Advances in Carbohydrate Chemistry and Biochemistry, Volume 49

Advances in Carbohydrate Chemistry and Biochemistry

Volume 49

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Advances in Carbohydrate Chemistry and Biochemistry Editor DEREK HORTON

Board of Advisors LAURENS ANDERSON J. GRANTBUCHANAN J. ANGYAL STEPHEN GUYG. S. DUTTON HANSH. BAER BENGTLINDBERG CLINTON E. BALLOU HANSPAULSEN JOHN S. BRIMACOMBE NATHANSHARON ROY L. WHISTLER

Volume 49

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9 8 7 6 5 4 3 2 1

CONTENTS PREFACE................................................................

vii

R e d Bognpr. 1913-1990 ANDRASLIPTAK.PALNANAsI. AND FERENC SZTARICSKAI Text

....................................................................

3

Jean Emile Courtois. 1907. 1989 FRANCOIS PERCHERON Text

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11

The Composition of Reducing Sugars in Solution: Current Aspects STEPHEN J . ANGYAL I. Introduction ........................................................ I1. Methods for Studying the Composition of Sugars in Solution ............... 111. Relative Stabilities of the Various Forms ................................ IV. Composition in Aqueous Solution: Aldoses .............................. V. Composition in Aqueous Solution: Ketoses .............................. VI. Composition in Aqueous Solution: Substituted and Derived Sugars .......... VII. Solutions in SolventsOther Than Water................................. VIII. TabulatedData .....................................................

19 20 22 25 27 28 31 32

Radical-Mediated Brominations at Ring Positions of Carbohydrates U S ZSOMSAK L ~ AND ROBERTJ. FERRIER

I. I1. 111. IV.

Introduction ........................................................ Radical-mediated Brominations........................................ The Regio- and Stereo-chemistry of the Reactions......................... Reactions of the Bromine-containing Products ........................... V . Conclusions ........................................................ VI . Addendum ......................................................... V

37 41 67 75

91 91

vi

CONTENTS 1. 4 . 3. 6.Dianhydrohexitols

PETERSTOSSAND REINHARD HEMMER I. I1. Ill . IV. V. VI .

Introduction ........................................................ 93 Nomenclature ...................................................... 96 Spectroscopic Properties. Structural Aspects. and Analytical Detection........ 99 Preparation of the Parent Compounds .................................. 119 Derivatives......................................................... 125 Applications........................................................ 158

Enzymic Methods in Preparative Carbohydrate Chemistry

SERGE DAVID.CLAUDINE AuGB. AND CHRISTINE GAUTHERON I. I1. 111. IV . V. VI . VII . VIII. IX .

Introduction ........................................................ Immobilization ..................................................... Aldol Additions and Other C-C Bond-forming Reactions .................. Phosphorylations.................................................... Glycosylations with Transferases ....................................... Transfer Reactions Catalyzed by Glycosidases............................ Miscellaneous Syntheses in Aqueous Solution ............................ Enzymes in Organic Solvents.......................................... Addendum .........................................................

176 180

189 207 218 231 234 235 236

Structure of Collagen FibriLAssaciated. Small Proteoglycans of Mammalian Origio HARIGARGAND NANCYLYON Introduction ........................................................ Structure of Different Glycosaminoglycans............................... Carbohydrate- Protein Linkage Regions ................................. Isolation and Fractionation of Small Proteoglycans........................ M, of Small Proteoglycans. Their Protein Cores. and Glycosaminoglycan Chains ............................................................ VI . N-Terminal Sequence of Small Proteoglycans ............................ VII. Amino Acid Sequence. Analysis of the Small Proteoglycan Core Protein. Deduced from Cloned cDNA .......................................... VIII. Biosynthesisof Small Proteoglycans .................................... IX . Biological Roles of Small Proteoglycans ................................. X . Addendum .........................................................

239 240 240 243

........................................................... AUTHORINDEX

263

SUBJECTINDEX...........................................................

279

I. I1. I11. IV . V.

244 251 254 256 258 260

PREFACE Tribute is paid here to the contributions in the carbohydrate field of two notable figures, Rezsi3 Bognk and Jean Emile Courtois, in articles respectively furnished by A. Liptik, P. NhnBsi, and F. Sztaricskai (Debrecen), and by F. Percheron (Pans). Analysis of the tautomeric compositions of reducing sugars in solution by classical polarimetric methods has inherent limitations, but n.m.r.-spectroscopic methods have greatly enhanced our ability to monitor and quantitate such mobile interconversions of sugars. An excellent overview of developments in this field was presented by s. J. Angyal (Kensington, N.S.W., Australia) in Volume 42. However, the rapid progress of new research, with the advent of more sophisticated spectrometers and techniques of data manipulation, has provided the motivation for a supplement, prepared again by Angyal, which updates and complements his earlier chapter and is to be used in conjunction with it. The synthetic proceduresavailable to the carbohydrate chemist have been largely dominated by standard reactions proceeding by heterolytic processes within a chiral matrix. The preparative utility of radical-mediated reactions has, however, been amply demonstrated in recent years. The chapter contributed here by L. Somsik (Debrecen) and R. J. Femer (Wellington), on bromination reactions of carbohydrates proceeding by radical processes integrates the literature related to Femer’s pioneering work in this area and underscores its excellent potential in synthesis. Continuing in the synthetic vein, S. David, C. AugC, and C.Gautheron (Paris) present a practical overview of the potential of enzymes as synthetic tools for the general organic chemist. Their chapter, with a well-selected variety of examples, should help the bench-level organic chemist to overcome the classic preconception that enzymes are exclusively the domain of the biochemist working with nanomolar amounts of material. The David AugC contribution should materially help in opening up the way for enzymes, both free and immobilized,to be used advantageously for preparative access to important and useful sugars and metabolic intermediates. P. Stoss (Dottikon, Switzerland) and R. Hemmer (Senden, Germany), in their articleon the 1,4 :3,6-dianhydrohexitols, provide the perspective of the industrial chemist and bring up to date a subject that was treated by Wiggins in Volume 5 of this series and by Soltzberg in the tabular material contributed in Volume 25. Theseanhydridesare ofconsiderable theoretical interest, but much of the rapidly burgeoning related research is recorded in the patent literature because of the wide practicalpotential manifested by these bicyclic diols.

...

vlll

PREFACE

Although the classic proteoglycans of cartilage tissue are now well characterized, considerably less is known concerning the “small proteoglycans” containing only one or two glycosaminoglycan chains on the protein core; their structures and biological roles are surveyed here by H.Garg and N. Lyon (Boston). It is with great regret that I record the passing on July 13, 1991 of R. Stuart Tipson in his 85th year. Dr. Tipson was a contributor to the first volume in this series and a member of the editorial team beginning with Volume 8 in 1954 until his retirement from the editorship at the completion of Volume 48 in 1990. A fuller survey of his life and scientific work is scheduled for an upcoming volume.

Columbus, Ohio August I991

DEREK HORTON

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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 49

R E Z S ~BOGNAR 1913- 1990

An extremely rich and comprehensive life and career ended in Debrecen, Hungary, on the evening of Sunday, February 4th, 1990, when Rezsd Bognir, Professor Emeritus of the Lajos Kossuth University of Debrecen passed away at the age of 77. Despite knowing for almost a year that he had been stricken with an incurable cancer, he went to work in his office until the very last days, making plans and engagingin organizational activities,as well as to learn. He spent the last days of his life in a guest-house and he carried his French language text-book there as he wanted to improve his French in the last months. This episode was characteristic of his whole life, but not of a man who takes leave of his life, a life that he could organizewith an imposing sense, leaving time for almost everything he deemed important. Rezd Bognk was born on March 7th, 1913 in the town of Hodmeziivasarhely, the capital of the poverty-stricken South-Eastem part of Hungary, the so-called Viharsarok (the “Stormy Corner”). This town used to be the center of the masses of poor peasantry fighting for work and a living. Although the Bogndr family never suffered from bread-and-butter worries, the solicitude of his father, Rezsd Bognar, a Presbyterian schoolmastercantor, and his mother Klara Hegedus, ensured an unclouded childhood to the little boy Rezsii, and the puritan life-style and the understanding and espousal of the problems of poor people were characteristic of the whole Bognar family, including Professor Bognk. He finished his elementary and secondary school studies in his home-town and obtained the certificate of final examination, required for attending university, in 193 1. His parents wanted the young Rezsd to stay close to home, and so they enrolled him to the University of Szeged (20 km away from the home-town) to learn to become a pharmacist. However, these studiesdid not satisfy the young man, who was primarily interested in technical and practical problems, and in the next year he moved to Budapest to continue his studies at the J6zsef.Nador University ofTechnica1and Economic Sciences,where he graduated in 1936 as a chemical engineer. 3

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LIPTAK et ai.

GCza Zemplen, the famous professor of this university, soon recognized the talent of his student, and invited him to join the Department of Organic Chemistry. However, the young Rezsii decided he would spend at least a short period of time in industry, and he worked for the Guttapercha Cs Gumi factory, but, one year later, he returned to the University and became the private assistant of Professor Gkza ZemplCn, the man who represented Organic Chemistry in Hungary in the first part of the 20th century. This outstanding scientist (see Vol. 14, p. I), who had extraordinary human features, had studied and practised organic chemistry in the laboratory of Emil Fischer in Berlin, and had obtained particularly important results in the field of the chemistry of carbohydrates. Rezsd Bognk proved to be an excellent student and disciple, and he made the most of his outstanding preparative capabilities. “Everything” crystallized in his hands, and this was especially important in those days, before the introduction of chromatographic techniques. Their firstjoint paper appeared in 1939,on the synthesis of primeverose and its derivatives [Ber. Deutsch. Chern. Ges., 72 (1939) 47-491,and it was followedby 2 1 papers up to 1944,primarily from the field of flavone and anthraquinone glycosides. Naturally, these glycoside syntheses required the preparation of numerous oligosaccharides. These studies comprised the basis of the Ph.D. thesis of Rezsii Bog&, presented in 1941 , in which the definitive syntheses of linarin (5-hydroxy-4’-methoxy-7-Prutinosyloxyflavone) and pectolinarin (5-hydroxy-6,4’-dimethoxy-7-~-rutinosyloxyflavone)were described. He lived in Budapest through the fighting and ravages of World War I1 and, after the march from the front, the restoration work of the Department of Organic Chemistry of the Technical University started under his leadership. Besides this work, most of the organizing and educational duties in the Department weighed heavily on Rezsii Bogn6r because of the foreign visiting-professorship, and later, due to the advanced stage of sickness of GCza Zempltn. Despite these manifold activities, the scientific career of Rezsii Bognar proceeded unbroken; in 1946,he qualified as privat-docent (after habilitation) and appointed a university professor in 1949.Together with G6za Zemplen, he was in 1948awarded the Kossuth Prize in the company of such world-famous persons as the composer ZoMn Kodily and the Nobel Prize laureate Albert Szent-Gydrgyi. The general scientificpublic considered Rezs6 Bognk to be the successor of G k a Zempltn at the Technological University in Budapest, and thus it is remarkable and surprising that he accepted the invitation of the Lajos Kossuth University of Debrecen to establish the Chair of OrganicChemistry, to organize the related educational duties, and to start scientific research. During the four decades of his activities in Debrecen he performed significantorganizing work in both education and research, was the Rector of the University for two election-cycles,and was appointed the Secretary-General of the Hungarian Academy of

R E Z S ~B ~ G N A R

5

Sciences. He also served as President of the Debrecen Local Committee of the Hungarian Academy of Sciences from its foundation in 1976 until his death in 1990. In Debrecen, he continued research on flavonoid compounds and carbohydrates, but with significantlychanged thematics. Completely new fields of natural products research were involved, such as isolation and chemical modification of opium alkaloids, isolation and structure elucidation of steroid- alkaloid glycosides, as well as research on antibiotics. In the field of the opium alkaloids, he made considerable efforts for the isolation of the accompanying minor alkaloids (codeine, thebaine, narcotine, narcotoline, and papaverine) in the form of industrially utilizable preparations. The partial hydrogenation of thebaine to dihydrothebaine allowed the preparation of the medicinally important dihydrocodeine. Detailed studies were performed on narcotine and narcotoline, and the total synthesisof narcotine was also elaborated. In the case of narcotoline, the elimination of the phenolic hydroxyl group was studied in particular. A new method was worked out for the synthesis of phthalidoisoquinoline alkaloids. With the morphine compounds, nucleophilic substitution of the 6-alkyl ethers and arylsulfonic esters of the ring-carbon atom and numerous derivativeshaving outstanding biological activity were synthesized.He also performed important studies in the field of steroid- alkaloid glycosides, including the isolation and structure elucidation of several new glycosides isolated from numerous Sofunurnspecies. Tometidenol, isolated in considerable quantities from Sofunurndufcurnaru L., proved to be a useful precursor for the synthesis of steroid derivatives having industrial importance. The research on flavonoids, started together with Zemplh, was continued in Debrecen, the primary aim being to modify the carbon skeleton, and the isolation of rutin on an industrial scale was also elaborated in Debrecen. From the synthetic studies on flavonoid compounds, the most important results were the separation of, and assignment of the absolute configuration to, the 3-bromoflavone isomers, obtained upon bromination of flavanone, and related studies, including conformational investigations, on the isomeric 3-hydroxyflavanone, 4-hydroxyflavone, 3-aminoflavanone, and 4aminoflavane.Procedures for the resolution of 4-aminoflavane and flavanone were also elaborated. He and his coworkers performed detailed investigationson the preparation and chemical transformations of the epoxides and aziridines derived from chalcones. His group also performed pioneering work in the field of the synthesis of flavonoids containing nitrogen and sulfur in the heterocyclic ring. In the second half of the 19503, his attention turned more and more to the antibiotic substances, and, in 1960, he founded the Antibiotic Research Group of the Hungarian Academy of Sciences. The first studies in the antibiotics field aimed at the synthesis of chloroflavonineand several analogs of

6

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novobiocin. The isolation and structural investigation of a few antibiotics (including desertomycin and flavofungin), that originated in Hungary, are also linked with the name of Rezsd BognSr. In the case of flavofungin, the assignment of a new structural unit (a pentaene chromophore conjugated with a lactone-carbonyl group) led to the recognition of a novel sub-groupof the pentaene macrolides. The zenith of the antibiotic research of his group was the elucidation of the structure of the glycopeptide antibiotics actinoidins A and B, and rktomycins (ristocetins)A and B. He and his associates were the first to isolate ristosamine, an important representative of the 3amino-2,3,6-trideoxyhexoses.This amino sugar and its stereoisomers (acosamine, daunosamine, and D-ristosamine)were synthesized by the BognSr group by the application of several methods, and the intermediates for these syntheseswere used for transformation into cyclitol derivativesby means of the Ferrier ring-transformation reaction. The amino sugars thus prepared were also utilized for the preparation of semisyntheticanthracycline glycoside antibiotics (such as daunomycin and carminomycin), as well as for aminocyclitolantibiotic analogues. In connection with the research on antibiotics, Professor Bognar returned in the last decade of his scientificactivity to one of the topics of his youth, namely, to the synthesisof oligosaccharides. He and his collaborators synthesized ristobiose (2-O-a-~-mannopyranosylD-glucose), ristotnose (0-a-L-rhamnopyranosyl-(1 3 6)-O-[a-~-mannopyranosyl-(1 2)]-~-glucose),ristriose [0-a-D-arabinofuranosyl-(1 2)0-a-D-mannopyranosyl-(1 + 2)-~-glucose],and a derivative of acobiose [2-0-(3-amino-2,3,6-~deoxy~-~-urabino-hexopyranosyl~~glucose]. Of these research topics, the most beloved one for Professor Bognhr was still the chemistry of carbohydrates. He was extremely productive in this field, and so, only the most important results of his contribution to carbohydrate chemistry can be discussed here. In the fifties, it was not entirely clear whether the secondary glycosylamines possess a glycosylic or a Schiff-basestructure. On the other hand, the simple preparation of such compounds offered the possibility of transforming sulfonamide derivatives having low water-solubility into more-soluble, and pharmacologically more effective, glycosylamine analogs. Rezsd BognSr connected the solution of the theoretical-structural problem with the demands of practice. By using p-aminosalicylic acid (PAS) and p-aminobenzenesulfonamide(PAB) as aglycons, the Bogniir group obtained glycosylamines having high water-solubility. Moreover, by extension of these studies to other aromatic amines, it was unequivocally proved that the derivatives produced were of glycosylic structure. In experiments with acetylated and methylated pyranoid derivatives the anomerscould be separated and isolated pure, and, upon U-deacetylation of the individual anomers, the a-and B forms of the unprotected glycosylaminescould be prepared.

-

-

RE&

BWNAR

7

Efforts to obtain glycofuranosylamineswere successful only in the case of methyl ether derivatives. It was also recognized that, in solution, theglycosylamines are always present in the form of anomeric mixtures, and the chance of obtainingone of the pure anomers by crystallizationis always determined by their physical characteristics. Systematic research on glycosylaminesled to observation of occurrence of the so-called transglycosylation reaction, and the mechanism of this transformation was studied and explained through the followingexamples, which have practical utility:

+

+ + g l y ~ ~ y l ‘ - O H glyc~~yl’-NH-R+ glyc~~yl-OH GIY~OSYI-NH-R GIYcosYI-NH-R + gly~o~yl’-NH-R’ glyco~yl-NH-R‘+ gly~~yl’-NH-R

-

GIYCOS~I-NH-RHZN-R’ 4 glyc~~yl-NH-R‘ H,N-R

+

Similar reactions could also be performed with the acetylated and the methylated derivatives. The real transglycosylation character of the proton-catalyzed process was unequivocally proved by demonstrating the intermediacy of a glycosylium ion. Studies on these very fast reactions allowed Rezsii Bognir to display his outstanding preparative skill; by proper choice, and change, of the experimental conditions, the equilibrium system could be completely shifted towards one direction, affording almost quantitative yields of the desired products. The Bognir team synthesized numerous glycosylated carbonic acid derivatives, of which the bis-glycosylcarbodiimidesare the most important. He was concerned with the reaction of sugars and amino acids for decades and investigated the structure of the products and the mechanism of the reactions. The most significant field of this research was the preparation of new thiazolidine and benzothiazoline derivatives, carrying a C-2 polyhydroxyalkyl side-chain, by means of the condensation of aldehydo sugars with mercapto-amino acids (L-cysteine and D-penicillamine) and o-aminobenzenethiol. The transformation of pentoses and hexoses into 2-furaldehyde and 5 4 hydroxymethyl)-2-furaldehyde,respectively, by the action of acids is a well-known reaction. Professor Bognar was long interested in ascertaining whether this reaction is reversible. With both a theoretical and a practical goal, the Bognar group then synthesized the DL forms of several important monosaccharides (xylose, ribose, and arabinose) from the aforementioned furan derivatives. By investigating the reaction of &,a-dihalo ethers with peracetylated sugars and acetylated glycosides, Rezsii Bognir recognized that these halogenating agents are extremely suitable for the synthesis of 0-acylglycosyl

8

ANDRAS LIPTAK et

a/.

halides, permitting the isolation of both anomers of the 1-halides. The reagents could also be applied for the selectivesplittingof oligosaccharide-type glycosides. As an example, from peracetylated rutin, the disaccharide component could be isolated in the form of acetyl-a-rutinosylchloride. During the last few years, this procedure has emerged as one of the most popular general methods for obtaining glycosyl halides, so much the more because benzylated or allylated sugars also readily give the sensitive, otherwise difficultly accessible 1-halides. The Bognar group successfully applied glycosyl cyanides for the synthesis of C-glycosyl heterocycles (C-nucleosides).During related studies, numerous 5-glycosyltetrazolederivatives were prepared, and, by means of their ring-transformation reactions, C-nucleoside-type 1,3,4-oxadiazoles and condensed heterocyclic compounds (triazolopyridines and triazolopyrimidines) were obtained. The latter derivatives are synthetic analogs of the antibiotic formicin. In recognition of his scientific activities, Professor Bognar was elected to membership on the editorialboard of severaljournals: Journal ofAntibiotics (from 1968), Organic Prep. Proc. International, Acta Chimica Hungarica, Magyar Kimiai Folybirat, and of the series Recent Developments in the Chemistry of Natural Carbon Compounds. Professor BognSlr’s contribution to the scientific literature totaled more than 400 publications, 30 patents, and several monographs. He worked as Visiting Professor for long periods at the universitiesof Dublin (Ireland) and Kiev (USSR). Many honors were conferred on him both in Hungary and abroad. He was awarded the Kossuth Prize twice (1948 and 1962), and honorary titles and medals, such as the JSlnos Kabay medal (1956),Purkyne medal (Czechoslovakia, 1964), Cyril1 and Method medal (Bulgaria, 1970), the Gold Medal of the Hungarian Academy of Sciences( 1982),and the GCza ZemplCn medal ( 1985). He was elected first corresponding member ( 1948) and then ordinary member (1953) of the Hungarian Academy of Sciences, member of the Bulgarian Academy of Sciences (1952) and the German Academia Leopoldina of Halle (1970). An honorary Doctor’s degree was conferred upon him by the University of Kiev (USSR, 1967)and the Lajos Kossuth University of Debrecen (Hungary, 1988). Professor Bognar was a well-known and prominent character at international scientific conferences. His kind and informal personality, great knowledge, and well considered, but never aggressive, logical arguments brought international recognition, not only to himself, but also to Hungarian carbohydrate chemists in general. Many of his former students and collaborators declared, and still declare, themselves disciples of the “Bognir-school.” He was very proud of his best students and coworkers, and he always helped and supported them, both in their scientific careers and pri-

REZSO BOGNAR

9

vate lives. The recognition and affection of his friends and collaboratorswere truly a life-giving support for Professor Bognhr. He enjoyed scientific successes, but never monopolized them. In 1962, he divided the money-prize of his second Kossuth Prize between his associates, saying that the reasons behind the high prize were the results they produced together. He always felt at home in Debrecen, and was able to resist invitationsto the beloved capital of Budapest, despite the many attractions of that metropolis. He was a warm, friendly, informal, and loveable man with ash-blue eyes and a youthful appearance, or as many of his friends recalled him, an altogether charmingperson. He bravely endured the ordeal of his last weeks with endless patience, and, instead of complaining, he still planned and thought of the future. In Professor RezsB Bognhr's person, the international scientific community has lost a scientistwith a wide intellectual horizon, who was also a great humanist.

ANDRASLIPTAK PAL NANASI FERENCSZTARICSKAI

ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 49

JEAN EMILE COURTOIS 1907- 1989

Born in Pans on March 6th, 1907, Jean Emile Courtois belonged to a family that had practised the pharmaceutical profession for three generations in Saulieu. It was in this small town ofthe Burgundian Morvan, not far from Dijon, that he attended Junior High School. After some delay in which to prepare for the entrance examination for the French Colonial School, he finally chose to undertake a pharmaceutical education, a decision which gave great satisfaction to his family. This education began with a one-year introductory course in a pharmaceutical dispensary, which in his case was the family one in Saulieu. Here, the young student had a rigorous initiation into the art, and learned the conscientiousnessof pharmaceutical practice, thanks to the kindly but firm solicitude of his father and his grandfathers, all of whom were pharmacists. The high concept of these practitioners of their mission towards their patients and the public was determining for J. E. Courtois, who, the next year, attended the FacultCde Pharmacie de l’Avenue de I’Observatoire in Paris. He was a brilliant pupil who, in 1930, was graduated as a pharmacist and simultaneously as Bachelor of Science in the FacultC des Sciences. In the same way, J. E. Courtois had undertaken a hospital career: received in 1927as an Interne in Pharmacy, he was named in 1932, after competitive examination, Pharmacist of the Paris Hospitals. At that time, these functions included the direction ofboth the pharmaceuticaldispensary and the clinical chemistry laboratory of a hospital. He continued in these functions until his retirement in 1978. It must be observed here that, in France, hospital functions may be associated with an academic position. Consequently, J. 8. Courtois, who was attracted to biological chemistry, entered the Faculty of Pharmacy in the laboratory headed by Paul Fleury, his master with whom a fruitful collaboration became established that was to last for many years. His academic career proceeded harmoniously: beginning as a practical instructor, he was later to become Head of Practical Training, Associate 11

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Professor, and Professor,and he eventuallyreplaced P. Fleury in the Chair of Biological Chemistry in 1955. This long career allowed Professor Courtoisto live alongsidethe development of modem fundamental biochemistry, as well as the applications of biology to medical diagnosis, from the ancient simple manual techniques to the use of the most sophisticated devices. His double career was interrupted twice: in 1939- 1940, during the Second World War, in which he served as “auxiliary pharmacist,” and from November 1944 to October 1945 when, as Captain Pharmacist in the Forces Fran@ses de I’IntCrieur, he finished his service in the war as a volunteer. During the German Occupation period,he took into his home, in Paris, some members of the “Rbistance” who were wanted by the Gestapo. In spite of his heavy professional occupations,Courtois established a very successful career as a researcher. This activity began in 193 1; he wrote a university thesis on the adsorption of sugars by metallic hydroxides in 1932. He obtained the Doctorat es Sciencesd‘Etat in 1938, with a thesis devoted to a kinetic study of some plant phosphatases. These enzymes retained his attention for some years, but the carbohydrates,from the chemical as well as the enzymic point of view, quickly became the favorite research topic of Professor Courtois. The chemical researches were directed towards three main aims. The first dealt with periodic acid oxidation. In 1928, L. Malaprade, at the University of Nancy, hoping to specify the effect of D-mannitol upon the acidity of periodic acid, observed that the carbon-carbon linkages ofthe polyol were cleaved, and showed that this was a general feature of the specificreaction of periodic acid with a-glycols. Then, P. Fleury had the premonition that this acid should be an invaluable reagent for analytical purposes. He described the utilization and determination of this remarkably selective oxidant, working under mild conditions of pH and temperature. Then began, and continued for more than twenty years, a long series of analytical and structural researches on carbohydrates by P. Fleury, J. E. Courtois, and their coworkers. It may be recalled that sodium periodate was not then readily available and, especially during the sad years of the Second World War, had to be prepared in the laboratory. Their main results may be summarized as follows. The periodic acid oxidation of polyols afforded a method for quantitative determination of these compounds, and it was demonstrated that the first reaction products are carbonyl compounds, themselves in turn degraded from their reducing end. After complete oxidation, it is possible to make an estimate of the consumption of oxidant, as well as of the formic acid and formaldehyde that are produced. The monosaccharides are attacked preferentially at the neigh-

JEAN EMILE COURTOIS

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boring reducing groups: sequentially, the aldoses give rise to their lower homologs, whereas, for ketoses, the oxidation can begin on either side of the carbonyl group, and proceeds along the carbon chain. Very successful experimentswere carried out on the oxidation of sucrose, one mole of which needs three moles of periodic acid, with the formation of one mole each of formic acid and a tetraaldehyde. The latter is oxidized by bromine to a tetracarboxylic acid; subsequent acid hydrolysis of the oxygen bridges affords a mixture of acids, all of which were isolated and identified. These results brought in 1943 a confirmation of the structure of sucrose which was discussed, and which gained the approval of C. S. Hudson who, before that, was a little doubtful about the furanoid form of the D-fructosyl group. Similar work was done later with trehalose. In the same way, J. 8. Courtoisobtained confirmation of the structure of rafhnose, and established that of stachyose. Applying periodic acid oxidation to reducing di- and oligo-saccharides having (1 -4) linkages, J. E. Courtois observed the “overoxidation” phenomenon, which was further extended by study of the oxidation of malonic, malic, and citric acids. Two heteroside structures, amygdaloside and vicianoside, were also studied with this reagent. The second topic examined by Professor Courtoisin carbohydratechemistry concerned the isolation and structural determination of a number of plant oligosaccharides in the series of the sucrose D-galactosides. The raffinose-stachyose family was completed by the isolation from Verbascum thupsiforme of the higher homologs, verbascose and ajugose, followed by a hepta- and an octa-saccharide. Ajugose had previously been described in Japan, but with an erroneous structure. The correct one was established by Courtois, and confirmed by using an Ajugu species cultured in Paris from seeds of Japanese origin. The botanical family of Caryophyllaceae was the subject of extensive research, leading to the discovery of other types of sucrose D-galactosides: the lychnose series, where the chain of D-galactosyl residues is linked at C-1 of the D-frUCtOSY1 moiety of sucrose, and that of isolychnose, where the oligogalactosidic chain is substituted at C-3 of the D-fructose. The compounds isolated contain up to five D-galactosyl units. The comparison of these results with those from the studies of phytophysiologists led to the conclusionthat the D-galactosidesof sucrose play an important role in plants, firstly as reserve carbohydrates, readily mobilized if needed, and secondly, the accumulation in plants of these highly soluble products of low molecular weight may favor their resistance to freezing. Also may be cited the isolation, from ViCia seeds, of galactinol, a galactoside of inositol, previously known only in beetroots; it is accompanied by a higher homolog, a digalactosyl-inositol. It has since been shown elsewhere

14

FRANCOIS PERCHERON

that galactinolis the first galactosederivativeappearing after photosynthesis, and that it seems to be a transient donor for the biosynthesis of sucrose D-galactosides. Polysaccharideswere the third subject of the chemical interest of Professor Courtois, in the field of D-mannose-containing glycans: P-D-(1 +4)-mannans from palm-tree seeds; orchid-tuber glucomannans, either from Syrian salep or from wild species in France, which appeared to have mainly P-D( 1 +4) linear structures, with D-glucosyl residues inserted among residues of the main D-mannan chain; and galactomannans from various leguminous seeds. The major structural data were obtained by classical determinations, namely, methylation and periodic acid oxidation. It may be recalled that, for the first time, the non-regular repetition of the a-D-galactosylresidues, substituted at 0-6 of the D-mannan backbone of the galactomannans, was demonstrated, using the enzymic reagents a-D-galactosidaseand P-D-mannanase. Confirmationwas afforded by more-sophisticated chemical means in other countries. With this utilization ofenzymes in structural studies, we arrive now at the second major subject of Professor Courtois’s activity in the carbohydrate field, namely, the glycosidases. Several cr-D-galactosidaseswere the subject of extensive studies which led to the demonstration of transglycosylation reactions. With the enzyme from coffee-bean, using phenyl a-D-galactoside as the donor, transfer was observed of the a-D-galactosylgroup to many hydroxylated acceptors, such as methanol, free sugars, and oligosaccharides.The rate of reaction was found to depend on the structure of the acceptor, and the transfer to occur preferentially on a primary alcohol, less usually on a secondary one. Thisdiscovery permitted the first in vitro biosynthesis of ra!€inose. The seeds of Pluntugo ovata contain two a-D-galactosidaseshaving different specificity. Using sucrose as the acceptor, one enzyme transfers the D-galactosyl group to the primary alcohol group of the D-glucosyl moiety, the other one to the primary alcohol on C-6 of the D-fructosyl moiety leading to planteose. The coffeebean a-D-galactosidase, using cellobiose as the acceptor, catalyzes three transglycosylation reactions, respectively to the hydroxyl group on C-6 or C-3 of the nonreducing unit, and C-3 of the reducing one. Analogous reactions catalyzed by the almond P-D-glucosidase allowed Courtois to suggest a generalization concerning the catalytic action of glycosidases, which is always a transfer reaction, hydrolysis occurring when the acceptor of the glycosyl group is the hydroxyl group of water. Such studies were extended to a-D-galactosidasesof various origins: intestinal bacteria, Penicillium species, germinated legume seeds (Vicia, Medicugo, and Trigonellum), molluscs, and mammalian kidney.

JEAN EMILE COURTOIS

15

The a,a-trehalase from various sources retained the attention of Courtois for many years. Specimens of this enzyme were purified from bacteria (Pseudomonas), insects (may-bug), porcine gut and kidney, and human kidney, in order to compare their properties. Trehalase is always a very specific enzyme, showing no transglycosylation activity, unusual properties for a glycosidase, and, up to 1975, its only known substrate was a,&trehalose. D-GIUCOS,as well as D-gluconicacid, does not inhibit its activity, but sucrose is a potent inhibitor for mammalian a,a-trehalases. This glycosidase is a most widely distributed enzyme that may play a primary metabolic role in organismsthat use a,a-trehalose as a reserve carbohydrate, such as insects. Vertebrate trehalases are strongly inhibited by phloridzin and phloretol; it is possible that this inhibition is involved in the renal diabetes induced by the injection of phloridzin, trehalase being implicated in the active transport or renal resorption of D-glucose. Professor Courtois and his coworkers also discovered trehalase activity in human serum. This activity decreases after kidney removal, as well as in liver cirrhosis, an argument for the renal, and mostly hepatic, origin of the enzyme of the serum. Another original research contribution of J. 8. Courtois dealt with the glycosidasesand glycanases from xylophagic insects; such insects are serious predators in the forests of many countries. This work was initiated after 1955, when many sprucesin the Morvan area were destroyed after a massive infestation by a coleopteron, Ips typographus.It was observed that this insect possesses an exceptional variety of enzymes, namely, oligosaccharidasesable to hydrolyze the intracellular or sap oligosaccharides,and a wide selection of glycanases allowing the hydrolysis of most of the polysaccharides entangled in the bark and the wood. This enzymic mix was studied at different stages of development of the insect (larvae, pupae, young adult, and adults), and this revealed a positive correlation between the enzymic activities and the nutritional activity. Such studies were also carried out on eighteen other insect species that are parasites of conifers, poplars, or oaks. He then attempted to ascertain if the glycosidases are synthesized by the insect itself, or are due to the presenceof micro-organismsfrom the intestinal flora or of symbiotic mycetomae. Indeed, it was possible to observe, in the digestive tract of the larvae of a specific parasite of a coniferous species (Halobius abietis), the presence of mycetomae from which was isolated the yeast Candida brumptii. Similarly, two bacteria (Achromobacter) and a Candida were identified in the digestive tract of Ips sexdentatus. These micro-organisms always revealed enzymic activities less elevated and less varied than those of the host insects. Moreover, breeding of several insect specieson wood or bark impregnated with antibacterial and antifungal drugs showed the disappearance of the micro-organisms, the enzymic activities

16

FRANCOIS PERCHERON

remaining unmodified. It was then possible to assess that the enzymic activities permitting the attack on trees were essentially due to the enzymes of the insect, the digestive flora being only a minor component. Besides his fundamental research in the carbohydrate field, the functions of Courtois as the head of a hospital laboratory for many years led him to publish a number of papers dealing with clinical chemistry, among which may be cited: determination of ethyl alcohol, proteins, acidic phosphatases, and trehalase in blood determination of the basic groups of proteins by phytic acid; study of the phytosoluble glycoproteins in biological fluids; and identification and determination of scyllitol in urine. Under the aegis of the International Pharmaceutical Federation, he participated in the standardization of the methods proposed for the assay of such enzymes as cellulases and hemicellulases. In all fields, these researches benefited from the remarkable qualities as an analyst, acquired by Courtois with P. Fleury, to which was added his acute faculty of interpretation. This intense activity materialized in about three hundred original papers and a hundred general reviews. Professor Courtois was a real head of a school; he contributed to the professional development of a great number of students. Some of them turned towards various aspects of the pharmaceutical profession, whereas many others succeeded in an academic career in France as well as in foreign countries, or at the Centre National de la Recherche Scientifique. They all kept a great attachment to him, and often became real friends with him. It is necessary to recall the major role played by Courtoisin the Soci6t6 de Chimie Biologique: being an active member of this society since 1930, he became in 1953 the General Secretary, a very time-consuming charge he assumed and continued to 1969, before becoming President in 1972. He was there faced by multiple tasks, particularly the organization of many meetings, colloquia, or congresses. Thus, he was in touch with the international elite in biochemistry, among whom he gained a great number of friends. This untiring activity led Professor Courtois to become involved with many international authorities where he worked at the highest level: these included the International Unions of Pure and Applied Chemistry, and of Biochemistry, the International Federation of Clinical Chemistry (of which he was the president from 1964 to 1968), the Federation of European Biochemical Societies (taking part in its foundation), and International Commissions of Nomenclature. In all these authorities, his experience, his common sense, and his characteristic optimism were greatly appreciated. It was the same in France, where multiple commissionsappealed to J. 8. Courtois: the Comites Nationaux de Chimie, de Biochimie, and de Biophysique, and commissions of the Centre National de la Recherche Scientifique. He was one of the founder members of the Groupe FranCaisdesGlucides. He

JEAN BMILE COURTOIS

17

sat for many years on the Commission Permanente de la Pharmacop6e,and served as an expert at the European Pharmacopoeia. Such important activities, not only strictly scientific,but also in the service of scientific communities, were rightfully recognized by many honors and distinctions in France and elsewhere. J. E. Courtois was an officer of the Ugion d‘Honneur and of the Ordre National du MCrite, commander of the Palmes Acadtmiques, commander of the Spanish Order of Alphonso X el Sabio, member of the AcadCmie National de Pharmacie since 1945, and he was elected to the Academie Nationale de Mtdecine in 1967. He was a foreign member of the Sciencesand Letters Academy of Oslo, a corresponding member of the Real Academia de Farmacia in Madrid, Doctor honoris CUUSQ of the universities of Madrid and Ghent, and this is an incomplete list! It may be mentioned that Professor Courtois’ reputation led him to many teaching assignments (Saigon, Hanoi, Montreal, Algiers) and to answer multiple invitations to give lectures in about sixty cities all over the world. Quite obviously, such success in various fields was not a matter of chance or of gratitude, but reflected the qualities of the man. To exceptional gifts of acute intelligence, J. 8.Courtoisadded, during his whole career, his working capacities, his analytical and also synthetic mind, his enthusiasm, and his taste for human relations; in a word, hisjoie de vivre,as well as his ability in any case to give preference to the pleasant aspects. Thanks to all these qualities, he counted only friends everywhere. The culture of J. 8. Courtois was not restricted to the scientific field. His erudition was extended to history and to all forms of art, ancient and contemporary. After 1978, being retired, he worked and published in archeology, under the aegis of the AcadCmie du Morvan and of the Societt des Archtologuesde 1’Yonne.Just as in his scientificwork, we find again here the qualities of Professor Courtois, his interpretive ability, and a taste.for unexpected and sometimes surprisingcomparisons,which constituted one of the attractions of his conversation. His love for archeology led him to study in the field the artifactsof Persian, Greek, or Roman people, from Persepolisto Delphi, from Agrigente to Leptis magna, not to forget the early Christian churches in Yugoslavia or in Soviet Armenia. Going through a museum or a monument with him was a real pleasure, because he was a reliable guide, able to correct the professional ones! This inclination for art was fulfilled by his numerous trips, where, besides biochemistry, were always added characteristic visits in each city, and his memory firmly retained everything. Those who had the privilege to live close to him know his unconditional attachment to Burgundy, sometimes marked with a little bias against the historical enemies of Burgundians. One could not forget his passion for shooting, which he was pleased to share with foreign colleagues, and for sports in general, especially for rugby. For a long time, he was a regular

18

FRANCOIS PERCHERON

spectator, sometimes noisy, of major competitions upon which he later commented vigorously. J. 8. Courtois personified humanism, and represented a type of personality, with vast erudition, that is rarely encountered in this day and age. J. E. Courtois mamed Gilberte Quinque, who was herself a pharmacist. They brought up five daughters, Micky, Marielle, Chantal, Marie-Aleth, and Isabelle, who were respectively graduated in pharmacy, law, history, medicine, and mathematics, and who gave them 13 grandchildren. The year 1989was marked by an exceptionally cruel ordeal, which Professor Courtois bore with exemplary courage: the tragic and unexpected death of his eldest daughter. Severelyattacked himselfby disease in August, he died on December 9th of that year. He now rests close to his daughter and to his ancestors, as he had wished, in his dear Burgundy in the cemetery of Saulieu, leaving to all those who knew him the memory of an outstanding and warm personality.

FRANCOIS PERCHERON

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL 49

THE COMPOSITION OF REDUCING SUGARS IN SOLUTION: CURRENT ASPECTS BY STEPHEN J . ANGYAL School of Chemistry. University of New South Wales. Kensington. N.S.W. 2033. Australia I . Introduction ......................................................... I1. Methods for Studying the Composition of Sugars in Solution ................ 2 Nuclear Magnetic Resonance Spectroscopy............................. 4 Other Methods .................................................... I11. Relative Stabilities of the Various Forms ................................. 1 The Pyranose Form ................................................ 2 The Furanose Form ................................................ 4 The aldehydo and k t o Forms........................................ 5 Hydrated Carbonyl Forms........................................... 6 Variation of the Composition with Temperature ........................ IV. Composition in Aqueous Solution: Aldoses ............................... 1 Aldohexosesand Aldopentoses ....................................... 2 Aldotetroses and Related Sugars...................................... V Composition in Aqueous Solution: Ketoses ............................... 1 Hexuloses and Pentuloses ........................................... 2. Heptuloses........................................................ VI. Composition in Aqueous Solution: Substituted and Derived Sugars ........... 1 Partially @Substituted sugars........................................ 2 AminoSugars ..................................................... 3 Thio Sugars ....................................................... 4 Branched-chain Sugars.............................................. 5 . Sugars Having Fused Rings .......................................... VII. Solutions in Solvents Other than Water .................................. VIII . Tabulated Data ......................................................

. . . . . . .

.

. . . . . . .

19 20 20 21 22 22 23 24 25 25 25 25 26 27 27 28 28 28 29 30 30 31 31 32

I . INTRODUCTION A chapter' in this Series. published in 1984. summarized o w knowledge of the composition of reducing sugars in solution. and tabulated results collected up to the end of 1983. Since then. data on this subject have been (1) S.J . Angyd. Adv. Carbohydr. Chem. Biochem.. 42 (1984) 15-68 .

20

STEPHEN J. ANGYAL

published at an increasing rate, presumably for two reasons. First, authors of research papers have become conscious of the importance of these data, and it has become increasingly common to describe not only the chemical shifts and coupling constantsin the spectra of reducing sugars but also the proportion of the various forms. Secondly, improvements in the methods and techniques used have made it easier to determine such composition data from n.m.r. spectra and, occasionally, by other methods. Increased interest in the subject has alsogiven rise to severalpapers in which the variation of the composition with the change of temperature or solvent was systematically investigated. Hence, it appeared worthwhile to bring the original chapter up to date by reviewing the recent advances and gathering the new data into additional Tables. This chapter is supplementary to the original one: it does not stand on its own. The same section headings and section numbers have been used (even though there have been no additions to several sections). The Tables are also numbered in the original way. In the Tables, sugars originally shown are listed only if additional or more accurate data have become available; these sugars are marked with an asterisk. Those not thus marked appear here for the first time. References to the original article’ are shown in square brackets, as in [p. 221. 11. METHODS FOR STUDYING THE COMPOSITION OF SUGARSIN SOLUTION

2. Nuclear Magnetic Resonance Spectroscopy Practically all of the new data have been obtained by nuclear magnetic resonance (n.m.r.) spectroscopy. It was stated’ in 1984 that this method cannot detect components that occur in equilibrium in very small proportions (< l%), such as the free and the hydrated carbonyl forms. This is no longer true: n.m.r. spectrometershave been considerably improved, and two important advances now allow the detection and measurement of components that occur in the range of 0.01 -0.1%. Allerhand and coworkers*developed an “ultra-high resolution” methodology by which, with some modification of the instrument and the usual operating technique, very small signals can be detected. Using 13C-labelled~-glucose,they determined’s4 the proportion of the furanose forms, and the free and the hydrated aldehyde form at six temperatures between 27 and 82”. The smallest of these values (2) A. Allerhand, R. E. Addleman, and D. Osman,J. Am.Chem.Soc., 107 (1985) 5809-5810; A. Allerhand and C. H. Bradley,J. Mum.Reson., 67 (1986) 173- 176. (3) C. Williams and A. Allerhand, Curbohydr.Rex, 56 (1977) 173- 179. (4) S. R. Maple and A. Allerhand, J. Am. Chem. Soc., 109 (1987) 3168-3169.

COMPOSITION OF REDUCING SUGARS

21

was only 0.0024%. The method is difficult to apply and is demanding of instrument time; for example, for the spectrum at 67”,75,200 scans were averaged. The method is suitable for any sugar, but has so far only been applied to ~-glucose. On the other hand, the use ofspecifically 13C-labelledsugars, developed by Barker and Serian~~i,~-” has been applied to many sugars; it is particularly useful when the label is in position 1. Labelling results in an 100-fold increase of the 13Csignal of the labelled carbon atom, making it possible to detect components occurring in very small proportions, down to 0.0 1%; for example, for riboset0at 25 0.05% of free aldehyde. These results are discussed in Sections II1,4 and 111,s. The composition of many aldoses and two ketoses has been determinedt4 by 13C-n.m.r.spectroscopy;the results agreed well with those from previous determinations made from IH-n.m.r. spectra.

-

O,

-

4. Other Methods

Gas - liquid chromatography(g.1.c.)of trimethylsilyl derivativeshas again been used to determine the composition of some dozen sugars in water and in pyridine.15 In the latter solvent, the results agreed well with previous determinations;in aqueous solution, however, some of the values for furanoses proved to be too high, and the values for idose (presumably mutarotating rapidly [p. 231) differ considerably from those obtained by n.m.r. spectroscopy. G.1.c. of the trimethylsilyl derivativeswas also used for studying16 the mutarotation of D-fructose. The composition data obtained for the major components agreed well with those given by n.m.r. spectro~copy,~~ but those for the a-pyranose (0.4-0.82% between 10 and 5 5 O ) are much (5) R. Barker and A. S. Serianni,Acc. Chem. Res., 19 (1986) 307-313. (6) A. S. Serianni, J. Pierce, S.-G. Huang, and R. Barker, J. Am. Chem. Soc., 104 (1982) 4037-4044. (7) J. R. Snyder and A. S. Serianni, J. Org. Chem.,5 1 (1986) 2694-2702. (8) J. R. Snyder and A. S. Serianni, J. Am. Chem. Soc.,11 1 (1989) 2681 -2687. (9) J. R. Snyder and A. S. Serianni, Curbohydr.Res., 163 (1987) 169- 188. (10) M. J. King-Moms and A. S. Serianni, J. Am. Chem. Soc..109 (1987) 3501 -3508. (1 1) J. R. Snyder and A. S. Serianni, Carbohydr.Res., 166 (1987) 85-99. (12) J. Wu, T. Vuorinen, and A. S. Serianni, Curbohydr. Res., 206 (1990) 1 - 12. (13) J. R. Snyder and A. S. Serianni, Carbohydr.Res., 210 (1991) 21-38. (14) R. Rrez-Rey, H. VClez Castro,J. Crernata Alvarez, L. Fernindez Molina, and J. Hormaza Montenegro,Rev.Cienc.Quim., 16(1985)225-227 [Chem.Abstr.. 107 (1987)237,141]. (1 5) M. Paez, 0. Martinez-Castro,J. Sam, H. Olano, A. Garcia-Rasz, and F. aura-Calixte, Chromatographia,23 (1987) 43-46. (16) M. Cockman, D.G. Kubler, A. S. Oswald, and L. Wilson, J. Curbohydr. Chem.,6 (1987) 181-201. (17) F. W. Lichtenthaler and S. Renniger, J. Chem. Soc., Perkin Trans, 2, (1990) 1489- 1497.

22

STEPHEN J. ANGYAL

smaller than the figures obtained by several authors from n.m.r.spectral data. G.1.c. also showed the presence ofthe keto form but the proportion thus obtained (0.22-0.36%) may also be too small. Working at low temperatures (0-4"), h.p.1.c. on a cation-exchange resin in the calcium form will separate the pyranose anomers of most of the aldo-hexoses and -pentoses16; under these conditions, mutarotation is slower than separation. The furanoses are not separated, because they interconvert too rapidly, but, at -25 to -45 the two furanose forms of D-galactose and L-fucose have been ~eparated.'~ Attempts to separate the various forms of sugars on a preparative scale [ p. 241 have not succeeded so far.2o O,

111. RELATIVE STABILITIES OF THE VARIOUS FORMS

1. The Pyranose Form

Further attempts have been made to explain and predict the proportions of the pyranose forms in solution. It is not difficult to calculate, by various methods, the relative free energies in vacuum or in inert solvents; it is not, however, easy to take the effect of solvation into account. Clearly, solvation has a substantial effect on the composition, and the variation ofthe dielectric permittivity between different solvents does not fully account for this effect. Tvarogka and KoZirzl developed a method whereby solvation is considered in calculating the Gibbs energy which encompasses electrostatic, dispersion, and cavity terms. The composition of g glucose in each of eleven solvents was calculated. In only three solvents were experimentaldata available, and these agreed reasonably well with the calculated figures. The variation of the composition with the change in temperature in aqueous solution was also well accounted for by the results of these calculations. AM 1 semi-empirical molecular-orbital calculationshave been carried out on several sugars in order to establish energy minima and favored conformations.22However, TvaroSka and Carterz3showed that this method does not provide the correct energy differences between anomers. The difficulty lies in the comparatively small energy differences (- 1 -2 W/mol) between anomers; much-more refined calculations are necessary for this to emerge (18) S. Honda, S. Suzuki,and K.Kakehi, J. Chromatogr., 291 (1984) 317-325. (19) M. Monyasu, A. Kato, M. Okada, and Y. Hashimom, Anal. Left.,17 (1984) 689-699, 1533- 1538.

(20) S. J. Angyal, unpublished results. (21) I. TvaroHka and T. KoZiir, Theor. Chern.Acta, 70 (1986) 99- 114. (22) R. J. Woods,W. A. Szarek,and V. H. Smith, Jr., J. Am. Chem. Soc.. 112 (1990) 47324741. (23) I. Tvaroska and J. P. Carver,Abstr. Pap. Znt. Carbohydr. Symp., 15th. Yokohama (1990) c 001.

COMPOSITION OF REDUCING SUGARS

23

from the background, statistical noise of the data. Similar calculations have not been carried out for furanoses, which constitute a much more difficult problem owing to the small energy-differencesbetween conformers and the small barrier between them. Most of these calculations have focused on water as a solvent; they have been summarized in a detailed review.” It seemsthat hydration is stereospecifi~,2~ and there appears to be strong support for Kabayama and Patterson’s original proposalM[p. 241 that equatorial hydroxyl groups are more strongly solvated,and therefore stabilized,than axial ones. An interesting example is &D-ribopyranose, which, in aqueous solution, exists as a mixture (- 1 :5 ) of the ‘C,and the 4CIforms. Increasing the temperature causes a lessening of the 4C1form, while the proportion of the ‘C4form does not alter.*’ Similarly, changing the solvent from water to dimethyl sulfoxide causes a substantial diminution of the proportion of the ,C,form, while that of the lC4 form actually increases. The 4C1form has three equatorial hydroxyl groups, whereas the ‘C4form has only one. An ingenious examination of the “hydrophilicity” of the eight aldohexoses (that is, their hydrophilic volume in water) allowed a rationalization of their a :p pyranose ratios in aqueous solution.** The hydration characteristics of carbohydrates in aqueous solution provide an intriguing and challenging problem.” Future research will need to explore further the specificity and the thermodynamics of the solvation of carbohydrates. Another approach, now being actively pursued,6 1 9 121329,30 targets on mechanistic investigations. In particular, overall and unidirectional rate-constants of anomeric changes are measured, and related to the structure of sugars and to solution conditions. Eventually, such information should shed light on the molecular factors affecting ring formation and ring-opening reactions, and, hence, on their equilibria. 9

9

9

2. The Furanose Form

Another example of the observation that the side chain attached to the anomeric carbon atom in ketoses affectsthe proportion of one furanose form [p. 291 is afforded by the series 1-deoxy-D-fructose:N-substituted l-amino(24) F. Franks and J. R. Grigera, WaferSci. Revs., 5 (1990) 187-289. (25) M. J. T i t , A. Suggett, F. Franks, S. Ablett, and M. J. Quickenden, J. Solution Chem., 1 (1972) 131-151. (26) M. A. Kabayama and D. Patterson, Can. J. Chem., 36 (1958) 563-573. (27) F. Franks, P. J. Lillford, and G . Robinson, J. Chem. SOC.,Faraday Trans. I, 85 (1989) 24 I 7 2426. (28) M. D. Walkinshaw, J. Chem. SOC.,Perkin Trans. 2, (1987) 1903- 1906. (29) J. Pierce,A. S. Serianni, and R. Barker, J. Am. Chem. Sot.., 107 (1985) 2448-2456. (30) J. R. Snyder and A. S. Serianni, J. Am. Chem. Soc., 1 I I (1989) 2681 -2687.

-

24

STEPHEN J. ANGYAL

1deoxy-~-fructoses~~: Dfructose: and 1,2-dideoxy-~-urubino-3-heptulose3*;as the side chain is increased from CH3to CH2NR, to CH20H, to CH2CH3,the proportion ofthep-furanose increases from 9 to 10- 14,to 20, to 31%.

4. The uldehydo and &to Forms

The proportion of the aldehydo and keto forms obtained by n.m.r.-spectral studies agrees well, in most cases, with those obtained earlier by circular dichroism [p. 211. The proportion of the acyclic form in equilibrium is to a large extent governed by the Thorpe- Ingold effect: the presence of substituents favors the formation of rings. One manifestation of this effect is the greater tendency of secondary, rather than primary, alcohols to form cyclic acetals [p. 351; the second alkyl group attached to the secondary alcohol becomes a ring substituent on acetal formation. Illustrations of this effect are the 0.2% aldehyde content at 30” of 3-C-rnethyl-~~-erythrose, compared with 2% for D-erythrose, and the 0.3% of 3-C-methyl-~-threose,compared with 0.96% for ~-threose.’~ It is even lower (0.1%) in 3,3-di-C-methyl-~~-glycerotetrose. (Data for these equilibria have also been determined at 60”.) Removal of the hydroxyl group from C-2 increases the aldehyde content at removal of the hydroxyl equilibrium: 3.1% for 2-deoxy-D-glycero-tetroseL3; group from C-3 has little effect. The ratio at e q ~ i l i b r i u m between ~~ 5-hydroxypentanaland tetrahydro-2hydroxypyran in D20at 43.7” is 4 :96 [in contrast to what was predicted on p. 301. However, hydroxyketones cyclize to a lesser extent. At 25”, in to tetrahydro- 1aqueous solution, the ratio34of 1,6-dihydroxy-2-hexanone (hydroxymethy1)pyranis 60 :40. This example illustrates the effect, on hemiacetal formation by the keto group, of a hydroxyl group on a neighboring carbon atom, because 6-hydroxy-2-hexanone is found only in the acyclic form [p. 301.There are now sufficient examples to illustrate how the accumulation of hydroxyl groups favors the cyclic forms: the keto content at equilibrium of “1 ,3,4,5-tetradeoxyhexuloseyy is 100%; that of “2,3,4-trideoxyhexulose”is 60%,of the 1deoxyhexuloses, 6%, and of the hexuloses, -0.3%.However,in dimethyl sulfoxide,the presence of a hydroxyl group on C-1 seems to have much less effect: there is 55% of the acyclic 1,6-dihy-

-

(31) A. G6mez-Shchez and M. de Gracia Garcia Martin, Carbohydr.Res.. 149 (1986) 329345. (32) I. I. Cubero and M. T. Plaza Lbpez-Espinosa, Carbohydr. Res., 173 (1988) 41 -52. (33) J. Buddrus, M. Jablonowski, and H. Brinkmeier, Justus Liebigs Ann. Chem., (1987) 547 - 548. (34) W. A. Szarek, D. R. Martin, R. J. RaIka, and T. S. Cameron, Can. J. Chem., 63 (1985) 1222- 1227.

COMPOSITION OF REDUCING SUGARS

25

droxy-2-hexanonein equilibrium, the same proportion as for 6-hydroxy-2hexanone. It appearsthat the presence of a hydroxymethyl group adjacent to the anomeric center in ketoses favors the cyclic forms in aqueous solution, probably by favorable solvation. 5. Hydrated Carbonyl Forms When there is a branch at C-3 in an aldose, the aldehydo form is hydrated to a much lesser extent than in an unbranched sugar: the branching causes a 1,3-parallel interaction with one of the hydroxyl groups of the gem-diol. Whereas the ratio of aldehydrol to aldehyde is 10: 1 for threose and 5 : 1 for erythrose, it is only 1.7: 1 and 1.5: 1, respectively, for their 3-C-methyl derivatives, and 0.4 : 1 for 3,3-dirnethyl-~~-glycero-tetrose.'~ When there is a keto group in a ring, for example, in the aldopyranose and aldofuranose forms of aldosuloses (see Section V,l), it is extensively hydrated unless hydration gives rise to a syn-axial O / / Ointeraction (for a discussion, see Ref. 35). In the aldofuranoseformsofpentos-2-~loses,~ the keto group is almost completely hydrated; in their aldopyranose forms, it is hydrated to a considerableextent. In this case,the tendency to form a hydrate is reinforced by the inductive effect of the neighboring anomeric center. The various forms of ~-ribo-3-hexosuloseare hydrated to a lesser extent.37

6. Variation of the Composition with Temperature The results of a detailed study of D-fructose and five O-glucosyl-substituted mfructo~es'~ confirmed that increasing the temperature increases the proportion of the furanose forms. This effect is much more noticeable in water and in pyridine than in dimethyl sulfoxide. In other ketoses, the proportion of the keto form also increases with increasing temperature.**

Iv. COMPOSITION IN AQUEOUSSOLUTION: ALDOSES 1. Aldohexoses and Aldopentoses The composition of D-glucose has been determined over a wide range of temperature by Franks and coworkers27and by Maple and Allerhand4(see Table 11). Both sets of data are self-consistent, but the a:ppyranose ratio recorded by Maple and Allerhand is considerably higher, for example, 39.4 k 0.8% of a-pyranose versus 35.5 k 1% at 37".These authors added 1 1% of 1,Cdioxane to the solutions they used for recording the I3C-n.m.r. (35) S. J. Angyal, D. Range, J. Defaye, and A. Gadelle, Curbohydr.Rex, 76 (1979) 121 - 130. (36) T. Vuorinen and A. S. Seriami, Curbohydr. Res., 207 (1990) 185-210. (37) P. E. Moms, K. D. Hope,and D. E. Kiely, J. Curbohydr. Chem.. 8 (1989) 515-530.

26

STEPHEN J. ANGYAL

spectra. Dais and Perlin3*showed that addition ofas little as 10%ofdimethyl sulfoxide to an aqueous solution of D-fructose considerably lowers the proportion of the p-pyranose form. Addition of 1,4-dioxane would probably have the same effect; hence, Maple and Allerhand's a-pyranose values are too high. In an earlier paper by Williams and Allerhand,3values were obtained which were about half-way between the aforementioned two sets of values (37.3 f 1.O% at 4 1 "); an unspecified proportion of 1,4-dioxane was used as the internal standard. This case should serve as a warning that compoundsadded as internal standardsor as deuterium locks should be kept to the minimum, so as not to affect noticeably the composition of sugars in the solution. 2. Aldotetroses and Related Sugars

During an investigation of the properties of furanoses, Serianni and coworkers6 studied the composition of sugars which cannot form pyranoses. Starting with the aldotetrose9 [p. 361, they studied the 5-deoxypentoses~ some 5-0-substituted p e n t o ~ e s ,and ~ . ~ the ~ 2-pent~loses.'~ All these compounds will be discussed in this Section. In the aldoses, the furanose having 0-1 and 0-2 cis is the less stable anomer, except for the xylu compounds where the trans form slightly preponderates. The ratio of anomers is large in erythrose and its homologs and small in threose and its homologs, owing to the cis arrangement of 0-1 and 0 - 3 in the less-stableisomer. This applies to the 5-deoxypentoses(Table 11); they are similar in the pentose 5-phosphates and the 5-0-methyl-pentoses. The proportion of the acyclic forms, however, is smaller for the pentoses than for the tetroses, because ring closure in the latter occurs on primary hydroxyl groups. As found typically for glucose and idose, nearly equal proportions of the two furanose forms were observed39for 5-deoxy-5-fluoro-~-glucose (45 :5 5 ) and -L-idose (47 :53). In the pentuloses,12the anomers with 0-2 and 0 - 3 cis are the more stable anomers, since in that case 0-3 and the side chain are trans. In both pentuloses (Table IV), the proportion of the anomers is about the same (3: 1) because 0-4 is cis to a substituent on C-2 in both. The proportion of the acyclic form (which is higher in ketoses than in aldoses in any case) is much higher because the ring is formed through a primary hydroxyl group. By comparison, the homomorphous 6-0-methyl-~-fructos~~ has only 3.6%of the ketu form in equilibrium at 40". (38) P. Daisand A. S. Perlin, Curbohydr.Res., 136 (1985) 215-223; 169 (1987) 159-169. (39) R. Albert, K. Dax, S. Seidl, H. Sterk, and A. E. Stiitz, J. Curbohydr. Chem.. 4 (1985) 5 13- 520.

COMPOSITION OF REDUCING SUGARS

27

V. COMPOSITION I N AQUEOUS SOLUTION: KETOSES 1. Hexuloses and Pentuloses

The compositions of solutions of D-fructose" and of I-, 3-, 4-, 5-, and 6-O-a-ghcopyranosy~-D-fmctoses in water, pyridine, and dimethyl sulfoxide were determined at several temperatures; all these data cannot be reproduced here. Similar, although not so extensive, data were obtained by Jaseja and coworkers,'" using two-dimensional n.m.r. spectroscopy;they did not observe the presence of any of the a-pyranose form. As in other instances,increasingthe temperature was found to favor the furanose forms, as does change to organic solvents. As noted previously [ p. 391, substitution has little effecton the composition, except at the 3 position, where it lessens the proportion of thep-pyranose form substantially.The curious behavior of the 3-0-glucosyl isomer, turanose, in pyridine is discussed in Section VII. If a sugar has two carbonyl groups, each can form pyranose or furanose rings; bicyclic forms sometimes result. An example is ~-threo-3,4-hexodiulose4*;because this compound is symmetrical, and pyranoses are not possible, only two forms are present at equilibrium. In aqueous solution, and also in dimethyl sulfoxide, 72% of the a,a- and 28% of the p,P-difuranose were found at 27". The a$ form, which would have two trans-fused five-membered rings, was not observed. In dimethyl sulfoxide, there is also a small proportion of the monocyclic furanose form present. If a sugar contains both an aldehyde and a ketone function, the aldehyde will form rings mainly; the composition can be quite complex. Eight monomeric forms (out of a possible 18)were identified in the spectra ofeach of the pentos-2-ulo~es.~~ The erythro isomer was found to consist, at 23" in D,O, of 4.5% of the a-aldopyranose and 20.5%of its hydrate, 4.5% of the p-aldopyranose and 38.8%of its hydrate. 10.9%of the a-aldofuranose hydrate, 14.290 of thep-aldofuranosehydrate, 5.2%ofthe a-ketofuranose hydrate, and I. 1% of the /?-ketofuranosehydrate. For the threo isomer, the composition, in the sameorder,wasfoundtobe0.9,59.8,1.0,29.1, 1.5,1.3, l.O,and5.4Yo.These compositions were also determined at 80". There are at least ten forms in an aqueous solution of ~-ribehexos-3of which eight have been identified. Most forms have a free or a hydrated keto group: at 23', there is 44Yo of the a-furanose and 1.5% of its hydrate; 22%of the p-pyranose and 12%of its hydrate; 590of the a-pyranose and 2% of its hydrate, 1% of a bicyclic p-furanose, and 8% of a dimer. The (40) B. Schneider, F. W. Lichtenthaler, G. Steinle, and H. Schiweck, Justus Liebigs Ann. Chem., (1985) 2443-2453. (41) M. Jaseja, A. S. Perlin, and P. Dais, Mum.Reson. Chem., 28 (1990) 283-289. (42) S. J. Angyal, D. C. Craig, and J. Kuszmann, Carbohydr. Rex, 194 (1989) 21 -29.

STEPHEN J. ANGYAL

28

n.m.r. spectra are complex and it is not certain that the minor components have been correctly assigned. The major components in the equilibrium of 6-deoxy-~-xylo-hexos-5u10se~~ are the a-aldofuranose (36%), the P-aldofuranose (28%), and the pyranose involving both the aldehyde and the ketone group (36%). The ketone group in the furanoses is not hydrated; the configuration of the pyranose is p-1,a-5(1R,5R).The same type of pyranose is the main component (67%)in an aqueous solution of ~-x~~o-hexos-5-ulose." The pentuloses are discussed in Section IV,3. 2. Heptuloses

has been deterThe composition of the four 1,2-dideoxy-3-heptuloses mined.32These compounds are similar to the hexuloses, the hydroxyl group on C- 1 having been replaced by a methyl group; their composition should be similar to those of the corresponding hexuloses, and, in most cases, this is true. For 1,2-dideoxy-3-lyxo-heptulose, however, and for its lower homolog, 1-deoxytagatose,the a-furanose form (in which 0-3 and the side chain are cis) is more stable than the P-furanose (with 0-2 and 0-3 cis). A study of the conformationscan rationalize this observation: in the p-furanose, there is a quasi-syn-axial interaction between 0-2 and 0-4 which is aggravated by the side chain, particularly if bulky. There is a similar effect in the P-pyranose, which also becomes a minor constituent of the equilibrium mixture. The composition values previously reported [p. 421 for 3-deoxy-~manno-2-octulosonic acid (Kdo) are incorrect. The correct values45in a 0.72 M solution of the ammonium salt at 20" are 64: 6 : 10 :20 and, in a 0.18Msolution, 60: 11:9:20. VI. COMPOSITION IN AQUEOUSSOLUTION: SUBSTITUTED AND DERIVED SUGARS 1. Partially 0-Substituted Sugars

Another example of substitution increasing the ratio of a-to P-pyranoses

[ p. 451 is 3-O-methyl-~-dtrose~: 25% a-and 32%P-pyranose, 43% a -tp-

furanose. (43) D. E. Kiely, J. W. Talhouk, J. M.Riordan, and K. Gray, J. Curbohydr.Chem., 2 (1983) 427-438. (44) J. M.Riordan, R. E. Harry-Okuru, J. W. Talhouk, and D. E. Kiely, Abstr. Pup. fnt. Curbohydr. Symp., 12th, Utrecht (1984) A 1.57. (45) P.A.McNicholas,M.Batley,andJ. W.Redmond, Curbohydr.Res.,146(1986)219-231. (46) J. J. Patroni, R. J. Stick, D. M.G. Tilbrook, B. W. Skelton, and A. H. White, Aust. J. Chem., 42 (1989) 2127-2141.

COMPOSITION OF REDUCING SUGARS

29

Several methyl ethers of D-glucose were studied by Reuben:' who found that the a : p ratio increases from 36 : 64 to 60 :40 for the 2,3,6-trimethyl ether. A carboxymethylgroup as substituentwas found to cause less change than a methyl group. Substitution on the 2-position caused greater change than in any other position. The same effect was observed in the 0-D-~~UCOsyl-D-glucoses: their composition was similar to that of glucose, except for the disaccharidelinked in the 2-position which has more a-than p-pyranose in equilibrium.48Similar results were obtained with some methyl ethers of ~-galactose?~ All the derivatives studied had a methyl group on 0-4 in order to preclude formation of the furanose forms. 4-O-Methyl-~-galactosehas 35% of the a-pyranose in equilibrium; the 2,4-dimethyl ether has 5 5 % the 3,4-dimethyl ether, 35%;and the 4,6-dimethylether, 39%.Further methylation was found to have very little effect. Replacement of a hydroxyl group by fluorine in the 2-position in glucose, but not in mannose, increases the proportion of the a-pyranose.50 Fructoses 0-substituted by glucosyl in various positions are discussed in Section V, 1. The presence of substituents outside the ring usually does not affect the composition, Thus, D-allose 6-phosphate, D-altrose 6-phosphate, and Dmanno-heptulose 7-phosphate have practically the same composition as the parent sugar^.^' 2. AminoSugars

4-Amino-4,6-dideoxy-~-mannosehydrochloride (perosamine) forms an

-45 :55 mixture of the a-and p-pyranose forms in solution.52For 2-amino-

2,6-dideoxy-~-glucose6-sulfonic acid, the ar:p pyranose ratio is 68 :32 at 20°, indicating [p. 471 that, in solution, the sugar is zwitterioni~.~~ Four 1-amino- 1-deoxy-D-fructoses,each having a different substituenton the nitrogen atom, were ~tudied.~' The nature of the substituent makes little difference to the composition, but, in each case, in contrast to D-fructose, there was no great preponderance of thep- over thea-furanose form; in some cases, the a-furanose was preponderant. Changing the solvent from D20to

(47) J. Reuben, Carbohydr. Res., 184 (1988) 244-246. (48) T. Usui, M. Yokoyama, N. Yamaoka, K. Tuzimura, H. Sugiyama, and S. Seto, Carbohydr. Res., 33 (1974) 105- 116. (49) E. B. Rathbone and A. M. Stephen, S. Afi. J. Sci., 69 (1973) 183. (50) N. Satyamurthy, G. T. Bida, H. C. Pudgett, and J. R. Barrio, J. Curbohydr. Chem., 4 (1985) 489-512. (51) F. P. Franke, M. Kapuscinski, and P. Lyndon, Carbohydr. Rex, 143 (1985) 69-76. (52) M. J. Eis and B. Ganem, Carbohydr. Res., 176 (1988) 316-323. (53) J. Fernandez-Bolaiios, I. M. Cadla, and J. Fernandez-Bolaiios Guzman, Curbohydr. Res., 147 (1986) 325-329.

30

STEPHEN J. ANGYAL

dimethyl sulfoxide causes a much larger increase of the P- than of the a-furanose form.

3. Thio Sugars Further data have been reported on the composition of thio sugars. For 5-thio-~-altrose,an a :P-pyranose ratio of - 2 :3 was founds4;for 5-thio-~all~se,~' - 1 : 1; and for 5-thio-~-rnannose,~~ 94 :6. For 4-thio-~-galactose~~ and for 6-deoxy-4-thio-~-galactose,~~ an a- to P-furanose ratio of 2 : 1 was found, confirming again that pyranoses do not normally occur in the equilibria of 4-thioaldoses. It was reporteds9that 5-thio-a-~-lyxoseand 5-thio-PD-arabinose do not mutarotate, and therefore constitute the main form in solution; however, it is well knowna that 5-thio sugars mutarotate very slowly at pH 4.4 and lower, but rapidly at pH >6.5, and it is not evident whether the pH was checked or controlled. Mutarotation was reported for 5-thio-a-~-fucose,but the change in rotation was small, and only the signals of the a-pyranose were detected in the n.m.r. spectrum.61 4. Branched-chain Sugars

The composition of D-apiose [p. 541that was 13C-labelledinthe 1-position was studied in detail'l: at 25 it was found to be 26% Ofa-D-c?@hrU-, 44% of P-D-erythro-, 16%of a-D-threo-. 14%ofp-D-threo-furanose,5 0.0 1Yo of aldehyde, and -0.1% of aldehydrol. Several other 3-C-(hydroxymethy1)aldoses have been investigated.62Like apiose, they can give rise to four furanose forms but, pyranoses also being possible, there are altogether six cyclic forms in equilibrium. At 27", 3-C-(hydroxymethy1)- glucose consists of 19% of a-pyranose, 32% of P-pyranose, 17Yo of a-(1,4)-furanose,2 1Yo ofp-(1$)-furanose, 3%ofa-( l ,3l)-furanose, and 8%ofp-( l ,3l)-furanose; the composition of 3-C-(hydroxymethyl)-~-xyloseis 35.5, 10.5, 15, 15, 8.5, and 15.5%;that of 3-C-(hydroxymethyl)-~-lyxose, 57, 13, 20, 6, 2.5, and 1.5; and that of 3-C-(hydroxymethyl)-~-ribose, 18.5, 70.5, 3.5, 4.5, 1, 1.5%, respectively. The compositions, at 30°,of 3-C-methyl-~~-erythrose (30.1% a-furanose, 69.4% P-furanose, 0.3% aldehyde, and 0.2% aldehydrol), 3-C-methyl-~~O ,

(54) (55) (56) (57) (58) (59) (60) (61) (62)

N. A. L. Al-Masoudi and N. A. Hughes, Carbohydr. Rex, 148 (1986) 39-49. N. A. L. Al-Masoudi and N. A. Hughes, Carbohydr. Res., 148 (1986) 25-37. R. J. Capon and J. K. MacLeod, Chem. Commun., (1987) 1200- 1201. 0. Varela, D. Cicero, and R. M. de Lederkremer, J. Org. Chem., 54 (1989) 1884- 1890. D. Cicero, 0. Varela, and R. M. de Lederkremer, Tetrahedron,46 (1990) 1131 - 1144. N. A. Hughes and N. M. Munkombwe, Carbohydr. Res.. 136 (1985) 397-409. C. J. Clayton and N. A. Hughes, Carbohydr. Res., 4 (1967) 32-41. H. Hashimoto, T. Fujimori, and H. Yuasa, J . Carbohydr. Chem., 9 (1990) 683-694. S. J. Angyal, Carbohydr. Res., 216 (1991) 171-178.

COMPOSITION OF REDUCING SUGARS

31

threose (55.0 :44.3 :0.5:0.3), and 3,3-dimethyl-~~-glycer&tetrose (29.0 :7 1.O :0.04 :0.1) were determined.13 They do not differ substantially from those of the parent, unbranched tetroses, except for the much lessened proportion of the acyclic forms (see Sections II1,4 and 111,5). The compositions have also been determined at 60 . The composition of 5-C-methyl-~-glucose~~ at 37" is 6.5 :92.0:0.7 :0.8%. O

5. Sugars Having Fused Rings

The composition of 2-C-spirocyclopropyl-2-deoxy-~-arabinose was found& (by 13C-n.m.r.spectroscopy)to be pyranoses, furanoses and acyclic forms in the ratios of 10.6:2.9: 1.0. This is, actually, not a sugar having a fused ring but one with a spiro structure. The proportion of the acyclic form is surprising; apparently the spiro arrangement introduces strain into both the pyranose and the furanose forms. IN SOLVENTS OTHERTHAN WATER VII. SOLUTIONS

A detailed studyI7of D-fructose and five a-mglucopyranosyl-substituted fructoses showed that, in pyridine, the proportion of the furanose forms is higher, and in dimethyl sulfoxide much higher, than in water. A curious exception is turanose (3-O~-~-glucopyranosyl-D-Fructose), which has a slightly lower proportion of the /3-pyranose form in dimethyl sulfoxide but a much higher one (88.3% at 20") in pyridine. Neither the other isomers, nor 3-O-methyl-~-fructose,show such behavior. The high stability of the /3-pyranose form may be due to a hydrogen bond between the two sugar moieties. In fact, in the crystal structure ofturanose,there is a hydrogen bond between 0-2of the glucose and 0-4of the fructose componenP; it is possible that this bond persists in pyridine but not in water or dimethyl sulfoxide. Idose is an exception to the rule that the proportion of the pyranoses is lower in organic solventsthan in water. In 1 : I dimethyl sulfoxide-acetone, the /3-pyranose content is much higher (Table VII) than in water,& for reasons as yet unknown. It is still not clear why the furanose content is generally higher in organic solvents than in water. The effect on solvation of the water structure [p. 241 has been proposed as an explanation; it seems to explain the interesting fact38 that addition of even a small proportion (<10%)of dimethyl sulfoxide to an (63) G. E. Driver and J. D. Stevens,Amt. J. Chem.,43 (19%) 2063-2081. (64) R. C. Petter and D. G. Powers, Tetrahedron Lett., 30 (1989) 659-662. (65) A. Newman, D. Avenel, and H. G. Pandraud, Acta Crystallogr..,Sect. B, 34 (1978) 242248. (66) J. Reuben, J. Am. Chem. Soc., 107 (1985) 5867-5870.

32

STEPHEN J. ANGYAL

aqueous solution of fructosedecreasesthe proportion of the Ppyranose form substantially,whereas as much as 30%of water can be added to a solution in dimethyl sulfoxide without changing the composition to any great extent; but, were only the water structure resposible, the composition in all nonaqueous solvents should be the same, and this is not so. The formation oftwo intramolecular hydrogen bonds was claimed to stabilize the /3-fructofuran.~~ the proportion of the a-furanose ose form in dimethyl s u l f o ~ i d eHowever, form increases to the same extent when changing from water to dimethyl sulfoxide; and if one of the hydroxyl groups in fructose is blocked, the increase in the /3-furanoseform remains large when the sugar is dissolved in dimethyl sulfoxide. In the case of ldeoxy-D-fructose, the proportion of the a-furanose increases so much more than that of the b-form that it becomes greater in dimethyl sulfoxide solution than that of the &formm (see Table VII). Clearly, solvation by different solvents stabilizes different forms of a sugar to a different extent, but we have, as yet, but little understanding of this process. VIII. TABULATED DATA The Tables have been set up under the same headings as in the original article [pp. 63-68]. Compounds marked by an asterisk had already been listed there. Improvements in the quantitative evaluation of n.m.r. spectra now justify listing some of the data to within one decimal, as an accuracy of +OS% can now readily be achieved.

(67) (68) (69) (70)

T. L. Mega, S. J. Cortes, and R. L. Van Etten, J. Org. Chem., 55 (1990) 522-528. T. E. Walker, D. S. Ehler, and C. J. Unkefer, Carbohydr.Res., 181 (1988) 125- 134. C. Du Mortier and R. M. de Lederkremer, J. Carbohydr.Chem., 3 (1984) 219-228. P. Bravo, M. Fngeno, G. Fronza, A. land, and G. Resnati, Tetrahedron, 46 (1990)

997- 1008. (71) S. J. Cortes, T. L. Mega,and R. L. Van Etten, J. Org. Chem., 56 (1991) 943-947. (72) C. 0. Jeroncic, H. F. Cirelli, and R. M. de Lederkremer, Carbohydr. Res., 167 (1987) 175-186. (73) S. Ronniger, Doctoral Thesis, Technische Hochschule Darmstadt, 1990. (74) T. Suami, K. Tadano,T. Iimura, and H. Tanabe,Curbohydr.Rex. 135 (1985) 319-323. (75) B. Coxon, J. Carbohydr.Chem., 3 (1984) 525-543. (76) B. Coxon, Mugn. Reson. Chem., 24 (1986) 1008- 1012.

TABLES I1 AND III The Composition (%) of Aldoses and Some of Their Deoxy Derivatives in D,O Aldose

Temperature (degrees)

Glucosec”

W

W

Idose* Mannose* Talose.* 2-Deoxy-arabinehexose* 3-Deox y-arabino-hexose 2,3-Dideoxy-3-fluoroarabino-hexose -, lyxo-hexose -, ribo-hexose -, xylehexose 5-Deoxyarabinose 5-DeOxylyXOSe 5-DeOXyriboSe 5-DeOXyXylOSe 2-Deoxy-erythro-pentosec” mglycero-D-ido-Heptose*a 3-Deoxy-gluceheptose

-

20 27 82 30 21 28 25 30

B

34 38.8 40.1 35.9 68.0 41.0 49 56.6

66 60.9 58.5 33.4 32.0 29.0 49 25.7

-25 25 25 25 21

30 27

* Data for other temperatures were also given. At 31‘.

pyraoose a-

--

22 65 22 65

40 24.4 20

--7835 --7835 40.5 50.8 61

FlWaIJOW

a-

Aldehyde

Aldehydrol

0.14 0.60 13.5

0.15 0.69 16.5

0.0024b 0.019 0.1

0.0045 0.022 0.5

18.5

11.6 0.5

0.03

0.03

1

17.6

63.2 78.0 31.8 54.0 11

8.7 4

-

36.0 20.3 67.2 42.9 8.5 15.5 15

0.2 0.2 0.1 0.4

0.6 1.5 0.8 2.7

0.06

0.6

References 27 4 4 7 67 8 68 69 70 70 70 70 9 9 9 9

71 7 72

TABLES IV, V, AND VI The Composition (To) of Aqueous Solutions of Ketoses and Amino Sugars

Furanose

keto

a-

B

a-

B

Form

3.1 2.3 1.4 1.2 1.9

61 51.0 70.9 47.3 73.4 98.1

3 11.8 5.7 14.5 9.8

30 34.1 21.1 36.8 15.6

19.7 16.1 13.1 20.2 62.8 18.1 13 25 38

80.3 81.8 86.0

PyranOSe sugar

1-@Methylfructose 3-O-Methylfru~tose*4~ 1-~-a-D-Glucopyranosylfructose 3-O-~~-~-Glucopyranosylh.Ictose* 4-O-cu-~-Clucopyranosylfructose* 5-o-LY-D~lUCOp~UOSy~f~~O~

6-O-~~-~-Glucopyranosyl~ctose Fructose 6-phosphate Fructose 1,6-bisphosphate 1-Deoxytagatose' erythro-2-Pentulose" threo-2-Pentulose*'

1,2-Dideoxy-arubino-3-heptulose

-,lyxo-3-heptulosed -, ribo-3-heptulose -, xyb3-heptulose 2-Amino-2-deoxygulosee, hydrochloride

20 20 20 20 20 20 20 6 6 32 20 26 32 32 32 32

73.5 75 27 100

-75

6.3 41.5 19 -25

-

20.4 62.3 37 10

-

2.2 0.9 16.8 19.6 8.5

6 -

References 41 73 17 17 17 17 17 29 29 32 12 12 32 32 32 32 74

a Data for other temperatures were also given. The data previously cited [p. 431 are incorrect.cAlso,a s mallproportion ofthekfo form. Also,a small proportion of the ppyranose, the pfuranose,and of the kpto form.

TABLE VII Composition (Yo) of Solutions of Sugars in Solvents Other than Water Temp. Sugar Galactose*a Glucose*k

ambient

17 ambient

(I

W Ih

(degrees)

Idose Mannose*O 2,6-Dideoxy-~-ribo-hexose*

24 ambient

27

25 30 20 20 3-0-c~-~-Glucopyranosyl-~-fructoseb 20

Ribose*b 1-0-Methylfructose 3-0-Methylfru~tose*~

20 gulucfo-Heptulose 1-Deoxyfructose

20 27

Pyranose Solvent

a-

jk

46 31 45 55 45 53 26.5 56 77 20 11.2 67.3 12.5 64.2 18 57 20 8 26 3 20 1.3 88.3 2.8 36.9 38 4 46

Furanose

a5 0.6 10.5 2.5 8.4 9.3 6 18 25 30 3.4 23.4 23 21

Carbonyl form

18 1

7 0.5 13.0 14.0 19 61 41 47 7.0 36.9 38 16.5

0.1

10.5

References

15 27 15 66 15 75 76 27 41 17 17 17 17 41 20 ~

~

By gas-liquid chromatography of the trirnethylsilyl derivatives. Data were also given for other temperatures. In 50% solution, 48 :52. From the IH-n.m.r. spectrum. *From the 'T-n.m.r. spectrum. a

This Page Intentionally Left Blank

ADVANCES rN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL. 49

RADICAL-MEDIATED BROMINATIONS AT RING POSITIONS OF CARBOHYDRATES

BY LAsz~6SOMSAKAND ROBERTJ. FERRIER

.

Department of Organic Chemistry.Lujos Kossuth University. H.4010 P . 0. Box 20. Debrecen Hungary; Department of Chemistry. Victoria University of WeNington. P. 0. Box 600. Wellington.New Zealand

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.

I Introduction .......................................................... I1 Radical-mediated Brominations .......................................... 1. General: Reaction Conditions and Suitable Compounds .................. 2. Hexuronic Acid Derivatives.......................................... 3. Peracylated Aldoses ................................................ 4. Anhydropyranose and Anhydrofuranose Derivatives ..................... 5 . Glyculose and Glycosulose Derivatives................................. 6. Glycosyl Cyanide Esters ............................................. 7. C-Glycosylbenzeneand C-GlycosylheterocycleEsters..................... 8. Glycosyl Halide Esters .............................................. 9 HexopyranosideEsters .............................................. 10. Phenyl 1-ThiohexopyranosideEsters................................... I I Miscellaneous Compounds .......................................... I11. The R e g b and Stereo-chemistry of the Reactions ........................... I . The Regicchemistry of the Reactions .................................. 2. The Stereochemistryof the Reactions.................................. IV Reactions of the Bromine-containing Products .............................. I Substitution Reactions .............................................. 2 EliminationReactions .............................................. V Conclusions .......................................................... VI Addendum ...........................................................

.

.

.

. . .

. .

37 41

41 42 45 51 54 57 59 60 62 64 65 67 67 71 75 75 85 91 91

I . INTRODUCTION Free-radical reactions. for so long the Cinderellas of organic chemistry because of lack of control over their selectivity. have in recent years frequently emerged as simple. efficient. and novel means for effecting molecular transformations. and they are now at the forefront of synthetic methods. Previous emphasis was on the physicochemical investigations of reactions from which developed appreciation of such factors as the regioselectivityof 37

. .

Copyfight8 I99I by Academic Raa, Inc All rightaofreproduction in any form m t r v u l

38

LASZLO SOMSAK AND ROBERT J. FERRIER

radicals in substitution' and addition* reactions of organic compound^,^.^ and the theoretical and preparative aspects of substituent effects in radical chemi~try.~ The methods which have emerged have led to powerful new procedures for forming carbon - carbon bonds both inter- and intra-molecularly, for selectively cleaving such bonds, and for novel, functional-group transformations$- lo Some of this more recent synthetic work was developed by use of carbohydrate compounds,6-10and an appreciable number of photochemical radical reactions have been applied in the series," but a high proportion ofcarbohydrate free-radical research has been based on the investigation of the products of radiolysis12or of chemical initiating species in s~lution.'~ Except for photochemical intramolecular cyclization of 3-oxoalkyl glycosides which result in spiro-bicyclic acetalsI4and of the hypoiodites of the corresponding 2- or 3-hydroxyalkyl glycosides which give spiro-orthoester derivative^,'^ both substitution processes resulting from abstraction of H-1, it seems that few examples have been reported of efficient and direct radical-induced substitution reactions occurring at carbohydrate carbon atoms. Features of the chemistry of radicals at the anomeric center have been the subject of a specific review.16 The aim of the present article is to survey a radical-mediated reaction by which bromine atoms may be substituted directly into some carbohydrate derivatives at ring positions by the following general mechanism.

J. M. Tedder, Tetrahedron,38 (1982) 313-329. J. M. Tedder and J. C. Walton, Tetrahedron. 36 (1980) 701 -707. J. M. Tedder, Angew. Chem., Znt. Ed. Engl.. 21 (1982) 401 -410. A. L. J. Beckwith, Tetrahedron, 37 (1981) 3073-3100. H. G. Viehe, Z. Janousek,and R. Merenyi (Eds.), Subscituenr Efects in RudicalChemistry, Reidel, Dordrecht, 1986. (6) B. Giese, Angew. Chem., Int. Ed. Engl., 24 (1985) 553-565. (7) B. Giese, Radicals in Organic Synthesis:Formntion of Carbon- Carbon Bonds, Pergamon, Oxford, 1986. ( 8 ) B. Giese (Ed.), Tetrahedron Symposia in Print No. 22, Selectivity and Synthetic Applications of Radical Reactions, Tetrahedron,41 (1985) 3837-4302. (9) M. Ramaiah, Tetrahedron, 43 (1987) 3541 -3676. (10) D. P. Curran, Synthesis, (1988) 417-439,489-513. ( I 1) R. W. Binkley, Adv. Carbohydr. Chem. Biochem., 38 (1981) 105- 193. (12) C. von Sonntag, Adv. Carbohydr. Chem. Biochem., 37 (1980) 7-77. (1 3) M. Fitchett and B. C. Gilbert, J. Chem. Soc.. Perkin Trans. 2, (1986) 1 169- 1 177. (14) G. Remy, L. Cottier, and G. Descotes, Can. J. Chem., 61 (1983) 434-438. (15) J.-P. Praly, G. Descotes, M.-F. Grenier-Loustalot, and F. Metras, Carbohydr. Res., 128 (1984) 21-35. (16) G. Descotes, J. Curbohydr. Chem.. 7 (1988) 1 -20.

(1) (2) (3) (4) (5)

RADICAL-MEDIATED BROMINATIONS

Br

\ /

Br

.A C / \ I

39

+Br

\

OR

The survey is intended to cover the published literature on the subject as fully as possible. Although the four halogens are well known as constituents of a wide range of carbohydrate compounds, radical-mediated halogenations have, in most cases, been realized only with bromine. There are, however, some examples of chlorinations; to the best of our knowledge, there are no reported fluorinations or iodinations which occur by direct, radical-mediated processes. The radical-mediated reactions by which bromodeoxy carbohydrates are obtainable from benzylidene acetalsl7-I9are not considered. That bromine atoms can be introduced directly at C-5 of some pyranoid compounds, at C-4 of some furanoid derivatives, and at chemically related centers of certain other monosaccharide compounds was discovered following the finding by Dr. Richard Furneaux, during his Ph.D. studies in Wellington, that phenyl tetra-0-acetyl- l 4hio-P-D-glucopyranoside (1) is conCI120Ac

I

CI120Ac

I

AcOQPh

OAc

1

verted into the enone phenyl 2,4,6-tri-O-acetyl-I -thio-D-eryZhro-hex-1enopyranosid-3-ulose (2) and its 2-0-bromoacetyl analog (3) by treatment with N-bromosuccinimide in refluxing carbon tetrachloride, provided that the reaction is carried out under light or in the presence of a radical initiator (17) S. Hanessian and N. R. Plessas, J. Org. Chem., 34 (1969) 1035- 1058. (18) J. S. Chana, P. M. Collins, F. Farnia, and D. J. Peacock, J. Chem. SOC.,Chem. Commun., (1988) 94-96. ( I 9) P . M. Collins, A. Manro, E. C. Opara-Mottah, and M. H. Ali, J. Chem. SOC.,Chem. Commun.. (1988) 272-274.

L A S Z L ~SOMSAK AND ROBERT J. FERRIER

40

such as benzoyl peroxide.20 When the reaction was applied to methyl (phenyl tri-0-acetyl- 1-thio-&D-glucopyran0sid)uronate (4), as well as the

ksph -

expected enone 5, the product of bromine substitution at C-5, namely, methyl [phenyl (SR)-tri-O-acetyl-5-bromo-1-thio-~~-glucopyranosid]uronate (6),was isolated crystallinez1in 12% yield (see Scheme 1). o

p

h

OAc

+

QPhC0,Mc

OAc

ACO

AcO

AcO OAc OAc

OAC

4

5

6

SCHEME1

It was envisaged that the enones were produced following abstraction of H- 1 (a process facilitated by the ability of sulfur atoms to stabilizeradicals on bonded carbon centers), radical bromination, elimination of hydrogen bromide to give substituted glycals, allylic bromination at C-3, and loss ofacetyl bromide. In the formation of compound 6, hydrogen abstraction from C-5 was deemed to compete with that from C- 1, and to lead to substitutionat the former site with the formation of a relatively stable product. At the time of the finding of the reaction which led to compound 6, formation of 6 was not surprising, because radical brominations at carbon atoms adjacent to ester carbonyl groups were well knownYuand hydrogen abstraction from such centers as C-5 of compound 4, which provide captodative radical stabilization (high delocalization resulting when electrondonating and electron-withdrawing groups are concurrently present; see Section 111, 1b), is particularly fav0red.2~ What was to emerge was the finding that several pyranoid and furanoid carbohydrate compounds that lack such radical-stabilizingcomponentsas carbonyl groups also undergo radical bromination at the non-anomeric centers bonded to the ring-oxygen atoms by highly selective processesto give relatively stable products. In some cases, the stability is very high; a sample of crystalline penta-O-benzoyl-S-bromo-~-~glucopyranose has remained unaltered in a sample tube at room tempera(20) R.J. Femer and R. H. Fumeaux, J. Chem. Soc.,Perkin Trans. 1, (1977) 1993- 1996. (21) R. J. Femer and R. H. Furneaux, J. Chem. Soc.,Perkin Trans. I , (1977) 1996-2OOO. (22) N. P. Buu-Hol and P. Demerseman, J. Org. Chem., 18 (1953) 649-652. (23) H. G. Viehe, Z. Janousek, R. Meknyi, and L. Stella,Acc. Chem.Res., 18 ( 1985) 148 - 154.

RADICALMEDIATED BROMINATIONS

41

ture for a decade, and a related 5-bromouronate peracetate can be purified by sublimation even at atmospheric pressure.21 This article deals with the state of knowledge of ring photobromination of carbohydrate derivatives about 12 years after the phenomenon was first observed. The reaction is very unusual in providing means by which hydrogen atoms bonded to carbon atoms ofcyclic carbohydratederivativesmay be substituted directly and, in many cases, affording selectivity at ether carbon atoms in preferenceto the dioxygenated anomeric centers. Some aspectsof it have been reviewed from the standpoint of the operation of the captodative effect.24 11. RADICAL-MEDIATED BROMINATIONS 1. General: Reaction Conditions and Suitable Compounds

Radical-mediated brominations of carbohydratederivatives have usually been conducted in refluxing carbon tetrachloride, under a tungsten ( 150250 W) or heat (250-450 W) lamp, with either N-bromosuccinimide (1.25 molar equivalents) or bromine (2 - 5 equivalents) as the source of halogen. Addition of bromotrichloromethane to the carbon tetrachloride can be adv a n t a g e ~ ~Substrate ~ . ~ ~ concentrations have ranged from 0.02 to 0.2 mol. 1 - I and have depended to some degree on solubility factors, and ordinary laboratory glassware has most often been used. It must be emphasized that the conditions employed for most of the reactions reported to date have been selected somewhat arbitrarily, and no adequate studies have been camed out to assess the significance of all of the variables. For example, it is not fully known why apparently similar experiments camed out on different scales have sometimes given inconsistent results. Suspicions that decreases in light flux with increasing solution bulk have led to the use of “lollipop” reaction vessels for larger-scale reactions. The effects of the use of such chemical radical-initiators as benzoyl peroxide together with, or instead of, light sourcesare incompletelyassessed,and even the grounds for selection as between N-bromosuccinimide and bromine as reagent are so poorly appreciated that current best advice should be to “try each.” Bromine generates hydrogen bromide as a by-product that can react further with substrates possessing acid-sensitive groups, but nevertheless, as for the reaction of penta-0-benzoyl-P-D-glucopyranose, it can be the reagent of choice.26In other cases, the effects of the hydrogen bromide can be damaging, but can be minimized by use of an acid scavenger such as barium or (24) L. Somsik, Magv. Kkm.Lapju, 43 (1988) 219-225 [Chem.Abstr., 110 (1989) 135,5701. (25) J.-P. Praly, L. Brard, G . Descotes, and L. Toupet, Tetrahedron. 45 (1989) 4141 -4152. (26) R. J. Femer and P. C. Tyler, J. Chem. Soc..,Perkin Trans. I , (1 980) 1528- 1534.

LASZLO SOMSAK AND ROBERT J. FERRIER

42

potassium carbonate. In some instances, more-selective reaction may be obtained by using N-bromosuccinimide, as, for example, with tetra-0-acetyl-/?-~-xylopyranose.~~ Hydroxyl groups are always protected prior to reaction with bromine radicals, and derived esters have proved suitable. Benzoates are particularly useful and are preferable to acetates, which are susceptible to methyl-group bromination, particularly when the acetoxyl groups are bonded to carbon atoms in the a-relationship to carbon radicals. Conceivably,this susceptibility can be accounted for as follows. I I -c-cI

0

0 \/

- I

I I -c-c0,

o,

I I r;, -c-c-

\ - I

I t e’, -c-c-

\ - I

\ - I

I I -c-c-

C

I

Methyl ethers have been employed, but alkyl glycosides may be unstable following hydrogen abstraction from the anomeric center (see Section 11,9). Benzyl ethers and benzylidene (and other aldehyde-based) acetals, which themselves undergo ready radical brorninatior~,~’~ can be expected to be entirely unsuitable protecting groups. Ketone-derived acetals, on the other hand, should be stable, at least in the absence of acid, and a few examples of successful brominations in their presence are reported in Section II,4. 2. Hexuronic Acid Derivatives

As indicated in the Introduction, radical bromination at a ring-carbon atom of a carbohydrate was first encountered following reaction of the methyl uronate thioglycoside 4 with N-bromosuccinimide in refluxing carbon tetrachloride under a heat lamp.*’ In this case, dominant competitive reaction occurred at the anomeric center, but, when substrates having substituents at this position (other than the radical-stabilizing sulfur) were examined, the reactivity at C-5 was relatively enhanced, and C-5-brominated products were preponderant. Thus, methyl tri-O-acetyl-2,6-anhydro-~-gulonate (7), which has no radical-stabilizing substituent at the “anomeric center,” gave mainly methyl tri-0-acetyl-a-L-xylo-hexulopyranosylonate bromide (8) (47%, crystalline)21(see Scheme 2), illustrating the practical potential of the reaction, as the product can be readily converted into Lascorbic acid.21.28 (27) R. J. Femer and P. C. Tyler, J. Chem. SOC.,Perkin Trans. 1, (1980) 2767-2773. (27a) R. W. BinMey and D. G . Hehemann, J. Org. Chem.. 55 (1990) 378-380. (28) R. J. Femer and R. H. Furneaux, J. Chem. SOC.,Chem. Commun., (1977) 332-333.

43

RADICAL-MEDIATED BROMINATIONS

I

OAc

OAc

8

7 SCHEME 2

Introduction of an acetoxyl group at C-1 (giving methyl tetra-0-acetyl-/3D-glucopyranuronate,9) did not reactivate the anomeric center, and methyl (5R)-tetra-O-acetyl-5-bromo-~-~-ghcopyranuronate (10) was obtained in good with N-bromosuccinimide*' or excellent (with bromine)= yield (see Scheme 3). On the other hand, the a-acetate 12 reacted significantly more

OAC

9

~

A

COZMe

AcO

OAc

OAc

10 SCHEME3

11

slowly to give the 5-bromide 14 in low yield, together with a dibromo analog having a second bromine atom in one of the acetyl ester groups.26The significant difference in reactivities of the anomers was attributed to steric hindrance to attack by bromine at H-5or C-5by the axial acetoxyl group at C- 1;this and other steric factors are discussed further in Section III,2. Several other methyl /3-D-glucopyranosyluronicderivatives, including glycosides, have given 5-bromo products in an isolated yield of - 5096,as indicated in Table I. It is noteworthy that, while methyl tetra-0-acetyl-P-D-glucopyranoside reacts at the anomeric center (see Section 11,9), the corresponding methyl uronate derivative (13) undergoes selective bromination at C-5, to giveM (29) R.J. Ferrier and P. C. Tyler, J. Chem. Soc., Chem. Commun., (1978) 1019- 1020. (30) T. Chiba and P. Sinay, Curbohydr. Res., 151 (1986) 379-389.

c

LASZLO SOMSAK A N D ROBERT J. FERRIER

44

(-1: C0,Me

C02Me

I

I

AcO

I

OAc

OAc

12 (R1 13 (R1

- H, RZ

=

14 (R1 15 (R'

OAc)

- OMe, R2 - A)

-

H, R 2

- OAC)

OMe, R 2

-

H)

compound 15, illustratingthe significanceof the captodative stabilizationof the radical formed at this center. In similar fashion, reaction of 1,6-anhydro2,3di-0-benzoyl40(methyl 2,3,4-tri-O-benzoyl-~-~-glucopyranuronate)-P-D-glucopyranose(16) occurred preferentially at C-5 of the uronate moiety to give compound 17 (47%),but the dibromide 18 was also produced in minor proportion^.^^ TABLEI 5-Bromides Produced from Hexuronic Acid Derivatives Substrate

Product

References

6'

12b

8

47c 68c 18

21 21 21 26 30 30 30 30

C0,Me

F02Me

I

Yield (%)

R

-

)

R3 C OR2

4

R' P-SPh

7

H

9 12

P-OAC CU-OAc

13

&OMe P-OMe P-OMe &OAC

RZ Ac Ac Ac Ac Ac

R3 OAc OAc OAc OAc OAc

10 1Y 15

48

Me Me

OMe

4Y

OAc

46b

Ac

H

476

Tnxiuced as a minor product, together with compound 5. holated by chromatography. CIsolated by crystallization.dProduced together with 32% of a bromoacetyl derivative.

(31) Y . Ichikawa and H. Kuzuhara, Curbohydr. Res., 115 (1983) 117- 129.

RADICAGMEDIATED BROMINATIONS

BzO

45

I

OBz

16 17 18

-H (R1 - H (R1 - Br (R1

, RZ , R2 , R2

-H

)

- Br)

=

Br)

Photobromination of methyl tetra-0-acetyl-a-L-idopyranuronate (11)afforded the 5-bromo-/3-~-g/uco compound 10 in 63% isolated yield, indicatingmthat epimers 9 and 11react by way of a common C-5 radical intermediate (see Scheme 3). Methyl tetra-0-acetyl-P-mgalactopyranuronate may be converted into the analogous (5R)bromide by use of bromine or N-bromosuccinimide. In the former case, small proportions of a bromoacetyl product are also formed.32 It is also noteworthy that the stabilities of the bromine-containing members of this series vary considerably; compound 10 can be sublimed, even at atmospheric pressureyz1 whereas the 4-deoxy-analog ism “very unstable.”

3. Peracylated Aldoses a. Pyranose Derivatives.-After the finding that hexopyranuronic acid derivatives may be brominated at C-5, it was observed that some hexopyranose compounds may undergo analogous sub~titution.~~ Thus, for example, on treatment on a 200-rng scale with N-bromosuccinimide under light, penta-0-acetyl-/3-D-glucopyranose (19) gave the 5-bromide 20 almost exclusively, a yield of 50% being obtainable by direct crystallizationand > 80% by chromatographic purifi~ation.~~ On a 5-g scale, the reaction required longer times (presumably because of decreased mean irradiation-intensity) and the main product, although still obtainable in good yield, was contami-

-

(32) L. S o m a , unpublished observations. (33) R. Blattner and R. J. Ferrier, J. Chem. SOC.,Perkin Trans. I , (1980) 1523- 1527.

+

0 -G

0

z

c

m

Ld

a

V

rn Ld

2

46

Q' +

+

0

4

g

RADICAL-MEDIATED BROMINATIONS

41

nated by analogs (21) containing mono-, di-, and tri-bromoacetyl groups which, it is presumed, were formed by way of a 4,5-acetoxonium ion, or equivalent bicyclic radical species. Use of bromine as brominating agent likewise afforded the 5-bromide (20) when the reaction was camed out on a small scale, but also, the major by-products 22 - 24 when applied on a larger scale (see Scheme 4). Glycosyl bromides 22 and 23 can be accounted for as being products of reaction of hydrogen bromide with the glycosyl acetates 20 and 19, respectively, while the gem-dibromide 24 probably resulted from photobromination of tetra-0-acetyl-P-D-glucopyranosylbromide (see Section 11,8). That hydrogen bromide was largely responsible for these complications was established by the observation that the 5-bromide was produced in 90%yield when the larger-scale bromine-induced reaction was repeated in the presence of potassium ~ a r b o n a t e . ~ ~ * ~ ~ When penta-0-acetyl-a-D-idopyranose(25) was subjected to radical bromination using N-bromosuccinimide as source, it gave, in good yield, pentaO-acetyl-5-bromo-~-~-glucopyranose (26) (see Scheme 5), which is the en-

-

AcO

Br

7

AcO

Acd

Iy

OAc

25

26 SCHEME 5

antiomer of the product 20 obtained from the P-D-gluco isomer (19), and it follows that the esters 19 and 25 reacted by way of the enantiomeric radicals which brominated axially.33It can likewise be concluded that compound 19 and its C-5 epimer, penta-0-acetyl-a-L-idopyranose,afford 20 by way of a common radical. It was then found that a tertiary center is not required at C-5, and that tetra-0-acetyl-P-D-xylopyranose (27), treated with radicals derived from Nbromosuccinimide, affords a mixture of (55‘)- and (SR)-tetra-O-acetyl-5bromo-P-D-xylopyranose(28 and 29; see Scheme 6), which is in accord with, but does not necessarily follow from, the finding that 27 exists in solution in both chair conformations, each of which is subject to axial hydrogen abstraction and subsequent axial br~mination~’.’~ (see Section 1142). In this (34) L. Somshk and E. Tarcsa, unpublished results. (35) R. J. Ferrier, S. R. Haines, G. J. Gainsford, and E. J. Gabe, J. Chem.Soc., Perkin Trans. I , (1984) 1683- 1687.

LASZL~SOMSAK AND

48

-

ROBERT J. FERRIER BK

OAc

+

AcO -0AC AcO

AcO

OAc

OAc

OAc

OAc

27

29

28 SCHEME 6

way, the pentose derivative behaves differenfly from the conformationally more-discrete hexopyranose compounds so far examined, none of which have given identified mixed epimeric bromides. In keeping with findings in the hexuronic acid series (see Section 11,2), hexopyranose peresters having axial substituents at the anomenc center react appreciably more slowly than do compoundswith such groups equatorial; they give complex sets of products from which 5-bromo-a compounds have been isolated in only modest fields.%*%As with the hexuronic acids, this observation can be attributed to steric inhibition of axial attack by bromine at C-5 or at H-5. Results obtained to date in this series are summarized in Table 11. TABLEI1 5-Bromides Produced from Hexopyranose Esters Substrate

Product

Yield (%)

References

50", 826 2 1" 43"C, 1 P . d 226 49 4fja, 30 (+ 33% of c-5epimer)b

29,33 34 26,29 26 34 21,29,35

8' ' O R4 OR' O

19

27 a

Rt R2 B-AC AC CU-ACAC B-Bz BZ &-Bz BZ B-AC AC 8-AC AC

R

OR2 R3 R4 OAC H OAC H H OBZ H OBZ OAC H H OAC

t

r

OR2 D O

R

f

OR2 R3 CHZOAC CHZOAC CH~OBZ CHZOBZ CHZOAC H

20

28 (+ 29)

Isolated by crystallization. * Isolated by chromatography. N-Bromosuccinimide. d Bromine.

RADICAGMEDIATED BROMINATIONS

49

Given the facts that #?-D-glucopyranosylesters react favorably at C-5 and that a-D-glucopyranosyl derivatives are less reactive, it was predicted that p-maltose octaacetate would undergo bromination preferentially at C-5 of the reducing moiety, and so it transpired. Compound 30 was obtained with unknown selectivity,except that the unfractionated products gave the corresponding exo-alkene in 12% overall yield after zinc - acetic acid treatment followed by ~hromatography.~~

AcO

OAc OAc

b. Furanose Derivatives.-Because radicals are formed by hydrogen abstraction at the ether positions of tetrahydrofuran more readily than is the case at the comparable positions of tetrahydropyran,3’ it was to be expected that photobromination would occur in suitable furanoid compounds, and, when 1-0-acetyl-2,3,5,6-tetra-O-benzoyl-~-~-~ucofurano~ (31)or the Dgalacto isomer 32 was treated with bromine in the presence of potassium carbonate, 1-0-acetyl-2,3,5,6-tetra-O-benzoyl-4-bromo-~-~-g~acto~ (33) and -glucose (34)were produced in the ratio of 4 1 :9 from each, indicating the presence3*in the reactions of a common C-4 radical (see Scheme 7). A convenient feature of these bromides was that the minor component underwent selective hydrolytic debrominationto an aldos-4-uloseproduct, probably with the participation of the trans-related 3-0-benzoyl group, when the mixture was subjected to chromatography on silica gel, which allowed easy isolation of the main bromide 33 in 72% yield. Similar reaction of 1-O-acetyl-2,3,5-tri-O-benzoyl-~-~-n~se (36)on a small scale and in a cooled reactor gave the product of direct substitution, namely, 1 -0-acetyl-2,3,5-tn-O-benzoyl-4-bromo-~-~-nbose (37),but, on a larger scale, or without cooling, an equilibrated mixture of the rib0 and the L-Z~XO bromides (37and 38)was produced (see Scheme 8). The former, with the benzoyloxy group at C-3 and the bromine atom cis-related, was readily (36) R. Blamer, R. J. Femer, and P. Prasit,J. Chem.Soc., Chem. Comrnun.,(1980)944-945. (37) V. Malatesta and K.U. Ingold, J. Am. Chem. Sm., 103 (1981) 609-614. (38) R. J. Femer and S. R. Haines, J. Chem. Soc., Perkin Trans. 1, (1984) 1675- 1681.

LASZLO SOMSAK AND ROBERT J. FERRIER

50 BzOH

I BzOI12$

uO" I

HCOBz

I

34

33

I

oBz

CH20Bz

32

I

HCOBZ

I CII,OBz

OBz

35 SCHEME 7

isolated, because, as with compound 34,the latter was hydrolyzed during column chromatography. Previous studies had indicated that, in general, although some differences were sometimes observable, the products formed by photobromination of carbohydrate derivatives using bromine or N-bromosuccinimide were similar. When compound 31 or 32,however, was treated with the latter reagent, a major difference was found, and the main product (74% isolated) was the orthoamide35,formed, it was concluded, by way of the bromides 33 and 34 and, thence, a cyclic 3,4-benzoxonium ion.35Support for this route was obtained by observing that treatment of a mixture of the bromides with N-bromosuccinimidein refluxing carbon tetrachloride without irradiation resulted in their complete conversion into the orthoamide35. N-Bromoacet-

RADICAL-MEDIATED BROMINATIONS

36

37

51

38

SCHEME 8

amide and N-bromophthalimide together with 31 or 32 under photolytic conditions afforded analogous orthoamide derivatives. Reaction of the ribofuranose ester 36 with N-bromosuccinimide again resulted in C-4 substitution, to give the bromides 37 and 38 in the ratio of 3:2, and compound 35 and close analogs remain the only orthoamides encountered during these studies. An example of the photobromination of a nucleoside derivative is given in Section 11, 1 1. 4. Anhydropyranose and Anhydrofuranose Derivatives a. 1,6-Anhydrohexopyranose Derivatives. -Because abstraction of equatorial hydrogen atoms at C- 1 or C-5 is required (see Section 111, la), and because the formation of bridgehead radicals would be involved,391,6-anhydroaldohexose derivatives do not undergo photobromination within the hexose rings. Instead, they react with high stereoselectivity and with complete regioselectivity at C-6, to give the exo-monobromides. 2,3,4-Tri-0acetyl-1,6-anhydro-~-~-glucose (39) thus gives (6S)-2,3,4-tri-O-acetyl-1,6anhydro-6-bromo-~-~-glucose (40)essentially specifically(67%was isolated crystalline)40and, likewise, the corresponding monobromotribenzoate(41) was obtained in 78% yield.40*41 No products of endo-monobromination have been encountered, but the exo-bromides can be induced to give geminally substituted dibromides (for example, 42) by using prolonged reaction-tirne~,~?~~ and, in some cases, but not others, 6,6-dibromides appear as significant products at early stages in (39) J. March, Advanced Organic Chemistry, 3rd. ed., Wiley, New York, 1985, pp. 167, 616. (40) R. J. Femer and R. H. Furneaux. Ausf.J. Chem., 33 (1980) 1025- 1036. (4 I ) H. Ohrui, H. Honk, H. Kishi, and H. Meguro,Agric. Biol. Chem..47 (1983) 1 10 1 - I 106. (42) H. Hori, T. Nakajima, Y. Nishida, H. Ohrui, and H. Meguro, J. Cur6uhydr. Chem., 5 ( I 986) 585 -600.

LkSZL6 !SOMSAK AND ROBERT J. FERRIER

52

the reactions. An interesting, comparative study of the 1,6-anhydro-~-aldohexose tribenzoates has allowed a rationalization of this phenomenon by revealing that dibromides are not formed initially from the stereoisomers having axial ester groups42at C-3.Thus, from these compounds, the crystalline 6-exo-monobromotribenzoateshave been isolated as the sole products, as follows:gluco, 78%;40,41manno, 92%;42galacto,86%43and talo, 87%.42On the other hand, under the same conditions, lY6-anhydro-2,3,4-tri-O-benzoyl-j?-D-allose (43) gives 32% of the 6,6dibromide (44), together with 68% of the e~o-rnonobrornide~~ (45). Comparison of the results for the D-alloand D-gluco-benzoatesclearly indicated that the axial ester group at C-3 in the latter case impedes the abstraction of the endo-H-6, but inversions of configuration at C-2 or C-4, or both, of compound 43 also have an impeding influence. The altro and gulo isomers, which have inverted stereochemistry at C-2 and C-4, respectively, give 22 and 19%of the dibromides,while the id0 compound, which has the alternativestereochemistry at C-2 and C-4, affords the exo-monobromide on its K'

39 40 41 42

R1

R2

R3

H Br Br Br

H H H

Ac

Br

Ac

Bz Ac

43 44 45

R1

R2

H Br Br

H Br H

Extended reaction times can lead to the production of the 6,6-dibromides in considerable proportions, not only from such compounds as 1,6-anhydro-2,3,4-tri-O-benzoyl-~-~-gulose (equatorial ester group at C-3),42but also from such less reactive compounds as, for example, 2,3,4-tri-O-acetyl-1,6anhydro-p-D-glucose.40 This stereoselectivebromination at C-6 of 1,6-anhydrohexoseesters has opened methods for the production of specifically deuterated hexose com(43) H.Ohrui, Y. Nishida, and H.Meguro, Agric. Biol. Chem., 48 (1984) 1049- 1053.

53

RADICALMEDIATED BROMINATIONS

pounds (seeSection IV,1a), and methyl (6S)-2,3,4-tri-O-ben~oy1-6-~H-/3-~glucopyranoside, prepared following reductive deuteration of compound 41, and subjected to glycosylation, has given access to the specifically labeled4 methyl p-D-isomaltoside (46). Similarly labelled methyl p-D-maltoside (47), also required for conformational studies, was, however, obtained from the 6-ex0 product (48) of photobromination of the corresponding disaccharide derivative. In this case,the selective halogenation gave the crystalline bromide in 64%yield,44but, when applied to the p-linked analog, hexa-O-acetyl- 1,6-anhydrolactose(49), the reaction was unsuccessful, affording 20%of the product of anomerization at the inter-unit bond, together, we speculate, with products of bromination at C-1 of the nonreducing D-galactopyranosyl ring.4s (See Section 11,9). The successful photobromination of a uronic acid-containing anhydrodisaccharide is noted in Section II,2. CH-OH

CH.011 110

0

HO OMe "

Q

O

O HM

e

OH

46

47

AcO' A

c

O

q

AcO

o

H OAc

48

b0rYc

AcO

OAc

49

The negative results obtained on photobromination of 1,6-anhydr0-2-0benzoyl-3,4-O-isopropylidene-~-~-galactose and its 3,4-O-cyclohexylidene analog are more difficult to rationalize, especially as the authors used N-bro(44) K.Bock and H.Pedersen, Acta Chem. Scund., Ser.B, 42 (1988) 190- 195. (45) K. Adelhorst, K. Bock,H. Pedexn, and S. Refn, Acfn Chem. Scand., Ser. B, 42 (1988) 196 -20 1.

L A S Z L ~SOMSAK AND ROBERT J. FERRIER

54

mosuccinimideto suppressthe production of acid during the reactions, and, under these conditions, obtained a product of selective substitution from 1,5-anhydro-2,3-O-isopropylidene-~-~-nbose~ (see next Section). The photobromination of a relevant cyclohexane derivative is noted in Section 11,l I . b. 1,5-Anhydropentofuranose Derivatives. -In our experience, and as indicated in the previous Section, some isopropylidene acetals of cyclic carbohydrate derivativesare unstable towards photobromination. However, following treatment with N-bromosuccinimide and column chromatographic purification of the products, 1,5-anhydro-2,3-0-isopropylidene-/3D-ribose gives4650%of the crystalline(5s)-1,5-anhydro-5-bromo-2,3-O-isopropylidene-P-D-ribose (50). Similar treatment of its 2- and 3-C-methyl derivatives and of 1,5-anhydro-2,3-O-isopropylidene-~-lyxose gave analogous crystalline products (5 1, 68 and 30% yield, re~pectively),~’ and the relative stabilitiesof these and their precursors can be consideredto relate to the stabilities of their trioxabicyclo[3.3.0loctane components.46However, 1,5-anhydr0-2,3-di-O-benzoyl-~-~-arabinose and -P-D-xylose also afford relatively stable exo-5-bromides (51 and 52) in 57 and 43% yield, respectively, illustrating that their dioxabicyclo[2.2.llheptane rings have sufficient independent ~tability.~’ Br

0

0

‘cxi 50

2

R1 R2 R3 R4 51 II OBz OBz H 52 OBz I i H OBz

5. Glyculose and Glycosulose Derivatives

Lichtenthaler and his coworkers, recognizing that pyranoid compounds bearing carbonyl groups at C-2 or C-4would be subject to captodatively (46) H. Ohrui, T. Misawa, and H. Meguro, Agric. Biol. Chem., 48 (1984) 1825- 1829. (47) H. Ohrui, T. Misawa, H.Hori, Y. Nishida, and S. Meguro, Agric. Biol. Chem., 5 1 ( I 987) 81-85.

RADICAL-MEDIATED BROMINATIONS

55

stabilized radical substitution-reactionsat C- 1 and C-5,respectively, submitted 1,5-anhydro-3,4,6-tn-O-benzoyl-~-fructose (53) and the aldos-4dose derivative (59)to photobromination, and obtained tri-0-benzoyl-a-Darubino-hex-2-ulopyranosyl bromide (56) and tri-0-benzoyl-5-bromo6-deoxy-~-~-xylo-hex-4-ulopyranose (60) in 78 and 90% yield, respectively (Scheme 9). Likewise, the a anomer of compound 59 reacted just as readily

BK

59

60 SCHEME9

and efficiently, to give the crystalline a anomer of compound 60 (87%), despite the product’s having cisrelated axial ester and bromo groups at C-1 and (2-5,r e s p e ~ t i v e l y . ~ ~ - ~ ~ Even forcing conditions did not cause the anhydro-D-fructosederivative 53 to undergo photochlorination with N-chlorosuccinimide, but, with sulfury1 chloride- azobis(isobutanonitri1e)in refluxing carbon tetrachloride, it gave several products, one of the major being the crystallinea-chloro analog of the bromide 56 (14% isolated).m Oximo groups cannot themselves be used instead of the carbonyl functions in reactions of this kind, because, with N-bromosuccinimide or bro(48) F. w. Lichtenthalerand P. Jarglis,Angew. Chem., Int. Ed. Engl., 21 (1982) 625-626. (49) F. W. Lichtenthalerand P. Jarglis,Angew. Chem. Suppl. (1982) 1449- 1459. (50) F. W. Lichtenthaler,P. Jarglis, and W. Hempe, JustusLiebigs Ann. Chem.,(1983) 19591972.

.

U s Z L 6 SOMSAK AND ROBERT J. FEWER

56

mine, they undergo oxidation; derivativeshaving the oximo group acylated have proved, however, to be most suitable, and the brominated products to be very valuable as glycosylating agents. Thus, the 0-benzoyl(54) and 0-p nitrobenzoyl(55)derivatives of compound 53 give the a-glycosyl bromides 57 and 58, respectively, in very high yield^.^*-^ Importantly, this halogenation process can be extended to members of the disaccharide series, and the lactose, maltose, and cellobiose derivatives 61, 62, and 63 afford the crystalline a-glycosylbromides 64,65, and 66 in almost quantitative yields (see Scheme 1O).51 As these 0-benzoylated oximes are CIf,ODz

CH,OBz

0

NOBz

NOBz OBz

OBz nonreducing units.

61

62 63

p - D -galacto a-o-glucoP-O-glUCO-

64 65 66

SCHEME 10

easily made from the readily available, corresponding hydroxyglycal benzoates, and because the bromides serve as useful disaccharide glycosylating agents, to afford, for example, 2-amino-2-deoxyhexose-containingdisaccharide glycosidesS2and trisac~harides~~ (see Section IV, 1b), this development constitutes a very significant step forward in the synthesis of specific oligosaccharides. The anhydropentulose oxime derivative 67 gives in high yield the P-bromide 68, which, like related pentose compounds, adopts the conformation having all of the substituentsat the secondary centers in the axial orientation (see Scheme 1l).so Photobromination of the enolone 69 is of interest, because radicals formed at C-1or C-5 (carbohydratenumbering)are both subject to captoda(51) F. W. Lichtenthaler, E. Kaji, and S. Weprek, J. Org. Chern.. 50 (1985) 3505-3515. (52) E. Kaji, F. W. Lichtenthaler,T. Nishino, A. Yamane, andS. Zen,Bull. Chem.SOC.Jpn., 61 (1988) 1291- 1297. (53) F. W. Lichtenthaler and E. Kaji, JusfusLiebigs Ann. Chem., (1985) 1659-1668.

57

RADICALMEDIATED BROMINATIONS

0-

BZO

NOBz

NOBz

BzO

68

67 SCHEME1 1

tive stabilization. Its reaction at the latter center to give mixed epimers from which crystalline 71 was isolated in 5 1% yield established that the keto function selectively favors the vinylogous radical rather than that at the adjacent center which also carries the disfavoring benzoyloxy group (see Section 111, 1). Compound 70 gives mainly di-0-benzoylkojic acid (72) by way of the 5-bromo product of substitution.50

c? CH20Bz

CH20Bz

I

I

-

I

Q

BzO QBz

BzO

69 R OBZ 70 R - H

CH,OBz

OBz

71

72

Somewhat surprisingly, particularly in view of the proposed means of formation of the glycosid-3-ulose derivative 2, I ,5-anhydro-2,3,4,6-tetra-Obenzoyl-D-arubino-hex-1-enitol (tetra-O-benzoyl-2-hydroxy-~-glucal) does not undergo bromination at the allylic C-3 atom; the only products isolated followingattempted photobrominationwith N-bromosuccinimidewere two dibromides produced by addition reactions.50 6. Glycosyl Cyanide Esters

Cyclic carbohydrates bearing a cyano group at the anomeric center have, at this position, captodative radical-stabilizinggroups similar to those at C-5 of the hexopyranuronic acid compounds (see Section 11,2). In consequence,

LASZLO SOMSAK AND ROBERT J. FERRIER

58

they undergo regioselective photobromination at C- 1, tetra-0-acetyl-P-Dglucopyranosylcyanide (73)giving the crystalline, axially brominated tetra0-acetyl-1-bromo-P-D-ghcopyranosyl cyanide (74) in 83% yield (see Scheme l 2).48*49 Significantly,in the respective cases of the acetylated D-gulacto- and D-arubino-pyranosylcyanides, the analogous products (75 and 76) are formed, also with high efficiency, from each anomer of the starting

material^.^^^^^

& AcO

AcO OAc

Br

73

74 SCHEME12

Tn-0-acetyl-P-D-ribopyranosyl and -xylopyranosyl cyanides give - 2 : 1 mixtures of 1-bromo-/?-and -cy-D-glycosyl cyanides, the anomers being of comparable stabilities in these case^.^^,^^ Br

AcO

MCN I

AcO AcO

75

ir

I AcO

OAc

76

Tetra-0-acetyl-a-D-mannopyranosyl cyanide might be expected to yield the a-glycosyl bromide in particularly high yield, but the recorded figure56is 49%. Although it is noteworthy that this represents one of the few compounds to react by abstraction of an equatorial hydrogen atom, which is a relatively disfavored procedure (see Section 111,2), this factor cannot be held wholly responsible, because, for example, tetra-0-acetyl-a-D-galactopyranosyl cyanide underwent very selective and efficient photobromination (see preceding).54 (54) L. Somsik, G . Batta, and 1. Farkas, Curbohydr. Res.. 124 (1983) 43-51. (55) L. Somstik, G. Batta, and I. Farkas, Curbohydr. Res., 106 (1982) c4-c5. (56) L. Somsik, I. Bajza, and G. Batta, Justus Liebigs Ann. Chem., (1990) 1265- 1268.

RADICAL-MEDIATED BROMINATIONS

59

Determination of the structure of tetra-O-acetyl-1-bromo-p-D-galactopyranosyl cyanide by X-ray diffraction analysiss7provided an unambiguous reference for the assignment of configuration at the anomeric centers of compounds of this category, and circulardichroism can be used for comparative analysis. Thus, the reference compound and other 1-bromo-P-D-glycosyl cyanides (that is, 1-cyano-a-D-glycosylbromides) show a positive Cotton effect at 193 nm, whereas the anomers (for example, 76) exhibit negative effectss8 7. C-Glycosylbenzene and C-Glycosylheterocycle Esters Because the aromatic rings ofcompounds ofthis class render the anomeric centers benzylic, and also conceivably subject to captodative radical stabilization, photobromination results might be expected to correlate with those observed for the glycosyl cyanides (see Section 11,6). Reaction of tetra-0-acetyl-PD-glucopyranosylbenzene (77)with bromine radicals at room temperature gives a complex set of products; at -3O", however, only one is formed, but, because of its instability, it has not been isolated or derivatized.As was ~peculated,~~ these observationsare consistent with the product having been that of substitution at C-1, that is, a benzylic, a tertiary, and an a-oxygenatedbromide which would be unstable for any one of these reasons, far less for all three.

AcO

Me

AcO

AcO OAc

AcO

77

With heterocyclic compounds, observation of anomeric-carbonbromination was first made during attemptsa to halogenate the methyl group of compound 78. Instead, the crystalline compound 5-methyl-2-(tetra-O-acetyl- 1-bromo-/?-D-galactopyranosyl)-1,3,4-0xadiazole (79) was obtained in 75% yield,54and similar yields were found during the analogous reactions of the oxadiazole C-glycosyl compounds having bromomethyl and trifluoro(57) L.Pirkinyi, A. E l m i n , L. Sornsik, and I. Farkas, Curbohydr. Res., 168 (1987)1-5. (58)I. Farkas, G.Snatzke, and L. Somsiik, Jusrus Liebigs Ann. Chem., (1989)599-600. (59) J. N.BeMiller and L. H. Muenchow, Carbohydr. Rex, 28 (1973)253-262. (60) I. F. SzaM, L. Somsiik, and I. Farkas, Acra Chim. Hung., I15 (1984)319-325.

LASzL6 SOMSAK AND ROBERT J. FERRIER

60

methyl substituent groups in place of the methyl group of compound 78. Acetylated P-D-xylopyranosyland a-D-arabinopyranosylanalogs of this glycoside gave mixed products of photobromination, in keeping with the relative chemical and conformational instabilities of many pentose derivatives. However, the 2-(tn-O-acetyl-1-bromo-a-~-arabinopyranosyl)-5-(tnfluoro-

wyJ

AcO

@XCF3

AcO

OAc

OAc

80

0

AcO AcO

BK

/

81

methyl)-1,3,4-oxadiazole(80)was isolated in 50%yield in the course of the work, and crystalline 2-(tetra-O-acetyl-1-bromo-8-D-galactopyranosy1)benzothiazole (81)was obtained in somewhat higher yield.u 8. Glycosyl Halide Esters The first suspicion that acyl glycosyl halides could be subject to radical bromine substitution was raised during photobromination studies of penta0-acetyl-P-D-glucopyranose (see Section II,3) which, when bromine was used as radical source, gave mainly the product of reaction at C-5, but also two dibromides bearing, respectively, the halogen atoms at C-1 and C-5 and both at the former site. Tetra-0-acetyl-cw-mghcopyranosylbromide was also present in small proportion^.^^ Because these by-products were not formed when the reaction was carried out in the presence of potassium carbonate, it could be concluded that hydrogen bromide had led to the formation of the two compoundsbearing one bromine atom at the anomeric position. How the 1, I-dibromide was formed was not clear; it did not appear to have arisen by substitutionfrom the tetra-0-acetyl-cw-mglucosylbromide, because this compound was unreactive under the reaction conditions, but it was speculated that the p-glycosyl bromide could have been the precursor. Subsequent studies by Descotes and ~ o w o r k e r shave ~ ~ . ~given ~ some credibility to this possibility, and the other, namely, that C-1 photobromination of the pentaacetate was followed by hydrogen brominolysis of the anomeric ester group, is devalued by the apparent lack of a 1-bromoproduct following photobromination in the absence of hydrogen bromide. The French workers demonstrated that tetra-U-acetyl-8-D-glucopyranosyl chloride(82) reacts with bromine radicals derived from N-bromosuccini(61) J.-P. Praly and G. Descotes, Tetrahedron Left..28 (1987) 1405- 1408.

RADICALMEDIATED BROMINATIONS

61

mide, to give tetra-0-acetyl- 1-bromo-P-D-glucopyranosyl chloride (U), which can be stored for considerable times at room temperature, in 65% isolated yield, and whose structure was established by X-ray diffraction a n a l y s i ~ .From ~ ~ * ~the ~ reaction products, tetra-O-acetyl-5-bromo-&~-glucopyranosyl chloride (86) was also obtained in 14% isolated yield (see Scheme 13). Parallel work on tetra-O-acetyl-/3-D-glucopyranosyl fluoride CII,OAc

UAC

(83) resulted in the formation of the analogous products tetra-0-acetyl-1bromo-~-D-glucopyranosylfluoride (85) and tetra-O-acetyl-5-bromo-/3-~glucopyranosyl fluoride (87), but, in this case, their ratio was reversed, with the former being isolated in only 4%yield, while the 5-bromide dominated (56%), showing that one factor controlling the reaction is the nature of the halogen atom of the starting materials. A further very important factor is the orientation of the initial anomeric halogen atoms. Thus, the a anomers of compounds 82 and 83 do not undergo abstraction of their equatorial anomenc hydrogen atoms. Both compounds react very much less readily than do their anomers, and no products of direct substitution at C-1are formed. Rather, the former, tetra-0-acetyla-D-glucopyranosylchloride (and the correspondingbromide), gives 1,2-dibromides, presumably formed by bromine addition to the acetylated hydroxyglycal, while the latter ultimately gives the 5-bromide 88 (57% isolated).

AcO AcO

88

62

LASZLd SOMSAK A N D ROBERT J. FERRIER

Configurational inversion at C-2 of compound 82 has no major effect on the products of photobromination, tetra-0-acetyl-p-D-mannopyranosyl chloride giving the C-2 epimers of compounds 84 and 86 in 72 and 12% yield, re~pectively.~~ Some of the few literature examples of photochlorination of carbohydrate derivatives relate to compounds of this series. On treatment with sulfuryl chloride in the presence of azobis(isobutanonitri1e) in refluxing carbon tetrachloride, tetra-O-acetyl-P-D-glucopyranosyland -mannopyranosyl chloride giveZSproducts of monosubstitution of chlorine at C-1 and at C-5.

9. Hexopyranoside Esters Only a few studies have been carried out on the reaction of acylated glycosides with bromine radicals, and only a small number of the compounds studied have given identified products but, nevertheless, tentative generalizationscan be made. Firstly, no pyranoside ester having an axial aglycon has given an identified brominated product: methyl a-D-glucopyranoside tetraacetate and tetrabenzoate, the corresponding D-galactose derivatives, methyl tetra-0-acetyla-D-mannopyranoside,and methyl tri-0-acetyl-P-D-arabinopyranoside can all be recovered in considerable proportions after prolonged reaction times, and the only compounds formed appear to be products of decomposition.62 This concurs with an earlier observation that only complex reactions giving no discrete products occur with methyl a-D-glucopyranoside esters.40The relative stability of compounds of this category permits the protection of axially bonded glycosidic groups of pyranoid compounds, and diverts reaction elsewhere in complex compounds containing such moieties. Thus, the nonreducing unit of fi-maltose octaacetate is largely unreactive, whereas bromination occurs at C-5 of the reducing ring, and compound 30 is produced (see Section 11,3). Similarly, a 1,6-anhydromaltosederivative reacted selectivelyat C-6 of the reducing unit to give compound 48 (see Section 11,4). Secondly, pyranosides having equatorial aglycons may react at C- 1 or at C-5, depending largely on the nature of the aglycons. Because methyl P-Dglucopyranosidetetraacetate reacts faster than does penta-O-acetyl-p-D-guc0pyranose,6~ it was concluded that the anomeric center takes part in reaction of the former and, subsequently,62small proportions of a bromolactone were isolated from the reaction products. In the case of the corresponding tetrabenzoate89, however, a less-complexset of compoundswas formed and tetra-O-benzoyl-2-bromo-~-glucono1,s-lactone (90) was isolated63from them in 48% yield (see Scheme 14). Lack of reaction of ~-glucono1,5-lac(62) L.Somshk and E. Tarcsa, unpublished results.

63

RADICALMEDIATED BROMINATIONS

CH,OBz

CH,OBz

I

I

OB z

OBz

89

90 SCHEME 14

tone tetrabenzoate under photobromination conditions62precludes it as an intermediate in the formation of compound 90, which most probably arose by loss of hydrogen bromide from an initial 1-bromide,followed by bromine addition to the substituted glycal, and collapse of the anomeric bromomethoxy system. The (S) configuration was tentatively assigned to C-2 on the basis that the brominated lactone is slightly more levorotatory than is tetraO-benzoyl-D-glucono-I ,5-lact0ne.~~ In an attempt to divert reaction from C-1 to C-5 in compounds of this category, phenyl tetra-O-acetyl- and -0-benzoyl-PD-glucosidewere examined, and both gave the respective 5-bromides (91 and 92) in modest yield after chromatography.Thus, it appears that the phenoxyl group is less effecCH,OR~ t

OR2 91 92

(R1

=

H,

(R1 = H, 93 (R' = NO,,

R2

= Ac)

R2 = Bz) R2 = A c )

(63) R. J. Femer and P.C. Tyler, J. Chem. Soc., Perkin Trans. 1, (1980) 2762-2766.

LASZL6 SOMSAK AND ROBERT J. F E W E R

64

tive than is the methoxyl group in stabilizing radicals at the anomeric center, and is less able to promote reaction at this site. It follows that electron-withdrawing groups on the benzene ring should be more effective in diverting reaction towards C-5, and the only result available suggeststhat this may be so: the yield of the 5-bromide 93 went to 57% (chromatographicseparation) when pnitrophenyl tetra-0-acetyl-P-D-ghcopyranosidewas photobrominated.63 Glycosides having readily substituted groups within their aglycons react selectively at these sites. Benzyl compounds thus give a-bromobenzylproducts which react further to afford glycosyl halide analog^,^^*^ and alkyl groups bonded to the aromatic rings of aryl glycoside esters are converted into a-bromoalkyl gr0ups.6~ 10. Phenyl 1-ThiohexopyranosideEsters

Very efficient reaction occurs when phenyl 1-thio-P-D-glucopyranoside and -D-galactopyranoside tetrabenzoates (94 and 95)are treated in refluxing carbon tetrachloride with N-bromosuccinimide under a heat lamp, and, within 15 minutes, they are converted into products from which the enones 96 and 97 have been obtained by direct crystallizationin 76 and 83%yield, respectively (seeScheme 15).20

94

(D-glUCO-)

95

(o-galacto-)

96

(D-erythro-)

97

(D-threo-)

SCHEME15

Although there is no direct evidence on the point, the reactions leading to the enones 96 and 97 are no doubt initiated by hydrogen abstraction from C- 1, the resultant radicals being particularly stabilized by the sulfur atoms. (64)

H.Hashimoto, M.Kawa, Y.Saito, T.Date, S. Horito, and J. Yoshimura, Tefruhedron

Lett., 28 (1987) 3505-3508. (65) B. Helferich and K. H.Rullmann, J. Prakt. Chem., 1 1 (1960) 233-238.

RADICALMEDIATED BROMINATIONS

65

Consistent with this concept are the observations that phenyl 14hio-a-Dglucopyranoside tetrabenzoate, having H- 1 equatorial, also gives the enone 96 (7 1% isolated directly) on photobromination, and that the reaction is appreciably slower than is that of the anomer 94, having H-1axial (see Section 111,2). Bromine substitution at the anomeric center could be followed by loss of hydrogen bromide, to give 1,2-disubstituted glycals, and allylic hydrogen abstraction at C-3 could be the subsequent step leading to the products 96 and 97. Although the acetate analogs of compounds 94 and 95 give the related enones, as expected, the reactions of these esters are complicated by the concurrent formation of bromoacetates which cocrystallize with the triacetates, and the mixed crystals, obtainable directly in yields of 40%, contain 25% of the brominated products (see Section I). The reaction of phenyl 1-thio-a-D-glucopyranosidetetraacetate also gives the expected enone (2) and its 2-bromoacetyl analog (3) but, as in the benzoate series, the rate of reaction is considerably lowered. It was envisaged that selectivebromination of the acetoxyl group at C-2 occurs during the photobromination by way of a 1,2-acetoxonium ion or a bicyclic radical analogm (see Section I1,l).

-

11. Miscellaneous Compounds

Carbohydrate analogs that have been produced by direct photobromination reactions are the nucleoside derivative 1,N-dibenzoyl-2’,3’,5’-tri-Oben~oyl-4’-brornoadenosine~~ (98) (compare Section II,3b) and the cyclohexane derivative (lS,2S,3R,4S,5S,7R)-l-acetoxy-2,3,4-tetra-O-be.nzoyloxy-7-bromo-6-oxabicyclo[3.2. l]octaneM(99) (compare Section II,4a). NBz

BzO

OBz

98 (66) R. Blattner and R. J. Femer, Curbohydr.Res.. 150 (1986) 151 - 162.

99

'f

FI1,OR

&

CH,OBz

Bz

OBz

BzO

R

0, /o

BzO

CNez

50

Bz

51

Ac

Bz 69 AcOCH,

73

78

R~OCH,

AcOCH2

" Ac

Ac Ac

S

F OAc

R

z

S

o

O

R

1

ORz

(gB2 (gW (y RQ.1. -Representative compounds which, on photobmmination, afford isolable products with bromine atoms in place of the identifiedhydrogen atoms. [(a) R' H,OMe,OAc, R2= Me@, /I anomem much mom reacthq same products are formed fiom C-S epimq (b) R Ac,Bz;j? anomem much mom reactive;same producta are formed from CS epimm, (c) mixed C-5 epimeric bromides are form&, (d) mixed C-4 epimeric bromides are forme, (e) R Ac,& confisurationrrat G2-C4 may (f) X 0,NOBz; (g)reect at C-1and CS; the a anomers react much less d y and give no products ofCl bromination; (h) R' H,NOz ;R2 AC,BZ].

-

-

-

RADICALMEDIATED BROMINATIONS

61

111. THEREGIO- AND STEREO-CHEMISTRY OF THE REACTIONS

In Section 11, the results obtained on radical bromination of many carbohydrate derivativeswere described without considerationof the factors upon which the substitution processes were dependent; these are summarised in Figure 1 by indicating representative vulnerable compounds which give isolable bromoderivatives and, within their structures, the most readily replaceable hydrogen atoms. No systematic studies involving thorough analysis of the reaction variables and the mixed products formed have been carried out in order to enable an assessmentof these factors, and the conclusions that may be drawn come largely from data relating to the formation of single bromo-products from particular substrates- often,but not always, with good efficiency.From this rather weak base, it is neverthelesspossible to identifjl the factors that dominate in the determination of the general reactivity of compounds and of the main products of bromine radical substitution, and thereby to make predictions as to the possible susceptibilityof any single compound and the nature of its main photobromination product. 1. The Regiochemistry of the Reactions

Given the presence of stable protecting groups on the hydroxyl functions of cyclic carbohydrate derivatives, photobrominations occur at the ether ring positions with facilities which depend upon the relative rates of hydrogen abstractions from the available sites6' (see Scheme 16). These,in turn,

'Br

are dependent on two closely related factors the influences of which are clearly apparent in the results reported in W o n 11, that is, the relative ease with which the susceptible hydrogen atoms (on each of the carbon atoms adjacent to the ring-oxygen atoms) may be abstracted, and, secondly, the (67) M.L Poutsma, in J. K.Kochi (Ed.)FreeRadicaLr.Wiley, New York, 1973, Vol. II,pp. 164-169.

68

L h Z L 6 SOWk AND ROBERT J. FEEUUER

stabilizing effects of the relevant substituents (X)on the developing free-radial intermediates. In addition, the kinetic stabilization of these intermediates by large (particularly axial) ring-substituentsmay be a factor in particular cases. a. Hydrogen Atom Abstraction.-Homolytic carbon- hydrogen bond cleavage is facilitated by adjacent heteroatoms, particularly when they provide coplanar electron lone-pairs for stabilization of the developing radical intermediates.In tetrahydrofuran and tetrahydropyran, therefore, hydrogen abstraction occursat the ether carbon atoms,3' and all of the bromine substitution reactions recorded in Section I1 occur likewise at C-1 or C-5 for pyranoid derivatives, and at C-1 or C-4 for furanoid derivatives, showing that the oxygen-bonded substituents elsewhere on the rings do not permit competitiveradical formation. On the grounds that, in most cases reported, the hydroxyl groups of the parent carbohydratescarry acyl substituents, it may be tentativelyconcludedat this stage that acyloxy groups are not, at least in comparison with the ring-oxygenatoms, potent radical-stabilizingcarbon substituents. This important point, and others relating to radical stabilization at particular centers, are developed in the next Section. The regiochemistry of the products is also dependent on the stedc availability of the susceptible hydrogen atoms at the centers adjacent to the ring-oxygen atoms and, for example, D-@UCOpyranOSylcompounds with propensity to react by hydrogen abstraction at C-5 are appreciably stabilized when the anomeric substituents are axial (a anomers). For this reason, methyl t e t r a - O - a c e t y l - c u - D - @ u c o p ~ ~and o ~ ~ penta-0-acetyl- and penta-~benzoyl+x-D-glumpyranose are much less reactive than are their &uubgs and are poor substrates generally. This factor can be turned to advantage and used to divert reaction from a-D-ghcopyranosyl units in complex compounds: @-maltoseoctaacetate therefore reacts selectively at C-5 of the reducing residue, to give compound 30, and the 6-bromo derivative 48 can be produced directly in moderate yield, the anhydro-ring methylene group being Considerably more reactive than either C-1 or C-5 of the tetra-O-acetyl~-D-glucopyranosyl unit. A further stereochemical factor (discussed next) that has bearing on the regiochemistry of some photobrominations is the relative difficulty with which equatorial hydrogen atoms on pyranoid rings may be abstracted. In the cases of the t&%acylated 1,6-anhydrohexopyran~d o n is diverted to G6,becausethe anhydrobridge holds the pyranoid ring in the lC, conformation, in which H-1 and H-5are both equatorial.

b. Radical Intermediate Stabilization. -a-Hetero-atoms stabilize carbon fiee-radicals? and, for this reason,all products reportedfiom radical

RADICALMEDIATED BROMINATIONS

69

brominations of cyclic carbohydrate derivatives (see Section 11) have bromine atoms at carbon atoms bonded to the ring-oxygen atoms (illustrating, in the case of esterified compounds, that acyloxy groups do not stabilize radicals as efficiently as do the ether-linked ring-oxygen atoms; see later). The many carbohydrate compounds to have been examined (see Section 11) clearly raise the interesting question of the combined effects of the ringoxygen atoms and of the other substituentson radicals formed at C- 1 and at C-4 and C-5 for furanoid and pyranoid compounds, respectively. The several acetylatedP-D-glucopyranosylderivatives(100)to have been studied are particularly suitable compounds for examination in this regard, since, because of their near-symmetry, the effectsof the anomeric substituent (X)in respect of its ability to facilitate photobromination at C- 1 can be compared closely with the effect of the substituent (CH,OAc) at C-5. It is fully recognized that thermodynamic stabilization of carbon radicals is provided by both electron-donating and electron-accepting bonded groups,23as can be accounted for on the basis of delocalization.

Frontier orbital theory leads to the conclusion that interaction between the unperturbed radical orbital (SOMO) and the LUMO orbital of an acceptor results in lowering the energy ofthe singly occupied orbital of the substituted radical, to give a more-electrophilic species, while, in the case of electrondonating substituents,three electron transitions are involved, to give morenucleophilic species, with the singly occupied y2being of higher energy than the initial SOMO level. Furthermore, particularly effective thermodynamic stabilization of carbon radicals occurs in the “captodative” situation, that is, when electrondonating are involved together with electron-withdrawinggroups to provide a high degree of delocali~ation.~~

This factor is shown to be very important in several of the examplesgiven in Section 11: the compounds described in Section II,2 showing enhanced activity at C-5, those in II,6 enhanced activity at C-1, and those in 11,5, enhanced activity at C- 1 or C-5, according to whether the carbonyl or oximo groups are at C-2 or C-4 of pyranoid derivatives. Captodativeradical stabili-

70

LASZLO SOMSAK AND ROBERT J. FERRIER TABLE111 Relative Radical Stabilization Factors (RRSA for Radicals at Positions Adjacent to Ring Oxygen Atoms of Cyclic Carbohydrate Derivatives’

F H OAc CH3(CH,0AcY c1 OMe

- 1.4

0 0.9 2.3 2.4 4.5

3.3 4.5 5.3 6.5 6.6 8.3

OPh C0,Me CN COMe SPh

4.9 7.9 8.6 10.2 10.7

8.7 11.3 11.9 13.2 13.7

”Xrepresents substituentsat the anomeric Center or at C-5 ofpyranoidorC4 of furanoid compounds; 0 represents the ring-oxygen atoms. Relative radical stabilizationfactors for the X suhstituentsare given (RRS,), and the factors for the combined effects of 0 and X (RR&,,) were calculated from the expression 0.03RRS0,x = I-( I-0.03RRSoM,) 1-0.03RR&).’O bRR&na is taken as RR&,.
zation has been the subject of much experimental and theoretical work, although it is not accepted as being universally ~ a l i d . ~ * , ~ ~ In Table 111, carbon radical stabilization factors are cited for a range of relevant a-substituent groups, and from them were calculated the corresponding factors for each in combination with oxygen atoms to give relative stabilizations to be expected at substituted positions adjacent to carbohydrate ring-oxygen atoms.’O These are also given in Table 111, and the results noted in Section I1 can now be examined in the light ofexpectationsbased on these values. From Table 111, it is to be expected that acetal centers having two donor atoms available to stabilize radicals are more prone to hydrogen abstraction than are such ether centers as C-5 of pyranoid derivatives. This is consistent with common experience and with the observation that methyl P-D-~~ucopyranoside tetrabenzoate reacts with bromine atoms preferentially at C- 1 (see Section 11,9).Contrary to expectations based on Table 111, however, the analogous phenyl glycoside ester appears (on the basis of incomplete evidence) to undergo more reactions at C-5, but, in this case, conceivably, reaction is diverted from C- 1 by the bulk of the aglycon and by the lessened ability of the unshared electron-pairs on 0-1 to stabilize electron density at C- 1 consequent upon some delocalization into the benzene ring. In the case (68) A. R. Katritzky, M. C. Zerner, and M. M. Karelson, J. Am. Chem. Soc., 108 (1986) 72 13-72 14. (69) D. J. Pasto, J. Am. Chem. SOC.,110 (1988) 8164-8175. (70) R. Merknyi, Z. Janousek, and H. G. Viehe, in Ref. 12. pp. 301 -324.

RADICALMEDIATED BROMINATIONS

71

of methyl P-D-glucopyranosiduronic esters with the strong stabilizinggroups (Table 111)at C-5, reaction is diverted to that position (see Section 11,2),and alternatively, sulfur has excellent electron-delocalizingcapacity, and, consequently, 1-thio-P-D-glucopyranosideesters react readily at the anomeric center (see Section I1,lO). The cases of the phenyl 1-thio-P-D-glucopyranuronoside esters (for example, 4) are noteworthy because they show that the captodative stabilization provided at C-5 is comparable to that afforded at the anomeric centers by the ring-oxygen atom and the phenylthio group. The case of the acylated hexopyranosyl halides (see Section II,8) is of particular significance, because only in this series has an indication been gained of the proportions of isomeric brominated products formed. From Table 111, it would be expected that tetra-0-acetyl-P-D-glucopyranosyl fluoride would generate a more-stable intermediate at C-5 than at C-1, and, consistent with this presumption, the respective bromofluorides were isolated in 56 and 49/0yield, respectively. Conversely, the corresponding glycosyl chloride, having a halogen atom with higher energy lone-pairs better able to stabilize C- 1 radicals than those of the fluorine atom, should react in the reverse manner (compare RRSa, RRS,,), and the C-5 and C- 1 bromides were obtained in 14 and 65% yield, respectively. For the acetoxyl group, the value in Table 111is only marginallyin keeping with the fact that P-D-glucopyranose peresters react with high selectivity at C-5, and the discovery that tetra-0-acetyl-P-D-xylopyranosealso reacts preferentially at C-5 suggested that the RRS,, value for acetoxyl should be negative, although the size of the group could have a bearing on the lack of reactivity at C-1. In some series of radical-stabilizingabilities of groups, the acetoxyl group is accorded a lower status, and the evidence from the present analysis suggests that its RRS factor should be negative. Substitution at C-1 or C-5 of pyranosyl compounds (or C-1 or C-4 of furanosyl compounds, although none seem to have been examined)by such electron-acceptinggroups as carbonyl or nitrile, or by introduction of a keto or related function at C-2 or C-4 of pyranosyl compounds (C-2 or C-3 of furanoids), provides anomeric or C-5 (C-4)radicals with “captodative” stabilizationZ3which, quantitatively (see Table III), can be expected to be very significant. In keeping with this hypothesis, all of the uronic acid derivatives, the glycosyl cyanides and pyranosid-2- and -4-ulose compounds that have been examined (see Section 11) react readily, and with high selectivity, at their respective captodatively stabilized radical centers. 2. The Stereochemistry of the Reactions

a. Hydrogen Atom Abstraction.-An important stereochemical factor governs hydrogen-atom abstraction from C-1 or C-5 of pyranoid compounds; it is well established that substituted tetrahydropyranshaving equa-

L A S Z L ~SOMSAK AND ROBERT J. FERRIER

72

torial substituents and an axial hydrogen atom at C-2 undergo radical abstraction of the latter 10- 16 times as readily as do their anomers having This phenomenon can be accounted for by equatorial C-H bonds.37*71*72 invoking the anomenc effect, that is, the facilitation,by the axial non-bonding electron-pair of the ring-oxygen atom, of the breaking of the antiperiplanar carbon - hydrogen bond (101), and relevant results reported in Section I1 are consistent with this generalization: acylated glycosyl cyanides (see Section 11,6), halides (11,8),glycosides (11,9), and phenyl 1-thioglycosides (11,lO)having equatorial C-1-H bonds (for example, a-D-glucopyranosyl compounds) all react by hydrogen abstraction at C-1 appreciably more slowly than do their counterparts having axial hydrogen atoms.

OAc

100

101

102

b. Conformations of the Radical Intermediates.-Radicals at pyranoid anomeric centers may be considered in the first approximation to be axial a-speciesthat are stabilized by interaction between the axial lone pairs of the ring-oxygen atoms and the half-filled orbitals at C-1 (102). Giese and his coworkershave, however, shown by e.s.r. spectroscopicmethods that substituted D-glucopyranosyl radicals, which have been studied extensively, do not adopt the ‘C, conformation but are distorted into (approximately)the 2B5shape (103), as this is further stabilized by having the C-2-0 bond also coplanar with the single-electronorbital. Their e m . studies also led them to conclude that the tetra-0-acetyl-D-glucopyranosylradical is planar and a in ~ h a r a c t e r . D-Mannopyranosyl ~~.~~ radicals (104), which have an axial C-2 0 bond in the undisturbed ‘C, conformation, do not require such distortion in order to achieve this further stability, and are particularly readily formed, tetra-0-acetyl-a-D-mannopyranosyl chloride undergoing chlorine-atom loss 7.8 times faster than does the a-mglucopyranosyl isomer. In related fashion, C-5 radicals would be expected to have energy minima with C-4-0 (71) K. Hayday and R. D. McKelvey, J. Org. Chem., 41 (1976) 2222-2223. (72) R.D. McKelvey and H. Iwamura,J. Org. Chem., 50 (1985) 402-404. (73) J. Dupuis, B. Giese, D. RUegge, H. Fischer, H.-G. Korth, and R. Sustmann,Angew. Chem., Int. Ed. Engl,, 23 (1984) 896-898. (74) H.G.Korth, R.Sustmann,J. Dupuis,and B.Giese,J. Chem.Soc.,Perkin Truns.2, (1986) 1453- 1459.

RADICALMEDIATED BROMINATIONS

73

bonds axial, that is, the ~-glucopyranose-5-yland D-galactopyranose-5-yl specieswould favor the B, (105) and (106)forms, respectively,and e.s.r. data on the former are consistent with this e ~ p e c t a t i o n . ~ ~

,

OR 104

103

RO

X

&OR

OR

R

o

bOR

R

RO 105

106

c. The Stereochemistry of the Products. -Although carbon radicals at C-2, C-3, and C-4 of glucopyranosederivatives react preferentiallywith, for example, acrylonitrile,to give equatorially substitutedadducts [but this does not apply to isomers with (particularly)two axial substituents in the p-relationship to the radical centers76],it is well establishedthat oxygen-stabilized species at the anomeric center (and at C-5) exhibit a strong tendency for reaction in the axial mode. In this way, anomeric radicals obtained from such compounds as tetra-0-acetyl-a-D-glucopyranosylbromide (23),on treatment with tributylstannane in the presence of a radical initiator, add to acrylonitrile to give the a-linked C-glycosyl compound 3-(tetra-O-acetyl-aD-glucopyranosy1)propanonitrile(107) with good selectivity (see Scheme 17).6,77In related fashion, radical desulfurization of the thio-orthoester de(75) H.G. Korth, R. Sustmann, K. S. Grihinger, M. Leisung, and B. Giese, J. Org. Chem., 53 (1988)4364-4369. Thispaperdescribesac-5 radicalashavingbeenderivedfrommethyl tetra-O-acetyl-5-bromo-cu-D-glucopyranoside. In fact, the precursor was penta-0-acetyl5-bromo-&~-glucopyranose (Professor B. Giese, personal communication). (76) B. Giese,Angew. Chern.,Inl. Ed. Engl., 28 (1989) 969-980. (77) B. Giese, J. Dupuis, M. Leising, M. Nix, and H. J. Lindner, Curbohydr.Res., 171 (1987) 329- 341.

L A S Z L ~ SOMSAK AND ROBERT J. FERRIER

74

'"VH CHIOAc

CH,OAc Bu3SnH AlBN AcO

AcO

CH2-CHCN

AcO

Br

OAc

107

23 SCHEME 17

rivatives 108, which are obtainable from the corresponding substituted aldono- l ,5-lactone and hence the thionolactone, give, with the same reagent, good yields of the products derived from the radicals 109 which abstract hydrogen from the reagent by the axial mode and thus provide a novel approach to the synthesis of a range of ~-D-glucopyranosides(110) (see Scheme 18). The stereoselectivityof the reaction of the radical intermediates with hydrogen atoms is78 10:1.

-

CHzOBn '

" BnO

~

Cf20Bn S

M

OR

e BulSnH AlBN

Brio

r m o CH,OBn

BnO

OR

BnO

108

109 SCHEME 18

110

Anomeric radicals (and, it may be assumed, radicals at C-5 of pyranoid compounds) therefore exhibit a strong kinetic, anomeric effect, with the uncoupled electron expressing its reactivity with marked axial preference: photobrominationproducts therefore have the bromine atoms in the kinetically and thermodynamically favored axial orientation (see Section 11). The case of the products derived from tetra-0-acetyl-P-D-xylopyranose is particularly noteworthy, because this compound affords epimeric bromides at C-5. Reaction with N-bromosuccinimidegives the (S) and (R) products (28 and 29;see Scheme 6) i d 5the ratio of 3 :2, which is similar to the ratio of the 4C1and 'C,,conformations of the initial tetraacetate in solution,7* and although this does not prove that the axial hydrogen atoms on C-5 were abstracted from each of these conformations to give two axial radicals and, hence, the axial bromides, the findings are consistent with this possibility. (78) D. Kahne, D. Yang, J. J. Lim, R.Miller, and E.Paguaga,J. Am. Chem. Soc.,110 (1988) 87 16-8717. (79) P.L.Durette and D. Horton, J. Org. Chem., 36 (1971) 2658-2669.

RADICALMEDIATED BROMINATIONS

15

It follows that epimers that differ stereochemically at the sites of photobromination give the same products and, thus, that penta-0-acetyl-P-Dglucopyranose (19) and its C-5 epimer, that is, the a-L-idopyranose ester, afford the same C-5 bromide (see Section II,3),” and that a common C-1 a-bromide is produced from the epimerictetra-0-acetyl-D-galactopyranosyl cyanides (see Section II,6).54 These products, and many others reported in Section 11, are the thermodynamicallyfavored epimers, and, although there is only scant evidence on the point, they are probably not formed after equilibration processes, but by kinetically controlled reactions involving preferential axial attack at radical centers which inherently react in this way and which, with bromine, are further encouraged to do so by the strong anomeric effect which develops en route from the radical to the transition state leading to the products. The recognition that epimers can give common radicals has been used to advantage in synthesis (see Section IVY1c). IV. REACTIONS OF THE BROMINE-CONTAINING PRODUCTS

Not enough data are available for the completeassessment ofthe chemical reactions undergone by the products of photobromination of carbohydrate derivatives, but the following observations permit an appreciation of the current state of knowledge of their chemistry and of the potential of these compounds as synthetic intermediates. 1. Substitution Reactions a. Substitution by Hydrogen.-There are only a few data available on the reactions undergone by 5-bromohexopyranoid compounds with lithium aluminum hydride, but it is now established that such reactions occur, at CH,OBZ

I

OBZ

111

OAc

112

LASZLC) SOMSAK A N D ROBERT J. FERRIER

76

least to a considerable degree, with inversion of configuration and thus provide, for example, access to compounds of the L series from more-common derivatives having the D configuration. When, for example, phenyl tetra-0-benzoyl-p-D-glucopyranoside (111) was subjected to photobromination, and the unfractionated products were reduced with lithium aluminum hydride and the products then acetylated, phenyl tetra-0-acetyl-cu-Lidopyranoside (112) was obtained, after chromatography, in 26% ~ield.6~ Although, quantitatively, this was less than satisfactory, the procedures are relatively simple to apply and the starting material is readily available. In" (115) related work, a yield of 46% of ethyl tetra-0-acetyl-a-L-idopyranoside was obtained from the 5-ChlOrOderivative 114, which was prepared from the photobromination product 113 as indicated in Scheme 19.26Reduction of CK2OBz

: ::iz:z'" ___)

3)

OBz

bEt CH,OBz

HeI, Ag20

CH,OBz

___, AcOH.HC1

OBz

1)___) LiAlH, 2) AC 20,Cg Hg N

OAc

OBZ

113

114

115

SCHEME19

1,5-anhydro-5-bromo-2,3-O-isopropyl~dene-~-~-~bose (50) with lithium aluminum hydride occurred with extremely poor selectivity,* but lithium triethylborodeuterideafforded a means of effectinga displacementwith very high stereoselectivity,and gave access to (5R)-~-(5-*H)ribose.~ Tributylstannane is also an effective, reductive debrominating reagent, but it is not a simple alternative to lithium aluminum hydride because, rather than effecting nucleophilic displacement of bromide, it requires the conjoint use of a radical initiator (usually azobis-isobutanonitrile, AIBN) and causes the formation of radical intermediates which, in the pyranoid series, would be expected to react with hydrogen atoms at axial sites (see Section 111,2c), In this way, 1- or 5-bromo-~-ghcopyranosylderivatives would be expected to be reduced largely with retention of configuration, an expectation strengthenedby a novel synthesis ofp-D-glucopyranosidesfrom alkoxy-G 1 radicals (see Section 1142~).When, however, the 5-bromouronates 116- 120 were treated with this reagent, they gave products from which D-glum and ~ - i d isomers o were isolated, as shown in Scheme 20.30 (80) H. Ohrui, T.Misawa, and H. Meguro, Agric. Biol. Chem., 49 (1985) 239-240.

RADICALMEDIATED BROMINATIONS

77

C02Me

CO Me I

I

OR2

OR2

OR

%

% -

R1

RZ

R3

R4

116

OAc

Ac

OAc

H

64

28

117

OMe

Ac

OAc

n

45

38

118

OHe

Me

OMe

H

42

35

119

OMe

Me

OAC

H

44

37

120

H

Ac

OAc

H

44

34

121

OAc

Ac

H

H

94(not isolated)

122

OAc

Ac

H

OAc

high

6

SCHEME20

Clearly, therefore, the anticipated high stereoselectivitydid not eventuate in these cases, but, when the reaction was camed out in the absence of a C-4 substituent(as in compound 121),the reaction became almost stereospecific in favor of the mglucuronic acid product, strongly suggesting that, with compounds 116 - 120, the tributylstannane was impeded from delivering the hydrogen atom to the a-side of the C-5 radicals bymthe substituentson C-4. Treatment of methyl 1,2,3,4-tetra-~-acety~-5-bromo-~-~-galacturonate (122) with tributylstannane likewise gave the D-galacturonate with extremely high ~electivity,)~ thus corroborating this conclusion and being consistent with the known anti-directing effects of /I-substituents situated on pyranoid rings.76Apparently, in the radicals derived from compounds 116120, which adopt conformations close to (105; see Section 111,2b), the substituent groups on C-4 inhibit the approach of the tributylstannane, but they did not similarly impede the generation of the precursor Sbromides, which are obtainable with high selectivity. Likewise, the substituentsat C-2 in the orthothioesters 108 did not impede the attack of tributylstannane from the axial &direction. The rationalization of the available data is, therefore, very complex, but the formation of the C-5 axial bromides may be highly selective, primarily because the products are thermodynamically controlled, and, conceivably, captodative radicals are more susceptible to

,&

LASZL~SOMSAK

78

AND ROBERT J. FEWER

stereochemical impedance by P-oxygen substituents than are alkoxy-bonded radicals. Consistent with this possibility is the observation that 1-cyano-a-Dgalactopyranosyl bromide derivatives are reduced under radical conditions to give thep-cyanides mainly, but with only poor selectivity.81In the D-mannose series, however, the p-nitrile was formed, as expected, exclusively.81* Radical reductive debromination of 1,5-anhydro-5-bromopentoseand 1,6-anhydro-6-bromohexopyranose derivatives has been of particular value, as it has given access to pentose and hexose compounds with stereospecific deuterium labelling at C-5 and C-6, respectively. In this way, for example, and by use of tributyltin deuteride, ( 6 9 - I ,6-anhydro-2,3,4-tri-O-benzoyl-5bromo-P-D-glucose (41) gave the (659 compound 123 and, hence, access to specifically labelled D-glucose, and the (R)-labelled form of 123 was made similarly from the C-6-deuterated bromide 41 by reduction with tributyl~tannane.~' Radical reductions of all 1,6-anhydro-2,3,4-tri-O-benzoyl-6bromo-D-hexopyranoses have been found to O C C U ~ ~ I with - ~ ~ high efficiencies and with stereoselectivities greater than 80% and often approaching 100%. In the 1,5-anhydropentose series, 1,5-anhydro-5-bromo-2,3-0-isopropylidene-P-D-ribose(SO) gave the ( 5 s ) and (5R)compounds 124 and 125 in the ratio of 17 :3 when treated with tributyltin deuteride in the presence of AIBN, and the ratio became 22 :3 on use of triphenyltin deuteride.* In this series, the presence ofa C-3 substituent in the cis-relationshipto the anhydro ring leads to stereospecific reduction, with the exclusive formation of (559 products when tributyltin deuteride is used. OAc

(-y I

tI

fw

BzO

I

OBz

123

0

0 CNe,

\ /

124 125

(K' (R'

-

-

,ti,

OAc

K~

H , KZ

- H) -

211)

126 127

(R (R

-

O A ~ )

11)

Ohrui and coworkers8*exploited the highly selectively deuterated sugars available from the bromo compounds by, for example, synthesizing a-and &( 1 +6)-linked hexose disaccharides with deuterium incorporated selectively within the methylene group involved in the interunit linkage, thereby (81) L. Somsik, G. Batta, and I. Farkas, Tetrahedron Left.,27 (1986) 5877-5880. @la) L. Som&, I. Bajza, and G. Batta, unpublished results. (82) H. Ohrui, Y.Nishida, M. Watanabe, H. Hori, and H. Meguro, Tetrahedron Lett., 26 (1985) 3251 -3254.

RADICALMEDIATED BROMINATIONS

79

allowing 'H n.m.r. means of studying rotamer states about the C - 5 4 - 6 bonds. They also used D-ribose specifically labelled at C-5 in a synthesis of specifically labelled, and therefore chiral, gly~ine.*~

b. Nucleophilic Substitutions by Nucleophiles other than Hydride. Nucleophilic displacement of the halogen atoms from the products of photobromination of carbohydrate derivatives is complicated by the concurrent formation of epimers and of products of elimination reactions. Few reactions have been studied more carefully than is required for the isolation and characterization of the main products, and only a rather incomplete appreciation of this set of chemical processes is available. One of the few efficient reactions to have been reported of 5-bromohexose derivatives is the acetolysis [mercury(II) acetate in acetic acid] of methyl tetra-U-acetyl-5-bromo-~-~-glucopyranuronate (lo), which affords the crystalline methyl 5-acetoxy-tetra-U-acetyl-a-~-idopyranuronate (126); this was assigned the inverted configuration at C-5 on the grounds of a major optical rotational change during its formation. Likewise, methyl tetra-0acetyl-/3-L-xylo-hexulopyranosonate(127) is obtained in excellent yield from the bromide 8, and it, too, is dextrorotatory and is produced from a levorotatory bromide, conceivably by way of a cyclic 4,5-acetoxonium ion. Compound 127 also differs in expected ways from its known C-5 epimer, and, on deprotection, affords L-ascorbic Methanolysis of penta-O-benzoyl-5-bromo-~-~-glucopyranos, using silver oxide and methanol, gives a complex set of products, but hydrolysis in the presence of this solid affords the 5-hydroxy analog 128 which, in aqueous media, equilibrates with the 5-ulose 129; this loses benzoic acid, and the resulting aldehyde recyclizes, to afford 2,3,4,6-tetra-O-benzoy1-5-hydroxyP-D-glucose (130) (see Scheme 21).26

128

129 SCHEME 21

130

Hydrolysis studies of (5S)-tetra-O-acetyl-5-bromo-~-~-xylopyranose (28) showed it to be much less susceptible than is tri-0-acetyl-cu-D-xylopyranosyl bromide, presumably because the acetoxyl group at C-1 inhibits the partici(83) H.Ohrui, T.Misawa, and H. Meguro, J. Org. Chem., 50 (1985) 3007-3009.

80

L A S Z L ~SOMSAK AND ROBERT J. FERRIER

pation ofthe unshared electronson the ring-oxygenatom in the ionization of the carbon- bromine bond. However, it does undergo hydrolysis, and also displacement of bromide by acetate, thioacetate, azide, and methoxy nucleophiles, to give compounds 131 - 134, which were isolated in 58, 23,26, and 91% yield, respectively, and extension of these studies led2’ to the morecomplex and unusual compounds 135- 138. X

OAc

X -

131 132 133 134

OAc

SAC

N, OMe

In the 4-bromofuranoid series, l-O-acetyl-2,3,5,6-tetra-O-benzoyl-4bromo-/h-ghcose (34)in admixture with the galucto epimer (33)is quickly hydrolyzed selectively (presumably because the trans-related benzoyloxy group at C-3 facilitates the displacement of the bromine atom), and this permits convenient isolation of the latter. On treatment with silver fluoride in acetonitrile, this D-gulactobromide givesthe D-glwo product 139 in good yield and, with silver tetrafluoroborate in diethyl ether containing boron trifluoride, also this compound and the C-4epimer3*in the ratio of 3 : 1. There was particular interest in the analogous reactions of 1-0-acetyl2,3,5-tri-O-benzoyl-4-bromo-~-~-ribose (37), because of the potential to prepare fiom it compounds related to nucleocidin, which is a 4’-fluoroadenosine derivative having antitrypanosomal activity. With silver fluoride in acetonitrile, this bromide also reacted mainly with inversion of configuration, and gave the L-I~XOfluoride 140 (53% isolated yield), whereas, with silver tetrafluoroborate, much more of the more interesting 1-0-acetyl2,3,4-tri-O-benzoyl-4-fluoro-~-~-ribose (141) was obtained.38

RADICAL-MEDIATED BROMINATIONS

Q7Qc AcO

AcO OAc

81

QH2o/c\

AcO OAc

OAc

135

136

OAc

OAc

137

138

Reaction of the photobromination product 40 of tri-O-acetyl-l,6anhydro-j?-rnglucopyranose under conditions of kinetic control gives mainly the endo-productsof displacementwith inversion of configuration;thus, potassium thiophenolate affords compound 142 in 73%yield, and methanolysis BzOI1,C I

OBz

BzO

OBz

140

BzO

OBz

LASZLO SOMSAK AND ROBERT J. FERRIER

82

gives the methyl acetal 143 and its epimer in the ratio of 9 : 1. When, however, the initial products may epimerize, the thermodynamically favored em-isomerspreponderate, and the acetoxy and iodo derivatives 144 and 145 are obtainable from the bromide 40 on treatment with acetic anhydride and boron trifluoride etherate, and with sodium iodide in refluxing acetone, respectively."

R13

AcO

I

I

OAc

R1

R2

40 142 143 144

Br H

H SPh

H

OMe

OAc

H

145

I

H

As shown in Section 11,5, substituted glyc-2-ulopyranosyl bromides and their oximes are obtainable by photobromination of the corresponding anhydroketose compounds having a methylene group at the pro-anomeric center; the bromoketones are also obtainable efficiently from 2-hydroxyglycal esters by treatment with N-bromosuccinimide and methanol in dichloromethane.*' As they are modified glycopyranosyl halides, these haloketones are subject to nucleophilic displacement at C-I, to give aldos-Zulosides which afford aldopyranosides on reduction. In this way, bromoketone (56) givesp-glycosides(146) which, on reduction with sodium borohydride, afford &D-mannopyranosides (147) with high stereoselectivity (see Scheme 22). Extensions of this approach using disaccharide glycosylatCH,OAc

CH,OAc

BzOQr

0

-Bzo&

0

54

146

147

SCHEME 22 (84) F. W. Lichtenthaler, E. Cuny, and S. Weprek, Angew. Chem., Int. Ed. Engl.. 22 (1983) 891 -892.

RADICALMEDIATED BROMINATIONS

83

ing agents have led to an ingenious synthesis of 0-P-D-galactopyranosyl( 1+4)-O-P-~-mannopyranosyl-(1-6)-~-galactose (from lactose)53and to

methyl 4-0-(2-acetam~do-~-deoxy-~-~-mannopy~nosyl)-~-~-~ucopyrano~ide.~~ Reaction of several acetylated 1-bromo-B-D-glycopyranosyl cyanideswith mercury(11) acetate in acetic acid-acetic anhydride gave the peracetyl P-Dald-2-ulopyranosyl nitriles (for example, compound 148)as the major produ c t ~From . ~ ~the precursor ofcompound 148, the thioglycosides149 and 150 were also obtained by nucleophilic displacement reactions.86 On heating in carbon tetrachloride with tetrabutylammonium bromide, compounds 74 and 75, that is, the products of photobromination of tetra-0-

CH20Ac

I

OAc

bAC

148

149 CH,QAc

CH 20Ac

I

I

OH

150

OAc

83

R'

R2

C1

Rr

151 F 152 F

CL

153

OMe

ONe

F

( 8 5 ) L. Sornsik, G. Batta, and I. Farkas,Curbohydr. Res., 132 (1984) 342-344. (86) L. Somsik, G. Batta, 1. Farkas,L. Piukinyi, A. Kglrnin, and A.Sornogyi,J. Chem. Res., (S) (1986) 436-437, (M)(1986) 3543-3566.

84

L A S Z L ~SOMSAK AND ROBERT J. FERRIER

acetyl-/3-D-gluco-and -galacto-pyranosylcyanide, respectively, undergo epimerization to the extent of 10 f 2%, from which it has been calculatedE7that the anomeric effect for the cyano-group is 7.5-9.7 kJ.mol-'. Selectivereaction of the bromochloro compound 83with silver fluoride in acetonitrile gave a good yield of the product of nucleophilicdisplacement of bromide with inversion of configuration, namely, 151, and treatment with an excess of the reagent affordedz5the gemdifluoride (152). With alcoholsin the presence of silver tnflate, compound 83 affords access to orthoesters (for example, the dimethyl compound 153), and with diols spiro-analogs (for example, 154, which has the orthoester structural feature of the orthosomycins), are obtained.EE

154

c. Radical Reactions h d i n g to Substitutions.-As has been described in Section III,2c, acylated hexopyranosyl bromides on treatment with tributyltin hydride in the presence of a radical initiator afford glycosyl radicals which add to electron-deficientdouble bonds to give, with good stereoselectivity, axial C-glycosyl compounds which may be regarded as products of substitution of the bromine. However, treatment in this manner of the tetra-U-acetyl-5-bromo-~-~-xy~opyranoses (28 and 29)with acrylonitrileafforded similar proportions of the mixed epimers 155 and 156 which, in keeping with expectations based on experience with penta-0-acetyl-/3-D-glucopyranose and its 5-epimer (see Section II,3a), both gave the same compound 157 on photobromination. This was selectively reduced back to the expected Dgluco compound having axial H-5 by treatment with tributyltin hydride,

(87) L. Som& and M.Szab6, J. Carbohydr. Chem., 9 (1990) 755-759. (88) J. P.Praly, L. Brard, and G. Descotes, Tetrahedron Lett., 29 (1988) 265 1-2654.

RADICALMEDIATED BROMINATIONS

85

OAc

155

OAc

A

AcO c

O

28,29

7

&

AcO AcO

h OAc OAc

157

Bu3SnH

OAc

OAc

156 SCHEME 23

and, in this way, compound 156 was obtained with good efficiency (see Scheme 23).89 2. Elimination Reactions

In accord with the reactions undergone by, for example, tetra-O-acety1-aD-glucopyranosylbromide (23)with bases, and with zinc -acetic acid, which give tetra-0-acetyl-1,5-anhydro-~-arabino-hex1-enitol (“tetra-O-acetyl-2hydroxy-D-glucal”)(158) and tn-0-acetyl- 1,5-anhydro-2-deoxy-~-arabinohex- 1-enitol (“tri-0-acetyl-D-glucal”) (159), respectively (see Scheme 24), CIIZOAc

AcO OAc

158

OAc

23

159

SCHEME 24

(89) R. Blattner, R. J. Femer, and R. Renner, J. Chern.SOC.,Chern. Comrnun.,(1987) 10071008.

AcO W

m

OAc

160

OAc

10

OAc

OAc

161 SCHEME25

9

OAc

11

RADICALMEDIATED BROMINATIONS

87

the bromine-containingproducts of photobromination lose the elements of hydrogen bromide and of acetyl hypobromite when treated with these reagents. Thus, the 5-bromouronate compound 10 gives compounds 160 and 161 as main products, 53% and 62% of each, respectively, having been isolated following separate reaction with 1,5-diazabicyclo[5.4.01undec-5ene and with zinc-acetic acid (see Scheme 25).w The Q! anomer of the benzoylated analog of compound 10 gives a similar yield of the anomer of benzoyl analog alkene 161 with the latter reagent,gDand closer examination of the conversion 10- 161 has shown that this main product is accompanied by methyl tetra-0-acetyl-P-D-glucopyranuronate(9) and the isomeric L-iduronate (1 l),the three products being formed in the ratios 83 :9 :8 ('H n.m.r. analysis). Analogously, the D-gulactoisomer of compound 10 affords the alkene 161 and the P-D-galacto- and a-L-altro- products of reductive debromination in3*the ratios 67 : 13 :20. In similar fashion, (5s)- 1,2,3,4-tetra-~-acetyl-5-brom0-/3-~-xylopyranose (28) gave2' the alkenes 162 and 163 as major products on treatment with DBU and zinc-acetic acid, respectively (see Scheme 26).

qoAc4 q +qQ OAC

AcO ACO

OAc

QAc

162

28

OAc

163

SCHEME 26

The situation is more complicated in the case of 5-bromohexopyranose derivatives, because products of both endo- and exo-elimination may be formed, and available evidence indicates that, with 5-bromo-P-~-glucopyranose esters, base-catalyzed elimination favors the production of endo-alkenes following loss of axial hydrogen and bromine atoms. Alternatively, treatment with zinc - acetic acid gives, mainly, the products of exo-elimination.w From the acetate 164 (R = Ac) and the benzoate 164 (R = Bz), the 4-enes (165, R = Ac, Bz) were both obtained in 65% yield following treatment with 1,5-diazabicyclo[5.4.0]undec-5-ene, whereas zinc - acetic acid afforded the 5-enes (166, R = Ac, Bz) in 59 and 67%yield. The isomeric endo products 167 (R = Ac, Bz) were isolated in 15 and 1 1% yield, and, from the (90) R. Blattner, R. J. Femer, and P. C. Tyler, J. Chem. Soc.,Perkin Trans. 1, (1980) 15351539.

88

LASZLO SOMSAK A N D ROBERT J. FERRIER ROCH2

CH2GR

I

Q

RG

RoQ

OR

165 R05H2

OR

164

RGCH

+

+

OR

OR

167

166

OR

168

SCHEME 27

reaction of the pentaacetate 164 (R = Ac), small proportions of the alkene (168, R = Ac), derived by exo-elimination of hydrogen bromide, were also obtained (see Scheme 27).33*90 When treated with sodium cyanide, sodium benzoate, or cesium fluoride in N,N-dimethylformamide, compound 164 (R = Bz) did not undergo substitution of the bromine, but again gave the product of endo loss of hydrogen bromide (165, R = Bz). However, with sodium thioacetate in this solvent,or sodium iodide in refluxing acetone, the main product, which could only be isolated in low yields, was the isomer 168 (R = Bz), formed by loss of hydrogen bromide in the ex0 sense.g0Perhaps, it was speculated, these stronger nucleophiles did effect substitution, conceivably with inversion of configuration, to give unstable products which underwent exo-elimination. Because 6-deoxyhex-5-enopyranosederivatives can be readily converted into 2-deoxyinoso~es,~~ 5-bromides (for example, 164) give useful access to such carbocyclic compound^.^^ In this way, octa-0-acetyl-P-maltose was converted by treatment with zinc-acetic acid into the 5-ene by way of the corresponding 5-bromide 30 and thence into the pseudo-disaccharide compound (169) which is related to componentsof members of the aminoglycoside antibiotic series.% (91) R. J. Fenier, J. Chem. Soc., Perkin Trans. 1, (1979) 1455- 1458. (92) R. Blattner, R. J. Femer, and S. R. Haines, J. Chem. Soc., Perkin Trans. I , (1985) 2413-2416.

89

RADICALMEDIATED BROMINATIONS

CH,OAc

Mixed bromides 33, 34 formed from either 1-0-acetyl-2,3,5,6-tetra-Obenzoyl-P-D-glucose or -galactose, also undergo preferential endu-elimination on treatment with DBU, and give the alkene (170).As in the pyranose series, exo-eliminationis favored with use ofzinc- acetic acid, and geometric isomers (171) are the main products and are formed93together with the 3-deoxy-3-ene (172). OAc

BzOH

c -c

B z O A z C ~ - OBz

BzO

Bzo

OBz

170

OBz

171

OBz

172

Acetylated 1-bromoglycopyranosyl cyanides, treated with DBU give, as expected,products of the loss of hydrogen bromide. In this way, for example, the D-gluco-compound 74 affords the fully substituted glycal 173, but the elimination was effected more satisfactorily by use of mercury(11) cyanide and catalytic silver tosylate. On the other hand, the “cyanoglycal” 174 was obtained in excellent yield by use of zinc and refluxing benzene in the presence of triethylamine or pyridine (see Scheme 28), while the more generally used zinc - acetic acid gave poor conversion, because of the concurrent production of the epimeric glycosyl cyanide^.^^.^^ Treatment of tetra-O-acetyl-1-bromo-P-D-glucopyranosylchloride (84) with 1,4-diazabicycl0[2.2.2]octanecausedZSpreferential dehydrobromination and the production of 2,3,4,6-tetra-O-acetyl-~-arahu-hex1-enopyranosyl chloride (175) (see Scheme 29). (93) R. J. Femer and S. R. Haines, J. Chem. SOC.,Perkin Trans. 1, (1984) 1689- 1692. (94) L. S o m a , Curbohydr. Res., 195 (1990) CI -C2.

a S Z L 0 SOMSAK AND ROBERT J. FERRIER

90

I

OAc

OAc

173

74

174

SCHEME 28

CII,OAc

Q:-

AcO

l

c

+

-

)

o

c

CH,OAc

A

OAc OAc

83

175 SCHEME 29

&Po -;,t)-. CH,OBz

CH2013z

BzO

OBz

OBz

90

176 SCHEME30

RADICALMEDIATED BROMINATIONS

91

Reaction of the bromolactone 90, the product of photobromination of methyl tetra-0-benzoyl-P-D-glucopyranoside, with sodium iodide in acetone affords632,4,6-tri-0-benzoyl-3-deoxy-~-erythro-hex-2-enono1,5-lactone (176) in good yield (see Scheme 30). V. CONCLUSIONS

Radical-mediated bromination reactions of carbohydrate compounds are, in appropriate circumstances, selective and efficient processes, but several criteria must be met for these advantages to be claimed: appropriate substituent groups must be used, and various electronic and stereochemical needs must be satisfied. At present, only a limited range of compounds necessary to define these needs has been examined, and many important carbohydrate classes, such as the nucleosides and nucleotides, glycosylamines, oligo- and poly-saccharides, furanoid compounds, and compounds having heteroatoms other than oxygen in the ring remain largely unexplored. The field is rich in offering opportunities for the study of the fundamental aspects of radical reactions, in particular the electronic and steric influences of radical substituent groups. Because bromine within organic compounds is subject to heterolytic elimination and substitution reactions and also to homolytic cleavage, the bromides obtained by the procedures described in this article offer much scope for developments in synthetic chemistry. VI. ADDENDUM Mild photobromination proceduresg5cause benzyl glycosides to be converted into the corresponding glycosyl bromides. The bromoketone 56 reacts to give an extensive range of glycosylated compounds with good selectivityg6and the 1-bromo-P-D-glucopyranosyl chloride 81 provides a means for synthesizing 1-azido-1-glycosyl =idesg7 and glycosyl orthoestem9* A further report has appeared on the conversion of glycosyl cyanobromides (such as 74) to fully substituted alkenes; mercury(I1) cyanide- silver (95) P. M. Collins, P. Premaratne, A. Manro, and A. Hussain, TetrahedronLett., 30 (1989) 4721 -4722. (96) F. W. Lichtenthaler, S. Schwidetzky, and K. Nakamura, Tetrahedron Lett., 31 (1990) 71 -74. (97) J.-P. Praly, Z. El Kharraf, and G . Descotes, J. Chem. SOC.Chem. Commun., (1991) 431 -432. (98) J.-P. Praly, Z. El Kharraf, P.-J. Comnger, L. Brard, and G . Descotes, Tetrahedron, 46 (1990) 65-75.

92

LASZLO SOMSAK AND ROBERT J. FERRIER

triflatein nitromethaneis particularlyeffective. Use of mercury(I1)acetate in dimethyl sulfoxidegave, on the other hand, the correspondingperacetylated aldonola~tones.~~ Whereas phenyl 1-thiohexopyranosideesters, on photobromination,give 1-en3-ones (Section 11, lo), the correspondingacetylatedPD-glumpyranosyl sulfoxides, on irradiation in carbon tetrachloride containing N-bromobromide. The succinimide, react to give tetra-0-acetyl-a-D-glucopyranosyl further oxidized sulfone, on the other hand, undergoes hydrogen substitution to afford the sulfones having axial bromine atoms at C-1 (48%)and C-5 (38%).loo Appropriate bromides, made by photobromination procedures, have been used as sources for the e m . study of acetylated 1-cyano- and 1-chlorohexopyranos-1-yl radicals and acetylated pentopyranos-5-yl radicals and their 5-acetoxymethyland 5-methoxycarbonyl analogues.1oi Photobromination of methyl (tri-0-acetyl-j?-D-glucopyranosyl fluoride)uronate, followed by radical reduction of the derived Sbromide, gave access to the corresponding glycosyl fluoride of the P-tido series.1o2 Substituted glycosyl azides give high yields of the corresponding N-bromoimidolactones under photobromination condition^.'^' The preparations and properties of some of the compounds noted in this chapter have been collected within a compendium on “C-radicals”.’04

(99) L. Somsik, E. Papp, G. Batta, and 1. Farkas, Carbohydr. Res., 21 1 (1991) 173- 178. (100) J.-P. Praly and G. Descotes, TetrahedronLett., 31 (1990) 1133- 1136. Korth, J.-P. Praiy, L. Somsik,andR. Sustmann, Chem. Ber., 123(1990) 1155(101) H.-G. 1160. (102) Ya. V. Voznyi, I. S. Kalicheva, A. A. Galoyan, and N. B. Gusina, Bioorg. Khim.. 15 (1989) 1411-1415. (103) J.-P. Praly, C. Di Stefano, L. Somsik, and G. Descotes, J. Carbohydr.Chem.,(1991) in PESS.

(104) J. 0.Metzger, in M. Regitz and B. Giese (Eds.) Methoden Org. Chem. (Houben- Weyo, Thieme, Stuttgart, 1989, Vol. E19a, pp. 340-346.

.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL 49

1 4 :3.6.DIANHYDROHEXITOLS

BY PETER STOSS*AND REINHARD HEMMER** Chemical Research and Development. Heinrich Mack Nachj; Chemical Pharmaceutical Company. 0-7918 Illertissen. Germany I. Introduction .......................................................... I1. Nomenclature., ....................................................... 1. Sugar-derivedNames ................................................ 2. BridgedSystems .................................................... 3. FusedSystems ...................................................... 111. SpectroscopicProperties. Structural Aspects, and Analytical Detection .......... 1. Spectroscopic Properties.............................................. 2. StructuralAspects ................................................... 3. Analytical Behavior, Detection. and Determination ....................... IV. Preparation of the Parent Compounds ..................................... V . Derivatives ........................................................... 1. Esters ............................................................. 2. Ethers ............................................................. 3. Deoxy Derivatives................................................... 4. OxidationProdu cts .................................................. VI. Applications .......................................................... 1. Chemical Uses...................................................... 2. Pharmaceutical Uses ................................................. 3. Technical Applications...............................................

93 96 96 97 98 99 99 114 117 119 125 125 135 146 155 158 158 161 167

I. INTRODUCTION More than a century has now elapsed since Fauconnier. in 1884. described 1.4 :3.6dianhydro-~.mannitolas the first member of the title series. Thiswas followedby a long period of silence. with only some sporadic papers

.

( I ) A Fauconnier. Bull . SOC. Chim. Fr., 41 (1884) 119-125 .

* Present address: Research and Development. EMS-DOTTIKON AG. CH-5605 Dottikon. Switzerland. nPresent address: Centre for Solar Energy and Hydrogen Research. Rittinghausstr. 2. D-79 I3 Senden. Germany. 93

Copynsbt b 1991 by Academic Press, Inc. AUrightsofrepmiuctiminanyform mewed.

94

PETER STOSS AND REINHARD HEMMER TABLE I Chemical Abstracts References on 1,4:3,6Dianhydrohexitols

Volume Coll. Vol. 8

9 10 11 Vol. 106/107 108/109 110

a

Period Covered

Number of references

1967-71 1972-76 1977-81 1982-86 1987 1988 1989 Total

58 82 154 406 94 94 49

937

Average per year

11.6 16.4 30.8 81.2 94 94 98"

Estimated.

on them. Then, a flood of experimental work appeared in the chemical literature during the 1940's and 1950's, when intensive investigations on all of the possible isomers of 1,4 :3,6-dianhydrohexitols were conducted, their structural properties ascertained, and the synthesis of numerous derivatives performed. Later, interest declined to a much lower level. However, an increasing number of contributions in subsequent years shows that considerable attention is again being directed towards this special class of carbohydrate-derived molecules. The sharp rise in the number of papers and patent applications is evident on inspection of the citations in Chemical Abstracts. For the period from 1967, when C.A. started their online file, until 1989, a nearly exponential trend in publications is obvious (see Table I). For comparison, the ten-year period from 1957 to 1966 contains only about 80 relevant C. A. references, which is an average of eight per year. In view of this, a new overview of developmentsin this area is warranted, especiallyas no special summary on this subject has been published for some time. A comprehensive article*in a previous volume of this series appeared in 1950. Subsequently,a brief discussion of 1,4 :3,6-dianhydrohexitolswas incorporated in a contribution on the stereochemistry of cyclic derivativesof carbohydrate^,^ and they were treated exhaustively within a summary of alditol anhydrides that contained tables of physical properties of derivative~.~ (2) L. F. Wiggins, A h . Carbohydr.Chem., 5 (1950) 191-228. (3) J. A. Mills, A&. Curbohydr. Chem., 10 (1955)1-53. (4)S. Soltzberg,Adv. Curbohydr. Chem. Biochem.,25 (1970)229-283.

1,4:3,6-DIANHYDROHEXITOU

95

A special survey of the preparation of 1,4 :3,6-dianhydro-~-glucitol and its derivatives was published in a Polish paperS in 1973. The outstanding importance of the D-glucitol isomer as compared with the dianhydrides of mannitol and iditol was further documented by two additional reviews dealing in particular with its preparation, properties, and application^.^.' Differences in the behavior of the exo- and endo-position of 1,4 :3,6-dianhydrohexitolswas mentioned in an article summarizingthe relative reactivities of hydroxyl groups in carbohydrates.*Finally, it should be pointed out that the compoundsunder consideration are also referred to in Rodd’s Chemistry of Carbon Compoundsgand in Beilstein S Handbook of Organic Chemistry.*O In addition to the ongoing activities in this area around the world, from both a scientificand practical viewpoint, another aspect merits emphasis: the 1,4 :3,6-dianhydrohexitols belong to the so-called “biomass-derived substances” obtainable from natural products, and are thus classified as “regenerable resources.’’ The following report links up with that of Soltzberg? and includes the literature from about 1969 to 1990. It comprises a period of nearly 20 years, and tries to cover major aspects of the chemistry of both the parent compounds and their derivatives,with referenceto their manifold applications in research and industry. Not covered by this article are papers dealing solely with pharmacology, toxicology, pharmacokinetics, pharmaceutical dosage formulations, and clinical investigations of isosorbide dinitrate and its mononitrate metabolites. These aspects, which are partially responsible for the fast-growing number of publications, would be better reserved for a separate survey having a more medicinally oriented point of view. The literature referred to in ChemiculAbstractsuntil Vol. 1 10(first halfof 1989) is completely covered, with the aforementioned exceptions. In addition, some subsequent original papers dating from 1988and 1989, for which no C. A. reference exists so far, are further recorded, as they were accessible to us. (5) W. Jasinski and S. Ropuszynski, Przegl. Nauk. Inst. Technol. Org. Tworzyw Szrucznych Politech. Wroclaw, 12 (1973)3-29;Chem. Abstr. 79 (1973)146,746. (6) F. Jaquet, A. Gaset, and J. P. Gorrichon, I n j Chim., 246 (1984)155- 158; Chem. Abstr., 101 (1984)73,012. (7)G . Fleche and M. Huchette, Staerke, 38 (1986)26-30. (8) A. H.Haines, Adv. Carbohydr. Chem. Biochem., 33 (1976)I 1 - 109. (9)L.Hough and A. C. Richardson, in S. Coffey (Ed.), Rodd’s Chemistry ofcarbon Compounds, 2nd edn., Vol. I,Part F,Elsevier, Amsterdam, 1967,pp. 1-64. (10) Beilstein, Handbook of Organic ChernistV#4th edn., Springer. Berlin, I , 540-541; ]/I, 284-285; 1/11, 611; 19/11,94; 19/111/1V,121-122,689-691, 989-999, 4185-4188, 4210-421 1; 19/3/V201-205.

PETER STOSS AND REINHARD HEMMER

96

11. NOMENCLATURE

1. Sugar-derived Names

The most widely used terms in this class of compounds are based on sugar nomenclature, which reflects their properties as polyhydric alcohols. The numbering of the ring system in this case retains that of the parent sugar. For glucose (1)as startingmaterial, itself obtainable from starch, carbon atoms 2 to 6 remain unchanged during hydrogenationto D-glucitol (sorbitol, ,r o

H ‘lc

I H*COH I

H03CH I H %OH

HO

HO

- 6P&j; H2

-2

H

H20 c

HO

OH

1

H5COH I

%H~OH

1

3

2

2), and dehydration of 2 then gives 1,4 :3,6-dianhydro-~-glucitol (3). As a trivial term for 2 is sorbitol, 3 is frequently (albeit incorrectly) called 1,4 :3,6-dianhydrosorbitol. The same numbering applies for both of the other diastereoisomeric 1,4:3,6-dianhydrohexitols, respectively derived from D-mannose by way of D-mannitol and from L-fructose by way of (4) L-iditol; these are consequently named 1,4 :3,6-dianhydro-~-mannitol and 1,4 :3,6-dianhydro-~-iditol(5). OH

HO

@ OH

4

& A :

OH 5

1,4 :3,6-DIANHYDROHEXITOU

97

In addition to the foregoing classification, compounds 3,4, and 5 have been frequently referred trivially to as “isosorbide” (3), “isomannide” (4), and “isoidide” (5), respectively.This kind of terminology is employed in the following treatment whenever accuracy will not suffer. In the following formulas throughout this article, the angular hydrogen atoms on C-3 and C-4are omitted for reasons of easier drawing, but are always arranged in the same exo-position in relation to the two-ring system, as depicted in formulas 3,4, and 5 exceptions are indicated separately. On the one hand, this type of description indicates the close relationship to sugar chemistry and seems appropriate where it is unequivocal. In addition, when using the carbohydrate nomenclature, the stereochemical features are already included within the names, and no additional terms are necessary in order to define them. For example, the name 1,4 :3,6-dianhydro-~-glucitol implementsthe configuration of all four c h i d centers of the molecule, and the cis-fusion of the two rings, as well as the position of the 2-substituent being ex0 and that of the 5-substituent being endo. Although, sugar nomenclature is generally preferred by carbohydrate specialists, the systematic “Geneva” rules of organic chemistry may also be employed. Thus, in cases where clarity might suffer, or the sugar-derived names become too sophisticated, one of the following alternatives may be advantageous. 2. Bridged Systems

As 1,4:3,6-dianhydrohexitolsare bicyclic systems, the appropriate ringindex nomenclature may also be used. The numbering of the atoms in such names differs from that used in the sugar-derived names. Furthermore, the stereochemistry for each of the anomeric centers has to be separately defined. The compounds under discussion have a 2,6-dioxabicylo[3.3.010~tane framework, shown in Formula 6, and incorporates hydroxyl groups on C-4and C-8.

6

According to this system, the diols 3,4, and 5 are respectivelynamed ( I R, 4R,5R,8S)-2,6-dioxabicyclo[3.3.0]octan-4,8-diol(3), (lR,4R,5R,8R)-

98

PETER STOSS AND REINHARD HEMMER

2,6-dioxabicyclo[3.3.0]octan-4,8-diol(4), and (1R,4S,5R,8S)-2,6-dioxabicyclo[3.3.0]octan-4,8-diol(5). Other possibilities, less exact but used in the literature, are cis-2,6-dioxabicyclo[3.3.010ctan-4-exo-8-endo-diol or 4-exo-hydroxy-8-endo-hydroxy-cis2,6-dioxabicyclo[3.3.0Joctanefor 3, and the appropriate modifications for 4 and 5. When the structures of derivatives of the parent compounds depart more and more from those of the original sugars, the bridged-system nomenclature may be advantageouslyapplied over the carbohydrateterms. Thiscould be the case when one, or both, of the OH groups on C-4 and C-8 is (are) absent, as in the deoxy series (see Section V,3), and also in the oxidation products (see Section V,4), where sugar-derived names become complex.

3. Fused Systems Apart from both of the aforementioned possibilities,and according to the constitution ofthe two fused tetrahydrofuran rings, this class may further be designated as anellated or fused-ringsystems. In this instance, a numbering different again from both of the foregoing is used; as demonstrated in formula 7. The stereochemistry of the ring fusion and that of the hydroxyl substituents,which here occupy positions 3 and 6, have again to be specified, along with the parent name. Thus, 3 would be designated (3R, 3aR, 6S, 6aR)-hexahydrofuro[3,2-b]furan-3,6-diol.

7

Interestingly, the Beilstein Handbook (Ref.’fOfused this type of labelling in the I11-1V supplement, while, for supplement V, a change to sugar terms is evident. Originally,the trivial names isosorbide, isomannide, and isoidide were used in the main volume, as well as in Supplements I and 11.It should, however, be pointed out that “Beilstein names” in supplement 111-IV are based on an older stereochemical reference system according to which 3 is (3uR)-(3ur, 6ac)-hexahydrofuro-[3,2-b]furan-3~,62-diol. ChemicalAbstracts prefers the sugar terms, especially in the trivial forms; however, in some instances, the bicyclo nomenclature is also applied. Surveying the whole original literature, preference is doubtless given to carbohydrate names with frequent use of the trivial isohexide terms, followed by the bridged-systems labeling. The fused systems names are not in vogue.

I ,4 :3,6-DIANHYDROHEXITOL-S

99

111. SPECTROSCOPIC PROPERTIES, STRUCTURAL ASPECTS, AND ANALYTICAL DETECTION 1. Spectroscopic Properties

a. Ultraviolet Spectra and Chiroptical Properties.-Because of the lack of a chromophoric group, normal isohexides do not exhibit characteristic absorption bands in the ultraviolet region. Except for chromophoricsubstituents (not discussed here), only for the monoketones having the carbonyl group in the 2-position (8) and the diketone (9), both derived from isosorbide, is an absorption band, at 267 nm (in water), observed.''

% 0

OH

9 8

Isohexide mono- (10- 13), as well as di-, nitrates (14-16) show a weak, positive dichroic band at 265 nm (n+D* transition)and a second, stronger one near 228 nm, which is positive for endo-R-nitrato and negative for em-S-nitrato groups. For isosorbide dinitrate (14), both dichroic bands are positive.'s13 HO

HO

b 1

OaN>

OH

11

10

b i

13

12

0,NO

HO

ON02

% % ONO,

bN02

15

14

ON02

( 1 1) A. Jacquet, R. Audinos, M. Delmas, and A. Gaset, Biomass, 6 (1985) 193-209. (12) R. E. Barton and L. D. Hayward, Cun.J. Chem., 50 (1972) 1719- 1728. (13) L. D.Hayward and S. Claesson, J. Chem. Soc.. Chem. Commun.. (1967) 302-304.

100

PETER STOSS A N D REINHARD HEMMER

The corresponding isohexide dinitrites in solution in acetonitrile give more-complexcircular dichroism spectra. Here, from the R configurationof the nitrito group, there is observed14a positive c.d. band: for isosorbide dinitrite at 322,331,337,342,348,356,369,385, and 398 nm, for isomannide dinitrite at 310,320,330,337,342,349,357,370, and 385 nm, and for isoidide dinitrite, at 337, 348,360,375, and 391 nm. In the presence of isosorbide, and of isomannide, as a chiral environment, optical rotation is induced in symmetric carbonyl and nitro compounds, where it can be detected as circular dichroism (here the n-D* transition) in the ultraviolet spectra of the appropriate solutions. The rotational strengths of the induced c.d. dependI5on the solvent, the temperature, and the concentration.

b. Infrared Spectra.-The infrared spectra of unsubstituted isohexides show only a few characteristicabsorption bands. Complete spectra have not been presented in the literature. The unsubstituted parent compound cis-2,6-dioxabicyclo[3.3.0]octane (6) was characterized,16without assignment, by five absorption bands at 1 1 10,1060, 1040, 1020,and 900 cm-l (in CC1,). The monoketone 8 and the diketone 9 are characterized" by a carbonyl stretching-vibrationband at 1765 cm-I (KBr and acetonitrile). The diazides of four isohexides, namely, those of D-isosorbide (17) (2110 cm-'), D-isomannide (18) (2110 cm-'), L-isomannide (19) (2120 cm-l), and L-isoidide(20)(2100cm-I ) differ slightlyl'in the position of their azido group vibration (KEir). The infrared spectra (benzene solution)of the isohexide nitrates, as well as of their mixed nitric and p-toluenesulfonic esters, are well established. The positions of the v, and the vsnn bands for the nitrato group are remarkably constant, at 1645 f 3 cm-I and 1282 f 1 (endo), and 1274 k 1 cm-I (14)L.D. Hayward and R. N. Totty, J. Chem. Soc., D, (1969) 997-998. (15) L. D. HaywardandR. N. Totty, Can.J. Chem., 49 (1971)624-631. (16) M. L. J. Mihailovic, S. Konstanntinovic, and S. Dokic-Mazinjanin, Glus. Hem. Drus., Eeogrud, 41 (1976) 281-285. (17) J. Kuszmann and G. Medgyes,Curbohydr. Res., 85 (1980) 259-269.

1,4:3,6-DIANHYDROHEXITOLS

18

19

101

20

17

(exo). The vNo band is observed, for all compounds so analyzed, at 843 f 3 cm-'. The ratios of these bands are 1.5 : 1.O: 1.O. No shift occurs on changing the solvent. The constant position ofthe vm vibration band can be used1*to decide whether the ex0 or endo position is filled. The two isomers (10 and 11) of isosorbide mononitrate may also be distinguished by the different strength of the hydrogen bridges from the 5-endo- or 2-exo-OH group to the oxygen atom in the opposite ring. The 2-exo-mononitrate (free 5-OH group) shows Av = v(OH),-v(OHLd = 148 cm-', whereas the Av for the corresponding 5-endo-mononitrate is onlyL982 cm-l (spectra recorded for 5 mM solutions in CCl,). The strong TABLEI1 Infrared Data for Isohexide Derivatives

NO* Free Bonded Asym. Sym. OH

Compound

3625 Isosorbide 2-0-acetyl2-0-acetyl-5-0-mesyl2-chloro-2deoxy2-O-acetyl-5-chloro-5-deoxy- 3688 2-nitrate 3688 5-nitrate 2J-dinitrate Isomannide 2(5)-0-acetyl2(5)-0-acetyl-5(2)-0-mesylIsoidide 2(5)-0-acetyl-5(2)-chloro5( 2)deoxy2(5)-chloro-2(5)deoxy-

3620

3562 3555

c1

-

-

3555

680-755 685-755

-

-

3540 3605

1645 1645 1650

-

Others

References

-

3560 3560 -

-

680 755 680 - 755

21 21 21 21 21 19 19 20 21 21 21 21 21

( 1 8) L. D. Hayward, D. J. Livingstone, M. Jackson, and V. M. Csizmadia, Can. J. Chem.. 45

(1967) 2191 -2194. (19) M. Anteunis, G. Verhegghe, and T. Rosseel, Org. Mugn. Reson., 3 (1971) 693-701.

PETER STOSS AND REINHARD HEMMER

102

sharp band of the nitric ester group in isosorbide 2,5-dinitrate (14), at - 1645 crn-l, is suitable for detection and quantitative determinationof this compound in pharmaceutical formulations.20 The carbonyl vibration, at 1735 cm-l, of 2-0-acetylisohexide derivatives turns out to be insensitive as regards other substituents in these molecules.21 Without assignment, the infrared data for D-isomannidemonooleatewere reportedz2 in a patent: 1020m, 1060m, 1080m, 1 120m, 1 170m, 1240m, 1470m, 1650vw, 1740s, 2850s, 2920s, 3000m and 3444m. All relevant infrared data are summarized in Table 11.

c. Nuclear Magnetic Resonance Spectra.- H-N.m.r. spectroscopicdata for the three unsubstituted i s o h e x i d e ~ , 2their ~ ~ ~ *diacetates," ~~ dimesylate^,^^,^^ diammoniumdideoxy salts,23dito~ylates,~~ dideoxydiazide~,~~,~~ and other a~ylates:~as well as some mixed 2,5-0-dis~bstituted~'*~~~~ and 2(3-0or 2(5)-deoxy monosubstituted isohexides,21have been reported. Fewer such compounds have thus far been characterized by 13C-n.m.r. spectroscopy.Thus, data were reported for isosorbide, 1,21*27isomannide, ls2* the three isohexide dirnesylate~,2'2~ the 2-deoxy-2-iodo 5-mesylate,2' the dideoxydiiodides of isosorbide and isoidide,21*m isomannide ditosylate, and bis(2,5-dideo~y-2,5-diphenylphosphino)isoidide,~~ of mixed substituted acetates, and of monochloromonodeoxy compounds.21 Most of these investigations dealt with the elucidation of stereochemical relationshipsof this ring system,17,19~21~24~26,29 the examination of substitution p a t h ~ a y s , ~and ' , ~the ~,~ complexation ~ behavior toward alkali-metal ions,26 some different ammonium i0ns,2~.~' and such lanthanide chelates217as Gd(dmp), and Eu(fod), . (i) 'H-N.m.r. Spectra.-It has been shown by 'H-n.m.r. spectroscopy that only the cis isomer of 2,6-dioxabicyclo[3.3.O]octane (6) is formed during the treatment of 2-tetrahydrofuraneethanolwith lead tetraacetate.l6 D. Woo, J. K. C. Yen, and P. Sofronas, Anal. Chem., 45 (1973) 2144-2145. J. C. Goodwin, J. E. Hodge, and D. Weisleder, Carbohydr. Res., 79 ( I 980) 133 - 14 1. R. J. Tull (Merck & Co., Inc.), DE 2,249,831 (1972); Chem. Absrr., 81 (1974) 13,752. J. Thiem and H. Lueders, Makromol. Chem., 187 (1986) 2775-2785. F. J. Hopton and G. H. S. Thomas, Can. J. Chem., 47 (1969) 2395-2401. J. Thiem and H. Lueders, Staerke, 36 (1984) 170- 176. J. C. Metcalfe, J. F. Stoddart,G. Jones, T. H. Crawshaw,A. Quick, and D. J. Williams, J. Chem. Soc., Chem. Commun.,(1981) 430-432. (27) J. A. Peters, W. M. M. J. Bovee, and A. P. G. Kieboom, Tetrahedron,40 (1984) 2885-

(20) (2 1) (22) (23) (24) (25) (26)

2891. (28) (29) (30) (3 1 )

J. Bakos, B. Heil, and L. Marko,J. Organomet. Chem., 253 (1983) 249-252. P. Sohar, G. Medgyes, and J. Kuszmann, Org. Magn. Reson., 11 (1978) 357-359. J. M. Sugihara and D. L. Schmidt, J. Org. Chem., 26 (1961) 4612-4615. J. C. Metcalfe, J. F. Stoddart,G. Jones, T. H. Crawshaw, E. Gavuzzo,and D. J. Williams, J. Chem. Soc., Chem. Cornmun.,(1981) 432-434.

1,4 :3,6-DIANHYDROHEXITOLS

103

The protons on C-1 and C-5 (S4.56, sym. multiplet) are equivalent. For the methylene groups at C-3 and C-7, the signal at S 3.80 occurs as a triplet, whereas the neighboring groups at C-4 and C-8 show a symmetrical multiplet splitting at S 2.01. The following coupling constants"j were measured: 3J8,,= 3J4,5 = 3.2 Hz, and 3J3,,= 3J,,8 = 6.8 Hz. The conformations of D-isosorbide (21), D-isomannide (22), and L-isoidide (23), and of their diacetyl and dimesyl derivatives, have been studied in great detail.24

21

22

23

a X=H b X=OCOCH, c X=OS02CH,

The two molecules (22 and 23) having a C2 axis through the C-3-C-4 bond constitute an ABXYY' spin system, whereas the protons in the isohexide 21 occur as an ABXYZ spin system. In 22 and 23, the H-2 and H-5 atoms couple with those of the neighboring hydroxyl groups. For the endo-OH, this coupling is twice that observed for the exo-OH group. The magnitude of 3J depends on the orientation of the hydrogen atom of the hydroxyl group to the vicinal hydrogen atom on C-2 or C-5. Between H-2 and H-4, as well as between H-3 and H-5, a small, long-range coupling is observed in 21c between H-3 and one ofthe geminal protons (namely, H- 1 ,). The split signal of this H- 1 atom occurs at a field lower than that of the geminal neighboring atom H- 1 ., Based on these observations, the assignment of H-1 A and H- 1 in similar molecules can be carried out in the same way. The geminal coupling constant between H-1, and H-1, is sensitive to substitution effects. Replacing the hydroxyl group by an acetoxy or a mesyloxy group causes a diminution in Jm . The magnitude of change is greater when the substituent is in the ex0 than when it is in the endo position. From the data, especially the coupling constants given in the original paper, it was concluded that only two distinct conformations are acceptable for either ring. The first of these is the envelope conformation, Structure 1, where C-3 is displaced from plane I, and C-4 from plane 11. The second is the twist (T) conformation, in which C-2 and C-5 are symmetrically displaced

,

,

PETER STOSS A N D REINHARD HEMMER

104

STRUCTURE 1

below and above plane I, defined by C- 1 ,(2-4, and the oxygen atom between them. Similarly, C-4 and C-5 stand out from plane 11, as shown in Structure 2. However, the coupling constants estimated for the compounds showed" that a mixed conformation between the envelope and the twist form for the two rings is present here. The use of a generalized Karplus relation was the basis for comparison between calculated and experimental vicinal proton - proton coupling con-

STRUCTURE 2

stants of isosorbide (in acetone-d6). The significant deviation between calculated and experimentally obtained values for couplings between H-5 and B Hz H-6,, (7.3 Hz measured versus 10.7 Hz calc.) and H-5and H - ~ (6.4 measured versus 8.4 Hz calc.) was explained by assuming displacement of C-5 from the plane C-4-C-3 -0-6-C-6 in the endo direction, thus diminishing the vicinal coupling27between H-5 and H-6* and H - ~ BTable . I11 presents the coupling constants between protons in the three i~ohexides.~~ The stereochemical behavior of the 2,5-diazido-2,5-dideoxyisohexides 17-20 was also derived from their 'H-n.m.r.-spectral data (CDCI,). From them, conclusionsregarding their conformationswere drawn. In accordance with its symmetry and the lack of any coupling between H-2 and H-5, H-3 H-4 in 20 appears as a sharp singlet at 6 4.55 (CDCI,), (4.24 in2, C6D6). A double envelope structure having two axial azido groups was derived. For 2,5-diazido-~-and -L-isomannide(18and 19),an opened twist conformation, leadingto two quasi-diequatonallyoriented azido groupswas

+

1,4 :3,6-DIANHYDROHEXITOLS

105

TABLE III Proton Coupling Constantsn of the IsohexldeP C o u p l i const.nts(Hz)Between protoas Compound

1,lB

1,2

1 ~ 2 23

34

45

56,

56.

6,6,

Isosorbide

-8.5

3.4 3.3 2.9 5.1 3.8

0.5 <1 0.8 5.4 0.7

4.4 4.2 4.7 5.4 3.8

4.7 4.5 4.1 5.4 0.5

5.8 7.3 10.7 5.1 0.7

5.9 6.4O 8.4 5.4 3.8

-9.5

calc.27

Isomannide Isoidide a

-9.2 -9.5

0.8
-9.2 -9.5

Solvent: acetone-d6.

suggested. A vkinal coupling between H-3 and H-2 (H-4 and H-5) with 3J

-2 Hz, and a long-rangecouplingbetween H-3 and H-6, + H-6, (H-4 and H-1,+ H-1), leadsI7to a broadening of the signal of H-3 and H-4 at S 4.70

(3.97 in2’ C,D,). The spectral data for 17 are consistent with a double envelope form for the fused-ring system, in which the azido group at C-5 is - 3 Hz; axially oriented. Signals for H-3 and H-4 appear at S 4.45 (d; 3J3r 3J2,30Hz)and4.8 (dd;3J,,5-3J3,4-3 Hz)17(3.85and4.12 forH-3 andH-4 in2’ CsDs. A complete set of values for shifts and coupling constants were reported23;see Table IV). Similar structural features were found for compounds 24 - 26. Thus, the H-3 and H-4 signals of 26a appearas a singlet at 64.80, and in the case of 25a, TABLE IV ‘H-ChemicslShifts (6)and Gedd (* and .I) Vicinal (3J)Coupling Constants for= the 2.5-Diazido-2,5-dideoxy isohexides 17,18,19, and 20

2,S-Diazido2,s-dideoxyisosorbide

A 10.2

Cc dd

3.59 d

3.8 3.33 d

A 10.0 B 3.46 C dd

3.26 dd

3.40 dd

B 3.52 Cm

3.52

3.08

3.97

m

m

m

B 3.50 isomannide

isoidide

1.6

4.5 5.2 3.85 4.12 d dd

6.6 2.93 ddd

6.4 3.26 dd

9.2 Hz 3.15 dd

4.24

1.6 3.40 dd

4.1 3.46 dd

10.0 Hz 3.26 dd

3.97

3.08

3.52

m

m

m

3.52 m

4.1

Chemical shift. Coupling Constant. Multiplicity.

S

4.24 s

PETER STOSS AND REINHARD HEMMER

1 06

as the A part of a symmetric AA'XX' multiplet pattern at 6 4.75. For 24a, these protons give two signals, namely, a doublet at 64.70 and a triplet at 6 4.85. The H-3 and H-4 signals for 26b appear as a singlet at S 5.20. In the spectrum of the asymmetric 24b, a m~ltiplet*~ at S 4.90 occurs.

24

25

26

a R, = R, = 0S02CH3 b R,=R2=I

In contrast to the data for the unsubstituted isohexidesand their diacetates and dimesylates, there are characteristic differences in 3J5,6Aand *JIMrespectively: 2J6B for the endo and exo isomers of isosorbide mononitrates (10, 11) [ ,J5,6 (eX0) 5.5 HZ, 3J5,a(endo) 3.2 HZ; 2J6&6~(eX0) 9.6 HZ, ' J 6 4 6 ~ (endo) 11.6 Hz;solvent, CDCI,]. From the solvent-induced shift values observed (for the ex0 and endo isomers)caused by change ofthe solvent from chloroform to pyridine, a specific solvation between the pyridine nucleus with only the (exo) OH-2 group was assumed. In pyridine, the H- 1, H-2, and H-3 atoms of the (endo) 5-nitrate are situated in the deshieldingzone of the pyridine ring, leading to a shift difference19 of at least -0.2 p.p.m. The TABLEV 'H-N.m.r. data for Isosorbide Mono~itrates~~ Hydrogen atoms

Compound

Isosorbide 5-nitrate (endo) 2-nitrate (exo)

a

14s

2

3

4

5

Aa 3.92d Bb4.12m 4.02m A 4.15d

4.35t 4.54dd

4.39d 4.59d

4.98t 5.12t

5.34dt 5.41dt

5.39t

4.57d

4.65t

4.33q

B 4.24m 4.10m

5.41dd

4.58d

4.64t

4.39dq

In cDc1,. In pyridine-dl.

6,

3.98 3.97 3.83m 3.91 3.65m 3.91 3.79m

1,4 :3,6-DIANHYDROHEXITOLS

107

chemical shifts for the isosorbide mononitrates are summarized in Table V. Somewhat different shifts and coupling constants are given in reference 32. The isoidide structure of the bidentate phosphane 27 was derived from the single signal in its 31P-, as well as from its I3C-, n.m.r. spectrum. Here,

the C2symmetry is the origin of the identity of the chemical-shiftvalues2*of C-l/C-6, C-2/C-5, and C-3K-4 (see Table VI). Big 1,4:3,6-dianhydro-~-mannitolo)-30-crown10 (28) is characterized by its 'H-n.m.r. spectrum. In CDCI,, the four hydrogen atoms at the two bridgehead carbon atoms ofthe fused five-membered rings show a multiplet, centered at 6 4.66. The hydrogen atoms vicinal to the four endo-oriented oxygen atoms connecting the crown chain occur as a multiplet, centered at S 4.22, coupling26with one, or both, of the hydrogen atoms of the vicinal methylene group, 3J 7.9 Hz.

28

Studies of the conformational behavior at temperatures down to - 110" indicated that a ring-inversion process, possible in chloroform solution, is revealed by a broadening of the I3C-signalsof C-2 and C-5 (67 1.9),as well as those26of the bridging atoms C-3 and C-4 (6 8 1.3). Compound 28 is able to form 1 : 1 : 1 complexes with organic primary ammonium salts and water, or, alternatively, with a primary amine. In the (32) 0.De Lucchi, A. Angius, F. Filipuzzi, G. Modena, and E. Camera, Gazz. Chim. Itul., I 17 (1987) 173-176.

PETER STOSS A N D REINHARD HEMMER

108

TABLEVI 'H Chemical shifts for the Ring System of Isobexide Derivatives Chemical shifts Compound Isosorbide 2-0-acetyl-

A" A

B

2,5-di-O-acetyl2,5-di-O-mesyl2-chloro-2deoxy-

A" A"

2-0-acetyl-5-0mesyl2,5-diamino-2,5dideoxy* 2 HCI

2,5-di-O-aCetyl2-0-acety1-5-0mesyl2,5-diamino-2,5dideoxy2 HCl

-

References

3.14 4.00 m 3.90 4.00

3.18

4.16 5.16

5.09 5.14

3.31 3.54 dd 3.15 3.80

3.63 3.86 dd 3.87 3.95

24 21

5.12 5.06

4.44 4.59 t 4.16 4.89

4.08 4.25

3.94 4.1 1

4.26 4.45 d 4.42 4.65 4.56 d

4.68 t

4.24 m

3.54 dd

3.86 dd

21 21

m

9

24 24

4.15 d

4.61 q

4.76 t

3.44

m

A

4.0 1

5.19

4.49

4.81

5.03

3.84

21

AC

4.14 dd

4.01 dd

3.93 ddd

4.19 d

4.88 dd

3.94 ddd

4.10 dd

3.75 dd

23

3.6 1 d 3.1 1 dd 3.91 3.96

4.1 1 d 4.83 d 4.23 5.10 9 4.91

3.69 d 4.32 d 4.48 4.46

4.35 ddd 4.52 ddd 4.23 4.30

3.33 dd 3.50 dd 3.69 3.79

2.83 dd 3.34 dd 3.97 3.5 1

4.54

4.16 dd 4.18 dd 4.48 4.65 t 4.54

23

A B A"

3.45 dd 3.49 dd 3.69 4.10 m 3.60

A

3.89

5.03

B

m

m

4.64 m

4.64 m

AC

4.19 m 3.82 m 4.65 4.54 4.38

4.19 m 3.82 m 4.65 4.54 4.38

4.00

Ab

4.21 dd 3.55 dd 3.88 3.18 3.55

A B

4.01 d

4.29

4.63 d

4.76 d

Ab

3.12 m

5.08

4.62 m

4.62 m

Ab Ab A"

B Ab

2-&&yl-5chloro-5deoxy-

H4,,

3.86

B

2-chloro-2deoxy-

HdA

m

Ab

Isoidide

H-5

3.66

B Isomannide 2-0-acetyl-

H4

Ab B

B 2,5-di-O-t0~yl-

H-3

4.32

B 2,5-di-O-chloroformyl-

H-2

4.1 1 d

A

B 2-O-acetyl-5chloro-5deoxy-

H-lA H-1.

Aa

B

m

m 3.86

3.19 dd 3.42 dd 3.96 3.8 1

4.00

m 4.5 1

m 5.02 4.25 3.55

m

m

t

m

23 24 21

m

m

m

4.9 1

3.60

3.86

24

5.03

3.89

21

m

m 4.2 1 dd 3.55 dd 3.88 3.18

3.19 dd 3.42 dd 3.96 3.8 1 3.55

23

4.29 m

3.85 d

21

4.06 d

3.12

21

m 4.5 1 m 5.02 4.25 3.55

m

23 24 24 23

1,4: 3,6-DIANHYDROHEXITOLS

109

TABLE VI (continued) -

Chemical shifts

H-lA H-1,

Compound 2,S-di-0-acetyl2,5-diamino-2,5dideoxy* 2HCl

H-5

HdA

HdB

References

3.92

5.12

4.56

4.56

5.12

3.80

3.92

24

A‘

4.08 m

3.92 m

3.92 m

4.88 s

4.88 s

3.92 m

4.08 m

3.92

23

3.66 d 3.90 3.25 dd

3.76 5.18 4.61 d d d d 3.99 5.02 4.76 3.70 4.83 4.51 dd dd s

4.66 d 4.76 4.51 s

5.38 5.03 d d d 5.02 3.90 4.83 3.25 dd dd

3.82 d 3.99 3.70 dd

5.96 d

4.63 dd

4.32 d

3.25 d

3.12 dd

Ab A* Ab

B 1,4:3,6-dianhydro5-azido-2,5-dideoxy-~-xyl+ hex- 1-enitol

H-4

3.80

B 2,5-di-Omesyl2,5-di-trto~yl-

H-3

Aa

B 2-0-acetyl-5-0bemoyl-

H-2

Ab

B

5.04 dd

3.55 d

m 23 24 23

23

complexed state, the multiplet of H-3 and H-4 is shifted upfield for a nearly constant” 0.1 p.p.m. ‘H-N.m.r. spectroscopy showed that isohexides can act as bidentate ligands for Gd(dmp), (dmp, 2,2,6,6-tetramethylheptane3,5-dionate) and Eu(fod), (fbd, 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5octanedionate). The erythro configuration (endo-OH) exhibits a high selectivity for complexation of the 0 - 1 -C-4-C-5 -OH unit (see Structure 3). Only a low associationconstant was found for isoidide, where this part of the molecule exists solely in the threo configuration.” In Table VI, the ‘Hchemical shifts for the isohexidering system of some derivativesare summarized.

Ln

I11

(ii) I3C-N.m.r. Spectra.-An attempt was to establish, for different substituted isohexides, a conformational relationship from the

110

PETER STOSS AND REINHARD HEMMER

I3C chemical-shift differences, especially at the C-2 and C-5 (a)atoms, as well as at C-3 and C-4 (p). Using the field effect, which causes diamagnetic shifts, especially at these two pairs of carbon atoms, it was confirmed that, for a given substituentat position 2 or 5, the shift depends mainly on whether it is in the exo or the endo orientation. (see Structure 4.)

STRUCTURE 4

Generally, lower shift values (downfieldshifts)are measured for groups in the em, compared with those in the endo, position.21*29 For mixed 2,5-disubstituted isohexides, the situation is not so clear in each case?' The I3Cchemical shifts of the isohexidesand selected derivatives are summarized in Table VII. d. Mass Spectra.-Mass spectrometric (electron impact, e.i.) data were reported'1,33~u for the three unsubstituted isohexides 3-5, especially D-isomannide,35their 0-deuterated isomers,33the mono- (8)and1' di-ketone (9), some mono- and di-aroyl derivative^,^^ and isosorbide 5-nitrate3' (11).The isomannide cation, formed by dehydration reactions, was also found to be present, as evidenced in the e.i. mass spectra of some other mannitol derivative~.~~ Mass-spectrometric analysis has been used to detect isosorbide and its 5-nitrate in human urine and plasma probes.34An assay for the quantitative determination of isosorbide 5-nitrate and its D-glucosiduronicester, and that of isosorbide itself, using g.1.c. -m.s. techniques with a triple-stage, quadrupole mass spectrometer, has been rep~rted.'~ A combination of g.1.c. and (33) N. S. Vul'fson, 0. S. Chizhov, and L. S. Golovkina, Izv. Akad. Nauk SSSR, Ser. Khim., (1969) 168-174; Chem. Abstr., 71 (1969) 25,794. (34) S. G. Wood, B. A. John, L. F. Chasseaud, R. M. Major, M. E. Forrest, R. Bonn, A. Dam&, and R. F. Lambe, Arzneim. Forsch., 34 (1984) 103 1- 1035. (35) J. Szafranek and A. Wisniewski, J. Chromatogr., 161 (1978) 213-221. (36) R. Endele and M. Senn, Znt. J. Mass Spectrom. Ion Phys., 48 (1983) 81 -84.

TABLEVII IF Chemical Shifts of Isohexides and Derivatives in CDCI,'versus Me, Si as Internal Standard

'FChemical shifts (ppm) Compound Isosorbide Me,SO acetone-d6 2,5-di-O-mesyl2-0-acetyl 5-O-acetyl-2-chloro-2deoxy2-chloro-2deoxy2,5-dideoxy-2,5-diiodo2deoxy-2-iodo-5-0-mesylIsomannide 2,5-di-O-mesyl2,Sdi-o-tosyl2deoxy-2-iodo-5-0-mesyllsoidide 2,5-di-O-mesyl2,5dideoxy-2,5diiodo2-chloro-2dideoxy2deoxy-2-iodo-5-0-mesylbis(2,5-diphenylphosphino-2,5-dideoxy)CHydroxy-2,6dioxabicyclo-[3.3.0]octan-8-one (Me83 2,6-Dioxabicyclo[3.3.O]octane-4,8dione (Me,SO) Unless otherwise noted

C-1

C-2

C-3

C.4

C-5

Cd

References

75.0 75.0 76.2 75.8 71.7 73.7 75.8 76.0 76.6 77.7 75.1 73.9 71.6 70.0 76.1

76.6 75.5 77.0 76.6 80.5 78.6 60.7 21.7 21.7 25.9 71.9 73.9 79.9 18.2 21.1

88.2 87.5 88.7 88.2 81.9 85.8 88.9 82.8 82.8 90.4 82.0 83.0 81.6 80.0 82.6

81.7 81.3 82.2 81.7 87.1 82.1 80.5 89.2 89.2 80.0 82.0 83.0 81.6 80.0 79.6

72.3 71.0 73.3 72.3 84.1 72.5 74.0 26.9 26.9 78.5 71.9 73.9 19.9 78.2 79.3

73.4 72.0 72.7 73.4 74.5 73.5 70.5 78.1 78.1 70.9 75. I 73.0 71.6 70.0 71.0

11,21 11 27 21 29 21 21 21 29 29 11,21 28 21,29 28 29

73.2 77.6 75.3 77.6 71.2

84.0 26.5 60.5 25.4 44.4

86.4 88.5 88.1 90.1 87.6

86.4 88.5 81.5 85.1 87.6

84.0 26.5 75.6 82.2 44.4

73.2 77.6 74.8 73.6 71.2

29 29 21 29 28

74.7 70.0

75.3 209.6

81.1 78.8

18.5 78.8

21 1.5 209.6

69.7 70.0

11 11

PETER STOSS AND REINHARD HEMMER

I12

e.c.d.-negative mass ion spectrometry has been used for the analysis of isohexide nitric esters3’ The unsubstituted isohexides differ only in the steric arrangement of the substituents at C-2 and C-5. Their spectra (reproduced in the original paper) contain a noticeable peak of the molecular ion with m/z 146, and peaks with m/z 128 (M - H,O)+, 1 1 1 (M - H 2 0 - OH)+, 102 (M CH2CH=OH)+, 98 (M - H 2 0 - CH20)+,the ion m/z 69 of protonated furan, and further peaks with m/z 103,102,86,85,73,and 44. The spectrum of isoidide also contains the peak of the ion (M l)+. The main differences in the spectra are found in the heights of the appropriate peaks. The mechanism of fragmentationfor the three isohexideswasdiscussedin great detail in

+

m

HO

-I+

A

+

Jm/z

146

oo>H

HO

c + HO’

5 C H z O

HO

OH

m / z 103

* O H 7 m / z ;6

0

l + CH -CZO

m / z 58

+

m / z 69

dOH1’

m/z 43

m / z 43 SCHEME1 (37) J. C. Signall, N. W. Davis, M. Power,M. S. Roberts,P. A. Cossum,and G. W. Boyd, Anal. Chem. Symp. Ser., 7 (198 1) 1 I 1 - 122.

1,4 :3,6-DMNHYDROHEXITOJ.S

113

the original paper.j3 The most important fragmentation steps’‘-3334 are shown in Scheme 1. For the deuterated compounds, the appropriate fragments having the corresponding higher mass were observed.33 The nature of the fragmentation is substantiallychanged in the case of the monobenzoylatedisohexides.The highest mass, observed at m/z 207, corresponds to the fragment 29, following A in Scheme 1. A further way of +

29

degradation leads” to formation of the ions m/z 128 and 85 (see Scheme 2). The spectra of the dibenzoylated isohexides all show a peak of the ion m/z 249, corresponding to (M - C,H5CO)+. Normally, the peak for (M -

OCOCsHS m / z 85

m / z 128 SCHEME 2

C&COOH)+, with m/z 232, occurs with high intensity. Elimination of the second benzoyl group creates3) an ion with m/z 127. The fragmentation behavior of isosorbide Snitrate (11) is well established, as characteristic fragments” occur (e.i. method; see Scheme 3). The c.i. mass spectrum ofthis compound also contains” a quasimolecularion (M 1) with m/z 192. The

+

HO

+

t

HO

m/z 127

m / z 85 SCHEME3

m/z 69

m / z 43

PETER STOSS AND REINHARD HEMMER

I14

U

m/z 144

m / z 86

b.4' m/z 56

U

m/z 84

m / z 142

1

D-6

m / z 55 SCHEME4

e.i. mass spectra of the mono- (8) and di-ketone and (9), depicted in the original paper, show similar fragmentation pathways'' (see Scheme 4). A collisionallyactivated mass spectrum of an isohexide whose exact structure was not given led38to the main fragments m/z 145 (M-H)-, 143 (M-H-2)-, 1 15 (M-H-30)-, and 10 1 (M-H-44)-. 2. Structural Aspects

Four X-ray investigations of isohexide derivatives have been p~blished.'~-~* The oldest of them gave very little information about structural details. Two crystallographically independent molecules of 1,4:3,6dianhydro-2-O-(pbromophenylsulfonyl)-~-gluc~tol 5-nitrate (30), having the same conformation, are in the asymmetric unit. The value of the angle between the two fused, five-membered rings is 1 10".The dihedral angle between the two rings cannot be given precisely,

-

(38) W.C. Brumley, D. Andnejewski, and J. A. Spohn, Org. Mass Spectrow., 23 (1988) 204-2 12. (39) A. Camerman, N.Camerman, and J. Trotter, Acta Crystallogr.,19 (1965)449-456. (40)H. Van Koningsveld,J. A. Peters, and J. C. Jansen,Acta Crystallogr.,Sect. C: Crysz.Struct. Commun.,40 (1984)519-521. (41) F.W.B. Einstein and K. N. Slessor, Acta Crystallogr.,Sect. B, 31 (1975)552-554. (42) R.Hemmer, P. Stoss, P. Merrath, and M. Kasper, unpublished results.

1,4 :3,6-DIANHYDROHEXITOLS

115

30

because they cannot be defined by a plane unique for each of them (R = 0.157). However, the two rings are significantlynonplanar. Consequently, if the C-3- C-4 - C-5 -0-8 and C-2 - C-3-C-4 -0-7 atoms form the two relevant planes, C- 1 C-6 are each displaced from them by -0.05 nm. The crystal data are39monoclinic, space group P2, (C;); a = 26.16 k0.04, b = 11. I 1 +0.02,c=5.34f0.01 A;/3=9O0 1 6 ' f 5 ' ; Z = 4 . The structure of 1,4 :3,6-dianhydro-~-glucitol (see Structure 5, taken from Ref. 40; atom numbering as in formula 33) is well established. An X-ray analysis of the molecular structure of the molecule, recorded at 100 K (R = 0.026), has been published.40 The geometry of the ring system of 3 differs significantly from that in 30. In3, thetwoplanesdefined by C-6-0-8-C-3-C-4andC-3-C-4-0-7-C-6 are nearly perpendicular (87") to each other. In contrast to 30, C-2 and C-5 are displaced here from the planes (58 and 59 pm, respectively). Between the oxygen atom in the 5-end0position and 0-7 in the same molecule and 0-8 in the neighboring molecule, hydrogen bonds (length 242 and 199 pm respectively) exist. Further important angles are C-2-C-3-0-8, 110";0-8-C-4(2-5, 110.5"; 0-7-C-3-C-4-0-8, 92"; and C-2-C-3-C-4-C-5, - 140.1'. The crystal data are orthorhombic, space group P22222,;a = 5.3 13(7),b = 7.006(3), c = 17.174(5); and 2 = 4. The molecular structures of the two diastereoisomers of the 5-0-acyl substituted isosorbide derivative 5-[ 1,4-dihydro-3-(methoxycarbonyl)-2,6-

.

0 0

OH STRUCTURE 5

PETER STOSS AND REINHARD HEMMER

116

dimethyl-4-(2-nitrophenyl)-5-pyridylc~bonyl]isosorbide (31 and 32)were elucidated. In 31 the c h i d C-atom in the dihydropyridine ring has the S configuration; in its diastereomer, the opposite configuration,R, is present. The planes of the two fused rings in the isosorbide part of this molecule form4*an angle of 120".

-

31

32

1,4 :2,5 :3,6-Trianhydro-~-mannitol (33), an isomannide derivative in which the two endo hydroxyl groups form an intramolecular ether bridge, consists of three fused tetrahydrofuran rings. An X-ray analysis (R0.049) showed that the molecule has a twofold symmetry axis through the bridging Q

33

oxygen atom and the center of the C-3 - C-4 bond of the isomannide ring system. The furan ring formed by 0-9- C-2 - C-3-C-4 - C-5 has a perfect T, conformation, wherein G 3 and (2-4 are displaced 54.3 pm out of the plane. The other furan ring, C- 1 -C-2 -C-3 -C-4 -0-7, has the T2conformation, wherein C-3 is above (0.62.2 pm), and C-2 is below (0.26.4 pm), the plane. Some important angles are H-3-C-3-C-4, 118.6";C-2-C-3-C-4, 91.2"; C-2-0-9-C-5, 103.0";and 0-9-C-2-C-3, 101.8".The crystal data are4' tetragonal, space group P4,2,2; at 100 K, a = b = 5.988(1), c = 15.840(3) A;Z = 4. The X-ray analysisz6of the 30-crown-10 derivative (28) of D-isomannide and its I : I complex with S- I-ethylphenylammonium perchlorate

1,4:3,6-DIANHYDROHEXITOl-S

117

and water3' has been briefly reported, but no detailed stereochemicalinformation was given for the isohexide part of these macrocycles.

3. Analytical Behavior, Detection, and Determination Isosorbide (3),isosorbide h i t r a t e (ll),and D-glucitol(34) can be readily separated by thin-layer chromatography using one of the following solvent mixtures: a, 5 :4 : I (v/v) chloroform-methanol-aceticacid orM6,4 : 1 (v/v) isopropyl alcohol-conc. ammonia. In another method, 1 : 1 toluene-ethyl acetate as the mobile phase and permanganate- metaperiodate as a chromogenic spray reagent for identification have been used for separation and identification of the three isosorbide nitrates and i~osorbide.~~ Monoacetates, mononitrates, and mixed acetate-nitrates of isosorbide can be separated on silica plates by using benzene - ethyl acetate - isopropyl alcohol or dichloromethane- diisopropyl ether - 1,Cdioxane- isopropyl alcohol mixtures.44The different separation behavior of isosorbide 2- and 5-nitrate on silica gel is attributed to the different strengths of the intramolecularhydrogen-bridge~'~ in these molecules.1s A high-performance, liquid chromatography (h.p.1.c.) assay (reversed phase, C,, column, methanol- water-acetate buffer) for isosorbide dinitrate along with the two mononitrates in pharmaceutical formulations has been des~ribed.4~ A similar method can be found in Ref. 44. Isosorbide dinitrate can be determined by reversed-phase h.p.1.c. (CISphase) using4 methanol as the mobile phase and detection at 2 18 nm; for other methods, see Refs. 47 - 49a. Among other organic nitrates, isosorbide dinitrate can be selectivelydetected by h.p.1.c. by first using a photolysis step, followed by an electrochemical detection m e t h ~ d . ~ ~ ~ ~ ~ (43)M.Carlson and R. D. Thompson, J. Chromatogr.,368 (1986)472-475. (44) N.Dimov, N.Agapva, S. L e i , and I. Yanachkov, J. Chromatogr..,285 ( 1984)5 15- 5 17. (45)M. Carlson, R.D. Thompson, and R. P. Snell, J. Chromutogr.Sci., 26 (1988)574-578. (46)H.L.Bhalla and J. E. Khanolkar, Indian Drugs, 22 (1985)541 -543. (47)J. B. F. Lloyd, Proc. Int. Symp. Anal. Defect.Explos., (1983)31-39;Chem. Abstr., 102 (1985)81,158. (48)N.Mizuno, C.Shimizu,E. Morita,D. Shinkuma,and Y. Yamanaka, J. Chromatogr.,264 (1983)159-163. (49)W.C.Yu, E. U. Goff, and D. H. Fine, Proc. Inf. Symp. Anal. Defect. Explos., (1983) 329-340;Chem.Abstr., 102 (1985)142,685. (49a) W.Y. Liu, Yuoxue Xuebuo 24 (1989)797-800,Chem. Absfr., 112 (1990)104,962. (50) I. S. Krull, X.D. Ding, C. Selavka, K. Bratin, and G. Forcier, J. Forensic Sci., 29 (1984) 449-463. (5 1) I. S.Krull, C. Selavka, X. D. Ding, K. Bratin, andG. Forcier,Proc. Znt. Symp. Anal. Defect. Explos., (1983)1 1 -29;Chem. Abstr., 102 (1985)97,782.

118

PETER STOSS AND REINHARD HEMMER

For isohexide nitrates gas - liquid chromatographic separation methods using OF-1 (Ref. 52), OV-101(Refs. 53-59), OV-17(Ref. 60),or OV-210 (Ref. 6 1) columns have been reported. Most of the methods described are used to detect traces of nitrates, as well as of their metabolites, in urine and plasma probe^.^^-^^ All known isohexide nitrates can be measured in mix~~.~~ detection was tures by using a DB-5capillary c ~ l u m n .Electron-capture applied for the g.1.c. determinationof isosorbide dinitrate7IPand its metabolit e ~ . ~ A g.1.c. method (OV-101)for determining isosorbide in foods, using its trimethylsilyl derivative^,^^ has been reported. D-Isomannide and 2(5)chloro-2(5)-deoxyisomannidecan be separated from a series of other sugar derivatives of mannitol on an OV-101 column.3s Other methods have also been used for quantitative determination of isohexide nitrates, including c ~ l o n r n e t r y , ~infrared ~ - ~ ~ spectroscopy,20 n.m.r. spectro~copy,~~ and p o l a r ~ g r a p h y . ~ ~ * ~ ~ - ~ (52) M. T.Rosseel and M. G. Bogaert, J. Chromatogr.,64 (1972) 364-367. (53) K.H.Goebbeler, Pharm. Ztg., I16 (1971) 961 -962. (54) H. Laufen, F. Scharpf, and G. Bartsch, J. Chromatogr., 146 (1978) 457-464. (55) A. Sioufi and F. Pommier, J. Chromatogr.,229 (1982) 347-352. (56) M. Ahnhoff and G. Holm, Proc. Int. Symp. Capillary Chromatogr.,4th, (1981)673-686; Chem. Abstr., 97 (1982) 207,590. (57) A. Sioufi and F. Pommier, J. Chromatogr.,305 (1984) 95- 103. (58) N. Ruseva, N. Dimova, G. Spirov, and M. Yurovska, J. Chromatogr.,295 (1984) 255258. (59) N. Ruseva and N. Dimova, Farmutsiya,35 (1985) 1-4; Chem.Abstr., 104 (1986) 75,168. (60) M. T.Rosseel and M. G. Bogaert, Anal. Chem. Symp. Ser., 3 (1980) 59-63. (61) D. G. h e , R. N. Johnson, and B. T.Kho, J. Assoc. Of Anal. Chem., 60 (1977) 13411344. (62) I. W. F. Davidson, F. J. Dicarlo, and E. 1. Szabo, J. Chromutogr.,57 (1971) 345-352. (63) E. Doyle, L. F. Chasseaud, and T. Taylor, Biopharm. Drug Dispos., I (1980) 141- 147. (64) J. Halkiewicz and W. Sawicki, Pharmazie, 43 (1988) 865-866. (65) M. T. Rosseel and M. G. Bogaert, Biochem. Pharmacol.,22 (1973) 67-72. (66) Y. Santoni, P. H. Rolland, and J. P. Cano, J. Chromatogr.,306 (1984) 165- 172. (67) A. J. Woodward, P. A. Lewis,and J. Maddock, Methodol.Surv.Biuchem.Anal., 14(1984) 369-370. (68) M.T.Rosseel and M. G. Bogaert, J. Pharm. Sci.. 62 (1973) 754-758. (69) J. 0. Malbica, K. Monson, K. Neilson, and R. Sprissler, J. Pharm. Sci., 66 (1977) 384386. (70) D. Lutz,J. Rasper, W. Gielsdorf, J. A. Settlage, and H. Jaeger, Glass Capillury Chromatogr. Clin. Med. Pharmacol.,(1985) 497 - 5 10. (71) D. Lutz, J. Rasper, W. Gielsdorf, J. A. Settiage, and H. Jaeger, J. High Resolut. Chromatogr. Chromatogr.Commun.,7 (1984) 58-65. (71a) B. A. Shah and H. P. Tipnis, Indian Drugs 27 (1990) 481 -483. (71b) G. Michel, L. Fay, and M. Prost, J. Chromatogr.493 (1989) 188- 195. (72) G. Schneider,E. Hieke, and W. Baltes, 2.Lebensm.-Unters.Forsch., 183( 1986) 199-204. (73) H. L. BhaUa and J. E. Khanolkar, Indian Drugs, 21 (1984) 158- 159. (73a) D. M. Shingbal and U. G. Barad, Indian Drugs 22 (1985) 607-608.

1,4:3,6-DIANHYDROHEXITOLS

119

Differential scanning calorimetry can be used to detect isosorbide dinitrate in the presence of various proportions of other isohexide mononitrates in pharmaceutical formulations*’and for testing its hazardous characteristics.82A complete analytical profile for isosorbide dinitrate, detailing spectroscopic and other physical properties, as well as useful analytical methods, has been reported.83 IV.

PREPARATION OF THE

PARENTCOMPOUNDS

From among the known isohexides(see Section 11),isosorbide (3) is that of the highest importance, not least because of the pharmaceutical use of its nitrates and the good solvent properties of its dimethyl ether. All methods leading to isohexides start from the appropriate hexitol, that is, D-glucitol(34), D-galactitol(33, D-gulitol(36), D-mannitol(37),D-talitol (38), D-allitol(39),or miditol(40), or their respective enantiomers, by using different procedures (acidic media, in general) for dehydrating the substrate^.^^-^^ The putative steps leading to isosorbide from D-glucitol(34) are outlined in Scheme 5 . G. Bongiovanni, C. Giani, F. Innocenti, M. Maccari, E. Minet, and L. Pogliano, Boll. Chim. Farm., 123 (1984)14-35. E. U. Go6 W. C. Yu,and D. H. Fine, Proc. Int. Symp. Anal. Detect. Explos., (1983) 159- 168;Chem. Abstr., 102 (1985)97,784. S.N.Chiarelli, M. T. Rossi, M. T. Pizzorno, and S . M. Albonico, J. Pharm. Sci., 71 ( 1982) 1178- 1180. A. B. Grigor’ev, L. T. Chistotinova, and M. K. Polievktov, Khim.-Farm. Zh., 7 (1973)

50-52;Chem. Abstr., 78 (1973)164,156. B. Persson and L. Rosen, Anal. Chim. Ada. 123 (1981)115- 123. S . Silvestri, Pharm. Acta Helv.,50 (1975)304-307. W. R. Turner and R. S. Lenkiewicz, J. Pharm. Sci., 65 (1 976) 1 18- 121. W. Waechter, B. Snepanik, and G. Codes, Acta Pharm. Technol., 30 (1984)17-23. B. K.M. Murali, V.Ganesan, K. B. Rao, and V. K. Mohan, J. Hazard. Muter., 3 (1979) 177- 182. L. A. Silvieri and N. J. De Angelis, Anal. ProJilesDrug Subst., 4 (1975)225-244. L.A. Hartmann (Atlas Chem. Ind. Inc.), U.S. Pat. 3,160,641 (1961);Chem. Abstr.. 62 (1965)9,227. H.salzburg, M. Hajek, and H.Meyborg(BayerAG), DE 3,229,412(1982);Chem.Abstr., 100 (1 984)176,241. L. W. Wright and J. D. Brandner (Atlas Chem. Ind. Inc.), U.S. Pat. 3,023,223(1960); Chem. Abstr., 57 (1962)45,43i. L. A. Hartmann (Atlas-Chem. Ind. Inc.), U.S.Pat. 3,454,603(1966);Chem. Abstr., 73 (1970)35,703. (88) L. A. Hartmann (Atlas Chem. Ind. Inc.), U.S.Pat. 3,484,459(1967);Chem. Abstr., 72 (1970)101,059. (89)B. J. Arena (UOP Inc.), US.Pat. 4,313,884(1980);Chem.Abstr.. 96 (1982)163,115. (90)J. Defaye and C. Pedersen (Beghin-SayS. A.), WO 89,00,162(1987);Chem. Abstr., 1 1 I (1989)39,828.

120 CH,OH

I

HCOH

I

HOCH

I I HCOH I C$OH HCOH

34 CH,OH

PETER STOSS AND REINHARD HEMMER CH,OH

I I HOCH I HOCH

HCOH

1

HCOH

36

CH,OH

I HCOH

38

HCOH

35

1 HCOH

I

I I HOCH I

HCOH

I CHzOH

I HCOH I HCOH 1

CH,OH

I

HCOH

I CH,OH

I HOCH I HOCH I HOCH

CH,OH

HCOH

I

CH,OH

I I HOCH I HCOH I HOCH

HCOH

I

CH,OH

37

CH,OH

I

HOCH

I 1

HCOH HOCH

I

HCOH

I

CH,OH

CH,OH

39

40

Protonation occurspreferentially at the primary hydroxyl group. The first dehydration step can also take place between the 3- and 6-position, leading to the 3,6-monoanhydro derivative 41, The second water-elimination step from the 1,4-, as well as from the 3,6-, anhydro-D-glucitol, leads to the formation of D-isosorbide. However, kinetic studies showedwthat the proportion of the 3,banhydro isomer is low compared to that of the 1,4-anhydride. An investigation giving similar results is described in Ref. 95. For (1s)-1-ZH-D-mannitol(42) as the starting material, it has been elucidated that, during dehydration to the 1,Canhydro- (43) or 3,6-anhydromannitol (44), and, subsequently, to the ( 1R)-l-2H-isomannide(45), an (91) H. Salzburg, H. Meyborg, and H. Ziemann (Bayer AG), DE 3,111,092 (1981); Chem. Abstr., 98 (1983) 54,396. (92) J. Feldmann, H. Koebernick, and H. U. Woek ( M a i Z e GmbH), ~ DE 3,041,673 (1980); Chem. Absfr.,97 (1982) 163,415. (93) R. Barker,J. Org. Chem., 35 (1970) 461-464. (94) K. Bock,C. Pedersen,and H. Th0gersen.AcfaChem. Scmd.,Sm.B, 35 (1981) 441 -449. (95) S. Krauze, E. Gromadzinska,and J. Ojrzanowski,Acre Pol. Pharm., 43 (1986) 41 1-415.

1,4 :3,6-DIANHYDROHEXITOLS

-& +

"27

-

H

HZ:; I H2°

HCOH

I

H+

34

-2-

-

HOCH H~OH

I

1

F-

HO H HCQH I

I

HCOH

HCOH

I

I

CHZOH

CHZOH

Ho,

121

OH

0

kv'

' H'

OH

OH

HO

SCHEME 5

inversion at the primary (C-1 and C-6)atomsoccurswThe reactionpathway is shown in Scheme 6. Important sources of side reactions during the dehydration processes are (1) formation of the 2,Sanhydro isomer, which cannot be converted into OH

41

PETER STOSS AND REINHARD HEMMER

122

HO D

-

HO

"T

OH OH

43

OH

ix;..

HOCH

I

HOCH I HCOH

-H

L

D

HO?

D

OH

1

45

HCOH

I

H2COH

' O H qOH

42

44

SCHEME6

isosorbide (3), and (2) the intramolecular elimination of water between isohexide molecules, leading to higher molecular units. For the preparation of isohexides, especially in order to enhance the yield and to avoid side reactions, various modifications of the reaction conditions have been employed. The most important methods are summarizedin Table VIII. The velocity of the dehydration steps in this reaction, leading from 1,Canhydro derivatives of D-iditol(40), D-gulitol(36), D-glucitol(34),and D-mannitol(37) to the 1,4 :3,6-dianhydro compounds isoidide (5) and isomannide (4) depends93on whether the hydroxyl group on C-5 is endo (as in D-mannitol and D-glucitol)or e m (asin D-iditol and D-gulitol) (see Scheme 5 and Structures 6,7, and 8). Thus, the formation of 1,4 :3,6-dianhydrohexitols from the 1,Canhydro derivatives40 of D-iditol and 36 of D-gulitol is -40 times faster than from93those (34)of D-glucitol and (37)D-mannitol. For a ~

M+

c

'I _ -/, t 0

1 0 1 ,

'

"*o

--

101

M+

STRUCTURE6

I

TABLEVIII Reaction Conditions for Isohexidp Syntheses Yield (To) EdUCt

Reaction conditions

molten d u c t , Amberlite IR- 120,0.5 h at 170" and 1.33 kPa molten duct, ionexchange resin, 2 h at 140" 2 and 3 kPa pTsA," toluene, 12 h at 1 12 Dean - Stark a p 3 paratus acetic acid, HCI, 4 MPa 4 formic acid, HF, 3 h at 40" 5 Wofatit KPS, toluene, 5 h at 110" 6 H,PO, or H,SO,, boiling toluene 7 aq. solution, Ni catalyst 5% NiCI,, H, at 225" 8 and 4.8 MPa Amberlite IR- 120, 1,4-dioxane-ethyl acetate, 9 24 h, reflux for conditions, see No. 3 10 D-Mannitol for conditions, see No. 2; 5h 11 acetic acid, HCI, 0.5 h at 140" 12 formic acid, HF, 0.3 h at 20" 13 for conditions, see No. 9 14 Dowex 50 W-X2, special procec -re 15 3,4-Di-O-mes). D-mannitc 15a 3,4-Di-O-tosyl-~-mannitolBa(OMe),, MeOH, 2 h at 25" pTsA," 0.5 h at 125" and 3.6 Wa I6 L-Iditol pTSA," 2 h at 140", and 4.7 kPa 17 DL-Tditol pTsA," 120-150" at 10.0 kPa 18 9:16 Allitol-mannitol

IS

IM

I1

References

93

-

-

94

91

-

-

92

65 84

-

-

57

-

-

73 83

-

-

-

-

96 91 90 100

28

-

-

89

39 -

35 76 65 55 35

-

21 96 92 91 90 21 97 99 88 87 87

1 D-Glucitol

a

-

-

-

8 37'

-

-

-

-

-

100

91 41 30

-

11, isoidide; IM, isomannide; IS, isosorbide. pToluenesulfonic acid. T h e exact amount was not determined.

101

PETER STOSS AND REINHARD HEMMER

124

STRUCTURE 7

STRUCTURE 8

detailed stereochemical pathway of the dehydration reaction of hexitols, see Ref. 96. 3,4-Di-O-mesyl-~-mannitol (46) can be converted under acidic conditions (Dowex 50W-X2 cation-exchangeresin) by way of lY4-anhydro-3-Omesyl-D-talitol (47) into D-isoidide (49) in almost quantitative yield. On using triethylamine- water, 2,3 :4,5dianhydro-~iditol(48)is form&, from this, on treatment with an acid ion-exchanger, D-isoidide (49) is formed in only 20%yield (see Scheme 7). From these observations, it wa~decided~'.~* that the 2,3:4,5-dianhydro isomer 48 cannot be an intermediate in the conversion of 46 into 49. Similar results were found for the reaction of 46 or 3,4-di-O-ptolylsulfonyl-~-mannitol( pCH3C6H,S02-instead of CH3S02in 46) with barium methoxide in methanol.99 Pyrolysis of cellobiitol at 350" in v m o yields -32% of levoglucosan ( 1,6-anhydro-~-~-glucopyranose) and 70% of a mixture of D-glucitol and its anhydridesw*Preparation of mono- and di-anhydrohexitols, mixed with linear polyols and polymers, were claimed by application of particular noble metal catalysts, coated with copper, on catalytic hydrogenation Of D-glUCitOl and ~ - m a n n i t o l . ~ ~

-

-

(96) Z. Cekovic, J. Serb. Chem..51 (1986)205-211. (97) D. R. Hicks and B. Fraser-Reid, Can. J. Chem., 52 (1974) 3367-3372. (98) B. Fraser-Reid and D. R. Hicks, J. Chetn. SOC.,Chem. Commun.. (1972) 19-20. (99) R. S. Tipson and A. Cohen, Curbohydr.Res., 7 (1968) 232-243. (99a) T. L. Lowary and G. N. Richards, Curbohydr. Res. 198 (1990) 79-89. (99b) J. Barbier, J.-P. Boitiaux, P. Chaumette, S. Lepoq, J.X. Menezo and C. Montassier, (Institut FranGais du Petrol/Groupment d'hteret Economique dit: Sucre Recherches et Developpements) EP 380,402 (1989); Chem. Abstr., 114 (1991) 143,915.

1,4 :3,6-DIANHYDROHEXITOLS

125

'CH20H[

I HO~CH

I

Ms03CH

H+

I

H4COMS I

H5COH

I

~ C H ~ O H

CH 20H I

-

o,YH 'CH

Hi

I

HC \ I

HC'

yield,

20% HO

0

I

49

CH 2 0 H 40

SCHEME I

Treatment of 2,6-dibromo-~-glucitolwith aqueous potassium hydroxide in 74% yield via rearrangement through gave 1,4 : 3,6-dianhydro-~-glucitol, epoxide migration. During this reaction all four chiral C-atoms become inverted, thus generating the L-enantiomer of isosorbide.* V. DERIVATIVES

1. Esters

a. Esters with Carboxylic and Sulfonic Acids. -The relative reactivities of hydroxyl groups in carbohydrate derivatives have been discussed in a review." (99c) K.Bock, I. M.Castilla, I. Lundt, and C . Pedemn, Acta Chem. Scand., 43 (1989)264268.

126

PETER STOS AND REINHARD HEMMER

Among the isohexides, isosorbide (3) has two hydroxyl groups and these are in different stericsurroundings;that is, the OH-2 group in the exoand the OH-5 group in the endo position of the bicyclic ring-system, Many attempts have been made to control the regioselectivity of reactions at these positions. In 3, an intramolecular hydrogen bond between the endehydroxyl group on C-5 to the ring-oxygen atom between C-1 and C-4 was confirmed by spectroscopic methods (see Section 111). The hydrogen bond enhances the nucleophilic properties and also the reactivity of the OH-5 group in respect to the exo-hydroxyl group on C-2. From the examples in Table IX, it may be recognized that there are some reaction conditionsallowingreactions wherein the 2-ex0 position is more or less favored in respect to the 5-endo position, but the results do not allow a real prediction of the reaction pathway leading to only one of the desired products. It seems clear that conditions which enhance the nucleophilic character of the OH-5 group favor an increasing yield of 5-acylated products (see examples 3,6,7, 13 - 15 and I7 in Table IX). As activating and activated intermediates, such structures as those in Structure 6 have been proposed.loZ A preference for the exo-2-hydroxylgroup is also found (seeexamples 1,2, 4,8,10- 12and 16 in Table IX). Because of the heterogeneity of the reaction conditions, a clearly recognizable reason for this preference does not exist. It seems that mainly the fact of strong steric hindrance and the lack of activation of the endo-5-hydroxyl group lead to a preference for the exo-2-hydroxyl group. Example 15 in Table IX shows a result which is explicable by such a dianionic structureLo2as that depicted in Structure 6, where the attacking electrophile reacts with the less sterically hindered (and more dissociated?) ex0 group, but change of the solvent from the less coordinating N,N-dimethylformamideto the coordinating 1,Zdimethoxyethane reverses the proportions of the endo and ex0 products. An alternative explanation starts from the fact that acyl- (5Oa) and sulfonyl- (Sob)pyridinium (50) ions are effective acylating agents whereby the acylating group of the salt 50 is oriented to the proximity of that hydroxyl group, which is activated by polar interactions between the neighboring oxygen atoms with the positively charged nitrogen atom of the pyridine ring. 102,103 ( 1 0 ) S. Ropuszynski, H. Matyschok, and M. Rzepka, Przem. Chem.. 48 (1969) 665-668; Chem. Abstr., 72 (1970) 79,370. (101) J. Plucinski, W. Durda, and S. Sinicka, Przegl. Nauk. Inst. Technol. 0%.Tworzyw Sztucznych Politech. Wroclaw, 10 (1971) 3- 14; Chem. Abstr., 76 (1972) 14,835. (102) G. Le Lem, P. Boullanger, G. Descotes, and E. Wimmer, Bull. Soc.Chim. Fr., (1988) 567-570. (103) S. A. Abbas and A. H. Haines, Carbohydr.Res., 39 (1975) 358-363.

TABLE IX Acylatiod of Isosorbide Example No.

Acylation Reagent

1

1-benzoylimidazole benzoyl cyanide benzoyl chloride

2 3 4 5

acetic anhydride

6 7 8 9 10 11

12 13 14 15 16

17

acetic acid propanoic acid pivalic acid benzoyl chloride acetyl chloride pivaloyl chloride

Acylated IS Reaction conditions chloroform, reflux, 10 h acetonitrile, Net,, 0.5 h pyridine, 10 h at 20" Bu,N+CI-, benzene, 20% NaOH pyridine, 24 h at 25" pyridine. HCI, 2 h at 25" PbO, 20-40 h at 20" basic catalyst, 120-40" pyridine, 24 h at 20" CH2C12, DCCD, 4-DMAP, 3 h 0" 42 h 5h pyridine, CH2C12,4 h at - 80" 2.5 h 2.2 equiv. NaH, hexane, DMF, 2.5 h at 0" 2.2 equiv. NaH. hexane, DME, 1.5 h at 25"

2-0 1.4 1.16 I 3.8 1.7 1

15.2 1.43 5.7 36 12 I .3 1 1

5.5

I

5-0

2,5-0

Ref

1.0 1.0 3.8 1.4

1.35 1.57 3.3

1

3.3 3.9 1 4

103 103 103 108 109 109 104 i04 102 110 110

4 1

110 110

-

102 102 102 102

3.5 16 1

1 1

12.5 5 10

I 2.5

1

1

1

-

"Bu = C&; DCCD = dicyclohexylcarbodiimide,CDMAP = 4-dimethylaminopyridine; DMF = N,N-dimelhylformamide, DME = 1,2-dimefhoxyethane.

128

PETER STOSS AND REINHARD HEMMER

R a Z=C b Z-SO

50

In connection with the elaboration of specific procedures for the manufacture of both of the isosorbide mononitrates, methods were developed to shift, with high regioselectivity, the course of the acylation reaction either towards the 5(endu) or the 2(exo) position, depending on the conditions applied.104-106 Thus, isosorbide 5-acylates are obtained in 70-85% yield when 3 is treated with the appropriate acid anhydride in the presence of lead(I1) oxide or acetate for 20 to 40 h at room temperature. To avoid transesterifications to the appropriate 2-acylate, the workup procedures, especially during the distillation, have to be conducted under strictly neutral conditions. The contrary effect, a remarkable enrichment by the isosorbide 2-acylate (up to 90%)is realized by a transesterificationprocess mediated by a small proportion of sodium or potassium carbonate, hydroxide, or methoxide, which are added to the acylation mixture from isosorbide at 120140", and subsequent removal of the 2-acylate by vacuum distillation.Iw These two regioselectiveacylation methods may be used for aliphatic, as well as for aromatic, carboxylic acid anhydrides. Isosorbide 2-acetate may also be prepared by transesterification of equimolar amounts of isosorbide 2,S-diacetate and isosorbide in the presence of acidic or basic catalysts (KOH orptoluenesulfonic acid, for 1 h at 140",and d i ~ t i l l a t i o n ~of ~ Jthe ~ ' 2-acetate at 100" at 27 kPa; 89%). Subsequent esterification of the resulting isosorbide mononitrates with acyl and aroyl halides P. Stoss, P. Merrath, and G. Schliiter, Synthesis, (1987) 174- 176. P. Stoss(HeinrichMackNachf.),DE 3,124,410( 1981); Chem.Abstr.,98 (1983) 161,103. P. Stoss (Heinrich Mack Nachf.), EP 57,847 (1981); Chem. Abstr., 98 (1983) 54,395. K. Schoenafinger(Cassella AG), DE 3,128,102 (1981); Chem. Abstr., 99 (1983) 22,845. W. Szeja, J. Chem. SOC.,Chem. Commun.. (1981) 215-216. K. W. Buck, J. M. Dwbury, A. B. Foster, A. R. Perry, and J. M. W e b k , Curbohydr. Rex, 2 (1966) 122-131. (1 10) Z. Cekovic and Z. Tokic, Synthesis, (1989) 610-612.

(104) (105) (106) (107) (108) (109)

1,4 :3,6-DM"DROHEXITOLS

129

has been des~ribed.~~Jl I 2,5-Di-O-acetyl-~-isomannide was obtained'l2 in 60%yield from 1,4-anhydro-~-mannitoI(43) by treatment with HBr-saturated acetic acid for 1 h at 20". In the course of a stereospecificsynthesis of was obtained in 41% (+)-muscarhe, 2,5-di-O-benzoyl-~-isomannide yield during acid-catalyzed cyclization of D-mannitol. lH-N.m.r. data and the optical rotation were provided.112.Perfluoroalkylated mono- and di-esters of D-isomannide and D-isosorbide were prepared and shown to display moderate surfactant activities. IZb Kinetic studies of the esterification of isohexides were camed out for long-chain fatty acids.1wJ13-116 Search for optimal reaction conditions for these processes has been d e s ~ r i b e d . ~ ~ ~ - ~ ~ Some special esters of isohexides (prepared conventionally, however) have been described, including diestersof several long-chain fatty acids121~1zz and dicarboxylic acids,123 isomannide-mono-oleate, isosorbide dinicotinate,lZ5and perfluorinated long-chain fatty acid diesters of isosorbide and isomannide.lZ6 Bis(chloroformate)s of isosorbide and isomannide, and the

-

(1 1 I ) P. Chiesi and V. Servadio (Chiesi Farrnaceutici S. p. A.), EP 290,885 (1987); Chem. Abstr., 11 1 (1989) 39,821. (1 12) C. Pedersen, K. Bock, and I. Lundt, Pure Appl. Chem., 50 (1978) 1385- 1400. (1 12a) A. M. Mubarak and D. M. Brown, J. Chem. SOC.,Perkin Trans 1. (1982) 809-8 13.

(1 12b) L. Zarif, J. Greiner, and J. G. Rim, J. Fluorine Chem. 44 (1989) 73-85. ( 113) W.Jasinski and S. Ropuszynski, Przegl. Nauk. Inst. Technol. Org. Tworzyw Sztucznych Poljtech. Wrocluw, 12 (1973) 75-96; Chem. Abstr., 79 (1973) 146,767. (114) W. Jasinski and S . Ropuszynski, Chem. Stosow., 17 (1973) 83-99; Chem. Abstr., 79 (1973) 53,702. ( 1 15) H. Matyschok and S . Ropuszynski, Przegl. Nauk. Inst. Technol. Org. Tworzyw Sztucznych Politech. Wroclaw, 13 (1973) 377-387; Chem. Abstr., 81 (1974) 3,311. (116) S. Ropuszynski and W.Jasinski, Przem. Chem., 52 (1973) 96-99; Chem. Abstr., 78 (1973) 158,873. ( 1 17) W. Jasinski and S. Ropuszynski, Przegl. Nauk. Inst. Technol. Org. Tworzyw Sztucznych Politech. Wroclaw,12 (1973) 97- 114; Chem. Abstr., 80 (1974) 61,387. (1 18) W. Jasinski and S . Ropuszynski,Przegl. Nauk. Inst. Technol. Org. Tworzyw Sztucznych Politech. Wroclaw, 12 (1973) 31-74; Chem. Abstr., 79 (1973) 146,765. (1 19) S. Ropuszynski, J. Perka, and W.Jasinski, Przem. Chem., 48 (1969) 340-343; Chem. Abstr., 71 (1969) 40,581. (120) S. Ropuszynski, W. Jasinski, and J. Perka, Przem. Chem., 49 (1970) 222-225; Chem. Abstr., 73 (1970) 56,336. (121) Res. Djscl., 158 (1977) 45-47; Chem. Abstr., 88 (1978) 14,243. (122) W. H. Knightly (Atlas Chem. Ind. Inc.), U.S.Pat. 3,394,009 (1964); Chem. Abstr., 69 (1968) 67,666. (123) Courtaulds Ltd.,NL 6,405,497m (1963); Chem. Abstr., 62 (1965) 10,587h. (124) R. F. Czaja and R. J. Tull (Merck and Co., Inc.), G.B. Pat. 1,374,325 (1972); Chem. Abstr., 83 (1975) 43,684. ( 125) Aspro-Nicholas Ltd., FR M 23 18 ( 1962); Chem. Abstr., 6 1 ( 1964) 7 15c. (126) B. Charpiot, J. Greiner, M. Le Blanc, A. Manfredi, J. Riess, and L. Zarif (Atta), EP 255,443 (1986); Chem. Abstr., 110 (1989) 75,969.

130

PETER STOSS AND REINHARD HEMMER

manufacture of polycarbonates and polyurethanes from them, have been d e ~ c r i b e d . ~ ~ .Starting ~ ~ J ~ ’from J ~ ~ its aminocrotonates, cardiovascularlyactive 1,4-dihydropyridine-3(5)-carboxylicacid esters of isohexides have been prepared.129J30 Sulfonic acid esters of isohexides, which are useful educts for several substitution reactions, can be prepared principally in the same way as their carboxylic analogs, starting with the isohexide and the appropriate sulfonyl chloride in the presence of a 2-0-Acylisosorbide 5-sulfates are formed13*by reaction of SO3with long-chain (C, -C 16) 2-0-acylisosorbides in 1,4-dioxane-dichloromethane during 5 h at -40”. b. Esters with Phosphoric Acid. -Isohexide derivatives of phosphoric acids were not described in earlier reviews. Therefore, older articles are also referred to here.

51

52a 2-endo 52b 2-ex0

(127) H. Medem, M. Schreckenberg, R. Dhein, W. Nouvertne, and H. Rudolph (Bayer AG), DE 2,938,464 (1979); Chem. Abstr., 95 (1981) 44,118. (128) J. Thiem and H. Lueders, Polym. Bull., 11 (1984) 365-369. (129) P. Stossand M. Leitold (Heinnch Mack Nachf.), DE 3,248,548 (1982); Chem.Abstr., 102 (1985) 149,715. ( 1 30) K. Schoenafinger,H. Bohn, P. A. Martorana, and R. E. Nitz (CassellaAG), DE 3,43 1,152 (1984); Chem. Abstr., 105 (1986) 114,915.

1,4:3,6-DIANHYDROHEXITOLS

131

Phosphoric acid esters of the type depicted in 51 were described in a Polish patent. 133 Isomannide and isosorbide bis[2,5-(dipropylphosphite)] (52a and 52b) have been obtainedlM by transesterification of propyloxydipropylphosphane (53) or dipropylamidodipropylphosphane (54) with the isohexide for 5 h at 150" and 142 kPa.

55

55a: 2-endo 55b: 2-exo

X = halogen

Reaction of 52 with benzyl chloride opens134a new way to the deoxyhalogenoisohexides (55). The oxidation of 52 with air or sulfur transforms the phosphinites into the phosphonates (56a) and thiophosphonates (56b), respectively (see Scheme 8). With 15% sodium hydroxide, the phosphonates X V 3 H 7

b

0-P \,C3H7 II

56

56a

X C3H7

X=O

56b X = S SCHEME8

(131)V. P. Araya, IndiunJ. Chem.,Sect. B, 16 (1978)153-155. (132)Y.Saheki, K.Negoro, and T. Sasaki, J. Am. Oil Chem. Soc.,63 (1986)927-930. (133)S. Ropuszynski and W. Jasinski (Politechnika Wroclawska), PL 68,094(1969);Chem. Absfr.,80 (1974)146,476.

PETER STOSS AND REINHARD HEMMER

132

can be readily hydrolyzed to the free isohexide in almost quantitative ~ie1d.l~' The bis(tetramethy1phosphoricdiamide) ester (57) of isosorbide has been obtained by reaction of 3 with hexamethylphosphrictriamide. Transamination with ethyleneimine leads13sto the tetrakis(aziridide)58 (see Scheme 9).

CN'P'

N'1 1 0

OPZ

4Tw bPZ 57

58

a: Z=NMe2 b z=NEt, SCHEME 9

An acidic monosubstituted isosorbide phosphinite (a), whose position of substitution is not clear, is formed136from isosorbide and 59 (see Scheme 10). HO

,OH CH3-P

3

5 h;

175" OEt

\

b

b?

,CHj

OP

59

'OH

60

SCHEME 10 ( 1 34) K. A. Petrov, E. E. Nifant'ev, A. A. Shchegolev, and N. A. Khudyatsev, Zh. Obshch.

Khim., 32 (1962) 3074-3080; Chem. Abstr., 58 (1963) 11,456a. (135) E. E. Nifant'ev, A. I. Zavalishiaa, and M. R. Ter-Ovanesyan, Zh. Obshch. Khim., 39 (1969) 360-365; Chem. Abstr., 70 (1969) 114,890. (136) K. A. Petrov, E. E. Nifant'ev, A. A. Shchqolev, and A. P. Tuseev, Zh. Obshch. Khim., 34 (1964) 690-693; Chem. Abstr., 60 (1964) 14,579h.

1,4:3,6-DIANHYDROHEXITOU

133

c. Esters of Nitric Acid.-Isohexides form mono- and di-nitrates when treated with nitric acid. In most of the published proceduresfor the synthesis of mononitrates, an isohexide 2-acylate (mostlythe 2-acetate) is treated with mixtures of nitric acid (of different concentrations, 65 - 100%)with acetic acid or acetic anhydride, or both the substrate is dissolved in d i c h l ~ r o m e t h a n e , ~c~h”l- o~ r~o~ f o ~ m ,acetic ~ ~ ~acid, - ~ ~or mixtures with acetic anhydride,145-152 or used without a ~ ~ l ~ e normally n t , ~at -~15~to ~ ~ ~ ~ 30 After a workup step, where, in most cases, the ac(et)ylgroup is hydrolyzed with a basic medium (sodium hydroxide or methoxide, or potassium hydroxide or carbonate), isohexide mononitrates are isolated. (The reaction

+

O.

(1 37) S.Krauze, E.Gromadzinska, J. Oj~zanowski, J. Grelewicz, E. Kdczycki, J. Frize, and E.

Olejniczak (Osrodek Badawczo-Rozwojowy Przemyslu Barwnikow “Organika”), PL 138,011 (1984);Chem. Abstr., 110 (1989)154,806. (138)K. Schoenafinger(CassellaAG),DE 3,117,612(1981);Chem.Abstr., 98 (1983)179,822. (139)T.Gizur, K.Harsanyi, L. Demeter, J. Vikar, and Z. Vincze (Richter Gedeon), HU 42,498(1985);Chem. Abstr., 109 (1988)129,586. (140) W. Loesel, 0.Roos, and F. Esser (Boehringer Ingelheim KG), DE 3,123,719(1981); Chem. Abstr., 99 (1983)38,772. (141)Sanol Schwarz-Monheim GmbH, DE 2,903,927 (1979); Chem. Abstr., 93 (1980) 239,846. (142)K. Messing, S. S. Chattejee, and B. L. Gabard (Dr.Willmar Schwabe GmbH), EP 44,940(1980);Chem. Abstr., 96 (1982)218,188. (143)K. Messing and S. S. Chattejee (Dr. Willmar Schwabe GmbH), EP 44,928 (1980); Chem. Abstr., 96 (1982)200,111. (144)K. Messing and S. S. Chattejee (Dr. Willmar Schwabe GmbH), EP 44,927 (1980); Chem.Absfr.,96 (1982)218,190. (145)C. H. Chou and G. S. Myers (American Home Products Corp.), U.S. Pat. 4,065,488 (1977);Chem.Abstr., 88 (1978)121,660. (146)W. Dvonch and H. E. Album (American Home Products Corp.), DE 2,221,080(1971); Chem.Abstr., 78 (1973)43,942. (146a)J. Bron, G. J. Sterk,J. F. van der Wed, and H. Timmerman (CedonaPharmaceuticalsB. V.) EP 359,335 (1988);Chem. Abstr. 113 (1990)184719. (147)K. Messing and S. S. Chattejee (Dr. Willmar Schwabe GmbH), EP 44,931 (1980); Chem. Abstr., 97 (1982)24,163. (148)A. Gallardo Camera (Fordonal S. A.), ES 518,807 (1983);Chem. Abstr., 101 (1984) 91,399. (1 49) T.Ito, S.Ishiguro, F. Shimada, and K. Ishibashi (Toshin Chemical Co.,Ltd.), EP 143,507 (1983);Chem.Abstr., 104 (1986)6,138. (150)K. Lauer and E. Kiegel (Boehringer Mannheim GmbH), DE 3,028,873(1980);Chem. Abstr., 96 (1982)143,258. (151)L.D. Hayward, US. Pat. 4,721,796(1983);Chem. Abstr., 109 (1988)6,893. ( 1 52) K. Saito and T. Masuko (Tao Eiyo Kagaku Kogyo Co., Ltd.), Jpn. Pat. 83 18 385 (198I); Chem. Abstr., 99 (1983)53,726. (153)K.Schoenafinger (Cassella AG), EP 64,194(1981);Chem. Abstr., 98 (1983)179,822. (154) P.StossandM.Leitold(HeinrichMackNachf.),DE 3,602,067(1986);Chem.Abstr., 107 (1987)176,017. (155) P. Stoss (HeinrichMack Nachf.), DE 3,102,947(1981);Chem.Abstr., 98(1983)54,395.

PETER STOSS AND REINHARD HEMMER

134

most reported leads to the formation of isosorbide 5-nitrate.) In some cases, the resulting isosorbide mononitrate mixtures must be separated chromatographically.146~152 Aminodeoxyisohexides were nitrated107J42-144 with a reagent prepared from fuming nitric acid, sulfuric acid, and urea at - 15 '. Also, isohexides substituted with a purine base have been nitrated143with nitric acid- urea. Some other methods for preparing isohexide mononitrates have also been reported. Thus, isosorbide dinitrate can be partially hydrolyzedby treatment with 4 N hydrochloricacid for 192 h at 37 .The separation of the 24x0 and 5-end0 nitrates was carried out by chromatography on silica gel.19 Isosorbide 2-nitrate (10) has been prepared by nitration of isomannide 2-trifluoromethanesulfonate(61) with the phase-transfer reagent tetrabutylammonium nitrate in boiling acetone'56(see Scheme 1 l), or by regioselecO

HO

b

[N( C 4 H 9 ) 41+N03c

10

acetone

b

OS02CF3

61

SCHEME 11

tive 5-acylation and subsequent nitration at the 2-positi0n.'~~ The reagents FeSO,. 7 H 2 0(8 1% of 2-11itrate),~~J~' CuC1, (45% of 2-nitrate),I5l7powdered zinc (75% of 5-nit~ite),'~J~~ palladium-on-carbon in the presence of NiC1, (6 1% of h ~ i t r a t e ) ,hydrazine '~~ hydrate,159or N,N-dimethylhydrazine(with no significant enrichment by one of the possible mononitratesg2)have been used to cleave isosorbide dinitrate into the mononitrates named. Sodium isosorbide hitrate, containing various different proportions of crystal water, was prepared in 46% yield by adding fuming nitric acid to isosorbide dissolved in a mixture of acetic acid, acetic anhydride, and ben( I 56) Sanol Schwan-Monheim GmbH, DE 2,903,983 (1979); Chem. Abstr., 94 (198 I ) 175,445. (157) E. Camera, 0. De Lucchi, F. Filipuzzi, and G. Modena (Consiglio Nazionale delle Ricerche), EP 266,450 ( 1986); Chem. Abslr., 109 (1988) 23,3 13. ( I 58) E. Camera, F. Filipuzzi, 0. De Lucchi, and G . Modena (Consiglio Nazionale delle Ricerche; Dinamite S.p. A.), EP 201,067 (1985); Chem. Abstr., 106 (1987) 67,625. ( I 59) J. M. Emeury and E. Wimmer ( M e t e Nationale des Poudres et Explosifs), EP 59,664 (1981); Chem. Abstr., 99 (1983) 5,968.

1,4:3,6-DIANHYDROHEXITOLS

135

zene at 30°, and treating149the resulting mixture (after neutralization to pH 7) with 30% NaOH. Optimum conditions for the preparation, purification, and crystallization of isosorbide dinitrate have been e1ab0rated.l~-lci3 The bioconversion of isosorbide dinitrate into the mononitrates by various micro-organisms has been investigated.164J*165 Stable inclusion complexes of isosorbide 5-mononitrate using cyclomaltoheptaose166J66a have been prepared. 2. Ethers a. Alkyl Ethers.-As a general principle, the alkylation of 1,4 : 3,6-dianhydrohexitols is not as easy to achieve as it appears at first sight. With the exception of some “simple” alkylating agents, such as methyl iodide, dimethyl sulfate, ally1 bromide, benzyl bromide, and the like, building up an ether linkage requires special reaction conditions in order to give satisfactory yields. Of course, dialkylation products are easier to prepare and isolate than are monoalkyl derivatives. Attempts to achieve favored monoalkylation have always resulted in mixtures of starting material and mono- and di-alkylation products. The situation is further complicated in the case of isosorbide as the substrate; two different mono-ethers are formed, corresponding to the 2(exo) and Sfendo) positions. Thus, in contrast to a regioselective monoacylation,lW a similar monoalkylation of isosorbide has not as yet been observed. As a consequence, in most cases, the preparation of dialkyl isohexides is described in the literature. Some mono ethers have also been prepared, bearing different acyl groups on the second hydroxyl group of the ring system. Only very few examples of monoalkyl derivatives lacking further substituents are known. (160) E.Gromadzinska,S.Krauze, andJ. Ojrzanowski,ActaPol.Pharm., 43 (1986)416-419; Chem. Abstr., 106 (1987)214,246. (161)J. Feldmann, H. Koebernick, K. Richter, and H. U. Woelk (Maizena GmbH), DE 3,230,349(1982);Chem. Abstr., 101 (1984)7,594. (162) R. Nec, CS 216,771 (1981);Chem. Abstr.. 102 (1985)79,286. (163)J. Ojrzanowski,E. Gromadzinska,and S. Krauze, ActaPol. Pharm., 43 (1986)567-571; Chem. Abstr., 108 (1988)222,008. (164)M. Lenfant, J. Ropenga, and E.Wimmer (CentreNational de la Recherche Scientifique; SocieteNationaledesPoudresetExplosifs), EP252,855(1986);Chem.Abstr., I08(1988) 185,278. (164a) J. S. Ropenga and M. Lenfant, Appl. Microbiol. Biotechnol., 27 (1988)358 - 36 1. (165)J. S. Ropenga and M. Lenfant, Appl. Microbiol. Biofechnol.,26 (1987)1 1 7- 119. (166)Tao Eiyo Kagaku Kogyo Co., Ltd., Jpn. Pat. 84 93 032 (1982);Chem. Absfr., 101 (1984) 230,946. (166a) K.Uekama, K.Oh, T. Ine, M. Otagiri, Y. Nishimiya, and T. Nara, Int. J. Pharm. 25 (1985)339-346.

PETER STOSS AND REINHARD HEMMER

136

In the mid-sixties, Atlas Chemical Industries investigated the reaction of epoxideswith isohexides. Transformationof isosorbide (3) by ethylene oxide affords polyoxyethylene isosorbide (62), which was treated with oleic acid to afford the corresponding diesters (63), and these were further transformed into epoxidized products 16' (64) (see Scheme 12). On treating all three iso-

62

R1

=

64

63

(CH2)7CH=CH (CH2),CH3 YO,

R2 = (CH2)7CH-CH(CH2)-/CH3

SCHEME 12

hexides (65) with epichlorhydrin (66), low yields of the corresponding bisglycidyl ethers (67) result168(see Scheme 13). Oxyethylation of isosorbide monoesters gives 5-0-acyl- 1,4 :3,6-dianhydro-~-glucitol2-ethylene oxide polycondensates (68 - 70). The reactivity of the three isosorbide esters investigated was found169to decrease in the sequence octadecanoate > dodecanoate > oleate. The poor reactivity of isohexides towards epichloroHO

65

67

SCHEME 13 (167) J. W. Le Maistre and E. C. Ford (Atlas Chem. Ind., Inc.), U.S. Pat. 3,225,067 (1962); Chem. Abstr., 64 (1966) 9,904. (168) J. D. Zech and J. W. Le Maistre (Atlas Chem. Ind., Inc.), U.S. Pat. 3,272,845 (1963); Chem. Abstr., 65 (1966) 20,205. (169) S. Ropuszynski and W. Jasinski, Przegl. Nauk. Znst. Technol. Org. TworzywSztucznych Politech. Wroclaw, 3 (1971) 15-38; Chem. Abstr., 76 (1972) 47,613.

1,4:3,6-DIANHYDROHEXITOLS

137

hydrin has been confirmed. It turned out to be advantageous to apply a two-step sequence for the synthesis of 72 from 71 by reaction with allyl bromide (73)and subsequent epoxidation of the allyl intermediate 74 with rn-chloroperoxybenzoic acid (75). 0-(2,3-Epoxypropyl)isohexides(72) were

m

RO

I

:

6 L O W

68 R = octadecanoyl 69 R=dodecanoyl 70 R = oleoyl

then used 170~171to generate the so-called “p-blocker side-chain” in compounds 76 containing different amine residues (see Scheme 14). R’0

R’0

H N R ~ R ~

i-l

-

71 OH

$4 72 R’O

73

R = H, NO,

0 74

76

OH

SCHEME 14

Epoxides 72 may also act as starting materials for hybrid structures 77 containing an isohexide and a glyceryl moiety, with nitric ester functions at different positions.172 Additional monoethers of isosorbide 2- and 5-nitrate 78 were synthesized by reaction of the free hydroxyl group with any of several alkyl iodides in the presence of freshly prepared silver oxide. The yields were low, as u ~ u a l . Amongst ’~ a larger series of different alcohol (170)P.Stoss and M. Leitold (Heinrich Mack. Nachf.), DE 3,421,072(1984);Chem. Abstr., 106 (1987)18,986. (171)P. Stoss, M. Leitold, and R. A. Yeates (Heinrich Mack Nachf.), EP 319,030 (1987); Chem. Abstr., 112 (1990)210,988. ( 172) P.Stoss, G.Schlueter, and R. Axmann, Arzneim. Forsch., 40 ( 1990) 13- 18.

PETER STOSS AND REINHARD HEMMER

138

77

78

nitrates, three benzyl ether derivatives of isosorbide 5-nitrate were mentioned.'& A different approach was used by messing and Chatte~jee.'~~ Isosorbide 5-methanesulfonate (79) reacted with 4-chlorophenol, affording the Walden-inverted isoidide derivative 80, which was transformed into the appropriate nitrate 81 (see Scheme 15).

-

-

OH

-

m - cq 0-CgHqC1-P

0-C6H4 C1-p c

ONOZ

OH 80 SCHEME15

79

81

In the course of the synthesis173of oxaprostaglandinsfrom 1,4: 3,6-dianhydro-D-glucitol, the latter was first monotosylatedat the 5-position and the ester benzylated, to afford 82. Elimination of the tosyl oxy group under special conditions yields the enolethers 83 and 84 (see Scheme 16) as a 2 : 1 mixture which can be separated by column chromatography. OTs

Me3COK

-

Me2S0

OCH2C6H5

82

OCHZC6 H5 83 SCHEME16

OCH2C6H5 84

(173) J. Thiem and H. Lueders, Justus Liebigs Ann. Chem., (1985) 2151 -2164.

I ,4 :3,6-DIANHYDROHEXITOLS

139

Another series of monoakylated isohexides (87) has been prepared as nucleoside analogs, with the bicyclic carbohydrate being linked like a glycoside, replacing the normal sugars D-ribose or 2-deoxy-~-erythro-hexose.174,175 For their preparation, isohexide monoacylates (85), previously synthesized with high regioselectivity,'04 were chloromethylated to 86, and subsequently reacted with numerous pyrimidine and tnazole bases to yield 87 (see Scheme 17).

86

85

87

SCHEME 11

Etherification of isohexides with substituted-benzylchloride in aqueous sodium hydroxide, or by means of sodium hydride in dimethyl sulfoxide, yields mixtures of mono- and bis-ethers, which can be conventionally separated by distillation or by column ~hromatography.'~~ The preparation of some phenyl ethers was also described, using the tosylate-phenoxide exchange reaction. Monoethers (88)synthesized in this way were transformed into carbamates (89) by reaction with sodium cyanide- trifluoroacetic acid (see Scheme 18). RO

RO

%

OH

OCONH~

89

88 R = subst. phenyl or subst. benzyl SCHEME18

(174) P. Stoss and E. Kaes (Heinrich Mack. Nachf.), DE 3,606,634 (1986); Chem.Abstr., 108 (1988) 38,315. ( 175) P. Stoss and E. Kaes, Nucleos. Nucleot., I ( 1988) 2 13 - 225. (176) J. W.LeMaistreandT.P.Mori(ICIAmericas,Inc.),U.S.Pat.4,169,152(1977);Chem. Abstr., 92 (1980) 94,676.

PETER STOSS AND REINHARD HEMMER

140

For lower dialkyl isohexides (go), especially 2,5-di-O-methylisosorbide, which is used as solvent for organic reactions or for pharmaceutical dosage formulations, several manufacturing processes have been reported. The most widely applied method, consisting of the reaction of the freediols (65) with dimethyl sulfate or methyl iodide, was improved by employing special conditions (see Scheme 19). Thus, acetone in the presence of 50% aqueous RO L

R2S04 65

or RI

SCHEME 19

sodium hydroxide was employed177as a solvent in the production of 90 (R = CH,) by methylation of isosorbide with dimethyl sulfate. An improved yield was claimed for use of tert-butanol as the solvent for isosorbide and simultaneous addition of aqueous sodium hydroxide and dimethyl sulfate.17* The application of phase-transfer conditionsfor the successfulsynthesisof di-0-methylisosorbide was demonstrated.179 The same group investigated the alkylation of isosorbide with a series of alkyl bromides, involving a solid-liquid phase transfer in weakly hydrated organic mixtures.lsO A different approach for the synthesis of di-0-methylisosorbideand other lower di-0-alkylated derivatives, using chloromethane in different solvent systems, with or without the aid of additional phase-transfer catalysts, was the subject of a patent application.18* In addition,a one-vessel dehydrationmethylation reaction starting from D-glucitol was mentioned. Mixtures of mono- and di-0-methylisosorbide resulted on alkylation of the diol with

(177) R. L. Hillard and I. D. Greene (American Cyanamid Co.), US.Pat. 4,322,359 (1981); Chem. Abstr., 97 (1982) 6,732. (1 78) M. Maurer, W. Orth, and W. Fickert (RuetgerswerkeAG), DE 3,521,809 (1 985); Chem. Abslr., 106 (1987) 120,177. (179) D. Achet, D. Rocrelle, I. Murengezi, M. Delmas, and A. Gaset, Synthesis, (1986) 642643. (180) D. Achet, M. Delmas, and A. G w t , Biomass, 9 (1986) 247-254. (181) W. M. Kruseand J. F. Stephen(IC1Americas, Inc.), EP92,998( 1982);Chem.Abstr., 100 (1984) 103,815.

1,4:3,6-DIAMiYDROHEXITOLS

141

dimethyl carbonate in the presence of a base as catalyst.1822,SDi-O-pentylisosorbide, characterized as a liquid by an optical rotation value, was mentioned in a contribution183;however, its preparation was referred to an unpublished paper. A Russian group described an example of an unsaturated ether formed by treating isosorbide (3)and isomannide (4) with acetylene. By hydrogenationofthe vinyl ethers(91), 2,5-di-O-ethylisohexides (92) were obtained'" (see Scheme 20).

3,4

OCH=CH 2

-

OEt

HCzCH

KOH-1,4-Dioxane

~CH=CH~

OEt

91

92 0

0-CH=CH-0-C-C, If //CH2

A COZH

CH3

91 2

%

JI

0-CH=CH-O-C-C

93

4'CHJ 3 2

0

SCHEME20

Reaction of 91 with methacrylic acid gives rise to double unsaturated side-chain compounds (93);these were subjected to polymerizati~n.~~~ Subjecting isosorbide to etherification with a,w-dihaloalkanes, mono- and dialkyl derivatives were obtained. These could not be transformed into polyethers. The use of trans-l,.l-dichlorobutenelead to oligomers up to the (182) J. N. Greenshields (ICI Americas, Inc.), U.S. Pat. 4,770,871 (1987); Chem.Absfr., 110 (1989) 63,514. (183) V . Vill, F. Fisher, and J. Them, 2. Nufudorsch..TeilA, 43 (1988) 1119- 1125. (184) B. I. Mikhant'ev, V. L. Lapenkov, and A. I. Slivkin, Zh. Obshch. Khim., 42 (1972) 2302-2303; Chem. Abstr., 78 (1973) 72,485. (185) V. L. Lapemkovand A. I. Slivkin,Monomery Vysokomol.Soedin.,(1973) 73-77; Chm. Abstr., 81 (1974) 37,869.

PETER STOSS AND REINHARD HEMMER

142

heptamer.lESa The dipicrate (94) of isomannide was first described IE6 in 1971. Pentafluorophenyl ethers 95 and 96 were formed by reaction of the diols with hexafluorobenzene.lE7

:xz

R’O

OZN

\

OR2

95

R’ = H, R2 = C,F,

Finally, isosorbide was tritylated to afford both of the monotrityl isomers, which were separated chromatographically.The 2-0-trityl derivative97 was then used for the preparation of isosorbide 5-nitrate (11) by way of intermediateI5*98 (see Scheme 21). Earlier reports on both of the 2,5-di-O-trityl OH

@ -

OTr

OTr 97

98 SCHEME 21

derivatives of isosorbide and isomannidewere corrected on repetition of the synthesis under more appropriate conditions, and unambiguous characterization of these compounds188was achieved. As they are diols, isohexides can be used to act as starting materials for crown ether derivatives. This type of application was first reported in 1981, when the isomannide compound 28 was prepared and investigated for its conformational behavior26and complexing properties.” During investigation of chiral crown ethers and podands containing one or two isomannide moieties, the alkylation behavior of this bis-endo-oriented diol was studied in (185a) J. Thiem, T. Hiirining, and W. A. Strietholt, Starch/Sidrke 41 (1989) 4- 10. (186) M. L. Sinnott and M. C. Whiting, J. Chem. Soc., B, (1971) 965-975. (187) A. H. Haines and K. C. Symes, J. Chem. Soc.. Perkin Trans. 1, (1973) 53-56. ( I 88) P. A. Finan and J. P. Reidy, J. Chem. Rex, Synop., (1989) 69.

1,4 : 3,6-DIANHYDROHEXITOLS

143

more detail. Bulky substituents seem to impede dialkylation because of steric hindrance. By using a superbasic medium, these difficulties could be overcome.189*190 Alkylating isomannide with 2-(2-bromoethoxy)tetrahydropyran under these conditions, followed by deprotection, treatment with thionyl chloride, and condensation of the product with isomannide or 8-hydroxyquinoline yields compounds 99-102,and other derivativeswere prepared in this way. Following this, additional podands were prepared,'* and investigations of the complexation ability of 101 with different chiral ammonium salts were performed.IwbSeveral congeners of the podands 100 display affinity to Na and Li cations, but scarcely bind K ions.'*

99

A novel approach to the production of chiral, polymeric, crown ethers incorporating isomannide was developed by a Japanese group. The optically active divinyl ether 103 was polymerized with cationic catalysts to afford 104, consisting of only cyclic constitutional units.lgl In addition, another crown ether (lOS),containing five ethylenedioxy moieties, was prepared. It is worth mentioning that the crystal and molecular structure of the known intramolecular ether 1,4 : 2,5 :3,6-trianhydro-~-mannitol(33), consisting of three fused furanoid rings, was the subject of a

(189) E. A. El'perina, R. 1. Abylgaziev, M. I. Struchkova, and E. P. Serebryakov, Zm.Akad. Nauk SSSR, Ser. Khim., (1988) 627-632; Chem. Abstr.. I 1 1 (1989) 58,204. (190) E. A. El'perina, R. I. Abylgaziev, and E. P. Serebryakov, In. Akad. Nauk SSSR. Ser. Khim., (1988) 632-637; Chem. Abstr.. 11 I (1989) 58,205. (190a) E. A. El'perina, E. P. Serebryakov, and M. I. Struchkova, Heterocycks 28 (1989) 805812. (190b) M. I. Struchkova, E. A. El'perina, R. I. Abylgaziev, and E. P. Serebryakov, Izv. Akad. NaukSSSR,Ser Khim. (1989) 2492-2500; Chem. Abstr., 112 (1990) 217,371. ( 1 90c) M. I. Struchkova, E. A. El'perina, L. M. Suslova, R. I. Abylgaziev, and E. P. Serebryakov, I n .Akad. Nauk SSSR, Ser. Khim. (1989) 2501-2504; Chem. Abstr., 112 (1990) 166,236. (191) T. Kakuchi, T. Takaoka, and K. Yokota, Mucrornol. Chem., 189 (1988) 2007-2016.

144

PETER STOSS AND REINHARD HEMMER

b

100

c

R =

R , R =

101

n n m

oh

~owoL/o

0

102

103

I ,4 :3,6-DIANHYDROHEXITOLS

145

105

b. Silyl Ethers.-To date, there is only one report involving the preparation of silyl ethers ofadianhydrohexitol.Compounds106 and 107 have been RO

-

R = H,COCH,

107

PETER STOSS A N D REINHARD HEMMER

146

obtained by treatment of isomannide or its diacetate with diethoxydimethylsilane.lg2During gas chromatography- mass spectrometricdetermination of isosorbide 5-mononitratein human serum, a silylation reaction was used. The presence of pyridineand heating at 80' lead to formation of a trimethylsilyl ether not only at C-2, where a free hydroxyl is present, but also at C-5, where the nitro group is r e p 1 a ~ e d . l ~ ~ 3. Deoxy Derivatives

a. Mono- and Di-unsubstituted.-Only a few articles on mono- and dideoxyisohexides, saturated as well as unsaturated, have appeared in the literature. As the earlier work was not fully covered by Soltzberg's article$ it seemed reasonable to include the few missing papers in the present article. A small amount of an unsaturated amine derivative 110 was isolated by Cope and Shenlg3;it was probably formed as aresult ofthe elimination of the less reactive tosylate group of isosorbide 2,5-ditosylate(108)under the influence of the dimethylamine reagent at 120" (see Scheme 22).

OTs

109 SCHEME 22

108

110

At 165 both tosyloxy groupsin 108were replaced with inversion, and the bis(dimethylamin0)-D-ghcitol derivative was obtained. On heating isosorbide (3)or its diacetate (111)in the presence of such dehydrating agents as aluminum oxide in a Pyrex-glass tube above 400°, the doubly unsaturated compound 112 is formed in - 50% yield1%(see Scheme 23). A further example of an unsaturated isohexide was publishedL73 in 1985. When 2-0-benzyl-5-0-tosylisosorbide(82) was subjected to elimination by potassium tert-butoxide, a 2 : 1 mixture of the corresponding benzylated O,

(192) B. Pavare, 0. Lukevica, and L. Maijs, Latv. PSR Zinat. Akad. Vestis,Kim. Ser., (1973) 234-238; Chem. Abstr., 79 (1973) 137,228. (192a) P. Zuccaro, S. M.Zuccaro, R. Pacifici, S. Pichini, and L. Boniforti,J. Chromatogr. 525 (1990) 447-453. (193) A. C . Cope. andT. Y. Shen, J. Am. Chem. Soc., 78 (1956) 3177-3182. (194) H. Hopff and A. Lehmann (DEGUSSA), DE 952,092 (1955); Chem. Abstr., 53 (1959)

2,2526.

1,4:3,6-DIANHYDROHEXITOLS

147

a- 1 OAc

Ac20

3

400"

-

\

500'

112

C

SCHEME 23

enol ethers83 and 84 was obtained (see Scheme 17). Experimentsto demonstrate the isomerization of compound 84, which is of limited stability even at -20°, failed. Also, as a result of a partial elimination of the exo-tosylate 108 by azide, which is a sterically hindered reaction, the unsaturated azido isohexide 113 was ~ b t a i n e d * (see ~ J ~Scheme ~ 24).

-

_N3

NaN3

108

113 SCHEME 24

The synthesis of the unsubstituted dideoxyisohexide parent compounds was performed by Cope and Shen.196J97 Isomannide dichloride (114) has been converted into ~-cis-2,6-dioxabicyclo[3.3.O]octane (115)by hydrogenolysis. The L enantiomer 117 was obtained by reaction of D-1,6-diacetoxy3,4-hexanediol ditosylate (116) with sodium methoxide (see Scheme 25). Presumably, compound 115 has also been isolated, as an extremely volatile liquid, in 15%yield from a complex mixture of other bicyclic ethers by oxidation of 1,6-hexanediol (118) with lead t e t r a a ~ e t a t e ~(see ~ ~ Scheme ,'~

(1 95) H. Lueders, Ph. D. Thesis, University ofHamburg, Federal Republic ofGermany (1984). (196)A.C.CopeandT.Y.Shen, J. Am. Chem. Soc., 78(1956)5916-5920. (197)A.C.CopeandT.Y.Shen,U.S.Pat.2,932,650(1960);Chem.Abstr., 54(1960)24,7996. (198)V.M. Micovic, S.Stojcic, S.Mladenovic, and M. Stefanovic, TetrahedronLett., (1965) 1559-1563. (199)V.M. Micovic, S.Stojcic, M.Bralovic, S.Mladenovic, D. Jeremic, and M. Stefanovic, Tetrahedron, 25 (1969)985-993.

PETER STOSS AND REINHARD HEMMER

148

a H

‘b

*

H2 Ni Raney

tl

H

114

115

H NaOCH3 Ac 0

OAc

H

116

117 SCHEME 25

HO

Pb ( O A c ) -

.

=

P b (OAc)

*-

115

/

OH

118

119 SCHEME 26

26). The authors confirmed the structure of a cis-lY4-dioxaperhydropentalene on the basis of the IH-n.m.r. spectrum, without mention of the D or L configuration. A similar approach was used by Mihailovic and coworkers.16When 2-tetrahydrofuranethanol(ll9) was treated with lead tetraacetate, an intramolecular ring-closure occurred, to give a 45% yield of (R,R)-czs-2,6-dioxabicyclo[3.3.0]octane (115), together with seven other compounds in minor proportions. Compound 115 was prepared from D-mannitol by following the established procedure of Cope and Shen,193and was used as an intermediate for the first synthesis of thiacy~lodeca-4,7-diene.~ Finally, several OH H O - 0 I

HO

\

OCHZR

- 4 4

9 Hd

bCHZR

-a ‘r

OCH2R

121

120

122

SCHEME 27 (200) V. Cere,E. Dalcanale, C.Paolucci, S. Pollicino, E. Sandri, L. Lunazzi, and A. Fava, J. Org. Chm.,47 (1982) 3540-3544.

1,4:3,6-DIANHYDROHEXITOI-S

149

monodeoxy derivatives(122) have been synthesized,for use asherbicides, by a ring-closure reaction starting from substituted diols 121 preparedz0' in a multi-step sequence from tetrahydrofurandiols 120 (see Scheme 27).

b. Halogens.-Examples of the direct halogenation of 1,4: 3,6-dianhydrohexitols during the reported period are rather rare. More frequently, nucleophilic displacement of such other substituents as mesylates or tosylates by halogens have been applied. An interesting contribution using the Arbusov reaction appeared from a Russian groupmz;they studied 1,4 :3,6dianhydro-D-glucitol 2,5-bis(tetraethylphosphorodiamidites) as examples for replacement of secondaryhydroxyl groupsby halogens in carbohydrates. When the appropriate isomannide analogue of (57) reacted with benzyl chloride at 130°, a 79% yield of the dichloro-L-isoidide (123) resulted (see Scheme 28). In contrast, no dihalogeno derivative was isolated from the isosorbide derivative. c1

67b*

C6H5CH2C1

t

Cq

130"

Cl ' bis(endo)isomer

123 SCHEME28

During gas-liquid chromatographic- mass spectrometric analysis of the acid-catalyzed dehydration reaction of D-mannitol, 1,4 :3,6-dianhydro-2chloro-2-deoxy-~-man~tol was found among the reaction products.3sCon~ according to which 2,5-di-endo trary to the postulated S N mechanism, oriented leaving-groups are substituted by different nucleophiles, resulting L-iditol), an isomerhation takes in 2,Sdi-exo derivatives(D-mannitol place in the presence of sodium iodide.203Reaction of 1,4 :3,6-dianhydro2,5-di-O-mesyl- (124) and -tosyl-D-mannitolwith sodium iodide gave a 1 : I mixture of 2,5-dideoxy-2,5-diiodo-~-iditol (126) and -D-glucitol (128). 1,4 :3,6-Dianhydro-2-deoxy-2-iodo-5-O-rnesyl-~-gluc~tol (125) and the corresponding D-mannitol derivative 127 are formed as intermediates (see Scheme 29). This unusual isomerization reaction is restricted to starting

-

(201)K.M.Sun (Shell Int. Res.), EP 264,978(1987);Chem. Abstr., 109 (1988)68,856. (202)E. E. Nifant'ev, M. P. Koroteev, and N. S. Rabovskaya, Zh. Obshch. Khim.,43 (1973) 1806- 181 1; Chem. Abstr., 79 (1973)137,414. (203)J. Kuszmann and G. Medgyes, Curbohydr. Rex. 64 (1978)135- 142.

PETER STOSS AND REINHARD HEMMER

150

OMS

tqNa I

-

I 125

't

T

OMS

cq

124

I

127 SCHEME 29

materials having the manno (bis-endo) configuration. Other nucleophiles, such as benzoate, bromide, phthalimide, or thiobenzoate, behave normally. The configuration of these isomers was determined unambiguously by l3C-n.m,r. spe~troscopy.~~ Configurational inversion in chlorine displacement of methanesulfonatesof isomannide and isosorbide, affording chlorodeoxyisosorbide and chlorodeoxyisoidide was confirmed by "C-n.m.r. investigations.*' A process for the chlorination of alcohols by causing the alcohol to react with triphenylphosphaneoxide and thionyl chloridewas also demonstrated for isosorbide among other substrates.2wIt is worth mentioning that, on brief treatment of 1,4-anhydro-~-glucitoland -D-mannitol with and -manhydrogen bromide in acetic acid, only 2,5-di-O-acetyliso-sorbide nide could be isolated, whereas, after prolonged reaction, only brominated ring-opened products result. No 1,4 :3,6-dianhydro-bromohexitolswere detected.l A study of alkyllithium-promotedring fissions of dideoxydihaloisohexides,as a source for several homochiral synthons, was published.205* 121205

c. Amines.-The exchange of the sulfonyloxy groups of sulfonylated 1,4 :3,6-dianhydrohexitolsby nucleophiles, including amines, was discussed

briefly in an earlier article.' It is known that a considerable difference in the ease of reaction exists between endo and e m displacement,and attention has (204) E. A. OBrien, T. OConner, M. R. J. Tuite, and L. High (McNeil Lab., Inc.), GB 2,182,039 (1985); Chem. Absfr., 108 (1988) 187,199. (205) K. Bock, P. Gammeltoft, and C. Pedersen, Acta Chem. Scand. Ser. B, 33 (1979) 429432. (205a) V. Cere, C. Paolucci, S. Pollicino, E. Sandri, and A.Fava, Tetrahedron Lett. 30 (1989) 6737 -6740.

1,4:3,6-DIANHYDROHEXITOLS

151

to be paid to possible Walden inversion. In the meantime, a large number of different amino-substituted isohexides have been synthesized, albeit described in only a few publications. The aim of most of this work was the generation of potential new drugs, or monomers for synthesizing polymeric compounds. The last-mentioned application was the aim of a patent206dating from 1971. 4,8-Diamino-2,6-dioxabicyclo[ 3.3.0]octanes, endo-endo as well as endo-em isomers, were used as starting materials for polyamides. First, the diamines 129 were transformed into their salts with various dicarboxylic acids, and these were polymerized to compounds 130 containing acylated amino moieties as monomeric building blocks (see Scheme 30).

-+

NN-CO- ( C H 2 j n - C 0 . .

COZH

$ 1

NH2

+

(CH214 CQ2H

-

formation

.

M-cc- ( c H 2 ) n - C 0 .

129

..

130

SCHEME 30

A similar approach was used by Thiem and L ~ e d e r s ~ ' for . ~ ~generating J~~ polyurethanes 132 and 133 from 129 or aliphatic diamines and 1,4:3,6dianhydrohexitol-derivedbis(ch1oroformates) 131. A preparation for 129, starting from mesylates and proceeding by way of azides, was provided in this connection (see Scheme 3 1). On evaluating the behavior of 1,4 :3,6-dianhydro-2,5-di-O-mesyl-~mannitol(l24) towards different nucleophiles, the doubly exchanged product 134 was mainly obtained,2o3in addition to a small proportion of the D-glucitol derivative 135 (see Scheme 32). Mesylates and tosylates of isosorbide and isomannide undergo nucleophilic displacement with a number of primary and secondary amines, as described in a pharmaceutically oriented p~blication.'~' Several patents describe the preparation of numerous aminodeoxyisohexide derivatives bearing an additional nitric ester group at the 2 or 5 position of the isohexide ring-~ystem.'~~144,207 Among these, purines and purine-alkylamines were (206)L. P.Friz, G. Anzuino, and D. Schiattarella(Montedison Fibre S. p. A.), D E 2,262,319 (1971);Chem. Abstr., 79 (1973)137,869. (207) K.Klessing (Dr.Willmar Schwabe GmbH), EP 44,932 (1980); Chem. Abstr., 97 (1982) 110,337.

PETER STOSS A N D REINHARD HEMMER

152

1,

NH-c-0

mOCOCl

tI

0

132

&OCl

5

131

0

0-C-NH-

..

(CHZ),-"El.

H2N- (CH21,-NHZ

SCHEME 31

124

PhthNk

-

N-Phth

OMS

& j + -N-Phth

134

-

N-Phth

135

SCHEME 32

used as amine moieties. They were synthesized for their potential application as cardiovascularagents. The same applies to a large number of piperazine-substituted deoxyisohexide nitrates, which have recently been synthesized via mesylates.wIO (207a) F. Suzuki, H. Hayashi, T. Kuroda, K. Kubo, and J. Ikeda (Kyowa Hakko Kogyo Co.) EP 393574 (1989); Chem. Abstr.: no reference up to Vol. 115 (1991) No. 2.

1,4:3,6-DIANHYDROHEXITOLS

153

d. Thio Derivatives.-Among the few examples in the literature, there are no unsubstituted mercaptans and no S-alkyl derivatives; only acylated thio derivativeshave been described. From I ,4:3,6-dianhydro-~-mannitol octaethyldiamidophosphite(bis(endo)isomer of 57b), by reactionwitb benzyl bromide, the diphosphonium salt 136, a compound that can be transformedm into 1,4 :3,6-dianhydro-2,5-dideoxy-2,5-(dithiocyano)-~-iditol (137) was obtained (seeScheme 33).

SCN

PhCH2Br 67b'

*

-

0-P-NEt2

SCN

1

CH2 Ph

137

136 * bis(endo)isomer

SCHEME 33

1,4:3,6-Dianhydro-2,5-di-i-O-bemoyl-2,S-dithio-~-iditol (138), together with 1,4:3,6d~nhydro-2-9benzoyl-5-~-methylsulfonyl-2-thio-~-glucitol (139) was preparedm3from isomannide 2,5-dimesylate (124) (see Scheme 34). On use of a longer reaction time, compound 138 was the sole product. OMS

SBZ c C6H5COSK

124

SBZ 138

SBZ 139

SCHEME 34

(208)

N. K. Kochetkov, E. E. Nibt'ev, and M.P.Koroteev, D&. A M . Nauk SSSR,194 (1970)587-590;Chem. A h . , 74 (1971)76,608.

PETER STOSS AND REINHARD HEMMER

154

e. Azides. -In 1980, all three 2,Sdiazides of 1,4 :3,6-dianhydro-2,5dideoxyhexitols were prepared for the first time.17JwReaction of 124 or the appropriate ditosylate With sodium azide for 2 h at 120” afforded the 2,5diazido-L-isoidide derivative 20. When the 2,5-di-O-mesyl- or -tosyl-D-isosorbide derivative (tosyi derivative 108)was similarly treated, the reaction temperature had to be increased to the boiling point of the N,N-dimethylformamide used as the solvent, and a reaction time of 4.5 h was needed in order to complete the replacement of both ester groups to afford 17. Attempts to replace the 2,5-situated mesyloxy groups in the L-isoidide derivative by azide were unsuccessful, as the starting material remained unchanged. Therefore, a “reversed” synthesis was carried out for 18, by first introducing the azido groups at C-2 and C-5 in properly substituted, acyclic hexitols 140, and then closingthe anhydro ring. Both the D and L diazidoisomannides were synthesized by a similar strategy (see Scheme 35). Compounds 20 and 17 were later prepared by an identical reaction, and 18 was also obtained from the ditosylate of ~-isoidide.~’J~~ These azides were then submitted to hydrogenolysis to afford the corresponding diamines.

NaN3

124

& -

2 h / 120°

NaN3

ioa

L

4.5 h / 160”

N3

17 MsO

N3

to N3

H O / ”

OH NaOCH3-

N3

OMS

A

N3

ia

140 SCHEME 35

(209) J. Kuszmann,G. Medgyes, F. Andrasi, and P. Berzsenyi (GyogyszerkutatoIntenet), HU 20,368 (1980); Chem. Absfr., 96 (1982) 200,099.

1,4:3,6-DIANHYDROHEXITOLS

155

f. Phosphanes. -Only one example of the preparation of a phosphorussubstituted isohexide derivative has been published since 1970.The chiral 27 has been prediphosphane of 1,4 : 3,6-dianhydro-2,5-dideoxy-~-iditol pared28from isomannide by way of its ditosylate 141 (see Scheme 36). LLP ( P h z )

*

27

b OTs

141 SCHEME36

g. C-Nitro Compounds. -The first example of dideoxyisohexideC-nitro compounds, in addition to the widely known nitrate esters, have been prepared recently. exo-4,8-Dinitro-, exo-4,4,8-trinitro-, and exo-4,4,8,8-tetranitro-2,6-dioxabicyclo[3.3.O]octanes resulted from reaction of 20 via diamine 129 (exu-amino bonds). All of the nitro derivatives had explosive properties. However there was no indication concerningtheir phannacological behavior.209a 4. Oxidation Products

The first example of an oxidation reaction with defined isolated products in the 1,4 :3,6-dianhydrohexitol series was reported210in 1963. Catalytic oxidation with oxygen in the presence of Adams' catalyst transformed and -D-glucitol(3) into the correspond1,4 : 3,6-dianhydro-~-mannitol(4) ing monoketones (8a) and (8b), respectively, whereas the L-iditol epimer (5) remained unchanged. As in the case of 3, only the monoketone was observed,this type of reaction turned out to be stereospecific, affecting only endo-disposed hydroxyl groups. This conclusion was substantiated by a longer reaction time for 4, which contains two endo hydroxyl groups, affording the diketone 9 (see Scheme 37). In addition to the parent ketones, some derivatives(such as the 2,4-dinitrophenylhydrazonesand ptolylsulfonylhydrazones) have been prepared by the same authors, and this work was covered by a patent?" A more detailed investigation of the major reaction parameters influencing the platinum-catalyzed oxidation of isosorbide (3)was undertaken2I2in (209a) T.G . Archibald and K. Baum, Synth. Commun. 19 (1989) 1493- 1498. (210) K. Heyns, W.-P. Trautwein, and H. Paulsen, Chem. Ber., 96 (1963) 3195-3199. (21 I ) Atlas Chem. Ind., Inc., FR 1,426,204(1963); Chem. Absfr.,65 (1966) 15,490. (212) F. Jacquet, C. Granado, L. Rigal,and A. Gaset, Appl. Cutul., 18 (1985) 157-172.

PETER STOSS A N D REINHARD HEMMER

156

HO

to A

OH

4

HO

tq OH

3

rn

HO c

-

02/Pt

-

no r e a c t i o n

OH 5

SCHEME 37

1985. The reaction was then optimized for maximum yield ofthe monoketone21h 8b. In contrast to those results, no useful discrimination between endo and ex0 hydroxyl groups could be found when employing ruthenium tetraoxide as an oxidant?” Compounds 4,3, and 5 each affords the diketone 9 exclusively; this was isolated as the bis(2,4-dinitrophenylhydrazone). During a search for additional synthetic possibilities, a French group an electrochemical oxidation of isosorbide to provide the monoketone 8b,accompanied by small proportions of the diketone 9. This greater susceptibility of the endo hydroxyl function to anodic oxidation was confirmedZLS by demonstrating that isomannide (4) was transformed into 8a and 9. (212a) F. Jacquet,L. Rigal, and A. Gaset, J. Chem. Techno[.Biotechnol.48 (1990) 493-506. (2 13) P.M. Collins,P. T. Doganges, A. Kolarikol, and W.G. Overend, Carhhydr. Res.. 1 1 (1969) 199-206. (2 14) F. Jacquet, A. Gaset,J. Sirnonet, and G.Lacoste,Elektrochim. Aaa, 30 ( 1985) 477-484. (215) G. Fleche, A. Gaset,and F. Jacquet (Roquette FreresS. A.), ElJ 125,986 (1983); Chm. Absrr., 102 (1985) 69,340.

1,4 :3,6-DIA"YDROHEXITOU

157

Monoketones containing additional ex0 or endo nitric ester groups have been synthesized.'" Starting from the appropriate nitrates 11 and 10, pyridinium chlorochromate as the oxidizing agent has been used for their preparation. The endo and the ex0 hydroxyl function were oxidizedin comparable yield. The opposite reaction sequence, by nitration of the monoketones 8a and 8b, was also successfully employed (see Scheme 38).

-

HNO) 8a \\ 0

OH 11

142

HO

to

-

-

0

HNOj

-

8b

;NO2

ON02

10

143

SCHEME 38

Compounds 142 and 143 could be transformed into a variety of derivatives, such as acetals, oximes, semicarbazones, and hydrazones.l' Other types of monoketonederived products, especially oxime ethers 144 containing a 3-amino-2-(hydroxypropyl)side-chain, have been synthesized and evaluated as potential drugs216(see Scheme 39).

1 42,

143

-

ONOZ

to

\\N - O T N /

OH

1

'R2

144

SCHEME 39

(216)P.StmandM.Leitold(HeinrichMackNachf.),DE3,704,604(1987);Chem. Abstr., 110 ( 1989) 75,47 1.

PETER STOSS AND REINHARD HEMMER

158

On submitting D-isomannide 2,5-dinitrate (15) to flash vacuum thermo(145) was obtained as the main lysis, 1,4 :3,6-dianhydro-~-mannopyranose product (75 - 80%yield), accompanied by 5 - 10%ofthe monoketone 8a. In contrast, when D-isosorbide 2,5-dinitrate (14) was treated under the same conditions, a 6 :3 : 1 mixture consisting2”of 8a, 145, and its D-gluco epimer 146resulted (see Scheme 40). These resultscould be explainedby a restricted number of rearrangements ofintermediateradicals, with inversion or retention of configuration, respectively. By reduction with sodium borohydride, 8a was stereoselectively converted into D-isomannide (4). ON02

tQ

< -

0 400-450’

c 6 . 7 Pa

OH

+

aa

ON02

15

145

400-450’ c

+ & h + 1 4 5 +

6 . 1 Pa

146

14

SCHEME 40

VI. APPLICATIONS 1. Chemical Uses

Under mild conditions, isosorbide (3)is converted into 1,6-dichloro-1,6dideoxy-D-glucitol (147) by reaction with boron trichloride (Scheme 4 1). Compound 147, which was not isolated in pure form, was allowed to react with benzaldehyde to afford 2,4-O-benzylidene- 1,6-dichloro-1,6ddeoxyD-glucitol(148)and 2,4 :3,5-di-O-benzylidene-1,6-dichloro-1,6-dideoxy-~glucitol(l49). By this method, the two tetrahydrofuran rings are cleaved in a very mild manner.218 (217)J. G.Batelaan,A. J. M.Weber, and U. E. Wiersum, J. Chem. Soc.,Chem. Commun., ( 1987) 1397- 1399. (218)M.A. Bukhari, A. B. Foster, and J. M. Webber, Carbohydr. Res., 1 (1966)474-481.

1,4 :3,6-DIANHYDROHEXITOLS

I59

-

to -

'CHZCl

OH

OH

C6 H CHO

BClj

C H 2 C 1 2 , 30 h at - 8 0 " -rt

b H CH2C1

3

147

CH2Cl

C1-CH2

148

149

SCHEME 41

Isosorbide (3)as a starting material from the "chiral pool" is the educt for the ten-step enantioselective ~ynthesis''~of 11deoxy-8-epi-1 1-0xaprostaglandin Fa (150a) and its (15R)diastereoisomer (150b) (see Scheme 42). (R,R)-cis-2,6-Dioxabicyclo[3.3.0]octane (115) was the starting compound

w

--

OH

OH

10 steps

OH

3

150

a:

R'

=

b:

R'

= OK, R2 = H SCHEME 42

H, R~ = OH

PETER STOSS AND REINHARD HEMMER

160

151

115

SCHEME 43

for the c h i d (E,E)-thiacyclodeca4,7diene(151a)and its 3-methyl derivative (151b);the latter hasm a helical shape (see Scheme 43). Isosorbide (3)and isomannide (4)act as chiral auxiliaries for the sodium borohydride reduction of some prochiral ketones; optical yields of up to 20% were achieved. It seems that the isohexides form chiral complexes with sodium borohydride, whereby the chiral information is transferred to the ~ubstrate.2~~ Optical active alcohols were obtained by reduction of appropriate ketones with sodium or lithium borohydride in the presence of isosorAsymmetric reduction of propiophenone using sodium borohydride, modified with (+)-camphoric acid and isosorbide, resulted in (S)-phenylethylcarbinolin 35% enantiomeric excess.219b Similar results were obtained with lithium aluminum hydride, using 4 as the c h i d ligand, by complexing the dihydridoaluminate- ketone adduct in the transition state, as shown*%in Structure 9. In this case, the optical yield stays below 5%. In all cases described, the S isomer is formed in excess.?20

STRUCTURE 9

The chiral crown ethers 101,102,104,105, and 152,and podands 100and 153 were formed from isomannide by introducing ethylene glycol units as (219) A. Hirao, H. Mochizuki, S. Nakahama, and N. Yamazaki, J. Org. Chem., 44 (1979) 1720- 1722. (219a) Teijin Ltd. JP 80,120,526 (1979); Chem. Abstr., 94 (1981) 156,521. (219b) H. H. zoorOb, Egypt. J. Chm. 29 (1986) 333-338. (220) N. Baggett and P. Stribblehill, J. Chem. Soc.,Perkin Trans. I , (1977) 1123- I 126.

1,4:3,6-DIANHYDROHEXITOLS

161

152 153

N &

bridges over one molecule, or between two molecules of i~omannide,3~J~~191 or between isomannide and other heterocyclic end-groups.IE9 Compounds 28 (ref. 31), 105 (ref. 19l), and 104were found to be capableof chiral recognition of saltsof racemic primary amines” and a-amino acids,191 forming host -guest complexes, mainly hydrogen bridges between the ammonium group and the lone pairs of the surrounding ether oxygen atoms. An isohexide monoester (not specified) has been used as an adjuvant for the synthesis of organic sulfides and oligosulfides.221 2. Pharmaceutical Uses

The continuous worldwide application of isosorbide dinitrate and, since the early eighties, of isosorbide 5-mononitrate also, has given rise to a very great number of publicationson different aspects of these drugs. It would be beyond the scope of this report to include all of the work on these two important compounds. The present synopsis is therefore restricted to other novel isohexidederivativesand to some new pharmaceuticalapplicationsof known compounds. Because of the known vasodilating activity of isohexide nitric esters, several attempts were made to improve or modify these compounds by introducing additional substituents at the second hydroxyl group. On the one hand, this type of molecular modification would influence the polarity and (221)

R. Kolta, K.Mihalszky, I. Cseko, G.Lelki, P. Szalay, and D.Fazekas (Herceghalmi Kiserleti Gazdasag), HU 39,423(1984);Chem. Absfr.,107 (1987) 58,501.

162

PETER STOSS AND REINHARD HEMMER

lipophilicity of the derivativesand could therefore cause a different biological response. On the other hand, certain new substituents could themselves contribute to the biological activity and thus generate drugs having a new profile of action. With this intent, the acetates (R = COCH,, 78a), carbamates (R = CONH2, 78b), sulfamates (R = S02NH2, 78c), and ethyl ethers (R = C2H,, 78d) of isosorbide 2- and 5-nitrate were investigated, and found to be similar in activity to the parent compounds.146Several isohexide nitrates further substituted by purine bases 154 were prepared as potential cardiovascular agents.143

R-

ONOp 154

Additional aliphatic amine derivatives (155), including*42the so-called “/3-blockerside-chain”of aryloxypropanolamines(156), and also those having purinalkylamines as substituents144 (157), exhibit useful therapeutic activities. One compound in this series has undergone advanced clinical investigations under the international nonproprietary name (INN), “teopranitol.” Nicotinic esters (R= 3-pyridylcarbony1, 78e), 4-chlorophenyl ethers (R = 4-C1-CaH4,78f), and mesylates (R = S02CH,, 78g) of all three epimeric isohexides have been the subject of a patent appli~ati0n.l~’

155

1,4:3,6-DIANHYDROHEXITOLS

163

OH 157

156

An interesting acyl moiety containing the well known calcium channel blocking dihydropyridine structure was introduced into isohexides (R = H) and their nitrates (R = NO,), giving rise129to compounds of type 158. Somidipine(INN) (31)is one derivativeof this seriesthat is at present under clinical investigation.u2 Identical structureswere later used by other groups as intermediates to generate optically active dihydropyridinecalcium antagonistsImby way of transesterification of compound 158.It has been demonstrated that pharmaceuticals consisting of compounds 158 and congeners,

H 158

0-NH 159

combined With isohexidemononitratesor glycerol nitrates, exhibit favorable effectsagainst angina pectoris.2228 Among a number of other compounds, a few isosorbide sydnonimine derivatives 159 appeared in a cardiovascularoriented report.223 (222) P.Stoss, R.Hemmer, and P.Memth (Heinrich Mack Nachf.), DE 3,906,267(1989); Chew.Abstr., 114 (1991)81,807. (222a) P. Stoss, M. Leitold (H. Mack Nachf'.) EP 361,156 (1988);Chm. Abstr., 114 (1991) 240,625. (223)K.Schoenalinger, R. Beyerle, H. Bohn, M. Just, P. Martorana, and R. E. Nitz (Cassella AG), DE 3,526,068(1985);Chem. Absfr., 106 (1987)144,012.

164

PETER STOSS A N D REINHARD HEMMER

Using isosorbide 5-nitrate (11) as part of such special amino-substituted novel /?-blocker structures, compounds 160 were claimed to be useful for treatment of heart and circulatory disorders.u4 In contrast to the foregoing

compounds of an acylated isosorbide, isohexides were ether-linked and U S ~ ~ ~ ~ as O Jphenoxy ' ~ equivalentsin oxypropanolamine-likederivatives76.

Isosorbide 2- and 5-nitrates substituted at the second hydroxyl group by a number of aliphatic, aromatic, and cinnamic acids (78, R = various acyl groups) have been claimed to be useful in vasodilating therapy."' However, it was confirmed by the authors that there seems to be no direct correlation between lipophilicity and therapeutic activity, and that structure-activity relationships in the isosorbide nitrate area are more complex than that. An oxidized stage of isohexide nitrates, wherein the remaining hydroxyl group is transformed into the ketone, gives rise to a number of derivatives 161, such as oximes, semicarbazones, acyclic and cyclic acetals, and hydrazones. lS4 Among them, 3-amino-2-hydroxypropyl-substituted oxime ethers

161

OH 162

162 exhibit interesting hybrid properties as organic nitrates and /?-blocking

In an application dealing with antithrombotic and antihypertensivecompositions, the disulfite 163 ofisosorbidewas mentioned as one ofthe possible active ingredients.225Cardiovascularactivities were also claimed for a large (224) H. Simon, H. Michel, W. Bartsch, and K. Strein (Boehringer Mannheim GmbH), DE 3,512,627 (1985); Chern. Abstr., 106 (1987) 49,604. (225) B. K. Martin (T and R Chemicals,Inc.), EP 113,235 (1983); Chem. Abstr., 101 (1984) 157,677.

1,4 :3,6-DlANHYDROHEXITOLS

165

O-S-OH

0 163

number of piperazine-substituteddeoxyisohexide nitrates,ma and for three isosorbide 5-nitrate benzyl ether derivatives.146o In addition to the cardiovasculararea, to which all of the aforementioned derivativesbelong, isohexides have also been elaborated for other pharmaceutical indications. Several mono- and di-0-alkylisohexides, as well as monoalkylisohexide carbamates, were tested for anticonvulsant activity. 176 During an investigation of the hypnotic properties of 2,5-diazido-2,5-dideoxyisohexides (17-20), the L-isomannide derivative turned out to be comparable to the known gluthethimide, whereas the D-antipode was completely inactive. The D-glucitol compound showed significant hypnotic activity, but the L-isoidide derivative, none.”J@’ Among 138 examples of various sulfamoyl compounds, three isohexide derivativeswere mentioned. Their preparation started from corresponding free hydroxyl compounds by reaction with sulfamoyl chloride, which itself had been generated in situ from chlorosulfonyl isocyanate. They were claimed for treatment of chronic arthritis and osteoporosis.225a A number of isohexide mono- and di-amines (164) failed to exhibit antitumor activity. A few of them are weak antiflammatories, albeit rather X

0’

-NR’R* 154

t 0 ~ i c . l Potential ~’ antitumor and antiviral properties were the aim of the nucleoside-like compounds 87 containing several pyrimidine, triazole, and (225a) Y. S. Lo,J. C.Nolan,D. A. Walsh, and W. J. Welstead Jr., (A. H. Robins Co.) EP 403,185 (1989). Chem.Abstr.: no reference up to Vol. 115 (1991) No. 2.

166

PETER STOSS AND REINHARD HEMMER

165

imidazole nucleobases.174~175Some monodeoxyisohexide ethers 165 substituted with phenyl groups (R) were found to be useful as herbicides.m1 Nitroxide radical-forming agents, for example, all seven isohexide monoand di-nitrates, were claimed as ingredientsin topical formulations for stimulation of hair The widespread applicability of di-0-methylisosorbide as a medium for chemical reactions or as a solvent for pharmaceutical formulations is well documented. In some cases, an additive synergism of the solvent and the solutewas observed. Sometypical examples mentioned include that it acts as a solvent for muscle-relaxant drugs, which are otherwise difficultly soluble,226and is used for topical and other types of pharmaceutical formulati0ns,227J28 transdermal controlled-release films229and tapes:w anthelmintic sol~tions,2~* antimycotic emul~ions,2~~ and for the treatment of skin disorders, such as eczema.233 The antifungalactivity of thiabenzazoleagainst Penicillium digitatum was found to be enhanced by adding various carbohydrate esters of fatty acids. Among them, isosorbide monododecanoate was moderately active?” Isomannide mono-oleate has frequently been applied to generate highly stable water-in-oil type emulsions which could act as useful adjuvants for vaccines to enhance the efficacy of incorporated antigen^.^^^-*^^ Isosorbide mono- or (225b)P. H.Proctor (P. H. Proctor) EP 327,263(1988);Chem. Abstr., 112 (1990)204,461. (226) R. 0.Beauchamp,Jr., J. W. Ward, and B. V. Franko(A. H. Robins, Co.,Inc.), US.Pat. 3,699,230(1971);Chem. Abstr., 78 (1973)20,197. (227) J. L. Chen and J. M. Battaglia (E. R. Squibb and Sons, I c . ) , U.S. Pat. 4,082,881 (1976); Chem. Abstr., 89 (1978)117,860. (228) J. C.Dederen, Expo.-Congr.Int. Technol.Pharm., 3rd, (1983)335-336;Chem. Abstr., 103 (1985)76,165. (229) M.Dittgen and R. Bombor (Ernst-Moritz-Amdt-UniversityGreifswald), D D 217,989 (1983);Chem.Abstr., 103 (1985)147,168. (230)Y.Ito, T.Horiuchi, and S . Otsuka (Nitto Electric Ind. Co., Ltd.), Jpn. Pat. 86,221,121 (1985);Chern.Abstr., 106 (1987)107,914. (231) M.R.Clark and A. Lewis (May and Baker Ltd.), DE 3,442,402(1983);Chem.Abstr., 103 (1985) 183,558. (232)M.Wischniewski and L. Feicho (Kali-Chemie Pharma GmbH), DE 3,600,947(1986); Chern.Abstr.. 108 (1988)26,961. (233) L.A.LuzziandJ.K.Luzzi,U.S.Pat.4,711,904(1986);Chem.Abstr., 108(1988)82,146. (234) Y.Nishikawa and M. Ohkawa, Chem. Pharm. Bull., 36 (1988)3216-3219. (235) R. J. Tull (Merck and Co.,Inc.), DE 2,249,831(1927);Chem. Abstr., 81 (1974)13,752.

1,4 :3,6-DIANHYDROHEXITOLS

167

di-alkyl ethers, or mixtures thereof, with preference for the dimethyl compound, were claimed as being useful for dentifrice formulations.182*240 It is worth mentioning that the explosiveproperties of the vasodilatordrug isosorbide 2,5-dinitrate can be overcome by forming its 1 : 1 complex with cyclomaltoheptaose.21

3. Technical Applications a. Food. -The sensory properties(sweetnessand bitterness)of isosorbide and isomannide, among those of other carbohydrate derivatives, have been discussed on a molecular basis.242 Isosorbide dimethyl ether ( M a ) is used as an ingredient in the manufacture of chewing gums, chewable tablets, hard candies, and nougat products.240

Isosorbide dipropanoate (166b) is used as an effectivesoftening agent, as well as a fungistat, when incorporated into bakery products.122Ethoxylated fatty acid esters of isosorbide are used as conditioners in bread making.243 Isosorbide acts as an all-purpose, plastic, shortening material in the manufacture of cakes, icings, and cream fillings, producing excellent moisture retention and aeration properties.244A process for preparing benzaldehyde and acetaldehydetakes place in presence of water and a nonionic emulsifier. The latter containing mixtures of “sorbitan-” and isosorbide-fatty acid esters2*”.Moderate surface activities were reported on perfluoroalkylated mono and di-esters of isomannide and isosorbide.112b (236) Merck and Co., Inc., Jpn. Pat. 74 72 285 (1972); Chem.Abstr., 85 (1976) 21,766. (237) A. F. Woodhour and M. R. Hilleman (Merck and Co.,Inc.), US. Pat. 3,983,228 (197 1); Chem.Absrr., 85 (1976) 182,397. (238) M. Midler, Jr. and E. Paul (Merck and Co., Inc.), U.S. Pat. 4,073,743 (1975); Chem. Absrr., 88 (1978) 197,630. (239) B. Brancq and L. De Philippe (ProduitsChimiques de la Montagne Noir), FR 2,501,526 (1981); Chem. Abstr., 98 (1983) 59,877. (240) M. J. Lynch (ICI Americas Inc.), U.S.Pat. 4,585,649 (1984); Chem. Abstr., 105 (1986) 66,28 I . (241) M. Low,L. Kisfaludy, A. Vikman, J. Szejtli, I. Stadler,1. Gemesi, I. Kolbe, G. Hofhann, M. Gyannathy, and G. Hortobagy (Richter Gedeon), HU 37,801 (1984); Chem.Abstr.. 106 (1987) 143,984. (242) C. K. Lee and G. G . Birch, J. Food Sci., 40 (1975) 784-787. (243) R. K. Langhans (ICI Americas Inc.), U.S. Pat. 3,859,445 (197 I); Chem.Absrr., 82 (1 975) 154,058. (244) D. T. Rusch (AtlasChem. Ind., Inc.), U.S. Pat. 889,005 (1970); Chem.Abstr., 75 (1971) 117,364. (244a) A. 0.Pittet, R. Muralidharaand A. L. Liberman (Internat. Flavorsand Fragrances I c . ) U.S.Pat. 4,683,342 (1987); Chem. Absfr., 1 10 (1989) 22,530.

168

PETER STOSS AND REINHARD HEMMER

Isosorbide mono(tetradecanoate) (166d) prevents the denaturation of ground fish during freezing.z45Mixed ether-ester-substitutedisohexides (especially those of isosorbide) are used as flavor enhancers.z46

R20

b OR'

166

a R'=R2=CH 3 b R1 = R2 = COCH,CH, c R' or R2 = C0(CH2)&H3 d R' or R2 = CO(CH2)12CH3 e R' = R2 = COCH(C2HS)C4H, f R' = R2 = C0(CH2)&H3 g R' = R2 = CO(CH2)&H3 h R1= R2 = CO(CH2)&H3

i R' = H, RZ= Co(CH2),CH
R'

= H; R2 = CO(CH2)IICH=CH(CH2)7CH3

b. Cosmetics. -Long-chain fatty acid diesten of isosorbide (forexample, 166e)are used as base materials in cosmetic formulation^.*^^^^^^-^^ A physical testing method for the emulsifying and dispersing properties has been described.13zDi-0-methylisosorbideis used as a plasticizer in antiperspirant sticks.zs'

c. Other Applications.-Isohexides are used as chain extenders in the manufacture of polyurethane elastomers derived from 1,5-di(isocyanat0)naphthalene.~~~J~~ Isosorbide is used as a link between poly(ether)poly(ester)diolsand 1,5-di(i~ocyanato)naphthalene.*~~~~~ (245) H. Amano, C. Yoshida, and A. Nakamura (Kao Soap Co., Ltd.), Jpn. Pat. 80 07 017 (1978); Chem.Abstr., 93 (1980) 69,076. (246) C. Wiener and A. 0. Pittet (Intemat. Flavors and Fragrances Inc.), U.S.Pat. 4,617,4 19 (1985); Chem.Abstr., 106 (1987) 4,034. (247) Nihon Surfactants Industry Co., Ltd., Jpn. Pat. 84 175 408 (1983); Chem. Abstr., 102 (1985) 67,233. (248) M. Ochiai and T. Ozawa (Pola Chem. Ind. Co., Ltd.), Jpn. Pat. 78 45 379 (1975); Chem. Abstr., 90 (1979) 209,946. (249) H. Stuehler, E. Krempl, and A. Oberhauser (Hoechst AG), DE 3,119,553 (1981); Chem. Abstr., 98 (1983) 161,109. (250) Y .Greiche, P. Hartmann, and J. Kohler (Wella AG), EP 302,265 (1987); Chem. Abstr,, 1 1 1 (1989) 120,611. (25 I ) N. Geria (Bristol-MyersCo.), GB 2,139,496 (1983); Chem. Abstr., 102 (1985) 119,437. (252) M. Barnes and F. Fassbender (Bayer AG), DE 3,233,086 (1982); Chem. Abszr., 101 (1984) 24,838. (253) H. Meyborg, K. Wagner, J. M. Barnes, and H.Salzburg (Bayer AG), DE 3,111,093 (1981); Chem. Abstr., 98 (1983) 55,354. (254) M.J. Barnes and W. Betz (Bayer AG), DE 3,437,915 (1984); Chem. Abstr., 107 (1987) 40,98 1.

1,4 :3,6-DIANmROHEXITOLS

169

Isohexides exhibit excellent properties as plasticizers for polyvinyl alcohol polymers.255Compounds obtained from isosorbide and trialkylaluminum proved to be highly active cocatalysts for polymerization of alkenes. Such derivatives,which were supposed to be oligomeric U-aluminum-isosorbides, are of glass-likeappearance and exhibit pyrophoric properties.255a Isosorbide is a component of mixtures used for water-based pigment inks, having excellent dispersion stability, which is necessary for ink-jet printing.256*ZS7 Long and branched-chain fatty acid diesters of isosorbide (especially the diheptanoate 166f) can be used as solvents for color couplers, as well as stabilizers of color images in photographic light-sensitive materials. lZ1 Different diacylisohexideshave been used as components in silver halide emulsions for photographic material.2S7~2s7b Isosorbide di(octadecan0ate)(166g) and di(docosanoate)(166h) dissolved in cyclohexanecan act as antiblocking agents and as a lubricant coating for aluminum sheets.2s8 Isosorbide mono-oleate (166i) can be used as a dispersant for carbon, titanium dioxide, and other 2-Phosphatesof fatty acid esters of isosorbide, as well as their appropriate sodium salts, are used as excellent dispersant and emulsifying agents.' l7 By effectinga homeotropic arrangementof liquid-crystal molecules, small proportions of isosorbide monocarboxylates (alkyl group of the fatty acid chain ranging from C,,, to C,) eliminate the cloudiness present in liquidcrystal devices, even at zero voltage.260A number of long-chain fatty acid esters of jsohexides were studied as dopants for liquid-crystal mixtures. Fastswitching ferroelectric cells could be realized, and electroclinic effects were observed.z60.

(255) H. Salzburg, K. Reinking, and F. Kleiner (Bayer AG), DE 3,347,075 (1983); Chem. Abstr., 103 (1985) 196,882. (255a) W . Kaminsky and H. Miidler (Hoechst A G) EP 307,877 (1987); Chem. Abstr., 1I 1 (1989) 78,812. (256) Pentel Co., Ltd., Jpn. Pat. 85 72 968 (1983); Chem. Abstr., 103 (1985) 125,228. (257) Fuji Photo Film Co., Ltd., Jpn. Pat. 82 57 762 (1980); Chem. Abstr., 97 (1982) 74,093. (257a) T. Yagi and Y. Yamada (Konishiroku Photo Industry Co., Ltd.) Jpn. Pat. 87 173 457 (1986); Chem. Abstr., 108 (1988) 85,267. (257b) T. Yagi and Y. Yamada (Konishiroku Photo Industry Co., Ltd.) Jpn. Pat. 87 173 456 (1986); Chem. Abstr., 108 (1988) 85,268. (258) F. A. Hughes (Atlas Chem. Ind., Inc.), U.S. Pat. 3,468,701 (1966); Chem. Abstr., 72 (1970) 4,958. (259) Nihon Surfactants Industry Co., Ltd., Jpn. Pat. 84 177 122 (1983); Chem. Abstr., 102 (1985) 115,553. (260) A. Monyama, M.Fukai, K.A d , and K.M o n (MatsushitaElectric Ind. Co., Ltd.), Jpn. Pat. 74 74 681 (1972); Chem. Abstr., 82 (1975) 18,790. (260a) V.V i , F. Fischer, and J. Thiem,Z. Nuturforsch.,A: Php. Sci. 44 a (1989) 675-679.

PETER STOSS AND REINHARD HEMMER

170

0

?& 'L'

t10 -

OH

% The isosorbide diester 167 acts as an excellent antioxidant and heat stabilizer for several polymers.261 Isosorbide or isomannide derivatives 168 0-alkylatedwith certainphenyland heteroaryl-methylgroups at the endo position are used as pre-emergence herbicides for grass culture.2o1 Ar I

OH 168

Ar =

-Q

-@

;X=CI , F

X

(261) J. F.Stephen, J.H.Smith,andM.H.Meshreki(ICIAmericas,Inc.),U.S.Pat.4,613,638 (1985); Chem. Abstr., 106 (1987) 68,267.

1,4:3,4-DIANHYDROHEXITOLS

171

Optically black hydrocarbon films may be formed in aqueous media fiom solutions of isosorbide mono-oleate ( M i ) and its mono(truns-docosenoate) (166j) and phospholipids in decane.262 By addition of each of several diesters of isosorbide, isomannide, and isoidide to a nematic phase, cholesteric phases can be produced. All compounds exhibit a large twisting power. In the cholesteric phase, helix inversion, large or small temperature-dependenciesof the pitch, and broad blue phases were achieved.lE3 Attempts have been made to study the hazard potential of pure isosorbide dinitrate and mixtures of it with various proportions of lactose.82

d. Polymers Containing Isohexide Moieties (a Selection).-(i) Ethoxides.-Ethoxylated isosorbide monoesters (for example, 169)having long-

169

chain alkyl groups have been tested for their “Tween”-analogproperties.169 Compounds such as 170, esterified with long-chain carboxylic acids, are useful as plasticizers for vinyl-resin compositions.16’ 0

0 170

(262) G.M. W. Cook, W.R.Redwood, A. R. Taylor, andD. A. Haydon, Kolloid-Z. Z. Polym., 227 (1968) 28-37.

172

PETER STOSS AND REINHARD HEMMER

(ii) Polyurethanes, Polycarbonates, and Polyamides.-Homogeneous urethane rubbers or foams can be prepared by using isohexides, especially isosorbides, as chain extender^.^^^**^^ The resulting polymers are suitable for the production of films and coatings and as molded articles and foams.263A special sort of urethane (132) was prepared from the bis(ch1oroformate) (131) of isosorbide and 2,5-diamino-2,5-dideoxyisohexides(129) (see Scheme 31). Other types of a,&amines were also used. The interfacial polycondensationleads to polymers having an average molecular weightz3Js of 3000. For the manufacture of a transparent, tough, thermally stable polycarbonate, the bis(ch1oroformate)sof isosorbide and isomannide were copolymerized with such dihydroxy compounds as dihydroxybenzenes and aliphatic diols by using the interfacial condensation method.1z7 Difunctionalized phenolic compounds of the general formula 171 were used to prepare, from isohexides and phosgene, polycarbonates having enhanced stability against electric-current

-

OH t 71

Long-chain a,w-dicarboxylic acids have been condensed with diaminoisohexides, forming linear polyamides suitable for producing fibers having a silklike texture and higher moisture-absorption properties than nylon-like polymers.206

(263) S. K. Dirlikov and C. J. Schneider (Dow Chemical Co.), U.S.Pat. 4,443,563 (1983); Chem.Absrr., 101 (1984) 24,146. (264) H. Medem, M. Schreckenberg, R. Dhein, W. Nouvertne, and H. Rudolph (Bayer AG), DE 3,002,762 (1980); Chem. Abstr., 95 (1981) 151,439.

1,4:3,6-DIANHYDROHEXITOL.S

173

Polyesters synthesized from terephthaloyl chloride and isohexides were prepared, and characterized by differential scanning calorimetry, n.m.r. spectroscopy, and viscosity mea~urement.~~**~* Another “polyester,” prepared by copolymerization of (2-hydroxyethy1)methacrylatewith isomannide dimethacrylate (172), forms a hydrogel when allowed to swell in isotonic sodium chloride solution. This gel has useful properties for manufacturing contact-lens material.265 Isosorbide 2,5-dimethacrylate was used for preparation of templateimprinted vinyl and acrylic ACKNOWLEDGMENT The authorsare gratefulto C. Unger for undertakingthe task of compiling the numerousdata from original papers, patents, and Chemical Abstracts references.

(265)G.Kossmehl, N. Klaus, and H. Schaefer, Angew. Macromol. Chem., 123/124(1984) 241-259. (266)G.Wulff, J. Vktmeier, and H. G. Poll,Macromol. Chem., 188 (1987) 731-740.

This Page Intentionally Left Blank

ADVANCES IN CARBOHYDRATE CHEMISTRY AM) BIOCHEMISTRY, VOL. 49

ENZYMIC METHODS IN PREPARATIVE CARBOHYDRATE CHEMISTRY BY SERGE

DAVID,CLAUDINE AuGE,

A N D CHRISTINE

GAUTHERON

lnstitut de Chimie Molkculaire d’Orsay, UniversitP Paris-Sud, Bt 420, F-91405 Orsay ckdex, France 1. Introduction.. ....................................................... 1. The Interest of Enzymic Methods. . .......................... 2. Difficulties in Defining the Scope o f t e .......................... ..................... 3. Definitions and Abbreviations. 11. Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

....................................

176 176 177 177 180 180

2. Agarose ...................................... 4. Silica Gel-Glutaraldehyde

.....

..............................

188

111. Aldol Additions and Other C-C, Bond-forming Reactions .................. 189 1. General Considerations .............................. .......... 189

..........

3. Syntheses with Sialyl Al 4. Transketolase and Othe IV. Phosphorylations. . . . . . . .

194

....................

207

................................

213

2. Sugar Phosphates . . . . . . . . . . . . . . 4. “Nucleotide-Sugars”

...

I . General Considerations 2. Galactosylation. ...........................

VIII. Enzymes in Organic Solvents.. ..... . . . . . . . . . . . . . . . 235 IX. Addendum .......................................................... 236

175

Copynght 0 1991 by Academic Rcss, Inc. All rights of reproduction in any form rscrved.

176

SERGE DAVID ef al.

I, INTRODUCTION 1. The Interest of Enzymic Methods

Natural sugars are very active metabolites in cells, and so it would be expected that enzymes would be useful at some steps in their in vitro processing. The organic chemist, however, foresees difficulties: enzymology has evolved through effortsto understand life phenomena, not to help in organic synthesis,and enzymes are rare, or costly, or need to be isolated by unfamiliar techniques. Coenzymes, which are often necessary, are in most cases complicated and costly molecules,and their preparation on a large scale may be exceedinglytedious. Partly for these reasons, and partly because, from the biochemist’s point of view, there was no obvious necessity to scale up the process, the scale of most enzymic reactions reported in the biochemical literature, from the nanomole to the micromole, is definitely too low to have any preparative significance. It is true that, sometimes, only very small amounts indeed of oligosacchandes are needed, for instance to trigger the manufacturing of large amounts of monoclonal antibodies by molecular biology techniques. However, the normal goal of organicsynthesisis to make compounds available in quantity. Another inconvenience of enzymic reactions appears in glycosylation with stoichiometric amounts of “nucleotidesugars.” The result of the coupling reaction is the accumulation, in the medium, of the corresponding free nucleotide, which may prove inhibitory to transferases at millimolar concentrations. The now-classical solution to these problems is to attach the enzyme to a suitable polymer which is used as an aqueous suspension. When the reaction is finished, the enzyme is separated from the products by filtration, and, not infrequently, may be used again many times. In the operation of the D-galactosylation cycle described in SectionV,2, only catalyticquantitiesof nucleotide-sugars are necessary, as they are constantly regenerated in the medium by the interplay of appropriatesubstrateswith other enzymes, also present in the immobilized state. Immobilization has other advantages: it can slow enzyme deactivation by inhibiting protease attack and minimizing shear, interfacial,temperature, or solvent denaturation. As for the scarcity of some potentially very useful enzymes, it may be only a temporary problem. The development of cloning techniques, and probably the very increase in demand will result in lower prices. One spectacular instance is sialyl aldolase (see Table I). Industrial production of this enzyme by the gene-cloned strain of Eschmichiu coli has been Sialylaldolaseis now available from Toyobo at a moderate price. (1) T. Uwajima and K. Aisaka, Eur. Pat. Appl. EP 164, 754 (18/12/85); Chem. Abstr., 104 (1986) 143,165j. (2) K.Aisaka, S.Tamura, Y.Arai, and T. Uwajima, Biofechnol.Lett., 9 (1987) 633-637.

ENZYMIC PREPARATION OF CARBOHYDRATES

177

Previous accounts of enzymic methods in organic synthesis include four reviewP and a chapter in a book,’ while immobilization of enzymes is discussed at length in three volurness-lo of Methods in Enzymology. The older literature may be found in an earlier volume of the same collection.I1 2. Difficulties in Defining the Scope of the Article The boundaries of this article are difficult to delimit. Immobilized enzymes cannot only be considered, for, in the literature, there are instances of relatively high-scale preparation with soluble enzymes, and immobilization may have little advantage in working with such enzymes as the glycolysis aldolase, which are very common and need no coenzyme. It is also difficult to give a precise definition of such words as “preparative scale.” Many “purely organic,” multistep syntheses of oligosaccharidesend with no more than 5 mg, or, indeed, 1 mg, of product, while certain biochemical groups have reported preparations on a definitely higher scale with the use of soluble enzymes. We are clearly conscious that there is a measure of arbitrariness in our selection of papers for discussion. 3. Definitions and Abbreviations

Table I is a list of all the enzymes that will be mentioned in this article. Neither the E.C. number nor the nomenclature name is convenient for repeated use in a discussion. Therefore, for the running text, we shall use mostly the time-established trivial names. We shall use 1 to 3-letter symbols in the Tables. For these, we have followed common practice as much as possible, but we had to innovate in some cases in order to avoid confusion. We have adhered to this definition of the unit (U) of enzymic activity: the amount of enzyme which catalyzes the transformation of one micromole of substrate per minute under the best possible conditions. Concerning the G. M. Whitesides and C. H. Wong, Angew Chem., Int. Ed. Engl., 24 (1985) 617-638. J. B. Jones, Tetrahedron.42 (1986) 335 1 - 3403. V. N. Shibaev, Bog. Khim. Uglevodov,(1985) 149- 173. C. H. Wong, Science, 244 (1989) 1145- 1152. C. H. Won& in M. P. Schneider (Ed.),Enzymes as Catalysts in Organic Synthesis, Reidel, Dordrecht, Holland, 1986, pp. 199-216; compare E. J. Toone, E. S. Simon, M. D. Bednarski, and G. M. Whitesides, Tetrahedron, 45 (1989) 5365-5422. (8) Methods Enzymol., 135 (1987), “ImmobilizedEnzymes and Cells, Part B,” K. Mosbach

(3) (4) (5) (6) (7)

(Ed.), Academic Press,New York. (9) Methods Enzymol., 136 (1987), “ImmobilizedEnzymes and Cells, Part C,” K. Mosbach

(Ed.),Academic Press, New York. (10) Methods Enzymol., 137 (1 987), “ImmobilizedEnzymes and Cells, Part D,” K. Mosbach (Ed.),Academic Press, New York. (1 1) Methods Enzymol., 44 (1976), “Immobilized Enzymes,” K. Mosbach (Ed.),Academic Press, New York.

TABLE I EC Number

Systematic name

Oxidoreductases 1.1.1.8 sn-Glycerol 3-phosphate: NAD+ 2-oxidoreductase 1.1.1.44 6-Phosphogluconate: NAD+ 2-Oxidoreductase 1.1.3.9 Galactose oxygen 6-oxidoreductase 1.4.1.3 L-Glutamate : NAD+ oxidoreductase Transferases Sedoheptulose 7-phosphate : Dglyceraldehyde 3-phosphate 2.2.1.1 glycolaldehydetransferase 2.4.1.7 Sucrose orthophosphate a-Dglucosyltransferase 2.4.1.13 UDP-glucose: fructose 2-glucosyltransferase 2.4.1.19 Cyclodextrin (u-( l4)-glucosyltransferase uDP-@aCtOSe : D-GlUCOSeB( 14)-&ctOSyl tranSferase 2.4.1.22 CMP-NeuSAc : &D-Galp(1 4 ) - ~ - G l c N A ca42-6)2.4.9 1.1 sialyltransferase 2.4.99.4 CMP-NeuSAc: &D-Galp( 1+3)+-GaINAc ~14243)sialyltransferase 2.4.99.5 CMP-NeuSAc: &DGalp[1+3(4)]~-GalNAc a-(2+3)sialyltranferase 2.7.1.1 ATP : whexose 6-phosphotransferase 2.7.1.11 ATP : fructose 6-phosphate 1-phosphotransferase 2.7.1.19 ATP: ribulose 5-phosphate I-phosphotmnsferase 2.7.1.20 ATP : adenosine 5’-phosphate ATP : glycerol 3-phosphotransferase 2.7. I .30 2.7.1.40 ATP : pyruvate 2-0-phosphotransferase 2.7.2.1 ATP : acetate phosphotransferase

Common name

Abbreviation

glycerophosphatedehydrogenase

GD

phosphogluconate dehydrogenase galactose oxidase glutamate dehydrogenase

PGD

tramketolase sucrose phosphorylase sucrose synthetase

Go GLD TK SP

ss

galactosyl tramferase sialyltransferase

GT STA

sialyltransferase

STB

sialyltransferase

STC

hexokinase 6-ph0~ph0fruct0ki~~ phosphoribulokinase adenosine kinase glycerokinase pyruvate kinase acetokinase

HK PFK PRK ADK GK PK

AK

2.7.4.3 2.1.4.4

ATP: AMP phosphotransferase ATP : nucleoside monophosphate phosphotransferase

2.7.5.1

cu-~-Glucose1,6-diphosphate a-~-glucose1-phosphate phosphotransferase Polyribonucleotide: orthophosphate nucleotidyl transferase UTP : CY-D-~~UCOS~ 1-phosphate uridyltransferase CMP-acetylneuraminicacid synthetase

2.7.1.8 2.1.7.9 2.7.7.43 Hydrolases 3.1.1.3 3.1.4 3.1.21.1 3.2.1.22 3.2.1.23 3.2.1.24 3.2.1.37 3.6. I. 1 3.4.4.16 Lyases 4.1.2.13

4.1.2.16 4.1.3.3 Isomerases 5.1.3.1 5.1.3.2 5.3.1.1 5.3.1.5 5.3.1.6

Triacylglycerol acyl hydrolase Deoxyribonucleate 5'-oligonucleotidohydrolase a-D-Galactoside galactohydrolase fidalactoside galactohydrolase cu-DMannoside mannohydrolase 1 4-Po-Xylan xylohydrolase Pyrophosphate hydrolase

-

Fructose 1,6-bisphosphate D-glyceraldehyde 3-phosphate lY= 3-Deoxy-~-mannc~octulosonate 8-phosphate synthetase N-Acetylneuraminatepyruvate lyase

adenylate kinase nucleoside monophosphate kinase phosphoglucomutase polynucleotide phosphorylase UDP-CIc-pyrophoSphoryylase CMP-NeuSAc synthetase

lipase nuclease PI deoxyribonuclease I a-galactosidase pgalactosidase a-mannosidase j?-xylosidase inorganic pyrophosphatase protease N subtilisin aldolase Kdo-synthetase Sialyl aldolase

Ribulose 5-phosphate 3epimerase UDP-glucose 4epimerase D-Glyceraldehyde 3-phosphate ketol isomerase

triose phosphate isomerase

Ribose 5-phosphate ketol isomerase

glucose isomerase phosphoriboisomerase

AYK

NK M POP UP

cs N

N D AGA BGA AMA IP

A

Ks SA

E TPI GI PRI

180

SERGE DAVID et al.

information given in Tables I11 to X,we felt bound to report, or recalculate, yields and scales from the original papers as precisely as reasonable. However, we warn the reader that many of the reactions summarized in these Tables, not developed under the constraintsof industrial practice, are probably not optimized. We describe a few procedures in detail. Our intention is to allow the reader to grasp more fully the practical aspects of this specializedchemistry, which is not currently very well understood, without having to interrupt his reading to go to the library. Of course, the original papers, all readily available, should be consulted by chemists planning repetition of these preparations. 11. IMMOBILIZATION

1. General

The successful conversion of g glucose into D-fmctose on the industrial scale with immobilized D-glucose isomerasewas a brilliant demonstrationof the value of this kind of approach. Then followed a huge technical literature on enzyme immobilization, reviewed in Ref. 9 (page 353). We shall here restrict ourselves to the methods which have been utilized in the syntheses outlined in Tables I1 to X.We suggest to readers interested in theses techniques that they first use these methods. Ifthey prove unsatisfactory, asthere is a plethora of alternatives, other techniques, described in Refs. 8 - 10, may be tried: a majority of readily available carbohydrate enzymes have been immobilized, often in several different ways. It is necessary to know the efficiency of the coupling process, that is, the ratio of immobilized activityto that initially present in the solution. For this, the common protocol given in textbooks of enzymology may be followed, with the restriction that these methods were generally devised for soluble enzymes. Provision must be made for gentle stirringof the suspension,in, for instance, the cell ofthe ultraviolet spectrophotometer.In most cases, there is a loss of activity on immobilization, a fact that may seem distressingwhen it happens as the conclusion of a laborious purification procedure. Despite this, immobilization may be advantageousin the long run, even with a 70% loss in activity: the soluble enzyme may be so unstable as to degrade itself even during the enzymic reaction (indeed, biochemists generally add it in excess) and, in any case, it cannot be recovered, while recovery ofthe immobilized enzyme involves only a filtration. In some instances, the total activityof a soluble preparation is increasedon immobilization, probably because the process removes, or neutralizes in some way, an inhibitor. This is observed with concentrates of CMP-Neu5Ac synthetase.

ENZYMIC PREPARATION OF CARBOHYDRATES

181

Another figure of practical value is the activity per mL of the gel. The consequence of a small concentration of enzyme is the need to work with a great volume of gel, and thus a great volume of solution, and an unfavorable dilution of substratesand coenzymes. This precludes recourse to an excessof gel in the immobilization reaction with the hope of exhausting the soluble activity. Impure enzymes raise a similar problem. The organic chemist who needs to isolate an enzyme may well consider that purification to homogeneity is very laborious, wasteful of activity, and, generally speaking, foreign to his field of competency; but, in the immobilization process, foreign proteins in an impure sample are immobilizedas well, decreasingthe number of active sites available to the enzyme. The result will be a loss of specific activity. A common practice is to conduct the immobilization procedure in the presence of species that occupy the active site of the enzymes, such as substrates, cofactors, reversible competitive inhibitors, or products, at concentrations preferably above their Michaelis or inhibition constants. Such a precaution is not necessary for the immobilization of CMP-NeuSAcsynthetase. 2. Agarose

Agarose is a natural polymer of D-galactose and 3,6-anhydro-~-galactose residues. Agarose gels having spherical particles are sold in the swollen state, suspended in water or buffer containing 0.02% of sodium wide as a bacteriostatic agent, under the trade names Sepharose (Pharmacia, Sweden), U1trogel (IBF, France), and Bio-Gel A (Bio-Rad Labs, USA). Sepharose4B and Ultrogel A4 are an -4% concentration of agarose in the form of swollen particles, with sizes 40 - 190 and 60 - 140 pm, respectively. Agarose must be first activated by treatment with cyanogen bromide. Then, it can build covalent linkages with any molecule having a basic primary amino function, including the free amino group in proteins (see Scheme 1). Obviously, neither on the buffer molecules, nor anywhere else in the system, should there be amino groups (which would also react with activated agarose). Activation by cyanogen bromide,I2introduced by Porath and coworkers in 1967, appears still to be a favorite method. Cyanogen bromide first gives a cyanate, which may react with a neighboring hydroxyl group to give a cyclic imidocarbonate.This is presumed to be the most reactive speciestowardsthe enzyme, which is then attached with displacement of NH, . Direct reaction of the cyanate with one amino group of the enzyme also covalently binds the enzyme, with the formation of an isourea. Other transformations lead to (12) J. Porath, R. Axen, and S. Ernback, Nufure,215 (1967) 1491- 1492.

z

0

w

w

+

cir

z

I

+ -__*

I

z? z w

+

I

ph

B

0

(r

ENZYMIC PREPARATION OF CARBOHYDRATES

183

derivatives having little or no activity. According to Kohn and Wilchek,13 the constitution of agarose is not favorable to the building of imidocarbonates, and the linkages to enzyme may be mainly of the isourea type. The enzyme-agarose conjugate, a gel, is stored as a suspension in the immobilization buffer. This gel is rather mechanically fragile. Magnetic stirrers should be avoided, and the contents of reaction vessels gently stirred on a rotary shaker. Attention is drawn to the poisonous nature of cyanogen bromide. Agarose gels are excellent at the laboratory level, but their high cost precludes their use in Industry. The cheaper “Trisacryl,” an all-synthetic polymer having similar properties, has gained wide acceptance for technical applications.l4 To illustrate enzyme immobilization on agarose, we have purposely selected instances of enzymes prepared in an organic chemistry laboratoryand not purified to h~mogeneity,’~ as in the preparation of immobilized cytidine-monophosphate-Nacetylneuraminicacid synthetase by Augk and coworker~.’~ Two calf brains (600 g, 40 U) were homogenized with 0.01 M sodium pyrophosphate (1 L) in a Waring Blendor. The homogenate was centrifuged, and the pellet was extracted twice with 0.4 M KC1 (400 mL). After centrifugation at 30,000 gfor 20 min, each supernatant liquor from the KC 1 extractions was separately precipitated with ammonium sulfate according to Higa and Paulson.16The precipitate was taken up in the buffer used for the immobilizationstep (0.1 MNaHCO,, pH 8.8, containing0.5 M NaC1). A quarter of the CMP-Neu5Acsynthetase (100 mL, 5 U) was stirred overnight at 4”under nitrogen with Ultrogel A4 (50 mL) freshly activated with BrCN (100 mg per mL of gel). The gel was successively washed with M NaCl, twice-distilled water, and 0.1 M Tris buffer, pH 9, containing 3 mM 2-mercaptoethanol, and then stored in suspension in this buffer (enzymic activity bound to agarose: 184 mU/mL of gel). The activity of immobilized CMP-Neu5Ac synthetase was determined by the thiobarbituric acid assay, using the standard procedure described for the solubleenzyme.l7 In this case, immobilization almost doubled the available activity. Another example is the preparation of immobilized Galp-P-(1-4)GlcpNAc-a-(2+6)-sialyltransferase by Augk and coworkers.Is Glassware was siliconized. Column fractions were collected in plastic tubes. Porcine liver (500 g) was homogenized with 25 mMNa cacodylatebuffer containing 20 mh4 MnC1,. The pellet was extracted twice with Triton X-100, and each (13) (14) (15) (16) (17)

J. Kohn and M. Wilchek, Anal. Biochem.. 115 (1981) 375-382. E. D. J. Brown and J. Touet, J. Chem. Res., 5 (1979) 290-291. C. Aug6, C. Gautheron, and R. Fernandez, Curbohydr. Rex, 200 (1990) 257-268. H. H. Higa and J. C. Paulson, J. Biol. Chem., 260 (1985) 8838-8849. E. L. Kean and S. Roseman, Methods Enzymol., 8 (1966) 208-215.

SERGE DAVID ef al.

184

extract was adsorbed to a column (4.5 X 6 cm) of CDP-hexanolamineagarose (10pmol/mL of gel), according to the procedure described for the rat-liver enzyme.** The GalpP-( 1+4)-GlcpNAc-a-(2-6)-sialyltransferase was eluted from the column with 30 mM Na cacodylate buffer (pH 5.8) containing 1.5 mM NaC1, 1% of Triton X- 100, and 25% of glycerol. Fractions containing enzymeactivity were pooled (1 50 mL) and dialyzed against 10 mM Na cacodylate buffer (pH 6.5; 2 X 4 L) containing 1% of Triton X-100and 25% of glycerol. The dialyzate was then applied to a column (2.5 X 14 cm) of CDP-hexanolamine-agarose (2 ,umol/mL of gel). The enzyme was eluted with a linear CDP gradient (0- 2 mM) in 30 mM Na cacodylate buffer (pH 5.8) containing 75 mA4 NaC1, 1% of Triton X-100, and 25% of glycerol. The pooled enzyme (50 mL) was 4-fold diluted in 10 mMNa cacodylate buffer (pH 5.5) containing 25 mM NaCl and 1% of Triton X-100, and then loaded onto a column (0.4 X 4 cm) of SP-CSO Sephadex equilibrated in the same buffer. The column was washed with this buffer (5 mL), and the enzyme was eluted from the column with 30 mMNa cacodylatebuffer (pH 6.0) containingMNaC1, 1% ofTriton X- 100,and 25% of glycerol. Fractions containing the partially purified sialyltransferase were pooled (8 mL), and dialyzed during 3 h against 0.1 MNa phosphate buffer (pH 7.8; 2 X 100 mL) containing 25% of glycerol. Prior to dialysis,the dialysis membrane had been saturated with bovine serum albumin, and bovine serum albumin (4 mg) was added to the enzyme preparation. The dialyzate was stirred overnight at 4", under nitrogen, with Ultrogel A4 (0.25 vol.) freshly activated by BrCN (60 mg per mL ofgel), in the presence of 1 mMCDP. The gel was successively washed with twice-distilledwater and 10 mA4 Na cacodylate buffer(pH 6.5)containing0.5 MNaCl, l%ofTriton X-100, and 25% of glycerol (enzymic activity bound to agarose: 0.1 U/mL ofgel). The gel was stored at -20" after addition of ice-cold glycerol (0.5 vol.). Before utilization, the immobilized preparation was filtered on a sintered glass septum in order to remove the storage buffer, and washed with the buffer to be used for enzymic incubation. The activity of immobilized Galpp-( 1 +4)-GlcpNAca-(2+6)-sialyl transferase was determined by radiochemical assay. The assay mixture (200 pL), continuously stirred during incubation at 37 contained 4 pmol of N-acetyllactosamine, 200 pg of bovine serum albumine, 50 pL of gel, and 42 mmol of CMP-[I4C]Neu5Ac(40,000 cpm) in 40 mA4 O,

(18) J. Weinstein, U. de Souza-e-Silva,and J. C. Paulson, J. Biol. Chem., 257 (1982) 13,835 13,844. ( 19) C. AugC, C. Mathieu, and C. MCrienne, Carbohydr.Res., 15 1 ( 1986) 147 - 156.

(20) C. A@, S. David, C. Gauthemn, A. Malleron, and B. Cavayk, New J. Chem.,12 (1988) 733 144.

-

TABLE Il Immobhtion of Enzymes on Agarow Enzyme

mg/d

Galactosyl transferase Pyruvate kinase Nucleoside monophosphate k i n a UDP-Glc-~ophoSpho~ylase Inorganic pyrophosphatase UDP-glucose kpimerase Sialyl aldolase CMP-NeuAc synthetase Sialyl transfetase

10 0.6 5

0.3 0.2 0.4 0.25

Material for the protection of the active site (mM)

Yield (%)

Unit/mL

References

UDP-Glc (1); GlcNAc (5); MnCl, (25) ADP (0.4); “PEP (1); M a , (10) ATP (1); CMP (0.5) UDPGlc ( 1.5); PPi ( I .5) Pi(I);PPi(l) UDPGlc (0.5); N A P (0.005) Pyruvate (40)

26-36 71-88 30 -40 31-41

0.25 100

CDP (1)

25-70

19 19 15 19 19 19 20 15 15

6 2.5

41-51

37-46 60 180

0.3 6.71 40 0.6 4 0.18 0.1

186

SERGE DAVID et al.

Na cacodylate buffer (pH 7.5) containing 2.5% of Triton X-100.The radioactive product formed was quantitated after elution with water from a Pasteur pipet column of Dowex-1 X8 (PO,HZ-, 200 - 400 mesh). Table I1 illustrates immobilization on agarose. Agarose, or any polymer having primary alcoholic functions, may also be activated by the conversion of its CH20H groups into their 2,2,2-trifluoroethanesulfonates.This introduces good leaving-groups(“tresylates”), which are displaced by an amino group from the enzyme with the formation of a covalent C -N bond?’

3. Poly(acry1amide) Gels This method, developed by Whitesides’ group, is outlined in Scheme 2. PAN is prepared by free-radical polymerization of a mixture of acrylamide and N-(acry1oxy)succinimideinitiated with azoisobutanonitrile. The procedure for enzyme immobilization is based on the simultaneous reaction of three components in neutral, buffered, aqueous solution at room temperature: the PAN polymer, triethylenetetramine, and the enzyme E-NH2. The diamine is first introduced in such a quantity that 15% of the active sites on the polymer remain available to the enzyme. Reaction of the diamine with the active ester groups of the PAN crosslinks the polymer chains, and forms an insoluble gel connected through amide groups. Then the enzyme is added, before the gel point, or very near to it (about one minute), in order to avoid parasitic reactions. Reaction of the amino functions of the enzyme with residual active esters covalently links the enzyme to this gel through additional amide linkages. The success of the method rests on the fact that the enzyme is introduced before gel formation. Because it is originally dissolved in a homogeneous solution containing the reactive polymer, and because the entire volume of solution is transformed into a gel, the enzyme is completely and uniformly distributed throughout the gel. The cross-linking reaction generates very little heat, and shows no tendency to deactivate enzymes thermally. As in many cases, the enzyme should be immobilizedin the presence of species which occupy its active site, and of a reducing agent such as 1,4-dithiothreitol,sometimes under an inert atmosphere, with degassed solutions. The final gel is ground, and washed. It is broken into small particles, and suspended in the reaction mixture, with stirring. Modifications are necessary for use in columns. For instance, for the immobilization of 710 U of hexokinase,?2PAN-450, a polymer carrying 450pmol ofactive ester group/g, was quickly dissolved in (21) K. Ndsson and K. Mosbach, Methods Enzymol., 135 (1987) 65-78. (22) A. Poll&, H. Blumenfeld,M. Wax, R. L. Baughn, and G. M. Whitesides, J. Am. Chem. SOC.,102 (1980) 6324-6336.

ENZYMIC PREPARATION OF CARBOHYDRATES

00

I87

0

N

I

J"

HN

I

CONH-E

I

SCHEME2.-Formation of Cross-linked PAN Gels Containing Immobilized Enzymes.

0 . 3 M Hepes buffer, pH 7.5, containing MgCl,, D - ~ ~ U C O Sand ~ , ADP. 1,CDithiothreitol and tnethylenetetramine were added with vigorous stirring, and, 30 s later, a solution of hexokinase was added. In less than 2 min, the mixed solutions set to a transparent, resilient gel. Blending of the gel in a Waxing Blendor at controlled speeds converted it into a suspension of particles having 100fim diameter which were washed several times by centnfugation. The activity of a gel prepared in this way from 3 g of PAN-450 and 1 160 U of hexokinase was 7 10 U (6 1% yield). Table I11 in Ref, 22 lists a number of enzymes that have been immobilized in this way. We shall cite here the specific activities(given in parentheses, in U per mL of gel) of those that are more or less concerned with carbohydrate

-

188

SERGE DAVID et al.

transformation: glycerophosphate dehydrogenase (0.7),6-phosphogluconic dehydrogenase(33, glutamate dehydrogenase(34), hexokinase ( 109, phosphoribulokinase(4.0), adenosine kinase (0.4), glycerol kinase (0.2), pyruvate kinase (lo), acetate kinase (67), adenylate kinase (92), nucleoside monophosphate kinase (0.25), phosphoglucomutase ( 12), UDP-Glc-pyrophosphorylase (OS),aldolase (15), ribose 5-phosphate isomerase (67), and glucose 6-phosphate isomerase (39). Also immobilized on PAN gels were galactosyltransferase(2.3);23nuclease PI(not given);24inorganic pyrophosphatase (120);23and UDP-glucose 4-epimerase ( 5 .3).23 4. Silica Gel-Glutaraldehyde Inorganic support-materials have been shown to be excellent carriers for immobilizedenzymes. Obviously,a large surface of contact is necessary, and consequently, the material should be highly porous; however, the pores must be wide enough not to interfere with the diffusion of enzymes and reagents, and thus, the material should fulfil contradictoryrequirements. In the case of glass, or silica gel, the surface, carefully cleaned, is first treated with (3-aminopropyl)triethoxysilane,between pH 3 and 4, and then with glutaraldehyde in a phosphate buffer. The enzyme (E-NH,) is bound to the surface by the formation of a Schiff base between one of its free amino groups and the free aldehyde group of glutaraldehyde (see Scheme 3).25 5. Dialysis Bags

In this technique, the enzyme solution is put inside a dialysis bag which is then immersed in a solution of substrate, or cofactors. Small molecules can diffise through the wall of the bag and react in the presence of the enzyme, while products, if also small molecules, diffise into the outside solution, where they may be recovered. This technique has been used in syntheseswith sialyl aldolase, Kdo-synthetase, the common aldolase, a mixture of hexokinase and pyruvate kinase, a-(2-*6) sialyl transferase,26a mixture of pyruvate kinase and adenylate kinase?' and CMP-NeuSAc synthetase.28

C. H. Wong, S.L. Haynie, and G. M. Whitesides, J. Org. Chem., 47 (1982) 5416-5418. W. E. Ladner andG. M. Whitesides, J. Ore. Chem., 50 (1985) 1076-1079. H. H. Wheetall, Methods Enzymol., 44 (1976) 134- 148. M. D. Bednarski, M. K. Chenault, E. S.Simon,andG. M. Whitesides, J.Am. Chem. Soc., 109 (1987) 1283- 1285. (27) E. S. Simon, M. D. Bednarski, and G. M. Whitesides, Tetrahedron Lett., 29 (1988) 1123- 1126. (28) E. S. Simon, M. D.Bednarski, and G. M. Whitesides, J. Am. Chem. Soc., 110 (1988) 7159-7163.

(23) (24) (25) (26)

ENZYMIC PREPARATION OF CARBOHYDRATES

I I

I I

I

0

0

-0-Si-OH

189

0

I

-0-Si-0-Si-(CH,),NH,

I I -0-Si-OH I

I

I I

0

(EtO),Si(CH,),NH,

0

b

-0-

0

I

Si -0 -Si -(CH,),NH,

I I 0 I I -0-Si-0-Si-(CH,),NH, I I 0 0 I I 0

0

I -0-Si-OH I 0 I

+ CHO(CH,),CHO

+ E-NH,

1

R-Si-(CH,),-N=CH-(CH2),-CH0

______,

I

1

R-Si-(CH,),-N=CH-(CHJ

I

,-CH=N-E

SCHEME3. -Immobilization on Functionalized Silica Gel by Means of Glutaraldehyde.

111. ALDOLADDITIONS AND OTHER C - C BOND-FORMING REACTIONS 1. General Considerations

Nature builds carbon -carbon bonds essentially by aldol or Claisen-type reactions, both involving a carbonyl group as an electrophile,and an enol or enolate as a nucleophile. A less-frequent reaction bears a formal analogy to the Claisen acyloin condensation. The aldol reaction of carbohydrates always involves an aldehyde as the electrophile,and an aldehyde or ketone as the nucleophile (see Eq. 1). R-CHO

+ -CH-CO-

R-CHOH-CH-CO-

(1) The enzymes which catalyze this reaction, the aldolases, are members of the general group called lyases (see Table I). They have been isolated from many living cells, and vary in specificity.The reader will find, in Methods of 4

SERGE DAVID et al.

I90

Enzymology,,29techniques of isolation, and descriptions of a number of them. Apparently, only three have been considered for preparative chemistry, that is, aldolase, sialyl aldolase, and Kdo synthetase. However, whole cells of some strains of Escherichzu coli have been used as sources of “fuculose” 1 -phosphate aldolase (E.C. 4. I .2.17) or “rhamnulose” 1-phosphate aldolase (E.C. 4. I .2. 19).30Extraction, and concentrationto a suitable degree of homogeneity, of noncommercially available aldolases are not difficult. The examination of their synthetic possibilities could be very rewarding for we already observe that the wealth of chemicals prepared with the help of aldolase and sialyl aldolase far exceeds what they make in Nature. Still, not any aldehyde, however hydrophilic, is a substrate for aldolases. In the other mode of formation of carbon - carbon bonds in Nature which we shall consider, the overall reaction corresponds to the nucleophilic addition of a carbonyl onto an aldehyde (see Eq. 2). R-CHO

+ R-CO-CH,OH

R-CHOH-CO-CH,OH

+ ...

(2) This is reminiscent of the “Umpolung” reactions of organic chemistry. As the group CO-CH20H is transferred from a donor molecule (R’-CO-CH,OH) to the aldehyde (R-CHO), the corresponding enzymes are classified among transferases. The use of transketolase will be considered in this Section. +

2. Syntheses with the Glycolysis Aldolase

This enzyme catalyzes between sugar phosphates a reversible reaction which is a step in the synthesis and degradation of D - ~ ~ U C O in S ~ cells (see Scheme 4).

Po

+

H-C-OH

I

CH2OPO3H2

FPOBH2

co I

&

CH2OH HO

SCHEME 4. -The Reversible Reaction Catalyzed by the Glycolysis Aldolase.

This reaction has often been utilized in the degradative direction, as a preparation of D-glyceraldehyde3-phosphate.An equimolecularquantity of (29) MethodsEnzymol.,42 (1 975) 223 -297 “Carbohydrate Metabolism, Part C,” W. A. Wood (Ed.),Academic Press, New York. (30) D. G. Drueckhammer,J. R. Dumachter, R. L. Pederson, D. C. Crans, L. Daniels, and C. H. Won& J. Org. Chem., 54 (1989) 70-77.

ENZYMIC PREPARATION OF CARBOHYDRATES

191

"dihydroxyacetone" ( 1,3-dihydroxy-2-propanone) phosphate is formed at the same time. The addition (to the medium) oftriose phosphate isomerase, which catalyzes the equilibrium in Scheme 5, allows a complete conversion of D-fructofuranose 1,Bbisphosphateinto D-glyceraldehyde 3-phosphate. CHO I

I

HCOH

I

CHlOPO3H,

SCHEME5. -The Equilibrium Catalyzed by T r i m Phosphate Isomerase.

The removal of the aldehyde, for instance as partner to an aldol reaction catalyzed by the same aldolase, displaces the equilibrium to the right. Both these enzymes are commercially available and inexpensive. Probably for this reason, they have almost always been utilized in the soluble state, and in great excess. However, it may be observed in Table 111 that these enzymes are much more efficient when3*immobilized on PAN. Aldolase requires dihydroxyacetone phosphate as ketonic partner, but will accept each of a wide collection of aldehydes as sub~trates.~~ In all cases, the newly built, vicinal diol has the D-threo configuration (see Scheme 6).

CH20P03Hz

I p" +I" R

CH2OH

#

"7" R

SCHEME&-The D-threo Configuration of the Newly Built Vicinal Diol in the Reaction Catalyzed by the Glycolysis Aldolase.

Table I11 gives preparations of six-, seven-, and eight-carbon ketoses, and their methylated, deoxygenated, azido, amido, and branched-chain denvatives. All these are first obtained as ketose phosphates, but this is not a severe restriction, for, apparently, enzymic hydrolysis with phosphatases is always successful. Phosphatases constitute a versatile group of enzymes, easily (31) C. H. Wong and G. M. Whitesides, J. Org. Chem., 48 (1983) 3199-3205. (32) M. D. Bednarski, E. S. Simon,N. Bischotberger, W. D. Fessner, M. J. Kim, W. Lees,T. Saito, H. Waldmann, and G. M. Whitesides, J. Am. Chem. Soc.,I 1 1 (1989) 627-635.

TABLEID Condensations Catalyzed by the Common A l d o W Product

Aldehyde

Reactions with 1,3-dihydroxyacetme phosphate None added fructose 1,6-bisphosphate 2,3-Dihydroxypropanal D-fructose L-sorbose ( 1:1) 2-OMethyl-~-glyceraldehyde 5-O-methyl-~-fructose ribose 5-phosphate D-gIycereD-ultrdose 1,8-bisphosphate

+

Reactions with fructose 1,6-bisphospbate Propanal 5,6dideoxy-~-threehexulose I-phosphate (Rk2-Hydroxypropad 3-H ydroxypropanal

3-(Tritluoroacetamido) proPand 3-Azido-2-hydroxypropanal 3-Hydr0~yb~tand 3-Hydroxy-3-methylbutanal 2-(Hydroxymethyl)4pntend ribose 5-phosphate Tetrahydropyranyloxyacetaldehyde Benzyloxyacetaldehyde

6 d e 0 ~ y - ~ - f r u 1-phosphate ~t0~ 5deoxy-~-threo-hexulm1-phosphate

5,6dideoxy-6-trifluoroacetamido-~~hree

Scale (-01)

40 1 4 0.5 1100

6 6 1

hexulose 6azido-6deoxy-~-uru~~~~hexulose 5,7dideo~y-~-~&h~td0~e 5,7dideoxy-6-C-methyl-~-threeheptulose 5-allyl-5deoxy-~-xylo-hexulose D-gIycercFD-ultrooctulose I ,I-bisphosphate 5- 0-tetrahydropyranyl-D-f~reepntose 5- 0-benzyl-D-threo-pentose

10

(%)b

Units per mmol

References

80 90 56 82d

A': 1.7;TPIc: 3 AC:8 Ac: 14 A: 100

31 31 32 33

13 62 42 41

A' 1.4; P I c : 1.8 A:@, TPI:83 A:28;Tl'I:83 A:240, TPI:400

32 34 34 35

71 97 50 50 61d 73

A:210, TPI:350 A:80; TPI:60 A:380 TPI:90 A:350;TPI:500 A:33; TPI:170

35 35 35 35 33 32

75

AC:17; TPI:3.5

32

Yield

Reactions with soluble enzymes, in the presence ofhose-phosphate isomerase, unless otherwise stated. Yields, calculated on dihyclroxyacztone phosphate. Enzymes immobilized in PAN gels. Yield, calculated on the pentose phosphate.

ENZYMIC PREPARATION OF CARBOHYDRATES

193

available, with broad specificities. For this reason, dephosphatation steps have not been explicitly reported in Table 111. However, dihydroxyacetone phosphate is needed. An original solution to this problem, not yet fully exploited, has been to replace it by a mixture of dihydroxyacetone and arsenate.w Probably this results in the formation of a labile arsenate, ,which is recognized,and converted into CH,OH--0-CH,O-AsO,H, a ketose arsenate. This is quickly hydrolyzed, in the medium, to the free ketose. Another restriction of the aldol reaction is that it gives ketoses. While isomerization to aldose may sometimes succeed with the free sugars in the presence of glucose isomerase,” starting with phosphates and phosphoglucose isomerase3’may be a safer procedure. An equimolar mixture ofdihydroxyacetonephosphateand 2,3-dihydroxypropanal is converted in the presence of PAN-immobilized aldolase (7 U/mmol), in quantitative yield, into an equimolar mixture, after dephosphatation, of D-fructose and L-sorbose (see Scheme 7).31This is what would CH,OH CHO

I I

HCOH

CH,OPO,H,

+

I I

co CH,OH

CH,OH

I I

co HOCH

I

HCOH

I I

HCOH CH,OH

CO

CHO

I

HOCH

1

CH,OH

+

co 1

CH,OH

I 1

HOCH HCOH

I

HOFH I

CH,OH

SCHEME 7.-The Equimolar Mixture of *Fructose and L-Sorbose Obtained from DL -2,3Dihydroxypropanal in the Reaction Catalyzed by the Glycolysis Aldolase.

(33) M. D. Bednarski, H. J. Waldmann, and G. M. Whitesides, TefruhedronLett., 27 (1986) 5807 -58 10.

(34) J. R. Durrwachter, D. G. Drueckhammer, K. No&, H. M. Sweers, and C. H. Won& J. Am. Chem. Soc., 108 (1986) 7812-7818. (35) J. R. Dumachter and C. H. Won& J. Org. Chem.,43 (1988) 4175-4181.

SERGE DAVID et d.

194

be expected from the addition of the two reactions depicted in Scheme 7. However, condensation of 3-hydroxybutanal or 2-( hydroxymethyl)-4-propenal, in great molar excess, affords only one of the two possible isomeric ketoses. This is considered to be a consequence of the reversibility of the aldol reaction, accumulating in the end the ketose with smallest conformational free-energy (see Scheme 8). Thus was obtained 5,7-dideoxy-~-xyb y2OH

a3

(i””

p e e

r CH,

HOCH II HCOH

I

FHZ

-

H &

e

CH20H OH

CHOH

I

CH3

SCHEME 8.-The Formation of the Thermodynamically More-Stable product in the Aldol Addition of ~~-3-Hydroxybutanal, as a Consequence of the ReversibleNature of the Reaction.

h e p t u l o ~ e .3-Hydroxybutanal ~~ was prepared in situ by hydrolysis of its dimethyl acetal (3.8 mmol) in dilute, aqueous HC1 at room temperature. D-Fructofuranose 1,6-bisphosphatetrisodium salt (0.8 mmol) was added, and the pH was adjusted to 7.0 with NaOH. The mixture was kept for 15 h in the presence of aldolase and triose phosphate isomerase. Then, the phosphates were converted into their barium salts with BaCl, at pH 7.3, precipitated by addition of acetone, and separated and washed by centrifugation. Treatment of the Ba salts as an aqueous suspension with Dowex-50 (H+) ion-exchangeresin regenerated the free acids. These were dephosphorylated by incubation at pH 4.5 for 4 days in the presence of phosphatase. Chromatography on a Dowex-50 (Ba”) column separated the heptulose ( 1.6 mmol, 97%). Methyl 2-acetamido-4-oxobutanoate was condensed with dihydroxyacetone phosphate as a key step in a synthesis of 3deoxy-~-arabino-heptulosonic acid 7-pho~phate.~~

3. Syntheses with Sialyl Aldolase a. General. -The biosynthesis ofN-acetylneuraminicacid (2), an almost exclusive prerogative of the animal kingdom, occurs by aldol addition between phosphorylated substrates, mediated by a special ald~lase.~’ This is (36) N. J. Turner and G. M. Whitesides, J. Am. Chem. Soc., 1 1 1 (1989) 624-627, (37) R. Schauer, Adv. Carbohydr.Chern. Biochem., 40 (1982) 131 -234.

ENZYMIC PREPARATION OF CARBOHYDRATES

195

not the enzyme that is going to be dealt with, namely, sialyl aldolase, which catalyzes a reversible condensation between unphosphorylated substrates, N-acetylmannosamine (1) and pyruvate (see Scheme 9). HlCOH

+ OH

1

CH,COCO?H

#

H

2 + 0

OH

OH

AcN H

OH

2

SCHEME9. -The Reversible Addition Catalyzed by Sialyl Aldolase.

This enzyme, found in some bacteria, such as E. coli or Clostridium perfringens, has only a catabolic function in cells (see Scheme 9, left to right). It is commercially available, the cloning has been and, in any case, the price of the unit thereof is steadilydecreasing. An excess of pyruvate displaces the equilibrium in the synthetic direction, as indicated by most of the yields given in Table IV. The only acceptable natural source of N-acetylneuraminic acid is edible birds’ nest, surely an expensive luxury food. Although chemical syntheses are known (summarized in Ref. 20), a direct preparation seemed desirable. Furthermore, many derivatives of N-acetylor N-glycolyl-neuraminicacid, such as acetates, lactates, and ethers, occur in animal cells. Some are labile in acidic media, and it is therefore difficult to isolate, or even estimate, them by extraction. They seem endowed with interesting biological properties, and deserve closer investigation. Although the specificity of the enzyme is not completely known, some indications may be drawn from the yields obtained with it in preparative chemistry. Starting from the “normal” substrate N-acetylmannosamine (2-acetamido-2-deoxy-~-mannose,1) we can make the following observations: the 2-epimer, N-acetylglucosamine (3) is not a substrate. On the other hand, modification of the substituent at C-2, while retaining the D-manno configuration, gave good substrates, some of them, like D-mannose, better than N-acetylmannosamine itself. Thus, 2-deoxy-~-arabino-hexose (4), Dmannose (5), and 2-deoxy-~-mannoseshaving the following substituents at C-2: NHCOCH20H(6), NHCOCH,OAc (7), N, (8), and Ph (9),gave excellent yields of condensation products, respectively 10 and 11 (Refs. 41, 42, 44), 12 (Ref. 20), 13 (Ref. 20), 14 (Ref. 43), and 15 (Ref. 44). The reactivity of 2-deoxy-2-C-phenyl-~-mannose is remarkable, for this is surely a case of extreme bulk for a substituent. Likewise, such functional derivatives of N-acetylmannosamine as the 6-acetate(16),the 6-(~-lactate)17,and the 4- and 6-methyl ethers, 18 and 19, condensed with pyruvate, gave,20in good yields, acids 20, 21, 22, and 23.

TABLE N Naturally Occurring Sialic Acids and Related SugaW Scale

Acids

N-Acetylglucosamine, N-acetylmannosamine, 1:1 mixture NGlycolylglucosamine, N-glycolylmannosamine, 2 :3 mixture Derivatives Of 2-nmin0-2de0~y-Dmannose N-Acetyl-6-0-acetyl N-A~etyl-6-O-[(S>(2hydroxypropanoyl)] N-Acetyl4 0-methyl N-Acetyl-6- 0-methyl 6- O-Acetyl-N-glyCOlyl N-(2-Acetoxyacetyl) Other compounds DArabinose D-Lyxose D-Xylose D-GlUCOSe 2-Deoxy-~-arabino-hexose 4-Deoxy-mlyxo-hexose D-Mannose 2-Azido-2deoxy-~-mannose 2-Deoxy-2-C-phenyl-~-mannose

mmol

Yield 96

Units/ mmol

Ref.

N-acetylneuraminicacid

5

67

I

20,38,39

N-glymlylneuraminicacid

1

61

1

20,39

N-acetyl-9- O-acetylN-acetyl-9-0-(L-lactyl>

4 0.6

67 53

16 24

20,39,40 20

N-acetyl-7- O-methylN-acetyl-9- O-methyl9- 0-acetyl-N-glyCOlyl N-(2-acetoxyacetyl)Derivatives of 3deoxyoctulosonic acid ~ m n o - ( K d oand ) mgluco0-galactoD-gulo Derivatives of 3-deoxynonnlosoniracid Dglycero-Dgulo5-deoxy-D-gluco7de0~y-Dgalac0Dglycero-D-galacto5-Azido-5deoxy-~gl~ero-~galacto 5-Deoxy-5-C-phenyl-mglpxroDgalacto

0.6 0.3 1.3

59 59 63

20 20

0.25

50

12 14 6 12

1

1 1

35 66 18

12 14 20

4 1,42 41,42 41,42

1 1 1 1 1 I

28 36 67 84 78 76

16 6 12 15 12 8

4 1,42 4 1,42 4 1,42 4 1,42 43

Derivatives of neuraminic acid

* condensationswith pyruvate in the pre-senceof N-acylneumnkte pyuvate lyase immobibd on agarose.

u)

20

44

ENZYMIC PREPARATION OF CARBOHYDRATES

197

R=H R=OH R = NHCOCH2OH R=NHCOCHzOAc 8 R=N3 9 R=Ph 4 5 6 7

3

10 R = H 11 R = O H 12 R = NHCOCH2OH 13 R = NHCOCH~OAC 14 R = N , 15 R = P h

16 17 18 19

R' R' R' R'

= H, R2 = Ac = H, R2 = (S)MeCHOHCO = Me, R2 = H = H, R2 =Me

Mannose reduced at C-4 (24), or truncated to D-lyxose (giving 25), gave41*42944 nonulosonic acid 26 and octulosonic acid 27. (38) C. Auge, S.David, and C. Gautheron, TetrahedronLat., 25 (1984) 4663-4664. (39) S. David and C. Auge, Pure AppL Chem.,59 (1987) 1501 1508. (40) C. Auge, S. David, C. Gautheron, and A. Veyrikes, TetrahedronLett., 26 (1985) 24392440. (41) C. Auge and C. Gautheron, J. Chem. Soc., Chem. Commun..(1987) 859-860. (42) C. Auge, B. Bouxom,B. Cavayt, and C. Gautheron, TetrahedronLett., 30 (1989) 22172220. (43) C. Augt, S. David, and A. Malleron, Curbohydr. Res.. 188 (1989) 201 -205. (44) C. Aug6, C. Gautheron, S. David, A. Malleron, B.Bouxom,and B.Cavaye, Tetrahedron, 46 (1990) 201-214.

-

SERGE DAVID et al.

198

P”

R20

OR’

Ac

R’ = R2 = H, R3 = AC 21 R‘ = R2 = H, R3 = (S)MeCHOHCO 22 R‘ =Me,RZ =R3 = H 23 R’ = R2 = H, R3 = M e 20

OH 24 R1 =H, R2 = CH20H 25 R’ =OH,R~ = H HO

QH

. OH

26 R’ = H, R2 = CH@H 27 R’ =OH, R~ = H

The sensitive positions are OH-3 and OH-5. The 5-methyl ether (28) of N-acetylmannosamine gave only an insignificant yield of 29, one of the starfish sialic acids.” Another starfish sialic acid, 30, expected to give 2deoxy-2-glycolamido-5-O-methyl-~-mannose in the presence of sialyl aldolase, was inert under these conditions.4sAlso, the enzyme is deeply affected in the presence of C-3epimers. In contrast with the satisfactory substrate ~-lyxose,D-arabinose reacts sluggishly, finally giving 19% of the “normal” product 31, which has on the correspondingcarbon atoms the same configuration, but not the same conformation, as N-acetylneuraminicacid, but also, (45)

L.Warren, Biochim. Biophys. Acta, 83 (1964) 129- 132.

ENZYMIC PREPARATION OF CARBOHYDRATES

199

MeOCH

28

COR

29 R = M e 30 R=CH20H

31 d =H,R* =OH 32 R' =OH,R* = H

simultaneously affording 17% of 3deoxy-~-mann~-octu~osonic acid (32). Thus, the enzyme has 1 0 s t 4 ~the * ~specificity of the orientation at C-4. Consequently, what information we have on hand is that sialyl aldolase does not equilibrate sialic acids having two hexose units methylated at C-5, and that the enzyme worked poorly with a pentose which could not yield an octulosonic acid having the same pyranose ring conformation as N-acetylneuraminic acid. The reactions of D-glucose and D-xylose, not preparatively significant (see Table IV), are nevertheless interesting, as N-acetylglucosamine, with the same ring configuration, is definitely not a substrate.

200

SERGE DAVID ef ul.

Kinetic parameters for glucose and other usual sugars have been reported.46Kinetic and tracer studies of the equilibrium between N-acetylmannosamine and pyruvate, and N-acetylneuraminic acid shown in Scheme 9, from Brossmer et uL4' led them to the conclusion that substrate and product are both a-pyranose tautomers. b. Naturally Occurring Sialic Acids. -The preparative advantages of enzymic syntheseswould be problematic if it was necessary to use the costly, pure N-acetylmannosamine(1) as substrate or starting material for derivatization. However, in a mixture of N-acetylglucosamineand N-acetylmannosamine, prepared in an inexpensive way by alkaline epimerization of the former sugar, the enzyme selects the manno no compound. The product, being an acid, is easily separated from unreacted N-acetylgl~cosamine.~~~~ N-Acetylneuraminic acid4 (2) (AugE and coworkers).-2-Acetamido-2deoxy-D-ghcopyranose(3;85 g) was dissolved in water (400 mL); the pH of the solution was adjusted to 1 1 with 5 Msodium hydroxideand the solution was left for 1 day at room temperature. The mixture was de-ionized with Dowex 50-X8(H+) resin, and evaporated to dryness under vacuum. The residue was taken up with ethanol (300mL), and the mixture was treated on the steam bath, with stimng, to dissolvethe syrup. On cooling, 2-acetamido2-deoxy-~-glucopyranose (3)crystallized (66 g); the mother liquor was concentrated and a second crop of 3 (5 g) was obtained. Concentration of the second mother liquor gave a third crop (3.5g) of 3. The recovered 3 was re-treated in the same way. Both residual syrupsafforded a mixture of 1and 3 (total, 17.1 g) containing 88% of 1 according to n.m.r.-spectral analysis. Immobilized sialylaldolase(50mL ofgel, 68 U) was added to a mixture of 88% pure N-acetylmannosamine (20 mmol), sodium pyruvate (180mmol), 1,4-dithiothreitol(0.2 mmol), and sodium azide (20 mg) in 0.05 M potassium phosphate buffer, pH 7 (1 50 mL). The suspension was gently stirred under nitrogen for 4 d at 37', the reaction being monitored by t.1.c. in 7 :3 propanol- water. The gel was removed by filtration, washed with the buffer, and N-acetylneuraminic acid (2) was isolated by chromatographyon Dowex 1 X8 (HCOJ-) resin, using a gradient of formic acid as the eluant, in 66% yield. The gel was used in four successive runs. Starting from 17 g of 88% pure N-acetylmannosamine,the procedure allowed the synthesis of 14 g of N-acetylneuraminic acid (2). In the end, the recoveredgel retained 80%of its enzymic activity. M.J. Kim,W. J. Hennen, H. M. Sweers, and C. H. Won&J. Am.Chem.Suc., 110 (1988) 6481-6486. (47) W. Baumann, J. Freidenreich, 0 . Weisshaar, R. Brossmer.and H. Fnebolin, Biol. Chern. Hoppe-Seyler, 370 (1989) 141 149. (48) C. Augk and C. Gautheron, unpublished results. (46)

-

ENZYMIC PREPARATION OF CARBOHYDRATES

20 1

A mixture of N-glycolylmannosamine6 and N-glycolyl-D-glucosamineis prepared in the same way, and utilized in the synthesis of N-glycolylneura(16), precursor to the minic acid (12). 6-0-Acetyl-N-acetylmannosamine very important natural sialic acid 20, was prepared from N-acetylmannosamine either by selective chemical acetylationY4O or, in elegant fashion, by a protease-mediated acylation in an organic solvent.4 For the preparation of N-acylmannosamines substituted at 0-4 or 0-6,the general precursor 35 was synthesized from the benzyl pyranoside 33, easily available on the mole scale by conventional carbohydrate chemistry. Preparation of the imidazylate, with the very inexpensive sulfonyl diimidazole reagent,* followed by S N displacement ~ with azide, gave 34, reduced to the mannosamine 35 (see Scheme 10).

__t

OBn

OH 33

H

H I

34

35

SCHEME 10.- S N ~ Displacementof the Iddazylak with hide, Leading to a D-Mannosamhe Derivative.

Acylation of the amine 35, followed by O-deacetalation, gave 36,which was converted into its dibutyltin derivative.%Thisallowed selectiveacetylation, lactoylation, methylation, and benzylation on 0-6, to give the protected N-acetylmannosamines,37,38,39, and 40, respectively.Methylation of 40 gave the 4-methyl ether 41. Catalytic hydrogenolysis led to the free sugars, 16 to 19 (see Scheme 11). (49) S. Hanessian and J. M. Vat&le,Tetrahedron Lett., 22 (1981) 3579-3582. (50) S. David and S. Hanessian, Tetrahedron, 41 (1985) 643-663.

SERGE DAVID et al.

202

CH,

I I

HCOBr cO,CH*

-O H BnO

%

OBn

HOBnO

37

38

f

t HOCH,

H BnO

OBn

MeOCH,

O

mOBn

+ OBn

36

39

BnOCH,

BnOCH2

*

BnO "=OBn

40

M e oBnO -%%/oBn 41

SCHEME 1 1.-Chemical Synthesis of Differently Substituted iV-Acetyl-D-mannosamineDerivatives.

The 5-methyl ether of N-acetylmannosamine cannot be prepared by this route. The mglucofuranose 42 was obtained from the 5,6-&01 by selective benzylation by way of the dibutyltin derivative, followed by conventional methylation. It was converted into the benzyl glycoside 43 with benzyl alcohol under acidic conditions. Conversion into the amine 44and amide 45 followed the same path as in the pyranose series. Hydrogenolysisgavem 28 (see Scheme 12). Table IV gives a list of natural sialic acids and derivatives prepared with sialyl aldolase. Some properties of natural sialic acids have been reviewed3' in a Volume of this Series. c. 3 - D e O X y - D - g ~ ~ C e ~ ~ D - g U ~ ~ ~ ~ - n O n U ~Acid O S Oand n i C Other GlyculosonicAcids. -Here we havegatheredtogethersynthesesstartingwithprecur-

ENZYMIC PREPARATION OF CARBOHYDRATES

MeOCH

Q7 O-CMe2

42

203

MeOCH L_*

QBn OH 43

CH,OBn

I

MeOCH

MeOCH

44

45

SCHEME 12. -Chemical Synthesis of the Furanose Derivative of N-Acetyl-Dmannosamine.

sors not carrying amino groups. Only two are uncommon sugars. Compound 46 was prepared from 3,4 :5,6-di-O-isopropylidene-uldehydo-~glucose dimethyl acetal by displacement with azide of the derived imidazylate, and converted into 2-azido-2-deoxy-~-mannose (8) by acid hydroly~is.~~ The starting material for 2-deoxy-2-C-phenyl-~-mannose(9) was the known5’ 2,3-dideoxy-4,5:6,7-di-O-isopropylidene-3-C-phenyl-~munno-heptonate (47, R = H), which was oxidized to a mixture of alcohols (47, R = OH) by the Vedejs procedure.52 Ester 47 (R = OH) was reduced with lithium aluminum hydride to the diol, which was oxidized to the protected aldehyde-hexose with periodate. Deprotection in aqueous acetic acid gaveu 9. Among all the compounds listed in Table IV under the heading “Derivatives of 3-deoxy-2-nonulosonicacids,” the only one reported so far as a component of living cells is ~-deoxy-~-g~~cero-~-gu~uc~o-~-nonu~oson (51) I. W. Lawston and T. D. Inch, J. Chem. Soc., Perkin Trans. I, (1983) 2629-2635. (52) E. Vedejs, D. A. Engler, and J. E. Telschow, J. Org. Chem., 43 (1978) 188- 196.

204

SERGE DAVID et ul.

acid (ll),called “KDN” (better Kdn) by its disc~verers.~~ It was isolated on the pg scale from rainbow trout egg polysialoglycoprotein; it is exclusively located at the nonreducing end of the sialyl chains, and therefore may be involved in egg activation of salmonid fishes by protecting these chains against sialidases. The authors remarkeds3that it is cleaved to mannose and pyruvate by sialyl aldolase. This suggested its preparation enzymically. In view ofthe availability of D-mannose, and the high yield ofthe condensation, “Kdn” may now be prepared in one step on any desired scale.41

4. Transketolase and Other Enzymes Transketolase from common yeast (Succhuromyces cerevisiue) is commercially available, but it is possible to work with a partially purified enzyme, isolated with little expense from spinach leaves.” Transketolasecatalyzes the transfer of a hydroxyacetyl group, reversibly from a ketose phosphate, or irreversibly from hydroxypyruvate to an acceptor aldose, phosphorylated or not.55It requires thiamine pyrophosphateas a coenzyme, but only in catalyticamounts. In all the cases listed in Table V,the new c h i d center, C-3 of the ketose, has the L-glycero configuration. “Kdo-synthetase”catalyzesthe aldol addition of enolpyruvate phosphate with D-arabinose 5-phosphate (see Scheme 13), which gives 3deoxy-~manno-2-octulosonic acid 8-phosphate (Kdo 8-phosphate). Kdo is an important component of oligosaccharides of Gram-negative bacteria. (53) D.Nadano, M. Iwasaki, S.Endo, K. Kitajima, S. Inoue, and Y.Inoue, J. Biol. Chem., 26 1 (1986) 11,550-1 1,557. (54) J. Bolte, C. Demuynck, and H. Samaki, Tetrahedron Lett., 28 (1987) 5525-5528. (55) (a) F.Racker, A h . Enzymol., 15 (1964) 141; (b)F. Racker, in TheEnzymes,P.D. Eoyer, H. Lardy, and K. Myrbach (Eds.), Vol. 5, pp. 397-406, Academic Press, New York (1 96 1).

TABLEV Misoellaneous Carbon-Carbon Coupling Reactionsa

Product

Starting material

W e (mol)

Yield

(%I

UnitS/mmol

Refereaces

Condensations with hydroxypyrnvate and transketolase

Glycolaldehyde L-glycero-tetrulose 2-Hydrox~pmpanal 5deoxy-D-threo-pentdose 3-A~id0-2-hydroxypropanal 5azidO-5deoxy-D-rhreo-pen~~ 2,3-J3hydroxypropanal mthreo-pentulose fructose 1,6-bisphosphate D-rhreo-pentulose 5-phosphate ~AUose6-phosphate D-gfyCUo-D-altrO&ulO~ I-phoSphate DGlucose 6-phosphate mglycero-tbido-octulose8-phosphate Other condensations with enolpyruvate phosphate and syntheses mArabinose 3deoxy-~-g/ycero-~-~yxoyxo-octulosonic acid 8-phosphate

~-Fructose,ribose 5-phosphate'

3deoxy-~-urabino-heptulo~~c acid 7-phosphate

606

5 2.6

42b

7lC 24b

TK:

2

8od

TK:250

60"

TK:400

38

63d

HK:416; PK:4; KS: 1.3

59

5

69d

HKf:3.2; PKf:1.7;

60

0.1

0.1 0.05

TK: 18 TK: 38 TK:45 A:TK

T K ' : 2.6; w:1.3

54 54

56 54 57 58 58

* Reactions with soluble eazymes,unless stptedothenvk. * Yield from hydmxypymvate. Yidd, pmmmably&om the (R)component.'Yield &om startingaldehyde. text. JEnzymes immobilized on PAN gels.

SCc

SERGE DAVID el a/.

206

COzH

I

p" I

HCOH

1

HCOH

I

CHzOPO$Iz

c"'

COzH

HOCH

+

I c-oqH* II CH2

HOCH 4

I I HCOH I

HOCH

HCOH

I

CH,OPO& SCHEME13.-The Aldol Addition of Enolpyruvate Phosphate with D-Arabinose 5-Phosphate, Catalyzed by Kdo-Synthetase.

Kdo was prepared on the 38-mmol scale starting from D-arabinose, by the simultaneous operation of three enzymes in the same vessel. One is Kdo syntheta~e;~~ hexokinase catalyzes the phosphorylationS7of D-arabinoseby ATP (catalytic), and pyruvate kinase catalyzess8the regeneration of ATP with enolpyruvate phosphate. Such systems are described in more detail in Section IV.In this preparation, enolpyruvate phosphate serves two very different purposes, acting as a source of "high-energy" phosphate, and as a three-carbon donor.s9 Condensation of D-erythrose 4-phosphate with enolpyruvate phosphate in the presence of a specific synthetase gave 3-deoxy-~-arabino-2-heptulosonic acid 7-phosphate (see Scheme 14).60In the complete system, which involves the simultaneous operation of four enzymes mixed together, Dfructose is first phosphorylated by the hexokinase- ATP-pyruvate kinaseenolpyruvatephosphate system already described. Then, transketolase, utilized in the depdative direction, converts D-fructose 6-phosphate into D-erythrose 4-phosphateby the transfer of the CO-CH,OH fragment onto D-ribose 5-phosphate (giving a heptulose 7-phosphate). However, this is an (56) T. Ziegler, A. Straub, and F. Effenberger, Angew. Chem., Znt. Ed. Engl., 27 (1988) 7 16. (57) A. Mocali, D. Aldinucci, and F. Paoletti, Carbohydr. Res., 143 (1985) 288-293. (58) M. Kapuscinski, F. P. Franke, I. Flanigan, J. K. McLeod, and J. F. Williams, Curbohydr. Res., 140 (1985) 69-79. (59) M. D. Bednarslci, D. C. Crans, R. Dicosimo, E.S. Simon,P.D. Stein, and G. M. Whitesides, TetrahedronLett., 29 (1988) 427-430. (60) L. M.Reimer, D. L. Conley, D. L. Pompliano, and J. W.Frost, J. Am. Chem.Soc., 108 (1986) 8010-801 5.

207

ENZYMIC PREPARATION OF CARBOHYDRATES COz H

I

CHO

I

COzH

I

C-OPO,Hz

HCOH

ICH,OPO,Hz +

HCOH

p“

I

II

4

CHZ

r

HOCH

I

HCOH

I

HCOH

I

CH2OPO3HZ

SCHEME 14.-The Aldol Addition of EnolpyruvatePhosphate with mErythrose 4-Phosphate, Catalyzed by DHAP-Synthetase.

expensive route to D-erythrose 4-phosphate, as stoichiometric amounts of D-ribose 5-phosphate are needed, and stoichiometric amounts of unwanted heptulose phosphate are generated in the medium.

IV. PHOSPHORYLATIONS 1. General Considerations Following the logic of organic chemistry handbooks, we have considered the syntheses of sugars before their phosphorylation. The historical order is the reverse one: the preparation of sugar phosphates on the mole scale actually afforded the first demonstration of the high possibilities of immobilized-enzyme methods in fine chemistry. Together with the preparation of phosphates, there will be considered in the same section that of nucleotides, which are obligatory donors in enzymic phosphorylation, and of the socalled “nucleotide-sugars” which belong to the same chemical family. 2. Sugar Phosphates

a. General. -As is well known, the phosphorylation of a sugar is a preliminary to most metabolic conversions. The universal phosphorylating agent is adenosine triphosphate, ATP, which transfers one phosphate group to the substrate, in the presence of an enzyme called a kinase that is more or less specific for the substrate. The phosphorylation of the substrate involves the formation of an equivalent amount of adenosine &phosphate, ADP, generally not a phosphate donor. Normally, the phosphorylation of the substrate is associated with a reaction regenerating ATP, so that only catalytic amounts are necessary. This is outlined in Scheme 15, which illustrates

208

SERGE DAVID el al.

OPO3H,

SCHEME 15.-Enzymic Transfer of a Phosphate Group, Catalyzed by Specific Kinase and Coupled with Reaction Regenerating ATP.

the most popular regeneration system nowadays. Thus, the necessity for stoichiometric quantities of the very expensive ATP is avoided. Molar proportions as small as 1% have sometimesbeen used. Another general property of these systems is the absolute requirement for magnesium by all lcinases. b. Enzymes for the Phosphorylationof the Substrates.-Glycerol kinase is commercially available and inexpensive. The enzyme from S. cerevisiae has broader specificity, but is stable in solution only in the presence of glycerol. However, the immobilized enzyme loses no activity during six months6*at 4”. The S. cerevisiaeglycerol kinase catalyzes the phosphorylation of glycerol, “dihydroxyacetone,” L-glyceraldehyde, and simple diols that are more-distant relatives of the sugar f a m il~.~l*~l Phosphorylation is stereospecific. Glycerol, a prochiral molecule, is converted into optically pure sn-glycerol 3-phosphate, with the (R)configuration, and only the (S) enantiomer of 2,3-dihydroxypropanal reacts, to give L-glyceraldehyde 3phosphate. The classical enzymes of phosphorylation, the hexokinuses, have broad specificity, acting as well on ~-glucose,D-mannose, and ~ - f r u c t o s e . 6The ~~~~ yeast enzyme is utilized. 6-Phosphofructolcinaseand phosphoribulokinase create a second phosphate ester function on a sugar monophosphate. c. ATP Regeneration.-This is the phosphorylation of a terminal hydroxyl group in a pyrophosphate. Only two systems have been practically (61) D. C. Crans and G. M. Whitesides, J. Am. Chem.Soc., 107 (1985) 7019-7027. (62) A.Pollak,R. L.Baughn,andG.M.Wtesides,J. Am. Chem.Soc.,99(1977)2366-2367. (63) C.H.Wong,S.L.Haynie,andG.M.Wtesides,J.Am.Chem.Soc., 105(1983)115-117.

?4

8 8

X

0"

s u

bI

I

X u

+

b

2

5

N

0,

+

a k

c

8-2 I

I

Y r

8-2 rY

bI

gl c

N

I

5 2 b b u, 6

b

d

b

+

rY

2 10

SERGE DAVID ef a1

utilized (see Scheme 16). Acetyl phosphate is a chemical very easily prepared, either in ethyl acetateMor watef15solution. Transfer of phosphate to ADP occurs in the presence of acetate kinase, found in E. coli. However, because of the relative instability of acetyl phosphate in water, it must be added gradually to the vessel in case of long incubation periods. It appears to have been abandoned in favor of enolpyruvate phosphate, which is more stable in water solution, despite a more-complex syntheskMThe enzyme associated with enolpyruvate phosphate is the widespread pyruvate kinase, which is one of the key glycolysis enzymes.

d. Preparation of Pentose Phosphates with Systems of More than Two Enzymes.-Scheme 16 indicates that phosphorylating systems are essentially two-enzymesystems, a substrate-specifickinase, and a kinase for ATP regeneration. However, other enzymes may be associated to the kinases in the same vessel, either for the in situ preparation of substrate, or the further processing of product. In the preparation of ribulose (D-erythro-pentulose) 1,5-diphosphate, the substrate of the phosphorylation enzyme, namely, ribulose 5-phosphateYis obtained by the oxidative decarboxylation of ~ - g l u conic acid 6-phosphate with coenzymeNAD( P) as oxidant, and evolution of COz. The reduced coenzyme NADH(P) is oxidized back to NAD( P) with 2-ketoglutarate in the presence of NH, ,which is converted into glutamate, and is the final oxidant. The successful operation of this system demonstrated the possibility of preparing compounds on the mole scale with four immobilized enzymes6’ Alternatively, “ribulose” (D-erythro-pentulose) 5-phosphate may be isomerized to ribose 5-phosphate with pentose phosphate isomerase, but the same isomerase will convert D-ribose 5-phosphate into D-erythro-pentulose Sphosphate, the equilibrium being displaced by phosphorylation to the diphosphate (involving three enzyme systems). 3. Nucleotides Phosphorolysis of ribonucleic acid with polynucleotide phosphorylase gives a mixture of the diphosphates of the four common nucleosides, which are transformed into triphosphates with enolpyruvate phosphate and pyruvate kinase. This mixture may be used as such as a source of uridine triphosphate in the preparation of the nucleotide-sugar uridine 5’-(a-~-glucopyranosyl diphosphate) (“uridine-diphosphate-glucose,” UDP-Glc), or as a (64) D. C. Cransand G. M. Whitesides, J. Org. Chem., 48 (1983) 3130-3132. (65) R. J. Kazlauskas and G . M. Whitesides, J. Org. Chem., 50 (1985) 1069- 1076. (66) B. L. Hirschbein, F. P. Mazenod, and G. M. Whitesides, J. Org. Chem.,47 (1982) 3765 3766. (67) C. H. Wong, S. D. McCurry, and G . M. Whitesides, J. Am. Chem. SOC.,102 (1980) 7939-7940.

ENZYMIC PREPARATION OF CARBOHYDRATES

21 1

source of ATP in the preparation of glucose 6-pho~phate.~~ In the same way, the enzymic hydrolysis of deoxyribonucleic acid gives deoxyadenosine monophosphate, which can be phosphorylated to deoxyadenosine triphosphate. In the latter synthesis, the double phosphorylation is catalyzed by pyruvate and adenylate kinase, the phosphate donor being enolpyruvate phosphate.24A three-enzyme system, namely, adenosine kinase, adenylate kinase, and acetokinase,converts the very common chemical adenosineinto its most valuable triphosphate,ATP, with acetyl phosphate as the phosphate donor:68 Cytidine triphosphateis necessaryto the activationof N-acetylneuraminic acid (see Section V,3). Its preparati~n’~.~~ is given in Scheme 17. The not

Enolpymvate phosphate

’(

CDP

Enolpyruvatephosphate

Pyruvate

SCHEME 17.-Enzymic Preparation of CTP.

unduly expensive cytidine monophosphate (CMP) is phosphorylated to its diphosphate (CDP) in the presence of immobilized nucleoside-monophosphate kinase. The phosphate donor is ATP, which is regenerated with enolpyruvate phosphate and immobilized pyruvate kinase. Conversion of CDP into CTP must also be catalyzed by the same system, that is enolpyruvate phosphate and pyruvate kinase, and this creates a small problem, for this enzyme has much less affinity for CDP (K, near 5 mM) than for ADP (K, 0.1 mM),and so it must be added in excess. Stoichiometricamounts ofCMP and enolpyruvate phosphate, together with catalytic amounts ofATP, gave a (68) R. L. Baughn, 0. Adalsteinsson, and G. M. Whitesides, J. Am. Chem. SOC.,100 (1978) 304 - 306. (69) C. Augb and C. Gautheron, Tetrahedron Left.,29 (1988) 789-790.

TMLEVI !hgar Phosphates and Nucleotidesa

Sobstrate

product

Glycerol (2JF)Glycerol 2J-Dihydroxypropaxd 1,3-Dihydmxyacetone 1,fDihydroxyacetone D-RibOSe-5-phOSphate DGluconic acid 5-phosphate DGluconic acid 6-phosphate

~n-glycer~l I -phosphate (R)-(2-1T)Glycerol1-phosphate L-glyceraldehyde 3-phosphate 1,3dihydmxyac&onephosphate 1,3dihydroxyacetonephosphate D-erythro-pentdose 1,5-bisphosphate ribose 5-phosphate Derythro-pentulose 1,5-bisphosphate

lo00 20 1

D-FrUCtOSe

~ - f r u c t o6-phosphate ~ mglucose 6-phosphate D~~UC 6-ph0~phat& OS~ D - ~ ~ U C O6-phosphaW S~ 3-deoxy-3-fluoro-~-glucose6-phosphate 4deoxy4fluoro-~-6uctose 1,6-bisphophate ATp(24%),UTP(28%), GTP(30%), CTP ( 18%) ATP

200 lo00 700 3 20

DGlucose DGlucose fructose 6-phosphate

3-Deoxy-3-fluoro-D-@ucoglucose 4-Deoxy4fluoro-~-@ucose Ribonucleic acid Adenosine Adenosine 5’-(monothiophosphate) Deoxyribonucleic acid dAMP CMP CMP

(Sphadenosine 5’4 1-thiotriphosphate) dAMP dATP

CTP CTP

400 160 120 160 130

92 89 41 83 80 58 72 66

1

98 64 68 66 86 30

30 125

62 83

20 150 100 1 2

53 68 100 74

GK:0.7; AK:0.9 GK: 7; PK: 25 GK: 25; PK: 50 GK: 5; AK: 5 GK: 1; PK:7 RPI :8; PHK :7; AK :7 PGD:5; GD:5; RPI:5 PGD:6; PHK:6; AK:6; GD:6 HK:3;AK:2 HK: 1.3; AK: 1.2 HK: 0.5 PGI:33 HK: 14; PK: 18 HKd: 5000, F a d : FKd:2000 NPd:2; PP:0.3; PK:2 ADK:0.3; AYK:0.4; AK: 1.4 AK: 150; PK:250 NP:12; JY AYK:3; PK:6 NKc:3; PKc: 10 AYKf: 1400, PKf:600

61 31 31 61 31 67 67 67 31 62 63 31 70 71 63 68 72 24 24 39,69 27,28

Enzymes immobilized on PAN gel, unless stated otherwise. The actual phosphorylating agent, ATF’, is regenemted either by acetyl phosphate and acetokinase, or enolpyruvate phosphate and ppvate. Irinase. Phosphorylations by a mixture of the common ribonuclmtidetriphosphates. Not isolated. Soluble enzymes. Enzymes immobilized on agarosc.fEnzymesenclosed in a dialysis bag.

ENZYMIC PREPARATION OF CARBOHYDRATES

213

100% yield of CTP in 48 h. A solution of CTP is then obtained by filtration, and utilized directly in the next step. Thus, this synthesis involves three! enzymic conversions but only two enzymes. The same mixture of immobilized enzymes has been used many times without apparent loss of activity. It is very convenient not to be limited in the availability of CTP, for an excess is needed at the next stage to speed up the reaction. Cytidine triphosphate is still an expensive reagent, although several preparations, either by organic chemistry methods or fermentation with whole organisms, have been described in the patent literature. The authors’ experience is that the enzymic phosphorylation and diphosphorylation of nucleoside monophosphates is very efficient: the yields are nearly quantitative and the immobilized enzyme system appears reusable for at least three months. The reported preparations of phosphates and nucleotides are summarized in Table VI. 4. “Nucleotide-Sugars”

These compounds are glycosyl esters of nucleoside mono- or diphosphates. A number have been found in cells, but only two have so far been considered in the present context. The preparation of “uridine diphosphate glucose” (48) is possible independentof its further transformation(seeTable

48 VIII), but the relevant reactionsare more interestingwhen they constitutean integral part of a galactosylation cycle, and they will be described in Section (70) D. G. Drueckhamrner and C. H. Won& J. Org. Chem., 50 (1985) 5913-5916. (71) P.J. Card, W. D. Hik, and K. 0.Ripp, J. Am. Chm. Soc,108 (1986) 158-161. (72) I. R.Moran and G. M.Whitesides, J. Org. Chem.,49 (1984) 704-706.

".1

2 %HEME

J

I

1

49

18.-The Reaction Catalyzed by Cytidine MonophosphateN-AcetylneuraminicAcid Synthetase.

ENZYMIC PREPARATION OF CARBOHYDRATES

215

V,2. This is not the case with cytidine monophosphate N-acetylneuraminic acid (49) (see Scheme 18),the activated form of N-acetylneuraminic acid for sialoside synthesis, as no sialylation cycle has so far been achieved, and thus this precursor must be added to the system in stoichiometric quantity. Thus, the availability of 49 is still the limiting factor in the large-scale synthesis of sialosides. Free, unphosphorylated N-acetylneuraminic acid is directly converted into 49 by cytidine triphosphate in the presence of a synthetase (see Scheme 18). This enzyme is not commercially available for the time being, but calf brain is a good source,73and purification to homogeneity is not necessary. This synthetase accepts substrates other than N-acetylneuraminicacid, such as N-acetyl-9-0-acetylneuraminicacid (20), N-glycolylneuraminic acid (12), and, with less efficiency, "Kdn" (11). It is not possible to associate this synthetase to pyruvate kinase and nucleoside monophosphate kinase as a three-enzyme system in a single vessel, for CMP is degraded by this enzyme. This is not a severe problem: the crude solution of CTP obtained by the reactions of Scheme 17 is separated from the gel by filtration, and then, the sialic acid and the immobilized synthetaseare added. Immobilized inorganic pyrophosphataseis also added in order to drive to the right the equilibrium in Scheme 18, by decomposition of the product pyrophosphate (see Scheme 19).15,39,69

The preparation of cytidine monophosphate N-acetylneuraminic acid (49) was described by Aug6 and coworkers.15Immobilized nucleoside monophosphokinase (0.6 U ) and pyruvate kinase (10 U) were gently stirred at 37' under nitrogen with CMP (0.5 mmol), ATP (0.05 mmol), and enolpyruvate phosphate (1.5 mmol) in 0.1 MTris buffer (pH 7.5) containing 35 mM KCl, 2 mM MgClz, 3 mM 2-mercaptoethanol, mM thymol, and 0.1 m M EDTA. The reaction was monitored by t.1.c. on PEI-cellulosewith successive elutions with LiCl: 0.3 M ( 1 min), M ( 12 min), and 1.6 M(47 min). After 2 days, the gel was collected, and washed with 0.1 MTris buffer (pH 7.5), and the filtrate and washings were used without further treatment for CMP-sialic acid synthesis. Immobilized CMP-sialic acid synthetase (3.7 U)and inorganic pyrophosphatase (6 U) were added to the crude preparation of CTP (0.5 mmol), together with N-acetylneuraminic acid (0.5 mmol). The substrate was adjusted to 2 mMby dilution with 0.1MTris buffer (pH 9). The pH was adjusted to 9 and the MgCl, concentration to 35 mM. 2-Mercaptoethanol and thymol were kept at 3 mM and 1 mM, respectively, and the mixture was gently stirred at 37" under nitrogen. The reaction was monitored by t.1.c. on PEI-celluloseas described for the synthesis of CTP, and on silica gel (7 :3 1-propano1-water). After 10 h, the yield of 49 was 60% as (73) D. H. van den Eijnden and W. van Dijk, Hoppe-Seyler's Z. Physiol. Chem., 353 (1972) 1817.

-

SERGE DAVID et al.

216

u

Neu5Ac

P207H4

CMP-Neu5Ac

Enolpyruvate phosphate pyruvate

\

CDP

Enolpyruvate phosphate

IJyruvate

SCHEME

19. -Enzymic

Synthesis of Cytidine Monophosphate N-Acetylneuraminic Acid

Starting from CMP.

estimated by the thiobarbituric acid assay” and the reaction was stopped. The gel was collected, washed with 0.1MTris buffer (pH 9), and the filtrate and washings were combined, and purified by chromatography on a refrigerated column (3 X 45cm) of DEAE-Sephadex A-25 (HCO,). Elution with a gradient of 0 to 0.75Mtriethylammonium hydrogencarbonate (pH 7.8) gave 49 as its di(triethy1ammonium)salt (234 mg, 52%), RF 0.53 (7 :3 1(74) C. Aug6 and C. Gautheron, Colloque Int. Rkactifs Support&, Lyon, Juin 1982. (75) J. Thiem and W. Treder, Angew. Chem., Int. Ed. Engl., 25 (1986) 1096- 1097.

TABLE Vn NucleotideSogarsa Scale

Stnrting material

UMP, ~-glucosylphosphate ATP, GTP, CTP, UTP, ~glucosylphos-

NucleotideSugar

Yield

(-01)

(%I

1

6

92 97

AK:2.5;UP: 1; IP:4 upb: 10; Mb:10;I F : 10

74 63

0.5

60

PK:20;NK:1.2;CS:7

39,69

0.1

80

PK:20;NK:1.2;CS:6.5

69

0.5

52

PK:20;NK:1.2;CS:12

69

0.5

26

PK:20;NK: 1.2;CS:18

69

0.1

72

CSc

75

“uridine-diphosphate.-glum” “uridine-diphosphate.-~um”

Units/mmol

References

phate cytidine monophosphate-N-acetylneuraminic acid CMP, N-glycolylneuraminic acid cytidine monophosphate-N-glycolylneuraminic acid CMP, N-acetyC9-O-acetylneuraminicacid cytidine monophosphate-N-acetyl-90-acetylneuraminicacid CMP, 3 ~ e o x y - ~ - g I y c e r o - ~ g a l a c t c - ~cytidine o n ~ ~ monophosphate-3-deoxy-~glycerc-n-galactc-nonulosonic sonic acid acid W, N-acetylneuraminic acid cytidine monophosphate-N-acetylneuraminic acid

CMP,N-acetylneuraminic acid

a

Unless otherwise stated, enzymes were immobilized on agarose. Immobilized in PAN gel. Immobilized on silica gel-glutaraldehyde (a six-fold excess of <JTp WBS

Ufilizad).

218

SERGE DAVID el a/.

propanol-water); [a],20- 18" (c 1.9, water); 'H-n.m.r data (DzO):S 1.65 (m, 1 H, H-34, 2.05 (s, 3 H, NAc), 2.50 (dd, 1 H, J3,,%12.5, Jh4 5 Hz, H-3e), 5.97(d, 1 H, J,,z4.5 Hz,H-1 ofribose),6.1O(d, 1 H, J5,67.5Hz,H-5 of cytosine), and 7.97 (d, 1 H, H-6 of cytosine). The nucleotide-sialic acids 50,51, and 52 could be prepared in the same ~ay.6~ Table VII gives a list of nucleotide-sugars prepared with immobilized enzymes.

so R' = NHCOCH~,R~ = AC 51 R' = NHCOCH1OH. R2 = H 52 R' = O H , R ~= H

V. GLYCOSYLATIONS WITH TRANSFERASES

1. General Considerations Glycosylationsoccur in cells by the Leloir pathway, first demonstratedfor galact~sylation.~~ The glycosyl donor is a nucleotide-sugar, and the glycosylation step proper is catalyzed by a transferase. At the same time, a free nucleotide is released which may be used to regenerate the starting nucleotide-sugar in a few enzymic steps. Therefore, in principle, the role of nucleotides should only be catalytic. Only a limited number of nucleotide-sugars occur in cells, so that any one of them may be involved in different types of coupling. On the other hand, the transferase is highly specific,with respect to the glycosyl donor, the sugar acceptor, and the position and anomeric orientation of the coupling. Variations may be tolerated in the sugar units of the (76) L.F.Leloir, Science, 172 (1971) 1299-1303.

ENZYMIC PREPARATION OF CARBOHYDRATES

219

oligosaccharideacceptor not directly involved, but this part of the acceptor is by no means totally indifferent. Relativelyearly reports ( 1980- 1982)from Barker and his group described galact~sylation'~ and fuco~ylation~~ with soluble transferases. 2. Galactosylation

Scheme 20 shows the correspondingcycle, first reported for the synthesis of N-acetyllactosamine on the 10-g scale (R = H),u and later utilized in the

Enolpyruvate phosphate

SCHEME20. -The Multi-enzyme System which RegeneratesUDP-Galactose in situ for Enzymic D-Gdactosylation.

synthesis of many complex oligosaccharides (R = oligosaccharide residue). 15*19*79*80The transfer of a /?-D-galactopyranosylgroup from "uridinediphosphate-galactose" to 0 - 4 of a terminal, nonreducing residue of N-acetyl-/?-D-glucosamine,catalyzed by galactosyl transferase (GT) releases an equimolecular quantity of uridine diphosphate. This is enzymically phosphorylated to uridine triphosphate by enolpyruvate phosphate in the presence of pyruvate kinase. Another transferase, UDP-pyrophosphorylase (77) H. A. Nunez and R. Barker, Biochemistry. 19 (1980) 489-495. (78) P.R. Rosevear, H. A. Nunez,and R. Barker, Biochemistry, 2 1 (1 982) 142 1 - 143 1. (79) C. Augk, S. David, C. Mathieu, and C. Gautheron, Tetrahedronh i t . , 25 (1984) 14671470. (80) C. A@, C. Gautheron, and H. Pora, Carbohydr.Res., 193 (1989) 288-293.

220

SERGE DAVID et al.

(UP), catalyzesthe synthesis of the nucleotide-sugar, “uridine-diphosphateglucose”, from uridine triphosphate and a-D-glucosyl phosphate. This is a reversible reaction which must be displaced in the synthetic direction by the decomposition of its other product, pyrophosphate, which is hydrolyzed to inorganic phosphate with the help of inorganic pyrophosphatase (IP). The last step is the conversion of “uridine-&phosphate-glucose” into “uridinediphosphate-galactose”, catalyzed by epimerase (E). Broadly speaking, the system must be fed with a-D-glucosyl phosphate and the “source of energy” enolpyruvatephosphate, and it releases inorganic phosphate and pyruvate as by-products. All the enzymes utilized in this cycle are commercially available, and, among them, pyruvate kinase, UDPG-pyrophosphorylase, and inorganic pyrophosphatase are relatively inexpensive. However, we recommend the preparation of galactosyl transferase in the laboratory. For this, the only necessary addition to the usual equipment for organic chemistry is a refrigerated centrifuge. Carbohydrate chemists need no extensive practical knowledge in enzymology in order to concentrate 180 U of this enzyme from 2 L of cow colostrum.*l The five enzymes are immobilized separately on PAN or agarose gels. These gels are suspended in water, and the pH is maintained at its optimum value, 8.0, with pH-stat equipment. A 0.1 MTris buffer, pH 8.0, may also be used for small-scalepreparations, when an excess of salts may be tolerated in the work-up. The system is gently stirred at 30”. The complete reaction requires a few days with 2 U of immobilized transferase per mmol of substrate. Afier the reaction has stopped, the product is separated from the gels, which can generally be utilized again, either on the same substrate or another one. The galactosylated oligosaccharideis recovered from its solution by ion-exchangede-ionization, and this is followed by freezedrying. Starting material, if still present, is removed by chromatography on a column of silica gel. The reaction may slow down at 70% completion. The reason is the accumulation of an ionic inhibitor, possibly phosphate. In such a case, the solution separated from the gel is de-ionized, and mixed again with the samegel, and, of course, a fresh batch of ionic cofactors. Galactosyltransferaseintroduces a P-D-galactopyranosylgroup at 0-4 of a terminal, nonreducing 2-acetamido-2deoxy-~-~-glucopyranosyl unit in an oligosaccharide.The overall process is the building of a N-acetyl-p-lactosamine unit. While this may be done by block synthesis with an activated glucosamine derivative, deprotection and enzymic galactosylation appear to be a valuable alternative. In many cases, per-0-acetylated N-acetylglucosamine residues having the P-Dconfiguration may be introduced easily, From (81) R.Barker,K. W.Olsea,M.Shaper,andR.L.Hdl,J. Biol. Chem.,247(1972)7135-7147.

22 I

ENZYMIC PREPARATION OF CARBOHYDRATES

the corresponding chloride, with tin trifluoromethanesulfonate as promotor.82 The phthaloylation -dephthaloylation -N-acetylation sequence is then unnecessary. Furthermore, a free N-acetyllactosamine unit may be further glycosylated with another enzymic system (see Section V,3). Thus were synthesized oligosaccharides 53 and 54 (Ref. 79), 55 and 56 (Ref. 19), and 57 (Ref. 15) and glycopeptide 58 (Ref. 80) by the galactosylation of precursors prepared by organic chemistry procedures. Trisaccharide 53 was first recognized as the epitope of one of the blood-group I antigens in man, namely, I(Ma), but may well play a fundamental role in embryogeneskS3The free hexasaccharide corresponding to glycoside 56 is a trace component of human milk (5 mg/L).84Trisaccharide 57 and glycopeptide 58 were prepared as substrates for sialyltransferase (see Section V,3).

0

HO

Ho OH

53

Ho

w?s

HO

%OH

Ho

CHzOH

OH

OH

54

The galactosylation of the branched trisaccharide-glycoside59 raised an interesting problem. There are two reactive positions, one on each terminal, nonreducing P-N-acetylglucosamine residue. Delicate kinetic experiments indicated that the residue linked to the primary position of galactose was only marginally more reactive than the other one towards solublegalactosyl(82) A. Lubineau and A. Malleron, Tetrahedron Lett., 26 (1985) 1713- 1714; A. Lubineau, J. Le Gallic, and A. Malleron, ibid., 28 (1987) 5041-5044. (83) T. FeFzi, Blood Trans. Immunohaematol., 23 (1980) 563-577. (84) A. Kobata and V. Ginsburg, Arch. Biochem. Biophys., 150 (1972) 273-281.

SERGE DAVID er al.

222

p M p e o *

HO

OH

CH 2OH HO CHzOH 0 HO m CH20H a 0

&

CH2

O

56

HO

OH

I

57

OMe COzH I

HzNCH

58

OMe

ENZYMIC PREPARATION OF CARBOHYDRATES

223

HO

59

transfera~e.~~ The conditions of the immobilized enzyme reactor exaggerated this slight difference. We obtained only traces of hexasaccharide 56, even after 6 days. The only product was pentasaccharide 55, that is, the product of D-galactosylation of the N-acetyl-D-glucosamine residue linked to the primary position of D-galactose at the branching point. Structure 55 was proved by the two-dimensional, COSY proton-n.m.r. spectrum, which could be interpreted in a completely consistent manner. The progressive inhibition of D-galactosylation observed in other systems79may be the origin of this enhanced selectivity, as further D-galactosylation could be achieved, with fresh enzymes, giving 56. Table VIII summarizes the preparative D-galactosylations with immobilized enzymes so far reported.

3. Sialylation Sialyl residues in oligosaccharides are introduced by the reaction of cytidine monophosphate-N-acetylneuraminicacid (49) as the sugar donor with the appropriate substrate, in the presence of specific transferases. Three of these have been utilized in syntheses which may be considered to be “preparative.” None are readily available. The most common, which we have called STA (see Table I), catalyzes the transfer of a 5-acetamido-3,Sdideoxy-~-g~ycero-a-~-ga~acto-2-hexulopyranosonic acid unit (the a-D-pyranose form of N-acetylneuraminic acid) to the primary position of D-galactose in a N-acetyllactosamine residue.86 This enzyme also transfers N-acetyl-9-0-acetylneuraminicacid (20) and N-glycolylneuraminic acid (12) from the corresponding cytidine monophosphate derivatives.I6 The commercial enzyme is rather expensive, but pork liver from a butcher is a (85) W. W. Blanken, G. J. M. Hooghwinkel, and D. H. vanden Eijnden, Eur. J. Biochem., 127 (1982) 547-552. (86) J. C. Paulson, J. I. Rearick, and R. L. Hill, J. Eiol. Chem., 252 (1977) 2363-2371.

N-Acetyllactosamine N-Acety. ec f-i1 +4>/3-~&1cpNAc-(l+3)-~-Gal (54)d &LAMP-( 1 +4)-/3-~-GlcpNAc-(1+6)-~-Gal(53)d

fi~-Galp(I+4)-,T-~-GlcpNAc-(l+Z)-a-~Manp-(l +OMe) (57)d 1+4)-/3-~-Gl~pNAc-(l+~)-[BD-G~c~NAc-( 1+3)]-/3DGalp-(1+4)-,T-~-Glcp-(l+OMe) (55)d fidrmp( 1+4)-/3-~-GlcpNAc-(I +3)-[Bdrmp( 1 + 4 ) - f i ~ GlcpNAH 1 +6)]-/3-~-Galp-(l+4)-/3-L&lcp(l +OMe) (56)d fioGalp(l+4)-fi~-GlcpNAc-(l + A m ) (58)d

34 2 0.5 0.5

GT: 1; E:1; UP:1.2; M:3; IP:3.6; PK:4

85 30 70 70

GT:4; E:4; UP:+ IP:80; PK: 140 GT:4; E:4; UP:+ IP:80; PK: 140

0.05

36

GT :24; E :30; UP:46; PK: 290, IP:350

19

0.07

44

GT:24;E:30;UP:46;PK:290;IP:350

19

0.13

26'

GT:29; E:19; UP: 17; IP:100, PK:90

80

23 75 79 79 15

Unless otherwisestated, the galactose precursorwas Dglucosyl phosphate,the phosphorylatingagent was ATP, and the source ofenergy was enolpyruvatephosphk. Enzymes immobhed on PAN gel; Mucose 6-phosphate as precursor. Enzymes immobilized on silica gel-glutamldehyde. Enzymes immobilized on agarose. Isolation &cult.

ENZYMIC PREPARATION OF CARBOHYDRATES

225

good, inexpensive source,87where it is fairly abundant (60 U/kg). A homogeneous enzyme is not necessary for sialylation.From a suitable concentrate, the enzyme may be immobilized on agarose in good yield, after addition of 0.5 mg of bovine serum albumin per mL of extract, and dialysis against the immobilization buffer (0.1 M phosphate, pH 7.8; 25% of glycerol). Such preparations are stable for at least 5 months at 4",and may be utilized at least three times without noticeable loss of activity.l5 The second transferase (STB; see Table I) is also commercially available, and is still more expensive. It catalyses the transfer of N-acetylneuraminic acid to 0-3 of D-galactose in the terminal residue &~-Galp(1+3>wGalN A C .The ~ ~ third one (STC), so far does not appear to be at all easily available. It catalyzes the transfer of a N-acetylneuraminicacid residue to 0-3 of D-galactose in a B-~-Galp-[143(4)]-/3-~-GlcpNAcresidue. Most sialylations so far reported have been achieved with solubletransferases, and seldom on a more than 20-pmol scale (see Table IX),with the intention to prepare and describe sequences present in glycoproteins and glycolipids. Trisaccharide a-~-NeuSAc-(2 +3)-8-~-Galp( 1 ~)-P-DGlcpNAc-( 1 +OMe) was prepared with two different transferases, STB and STC.In our view, the greater efficiency of STB in this preparation deserves further investigation, as the reverse observation might have been expected in view of the known specificities of these enzymes. The one glycopeptide in Table IX, namely, 60 (Ref. 80), was prepared from the known922-acetamido- 1-N-(~-aspart-4-oyl)-2deoxy-~-~-~ucopyranosylamineby two enzymic steps, a D-galactosylation, to give intermediate 58, followed by sialylation.mIt is interesting that neither the wboxylate nor the amino group of the L-aspartamide moiety was inhibitory in these reactions. Compound 60 appears identical with a glycopeptideisolated from the urine of a patient suffering from aspartylglu~osaminuria.~~ In Table X are reported three syntheses with immobilized transferase STA.15 Comparison with the reactions of the same enzyme in Table IX outlines the advantages of immobilization: the scale has been raised, and much less activity is necessary. It is possible to work with a mole to mole ratio 18pg9

(87) D.H.vandenEijndenandW.E.C.M.Schiphorst,J.Bid. Chem.,256(1981)3159-3162. (88) J. E. Sadler, J. I. Rearick, J. C. Paulson, and R. L. Hill, J. Bid. Chem., 254 (1979) 4434 -4443. (89) J. Weinstein, U. de Souza-e-Silva,and J. C. Paulson, J. Biol. Chem., 252 (1982) 13,84513,853. (90) S. Sabesan and J. paulson, J. Am. Chem. Soc, 108 (1986) 2068-2080. (91) K. G. I. Nilsson, Curbohydr. Res., 188 (1989) 9- 17. (92) H.G. Garg and R. W.Jeanloz, Adv. Curbohydr. Chem. Biochem.,43 (1985) 135-201. (93) J. F.G. Vliegenthart, L. Dorland, and H. van Halbeek,Adv. Curbohydr. Chem.Biochem., 41 (1983) 209-374.

TABLEIX Siylations with Soluble Transferases product

Oligosacchnrides a-mNeuSAc-(2+6)-&~-GaIp(1-rOMe)

a-mNeuSA~-(2+6)-fi~-Galp-(l-r4)-&~-Glcp( 14OMe) a-~-NeuSAc-(2+6)-&~-Galp-( 1+4)-~-GlcNAc a-~-Neu5Ac-(2+6)-fioGalp( 1+4)-BDGlcpNAo(l +OMe)

N OI N

a-~-NeuSAc-(2+6)-&oGalp( 1-r4)-&~-GlcpNAc-(l+3)-fi~-Galp( 1 -4)-~-Glc a-~-NeuSAc-(2+3)-&ffialP( 1-+3)-a-~-GalpNAc-OR R = Et R = CH,CH,Br R = (CH,),CO,Me ~ - D - N ~ U ~ A C - ( Z + ~ ) - ~ ~ ~ + ~ ) - ~ ~1+OMe) D-G~C~NAC-(

Scale (Pol) 7 9 47 20 14

64 8 13 17 7 6 a-~NeuSAc-(2+3)-&~-Galp-(1+4)-&ffilcp(l +OMe) 9 a-~-Neu5Ac-(2+3)-&~-GaIp(l+4)-&ffilcpNAc-( 1 +OMe) 9 a-~-NeuSAc-(2+3)-fioGalp(l~4)-fi~-GlcpNA~l~3)-fiD-Galp(l+4)-~-Glc 7 a-~-NeuSAc-(2+3)-fioGalp(l~3)-&~-GlcpNAc-(l+3)-&D-GaIp(l+4)Glc 50 Clycopeptide a-~-Neu5Ac-(2+6)-fi~l+4)-/3-~-GlcpNAo(l +N)Asn (60) a

Yield with respect to the substrate. Yield with respect to cytidine monophosphat&”lneuramjnic gland.

Yield I”

Yield IIb

(%)

(%I

1 1 57 48 74

33 42 47 96

64 95 32 23 18 0.7 22 47 35 38

64 59 32 52 18 28

35

45

22 17 38

Units/ mmol

References

STA:32 STA:24 STA:53 STA:12 STA:21

90 90 75 90 90

STBc STB:2.5 STB:1 STB:2.3 STC:7 STC:8.5 STC:5 STC:6 STC:7 STA:lO

91 91 90 91 90 90 90 90 90 80

acid. A preparation correspondingto 65 g of porcine

TABLEX Sialyhtionswith I m m o b i i Sialyltransferases~ ~-

Nucleotide-sugar

R 4

c ~ Neu5Ac p (49)

CMP-Neu5,9Ac2(50)

Product

a-~-NeuSAc-(2--r6)-S~-Gallp(l+4)-/3-~Gl~pNAc-( 1-+2)-ol-~-Manp (1 4 O M e )(61) a-~-NeuSAc-(2+6)-/3-D-Galp(l -+4)-/3-~-GlcpNAc-(l+3)-[/3-&alp-( 1 - 4 ) /~-DCIC~NAC-( 1 --r6)]-B.~-Galp-(l-*4)-~~-Cilcp(l-.OMe) (62) a-~-Neu5,9Ac~-(2+6)-&DGalp( 1+4)-~-GlcpNAc(63)

Scale @mol)

Yield* (%)

Units/ mmol

References

100

46

4c

15

45

34

8.9

95

160

65

2.5'

15

~~

CMP NeuSAc :&&alp( 1 - + 4 ) - ~ l c p N A c a ~ 2 - 6 > y l t r a n s f ~ . Equimolecularamountsof substrate and coenzyme were used. After these couplings, the ~ ered enzyme preparation retained full activity.

~ o y -

228

SERGE DAVID et al.

HO 60

of precursor oligosaccharide to CMP-NeuSAc. Furthermore, it should not be forgotten that the enzymic gel may be used again at least three times, so that, in principle, a scale three times as high is within reach. The synthesis of tetrasaccharide-glycoside61 involved first the preparation¶by organic chemistry methods, of the known glycoside&D-GlcpNAe (1 +2)-ct-~-Manp( 1+OMe), followed by enzymic D-galactosylation to give 57, and then sialylation. Its sequence is a common feature of a class of glycoproteins. The free tetrasaccharide has been prepared by organic glycosidic coupling.94 Heptasaccharide 62 was obtained from hexasamharide 56 (see Section V,2). As in the case of D-galactosylation, enzymicsialylation turned out to be highly regioselective, leading to a single compound, whereas each galactose

(94) T. Kitajima, M. Sugimoto, T. Nukada, and T.Ogawa, Carbohydr. Res., 127 (1984) cl -c4; H.Paulsen and H. Ti&, ibid. 144 (1985) 205-229.

ENZYMIC PREPARATION OF CARBOHYDRATES

229

Ho

62

63

residue could theoretically be substituted. Heptasaccharide 62 corresponds to the product of sialylation of the D-galactose residue on the p-( 1-3) branch.9sIt is the methyl glycoside of a sialylhexasaccharide isolated from human milk.% The preparation of trisaccharide 63 illustrates the activation and enzymic coupling of the 9-acetate of N-acetylneuraminic acid. This involves the utilization of enzymes in a cascade of reactions which probably do not occur in cells: (a) synthesis of Neu5,9Ac2from the 6-acetate of N-acetylmannosamine with the catabolic sialyl aldolase, (b) activation with CMPNeuSAc synthetase,and (c) coupling. Acetylation in cells seems posterior to coupling. Terminal nonreducingN-acetyl-9-0-acetylneuraminicacid residues appear (95) C. Augk and C. Gautheron, unpublished results. (96) M. T. Tarrago, K. H. Tucker, H. van Halbeek, and D. F. Smith,Arch. Biochem. Biophys., 267 (1988) 353-362.

H

NeuSAc

+

g 0

P2°7H4

x \

o

'aoNHAC

H % ~ , ~ ~

/

\r

CMP-Neu5Ac CMP-Neu5Ac

Enolpyruvate phosphate

CDP

CMP

A

\

CHzOH

Enolpyruvate phosphate

Pyruvate

I

Ac OH SCHEME 21.-Cycle for Enzymic Sialylation which Should Allow in situ Regeneration of CMP-N-AcetylneuraminkAcid.

ENZYMIC PREPARATION OF CARBOHYDRATES

23 1

fairly widespread as part of the oligosaccharide epitope of some important antigens (see, for instance, Ref. 97). These reactions correspond to the right-hand side of Scheme 2 1. The complete Scheme would correspond to the overall synthetic route to sialosides, with the following balance: Neu5Ac

+ 2 CH2

-

+

C(OPO,H,)--CO,H R-OH sialoside f 2 CH$OCOZH P207H4

+

-B

It would be elegant to bring together the broken ends of the cycle, and make the four immobilized enzymeswork together in one vessel, so that the part played by CMP would be only catalytic,but such a cycle has not yet been reported. The difficulties come from problems of inhibition: sialyltransferases are inhibited by CTP and CDP (K, = 2. 10-5M).86 However, this inhibition was reported89to be relieved by the addition of MnCl,, and so the in situ regeneration of CMPNeuSAc should be feasible. 4, Glucosylation

Stoichiometric quantities of “uridine diphosphate glucose” were used, in the presence of a transfer enzyme, sucrose synthetase, in the soluble state (extraction given). Coupling with modified D-fructose gave sucroses modified on the D-fructosyl group, on the 1- 3-mmol scale. Thus were prepared 1I-deoxy- 1’-fluoro- (59%),98 4’-deoxy-4’-fluoro- ( 16%), and 1I-azido-1Ideoxy-sucrose ( 15%).’l 6-Deoxy-6-fluoro-~-glucosewas isomerized to 6deoxy-6-fluoro-~-fructosewith isomerase, and gave 6’deoxy-6’-fluorosucrose. VI. TRANSFER REACTiONS CATALYZED BY GLYCOSIDASES Glycosidases catalyze the hydrolysis of glycosidic bonds D-OZ

+ H20

+

D-OH

+ Z-OH,

D being a glycosyl group. The reaction occurs in two main steps, formation of a glycosyl enzyme, and transfer of the glycosyl group to a water molecule. D-0-Z

D-E

+ EH

+ HO-H

-P

+

+ ZOH D-OH + EH DE

(97) D. C. Gowda,G.Reuter, A. K. Shukla, and R. Schauer,Hoppe-Styler’s Z. Physiol. Chem., 365(1984) 1247-1253.G. N. Rogen,G. Herrler,J.C. Paulson,andH. D. Klenk,J. Eiol. Chem., 261 (1986) 5947-5951. (98) P. J. Card and W. D. Hitz, J. Am. Chem. Soc., 106 (1984) 5348-5350.

SERGE DAVID et al.

232

Other hydroxylated derivatives (A-OH), such as alcohols or sugars, are also possible acceptors: D-E

+ A-OH

-L)

D-0-A

+ EH,

so that the overall reaction is the transfer of the glycosyl group D from the donor molecule D-0-Z to one oxygen atom of the acceptor A-OH. D-0-Z+A-O-H~D-O-A+Z-OH

There are presumably two configurational inversions in such a mechanism, so that the anomeric configuration of the newly formed glycosidic bond is the same as that of the donor. Now that some glycosidasesare common and inexpensive,this scheme of glycosylation looks very attractive. Furthermore, if the acceptor is a polyhydroxylatedderivative, the reaction is regioselective, and there is a measure of control over that regioselectivity. However, yields are small,rarely exceeding 30% with respect to the donor molecule, and generally inferior, and the acceptor is added in 2-20-fold excess. At first sight, this would appear of little importance for practical purposes, for the starting sugars are often less expensive than other chemicals, such as solvents or chromatographyadsorbents, and it should not be forgotten that nonenzymic oligosaccharidesynthesis, with its many steps in the present state-of-the-art, gives small overall yields. In our view, the problem lies elsewhere: the simple derivatives obtained with glycosidaseswill be interestingas starting compoundsfor further syntheses, and thus needed in relatively large quantities. Then, separation from a great excess of sugars with like properties may be very expensive. Money and labor saved on one side may be wasted on the other. Thus, it is to be expected that these promising routes will achieve popularity when cheap separation procedures are evolved. Some significantresults are reported in Table XI. Lactose, a by-product of the dairy industries having a negligible value, acts as a source ofpD-galactopyranosyl groups in the presence of #b-galactosidase. a-D-Galactopyranosyl and a-D-mannopyranosylgroups are transferred from the corresponding pnitrophenyl glycosides in the presence of a-glycosidases. Such systems allow, for instance, a remarkably quick preparation of derivatives of the disaccharide a-D-Galp-(1 --* 3)-mGal, a sequence present in blood-group B substance, and not readily available because of its 1,Zcislinkage. (99) K. G. I. Nilssoo, Curbohydr. Res.. 167 (1987) 95- 103. (100) K. G.I. Nilssoo, Curbohydr.Res., 180 (1988) 53-59. (101) F. Bjarkliog and S.E.Godtfredsen, Tetrahedron,44 (1988) 2957-2962. ( 102) P.0.Larsson, L. Hedbys, S.Sveossoo,and K.Mosbach,Methods Enzymol., 136 (1 987) 230-233.

TABLEXI Pyranosyl Transfer with Glycosidases" Yield Acceptor

Product

(a) System:pnitrophenyl a-D-galactopyranoside a-mgalactosidase a-~-Galp(1+3)-a-~-G&ll +OC6H4N02-d4)1 a-D-Galpl1+OC,H4N0,-

d411

a-D-GalP(1+3)a-~-Galp( 1+OCHZCH=CHZ) (1 +OCHZCH=CHz) a-o-Galp(l-3)-a-~-Galp(l+OMe) a-PGalp(1+OMe) /3-~-Galp41+OMe) a--dmh(l+3)-/3-oGalp(l+OMe) (b) System: o-nitrophenyl j?-D-galactopyranoside, /?-D-galactosidase 1+OMe) /3-~-CAp-(l+3)-/3-~-Galp(l+OMe) 2,3-Epoxypropanol 1-~/3-~galactopyranosy1-[2(R,S),3-epoxypropol] (c) System: lactose,j?-D-galactosidase CH,=CH-CH,OH bDGalp(l+OCH,--CH=CH,) PhCH,OH bo-Galp(1+OCH,Ph) GalNAc /3-D-Galp( 1+6)~-GalNAc (d) System: pnitrophenyl a-D-mpnnopyranoside, a-mmannosidase a-~-Manp(1+2)-a-~-Manp( 1+OMe) cr-~-Manp(1+OMe) a-DGalp

a

Soluble enzymes, unless stated otherwk. Enzyme immobilized on tresyl-activated Sepharose.

Scale

(%)

U/mmol

12

60

1

99

18

45

0.5

100

28 17

6 28

2 0.5

99 99

17 28

400

1.5 5

101

31 14 25

17 240 7ob

100 15 1

100 100 102

15

37

10

99

10

References

(-01)

99

234

SERGE DAVID ef al.

VII. MISCELLANEOUS SYNTHESES IN AQUEOUS SOLUTION

Some lipases catalyze the selective hydrolysis of the anomeric acetate in peracetylated sugars. Thus were prepared 2,3,4,6-tetra-O-acetyl-~-galactopyranose, -D-glucopyranose, and -D-mannopyranose, 2-acetamido-3,4,6tri-O-acetyl-2-deoxy-~-glu~p~nose and -mmannopyranose, 2,3,4-tri-Oacetyl-L-rhamnoseand -L-fucose, and 2,3,5-tri-O-acetyl-~-ribofuranose and -D-xylofuranose on the 2-mmol scale, generally in good yields.lo3 These reactions were conducted in a 1 :9 mixture of N,N-dimethylformamideand 0.1 M phosphate buffer (pH 7) by stirring at room temperature in the presence of the lipase, and adjusting the pH to 7.0 with 1.0 M NaOH. Under similar conditions, methyl glycosides are 0-deacetylated on the primary position, affording, inter a h , methyl 2,3di-O-acetyl-cu-~ribofmnoside, P-D-ribofuranoside,and a-~-arabinopyranoside,~~~ and methyl 2,3,4-tri-0pentanoyl-cx-D-ghcopyranoside.104 Treatment of 3,6-di-O-butanoyl-~-glucose with the lipase from Candida cylindracea gave 3-O-butanoyl-~-glucose in 85% yield.lo5More information on acylation and deacylation, but this time in organic media, will be found in Section IX. We finish this Section with enzymic conversions that are difficult to classifyelsewhere:Takasweet, a commercialvariety of immobilizedglucose-isomerase, converts 6-0-methyl-~-fructoseand 6-deoxy-~-fructoseinto the gluco isomers in not very satisfactory yield.” A mixture of catalase (75 U/mmol) and glucose oxidase (80 U/mmol) oxidizes xylitol to ~-xylosein 5040 yield, on the 100-pmol scale.lMThe enzyme cyclodextrin a-(1 4 4 ) glucosyltransferase ( 1000 U, immobilized on silica gel-glutaraldehyde) preparation of cyclomaltohexaose (0.3 g), cyclomaltoheptaose

64 (103) W. J. Hennen, H. M.Sweers, Y.F. Wan& and C. M.Won& J. Org. Chem.. 53 (1988) 4939- 4945. (104) H.M. Sweers and C. H. Won& J. Am. Chem. Soc.,108 (1986) 6421-6422. (105) M. Thtrisod and A. M. Klibanov, J. Am. Chem. SOC.,109 (1987) 3977-3981. (106) R. L. Root,J. R. Dunwachter, and C. H. Won& J. Am. Chem. Soc.,107 (1985) 29972999.

ENZYMIC PREPARATION OF CARBOHYDRATES

235

(0.38 g), and malto-oligomers from a-D-glucopyranosyl fluoride (1 g).Io7 The same enzyme allowed the preparation of 12 mmol of the glycoside64 of ldeoxynojirimycin in 25% yield.’@

VIII. ENZYMESIN ORGANIC SOLVENTS*

Klibanov has summarized the principles of the technique in a short survey.lWEnzymes still work albeit, sometimes, in a different way, if a layer of “essential water” is somehow localized and kept on their surfaces, and the bulk water is replaced by an organic solvent. Thus, the enzymesare generally freeze-dried, and the solids suspended in an organic solvent, and then traces of water are added to ensure maximum activity. In this state, they show high conformational rigidity: heat-induced unfolding (denaturation)is hindered, and some are stable for hours at 100”.Because of this rigidity, they keep a “memory” of their previous state in water: freeze-drying in the presence of active-sitedirected molecules may yield more active conformations. The ionization state corresponding to the pH of the aqueous solution, which should be optimal, is also retained. The absence of water may have other advantages: for instance, lipases may act as esterification catalysts, a property obscured in water solution by the reverse, common hydrolytic reactions. However, it seems that empirical trials are still neceSSary in order to achieve a successful synthesis: several enzymes from different natural sources should be tested, and even enzymeshaving different specificities. For laboratory-scale preparations, the cost of such enzymes as the lipases from porcine pancreas (PPL), Cundida cylindruceu (CCL), and Chromobucterium viscosurn (CVL), and Protease N (Ammo) is negligible. Subtilisin, a protease, is much more expensive. Transesterificationof sugars and derivatives with such “active” esters as the acetate, butanoate, decanoate, or dodecanoate of 2,2,2-trichlorethanol allowed selective acylation. In this first way, ~-glucose,D-galactose, and D-mannose, in multigram quantities, gave the primary acylate in fair yield,Il0in pyridine solution, in the presence of PPL (70,000 U). This type of reaction was also selective with di- and tri-saccharides: thus, in

* The authorsare much indebted to Dr.Michel Thi5rkx.I for the preparation of this Section. (107) W. Treder, J. Thiem, and M. Schlingmann, Tetrahedron Left.,27 (1986) 5605-5608. (108) Y. Ezure,Agric. B i d . Chem., 49 (1985)2159-2165. (109) A.M. Klibanov, TIBS, (1989) 141-144. ( 1 10) M. Thinsod and A. M. Klibanov,J. Am. Chem. Soc., 108 (1986) 5638-5640.

SERGE DAVID et al.

236

N,N-dimethylformamide solution, in the presence of subtilisin, maltose, cellobiose, lactose, and (remarkably) maltotriose, gave, on the gram scale, fair yields of the primary butanoate in the nonreducing unit. Sucrose (1 3 g) gave, in 60%yield, the primary butanoate of the ghca moiety in the presence of the very cheapti1Protease N; transestefication from 2,2,2-trifluoroethyl acetate, in oxolane solution, in the presence of PPL, likewise gave the primary acetate of some methyl pentofuran~sides.~~~ Degueil-Castaing and coworkers1**introduced enol esters in transesterification, and these were also used in the carbohydrate field: N-acetylmannosamine and isopropenyl acetate in N,Ndimethylformamidesolution gave 2-acetamid0-6-O-acetyl2-deoxy-~-mannose, in the presence of protease N (1 g/mmol)." This acetate is the precursor of an important sialic acid." In a study of the selective esterifidon of hexoses already substituted at the primary alcoholic function, Th6risod and K l i b a n o ~ observed '~~ very suggestive differences in selectivity,according to the origin of the lipase. All these reactions were transesterification with 2,2,2-trichlorethyl butanoate, with 100,000 U of lipase per gram of substrate, in oxolane as the solvent. With 6-O-butanoyl-~-glucose as the substrate, lipase CVL catalyzed transesterification to 0-3, to give an 80% yield of 3,6-di-O-butanoyl-~-glucose, while PPL directed the reaction to 0-2, giving a 50% yield of 2,6-di-O-butanoyl-D-glucose. The 0-3 atom was only marginally favored over 0-2 with 6-O-butanoyl-~-galactoseand CVL, but a yield of 55% of 3,6di-O-butanoyl-D-mannose could be achieved from 6-O-butanoyl-~-mannose.Remarkably, 6-O-trityl-~-glucosewas a substrate of lipase CVL (only 20,000 U/gof substrate). Transesterification, followed by detritylation, gave a 90% yield of 3-O-butanoyl-~-glucose.

-

IX. ADDENDUM A large-scaleenzymic synthesis of the trisaccharidea-~-NeuSAc-(2+3)gal-( 1+3)-~-GlcNAc (65), the tumor-associated carbohydrateantigen CA 50, has been achieved.' l3 This is a further illustration of the cross reactivity and efficiency of STB. This sialyltransferase, like STA, was partially purified from porcine liver, accordingto a modification of Conradt's procedure.Ii4The initial rate measured for p-~-Gal-(1*3)-~-GlcNAcat saturating concentration was 18%of the one measured for the real substrate B-D(1 11) S. Rim, J. Chopineau, A. P.G. Kieboom, and A. M.Klibanov, J. Am. Chm. Sot., 1 10 (1988) 584-589. (1 12) M.Degueil-Castaing, B. De Jew, S. Drouillard, and B.Maillard, Tetrahedron Lett., 28 (1987) 953-954.

ENZYMIC PREPARATION OF CARBOHYDRATES

231

Gal-(1+3)-~-GalNAc. By using 0.7 U of STB, as a soluble preparation readily obtained from 300 g of porcine liver, the sialylation of p-D-Gal( 1 -3)-~-GlcNAc was performed on a one-mmol scale and sialylated trisaccharide 65 was obtained in 2 1% isolated yield. In this respect, the purification of reaction mixturesis still troublesome, especially because of the presence of Triton X-100; from our experience, the use of immobilized enzymes, eliminating the need for detergent, greatly facilitatesthe purification procedure. Concerning aldolases,the cloning of enzymesis becoming more and more common. Thus the bacterial fuculose-1-phosphate aldolase (EC 4.1.2.17) and 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) have been recently overexpressed in E. coli and their synthetic use has been e ~ a m i n e d . ~ ~ ~ . ~ ~ ~ ACKNOWLEDGMENTS This work was supported by the Centre National de la Recherche Scientifique and the Universiti de Paris-Sud at Orsay.

( 1 13) C. Augi, P. Francois, and A. Lubineau, Jacques Monod Conference on Chemistry, Bio-

chemistry and Molecular Biology of Glycoconjugates,Aussok, France, October 22- 21, 1990. (1 14) H. S. Conradt, K. Hane, and M. Mom, Proc. JapaneseGerman Symp. Sialic Acids,

Berlin, F.R.G., may 18-21 (1988) 104- 105. (1 15) A. Ozaki, E. J. Toone, C. H. von der Osten, A. J. Sinskey, and G. M. Whitesides, J. Am. Chem. Soc., 112 (1990) 4910-4911. (1 16) C. F. Barbas, Y-F.Wang, and C-H. Wong, J. Am. Chem. Soc., 112 (1990) 2013-2014.

This Page Intentionally Left Blank

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 49

STRUCWRE OF COLLAGEN FIBRILASSOCIATED, SMALL PROTEOGLYCANS OF MAMMALIAN ORIGIN BY HARIG. GARG*AND NANCYB. LYON#

* Department of Biological Chemistry and Molecular Pharmacology and # Department of Dermatology, Harvard Medical School at the Massachusetts General Hospital, and the Shriners Burns Institute, Boston, Massachusetts 02114 I. Introduction.. ....................................................... 11. Structure of Different Glycosaminoglycans. ............................... 111. Carbohydrate-Protein Linkage Regions. ................................. IV. Isolation and Fractionation of Small Proteoglycans. ........................ V. M, of Small Proteoglycans, Their Protein Cores, and Glycosaminoglycan Chains VI. N-Terminal Sequence of Small Proteoglycans ............................. VII. Amino Acid Sequence Analysis of the Small Proteoglycan Core Protein, Deduced fromClonedcDNA ................................................. VIII. Biosynthesis of Small Proteoglycans ..................................... 1. PrimaryCulture. .................................................. 2. Explant Culture ................................................... IX. Biological Roles of Small Proteoglycans .................................. X. Addendum. .........................................................

239 240 240 243 244 253 254 256 256 257 258 260

I. INTRODUCTION** All mammalian tissues contain proteoglycans (PGs), which consist of a single protein core containing one to >200 glycosaminoglycan chains attached through 0-P-D-xylopyranosyl-(1+3)-~-serine/~-threoninelinkage(s). The various types of PGs present in different tissues can be divided into two categories, namely, (1) cell-associated PGs and (2) extracellularmatrix PGs. Of the extracellular-matrix PGs, the cartilage PGs have been

** Articles on this subject that have appeared since this text was completed are listed in the Addendum starting on p. 260.

239

Chynghl 8 1991 by Academic Press, Inc. All rights of nproductionin m y form -&.

240

HARI G. GARG AND NANCY B. LYON

extensively discussed in Cell-associated PGs have also been described in comprehensive During the past decade, collagen fibrilassociated PGs have begun to be characterized. These PGs have been isolated from normal bone, cartilage, scar, skin and other connective tissues, and have been shown to contain a protein core to which 1 or 2 glycosaminoglycan (GAG) chains are attached. Their molecular weights, in distinct contrast to the high density cartilage PG, range from 70 to 140 kDa, and for this reason they are generally referred to as “the small PGs.” The small dermatan sulfate collagen fibril-associatedPGs from interstitialmammalian tissuesare the subject of this Chapter. 11. STRUCTURE OF DIFFERENT GLYCOSAMINOGLYCANS Glycosaminoglycans (GAGS) are unbranched chains having repeating disaccharide units, which, with the exception of keratan sulfate, contain an acid and a base. The KS disaccharide unit consists of a hexose and a base. The structures of the different classes of GAG disaccharide units are given in Fig. 1, and summarized in Table I. The GAG chains of chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin (HP) also contain one molecule of D-xylose and two molecules of D-galactose per molecule, along with acidic (Dglucuronic/L-iduronic) and basic (D-galactosamine/D-glucosamine)sugars. The migration of the GAG chains on cellulose acetate plates stained with Alcian Blue occursgin the following order HP < DS < HS < HA < CS. 111. CARBOHYDRATE - PROTEINLINKAGE REGIONS

The carbohydrate-protein linkages of chondroitin sulfate (CS) and dermatan sulfate (DS) in collagen-associated PGs, haveI0J1the general struc-

A.

(1) D. HeinegArd and Oldberg. FASEB J., 3 (1989) 2042-2051. (2) L. C. Rosenbergand J. A. Buckwalter, in K. E. Kuettner,R. Schleyerbach,and V. C. Hascall (Eds.), Articular Cartilage Biochemistry, Raven Press, New York, 1986, pp. 39-57. (3) K. E. Kuettner and J. H. Kimura, J. Cell Biochem., 27 (1985) 327-336. (4) A. R. Poole, Biochem. J., 236 (1986) 1- 14. (5) C. J. Handley, D. A. Lowther, and D. J. McQuillan, CellBiol. Znt. Rep., 9 (1985) 753-781. (6) V. C. Hascall, in V. Ginsburgand P. Robins (Eds.), Biology ofCarbohydrates, Wiley, New York, 1981, pp. 1-49. (7) E. Ruoslahti, Annu. Rev. CellBiol., 4 (1988) 229-255. (8) E. Ruoslahti, J. Biol. Chem., 264 (1989) 13,369- 13,372. (9) D. A. Swam, H. G. Garg, W. Jung, and H. Hermann, J. Invest. Dermatol., 84 (1985) 527-53 1. (10) L.-.k Fransson, Biochim. Eiophys. Ada, 150 (1968) 31 1-316. (1 1) F. Akiyama and N. %no, Biochim. Biophys. Acta, 674 (1981) 289-296.

COLLAGEN FIBRIL-ASSOCIATED SMALL PROTEOGLYCANS

r

CH,OSO,-

H o $

0

-

HO

OH

J n

Hyaluronan (HA)

L

NHCOCH,

OH

Keratan sulfate

(KS)

NHCOCH, OH

OH

-

i n

Chondroitin 4-sulfate (CS-4s)

Dennatan sulfate ( DS)

r CH,OSO,-

I

OH

OS0,1

Heparan sulfate (HS)

L

Heparin (HP)

FIG.1. -Structure of Merent Disaccharide Units of Glycosaminoglycans (GAGS).

HARI G. GARG AND NANCY B. LYON

242

TABLE I General Composition of the GlycosaminoglycansHaving the General Formula (A -B),, Glycosaminoglycan

Hyaluronan Chondroitin 4-sulfate Chondroitin 6-sulfate Dermatan sulfate Heparan sulfate Heparin Keratan sulfate cartilage cornea

A

B

Sulfate (moles/ disaccharide)

GlcA

GlcNAc

0

GlcA

GalNAc

GlcA

Linkage IaID

Mrmge

B

(degrees)

(kDa)

/3-( 1-3)

/3-( 1 4 )

-70

60- 10,000

0.2- 1.0

/3-(1-3)

/3-(14)

-30

5-50

GalNAc

0.2- 1.3

/3-(1+3)

/3-(14)

-19

5-50

IdoA (GlcA)

GalNAc

1.0-2.0

(~-(143)

/3-(14)

-59

15-40

IdoA (GlcA) IdoA (GlcA)

GlcNAc

0.2-3.0

( ~ - ( 1 4 ) a-(1+4)

+SO

5- 12

GlcNAc

2.0-3.0

a-(14)

(~-(14)

+48

7- 16

Gal Gal

GlcNAc GlcNAc

1.1-1.8 0.9-1.7

/3-(14)

/3-(1+3)

+45

8-12 4- 19

A

YH

OH OH

GlcA

Gal

Gal 1

COLLAGEN FIBRIGASSOCIATED SMALL PROTEOGLYCANS

243

ture D-G~cA-D-Gal- &Gal - D-Xyl- L-Ser/L-Thr (of protein), depicted in 1. However, other glycosaminoglycan (GAG)-protein linkages have also been found. The DS.GAG chain in calf ligamentum nuchae is attached to a L-lysine residue in the protein core.12 In adult human skin, the DS.GAG chain is linked by a D-xylosyl-L-alanine bond involving the C-terminus carboxyl group of L-alanine.I3 IV. ISOLATION AND FRACTIONATION OF SMALL PROTEOGLYCANS The general scheme of isolation and fractionation of small PGs from various mammalian tissues is summarized in Scheme 1. In addition to the extraction and purification procedure outlined in Scheme 1, the following buffers have also been used for extraction of PG

Mammaliantissue

c c

(1) Chopped ground in Wiley mill (under liquid N2)

(2) Extracted with 4 M guanidinium chloride

containing proteinase inhibitors

c

Supernatant fraction

4 (3) DEAE-celluloseion-exchangechromatography

c

Small PG fraction

I

7 (4) Purified by one or more of the following procedures (i) Cesium chloride density gradient, (ii) Differential ethanol precipitation, or (iii) Molecular-sieve chromatography

SCHEME I. -Isolation and Fractionation of Mammalian, Small Proteoglycans.

(12) M. 0.Longas and K. Meyer, Proc. Natl. Acud. Sci. USA., 79 (1982) 6225-6228. (13) M. 0. Longas and D. R. Azulay, Proc. Int. Symp. Clycoconjugates, 8th, (1985) V-32.

HARI G. GARG AND NANCY B. LYON

244

from tissue: 2 M calcium c h l ~ r i d e , ~ phosphatebuffered ~J~ saline,I60.05 M sodium acetate," 2-3 M magnesium chloride,18J90.15 -0.45 M sodium chloride,20-226 M urea,23and EDTA.24In certain cases, PGs were isolated from the extracts by precipitation with organic

V. U,OF SMALLPROTEOGLYCANS, THEIRPROTEIN CORES,AND GLYCOSAMINOGLYCAN CHAINS Two different species of PGs, namely, PG-I and PG-11,were first identified25in bovine cartilage by preparative SDS-PAGE. The two forms of PG were subsequentlyisolated26from bovine fetal skin and calf articular cartilage. Separation was achieved by using molecular-sieve chromatography methods: (a) Sepharose CL4B chromatography in 0.5 M sodium acetate, 0.02% sodium azide, pH 7 buffer (associative conditions), or (b) octyl-Sepharose chromatography, which separates the two types of PG based upon differences in the hydrophobic properties of the protein cores. Amino acid compositions of bovine, human, and other species of PG-I1are given in Tables 11, 111, and IV,respectively. The PG-Iamino acid compositions are given in Table V. Although PG-Iand PG-I1differ in amino acid content, both are high in L-leucine, le as par tic, and ~-glutamicacids. The structures of PG-Iand PG-11from different tissues are given in TablesVI and VII. The protein cores of PG-I1from human skin and scars, and fetal rat, are of small size. ( 14) T. H. M. S. M. Van Kuppevelt, H. M. J. Janssen, H. M. Van Beuningen,K . 4 . Cheung, M.

M. A. Schijen, C. M. A. Kuyper,and J. H. Veerlamp,Biuchim. Biophys.Acra, 926 (1987) 296- 309.

-

( 15) J. A. Purvis, G. Embery, and W. M. Oliver, Arch. Oral Biol., 29 ( 1984) 5 13 5 19.

(16) A. Oohira, F.Matsui, M. Matsuda, Y. Takida, and Y. Kuboki, J. Biol. Chem.,263 (1988) 10,240- 10,246. (17) S. P. Damle, L. CBster, and J. D. Gregory,J. Biol. Chem., 257 (1982) 5523-5527. (18) S. Onodera, Chem. Pharm. Bull.. 36 (1988) 4881-4890. (19) N. Fujii and Y. Nagai, J. Biochem. (Tokyo), 90 (1981) 1249-1258. (20) T. Nakamwa, E. Matsunaga and H. Shinkai, Biochem. J., 213 (1983) 289-296. (21) B. P. Toole and D. A. Lowther, Biochim. Biophys. Actu, 101 (1965) 364-366. (22) R. Fleischmajer, J. S. Perlish, and R. L. Bashey, Biochim. Biophys. Acta, 279 (1972) 265-275. (23) B. P. Toole and D. A. Lowther, Arch. Biochem. Biophys., 128 (1968) 567-578. (24) S. Sato, F. Rahemtulla,C. W. Prince, M. Tomana, and W. T. Butler,Cow. TissueRes..14 (1985) 65-75. (25) L.C. Roseuberg, H. U.Choi, L.-H. Tang, T. L. Johnson, S. Paul, C. Webber, A. Reiner, and A. R. Poole, J. Biol. Chem.. 260 (1985) 6304-6313. (26) H. U. Choi, T. L. Johnson, S. Pal, L.-H. Tang, L. Rosenberg, and P. J. Neame, J. Biol. C h . , 264 (1989) 2876-2884. (27) E. Matsunaga, H. Shinkai, B. Nusgens, and C. M. Lapiere, Collugen Rel. Res.. 6 (1986) 467-479.

TABLE II Amino Acid Composition(ResiauC/lOOO) of Bovine PG-II

12927 52 69 108 60 76 63 19 61 18 52 I13 23 32 71 29 25

124’* IW 51 42 60 67 105 100 12 17 93 72 49 53 14 9 58 58 13 12 55 63 115 121 24 28 31 33 66 77 24 28 46 30

* Loar M,

Fraction 10. “D not determined ’T = traa.

High M,

105.u 47 82 168 105 121 56 11 44 11 51 70

131m 45

ND”

ND”

22 57 24 25

35 77 26 27

-

71 104

76 73 51 10 57 10 79 129

12629 132% 39 38 68 75 108 85 69 83 63 81 49 43 13 0 59 53 9 14 6 0 4 8 122 147 34 29 33 33 80 51 21 30 28 32

123m 49 68 122 14 84 54 9 59 7 55 115 15 34 76 25 32

13% 46 68 98 81 68 51 12 59 12 67 135 30 33 52 30 32

123” 44 11 96 15 92 51 NDd 61

142” 58 99 148 61 I32 57 3 30

8

Tc

m w

53 131 30 33 57 28 49

38 70

33 112 24 30 61 26 21

Ty

24 67 30 35

145= 46 108 108 82 102 63

NDd 33

14532 50 98 I09 89 89 61 NDd 37 35 113 23 30 69 21 27

6832 63 I25 163 95 122 66 NDd 61 NDd 40 75 13 29 20 28 32

116“ 42 69 130

90 79 61 NTY 57 9 51 128 I3 32 60 28 35

1w33

86 98 141 92 68 61 5 54 11 31 85 20 31 48 25 31

12P 36 74 108 61 80 49 NDd 58 9 57 123 29 33 75 27 31

TABLE In Amino Acid Composition (Residue/1000) of Human PG-II !scar@

Amino Uterine acid ceniU”

Articular

-es

Placenta#

ASP

130

w

73

Thr Ser

45

ND

79 I10 77 85 53 17 50 3 43 122 20 33 68 25 41

ND ND ND ND

59 73 150 77 64 56

Glu

Pro GlY

Ala CYSas Val Met Jleu Leu Tyr Phe J-YS

His Arg a

ND ND

ND ND ND ND ND

ND ND

ND ND

ND = not determined. * T = trace

46 67 13 36 83 28 37 29 34 75

Fetal membranen

108 60 77 101

72 103 62 11 66 14 46 99 17 34 62 34 34

Gingival= 110

EpitheliumY Epidermis”

46 90 194 50 I19 79

113 52 66 129 86 1 20 77

145 50 87 I09 73 84 49

ND

ND

ND

47 3 31 62 21 27 53 10 38

48

58 11 45 121 12 41 67 26 36

Tb

43 93 20 21 63 I1

52

Dermis” 170 36 91 I38 124 107 56 3 63 1 3 45 61 5 21 8 39

Normal Hypertrophic Bone“ 112 46 72 104 97 88 68 12 64 20 32 99 16 37 65 24 44

107 40 76 114 88 109 65 14 47 9 40 90 22 46 66 23 44

133 52 75 122 70 94 50

ND 51

ND 42 109

10 29 54 20 42

COLLAGEN FIBRIL-ASSOCIATED SMALL PROTEOGLYCANS

247

TABLEIV Amino Acid Composition of Non-Human PG-I1 (Residue/lOOO) ~

Skin

ASP Thr Ser Glu

Pro GlY Ala CYSO.5 Val Met Ile

Leu TYr Phe LYS His Arg

(28) (29) (30) (31)

Skeletal Muscle

Rat'*

Pig"

RabbiP

152 46 64 111 81 63 46 24 44 21 61 116 31 27 63 20 31

128 42 65 104 72 76 49 15 66 6 56 127 23 29 87 23 32

83 35 82 97 49 156 55 3 36 9 28 67 22 18 39 23 28

E. Matsunaga and H. Shinkai, J. Invest. Dermatol., 87 (1986) 221 -226. C. H. Pearson and G. J. Gibson, Biochem. J., 201 (1982) 27-37. L. Coster and L.-A. Fransson, Biochem. J., 193 (1981) 143- 153. L. W. Fisher, J. D. Termine, S. W. Dejter, Jr., S. W. Whitson, M. Yanagishita, J. H. Kimura, V. C. Hascall, H. K.Kleinman, J. R. Hassell, and B. Nilsson, J. Biol Chem., 258

(1983) 6588-6594. (32) K. G.Vogel and D. HeinJ. Eiol. Chem., 260 (1985) 9298-9306. (33) T. R. Oegema, Jr., V. C.Hascall, and R. Eisenstein, J. Biol. Chem., 254 (1979) 13121318. (34) N. Uldbjerg, A. Malmstrom, G. Ekman, J. K. Sheehan, U. Ulmsten, and L. Wingerup, Biochem. J., 209 ( 1983) 497 - 503. (35) L. de 0. Sampaio, M. T. Bayliss, T. E. Hardingham, and H. Muir, Biochem.J., 254 (1988) 757-764. (36) M. Isemura, N. Sato, Y. Yamaguchi, J. Aikawa, H. Munakata, N. Hayashi, Z. Yosizawa, T. Nakamura, A. Kubota, M. Arakawa, and C.-C. Hsu, J. Riol. Chem., 262 (1987) 89268933. (37) M.J. Brennan, A. Oldberg, M.D. Pierschbacher, and E. Ruoslahti, J. Eiol. Chem., 259 (1984) 13,742- 13,750. (38) P. M.Bartold, 0.W. Wiebkin, and J. C. Thonard, Biochem. J.,21 1 (1983) 119- 127. (39) H. G.Garg, D. A. R. Burd, and D. A. Swann, Biomed. Res., 10 (1989) 197-207. (40) D. A. Swann, H. G. Garg,C. J. Hendry, H. Hermann, E. Siebert, S. Sotman, and W. Stafford, Collagen Rel. Res., 8 ( 1988) 295 - 3 13.

HAM G. GARG AND NANCY B. LYON

248

TABLE

v

Amino Acid Composition of Bovine and Human PG-I (Residue/lOOO)

Asp Thr Ser Glu Pro GlY Ala CYS0.5 Val Met Ileu

Leu Tyr Phe LYS His A%

137 40 61 94 75 65 54 11 53 14 41 165 32 42 43 24 41

137 36 61 85 83 60 43 0 51 14 45 174 34 33 50 30 51

116 46 77 114 67 15 47

ND 56

ND 46 120 27 31 70 51 42

ND = not determined.

In general, small PGs have low M, values compared to the high-density the GAG cartilage PG,65and, contrary to the high-density cartilage PG,66*67 chain in small PGs is attached to the NH2-terminusof the protein core, not the C02Hterminus. In PG-I, two DS.GAG chains are attached to the L-serine/L-threonine residues at the 5 and 1 1 positions,whereas, in PG-11, a single ~

(41) L. W. Fisher, G. R. Hawkins, N. Twos, and J. D. Termine, J. Biol. Chem., 262 (1987) 9702-9708. 88 (1980) 1793- 1803. (42) I. Miyamoto and S. Nagase, J. Biochem. (Tokyo)). (43) N. Parthasarathy and M. L. Tanzer, Biochemistry, 26 (1987) 3149-3156. (44) P. J. Roughley and R. J. White, Biochem. J-. 262 (1989) 823-827. (45) B. obrink, Biochim. Biophys. Acta, 264 (1972) 354-361. (46)H. Habuchi, K. Kimata, and S. Suzuki, J. Bid. Chem.. 261 (1986) 1031 1040. (47) H. G. Garg, E. P. Siebert, and D. A. Swann, Carbohydr. Res.. 197 (1990) 159- 169. (48) J. McMurtrey, B. Radhalrrishnamurthy, E. R Delferes, Jr., 0.S. Berenson, and J. D. Gregory, J. Bid. Chem., 254 (1979) 1621: 1626. (49) R.Kapoor,C.F.Phelps,L.Wer,andL.-A.Fransson,Eiochem.J..197(1981)259-268. (50) B. Radhakrishnamurthy, N. Jeansonne, and G. S. Bereuson, Biochim. Biophys.Acza, 882 (1986) 85-96. (51) B. G, J. Salisbury and W. D. Wagner, J. Biol. Chem., 256 (1981) 8050-8057. (52) J. D. Gregory, L. C&ster,and S. P. Damle, J. Biol. Chem., 257 (1982) 6965-6970.

-

COLLAGEN FIBRILASSOCIATED SMALL PROTEOGLYCANS TABLEVI of PG-I from Hum.a and Bovine Tissues

SN I-

W(kD.) PG

249

Core protein

GAG

GAG No.

IdoA (% of total uronic acid)

(%,I w/w)

Protein

2

81-84

31

26

44 44

2

25-29

29

2

25,“ 26 44

46

2

41

References

Skin Bovinefetal Articular cartilage Bovine calf Human

148

150 200

Bone Human

350

40 ~

a The data

are taken from the reference in bold print.

GAG chain is a t t a ~ h e d ~at* *the ~ ~4 position of the NH2-terminus. The number of GAG chains in small PGs vanes from one to nine, but, in general, PG-I1 contains only one to two GAG chains. Small PGs also contain Nlinked oligosaccharides,which are 1iberated’O by hydrazinolysis. These oligosaccharides are composed of di-and tri-antennary oligosaccharide struo tures of the complex type. In bovine articular cartilage PG-I, two N-linked oligosaccharides have been reported, in comparison to” PG-11. There is evidence72suggesting that the addition of free dermatan sulfate chains enhances multimerization of PG-11. (53) J. D. Gregory,S.P. Damle, H.I. Covington, and C. Cintron, Invest. Ophthulmol. Vis. Sci.. 29 (1988) 1413-1417. (54) J. R. Hassell, D. A. Newsome, and V. C . Hascall, J. Biol. Chem., 254 (1979) 12,34612,354. (55) L. Cbster, L.-A. Fransson, J. Sheehan, I. A. Nieduszynski, and C. F. Phelps, Biochem. J., 197 (1981) 483-490. (56) K. Murata and Y. Yokoyama, Biochem. Inf., 15 (1987) 87-94. (57) P. J. Roughley and R. W. White, Biochim. Biophys. Acta, 759 (1983) 58-66. (58) T. Honda, K.Katagiri, A. Kuroda, E. Matsunaga, and H.Shinkai, Collagen Rel. Res., 7 (1987) 171-184. (59) K. G. Vogel and S. P. Evanko, J. Biol. Chem.. 262 (1987) 13,607- 13,613. (60) M. Yanagishita, D. Rodbard, and V. C. Hascall, J. Eiol. Chem., 254 (1979) 91 1-920. (61) A. Oldberg, E. G. Hayman, and E. Ruoslahti, J. Biol. Chem.. 256 (1981) 10,847- 10,852. (62) T. Shinomura, K. Kimata, Y. Oike, A. Noro, N. Hirose, K.Tanabe, and S.Suzuki, J. Biol. Chem., 258 (1983) 9314-9322. (63) A. F&n and D. HeinBiochem. J.. 224 (1984) 47-58. (64) P. G. Scott, T. Nakano, C. M. Dodd, G.A. Pringle, and I. M. Kuc, Matrix, 9 (1989) 284 292. (65) V.C. Hascall and S. W.Sajdera, J. Biol. Chem., 245 (1970) 4920-4930. (66) D. Heinegiird and V. C. Hascall, Arch. Biochem. Biophys.. 165 (1974) 427-441.

-

HARI G. GARG AND NANCY B. LYON

250

TABLE VII Structure of PG-I1 Isolated from Different Tissues

w (a) Tissue

PC

GAG IdoA % (of Protein GAG Core protein No. total uronic acid) (% w/w) References0

Skin pis (i) (ii) Rat Fetal rat

Calf

2900 70 36 111-200 112

15

26 23

41-44

1

85

58 60 46

55

20

56

17-18

55

79-92

27

46 46

90

16

53-55 20 43

130 66

17.5 17.5

45 14, 21.5

20

Dermatosparactic

calf Newborn calf (i) large (ii) small cow Hum (i) Epidermis (ii) Dermis Human scar Normal Hypertrophic

0) (ii) Ligament cow

COW

75

61

28 28 29

90 90

32 18

39 39

22-17

90

23

40

23.5 29

22-17 22-16

90 13

23

40 47

30 16

60-70

48

29 11

50

23

136

39 78 I20

calf Heart valves cow Aorta

100-200

1

85

25

80

Human Cervix Human

Cornea Rabbit Monkey Sclera Bovine (i) large (ii) small Cartilage Bovine (i) large (ii) small

45 17 42 46 18, 19,20

42,4840 51

50-70 73 100-150

47 55

2-3

50

30

34

35

32 70

52,53 54

45 59

30,55 30,55

1

80-85

100 46

165-285 87- 120

44 47 - 44

20 52

25 25

COLLAGEN FIBRILASSOCIATED SMALL PROTEOGLYCANS

25 I

TABLEVII (continued) M, (a)

Human Tendon Bovine Fetal bovine Fetal membrane Human Folliculpr fluid fig Yolk sac tumor Rat Embryo

Chick

70-80

GAG

120

48 45

1-2

45

2

56

40 250-500

52

Lug 15-35 Bovine Skeletal muscle 95 21 Rabbit Bone 25 - 35 Bovine compact 80- 120 Fetal calf 80- 120 Growing rat 80- 120 Human fetal Bovine mature Temporomandibular joint disk Bovine >56

Protein

50

40-42 37

IdoA % (of

43

35,44,56,57

73

32,58 58,59 37

9

20

60

10

5

61

4-9

62 14

91

43

50

45 40

40

40 40 46

24 31 31 31,41 63

79

Whenever more than one reference is given, the data are taken from the reference in bold print.

(67) J. W. Stevens, Y. Oike, C. Handley, V.C. Hascall, A. Hampton, and B.Caterson, J. Cell. Biochem., 26 (1984) 247-259. (68) C. H. Pearson, N. Winterbottom, D. S.Fackre, P. G. Scott, and M. R.Carpenter, J. Biol. Chem., 258 (1983) 15,101-15,104. (69) R. K . Chopra, C. H. Pearson, G. A. Pringle, D. S.Fackre, and P. G. Scott, Biochem.J., 232 (1985) 277-279. (70) H. Shinkai, T. Nakamura, and E.Matsunaga, Biochem. J., 213 (1983) 297-304. (71) P. J. Name, H. U. Choi, and L. C. Rosenberg, J. Biol. Chem., 264 (1989) 8653-8661. (72) L.-A. Fratwon, L. Cbster, A. Malmstr6m, and J. K. Sheehan,J. Biol. Chem., 257 (1982) 6333-6338.

64

TABLEWI NH,-Tcrminrrl Amiao Acid Sequences of B o v h and Human PG-II Humpn

Bovine ResidueNo. Cprtilage” &lentn

SCaP

Skinu Tendo$*

Dermis” Epidermis” Normal Hypertrophic Fetalmembranem Bone41 CprtiLge”

ASP

ASP

Asp

Asp

Asp

ASP

A S P A S P

Glu Ala

Glu

Ala

Glu Ala

Glu Ala

ob

ob

ob

ob

GlY

Glu Glu A l a A l a A l a Ser Ser X“ Gly Gly Gly

G~Y

GlY

GlY

GlY

Glu Ala ? GlY

Glu Ala Xa Gly

Ile

Ile

Ile

ne

Ile

lle

Ile

Ile

Ile

Ile

GlY

Gly Pro Glu Glu

Gly Pro Glu Glu

Gly Pro Glu Glu

GlY

GlY Pro Glu

GlY Pro Glu

Ala

GlY

Glu

GlY Pro Glu

Glu

Glu

Val

Val

Val

Pro

Pro ASP

Pro

Val Pro ASP ASP

Val Pro

Val Pro

Asp

Asp

ASP

ASP

1

ASP

Asp

2 3 4 5 6 7

Glu Ala

Glu

Xa

ASP

8 9 10

Pro GlU Glu

11 12

Xa Phe

13

Pro

H i s H i s H i s Phe Phe Phe P r o P r o P r o

14 15 16 17 18 19 20 21 22 23

Glu

Glu

Val Pro Glu

Glu Val Pro Glu

ne

De

Glu Pro

Glu Pro

Glu

Pro

ASP Asp Arg

Asp Arg Asp(?)

Asp

ASP

Pro

Glu

Ala Xb Gly

Pro

Arg

Arg

Arg

ASP Phe

ASP Phe

Pro

Arg Asp

Glu Pro

Glu Pro

Pro Phe

GlU

Glu

Pro

ser(?)

ser

?

Pro

ser

Leu

Leu GlY

Leu GlY

Ser

Leu

Pro Val

Pro Val

AsPo Phe

Phe

Glu Pro

Glu

Phe

COLLAGEN FIBRIGAssoclATED SMALL PROTEOGLYCANS

253

VI. N-TERMINAL SEQUENCE OF SMALL PROTEOGLYCANS

A comparisonof the amino acid sequencingof the NH,-terminal regions of the core proteins of PG-I1 from bovine articular cartilage, sclera, skin, tendon, human skin and post-bum scars, fetal membrane, bone, and cartilage are shown in Table VIII. The A, -A, amino acid sequences for bovine and human tissue are identical, and thereafter, the sequences within the TABLEIX NH, -Terminal Amino Acid Sequences of Bovine Cartilage and Human Bone and cprtilaee PG-I

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

ASP Glu Glu Ala

Xa GlY Ala Glu Thr

Thr X" GlY Ile pro ASP Leu ASP Ser

Leu Pro Pro Thr Tyr Ser

ASP Glu Glu Ala Xa GlY Ala ASP

Thr X GlY Val Leu ASP Pro Asp

ser

Val Thr

pro

Ala Met

X = blank cycle. (73) L. Cbster, L. C. Rosenberg, M. Van der Rest,and A. R. Poole, J. Biol. Chem.,262 (1987) 3809-38 12.

254

HARI G. GARG AND NANCY B. LYON

bovine species remain identical; but in the human species, the NH,-terminus bone sequence has only some similaritiesin comparison to human-skin, scar, cartilage, and fetal-membranePG-I1protein cores. The glycosaminoglycan chain is attached& to an L-serine residue at position 4. The NH,-terminal sequences of PG-I derived from human bone and cartilage, and bovine are given in Table IX. The A,-A, amino acid sequencesofthe protein core of human bone and cartilage PG-I are identical to that of bovine cartilage; thereafter, the sequences of the next 12 amino acids are different for bovine PG-I compared to human PG-I. The glycosaminoglycan chain in PG-IappearsMto be attached at position 5 of the protein core. On the whole, there is some homology in the primary structure of the core proteins of PG-I and PG-I1 from different species, but there are also some striking differences, which may be related to their biological activities.

VII. AMINOACID SEQUENCEANALYSIS OF THE SMALL PROTEOGLYCAN CORE-PROTEIN, DEDUCED FROM CLONED cDNA The core protein of small PGs has been cloned74from a Agt 1 1 fibroblast cDNA library. Protein sequences of human bone PG-I and PG-I1 (Ref. 75) and bovine bone PG-I1 (Ref. 76) have also been deduced by the aforementioned approach. Comparison of the NH,-terminal amino acid sequence (obtained by using a gas-phase sequencer) of bovine skin and human skin/ bone PG-I1 revealed complete homology with the total protein core sequences of the same species. The derived protein-core sequences of human PG-I (biglycan)showed sufficient homology with the PG-I1(decorin);that is, 55Yo of the amino acids are identical, with others involving chemically similar amino acid substitution. These data suggest that the two protein cores may be the result of a gene duplication. Comparison of human PG-I and PG-I1 amino acid sequences (see Fig. 2) shows that PG-I and PG-I1 contain 368 and 359 amino acid residues, respectively. The PG-I and PG-I1 protein cores contain75a series of 10- 12 tandem repeats of 24 amino acid residues rich in L-leucine or L-leucine-like amino acids. Similar structural characteristics had been r e p ~ r t e d ~ for~ several - ~ ~ unrelated non-PG proteoglycan proteins. (74) T. Krusius and E. Ruoslahti, Proc. Nut/. Acad. Sci. USA, 83 (1986) 7683-7687. (75) L. W. Fisher, J. D. Termine, and M. F. Young, J. Biol. Chem., 264 (1989) 4571 -4576. (76) A. A. Day, C. I. McQuillan, J. D. Termine, and M. R. Young, Biochem. J., 248 (1987) 801-805. (77) T. Kataoka, D. Broek, and M. Wigler, CeN, 43 (1985) 493-505. (78) N. Takahashi, Y. Takahashi, and F. W. Putnam, Proc. Natl. Acud. Sci. USA, 82 (1985) 1906-1910. (79) M. Handa, K. Titani,L. Z. Holland,J. R. Roberts,and Z. M. Ruggen,J. Biol. Chem.. 26 1 (1986) 12,579- 12,585.

255

COLLAGEN FIBRIGASSOCIATED SMALL PROTEOGLYCANS

I MWPLWRLVSLLALSQALPFEQRGFWDFTLDDGPFMMNDEEASGADTSGVL

I I

I

I I I I I l l I I I I .........SGIGPEVP I

IIMKATIILLLLAQVSWAGPFQQRGLFDFMLEDEA

50 41

DPDSVTPTYSAMCPFGCHCHLRWQCSDLGLKSVPKEISPDTTLLDLQNN 100

I

I

Ill I 1111111111111 Ill

1l111111111

DDRDFEPSLGPVCPFRCQCHLRWQCSDLGLDKVPKDLPPDTTLLDLQNN

91

DISELRKDDFKGLQHLYALVLVNNKISKIHEKAFSPLWQKLYISKNHL 150

1 1

I l l I 1 I1 I I I I I I I I

11 11

II 111 I

KITEIKDGDFKNLKNLHALILVNNKISKVSPGAFTPLVKLERLYLSKNQL 141

VEIPPNLPSSLVDVRIHDNRIRKVPKGVFSGLRNMNCIEMGGNPLENSGF 200

1 1

I I

I I I I I I I I l l I 1 1 1 1 1 1 II

KELPEKMPKTLQELRAHENEITKVRKVTFNGLNaMIVIELGTNPLKSSGI 191 EPGAFDGL .~NYLRISEAKLTGIPKDLPETLNELHLDHNKIQAIELED~ 249

11111 IIIII

I II II I IIIII Ill

I

ENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVDAASL 241

LRYSKLYRLGLGHNQIRMIENGSLSFLPTLRELHLDNNKLARVPSGLPDL 299

I I11 I 1

Ill1 I I I I I I I I I I I I I l l I I

KGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEH 291 KLLQVVYLHSNNITKVGVNDFCPMGFGVKRAYYNGISLFNNPVPYWEVQP 349

1 IllllT Ill II IIII I

1 I 1 1 111 111 I 1 1 II

KYIQWYLHNNNISWGSSDFCPPGHNTKKASYSGVSLFSNPVQY'WEXQP 341 ATFRCVTDRLAIQFGNYKK

IIIIt 1 111 1 1 1 1 STFRCVYVRSAIQLGNYK.

368 359

FIG.2.-Compariwn of Human PG-I and PG-I1 Protein Core Amino Acid Sequences(data taken from Ref. 75). Abbreviations:A, Ma; C, Cys;D, Asp; E, Glu; F, Phe; G, Gly; H, His;I, Ile; K, Lys; L, Leu; M, Met; N,Asn; P,Pro;Q, Gln; R, Arg; S, Ser; T, W,V, Val; W,Trp; Y,Tyr.

HARI G. GARG AND NANCY B. LYON

256

VIII. BIOSYNTHESIS OF SMALL PROTEOGLYCANS 1. Primary Culture

Synthesis of PGs by primary culture from guinea-pig skinya3human skin,84-8ahuman dermals9and human gingival fibroblastsmhas been thoroughly investigated. In this system, both large and small PGs are biosynthesized. The newly synthesized PG obtained by using ~-['H]glucosamine precursor yields PG containing low proportions of radioactivity:' whereas the use of [35S]-S04precursor results in [35S]-PGsthat contain higher proportions of [35S]-radioactivity?1The majority of the [35S]-incorporation takes placea7in small PGs (mainly found in cell-culture media). When cells are cultured on collagen gels, the radioactivity is also incorporated in the m a t r i ~ . ~Disease-related ~.~* alterations in the synthesis of PG macromolecules have been r e p ~ r t e d . Influences ~ ~ - ~ ~ of such reagents as chlorate%and lipopolysaccharidewon PG biosynthesis have also been reported. Chlorate treatment leads to the formation of GAG chains having a widely varying degree of sulfation. Glycosaminoglycan synthesis, in culture, using fibroblasts from human human normal and hypertrophic scarywand embryonic chick (80) K. Titani, K. Takio, M. Handa, and Z. M. Ruggeri, Proc. Nutl. Acud. Sci. USA, 84 (1987) 56 10- 56 14. (81) C. Hashimoto, K.L. Hudson, and K. V. Anderson, Cell, 52 (1988) 269-279. (82) R. Reinke, D. E.Kwntz, D. Yen, and S. L. Zipursky, Cell, 52 (1988) 291-301. (83) T. Honda, E. Matsunaga, K. Katagiri, S. Fujiwara, and H. Shinltai, Eiomed.Res., 8 (1987) 175 183. (84) I. Carlstedt, L. CBster, and A. Malmstdm, Biochem. J., 197 (1981) 217-225. (85) L. Caster, I. Carlstedt, A. M a l m m , and B. Smstrand, Biochem. J., 222 (1984) 575582. (86) H. Larjava, J. Heino, T. Krusius, E. Vuorio, and M.Tammi, Eiochem. J., 256 (1988) 35-40. (87) J. GlW, M.Beck, and H. Kresse, J. Biol. Chem.,259 (1984) 14,144- 14,150. (88) W.Truppe and H. Kresse, Eur. J. Eiochem., 85 (1978) 351-356. (89) I. A. Schafer, L.Sitabkha, and M.Pandey, J. Eiol. Chem.,259 (1984) 2321-2330. (90) P. M. Bartold and S. J. Millar, Idect. Immun., 56 (1988) 2149-2155. (91) T. Nalcamura and H. Shinkai, J. Dermatof., 12 (1985) 489-497. (92) J. T. Gallagher, N. Gasiunas, and S . L. Schor, Eiochem. J., 215 (1983) 107- 116. (93) A. Eigavish and E. Meezan, Bimhem. Biophys. Res. Commun.,152 (1988) 99- 106. (94) Y. Shishiba, M. Yanagishita, and V. C. Hascall, Connect. Tiss.Res., 17 (1988) 119- 135. (95) S.Fukui, H. Yoshida,T. Tanaka, T.Sakano, T. Usui, and I. Yamashina, J. Eiol. Chem., 256 (1981) 10,313-10,318. (96) H. Greve, Z. Cully, P. Blumber& and H. Kresse, 3. Eiol. Chem.. 263 (1988) 12,88612,892. (97) J. E.Silbert,M.E. Palmer, D. E. Humphries,andC. K. Silbert, J. Eiol. Chem.,261 (1986) 13,397- 13,400. (98) I. Sj&berg,I. Carlstedt, L. Cbster, A. MalmstrBm, and L.-A. Fiansson, Eiochem. 3.. 178 (1979) 257-270.

-

COLLAGEN FIBRILASSOCIATED SMALL PROTEOGLYCANS

257

skinla fibroblasts, shows differencesin the proportions of incorporation of the radiolabel into different types of GAG. Several cytokines have been shown to modulate the synthesis and dejyadation of various connective-tissue components, including GAGS.Treatment of cultured human dermal fibroblasts with different human interferons (INF)resultslO1 in specific synthetic responses. INF a and p lead to concentrationdependent decreases in GAG production and collagenase production, with no effect on fibronectin synthesis. Human interferon gamma, on the other hand, leads to concentrationdependent increases in GAG and fibronectin, as well as in collagenase. Cultured human skin fibroblasts have also been treated with interleukin (IG1) a! and /3 and tumor necrosis factor (TNF)a and p, which, under the isolated in vifroconditions, lead to concentrationdependent increases in collagen, GAG, and collagenase, with inhibition of fibronectin.lo2However, the maximum increases in GAG synthesis stimulated by IG 1 and TNF may be overshadowed by catabolic effects. Several cell types, including synovial cells, dermal fibroblasts, and chondrocytes, have been stimulated by mononuclear-cellderivedIG 1 to produce high levels of proteogly~anase.~~~J~ 2. Explant Culture

Biosynthesis of small proteoglycans in organ culture from rat skin using [3sS]-S04(Ref. 46), bovine aorta using [35S]-S04and ['H]glucosamine (Ref. 105), and bovine tendon using [35S]-sulfate(Ref. 106) have been investigated, and the characteristicsofthe GAG chainsand protein cores have been studied. Retinoic acid has been used on cultured human skin explants, which respond by accumulating HA between keratinocytes.lm-'@ The dermis of skin explants also responds to retinoids with an apparent increase (99)K.Savage and D.A. Swann, J. Invest. Dermatol., 84 (1985)521 -526. (100)R.Evangelisti, G.Stabelhi, E. Becchetti, and P. Carinci, Cell Biol. Int. Rep., 13 ( 1989) 437- 446. (101)M.R.Duncan and B. Beman,Arch. Dennutof. Res., 281 (1989)1 1 - 18. (102) M.R.Duncan and B. Berman, J. Invest. Dermatol., 92 (1989)699-706. (103)M.Gowen, D.D.Wood,E. J. Ihrie, J. E. Meats,and R. G. G. Russell, Biochim.Biophys. Ada, 797 (1984)186- 193. (104)E. E. Golds, V. Santer, J. Killackey, and P. J. Roughley, J. Rheumaro[., 10 (1983) 861-871. (105)A. Schmidt, M.Prager, P. Selmke, and E. Buddecke, Eur. J. Biochem., 125 (1982) 95-101. (106)T. J. Koob and K. G. Vogel, Biochem. J., 246 (1987)589-598. (107) R. Tammi and M. Tammi,J. Ceff.Physiol., 126 (1986)389-398. (108)I. A. King, Biochim. Biophys. Acta, 674 (1981)87-95. (109) R. Tammi, J. A. Ripellino, R. U. Margolis, H. I. Maibach, and M.Tammi, J. Invest. Dermato[.,92 (1989)326-332.

258

HARI G. GARG AND NANCY B. LYON

in ground substance. This has been demonstrated using electron microscopy, and accompanies hypermetabolioappearing fibroblasts and other connective-tissue changes.II0 IX. BIOLOGICAL ROLESOF SMALLPROTEOGLYCANS Small proteoglycans are widely distributed throughout fibrous interstitial tissues, suggesting crucial roles in connective tissues. They are dynamic molecules that, through their abilities to interact with other major molecules such as collagen, appear to have critical roles in developrnentl1' and tissue modeling, and may play a role in the pathophysiology of disease processes. The binding characteristicsof small proteoglycans are multifaceted. Ionic interactions with other molecules are facilitated by way of the highly negatively charged glycosaminoglycan chains. This may permit electrostatic interactions with L-lysine and targuinine on collagen and also with such counter ions, as sodium, which then create an osmotiopressure gradient to draw and hold water molecules in tissues. GAG chains are also responsible for the ability of some DSPGs to self-associate to form multimeric PG comp l e x e ~The . ~ ~ protein core is also capable112of specific molecular interactions. The DSPG protein sequence shows homology with several nonconnee tive tissue proteins from several different species. Repeatingsequencesin the PG-I and PG-I1 protein core75are characterized by several conserved L-leucine residues and L-leucine-like amino acids found at locations that had previously been described for a highly varied group of non-proteoglycan proteins, including von Willebrand Factor-binding protein of the platelet membrane and yeast adenylate cyclase, where the repeating domain is thought to bind the enzyme to the cell membrane. Two Drosophilu proteins, chaoptin and toll, also exhibit these repeating domains, which are thought to interact with the plasma membrane and influence, respectively, morphogenesis of photoreceptor cells and dorsal-ventral pattern formation in the embryo.81s82A common theme in molecular interactions appears to be emerging in organisms as diverse as yeasts and mammals. It now appears that several non-proteoglycan proteins, which also have binding functions, contain the L-leucine-richtandem repeatsthat are also found in DSF'GS."-~~ These macromolecules may play a role in collagen fibril organization.ll*The core protein of PG-I1 binds collagen type I and type I1 in vitro and (110) L. H.Kligman, J. Am. Acud. Dermatol., 15 (1986) 779-785. (1 1 1) S. Vainio, E. Lehtonen, M. Jalkanen, M. Bernfield, and L. Saxen, Develop. Biol., 134 (1989) 382-391. ( I 12) T.R. Oegema, Jr., J. Laidlaw, V. C. Hascall, and D. D. Dziewiatkowski,Arch. Biochem. Biophys., 170 (1975) 698-709.

COLLAGEN FIBRILASSOCIATED SMALL PROTEOGLYCANS

259

affects the rate and diameter of fibril formation.ImFibromodulin, a 59 kDa protein, isolated from many connectivetissues, is structurally related to PGs and also contains similar L-leucine-rich tandem repeats.121 - lZ3 Like PG-11, fibromodulin delays collagen fibril formation and leads to fibrils with a thinner diameter. The binding to collagen seems to be at different locations, since PG-I1 and fibromodulin together have additive effects in the collagen fibrillation studies.123 The protein core appears to have very specific interactions with collagen. IZ4 Electron-microscopicstudies have revealed that DSPG-collagen interactions occur specificallyat the d- and e-bands in collagen.124The interaction depends on intact disulfide bridges on the protein core and, in collagen fibrillationstudies, is independentof the DS -GAG chain."' Biomechanical strength of collagen fibers probably also depends upon the PG-type I collagen interaction.' The physiology of reproduction may be associated with proteoglycan changes.''*'26 A role for DSPGs in dilation of the rat uterine cervix is suggested by a fourfold increase in DSPG levels in pregnancy, which decrease rapidly within the first postpartum day.127 PGs appear to play a rolegin wound healing and scar formation. PG levels fluctuate during wound healing. An initial increase in HA is replaced by increased levels of DSPG as the wound ages. DSPGs from articular cartilage have been shown to bind fibronectin non-covalently and to inhibit attachment and spreading of fibroblasts.129DSPG, the major PG in scars,is increasedgin hypertrophic scars and keloids. Many phases of wound healing and tissue remodelling may be affected by alterations in proteoglycans. (113) D.A.D.Pamy,M.H.Flint,G.C.Gflard,andA.S.Craig, FEBSLett., 149(1982) 1-7. (114) J. M.Snowden and D. A. Swann, Biopolymers, 19 (1980)767-780. (115)J.E.ScottandM.Haigh, Biosci.Rep., 5(1985)71-81. (116) J. E. Scott and C. R. Orford, Biochem. J., 197 (1981)213-216. ( 1 17) P. G.Scott, N. Winterbottom, C. M. Dodd, E. Edwards, and C. H. Peamn, Biochem. Biophys. Res. Commun., 138 (1986)I348- 1354. (118)A.K.Garg,R.A.Berg,F.H.Silver,andH.G.Garg, Biomuteriuls, 10(1989)413-419. (1 19) N. Uldbjerg and C. C. Danielsen, Biochem. J.. 251 (1988)643-648. (120) K.G. Vogel, M. Paulsson, and D. Heineghi, Biochem. J., 223 (1984)587-597. (121)D.Heinegfird, T.Larsson, Y. Sommarin, A. FranzCn, M. Paulsson, and E. Hedbom, J. Biol. Chem., 261 (1986)13,866-13,872. (122) A. Oldberg, P. Antonsson, K.Linblom, and D. Heinegilrd, EMBO J., 8 (1989)2601 2604. (123) E. Hedbom and D. Hein-, J. Biol. Chem., 264 (1989)6898-6905. (124)J. E. Scott, in E. Evered and J. Whelan, Eds., Functions of the Profeoglycuns,(Ciba Found. Symp. 124)Wiley, Chichester, 1980,pp. 104- 116. (125)C.A. Stephk and P. A. A m Comp. Biochem. Physiol.. 84B (1986)29-35. (126) N.Uldbjexg, Actu. Obstef. Gynecof. Scand., Suppl., 148 (1989)1-40. ( 127) R. Kokenyesi and J. F.Woessner, Jr., Biochem. J., 260 ( 1989)413-4 19. (128)S. A. Alexander and R. B. Donof, J. Surg. Res.. 29 (1980)422-429.

260

HARI G.G A R G AND NANCY B. LYON

X. ADDENDUM The following articles on the subject have appeared since this text was completed.

(1 30) G. Westergren-Thorsson, P. Antonsson, A. Malmstrtjm, D. Heinegiird, and A. Oldberg, The synthesis of a family of structurallyrelated proteoglycans in fibroblasts is differentiallyregulated by TGF-p, Matrix, l l (1991)177-183. (131) H.G. Gar&E. W. Lippay, D. A. R. Burd, and P. J. Neame, Purification and characterization of iduronic acid-rich and glucuronic acid rich proteoglycans implicated in human post-bum keloid scar. Carbohydr. Rex, 207 (1990)295-305. (132)G.M. Cerhchi, R. Coinu, P. Demuro, M. Formato, G. Sanna, M. Tidore, M. E. Tira, and G. Deluca, Structural and functional modifications of human aorta proteoglycans in atherosclerosis.Matrix, 10 ( 1990)362- 372. (1 33) M.C. Lane and M. Solursh, Primary mesenchyme cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan. Devel. Biol., 143 (1991)389-397. (1 34) C.K.Silbert, D. E. Humphries,M. E. Palmer,and J. E. Silbert,Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblast from normal and diabetic individuals. Arch. Biochem. Biophys., 285 (1 991) 137- 141. ( 1 35) K.Schwa, B. Breuer, and H.Kresse, Biosynthesisand propertiesofa further member of the small chondroitin/dermatansulfate proteoglycan family. J. Biol. Chem., 256 (1990)22,023-22,028. (1 36) M.R.Shetlar, C. L. Shetlar, C. W. Kischer, and J. Pindur, Implantsof keloid and hypertrophicscarsinto the athymic nude mouse: Changes in the glycosaminoglycans of the implants. Connect. Tissue Res., 26 (1991)23-36. (1 37) T. -K. Yeo,L. Brown, and H. F. Dvorak, Alterationsin proteoglycan synthesiscommon to healing wounds and tumors. Am. J.Pathol., 138 (1991)1437-1450. ( 138) D. T.Simionsescu and N. A. Kefalides, The biosynthesis of proteoglycans and interstitial collagens by bovine pencardial fibroblasts. Exp. CellRes., 195 (1991)171-176. (139) R.Fleischmajer, L.W. Fisher, E. D. MacDonald, L. Jacobs, Jr., J. S. ( 129) L. C. Rwnberg, H. U.Choi, A.R.Poole, K.Lewandowska, and L. A.Culp, in E.Evered

and J. Whelan Eds.,Functions of the Proteoglycans (Ciba Found. Symp. 124) Wdq, Chichester, 1980, pp. 47 - 68.

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Perlish, and J. D. Termine, Decorin interactswith fibrillar collagen of embryonicand adult human skin. J. Sfnrct.Biol., 106 (1991) 82-90. (1 40) G. Stocker,H. E. Meyer, C. Wagner, and H. Greiling, Purificationand N-terminal amino acid sequence of a chondriotin sulfate/dermatan sulfate proteoglycan isolated from intima/media preparations of human aorta. Biochem. J.,276 (1 99 1) 4 15 -420. (141) K. Takigaki, T. Nakamura, A. Kon, S.Timura, and M.Endo, Characterization of /l-D-xyloside-induced glycosaminoglycans and oligosaccharides in cultured human skin fibroblasts.J. Biochem. (Tokyo), 109 (1991) 514-519. (142) T. C. Register and W. D. Wagner, Heterogeneity in glycosylation of

dermatan sulfate proteoglycan core proteins isolated from human aorta. Connect. Tissue Res., 25 (1990) 35 - 38. (143) V. Vilim and J. Krajickova, Proteoglycans of human articular cartilage. Identification of several populations of large and small proteoglycansand of hyaluronicacid-bindingproteinsin successivecartilage extracts. Biochem. J., 273 (1991) 579-585. (144) A. Schmidtchen, I. Carlstedt, A. Malmstriim, and L. -A.Fransson, Inventory of human skin fibroblasts proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulfate proteoglycans. Biochem. J., 265 (1990) 289-300. (1 45) S.Inerot and I. Axelsson, Structure and composition of proteoglycans from humans annulus fibrosus. Connect. Tissue Res., 26 ( 1991) 47 63. (146) H. G. Garg, E. W. Lippay, E. A. Carter, M. B. Donelan, and J. P.

Remensnyder, Proteoglycan synthesis in human skin and bum explant cultures. Burns, 17 ( 199 1) in press. (147) H. G. Garg, E. W.Lippay, and P. J. Neame, Proteoglycansin human bum hypertrophic scars fiom a patient with EhIers-Danlos Syndrome. Carbohydr.Res.. in press. ACKNOWLEDGMENTS

This work was supported by research funds fiom the Shriners Hospitals for Crippled Children of North America. The authors gratefully acknowledge the editorial work of Sarah Niemczycki.

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AUTHOR INDEX Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A

Abbas, S. A., 126 Ablett, S.,23 Abylgaziev, R. I., 142, 143, 161(189, 190) Achet, D., 140 Adalsteinsson, O., 2 1 I Addleman, R. E.,20 Adelhorst, K., 53 Agapova, N., 117 Ahnhog M., 118 Aikawa, J., 247 Aisaka,K, 176, 195(1,2) Akiyama, F., 242 A l - M ~ u d i N. , A. L.,3 1 Albert, R., 27 Albonico, S. M., 119 Album, H.E., 133, 134(146), 137(147), 162(146) Aldinucci, D., 206 Alexander, S.A., 259 Ali, M. H.,39 Allerhand, A., 20,25(27), 27(3) Amano, H., 168 A ' .m P.A.,259 Anderson, K. V.,254,258(81) Andrasi, F., 154, 165(209) Andrzejewski, D., 114 Angius, A., 107, 130(32), 13432) Angyal, S.J., 19,20(1), 22,25,28,31,35(20) Anteunis, M., 101, 102(19), 106(19), 117(19), 134(19) Antonsson, P., 258 Anzuino, G., 151, 172(206) A A , Y., 176, 195(2) Arakawa, M., 247 Amy% V.P., 130(131), 131, 151(131), l65( 131) Archibald, T. G., 155 Arena, B. J., 1 19 A&, K.,169 Aspro-Nicholas Ltd., I29

Atlas Chem. Ind., Inc., 155 Audinos, R., 99, 102(1I), 110(1 l), 113(1 I), 114(1I), 156(11) Aug&,C., 183, 184, 195(20), 197, 198(20), 199(42,44),200(20, 38), 201(38, a), 201(48), 202(20), 203(43,44), 204(41), 21 1(39), 215(15,39,69), 216,218(69), 219(15), 223(79), 225(15, 80), 229, 236(40,48), 237 Avenel, D., 32 Axen, R., 181 Axmann, R., 137 Azulay, D. R., 243

B Baggett, N., 160 Bajza, I., 58,78(56), 89(56) Bakos, J., 102, 107(28), 155(28) Baltes, W., 118 Barad, U. G., 118 Barbas, C. F., 237 Barbier, J., 124 Barker, R.,2 1,23(6), 27(6,29), 119(93), 120, 122(93),219,220 Barnes, J. M.,168, 172(253) Barnes,M.,168 Barnes, M. J., 168 Barrio, J. R., 30 Bartold, P. M.,247,254(90), 255,256(90) Barton, R. E., 99, 150(12) Bartsch, G., 1 18 Bartsch, W., 164 Bashey, R. L.,244 Batelaan, J. G., 158 Batley, M., 29 Batta, G., 58, 59(54),60(54), 75(54), 78(56), 83,89(56) BatJ. M.,166

263

264

AUTHOR INDEX

Baughn, R. L., 186,208,21 1 Baum, K., 155 Baumann, W.,200 Bayliss, M.T.,247 Beauchamp, R.O.,Jr., 166 Becchetti, E.,256 Beck, M.,254(87), 255,256(87) Beckwith, A. L.J., 38 Bednarski, M. D.,188, 191, 193,206

Beilstein,95 BeMiller, J. N.,59,64(59) Berenson, G.S.,247 Berg, R.A., 258,259(118) Berman, B., 256 Bernfield, M.,257 Bemenyi, P.,154, I65(209) Beta,W.,168 Beyerle, R.,163 Bhalla,H.L., 117, 118 Bida, G.T.,30 Bignall, J. C., 1 12 Binkley, R. W.,38,42 Birch, G.G.,167 Bischofberger, N.,19 1 Bjbrkling, F.,232 Blanken, W.W.,223 Blattner, R.,45,47(33), 49,60(33), 65, 75(33), 85,87,88(33,36,90) Blumberg, P.,256 Blumenfeld, H.,186 Bock, K.,53, 120, 121(94),125,129, 150 Bogaert, M.G.,118 Bohn, H.,130, 163(130) Boh, P. A., 130 Boitiaux, J.-P., 124 Bolte, J., 204 Bombor, R.,166 Bongiovanni, G.,118(74),119 Boniforti, L., 146 Bonn,R., 110 Boullanger, P., 126 Bouxom, B., 197, 199(42,44),203(44) Bovee, W.M.M. J., 102, 104(27), 109(27) Boyd, G.W.,112 Bradley, C. H.,20 Bralovic, M.,147 Brancq, B., 167 Brandner, J. D.,119 Brard, L.,41,60(25),61(25),62(25), 84(25), 89(25)

Bratin, K., 1 17 Bravo, P., 35 Brennan, M. J., 247 Brinkmeier, H., 24 Broek, D.,254,258(77) Bron, J., 133, 136(146a), 165(146a) Brossmer, R., 200 Brown, D.M.,129 Brown, E. D.J., 183 Brumley, W. C.,114 Buck, K.W.,128 Buckwdter, J. A., 239 Buddecke, E.,257 Buddrus, J., 24 Bukhari, M. A., 258 Burd, D.A. R., 247,252(39) Butler, W.T.,244 Buu-HoI, N.P., 40

C

Camera, E., 107, 130(32), 134(32) Camerman, A., 114,115(39) Camerman, N.,114, 115(39) Cameron, T.S., 24 Cano, J. P., 1 18 Capon, R. J., 31 Card, P.J., 213,231(71) Carinci, P., 256 Carlson, M., 117 Carlstedt, I., 254(84, 85),255,256 Carpenter, M.R.,249,252(68) Carver,J. P., 22 Castilla, I. M.,30, 125 Caterson, B., 248(67), 249 Cavay6, B., 184, 195(20),197, 198(20),

199(42,47),200(20) Cekovic, A., 128 Cekovic, Z.,124 Cere,

V., 148, 150, 160(200)

Chana, J. S., 39 Charpiot, B., 129 Chasseaud, L. F., 110, 113(34),118 Chatterjee, S.S., 133, 134(142, 143, 144),

138(147), 151(142, 143, 144), 162(142, 143, 144, 147) Chaumette, P., 124 Chen, J. L., 166 Chenault, M.K.,188

AUTHOR INDEX Cheung, K-S., 243 Chiarelli, S.N., 119 Chiba, T., 43, 76(30), 77(30) Chid, P., 129 Chistotinova,L. T., 118(77), 119 chizhov,0.S.,110, 112(33) Choi, H. U., 244,248(26), 249,253(26), 259 Chopineau, J., 236 Chopra, R.K.,249 Chou, C. H., 133 Cicero, D., 3 1 Cintron, C., 248 Cirelli, H. F., 35 Clark, M.R.,166 Clayton, C. J., 3 1 Cockman, M., 21 Coffey, S.,95 Cohen, A,, 124 Collins, P. M.,39, I56 Conley, D. L., 206 Conradt, H. S.,237 b k , G. M.W., 171 Cope, A. C., 146, 147, 148(193) Cordes, G., 119 Cortes, S. J., 35 Cossum, P. A., 112 CBster, L., 243,244,247( 17), 248,249, 252(73), 254(84,85), 255,256 Cottier, L., 38 Courtaulds Ltd., 129 Covington, H. I., 248 Coxnon, B.,35 Craig, A. S.,258 Crans, D. c., 28, 190,193(30),206,208,210 Crawshaw, T.H.,102, 107(26), 109(31), 116(26), 117(31), 142(31), 161(31) Cremata Alvarez, J., 2 1 Cseko, I., 161 Csizmadia, V. M., 101, 117(18) Cuko, I. I., 24,29(32) Cully, Z., 256 Culp, L. A., 259 Cuny, E., 82 Curran, D. P.,38 Czaja, R.F., 129

265

Damle, S.P., 243,247( 17), 248 Daniels, L., 190 Danielsen, C. C., 258 Darragh, A., 110 Date, T., 64 David, S., 184, 195(20), 197, 198(20), 199(44), 200(20,38), 201(40), 202(20), 203(43,44), 211(39), 215(39), 219, 223(79), 236(40) Davidson, I. W. F., 118 Davis, N. W., 112 Dax,K.,27 Day, A. A., 254 De Angelis, N. J., 119 De Jew, B.,236 de Lederkremer, R. M., 35 De Lucchi, O., 107, 134, 130(32), 134(32) De Philippe, L., 167 de Souza-e-Silva, U., 184, 225(18), 231(89) Dederen, J. C., 166 Defaye, J., 25, 119 Degueil-Castaing, M.,236 Dejter, S. W., Jr., 244 Delferes, E. R.,Jr., 247 DelM.,99, 102(1l), 1 lO(1l), 113(1I), 114(11), 140, 156(11) Demerseman, P., 40 Demeter, L., 133 Demuynck, C., 204 Descotes, G., 38,41,60(25), 61(25,61), 62(25), 84(25), 89(25), 126 Dhein, R.,130, 172(127) Dicarlo, F. J., 118 Dicosimo, R., 206 Dimov, N., 117 Dimova, N., 118 Ding, X.D., 117 Dirlikov, S. K, 172 Dittgen, M., 166 Dodd, C. M.,249,258,259( 117) Doganges, P. T., 156 Dokic-Mazinjanin,S., 100, 102(16), 148(16) Donoff, R. B., 259 Dorland, L., 225 Doyle, E., 1 18 Driver, G. E., 32 Drouillard, S.,236 heckhammer, D.G.,190, 193(30), 2 13, 234(34) Du Mortier, C., 35

AUTHOR INDEX

266

Duncan, M. R., 256 Dupuis, J., 72, 73 Durda, W., 126 Durette, P. L., 74 Dumachter, J. R., 190, 193(30), 194(35), 234(34)

Duxbury, J. M., 128 Dvonch, W.,133, 134(146), 137(146), 162( 146)

Dziewiatkowski, D. D., 258

E Edwards, E., 258, 259(117) Effenberger, F., 206 Ehler, D. S.,35 Einstein, F. W. B., 114, 116(41), 143(41) Ek, M. J., 30 Eisenstein, R., 244 Ekman, G., 244 Elgavish, A., 256 El'perina, E. A., 142, 143, 161(189, 190) Embery, G., 243 Emeury, J. M., 134 Endele, R., 110 Endo, S., 204 Engler, D. A., 203 Emback, S., 18 1 Esser, F., 133 Evangelisti, R., 256 Evanko, S. P., 248 Ezure, Y.,235

F

Fackre, D. S., 251, 252(68) Farkas, I., 58, 59(54), 60(54), 75(54), 78,83 Famia, F., 39 Fassbender, F., 168 Fava, A., 148, 150, 16q200) Fay, L., 1 18 Fazekas, D., 161 Feicho, L., 166 Feizi, T., 22 1 Feldmann, J., 1 19(92), 120, 135 Femandez, R., 183 Femandez-Bolaiios,J., 30

Femandez-BolaiiosGuzman, J., 30 Fernbdez Moha,L., 2 1 Femer, R. J., 40, 41(21), 42(21, 28), 43(21, 29, 36), 45,47(27, 33), 48(26), 49, 50(35), 51,52(40), 60(33), 62(63), 63, 64(20, 63), 65(20, 38), 75(33), 76, 79(21,26,28), 80(27,38), 82(40), 85, 87(27), 88(33,36, W),89(93), 91(63) Fessner, W.D., 191 Fickert, W., 140 Fdipuzzi, F., 107, 130(32), 134(32) Finan, P. A., 142 Fine, D. H., 117, 118(75), 119 Fischer, F., 141, 169, 171(183) Fischer, H., 72 Fisher, G. R., 247 Fisher, L. W.,244,248(41), 252(41), 253(41), 254, 258(75)

Fitchett, M.,38 Flanigan, I., 206 Fleche, G., 95, 156 Fleischmajer, R., 244 Flint, M. H., 258 Forcier, G., 117 Ford, E. C.,136, 171(167) Forrest, M. E., 110 Foster, A. B., 128, 158 Franke, F. P., 30,206 Franko, B. V.,166 Franks, F., 23,25(27) Franois, P., 237 Fransson, L.-A., 242,244,247,248, 249,256 FranzCn, A., 249,258 Fraser-Reid, B., 124 Freidenreich, J., 200 Friebolin, H., 200 Frigerio, M., 35 Friz, L. P., 15 1, 172(206) Frize, J., 133 Fronza, G., 35 Frost, J. W., 206 Fuji Photo Film Co., 169 Fujii, N., 243 Fujimori, T., 3 1 Fujiwara, S., 254 Fukai, M., 169 Fukui, S., 256 Fumeaux, R. H., 40,41(21), 42(21,28), 51, 52(40), 64(20), 65(20), 79(21,28), 82(40)

AUTHOR INDEX

G Gabard, B. L., I34( 142) Gabe, E. J., 47,50(35) Gadelle, A., 25 Gainsford, G. J., 47,50(35) Gallagher,J. T., 256 Gallardo Carrera, A., 133 Gammeltofi, P., 150 Ganem, B., 30 Ganesan, V., 119, 171(82) Garcia Martin,M.de G., 24,30(3 1) Garcia-Rasz, A., 2 1 Garg, A. K., 258,259( 1 18) Garg, H. G., 225, 242, 247, 25 1(40), 252(39), 258,259(9, 118) Gaset, A., 95, 99, 102(I I), 110( 1I), 113(1 I), 114(11), 140, 155, 156(11),201(40) Gasiunas, N., 256 Gautheron, C., 183, 184, 195(20), 197, 198(20), 199(42,44),200(20,38). 201(48), 202(20), 204(41), 211,215(15, 69), 216,218(69), 219(15), 223(79), 225(15,80), 229,236(40,48) Gavuzzo, E., 102, 109(31), 117(31), 142(3 l), 16I( 3 1) Gemesi, I., 167 Geria, N., 168 Giani, C., 118(74), 119 Gibson, G. J., 244 Gielsdorf, W., 118 Giese, B., 38,72,73(6), 77(76) Gilbert, B. C., 38 Gillard, G. C., 258 Ginsburg, V., 221,239 Gizur, T., 133 Glaesson, S., 99 Gli)ssl, J., 254(87), 255,256(87) GinneZ-Sinchez, A., 24,30(31) Godtfredsen, S. E., 232 Goebbeler, K.H., I18 GOB,E. U., 117, 118(75),119 Golds, E. E., 257 Golovkina, L. S., 110, 113(33) Goodwin, J. C., 102, 109(21), 110(21), 15q21) Gorrichon, J. P., 95 Gowda, D. C., 231 Gowen, M., 257

267

Granado, C., 155 Gray,K., 29 Greene, I. D., 140 Greenshields, J. N., 140 Gregory, J. D., 243,247(17), 248 Greiche, Y., 168 Greiner, J., 129 Grelewicz, J., 133 Grenier-Loustalot, M.-F., 38 Greve, H., 256 Grigera, J. R., 23, 118(77) Grigor’ev, A. B., 119 Grtjninger, K. S.,73 Gromadzinska, E., 120, 133, 135 Gyarmathy, M.,167

H Habuchi, H., 247 Haigh, M., 258 Haines, A. H., 95, 125(8), 126, 141 Haines, S. R., 47,49, 50(35), 65(38), 80(38), 88, 89(93) Hajek, M.,119, 168(85) Halkiewicz, J., 118 Hamptom, A., 248(67), 249 Handa, M.,254,258(79) Handley, C., 248(67), 249 Handy, C. J., 239 Hane, K., 237 Hanessian, S., 39,201 Hardingham, T. E., 247 Hiiring, T., 141 Harry-Okuru, R. E., 29 Harsanyi, K., 133 Hartmann, L. A., 119 Hartmann, P., 168 Hascall, V. C., 239,244,248(65,66,67), 249,256,258 Hashimoto, C., 254,258(81) Hashimoto, H.,3 1,64 Hashimoto, Y.,22 Hassell, J. R., 244,248 Hawkins, G. R., 247,248(41), 252(41), 253(41) Hayashi, H., 152, 165(207a) Hayashi, N., 247 Hayday, K., 72 Haydon, D. A., 171

268

AUTHOR INDEX

Hayman, E. G., 248 Haynie,S. L., 188,208,211(63), 219(23) Hayward, L. D., 99, 100, 101, 117(18), 133, 150(112) Hedbom, E.,258 Hedbys, L., 232 Hehemann, D. G., 42 He& B., 102, 107(28), 155(28) Heineg;Zrd,D., 239,244, 248(66), 249, 252(32), 258 Heino, J., 254(86), 255 Helferich, B., 64 Hemmer, R., 114, 116(42), 163 Hempe, W., 55,56(50), 57(50) Hendry, C. J., 247,25 l(40) Hennen, W. J., 200,234,236( 103) Hermann, H., 242,247,251(40), 259(9) Herder, G., 23 1 Heyns, K., 155 Hicks, D. R., 124 Hieke, E., 118 Higa, H. H., 183,223(16) High, L., 150 HiU, R. L,, 220,223,225 Hdard, R. L., 140 Hilleman, M. R., 167 Hirao, A., 160 Hirose, N., 249 Hirschbein, B. L., 210 Hitz, W. D., 213,231(71) Hodge, J. E., 102, 109(21), 150(21) Hoffmann, G., 167 Holland, L. Z., 254,258(79) Holm, G., 118 Honda, S., 22 Honda, T., 248,254(83) Hooghwinkel, G. J. M., 223 Hope, K. D., 25,28(37) Hop!€, H.,146 Hopton, F. J., 102, 104(27) Hori, H., 51, 52(42), 54,78(42) Horilri, H., 51,52(41), 78(41) Honto, S.,64 Horiuchi, T., 166 Hormaza Montenegro, J., 21 Hortobagy, G., 167 Horton, D., 74 Hough, L., 95 HSU,C.-C., 247 Huang, S.G., 21,23(6), 27(6)

Huchette, M., 95

Hudson,K.L., 254,258(81) Hughes,F. A., 169 Hughes,N.A., 3 1 Humphries, D. E.,256 I

Ianni, A., 35 Ichikawa, Y.,44 M e , E.J., 257 Ikeda,J., 152, 165(207a) Inch, T. D., 203 Ingold, K. U., 49,68(37), 72(37) Innocenti, F., 1 18(74), 119 Inoue, S.,204 Inoue, Y.,204 Irie, T., 135 Isemura, M., 247 Ishibashi, K., 133, 135(149) Ishiguro, S., 133, 135(149) Ito, T., 133 Ito, Y., 166 Iwamura, H., 72 Iwasaki, M., 204 J

Jablonowski, M., 24 Jackson, M., 101, 117(18) Jaquet, A., 99, 102(11), l l q l l ) , 113(11), 114(11). 156(11) Jwuet,F.,.iss, is6 Jaeger, H., 1 18 Jalkanen, M., 257 Janousek, Z., 38,40,69(23), 70 Jansen, J. C., 114, 115(40) Janssen, H. M. J., 243 Jaquet, F., 95 Jarglis, P.,55,56(48,49,40), 57(50), 58(48, 49) Jaseja, M.,28 Jasinski, W.,95, 129, 131, 136, 137, l69( 117), 171(169) Jeanloz, R. W., 225 Jeansonne, N., 247 Jeremic, D., 147 Jeroncic, C. O., 35 John, B.A., 110, 1L3(34)

AUTHOR INDEX Johnson, R. N., I18 Johnson, T. L., 244,248(26), 253(26) Jones, G., 102, 107(26, 31), 116(26), 117(31), 142(31), 161(31) Jones, J. B., 177 Jung, W., 242, 259(9) Just, M.,163

K Kabayama, M. A., 23 Kaes, E., 139 Kahne, D., 74 Kaji, E., 56, 83(52, 53) Kakebi, K., 22 Kakuchi, T., 143, 161(191) WmBn, 59,83 Kaminsky, W., 169 Kapoor, R., 247 Kapuscinski, M.,30,206 Karelson, M.M.,70 Kasper, M.,114, 116(42) Katagiri, K., 248, 254(83) Kataoka,T., 254, 258(77) Kato, A., 22 Katritzky, A. R.,70 Kawa,M.,64 Kazlauskas, R.J., 210 Kean, E.L.,183,216(17) Khanolkar, J. E., 1 17, 1 18 Kho, B. T., 118 Khudyntsev, N. A., 132 Kieboom, A. P.G., 102, 104(27), 109(27), 236 Kiegel, E., 133 Kiely, D.E., 25, 28(37), 29 Killackey, J., 257 Kim, M. J., 191,200 Kimata, K., 247,249 Kimura, J. H., 239,244 King, 1. A., 257 Kim-Moms, M.J., 21 Kisfaludy, L., 167 Kishi, H., 51, 52(41), 78(41) Kitajima, K., 204 Kitajima, T., 228 Klaus, N., 173 Kleiner, F.,169 Kleinman, H. K., 244 Klenk, H. D., 23 1

269

Klessing, K, 133, 134(142, 143, 144), 138(147), 151(142, 143, 144), 162(142, 143, 144, 147) Klibanov, A. M.,234,235,236( 105) Kligman, L. H., 257 Knightly, W. H., 129 Kobata, A., 22 1 Kochetkov, N. K., 153 Koebemick, H., I19(92), 120, 135 Kohler, J., 168 Kohn, J., 183 Kokenyesi, R.,259 Kolarikol, A., 156 Kolbe,I., 167 Kolta, R., 161 Konstanntinovic, S., 100, 102(16), 103(16), 148(16) Koob, T. J., 257 Koroteev, M. P.,149, 153 Korth, HA.,72,73 Kossmehl, G., 173 KO&, T., 22 Krantz, D. E.,254, 258(82) Krauze, S., 120, 133, 135 Krempl, E., 168 Kresse, H., 254(87,88), 255,256(87) Krull, I. S., 117 Kruse, W. M.,140 Krusius, T., 254(86), 255 Kubler, D. G., 2 I Kubo, K., 152, 165(207a) Kuboki, Y.,243 Kubota, A., 247 Kuc, I. M.,249 Kuettner, K. E.,239 Kulczycki, E., 133 Kuroda, A., 248 Kuroda, T.,152, 165(207a) Kuszmann, J., 28, 100, 102(17), 105(17), 106(29), 109(29), 110(29), 149, 150(29), 151(203), 153(203), 154(17), 165(17,209) Kuyper, C.M. A., 243 Kuzuhara, H.,44

L Lacmte, G., 156 Ladner, W. E., 188, 21 l(24) Laidlaw, J., 258

AUTHOR MDEX

270

Lambe, R. F.,110 Langhans, R.K., 167 Lapenkov, V. L.,141 Lapiere, C. M., 244 Larjava, H., 254(86), 255 Larsso, T.,258 Larsson, P. O., 232 Lauer, K., 133 Laufen, H., 118 Lawston, I. W.,203 Le Blanc, M., 129 Le Lem,G., 126 Le Maistre, J. W., 136, 139, 165(176), 171(167)

Lee, C. K., 167 Lees, W., 191 Lehmann, A., 146 Lehtonen, E.,257 Leising, M., 73 Leisung, M., 73 Leitold, M., 130, 133, 137, 157(154), 163(129), 164(170, 171)

Lelki, G., 161 Leloir, L. F., 2 18 Lenfant, M., 135 Lenkiewicz, R.S., 118(80), 119 Leproq, S., 124 Lei, S., 117 Lewandowska, K., 259 Lewis, A.,166 Lewis, P. A., 118 Libeman, A. L., 167 Lichtenthaler, F.W., 2 I, 25( 17), 28, 32( 17), 55,56(48,49,50), 57(50), 58(48,49), 82, 83(52, 53) Lillford, P.J., 23,25(27) Lim, J. J., 74

Limura, T.,35 Linblom, K.,258 Lindner, H.J., 73 Liu, W.Y., 117 Livingstone, D. J., 101, 117(18) Lloyd, J. B. F., 117, 118(47)

Lo,Y.S.,165 Loesel, W.,133 Longas, M. O., 242,243 Low, M., 167 Lowary, T.L., 124 Lowther, D. A.,239,244 Lubineau, A., 221,237

Lueders, H., 102, 105(23), 130(23,25), 138, 146(173), 147(23), 151(23,25, 195), 154(23, 195), 159(173), 173(25, 128) Lukevica, O., 146 Lunazzi, L., 148, 160(200) Lundt, I., 125, 129 Lutz, D., 118 LuWi, J. K., 166 Luzi, L. A., 166 Lynch,M. J., 167 Lyndon, P., 30

M Ma&, M., 118(74), 119 MacLeod, J. K., 31 Maddock, J., 1 18 Miidler, H., 169 Maibach, H. I., 257 Ma$, L.,146 Maillard, B., 236 Major, R. M., 110, 113(34) Malatesta, V.,49,72(37) Malbica, J. O., 118 Malleron, A., 184, 195(20), 197, 198(20), 199(44), 200(20), 202(20), 203(43,44)

Malmstrom, A., 49,68(37), 244,249, 254(84, 85), 255,256

Manfredi, A., 129 Manro, A., 39 Maple, S.R.,20, 25(4) March, J., 5 1 Margok, R. U.,257 Marko, J., 102, 107(28), 155(28) Martin, B. K., 164 Martin, D. R.,24 Martinez-Castro, O.,21 Martorana, P., 130, 163(130) Martorana, P. A., 130, 163(130) Mathieu, C., 184,219(15), 223(79) Matsuda, M., 243 Matsui, F., 243 Matsunaga, E., 244, 248,249,254(83) Matyschok, H., 126, 129 Maurer, M., 140 Mazenod, F. P.,210 McCurry, S.D., 210 McKelvey, R. D., 72 McLeod, J. K., 206

AUTHOR INDEX

McMurtrey, J., 247 McNicholas, P. A., 29 McQuillan, C. I., 254 McQuillan, D. J., 239 Meats, J. E., 257 Medem, H., 130, 172(127) Medgyes, G., 100, 102(17), 105(17), 106(29), 109(29), 110(29), 150(29), 151(203), 153(203), 154(17), 165(17), 165(209) Meezan, E., 256 Mega, T. L., 35 Meguro, H., 5 1 52(4 1,42), 54,76(46), 78(41,42,43), 79 Menezo, J.-C., 124 Merck and Co., Inc., 167 MCrienne, C., I84,2 19(19) MerCnyi, R., 38,40,69(23), 70 Merrath, P., 114, 116(42), 128, 135(104), 139(104), 163 Meshreki, M. H., 170 Metcalfe, J. C., 102, 107(26), 109(31), 116(26), 117(31), 142(31), 161(31) Metras, F., 38 Meyborg, H., 119(91),120,168(85), 172(253) Meyer, K., 242 Michel, G., 1 18 Michel, H., 164 Micovic, V. M., 147 Midler, M., Jr., 167 Mihailovic, M. L. J., 100, 102(16), 103(16), 148(16) Mihalszky,K., 161 Mikhant'ev, B. I., 141 Millar, S. J., 254(90), 255, 256(90) Miller, R., 74 Mills, J. A., 94 Minet, E., 118(74), 119 Misawa, T.,54,76(46), 79 Miyamoto, I., 247 Mizuno, N., I17 Mladenovic, S., 147 Mocali, A,, 206 Mochizuki, H., 160 Modena, G., 107, 130(32), 134(32) Mohan, V. K., 119, 171(82) Monson, K., 118 Montassier, C., 124 Moran, I. R., 2 13

27 1

Mori, K., 169 Mori, T. P.,139, 165(176) Morita, E., 117 Moriyama, A., 169 Moriyasu, M., 22 Mom, M., 237 Moms, P.E., 25,28(37) Mosbach, K., 186,232 Mubarak, A. M., 129 Muenchow, L. H., 59,64(59) Muir, H., 247 Munakata, H., 247 Munkombwe, N. M., 31 M u d , B. K. M., 119, 171(82) Muralidhara, R., 167 Murata, K., 248 Murengezi, I., 140 Myers, G. S., 133

N Naadano, D., 204 Nagai, Y.,243 Nagase, S.,247 Nakahama, S.,160 Nakajima, T., 5 1, 52(42), 78(42), 256 Nakamura, A., 168 Nakamura, T., 244,247,249 Nakano, T., 249 Nara, T., 135 Neame, P. J., 244, 248(26), 249,253(26) Nec, R., 135 Negoro, K., 131, 168(132) Neilson, K., 118 Newman, A., 323 Newsome, D. A., 248 Nieduszynski, I. A,, 248 Nifant'ev, E. E., 131(134), 132, 149, 153 Nihon Surfactants Industry Co., 168, 169 Nilsson, B., 244 Nilsson, K., 186 Nilsson, K. G. I., 225,232 Nishida, Y.,51, 52(42), 54, 78(42,43) Nishikawa, Y., 166 Nishimiya, Y ., 135 Nkhino, T., 56,83(52) Nitz, R. E., 130, 163(130) Nix, M., 73 Nolan, J. C., 165

AUTHOR INDEX

272

Noro, A., 249 Nouvertne, W., 130, 172(127) Nozaki, K.,193,234(34) Nukada, T., 228 Nunez, H. A., 219 Nusgens, B.,244 0

Oberhauser, A., 168 O'Brien, E. A., 150 (Ibrink, B., 247 Ochiai, M., 168 OConner, T., 150 Oegema, T. R., Jr., 244,258 Ogawa, T.,228

Oh, K., 135 Ohkawa, M., 166 Ohrui, H.,5 1, 52(4 1,42), 54, 76(46), 78(4 1, 42,43), 79 Oike, Y.,248(67), 249 Ojrzanowski, J., 120, 133, 135 Okada, M., 22 Olano, H., 2 1 Oldberg, A., 239,247,248,252(37), 258 Olejnicak, E., 133 Oliver, W. M., 243 Olsen, K. w., 220 Onodera, S., 243 Oohira, A., 243 m - M ~ a hE. , C., 39 Orford, C. R., 258 Orth, W., 140 Osman, D., 20 Oswald, A. S., 21 Otagiri, M.,135 Otsuka, S.,166 Overend, W. G., 156 Ozaki, A., 237 Ozawa, T., 168

P Pacifici, R., 146 Paez, M., 21

paguaga,E.. 74 Pal, S.,244,248(26), 253(26) Palmer, M. E., 256 Pandey, M., 254(89), 255

Pandraud, H. G., 32 Paoletti, F., 206 Paolucci, C., 148, 150, 160(200) PArkhnyi, A., 83 P&khnyi, L., 59 Parry, D. A. D., 258 Parthasarathy, N., 247 Pasto, D. J., 70 Patroni. J. J.. 29 Pa&n, D., 23

Paul,E., 167 Paul, S.,244 Paulsen, H., 155,228 Paulson, J. C., 183, 184,223(16), 225, 231(89)

Paulson, M.,225( 18), 258 Paulsson, M., 258 Pavare, B., 146 Peacock, D. J., 39 Pearson, C. H., 244,249,252(68), 258, 259( 117)

Pedersen,C., 119, 120, 125, 129, 150 Pedersen, H., 53, 121(94) Pederson, R. L., 190, 193(30) Pentel Co., 169 pkez-Rey, R., 2 1 Perka, J., 129 Perlin, A. S.,27, 28, 32(38), 35(38) Perlish, J. S.,244 Perry, A. R., 128 Persson, B., 118(78), 119 Peters, J. A.. 102, 104(27), 109(27), 114, 115(40)

Petrov, K. A., 13I( 134), 132 Petter, R. C., 32 Phelps, C. F.,247,248 Pichini, s., 146 Pierce, J., 21,23(6), 27(6,29) Pierschbacher, M. D., 247,252(37) Pittet, A. O., 167, 168 Pizzorno, M.T., 119 Plaza Upez-Espinosa, M.T., 24,29(32) Plessas, N. R., 39 Plucinski, J., 126 Pogliano, L., 118(74), 119 Polievktov, M.K., 118(77), 119 Poll,H. G., 173 Pollak, A., 186,208 Pollicino, S., 148, 150, 160(200) Pommier, F., 118

AUTHOR INDEX Pompliano, D.L., 206 Poole, A. R., 239, 244, 252(73), 254, 259

Popuszynski, s., 95 Pora, H., 219,225(80) Porath, J., 181 Poutsma, M. L., 67 Power,M., 112 Powers,D. G.,32 F'rager, M., 257 M y , J.-P., 38,40,60(25), 61(25,61), 62(25), 84(25), 89(25) Prasit, P., 49,88(36) Prince,C. W., 244 Pringle, G. A., 249 Proctor,P. H., 166 Prost, M., I18 h e , D.G., 118 Pudgett, H. C., 30 PuMs, J. A., 243 Putnam, F.W.,254,258(78)

Q Quick, A., 102, 107(26) Quickenden, M. J., 23

R Rabovskaya, N. S., 149 Racker, F., 204 Radhakrishnamurthy, B., 247 Rafka, R. J., 24 Rahemtulla, F., 244 Ramaiah,M., 38 Range, D., 25 Rao, K.B., 119, 171(82) Rasper, J., 1 18 Rathbone, E. B., 30 Rearick, J. I., 223, 225 Redmond, J. W., 29 Redwood, W.R., 171 Refh, S.,53 Reidy, J. P., 142 Reimer, L.M., 206 Reiner, A., 244 Reinke, R., 254,258(82) Reinking, K., 169

273

Remy, G., 38 Renner, R., 85 Resnati, G., 35 Reuben, J., 30, 32 Reuter, G., 231 Richards, G. N., 124 Richardson, A. C., 95 Richter, K., 125 Riess, J., 129 Riess, J. G., 129 Rigal, L., 155, 156 Riordan, J. M.,29 Ripellino, J. A., 257 Ripp, K. G., 213,231(71) Riva, S.,236 Roberts, J. R., 254,258(79) Roberts, M. S., 112 Robins, P., 239 Robinson, G., 23,25(27) Rocrelle, D.,140 Rodbard, D., 248 Rogers, G. N., 23 I Rolland, P. H., 118 Ranniger, S.,21,25(71), 35 Roos, O., 133 Root, R. L., 234 Ropenga, J., 135 Ropuszynski, S., 126, 129, 131, 136, 169(117), 171(169) Roseman, S., 183,216(17) Rosen, L., 118(78), 1 19 Rosenberg, L., 244,248(26), 253(26) Rosenberp, L. C., 239,244,249,252(73), 254,259 Rosevear, P. R., 2 1 Rowel, M. T., 118 Rosseel,T., 101, 102(19), 106(19), 117(19), 134(19) Rossi, M. T., 119 Rougbley, P. J., 247, 248, 252(44), 253(44), 257 Rudolph, H., 130, 172(127) Ruegge, D.,72 Ruggeri, Z. M.,254,258(79,80) Rullmann, K.H., 64 Ruoslahti, E.,241,247,248,252(37), 254 Rusch, D. T., 167 Ruseva, N.,118 Russell, R. G. G., 257 Rzepka, M.,126

274

AUTHOR INDEX S

Sabesan, S., 225 SadIer, J. E., 225 Saheki, Y., 131, 168(132) !hito, T., 130, 168(132), 191 Saito, Y., 64 Sajdera, S.W., 248(65), 249 Sakano,T., 256 Salisbury, B. G. J., 119(91), 120, 248 %burg, H., 119, 120, 168(85), 169, 172(253) Samaki, H.,204 Sampaio, L. de O., 247 Sandri,E., 148, 150, 160(200) Sanol Schwarz-Monheim, 133, I34 Santer, V., 257 Santoni, Y., 118 Sam, J., 2 1 Sibstrand, B., 254(85), 255 Sasaki, T., 131 Sato, N., 247 Sato, S., 244 Satyamurthy, N., 30 Saura-Calixte, F.,2 I Savage, K., 256 Sawicki, W., I18 Saxen, L., 257 Schaefer, H.,173 Schafer, I. A., 254(89), 255 Scharpf, F., I 18 Schauer, R., 194,202(37), 231 Schiattarella, D., 151, 172(206) Schijen, M. M. A., 243 Schiphorst, W.E. C. M.,225 Schiweck, H., 28 Schleyerbach,R., 239 Schlingmaan, M.,234(107), 235 Schlueter, G., 137 Schliilter, G., 128, 135(104), 139(104) Schmidt, A., 257 Schmidt, D. L., 102 Schneider, B., 28 Schneider, C.J., 172 Schneider, G., 118 Schneider, M. P.,177 Schoenafinger,K., 128, 130, 133, 134(107), 163(130) Schor, S. L., 256 Schreckenberg, M., 130, 172(127)

Scott, J. E., 258,259 Scott, P. G., 249,252(68), 258,259( 117) Snepanik, B., I19 SeidI, S., 27 Selavka, C., 1 17 Selmke, P.,257 Senn, M.,110 Seno,N., 242 Serebryakov, E. P.,142, 143, 161(189, 190) Sekc A. S., 21,23(6, 12, 13), 25(12, 13), 27(6,9, 12,29), 28(36), 32(13) Servadio, V., 129 Seto, S., 30 Settlage, J. A., 118 Shah,B. A,, 118 Shaper, M.,220 Shchegolev, A. A., 131( 134), 132 Sheehan, J., 248 Sheehan, J. K., 244,249 Shen, T. Y., 146, 147, 148(193) Shibaev, V. N., 177 Shimada, F., 133, 135(149) Shimizu, C., 1 17 Shingbal, D. M.,1 18 Shinkai, H., 244,248,249,254(83), 255 Shinkuma, D.,117 Shinomura, T., 249 Shishiba, Y., 256 Shukla, A. K., 231 Siebert, E., 247,25 l(40) Siebert, E. P., 247 Silbert, C. K., 256 Silbert, J. E., 256 Silver, F. H., 258, 259( 1 18) Silveri, L. A., 119 Silvestri, S., 118(79), I19 Simon, E. S.,188, 191,206 Simon, H.,164 Sirnonet, J., 156 Sinaj, P., 43,45(30), 76(30), 77(30) Sinicka, S.,126 Sinnott, M. L., 141 Sinskey, A. J., 237 Siooufi, A., 1 18 Sipursky, S.L.,254 Sitabkha, L., 254(89), 255 Sjaberg, I., 256 Skelton, B. W.,29 Slessor, K. N., 114, 116(41), 143(41) slivkin, A. r., 141

AUTHOR INDEX Smith, D. F., 229 Smith, J. H., 170 Smith, V.H., Jr., 22 Snatzke, G., 59 Snell, R. P., 117 Snowden, J. M., 258 Snyder, J. R.,21, 23(6, 13), 25(13), 27(9), 32( 13) Sofronas, P., 102, 118(20) Sohar, P., 102, 106(29), 109(29), 1 lO(29) Soltzberg, S., 94,95(4), 146(4), 150(4) Sommarin, Y.,258 Somogyi, A., 83 Somdc, L., 41,45,47,48(34), 58, 59(54), 60(54), 62,63(62), 75(54), 77(32), 78(56), 83, 84, 87(32), 89(56,94) Sotman, S., 247,251(40) Spirov, G., 1 18 Spohn, J. A., 114 Sprissler, R., I18 Stabellini, G., 256 Stadler, I., 167 Stafford, W., 247,251(40) Staub, A., 206 Stefanovic, M., 147 Stein, P. D., 206 Steinle, G., 28 Stella, L., 40,69(23) Stephen, A. M., 30 Stephen, J. F., 140, 170 Stephens, C. A., 259 Sterk, G. J., 133, 136(146a) Sterk, H., 27, 133, 165(146a) Stevens, J. D., 32 Stevens, J. W., 248(67), 249 Stick, R.J., 29 Stoddart, J. F., 102, 107(26, 31), 116(26), 117(31), 142(31), 161(31) Stojcic, S., 147 Stoss, P., 114, 116(42), 128, 130, 133(105), 134(105), 135(104), 137, 139(104), 157(154), 163(129), 164(170, 171) Strein, K., 164 Stribblehill, P., 160 Strietholt, W. A., 141 Struchkova, M. I., 142, 143, 161(189) Stuehler, H., 168 Stiitz, A. E., 27 Suami, T., 35 Suggett, A., 23

275

Sugihara, J. M., 102 Sugimoto, M., 228 Sugiyama, H., 30 Sun, K. M., 149, 17q201) Suslova, L. M., 143 Sustmann, R.,72,73 Suzuki,F., 152, 165(207a) Suzuki, S., 22,247,249 Svensson, S., 232 Swann, D. A., 242, 247,251(40), 252(39), 256, 258,259(9) Sweers, H. M., 200,234,236( 103) Sweers, H. W., 193 Symes, K. C., 141 Synder, J. R.,21,23 Szabii, I. F., 59, 84 Szabo, E. I., 118 Szafranek, J., 110, 118(35), 149(35) Szalay, P., 161 Szarek, W. A., 22, 24 Szeja, W., 128 Szejtli, J., 167

T Tadano, K., 35 Tait, M. J., 23 Takahashi, N., 254,258(78) Takahashi, Y.,254, 258(78) Takaoka, T., 143, 161(191) Takida, Y.,243 Takio, K., 254,258(80) Talhouk, J. W., 29 Tammi, M., 254(86), 255, 257 Tammi, R., 257 Tamura, S., 176, 196(2) Tanabe, H., 35 Tanabe, K., 249 Tanaka, T., 256 Tang, L.-H., 244,248(26), 253(26) Tamer, M. L., 247 Tao Eiyo Kagaku Kogyo Co., 133, 134(152), 135, 142(152) Tarcsa, E., 47,48(34), 62,63(62) Tarrago, M. T., 229 Taylor, A. R., 171 Taylor, T., 1 18 Tedder, J. M., 38 Teijin Ltd., 160 Telschow, J. E., 203

AUTHOR INDEX

276

Ter-Ovanesyan, M. R., 132 Tennine, J. D., 244,247,248(4 l), 252(4 l), 253(41), 254(75) Th6risod, M., 234,235,236( 105) Thiem, J., 102, 105(23), 130(23, 25), 138, 141, 146(173), 147(23), 151(23,25), 154(23), 159(173), 169, 171(183), 173(25, 128), 216,234(107), 235 Tbgersen, H., 120, 121(94) Thomas, G. H. S., 102, 104(24) Thompson, R. D., 1 17 Thonard, J. C., 247 Tietz, H., 228 Tilbrook, D. M. G., 29 Timmerman, H., 133, 138(146a), 165(146a) T i p a H. P., 118 Tipson, R. S., 124 Titad, K., 254,258(79,80) Tokic, Z., 128 Tomana, M., 244 Toole, B. P., 244 Toone, E. J., 237 Totty, R. N., 100 Touet, J., 183 Toupet, L.,41,60(25), 61(25), 62(25), 84(25), 89(25) Trautwein, W.-P., 155 Treder,W., 216,234(107), 235 Trotter,J., 114, 115(39) Truppe, W.,254(88), 255 Tucker, K. H., 229 Tuite, M.R.J., 150 Tull, R.J., 102, 129, 166 Turner, N. J., 194 Turner, W. R., I18(80), 119 Tuross, N., 247,248(41),252(41), 253(41) Tuseev, A. P., 132 Tuzimura, K., 29 Tvaroska, I., 22 Tyler, P. C., 41, 42,43(26, 29), 47(27), 48(26), 62(63), 63,64(63), 76(26,63), 79(26), 80(27), 87(27), 88(90), 91(63)

U Uekama, K., 135 Uldbjerg, N.,244,258,259 Ulmsten, U., 244 Unkefer, C.J., 35

Usui, T., 30,256 Uwajima, T., 176, 195(1,2)

V Vainio, S., 257 Van Beuningen, H. M., 243 van den Eijnden, D. H., 2 15,223,225 Van der Rest,M.,252(73), 254 vanderWerf,J.F., l33,138(146a),165(146a) van Dijk, W., 215 Van Etten,R.L.,35 van Halbeek, H., 225,229 Van Koningsveld, H., 114, 115(40) Van Kuppevelt, T. H. M.S., 243 Varela, O., 31 Vat*le, J. M., 201 Vedejs, E., 203 Veerkamp,3. H., 243 Vlez Castro, H., 2 1 Verhegghe, G., 101, 102(19), 106(19), 117(19), 134(19) VeyreBrew, A., 197,201(40) Viehe, H. G., 38,40,69(23), 70 Vietmeier, J., 173 Vikar, J., 133 Vikman, A., 167 Vill, V., 141, 169, 171(183) Vincze, Z., 133 Vliegenthart, J. F. G., 225 Vogel, K. G.,244,248,252(32), 257,258 von der Osten, C. H., 237 von Sonntag, C.,38 Vul'son, N.S., 110, 1 13(33) Vuorinen, T., 21, 23(12), 25,27(12), 28(36) Vuorio, E., 254(86), 255 Vuorio, E., 254(86), 255

W Waechter, W., 119 Wagner, K., 168, 172(253) Wagner, W. D., 248 Waldmann, H.,1s Waldmann, H. J., 193 Walker, T. E., 35 Walkinshaw, M.D., 23 Walsh, D. A., 165 Walton, J. C., 38

AUTHOR INDEX

Wang, Y. F., 234,236(103) W a g , Y-F., 237 Ward, J. W., 266 Warren, L., 198 Watanabe, M., 78 Wax, M., 186 Webber, C., 244 Webber, J. M., 128, 158 Weber, A. J. M., 158 Weinstein, J., 184, 225(18), 231(89) Weisleder, D., 102,109(21), 110(21), 15q21) Weisshaar, G., 200 Welstead, W. J., Jr., 165 Weprek, S.,56, 82 Wheetall,H. H., 188 White, A. H., 29 White, R. J., 247, 252(44), 253(44) White, R. W., 248 Whitesides,G. M., 177, 186, 188, 191, 193(31), 194,206,208(31), 210, 211(24,63), 213, 219(23), 237 Whiting, M. C., 141 Whitson, M., 244 Wiebkin, 0. W., 247 Wiener, C., 168 Wiersum, U. E., 158 Wiggins, L. F., 94 Wigler, M., 254,258(77) Wilchek, M., 181 Williams, C., 20, 27(3) Williams, D. J., 102, 107(26), 109(31), 117(31), 142(31), 161(31) Williams, J. F., 206 Wilson, L., 2 1 Wimmer, E., 126, 134 Wingerup, L., 244 Winterbottom, N., 249,252(68), 258, 259( 1 17) Wischniewski, M., 110, 149(35), 166 Wiseniewski, A., 110, 118(35) Woelk, H. U., 119(92), 120, 135 Woessner, J. F., Jr., 259 Wong, C. H., 177, 188, 190, 191, 193(31), 194(35), 200, 208(31), 210,211(63), 213,219(23) Wong, C. M., 234,236( 103) Won& C-H., 237 Woo, D., 102, 118(20) Wood, D. D., 257 Wood, S.G., 110, 113(34)

277

Woodhour, A. F., 167 Woods, R. J., 22 Woodward, A. J., 118 Wright, L. W., 119 Wu, J., 21,23(6), 25(12), 27(12) Wulff, G., 173

Y Yagi, T., 169 Yamada, Y., 169 Yamaguchi, Y., 247 Yamanaka, Y., 117 Yamane, A., 56,83(52) Yamaoka, N., 30 Yamashina, I., 256 Yamazaki, N., 160 Yanachkov, I., 117 Yanagishita, M., 244,248,256 Yang, D., 74 Yeates, R. A., 137, 164(171) Yen, D., 254,258(82) Yen, J. K.C., 102, 118(20) Yokota, K., 143, 161(191) Yokoyama, M., 30 Yokoyama, Y., 248 Yoshida, C., 168 Yoshida, H., 256 Yoshimura, J., 64 Yosizawa, Z., 247 Young, M. F., 254,258(75) Young, M. R., 254 Yu, W. C., 117, 118(75), I19 Yuasa, H., 31 Yurovska, M., 118

2

Zarif, L., 129 Zavalishina, A. I., 132 Zech, J. D., 136 Zen, S.,56, 83(52) Zerner, M. C., 70 Ziegler, T., 206 Ziemann, H., 119(91), 120 Zipursky, S. L., 254,258(82) Zoorob, H. H., 160 Zuccaro, P.,146 Zuccaro, S. M.,146

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SUBJECT INDEX Azides, 1,43,6-dianhydmhexitols,I54

A ( lS,2S,3R,4S,5S,7R)- 1-Acetoxy-2,3,4-tetra-

~benzoyloxy-7-bromo-6sxabicyB clo[3.2. lloctane, synthesis, 65 N-Acetylmannosamine 2,4-O-Benzylidene-l,6-dichloro-1,6-difunctional derivatives, 195, 197-199 deoxy-D-glucitol, 158- 159 synthesis of derivatives, 20 1- 203 Bis-( 1,43,6dianhydro-~-rnnit010)-30N-Acetylneuraminicacid crown-10, 107- I08 biosynthesis, 194- 195 Bis(2,4dinitrophenylhydrazone), 156 synthesis, 200 m k ,RezsB, 3-9 1-0-AcetyI-2,3,5,6-tetra-O-benzoyl-4academic career, 4 bromO-gD-galactOse, synthesis, 49 antibiotic research, 5-6 I -O-Acetyl-2,3,4-tn-o-benzoyl-4-fluoro~~ C-nucleoside synthesis, 8 ribose, synthesis, 80- 8 1 glycosylamine research, 6 - 7 Acylation, isosorbide, 126- 127 honors, 8 Agarose, immobilization on, I8 1 - 186 reaction of cr,adihalo ethers, 7 - 8 Aldohexoses, 25-26 research on tlavonoid compounds and Aldol reaction,carbohydrates, 189- 190 carbohydrates, 5 Aldopentoses, 25-26 Branched-chain sugars, in solution, 30-31 Aldoses, in aqueous solution, 25-26 Brominations, see Radical-mediated broAldotetroses, 26 minations Alkyl ethers, 1,43,6-dianhydrohexitols, I-Brorno-D-glycosyl cyanides, synthesis, 58 135-145 Amino acids PG-I C composition, 248 Carbohydrate-protein linkage regions,proteNH,-terminal sequences, 253 PG-I1 oglycans, 242 - 243 Carbon radical stabilization Eactors, radicalcomposition, 244-247 NH,-terminal sequences, 252 mediated brominations, 70-7 1 sequence analysis, proteoglycans, 254- 255 Carboxylic acid, 1,4:3,6dianhydrohexitls 3-Amin~2-hydmxypropyl-substituted esters, 125-130 oxime ethers, 164 C-C bond-forming reactions, see Enzymic Amino sugars, in solution, 29- 30,34 methods I ,6-Anhydro-2-O-benzoyl-3,4-O-iwpropyli- Chiroptical properties, 1,43,64anhydrodene-/?-D-galactose, photobromination, hexitols, 99- 100 53-54 'T-N.m.r. spectra, 1,43,6-dianhydmhexitols, 109- 11 1 1,6-Anhydrohexopyranosederivatives, radiCollagen fibrils, proteoglycans role in orgacal-mediated brominations, 5 1 -54 nization, 258-259 1,5-Anhydmpentohranose derivatives, radiCosmetics, I ,4:3,6-dianhydrohexitolsapplical-mediated brominations, 54 Antitumor agents, isohexides, 165- 166 cations, 168 Aryloxypropanolamines,8-blocker sideCourtois, Jean Emile, 1 1 - 18 chain, 162 academic career, 11- 12 ATP regeneration, sugar phosphates, 208 archeological work, 17 I 8 210 glycosidase research, 14- 15

-

279

SUBJECT INDEX

280

glycosidases and glycanasesfrom xylophagicinsects, 15-16 honors and distinctions, 17 international organizations, 1 6- 17 periodic acid oxidation research, I2- 1 3 plant oligosaccharide research, 13- 14 role in Societk de Chimie Biologique, 16 Cytidine monophosphate N-acetylneuraminic acid enzymic synthesis, 21 5 -21 6 reaction catalyzed by, 2I4- 21 5 Cytidine triphosphate, enzymic preparation,

211,213

D 1 1 -Deoxy-8-epi- 1 1 -oxaprostaglandinFa, 159 3-Deoxy-~-arabino-2-heptulosonic acid 7phosphate, 206-207 3-DeOXy-D-gUfaCto-nOUulOsoniC acid, 202204 3-Deoxy-D-manno-2-octuctulosonic acid 8phosphate, synthesis, 204,206 ~ - D ~ o x ~ P ~ u27 ~os~s, Dialysis bags, immobilization in, 188

1,4:3,6-Dianhydro-2,5-O-benzoyl-2,5dithio-L-iditol, 153 1,43,6-Dianhydro-~-glucitol, 96-97,1 17 1,43,6-Dianhydro-2,5dideoxy-2,5-(dithiocyano)-L-iditol, 153 2,3:4,5-Dianhydro-~-iditol, 124- 125 1,4:3,6-Dianhydro-~-mannitol,96 1,4:3,6-Dianhydro-~-mannopyranose, 158 1,43,6-Dianhydrohexitols,93- 173 dkyl ethers, 135-145 j?-blocker side-chain, 137 bis-glycidyl ethers, 136 crown ethers, 143- 144 2,5diGethylisohexides, 141 isohexide etherihtion, I39 isosorbide 5-nitrate, 142 oxaprostaglandins, 138 pentafluorophenyl ethers, 141- 142 analytical behavior, detection, and determination, 117- 119 Chemical Abstracts references, 94 chemical uses, 158-161 cosmetics use, 168

deoxy derivatives amines, 150- 152 azides, 154 C-nitro compounds, 155 halogens, 149- 150 mono- and di-substituted, 146- 149 oxidation products, 155 - 158 phosphanes, 155 thio derivatives, 153 esters with carboxylic and sulfonic acids,

125-130 of nitric acid, 133- 135 with phosphoric acid, 130- 132 ethoxides, 171 food applications, 167- I68 as herbicides, 170 nomenclature, 96-98 bridged systems, 97-98 fused systems, 98 sugar-derived names, 96- 97 parent compound preparation, 119- 125

2,3:4,5dianhydro-~-iditol, 124- 125 isohexides, 1 22- I24 protonation, 120 ( 1R)-[1 -ZH]isomannide,1 20- 122 pharmaceutical uses, 161 - 167 antitumor agents, 165- 166 aryloxy propanolamines, I62 di-0-methylisosorbide, 166 isohexide nicotinic esters, 162 isohexide nitric esters, 16 1 - 162 isosorbide dinitrate, 16 1 isosorbide disulfite, I64- 165 isosorbide 5-mononitrate, 16 1 isosorbide sydnonimine derivatives, 163 somidipine, 163 as plasticizers, 168- 169 polymers containing isohexide moieties,

171-173 polyurethanes, polycarbonates and polyamides, 172- 173 preparation survey, 95 silyl ethers, 145 146 spectroscopic properties, 99- I 14 chiroptical properties, 99- 100 infrared spectra, 100- 102 mass spectra, 110, 112- 114 n.m.r. spectra, 102- 1 1 1 ultraviolet spectra, 99- 100

-

SUBJECT INDEX structural aspects, 114- 117 1,43,6-Dianhydro-2-S-benzoyI-5-0-methylsulfonyl-2-thio-~-glucitol, I53 1,4:3,6-Dianhydro-2-~brornophenylsulfOnyl)-D-glUcitOl 5-nitrate, 1 14-1 15 1,43,6-Dianhydro-~-iditol, 96 2,5-Diazido-2,5-dideoxyisohexides, 10 1, 104- I05 hypnotic properties, 165 1 ~-Dibenzoyl-2’,3’,5’-tri-O-benzoyl-4’-bromoadenosine, synthesis, 65 2,43,5-Di-0-benzylidene-1,6-dichloro-l,6dideoxy-D-glucitol, 158 1 ,CDichlor~1,6diideo~y-~-gl~cit0l,I58 159 Dichloro-L-isoidide, 149 2,5-Diideo~y-2,5-diiodo-Dglucitol 149- 150 2,5-Dideoxy-2,5aiiodo-~-i~tol, 149- I50 2,5-Di-O-ethylisohexides,141 1,2-Diideo~y-3-he~t~lo~e~, 28 5,7-Dideoxy-~-xylo-heptulose, synthesis, 194 Dideoxyisohexide C-nitro compounds, 155 Differential scanning calorimetry, 1,43,6dianhydrohexitols, 1 19 5-[1,4-Dihydro-3-(rnethoxycarbonyl)-2,6-dimethyl-4-(2-nitrophenyl>5-pyridylcarbonyllisosorbide, 1 15 - 1 I6 1,3-Dihydroxyacetonephosphate, reactions with aldolase, 192 Di-0-methylisosorbide, 140, 166 (RJ)-cis-2,6-Dioxabicyclo[3.3.O]octane, 147- I48 2,6-Dioxabicyclo[3,3,9]octaneframework, 97 DS-GAG chain, 242-243

28 1

glycosylations with transferases, see Glycosylation immobilization, 180- 189 agarose, 181-186 dialysisbags, 188 poly(acry1amide) gels, 186- 188 silica gel-glutaraldehyde, 188- 189 interest in, 176- 177 in organic solvents, 235-236 phosphorylations, 207-218 nucleotides, 210-213 “nucleotide-sugars”, 213 -218 sugar phosphates, 207-210,212 syntheses in aqueous solution, 234-235 transfer reactions, catalyzed by glycosi-

dases,231-233 Ethoxides, 1,43,6-dianhydrohexitols,171 Ethyl tetra-0-acetyl-a-bidopyranoside, synthesis, 76

F Food, 1,43,6-dianhydrohexitolsapplications, 167- 168 D-FIU~~OS~ from ~~-2,3,dihydroxypropnal, 193- 194 D-glucose conversion, 180 D - F I U ~ ~1,6-bisphosphate, OS~ reactions with aldolase, 192 Furanose derivatives, radical-mediated brominations, 49- 5 1 Fused rings,sugars, in solution, 3 1

C

E Elimination reactions,radical-mediated brominations, 85-91 Enzymic methods, 175-237 C-C bond-forming reactions, 189-207 aldol reaction, 189- 190 miscellaneous reactions, 205 syntheses with glycolysis aldolase, 190-

194 syntheses with sialyl aldolase, 194-204 tnlnsketolase, 204 207 definitions and abbreviations, 177

-

Galactosylation, with transferases, 219- 224 Galactosyltransferase, 220- 221 Gas-liquid chromatography I ,4:3,6dianhydrohexitols,1 18 sugars in solution, 21 -22 &D-Glucopyranosides, synthesis, 74

D-Glucose conversion into mhctose, 180 methyl ethers, 29 Glycerol kinase, S.cerevisiae, 208 N-Glycolylneuraminic acid, synthesis, 201 Glycolysis aldolase,syntheses, 190- 194 Glycopeptide, synthesis, 221

SUBJECT MDEX

282

Glycosaminoglycans composition, 24 1 Structure, 24 1-242 di~aCCharideunits, 240 24 1 synthesis, 256 G1ycosidases pyranosyl transfer with,232-233 transfer reactions catalyzed by, 231 -233 Glycosulose derivatives, radical-mediated brominations, 54 -57 Glycosylations, with transferases, 2 18-23 1 galactosylation, 2 19- 224 glycosylation, 23 1 sialylation, 223-23 1 C-Glycosylbenzeneesters, radical-mediated brominations, 59-60 Glycosyl cyanide esters, radical-mediated brominations, 57- 59 Glycosyl halide esters, radical-mediated brominations, 60 -6 1 C-Glycosylheterocycleesters, radical-mediated brominations, 59-60 Glyculose derivatives, radical-mediated brominations, 54 - 57

-

H Halogenation, 1,43,6-dianhydrohexitok, 149- 150 'H chemical shifts, isohexide derivative ring system, 108- 109 Heptasaccharide, synthesis, 228- 229 Heptuloses, 28 Herbicides, 1,43,6-diauhydrohexitok as, 170 Hexolrinase, immobilization, 186- 187 Hexopyranose esters, 5-bromides from, 48 Hexopyranoide esters, radical-mediated brominations, 62 - 64 Hexuronic acid derivatives 5-bromides from, 43-44 radical-mediated brominations, 42 -45 'H-n.m.r. spectra, 1,4:3,6-dianhydrohexitols, 102- 109 H.p.l.c., sugars in solution, 22 Hydrocarbon films, 1,43,64anhydrohexitols, 171 Hydrogen, substitution reactions,radicalmediated brominations, 75 -79 Hydrogen atom abstraction, radical-mediated brominations

regiochemistry, 67-68 stereochemistry, 7 1 - 72 3-Hydroxybutanal, condensation, 194 I

Idose, in solution, 31, 35 Infrared spectra, 1,4:3,6-dianhydrohexitols, loo- 102 Isohexide amino-substituted, 150- 15 1 derivatives 'H chemicalshifts, ringsystem, 108- 109 infrared data, 101 dialkyl, 140 esters, 129-130 etherification, 139 fragmentation, 1 12- 11 3 monoalkylated, I39 mono- and di-nitrates, 133- 134 nicotinic esters, 162- 163 phosphorus-substituted, 155 as plasticizers, 168- 169 preparation, 122- 124 proton coupling constants, 105 unsubstituted azido, 147 Isohexide mono- and di-amines, 165 Isohexide nitrates, 118 Isohexide nitric esters,vasodilation, 161- 162 ( 1R)1-%-Isomannide, 120- I22 Isosorbide, 126 acylation, 126 127 bis(tetramethy1phosphoroicdiamide), 132 diesters, cosmetics use, 168 disulfite, 164- 165 ethoxylated monoesters, 171 platinum-catalyzed oxidation, 155- 156 proton-proton coupling constants, 104 syndnonimine derivatives, 163 Isos~rbide2-acetate, 128 Isosorbide S-acylates, 128 Isosorbide di(docosanoate), 168 169 Isosorbide diheptanoate, 168- 169 Isosorbide dimethyl ether, 167 168 Isosorbide dinitrate, 1 17 pharmaceutids, 161 Isosorbide &(-oak), 168- 169 Isosorbide dipropanoate, 167- 168 Isosorbide mono(truns-docosenate),168,171 Isosorbide mononitrates

-

-

SUBJECT INDEX

‘H-n.m.r. data, 106 pharmaceuticals, 16I Isosorbide mono-oleate, 168- 169, 17I Isosorbide mono(tetradecanoate), 168 Isosorbide 2-nitrate, 134 Isosorbide 5-nitrate, 99, 117, 142 fragmentation, 1 I3 - I 14 Isosorbide phosphinite, monosubstituted, 132

283

N-terminal sequence, proteoglycans, 25 1 253 Nuclear magnetic resonance spectroscopy 1,4:3,6dianhydrohexitols,102- I10 sugars in solution, 20 -2 1 Nucleophilic substitutions, radical-mediated brominations, 79 - 84 Nucleotides, phosphorylation, 2 10-2 13 “Nucleotide-sugars”, phosphorylation, 2 13218

K Karplus relation, 104 Ketoses in aqueous solution, 28-29 preparations, 19 1 - 192

M &D-Mannopyranosides, synthesis, 82 D-M~UUOPYIZ~UOS~I radicals, 72-73 Mass spectra, 1,4:3,6dianhydrohexitols, 110, 112-114 Methyl 5-acetoxy-tetra-0-acetyla-L-idopyranuronate, synthesis, 79 - 80 Methyl fiisomaltoside, synthesis, 53 Methyl 4-0-(2-acetamido-2deoxy-8-D-mannopyranosyl)-a-D-glucopyranoside, synthesis, 83 S-Methyl-2-@etra-O-acetyl-I -bromo-fiD-galactopyranosy1)- I ,kxadiazole, synthesis, 59 Methyl(SR>tetra-O-acetyI-S-bromo-fiD-glucopyranuronate, synthesis, 43 Methyl tetra-0-acetyl-fiD-glucopyranuronate, synthesis, 87 Methyl tetra-0-acetyl-fiL-xylo-hexulopyranosonate, synthesis, 79-80 Methyl tri-0-acetyla-L-xyh-hexulopyranosylate bromide, synthesis, 42 -43 Methyl tetra-0-acetyla-L-idopyranuronate, photobromination, 45 Molecular-orbital calculations, pyranose forms of sugar in solution, 22-23 Monobenzoylated isohexides, fragmentation, 113

N Nitric acid, 1,43,6dianhydrohexitol esters, 133- 135

0

Oligosaccharides sialylations, 225 - 226 synthesis, 22 1 Oxaprostaglandins, I38 Oxidation products, 1,4:3,6dianhydrohexitols. 155-158

P PAN gels, cross-linked, 186- 187 Penta-0-acet yl-S-brorno-~~glucopyranose, synthesis, 45 - 49 Pentose, 5-0-substituted, 26 Pentose phosphates, preparation, 2 10 2-Pentuloses, 26, 34 Peracylated aldoses furanose derivatives, 49 - 5 1 pyranose derivatives, 45 - 49 radical-mediated brominations, 45 - 5 1 PG-I amino acid composition, 248 NH,-terminal amino acid sequences, 253 protein core amino acid sequences, 255 structure, 249 PG-I1 amino acid composition, 244- 247 NH,-terminal amino acid sequences, 252 protein core amino acid sequences, 255 structure, 250-251 Pharmaceuticals, 1,4:3,6dianhydrohexitols US, 161-167 Phenyl tetra-O-acetyla-L-idopyranoside, synthesis, 76 Phenyl 1-thiohexopyranosideesters, radicalmediated brominations, 64 -65 Phosphoric acid, 1,43,6-dianhydrohexitols esters, 130- I32

SUBJECT INDEX

284

Phosphorylations,enzymic methods, see Enzymic methods Poly(acry1amide) gels, immobilization on,

186-188 Polyamides, 1,4:3,6-dianhydrohexitols,

172- 173 Polycarbonates, 1,4:3,6-dianhydrohexitols,

172-173 Polyoxyethylene isosorbide, 136 Polyurethanes, 1,4:3,6-dianhydrohexitols,

172- 173 PrOteoglycanS, 239-259 amino acid sequence analysis, 254-255 biological roles, 257-259 biosynthesis explant culture, 257 primary culture, 254,256-257 carbohydrate-protein linkage regions,

242-243 isolation and fractionation, 243-244 M,values, 248-249 N-terminal sequence, 25 1-253 Proton coupling constants, isohexides, 105 Pyranose derivatives, radical-mediated brominations, 45-49 Pyranosyl transfer, with glycosidases, 232-

233

R Radical intermediate stabilization, radicalmediated brominations, 68- 71 Radical-mediated brominations, 37-9 1 1,6-anhydrohexopyranosederivatives,

51-54 1,s-anhydropentofuranosederivatives, 54 carbon radical stabilization factors, 70- 7 1 elimination reactions, 85-9 1 C-glycosylbenzene and C-glymsylheterocycle esters, 59-60 glycosyl cyanide esters, 57-59 glycosyl halide esters, 60- 61 glyculose and glycosulose derivatives, 5457 hexopyranoside esters, 62- 64 hexuronic acid derivatives, 42-45 hydroxyl group protection, 42 introduction at C-5,39 miscellaneous compounds, 65 peracylated aldoses, 45- 5 1

phenyl 1-thiohexopyranosideesters, 64-

65 reaction conditions and suitable compounds, 4 1 -42 regiochemistry, 67- 71 hydrogen atom abstraction, 67- 68 radical intermediate stabilization,68- 7I stereochemistty, 7 1- 75 hydrogen atom abstraction, 7 1- 72 products, 73-75 radical intermediate conformation, 72-

73 substitution reactions, 75-85 nucleophilic substitutions, 79- 84 radical reactions leading to, 84-85 substitution by hydrogen, 75- 79 Radical reactions, leading to substitutions, radical-mediated brominations, 84-85 Reducing sugars, in solution, 19- 35 aldohexoses and aldopentoses, 25-26,33 aldotetroses and related sugars, 26, 34 amino sugars, 29- 30,34 branched-chain sugars, 30- 31 furanose content in organicsolvents, 31,32 gas-liquid chromatography, 2 1 - 22 heptuloses, 28 hexuloses and pentuloses, 27-28 h.p.l.c., 22 n.m.r. spectroscopy, 20-21 partially O-SUbStitUted sugars, 28-29 relative stability aldehyde and keto forms, 24-25 composition vaxiation with temperature, 25 furanose form, 23-24 hydrated carbonyl forms, 25 pyranose form, 22-23 in solvents other than water, 31, 32,35 sugars having fused rings, 31 thio sugars, 30

Regiochemistry, radical-mediated brominati on^, 67- 71

S Sepharose, mechanism of activation, 181-

182 Sialic acids, naturally occumng, 195- 196,

200- 203

Sialosides,synthesis, 23 1

SUBJECT INDEX

285

Tetra-O-acetyl-5-bromo-~~-glucopyranosyl Sialyl aldolw, syntheses with, 194-204 chloride. synthesis, 6 1 N-acetylneuraminic acid, 194- 194 N-acetylmannosamine derivatives, 195, Tetra-0-acetyl-1-bromo-/h-glucopyranosyl 197- 199 fluoride, synthesis, 6 1 M t U d y OCCUhg *C acids, 195Tetra-O-aoetyl-5-bromo-/h-glucopy1anosyl fluoride, synthesis, 6 1 196,200-203 3deoxy-~-glycero-~-gagalact~nondosonic Tetra-Oacetyl-5-bromo-/h-xylopyranose, acid, 202 -204 47-48 elimination reactions,87 Sialylation, with transferases, 223, 225-23 I Tetra-Oacetyl-D-glucopyranosylradical, 72 heptasaccharide synthesis, 228 -229 immobilizedsialyltransfm, 225,2273Cretra-O-aoety1~-D-gl~PWWlW panonitrile, synthesis, 73- 74 228 sialoside synthesis, 230 -23 1 Tetra-O-benzoyl-2-bromo-P.glucono-13soluble transferases,225-226 lactone, synthesis, 62-63 tetrasaccharide-glycosidesynthesis, 228 2,3,4,6-Tetra-~benz0~1-5-by~~~-B-D-gl~trisaccharidesynthesis, 229 cose, synthesis, 79 Silica gel-glutataldehyde, immobilization Tetrasaccharide-glycoside,synthesis, 228 Thiabenzazole, antifungal activity, 166 on, 188-189 Silyl ethers, 1,4:3,6dianhydrohexitols,145- (E,E)-Thiacyclodeca-4,7-diene, 160 Thio sugars, in solution, 3 1 146 Sodium isosorbide 5-nitrate, 134- 135 Thorpe-Ingold e&ct, 24 L-Sorbose, from ~~-2,3,dihydroxypropanal, Transesterification,sugars, 235-236 193- 194 Transferases, glycosylations with, see GlycoSornidipine, 163 sylations Stereochemistry, radical-mediated brominaTransfer reactions,catalyzed by glycositions,7 1 -75 dase~,23 1-233 Substitution reactions,radical-mediated Transketolase, synthesis, 204-207 (6s?-2,3,4,Tri-O-acetyL1,6-anhydro-6brominations, 75 - 85 Sugar phosphates, 207 - 2 10,212 bromo-@-glucose, synthesis, 5 1 Tri-Gacetyl-1,5-anhydro-2deoxy-~-araenzymes for phosphorylation,208 ATF' regeneration, 208 - 2 10 bino-hex- lenitol, synthesis, 85 pentose phosphate preparation, 2 10 24Tri-0-acetyl-1-bromcwr-DarabinopyranSugars,See also Reducing sugars osyl)-5-(triauoromethyl)-1,3,4-oxadiatransesterification, 235- 236 zole, synthesis, 60 (SRtTn-O-acetyl-5-bromo-1-thio-fiD-gluSulfonic acid, 1,43,6dianhydrohexitols esters, 125-130 copyranosid)uronate,40 2,4,6-Tn-O-acetyl-I-thio-~-e~f~ro-hex1-enopyranosid-3-ulose, 39 1,42,5:3,6-Trianhydro-~-isomannide, 116 T Tri-Obenzoyl-5-bromo-6dmxy-gL-xyloTetra-0-awl- 1,5-anhydro-~-arabinoexhex-4-ulopyranose, synthesis, 55 lenitol, synthesis, 85 2,4,6-Tri-O-benzoyl-3deoxy-psrythro-hex2-(Tetra-&mtyl- l-bmmo-j3-D-galactopy2enono-1,5-lactone, synthesis, 90-91 ranosyl)benzothiazole, synthesis, 60 Tri-O-benzoyl-cu-~-arabino-hex-2-ulopyranTetra-0-acetyl-l-bromc+D-glucopyranosyl osyi bromide, synthesis, 55 Tributylstannane, 76-77 chloride elimination reactions, 89-90 Tnose phosphate isomerase, equilibrium catalyzed by, 191 synthesis, 6 1 ~ i & , 221,229,236-237 T e t r a - O - ~ l - l - b r o ~ o - j 3 - ~ U ~ p ~ oTsryi l~ ~ ~ h a synthesis, Turanose, 31 cyanide, synthesis, 58

SUBJECT INDEX

286 U

Ultraviolet spectra, 1,43,6dianhydmhexitols, 99- 100 Uridine diphosphate glucose, preparation, 213

V Vasodilation, isohexide nitric esters, 161 162 Vicinal diol, D-threo configuration, 19 1