i g h oxygen c o n t e n t i s due t o a number o f c y c l i c e t h e r s , a few h y d r o x y l g r o u p s , and methoxyl g r o u p s . The compounds a l s o have a l a r g e number of C - a l k y l ( m e t h y l , e t h y l ) g r o u p s . Carbon backbone l e n g t h s of C 2 4 , C 2 6 , C30, a n d C 3 6 have been n o t e d w i t h t h e publ i s h e d s t r u c t u r e s l o ; and a c o n s i s t e n t numbering s y s t e m f o r t h e p o l y e t h e r a n t i b i o t i c s h a s been r e c e n t l y p r o p o s e d l l . L a s a l o c i d and i t s a n a l o g s a r e t h e o n l y p o l y e t h e r a n t i b i o t i c s known t o
482 p o s s e s s an a r o m a t i c g r o u p . Dianemycin, A 2 0 4 A , A 2 8 6 9 5 A ( s e p t a m y c i n ) , and CP38295 a l l c o n t a i n g l y c o s i d e - l i k e branched t e t r a h y d r o p y r a n y l r i n g s . Highly r e a c t i v e h e m i k e t a l g r o u p s a r e p r e s e n t i n A204AyA28695A, CP38295, and lonomycin. The s t r u c t u r e s o f t h e p o l y e t h e r a n t i b i o t i c s have been e l u c i d a t e d a l m o s t e n t i r e l y by x-ray a n a l y s i s of t h e i r heavy metal s a l t s , I n t h e c r y s t a l l i n e s t a t e , t h e p o l y e t h e r molecule e x i s t s i n a c y c l i c c o n f o r m a t i o n w i t h t h e c a r b o x y l g r o u p on one e n d h e l d by a hydrogen bond t o a t e r t i a r y h y d r o x y l group on t h e terminal c y c l i c e t h e r . The a l k y l g r o u p s e x t e n d o u t s i d e t h e m o l e c u l e , i m p a r t i n g t h e hydrophobic p r o p e r t y ; t h e oxygen f u n c t i o n s a r e c o n c e n t r a t e d i n t h e c e n t e r . The c a t i o n i s h e l d by l i g a n d s w i t h i n t h e formed p o c k e t , r e n d e r i n g t h e s a l t v i r t u a l l y i n s o l u b l e i n water b u t v e r y s o l u b l e i n s o l v e n t s s u c h as c h l o r i n a t e d hydrocarbons, esters , k e t o n e s , a l c o h o l s , and benzene. The f r e e a c i d s and s a l t s have similar s o l u b i l i t y c h a r a c t e r i s t i c s .
Because t h e p o l y e t h e r s have many common p r o p e r t i e s , gene r a l i s o l a t i o n and p u r i f i c a t i o n p r o c e d u r e s w i l l be d i s c u s s e d b e f o r e g i v i n g s p e c i f i c examples of i s o l a t i o n p r o c e d u r e s . Monens i n , l a s a l o c i d , A 2 0 4 , n a r a s i n , A23187, and lonomycin w i l l b e discussed in d e t a i l later i n t h e chapter.
1.1 E x t r a c t i o n Because of t h e h i g h o r g a n i c - s o l v e n t s o l u b i l i t y of b o t h t h e f r e e a c i d s and s a l t s , s o l v e n t e x t r a c t i o n is t h e most p r a c t i c a l i s o l a t i o n p r o c e d u r e . The p o l y e t h e r a n t i b i o t i c s , when produced i n l o w - y i e l d i n g f e r m e n t a t i o n s , are o f t e n p r e s e n t i n b o t h t h e b r o t h f i l t r a t e a n d t h e m y c e l i a l f i l t e r cake; b u t as y i e l d s i n c r e a s e , most o f t h e a n t i b i o t i c a c t i v i t y i s c o n t a i n e d i n t h e f i l t e r cake, e s p e c i a l l y i f t h e b r o t h i s a l l o w e d t o s t a n d f o r a p e r i o d a f t e r h a r v e s t . The a n t i b i o t i c s are r e a d i l y removed from t h e f i l t e r cake by e x t r a c t i o n w i t h w a t e r - m i s c i b l e s o l v e n t s s u c h as methanol o r a c e t o n e o r w i t h w a t e r - i m m i s c i b l e s o l v e n t s s u c h as c h l o r o f o r m , e t h y l a c e t a t e , or benzene. The h i g h nonp o l a r i t y o f l a s a l o c i d a p p a r e n t l y a i d s i t s e x t r a c t i o n i n t o hexane u s i n g a S o x h l e t E x t r a c t o r . Although e x t r a c t i o n o f t h e whole b r o t h w i t h w a t e r - i m m i s c i b l e o r g a n i c s o l v e n t s h a s been used, e m u l s i o n s are o f t e n a problem. E x t r a c t i o n s are g e n e r a l l y carr i e d o u t on a l k a l i n e b r o t h s (pH 8 . 5 - 9 . 5 ) t o o b t a i n t h e s t a b l e s a l t s . The f r e e a c i d s can be o b t a i n e d by e x t r a c t i o n of t h e a c i d b r o t h (pH 3-51, however, t h i s e x t r a c t i o n must be done caref u l l y due t o t h e i n s t a b i l i t y of t h e f r e e a c i d s . The g l y c o s i d i c l i n k a g e s and h e m i k e t a l f u n c t i o n s a r e e s p e c i a l l y l a b i l e . Compounds c o n t a i n i n g h e m i k e t a l f u n c t i o n s form k e t a l s v e r y r a p i d l y w i t h lower a l c o h o l s and t h i o l s i n a c i d i c s o l u t i o n s , as r e p o r t e d f o r A204A12 and p o l y e t h e r i n A ( n i g e r i c i n ) 1 3 ; care m u s t be t a k e n i n t h e e x t r a c t i o n o f t h e f i l t e r cake t o keep t h e pH above 6 .O w i t h t h e s e a n t i b i o t i c s . D a r k g r e e n o r b l u e s o l u t i o n s w i l l res u l t when e x t e n s i v e d e g r a d a t i o n h a s t a k e n p l a c e . f o r m a t i o n of t h e d e g r a d a t i o n p r o d u c t s c a n a l s o b e f o l l o w e d by TLC. The k e t aLs o r e t h e r s formed are b i o l o g i c a l l y a c t i v e and r e t a i n t h e i r i o n o p h o r i c p r o p e r t i e s . E x t r a c t i o n of t h e whole b r o t h and a z e o t r o p i c d i s t i l l a t i o n with hexyl alcohol o r benzyl alcohol has been r e p o r t e d for monensin and w i l l b e d i s c u s s e d l a t e r .
b83 The s t r e p t o m y c e t e s which p r o d u c e p o l y e t h e r a n t i b i o t i c s q u i t e o f t e n p r o d u c e l a r g e amounts o f l i p i d - l i k e compounds which are e x t r a c t e d w i t h t h e a n t i b i o t i c s and c o m p l i c a t e f u r t h e r p u r i f i c a t i o n . Removal of t h e l i p i d - l i k e compounds h a s been accomp l i s h e d b y s e l e c t i v e e x t r a c t i o n w i t h n o n - p o l a r s o l v e n t s s u c h as h e x a n e o r p e t r o l e u m e t h e r s o r by s i l i c a g e l c h r o m a t o g r a p h y , u s i n g b e n z e n e t o wash o f f t h e o i l s , b e f o r e e l u t i n g t h e a n t i b i o t i c s w i t h more p o l a r s o l u t i o n s . A n o t h e r p r o c e d u r e f o u n d t o be u s e f u l i n removing t h e s e s u b s t a n c e s from t h e o r i g i n a l e x t r a c t i s t o a d s o r b t h e i m p u r i t i e s on a column o f d e c o l o r i z i n g c a r b o n a n d t h e n t o wash t h e column w i t h c o p i o u s amounts o f s o l v e n t . A l t h o u g h a number o f w a t e r - i m m i s c i b l e s o l v e n t s may be u s e d , c h l o r o f o r m a p p e a r s t o w o r k b e s t f o r t h i s method. T h i s p r o c e d u r e i s d i s c u s s e d f u r t h e r i n t h e monensin i s o l a t i o n s e c t i o n . D i k e t o p i p e r a z i n e s a r e a common unwanted i s o l a t i o n p r o d u c t i n l a b o r a t o r i e s c o n c e r n e d w i t h fermentation^^^. These d i k e t o p i p e r a z i n e s {most commonly a n h y d r i d e s o f p r o l i n e and o n e o f t h e b r a n c h e d - c h a i n amino a c i d s ( v a l i n e , l e u c i n e , o r i s o l e u c i n e ) } have similar s o l u b i l i t i e s t o t h e p o l y e t h e r a n t i b i o t i c s and, consequently, are l i k e l y t o c o - e x t r a c t . S i l i c a g e l chromatog r a p h y o r s e l e c t i v e c r y s t a l l i z a t i o n a r e u s e f u l methods o f s e p a r a t i n g t h e s e d i k e t o p i p e r a z i n e s from t h e p o l y e t h e r s .
1.2 Fractionation As w i t h most f e r m e n t a t i o n s , b o t h c l o s e l y r e l a t e d a n d unr e l a t e d a n t i b i o t i c s are o f t e n c o p r o d u c e d w i t h t h e m a j o r p o l y e t h e r a n t i b i o t i c . The p r o b l e m s o f s e p a r a t i n g a n d p u r i f y i n g e a c h component c a n b e c h a l l e n g i n g . The most common method employed t o s e p a r a t e p o l y e t h e r a n t i b i o t i c s i s b y a d s o r p t i o n column c h r o matography u s i n g s i l i c a g e l o r a l u m i n a . S i l i c a g e l columns u s i n g b e n z e n e and i n c r e a s i n g c o n c e n t r a t i o n s o f e t h y l a c e t a t e h a v e worked v e r y s a t i s f a c t o r i l y i n t h e s e p a r a t i o n s o f many p o l y e t h e r a n t i b i o t i c s , i n c l u d i n g m o n e n ~ i n ~A 2~0,b 1 6 , A2869517, a n d n a r a s i n f a c t o r s 1 * . C h l o r o f o r m - m e t h a n o l m i x t u r e s have a l s o b e e n u s e f u l . Alumina columns were u s e d i n t h e i s o l a t i o n o f s a l i n o m y c i n l 9 , K 3 5 8 ( g r i s o r i x i n ) 2 0 , a n d DE3936 ( l o n o m y c i n ) 2 1 . C o u n t e r c u r r e n t d i s t r i b u t i o n h a s been used t o s e p a r a t e t h e n a t u r a l l y o c c u r r i n g analogs of l a s a l o c i d A22.
1 . 3 Detection -
and a s s a y
The p o l y e t h e r a n t i b i o t i c s i n h i b i t g r a m - p o s i t i v e b a c t e r i a . S u i t a b l e t u r b i d o m e t r i c and c y l i n d e r - o r paper-disc-plate a s s a y s u s i n g B a C i C . t U A A u b t i C i A , Sancina C u t e a , o r S t n e p t o c O c c u A duecaCiA have b e e n d e s c r i b e d 5 3 . B i o a u t o g r a m s from p a p e r - c h r o m a t o g r a p h i c s t r i p s o r t h i n - l a y e r c h r o m a t o g r a p h i c p l a t e s c a n b e made on a g a r p l a t e s i n o c u l a t e d w i t h a n y o n e o f t h e a b o v e o r g a n i s m s .
When r e a c t e d w i t h a m i x t u r e o f v a n i l l i n i n 0 . 5 % s u l f u r i c acid i n a l c o h o l , p o l y e t h e r a n t i b i o t i c s y i e l d c o l o r e d s o l u t i o n s which c a n b e q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y . While t h e i n d i v i d u a l p o l y e t h e r a n t i b i o t i c s v a r y i n t h e i n t e n s i t y of c o l o r d e v e l o p e d , monensin can b e d e t e r m i n e d t o 1 ug/ml o r l e s s a t 5 1 8 nm. L a s a l o c i d i s u n i q u e i n t h a t i t c o n t a i n s a c r e s o t i c a c i d chromophore which causes a s i g n i f i c a n t f l u o r e s c e n c e i n v a r i o u s o r g a n i c s o l v e n t s ; a s p e c t r o f l u o r o m e t r i c method h a s b e e n d e v e l -
484 A gas-chromatographic oped w i t h a s e n s i t i v i t y o f 0 . 5 vg/ml. p r o c e d u r e was a l s o d e s c r i b e d f o r l a s a l o c i d . Details o f t h e a b o v e - m e n t i o n e d methods are d e s c r i b e d i n t h e r e s p e c t i v e a n t i biotic sections.
S i l i c a gel o r alumina t h i n l a y e r chromatography (TLC) has p r o v e n u s e f u l i n t h e i d e n t i f i c a t i o n of p o l y e t h e r a n t i b i o t i c s . D e t e c t i o n i s a c c o m p l i s h e d w i t h b i o a u t o g r a p h y o r s p r a y s . An e x t e n s i v e s u r v e y o f TLC-Rf v a l u e s f o r t h e s e a n t i b i o t i c s h a s n o t b e e n made, b u t s i m p l e s y s t e m s s u c h as e t h y l a c e t a t e ; e t h y l a c e t a t e : d i e t h y l a m i n e ( 9 6 : 4 ) ; ch1oroform:methanol ( 9 : l ) ; benzene: a c e t o n i t r i l e (1:l) are u s e f u l f o r i d e n t i f i c a t i o n p u r p o s e s . Our l a b o r a t o r y (unpublished data) has found t h a t t h e use of e t h y l a c e t a t e : e t h a n o l ( 2 0 : l ) as a d e v e l o p e r on Merck s i l i c a - g e l p l a t e s w i l l s e p a r a t e a number o f p o l y e t h e r a n t i b i o t i c s i n t h ? f o l l o w i n g order s t a r t i n g n e a r :he o r i g i n : g r i s e o r i x i n , n i g e r i c i n , d i a n e m y c i n , monensin B, monensin, n a r a s i n B , s a l i n o m y c i n , X 2 0 6 , n a r a s i n , A204A, l o n o m y c i n , A286958, A28695A, a n d l a s a l o c i d . L a s a l o c i d by v i r t u e o f i t s a r o m a t i c g r o u p i s t h e most n o n p o l a r compound. L a s a l o c i d and t h e polymethoxy compounds (A28695A, A28695B, A 2 0 4 , lonomycin, a n d CP38295) a r e c l u s t e r e d t o w a r d t h e solvent front. S u l f u r i c a c i d , v a n i l l i n - s u l f u r i c a c i d , and Dragendorff s p r a y s a r e v e r y u s e f u l i n d e t e c t i n g t h e a n t i b i o t i c s on TLC p l a t e s ; and, w h i l e t h e s e n s i t i v i t i e s t o t h e s e r e a g e n t s v a r y c o n s i d e r a b l y , many o f t h e compounds c a n b e detected a t 1 vg. TLC p l a t e s of p o l y e t h e r a n t i b i o t i c s s p r a y e d w i t h v a n i l l i n s u l f u r i c a c i d i n a l c o h o l i c s o l u t i o n a n d h e a t e d a t 100°C on a steam t a b l e e x h i b i t c o l o r s r a n g i n g f r o m o r a n g e t o r e d t o d a r k p u r p l e , d e p e n d i n g on t h e h e a t i n g time. L y s o c e l l i n g i v e s a b r i g h t yellow-green t o green c o l o r .
1 . 4 Literature
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28.
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29.
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34.
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39.
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52.
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53.
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54.
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Saito,
c
Table 1
(13
m
Polyether A n t i b i o t i c s
Antibiotic A130A
Molecular Formula C47H1 ' 8 7
3
Molecular Weight 851.1
Reference
Producinq Organism
S&cp.tom/Ces
hygfloAcopicLLs
23
A130 ATCC 21840
s&ep2htyCeb
RO 21-6150
hy@OACOp~cub
24
s t r a i n X14563 A204A
C49H84O 1 7
945.16
Factor 8 A2869 5A
C48H82016
915.16
S&eptonyceb
d u d NRRL 3384
16,25
Sakzptonyces
d b u d NRRL 3384
16,25
SZ&eptonyceb &us
NRRL 3883
sa2eptonyce.4 hyqno4cop.icub
Sep t amycin
17 26
NRRL 5678.
BL 580a*
27
sJ2ep.bnyceb hljqno4copicuA NRRL 5647
S&eptonyceb
Factor 8
a(bu.4
NRRL 3883
sa2epp.tomyceb hyglt04copicLLb
8L 5808*
17 27
NRRL 5647 Alborixin
c4 BH84' 14
885.17
sJ2ep.tonyceb aeblLd
28
CP 38295
C48H820 1 6
915.16
sa%Lp;tonyces
[email protected]
29
ATCC 31050 ~ ~ ~
Etheromycin ( C 20-12) D i anemycin
c 4 7" 78O 14
867.1
[email protected] ~ ~ Skeptonycu hlj@oAcopicw NRRL 3444
C
e 54 b
30,31
Table 1 (Continued) Antibiotic Grisorixin
Molecular Formula
Molecular Weight
h O H 6'8 10
708.9
Producing Organism
Reference 32
K358
20
I momy c i n
c 4 lH7Z09
708.99
K-41
C48H82019
963.16
L a i dlomy c i n Lasalocid A (X537-A)
37H62012 C34H5408
Statnephyces congtobutu
33
ATCC 31005
s.?ktp&m#Jced
hygfiOACOpicud
34
K-41 FERM-P1342 698.9
Staep.tonyces e a o c i d i c u var. Q b a 2 h o c ~ c u A ATCC 31175
590.77
Shepbnyces maLiensio
35,36 3,22
NRRL 3382 Factor 8
35H 56O 8
604.79
Factor C
C35H5608
604.79
Factor D
35H 5 6' 8
604.79
35H560 8
604.79
Factor
E
Go-Lasalocid A
Staepptonyces l?aAaLien,5A
3,22
NRRL 3382
Saeptonycen W&en,5&
3,22
NRRL 3382
ShnCp.tonryceA hatnbb
3,22
NRRL 3382
eaSmnai4
3,22
sfiepp.tonLjces 4aSaaensio
3,22
S&tptoycen NRRL 3382
c3 4H 5 408
590.7
NRRL 3382
-
Tsble 1 (Continued)
F
In
0
Antibiotic Lmomycin
Molecular Formula C44H76'1 4
Molecular Weight 829.1
Producing Organism
si2eptonyces nibasidi~icub
Reference 37
TM-481 ATCC 31051
strtep.tomyces hygnascopicuA
A218
34
FERM-PBZ8
strreptonyces hygnascopicu4
E m r i c i d (RP 31559)
38
D524367 NRRL 5787
saeptonyces
DE 3936
hygLo.4capicu4
21
9735-1 Lysocellin
C34H600 1 0
628.8
Monensin
C36H62011
670.89
Factor B
C35H600 11
656.87
Factor C
C37H64011
684.9
Factor D
c3f16401 1
684.9
Narasin (A280B6A)
C43H7201 1
765.01
Factor 8
C43H70° 1 1
763.01
C44H74O 11
779.0 3
Factor D
St~t~ppt4myce~ cacao4 var.
39,40
Strteptonyces &numanen&
15,41
awndb K-9 MetATCC 15413
strtepanyces CinnamonenAis
15,41
ATCC 15413
S&pd3nyces
csvuuunonenkib
15,41
CinnamoneUG
15,41
ATCC 15413
S&p.tonyces ATCC 15413
StrtepZonyces a r ~ r e o 6 d e n ~ NRRL 8092 o r NRRL 5758 SikepZayces auneoddens
18,42 18,42
NRRL 8092 or NRRL 5758
Szteptonyces arYreo~&en4 NRRL 8092 or NRRL 5758
18,42
Table 1 (Continued) Antibiotic Nigericin
Molecular Formula C40H6801 1
Molecular Wei#t 724.9
Producing Organism
S&tnep.tomyceb sp.
x-464
StJttnepp.tOycU X464
Polyetherin A
S.&tnephlYCeb
Reference 2,43 3
hijQhOACOpiCW
44
E-749 H e l i x i n C**
StJteptOyceb sp.
K-178”
Sa2tnep.toyceb &bud
Azalomycin M+*
S.&epP.tonyCU hyg4o~copicu6
47
Duamy tin**
Sa2tnepp.tOyceA fyg/tobcopicud duanyceth.6
48
StAtnepbyceb g a e e i w u d
49
RP 30504
45 K-178
46
DS 25881 NRRL 5785
Salinomycin
C42H70011
750.98
S a 2 e p . t o y c ~&bud
Salinomycin A I1
C42H68011
748.98
StJttpp.tOnyce6 aLbu.3 ATCC 21838
X-206
C47H82014
871.13
StJttnep2hyCU X-206
ATCC 21838
19, SO
51
3,52
%L S -@a€i and EL-5808 cannot be separated from A28695A and A286958, r e s p e c t i v e l y , by the chromatographic procedures used a t t h e L i l l y Research Laboratories. *+tlelixin C, K-178, and azalomycin M cannot be separated from n i g e r i c i n by t h e chromatographic procedures used a t t h e L i l l y Research Laboratories. ***Duamycin has been r e p o r t e d t o be the same 83 n i g e r i c i n i n a p e r s o n a l communication from Kaken Chemical Company, Ltd.
49 2 2. Monensin 2.1 Introduction In 1967, the isolation' and anticoccidial activity2 of monensimwere first reported. After several years of testing, monensin was shown to be a safe and efficacious agent for the control of coccidiosis in poultry, This antibiotic was the first of the polyether group to be marketed. 2.1.1
Producing organism
Stxep.tomyceA CinnamonenAiA ATCC 15413'. 2.1.2
Chemical and physical properties'
Mone ns in : C36H62011 mol. Wt. 670.89 m.p. 103-105°C {a)65+47. 7'
(csl, methanol)
pKa 6.65 (66% dimethylformamide) Monensin, sodium salt: C36H61011Na m01.
Wt.
692.87
m.p. 267-269OC {a1$5+57.30 (c=l, methanol). 2.1.3
Structural formula3
R, =CHIMe)C02H,R2*Et Factor B R, 1CH 1 Me)CO$i ,R2 = Me Factor C R, 1 tCH21,CO$ ,R2. Me 2.2 Summary of. biological activities and uses Momnrin
Monensin is active i n v i t n o against gram-positive bacteria, mycobacteria, and fungi, but it is not effective against bacterial infections in mice when administered parenterally o r orally'. The oral LD50 values for monensin are 43.8 mg/kg in mice and 284 mg/kg in chicks'. In chickens, monensin is active againet infections caused by the six ma'qr coccidial species: E.tmen.ta t e n e l l a , E , n e c a t ~ x ,E. b n u n e d + , E. a c e n v u l d n a ,
49 3 E . maxima, and E . m i v a t i . Mixed infections containing the above species as well as E. hagani and E. pltaecox are also controlled by monensin. The antibiotic reduces or prevents mortality, intestinal and/or cecal lesions, reduces numbers of oocysts passed, and improves weight gains and feed efficiencies2. Monensin is also effective in the control of coccidiosis in turkeys, sheep, cattle, and rabbits.
The recommended dosage of monensin is 100 to 120 ppm in chicken feed4. The sodium salt of monensin is marketed under the trademark CobanR for this use. Monensin increases the efficiency of feed utilization in ruminant animals. In v i t l t o and i n v i v o tests show that monensin alters rumen fermentation. Measurements of the volatile fatty acid content in the rumen fluid indicate that the molar ratio of propionic acid is increased and the molar ratios of acetic and butyric acids are decreased5. Increased propionic acid production results in more efficient energy conversion than does either acetic or butyric acid production. Rumensi# is the trademark for monensin, sodium salt, marketed for use in beef cattle rations. At levels of 50-200 mg/head/day in pasture cattle, the antibiotic improves rate of gain and feed efficiency6. In feedlot cattle, a level of 30 g/ton of feed is reccommended7.
2.3 Extraction, separation and 2.3.1
purification
Isolation method No. 1 1 , *
The fermentation broth is filtered with 2 % (w/v) Hyflo Supercel (Johns-Manville Co., New York). The filtrate is adjusted to pH 3 with 5N HC1 and is extracted twice with one-half volume of chloroform. The chloroform extracts are combined, concentrated to a low volume, and passed over a carbon column (Type CAL, 12 x 40, Pittsburgh Activated Carbon Co., Pittsburgh, PA) to remove color and impurities. Elution is continued with chloroform until the eluate gives a negative reaction with vanillin-H2S04 spray on silica gel thin layer plates. This carbon purification step has proven to be quite useful in removing impurities, particularly lipid-type impurities, from a number of polyether antibiotic extracts to permit crystallization without further purification. Although a number of water-immiscible solvents can be used for the carbon chromatographic step, chloroform appears to work best for this method. The chloroform eluate is concentrated to dryness and the residue is dissolved in warm methano1:water (10:l) and chilled for crystallization. The crystalline monensin complex is separated by centrifugation or filtration, washed with cold distilled water, and dried i n v a c u o . The crystals are recrystallized from diethyl ether: petroleum ether (1:l). The sodium salt is isolated by adjusting the broth filtrate to pH 8.5 with 1N NaOH before the chloroform extraction and proceeding with the purification as described for the acid form of monensin.
49 4
The i s o l a t i o n p r o c e d u r e i s a l t e r e d f o r h i g h e r - p r o d u c i n g f e r m e n t a t i o n s ( e . g . , 5 0 0 u g h 1 o r h i g h e r ) . Most o f t h e monens i n w i l l p r e c i p i t a t e i f t h e fermentation b r o t h i s allowed t o s t a n d f o r a n h o u r a t room t e m p e r a t u r e b e f o r e f i l t r a t i o n . The a n t i b i o t i c a c t i v i t y can t h e n be r e c o v e r e d from t h e f i l t e r cake. The low water s o l u b i l i t y of p o l y e t h e r a n t i b i o t i c s i n t h e a c i d o r s a l t form makes t h i s p r o c e s s v e r y p r a c t i c a l as f e r m e n t a t i o n y i e l d s i n c r e a s e . The m y c e l i a l f i l t e r cake i s e x t r a c t e d twice w i t h methanol ( o n e - h a l f of t h e o r i g i n a l b r o t h volume). The methanol e x t r a c t s are combined and c o n c e n t r a t e d i n vacuo t o remove t h e methanol. The aqueous s o l u t i o n i s a d j u s t e d t o pH 3 o r pH 8.5, depending on w h e t h e r t h e a c i d o r s a l t form i s d e s i r e d , and t h e p u r i f i c a t i o n i s performed as d e s c r i b e d p r e v i o u s l y . Direct c r y s t a l l i z a t i o n can be a c h i e v e d d u r i n g t h e c o n c e n t r a t i o n of t h e m e t h a n o l i c e x t r a c t s i f t h e f e r m e n t a t i o n y i e l d s a r e s u f f iciently high. 2.3.2
I s o l a t i o n method No. 2 9
An a l t e r n a t i v e i s o l a t i o n p r o c e d u r e i n v o l v i n g a z e o t r o p i c d i s t i l l a t i o n can be used f o r h i g h p o t e n c y monensin fermentat i o n s . The p r o c e s s i n v o l v e s d i s t i l l a t i o n of t h e water from t h e f e r m e n t a t i o n b r o t h by f o r m i n g an a z e o t r o p i c m i x t u r e w i t h a s o l v e n t s u c h as b e n z y l o r n-hexyl a l c o h o l , u s i n g s u f f i c i e n t e x c e s s of t h e s o l v e n t u n t i l o n l y t h e o r g a n i c s o l v e n t p h a s e r e m a i n s . The mycelium and o t h e r i n s o l u b l e s a r e removed, and monensin i s r e c o v e r e d from t h e l i q u i d p h a s e by e v a p o r a t i o n and c r y s t a l l i z a t i o n . T h i s method i s i l l u s t r a t e d by t h e f o l l o w i n g examples: A. To 1 1 o f monensin whole b r o t h , a s s a y e d a t 2 . 2 6 mg of a c t i v i t y p e r m l , i s added 1 , 5 0 0 m l o f n-hexyl a l c o h o l . The m i x t u r e i s a z e o t r o p i c a l l y d i s t i l l e d a t 98OC a t a t m o s p h e r i c p r e s s u r e u n t i l o n l y t h e s o l v e n t p h a s e r e m a i n s . The s l u r r y i s c o o l e d , f i l t e r e d , and t h e s o l i d s are washed w i t h 1 0 0 m l o f f r e s h nh e x y l a l c o h o l . The f i l t r a t e i s e v a p o r a t e d under vacuum a t a b o u t 30DC u n t i l o n l y an o i l p h a s e r e m a i n s . To t h e o i l i s added 500 m l o f methanol and t h e m i x t u r e i s a l l o w e d t o s e p a r a t e i n t o a n o i l and methanol p h a s e . The methanol p h a s e c o n t a i n s a b o u t 84% of t h e o r i g i n a l monensin a c t i v i t y . The monensin i s r e c o v e r e d by c o n c e n t r a t i n g t h e methanol s o l u t i o n and a d d i n g water. The c r y s t a l l i n e monensin i s r e c o v e r e d by f i l t r a t i o n .
8. A c o n t i n u o u s f a l l i n g f i l m e v a p o r a t o r i s c h a r g e d w i t h 35 1 of b e n z y l a l c o h o l . The e v a p o r a t o r i s o p e r a t e d under a vacuum of 2 7 . 5 i n c h e s o f mercury a t 5OoC w h i l e t h e s o l v e n t i s c i r c u l a t e d i n t h e s y s t e m a t a r a t e of f o u r g a l l o n s p e r m i n u t e . To t h e e v a p o r a t o r a r e c o n t i n u o u s l y c h a r g e d 5 0 1 p e r h o u r of monensin whole b r o t h c o n t a i n i n g 8.75 mg of a c t i v i t y p e r m l and 2 7 1 p e r hour o f f r e s h b e n z y l a l c o h o l . The a z e o t r o p e i s condensed o v e r h e a d , and t h e s l u r r y of d r y s o l i d s and monensin-rich s o l v e n t i s d i s c h a r g e d p e r i o d i c a l l y from t h e e v a p o r a t o r t o maint a i n a c o n s t a n t volume of a b o u t 35 L i n t h e e v a p o r a t o r body. The s o l v e n t i s d e c a n t e d from t h e s o l i d s and a s s a y e d f o r monens i n a c t i v i t y . C r y s t a l l i n e monensin i s r e c o v e r e d from t h e s o l v e n t by t h e p r o c e s s d e s c r i b e d i n method ( a ) . The o v e r a l l r e c o v e r y of monensin by t h i s p r o c e s s i s 9 0 % from whole b r o t h t o the solvent e x t r a c t .
49 5 2.3.3
S e p a r a t i o n of i n d i v i d u a l f a c t o m l o
Monensin f a c t o r s can b e s e p a r a t e d by s i l i c a g e l chromatog r a p h y o r p r e p a r a t i v e TLC. S i l i c a g e l column c h r o m a t o g r a p h y i s t h e most p r a c t i c a l s t e p . TWO u s e f u l methods a r e d e s c r i b e d b e low * A. Monensin m i x t u r e ( 1 . 8 5 g ) i s d i s s o l v e d i n 25 m l c h l o r o f o r m and a p p l i e d t o a 4 . 5 x 75 c m column o f s i l i c a g e l ( 0 . 0 8 mm, Brinkmann I n s t r u m e n t s , I n c . , W e s t b u r y , NY) p r e p a r e d i n c h l o r o f o r m . The column i s e l u t e d w i t h c h l o r o f o r m a t a f l o w r a t e o f 0 . 9 ml/min, c o l l e c t i n g 2 2 m l f r a c t i o n s . E l u t i o n of t h e f a c t o r s i s m o n i t o r e d by s i l i c a g e l TLC, u s i n g e t h y l a c e t a t e a s t h e dev e l o p i n g s o l v e n t a n d 3% v a n i l l i n - 0 . 5 % HzSOt+ o r HzSOt, s p r a y s . F a c t o r C i s e l u t e d f i r s t , t h e n f a c t o r D, f o l l o w e d b y monensin ( f a c t o r A) and t h e n f a c t o r B . S i m i l a r s e p a r a t i o n is achieved u s i n g e t h y l a c e t a t e i n p l a c e o f c h l o r o f o r m as t h e e l u t i n g s o l vent. F r a c t i o n s c o n t a i n i n g more t h a n o n e f a c t o r are combined a n d r e c h r o m a t o g r a p h e d . The f r a c t i o n s c o n t a i n i n g a s i n g l e f a c t o r are combined, c o n c e n t r a t e d i n vacuo t o a n o i l , a n d d i s s o l v e d i n w a r m a c e t o n e . Water i s a d d e d t o 1 / 3 volume f o r c r y s t a l l i z a t i o n . The c r y s t a l s a r e f i l t e r e d , washed w i t h w a t e r , and d r i e d i n v a c u o . B. A m i x t u r e o f m0nensi.n A a n d B ( 4 . 1 g ) , d i s s o l v e d i n b e n z e n e w i t h a small amount o f c h l o r o f o r m a d d e d t o c o m p l e t e l y d i s s o l v e t h e s a m p l e , i s a p p l i e d t o a 2 . 7 x 1 4 0 c m column o f s i l i c a g e l (Grace Grade 6 2 , W . R . G r a c e , Davison Chemical D i v . , B a l t i m o r e , MD) p r e p a r e d i n b e n z e n e . The column i s washed w i t h benzene and developed w i t h b e n z e n e : e t h y l a c e t a t e ( 6 : 4 ) , c o l l e c t i n g 50 m l f r a c t i o n s . E l u t i o n i s f o l l o w e d by TLC. Factor A is p r e s e n t i n f r a c t i o n s 2 3 t o 4 5 , a m i x t u r e of f a c t o r s A a n d B i n f r a c t i o n s 5 0 - 6 7 , a n d f a c t o r B i n f r a c t i o n s 68-87. The r e s p e c t i v e f r a c t i o n s a r e combined a n d c o n c e n t r a t e d i n vacuo t o an o i l . The o i l i s d i s s o l v e d i n 75 m l o f h o t a c e t o n e , and 50 m l water i s a d d e d . The v e s s e l i s a l l o w e d t o s t a n d u n c o v e r e d a t room t e m p e r a t u r e f o r c r y s t a l l i z a t i o n . The c r y s t a l s are f i l t e r e d , washed w i t h water, a n d d r i e d i n v a c u o . T y p i c a l y i e l d s o b t a i n e d are: 3.62 g monensin A , 1 7 6 mg o f t h e m i x t u r e , a n d 1 1 8 mg f a c t o r B.
I n t h e a u t h o r s ’ e x p e r i e n c e , method B i s t h e p r e f e r r e d s e p a r a t i o n p r o c e d u r e f o r r e c o v e r i n g monensin f a c t o r s A a n d B . S e p a r a t i o n u s i n g p r e p a r a t i v e s i l i c a g e l TLC p l a t e s h a s been a c c o m p l i s h e d w i t h d e v e l o p i n g s o l v e n t s s u c h as e t h y l a c e t a t e or d i e t h y l e t h e r : a c e t i c a c i d ( 9 7 : 3 ) . 2.3.4
Methods f o r d e t e c t i o n a n d a s s a y
Monensin i n h i b i t s gram p o s i t i v e b a c t e r i a , a n d m i c r o b i o l o g ical a s s a y s have been developed t o determine f e r m e n t a t i o n y i e l d s and s a m p l e p u r i t y . A d i s c - p l a t e a g a r - d i f f u s i o n method u s i n g B a c i l l u h b u b t i l i . 4 ATCC 6633 i n pH 5 . 5 a g a r h a s a d o s e - r e s p o n s e r a n g e of 6.25-50 p g / m l l l w h i l e a c y l i n d e r - p l a t e a g a r - d i f f u s i o n method was d e s c r i b e d w i t h a r a n g e of 0.25-2.0 u g / m l I 2 . An a u t o mated p h o t o m e t r i c method u t i l i z i n g t h e i n h i b i t i o n of S a t n e p t o coccu.4 d a t c a l i b ATCC 8043 was r e p o r t e d t o have a r a n g e of
49 6 0.1-0.4
pg/m113,
S i l i c a g e l TLC works e x t r e m e l y w e l l f o r monensin and rel a t e d f a c t o r s . A number of s o l v e n t s y s t e m s w i l l s e p a r a t e t h e factors. These i n c l u d e : e t h y l a c e t a t e ; e t h y l a c e t a t e : d i e t h y l amine ( 9 6 : 4 ) , d i e t h y l e t h e r : a c e t i c a c i d ( 9 7 : 3 ) ; c a r b o n tetrac h 1 o r i d e : m e t h a n o l ( 9 5 : 5 ) . A s e n s i t i v i t y of a b o u t 1 pg monensin can be o b t a i n e d w i t h s p r a y s s u c h as 3% v a n i l l i n i n 1 . 5 % s u l f u r i c a c i d i n e t h a n o l , h e a t i n g t h e s p r a y e d p l a t e a t 1 0 0 ° C f o r 5 min. t o y i e l d an i n t e n s e r e d s p o t ' . Developed TLC p l a t e s can a l s o b e u s e d f o r b i o a u t o g r a p h i c measurement u s i n g a n a g a r i n o c u l a t e d w i t h B . d u b . t i L i 4 ATCC 6633 i n an a ar s p r a y a n d o v e r l a y t e c h n i q u e d e s c r i b e d by K l i n e and G o l a b f 4 . S h o r t e x p o s u r e o f a dev e l o p e d p l a t e d i r e c t l y o n t o t h e s u r f a c e of an i n o c u l a t e d agar p l a t e f o l l o w e d by i n c u b a t i o n of t h e a g a r p l a t e a l s o works w e l l .
A c o l o r i m e t r i c method15 h a s b e e n r e p o r t e d f o r t h e d e t e r m i n a t i o n of monensin i n f e r m e n t a t i o n b r o t h s , c r y s t a l l i n e samp l e s , f e e d s and premixes. T h i s method employs 3% v a n i l l i n i n 0 . 5 % s u l f u r i c a c i d i n m e t h a n o l as t h e c o l o r a g e n t ; t h e c o l o r is d e v e l o p e d i n a 6OoC water b a t h f o r 2 5 min. a n d a b s o r b a n c e a t 5 1 8 nm i s d e t e r m i n e d . C r y s t a l l i n e monensin i s used as t h e s t a n d a r d . The method is r e p o r t e d t o be s i m p l e , f a s t , s e n s i t i v e (1 p g / m l ) , a n d r e p r o d u c i b l e . A TLC-spectrodensitometric p r o c e d u r e ( u n p u b l i s h e d d a t a of E . Boothe, E l i L i l l y and Company) h a s been d e v e l o p e d f o r meas u r e m e n t of monensin B c o n t e n t i n monensin s a m p l e s and would be a p p l i c a b l e t o o t h e r f a c t o r s i f p r e s e n t . The s a m p l e s a r e c h r o matographed on Merck p r e c o a t e d s i l i c a - g e l p l a t e s ( w i t h o r w i t h o u t f l u o r e s c e n c e i n d i c a t o r ) which a r e s c o r e d , a n d s a m p l e s are a p p l i e d t o e v e r y o t h e r l a n e . The p l a t e s are d e v e l o p e d i n d i e t h y l e t h e r : g l a c i a l a c e t i c a c i d ( 9 7 : 3 ) . The c o l o r i s d e v e l o p e d by l a y i n g t h e p l a t e f a c e down i n a s o l u t i o n of 3 % v a n i l l i n i n 3 % s u l f u r i c a c i d i n m e t h a n o l i n a f l a t s h a l l o w p a n , t h e n removing t h e p l a t e and p l a c i n g i t h o r i z o n t a l l y i n a TLC p l a t e holder t o dry f o r 1 h r i n a d r a f t . Alternatively, the p l a t e can be d r i e d i n a 37OC oven f o r 2 0 m i n . Excess v a n i l l i n i s wiped o f f t h e back of t h e p l a t e a n d t h e p l a t e i s s c a n n e d w i t h i n an h o u r . A S c h o e f f e l SD 3000 S p e c t r o d e n s i t o m e t e r w i t h AvtoLab i n t e g r a t o r a n d r e c o r d e r i s used t o scan e a c h l a n e a t 480 nm. The a r e a u n d e r e a c h peak i s used t o c a l c u l a t e p e r c e n t a g e s . A t an a t t e n u a t i o n s e t t i n g of 0 . 2 , t h e l i m i t of d e t e c t i o n of monensin f a c t o r s i s 0 . 0 2 pg i n t h e r e f l e c t a n c e mode and 0 . 0 1 p g i n t h e transmittance mode. I n t h e p r e s e n c e o f monensin, monensin B can b e d e t e r m i n e d down t o 0 . 0 5 % .
2.4 Literature
Cited
1.
M . E. Haney, J r . and M. M . Hoehn, A n t i m i c r o b . Agents Chemother.-1967 ( 1 9 6 8 ) 349-352.
2.
R . F. Shumard and M. E . C a l l e n d e r , A n t i m i c r o b . Agents Chemother - 1 9 6 7 ( 1 9 6 8 1 369 -377.
3.
J . W . Chamberlin and A . A g t a r a p , Org. Mass S p e c t r o m e t r y 3 ( 1 9 7 0 ) 271-285.
.
49 7 4.
W . M. R e i d , L. K o w a l s k i a n d J . Rice, P o u l t r y S c i . 5 1 ( 1 9 7 2 ) 139-146.
5.
L. F. R i c h a r d s o n , A. P. R a u n , E . L. P o t t e r , C . 0 : C o o l e y a n d R. P. R a t h m a c h e r , J. Animal S c i . 43 ( 1 9 7 6 ) 657-664.
6.
E . L . P o t t e r , C . 0 . C o o l e y , L . F. R i c h a r d s o n , A. E. Raun and R. P . R a t h m a c h e r , J. A n i m a l S c i . 113 ( 1 9 7 6 ) 665-669.
7.
A. P. Raun, C. 0 . C o o l e y , E . L. P o t t e r , R . P . R a t h m a c h e r a n d L. F. R i c h a r d s o n , J . A n i m a l S c i . 43 ( 1 9 7 6 ) 6 7 0 - 6 7 7 .
8.
M. E. H a n e y , M. M . Hoehn a n d J. M . M c G u i r e , U.S. 3 , 5 0 1 , 5 6 8 ; March 1 7 , 1 9 7 0 .
9.
B r i t i s h P a t e n t 1,296,584;
Patent
November 1 5 , 1 9 7 2 .
10.
M . Gorman, J. W .
C h a m b e r l i n a n d R . L. H a m i l l , A n t i m i c r o b . A g e n t s Chemother.-1967 ( 1 9 6 8 ) 363-368.
11*
W . M. S t a r k , N . G . Knox a n d J . E . W e s t h e a d , A n t i m i c r o b . A g e n t s C h e m o t h e r . - 1 9 6 7 ( 1 3 6 8 ) 353-358.
12.
R . M . K l i n e , R. E . S t r i c k e r , J. D . C o f f m a n , H . B i k i n , R . P . R a t h m a c h e r , J . Assoc. O f f i c i a l A n a l y t i c a l C h e m i s t s 5 3 ( 1 9 7 0 ) 50-53.
13.
F . W. K a v a n a g h a n d M. W i l l i s , J. Assoc. O f f i c i a l A n a l y t i c a l C h e m i s t s 55 ( 1 9 7 2 ) 1 1 4 - 1 1 8 .
14.
R . M. 411.
15.
T. G o l a b , S . J . B a r t o n a n d R . E . S c r o g g s , J . Assoc. O f f i c i a l A n a l y t i c a l C h e m i s t s 56 ( 1 9 7 3 ) 1 7 1 - 1 7 3 .
K l i n e a n d T . G o l a b , J . C h r o m a t o g r a p h y 1 8 ( 1 9 6 5 ) 409-
49 8
3. L a s a l o c i d Complex (X-537Ay R0-2-2985)
3.1 Introduction L a s a l o c i d was o n e of t h e f i r s t p o l y e t h e r a n t i b i o t i c s t o b e i s o l a t e d i n 1 9 5 1 l . The complex c o n s i s t s o f t h e m a j o r f a c t o r y l a s a l o c i d A, a n d m i n o r f a c t o r s , i & o - l a s a l o c i d A , l a s a l o c i d B , C , D, and E . I n r e c e n t y e a r s t h i s a n t i b i o t i c h a s proved t o b e a unique b i o c h e m i c a l t o o l b e c a u s e o f i t s a b i l i t y t o t r a n s p o r t monovalent and d i v a l e n t c a t i o n s as well as o r g a n i c arnines t h r o u g h b o t h a r t i f i c i a l a n d b i o l o g i c a l membranes2. 3.1.1
P r o d u c i n g organ-
S&tneptornycea h & a . & k n 4 i A NRRL 3 3 8 2 3 . 3.1.2
Chemical and p h y s i c a l p r ~ p e r t i e s l ’ ~
Lasalocid A C ~ L , H ~ I + mol. O~ wt.
m.p.
590.8
110-114°C -7.55’
(c.1,
methanol)
Xmax 248 nm
(E
6750)
318 nm
(E
4200)
3.1.3
S t r u c t u r a l formula5
LOWlocd A
Laralocid B C
D E
499
3.2 Summary
of b i o l o g i c a l a c t i v i t i e s a n d u s e s
L a s a l o c i d e x h i b i t s in v i 2 2 0 a c t i v i t y a g a i n s t g r a m - p o s i t i v e b a c t e r i a . The LD50 v a l u e i n mice, i . p . i s 4 0 mg/kg When i n c o r p o r a t e d i n t o p o u l t r y f e e d a t a l e v e l of 0.003-0.006% by weight, l a s a l o c i d w i l l c o n t r o l c o c c i d i a l i n f e c t i o n s 3 . In addit i o n , s w i n e d y s e n t e r y , c a u s e d by t h e s p i r o c h e t e , Taeponema h y o d y A e n t e a i a e , i s c o n t r o l l e d by t h e a d d i t i o n of l a s a l o c i d t o f e e d a t a l e v e l o f 0.0055-0.055% by w e i g h t 6 .
.
All of t h e p o l y e t h e r s show i o n o p h o r e t i c p r o p e r t i e s . Lasa l o c i d can be considered a broad-spectrum ionophore because o f i t s a b i l i t y t o t r a n s p o r t a l k a l i metal i o n s , d i v a l e n t c a t i o n s , and o r g a n i c a m i n e s 2 . Calcium b i n d i n g and t r a n s p o r t by l a s a l o c i d r e s u l t s i n a c t i v i t y i n a v a r i e t y o f b i o l o g i c a l s y s t e m s 2 . The p o s i t i v e i n o t r o p i c a c t i v i t y o f l a s a l o c i d h a s prompted s u g g e s t i o n s of t h e r a p e u t i c u s e f u l n e s s i n c a r d i o v a s c u l a r problems7.
3.3 E x t r a c t i o n , 3.3.1
s e p a r a t i o n and p u r i f i c a t i o n
Lasalocid isolation'
The b r o t h ( 2 0 4 L ) i s f i l t e r e d w i t h t h e a i d o f H y f l o S u p e r C e l , and t h e f i l t r a t e i s d i s c a r d e d . The wet m y c e l i a l c a k e i s s u s p e n d e d i n 1 0 0 L of n - b u t y l a c e t a t e , and t h e m i x t u r e s t i r r e d o v e r n i g h t ( e t h y l a c e t a t e i s a l s o u s e f u l as a n e x t r a c t i n g s o l vent). The m i x t u r e i s f i l t e r e d , a n d t h e water p h a s e i s s e p a r a t e d and d i s c a r d e d . The b u t y l a c e t a t e p h a s e i s c o n c e n t r a t e d i n vacuo t o 3 L., washed w i t h 1 0 % sodium c a r b o n a t e , and d r i e d o v e r a n h y d r o u s sodium s u l f a t e . The b u t y l a c e t a t e s o l u t i o n i s t h e n c o n c e n t r a t e d t o 300 m l and t h e r e s u l t i n g c o n c e n t r a t e i s d i l u t e d w i t h 350 m l o f p e t r o l e u m e t h e r ( b . p . 5 0 - 6 G ° C ) t o p r e c i p i t a t e t h e c r u d e l a s a l o c i d ( 4 1 g). The s o l i d o b t a i n e d i s p l a c e d i n t h e t h i m b l e o f a S o x h l e t a p g a r a t u s and e x t r a c t e d w i t h 4 k o f p e t r o l e u m e t h e r ( b . p . 5 0 - 6 0 C) f o r 4 0 h r . The e x t r a c t i s conc e n t r a t e d i n vacuo t o d r y n e s s a n d t h e c r y s t a l l i n e r e s i d u e i s s u s p e n d e d i n 1 0 0 m l of p e t r o l e u m e t h e r , f i l t e r e d , and d r i e d t o y i e l d 2 4 g o f l a s a l o c i d s a l t . The m o t h e r l i q u o r i s c o n c e n t r a t e d t o y i e l d a s e c o n d c r o p of c r y s t a l s . The c r y s t a l l i n e s a l t i s t h e n r e c r y s t a l l i z e d from d i e t h y l e t h e r - p e t r o l e u m e t h e r . The sodium s a l t of l a s a l o c i d can b e c o n v e r t e d t o t h e f r e e a c i d by d i s s o l v i n g i t i n d i e t h y l e t h e r and w a s h i n g w i t h d i l u t e s u l f u r i c a c i d . The e t h e r e x t r a c t i s c o n c e n t r a t e d i n vacuO t o an o i l , and t h e o i l i s d i s s o l v e d i n e t h a n o l f o r c r y s t a l l i z a t i o n of t h e l a s a l o c i d (m.p. 100-1G9°C). The sodium s a l t can be made from t h e f r e e a c i d by d i s s o l v i n g i t i n d i e t h y l e t h e r and w a s h i n g t h e e t h e r s o l u t i o n w i t h aqueous sodium c a r b o n a t e . The e t h e r s o l u t i o n i s c o n c e n t r a t e d i n vacuo t o a r e s i d u e from which t h e l a s a l o c i d sodium s a l t (m.p. 168-171'C) can b e c r y s t a l l i z e d by u s i n g b e n z e n e - l i g r o i n . Barium a n d p o t a s s i u m s a l t s c a n b e p r e p a r e d i n a similar manner.
500
3.3.2
I s o l a t i o n of i 4 a - l a s a l o c i d A a n d l a s a l o c i d f a c t o r s B , C, D, and E3.
The f i n a l mother l i q u o r ( p e t r o l e u m e t h e r s o l u t i o n ) from t h e c r y s t a l l i z a t i o n of 1 0 kg of l a s a l o c i d A sodium s a l t c o n t a i n s a m i x t u r e of l a s a l o c i d A , l a s a l o c i d i s o m e r i c homofogs B , C , , D , and E , and t h e a n a l o g 1 4 0 - l a s a l o c i d A. T h i s mother l i q u o r is c o n c e n t r a t e d t o a s o l i d r e s i d u e and t h e n d i s s o l v e d i n 1 6 0 m l of t h e mixed p h a s e s of n - h e p t a n e : e t h y l a c e t a t e : m e t h a n o l : w a t e r ( 2 7 : 18:18:2) and chromatographed on a 2 0 0 t u b e ( e a c h 8 0 m l c a p a c i t y ) c o u n t e r c u r r e n t d i s t r i b u t i o n a p p a r a t u s by p l a c i n g t h e a n t i b i o t i c s o l u t i o n i n t h e f i r s t two t u b e s . After 380 t r a n s f e r s , t u b e s were a n a l y z e d b y UV, s i l i c a g e l TLC, and pyrolysis/GLC and comb i n e d as f o l l o w s : Fraction
la lb lc
Tubes Combined 135-185 110 -134 60-90
Laealocid Components D,E A -ibO-lasalocid A
The LAO-lasalocid A ( N a - K s a l t s ) f r a c t i o n i s d i s s o l v e d i n methylene c h l o r i d e and t h e s o l u t i o n washed w i t h 0.1N H C I t o conv e r t i t t o i . 4 o - l a s a l o c i d A f r e e a c i d . The m e t h y l e n e c h l o r i d e solution is concentrated t o dryness t o y i e l d the f r e e a c i d (m.p. 203'C). The sodium s a l t of i b o - l a s a l o c i d A can b e p r e p a r e d from t h e f r e e a c i d by d i s s o l v i n g t h e a c i d i n methylene c h l o r i d e and washing t h e s o l u t i o n w i t h a s a t u r a t e d aqueous s o l u t i o n of sodium c a r b o n a t e . The methylene c h l o r i d e s o l u t i o n i s c o n c e n t r a t e d i n Vacua w i t h hexane t o y i e l d c r y s t a l l i n e i d o - l a s a l o c i d A sodium s a l t (m.p. 183-185'C). O t h e r s a l t s can b e p r e p a r e d by similar procedures. I s o l a t i o n of l a s a l o c i d D and E i s a c h i e v e d by c o u n t e r c u r r e n t d i s t r i b u t i o n o f f r a c t i o n la. The r e s i d u e from f r a c t i o n l a i s d i s t r i b u t e d i n a 500-tube ( 1 0 m l u p p e r a n d 10 m l l o w e r p h a s e ) CCD i n s t r u m e n t u s i n g t h e s o l v e n t s y s t e m n - h e p t a n e : e t h y l acetate:ethanol:water:glacial a c e t i c a c i d ( 1 0 : 5 : 9 : 3 : 1 ) . By r e c y c l i n g t h e t o p p h a s e , 3,800 t r a n s f e r s a r e made and t h e t u b e s are a n a l y z e d b y p y r o l y s i s / G L C . Tubes 1 1 0 - 1 6 0 a r e p o o l e d , conc e n t r a t e d t o d r y n e s s , and c r y s t a l l i z e d from aqueous 2 - p r o p a n o l The c r y s t a l l i n e t o y i e l d c r y s t a l l i n e l a s a l o c i d E (rn.p. 9 0 ° C ) . sodium s a l t is made by d i s s o l v i n g l a s a l o c i d E i n m e t h y l e n e c h l o r i d e and washing w i t h s a t u r a t e d aqueous Na2C03. The methyl e n e c h l o r i d e p h a s e i s e v a p o r a t e d w i t h a d d i t i o n o f hexane t o y i e l d t h e c r y s t a l l i n e l a s a l o c i d E sodium s a l t (m.p. 181-182°C). Tubes 2 4 1 - 3 0 0 are p o o l e d , c o n c e n t r a t e d t o d r y n e s s , a n d c r y s t a l l i z e d from aqueous 2 - p r o p a n o l t o y i e l d l a s a l o c i d D , cont a i n i n g 2 - p r o p a n o l as s o l v e n t of c r y s t a l l i z a t i o n (m.p. 1 0 2 104'C). L a s a l o c i d B and C are i s o l a t e d from t h e p r e p a r a t i o n o f c r u d e c r y s t a l l i n e l a s a l o c i d A o b t a i n e d i n p r o c e d u r e 1 by c o u n t e r -
501 c u r r e n t d i s t r i b u t i o n . F o r t y - f o u r grams o f l a s a l o c i d A is d i s t r i b u t e d i n t h e 2 0 0 - t u b e CCD i n s t r u m e n t u s i n g t h e s o l v e n t s y s tem n - h e p t a n e : e t h y l acetate:ethanol:water:acetic a c i d ( 1 0 : 5 : 9 : 3:l). Tubes 86-130 are p o o l e d and c o n c e n t r a t e d t o y i e l d 4 . 5 8 g of a m i x t u r e e n r i c h e d i n l a s a l o c i d B a n d C . T h i s m i x t u r e i s r e d i s t r i b u t e d i n a 5 0 0 - t u b e CCD i n s t r u m e n t u s i n g t h e same s o l v e n t s y s t e m . A f t e r 2,000 t r a n s f e r s , t h e t u b e s a r e a n a l y z e d as d e s c r i b e d e a r l i e r , a n d t u b e s 200-400 c o n t a i n i n g l a s a l o c i d A a r e decanted o f f and f r e s h s o l v e n t i s added. A f t e r an a d d i t i o n a l 2,000 t r a n s f e r s , t h e t u b e s a r e a n a l y z e d and t h e a p p r o p r i a t e p o o l s made. Tubes 450-490 c o n t a i n i n g l a s a l o c i d B a r e concent r a t e d t o d r y n e s s i n vacua a n d c r y s t a l l i z e d from a q u e o u s 2-prop a n o l t o y i e l d 230 mg of l a s a l o c i d B (m.p. 86-87'C). Tubes 71-80 a r e c o n c e n t r a t e d t o d r y n e s s i n vacua a n d c r y s t a l l i z e d from a q u e o u s 2 - p r o p a n o l t o y i e l d 360 mg o f l a s a l o c i d C c o n t a i n i n g 2 - p r o p a n o l a s s o l v e n t o f c r y s t a l l i z a t i o n (m.p. 9 7 100OC). 3.3.3
D e t e c t i o n and a s s a y
L a s a l o c i d A a n d t h e minor f a c t o r s B , C , D, E , a n d i A a l a s a l o c i d A can b e d e t e c t e d b y b i o a u t o g r a p h y u s i n g o r g a n i s m s s u c h as 8 a C i e k h A d u b t i l i b o r BacitLud s p . TA (ATCC 2 7 8 6 0 ) . The l a s a l o c i d s c a n b e d e t e c t e d on s i l i c a g e l 60F-254 ( E . Merck, D a r m s t a d t , Germany) TLC p l a t e s by s c a n n i n g w i t h a s h o r t w a v e UV lamp o r s p r a y i n g w i t h a 1:l s o l u t i o n o f c o n c . s u l f u r i c a c i d i n methanol and c h a r r i n g t h e p l a t e s a t l0O'C t o y i e l d b l a c k s p o t s 5 . The l a s a l o c i d s are less s e n s i t i v e t o v a n i l l i n - s u l f u r i c a c i d s p r a y t h a n many o f t h e o t h e r p o l y e t h e r a n t i b i o t i c s . S e p a r a t i o n of l a s a l o c i d A and LAO-lasalocid A i s achieved on s i l i c a g e l TLC p l a t e s u s i n g b e n z e n e : m e t h a n o l ( 9 : 1 ) , b u t t h i s s y s t e m w i l l n o t s e p a r a t e t h e homologs f r o m l a s a l o c i d A . Separat i o n o f t h e homologs from l a s a l o c i d A c a n b e o b t a i n e d by u s e of t h e f o l l o w i n g s y s t e m s b u t no s e p a r a t i o n o f t h e homologs from 2-propano1:acetic a c i d : e a c h o t h e r h a s b e e n s u c c e s s f u l by TLC: cyclohexane (5:9:86); methano1:octanoic acid:n-hexane (5:6:190); and 2-propano1:acetic acid:n-hexane (3:3:1915. The r e l a t i v e b i o l o g i c a l a c t i v i t y of t h e l a s a l o c i d s c a n b e d e t e r m i n e d u t i l i z i n g 8 a c i l . l ~ s~p . TA (ATCC 27860) i n a microb i o l o g i c a l c u p - p l a t e a g a r d i f f u s i o n a s s a y i n which t h e compounds g i v e p a r a l l e l d o s e - r e s p o n s e c u r v e s . F a c t o r s C , D , a n d E e x h i b i t 1 . 6 - 1 . 8 times h i g h e r a c t i v i t y t h a n l a s a l o c i d A w h i l e L d o - l a s a l o c i d A i s o n l y 0 . 4 times as a c t i v e and f a c t o r B i s 0 . 9 times as a c t i v e . The above a s s a y method c a n b e u s e d f o r d e t e r m i n i n g t h e t o t a l a c t i v i t y i n t h e b r o t h , b r o t h e x t r a c t s , and purification fractions5. A s p e c t r o f l u o r o m e t r i c a s s a y u t i l i z i n g t h e f l u o r e s c e n c e of l a s a l o c i d i n o r g a n i c s o l v e n t s s u c h as e t h y l a c e t a t e , b u t y l acet a t e , t e t r a h y d r o f u r a n , dimethoxymethane, a n d d i o x a n e h a s b e e n d e v e l o p e d . Of t h e s e , e t h y l a c e t a t e i s t h e s o l v e n t o f c h o i c e due t o i t s e f f i c i e n t e x t r a c t i n g p r o p e r t i e s . L a s a l o c i d e x h i b i t s h i g h and r e p r o d u c i b l e f l u o r e s c e n c e i n t h i s s o l v e n t , a n d t h e b a c k g r o u n d f l u o r e s c e n c e o f e t h y l a c e t a t e i s low. The f l u o r e s -
502 cence response of t h e s t a n d a r d is l i n e a r o v e r a range o f 0 . 1 2 0.48 ug/ml. Methods f o r d e t e r m i n a t i o n of l a s a l o c i d i n f e e d s e a n d i n b l o o d g h a v e b e e n r e p o r t e d . S p e c t r o f l u o r o m e t r i c methods would l i k e l y b e a p p l i c a b l e t o b r o t h , m y c e l i a l e x t r a c t s , o r p u r i fication fractions,
A g a s - l i q u i d chromatographic (GLC) d e t e r m i n a t i o n of lasal o c i d l 0 h a s b e e n d e v e l o p e d f o r whole f e r m e n t a t i o n b r o t h s a n d p u r i f i e d s a m p l e s i n which l a s a l o c i d is q u a n t i t a t i v e l y c l e a v e d a t h i g h t e m p e r a t u r e s (300°C) t o t h e r e t r o a l d o l k e t o n e a n d two dihydronaphthols. The amount of r e t r o a l d o l k e t o n e formed i s measured u s i n g m e t h y l b e h e n a t e as t h e i n t e r n a l s t a n d a r d . The thermolysis-GLC method compares f a v o r a b l y w i t h t h e c u p - p l a t e a g a r d i f f u s i o n method. The GLC method h a s b e e n r e c e n t l y a p p l i e d t o t h e a n a l y s i s o f t h e l a s a l o c i d complex5 e x t e n d i n g t h e a n a l y s i s t o i n c l u d e t h e d i h y d r o n a p h t h o l s which a r e a l s o formed i n t h e cleavage reaction.
3.4 L i t e r a t u r e
Cited
1.
J . B e r g e r , A. I . R a c h l i n , W . E . S c o t t , L. H . S t e r n b a c h and M . W . G o l d b e r g , J . h e r . Chem. SOC. 73 ( 1 9 5 1 ) 5295-5298.
2.
B . C . Pressman a n d N . T. deGuzman, Ann. N . Y . Acad. S c i . 264 ( 1 9 7 5 ) 373-386.
3.
J. B e r g e r , U.S.
4.
J. W . W e s t l e y , R . H . Evans, J r . , T . Williams and A. S t e m p e l ,
P a t e n t 3 , 7 1 9 , 7 5 3 ; March 6 , 1 9 7 3 .
Chem. Commun. ( 1 9 7 0 ) 71-73. 5.
J. W . W e s t l e y , W . Benz, J . Donahue, R . H. E v a n s , Jr., C . G . S c o t t , A. S t e m p e l a n d J. B e r g e r , J. A n t i b i o t i c s 2 7 ( 1 9 7 4 ) 744-753.
6.
R . E . M e s s e r s m i t h , U.S.
7.
P . C. G i l l e t t e , R. G . Munson, R . M. P e d i a t r i c Res. 1 0 ( 1 9 7 6 ) 570-574.
8.
M. Osadca a n d M . A r a u j o , J . Assoc. O f f . A n a l . Chem. 57 ( 1 9 7 4 ) 636-641.
9.
M . A . B r o o k s , L . D ' A r c o n t e , J. A. F. d e S i l v a , G . Chen a n d C. Crowley, J. Pharm. S c i . 64 ( 1 9 7 5 ) 1874-1876.
10.
P a t e n t 3 , 9 4 7 , 5 8 6 ; March 30, 1 9 7 6 .
L e w i s a n d A. S c h w a r t z ,
J. W . W e s t l e y , R . H . Evans, Jr. and A . S t e m p e l , A n a l . Biochem. 59 ( 1 9 7 4 ) 574-582.
503
4. Narasin ( A 2 8 0 8 6 A ) 4.1 Introduction Narasin, the major factor of a polyether antibiotic complex, is structurally similar to salinomycin’. Narasin differs from salinomycin only in the presence of an additional methyl group on the tetrahydropyran ring adjacent to the carboxyl group. In addition to narasin (factor A ) , the fermentation produces smaller amounts of biologically active factors B and D and the biologically inactive methyl ester of narasin. Narasin in feed controls coccidiosis in poultry. 4.1.1
Producing organism2
S t J t e p t o m y C e A auJteodacienA NRRL 5 7 5 8 or NRRL 8092, 4.1.2
Chemical and physical properties2
C43H7,O,,
mol. wt. 7 6 5 . 0 1
m.p. 98-100°C -54’
(c.0.2,
methanol)
pKa 7 . 9 (80% dimethylformamide) 4.1.3
Structural formula3
The stereochemistry of the narasin factors has not been determined. Because narasin exhibits a CD spectrum virtually identical to that of salinomycin, it might be assumed that the stereochemistry of both compounds is the same.
HOOC
Narssin A:
Factor 6 : Factor D:
= R2 = Me R = 0, R 1 = R2 = Me R = OH, R 1 = Me, R2 = E t
R
OH, R 1
or Rl = E t , R 2
4.2 Summary
=
Me
of biological activities and uses2
Narasin is active i n v & 2 0 against gram-positive bacteria, mycobacteria, fungi, and viruses. It is also effective against a broad spectrum of anaerobic bacteria in a range of ~ 0 . 5to 16 ug/ml and against Mycoptabma species at 6.25-12.5 pg/ml. The
504
LD,,
( i , p , ) i n mice i s 7 . 1 5 mg/kg. Narasin c o n t r o l s c o c c i d i o s i s c a u s e d . b y Eimeaia s p e c i e s i n a number of d o m e s t i c and l a b o r a t o r y a n i m a l s . When i n c o r p o r a t e d i n t o t h e f e e d a t l e v e l s o f 40-120 ppm, n a r a s i n is e f f e c t i v e a g a i n s t c o c c i d i a l i n f e c t i o n s i n c h i c k ens. N a r a s i n improves f e e d u t i l i z a t i o n e f f i c i e n c y i n r u m i n a n t animals. The a n t i b i o t i c a f f e c t s t h e rumen f e r m e n t a t i o n by i n c r e a s i n g t h e c o n c e n t r a t i o n of p r o p i o n a t e and d e c r e a s i n g t h e conc e n t r a t i o n of a c e t a t e a n d b u t y r a t e i n t h e rumen f l u i d , t h e r e b y improving energy conversion i n t h e animal.
4.3 S e p a r a t i o n 4.3.1
and p u r i f i c a t i o n methods
I s o l a t i o n o f c r u d e n a r a s i n complex
The n a r a s i n f e r m e n t a t i o n b r o t h c o n t a i n s n a r a s i n A as t h e m a j o r f a c t o r , and small amounts o f f a c t o r s B a n d D, as w e l l as t h e methyl ester o f n a r a s i n A. The f e r m e n t a t i o n b r o t h ( 6 0 1 ) i s a d j u s t e d t o pH 3 w i t h d i l u t e H C 1 and i s f i l t e r e d w i t h 3 % f i l t e r a i d (Hyflo S u p e r c e l , Johns-Manville, New Y o r k ) . The f i l t r a t e i s d i s c a r d e d . The m y c e l i a l f i l t e r c a k e is t h e n e x t r a c t e d w i t h 30 L o f m e t h a n o l . During t h e e x t r a c t i o n 1 . 5 6 kg o f NaHC03 i s a d d e d t o t h e m i x t u r e w i t h s t i r r i n g i n o r d e r t o r a i s e t h e pH. The m i x t u r e i s f i l t e r e d , and t h e f i l t e r c a k e i s e x t r a c t e d a g a i n w i t h 30 R o f m e t h a n o l . The m e t h a n o l e x t r a c t s are comb’ined a n d c o n c e n t r a t e d i n vacuo t o remove t h e m e t h a n o l . The r e s u l t i n g a q u e o u s s u s p e n s i o n i s a d j u s t e d t o pH 7 . 5 w i t h d i l u t e H C 1 a n d e x t r a c t e d twice w i t h e t h y l a c e t a t e ( 7 1 e a c h ) . The e t h y l a c e t a t e e x t r a c t s are combined and c o n c e n t r a t e d i n vacuo t o an o i l y r e s i d u e . The r e s i d u e i s d i s s o l v e d i n 1 , 5 0 0 m l a c e t o n e , 1 , 5 0 0 m l o f water i s a d d e d , and t h e s o l u t i o n i s a d The s o l u t i o n i s a l l o w e d t o j u s t e d t o pH 3 w i t h d i l u t e H C 1 . s t a n d f o r 1 h r and t h e p r e c i p i t a t e w h i c h forms i s f i l t e r e d o f f . The p r e c i p i t a t e i s d i s s o l v e d i n 1 , 5 0 0 m l o f a c e t o n e , 4 0 0 m l o f water i s a d d e d , and t h e s o l u t i o n i s a l l o w e d t o s t a n d f o r 1 6 h r f o r c r y s t a l l i z a t i o n t o o c c u r . The c r y s t a l s are f i l t e r e d off and d r i e d i n vacuo t o y i e l d a c r u d e c r y s t a l l i n e p r o d u c t c o n t a i n i n g n a r a s i n A, B , D, and o t h e r c r y s t a l l i n e i m p u r i t i e s . 4.3.2
P u r i f i c a t i o n o f n a r a s i n complex
S e p a r a t i o n o f n a r a s i n f a c t o r s from t h e i n a c t i v e n a r a s i n m e t h y l e s t e r and o t h e r i m p u r i t i e s i s a c h i e v e d b y s i l i c a g e l column c h r o m a t o g r a p h y . The c r u d e c r y s t a l l i n e p r o d u c t (40 g ) i s d i s s o l v e d i n 2 5 0 m l o f b e n z e n e and a p p l i e d t o a 9 x 120 c m c o l umn of s i l i c a g e l (Grace Grade 6 2 , W. R . Grace, Davison Chemic a l D i v . , B a l t i m o r e , MD) p a c k e d i n b e n z e n e . The column i s e l u t e d s u c c e s s i v e l y w i t h 4 0 R e a c h o f : b e n z e n e ; b e n z e n e : e t h y l acetate ( 9 : l ) ; benzene:ethyl a c e t a t e (4:1), benzene:ethyl a c e t a t e ( 7 : 3 ) ; b e n z e n e : e t h y l a c e t a t e (1:l); and e t h y l a c e t a t e . One l i t e r f r a c t i o n s a r e c o l l e c t e d and t h e e l u t i o n i s m o n i t o r e d by s i l i c a g e l TLC. F r a c t i o n s 83-92 from t h e b e n z e n e : e t h y l a c e t a t e ( 4 : l ) e l u t i o n c o n t a i n n a r a s i n A m e t h y l e s t e r , which i s m i c r o b i o l o g i c a l l y i n a c t i v e b u t c a n b e d e t e c t e d by i t s i n t e n s e p u r p l e c o l o r w i t h v a n i l l i n - H 2 S ? , + s p r a y . N a r a s i n A m e t h y l e s t e r can b e r e c o v e r e d by c o n c e n t r a t i n g t h e f r a c t i o n s t o d r y n e s s , a n d t h e n
50 5 dissolving the residue in 200 ml acetone. Eighty ml of water is added, and the solution is allowed to stand overnight for crystallization. The crystals are filtered and dried to yield 4.3 g of narasin A methyl ester (map. 160-162'C). The narasin complex is eluted by the benzene:ethyl acetate (7:3) solution. These fractions are combined and concentrated to dryness. This residue is dissolved in 500 ml of acetone and 500 ml of water is added. The pH is adjusted to 4 with dilute HC1, and the mixture is stirred for 1 h r . The precipitate which forms is filtered off and crystallized from acetone (500 m1)-water (180 ml). The crystals are filtered and dried to yield about 20 g of a crystalline mixture of narasin factors A, B, and D. 4.3.3
Separation of narasin A, B , and D
Separation of the narasin factors is accomplished by chromatography on a fine mesh silica gel column. The crystalline mixture (18 g) is dissolved in 50 ml of benzene and applied to a 7 x 100 cm column of silica gel ( E . Merck, Darmstadt, Germany, Grade 60, finer than 230 mesh, ASTM) packed in benzene. The column is eluted successively at a flow rate of 90 ml/hr with: 12 L of benzene: 12 L of benzene:ethyl acetate (9:l); 12 E. of benzene:ethyl acetate (4:l); and 32 .E of benzene:ethyl acetate (7:3). The elution is monitored by silica gel TLC using ethyl acetate:diethylamine (95:s) as the developing solvent, which separates all three factors. Narasin B is eluted first with the benzene:ethyl acetate (7:3); these fractions are combined, concentrated to dryness, and crystallized from acetone:water (1:l) to yield about 4 mg of narasin B (m.p. 150-153'C). Narasin D is eluted next with the benzene:ethyl acetate (7:3) and followed by narasin A in the same system. The fractions containing only narasin D are evaporated and crystallized from acetone:water (1:l) to yield 135 mg of narasin D (m.p. 9 6 - 9 8 ' C ) . The next series of fractions yield 160 mg of factor D and a small amount of narasin A . The fractions containing only narasin A are evaporated, and crystallization from acetone:water (1:l) yields 4 g of crystalline narasin A . The amounts of the individual factors recovered will depend on the composition of the starting material. The yields given above are typical examples. 4.3.4
Preparation of the sodium salt of narasin
The sodium salt of narasin A is prepared by dissolving. 500 mg of the narasin A (acid form) in 50 ml of acetone; 50 ml of water is added, and the pH of the solution is adjusted to 10.5-11.0 with 5N NaOH. The solution is stirred for 1 hr and then extracted with ethyl acetate. The ethyl acetate extract is evaporated to dryness i n vacuo and is precipitated from acetone:water to yield 378 mg of narasin A sodium salt. The salt can be crystallized from acetonitrile (rn.p. 158-160'C). An alternative procedure, shown below, even though complicated, has worked consistently. Five g of narasin A (acid) is dissolved in a mixture of 200 ml of 2.5N NaOH and 200 m l
506 d i o x a n e and s t i r r e d f o r 2 h r . The r e a c t i o n m i x t u r e i s d i l u t e d w i t h 2 0 0 m l o f water and 2 0 0 m l e t h y l a c e t a t e a n d s t i r r e d slowly. The o r g a n i c p h a s e i s s e p a r a t e d and washed w i t h 2 0 0 m l o f water. I f an e m u l s i o n o c c u r s , as o f t e n h a p p e n s , 300 m l e t h y l a c e t a t e a n d 120 m l acetone a r e a d d e d t o b r e a k t h e e m u l s i o n . The o r g a n i c p h a s e i s d r i e d o v e r a n h y d r o u s Na2SO4 a n d a l l o w e d t o The s t a n d o v e r n i g h t a t room t e m p e r a t u r e f o r c r y s t a l l i z a t i o n . c r y s t a l s are f i l t e r e d o f f ; t h e m o t h e r l i q u o r is c o n c e n t r a t e d t o d r y n e s s i n vacuo; and t h e r e s i d u e i s d i s s o l v e d i n 1 5 m l a c e t o n i t r i l e b y h e a t i n g . The a c e t o n i t r i l e s o l u t i o n i s c h i l l e d , s e e d e d w i t h t h e c r y s t a l s c o l l e c t e d previously, and t h e c r y s t a l l i z a t i o n i s c o m p l e t e d o v e r n i g h t a t room t e m p e r a t u r e . The c r y s t a l s a r e f i l t e r e d and d r i e d in vacuo. The m o t h e r l i q u o r i s f u r t h e r conc e n t r a t e d t o y i e l d a d d i t i o n a l crops of c r y s t a l s t o give a f i n a l y i e l d o f 2 . 7 g n a r a s i n A , sodium s a l t , (m.p. 158-160OC). T h i s p r o d u c t can b e r e c r y s t a l l i z e d from a c e t o n i t r i l e . 4.3.5
Detection and a
m
The n a r a s i n f a c t o r s can be d e t e c t e d by b o t h m i c r o b i o l o g i c a l and c h e m i c a l methods. Bioautograms o f TLC p l a t e s and p a p e r c h r o m a t o g r a p h i c s t r i p s c a n r e a d i l y b e made from BacLeLud A u b UCi-5 ATCC 6 6 3 3 i n o c u l a t e d a g a r p l a t e s . A s e n s i t i v i t y of 0 . 1 pg and l e s s can be o b t a i n e d . A d i s c - p l a t e a g a r d i f f u s i o n a s s a y u s i n g B. - 5 u b t i l i A h a s b e e n d e v i s e d w i t h a s e n s i t i v i t y of a b o u t 2 pg/ml (J, Westhead, L i l l y R e s e a r c h L a b o r a t o r i e s , u n p u b l i s h e d d a t a ) . A more d e s i r a b l e a s s a y h a s been d e v e l o p e d u t i l i z i n g Staphy~ococcu-5aulltu-5 NRRL 8-314 i n a semi-automated t u r b i d i metric s y s t e m w i t h a s e n s i t i v i t y r a n g e o f 1 . 0 - 5 . 0 vg/ml4. The v a n i l l i n - s u l f u r i c a c i d a s s a y method d e s c r i b e d i n t h e monensin s e c t i o n i s a p p l i c a b l e t o t h e d e t e r m i n a t i o n of n a r a s i n . TLC a n d p a p e r chromatography a r e b o t h u s e f u l i n t h e s e p a r a t i o n and det e c t i o n of n a r a s i n f a c t o r s . Representative paper chromatographic s y s t e m s a n d R+ v a l u e s d e t e c t e d by 8. d u b t i e i a b i o a u t o g r a p h y are g i v e n below: Rf Values
D
Factor A
Factor B
0.41
0.16
0.26
Water s a t u r a t e d with MIBK* p l u s 2% p - t o l u e n e s u l f o n i c a c i d and 1%p i p e r idine.
0.54
0.46
0.36
Water:methanol: acetone (12:3: 1) a d j u s t e d t o p H 10.5 with NH40H and then lowered t o pH 7.5 w i t h H3POb.
0.48
0.36
0.29
1%MIBKU, 0.5% NH40H i n water.
0.15
0.33
0.25
17.4 g K2HP04, 30 m l e t h a n o l p e r l i t e r o f water.
0.24
0.51
0.26
Benzene s a t u r a t e d w i t h water.
Factor
Solvent System
-
*Methyl I s o b u t y l Ketone.
S e v e r a l r e p r e s e n t a t i v e s i l i c a g e l TCL s y s t e m s a n d Rf v a l u e s as d e t e c t e d b y s p r a y and b i o a u t o g r a p h y a r e shown h e r e :
50 7
Rf Values Factor A
Factor B
Factor D
Solvent System
0.24 0.54
0.42 0.34
0.26 0.66
6enzene:ethyl acetate ( 3 : 2 ) , Ethyl acet8te:diethylamine (95:5).
0.19
0.33
0.20
Ethyl acetate,
N a r a s i n A methyl e s t e r h a s a n R f of 0.53 i n t h e e t h y l acet a t e s y s t e m as d e t e c t e d by s p r a y i n g ; w h e r e a s , no zone i s det e c t e d on a b i o a u t o g r a m . P l a t e s s p r a y e d w i t h v a n i l l i n (3%)-0.5% H2SOI, i n methanol d e v e l o p t h e b e s t b l u e c o l o r w i t h l e s s background c o l o r when t h e g l a t e s are h e a t e d on a steam t a b l e r a t h e r Re-spraying t h e p l a t e s w h i l e w a r m t h a n i n an oven a t 1 0 0 C. and t h e n r e h e a t i n g i n t e n s i f i e s t h e c o l o r . The e t h y l a c e t a t e : d i e t h y l a m i n e ( 9 6 : 4 ) s y s t e m c a n b e u s e d i n a q u a n t i t a t i v e TLC method as d e s c r i b e d f o r monensin t o d e t e r m i n e t h e p e r c e n t a g e of n a r a s i n f a c t o r s u s i n g a S c h o e f f e l SD3000 S p e c t r o d e n s i t o m e t e r . C e l l u l o s e TLC p l a t e s r u n i n w a t e r : methano1:acetone ( 1 2 : 3 : 1 ) ( a d j u s t e d t o pH 10.5 w i t h N H k O H , t h e n lowered t o pH 7 . 5 w i t h H C 1 ) show t h e f o l l o w i n g Rf v a l u e s as d e t e c t e d on b i o a t u g o r a p h y p l a t e s : A = 0 . 4 6 ; B = 0 . 4 0 ; D = 0 . 3 1 .
4.4 Literature
Cited
1.
Y . M i y a z a k i , M. S h i b u y a , H . Sugawara, 0 . Kawaguchi, C . H i r o s e , J. Nagatsu and S . E s u m i , J . A n t i b i o t i c s 2 7 ( 1 9 7 4 ) 814-821.
2.
R . L. H a m i l l , D. H. Berg and M . M . Hoehn, B e l g i a n P a t e n t 830043; December 1 0 , 1 9 7 5 .
3.
J . L. O c c o l o w i t z , D. H . B e r g , M . Debono and R . Biomed. Mass S p e c t r o m e t r y 3 ( 1 9 7 6 ) 272-277.
4.
L . D. Boeck, M . M . Hoehn, R . E . K a s t n e r , R . W . W e t z e l , N . E . Davis and J . E . Westhead, t o b e p u b l i s h e d , Developments i n I n d u s t r i a l M i c r o b i o l o g y , Vol. 1 8 .
L. H a m i l l ,
50 8
5 . Lonomycin
5.1 Introduction I n 1975 and 1 9 7 6 f o u r d i f f e r e n t g r o u p s of i n v e s t i g a t o r s r e p o r t e d t h e i s o l a t i o n of a new p o l y e t h e r a n t i b i o t i c . The d e s i g n a t i o n s A128l, lonomycin ( T M - 4 8 1 ) 2 , e m e r i c i d (RP 3155913, and DE 3936'+ w e r e u s e d f o r t h i s a n t i b i o t i c . I d e n t i c a l s t r u c t u r e s h a v e been p u b l i s h e d f o r e m e r i c i d , lonomycin, a n d DE 3936. T s u j i et h a v e presumed t h a t A 2 1 8 and lonomycin a r e i d e n t i c a l a f t e r a comparison of I R s p e c t r a . 5.1.1
Producing organisms
S t a e p t o m y c e a ~ i b o ~ i d i d i c uTM-481 d ATCC 310512 ; S f A e p t o -
mycea h y g x o A e o p i c u n FERM-P92E1 ; SfAeptomycsA h y g a o d c o p i c u h DS 24367 NRRL 57873 and S&ctneptOmyceA hygAoACOpiCuA 9735-14. 5.1.2
Chemical a n d p h y s i c a l p r o p e r t i e s 5
Lonomycin, f r e e a c i d C44H76014
mol. w t .
829.1
pKa 6 . 5 ( 6 6 % d i m e t h y l f o r m a m i d e l Lonomycin, sodium s a l t C4,+H75OI4 m o l .
m.p.
1 ~ 8 - 1 8 9 ~ ~
{r~}k5
5.1.3
w t . 851.1
+47O ( c = 1 , m e t h a n o l ) Structural
E m e r i c i d (Lonomycin, A218)
5.2 Summary
of b i o l o g i c a l a c t i v i t i e s and u s e s
DE-3936 i s a c t i v e i n v i a 2 0 a g a i n s t g r a m - p o s i t i v e b a c t e r i a , m y c o b a c t e r i a , and mycoplasma. The LD,, v a l u e s i n mice f o r DE3936, sodium s a l t a r e as f o l l o w s : 4 5 . 8 mg/kg, o r a l ; 1 3 . 0 mg/kg, i . p . ; 3 7 . 5 rng/rng, s . c . ~ . B e n a z e t e t have r e p o r t e d on t h e a n t i c o c c i d i a l a c t i v i t y o f emericid i n c h i c k e n s a n d r a b b i t s . A t l e v e l s o f 0 . 0 0 6 3 t o 0 . 0 1 % i n t h e f e e d , e r n e r i c i d was e f f e c t i v e i n c o n t r o i l i n g b o t h s i n g l e s p e c i e s and mixed i n f e c t i o n s of
509
Eirnehia i n c h i c k e n s . A t a l e v e l o f 0.0025% i n t h e f e e d , e m e r i c i d c o n t r o l l e d i n f e c t i o n s of EirneAia h t i e d a e i n r a b b i t s .
5.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
The i s o l a t i o n methods r e p o r t e d f o r A 2 1 8 '
and e m e r i c i d 1 °
are s i m i l a r t o t h e g e n e r a l methods p r e v i o u s l y d i s c u s s e d . Sephad e x LD20 i s u s e d i n t h e p u r i f i c a t i o n o f l o n o m y c i n 5 a n d DE-393fj4. The d e t a i l s o f t h e i s o l a t i o n method f o r DE-3936 are g i v e n below. 5.3.1
I s o l a t i o n method f o r D E - 3 9 3 6 4
The f e r m e n t a t i o n b r o t h i s f i l t e r e d and t h e f i l t r a t e i s e x t r a c t e d a t pH 7 w i t h b e n z e n e . The m y c e l i a l c a k e i s e x t r a c t e d w i t h m e t h a n o l and t h e m e t h a n o l i c e x t r a c t i s c o n c e n t r a t e d i n vacuo t o an a q u e o u s s o l u t i o n which i s t h e n e x t r a c t e d w i t h b e n z e n e . The two b e n z e n e e x t r a c t s a r e combined a n d c o n c e n t r a t e d in vacuo t o an o i l . The o i l i s d i s s o l v e d i n b e n z e n e a n d p u r i f i e d o v e r a n a l u m i n a column p a c k e d i n b e n z e n e . The column i s washed w i t h b e n z e n e , t h e n e t h y l a c e t a t e , and t h e a c t i v i t y is e l u t e d w i t h e t h y l a c e t a t e : m e t h a n o l ( 1 : l ) . The a c t i v e f r a c t i o n s are combined, c o n c e n t r a t e d i n vacuo and c h r o m a t o g r a p h e d o v e r a column o f Sephadex LH-20 ( P h a r m a c i a F i n e C h e m i c a l s , P i s c a t a w a y , N J ) i n m e t h a n o l . The a c t i v e f r a c t i o n s are combined a n d c o n c e n This residue is dissolved t r a t e d i n vacuo t o a n o i l y r e s i d u e . i n b e n z e n e a n d c h r o m a t o g r a p h e d t w i c e o v e r a s i l i c a g e l column packed i n b e n z e n e u s i n g a s o l v e n t s y s t e m of a c e t o n e : b e n z e n e i n g r a d i e n t from 1 : 2 0 t o 1 : 5 . A c t i v e f r a c t i o n s a r e c o n c e n t r a t e d i n vacuo t o d r y n e s s a n d t h e r e s i d u e i s c r y s t a l l i z e d from b e n z e n e - p e t r o l e u m e t h e r t o y i e l d t h e s o d i u m - p o t a s s i u m s a l t o f DE-3936. 5.3.2
Detection and a s s a y 4
DE-3936 i s c h r o m a t o g r a p h e d on TLC s i l i c a g e l p l a t e s u s i n g e t h y l a c e t a t e : b e n z e n e (1:l) a s t h e d e v e l o p e r a n d v a n i l l i n S u l f u E i c a c i d i n e t h a n o l as t h e s p r a y r e a g e n t . Under t h e s e c o n d i t i o n s , DE-3936 h a s a n Rf v a l u e of a b o u t 0 . 2 . 5.4 L i t e r a t u r e -
1.
Cited
T s u j i , K . Nagashima, M . K o b a y a s h i , Y . W a k i s a k a , Y . Kawamura, S . Kozuki and M . Mayama, J . A n t i b i o t i c s 29 ( 1 9 7 6 ) N.
10-14. 2.
J. Sawada, S . Omura, M . S h i b a t a and S . Machida, U.S.
Patent
3,950,514; A p r i l 1 3 , 1 9 7 6 . 3.
J . F l o r e n t , J. L u n e l and D . Mancy, German P a t e n t 2 , 5 0 9 , 6 5 3 ; S e p t e m b e r 11, 1 9 7 5 .
4,
M . Ohshima, N . I s h i z a k i , K . Abe, M. U k a w a , Y . Marumoto, K . N a k a t s u k a , T. H o r i u c h i , Y . Tonooka, S. Y o s h i n o a n d N. Kanada, J. A n t i b i o t i c s 2 9 ( 1 9 7 6 ) 354-365.
510 Omura, M. S h i b a t a , S . Machida a n d J. Sawada, J. A n t i b i o t i c s 29 (1976) 15-20.
5.
S.
6.
N. O t a k e a n d M. Koenuma, T e t r a h e d r o n L e t t . (1975) 41Q7-4150.
7.
C. R i c h e a n d C . P a s c a r d - B i l l y , J . Chem. S O C . , Chem. Commun. (1975) 951-952.
8.
K . Yamazaki. K . Abe a n d M . 91-92,
9.
F. B e n a z e t , J. R . C a r t i e r , J . F l o r e n t , C . J o h n s o n , J. L u n e l and D. Mancy, i n J. D. Williams a n d A . M. Geddes ( E d i t o r s ) , Chemotherapy, Vol. 6 , P a r a s i t e s , Fungi a n d V i r u s e s , Plenum Press, N e w York and London, 1976, pp. 91-96.
10.
L. N i n e t , F. B e n a z e t , D. Mancy, A. Abraham, Godard, M . Moreau, R . E x p e r i e n t i a 32 (1976)
Sano, J . A n t i b i o t i c s 29 (1976)
H . D e P a i r e , J. F l o r e n t , J. L u n e l , J. R . C a r t i e r , N . d e c h e z e l l e s , C. T i s s i e r and J . Y . L a l l e m a n d , 319-321.
511
6.1 Introduction A204A is the major factor of a polyether antibiotic complex showing structural similarities to nigericin and grisorixin. Several of the alkyl ether derivatives of A204A have been made and tested. These ether derivatives have similar biological activities to the parent compound but are less toxic. 6.1.1
Producing organism
Sa2eptomyced albud NRRL 3 3 84 l. 6.1.2
Chemical descriptionlY2
A2 0 4A
C49H84017 mol. wt. 945.16 m.p. 96-97'C t68.1'
(c-2,
methanol)
pKa 6.1 (66% dimethylformamide) A204A, sodium salt C4,Hs3017Na
mol. wt. 967.0
m.p. 178-179'C t54.9' 6.1.3
(c.2,
methanol)
Structural formula2
Antibiotic A 2 0 4 A 6 . 2 Summary of --
biological activities and uses
A204A inhibits gram-positive organisms i n u i t n o . When incorporated into feed at a level of from 9 to 45 g/ton, A204A will control coccidiosis in poultry1. A204A shows similar activity to other polyether antibiotics in increasing feed
51 2 e f f i c i e n c y i n ruminants3.
The LD50 v a l u e i n mice i s 9 . 2 mglkg
0ra14.
The a l k y l e t h e r s o f A204A a r e c o n s i d e r a b l y l e s s t o x i c t h a n t h e p a r e n t compound5. LD50 v a l u e s (mice, o r a l ) f o r t h e s e d e r i v a t i v e s are: m e t h y l e t h e r , 43 mg/kg; e t h y l e t h e r , 1 1 5 mg/kg; n - p r o p y l e t h e r , 3 8 mg/kg; and i s o p r o p y l e t h e r , 20-36 mg/kg. The e t h e r d e r i v a t i v e s are similar t o t h e p a r e n t compound i n b i o l o g i c a l a c t i v i t i e s , i . e . , a n t i b a c t e r i a l , a n t i c o c c i d i a l , and increasing feed efficiency.
6.3 Separation 6.3.1
and p u r i f i c a t i o n methods
I s o l a t i o n o f A204 complex'
The f e r m e n t a t i o n b r o t h i s f i l t e r e d w i t h t h e a i d of HyfloS u p e r c e l ( J o h n s - M a n v i l l e Corp., New Y o r k ) . The m y c e l i a l f i l t e r c a k e i s e x t r a c t e d t h r e e times w i t h o n e - h a l f volume of 5 0 % aqueous methanol. The combined e x t r a c t s are c o n c e n t r a t e d i n V Q C U O t o remove t h e m e t h a n o l , and t h e aqueous s o l u t i o n i s combined with t h e b r o t h f i l t r a t e . This s o l u t i o n is a d j u s t e d t o pH 8 . 5 and e x t r a c t e d two times w i t h h a l f volumes o f e t h y l a c e tate. The combined e x t r a c t s a r e c o n c e n t r a t e d i n V Q C U O t o d r y n e s s t o y i e l d c r u d e A 2 0 4 s a l t ( m i x t u r e o f sodium and p o t a s s i u m salts). The c r u d e s a l t i s d i s s o l v e d i n c h l o r o f o r m ( 1 0 ml/g of s o l i d s ) and t h i s s o l u t i o n i s p a s s e d o v e r a c a r b o n column, P i t t s b u r g h T y p e CAL, 1 2 x 4 0 ( P i t t s b u r g h A c t i v a t e d Carbon Co., P i t t s b u r g h , PA) ( 2 g c a r b o n / l g of A2041 i n c h l o r o f o r m i n o r d e r t o remove c o l o r and o i l y i m p u r i t i e s . The c h l o r o f o r m e l u t i o n i s c o n t i n u e d u n t i l a l l t h e A204 h a s b e e n washed o f f as i n d i c a t e d The c h l o r o f o r m e l u a t e i s by a n e g a t i v e r e a c t i o n t o v a n i l l i n . c o n c e n t r a t e d i n V ~ C U Ot o d r y n e s s a n d t h e r e s i d u e i s d i s s o l v e d i n a small volume o f w a r m m e t h a n o l . The s o l u t i o n i s f i l t e r e d The c r y s t a l s a n d t h e f i l t r a t e is c h i l l e d f o r c r y s t a l l i z a t i o n . a r e r e c r y s t a l l i z e d by d i s s o l v i n g i n a c e t o n e , a d d i n g o n e - h a l f volume o f water, and a l l o w i n g t o s t a n d a t room t e m p e r a t u r e . The c r y s t a l l i n e p r o d u c t i s a m i x t u r e o f t h e sodium and p o t a s s i u m s a l t s of A204A a s w e l l as a small amount of t h e A204B s a l t s . The a c i d form of A204 can be o b t a i n e d by a d j u s t i n g t h e pH o f t h e o r i g i n a l aqueous s o l u t i o n t o 3 w i t h H C 1 b e f o r e t h e e t h y l a c e t a t e e x t r a c t i o n and p r o c e e d i n g as o u t l i n e d f o r t h e p u r i f i c a tion. T h i s s t e p , however, can r e s u l t i n d e g r a d a t i o n o f t h e a c i d form, and t h e c r y s t a l l i z a t i o n from m e t h a n o l can r e s u l t i n f o r m a t i o n of t h e m e t h y l e t h e r o f A 2 0 4 . The a c i d form i s b e s t p r e p a r e d from t h e s a l t by d i s s o l v i n g t h e s a l t i n d i o x a n e , addi n g a volume o f water; a n d a d j u s t i n g t h e pH t o 4 w i t h d i l u t e H C 1 , and c o n c e n t r a t i n g i n V Q C U O t o remove t h e d i o x a n e . The res u l t i n g a q u e o u s s u s p e n s i o n i s e x t r a c t e d w i t h c h l o r o f o r m , and t h e chloroform e x t r a c t is c o n c e n t r a t e d i n VQCUO t o dryness. The r e s i d u e i s c r y s t a l l i z e d from d i e t h y l e t h e r . The c r y s t a l l i n e sodium, p o t a s s i u m , and ammonium s a l t s o f A204 are p r e p a r e d by d i s s o l v i n g 1 g of t h e a c i d form i n 1 0 m l a c e t o n e , a d d i n g 1 0 m l of w a t e r , and a d j u s t i n g t h e pH t o 9 w i t h
513
t h e r e s p e c t i v e hydroxide. The m i x t u r e i s warmed s l i g h t l y t o c l a r i f y and a l l o w e d t o s t a n d a t - 1 5 O C u n t i l c r y s t a l l i z a t i o n i s c o m p l e t e . The c r y s t a l s are f i l t e r e d a n d r e c r y s t a l l i z e d from a q u e o u s e t h a n o l o r aqueous a c e t o n e . 6.3.2
S e p a r a t i o n o f A204 f a c t o r s 1
The A204 s a l t ( 1 0 0 g ) c o n t a i n i n g A 2 0 4 A and a small amount of A204B i s d i s s o l v e d i n 5 0 0 m l of b e n z e n e : e t h y l a c e t a t e ( 7 : 3 ) and t h e s o l u t i o n i s chromatographed o v e r a column c o n t a i n i n g 2 kg of s i l i c a g e l (Grade 6 2 , W. R . Grace, Davison Chemical D i v . , B a l t i m o r e , MD) u s i n g b e n z e n e : e t h y l a c e t a t e ( 7 : 3 ) as t h e e l u t i n g s o l u t i o n . The e l u t i o n was f o l l o w e d by s i l i c a g e l TLC w i t h e t h y l a c e t a t e as t h e d e v e l o p i n g s o l v e n t a n d s u l f u r i c a c i d s p r a y f o r d e t e c t i o n . A204A i s e l u t e d f i r s t . A f t e r approximately 32 9. of e l u a t e are c o l l e c t e d , t h e s o l v e n t i s changed t o b e n z e n e : A204A-rich f r a c t i o n s a r e e t h y l a c e t a t e (1:l) t o e l u a t e A 2 0 4 B . combined, c o n c e n t r a t e d t o d r y n e s s and t h e r e s i d u e i s c r y s t a l l i z e d from a c e t o n e - w a t e r t o y i e l d t h e mixed s o d i u m - p o t a s s i u m s a l t of A204A. Mixed s o d i u m - p o t a s s i u m s a l t s of A204B are r e c o v e r e d i n a similar manner. T h i s p r e p a r a t i o n ( 3 g ) i s r e c h r o m a t o g r a p h e d on a 2 x 4 5 cm s i l i c a g e l column d e v e l o p e d w i t h b e n z e n e : e t h y l a c e t a t e ( 7 : 3 ) i n o r d e r t o s e p a r a t e A 2 0 4 B from A204C a n d o t h e r minor factors. The column i s d e v e l o p e d a t a r a t e of 11 m 1 / 1 5 min. The e l u t i o n i s m o n i t o r e d by TLC and t h e A204B-rich f r a c t i o n s (no. 5 0 - 8 4 ) a r e combined a n d c o n c e n t r a t e d t o d r y n e s s . The r e s i d u e i s c r y s t a l l i z e d from a c e t o n e - w a t e r t o y i e l d 780 mg of A204B mixed s o d i u m - p o t a s s i u m s a l t (m.p. 1 7 7 - 1 7 9 O C ) . O t h e r A204 f a c t o r s are recovered i n l a t e r f r a c t i o n s . 6.3.3
P r e p a r a t i o n o f A204A m e t h y l e t h e r d e r i v a t i v e from A204A sodium s a l t 5
A n t i b i o t i c A204A sodium s a l t ( 2 0 g ) i s d i s s o l v e d i n metha n o l (1 9 . ) and w a t e r ( 5 0 0 m l ) i s added s l o w l y . T h i s s o l u t i o n i s a d j u s t e d t o pH 3.0 by t h e a d d i t i o n o f 6N h y d r o c h l o r i c a c i d . The r e s u l t i n g s o l u t i o n i s s t i r r e d f o r 1 h r a n d t h e n i s e x t r a c t e d w i t h an e q u a l volume o f c h l o r o f o r m . T h i s c h l o r o f o r m e x t r a c t i s e v a p o r a t e d t o d r y n e s s u n d e r vacuum. The r e s u l t i n g r e s i d u e ( 2 g ) i s d i s s o l v e d i n 1 0 m l o f a b e n z e n e : e t h y l a c e t a t e ( 7 : 3 ) m i x t u r e , and t h i s s o l u t i o n i s c h r o matographed on a 2 x 5 1 cm s i l i c a g e l column ( M . Woelm, Eschwege, Germany), e l u t i n g w i t h t h e same b e n z e n e : e t h y l a c e t a t e s o l v e n t m i x t u r e i n o r d e r t o s e p a r a t e t h e p r o d u c t from u n r e a c t e d A 2 0 4 A . E l u t i o n i s m o n i t o r e d by TLC. F r a c t i o n s c o n t a i n i n g A204A m e t h y l e t h e r d e r i v a t i v e a r e combined and e v a p o r a t e d t o d r y n e s s u n d e r vacuum. A204A m e t h y l e t h e r d e r i v a t i v e ( 1 . 4 g l 0 i s c r y s t a l l i z e d from m e t h a n o l - w a t e r , m.p. 1 1 5 - 1 1 7 ° C , +75 ( c f l , methanol); pKa ( 6 6 % aqueous d i m e t h y l f o r m a m i d e ) 7 . 1 . 6.3.4
P r e p a r a t i o n o f A204A m e t h y l e t h e r d e r i v a t i v e from A204A3
A n t i b i o t i c A204A i n t h e a c i d form ( 4 0 0 mg) i s d i s s o l v e d i n m e t h a n o l ( 1 0 m l ) , and water ( 5 m l ) i s added. A f t e r b e i n g
514 a l l o w e d t o s t a n d 1 h r , t h e s o l u t i o n i s e v a p o r a t e d u n d e r vacuum. The r e s u l t i n g r e s i d u e i s chromatographed as d e s c r i b e d i n method 3 t o g i v e A204A m e t h y l e t h e r d e r i v a t i v e . 6.3.5
D e t e c t i o n and a s s a y
A 2 0 4 f a c t o r s can b e a s s a y e d by d i s c - p l a t e a g a r d i f f u s i o n methods u s i n g B. A u b t i L i A as t h e a s s a y o r g a n i s m . B i o a u t o g r a m s can be made from p a p e r c h r o m a t o g r a p h i c s t r i p s a n d TLC p l a t e s u s i n g B . ~ u b t i t i t ias ~ t h e d e t e c t i n g o r g a n i s m . Chemical and m i c r o b i o l o g i c a l methods d e s c r i b e d i n t h e monensin s e c t i o n a r e g e n e r a l l y a p p l i c a b l e t o t h e A204 f a c t o r s .
Two p a p e r c h r o m a t o g r a p h i c s y s t e m s f o r A 2 0 4 are g i v e n be low : Solvent
Rf -
Water: n-propanol ( 9 :1)
0.87
Water :methanol :acetone (12:3:1) s o l u t i o n a d j u s t e d t o pH 10.5 w i t h NH40H and then the pH i s lowered t o pH 7.5 with H~POI,
0.33
P a p e r chromatography g i v e s e s s e n t i a l l y n o r e s o l u t i o n o f t h e complex i n t o i t s i n d i v i d u a l f a c t o r s , b u t t h i s r e s o l u t i o n can be a c c o m p l i s h e d by TLC. Using e t h y l a c e t a t e as t h e d e v e l o p i n g s o l v e n t on s i l i c a g e l p l a t e s , t h e f o l l o w i n g R f v a l u e s are o b t a i n e d : -A204A, 0 . 8 2 ; A 2 0 4 B , 0 . 7 5 ; A204C, 0 . 6 4 . A developing s o l u t i o n c o n s i s t i n g of b e n z e n e : e t h y l a c e t a t e (1:l) s e p a r a t e s A204A and i t s e t h e r d e r i v a t i v e s w i t h a p p r o x i m a t e Rf v a l u e s o f A204A, 0 . 2 3 ; A204A m e t h y l e t h e r , 0 . 3 0 ; A2OliA e t h y l e t h e r , 0 . 4 1 ; A204A n - p r o p y l e t h e r , 0 . 5 1 . B e n z e n e : a c e t o n i t r i l e (1:l) i s a l s o a n e x c e l l e n t d e v e l o p e r f o r s e p a r a t i o n of A204 f a c t o r s and d e r i vatives. D e t e c t i o n i s a c c o m p l i s h e d by b i o a u t o g r a p h y u s i n g B. A u b t i e i d as t h e d e t e c t i n g o r g a n i s m o r by u s e of t h e f o l l o w i n g
s p r a y r e a g e n t s : 3% v a n i l l i n and 1 . 5 % s u l f u r i c a c i d i n m e t h a n o l f o l l o w e d by h e a t i n g t h e p l a t e a t 100°C f o r 5 min; s u l f u r i c a c i d f o l l o w e d by h e a t i n g t h e p l a t e a t 1 0 0 ° C f o r 5 min; o r Dragend o r f f ' s reagent.
6.4 Literature
Cited
1.
R . L . H a m i l l a n d M. M . December 5 , 1 9 7 2 .
2.
N. D . J o n e s , M . 0. Chaney, J . W . Chamberlin, R . L. H a m i l l a n d S . Chen, J . A m e r . Chem. SOC. 95 (19731, 3399-3400.
3.
A. P. Raun, U.S. P a t e n t 3,794,732;
4.
H. M. Worth, D. B. Meyers, W. R. Gibson a n d G . C. Todd, Antimi c r o b Ag Chemother. - 1 9 7 0 ( 19 7 1 1 3 57-36 0.
.
.
Hoehn, U.S.
P a t e n t 3,705,238;
F e b r u a r y 26, 1 9 7 4 .
515 5.
R.
L. Hamill, U.S. P a t e n t 3 , 9 0 7 , 8 3 2 ;
Se pte mbe r 2 3 , 1975.
516 7 . A23187
7.1 Introduction A23187 i s a n i o n o p h o r e q u i t e s p e c i f i c f o r d i v a l e n t c a t i o n s . This a n t i b i o t i c i s a monocarboxylic a c i d c o n t a i n i n g a s p i r o r i n g s y s t e m c o n s i s t i n g o f two t e t r a h y d r o p y r a n r i n g s . Howe v e r , A23187 i s n o t a t y p i c a l p o l y e t h e r a n t i b i o t i c s i n c e i r s s t r u c t u r a l f e a t u r e s also include a s u b s t i t u t e d benzoxazole r i n g a n d a n a - k e t o - p y r r o l e r i n g . A23187 s h a r e s t h e s o l u b i l i t y p r o p e r t i e s o f t h e p o l y e t h e r s , s i n c e b o t h t h e a c i d a n d salt f o r m s are w a t e r - i n s o l u b l e , b u t s o l u b l e i n o r g a n i c s o l v e n t s . Producing orpanism
7.1.1
S t a e p t o r n y c e d chaa.t.teudid N R R L 3 8 8 2 l . Chemical and p h y s i c a l p r o p e r t i e s l Y 2
7.1.2
CZ9H37N3O6
m.p.
mol. w t .
523.6
181-182OC
Ia}C5 -56'
(C.1,
chloroform)
Amax ( e t h a n o l ) 2 0 4 nm, E:$,
= 435
= 405 1cm 2 7 8 nm, E1% = 2 9 0 1cm
2 2 6 nm, El%
= 270
285 ( s h ) nm,
1% 380 nm, Elcm pKa 6 . 9 7.1.3
50
(90% dimethyl sulfoxide)
S t r u c t u r a l formula2
'NHMe CO2H
517
7 . 2 Summary
of b i o l o g i c a l a c t i v i t i e s and u s e s
A23187 e x h i b i t s i n v i t . 4 0 a n t i m i c r o b i a l a c t i v i t y a g a i n s t gram-positive b a c t e r i a and fungi. The L D 5 0 v a l u e i n mice ( i . p . 1 is 5 . 8 mg/kgl. A23187 h a s f o u n d b r o a d a p p l i c a t i o n a s a b i o c h e m i c a l t o o l b e c a u s e o f i t s h i g h s e l e c t i v i t y as a c o m p l e x i n g a g e n t f o r d i v a l e n t c a t i o n s 3 . Calcium p l a y s a r o l e i n s t i m u l u s - s e c r e t i o n c o u p l i n g and A23187 h a s b e e n w i d e l y u s e d t o i n v e s t i g a t e t h i s p r o c e s s . For i n s t a n c e , A23187 s t i m u l a t e s h i s t a m i n e r e l e a s e f r o m mast c e l l s 4 , i n d u c e s s e c r e t i o n from r a b b i t l a c r i m a l g l a n d 5 , and i n c r e a s e s t h e r a t e o f enzyme s e c r e t i o n i n t h e r a t p a n c r e a s 6 . O t h e r u n i q u e u s e s for A23187 i n c l u d e a c t i v a t i o n o f u n f e r t i l i z e d e g g s 7 and i n v e s t i g a t i o n of d i s o r d e r s of p r o t e i n s y n t h e s i s i n m u s c l e c e l l s from m u s c u l a r d y s t r o p h y p a t i e n t s 8 .
7.3 Separation,.
e x t r a c t i o n and p u r i f i c a t i o n
A23187 i s c o p r o d u c e d w i t h t u n i c a m y c i n , a g l u c o s a m i n e c o n t a i n i n g a n t i b i o t i c f i r s t r e p o r t e d i n 1 9 7 1 9 . Both a n t i b i o t i c s a r e f o u n d a l m o s t e x c l u s i v e l y i n t h e mycelium. Two p r o c e d u r e s a r e given here; t h e first provides f o r the e x t r a c t i o n of both a n t i b i o t i c s , and t h e second procedure a l l o w s f o r p r e f e r e n t i a l e x t r a c t i o n o f A23187. 7.3.1
I s o l a t i o n method N o . 2
The f e r m e n t a t i o n b r o t h ( 5 0 L ) i s f i l t e r e d w i t h H y f l o S u p e r c e l ( J o h n s - M a n v i l l e C o r p . , New Y o r k ) , and t h e f i l t e r c a k e i s washed w i t h w a t e r . A f t e r d i s c a r d i n g t h e f i l t r a t e a n d w a s h , t h e f i l t e r cake i s s u s p e n d e d i n 8 . 5 1 of m e t h a n o l , s t i r r e d a t room t e m p e r a t u r e f o r o n e - h a l f h o u r a n d f i l t e r e d . Two more s i m i l a r e x t r a c t i o n s a r e done t o remove m o s t o f t h e t u n i c a m y c i n a n d a s m a l l p o r t i o n o f A23187. The c a k e i s t h e n e x t r a c t e d t h r e e t i m e s by s t i r r i n g i n 11 of h o t methanol (60-65'C) and f i l t e r i n g . The f i l t r a t e s from t h e h o t m e t h a n o l a r e combined, c o n c e n t r a t e d t o a b o u t 4 P. a n d c o o l e d t o c r y s t a l l i z e t h e mixed c a l c i u m magnesium s a l t of A23187 w h i c h c o n t a i n s a small amount o f t u n i camycin. The c r y s t a l s a r e f i l t e r e d o f f a n d d r i e d . Tunicamycin p r e s e n t i n t h e m o t h e r l i q u o r c a n b e p r e c i p i t a t e d by a d d i t i o n o f a c e t o n e . An a d d i t i o n a l amount o f A23187 i s r e c o v e r e d from t h e room t e m p e r a t u r e m e t h a n o l i c e x t r a c t s by c o n c e n t r a t i n g t h e e x t r a c t s i n vucuo t o a n a q u e o u s s o l u t i o n , a d j u s t i n g t h e pH t o 1 0 w i t h NaOH, and e x t r a c t i n g w i t h e t h y l a c e t a t e . Tunicamycin r e m a i n s i n t h e a q u e o u s p h a s e and c a n b e e x t r a c t e d i n t o n - b u t a n o l a t pH 3. The e t h y l a c e t a t e e x t r a c t i s c o n c e n t r a t e d t o a n o i l and t h e o i l i s d i s s o l v e d i n a small volume o f m e t h y l e n e c h l o r i d e . Methanol i s added s l o w l y t o t h i s s o l u t i o n u n t i l c r y z t a l l i z a t i o n s t a r t s , and t h e m i x t u r e i s c h i l l e d o v e r n i g h t a t 5 C f o r complete c r y s t a l l i z a t i o n . The mixed calcium-magnesium s a l t s o f A23187 a r e f i l t e r e d a n d d r i e d . C a u t i o n s h o u l d b e t a k e n d u r i n g t h e i s o l a t i o n p r o c e d u r e as h a n d l i n g o f l a r g e volumes o f conc e n t r a t e d s o l u t i o n s of A23187 c a n r e s u l t i n s k i n a n d e y e i r r i t a tion. Twenty grams of t h e mixed c r y s t a l l i n e Ca-Mg s a l t s of A23187 c o n t a i n i n g t u n i c a m y c i n a r e f u r t h e r p u r i f i e d by d i s s o l v i n g
518 t h e s o l i d i n 9 0 m l of benzene and chromatographing t h e s o l u t i o n o v e r a s i l i c a g e l (Grade 62, W . R . Grace, D a v i s o n Chem. D i v . , Baltimore, MD) column ( 3 x 1 0 7 cm) p a c k e d i n b e n z e n e . The c o l umn i s washed w i t h 2.5 a o f b e n z e n e t o remove i m p u r i t i e s , a n d A23187 i s e l u t e d w i t h b e n z e n e : e t h y l a c e t a t e (95:5). The e l u t i o n i s m o n i t o r e d b y s i l i c a g e l TLC. F r a c t i o n s c o n t a i n i n g A23187 w i t h some t u n i c a m y c i n are combined, c o n c e n t r a t e d t o a r y n e s s i n v a c u o , and d i s s o l v e d i n h o t m e t h a n o l . The m e t h a n o l s o l u t i o n i s c o o l e d f o r c r y s t a l l i z a t i o n t o o c c u r a n d c r y s t a l s of t h e mixed Ca-Mg s a l t o f A23187 are f i l t e r e d o f f , washed w i t h c o l d metha n o l , and d r i e d i n v a c u a . The t u n i c a m y c i n i s p r e s e n t i n t h e m e t h a n o l f i l t r a t e and is p r e c i p i t a t e d by t h e a d d i t i o n o f a c e tone. 7.3.2
I s o l a t i o n method No. 2
The m y c e l i a l f i l t e r c a k e i s washed w i t h water and d r i e d w e l l on t h e f i l t e r f u n n e l u s i n g vacuum. The cake i s e x t r a c t e d twice w i t h c h l o r o f o r m ( o n e - h a l f of t h e o r i g i n a l b r o t h volume) by s t i r r i n g f o r o n e - h a l f h o u r and f i l t e r i n g . The c h l o r o f o r m e x t r a c t s , which c o n t a i n A23187 and no t u n i c a m y c i n , are combined a n d c o n c e n t r a t e d i n vacuo t o d r y n e s s . The r e s i d u e i s d i s s o l v e d i n c h l o r o f o r m and c h r o m a t o g r a p h e d o v e r a s i l i c a g e l column (10 a of Grace Grade 62) p a c k e d i n c h l o r o f o r m . The column i s washed w i t h c h l o r o f o r m t o e l u t e A23107, l e a v i n g most o f t h e i m p u r i t i e s a d s o r b e d . The f r a c t i o n s c o n t a i n i n g o n l y A23187 as d e t e c t e d by s i l i c a g e l TLC are combined and c o n c e n t r a t e d t o d r y n e s s . The r e s i d u e i s d i s s o l v e d i n a minimum of h o t m e t h a n o l and t h e s o l u t i o n i s c h i l l e d f o r c r y s t a l l i z a t i o n . The y e l l o w c r y s t a l s of t h e mixed Ca-Mg s a l t s of A23107 are f i l t e r e d o f f , washed w i t h c o l d m e t h a n o l , a n d d r i e d . 7.3.3
P r e p a r a t i o n of t h e a c i d form o f A23187
The Ca-Mg s a l t of A23187 i s c o n v e r t e d t o t h e a c i d form by d i s s o l v i n g 1 g i n 5 0 m l of e t h y l a c e t a t e and e x t r a c t i n g t h e s o l u t i o n s e v e r a l times w i t h e q u a l volumes of 0.1N H C 1 . The e t h y l a c e t a t e p h a s e i s c o n c e n t r a t e d t o an o i l , a n d t h e r e s i d u e i s d i s s o l v e d i n b e n z e n e . The b e n z e n e s o l u t i o n i s c o n c e n t r a t e d t o dryness i n v a c u a , r e d i s s o l v e d i n benzene, and r e c o n c e n t r a t e d t o dryness. These s t e p s a r e r e p e a t e d w i t h m e t h a n o l t o f i n a l l y o b t a i n a d r y y e l l o w t o t a n powder. The A23187 a c i d i s p u r i f i e d by d i s s o l v i n g t h e powder i n c h l o r o f o r m and c h r o m a t o g r a p h i n g t h e s o l u t i o n o v e r a P i t t s b u r g h Type 20/50 ( P i t t s b u r g h A c t i v a t e d Carbon Co., P i t t s b u r g h , P A ) c a r b o n column (20 m l o f c a r b o n p e r 1 g of powder) i n c h l o r o f o r m . C h l o r o f o r m washes are c o n t i n u e d u n t i l n o more A23187 i s d e t e c t e d . The c o l o r l e s s f r a c t i o n s c o n t a i n i n g A23187 a c i d a r e comb i n e d and c o n c e n t r a t e d t o d r y n e s s . The r e s i d u e i s d i s s o l v e d i n a minimum o f h o t m e t h a n o l and c h i l l e d a t 5OC f o r c r y s t a l l i z a t i o n t o o c c u r . The c o l o r l e s s c r y s t a l s a r e f i l t e r e g , washed w i t h c o l d m e t h a n o l , and d r i e d i n vacuo (mp 181-182 C). The m o t h e r l i q u o r w i l l y i e l d f u r t h e r c r o p s upon s t a n d i n g a t 5OC.
519 7.3.4
D e t e c t i o n and a s s a y
A23187 h a s w e a k a n t i b a c t e r i a l a c t i v i t y which c a n b e u t i l i z e d f o r bioautograms and b i o a s s a y . B U C i & ? U A dubt.Ll;iA can b e u s e d as t h e d e t e c t i n g o r g a n i s m f o r b i o a u t o g r a m s b u t i s i m p r a c t i c a l f o r d i s c - a g a r p l a t e a s s a y as l i t t l e o r n o z o n e s i z e d i f f e r e n c e i s observed o v e r a wide c o n c e n t r a t i o n r a n g e ( e . g . , 1 0 1 , 0 0 0 pg/ml). A semi-automated t u r b i d i m e t r i c b i o a s s a y u s i n g S&zphy&coccuA aulleud NRRL B-314 h a s b e e n d e v e l o p e d (J. Westhead, L i l l y Research L a b o r a t o r i e s , i n p r e p a r a t i o n ) for p u r i f i e d s a m p l e s as w e l l as b r o t h e x t r a c t s w i t h a s e n s i t i v i t y r a n g e o f 0 . 4 - 1 . 0 pg/ml. A b s o r p t i o n a t 278 nm a n d t h e h i g h f l u o r e s c e n c e p r o e r t i e s of A23187 h a v e b e e n u s e d i n o u r l a b o r a t o r i e s a n d o t h e r s l g f o r very s e n s i t i v e assays. Thin l a y e r c h r o m a t o g r a p h y h a s b e e n more u s e f u l t h a n p a p e r c h r o m a t o g r a p h y f o r d e t e c t i o n of A23187 a n d t u n i c a m y c i n . U s i n g c e l l u l o s e p l a t e s (aluminum s u p p o r t , E . Merck, D a r m s t a d t , Germany) w i t h n - b u t a n o l s a t u r a t e d w i t h water as t h e d e v e l o p i n g s o l v e n t a n d 8 . A u b t i L i A on a m i n i m a l a g a r medium as t h e t e s t o r g a n i s m , R f v a l u e s o f a b o u t 0.9 f o r A23187 a n d 0 . 3 f o r t u n i c a m y c i n are o b s e r v e d . A p p r o x i m a t e l y t h e same Rf v a l u e s a r e o b s e r v e d u s i n g p a p e r c h r o m a t o g r a p h y w i t h t h e same s o l v e n t system. S i l i c a g e l TLC i s v e r y u s e f u l f o r d e t e r m i n i n g t h e p u r i t y of A23187 by v i s u a l m e t h o d s . B i o a u t o g r a m s u s i n g s i l i c a g e l p l a t e s h a v e n o t worked as w e l l as b i o a u t o g r a m s o f cellulose plates. A number o f s o l v e n t s y s t e m s are a p p l i c a b l e t o s i l i c a g e l TLC a n d t h e s e i n c l u d e : m e t h a n o l , c h l o r o f o r m : Visual detection m e t h a n o l ( Q : 6 ) , b e n z e n e : e t h y l a c e t a t e (1:l). c a n b e p e r f o r m e d b y : s h o r t w a v e l e n g t h U V , l o n g wavehength UV, H ~ S O L ,s p r a y f o l l o w e d by h e a t i n g of t h e p l a t e a t 1 0 0 C f o r 5 min, c h l o r i n e chamber e x p o s u r e f o l l o w e d b y o - t o l i d i n e - K I i n a c e t i c a c i d s p r a y , o r TLC s c a n n e r ( S c h o e f f e l S p e c t r o d e n s i t o m e t e r SD 3 0 0 0 ) a t 280 nm. 7.4 Literature -
Cited
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R. M. Gale, C. E . H i g g e n s a n d M . M. 3 , 9 2 3 , 8 2 3 ; December 2 , 1 9 7 5 .
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M. 0. Chaney, P. V . Demarco, N . D . J o n e s a n d J . L . O c c o l o w i t z , J . Amer. Chem. S O C . 9 6 ( 1 9 7 4 ) 1832-1933.
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C. P h o l p r a m o o l a n d V . ( 1 9 7 6 ) 381-388.
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S . E i m e r l , N . S a v i o n , 0 . H e i c h a l a n d 2. S e l i n g e r , J. B i o l Chem. 249 ( 1 9 7 4 ) 3991-3993.
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R . A. S t e i n h a r d t , D . E p e l , E . J . C a r r o l l , J r . a n d R . Y a n a g i m a c h i , N a t u r e (London) 2 5 2 ( 1 9 7 4 ) 41-113.
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V. I o n a s e s c u , H . Zellweger, R . I o n a s c e s c u , C. L a r a - B r a u d a n d P. C a n c i l l a , A c t a N e u r o l . S c a n d i n a v . 5 4 (1976) 241-247.
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A . T a k a t s u k i , K. A r i m a a n d G . (1971) 215-223.
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Tarnura, J. A n t i b i o t i c s 2 4
D . R . P f e i f f e r , P. W. R e e d a n d H . A. L a r d y , B i o c h e m . 1 3 ( 1 9 7 4 ) 4007-4013.
Acknowledgement T h e a u t h o r s a r e g r a t e f u l t o Mrs. P h y l i s Hager f o r h e r c a p a b l e a s s i s t a n c e i n p r e p a r a t i o n of t h e m a n u s c r i p t .
521 S i d er o c h r o m e s Hubert Maehr Chemical Research Department Hoffmann-La Roche I n c . , N u t l e y . N e w J e r s e y
.
1 2
.
........................................ .............................
Introduction Literature cited
1.1 -
525 526
Albomycin. Grisein a n d r e l a t e d a n t i b i o t i c s Introduction Biological activities
2.1 2.2 2.2.1
2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.3 2.3.1 2.3.1.1 2.3.1.2 2 . 3 . l .3 2.3.1.4 2.3.1.5 2.3.2 2.3.3 2.3.4 2.3.4.1 2.3.4.2 2.3.4.3 2.3.5 2.3.5.1 2.3.5.2 2.3.5.3 2.3.5.4 2.3.5.5 2.3.5.6
................................. 5 2 0 ........................ 5 2 8 G r i s e i n .................................... 528 530 A n t i b i o t i c 3510 ............................ 530 A l b o m y c i n s ................................. A n t i b i o t i c s L . A . 5352 a n d L . A . 5 9 3 7 ........ 5 3 1 531 A l v e o m y c i n ................................. A n t i b i o t i c s Ro 5 - 2 6 6 7 . R o 7-7730 a n d RO 7 - 7 7 3 1 .................................. 531 I s o l a t i o n .................................... 531 G r i s e i n .................................... 531
......... 5 3 1
Preparation of crude concentrate Purification of grisein by distribution between aqueous a c i d a n d phenolchloroform P u r i f i c a t i o n of g r i s e i n b y p a r t i t i o n c h r o m a t o g r a p h y on s i l i c a g e l P u r i f i c a t i o n of g r i s e i n by p a r t i t i o n c h r o m a t o g r a p h y on c e l l u l o s e p o w d e r P u r i f i c a t i o n o f g r i s e i n by C r a i g distribution., Albomycin A n t i b i o t i c s L.A. 5352 a n d L.A. 5 9 3 7 Alveomycin Preparation of crude concentrate P u r i f i c a t i o n of crude alveomycin b y c h r o m a t o g r a p h y on s i l i c a g e l P u r i f i c a t i o n of c r u d e a l v e o m y c i n b y phenol-chloroform e x t r a c t i o n A n t i b i o t i c s Ro 5-2667. Ro 7-7730 and RO 7 - 7 7 3 1 P r e p a r a t i o n of c r u d e c o n c e n t r a t e w i t h c h a r c o a l (Procedure A) Preparation of crude concentrate with A m b e r l i t e XAD-2 ( P r o c e d u r e 8 ) Preparation of crude concentrate with A m b e r l i t e IRA-401-S ( P r o c e d u r e C). P u r i f i c a t i o n of crude a n t i b i o t i c with A m b e r l i t e IRA-401-S a n d Amberlite XAD-2 ( P r o c e d u r e D ) P u r i f i c a t i o n b y c h r o m a t o g r a p h y on Dowex 50W-X2 ( P r o c e d u r e E ) P u r i f i c a t i o n b y r e c h r o m a t o g r a p h y on Dowex 50W-X2 ( P r o c e d u r e F )
............................... 532 ............. 532 ....... 532 ........................... 532 .................................. 5 3 4 ........ 5 3 4 ................................. 5 3 4
......... 5 3 4
.............5 3 4
............. 535 .................................. 5 3 5 ...................5 3 5 ............ 5 3 5 ....... 536 ...................... 536 ..................... 5 3 7 ...............5 3 8
522
2.
Albomycin, G r i s e i n a n d r e l a t e d a n t i b i o t i c s ( c o n t i n u e d ) 2.3.5.7 P u r i f i c a t i o n b y c h r o m a t o g r a p h y on Rexyn 539 102 ( P r o c e d u r e G I . . . . 2.3.5.8 P u r i f i c a t i o n b y c h r o m a t o g r a p h y o n CM 539 S e p h a d e x C-25 ( P r o c e d u r e HI..... 2.3.5.9 S e p a r a t i o n of a n t i b i o t i c s Ro 5-2667 a n d R o 7-7730 b y p a r t i t i o n c h r o m a t o g r a p h y (Procedure I)... 540 Chromatographic i d e n t i f i c a t i o n of albomycin, 2.4 541 g r i s e i n and r e l a t e d a n t i b i o t i c s . . 2.5 Literature Cited..... 542
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3.
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................................ ......................... ....................... .......................... ................................... ................ ...... ......................... ................................. .............................
Danomycin Introduction Producing organisms 4.1.2 Chemical d e s c r i p t i o n 4.2 Biological a c t i v i t y Isolation.... 4.3 P r e p a r a t i o n o f c r u d e danomycin 4.3.1 4.3.2 P u r i f i c a t i o n of c r u d e d a n o m y c i n b y p h e n o l chloroform e x t r a c t i o n and C r a i g - d i s t r i b u tion. 4.4 Chromatographic c h a r a c t e r i z a t i o n o f danomycin 4.5 Literature Cited..
4.1 4.1.1
-
-
-
5.
............ ........................
succinimycin Introduction. Producing organism 3.1.2 Chemical d e s c r i p t i o n 3.2 Biological activity 3.3 Isolation. 3.3.1 P r e p a r a t i o n of c r u d e s u c c i n i m y c i n s u l f a t e w i t h charcoal a n d Dowex 50. 3.3.2 P u r i f i c a t i o n of c r u d e s u c c i n i m y c i n s u l f a t e by p h e n o l - c h l o r o f o r m e x t r a c t i o n . . . . . . 3.3.3 Preparation of pure succinimycin by Craig-distribution 3.4 Chromatographic c h a r a c t e r i z a t i o n of succinimycin 3.5 Literature Cited
3.1 3.1.1
-
4.
.........................
................................. ......................... ....................... .......................... ................................ ............. ...................................... ...........................
Ferrimycins Introduction.... Producing organisms.. Chemical d e s c r i p t i o n 5.1.2 Biological a c t i v i t y . , 5.2 Isolation..... 5.3 5.3.1 Preparation of crude ferrimycin w i t h charcoal.. 5.3.2 Preparation of crude ferrimycin with A m b e r l i t e IRC-50.. 5.3.3 P u r i f i c a t i o n of c r u d e f e r r i m y c i n b y p r e c i p i t a t i o n with phenol.. 5.3.4 S e p a r a t i o n o f f e r r i m y c i n s A and B by p a r t i t i o n c h r o m a t o g r a p h y on c e l l u l o s e . .
5.1 5.1.1
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-
............................. ...................... ....................... ........................ ............................... ................................. ......................... ................ ....
544 544 544 544 544
544 545
545 545 5 46
547 547 547 547 548 548
548 549 549 550 550 550 550
551 552 552 552
553
523 5.
Ferrimycins (continued) 5.3.5 5.3.6 5.3.7 5.3.8
5.4 5.5 6.
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-
................................ ........................ ...................... ......................... ............................. ........................ ...............
Gluconimycin Introduction 8.1.1 Producing organism 8.1.2 Chemical d e s c r i p t i o n 8.2 Biological activity 8.3 Isolation...... 8.4 Chromatographic c h a r a c t e r i z a t i o n of g l u c o n i m y c i n b y PC on Whatman No. 1 p a p e r . . . , , Literature Cited.. 8.5 8.1 -
9.
................................ ........................ ...................... ......................... ........................... ........................ .......................
A n t i b i o t i c ASK-753 Introduction Producing organism 7.1.2 Chemical d e s c r i p t i o n 7.2 Biological a c t i v i t y 7.3 Isolation...... 7.4 Chromatographic c h a r a c t e r i z a t i o n of a n t i b i o t i c ASK-753.. 7.5 Literature Cited............. -
7.1 7.1.1
8.
........................ ........................ ...................... .................. .............................,... ................
A n t i b i o t i c NRCS-15 Introduction 6.1.1 Producing organism 6.1.2 Chemical d e s c r i p t i o n 6.2 Biological activity 6.3 Isolation........ 6.4 C h r o m a t o g r a p h i c c h a r a c t e r i z a t i o n of a n t i b i o t i c NRCS-15.. 6.5 Literature Cited.....
6.1 -
7.
Separation o f ferrimycins A and B by Craig distribution...... P u r i f i c a t i o n o f f e r r i m y c i n A by chromatog r a p h y on Dowex 50 S e p a r a t i o n o f f e r r i m y c i n s A, a n d A2 b y Craig d i s t r i b u t i o n . . S e p a r a t i o n of f e r r i m y c i n A, a n d A, b y p a r t i t i o n chromatography Chromatographic c h a r a c t e r i z a t i o n of ferrimycins Literature Cited............
................................ ........................ ...................... ......................... .............................
. . . . . .. .. . . .. . . . . . .. . . . .
Ferramidochloromycin Introduction Producing organism Chemical d e s c r i p t i o n 9.1.2 Biological a c t i v i t y 9.2 Isolation.. 9.3 C h r o m a t o g r a p h i c c h a r a c t e r i z a t i o n of 9.4 f e r r a m i d o c h l o r o m y c i n b y d e s c e n d i n g PC.. 9.5 Literature Cited. 9.1 9.1.1
-
................................ ........................ ...................... ......................... ................................. . .. ..
............................
553 553 554 554 554 557 558 558 558 558 559 559 559 560 560 560 560 560 561 561 562 562 562 562 562 562 563 564 564 564 564 564 564 565
524
10.
Sideramines Introduction............................, 10.1.1 Sideramines produced by fungi and containing three N5-hydroxyornithine moieties.. Sideramines produced by actinomycetes 10.1.2 and containing three amino-hydroxyaminoalkane moieties... 10.2 Biological activities Isolation..... 10.3 10.3.1 Ferrichrome-type sideramines....... 10.3.2 Ferrioxamines... 10.3.2.1 Crude ferrioxamine.. 10.3.2.2 Preliminary separation by Craigdistribution.. Ferrioxamine B by ion-exchange 10.3.2.3 chromatography 10.3.2.4 Ferrioxamine A by ion-exchange chromatography.. 10.3.2.5 Ferrioxamine A and B by Craigdistribution.... 10.3.2.6 Separation of ferrioxamines A 1 and A 2 as their N-acetyl derivatives.. , 10.3.2.7 Ferrioxamine C by ion-exchange chromatography.................... 10.3.2.8 Ferrioxamines D1, Dz, E and F by ionexchange chromatography..... 10.3.2.9 Ferrioxamine G by ion-exchange chromatography..... 10.3.2.10 Nocardamine....... 10.4 Chromatographic characterization of sideramines. 10.5 Literature Cited
-
566
.................... ,........ 5 6 6 ................ 55 77 31 .................... ........................... 5 7 3 .... 5 7 3 ....................... 574 ....,,........... 5 7 4
.......................
574
.......................
575
..................... 5 7 5 ... .................. 575 .. 576 ...
11.
576
......... 5 7 6 .................. 55 77 77 ................... ............................. ......................... 55 87 07 Solvent systems used for Chromatography and Craig-distribution............................... 583
525
1. Introduction Siderochromes are natural products with three hydroxamic acid groups per molecule constituting a sexadentate metalchelating site with distinct preference for Fe(II1) ions. The Fe(II1) chelates are very stable and most siderochromes are isolated in Fe(II1)-chelated form with characteristic absorption maxima at 420-440 nm or are readily converted into these forms with Fe(II1) ions’. Siderochromes with antibiotic activity are termed sideromycins and have been found exclusively as metabolites of actinomycetes, they show cross resistance among each other, induce resistance of sideromycin-sensitive microorganisms rapidly and are antagonistically affected by sideramines, which are siderochromes with microbial growth-factor activity. This definition does obviously not include a number of iron-containing peptide antibiotics which lack the trihydroxamate structure such as antibiotics NRCS-15 and ASK-753, gluconimycin and ferramidochloromycin; for this class of compounds the term “pseudosideromycin” was proposed2. The interrelationship between sideromycins and sideramines, their role in microorganism^^-^, and the chemistry of siderochromes1’6-* was reviewed. The competitive antagonism between sideromycins and sideramines was studied by Zahner furnishing a qualitative and quantitative assay system for both class‘es of siderochromes ,6. In addition to these biological assay methods f o r sideromycins and sideramines, siderochromes in general can be visually recognized and quantitatively determined by the orange to brown-red color of the Fe(II1)-trihydroxamate function. This inherent pigment, as well as the blue color-reactions with Hoppe reagentlo proved useful in FeC13-K3FeCCN)69 or Reindel the course of isolation work.
-
The isolation of siderochromes from fermentation broths was accomplished most often by adsorption onto charcoal and elution with aqueous organic solvents. Certain ionic siderochromes could be isolated with ion-exchange resins such as Amberlite IRC-50, Amberlite IRA-401S, Dowex 50 or similar resins. Siderochromes are usually very soluble in water, somewhat less soluble in phenol, dimethylformamide, dimethylsulfoxide, glycol, methanol, pyridine and benzyl alcohol, but rarely soluble in less polar solvents. As a result, isolation and purification by conventional solvent extraction is generally not feasible. Based on the pioneering work by Kuehl and coworkersll concerned with the isolation of grisein, however, mixtures of phenol and chloroform were used successfully and almost universally for the extraction of siderochromes from aqueous solutions or salt-containing fractions obtained in the course of further purification. Addition of ether and water to the phenol-chloroform phases extracted the siderochromes into the aqueous phases which were freed of phenol by additional ether extraction and freeze-dried to afford the crude siderochromes as amorphous powders. An important advancement in the isolation of siderochromes, either from culture filtrates or from salt-containing chromatographic fractions, was achieved in our laboratory by the use of
526 non-ionic, macroreticular polystyrene r e s i n s . Of t h e s e , Amberl i t e XAD-2 p r o v e d t o be most u s e f u l and e c o n o m i c a l and was s t u d i e d most e x t e n s i v e l y i n t h e c o u r s e o f t h e i s o l a t i o n o f a n t i b i o t i c s Ro 5-2667, Ro 7-7730, Ro 7-7731 and a l b o m y c i n . A l l s i d e r o chromes t e s t e d c o u l d b e a d s o r b e d o n t o t h e r e s i n from aqueous s o l u t i o n and e l u t i o n was g e n e r a l l y c o n d u c t e d w i t h aqueous e t h a n o l o r aqueous e t h a n o l c o n t a i n i n g small q u a n t i t i e s o f ammonium h y d r o x i d e . The o n l y r e q u i r e m e n t f o r s u c c e s s f u l a d s o r p t i o n i s t h e absence of o r g a n i c s o l v e n t s i n t h e siderochrome s o l u t i o n . Thus, t h i s t r e a t m e n t can b e u s e d a d v a n t a g e o u s l y i n p l a c e o f t h e i n i t i a l charcoal adsorption s t e p o r t h e unpleasant phenolchloroform e x t r a c t i o n s previously used f o r p u r i f i c a t i o n and desalting. Crude s i d e r o c h r o m e s were p u r i f i e d and s e p a r a t e d i n t o i n d i v i d u a l components by p a r t i t i o n Chromatography on c e l l u l o s e , s i l i c a g e l a n d Sephadex, by i o n - e x c h a n g e chromatography on Dowex 50-X2, o r similar r e s i n s , w i t h a c e t a t e b u f f e r s , a n d e s p e c i a l l y by C r a i g - d i s t r i b u t i o n w i t h s o l v e n t s y s t e m s 79, 82, 83 a n d 8592. A l t h o u g h s t r u c t u r a l l y complex, s i d e r a m i n e s are composed o f r e p e a t i n g b u i l d i n g b l o c k s of i d e n t i c a l o r similar s t r u c t u r e s i n d e f i n e d p a t t e r n s 1 . They a r e r e l a t i v e l y s t a b l e a n d can o c c a s i o n a l l y b e i s o l a t e d i n c r y s t a l l i n e form. S i d e r o m y c i n s , on t h e o t h e r hand, a p p e a r t o b e much more c o m p l i c a t e d c h e m i c a l l y , uns t a b l e a n d f a r more d i f f i c u l t t o i s o l a t e i n p u r e form. I t i s , t h e r e f o r e , n o t s u r p r i s i n g t h a t no c r y s t a l l i n e s i d e r o m y c i n was e v e r obtained. Impurities a r e i n v a r i a b l y of p e p t i d e n a t u r e so t h a t t h e a m i n o - a c i d c o m p o s i t i o n o f a h y d r o l y z a t e as c h e m i c a l c r i t e r i o n of p u r i t y h a s t o b e i n t e r p r e t e d w i t h c a u t i o n and i s only s i g n i f i c a n t a f t e r meticulous p u r i f i c a t i o n s .
1.1 L i t e r a t u r e
Cited
1.
H . Maehr, P u r e Appl. Chem. 28 (1971) 603-636.
2.
J. BBrdy, Adv. Appl. M i c r o b i o l . 1 8 (1974) 309-406.
3.
F. Kniisel, J . Nuesch and H . J . T r e i c h l e r , Naturwiss. 5 4 (1967) 242-274.
4.
J. Niiesch and F. Knilsel, i n : A n t i b i o t i c s Vol. 1, D. G o t t l i e b and P . D . Shaw, E d s . , S p r i n g e r V e r l a g , N e w York, 1967, 499541.
5.
F . Knusel and W . Zimmerman, i n : A n t i b i o t i c s Vol. 3, J . W . Corcoran a n d F . E . Hahn, E d s . , S p r i n g e r V e r l a g , N e w York, 1975, 653-667.
6.
W.
7.
J. B. Neilands, i n : Inorganic Biochemistry, G . I . Eichhorn, E d . , Vol. 1, E l s e v i e r , N e w York (1973) 167-202.
8.
T. Emery, Adv. Enzymol. 35 (1971) 135-185.
K e l l e r - S c h i e r l e i n , V. Prelog and H. Zahner, F o r t s c h r . Chem. Org. N a t u r s t o f f e 22 (1964) 279-322.
527 9.
G. M. B a r t o n , R . (1952) 249-250.
S . E v a n s a n d J . A . F. G a r d n e r , N a t u r e 1 7 0
10.
F. R e i n d e l and W . H o p p e , Chem. Ber. 8 7 ( 1 9 5 4 )
11.
F. A. K u e h l , Jr., M . N . B i s h o p , L . C h a i e t a n d K . F o l k e r s , J . h e r . Chem. SOC. 7 3 ( 1 9 5 1 ) 1 7 7 0 - 1 7 7 3 .
1103-1107.
52 8 2 . Alb.omycin, G r i s e i n a n d R e l a t e d A n t i b i o t i c s
2 . 1 Introduction Grisein, discovered already i n 1 9 Q 7 l , i s the f i r s t sideromycin r e p o r t e d . A number of s u b s e q u e n t l y d e s c r i b e d s i d e r o m y c i n s , which are summarized i n T a b l e 1, are o b v i o u s l y r e l a t e d t o g r i s e i n , b u t d e f i n i t e i d e n t i t y w i t h g r i s e i n c o u l d n o t b e establ i s h e d i n any case. These a n t i b i o t i c s are u n s t a b l e , n o t c r y s t a l l i n e , a n d d i f f i c u l t t o p u r i f y . They are g e n e r a l l y v e r y s o l u b l e i n water a n d phenol, s l i g h t l y s o l u b l e i n benzyl a l c o h o l , dimethylformamide, methanol and p y r i d i n e , b u t v i r t u a l l y i n s o l u b l e i n less p o l a r solvents. The a l b o m y c i n s 4 a n d a n t i b i o t i c s Ro 5-2667, Ro 7-7730 and Ro 7-773114, i d e n t i c a l w i t h a l b o m y c i n s 62, 6 1 a n d E , r e s p e c t i v e l y , have been o b t a i n e d i n p u r e form a n d were s t u d i e d chemi c a l l y t o some e x t e n t . P r o p o s e d s t r u c t u r e s f o r a l b o m y c i n 4 Were shown t o be i n c o r r e ~ t and ~ ~ ’f i~n a~ l s t r u c t u r e s a r e n o t as y e t known. A l l t h r e e a l b o m y c i n s have t h e same b a s i c s t r u c t u r e cont a i n i ng i n t e l r a l i a , t h pee N -ace t y 1- N - h y d r o x y -L-orn i t h i n e moieties c h e l a t i n g F e ( I I I ) , one s e r i n e r e s i d u e , a s u l f u r atom a n d a p y r i m i d i n e m o i e t y g i v i n g r i s e t o c h a r a c t e r i s t i c UV s p e c t r a 1 4 as shown i n T a b l e 2 .
,
A n t i b i o t i c s of t h e grisein-albomycin type r e p r e s e n t a d i s t i n c t g r o u p o f s i d e r o m y c i n s r e a d i l y i d e n t i f i e d by R f - v a l u e s b e tween 0 - 0 . 2 0 (PC) i n s o l v e n t s y s t e m 5 2 a n d a r e r e f e r r e d t o as members of Group I o f B i c k e l ’ s c l a s s i f i c a t i o n s y s t e m l o . Known f o r i t s i n s t a b i l i t y 4 , albomycin 6 2 is t h e major a n t i b i o t i c i n t h e f e r m e n t a t i o n b r o t h s a n d g i v e s r i s e t o two b i o l o g i c a l l y a c t i v e d e g r a d a t i o n p r o d u c t s , albomycin 6 and E , Albomycin and g r i s e i n are most p r o b a b l y i d e n t i c a l ; t h e y have v e r y s i m i l a r b i o l o g i c a l p r o p e r t i e s a n d t h e p u r e s t s a m p l e o f g r i s e i n 1 8 is i d e n t i c a l w i t h a n t i b i o t i c Ro 7-7730 ( a l b o m y c i n 6 1 ) i n terms of UV The i s o l a t i o n o f t h e 6 1 comp o n e n t from g r i s e i n would n o t be s u r p r i s i n g i n view o f t h e l e n g t h y p r o c e d u r e s employed i n t h e p u r i f i c a t i o n o f g r i s e i n which would a c t u a l l y be e x p e c t e d t o c a u s e t h e t r a n s f o r m a t i o n o f t h e 6, t o t h e 6 1 component. F i n a l l y , t h e l i k e l i h o o d of i d e n t i t y of albomycin a n d g r i s e i n i s s u s t a i n e d b y amino a c i d a n a l y s e s 4 a n d chromatographic comparisons3.
2.2 Biological a c t i v i t i e s 2.2.1
Grisein
The c r u d e a n t i b i o t i c i s a c t i v e a g a i n s t c e r t a i n gramp o s i t i v e b a c t e r i a , s u c h as BUCLLLUA d u b t d t i d a n d S t a p h y L o c o c c u 4 aulreud, as w e l l as c e r t a i n g r a m - n e g a t i v e b a c t e r i a , n o t a b l y E d c h e d c h i a C O I L . I t s a n t i b a c t e r i a l s p e c t r u m i s much more l i m i t e d than t h a t of e i t h e r streptomycin o r s t r e p t o t h r i c i n . Unlike s t r e p t o m y c i n , g r i s e i n shows n o a c t i v i t y a g a i n s t BacLILud Ceheud
TABLE 1:
ANTIBIOTIC
A n t i b i o t i c s o f t h e Grisein-Albomycin
PRODUCING ORGANIS
Family
REMARKS
Grisein
Stn?ptomyceswiseus Waksman et F n r i c i
Probably identical with albomycin (3,4),cross.resistant with albomycin and viomycin (3)
Antibiotic 3510
SDSptomycesgriseus
arisein-like but not cross-resistantwith arisein (2)
Albomycin 6
s b g p ~ y c e gris9m s
degradationproduct of albomycin resistant with grisein
Wakman et Henrici Albomycin 62
l A c t i m y c e s subtrqpicm Kudrina et Kochetkova)
62,biologically active, cross-
major antibiotic component, cmss-resistantwith gisein
can behave as antibiotic at 28" and ferrichrome-like a t higher temp wature (8)
Albomycin E
5 -
6,7
-
Antibiotic A-1787
Streptomyces IA 1707
probably identical with albomycin (PC) (10)
-
Antibiotic LA-5352
Saepmmyces LA-5352
cross-resirtant with LA4937 and albomycin, unstable in phenol
11 -
Antibiotic LA-5937
Streptornyces bobilia vw. sparifkans
iron.free; cross-resistantwith LA-5352and albomycin, basic wbstance, sli&tly soluble in water, neg. ferric chloride test, probably not a sideromycin
1.12
Antibiotic ETH 10073
Sneptomyces griswr
probably identical with albomycin (PC)
10 -
Alveomycin
Strwmmyces sd 094
probably identical with albomycin (PC)
13
Antibiotic Ro 7-7731
-
identical with albomycin 6, (15)
Antibiotic Ro 5-2667 Antibiotic Ro 7-7730
9
Streptornycesgriseus VBT. X-2455
identical with albomycin 6, (15)
identical with albomycin € (15); can antagonize the activity of Ro 5-2667 and Ro 7-7730 (16)
14
vl
N
co
5 30 TABLE 2
UV Maxima o f Albomycins
CHRWOmORE
ANTIBIOTIC
PH 1
PH 7
Albomycin 6, (Antibiotic Ro 7-7730)
a2
281 ~128).420 128)
Albomyein (Antibiotic Ro 6m71
AlbomVcin E (Antibiotic Ro 7-7730
279 (1071, 440 1131
279 11061, 423 (21)
( B . mycoided) a n d , i n c o n t r a s t t o s t r e p t o t h r i c i n , i s i n a c t i v e a g a i n s t f u n g i , Both g r i s e i n and s t r e p t o m y c i n are i n a c t i v e a g a i n s t AcinetobacteA calcoaceticu.4 ( B o d e n h e i m e r ' s b a c i l l u s ) . G r i s e i n i s o f low t o x i c i t y , a n d i s a c t i v e in v i u 0 a g a i n s t Rapid development o f a n t i b i o t i c r e s i s t a n t b a c t e r i a l is c h a r a c t e r i s t i c f o r g r i s e i n as w e l l as f o r a l l sideromycins.
S. d c h o t t m u e l l e t i a n d S. auReuA1.
2.2.2
A n t i b i o t i c 351.0
Although v e r y s i m i l a r t o g r i s e i n t h i s a n t i b i o t i c i s a c t i v e a g a i n s t Acinetobactea CaJ?CoaC~tiCuA5. 2.2.3
Albomycin
Albomycin6 i n h i b i t s growth o f g r a m - p o s i t i v e c o c c i , c h i e f l y pneumococcus, s t a p h y l o c o c c u s , s t a p h y l o c o c c i r e s i s t a n t t o o t h e r a n t i b i o t i c s a n d Klebdiella pneumoniae ( F r i e d l H n d e r ' s b a c i l l u s ) . I t i s a c t i v e a g a i n s t a number of g r a m - n e g a t i v e b a c t e r i a s u c h as Edcheaichia C O U , KlebdielLa a n d t h e c o l i - d y s e n t e r y g r o u p a n d , s i m i l a r t o g r i s e i n , albomycin i s i n a c t i v e toward BacllLuA CeReud. Albomycin p r o t e c t e d mice i n f e c t e d w i t h Tneumococci (1 mg/kg, s C ) a n d K l e b d i e l e a pneumoniae ( 3 5 ug/kg, s c
.
Albomycin was u s e d i n c l i n i c a l p r a c t i c e i n t h e S o v i e t Union a g a i n s t i n f e c t i o n s c a u s e d by p a t h o g e n i c c o c c i , f o r t h e t r e a t m e n t of pneumonia, s e p t i c c o m p l i c a t i o n s of d y s e n t e r y a n d m e a s l e s , v a r i o u s i n f e c t i o n s c a u s e d by p e n i c i l l i n - r e s i s t a n t
531 b a c t e r i a , r e l a p s i n g f e v e r due t o S p . Aogdianum ( d o s a g e f o r a d u l t s : 5 . 2 mg i n t r a m u s c u l a r l y twice a day f o r 7-12 d a y s ) , p e r i t o n i t i s and p r o s t a t i t i s 1 7 . Albomycin i s t o l e r a t e d w e l l i n l a r g e d o s e s ( u p t o 7 0 mg/ kg g i v e n s u b c u t a n e o u s l y o r i n t r a v e n o u s l y ) . The d i s c o n t i n u a t i o n o f i t s u s e was p r i m a r i l y d u e t o t h e r a p i d d e v e l o p m e n t o f b a c t e rial resistance. 2.2.4
A n t i b i o t i c s L. A .
5352 a n d L . A .
5937
Both a n t i b i o t i c s are a c t i v e i n v i t a 0 a g a i n s t g r a m - p o s i t i v e b a c t e r i a b u t i n a c t i v e a g a i n s t gram-negative b a c t e r i a and f u n g i , I n f u r t h e r c o n t r a s t t o g r i s e i n and a l b o m y c i n , a n t i b i o t i c s L .A. 5352 and L.A. 5937 are v e r y a c t i v e a g a i n s t S a n c i n a - h ~ t e a ~ ~ ~ ~ A n t i b i o t i c L.A. 5352 i s a c t i v e i n v i v o a g a i n s t ViptococcuA Both a n t i b i o t i c s L.A. 5352 a n d L.A. 5937 showed t h e same low t o x i c i t y o f a l b o m y c i n a n d g r i s e i n a n d were n o t e d f o r r a p i d r e s i s t a n c e development i n b a c t e r i a l l .
pneUmOniae.
2.2.5
Alveomycin
S i m i l a r t o a l b o m y c i n a n d g r i s e i n , t h i s a n t i b i o t i c i s act i v e i n v i t a 0 a g a i n s t g r a m - p o s i t i v e and g r a m - n e g a t i v e b a c t e r i a . A p o t e n t i a t i n g s y n e r g i s m i n V i a 0 was r e p o r t e d between a l v e o mycin a n d p e n i c i l l i n ( s t a p h y l o c o c c i ) . The a c t i v i t y o f s t r e p t o mycin and c h l o r a m p h e n i c o l was e n h a n c e d by a l v e o m y c i n ( E . coli)l3. 2.2.6
A n t i b i o t i c s Ro 5-2667,
Ro 7-7730 a n d Ro 7-7731
A n t i b i o t i c Ro 5-2667 ( a l b o m y c i n 6 2 ) i s t h e m a j o r b i o a c t i v e component i n t h e f e r m e n t a t i o n b r o t h e x h i b i t i n g i n v i a 0 a c t i v i t y a g a i n s t gram-positive and gram-negative b a c t e r i a , n o t a b l y S. auReuA. D u r i n g t h e i s o l a t i o n a n d upon s t o r a g e Ro 5 - 2 6 6 7 decomposes more o r l e s s e x t e n s i v e l y t o g i v e r i s e t o Ro 7-7730 ( a l b o m y c i n 6 1 ) a n d Ro 7-7731 ( a l b o m y c i n E ) . A n t i b i o t i c Ro 7 7730 h a s an a n t i b a c t e r i a l s p e c t r u m c o m p a r a b l e t o t h a t o f Ro 5 2 6 6 7 w i t h t h e e x c e p t i o n o f a weaker a c t i v i t y a g a i n s t S . auReud14. A n t i b i o t i c Ro 7-7731 is a c o n s i d e r a b l y w e a k e r a n t i b i o t i c t h a n Ro 5-2667 a n d Ro 7-7730 a n d , i n f a c t , i s a b l e t o a n t a g o n i z e t h e a c t i v i t y of Ro 5-2667 a n d R o 7-7730 u n d e r c e r t a i n c o n d i t i o n s 1 6 . A n t i b i o t i c Ro 5-2667 was a c t i v e i n mice by t h e s u b c u t a n e o u s r o u t e a g a i n s t Ktebbietla pneumoniae ( C D , , 0 . 1 6 mg/kg) a n d D i p t o coccu4 pneumoniae ( C D 5 0 0 . 1 3 mg/kg).
2.3 I s o l a t i o n 2.3.1 2.3.1.1
Grisein P r e p a r a t i o n of crude concentrate’*
The f e r m e n t a t i o n b r o t h ( 2 4 7 0 a , 2 0 0 u n i t s / m l I 2 was a c i d i f i e d t o pH 2 . 5 - 3 . 0 , mixed w i t h d i a t o m a c e o u s e a r t h ( 2 3 k g ) a n d filtered. The f i l t e r cake was washed w i t h w a t e r (380 I I ) , f i l t r a t e a n d w a s h i n g s were combined, a d j u s t e d t o pH 7 . 5 - 8 . 0 a n d
5 32 f i l t e r e d . The f i l t r a t e was s t i r r e d w i t h c h a r c o a l ( N o r i t A , 1 1 . 4 kg) f o r 30 min. w i t h a m i x t u r e of water ( 4 5 0 a ) a n d p y r i d i n e ( 1 5 0 R ) a n d t h e s u s p e n s i o n was f i l t e r e d . The c h a r c o a l cake The combined was washed w i t h w a t e r - p y r i d i n e , 4 : 1 v / v , 150 R. e l u a t e and w a s h i n g s were e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e a t 110' t o ca. 25 e . - T h i s c o n c e n t r a t e was d i l u t e d w i t h m e t h a n o l ( c a . 2 5 0 a ) , c l a r i f i e d by f i l t r a t i o n and added t o e t h e r ( c a . 8 3 R), The r e s u l t i n g p r e c i p i t a t e ( 1 . 9 k g ) had a m i c r o b i o l o g i c a l potency of ca. 320 units/mg. Powdered g r i s e i n c o n c e n t r a t e ( 1 3 . 5 kg, 540 u n i t s / m g ) was e x t r a c t e d with f o u r 5 3 - l i t e r p o r t i o n s of methanol, t h e e x t r a c t e d r e s i d u e had d o u b l e d a c t i v i t y w i t h a 5 0 % y i e l d o f r e c o v e r y . 2.3.1.2
P u r i f i c a t i o n of c r u d e g r i s e i n by d i s t r i b u t i o n between aqueous a c i d and p h e n o l - c h l o r o f o r m L 8
The p r o c e d u r e i s o u t l i n e d i n C h a r t I . c o n t a i n i n g c a . 1 2 % water was u s e d . 2.3.1.3
Liquefied phenol
P u r i f i c a t i o n of g r i s e i n by p a r t i t i o n chromatog r a p h y on s i l i c a g e l ' u
G r i s e i n ( 3 . 2 6 g , 45000 u n i t s / m g ) was d i s s o l v e d i n 3 4 % p h e n o l - c h l o r o f o r m ( v / v , 45 m l ) ; p r e v i o u s l y s a t u r a t e d w i t h c i t r a t e b u f f e r ( 0 . 1 M y pH 4 . 6 ) , and d i l u t e d w i t h c h l o r o f o r m t o a volume o f 9 0 m l . T h i s s o l u t i o n was chromatographed on a column of s i l i c a g e l ( 1 2 0 g ) w e t t e d w i t h c i t r a t e b u f f e r ( 8 4 m l ) , by development w i t h 1 7 % p h e n o l - c h l o r o f o r m ( v / v ) . Grisein ( 0 . 4 g, c a . 150030 u n i t s / m g ) was i s o l a t e d from t h e d a r k - r e d f r a c t i o n s ( 1 6 3 m l ) by e x t r a c t i o n w i t h p h e n o l - c h l o r o f o r m ( l : l , v / v ) . The e x t r a c t was washed w i t h w a t e r , d r i e d by f i l t r a t i o n t h r o u g h p a p e r , d i l u t e d w i t h p e t r o l e u m e t h e r c o n t a i n i n g a small amount o f d i e t h y l e t h e r a n d e x t r a c t e d e x h a u s t i v e l y w i t h water. The w a t e r s o l u t i o n was e x t r a c t e d w i t h e t h e r t o remove p h e n o l a n d then concentrated t o dryness. 2.3.1.4
P u r i f i c a t i o n o f F r i s e i n by p a r t i t i o n chromatography on c e l l u l o s e powder3
Crude g r i s e i n (1.1 g , 2 2 0 0 0 u n i t s / m g ) i n 4 0 m l o f u p p e r p h a s e was c h r o m a t o g r a p h e d w i t h s y s t e m 5 2 on a column of p u l v e r i z e d p a p e r ( 8 0 0 g ) w e t t e d w i t h l o w e r p h a s e . The e f f l u e n t was m o n i t o r e d by a b s o r b a n c y a t 4 2 5 nm a n d t h e y e l l o w e f f l u e n t c o l l e c t e d i n 2 5 m l f r a c t i o n s . C o n c e n t r a t i o n and f r e e z e - d r y i n g o f f r a c t i o n s 7 1 - 1 5 0 y i e l d e d a g r i s e i n p r e p a r a t i o n w i t h 60,000 u n i t s / mg
-
2.3.1.5
P u r i f i c a t i o n of g r i s e i n by C r a i g - d i s t r i b u t i o n 1 8
R e p e a t e d C r a i g - d i s t r i b u t i o n of p u r i f i e d g r i s e i n (150COC u n i t s / m ) w i t h s y s t e m 9 1 employing i n s u c c e s s i o n 1 5 , 1 0 a n d 8 stages ultimately yielded a g r i s e i n preparation with 3 O G O O O u n i t s / m g w i t h a UV s p e c t r u m e s s e n t i a l l y i d e n t i c a l w i t h t h a t of p u r e albomycin f j 1 1 4 . G r i s e i n was i s o l a t e d a f t e r e a c h of t h e s e t h r e e d i s t r i b u t i o n s from t h e combined peak f r a c t i o n s , as d e t e r mined by t h e a b s o r b a n c y a t 4 2 0 nm, by e x t r a c t i o n w i t h p h e n o l -
533
-
CHART I Purification o f Crude Grisein GRlSElN (300Q, 750 unitdmg) IN WATER (21) d j . pH to 1.0 12.5 N HCI )
add phenol-ehloroform (2I, 1:1, v/v), stir for 5 minutes
SPENT LOWER PHASE
UPPER PHASE
I
repeat phenol-chloroform exnsction
I
I
1
SPENT LOWER PHASE
UPPER PHASE
neutralize to pH 7 (NaHCOJ
extract by stirring with two 1.bliter portions of phenolchloroform (3:10, v/v)
COMBINED LOWER PHASES
SPENT UPPER PHASE
stir with weter (400 ml) and add dil. HCI in small portions until D H 1.0 is readved
I
SPENT LOWER PHASE
I
UPPER PHASE
I
repeat aqueous extraction atpH 1
I
~
I COMBINED AQUEOUS PHASES
SPENT LOWER PHASE
dj. to pH 2.0 (NaHCO,)
extract with five 1OOrnl portions of phenol.chloroform (l:l, v/v)
COMBINED’ EXTRACTS
SPENT ~QUEOUSPHASE
filter through paper and treat with large volume af petroleum ether fontaining little ether and extrect
COMBINED AQUEOUS EXTRACTS
wash several times with ether concentrate to a volume of 5 ml end add 2-propanol (80 mll
GRlSElN 11.33 g. 65000 unitdw, 38% yield)
SPENT ORGANIC PHASE
5 34 Employing a m a t e r i a l as c h l o r o f o r m as d e s c r i b e d p r e v i o u s l y . p r e p a r e d v i a p r o c e d u r e 2 . 3 . 1 . 4 , C r a i g - d i s t r i b u t i o n i n a 39-tube e x p e r i m e n t w i t h s y s t e m 9 1 gave g r i s e i n w i t h C = 0 . 2 6 3 . 2.3.2
Albomycin
Crude albomycin was o b t a i n e d by t h e c h a r c o a l method, opt i m u m d e s o r p t i o n ( u p t o 6 0 % ) was a c h i e v e d a t pH 7 . 0 w i t h 80% a c e t o n e and 9 0 % 2 - p r o p a n o l employing volumes 5 times as l a r g e as t h e c a r b o n c a k e
g.
Chromatography on a column (1.1 c m x 5 0 c m ) o f s u l f o n a t e d p o l y s t y r e n e r e s i n ( Z e r o l i t e 2 2 5 , N H 4 + , 2.5% d i v i n y l b e n z e n e ) w i t h 0 . 2 M ammonium a c e t a t e b u f f e r , pH 4 . 2 , ( 6 0 0 m l ) gave s e v e r a l f r a c t i o n s ; t h e major a n t i b i o t i c - z o n e c o m p r i s i n g a l b o m y c i n s 6 1 a n d 6 2 emerged i n t h e e f f l u e n t - v o l u m e i n t e r v a l o f 500-600 m l . Albomycin E was e l u t e d w i t h ammonium a c e t a t e b u f f e r , pH 7 . 5 (1200 m l ) . The p u r i f i e d albomycin p r e p a r a t i o n s were o b t a i n e d by removal o f ammonium a c e t a t e u n d e r r e d u c e d p r e s s u r e below 50°20.
A p a r t i a l s e p a r a t i o n o f a l b o m y c i n s 6 1 a n d 6 2 was a c h i e v e d on a Z e r o l i t e 225 X 2 column ( N H h + , 1 0 0 - 2 0 0 mesh) w i t h g r a d i e n t e l u t i o n commencing w i t h 0 . 2 M ammonium f o r m a t e b u f f e r , pH 3 . 5 , a n d c h a n g i n g t o 2 . 0 M ammonium a c e t a t e b u f f e r , H 8 . 5 . Albomycin E was e l u t e d w i t h 2 M ammonium h y d r o x i d e 2 P . Albomycin 6 2 was r e c o g n i z e d as t h e main a n t i b i o t i c whose d e c o m p o s i t i o n gave r i s e t o t h e r e m a i n i n g b i o l o g i c a l l y a c t i v e components. 2.3.3
A n t i b i o t i c L.A. 5352 a n d L.A.
593711y12
Both a n t i b i o t i c s were i s o l a t e d by t h e same p r o c e d u r e . The f e r m e n t a t i o n b r o t h was f i l t e r e d a t pH 7 . 0 - 7 . 5 , sodium c h l o r i d e was added ( 2 0 % , v / v ) t o t h e f i l t r a t e , t h e pH a d j u s t e d t o 8 . 5 a n d t h e s o l u t i o n e x t r a c t e d t h r e e times w i t h 2 0 % b e n z y l a l c o h o l . The o r g a n i c p h a s e was t h e n r e p e a t e d l y e x t r a c t e d w i t h 0 . 1 N s u l f u r i c a c i d , d r i e d ( s o d i u m s u l f a t e ) a n d mixed w i t h d i e t h y l e t h e r t o p r e c i p i t a t e t h e a n t i b i o t i c i n c r u d e form. 2.3.4 2.3.4.1
Alveomycin P r e p a r a t i o n of c r u d e c o n c e n t r a t e
The f i l t e r e d f e r m e n t a t i o n b r o t h (500 L, 40 u n i t s / m l ) was s t i r r e d w i t h c h a r c o a l ( 2 k g ) f o r 30 min. a t pH 7 . 0 , t h e c h a r coal cake was washed w i t h a c e t o n e ( 5 0 % ) a n d c h l o r o f o r m ( 2 0 t ) and e l u t e d twice w i t h a c e t o n e - w a t e r c o n t a i n i n g 1 . 5 % ammonia 55:45, v / v , 1 0 a ) . The combined e x t r a c t s were c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e t o a volume o f 3.5 a a n d t h e c o n c e n t r a t e was f i l t e r e d i n s u c c e s s i o n t h r o u g h two i o n - e x c h a n g e - r e s i n c o l umns ( L e w a t i t M I H , OH-, 6 % a n d L e w a t i t C N O , H + , 2 a ) t a k i n g 1 - l i t e r f r a c t i o n s . The peak f r a c t i o n s ( 3 a , 3 2 0 0 u n i t s / m l ) were c o n c e n t r a t e d a n d f r e e z e - d r i e d t o g i v e c r u d e a l v e o m y c i n as a brown powder (140 g , 7 0 u n i t s / m g , 5 0 % y i e l d ) . 2.3.4.2
P u r i f i c a t i o n o f c r u d e a l v e o m y c i n by chromatog r a p h y on s i l i c a g e l L 3
535 Crude a l v e o m y c i n ( 1 4 g , 70 u n i t s / m g ) was d i s s o l v e d i n water ( 9 . 4 m l ) , mixed w i t h 2 - p r o p a n o l ( 1 0 . 6 m l ) a n d f i l t e r e d . The s o l u t i o n was c h r o m a t o g r a p h e d on a column o f s i l i c a g e l ( 5 c m x 50 cm), p r e v i o u s l y washed w i t h water, by d e v e l o p m e n t w i t h 2-propanol-water (53:47, v / v ) . F r a c t i o n s (SO m l e a c h ) w i t h more t h a n 1500 u n i t s / m l were combined, c o n c e n t r a t e d a n d f r e e z e d r i e d t o g i v e a brown-red powder ( 1 . 3 g, 700 n i t s / m g , 93% y i e l d ) , XH20 277 nm ( E $ i m 51) and 420 nm (Ei!m 11). max 2.3.4.3
P u r i f i c a t i o n o f c r u d e a l v e o m y c i n by p h e n o l chloroform e x t r a c t i o n L 5
A s o l u t i o n of crude alveomycin ( 7 0 g , 70 units/mg) i n water ( 1 . 5 II) was e q u i l i b r a t e d w i t h b u t a n o l - p h e n o l ( 4 : 1 , v / v ) , t h e aqueous p h a s e was e q u i l i b r a t e d w i t h p h e n o l - c h l o r o f o r m ( 4 : 7 , v / v , 1 . 5 i ) , t h e c h l o r o f o r m - p h e n o l p h a s e was d i l u t e d w i t h a 3f o l d volume of e t h e r a n d e x t r a c t e d w i t h water ( 2 x 5 0 0 m l ) . The combined a q u e o u s p h a s e s were e x t r a c t e d w i t h e t h e r a n d t h e a q u e o u s p h a s e c o n c e n t r a t e d a n d f r e e z e - d r i e d t o y i e l d a brown powder ( 1 2 g , 350 u n i t s / m g , 8 6 % ) . 2.3.5 2.3.5.1
A n t i b i o t i c s Ro 5 - 2 6 6 7 ,
Ro 7-7730 a n d R o 7-773114
P r e p a r a t i o n of crude c o n c e n t r a t e with c h a r c o a l (Procedure A)
The f e r m e n t a t i o n b r o t h ( 1 1 2 0 e l was f i l t e r e d a t pH 4 . 0 , w i t h t h e a i d o f H y f l o S u p e r c e l ( 3 0 k g ) , t h e pH o f t h e f i l t r a t e was a d j u s t e d t o 7 . 0 , t h e f i l t r a t e s t i r r e d f o r 30 m i n u t e s w i t h c h a r c o a l ( D a r c o G - 6 0 , 1 . 5 % w/v) a n d f i l t e r e d . The c h a r c o a l cake was washed w i t h water ( 2 x 2 0 0 I I ) a n d e l u t e d twice w i t h a c e t o n e - w a t e r ( l : l ,v / v , 230 - ) , The combined a q u e o u s e x t r a c t s were c l a r i f i e d by f i l t r a t i o n , c o n c e n t r a t e d t o a volume of ca. 20 a a n d e x t r a c t e d o n c e w i t h 1 - b u t a n o l ( 2 0 L ) t o remove l i p o p h i l i c matter. The a q u e o u s p h a s e was c o n c e n t r a t e d f u r t h e r a n d f r e e z e - d r i e d t o y i e l d a dark-brown powder ( 5 5 0 g , 4 u n i t s / m g , ca. 6 0 % y i e l d ) . A l t e r n a t i v e l y , t h e c o n c e n t r a t e d aqueous phase was u s e d d i r e c t l y f o r f u r t h e r p u r i f i c a t i o n . ( P r o c e d u r e D). A n t i b i o t i c a c t i v i t y was f o l l o w e d by t h e p e n i c i l l i n - t y p e c u p - p l a t e a g a r d i f f u s i o n a s s a y w i t h S e A A a t i a s p . as t e s t o r g a n ism. The c o n c e n t r a t i o n o f a n t i b i o t i c r e q u i r e d t o p r o d u c e an i n h i b i t i o n zone o f 25 mm i s o n e u n i t / m l . 2.3.5.2
P r e p a r a t i o n of c r u d e c o n c e n t r a t e w i t h A m b e r l i t e XAD-2 ( P r o c e d u r e B )
1. P u r i f i c a t i o n o f new A m b e r l i t e XAD-2. A m b e r l i t e XAD-2 i s a m a c r o r e t i c u l a r , n o n - i o n i c , s t y r e n e - d i v i n l y b e n z e n e copolymer a n d is s u p p l i e d by t h e m a n u f a c t u r e r w i t h sodium c h l o r i d e a n d sodium c a r b o n a t e as p r e s e r v a t i v e s . The r e s i n i s s u s p e n d e d i n m e t h a n o l , s o a k e d f o r 3 0 min, t r a n s f e r r e d t o a column a n d washed w i t h water u n t i l t h e e f f l u e n t i s s a l t - f r e e . The r e s i n i s now washed w i t h a c e t o n e , i n a n upflow d i r e c t i o n . A f t e r r e m o v a l of a i r b u b b l e s t h e a c e t o n e wash i s c o n t i n u e d , e i t h e r i n downflow o r upflow d i r e c t i o n , u n t i l a s a m p l e o f t h e column e f f l u e n t ceases t o g i v e a m i l k y t u r b i d i t y upon d i l u t i o n w i t h water. The r a t e
5 36 o f t h e a c e t o n e wash s h o u l d be slow, p r e f e r a b l y n o t e x c e e d i n g a l i n e a r f l o w r a t e o f 0 . 7 cm/min. The r e s i n i s f i n a l l y washed w i t h a t l e a s t 5 column volumes of water t o remove a c e t o n e a n d i s then ready f o r use. 2. R e g e n e r a t i o n o f A m b e r l i t e XAD-2. The s p e n t r e s i n i s washed-with m e t h a n o l c o n t a i n i n g 1 0 % conc. h y d r o c h l o r i c a c i d u n t i l the effluent is colorless. The column i s s u b s e q u e n t l y washed w i t h w a t e r u n t i l t h e e f f l u e n t i s n e u t r a l . 3. D e c o l o r i z a t i o n o f A m b e r l i t e XAD-2. After repeated a d s o r p t i o n a n d e l u t i o n c y c l e s , t h e r e s i n u s u a l l y remains d i s colored a f t e r regeneration. More o r l e s s c o m p l e t e d e c o l o r i z a t i o n i s p o s s i b l e by s o a k i n g t h e r e g e n e r a t e d a n d water-washed r e s i n i n a 1%s o l u t i o n o f sodium h y p o c h l o r i t e f o r a day f o l lowed by e x t e n s i v e water washes. 4. Broth treatment. The b r o t h ( 2 a , 2 . 6 u n i t s / m l ) was f i l t e r e d a t pH 5 . 0 and t h e f i l t r a t e p a s s e s t h r o u g h a column c o n t a i n i n g r e g e n e r a t e d A m b e r l i t e XAD-2 r e s i n ( 1 0 0 m l , l i n e a r f l o w - r a t e 1 - 2 cm/min). The column was t h e n washed w i t h w a t e r (500 m l ) a n d t h e a n t i b i o t i c a c t i v i t y e l u t e d w i t h e t h a n o l - w a t e r ( 2 : 8 , v / v , 250 m l ) . The a l c o h o l i c e f f l u e n t was c o n c e n t r a t e d t o a volume o f 20 m l and f r e e z e - d r i e d t o a f f o r d dark-brown s o l i d s ( 1 . 0 5 g , 3.9 u n i t s / m g , 80% y i e l d ) .
C o n c e n t r a t i o n of t h e a l c o h o l i c c o l u m n - e f f l u e n t t o a volume Qf 1 0 0 m l , e x t r a c t i o n o f t h e c o n c e n t r a t e w i t h 1 - b u t a n o l ( 1 0 0 m l ) a n d c o n c e n t r a t i o n a n d f r e e z e - d r y i n g gave a brown powder ( 0 . 4 6 g , 6.4 units/mg, 58% y i e l d ) . 2.3.5.3
P r e p a r a t i o n o f c r u d e c o n c e n t r a t e w i t h Amberli t e I R A 401-S r e s i n ( P r o c e d u r e C)
A column c o n t a i n i n g A m b e r l i t e I R A 401-S ( 5 6 0 m l ) was washed, i n s u c c e s s i o n , w i t h 1 M p h o s p h o r i c a c i d ( 5 . 6 a ) , a l k a l i n e p h o s p h a t e s o l u t i o n ( 0 . 2 5 m o l a r i n t r i s o d i u m p h o s p h a t e and 0 . 7 5 m o l a r i n sodium h y d r o x i d e , 2 . 5 a ) a n d water ( 1 . 7 a ) . The f e r m e n t a t i o n - b r o t h f i l t r a t e ( 1 9 a , pH 8 . 7 u n i t s / m l ) was p a s s e d t h r o u g h t h e column f o l l o w e d by water ( 2 . 8 2 ) a n d 0.5 M monosodium p h o s p h a t e 0.5 M p h o s p h o r i c a c i d ( 9 : 1 , v / v , 5 . 6 $1 e l u ting the a n t i b i o t i c activity. The a c i d i c e f f l u e n t as w e l l as a l l a c i d i c w a t e r - w a s h e s were a d j u s t e d t o pH 5.0 a n d p e r c o l a t e d t h r o u g h a column of A m b e r l i t e XAD-2 ( 6 0 0 m l ) p r e v i o u s l y r e g e n e r a t e d a n d washed w i t h water ( P r o c e d u r e B). The A m b e r l i t e XAD 2 column was washed w i t h water u n t i l t h e e f f l u e n t was f r e e o f p h o s p h a t e i o n s and e l u t e d w i t h e t h a n o l - w a t e r ( 3 : 7 , v / v , 2 . 8 1). C o n c e n t r a t i o n o f t h e a l c o h o l i c e f f l u e n t a n d f r e e z e - d r y i n g gave a brown powder ( 4 . 5 g , 2 3 u n i t s / m g , 78% y i e l d ) .
-
2.3.5.4
--The
(SO& XAD-2
P u r i f i c a t i o n of crude a n t i b i o t i c with Amberlite I R A 401-S a n d A m b e r l i t e XAD-2 P r o c e d u r e D , ( F i g u r e 1)
b o t t o m o f a column c o n t a i n i n g A m b e r l i t e I R A 401-S 2 a ) was c o n n e c t e d t o a column c o n t a i n i n g A m b e r l i t e (10 a ) a n d 0 . 2 M ammonium s u l f a t e ( 1 2 a ) was p a s s e d
,
537
t h r o u g h b o t h columns. A s o l u t i o n of c r u d e a n t i b i o t i c , as obt a i n e d a c c o r d i n g t o p r o c e d u r e A , was mixed w i t h a c o n c e n t r a t e d s o l u t i o n c o n t a i n i n g 1 3 g of f e r r i c c h l o r i d e , f i l t e r e d , d i l u t e d w i t h 0 . 4 M ammonium s u l f a t e ( 6 8 ) and a l l o w e d t o p e r c o l a t e The t h r o u g h b o t h columns a t a l i n e a r f l o w - r a t e of 1 - 2 cm/min. columns were t h e n washed w i t h 0 . 2 M ammonium s u l f a t e ( 1 3 . 5 a ) and water ( 2 3 a ) . An a p p r e c i a b l e amount of c o l o r e d matter r e 'mained a d s o r b e d on t h e A m b e r l i t e I R A 401-S column, whereas some pigments were e l u t e d as i l l u s t r a t e d i n F i g u r e 1. The column s e r i e s was d i s c o n n e c t e d and t h e a n t i b i o t i c s u b s t a n c e e l u t e d from t h e A m b e r l i t e XAD-2 column w i t h water c o n t a i n i n g 1 8 % e t h anol. The b i o l o g i c a l l y a c t i v e f r a c t i o n s were combined, concent r a t e d and f r e e z e - d r i e d t o a f f o r d a brown powder ( 8 8 g; 2 0 units/mg, 80% y i e l d ) . COLUMN I 8AMBERLITE IRA 401-5 ( 7 . 6 c m ~ 4 4 c m l COLUMN II I AMBERLITE XAD-2 ( I O c m X 127Cm)
k
COLUMNS
I and I1
&!-COLUMN
1-
I
t 0
z a
m 0:
0 v)
m
a
LITERS THROUGH COLUMN FIGURE 1
2.3.5.5
P u r i f i c a t i o n by chromatography on Dowex 5OW-XZ P r o c e d u r e E , ( Fi g u r e 2 ) t
A column of Dowex 5OW-XZ ( N a , 5 0 - 1 0 0 mesh, 6 . 6 cm x 7 6 c m ) was washed w i t h 0 . 2 M monosodium p h o s p h a t e s o l u t i o n ( 8 a )
and c h a r g e d w i t h a c o n c e n t r a t e d aqueous s o l u t i o n of an a n t i b i o t i c p r e p a r a t i o n as o b t a i n e d by P r o c e d u r e D. Column development was i n i t i a t e d w i t h 0 . 2 M monosodium p h o s p h a t e t o e l u t e c o l o r e d m a t t e r and an i n s i g n i f i c a n t q u a n t i t y of b i o l o g i c a l l y a c t i v e material. A s t h e e f f l u e n t became l i g h t e r i n c o l o r , column 0.2 M development was s t a r t e d w i t h 0 . 2 M monosodium p h o s p h a t e disodium p h o s p h a t e ( l : l , v / v ) p a r t i a l l y s e p a r a t i n g a d a r k pigment
-
538 from t h e major a n t i b i o t i c zone. The a n t i b i o t i c f r a c t i o n s were combined a n d d e s a l t e d by p a s s a g e t h r o u g h a column of A m b e r l i t e XAD-2 ( 1 . 5 a ) p r e v i o u s l y r e g e n e r a t e d and washed w i t h water. The A m b e r l i t e column was washed w i t h water u n t i l t h e e f f l u e n t was s a l t - f r e e , t h e a c t i v i t y e l u t e d w i t h e t h a n o l - w a t e r ( 2 . 5 : 7 . 5 , v / v ) and worked-up as u s u a l t o g i v e a brown powder ( 1 4 g, 1 0 0 u n i t s h g , 80% y i e l d ) .
0 0
- IS0
1700
-
16.0-
-*
N 4.00 0
-
NoH2PO4-
0.2&N0H2PO4 -02&N02HPO4 , I ' l , W / v
I
3.00-
m
a 0
v)
2.00
0
b
0
0
-
*
I
1
0
0
'
0
q
-
2. 0
0 0
O
0
o 01
-
-I 4
20
t \
0
o I
I
0
I II
0
O 1.00-
0
I
0
0
- 30 2 0
I I I
0
0
r D
I
-
40
0
I I I I
0
4 0
0
I
-0.24
Y)
m
-100
0
I
E
f
: .I.- B
0
-
O
0
10
5
0 I
I
I
.
I
I
FIGURE 2
2.3.5.6
P u r i f i c a t i o n by rechromatography on Dowex 5OW-X2 P r o c e d u r e F , (Fi g u r e 3 )
+
A column o f Dowex 50W-X2 (Na , 2 0 0 - 4 0 0 mesh,4.5 cm x 4 8 cm), p r e v i o u s l y washed w i t h 0 . 2 M monosodium p h o s p h a t e , was c h a r g e d w i t h a p r e p a r a t i o n ( P r o c e d u r e E) d i s s o l v e d i n a minimum amount of 0.2 M monosodium p h o s p h a t e and a d j u s t e d t o pH 3 . 5 with phosphoric acid. Column development w i t h 0 . 2 M monosodium p h o s p h a t e e l u t e d a d a r k pigment and v e r y l i t t l e a n t i b i o t i c act i v i t y . E l u t i n g w i t h a m i x t u r e o f e q u a l volumes of 0 . 2 M monosodium p h o s p h a t e and 0 . 2 M disodium p h o s p h a t e c a u s e d a d a r k brown zone t o t r a v e l r a p i d l y t h r o u g h t h e column, p a s s i n g t h e a n t i b i o t i c zone which, i n t u r n , a l s o moved f a s t e r due t o t h e b u f f e r change. D e s a l t i n g of t h e m a j o r a n t i b i o t i c f r a c t i o n s on an A m b e r l i t e XAD-2 column ( c a . 5 0 0 m l ) i n t h e u s u a l f a s h i o n gave a red-brown powder (4.2 g , 300 u n i t s / m g , 9 0 % y i e l d ) .
5 39 COLUMN: DOWEX S O W - X 2 , 200-400 MESH (4.5cmx 4 8 c m )
I
a
I
200
500
1000
Is00
ml
2000
2500
3000
3500
4OOO
THROUGH COLUMN
FIGURE 3
2.3.5.7
P u r i f i c a t i o n by c h r o m a t o g r a p h y o n Rexyn 1 0 2 ( P r o c e d u r e GI
A column o f a c a r b o x y l i c a c i d i o n - e x c h a n g e r e s i n ( 4 . 9 c m x 55 c m , Rexyn 1 0 2 Na', 200-400 mesh) was e q u i l i b r a t e d w i t h 0 . 2 M s o d i u m p h o s p h a t e b u f f e r ( 0 . 2 M monosodium p h o s p h a t e 0.2 M d i s o d i u m p h o s p h a t e , 3 2 : 1 , v / v ) a n d c h a r g e d w i t h a s o l u t i o n of p u r i f i e d a n t i b i o t i c (300 mg, P r o c e d u r e F ) i n a minimum amount of b u f f e r . Column d e v e l o p m e n t w i t h t h e same s o l v e n t e l u t e d a p i g m e n t w i t h m i n o r b i o l o g i c a l a c t i v i t y ( 4 3 0 r n l ) followed by t h e m a j o r a n t i b i o t i c m i x t u r e c o n s i s t i n g o f Ro 5-2667 a n d Ro 7-7730 ( 8 2 0 m l ) a n d f i n a l l y Ro 7-7731 ( 3 3 0 0 ml). Desalting of t h e m a j o r a n t i b i o t i c f r a c t i o n g a v e a b r o w n - r e d powder ( 1 6 5 mg, ca. 500 u n i t s / m g , ca. 9 2 % ) .
-
2.3.5.8
P u r i f i c a t i o n by c h r o m a t o g r a p h y on CM-Sephadex C-25 Procedure H , ( F i g u r e 4)
CM-Sephadex C-25 (100 g , 4.5 meq/g) was s u s p e n d e d i n 0 . 2 M ammonium p h o s p h a t e b u f f e r ( 0 . 2 M monoammonium p h o s p h a t e 0 . 2 M diammonium p h o s p h a t e , 1 6 : 1 , v / v ) , e q u i l i b , r a t e d w i t h t h e b u f f e r a n d f i l l e d i n t o a column o f 4 . 1 c m d i a m e t e r t o g i v e a b e d - h e i g h t of 52.5 cm. The m a t e r i a l o b t a i n e d b y P r o c e d u r e G ( 0 . 5 g ) , was c h r o m a t o g r a p h e d as i l l u s t r a t e d i n F i g u r e 4 . Des a l t i n g , c o n c e n t r a t i o n a n d f r e e z e - d r y i n g o f t h e major z o n e g a v e a v i r t u a l l y p u r e m i x t u r e o f a n t i b i o t i c s Ro 5-2667 a n d Ro 7-7730 (600-800 u n i t s / m g ) .
-
540
10 0
-
0
ANTIBIOTICS and 0
*0 5 m =0 v)
1.50
-
1.25
-
1.00
-
0.50
-
0.25
-
0 --n
Ro7-7730
0
0
ABSORBANCY (425 nm)
0.75 -
U
RoS - 2667
0
ANTIBIOTIC Ro7-7731
0 0
00
’
0
O O
I
ml T H R O U G H COLUMN
FIGURE 4
2.3.5.9
S e p a r a t i o n of a n t i b i o t i c s Ro 5-2667 and Ro 7-7730 by p a r t i t i o n chromatography (Pr o c e d u r e I )
A s l u r r y of Sephadex 6-25 s u p e r f i n e (100 g) i n t h e lower p h a s e of s y s t e m 6 5 was used t o p r e p a r e a column of 5 c m diamet e r . The packed column was washed w i t h t h e upper p h a s e of s y s tem 6 5 under s l i g h t p r e s s u r e u n t i l t h e e f f l u e n t was e s s e n t i a l l y f r e e o f s t a t i o n a r y ( l o w e r ) p h a s e . The a n t i b i o t i c m i x t u r e ( 1 . 0 g) was d i s s o l v e d i n a minimum q u a n t i t y of lower p h a s e and d r y Sephadex 6-25 was added t o make a t h i c k p a s t e which was l a y e r e d on t o p of t h e column. Development w i t h t h e upper p h a s e of s y s t e m 6 5 e l u t e d a n t i b i o t i c Ro 7-7730 f o l l o w e d by R o 5 - 2 6 6 7 , as two red-brown bands which were d i l u t e d w i t h p e t r o l e u m e t h e r ( c a . 1 0 % o f t h e o r i g i n a l volume) and e x t r a c t e d r e p e a t e d l y w i t h small p o r t i o n s of water u n t i l t h e orange-brown pigments were removed from t h e o r g a n i c p h a s e s . The aqueous e x t r a c t s were c o n c e n t r a t e d under reduced p r e s s u r e t o remove o r g a n i c s o l v e n t s , d e s a l t e d as u s u a l and f r e e z e - d r i e d . Both a n t i b i o t i c s had b i o p o t e n c i e s of ca. 800 units/mg.
541
2.4 Chromatographic
i d e n t i f i c a t i o n o f albomycin, g r i s e i n and r e l a t e d a n t i b i o t i c s TABLE 3 Comparison of Albomycin and G r i s e i n by Ascending PC, Whatman No. 1 Paper, Bioautography w i t h E. c o s 3 Rf Values
S o l v e n t System
Albomycin (0.4 ps) 0.14, 0.83 0.68 6.79 0.79, 0.71
55 56 32 64 95 39
0.33,
G r i s e i n (0.5
0.46
0.14, 0.83 0.68 0.78 0.97 0.75
0.92
0.37,
pg)
0.46
TABLE 4 Comparison o f Albomycin with A n t i b i o t i c s L.A. 5352 and L.A. 5937 by PC, Whatman No.1 Paper, Bioautoqraphy w i t h ~CnococcudawlUA11’12 Rf Values Solvent System
L.A.
3
0.80 0.75 0.87 0.88 0.17 0.54, 0.35,
32 95 56 18 55 57
5352
0.72 0.45
L.A.
0.14, 0.07,
5937
0.80 0.75 0.83 0.90 0.20 0.37, 0.17,
Albomycin 0.88 0.78 0.84 0.83 0.02 0.53 0.02,
0.46 0.31
0.32 TABLE 5 Comparison o f Albomycin w i t h A n t i b i o t i c A 1787 by PC9
Rf Values Solvent System
74 58 59 77
A 1787
0.50, 0.11, 0.86 0.58,
0.57 0.2
0.70
Albomycin 0.50, 0.10, 0.92 0.56,
0.57 0.19 0.68
0.2,
542
TPBLE 6
Comparison o f Albomycins with A n t i b i o t i c s Ro 5-2667, Ro 7-7730 and Ro 7-7731, TLC, W-polygrarn Cel 300 Precoated P l a s t i c Shsets15
Rf values Albomycin 62, RO 5-2667
Solvent System
25* 26*
Albornycin 61, RO 7-7730
0.17 0.48 0.73 0.34 0.63 0.50 0.11
0.06 0.30 0.65 0.26 0.52 0.40 0.03
37 52 74 75 81
Albomycin E , RO 7-7731
0.04 0.08
-
-
-
*For development w i t h systems 25 and 26, p l a t e s were p r e t r e s t e d by d i p p i n g i n t o 0.2 M ammonium s u l f a t e s o l u t i o n and d r y i n g a t room temperature. TABLE 7 Comparison o f Albomycin and Alveornycin by R a d i a l PC, Bioautography with S. (vreu13
Rf Values Solvent System 50 51 52 76
2.5 L i t e r a t u r e
Albomycin
Alveomycin
-
0.85 0.50 0.13 0.50
0.50
Cited
1.
D. M. R e y n o l d s , A. S c h a t z a n d S . A. Waksman, P r o c . S O C . E x p t . B i o l . Med. 6 4 ( 1 9 4 7 ) 5 0 - 5 4 .
2.
D. M. R e y n o l d s a n d S . A . Waksman, J. Bact. 5 5 ( 1 9 4 8 )
739-
752. 3.
E . 0. S t a p l e y a n d R. E. Ormond, S c i e n c e 1 2 5 ( 1 9 5 7 ) 5 8 7 - 5 8 9 .
4.
J. Turkova’,
5.
W.
V
0. M i k e E a n d F . Sorm, C o l l . Czech. Chem. Commun. 3 1 (1966) 2444-2454.
Garson a n d S . A. Waksman, P r o c . Nat. Acad. S c i . 3 4 ( 1 9 4 8 )
2 32-2 39.
6.
G . F. Gauze a n d M . G . B r a x n i k o v a , N o v o s t i Med. Akad. Med. Nauk SSSR 2 3 ( 1 9 5 1 ) 1, 3.
543 7.
R. Hatter, Giorn. M i c r o b i o l . 11 (1963) 191-246.
8.
0 . Mikes, J. Turkcwa-and F. Sorm, C o l l . Czech. Chem. Commun. 2 8 (1963) 1747-1760.
9.
H. Thrum, N a t u r w i s s . 4 4 (1957) 561-562.
W
10.
H. B i c k e l , E. Gaumann, W. K e l l e r - S c h i e r l e i n ,
11.
P . S e m i a n d M. T. Timbal, A n t i b i o t i c s and Chemother. (1959) 160-166.
12.
B r i t i s h P a t e n t No. 920,799; March 13, 1963.
13.
G . S c h m i d t - K a s t n e r a n d J. Schmid, Med. und Chem., Abhandl. Med. -Chem. F o r s c h u n g s s t a e t t e n F a r b e n f a b r i k e n Bayer AG, V I I (1963) 528-529, V e r l a g Chemie, W e i n h e i m / B e r g s t r a s s e .
14.
H. Maehr and J. B e r g e r , B i o t e c h n o l . Bioeng. 11 (1969) 1111-1123.
15.
H. Maehr and R . P i t c h e r , J . A n t i b i o t i c s 2 4 (1971) 830-834.
16.
H. Maehr, P u r e Appl. Chem. 28 (1971) 603-636.
17.
G. F. Gauze, B r i t . Med. J. 2 (1955) 1177-1179.
18.
F. A. Kuehl, J r . , M. N. B i s h o p , L . C h a i e t and K . J. h e r . Chem. S O C . 73 (1951) 1770-1773.
19.
M. G . B r a z n i k o v a and L . P. Khayurova, A n t i b i o t i k i 3 (1958) 54-58.
20.
M.
V. P r e l o g , E . V i s c h e r , A. W e t t s t e i n and H . Z l h n e r , E x p e r i e n t i a 16 (1960) 129-133.
G . B r a z n i k o v d , 0 . Mikes a n d N . N. 22 (1957) 1 0 4 - 1 1 0 .
9
Folkers,
Lomakina, B i o c h e m i s t r y
(MOSCOW)
21.
J. Turkova, 0. Mikes a n d F. zorm, A n t i b i o t i k i 7 (1962) 878-883.
3. Succinimycin
3.1 Introduction 3.1.1
P r o d u c i n g orEanisms
S u c c i n i m y c i n i s e l a b o r a t e d by St4eptomyceA o l i u o c h n o m o The a l l e g e d p r o d u c t i o n o f s u c c i n i mycin by v a r i o u s s p e c i e s o f S t 4 e p t o r n y c e ~a u n e o 6 a c i e n ~ 2 a p p e a r s t o require f u r t h e r confirmation.
gene4 Waksman e t H e n r i c i l .
3.1.2
Chemical d e s c r i p t i o n 1
S u c c i n i m y c i n a c e t a t e i s a d e e p r e d - o r a n g e y amorphous powd e r y C 45.0-46.2%, H 6.6-7.0%, N 8.1-8.9%, i t i s s o l u b l e i n p o l a r s o l v e n t s o n l y , shows maximum s t a b i l i t y a t pH of ca. 6 a n d i s r a p i d l y i n a c t i v a t e d a t o r below pH 4. Hydrolysis of succinimycin with barium hydroxide y i e l d e d , s u c c i n i c a c i d , c a d a v e r i n e , p r o l i n e , ammonia a n d methylamine. UV a n d I R s p e c t r a of s u c c i n i m y c i n a c e t a t e were reported5.
i n t e / r alia,
I n c o n t r a d i c t i o n t o a s t a t e m e n t by H . Umezawa e t s u c c i n i m y c i n b e l o n g s t o t h e s e c o n d g r o u p of s i d e r o m y c i n s i n B i c k e l ’ s c l a s s i f i c a t i o n 4 ; c o m p a r a t i v e PC-data w i t h s y s t e m 52 showed t h a t s u c c i n i m y c i n h a $,cn R f - v a l u e h i g h e r t h a n g r i s e i n and lower than ferrimycin A
.
3.2 B i o l o g i c a l
activity
The a c t i v i t y o f s u c c i n i m y c i n i s r e s t r i c t e d t o a narrow r a n g e of g r a m - p o s i t i v e o r g a n i s m s p a r t i c u l a r l y c e r t a i n s t r a i n s o f s t a p h y l o c o c c i a n d s t r e p t o c o c c i . S u c c i n i m y c i n was n o t e d f o r r a p i d r e s i s t a n c e development o f s u s c e p t i b l e m i c r o o r g a n i s m s , e s p e c i a l l y s t a p h y l o c o c c i , which were t h e n c r o s s - r e s i s t a n t w i t h grisein.
-3.3
Isolation’ 3.3.1
Preparation of crude succinimycin s u l f a t e with c h a r c o a l a n d Dowex 50
The b r o t h f i l t r a t e ( 2 6 a ) was a d j u s t e d t o pH 5 . 6 , a g i t a t e d w i t h Darco G-60 (260 g ) a n d C e l i t e 545 ( 2 6 0 g) f o r 1 5 min. a n d f i l t e r e d . The f i l t e r c a k e was washed w i t h 1 5 % aqueous a c e t o n e ( 2 6 e ) a n d water ( 2 6 a) a n d s u s p e n d e d i n 50% a c e t o n e ( 1 3 a ) t o e x t r a c t t h e a n t i b i o t i c . The e x t r a c t was a d j u s t e d t o pH 5 . 7 and p a s s e d t h r o u g h Dowex 5 0 - X 1 “ a t , 220 m l ) . The r e s i n column was washed w i t h water and t h e a n t i b i o t i c e l u t e d w i t h 5 % sodium s u l f a t e s o l u t i o n ( 2 . 5 k). The e f f l u e n t was a d j u s t e d t o pH 5 . 5 , s t i r r e d f o r 10-15 min. w i t h Darco G-60 ( 1 8 g ) a n d C e l i t e 545 ( 1 8 g) a n d f i l t e r e d . The f i l t e r c a k e was washed w i t h water u n t i l i t was s a l t - f r e e , t h e n w i t h 1 5 % aqueous m e t h a n o l ( 6 0 0 m l ) and f i n a l l y e l u t e d b a t c h - w i s e w i t h 50% aqueous a c e t o n e ( 1 . 2 a , pH a d j u s t e d t o 3.5 w i t h s u l f u r i c a c i d ) . The aqueous a c e t o n e e x t r a c t was i m m e d i a t e l y a d j u s t e d t o pH 5 . 5 ( A m b e r l i t e IR-45,
545 free-amine f o r m ) , f i l t e r e d , c o n c e n t r a t e d and f r e e z e - d r i e d t o y i e l d c r u d e s u c c i n i m y c i n s u l f a t e ( 0 . 5 g , ca. 2 5 % p u r e ) . 3.3.2
P u r i f i c a t i o n o f c r u d e s u c c i n i m y c i n s u l f a t e by phenol-chloroform e x t r a c t i o n
Crude s u c c i n i m y c i n s u l f a t e ( 2 5 g ) was d i s s o l v e d i n water ( 6 0 0 m l t o t a l v o l u m e ) , mixed u n d e r s t i r r i n g w i t h 2 - p r o p a n o l ( 2 . 6 2 1 1 , allowed t o s t a n d ( 1 6 h r . 0-5') and f i l t e r e d ( C e l i t g 5 4 5 ) . The f i l t r a t e was a d j u s t e d t o pH 6 , c o n c e n t r a t e d a t 30 t o remove 2 - p r o p a n o l , mixed w i t h s o d i u m s u l f a t e ( 4 7 . 3 g ) a n d b r o u g h t t o a volume of 1 6 6 5 m l w i t h water. T h i s s o l u t i o n was e x t r a c t e d w i t h c h l o r o f o r m - p h e n o l (7:1, v/w, 5 x 3 0 0 ml) w i t h p e r i o d i c a d j u s t m e n t s t o m a i n t a i n t h e pH b e t w e e n 5.6 a n d 6 . 0 . The o r g a n i c e x t r a c t s were d i s c a r d e d . The a q u e o u s p h a s e (pH 5 . 6 ) was e x t r a c t e d w i t h c h l o r o f o r m - p h e n o l ( l : l , v/w, 4 x 300 m l ) w i t h t h e pH m a i n t a i n e d b e t w e e n 5.2 a n d 6 . 0 . The combined o r g a n i c ~ x t r a c t s w e r e washed w i t h water ( 2 x 130 m l ) c o n c e n t r a t e d a t 30 t o a volume o f 500 m l , d i l u t e d w i t h e t h e r ( 1 . 5 fi) a n d e x h a u s t i v e l y e x t r a c t e d w i t h 1 0 0 m l p o r t i o n s of water. The combined a q u e o u s e x t r a c t s w e r e washed w i t h e t h e r a n d w i t h h e p t a n e t o remove p h e n o l , a d j u s t e d t o pH 5 . 8 , c o n c e n t r a t e d a n d f r e e z e d r i e d t o g i v e p u r i f i e d s u c c i n i m y c i n s u l f a t e ( 4 . 7 g , ca. 75% p u r e ) . A s o l u t i o n o f t h i s m a t e r i a l ( 1 . 5 g ) was p a s s e d t h r o u g h a column o f Dowex 1 - X 2 ( a c e t a t e f o r m ) ; f r e e z e - d r y i n g o f t h e e f f l u e n t y i e l d e d s u c c i n i m y c i n a c e t a t e ( 1 . 4 g , ca. 7 5 % p u r e ) . 3.3.3
P r e p a r a t i o n o f p u r e s u c c i n i m y c i n by C r a i g distribution
P u r i f i e d s u c c i n i m y c i n a c e t a t e ( 1 . 8 g ) was s u b j e c t e d t o 9 9 t r a n s f e r s i n s y s t e m 82 e m p l o y i n g 40 m l o f u p p e r a n d l o w e r p h a s e per tube. The a n t i b i o t i c was t r a n s f e r r e d t o t h e a q u e o u s p h a s e s by a d d i t i o n o f 1 0 0 m l o f e t h e r t o e a c h t u b e , t h e p e a k a c t i v i t y ( t u b e s 4 3 - 5 7 , Baci&hA d u b t i e i d as t e s t o r g a n i s m ) was o b t a i n e d by e x t r a c t i o n of t h e combined l o w e r p h a s e s w i t h c h l o r o f o r m p h e n o l (l:l, v/w, 3 x 1 2 5 m l ) a n d w a s h i n g o f t h e combined The o r g a n i c p h a s e was o r g a n i c e x t r a c t s w i t h water ( 2 x 30 m l ) . d i l u t e d w i t h e t h e r ( 1 . 6 fi) a n d t h e a n t i b i o t i c was e x t r a c t e d w i t h water ( 1 5 5 - m l ) . The a q u e o u s e x t r a c t was p a s s e d t h r o u g h Dowex 1 - X 2 (AcO , 1 1 5 ml) a n d t h e e f f l u e n t f r e e z e - d r i e d . The r e s u l t i n g p r o d u c t ( 4 9 0 m g , ca. 9 5 % p u r e ) was s u b j e c t e d s i m i l a r l y t o 49 t r a n s f e r s , t u b e s 1 9 - 2 9 y i e l d e d h i g h l y p u r i f i e d , amorphous s u c c i n i m y c i n a c e t a t e (260 m g ) , Amax 430 (pH 7 . 0 , El!m 22.8). D e s f e r r i s u c c i n i m y c i n s u l f a t e ( C > 3) and succinimycin s u l f a t e ( C = 0 . 2 6 ) were a l s o c h a r a c t e r i z e d by C r a i g - d i s t r i b u t i o n i n system 90.
3.4 C h r o m a t o g r a p h i c
characterization of succinimycin' TABLE 8
Descending PC o f Succinimycin on Whatmen No. Solvent System
52 63 62
1 Paper
Rf Values
0.42 0.52 0.40
3.5 L i t e r a t u r e
Cited
1.
T . H . H a s k e l l , R . H . Bunge, J . C . F r e n c h a n d Q . R . B a r t z , J. A n t i b i o t i c s ( T o k y o ) S e r . A . 1 6 ( 1 9 6 3 ) 67-75.
2.
W . K e l l e r - S c h i e r l e i n , V . P r e l o g a n d H . Z ghne r; F o r t s c h r . Chem. Org. N a t u r s t o f f e 2 2 ( 1 9 6 4 ) 279-322.
3.
H . Ogawara, K . Maeda a n d H . Umezawa, J . A n t i b i o t i c s (T okyo) S e r . A . 1 9 ( 1 9 6 6 ) 190-193.
4.
H . B i c k e l , E . Glrumann, W . K e l l e r - S c h i e r l e i n , V . P r e l o g , E . V i s c h e r , A. W e t t s t e i n a n d H . Z a h n e r ; E x p e r i e n t i a 1 6 ( 1 9 6 0 ) 129-133.
5.
T. H. H a s k e l l , J . E h r l i c h , J. C. F r e n c h , R . H. Bunge a n d R. I . P i t t i l l o , U.S. P a t e n t 3 , 2 1 0 , 2 4 6 ; O c t o b e r 5 , 1 9 6 5 .
6.
T. H . H a s k e l l , p e r s o n a l c o m m u n i c a t i o n .
547 4 . Danomycin
4.1 Introduction 4.1.1
Producing organisms
StAeptomycea albaduncu4 n o v . s p . , s t r a i n No. 1 3 2 4 6 l , SZAeptomycea No. B 3 2 Q 2 . 4.1.2
Chemical
The a n t i b i o t i c i s a r e d d i s h compound of p o l y p e p t i d e n a t u r e , C 46.33, H 6.98, N 8.79 a n d Fe 3 . 5 3 % ? pKa 4 . Q 2 , 7.95, 1 0 . 3 , n e u t . e q u i v . 1 8 0 0 , m.p. 163-165' ( d e c . I 2 , i t i s s o l u b l e i n water a n d
a q u e o u s , l o w e r a l c o h o l s as w e l l as p h e n o l - c h l o r o f o r m m i x t u r e s . A c i d h y d r o l y s i s of danomycin y i e l d e d a c e t i c a c i d , s u c c i n i c a c i d , ammonia, c a d a v e r i n e ( 3 m o l e s ) and some 1 7 amino a c i d s . Danomycin c o n t a i n s a c h a r a c t e r i s t i c UV-chromophore ( T a b l e t h e maximum a t 270 nml was n o t f o u n d i n l a t e r p r e p a r a t i o n s 2 . I R s p e c t r a o f danomycin were p u b l i s h e d l Y 2 . 91,
TABLE 9 UV and Visible Light-absorption Maxima o f Danomycin2
Sol vent Water 0.1 N H C 1 0.1 N NaOH
325 (211, 420-440 (16.5) 325 (18), 440-460 (13.0) 325 ( s h . , 20), 410-430 (15.0)
I n a c o m p a r i s o n w i t h f e r r i m y c i n , danomycin e x h i b i t s a d i s t i n c t l y lower Rf v a l u e i n s y s t e m 52 ( T a b l e 3 ) p l a c i n g danomycin i n t o Group I 1 of B i c k e l ' s c l a s s i f i c a t i o n o f s i d e r o m y c i n . Umezawa e t a-!. a s s i g n e d danomycin, p r o b a b l y i n c o r r e c t l y , t o Group 1112.
4.2 Bioloaical
activity]
Danomycin i s a c t i v e b o t h i n V ~ V Oand i n vi.7%0 a g a i n s t I t is highly active against c e r t a i n gram-positive b a c t e r i a . d i p l o c o c c i and s t a p h y l o c o c c i i n c l u d i n g s t r a i n s r e s i s t a n t t o commonly u s e d a n t i b i o t i c s a n d v i r t u a l l y i n a c t i v e a g a i n s t gramn e g a t i v e b a c t e r i a , m y c o b a c t e r i a , f u n g i a n d some g r a m - p o s i t i v e b a c t e r i a s u c h as s t r e p t o c o c c i , l a c t i c b a c i l l i a n d S . a l b u b . Grisein-cross-resistance was d e m o n s t r a t e d w i t h S. a u R U A 209P a n d s p o n t a n e o u s r e s i s t a n c e t o w a r d danomycin was n o t e d f o r s e v eral b a c t e r i a . mg/kg,
The t o x i c i t y o f danomycin i n mice i s v e r y low (LD50 3250 i v ) , t h e i n v i v o d a t a are summarized i n T a b l e 1 0 .
54 8 TABLE 10
Chemotherapeutic Ef f ect o f Danomycin on Mice Infection
2. auReu.5 Smith s t r a i n
Administration o f Druq CD,, sc, danomycin
danomycin sc, penicillin G PO, p e n i c i l l i n G
0,015 0.78 0.3 0.85
s c , danomycin P O , danomycin
0.075 0.95
PO,
2. auheud
BX-1633-2 (virulent, penicillinresistant strain 1
(mg/kg)
4.3 I s o l a t i o n 4.3.1
P r e p a r a t i o n o f c r u d e danomycin]
The b r o t h was f i l t e r e d a t pH 6 . 0 , t h e f i l t r a t e s t i r r e d w i t h c h a r c o a l ( 0 . 5 % w/v) a t pH 7-8 f o r 30 min., and t h e f i l t e r e d c a r b o n cake washed t h r e e times w i t h water a n d t h r e e times w i t h 50% m e t h a n o l . The a c t i v i t y was e l u t e d w i t h b u t a n o l - s a t u r a t e d w a t e r employing 1 / 8 o f t h e o r i g i n a l b r o t h - v o l u m e . Concentration and f r e e z e - d r y i n g y i e l d e d a brownish powder o f a p p r o x i m a t e l y 1% purity
.
An a q u e o u s s o l u t i o n o f t h i s c r u d e p r e p a r a t i o n was a d s o r b e d on a column of c h a r c o a l which was washed w i t h water and 5 0 % m e t h a n o l ; 6 0 % a c e t o n e e l u t e d t h e a n t i b i o t i c . The b i o l o g i c a l l y a c t i v e f r a c t i o n s were c o n c e n t r a t e d t o small volume t h e a n t i b i o t i c was p r e c i p i t a t e d w i t h a c e t o n e t o g i v e a p r e p a r a t i o n I n a c t i v e m a t e r i a l c o u l d be removed by e t h a n o l of 1 5 - 2 0 % p u r i t y . p r e c i p i t a t i o n of a c o n c e n t r a t e d aqueous s o l u t i o n r e s u l t i n g i n 25-30% p u r i t y o f t h e p r o d u c t .
,
4.3.2
P u r i f i c a t i o n o f c r u d e danomycin by p h e n o l c h l o r o f o r m e x t r a c t i o n and C r a i g - d i s t r i b u t i o n ]
An aqueous s o l u t i o n of c r u d e danomycin was e x t r a c t e d w i t h The e x t r a c t was washed w i t h 0 . 5 % phenol-chloroform (l:l,v / v ) . sodium h y d r o g e n c a r b o n a t e s o l u t i o n , 0 . 0 1 N h y d r o c h l o r i c a c i d a n d water. The a n t i b i o t i c was t r a n s f e r r e d t o t h e a q u e o u s p h a s e by t h e a d d i t i o n o f 2 volumes o f e t h e r - p e t r o l e u m e t h e r ( l : l , v/v). The a q u e o u s p h a s e was f r e e d o f p h e n o l by e t h e r e x t r a c t i o n ; c o n c e n t r a t i o n and f r e e z e - d r y i n g y i e l d e d a r e d d i s h - o r a n g e s o l i d of 65-80% p u r i t y . I n a similar e x p e r i m e n t , danomycin was e x t r a c t e d with chloroform-phenol, 1 : 7 , v/v2. P u r e danomycin was p r e p a r e d by C r a i g - d i s t r i b u t i o n employi n g s y s t e m 8 8 ( C = 0 . 3 3 ) l o r 83 ( C = 0 . 0 8 ) Alternatively, f i n a l p u r i f i c a t i o n was a c h i e v e d by p a r t i t i o n chromatography on c e l l u l o s e w i t h s y s t e m 7 4 ( c o m p o s i t i o n : 60:4:30, v / v ) . Crude danomycin ( 1 5 7 mg) was c h r o m a t o g r a p h e d on a column (3 cm x 6 8 cm) c o n t a i n i n g 1 2 0 g o f c e l l u l o s e a n d f u r n i s h e d 1 2 . 9 mg o f p u r i f i e d product2.
.
5 49
4.4 Chromatographic
characterization
TABLE 11 PC o f Danomycin and F e r r i m y c i n A l l Rf Values
Solvent System
Danomycin
3 22 1 52 57 38 18 16 61 60
Ferrimycin A 1
0.95 0.85
0.60 0.65 0.05 0.40 0.73 0.45
1.00 0.23 0.50 0.65 0.05 0.95 0.50 0.30 0.05 0.35 0.85
31 55 95
-
TABLE 12 TLC of Danomycin on Cellulose Powder2 Solvent System 52
81* 2 22 57 37 74 32 75
Rf Values o f Danomycin 0.37 0.52 0.98 0.90 0.75 0.72 0.71 0.84 0.35
*P l a t e p re t re atmnt w i t h acetone -w a t e r-s a t ur at e d s o d i urn c h l o r i d e , 16:3:1, v/v.
4.5 L i t e r a t u r e
Cited
1.
H. T s u k i u r a , M. O k a n i s h i , T. Ohmori, H . Koshiyama, T . M i y a k i , H . K i t a z i m a a n d H. Kawaguchi, J . A n t i b i o t i c s (Tokyo) S e r . A 17 (1964) 39-47.
2.
H. Ogawara, K . Maeda a n d H. Umezawa, J. A n t i b i o t i c s ( T o k y o ) S e r . A 19 (1966) 190-193.
550 5 . Ferrimycins
5.1 Introduction 5.1.1
Producing organisms
S,?keptornyceA g n i ~ e o & & 2 v u( K ~ r a i n s k y ) Waksman e t H e n r i c i , s t r a i n ETH 9578 (NRRL 2 7 1 7 ) l v 5 ; StJteptomyceA gniAeodlavUA, s t r a i n ETH 1 5 3 1 1 2 ; S , ? k e p t o m y c e A tavendulae (Waksman et C u r t i s ) Waksman e t H e n r i c i , s t r a i n ETH 1 4 6 7 7 2 ; StJteptomyceA Lavcndulae, s t r a i n ETH 21510’; S 2 2 e p t a m y c e A galLlaeuA, s t r a i n ETH 1 8 8 2 2 l . 5.1.2
Chemical d e s c r i p t i o n 3 9 4
F e r r i m y c i n A l d i h y d r o c h l o r i d e : C41Hs5014NloFe. 2 H C 1 , pKAEs 4.11, pK$& 7.92 ( e q u i v . w t . 1 1 2 9 1 , pKBEs ca. 1 1 . 4 , 1% (Elcm) 229 ( 3 1 7 1 , 319 ( 2 8 . 3 1 , 430 ( 2 6 . 4 ) nm. I R s p e c t r u m 3 , NMR s p e c t r u m 4 . F e r r i m y c i n A2 d i h y d r o c h l o r i d e : C 45.78, H 6 . 7 7 , N 1 2 . 7 5 , ( C I C H 3 2.16, C1 6 . 2 3 , Fe 5 . 2 9 % , pKAEs 4 . 0 4 , pK$Es 7 . 9 1 ( e q u i v . H O w t . 1086), pK$& ca. 1 1 . 5 , ( E $ b m ) 231 ( 3 2 8 1 , 3 1 9 (301, 435 ( 2 6 . 2 ) nm, I R s p e c t r u m 3 . The f e r r i m y c i n h y d r o c h l o r i d e s are v e r y s o l u b l e i n water, m e t h a n o l , 90% p h e n o l , p h e n o l - c h l o r o f o r m m i x t u r e s , d i m e t h y l formamide a n d 2 - m e t h o x y e t h a n o l , somewhat l e s s s o l u b l e i n a c e t i c a c i d , e t h a n o l , b e n z y l a l c o h o l and p y r i d i n e a n d i n s o l u b l e i n 1b u t a n o l , c h l o r o f o r m , e t h y l a c e t a t e a n d less p o l a r s o l v e n t s . Prolonged h e a t i n g with ninhydrin i n pyridine-butanol g i v e s a b l u e - v i o l e t c o l o r and a c i d i f i e d s o l u t i o n s of f e r r i m y c i n g i v e a p o s i t i v e Prussian-blue r e a c t i o n with potassium ferrocyanide. I n s t a b i l i t y is p r o n o u n c e d o v e r a w i d e pH-range; maximum s t a b i l i t y was o b s e r v e d w i t h i n t h e pH-range o f 3 - 4 a n d minimum s t a b i l i t y a t pH 7, somewhat improved s t a b i l i t y was n o t e d a b o v e pH 8. I s o l a t i o n work and b i o l o g i c a l a s s a y were c o m p l i c a t e d by d e g r a d a t i o n p r o d u c t s of f e r r i m y c i n e x h i b i t i n g s i d e r a m i n e a c t i v i t y 3 . Ferrimycin belongs t o t h e t h i r d group o f a n t i b i o t i c s i n B i c k e l ’ s c l a s s i f i c a t i o n ’ and i s i d e n t i c a l with pilosomycin5. F e r r i m y c i n A 1 i s t h e m a j o r a n t i b i o t i c component and was e x t e n s i v e l y s t u d i e d . The p r o p o s e d s t r u c t u r e 4 e x h i b i t s an Fe ( I I I I - b i n d i n g c e n t e r common t o f e r r i o x a m i n e s , e s p e c i a l l y f e r r i o xamines B a n d D1 and r e p r e s e n t s a s e x a d e n t a t e l i g a n d made up by t h r e e b i d e n t a t e h y d r o x a m a t o l i g a n d s p r o v i d e d by t h r e e N5-hydroxyN5-succinoyl-l, 5-diaminopentane m o i e t i e s i n l i n e a r c o n n e c t i o n 7 .
5.2 B i o l o g i c a l
activity6
Ferrimycin is a c t i v e p r i m a r i l y a g a i n s t gram-positive bact e r i a s u c h as m i c r o c o c c i , s t r e p t o c o c c i , c o r y n e b a c t e r i a and b a c i l l i , w i t h t h e e x c e p t i o n o f BaCiLlu.4 megathekium a n d S . i a e c a l i A , t h e i n h i b i t o r y e f f e c t d e p e n d i n g g r e a t l y on t h e medium u s e d . T h i s phenomenon was assumed t o b e d u e t o t h e i n s t a b i l i t y of f e r r i m y c i n w i t h i n t h e p h y s i o l o g i c a l pH-range a n d i t s decomposi-
551
OH
FERRlMYClN A, t i o n i n t o s i d e r a m i n e s which a n t a g o n i z e f e r r i m y c i n - a c t i v i t y . F e r r i m y c i n was 10-50 times more e f f e c t i v e t h a n p e n i c i l l i n G a n d 50-100 times more e f f e c t i v e t h a n e r y t h r o m y c i n when t e s t e d i n mice i n f e c t e d w i t h S. aLIRLud a n d SiY~tnep.tococcud p y o g e n e d . A g a i n s t pneumococcal i n f e c t i o n s i n mice, t h e LD5o f o r a s i n g l e p a r e n t e r a l o r o r a l d o s e was 0 . 3 o r 3 g / k g , r e s p e c t i v e l y , b u t a t h e r a p e u t i c e f f e c t was a l r e a d y a c h i e v e d a t d o s a g e s of 0 . 3 - 3 mg/ kg
.
A d m i n i s t r a t i o n o f p i l o s o m y c i n A (1 mg/kg, s c ) t o mice i n f e c t e d w i t h S t a p h y t o c o c c u d pyogened, Pncumococcud Type 111, o r SLtep.tococcuA h e m o l y t i c u d , c a u s e d 80-100% s u r v i v a l ; w i t h f i v e i n j e c t i o n s of 0 . 1 mg/kg,60% s u r v i v a l was o b s e r v e d . S i m i l a r e f f e c t s were o b s e r v e d w i t h o r a l a d m i n i s t r a t i o n when t h e d o s a g e was i n c r e a s e d t e n f o l d . P i l o s o m y c i n B e x h i b i t e d t h e same h i g h a c t i v i t y i n V ~ V Oa g a i n s t Pneumococcud Type 111. F e r r i m y c i n a n d p i l o s o m y c i n have v e r y low t o x i c i t y ; p i l o s o mycin, f o r i n s t a n c e , was t o l e r a t e d by mice a t l e v e l s o f 500 mg/ kg, s c 5 . A s a t y p i c a l sideromycin, f e r r i m y c i n is c r o s s - r e s i s t a n t w i t h g r i s e i n , i t s i n s t a b i l i t y a n d t h e r a p i d development o f r e s i s t a n t b a c t e r i a precluded i t s c l i n i c a l use. The c o m p e t i t i v e a n t a g o n i s m between f e r r i m y c i n a n d f e r r i o xamines was u s e d f o r q u a n t i t a t i v e a s s a y i n t h e form of a modif i e d Boni f as - t e s t ' 2.
5.3 Isolation
552 P r e p a r a t i o n of c r u d e f e r r i m y c i n w i t h c h a r c o a l 3
5.3.1
The b r o t h f i l t r a t e ( 6 0 0 a ) was mixed w i t h H y f l o - S u p e r c e l ( 5 . 5 k g ) , a d j u s t e d t o pH 4.0 w i t h 2 N h y d r o c h l o r i c a c i d ( 2 . 5 2 ) and f i l t e r e d . The f i l t e r cake was washed w i t h water (100 R ) and t h e c l e a r f i l t r a t e was s t i r r e d f o r 4 5 min. w i t h N o r i t ( 7 k g ) p r e v i o u s l y washed w i t h 1 N h y d r o c h l o r i c a c i d and water. The c h a r g e d N o r i t was removed by f i l t r a t i o n w i t h t h e a i d o f H y f l o S u p e r c e l and was washed w i t h water ( 2 x 2 0 0 a ) . The a n t i b i o t i c was e l u t e d b a t c h -w i s e w i t h 1 b u t a n o 1-methanol ace t i c a c i d -w a t e r (2:1:1:2, v / v ) by s t i r r i n g f o r 1 h r . The e l u t i o n was r e p e a t e d , t h e e x t r a c t s ( 1 4 0 L, 4 6 a ) were combined and a d d i t i o n o f 1b u t y l a c e t a t e (96 a > gave two p h a s e s . The o r g a n i c p h a s e was washed w i t h water t o y i e l d a t o t a l o f 6 5 a of aqueous p h a s e which was e x t r a c t e d w i t h 1 - b u t a n o l - 1 - b u t y l a c e t a t e (1:2, 72 a ) , e t h y l a c e t a t e ( 4 8 a ) and e t h e r ( 2 4 a ) . The r e m a i n i n g aqueous p h a s e ( 4 4 a ) was c o n c e n t r a t e d ( < 30') and f r e e z e - d r i e d t o a f f o r d a brown, h y g r o s c o p i c powder ( 5 0 9 g ) .
-
-
A s o l u t i o n of t h i s material ( 3 3 8 g) i n water ( 1 . 5 a ) was e x t r a c t e d w i t h p h e n o l - c h l o r o f o r m (l:l, w / v , 1 6 , 5 0 0 m l , 5 0 0 m l ) a f t e r a d d i t i o n of ammonium s u l f a t e (150 g ) . The o r g a n i c p h a s e was washed w i t h 1 N h y d r o c h l o r i c a c i d ( 7 5 0 m l ) , f i l t e r e d t h r o u g h C e l i t e and s t i r r e d w i t h w a t e r ( 6 0 0 m l ) , e t h e r ( 4 a ) a n d p e t r o l e u m e t h e r ( 4 a ) . The s e p a r a t e d aqueous p h a s e was washed w i t h e t h e r t o remove p h e n o l and f r e e z e - d r i e d t o y i e l d a brownr e d powder w i t h a b i o p o t e n c y ca. 1 0 0 t i m e s h i g h e r t h a n f r e e z e dried broth f i l t r a t e . a r a t i o n of crude f e r r i m y c i n w i t h Amberlite The f e r m e n t a t i o n b r o t h ( 3 0 0 a , 3000 u n i t s / m l ) was a d j u s t e d t o pH 4 . 0 w i t h 1 0 % o x a l i c a c i d and f i l t e r e d w i t h H y f l o S u p e r c e l . The f i l t r a t e was p a s s e d t h r o u g h a column o f A m b e r l i t e I R C 50 ( c a . 7 0 % Na+, 10 cm x 8 0 cm) a t a l i n e a r f l o w - r a t e o f 1 5 cm/min. The column was washed w i t h water (30 a ) and 8 0 % metha n o l ( 6 0 a ) . The a n t i b i o t i c was e l u t e d w i t h methanol 1N h y d r o c h l o r i c a c i d ( Q : lv,/ v , 3 7 1 ) . The e f f l u e n t was a d j u s t e d toopH 4 . 5 w i t h sodium h y d r o x i d e and c o n c e n t r a t e d t o 2 a below 30 , The f i l t e r e d c o n c e n t r a t e was e x t r a c t e d w i t h p h e n o l c h l o r o f o r m (l:l, 500 m l ) , t h e o r g a n i c p h a s e was washed w i t h 0 . 0 1 N h y d r o c h l o r i c a c i d c o n t a i n i n g 1 0 % sodium c h l o r i d e , f i l t e r e d t h r o u g h C e l i t e , s t i r r e d under c o o l i n g and mixed w i r h H y f l o - S u p e r c e l ( 3 5 g ) , e t h e r ( 1 . 5 a ) a n d p e t r o l e u m e t h e r (1 11). The m i x t u r e c o n t a i n i n g p r e c i p i t a t e a n d f i l t e r a i d was washed e x h a u s t i v e l y w i t h e t h e r t o remove p h e n o l and e l u t e d w i t h l i t t l e methanol. The r e s u l t i n g e l u a t e was e v a p o r a t e d t o y i e l d c r u d e f e r r i m y c i n (36 g, 1 2 0 0 0 units/mg $.ca. 6 0 % y i e l d ) . Pilosomycin was i s o l a t e d i n a similar f a s h i o n
-
5.3.3
,
P u r i f i c a t i o n of c r u d e f e r r i m y c i n by p r e c i p i t a t i o n with phenolJ
The sodium c h l o r i d e c o n t a i n i n g c o n c e n t r a t e , o b t a i n e d as d e s c r i b e d i n s e c t i o n 5 . 3 . 2 by c o n c e n t r a t i o n of t h e n e u t r a l i z e d A m b e r l i t e IRC-50 e f f l u e n t , was mixed under s t i r r i n g w i t h H y f l o -
553 S u p e r c e l ( 1 k g ) a n d p h e n o l - w a t e r (ll:l, 1 . 8 1 1 ) was a d d e d o v e r a 2 h r p e r i o d . The m i x t u r e was s t i r r e d f o r an a d d i t i o n a l 30 min. and t h e n f i l t e r e d . The f i l t e r c a k e was washed on t h e f i l t e r b a t c h w i s e a n d c o n s e c u t i v e l y w i t h e t h e r a n d a c e t o n e and f i n a l l y with chloroform. The m o i s t f i l t e r cake was a d d e d u n d e r s t i r r i n g t o p h e n o l - c h l o r o f o r m (l:l, w / v , 3 a ) , s t i r r i n g was c o n t i n u e d f o r 1 h r a n d c h l o r o f o r m ( 1 5 a ) was a d d e d o v e r a o n e h r period. The s u s p e n s i o n was f i l t e r e d , t h e cake was washed w i t h p h e n o l - c h l o r o f o r m (l:ll, w / v , 2 a ) a n d t h e combined f i l t r a t e s ( 2 2 a ) w e r e c o n c e n t r a t e d t o 4 e . The c o n c e n t r a t e was added dropwise t o a s t i r r e d mixture o f e t h e r ( 4 a ) , petroleum e t h e r ( 8 2 ) a n d H y f l o - S u p e r c e l ( 4 0 0 g ) . The m i x t u r e was f i l t e r e d ¶ t h e c a k e washed w i t h e t h e r ( 2 e ) and a c e t o n e (1 e ) and e l u t e d batchwise w i t h methanol ( 3 x 1 . 5 L) w i t h i n t e r m i t t e n t f i l t r a t i o n . The combined f i l t r a t e s y i e l d e d p u r i f i e d f e r r i m y c i n (88 g, 6 0 0 0 0 u n i t s / m g ) upon c o n c e n t r a t i o n . 5.3.4
S e p a r a t i o n o f f e r r i m y c i n s A a n d B by p a r t i t i o n c h r o m a t o g r a p h y on c e l l u l o s e 3
6 0 0 0 0 u n i t s / m g ) o b t a i n e d as Purified ferrimycin (75 g, d e s c r i b e d i n t h e p r e c e d i n g s e c t i o n was c h r o m a t o g r a p h e d on a c e l l u l o s e column ( 9 . 9 cm x 1 4 0 c m , 3 . 4 kg of c e l l u l o s e powder, Whatman S t a n d a r d G r a d e ) w i t h t h e u p p e r p h a s e of s y s t e m 5 2 t o which 1 0 % 1 - b u t a n o l h a d b e e n a d d e d . The column was p a c k e d d r y , prewashed w i t h t h e m o b i l e p h a s e and c h a r g e d w i t h a m i x t u r e p r e p a r e d by s t i r r i n g c e l l u l o s e ( 1 5 0 g ) , s o l v e n t (750 m l ) and f e r r i mycin ( 7 5 g ) f o r 2 h r . F e r r i m y c i n B ( 4 3 0 0 u n i t s / m g ) was e l u t e d b e f o r e A (110000-300000 u n i t s / m g ) , t h e a n t i b i o t i c a l l y a c t i v e f r a c t i o n s were a g i t a t e d w i t h p e t r o l e u m e t h e r , t h e r e s u l t i n g a q u e o u s p h a s e s washed w i t h b e n z e n e a n d f r e e z e - d r i e d .
P i l o s o m y c i n ( 7 0 0 mg) was c h r o m a t o g r a p h e d on c e l l u l o s e powder ( 1 2 7 g ) w i t h t h e same m o b i l e p h a s e , p i l o s o m y c i n was e l u t e d b e f o r e p i l o s o m y c i n A and worked up as d e s c r i b e d a b o v e 5 . 5.3.5
S e p a r a t i o n of f e r r i m y c i n s A a n d B by Craigdistribution
P u r i f i e d f e r r i m y c i n was s e p a r a t e d i n t o components A ( t u b e s 4 0 - 7 0 , 220000-600000 u n i t s / m g ) and B ( t u b e s 7 5 - 1 0 0 , 1 1 0 0 0 0 units/mg) a f t e r 1 1 5 t r a n s f e r s with system 89. The corr e s p o n d i n g t u b e c o n t e n t s were combined, e x t r a c t e d w i t h an e q u a l volume of p e t r o l e u m e t h e r and t h e a n t i b i o t i c s were i s o l a t e d from t h e a q u e o u s p h a s e s by p h e n o l - c h l o r o f o r m e x t r a c t i o n i n t h e u s u a l fashion3. P i l o s o m y c i n was s u c c e s s f u l l y p u r i f i e d a n d r e s o l v e d by C r a i g - d i s t r i b u t i o n i n s y s t e m s 79 a n d 9 2 5 . A f t e r 9 9 t r a n s f e r s , t u b e s 1 2 - 2 0 c o n t a i n e d p r e d o m i n a n t l y component A w h e r e a s t u b e s 2 1 - 4 0 were e n r i c h e d w i t h component B ( s y s t e m 7 9 ) . 5.3.6
P u r i f i c a t i o n o f f e r r i m y c i n A by c h r o m a t o g r a p h y on Dowex 5 0 5
F e r r i m y c i n A ( 2 g , 3 1 0 0 0 0 u n i t s / m g ) , o b t a i n e d by C r a i g d i s t r i b u t i o n as d e s c r i b e d , w a s c h r o m a t o g r a p h e d a t 17' on a column
554
of Dowex 50W-X2 ( 3 cm x 70 cm, NH4+, 1 0 0 - 2 0 0 m e s h ) , p r e v i o u s l y e q u i l i b r a t e d a n d d e v e l o p e d w i t h 0 . 2 M ammonium a c e t a t e b u f f e r , Gradient e l u t i o n pH 4 . 5 0 , a t a l i n e a r f l o w - r a t e o f 0 . 2 3 cm/min. i n v o l v i n g a o n e - l i t e r m i x i n g chamber a n d a d d i t i o n o f 2 M ammonium a c e t a t e b u f f e r , pH 4 . 5 0 , was s t a r t e d a f t e r a t o t a l d e v e l o p i n g time o f 20 h r a t which time ca. 1920 m l e f f l u e n t had been collected. F e r r i o x a m i n e B ( 1 6 mg) was c o n t a i n e d i n e f f l u e n t volume 3 . 6 - 4 . 0 a . The f r a c t i o n s were worked up as d e s c r i b e d i n The e f f l u e n t volume 7 . 2 - 7 . 8 a c o n t a i n e d f e r r i section 5.3.2. mycin A 1 and A 2 ( 4 4 0 mg, 6 5 0 0 0 0 u n i t s / m g ) w h e r e a s t h e volume 7.8-8.6 c o n t a i n e d predominantly f e r r i m y c i n A,. 5.3.7
S e p a r a t i o n of f e r r i m y c i n s A 1 a n d A? by CraigdistributionJ
F e r r i m y c i n s A , , A 2 a n d s i d e r a m i n e 2 were p a r t i a l l y s e p a r a t e d i n s y s t e m 83 a f t e r 33 t r a n s f e r s ( T a b l e 161, a q u a n t i t a t i v e s e p a r a t i o n s h o u l d be a c h i e v e d a f t e r 465 t r a n s f e r s as j u d g e d from the published data. 5.3.8
S e p a r a t i o n o f f e r r i m y c i n A 1 and A 2 b y p a r t i t i o n chromatographys
A m i x t u r e of f e r r i m y c i n A l a n d A2 ( 4 0 0 - 6 0 0 mg, 900000 u n i t s / m g ) c o n t a i n i n g p r e d o m i n a n t l y A l , was chromatographed on a column o f Whatman c e l l u l o s e powder ( 5 . 5 c m x 87 c m ) w i t h system 80. The d r y - p a c k e d column was p r e v i o u s l y washed w i t h m e t h a n o l 5 . 3 a ) f o l l o w e d by tw a t e r - s a t d . sodium c h l o r i d e s o h . ( 5 : 2 : 3 , b u t a n o l - 0 . 0 2 M h y d r o c h l o r i c a c i d - s a t d . sodium c h l o r i d e ( 2 : 1 : 1 , 3 $ 1 and w i t h t h e o r g a n i c p h a s e o f s y s t e m 80 ( 4 . 3 a ) which a l s o s e r v e d as m o b i l e p h a s e . The m a j o r f r a c t i o n c o n t a i n e d p u r e f e r The e f f l u e n t was e q u i l i b r a t e d w i t h an e q u a l v o l rioxamine A l . ume of e t h e r a n d 1 / 1 0 volume of w a t e r , f e r r i o x a m i n e A 1 ( l o 6 u n i t s / m g ) was i s o l a t e d from t h e a q u e o u s p h a s e by p h e n o l - c h l o r o form e x t r a c t i o n as d e s c r i b e d i n s e c t i o n 5 . 3 . 2 .
F e r r i m y c i n A2 ( l o 6 u n i t s / m g ) was o b t a i n e d s i m i l a r l y from a n A2-enriched f r a c t i o n ( s e c t i o n 5 . 3 . 6 ) a f t e r p r e v i o u s removal of s i d e r a m i n e Z by e l e c t r o p h o r e s i s .
5 . 4 Chromatographic ch,aracterization
o f ferrimycins
TABLE 13 Comparison o f Ferrimycins w i t h Other Basic, Water-soluble A n t i b i o t i c s by PC3 Antibiotic Ferrimycin A Ferrimycin B Streptomycin s u l f a t e Neomycin B s u l f a t e Neomycin C s u l f a t e Kanamycin s u l f a t e Viomy c i n s u l f a t e
System 52 0.40 0.55 0.04 0 0
D 0
Rf Values System 38 0.42 0.52 0.25 0.04 0.04 0.08 0.06
System 96 0.78 0.78 0.33 0.10 0.10 0.17 0.13
555 TABLE 14 Comparison o f F e r r i m y c i n A w i t h Other Sideromycins by TLC on C e l l u l o s e 8 Rf Values System 57 System 26"
S i derorny c i n A n t i b i o t i c from Tu 17 A n t i b i o t i c A 22765 Ferrimycin A *Plate-pretreatment
0.34 0.53 0.75
0.53 0.79 0.83
w i t h 0.2 M ammonium s u l f a t e s o l n . TABLE 15
Comparison o f Ferrimycins with Other Siderochromes by PC3
Rf Values* Antibiotic
i n System El**
Ferrimycin A 1 F e r r i m y c i n A2 Sideramine Z Albomycin Ferrioxamine B Ferrichrome
0.59 0.47 0.83 0 0.31 0.22
*Estimated from drawing. **Paper p r e t r e a t e d w i t h acetone-water-satd. chloride soln.
sodium
TABLE 16 C r a i g - d i s t r i b u t i o n of F e r r i m y c i n s i n System 833 Antibiotic Ferrirnycin Al F e r r i m y c i n A, Sideramine Z
D i s t r i b u t i o n Number ( C )
0.372 0,175 2.18
F e r r i m y c i n A l was compared by PC w i t h danomycin i n 6 s o l v e n t systems (cf. S e c t i o n b $ Table 11).
TABLE 1 7
Comparison of Pilosomycin A and B with Other A n t i b i o t i c s by PC5 Antibiotic Pilosomycin base A Pilosomycin base B s t r e p tomy ces Ristocetin A Ristocetin B Neomycin B Viomycin Ch l o r te t racy c l i n e Oxyte t r a c y c l i n e Actinomycin J Cyclose rim Grisein
18 -
0 0 0 0 0 0 0 0
0 0.92 0.07 0
52 0.49 0.63 0.05 0.05 0.17 0.02 0.02 0.66 0.55 0.92 0.32 0.22
Rf Values i n S o l v e n t Systems 34** 19 20 49 -
-
0.34 0.58 0.22 0 0.02 0.22 0.03 0.72 0.62 0.93 0.39 0.11
-
-
0.05 0.15 0 0 0 0 0 0-1*** 0-1*** 0.92
0.32 0.32 0 0.10 0.22 0 0 0-1*** 0-1*** 0.86
0
0.12
-
-
0.47 0.47 0.22 0.14 0.12 0.04 0.05 0.49 0.42 0.91 0.43 0.36
*Distance o f a n t i b i o t i c t r a v e l e d (cm) on continuous chromatography. *Whatman No. 4 p a p e r p r e t r e a t e d with 0.95M sodium s u l f a t e and 0.05 M sodium hydrosulf ate s o l u t i o n . ***Antibiotic d i s t r i b u t e d o v e r e n t i r e p a t h . ( ) P o s i t i o n poorly d e f i n e d .
43 -
94 -
0.74 0.74 0.07
2.7* 7.6* 0
-
0.02
0
0 0
0 0-1*** (14.5)* 0-1*** (8.8)* 0.94 27* 0.61 0.69 1*
557
5.5 L i t e r a t u r e 1.
Cited
H. B i c k e l , E. Gbumann, W . K e l l e r - S c h i e r l e i n , V . P r e l o g , E . V i s c h e r , A. W e t t s t e i n a n d H. Z a h n e r , E x p e r i e n t i a 1 6 (1960) 129-133.
2.
H. Zdhner, R . H u t t e r a n d E . B a c h m a n n , A r c h . M i k r o b i o l . ( 1 9 6 0 ) 325-349.
3.
H. B i c k e l , E . Gbumann, G . N u s s b e r g e r , P . R e u s s e r , E. V i s c h e r , W . Voser, A. W e t t s t e i n a n d H. Z a h n e r , Helv. Chim.
36
A c t a 4 3 ( 1 9 6 0 ) 2105-2118. 4.
H. B i c k e l , P. Mertens, V . Prelog, J. S e i b l and A . Walser, T e t r a h e d r o n , S u p p l . 8, P a r t I ( 1 9 6 6 ) 171-179.
5.
H. Gdumann, H. B i c k e l a n d E . V i s c h e r , U.S. May 8 , 1 9 6 2 .
6.
W.
7.
H. Maehr, P u r e A p p l . Chem. 28 ( 1 9 7 1 ) 6 0 3 - 6 3 6 .
8.
H. D i e k m a n n , A r c h , M i k r o b i o l . 7 3 ( 1 9 7 0 ) 6 5 - 7 6 .
P a t e n t 3,033,760;
S a c k m a n n , P . R e u s s e r , L . N e i p p , F . K r a d o l f e r a n d F. Gross, A n t i b i o t i c s a n d C h e m o t h e r a p y 1 2 ( 1 9 6 2 ) 3 4 - 4 5 .
558 6 , A n t i b i o t i c NRCS-15
6.1 Introduction 6.1.1
Producing organism
S t J M p t o m y c e A s t r a i n NRC-S-15.
6.1.2
Chemical d e s c r i p t i o n
Amorphous, b u f f p l a t e s , m.p. 149-150°, C 55.82, H 6 . 3 3 , N 8 . 0 8 , C1 2.52 and Fe 1 . 6 1 % . The a n t i b i o t i c is a p s e u d o s i d e r o mycin a n d e x h i b i t s i n d i c a t o r - l i k e p r o p e r t i e s , i t i s y e l l o w i n a c i d and r e d i n a l k a l i n e s o l u t i o n . I t i s s o l u b l e i n water, a l c o h o l , esters, acetone and chloroform, s l i g h t l y s o l u b l e i n d i e t h y l e t h e r and i n s o l u b l e i n petroleum e t h e r , gives p o s i t i v e tests with ninhydrin, potassium thiocyanate and potassium f e r r o c y a n i d e and n e g a t i v e t e s t s w i t h a c i d i c a n d a l k a l i n e p o t a s s i u m permanganate, Molisch, Fehling, Millon, Sakaguchi, f e r r i c chlor i d e and i o d i n e s o l u t i o n s . H y d r o l y s i s w i t h 6 N h y d r o c h l o r i c a c i d a t 105’ f o r 24 h r l i b e r a t e d l y s i n e , a s p a r t i c a c i d , g l u t a m i c acid, threonine, alanine, tryptophan, valine, leucine, o x a l i c , t a r t a r i c a n d maleic a c i d as judged by PC. The I R s p e c t r u m showed c h a r a c t e r i s t i c b a n d s a t 2400, 2950, 1 6 6 0 , 1 4 6 0 , 1 3 6 0 , 1 2 8 0 , 1250, 1 1 0 0 , 830 a n d 750 cm-1; t h e UV maxima a r e g i v e n i n T a b l e 18. TABLE 1 8 UV Maxima o f A n t i b i o t i c NRCS-15 So 1VB nt
‘max i n nm
(pi 1 lcm
95% e t h a n o l
210 (122.5)
95?6 ethanol-0.1N hydrochloric acid
208 (112.3)
95% ethanol-0.lN sodium hydroxide
222 (1212.5)
6.2 Biological
activity
A n t i b i o t i c NRCS-15 i s m a i n l y a c t i v e a g a i n s t g r a m - p o s i t i v e b a c t e r i a , weakly a c t i v e a g a i n s t g r a m - n e g a t i v e b a c t e r i a a n d i n a c t i v e a g a i n s t y e a s t s a n d molds. The a n t i b i o t i c was a c t i v e i n mice i n f e c t e d w i t h S. (ZUhLW, Smith s t r a i n , a t l e v e l s of 1 2 . 5 mg/kg w i t h t o x i c m a n i f e s t a t i o n s a t 6 0 mg/kg and LD5, ( s c ) of 50 mg/kg. Ferrioxapine B f a i l e d t o antagonize t h e a c t i v i t y of a n t i b i o t i c NRCS-15 a g a i n s t B a c i l l u d d u b t i l i d . T h i s p r o p e r t y a n d t h e a b s e n c e o f t h e b r o a d a b s o r p t i o n maximum a t 420-440 nm shows t h a t a n t i b i o t i c NRCS-15 i s p r o b a b l y n o t a F e ( I I 1 ) hydroxamate.
559
6.3 Isolation The b r o t h was e x t r a c t e d a t pH 8 . 0 - 8 . 5 w i t h c h l o r o f o r m , t h e c o n c e n t r a t e d e x t r a c t was r e d i s s o l v e d i n c h l o r o f o r m a n d chromatographed on a column o f a c t i v a t e d c h a r c o a l by development w i t h 7 0 % aqueous e t h a n o l . The b i o l o g i c a l l y a c t i v e f r a c t i o n s were p o o l e d , c o n c e n t r a t e d t o a n o i l y r e s i d u e a n d d i s s o l v e d in m e t h a n o l . An i n a c t i v e , w h i t e p r e c i p i t a t e was removed by f i l t r a t i o n , t h e f i l t r a t e was c o n c e n t r a t e d t o d r y n e s s , r e d i s s o l v e d i n c h l o r o f o r m o r e t h y l a c e t a t e a n d p r e c i p i t a t e d by t h e a d d i t i o n of petroleum e t h e r .
6 . 4 Chromatographic
c h a r a c t e r i z a t i o n of a n t i b i o t i c NRCS-15 TABLE 19
Chromatographic Behavior o f A n t i b i o t i c NRCS-15 on Descending PC, Bioautographic Detection w i t h
BacLtQUb .&.ti.eid Solvent System 14
13 48 68 24 4 52 5 72 7 68 3 70 6
11 27 30 9 29 12
1 45 28
R f Values* 0 0 0.72 0.81 0.85 0.87 0.88 0.88 0.88 0.88 0.89 0.90 0.91 0.92 0.93 0.93 0.95 0.95 0.96 0.96 0.95 0.97 0.97
*Estimated from drawing.
6.5 Literature 1.
Cited
B . M . Haroun, J. A n t i b i o t i c s (Tokyo) 2 7 ( 1 9 7 4 ) 1 4 - 1 9 .
560 7 . A n t i b i o t i c ASK-753
7 . 1 Introduction 7.1.1
P r o d u c i n g organism
StJtep.tomyceA AS -K- 7 5 3 . 7.1.2
Chemi c a l de s c r i p t i on
Light b u f f p l a t e s , d e c . >80°, C 5 4 . 6 4 , H 6 . 9 8 , N 6 . 9 2 , 0 2 8 . 9 6 and Fe 2 . 5 % . The a n t i b i o t i c i s s o l u b l e i n a l c o h o l s , a c e t o n e , c h l o r o f o r m , d i e t h y l e t h e r , h a r d l y s o l u b l e i n water, e t h y l and b u t y l a c e t a t e and i n s o l u b l e i n p e t r o l e u m e t h e r ; i t r e d u c e s a l k a l i n e and a c i d i c p o t a s s i u m permanganate s o l u t i o n b u t g i v e s n e g a t i v e Molisch, F e h l i n g , n i n h y d r i n , M i l l o n , Sakaguchi and f e r r i c g h l o r i d e t e s t s . H y d r o l y s i s w i t h 6 N h y d r o c h l o r i c a c i d at 105 f o r 2 4 h r l i b e r a t e d l y s i n e , glutamic a c i d , aspart i c a c i d , g l y c i n e , a l a n i n e , l e u c i n e , two u n i d e n t i f i e d amino a c i d s , g l u c o n i c , c i t r i c , f u m a r i c and a - k e t o g l u t a r i c a c i d . The I R s p e c t r u m 1 showed c h a r a c t e r i s t i c bands a t 3 5 0 0 , 1 6 7 0 , 1480 and 837 c m - 1 ; t h e UV maximal a r e summarized i n Table 20. TABLE 20 UV Maxima o f A n t i b i o t i c ASK-753
1%
7.2 Biological
Solvent
Amax i n nm fElcm)
Ethanol
270 (120.71, ca. 340 sh (89)
Acidic ethanol
270 (120)
Alkaline ethanol
287 (135.2)
activity
A n t i b i o t i c ASK-753 i s a c t i v e p r i m a r i l y a g a i n s t gram-posit i v e b a c t e r i a , e s p e c i a l l y Coaynebacteaium hoddmanii, most b a c i l l i and c o c c i . Although v e r y a c t i v e a g a i n s t PAeudomonaA aeaug h I O A U D 1 2 9 , i t i s l e s s a c t i v e a g a i n s t gram-negative b a c t e r i a and n o n - f i l a m e n t o u s f u n g i . The CDS0 f o r mice i n f e c t e d w i t h S . auaeu6 A 3 2 1 ( a p e n i c i l l i n s e n s i t i v e s t r a i n ) was 10 mg/kg, whereas t h e LD5o was 58 mg/kg ( i p ) . The a n t i b i o t i c a c t i v i t y of t h i s compound was n o t a n t a g o n i z e d by f e r r i o x a m i n e B ; a n t i b i o t i c ASK-753 a p p e a r s t o l a c k a b s o r p t i o n i n t h e v i s i b l e l i g h t and i s t h u s r e g a r d e d as a pseudosideromycin.
7.3 Isolation The b r o t h f i l t r a t e was e x t r a c t e d w i t h c h l o r o f o r m a t pH 7 . 5 - 8 . 0 , t h e e x t r a c t was washed r e p e a t e d l y w i t h p h o s p h a t e b u f f e r pH 4 . 0 , removing most pigments and o t h e r i m p u r i t i e s . The con-
561 c e n t r a t e d c h l o r o f o r m e x t r a c t s were r e d i s s o l v e d i n c h l o r o f o r m e t h a n o l ( 9 9 : 1 , v / v ) and chromatographed on a column of Sephadex LH-20 w i t h t h e same s o l v e n t m i x t u r e as m o b i l e p h a s e . The a n t i b i o t i c moved as a b u f f - c o l o r e d band which y i e l d e d t h e a n t i b i o t i c as b u f f p l a t e s .
7 . L, C h r o m a t o g r a p h i c
c h a r a c t e r i z a t i o n o f a n t i b i o t i c ASK-753 TABLE 2 1
Descending PC o f A n t i b i o t i c ASK-753 w i t h Whatman No. 1 Paper and Bioautography with Ba&UtuA .bub.ti.!A1’2 Solvent System
R f Values*
14 11 52 66 71 1
10 8 40 9 12 46
0 0.14 0.68 0.71 0.75 0.80 0.85 0.86 0.87 0.88 0.90 0.95
K a t i m a t e d from drawing.
7.5 L i t e r a t u r e
Cited
1.
I . R . S h i m i , G . M . Imain and B . M . Haraun, J . A n t i b i o t i c s (Tokyo) 2 2 ( 1 9 6 9 ) 1 0 6 - 1 1 1 .
2.
I . R. S h i m i , p e r s o n a l communication.
562 8 . Gluconimycin
8 . 1 Introduction 8.1.1
Producing organism
S.theptomyce.4 AS 9 . 8.1.2
Chemical d e s c r i p t i o n
Orange powder, m.p. 2 0 0 - 2 0 2 O , C 5 5 . 7 6 , H 6 . 5 5 , N 1 0 . 8 8 , Gluconimycin i s s o l u b l e i n w a t e r , a l c o h o l s , a c e t o n e , e t h y l and b u t y l a c e t a t e and c h l o r o f o r m b u t i s i n s o l u b l e i n petroleum e t h e r . I t reduces a l k a l i n e potassium permanganate b u t g i v e s a n e g a t i v e r e a c t i o n w i t h a c i d i c p o t a s s i u m permanganate, Molisch, F e h l i n g , n i n h y d r i n , M i l l o n , Sakaguchi and f e r r i c g h l o r i d e s o l u t i o n s . Hydrolysis with 6 N hydrochloric a c i d a t 1 0 5 f o r 2 4 h r g a v e g l u c o n i c a c i d and s i x n i n h y d r i n p o s i t i v e compounds; one of them was i d e n t i f i e d by PC as g l y c i n e , a n o t h e r as l e u c i n e o r i s o l e u c i n e . I R and UV s p e c t r a were reported', 0 2 5 . 4 1 and Fe 1 . 4 0 % .
8.2 Biological
activity
Gluconimycin i s a c t i v e i n v i a 2 0 a g a i n s t many g r a m - p o s i t i v e and g r a m - n e g a t i v e b a c t e r i a and f u n g i b u t e x h i b i t e d t o x i c i t y i n mice a t d o s a g e s g r e a t e r t h a n 6 m g / k g l . I t i n t e r f e r e s w i t h a s p a r t a t e c a r b a m o y l t r a n s f e r a s e and h e n c e w i t h t h e f o r m a t i o n of thymine2. 8.3 Isolation The f i l t e r e d b r o t h was e x t r a c t e d w i t h e t h y l a c e t a t e a t pH 7 . 0 , t h e e x t r a c t was washed w i t h sodium c a r b o n a t e b u f f e r a t pH 1 0 . 0 , s e v e r a l t i m e s w i t h w a t e r and was c o n c e n t r a t e d t o d r y ness. The r e s i d u e was r e d i s s o l v e d i n w a t e r a n d g l u c o n i m y c i n c o u l d be e x t r a c t e d w i t h c h l o r o f o r m . A d d i t i o n of p e t r o l e u m e t h e r t o t h e c o n c e n t r a t e d chloroform e x t r a c t p r e c i p i t a t e d gluconimycir as a y e l l o w i s h brown powder.
8 . 4 Chromatographic
c h a r a c t e r i z a t i o n of g l u c o n i m y c i n by PC TABLE 22
S o l v e n t System
Rf Values
1
0.75 0.95 0.48 0.91 0.89 0.89 0.89 0.96 0.00 0.94 0.95
11 66 12 40
67 71
73 14 47 52
563
8.5 L i t e r a t u r e
Cited
1.
I . R . S h i m i a n d A . Dewedar, A r c h . M i k r o b i o l . 5 4 ( 1 9 6 6 ) 246-252.
2.
I . R. Shimi, p e r s o n a l communication.
564 9.
Ferramidochloromycinl 9 . 1 Introduction -
9.1.1
P r o d u c i n g organism
StheptomyccA AS 1 3 , 9.1.2
Chemical d e s c r i p t i o n
S m a l l b r o w n i s h - g r a y p l a t e s , m.p. 99’ ( d e c . ) , C 4 5 . 6 , H 6 . 0 , N 9 . 9 , C 1 2 . 0 2 , S 0.99 a n d F e 11.3%, c o r r e s p o n d i n g t o C127H201C12Fe7N24070S. F e r r a m i d o c h l o r o m y c i n i s s p a r i n g l y s o l u b l e i n water, s o l u b l e i n a l c o h o l s b u t i n s o l u b l e i n e t h y l - a n d b u t y l a c e t a t e s , benzene, d i e t h y l e t h e r , chloroform and petroleum ether. IR and UV s p e c t r a were r e p o r t e d ’ , F e r r a m i d o c h l o r o m y c i n g i v e s a p o s i t i v e n i n h y d r i n t e s t and r e d u c e s p o t a s s i u m permanganate b u t shows no r e a c t i o n w i t h Fehl i n g s o l u t i o n or Molisch, Millon and Sakaguchi reagents. Hydrol y s i s with 6 N hydrochloric a c i d a t 1 0 5 f o r 24 hr l i b e r a t e d 7 amino a c i d s ; 6 of them were i d e n t i f i e d by PC as g l y c i n e , a l a n i n e , c y s t e i n e , v a l i n e , l e u c i n e and l y s i n e .
9.2 Biological
activity
F e r r a m i d o c h l o r o m y c i n i s a c t i v e p r i m a r i l y a g a i n s t gramp o s i t i v e b a c i l l i and c o c c i and e x h i b i t s a n t i f u n g a l a c t i v i t y a g a i n s t C a n d i d a aLb.icanA a n d , t o a l e s s e r e x t e n t , a g a i n s t
SacchatomyceA C e t e v i A i a e . An LD50 v a l u e o f 50 mg/kg ( m i c e , i v ) h a s b e e n r e p o r t e d and no a c u t e t o x i c i t y was o b s e r v e d a t d o s a g e l e v e l s o f 1 5 0 mg/kg by t h e s c r o u t e , 9.3 I s o l a t i o n The f i l t e r e d b r o t h was e x t r a c t e d w i t h 1 - b u t a n o l a t pH 3 . 0 , t h e e x t r a c t was washed r e p e a t e d l y w i t h c a r b o n a t e b u f f e r , pH 9 . 0 , and w i t h water and was c o n c e n t r a t e d t o d r y n e s s . The r e s i d u e was e x t r a c t e d w i t h e t h a n o l , t h e r e s u l t i n g s o l u t i o n was f i l t e r e d and d r i e d t o g i v e a n amorphous powder which was washed w i t h e t h y l a c e t a t e , b u t y l a c e t a t e and c h l o r o f o r m , r e d i s s o l v e d i n e t h a n o l and a l l o w e d t o c r y s t a l l i z e .
9.4 C h r o m a t o g r a p h i c
c h a r a c t e r i z a t i o n of ferramidochloromycin
by d e s c e n d i n g PC TABLE 23 S o l v e n t System
Rf Values
1 3 12 23
0.05 0.0 0.0 0.0
10 7
0.76
38
0.89 0.48
17 52 57
0.85 0.23 0 -45
565
9.5 Literature 1.
Cited
I . R . S h i m i and S . Shoukry, J . A n t i b i o t i c s (Tokyo) S e r . A 19 (1966) 110-114.
566
10
-
Sideramines
10.1 Introduction I n c o n t r a s t t o t h e s i d e r o m y c i n s , s i d e r a m i n e s have been found i n a l a r g e v a r i e t y of microorganisms and even i n p l a n t s a n d h a v e b e e n s t u d i e d r a t h e r s u c c e s s f u l l y . A summary of t h e most i m p o r t a n t s i d e r a m i n e s , and t h e i r r e s p e c t i v e p r o d u c e r s and p r o p e r t i e s is given i n Table 24. According t o t h e i r s o u r c e and chemistry t h e major s i d e r a m i n e s c a n b e d i v i d e d i n t o two g r o u p s :
10.1.1
S i d e r a m i n e s p r o d u c e d by f u n g i a n d c o n t a i n i n g t h r e e N>-hydroxyorni t h i n e m o i e t i e s
F u s a r i n i n e B (1) a n d f u s i g e n ( 2 ) a r e c l o s e l y r e l a t e d c h e m i c a l l y a n d a r e d i s t i n g u i s h e d by Two o r t h r e e e s t e r b o n d s , respectively, linking three cia-fusarinine unitsz8.
FUSARININE B Fe ( I l l ) - CHELATE
(1)
FUSIGEN
(2)
Coprogen ( 3 ) a n d c o p r o g e n B ( 3 ) are composed of t h r e e t m n d - f u s a r i n i n e u n i t s , two o f which a r e l i n k e d t o form a d i k e t o p i p e r a z i n e g i v i n g r i s e t o a d i m e r i c - a c i d m o i e t y ( E l . ThanAf u s a r i n i n e and d i m e r i c a c i d have i n f a c t been found i n c u l t u r e f i l t r a t e s o f f u n g i p r o d u c i n g c o p r o g e n B4. I n t e r e s t i n g l y , t h e r a t e o f F e ( I I 1 ) - c h e l a t e u p t a k e b y Neu/toApOha ChaAAU c o u l d b e e n h a n c e d by r e p l a c e m e n t o f t h e N - a c e t y l g r o u p o f c o p r o g e n w i t h o t h e r a c y l g r o u p s 3 0 . Coprogen e x h i b i t s c a . 1% of t h e a c t i v i t y of ferrichrome i n t h e antagonism t e s t w i t h Staphylococcud
auReuA 1.
TPBLE 24
I
I
I I I I
Arthmbacwrpaau
Bcillusmap.U~niWn
Arpqi/lus nidulnr Awm%+llus h w n b l a 10 9
Ferrichwsin
Q9n4BF$o14Fe
10
10
FerrihromeA
q 1 y B N g o M F s 1.11.26
29
11
Ferrirubin
q1H&&7Fe
1.11
11
12
Fnrirhcdin
qlkN&Fe
1.11
11
1
28.29 28,28, 30
11
29.26
28,29
!itrunurn unknwm. micmbisl
m-
(hctw with farioumine activity
Aq*rgilusdh Aqmqillus ~(MM
Micmbial mwth-factor. rideromkcin antagonin Micmbd pmwth-factor.rideromycin antagonist
Ustilw & r r o p n a
No oppplrentbiological ativity
Paicillium vailbie SMcwla ap Prrcilomycsr v r i o t i
Microbial wowlh-fnctor. sideromycin
~
Microbial growth-fanor.ridsromycin antagonist
I
I varrim/or m
Alp.rpUlur niduhs
1
ImmnqsOR .esm, D e l M i i i r i o n huw. a n amagonizeantibiotic activitv of fsrrimycin A
m n i m
I
Albomycin
Griain
1. 10 1.10
-
3.10
1, 10
TABLE 24 (continued) SIDERAMWE
WWOSlTION
FerrioxamineAl
G4H43NbogFa
FenioxamirW~ I+3&1N&Fe
IR
mmuciffi OR(ILJIISTA
13
31.32.33
13
31,32,33
-
15
FerrioxamineB
31,32
+ t$+ iN j &Fe
33-34
FmioxaminsD1
%&7N&gFe
Ferrioxamim D2
Q6&WgFc
14
13
18
,EF.
aicmbial aowh-factor. sideromycin ~ n ankt
2.
l i a o b i i l gmwd-facmr. sideromycin anagonin. cowaim 2 I-minoehvdroxvlmlmbutana moietii
12.
fiiwbial growth-factor. sidsmmvcin intaoonin
I. 6 12.
I3
K
12
31,32
Fenioamine C
16
strepmnycsrpilaur
SIMROMYCIN 4NALOG
REMARKS
31.32, 33,34
Wicmbial gowthfmor. demmycin mtagQnirt
31,32
Wiaabial g h f s c t w . ridnmvcin antqonist
31,32,34
Micmbial Wowthfactor. ridsromycin
antagmist
:nrimycin
12. 14.1
12. 13
1.6.
12,
25. 20
FerrioxamineF
17 ~
18
-1
Nooldmine
@7H48N$)g
39
31
Miaobiil gowthfactor. sidsrmycin magnin, nructuro unknown. basic, BiVa poritiw ninhydrin-mst
31.33
Cqxoducad with fenioxamine
B
Has antibiotic activity, identical W i t h dafeniferrioxanins E
12
20.16
24
17.18 19,20 21,39
-
569
4t' 0
H N
COPROGEN : R = COCH3
(3)
COPROGEN B : R = H
(4)
Containing N5-hydroxyornithine m o i e t i e s , r h o d o t o r u l i c a c i d ( S ) and d i m e r i c a c i d ( 6 ) a r e dihydroxamic a c i d s and a r e , t h e r e f o r e , n o t f o r m a l members o f s i d e r a m i n e s . Whereas S p o s s e s s e s some of t h e b i o l o g i c a l p r o p e r t i e s of s i d e r a m i n e s and r e s e m b l e s s c h i z o k i n e n 5 i n L a n k f o r d ' s Bacillu4 t e s t s y s tern5, 5 i s known as a f e r r i m y c i n - and albomycin a n t a g o n i s t 4 .
RHODOTORULIC D l M E R l C ACID
ACID :
R =
H
:
R = CH3
(5)
~ ~ ~ 2 - C H 2 (6) 0 H 3
A unique group of s i d e r a m i n e s of f u n g a l o r i g i n a r e t h e f e r r i c h r o m e - t y p e h e x a p e p t i d e s 1-12.F e r r i c h r o m e (1) was s t u d i e d f i r s t , i t s s t r u c t u r e was i n v e s t i g a t e d 3 1 and t h e a s s i g n m e n t conf i r m e d by I n c o n t r a s t t o 2, f e r r i c h r o m e A (?) a p p a r e n t l y l a c k s g r o w t h - f a c t o r a c t i v i t y and was t h e f i r s t s i d e r o chrome t o r e c e i v e a c r y s t a l s t r u c t u r e a n a l y s i s r e v e a l i n g t h e absolute c ~ n f i g u r a t i o n ~ ~ .
5 70.
s-5
0
K
a
f a I
0 LL
5 71 The amino a c i d a r r a n g e m e n t o f s i d e r a m i n e s o f t h e f e r r i chrome t y p e a p p e a r s t o b e u n i q u e i n t h a t t h e t h r e e N5-hydroxyo r n i t h i n e moieties a r e linked i n sequence; consequently, f e r r i c r o c i n (81, f e r r i c h r y s i n (lo), f e r r i r u b i n (11)a n d f e r r i r h o d i n (12)d i f f e r o n l y w i t h r e s p e c t t o t h e t h r e e r e m a i n i n g a m i n o a c i d s a n d t h e a c y l g r o u p s a t t h e hydroxyamino f u n c t i o n s o f t h e N 5 hydroxy-ornithine u n i t s 2 . I n t e r e s t i n g l y , f e r r i r h o d i n and f e r r i r u b i n c o n t a i n N 5 - h y d r o x y o r n i t h i n e m o i e t i e s i n t h e form o f c h f u s a r i n i n e a n d a a n 4 - f u s a r i n i n e , r e s p e c t i v e l y , which may a c t u a l l y s e r v e as b i o c h e m i c a l p r e c u r s o r s 7 . A l t h o u g h a v a r i e t y o f a c y l m o i e t i e s a r e e n c o u n t e r e d among the ferrichrome-type sideramines, only a c e r t a i n a c y l type is p r e s e n t i n a given sideramine. Ferrichrome, f e r r i c r o c i n and f e r r i c h r y s i n are d i s t i n g u i s h e d by N5-acetyl-N5-hydroxyornithine u n i t s and t h u s resemble albomycinZ8 i n t h i s s t r u c t u r a l a s p e c t . S i d e r a m i n e s o f t h e f e r r i c h r o m e - t y p e show c o m p l e t e a n d c o m p e t i t i v e a n t a g o n i s m of a l b o m y c i n w i t h b o t h g r a m - p o s i t i v e a n d g r a m - n e g a t i v e b a c t e r i a b u t a n t a g o n i s m o f f e r r i m y c i n was o n l y s e e n w i t h g r a m - p o s i t i v e b a c t e r i a s u c h as S t a p h y t o c o c c u 4 QuReuA S i d e r a m i n e s 9, 11 a n d l2, p o s s e s s i n g a c y l g r o u p s o t h e r t h a n a c e t y l a n d t h u s d z e r g i n g f u r t h e r from t h e s t r u c t u r a l f e a t u r e s o f a l b o m y c i n , a r e e i t h e r i n a c t i v e o r much l e s s a c t i v e i n t h e a n t a g o n i s m t e s t t h a n t h e i r a c e t y l - b e a r i n g c o u n t e r p a r t s 2, 8 and 1 0 . More s p e c i f i c a l l y , t h e a n t a g o n i s t i c a c t i v i t y o f 2 a n d gy a i n s t f e r r i m y c i n A i s ca. 1 0 % o f t h a t e x h i b i t e d by 1, 8 a n d ,f’ll.
-
10.1.2
S i d e r a m i n e s p r o d u c e d by a c t i n o m y c e t e s a n d c o n t a i n i n g t h r e e amino- hy droxyami n o a l k a n e mo i e t i e s
The r e p r e s e n t a t i v e s o f t h i s g r o u p o f s i d e r a m i n e s a r e d e s i g n a t e d as f e r r i o x a m i n e s a n d w e r e s u b j e c t t o e x t e n s i v e b i o l o gica16’26 and chemical2 i n v e s t i g a t i o n s . Surprisingly, ferrioxamines a n t a g o n i z e t h e a n t i b i o t i c a c t i v i t y o f s i d e r o m y c i n s o f t h e a l b o m y c i n - g r i s e i n t y p e o n l y a g a i n s t gram-posi t i v e b a c t e r i a z 6 . S t r u c t u r a l l y s i m p l e r a n d q u i t e d i f f e r e n t from f e r r i c h r o m e type sideramines, t h e ferrioxamines resemble f e r r i m y c i n w i t h r e s p e c t t o t h e i r o n - b i n d i n g c e n t e r . F e r r i o x a m i n e s are chemic a l l y c l o s e l y r e l a t e d and f a l l i n t o d i s t i n c t groupsz8 according to: 1. L i n e a r i t y o r c y c l i c i t y o f t h e c a r b o n - n i t r o g e n skeleton. 2. P r e s e n c e o f 1-amino-4-hydroxyaminobutane o r l - a m i n o 5-hydroxyaminopentane m o i e t i e s . 3. Nature o f t h e t e r m i n a l of t h o s e w i t h l i n e a r carbonnitrogen skeleton.
(s)
a n d D 2 (2) a r e d i s t i n g u i s h e d by Ferrioxamines A1 t h e p r e s e n c e o f o n e 1-amino-4-hydroxyaminobutane a n d two 1amino-5-hydroxyaminopentane u n i t s p e r m o l e c u l e ; A 1 i s l i n e a r a n d c o n t a i n s a t e r m i n a l a c e t y l g r o u p w h e r e a s D, i s c y c l i c w i t h three succinyl groups p e r molecule.
572
FERRIOXAMINE A ,
FERRIOXAMINE 0 2
(2)
Ferrioxamines B and D, (16) 3re most c l o s e l y r e l a t e d t o f e r r i m y c i n , e x h i b i t i n g l i n e a r skeletons, x r e e 1-amino-5-hydroxylarninqentene residues, and a t e r m i n a l a c e t y l group p e r molecule,
FERRIOXAMINE B : R = H FERRIOXAMINE
D,
3
R = COCH,
(15) (16)
573
Ferrioxamines G (17) and E (18) differ only with respect to linearity and cycliciTy, respec-f-ively.
FERRIOXAMINE G
NOCARDAMINE Fe (IlI)-CHELATE (FERRIOXAMINE E 1
10.2 Biological activities The biological properties of sideramines have been reviewed, pertinent references and principal biological activities are contained in Table 24. Of particular interest, however, is nocardamine, originally detected because of its antibiotic proper tie^'^,
Nocardamine is active specifically against mycobacteria and is inactive against staphylococci, streptococci, coli-, typhi- and dysentery bacteria as well as yeasts and fungi. The antibiotic action of nocardamine is primarily of bacteriostatic nature; bacteriocidal activity was only observed at high drug doses. Deferri-ferrioxamine B inhibits E. c o C i mobility, reversing the stimulating effect of cyclic adenosine37 and its Nmethanesulfonyl derivative (Desferal) is used in the treatment of iron intoxication and iron-storage diseases such as haemochromatosis and haemosiderosis. 10.3 Isolation 10.3.1 Ferrichrome-type sideramines
The culture filtrate was mixed with enough aqueous ferric sulfate solution to assure the complete Fe(II1)-chelation of all
574 s i d e r a m i n e s p r e s e n t and was e x t r a c t e d w i t h p h e n o l - c h l o r o f o r m , 1:1, w / v . The combined o r g a n i c e x t r a c t s were d i l u t e d w i t h c a . t w i c e t h e i r volume of e t h e r and a d d i t i o n of water p e r m i t t e d t h e e x t r a c t i o n of t h e s i d e r a m i n e s i n t o t h e aqueous p h a s e . The aqueous phase was e x h a u s t i v e l y e x t r a c t e d w i t h e t h e r t o remove p h e n o l , c o n c e n t r a t e d and f r e e z e - d r i e d t o y i e l d c r u d e s i d e r a m i n e . F e r r i c h r o m e and f e r r i c h r o m e A c o u l d be e x t r a c t e d i n a similar f a s h i o n from ammonium s u l f a t e s a t u r a t e d b r o t h f i l t r a t e w i t h benzyl alcohol29. F u r t h e r p u r i f i c a t i o n was a c h i e v e d by C r a i g - d i s t r i b u t i o n w i t h s y s t e m s 8312, 8912, 8611 o r 871°. D i s t r i b u t i o n numbers a r e summarized i n Table 28. To i s o l a t e t h e p u r e s i d e r a m i n e s from t h e a p p r o p r i a t e t u b e s , t h e p o o l e d C r a i g - f r a c t i o n s were d i l u t e d w i t h e t h e r t o f o r c e t h e pigments i n t o t h e aqueous phase which was s e p a r a t e d . A d d i t i o n a l aqueous e x t r a c t s were added t o t h e o r i g i n a l aqueous p h a s e , 1 0 % sodium c h l o r i d e ( w / v ) was added and t h e s i d e r a m i n e e x t r a c t e d w i t h p h e n o l - c h l o r o f o r m 1:1, w/v. The e x t r a c t s were washed w i t h 1 0 % aqueous sodium c h l o r i d e s o l u t i o n , c l a r i f i e d w i t h C e l i t e , d i l u t e d w i t h a t w o - f o l d volume of e t h e r and shaken w i t h s e v e r a l p o r t i o n s of water. The combined aqueous p h a s e s were r e p e a t e d l y e x t r a c t e d w i t h e t h e r and concent r a t e d t o d r y n e s s . Some sideram.ines c o u l d be o b t a i n e d i n c r y s t a l l i n e form from s o l v e n t s such a s methanol o r e t h a n o l . 10.3.2 10.3.2.1
Ferrioxamines Crude f e r r i o x a m i n e 1 2
The b r o t h ( 2 0 a ) was mixed w i t h H y f l o - S u p e r c e l (400 g ) and 1 0 % aqueous f e r r i c s u l f a t e s o l u t i o n (200 m l ) , s t i r r e d and filtered. The f i l t r a t e was e x t r a c t e d w i t h p h e n o l - c h l o r o f o r m ( l : l ,w / v , 2 a f t e r a d d i t i o n of sodium c h l o r i d e ( 3 . 6 k g ) , t h e e x t r a c t was d r i e d (sodium s u l f a t e ) and added t o a s t i r r e d s u s p e n s i o n of H y f l o - S u p e r c e l ( 2 0 0 g ) i n e t h e r ( 2 a ) and p e t r o l e u m e t h e r (10 1 ) . The r e s u l t i n g s o l i d s were f i l t e r e d o f f , washed w i t h e t h e r ( 2 k ) and e l u t e d r e p e a t e d l y w i t h methanol ( 5 x 6 0 0 ml). E v a p o r a t i o n of t h e e x t r a c t s y i e l d e d c r u d e f e r r i o x a m i n e as a brown-red powder (10 g ) , 10.3.2.2
P r e l i m i n a r y s e p a r a t i o n by C r a i g - d i s t r i b u t i o n ’ *
Crude f e r r i o x a m i n e ( 4 g ) was d i s t r i b u t e d i n s y s t e m 8 9 ( 7 9 t r a n s f e r s , 1 0 0 m l upper and 1 0 0 m l lower p h a s e ) y i e l d i n g 5 f r a c t i o n s ( T a b l e 2 5 ) a s d e t e r m i n e d by t h e B o n i f a s t e s t and a b s o r b a n c i e s a t l t 2 5 nm ( 2 m l of upper and lower phase were t a k e n from a g i v e n t u b e , d i l u t e d w i t h 3 2 m l of methanol and used f o r both tests). The f e r r i o x a m i n e s were i s o l a t e d from t h e p o o l e d t u b e c o n t e n t s by a d d i t i o n o f an e q u a l volume of p e t r o l e u m e t h e r f o l lowed by washing of t h e deep-red aqueous phase w i t h c h l o r o f o r m and e x h a u s t i v e e x t r a c t i o n w i t h phenol-chloroform ( l : l ,w/v) a f t e r a d d i t i o n o f sodium c h l o r i d e t o t h e aqueous phase ( l o % , w/v). The p h e n o l - c h l o r o f o r m e x t r a c t was washed r e p e a t e d l y w i t h 0 . 0 1 M h y d r o c h l o r i c a c i d c o n t a i n i n g 1 0 % sodium c h l o r i d e , f i l t e r e d through a s h o r t column of C e l i t e ( 2 0 g ) and mixed w i t h
575 TABLE 25
Craig-Distribution o f Crude Ferrioxamine After 79 T r a n s f e r s (System 89) F r a c t i o n No.
Tube No. 0-5 6-18 19-32 33-62 63-80
Major Ferrioxemines
-
I I1 I11 IV
A1sA2
B
C
v
D 1 f D2, E, F
H y f l o - S u p e r c e l ( 8 5 g ) , e t h e r ( 5 0 0 m l ) a n d p e t r o l e u m e t h e r (1 a ) by s t i r r i n g a t 0 The s o l i d s w e r e washed w i t h e t h e r a n d e l u t e d w i t h methanol. E v a p o r a t i o n of t h e e x t r a c t y i e l d e d t h e i n d i v i d ual ferrioxamine fractions.
.
10.3.2.3
F e r r i o x a m i n e B by i o n - e x c h a n g e c h r o m a t o g r a p h y 1 2
S t a r t i n g m a t e r i a l : F r a c t i o n No. I11 ( T a b l e 2 5 1 , 865 mg. Column: Dowex 50W-X2, 1 0 0 - 2 0 0 mesh, 30 mm x 6 6 0 mm. M o b i l e p h a s e : Ammonium a c e t a t e b u f f e r , 0 . 2 M , pH 4 . 6 ; a f t e r 72 h r change t o g r a d i e n t e l u t i o n u s i n g a 1 - l i t e r m i x i n g chamber f e d b y ammonium a c e t a t e b u f f e r , 2 . 0 M , pH 4 . 7 . Flow r a t e : 0 . 2 3 cm/min. 10.3.2.4
F e r r i o x a m i n e A by i o n - e x c h a n g e c h r o m a t o g r a p h y 1 2
F r a c t i o n No. I1 ( T a b l e 251, 5 g . S t a r t i n g material: Column: Dowex 50W-X2, 100-200 mesh, 53 mm x 9 0 0 mm. Mobile p h a s e : Ammonium a c e t a t e b u f f e r , 0 . 1 M , pH 4 . 6 ; a f t e r e l u t i o n of c a . 3 . 4 a c h a n g e t o g r a d i e n t e l u t i o n u s i n g a 4 - l i t e r mixing chamber f e d by ammonium a c e t a t e b u f f e r , 0 . 9 M , pH 4 . 6 0 , a n d by ammonium a c e t a t e b u f f e r , 1 . 7 5 M , pH 4 . 7 , a f t e r e l u t i o n o f ca. 1 0 . 5 a . Flow r a t e : 0 . 0 8 3 cm/min. F e r r i o x a m i n e A was p r i m a r i l y l o c a t e d i n t h e e f f l u e n t volume b e t w e e n ca. 1 0 . 6 - 1 1 . 3 a ; t h e f o l l o w i n g l i t e r of e f f l u e n t c o n t a i n e d a m i x t u r e o f f e r r i o x a m i n e s A and B . 10.3.2.5
F e r r i o x a m i n e s A and B by C r a i g - d i s t r i b ~ t i o n l ~
The m i x t u r e o f f e r r i o x a m i n e s A a n d B , as o b t a i n e d by c h r o m a t o g r a p h y on Dowex 5OW-X2, was s e p a r a t e d by C r a i g - d i s t r i b u t i o n e m p l o y i n g s y s t e m 85. I n s t r u m e n t : 200 t u b e s . Mode: Recycling process, 874 t r a n s f e r s . Z i o x a m i n e A h y d r o c h l o r i d e : C = 0.12 ( t u b e s 71-116,
r* = 9 1 )
Ferrioxamlne B hydrochloride: C = 0.21 (tubes 130-173,
r* = 1 5 1 ) .
576 The s i d e r a m i n e s were i s o l a t e d f r o m t h e C r a i g - f r a c t i o n s i n t h e same f a s h i o n as d e s c r i b e d f o r f e r r i c h r o m e - t y p e s i d e r a m i n e s . 10.3.2.6
S e p a r a t i o n o f f e r r i o x a m i n e s A 1 a n d A 2 as t h e i r N-acetyl d e r i v a t i v e s L 5
F e r r i o x a m i n e A h y d r o c h l o r i d e ( 6 0 0 mg) a n d a n h y d r . s o d i u m a c e t a t e ( 1 0 0 mg) w e r e d i s s o l v e d i n m e t h a n o l ( 5 m l ) a n d a c e t i c a n h y d r i d e ( 5 m l ) was a d d e d . The m i x t u r e was e v a p o r a t e d t o d r y n e s s a f t e r 1 4 h r a n d c o u l d b e s e p a r a t e d by C r a i g - d i s t r i b u t i o n i n s y s t e m 85 a f t e r 339 t r a n s f e r s . N-acetylferrioxamine Al: N-acetylferrioxamine 10.3.2.7
A2:
C = 1 . 3 1 ( t u b e s 179-208, r* 193) C = 0 . 7 3 ( t u b e s 133-158, r* 143)
F e r r i o x a m i n e C by i o n - e x c h a n g e c h r o m a t o g r a p h y l 2
S t a r t i n g material: F r a c t i o n N o . I V ( T a b l e 2 5 ) , 1 . 4 8 g . Column: Dowex 50W-X2, 100-200 mesh, 53 mm x 9 0 0 mm. Development: Ammonium a c e t a t e b u f f e r , 0 . 1 M, pH 4 . 6 ; a f t e r e l u t i o n o f ca. 2 7 a c h a n g e t o g r a d i e n t e l u t i o n u s i n g a 4 - l i t e r m i x i n g chamber f e d by ammonium a c e t a t e b u f f e r , 0 . 9 M , pH 4 . 6 0 , a n d 1 . 7 5 M b u f f e r , pH 4 . 7 , a f t e r e l u t i o n o f 36 k. F e r r i o x a m i n e B ( c a . 2 9 . 3 - 3 1 . 1 k ) emerged f r o m t h e column immediately b e f o r e f e r r i o x a m i n e C (ca. 32.8-33.8 a ) . 10.3.2.8
Ferrioxamines D1,D2, chromatographyiz
E a n d F by i o n - e x c h a n g e
S t a r t i n g material: F r a c t i o n No. V ( T a b l e 2 5 ) . Column: Dowex 50W-X2, 1 0 0 - 2 0 0 mesh, 5 6 . 6 mm x 1 5 0 mm. Development: 0 . 1 M ammonium a c e t a t e b u f f e r , pH 4 . 7 , ca. 6 0 a ; 0 . 2 M ammonium a c e t a t e b u f f e r , pH 4 . 7 , ca. 6 0 k; 0 . 6 M ammonium a c e t a t e b u f f e r , pH 4 . 7 , c a . 6 0 8 ; 1 . 8 M ammonium a c e t a t e b u f f e r , pH 4 . 7 , ca. 2 5 0 & . Flow r a t e : c a . 0 . 5 3 cm/min. ( 2 - 3 k / h r ) . F r a c t i o n s of 5 k e a c h were c o l l e c t e d , e f f l u e n t volume 1 0 - 2 5 & ( f r a c t i o n s 2-51 c o n t a i n e d m o s t l y f e r r i o x a m i n e s D,, D 2 a n d E (44 g ) , t h e volume 240-275 & ( f r a c t i o n s 4 8 - 5 5 ) p r i m a r i l y f e r r i o x a m i n e F ( 8 . 4 g). F r a c t i o n s 2 - 5 were r e c h r o m a t o g r a p h e d as d e s c r i b e d i n section 10.2.3.4 but without gradient e l u t i o n . Ferrioxamines D 1 a n d D 2 a p p e a r e d i n t h e e f f l u e n t volume 1 . 8 - 2 . 1 k, f e r r i o x amine E i n 2 . 5 - 3 . 0 k. The m i x t u r e o f f e r r i o x a m i n e D 1 a n d D 2 ( 9 8 mg), i s o l a t e d f r o m t h e e f f l u e n t , was d i s s o l v e d i n water ( 1 0 0 m l ) , t h e s o l u t i o n was s a t u r a t e d w i t h sodium c h l o r i d e a n d e x t r a c t e d twice w i t h a n e q u a l volume o f c h l o r o f o r m . The d r i e d c h l o r o f o r m e x t r a c t ( s o d i u m s u l f a t e ) g a v e f e r r i o x a m i n e D1 on e v a p o r a t i o n ( 6 9 7 mg). The a q u e o u s p h a s e was e x t r a c t e d e x h a u s t i v e l y w i t h c h l o r o f o r m a n d worked up by p h e n o l - c h l o r o f o r m e x -
577 tractions to furnish ferrioxamine D2 (95 mg). Fractions 48-55 were rechromatographed as described in section 10.3.2.4 starting with 0.5 M ammonium acetate, pH 4.7, and changed to gradient elution with 4-liter mixing chamber fed by 1.75 M ammonium acetate buffer, pH 4.7, after elution of ca. 25 a . Ferrioxamine F was isolated from the effluent volume of 30.8-33.0 a . Two Craig-distributions in 1-butanol-water (19 and 29 transfers) were used for final purification. 10.3.2.9
Ferrioxamine G by ion-exchange chromatographyz4
Starting material: Crude ferrioxamine B preparation ( 4 g). Column: Dowex 50W-X2, 100-200 mesh, 30 mm x 570 mm. Development: Ammonium acetate buffer 0.1 M, pH 4.6-4.7, eluting ferrioxamine G , and ammonium acetate buffer, 0.3 M, eluting ferrioxamine B. 10.3.2.10
N~cardamine’~
Whole fermentation broth (22 1 ) was stirred with 1-butanol (10 a ) and the phases were separated by centrifugation. The aqueous phase was extracted four times with 5 L of 1-butanol each, the butanol extracts, ranging from wine-red to yellow, were combined and concentrated under reduced pressure. The resinous, dark-red residue was triturated with ether to afford a powder (ca. 22 g). The crude preparation was dissolved in a 20-fold quantity of hot water and the turbid solution filtered through talcum retaining red-brown resins. Nocardamine was deposited as needles from the yellow to light brown-red filtrate, (7.7 g). Further purification was possible by recrystallization from a 50-fold quantity of hot water or water-saturated 1-butanol; strongly colored solutions could be decolorized with very smail quantities of charcoal. Pure nocardamine showed m.p. 183-184
.
10.4 Chromatographic characterization of sideramines TABLE 26 Chromatographic Characterization o f Coprogens, Dimeric Acid and Tkm&fusarinine by TLC on S i l i c a g e l G4
Rf VdUeS Compound Coprogen Coprogen B Coprogen C Dimeric. a c i d Fe(1II) complex ‘Taw-fusarinine Fe( 111) complex
System 35 0.27
System 36
System 54
System 42
0
0.76 0.45,0.56 0.84
0.18 0.07 0.04
0.25 0.11 0.18
0
0.80
0.07
0.13
0
0.73
0.02
0.05
0.18
578 TABLE 27 Chromatographic C h a r a c t e r i z a t i o n o f R h o d o t o r u l i c A c i d by PC on Whetmen No. 1 Paper5 Solvent System
R f Values
93 41 54
0.10 0.73 0.63 0.62 0.42 0.52
81** 18 44 TABLE 28
P C 1 and C r a i g - d i s t r i b u t i o n 1 ' lo' l1 of
Various Sideramines
Rf Values S i deramine Ferrichrome A Ferrichrom F e r r ic h r y s i n Ferricrocin Coprogen Fe r r irubin Ferrirhodin
C Values
System 54* System 81**
0.28 0.26 0.29 0.43 0.51 0.61
-
System 86
-
1.19 1.0 0.62 0.55
0.30 0.34 0.28 0.55 0.73 ca. 0.9
System 87
1.17 0.61
-
1.32
-
5.1 7.3
7.3
TABLE 29 PC o f Various Sideramines
Rf Values Sideramine Fe r r ichrome Ferrichrome A F e r r i ch r y s i n F e r r ir u b i n F e r r ir h o d i n
System 54" 0.28 0.28 0.26 0.52 0.61
System 81** 0.29 0.38 0.34 0.81
0.88
TABLE 30
PC o f F e r r i c h r ~ m e a ~ ~ Rf Values Sideramine Ferrichrome Ferrichrom A
System 52 0.28 0.03
System 15 0.77 0.45
5 79 .-TABLE 31
Physico-chemical Data o f Various Ferrioxamines l 2 ' 2 o 2 6 Craig Distri bution C-value i n Syst. 83
-
Ferrioxamine
Paper Electrophoresis
A
9.7
8 C D1 02
9.1 8.4 0
-
E
F C
0 0
Rf Values (PC)
Syst.
Syst.
Syst.
0.35
0.21
0.44
0.29
0.45 0.53
0.54
0.37
0.73 0.64
0.48
-
0.59 0.80 0.26
-
52 81** 21 -
0.72
0.68
8.6
0.63 0.40
Absorbency a t 430 nm $7; l c m (H70) 37 39 39
0.11 0.23 0.49 1.80 0.86 1.59 3.12 0.40
-
-
Pk * MCS 9.89 9.74 8.88
634
9.75 5.79; 10.53
695
-
44
-
42 34
-
TABLE 32 Deacending PC o f Various Ferrioxernines2
Rf Values Ferrioxamine
System 53
System 2 1
0.32
0.45 0.53
0.42
-
0.59 0.71
-
0.84 TABLE 33 PC end C r a i g - d i s t r i b u t i o n o f Various Ferrioxamines and D e r i v a t i v e s 2 4 Bl 3 Rf values
Ferrioxamine
G HC1 B HC1 N-acetyl N-acetyl ester Dl A N-acetyl N-acetyl
System 52
0.40 0.43 G G,
0.77
0.26 0.32 0.86
i n System 85
0.21
-
Me 0.68 0.72
A1 A2
C Values
System 81%'
-
0.76 0.77 0.25 0.68 0.60
Equiv.
wt. -
0.12 1.31 0.73
704 762
-
580 TABLE 34 Comparison o f Various Ferrioxsmines by PC and TLCZ5
R,= Values Ferrioxemine
TLC System 54 System 36
B D1 D 1 *** E***
0.05 0.12 0.12 0.10
0.32 0.52
-
0.55
PC System El**
System 52
0.21
0.28 0.59 0.72 0.60
-
-
*Ascending development. **Upper phase, descending development, paper impregnated w i t h acetone-water-satd. sq. sodium c h l o r i d e s o l u t i o n , 6:3:1, v/v. ***Sample d i s s o l v e d i n a c e t i c acid.
10.5 Literature
Cited
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H . Z a h n e r , W . K e l l e r - S c h i e r l e i n , R . H G t t e r , K . HessL e i s i n g e r a n d A. D e e r , A r c h . M i k r o b i o l . 45 ( 1 9 6 3 ) 1 1 9 - 1 3 5 .
2.
W. K e l l e r - S c h i e r l e i n , V. P r e l o g a n d H. Zdhner, F o r t s c h r . Chem. Org. N a t u r s t o f f e 22 ( 1 9 6 4 ) 2 7 9 - 3 2 2 .
3.
J. B. N i e l a n d s , i n : I n o r g a n i c B i o c h e m i s t r y , G . I . Ed. Vol. 1, E l s e v i e r , N e w York ( 1 9 7 3 ) 1 6 7 - 2 0 2 .
4.
H . Diekmann, Arch. M i k r o b i o l .
5.
C . L. A t k i n a n d J . B . N i e l a n d s , B i o c h e m i s t r y 7 ( 1 9 6 8 ) 3734-3739.
6.
H. Z S h n e r , E . B a c h m a n n , R . Hiitter a n d J. N c e s c h , P a t h o l . M i c r o b i o l . 25 ( 1 9 6 2 ) 708-736.
7.
J. M. S a y e r a n d T. F. Emery, B i o c h e m i s t r y 7 ( 1 9 6 8 ) 1 8 4 - 1 9 0 .
8.
H . Diekmann a n d H . Zdhner, E u r . J . B i o c h e m . 218.
3 (1967) 213-
9.
W. K e l l e r - S c h i e r l e i n a n d H. ( 1 9 7 0 ) 2035-2044.
Chim. A c t a 5 3
Eichhorn,
73 ( 1 9 7 0 ) 65-76.
Diekmann, Helv.
10.
W . K e l l e r - S c h i e r l e i n a n d A. DeGr, H e l v . Chim. A c t a 4 6 ( 1 9 6 3 ) 1907-1920.
11.
W. K e l l e r - S c h i e r l e i n ,
12.
H . B i c k e l , R . B o s s h a n d t , E . Gaumann, P . R e u s s e r , E . V i s c h e r , W . Voser, A. W e t t s t e i n a n d H . Z z h n e r , H e l v . Chim. A c t a 4 3 (1960 1 2118-212 8.
H e l v . C h i m . A c t a 46 ( 1 9 6 3 ) 1 9 2 0 - 1 9 2 9 .
581 K e l l e r - S c h i e r l e i n , P . Mertens, V . P r e l o g a n d A. Walser, H e l v . Chim. Acta 4 8 ( 1 9 6 5 ) 7 1 0 - 7 2 3 .
13.
W.
14.
W . K e l l e r - S c h i e r l e i n a n d V . P r e l o g , H e l v . Chim. Acta 44 ( 1 9 6 1 ) 709-713.
15.
T. Emery, Biochem. 5 ( 1 9 6 6 ) 3694-3701.
16.
V . P r e l o g a n d A. Walser, H e l v . Chim. Acta 45 ( 1 9 6 2 ) 1 7 3 2 1734.
17.
A. S t o l l , A. B r a c k a n d J. R e n z , B a k t . 1 4 ( 1 9 5 1 ) 225-233.
18.
A. S t o l l , J. R e n z a n d A. B r a c k , H e l v . Chim. A c t a 34 ( 1 9 5 1 ) 862-873.
19.
R . F. C . B r o w n , G . B i i c h i , W . K e l l e r - S c h i e r l e i n , V . P r e l o g a n d J . R e n z , H e l v . Chim. Acta 4 3 ( 1 9 6 0 ) 1 8 6 8 - 1 8 7 1 .
20.
W. K e l l e r - S c h i e r l e i n ( 1 9 61) 1 9 81- 1 9 8 5 .
21.
D. v a n d e r H e l m a n d M. P o l i n g , J. A m e r . Chem. S O C . 9 8 ( 1 9 7 6 ) 82-86.
22.
M . 0 . B u r t o n , F . J. Sowden a n d A. G . B i o c h e m . 32 ( 1 9 5 4 ) 4 0 0 - 4 0 6 .
23.
A . G. 245.
24.
W. K e l l e r - S c h i e r l e i n ( 1 9 6 2 ) 590-595.
25.
A. M i l l e r a n d H . 263.
26.
H . Z z h n e r , R . Hfitter a n d E . B a c h m a n n , A r c h . M i c r o b i o l . ( 1 9 6 0 ) 325-349.
27.
B. R . B y e r s , M. (1967) 286-294.
28.
H . M a e h r , P u r e A p p l . Chem.
29.
J . A. G a r i b a l d i a n d J. B . N i e l a n d s , J . Amer. Chem. S O C . 77 ( 1 9 5 5 ) 2 4 2 9 - 2 4 3 0 .
30.
J. E r n s t a n d G. 2 71-2 8 2 .
31.
S . J . R o g e r s , R . A . J. W a r r e n a n d J. B . N i e l a n d s , N a t u r e 200 ( 1 9 6 3 ) 1 6 7 .
32.
W. K e l l e r - S c h i e r l e i n ( 1 9 6 9 ) 603-610.
L o c h h e a d a n d M.
and V.
Schweiz. Z e i t s c h r . P a t h .
P r e l o g , H e l v . Chim. Acta 4 4
L o c h h e a d , Can. J.
0. B u r t o n , S o i l S c i .
82 ( 1 9 5 6 ) 2 3 7 -
a n d V . P r e l o g , H e l v . Chim. Acta 45
Z g h n e r , A r c h . M i k r o b i o l . 62 ( 1 9 6 8 ) 2 5 7 -
V. Powell and C. E.
36
L a n k f o r d , J. B a c t . 9 3
28 ( 1 9 7 1 ) 603-636.
Winkelmann, A r c h . Microbiol. 1 0 0 ( 1 9 7 4 )
a n d B . M a u r e r , H e l v . Chim. A c t a 52
5 82 33.
A. Z a l k i n , J. D. F o r r e s t e r a n d D. H . T e m p l e t o n , J . Amer. Chem. SOC. 88 (1966) 1810-1814..
34.
J. NUesch a n d F. KnUsel, i n : A n t i b i o t i c s Vol. 1, Mechanism of a c t i o n , D. G o t t l i e b a n d P. D . Shaw, Ed., S p r i n g e r - V e r l a g N e w Y o r k I n c . 1967, pg. 499-541.
35.
H. Diekmann, Arch. M i k r o b i o l . 58 (1967) 1-5.
36.
T. Emery and J. B . N i e l a n d s , J . Amer. Chem. SOC. 82 (1960) 365 8-3662.
37.
H. B. Maruyama, H. Azuma, Y . Kotch a n d Y . S u h a r a , Antimicrob. A g e n t s and Chemotherapy (1975) 377-380.
38.
K. B . M u l l i s , J . R . P o l l a c k and J . B . N i e l a n d s , Biochemi s t r y 1 0 (1971) 4894-4898.
39.
H. Maehr, W . Benz, J . S m a l l h e e r and T. H . W i l l i a m s , Z. N a t u r f o r s c h .
T e i l B 32 (1977) 937-942.
583 11. S o l v e n t s y s t e m s u s e d for c h r o m a t o g r a p h y and C r a i g distribution System
No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
22
23 24 25 26 27 28 29 30
31 32 33 34 35 36
37 38 39 40 41 42 43 44 45 46 47 48 49 50
Conponent s water water c o n t a i n i n g 1.5% ammonium c h l o r i d e water c o n t a i n i n g 3 1 ammonium c h l o r i d e water c o n t a i n i n g 0.5% sodium carbonate water c o n t a i n i n g 0.5% disodium hydrogen phosphate 2N h y d r o c h l o r i c a c i d methanol e t h y l alcohol butanol acetone e t h y l acetate chloroform d i e t h y l ether petroleum e t h e r 75% p y r i d i n e OU% phenol 1-butanol-water water-saturated 1-butanol water-saturated 1-butanol c o n t a i n i n g 2% p-toluenesulfonic acid water-saturated 1-butanol c o n t a i n i n g 2% p i p e r i d i n e water-saturated 2-butanol c o n t a i n i n g 2 1 t r i c h l o r acetic acid ace tone-water e t h y 1 ace t ate-w a t e r chloroform-water 2 -p ropanol-water 2 -prop ano 1-w a t e r ch l o r o f orm-ammoni a chloroform-acetic a c i d e t h y l acetate-ammonia e t h y l acetate-acetic acid benzene -methanol methanol-0.1 N h y d r o c h l o r i c a c i d ethanol-2% aq. sodium c h l o r i d e 80% e t h a n o l c o n t a i n i n g 1.51 sodium c h l o r i d e 1-p rop anol- conc ammoniurn hy d r o x i de dioxane-0.33 M a c e t i c a c i d ethanol-water (2:1, v/v) + 2% sodium c h l o r i d e e t h a n o l - w a t e r (3:1, v/v) + 2% sodium c h l o r i d e ace tone-water-ace t i c a c i d ch l o r o f orm-e th an o l -water methan o I-wate r -die t h y 1amine 1-propanol-ace t ic e c i d-w ate r butanol-e thanol-water l 4 . ut anol-e than 01-wa t e r butanol-ethanol-water 1-butanol-ethanol-water butanol-petroleum ether-water butanol-petroleum ether-water butanol-pyridine-water butanol-phenol-water
.
Conposit i o n
1:1, v/v
-
1:1, v/v 1:1, v/v 1:1, v/v 7:2, v/v 7:3, v/v 1:1, v/v 1:1, v/v 1:1, v/v 1:1, v/v 4:1, v/v 3:1, v/v 3:1, v/v
-
100:24, v/v 1:2, v/v
-
60:37: 3, v/v 2:1:1, v/v 20: 5 : 1, v/v 4:1:1, v/v 1:1:2, v/v 4:1:5, v/v 2:1:2, v/v 2:1:1, v/v 2:1:1, v/v 2:1:2, v/v 6:4:3, V/V 3 :3 :4, v/w /v
5 84 System No.
Components
51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 7.5 76 77 70 79
butanol-aminopropanol-water 1-butanol-acetic acid-water system 52, upper phase with 5% 1-butanol added 1-butanol-acetic acid-water 1-b u t a n o l - a c e t i c acid-w a t e r 1-butanol-acetic acid-water l - b u t a n o l - a c e t i c acid-water 1-butanol-acetic acid-water 1-but anal-ace t i c a c i d-w a t e r 1-bu tanol-me thanol-w ate r system 60 contg. 1.5% methyl orange P-propanol-0.2 M sodium acetate b u f f e r , pH 6.0 ethanol-0.05 M sodium acetate b u f f e r , pH 6.0 1-propanol-2.5% sodium c h l o r i d e - a c e t i c a c i d 1-butanol-2-propanol-0.2 M ammonium s u l f a t e e t h y l acetate -e th anol -w ate r e t h y l acetate-petroleum ether-water e t h y l ace tate-pe troleum e t h e r -w ate r c h l o r o f orm-ethanol-water chloroform-ethanol-water chloroform-petroleum ether-water ch l o r o form-pe t r o l e um e the r-water petroleum ether-ethanol-water l-propanol-py r idine -WB t e r
a0
t-butanol-0.001 N h y d r o c h l o r i c acid-satd. aq. sodium c h l o r i d e t-butanol-0.004 M h y d r o c h l o r i c acid-satd. aq. sodium c h l o r i d e benzyl alcohol-1.75 M sodium acetate b u f f e r , pH 5.7
81 a2 83 84
85 86
87 88
89 90
l-pentanol-pyridine-water phenol-butylace tate-water l - p r o p a n o l - a c e t i c acid-water 1-propanol-acetic a c i d - w a t e r see.-butanol-0.1 N ammonium acetate b u f f e r , pH 4.60
benzyl alcohol-1-butanol-0.001 N hydrochloric acid-satd. aq. sodium c h l o r i d e b e n z y l alcohol-1-butanol-0.001 N hydrochloric acid-satd. aq. sodium c h l o r i d e N hydrochloric benzyl alcohol-1-butanol-0,001 acid-satd. aq. sodium c h l o r i d e b e n z y l alcohol-1-butanol-0.001 N hydrochloric acid-satd. aq. sodium c h l o r i d e N hydrochloric benzyl alcohol-1-butanol-0.001 acid-satd. aq. sodium c h l o r i d e benzyl alcohol-1-butanol-0.01 N hydrochloric acid-20% sodium c h l o r i d e benzyl alcohol-1-butanol-0.02 N hydrochloric acid-satd. aq. sodium c h l o r i d e t-butanol-0.1 M sodium acetate b u f f e r , pH 5.6, contg. sodium s u l f a t e (142 g/E)
Conposition 25:1:25, v/v 4:1:5, v/v
-
4:1:1, 4:1:2, 1:1:2, 2:1:1, 5:1:4, 1:2:1, 4:1:2,
-
v/v v/v v/v v/v v/v v/v v/v
7:3, v/v 8:2, v/v 10:8:1, v/v 2:1:1, v/v 2:1:1, v/v 2:1:1, v/v 2:1:2, v/v 2 :1: 1,.v/v 2:1:2, v/v 2:1:1, v/v 2:1:2, v/v 2:1:1, v/v 60:4:40, v/v 7:7:6, v/v 5:4:9, w/v/v 60:4:40, v/b 60:4:40, v/v t o make 2 equivoluminar phases 2:1:1, v/v 2:1:1,
v/v
t o make 2 equivoluminar phases 5:10:15:3, v/v 15 :5 :15 :3, v/v 15:10:15:3, v/v 9:9:15:5, v/ v 9:9: 15 :15,
v/v 20 :10: 3: 30, 10: 5 :15 :3, v/v 2:3, v/v
5 85 System No.
Composition
Component s
91
phenol (cryst.)-chloroform-phosphate-buffer, pH 6.7
92
0.1 N ammonium acetate b u f f e r , pH 4.6 of phenol i n chloroform
93 94
t-bufanol-butanone-water-die thy lamine b u t a n o l - b u t y l acetate-acetic acid-water
95
1-but anol-e t h a n o l -acet ic a c i d-w ate r
96
1-propanol-py r idine - a m t i c a c i d-w a t e r
-
2M,
10% s o h .
phenol (10 g) d i l . w i t h chloroform t o 100 ml and b u f f e r t o make 2 e q u i voluminar phases t o make 2 equivo lumin a r phases 10:10:5:1, v/v 10: 3 : 1 . 3 : 1 4 . 3 , v/v 25 :25 :3: 4 7 , v/v 15: 10 :3: 12, v/v
This Page Intentionally Left Blank
587
S tr ept amih e-Con tain ing Pn t i b i ot i cs
.
D Perlman and Yasuaki Ogawa School of Pharmacy. University of Wisconsin. Madison. Wisconsin and Central Research Laboratories. Meiji Seika Kaisha. Yokohoma. Japan
.
1
Streptomycin
1.1 Introduction ................................... 1.1.1 Producing organisms..........................
................... .......................... ..................... ........ ...............................
1.1.2 Brief chemical description 1.1.3 Structural formulae 1.2 Summary of therapeutic use 1.3 Extraction. separation and purification 1.4 Literature cited
c _
.
2
589 589 589 589 591 591 597
Mannosidostreptomycin Introduction Producing organisms Brief chemical description Extraction. separation and purification Literature cited
2.1 2.1.1 2.1.2 2.2 2.3 -
...................................
600 .......................... 600 ................... 600 ........ 6 0 0
............................... 602 3 . Dihydrostreptomycin 3.1 Introduction................................... 603 603 3.1.1 Producing organisms .......................... 3.1.2 Brief chemical description ................... 603 3.2 Summary of therapeutic use ..................... 603 3.3 Extraction. separation and purification ........ 603 3.4 Literature cited ............................... 605 -
.
4
Hydroxystreptomycin Introduction Producing organisms 4.1.2 Brief chemical description 4.2 Summary of therapeutic use 4.3 Extraction. separation and purification 4.4 Literature cited
4.1 4.1.1
_c
5
.
................................... ..........................
................... ..................... ........ ...............................
Mannosidohydroxystreptomycin Introduction Producing organisms Brief chemical description Extraction. separation and purification Literature cited
5.1 5.1.1 5.1.2 5.2 5.3 -
606 606 606 606 606 607
...................................
608 .......................... 608 ................... 608 ........ 608
............................... 608 6 . Zygomycin B 6.1 Introduction ................................... 609 6.1.1 Producing organisms .......................... 609 6.1.2 Brief chemical description ................... 609 6.2 Extraction. separation and purification ........ 609 6.3 Literature cited ............................... 610 -
588 7.
8.
Bluensomycin Introduction................ Producing organisms 7.1.2 Brief chemical d e s c r i p t i o n . . 7.1.3 Structural formulae.......................... 7.2 Therapeutic use......... E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n . . 7.3 7.Q Literature cited.
...................611 .......................... .....,..,........ 6611 611 11 ...,............. ...... 611 .... . . 612 ... ........................... 613 Actinospectacin 8.1 I n t r o d u c t i o n . . . . . . ...... ....................... 614 8.1.1 P r o d u c i n g o r g a n i s m s .......................... 614 8.1.2 Brief c h e m i c a l d e s c r i p t i o n ..,................ 6 1 4 8.1.3 S t r u c t u r a l formulae.................. ......... 614 8.2 Summary of t h e r a p e u t i c use.....................614 8.3 E x t r a c t i o n , s e p a r a t i o n a n d p u r i f i c a t i o n . . .... , . 615 8.4 Literature cited..........,... ................. 616 7.1 7.1.1
5 89
Although t h e ' s t r e p t a r n i n e d e r i v a t i v e s
'
form a r e l a t i v e l y
small group o f t h e 'Aminoglycoside A n t i b i o t i c s ' , t h e i r p u r i f i c a t i o n h a s been more i n t e n s i v e l y s t u d i e d than t h a t of o t h e r small g r o u p s s i n c e t h e i r major member, STREPTOMYCIN, was one of t h e f i r s t of t h e s t r e p t o m y c e t e - p r o d u c e d a n t i b i o t i c s t o be w i d e l y used c l i n i c a l l y f o r many t y p e s of major i n f e c t i o n s . This e x t e n s i v e c l i n i c a . 1 use r e s u l t e d i n time i n an a w a r e n e s s of a l a r g e number of c l i n i c a l l y s i g n i f i c a n t s i d e - e f f e c t s . And, t h i s l e d i n t u r n , t o t h e e x a m i n a t i o n of t h e p u r i f i c a t i o n methods i n an a t t e m p t t o d e t e r m i n e whether n o n - a n t i b i o t i c components cont a m i n a t i n g t h e a n t i b i o t i c p r e p a r a t i o n s were r e s p o n s i b l e f o r t h e s e u n d e s i r a b l e s i d e - e f f e c t s , and w h e t h e r i f t h e s e i m p u r i t i e s were e l i m i n a t e d by more e f f e c t i v e r e c o v e r y methods t h e s i d e e f f e c t s would be s i g n i f i c a n t l y r e d u c e d . U n f o r t u n a t e l y t h i s e l i m i n a t i o n of s i d e - e f f e c t s was n o t c o m p l e t e l y s u c c e s s f u l . However, a s a b y - p r o d u c t of t h i s e f f o r t we do have a number of methods of p u r i f i c a t i o n of s t r e p t o m y c i n . I n t h i s c h a p t e r we w i l l g i v e major a t t e n t i o n t o t h o s e methods which have been w i d e l y u s e d , e . g . , on a commercial s c a l e , and a l l o c a t e l e s s s p a c e t o t h o s e methods which have n o t been s o e x t e n s i v e l y t e s t e d .
,
1. S t r e p t o m y c i n
1.1
Introduction P r o d u c i n g organisms
1.1.1
S t r e p t o m y c i n was i n i t i a l l y s e l e c t e d f o r s t u d y on t h e b a s i s of i t s a c t i v i t y a g a i n s t gram-negative b a c t e r i a and mycobacteria. I t i s produced by s e l e c t e d s t r a i n s o f S. g Z i A e u d l , S bikinienAi42 S. olivaceud S rna4huen4iA4, S ZameuA5, S g a l b u 4 6 , and S . e,zyth&ochAornogene& v a r . n a n u t o e n ~ i 4 when grown i n a p p r o p r i a t e media.
.
.
1.1.2
.
.
B r i e f chemical d e s c r i p t i o n
The e m p i r i c a l f o r m u l a f o r s t r e p t o m y c i n i s C ~ l H 3 9 N 7 0 1 2 ~ and t h e compound i s a s t r o n g b a s e . A v a r i e t y of s a l t s have been c l i n i c a l l y u s e f u l i n c l u d i n g t h e t r i h y d r o c h l ~ r i d e ~t h e s e s q u i s u l f a t e l O ' l l and t h e calcium c h l o r i d e double s a l t I 2 . { O t h e r s a l t s i n c l u d i n g t h e trihydrobromide", t h e t r i n i t r a t e l l , and t h e s e s q u i p h o s p h a t e l l have been p r e p a r e d i n c r y s t a l l i n e form b u t have n o t been found t o be c l i n i c a i l y u s e f u l . } The r o t a t i o n of t h e t r i h y d r o c h l o r i d e i s { ~ 1 ~ ~ - 8 6 .(1C 1, HzO)ll. S t r e t o mycin shows o n l y end a b s o r p t i o f ? i n t h e r a n g e 2 3 0 t o 4 0 0 nmY3. bioassay (using both gram-positive The p o t e n c y i s d e t e r m i n e d and g r a m - n e g a t i v e b a c t e r i a o r c o n v e r s i o n t o m a l t o l 1 4 and estimation colorimetrically. The t r i h y d r o c h l o r i d e c o n t a i n s 8 9 1 pg b a s e p e r mg".
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1.1.3
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S t r u c t u r a l formulae
Its s t r u c t u r a l relationships t o o t h e r streptomycins a r e shown i n F i g u r e s 1 and 2 .
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II
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STREPTOMYC I N N-DEMETHYLSTREPTOMYCIN HYDROXYSTREPTOPWC I N MANNOS IDOSTREPTOMYCI N MANNOS IDOHYDROXYSTREPTOMYC I N D I HYDROSTREPTOMY C I N
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FIGURE 1 Structural Relationships Among the Streptomycins
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f P H H H
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591 Dihydro drrivotivrr o f
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N-mrthyl- L glucoromine D-manme
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r t r r ptobioramim Strrpt omycin
I Monnoridortrr~tomvcin Hydroxyrtrrptomycin
I
Monnoridohydroxyrtreptomycin
FIGURE 2
An a n a l y s i s o f t h e s t r u c t u r a l r e l a t i o n s h i p s among the streptomycins
1.2
Summary o f t h e r a p e u t i c u s e
S t r e p t o m y c i n i s c l i n i c a l l y u s e d f o r t r e a t m e n t of t u b e r c u l o s i s , s y s t e m i c i n f e c t i o n s by g r a m - n e g a t i v e b a c t e r i a , and b a c t e r i a l i n f e c t i o n s of t h e u r i n a r y t r a c t . The u s u a l d o s e i s 0 . 5 t o 1 . 0 g p e r day by i n t r a m u s c u l a r i n j e c t i o n . I t has a l s o f o u n d u s e i n c o n t r o l l i n g p l a n t p a t h o g e n i c g r a m - p o s i t i v e and g r a m - n e g a t i v e b a c t e r i a and h a s i n f r e q u e n t l y been u s e d as an a n i m a l f e e d s u p p l e m e n t where i t a c t s as a growth p r o m o t a n t f o r p o u l t r y , swine and c a t t l e .
1.3
E x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n
The methods u s e d t o r e c o v e r p u r e s t r e p t o m y c i n s a l t s from f e r m e n t e d media t a k e a d v a n t a g e of t h e f o l l o w i n g p r i n c i p l e s : 1. S e l e c t i v e a b s o r p t i o n on a c t i v a t e d c h a r c o a l a n d s u b sequent e l u t i o n with a c i d i c lower a l c o h o l s o l u t i o n s * 9 u y 1 5 - 2 1 .
2. S e l e c t i v e a d s o r p t i o n by c a t i o n exchange r e s i n s folfowed by e l u t i o n w i t h d i l u t e m i n e r a l a c i d s o l u t i ~ n s ~ ' ~ ~a n- dz ~ ;
592
3. S e l e c t i v e p r e c i p i t a t i o n as t h e s i l i c o t u n g s t a t e 2 6 t h e 8-am~no-7-p-nitrophenylazo-2-phenylazo-l-naphth01-3~6d i ~ u l f o n a t e ~o r~ ,t h e p-(2-hydroxy-l-naphthylazo)-benzene s u l f o n a t e 2 e , and s u b s e q u e n t c o n v e r s i o n t o s t r e p t o m y c i n t r i h y d r o c h l o r i d e or s t r e p t o m y c i n s u l f a t e . The c r u d e s t r e p t o m y c i n r e c o v e r e d by t h e s e p r o c e d u r e s may be p u r i f i e d f u r t h e r by: A.
Chromatography on a l u m i n a 8 * 1 7 * 2 9 - 3 2 ;
B. P r e c i p i t a t i o n as c r y s t a l l i n e oxime ( a n d s u b s e q u e n t con ve rs i on t o s t r e p tomy c i n s u l f a t e 3 3 ; C. Selective p r e c i p i t a t i o n a s the helianthate4 the p h o ~ p h o t u n g s t a t e ~t h~e p ~ l y h a l o p h e n o l a t e ~t h~e, d i p h e n y l - 4 sodium m o n o s u l f a t e l 6 , t h e p i ~ r a t e t~h e~ r~e i~n ,e ~ k a t e ~t h~e, 5-methyl-$-naphthalene s u l f o n a t e 6 , o r t h e t e r g i t a t e 3 9 and subsequent conversion t o the trihydrochloride o r s u l f a t e ;
D. Formation of t h e S c h i f f b a s e (and s u b s e q u e n t convers i o n t o s t r e p t o m y c i n s u l f a t e ) 1 0 '40 4 1 ; E. Solvent e x t r a c t i o n u s i n g a water-immiscible s o l v e n t w i t h an o r g a n i c a c i d as a c a r r i e r ( a n d s u b s e q u e n t back e x t r a c t i o n i n t o aqueous s o l u t i o n ) 4 2 - 4 ' + ; o r
F. Solution of the streptomycin trihydrochloride i n methanol f o l l o w e d by c o n v e r s i o n t o t h e m e t h a n o l - i n s o l u b l e s t r e p tomycin s u l f a t e upon a d d i t i o n of t r i m e t h y l a m i n e s u l f a t e t o t h e s o l u t i o n 45
.
I n many i n s t a n c e s t h e s t r a t e g y h a s i n v o l v e d c o m b i n a t i o n s of s e v e r a l p r o c e d u r e s . An example o f one u s i n g c h a r c o a l a b s o r p t i o n , p r e c i p i t a t i o n of t h e p i c r a t e and t h e h e l i a n t h a t e , and f i n a l l y chromatography on alumina i s t h e p r o c e d u r e summarized by Kuehl e t The f e r m e n t e d medium was f i l t e r e d t h r o u g h a t h i n pad of f i l t e r c e l t o remove suspended s o l i d s , and t h e f i l t r a t e w a s t r e a t e d w i t h 7 . 5 g of N o r i t e p e r l i t e r . T h i s s u s p e n s i o n was s t i r r e d f o r one h r a t room t e m p e r a t u r e and f i n a l l y f i l t e r e d . The a b s o r b a t e was t h e n washed by s t i r r i n g w i t h 1 l i t e r o f e t h a n o l p e r 1 0 0 g o f N o r i t e f o r 45 min. The N o r i t e was removed by f i l t r a t i o n and d r i e d i n uacuo a t room t e m p e r a t u r e . The s t r e p t o m y c i n was e l u t e d w i t h 0.8 N f o r m i c a c i d i n T h i s was a c c o m p l i s h e d by s u s p e n d i n g t h e N o r i t e i n a volume o f . e l u a t e e q u i v a l e n t t o 1 5 % o f &he volume o f t h e b r o t h t r e a t e d . A f t e r s t i r r i n g f o r 4 h r s a t 45 , t h e N o r i t e was removed by f i l t r a t i o n and t h e f i l t r a t e c o n c e n t r a t e d i n vacuo t o 1% of i t s o r i g i n a l volume. F i v e volumes of m e t h a n o l were added f o l l o w e d by f o u r volumes o f a c e t o n e . The r e s u l t i n g p r e c i p i t a t e was removed by c e n t r i f u g i n g , washed w i t h e t h y l e t h e r , and d r i e d . i n uacuo. 1:1 methanol-water.
F u r t h e r p u r i f i c a t i o n was o b t a i n e d by p r e c i p i t a t i o n w i t h
59 3
p i c r i c a c i d f o l l o w e d by c o n v e r s i o n t o t h e h y d r o c h l o r i d e . Crude s t r e p t o m y c i n and an e q u a l w e i g h t of p i c r i c a c i d were added t o w a t e r a t 40 g of e a c h p e r l i t e r and t h e m i x t u r e was s t i r r e d and h e a t e d u n t i l a l l s o l i d s were d i s s o l v e d . The s o l u t i o n was c o o l e d t o a b o u t 5 and a l l o w e d t o s t a n d f o r s e v e r a l h o u r s . The s l u d g e o f s t r e p t o m y c i n p i c r a t e and f r e e p i c r i c a c i d was s e p a r a t e d from t h e s u p e r n a t a n t s o l u t i o n by d e c a n t a t i o n and d i s s o l v e d i n warm methanol c o n t a i n i n g an excess o f 2 . 5 N H C 1 . The h y d r o c h l o r i d e was p r e c i p i t a t e d by p o u r i n g t h e s o l u t i o n i n t o 10 volumes o f ethyl ether. The p r e c i p i t a t e was d i s s o l v e d i n methanol and r e p r e c i p i t a t e d with e t h y l e t h e r . A f t e r drying i n vacuo, the s t r e p t o m y c i n h y d r o c h l o r i d e a s s a y e d 1 0 0 t o 2 0 0 ug p e r mg. The s t r e p t o m y c i n h y d r o c h l o r i d e was f u r t h e r p u r i f i e d by chromatography on acid-washed aluminum o x i d e i n a r a t i o of 10 t o 30 g of a d s o r b e n t t o 1 g s t r e p t o m y c i n h y d r o c h l o r i d e concent r a t e . The columns were f i l l e d w i t h t h e a d s o r b e n t and m e t h a n o l , and t h e s o l v e n t was a l l o w e d t o d r a i n u n t i l a l a y e r o f s o l v e n t above t h e a d s o r b e n t was a p p r o x i m a t e l y 1 t o 2 mm deep. The c r u d e s t r e p t o m y c i n h y d r o c h l o r i d e was d i s s o l v e d i n methanol a t a conc e n t r a t i o n of 1 0 t o 5 0 % and t h e s o l u t i o n was a l l o w e d t o flow t h r o u g h t h e column e i t h e r by g r a v i t y o r under p r e s s u r e of 10 t o 2 0 mm of mercury. When t h e s o l u t i o n was a l l i n t h e a d s o r b e n t l a y e r , methanol was added and a l l o w e d t o p e r c o l a t e e i t h e r by The e l u a t e s were t e s t e d f o r t h e g r a v i t y or under p r e s s u r e . p r e s e n c e of s o l i d s by t h e a d d i t i o n of a few d r o p s of t h e e l u a t e t o 5 m l o f a c e t o n e o r e t h y l e t h e r . When p r e c i p i t a t i o n o f s o l i d s a p p e a r e d i n t h e t e s t , t h e c o l l e c t i o n of f r a c t i o n s was begun. The e l u a t e s were c o n c e n t r a t e d t o a b o u t 1 / 1 0 o f t h e o r i g i n a l volume and poured i n t o a c e t o n e or e t h y l e t h e r t o p r e c i p i t a t e t h e s t r e p t o m y c i n which was t h e n c o l l e c t e d by f i l t r a t i o n or cent r i f u g a t i o n and d r i e d i n v a c u o . The p o t e n c y o f t h i s p r o d u c t was u s u a l l y 4 0 0 t o 6 0 0 ug s t r e p t o m y c i n p e r mg (or n e a r l y 3 times t h a t of t h e s t a r t i n g m a t e r i a l ) . Chromatography on a m i x t u r e o f Darco G - 6 0 and f i l t e r p a p e r p u l p was shown t o be e q u a l l y e f f e c t i v e a s a method o f p u r i f i c a t i o n of t h e crude streptomycin h y d r o c h l o r i d e . T r e a t m e n t of samples of s t r e p t o m y c i n h y d r o c h l o r i d e which showed an a c t i v i t y o f 4 0 0 pg p e r mg (or h i g h e r ) w i t h t h e sodium s a l t o f h e l i a n t h i n e ( m e t h y l o r a n g e ) o r of p-(Z-hydroxy-l-naphthylazof-benzenesulfonic a c i d ( o r a n g e 11) y i e l d e d t h e corresponding c r y s t a l l i n e s a l t s : A 2 6 . 3 g of s t r e p t o m y c i n h y d r o c h l o r i d e ( 5 0 0 ug p e r mg) was d i s s o l v e d i; 390 m l of methanol and t h e s o l u t i o n h e a t e d t o a p p r o x i m a t e l y 5 5 , T h i s was mixed w i t h a s o l u t i o n c o n t a i n i n g 20.8 g of methyl o r a n g e i n 1 4 5 6 m l of w a t e r (which had been h e a t e d t o 7 5 ' ) . After standing overnight at 10' t h e m i x t u r e was c e n t r i f u g e d and t h e c r y s t a l l i n e p r o d u c t was washed w i t h a p p r o x i m a t e l y 2 0 0 m l o f water. R e c r y s t a l l i z a t i o n of t h e h e l i a n t h a t e was accomplished by d i s s o l v i n g i t i n 3 l i t e r s of h o t 33% aquezus m e t h a n o l and a l l o w i n g t h e s o l u t i o n t o s t a n d o v e r n i g h t a t 10
.
The h e l i a n t h a t e was t h e n removed by c e n t r i f u g i n g and washed w i t h 2 0 0 m l o f water and 2 0 0 m l e a c h o f i s o p r o p a n o l , a c e t o n e , and e t h y l e t h e r . The w e i g h t of t h e p r o d u c t was 32 g
594
(87% y i e l d ) . T h i s was c o n v e r t e d t o t h e h y d r o c h l o r i d e by d i s methanol m i x t u r e ( r a t i o 1:26, solving i t i n concentrated HC1 v / v ) a t a l e v e l o f 2 9 g p e r 465 ml of m i x t u r e . A f t e r t h e mixt u r e was t h r o u g h l y s t i r r e d , t h e s u s p e n s i o n was f i l t e r e d t h r o u g h a t h i n l a y e r of Darco G-60 and t h e f i l t e r cake was washed w i t h 85 m l o f methanol. The f i l t r a t e and washings were poured i n t o 4500 m l of a c e t o n e and t h e m i x t u r e c e n t r i f u g e d . The c o l l e c t e d s t r e p t o m y c i n h y d r o c h l o r i d e was washed w i t h a c e t o n e and d r i e d . The y i e l d was a b o u t 9 7 % and t h e p o t e n c y a b o u t 785 ug p e r mg.
-
The ion-exchange p r o c e d u r e o f Howe and P u t t e r 2 2 i s t h e most w i d e l y used p r o c e s s f o r t h e p u r i f i c a t i o n of a m i n o g l y c o s i d e a n t i b i o t i c s i n 1 9 7 6 . An example t a k e n i n p a r t from t h e i r p a t e n t i s as f o l l o w s : A g r a n u l a r copolymer of m e t h a c r y l i c a c i d and d i v i n y l benzene c o n t a i n i n g 5% d i v i n y l b e n z e n e (known as Amberlite I R C 5 0 ) was s l u r r i e d w i t h water and t h e n added t o a s u i t a b l e column t o g i v e a bed a b o u t 3 i n c h e s deep and 50 m l i n t o t a l volume. The r e s i n was washed w i t h 2 . 5 N H C 1 and t h e n w i t h water. I t was t h e n c o n v e r t e d t o t h e ammonium c y c l e by t h e a d d i t i o n of 2 . 5 N N H k O H , f o l l o w e d by washing a g a i n w i t h water. Swelling occurred when t h e r e s i n was c o n v e r t e d t o t h e ammonium c y c l e , g i v i n g a column w i t h a r e s u l t i n g bed h e i g h t of a p p r o x i m a t e l y 5 i n c h e s . The column was t h e n back washed w i t h water u n t i l t h e pH of t h e e f f l u e n t l i q u i d was a b o u t 1 0 . 5 . The f i l t e r e d b r o t h o b t a i n e d from a s t r e p t o m y c i n - p r o d u c i n g f e r m e n t a t i o n was a d j u s t e d t o pH 7 and p a s s e d o v e r t h e column a t a r a t e s u c h t h a t t h e b r o t h was i n c o n t a c t w i t h t h e r e s i n f o r a b o u t 1 min. A t t h e end of t h e abs o r p t i o n c y c l e t h e column was washed w i t h water and e l u t e d w i t h 1 . 0 t o 1 . 5 N HC1. The e l u t e d f r a c t i o n s were immediately neut r a l i z e d w i t h 2 . 5 N NaOH t o pH 7 t o pH 8. The n e u t r a l i z e d e l u a t e f r a c t i o n s g i v i n g a p o s i t i v e Sakaguchi t e s t were p o o l e d , c o n c e n t r a t e d i n vacuo t o a small volume, and d i l u t e d w i t h metha n o l . The p r e c i p i t a t e d s t r e p t o m y c i n t r i h y d r o c h l o r i d e was c o l l e c t e d by f i l t r a t i o n and d r i e d i n vacuo. The r e c o v e r y of s t r e p tomycin i n t h e f e r m e n t e d medium was a b o u t 6 0 % , and t h e p o t e n c y was 500 pg p e r mg. Although t h e o r i g i n a l p r o c e s s recommended u s e of f i l t e r e d f e r m e n t e d medium, i t can be a l i e d t o u n f i l t e r e d m e d i u m u s i n g t h e up-flow f e e d t e c h n i q u e g g . A s e c o n d example i s summarized i n t h e f o l l o w i n g flow
sheet: 64.8 1 fermented b r o t h (360 pg p e r m l ) IRC-50 r e s i n (sodium form) f l o w r a t e : 150 m l per min through
I
4.3 ug p e r m l
washed with 1 1 water e l u t e d with 1 1 1 . O N HC1
f i r s t column 670 m l a t 25 mg streptomycin p e r ml
+
second ~ o l u m 235 m l a t 28 mg streptomycin p e r m l
595
-
c o n c e n t r a t e d almost t o dryness add 100 m l methanol
filter
precipitate (discard)
precipitate 26.3 g w i t h 650 pg s t r e p t o mycin per mg
add 500 m l acetone
filtrate (discard)
L. add 81 m l methanol add 23.6 g Carl2
s t i r f o r 40 h r s filtrate p r e c i p i ft ai lt tee r (wash with 10% Car12 soln., methanol and ethanol
(discard)
I
dry i n vacua
1
17.5 g streptomycin calcium c h l o r i d e s a l t with 780 pg streptomycin P e r mg
The crude s t r e p t o m y c i n p r e p a r a t i o n s o b t a i n e d by t h e i o n exchange p r o c e s s can be p u r i f i e d by s e v e r a l p r o c e d u r e s i n c l u d i n g f u r t h e r i o n exchange t r e a t m e n t , c o n v e r s i o n t o t h e oxime (which i s back c o n v e r t e d t o t h e s u l f a t e ) , and f o r m a t i o n of a S c h i f f base :
1. Friedman e t recommended a b s o r p t i o n on a c a r b o x y l i c a c i d i o n exchange r e s i n (Amberlite XE-89) and e l u t i o n w i t h 0 . 3 6 N H~SOL,. The s t r e p t o m y c i n - c o n t a i n i n g f r a c t i o n s were p o o l e d and p a s s e d o v e r a t h i r d i o n exchange column c o n t a i n i n g a s u l f o n a t e d copolymer of p o l y s t y r e n e and d i n v i n y l b e n z e n e (Dowex 50-Xl6) t o remove t h e e x c e s s a c i d . The e f f l u e n t was t h e n neut r a l i z e d w i t h Ba(OHl2 s o l u t i o n and t h e p r e c i p i t a t e d Bas04 removed by f i l t r a t i o n . The s t r e p t o m y c i n - c o n t a i n i n g s o l u t i o n was c o n c e n t r a t e d t o . a small volume i n v a c u a , t r e a t e d w i t h a small amount of a c t i v a t e d c h a r c o a l ( t o remove c o l o r ) and mixed w i t h s e v e r a l volumes of methanol. The p u r e s t r e p t o m y c i n s u l f a t e p r e c i p i t a t e d and was c o l l e c t e d by f i l t r a t i o n and d r i e d . I t had a p o t e n c y o f 790 pg p e r mg. 2.
H e u ~ e rs u~g ~ g e s t e d t h a t f o r m a t i o n of t h e c r y s t a l l i n e
596
s t r e p t o m y c i n oxime s a l t can be a u s e f u l i n t e r m e d i a t e i n t h e f u r t h e r p u r i f i c a t i o n . He took t h e n e u t r a l i z e d s t r e p t o m y c i n - c o n t a i n i n g e l u a t e from t h e IRC-50 column and c o n c e n t r a t e d i t in vacuo and added methanol t o t h e c o n c e n t r a t e . The p r e c i p i t a t e d i n o r g a n i c p h o s p h a t e ( o r i g i n a t i n g from t h e f e r m e n t a t i o n medium) was removed by f i l t r a t i o n and an e x c e s s o f hydroxylamine s u l f a t e was added. The a c i d released was n e u t r a l i z e d w i t h t r i m e t h y l amine s o t h a t t h e pH of t h e s o l u t i o n was 5.8 and t h e s o l u t i o n was s t i r r e d f o r 1 h r . A f t e r s t a n d i n g o v e r n i g h t , t h e c r y s t a l s o f s t r e p t o m y c i n oxime were c o l l e c t e d by f i l t r a t i o n and d r i e d . These c r y s t a l s were c o n v e r t e d t o s t r e p t o m y c i n s u l f a t e by 2 0 g of s t r e p t o m y c i n ogime s u l f a t e t h e following procedure: c r y s t a l s were d i s s o l v e d it 200 m l of water a t 45 t o 50'. The s o l u t i o n was c o o l e d t o 30 and a d j u s t e d w i t h 3.6 N H ~ S O L ,t o pH 2.0. It was t h e n p l a c e d under vgcuum d i s t i l l a t i o n a t 28 t o 32 mm Hg a t a p o t t e m p e r a t u r e o f 2 8 t o 30' and 2 0 ml o f f o r m a l i n ( d i l u t e d t o 400 m l w i t h d i s t i l l e d water) was added o v e r a 8 h r p e r i o d a t a r a t e of 50 m l p e r h r . A f t e r d i s t i l l a t i o n , t h e conc e n t r a t e ( a b o u t 65 m l ) was mixed w i t h 5 volumes of methanol and a d j u s t e d t o pH 5.0 by t r e a t m e n t w i t h an a n i o n exchange r e s i n ( A m b e r l i t e IR-45). The p r e c i p i t a t e was removed by f i l t r a t i o n and t h e f i l t r a t e was t r e a t e d w i t h 3 g of a c t i v a t e d carbon (Darco G - 6 0 ) and f i l t e r e d . The f i l t r a t e was d i l u t e d t o 50 m l and t h e n l y o p h i l i z e d y i e l d i n g about 1 7 . 2 g of p u r i f i e d streptomycin s u l fate. 3. Heuser41 a l s o s u g g e s t s as an a l t e r n a t i v e t h e format i o n of a S c h i f f b a s e w i t h v a r i o u s amines. H e c a r r i e d o u t t h i s as f o l l o w s : 66 m l of s t r e p t o m y c i n h y d r o c h l o r i d e e l u a t e from t h e IRC-50 column w i t h a p o t e n c y o f 2 6 0 mg s t r e p t o m y c i n p e r m l were mixed w i t h 8 0 m l of methanol c o n t a i n i n g 1 2 t1 o f d i b e n z y l m e t h y l amine. The m i x t u r e was h e a t e d t o 4 0 t o 45 a t t h e s t a r t t o i n i t i a t e c r y s t a l l i z a t i o n . A f t e r t h e a d d i t i o n was c o m p l e t e d , The t h e pH was a d j u s t e d w i t h t r i e t h y l a m i n e t o pH 7.3 t o 9 . 0 . s u s p e n s i o n was a g i t a t e d f o r 30 min. and t h e m i x t u r e s t o r e d o v e r The c r y s t a l s were c o l l e c t e d by f i l t r a t i o n n i g h t a t '5 t o 10'. and washed w i t h 5 0 m l o f 60% aqueous methanol and t h e n w i t h a b s o l u t e methanol. The c r y s t a l s were t h e n d r i e d and 25.5 g of streptomycindibenzylamine were r e c o v e r e d .
T h i s w a s c o n v e r t e d t o s t r e p t o m y c i n s u l f a t e by t h e f o l lowing s t e p s : 5 g of t h e streptomycindibenzylamine were s l u r r i e d i n a m i x t u r e of 50 m l c h l o r o f o r m and 50 m l of water w i t h a d d i t i o n o f 14.4 N H ~ S O L ,t o a d j u s t t h e pH t o 7.0 t o 7.5. The aqueous l a y e r was s e p a r a t e d and r e - e x t r a c t e d w i t h 25 m l o f c h l o r o f o r m . It was t h e n t r e a t e d w i t h 0 . 6 g a c t i v a t e d c h a r c o a l and f r e e z e d r i e d t o y i e l d a b o u t 3.5 g s t r e p t o m y c i n s u l f a t e w i t h a potency of 785 vg p e r mg. Heuser h a s a l s o u s e d B-phenethylamine i n s t e a d of t h e dibenzylmethylamine w i t h e q u a l success. 4. Rhodehamel e t U k ? . 1 0 ' 4 0 describe a solvent extraction p r o c e d u r e which t h e y used t o p u r i f y t h e s t r e p t o m y c i n - c o n t a i n i n g e l u a t e from t h e IRC-50 column. They p r e s e n t t h e f o l l o w i n g examp l e : 5 l i t e r s of s t r e p t o m y c i n h y d r o c h l o r i d e s o l u t i o n a s s a y i n g
597 350 mg s t r e p t o m y c i n p e r m l a n d h a v i n g a pH o f 7 were e x t r a c t e d w i t h a n e q u a l volume o f h e x y l a m i n e . The two p h a s e s w e r e s e p a r a t e d a n d t h e w a t e r p h a s e was e x t r a c t e d w i t h a f u r t h e r 1 . 3 l i t e r s of h e x y l a m i n e . The combined h e x y l a m i n e e x t r a c t s were t r e a t e d w i t h a m i x t u r e o f 6 l i t e r s o f water a n d 18 l i t e r s o f c h l o r o f o r m . The m i x t u r e was w e l l s t i r r e d a n d was t h e n a l l o w e d t o s e p a r a t e i n t o two l a y e r s . The water l a y e r ( c o n t a i n i n g t h e s t r e p t o m y c i n ) was t h e n e x t r a c t e d w i t h a m i x t u r e of 3 l i t e r s of c y c l o h e x a n o n e The a n d 6 l i t e r s o f a m y l a c e t a t e t o remove a n y r e s i d u a l amine. s t r e p t o m y c i n - c o n t a i n i n g water s o l u t i o n was t h e n a d j u s t e d w i t h H~SOL, t o pH 6 . 0 a n d l y o p h i l i z e d . 2 . 3 k g o f s t r e p t o m y c i n s u l f a t e a s s a y i n g 500 pg p e r mg were r e c o v e r e d . Among t h e a l t e r n a t i v e s t o t h e h e x y l a m i n e were 2 - a m i n o h e p t a n e , b e n z y l a m i n e , o c t y l a m i n e , and 2-ethylhexylamine. While s t r e p t o m y c i n i s u s u a l l y t h e m a j o r a n t i b a c t e r i a l substance i n t h e f e r m e n t a t i o n s by t h e streptomycin-procuding S . ghi4euA s t r a i n s , m a n n o s i d o s t r e p t o m y c i n i s s o m e t i m e s f o u n d i n s i g n i f i c a n t amounts32. I t can be s e p a r a t e d from s t r e p t o m y c i n by s e v e r a l p r o c e d u r e s (see s e c t i o n 2 ) a n d t h e s t r e p t o m y c i n s u l f a t e now u s e d i n c l i n i c a l t r e a t m e n t of human i n f e c t i o n s i s ess e n t i a l l y pure. P e r e i r a 4 7 found a d d i t i o n a l s t r e p t o m y c i n - r e l a t e d s u b s t a n c e s i n some f e r m e n t e d m e d i a a n d p u r i f i e d t h e s e by i o n e x c h a n g e c h r o m a t o g r a p h y u s i n g CG-50 (Na f o r m ) w i t h a g r a d i e n t e l u t i o n by 0 . 5 M t o 1 . 5 M N a C l s o l u t i o n .
1.4
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600
2 . Mannosidostreptomycin 2 . 1 Introduction -
Mannosidostreptomycin was o r i g i n a l l y d e t e c t e d as a 'cont a m i n a n t ' i n p u r i f i e d p r e p a r a t i o n s o f impure s t r e p t o m y c i n ' , and t h e i n t e r e s t was t o e l i m i n a t e i t from t h e more d e s i r a b l e s t r e p tomycin2 r a t h e r than t o i s o l a t e i t as a p u r e s u b s t a n c e . (The s t r u c t u r a l r e l a t i o n s h i p s a r e shown i n F i g u r e s 1 and 2 ) . 2.1.1
P r o d u c i n g organisms
I t can be e n z y m a t i c a l l y c o n v e r t e d t o s t r e p t o m y c i n 3 and c e r t a i n s t r a i n s of S t A e p t o m y c e A g A i A e u A have t h i s c a p a c i t y 3 y 4 . Mannosidostreptomycin h a s n o t been r e p o r t e d as a p r o d u c t of c u l t u r e s s y n t h e s i z i n g S t r e p t o m y c i n o t h e r t h a n S. g R i A e u A , e .+. Sthttrcptomyceb b i h i e f l i f l A i d b u t p r o b a b l y o c c u r s i n t h e s e o r g a n i s m s too. 2.1.2
B r i e f chemical d e s c r i p t i o n
The e m p i r i c a l f o r m u l a f o r mannosidostreptomycin i s C 2 7 H 4 9 an$ t h e m e l t i n g p o i n t of t h e t r i h y d r o c h l o r i d e d i h y d r a t e i s 179-182 ( d e c . ) and t h a t of t h e anhydrous t r i h y d r o c h l o r i d e i s 1 9 0 - 2 0 0 ° ( d e c . b 6 . The r o t a t i o n of t h e t r i h y d r o c h l o r i d e i s {alk0'6 i s - 5 4 . 1 ( c 1, H20). Mannosidostreptomycin shows no a b s o r p t i o n maxima i n t h e r a n g e 2 2 0 nm t o 4 0 0 nm.
N70175r
2.2 Extraction
s e p a r a t i o n and p u r i f i c a t i o n
Three methods have been p r o p o s e d f o r i s o l a t i o n of and s e p a r a t i o n of mannosidostreptomycin from s t r e p t o m y c i n : The i n i t i a l method i n v o l v e d Chromatography' and t h i s was f o l l o w e d by a method b a s e d on s o l v e n t The t h i r d method h a s u t i l i z e d ion-exchange chromatography of complexesg. Analyt i c a l p r o c e d u r e s have f o c u s e d on i n d e p e n d e n t l y d e t e r m i n i n g s t r e p t o m y c i n and mannosidostreptomycin i n m i x t u r e s of t h e two 7 , l O Y 11.
I n t h e i r i n i t i a l s t u d i e s F r i e d and utilized chromatography on alumina of s t r e p t o m y c i n - c o n t a i n i n g p r e p a r a t i o n s which showed a l o w e r t h a n e x p e c t e d b i o p o t e n c y b a s e d on t h e m a l t o l c h e m i c a l a s s a y . Ten grams o f s t r e p t o m y c i n complex ( 4 0 0 pg/mg) was d i s s o l v e d i n 1 0 0 m l of 8 0 % methanol and p e r c o l a t e d a t a flow r a t e of between 8 0 and 1 0 0 d r o p s p e r min t h r o u g h a column of 3.2 cm d i a m e t e r c o n t a i n i n g Q50 g of alumina which had been p r e v i o u s l x washed w i t h d i l u t e H~SOL, t o a pH o f 4 . 5 and reactivated a t 150 The column was washed w i t h 8 0 % methanol and t h e e f f l u e n t s o l u t i o n was t e s t e d by means o f t h e Sakaguchi r e a c t i o n . When 4 9 0 m l o f s o l v e n t had p a s s e d t h r o u g h t h e column t h e Sakaguchi t e s t became p o s i t i v e and t h e e f f l u e n t s o l u t i o n was c o l l e c t e d i n 5 f r a c t i o n s t o t a l l i n g 1 0 8 0 m l . Water was subs t i t u t e d t h e n f o r 8 0 % methanol as t h e e l u t i n g a g e n t and two more f r a c t i o n s were c o l l e c t e d . The s t r e p t o m y c i n was r e c o v e r e d from t h e e l u a t e s by d i s t i l l i n g o f f t h e methanol in V U C U O , f i l t e r i n g t h e r e s u l t i n g aqueous s o l u t i o n s and l y o p h i l i z i n g them. Those f r a c t i o n s w i t h h i g h m a l t o l v a l u e s and low b i o a s s a y s were p o o l e d and c o n v e r t e d t o t h e t r i r e i n e c k a t e s a l t : 1 9 . 3 g of t h e p o o l e d
.
601
f r a c t i o n was d i s s o l v e d i n water and t h e s u l f a t e i o n ( w h i c h had b e e n e l u t e d from t h e H2SO4 washed a l u m i n a was r e p l a c e d by c h l o r i d e i o n by means of 2 % B a C 1 2 s o l u t i o n ) . The Bas04 p r e c i p i t a t e was removed by f i l t r a t i o n o v e r a b e d o f Darco G - 6 0 , a n d t h e f i l t r a t e d i l u t e d w i t h water t o a volume o f 380 m l . To t h i s s o l u t i o n was a d d e d a t 40' a s o l u t i o n of 2 5 . 1 g r e i n e c k e s a l t i n 6 0 0 m l of water a n d t h e m i x t u r e was a l l o w e d t o c o o l v e r y s l o w l y . Upon s t a n d i n g a t room t e m p e r a t u r e f o r 1 5 h o u r s , a c o p i o u s p r e c i p i t a t e f o r m e d , which was r e d i s s o l v e d by warming t o 40' l e a v i n g a s m a l l amount o f amorphous and s a n d y c r y s t a l l i n e m a t e r i a l . T h i s was removed b y f i l t r a t i o n a n d t h e f i l t r a t e was a l l o w e d t o c r y s t a l l i z e s l o w l y a t room t e m p e r a t u r e . R e c r y s t a l l i z a t i o n of t h e r e s u l t i n g material a t room t e m p e r a t u r e y i e l d e d 6 . 9 g mannos i d o s t r e p t o m y c i n t r i r e i n e c k a t e which c o n t a i n e d l e s s t h a n 1 . 5 % streptomycin. The m a n n o s i d o s t r e p t o m y c i n t r i r e i n e c k a t e was c o n v e r t e d i n t o t h e m a n n o s i d o s t r e p t o m y c i n t r i h y d r o c h l o r i d e by a d d i n g a q u e o u s s i l v e r n i t r a t e t o a w a r m s o l u t i o n of t h e t r i r e i n e c k a t e . The p r e c i p i t a t e d s i l v e r r e i n e c k a t e was removed b y f i l t r a t i o n and 2 % B a C 1 2 s o l u t i o n was a d d e d u n t i l e x c e s s was o b t a i n e d ( a s shown by t e s t s w i t h H~SOL, and a d d i t i o n a l B a C 1 2 ) . The m i x t u r e was f i l t e r e d t h r o u g h Darco G-60 and t h e f i l t r a t e l y o p h i l i z e d . The p u r e mannosidostreptomycin t r i h y d r o c h l o r i d e can be c r y s t a l l i z e d i f desired14. The s e c o n d method o f s e p a r a t i n g m a n n o s i d o s t r e p t o m y c i n from s t r e p t o m y c i n i n v o l v e s t h e u s e of C r a i g ' s l i q u i d - l i q u i d c o u n t e r c u r r e n t d i s t r i b u t i o n p r i n c i p l e which T i t u s and F r i e d 1 h a d u s e d t o show t h e c o n t a m i n a t i o n of s t r e tomycin p r e p a r a t i o n s by mannos i d o s t r e p t o m y c i n . O'Keefe e t used a borate-phosphate buff e r (pH 0 . 7 5 ) as t h e s t a t i o n a r y p h a s e a n d l a u r i c a c i d i n P e n t a s o l R as t h e moving p h a s e . (When t h e two s o l u t i o n s were s t i r r e d t o g e t h e r t h e pH of t h e two p h a s e s was a d j u s t e d t o pH 7.15 u s i n g N a O H ) . O p e r a t i o n s were c a r r i e d o u t u s i n g 11 onel i t e r s e p a r a t o r y f u n n e l s w i t h 495 m l o f s o l v e n t and 450 m l of b u f f e r i n e a c h f u n n e l . Each f e e d i n c r e m e n t c o n s i s t e d o f 4 . 5 g o f s t r e p t o m y c i n complex ( c o n t a i n i n g 6 7 % s t r e p t o m y c i n a n d 3 3 % mannosidostreptomycin). 9 3 % o f t h e s t r e p t o m y c i n was r e c o v e r e d The p r o c e s s i n p u r e form and 9 9 % of t h e m a n n o s i d o s t r e p t o m y c i n . was s c a l e d - u p ( b y u s i n g c e n t r i f u g e s i n s t e a d o f t h e s e p a r a t o r y f u n n e l s ) i n t h e p i l o t p l a n t s i z e equipment15. The t h i r d method u t i l i z e d i o n - e x c h a n g e c h r o m a t o r a p h y of t h e streptomycin-mannosidostreptomycin b o r a t e complex :
fi
Crude s t r e p t o m y c i n h y d r o c h l o r i d e , a s s a y i n g 525 mcg/mg a n d c o n t a i n i n g 4 % m a n n o s i d o s t r e p t o m y c i n was d i s s o l v e d i n water t o g i v e a c o n c e n t r a t i o n o f 4 . 3 g p e r l i t e r . T h i s s o l u t i o n was p a s s e d t h r o u g h a column o f A m b e r l i t e IRC-50 i o n e x c h a n g e r e s i n (Nat c y c l e ) o f 1 3 m l c a p a c i t y u n t i l t h e s a t u r a t i o n of r e s i n was obtained. The column was r i n s e d w i t h d i s t i l l e d water and t h e n washed a t a r a t e o f 5 m l 2 . 5 % b o r i c a c i d s o l u t i o n p e r min. The m a n n o s i d o s t r e p t o m y c i n was l o c a t e d i n t h e f i r s t 4 0 0 m l o f t h e e l u a t e and c o u l d b e r e c o v e r e d by r e a b s o r p t i o n on A m b e r l i t e I R C 5 0 (Na4 c y c l e ) and e l u t i n g w i t h H2SO4. P e r e i r a 1 6 h a s u t i l i z e d ion-exchange chromatography t o
602 p u r i f y streptomycin, mannosidostreptomycin, and r e l a t e d bases from a c r u d e m i x t u r e . H e f o u n d A m b e r l i t e CG-50 t y p e I1 (200 t o 400 mesh) b e s t f o r t h i s p u r p o s e . H i s columns were 2.54 x 76 cm a n d t h e r e s i n was i n t h e sodium c y c l e . 1.5 g o f t h e m i x t u r e was a p p l i e d t o t h e column a n d 0.50 t o 1.20 M N a C l g r a d i e n t was u s e d as e l u a n t . The flow r a t e was 1 . 4 ml/min. a n d 20 m l f r a c t i o n s were c o l l e c t e d . F i v e m a l t o l - p o s i t i v e compounds were o b t a i n e d , a n d m a n n o s i d o s t r e p t o m y c i n was w e l l s e p a r a t e d from t h e o t h e r compounds.
2.3 L i t e r a t u r e
Cited
1. J. F r i e d a n d E. T i t u s , J. B i o l . Chem. 1 6 8 (1947) 391-392.
2. A. L. Demain and E . I n a m i n e , Bact. Rev. 34 (1970) 1 - 1 9 . 3. D. Perlman and A. F. L a n g l y k k e , J . A m e r . Chem. Soc. 70 (1948) 3968. 4 . A. F. Langlykke a n d D. P e r l m a n , U.S. Jan. 3, 1950.
P a t e n t 2,493,489;
5. J. F r i e d a n d E . T i t u s , J . Amer. Chem. Soc. 70 (1948) 36153618. 6. J. F r i e d and H. E. S t a v e l y , J. h e r . Chem. S o c . 74 (1952) 5461-5468. 7. E. T i t u s and J . F r i e d , J. B i o l . Chem. 1 6 8 (1947) 393-394. 8. A. E. O ’ K e e f f e , M. A . D o l l i v e r a n d E. T. S t i l l e r , J. h e r . Chem. Soc. 71 (1949) 2452-2457. 9. 0. B. F a r d i g a n d I . R . Hooper, U . S . 13, 1965. 10. W.
P a t e n t 2,717,892; S e p t .
B.. Emery and A. D. Walker, A n a l y s t 74 (1949) 455-457.
11. J, A. Kowald and R . B . McCormack, A n a l . Chem. 21 (1949) 1383-1384. 12. G. W. E . P l a u t and R. B . McCormack, J. Amer. Chem. S o c . 71 (1949) 2264-2265. 13. E. T i t u s a n d J . F r i e d , J . B i o l . Chem. 174 (1948) 57.
14. L. J. H e u s e r , M. A. D o l l i v e r a n d E. T. S t i l l e r , J. Amer. Chem. SOC. 70 (1948) 2833-2834. 1 5 . C. R. Bartels and G . Keliman, Chem. Eng. P r o g . 45 (1949)
589-594. 1 6 . J. N. P e r e i r a , J . Biochem. M i c r o b i o l . Tech. Eng. 3 (1961)
79-85.
603 3. Dihydrostreptomycin
3.1 Introduction 3.1.1
Producing organisms
Although most of the dihydrostreptomycin used in clinical therapy has been produced by chemical reduction of streptomycin 1’2, some dihydrostreptomycin has been produced on a commercial scale by direct fermentation processes. Dihydrostreptomycin is biosynthesized by Sthcptomyceo h u m i d u ~and ~ ~two ~ unidentified streptomycetes M43-05026 and M43-052275. It is also known as antibiotic 235724. 3.1.2
Brief chemical description
Dihydrostreptomycin has an empirical formula of C 2 1 H 4 1 N 7 Its structural relationships to the other streptomycins are shown in Figures 1 and 2. The trihydrochloride and sesquisulfate are very soluble in water, and slightly soluble in most organic solvents. The potency is determined by bioassay6 with the sesquisulfate containing 800 pg base per mg. Ol2.
3.2 Summary of
therapeutic use
Dihydrostreptomycin is clinically used for treatment of .tuberculosis, systemic infections by gram-negative bacteria, and bacterial infections of the urinary tract. The usual dose is 0.5 to 1 g per day by intramuscular injection.
3.3 Extraction, separation and purification Several methods have been suggested for recovery of dihydrostreptomycin from media fermented by S. humidud. Those de’ ~ ’ ~ vised by Tatsuoka e t ~ 1 . ~ include: 1.
Purification process with activated carbon: Cultured Broth
1
Filtrate
4
a d j u s t e d t o pH 8.5 adsorbed on 1%a c t i v a t e d C
Waste b r o t h
Activd. Carbon extd. with 30, 10 vols. HC1-MeOH a t pH 2.0 Extract
4I
Waste carbon
concd. t
NaCl
Ln
adjusted t o pH 7.0 with N NaOH
vucuo
Conce’n t r a t e acetone added
604
P u r i f i c a t i o n p r o c e s s u s i n g I R C - 5 0 ion-exchange r e s i n .
2.
Cultured broth
I
Filtrate Oxalic a c i d added Ca oxalate
a d j u s t e d t o pH 7.0-8.0
adjuated t o pH 8.0 with N NaOH H7.O
I
N.HC1
N.HC1
I
I
1
I
I
I
I
I
I
Effluent Washings Eluate
Effluent Washings Eluate Ambe r l it e IR-45 ( OH) concd.
concd.
i n vacuo
in vacuo
Concentrate
Concentrate
3.
1
Ambe r l it e IRC-50(Na)
Amberlite IRC-SO(H)
P u r i f i c a t i o n p r o c e s s using o r g a n i c s o l v e n t . CuLt-tuped b r o t h
I
Filtrate extd. with n-BuOH a t pH 7.5, c a r r i e r : l a u r i c a c i d
I
n-BuOH
Waate b r o t h
-
HCl-HZO added Water l a y e r (pH 2.0) B
Water l a y e r ether l a u r i c acid
n-E(uOH H20 added n-BuOH
adjusted t o pH 6.5 w i t h N NaOH concd. in V ~ U O Con’cent r a t e
Chromatography on a c t i v a t e d carbon was u s e d t o p u r i f y t h e d i h y d r o s t r e p t o m y c i n f u r t h e r . The size of t h e a c t i v a t e d c a r b o n was e s p e c i a l l y i m p o r t a n t and t h a t w i t h i n t h e r a n g e 1 5 0 t o 1 8 0 mesh was f a v o r e d . The a n t i b i o t i c was e l u t e d u s i n g d i s t i l l e d water All d i h y d r o s t r e p t omycin -con t a i n i n g f r a c t i o n s w e r e combined and c o n c e n t r a t e d i n V ~ C U Oa l m o s t t o d r y n e s s . Dehydrat i o n by a c e t o n e gave t h e amorphous d i h y d r o s t r e p t o m y c i n t r i h y d r o -
.
605
chloride. T h i s was c o n v e r t e d t o t h e c r y s t a l l i n e d i h y d r o s t r e p t o mycin t r i h y d r o ~ h l o r i d e ~ .
3.4 L i t e r a t u r e
Cited
1. Q. B a r t z , J. C o n t r o u l i s , H . M . C r o o k s a n d M . J . Amer. Chem. SOC. 6 8 ( 1 9 4 6 ) 2 1 6 3 - 2 1 6 6 .
C. R e b s t o c k ,
2 . J. F r i e d a n d 0. W i n t e r s t e i n e r , J . Amer. Chem. S O C . 6 9 ( 1 9 4 7 ) 79-86. 3 . S. T a t s u o k a a n d S. H o r i i , P r o c . J a p a n A c a d . S c i . 39 ( 1 9 6 3 ) 314-318. 4 . S. T a t s u o k a , T . K u s a k a , A. M i y a k e , M . I n o u e , H . H i t o m i , Y . S h i r a i s h i , H . I w a s a k i a n d M. I m a n i s h i Chem. P h a r m . B u l l . 5 ( 1 9 5 7 ) 343-349.
,
5 . F. K a v a n a g h , E . G r i n n a n , E. A l l a n s o n a n d D. T u n i n , A p p l . Microbial; 8 ( 1 9 6 0 ) 1 6 0 - 1 6 2 . 6 . D. C. Grove a n d W. A. R a n d a l l . A s s a y M e t h o d s of Antib o t i c s . A L a b o r a t o r y Manual. Medical E n c y c l o p e d i a , I n c . , N e w Y o r k p. 3 4 - 4 3 , 1 9 5 5 . 7 . K. N a k a z a w a , M. S h i b a t a , K . T a n a b e a n d H . Yamamoto, U S. P a t e n t 2,931,756; A p r i l 5, 1960. 8 . S. T a t s u o k a , T. K u s a k a , M . M i y a k e , M. I n o u e , Y. S h i r a s h i , H. I w a s a k i a n d M. I m a n i s h i , U.S. P a t e n t 2 , 9 5 0 , 2 7 7 ; Aug. 2 3 , 1960. Japan P a t e n t 7,044; June 8, 1961. Japan P a t e n t 7,199; J u n e 1 0 , 1961. Japan P a t e n t 8,947; June 28, 1961. Japan P a t e n t 9,195; June 30, 1961. 9 . F. J . Wolf, E . T. E l m e n d o r f , R . G. D e n k e w a l t e r a n d M . T i s h l e r , S c i e n c e 1 0 9 (1949) 515-516.
606
4. Hydroxystreptomycin
4 . 1 Introduction 4.1.1
P r o d u c i n g organisms
The a n t i b i o t i c i s produced by S Z n e p t o m y c e d g&ideocanneud NRRL B-1067 and NRRL B-10681'2, S t n e p t o m y c e d n u b n i & e t i c u l i 3 " + and S t n e p t o m y c e d ~ u b ~ u t i l uwhen ~ 5 grown i n a p p r o p r i a t e media. I t i s a l s o known as r e t i ~ u l i n ~ and ' ~ NA-232-M17. 4.1.2
B r i e f chemical d e s c r i p t i o n
The p u r i f i e d m a t e r i a l i s a b a s e w i t h t h e e m p i r i c a l f o r m u l a C,1;39N7013' and t h e r o t a t i o n of t h e t r i h y d r o c h l o r i d e i s -95 ( C 1, H20). There i s o n l y end a b s o r p t i o n i n t h e r a n g e ?20 nm t o 400 nm o f t h e s p e c t r u m . The s t r u c t u r e and r e l a t i o n s h i p s of h y d r o x y s t r e p t o m y c i n t o t h e o t h e r members of t h e s t r e p t o m y c i n group are shown i n F i g u r e s 1 and 2 .
4.2 Summary
of t h e r a p e u t i c u s e
%aCik?eUAd u b t i l i d h a s been used i n t h e a g a r - d i f f u s i o n b i o a s s a y f o r hydroxys t r e p t o m y c i n 2 7 , Although h y d r o x y s t r e p t o mycin i n h i b i t s t h e growth of g r a m - p o s i t i v e and gram-negative b a c t e r i a i n v i t & o and i n V ~ V Ot h e t o x i c i t y i s v e r y similar t o t h a t of s t r e p t o m y c i n , and h y d r o x y s t r e p t o m y c i n h a s n o t been u s e d f o r t h e r a p y o f human i n f e c t i o n s g .
li.3 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n
S t o d o l a e x a L . * used t h e f o l l o w i n g p r o c e d u r e t o i s o l a t e h y d r o x y s t r e p t o m y c i n from media f e r m e n t e d w i t h S . QAideocanncuA: To e a c h l i t e r of f i l t e r e d c u l t u r e l i q u o r was added 11 g of Nuchar C-250N and t h e m i x t u r e s t i r r e d f o r 3 0 min. The carbon was removed by f i l t r a t i o n and washed i n s u c c e s s i o n w i t h 1 l i t e r o f water, 1 0 0 m l of 5 0 % aqueous e t h a n o l , and 1 0 0 m l of a b s o l u t e methanol. The a i r d r i e d c a r b o n was s t i r r e d f o r 5 min. w i t h s u f f i c i e n t methanol - H C 1 m i x t u r e ( p r e p a r e d by a d d i n g 0.5 ml o f c o n c e n t r a t e d H C 1 t o 1 l i t e r of m e t h a n o l ) t o p e r m i t s t i r r i n g . A f t e r f i l t r a t i o n t h e f i l t r a t e was c o n c e n t r a t e d i n Vacua t o onef o u r t h volume and added t o 10 volumes of a c e t o n e . The p r e c i p i t a t e was c o l l e c t e d and d r i e d i n vacua t o g i v e a somewhat hygros c o p i c t a n powder a s s a y i n g a b o u t 30 t o 4 0 % h y d r o x y s t r e p t o m y c i n . T h i s c r u d e p r o d u c t (25 g ) was d i s s o l v e d i n 50 m l o f water and t h e pH a d j u s t e d t o 5.8. The i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n and t h e s o l u t i o n p l a c e d on a 6.5 and b 5 c m c o l umn o f acid-washed Harshaw alumina ( a d j u s t e d t o pH 4.7). Eighty p e r c e n t aqueous methanol was used f o r development of t h e chromatogram and 1 0 0 m l f r a c t i o n s were c o l l e c t e d . About 75% of t h e b i o a c t i v i t y was o b t a i n e d i n f r a c t i o n s 9 through 16 and t h e methanol i n t h e s e f r a c t i o n s was removed i n vacuo. The r e s u l t i n g aqueous s o l u t i o n s were l y o p h i l i z e d t o w h i t e powders t h e p u r i t i e s of which r a n g e s from 80 t o 9 0 % . T h e crude h y d r o x y s t r e p t o m y c i n was f u r t h e r p u r i f i e d by p r e p a r a t i o n of t h e h e l i a n t h a t e as f o l l o w s : 1 . 5 4 g of c r u d e hydroxy-
60 7
s t r e p t o m y c i n o f a b o u t 80% p u r i t y was d i s s o l v e d i n 4 4 m l of 50% aqueous m e t h a n o l . 2 . 2 g of methyl orange d i s s o l v e d i n 150 m l of water ( a t 55') were added t o t h i s s o l u t i o n and t h e r e a c t i o n m i x t u r e was c o o l e d i n t h e r e f r i g e r a t o r o v e r n i g h t . The c r y s t a l s of t h e h y d r o x y s t r e p t o m y c i n m e t h y l o r a n g e complex were s e p a r a t e d by c e n t r i f u g a t i o n and washed twice w i t h water. R e c r y s t a l l i z a t i o n from 3 3 % aqueous methanol gave 1 . 8 g o f geddish-brown c r y s t a l s of h e l i a n t h a t e which were d r i e d a t 1 Q Q i n vacuo t o c o n g ta n t weight. The h e l i a n t h a t e s t a r t e d t o d a r k e n a t a b o u t 2 2 0 and s l o w l y c h a r r e d w i t h o u t m e l t i n g . I t s X-ray d i f f r a c t i o n p a t t e r n c o u l d s c a r c e l y be d i s t i n g u i s h e d from t h a t o f s t r e p t o m y cin helianthate.
-
P u r e h y d r o x y s t r e p t o m y c i n t r i h y d r o c h l o r i d e was p r e p a r e d by t a k i n g 700 mg of t h e p u r e h e l i a n t h a t e a n d g r i n d i n g i t v i g o r o u s l y f o r 1 min. w i t h 11 m l o f a m e t h a n o l i c H C 1 m i x t u r e (1 m l of conc e n t r a t e d H C 1 added t o 50 m l of m e t h a n o l ) . The s o l u t i o n was f i l t e r e d t h r o u g h a pad of Darco G-60 and t h e c o l o r l e s s f i l t r a t e was added d r o p w i s e t o 150 m l o f e t h y l e t h e r . The w h i t e p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and washed twice w i t h e t h y l 2 6 8 mg o f t h e h y d r o x y s t r e p t o m g c i n t r i h y d r o c h l o r i d e were ether. r e c o v e r e d a f t e r d r y i n g i n vacuo a t 8 0
.
4.4 Literature
Cited
1. R . G . B e n e d i c t , F. H. S t o d o l a , 0. L . S h o t w e l l , A. M. and L. A. L i n d e n f e l s e r , S c i e n c e 1 1 2 ( 1 9 5 0 ) 77-78.
Borud
2 . R . G . B e n e d i c t , L. A . L i n d e n f e l s e r , F. H . S t o d o l a a n d D. H. T r a u f l e r , J. Bact. 6 2 ( 1 9 5 1 ) 487-497. 3 . S. Hosoya, M. Soeda, N. Med. 2 0 ( 1 9 4 9 ) 327-335.
Komatsu and Y . Sonoda, J a p a n J. Exp.
4 . S. Hosoya, M. S o e d a , N . Komatsu and Y . Sonoda, J . A n t i b i o t i c s 3 (Supp.A) ( 1 9 5 0 ) 66-71. 5. T. Arai, S. Kuroda, S . Yamagishi and Y . Katoh, J . A n t i b i o t i c s 1 7 ( A ) ( 1 9 6 4 ) 23-28. Hosoya, M. Soeda, N . Komatsu, N . Hara and T . Yamaguchi, J. A n t i b i o t i c s 6 ( A ) ( 1 9 5 3 ) 1 0 2 ; 6 ( B ) ( 1 9 5 3 ) 61-66.
6 . S.
7. W. E. Grundy, A. L. Whitman, M . E . Hanes and J . C. S y l v e s t e r , A n t i b i o t i c s and Chemotherapy 1 ( 1 9 5 1 ) 309-317. 8 . F. H. S t o d o l a , 0. L. S h o t w e l l , A. M. Borud, R . G . B e n e d i c t and A. C. R i l e y , J . h e r . Chem. SOC. 73 ( 1 9 5 1 ) 2290-2293.
9 . W . E. Grundy, J . R . Schenck, R. K . C l a r k , M . P. H a r g i e , R . K . R i c h a r d s and J . C . S y l v e s t e r , Arch. Biochem. 28 ( 1 9 5 0 ) 150152.
60 8
5. Mannosidohydroxystreptomycin
5.1 I n t r o d u c t i o n 5.1.1
Producing organisms
T h i s a n t i b i o t i c i s p r o d u c e d by a n u n i d e n t i f i e d s t r e p t o m y cete which a l s o p r o d u c e s h y d r o x y s t r e p t o m y c i n , s t r e p t i d i n e , a n d toyokamycin 2. 5.1.2
Brief chemical d e s c r i p t i o n
L i t t l e i n f o r m a t i o n i s a v a i l a b l e on t h e b i o l o g i c a l a c t i v i t y l . The c h e m i c a l r e l a t i o n s h i p s t o t h e o t h e r s t r e p t o m y c i n s i s shown i n F i g u r e s 1 and 2 .
5.2 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n
The mannosidohydroxystreptomycin was p u r i f i e d 2 by s t a n d a r d a b s o r p t i o n - e l u t i o n p r o c e d u r e s on c a t i o n e x c h a n g e r e s i n s t o g i v e a m i x t u r e o f s u l f a t e s which were s e p a r a t e d by chromatoThe g r a p h y on a column o f a c t i v a t e d c h a r c o a l and f i l t e r - a i d . e l u t i o n was c a r r i e d o u t w i t h d i s t i l l e d water f o l l o w e d by 0 . 1 N H2S04 i n 50% a q u e o u s m e t h a n o l . The f o l l o w i n g amounts were obt a i n e d from 2 0 l i t e r s o f f e r m e n t e d medium: 1 . 6 g s t r e p t i d i n e s u l f a t e ; 5 . 8 g h y d r o x y s t r e p t o m y c i n s u l f a t e ; 6.4 g mannosidoh y d r o x y s t r e p t o m y c i n s u l f a t e ; a n d 1 . 2 g toyokamycin. The mannos i d o h y d r o x y s t r e p t o m y c i n was p u r i f i e d f u r t h e r t h r o u g h t h e crys t a l l i n e B - n a p h t h a l e n e s u l f o n a t e and i s o l a t e d as t h e h y d r o c h l o r ide.
5.3 L i t e r a t u r e
Cited
1. C. S p a l l a , L. T o g n o l i , A. G r e i n and G . Canevazzi i n Biogenes i s o f A n t i b i o t i c S u b s t a n c e s . E d i t e d by Z. Vanek and Z. H o s t a l e k , Czechoslovak Academy of S c i e n c e s , P r a g u e , p . 2712 7 9 , 1965. 2 . F. Arcamone, G. C a s s i n e l l i , G. d’Amico a n d P. O r e z z i , E x p e r i e n t i a 2 4 ( 1 9 6 8 ) 441-442.
609 6 . Zygomycin B
6 . 1 Introduction 6.1.1
Producing organisms
T h i s a n t i b i o t i c i s p r o d u c e d . by SLteptomyceb pulueaaceub a l o n g w i t h zygomycin A ( a member o f t h e neomycin g r o u p 1 ) . I t i s a l s o known as 4 5 4 4 9 ~ 2 . 6.1.2
B r i e f chemical d e s c r i p t i o n
The p u r i f i e d m a t e r i a l i s a b a s e and t h e s u l f a t e i s v e r y water s o l u b l e . The m e l t i n g p o i n t o f t h e f r e e b a s e i s 178'-188' and t h i s material g a v e t h e f o l l o w i n g a n a l y s i s : C , 3 4 . 2 9 % ; H , 6 . 3 7 % ; and N 12.006% ( a s s u l f a t e ) 2 . The r o t a t i o n of t h e s u l f a t e was I a l i 2 - 8 4 ( c 1, MeOH) a n d t h e r e was o n l y e n d a b s o r p t i o n i n t h e r a n g e 2 2 0 nm t o 4 0 0 nm. The p u r i f i e d material g a v e a p o s i t i v e S a k a g u c h i t e s t and n e g a t i v e Elson-Morgan, n i n h y d r i n , and m a l t o l t e s t s . Zygomycin B i s d i s t i n g u i s h e d f r o m d i h y d r o s t r e p t o m y c i n and dihydrodeoxystreptomycin on t h e b a s i s of X-ray d i f f r a c t i o n p a t t e r n s and mixed m e l t i n g p o i n t d e p r e s s i o n .
6.2 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
The p r o c e d u r e s u s e d f o r t h e i s o l a t i o n of zygomycin B i n v o l v e d t h e f o l l o w i n g 2 ,3 : The f i l t e r e d b r o t h was t r e a t e d w i t h o x a l i c a c i d t o remove calcium i o n a n d t h e s t i l l r e m a i n i n g calcium i o n was complexed by a d d i t i o n o f ethylenediaminetetraacetate. The s o l u t i o n was t h e n a d j u s t e d t o pH 7 t o 8 , a n d t h e a c t i v e components were a b The r e s i n was s o r b e d on A m b e r l i t e I R C - 5 0 r e s i n (Na' c y c l e ) . washed w i t h water and t h e zygomycins e l u t e d w i t h 0 . 5 t o 1 N H C 1 . The e l u a t e was c o n c e n t r a t e d i n v a c u o , c h r o m a t o g r a p h e d on a c t i v a t e d c h a r c o a l , a n d e l u t e d w i t h water. The i n o r g a n i c s a l t s were found i n t h e f i r s t f r a c t i o n s , f o l l o w e d b y n i n h y d r i n - p o s i t i v e f r a c t i o n (zygomycin A ) , and t h e n a S a k a g u c h i - p o s i t i v e f r a c t i o n (zygomycin B). The zygomycin A a n d B c o n t a i n i n g f r a c t i o n s were c o n c e n t r a t e d s e p a r a t e l y , a n d upon t h e a d d i t i o n of a c e t o n e t o e a c h c o n c e n t r a t e , zygomycins A and B h y d r o c h l o r i d e s p r e c i p i t a ted. P a p e r c h r o m a t o g r a p h y o f t h e B p r e p a r a t i o n showed c o n t a m i n a t i o n w i t h zygomycin A and t h i s B p r e p a r a t i o n was p u r i f i e d f u r t h e r as f o l l o w s : The c r u d e s a l t was d i s s o l v e d i n water, a b s o p b e d on I R C - 5 0 r e s i n (NH4' c y c l e ) , and e l u t e d w i t h a b o u t 5 % a q u e o u s N H 4 0 H ( w h i c h washed o u t zygomycin A and n o t B). The r e s i n column was t h e n washed w i t h water and e l u t e d w i t h 0 . 5 N H C 1 , a n d t h e res u l t i n g B-containing f r a c t i o n , a f t e r c o n c e n t r a t i o n in vacuo, was c h r o m a t o g r a p h e d on a c t i v a t e d c h a r c o a l . The chromatogram was d e v e l o p e d w i t h water, and i n o r g a n i c s a l t s were e l u t e d f i r s t f o l l o w e d by t h e S a k a g u c h i - p o s i t i v e f r a c t i o n . The l a t t e r was c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e and a c e t o n e was a d d e d t o t h e c o n c e n t r a t e . The zygomycin B h y d r o c h l o r i d e p r e c i p i t a t e d .
610 T h i s was c o n v e r t e d t o t h e s u l f a t e b y t h e p r o c e d u r e s u s e d f o r c o n v e r s i o n of s t r e p t o m y c i n h y d r o c h l o r i d e t o t h e s u l f a t e .
6.3 Literature
Cited
1. E . H i g a s h i d e , T. K a n z a k i , H . Yamamoto, M. S h i b a t a a n d K . N a k a z a w a , A g r . B i o l . Chem. 2 5 ( 1 9 6 1 ) 1 8 8 - 1 9 9 . 2 . H . H i t o m i , S. H o r i i , T . Y a m a g u c h i , M . I m a n i s h i a n d A. M i y a k e , J. A n t i b i o t i c s 1 4 ( A ) (1961) 6 3 - 6 7 . 3. K . N a k a z a w a , M. S h i b a t a , E . H i g a s h i d e , T . K a n z a k i , H. Y a m a rnoto, A. M i y a k e , H. H i t o m i , S . H o r i i , T. Y a m a g u c h i , T. A r a k i , K . T s u c h i y a , T. Oka a n d A. Imai, J a p a n P a t e n t 1 5 , 3 0 0 ; S e p t . 27, 1962.
6 11
7 . Bluensomycin
7.1 Introduction 7.1.1
Producing organisms
T h i s a n t i b i o t i c i s p r o d u c e d by StAeptomyceA b l u e n n i b v a r . and Stheptomyceo hygaoncopicud forma gleboaub2 when grown i n a p r o p r i a t e m e d i a . I t i s a l s o known as U - 1 2 8 9 8 l a n d glebomycin
bluenoin
s.
7.1.2
B r i e f chemical d e s c r i p t i o n
The p u r i f i e d m a t e r i a l i s a b a s e w i t h a pKa of 7 . 6 l and i s o v e r y s o l u b l e i n water. The m e l t i n g p o i n t o f t h e s u l f a t e i s 2 0 5 (dec.). The e m p i r i c a l formul;: i s C21H39N5014. The r o t a t i o n of t h e h y d r o c h l o r i d e i s { a ) 2 0 - 8 9 ( c 1, H20). T h e r e i s o n l y e n d a b s o r p t i o n i n t h e r a n g e 9 1 0 nm t o 4 0 0 nm of t h e s p e c t r u m
.
7.1.3
S t r u c t u r a l formulae
The s t r u c t u r a l f o r m u ~ a ~ -i ~s :,
0 II OCNH
HO HzNCHN iH
HO
7 . 2 Therapeutic
OH
NHCH3
use
Bacillus n u b . t i l i A was u s e d f o r b i o a s s a y o f b l u e n s o m y c i n by t h e a g a r d i f f u s i o n t e c h n i q u e 1 y 6 . A l t h o u g h b l u e n s o m y c i n i n h i b i t s t h e growth o f gram-posi t i v e i t h a s n o t been u s e d f o r c l i n i c a l and g r a m - n e g a t i v e t r e a t m e n t of i n f e c t i o n s .
612
7.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
Two p r o c e d u r e s have been u s e d f o r i s o l a t i o n o f bluensomyThat d e v i s e d by Bergey e t a t . ? was c i n from f e r m e n t e d media. summarized as f o l l o w s : 5,350 l i t e r s o f f e r m e n t e d medium were t r e a t e d w i t h 1 6 kg o f o x a l i c a c i d a n d a d j u s t e d t o pH 2.9 ( w i t h HzSOh). The b r o t h was f i l t e r e d w i t h 3 % (w/v> H y f l o S u p e r Cel a n d t h e c a k e washed w i t h 5 0 0 l i t e r s of water a n d d i s c a r d e d . The comblned c l e a r b r o t h and wash was a d j u s t e d t o pH 7 . 8 t o 8 . 0 w i t h N a O H , c l a r i f i e d by f i l t r a t i o n t h r o u g h D i c a l i t e No. 4200 a n d p a s s e d downf l o w t h r o u g h 9 c u b i c f e e t o f A m b e r l i t e IRC-50 r e s i n (Na' c y c l e , 2 0 t o 50 m e s h ) . The flow r a t e was 19 l i t e r s / m i n . The s p e n t f i l t r a t e was d i s c a r d e d a n d t h e r e s i n was washed w i t h water. The a n t i b i o t i c was e l u t e d by s t i r r i n g t h e r e s i n 4 t i m e s w i t h 2 0 0 l i t e r s of w a t e r a t pH 1 . 2 t o 1 . 5 (HzSO,). The e l u a t e s were p o o l e d and a d j u s t e d t o pH 6 . 4 ( N a O H ) , c o n c e n t r a t e d t o a p p r o x i m a t e l y 0 . 5 volume, a n d t r e a t e d w i t h a c t i v a t e d c a r b o n ( D a r c o G-60). The q u a n t i t y of c a r b o n e q u a l e d t h e w e i g h t o f d i s s o l v e d s o l i d s . The c a r b o n was removed by f i l t r a t i o n , washed w i t h w a t e r , and e l u t e d b a t c h w i s e w i t h 4 0 0 l i t e r s o f 1 5 % a c e t o n e i n water. The e l u a t e s were c o n c e n t r a t e d i n vacuum a n d f r e e z e d r i e d . The f r e e z e - d r i e d material weighed 1 , 8 1 1 g and a s s a y e d 3 30 ug/mg ( a s s a y s t a n d a r d was bluensomycin s u l f a t e ) . A m i x t u r e c o n t a i n i n g 1 6 . 5 kg e a c h o f Darco G - 6 0 a n d D i c a l i t e No. 4 2 0 0 i n 1 2 0 l i t e r s o f w a t e r was p o u r e d i n t o a 1 4 i n c h d i a m e t e r c h r o m a t o g r a p h i c column a n d packed t o a c o n s t a n t bed h e i g h t o f a p p r o x i m a t e l y 4 f t . D r i e d , c r u d e bluensomycin s u l f a t e ( 2 , 7 0 7 g ) , o b t a i n e d by t h e I R C - 5 0 i s o l a t i o n p r o c e d u r e f o l l o w e d by c a r b o n a b s o r p t i o n , was d i s s o l v e d i n 6 , 6 0 0 m l o f w a t e r , a d j u s t e d t o pH 3 . 5 w i t h HzSO4, and packed i n t o t h e p r e p a r e d c a r b o n column. The column was washed w i t h 150 l i t e r s of w a t e r and t h e e l u t e d w i t h 300 l i t e r s of 1 0 % a c e t o n e i n water. The most p o t e n t f r a c t i o n s were combined, c o n c e n t r a t e d , c l a r i f i e d by f i l t r a t i o n , and f r e e z e - d r i e d t o y i e l d 4 9 0 g o f bluensomycin s u l f a t e , a s s a y i n g a p p r o x i m a t e l y 1 , 0 0 0 pg/mg. This preparation was u s e d f o r p r e p a r a t i o n o f t h e d i h y d r o c h l o r i d e .
The p r o c e d u r e u s e d by Miyaki following flow s h e e t :
e t at.* i s summarized i n t h e
Cultured broth f i l t e r pH 5
I
broth f i l t r a t e
I
Amberlite I R C l 5 0 (Na'
I
cycle)
0.1 N HC1
eluate
concd. h uucuo
613
1
concentrates
PH 8
carbon chromatography washed w i t h 0.05% NH40H washed w i t h water washed with a c i d i c 50% M OH
1
eluate pH 6
concd.
in
VUCUO
l y o p h il i z e
I
I
anhydrous k M
I
residue
k O H solution
4-7 acetone
s o l v e n t waste
precipitate
I
dried
glebomycin h y d r o c h l o r i d e
7.4 L i t e r a t u r e
Cited
1. D. J. Mason, A. D i e t z a n d L. J. H a n k a , A n t i m i c r o b . A g . Chemothar. 1 9 6 2 (1963) 607-613. 2 , T. Ohmori, M. O k a n i s h i a n d H . K a w a g u c h i , J . A n t i b i o t i c s 1 5 ( A ) ( 1 9 6 2 ) 21-27. 3 . B . B a n n i s t e r a n d A. D . A r g o u d e l i s , J. Amer. Chem. SOC. (1963) 119-120. 4 . B . B a n n i s t e r a n d A. D. ( 1 9 6 3 ) 234-235.
5 . M. H . G . Munro and K .
85
A r g o u d e l i s , J. Amer. Chem. S O C . 85
L . R i n e h a r t , i n press. ( 1 9 7 6 ) .
6 . M . O k a n i s h i , H . K o s h i y a m a , T . Ohmori, M . M a t s u z a k i , S . O h a s h i , a n d H. K a w a g u c h i , J. A n t i b i o t i c s 1 5 ( A ) ( 1 9 6 2 ) 7 - 1 4 . 7. M .
E: B e r g e y , T. E . E b l e , R . R . H e r r , C . M . L a r g e a n d B . B a n n i s t e r , A n t i m i c r o b . Ag. Chemother. 1 9 6 2 ( 1 9 6 3 ) 6 1 4 - 6 1 8 .
8. T. M i y a k i , H . T s u k i u r a , M. Wakae a n d H . K a w a g u c h i , J. A n t i b i o t i c s 15(A) (1962) 15-20.
614 8. A c t i n o s p e c t a c i n
8.1 Introduction 8.1.1
Producing orpanisms
T h i s a n t i b i o t i c i s p r o d u c e d by Staeptomyced d p e c t a b i l i d NRRL B - 2 4 9 4 l a n d S t 4 e p t O m L j C e d ~ . t a v o p e n d i c u d NRRL B-28202 when It is i d e n t i c a l with the antibiogrown i n a p p r o p r i a t e media. t i c known as M-1412. 8.1.2
B r i e f chemical d e s c r i p t i o n
The p u r i f i e d material i s a b a s e w i t h pKa's o f 6.95 a n d 8.70, and t h e s u l f a t e i s v e r y s o l u b l e i n water. The m e l t i n g p o i n t o f t h e h e x a h y d r a t e i s 650-7204 a n d o f t h e t e t r a h y d r a t e 185' ( d e c . ) . The m o l e c u l a r w e i g h t of t h e base is 346 a n d t h e e m p i r i c a l f o r m u l a i s C14H24N207. The r o t a t o r y d i s p e r s i o n meas u r e m e n t s show i t t o b e g e n e r a l l y d e x t r o r o t a t o r y w i t h an t 7 . 6 ' ( c 1, H2OI4. A c t i n o s p e c t a c i n s u l f a t e shows n o d i s t i n c t i v e u l t r a v i o l e t a b s o r p t i o n i n water b e t w e e n 220 and 4 0 0 nm5. 8.1.3
S t r u c t u r a l formulae
The s t r u c t u r a l f 0 r m u l a 3 ' ~ ' 6 is:
NHCH3 I
OH 8.2 Summary
o f t h e r a p e u t i c use
Kf!QbAie&z pneumoniae ATCC 1 0 0 3 1 i s u s e d f o r b i o a s s a y by e i t h e r the agar diffusion o r the turbidimetric techniques7.
In v i t l r o s t u d i e s h a v e shown a c t i n o s p e c t a c i n t o b e a c t i v e a g a i n s ' t most s t r a i n s of Neiadeaia gonomhaeae ( m . i . c . r a n g i n g from ~ 7 . 5t o 20 pg/ml) a n d t h e a n t i b i o t i c i s used c l i n i c a l l y t o c o n t r o l t h e s e i n f e c t i o n s . The u s u a l d o s e f o r a c u t e g o n o r r h e a l u r e t h r i t i s a n d p r o c t i t i s i n t h e male i s 2 g i n j e c t e d d e e p i n t o t h e u p p e r o u t e r q u a d r a n t o f t h e g l u t e a l m u s c l e . The d o s e f o r acute g o n o r r h e a l c e r v i c i t i s and p r o c t i t i s i n t h e female is 4 g
615 i n j e c t e d i n t o two g l u t e a l i n j e c t i o n s i t e s . A c t i n o s p e c t a c i n i s n o t e f f e c t i v e i n t h e t r e a t m e n t of s y p h i l i s .
8.3 E x t r a c t i o n ,
s e p a r a t i o n and p u r i f i c a t i o n
Two p r o c e s s e s have been d e s c r i b e d f o r i s o l a t i o n o f a c t i n o s p e c t a c i n from f e r m e n t e d media: That u s e d by Bergey e t 4 . 8 l i t e r s of c u l t u r e b r o t h c o n t a i n i n g 1 . 2 3 mg/ml of a c t i n o s p e c t a c i n ( a s f r e e b a s e e q u i v a l e n t ) was f i l t e r e d a t h a r v e s t (pH 7 . 4 ) w i t h 3% (w/v) of D i c a l i t e 4 2 0 0 f i l t e r a i d and t h e f i l t e r c a k e was washed w i t h 480 m l o f water. The combined c l e a r b e e r and water wash, c o n t a i n i n g 1 mg/ml a c t i n o s p e c t a c i n was p a s s e d up-flow t h r o u g h a column c o n t a i n i n g 96 m l of A m b e r l i t e IRC-50 (H' c y c l e ) ( 1 6 t o 50 mesh) a t a flow r a t e of 1 2 ml/min. A f t e r t h e c l e a r b e e r was p a s s e d t h r o u g h , t h e r e s i n was washed w i t h 2 0 0 m l of water and t h e s p e n t e f f l u e n t a n d wash were d i s c a r d e d . The a n t i b i o t i c was e l u t e d by s t i r r i n g t h e r e s i n i n 4 0 0 m l o f water f o r 30 min. a t pH 1 . 8 (H,SOL,). The r e s i n was removed by f i l t r a t i o n and washed w i t h 1 0 0 m l o f water. The wash and t h e e l u a t e were p o o l e d and t h e excess a c i d p r e s e n t removed by t i t r a t i n g t h e combined e l u a t e and wash t o pH 6.0-7.0 w i t h Dowex 2 x 8 (OH-cycle). The r i c h s o l u t i o n w a s o f i l t e r e d and t h e e l u a t e c o n c e n t r a t e d i n vaCuO a t l e s s t h a n 50 a n d l y o p h i l i z e d . The f r e e z e - d r i e d p r o d u c t weighed 1 2 . 8 g and a s s a y e d 315 pg/mg ( 6 9 % r e c o v e r y from t h e f e r m e n t e d medium).
The c r u d e a c t i n o s p e c t a c i n s u l f a t e was t h e n chromatographed on Darco G-60 (column p r e p a r e d by mixing 375 g Darco G-60 w i t h 375 g of C e l i t e 545 f i l t e r a i d and p o u r i n g t h e w e t m i x t u r e i n t o a 2 i n c h column t o g i v e a packed l e n g t h o f 4 1 i n c h e s ) : 75 g o f sample were d i s s o l v e d i n 1 0 0 m l of water and t h e s o l u t i o n a p p l i e d t o t h e column. The column was washed w i t h 4 l i t e r s o f water. I t was t h e n e l u t e d w i t h 1%aqueous a c e t o n e . The a c t i v e f r a c t i o n s were p o o l e d and l y o p h i l i z e d t o g i v e 7.5 g of material a s s a y i n g 543 pg/mg (18% r e c o v e r y of a c t i v i t y from t h e l y o p h i l i zed powder). Four g of t h e carbon-chromatographed m a t e r i a l were d i s The s o l s o l v e d i n 1 0 ml of watzr and c l a r i f i e d by f i l t r a t i o n . u t i o n was h e a t e d t o 50 and 2 0 0 m l of 95% E t O H was added s l o w l y w i t h s t i r r i n g . The s o l u t i o n was s t i r r e d f o r 30 min and t h e n c o o l e d a t 5' o v e r n i g h t . The c r y s t a l s were c o l l e c t e d by f i l t r a 2.6 t i o n and washed w i t h 5 % aqueous E t O H f o l l o w e d by 9 5 % E t O H . g of c r y s t a l s (630 pg/mg) were r e c o v e r e d a f t e r vacuum d r y i n g . R e c r y s t a l l i z a t i o n from aqueous a c e t o n e gave a c t i n o s p e c t a c i n s u l f a t e t e t r a h y d r a t e a s s a y i n g 6 6 2 pg/mg ( e s s e n t i a l l y p u r e a c t i n o spectacin sulfate). The o v e r a l l r e c o v e r y from t h e f e r m e n t e d medium was a b o u t 1 4 % . The s e c o n d method summarized by S i n c l a i r and W i n f i e l d 8 included: T r e a t m e n t of t h e whole b e e r w i t h s u f f i c i e n t o x a l i c a c i d t o p r e c i p i t a t e t h e calcium i o n w h i l e a t t h e same time t h e pH was m a i n t a i n e d a t pH 6-7 w i t h NH40H. A f t e r f i l t r a t i o n ( w i t h t h e h e l p of f i l t e r a i d ) , t h e c l a r i f i e d b e e r was p a s s e d o v e r a
616
column of IRC-50 ( N a ' c y c l e ) r e s i n . (Note: One l i t e r o f I R C 50 (Na' c y c l e ) h o l d s a b o u t 1 8 g a c t i n o s p e c t a c i n b a s e ) . A f t e r washing t h e column w i t h w a t e r , t h e a n t i b i o t i c was e l u t e d w i t h 0 . 5 N H C 1 , and t h e pH o f t h e c o l l e c t e d f r a c t i o n s was a d j u s t e d t o pH 6 . The f r a c t i o n s w i t h h i g h b i o l o g i c a l a c t i v i t y were comb i n e d and e v a p o r a t e d t o a small volume. The a n t i b i o t i c p r e c i p i t a t e d along with t h e NaC1. The p r e c i p i t a t e was e x t r a c t e d w i t h MeOH ( w h i c h d i s s o l v e d t h e a c t i n o s p e c t a c i n and l e f t most of t h e N a C l b e h i n d ) and t h e MeOH was e v a p o r a t e d c a r e f u l l y ( w i t h a d d i t i o n of some w a t e r a t t h e e n d ) . The a c t i n o s p e c t a c i n h y d r o c h l o r i d e c r y s t a l l i z e d from t h e c o o l e d c o n c e n t r a t e d s o l u t i o n . The p r o d u c t t h u s o b t a i n e d was a t l e a s t 85% p u r e , and i t was u s u a l l y n e c e s s a r y t o r e d i s s o l v e i t i n MeOH and d e c o l o r i z e w i t h c a r b o n b e f o r e r e c r y s t a l l i z i n g . The a c t i n o s p e c t a c i n b a s e and i t s s a l t s a r e v e r y s o l u b l e i n w a t e r and p y r i d i n e , and m o d e r a t e l y s o l u b l e i n MeOH. They are almost i n s o l u b l e i n o t h e r common o r g a n i c s o l v e n t s . 8.4 L i t e r a t u r e -
Cited
1. D. J . Mason, A. D i e t z a n d R . M . t h e r a p y 11 ( 1 9 6 1 ) 1 1 8 - 1 2 2 .
S m i t h , A n t i b i o t i c s and Chemo-
2 . T. J . O l i v e r , A. G o l d s t e i n , R . R . Bower, J . C . H o l p e r a n d R . H. O t t o , A n t i m i c r o b . Ag. Chemotherap.-1961 ( 1 9 6 2 ) 495-502.
3. H . Hoeksma, A. D. A r g o u d e l i s a n d P. F. W i l e y , J . Amer. Chem. SOC. 84 ( 1 9 6 2 ) 3212-3213. 4 . P. F. W i l e y , A. D . A r g o u d e l i s and H. Hoeksma, J . Amer. Chem. SOC. 85 ( 1 9 6 3 ) 2652-2659.
5 . M. E . B e r g e y , T. E . E b l e a n d R . R. H e r r , A n t i b i o t i c s and Chemotherapy 11 ( 1 9 6 1 ) 661-664. M. S t r o s h a n e , M. T a n i g u c h i , K . L. R i n e h a r t , J . P. R o l l s , W . J. Haak a n d B . A . R u f f , J. h e r . Chem. S O C . 9 8 ( 1 9 7 6 ) 3025-3027.
6 . R.
7 . W . T. S o k o l s k i .in A n a l y t i c a l M i c r o b i o l o g y . E d i t o r , F. Kavanagh, Academic P r e s s , I n c . , v o l . 11, 339-341, 1 9 7 2 .
8. A. C . S i n c l a i r and A . F. W i n f i e l d , A n t i m i c r o b . Ag. Chemot h e r a p . - l 9 6 1 ( 1 9 6 2 ) 503-506.
617 Streptothricins and Related Antibiotics
. .
A S Khokhlov Shemyakin Institute of Bioorganic Chemistry. USSR Academy of Sciences. Moscow
1
.
2
.
General introduction
...............................
625
Streptothricins F. E. D. C. B and A (usual streptothricins) 2.1 Introduction 2.1.1 Producing organisms 2.1.2 Brief chemical description 2.1.3 Structural formula 2.2 Summary of therapeutic use 2.3 Extraction. separation and purification
................................. ........................ 630 630 ................. 630 ......................... ................... 632 636
...... 641 3 . Properties of individual streptothricins 3.1 Streptothricin F ............................. 652 Introduction ............................... 652 3.1.1 3.1.2 Producing organisms ........................ 652 3.1.3 Brief chemical description ................. 652 652 3.1.4 Structural formula ......................... 3.1.5 Summary of therapeutic use ................. 653 3.1.6 Extraction. separation and purification .... 653 3.2 Streptothricin E ............................. 653 653 3.2.1 Introduction ............................... 3.2.2 Producing organisms ........................ 653 3.2.3 Brief chemical description ................. 653 653 3.2.4 Structural formula ......................... 3.2.5 Summary of therapeutic use ................. 654 3.2.6 Extraction separation and purification .... 654 Streptothricin D ............................. 654 3.3 Introduction ............................... 654 3.3.1 3.3.2 Producing organisms ........................ 654 3.3.3 Brief chemical description ................. 654 654 Structural formula ......................... 3.3.4 3.3.5 Summary of therapeutic use ................. 654 Extraction. separation and purification .... 655 3.3.6 Streptothricin C ............................. 655 3.4 3.4.1 Introduction ............................... 655 655 Producing organisms ........................ 3.4.2 Brief chemica7 description ................. 655 3.4.3 655 Structural formula ......................... 3.4.4 Summary of therapeutic use ................. 655 3.4.5 -
.
3.4.6
3.5 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 3.5.6
.... 655 ............................. 656 ............................... 656 ........................ 656 ................. 656 ......................... 656 ................. 656
Extraction. separation and purification Streptothricin B Introduction Producing organisms Brief chemical description Structural formula Summary of therapeutic use Extraction. separation and purification
.... 656
618 3
.
4
.
Properties of individual streptothricins (continued) Streptothricin A. Introduction 3.6.2 Producing organisms 3.6.3 Brief chemical description 3.6.4 Structural formula 3.6.5 Summary of therapeutic use 3.6.6 Extraction. separation and purification 3.7 Streptothricin X 3.7.1 Introduction 3.7.2 Producing organism 3.7.3 Brief chemical description 3.7.4 Structural formula 3.7.5 Summary of therapeutic use 3.7.6 Extraction. separation and purification
............................ 656 ............................... 656 656 ........................ ................. 657 657 ......................... 657 ................. .... 657 657 ............................. 657 ............................... ......................... 657 ................. 657 657 ......................... 658 ................. .... 658
3.6 3.6.1
Antibiotics
-
mixtures of usual streptothricins F.
E. D. C. B and A
Q-1 4.1.1
..................... 659 659 ............................... 659 ......................... 659 ................. 659 ......................... ................. 660 purification .... 660 660 ............................... 660 ............................... 660 ......................... ................. 660 660 ......................... 660 ................. purification .... 660 ............................ 660 660 ............................... ......................... ................. 661 661 ......................... 661 ................. 661 purification .... 661 661 ................................. ............................... 661 ......................... ................. 661 661
Streptolin (streptoline) Introduction 4.1.1.1 Producing organism 4.1.1.2 Brief chemical description 4.1.1.3 Structural formula 4.1.2 Summary of therapeutic use 4.1.3 Extraction. separation and 4.2 Antibiotic 136 4.2.1 Introduction 4.2.1.1 Producing organism 4.2.1.2 Brief chemical description 4.2.1.3 Structural formula 4.2.2 Summary of therapeutic use 4.2.3 Extraction. separation and 4.3 Streptothricin VI 4.3.1 Introduction 4.3.1.1 Producing organism 4.3.1.2 Brief chemical description 4.3.1.3 Structural formula 4.3.2 Summary of therapeutic use 4.3.3 Extraction. separation and 4.4 Roseothricin 4.4.1 Introduction 4.4.1.1 Producing organism 4.4.1.2 Brief chemical description 4.4.1.3 Structural formula 4.4.2 Summary of therapeutic use 4.4.3 Extraction. separation and 4.5 Pleocidins m . 1 Introduction 4.5.1.1 Producing organism 4.5.1.2 Brief chemical description 4.5.1.3 Structural formula 4.5.2 Summary of therapeutic use 4.5.3 Extraction. separation and
......................... ................. purification .... ................................... ............................... ......................... ................. ......................... ................. purification ....
662 662 662 662 662 662 662 663 663 663
619 4
.
Antibiotics .mixtures of usual streptothricins F. E. D. C. B and A (continued) 4.6 Geomycin (geomycins) 4.6.1 Introduction 4.6.1.1 Producing organism 4.6.1.2 Brief chemical description 4.6.1.3 Structural formula 4.6.2 Summary of therapeutic use 4.6.3 Extraction. separation and purification 4.7 Grasseriomycin 4.7.1 Introduction 4.7.1.1 Producing organism 4.7.1.2 Brief chemical description 4.7.1.3 Structural formula 4.7.2 Summary of therapeutic use 4.7.3 Extraction. separation and purification., 4.8 Grisin (grisemin) 4.8.1 Introduction 4.8.1.1 Producing organism 4.8.1.2 Brief chemical description 4.8.1.3 Structural formula 4.8.2 Summary of therapeutic use 4.8.3 Extraction. separation and purification 4.9 Racemomycins A . B. C. D and E 4.9.1 Introduction 4.9.1.1 Producing organism 4.9.1.2 Brief chemical description 4.9.1.3 Structural formula 4.9.2 Summary of therapeutic use 4.9.3 Extraction. separation and purification 4.10 Mycothricins 4.10.1 Introduction 4.10.1.1 Producing organism 4.10.1.2 Brief chemical description 4.10.1.3 Structural formula 4.10.2 Summary of therapeutic use 4.10.3 Extraction. separation and purification 4.11 Phytobacteriomycin 4.11.1 Introduction 4.11.1.1 Producing organism 4.11.1.2 Brief chemical description 4.11.1.3 Structural formulae 4.11.2 Summary of therapeutic use 4.11.3 Extraction. separation and purification 4.12 Polymycin 4.12.1 Introduction 4.12.1.1 Producing organism 4.12.1.2 Brief chemical description 4.12.1.3 Structural formula 4.12.2 Summary of therapeutic use 4.12.3 Extraction. separation and purification 4.13 Nourseothricins A and 8 4.13.1 Introduction 4.13.1.1 Producing organism 4.13.1.2 Brief chemical description 4.13.1.3 Structural formula
-
-
-
-
-
......................... 663 ............................... 663 ......................... 663 ................. 663 ......................... 664 ................. 664 .... 664 ............................... 664 ............................... 664 ......................... 665 ................. 665 ......................... 665 ................. 665 . . 665 ............................ 665 ............................... 665 ......................... 665 ................. 665 ......................... 665 ................. 665 .... 666 ................ 666 ............................... 666 ......................... 667 ................. 667 ......................... 667 ................. 667 .... 667 ................................. 667 ............................... 667 ......................... 667 ................. 667 ......................... 668 ................. 668 .... 668 ........................... 668 ............................... 668 ......................... 668 ................. 668 ........................ 668 ................. 668 .... 668 .................................... 669 ............................... 669 ......................... 669 ................. 669 ......................... 669 ................. 669 .... 669 ...................... 6'69 ............................... 669 ......................... 670 ................. 670 ......................... 670
620
4.
Antibiotics - mixtures of usual streptothricins F, E, D, C, B and A (continued) 4.13.2 Summary of therapeutic use................ 4.13.3 Extraction, separation and purification. 4.14 Virothricin. 4.14.1 Introduction........ 4.14.1.1 Producing organism 4.14.1.2 Brief chemical description 4.14.1.3 Structural formula........................ 4.14.2 Summary of therapeutic use................ 4.14.3 Extraction, separation and purification.. 4.15 Yazumycin (yazumycins) 4.15.1 Introduction ,, 4.15.1.1 Producing organism.. 4.15.1.2 Brief chemical description 4.15.1.3 Structural formula........................ 4.15.2 Summary of therapeutic use.. 4.15.3 Extraction, separation and purification.. 4.16 Boseimycin 4.16.1 Introduction , 4.16.1.1 Producing organism., , 4.16.1.2 Brief chemical descrip ion................ , , 4.16.1.3 Structural formula.. 4.16.2 Summary of therapeutic use................ 4.16.3 Extraction, separation and purification.. 4.17 Akimycin 4.17.1 Introduction 4.17.1.1 Producing organism. 4.17.1.2 Brief chemical description Structural formula.. 4.17.1.3 , 4.17.2 Summary of therapeutic use................ 4.17.3 Extraction, separation and purification 4.18 Antibiotic A-249............................ , , , Introduction.. 4.18.1 4.18.1.1 Producing organism 4.18.1.2 Brief chemical description , , ,, 4.18.1.3 Structural formula.. 4.18.2 Summary of therapeutic use................ 4.18.3 Extraction, separation and purification.. , , , , 4.19 Antibiotic A-8265.. , , , 4.19.1 Introduction ,, , 4.19.1.1 Producing organism., , , , , 4.19.1.2 Brief chemical description 4.19.1.3 Structural formula........................ 4.19.2 Summary of therapeutic use.....,.......... 4.19.3 Extraction, separation and purification.. , 4.20 Antibiotic A 4788........................... 4.20.1 Introduction 4.20.1.1 Producing organism 4.20.1.2 Brief chemical description 4.20.1.3 Structural formula., , 4.20.2 Summary of therapeutic use................ 4.20.3 Extraction, separation and purification
-
-
-
-
670
70 70 ................................ . . 66670 ...................... 670 ........................ ................ 670 670 670 . 671 671 ...................... 671 ....... ....................... . .. . . . . ... .. . . . . . .. . 671 ................ G71 671 . .. . ... . . . . . . .. 671 .............. ................... 671 671 671 .......... ................... . .. . . . . . . .. . . . . . ... 671 672 . . .. .. . . .. . . .. . . . .. 672 672 . 672 .................................... 672 .............................. 672 ....................... 672 672 ...................... . .. . ...... .. . . . 672 672 ... 673 673 . .. . ........................ . . . .. .,. . . . . .. . . . . . . . 673 673 673 ................ . . . . . . . .. . .. . . . . . 673 673 . 673 . .. . . . . . . . . . . . . . .. 673 673 .............................. . . ................. . . . .. . . . . . . . 673 674
.............................. ........................ . . . . . .................. . . . . .. . .. . . . . . ...
674 674 674
674 674 674 674 674 674 674
621
4
.
Antibiotics
. mixtures
of usual streptothricins F.
E. D. C. B and A (continued)
...........................
Antibiotic A 3698 Introduction 4.21.1.1 Producing organism 4.21.1.2 Brief chemical description 4.21.1.3 Structural formula 4.21.2 Summary of therapeutic use 4.21.3 Extraction. separation and 4.22 Antibiotic A 3885 t.22.1 Introduction 4.22.1.1 Producing organism 4.22.1.2 Brief chemical description 4.22.1.3 Structural formula 4.22.2 Summary of therapeutic use 4.22.3 Extraction. separation and 4.23 Antibiotic A 3967 4.23.1 Introduction 4.23.1.1 Producing organism 4.23.1.2 Brief chemical description 4.23.1.3 Structural formula 4.23.2 Summary of therapeutic use 4.23.3 Extraction. separation and 4.24 Antibiotic 4714-12 4.24.1 Introduction 4.24.1.1 Producing organism . 4.24.1.2 Brief chemical description 4.24.1.3 Structural formula 4.24.2 Summary of therapeutic use 4.24.3 Extraction. separation and 4.25 Antibiotic 11-8 4.25.1 Introduction 4.25.1.1 Producing organism 4.25.1.2 Brief chemical description 4.25.1.3 Structural formula 4.25.2 Summary of therapeutic use 4.25.3 Extraction. separation and 4.26 Antibiotic 4562-3 m . 1 Introduction 4.26.1.1 Producing organism 4.26.1.2 Brief chemical description 4.26.1.3 Structural formula 4.26.2 Summary of therapeutic use 4.26.3 Extraction. separation and 4.27 Antibiotic 4786-14 4.27.1 Introduction 4.27.1.1 Producing organism 4.27.1.2 Brief chemical description Structural formula 4.27.1.3 Summary of therapeutic use 4.27.2 Extraction. separation and 4.27.3 4.21
4.21.1
-
-
675
.............................. 675 ......................... 675 ................ 675 675 ........................ ................ 675 purification ... 675 ........................... 675 .............................. 675 ........................ 675 ................ 675 ........................ ................ 676 676 purification ... 676 ........................... 676 .............................. 676 ........................ 676 ................ 676 ........................ 676 ................ 676 purification ... 676 .......................... 676 .............................. 676 ........................ 677 ................ 677 ........................ 677 ................ 677 purification ... 677 ............................. 677 .............................. 677 ........................ 677 ................ 677 ........................ 677 ................ 677 purification ... 678 ........................... 678 .............................. 678 ........................678 ................ 678 ........................ 678 ................ 678 purification ... 678 .......................... 678 .............................. 678 ........................ 678 ................ 678 ........................ 678
................ purification ...
679 679
622
.
4
Antibiotics .mixtures of usual streptothricins F. E. D. C. B and A (continued) 4.28 Antibiotic 4850-16 4 . 1 Introduction 4.28.1.1 Producing organism 4.28.1.2 Brief chemical description 4.28.1.3 Structural formula 4.28.2 Summary of therapeutic use 4.28.3 Extraction. separation and purification 4.29 Antibiotic 5438-10 4.29.1 Introduction 4.29.1.1 Producing organism 4.29.1.2 Brief chemical description 4.29.1.3 Structural formula 4.29.2 Summary of therapeutic use 4.29.3 Extraction. separation and purification Antibiotic 5491-17 4.30 4.30.1 Introduction 4.30.1.1 Producing organism 4.30 1.2 Brief chemical description 4.30.1.3 Structural formula 4.30.2 Summary of therapeutic use Extraction. separation and purification.,. 4.30.3
.
5
.
.......................... 679 .............................. 679 ........................ 679 ................ 679 ........................ 679 ................ 679 ... 679 .......................... 679 .............................. 679 ........................ 679 ................ 679 ........................ 680 ................ 680 ... 680 .......................... 680 .............................. 680 ........................ 680 ................ 680 ........................ ................ 680 680 680
Scarcely studied antibiotics. related to streptothricins 5.1 Actinorubin Amycin 5.3 Bagacidin 5.4 Bulging factor 5.5 Evericin 5.6 Ferromycin 5.7 Fuscomycin 5.8 Grisamine 5.9 Lavendulin 5.10 Lemacidin 5.11 Luridin 5.12 Musashimycin 5.13 Neonocardin Neothricin 5.15 Novomycin 5.16 Phagomycin 5.17 Roseocitrin A. 5.18 Roseocitrin B 5,19 Solemycin Streptin 5.21 Streptothricin B1 5.22 Streptothricin B2 5.23 Streptothricin Type 3 5.14 Virusin 16-D9. 5.15 Antibiotic A-6 5.26 Antibiotic A - 2 0 5.27 Antibiotic A-365 5.28 Antibiotic A-7907 5.29 Antibiotic B-637 Antibiotic EI5
-
-
-
.................................. 681 681 ....................................... 681 .................................... 681 ............................... 682 ..................................... 682 ................................... 682 .................................... 682 .................................... 682 ................................... 683 .................................... 683 ...................................... 683 ................................. 683 .................................. 683 ................................... 683 .................................... 684 ................................... 684 ............................... 684 ................................ 684 .................................... 684 ..................................... 685 ............................ 685 ............................ 685 ........................ 685 ............................... 685 ............................... 685 .............................. 686 ............................. 686 ............................ 686 ............................. 686 ...............................
62 3
5
.
Scarcely studied antibiotics. related to strep tothricins ( continued) 5.31 Antibiotic F-20 5.32 Antibiotic P-9 5.33 Antibiotic 5-15-1 5.34 Antibiotic SOB-? 5.35 Antibiotic 2-229 5.36 Antibiotic 539 5.37 Antibiotic 58?/13 5.38 Antibiotic 24010 B-1 5.39 Antibiotic from an actinomycete No -
-
6
.
............................. .............................. ........................... ............................ ............................ .............................. ........................... ........................ . 4279 ....
Streptothricin-like antibiotics Antibiotic LL-AC 541 Introduction 6.1.1.1 Producing organisms 6.1.1.2 Brief chemical description 6.1.1.3 Structural formula 6.1.2 Summary of therapeutic use 6.1.3 Extraction. separation and 6.2 Antibiotic deformimino-LL-AC 6.2.1 Introduction 6.2.1.1 Producing organism 6.2.1.2 Brief chemical description 6.2.1.3 Structural formula 6.2.2 Summary of therapeutic use 6.2.3 Extraction. separation and 6.3 Antibiotic LL-AB 664 6.3.1 Introduction 6.3.1.1 Producing organism 6.3.1.2 Brief chemical description 6.3.1.3 Structural formula 6.3.2 Summary of therapeutic use 6.3.3 Extraction. separation and 6.4 Antibiotic deformimino-LL-AB 6.4.1 Introduction 6.4.1.1 Producing organism 6.4.1.2 Brief chemical description 6.4.1.3 Structural formula 6.4.2 Summary of therapeutic use 6.4.3 Extraction. separation and 6.5 Antibiotic RAB-159 6.5.1 Introduction 6.5.1.1 Producing organism 6.5.1.2 Brief chemical description 6.5.1.3 Structural formula 6.5.2 Summary of therapeutic use 6.5.3 Extraction. separation and 6.6 Antibiotic ST-701 6.6.1 Introduction 6.6.1.1 Producing organisms 6.6.1.2 Brief chemical description 6.6.1.3 Structural formula 6.6.2 Summary of therapeutic use 6.6.3 Extraction. separation and
6.1 6.1.1
686 686 687 687 687 68’7 687 688 688
689 ........................ .............................. 689 689 ....................... ................ 689 689 ........................ ................ 689 purification ... 690 541 ............ 691 691 ............................... 691 ........................ ................ 691 691 ........................ ................ 691 purification ... 691 691 ........................ 691 .............................. 692 ........................ ................ 692 692 ........................ ................ 692 purification ... 692 664 ............ 694 694 .............................. 694 ........................ 694 ................ 694 ........................ ................ 694 purification ... 694 695 .......................... 695 .............................. 695 ........................ ................ 695 695 ........................ ................ 695 purification ... 695 695 ........................... 695 .............................. 695 ....................... ................ 695 695 ........................ ................ 696 purification ... 696
624
6.
Streptothricin-like antibiotics (continued) 6.7 Sclerothricin 6.7.1 Introduction 6.7.1.1 Producing organism 6.7.1.2 Brief chemical description Structural formula 6.7.1.3 6.7.2 Summary of therapeutic use 6.7.3 Extraction. separation and purification Racemomycin 0 6.8 6.8.1 Introduction Producing organism 6.8.1.1 6.8.1.2 Brief chemical description Structural formula 6.8.1.3 Summary of therapeutic use 6.8.2 6.8.3 Extraction. separation and purification 6.9 Fucothricin 6.9.1 Introduction Producing organism 6.9.1.1 6.9.1.2 Brief chemical description 6.9.1.3 Structural formula Summary of therapeutic use 6.9.2 6.9.3 Extraction. separation and purification 6.10 Antibiotic R4H 6.10.1 Introduction 6 . 1 0 . 1 . 1 Producing organism 6.10.1.2 Brief chemical description Structural formula 6.10.1.3 Summary of therapeutic use.. 6.10.2 6.10.3 Extraction. separation and purification
............................... 696 .............................. 696 ........................ ................ 66 99 77 ........................ 697 ................ 697 ... 66 99 78 ............................... .............................. 698 ........................ 698 ................ 698 ........................ 698 ................ 698 ... 699 ................................. 700 .............................. 700 ........................ ................ 77 00 00 ........................ 700 ................ ... 77000 0 .............................. 701 .............................. 701 ........................ ................ 77 00 11 ........................ 701 .............. ... 77 00 11 7 . Literature Cited ................................... 702
625
1. G e n e r a l I n t r o d u c t i o n S t r e p t o t h r i c i n , t h e f i r s t member o f a n t i b i o t i c g r o u p u n d e r c o n s i d e r a t i o n , was d e s c r i b e d by S. A . Waksman a n d H . B . Woodruff i n 1 9 4 2 l , i . e . , i t was one of t h e e a r l i e s t a n t i b i o t i c s w i t h b r o a d a n t i m i c r o b i a l s p e c t r u m from a c t i n o m y c e t e s . P o s s e s s ing not very high acute t o x i c i t y , it i n h i b i t e d very s t r o n g l y g r a m - p o s i t i v e , g r a m - n e g a t i v e and a c i d f a s t b a c t e r i a , as w e l l as some f u n g i . The d i s c o v e r y o f s t r e p t o t h r i c i n h a s a r o u s e d g r e a t i n t e r e s t and was a l i n k i n a c h a i n o f i n v e s t i g a t i o n s which h a s l e d t o t h e d i s c o v e r y a n d b r o a d u s e i n m e d i c i n e and o t h e r b r a n c h e s of human p r a c t i c e of many u s e f u l a n t i b i o t i c s . In 1958 t h e a u t h o r , P r o f e s s o r S . A. Waksman, h a s e v a l u a t e d t h i s d i s covery f o r t h e f u r t h e r r e s e a r c h i n t h e a n t i b i o t i c f i e l d i n t h e following words2: " S t r e p t o t h r i c i n , a b a s i c , w a t e r - s o l u b l e compound, n o t t o o t o x i c , a n d a c t i v e a g a i n s t b o t h g r a m - p o s i t i v e and g r a m - n e g a t i v e b a c t e r i a , was i s o l a t e d from a s t r a i n o f S t h e p t O m L j C e A t a u e n d u k e i n 1942. The i s o l a t i o n of t h i s a n t i b i o t i c may b e c o n s i d e r e d o f g r e a t h i s t o r i c a l s i g n i f i c a n c e , s i n c e i t p o i n t e d a way t o a new t y p e o f d e s i r a b l e a n t i m i c r o b i a l a g e n t . Had no o t h e r , l e s s t o x i c compound been soon i s o l a t e d , i t would have d e f i n i t e l y f o u n d a p l a c e i n chemotherapy. I t appeared t o supplement p e n i c i l l i n i n i t s a c t i v i t y on d i f f e r e n t m i c r o o r g a n i s m s . S i n c e s t r e p t o t h r i c i n was a c t i v e n o t o n l y a g a i n s t g r a m - p o s i t i v e and g r a m - n e g a t i v e b a c t e r i a , b u t a l s o a g a i n s t f u n g i and a c i d - f a s t b a c t e r i a , i t may b e c o n s i d e r e d as t h e f i r s t " b r o a d - s p e c t r u m " a n t i b i o t i c , i f t h i s term i s t o b e used i n a t r u l y e t y m o l o g i c a l s e n s e " . I n a s h o r t time a f t e r t h e s t r e p t o t h r i c i n d i s c o v e r y a g r e a t number of r e l a t e d a n t i b i o t i c s was d e s c r i b e d by many s c i e n t i s t s in d i f f e r e n t countries: s t r e p t o l i n 3 , roseothric n4, p l e o c i d i n 5 , geomycin6, g r a s ~ e r i o m y c i n ~g,r i s i n ( g r i s e m i n ) * racemomycin ( a n t i b i o t i c No. 22919, m y c o t h r i c i n l O , p h y t o b a c e r i o m y c i n l l , polymycin12 and o t h e r s . T h e r e f o r e an i m p r e s s i o n was b o r n t h a t t h e s t r e p t o t h r i c i n g r o u p was r a t h e r l a r g e a n d cont a i n e d a g r e a t number of d i f f e r e n t a n t i b i o t i c s e s p e c i a l l y s i n c e many d e s c r i b e d p r e p a r a t i o n s c o n s i s t e d of s e v e r a l a c t i v e compone n t s . However, i t t u r n e d o u t f u r t h e r , t h a t r e a l l y many c l a i m e d a n t i b i o t i c s were i d e n t i c a l w i t h e a r l i e r d e s c r i b e d o n e s o r were m i x t u r e s o f t h e same components i n s l i g h t l y v a r y i n g p r o p o r tions". P a p e r chromatography p l a y e d a v e r y i m p o r t a n t p a r t i n t h e comparison of d i f f e r e n t s t r e p t o t h r i c i n p r e p a r a t i o n s . Various methods o f p a p e r c h r o m a t o g r a p h i c i n v e s t i g a t i o n of s t r e p t o t h r i c i n s were p r o p o s e d i n t h e c o u r s e o f t h e i r s t u d i e s ( s e e e . g . p a p e r s 6 , 1 0 , 1 4 - 1 8 and s p e c i a l r e v i e w s 1 9 , 2 0 1 , b u t o n l y i n 1958 M . I . Horowitz and C . P . S c h a f f n e r have f o u n d c o n d i t i o n s s e c u r i n g t h e p r o p e r s e p a r a t i o n of d i f f e r e n t s t r e p t o t h r i c i n s m i x t u r e s 2 1 . The a u t h o r s s t u d i e d a number o f s t r e p t o t h r i c i n % A review o f s t m p t o t h r i c i n s t u d i e s and a r a t h e r f u l l b i b l i o g r a p h y o f papers which appeared b e f o r e 1960 wss given i n Chapter XV "Streptot h r i c i n s " , i n a comprehensive monograph on a n t i b i o t i c s chemistry, p u b l i s h e d i n 196113.
626 preparations by circular paper chromatography in the solvent system 1-propanol-pyridine-acetic acid-water (15:10:3:12) and demonstrated that almost all tested preparations were mixtures of components with similar Rf values. The close mobilities i.n only one solvent system were, of course, insufficient to be considered as decisive evidence of identity of fractions with similar Rf of components, which were found in different strepto thricin preparations. It was quite necessary to isolate them in a homogeneous state and accomplish the comparative investigation of separated components. Using the same solvent system and column chromatography on cellulose, the authorsz1 isolated four pleocidin components in rather small quantities, which did not separate further when treated similarly and differed in Rf values. The authors have mentioned that these components differed also in optical activity and antimicrobial action but the corresponding data were not published. In spite of a number of grave shortcomings (necessity to work with voluminous eluates, containing up to 25% of pyridine, cumbersome analysis, a rather high inactivation, unsatisfactory separation of components a.o.1 this method was for some time the sole one which gave the possibility of isolating individual components (even if in small quantities) in rather pure state, However, its shortcomings prevented the use for preparation of separate components in amounts insuring the studies of their properties and structure. Therefore a method of ion exchange chromatography of streptothricins on carboxy methyl cellulose with elution in NaCl gradient was very useful; it was developed in 1964-1965 by A. S. Khokhlov and P. D. re she to^^^'*^. It allowed the separation of streptothricin components rather quickly and with good yields. The possibility of a simple quantitative analysis of components in fractions by measuring the UV end absorption (at 220 nm) was among the serious merits of this method. A . S. Khokhlov and P. D. Reshetov investigated a number of streptothricin antibiotics by this method and concluded all the studied preparations to be the mixtures of six individual streptothricins (A, B, C, D, E and F ) , which were eluted at different, strictly definite, NaCl concentrations. Component A was the slowest one; component F had the highest mobility. A similar system of indexation was used by other scientists too. Some physico-chemical and biological properties of all six individual streptothricins were studied. The components from different antibiotics which were eluted by the same NaCl concentration were shown to be identical. Later, A. S. Khokhlov and co-workers elucidated the structure of all isolated s t r e p t o t h r i c i n ~ ~ ~ -All ~ ~ .six streptothricins were found to contain one residue of peculiar heterocyclic @-amino acid streptolidine (roseonine, geamine) and one residue of d-gulosamine, but they differ in number of B-lysine residues in the molecule; from 1 (in streptothricin F ) to 6 (in streptothricin A). The question of structure elucidation of streptothricins will be discussed in more detail in the second part of this chapter.
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The s t u d i e s o f v a r i o u s s t r e p t o t h r i c i n p r e p a r a t i o n s by i o n e x c h a n g e c h r o m a t o g r a p h y w i t h s a l t g r a d i e n t h a v e shown s t r e p t o t h r i c i n ( d i s c o v e r e d by S . A . Waksman and H . B . W o o d r u f f ) l a n d g r a s s e r i o m y c i n ? t o c o n s i s t a l m o s t e n t i r e l y o f component F. S t r e p t o l i n seemed t o b e a m i x t u r e o f components D a n d F w i t h p r e v a l e n c e of t h e f o r m e r . P l e o c i d i n a n d racemomycin w e r e shown t o b e similar m i x t u r e s o f components F , E , D a n d C . The p r o p e r t i e s o f s i x i n d i v i d u a l s t r e p t o t h r i c i n s , f o u n d i n d i f f e r e n t p r e p a r a t i o n s , a r e d e s c r i b e d i n s e c t i o n 2 and i n S e c t i o n 3 t h e component c o m p o s i t i o n s o f a number o f a n t i b i o t i c s ( p u b l i s h e d u n d e r d i f f e r e n t names a n d i n d e x e s ) a r e d i s c u s s e d , which a r e v a r i o u s m i x t u r e s o f s t r e p t o t h r i c i n s A-F.
Later, d i f f e r e n t m o d i f i c a t i o n s of s t r e p t o t h r i c i n i o n exc h a n g e c h r o m a t o g r a p h y w e r e d e s c r i b e d . A v a r i a n t of t h e method was p r o p o s e d by Taniyama a n d c o - w ~ r k e r s ~i ~n , w h i c h a v o l a t i l e p y r i d i n e - a c e t a t e b u f f e r was i n t r o d u c e d i n s t e a d o f N a C 1 , a l t h o u g h i n t h i s case t h e a n a l y s i s o f f r a c t i o n s was more cumbersome a n d t h e d a n g e r o f i n a c t i v a t i o n was g r e a t e r . The method o f i o n e x c h a n g e c h r o m a t o g r a p h y was u s e d s u c c e s s f u l l y f o r s e p a r a t i o n o f some s t r e p t o t h r i c i n s on a w e a k l y a c i d i c c a t i o n e x c h a n g e r e s i n A m b e r l i t e CG-50 w i t h e l u t i o n i n K C 1 g r a d i e n t . The s t r e p t o t h r i c i n c h r o m a t o g r a p h y on columns w i t h d e x t r a n g e l L H - 2 0 a n d f o l l o w i n g e l u t i o n w i t h 1 0 % a q u e o u s m e t h a n o l was a new and sometimes u s e f u l p r o c e d u r e , e l a b o r a t e d by H . Taniyama and co-workers34. The a p p l i c a t i o n of t h e l a s t method h a s res u l t e d i n t h e p o s s i b i l i t y t o show, f o r e x a m p l e , t h a t y a z u m y c i n , c l a i m e d e a r l i e r t o b e a new a n t i b i o t i c , was r e a l l y a m i x t u r e o f usual streptothricins. U n f o r t u n a t e l y , a g r e a t number o f s t r e p t o t h r i c i n p r e p a r a t i o n s w e r e n o t s t u d i e d by i o n e x c h a n g e c h r o m a t o g r a p h y u n d e r s t a n d a r d c o n d i t i o n s , s e c u r i n g well reproduced r e s u l t s . Theref o r e i t was v e r y d i f f i c u l t t o j u d g e w h e t h e r some p r e p a r a t i o n s were u s u a l s t r e p t o t h r i c i n s o r new s t r e p t o t h r i c i n - l i k e compounds, which d i f f e r e d from u s u a l s t r e p t o t h r i c i n s i n some s t r u c t u r a l peculiarities. A number of s t r e p t o t h r i c i n - l i k e a n t i b i o t i c s were d i s c o v e r e d a n d s t u d i e d a f t e r 1 9 6 7 ; t h e y d i f f e r from s t r e p t o t h r i c i n s A-F i n t h e s t r u c t u r e o f some m o l e c u l a r p a r t s . A t present t h e existence at l e a s t of t h e following s t r e p t o t h r i c i n l i k e a n t i b i o t i c s seems t o b e p r o v e d :
1. An a n t i b i o t i c , which c o n t a i n s a l o n g w i t h s t r e p t o l i d i n e a l s o N-methyl-a-D-gulosamine w i t h c a r b a m o y l g r o u p i n g a n d formi m i n o g l y c i n e . I t was d e s c r i b e d as a n t i b i o t i c LL-AC 5 4 1 3 5 - 3 7 a n t i b i o t i c BY-8139 a n d c i t r o m y ~ i n ~ ~ ’ ~ ~ I a n t i b i o t i c E-749 2. An a n t i b i o t i c deformimino-LL-AC 541, w h i c h d i f f e r s from t h e p r e c e d i n g o n e i n t h e a b s e n c e o f a f o r m i m i n o - g r o u p at glycine residue42. 3. A n t i b i o t i c , w h i c h c o n t a i n s i n c o n t r a s t t o two p r e v i o u s l y mentioned a n t i b i o t i c s , N - m e t h y l - s t r e p t o l i d i n e i n s t e a d
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of s t r e p t o l i d i n e . I t was d e s c r i b e d as a n t i b i o t i c LL-AB and a n t i b i o t i c B D - 1 2 3 9 ,
66437y43
4. An a n t i b i o t i c deformimino-LL-AB 6 Q , which d i f f e r s from t h e p r e c e d i n g one i n t h e a b s e n c e o f a formimino g r o u p a t g l y c i n e residue42.
5 . S c l e r o t h r i c i n , which c o n t a i n s N - m e t h y l - s t r e p t o l i d i n e , g l y c i n e a n d N-methyl-hexosamine w i t h o u t a carbamoyl g r o u p ; i t d i f f e r s a l s o from o t h e r u s u a l s t r e p t o t h r i c i n s a n d s t r e p t o t h r i c i n l i k e a n t i b i o t i c s i n t h e a b s e n c e of a l a c t a m r i n g 4 4 . 6. An a n t i b i o t i c , which d i f f e r s from a l l above-mentioned compounds i n t h e p r e s e n c e o f N-methyl g l y c i n e ( s a r c o s i n e ) . I t was d e s c r i b e d as a n t i b i o t i c SF-70145 and a n t i b i o t i c LL-BL 1 3 6 4 6 .
B e s i d e s t h e s e compounds two o t h e r a n t i b i o t i c s were d e s c r i b e d i n t h e l i t e r a t u r e , d i f f e r i n g from s t r e p t o t h r i c i n s A-F i n t h e s t r u c t u r e of t h e i r amino s u g a r m o i e t i e s : f u ~ o t h r i c i n ~ ~ , ~ ~ (which was c l a i m e d t o c o n t a i n f u c o s a m i n e i n s t e a d of g u l o s a m i n e ) a n d racemomycin 0 (which was c l a i m e d t o c o n t a i n d-glucosamine and a l i p h a t i c racemonic aldehyde)4g. A n a t u r a l d e r i v a t i v e of s t r e t o t h r i c i n F ( o r racemomycin A ) i s m e n t i o n e d ( a n t i b i o t i c RQH)Fo, The l a t t e r was f o u n d t o form s t r e p t o t h r i c i n F a f t e r mild h y d r o l y s i s . P r o f . H. Taniyama c o r p e s p o n d e d w i t h t h e a u t h o r of t h e p r e s e n t r e v i e w and i n d i c a t e d t h a t h e h a d i s o l a t e d a new s t r e p t o t h r i c i n - l i k e a n t i b i o t i c t e n t a t i v e l y named R A B - 1 5 g 5 1 . T h i s compound r e s e m b l e d a n t i b i o t i c LL-AB-664 ( B D - 1 2 1 , b u t had n o a m i d i n o f u n c t i o n . A n t i b i o t i c R A B - 1 5 9 seemed t o be a n i n t e r m e d i a t e i n LL-AB 6 6 4 b i o s y n t h e s i s . Some o f t h e s u b s t a n c e s m e n t i o n e d , however, were d e s c r i b e d r a t h e r b r i e f l y ; t h e i r s t r u c t u r e s were n o t e l u c i d a t e d a n d , t h e r e f o r e , t h e r e i s some d o u b t i n t h e i r e x i s t e n c e as i n d e p e n d e n t substances. The q u e s t i o n may b e even more d i f f i c u l t , b e c a u s e t h e s t r e p t o t h r i c i n p r o d u c i n g s t r a i n s changed t h e i r p r o p e r t i e s r a t h e r e a s i l y d u r i n g t h e i r s t o r a g e and p r o d u c e , a f t e r some t i m e , a n t i b i o t i c m i x t u r e s w i t h d i f f e r e n t r a t i o s of components. Such a phenomenon was o b s e r v e d i n t h e c a s e o f Actinomyceh p o l y m y c i n i i n t h e e a r l y e x p e r i m e n t s i t gave m i x t u r e s ( p o l y m y c i n ) , which c o n t a i n e d almost o n l y s t r e p t o t h r i c i n s A and B , b u t l a t e r t h e r e l a t i v e c o n t e n t o f components w i t h s h o r t e r p e p t i d e c h a i n s ( C , D ) was i n c r e a s e d . S i m i l a r phenomena may h a v e p l a c e i n t h e c a s e of racemomycin 0 and some o t h e r a n t i b i o t i c s .
-
I n t h e l i t e r a t u r e and p a t e n t s of d i f f e r e n t c o u n t r i e s t h e r e were d e s c r i p t i o n s o f a r a t h e r g r e a t number o f a n t i b i o t i c s which r e s e m b l e d s t r e p t o t h r i c i n s . As a r u l e , t h e y were v e r y l i k e l y i d e n t i c a l t o t h e u s u a l s t r e p t o t h r i c i n s or t h e i r m i x t u r e s , b u t i n s e v e r a l c a s e s t h e y might be m i x t u r e s , which c o n t a i n e d a l o n g w i t h u s u a l s t r e p t o t h r i c i n s known o r even new s t r e p t o thricin-like antibiotics. They a r e r e f e r r e d t o r a t h e r b r i e f l y i n S e c t i o n 3 of t h i s c h a p t e r . I n S e c t i o n 4 t h o s e a n t i b i o t i c s w i l l be d i s c u s s e d i n v e r y s h o r t f o r m , which were d e s c r i b e d w i t h o u t d e t a i l s b u t were i n c l u d e d i n t h e g r o u p of s t r e p t o t h r i c i n s
629
by t h e s c i e n t i s t s who h a d d i s c o v e r e d them, o r b y a u t h o r s of r e v i e w s and monographs ( s o m e t i m e s w i t h o u t s o l i d g r o u n d ) , as w e l l as some compounds v e r y s i m i l a r t o s t r e p t o t h r i c i n s .
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2. Streptothricins F, E, D,C,
B and A (usual. streptothricins)
2.1 Introduction As stated above, most known streptothricin preparations were mixtures (in different ratios) of six individual streptothricins (F, E, D, C, B and A ) . Since these compounds are very similar in their properties, their separation was a rather difficult task and therefore the studies of their biological properties and attempts to use them in practice were accomplished as a rule on crude preparations which contained several components. The differences in the properties of individual streptothricins F, E, D, C, B and A are not very sharp and the results of their biological investigations depend strongly on slight alterations in experimental conditions. Therefore only data obtained in experiments on direct comparison of different streptothricins under the same and strictly controlled conditions may be of serious importance for comparative evaluation of individual streptothricins. Taking this into consideration, in this part of the chapter some general problems of usual streptothricins studies will be discussed and then each individual streptothricin will be characterized. 2.1.1
Producing organisms
Streptothricins are produced by different strains of ~ a v e n d ~ L a e l1l ~r 158 ~y 5~0 -~6 0 , includin S LavenduLae var. vinothnicinua61, S. n o ~ e o c h ~ o m o g e n e a1‘f+r~6 i - 6 5 , S. n a c e m o ~ h n o m o g e n e h 3 3 ’3 4 r 6 6 r 6 7 , S. x a n t h o p h a e ~ 6 6 ~ 1 7 ~ 6 ~ - S. 70, n o u ~ h e ~ ~A c~t i ’ n o~ m y~ ce, h g n i h e u d 8 ’ 7 3 , A. p ~ , t y m y c i n i ~ ~A’. ~ ~ , and by a number of non-identified actinomycetes Stneptomyced
3,77-80.
2.1.2
Brief chemical description
Streptothricins are basic substances, well soluble in water, less in lower alcohols and insoluble in non-polar organic solvents. Depending on a number of B-lysine residues in a molecule they have from 2 to 7 free amino groups (basicity from 3 to B), which is easy to determine by paper electrophoresis of their products of dinitrosulphophenylation (Table l ) 2 4 . Since in most cases streptothricins are produced as mixtures, paper chromatography plays an important part in their characterization. Circular chromatography in the solvent system 1-propanol-pyridine-acetic acid-water (15:10:3:12) or l-butanolpyridine-acetic acid-water (15:10:3:12) is the most widespread v a r i a n t ~ 8 - 2 3 y 3 3 ’ 4 7 y 5 4 y 7 ~ y 7 ~ y A~ ~ -reat ~ 4 . number of other solvent systems were proposed 14- 1 6 Y 19 9 4 0 Y 3 4 Y 57967 Y 7 1 Y 7 2 Y 7 7 Y 82 Y 8 5 but they have no advantages in comparison with the two mentioned A s stated above, the ion-exchange chromatography of streptothricins on carboxy methyl cellulose in NaCl gradient had great importance in separation and characterization of these antibiotics. The characteristics for each streptothricin-eluting salt concentrations are shown also in Table 123,2‘+,
1 Table
Some Properties o f Individual Strep.toChricins
Streptothricins Properties
-F
Eluting NaCl concentration
Rf value i n solvent system: n-prop anol-py r i dine-acetic acid-water ( 15 :10 :3: 12) Optical activity {a}D
E
-D
-C
B -
-A
0.19 M
0.22 M
0.25 M
0.30 M
0.34 M
0.36 M
hydrochloride
0.48
0.40
0.35
0.29
0.24
0.20
sulphate
0.43
0.36
0.32
0.21
0.20
0.11
- -40'
-9.20
-36'
-22O
-12'
-9.6'
Number o f residues: streptolidine gulosamine 8-lysine
1 1 2
1 1 3
1 1 4
1 1 5
1 1 6
Nunber o f free amino groups
3
4
5
6
7
4
5
6
7
8
Nuher o f basic groups
3
6 32
2.1.3
S t r u c t u r a l fprmula
E l u c i d a t i o n of t h e s t r u c t u r e of s t r e p t o t h r i c i n s demanded t h e l o n g e f f o r t s of numerous s c i e n t i s t s from many c o u n t r i e s . S t r e p t o t h r i c i n F was t h e f i r s t member of t h i s g r o u p , which s t r u c t u r e was e s t a b l i s h e d . The most i m p o r t a n t s t e p i n t h e c h e m i c a l s t u d i e s of s t r e p t o t h r i c i n was i t s a c i d h y d r o l y s i s , t h e p r o d u c t s of which were t h e n i s o l a t e d and s t u d i e d . They a r e shown on F i g u r e 1. The s t r u c t u r e s o f a l l t h e s e d e g r a d a t i o n p r o d u c t s were f u l l y e s t a b l i s h e d and a l m o s t a l l of them were synthesized. The s i m p l e and c o n v e n i e n t methods f o r t h e i r q u a n t i t a t i v e a n a l y s i s were e l a b o r a t e d by means of ion-exchange chromatography2 3 y 86 787.
y 2
Vl
OH
NHZ
m lI
NH,
CH2-0
"Q &
HNYN V
NH,
IV
I s o l a t i o n of B - l y s i n e ( i s o l y s i n e ) ( V I ) f r o m s t r e p t o t h r i c i n and s t r e p t o l i n h y d r o l y s a t e s and i t s s t r u c t u r a l s t u d i e s were p e r formed i n 1952-1954 by c o l l e c t i v e e f f o r t s of many s c i e n t i s t s 58788-91. P r a c t i c a l l y a t t h e same time t h i s amino a c i d was found i n a c i d h y d r o l y s a t e s of viomycin, which d o e s n ' t b e l o n g t o t h e s t r e p t o t h r i c i n a n t i b i o t i c s g 2 . Almost s i m u l t a n e o u s l y t h e s y n t h e s i s of 8 - l y s i n e was a c ~ o r n p l i s h e d ~ ~ ~ ~ ~ .
633
The s t r u c t u r e of g u l o s a m i n e (111) was e s t a b l i s h e d i n 1 9 5 6 9 3 and c o n f i r m e d by s y n t h e b i ~ ~ ~ The - ~ ~ s y.n t h e s i s of anhyd r o g u l o s a m i n e ( I V ) was also p e r f o r m e d g 7 . The s t r u c t u r e d e t e r m i n a t i o n o f s t r e p t o l i d i n e was more d i f f i c u l t . Many p a e r s o f Americang8, B r i t i s h 8 3 y 9 9 , J a p a n e s e l o o - 1 0 3 , and Germanlt4y105 a u t h o r s were d e v o t e d t o s t u d i e s o f t h i s compound. Its correct 1 and. t h e~n ~ f o r m u l a ( V ) was g i v e n i n 1 9 6 1 by H. Carter e t ~ c o n f i r m e d by X-ray c r y s t a l l o g r a p h y l o 6 . S t r e p t o l i d i n e , was s y n t h e s i z e d o n l y i n 1 9 7 2 s i m u l t a n e o u s l y by two g r o u p s of s c i e n t i s t ~ ~ The ~ ~d e-t e r~m i n~a t i~o n . o f t h e s t r u c t u r e of N-guanstreptolidyl-gulosaminide (11) p l a y e d a v e r y r o m i n e n t r o l e i n t h e e l u c i d a t i o n of s t r e p t o t h r i c i n s t r u c t u r e l l g . Similar but somewhat d i f f e r i n f o r m u l a e f o r t h i s s u b s t a n c e were p r o p o s e d by o t h e r The t o t a l s t r u c t u r e of s t r e p t o t h r i c i n ( I ) was e l u c i d a t e d i n 1 9 6 1 by E . E . Van Tamelen, J . R . Dyer, H. A. Whaley, H. E . C a r t e r and G . B . W h i t f i e l d , J r . 1 1 2 , A s i m i l a r f o r m u l a was p r o p o s e d by A . W . Johnson and J . W . Westiey83. The f o r m u l a ( I ) i s now a g e n e r a l l y a c c e p t e d o n e , b u t t h e l o c a t i o n of a carbamoyl g r o u p i n g a t t h e s i x t h c a r b o n atom o f t h e gulosamine r e s i d u e i s not s t r i c t l y proved. D. B. B o r d e r s 4 2 s u p p o s e d t h a t s t r e p t o t h r i c i n F, as a number o f l a t e r s t u d i e d s t r e p t o t h r i c i n - l i k e a n t i b i o t i c s , contains t h i s group r a t h e r a t t h e t h i r d o r f o u r t h c a r b o n atom of t h e amino s u g a r m o i e t y . If i t i s s o , t h e f o r m u l a (Ia) c o r r e s p o n d s b e t t e r w i t h t h e s t r u c t u r e of s t r e p t o t h r i c i n F .
CH20H
RoQ R'O H R'O
NH NH
I
COCH, CHCH,CH,CH,NH,
I NH2 R,R' = H,CONH,
Ia Although t h e s t r u c t u r a l s t u d i e s o f some o t h e r s t r e p t o t h r i c i n s were s t a r t e d s i m u l t a n e o u s l y w i t h i n v e s t i g a t i o n s o f s t r e p t o t h r i c i n F , t h e y were c o m p l e t e d much l a t e r . The s t r u c t u r a l f o r m u l a e , p r o p o s e d f o r some s t r e p t o t h r i c i n s , t u r n e d o u t l a t e r t o be n o t q u i t e c o r r e c t b e c a u s e o f d i f f e r e n t r e a s o n s . E i t h e r t h e a n t i b i o t i c p r e p a r a t i o n s t a k e n f o r s t u d i e s , were
634
i n s u f f i c i e n t l y p u r e , o r t h e r e were g r e a t d i f f i c u l t i e s i n c a r r y i n g o u t some t r a n s f o r m a t i o n s of s t r e p t o t h r i c i n s , caused by p o o r s o l u b i l i t y of r e a c t i o n p r o d u c t s . T h e r e f o r e i n many c a s e s t h e r e a c t i o n s were i n c o m p l e t e and a n a l y s i s of p r o d u c t s of s u c h reactions led t o erroneous conclusions. I n 1 9 6 5 , P. D. Reshetov, C . A . Egorov and A . S . Khokhlov i n v e s t i g a t e d t h e p r o p e r t i e s of s i x i s o l a t e d i n d i v i d u a l s t r e p t o t h r i c i n s l 9 , i n p a r t i c u l a r , t h e q u a n t i t a t i v e c o n t e n t s of r e s i dues of s t r e p t o l i d i n e , gulosamine + anhydrogulosamine and $ l y s i n e ( s e e T a b l e 1) and s u g g e s t e d t h a t t h e s t r u c t u r e of s t r e p t o t h r i c i n s E-A may b e e x p r e s s e d by t h e g e n e r a l f o r m u l a (VII). D i s t i n c t i o n between d i f f e r e n t s t r e p t o t h r i c i n s E-A depends o n l y on t h e number of $ - l y s i n e r e s i d u e s i n t h e i r p e p t i d e c h a i n s : from 2 i n s t r e p t o t h r i c i n E t o 6 i n s t r e p t o t h r i c i n A . I t was s u g g e s t e d t h a t a l l s t r e p t o t h r i c i n s have t h e same s t r e p t o l i d y l gulosamine moiety and t h e amino a c i d r e s i d u e s i n t h e i r p e p t i d e c h a i n s a r e c o n n e c t e d by €-amino g r o u p s .
NH
HO
I
(COCH, CHCH2CH2CH2 NH InH
vn
HOQ CHzOH
HO
NH,
I
NH,
OH
-CH,
Vm
NH,
I t i s p o s s i b l e t o c o n s i d e r as t h e f i r s t c o n f i r m a t i o n of t h i s f o r m u l a t h e i s o l a t i o n from p a r t i a l h y d r o l y s a t e s of s t r e p t o -
t h r i c i n D of a d i p e p t i d e 6 - l y s y l - B - l y s i n e ,
which s t r u c t u r e was
6 35
s t r i c t l y p r o v e d by isomeric d i p e p t i by H . Taniyama and t h r i c i n E ) and was
i t s comparison w i t h s p e c i a l l y s y n t h e s i z e d d e ~ ~ ~ The - ~ ~same . p e p t i d e was i s o l a t e d l a t e r co-workers from racemomycin C ( = s t r e p t o synthesized according t o o t h e r schemes113-116.
The more g e n e r a l p r o o f of c o r r e c t n e s s o f t h e p r o p o s e d f o r m u l a e f o r a l l s t r e p t o t h r i c i n s E-A was g i v e n by A. S . Khokhlov and K . I . S h u t o v a . They i s o l a t e d from t h e p r o d u c t s o f m i l d h y d r o l y s i s of e a c h s t r e p t o t h r i c i n ( E - A ) t h e one a n d t h e same stre t o l i d y l gulosaminide ( X I ) and corresponding p e p t i d e s ( V I X I ) 30-3g. A l l p e p t i d e bonds i n t h e l a t t e r compounds were shown t o be b u i l t by c-amino g r o u p s o f B - l y s i n e r e s i d u e s , w h e r e a s t h e i r 0-amino g r o u p s were f r e e . It allows the p o s s i b i l i t y t o assure t h a t f o r m u l a ( V I I ) e x p r e s s e s c o r r e c t l y t h e s t r u c t u r e of a l l streptothricins. However, as s t a t e d a b o v e , i n t h e s t r u c t u r a l formula of s t r e p t o t h r i c i n F t h e r e i s no d e c i s i v e evidence f o r t h e l o c a t i o n of a carbamoyl g r o u p a t t h e s i x t h c a r b o n atom of t h e gulosamine moiety. I t i s t h e r e f o r e p o s s i b l e t o assume t h a t
0 H Q;N<
OH
i n s t r e p t o t h r i c i n s E-A t h i s g r o u p i s l o c a t e d a l s o a t t h e t h i r d
or f o u r t h c a r b o n atom o f t h e g u l o s a m i n e r e s i d u e : i n t h i s case, t h e s t r u c t u r e o f a l l s t r e p t o t h r i c i n s E-A may be e x p r e s s e d b e t t e r by t h e g e n e r a l f o r m u l a ( V I I a ) . R e c e n t l y , a s e r i e s o f p a p e r s was p u b l i s h e d 3 0 y 1 1 7 - 1 2 4 d e v o t e d t o i n v e s t i g a t i o n s on p h y s i c o - c h e m i c a l p r o p e r t i e s o f t h e u s u a l s t r e p t o t h r i c i n s and on p r e p a r a t i o n o f t h e i r d e r i v a t i v e s ( N - a c e t y l , a m i n o - a c i d and o t h e r d e r i v a t i v e s ) i n c l u d i n g t h e e l a b o r a t i o n o f a method, g i v i n g t h e p o s s i b i l i t y t o t r a n s f o r m s t r e p t o t h r i c i n s F, E and D i n t o t h e i r n e x t h i g h e r homologue by a d d i t i o n of a L-0-lysine r e s i d u e 1 2 4 .
636
0
OR'
NH
I
( C O C b CHCH2 CHZCH, NH 1" H
I
NH2
VII a R, R' = H,CONH2
n = 2,3,4,5,6
I n t h e course of b i o g e n e t i c i n v e s t i g a t i o n o f s t r e p t o t h r i c i n s , 0 . I . V o r o n i n a , I . I . Tovarova and A . S. K h o k h 1 0 v l ~ ~ 129 s t a t e d t h e following g e n e r a l r e g u l a r i t i e s : A. S t r e p t o t h r i c i n s are formed by o t h e r ways t h a n p r o t e i n s of t h e p r o d u c i n g m i c r o o r g a n i s m s , C h l o r a m p h e n i c o l d o e s not i n h i b i t streptothricin biosynthesis.
B. A l l t h e s t r e p t o t h r i c i n s a r e b u i l t t h e same way: s t r e p t o t h r i c i n F i s formed f i r s t , t h e n t h e 8 - l y s i n e r e s i d u e s a r e a t t a c h e d t o i t . B - l y s i n e i s formed from a - l y s i n e by t r a n s f e r o f an amino g r o u p w i t h o u t a t r a n s a m i n a t i o n s t a g e . R e c e n t l y t h e J a p a n e s e s c i e n t i s t s o b t a i n e d new d a t a conc e r n i n g b i o s y n t h e s i s of racemomycin A ( s t r e p t o t h r i c i n F ) 1 3 0 - 1 3 1 . They d e m o n s t r a t e d t h a t g l u c o s e and a - l y s i n e i n c o r p o r a t e d v e r y w e l l i n t o g u l o s a m i n e and B - l y s i n e m o i e t i e s of a n t i b i o t i c res p e c t i v e l y , whereas a r g i n i n e incorporated i n t o s t r e p t o l i d i n e o n l y i n low amounts. A c e t i c a c i d i n c o r p o r a t e d more i n t o s t r e p t o l i d i n e t h a n i n o t h e r p a r t s of t h e s t r e p t o t h r i c i n molec u l e . C a r b o n a t e showed a p r e f e r e n t i a l i n c o r p o r a t i o n i n t o a carbamoyl g r o u p . These a u t h o r s o b s e r v e d t h e s e v e r e i n h i b i t i o n of s t r e p t o t h r i c i n F b i o s y n t h e s i s by g l u c o s e , w h e r e a s maltose c a u s e d a m i l d s t i m u l a t i o n of a n t i b i o t i c f o r m a t i o n .
2 . 2 Summary
o f t h e r a p e u t i c use
AS s t a t e d a b o v e , s t r e p t o t h r i c i n s a t t r a c t e d g r e a t a t t e n t i o n of s c i e n t i s t s immediately a f t e r t h e discovery of s t r e p t o t h r i c i n F through t h e i r s t r o n g i n h i b i t o r y a c t i o n of gram-posit i v e , g r a m - n e g a t i v e and a c i d f a s t b a c t e r i a , as w e l l as some pathogenic fungi. Several s t r e p t o t h r i c i n preparations contained the a n t i b i o t i c s with longer peptide chains (streptothricin A,
Table 2
Antimicrobial A ctivity o f Individual S t r e p t o t h r i c i n s Minimal I n h i b i t o r y Concentration (mcg/rnl) o f S t r e p t o t h r i c i n
-F
-E
-D
-C
-B
-A
6-12
3-5
0,5-1
0,5-07
0,9-1
1
15
5
0,3-0,4
0,3
092
0,03
5
5
0,05-0,1
0,07
0,05
0,15
6-9
3-5
0,5-1
0,5-1
0,5-1
1-3
18-37
19-25
12-25
9-18
9-18
-
195
-
50 7 100
20-50
-
1,5
100
50-100
25-37
9-18
9
9-25
2-5
1,5-4
1-2
195
Products o f I n a c t i v a t i o n
E A c h e h i c h L ~COUB
750
150
30
638
s t r e p t o t h r i c i n B ) were shown l a t e r t o have d e f i n i t e a n t i v i m 1 a c t i o n ( m y c o t h r i c i n , geomycin, p o l y m y c i n ) . However i n h e r e n t i n a l l s t r e p t o t h r i c i n s are d e l a y e d t o x i c i t y and n e p h r o t o x i c i t y which p r e v e n t e d them from b e i n g u s e d i n m e d i c i n e , e s p e c i a l l y as a number o f l e s s t o x i c a n t i b i o t i c s o f b r o a d a n t i m i c r o b i a l s p e c trum ( s t r e p t o m y c i n , c h l o r a m p h e n i c o l , t e t r a c y c l i n e s ) were d i s c o v e r e d r a t h e r soon t h e r e a f t e r . Thus, i n s p i t e o f s e v e r a l e a r l y papers, devoted t o medical u s e of s t r e p t o t h r i c i n s , i t i s possib l e t o c o n c l u d e t h a t t h e y have n o r e a l use i n m e d i c i n e . T h e i r use i n a g r i c u l t u r e seems somewhat more p r o m i s i n g a l t h o u g h l i m i t e d , b o t h f o r s u p p r e s s i o n of some p h y t o p a t h o g e n i c b a c t e r i a a n d f o r a d d i t i o n i n t o animal feed. The d a t a c o n c e r n i n g a n t i m i c r o b i a l a c t i v i t y of d i f f e r e n t s t r e p t o t h r i c i n p r e p a r a t i o n s i n u i t h o were p u b l i s h e d i n a g r e a t number of p a p e r s , b u t t h e i r q u a n t i t a t i v e comparison i s a v e r y d i f f i c u l t t a s k because of d i f f e r e n c e s i n s t r a i n s used, e x p e r i mental c o n d i t i o n s and o t h e r reasons. Therefore t h e comparative d a t a are d i s c u s s e d as f o l l o w s , which were o b t a i n e d by t h e same authors under s t r i c t l y c o n t r o l l e d conditions. So, I . D. Ryabova, P . D . R e s h e t o v , G . L. Zhdanov and A. S . K h o k h l ~ v lhave ~ ~ compared a c t i v i t y o f i n d i v i d u a l s t r e p t o t h r i c i n s a g a i n s t a number o f m i c r o o r g a n i s m s (see T a b l e 2 ) . As one can see i n t h i s t a b l e , s t r e p t o t h r i c i n s F - A a r e v e r y similar i n g e n e r a l t o each o t h e r i n t h e i r a n t i m i c r o b i a l spectrum, b u t a n t i b i o t i c s w i t h l o n g e r p e p t i d e c h a i n s ( A , B , sometimes C ) a r e more a c t i v e t h a n s t r e p t o t h r i c i n s F a n d E , e s p e c i a l l y a g a i n s t
t h e y e a s t s Candida albicanA and Sacchaaomyced c e a e v i d i a e . S i m i l a r d a t a were p u b l i s h e d l a t e r by H . Taniyama and co-workers 3 3 on s t r e p t o t h r i c i n F (racemomycin A ) , s t r e p t o t h r i c i n E (racemomycin C ) , s t r e p t o t h r i c i n D (racernomycin B) a n d s t r e p t o t h r i c i n C (racemomycin D ) ( T a b l e 3 ) . Table 3
Antimicrobial A c t i v i t y o f Racemomycin Sulphates A -
-C
B -
1
0.3 12.5 25 1.56 25 6.25 3 6.25
0.3 1.56 6.25 0.39 12.5 1.56 3 1.56 100 25 1 10
25 0.78 3.12 100 3.12 3 3.12 100 50-10 1 10 100 30 30
1 3 3 3
100 50 1 10 30 30 30 0.1 10 0.3 10
100 30
30 0.03 10 0.1 3
639
The problem of s t r e p t o t h r i c i n a c t i v i t y a g a i n s t microorganisms r e s i s t a n t t o s t r e p t o m y c i n was t h e s u b j e c t of s p e c i a l investigation^^^^'^^^. Low c r o s s r e s i s t a n c e was shown t o e x i s t between s t r a i n s r e s i s t a n t t o s t r e p t o t h r i c i n s and t o s t r e p t o ~ n y c i n ’ ~ ~Very . h i g h r e s i s t a n c e e x i s t s between d i f f e r e n t s t r e p t o t h r i c i n s ( i n p a r t i c u l a r F, E , D and C)133. The d i s t i n c t i o n s between d i f f e r e n t s t r e p t o t h r i c i n s a r e much more profound i n t h e i r a n t i v i r a l a c t i v i t y 1 3 4 , d e t e r m i n e d a c c o r d i n g t o i n h i b i t i o n of t h e h e m a g g l u t i n a t i o n r e a c t i o n ( T a b l e 4). Table 4 I n h i b i t o r y A c t i o n o f S t r e p t o t h r i c i n s ~7 I n f l u e n z a Virus S t r e p t o t h r i c i n amount i n mcg/embrio
St r e p t o t h r i c i n
-5 -10 -25 - 50- 100 -
250
Average t i t e r o f h emagglutina t i o n re a c t i o n (76 o f c o n t r o l ) 2.34
-
0.61 25.1
0.19
12.5
-
0.14
-
6.35
1.58
29.40 13.40
-
-
28.80
-
-
-
-
10.3
4.34
14.4
5.34
-
-
-
31.7
The s t r e p t o t h r i c i n s w i t h l o n g c h a i n s (B and A ) s t r o n g l y excelled i n t h i s case the a n t i b i o t i c s with s h o r t e r peptide m o i e t i e s . S i m i l a r d a t a r e l a t e d t o s t r e p t o t h r i c i n s F , E , D and C (racemomycins A , C , B and D) were p u b l i s h e d l a t e r by J a p a n e s e authors33 (Table 5 ) . Table 5 A n t i v i r a l A c t i v i t y o f Racemomycins* Concentration (mcg/ml)
300
3
2
Racemomycin A
1.0
1.0
3.5
Racemomycin C
1.0
1.0
1.0
Racemomycin B
1.0
1.0
1.0
2.0
A n t i b i o t i c (Sulphate)
1
Control
6.0
6.3
6.5
5.0
5.5
7.0
5.5
7.0
*The t i t e r o f hemagglutination r e a c t i o n was measured. The l o g a r i t h m o f t h i s value i s given i n the t a b l e .
There a r e i m p o r t a n t d i s t i n c t i o n s between i n d i v i d u a l which a r e i n d i r e c t streptothricins in their dependence on a 5 - l y s i n e r e s i d u e number i n t h e m o l e c u l e , S O s t r e p t o t h r i c i n F (racemomycin A ) i s t h e l e a s t t o x i c compound and s t r e p t o t h r i c i n A h a s t h e h i g h e s t t o x i c i t y . The a n t i t u m o r a c t i o n i n t h e c u l t u r e o f human c a n c e r c e l l s was found i n s t r e p t o t h r i c i n p r e p a r a t i o n s w i t h a h i g h c o n t e n t of components A and B ( p 0 1 y r n y c i n ) l ~ ~ .
640 Table 6
T o x i c i t y o f Racemomycins
LDso (mg/kg)
Antibiotic (Sulphatel
Reference
Racemomycin A
300
33
Racemomycin C
26
33
Racemomycin 8
below 10
33
Racemomycin E
7
148
12
33
Crude p r e p a r a t i o n (M+C)
R a t h e r d i f f e r e n t a p p r o a c h e s were used f o r s o l u t i o n o f t h e h a r d problem o f s t r e t o t h r i c i n mode of a c t i o n . C. Z i m m e r , H . T r i e b e l and H . ThrumP36 showed i n 1 9 6 7 t h e a b i l i t y of s t r e p t o t h r i c i n s t o s t a b i l i z e t h e DNA s e c o n d a r y s t r u c t u r e a g a i n s t t h e r mal d e n a t u r a t i o n in v i t 4 0 . The a u t h o r s 1 3 6 s u g g e s t e d t h a t a t low i o n i c s t r e n g t h s t r e p t o t h r i c i n s a r e bound l o o s e l y t o t h e n e g a t i v e l y c h a r g e d p h o s p h a t e groups o f DNA; t h i s phenomenon was supposed t o be t h e r e a s o n f o r t h e i r a n t i m i c r o b i a l a c t i o n . were s t u d y i n g t h e I n 1 9 7 1 T. K . Misra and R . K . mode of a c t i o n of boseimycin which, a c c o r d i n g t o H . Taniyama, Y . Sawada e t ae.e2 i s a m i x t u r e of u s u a l s t r e p t o t h r i c i n s . They b e l i e v e d t h a t boseimycin a f f e c t e d t h e t r a n s l a t i o n l e v e l o f t h e p r o t e i n s y n t h e s i z i n g s y s t e m of b a c t e r i a . There i s o n l y a t r a n s i e n t i n h i b i t i o n o f p r o t e i n s y n t h e s i s r a t h e r t h a n any i r r e p a r a b l e damage caused t o c e l l u l a r components137.
I n 1973 G. N . T e l e s n i n a , I . D. Ryabova, K . I . Shutova and A. S. Khokhlov p u b l i s h e d a p a p e r 1 3 8 on s t u d i e s of i n h i b i t i o n of p r o t e i n b i o s y n t h e s i s by d i f f e r e n t s t r e p t o t h r i c i n s i n N i r e n b e r g c e l l - f r e e s y s t e m c o n t a i n i n g E . c o l i B ribosomes*. Corresponding d a t a are g i v e n i n t a b l e 7 . Table 7 I n h i b i t i o n of P r o t e i n Biosyntheais by D i f f e r e n t S t r e p t o t h r i c i n s and Their "Products o f I n a c t i v a t i o n " i n C e l l - f r e e System f r o m E. COUB ( i n 4: t o c o n t r o l ) Concentration (mcmol/l) Compound Streptothricin B Streptothricin D St r e p tot h r i c i n F Products o f i n a c t i v a t i o n of Str. B D F Chlor ampheni col
3.13 25 200 400 800 1600 3200 7.1 7.3 6.5
-
24.6 22.7 58.7
60.7 64.7 70.5
93.6 79.8 74.3
98.0 98.5
-
80.1
86.1
95.1
-
48.6 49.3 46.9
60.5 59.7 57.8 98.5
68.9 74.6 69.6
-
-
-
-
27.7
67.8
79.6
-
-
75.0
-
-
*The p r e l i m i n a r y data were r e p o r t e d by A.S. Khokhlov a t A n t i b i o t i c P r e Symposium h e l d i n Riga, On June 19 and 20, 1970139.
6 41
The i n h i b i t i o n o f p r o t e i n b i o s y n t h e s i s depended s t r o n g l y on B - l y s i n e c o n t e n t i n s t r e p t o t h r i c i n s ; t h e a c t i v i t y o f s t r e p t o t h r i c i n B i s commensurate w i t h t h a t o f c h l o r a m p h e n i c o l , e . g . p r o t e i n b i o s y n t h e s i s i s i n h i b i t e d i n 9 8 - 9 9 % by 4 0 0 mc m o l / l of c h l o r a m p h e n i c o l and by 8 0 0 mc m o l / l of s t r e p t o t h r i c i n B . The i n h i b i t i o n o f p r o t e i n b i o s y n t h e s i s was a l m o s t e q u a l i n t h e a b s e n c e and i n t h e p r e s e n c e o f a r t i f i c i a l m a t r i x ( p o l y - U ) . so? t h e mode o f s t r e p t o t h r i c i n a c t i o n i s c o n n e c t e d w i t h t h e a b i l i t y t o i n h i b i t p r o t e i n b i o s y n t h e s i s d i r e c t l y on r i b o s o m a l l e v e l . A n t i b a c t e i i a l l y i n a c t i v e products of mild a c i d i n a c t i v a t i o n of s t r e p t o t h r i c i n show a n e f f e c t on p r o t e i n b i o s y n t h e s i s s i m i l a r t o t h a t of t h e p a r e n t a n t i b i o t i c s . Probably, t h e d i s t i n c t i o n i n a c t i o n o f t h e s e p r o d u c t s on i n t a c t m i c r o b i a l c e l l s and on a r i b o s o m a l s y s t e m , p r e p a r e d from t h e s e c e l l s , d e p e n d s on t h e i r d i f f e r e n t p e n e t r a t i o n s t h r o u g h b a c t e r i a l c e l l walls.
2.3 Extraction,
s e p a r a t i o n and p u r i f i c a t i o n
E x t e n s i v e m a t e r i a l on i s o l a t i o n o f c r u d e p r e p a r a t i o n s and on methods of t h e i r s e p a r a t i o n i n t o i n d i v i d u a l components were c o l l e c t e d d u r i n g a l o n g p e r i o d o f s t u d i e s of s t r e p t o t h r i c i n s { a n d c o n t a i n e d d i f f e r e n t m i x t u r e s o f s t r e p t o t h r i c i n s A-F ( w h i c h were d e s c r i b e d m a n i f o l d u n d e r i n d e p e n d e n t n a m e s ) ] . The c r u d e s t r e p t o t h r i c i n p r e p a r a t i o n s were o b t a i n e d as d e s c r i b e d i n t h e m a j o r i t y of p a p e r s by d i f f e r e n t c o m b i n a t i o n s o f similar methods of p u r i f i c a t i o n : a d s o r p t i o n on a c t i v e c a r b o n and f o l l o w e d by e l u t i o n w i t h a c i d i f i e d aqueous methanol o r a c e t o n e ; a d s o r p t i o n on c a t i o n e x c h a n g e r e s i n s and d e s o r p t i o n w i t h d i l u t e a c i d s ; p r e c i p i t a t i o n with p i c r i c a c i d , methyl orange o r Reinecke’s s a l t ; c o n v e r s i o n t o s u l p h a t e s o r h y d r o c h l o r i d e s , I n many c a s e s t h e s e s t a g e s were r e p e a t e d i n one o r a n o t h e r o r d e r . R a t h e r p u r e c r u d e p r e p a r a t i o n s were o b t a i n e d i n t h i s manner; however, t h e y c o n t a i n e d as a r u l e , m i x t u r e s of s t r e p t o t h r i c i n s F, E , D, C , B and A i n d i f f e r e n t r a t i o s . Only i n t h e cases o f some p r o ducing s t r a i n s ( g i v i n g , as a r u l e , s t r e p t o t h r i c i n F) a p r a c t i c a l l y homogeneous s u b s t a n c e was i s o l a t e d a t t h i s s t a g e o f p u r i fication. I n t h e overwhelming m a j o r i t y o f cases t h e m i x t u r e s of s t r e p t o t h r i c i n s were o b t a i n e d as a r e s u l t o f s u c h o p e r a t i o n s . The s e p a r a t i o n methods w i l l b e d i s c u s s e d a t t h e end o f t h i s part. S e v e r a l e x a m p l e s of i s o l a t i o n o f c r u d e s t r e p t o t h r i c i n p r e p a r a t i o n s a r e g i v e n as f o l l o w s . EXAMPLE 1
-
Isolation of streptothricin60
S t r e p t o t h r i c i n p r e p a r a t i o n s o f h i g h p u r i t y were o b t a i n e d as a r e s u l t o f c o n s e c u t i v e a p p l i c a t i o n o f t h e f o l l o w i n g m e t h o d s : a d s o r p t i o n on a c t i v e c a r b o n , e l u t i o n w i t h a q u e o u s m e t h a n o l , a c i d i f i c a t i o n by f o r m i c a c i d , p a r t i a l c o n c e n t r a t i o n o f e l u a t e s in vacuo, p f e c i p i t a t i o n w i t h a c e t o n e , f o r m a t i o n of p i c r a t e , i t s c o n v e r s i o n i n t o h y d r o c h l o r i d e , c h r o m a t o g r a p h y of h y d r o c h l o r i d e on aluminium o x i d e , and p r e p a r a t i o n o f h e l i a n t h a t e . D e t a i l e d d e s c r i p t i o n s of t h e s e s t a g e s of p u r i f i c a t i o n f o l l o w . A. P u r i f i c a t i o n by a d s o r p t i o n on a c t i v e c a r b o n . 7 . 5 g/R o f a c t y v e c a r b o n ( N o r i t e ) were added t o t h e f i l t e r e d b r o t h o f
642
S&ep.tomyces C a v e n d u l a e . The s u s p e n s i o n was s t i r r e d f o r o n e The a d s o r b a t e was h o u r a t room t e m p e r a t u r e a n d t h e n f i l t e r e d . washed by s t i r r i n g w i t h 1 l i t e r o f e t h a n o l p e r 1 0 0 g o f N o r i t e The Norite was f i l t e r e d o f f and d r i e d i n vacuo a t f o r 45 m i n . room t e m p e r a t u r e . S t r e p t o t h r i c i n was e l u t e d w i t h 0 . 8 N f o r m i c a c i d i n 5 0 % a q . m e t h a n o l by s u s p e n d i n g t h e N o r i t e i n a volume o f e l u e n t e q u i v a l e n t t o 15: of t h e volume of b r o t h t r e a t e d . A f t e r s t i r r i n g 4 h r . a t 45 , t h e N o r i t e was removed by f i l t r a t i o n and t h e f i l t r a t e c o n c e n t r a t e d i n vacuo t o 0 . 0 1 of i t s o r i g i n a l volume, F i v e volumes of m e t h a n o l were a d d e d , f o l l o w e d by f o u r times t h e t o t a l volume of a c e t o n e . The r e s u l t i n g p r e c i p i t a t e was removed by c e n t r i f u g i n g , washed w i t h e t h y l e t h e r , and d r i e d i n v a c u o . The c o l o r of t h e p r o d u c t v a r i e d from a l i g h t t a n t o brown. B. P u r i f i c a t i o n of s t r e p t o t h r i c i n b p r e c i p i t a t i o n w i t h icric-acir I t was f o u n d t h a t a y e f l o w g:mmy p r e c i p i t a t e was E b t a i n e d when aqueous s o l u t i o n s o f s t r e p t o t h r i c i n were t r e a t e d with p i c r i c acid. The p r e c i p i t a t e c o u l d b e p u r i f i e d by d i s s o l v i n g i t i n methanol and adding e t h y l e t h e r t o p r e c i p i t a t e t h e c r u d e s t r e p t o t h r i c i n p i c r a t e as a y e l l o w powder. To conv e r t t h e c r u d e p i c r a t e t o t h e h y d r o c h l o r i d e i t was d i s s o l v e d i n m e t h a n o l , a c i d i f i e d w i t h H C 1 and p o u r e d i n t o e t h y l e t h e r c a u s i n g p r e c i p i t a t i o n of t h e hydrochloride. I t is possible t o avoid the s t a g e of p i c r a t e i s o l a t i o n . C. Chromatogra h i e p u r i f i c a t i . t o t h r i c i n on aluminium o x i d e . AciPd-washed aluminium o x i d e was used i n a r a t i o o f 10-30 g o f a d s o r b e n t t o 1 g o f c r u d e s t r e p t o t h r i c i n hydrochloride. The column was f i l l e d w i t h t h e a d s o r b e n t a n d m e t h a n o l . The s o l v e n t was a l l o w e d t o d r a i n u n t i l a l a y e r o f s o l v e n t above t h e a d s o r b e n t was c a . 1 - 2 mm d e e p . The c r u d e s t r e p t o t h r i c i n h y d r o c h l o r i d e was d i s s o l v e d i n m e t h a n o l a t a c o n c e n t r a t i o n o f from 1 0 - 5 0 % a n d t h e s o l u t i o n was a l l o w e d t o f l o w t h r o u g h t h e column e i t h e r by g r a v i t y or u n d e r p r e s s u r e o f 1 0 - 2 0 mm of m e r c u r y . The e l u t i o n was a c c o m p l i s h e d by m e t h a n o l . The e l u a t e s were c o n c e n t r a t e d t o a b o u t 1 0 % o f t h e o r i g i n a l v o l ume and p o u r e d i n t o a c e t o n e o r e t h e r t o p r e c i p i t a t e s t r e p t o t h r i c i n hydrochloride. D. Formation of s t r e p t o t h r i c i n h e l i a n t h a t e . 2 0 , g of c r u d e s t r e p t o t h r i c i n h y d r o c h l o r i d e were d i s s o l v e d i n 2 0 0 m l o f m e t h a n o l a n d a h o t s o l b t i o n of 1 6 g o f m e t h y l o r a n g e i n 160 m l of w a t e r was added w i t h c o n t i n u o u s s t i r r i n g . A f t e r s t i r r i n g f o r 1 h r , t h e m i x t u r e was k e p t 4 h r a t 5 The o r a n g e - c o l o r e d p r e c i p i t a t e o f s t r e p t o t h r i c i n h e l i a n t h a t e was r e t o v e d b y c e n t r i The s o l u t i o n f u g i n g and d i s s o l v e d i n 200-ml of m e t h a n o l a t 65 was f i l t e r e d w h i l e h o t , and 2 0 0 m l of h o t w a t e r was adged. Scratching induced c r y s t a l l i z a t i o n . After 1 2 hrs at 5 , t h e c r y s t a l s were removed by f i l t r a t i o n , washed o n c e w i t h water, a n d d r i e d i n vacuo t o y i e l d 1 7 . 1 5 g o f s t r e p t o t h r i c i n h e l i a n thate.
.
.
E. C o n v e r s i o n of s t r e p t o t h r i c i n h e l i a n t h a t e t o s t r e p t o t h r i c i f i hydrochloride. 7 g o f h e l i a n t h a t e were added t o 30 m l o f m e t h a n o l . f o l l o w e d by 1 . 2 m l o f c o n c e n t r a t e d H C 1 d r o p w i s e with constant s t i r r i n g . - A f t e r t r i t u r a t i n g thoroughly, t h e viol e t c o l o r e d m i x t u r e , which had a pH o f 4 . 0 , was f i l t e r e d
643 t h r o u g h a 1 g pad o f Darco G-60 and t h e Darco was washed w i t h t h r e e , 3 m l p o r t i o n s of methanol. The c o l o r l e s s f i l t r a t e was t r e a t e d immediately w i t h 1 7 5 m l of a c e t o n e and t h e r e s u l t i n g p r e c i p i t a t e removed by c e n t r i f u g i n g . T h e o p r e c i p i t a t e was washed once w i t h a c e t o n e , and d r i e d a t 2 5 i n uacuo t o y i e i d 2 . 5 5 g of w h i t e powder w i t h o p t i c a l a c t i v i t y I C X I ~+ ~- 5 1 . 3 (Cs1.4, H20). EXAMPLE 2
-
P u r i f i c a t i o n of a n t i b i o t i c A 8265 ( a m i x t u r e of streptothricinslU5
The b r o t h (300 g a l l o n s 2 1 , 3 5 0 a ) of StJLtnep.tOtnyCtned A 8 2 6 5 was c l a r i f i e d by f i l t r a t i o n through k i e s e l g u h r a t pH 5 . Biol o g i c a l l y a c t i v e m a t e r i a l was a d s o r b e d from t h e f i l t r a t e on a c t i v e c h a r c o a l and e l u t e d w i t h m e t h a n o l i c HC1 a t pH 3 . A f t e r f i l t r a t i o n , n e u t r a l i z a t i o n and e v a p o r a t i o n , w a t e r was added, and t h e s o l u t i o n f r e e z e - d r i e d t o g i v e a c r u d e s o l i d ( 7 5 0 g ) , which was a c t i v e a g a i n s t S t a p h y l o c o c c u d auReud. F o r f u r t h e r p u r i f i c a t i o n t h e c r u d e p r e p a r a t i o n (100 g ) was t r e a t e d w i t h water ( 1 2 0 m l ) and s t i r r e d f o r 1 h r ; t h e s o l i d was f i l t e r e d o f f and washed w i t h water ( c a . 5 0 m l ) , and t h e combined f i l t r a t e and washings were t r e a t e d w i t h s a t u r a t e d p i c r i c a c i d s o l u t i o n ( 2 k). After being kept overnight, the mixture deposited a gummy p r e c i p i t a t e which was s e p a r a t e d by d e c a n t a t i o n and d i s s o l v e d i n a c e t o n e (80 m l ) . S a t u r a t e d aqueous p i c r i c a c i d ( 7 5 m l ) was added t o c a u s e i n c i p i e n t t u r b i d i t y , f o l l o w e d by more p i c r i c a c i d (10 m l ) . The s o l u t i o n , d e c a n t e d from t h e gummy p r e c i p i t a t e , was poured i n t o s a t u r a t e d p i c r i c a c i d ( 2 a ) and w a t e r ( 2 0 0 m l ) and k e p t a t 0' o v e r n i g h t . The r e s u l t i n g gummy p r e c i p i t a t e was c o l l e c t e d and r e p r e c i p i t a t e d by t h e same proc e d u r e t h r e e more times, t o y i e l d a y e l l o w s t i c k y s o l i d . This was t r i t u r a t e d w i t h e t h a n o l and t h e s o l i d formed ( 1 . 2 g ) was washed w i t h e t h a n o l and e t h e r . I t c o u l d n o t be c r y s t a l l i z e d . The p i c r a t e ( 1 0 g ) was d i s s o l v e d i n a c e t o n e ( 5 0 m l ) and t r e a t e d w i t h c o n c e n t r a t e d H C 1 ( 5 m l ) which c a u s e d immediate p r e c i p i t a t i o n of a p a l e brown h y d r o c h l o r i d e . T h i s was s e p a r a t e d , d i s s o l v e d i n water ( 1 0 m l ) , and r e p r e c i p i t a t e d w i t h acet o n e . The h y d r o c h l o r i d e was a g a i n removed and t h e o p e r a t i o n r e p e a t e d u n t i l a l l of t h e p i c r i c a c i d had been removed and t h e aqueous s o l u t i o n was a l m o s t c o l o r l e s s . I t was $hen f r e e z e - d r i e d g i v i n g a h y g r o s c o p i c s o l i d ( 5 . 3 g), {a}2a = - 2 7 I t was f u r t h e r p u r i f i e d by chromatography of a m e q h a n o l i c s o l u t i o n of t h i s p r o d u c t ( 5 g ) on acid-washed aluminium o x i d e ( 1 0 0 g), t h e e l u t i o n b e i n g e f f e c t e d w i t h methanol. The b i o l o g i c a l l y a c t i v e = -40'. h y d r o c h l o r i d e ( 1 . 5 g ) had
.
EXAMPLE 3
-
P u r i f i c a t i o n of ~ t r e p t o l i n " ~ ~
One hundred k of b r o t h (which a s s a y e d 3 0 , 0 0 0 u n i t s p e r m l ) was a c i d i f i e d t o pH 3 w i t h H2S04, mixed w i t h 2 5 0 g of Darco G-60 and f i l t e r e d w i r h Hyflo Super-Cel t h r o u g h a p l a t e and frame f i l t e r p r e s s . The f i l t r a t e was n e u t r a l i z e d t o pH 7 . 5 was shown later, s t r e p t o l i n was a mixture of d i f f e r e n t s t r e p t o t h r i c i n s w i t h a prevalence of s t r e p t o t h r i c i n D and s t r e p t o t h r i c i n F.
3 As
644
w i t h N a O H and a g a i n f i l t e r e d . The a n t i b i o t i c was t h e n a d s o r b e d on 1 , 5 0 0 g of Darco G-60 a n d 5 0 0 g o f H y f l o S u p e r - C e l was mixed with the adsorbate. The m i x t u r e was washed w i t h water. About 5 % o f t h e a c t i v i t y was l o s t i n t h e s e s t e p s . The c a k e was e l u t e d by c y c l i n g 1 8 R o f 7 % a c e t o n e f o r 3 0 min. and a d d i n g d i l u t e H2S04 s u c h t h a t pH 2 was m a i n t a i n e d . T h i s o p e r a t i o n was r e p e a t e d t w i c e w i t h new b a t c h e s of a c e t o n e . The t h i r d e l u a t e was h e l d f o r e l u t i o n o f s u b s e q u e n t b a t c h e s . The f i r s t two e l u a t e s , which c o n t a i n e d a b o u t 75% of t h e a d s o r b e d a c t i v i t y , were mixed w i t h two volumes o f a c e t o n e . The gum which s e t t l e d on s t a n d i n g was d i s s o l v e d i n 6 0 0 m l of water, a d j u s t e d t o pH 2 w i t h H,S04 and p r e c i p i t a t e d w i t h 3 m l o f m e t h a n o l , F o r t y f i v e g of c r u d e s u l p h a t e which a s s a y e d 30,000 u n i t s p e r 1 mg was o b t a i n e d , g i v i n g an o v e r a l l r e c o v e r y o f 4 5 % . Twenty f i v e g o f p r e p a r e d c r u d e s u l p h a t e were p r e c i p i t a t e d from water w i t h 5 0 g o f ammonium r e i n e c k a t e . The p r e c i p i t a t e was c r y s t a l l i z e d s l o w l y from 400 m l of w a r m 40% e t h a n o l . Twenty f o u r g o f c r y s t a l l i n e s t r e p t o l i n r e i n e c k a t e a s s a y i n g 1 9 , 0 0 0 u n i t s p e r 1 mg were o b t a i n e d . Ten g of s t r e p t o l i n r e i n e c k a t e were c o n v e r t e d t o t h e s u l p h a t e by d i s s o l v i n g i n 250 m l of m e t h a n o l and p r e c i p i t a t i n g w i t h 0 ml of 2.6 N H2SO+. The p r e c i p i t a t e was d i s s o l v e d i n water, d e c o l o r i z e d w i t h a c t i v e c a r b o n and r e p r e c i p i t a t e d w i t h m e t h a n o l . Four g o f w h i t e amorphous s u l p h a t e which a s s a y e d 43,000 u n i t s p e r 1 mg was o b t a i n e d , = -20' ( c = 1 . 8 ) . C r y s t a l l i n e h e l i a n t h a t e o f s t r e p t o l i n ( 6 . 1 g ) was obt a i n e d from 3 g o f s u l p h a t e a c c o r d i n g t o 3 and c r y s t a l l i z e d from methanol. One g o f h e l i a n t h a t e was c o n v e r t e d t o t h e s u l p h a t e by s l u r r y i n g w i t h t h r e e s u c c e s s i v e 100 m l p o r t i o n s of 0 . 5 M t r i e t h y l - a m i n e s u l p h a t e i n 9 0 % m e t h a n o l . The r e s u l t a n t y e l l o w p r o d u c t was washed w i t h m e t h a n o l , d i s s o l v e d i n 10 m l o f water, d e c o l o r i z e d w i t h a c t i v e c a r b o n and p r e c i p i t a t e d w i t h 5 0 m l o f To m e t h a n o l ; 0 . 4 1 g o f amorphous w h i t e s u l p h a t e was o b t a i n e d . p r e p a r e a p u r e r s u l p h a t e by t h i s method t h e h e l i a n t h a t e was c r y s t a l l i z e d many times from 5 0 % m e t h a n o l . F o r t h i s p u r p o s e 1 g o f h e l i a n t h a t e was d i s s o l v e d i n 500 m l o f 5 0 % m e t h a n o l a t 55' and a l l o w e d t o c o o l s l o w l y t o room t e m p e r a t u r e . The c r y s t a l s which s e p a r a t e d were s i m i l a r l y r e c r y s t a l l i z e d f i v e times. The h e l i a n t h a t e o b t a i n e d by t h i s p r o c e d u r e h a d c o n s t a n t p r o p e r t i e s for the last three crystallizations. Such h e l i a n t h a t e was conv e r t e d t o amorphous s u l p h a t e of s t r e p t o l i a A, which a s s a y e d 32,000 u n i t s p e r 1 mg a n d had l a ) 2 5 -20
EXAMPLE 4
-
D
.
I s o l a t i o8n1 - 0
These v e r y s i m i l a r a n t i b i o t i c m i x t u r e s were i s o l a t e d from c u l t u r e b r o t h s of two a c t i n o m y c e t e s (No. 3 7 1 6 a n d No. 37171, b e l o n g i n g t o t h e S,t%ep.tornyctd t a u e n d u l a e g r o u p . The b r o t h was a d j u s t e d t o pH 2 . 5 w i t h H C 1 , t r e a t e d w i t h 2 % o f Darco G-60 f o r 3 0 min and f i l t e r e d . The f i l t r a t e was a d j u s t e d t o pH 7 . 5 w i t h 1 N N a O H , t r e a t e d w i t h 2 % o f Darco G-60 w i t h c o n s t a n t s t i r r i n g f o r 3 0 min a n d a g a i n f i l t e r e d . The e l u -
645
t i o n was e f f e c t e d w i t h 75% a c i d i f i e d e t h a n o l f o r 30 min w i t h constant agitation. The e l u a t e was a d j u s t e d t o pH 7 . 0 w i t h A m b e r l i t e I R 4 5 r e s i n ( O H - f o r m ) , c o n c e n t r a t e d i n uacuo t o 2 0 % of t h e o r i g i n a l volume a n d p r e c i p i t a t e d w i t h 1 0 volumes o f acet o n e . The p r e c i p i t a t e was d i s s o l v e d i n water a n d f r e e z e - d r i e d .
EXAMPLE 5
-
P u r i f i c a t i o n of s t r e p t o t h r i c i n V159
The c u l t u r e b r o t h o f SLteptorngced lavendulae 3 5 1 ’ 6 was a c i d i f i e d t o pH 2 . 0 - 2 . 5 w i t h c o n c e n t r a t e d H C 1 , s t i r r e d w i t h Darco G-60 ( 5 g/!L) f o r 30 min. a n d f i l t e r e d . The f i l t r a t e was a d j u s t e d t o pH 7 . 0 - 7 . 3 w i t h 4 0 % N a O H , a n d s t i r r e d f o r 30 min. w i t h N o r i t e A ( 1 0 g/!L). The a d s o r b a t e was washed w i t h 9 5 % e t h a n o l a n d a l l o w e d t o d r y o v e r n i g h t a t room t e m p e r a t u r e . The a c t i v e s u b s t a n c e was e l u t e d w i t h a m e t h a n o l i c s o l u t i o n of f o r mic a c i d ( a s i n e x a m p l e 1) y i e l d i n g t h e f o r m a t e o f s t r e p t o thricin V I . The f o r m a t e was c o n v e r t e d t o t h e p i c r a t e and t h e n t o the hydrochloride. As s t a t e d a b o v e , t h e s e methods o f i s o l a t i o n and p u r i f i c a t i o n a l l o w e d one t o o b t a i n t h e p r e p a r a t i o n s which c o n t a i n e d as a r u l e , m i x t u r e s o f d i f f e r e n t s t r e p t o t h r i c i n s F-A. T h e r e were a number o f p a p e r s d e v o t e d t o e l a b o r a t i o n o f methods f o r s e p a r a t i o n of these mixtures. I t i s w o r t h y o f m e n t i o n t h a t wides p r e a d methods o f s e p a r a t i o n o f s i m i l a r compounds s u c h as c o u n t e r - c u r r e n t d i s t r i b u t i o n i n v a r i o u s s o l v e n t systems t u r n e d o u t t o b e r a t h e r i n e f f e c t i v e i n t h e c a s e of s t r e p t o t h r i ~ i n s ~ ~ ~ ’ l4I. Sometimes t h e s t r e p t o t h r i c i n p r e p a r a t i o n s a p p e a r e d t o b e homogeneous a c c o r d i n g t o t h i s m e t h o d , b u t were r e v e a l e d t o cont a i n two or more components when t h e y were s t u d i e d by p a p e r chromatography i n t h e p r e v i o u s l y mentioned s o l v e n t systems.
Many p r o c e d u r e s were p r o p o s e d t o s e p a r a t e t h e s t r e p t o t h r i c i n mixtures i n t o individual a n t i b i o t i c s . The f i r s t method, which p e r m i t t e d t h e i s o l a t i o n i n p r e p a r a t i v e amounts ( a l t h o u g h i n r a t h e r small q u a n t i t i e s o f t h e i n d i v i d u a l s t r e p t o t h r i c i n s ) from m i x t u r e s , c o n s i s t e d o f column p a r t i t i o n c h r o m a t o g r a p h y on c e l l u l o s e i n t h e same s o l v e n t s y s t e m , l - p r o p a n o l - p y r i d i n e a c e t i c a c i d - w a t e r ( 1 5 : 1 0 : 3 : 1 2 ) , which appeared t o b e v e r y e f f e c t i v e f o r a n a l y s i s o f s t r e p t o t h r i c i n m i x t u r e s by c i r c u l a r p a p e r c h r o m a t o g r a p h y z 1 . T h i s method was a p p l i e d f i r s t t o s e p a r a t i o n o f p l e o c i d i n -- a m i x t u r e c o n t a i n i n g m a i n l y s t r e p t o Later t h i s method was u s e d f o r t h e s e p a t h r i c i n s D , E, a n d F. r a t i o n of o t h e r a n t i b i o t i c s of t h e group under c o n s i d e r a t i o n . EXAMPLE 6
-
I s o l a t i o n of i n d i v i d u a l s t r e p t o t h r i c i n s from pleocidinZ
U s i n g 23 gram p o r t i o n s , 4 1 4 g of d r y c e l l u l o s e powder (Whatman a s h l e s s , s t a n d a r d g r a d e ) were p a c k e d and tamped i n t o a g l a s s column ( 5 9 x 5 c m ) . The p a c k e d h e i g h t was 5 2 c m . A s a m p l e o f p l e o c i d i n h y d r o c h l o r i d e ( 5 2 9 mg) was d i s s o l v e d i n 1 2 m l o f s o l v e n t m i x t u r e , a n d t h e r e s u l t i n g s o l u t i o n was a d d e d t o t h e t o p of t h e a d s o r b e n t and allowed t o flow through under g r a v i t y . A s t h e l e v e l of s o l u t i o n dropped t o t h e t o p of t h e a d s o r b e n t , t h r e e 1 5 m l p o r t i o n s o f t h e s o l v e n t s y s t e m were a d d e d i n s u c c e s s i o n . An a d d i t i o n a l 4 0 m l o f t h e s o l v e n t s y s t e m
646
were added, and a 1 l i t e r s e p a r a t o r y f u n n e l c o n t a i n i n g t h e s o l v e n t system was a t t a c h e d t o t h e column t o s e r v e as a r e s e r v o i r f o r c o n t i n u o u s f e e d . A flow r a t e was m a i n t a i n e d c a . 48 m l p e r h r . F r a c t i o n s of 1 3 m l were c o l l e c t e d and a n a l y z e d by n i n h y d r i n s p o t t e s t and by c i r c u l a r chromatography. The r e s u l t s of t h e c i r c u l a r chromatograms s e r v e d as g u i d e s f o r p o o l i n g t h e a p p r o p r i a t e f r a c t i o n s . The p o o l e d column e f f l u e n t was p r o c e s s e d by e x t r a c t i o n w i t h an e q u a l volume of e t h y l e t h e r , and t h e p r e c i p i t a t e d aqueous p h a s e which c o n t a i n e d t h e a n t i b i o t i c was f r e e z e d r i e d . The a n t i b i o t i c r e s i d u e was f u r t h e r p u r i f i e d by t h e f o r m a t i o n , r e c r y s t a l l i z a t i o n , and r e c o n v e r s i o n of t h e h e l i a n t h a t e s a l t d e r i v a t i v e s . The r e s u l t s o f t h e e x p e r i m e n t are g i v e n i n t a b l e 8. Table 8 Chromatogrsphy o f Pleocidin Hydrochloride on a Column o f Cellulose Powder Fraction Eluate Volume (ml)
Components Pigments
0-455
+
t r a c e s o f ptiper
456-610
Pleocidin I
611-715
Traces o f ple oc idin I I1
716-931
Pleocidin I1
932-1022
1435-1889
Traces o f ple oc idin I1 + ple oc idin I11 Pleocidin I11 Traces o f ple oc idin I11
1890-20 84
Pleocidin IV
1023-1434
+ pleocidin
There are no d a t a i n t h e p a p e r z 1 c o n c e r n i n g o p t i c a l a c t i v i t y and e l e m e n t a l a n a l y s i s which c o u l d be i n d e p e n d e n t e v i d e n c e of homogeneity and n a t u r e of i s o l a t e d f r a c t i o n s . EXAMPLE 7
-
I s o l a t i o n of i n d i v i d u a l s t r e p t o t h r i c i n s from antibiotic A 8265u3
S t a n d a r d a s h - l e s s c e l l u l o s e ( 3 , 8 0 0 g ) was dry-packed i n a column (diam. 1 0 cm), and tamped down a f t e r e a c h a d d i t i o n (10 g ) . The f i n a l l e n g t h of t h e column was 86 c m . A n t i b i o t i c A 8265 s u l p h a t e ( 8 g ) was d i s s o l v e d i n l - p r o p a n o l - p y r i d i n e a c e t i c a c i d - w a t e r (15:10:3:12) ( 2 0 m l ) and a p p l i e d t o t h e c o l umn. A f t e r 1 6 h r , s o l v e n t s t a r t e d t o flow from t h e column a t t h e r a t e o f 9 0 m l / h r , and f r a c t i o n s ( 4 5 m l ) were c o l l e c t e d . The f i r s t 30 were a l l d a r k brown b u t t h o s e from 31 onwards were c o l o r l e s s . Each was e x t r a c t e d w i t h e t h e r ( 5 0 m l ) and t h e aqueous p h a s e ( 5 m l ) s e p a r a t e d and f r e e z e - d r i e d . The s o l i d s o o b t a i n e d was d i s s o l v e d i n w a t e r ( 0 . 5 m l ) and chromatographed on p a p e r . A s o l u t i o n of a n t i b i o t i c A 8265 s u l p h a t e ( 5 0 mg/ml) was used as a c o n t r o l and t h e chromatogram was developed for 1 6 h r by d e s c e n d i n g chromatography, t h e same s o l v e n t b e i n g u s e d . From
647
t h e s e chromatograms f r a c t i o n s g i v i n g i d e n t i c a l n i n h y d r i n - p o s t t i v e s p o t s were p o o l e d and f r e e z e - d r i e d . A f t e r 300 h r t h e e l u e n t no l o n g e r c o n t a i n e d n i n h y d r i n - p o s i t i v e compounds a n d t h e e l u t i o n was s t o p p e d . A l l t h e b u l k s o l i d s were d i s s o l v e d i n water ( 5 0 m g / m l ) a n d a p p l i e d t o c i r c u l a r p a p e r chromatograms. The r e s u l t s o f t h e s e p a r a t i o n are shown i n t a b l e 9 . Table 9 Separation o f A n t i b i o t i c A 8265 by Chromatography on Cellulose
Rf
Fraction Nwnber 1-30 31-47 148-190 220-256
383 mg
0.43
655 mg
F + E
354 mg
0.36
E
527 mg
0.32
D D + C
0.27
C + B
0.20 0.20, 0 . 1 7 0.17
463-570
193 mg
1407 mg 520 mg 635 mg
C
0.27, 0.20
372-419 420-462
F F
0.32, 0.27
311-330 331-371
0.43 0.43, 0.36
191-215 257-310
Corgonen ts
8
273 mg
B + A
199 mg
A
180 mg
T h u s , from 0 g o f o r i g i n a l m i x t u r e , 3 . 5 g o f i n d i v i d u a l s t r e p t o t h r i c i n s ( w h i c h demanded, however, a d d i t i o n a l p u r i f i c a t i o n ) a n d 2 . 6 g o f b i n a r y m i x t u r e s were o b t a i n e d a f t e r l o n g a n d v e r y cumbersome work. The f u r t h e r p u r i f i c a t i o n o f p r e p a r a t i o n s s o o b t a i n e d was a c c o m p l i s h e d by t h e i r c o n v e r s i o n i n t o corresp o n d i n g p i c r a t e s and t h e n i n t o h y d r o c h l o r i d e s . T h i s method of s e p a r a t i o n of s t r e p t o t h r i c i n m i x t u r e s s u f f e r e d from many g r a v e s h o r t c o m i n g s : i t t o o k v e r y l o n g t i m e , demanded a s p e c i a l cumbersome a n a l y s i s , a n d u s e d l a r g e amounts of p y r i d i n e . A l t h o u g h i n t h e p a p e r s from which t h e e x a m p l e s 6 and 7 are t a k e n , t h e r e was n o m e n t i o n c o n c e r n i n g i n a c t i v a t i o n of s t r e p t o t h r i c i n d u r i n g s u c h l o n g e x p e r i m e n t s , b u t i t r e a l l y existed. The methods o f s e p a r a t i o n o f s t r e p t o t h r i c i n m i x t u r e s on i o n e x c h a n g e s were f o u n d t o b e much more c o n v e n i e n t . The c h r o matography o f aqueous s o l u t i o n s of s t r e p t o t h r i c i n m i x t u r e s on c a r b o x y m e t h y l c e l l u l o s e w i t h a N a C l c o n c e n t r a t i o n g r a d i e n t is t h e s i m p l e s t t e c h n i q u e 2 2 - 2 6 . I t was p o s s i b l e t o s e p a r a t e v e r y complex m i x t u r e s ( c o n t a i n e d up t o a l l s i x n a t u r a l s t r e p t o t h r i c i n s ) w i t h t h i s method: e a c h s t r e p t o t h r i c i n was e l u t e d by a s t r i c t l y d e f i n e d s a l t c o n c e n t r a t i o n . S t r e p t o t h r i c i n F was e l u t e d by a N a C l c o n c e n t r a t i o n e q u a l t o 0 . 1 9 M , E 0.22 M, D 0 . 2 5 M, C 0 . 3 0 M; B 0 . 3 4 M and A 0.36 M. An i m p o r t a n t
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648 a d v a n t a g e o f t h i s method was t h e p o s s i b i l i t y of a s i m p l e a n d a c c u r a t e a n a l y s i s o f f r a c t i o n s : i t was v e r y e a s y t o d e t e r m i n e a n t i b i o t i c c o n t e n t i n e a c h f r a c t i o n by e n d a b s o r b t i o n a t 2152 2 0 nm. T h i s method o f c o n t r o l was shown23 t o c o i n c i d e q u i t e w e l l w i t h n i n h y d r i n o r b i o l o g i c a l a s s a y . The s e c o n d merit o f t h e method was i t s r a p i d i t y t h e whole e x p e r i m e n t on chromatographic s e p a r a t i o n of s t r e p t o t h r i c i n mixture took l e s s than 8 h r (compare w i t h t h e p r e v i o u s method.). I n g e n e r a l , t h i s method r e p r o d u c e d s o w e l l t h a t i t was u s e d as a s t a n d a r d a n a l y t i c a l p r o c e d u r e t o d e t e r m i n e , f o r example, t h e q u a n t i t a t i v e a l t e r a t i o n s i n c o n t e n t o f d i f f e r e n t s t r e p t o t h r i c i n s (A-F) i n c o u r s e of t h e i r b i o s y n t h e ~ i s ~ ~ ~ .
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EXAMPLE 8
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S e p a r a t i o n of s t r e p t o t h r i c i n m i x t u r e s by i o n exchange chromatography on c a r b o x y m e t h y l c e l l u l o s e 2 4
A s a r u l e , c r u d e s t r e p t o t h r i c i n p r e p a r a t i o n s were u s e d f o r s e p a r a t i o n , which were p r e v i o u s l y p u r i f i e d a t t h e l a s t s t a g e by c o n v e r s i o n t o p i c r a t e s and t h e n t o h y d r o c h l o r i d e s ,
Ion e x c h a n g e chromatography was a c c o m p l i s h e d i n g l a s s columns ( 0 . 9 x 40 c m ) . The c a r b o x y m e t h y l c e l l u l o s e ( S e r v a E n t w i c k l u n g s l a b o r - H e i d e l b e r g ) w i t h c a p a c i t y o f 0.55 e q u i v . / g o r c a r b o x y m e t h y l c e l l u l o s e home-made i n t h e l a b o r a t o r y a c c o r d i n g t o a p r o c e d u r e 1 4 2 i n t h e N a t form was u s e d a6 t h e Fractions of a d s o r b e n t . The e l u t i o n r a t e was ca. 15-20 m l / h r . ca. 5 m l were c o l l e c t e d 2 2 . The m i x t u r e t a k e n f o r s e p a r a t i o n ( 2 0 - 3 5 mg) was d i s s o l v e d i n 0 . 5 m l of w a t e r . In the case of s e p a r a t i o n of l a r g e r amounts o f s t r e p t o t h r i c i n m i x t u r e s ( 4 - 1 3 g ) a column 6 x 9 0 cm (volume ca. 2 a ) was u s e d . The e l u t i o n r a t e i n t h i s c a s e was ca. 200-250 m l / h r a n d f r a c t i o n volume ca. 250300 m l . F r a c t i o n s were a n a l y s e d by a b s o r p t i o n a t 2 2 0 nm, p o o l e d and t r e a t e d f u r t h e r a c c o r d i n g t o t h e f o l l o w i n g method ( d e s c r i b e d i n example 9 ) f o r t h e i s o l a t i o n of s t r e p t o t h r i c i n B (polymycin B) from a c r u d e p r e p a r a t i o n o f polymycin. EXAMPLE 9
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I s o l a t i o n o f s t r e p t o t h r i c i n B from f r a c t i o n s obt a i n e d i n t h e c o u r s e of i o n e x c h a n g e chromatography of polymycin
The t o t a l volume o f f r a c t i o n s c o n t a i n i n g polymycin 8 , i n t h e c a s e of a s e p a r a t i o n on a column 6 x 9 0 cm, was ca. 5 R. I t was d i l u t e d t h r e e times w i t h w a t e r and was p a s s e d t h r o u g h a small column ( 1 . 5 x I( cm) w i t h A m b e r l i t e I R C - 5 0 r e s i n i n N a + form ( s i z e o f p a r t i c l e s ca. 80-100 m i c r o n s ) w i t h a r a t e ca. 500 ml/hr. A s sodium i o n was d i s p l a c e d by a n t i b i o t i c , t h e l i g h t p i n k c o l o r of t h e c a t i o n i c e x c h a n g e r changed t o p u r e w h i t e , s o i t was v e r y e a s y t o d e t e r m i n e t h e zone o f s a t u r a t i o n v i s u a l l y . A d s o r p t i o n was recommended t o f u l l s a t u r a t i o n of t h e A f t e r t e r m i n a t i o n of a d s o r t i o n , t h e rer e s i n with a n t i b i o t i c . m a i n i n g sodium i o n s were d i s p l a c e d w i t h 0.01-0.02 N a c e t i c a c i d a t a r a t e c a . 2 0 0 m l / h r t i l l t h e a p p e a r a n c e of s t r e p t o t h r i c i n B in the eluate. Thereupon t h e a n t i b i o t i c was d e s o r b e d w i t h 0 . 0 1 N HC1 ( r a t e o f d e s o r p t i o n ca. 5 0 - 1 0 0 m l / h r ) . The c o m p l e t e n e s s of d e s o r p t i o n was d e t e r m i n e d by r e a c t i o n w i t h sodium phosphotungstate. The n e u t r a l and a c i d e l u a t e s ( c a . 50 m l of e a c h ) were c o l l e c t e d s e p a r a t e l y . The a c i d e l u a t e was a d j u s t e d t o pH
6 49
5-6 with Amberlite IR-45 in the OH- form, pooled together with neutral eluate, and evaporated to dryness in the presence of abs. ethanol. Amorphous hygroscopic streptothricin B hydrochloride was stored i n uacuo over P205. For further purification, 600 mg of streptothricin B hydrochloride isolated as above were dissolved in 5 ml of water, added to a hot solution of picric acid (1 goin 20 ml of water) and held overnight in refrigerator at ca. 4 The resulting gummy precipitate was collected, dissolved in 5-6 ml of aqueous acetone (ca. 10% of water). The undissolved residues were removed by centrifugation, and the picrate was precipitated by 25-30 ml of ether. The gummy precipitate was redissolved in 5-6 ml of aqueous acetone, diluted three times with aqueous methanol (3% of water) and was passed through a column (1.2 x 4 cm), composed of three layers (from bottom): 1) ca. 2 ml of mixture of active carbon with celite 545; 2 ) 2 ml of celite and 3 ) ca. 4 ml of carefully washed fine-ground(400-600 mesh) Amberlite IRA-400 in C1- form. Streptothricin B hydrochloride was eluted by aqueous methanol. The eluate was dried in the presence of abs. ethanol; yield was ca. 520 mg. To prepare oxalate or sulphate the aqueous solution of streptothricin B hydrochloride was passed through a column (0.6 x 4 cm) with carefully washed fine-ground (400-600 mesh) Amberlite IRA-400 in the oxalate or sulphate form, respectively.
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In 1971 H. Taniyama and co-workers33 proposed a variant of ion exchange chromatography on carboxymethyl cellulose by introducing volatile pyridine-acetate buffer instead of NaC133. This alteration simplified somewhat the further purification of fractions (although the authors did not describe it in detail), but simultaneously caused a number of difficulties and shortcomings: the necessity to use large amounts of pyridine, the long duration of the separation process and therefore increased danger of inactivation. Besides, the use of pyridine-acetate buffer excludes the possibility of application of a very simple method of fraction analysis by means of measuring end absorption at 215-220 nm. Thus the merits of the proposed variant hardly exceed its shortcomings. Examples of using this method for separation of streptothricin mixtures follow. EXAMPLE 10
Separation of the mixture of racemomycins A, C, B, and D by ion exchange chromatography on carboxy-cellulose in gradient of pyridine-acetate buffer3
A crude preparation of racemomycin mixture (A, C, B and D) which contained ca. 30% of inorganic impurities was used for separation. As mentioned above, racemomycins A, C, B and D were identical to streptothricins F, E, D and C, respectively. Chromatography was accomplished on a column 8 x 20 cm. The carboxymethyl cellulose (Brown Co.) (capacity 0.77 meq./
