Advances in Carbohydrate Chemistry, Volume 21

ADVANCES IN CARBOHYDRATE CHEMISTRY VOLUME 21 Advances in Carbohydrate Chemistry Editor MELVILLE L. WOLFROM Associate...

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ADVANCES IN CARBOHYDRATE CHEMISTRY

VOLUME 21

Advances in Carbohydrate Chemistry Editor MELVILLE L. WOLFROM Associate Editor R. STUART TIPSON Board of Advisors W. W. PIOMAN

It. C. HOCKET~

ROYL. WHISTLEB

Board of Advisors for the British Isles SIB EDMIIND H I ~ T

STANLEY PEAT

MAURICE STACEY

1966

Volume 21

ACADEMIC PRESS REPLICA REPRINT

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or Haroourt Breoe Jovanovloh. Publlehere

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This is an Academic Press Replica Reprint reproduced directly from the pages of a title for which type, plates, or film no longer exist. Although not up to the standards of the original, this method of reproduction makes it possible to provide copies of books which otherwise would be out of print.

PRINTBD IN THE UNITED STATES OF AMERICA

8182

9 8 7 6 5 4 3 2

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the Authors’ contributions begin.

A. R. ARCHIBALD, Microbiological Chemistry Research Laboratory, Department of Organic Chemistry, University of Newcaslle uponTyne, England (323)

J. BADDILEY, Microbiological Chemistry Research Laboratory, Department

of Orgunic Chemistry, University of Newcastle upon Tyne, England (323)

K. VENKATRAMANA BHAT,Department of Chemistry, Georgetown University, Washington, D. C. (273) 0. S. CHIZHOV, Institute for Chemistry of Natural Products, Academy of SdenCeS, MO~COW, U. S. S. R. (39) KARLFREUDENBERG, 6800 Heidelberg, Wilckensstrasse 34, Germany (1) I . J. GOLDSTEIN, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan (431)

JOHNW. GREEN,The Institute of Paper Chemistry, Appleton, Wisconsin (95)

STEPHENHANEBSIAN, Research Laboratories, Parke, Davis & Company, Ann Arbor, Michigan (143) H. W. HILTON,Experiment Station, Hawaiian Sugar Planters’ Association, Honolulu, Hawaii (377) 0

T. L. HULLAR,Department of Medicinal Chemistry, School of P h a m c y , State University of New York at Buflalo, Bufalo, New York (431)

N . K. KOCHETKOV, Institute for Chemistry of Natural Products, Academy of Sciences, Moscow, U . S. S. R. (39) J. A. REINDLEMAN, JR., Northern Regional Research Laboratory, Northern Utilization Research and Development Division, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois (209) W. WERNERZORBACH, Department of Chemistry, Georgetown University, Washington, D. C. (273) V

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PREFACE The editors herewith present the twenty-first volume in this serial publication. To celebrate our “coming of age,” we are proud to offer a review of the contributions of Emil Fischer to carbohydrate chemistry, by one of his students, Professor Karl Freudenberg. In translation, some of the fine expression and style of the original German may have been lost, yet the review is nevertheless an outstanding evaluation of Fischer’s contributions to the fundamentals of modern carbohydrate chemistry. Current organic chemistry is ever utilizing new instrumentations and techniques that have originated in physics and have been perfected by the modern instrument fabricator. The newest instrument to impinge upon the carbohydrate field is the mass spectrometer, and a review of current work on its use in this area is made by Kochetkov and Chizhov (Moscow). This volume contains two chapters which update topics presented in earlier onea. The chemistry of the deoxy sugars has been expanded considerably since the review in Volume 8 by Overend and Stacey (Birmingham), as is attested by the chapter by Hanessian (Ann Arbor). The article on synthetic cardenolides, or cardiac glycosides, by Zorbach and Bhat (Georgetown) is an extension of related topics previously reviewed by Elderfield (Volume 1) and by Reichstein and Weiss (Volume 17). The discussion of chemical synthesis of polysaccharides, by Goldstein and Hullar, is another contribution to those chapters on carbohydrate polymers which have appeared in this serial publication. Green (Appleton) reviews the generally neglected topic of glycofuranosides. Inorganic chemistry is included in the chapter on complexes of alkali metals and alkalineearth metals with carbohydrates by Rendleman (Peoria). Finally, two topics in biochemistry are reviewed by Archibald and Baddiley (Newcastle) and by Hilton (Honolulu), the former being concerned with the teichoic acids, and the latter with the effectsof plant-growth substances on carbohydrate systemd in plants. The Subject Index for this as well as for the preceding volume has been prepared by Dr. L. T. Capell, long associated with Chemical Abstracts and an internationally recognized authority on organic nomenclature. ColumbuR, Ohio Gaithersburg, Md. Nowmber, 1866

M. L. WOLFROM R. STUARTTIPBON

vii

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CONTENTS LIST OF CONTRIBUTOR^ .................................................... PREFACE .................................................................

v vii

Emil Fiacher and Hie Contribution to Carbohydrate Chemistry

KARLFREUDENBERQ I . Introduction ........................................................ I1 Emil Fischer and His Scientific I11 The System of the Monoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I V . Development and Extension of V . Ex+uensionof the System, and Tr ......... V I . Oligo- and Poly-saccharides.... ......... V I I StericSeri es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Insights into Stereochemistry and Its Relation to Biochemistry. . . . . . . . . . . IX . GeneralReferencea ..................................................

.

2

.

8

.

27 32 34

16

38

MMSSpectrometry of Carbohydrate Derivatives

.

.

. .

N K . KOCH~TXOV AND 0 S CHIZHOV

I Introduction ........................................................ I1. The Basic Principles of Mass Spectrometry of Organic Compounds . . . . . . . . I11. M w Spectra of Carbohydrate Derivatives ............................. IV Conclusion.......................................................... Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

39 40 46 92

93

The Glycofuranosides

.

JOHN W GREEN

. .

I Introduction ........................................................ I1. Conformation of the Glycofuranosides................................. 111 Formation of Glycofuranosides in Acidic Methanol . . . . . . . . . . . . . . . . . . . . . . IV. Preparation of Glycofuranosidea from Dithioacetals ...................... V . General Preparative Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Structure of Glycofuranosidea. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

95

96 101 112 121 127 137 140

Deoxy Sugus STEPHEN HANESSIAN

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

ix

143 144

CONTENT8

X

I11. Monodeoxy Sugars .................................................. I V Dideoxy Sugars ..................................................... V . Trideoxyhexwes..................................................... VI . Chromstog;raphy.................................................... V I I . Nuclear Magnetic Resonance Spectroscopy............................. VIII MeesSpectrometry., ................................................

.

.

145 183 196 197 201 201

Complexes of Alkali Metals and Alkaline-earth Metals with Carbohydrates

.

.

J. A RENDLEMAN. JR

. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 . Complexes of Carbohydrates with Metal Salk . . . . . . . . . . . . . . . . . . . . . . . . . .211 . Complexes from the Interactioniof Carbohydrates with Metal Bases. . . . . . 237 . Alcohohtea from Reactions, in Liquid Ammonia, of Carbohydrates with

I I1 I11 IV

Alkali Metale. Alkalinwarth Metals. and Alkali Metal Amides . . . . . . . . . 269 Synthetic Cardenolides

w . WERNER

zORBACH

AND

K

.

VENKATBAMANA

BUT'

. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !273 . General Methodology Employed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 . Synthesis of Individual Glycosides..................................... 281 . Contribution of the Carbohydrate Component to Physiological Activity . . . . 311 . Table of Synthetic Cardenolidee............... . . . . . . . . . . . . . . . . . . . . . . . . 318

I I1 I11 IV V

The Teichoic Acids

.

.

A R . ARCHIBALD AND J BADDILEY

I . Introduction ........................................................ 323 I1. Surface Structurm of Qram-positive Bacteria ........................... 324 IIX, Discovery of the Teichoic Acids ....................................... 326 I V The Hydrolysis of Eaters of Phosphoric Acid ............................ 328 V Membrane Teichoio Acids ............................................ 332 V I Wall Teichoic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 VII Teiohoic Acids of Actinomycetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 VIII . The Looation of Teichoic Acida in Relation to Cell Structure . . . . . . . . . . . . . . 365 372 IX . Biosynthesis........................................................

. .

. . .

The Effects of Plant-growth Substances on Carbahydrrte Systems

. .

H W HILTON

I. Introduction ........................................................ I1. Indole-3-acetic Acid and 1-Naphthaleneacetic Acid. ..................... I11 Plantgrowth Substances Used aa Herbicides ...........................

.

377 381 392

xi

CONTENTS

IV. Glycosidea and Other Carbohydrate Derivatives as Plant-growth Substances V. Gibberellins and Kinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI The Effects of Plant-growth Subtenca on Sugarcane.................... VII . A b h i o n a n d nipening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

408 416 421 429

Chemical Synthesis of Polysaccharides

.

I . J . GOLDSTEIN AND T. L HULLAR I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Condensation Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Addition Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . IV Methods of Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Applications of Synthetic Polysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

AUTHORINDEXFOR VOLUME 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VOLUME 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CUMULATIVE AUTHOR INDEX FOR VOLUMES 1-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CUMULATIVE SUBJECT INDEX FOR VOLUMES 1-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EFLRATA ................................................................. STJBJsCT INDEX FOR

431 434 477 491 507 513 538 655 562 572

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EMIL FISCHER AND HIS CONTRIBUTION TO CARBOHYDRATE CHEMISTRY* BY KARLFREUDENBERG Heidelberg, Gennany

DEDICATED TO THE MEMORY OF MAXBERGMANN (1886-1944) I. Introduction. ....................................................... 11. Emil Fischer and His Scientific Work. ................................. ................. 111. The System of the Monoees.. .................I. . IV. Development and Extension of the System.. ........................... 1. Beginnings: The Sugars and Phenylhydrazine. . . . . . . . . . . . . . . . . . 2. Additional Methodical Approaches. ........................... 3. Synthesis of Glucose (1890). ....................................... 4. The Configuration of Glucose.. ..................................... 5. The Configuration of Galactose. .................................... V. Extension of the System, and Transformations of the Sugars.. . . . . . . . . . . . . 1. General........................... ... 2. AminoSugere .................................................... 3. Glucal and Deoxy Sugars.. ..................... 4. Functional Derivatives and Their Application. . 5. Glycosides.. ..................................................... 6. Nucleosides and Other N-Glycosyl Compounds. . . . . . . . . . . . 7. Acyl Derivatives; Acyl M VI. Oligo- and Poly-saccharides. . . . . . . .................... 1. General ........................................................... 2. Di- and Tri-saccharides. . VII. Skric Series. ....................................................... VIII. Insights into StereochemiRtryand Its Relation to Biochemistry, . . . . . . . . . . IX. General References. .................................................

* Translated

2 2 8 10

12 13

15 16

17 21

27 32 34 38

from the German by Gerhart Schwab, with the assistance of Edward

W.Koos, Dr. J. M. Harkin, and the editors. The frontispiece is from a hitherto unpub-

lished portrait, the original of which ie to be found in the Institute of Organic Chemistry of the University of Erlangen, Germany. In this biographical notice, we shall maintain, in English translation, some of the nomenclature employed by Emil Fiwher. The names used by Fischer sufficed to meet the needs of the structures as then known, and have served as a solid basis for modern carbohydrate nomenclature, which, however, requires the definition of structural features unknown to Fischer.

2

KARL FREUDENBERQ

I. INTRODUCTION Emil Fischer himself collected, and edited, the greater part of his own work in the carbohydrate field in hie well-known book “Untersuchungen uber Kohlenhydrate und Fermente.” This volume was completed in 1908. Subsequent *ark was compiled under the same title by Max Bergmann, Fischer’s student and faithful assistant; this second volume was published in 1922. Both volumes contain a list of the titles and places of journal publication of his papers, as well as a subject index to them. The present author accordingly considers it unnecessary to cite references here. In a few instances, work is described which appeared subsequent to 1922.

11. EMILFISCHER AND HIS SCIENTIFIC WORK When Emil Fischer died in August, 1919, at the age of 67 years, he bequeathed a lifework of rare comprehensiveness. He had contributed greatly to our knowledge of the material world, and especially to that of organisms. He refocused the thinking in organic chemistry back to its starting point, the world of animated Nature. Emil Fischer was born on October 9th, 1852, and grew up in Euakirchen, near Cologne, Germany. His father was a highly successful business man, and was versatile, full of vitality and practical wisdom, and the posseasor of a cheerful outlook on life. His mother came from the Poensgen family, and was the calmer, more contemplative partner in the happy marriage; her son,inherited much of her nature. From his father, he learned to persevere in working toward his goals, to make quick decisions, and grasp good opportunities. His father first apprenticed him in the timber business, but this experiment failed because of the young apprentice’s antipathy toward this career and because of his persistent illnessdue to a chronic stomach disorder. At long last, Emil waa allowed to study. His first semesters in KekulcYs laboratory in Bonn did not satisfy the ambitious, young chemistry student. In the Fall of 1872, he transferred to Adolf Baeyer and F. Rose in Strassburg. In spite of having these excellent teachers, here as well as in Bonn, he was depressed by the abundance of disorganized material which he despaired of mastering, as he once told me in later years. The clear organization of physics, this older, more mathematical, and already better formed sister science, m taught by Kundt, made him hesitate as to which of the two discipline8 he should choose. But Baeyer won. Fischer’s doctoral work, too, was not without those crises which are often of benefit and which tend to beset primarily the seriouR student. Baeyer’s established ability to evaluate the personalities of young men-he himself possessed a magnificent, harmonious personality-caused him to recognize the worth of the young doctor. He entrusted to him the instruction of the student laboratory

EMIL FISCHER AND HIS SCIENTIFIC WORK

3

course in organic chemistry. Here, in an attempt to save a student’s unsuccessful experiment, Emil Fischer made his first discovery-phenylhydrazine. It was with a thesis on the substituted hydrazines that he qualified in 1878 for the rank of Privatdozent in Munich, whence he had followed his teacher Baeyer. In 1882, at the age of 30 years, he became full Professor and Director of the Chemical Institute at the University of Erlangen; a t the age of 33, Professor in Wtirzburg; and, in 1892, when 40 years old, Professor in Berlin. Very early, he developed his characteristic manner of working. He worked with many selected coworkers, subordinating each individual problem to the clearly envisaged general goal, and, thus, worked out the whole. Adolf von Baeyer accomplished his excellent work with fewer coworkers, whom he used to retain for many years on end; Baeyer might be compared to the leader of a reconnaissance patrol with a trained eye for the terrain and the possibilities of the pathways. In contrast, Emil Fischer was the clever tactician who proceeded on a broad front, here gaining ground very quickly, there lagging back cautiously, until that which lay behind was all in his safe possession. He knew how to make good use of the great advantages of the research program required for doctoral candidates at German universities. Teamwork, as he developed. it, demands a superior leader. It was magnificent to work under his direction and yet independently; as a research instructor, he blazed the trail for the generations succeeding him. Nevertheless, obstacles that slowed down his work arose at times. Chronic phenylhydrazine poisoning had already troubled him during his years in Erlangen. This he overcame, but it reappeared at intervals in Wurzburg and, later, in Berlin. In Wursburg, the need for a new institute caused him much extra work. For him, the practical man, the planning was easy; but he had to fight hard to obtain the requisite funds. This challenge presented new and time-consuming problems, but these were successfully mastered by the efficient businessman’s eon. Later, in Berlin, the same Ptruggle was repeated, but again he emerged the victor. In his construction of them chemistry institutes, too, he again set an example for his successors to follow. His years in Wiirzburg, although full of work and struggle, were nevertheless the happiest of his life. There, in Wurzburg, he married his beloved wife, Agnes Gerlach, who bore him three sons. In the summer of 1892, although reluctant to abandon Wiirzburg, of which he had grown so fond, he accepted an appointment to the greatest professional chair that Kaiser Wilhelm’s Reich had to offer, the chair of chemistry in Berlin, vacated by the death of August Wilhelm von Hofmann. Althoff, the director of the Universities Section in the Prussian Ministry of Education, promised the now forty-year-old Fischer a large new Institute,

4

KARL FREUDENBERG

However, it waR only after much controversy that the latter wai occupied eight yearn later. Out of this conflict with Althoff grew mutual respect and friendship. The Inetitute waR magnificently designed for the standards of that time. It was destroyed during World War 11, but was rebuilt essentially in its original form; and the design of the Institute is, today, still a practical one. Emil Fischer preferred to think synthetically, rather than analytically, Unlike 80 many of his contemporaries, he never gave unbridled rein to his synthetic efforts, nor did Be fall into the temptation of purposeless synthesis. He always remained a true scientist-a student of Nature. He thought in broad correlations, and applied his skill to fundamental problems. Whereas it was said, with some exaggeration, that the congenial Victor Meyer had work done on a different subject on each individual laboratory bench, Emil Fischer proceeded systematically toward distant goals. Theoretical questions played a minor role in his thoughts, although he proved to be a great scientific thinker when he systematically developed and consolidated the sugar series. There were many who were better read than he, but no-one who had more practical experience. He loved the intimate conversation, and I remember many a blissful hour, when he returned to the laboratory at night and sat down to chat, or when he interrupted our writing work so that we might have a simple meal in his villa on the Wannsee. Still clear to me are the memories, of the simplicity of this great man,his respect for nature, and his modesty in his efforts to understand it. The illness and early death of his wife, and the loss of two sons in the First World War, saddened the life of the now lonely man. Yet he lived to see the beginning of the successful Rcientific career of his son Hermann.1 He mastered the administrative burdens of his large Institute with typical competence. Until about 1910, he gave the introductory lectures in inorganic chemistry, with many demonstration experiments. Fischer’s lectures were distinguished by harmony between the spoken word and the experiment, and between objective earnestness and stimulating perfect diction. About that time, 1910, his duties became nearly overwhelming. I once heard him remark-although this statement wa.s not intended to be taken literally-“the move to Berlin was the mistake of my life.” The plan for the “Kaiser-Wilhelm Gesellschaft” matured. The idea was created by Fischer together with his fricnd Adolf Harnack. Fischer was the adviser to the minister Althoff, and to his successor, on questions in the area of chemistry. When World War I began, Fischer was one of tho first to point out, tho t,otdly ~nndoqunt,ceconomic! prepnrstion of Germany. He brought

EMIL FISCHER AND HIS SCIENTIFIC WORK

5

the industrial leaders of the Ruhr district together, and showed them the seriousness of the situation. Extended travel and the chairmanship of committees on the economics of the war were too much for him. When I talked to him in the spring of 1916, during a military furlough of mine, he was aware of the impending, unfortunate outcome of the war. Knowing what ww going to occur, he lived to see the ddbbcle, and he understood the consequences for his countrymen. Nevertheless, he continued his studies with the little group of students who were left-among them, Max Bergmann-until, in August, 1919, the old ailment reappeared and brought the end. Even such a talented life of investigation has its high-lights and shadows. Periods of impetuous progress were followed by years of moderate advances, for example, at the end of the first decade of this century, when his work on the proteins came to a stop because of experimental difficulties insurmountable at that time. It is greatness, too, to bear such periods of lesser productivity calmly and without becoming disconcerted. Emil Fischer’s life wm based on responsibility: responsibility for the austerity and purity of his work and its aims-responsibility for the university as an important organ of our cultural and economic life-and responsibility for each of his students, who always found him ready for discussion and consultation, whenever a question arose concerning their professional goals. He was a civil servant, but no regulations could command the performance of duties he imposed upon himself and on which his life waa based. His influence far exceeded his scientific work and its material consequences. This great individual was a man of inflexible veracity and simplicity, fully devoted to his mission and, withal, a sensitive and shy personality, respectful but commanding respect. Only from such a man can arise the great influences which I am trying to describe. When Justus Liebig’s university studies began about 1820, only irregular courses were available. When Robert Bunsen first went to a university in 1830, he found somewhat more organization in what was presented to him. Emil Fischer registered at the University of Bonn in 1871, when he was nineteen years of age, and in contrast to Liebig and Bunsen, was confronted with chemistry as a clearly defined science, offered in well-ordered courses. This is why Emil Fischer was more influenced by the school which molded him, especially since Adolf Baeyer soon became his teacher. Fischer’s Ph.D. research was carricd out at Stramburg, on fluorescein, and brought him close to the rapidly Moomirig chcmixtry of dyes. Although the great tradition of irivcstigtttion on uric acid niid related compounds had led from .J. Lichig arid F. Wijhler through A. Rwyer to A. Strecker, Baeyer’s group was Atill well familiar with this ficld, and this apparently produced the stimulation which brought about Fischer’s decision, in 1881, to work on

6

KAHL FREUDENBERG

the purines, as he later called them. All other topics which he took up and studied, intensively originated from his own initiative. For ten years, he remained within AdoH Baeyer’s sphere of action. Many a scientist in such an environment becomes dependent on his excellent teacher and cannot fight his way to independence.It was quite the contrary with Emil Fischer. He had discovered phenylhydrazine on his own in his early years, and his thorough study of its reactions led to work on indole; his return later to the ttiphenylmethane dyes was also influenced by the discovery of phenylhydrazine. His first work on sugars, which appeared in 1884, likewise resulted from his studies on phenylhydrazine and the osazones formed by it. Here he was in new territory into which only few had ventured before him, The sugar investigations were continued in Wbzburg, and later in Berlin, with typical energy. It was not a large step from the carbohydrates, their stereochemistry, and the enzymes acting on them, to the amino acids and proteins. It is understandable that the creator of carbohydrate chemistry desired to execute equally good work with the proteins, which presented new tasks for the practised stereochemist. A chance observation on tyrosine led down a narrow path to the phenolic carboxylic acids and thence to the gallotannins, where he encountered the sugars again. One guiding principle behind all his larger projects was the aasumption of repeated interlinking of bifunctional molecules. Thie assumption was realized in the polysaccharides, the proteins, and, to some extent, the tannins. In synthesizing the galloylgallic acids, he encountered the phenomenon of acyl migration, and from this m s e a plan to investigate the fats. Only a few publications on this topic had appeared when he died. When the main fundamental work in one field was done, he moved on to a new one. Only the carbohydrates and their reciprocal relations to the enzymes, as well as the resulting stereochemical problems, fascinated him even to his last days. The greater part of Emil Fischer’s enormous work was directed to entire groups of natural compounds, which he treated fundamentally and, for his time, most thoroughly. The finding that phenylhydrazine forms readily isolable, characteristic compounds with the sugars led him into the sugar field, which had until then been confused and which confronted experimenters with unusually difficult prwtical problems. He discovered the connections between the many kinds of sugars and discovered new ones, using phenylhydrazine again and again, but also applying other methods. He utilized the stereochemistry of the sugars to develop the theory of Van’t Hoff and Le Be1 on the spatial arrangement of atoms, and this led to unanticipated consequences. Here he proved himmlf to be a scientific thinker of the highest rank. In nightly walks through old Wlirzburg, he pondered over the laboratory work of the day, creating and classifying spatial conceptions. A new

EMIL FISCHER AND HIS SCIENTIFIC WORK

7

phase in the chemical observation of Nature was beginning. A new leader was arising to replace the aging Baeyer. In Berlin, the work on the purines was brought to a close, and that on the sugars was continued; indeed, Fischer carried it on throughout his 27 years in Berlin. At the same6:time,he turned to the then central problem of biochemistry, the proteins. On the basis of his experience in the fields of carbohydrates, proteins, and enzymes, Fischer repeatedly pointed out that synthetic chemistry should follow the lead of Nature and develop milder methods. At the same time, he laid the ground-work for a more important finding, The proteins showed very clearly the consequence of the bifunctional nature of their building units, the amino acids. The simple sugars are also bifunctional and are able to combine with themselves into larger compounds. By studying the sugars, amino acids, and proteins, Emil Fischer came to understand and to establish experimental evidence as to the manner in which natural compounds of high molecular weight are built up. This assuredly was his greatest and most important discovery. Organic chemistry developed from pharmacy 200 years ago. Carl Wilhelm Scheele, and, after him, especially French and German pharmacists, began to isolate and investigate natural compounds from plants. About 70 years later, around 1835, synthetic organic chemistry began. From then on, whatever living Nature had to offer to the organic chemist was supplemented and, from time to time, even surpassed by synthetic products, then being discovered aa one new group after another. Synthesis consequently became an end in itself in many laboratories. New methods were developed, leading to an ever-increasing command over materials. The systematic classification of organic compounds was placed in the foreground, and many an organic chemist lost contact with general problems. This course was altered by a few men in the last quarter of the nineteenth century-and a leader among them was Emil Fischer. With the aid of the abundant newly developed organic syntheses, he attacked the problems of natural compounds, not in the form of fortuitously encountered parts of the metabolism of plants or animals, but in that of the fundamental material basis of the living cell. Under his leadership, synthetic and theoretical chemistry was reunited with biochemistry, and a broad scientific basis was restored to organic chemistry. The investigation of natural compounds received an enormous impetus. Biochemistry was established in the chemical research laboratories. It would be misleading to believe that the impact of such an innovator would be limited to his own field. The organic chemical industry began its rise 100 years ago with the synthesis of dyes. There have always been mutual relationships between the laboratories of industry, directed toward production, and those of the universities, which have limited themselves

8

KARL FREUDENBEltQ

increasingly to basic research. Industry absorbs the results obtained in pure science in the universities. Consequently, the influence of such a man as Emil Fischer is not restricted to the laboratories and libraries of the learned, but rather is transferred step by step to the industrial economy, often in t i rapid, sometimes in a slow development. An example of this is encountered in Fischer’s discovery of the bifunctional interlinking of the units in polymeric compounds. Industry and economy shape the lives of individuals and peoples. We can thus appreciate the influence of a creative mind such as Emil Fischer in the scientific world, in scientific thinking, and in the education of generations of chemistry students. We detect it in the laboratories and offices of industry; and we encounter it i t hospitals, in produce markets, in agriculture, and in the household.

111. THESYBTEM OF THE MONOSES The historical aspects of Emil Fischer’s fundamental conventions for writing stereoformulas in a plane have been thoroughly delineated.2 It may be mentioned that the perspective formulas generally attributed to W. N. Haworth were actually a revival of J. B8ese!enJs cyclic formulas.* Van% Hoff had developed a method for representing a sequence of asymmetric centers, such as are uniquely encountered in the sugars, by a system involving tetrahedral geometric forms combined with an algebraic sign denoting right- or left-handedness as established by the observer’s position. This system was bssed on Van’t Hoff’s erroneous concept of optical superposition, which was corrected only in 1931.‘ Emil Fischer found Van’t Hoff’s method unsuitable for his purpose. He, therefore, daringly proceeded to picture the actual spatial models mentally, and invented the two-dimensional projection formulas based upon certain conventions.6This found immediate acceptance, and has since been employed universally. His method of distinguishing antipodes, by means of the prefixed letters d and I , originated partly from genetic relationships and partly from the measwed optical rotation, designated today by (+) and (-). Some years after Fischer’s work, d and I were replaced by the prefixes D and L in order to correlate configurations more closely in a system based relatively on his conventional standards. Emil Fischer suggested the now well-known family tree (see Fig. 1) of the D-seriee of the aldoms, based upon their relationship to n-glyaeraldehyde, despite the fact that this aldehyde and a numbq of other sugars were not known in his time. Figure 1 deviates in (2) C. 8.Hudson, This S m h , 8, 1 (1948). (3) J. BOeaeken, Bet., 46, 2612 (1913). (4) K.Freudenberg and W. Kuhn, Bet., 64, 703 (1931). (5)1 E. Fiecher, Bet., 24, 1836 (1891).

Yo ECOH I EY EOCE I

ECOH J&E

D-(+)Galactose (14)

ECO I

T

"OF" ECOH I E,COE

D-(+)-TPlo.e (15)

HCO I HOCH 1 ECOH I

ECO I I

ECOH

&OH D-(-)-ErgthrOSe

(1)

HCO I HCOH I

E&OH D-(+)-Qy

ceraldebyde (1)

ho. 1.-The

D

Series of the Aldoses. (The rotatory signs in parentheses refer to the equilibrated solutions in water.)

10

KARL FREUDENBERQ

another point from the original nomenclature of E. Fischer. He had originally assigned the prefix 1 to the gulose (12), i d o s (13), xylose ( 6 ) , threose (3) series, to which we iiow amign the prefix D. Such a change was propoaed by M. A. Ibsanoff6 who pointed out that a set of configurational relationships might be inconsistent unless a single asymmetric center was used as a reference standard, namely, that of Dglyceraldehyde. His conclusions were independent of the actual fulfilment of all the experimental steps later completed by A. Wohl and Momber and others. The correction waa accepted and was substantiated' after Wohl had prepared D-(+)glyceraldehyde. In 1891, Fischer agreed to a numbering of the carbon atoms that begins with the most highly oxidized carbon atom. In the case of the dicsrboxylic acids and sugars alcohols, which are structurally symmetrical, such numbering is not unequivocal. Fischer's acyclic formulas were later adapted to the ring forms, when the existence of these became established. In analogy to the common y-lactones, Fischer and the older chemists wrote the sugar rings as furanoid systems. It was only after Emil Fischer's death that W. N. Haworth showed that the six-membered pyranosc rings are more stable, and occur more often, than those of the fivemembered furanoses. The need for such ring formulas dates back to B. Tollens and his m u t e rotation studies. E. Fischer agreed with such cyclic representations for the glycosides, acetates, and acetohalogenoses without reservation; for the free sugars, however, he retained the right to make me of the acyclic, aldehyde formulas whenever this was sufficient to represent reactions, or whenever it was clearer, as, for example, in the representation of thr configurationd relationships of the monoses.

IV. DEVELOPMENT AND EXTENSION OF THE SYSTEM 1. Beeinnings :The Sugars and Phenylhydrazine

As remarked above, in the early eighties, the field of the sugars was not very inviting to the experimentalist, and Fischer would not have decided to enter it had it not been for his discovery of phenylhydrazine, a new and powerful tool to use in sugar chemistry. After the osazones had been discovered, their formation was investigated more closely, and it was found that the first stage was the formation of the phenylhydrazones; among these, the pheqylhydrazone of mannose waa outstanding because of its low solubility, and it was used to identify and to crystallize mannose, which could be obtaincd from it by treatment with aldehydes or acids. Manno,se is (6) M.A. Roaanoff, J . Am. Chem. Soc., 28, 114 (1906). (7) A. Wohl and K.Fraudenberg, Bet., 66, 309 (1923).

EMIL FISCHER AND HIS SCIENTI~ICWORK

11

ucacmihlc by oxidation of muiitiitol, hut it w&nmade readily avtlilable by tho tiydrolyni~of vcgctablc ivory turnings. Pentows and hcxoscn could be distinguished readily with phenylhydrazine. The compound hitherto called “isodulcitol” wae identified as a sugar by means of phenylhydrazine, and was designated ae rhamnose from that time on. Glucose and fructose (which H. Kiliani had discerned to be an aldohexose and a ketohexose, respectively) and mannose were found to give one and the same phenylosazone; the three sugars, therefore, have the same configuration at carbon atoms 3,4, and 5. On treatment with strong acid, the osazones form the osones, which were recognized aa being 2-ketoaldoses by their reaction with o-phenylenediamine. With phenylhydrazine, the osones reform osazones. Zinc dust and acetic acid converts glucosone into fructose. Generally, the aldehyde group of osones is reduced first. Thus, both glucose and mannose could be converted into fructose by this route. By utilizing previous work of H. Kiliani, Fischer oxidized aldoses to aldonic acids and, by further oxidation, to the dibaaic acids; for example, gluconic acid was converted into saccharic (glucaric) acid, and galactonic acid into mucic (galactaric) acid, both of which are tetrahydroxyadipic acids. The pentose which Kiliani recognized as arabinose yielded, by way of arabinonic acid, a trihydroxyglutaric acid. This and other acids were characterized by Fischer phenylhydrazides, from which the respective acid could be regenerated with baryta. Occasionally, the acids were also isolated as their cadmium salts. (p-Bromophenyl)hydrazine, asymmetrical l-methyl-l-phenylhydrazine, and 1,ldiphenylhydrazine were also used for the formation of hydrazides or hydramnes. 2. Additional Methodical Approaches

H. Kiliani, aa Fischer always emphatically acknowledged, discovered and developed the method of building up the aldose series by the cyanohydrin reaction to give nitriles; from the nitrile, the next higher aldonic acid could then be prepared. In 1890, A. Wohl, working in Fischer’s Berlin laboratory, elaborated the dehydration of an aldose oxime to the nitrile, from which the next lower aldose could be prepared by loss of hydrocyanic acid. Fischer exploited the possibilities of sugar extension and degradation afforded by the use of these two important methods. He himself found that aldonolactones could be reduced with sodium amalgam to the aldoses. Thus, the way was opened to proceed from any pentose to the next higher aldose, which, as he soon showed, always arose in two stereoisomeric forms because of the introduction of a new asymmetric center. Another novel method created was the epimerization of aldonic

12

KARL FREUDENBERO

acids caused by heating them in tertinry amines. This steric rearrangement results in an equilibrium involving the two steric positions of the hydroxyl group on the carbon atom adjacent to the caxboxyl group. These methods, together with the customary (but coristantly improved) methods of oxidation and reduction, comprised the preparative tools of Lsugar chemistry before the turn of the century.

3. Synthesis of Glucose (1890) I n 1861, A. Butlerow had heated polyoxymethylene with lime-water, and had obtained a sirup which had a sweet taste and showed characteristic sugar reactions. Twenty-five years later, 0. L6w improved this reaction by using formaldehyde and lime-water at room temperature; but his product, too, could not be adequately characterized by analysis. ’At this point, Emil Fischer began his work. By means of phenylosaeone formation, he demonstrated that both workers had obtained a mixture of sugars. From this mixture, he obtained a crystalline phenylosazone having properties that suggested that it could be glucose phenylosazone. The yield of this phenylosazone was remarkably improved when acrolein dibromide was used instead of formaldehyde. After careful treatment with cold baryta water, Fischer isolated a bromine-free material from which he obtained the phenylosazones of two isomeric hexoses which were named a- and ,3-acrose. Even better results were obtained from the condensation starting with “glycerose,” which is formed from glycerol by oxidation with bromine in the presence of sodium bicarbonate, and which is a mixture of glyceraldehyde and dihydroxyacetone. From the condensation mixture, he again obtained the two acroae phenylosazones. The “a-acrosazotie” obtained was, apart from its optical inactivity, strikingly similar to glucose phenylosazone. Cautious reduction of the a-acromne afforded a sweet sirup which, on further reduction, yielded a beautifully crystalline hexahydric alcohol, a-acritol, which exhibited such a striking similarity to mannitol that it could well be supposed to be its inactive form. However, one kilogram of glycerol yielded only 200 milligrams of a-acritol. Because of this low yield, another route to it, starting from the natural sugttrs, was sought. Natural Dmtbnnose is the aldehyde of natural D-mannitol, and is transformed by the action of bromine water into D-mannonic acid, which was isolated a8 its phenylhydrazide. The acid was regenerated from the phenylhydrazide and isolated as its crystalline lactone. Kiliani had obtained the enantiomorph of this lactone on applying the cyanohydrin reaction to natural barabinose. A mixture of both lactones formed a racemate. Then, by taking recourse to his newly discovered reduction of the lactones to the aldoses, a reaction which Fischer designated the most significant in the

EMlL FIBCHER AND HIS SCIENTIFIC WOltK

13

entire series, he, now secured D-, t,and Dcmaiuiose, and, from them, the corresponding mannitols. a-Acritol was shown to be DL-ni:ititiitol. .is noted above, the a-acritol arose froni a-ncrose, which, in turn, hid beeii obt:iiud by the reduction of a-acrosone. Sirice the reduction of an omne leads to a ketose, a-acrose must have been Dcfructose; and this conclusion was confirmed by the formation of levulinic acid from crude a-acrose with acids, and by the fact that cy-acrose was partially fermented by yeast, leaving dextrorotatory cfructose. Emil Fischer compared the above work with the construction of a tunnel: “If the mountain is not too broad, one can dig through in one direction. Otherwise, the engineer has to start the work from the opposite side, too. However, the engineer is fortunate, in that he can determine the point of attack by exact measurements and has the certainty of bringing both parts together in the massive interior. Our science is, unfortunately, still far from being deductive enough to permit calculations like that. The chemist can, therefore; count himself lucky if he digs his way through the material from the opposite point8 and finds the connection in the interior by several zig-zag paths.” Now the way wm paved for further work. DLMannose was oxidized to DL-mannonic acid, and its two antipodes were separated by means of brucine. The acids were rearranged to D- and L-gluconic acid by the action of a tertiary amine. D- and L-Glucose were then obtained from the lactones. Also, D-, L-, and Dcmannose too were now obtainable synthetically. The nature of /3-acrose was not explained until later; it is Dcsorbose, and is formed, together with DL-fructose, by the condensation of “glycerose,” the mixture of glyceraldehyde and dihydroxyacetone. With respect ,to the natural hexitol sorbitol, which is formed from D-glucose, D-fructose is 2-keto-sorbito1, and L-sorbose is 5-keto-sorbitol. Many decades later, H. 0. L. Fischer extended his father’s work by preparing D-fructose and D-sorbose through an aldol reaction from a mixture of D-glyceraldehyde and dihydroxyacetone? 4. T h e Configuration of Glucose

The question of the configurations of the sugars, and of methods for eqtabliahiag them, W ~ first S conceived during the course of the synthesis of glucosc described above. The work fell into two distinct divisions: the first concerned the mannitol series, to which glucose belongs; and the second, the dulcitol series, which has galactose as the central compound. In Fischer’s words: “The difference between the mannitol and the dulcitol series lies in the fact that the two innermost carbon atoms in inannose are arranged (8) €3. 0.L. Fiffiher and E.Baer, Helu. Chim. Ada, 19, 519 (1936).

KARL FRI!JUDENBDRQ

14

differently, whereas those in the dulcitol series are arranged similarly. Since, initially, all changes were performed on the outer carbon atoms, it was very difficult to get to the dulcitol series from the mannitol series.” Repeatedly, difficulties arose, because of supernumerary substances which did not fit into the system, such as isosaccharic acid and chitonic acid, which finally both proved to be furanoid derivatives. In the synthesis of glucose, only a few hints regarding its configuration were found, but the problem was completely elucidated between 1891 and 1894. The key to Fischer’s solution lay in the transformation of the monose8 into alditols or into dicarboxylic acids, thus making the ends of the carbon chains alike. Thereby, a few of the monoses lose their optical activity, and some pairs lead to identical products. He designated some of these dicarboxylic acids (or tetrahydroxyadipic acids) “mucic acids” (for example, galactaric acid as “mucic acid,” and allaric acid as “allomucic acid”) and others as “sacchazic acids” (for example, D-glucaric acid as “D-saccharic acid,” and idaric acid as “idosaccharic acid”). During the work on the Rynthesis of glucose, mannose, and fructose, so many observations had been made that the existence, and the symmetry relations, of the still unknown monoses and their acids and alcohols could be predicted with certainty. This theoretical work, based upon the theories of Van’t Hoff and Le Bel, enabled Fischer even to include in the following derivation such members of the sugar group (and their derivatives) aa were actually discovered only later. The following exposition, in contrast to the exact historical development, avoids all of the detours into which Fischer wm forced before he visualized the formulation of the pertinent questions and the definition of his objectives. In addition, Fischer’s original papers are written very concisely, and, for good comprehension, they require thorough familiarity with the subject! D-GlUCORe and its epimer, D-mannose, each give an active dicarboxylic acid, namely, Pglucaric acid and D-mannaric acid; hence, neither of these two sugars can have the configuration assigned to the two epimeric systems allose-altrose (8, 9) and galactose-talose (14,IS) [ ( 8 ) and (14)provide inactive acids]. Among the four remaining sugars (formulas 10 to 13), (11) (mannose) and (13) (idose) are out of the question as the proper configuration for glucose, since they give different dicarboxylic acids which are not formed by any other sugars. On the other hand, D-glucaric acid is formed from (10) (D-glucose), as well as from the enantiomorph of (12) (that is, L-gulose). Fischer had this gulose available. D-Glucaric acid forms a 1,Clactone which was reduced, by way of bgulonic acid, to tgulose; and, conversely, this sugar could be oxidized back to Dglucaric acid. (9) For a historical review of these researches, me C.S. Hudson, J . Chem. Edw., 18, 353 (1941).

EMIL FISCHEB AND HIS SCIENTIFIC WORK

15

I n order to make a final decision between glucose and gulose he had to examine the related pentosee, D-arabinose (5) and D-xylose (6). Arabinose-as could be shown by chain extension and degrading of glucose and mannose-forms a portion of both of these hexoses; its configuration c m be neither that of ribose, nor that of xylose, because, in contrast to arabinose, these pentoses forni inactive dicarboxyljc acids. The formula (7)(lyxose) is ruled out for arabinose, since a one-carbon addition to it leads to an inactive (galactaric) acid and an active (talaric) acid. Consequently, D-arabinose must have formula (5) ; and with that conclusion, (10)had been derived for D- (+)-glucose-but with one restriction. Fischer had to choose between (lo) and its enantiomorph; he chose (10), and wrqs aware of the arbitrary nature of this decision. The same decision fixed the form of all the other series-related sugars represented in Fig. 1. That Fischer had accidentally chosen the correct absolute configuration was not realized until after his death. 5. The Configuration of Galactose

The question of the configuration of galactose was solved in 1904. There still remained the choice between the dicarboxylic acids derived from (8) or (14)for the configuration of galactaric acid. The conversion of this acid into racemic (Dctartaric) acid favored the selection of the dicarboxylic acid derived from (14),but Fischer did not consider this observation to be definitive proof. Galactose forms two galactoheptonic acids which, on oxidation, give two optically active pentahydroxypimelic acids. This would not be the case with ( 8 ) , as one acid would be a meso form. Therefore, galactaric acid must arise from ( 14) or its enantiomorph. Natural galactose is, then, either (14)or its enantiomorph. A decision on this question was found during the work on L-rhamnose (17) (see Fig. 2),a natural C-methylpentose (Bdeoxyhexose) whose configuration at C-5 was then unknown (a circumstance which need not interfere with our considerations). Will and Peters had found (in 1889) that, on oxidation with nitric acid, this sugar loses its C-methyl group, to give the same optically active trihydroxyglutaric acid (16) that Kiliani had obtained from natural I,arabinose (the enantiomorph of 5) ; accordingly, mhamnose is either (17) or its epimer at C-5. Fischer produced an “a-” and a “&rhamnohexonic” acid by applying the cyanohydrin reaction to rhamnose; the ‘‘b” form could also be obtained by epimerization of the “cr-”acid. The “cr-”acid formed galactaric acid (19)on treatment with nitric acid. Only rhmnose of configuration (17) (or its C-5 epimer) can give a rhamnohexonic acid from which galactaric acid can be formed. Therefore, a-rhamnohexonic acid has the configuration (18)(or its C-5 epimer) . The epimer (20) gives

16

XARL FREUDENBERG

L-talaric acid, which could also arise from btaloae or caltrose. It was possible to eliminate the latter alternative, because talose was known to be the epimer of galactose. bTalose thus has formula (22), and its enantiomorph is epimeric with &galactose. Therefore, wgalactose has formula ( 14).

V. EXTIWSION OF THE SYSTEM, AND TRANSFORMATIONS OF THE SUGARS 1. General

Fischer himself considered the above synthesis of glucose, and the determination of the steric structures of glucose and galactose, to be a roundedoff piece of research. We have not shown the detours and obstacles which detain4 the experimentalist. The paths, the concepts adopted, and even the aims to be achieved could be determined only aa the work progressed. A survey will now be given of other results, obtained concurrently with the above, but this survey will necessarily be limited to the more important findings.

EMIL FISCHER AND HIS MCIENTIFIC WORK

17

The framework created for the interrelationships of the hexoses and pentoses, and their derived alcohols and mono- and di-carboxylic acids, revealed gaps which were all closed in the course of time, essentially by means of the preparative methods already described. Among these grips was &ribose, which P. A. Leveiic ltiter found in the nucleic widcr. Other natural compounds found their placrs in the system. Adonitol, found by E. Merck, proved to be the pentitol related to D-ribose; G. Bertrand isolated D-iditol, previously synthesized by Fischer, from Sorbue aucuparia. Fischer synthesized natural perseitol, discovered by Maquenne, and recognized it as one of the mannoheptitols. Another polyhydric alcohol, volemitol, found in a mushroom by E. Bourquelot, was shown by Fischer to be a heptitol. Ascending from the hexoses, he obtained heptoses, octoses, and nonoses. A. Wohl proceeded, by means of his method of degradation, from arabinose and xylose to the tetroses; he synthesized D-(+)-glyceraldehyde,1° which was the basis of the system. Rhamnonic acid was epimerized by Fischer to epirhamnonic acid, and this was reduced to cepirhamnose, which was recognized to be 6-deoxy-L-glucose. He also worked on the C-methylpentose chinovose, but this was not established11as being 6-deoxyD-glucose until after his dcath. Whereas “acetobromoglucose” and its analogs are dealt with under the acyl derivatives of the sugars, “acetodibromoglucose” (2 ,3,4-tri-O-acetyl6bromo-6deoxy-~-g~ucosy~ bromide), isolated in 1902, will be discussed here because of its involvement with C-6 of D-glucose. It is obtained by the prolonged action of hydrogen bromide on D-glucose pentaacetate. With methanol, the dibromo compound gives the acetylated methyl glycoside of the 6bromohydrin; and this, with base, affords the methyl glycoside of an anhydroglucose which shows no mutarotation and exhibits a slow, but positive, fuchsin-sulfur dioxide test. Fischer proposed the structure of a 5 , 6 or a 3,6-anhydride for this compound. The latter alternative was later proved t o be correct. The anhydro sugar forms a phenylosaxone, and can be reduced to an anhydrosorbitol. Hydrogenolysis of the bromohydrin yields a C-methylpentose (6deoxy-~-glucose)which is the enantiomorph of the G epirhamnose (obtained by epimerization and subsequent reduction of rhamnonic acid). Thus, the configuration at C-5 of natural rhamnose (6deoxy-L-mannose) was elucidated. 2. Amino Sugars

Ntrturtil glucon:iiniiic gcivc? glucLonc!phenylosazor~o.D-Arubinose treated wi 111 hydrociy:iriic! :wid rtlid nn~moniiiformcd n nitrile which could be (10) A. Wohl and F. Momlwr, Ber., 47, 3340 (1914);60, 455 (1917). (11) K.Freudenberg and K. %Whig, Ber., 82, 373 (1929).

18

KARL FREUDENBERQ

hydrolyzed to D-glucosaminic acid. Hence the glucosamine obtained from chitin is a 2-amino-2-deoxyhexos, being 2-amino-2deoxy-~-glucose,as ww found much later.I2Fischer found that nitrous acid deaminates glucosamine and its related acid, furanoid 2 ,banhydro derivatives being formed. Starting from glucal,la he also obtained an amino hexose which he called epiglucosamine. Much later, other workers established that this compound l-hino-ldeoxy- D-fructose was obtained by is 2-amino-2-deoxy-~-altrose. reduction of glucose phenylomzone. Acrose phenylosazone behaved similarly, yielding 1-amino-ldeoxy-Dbfructose. 6-Amino-gdeoxyglucose was produced by +heaction of ammonia on the methyl glucoside 6-bromohydrin.

3. Glucal and Deoxy Sugars In 1914, Fischer discovered another transformation of the sugars on treating the “twetohalogenoses” with zinc dust. Glucose thereby forms glucal (24). The reaction is general for the aldoses. One molecule of glucal

D-G~uc~~ (24)

takes up two atoms of hydrogen or bromine. The triacetate of dibromoglucal can be transformed into the monobromohydrin of a hexose, which gives glucose phenylosazone. On treatment with ozone, and subsequent deacetylation, D-glucal tiacetate yields D-arabinose. Tri-O-acetylglucal dibromide reacts with methanol and silver carbonate to give two methyl glycosides of the bromohydrin, both of which form the same methyl 2-deoxyglucoside on reduction with sodium amalgam; (12) Sibungaber. Heidelberger Akad. Wiaa., 9 Abh. (1931). Here it is shown that glucoAsminic acid exhibits optical properties corresponding to a n-a-amino acid; 0. Lutz rrnd R. R. Jirpumns, Ber., 66, 784 (1932); P. Pfeiffer and W. Christeleit, 2. Phyeid. Cham., 247, 282 (1037); W. N. Haworth, H. W. G. Lake, end S. Pqat, J . C h . Soc., 271 (1939); W. 0. Cutler and S. Peat, aBd., 782 (1939); M. L. Wolfrorn, R. U. hrnieux, and 53. M. Olin, J . A m . Chem. Soc., 71, 2870 (1949). Those lwt autliow related gluconanrine to natural ( + ) - s h i n e by methrxb not involving operations on the symmetric center bearing the amino group. (13) See next Section.

EMIL FISCHER AND HIS SCIENTIFIC W O R K

19

this glucoside is hydrolyzed neither by yeast extract nor by emulsin. However, after deacetylation and mild treatment with acid,2deoxyglucose is formed. These experiments revealed a mute to the 2deoxy monoses, substances which later became very important. No noticeable amounts of halogen can be split off from one of the above methyl glucosidebromohydrins by ammonia, even under drastic conditions. The other affords the aminohexose mentioned. Some of the experiments described above can also be effected with the corresponding chloro compounds. 4. Functional Derivatives and Their Application a. Compounds with Acetone.-With acetone and a small proportion of mineral acid, fructose and arabinose form “diacetone” (di-0-isopropylidene) compounds; rhamnose yields a monoacetone compound; and glucose gives a mixture of a mono- and a di-acetone derivative. Later, Fischer employed 8-=glucose for this reaction, as this anomer dissolves more quickly in the reaction mixture than the common a-D form. The monoacetone compound from glucose could be produced from the diacetone compound by mild, partial hydrolysiswith acid. Fischer did not speculate on the constitution of the acetone compounds. As they would not reduce Fehling solution, he assumed that their carbonyl group was involved in the condensation. Alditols, such as mannitol and erythritol, form compounds with three or two molecules of acetone; in aqueous acetone, they combine with only two or one. In all cases, the sugar or alditol could be regenerated with dilute acids. In later work, Fischer used these acetone compounds for the preparation of partially acylated sugars and alditols. b. Compounds with Benza1dehyde.-Benzylidene compounds obtained from alpitols were used for the isolation and characterization of alditole, as well as for their purification, since the parent substanceswere readily regenerated from them. With alditols containing an odd number of hydroxyl groups, one hydroxyl group always remained free; and sometimes, several did not react. Even the hydroxyl groups of alditols having an even number sometimea did not react completely with benzaldehyde. In one case, the monobenzylidene acetal of a glucoheptitol, isomerism was observed which Fischer believed might have arisen from the new asymmetric center formed on the benzylidene carbon atom. The benzylidene compounds, like the acetone compounds, served for the synthesis of partially acylated derivatives. c. Mercaptals.-Fischer found that aldoses react with alkanethiols (and also, a-toluenethiol), in concentrated mineral acid solution, to form acyclic mercaptals (dithioacetals). Cyclic mercaptals were obtained with ethanedithiol and 1,3-propanedithiol. Fischer used these derivatives for

20

KARL FREUDENBERG

the isolation of sugam, as they are formed readily and are, for the most part, easy to crystallize. Today, they form the starting point for certain acyclic derivatives of the sugars. d. Acetohalogeno Sugars; 0rthoacetates.-When Fischer entercd this field, in 1901, the common form of acetobromoglucose ([a]D +199” in chloroform) was known. Its discoverers, W. Koenigs and E. Knorr, had found that it could be converted into methyl Bglucoside and into 8-glucose pentaacetate. Fischer prepared acetobromoglucose by starting from a- and 8-D-glucose pentaacetates. He concluded that, in one of these reactions, a Wdden inversion had occurred. He greatly simplified the preparation of acetobromoglucose by treating the acetylated sugar with hydrogen bromide in acetic acid. He designated the bromo sugar derivative “&acetobromoglucose”; today, we know that a Walden inversion always occurs &.wing the reaction with alcohols, and this bromo derivative is now assigned to the a-D series. The reaction waa extended to other sugars. An analogous chloro (and bromol tetraacetyl derivative w a obtained from galactose. A crystalline hepta-0-acetylmaltosyl chloride was synthesized; the bromide did not crystallize well. By using hydrogen halide in acetic acid, acetobromolactose and acetobromocellobiose, aa well as aceto-iodoglucose and aceto-iodocellobiose were prepared. Representatives of the &acetohalogeno sugars are likewise known. Glucose pentabenzoate was synthesized, and transformed into the benzoylated bromo compound. Fischer obtained a crystalline quarternary salt from acetobromoglucose and pyridine. An anomaly appeared in the rhamnose series. When acetobromorhamnose was treated with methanol, and the product was deacetylated with alkali, a “methyl rhamnoside acetate” wm obtained which had retained an acetate group not saponifiable by alkali; Fischer named this compound y-methyl rhamnoside monoacetate. It was not until 1930 that it wm shown to be the lJ2-(methyl orthoacetate) (25). Today, more orthoaoetates of the same kind are known in the sugar series.

L-Rhamnose 1,2(methyl orthoacetate) (25)

EMIL FIBCHER AND HI8 SCIENTIFIC WOXK

21

5. Glycosides

The many natural compounds of the sugars with alcohols and phenols present a challenge to the synthetic chemist. In 1879, A. Michael had synthesized the first glucoside by allowing acetochloroglucose to react with a phenol. The course of this reaction was rather abstruse, and the @-Dglucoside waa produced in only low yield. In 1893, Fischer found a method of synthesizing alkyl glycosides by saturating a mixture of a sugar and an alcohol with hydrogen chloride. When the sugar was too difficultly soluble in the mixture, he found that he could use its acetylated or acetochloro derivative just as well. Using this reaction a t room temperature, Fischer obtained glycosides (not all crystalline) of aldoses, fructose, and glucuronic acid with different alcohols and hydroxy acids. He favored the cwrently accepted, cyclic hemiacetal structure for the glucosides, but formulated them with the furanoid ring for glucose proposed in 1883 by B. Tollens. w. N. Haworth later established that the glucose residue in such glucosides is present in the form of a pyranoid ring. In 1908, he wrote concerning the methyl at-wglucoside discovered by him in 1893 and its predicted fl anomer found a year later by Alberda van Ekenstein: “After I observed (1894)their characteristic behavior toward emulsin and yeast, I designated the two substances as a- and &methyl-& glucosides, and extended this distinction to the entire class of glucosides. Immediately after the discovery of the alkyl glucosides, I called attention to their structural relation to the two then-known glucose pentaacetates, and advanced the opinion that these are not structural isomers but stereoisomers. I extended the concept of the glucosides to the polysaccharides.” In 1895, Fischer reported a greatly improved glycoside synthesis. He heated a mixture of the sugar with the alcohol containing one percent or less of hydrogen chloride, and continued the heating until the Fehling reduction was no longer positive. Better yields were obtained by this procedure, especially with the acid-labile sugars such as fructose. Again, an anomeric mixture was obtained from glucose, together with an amorphous, readily hydrolyzable product, which he considered to be the acyclic dimethyl aoetdtl of glucose. It was not until 1914 that he succeeded in obtaining this substance in analytically pure condition (by distillation at 0.2 mm) and thus established that here, also, he had a monomethyl compound. Hc named the new glucoside “methyl y-glucoside”; in this name, the prefix had no steric or constitutional significance. About ten years later, W. N. Haworth demonstrated that this product is an anomeric mixture of methyl D-glucofuranosides. Glycosides could also be prepared from ketoses by using the new method. Fischer had also utilized the acetohalogeno sugars in a Koenigs-Knorr

22

KARL FltEUDlCNBEltO

type of condensation with alcohols, using either silver oxide or silver carbonate, and had thus obtained alkyl glycosides. For phenolic glycosides, he employed the alkali phenoxide in dry form, or in solution in water or acetone; glucosides of phenol, resorcinol, phloroglucinol, and 2,4 ,6-tribromophenol were obtained in this way. In his later years, Fischer concerned himself with improvements in this synthesis of glycosides, which had always produced the &D form mainly. By addition of quinoline to the reaction mixture, he obtained an anomeric mixture of glycosides from which the difficultly available C~-Dforms could sometimes be obtained by fractional recrystallization. In the reaction with phenols, he found that some deacetylation may occur, so that improved yields could be obtained by reacetylation procedures. Fischer’s faculty for observation, and his perseverance, led to glycoside syntheses that were more and more complicated, with respect to both the sugar and the aglycon. There is now no difficulty in transforming such disaccharides as maltose or lactose into their acetobromo compounds and, thence, into glycosides. When acetobromoglucose was dissolved in ether, and treated with an aqueous, alkaline solution of benzenethiol, phenyl 1-thioglucoside was obtained; acetobromolactose waa also utilized successfully in this type of synthesis. The cyanogenetic glycoside amygdalin (26) has long been of interest to

&H Amygdalin (a6)

chemists. It is now known to be the B-gentiobioside of the nitrile of D-( -)mandelic acid (27). In 1894, Fischer found that only one molecule of

D- (-1 -Mandel lc acid

(27)

EMIL FISCHER AND HIS SCIENTIFIC WONK

23

glucose WL~Bremoved from amygdalin by yeast extract-a surprising result, as both linkages present are now known to be Bwglucosidic. There remained, after digestion with yeast, a substance which Fischer designated as “Z-mandelonitrile glucoside.” In 1917, E. Fischer and M. Bergmanil treated ethyl dZ-mandelate with acetobromomoglucose and silver oxide, and obtained a diastereoisomericmixture of ethyl O-~-glucosyl-d-mandelate and ethyl 0-D-glucosyl-Lmandelate. This was transformed, through the acetylated amides, into the acetylated nitrile mixture, which was separated by fractional ,recrystallization into the glucosides of (+)- and ( -)mandelonitrile. The latter was identical with that obtained from amygdalin. The former corresponded to sambunigrin, which h d been found in Nature by Bourquelot and Danjou (1905) and which, on hydrolysis with acid, gave c (+)-mandelic acid. The same route was adopted in 1919 to effect the synthesis of natural linamarin, which was known to be the glucoside of acetone cyanohydrin. The simplest member of the series, glycolonitrile glucoside, was also prepared.

6. Nucleosidee and Other N-Glycosyl Compounds Stimulated by the work of P. A. Levene and W. A. Jacobs on the cleavage products of the nucleic acids, Fischer (mainly working together with B. Helferich) published, from 1914 onwards, research on wglucosyl derivatives of purines in which the sugar was attached either to a nitrogen atom or to an oxygen atom. Fischer’s experience with the purines was thereby combined with his knowledge of the sugars. His method relied on the reaction between the acetobromo sugar and salts of the purines, especially the silver salts, in nonaqueous media at elevated temperatures. The N-glucosyltheophylline prepared in this way, through the tetraacetate, had the glucose residue in the 8-D configuration linked to the nitrogen atom at position 9. Linkage to oxygen is here not possible. In contrast, the glucosyltheobromineis hydrolyzed by water at 20’. Only the pyrimidine nucleus comes into question as the position of attachment of the sugar in this compound, and for this linkage, only the nitrogen atom at position 1 or the oxygen atoms at 2 or 6 are possible; a similar situation exists for 1,3 ,’l-trimethyluric acid. Fischer and Helferioh devised a new method for the preparation of glucosyhdcniiie. Thcy started witb dic:hloroadenine, glucosylated it, and replaced the chlorine atoms by hydrogen (by the action of hydrogen iodide and phosphonium iodide). The AT-glucosyladenineso obtained had the sugar residue attached to the nitrogen atom at position 7 or 9 (it was later shown to be at 9). The dichloro-N-gluctosyladerliliewas transformed into a monochloro-N-glucosyladenine. On treatment of this compound

24

KAHL FHEUDENBERQ

with ammonia, followed by nitrous acid, a crystalline glucosylpurine, probably a derivative of guanine, wm obtained. Galactosyl and rhamnosyl derivatives of the purines were also prepared. A further step toward the synthesis of nucleotides was taken in 1914, when Fischer succeeded in phosphorylating N-glucosyltheophylline with phosphoryl chloride and pyridine. The crystalline product obtained bore the phosphate group’on the glucose portion. The substances mentioned above illustrate how Fischer repeatedly performed syntheses of glycosyl combinations wherein the aglycon moiety was varied. The number of such examples was increased in 1914.Starting from silver succinimide and acetobromoglucose, a tetrwetate was obtained which , on deacetylation with ammonia, led to N-8-D-glucosylsuccinimide. Acetobromoglucose and silver thiocyanate gave D-glucosyl thiocyanate which, with ammonia, produced N-glycosylthiourea. When the same reactions were effected with silver cyanate, a N-D-glucosylurea was obtained, which was identical with that prepared in 1903 by N. Schoorl directly from >glucose and urea. ‘7. Acyl Derivatives; Acyl Migration; Gallotannins

In the foregoing Sections, we have frequently mentioned the acetates and other esters, such as the pentaacetates of the hexoses and octaacetates of the disaccharides. It is appropriate to summarize here Fiucher’s work on the partially acylated sugars and alditols. He encountered some of these compounds accidentally; but, when their relation to natural compounds became apparent, he prepared them by intent, using constantly improved methods. When acetohalogeno sugars are treated with moist silver oxide, acetates are produced having the C-1 hydroxyl group free; some of these exhibit mutarotation. Hexose tetraacetates, rhamnose triacetate, and partially acetylated disaccharides were obtained in this manner. Among the disaccharide derivatives, maltose heptaacetate ( 1910) was outstanding, because its ease of crystallization was so great that it could be used for the identification of this disaccharide. Glucose tetrabenmate was synthesized from the benzoate analog of acetobromoglucose. In nearly all other :cases, isopropylidene derivatives were used. “Diacetoneglucose” formed a monobenzoate from which the acetone groups could be removed by dilute acids. The glucose monobenzoate so produced is isomeric with, or may possibly be identical with, a product obtained from vacoiniin, which C. Griebel had isolated in 1010 from cranberries (Vac& n h m viti8 idea). As noted previounly, one ttcetoiie residue is very readily removed from

EMIL FISCHER AND HIS SCIENTIFIC W O R K

25

diacetoneglucose; the three free hydroxyl groups (now known to be at C-3,C-5, and G O ) in the monoacetone compound were benzoylated. After removal of the remaining acetone residue, a glucose tribenzoate was obtained which, from carbon tetrachloride, crystallieed with one molecule of carbon tetrachloride. The original monoacetone tribenmate could be regenerated from this product. Fischer obtained (in 1915-1916) many other partially ,acylated products from isopropylidene derivatives of dulcitol, mannitol, and erythritol. In the reaction of acetobromorhamnose with methanol, Fischer obtained, among other products, a sirupy substance (not well defined) that he considered might have arisen by a change in ring size, with accompanying acyl migration. Acyl migration was first established (in 1911) on a sound experimental basis in his synthesis of digaJlic acid (28) , and was later recognized

HO

OH

OH m-Digallic acid (2 8)

to occur frequently with partially acylated alditols. Thus, two forms of dictcetonedulcitol were obtained, from which two different dibenzoates, which were partially interconvertible, were produced. He explained these migratione by what is now known as neighboring-group participation.

I

I

I

HCO’

I

I

HCOH

I

HCOH

‘OH

I

HC-C-R

II d0

Fischer once termed hexoses “monotonous” because all five of the available hydroxyl groups give derivatives, with little differentiation in reactivity. He broke the “monotony” mainly through use of the isopropylidene derivatives, and thereby prepaxed the way for later developments,

26

KARL FREUDENBERG

Nature provides us with many glycoside esters in which the acyl group is attached to the sugar portion; an example is thc compound vacciniin already mentioned. However, the largest group of natural acyl derivutivcs of sugars is to be found in the gallot.anninsand related substances. After Fischer had found (from 1912 onwards) that the two most iniportant types of gallotannin , namely, the Turkish and Chinese gnllotannins, contain a small proportion of glucose which is difficultly separable with acids from its combination with gallic acid, he esterified a-D-glucose with tris-0-(methoxycarbony1)galloyl chloride in quinoline. After removal of the methoxycarbonyl groups with alkali, a tannin WIM produced which consistd essentially of penta-0-galloyl-wglucose. Since the same product arose from both a- and j3-D-g1ucoseJ a tetra-0-galloyl-D-glucose was also considered as another possibility for the product. On using tri-0-acetylgal\oyl chloride in the same sequence, different products were obtained from the D-glucose anomers (1914). The synthetic tannin had roughly the same content of glucose and gallic acid as had the Turkish gallotannin (from the galls of Quercus infectoh) , However, the natural product contains several percent of bonded ellagic acid (29) and, furthermore, after methylation14 and hydrolysis, it yields,

HO

Ellagic acid (29)

beside8 tri-0-methylgallic acid , a small proportion of di-0-methylgallic acid. Thus, Turkish tannin is not a simple ester of D-glucose with gallic acid. Nevertheless, most of the hydroxyl groups of its D-glucose residue are esterified with gallic acid. For comparative purposes, the anomers of D-glucose pentakis (tri-0-methylgallate) were prepared ; the &D form crystallized. The gallotannin from Chinese leaf galls (Rhus semidata) contains 8-10 gallic acid residues for each glucose residue. A large part of the gallic acid (14) The requisite treatment with diazomethane must be done in the nbsence of methanol, as otherwise the diaaomethane catalyzes a degrdative de-esterification (1914); K.Freudenberg, “Die Chemie der netfirlichen Gerbstoffe,” Springer, 1920.

EMIL FISCHER AND HIS SCIENTIFIC WORK

27

is esterifid, at the met&hydroxyl group,,with a second gallic acid residue. u-Gluccse was then successfully esterified with five equivalents of mdigallic acid (28), employing the methoxycarbonyl or, better, the acetyl group as the protecting group. This synthetic tannin showed great similarities to natural Chinese gallotannin, which, however, has a constitution that is not so regulax D-G~UCOS~ pentakis( penta-O-methyldigallate) was also synthesized, A tri-0-galloyl-D-glucose, prepared by way of monoacetoneglucose, showed. the properties of a tannin. It consisted, in the main, of 3,4,5-tri-Ogalloyl-D-glucofuranose.In 1918, an amorphous mono-O-galloyl-D-glucose was obtained from diacetoneglucose; it did not have tanning properties. After the elucidation of the structure of diacetoneglucose (in 1923), this compound .,odd be regarded as 3-O-galloyl-~-glucose.A mono-O-galloylD-fructose, prepared analogously, was crystalline. From acetobromoglucose and the silver salt of tri-0-acetylgallic acid, with subsequent deacetylation, there w8s prepared a crystalline D-glucosyl gallate; this did not have the properties of a tannin. Hamameli tannin and naturally occurring chebulinic acid wers found to contain sugars esterified with gallic acid (1912).

VI. OLIQO-AND POLY-SACCHARIDES 1. General Monosaccharides “give rise to all polysaccharides in one and the same way, namely, by loss of water, to form residues which condense to a larger system. In the reverse sense, all of the complicated carbohydrates can revert to the simple sugars by hydrolysis. The polysaccharides include sucrose and milk sugar (lactose), as well as the various gums and waterinsoluble materials, such as starch and cellulose.” Considering the great number of monosaccharides, the great variety found in the polysaccharides can be readily understood. Fischer’s investigations of the glucosides led “to the surprising result that, in principle, there is no difference between the glccosides and the complicated carbohydrates.’ The latter are simply to be considered as glucosides of the sugars. This is in harmony with the fact that they are cleaved by acids and enzymes, and also with the results 90 far obtained by synthesis in this difficult area.” When Fischer referred to polysaccharides, he included in that designation di- and tri-saccharides ( 1894) , The name “oligosaccharide” was introduced later, by B. Helferich. Fischer was aware of the fact that the linkage from monnse to monose is glycosidic in nature, and he consequently adopted the cyclic acetal formulas suggested by B. Tollens. He well knew that, in such tionreducing disaccharides as sucrose and trehalose, the hydroxyl group8 of the hemiacetal function are involved in the glycosidic linkage,

28

KARL FREUDENBEHO

He formulated the oxygen-containing ring in the furanoid form type, as w&s customary in those days; the predominance of the pyranose ring (W. N. Haworth, 1926) waa still unknown. As with the simple glycosides, he differentiated between u- and 8-glycosidic linkages from monose to monose. During Fischer’s lifetime, stuchyose, a tetraaaocharide, wns the highest “polysaccharide” of fairly well known structure; beyond this was unexplored territory. It was known only that cellulose, starch, and glycogen afford glucose diaaccharides and dextrins, and that they contain glucosidic linkages and have high molecular weights. Recalling the formation of his “isomaltoas” by treatment of glucose with hydrochloric acid, Fischer stated that, if this treatment is prolonged, “the synthetic process exceeds the formation of isomaltose, and more complicated compounds arise which can be compared to dextrins. The results obtained here may be poor, but suffice for demonstrating, in principle, the possibilities of the synthesis. Certainly, a long way lies ahead before starch or cellulose can be made artificially--but one can already be assured that the aim is not inaccessibly high.” It is evident that, at that time, he already considered that the true polysaccharides already mentioned are the higher members of a series starting with the di- and tri-saccharides. The situation was the same as that found later with the proteins. Starting from the amino acids, he proceeded, by way of the oligopeptides, to an octadecapeptide and thus reached the group of the peptones. For lack of experimental evidence, he wm forced to disregard whether enlargement of the “chains” (the expression was first used in this sense by Tollens), and, as a result, a large molecular size, sufEced to explain the properties of the proteins, starch, and cellulose, or whether some unknown constitutional principles were involved. The inability of starch, cellulose, and glycogen to reduce Fehling solution to a stoichioinetrically detectable extent proved, in Fischer’s time, to be an inburmountable obstacle to every speculation; and so, Fischer concentrated his attention on the oligosaccharides. 2. Di- and Tri-saccharides a. Natural Compounds.-hLaetose forms a phenylosazone and, from this, an omne. With dilute acids, the osazone yields an anhydride (1887). Hydrolysia of lactosone gives glucosone and galactose. WGlucose, therefore, constitute8 the reducing portion of lactose (1888).Five years later, Fischer assigned a ring structure to the galactose portion, and, in 1908, extended this to the glucose portion. Which oxygen atom of the glucose moiety carried the galactose residue remained an open question. Aldobjow form acetohalogeno compounds when their octaacetates are

EMIL FIYCHER A S D HIS SCIENTIFIC WORK

29

treated with hydrogen halide in glacial acetic acid. Experiments designed to produce a tetrasaccharide of the trehalose type from acetobromolactose (with silver carbonate) led to a mixture containing the desired product and lactcse (1910). In 1896, Fischer developed the “pfienylhydrazine test” for the detection of hydrolytic scission of disaccharides, especially by enzymes; this depends on the fmt that the phenylosazones of disaccharides are soluble in hot water, whereas those of the monosaccharides are not. Lactose is hydrolyzed by emulsin (1894) and by lactase; it is not fermentable by yeast, and is unaffected by invertase (1894). An extract of the small intestine of horses and cattle, especially from young animals, hydrolyzes lactose (1896). The action of enzymes on lactose allowed it to be classified, along with cellobiose and maltose, with the “normal” (and not the y-type of) methyl glucoside (1914). In the discussion of maltose, the relationship of lactose to the @-serieswill Be mentioned later. Sucrose yields glucose phenylosazone, only, after hydrolysis (1894). Fuming hydrochloric acid produces (chloromethy1)furaldehyde (1914). The hydrolysis of sucrose by acids was found to be about half as fast as that of “methyl y-glucoside,” and very much faster than the hydrolysis of the anorneric forms of the “normal” methyl glucosides. After Purdie and Irvine (in i905) had obtained from methylated sucrose the same tetramethyl ether of glucose as from the “normal” methyl glucosides, Fischer concluded (in 1914) that the similarity between sucrose and his “methyl y-glucosids” which he had noted could not reside in the glucose portion of sucrose. Sucrosein fermented by nearly all yeasts (1894,1898). Inversion precedes fermentation (1895). Of various animal secretions tried, the only one effective in cleaving sucrose was that from the mucous membrane of the small intestines of several animals (1896). Sucrose was found to be unaffected by emulsin (in 1894). An approximhtely correct formula for sucrose had been published in 1883; its shortcomings were the incorrect ring size for the glucose residue and the uncertainty regarding configurations at the interlinked carbonyl groups. Fischer performed a few experiments with the natural disaccharide trehalose. It does not react with phenylhydrazine. A diastase from .green malt has no action on it; Frohberg yeast has a weak action (1895). Carp blood hydrolyzes it rapidly, in contrast to the blood of other fishes. Extracts of the mucous membrane from the small intestines of horses and cattle are also active (1906). A. Xalanther, in Fischer’s laboratory, found, in 1898. that wine yeasts also hydrolyze this disaccharide. On the basis of reports in the literature, Fischer considered gentiobiose to be an O-&D-glucosylglucose (see cellobiose, p. 30).

30

KARL FREUDENBERG

The nctturally occurring trisaccharide rafinose (melitriose) is hydrolyzed as easily as sucrose, by nearly all yeasts, into fructose and the disaccharide melibiose (Scheibler and Mittelmeier, 1889). The fructose part appears to be combined in the same manner aa in sucrose (Fischer, 1898). Melibiose is hydrolyzed to glucose and galactose by emulsin or, before fermentation, by bottom yeast (1902). The same enzymes split melibiosone into glucosone and galactose; with phenylhydrazine, the resulting glucosone forms glucose phenylosazone in the cold, and the galactose gives galactose phenylosazone on heating (1902) ; therefore, melibiose is an O-galactosylglucose. From acetochlorogalactose, glucose, and sodium hydroxide in aqueous alcohol, a synthetic O-galactosylglucose was obtained which Fischer thought waa melibiose (1902) ; perhaps, it waa actually synthetic lactose (Schlubach and Rauschenberger, 1926). Fischer confirmed the disaccharide formulation for turanose by osazone formation (1894). Turanose is formed from the trisaccharide melezitose, which is fermentable with yeast.

b. Derived Disaccharides.-In contra& to the oligosaccharides discussed above, maltose is a product that does not occur in Nature as such, but only arises by the action of enzymes on starch or glycogen. As it is a reducing disaccharide of glucose, maltose forms both a phenylosazone and an osone. Treatment of the osone with an aqueous extract of brewers’ yeast liberates glucosone, which reacts with phenylhydrazine in the cold to produce glucose phenylosazone (1902) ; after filtration and warming, another portion of glucose phenylosaaone was obtained (from the glucose). Maltohionic acid is hydrolyzed by acids to a mixture of glucqnic acid and glucose (1889). A characteristic derivative of maltose is the readily crystallizable mdtose heptaacetate previously mentioned. Maltose is fermented by nearly all yeasts and, like sucrose, is hydrolyzed by invertase. It is unaffected by emulsin. The parallel between methyl a-D-glucoside and maltose is unmistakable (1898). Maltose and lactose exhibit differences similar to those shown between methyl a-D-glucoside and methyl p-Dglucoside (1894). Cellobicse is readily obtainable through its octaacetate, which is prepared by the aoetolysis of cellulose. It served Fiseher repeatedly as a counterpart to maltose end lactose. The acetobromo and aeetoiodo compounds crystallize weil; the former was used for the preparation of the heptaacetate and many cellobiosides, among them, those of glycolic acid and its amide and nitrile. With silver oxide, it gave the acetate of a nonreducing tetrasaccharide; but it could not be obtained in pure condition. Most of the reactions establish& for glucal could also be effected with cellobial (with G. ZemplCn, 1910).

EMIL FISCHER AND HIS SCIENTIFIC WORK

31

Cellobiore and its &glycosides were hydrolyzed to glucose by emulsin. Cellobiosone and hydrocellobial were also cleaved by this enzyme. Extracts of Aspergillus niger and Kefir (a Caucasian micro-organism that ferments milk) hydrolyzed cellobiose; yeast extract was inactivc. Cellobiose behaves toward eniulsin and yeast extract “like ycntiobioae and (Fischer’s) ‘isomaltose’and has some similarities to lactose.” Furthermore, cellobiose, “isomaltose,” and gentiobiose have the same configuration of their glycosidic linkage; maltose has the other. Fischer expressed the imminent conclusion that cellobiose, lactose, and gentiobiose have a p-D glycosidic linkage only with reservation, because pure, individual enzymes were not at, hand (1909). c. Synthetic Disaccharide8.-Although Fischer’s isomaltose is now known to have been a mixture,16 and the name now designates a pure disaccharide prepared by other methods, there is little doubt that his preparation did contain this substance. He described a phenylosazone. He made his preparation (1890, 1894, 1895) by the action of concentrated hydrochlcric acid on glucose. Impurities that were precipitated on adding alcohol were discarded. Addition of ether to the mother liquor precipitated a mixture which was subjected to yeast fermentation. The phenylosamne was obtained from the nonfermentable residue. More directed syntheses of disaccharides were effected by treating a solution of an acetochloro sugar in ether with a solution of a second sugar in aqueous alcohol containing alkali.16 In this manner, an O-glucosylgalactose, an 0-galactosylglucose (mentioned previously), and an O-galactosylgalactose were obtained, all more or less pure. All three formed phenylosazoiies. Xone of the three was in any marked way fermented by top yeast, s3 that, in this manner, admixed monosaccharides could be removed. On the other hand, bottom yeast fermented the 0-glucosylgalactose and the 0-galactosylglucose, but not the 0-galactosylgalactose. Emulsin hydrolyzed all three. Isolactose is a disaccharide arising when a mixture of glucose and galactose is treated with Kefir extract (1902). I t is different from lactose and melibiose, and forms a crystalline phenylosazonk. When acetobromoglucose was treated with moist silver oxide, a mixture of brominefree acetates was formed from which was obtained, besides glucose tetraacetate, a small proportion of u, crystalline disaccharide octa-

(15) A. Thompmn, K. Anno, M. L. Wolfrom, and M. Inatome, J . Am. Chem. Soc., 76, 1309 (1854); A. Thompson and M. L. Wolfrom, ibid., 76, 5173 (1954). (16) “On introducing acetochloroglucose into an alkaline solution of a monosaccharide in aqueous alcohol, apart from thcir condensation, partial removal of acetyl groups, in the form of ethyl acetate, occurs.” This was the first indication of transesterification by alkoxide (1902), a reaction that later achieved great importance.

32

KARL FltEUDENBEHGI

acetate. From the octaacetate, a sirupy disaccharide (called isotrehalose) was generated. This disaccharide does not reduce Fehling solution, and, in contrast to natural trehalose, is levorotatory. After hydrolysl, it react’s with phenylhydrazine to give glucose phenylommne only. I t therefore has the constitution of trehdoae, but differs in the configuration of its glucosidic linkage. VII. STERIC SERIES

E. Fischer wrote in 1896: “The niost serious discrepancy in the stereochemical system of the sugrtr group at this time is CRW by the uncertainty concerning the configuration of dextrorotatory tartaric acid.”. Since all stereochemical considerations originate from tartwic acid, and , furthermore, since this acid bears simple relationships to malic acid (34), asparagine, etc., I have tried . .again and again to resolve the problem.” It w a successfully ~ solved with the aid of rhamnose (17).The configuration at (3-2, C-3, and C-4 of this sugar had been elucidated at that time. Rhamnose “can be degraded to a methyltetrose (30)by the excellent method of Wohl. If the methyltetrose is finally oxidized with nitric acid, d-tartaric acid (31)is formed. Since, under the same conditions, Gtrihydmxyglutsric acid @e w u referring to tarubiino-trihydroxyglutaric acid (la)] is formed from rhamnose (17),and muck (galactaric) acid (19)is formed from rhamnohexonic acid (18) ,and furthermore, since, in all cases, , . .the methyl group is lost, it seems reasonable to explain the conversion of the methyltetrose into tartaric acid in the same manner. . .,’

..

...

.

.. .

COaH I HCOH I HOCH

Aw

(+)-Tartaric acid (91)

.

CO,H I HOCH I HYOH COaH

(-)-Tartar Ic

acid

(32)

yaH HVOH

4 COSH

meso-

Z0,H

Tar-

D-(+)-

taric, acid (33)

(34)

Malic acid

It now became clear that the (+Nwtrtric acid produced by oxidation of Dsnocharic (mglucaric) acid is formed from its first four carbon atoms, and that racemic tartaric acid (31 32) is produced in the same manner, from mucic (galactaric) acid (19).The choice of formula (31) for (+)-

+

(17) Recher oalled it &tartaric acid.

EMIL FISCHEa AND HIS SCIENTIFIC WORK

33

tartaric acid (rather than 32) follows automatically from the decision already made (between enantiomorphous forms) for the D-glucose projection formula; we now know that this choice conformed to the absolute conGgunttion established later. Fischer had foreseen that the configuration of glucose could be derived from that of the tartaric acids (1894); but this prophecy was rigorously fulfilled only after his death, when M. Bergmann found, in 1921, that n-glucaroditlmide gives (-)-tartaric acid by way of the dialdehyde. Erythrib1 (which is meso) arises from &glucose by way of D-arabinonic acid and D-erythrose (0.Ruff, 1900);the latter can be oxidized to meso-tartaric acid (S.Przyhytek, 1884). (+)-Tartaricacid (31)gives information about the configurations at C-2 and C-3 of D-glucose (lo), (-)-tartaric acid (32) about those at C3 and C-4, and meso-tartaric acid (33) about those at G 4 and G5. (+)-Malic acid is formed by treatment of (+)-thrtaric acid with hydriodic acid (W. Bremer, 1876) and, hence, formula 34 was assigned to It. E. Fischer later classified D-( -)-glyceric acid in this series, and A. Wohl and F. Momber included D-(+)-glyceraldehyde. When Fischer decided to create a system for the a-substituted acids, he wittingly deviated from the convention for the system of sugm and took C-2 (C-u) of the a-hydmxy acids aa the reference carbon atom. He them fore classified (+)-tartaric acid as d (later changed into D). In doing &I, he proceeded from the consideration that ite a-carbon atom corresponds to that in gluconic acid and that, as a twofold a-hydroxy acid, it contains the group of the a-monohydroxy fatty acids twice over, as may be seen by rotating the lower half of its projection formula through 180'. In 1894, Fischer wrote concerning the programmatically demanded extension Gf the configurational theory beyond the sugars to the rr-substituted carboxylic acids and all the other aliphatic compounds containing asymmetricdly substituted carbon atoms: "A vast new field of experimental research has been operied, and since its treatment must ultimately always be connected to the sugars, it will undoubtedly furnish not only a general spreochemicd system for the optically active aliphatic compounds but also mmy new results on the chemicd transformations of the carbohydretes whioh can be of special interest for the chemistry of plants and animals." This prediction haa been fulfilled to an unexpected degree. Today,the configurational theory dominates organic chemistry. l+htural tartaric acid is dextrorotatory in water at the D-line of sodium and has twc asymmetric centers, possessing a rotating axis of symmetry, aa dso do@ mannitol; thus, positions 2 and 3 are chemically and sterically equivalent dthough apparently of opposite configurational sign. Fischer regarded (+)-tartaric acid as the very prototype for the D configuration of the arhydroxy acids. He extended the D designation to all of the a-substi-

34

KARL FREUDENBERG

tuted carooxylic acids (for example, the amino and chloro derivatives) having the configuration

This convention for such acids has been generally accepted. Only one exception has been made in recent years to this convention for denoting the ccnfigurations of a-substituted acids, and curiously, this is with the basic member of the series itself, namely (+)-tartaric acid. If this acid is considered, not as an a-hydroxy acid but, reverting to the sugars, as a sugar derivstive, then it has, of course, to be named I,-(+)-threaric acid.18 This nomenclature is currently preferred by nomenclature commissions; I suggest letting future developments make the final choice between D-(+)-tartaric acid and L(+)-threaric acid for (+)-tartaric acid. Fiscter’s statement, cited above, that aspartic acid bears a simple relationship to the a-hydroxy acids and, consequently, to the sugars, was made with the explicit reservation that no Walden inversion may occur on deaminatioii with nitrous acid (1896). This condition could not be retained. Furthermore, his conclusion that acetobromoglucose belongs to the / 3 - ~ series because it forms j3-D-glucosides is also inadmissible. It is amazing that these two cases seem to be the only ones in which he was misled by the then partially unknown, sometimes unfathomable phenomena of inversion.

VIII.

INSIGHTS INTO STEREO~HEMIGTRYAND

ITSRELATION TO BIOCHEMISTRY It was emphasized above that, during the course of his experimental work, Emil Fischcr had to create new concepts on which to base further exploratory research. A few examples of this will now be cited. In 1894, Fischer wrote: “It will probably be possible to obtain all hembers of the sugar group by a combination of the cyanohydrin reaction with the reduction of lactones, as .won as we have succeeded in finding the two optically activo forms of glyc!eraldohyde. All observations agree with the immcriamc forcseeri by Van’t Hoff, ttbove all the dieappearanca of 1:eomrrs if the molccule becomes constitutionally symmetric. This iricludea the transformation of different stereoisomers into one and the same substance if one of several asymmetric renters is abolished.” An example of this is (18) See Rule 28, J . Chem. Soc., 5117 (1952); Rule 29, J . Org. Chsm., 18, 288 (1903).

EMIL FISCHEH AND HIS SCIENTIFIC WORK

35

the forination of the same omzone from *mannose and *glucose. This proce~swaw new. In the iiiitial confusion, it was the great achievement of Emil Fischer to disengage himself from Van% Hoff ’8 method of writing configurational formulas, and to go back to the ordered tetrahedral models themselves, for which he had to invent a projection of the steric arrangement onto the plane of depiction. Initially. only the nitrile of L-mannonic acid was found on addition of hydrocyanic acid to Larabinose; this acid retained the original arabinose in the asymmetry centers 3,4,and 5.The new center of asymmetry created in this way at C-2 was first considered by Fidcher-to be racemic. This would have meant that the Gmannonic acid should be a partial racemate; however, attempts to separate it into two stereoisomers failed. The idea of a partial racemate led to the question as to whether Lmannonic acid and wgluconic acid (which are enantiomorphous on carbon atoms 3,4,and 5) could form such a partial racemate (which would still be optically active). Such a compound could not be isolated, but (‘negative results have only limited vdues as proof.” Soon afterwards, the bmannonic acid was recognized to be a homogeneous substance, and hence an unequivocal configuration had to be assigned to C-2 of its molecule. This meant that the three &symmetriccenters brought in by the arabinose had determined the configuration of the new asymmetric center produced in the cyanohydrin synthesis. In 1898, when a small amount of the nitrile of Lgluconic acid was found in the same reaction mixture, he wrote: “The simultaneous formation of two stereoisomeric products on the addition of hydrocyanic acid to aldehydes, which has been observed here for the first time, is quite noteworthy, theoretically as well as practically”; and, in 1894: ‘(These observations are, to my knowledge, the first definitive evidence that further synthesis with asymmetric systems proceeds in an asymmetric manner. Although this statement does not at all contradict theory, it is by no means a consequence of it.” Not until then could he attempt to derive the configurations of the individual sugars, “encouraged by the excellent agreement with theory of progressing observations which often appeared surprising to me.” The concepts thus obtained touch on the phenomenon of “assimilation.” Fischer wrote in 1890: “Chemical synthesis leads. . . to optically inactive acrose. In contrast to this, only active sugars have so far been found in plants. No known fact contradicts the supposition that the plant produces. . . first the inactive sugars which it then resolves, and uses the member8 of the d-mannitol series to build up starch, cellulose, inulin, and the liko, while using their optiattl i,wmers for other purposes, now unknown. . . Since then, I havo attempted it1 vain to find Z-glucose or

.

36

KARL FHEUDENBEM

Lfructom in leaves.” In 1894: “It is impossible to doubt that &glucosc and &fructose or their polysaccharides are produced predominantly, if not exclusively, by assimilation. In accord with the precepts of Pasteur, one notea a marked differencebetween the natural and the laboratory synthesis. As the latter glways yields inactive products, it may be said to proceed in a symmetrical manner. In reality, though, this is no longer true for campounds having several asymmetrically substituted carbon atoms. If the synthesis of hexoses from formaldehyde or glycerose or acrolein dibromide were to ttike place in a totally symmetrical manner, the chances of formation for each of the 16 isomeric aldoses or 8 ketoses would be equal. However, despite all efforts, I have not succeeded in finding another one of the known hexoses (naturally, in the racemic form) besides a-acrose.” A t that time, Fischer still considered the &acme (Dcsorbose) to be a sugar having an anomalous carbon chain. “It therefore, follows that particular configurations are preferred in chemical syntheses, and that there are equal chances for the mirror images only. . I have found that, once a molecule is asymmetric, its extension also proceeds in an asymmetric sense. If one should then consider that the mannononose, formed from mannosa by the asymmetric, three-fold addition of hydrocyanic acid, could be cleaved back into the ohginal hexose and the three-carbon addition product, it would be found that the latter would be an optically active system. The one active molecule would then have given rise to a second active one. It seems to me that this concept offers a simple solution for the enigma of natural asymmetric synthesis. According to the plant physiologists, carbohydrate formation takes place in the chlorophyll granule, which itself consists entirely of optically active substances. I can imagine that the formation of carbohydrates is preceded by the generation of a compound of carbonic acid or formaldehyde with those substances; and that then, since the combination is already asymmetric, the condensation to give the sugars also takes place in an asymmetric fashion. The final sugar would then be releaeed from the combination, and would later be used by the plant, as is known, to produce all the other organic components. Their asymmetry can thus be readily explained by the nature of the matwid from which they were produced. Of course, this also forms new chlorophyll nuclei which, in turn, produce active sugar. In this manner, the optical activity propagates from molecule to molecule, as life itself does from cell to cell. It iR, therefore, not necessary to attribute the formation of optically activa suhstmcas i n the plant to asymmetric foices lying outside the organism, as Pasteur had supposed. . . . This concept completely climinates the difference between natural and artificial synthesis. The advance of science has rcmoved this last chemical hiding place for the once 90 highly esteemed vis vitalia. Now we can produce active molecules

..

EMIL FISCHER AND HIS BCIENTIFIC WORK

37

artificially without the aid of an organism. Nevertheless, there is still one eamntial difference between chemical and natural synthesis. Laboratory experiments always initially produce an inactive product which must be resolved by special operations, whereas the process of assimilation leads directly to exclusively active sugars.” Emil Fischer could be highly astonished at unexpected results. This was also true of the thesis worked out by him that the configurations of individual groups in stereoisomeric substances can cause profound differences in their chemical, physical, and biochemical behavior. In contrast to all the other polyhydroxy dicarboxylic acids that he knew, mannaric acid reduces Fehling solution and shows a yellow coloration on heating with alkali. He was at first reluctant to believe that the constitution he had to assign to it was correct, although this was later established unequivocally. The differences in the chemical behavior of stereoisomers are especially striking when they react with other asymmetric systems. This applies especially to enzymes and, consequently, to biochemical reactions in general. An example is methyl 6deoxy-&D-g1ucopyranosidel which is hydrolyzed by emulsin, whereas the closely related methyl b-D-xylopyranoside is not attacked. In 1894, he wrote: ‘‘From the observations under discussicn, which hitherto could not be made to the same extent in any other group, it clearly follows that the same kind of isomerism also affects chemical transformations and brings about differences which are at least aa large as those found with unsaturated and cyclic stereoisomers.Alcoholic fermentation takes place with glucose, mannose, galactose, and fructose, which are all hexoses. However, the observation that glyceraldehyde and dihydroxyacetone, as well as mannononoseJl0can also be fermented shows the surprising fact that the most ordinary functions of a living organism depend more on the molecular geometry than on the composition of its nutrients. This represents a significant extension of Pasteur’s observation that micro-organiams alter only one of two enantiomorphs, and reminds one of a statement of Pasteur’s relating the different tastes of the two (stereo)isomeric asparagines to the asymmetry of neural substance. The action of enzymes and yeasts involves a far-reaching chemical process which takes place readily or not at all, depending on the configuration of the substrate upon which they act. Here, apparently, the geometric structure has such a profound influonce on the action of chemical affinities that it seemR to me permissible to compare the two molecules under reaction with B key and lock. If one wnrits to do justice to the kriowri fact that several yeasts can ferment a larger number of hexoses than other yeasts, the picture could be completed by the differentiation between a main key (19) Tlint thc mannononose waa fermentable was shown later to be an error.

38

KARL FIU!XJDENL)EItCf

and special keys.” Feeding experiments, carried out by others with isomeric sugars, showed that “the fermentable sugars are also the true precursors of glycogen. Moreover, it can be concluded from this that the organism can produce glucDse from its isomers fructose, mannose, and galactose. In the reverse manner, galactose, a component of lactose, is most probably produced by mammals from nutrient glucose.” A comment made by E. Fischer-probably incidentally-in 1904, is characteristic of the manner of thinking of the great synthetic chemist, and may serve as a final observation: “Only 6 of the 32 heptoses and only 2 of the 128 aonoses have been prepared. But, since these compounds have not yet been round in NaturePo and are, therefore, of only minor interest, their systematic elaboration may be left for a later period.”

IX. GENERALREFERENCES 1. E. Fischer, “Erinnerungen aus der Stramburger Studentenzeit (1872-1875),” in “Gemmelte Werke,” A. von Bseyer, ed., Vieweg und Sohn, Braunschweig, 1905, VOl. 1, p. xxi. 2. E. Fischer, “Untersuchungen ilber Kohlenhydrate und Fermente (1884-1908),” J. Springer, Berlin, 1909. 3. Fischer, E., “Untersuchungen Uber Depside und Cerbstoffe (190&1919),” J. Springer, Berlin, 1919. 4. A. von Harnack, “Grabrede flLr Emil Fischer,” 1919 (personally distributed). 6. C. Harries: B. Abderhalden, A. von Weinberg, E. Trendelenberg, and L. Lewin, “Dem Andenken an Emil Fhher,” N~um‘8amchqften,7, 841 (1919). 6. N. 0. Forster, “Emil Fischer Memorial Lecture,” J . C A n . Soc., 117, 1157 (1920).

7. K. HOWC~, “Emil Fischer, sein Leben und sein Werk,” Deutsche Chemische Geaellachaft, Berlin, Ber. (Special Issue), 1921. 8. E. Fieaher, “Aus Meinem Leben,” M. Bergmann, ed.,J. Springer, Berlin, 1922. 9. E. Fischer, “Untemchung ilber Kohlenhydrate und Fermente, I1 (1908-1919),” M. Bergmann, ed., J. Springer, Berlin, 1922. 10. M. Bergmann, “Emil Fischer,” in “Das Buch deutscher Chemiker,” 1930, Vol. 2. 11. B. Helferich, “Emil Fiaoher eum 100 Geburtetag,” Angeu. Chem., 66, 45 (1953). 12. K. Frendenberg, “Emil Fischer, ein Wegberaiter der Biochemie,” in “Foracher und Wissenechbftler im Heutigen Europa,” H. Schwerte and W. Spenpler, eds., G. Stalling, Oldenberg, 1955, Vol. 1, p. 158. 13. K. Freudenberg, “Ed1 Fischer” (Lecture), Jahresh. Heidelberger Akad. Wiss. 1945/66, 161 (1959).

14.

K. Freudecberg, “Emil Fischer,” in “Neue Deutsche Biographie,” Historkche

Kommiseion bei der Bayerischen Akademie der Wimnschaften, ed., Duncker und Humblot, Berlin, 1961, Vol. 5, p. 181. 16. B. Helferich, “Emil Fischer,” in “Great Chemists,” E. E. Farber, ed., Interecience, New York, N. Y.,1961, p. 983. (20) Later, some of the aldoheptoses (and wtuloees and nonulosrts) were actually

found in Nature.

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

BY N. K. KOCHETKOV A N D 0.S. CHIZHOV Instilute for Chemistry of Natural Produck, Academy of Seiencee, Moeoow, U. S. S. R.

I. Intioduction .......................................................... 11. The Basic Principles of Maas Spectrometry of Organic Compounds.. . . . . . . . . 1. Principal Designs of Maas Spectrometer, and the Physical Principles Involved. ..................... 2. Treatment of the Mass Spectra ...................... 3. Saope and Limitations of Mass Spectrometry. The Principles of Interpretation of Mass Spectra.. ......................................... 111. Maas Spectra of Carbohydrate Derivatives. . ............. 1. General Remarks.. .................... ............. 2. Monocyclic Derivatives: Pyranoid and Furanoid . . . . . . . . . . . . . . . . . 3. Monosaccharide Derivatives Having Fused Rings. 4. Acyclic Derivatives of Monosaccharides. . . . . . . . . . . . . . 5. Mass Spectra of Miscellaneous Carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusion. . . . . . . . . . . . . . . . . . . .

39 40

43

43 46 46

90

I. INTRODUCTION The present article deals with the applications of mass spectrometry to the structural analysis of carbohydrate derivatives. The mass-spectral techniqge has now become a useful supplement to chemical methods, and provides ready solution of a variety of problems which were a stumbling block in the classical approaches. The principles of mass spectrometry of organic compounds are described in a number of excellent reviews and monographs.l-‘ Hence, we shall not attempt to present here any theory of the mass-spectral method that is more comprehensive than that necessary for understanding the discussion. The mess-spectral method was first applied to carbohydrate derivatives in 1958, when Reed and coworkers6reported the mass spectra of D-glucose, I

(1) J. H. Beynon, “MBSRSpectrometry arid its Applicatiom to Organic Chemistry,”

IClsevier, Am~terdnm,Holland, I!)On. (2) I<. 13iemunn, ‘‘M~LRN 8pec4mmetr.y,” M d irtLw-Hill, New York, N. Y., 1962. (3) 11. !htlzikiewirz, (1. I)jenwwi, ancl I ). ll, WilliamR, “Interpretntioii of MRRR Spectra of 0rg;unic Compoiindw,” Jlolden - I k y , Ha11 Frltnciwco, Calif., 19G4. (4) K. Bismtrnn, Angeui. Cheni., 74, 102 (1962). ( 5 ) P. A. Pinan, R. I. Reed,and W. Snodden, (%en&.Inn. (Idondon),1172 (1958).

39

N. K. KOCHXTKOV AND 0. 8. CHIZHOV

40

D-galactose, methyl a- and 8-D-glucopyranosides, and a number of disaccharides, thus demonstrating that mass spectrometry is useful in the field of carbohydrate chemistry. In this study, the appearance potentials of CsHl~O~e ions were measured, and the results interpreted in terms of greater stability of the B-(D or L) (equatorial) anomer with respect to the CP(D or L) (axial) one in the CA conformation. More-systematic investigations of carbohydrate derivatives were started about four years later, when a number of publications appeared almost simultaneously from the laboratories of the Institute for Chemistry of Natural Products of the USSR Academy of Sciences,6the Massachusetts Institute of Technology,’ the University of Glasgow,R and the University of HamburgnA review summarizing the advances in the mass spectrometry of carbohydra Zes has been published,1° but it is already inadequate, because of extremely rapid progress in the field. Another survey“ is a very brief summary of the results published in a few papers dealing with the problems discusaed below.

11. THEBASICPRINCIPLES OF MAS^ SPECXROMETRY OF O R O ~ ICOMPOUNDS C 1. Principal Designs of Mass Spectrometer, and the Physical Principlee Involved

The mma spectrometry of organic compounds is based on fragmentation of organic molecules under electron impact, and differentiation of the resulting particles by use of the mass-to-charge relation. We shall not describe here the differences among several types of mass spectrometer especially designed for structural analysis of organic compounds, but shall only give general principles which are necessary for understanding how a mass spectrum arises. (6) N. K. Kochetkov, N. 5. Wulfson, 0.8.Chizhov, and B. M. Zolotarev, DokZ. A b d . Nauk SSSR, 141, 1369 (1962). (7) K. Biemana, H. K. Schnoee, and J. A. McCloskey, Chem. Ind. (London), 448

(1963). R. I. Reed, W. K. Reid, and J. M. Wilson, Aduan. Mae8 Specfrometry, Proc. Con., &ut, Oxford, 1961, Vol. 2, pp. 420-422. (9) H. Soharmam, Ph. D. Thesis, University of Hamburg, Germany, 1962. (10) H. Budrikiewica, C. Djehei, and D. H. William, ‘‘Structure Elucidation of Naturd Produota by M w Spectrometry,” Holden-Day, Ban Frmcisco, Calif., 1964, Vol. 3, pp. 203-240. (11) R. J. Ferrier and N. R.Williams, Chem. Znd. (London), 1696 (1964). (8)

M A R S RPEf?l’ROMETRY

41

OF CARROHYDRATE DERIVATIVES

A mam spectrometer consists of the following major parts: (a) inlet system; (b) ion source; (c) analytical system, and (d) amplifier-registration system. a. The Inlet Syetem.-This servw for introducing the sample into the instrument and for its evaporation. This part of the instrument is to be maintained a t a constant temperature (up to 300’) in a vacuum of about 10-6 mm. Hg, to evaporate substances having a low volatility. The inlet system is usually made of stainless steel. However, it has occasionally been observed that this material catalyzes the pyrolysis of some types of compound; therefore, a glass inlet system is often prelerred. The sample passes into the ion source from the heated reservoir through a diaphragm having a minutp hole (about 1-5p) which provides a constant rate of vapor flow. Special devices for the direct introduction of the sample into the ionsource chamber have been designed for substances that are unstable or have a very low volatility. Constancy of the rate of vapor flow is provided in this particular case by the constant evaporation temperature and by the presence of condensed phase.

b. The Ion Source.-This serves for exposing the substance under investigation to electrons having energy ranging from 5 to 70 eV, the latter being mmt commonly applied. The ionization potential of the majority of organic substances ranges between 7 and 12 eV. Electron impact on the organic molecules usually results in elimination of one electron (more rarely, two) and, thus, in the formation of a positively charged ion-the so-called “molecular” or “parent” ion-designatedI1* as M or P The molecular ion is subsequently involved in the two following types of reaction : (i) fragmentation and (ii) rearrangembnt.llb (i) The fragmentation process is the decomposition of the M ion, which is usually an ion-radical having an odd number of electrons, into two or more particles, one of which is charged and the other or others neutral @,

@

@

(lla) An electron may also “adhere” to the molecule, giving rise to a negative ion. The probability of this event, however, is less by far, and the yield of negative ions is many times lower than that of positive ones. Nevertheless, a number of publicationb (eee Reference 3, pp. 44-47) are concerned with masa spectral studies of negative ions. Thie approach has not yet been developed for carbohydrate derivatives. (Ilb) Collision of two charged particles may result in an intermolecular reaction giving rise to a new ion composed either of two whole particles or of their fragments. The intensitiea of the corresponding peaks of the maw spectrum are proportional to the square of the vapor pressure of the sample in the ion source. This differentiatea such ions from the usual ones, which are present in concentrations proportional to the vapor pressure.

N.

42

I(.

KOCHETKOV AND 0. 8 . CHIZHOV

(molccules having ail wen riumhr of clcctrons, or radicals having an odd number of c!lcctrorm). The char,q!tl fragments formed from the molecular ion may aim rlcwmponc furtjhor, prodiiciny Rmallor ions. (ii) If iwolviny tiot OIJIY clcl~vltgcof the boiids present in the startirig molecule, but alw, the formatioii of ricw ones, the fragmentation is termed rearrangement. Ioris produced by rearrangement are ofteii very stable, and give rise to intense peaks in the mass spectrum, The above considerations can be illustrated by the following scheme, where ABC is a molecule consisting of the moieties A, B, and C.

/ A C '

+B'

/+A-B-C

A-B-C-A~

+ BC.

The abundance of an ion depends on: (i) its stability, (ii) the stability of neutral prtrticles formed contemporaneously with the ion, and (iii) the energy of the bond(s) cleaved during formation of the ion. The stability of ions and radicals is determined by the usual structural features long known in organic chemistry: for example, tertiary ions and radicals are thermodynamically favored over secondary and primary ones; and conjugation with a double bond, an aromatic system, or a pair of r-electrons of a hetero atom increases the stability of ions and radicals. Therefore, rupture of the bonds takes place in accordance with the rules mentioned above, and there is a correlation between the structure and the maas spectrum of the compound that makes possible the study of structures of organic molecules by m a n s of the mawspectral method. C. Analysis of the Ions Arising in the Ionic Source.-For a mass spectrum, this is accomplished as follows: the potential applied pushes the ions out of the ion-source chamber; the beam obtained is focused, accelerated in an electrical field, and directed into a magnetic one.110The deviation of ions in the magnetic fidd is the greater, the less is the mass of the particle and the greater its charge. At any given magnetic-field tension, particles of a given mass-to-charge ratio ( m / e ) reach the collector. d. Recording and Measurement of a Mass Spectrum.-These are carried out as follows. The collector system registers the intensity of the (llc) Focusing is performed according either to direction or energy. Doublefocusing instruments have also become available; these are gradually displacing the usual ones.

MAS6 SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

43

ionic current, which is directly proportional to the iiunihcr of ions. Thc ionic current is subsequently amplified, and registered by a recording potentiometer or an oscillograph. If ion a gives rise to another one ( b ) and to a noncharged particle (c) on the way from the ionic-source chamber to the magnetic field, the registering recorder writes a diffuse peak of low intensity, a so-called “metastable” peak. Its position in the mass spectrum is related to those of peaks a and b by the equation: m = b2/a.

The identification of metastable peaks is of great importance, being a direct, experimental demonstration of the process ae-+be

+ c.

2. Treatment of the Mass Spectra Obtained The peaks of the mass spectrum often differ by a factor of several hundred in their intensity. Therefore, the registering system of a mass spectrometer records the spectrum simultaneously by several channels which differ in the magnitude of amplification. On the other hand, the intensity of the peaks is also dependent on the amount of substance introduced into the ion source. Hence, it is necessary to subject the spectrum obtained to additional treatment, which consists of relating all the peak intensities to that of a reference one. The most intense, or “major,” peak of the spectrum is commonly taken as the reference. The relative intensity is independent of the amount of substance, and all of the peaks can be expressed in one scale. The peak intensities may be also expressed in per cent of the total intensity of all the peaks of the mass spectrum; this form is preferred when the mass spectra of several related compounds are to be compared, because the total peak intensity of the mass spectra of related compounds varies insignificantly, whereas the intensity of the (‘major” peak may, in this case, change very strongly.

3. Scope and Limitations of Mass Spectrometry. The Principles of Interpretation of Mass Spectra One of the major advantages of the mass-spectrometry method is that only a very small amount of a substance is required. The measurement of a mass spectrum is made on about 0.1 mg. of substance (and, sometimes, on even sribller amounts, down to 0.1-1.0 p g . ) . Moreover, the major limitation here is not the sensitivity of the method, but the difficulties in manipulating such small samples. Mass Epectrometry is used in organic chemistry for (a) molecular-weight

44

N. K. KOCHETKOV AND 0. 8. CHIZHOV

determination, (b) elementary analysis, (c) determination of functional substituenta, and (d) studying the structure of the molecule. a. Molecular-weight

Determination.-By

measurement of the

m/e value for a molecular ion, tho maes-spectral method permits thc determination of molecular weights up to ~2~ mass uiiits (ni. u.).

Compounds having greater molecular weights seldom provide sufficient vapor pressure. Special methods have been elaborated for identification of the moiecular ion (Ma) among the fragment ions (see, for example, Reference 4). Unfortunately, owing to the extreme instability of the carbohydrate molecule, thc molecular ion can only occasionally be traced in the mass spectra of cerbohydrate derivatives. b. Elementary Analysis.-Two methods for elucidating the elementary compositiou from the data of a mass spectrum have been proposed. The first is based on the relative intensities of isotopic peaks, and the other, on the position of the M a peak measured with an accuracy of 0.001 m.u. (see, for exaniple, Reference 1). The second method needs an instrument having &n extremely high resolving power (a double-focusing mass spectrometer). Special tables‘ have been computed for substances of molecular weight up to 250 mass units. A method for calculating the empirical formula on the basis of an accurately determined molecular weight has also been proposed foz compounds of greater molecular weight.I2 Analogous methods are used for memuring the molecular weight of fragmenta, precise determination of the latter sometimes considerably facilitating interpretation of the mass spectra. c. Determination of Functional Groups and Substituents.Interpretation of the mass spectrum of an unknown substance usually starts with a consideration of the high mass-number region, beginning from the M e ion. The peaks having m/e values immediately following that of the M eion can be presented as M - R, where R is the part of the moleculc eliminated. For example, the presence of peak M-17 (OH) or M-18 (HzO) is usually attributed to the presence of a hydroxyl group;‘of peaks M-15 (CHa), to the presence of -C-CH8 groupings; of peaks M-31 (CHoO) or M-32 (CHaOH), to the presence of a methoxyl group; of M-29, to elimination of - C H 4 or CpHs; of M 4 2 , to elimination.of ketene ( C H A P O ) ; of M 4 3 , to elimination of acetyl (CHaCO); of M-59, to elimination of acetoxyl (CHaCOZ) ; and of M-60, to elimination of acetic acid (CHaCOZH) or methyl formate (HCO&H3). For resolving (12) J. Lederberg, “Computation of Molecular Formula for Mass Spectrometry,” Holden-Day, San Francisco, Calif., 1964. See also, Ref. 10, pp. 279-297.

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

45

isobaric fragments (having the same molecular weight but different elementary composition), mass spectra of high resolution, or isotopic labels, are used. Lastly, attention should be paid to the presence in the spectrum of M-R peaks (with R corresponding to an improbable, or even impossible, combination of atoms) from M-3 to M-12, M-19, M-22, M-35, and so on. The presence of such peaks is usually regarded as an indication of impurity in the sample. d. The Principles of Interpretation of Mass Spectra.-As already mentioned, several methods can be applied for elucidation of the structure of a fragment (or of several isobaric fragments) corresponding to a peak at a given m/e value. The elementary composition of the fragment may be calculated from the high-resolution spectrum. Introduction of an isotopic label or of substituents (“chemical labels”) into known positions of the molecule may produce indication of the presence of the corresponding part of the molecule in the fragment under investigation. If the label is not incorporated by the corresponding fragment, the peaks remain in the same positions, tiit a shift of the peak indicates incorporation of the label. Identification of metastable peaks can serve as a basis for tracing the genetic relationship between a series of fragments, and thus considerably facilitates interpretation. e. Limitations.-Three types of limitation are involved : those caused by (i) volatility of substances, (ii) the design of the instrument, and (iii) the chemical nature of the processes taking place in the mass spectrometer. It is usually possible to measure mass spectra with the usual type of mass spectrometer if the substance provides a vapor pressure of mm. Hg (or more) a t 150-250”. If the substance has a lower volatility, it is usually either converted into a more volatile derivative or investigated by use of an instrument so constructed that the sample may be introduced directly into the ion source. In the latter case, volatilities down to lo-’ mm. Hg appear sufficient. The separation of ions in the mass spectrometer is caused by the action of a magnetic field At increasing mass numbers, the distance between the M and the M+1 peaks becomes smaller. The usual instruments provide a good resolution in the region up to 600-700 mass units. Special, highresolution, mass spectrometers completely remove this limitation, and the only one remaining appears to be the volatility factor. The third type of limitation follows from the previously mentioned fact that the relative intensity of the peak is primarily determined by the free energy of (a) the corresponding fragment and (b) the neutral particles

N. K. KOCHETKOV AND 0. 8. CHIZHOV

46

arising duriiig its formation; to a far smaller extent, it depends on the energy of t.he bonds of the molecule cleaved. As only the latter is sensitive to steric effects, the mawspectral method appears mainly valid for deducing the structure of compounds (and, to a limited extent, for studying their stereochemistry)

.

111. MASSSPECTRA O ~ TCARBOHYDRATE DERIVATIVES 1. General Remark8

Mono-, oligo-, and (certainly) poly-saccharides baing thermlly unstable and practically nonvolatile, mass-spectral studies on them have been performed on their more volatile derivatives, such as, methyl ethers, acetates, and alkylidene derivatives. The characteristic feature of almost all masa spectra of carbohydrate derivatives studied is the absence of the peak of the molecular ion. The steric dieerences are occasionally reflected only by changing relative intensities of some of the peaks, and, hence, the mass-spectral method cannot at present claim any wide application to stereochemical studies in the field of carbohydrate derivatives, although such a possibility may develop after progress has been made in the techniques of mass spectrometry. On the other hand, the method permits ready solution of a wide variety of structural problems. Among these are: choice between the acyclic and cyclic forms; choice between furanoid and pyranoid f o m ; determination of the positions of substituents (for example, the position of hydroxyl groups in partially methylated monosaccharides, the position of linkage of the monosaccharide residues in disaccharides, the position of the methylene group in deoxy sugars and of the amino group in amino sugars) ; choice between aldoses and ketoses; and determination of the number of carbon atoms in the major chain (for example, choice between pentoses and hexoses). The cyclic forms-pyranoid and furanoid-have been the most extensively studied among the derivatives of monosaccharides. The mass spectra of these compounds have been interpreted on the basis of numerous analyses using labeled analogs The compounds investigated in the greatest detail are probably the methyl ethers.l*-U As regards the studies made on acetates,1oalthough these were performed without selective labeling of all (13) N. K. Kochetkov, N. S.Wulfson, 0.8. Chiehov, and B. M. Zolotarev, Tetrahedron, (1963) ; Tzu. A M . Nauk SSSR, Ser. Khim., 776 (1966). 19, (14) K. Heyne and D. Mtiller, Tetrahedron, 81, 66 (1965). (15) N. K. Kochetkov and 0 . 8 . Chizhov, Tetrahedron, 81, 2029 (1965). (16) K. Biemann, D. C. Ddongh, and H. K. Schnoes, J . Am. Chem. Soc., 86, 1736 (1903).

MASS SPECTROMETBY OF CARBOHYDRATE DERIVATIVES

47

of the sugar chaiii, the fragmentation patterns proposed also appear to be convincing. Therefore, hi discussiori of the fragmentation patterns, we shall concentrate on the results obtained with these two types of derivative. Alkylidene derivatives and anhydro sugars will be discussed next, as the presence of additional rings bears strongly on the fragmentation of pyranoid and furanoid rings. Finally, the mass spectra of acyclic carbohydrate derivatives will be considered: in many aspects, these differ essentially from those of the cyclic form. In accordance with the proposal of Budzikiewicz, Djerassi, and William~,~ we shall denote the shift of a pair of electrons as and of a single electron as -. The seriw of ions related by similar structure or common origin will be denoted as capital letters (A, B, C, . . . Y, 2, AA, BB, CC, . . .). The subscript numeral will denote the number of steps needed for the formation of the fragment from the molecular ion, and the superscript numeral, the ordinal number of the isomeric ion. The lowest ordinal number is given to the isomer having the substituent at the carbon atom of lowest ordinal number or, respectively, containing the lowest ordinal carbon atoms.15 For example, the symbol Af means that the ion arises as the result of a two-step transformation of M EI ion, being the second of several possible isomers. The lowest ionization potential must be characteristic of oxygen atoms at C-1, the ring-oxygen atom, and the glycosidic oxygen atom. In his pioneering studies, Reede suggested that the ionization of a carbohydrate molecule begins from the glycosidic center. We assume that the molecular ion is formed by removal of an electron from the ring-oxygen atom (1, 2), and all the data available may be reasonably interpreted on the basis of this assumption.

c,” 2. Monocyclic Derivativest Pyranoid and Furanoid a. Fragmentation of the Pyranose and the Furanose Ring in Monosaccharides.-The major fragmentation of pyranoid derivatives (and, also, of rather similar ones of furanoses) after electron impact is

48

N. E. EOCHETKOV AND

0 . 8. CHIZHOV

best illustmted by the mass spectrum of methyl 2 , 3 ,4-tri-O-methyl-p-~a r a b i n ~ p ~ n o s i d epresented , ' ~ ~ ~ ~in~ Fig. ~ 1. As mentioned previously, the ioniaation is assumed to begin with the removal of an electron from the ring-oxygen atom. The degradation of the pyranoid ring is usually initiated by clesvege of one of the bonds occupying the position 19 to this oxygen atom.The C-1 to C-2 bond gives rise to peaks of the series C, D, F, H, K ; the C-4 to C-5 bond, to peaks of the series J, H, B, D; and the C-1 to 0 bond (glycosidic), to peaks of series A. This is followed by rupture of one of the 4 C - 5 to 0 ring, for ions of the C, K, and H series; and the C-1 to 0 ring for ions of the B and H series) or of y-bonds (C-4 to C-3 for ions of the H and K series; C-3 to C-2 for ion Ha a2d related ions).

FIQ. I.-Msss Spectrum of Methyl 2,3,eT~~methyl-&lrarabinopyrsnoside.

The subsequent degradation of the fragments initially formed proceeds through elimination of CHaOH, CHaO-, CHa., CHzOCHa, or (CH8O)zCH.; the formation of ions producing the most intense peaks of the mass spectrum often invalves1*J6J7migmtion of a methoxyl group from C-3 to C-1. Ions of the A, C, and F series tend to eliminate a methoxyl group from C-3 and to retain methoxyl groups a t C-2 and C-4; those of the H and G series, to retain methoxyl groups at C-2 and C 3 ; those of the D and J series, to retain methoxyl groups a t C-1 and C-3; and the ion K1,to retain a methoxyl group at C-4.

Ions of the A series are produced by Ioss of substituent from C-1, with subsequent, stepwise elimination of other substituents. This series is represented by peaks a t m/e 175 (Al), 143 (A:, A:, A:) and 111 (A:, A:, A!). (17) N. IC. Kochetkov, 0. S. Chhhov, and B. M. Zolotarev, Izu. Akad. Nauk SSSR, Sw. Ichim., in prees.

49

MASS BPECTROMDTRY OF CAHBOHYDRATE DERIVATIVE&

The formation of these ions is explained by the following scheme:

Ax,m/e

4

175

A:,,m/e 111

AD

The ions A: and A: produce a relatively minor contribution to the intensity of the peaks at m/e 143,thus indicating favored elimination from (2-3. Ion B1 (m/e 176), the parent ion of the B series, is formed by elimination of C-5 and oxygen as formaldehyde:

I

OM0

L

I

OMe

.

AMe Bl,m/e 176

Another way is possible:

Bl,m/e 176

It decomposes subsequently either by elimination of a hydrogen atom and the > CHOCHI p u p as the CHzOCHa radical, to give rise to ion

50

Bt

N. K. ICOCHETKOV A N D 0. 8. CHIZHOV

(m/e 131) :

a

0 M@=CH-

OMe H-OMe H-OM0

__c

=CH-OMe

+ CKOMe

B,,m/e 191

or by producing ions F:, J: or J:: ?M=

0 0 MeO-CH=OMe

-----+

+ MeOCH=CH-6HOMe

J:, m/e 75

a3

-

MdHOMe

CB

+ M@=CH-CH=CH-OMe q,m/e

101

Series C. The next important series is initiated by cleavage of the C-1 to C3 bond:

-

8

- HCO&Ie

[ e O cOMe y O M e

M e O OMe q : M e

i

OMe

OMe

M@

OMe C , , m / e 148

M?

< :qoMe MeOH

Me0 Or

(OMe

Or

MeO

Ale

OMe

Me

H

51

MASS SPECTNOMETHY OF CAliBOHYDlZATE DElUVATlVES

The ion-radical C1 (m/e 146) initially formed is not traced in the mass spectrum; bein'g extremely unstable, it must immediately lose one of the methoxyl groups as CH30*,giving rise to isomeric ions Ci, C:, and C: with m/e 115, the major contribution to the corresponding peak being provided by the ion C:.

The ion8 of the D, F,and J seriea. Another pathway of fragmentation of the M1 ion leads to the ion of D1 (m/e 105). Subsequent destruction of the D1 ion gives rise to a new type of ions (J series). 0 M a p O M O

-

0 OMe

MeO-CH=CH-CH-OMe

+ c~&=~-A~fbaae

/

rOMe 'CH-OMe

D,,m/e

105

CI&O f MeO-CH=OMe J:,m/e 75

This pathway is probably the major one of those giving rise to ion J: (m/e 75). The fragmentation of the M1@ion with cleavage of the same bondings but with different distribution of charges on the fragments produced leads to ion F: ( m/e 101) 0 MeO-CH=CH-CH=OMe

q , m / e 101

+

___1c

CH,O

+ CH(OM~),

This process is evidently the major one of those affording the ion I?:. Ion F: contributes about 80% of the total intensity of the m/e 101 peak. About 12% of this peak intensity is contributed by ion G::

OMe

N. K. KOCHETKOV AND 9. 8. CHIZHOV

52

H and K series. A conjugated electronic shift gives rise to series H and K, depending on the charge localization:

OMe

C q O t MeO-CH=CH-OMe

i

[MeO-CB=CH-OMe]

Hi, m/e

@

88

OM0

-

CqO

+ MeO-CH=CH-OMe

t [MeO-CH=CH-OMe] I

@

*

mfe 88

OMe

HCO#e

+ MeO-CH=CH

-0Me

+ [MeO-CH=C€&,]

@

*

X,,m/e 58

The other possible murce of ions Hi and H: is the cleavage of ion B1in two, The grtmtest contribution, about SO%, to the intensity of peak m/e 88 is provided by ion Hi. Ions Hi,Hi,and H: eliminate a CH,radical, affording ions HI(m/e73) : Me-O-CH-CH u

+

OMe + CH,.

+0

-

@

CH-CH

= OMe

HI, mle 73

The above-outlinctl, fragmentation patterns have been deduced14J?from amthe mss spectra of methyl 2 J ,4-tri-O-methyl-pC.~-arabii~opyrar~oside logs containing CDsO groups at (3-2, (2-3, and (3-4. These fragmentation patterns may now serve m a basis for deciding the position(s) of CD90 group(s) from the characteristic shifts iri the positioii of some peaks of the

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

53

mass spectrum. This approach is used for the structural analysis of partially methylated monosaccharides (see Section 111, 2d, p. 71). The character of the'fragmentation of the pyranoid ring is only slightly affected by the nature of substituents on oxygen atoms, although the degradation of acetates ft8 compared with that of methyl ethers differs in the weakening (or even disappearance) of some of the characteristic directions and in intensification of others. This may be illustrated by the mass spectrum of methyl 8-Bxylopyranoside triacetate.ls This spectrum contains peaks of the A (m/e 259, 139, 97), C (m/e 230, 170), and F (m/e 157, 115, 73) series. The structures of the corresponding ions are analogous to those obtained from methyl tri-O-methyl++arabinopyranoside. The peaks of ions belonging to the B, D, H, J, and K series are absent from the mass spectrum. A characteristic featwe of the mass spectra of the polyacetates is the presence of ion M+43 formed by intemction of ion CHoCO@with the starting, neutral molecule. The mass spectra of acetates are alw, characterized by the presence of triacetoxonium (m/e 145) and diacetoxonium (m/e 103) ions: + + Ac-O-Ac

A&Ac

I

Ac

I H

m/e 145

m/s 103

The most intense peak in the spectrum of acetates is that of the acetyl ion CHsCO* (m/e 43)) whereas the corresponding ion CHsO in the methyl ether series is unstable and produces only a small peak. Elimination of methoxyl is possible in only two ways, as CHsO. or CHsOH, but acetoxyl is Bliminated in four ways, as CH&OzH, CHsCOO-, CH&O*, or CHp==C==O. The last is most characteristic of the unsaturated species fornied by the elimination of C H I C O :~ ~ @

Hence, several peaks appear in the mass spectrum of acetates, in place of a single peak in the spectrum of a methyl ether, thus complicating the interpretation. On the other hand, acetates, 8 s mentioned above, are involved in a smaller number of degradation pathways. (18)

K.Biemann and D. C. Ddongh, J . Am. Chem. Soc.,

86, 2289 (1963).

54

N. K. KOCHETKOV AND 0 . 8. CHIZHOV

The mass speclra of methyl glycosides having unprotected hydroxyl groure have beeti invcstigated less than those of carbohydrate methyl ' he data obtained in different laboratories are someethers and acetates. 1 what contradict.ary.*s"O However, it may be considered that the fragmentation of mcthyl glyoopyranosides must resemble that of methyl ethers even more than does that of acetates. Thus, the mass spectrum of methyl 8-L-arabinopyranoside contains peaks at m/e 133 (peak At analog) , m/e 73 (analog of F1 and G1), m/e 60 (amlog of HI) , and so on. The characteristic feature of the maas spectra of methyl glycopyranosides is the presence of peaks M+1. The general similarity of the fragmentation patterns of carbohydrate methyl ethers, acetates, and unprotected methyl glycosides makes possible 8 for the identification of partially methylated the rse of ~ & 8 spectrometry monosaccharides. The fragmentation pattern of acetated8 of monomethyl derivatives tlre very similar to those of completely acetylated sugars; mass spectra of both the latter and the former contain peaks of the A, C, and F series. Ao mentioned above, iona of these series tend to eliminate the substituent from C 3 . Therefore, a characteristic difference is observed: for 3-O-methyl-~-xyloseacetate, the CI and F1 peaks are situated at m/e 170 and 157 (elimination of CHaO), but the same peaks in 2-O-methyl-~-xylose acetate are shifted 28 mass units (the difference between CHsO and CHsC02) to m/e 142 and 129, respectively.18 Accumulation of methyl groups in the molecule leads to a change in the mass spectrum, that is, in the appearance of the H and K series, characteristic of permethyl ethers. Thw fact is explained by the greater electrondonating power of the CHaO group compared with that of the acetoxyl group, which facilitates the cleavage of adjacent carbon-carbon bonds and the formation of stable ions. A more detailed account of the mass spectra of partially methylated monosaccharides is given in Section 111, 2d (see p. 71).

b. The Effect of Substituents on the Mass Spectrum. (i) Subetituents at C-S.-On proceeding from pentopyranoses to hexopyranosea, characteristic changes occur in the mass spectra; these changes serve as a basis for referring the compounds to one of the two classes. The general fragmentation pattern of hexopyranoses has much in common with that of pentopyranosos. For the methyl ethers,l4 the peBks of the B, F, G, H, arid J Rerim retain their poaitions, the only difference being that peak m/c! 88 (HI) becomw morc iritensc than that at m/e 101 (Fl, GI). The p e a b of ions of the A, C, D, and K series containing C-5 are shifted 44 mass units toward greater m/e values, this shift corresponding to substitution of the CHaOCHr group for the hydrogen atom. (19) K. Heyns and H. Scharmann, Ann., 677, 183 (1903).

(20) P. A. Finan, R. I. Reed, W. Snedden, and J. M. Wilson, J . Chem. Em.,6946 (1903).

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

55

Series E, Besides this, a new series of peaks (E) of low intensity appears, corresponding to ions formed by elimination of the side chain:

I

OMe

E,, m/e 111

E,,m/e

141

Aimlogous phcnomeiia take place in the case of acctates:

m/e 289 (M-73)

(ii) Substituents at C-1.-As already mentioned, the fragmentation of carbohydrate molecules in cyclic forms is believed to be initiated by ionbation of the ring-oxygen atom. Hence, it m a y be anticipated that

N. K. K0CHB)TKOV AND 0. 8. CHIZHOV

56

substituents at C-1 will bear most strongly upon the fragmentation pattern. Among the pennethyl ethers, Dglucose derivatives have been studied the most extensively: the mass spectra have been reported for methyl 2 ,3 ,4 ,6tetra-0-methyl-a- and @-D-glucosides, phenyi tet-0-methyl+ and @-Dglucopyranoaides,Mpl'and l-0-acetyl-2 ,3,4 ,6-tetra-0-methyl-@-~gluco~e.~~

(3) (4) (5)

R R R

=

H,

R' = OMe

= OMe, R' = H = H, R' = OPh

(6) R = OPh, R' = H (7) R = OH, It' = H (8) R = OAC, R' = H

The maas spectra of compounds (3) and (4) contain identical sets of peaks. The marked difference between them consisb of the different ratio of intensities of peaks m/e 176 (BI) and m/e 187 (AS). The a-D anomer (3) produces the A* peak about twice as intense as BI, a reversed relationship being found with the &D anomer (4). This may be explained by the fact that, in the CA conformation of the CY-D anomer, the methoxyl group at C-1 occupies the sterically unfavorable axial position, so that its elimination (leading to fragments of the A series) is facilitated (compare the rate of solvcdysis of axial and equatorial ptoluenesulfonates or halides,a the so-called "steric acceleration"). An analogous regularity is observed with other anomeric pairs of permethylated methyl glycosides, such as the methyl tetra-0-methyl-D-galactopyranosides and the methyl tetra-0methyl-D-mannopyranosides,l*~zl This phenomenon can thus be used for assigning the configuration at C-1 in methyl glycopyranosides if the conformation is known. The anomeric phenyl glycosides (5) and ( 6 ) afford practically identical mass spectra. A characteristic feature of the mass speotra of the phenyl glycosides is the strong increase of intensity of the series A fragments (m/e 219, 187, 155) and of the Ed (m/e 111) fragment. These ions are formed by elimination of the phenoxyl grouping as phenol or as the CsHbO. radical; the latter is rather stable, due to interaction of the odd electron with the beneene ring. At the same time, peaks of the series B, C, D, and E (except Ed) become less intense, or even disappear completely from the mass spectrum. The decomposition of compound (7), having a free reducing.group, is (21) N. K. Kochetkov, N. 8.Wulfeon, 0. 8.Chirhov, and B. M. Zolotarev, Dokl. Alcad. Nouk S88R,161, 336 (1963). (22) 8.Whtein and N.J. Holnea, J . Am. Chem. Soc., 77,5562 (1955).

57

MASS SPECFROMETBY OF CARBOHYDRATE DERIVATIVES

essentially different from that of the corresponding glycosides.laV2a The 8pcctrum contains a peak of low intensity at m/e 234 (M-2), and the formation of the most intense peaks of the spectrum can be explained by the decomposition of the componding ion:

OM0

OM0

/MaQ@

m/e 234

- hi0

MmH,

MeOCH,

--

Q@

MeO

OMe

-MeoH

-

m/e 141

OMe

m/e 205

m/e 173

There is also a n intense peak a t m/e 130 which must also be due to the decomposition of ion m/e 234 :

- O=C-CH,OMe MeO G

o

OMe I MeO=CH-CH-CH-CH=O I OMe

-[

OMe m/e 161

m/e 234

_/_/_/_ - MeO'

OMe

[MeO-CH=CH-&H-CH=O]'O m/e 130

- CH=O

@ Ma-CCH=CH-CH=OMe q , m / e 101

Elimination of CHO from this fragment gives rise to ion F:, m/e 101. (23)B. M. Zolotarev and 0.S. Chiehov, unpublished data.

1

58

N. K. KOCHETEOV AND 0 .

8. CHIZHOV

Beside8 thew peaks, the ma^ spectrum of (7) reveals the presence of the A series [m/e 218 (M-18), m/e 187 (218-31), and m/e 186 (218-32)], of the E series (m/e 191,159,141, and lll), of the C series (m/e 159, 127), and of the H series (m/e 88, 75). The peaks of these series are of low intensity. The ions of the B series and D1 are abseiit. Hencc?, the iiutjor directioii of the fragmentation of reducing sugars must be dehydrogeiiatioii involviug, most probably, opening of the ring. Analogous processes are naturally impossible with 1-0-substituted derivatives. The m m spectra of the monosaccharides having a free hydroxyl group at C-1 are characterized by a rather poor reproducibility of the relative intensities; this must be due to the lower thennostability of these compounds compared with that of the 1-O-aubstituted derivatives. For this reason, analytical application to this type of compound does not seem very promising.lc The maw spectrum of (8) is characterized by an extremely low intensity of the series A, C,E,H, and J, and by the absence of series B and D due to the lower electron-donating power of the CHGO group compared with The ions of the series C, D, H, and J could indeed be formed that of CH80. after cleavage of the C-1 to C-2-bond leading to an ion-radical of the

MIetype. Substitution of the OCHa grouping for OCOCHa in this ion must lower its stability. The fragment FI, its formation being much less affected by this substitution, gives rise to the major peak of the spectrum.

d!H=OMe

@

The diminished intensity of the A Reries is most probably due to the decreased Rtability of CHaCOO. compared with CHsO-,and the low intensity (24) B. M. Zolotarev and L. 8.Golovkina, unpublished data.

MASS SPECTROMETHY OF CAHBOHYDRATE DERIVATIVES

59

of the E series is due to lower stability of the ion E? compared with ion El:

bMe

OMe

~ , A a , m / ea33

El,m/e 205

Analogouo regularities are also observed with sugw acetates.16Thus, the intensity of the A1 (m/e 331) fragment increases in the sequence ( 9 ) < (10) < ( l l ) ,being, respectively, 1.5, 3.0, and 30 % of the m/e 115 peak.

R' (0) R = OAc, R' = H (10) R = OMe, R' = H

(11)R=OPh, R ' = H R' = OAc

(12) R = H,

Also, the A1 peak ( m / e 331) in the spectrum of a-D-glucose penbcetate (12) is more intense than in that of the &D anomer (9). An analogous phenomenon has also been observed with D-mannose pentascetate and saltrose pentaacetate. The increase in intensity of the A1 peak in the mass spectra of CPD( or L) anomers may be explained as due to the unfavorable steric position of the OAc group a t C-1, analogous to that in the methyl ethers.

(iii) Configurations at C-2 to C-S.-As a rule, the mass spectra of stereoisomers differ only in the relative intensity of some of the peaks, this difference often not exceeding the experimental error13J6given by modern mass spectrometers. The behavior of isotopically labeled methyl tetra-0-m2thvl-a-wglucoand -8-n-galactopyranosidela,I6suggests that the fragmentation of these two stereoisomers proceeds in an essentially similar manner, the contributions of isomeric ions to the corresponding peak intensities being of the same order of magnitude for both. Hence, it may be anticipated that the evidence obtained by studying labeled analogs of one of the stereoisomers in the monosaccharide series may also be used for elucidating the position of the label in the other stereoisomers (see Section 111, 2d, p. 71).

60

N. K. KOCHETKOV AND

0 . 8. CHIZHOV

Applioation of mass spectrometry to stereochemical analysis in the monosaccharide series is limited by the lack of experimental studies, by the absence of a generalizing, theoretical concept based upon modern confoimational analysis, and by lack of precise knowledge of the mechanism and stereochemistry of the reactions taking place in the maw spectrometer, The only investigation in which a thorough study has been made of the effect of the stereochemical features of carbohydrates upon their mass spectra is that of Heyns and Scharmann," who measured the mass spectra of a number of permethylated methyl pentopyranosides, namely, methyl tri-O-methyl-&D-ribo- (13),-arabino-( 14), -xylo- (15), and -1yxo-pyranosides (16),and of methyl tri-O-methyl-ar-D-lyxop~Tanoside (17). OMe Md%OMe

@OMe OMe Me0

Me0 lKeOM-0Me e0 Me0

(16)

k (16 R 0 H, R'= OMe (17j R = OMe, R' = H

It was demonstrated that the highest intensity of the peak B1 (m/e 176) is observed with (13)and (16),having three methoxyl groups cis to each other. All the other isomers produce a less intense peak a t this m/e value. The higheet intensity of peaks F1 and J (m/e 101 and 75) has been observed with (13) and (14). It is somewhat lower with (17)and lowest with (15) and (16).Hence, the formation of these ions is facilitated when the methoxyl groups at C-1 and C-3 are trans to each other. Peak HI(m/e 88) is the most intense in the mass spectra of (16)and (17),suggesting that the formation of the corresponding ion is favored by the 2,3-cis and 3,Ctrans configurations. The intensity of this peak is considerably reduced in the maas spectrum of riboside (13),where the methoxyl groups at C-3 and C-4 are cis. The intensity becomes still less in the mmis spectra of xyloside (IS)and arabinoside (13),where the methoxyl (25)

K.Heyne and H.Scharmann, T e - m ,

21, 607 (1965).

MASS SPECTROMETRY OF CARBOHYDUTE DERIVATIVES

61

groups a t C-2 and C-3 are trans. The orientation of the methoxyl group at Gl and C-2 is evidently of no impqrtance. The difference in the relative intensities can be used for distinguishing However, niore between all four methyl tri-0-methyl-#?-D-peiitopyranosides. definite identification needs consideration of a iiumber of the less intense peaks. A characteristic feature of the mass spectrum of the &D-lyxoside (16) which differentiates it from all the other isomers is the position of its major peak a t m/e 88. j3-DRiboside (13) may be recognized by the extremely low intensity of the m/e 175 peak. @-D-Arabhoside(14) can be distinguished from @-D-xyloside(IS)by the ratio of the intensities of peaks m/e 143 and 131: the former is greater than the latter for (14), and smaller than the latter for (15). Unfortunately, analogous data which could confirm the empirical regularities outlined above are not yet available for the a-(D or L) anomers of the permethylated methyl pentopyranosides. A dependence between the relative intensity of a number of peaks and the configuration at C-2 to C-5 has also been found for the permethylated methyl hexopyranosides.'O For example, the D1peak (m/e 149) in the is more intense spectrum of methyl tetra-0-methyl-cu-D-galactopyranoside than in that of methyl tetra-0-methyl-a-D-glum and -manno-pyranosides, and the peak m/e 145 has the highest intensity in the latter and is considerably diminished in the spectra of the two former compounds. The interpretation of this fact presented in the original paper1*is wrong, because the structures proposed for ions m/e 149 and 145 were incorrect (see References 13 and 15). (iv) The ring size.-The size of the ring in a monosaccharide bears strongly upon its mass spectrum, the characteristic differences providing a firm basis for distinguishing between pyranoid and furanoid derivatives,' ,'I,I8 s l 6 ,18,26 The most marked are the differences in the positions and intensities of peaks of the E series; these peaks are due to ions formed by fission of the side chain. As already mentioned, peaks of this series in the mass spectra of hexopyranoses are of low intensity. The corresponding fragments arise after cleavage of the C-5 to C-6 bond, for example, in @-D-galactopyranose pentagcetate : AcOCH,

0.

8

-

ACoQ

ACQ

OAC

OAC

E,,m/e 317

62

N. K. KOCHETKOV AND 0. 8. CHIZHOV

The isomeric &u-galactofuraiiose pentacetate gives rise to fragment E, by cleavage of the C-4 to (2-5 bond, and, therefore, the ion has a lower maas number:

Moreover, the peaks of the E series in the mass spectra of furanoses have an increased intensity, the h i o n of the side chain from the five-membered ring leading to planar oxinium ion, thermodynamically favored over the analogous ion having a six-membered ring.%

As revealed by the maas spectra of tetrahydrofuraldehyde diacetate

&t+ and truns-2,3-diacetoxytetrahydppns (19) ,= the formation of fragments of the E series may be complicated by rearrangements. As expected, the mass spectrum of (18) contains an intense peak at m/e 71 :

(18) and of

Surprisingly, the mass spectra of the cis and trans isomers of (19) appear to be practically identical with that of (18). The formation of the m/e 71 fragment may be explained by the following rearrangement:

The mass spectra of hexopyranose pentaacetates16contain small peaks a t m/e 245. An analogous peak a t m/e 187 in the mass spectrum of 2deoxy-~(25s) Compare, for example, the rates of hydrolysis of furanoaides and pyranoaides. (26) M. Venugopalan and C. B. Anderson, Chum. Znd. (London), 370 (1964).

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

63

or -~umbino-hexopyra.nosetetraacetatel" has a somewhat greater intensity. The formation of these ions may be explainedu by an analogous rearrangement:

-

- CH-C&-OAc

AcO'

m/e 187

Hence, the size of ring of monomccharides must be deduced from their mass spectra with caution, because of the possibility of rearrangements, especially in the case of derivatives of deoxy sugars. However, there still remain a number of characteristic features (not yet mentioned) which differentiate the mass spectra of furanoid from those of pyranoid compounds. For example, the mass spectrum of methyl tri-0methyl-a,&carabinofuranoside (see Fig. 2) contains no D and no BIions, their formation being structurally impossible. The peak a t m/e 88 is of low intensity, as the formation of both of the m/e 88 fragments (Hi and Hi) proceeds ccncomitantly with the formation of unfavored carbenes:

MeO-CH&H=O

-

QOMe MeOH,C

+

CH-OMe

+

OMe

[MeO-CH=CH-

Hi,m/e

OMe] ' @ 88

As already mentioned, several isomeric ions correspond td the peak at m/e 101. The major one of them in the methyl ether of the pyranoside series is the F: ion containing methoxyl groups of C-2 and C-4. An anal-

N. IC. KOCHETKOV AND

64

0. 8. CHIZKOV

(126 100 ’I

,

50

Fro. 2.-Mase

.

.

.

.

100

_ . . .

Spectrum of Methyl 2,3,BTrGO-methyl-cu,&ofur~oeide.

ogous ion cannot be formed in the fumnoside series, and more than 80% of the m/s 101peak is contributed by fragment Gfcontaining the methoxyl groups of C-2 and C3.This fragment contributes no more than 25% of the intensity in the pyranoside series. Although the characteristic differencm discussed above permit ready distinction between furanose and pyranose derivatives, their fragmentation patterns have much in common. The fragments of the A, C, and J series are formed in a s i m h r manner from both of the two types of derivatives, although there is a difference in the contribution of some of the isomeric iona,u*fl (v) Deoxy sugars.-The presence and the position of a methylene or C-methyl group in the chain of a carbohydrate molecule may be established on the basis of its maas spectrum. The effect of the methylene or C-methyl group on the mass spectrum may be illustrated by those of the permethyl ethers of methyl 2-deoxy-(20) , 3deoxy-( 21) , and ,&deoxy-“glucosides” (22) a and by t h t of methyl tri-O-methyl-fl-D-fucopyranoside(23) The presence of a “deoxy unit” bears first of all upon the directions of fragmentation involving adjacent bonds; the cleavage of these bonds proceeds less readily, as the particles of the RCHl* or RCHZ type are less stable than those of the RCH-0 WHa, or RCH-OCHa type. The general stabhation of the molecule is revealed by the presence of the M* ion pwk, its intensity decreasing in the sequence (20) > (21) > (22). To understand this phenomenon, it must be remembered that the fragmen.1619g

(27) 0. 8.Chihov a d N. F. Madudina, Zzu. A M . Nauk SBSR, Set. Khim., in pme. (28) N. K. Kochetkov, 0. 8. ChiShOV, and B. M. Zolotarev, Dokl. A M . Nauk S8SR, 165, 568 (1966).

(a)N. K.Koohetkov, 0. S. Chiihov, B. M. Zolotsrev, and V. Sh. Sheinker, lau. AM. Nauk SSSR, in press.

MA88 SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

65

tation of monosaccharide methyl ethers involves, first of all, cleavage of the C-1 to C-2 bond (the serim C, D, F, H, G,and J producing the most intense peaks).

OMe

OMe

The mass spectrum of the 2-deoxy compound (20) is characterbed by the absence of the C and D series, by a diminished intensity of the J peak (m/e 75), and by an increasing intensity of the E series. The formation of the fragments of the latter series is sterically favored because of the absence of substituents at (2-2. Characteristic features of the spectrum of the 3-deoxy compound (21) are the absence of the D1 peak, the diminished intensity of the C, H, and J series, and the high intensity of peaks corresponding to fragments Kl (m/e 102) and FZ (m/e 71). The mass spectrum of the 4deoxy compound (22) differs in showing the presence of the Dl peak (m/e 149), in the high intensity of the C and J series, and in a considerable decrease of intensity of the K1(m/e 102) and FI (m/e 101) peaks. As expected, the spectrum of the 6deoxyhexose (23)contains no peaks of the E series; however, the B series appears (m/e 176, 131), the peaks of the F, C,H, and J series have the same intensities and positions as with permethylated methyl hexosides, and the peaks of fragments involving C-5 and C-6 are shifted 30 mass units to lower m/e values compared with the peaks of permethylated methyl hexosides (DI, m/e 119; C2, m/e 129, and so on).

N. K. KOCHETKOV AND 0. 8. CHIZKOV

66

Hence, a number of characteristic features in the mas8 spectra of these four compounds apparently differentiate them from each other. The maaa spectra of acetates of deoxy sugars have been investigated in less detail: the masa spectra have been measured16only for f3-deoxy- and Zdeoxy-D-"glucoae."The same type of fragmentation has been demonstrated. (vi) Ketoses.-The structural difference between isomeric aldopyranoses and ketopyranoses causes a difference between their mass spectra sufficient to permit distinguishing between the two types of compounds. Mass spectra have been measured for ketose acetates only,16 and the most characteristic feature of these maas spectra is the formation of a new series of fragments having no analogy in the maaa spectra of other types of carbohydrate derivatives.

I Aco

0

-= -&OH

1

m/e 186

The fragments of the C series expected have the aame structures and m/e values as in the spectra of pentosea, and are shifted 72 mam units (the difference between H and CH20COCHs) to lower m/e values compared

with those of ieomerio aldohexoses. This feature may also serve to disQ ACO G

O

A

ACA

E,,m/e 317 (Irucbse)

c

(=J

ACO

OAc

E,, m/e 317 (glucose)

67

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

tinguish between ketoses and aldoses. The fragments of all of the other series (A, F, E) produce peaks a t m/e values the same as in the mass spectra of aldohexoses, although the fragments of the E series of ketoses have a structure different from those formed from aldoses. (vii) Amino sugars.-The maas spectrum of methyl 2-acetamid+2deoxy-3,4 ,6-tri-O-methyl-a-~-glucoside(methyl N-acetyl-tri-0-methyl-& has much in common with that of methyl tetra-0-methylglucosaminide)gO a-D-glucopyranoside and contains the same series of peaks, except D1. The A shies involves peaks a t m/e 246 (At) , 214 (Az) , 182 (A3) and 140 (A:). The latter is formed from A1 by elimination of ketene, which is characteristic of N-and 0-acetyl derivatives.

- cs=c=o

As,m/e 102

The B series involves fragments B1 (m/e 203) and B8 (m/e 172), the latter arising by elimination of CHaO. from C-3.

M&-dH-

OMe I CH -CHI

8 CH=OMe

NH-AC

Bl,m/e 203

-

8

MeO-CH=

CH -CH -CH=OMe I NH-AC

%,rn/e 172

The peak a t m/e 203 (BI) is more intense than that at m/e 214 (A2). Hence, the regularity observed for permethylated glycosides of simple hexoses (see Section 11IJ2bJii)does not hold for amino sugars. The ions of the C series are formed from amino sugars by elimination of methanol (instead of the CHaO. radical in the case of neutral hexosides). Subsequently, the fragment eliminates ketene and, at last, the CH30CH2. radical. This sequence is not observed with the neutral hexosides, indicating the greater stability of the ammonium compared with the oxonium ion. (30) N.

K.Kochetkov, 0.S. Chizhov, and B. M.Zolotarev, Carbohydrate Re&, 2,

( 1966).

89

N. K. KOCHETKOV AND

68

0 . 8. CHIZHOV

The E series monaista of ions m/s 232 (HI), 200 (E*), 168 (EJ),126 (EBI), and 138 (EJ, The fragments of the E series of amino sugars eliminate the CH80 group from C-4, and not (like those of the hexose series) from C-3.

h

MeOhC

C

0

-q. L'm Ii

(Cl,)",

mle

143

The other characteristic feature is the presence of an intense Eopeak which is not observed for the neutral hexosee because of the instebility of the corresponding ion.

E,,m/o 253

Ea,m/e 200

E,,m/e 188

E,,m/e

E;,m/e

158

128

MASS SPFCPROMICTRY OF CARBOHYDRATB DERIVATIVES

69

At present, it seems very difficult to explain ressonably these peculiarities of the fragmentation of amino sugars. As with the neutral hexosides, the most intense peaks are those of the F, H,G, and J series, The peaks of ions containing C 2 (F!, G:, G:, Hi, Hi)are shifted 27 mass units, and appear a t m/e 128 (F;,G:, G:) and m/e 115 (Hi, Hi). The peaks at m/e 101 (Fi, G:) and m/e 88 (Hi) are, as expected, of low intensity. Elimination of ketene from ions m/e 128 and m/e 115 leads to fragmente m/e 86 and 73, respectively. The peak m/e 73 is the most intensive one in the spectrum:

[ Lg2=o MeO-CH=CH

*@

$.

MeO-CH,-CH=NH

OW’or

%‘€I]

(@)’,m/e 73

The mass spectra of the fully acetylated amino sugar1@ are characterized by (a) the absence of acetoxonium fragments m/e 103 and m/e 145, (b) low intensity of the A series, (c) increasing intensity of the C series, and (d) a number of other peculiarities caused by the greater electrondonating power of the acetamido grouping compared with that of the acetoxyl group. c. Dieaccharides.-The mass spectra of the methyl glycosides of disaccharide methyl ethers containing ( 1 4 2 ) , ( 1 4 ) , or ( 1 4 ) bonding have been investigated, Their fragmentation proceeds in a manner essentially similar to that of methyl ethers of The two monosaccharide moieties are degraded independently; this leads to the formation of two types of fragments. The fragments formed by degradation of the “a” ring are found in the mass spectra of all of the disaccharides investigated. The fragments arising from the “b” ring are characteristic: their presence or absence serves as an indication of the type of l i g e between the monosaccharide residues in the (1-2) , ( 1 4 3 ) , ( 1 4 ) , and ( 1 4 ) linked disaccharides.

Methyl O-hexopyranosyl-(142) - and - ( 1 4 ) -hexopyranosides produce characteristic peaks at m / e 380 and 305. The latter also produce an additional peak at m/e 161. The absence of the m/e 161 peak from the spectra (31) N. K. Kochetkov, 0. 9. Chiehov, and 168, 686 (1984).

14,

A. Poliakove, Dokl. A h a . Nauk SSSR,

70

K. ICOCHETKOV AND 0.

N.

P. CHIZHOV

of 0-hexosyl- ( 1-*2)-hexosidea, explained by the following scheme, may serve to distinguish between the ( 1 4 2 ) and ( 1 4 ) isomers.

@

PMe

mrokdB-CH-

Q OMe Hex-O=CH-hi-CH-~H-OMe bMe

bB,, m/e 300

bB,,m/e 900

OM0 Hex* f O = C H - h -

Q

Q

1

Hex- O= CH-CH=CH-OIUe

bB,,m/e 161

bFf,m/e 306

6-Hex

where “Hex” is tetra-0-methylhexopymnosyl. The mass speatra of 0-hexosyl-( 14)-hexosides contain a characteristic peak at m/e 353: ShOCH,

-

QoMe

H

MeO-CH=CH--dH-OMe+ 101 mom unite

- -

8

Hex 0 CH,- CH=O-CH(OMe), bD,,m/e 353

OMe

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

71

It should be mentibned that analogous elimination of 101 mass units from the “a” ring is not traced, as the spectra of (142)- and (14)-hexosylhexosides contain no peak a t m/e 353; this may be due to rapid degradation of the corresponding ion, leading to ion m/e 279, the corresponding peak being a very intense one in the spectrum of methylated disaccharides: 0 [M~OC&-C€I=OJCH-OGI] CbMe

aD,,m/e 353

-

@

MeO=CH-OGI

aJ:,m/e 270

where G1 is the methyl tri- 0-methyl-D-glucoside residue. The stereochemistry of the glycosidic bond and that of the monosaccharide residues is reflected by the intensities of the peaks of several ions; for example, the intensity of the m/e 187 peak of O-galactopyranosylgluoopyranoses is less than that of the m/e 219 peak, alreversed ratio being the case for 0-glucopyranosylglucopyranoseg. This fact may be interpreted in terms of increasing interaction between the cis substituents a t C 4 and C-5 on going from the aAt ion (m/e 219) to the still more planar aA: (m/e 187)

’OMe pA,,m/e a19

a&,m/e 187

d. Identification of Partially Methylated Monosaccharides as their Deuterlomethyl Derivatives.-Identification of partially methyllated monosaccharides by means of mass spectrometry of their acetates has been discussed in Section III,2a. This method is, however, inconvenient, as the mass spectrum of partially methylated monosaccharide acetates can only he qualitatively interpreted, NO tthata considcrahlr number of authentic Ramplee muNct be studied before a definite coiiclusioii can be reached. Auother approach to this important problem is possible. Detailed investigation of the m a s spectra of permethylated methyl glycosides of the p e n t o ~ e , ~ hexose,laJ6 ~ J ~ ~ ~ 7 f3deo~yhexose,*~~~ and 2-amino2-deoxyhcxosca0series, using compounds specificallylabeled with trideuteriomethyl groupings in all of the positions, served as a basis for elucidating

72

N. K. KOCHETKOV AND 0 . 1. CHIZHOV

the structure of ions corresponding to all of the important peaks of a number of m&88 spectra and for evaluating the contribution of isomeric ions to the peak intensities. The data obtained pennit the solution of the inverse problem, namely, oalculation of the m&88 spectra, including the position of peaks and their relative intensities, for permethyl ethers containing trideuteriomethyl groupings in all of the possible positions, on the bwis of the known structure of ions and their contributions to the peak intensities. These calculated spectra for trideuteriomethyl derivatives of methyl pentoaides,1bJ7.as hexosides,u*=6deoxyhexosidesJu.”0and 2-amin0-2deoxyhexosidesMare presented as Tables (see, for example, Table I, containing data for D-glucose derivatives). The calculated spectra indicate that trideuteriomethyl derivatives of each of the compounds of the abovementioned series will produce a characteristic set of peaks, differing from the set for all of the other isomers (compare, for example, Tables 3, 6, 9, and 12 in Referenoe 15). Hence, the number and the positions of trideuteriomethyl groupings in permethylated methyl glycosides can be determined by comparing the experimental spectrum with the calculated ones presented in such Tables. It is important that the stereochemistry of the monosaccharide has no baring on such an identification, as revealed by the Tables calculated on the basis of experimental data obtained for labeled analogs of methyl tetrt+O-methyl-cu-wglucopyranosidemand methyl t e t m O-methyl-pD-gahctop yranoside.u On this basis, the authors of the present review proposed the following standard procedure for the identification of partially methylated monosaccharides obtained during the structural analysis of polysaccharides and other carbohydrate-containing compounds by the methylation method.8z The partially methylated monosaccharide is converted into the methyl glycoside and then (trideuteriomethy1)ated with CDJ or some other convenient methylating agent. The position of the trideuteriomethyl labels in the permethylated derivative obtained, which is the same as that of the free hydroxyl groups in the monosaccharide started with, is determined by comparing its mass spectrum with the tabulated data. If the polpccharide contttine several monosaccharide residues of the same class (for example, several hexose residues) , the partially methylated monosaccharide is first related to the parent monosaccharide by determination of the constants of the oompound obtained after exhaustive (trideuteriomethyl) ation. This relation appears rather simple, as the permethylated glycosides are very well known,and the corresponding authentic samples are readily available. The advantage of this approach to the identification of partially methylated monosaccharides involves the fact that no preparation of difficultly (32) N. K.Koohetkov and 0.8.Chuhov, Biochim. Biophys. Acla, 88, 134 (1964).

Taam I MaseSpectra of Methyl T e t r a - 0 3 m e t h y l - a - ~ g l ~ ~ p y - ~ i d e ( M T M G ) a n d i t e ~ o ~ rValues m / e and Relative Intensities

Positions of CDI Groupings MTMG 6

4

219 205 187

222 205

222

190

176 173 159

176 173 162

190 (84) 187 (16) 179 176 162

149 145 131

152 145 131

101

104 (10) 101 (90)

88

45

88

208

149 148 134 (60) 131 (40) 104 (70) 101 (30) 91 (16)

88 (sa)

3

222 208 190 (25) 187 (75) 179 173 159 (50) 162 (50) 152 148 134

2

293

222

225

208

211 193(16) 190(84) 182 176 162

190(90) 187(10) 179 176 159(50) 162 (50) 149 148 134 104(90) lOl(10) 91(84) 88(16)

152 151 137 107(30) l04(60) lUl(10) 94U9) 91(21)

e(30) a(70)

45(W

294

296

394

225 211 193g5) 190(%)

225 208 193(90) lsO(10)

225 211 193(10) lW(90)

182 179 165(50)

179 176 165(50)

182 176 165(50)

162(50) 149 151 137(60) 134 (40) 107(60) lOa(40)

162(50) 152 148 134

162(50) 152 151 137(60) 134 (40) 104 (95) lOl(5)

104

91

9l(Sa) SS(16)

48(30)

48(80) 45(20)

45(70)

W(16) 91(79) 88 (5) 48(15) 45(85)

336 225

208 193(25) lW(75) 179 173

165(50) 162(50) 155 148 134

m

436 225 208 193(84) lW(16)

179 176 165 lS2 148 134(60) 131(40) 107 (10)

104 (35) lOl(65)

104(60)

91(95) 88(5)

lOl(30) 91(16) 88(84)

48(60) 45(50)

&(So) &(50)

v

4TI 1 4

s

P

0

X

d

$

z5

5

N. K. KOCHETKOV AND 0.

74

8. CHIZHOV

available, authentic samples is needed. Moreover, an extremely small amount of the substance and a minimum of working time are needed for the analysis, and the procedure is the fist really standard one of those ever prdposed for the purpose. This approachaP has been applied for the ideritificatioii of partially methylated mannoses, in the course of the structural analysis of Cundida 2ipolgticus mannan.88 3. Monosaccharide Derivatives Having Fused Rings

As already mentiosed (see p. 47), the presence of additional rings may prevent the formation of the corresponding fragments after cleavage of some of the bonds in the pyranose or furanose ring, the group of atoms to be eliminated still remaining attached to the charged part of the molecule. This leads to the disappearanoe, from the mass spectrum of monosaccharide derivatives containing fused ring systems, of some of the series. On the other hand, the additional rings cause groupings that are situated far apart in the monocyclic molecule to approach each other, thus permitting new types of reactions, especially rearrangements. a, Monosaccharide Ieopropylidene Derlvatives.-It is well known that the structure of isopropylidene acetals depends on the stereochemistry of the parent monosaccharides. Therefore, the isopropylidene acetals of various stereoisomericmonosaccharides, such as D-glucose, D-galactose, and D-mannose, are structural isomers, and their mass spectra differ considerably from each other, whereas practically identical spectra are produced by the corresponding aoetates or methyl ethers. This may be useful in the application of mms spectrometry to stereoohemical studios in the carbohydrate field. A typiaal representative of the group is 1,2 :3,4-di-O-impropylidene-~gshotopyranose (24) (see Fig. 3). R

(24)

-

R C&OH

I

Me (25) R

-

H

(at?)

R = CH,

(38) A. I. Usov, Report at IX Meeting of Mendeleev Society of the U. S. 8. R., Kiev, U.S. 8.R.,1066. (34) D. C. Ddongh and K. Biemann, J . Am. Chem. Soc., 86, 87 (1964).

75

MASH BPECmtOMETttY OF CAICROHYDICATE DEHIVATIVEB 'I

C

.c

R

FIG.3.-Msaa Spectrum of 1,2:3,4-Di-O-ipropylideneD-&utme DeJongh and K.Biemenn, J . Am. C h . ~ o c . ,86, 67 (1964)l.

[from D. C.

The ion of highest m/e value is that a t m/e 245 (M-15) formed from the molecular ion by elimination of one of the methyl groups. Structure 0 is propo@ for the ion, The corresponding intense peak may be used to determine the molecular weight of the compound. The subsequent fragmentation involves elimination of 58 (acetone), 60 (acetic acid), and 58+60 mass units. It has been proposed that ions R and Q may undergo ring enlargement, although there is no proof for such a reaction: 0

-

1 W

Me

O

Me

\

Me

Qm/e 187

Me

O,m/e 245

- ACOH

- AcOY

Me R',m/e 127

R,m/e 127

The ion having m/e 113 corresponds to ion G: or G:.

P,m/e 186

N. K. ICOCHE'I'ICOV AND 0. 8. CHIZHOV

76

IO

v

Me

Me

P

Me

Me

Me

Me O x Me 0 G i , m / e 113

The ions having m/e 100 correspond to ions H: and H:. Elimination of the CHs. radical gives rise to ion m/e 85 (Hz)'.

I Me

(I#)' or (H:)',m/e

85

The C series is represented by the m/e 99 fragment and by the ones arising from it after elimination of Hz0, CO, or CHaO, having m/e 81, 71,and 69, reepectively. (25) and The fragmentation of 1,2 :3,4di-O-isopropylidene-~-arabinose

1,2 :3,4di-O-isopropylidene-a-fucopyranose (26) proceeds in a similar manner. All tho C-8containing fragments produce peaks shifted 30 mass units for (25) and 16 maas units for (26) to lower mass numbers, and the fragmente arising from (C:)' are absent, as their formation needs the presence of a hydroxyl group at C-6. D-Fructose affords two ieopropylidene acetals-2 :3,4 :5-di-0-isopropylidene-(27) and 1:2,4: bdi-0-isopropylidene-D-fructopyranose(28). Compound (27) differs from (24) in the position of the hydroxymethyl group. As this group is situated a t the anomeric center (C-2), its fission

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

leads to the formation of a stable ion M-31 decomposes, eliminating acetone.

77

(m/e299), which subsequently Ma

M e t 0

Me

The H i and (Hi)’ ions from (27) have m/e values 130 and 115, due to the presence of the hydroxymethyl group. The mass spectrum of (28) contains no fragments which could arise due t~ the presence of an unprotected hydroxymethyl group (m/e 130, 115, M-31, M-31-58), but contains two peculiar peaks having no analogs in the other mass spectra (m/e 117,m/e 72):

de

The above example demonstrates the potentialities of mass spectrometry as a method for elucidating the position of isopropylidene groupings. This problem has often to be solved in synthetic monosaccharide chemistry, especially when the compound may form several isomeric isopropylidene derivatives and the chemical methods are tedious and, sometimes, unreliable. The fragmentation of 1 ,2 :5,6-di-0-isopropylidene-a-~-glucofuranose (29) or 2 , 3 :A, 6di-0-isopropylidene-D-mannofuranose starts with cleavage of the bond between C-4 and C-5. The charge is primarily retained by the SIfragment, and El produces a less intense peak. The potentialities of modern physiml methods are well illustrated by the discovery of a new di-0-isopropylidene-D-galactose (30) For many years, it had been commonly accepted that only (24) is formed in the reaction of acetone with wgalactose. Gas-liquid chroma-

78

N. K. KOCHETKOV AND 0. 8. CHIZHOV

togmphy clemollstrated a small contamination of the reactiori product, and the additional compound was kthted by this method. The ma^ spectrum

-

:x;Qo

Me

O+Me Me (29)

+Go 8

t Me

O

Me SI,m/e 101

E,,m/e 150

of the compound was practically identical with that of (29), thus indicating that the only possible structure was (30).

The maas spectrum of 1,2:4,5-dicO-ktpropylidene-D-xylofuranose(31) contains, besides peaks m m n to all the mass spectra of di-isopropylidene

acetsls of the monosaccharides (M-15, M-15-58, M-15-60, M-155 8 4 , eta.), only one characteristic peak, at on/e 129. Structure TI is tentatively assigned to the corresponding fragment. The fragments El and

MASS SPECl‘ROMEmZY OF CAHBOHYDRATE DERIVATIVES

79

S1 cannot form from (31), and related peaks are absent from the mass spectrum. b. Alkylidene A c e t a W of Polyhydric Alcohols.-Although the data concerning the mass spectra of alkylidene derivatives of polyhydric alcohols are at present rather fragmentary, it may be expected that this class qf compounds will be the most convenient for the study of the stereochemistry of polyhydric alcohols. (i) Isopropylidene Aceta1s.-The fragmentation of isopropylidene acetals of polyhydric alcohols closely resembles that of isopropylidene acetals of monosaccharides, but is less complicated. One of the most intense peaks in the mass spectra of 0-isopropylidenealditols is that corresponding to fission of the methyl group from the molecular ion (the peak of the molecular ion is not traced in these spectra). This ion, analogous to the “0” fragment (see Section III,3a) forms, in the mass spectrum, a peak a t m/e 117 for 0-isopropylideneglycerol,at m/e 187 for di-0-isopropylidene-Dthreitol, and at m/e 287 for tri-O-isopropylidene-D-mn~tol.Like ion “0,” this fragment may decompose by two pathways, one involving fission of acetic acid, and the other, of acetone. The ions formed (analogs of P and Q, m/e 129 and 127 for di-O-isopropylidene-D-threito1, and m/e 229 and 227 for tn-O-jsopropylidene-D-mannitoI) may, in turn, eliminate an acetone or acetic acid molecule, to give rise to ion analogs of R (m/e 69 for di-0isopropylidene-D-threitol, and m/e 169 for tri-0-isopropy1idene-Dmannitol) . All the isopropylidene acetals of alditols produce ions m/e 101 (analog of Sl), m/e 59 (protonated acetone) , and m/e 43 (CH:CO+

or

C H d e H ) .

0-

Ion m/e 201 is also registered in the mass spectrum of tri-0-isopropylideneD-mannitol, the low intensity of the corresponding peak being probably due to its conversion into ion m/e 143 by elimination of an acetone molecule.

Me

Me m/e 201

(35) 0. S.Chizhov, L. S. Golovkins, and N. S. Wulfeon, in preae.

80

N. K. KOCHElTKOV AND

0 . B. CHIZHOV

(ii) Methylene, Ethylidene, and Benzylidene AcetaW of Polyhydric Alcohols.-Although the presence of five-membered dioxolane rings L c h c t e r i s t i c of isopropylidene metals, methylene, ethylidene and bemylidene metals more readily form six-membered rings of the 8, BC, or f l type." Typical features of the m&88 speotra of this type of compound may be illustrated by those of di-0-methylene, di-0-ethylidene-, and di-0-bexuylidene-D-threitd (32a,b,c). R I

a, R m H

b, R = M s

C,

R=Ph

(W The M+1, M, M-1 triplet is characteristic of these spectra. The most intense peaks are formed by M-1 ions. The peak of the molecular ion and the M+l and M-1 peaks have been detected in the spectra of a wide variety of 0-alkylidenealdits, so that the molecular weight can often be directly determined by Uaing the method. The fragmentation of compounds (32a-c) proceeds by the following major pathways, involving (i> fission of the R. radial, the most ready in (32b) [m/e 159 (b) m/e 221 (c)]; (ii) fission of the aldehyde RCHO; the intensity of this reaction drops in the sequence (32a) > (32c) > (32b) [m/e 116, (a) m/e 192 (c)]. The M-RCHO ion formed loses 30 mass units, moat probably, in the form of C H A [mle 86 (a) , m/e 162 ( c ) ] . It may appear also that the formation of m/e 86 and m/e 162 fragments proqeeda independently by deoomposition of ions M-1 or M-R.

(36) 8.A. Barker and E.J. Bourne, Advan. Carbohydrute Chem., 7, 137 (1952).

MASS SPECTBOMETILY OF CARBOHYDRATE DERIVATIVES

81

(iii)Cleavage of the molecule into “two halves.” One of the possible modes of such cleavage is aa follows.

“0.5 M”

This rather unexpected process is observed in each of the three cases [m/e 73 (a), m/e 87 (b), m/e 149 (c)]. The spectra of benzylidene acetals contain intense peaks of aromatic ions (m/e 77, CsHse; m/e 91, C7H7@;m/e 105, CsHs-Ce=O; m/e 106,

CeHsCHeO $; m/e 122, 0.a

/ COHbC

,

\

OH

and so on). Maes spectra of a number of other O-alkylidenealditols have been measured, and they do not contradict the above considerations. However, the data available are not yet sufficient for more-detailed interpretation of the effects of structure and stereochemistry in terms of the fragmentation patterns. c. Anhydro S ~ g a r s . ~ ~ * ~ 7 - Tmass h e spectrum may indicate the size and position of the anhydro rings in anhydro sugars. For example, the mass spectra of 1,&anhydro sugars differ considerably from those of 3,6anhydro sugars, and the latter are strongly dependent on the furanose or pyranose nature of the monosaccharide ring. These regularities will be illustrated by the methyl ethers of levoglucosan (1,6anhydr0-/3-~-glucopyranose, 33), methyl 3,6-anhydro-a-D-glucopyranoside(34), and methyl 3,6-anhydro-a-D-glucofursnoside(35). The maas spectrum of (33) is the most similar to that of methyl tetra-0methyl-a-D-glucopyranoside,although the presence of an additional ring iu this compound brings about some peculiar features in its fragmentation. The mass spectrum of (33) contains no peaks of the A and E series, and lacks.ion B1,cleavage of C-1-0, C-5-C-6 or of C-1-0 and C-4-C-5 being (97)

N. K. Kochetkov, 0. 8. Chuhov, and B. M. Zolotarev, Chem. Nat. Prod. (Taehkent), 152 (1966).

82

N. K. EOCHIDTKOV AND 0.

8. CHIZHOV

not yet enough for elimination of the corresponding fragments. The ions B8 (m/e 145) and Ba (m/e 131) are formed normally.

Q

Q (

OMe

Me

MmQ

Me0

OMe

OMe

OMe

OMe

OMe

(34)

(33)

(35)

Cleavage of the C-1 to C-2 bond affords several series of structurally similar ions of m/e 175, 159, 143, 127, end 95, somewhat analogous to the C series. The Dr ion of m/e 103 may be tentatively proposed to have the above structure. The other ions have mass numbers the same as in the mass spectra of methylated methyl hexosides: F: and G: (m/e lOl), H: and Hi ( m/e 88), HI (m/e 73), and Fz (m/e 71).

M e QMe 0

G o

Me0

C, ,m/e 175

OMe

OMe

Cl,m/e 176

C,”,m/e 159

/

J

I

D, ,m / e 103

OMe

OMe

C;,m/e 143

c,”,1n/e 127

CS,m/e 85

The mam spectrum of (34) retains the peak of the A and E series (isomeric for these compounds), ion D1 (m/e 103), atld F: (m/e 101). More characteristic of the (34) fragmentation pattern are new processes not observed with the monocyclic compounds and (331. The rupture of the C-5-C-6 bond takes place, followed by C-54-1rearrangement, and subsequent fragmentation giving rise to ions of the L, M, and N series, as &own.

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

83

.o

I

OMe

M,,m/e 00

Ln,m/e 143

The most intense peaks of the maw spectrum of (35) are due to the following fragmentation rmctions.

--OMe -C&O

k

he

+ HC0,Me + [MeO-C€I=CH-CH=CH-OMe] m/e 114

@

'

84

N. K. KOCHETKOV AND 0. 8. CHIZHOV

An atlalogous mechanism which affords the ion of m/e 86 occurs with

1,4:3 ,6-dianhydmD-glucitolo1.”

Hence, 1,B-anhydro sugars behave similarly to the ordinary methyl hexosides; 3,6anhydro sugars decompose through mjgration of the CHzO group from C-5 to C1, and subsequent fragmentation to give the L, M, and N series; and the cis-dioxapentalane system of 3,6anhydroglycofuranosidea (and of related 1,4:3,&dianhydroddito1s) produces a stable, four-carbon fragment containing C-2, C-3, C-4, and C-5 with the substituenta, by conjugated transfer of electrons. The 3 ,&anhydro sugars having free hydroxyl groups20such as methyl 3,6-anhydro-aand -&~-gluco-, -ar-D-galauto- and -u-mmannO-pyranoside, may, under the conditions of maes spectrometry, be destroyed not only because of electron impact, but also because of considerable thermal decomposition. This is, probably, the reason why their maes spectra are so difficult to interpret. The peak of highest 111&88 number is A1 (or Ep) at m/e 145 (M-31). The other peaks of the spectrum (m/e, 115, 102, 85, 74, 69, 60, 57, and 55) have no arialogs in the maas spectrum of methyl 3,6-anhydro2,4di-O-methyl-a-D-glucopyranoside.The authors proposed a scheme for their formation as one of the possibilities, not excluding other, more complicated and unusual, rearrangements. This scheme appears rather unconvincing from the viewpoint of modern considerations concerning the fragmentation of organic compounds after electron impact. 4. Acyclic Derivatives of Monosaccharides

Acyclic compounds decompose in a manner less complicated than that of the cyclic monoeaccharides because of elimination of such factors (affecting the spectrum) as sine of the ring, and because of an even smaller effect of the stereochemical differences. For this reason, the use of acyclic derivatives is most reasonable when the molecular weight of the monosaccharide, the position and sire of specific groupings (for example, the position of the “deoxy unit” or acetamido grouping), atid the position of the carbonyl group are to be determined. The clam includes alditol derivatives (such as acetates and methyl (38)0. S. Chichov, L. 9. Golovkins, and N. 6.Wulfaon, unpublished data.

MASS SPECTROMETRY OF CAHBOHYDRATE DERIVATIVES

85

ethers) and those of aldehydo and keto forms of monosaccharides (and their acetates and dithioacetals) . The fragmentation of aldehydo and keto derivatives begins with the ionization of the carbonyl or potential carbonyl grouping. a. The Aldehydo and Keto Forms of Monosaccharide Derivatives. (i) Thioacetals.m-The maas spectra of thioacetals contain relatively intense peaks of molecular ion, so that the molecular weight of a monosaccharide can be directly determined using the mass-spectral method. This is an important advantage, as, in the monosaccharide series, the molecular weight can be determined by means of mass spectrometry only indirectly for almost all of the compounds except this type of derivative. A typical representative of the class, D-arabinose diethyl dithioacetal tetraacetate, will be considered. The molecular ion of this compound decomposes along several pathways. As with the sugar acetates, the first series of fragments is that represented by ions differing from each other in 60 (acetic acid) or 42 (ketene) mass units. The Jirst fragment of the series (UI)has m/e 364 and is formed by fission of acetic acid from the molecular ion. The second series (V) starts with the ion m/e 363 (VJ arising by elimination of the CZH& radical. This ieries is presented by ions m/e 303 (Vz), 261 (V3),201 (V,), and 159 (Vb). The parent ion of the third series (W) has an m/e 275 (WA, and is formed by fission of the ethyl group and of two molecules of acetic acid per molecule. The series includes ions m/e 215 (WZ)and m/e 173 (W3).

EfBucK @

OAc

W,,m/e 275

One of the most intense peaks of the mass spectrum (XI) of the compound is situated a t m/e 135; the corresponding ion is formed by cleavage of the C-14-2bond.

.o EtS CH-SEt AcOCH t HthOAC HhOAc

s;

LH,OA~

A C ~ H ' 8 Et-S=CH-S-Et

+

HCOAc H~OAC

X,,rn/e

136

(39) D. C. Ddongh, J . Am. Chem. Soc., 86, 3149 (1904).

CH,OAc

N. K. KOCHETKOV AND 0. 8. CHIZHOV

86

The subsequent decomposition of ion XI affords fragments m/e 107 ( E t S - - C H 4 @H),75 ( C H A @-Et) , 61 (EtS@) , and 45 ( C H d @). Thejijth eerie8 (Y)starts with ion m/e 218 (Y1) formed by cleavage of the C3-C-4 bond, followed by elimination of the aoetoxyl group from (2-2. Subsequent elimbtion of ketene gives rho to ion m/e 177 (Yf).

HI+ CH,OAc

Y,,m/e 219

Y l , m f e 177

The ion m/e 104 (21)is formed by cleavage of the (2-24-3 bond and subsequent elimination of ketene from V1.

___c

Z,,m/e 104

d OI A C ChOAc

-4

The other product of ion V1 decomposition, the ion with m/e 259 (AAII, is formed aa follows.

E y p @ Ac

ACO OAc

V,,m/e 303

-

AcO

OAc OAc

AcO

OAc OAC

AA,, m/e 259

The A A series also includes ions m/e 139 (AAd and 97 (AAa). Besides the peaks mentioned above, the mass spectrum of D-arabinose diethyl dithioacetd tetraacetate reveals fragments obtained by cleavage of C-2413,(2-34-4, and C-Q-C-5 bonds. The intensity of the peaks of these fragments, especially of that at m/e 217, is low. Peaks of low intensity are also produced by acetoxonium ions AcreOH (m/e 103) and AcaO@(m/e 145). The V and AA series in the mam apectra of 2-deoxy-D-"glucoseJ' diethyl dithioacetsl and Grhamnose diethyl dithioacetsl are shifted 14 mass unit,

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

87

to greater mass numbers. The most marked difference between the two compounds is the absence of X1ion (m/e 135) ip thqspectrum of the 2-deoxy compound, due to very low stability of radical (36) which ought to be formed along with ion X1 from the molecular ion; YIand Yn ions are also absent.

HCOAc I CH,OAc (36)

The mass spectra of aldohexose diet.hy1 dithioacetal pentaacetates resemble those of the corresponding pentose derivatives, but differ in the position of the U, V, W, and AA series, which are situated 72 mass units higher, due to addition of a CHOAc unit to the molecule. The major pathway of fragmentation of D-fructose diethyl dithioacetal pentaacetate is connected with production of the U and V series. The series Y and AA are of low intensity, and the X1 fragment is absent. Hence, the maw spectra of diethyl dithioacetal acetates may serve to distinguish between aldopentoses, aldohexoses, ketohexoses, 2-deoxyhexoees, and 6deoxyhexoses. Diethyl dithioacetals have been used for determining the position of the acetamido group in acetamido sugars by the mas-spectral meth0d.N A characteristic feature of the mass spectra of 2-acetamido-2-deoxyand m/e 268 aldose diethyl dithioacetals is the presence of m/e 177 (Yz) (Ul) fragments due to elimination of the acetamide molecule from C-2. 3-Acetamido-3-deoxy compounds do not eliminate acetamide, and their mass spectra reveal the YZion a t m/e 218. 5-Acetamido-Sdeoxy-pentoses and 6-acetamido-6-deoxy-hexoses differ from the 3-amino sugars in the position of the Ya fragment (m/e 177), and from 2-acetamido sugars in the position of the U1 fragment (m/e 279 for pentoses, m/e 309 for hexoses) which arises here by elimination of a molecule of water from C-14-2. The mam spectra of compounds having a terminal amino group contain, also, a number of peaks corresponding to fission of the C-14-2, C-2-(3-3, C-34-4, or C 4 G 5 bonds, with retention of charge on the nitrogen-containing fragment (m/e 162, 132, 102, and 72). 2,6-Diacetamido-2 ,B-dideoxyand of the series hexoses differ in the presence of the m/e 177 peak (Yz) (40)D. C.DeJongh and 8.Haneesian, J . Am. Chem. he., 8, 1408 (1986).

88

N. K. KOCIIICTKOV AND 0 . 8. CHIZHOV

m/e 132, 102, and 72 pectks, characteristic of the terminal acetamido group. 3,6Dketamido-3 ,&dideoxy-hexoses can be identified by the peaks m/e

218 (Yz) and M-18 (UI), characteristio of 3-amino sugars, and by peaks m/e 132, 102,and 72, which show the presence of the terminal acetamido PUP. The peak of the molecular ion is, SurpriSmgly, absent from the mass spectra of fully acetylated ethylene dithioacetals." Their fragmentation consish principally of elimination of CHaCOZH, CHaCOs., CHaCO., and ketene in various combinations. The most intense peaks of the spectra are those at m/e 43 (CHaCO@),445 ( C H a , 61 (EtS@), and XI (m/e 105): @>

X,,m/e 105

Fully acetylated diethyl dithioacetals are to be preferred to ethylene dithioacetals for analytical applications, as the mass spectra of the latter do not contain the peak of the molecular ion, and provide no information additional to that which can be drawn from the spectra of the former.

(ii) Aldehydo aoetates."The

typical features of the mass spectra of compounds may be cohveniently illustrated by that of of this group aldehyde-D-arabinose tetraacetate. The molecular ion is not revealed by this spectrum, but peaks M+l and M+43 are detected. The corresponding ions are due to bimolecular reactions. The most intense peak of the spectrum is that at m/e 43 (CHICO@).Some of the seriea correspond to those detected in the spectra of dialkyl dithioacetals (U'-series, m/e 259, 139, 97). Other series have no analogy. The BB se~ieebegins with the m/e 170 fragment. Subsequent fission of ketene leads to ions of m/e 128 and 86. .$

+ AcOH + AcO-CHO AcO H

m/e 170, BB,

(41) D. C. Ddongh, J . Am. Chem. Soc., 86,4027 (1964). (42) 1). C. Ddongh, J . Org. Chem., SO, 463 (lQ66).

89

MASS BPECPROMETRY OF CARBOHYDRATE DERIVATIVES

The CC series consists of three-carbon stom fragments m/e 217, 157, 115, and 73,which are formed mainly from C-2, (3-3, and (24, and, to a lesser extent, from C-3, C-4, and C-5. 8 CH-OAC FOAc ChOAc

- - -E"

m/e 217, CC,

63

Q

Q

CH-OAC I CH I1 CH-OAC

YH-OAC

CH-OH I

7%

H-OH

CH=O

m/e 115,

m/e 157, CC,

cc,

m/e 79, Cc,

The fragments of the DD series arise due to fission of the formyl group ( C H = O ) and subsequent elimination of AsO, AcOH, and ketene (m/e 289, 187, 127, and 85). ?H=O

-

@*

AcOCH I HCOAc I HCOAc h&OAc

8

I

AcTH HCOAc. I HCOAc I CH,O AC

- Ac,O

AcO

@

AcO

DD, ,m/e 289

DD,,m/e 187

The EE1ion is formed by the following reaction, characteristic of acyclic aldehydes.

-

$ - & y ~ ~ OCA ACO

[AcO-CH=CH-OH]'O EE,,m/e 102

OAc

Besides these series, the mass spectra of the aldehyde-aldose peracetates reveal the presence of stable ions of m/e 69 and 68 (furan and protonated furan); the mechanism of the formation of these ions remains rather obscure.

b. Alditol Acetates.4-A characteristic representative of this type of compound is D-glucitol hexaacetate, which, like other members of the class, is decomposed by a relatively simple fragmentation pattern. The molecular ion is not traced by the mass spectrum of D-glucitol hexaacetate. The heaviest ion of the spectrum is that at m/e 375 (M-59). The other (43) 0.8.Chiahov, L. S. Golovkina, and N. S. Wulfson, in press.

90

N. K. K0CHWI"I'OV AND

0 . 13. CHIZHOV

peaks of this mass spectrum correspond to ions formed by primary fissions, aa follows,

or by fission of AcOH and ketene from these primary fragments of m/e 361, 289, 217, and 145. Analogous behavior is also characteristic of pentitol and tetritol peracetates. The intensity of primary fragments increases with decreasing molecular weight, so that even the M @ion is detected in the mms spectrum of glycerol triacetate. The presence of the methylene group inhibits the cleavage of the neighboring bondings. Thus, in the mass spectrum of 1,2,4butanetriol triacetate, the intensity of the peak at m/e 145 is strongly diminished due to inhibition of splitting of the C - 2 4 3 bond. Hence, the mass spectra of alditol acetates may be used to solve problems, just m for the monosaccharide series; for example, for determination of molecular weight (indirectly, from the position of M-59 and M-73), of the position of the deoxy unit, and 80 forth. 5. Mass Spectra of Miscellaneous Carbhydrates

Finan and Reed4 measured the mass spectra of permethyllaminaran, and detected a series of fragments differing by 204 mass units. The authors ascribe this series of fragments to sequential fission of the 2,4,6tri-Omethyl-D-glucose residues which form the chain of the polymer. The mass spectra of nucleosides have been studied by Biemann and McCloskey." For all of the compounds of the series, the most intense peak corresponds to fission of the sugar (S)-base (B) bond, and one or two hydrogen atoms migrate from the sugar hydroxyl groups to the base residue. The B+1 iQn prevails in the mass spectra of 2-deoxynucleosides. A relatively intense peak of the carbohydrate moiety is observed for the pyrimidine nucleosides. Purine nucleosides produce no charged carbohydrate residue. The fragment B+30 has the structure HO@=CH-B, and the fragment M-89 has the structure [R--.CHCH*-B]@, where R H or OH.

-

(44)P. A. Finan and R. I. Reed, Nature, 184, 1886 (1859). (46) K. Biemann and J. A. McCloekey, J . Am. C h . h e . , 84, 2006 (1962).

MAW 8PECTHOMETIZY OF CAltBOHYDRATE DERIVATIVES

91

spectra* of stereoimneric iIiositols contain 110 pcak of the XI@ The ion, but only the M+l peak ( m / c 181).lhsion of water from this ion gives the m/e 163 fragmeut, which produces the most intense peak with compounds containing H and OH in the cis position a t C-1and C-4. Hence, it may be tentatively proposed that 1,4cis elimination of water takes place, and the ion formed has the structure shown. This ion gives rise to fragment m/e 144, and subsequently to m/e 102. The latter readily eliminates a HO HO

HO m/e 163

HO m/e 144

m/e 102

formyl group, to give rise to C3Hs0 (m/e 73), which is the most intense ion of the spectrum. It has been shown that, when 5-amino-5-deoxy-~-xylopyranose (37) is exposed to an acidic medium, it gives rise to 3-hydroxypyridine and to an optically ective compound which, it was proposed, might be l-amino-l,5anhyrln>-l-deoxy-D-threo-pentulose(38) Structure (38) has been con-

firmed, nut only by other methods, but also by the mass spectrum of the compound itself and by that of the l-acetamidotri-0-acetyl-1,5-anhydro-ldeoxyxylitol (39) obtained by reduction and subsequent acetylation of compound (38). The mass spectrum of (38) contains peaks at m/e 131 (M ion), at m/e 113 (M-18, the presence of OH) , m/e 103 (M-281, and m / e 85 (113-28). The expulsion of 28 mass units (CO molecule) is characteristic of cyclic ketones. The major peak of the spectrum corresponds to the CH2-N @H=CH2 ion-radical. The major fragmentation pathway of (39) is consecutive elimination of AcOH and ketene, leading to the 3-hydroxypyridinium ion. This transformation is analogous ta that of (37) caused by acid. (46)

H.Pauluen, Ann., 688, 187 (1965).

92

N. K. KOCJHFI'KOV AND 0 .

8. CHIZKOV

IV. CONCLUSION The rather considerable evidence accumulated to date reveals the great potentialities of mass spectrometry. As mentioned above, mass spectrometry may be applied to the detcrmhlation of molecular weights, to clucidating the position of substituents (such mi --O,CHs, H- in deoxy sugars, or -NHI in amino sugars) ,to deciding the sbe of thd ring, and, sometimes, to deducing the stereochemistry. Each of these problems to be solved needs the proper choice of derivatives. As revealed by the data available, the type of compound closest to the ideal for structural analysis of monosaccharides is the class of dialkyl dithioacetals or their acetates: their mass spectra contain a considerable peak due to molecular ion, and their fragmentatiop patterns are simple enough (due to the absence of a sugar ring) and specific enough to permit determhation of the position of substituents on the basis of the position of peaks. Thus, elimination is characteristic of the C-2-substifuentsJwhereas substituents a t C-3 tend to be retained, producing a peculiar difference between the mass spectra. However, the m&88 spectra of dialkyl dithioacetab provide almost no information regarding the stereochemistry of the monosaccharide molecule. Methyl ethers and acetates are convenient for elucidation of the size of the ring. The former are advantageous because of their simpler fragmentation patterns, permitting more ready interpretation of the spectra; the latter are usually more readily available. The mass spectra of acetates and of methyl ethers may sometimes be applied to the solution of stereochemical problems. I n order to decide stereochemical problems, alkylidene acetals of carbohydrates ohould be studied, the structure of these compounds being dependent on the stereochemistry of the parent monosaccharide molecule. Since the structure of the alkylidene acetal can sometimes be elucidated from the mass spectrum, the latter also provides evidence regarding the stereochemistry of the carbohydrate molecule started with. However, two other applications of mam spectrometry to carbohydrate chemistry seem the most important and promising. These are: the identification of partially methylated monosaccharides, and structural analysis of di- and oligo-saccharides (and penetration of this new technique into polysaccharide chemistry). As for the first application considered in Section I1IJ2d, the method for identification of methyl ethers using trideuteriomethyl. derivatives has already baen demonstrated to be valid for all of the major types of monosaccharides, and is now receiving practical application.88 We hope that the increasing availability of mass spectrometers will facilitate the introduction

MASS SPECTROMETRY OF CAItBOHYDRATE DERIVATIVES

93

of the niethod 8s a more routiiie practice; this would bc mi iinpc’tus to t,hc structural analysis of polysaccharidcs and other carboli~vdiutc.-rotIt.Ltiiriii~ polymers by means of the niet,hylatiotiprocedure. The second direction of future work, the structural analysis of oligosaccharides, despite some advances already made, needs fuiidamental investigation. First of all, mass spectra must be recorded and interpreted for tri-, tetra-, and, perhaps, penta-saccharides, Furthermore, the use of the methyl ether group is not always convenient, owing to certain difficulties connected with‘the methylation procedure. Hence, a new type of protective grouping, more readily introducible and affording volatile products, must be sought. At the same time, this new type of Frotective grouping must not enormously increase the molecular weight. However, the data available suggest that the results obtainable from mass spectrometry will considerably facilitate the structural analysis of oligosaccharides and thus permit direct investigation of the products of partial hydrolysis of polysaccharides on the oligomer level.

ADDENDUM During the past year, a number of important papers on the subject have appeared. Heyns and MUller(’ have published new data on the mass spectrometry of methyl ethers of a number of amino sugars. Data on the mass spectra of dialkyl dithioacetals having free hydroxyl groups were obtained by DeJongh.ls These data were used further@for detection of the positions of amino groups in amino sugars. More-detailed data on the mass spectra of some anhydro sugsrs also appeared.1° In connection with investigations on the mass spectra of oligosaccharides, those of trimethylsilyl ethers of mono- and di-saccharides were reported.61The results obtained indicated that these ethers are very suitable for mass-spectral studies of oligosaccharide structwes. (47) K. Beyns and D. Mtiller, Tetrahedron, 21, 3151 (1965); Tetrahedron Lethru, 449, 617 (19G6). (48) n. C. Ddongh, J . Org. Chem., 80, 1563 (1965). (49) D. C. Udongh and S. Hanessian, J . Am. Chem. Soc., 87,3744 (1965). (SO) K. Heyns and H. Scharmann, Carbohydrate Re.?., 1, 371 (1966). (61) N. K. Kochetkov and 0. 5. Chizhov, Intern. S y n p . Chem. Nut. Prod., ZV,Stockholm (June, 1966).

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BY JOHNW. GRBEN The Institute of Paper Chemislry, Applebn, Wiseonsin I. Introduction.. . . . . . .. . . . . . . . . . . 95 11. Clonfomtion of the ......................... 96 111. Formation of Glycofuranosideain Acidic Methanol.. .... . . . . . . . 101 101 1. Isolation of Products.. ............................................. 2.Hin9tics .......................................................... 106 . . . . . . . . . . . . 112 IV. Preparation of Glycofuranosides 1. Isdation of Products.. . . . . . . . . . . . . . . . . . . . . . . . . . 112 2. Alkyl 1-Thioaldofuranoaidea . 3. Mechmism.. ................ .......... . . . . . . . . . . . . 117 V. General Preparative Methods. . 1. Preparation from Furanom Esters and Halides.. ...................... 121 2. Glycofuranoaylamines.. . . . . . ..?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 3. M k e l l a ~ ~ w u Preparations. a ........................................ 125 VI. Structure of Glycofuranosides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 1. Application of Isomtation to Furanoid Structurea.. .................... 127 2. Acid Hydrolysis of Glycofuranosidee ......................... 3. Oxidation of Glycol Group.. ....................... 4. Ring Stability.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. General ............................................................. 137 1. Action of Alkali.. ................................. 2. Formation of Furanose Polymers. ................................... 138 3. Natural Occurrence of Furanose Residues.. ........................... 139 VIII. Tabla .............................................................. 140

I. INTRODUCTION In 1914, Emil Fischerl isolated a crude, sirupy “gamma” methyl D glucoside that was isomeric with the known, crystalline methyl) a- and &D-glucosides. It was not until 1929 that two “gamma” glycosides, ethyl a- and 8-D-glucofuranoside, were first obtained in pure, crystalline condition by Haworth and Porter.* The isolation (and the determination of structure of these compounds by methylation techniques) was one of the high points in the history of structural analysis in carbohydrate chemistry. The preparation of pure glycofuranosides from complex reactionmixtures was very difficult before the advent of chromatographic tech(1) E. Fischer, Bm., 47, 1980 (1914). (2) W.N. Haworth and C. R. Porter, J . C k m . Soc., 2796 (1929).

95

96

JOHN W. GREEN

niques. In 1937, only eight crystalline compounds of this kind were known. In 1938, Pacm and Green' devolopcd B specific synthesis for aldofuranosides, from ths corresponding dialkyl dithioacetals, and were able to prepare eight more crystalline, furpnoid glycosides. However, during the paat twenty years, various chromatographic techniques have been introduced, and now the furanosides can be readily separated from rather complex mixtures; consequently, their availability now depends to a great extent on the diligence of the worker. Expansion of work on model compounds in the field of nucleic acids has added greatly to the impetus to synthesize compounds that, only thirty-five years ago, were laboratory curiosities. The aclcrcity of studies of glycofuranosidea in the literature may be attributed to aeveral factors: their lability to acid (excepting pyrimidine nucleoaides), their formation in complex mixtures with unwanted byproducta, their low melting points, and sometimes, their reluctance to crystallize. In the present Chapter, Tables XII-XIV list most of the knQwn crystalline glycofuranosides, together with some that are presumably pure but have not yet been crystallired. Certain other articles in this Series have discussed the glycofuranosides in part;M preparative methods for several of them have been reviewed elsewhere?

11. CONFORMATION OF THE GLYCOFURANOSIDES Until 1961, the furan ring had been considered to be planar, and sugar derivatives having a fivsmembered sugar ring were usually represented by Fischer projection formulas or Haworth planar (perspective) formulaa. The traditional Haworth formula for methyl a-Parabinofuranoside is shown in formula (l),and, in this Chapter, this type of formula will mainly be used. The groups on adjoining carbon atoms in a planar, furanoid ring w t u d y bear a 0" or a 120' relationship to each other; they are either whol!y cie or wholly trans, and, in the eiS configuration, there is an eclipsed relationship that produces strain in the ring, in contrast to the staggered rellttionship observed in the chair conformations of pyranoid sugars. The nonbonded interaction produced by such eclipsed groupe has been termed (3) E.Pacau'and J. W. Green, J . Am. C h . &c., 68, 1823 (1936). (4) J. Conchie, G. A. Lewy and C. A. Mamh, Aduon. Carboh#drute C h . , 12, 167 (1687). (6) F.Shafiasdeh, Advan. Carbohgdrufe C h . ,18,9 (1968). (6) D.Rorton and D. H. Hutson, Advcm. Curhhydruie Chsm.,18, 123 (1963). (7) (a) E. L. Hi& and E. E. Peroivd, MeUIods Curbohydrub C h . , 1, 349 (1963); (b) E. Pacau, ibid., 2, 364 (1963);(c) D.Horton, $bid., 0, 368 (1963).

THE GLYCOFUBAXOSIDES

97

I-strain: and is appreciable when the groups concerned are polar in nature, as for OH, CHzOH, or CHOH-C!H,OH. Interactions of such groups at C-1or C-4 with the ring-oxygen atom are considered to be at a minimum.* Fomula (2) ie a pictorial representation of the planar conformation of

methyl a-D-arabinofuranoside; the ring is a plane, but the subsdituent groups are shown at 0" and 120"to each other. This planar concept of the furanoid ring had been maintained until 1961. However, work on the D-ribofuranose moiety in the nucleic acids, performed with the aid of nuclear magnetic resonancelb12 and of x-ray diffraction,*sJ' showed a flexing or puckering of the ring. This relieves the strain of the planar conformation and can be accomplished by moving either one or two atoms (carbon or oxygen) out of the plane. When one atom is exoplanar, an envelope conformation is obtained; this is referred to as a V conformation, which is specified by a subscript or a superscript numeral denoting which exoplanar carbon atom is below or above the plane. The side bearing C-5 is considered to be the upper side of the plane in the D series, and the lower side in the L series. Formula (4) shows a V2 conformation, C-2 being below the plane. Formulas (5) and (6) have V8 and V2 conformations, respectively. A twist conformation results when two ring atoms are exoplanar; such conformations are defined by the letter T with appropriate subscripts or superscripts. Formula (3) depicts methyl a-Darabinofuranoside (3) in the T2 conformation. Formulaa (3) to (8) show the various conformations for the eight methyl D-aldopentofuranosides;the favored conformation of each, with the number (8) H. C. Brown, J. H. Brewster, and H. Shechter, J . Am. Chem. Soc., 75, 467 (1954); see a h , F. Shafbadeh, Ref. 5, p. 23. (9) There hao been Borne consideration of the oxygen atom aa being tetrahedral in nature; me H. A. Bent, Chem. Rev., 61, 275 (1961). (10) R. U. Semieux, Can. J . Chem.,59, 116 (1961). (11) C. D. Jardebky, J. Am. Chem. ISOC., 68, 2919 (1961). (12) See L. D. Hall, Advan. Carbohydrate Chem., 19, 51 (1964), eapecially pp. 75-78. (13) M. Spencer, Acts Cry8f., 8, 325 (1959). (14) See G. A. Jeffrey and R. D. Rosenstein, Advan. Carbohydrate Chem., 19, 7 (1964), especially pp. 13-15.

JOHN W. GREEN

98

of non-bonded interactions is given in Table I. It will be noted that the favored conformation of methyl a-D-arabinofuranoside is the most stable of them all, and has no interactions, whereas that of methyl Bwlyxofuranwide is unstable in comparison, having three interactions despite a flexing of the ring. This listing of favored conformations is still tentative; the conformations were suggested on the baais of possible interactions.lb Nuclear magnetic resonance data have confirmed that the Va conformation is favored for methyl &wribofuranwide.la

,OMe

(7)

(8)

In Table I, the more stable anomer is listed first. In each of the four camea, this anomer is the trans-cl-ca-aldofuranoside, having a trans relationship of the aglycon group to the hydroxyl group at C-2. Thus, the &D anoniers of methyl D-ribofuranoeide and methyl Dxylofuranoside are listed first, even though the interactions for the latter are equal. Later in this Chapter, the offeeat of this conformational stability will be discussed in relation to formation of products A t present, it suffices to mention that there are two factors to be considered, the C-1and C-2 interactions, and the overdl interactions (C-1 and C-2, C-2 and C-3, C-3 and 0 4 ) . The (16) C.T. Bishop and F. Cooper, Can. J . C b m . , 41, 2743 (1963). (16) R. U.Lemieux and R. Nagarajan, Can. J . Chem., 42, 1270 (1964).

99

THE OLYCOFURANOSIDES

TABLE I Favored Conformation of the Methyl D-AldopentofuranoaidedK Methyl D-aldo- Formula Favored pentofurenoaide conformation

Hydmxyl interactions located at

-

cr-ArsbinO

B anomer

total no.

G 1 and G 2

&Rib0 a anomer

G 2 and C-3 Gl and G2; C-2 and C-3

8-Xylo u anome-

G3 and C-4 G3 and G 4

a-Lyxo 8 anomer

C-2 and G 3 ; C-3 and C-4 G 1 and C-2; C-2 and C-3; C-3 and G 4

order of stability : trans-C1-C2aldofuranosides > c i s 4 l-Ca-aldofuranosides seems to hold very definitely in acid and neutral systems; the order seems to be reversed in alkaline systems. The order of stability: warabinofuranosides > D-ribofuranosides > wxylofuranosides > wlyxofuranosides, has also been demonstrated in acid systems to a certain extent; this order is not so important aa the effect of the C-1 and C-2 interactions alone. The conformations given above have only C-2 and C-3 exoplanar. This provides tho best solution for relieving interactions; very little interaction should occur between C-1 or C-4 in the plane and the ring-oxygen atom. Calculationss7 derived from x-ray diffraction data for nucleic acids and sucrose have shown that C-2 or C-3 is the exoplanar atom for all examples studied. However, where the endocyclic bonds of C-2 or C-3 are prevented (by substitution at the exocyclic bonds) from flexing, other conformations can result. Thus, in both methyl 2,3-anhydro-a- and- 8-wribofuranoside, the four ring-carbon atoms are held in a plane by the anhydro group, and the ring-oxygen atom is exoplanar ; nuclear magnetic resonancela shows no proton-proton coupling between C-1 and C-2, or between C-3 and C-4. Similar data have been reported for other 2,3-anhydroaldo-pento- and -hexo-furanosides. Whereas anglea of 0' and 120' were projected for the angles of the cisand tram- hydroxyl groups to the planar furanoid ring, probable values of 50' have been reported for cis-hydroxyl groups, and 75' or 160" for truns(17) M. Sundsralingam, J . Am. C h m . Sor., 87, 599 (1966). (18) L. L). Hall, Chem. Znd. (London), 960 (1983).

100

JOHN

W. GltEEN

hydroxyl groups; these values are for envelope (V) conformation^.^' It is considered thph a value of 75’ is more reasonable for trans-hydroxyl groups where reactions of such hydroxyl groups are slow, aa with periodate. If complete rexistance to a reagent is shown, the possibility of an angle of 160’ should be considered. An angle of 150” has been found in one of the Dfruc tof uran ose residues in 1’ ,2-anhydro-l-0- (a-wfructofuranosyl) -&Dfructofuranose : the a-D-furanoid ring is rigidly locked, and is very resistant to periodate oxidation.I6Capon and ThackerlBahave shown, for the methyl aldofurariosides of D-glucose, D-galactose, D-xylose, and carabinose, that the anomers can be clearly distinguished by their nuclear magnetic resonance spectra. The lJ2-truns compounds all have J values of 2 C.P.S. or less; the values for the 1,2-cis compounds are in the 4.04.5 C.P.S.range. In the Eormation of glycofuranosides from the aldopentoses, the oxygen atom attached to C-4, the configuration of which defines the chirality (D or L family) of the given sugar, is included in the ring; therefore, the hydroxymethyl group on C-4 will be in roughly the same position, relative to the plane of the ring, for all of the D-aldopentofuranosides and for all of the ~aldopentofuranosides.le In the aldohexofuranosides, C-5, the “configurational” or reference carbon atom, is not included in the ring, and so the bulky dihydroxyethyl (CHOH-CHaOH) group attached to C-4 does not necessarily have, for the D series MI a group, and the L series aa a group, the same position in reference to the ring. For half of the Paldohexoses, in methyl 8-D-glucofuranoside (9) and methyl a-wmannofuranoside (lo), for example, the exocyclic Wdihydroxyethyl) group is above the plane, as for the (3-4 group of the D-aldopentofuranosides. For the other half of the wddohexoseb, for example, in methyl Swgalactofuranoside (1l), this large group is below the plane, and these glycosides are 4-C-substituted taldopentofuranosides; thus, (11) resembles methyl a-barabinofuranoside (12).

The reasoning for the respective conformational stabilities assigned t o the aldopentofuranosides listed in Table I can be extended to the aldohexofuranosides and the ketohexofuranosides. The introduction of a twocarbon group instead of a one-carbon group at C-4 of the aldohexofuranosides may have some effect on the C-3, C-4 interaction, but it seems to be minor. The glycofuranosides of n-galactose and 6-deoxy-~-galactose b-fucose) may be compared with those of the carabinofuranosides, those of D-glucoRe and D-glucurono-6,%lactone with the D-xylofuranosides, and those of D-mannosc wid O-tlooxy-L-rnniitioso (L-rhnninose) with the Dnricl L-IyxcifiirnrioRitles, respectively. (18s) l.3, Ciy)oii rttid I). Thncker, Proc. ( ’ h p m . Soc., 369 (1964). ( l e ) ThiH inclunion of the oxygen atam of the “u-hydroxyl” or “lphydroxyl” group at (2-6 within the ring occurs for both sldohexo- and aldopento-pyranoeides, and 80 no difficulty occure in comparing the various inembers of these seriee,

101

THE QLYCOFURANOQDES

q

HOH,C OH

HCOH HO&L (11)

OH (12)

In the case of the methyl D-fructofuranosides, there is a relationship to the warabinofuranosides, with a different type of C-1, C-2 interaction. Here, the hydrogen atom on C-1 has been replaced by a hydroxymethyl group, and the difference in the C-2 interactions with’the C-3 hydroxyl group should be small for the anomers; the effect of the aglycon group (OMe) and the hydroxymethyl group will probably be similar. 111. FORMATION OF GLYCOFURANOSIDES IN ACIDICMETHANOL This reaction, a brief glycosidation of a sugar with methanol at room temperature, with hydrogen ion as the catalyst, is the Fischer reaction.’ For preparative purposes, it involves the isolation of kinetic products (glycofuranosides),formed at a relatively high rate, before their subsequent conversion, at a much lower rate, into the thermodynamically more stable products (glycopyranosides). The reaction ie usually performed at a low temperature for a short period of time, with a low concentration of acidic catalyst. The reaction time ia often monitored by following changes in optical rotation or decrease in reducing power, or by chromatography. Use has been msde of protecting groups suitably located to prevent the formation of the pyranosides. The presence of another ring (lactone or anhydro) often stabilizes the furanoside. 1. Isolation of Products It WBR from such n remtion mixture (obtained by shaking 20 g. of CY-Dglucose with 400 g. of methanol nontrtiriing 1% of hydrogen chloride for 15 hours at room temperature, neutralizing with silver carbonate, evaporating the solution, and extracting the resulting sirup with ethyl acetate) that Fischer isolated his methyl “gamma” D-glucoside, a nonreducing sirup that waa readily hydrolyred by dilute acids. This impure material (and

102

JOHN

W. GREEN

other "gamma" glycosides) had been methylated and converted into known, crystalline productsm before any crystalline glycofuranosides had been isolated. Hence, the ring structure of this impure gamma glucoside was demonstrated to be furanoid before fractionation into pure components had been aohieved. Levene and coworkers,a' using the ease of hydrolysis (10 minutes in 0.1 N hydrochloric acid at 100") of the aldofuranosides aa an analytical tool, studied the rate of glycoside formation (see Fig. 1) and confirmed the preliminary formation of aldofuranosides and subsequent conversion into aldopyranosides. The first crystalline furanoside waa prepared by Haworth and P0rter.l A protecting technique was used, starting with 1,2-0-isopropylidene-a-~glucofuranose; this compound was converted into the 5,6-carbonate, and the isopropylidene group waa removed with ethanolic hydrogen chloride, with formation of the two ethyl wglucofuranoaide 5 ,&arbonatee. These were separated by fractional recrystallization and each waa individually saponified to the D-glucofuranoside; the CY-D anomer was obtained aa a crystalline compound. The &D anomer was a hygroscopic solid, but it was later obtained in crystalline form by Phillips.g* (Methyl a-wglucofuranoside waa obtained in a similar manner.a*) Methyl a-wmannofuranoside wm prepared by a somewhat similar technique, starting from D-mannofuranose 2,3;5,&dicarbonate2'; the glycoside was also prepared directly, in good yield,28 by methyl glycosidation on seeding with a crystal obtained by the protecting techniq~e.~'Thus, methyl a-D-mannofuranoside may be considered to be the first ddofuranoside to be Crystallized directly from a Fischer reaction mixture. Although the f h t crystalline glycofuranoside waa prepared in ethanolhydrogen chloride, most of those subsequently prepared have been formed in acidio methanol, without the aid of protecting groups. Methyl CY-Dfructofuranoside waa prepared'd by allowing a solution of D-fructose in methanolic hydrogen chloride to reach its maximum dextrorotatory value, and then fermenting the unreacted sugar by treating the mixture with bakers' yeast. The sirupy mixture of fructosides remaining was fractionated (20) W. N. Haworth, E. L.Hirat, and E. J. Miller, J . Chem. Sw., 2436 (1927); H. D. K. Drew, E,H. Goodyear, and W. N. Haworth, ibid., 1337 (1927); C. F. Allpress, W. N. Haworth, and J. J. Inbter, ibid., 1234 (1927); W. N. Haworth end C. R. Porter, ibid., 616 (1928); P.A. Levene and Q. M.Meyer, J . Bwl. Chem., 76, 809 (1928); H. G.'Bott, E. L. Hirst, and J. A. B. Smith, J . Chem. Iqoc., 659 (1930). (21) P. A. Levene, A. L. Raymond, and R. T. Dillon, J . Biol. Chem.,96, 699 (1932). (22) D. D.Phillip, J . Am. Chsnt. floc., 76, 3598 (1954). (23) W. N. Haworth, C. R. Porter, and A. C. Waine, J . Chem. floc., 2254 (1932)(24) W. N. Haworth and C. R. Porter, J . Chem. Soc., A49 (1930). (26) W. N. Haworth, E. L. Hirst, and J. I. Webb, J . Chem. floc., 651 (1930). (26) C. B. Purves and C. 8.Hudmn, J . Am. Chem. Iqoc., 66,708 (1934).

THE OLYCOFURANOSIDES

looo-Lyxose/

103

-----I*-

--. 500

-Free wgor ----------

/@@

500

50

Time (hours)

Pyranosids Furonosib

Fro. 1.-Percentages of Free Sugar, Furanwide, and Pyranoaide during Glymide Formation at 25" in Methyl Alcohol Containing 0.5 per cent of Hydrogen Chloride.*'

with ethyl acetate, and a 10% yield of the crystalline product was finally obtained. Methyl am-arabinofuranoside was also prepared" by allowing the acidic methanol solution to reach its maximum dextrorotation; an ether extraction was used to obtain a 9% yield of product. The separation of glycosides on a cellulose column was first applied to a mixture of pyranosides**;subsequently, separations were achieved for the methyl sfructofuranosides and D-gdactofuranosides.*O This technique waa soou applied to many other mixtures; it permits not only the isolation of crystalline products, but also a more quantitative evaluation of the formation of g l y c o f u r a n o s i d e ~ . ~ ~ ~ (27) E. M. Montgomery rriitl C . S. IIutlwnri, J . Am. Chem. Soo., 69, M)2 (1937). (28) L. Trough, J. h'. N. J011ep1,atid W. 11. Wadmim, J . C h .Soc., 1702 (1950). (29) I. Auge8tad, E. Berner, and E. Weigner, Chem. Zd. (London), 376 (1953). 130) C. E. Ballou and H. 0. L. Fisoher, J . Am. Chem. SOC.,75,4605 (1953). (31) I. Augestad and E. Berner, Actu Chem. Scand., 8, 261 (1954). (32) D. F. Mowery and G. R. Ferrante, J . Am. Chena. SOC.,76, 4103 (1964). (33) G. P..Barker and D. C. C. Smith, J . Chem. h e . , 2161 (1954). (34) W. M. Watkins, J . C h . Soc., 2064 (1955).

104

JOAN W. UIEEEN

A large amourit of data has been accumulated during the preparation of the various glycofuranosides, and some of this is given in Table 11. These data are to a great extent qualitative, and the purpose of giving them here is only to show the preponderant anomer formed under certain conditions of acidic methyl glycosidation. Most of the data are derived from the results of chromatographic separations; some of them have been obtained by gas chromatography.l6 In almost every case, the preponderant anomer is the trans-l ,2-glycoside; the interaction between its aglycon group and the C-2 hydroxyl group is much leas than for the cis-1,2 anomer. Conformational stabilities for the aldopentofursnosides have already been discussed (see Section I1 and Table I). To 8 certain extent, the conformational stabilities of the aldohexofuranosides are comparable to those of the corresponding pentofuranosides; but the former have a bulkier group at C-4 to cause interactions that lower the stability. For the wfructofuranosides, there is apparently little difference between interactions of either the aglycon methyl group or the hydroxymethyl group (on C-2) with the C-3 hydroxyl group; the anomers are formed in approximately equal yields. The high yields obtained indicate the conformational stability of the D-fructofuranoside structure, which is very similar to tSat of the Darabinofuranosides. Although hydrogen chloride has been the catalyst principally employed in the Fischsr method, other acids, notably sulfuric acid, have been used, ae well as acidic, ion-exchange resins.8bag The advantage of the latter reagents is that the insoluble resin can be readily filtered from the reaction solution. It is held that the replacement of soluble acids by insoluble resins does not appreciably alter the formation of the glycofuranosides. The glycosides formed from the tetroses are, of necessity, furanosides. (36) E. M. Osman, K. C. Hobbs, and W. E. Waletan, J . Am. C h . Soc., 75,2726 (1961). (36) J. E. Cadotte, F. Smith, and D. Sprieaterbach, J . Am. C h .SOC.,74, 1601 (1962). (37) W. H. Wadman, J . Chcm. rSoc., 3061 (1962). (38) G. R. Dean and R. E. Pyle, Britiah Pat. 670,480 (1962); Chem. Abetracfs, 46, 9332 (1962). (39) D.F. Mowery, J . Ah. C h .Boc., 77, 1667 (1966). (40)J. N. B u i r and A. 8.Perlin, Can. J . C h . , 88,2217 (1980). (41) R. Barker and H. G . Fletcher, Jr., J. Org. C h m . , 26, 4605 (1961). (42) C. T. Biohop and F. P. Cooper, Can. J . Chsm., 40, 224 (1980). (43) 0. KjoelSerg and 0. J. Tjelveit, Acta Chem. Scand., 17, 1641 (1963). (44) P. W. Auetin, F. E. Herdy, J. G. Buchanan, and J. Baddiley, J . Chem. SOC.,6360 ( 1963). (46) D.F. Mowery, Jr., Method8 Carbohydrals C h . , 2, 328 (1963), (46) I). F. Mowery, Jr., J . Org. Chsm., 96, 3484 (1961). (47) J. Cf. Uarclirier and E. E. Percivnl, J . Chm. ~ o c . ,1414 (1958). (48) I. Auptad and E. Berner, Acb C h . Swnd., 10,811 (1966).

TABLE I1 Formation of Glycofuranosides in Acidic Methmol

Sugar

D-ED-Threose L-Arsbine D-Arabmaee D-Ribose D-Xylose

D-Lyxose

D-GlucOse D-Galactose

D-Mannose DFructose L-Fucose

GRhamnose ~-G~ucuron+6,3-lactone

Normality of HCI

0.6 0.6 0.01 0.a328 0.0028 0.28 [0 .18N H2SOd 0 .OO28 0.0036 0.14 0.00% 0.04 [O. 18 N Ha04 0.0037 cO.36 N H W I [DoweX-50 resin CDowex-50 resin 0.5 0.04 0.22

0.0055 0.14 [Nalcite HCIt resin

Temp., 'C.

25 25 65 35 35 25

4 35

65 25 35 65 25

65 65

65 65 25

65 15 65 25 65

Time, min.

Product formation Predicted References 0 Typeof snomer Anomer Anomer data" a

5 5

11 3

180

56 1 1

c

50 14 9 6 0 2 0 1

-

360 L

D

22

1 large 54

840 5 360@) 14 120 0.35 50 15 0.61 12 41 D 17 21 4Mw) 30 470 4200 2 180 2

-

65 2 22

0.32 3.3 16 80 1.69 45

1.70

small 3

9 54 1.05 3 0.34

45 36 44

-

62 72

A B A C C A B C A

C C A

A A

B A C A A

A A A A

B a

a a

B B B B B B a a

B B B

40

40

31 15 15 33 411 15 31 42 15 43

441 31

a

451

Q

461

B B a

29 43 47 48

B B

351

n

a Key: A, % of product isolated; B, ratio of products, determined by chromatography; C, relative rate of formation; D, reaction allowed to proceed to constant rotation.

z

M

JOHN W. GREEN

106

The ready formation of furanosides from wglucurono-6,3-lrtctone is aided by the presonce of the laotone ring. Mixtures of the anomers of glycofuranosides have often been used as starting materials for various syntheses, instead of the individual anomers. Thus, methyl a!,P-wribofuranoaide is often encountered in the literature of the nucleic acids. Such mixtures can be converted into the esters, or into the glycofuranosyl halides, and then into the individual glycofuranosides. Some of these applications will be discussed later (see pp. 121). 2. Kinetics

Several kinetic studies have been made, since the initial work of Levene and coworkers,*1which have established the importance of the anomerization of the furanosides, and the reversibility of the conversion of furanosides into pyranosides. However, this work has thus far been confined to the four aldopentoses and to two aldohexoses. With the d d of column chromatography, MoweryM studied the rate of formation of furanosides and pyranosides from Parabinose. The rate of furanoside formation ww extrapolated back to zero time, and an initial rate was obtained (see Table 111).The less stable anomer, the bfuranoeide, waa initially formed to the grertter extent; in the final equilibrium mixture, the more stable (u-D anomer waa preponderant. Tmm I11 Rats of Formation. of Aldooidea in Acidic Methanol'" Sugar

*Mannose

tArabinose

Glyaoride

Oridnal rate mnstant

Equilibrium Solution cornpodtion, time, houm

%

rrfunrnoeide @-furanosida a-pmoeide &pyrenoside

0.61 0.34 0.12 0.03

2 2 89

a-furanaside Bfuranoeide a-pyranoside ppyranoside

1.3 2 .o 0.15 0.16

23

7 8 24

45

72 72 72 72 24 24 24 24

a Theee v d u a M e r from those in Table 11, in that the rates are extrapolated back to mro time a i d do not ne-rily reprwnt the overall rate for the reaotion; nor do they represent anomerieation. b t a oonstsnte are first order, given with time (in hours) and oommon logarithms. Reaction conditions: Dowex-60 reain and methanol at 65'.

THE C)LYCOFURANOBIDES

107

Extrapolation of rates back to zero time also showed a small but definite formation of pyranosides from the free sugar, in addition to conversion from the furanoddes. The reversibility of the conversion of furanosides into pyranosides waa definitely demonstrated by obtaining the final equilibrium composition from two directions, namely, from the a-Dpyranoside and from the free sugar. A detailed study of the methyl glycosidation of the four aldopentoses has been made by Bishop and C o 0 p e r , 1 with ~ ~ ~ ~the aid of gas chromatography of the esters of the resulting pentofuranosides and pentopyranosides. Three competing reactions were established for the formation of the furanosides: (1) an irreversible formation of furanosides from the free sugars; (2) an anomerization of the furanosides; and (3) conversion of the furanosides into the pyranosides. (A fourth reaction, anomerization of the pyranosides, will not be considered here.) The kinetic data are given in Table IV, and the compositions of the equilibrium mixtures are given in Table V. I n both of these Tables, the data are given for the four pentoses in the same order as in Table I, that is, in the order of their conformational stability. The furanosides having the lowest number of nonbonded interactions are formed most rapidly, have a lower rate of anomerization, and a lower rate of conversion into the pyranosides. There is also a higher percentage of these more stable furanosides in the final equilibrium mixtures. The reverse holds for those glycofuranosides having a greater number of interactions. Thus, we find a much higher rate of anomerization for the methyl D-lyxofuranosides (in contrast to the D-arabinofuranosides), and only a small proportion of the a-wlyxofuranoside and no j3-D-lyxofuranoside in the h a 1 equilibrium mixture. In all cases, the rate of anomerization is very high, relative to the rate of conversion into pyranosides; this rate of anomeriaation is the major factor in determining the relative yields of the a- and ,9-furanosides from a reaction mixture, and not the relative proportions of these two anomers initially formed from the free sugar. The ratio of anomers waa found to be approximately constant throughout the course of the reaction (1.7:l for the j3- and a-~-xylofuranosides).~* Only with *lyxose watj there any evidence of pyranoside formation early in the reaction. A slight reverse reaction, furanoside to pentose, was also noted for this sugar. This behavior is in agreement with the low conformational stability of the D-lyxofuranosides. However, no initial formation of maxabinopyranosides was noted. The more probable formation of furanosides as compared with pyranosides, especially at lower temperatures, has been discussed by Shafizadeh.' Specific alignment and orientation of the group on C-5 is required

TABLE IV Kinetic Data for Methyl G l y d d a t i o n of Mdopentoeee"

--i

226 57 19

homerhation of fursn-osidess 4

a+8 *a

1.39 11.3 49

very malI

Conversion into pyrauusided h

Furanogide

pyranoaide *a

0 .m 0.069 0.12 0.84

5; 4.35 3 -4

29 very 0.11 0.00s 0 .o004 0.001

5.74 15.2 78

0.32 3.3 1.69

-695 740 -320

verylerge 7.9 8.8 320

6-50

-1260 -1330

-3530

-4080

The reactions were performed in methanol containing 0.0028 d l HCl at 35". * This rate constant could not be determined, because of the low solubility of ~arabimosein methanol; its value is considered to be very large, based on the amount of furanoside at equilibrium. K = v]/[a]. K = [arpyranorside] CBpyrsnoside]/[~-furanoside] C&furandde].

a P Eg

TEE ffLYCOFURANOSIDES

109

TABLE V D-AldopentofuranosideComposition at Equilibrium" Sugar"

a-Furanoside, PFuranoside,

96

%

Total furanoeide,

% . DArabinc-se D-Ribose D-Xylose D-Lyxose 3-O-Methyl-D-arabmose 20-Methyl-warabinose 2,3-Di-O-methyl-~-arabinose ;U)-Methyl-D-xylose 2-0-Methy 1-wxylose 2,3-Di-O-rnethyl-D-xylose ~

a

6.8 17.4 3.2 0

21.5 6.2 1.9 1.4

28.3 22.6 5.1 1.4

50.7

66.7 75.4 9 .o 12.8 16.4

_ _ _ _ _ _ _ ~ ~ ~ ~

Reaction conditions: 2% of sugar in 0.28 M MeOH-HCl at 35".

for the formation of pyranosides, in addition to the alignment of the carbon atoms also required for formation of the furanoside ring. The rates of methyl glycosidation are approximately proportional to the concentration of acid catalyst. In the early part of the glycosidation, a single reaction is predominant and can be characterized as first order. A cyclic, carbonium intermediate (13) wiw proposed for the mechanism of the anomeriration of the furanosides. D-XylOSe

HO

Hpy=xpH -

HOH,C,

HI

,OH

H

O

H

,

HoTQ(MF OH

Ho,c'H+

1-P

(1s)

Methyl (I-Dxy lofur anoside

C

!

~

M

OH

MeOH

+I@\ Methyl

p 8-D-

xylofuranoeide

The effect of adverse interactions is shown even more strongly in the case of the mono- and di-methyl ethers of Darabinose and D-xylose (see

~

110

JOHN W. QBIDEN

Table V). The distances between trans-hydroxyl groups (equatorialequatorial) in pyranosidos is 2.8 A.; for the truns-hydroxyl groups in furanosides, this distance is greater, namely, 3.44 A. This mems that there is a greater increase in adverse interations contributed by the bulky methoxyl groups in the pyranosides than for the furanosides. This relatively greater stability of the ethers of the furanosides is shown by their higher proportion in the final equilibrium mixtures, as compared with the unmethylated sugar. A kinetic study of the methyl glycosidation of wmannose was also made by M~wery.'~ The more stable anomer, the a-D-mannofuranoside, was formed at a higher initial rate (see Table 111); the proportions of both furanosides in the final equilibrium mixture wm too small to permit accurate comparison of isomer distribution. The conformational stability of the D-mannofuranosides may be compared with that of the wlyxofuranosides; the furanoid structures are similar, except for the bulky two-carbon group at C-4 of the hexoside. This similarity is shown in the very small proportion of wlyxofuranosides (see Table V) and of D-mannofuranosideo (see Table 111) in the final equilibrium mixtures, and also in the initial formation of wmannopyranosides" and of wlyxofuranosides.16 Overend and coworkers'@have studied the action of acidic methanol on methyl &wglucofuranoside, with two concentrations of acid catalyst (see Table VI). The rapid anomerization in methanol under the conditions studied gave only furanosides. Use of 14C-labeled methanol gave labeled furanosides, showing participation of the solvent in the anomerisation. These authors suggested the reaction (14) (15) to explain the role of methanol.

H

HO€I,C

d

q

HOMe

HOH, HdQEEe

+ MeOH

L 7

HMe

H (14)

OH (15)

The slower conversion of the anomeriaed D-glucofuranosides into wglucopyranosides waa studied with a higher concentration. of acid. The concentration of furanosides waa followed by determining the aqount of formaldehyde formed by periodate oxidation. It waa concluded that the same acyclic ion (16) is formed from both furanoid anomers, and that ring closure gives a mixture of pyranosides similar to that found in the equilibrium mixture of pyranosides. (48) B. Capon,G. W. Loveday, and W. G. Overend, Chsrn. Ind. (London), 1537 (1962).

THE QLYCOFURANOSIDES

111

TABLEVI Action of Acidic Methanol on Methyl D-GlucofuranoaidesU H e m tion

Anomerisationb Conversion into pyranosidesc

loSk (in em.-') at

Molarity of acida

0.10 2.0

25.0"

35.0"

1.05 0.748

2.88

35.2"

40.1"

2.65

3.25 10.2

45.0"

Methsuesulfonic acid. b The substrate is the &D-furanoside; the data are for 10' (kl kl), determined polarimetrically; the anomeriaed mixture contains f33% of the fl and 37% of the a form. 0 The substrate is the anomerised mixture; the data are for 106 h,the overall rate co-t, determined from the formaldehyde formed on oxidation with periodate. 0

+

The composition of the anomerized D-glucofuranosides (63% of /3: 37% of a) has exactly the same ratio (1.7:l) as that observed by Bishop and Cooper16 for the mxylofuranosides. This excellent agreement can be correlated with the structure of these hexofuranosides and pentofuranosides; presumably, their conformational stabilities are very similar.

Capon and T h a ~ k e r ' @ have ~ demonstrated the formation of aldofuranosides from acyclic acetals of D-glucose and &galactose in 0.05 M hydrochloric acid at 35'. The yields (kinetically controlled) of furanosides for the two acetals were 98 and 71%, respectively, showing the successful competition, in each case, of the C-4 hydroxyl group over the solvent. The authors have suggested a mechanism of simultaneous ring-closure and breaking of the acetal bond; the rate constant for the D-glucose acetal is much greater than that for the D-galactose acetal. This variation in the nucleophilicity of the C-4 hydroxyl group is also discussed on p. 120. (49a) B. Capon and D. Thacker, J . Am. Chem. Soe., 87, 4199 (1966). (49b) Unpubliihed work of A. A. Court (quoted in above reference).

112

JOHN W. QRPJEN

IV. PREPARATION OF GLYCOFURANOSIDEB FROM DITHIOACETALS

1. Isolation of Products This method, developed by Pacsu and Green,a consists in treating a sugar dialkyl dithioacetal (17) with niercuric chlorido and yellow iiicrcurio oxide in a chosen alcohol; the reaction is irreversible, and gives a mixture of the anomeric glycofuranosides (18) and (19). The reaction is maintained neutral, snd there is no anomeriaation end no oonversion of furanosides into pyranosides. The method has been applied to most of the common sugarB, with the exception of D-xylose. It is not applicable to =glucose, because a stable &yl l-thio-a-wglucofuranoside is formed. The reaction is valuable aa a preparative method, and products have often been crystdl i d directly from the reaation mixture. In a few cmes, acetals have been obtained instead of the glycofuranosides. H EtSCSEt

HAOH I

YHOH THOH

YOH C%OH (17)

(18)

(19)

where Y = -CHOH-CH,OH

As the glycofuranosides are the final thermodynamic products, there is no need to control the reaction conditions closely, unlike the isolation of kinetic products in the Fischer method. Constant agitation is needed, to ensure reaction of the insoluble mercuric oxide, and a desiccant (Drierite) is wed for removing the water aa it is formed. In most of the examples cited in this review, the diethyl dithioacetal is used as the Btarting material. In Table VII is given B list of products obtained by this method; the preponderant anomer obtained is the trans-1 ,2-glycofuranoside (IS), an effect similar to that noted for products formed by the Fischer method (see Table I1 on p. 105). The data in Table VII are limited in scope; the yields are mostly preparative, and do not represent quantitative recoveries. In only four of the examples cited are the yields above 50%. The 91% yield of the anomers of ethyl Grhamnofuranoside was obtained by separation on a chromatographic column, and represents the only quantitative information on this method. In all other examples, the products were isolated by crystallisation techniques, and it has often been assumed that the crystalline product isolated, often in low yield, had been the preponder-

113

THE GLYCOPCJIUSOSIDES

TABLE \.?I Glycofuranosides Formed from Dithioacetals

Glycofuranoside

Anomer

Yield, %

Predicted References anomer

GArabino furanoside methyl ethyl

a

14

a

24

a a

50 50

n-Lyxofuranoside methyl

a

61

U

51

B B

20

B B

50 3,52 53

n-Galactofuranoside methyl ethyl ethyl prowl benzyl D-Mannofuranoside methyl methyl ethyl ProPYl ieopropyl methyl 2-0-methyl-

a

B B a

B

70 5 23

12 64 13

B B B a a a

a

higha high0 higha

ff

41

a

r.,-Rhamnofuranoside ethyl ethyl

a

56 36

a

B

Methyl n-fructofuranoside

a

Methyl 2-acetamido-Zdeoxy-Dglucofuranoside

B

a

a

a a

50 50 54 54 54 54 54 55

a

5G 56

low

-

57

high"

B

58

a High yields are assumed from the contextij of the articles; no numerical data were given.

(50) J. W. Green and E. Paceu, J. Am. Chem. Soc., 60, 2056 (1938). (51) M. Nys and J. 1.' Verheijden, Bull. Soc. Chim. Belg., 69, 57 (1960). (52) J. W.Green i r r d JC. I'acsu, .I. Am. Chem. Soc., 80, 1205 (1937). (63) J. W. Gram arid E. Pwsu, J . A m . C'hem. Soc., 69, 2569 (1937). (54) A. Scut1srlr;oocI uiicl 14:. I'w~11, J . .4 v b . (.'htsm. S~JC., 62, 203 (1940). and H. M. Triater, J . A m . C/iuni. Soc., 68, 925 (1941). (55) E. PIL(:NU (56) J. D. Ueerdes, B. A. Lewis, It. Montgomery, and F. Smith, Anal. Chem., 26, 264 ( 1954). (57) E. Paceu, J. Am. Chem. Soc., 61, 1671 (1939); 60, '2277 (1938). (58) M. W. Wliitehouse and P. W. Kent, Tulruhedron, 4, 425 (1958).

114

JOHN W. GREEN

ant component in the reaction mixture, although this assumption is not necessarily valid. The formation of the trans-l,2-glycofuranosideais effected in neutral solution, where anomerization cannot occur. Treatment of 3 ,4-O-isopropylidene-2,S-di-O-methyl-~-rhamnosediethyl dithioacetal with mercuric chloride in boiling methanol gave6ga 61% yield of the crbfuranoside; no trace of the &-I, anomer was found. In this experiment, with an acidic solution (no mercuric oxide present), anomerization could have led to the formation of the more stable anomer. In the normal reactions cited, with maintenance of a neutral solution, no anomerization can occur, and yet the more stable anomer, presumably the kinetic product, is still the major anomer formed. Although dithioacetals of glucose do not form the wglucofuranosides, those of Zdeoxy-Parabino-hexose do; Stacey and coworkersm were able to convert the dibenzyl dithioacetal into a mixture of the methyl 2-deoxywarabino-hexofuranosides. Thus, the C-2 hydroxyl group has a bearing on the course of the reaction. 2. Alkyl LThioaldofuranosides

The l-thioglycosides have been reviewed in detail by Hortoh and Hutsono; the present Section deals only with the l-thioaldofuranosides, mostly in relation to their preparation from dithioacetals. The formation of these products from the dialkyl dithioacetals falls into two categories. The first group comprises compounds readily formed (or isolated), and is confined to derivatives of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-~-glucose,and wribose. The products are obtained in high yield; the formation is generally aacomplished in aqueous solution, with one mole of mercuric chloride per mole of dithioacetal, and the solution is kept neutral. The second category involves derivatives of lower stability-those of D-galactose and 6-O-benzoyl-D-arabinose ; here, similar modes of prepare tion are used, but the yields are much lower. In 1916, Schneider and Seppo1prepared ethyl l-thio-a-glucofuranoside by treating an aqueous solution of one mole of the diethyl dithioacetal with one mole of mercuric!chloride, and maintaining neutrality by neutralizing the hydrochloric acid formed with aqueous Rodium hydroxide. The product wm regarded by them as a “normal” ( L e . , pyranoid) l-thioglyB.Foster, J. Lehman, and M. Stacey, J . Chem. sbc., 4649 (1961). (60)W. G. Overend, M. Stacey, aid J. StanCk, J . Chem. Soc., 2841 (1049). (61) W. ScLeider and J. Sepp, Ber., 49, 2054 (1916). (MI) A.

THE GLYCOFURASOSIDES

115

cwide,O although they remarked that it was more readily hydrolyzed than the BD anomer prepared from tetra-0-acetyl-a-mglucopyranosyl bromide. The furanoid nature of the former compound was shown60 by its eaw of acid hydrolysis and isorotation values, and was further confirmed by periodate oxidation.ea A standard method of preparation of this l-thioaldofuranoside, and of others, was established by Pacsu and Wilson'J'; it consists of performing the reaction (20)-421) in aqueous solution, with H EtSCSEt I HCOH I CHOH

LHOH I CHOH I CH,OH

(20)

0 . 5 HgCI,

+ 0 . 5 HgO

Y,H

qsEt + HgClSEt + 0.5 H,O

H, OH

HO

(21)

where Y = -CHOH- CH,OH

0.5 mole of mercuric chloride per mole, and excess of mercuric oxide to

maintain a neutral solution (and to generate another half mole of mercuric chloride). In contrast to the preparation of the aldofuranosides, the alkyl group of the 1-thioaldofuranoside is the alkyl group of the original dialkyl dithioacetal. As the solvent does not participate in the formation of the 1-thioaldor'uranoside, it does not have to be nucleophilic; thus, acetone has been used."s Cadmium carbonate may also be employed, instead of mercuric oxide, to maintain neutralitye6; here, one mole of mercuric chloride per mole of dithioacetal is needed. The methyl, propyl, and bengl 1-thio-a-wglucofuranosides were preparede' by the original method (with neutralization by sodium hydroxide). Use of the method of Pacsu and Wilsone4gave the methyl, ethyl, propyl, and isopropyl 1-thio-a-D-ribofuranosidesin yields ranging from 65 to 80%. Sodium (methyl 1-thio-a-D-g1ucofuranosid)uronate and the ethyl and propyl analogs were obtained6' similarly from the sodium salt of the (62) It is iiiteresting that Schneider and Seppal gave the correct formula for their product,

and showed a furanoid ring; it waa not until a decade later that the pyranoid ring was accepted for the "normal" glycosidea. (63) M. L. Wolfrom, S. W. Waisbrot, D. I. Weisblat, and A. Thompson, J . Am. Chem. Soc., 66, 2063 (1944). (64) E. Pacsu and E. J. Wilson, Jr., J . Am. Chem. Soc., 61, 1450 (1939). (65) E. J. Reist, P. A. Hart, L. Goodman, and B. R. Baker, J . Am. Chem. Soc., 81, 5176 (1959). 166) H. Zinner, A. Koine, and H. Nima, Chem. Ber., 93, 2705 (1960). (07) Y, Nitta and A. Momoee, Yakugaku Zasshi, 82, 574 (1962); Chem. Ab&ack, 68, 4631)(1963).

116

JOHN W. ORIOEN

corresponding dithioacetal. The anomers of ethyl 2-acetamido-2-deoxyl-thio-D-gldcofuranoside were also prepared,(n the CY-D anomer in 56% yield and the BDanomer in lower yield (as the triacetate). Wolfrom and coworkers~+71were able to prepare various l-thio-a-P galactofuranoeides, generally isolated as the acetates, after purification by column chromatography. Thus, the Paceu arid Wilson method gave sirupy ethyl l-thio-a?.D-galactofuanoside,and a crystalline acetate. This product was also obtained by treatment of the dithioacetal with dilute hydrochloric acid and then mercuric oxide. Ethyl Zacetamido-2-deoxy-l-thio-~-~galactofuranoside w&s prepared in 32% yield, and the P-D anorner in 3% yield. Whereaa ethyl l-thio-/3-D-arabinofuranosidecannot be prepared directly, the 5-0-benzayl diethyl dithioacetal gave 38% of ethyl 5-O-benzoyl-l-thio&r+arabinofuranoside, which was debeneoylated to the desired product. The &benzoate of ethyl l-thio-a-D-ribofuranosideWBB similarly prepared.w Two other compounds, ethyl 2-tlcetamido-2-deoxy-l-thio-8-c~abinofuranoside" and ethyl 2-acetamido-2-deoxy-l-thio-cr~-xylofurq~ide,7~ were prepared from the corresponding D-galacto and Dgluco analogs by periodate oxidation, and subsequent borohydride reduction of the product. Zinner and coworkers studiedw the possibility of formation of l-thioaldofuranosidss from other sugar dithioacetals, and noted that, both with Pmannose dithioacetals and D-xylose dithioacetals, only the free sugar waa formed ;no thiofuranosides were obtained. With Plyxoae dithioacetals, both the free sugar and the l-thiofuranoside were formed: the latter, detected in only small amounts (on a paper chromatogram), w w not iaolated. All of the major products obtained, starting from the dithioacetals, have a cis relationship of the alkylthio group and the hydroxyl (or 2-acetamido-2deoxy) group at C-2. This formation of a atable, cis derivative is reminiscent of the similar relative stability of certain mylglycosyl halides." A corresponding effect has been noted for the l-thioaldopyranosides. Ethyl 2-acetarnido-3,4,6-tri-O-acetyl-2-deoxy1-thio-a-D-glucopyranoside is resistant to the action of mercuric chloride in hot, neutral, methanol solution, whereas the B-D anomer is readily converted into the 8-wgluco(68) M. L. Wolfrom, S. M. O h , and W.J. Polglase, J . Am. Chem. Soc., 73, 1724 (1960). (69) M. L. Wolfrom, Z. Yoaiiawa, and B. 0. Julieno, J . Org. Chem., 34, 1629 (1969). (70) M. L. Wolfrom, P. McWain, R.Pqnucco, and A. Thompon, J . Org. Chem., 39, 464 (1964). (71) M. L. Wolfrom and 2.Yo&awa, J . A m Cheni. Soc., 81, 3474 (1969). (72) M. L. Wolfrom and Z. Yodaawa, J . Am. Chern. Soc., 81, 3477 (1969). (73) M. t. Wolfrom and K. Anno, J . Am. Chem. Soe., 76, 1038 (1953). (74) L. J. lluyne~find F. €1. Newth, Aduaw. Carhh&de Chem.,10, 207 (1956).

THE GLYCOFURANOSIDES

117

pyranoside?6 Again, ethyl l-thio-p-Pmannopyranoside has been sh0w11~~ to be resistant to the action of mercuric chloride in methanol. Both of these nonreactive 1-thioaldopyranosides have this cis relationship. Conversely, whereaa dithioacetals of wribose afford the l-thio-D-ribofuranosidesreadily, those of 2-deoxy-Dglthro-pentose do not, but give the free sugar and unchanged dit hioacet a1 instead .m It is interesting that such resistance is encountered in a compound having a cis relationship and the alkylthio group; the latter is a leaving group which leaves readily, whose displacement should be facilitated by mercuric chloride. This effect will be discussed in more detail in the following Section. 3. Mechanism

A series of possible pathways for the formation of aldofuranosides (25) and of l-thioaldofuranosides (26) from dithioacetals (22) is shown on p. 118. Pathway AC leads to the aldofuranoside through an acyclic monothioacetal (23); the latter can also afford the diacetal (24) by pathway B . Pathway D leads directly from the dithioacetal to the l-thioaldofuranoside. A third pathway, EF, in aqueous solution, leads to the free sugar (28). Pathways C' and F' show alternative formations of the aldofuranoside and the free sugar, respectively, starting from the l-thioaldofuranoside. PacsuS7had proposed the possibility of an acyclic intermediate, a monothioacetal (23), in the formation of aldofuranosides and l-thioaldofuranosides; this suggestion was based on the isolation of acetals as minor products formed from the dithioacetals. Wolfrom and c~workers,?~ starting with such a monothioacetal, prepared from unrelated systems, showed that such a pathway is valid for &galactose. A very high yield (85-9001,) of ethyl 8-Pgalactofuranoside was obtained from wgalactose diethyl monothioaeetal; the yield was much higher than that obtaineds2directly from the dithioaeetal. For &glucose, the formation of ethyl l-thio-a-D-glucofuranoaide from a S-ethyl-O-methyl monothioacetal was not demonstrated; instead, a 95% yield of methyl &D-glucofuranoside was obtained, again demonstrating that pathway C had been followed. It may also be pointed out that the conversion of the monothioacetal into the l-thioaldofuranoside is improbable, as it involves the preferential displacement of an alkoxy group, instead of an ethylthio group; the former is a leaving group that leaves with difficulty, and the latter leaves wit,h ease, being acidic in nature. (75) L. Hough and M. I. Taha, J . Cheni. Soc., 2042 (1956). (76) J. Fried and L). E. Walr, J . Am. Chem. Soc., 71, 140 (1949). (77) M.L. Wolfrom, D. I. Weisblat, and A. R. Hanze, J . Am. Chew. Sor., 66, 2065 (1944).

JOHN W. GHEEN

118

Pathway C' has been demonstrated for both the ethyl and benayl 1-thio-a-wglucofuranosides;these compounds were uonverted,'* in ethanol

PR HtSEt H OH I

HT:H (29)

\ B

CHOH H t O H

(24)

at 70°, into 8 nonreducing sirup, presumably ethyl D-glucofuranoside,which W&B not investigated further. A mechanism is here proposed which is speculative in nature, but which affords a possible explanation of the reactivity of the acyclic monothioacetal (23) and the lack of reactivity of the 1-thioaldofuranoside (26). It

119

THE GLYCOFURANOSIDES

in b w d on the participation of the bulky, mercuric chloride molecule in the tranuition state for the reaction of the monothioacetal (30),the dithioacetal (29), or the 1-thioaldofuranoside (31) with a nucleophile (which may be an alcohol, water, or the C-4 hydroxyl group of the aldohexose or aldopentose concerned). This nucleophilic displacement of the ethylthio (or other ttlkylthio) groups from the dithioacetal or from subsequent products is aided by the mercuric chloride molecule. Shafizadeh' has suggested that a unimolecular (SNJ reaction is operative. In this type of reaction, the rate-determining step would be the formation of a carbonium ion, with the removal of the ethylthio group; subsequent attack on this ion by the nucleophile would be rapid. In an SN2 reaction, the removal of the ethylthio group and the attack by the nucleophile would be simultaneous. The SN1 reaction seems more probable here. (1) For the transition states (29) and (30) of the acyclic thioacetals, RO H \ / Et:---C---HOR HAOH HgC1, I

Etp---y---HOR Et\ /H 1'

&Cl*

\

HCOH

1

(30)

(29)

there is free rotation of the bond between C-1 and C-2, and the possibility of any steric interference by the C-2 hydroxyl group with the bulky mercuric chloride molecule is readily alleviated by rotation around this bond. So, the displacement of the ethylthio group, to form a carbonium ion, is readily accomplished, and steps A , B, or D should be fairly rapid. A similar possibility may be proposed for steps E and F. (2) With the cyclic 1-thioaldofuranoside, there is restricted rotation about the carbon-carbon bonds. The inclusion of the mercuric chloride molecule in the transition-state complex (31) of this cis compound would

Q \SEt

(31)

be severely hindered by tlic acljttaciit C-2 hydroxyl group. Reactions of the (68-1,2)-l-thioaldofuranoside with nucleophiles would then be relatively slow. The frequent isolation of 1-t,hioaldofuranosidesfrom reaction systcnis coiitaitiing either alcohols or watcr shows that rettctions C' and F'

120

JOHN W. QlZElN

do not occur readily. (The rapid, acid hydrolysis of l-thioddofuranosidea may be explained by the much smaller steric requirements of the proton; no such crowding will occur.) (3) The favored formation of (ch-1 ,2)-l-thioaldofuranosides and of tram-1 ,%aldofuranosides is more difficult to explain. One possibility is 811 orientation of the intermediate carbonium ions (32) and (33). There may

be more repulsion between the alkoxy group at C-1 and the adjacent hydroxyl group at C-2 than there is between the ethylthio group and the hydroxyl group. There seems at present to be no really adequate explanation here; in,each caae, the attacking nucleophile is the C-4 hydroxyl group (in order to effect ring closure). (4) There is also no adequate explanation of the fact that path A or E is observed with mme sugars (*galactose, n-mannoae, D or Irarabinoee, wxylose, and D-lyxose) and path D with others (wglucoae, n-glucuronic acid, and wribose). The C-2 hydroxyl group haa been ahown to have an effect in the c&ge of D-glucose. The l-thio-cr-D-glucofurmoside is readily formed from the dithioacetal, but 2-deoxy-wurubino-hexose dithioacetal gives only free sugar and unconverted dithioacetal under the same conditions. In methanol, the methyl 2-deoxy-D-urubino-hexofuranosidesare readily formed from the dithioaaetal, in contrast to the behavior of the D - ~ ~ U C O dithioacetal. S~ It is not yet known whether pathway D-c' or pathway A-C is being obaerved for the Zdeoxy-n-arubino-hexose system. Work by Capon and Thacker4*l on the formation of aldofuranoaides from the corresponding acyclic dialkyl metals in aqueous acid haa suggested that the C-4 hydroxyl group of the n-glucose derivative is much more nucleophilic than that of the wgalactose derivative, and that both of these groups are able to compete successfully with the water present aa a solvent; thus, ring closure to the furanoside occurs in preference to formation of a free sugar. This concept might be extended to the dialkyl dithioacetals. For wglucose dialkyl dithioacetds, the C-4 hydroxyl group successfully displaces a thioalkyl group at C-1, in competition with the solvent (either an alcohol or water), and the resulting product is a l-thio-Dglucofuranoside. With the D-galactose derivative, the C-4 hydroxyl group is, presumably, weaker niicleophile than the solvent, and cannot compete sucaendully; the

THE GLYCOFURANOSIDES

121

product is a mixed monothioacetal which is subsequently convert,ed into the D-gaJactofuranoside.

V. GENERAL PREPARATIVE METHODS 1. Preparation from Furanose Esters and Halides

The furanose acetates and bensoates may be used to advantage in the preparation both of alkyl and aryl aldofuranosides. The first synthesis of a wgalactofuranoside was achieved by Schlubach and Mei~enheimer~~; treatment of 2 ,3 ,5 ,6-tetra-O-acetyl-wgalactofurmosewith ethyl iodide and silver oxide gave ethyl 8-D-galactofuranoside. Application of this reaction may also be made to the l-thiofuranosides. Reaction of penta-0benzoyl-@-D-glucofuranosewith 2-methyl-2-propanethiol and zinc chloride gave the tertbutyl l-thio-a- and -fl-D-glucofuranosides in 48 and 25% yields, respectively.7e The aryl aldofuranosides have generally been prepared by fusing the furanose acetate with the appropriate phenol in the presence of ptoluenesulfonic acid. Tsou and Seligmanso prepared phenyl and Znaphthyl 8-D-ghcofuranosiduronolactonein this way, and Ishidate and MatsuP obtained the p-nitrophenyl P-D analog. The phenyl p-sfuranosides of wxylose, carabinose, D-glucose, and wgalactose were prepared similarly by Lindterg and coworkers.82~89 p-Chlorophenyl 8-D-ribofuranoside was the product from fusion of either the a- or 8-wribofuranose tetraacetate with p-chlorophenol and p-toluenesulfonic acid, but use of zinc chloride gave the a-wribofuranoside in~tead.~' Fletchsr and coworkers have utilized the poly-0-acylaldofuranosyl halides. Treatment of crude tri-0-bensoyl-wribofuranosyl bromide with sodium phenoxide in 1,2-dimethoxyethane, gave phenyl 0-wribofuranosidee6; use of sodium methoxide gave methyl /3-D-ribofuranoside.88 (78) H.H. Schluhach and X. Meisenheimer, Ber., 67, 429 (1934). (70) H. B. Wood, Jr., H. W. Diehl, and H. G. Fletcher, Jr., J . Org. Chem., 29, 461 (1964). (80)K . 4 . Teou and A. M. Seligman, J . Am. Chem. Soc., 74, 5606 (1952); 76, 1042 (1953). (81) M. Ishidata and M. Mateui, Yakugaku Zaeahi, 82, 662 (1962); Chem. Abstracta, 68, 4639 (1963). (82) H. Borjeeon, P. Jerkeman, and B. Lindberg, A c h Chem.Scad., I T , 1705 (1963). (83) P.Jerkemsn and B. Lindberg, Ada Chem. Scud., 17, 1709 (1963). (84) T. Shimidate, Nippon Kagaku Zaaahi, 88, 214 (1962); Chem. Abafracia, 69, 6498 ( 1963). (85) E. Vie and H. G. Fletcher, Jr., J . Am. Chem. SOC.,79, 1182 (1957). (86) C. Pederaen and H. G. Fletcher, Jr., J . Am. Chem. SOC.,84, 941 (1950).

122

JOHN W. GREEN

Methyl a-xdyxofuranoside was preparedw from tri-O-benroyl-wlyxofuranosyl bromide. PedersetP treatcd tetra-0-benzoyl-cr-mlyxopyranose with hydrofluorio wid and obtained tri-0-benroyl-cu-Plyxofuranosyl fluoride; reaction of the residual sirup, obtained from the mot,her liquor, with methanol and sodium hydrosido gave niet hyl cu-mlysofurlinc~idc. Although the neighboring-group c?!€ect8 gave the knm-1 ,2-glycwide in thc above examples, Barker and Fletcher“ found that 2,3,fi-tri-O-benzylPribofuranosyl bromide and methanol, with silver carbonate, gives mainly methyl a-mribofuranoside. Perline0 converted 5 ,&di-O-acetyl-a-wmannofuranosyl bromide, with sodium hydroxide in methanol, into methyl a-Dmannofuranoside in high yield; treatment of the bromide with methanol and silver oxide gave, instead, a sirup tentatively identified aa the 5,6diacetate 2 ,3-carbonate of methyl Pmmannofuranoside. 2. Glycofuranosylamines The subject of glycosylamineshas already been reviewed in thia Series,W and the nomenclature of these compounds, formerly called N-glycosides, amidea, anilides, etc., was discussed. The poly-0-acylglycofuraosyl halides have been valuable for use in oondensation resations with purines and pyrimidines, in order to prepare the naturally occurring nucleosides and various isomers containing sugar residues other than the Pribofuranosyl and 2-deoxy-mqthro-pentofuranosyl residues normally found. This subject, also, has been thoroughly reviewed in this Series.B1-04Such condensations are generally carried out either with the silvergsor the chloromercuri saltwof the nitrogenous base, and the furanoid structure of the products has been thoroughly established. The “trans” rule of Tipsonw has been used to explain the configuration of the products. Various epimerizations and other transformations of the sugar residues have been performed, primarily with the aid of the 2,3anhydro derivatives; the chemistry of methyl 2 ,3-anhydro-~-lyxofurano(87) A. K. Bhlrttaoharya, R, K. Ness, and H. G. Fletcher, Jr., J . Otg. Chem., 48, 428 (1983). (88) C. Pedersen, Acfa Chem. Scad., 18,BO (1904). (89)A. 8.Perlin, Can. J . Chem., 49, 1306 (1904). (90)a. P. Ellie and J. Honeynan, Advon. CarbohydrofeChem.,10,95 (1965). (91) R. 8. Tipaon, Advan. CarbohydrafeChem. 1, 193 (l&). (92) cf. R.Barker, Advan. Carbhydmb Chsm.,11,286 (1950). (98) J. J. Fox and I. Wempen, Advan. Carbohydrah Chetn., 14, 283 (1969). (94)J. A. Montgomery and H. J. Thomes, Advan. Corbohyhfe Chem.,17,301 (1962). (96) E.Fiecher and B. Helferiob, Ber., 47,210 (1914). (90)J. Davoll and B. A. Lowy, J . Am. C h . &c., 78, 1060 (1961). (97)R. 8. Tipeon, J . Ei01. C h . ,180, 66 (1939).

THE GLYCOFURANOSIDES

123

deg’ h a been developed as a preliminary to such reactions with the nucleosidcs. These transformations have been described in detail by Montgomery and The 2 ,2‘-anhydro derivatives of the nucleosides have also been used; in this type of derivative, the anhydro ring may be broken without inversion at the C-2 hydroxyl group of the sugar. The lability of the 2deoxy sugars originally necessitated use of methods involving the introduction of the methylene group at C-2 of the nucleoside, instead of starting with the difficultly available di-O-acyl-2-deoxy-Deylthro-pentofuranosyl halides. This problem was finally solved by use of the less reactive chlorides. Zorbach and Payneggaprepared a 2 ,6-dideoxyD-ribo-hexopyranosyl chloride 3,4-di-p-nitrobenzoate by treatment of the 1,3 ,Ptri-pnitrobenzoate with hydrogen chloride in dichloromethane. The first 2-deoxy-ddofuranosyl chloride was made by Fox and coworkersegbby treating the 3,B-di-ptoluoyl ester of methyl 2-deoxy-~erythro-pentofuranoside with hydrogen chloride in acetic acid : condensation with monomercuri-thymine and subsequent deacylation gave 60yo of thymidine and 4% of the a-Danomer. Ness and Fletcher’oo synthesized 2-deoxyadenosine from chloromercuri-6-benzamidopurine and 2-deoxy3,5di-O-p-nitrobenzoyl-D-erythro-pentofuranosylchloride : the latter compound was prepared by the method of Zorbach and P a ~ n e . ~No ~ ’steric control (“trans” rule) is, of course, observed with 2-deoxy-werythro-pentose or other 2-deoxy sugars. A new route, from l-thio-waldohexofuranosides, was developed by Wolfrom and coworkers; ethyl l-thio-a-D-glucofuranosidewas converted by chlorine into the chloride,lol and this wm condensed with the chloromercuri derivative of a 2 ,6-diacetamidopurine to give, on partial deacetylaD-Galactofuranosyl anation, a 2-acetamido-9-~-~-glucofuranosyladenine.~~ logs were also prepared. In 1946, Berger and LeeIo2heated &ribose with aniline in ethanol, and obtained a crystalline product to which they ascribed an a-D-furanoid structure. This N-phenyl-a-D-ribofuranosylamineshowed mutarotation in water, and was hydrolyzed by water, by aqueous acid, and by alkali. It was distinguished from the pyranoid isomer by differences in optical rotation and mutarotation. The furanoid structure was allegedly established (98) B. R. Baker, R. E. Schaub, and J. H. Williams, J . Am. Chem. Soc., 77, 7 (1955). (99) J. A. Montgomery and H. J. Thomas, Ref. 94, pp. 313-326 and 331-335. (!)Ha) W. W. Zorbach and T. A . Payne, Jr., J . A m . Chem. Soc., 80, 5564 (1958). (Wb) M. Hoffer, R. Duwchinsky, J. J. Fox, and N. Yung, J . Am. Chem. SOC.,81, 4112

(1959). (100) R. K. Neas and H. G. Fletcher, Jr., J . Am. Chem. Soc., 82, 3434 (1960). (101) M. L.Wolfrom and W. Groebke, J . Org. C h . , 28,2986 (1963); F. Weygand and H. Ziemann, Ann., 667, 179 (1962). (102) L. brger and J. Lee,J . Org. C h . ,11, 75 (1946).

1%

JOHN W. QRElN

by the preparation of a trityl derivative; periodate oxidation and methylation techaiques could not be applied,because of occurrence of extensive decomposition under the conditions used. Todd end coworkers reported108 that N-phenyl-D-ribopyranoaylamine is isomerieed by boiling ethanol to give the furanoid isomer. The rate of hydrolysia of the furanoaylmine was studied by Stacey and coworkera.m' Other N-arylglycosylamines have been prepared. Kuhn and Kirschenlohr'" isolated N-benzyl- and N-phenyl-carabinosylamine.Berezovskii and coworkerP prepared the N-(3 ,P.xylyl) derivatives of D-xylosylamine and D-arabinosylamine, and, from the reaction of 3 ,4 ,5-trimethylaniline with each of the aldopentoaes, obtained products whose respective configurations were cu-Darabinofuranosyl, cu-D-ribofuranosyl, &~-lyxofuranoayl, and @-Dxylofuranosyl.All of these products showed mutarotation in solution. Hockett and Chandler107 prepared a N-D-glucofuranosylacetamide (N-acetyl-Pglucofuranosylamine)as a product from the Wohl degradationIo8 of hexa-O-acetyl-D-gZuco-D-gubheptononitrile. This compound, having an amide grouping, is more stable t h w the arylglycosylamines discussed previously, and its rate of mutarotation is lower. A N-D-ribofuranosylurea was prepared by Benn and JoneslOg; the structure of this compound was inferred from its optical rotation and its relative mobility on a paper chromatogram. Considerable over-oxidation waa encountered in periodate oxidation of both of these compounds. Baddiley and coworkers1mprepared a D-ribofuranosylamine by condensing sodium azide with 2 ,3 ,6-tri-O-benzoyl-~-ribofuranosylchloride in acetonitrile and catalytically reducing the resulting glycosyl azide. The unstable amine waa converted, by reaction with (benzy1oxy)carbonylglycyl ethyl carbonate and subsequent debenzoylation and hydrogenolysis, into the anomers of N-glycyl-D-ribofuranosylamine.Each anomer wm oxidized (103) G. A. Howard, G. W. Kenner, B. Lythgoe, and A. R. Todd, J. Chem. Soc,, 856 ( 1946). (104) K. Butler, S. Lalsnd, W. G. Overend, and M. Stacey, J. Chem. Soc., 1433 (1960). (106) R. Kuhn and W. Kbachenlohr, Ann., 800, 116 (1956). (106) V. M. Berezovskii and V. A. Kurdyukovs, Do&. AM. Navk SSSR, 76, 839 (1961); C h m . Akhack, 46, 8464 (1961); V. M. Berezovakii, E. P. Rodionova, and L. I. Strel'chunas, Zh. Okhch. Khim., 44, 628 (1964) ; Chem, Abubacb, 48, 10696 (1964); V. M. Berezovslrii and E. P. Rodionova, Zh. Obshch. Khim, 26, 746 (1966); Chcm. Absfrack, 60, 146S7 (1966). (107) R. C.Hockett and L. B. Chandler, J. Am. Cham. SOC.,66, 067 (1944). (108) V. Deulofeu, Aduan. Carbohydrate Chcm.. 4, 110 (1949); V. Deulofeu and J. 0. Def~rrfbri,Analse Asoc. @im. Arg., 88, 241 (1960); Cham. Abelracfs, 46, 5110 (1961); J . Org. Chcm.,17, 1087 (1962). (109) M. H. Benn and A. 13. Jonep, J. Chem. Soc., 3887 (1960). (110) J. Baddiley, J. G. Buohanan, R. Hodgee, and J. F. Prenaott, J. Chem. SOC.,4769 (1967).

THE GLYCOFURAXOSIDES

129

successfully with periodate to establish its structure. The same diddeliydc was obtained from the j3-~anonier and from iV-glycyl-j3+glucopyranosylamine. The 2 ,3,5-tri-O-ben~l-D-ribosylamilie mentioned above was condensed by Shaw and Warrenerll' with a substituted acrylamide, to give uridine. Ellis and Honeyman" have raised strong doubts as to the validity of the structural determinations that have been applied to glycosylamines; these doubts are based on the fact that such compounds are readily isomerized, as evidenced by their mutarotation. The formation of a trityl ether cannot be considered valid evidence for the presence of a primary hydroxyl group in the compound tritylated, and periodate oxidation, which is often excessive, may lead to faulty conclusions. 3. Miscellaneous Preparations

A general route to alkyl D-glucofuranosides was initiated by ReeveP; this consisted in the reduction of methyl 2,5-di-O-methyl-~~-wglucofuranusidurono-6, 3-lactone with lithium aluminum hydride to give methyl 2,5-di-O-methyl-~~-~-glucofuranoside. The procedure was developed into a general method by D. D. Phillips,22with the preparation of methyl CY- and Bwglucofuranoside, and ethyl p-D-glucofuranoside; the reducing agent he employed was sodium borohydride.1128A significant method for the reduction of an acylated aldono-l,4-lactone to the acylated furanose employsll28 This procedure has been used especially bis( 1 ,Zdimethylpropyl)borane.112b in nucleoside synthesis with hexoses.1120 As a modification of the Fischer method, alcohols other than methanol have been used. Purves and Hudson*1aprepared benzyl a-wfructofuranoside by alcoholysis of methyl a-D-fructofuranoside in benzyl alcohol containing hydrogen chloride; the product, isolated aa the tetraacetate, waa unaffected by invertase. Treatment of %ribose with benzyl alcohol-hydrogen chloride until the reducing power had practically disappeared gave benzyl a similar procedure reported1148 gives 49% of product. &~-ribofuranoside~~'; An anomeric mixture of isopropyl wglucofuranosides was obtained1l6 (111)G.Shaw and R. N. Warrener, J . Chem. SOC.,2294 (1958). (112) R. E.ItBevea, J . Am. Chem. Soc., 78, 934 (1954). (1124 P. Kohn, R. H. Semaritano, and L. M. Lerner, J . Am. Chem. Soc., 88, 1457 (1964). (112b) G. Zweifel, K. Nagase, and H. C . Brown, J . A m . Chem. SOC.,84, 190 (1962). (112~) P. Kohn, R. H. Samaritano, and L. M. Lerner, J . Am. Chem. SOC.,87, 5475 (1965);J . Org. Chem., 31, 1603 (1966). (113) C.B. Purves and C. S. Hudson, J . Am. Chem. SOC.,60, 49 (1937). (114)R. K.Netu and H. G. Fletcher, Jr., J . A m Chem. SOC.,76, 3289 (1953). (114s)K. Heyns and J. h n z , C h .Ber., W, 348 (1961). (116)J. Kiss and H. Spiegelberg, Helu. Chim. Acta, 47, 398 (1964).

126

JOHN W. GREEN

from 3 ,5,6-tri-0-beneoyl-1 ,2-O-isopropylidene-a-~-glucofuranose in acidic isopropyl alcohol. The first preparation of crystalline methyl a-wmannofuranoside wt18 made by Haworth and Porter,%' by treating D-mmnofuranose 2 ,3 :5,6&carbonate with diasomethane, a neutral alkylating agent. Use of an alkalime reagent reverses the anomer distribution obtained with other reagents. Walker, Gee, and McCready116applied the Kuhnreagents (silver oxide and methyl iodide in N,Ndimethylformamide) to aeverd sugars, and analyzed the completely methylated glycosides by gas chromatography. D-Xylose, wmannose, and wglucose gave only traces of furanosides, and carabinose gave about 14% of an unresolved mixtwe. DGalactose gave Soyoof a- and 10% of Bfuranoside, and wgdacturonic acid gave 93 and 7%, respectively. Thus, a preponderance of the cis-l ,2-glycoside is'obtained in each instance. D-Fructose was converted into a mixture of 19% of a- and Slyo of 8-furanoside, a decided shift from the ratio observed in acidic reagents. The preponderance of furanoside had been noted by Haworth and coworkers'" in the methylation of wgalactose with dimethyl sulfate and alkali. A biochemical approach has been utiliaed in the preparation of &Dfructofuranosides. Invert-catalyzed transglycosylation118 of sucrose in methanol gives methyl Bwfructofuranoside; use of ethanol gives ethyl fl-D-fructofuranoside.ll@ Bacon and Edt$mqlm treated an tiqueous solution of glycerol with sucrose and invertase in order to prepare an 0-D-fructosylglycerol. Presumably, all of these compounds are fructofuranosides. Purves and HudsonMJ1*used the hydrolyzing power of invertaae to prepare methyl and benzyl a-D-fructofuranoside; wfructose and the &D anomer were fermented from the reaction mixture by the enzyme, thus allowing the ready isolation of the invertaae-resistant a-Danomer. This biochemical technique has been extended to the preparation of many di- and oligo-saccharides. The a-D-fructofuranoeyl derivatives of 2-deoxyD-@rabino-hexose(8-linked)lg1 and of wxyloseL** have been prepared. (118) H.C;. Walker, Jr., M. Gee, and R. M.McCready, J . Org. Chcm., 27,2100 (1982). (117) W. N. Haworth, D. A. Ruell, and G . C. Weatgarth, J . Chem. SOC., 126, 2488

(10%); J. Pryde, J . c h .SOC.,118, 1808 (1023). (118) T.Miwa, Koeo Kagoku Shiumpoaiumu, 8, 57 (1053);Chem.Abakaefs,47, 10676 (1983);K.Iahiaawa and T. Miwe, ibid., 9, 40 (1964);C h . Abuhrrcle,48, 7083 (1064). (119) A. I. Oparin and M. S. Bradinakaya, DoM. A M . Nauk SSSR,89, 631 (1953); Chem. Abatracb, 47,8113 (1863);M.8. Bardineksya, Tr. Komissii And. Khim., Akad. Nauk SSSR,Inel Gmkhim. iAnal. Khim., 6, 486 (lo&); Chem. Abahrrcb, 50, 13122 (1088).. (120) J. 5. D.Baaon and J. Edelman, Arch. Biochem., 18, 467 (1960). (121) G.A. Barber, J . Am. Chem. Soc.,81,3722 (1960). (122) a.Avigad, D.S. Feinpld, and 8.Herrtrin, Bdoclrim. Biophgu. Acb,PO, 129 (1068); Chom. AbaWcb, 50,11387 (1953).

THE GLYCOFURAKOSIDES

127

deveral oligosaccharides containing wfructofuranose residues have been as well as an a-D-glucopyranosyl ~-galactofuranoside.~** Special examples of stable glycofuranosides will be discussed in Section VI,4, but mention may be made here of two distinctive glycofuranosides. in Ballou and Fischera refluxed di-0-isopropylidene-D-~~~~~o-hexodialdo methanolic hydrogen chloride, and obtained a 20% yield of a compound containing two furanoside rings, namely, the dimethyl a ,d i u r a n o s i d e of =manno-hexodialdose. It waa identified by methylation, by its strong dextrorotation, and by its rate of hydrolysis by 0.01 N hydrochloric acid. Finan and Warren126 conducted a Koenigs-Knorr condensation of benzyl 3,5,6-tri-O-benzyl-a-~-glucofuranosidewith tetra-0-acetyl-a-Dglucopyranosyl bromide, and obtained a derivative in which the reducing residue of aophorose was present aa its benzyl furanoside.

VI. STRUCTURE OF GLYCOFURANOSIDES The uniqueness of the aldofuranosides, aa compared to the aldopyranosides, waa recognized initially through the difference in rates of hydrolysis by acid. This difference WBB confirmed by the use of methylation techniques; the latter have, to a great extent, been supplanted by the periodate oxidation of Jackson and HudsonlZ6and by oxidation with lead tetraacetate. Hudson's rules of isorotation were also employed to a great extent. The present discussion will deal mostly with hydrolysis and oxidation, although isorotation will receive consideration.

1. Application of Isomtation to Furanoid Struetures One of the primary functions of the rules of isorotation of Hudsonlfl was the demonstration that two glycosides can be a- and /3-anomers; this waa applied by Haworth and Portera to ethyl a- and &D-glucofuranosides; the difference in rotation of these two compounds is 184', whereaa that for the ethyl D-glucopyranosides is 183.7'. Similarly, establishment of a furanoid ring in a compound of undetermined structure waa applied by Green and PacsuS2to ethyl 1-thio-a-.p glucofuranoside; the observed specific rotation of this compound in aqueous (123)

(124) (126) (126) (127)

J. 8. D. Bacon and D. J. Bell, J . Chem. Soc., 2628 (1963); 8. A. Barker and T. R. Camngton, ibid., 2126 (1964); D.Grose, P. H. Blanohard, and D. J. Bell, ibid.,

1727 (1964); P. J. Allen and J. 8. D. Bacon, B i d e m . J., 88, 200 (1966); 8.A. Barker, E. J. Bourne, and 0. Theander, J . C h . Soc., 2064 (1967); G. Avigad, J . Bio2. Chem., 329, 121 (1967). E. J. Bourne, J. Hartiigan, and H. Weigel, J . C h .Soc., 1088 (1961). P.A. Finan and C. D. Warren, J . C h . Soc., 6229 (1963). E. L. Jackson and C. 8. Hudmn, J . Am. Chem. Soc., 69, 994 (1937). C. 8.Hudmn, J . Am. Chem. SOC.,91, 66 (1909).

128

JOHN W. GRBEN

solution was +120.7' and the theoretical value, calculated from data for Haworth's ethyl D-glucofuranosides,l and for the known ethyl 1-thi0-fl-Dglucopyranoside, prepared by Schneider and SeppJo1wm +120.7". Pacsu and coworkers60*Malso found good agreement between the calculated and oberved optical rotations for alkyl D-galactofuranosidea, barabinofuranosides, and wmannofuranosides, A final example is the work of Wolfrom and Shafizadehl*aon the structure of sucrose, for which Hudson had given an (Y-D configuration for the Dglucopyranosyl moiety, based on the results of invertasecatalyzed hydrolysis. These workers,l*ausing data from the methyl hfructofuranosides, demonstrated that this hydrolysis is not accompanied by a Walden inversion and that the original work of Hudson waa correct. 2. Acid Hydrolysis of Glycofuranosides The relatively rapid hydrolysis of glycofuranosides by acids was the first property used for differentiating these compounds from the more normally encountered, more stable glycopyranosides. In Tables VIII and IX are assembled data from the literature; the rate constants are given in set.-' and common logarithms.120The conditions of acid ooncentration and temperature varied; in the data r e p ~ r t e d , ' ~the * ~conditions ~ were the same, and so the rate constants can be compared. Heidt and P u r v e ~used ~ ~a term, k* = k/[H], to eliminate differencesin the concentration of acid used. The data in Table VIII are presented in the order of the conformational stability observed fop methyl glycosidation (see Table I on p. 99 and Table I1 on p. 106).In each of the three cmes where data for both anomers are available, the trans-1,2 anomer is the more stable, and a lower rateconstant is observed. This is the same pattern as that observed in Table 11. However the order of conformational stability given earlier is not observed here. The arabinofuranosides show the maximum stability, and the wgalactofuranosides and cfucofuranoeides, having similar conformations, show a similar stability, However, the furanosidea of ~-lyxoee,wmannose, and brhamnose show an unexpectedly high stability which is almost aa great as those of the furanosides of the first-mentioned sugars. These data lead to the conclusion that the conformational stability for tranaition complexes (128)M. L. Wolfrom and F. Sh&adeh, J . olg. C h . ,21,88 (1956). (129) The dimemions wed for rate constants in the literature vary, being given in eeconds, in minutes, and even in h o w ; normally, common (decimal) logarithms 81x9 used, although ocoaaionally natural 10a;srithms rn employed. Since common logarithma have been uaed in other articles in thin €jerk,the practice ie being

continued.

(130) L. J. Heidt and C. B. Purves, J . Am.

C h .Soc., 68, 1385 (1044).

THE GLYCOFUHASOSIDES

129

TABLE1111 Hydrolysis0 of Some Methyl A l d o f u r a n ~ s i d e a ~ ~ ~ * Methyl furanoeide of

Anomer

106 k (am.-')

a

1.5 7.7

B

B

49

B

54 92

a

-

11.3 46 0.89 7.5

B a

B

2.4 34

a

3.6

a

0

1.1 3.5

The reaction conditions were: 1.0 N hydrochloric acid at 20".

encountered in the hydrolysis mechanism is not necessarily similar to that encountered in the mechanism of methyl glycosidation.la1 In Table IX are presented data for the hydrolysis of aldofuranosides and of fructofuranosides, together with those for some pyranosides as reference compounds. The fructosides have been explored in great detail by Purves and coworkers2sJ1aJa2-4; the difference in rate of acid hydrolysis of the D-fructofuranosides and Dfructopyranosides is much less than the differences observed for the two kinds of aldosides. The activation energies foynd by Purvesl**are very similar for furanoid and pyranoid derivatives, and it WM concluded that the small difference in these energies (or in the rate constants) could not be used to distinguish between the two types of ring structure. (131) B. Capon and W. G. Overend, Aduan. Carbohydrate C b . ,16, ll(eee especially p. 34) (1960). (132) C. B. Purves, J . Am. Chem. Soc., 66, 1969 (1934).

JOHN W. GREEN

130

TAB- IX Acid Hydrolyda of S o m e Alkyl Glycofuranoridea and Glycopyranosidea Glycoride

Methyl 2-deoxy-u ,&wrabinOhexofuranomde Benzyl a-D-fructofurenoside Benzyl a-D-fruotopyranoeide Methyl a-D-fructofuranoeide Methyl a-wfructopyranoaide Methyl a-wgltwofuranoiide Ethyl crwglucofuranoside B anomer Sucrose Methyl a-D-mannofuranoside Ethyl 8-D-gelaotofuranoside u anomer Methyl a-warabinofuranoside Methyl 2 ,6di-O-methyl-w gluooaiduronolactone Benzyl 1-thio-a-D-glucofuranoeide Ethyl 1-thio-a-D-glucofuranoside Methyl a-D-arabinopyranoside Methyl 8-p.glucopyranoeide Methyl a-D-mannopynmoside Methyl a-wgalactopyranoside Benzyl a-D-fructofuranoeide Methyl a-D-fructofuranoaide Sucroae Methyl &D-fructopyranoside Ethyl pD-gshctofuranoside Ethyl 8-D-galactopyranoside Ethyl 8-wglucopyranoaide

Normality Temp., of Acid

OC.

10% (-.-I)

References

0.006

16

1700

60

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0 -01 0 -01 0.01 0.01 0.01

40

150 20

66-60 65-80 65-60 96-100 95-100 96-100 98 88 100 100

134 134 134 134 E3 23

0.01

100

0.01 0.01 0.01 0.01 0.01 0.26 0.26 0.26 0.33 0.33

100 100 96-100 95-100 95-100 20 20.2 20 20 20 19.8

2

.o

2 .o 2 .o 2 .o

40

50 50

300 100 128 380 207

890 830 260 133 133 113 100

23 137 137 137 50

m

27 138

10.6

64

9 .a

62 27 137 137 137 113 132 113 133 133 136 136 136 136

10.7 6 1.7 3-8 1%

58

80

7 21.6 16 .O 26.6 2880

60 60

12.6

m

(133) C. B. Purvea and C. 8.Hudson, J . Am. Chem.Soc., 68, 1170 (1937). (134) L. J. Heidt and C. B. Purvee, J. Am. Chsm. doc., 60, 1206 (1938). (135) W. 0.Overend, C. W. Rees, und J. S. Sequeira, J . C h . Soc., 3429 (1062). (136) F. A. Img, J. 0.Pritchard, and F. E. Stufford, J. Am. Chem. Soc., 78, 2362 ( 1867). (137) W.N. Haworth and E. L. Riret, J. Clmr. Soc., 2616 (1930). (138) R.E. Reeves, J . Am. Chem. Soc., 62,1616 (1940).

131

THE OLYCOFURANOSIDES

An entropy value for the acidic hydrolysis of ethyl BD-galactofuranoside by Overend and coworkers."6 The negative value (- 7.1 e.u.) waa interpreted aa being diagnostic of an A-2 mechanism, in contrast to the positive values (+13.7 e.u., mean value) and A-1 mechanism found for a large number of pyranosides. The proposed intermediate is shown in formula, (34). It waa considered that the planar, furanoid ring is capable of

WBR reported

accommodating a crowded transition-state. Entropy values have now been reported for several methyl aldof~ranosides*~* (see Table X); these are also negative, and thus support the A-2 mechanism. The rapid hydrolysis of ethyl BD-galactofuranoside, in comparison with the slower hydrolyses of the pyranosides, was attributedlss to the much lower activation energy (22.7 kcal.) compared with an average value of 31.6 kcal. found for the pyranosides. It is interesting that sucrose, having a fructofuranose residue, is hydrolyzed by an A-1 mechanism; a positive entropy value (+9.7 e.u.) has been reported.la6 TABLEX Entropies of Activation for Acid Hydrolysis of Methyl Aldoaides Methyl Aldoside

AS for the furanoside,a e. u.

a-D-Gluco B-D-G~uco a-D-Gslacto

&D-Galacto a-D-Xy lo P-D-XylO 4 Detern~ined'~* in 1 M HClO, at 25". ~niiied'gb in 1 M HClOd st 25".

-11.1

-9 .oE -9.4 -8.7 -8.3 -8.8

AS for the pyranoride,b e. U .

+14.8 +16.5 4-17.7 +13.3 +15.7 -1-17.5

* Determined'Jb in 2 N

HCI at 60".0 Deter-

132

JOHN

W. GREEN

3. Oxidation of Glycol Groups Two reagents have been used in determining the number and configuration of the glycol groups in glycofuranosides. The action of p e r i ~ d a t e ' ~ ~ is generally nonstereospecific, and the use of this oxidant i R confined mostly to stoichiometry. Lead tetraacetate" is more stereospecific in its nctioii on cis- and trans-glycol groups, and rate studies are generally made of its action. The consumption of one mole of oxidant per mole, with no formation of formic acid, is used to detect a furanoid structure in aldopentofuranosides. The consumption of two moles of oxidant per mole, with the formation of one mole of formaldehyde and no formic acid, is used in the detection of aldohexofuranosides. Overoxidation sometimes occurs. leaves only one Periodate oxidation of the ~-aldotetrofuranosides~~ optically active center, at the original C-1, and both &D anomers, (35) and (36),give a product (37)of [ a ] ~ -116 to -119O, whereas the CT-D

anomers give a product of [ a ]113 ~ to 120'. Similar data have been obtained for the D-aldopentofuranosidda6:here, there is also an asymmetric center left at the original C-4, as shown in (39).In both instmces, only one mole of oxidant is consumed per mole, and no formaldehyde or formic acid is formed. In the case of the D-aldopentofuranosides, for example, (38), the dialds hyde (39) can be equated with the same dialdehyde formed from an aldohexopyranoside, for example, (W),and hence the anomeric centers of

(139) J. M. Bobbitt, Advan. Carbohydrale ClLent., 11, 1 (1956). (140) A. 8. Perlin, Advan. Carbohydmk Chem., 14, 1 (1959).

THE GLYCOFURANOSIDES

133

the furanoside and the pyranodde are related. This has been demonstrated with the methyl D- and tarabinofuranosides,l% methyl a-wxylofurano~ide,"~ met hy 1 8-wrihof~ranoside~~'~ and N-glycyl-bwribofuranosylamine."o For such comparisons, it is not necessary that the products be isolated.141. Phenyl 8-D-ribofuranoside was equated with phenyl 8-wglucopyranosides6; with an excess of sodium metaperiodate, 0.313 M solutions of each gave constant, observed rotations of - 1.68" (after 3 minutes for the D-ribofuranosidr and after 6 hours for the D-ghcopyranoside). Similar results were obtained for benzyl 8-D-ribofuranoside and benzyl P-Dglucopyranoside.110 The aldotetro- and aldopento-furanosides each have a vic-diol grouping at C-2, C-3, and the rate of oxidation of these 2, 3diols by lead tetraacetate is determined by the cis or trans configuration, and, to a smaller extent, by the cis or trans relationship of the aglycon group to the adjacent C-2 hydroxyl group (that is, the amount of C-1, C-2 nonbonded interaction). The rate of oxidation of methyl CY- and 8-werythrofuranosides is very high compared with that of the D-threofuranosides, and is complete in 45 seconds at 0" (0.01 M sol~tion).'~ The rate of oxidation of very dilute solutions (0.OOOl M ) may be measuredi4zby spectral determination of Pbw at 280-300 mp; the rate for the CY-Danomer, having a cis-l,2 relationship of the aglycon and C-2 hydroxyl groups, is 3.5 times that of the @-D anomer. The slower oxidation of the methyl D-threofuranosides was determined at normal concentrations&; the rate for the 8-Danomer, having a C-1, C-2 interaction, is six times that of the a-Danomer. For the aldohexofuranosides, there are two sets of glycol groups, namely, a 2 ,3-glycol in the ring, and a 5,6-glycol outside the ring. It has been established that, with lead tetraacetate, the rate of oxidation is in the order cis8 ,3-glycol > 5 ,6-glycol > trans-2, 3-glycol. Thus, one mole of methyl a-wmannofuranoside (41) reacts very rapidly with one mole of oxidant14a;the second mole is consumed very slowly, and formaldehyde is formed only in small amount. This action is attributed to the fast oxidation of the cis-2,3-glycol; the subsequent, slow reaction of the 5,6-glycol of the dialdehyde (42) is attributed to the formation of hemicetals between these hydroxyl groups and the aldehyde groups. A similar type of reaction was observed with methyl 8-wgulof~ranoside.~~~ (141) E. Berner and 0. Kjoelberg, Ackr Chem. Scad., 14, 909 (1960). ( 1 4 1 ~ )However, we T. L. Hullar and F. Smith [J. 078. Chem., 31, 1657 (1966)] for rulwquent horohydride reduction, and cmvomion of tha rcsult,ing alcohols into the triH-p-nitrot)enzoateR. (142) A. S. Perlin and 8.Suriiki, Can. J . C h m . , 40, 1226 (1962). (143) R. C. Hookett, M. H. Nickernon, and W. H. Reeder, 111, J . Am. Che7-n. Soc., 66, 471 (1!)44). (144) H. Q. Fletcher, Jr., H. W. Diehl, and R. K. Nem, J . Am. Chem. Soc., 76, 3029

(1964).

JOHN W. QREEW

134

In contrast, ethyl 8-D-galactofuranoside (43) is oxidized at a lower but steadier rate1'*; more than two moles of oxidant are consumed per mole, and one mole of formaldehyde is formed. The 5,6-glycol is attacked first, to give (44), and then the truns-2,3-glycol is attacked, to afford the trialdehyde (45). Compound (44) cannot give a hemiacetal. The consumption

HOH& (49)

(44)

(45)

of more than two moles of oxidant per mole is attributed to the oxidation of the malonaldehyde grouping in (45) ; such compounds have been shown to be unstable to oxidants for glycols. Although periodate is less specific in its attack, use of only one mole of the oxidant per mole of an aldohexofuranoside having a 2,3-truns-glycol grouping gives one mole of formaldehyde, with cleavage of the C-6 to C-6 bond."' Subsequent reduction of the oxidation product with sodium borohydride gives an Jdopentofuranoside aa a product, and this procedure has been utilized to co-relate aldohexofuranosides with aldopentofuranosides.14* Methyl a- and &wgalactofuranoside were converted into the corresponding L-arabinofuranosides, and the methyl a-and &~-glucofuranosides into the Pxylofuranosides. Identity of products was shown by optical rotation, hydrolysis to the free pentose, or further oxidation of the pontofuranoside and isolation of glycolaldehyde as the (2,ainitrophenyl)hydraaone benzoate. Similar, controlled oxidation and subsequent reduction converted phenyl &.D-glucofuranosideinto the 8-D-xylofuranoside, and phenyl@-wgalactofuranosideinto the a-L-arabinofuranoside. ** Mitra. and Per1inlMemphasized the rapid oxidation of furanosides by lead tetraacetate. In 150 minutes, 0.88 mole of this oxidant w a consumed ~ by one mole of methyl 8-o-fructofuranoside, whereaa only 0.21 mole of (145) 0. Kjoelberg, Acfa C h .Soand., 14, 1118 (1960). (146) A. K.Mitra and A. 9. Perlin, Can. J . C h . ,87, 2047 (1959).

135

THE GLYCOFURAXOSIDES

perirnlatc was consumed. A reversal of the relative speeds for the two oxidants was uhown with methyl a-wglucopyranoside, and a corresponding selective oxidation was shown for the two sugar residues in sucrose. 4. Ring Stability

The stability of the furanoid ring may be altered in three ways. First, the polar nature of the groups at C-2 and C-3 can be altered. Secondly, another five-membered ring can be introduced into the molecule. Finally, the oxygen atom in the ring or attached to the aglycon group can be replaced by a different atom. Any or all of these transformations may raise or lower the stability of the aldofuranoside. The C-2 hydroxyl group has a very strong stabilizing effect' on the group at C-1. Replacement of this group by a more polar group increases this effect ; for example, methyl 2-O-p-tolylsulfonyl-cr-~-ribofuranoside~~~ is very stable, being unchanged by 0.33 N sulfuric acid during eight hours at 70". Conversely replacement of this hy droxyl group by a hydrogen atom results in less stable derivatives; for example, methyl 2-deoxy-~-erythropentofuranoside is completely hydrolyzedm by 0.005 N hydrochloric acid during three minutes at 100". The rate of hydrolysis of methyl 2-deoxya,/?-warabino-hexofuranosidedOin 0.005 N acid at 15" (see Table IX, p. 130) was found to be fifteen times that for methyl a+arabinofuranosiden under much more drastic conditions. Replacing the hydroxyl groups at both C-2 and C-3 by hydrogen atoms creates an even greater instability; Stacey and coworkers104were unable to prepare ethyl 2 ,3-dideoxy-werythrohexofuranoside, because of its great lability. Replacement of the C-3 hydroxyl group by a hydrogen atom, with retention of the stabilizing hydroxyl group at C-2, removes nonbonded interactions. 3-Deoxy-~-lyxo-hexose forms only furanosides with acidic methanol, at room temperature, under reflux, or1(*at 100". The removal of interactions, at C-2, C-3, and a t C-3, C-4, is far more effective than the loss of the C-3 hydroxyl group as a stabilising influence on the relatively distant group a t C-1. For the Z-deoxy derivatives, the loss of the hydroxyl group affects the stability to a greater degree than the removal of nonbonded interactions. The introduction of a second, five-membered ring haa already been discussed in reference to the ready formation of furanosides from Dgluaurono-(i,3-1a(!to1ic.~~~ The ratc (;onstant,given in Table VIII for methyl 2 , R-tli-O-iucl,liyl-cu-D-glucofuraiioxidurorio-O, 3-lactone is much smaller than thst for cthyl C3-D-Rlucofurrttiouidc. The effect is also shown in the ready (147) I). M. Hrowii, (1. I). Fenner, I). I. McCrath, and A. R. Todd, J . Chem. Soc., 1448 (1954).

(148) (149)

P. Weygand and H. Wols, Chem. Ber., 86, 466 (1952). L. N. Owen, S. Peat, and W. J. G . Jones, J . Chem. Soc.,

339 (1941).

130

JOHN W. (IItMEN

cowereion of tho mathyl pyranosidoe of 3, &anhydro-~-gluco~c~~ and of 3, B-anhydro-~-mannosc'" into tho more stable furanosides, by acidic in 0.1 methanol. Methyl 3, S-anhydro-2-deo~y-warabino-hexopyranoside~~~ N sulfuric acid givos a 70% yield of the furanoside; this is a marked incrciisc in stability over that of the 2-deoxy-warabino-hoxofuraiimide. Nicholas and coworkers16* converted methyl 3,0 :3', cj'-dianhydro-flcellobioside in acidic methanol into methyl 3, &anhydro-a-D-glucofuranoside; some fl-D anomer wa8 also obtained. This stabilizing effect of another ring has also been shown in thermal reactions. Bishop, Cooper, and Murray16*injected methyl 3,6-anhydro-a-~mannopyranoside ihto a gas chromatograph, operatigg at a column temperature of 225', and found 75% conversion into the furanoside. In some instances, another ring may introduce an * instability factor. Methyl and ethyl 2,5-anhydro-tarabinofuranosideshave been prepared,lM but are very unstable: they decompose in aqueous solution. The steric requirements of 3,Banhydro-=galactose are even more adverse : treatment166 of a"dialky1 dithiqacetal of this sugar under conditions designed to give furanosides (with methanol, mercuric chloride, and mercuric oxide) gave only the methyl pyranoside. Stabilising rings may also be created by forming 0-isopropylidene acetals. Condensation of methyl wribopyranoside with acetone wa8 shown by Levene and Stiller'w to give equal amounts of the 2,3-O-isopropylidene furanoside and the 3,4-O-isopropylidene pyranoside ; Barker and Spoors,167 investigating this reaction further, concluded that the isomerization occurs after the condensation with acetone. Several 4- and Sthioaldoses have now been prepared. Both 4-thio-~and +ribose form Cthiofuranosides in hot acidic methano1.168 In contrast, bthio-carabinose and 5thio-L-idose form 5-thiopyranoside~.~~~-~~~ In thew (150) W. N. Haworth, L. N. Owen, and F. S,mith, J . C h . doc., 88 (1941). (151) A, B. Foster, W. G. Overend, M. Stacey, and G. Vaughan, J . C h . sbc., 3367 ( 1964). (152) F. H. Newth, S. D. Nicholas, F. Smith, and L. F. Wiggins, J . Chem. Soc., 2550 (1949). (153) C. T. Bishop, F. P. Cooper, and R. K. Murray, Can. J . Chsm., 41, 2246 (1963). (154) M. Cifonelli, J. A. Cifonelli, R. Montgomery, and F. Smith, J . Am. Chem.,Soc., 77, 121 (1966). (165) H. Zinner, K.-H. Btark, E. Michalzik, and H. Kristen, Chsm. Ber., 06, 1391 (1962). (186) P. A. Ihvene and E. T. Stiller, J . Biol. Chsm., 100,421 (1934). (157) G.It. Barker and J. W. Sporrs, J . Chsm. Soc., 1192 (1956). (168) E. J. Reist, 1).E. Gueffroy, and L. Goodman,J . Am. C h .doc.,86,6658 (1964);

R. L. Whistler, W. E. Diak, T. R. Ingle, R. M. Rowell, and B. Urbas, J . Org. Chem., 20, 3723 (1964). (150) R.1,. WhiRtler, M. S.Feather, and D. L. Itigles, J . Am. Chem.Sor., 84, 122 (1962); It. L. Wliilitler and R. M. Rowell, J . Org. Chem., 20, 1250 (1964). (180) L. Goodman and J. E. Christensen, J . fhg. Chem., 20, 1787 (1964).

137

THE OLYCOFUHANOSIDES

three examples, the ring contains the hetero atom. In the formation of the ring of a glycoside, there is a nucleophilic attack by the C 1 or C-5 hydroxyl group on the protonated C-1; the nucleophilicit,y of these two groups is, apparently, very similar. The -8H group is a stronger nucleophile than the -OH group,1a1and so, nucleophilic attack by this group predominates, so that the sulfur atom becomes included in the ring. The lower rates of hydrolysis (see Table IX) found for l-thio-wglucofuranosidea are attributable to the lower basicity of the sulfur atom,161and thus, less protonation of the 1-thio-wglucoside occurs in acid solution, in comparison to the wglucofuranosides.

VII. GENERAL 1. Action of Alkali

Jansen and LindberglB2have determined the effect on various aldosides of 10% sodium hydroxide at 170'; the rate constants are given in Table XI. The aldofuranosides are much less stable in alkali than are the corresponding aldopyranosides, and the trans-1 ,Zanomers are less resistant than the cis anomers. The lower stability of the trans-glycosides has been attributed to the formation of a 1,2-anhydrideJ with elimination of the methoxyl group. TABLBI XI Alkaline Hydrolysis' of Methyl Aldonides'" lWk (sec.-I)

Methyl glycoside

Furanoside

Pyranoside

8.9

2.8 0.78 0.33

a-L-Arabino j3 anomer a-D-xylO j3 anomer u-D-GahCtO j3 anomer o-D-Gluco j3 anomer a-D-manno j3 anomer 0

-

2.2 above 28 2.2 7.8

-

above 28 8.3

-

1.60

0.28 1.6

0.28 0.70

0.78 03 0

With 10% sodium hydroxide at 170".

The high rate-constants found for the D-glucofyanosides and ~-xylofuranosides were unexpected. No correlation is observable of the relative stability of the various aldofuranosides with the order shown in acidic (161) D. Horton and D. H. Hutson, Ref. 6, see especially p. 124. (182) J. Jansen and B. Lindberg, Acfa Chem. Seam#., 14, 2061 (1960).

138

JOHN W. OHPEN

methanolysis or acidic hydrolysis. There ie, however, a correlation of the greater alkali stability of the cis arromers arid the high yields of cis anomers of methylated aldofuranmides formed in the Kuhir mcthylatioii reaction."* Also, a sorrelation can be made with the absorption on btuia, ioii-exchunge rosins; Bnddiley aiid coworkcrd4 foutrd that, ftiriwolcidcs, uxpwitilly tlw j3 anoniers, are more slowly eluted t,han tho pyrunoddee from such resins. The aryl aldofuranosides show a greater lability to alkali than the alkyl aldofuranosides. Phenyl 0-~xylofuranosideis very labile to alkali.82Treatwith dilute alkali ment of Znaphthyl ~~-glucofuranosidurono-6,3-lactone opens the lactone ring, and rapid hydrolysis occurs.BoIshidate and Matsuial is most noted that pnitrophenyl ~-~glucofuranosidurono-6,3-lactone sensitive to alkali, the amide next, and then the pyranoid derivatives; toward acids, the relative sensitivities of the lactone and the amide are reversed. 2. Formation of Furanose Polymers

Many of the methyl glycofuranosidea form polymers when distilled under reduced pressure. Bott, Hirst, and Smithleanoted such polymerizathey tion during the distillation of methyl tri-0-methyl-D-lyxofuranoside; obtained a high-boiling fraction, which changed to a solid melting at 77O, believed to be a dipentose polymethyl ether. This behavior has also been 2-deoxynoted with the fnethyl furanosides of Zdeoxy-~-arabino-hexose,~6~ D-lyxo-hexose,le6 and 2-deoxy-D-erythro-pentose.l~Il~The hard, glassy products obtained are believed to consist of 5 to 7 residues] linked through C-1 and the primary hydroxyl groups. In heating the polymer of 2-deoxy-~erythro-pentose with acidic methanol, the methyl furanoside wm re-formed. The benzyl furanosides behave similarly to the methyl furanosides. Peat and coworkerslg investigated the reversion products formed by the action of acid on &glucose and found, by periodate oxidation] that tho mixed anhydrides contain 18 to 47% of material having a furanoid structure. Some 1,6-anhydro-&~-glucofuranose was isolated. Other sugars apparently give only pyranoside reversion products. Dutton and Unrau168studied synthetic polymers obtained by the technique of heating the sugar with phosphorous acid.l" These products (163) H. G . Bott, E. L. Hirst, and J. A. B. Smith, J . Cham. Soc., 668 (1930). (164) I. W. Hughes, W. 0.Overend, and M. Stacey, J . Chem. Soc., 2846 (1949). (165) M. Stacey and W. G. Overend, British Pat. 884,689 (1953); Chem. Absfracfs, 47, 6166 (1963). (166) W.G. Overend, F. Shefiaadeh, and M. Stacey, J. Chem. Soc., 994 (1951). (167) S. Pest, W. J. Whelan, T. E. Edwards, and 0. Owen, J . Chem. Soc., 586 (1968). (168) G. G. S. Dutton and A. M. Unrau, Can. J . Chen., 40, 2105 (1962); 41, 2439 (1963); 481 924 (1966). (169) P. T. More, J. W. Wood,P. Msury, and B. G. Young, J. Am. Chsm. Soc., 80, 693 (1968).

THE OLYCOFUIlANOSIDES

139

were all highly hranc:hwl. Mothylation whowed that, in a xylan, 27% of the ititernal rc~idiiw,uric1 :$tif& of thc tn!rrninal rcsitlues, arc furtinoid. For a gluean, 201% of the tortnintti residucx arid wme of the internal re&lueu were in the furanose form. An arabinan also contained a large proportion of furanoid residues. 3. Natural Occurrence of Furanose Residues

Four sugars quite frequently occur as furanose residues in Nature. These sugars are wfructose, carabinose, D-ribose, and Zdeoxy-~-erythro-pentose D-Galactose, L-fucose, and Dpsicose have also besn found naturally in the furanose form. The /3-D-fructofuranose residue is present in sucrose17o;it also occurs in the levans and in inulin, as (2 + 1) and (2 + 6)-linked p01ymers.l~'The a-Larabinofuranose residue occurs in arabinan, generally combined with Dgalactopyranose residues17*:the backbone of such a polymer is (1 5)linked, with (1 + 3)-linked side-chains. This sugar residue also occurs as a side chain in hemicelluloses, often linked to D-arabinopyranose residues.178 The p-D-ribofuranosyl and 2-deoxy-~-~-e~i!h~o-pentofuranosy~ groups have already been discussed as the sugar residues in the n u c l e o ~ i d e s . ~Oc~-~~ casionally, an a-wribofuranosyl residue is found, as in a-riba~ole.~~' A galactan of short chain-length, containing &wgalactofuranose residues, has been r e ~ 0 r t e d . lL-Fucofuranose ~~ has been found as a residue in a polysa~charidp.'~~ An antibiotic substance, 6-amino-9-D-psicofuranosylpurine has also been in~estigated.'~~ The isolation of oligosaccharides after graded, acid hydrolysis of these polysaccharides is often difficult. Aspinall and Nicolson17B oxidized the primary hydroxyl group of the arabinofuranose residue in a European larch galactan, and obtained a (6-D-galactose L-arabinofuranosid)uronic acid. This strengthening of the furanoside linkage was also applied to an arabin~xylan.'~~ (170) I. Levi and C. B. Purves, Advan. Carbohydrak Chem., 4, 1 (1949). (171)E.J. McDonald, hoban. Carbohydrale Chem., 2, 253 (1946). (172)A. E. Goodban and H. S. Owena, J . Polymer Sci., 2S, 825 (1957). (173) G. 0.b i n a l l , Aduan. Carbohydrate Chem., 14,429 (1959). (174) N.G.Brink, F. W. Holly, C. H. Shunk, E. W. Peel,J. J. Cahill, and K. Folkere, J . Am. Chem. SOC.,72, 1866 (1960). (175)P: W. Clutterbuck, W. N. Haworth, H. Raietrick, G. Smith, and M. Stacey, Biochem. J., 28, 94 (1934);W.N. Haworth, H. Raiatrick, and M. Stacey, ibid., 31,640 (1937);P.A. J. Oorin and J. F. T. Spencer, Can. J. Chem., 31,499 (1959). (176)G. 0.Aepinall, R. S. P. Jamieson, and J. F.Wilkinson, J. Chem. SOC.,3483 (1956). (177)W. Schroeder and H. Hoeksema, J . Am. Chem. SOC.,81, 1767 (1958). (178)G. 0.Aspinall and A. Nicolson, J. Chem. ~ o c . ,2SO3 (1960). (179) G. 0.Aspinall and I. M. Cairncroaa, J . Chem.Soc., 3877, 3998 (1960).

140

JOHN W. GREEN

The above sugar residues have furanoid rings of high conformational stability, and their natural occurrence may be attributed to this factor. The various polymers also possess free primary hydroxyl groups, which may confer tz certain amount of water solubility upon them. These primary hykboxyl groups’would not be available in an aldoyentopyrauoid structure.

VIII. TABLES Tables XI1 to XV list the melting point and specific optical rotation of some methyl glycofuranosides, phenyl aldofuranosides, alkyl glycofur. anosides, and ethyl 1-thioaldofuranosidea. Tasm XI1 Melting Point and Specific Optical Rotation of Methyl Glyoofuranosides Methyl glycofuranoclide of

a-D-EqthroSe fl anomer a-D-Threw fl anomer a-tArabinose anomer a-D- Arebinom 4 anomer U-D-LYXOS~

M. p., ‘C. Simp sirup sirup Simp 520

58 66-7. 66-7 93-4 SiNP

4 anomer

79-80 84 46 91-2 69 63 sirup 100-1 118-19 46-7 80.6-1

[ a ] ~ ,degrees (water)

+133 148 +97 193 -128 +118 +123 -119 1366 +131 +146.90 +146

-

-

+

-50

-62.40

+182

-89.5 +lo4 -112 +ll8 1366 -77 108

-

+113

-112.6 +93.05 +91.64

@ anomer 0

-50.4

Hygroscopic. * Benzene aa solvent. * Methanol 88 solvent.

(180) J. 8.D. Becon and D.J. Bell, J . C h . Soc., 81181 (1957).

References

40 40 40 40 31 31 27 31 51 87 33 15 48,41 33 31 31 29 29 23 23 23 144 24 54 26 26 180

-

141

THE OLYCOFURANOSIDES

TABLE XI11 Melting h i n t and S p i f i c Optical Rutotion of Some Yhenyl Aldofuranogides Phenyl furanoside of

&L-Arabinose &n-Ribose &D-XyloSe 8-D-Galactose ~~-D-GIucoM fl-~-G~ucofuranurono-6,3lactone, diacetate 0

M. p., OC. 63-6 106-7 114-16 82-3 79-80 188-9

[ a ] ~ degreea , (water)

- 159 -99 - 128 - 148

-142 +74.50

References

82 85 82 83 83 80

In chloroform.

TABLE XIV Melting Point and Specific Optical Rotation of Some Miscellaneous Alkyl Glycofuranosides Furanoside

Ethyl a-tarabinoBenzyl BD-riboEthyl a-D-galactofl anomer Propyl fl-wgalactoBenzyl &n-gslactoEthyl CU-D-~~UCOB anomer Ethyl a-n-mannoPropyl Cr-D-rnannoIsopropyl a-n-mannoMethyl a-n-gluco~idurono-6,3lactone fl anomer Benryl WD-fructoMethyl a-L-rhamnoEthyl a-L-rhamnofl anomer Methyl a-L-fUcofl anomer Methyl bdeoxy-u-D-xyloMethyl 2,3-anhydro-a-n-lyxofl anomer

M. p.,

OC.

48-9 95-6 134-40 85-6 89-90 80-1 82-3 61-3 90 98 82-4 148-9 138-9 89 62 56.5-7.5 21-4 127-8 sirup 83-5 80-2 74-5

[ a ] ~ degrees , (water)

References

-116 -60.5 +92 - 102

50 114 53 52 50 50 2 22 54 54 54 35

-1w -96

+lo6 -76 105 +96 +96.7 149

+

+

-57 45.7 -98.6 -98 105 -108 +113 149.I4 67 - 102

+

149 113 48 56 56 47 34 181 98 98

In chloroform.

(181) K.J. Ryan, H. Araoumanian, E. M. Acton, and L. Goodman, J . Am. Chem. Soc., 88,2497(1964).

JOHN W. Q R B W

142

TABLE XV Melting Point and Specltia Optical Rotation of Some Ethyl 1-Thioaldofuranwider Ethyl 1-thiofuranoeideof

M. p., "C.

[ah,degreca

ReCeroncer

(water) ~

a-~-Ar~bin~ee

fl anomer a-D-Ribose a-D-Galactose a-~-Gluc~ fl anomer a-D-Glucofursnuronat, sodium salt ZAcetamida-Zdeoxy-&Irarabinme ZAcetamido-2deoxy-a-~-xylow ZAcetamido-Zdeoxy-cu-D-gahctow fl anomer 2.Acet.amido-2-deoxy-a-o-glucose fl anomer, triacetste

* In methanol. b In chloroform.

02-3 49-60 71-2 sirup

153 8iNP

210-2 127-9 157-8 81-3 108-10 1 19-20 174.80

+24@ -la@ +176 +124 +121 -104 +110

05 65 06 69 64 64 67

+172 +222

72 73 71 71 08 68

+1M)

- 1350 +170 -4!2b

DEOXY SUGARS BY STEPHEN HANESSIAN Research Labomtwiee, Parke, Davis & Company, Ann Arbor, Michigan I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 11. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 111. Monodeoxy Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 1. ZDeoxy Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 2. 3-Deoxy Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 3. 4DeoxySugars.. ................................................. 166 . . . . . . . . . . . . . . . . 167 4. 5-Deoxy Sugars.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Terminaldeoxy Sugars.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 IV. Dideoxy Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 1. 2,BDideoxyhexoses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 2. 3,6Dideoxyhexoses. ..................... ...................... 187 ...................... 190 3. 4,6Dideoxyhexoses. ..................... 4. 5,6Dideoxyhexoses... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 5. Other Dideoxy ....... - Sugars. V. Trideoxyhexoses.... VI. Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 1. Paper Chromatography... . . 2. Thin-layer Chro 3. Ionophoresie.. . . . . . . . . . . . . . . . . . . . . . . . . . 200 4. Gas-Liquid Chromatography... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 . . . . . . . . . . . . 201 VII. Nuclear Magnetic Resonance Spectroscopy VIII. Msss Spectrometry.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

I. INTRODUCTION The deoxy sugars, long known as components of natural products, are an important class of carbohydrates. Unlike other classes, many deoxy sugars confer unique biological properties on the natural substances of which they are a part. The commonly recognized members that have received attention from the biological standpoint have been the terminal-deoxy hexoses and the dideoxy-hexoses occurring as components of cardiac glycosides and as antigenic determinants in bacterial polysaccharides. Several unusual deoxy sugars have been isolated from other natural products in recent years. The advent of modern physical methods and the adaptation of new synthetic reactions to carbohydrates have greatly facilitated the elucidation of the structure of these uncommon deoxy sugars. Reviews on this subject have 143

144

STEPHEN HANEBBIAN

been very selective arid few in number. The deoxy sugars of the cardiac glycosides were reviewed by Elderfield’ in the first Volume of this Series and, more recently, by Reichstein and W e k a Other reviews on these compouiidtl have been published elsewhere.“’ The chemistry of the 2-dcoxy sugarv was discussed by Overend and Stticoys in Voluiiic 8 of this Scriw, and that of 2-deoxy-~-erylhro-petitotle was reviewed by the same uuthors’ in 1955. The chemistry and biology of the 3 ,O-dideoxyhexoses have been outlined in two article^.^^^ In recent years, significant advances have been made in our knowledge of the biosynthesis and metabolism of deoxy sugars.lo The objective of the present article is to discuss the natural occyrreme, synthesis, and chemical and physical properties of the deoxy sugars, and to outline chromatographic and physicochemical methods for their separation, characterization, and identification. Although the term deoxy sugar is often used loosely, the present review will be restricted to the openchain deoxyaldoses and deoxyketoses. Biochemical aspects of deoxy sugars, such as their biosynthesis, metabolism, and other biological transformations which might involve them, will not be discussed. Despite these obvious but unavoidable shortcomings, it is hoped that, with the emphasis on the chemistry of the deoxy sugars, the article will nevertheless be useful to the reader.

11. NOMENCLATURE The nomenclature adopted in this review will conform with the rules of carbohydrate nomenclature established cooperatively by the British and American committees.108The deoxy sugars have been subjected to a great deal bf misleading naming; the main difficulties have been occasioned by the unwarranted practice of assigning a name on the basis of source m a terial or preparation rather than on the structure of the sugar itself. The complexity of the nomenclature in this class of sugars has been discussed previously.@Although the present rules provide a simple, consistent system R. C. Elderfield, Advan. Carbohydrate Chem., 1, 147 (1945). T. hiohstein and E. Weiss, Advan. Carbohydrate C h . ,17,65 (1962). H. Heuaser, Forlschr. Chem. Org. Nalursbfe, 7,87 (1950) T. Reichstein, Angew. Chem., 08, 412 (1951). C. Tamm, Forbchr. Chem. Org. Naturstofle, 18, 137 (1956). (a) W. G. Overend and M. StSfiRY, Advan. Carbohydrate Cheni., 8 , 45 (1053). (7) W. G. Overend and M. Stscey, in “The Nucleic Acids,” E. Chargaff and J. N . Davidson, eds., Academic Press Ino., New York, N.Y., Vol. 1, 1955, p. 9. (8) 0. Weetphd and 0.Lfideritz, Angew. Cham.,72,881 (1960). (9) 0. Ltideritz, Bull. SOC.Chim. Biol., 41, 1355 (1960). (10) L.Claser, Phyeiol. Rev., 43,215 (1963). (108) J . Org. Cham., 28,281 (1963). (1) (2) (3) (4) (5)

PEOXY GUGAHS

145

for naming deoxy sugars, the efforts made to unify the nonienrlature systein of this class are impaired by those scientists who, presumably for reasons of supposed simplicity, continue to use unsatisfactory names. The term deoxy indicates the replacement of a hydroxyl group by a hydrogen atom. In naming a deoxy sugar, the prefix “deoxy” appears before the name of the sugar, preceded by the appropriate numeral indicating its position. The configuration of the sugar is denoted by the group prefix (such as eryulro and ribo) indicating the number of asymmetric centers, which should be consecutive but need not be contiguous. The following are some examples of correct nomenclature; incorrect names are given in parentheses: methyl 2-deoxy-a-~-arabino-htxopyranoside(methyl 2-deoxy-cu-~-glucoside); 3deoxy-~-ribo-hexose (3deoxy-~-glucose); 4deoxy-D-xyb-hexose (4deoxy-~-glucose); methyl 2,3-dideoxy-ar-~-erythrohexopyranoside (methyl 2,3-dideoxy-a-~-glucoside). The accepted trivial names rhamnose and fucose will be used in this Chapter. 111. MONODEOXY SUGARS

1. 2-Deoxy Sugars a. General Considerations.-Since this subject has been dealt with extensively in previous reviewsJ2-’ emphasis will be laid on the newer developments in the synthesis and characterization of Zdeoxy sugars. A Zdeoxy-3-O-methylpentose (corchsularose) was isolated, as the crystalline phenylhydrazone, from corchsularinJ1la bitter principle from the alcoholic extract of jute seeds. According to this investigation, the structure of the pentose derivative was adequately corroborated by the isolation of methoxysuccinic acid after permanganate oxidation. It seems remarkable, however, that a 2-deoxypentose derivative should withstand the conditions of hydrolysis (concentrated hydrochloric acid) which were employed to liberate it. Among the capbohydrate analogs which have been tested as glycolytic inhibitors, only 2-deoxy-~-ribo-hexose and 2-deoxy-~-xylo-hexose have shown significant inhibition in the glycolysis of human leucocytes, human leukemic cells, and a number of animal tumors.1aThis class of deoxy sugars and their derivatives should, therefore, be selected candidates as potential antimetabolites for cancer chemotherapy. Two new nucleosides, 9- (ZdeoxyD*rabino-hexopgranosyl) adenine and its 2,3-unsaturated analog, were found1*to be highly effective against AK leukemia 120. (11) M. A. Khalique and M. D. Ahmed, J . Org. Chem., lS, 1523 (1954). (12) J. Lmzlo, B. Landau, K. Wright, and D. Burk, J . Natt. Cancer Inst., 21,475 (1958). (13) J. J. K. Novak and F. germ, Colleclwn Czech. Chem. Commun., 27,904 (1962).

146

STEPHEN HANESSIAN

Considerable progress has been made in the past few years in obtaining crystalline esters, such aa the benzoates,+l6 acetates,17 and pnitrobenzoates,*30 of Zdeoxy sugars (and, in particular, of 2-deoxy-~-erythropentose) , The availability of these important intermediates as pure, crystalline substalices potentiates their utilization as precursors of the corresponding glycosyl halides and, thence, of various glycosides. The utility of crystalline p-chlorobenzoyl and ptoluoyl esters of Zdeoxy-D-erythro-pentofuranoeyl chloride for ready coupling with mercuripyrimidines was shown by Fox and coworkers.?1I n connection with the etudy of acid-catalyzed anomerizations, BonneP obtained pure 2-deoxy-a-~-ara~nohexopyranose tetraacetate and the corresponding B-D anomer by fractional recrystallization. Acetylation with hot acetic anhydride and sodium acetate led to a product containing 65% of the CY-Danomer. Although 2deoxy-~-erz~thropentose was recognized by Levene as being a component of one kind of nucleic acid over 35 years ago: only in the past few years has the role of this sugar and its phosphate esters been the target of many investigators. The phosphate esters, which are important intermediates in biological transformations, have been synthesized by strictly chemical means. Prepared by well established phosphorylation methods, the 5-phosphates have been reported from several laboratories.u-s Comparison of the physical properties of the momeric 2-deoxy-~-erythro-pentosylphosphates" with those of the natural ester showed that the natural anomer has the (Y-D configuration. Although the characterization of bugars and their derivatives as (substituted) hydrazones requires relatively mild conditions, an unexpected reaction was observedn when 3,5-di-O-benzoyl-2-deoxy-~-eryerythro-pentose (1) (14) H. Zinner end H. Nims, C h n . Ber., 01,1657 (1958). (16) H.Zinner, H.Nims, and E. Wittenburg, Clum. Bet-., 98,340 (1960). (16) C. Pedersen, H. W. Diehl, and H. G . Fletcher, Jr., J . Am. C h . SOC.,82, 3425 (1960). (17) H. Venner and H. Zinner, Chem. Ber., 98, 137 (1960). (18) W. W. Zorbach and T. A. Payne, J . Am. C b m . Soc., 80,5564 (1958). (19) R. K. New and H. G . Fletcher, Jr., J . Am. Clum. Sm.,82,3434 (1960). (20) W. W. Zorbach and G . Pietsch, Ann., 666,28 (1962); W. W. Zorbach and W. Birchler, Ann., 890, 118 (1963); K. V. Bhat and W. W. Zorbach, Carbohydrate Ree., 1, 93 (1986). (21) M. Hoffer, R. Dumhinsky, J. J. Fnx, and N. Yung, J . Am. C h n . Soc., 81,4112 (1959). (22) W. A. Bnnnw, J . Org. Chem., 26, (308(1961). (28) R. Allerton, W. G . Overend, and M. Stacey, Chem. I d . (London), 963 (1962). (24) T. Ukita and K. Nagasawe, Chem. Pharm. Bull. (Tokyo), 7,655 (1959). (25) D. L. MacDonald and H. G . Fletcher, Jr., J . Am. Chem. SOC.,81,3719 (1959). (26) D. L. hfacDonlrld and H. Q. Fletcher, Jr., J . Am. Chem. SOC.,84,1212 (1962). (27) M. G. Blair, D. Lipkin, J. C. Sowden, and D. R. Strobach, J . Org. Chem., 26, 1079 (1960).

DEOXY SUGARS

147

ww treated with l-benzyl-l-phenylhydrazine.An unsaturated hydrazone (2) waa formed by an a,~eliminationof the benzoyloxy group from G 3 ,CaK

OH

HC=N-N I HC ‘CH*CaH, HL! I HCOH

Bz0H9cP I

Bz

CH,OIjz

(1)

(2)

and a hydrogen atom from C-2. The product was reduced to the corresponding 2,Q-dideoxy derivative, debenzoylated, and finally degraded to succinic acid. Another example of based-catalyzed elimination in the 2-deoxy sugar series is that of 1-[2-deoxy-3-0-( methylsulfonyl)-5-O-trityl-p-~-erythropentofuranosyl]uracil and 2’ ,3-anhydro-l- (2deoxy-5-O-trityl-p-~-threopentofuranosyl) uracil, which, when treated with potassium tertbutoxide in methyl sulfoxide, give a 70% yield of a 2,3-unsaturated nucleoside.a8 A stepwise, chemical degradation reported20 for the 2-deoxy sugars permits each of the carbon atoms to be isolated, eventually, as barium carbonate, and their radioactivity, if any, to be determined. Yields as high as 90% (or even higher) of the available carbon may be obtained. In this method, the aldose is converted into the alditol, which is oxidized with periodate, to give three readily differentiatable products (namely, formaldehyde, formic acid, and 3-hydroxypropionaldehyde) which are, in turn, further degraded.

b. Syntheeis.-Several novel approaches to the synthesis of 2-deoxy sugars have been reported. However, some of the standard methods (such as the glycal method, the reductive cleavage of epoxides, and the FischerSowden method) are still in use. These general methods have been reviewed in detail.‘*sOThe glycals and their reactions have been reviewed by Helferich.81 Several of the methods included herein are described in detail and in revised form in the Methods in Carbohydrate Chemistry series.82 (28)

J. P.Horwitz, J. Chua,I. L.Klundt, M. A. DaRooge, and M. Noel, J . Am. Chem.

Soc., 88, 1896 (1964). (29)A. M.Unrau and D.T.Canvin, Can. J . Chem., 41,607 (1963). (30) J. C.Sowden, Aduun. Curbohydrute Chem., 6,291 (1951); see also, W. W. Zorbach and A. P. Ollapally, J . Org. Chem., 29, 1790 (1964). (31) €3. Helferich, Aduun. Curbohydrute Chem., 7, 209 (1952). (32) Methods Carbohydrate Chem., Vols. 1and 2 (1963).

148

STEPHEN HANEBBIAN

(i)The Glycal Method.-This method, which is considered to be the most direct route to Zdeoxy sugars, was first reported by Emil Fischer.aa It involves the conversion of an acetylated glycosyl halide into an acetylated glycal by the action of zinc dust in acetic aaid. The acctylated deoxy sugar is then obtained by mild treatment of the glycal with aqueous acid at low temperatures. This method has been the subject of extensive iuvestigation, particularly as regards improving yields and minimizing sideproducts. Iselin and Reichsteinw have obtained improved yields of the acetylated glycals by omitting the isolation of the acetylated aldosyl bromides. Although universal, the glycal method suffers from the disadvantage of affording low yields in certain cases and failing in others. An extreme caae was the failure of the glycal method in the synthesis of 2-deoxy-3-O-methyl-~-ribo-hexose.~ Prinssd attributed the variable yields to steric effects associated with the substituenta and with the configuration of the original aldose. According to the mechanism proposed" for the formation of glycals, two possible by-products could be the anhydroalditol (3) and the aldose peracetate (4), as shown in the Rcheme below:

AcOQ@

AAcoqc@ AcOQOR

0 0

AcO

R

-

(4)

OH, OAc

Ac

bAc

A detailed study of the mechanism of glycal formation and subsequent transformations in the light of modern concepts of nonclassical carbonium iona might prove uReful. Many years ago, use was made of glycals for the (33) (34) (35) (30) (37)

E. Fischer, Ber., 47, 196 (1914). B. Iselin and T. Reichstein, Helu. Chim. .Acb, 27, 1200 (1944). C. A. Grob and D. A. Prins, Helu. Chim. Acb, 28, 840 (1946). D.,A. Prins, Helv. Chim. Acla, 99, 1 (1946). D. A. Prim and R. W. Jeanlos, Ann. Rev. B i o c h . , 17,67 (1948).

DEOXY SUGARS

149

general synthesis of 2deoxy sugars.' 3,4,6-Tri-O-acetyl-~-g1ucal, on treatment with bromine or chlorine, afforded the respective 3,4,G-tri-O4cetyl2deoxy-2-halo-hexopynosyl halides, which were then converted into methyl 2-deoxy-~-arabino-hexopyranoside. An extension of this method was reported by Vargha and Kuszmann,* who treated di-0-acetyl-narabinal with chlorine in carbon tetrachloride, and obtained a mixture of 3,4-di-O-acetyl-2-chloro-2-deoxypentopyranosyl chlorides. This mixture was converted into a mixture of anomeric methyl glycosides which, on treatment with hydrogen and Raney nickel, afforded crystalline methyl 2-deoxy-a-D-erythro-pentopyranoside. Mild hydrolysis with aqueous beneoic acid the? gave a 70% yield of crystalline 2-deoxy-D-erythra-pentose (2530% from D-arabhose) . The methoxymercuration of glycals has been another potential synthesis investigated by two g r ~ u p s ~ it~ ~constitutes ~"; of 2-deoxy sugars. Thus, Schwarz and cow0rkem4~have obtained good from the 2-acetoxyyields of methyl 2deoxy-j3-~-arabino-hexopyranoside mercuri derivatives. A synthesis of 2-deoxy nucleosides involves the acidcatalyzed condensation of acetylated glycals with purines.'* This method is based OD.previous experiences with the condensation of 2,3-dihydropyran with certain purines."

(ii) The Fischer-Sowden Method.-One of the earliest synthetic applications of carbohydrate nitro-olefins was their successful conversion into 2-deoxy sugars. H.0. L. Fischer and Sowden4' showed that condensation of an aldose with nitromethane yields a mixture of epimeric C-nitro alcohols which can be acetylated and the acetates converted into an acetylated nitro-olefin by treatment with sodium bicarbonate in a nonpolar solvent such as benzene. The olefinic derivatives can be selectively hydrogenated to 1,2dideoxy-1-nitroalditolswhich, in the form of their aei salts, undergo the Nef reactions0to give the corresponding 2-deoxy sugars. Crystalline 2-deoxy-~-arabino-hexosewas obtained by this method, and purified through its benzylphenylhydrazone.44 Essentially similar results have been obtained by Stacey and coworkers,&who were able to isolate the crystalline sugar directly. The synthesis of 2-deoxy-D-erythro-pentose was also reported by this method.q Good yields are obtained at all stages (38) E. Fischer, M. Bergmann, and H. Schotte, Ber., 69,509 (1920). (39) L. Vargha and J. Kuszmann, Chem. Ber., 96,411 (1963). (40)G. R. IngliR, J . C. P. Rchwarz, and L. McLaren, J . Chem. Soc., 1014 (1962). (41).'1 Mnnolopoulos, M. Mdnick, and N. N. Lichtin, J . Am. Chem. SOC.,84, 2203 (1 9G2). (42) W. A. 13owles and R. K. Robins, J . Am. Chem. Soc., 86, 1253 (1964). (43) R. K. Rohina, E. F.Godefroi, E. C. Taylor, L. R. Lewis, and A. Jackson, J . Am. Che?tl. SOC.,88,2574 (1961).

L.Fischer and J. C. Sowdon, J . Am. Chem. Soc., 69, 1048(1947). (45) W.U. Overend, M. Stacey, and J. Stanek, J . C b m . Soc., 2841 (1949). (46) J. C: Sowden, J . Am. Chon.Soc., 71, 1897 (1949); 72,808 (1950). (44) H. 0

150

STEPHEN IIANESSIAN

in the synthesis of the 2-deoxypentose, and Sowdenaohas pointed out that,, for preparative purposes, isolation of intermediates may be omitted. Although thie practice would be satisfactory up to the acetylated nitro-olefin stage, particular care should be taken in the selective hydrogenation of the olefinic double bond, since this select ivity apparobtly 3optmdx markcdly on the solvent employed.a The Nef reaction," which gives rim to thr\ acid-labile 2deoxy sugars, requires a strongly acid medium, and should be conducted at low temperature. After considering the several methods that were available for the synthesis of 2-deoxy-~-e~ythro-pentose, Murray and Butler'? chose the FischerSowden method for the preparation of 2-deoxy-D-erythro-pentose-l-"C. The source of the D-erythrose was 4,6-0bensylidene-D-glucitol. In pilot experiments employing an excess of radioactive nitromethane without isolation of the intermediates, yields of 2-deoxy-n-erylhro-pentose-1-14C of about 30% (based on starting material) were obtained." (iii) From Anhydro Sugars.-The cleavage of 2 ,3-anhydroaldose derivatives with various reagents constitutes another method for the introduction of a deoxy function at C-2. An epoxide ring may open in two possible ways when attacked by a nucleophile. The stereoselectivity of ring opening, discussed later, depends on conformational factors. Selection of a suitable uucleophile capable of being reduced at a later stage of the synthesis would provide a convenient way of introducing a deoxy function into the molecule; this forms the basis of a method developed some twenty years ago by Reichstein and coworkers" for the preparation of 2deoxy sugars. Treatment of methyl 2 ,3-anhydro-4,6-0-benzylidenoa-D-allopyranoside in refluxing methanol with sodium thiomethoxide afforded, after chromatobenzylidene-2-S-methyl-2-thio-ar-~graphic purification, methyl 4,6-0altmside. Hydrogenation of the latter converted it into methyl 2-deoxya-D-ribo-hexopyranoside. The position of the deoxy function was proved'g by methylation of the product of ring opening, followed by reduction, hydrolysis, oxidation, and isolation of L-(-) -methoxysuccinic acid arising from C-1 to C-4 of the original hexose. The generality of the opening of the 2,&snhydro ring by thioalkoxide ions as a route to deoxy sugars is dependent on conformational factors3.*~w~61 The anhydro sugar derivatives comnionly used are fixed as fused, cis- or tranalinked, 4 ,&O-benzylidene acetals. The mode of epoxide ring opening follows the Fiirst-Plattner rule,*6 and results in the formation of the 2,3diaxial derivative. Of the four (47) (48) (49) (60) (61) (62)

D. H. Murray and G. C. Butler, Can J . Chem., 87, 1776 (1969). R. W.Jesnloz, D. A. Prins,and T. Reichstein, Ezperientia, 1, 336 (1946). R. W.Jeanloz, D. A. Prins, and T. Reichetein, Helu. Chim. Ada, 29, 371 (1946). W. G. Overend and G. Vaughan, Chent. Znd. (London), 996 (1966). F. H. Newth, Quart. Rev. (London), 18,30 (1969). A. mr8t and P.A. Plettner, Helu. Chim. Ada, 82, 276 (1949).

DEOXY SUGARS

151

methyl 2,3-auhydrd, ~ ~ ~ ~ y l i ~ e I i ~ psiblc, ~ h e ody s o ~ ~ ~ the Meoxy sugars possessing the ribo and xylo configurations*can be obtained by ring opening with a thioalkoxide. The derivatives available for synthmis are thus methyl 2 ,3-anhydro-4 ,6-O-ben~ylidene-ar-Plo~ide~~~~ and methyl 2,3-snhydro-4,6-0-benxylidene-a-~-guloside.~ The stereochemical predictions regarding the ring opening of sugar epoxides cannot be made with certainty for molecules which lack a stabilizing, fused ring. It had been maintained that the generalized rules of epoxide opening were not applicable to 2 ,3-anhydropentosides, and that attempts to convert these into 2-deoxypentosides afforded, instead, the oorregDoqding 3-deoxypentosides. However, Casini and Goodmans6 have now described the first example of a predominant attack by a nucleophile at C-2 3f an anhydropentoside. Treatment of methyl 2,3-anhydro-b-~lyxofuranoside with sodium a-toluenethioxide afforded a mixture of the correeponding 2- and 3-benxylthio derivatives, which could be separated as their p-nitrobenzoates. Reductive desulfurixation and hydrolysis afforded 2-deoxy-~-threo-pentoseas a sirup that was characterized as the crystalline benzylphenylhydrazone. Successful conversion of a 2,3-anhydropentoside into a 2-deoxypentoside by an indirect procedure has been reported by Baker and coworkers.67Ring cleavage of methyl 2,3-anhydro-j3-~-ribofuranoside with sodium ethanethioxide was found to proceed as expected. to give the 3-S-ethyl-3-thio-~-xylosederivative (5). On treatment of (5) with ptoluenesulfonyl chloride, a mixture of 2-chloro-3- S-ethyl-3-thio- and 3-chloro-2-S-ethyl-2-thio-pentoseswas formed, instead of the expected 2-p-toluenesulfonate. Acetolysis of the mixture gave methyl 3,5-di-0acetyl-2-S-ethyl-2-thio-~-~-arabinofuranoside (7) by way of an epi0 Desulfurization sulfonium intermediate (6) having the ~ 4 y i - configuration. with h n e y nickel, followed by acid hydrolysis, afforded 2-deoxy-~-eryfhropentose.

whcrc Ts is p-tolyltwlfonyl. (53) G. J. Robertson and C. F. Griffith, J . Chem. Soc., 1193 (1935). (54) N.K.Richtmyer and C. S. Hudson, J . Am. Chem. Soe., 63, 1730 (1941). (55) E. Sorkin and T. Reichstein, Helv. Chitn. A d a , 28, 1, 662, 9.20 (1045). (56)G.Cllsini and L. Goodman, J . Am. Chcm. Soc., 86, 1.127 (1964). (57) C.D.Anderson, L. Goodman, and B. R. Baker, J . Am Chem SOC.,81,898(1959).

152

STEPHEN HANBSBIAN

In 1948, Prink introduced a new niethod for the reduction of sugar epoxides to deoxy sugars by employing lithium aluminum hydride. It was subsequently found that the generalizations drawn for the ring opening of the anhydrohexosides are applicable to opening by hydride ion. This niethod is coiisidered to be nmre praaticd and low lrttmriouw tlran tlw thioxide met,hod. Schniid and I<arn?ld@have ijhown that tlic rwction of crtrbobydrate ptoluenesulfoiiates with lithium duminum hydride depends on the poeition of the ester function.o0Primary ptoluenesulfonates, except for 1-0-p-tolylsulfonylketoses, are invariably desulfonyloxylated to the deoxy compounds. Secondary ptoluenesulfonates undergo, even under prolonged treatment, simple desulfonylation to regenerate the secondary alcohol. -CHt4SO*R

-CH,

+

I -CHOH

I

-CH--OSOsR

+ RSOaH

4

+ WOsH

In a study of the cleavage of sulfonic esters of carbohydrates with lithium aluminum hydride, Allerton and Overendo' reported that treatment of with this methyl 3,4-O-isopropylidene-2-0-p-tolylsulfonyl-~-~ar~binoside hydride in dry ether afforded, after complete acid hydrolysis, only Garabinose, indicating mere ester cleavage. With methyl 2-0-ptolylsulfonyl-13-carabinoside, however, the preponderant product was methyl 2-dcoxy-8-1.-erylhro-pentoside and methyl 8-carabinoside, with a small proportion of the 3-deoxy derivative. Since, from paat experience, hydrogenolysis of epoxideswas known to give the 3-deofrypeqtosides,the formation of a ccnsiderable proportion of the 2-deoxy derivative by the ring opening of an intermediate, 2,3-anhydro derivative was ruled out; instead, a direct, reductive cleavage of the secondary p-tolylsulfonyloxy group at C-2 was assumed to have taken place. Allowing for minor steric effects, the corresponding 3,4-0-isopropylidene acetal would be expected to undergo the =me type of cleavage also, to give a 2deoxy sugar derivative. Another reductive method involves the use of Rrtney nickel; the direction of ring opening is, however, such that the products are invariably 3-deoxy sugar derivatives. Epoxide rings in sugars have been opened by the action of halogen acids. Both possible products of ring scission were observed in the reaction of methyl 2,8-anhydro-Q, fJ-O-benzylidene-a-~-alloside6~~~~ with hydrobromic (66) D. A. Prins, J . Am. Chem. Soc., 70,3965 (1948). (69) H. Bchmid and P. Karrer, Helu. Chim. Acta, ST, 1371 (1949). (80)R. 8.Tipson, Advan. Carbohydrate Chern., 8 , 107 (1963). (61) R. Ailerton and W. G. Overend, J . Chem. Soc., 3020 (1954).

DEOXY SUGARS

153

acid.“ ‘l’hc preponderant product was the 3-bromo derivative, so this method has no practical value for the synthesis of 2deoxy sugars. (iv) By Degradation of Higher Sugars.-Under this general title are included methods utilizing oxidative glycol cleavage, Ruff degradation, and elimination reactions. In preliminary experiments, Gorin and Jonese3partially oxidized 3-deoxyD-m’bo-hexose at C-1 to C-2 with periodate, and obtained a-deoxy-~erythro-pentose (as the “anilide”64) in 29% yield. Rembaraa6treated one mole of 3-deoxy-~-arabino-hexosewith one mole of lead tetraacetate in benzena, and obtained 2-deoxy-~-erythro-pentoseas the crystalline “anilide” in 60% yicld. The same selective cleavage between C-1 and C-2 of 3-deoxywxylo-hexose was effected& to give 2-deoxy-~-threo-pentose.Szab6 and coworkersB6obtained 2-deoxy-~-threo-pentose5-phosphate (as the crystalline barium salt) by the selective cleavage of 3-deoxy-~-xylo-hexose 6-phosphat’ewith sodium periodate. The first application of the Ruffs7degradation in the deoxy sugar series was repcrted by Kdiani and Naegeli,sEwho converted a 3deoxyhexonic acid into a crystalline 2-deoxypentose which had constants similar to those reported years later by Levene and Moriss for 2-deoxy-~-erythro-pentosc. Sowden70 found that a combination of Nef’s alkaline isomeri~ation~’ of aldoses (to the corresponding3-deoxyaldonolactones) with the modification of Ruff’s degradation readily afforded 2-deoxypentoses, The same sequence was reported independently by Richards.7a Further refinements of this general method have resulted in a simplified pro~edure’~ for obtaining large quantities of 2-deoxy-~-erythro-pentose.The method involves the treatment of D-glucose with calcium hydroxide, and submitting the resulting metasaccharinic acids (without isolation) to degradation in the presence of hydrogen peroxide and ferric acetate. The crude pentose is isolated as the “anilide” in 6 4 % overall yield, and is regenerated from the latter in high yield. Although the yields are low, the procedures involved are simple, (62) F. H. Newth, W. G. Overend, and L. F. Wiggins, J . Chern. Soc., 10 (1947). (63) P. A. J. Gorin and J. K. N. Jones, Nature, 172, 105 (1953). (64) R. E. Deriaz, W. G. Overend, M. Stscey, E. G. Teece, and L. F. Wiggins, J . Chem. Soc., 1’379 (1949). (65) G. Itembars, Chem. Ber., 96, 1565 (1962). (66) K. Antonakis, A. Dowgiallo, and 1,. SsaM, Bull. Soc. Chint. France, 1355 (1962). (67) 0. Ruff, Ber., 94, 1362 (1901). (68) H. Kiliani and H. Neegeli, Ber., 36,3628 (1902). (69) P. A. Levene, L. A. Mikeska, and T. Mori, J . Biol. Chem., 86, 785 (1930). (70) J. C. Sowden, J . Am. Chem. Soc., 76,3541 (1954). (71) J. U. Nef, Ann., 376, 1 (1910). (72) G. N. Richards, Chem. Znd. (London), 1035 (1953). (73) H. W. Diehl and H. G. Fletcher, Jr., Biochem. Prepn., 8,49 (1961).

154

STEPHEN HANESSIAN

axid tho starting material is cheap. V e n i ~ eobtaiiied r~~ 3deoxypeutonic acids from tarabinose and D-xylose, respectively, by reaction in aqueous alkali at 100' under nitrogen. Degradation of the resulting aldooic acid salts gave crystalline 2deoxy-Pglyccru-tetrose (10-117$ froni tho ponbosu) , 111 the sa111ereport,, Ve~iiicr~~ claimed the isolutioti of 8 (yuitrophotyL)o~rrr,u~', 1n.p. Q ~ Y - % ~ O O (50' ing.) froin the reactioii of 2-deoxy-u-erUlhro-peiito~ (1 g.) with an excess of (pnitropheny1)hydrasiiie in acetic acid. It is not unlikely that a slight contamination (less than 0.5%) by the starting 2-deoxypentose could have been responsible for the production of the osalione. The Ruff degradation has been applied76 to substituted, deoxy sugar derivatives to give correspondingly substituted 2-deoxypentoses. Thus, calcium 6-O-benzyl-~-gluconatewas subjected to degradation in the presence of barium acetate, ferric sulfate, and hydrogen peroxide to give sirupy 5-0-beneyl-2-deoxy-~-eryth~o-pentose. Similarly, 3 ,5-0-benzylidene-2-deoxy-o-ery#m-pentose was obtained from the appropriate metasrlccharinic acid. That the general conditions of degradation were suitable in the presence of a phosphate eater function was demonstrated by Szab6 and coworker^.^^ Reaction of 1,2-0-isopropylidene-3-0- (methylsulfonyl) -Dglucose with diphenylphosphorochloridate afforded the 6- (diphenylphosphate) , which apparently decomposed instantaneously in the presence of water. This phenomenon, which was attributed to transesterification with the GE,hydroxyl group to give a cyclic phosphate, was not observed when the C-5 hydroxyl group was substituted. Hydrolysis was, therefore, effected in dry benzene containing a cation-exchange resin. The resulting 3-0(methylsulfonyl)-D-glucose 6-phosphate (barium salt) was converted into metasaccharinic acid &phosphate with barium hydroxide, and this was degraded to 2-deoxy-~-erythro-pentosebphosphate.2a-'"6Crystalline 2-deoxy-D-threo-pentose has been obtained in 35-41 % yield from 3-deoxy-Dxylo-hexose by way of the oqrresponding calcium hexonate." D-Glucose substituted a t C-3 with various groups has been converted into 2-deoxy-~-erythro-pentoseby Revera1 investigators, SmithTsreported that, ( 8 ) is treated with 2 when onc mole of 3-0-(methy~su~fony~)-~-glucoee moles of sodium hydroxide a t room temperature, a rapid reaction occurs, to give the 2deoxypentose, D-arabinal, and formate (the last two being detected chromatographically). By partition chromatography on cellulose, (74) H. Venner, Nalumissenschafh, u),278 (19511); Chem. Ber., 90, 121 (1957). (75) J. Kenner and G. N. Richards, Brit, Pat. 768,250 (1057); Chum. Abstracts, 61, 16529 ;1957). (76) S. Lewak, R. Derache, und L. Szab6, Compt. Rend., 248, 1837 (1959). (77) H. Zinner, G. Wulf, and R. Heinatz, Chem. Ber., 97,3538 (1031). (78) D. C. C. Smith, Chem. Ind. (London), W (1955).

155

DEOXY SUGARS

a .5.5yGyield of the dcoq-pexitose (baed on the starting wgliicose d~rim-

tive) wm obtained. A mechanism may be formulated as follows: O Y O \ HC=CHOH

I

OH

H c=o I CH,

OH I

HCOH (!!&OH

Later, employing 1 ,4 ,6-tri-0-acetyl-3-0- (methylsulfonyl) +glucose, Smith79 reported the isolation of the enolate intermediate ( 9 ) as a sirup. A re-inveetigation of this reaction by Hardegger and coworkers,80who cited analogies in the steroid field, revealed that the alkaline degradation could be conducted in the pH range 8-9 at 45" for 2 hr., optimal yields of 4749% of the deoxypentose being produced. Furthermore, examination of the mother liquors provided no indication of the presence of D-arabinal, as originally suggested by Smith.T8 Since the yields of the required intermediates are very good, 100-200 g. of 2-deoxy-n-erythro-perltose could be prepared in one o p e r a t i ~ nAccording . ~ ~ ~ ~ ~to these authors,aothe yield of the deoxypentose was only 20-26% when 3-O-p-tolylsulfonyl-r>-glucosewas used. Kenner and Richardss1 studied the effect of the 3-substituent on the behavior toward alkali, and found, in contrast to the results of Hardegger and cowr)i'kers,80that, the 3-p-toluenesulfonate is an excellent source of 2-deoxy-~-er!ithro-pei~~,ose. Treatment of this ester with dilute lime-water (0.04N ) a t 25" resulted in a rapid reaction, with formation of the crystalline deoxypentose in 76% yield.s1 The action of limewater was founds1to be (7!)) D. C. C. Smith, J . Chem. Soc., 2690 (1957). (80)E. fisrdagger, M. Schellenbnum, R. Wyler, and A. Ziilst, Helv. Chim. Ada, 40, 1815 (1957). (81) J. Kenner and G . N. Richards, J . Chem. Soc., 3019 (1957).

156

STEPHEN HANESSIAN

more rapid than that of 2 N sodium hydroxide, showing the importance of the cation in this reaction. The preference for the ptolylsulfonyl, rather than the methylsulfonyl, derivative was based on the lower yield (65%) obtained from the latter.s1 A simplified method for the preparat,ioii of 2deory-o-erylhro-pentosefrom ( 8 ) was described by Rcroiido uitd Riirdcrknecht.*? The use of sodiuni carbonate for the dcgiadatioii wtts fouid to give particularly high yields (4740%) of the crystalline 2-deoxypentosc. A specific anion effect during the degradation was observed by these authors.s2The order of effectiveness of anions was CO, > HP04 > PO4 > SO, > Cl. Although the reactions discussed above provide an elegant synthesis for 2-deoxypentoses, several aspects are still riot settled. The variable effects of the 3-sulfonic esters and the apparent mtionic and anionic effects await explanation. Weygand and Woh88degraded 3-deoxy-~-xylo-hexoseoxime in the presence of 1-9uoro-2, .l-dinitrobemene, and obtained 2-deoxy-~-threo-pentose. The MacDonald-Fischefi degradation was used by Hough and T a y l o i for obt'aining 2-deoxy-~-erylhro-pentosefrom 3deoxy-D-ribo-hexose diethyl dithioacetal. (v) Other Methods.-F€ougha outlined a method for the synthesis of 2-deoxy-~-gylhro-pentosewhich involves the reaction of 2 ,3-0-isopropylidene-D-glyceraldehydewith allylmagnesium bromide. The resulting 5 ,6-0-isoyropylidene-l-hexene-~-erythro-4 ,5,6-triol, obtained in excellent yield, was hydroxylated to a mixture of products. Periodate oxidation of the hexitol derivatives, followed by hydrolysis, afforded the 2-deoxypentose. As essentially one deoxypentose was obtained, it appears that the Grignard synthesis is asymmetric in this case, since the product consists largely of the erylhro isomer. This attractive synthesis, which could be applied to a variety of deoxy sugars using different Grignard reagents, has not yet been exploited. The reactions of carbohydrate derivatives with certain organometallic reagents (Grignard and Friedel-Crafts processes) have been reviewed in this Series and elsewhere.syThe attempted application of a Chugaev reaction to sugar xanthates was reported by Wolfrom and Foster." It was found, (82) E. Recondo and H. Rinderknecht, Helv. Chim. Acta, 48, 1663 (1980). (83) F.Weygand and H. Wolr, Chem. Ber., 86,256 (1952). (84) D. L. MaoDQnald 8nd H. 0. L. Fischer, J . Am. Chem.Soc., 74, 2087 (1952). (&) L. Hough and T. J. Taylor, Chem. Znd. (London), 875 (1864) (86) L. HoLgh, Chem. Id.(London), 406 (1961). (87)(a) W. A. Bonner, Aduan. Carbohydrate Chem., 6, 261 (1961); (b) S. Haneseian, Ph.11. Dissertation, The Ohio Stab University (1960); Diseertdion Abslr., 31, 3268 (1961). I (88)M. L. Wolfrom and A. B. Foster. J . Am. C h .Soc.. 78. 1399 (1966).

DEOXY SUGARS

157

however, that a thermal rearraugerueut o c c d , Luted of the rypected elimination. A low yield of 2deoxy-D-erythro-piitose could be obtaiiicd from the rearranged 2-5- (methylthiocarbonate) . Another relatively unexplored reaction that might lead to a potential synthesis of 2-deoxy sugars is the selective acetylation of sugar dithioacetals and the subsequent reaction with a mercaptan. Thus, if 3,4,5,6-tetra-0-benzoyl-~-g1ucose diethyl dithioacetal is treated with ethanethiol and hydrochloric acid, a 2-S-ethyl-2-thio derivative result~P;this has been converted, in a number of steps, into 2-deoxy-~-urubino-hexosein good yield.w Similarly, 3,4,!j-tri0-bewmyl-wxylose diethyl dithioacetaP has been converted into a 2-S? ethyld-thio derivative,s2 but no attempt at subsequent conversion into a 2-deoxy-~-threo-pentosewas reported. To provide proof for the position of the cewly introduced ethylthio function, the product was reductively desulfurized to 1,2-dideoxy-u-threo-pentitol.The configuration at C-2 in these two ethylthio derivatives is still unknown. A synthesis of 2deoxym-erythro-pentose from 1-methoxy-1-buten-3-yne was described by Weygrtnd and Leube.ea 2. 3-Deoxy Sugars a. General Considerations.-To date, only one naturally occurring 3-deoxy sugar (namely, 3-deoxy-~-erythro-pentose) has been isolated. There has, however, been considerable controversy concerning the identity of this sugar, as will be outlined. The antibiotic substance cordycepin, an adenine nucleoside, was reportede4to contain a branched-chain sugar (cordycepose) having the molecular formula CsHlOO~. Cordycepose forms a crystalline (pnitrophenyl) osazone, indicating that there is no deoxy function at C-2. A crystalline phenylhydrazide from cordyceponic lactone allegedly did not conform with any of the theoretically possible, open-chain, 3-deoxypentonic phenylhydrazides. The riucleoside did not react with periodate, and theyefore a 3-deoxy structure was possible. Based on the above observations, a 3 deoxy-3-C- (hydroxymethyl)aldotetrose structure was assigned to cordy~epose.~' A supposed synthesis of Dbcordycepose was reported by Raphael and Roxburgh,gsstarting with diethyl 2-( diethoxyethyl)malonate. The (p-nitrophenyl) osasone from the synthetic productg6 seemed to be identical with an authentic sample prepared from the natural sugar.94 Following the publication of this supposedly overwhelming evidence in (89) P. Brig], H. Miihlschlegel, and R. Schinle, Ber., 64, 2921 (1931). (90)B.R. Bolligcr and M. D. Schmid, Helv. Chim. Acfa, 54, 1671 (1951). (91) M. L. Wolfrom and W. von Bebenburg, J . Am. Chem. Soc., 81, 6705 (1950). (92) M. L. Wolfrom and W. von Bebenburg, J . rlm. Chem. Soc., 82,2817 (1960). (93) F. Weygand and H. Leube, Chem. Ber., 89, 1914 (1958). (94) H. R. Bentley, K. G. Cunningham, and F. G. Spring, J . Chem. Soc., 2301 (1951). (95) R. A. Raphael and C. M. Roxburgh, J . Chem. SOC.,3405 (1955).

158

STEPHlN HANEBSIAN

favor of the branched-chain structure proposetl for aordycepose, a (%deoxypentosyl) adeninea obtaiiied from cultures of .4apergillus nidulans (Eidam) Wint. was found to be identical with cordycepiiiw and with a synthetic with respect to sample of 9-(3-depxy-~-er~lhro-peritofurariosyl)adenine~ nuclear magnetic remiance, infrared, a i d othor physioal data. Tlra rvrorukrl melting points for the (piiitrophrny1)oeasoticlJ froin 3-dwxy-I)- lttiti -L-erylhro-pentose, namely, m.p.b71m253-255.5O andm 254-250' (decomp.) , respectively are fairly close to that [2W0(decomp.)] reportedM for the osazone obtained from the natural product which partly explains the reasons for the ambiguity. The corrected structure of cordycepin has been corroborated- by mass-spectral studies on natural snd syntheticw cordycepin. Chemical evidence in favor of the 3-deoxy-wsrythro-pentose structure was obtained from degradative studies on W-labeled cordycopose.gg* Cordycepin has also been isolated from a new strain of Cordycepa militaria, and has been identified by infrared Many reactions of 3-deoxyhexoses involve their degradation by various means to Zdeoxypentoses, as discussed in the previous Section. The synthesis of 3-deoxy-~-m'bo-hexose6-phosphate for testing as a potential antimetaholite in cancer chemotherapy was reported by Dahlgard and Kaufmann.lmThe anomeric 3-deoxy-~-xylo-hexopynosylphosphates have been obtained101 as the crystalline barium salts from the corresponding acetylated glycosyl bromides; it was observedlol that the @-D anomer is hydrolyzed .more rapidly than the CY-Danomer by 0.02 N hydrochloric acid at 15'. A synthesis of 3-deoxy-~-ergthro-pentosc? 5-phosphate from Sdeoxywribo-hexose was described by the S ~ a b b sAlkaline . ~ ~ ~ treatment of pentose 3, &(hydrogen phosphates) produaes a mixture of 3-deoxy-threo- and erythro-pentonic acid

b. Syntheei8.-By far the moat commonly used method for the synthesis of 3-deoxy sugars involves the opening of anhydro rings by nucleophiles (98) E. A. Knckzka, N . R. Trenner, B.Arieon, R. W. Walker, and K. Folkers, Biochem. Biophye. Res. comm?4n.,14, 468 (1084). (97) W. W. Lee, A. Benitaa, C. D. Anderson, L. Goodman, and B. R. Baker, J . Am. Chem. Soc., 88, 1906 (1981). (98) P. W. Kent, M. Btaceg, and L. F. Wiggins, J . Chsm Soc., 1232 (1959). (99) 9. Mukherjee and A. R. Todd, J . Chem. Soc., 989 (1949). (99a) R. J. Suhadolnik and J. G. Cory, Biochim. Bwphys. Acta, 81, 861 (1964). (99b) S. Frederiksen and H.Malling, Biochim. Biophyu. Ada, 86, 189 (1965). (990) 8. Hanmdan, D. C. Ddongh, and J. A. McCloskey, Biochim. Bwphys. Acta. 117, 480 (1sSe). (100) M. Dahlgmrd and E. Kaufmann, J . Org. Chsm., 46,781 (1960). (101) K. Antonakis, Compf.find., 468,3611 (1964). (102) P. Sra'o6 and L. Saab6, J . C h . Soc., 2944 (1986). (102a) W. Jachymcayk, L. Menager, and L. Szab6, Tetrahsdrm, 41, 2049 (1985).

DEOXY SUGARS

199

aapable of being subsequently reduced, by hydride ion or by hydrogenolysis, to the deoxy function. Several other new methods are available, but have not yet been extensively tested. (i) From Anhydro Sugars.-Provided that the anhydro ring has the requisite stereochemistry (as discussed in the previous Section), such nucleophiles as methoxide or thiomethoxide ions attack a 2,3-anhydro ring predomimntly at C-3, in a stereoselective manner. Reaction of methyl with sodium thiometh2,3-anhydro-4 ,6-O-benzylidene-fl-~-talopyranoside oxide in methanol afforded the corresponding 3- S-methyl-3-thio derivative in quantitative yieldlo*; reductive desulfurization of this gave methyl 3-deoxy-4 ,6-O-benzylidene-~-lyxo-hexopyranoside.In the -me year, Mukherjee and Toddw reported the synthesis of methyl 3-deoxy-cergthropentoside by a similar procedure, A number of such applications, using different thioalkoxides, have since been reported. Of particular interest is the ring opening of 2,3-anhydropentofuranosidederivatives which leads to a predominant attack at C-3. The synthesis of 3deoxy-~-erythro-pentose from methyl 2, 3-anhydro-/3-D-ribofuranosideJby way of the 3-S-ethyl-3thio derivative, is a typical example.b7 An exception to the generalized behavior in the anhydro-ring opening of furanosides is found in the reaction of methyl 2,3-anhydro-fl-D-lyxofuranoside with sodium a-toluenethioxide.MSurprisingly, predominant attack occurs a t C-2, resulting in a 3:2 ratio of the 2-S-benzyl-2-thio- and 3-S-benzyl-3-thio-pentosides(isolated as the crystalline pnitrobenzoates) . A nucleophile which has some advantages from the practical standpoint is the thiocyanate ion. The resulting ring-cleavage products are amenable to*reduction in the presence of Raney nickel, or other suitable agents, to the corresponding deoxy sugar derivatives. An added asset associated with the use of thiocyanate ion is its good nucleophilicityand the facile characterization of the reaction products by virtue of the familiar infrared bands for SCN a t 2135-2170 cm.-'. The reductive cleavage of epoxides is one of the earliest and most universal routes to 3-deoxy sugars. All past experience has shown that the catalytic hydrogenation of substituted 2,3-anhydrohexosides affords, irrespective of the stereochemistry of the epoxide ring, a 3-deoxyhexoside. Frequently, a benzylidene protecting group is removed during this reactionsb or is hydrogenated to the 4,6-0-cyclohexylidene acetal. 3-Deoxy-~-arabinoand 3-deoxy-Plyxo-hexosela have been hexose,lo4:3-deoxy-D-x:ylo-hexose,1a syuthesizsd by this procedure. Crystalline :3-deoxy-D-ribo-hexose10'Jwas (103) M. Gut, 1).A. Prim, and T.Reichstein, Helu. Chim.Aciu, 90,743 (1947). (104) H . R. Bolliger and D. A. Prins, Helv. Chim.Ada, 29, 1061 (1946). (106) H. Kuber and T. Reichstein, Helv. Chim. Ada, 81, 1645 (1948). (106) J. W. Prntt and N. K. Richtmyer, J . Am. Chem. Soc., 79, 2597 (1957).

160

STEPHEN HANESSIAN

obtained by the catalytic hydrogenation of methyl 2,3-anhydro-4 ,GObenzylidene-a-D-alloside,followed by acetolysis and deacetylation. Induced acid treatment converted the 3-deoxyhexose into its 1 ,&anhydride to the extent of lo%,compared to 20% for the 8-deoxy-tarabitw auulog. The fuilurt) of soma of the related, iwnieric, dcwxy s u g m to ctptdliw i w y ho trttributablc to the presence of variable proportions of their 1,O-anhydridcs. Indeed, Pratt and Richtmyer'Q suggested that the C-3 substituent plays an important role in controlling the point of equilibrium. A useful, practical method for separating the resulting methyl 3-deoxyhexosides from other by-products consists in re-benzylidenation of the crude, reaction product and isolstion of the crystalline methyl 4 ,6-0-benzylidene-3-deoxyhexosides. In a re-exa~ination~~' of the hydrogenolysisabof methyl 2 ,3-anhydro-4,6-0benzylidene-u-D-allosidein the presence of h n e y nickel, it was found that the 2-deoxy- and 2,3-dideoxy-hcxosides are formed (in addition to the previously reporteda 3-deoxy derivative). Ionophoresis in borate buffer proved useful in establishing the heterogeneous nature of the reaction.lOl Since the introduction of lithium aluminum hydride for the cleavage of sugar epoxides," its use in this route to 3-deoxy sugars has become the method of choice. Thus, treatment of methyl 2,3-anhydro-4 ,6-0-benzylidene-u-D-mannopyranoside with lithium aluminum hydride affords methyl 4,6-0benzylidene-3deoxy-u-D-arabinehexopyranosideas the sole product in good yieldw; this product was also prepared by this multi-step procedure some years later.'m Hydrolysis with dilute acid, however, afforded sirupy 1,&anhydm-3-deoxy-/3-~-urubin~hexopyranose. Equilibration studieslm in dilute acid gave a mixture whose optical rotation properties indicated that it consisted of a 7:3 mixture of 3-deoxy-~-arab&no-hexose with its 1,6 anhydride; this permitted the calculation of the optical rotation of 3-deoxyD-arabino-hexose as being [&ID47 f lo.It became evident that the same deoxy sugar obtained as a sirup by Bolliger and PriqP by acid treatment contained a considerable proportion of the levorotatory 1,6anhydride. RembarzlQ was successful in obtaining crystalline 3deoxy-&~-arabinohexose in 8 total yield of 61% by acid hydrolysis of the corresponding glyooside; the 1,6-anhydride was found in the mother liquors. It is interesting to note that the initial value- of [ ~ J D 46.1' agreers remarkably well with that calculated from the equilibration &udiea.1m Two anomeric forms of 3-deoxy-D-ribo-hexose have been obtained in crystalline condition by Anetm; the high rate of mutarotation of the u-D anomer suggested that it has a furanose ring. (107) F. J. Hedgley, a. Overend, and R.A. C. Rennie, J . Chem. rsoC., 4701 (1903).

+

v.

(108) 0.Reknbers, Chem. Ber., 98,622 (1980). (108) E.F. L.J. Anat, C h . Ind. (London), 345 (1960).

DEOXY YIlGAHS

161

The preparatiouof M e o x y sugars may also bc d r i c w x i by Ity,yrtnyyc~alysis of the 3 ,hpoxides. This type of ring cleavage is not so storcosclcctivc as that of the 2,3-epoxides, and the mixture of 3- arid 4-dcoxy sugar derivatives resulting has to be separated. Methyl 3,4-anhydro-cu-~-galactopyranoside,l10on treatment with Raiiey nickel and hydrogen, affords a mixtuie of the 3- and 4-deoxy glycosides, from which methyl 4,6-0-benzylidene-3-deoxy-u-~-xylo-hexopyranoside may be obtained by rebenzylidenation of the crude mixture.111 Treatment of the @-D-glycoside1l2(10) with lithium aluminum hydride gave a 73y0yield of crystalline methyl 3-deoxy@-D-xylo-hexoside (12) and a 5y0 yield of the corresponding 4-deoxy derivative (1l).1laReduction with Rmey nickel, on the other hand, re-

sulted in the formation of only 10% of the 3-deoxy and 30% of the 4-deoxy derivative.118The ratio of products was found to depend on the age of the catalyst. These authors11sreported the isolation of 3-deoxy-~-xy20-hexose as an oil, and its conversion, in theoretical yield, into the corresponding (2 ,Pdiitrophenyl) hydralrone and hexitol derivative. It follows that this sugar, which is related to D-galactose and wgulose, apparently has litt.le tendency toward 1,6-anhydride formation during acid hydrolysis. V i and Karrer114have found that treatment of methyl 4 ,&O-benzylidene-2,3- 0-p-tolylsulfonyl-cu-Dglucoside( 13) with lithium aluminum hy.(15) in dride provides methyl 4,6-0-benzylidene-3-deoxy-a-~-ribo-hexoside good yield. Since hydrogenolysis is possible at both C-2 and C-3 of the two possible 2,3-anhydro dcrivatives, only the a110 isomer (14) could give the 3-deoxy derivative by further reaction with a reagent. Indeed, none of the alternative 2-deoxy derivative was found. This transformation is, un(110) J. C. Buchanan, J . Chem. Soc., 2511 (1968). (111) E. J. Hedgley, 0. Memz, W.G. Overend, end R. A. C. Rennie, Chem. Ind. (London), 938 (1960). (112) A. hlitller, M. Mbricz, and G . Verner, Ber., 72,745 (1939). (113) M. Dshlgard, B. H. Chastain, and RuJen Lee Han, J . 078. Chem., 27, 929 (1962). (114) E. Vis and P. Karrer, Helu. Chim. Acta, 97,378 (1964).

102

8TEPHEN HANEIIBIAN

doubtetily, an isolated example, and it must depend on the orientation of substituents. That this is 80 was shown by Overend and coworkers*" by

OTe (19)

\P

PC"

phcHQMe- I%

PhCyQOMe

OH

0

(14)

(16)

applying the eame treatment to the corresponding 8-D-glycoside; the expected product was isolated in only 38% yield. Changing the solvent from tetrahydrofuran to pdioxane gave even lower yields. The by-product in these reactions was found to be methyl 4,6-0-benzylidene-2,3-dideoxy0-D-erythro-hexoside, presumably resultingl0l from C-0 fission at C-2 and c-3. Anhydro sugars have been treated with hydrogen halides to give 2- and 3-halo derivatives which are amenable to reduction and provide 3deoxy sugars,wJu Some years ago, Newth and coworkers116 observed that methyl 2 ,3-anhyd*o-Q,6-0-benz;ylidene-a-~-alloside reacts with methylmagnesium iodide to give, as the sole product, methyl 4,6-0-beneylidene-3-deoxy-3iodo-a-D-glucoside in 80% yield. Richards11Thas found the same behavior with the u-manno isomer when methyl- or phenyl-magnesium iodides are used. Treatment of the epoxide with ethylmagnesium iodide, however, produces, instead of thc expected 3-iodo derivative, methyl 4 ,6-0-benzyliden~~-cleoxy-a-n-~~f~hcxoRide dircctly (in 53% yield) , presumably as tho result of rductioii by hyclrogcii trniiflfcr. Tho rcactioii of sugar epoxides with orgniiomstallic rcageiitH has not, yet becii followed up. (115) R. Allerton Rnd W. (1. Ovt*rend,J . Chen. Soc., 1480 (1951). (110) F. H.Newth, G.N. Richards, and L. F. WigninR, J . Chem. Soc., 2350 (1950). (117) Q. N. Ric*hnrda,J . (Ihenr. Soc., 4511 (1964).

163

DEOXY SUGARS

(ii) From Vinylic Ethers.-The basecatalyzed elinlitlation of ccrtain tosyloxy functions in carbohydrates was demonstrated as early as 1922 by Freudenberg and Brauns.l18 From the reaction of 1,2:5,6-di-O-isopropylidene-3-O-p-tolylsulfonyl-~glucofuranose ( 16) with anhydrous hydrazine, they obtained, in addition to the 3-hydrazino derivative, 3-deoxy-1 ,2 :5 ,6di-0-~propylidene-~-erythro-hex-3-enofurar10se( 17). Hydrogenation of

H

c=o I

HCOH I

yH*

HOCH I HCOH I CH,OH (19)

(18)

(17) was originally assumed to have given a 3-deoxy-~-m'bo-hexosederivative. The product has been shown by Weygand and W O ~ to Z be ~~ 3deoxy-l,2 :5,6-di-O-isopropylidene-~-xyZo-hexofuranose( 18). The hydrogenation step therefore prqceeds in a stereospecific manner. These authorssa also obtained the same intermediates in 67% yield by effecting the elimination in xylene in t8hepresence of sodium carbonate. Catalytic reduction and hydrolysis of the product gave 3-deoxy-D-xylo-hexose (19). A modification" involves heating ( 16) with sodium carbonate at 210" under vacuum, and affords a 70-75% yield of the unsaturated intermediatc.

(iii) From Sugar XanthateH.-This method is currently claimed10'to be the mont, Ratisfactory for the preparation of 3-deoxy-D-ribo-hexose. (118) I<. I"rc:ndrnl)argturd I". Bruuns, Her., 66, 3233 (1922).

164

STEPHEN HANESSIAN

Freudenberg and Wolfllg found that the 3-methylxanthate of 1,2:5,6-diO-isopropylidene-a-D-glucofuranose,readily obtained from the parent compound, rearranges, on distillation a t atmospheric pretmre, to the 3-5[(methylthio)carbonyl]-3-thiohexofuranoae derivative (this compound has been assigned the gZwo configuration in the abseuae of airy o t l w migument) .mSeveral iniprovements have been reported involving the rearraiigp ment step. Cerng and coworkers,-ln in a series of papers, reported a 60% yield of the rearranged product and Overend and coworkerslm used diphenyl ether aa the solvent, and reported an overall yield of 48% in largescale preparations. Crystalline 3deoxy-a-~4bo-hexose was obtained ih 94% yield by graded hydrolysis of the reduced rearranged product. (iv) Other Methods.-Lindberg and Theanderl" reported that catalytic hydrogenation, in the presence of Adams catalyst and dilute acid, of methyl B-n-ribo-hexosid3-ulose affords 59% of methyl 3-deoxy-@-~-ribohexopyranoside (as the triacetate) . As the introduction of carbonyl functions into cyclic and acyclic carbohydrate derivatives1*4has been greatly facilitated in recent years, this unique and relatively mild method of introducing a deoxy function, originally used in the inoaose series,l%should be examined for other glyculoses and glycosuloses. Crystalline 3-deoxy-Derylh~o-hexos-3-ulose~~ has been obtained by the deamination of l-amino1,3-dideoxy-D-erythro-hexulose. On applying the Kiliani cyanohydrin synthesis to 2-deoxy-~-erythropentose, Wood and Fletcherln obtained crystalline 3-deoxy-~-ribo-hexose and 3-deoxy-~-urabino-hexose (as the dithioacetal) . Interesting comments concerning the course of the Kiliani synthesis were madc by these authors. Condensation1*7a of 2-deoxy-~erythr-pentose with nitromethane gave epimeric 1 ,3-dideoxy-l-nitrohuxitolswhich, after hydrolysis under Nef (119) K. Freudenberg and A. Wolf, Ber., 60,232 (1927). (120) M.b r n f and J. Pacdk, Chem. Liuty, 4B, 1848 (1966); Chem. Abslracte, 60, 9298 (1966). (121) M. (4ernf and J. Pacdk, Collection Czech. Chem. Commun., 21, 1003 (1956). (122) M. brn9, J. Pa&, and V. Jma, M m f u h . , 94,632 (1963). (123) B. Lindberg and 0 . Thesnder, Acla Chem. Scud., 18, 1228 (1959). (124) 0.Theander, Aduun. Carbohdrate C h . ,17,223 (1982); K.Onodei.a, 8.Hinmo, and N. Kaahimura, J . Am. C h .Soc., 87,4661 ( 1 W ) ; V.M. Parikh and J. K. N. Jones, Can J. C h . ,48,3462 (1966); B. R. Baker and D. Buss,J , Org. Chem., 80, 2604 (1966), and references cited thorein; W. Sows and G. H. S. Thomas, Can.J. Chem., 44, &78 (1966). (126) T. Posternsk, Helu. Chim. Acfu, 88,360 (1960). (126) R.Kuhn, H. J. Ham, and A. Secliger, Cheni. Rer., 94, 2634 (1901). (127) H. B. Wood, Jr., and H. G . Fletcher, Jr., J . Org. Chem., 26, 1909 (1981). (127a) n. H. Murrsy and J. Prokop, J . Phurm. Sci., 64, 1635 (1966).

165

DEOXY SUGARS

cnnditicns, produced a nli.rture of 3dwxy-wtiho-hexow :md 3 d w x y - D arabino-hexose. A method of great potential interest in the synthesis of deoxy sugars was first reported by Lee and El Sawi.'% In this adaptation of a method introduced by Rydon and coworkersle, a hydroxyl group (primary or secondary) is converted into an iodo function by using triphenyl phosphite methiodide under rather mild conditions. Thus, 1,2 :5,6di-O-isopropylidene-a-D-giucofuranose was claimed12sto have given the corresponding 3-iodo derivative which, after reduction and acetolysis, afforded aldehydo3-deoxy-D-ribo-hexose hexaacetate. In a re-examination of this reaction, Kochetkov and U S O V 'have ~ ~ demonstrated the vewatility of the method for preparing deoxy sugars. These workers showed that the product obtained from 1,2 :5,6-di- 0-isopropylidene-a-~-glucofuranose(20) and triphenyl phosphite dibromide is not the expected 3-bromo derivative, as origimlly postulated (for the iodo analog), but the 6-bromo derivative (21), because of migration of the 5 ,B-O-isopropylidene group. Treatment HaC,

,OCHp

HaC/\h

Go> -

w-

0-c-CH,

r q CqBr

0-c-CH,

cHa

I

A% (20)

CHa (a 1)

of methyl 2,5,6-tri-O-methyl-j3-~-glucofuranoside with triphenyl phosphite methiodide gave the corresponding 3-iOdO derivative, which was converted into 3-deoxy-~-ribo-hexoseby reduction in the presence of Raney nickel, followed by hydrolysis. This reaction is applicable in the presence of many of the protecting groups commonly used in carbohydrate chemistry. It appears, however, to be susceptible to steric hindramcc, because hindered hydroxyl groups are unreactive, even under drastic conditions.Ia0The products have to be carefully identified, as experience has shown that rearrangements involving the migration of acetal functions and acyl groups frequently occur. Although the SN2 type of attack is shown in many other cases, it should not bc taken for granted from thc mcchaniatic point of view; for obtaining (128) J. €4. Lee and N. N. El Sawi,Chew. Ind. (London), 839 (1960). (129) S. R . Landauer and H. N. Rydon, J . Chent. sbc.,2224 (1953). (130) N. K. Koahetkov and A. I. Usov, TPtrahedron, 10,973 (1963).

166

STEPHEN HANESSIAN

deoxy sugars, however, the stereochemistry of the attack is irrelevant. Brown and Jones1a0.have obtained 3-deoxy-3-iodo-l , 2 :5 , Mi-O-isopropylidene-D-glucofuranose by treatment of the C-3 epimeric Shydrasino derivative with iodine. The iodo intermediate was converted into the 3-deoxywm'bo-hexose8derivative by hydrogenation. The latter was also obtained in 3040% yield when the 3-hydrazino derivative ( d o ) was oxidized with ferricyanide or periodate in an alkaline medium. 3. 4-Deoxy Sugars a. General Considerations.-This class of deoxy sugars has not yet been found among naturally occurring substances. Their interest lies mainly in their general novelty as a class of deoxy sugars and in their physical and chemical properties. The presence of the 4-deoxy function eliminates the possibility of formation of a furanose ring; this feature could be very informative in the study of optical rotation behavior during equilibration and in the kinetics of hydrolysis of some glycosides.

b. Synthesis.-The opening of epoxides Beem once again to have attracted the most attention. Ward and Kentlalobtained a methyl 4-bromo4deoxy-c-~-lyxopyranoside by treating methyl 3,4-anhydro-&triboside with hydrobromic acid; reduction and hydrolysis then afforded 4-deoxy-cerythro-pentose as a sirup. Overend and coworkerslWJI1 prepared crystalline 4deoxy-~-zy20-hexose(26) by catalytic hydrogenation of methyl 3,4-anhydro-cu-~-galactoside~~~ and hydrolysis of the product. The same product was obtained by other investigators,ll* who found that, with the fl-D-glycoside (10) , Raney nickel affords higher proportions of the 4-deoxy derivative (11) than does lithium aluminum hydride. A synthesis of an interesting ketose derivative was reported by Jones and coworkers,132 who treated 3,4-anhydro-l, 2- 0-isopropylidene-cu-D-ribhexulose with lithium aluminum hydride, and obtaixied (almost exclusively) 4-deoxy1,2-O-isopropylidenec~-~-erythro-hexulose which they converted into 4deoxy-D-erbthro-hexdose. Another approach involves the acid-catalyzed ring(25) ,' which is cleavage of 1,6-anhydro-4-deoxy-fl-D-xglo-hexopyranose1aa obtained from the readily available (22) through the intermediates (23) and (24). Sirupy Pdeoxy-D-arabino-hexose,obtained similarly, was shown to be a mixture of the 1,6anhydro compound with about 25% of the 4-deoxy sugar, undoubtedly as a result of equilibration. (13Oa) D. M. Brown and G. H. Jon-, Chem. Commun., 561 (1965). (131) P. F. V. Ward and P. W. Kent, Nature, 170,936 (19521; J . Chem. Soc., 416 (1953). (132) P. A. J. Gorin,L. Hough, and J. K. N.Jones, J . Chem. Soc., 4700 (1954). (138) M. eernf, J. ParPk, and J. Stanik, Chmi. Ind. (London), 946 (1961); Collertion Czech. Chenb. Coiimun., 17, 04 (luoa).

167

DEOXY SUGARS

(25)

(26)

The method employing triphenyl phosphite methiodide, as adapted to carbohydrates,'ao has been used for the synthesis of protected 4-deoxy and sugars. From methyl 2 ,3-di-0-methyl-6-O-p-tolylsulfonyl-a-~-glucoside the corresponding D-galactoside, epimeric 4-iodo derivatives were obtained; these were both reduced to methyl 4-deoxy-2 ,3-di-0-methyl-b-0-p-tolylsulfonyl-a-D-xylo-hexoside. Investigations by Hough and coworkers134 have shown that 4-sulfonyloxy groups of aldopyranose derivatives can be displaced rather readily with thiocyanate and iodide ions; this constitutes a potential route to Cdeoxy sugars. Catalytic hydrogenation of methyl (methyl 4deoxy-/3-~threo-hex-4-enopyranosid) uroriate afforded methyl (methyl 4-deoxy-/3-~-urubino-hexopyranosid) uronate as the main product.]& Reduction of this compound gave a mixture of 4-deoxy-~-urubino-hexoseand its 1,8anhydride. Thus, the tendency of deoxy-urubino-hexopyranoses to give 1 ,6-anhydrideslo6 extends to the 4-deoxy sugars as well. A minor product of the hydrogenation186was assumed to be the epimeric (at C-5) methyl (methyl 4-deoxya-D-xylo-hexopyranosid) uronate. 4. 5-Deoxy Sugars a. General Considerations.-This class of carbohydratcs, like the 4-cleoxy wialogs, has not yet been found among natural products. The (134)J. Hill, L. Hough, and A . C. Richardson, Proc. Chem. Soc., 314 (1963). H.W. H. Schmidt and €1. Neukom, Tetrahedron I,etf.em, 2063 (1964).

(135)

168

STEPHEN HANESSIAN

5-deoxy sugars impose the restriction of a furanose ring structure on the molecule and increase the chances, should favorable conditions exist, for septanose-ring formation; however, it may be expected that the acyclic form would be favored. The synthetic methods, developed independently in several laboratories, involve novel approaches.

b. Synthesis.-The first 5-deoxy sugar wa8 f o b d in the mother liquors from a large-scale fermentation of L-sorbose by Acelobacter suboxydans. The crystalline j-deoxy-D-lhreo-hexulose'" thus produced has served as a valuable reference material (in the form of its phenylosazone)la6 for synthetic samples prepared some 15 years later. The first synthetic 5-deoxyhexose, namely 5-deoxy-D-xylo-hexose (29) , was described by Wolfrom and coworkers,lq who extended the hydrqboration reaction of Brown and coworkers18 to the carbohydrate series. Treatment of 5,6dideoxy-1 ,2-O-isopropylidene-a-~-xylo-hex-5-enofuranose (27) lag with sodium borohydride and aluminium chloride in bis (Zmethoxyethyl) etherlSs afforded crystalline 5-deoxy-l , 2-O-isopropylidene-~-xylo-hexofuranose (28). The structure of this product was unambiguously determined by nuclear magnetic resonance studies; it was shown, from the spectrum of the produat, that a terminal deoxy function is absent. Mild hydrolysis with acid of (28) gave sirupy (29), the phenylosazone of which was identical

?:I

P Q c

-

- G> -

I;cG

(27)

- -CH,

&

(28)

C&OH &-Ia 0

QOH OH

(2Q)

with a known aample.laeAfter Wolfrom and Whiteley's preliminary communication,laa Overend and coworkers"' disclosed another synthesis for (28) ; this irivolved the catalytic hydrogeiiolysis of 5,6-anhydro-l,2-0(186) P. Itegna, J . Am. Chem. Soc., 68, 246 (1947). (187)(a) M. L. Wolfrom and T. E. Whitely, Abstracts Papers Am. Chem. Soc. Meeting, 187, 20 (1960). (b) M. L. Wolfrom, K. Mstsuda, F. Komitsky, Jr., and T. E. Whikly, J . Org. Chem., 28, 3661 (1903). (138) H.C. Brown and B. C. Subba Rao, J . Am. Chem. Soc., 78, 2682 (1966);H.C. Brown, J. K.Murray, L.J. Murray, J. A. hover, and G. Zweifel, ibid., 82, 4233

OW).

(139)(a) H. Ohle and E. Dickhtiuser, Ber., 68, 2693 (1925);(b) J. K. N. Jones and J. L. Thompnon, can. J . Chem., 86, 955 (1967);(c) L. D. Hall, T,. Hough, nntl li, A. Pritchard, J . C h .Soc., 1637 (1961).

DEOXY SUGARS

169

isopropylidene-a-D-glucof uranose in the presence of Rantly niclir.1 in nwt 11anol a t 100' and 110 atmospheres of hydrogen. The liydrogetiolysis of thc terminal anhydro ring apparently depends on the nature of the catalyst and on the medium.'" In the presence of alkali, a terminal deoxy sugar is produced exclusively, but, in acidic media, the product also contains 1 ,2-O-isopropylidene-6-O-methyl-a-~-glucofumnose. The product of reduction of 6-O-benzoyl-1,2- O-isopropylid~ne-5-~-p-to~y~s~fony~-a-~-g~ucofuranose with a hydride, previously assumed,141without proof, to be a 6-deoxy derivative, has been shown"* to be (28). The hydroboration reactionl37J" has been extended in the carbohydrate series by Goodman and coworker~.~4~ Hydroboration of the 3-methanesulfonate of (27) affords 5-deoxy-1 ,2-O-isopropylidene-3-O-(methylsulfonyl)-a-D-zylo-hexose; this is de-estersed by methanolic potassium hydroxide to give (28), and the latter may be hydrolyzed to the free sugar (29) . Methyl 2 6-di-O-benzoyl-,5-deoxy-~-ribo-hexopyranoside was obtained'd2 (by an inversion) by the action of sodium benzoate in N , N dimethylformamide on methyl 2 6-di-O-benzoyl-5-deoxy-3-O-(methylsulfonyl)-a-u-zylo-hexopyranoside,and this was converted into U~OXY-Dribo-hexose (33).The above sequence of reactions illustrates the use of a neighboring benzoate group in aiding the solvolytic displacement reactions of secondary sulfonates in cyclic carbohydrate derivatives. Compound (28) was formed in low yield (9%) by the Raney nickel desulfurization of 3 ,6-di-O-acetyl-5- S-acetyl-1 ,2-0-isopropylidene-5-thio-~-idofuranose;an accompanying product, namely, 5 ,6-dideoxy-1 ,2-O-isopropylidene-~-q&ohexose, was formed in 57% yield. A Chugaev elimination reaction was successfully performed on the 5-S-methylxanthate (30)of methyl 2 ,3-0-isopropylidene-6-deoxy-~-~-allofuranoside.~~~ The product was shown, by nuclear magnetic resonance studies, to be the allylic olefin (31), not the vinylic olefin (34).It should be noted that the related elimination reaction in cyclic derivatives gives rearranged products." The rather unexpected selectivity obtained during the pyrolysis was explained by these authors1* on the basis of steric hindrance in the transition state required for the formation of (34).Hydroboration of (31), and acid hydrolysis of the product after fractionation, afforded (33).The presence of u-deoxyhexose side-products (40%) in the hydroboration reaction mixture prompted these workers to use a bulkier borane, namely, cis- (3-methyl-2-butyl) borane, in an effort to increase the stereoselectivity of the reaction; the yield of (32) was unaffected. )

)

(140) E. J. Hedgley, Private communication. (141) E. J. Reisq R. R. Spencer, and B. R. Baker, J . 0t.g. Cham., 23, 1757 (1958). (142) K. J. Ryan, H. Arzoumanian, E. M. Acton, and L. Goodman, J . Am. C h m . SOC., 86,2503 (1964).

170

STIPHEN HANESSIAN

uocH'-

ROCH

o

CqOH

&Q HO

OH

This reaction should be extended to other types of olefinic carbohydrates containing different substituents and ring sizes, in order to ascertain whether coordination of the reagent with one or more of the several oxygen functions in the molecule could possibly account for the variable selectivity. Another synthesis of (28), and thence of (29) in 25% overall yield from n-glucose, utilizes a @-eliminationreaction of 3-O-acetyl-l , 2-0-isopropylidene-BO-ptoly~s~fony~-6-~-trity~-a-~-g~ucofuranose by the action of sodium metk.oxide.1" An interesting variation in the processing of the interuranose mediate A-deoxy-1 ,2-0- isopropylidene-&O-trityl-~-x~b-hex-5-enof (35) m-as independently reported by Bucharian and Oakes.1"" Acid-catalyzed detritylation of (35) afforded a dialdose derivative (36)as the difuranose, with concomitant production of a deoxy function at Cb, as in the glycal series. Borohydride reduction then produced the knownlp] (28). Montgomery and Hewson'* have described yet another synthesis for this class of deoxy sugars. Treatment of methyl 2,3-0-isopropylidene-50-(p-nitrophenylsulfanyl)-8-D-ribofuranoside with sodium cyanide in N ,Ndimethylforniamide afforded the corresponding 5-cyano-5deoxy derivative (143) R,E. Gramera, T. R. Ingle, and R. L. Whistler, J . Ore. Chem., 29, 2074 (1984). (144) J. G.Buchanan and E. M. Oakes, Tetrubdron Letters, 2013 (1964); Carbohydrate Ree., 1, 242 (1966). (146)J. A. Montgomery and K. Hewson, J . 0t.o.Chem., 29,3436 (1964); J . Med. C h m . , 9, 234 (1966)

-

DEOXY BUGARS

171

in modemtc yield. Rduction and dizotization then gave methyl hleoxy2,3-O-isopropylidone~-~~~~hexofurtLnoside as the sole product which, on acid hydrolysis, afforded (33) as an oil.

‘ko>

S ci

CHOTr II

0-c-CH, I CHa (55)

Xi@

- ‘“QY

-

08)

O-C-CH, I CH,

(36)

5. Terminal-deoxy Sugars a. General Considerations.-The w-deoxyhexoses are the most widespread class of deoxy sugar derivatives in the plant and microbial world. The large volume of historical background and literature concerning their occurrence has been adequately documented in a textbook by S t a ~ S k l ~ ~ and, in part, in several reviews.1-6 There are many reports in the literature in which 6-deoxyhexoses isolated from natural substances have been “characterized” by chromatographic techniques only. In many other instances, however, they have been isolated in crystalline condition. A selection of the more recent reports of their occurrence will be stressed in this brief outline. To date, no derivative of 6deoxyidose has been found in natural products. A reasonable explanation for this, on the basis of the conformational instability147inherent in the idopyranoss structure, has been proposed.* One of the 6deoxy sugars earliest isolated, 6-deoxy-~-mannose(crhamnose) , was obtained by Rigaudla from the plant glycoside quercetin. Although sugars are seldom found in the free form in Nature, a paper14g report+$ the occurrence of free brhamnose in the roots of Datisca cannabinae. LRhamnose is an abundant member of bacterial polysaccharides obtained from Gram-negative bacteria.I6O It has also been isolatedl5l from mycoside (146) J. Stanlik, M. cernf, J. Kocourek, and J. Pa&, “The Monosaccharides,” Academic Prws Inc., New York, N. Y., 1963, p. 403. (147) R. I!: Reeves, Advan. Carbohydrate Chem., 6, 108 (1951). (148) L. Rigaud, Ann., 90,283 (1854). (149) J. A. Mikhailova, L. N . Efremova, and A. A. Pryanishnikov, Tr. Vses. Nuuchn.Issled. Inst. Khim. Reaktivov, as, 65 (1959); Chem. Absiracts, 06,2493 (1961). (150) M. R. J. Salton, Biochim. Biophys. Acta, 46, 364 (1960). (151) A. P. MacLennan, Bwchern. J., 82, 394 (1962).

172

STEPHEN HANESSIAN

C, a mixture of glycolipides produced by strains of Mycobaclerium avium. D-Rhamnose, rarely found in Nature, has been isolated from a capsular polysaccharide of Gram-negative bacteria.ua McKinnell and Percivall63 have isolated crystalline Irrhamnose from the hydrolyzate of a watersoluble polysaccharide from the green seaweed E’nleromorpha cmnpressa. The authors speculated that the majority of sulfate ester groups in algal polysaccharides are probably carried by the brhamnose residues. Another report described the isolation and identification of L-rhamnose from rabbit skin.u4 Although neutral sugars frequently occur in glycoproteins and blood-group substances, Lrhamnose had not been previously reported as a constituent of mammalian tissue. Another common 6-deoxyhexose1 6-deoxy-Lgalactose (cfucoue) was isolated from an bfucose-containing polysaccharidelfi in 1912. D-Fucose, on the other hand, was obtained by the hydrolysis of convolvulin,’66and characterized as the crystalline 2-benzyl-2-phenylhydrazorle; this sugar is a constituent of numerous polysaccharides and plant glycoside~.~J46 The immunological significance of D- and bfucose and their methyl ethers in polysaccharides of the blood-group substances has been discussed in detai1.u’ Several antibiotic substancesu8 have been shown to have a fucose or a fucose methyl ether as a constituent. Galmarini and Deulofeulb~ isolated crystalline 4-0-methyl-~-fucose (D-curacose) from the antibiotic curamycin produced in the cultures of Streptmnycee mra-coi; this constitutes the first example of the isolation of such 8 D-fucose derivative from natural sources. Another antibiotic substance, chartreusin,lWwas also shown to contain wfucose. Finally, crystalline 2,4di-0-methyl-~-fucose (labilose) has been isolated from labilomycin,161 an antimycobacterial antibiotic substance. Methanolysis of the antibiotic material afforded a mixture of glycosides which, on hydrolysis by aqueous acid, gave crystalline labilose. Demethylation with hydrobromic acid gave D-fucose, thus proving the (182) A. Markovitz, J . Biol. Chem., 287, 1707 (1902). (163) J. P.Mal
DEOXY SUGARS

173

presence of the D-galactose stemhemistry, an assignnient that was cow firmed by nuclear magnetic resonance studies on the glycosides. The alkaloid chinovinIa provided the first source of G-deoxy-n-glucose, which wm obtained as the crystalline phenylosazone from a hydrolyzate of ethyl q u ii io ~ o si d e Considerable .~~~ confusion existed at t,hat time concerning the identity of 6deoxy-~-glucose,since several names (isorhamnose, isorhodeose, epirhamnose, and quinovose, depending on the investigator and the source) were in use. The discrepancy in melting points of the phenylosazones from synthetic and natural samples of the sugar was noticed by Freudenberg and R a ~ c h i gwho , ~ ~prepared ~ several 6-deoxyhexose phenylosazones. 6-Deoxy-~-glucosehas now been isolated1&from Holoruthin A, a toxic principle of Actinopyga agassizi, a sea cucumber found in the Bahama Islands. I n addition to their occurrence in the plant glycosidesJ2crystalline 6-deoxy-D-u2 and - ~ - t a l 0 ~ e ~ have ~ J ~been 7 isolated from bacterial polysaccharides and from glycolipides.I616-Deoxy-~-gdose,6-deoxy-~-altrose, and 6deoxy-~-alloseand some of their methyl ethers have so far been encountered in the cardiac glycosides only.* The following crystalline compounds have been isolated from cardenolide and pregnan glycosides, and subsequently synthesized in the past two years : 6-deoxy-2-O-methyl-~6-deoxy-3-0-methyl-~-altrose (vallarose),1670 6-deoxyallose (ja~ose),~'37av~~7b 2 ,3-di-O-methyl-~-glucose~~~ and 6-deoxy-3-0-methyl-~-allose.~~~ Dion and coworkers1BB have isolated crystalline 6-deoxy-2,3di-O-methylD-allose (mycinose) from the hydrolyzate of the macrolide antibiotic chalcomycin. This unique sugar is related to 6-deoxy-~-allose,as shown by demethylation in the presence of boron trichloride and by nuclear magnetic resonance studies.la Mycinose has also been isolated from hydrolyzates of the antibiotic substances t y l o ~ i n and '~~ neutramy~in.'~~~ The 6-deoxyhexoses are conveniently separated from their aglycons after (162) C. Liebermann and F. Geisel, Ber., 16, 926 (1883). (163) E. Fischer and C. Liebermann, Ber., 26,2415 (1893). (164) K. Freudenberg and K. Raschig, Ber., 62,373 (1923). (165) J. 13. Chanley, R. Ledeen, J. Wax, R. F. Nigrelli, and H. Sobotka, J . Am. Chem. Soc., 81,5180 (1959). (166) A. P. MacLennan, Biochim. Biophys. Ada, 48,800 (1961). (107) C. 8.Cummins &lidH. Harris, J . Uen. Microbiol., 18, 173 (1958). (I67rt) P. Mtihlradt, E:. We&, and T. Reiahstein, Ann., 086, 253 (1965). (187b) T. Reichstein, Private comrnunicrttion. (167~)H. Kaufmann, W. Wehrli, and T.Reichstein, Helu. Chim. Ada, 48, 65 (1985). 1167d) H. Kaufmann, Helu. Chim. Ada, 48, 789 (1986). (187e) J. 5. Brimacornbe and D. Portsmouth, J . Chem. Soc., 499 (1966). (168) H. W. Dion, P. W. K. Woo, and Q. R. Bartr, J . Am. Chem. Soc., 84,880 (1962). (169) R. B. Morin and M. Gorxnan, Tetrahedron Lettera, 2339 (1964). (189s) M. P. Kunstmann and L. A. Mitschev, Expezieniia, 21, 372 (1965).

174

STEPHEN HANESSIAN

mild hydrolysis by acid, although acetolysis of polysaccharides containing Amylose 0-deoxyhexoses has been the preferred procedure in some has been converted into 6-deoxyarnylo~e~~~ by applying a series of sequential reactions that were developed for monosaccharides (namely, tritylation of the C-6 hydroxyl group, acetylation, detritylation, ptoluenesulfonation at the C-U hydroxyl group, desulfonyloxylation with sodium iodide, and reduction). The succw of these reactions, ingeniously carried out with this polysaccharide, should cast LL new outlook on the possibility of other synthetic applications thereof. Deoxyketoses are of rare natural occurrence. The antibiotic hygromycin'" has been shown to contain 6-deoxy-~-arabino-hexoseresidues, since reduction of the free sugar gave Bdeoxy-~-altroseand cfucose. Yungsten (anand coworkers178 reported the isolation of a 6deoxy-~-x~/Zo-hexulose gustose) from the antibiotic Angustmycin A, for which structure (37) was proposed.

isolated, from psicofuranine fermentations Hoeksema and a substance having properties identical with those of (37), and gave it the name decoynine. The absence of a C-methyl doublet in its nuclear magnetic resonance spectrum was in disagreement with the proposed structure (37). This led the latter workers to re-interpret the chemical evidence provided in support of the original structure,17*and a new formula for Angustmycin (decoynine) or 9- (6-deoxy-/3-n-erythro-hex-5-enulofuranosyl)adenine (38), waa put forward. (170) G. 0. Aspinall, A. J. Charlmn, E.L. Hiret, and R. J. Young, J . C b m . Soc., 1696 (1983). (171) B. J. Bines and W. J. Whelm, C h m . Ind. (London), 997 (1960). (172) A. D. Elbein, H Koffer, and H.R. Garner, Biochim. Biophyu. Acla, 66, 166 (1082).

H.Yungsfen, J . Antihbtice (Tokyo), I%. A , 8,244

(1868); and preceding papers. (174) K. Hoekeema, G. Slomp, and E. van Tamelen, TetruMrcm Leucte, 1787 (1984).

(173)

DEOXY SUGARS

175

b. Yynthesis.--Jlost of the possible w-deoxy-pentoses and -hexhave beer1 kiiowu for marly yeaan arid have been synthesized. Although mme wrly synthetic methods which, by present standards, are grossly outmoded will be outlined, emphasis will be laid on the newer, more practical methods elaborated during the past two decades. (i) Epimerization of Lactones.-Lacking ways of introducing deoxy functions into sugar molecules, chemists first attempted the epimerization of lactones obtained from known and naturally occurring analogs. However, a limitation set by the nonavailability of many 6-deoxyhexoses restricted the number of epimeric sugars that could be obtained by this method. In a typical example, Fischer and P i l ~ t y 'prepared ~~ D-arabinono-1 ,4lactone from D-ribono-1,4-lacteoneby heating the latter in pyridine. Utilizing this procedure, 6-deoxyhexono-l,4-lactones having the L - ~ ~ U C O , ~ ~ ~ D-ta20,1n ~ - t a l o ,D-manno,18n ~ ~ ~ J ~ ~ and D-a1tro1*l configurations were synthesized; these were then converted into the corresponding deoxyhexoses with sodium amalgam. The method of epimerization with pyridine was also applied to Dcglyceraldehyde, from which 1 ,%-dihydroxy-2-propanonewas isolated.1s2The general applicability of this process was demonstrated by extenditig it to the common a1do~es.l~~ Syntheses of crystalline 6-deoxy-L were based on this method. lyxo-hexuloselu and 5-deoxy-~-threo-pentulose~ (ii) Reductive Methods.-One of the earliest syntheses of crystalline 6-deoxy-~-glucoseinvolved the reduction of methyl tri-O-acetyl-6-bromo-Gdeoxy-a-D-glucoside with zinc dust and acetic acid.'* The availability of wdeoxy-wiodo sugar derivatives, through the reactioii of the corresponding w-ptoluenesulfonates with sodium iodide, permitted th/e synthesis of many o-deoxy sugars. The iodo function (and such groups as mercapto, thiocyanate, arid t,hiuronium) may be smoothly reduced with hydrogen and Raney nickel, or other catalysts, to a deoxy function. The method usually affords good yields, and is particularly useful for compounds that possess groups which are affected by other reducing agents. This widely used (175) (176) (177) (178) (179) (180)

E. Fiacher and 0. Piloty, Ber., 24,4214 (1891). E. Fischer and H. Herborn, Ber., 29, 1961 (1896). E. VotoEek and C. Kraur, Ber., 44,362 (1911). E. VotoEek and J. Cerverng, Ber., 48,658 (1915). J. Schmuta, Helu. Chim. Acta, 31, 1719 (1948). E. VotoEek and F. Valentin, Chem. Listu., 21, 7 (1927); Chem. Abstracts, 21,

(181) (182) (183) (184) (185) (186)

H. Iwadare, Bull. Chem. SOC.Japan, 17, 296 (1912). H. 0.1,. Fischer, C. Taube, and E. Bmr, Ber., 60, 479 (J9'27). S. Danilov, E. Venus-Danilova, and P. Shantarovitch, Bar., 63, 2269 (1930). J. Barnett and T. Reichstein, Helv. Chim. Acta, 20, 1629 (1937); 21, 913 (1938). P. A. J. Gorin, L. Hough, and J. K. N. Jones, J . Chem. Soc., 2140 (1953). E. Filccher and K.Zach, Ber., 46, 3761 (1912).

1969 (1927).

176

BTBPHEN HANE8IIAN

procedure has been employed in the syiithesis of w-deoxy derivatives of ~-galactose,:87 D-xylose,188 ~-arabino,se,~~~ l~-Zpw-hexulosr,l~bxylo-hexulose,lW D-urabirbo-hexUlose,~Q~ ~-glucosc,19~--1Qb u-nianiiose,L96~ - a l t r o s e , ~ ~ ~ J ~ ~ ~-ribose,1*f'-'UL-lyxose,m and niaiiy of their derivatives. McMiyl 2,:b anhyd~deo~y-8-D-ribofurarlouide~~1 aid the corrwpondiiy cY-u-lysoftu.uiioeideg02 have been obtaiiied by t.he reduct,ion of the corroaponding 5-iodo deriva t ives. Catalytic hydrogenation of acetals of certain unsaturated compoundsM3 also gives wdeoxy sugars. Freudenberg and RaschiglB4obtained a mixture of Dfucose and 6-deoxy-caltrose when 5,6-dideoxy-l, 2 :3,5-di-O-isopropyldene-~-x&-hex-5-enofuranose was reduced with hydrogen in the presence of a platinum catalyst and the product was hydrolyzed. The related 3 ,SObenzylidene a ~ e t a l ? on ~ ' treatment with hydrogen at 130 atmospheres in the presence of Raney nickel, afforded a mixture of 6-deoxy-l , 2-0-isopropylidene-Iridofuranose arid its 3,A-0-cyclohexylidene acetal, both of which gave 6-deoxy-~-idoseon acid hydrolysis.2m The reductive cleavage of wterminal anhydro sugar derivatives also gives wdeo;
(187) (188) (189) (190) (191) (192) (193) (194)

2201 (1948). (199) K. Folkem and C. H. Shunk, U. 8.Pat. 2,847,413 (1958); C. H. Shunk, J. B. Lavigne, and K. Folkers, J . Am. Chem.Soc., 27,2210 (1955). (200) H. M. Kissman and B. R. Baker, J. Am. Chem. Soc., 79,5534 (1957). (2004 R.K. Hulyalkar and M. B. Perry, Can. J . Chem.,4S,3241 (1965). (201) P.Chang and Y. T.Liu, H u Haueh ~ Haueh Pao, 28, 169 (1957); Chem. Abstracts, 62,6220 (1968). (202) P. Chnng and Y. T.Liu, Hwr Hewh Hewh Pao, 28, 68 (1967); Chem.Abstracts, 62, 12875 (1968). P. Chang and Y. Fan, ibid., 2S, 175 (1958); Chem. Abatracte, 69, 16220 (l9aS). (203) B. Helferioh and E. Himmen, Ber., 61, 1825 (1928). (204) B. Helferich and R. Mittag, Ber., 71, 1586 (1938). (205) A. 8. Meyer and T.Reiohstein, Helv. Chim. Acto, 29,139 (1946). (208)K.Freudenberg, H.Eich, C. Knoevenagel, and W. Westphal, Ber., 71,441 (1940). (207) A. f?. Meyer and T.Reichatein, Helv. Chim. Acto, 29,162 (1946).

DEOXY SUOAHS

177

With ths introduction of lithium aluminuni hydride for the reductioii of primary sulfonyloxy groups,6e**use of the iodo compounds as intermediates has been largely avoided, and o-sulfonyloxy groups have been converted directly into deoxy functions. This method cannot be used with derivatives containing groups susceptible to the reductant without effecting changes in these. Furthermore, if the reaction is sluggish, this method affords sulfur-containing by-products which have to be removed, for example, by chromatography. Reduction with this hydride has been used in the synthesis of D-fucose,6B 6 d e o x y - ~ - g l u c o s e , ~5-deoxy-~-xylose,~~~ ~~~~ 5deoxy-~arabinose,all and D-rhamnose,alzand certain derivatives, from the corresponding w-p-toluenesulfonates. The reductive cleavage of w-terminal anhydrides to a deoxy function by lithium alclminum hydride has been reported.21s (iii) From Higher and Lower Sugars.-The application of degradation reactions permitted early investigators to synthesize 5deoxypentoses from the 6deoxyhexoses available, The Wohl degradationz1' of suitably acetylated aldononitriles was used for the synthesis of 5-deoxy-~arabinose,~~~ 5-deoxy-~-lyxose,~~~ and 5deoxy~-riboae.~l~ The Ruff degradationjs7 involving interaction of a salt of an aldonic acid, ferric acetate, and hydrogen peroxide, was employed in the synthesis of 5-deoxy-~arabinose~l~ and 5-deoxy-clyxo~e.~~~ Micheelm obtained Sdeoxy-L-arabinose and +-ribose derivatives by the oxidation of L-rhamnal and 6-deoxy-~-allalderivatives, respectively, with ozone. In the methods outlined above, none of the deoxypentoses were isolated; they were characterized as crystalline 2-benzyl-2-phenylhydrazonesor as phenylosazones. Except for the synthesis of the ~ I y x analog,21e o which may conveniently be prepared from tfucose, all of the other methods have been replaced by more practical approaches. By appropriate use of protecting groups, the synthesis of 6-deoxy-a-~ (208) P. Kwrer and A. Boettcher, Helv. Chim. Acta, 36,570 (1953). (209) M. L. Wolfrom and S. Hanessian, J . Org. Chem., 27, 2107 (1962). (210) P. Karrer and A. Boettcher, Helv. Chim.Actu, 36, 837 (1953). (211) H. Zinner, K. Wessley, and H. Kristen, Chem. Ber., 92, 1618 (1959). (212) W.W. Zorbach and C. 0. Tio, J . Org. Chen., 26,3643 (1961). (213) E. J. Reiet, R. R. Spencer, and B. R. Baker, J . Org. Chem., 23, 1753 (1958). (214) A. Wohl, Ber., 32,3666 (1899); and previous papers. (215) E. Fiacher, Ber., 29, 1377 (1896). (216) E. VotoEek, Bet., 60,35 (1917). (217) K. Iwadare, Bull. Chem. Soc. Japan, 17,90 (1942). (218) 0. Ruff, Ber., 31, 2360 (1902). (219) Y. Wang, W. Lin, W. Yi, and T. Ku, Hua Hsueh Hsueh Pao, 26, 265 (1959); Ckm.Abetracls, 64, 18370 (1960). (220) I?. Micheel, Ber., 63,347 (1930).

178

STEPHEN HANESSIAN

glucose was achieved.221 A dithioacetal prepared from D-glyMTO-D-gUbhep tose was reductively desulfurized, and the product was converted into 3 ,5-O-benzylidene-l-deoxy-Dglycero-Dgulo-.Cleavage at C-B-C-7 with lead tetraacetate afforded a bepzylidene wetal which was hydrolyzed to &leoxy-L-glucose (in two crystdlinc inodifirtitLiune). The MacDoimld-Fisuher degradatioiiu has bccn uacd in the syiitlicsis of 5-deoxy-~arabinose~ from L-rhamnose, and it could be adapted to the preparation of other deoxy sugars. The cyanohydrin synthesis has, in several instances, been used to obtain higher sugars from wdeoxyaldose precursors. The initial product of the reaction is an o-deoxyaldono-1,4-lactoneJ which may be reduced to the next higher o-deoxyaldoses with sodium amalgam or sodium borohydride.aza The readily available Grhamnose was converted into a mixture of two 7-deoxyheptoses (one obtained crystalline) by Fisoher and Piloty.% This product was later shown to be 7-deoxy-~glycero-~-galacto-heptose.~~ It is of interest that the 7deoxyheptose gives a methyl 7-deoxyheptofuranosidc in addition to the anomeric methyl pyranosides. By utilizing the cyanohydrin reaction , Krauam prepared two 7-deolryheptoses from D-fucose. The syiithesis of crystalline 6-deoxy-~-gulosefrom 5-deoxy-~-xylose~ by the cyanohydrin reaction was reported by Levene and Compton*; however, the L enantiomorph, preparedm by the same sequence, was obtained 8s a sirup. Ae is often the case in the cyanohydrin synthesis, one of the two lactones formed may be preferentially separated by recrystallization. (iv) Epimerization and Inversion Reactions.-An elegant preparation of 6-deoxy-~-allose wm described by Levene and Compton,22awho found that treatment of 2 ,3-O-isopropylidene-5-O-ptolylsulfonyl-Lrhamnofuranose (39)with sodium methoxide gives methyl 6-deoxy-2 ,3-0isopropylidene-D-allofuranoside (41) in reasonable yield. The structure of this product was proved unequivocally by these Baker and coworkers*" have reexamined this reaction, and have suggested a possible (221)E. ZisdR, N. K. Richtmyer, and C, 8. Hudson, J . Am. Chem. Soc., 73, 4714 (1R51). (222) L. Hough and T. J. Taylor, J . Chem., SOL, 3544 (1955). (223) M. L.Wolfrom and K. Anno, J . Am. Chem. Soc., 74, 5583 (1962). (224) E.Fisoher and 0. Piloty, Em., 28,3102 (18QO). (225) E.L.JackRon and C. 8. Hudmn, J . Am. Chem. Sm., 76,3000 (1953). 43, 48'2 (1!)10). (220) C.Krimz, h., (227) P.A. Tlevene and J. Compton, J . Biol. Chem., 111, 335 (1935). (228) H.Muller and T.Reichstein, Helv. Chim. A&, 11,251 (1938). (229) P.A. Imene and J. Compton, J . B i d . Chem., 116, 169 (1936). (230)E.J. Reist, L. Goodman, It. R. Spencer, and B. R. Baker, J . A m . Chcm. Sot., 8O,SQO2 (1058).

1i!)

DEOXY SUGAR8

mwhankm, pmeediig through an anhydro interniediate counts for the inversion at c-l and C-5.

rH yk/)

CHsf)ocHI

H , Qpqc HQB

HO

__t

o\ / o

H/\cH, (39)

YC/O H,C’ ‘CH,

(a), that

YC-

-

HOIQCHS

O\

/o

C H,C/ ‘CH,

(40)

(4 1)

This interesting transformation from a readily available 6-deoxyhexose forms the basis for the ~ynthesis~~1 of 6-deoxy-2,3-di-O-methyl-~-allose (mycinose),la one of the carbohydrate moieties of the antibiotic chalcomycin. The synthesis merely involves the protection of the C-5 hydroxyl group in (41) by benzylation, followed by partial hydrolysis with acid, methylation of the product at the C-2 and C-3 hydroxyl groups, and debenzylation. By employing sodium benzoate in N ,N-dimethylformamide, Baker and coworkers282effected the inversion of C-5 in a sulfonic ester of (41), and obtained the epimeric ctalo derivative, which was then hydrolyzed to 6-deoxy-ctalose. A synthesis288involving the conversion of 5-deoxy-~-xylose into 5-deoxy-~-riboseby inversion at C-3 utilized methyl 2- O-benzoyl-5deoxy-3-0- (methylsulfonyi)-D-xylofuranoside. The identity of ‘the initial inversion-product (having the D-rib0 configuration) was elegantly demonstrated by vapor-phase chromatography. Replacement of the 2-benzoyloxy by a 2-benzyloxy group resulted in a markedly sluggish reaction; this accentuates the effect of a neighboring group participating in the displacement of the 3-0-methylsulfonyl group. This experiment also demonstrates that direct SN, displacement of the 3-0-methylsulfonyl group of a furanoside is possible. Jones and Nicholson234have observed that treatment of 2-0-sulfonyl sugars with base leads to epimerization at (3-2. In this way, 2-0-p-tolylsulfonyl-L-fucose (42) was converted into Bdeoxy-L-talose (44) in high yield. A possible intermediate might be the 1,Banhydro sugar (43). S.Brimacombe, M. Stacey, and L. C. N. Tucker, Proc. Chem. SOC.,83 (1964); J . Chem. Soc., 5391 (1964). (232) E. J. Reist, L. Goodman, and B. R. Baker, J . Am. Chem. Soc., 80, 5775 (1968). (233) K. J. Ryan, H. Arzoumanian, E. M Acton, and L. Goodman, J . Am. C h . (231) J.

Soc., 86,2497 (1964). (234) J. I<. N. Jones and W. H. Nicholson, J . Chem. Soc., 3060 (1955).

180

BTPPHEN HANPBBIAN

1

H@ O H - [ F J ] - H Q O H

HO

HO HO

(43)

H

HO (44)

(49)

(v) Other Methods.-A synthesis of crystalline 6-deoxy-~-allosehas been described by MicheePO that involves the hydroxylation of 6deoxy~-allalwith peroxybenzoic acid. In a study of the anhydro-ring cleavage of methyl 2 ,3-anhydro-6-deoxy-c~-talopyranoside, it was reported236that reaction with sodium methoxide affords a 10-15% yield of sirupy BdeoxyL-idose and a 70430% yield of the corresponding 3-methyl ether. Meyer and ReichsteirP had previously obtained crystalline 6-deoxy-bidose by a different route. A stereospecificsynthesis of 6-deoxy-tidose derivatives has been reported by Wolfrom and Hanessian.2M Treatment of 3-0-benzyl1,2-O-isoprcpylidene-ar-~-qjl~pentod~ldo-l ,4-furanose (45) with methylmagnesium iodide afforded a 70% yield of crystalline 3-0-benzyl-6-deoxy1,2-O-isopropylidene-&~-idofuranose(46) , the configuration of which was proved unequivocally. The stereospecificity in the Grignard reaction may be rationalized on the basis of Cram's rule,w which correlates and predicts the stereochemical direction in which a new asymmetric center is created adjacent, to an existing one in acyclic systems. Coordination of the magnesium with the carbonyl and ring oxygen atoms would result in a 5-membered precursor, such as (47), that would fix the transition state in the h i d o configuration and hence favor it over the alternative wgluco configuration. It waa also observed2" that hydrolysis of 6deoxy-l,2pOHC=O

/I

HaC\

' 0

M$, /'

\\

'*O/

A 48

H (45)

(4 6)

(47)

(236) G. Chsralamboue and E. E. Peroival, J . Chem. Soc., 2443 (1964). (236) M. L. Wolfrom snd 5.Hanemian, J . Osg. Chem., 97,1800 (1962). (237) D. J. Cram and F. A. At)d Elhafez, J . Am. Chetn.. Soc., 74, 6828, 6861 (1982); D. J. Cram and K. R. Kopecky, ibid., 81, 2748 (1959).

DEOXY SUGARS

181

impropylidene-cidofuranose with dilute acid or a catiori-exchatlt?;e win affords a mixture of Weoxy-cidose and 6-deoxy-cxylo-hexulose. This phenomenon, which can be partly explained in terms of the conformational instability147 of the idopyranose structure, has been observed more dramatically by Vargha*a for the idoses. It is noteworthy that, when 3-0benzyl-6-deoxy-cidose2a is hydrogenolyzed under neutral conditions, one prdduct, presumably 6-deoxy-cidose1 is formed which, by treatment with acid, unaergoes a (partial) change to the ketose.'"aaAnother example of the reaction of suitably protected dialdoses with Grignard reagents involves the reaction of the glycol-cleavage product from 3-O-acetyl-l , 2-0-isopropylidene-~-D-ccrabino-hexulosewith methylmagnesium iodide.2ag A Fjdeoxy-sthreo-pentulose derivative was reported to have been formed, by an asymmetric synthesis, since none of the erythro isomer was found. A synthesis of crystalline 6-deoxy-ctalo~e~~~ is based on the catalytic hydrogenation of methyl 6-deoxy-3 ,4-O-isopropylidene-a-~-lyxo-hexuloside," and hydrolysis to give methyl 6-deoxy-a-etaloside and, thence, 6-deoxy-ctalose. The preparative value of the triphenyl phosphite methiodide method1% for the synthesis of 6deoxyhexoses was pointed out by Kochetkov.lmaul Advantage can be taken of the fact that, in the presence of secondary hydroxyl groups that are relatively hindered, the primary hydroxyl group M c t s preferentially, as exemplified by the conversion of methyl 3,4-0isopropylidene,fI-D-galactosideinto the corresponding 6-iodo deri~ative.~41 According to Lee and NolanlN1*triphenylphosphine in refluxing carbon tetrachloride converts aliphatic primary and secondary alcohols into the corresponding chlorides in high yield. A similar reaction with 1,2 :3 ,4-di0-isopropylidene-D-galactopyranoseafforded, in good yield, 6-chlor6-6 Unlike the cme of deoxy-1 ,2 :3 ,4-di-O-isopropylidene-~-g~lactopyranose. triphenylphosphite methiodide (or dibromide ), acetals, esters, and adjacent hydroxyl groups in the sugar molecule are unaffected. The hydroboration of methyl 5,6-dideoxy-2 ,3-O-isopropylidene-~-erythro-hex-4enofuranoside (34)14* hm been extensively studied and shown to give a 6-deoxy-D-guloae derivative. The cis hydration of the propenyl system in (34) from the less-hindered side accounts for the stereosolectivity of the reaction. (238) L. Vargha, C h m . Ber., 87, 1351 (1954). (239) P. A. J. C h i n , L. Hough, and J. K. N. Jones, J . Chem. Em.,2699 (1956). (240) P. M. Collins arid W. G . Overend, Chem. Znd. (London), 375 (1963). (241) N. K. Koohetkov, J. Sci. Ind. Res. (India), W , 324 (1964). (241a) J. B. Lee and T. J. Nolan, Can. J . Cbm., 44, 1331 (1966). (242) H. Arroumanian, E. M. Acton, and 14. Goodman, J . Am. Chem. Sac., 88, 74 (1964).

182

BTEPHEN HANESSIAN

Halogal atoms have been placed at C-6 of substituted 6deoxy-hexoses by a variety of interesting ways, and the producta provide potential routes to 6deoxyhexosesl. Helferich and coworker@ reported a low yield of methyl Bchloro-6-deoxy-a-D-glucoaidewhich they prepared by reaction of the {suitably protected) corresponding 6-trityl ether with phosphorus pentachloride. SiQclairu' has provided a convenient procedure for the preparation of the 6-chloro derivative in yields of 30435% by allowing methyl a-D-glucopyranoside to react with sulfur monochloride, S&la, in N,N-dimethylformamide. A much better yield (73%) of the 6-bromo derivative waa reportedla in the reaction of methyl 2,3,4tri-O-benroyl6-O-trilyl-ol-~-altrsidewith phosphorus tribromido and bromine. The reaction of methyl glycopyranosides with sulfuryl chloride has also provided a route to 64hloro (and other monochloro, dichloro, and trichloro) derivatives." Schlubach and Wagenitzub observed that reaction of p-Dgalactopyranose pentaacetate with hydrogen bromide affords a 1,6-dibromo derivative whioh they converted into crystalline n-fucose in two steps. A new reaction in the carbohydrate seriess7 provides a general route to 6deoxyhexosee from readily available methyl 4,6-O-benrylidene-hexopyranosidee. This reaction is based on the observation by Marvell and Joncich'u that benzddehyde diethyl acetal is transformed into ethyl benzoate by the action of N-bromosuccinimide. The acetal from 1,2-cyclohexanediol has been shownaato give trans-2-bromocyclohexyl benzoate. Treatment of methyl 4,W-benaylidene-a-D-galactopyranoside(48) with slightly more than one molecular proportion of N-bromosuccinimide and an excess of barium carbonate in refluxing carbon tetrachloride gave2" an almost quantitative yield of methyl 4-O-ben~oyl-6-bromo-8deoxy~-~-galactoside (SO); this was converted into methyl a-D-fucoside (51) in good yield. This novel ring-opening is applicable to other 4 ,GO-benzylidenehexopyranosides. An attractive feature of the reaction is the selective benzoylation at C-4 in these glycosides. A plausible mechanism involves initial abstraction of the benzylic hydrogen atom by a free-radical reaction, to give the unstable bromoacetal (49);this intermediate then dissociates into a cyclic, arbonium-ion intermediate and bromide ion. The reaction, moat probably, then aasumes an ionic nature, and bromide ion attacks the more accessibleand less hindered primary carbon atom (C-6) to give the product. (243) B.Helferich,W.Klein, and W. Schtifer, Ber., 19,79 (1926). (244) H.B.Sinclair, J . Org. Chum., 80, 1283 (lW). (245) (a) B.Helferich, Ber., M, 1082 (1921).(b) H. Jennings and J. K. N. Jonea, Can. J . Chem., 48,2372 (1966) and previous papera. (246) H. H. Schlubach and E. Wagenita, Ber., 81,304 (1932). (247) 8.Haneseian, Carbohydrda &I., 1,86 (1986). (248)E.N. Marvell and M. J. Jonoich, J . Am. Chem. EM., 78, 973 (1951). (249)A. Rieche, E.Schmitz, W. Schade, and E. Beyer. Chem. Ber., W, 2920 (1961).

183

DEOXY SUGARS

(48)

/

(49)

OH (51)

I

OH (50)

&tension of this reacti0n~4~" to other types of cyclic benzylidene acetals provideg a general route to deoxy and deoxyhalo sugars from readily available intermediates. In general, internal acetals formed from secondary hydroxyl groups (furanosides, pyranosides) give isomeric deoxyhalo benaoates, reducible to various types of deoxy sugars. The point of attack of bromide ion on the acetal-ring carbon atoms can, however, be altered to some extent by introducing a participating function (eater, hydroxyl) near the metal bridge. The ring-carbon atoms of the intermediate benaoxonium ion are thus attacked intramolecularly by the nearby groups, to give rearranged ions. If 'the latter cyclic ion involves & primary carbon atom in the sugar portion, attack of bromide ion will usually occur at that carbon atom, to give w-deoxy-+halo sugar derivatives.

IV. DIDEOXY SUGARS 1. 2 ,6-Dideoxyhexoses

a. General Considerations.-Until recently, this class of dideoxy sugars was orily kiiowii t,o occur hi thc cardiac glycosidcs. All of the four possible 2 ,6-dideoxy-1~-hexosesor their methyl ethers, or both, have been found in these materials. Their chemistry, distribution, and properties have been reviewed exten~iveIy.~-~J46 Several 2 ,6dideoxyhexoses and their (249a)

S.Hailmian, dbetracls Papers Am. Chern. See. Meeting, 162, 2

9 (1966). ~

184

STEPHEN HANESSIAN

methyl ethers have, in the past few years, been encountered as components of other biologiaally significant substances. The lgxo and crrabino configurations seem to predominate in the dideoxyhexosesof several new antibiotic substances. The neutral sugar in 7-rhodomycin 111, isolated from Slteptomyces ~ U T ~ U T U Stamm, ~ S WBB shown to be 2,6dideoxy-~-lg~hexose(52).260 Methanolysis9*1of the intibiotic olivornycinJu*isolated from Streptomyces oliuoreticuli, was shown to give three methyl glycosides which, on hydrolysis with acid, give the free sugars, olivomycose, olivomose, and olivose. ~ -+ -23" (in water), was assigned a Olivomycose, m.p. 103--6O,[ a ] -13" branched-chain, trideoxyheptose structure,"' as it contains three hydroxyl groups and two C-methyl groups. Olivomose is not oxidized by periodic acid, and contains two hydroxyl groups and one methoxyl group, and it waa therefore assigned a 2 ,6-dideoxy-4-0-methylhexose structure.261 The D-Z~XO configurationN8w a ~ later assigned to it; this was based on the identity of methyl 3-0-methyl-olivomoside with synthetic methyl 2 ,6dideoxy-3,4-di-O-methyl-a-~-lyxo-hexoside. Olivose, originally supposed to have a 2 ,&dideoxyhexose structurelB1 was subsequently assigned the D-arabino configurationNa on the bash of o p t i d rotation studies on the corresponding methyl olivoside in cuprammonia. I n this study,"' the authors claimed that olivomose is similar tq chmmoae A, a component of the carcinostatic antibioticw chromomycin As. Differences in melting point and optical rotation were attributedul to impurities in chromose A. A fourth sugar, acetyloliose (3-0-acetyl-2 ,&dideoxy-PZyzo-hexose), has been isolated from olivomycin.m* Chromomycin Aa is the principal constituent of chromomycin,*b' a complex mixture of antitumor antibiotica produced by Stteplomycee g~iseus7 . Mild hydrolysis by acidm*" of chromomycin As produces four 2,6-dideoxy(280) H. Brockmann and T. Waehneldt, Natud8mcha,ftcnJ 48,717 (1961). (251) Y. A. Berlin, 8. E. Eeipov, M. N . Kolosov, M. M. Shemyakin, and M. G. Brashnikova, Tetrahedron Lettar8, 1323 (1984). (252) G. F. Gauee, R. 8.Ukholiia, and M. A. Sveshnikovn, Anfibiotiki, 7,34 (1962); M. 0.Brazhnikova, E. B. Kruglyak, I. N. Kovsharova, N. V. Konetantinova, and V. V. Proshlyakova, ibid., 7,39 (1962). (253) Y. A. Berlin, 8.E. Eaipov, M. N. Kolosov, M.M. Shemyakin, and M. G. Brazhnikova, Te~rahsoltonLetlere, 3513 (1964). (25338) Y. A. Berlin,8. E. Enipov, M. N. Kolosov, and M. M. Shemyakin, Tetrahedron Luffera, 1431 (1966). (254) M. Shi'oata, K. Tanabe, Y.Hamada, K. Nakasawa, M. Miyake, H. Hitomi, M. Miyamoto, and K. Mizuno, Penishirin Son0 Ta ~08cibzlS8hiteu,13, 1 (1960); K. Mizuno, J . Anlibiotics (Tokyo), I%. A, 16, 22 (1963). (255) M. Miyamoh, Y. Kawamatau, M. Shinohara, Y. Amhi, Y. Nakedairs, H. Kakienwa, K. Nakanishi, m d N. 8.Bhaccs, Tetrahedron Letleva, 693 (1963). (268) M. Miyamgto, Y. Kawamatsu, M. Shinohara, K. Nakaniehi, Y. Nakhira, md N. 8.Bhacca, Tetrahedron LeUer8,2371 (1964).

DEOXY SUGARS

185

hexoses, named chromose A, B, C, aud D, aud the aglycou chroruoinyciiioue. C h r o w A (53) wm shown to belong to the D series, and was identified

HO

as 2 ,6-dideoxy4O-methyl-~-lyxo-hexose by nuclear magnetic resonance,

chemical, and optical rotation data.= An unequivocal synthesis267provided definitive proof of its identity; the synthetic sugar was identical, in melting point, infrared spectrum, x-ray diffraction pattern, and chromatographic properties, with the natural sugar. The melting point of olivomose is higher than that of natural and synthetic chromose A by%' some 7-9". Chromose B was shown to be a 4O-acetylepimycarose, namely, 4-0-acetyl-2 ,6-dideoxy-3-C-methyl-~urabino-hexose, by chemical and nuclear magnetic resonance data.= Chromose C (54) and chromose D (55) were akigned the structures of 2,6-

HO&OH

(54)

(55)

dideoxy-v-D-urabino-hexose and 3-0-acetyl-2 ,6-dideoxy-~-lyxo-hexose, respectively, on the basis of nuclear magnetic resonance and chemiortl evidence, including date from periodic acid oxidation." The stereochemical aesignment for chromose D has been corroborated by synthesis of the sugar.= Nuclear magnetic resonance spectroscopy has been of particular value in the elegant structural elucidation of the chromoses. Some antibiotic substances related to ChrOmOmyCih have been reported, but their structures are still undetermined.266 The total structures of the chromomycinsm. and (257) J. S. Brimombe, D. Portsmouth, and M. Stacey, Chem. I&. (London), 1758 (19f34); J . Chem. Soc., 9880 (1964). (268) J. 8. Brimacombe and D. Portsmouth, C h . Znd. (London), 468 (1965); Carbohydrute &., l, 128 (1965). (258a) M. Miyamoto, Y. Kawemetsu, K. Kawashima, M. Shinohsra, and K. Nakaniahi, Tetrahedm Letters, 545 (1966).

186

STEPHBN HANDSSIAN

olivomycinsmb have been established. Further work on the structure of olivomycin,~brevealed the existence of four olivomycins, differing in the eater moieties which are attached to the sugar portions. It ww also shown that the olivomycins and chromomycins are, structurally, closely related. It appears that olivomose and chromose A, acetyloliose and chromose D, and olivose and chromose C are identical pairs.

b. Synthesis.-All of the eight possible 2,Bdideoxyhexoaea (in the and L series) have been prepared, mainly through the pioneering efforts of Reichstein and coworkers.a The synthetic methods are simple and, in some cases, involve the use of u-deoxy sugars, such as D- and Lrhamnose or D- and cfucgse. Thesa were converted into the corresponding, acetylated glycah, and the latter, by conventional methods, into the respective, crystalline dideoxy sugars, 2 ,6-dideoxy-~-arabino-hexose(Lcanarose) zbe and 2,Bdideoxy-cly~o-hexoae~~ For the isomers having the rib0 and xylo configurations, another familiar approach has been used. Methyl 2,3anhydro-4 ,6-O-benzylidene-ar-~-alloside~~~~~ was converted into the 4 ,6-dip-toluenesdfonate,2*1and this was reduced with lithium aluminum hydride, giving methyl 2,6dideoxy-4-0-ptolylsulfonyl-cu-Dm’bo-hexopyranoside, which was converted into crystalline 2 ,6-dideoxy-wribo-hexose (digitoxose). It is of interest that the hydride reduction of the intermediate methyl 2 ,%anhydro-4,6ai-O-ptolylsulfonyl-ar-D-alloside could be so controlled as to effect reduction of the anhydro ring only, producing a Zdeoxy sugar.20a The order of reactivity of the anhydride ring and the sulfonyloxy groups toward lithium aluminum hydride is, therefore, anhydro > primary ptoluenesulfonate > secondary ptoluenesulfonate. The synthesis of crystalline 2,6-dideoxy-wxyZo-hexose (boivinose) involved the hydride reduotion of methyl 2,3-anhydro-6-O-ptolylsulfonyl-cu-~-guloside, followed by aoid hydrolysis of the resulting methyl 2,6dideoxy-~-xylo-hexoside. A synthesi@a of 2 ,&dideoxy-~-arabho-h-hexoseinvolved, as the first step, the reductive desulfonyloxylation (with lithium aluminum hydride) of 2deoxy-6-0-p-to~y~8ulfonyl-~-ar&no-hexosediethyl dithioacetal. Several methyl ethers of the above dideoxy eugars have been synthesiaed,l essentially by the above methods, with introduction of a methoxyl group (usually 4t C-3) at a convenient stage of the synthesis. D

(2SEb) Y.A. Berlin, 8.E. Eeipov, M . N. Kolwv, and M.M.Shemyskin, Tetrahdmn

1643 (1900). B. Inelin and T. Reichateii, Helu. Chim. Acta, 97, 1148 (1944). B. Iselin and T. Reichstein, Helu. Chim. A&, 97,1200 (1944). H. R. Bolliger and P. Ulrich, Helu. Chim. Ada, M,93 (1952). H. R. Bolliger and M. Thtlrkauf, Helu. Chim. Acla, 86, 1420 (1952). H. R. Bolliger and T. Reichstein, Helu. Chim. A&, 86,302 (1963). W. W, Zorbaoh and J. B. Cieudelli, J . Org. C h . ,80,461 (1966). -8,

(269) (260) (261) (202) (283) (204)

DEOXY SUGARS

187

The syrithesiu,s7 nieritioiied earlier, OF ehmn~ose.I was cffectd, from the &p-toluenesdfonate of a suitably protected methyl ‘Ldmxy-dyxohexoside, by reductive dcsulfonyloxylatiori followed by hydrolysis. Some useful experimental conditions used in the synthesisw ofw chromovc D warrant comment. I t has been obscrved that treatment of nicthyl 2,Odideoxy-cu-o-ll/zo-hc?xopyntriosid~ with an equivalent amount of acctic anhydride in pyridine produces a mixture of two monoacetates in which the :%acetate (having an equatorial bond to the acetate group in the D-C1 conformation) preponderates. Acetylation with trimethyl orthoacetateza in the presence of mesitylenesulfonic acid gives initially a 3,4-0-(methoxyethylidene) acetal which decomposes to a mixture of monoacetates in which the $-acetate preponderates. Methylation and nuclear magnetic resonance studies validated the proposed identities of the above products. In future work, advantage should be taken of this relatively selective acetylation of an axial hydroxyl group in cyclic compounds having an axial+quatorial disposition of vicinal hydroxyl groups.

2. 3,6-Dideoxyhexoses a. General Considerations.-Four of the eight possible 3,6-dideoxyhexoses have been isolated from various lipopolysaccharides elaborated by Gram-negative b a ~ t e r i a . ~These ~ ~ *pyrogenic, *~~ protein-free lipopolysaccharides, isolated initially from four species of Salmonella, were shown to contain two neutral sugars, named a b e q u o ~ eand ~ ~ ?tyvelose,2B8 which could be distinguished from the remaining sugars by their high mobility on paper chromatograms. Structural studies on these sugars by Westphal and coworkers267 indicated the molecular formula CaHr2Ol and the presence of one aldehyde, one C-methyl, and three hydroxyl groups. Slow uptake of periodate and colorimetric test data led to the postulation of a 3,6-dideoxyhexose or a branched-chain structure for these Definitive structural proof, based on studies of controlled oxidation with periodate, in favor of the 3,6-dideoxyhexose structure was later provided.M0Thus, reaction of one mole with one mole of periodate effected cleavage between (265) C.B. Reese and J. E. Sulton, Proc. Chem. SOC.,214 (1964). (266)D. A. L. Davies, Advun. Carbohydrate Chem., 16,271 (19W). (287) 0.Westphall 0.Liideritz, I. Fromme, and N. Joseph, Angew. Chem., 66, 555 (1963);A. M.Staub and R. Tinelli, I3u.U. Soc. Chim. Bwl., 89, (Suppl.), 65 (1957); I. Fromme, 0.Liideritz, A. Nowotny, and 0. Westphal, Pharm. Actu Helv., 33, 391 (1958). (268) 0.Westphal, Angew. Chem., 84, 314 (1952);G.Pon and A. M. Staub, Bull. SOC. Chim. Biol., 84, 1132 (1952). (269) I. Fromme, K.Himmelspsch, 0. Liideritz, and 0. Westphal, Angew. Chem., 69, 643 (1957).

188

BTEPHEN HANEBBIAN

(2-1 arid C-2, yielding a 2 , ; i - d i d ~ ~ x y p ewhich n ~ ~gave ~ a positive Webb color-reaotion,nl charactcristic of Zdcoxy mgare. The isolation of acetaldehyde, and the absence of formaldehyde from a total oxidation, indicated an wdeoxy structure. The configurations of abequoae and tyvelose were later shown to be wxylo (56)m and warobino (57),"* respectively, by

synthesis. In 1958, two other 3,6dideoxyhexoses, paratose and colitose, were encountered in bacterial lipopolysaccharides. Paratoe@ was obtained by mild, acid hydrolysis of a polysaccharide extracted from S. paratyphii -4, and colitosenbwaa isolated from a hydrolyzate of the O-antigen in Escherichia coli. Periodate oxidation studies, coupled with colorimetric teats on the oxidation products, revealed the 3 ,&dideoxyhexose structures of these sugtirs. In the same year, paratose was identified as 3,6-dideoyD-ribo-hexose (58) "; the (pnitrophenylsulfonyl) hydrazonen6 of colitose was found to be enantiomorphic with the synthetic analog (J6).nnThe (4'-nitro-4-biphenylsulfonyl)hydraeones and (azobenzenesulfonyl)hydraeones of the 3 ,&dideoxyhexoses have been found to be convenient compounds for characterizing, in crystalline form, very small amounts of these sugars. Dideoxy sugars have also been found in the specific polysaccharidea of Paeteurella p s e ~ d o t u b e r c d o a i s . ~ Another 3 ,6-dideoxyhexose, ascarylose, was obtained by acid treatmentm of ascaroside B, one of the glycosides in ascarylic alcohol, an unsaponifiable glycolipide found in the membrane of the eggs of Parmcaria equorum. Periodate oxidattion of the aldonic acid from ascarylose, and further oxi(270) I. Fromme, 0.Liiderits, H. Stierlin, and 0. Westphal, Biochsm. Z., 330,53 (1968). (271) J. M.Webb and H. B. Levy, J . Biol. Chem., 213, 107 (1955);K.Himmelspech and 0.Westphsl, Ann., 668, 1sS (1963). (272) 0.Westphal and 6. Stirm, Ann., 620,s (1959). (273) C.Fouquey, J. Polonsky, and E. Lederer, Bdl. Soc. Chim. Francs, 803 (1959). (274) D.A. L.Daviee, A. M. Staub, I. Fromme, 0. Lilderita, and 0. Westphsl, Nature, 181,822 (lQ68). (275) 0. Ltideritz, A. M. Staub, 8. Stirm, and 0. Westphal, Biocham. Z., 390, 193 (1958). (276) C. Fouquey, J. Polonsky, E. Lederer, 0. Westphal, and 0. Liiderite, Nature, 182, 944 (1968). (277) C.Fouquey, J. Poionsky, and E. Lederer, BuU. Soc. Chim. Biol., 8g, 101 (1957).

DEOXY SUGARS

189

cltion of the C4-acid fragment, produced mlactic acid (from C-14'4) ; berm, a n-ylycm configurationnMa t C-2 ww deduced for ascarylose. The carabirbo coiifiguratiorl was subsequently assigned by synthesis of the enantiomorph.n* Ascarylose (3,Sdideoxy-carubino-hexosc) is thus the optical antipode of tyvelose (57). A reportno discusses the chemical structure of ascarosides A, B, arid C, as well as of their aglycons. Ascarylose has been obtained from microbial sources for the first time.280Only the c r i b 0 and D and c2yxo configurations have not yet been encountered in biological substances. Tho 3 ,6-dideoxyhexoses have corisiderable biological significance, as they have been shown to. contribute to the serological specificity of many Artificial antigens281have immunologically active polysac~harides,8-~J@~*@ been made to stimulate antibody production by coupling diazotized paminophenyl3 ,6dideoxyhexosides with a protein such as serum albumin through the tyrosine residues. Such artificial antigens containing determinant dideoxy sugars have interesting immunological properties which point to a wide area for further research. The liberation of these dideoxy sugars from the polysaccharides by treatment with acid (1 N sulfuric acid for 3-5 minutes a t 100") indicates that they are probably nonreducing end-groups. Extension of the hydrolysis time and use of strong mineral acids result in losses of the dideoxy sugarsl because, like the 2-deoxy sugars, they are quite labile to acids. In addition to characterization by way of the substituted hydrazones discussed earlier (see p. 188), they may be reduced with sodium borohydride arid converted into crystalline dideoxyalditolss~B~n* having characteristic, diff erentiatable physical properties. This class of dideoxy sugars may readily be identified, and quantitatively estimated, in the presence of other sugars, by specific, colorimetric procedures.no,n1bas2

b. Synthesis.-The majority of synthetic methods simply involve the introduction by conventional methods of a second deoxy function into 3- or 6-daoxyhexoses. The sequence of methyl 3-deoxyhexopyranoside + 6-ptoluenesulfomte + 6iodo derivative (or direct reduction with lithium aluminum hydride) -+ methyl 3,&dideoxyhexopyranoside-+ 3,6dideoxyhexose (applied to the appropriate 3-deoxyhexose) has been used in the synthesis of 3,6-dideoxy-D-m'bo-hexose(paratose) ,ns,na3,6-dideoxy-~-xyEo(tyvelose) .n8,=3 hexose (abequose) ,*2*p88 and 3,6-dideoxy-~-arabino-hexose (278) C. Fouquey, J. Polonsky, and E. Lederer, BuU. SOC.Chim. Biol. 40,315 (1958). (279) C. Fouquey, J. Polonbky, and E. Lederer, Bull. SOC.Chim. Biol., 44, 69 (1962). (1961). (280) D. A. L. Ihvies, Nature, "43 (%81) 0. Liideritz, 0. Westphal, A. M. Staub, and L. LeMinor, Nature, 188, 556 (1960). (282) M. A. Cynkin and G. Ashwell, Nature, 180, 155 (1960). (2&1) C. Fouquey, E. Lederer, 0. Luderitz, J. Polonsky, A. M. Staub, S. Stirm, R. Tinelli, and 0. Westphal, Compt. Rend., 240, 2417 (1958).

190

STEPHION HANESSIAN

The synthc&eof 3,6-dideoxy-tZyxo-hexosc,~~~* an unnatural isomer, was achieved by treating methyl 3,4a1ihydro-6-deoxy-ttalopyranoside with lithium aluminum hydride, and hydrolysing the resulting 3,Gdideoxyglycoside with acid. A cyanohydrin synthesis from 2,5-dideoxy-obthreopentose afforded, after reduction of the mixed lactones, a 52% yield of a mkture of 3, Bdideoxy-Dbxylo-hexose with 3,6-dideoxy-D&yxu-hexose; this was separated into the racemic components by chromatography.% A synthesis% of 3, Gaideoxy-maylo-hexose (abequose) utilked 3deoxy-Dxylo-hexose diethyl dithioacetal; this was selectively sulfonylated at the C-fl hydroxyl group, and this ester was converted into the desired wdeoxy derivative by mans of lithium aluminum hydridu. On hydrolysis, the sugar was obtained in 18% overall yield (based on the 3deoxyhexose). Antonakism has described another method of synthesis of abequose and its l-phosphate from 3deoxy-~-xylo-hexose;this was selectively p-toluenesulfonated at the C-6 hydroxyl group, the ptolylsulfonyloxy group wm replaced by iodine, and the product was reduced and then hydrolyzed. RembarzP has reported the synthesis of 3,6-dideoxy-~-arabino-hexose (tyvelose), starting from 3-deoxy-~-ar&nehexose dimethyl dithioacetal. He observed that, as in many other instances, direct reduction (with hydride) of the terminal ptolylsulfonyloxy group affords lower yields of product (32%) than those obtained by first converting it into the wiodo derivative (51%) with subsequent reduction. Four of the possible 3,6-dideoxyhexoses have been syntheaised; those having the barabino (ascarylose), bx$a (colitose), D - ~ ~ x oand , bribe configurations have not yet been prepared. 3. 4,6=Dideo~yhexosee a. General Conefderadons,-Representativea of this c h of rare dideoxy sugars have been discovered aa components of the antibiotic substances lankamycin and chalcomycin, and neu tramycin.lee. On .hydrolysis with acid, lankamycin gives a reducing sugar, lankavose, having a relatively high mobility on paper chromatograms and containing one methoxyl and one C-methyl group.= On methanolysis, the antibiotic chalcomycin affords a crystalline glycoside, methyl chalcoside, which, on acid hydrolysis gives (284) 0. Weatphal and E. Rude, Swiss Pet. 369,693; Chem. Abstracts, 68, 8034 (1963). (286) H. Zinner, B. Emet, and F. Kreienbring, Chem. Bet-., 96, 821 (1962). (288) K. Antonakie, Compt. Rund., 168, 6911 (1964); BuU. 8oc. Chim. France, 2112 (1966). (287) 0.Rembara, J . Prakt. Chem., l B , 319 (1963). (288) E. G s b a n n , R. Hlitter, W. Kelle&hierlein, L. Neipp, V. Prelog, end H. Zahner, Helu. Chim. Acto, 48, 801 (1960).

191

DEOXY SUGARS

a crystalline sugar, chalcose (59).B8This sugar was shown to coiitaiu oiie

methoxyl group and oric C-niethyl group. With hydrobroniic acid, it is demethylated to a m w didcoxy nugar which, on pcriodatc oxidation, H 7=0 $=O +¶ c=o

QOH

CHOH

OH (59)

I CH,

(60)

affords crotonal4ehyde. Chalcose can be oxidized with periodate-permanganate to a six-carbon lactone. Oxidation with nitric acid produces a crystalline diacid, identical with synthetic mono-0-methyl-L-threaric acid. These data confirm the trans relationship of C-2 and C-3 of chalcose. On the basis of the above evidence, a 4,6-dideoxy-3-O-methylhexosestructure was assigned to chalcose. The configuration was elegantly elucidated from nuclear magnetic resonance studiesm of the free sugar and its methyl glycoside, and the xylo assignment has been confirmed by several syntheses (chalcose). The D - X ~ ~configurO of 4,6-dideoxy-3-0-methyl-~-xylo-hexose tion was also substant.iated by Stacey and who converted desosamine,202 3,6-dideoxy-3- (dimethylamino) -D-xylo-hexose, into chalcose. The total structure of chalcomycin, including the position of attachment of the sugars, has been l\eported.agaLankavose has been shown, by a detailed c o m p a h n of chromatographic properties and infrared spectra,2o4to be identical with chalcose. A labile 4,6-dideoxy-g1!ycero-hex~s-2,3-diulose(actinospectose) (60) has been isolated, as the crystalline bis-[ (2 ,4-dinitrophenyl) hydrazone] of (289) P. W.K.Woo,H. W. Dion, and Q.R. Bartz,J . Am. Chem. Soc., 88,3352 (1961). (280) P. W. K. Woo,H. W. Dion, and L. F. Johnson, J . Am. Chem. Soc., 1066 (1962). (291) A. B. Foster, M. Stacey, J. M. Webber, and J. H. Westwood, Proc. Chem. Soc., 279 (1963). (292) C. H. Bolton, A. B. Foster, M. Stacey, and J. M. Webber, J . Chem. Soc., 4831 (1961); A. C. Richardson, Proc. Chem. Soc., 131 (1963). (293) P. W. K. Woo,H. W. Dion, and Q. R. Bartz, J . Am. Chem.Soc., 86,2724, 2726 (1964). (294) W. Keller-Schierlein and G. Roncari, Helu. Chim. Ada, 46, 138 (1962); 47, 78 (1964). (295) H. Hoeksema, A. D. Argoudelis, and P. F. Wiley, J . Am. Chem. Soc., 84, 3212 (1962). (296) A. L. Johnson, DieeetlaGion Abalr., 26,4958 (1965).

y,

192

BTEPHEN HANESSIAN

methyl actinospectoside, from hydrolyzates of the antibiotic actinospectaCin."w b. Synthesfs.--Siiice no synthetic 4 ,(iaideoxyhexosea were available prior to 1961, efforts toward synthesis have been directed to the preparation of the only biologically derived analog, namely 4,6-dideoxy-~z~Zo-hexose and its %methyl ether. These investigations were published independently from several laboratories in the &me year, and involve different approaches. All of the constants reported agree with those of natural chalcose and lankavose. The first of these methods involves the reaction of triphenyl phosphite methiodide with methyl 3 - ~ - m e t h y ~ - 2 - ~ p t o ~ y ~ ~ o n y l ~ - ~ - g l u c o p y r a n o side, hydrogenation of the resulting 4,6-diiodo derivative, and hydrolysis,~ adapted to monosaccharides by Kochetkov and coworkers.m In another approach,lgl ethyl 2 ,3-anhydro-4,&dideoxy-Pribo-hexopyranoside, obtained from desosamine, w w treated with sodium methoxide, to give a mixture of 2- and 3-methyl ethers. After fractionation and separation of side products,** the %methyl ether was hydrolyzed, to give crystalline chalcose. McNally and Overendm have described another synthesis, starting from methyl 4deoxy-~-x&hexoside.* The solvolytic displacement studies by Hough and coworkerslM on methyl 2,3-di-O-benzoyl-4 ,6 d i 0-(methylsulfonyl)-a-D-glucoside revealed that the 4-methylsulfonyloxy group can readily be displaced by various nucleophilesl. By employing potassium thiocyanate, a 4 ,Bdideoxy-4,6dithiocyanato-a-D-galactoside waa isolated in moderate yield; this was transformed into crystalline 4,6 dideoxy-D-xylo-hexoee. Overend and coworkers1mreported the conversion of methyl 4-deoxy-a-~-~yto-hexop~noside, by way of the 6-iodo derivative, into methyl 4,6-dideoxy-ar-~aylohexopyranoside in a low oveiall yield. 4. 5,6=Dideoxybexosee

a, General Coneideradona.-This class of deoxy sugars is a further group for which ring formation is restricted to the furenoid form. Naturally occurring members have not yet been encountered. b. Synthmie.-The first synthesis was reported by McSweeney and Wiggine,wl who converted 5,Sanhydro-1 ,2-O-isopropylidenec~-ghco(297) D.J. Mason,A. Diets, and R.M. Smith, Antibiot. Chsmdczsrcrpy, 11, 118 (1961). (288) N. K. Koahetkov and A. I. Ueov, Tsh.aht?dron Lc#srs, 610 (1963); Ztv. Akud. Nauk XBXRj Xer. Khim., 492 (1965); Cham. Abstracb, 68,1867 (1886). (299) A. B. Foster, T. D. Inch, J. Lehmann, M. Elteoey, and J. M. Webber, J . Chem. Bw., 2116 (1962). (300)S.McNally and W. G. Overend, C h .Id.(London), 2021 (1964). (301) G. P. McSweeney and L. F. Wir#rine, Nature, la,874 (1961).

DPOXY SUGARS

193

furanose into the 5,Wdeoxy trithiocarbonate derivative which, on reductive cleavage, gave 5,6-dideoxy-l, 2-O-isoplbpyl'ldene-a-~-xylo-hexofuranose, The free sugar, 5,6-dideoxy-~-xylo-hexose, was obtained by acid involves ~ ~ J * reductive desulfurization of h y d r o l y s i ~A. ~related ~~ synthe~is~~ the 5,gepithio derivative.m English and Levyao3showed that the product from the reaction of * 5 ,6-anhydro-3-0-bqnzyl-1 ,2-O-isopropylidene-c~glucofuranose with methyllithium is, as expected, not a C-methyl derivative but 3-0-benzyl-5 ,6-dideoxy-l , 2-O-isopropylidene-a-~-xyh-hex-5enofuranose, which was reduced to the 5,g-dideoxy derivative. The reaction of sodium iodide with vicinal primary and secondary sulfonyloxya0Ju or halom groups results in an elimination, with the fwmation of terminally unsaturated derivatives. In this way, 1,2-0-isopropylidene-5 ,6-di-0-p tolylsulfonyl-a-D-glucofuranosewas converted m b into the corresponding hex-5-enofuranose derivativelag (27) and thence into the dideoxy sugar. Analogous treatment of methyl 2,3,5,6-tetra-O-ptolylsulfonyl-~-~-galactofuranoside afforded 5,6-dideoxy-~-arabino-hexose through the intermediate formation of a terminally unsaturated glycoside.80eGoodman and coworkersl"*"z have obtained methyl 5,6-dideoxy-2 ,3-0-isopropylidene/3-Dhexosides having the lyxo and ribo configurations by hydrogenation of suitable, unsaturated derivatives. The chemical synthesW@of 5 ,6-dideoxy-D-threo-hexulosewas achieved by treating the glycol-cleavage product from 3-O-acetyl-l,2-O-isopropylidene-@-D-arabino-hexdo6ewith ethylmagnesium iodide, and then hydrolyzing the product. Since one stereoisomer, the D-threo, was isolated, it seems that a stereospecific addition to the carbonyl function had occurred. A coordinated intermediate2a6that would fix the transition stage in the threo configuration could probably account for this stereospecificity. 5. Other Dideoxy Sugars

The 2,3-dideoxy sugars have received the most attention among the remaining classes of dideoxy sugars. This interest stems from the fact that ethyl 2, 3-dideoxy-a-D-erythro-hexopyranosidesMis a readily available compound; it was found to be more labile to N hydrochloric acid a t 18' than the 2-deoxy analog, and it was concludedw that an increase in the number of methylene groups in the chain definitely increases the lability of the (302) (303) (304) (305) (308) (307) (308)

A. M. Creighton and L. N. Owen, Nature, 1024 (1960). J. English, Jr., and M. F. Levy, J . Am. Chem. Soc., 78, 2846 (1956). R. 8.Tipson and L. H. Cretcher, J . Org. Chem., 8, 95 (1943). H. Finkelstein, Ber., 43, 1528 (1910). D. J. Ball, A. E. Flood, and J. K. k. Jones, Can. J . Chem., 37,1018 (1959). M. Bergmann, Ann., 448, 233 (1925). K. Butler, S. Lnlund, W. G . Overend, and M. Stacey, J . Chem. Soc., 1433 (1950).

194

STEPHEN HANESSIAN

glycosidic linkage to acid. By reaction of ethyl 2,3-dideoxy4,6-di-O(methylsulfonyl)-u-D-erythro-hexopyranosid@' with sodium benzoate in N ,N-dimethylformamide, and subsequent debenmylation of the product, the wthreo isomer was obtainedamas a result of invemion at C-4. A synthesis of 2 ,3-dideoxy-cgEycmo-pentus@' involved hydrogenation of the pseudoglycal formed, as in the hexose series, from l 1 4 - d i - 0 ~ c e t y l - D - a r a b i n a l . ~ ~ ~ It was noted in this investigation that the dideoxypentose gives a very weak, blue color with the diphenylamine reagent, in contraat to 2deoxy sugars. Pyrimidine nucleosidera containing the 2 ,3dideoxy-~+gthro-hexose residue have been reported by Stevens and coworkers,*l' who effected an elimination between (presumably) tradicurirtl methylsulfonyloxy groups and iodine atoms, situated at C-2 and C-3 in a protected nucleoside, by treatment with an excess of sodium iodide. The resulting 2 ,&unsaturated nucleoside was converted intu 1- (2,3-dideoxy:&D-erythr+hexopyranosyl) Cethoxy-2- (1H)-pyrimidinone. Displacement of the 3-tosyloxy group in a protected 2-deoxyadenosine derivative with ethanethioxide, followed by desulfubtion, forms the basis for synthesking 9- (2 ,3-dideoxy-&Pglycwopentofuranosyl)purine An alternative synthesis utilizing the ethylthio and thiooyanate protecting groups has been Several new routes to 2 ,3-, 3,4-, and 5,6-unsaturated sugars (which are, in essence, potential syntheses for the respective dideoxy sugars) have now been developed.*l' A 2 ,Mideoxy-wythrepentose was reported by Zinner and Wigerta*&; it was obtained by reduative desulfonyloxylationof 2-deoxy5- O-ptolylsulfonyl-Pet~ro-pentosedimethyl dithiowtd. The formation of a 2 ,5dideoxypentuse from a 3 ,6-dideoxyhexose forms the basis of the quantitative estimation of the latter compounds by the Webb color reGoodmana1ahas described a synthesis of methyl 2 ,6-dideoxy-u-Derythro-pentofuranoside (and of the corresponding 3,5-dideoxypentoside) by way of thio intermediates.

-

(300) 8. Leland, W.G . Overend, and M. Stacey, J . C h . Soe., 738 (18M)). (310) A. B.Foster, R. Harrison, J, Lehmann, and J. M. Webber, J . Chum. sbc., 4471 (1W). (311) R.Allerton, W. Q, Overend, and M. Stacey, J . C h .Hoc., 266 (1962). (312) C. L.Sbvens, N. A. Nielaen, and P. Blumberge, J . Am. Chern. Hoc., 86, 1894 (1984). (313) M.J. Robine and R. K.Robins, J . Am. Chem. Hoc., 86,3S85(1964). (313a) Q. L. Tong, W. W. Lee, and L. Goodmen, J . Org. Chem., 80,2884 (lQ65). (314) J. C.ChristenRen and L. Goodman, J . Am. Chew. Hoe., 88,3827 (1961); N. F. Taylor and 0.M. Rig@, C h .I d . (bndon), 2OQ (1963);D. Horton and W. N. Turner, Tetrahedron h k T 8 , 2531 (1984);A. H. Haines, Chem. Id.(London), lQQl (1964);R. J. Ferrier, J . Chem. Soe., 6443 (1%); N. K. Kochetkov and B. A. DdtdeV, TetruhedNm, PI, 803 (1966);R.J. Ferrier, Aduun. Curbohydrats Chem., 40, 68 (1985);A. H.Haines, Carbohydrale Res., 1, 214 (1985). (316) H.Zinner and H.Wigert, C h . Ber., 99, a893 (1968). (318) L.Goodman, J . Am. Chm, Soc., 88,4167(1964).

DEOXY SUGARS

195

V. TBIDEOXYHEXOSES This unusual class of deoxy sugars has been encountered in several antibiotic substances. Although biogenetically unrelated to each other, all of them seem to have a 2,3,Btrideoxyhexose as their neutral sugar component. One of the most thoroughly studied of the antibiotic substances is amicetin. The structure of this antitubercular antibiotic was first investigated by Flynn and coworker~,~17 and later by Haskell and coworkers.a18 Unfortunately, incorrect interpretation of misleading data obtained by periodate oxidation led to the initial assignment of a 1,3 ,Btrideoxyketohexose s t r u c t ~ r e *for ~ ~the neutral sugar in this antibiotic substance. A detailed investigationamlater revealed that the sugar designated amicetose is a 2,3,6-trideoxyaldohexose (61). Thus, methanolysis of amicetin gives methyl amicetoside, C7HuOa, which may be converted into amicetose (2 ,4-dinitrophenyl) hydrazone; on periodate oxidation, this gives acetaldehyde (2,4&itrophenyl) hydrazone (from C-6 and C-5) and succinaldehyde bis[ (2,4dinitrophenyl) hydrazone] (from C-14-4). It has been reportedsmthat methyl amicetoside can be hydrolyzed almost immedifltely to the free sugar with 3 N hydrochloric acid at room temperature. In this connection, it may be noted that the nucleoside linkage between amicetose and cytosine in nucleoside (62)"' is stable for 24 hours at room temperature in N hydrochloric acid. This stability may be contrasted with the lability of the nucleoside linkage of the (2-deoxy-D-erythro-pentosyl)pyrimidines.aa The periodate oxidation data and later synthesea confirmed the 2,3,6trideoxyhexose structure and also permitted the assignment of a ~ - ~ y t h r ~ configuration to amicetose. The P-D configuration of the anomeric linkage in amicetin has been established by nuclear magnetic resonance studies on (62) obtained by chemical degradationm1of the antibiotic material, and this assignment has been confirmed by synthesis of the nucleoside.S28 was shown to be the A 2,3,&trideoxyhexose, namely, rhodinose (a), neutral sugar component in the antibiotic rhodomy~in.~~4 The structure was established by periodate oxidation, with isolation of acetaldehyde and (317) E. H. Flynn, J. W. Hinman, E. L. Caron, and D. 0. Woolf, Jr., J . Am. Chem.Xoc., 76,5867 (1953). (318) T. H. Hsskell, A. Ryder, R. D. Frohardt, S.A. Fuaari, 2.L. Jakubowaki, and Q. R. Bartz, J . Am. Cham.Xoc., 80,743,747 (1958). (319) C. L. Stevens, R. J. Gaaaer, T. K. Mukherjee, and T. H. Haakell, J . Am. Chem. Soc., 78, 6212 (1958). (320) C . L. Stevens, K. Nagarajan, and T. H. Haakell, J . Org. Chem., 27,2991 (1962). (321) 5.Haneaaian and T.H. Haakell, Tetraibedrm Lellere, 2451 (1964). (322) R. 8. Tipwn, qduan. Carbohydrate Chem., 1, 193 (1945). (323) C. L. Stevens, N. A . Nielsen, P. Blumberga, and K. G . Taylor, J . Am. Chem. Soc., 86,5695 (1964). (324) H. Brockmann and T.Weehneldt, Natunuiseenschaflen, 60,43 (1963); h ~ 8 f C h T . C%m. &a. Naturstoffe. 21. 121 (1963).

190

BTEPHblN HANESIIIAN

mwhaldehydu an for amicetoso. Rhodinosu wan asaigncd the tthreo configuration on the baais of optical rotation and other comparative datam this asejgnment w&c)substantiated by synthesis of the enantiomorph. Finally, a 2,3 ,&trideoxyhexoseisolated from the antibiotic streptolydiginm w&e found= to be identical with rhodinose by comparison of their (2,4dinitropheny1) hydracones.

(6 1)

(63)

(62)

The synthesis of a member of this clase of deoxy sugars was accomplied* in 1950. The then well-known ethyl 2,3-dideoxy-a-~-ergthr~ hex-%enopyranosidP waa reduced and the product was converted into ethyl 2,3,&trideoxy40- (methylsulfonyl)-a-~erylhro-hexopyranosideby way of the 4,6-dimethanesulfonate. During this work, it W M found that, when the dimetbnesulfonate of the unsatumtd derivative is treated with sodium iodide in the cold, EL selective displacement at G4 occurs, no doubt due to the activated allylic eystem, The reeulting Ciodo derivative could

(68)

(87)

(335) C. L.Stevens, P.BlumbergB, and D. 1,. Wood, J . Am. Chsm. Soc., 86,3592 (1884). (328) K.L. Rimhart, Jr., and D. B. Borders, J . Am. Chum. Soc., 86,4037 (1963).

DEOXY SUGAR8

197

be converted into ethyl 2,3,4-trideoxy-6-O-(methylsulfonyl) -a-~-gZyce~ohexopyranoside.PDThe above intermediates paved the way for the faeile syntheaism of amicetow and its Dthreo isomer. Sequential ptoluenesulfon& tion and acetylation of ethyl 2,3-dideoxy-eD-ergthro-hexopyranoside~*~ gave the 6ptoluenesulfonate (64) which was converted into amicetose (61) by way of the 6iodo derivative (65) and ethyl amicetoside (66). By a configurational inversion at C-4 of ethyl 2,3,64rideoxy40-(methylsulfonyl)-a-D-erythro-hexopyranoside(67), caused by treatment with sodium benzoate and N ,N-dimethylformamide, the D-threo derivative (68) was produced; this was hydrolyzed to give 2,3,6-trideoxy-~-threo-hexose, the D enantiomorph of (a), enantiomeric with rhodinose. This sequence of reactions had previously been recorded by Foster and coworkers.a* 2,3,6Trideoxy-Dcerythro- and -Dcthreo-hexose have also been obtained, as the (2 ,4-dinittrophenyl)hydrazones, from 1-bromo-4-hexene by a series of rea~tions.~’

VI. CHROMATOGRAPHY 1. Paper Chromatography

The paper-chromatographic properties of the common deoxy and dideoxy a book,azoand individual sugars have been treated in several reviews,1*a2s publications. Common solvent-systems are 6:4 :3 1-butanol-pyridine-water (Solvent A) ; 4:1:5 1-butanol-acetic acid-water (Solvent B) ; and l-butanol-ethanol-water (4: 1 :5, upper phase, Solvent C; or 3: 1 : 1, Solvent D). The four 2-deoxy-D-hexoses and the 2 ,Wideoxyhexosea may be separated as their borate complexes.880The use of 1-butanol-water on the one hand, and of 2-butanone-borate buffer on the other, usually provides adequate separation; and, by use of a combination of solvents, these deoxy sugars may be identified.** The use of buffered systems has proved highly advantageous in the separation and identification of the isomeric 6deoxyhexoses.ml Other systems, such as Solvent Am and 2:1:2 ethyl acetate(827) C. I,. fitevene, B. Croee, and T.Toda, Abstracts Paper8 Am. C h .SOC.Meeting, 141, 7N 1962). (328) G. N.Kowkabany, Aduan. Carbohydrate Chem.,9,303 (1964); C. Tamm, Fortachr. Chem. Org. Nalumtofe, 14, 71 (1967); K. Macek, in “Paper Chromatography,” I. M. HniR nn’d K. Mnrek, P~R.,Arndcmia Pmw h a . , New Ynrk, N. Y.,1963, p. 180. (329) Ref. 146, p. 937. (350) 2.Kowaleweki, 0.Schindler, and T. Reiahetein, Helv. Chim. Acta, Is, 1214 (1960). (331) M. T. Krauss, H.Jtiger, 0.gchindler, and T. Reichstein, J . Chromatog., S, 63 (1980). (332) A. P. MacLennan, H. M. Randall, and D. W . Smith, Anal. Chem., S1, 2020 (1969); Biochem. J., 80, 309 (1961).

TABLEI Pqer-cbosna-phic

Mobilities of Selected DUD=S u m

Mobiity. in solventb system' 1

'-7 0 -60

2

3

4

5

6

(14)

0.30(133)

l.OfP(331) O.IxP(331)

0.22(133)

0.95$(331) 1.02*(331)

0.30(107)

0.17(133) 0 -536(131)

0.28(132)

2.59+(137) 1.53$(185) 0.25(133)

0.71 (328) 0.73(277) 2.2$(239) 0 -71(324) ~~

~

*

With respect ta reference compounds; key: rhamnose; A tetra4-methylglucose; * giucose. b Solvents:l, l-butanol-acetic scidammonia-water (4050S:15); 4, l-butanol-water;3, l-butanol-pyrkbe-water (6:4:3); 4, l-butanol-t?thanol-water (4:1:6, upper p b ) ; 6, l-butanokthanol-water (40:ll:lg); 6, 1-butanol-aeetc acid-water (4:l:l). cReferenre~are given in psrentheses.

199

DEOXY SUQhBB

pyridinttwate9 give fair separations of the 6deoxyhexoses, and may be used in combination with the borate system. The 3,&lideoxyhexoses are poorly separated in several of the solvent systems recordedsBg.naJ"; the corresponding alditols are, however, separated somewhat better.ua Many of the common reagents used for the detection of sugars on paper chromatogmms may be employed for the deoxy sugars, although some specific reagents are available.~*sal Table I lists the mobilities of some representative deoxy sugars and, in particular, of some of the more unusual members. 2. Thin-layer Chromatography Of the various media used in thin-layer chmmatography, many are adaptable to the deoxy sugars and their derivatives. Some selected media, solvent systems, and types of derivatives are listed in Table 11. TABLE I1 Mobilitier, of Selected Deoxy Sugaro and Their Derivatives on Thin-layer Chromatograms Compound Rhamnoee

Admrbent buffered Kieselguhr Celitestsrch buffered Kieaelguhr silica gel G (act.)

ZDeoxy sugars Dideoxy hexoeee "rideox yhexoses

Rhamnose diethyl dithioacetal Deoxyhexose dialkyl dithioacetsls Dideoxyhexoee dialkyl dithioacetals Methyl fucoside Methyl dideoxyhexosides

Solvent0

Mobility

References

1

medium fast medium slow

334

336 336 337

medium

337

2 1 3 3 4 6 3

-

fast

-

326 338

366

6

medium

366

7

medium

337

6

slow

337

The ealventa: 1, ethyl acetete--2propanol-water (126:70:36); 2, -2propanol-water (90: 10); 3, benzenemethanol (10:3); 4, chloroform-acetone (1:7); 6, 1-butanol-acetic acid-ether-water (9:6:3: 1); 6, chloroform-2,2,4-trimethylpentane-methsnol(50: 16:6); 7,chloroform-methanol (10:2). 0

- _(333)

-

F.A . Idlerwood uiid M. A. Jermyn, Biochent. J., 48, 615 (1961). (334) E.Stahl and U.Kaltenbach, J . Chromatog., 6, 361 (1961). (335) B. Shaeha and R. L. Whistler, J. chromatog., 14, 632 (1964). (336) G. Weidemann and W. Fiacher, 2.Phyuiol. Chem., 886, 189 (1964). (337)S. Haneasian, unpublished observations. (338)G. W.Hay, B. A. Lewis, and F. Smith, J . Chromalog., 11, 479 (1963).

200

STNPHEN HANESSIAN

Mixtures of benzene and methanol, or of chloroform and methanol (in proportions usually ranging from 1O:l to 10:3,. by volume) have been found suitable for the examination of various deoxy sugars and their deriv&tivesby thin-lsyer chromatography on silica ge1.W When the d e rivative is' psrticularly fastmoving in the above system (for example, thioacetals of dideoxy sugars; and glycosidea), the addition of 2,2,4trimethylpentane,'as a third component, usually lowers the mobility of the derivrqtive. Conversely, acceleration is usually attained by increasing the proportion of methanol. Thin-layer chromatography of simple deoxy sugars and their derivatives on activated silica gel, using these organic solvents, is not particularly suitable for the separation of complex mixtures, but it is ideal for rapidly checking homogeneity and for following reactions in which a deoxy sugar or a derivative thereof is produced. A new, microcrystalline cellulose has been found to give much more rapid development than ordinary cellulose in thin-layer chromatography.m Although specific examplea using deoxy sugars have not been reported, mixtures of deoxy sugars should be amenable to separation on these cellulose layers by use of the solvent system conventionally employed in paper chromatography. The anomeric pairs of methyl tri-O-acetyl-2-deoxy-~-arubinoand - ~ - l y x o hexopyranoeides oan be separated on silica gel glatea uaing methyl aulfoxide as the stationary phw.w** 3. Ionophoreab In ionophoresis, too,many of the systems described for common sugars should be applicable to deoxy sugars." Stevens and coworkersa have separated the (2,4-dinitrophenyl)hydraeonea of the ~-erythroand Pthreo isomers of 2,3 ,&trideoxyhexose by electrophoresis on paper in 0.083 M borax at pH 9.2 by applying a constant voltage of 300 volta. Rhmnose and fucose have different mobilities in cetyltrimethylammonium borate buffer.M' By using borate buffer of pH 10, 4deoxy-~-xyZo-hexoaehadIM Mo 0.25 and 3-deoxy-~-ribo-hexosehad Mo 0.79. Goodman and coworkers'" used basic lead acetate, in which S-deoxy-rwylo-hexme and the corresponding *rib0 homer have M R l b 0.00 and 0.64, respectively.

4. Gas-Liquid Chromatography The application of gas-liquid chromatography to the qualitative and quantitative analysis of carbohydrate derivatives has been increasing (330) M. 11.Wolfrom, D. L.Patin, and R.M. de Lederkremer, C h m . I d . (London), 1085 (1864).

(339s) G. R.Inglis, J . C b W g . , 90, 417 (1965). (340)A. B. Fmter, Aduan. CurbohgdrM C h . ,is, 81 (1857). (341) R. Piraa unnd E. Cabib, J . Chronmkw., 6.03 (1802).

D m X Y SUGAR8

201

steadily.u2 The deoxy sugars, as their glycosidesJWacetates,a” niethyl to ethers,u trimethylsilyl ethers,agama(d and a ~ e t a l s ‘ ~ ~are . ~ ’anienable *~~ separation by this very sensitive technique. The availability of preparative instruments now permits the separation of relatively large quantities of complex mixtures of deoxy sugars.

VII. NUCLEAR MAQNETIC RESONANCE SPECTROSCOPY Since the publication of the first application of nuclear magnetic resonance to carbohydratesJM it has become almost standard practice, in discussing many carbohydrate topics, to include nuclear magnetic resonance data. The subject has been r e v i e ~ e d . ~ ~ An attempt will be made in Table I11 to list the recorded chemical shifts and coupling constants of hydrogen atoms that are part of a deoxy function, and to show their effect on adjacent hydrogen atoms in the molecule. The examples are so selected as to give data on deoxy functions in various positions and environments in the molecule. In addition to the valuable information it provides, aa to the stemhemistry and configuration in general, nuclear magnetic resonance spectroscopy can be particularly useful in the differentiation of certain isomeric deoxy sugars. A typical example is in distinguishing between 6-deoxy- and 5-deoxy-hexofuranose derivatives.1mTable I11 provides nuclear magnetic resonance data for some representative, deoxy sugar derivatives.

VIII. MASSSPECTROMETRY This rapidly growing tool for structural studies in the field of carbohydrate chemistry has been the subject of excellent reviews and a book.w*3s1 Initial studies of carbohydrates by mass spectrometry were confined to the acetals, methyl ethers, and acetates of the common sugars, owing to limitations imposed by the involatility of samples of other derivatives. (342) C. T. Bishop, Advan. Carbohydrate C h . ,18,95 (1964). (343) H.G. Jones and M. B. Perry, Can. J . Chem., 40,1339 (1962). (344) S. W. Gunner, J. K. N. Jonae, and M. B. Perry, Can. J . Chem., 88,1982 (1961). (346) G.0.Aspinall, J . C h .SOC.,1676 (1963). (346)E.J. Hedgley and W. G. Overend, Chem. I d . (London), 378 (1960). (347) H. G.Jones, J. K. N. Jones, and M. B. Perry, Can. J . C h . ,40,1669 (1962). (348) R.U.Lemieux, R. .K. Kullnig, H. J. Bernstein, and W. G. Schneider,J . Am. C h . Soc., 80,6098 (1988). (349)L.D. Hall, Ahan. Carbohydrate Chem., 18,51 (1904). (360) (a) R. J. Ferrier and N. R. Williams, Chem. Id.(London), 1696 (1904).(b) H. Budrikiewica, C. U j e r d , and D. H. Williams, “Structure Elucidation of Natural Products by Mass Spectrometry,” Holden-Day Inc., San Francisco, Calif., 1964,Vol 11. (c) See N. K. Kochetkov and 0. S. Chizhov, This Volume, p. 39. (361) D. C.DeJongh and 8.Hanesaian, J . Am. Chem. Soc., 87,3744 (lass), and referencea cited therein.

8 i3

TABLE 111 Data* for Some h x y Sugar Derivatives

Nuclear Magnetic R-cc

Compound

1 2

3 -07 -7.60

3

7.9

3 4 5

5.98 (lm) 7.58(lm), 8.21 (lm) 5.59(1t)

4 5

8 -07(29)

J1.r

5.0, J1.t 1 . 9

56

5.55(1m)

6

6 -21

Methyl 5deoxy-2,50-isopropy~dent+ b ~ b O - h e x O f ~ ~ &

5 5

8.17 (2m)

3-0-Benzyl-Moxy-1 ,2-O-isopropylidene-

6

8.80(3d)

Js.8

5.9

137

6 D e o x y ~ h ~ ~ - m a n n hexaacetate ose

6

8.87 (3d)

J6.r

5.8

137

Methyl 6-deoxy-2,4-di-O-methyl-u-~

6

8.7l(3d)

Js.5

6.1

161

6

8.64 ( 3 4

J K .6.0 ~

161

S.ZO(2q)

142

p+idofuranoae

gslsctoside 6 anomer

5.28 (Id) 8.29,7.89 8 -72(3d)

8.18 (2m) 8.73(3d)

316

7.95(1q), 9.0-8.42(lq)

290

6.01 (It)

142

8.40(2m) 9.20(3m) 1 6

3.77 (lm) 8.16 (3d) . .

All spectra were recorded in deuterimhloroform at 60 Mc with tetramethylsilane as standard. *The numbera in psrentheses refer to the number of hydrogen &me, and the letters indicate.the multiplicity: s, singlet; d, doublet; t, triplet; q, quadruplet, m, multiplet. 8 In DP, with extemal standard.

;d

%

9 2

STEPHEN HANESSIAN

Nevertheless, from the fragmentation patterna of these volatile derivatives, useful information could be obtained as to their molecular weight, ring size, and position of substitution. The Bdeoxy sugars (as the acetates or acetals),= as well as two, isomeric 3,4dideoxyaldopcntoae dicctates,gba have beon studied. Whereas the use of the abovo protecting groups is desirable when combining *liquid chromatography with mass spectrometry, interpretation of the spectra is often complibted by the formation of intermediate, cyclic, fragmentation produots. With the advent of commercial availability of mass spectrometers equipped with inlet systems which allow the direct introduction of samples of relatively low volatility into the ion source, the use of carbohydrate dithioacelals has shown marked advantages in the study of structural aspects of various carbohydrates.8M The spectra are less complex than those of the cyclic derivatives, and yet they are very sensitive to structure differences. These characteristics have been displayed in the study of dithioacetals of common monosaccharide^,^^ and in assigning tho position of amino functions in dithioacetals of amino sugarsu1and their deoxy analogs.al+"M The deoxy sugar dithioacetals have also been found to be particularly amenable to such study." The position of the deoxy function in isomeric deoxy nupr dithioacetals can be located, thus providing a powerful tool for elucidation of their structure and for cbracterization. It is also gratifying that determinationof a mass spectrum requires less than a milligram of material. In general, fragmentation patterns are not affected by stereochemical differences, and it is reasonable to assume that stereoisomeric deoxy sugars fragment by the same general pathway, with minor differences in the relative intensities of certain peaks. Among the important aspects related to the use of these derivatives are the considerations that the dithioacetals can conveniently be obtained from the free sugars or their glycosides by mercaptalation, and t h t the same derivative can be used for the assignment of part of the configuration of the sugar by application of the MacDonald-Ficher degradations" (which provides the next lower aldose). Both structure and stereochemistry could, theoretically, be obtained from the same intermediate. The maes spectra of isomeric methyl 2-, 3-, and 4-deoxy-tri-0-methylD-hexopyrmosides have been reportedJaoaand provide a convenient method of examining some of the deoxy eugars aa pyranoeide derivatives. (362)K. Biemann, D. C. Ddongh, and H. K. Schnoee, J . Am. Chem. Soc., 86, 1783 (1963); 86, 87 (1964). (363) M. Venugopalan and C. B. Anderson, C h . Id.(London), 370 (1984). (364) (a) D. C. Ddongh, J . Am. C k a . Soc., 86,3149 (1964); (b) 86,4027 (1964); (c) J . Org. Chm., SO, 1663 (1906). (366) D. C. DeJongh and S. Hanesrrian, J . Am. C h .sbc. 87,1408 (1966). (360) D. C. Ddongh and 8.Hanessian, J. Am. C h . 8oc. 88,3114 (1966). Koohetkov, 0. S. Chirov, and B. M. Zoloterev, Dokl. A&&. Nuuk SSSR, (3604 166, 609 (1966); Cham. Abstracb, 64, 0738 (1966).

w.

205

DEOXY SUGARS

Five peaks are. important in the xuw spectra of the dialkyl dithioacetals of the deoxy sugarsm (as well aa of the common pentoses and hexoses). These are fragments A, B, and C, the dithioacetgl portion [CH(SR)r]@ reeulting from C-1, and the remaining portion of the molecule [M (CH(SR)a)@I.They result from carbon-carbon bondcleavage on electron impact, with the production of charged and neutral fragmente.

-

F’ragment A R OH, m/e 177 R - H , m/e 161

Fragment B

Fragment C R = OH, m/e 105 R = H , m/e 89

The major differences in the mass spectra of the deoxy sugar dithioaceti& can best be understood in terms of the apparent reluctance of their molecular ions to produce primary radicals (neutral fragments) when the molecules undergo bond cleavage. All of the deoxy sugars studied afford molecular ion peaks, thus allowing the direct determination of the molecular weight, M, from the spectra. For the 2-deoxy sugars, bond cleavage between C-1 and (2-2 is retarded probably because the neutral fragment produced a t C-2 is a primary radical. This makes 2-deoxy, 2 ,3-dideoxy, 2, Gdideoxy, etc., sugars especially easy to recognize. Fragments B and C are intense in their spectra, but the dithioacotal fragment, [CH(SC*Hs)2]*, is absent.

- -8 - c ciH@cA)a 7% - - - - Fragment C, m/e 89 CHOH Fragment A - -/-+ bHOH

-

m/e 195

I

FHOH CHaOH

The 3-deoxy and 3 ,&dideoxy sugars are characterized by the formation of Fragments A and B and the dithioacetal fragment in their spectra. m/e 1 9 5 - - - - - - I Fragment A, m/e 161

-- -

cH(sca&)a

CHOH

+IHoH I I

I-4

+--

- - - - Fragment c

208

STEPHEN HANnSSIAN

Fragment C is, however, minimal or abeent, since it would result in an unfavorable situation of a primary radical at G3. The 4-deoxy and 4,bdideoxy eugrtre are not cleaved predominantly between C-3 aqd C-4 for the same F8&Iw)n(a primary radical at C-4) ;they therefore provide low-intensity peaks for Fragment A. Fragments B and C and the dithioacetal fragment are, however, pmmt.

pwU*

m/e 1 3 5 - - - - - c Fragment A -

-/--LI O B - - --HOH +-

p

Fragment C, m/e 105

H

CH,

The 0-deoxy sugars studied (6-deoxy and 5deoxy) have intense peaks for the dithioacetal fragment, and ,also break down to Fragmenta A, B, and C. wDeoxyhexose dithioacetals cannot be differentiated from 5-deoxyhexose dithioacetals. The deoxy function is, apparently, too far removed from the bonds most susceptible to cleavage to have a significant effect. Mass spectrometry can also be useful in the characterip;ation of terminal branched-chain deoxy s u g r t r ~ The . ~ spectrum of 6-deoxy-5-C-methyl+ zyZo-hexose diethyl dithioacetal" contains prominent peaks due to Fragmenta A, B, and C. Apprtrently, substitution beyond C-4 does not affect Tmm IV MPU Spectrometric Data for Some h x y Sugar Methyl Mthioaoetal8~ Compound

ZDeoxyhexotw 3-Deoxyhexosed 6-Deoxyhexoee BDeoxyhexotw S-Deoxypentose 2, BDideoxyhexose 2,&Dideoxyhexoee 3 ,BDideoxyhexose 4 ,BDideoxyhexose

270 242 270 270 240

264 2b4 284 254

106 136 136 136

-

135 136

133 177 177 17f

-

101

-

B

C

147 147 147 147 117 131 131 131 131

89 106 105 106 89

106

See referenoe 3640. A dash indicates that thie fragment in either absent or of minor relative intensity. 'Dimethyl dithioacetel.

DEOXY BUQARS

207

their formation. An intern peak at m/e 59 can be ascribed to C-5 and its mbstituenh. 0

C&-C-OH

I CEI(

m/e 59

Pertinent data on the fragmentation of deoxy sugar dithioacetals are summarized in Table IV. It is possible to corroborate certain assignments in the above spectra by examination of the fragmentation patterns of dithioacetals containing different alkyl groups. Thus, in proceeding from diethyl to dipropyl dithioacetals, all fragments containing C-1 should appear a t 28 mass units higher, owing to the two methylene functions, but the M-C-1 fragment does not change. Fragments A and C also suffer changes accordingly. In conclusion, the mass spectra of deoxy sugar dithioacetals can be interpreted in terms of ( a ) the five peaks discussed, ( b ) peak shifts of other dialkyl dithioacetals, and ( c ) metastable ion peaks, m*.These are important in relating certain peaks, and in ascertaining that the corresponding fragments are formed as a result of electron-impact processes and not by thermal decomposition. Unsubstituted acetals and methyl glycosides of deoxy sugars have also been investigated by mass spectrometry.2'Da~ssT It is possible to differentiate between 3-, 5-, and 6-deoxy-l , 2-0-isopropylidenehexofuranosesfrom their mass spectra. The differences in the mass spectra of methyl 3- and 4deoxypentopyranosides are also sufficient for their characterization. In the spectra of 2- and 3-deoxypentofuranosides,however, the differences reside in the relative intensities of certain peaks and do not allow definitive distinctions. (357)

D.C. De Jongh, Abelracls Papere Am. Chem. SOC.Meeting, 161, 3 4 (1906). ~

This Page Intentionally Left Blank

COMPLEXES OF ALKALI METALS AND ALKALINE-EARTH METALS WITH CARBOHYDRATES BY J. A. RENDLEMAN, JR. N o r t h h g h a l Research Laboratory, N o r t h Utilization Rsearrch and Develqnnent Divieion, Agricultural Reaarrch deroiclr, U.S.Department of Agtidure, Peoria, IUitsoia

I. Introduction.. ....................................................... 209 11. Complexes of Carbohydrates with Metal Salts.. .......................... 211 1. Proof of Existence.. ................................................ 211 2. Adducta of Alkali Metal Salts and Alkeline-earth Metal Salts.. .......... 215 220 3. Methods of Preparation.. ........................................... 222 4. IStoichiometry ..................................................... 6. hlvation.. ....................................................... 226 -6.Influence of Size of the Cation on Stability of the Complex. . . . . . . . . . . . . . 227 7. Effect of Complexing on Optical Rotation.. ........................... 228 8. Electrophoreeis..................................................... 231 9. structure of the complex.. .......................................... 236 111. Complexw from the Interaction of Carbohydrates with Metal Bases.. . . . . . . 237 I. R$8ctions in Aqueous Media.. ....................................... 241 2. Reactions in Anhydrous, Alcoholic Media. ............................ 265 264 3. Resotione in Nonhydroxylic Solvents. ................................ 265 4. Structure of Alcoholatea and Adducts. ................................ IV. Alcoholaka from Reactions, in Liquid Ammonia, of Carbohydrates with Alkali Metals, Alkaline-earth Metals, and Alkali Metal Amidee.. . . . . . . . . . . . . . . 269

I. INTRODUCTION Published information on the complexes of alkali metals and alkalineearth metals with carbohydrates has often been vague and difficult to interpret, largely because (a) ions of these metals do not readily form stable complexes in aqueous solution, and (b) the study of such weak interactions is beset with many analytical difficulties. Although the detection of complex-formation in solution is relatively easy, the determination of stability constants has not yet been accomplished. In the present article, a11 attempt is made to produce some order from the available data. The presentation on the complexes of metal salts will be largely restricted to salts having univalent anions. Complexes containing nonmetallic cations, such as quaternary ammonium ions, will be mentioned only when such information is believed relevant to the discussion of complexes of alkali metals and alkaline-earth metals. The terms “adduct” and “addition compound” are synonymous, and will be used to describe those complexes consisting of carbohydrate and metal salt (or base) bound together, prob209

210

J. A. HENDLEMAN, JR.

ably, by ion-dipole forces of attraction. In those reactions where a hydroxylic proton is removed from a carbohydrate, the term “alcoholate” will be wed instead of “alkoxide,” to differentiate between the oxyanion of a carbohydrate and that of aamonohydric alcohol. In no instance will a “carbohydrate alcoholate” signify a carbohydrate solvated with an alcohol. Von Lippmanl reviewed much of the work on carbohydrate complexes published before 1904, and, for such complexes,Vogel end Georg2 bompiled a comprehensive table based on work published before 1930. It is important to note that many of the chemical formulas suggested by early investigators for compounds formed by the interaction of carbohydrates with metal bases were mere assumptions, based on insuiilcient evidence. Similar tendencies exist in the literature even today. Scant attention has been given by chemists to possible practical applications for carbohydrate complexes and complexing behavior. Differences in the solubility or stability of complexes can, in some instances, permit the largescale separation of one polyhydroxy compound from another.a.4 Differences in electrophoretic mobility of carbohydrates in the presence of a salt can be used to separate mixtures on a emall scale and to assist in identifying a carbohydrate. The ability of metal salts to complex selectively with polyhydroxy compounds has been the basis of certain separations of carbohydrates by column chromatographyf There have been no studies to determine whether the chemical properties of carbohydrate-aalt complexes differ significantly from those of the corresponding uncomplexed carbohydrates. Should such differences exist, they may be of great benefit to the preparative chemist. An understanding of the requirements for forming complexes of polyhydroxy compounds is eseential for the biologist studying processes of cellular transport. Closely ordered arrangements of donor groups in cell membranes may be responsible for the selectivity observed for certain metals in active transport. The effect of dietary f&ctom, especially sugars and amino acids, on the gastrointestinal absorption of calcium and other alkaline-earth motah R i well documented.&’ Charley and Saltmad have proposed that thc mcchunism by which sugars promote (1) E. 0. von Lippmann, “Die Chemie der Zuckerarten,” Friedrich Vieweg und 6ohn, Braunsahweig, 1904. (2) H. Vogel and A. Qeorg, “Tabellen der Zucker und Ihrer Derivate,” Julius Springer, Berlin, 1931, pp. 378-397. (3) A. J. Wattem, R. C. Horkeft, and C. 8. Hudson, J . Am. CAm. h., 66, 2100 (1934). (4) 8.Bey, 2.Kldn. Md.,38, 305 (1900). (6)J. K. N. Jones and R. A. Wall,Can. J . Cham., 36, 2290 (1960). (6) J. T. Irving, “Calcium Metabolism,” Methuen and Co., London, 1967. (7) P. Fournier, Compt. Rad., 248, 3744 (1859). (8) P.Fournier, B. Susbielle, and Y. Dupuis, C m p t . Rend., OM), 1111 (1980). (9) P. J. Charley and P. Saltman, rscisnce, 189, 1208 (1963).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

211

the absorption of calcium involves the fonnat.ion of a sugar-calcium complex in which the CaN ion is readily available for transport.

11. COMPLEXES OF CARBOHYDRATES WITHMETALSALTS 1. Proof of Existence The existence of carbohydrate-salt adducts had been tacitly assumed for many years, with little effort made to provide proof thereof. Helderman'O attempted to obtain an answer to this question by determining solubility curves, at 30°, for threecomponent system containing salt, water, and either sucrose or wglucose. Among the salts employed were sodium chloride, potassium chloride, and potassium sulfate. Unfortunately, a faulty experimental procedure led to the erroneous conclusion that adduct formation does not occur at this temperature. Later studies of the D-glucose-sodium chloride-water system by Matsuurall and Tegge,12 and of the sucrosesodium iodide-water system by Wiklund'a furnished proof of the existence

0

2

3 0

.c 0

25-

f

I

OO

25Wt.

%

50 of

NoCl

FIO.l.-Solubility Relations11 in the System D-Glucose--Sodium Chloride-Water at 24". (10) W. D. Xelderman, Arch. Suikerind. Ned. India, 98, 1701, 2306 (1920). (11) 8. MatBUura, Bull. C b . SOC.Japan, 9, 44 (1927); J . Chem. SOC. Japan, Pure Chem. Sect., 48, 247 (1928). (12) G. Tegge, Sfacrke, 14, 269 (1962). (13) 0. Wiklund, Zwker, 8, 266 (1956)

212

J. A. HENDLEMAN, J12.

2C,2 HZ2O,, 3NaI *3H20.

Not, moles/100 moles of H,O

FIQ.2.4k1lubility Relationsla in the Syetem Suckm-&xh ‘umIodide-Water at 30’.

of 2 D-glucose.I\iaC1-HzO,2 sucrose*3NaI-3 HtO, and sucrose.NaI.2 HnO. The diagrams in Figs. 1 and 2 show, respectively, the solubility relations in these two systems. They demonstrate not only the presence of sugar-salt adducts, but also how the formation of complexes affects the solubility of both the carbohydrate and the salt. have extensively studied the reTegge” and Lebedev and lation between temperature and the stability of 2 D-glucose*NaC1*HzO in aqueous systems, T h e fact that this adduct crystallizes better than pure D-glucose from aqueous solution is importmt industrially. &Glucose may be recovered from its adduct in 77.1% yield by agitating the adduct with water at 5’ for 1 hour.14 The sodium chloride diasolvee, and the D-glucose remains as a crystalline solid. Dialysis experiments16have shown that Cn”, M P , Ba2@,and Srm form mluble chelates in aqueous alkaline solution with D-galactose, D-glucose, D-fructose, D-arabinose, D-ribose, maltose, and lactose. The absence of any precipitation of alkaline-earth metal hydroxide when an aqueous solution containing Dfructose and an alkaline-earth metal salt is made alkaline (14) N. V. Lebedev, Sb. Tr. &a. Nauchn.-Iaulsd. Inut. Wrolizn. i Sd’jitno-Spirt. Pront., 8, 144, 202 (1980); E. L. C$lsakova and N. V. Lebedev, ibid., 8, 220, 235 (lSe0); 9, 90, 102, 110, 134 (1961); N. V. hbedev, A. A. Bannikova, and M. N. Lyuhotakayn, ihid., 8, 170, 186 (leS0); N. V. Lebedev and A. A. Bannikova, ibid., 8, 126 (1960); B, 81 (1961); 11, 68 (1963); N. V. Lebedev, B. 0.Lyubin, and A. A. Bannikova, ibid., 9, 70 (1961); Qidrolitn. i Lesokhim. Prom., 11, 3 (1968); N. V. Lebedev, B. 0. Lyubin, and E. L. Glaikova, Rutwian Pat. 121,088 (1969); Chem. Abelmcia, 64, 4012 (1960); N. V. Lehedev, M. N. Lyubetskaya, and A. A. Bannikova, Rumisian Pat. 136,836 (1961); Chem. Abutmtu, 66, 14962 (1961). (16) P. D. Saltman and P. J. Charley, Frenoh Pat. 1,827,727 (1963).

A L W I I AND ALKALINE-EARTH METAL COMPLEXES

213

(pH 12) is additional evidence16 that alkaline-earth metal hydroxides form complexes with carbohydrates. Similarly, calcium carbonate is not precipitated when carbon dioxide is passed through an aqueous solution of sucrose and calcium oxide (or hydroxide).” When an aqueous solution of sucrose and calcium oxide is added to an aqueous solution of sodium aluminate, there is formed a water-soluble complex containing sucrose, aluminum, and calcium.’8 Phase studied9 of the ternary systems sucrosewater-barium oxide and sucrose-water-strontium oxide have shown the existence of sucrose-BaO, sucrose-3 BaO, sucrose-SrO,and sucrose.2’SrQ. In a phase study of the ternary system sucrose-water-sodium carbonate,20 the complex sucrose*Na&Oswas shown to exist. However, a complex of sucrose with potassium carbonate has never been observed. The specific optical rotation of many sugars and sugar derivatives is altered by the presence of metal salts, the alteration being the greater, the greater the concentration of the salt. This phenomenon, which is now generally attributed to the formation of adducts, will be considered later in more detail. Other physical phenomena that may be associated, at least partially, with complex formation are the effect of a salt on the viscosity of aqueous solutions of a sugar and the effect of carbohydrates on the electrical conductivity of aqueous solutions of electrolytes. Measurements have been made of the increase in viscosity of aqueous sucrose solutions caused by the presence of potassium acetate, potassium chloride, potassium oxalate, and the potassium and calcium salt of 54x0-2-pyrrolidinecarboxylic acid.*’ Potassium acetate has a greater effect than potassium chloride, and calcium ion is more effective than potassium ion. Conductivities of 0.010.05 N aqueous solutions of potassium chloride, sodium chloride, potassium sulfate, sodium sulfate, sodium carbonate, potassium bicarbonate, potassium hydroxide, and sodium hydroxide, ammonium hydroxide, and calcium sulfate, in both the presence and absence of sucrose, have been determined by Se1ix.n At a sucrose concentration of 15” Brix (15.9 g. of sucrose/lOO ml. of solution), an increase of 1O Brix in sucrose causes a 4% decrease in conductivity. Landt and BodeaZsstudied dilute aqueous solutions of potassium chloride, sodium chloride, barium chloride, and tetra(16) P. J. Charley, B. Sarkar, C. F. Stitt, and P. D. Saltman, Biochim. Biophp. Acb, 88, 313 (1963). (17) .I. Dubourg, “Sucrerie de BetteravcR,” J. R . Builliere, Paris, 1952, pp. 179-181. (18) E. Calvet, H. Thibon, and R. Ugo, Bull. Soc. Chim. France, 1346 (1965). (19) K. Nishizawa and Y . Hachihama, 2.E l e k t r o c h . , 36, 385 (1929). (20):K. Nishwawa and M. Amagaaa, J . Soc. Chem. Id., Japan, S8, Suppl. binding, 497 (1933). (21) P. Naffa and C. Frege, Sum. Franc., 100, 179, 207 (1959). (22) M.Selii, Listy Cukrouar., 88, 151 (1949-50). (23) E. Landt and C. Bodea, 2.Ver. Deut. Zucker-Id., 81, 721 (1931).

214

J. A. HENDLEMAN, JR.

ethylammonium picrate containing high concentrations of sucrose (up to 670j0).They, too, found that an increase in the sugar concentration causes a decrease in conductivity. To explain the influence of sucrose on conductivity, a theory was proposed in which 13ucrom molecules are assumed to associate in solution. Association would incretm as the concentration of iucrose increases and as the temperature decreases. Ions traveling between these aggregates would meet with less opposition the smaller the ion. J. M. Stokes, R. H. Stokes, and coworkers~4extensively studied the limiting conductances of electrolytes and the limiting mobilities of ions in aqueous solutions of sucrose, wmannitol, pentaerythritol, glycerol, and the polysaccharide Ecoll (average molecular weight, about 1W). Although there was a clear correlation between the solution viscosity and the ionic mobilities, there was no simple quantitative relation between them. A similar observation was made by Landt and Bodea,N who found that ionic migration in sucrose solution ip,more rapid than that predicted by the solution viscosity. Both the size of the ion and the size of the polyhydroxy compound affect the conductivity, In general, emall ions, such as K@ and Cle, are less retarded by the presence of polyhydroxy molecules than are large ions, such as Ca*@or N(C4Ha)4",the latter approaching more nearly the classical hydrodynamic behavior predicted by Stokes' law or Walden's rule. The viscosity of ficoll solutions is several times that of solutions containing the same percentage by weight of sucrose or other simple polyhydroxy compounds; yet, the limiting conductances of small ions in all of these solutions differ only slightly. It wm concluded from these etudim that ficoll molecules form loose networks that are to a large extent permeable to small ions. Unfortunately, the possible contribution of complexing between polyhydraxy compounds and salts haa been almost completely ignored in theoreticd treatments of conductivity data. The results of a conductivity study on locust-bean gum (a neutral polysaccharide) in aqueous salt solutions led Btuy and Halseys to conclude that there is no interaction between this carbohydrate and the many ions employed (HaJ Na@,K@,Ag@,Barn, OAce, Cle, N O,', SOP, and HSOP). Hydroxide ion was an exception. The electrolyte concentrations ranged from 0.0005 to 0.1 N,and the maximum concentration of polysaccharide was 0.0277 M (on a monosaccharide-residue baeis). The specific conductivity of each polysaccharidegalt solution waa identical to that of the corresponding salt solution containing no polysaccharide, strongly indicating the absertce of significant adduct formation under the conditions (24) J. M. Stokes and R. H. Stokes, J . Phye. Chm., 60, 217 (1950); 62, 497 (1958); B. J. Steel, J. M. Rtokea,,and R. H.Stokee, iW., 62, 1614 (1958); F. J. Kelly, R. Mille, and J. M. Stokoe, ibid., 64, 1448 ( I N ) ; R.H. Btokea and I. A. Weeks, Auelralian J . C'henr., 17, 304 (1864). (26) J. A. Barry and C. D.Bsbey, J . Phye. Cham., 67, 1698 (1883).

ALKALI AWD ALKALINE-EARTH METAL COMPLEXES

215

employed. Had it been possible to use higher concentrations of carbohydrate, mme complexing might, perhaps, have been observed. However, the physical limitations of low solubility and high viscosity made it impractical to exceed a polymer concentration of 0.5y0by weight. The isolation of crystals having a constant composition constitutes good evidence of adduct formation. Crystals of sucrose*NaI*2H20, several centimeters in length and of constant composition, have been isolated.16 Were adduct formation not actually involved, sucrose and sodium iodide would have been precipitated separately. In dilute ethanolic solutions of alkali metal salts, many simple carbohydrates unite with the salt to give an isolable crystalline solid of either 1 :1 or 2: 1 ratio of carbohydrate to salt. The frequency with which these ratios are obtained argues not only for the existence of true adducts, but also for a generally preferred stoichiometry at low concentration of salt. Traces of sodium ion in samples of potassium bromide and potassium iodide used in preparing pressed discs for infrared analysis can alter the normal spectrum of D-glucose, This alteration has been attributed* to the formation of NaBr - 2 D-glucose and NaI-2 D-glucose. The normal spectra of D-xylose, D-sorbose, and sucrose are also altered by the presence of sodium ion. However, alteration of spectra is not always predictable; lactose, cellobiose, mmannitol, and erythritol give normal spectra in potassium bromide discs containing trace amounts of sodium. Tipson and Isbelln have found that the spectra of certain aldoses and alkyl glycosides in Nujol mulls differ from the corresponding spectra in discs of potassium iodide. If such behavior can be shown to be due to the formation of a complex with the metal salt, detection of complex formation by means of infrared spectra may be a possibility. Lactose, sucrose, maltose, wgalactose, Dfructose, and many other carbohydrates are highly soluble in absolute methanol containing sufficient calcium chloride.* This high solubility strongly suggests the formation of carbohydratecalcium chloride complexes. Lactose-CaC12.4 MeOH crystallizes slowly from concentrated solutions of lactose and calcium chloride in methanol. Alcoholic salt solutions might, therefore, serve as solvents for sugar reactions, and for fractionating sugar mixtures by extraction or Crystallization. 2. Adducte of Alkali Metal Salts and Alkaline-earth Metal Salts

Tltblc! I rccords most of the known, isolated adducts of alkali metal salt8 rtnd alkaline-earth metal salts with polyhydroxy compounds. A (26)D.Gauthier, Compt. Rend., 198, 638 (1904). (27) V. C.Fsrrncr, C'hem. I d . (London), 1306 (1959). (27t~)R.S.Tipuon aiid IT. 9. Isbell, J . Res.Natl.Rur.Std.,64A,239(1960);66A,31(1962).

Tmm I Adducts of Alkali Metal Salts and Alkaliue-earth Metal %Its Carbohydrate (ligand)

salt

potsssium bromide potassium formate potassium iodide potsssium propionate Calcmmchloride ealciumchloride

Molar ratio,. l@md:salt

Solvent of solvation, molecdes/cation

Solvent

References

medium*

29

1:l 2: 1 2:l 2:l

29 29 29 29 29 30

2:l 1:l l:1 1:l 2:l

31 31

2:3

6.3 EX)

32

barium iodide

calcium bromide

2:l 1:1

2 Hto 4 Hto

ealcilrmchloride

2:l

2

33 33 33

2: 1 2: 1 2: 1 2:1

4 MeOH 2 HzO

3 HI0 3 HZO

1:l

none

1:l

m

28

33 33

33 EtOH

34

EtQH

3

P

tn.4

c

2

E* g

2

i;:

calcium chloride

1:1

3 H20

calcium chloride p0tasS;uumacetate sodium bromide

1:l 2: 1 2: 1 2: 1 2: 1 2: 1 2:1

? MeOH

sodium chloride sodium fluoride sodium fomte. sodium iodide

none HZO 0 . 1 EtOH

HO

H20

35

MeOH Py-EtOH-EtOAC H20 H20 EtOH EtOH HZO

36 37 38 39 36

2: 1 2:l 2: 1 2: 1

HO NMP-EtOH-Et& EtOH ;H&EtOH HtO EtOH

@-D-Clucopyranoside, methyl

potsssium acetate.

1:1

D-Gulose

calcium chloride

2: 1

a-D-culose

calcium chloride

1:l

a-DGulopyranoside, methyl

calcium chloride

1:l 2: 1 1:l 2:1

2 HZO 3H O 2 H20 none

1:l

H2O

2: 1

2 HtO

HtO

H&EtOH

28

11, 12, 14, 38, 40 38

36 3641

38 3,36

42

HtO

42s

Hz0 H&EtOH HtO-EtOH EtOH

43

HzO

43a

HzO-EtOH

43s

c tiU

*

E

1 P 3X

B 0

/3

anomer

calcium chloride

Heptopyranoside, ~ - g Z y c e t o a - ~ calcium chloride gulo-, methyl B anomer calcium chloride

0

43

$

43 43

k2

E l

E?

K

TABLE I (Cdinucd) Adducts of Alkali Metal Salts and Alkaline-earth Metal Salts Carbohydrate (ligand)

Salt

Heptose, ~ - ~ L p m - ~ ~ w g t z k cdcium b chloride

Molar ratio," ligand 4t

Solvent of solvation, molecules/ca tion

Solvent

References

mediumb

1:l 1:l

3 HrO 4 Hto

HrO-EtOH H&EtDH

44

45

44

Heptose, D s l l J w o - & ~

calcium chloride

1:1

2HP

H&-EtOH

htae

calcium chloride

1:l

7 HzO

HrO ;H&MeOH

28,46

a-wlyxopyranoside, methyl

d c i u m chloride

1:l

2HP 4 MeOH

Hfi-C&OH

43a 28

d c i u m chloride

2 Hto

d u m iodide

1:l 1:1 l:1 1:l

%Propanone, 1,3-dihydroxy-

sodium chloride

2: 1

Lrsorbosi?

calcium cblonde

sucrose

barium bromide barium chloride barium iodide

f-.

?

E2 W

potsasium acetate

1:1

MeOH

HeEtQH

47 47

EtDH-EW EtOH-EW

36 36

none

H20

48

1:l

2 HrO

Hto

49

2: 1 2:1 2: 1

-

HP

504 50" 50'

4H t o

0.2 EtOH

-

-

Ha

Hso Hto

E

5 -2: -

.;;

barium thiocyanate calcium bromide calcium iodide lithium bromide lithium chloride lithium iodide potassium acetate

2 HtO 3 HzO 3 HtO 2 HzO 2 HtO 2 H20 -

sodium propionate sodium thiosulfate strontium chloride strontium bromide

1:l 1:l 1:1 1:1 1:1 1:l 2: 1 1:2 1:1 1:l 1:l 1:l 1:l 1:1 1:l 1:l 2:3 2: 1 1:l 1:1 1:l

HzO 3 H20 3 HzO

HtO HtO HtO

a-D-Xylose

calcium chloride

1:1

3 HzO

H20

31

SD-Xylopyranoside, mcthyl

potassium acetate

1:l

-

EtOH

3

potassium iodide potasaium thiocyanate sodium acetate sodium bromide sodium carbonate sodium chloride sodium iodide

2 HtO HZ0

2 HzO

none 2 H20 none 2 HtO HtO

-

HtO HtO HIO HtO H20 H20 NMP-EtOH-EhO N MP-EtOH-EhO HZO H2O HoO HoO HzO

HtO HLeEtOH HnO

N MP-EtOH-EttO

a The ligand in an amylose adduct is the wglucose residue. In other adducts, it is the entire carbohydrate molecule. * Py = Zpyrrolidinone; NMP = N-methyl-Zpyrrolidinone; EtOH = ethanol; MeOH = methanol; EtOAc = ethyl acetate; EhO = ethyl ether. Gauthier did not offer any analytical data. Therefore, those results not corroborated by other investigators should be viewed with some skepticism.

Footnotes for Table I are located at the bottom of page 220.

d

0

5

F=

2!

r?

220

J. A. RENDLEMAN,

JR.

number of adducts reported in the literature have been intentionally omitted, because of either tho unreliabitity of published data or the lack of sufficient information on the combining ratio. Many investigators have neglected the possibility that their “products” were not true adducts, but actually mixtures of separate components. The water of hydration bound to carbohydrate adducts isolated from aqueous media is ordinarily found by subtracting the combined weight of the carbohydrate and the salt, as determined by analysis, from the total weight of a sample of the adduct. Many adducts prepared in, or recrystallized from, alcoholic media (anhydrous or aqueous) have never been analyzed for alcohol of solvation. 3, Methods of Preparation

More preparations, and attempted preparations, have been carried out in water than in any other solvent. Aqueous alcoholic media have been used occasionally. The low solubility of many carbohydrates and salts in alcohols and most other organic solvents is, perhaps, the reason why most investigators have generally avoided nonaqueous media. (29) F.R.Senti and L. P. Witnrruer, J . Polymer Sci., 9, 116 (1962). (30) W. C.Austin and J. P. Walsh, J . Am. Chem. Soc., 60,934 (1934). (31) J. K.Dale, J . Am. C h m . Soc., 60,932 (1934). (32)A. Hybl, R. E. Rundle, and D. E. William, J . Am. C h .Soc., e7, 2779 (1966). (33) R.H. Smith pnd B. Tollena, Ber., 88, 1277 (1900). (34)R. Kuhn, H.’Baer, and A. Gsuhe, Chcm. Bet., 88, 1136 (1956). (36) R. M. Hann and C. S. Hudson, J . Am. C h m . doc., 69,2076 (1937). (36) J. A. Rendleman, Jr., J . Org. Chcm., 81, 1889 (1908). (37) J. Stenhouse, Ann., 119, 286 (1884). (38)H. Traube, N e w s Juhrb. Minenrl. Gml., Beilage Bd. VIII, 610 (1893). (39) M. Hlrnig and M. Rosenfeld, Ber., 10, 871 (1877). (40) Q. Tegge and W. Kempf, Stosrke, 10, 103 (1968). (41) J. A. WtUng, German Pat. 196,sOa (1907);Chum. Zenfr., 79, I, 1688 (1908). (42)H. [J. Isbell and W. W.Pigman, J . Re8. Natl. Bur. dfd., 18, 141 (1937). (42a) H.8.Iabell, Bur. Srccndarde J . Reeearch 6,741 (1930). (43)H. 8.Isbell, Bur. Stundurda J . Raeurch, 8, 1 (1932). (43a) H.8.Isbell and H. L. Fruah, J . Rse. Nu& Bur. W., 94, 126 (1940). (44)E.M, Montgomery and C. 8.Hudson, J . Am. Chem. 8oc., 04, 247 (1942). (46)H. S.Isbell and H. L. Fruah, J . &a. Nutl. Bur. dld., 81, 163 (1943). (46)B.L. Herringtan, J . Dairy Sci., 17, 805 (1934). (47) J. K.Dale, J . Am. Chem. Sot., 61,2788 (1929). (48)L. M. Utkin, Biokhimiyu, 4, 800 (1939). (49) R.L.Whistler and R. M. Hixon, J . Am. Chcm. Soc., 60,‘728(1938). (50) D. Gauthier, Compt. Rend., 187, 1269 (1903). (Sl) T. M h d y and G. Vavrinecs, Zucker, 11, 648 (1968). (S2) W.Cockran, Nature, 157, 231 (1946). (63) C.H. Gill, J . C h .doc., 14, 269 (1871);Ber., 4,417 (1871). (64) F.H.C.Kelly, Znlem. Sugur J., 66, 128 (1966).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

221

a. Aqueous and Aqueous Alcoholic Media.-The usual procedure for preparing adducts of carbohydrates no larger than oligosaccharides is to evaporate, slowly, a solution containing carbohydrate and salt in the molar ratio approximating the ratio expected in the desired adduct. The length of time required for crystallization to commence is often great for purely aqueous solutions, and depends upon the temperature, rate of evaporation, rate of nucleus formation, stirring, and adduct solubility. The presence of ethanol in the medium facilitates crystallization. In the absence of ethanol, the time to commencement of crystallization may vary from 1 day to several years. Use of molar ratios of carbohydrate to salt either greater or lower than the ratio expected in the adduct being prepared sometimes leads to undesirable results. Adducts having more than one combining ratio are possible, particularly with carbohydrates of high molecular weight (oligosaccharides and polysaccharides) Furthermore, at certain ratios of carbohydrate to salt, it is possible for either the carbohydrate or the salt to begin precipitating prior to precipitation of the adduct. This could lead to erroneous conclusions concerning the actual composition of the adduct. An example of this possibility occurs in the work of Sharkov and Guzhavina,” who reported that D-glucose combines with potassium iodide, lithium chloride, lithium bromide, or lithium iodide, respectively, in the remarkably high ratio of 4:l. The ratios for the adducts of the lithium salts, particularly, are unrempable, because the surface area of the lithium ion is much too small to accommodate 4 multidonor ligands. After precipitation, many adducts may be washed with ethanol, and allowed to dry at room temperature. However, consideration must be given to the fact that, if contact of the adduct with the washing solvent is unduly prolonged, washing with ethanol can lead to a reduction in the number of molecules of hydration and, possibly, to a change in the molar ratio of carbohydrate to salt. There is very little information on the preparation of polysaccharide adducts. Senti and Witnauerm have reported the only polysaccharide adducts of a definite stoichiometric type. Their method consisted of equilibrating the alkali metal hydroxide adduct (see Section 111) of the polysacchaxide with the proper salt in 20-25% aqueous ethanolic medium. Absorption of salt, with concurrent displacement of hydroxide, increases with increase in concentration of the salt in the exchange medium. Above certain concentrations of the salt, absorption can become virtually independent of the concentration , indicating that maximum absorption has been attained and that a “stable” adduct of definite stoichiometric compo-

.

(66) V. I. Sharkov and V. Guzhavina, Zh. Prikl. Khdm., 81, 1759 (1958).

222

J. A . HINDLEMAN, JIL

sition has bcori formd. There are indications that varying the salt concentration w y , in some instances, permit the imlation of more than one “stable” adduct. Tho term “stable” is used loosely, and describes adducts whose combining ratios vary only slightly over a rather wide range of salt concentration. Neutraliaation of a potassium hydroxide adduct of amylose with gweous carbon dioxide leads to a potaseium hydrogen carbonate adduct?# This type of reaction,which is rapid in a moist atmosphere, could possibly be used in the preparation of bicarbonate adducts of other carbohydrates.

b. Anhydrous Alcoholic Media ,-Only the simple carbohydrates, including oligosaccharides,have yet been studied in anhydrous media. The best procedure to use for preparing adducts is determined largely by the respective solubility of carbohydrate, salt, and adduct; thus, no specific procedure can be outlined that would be generally applicable. The simple addition of an alcoholic salt solution to an alcoholic carbohydrate solution might be all that would be necessary to effect precipitation of an adduct. Ethanol is better than methanol as a preparative medium, because of the lower solubility, and perhaps greater stability, of the adduct in the former. Addition of ether to an ethanolic solution of oarbohydrate and salt facilitates precipitation; however, the addition must be cautious, to prevent total precipitation. The laotams Zpyrrolidinone and N-methyl-2-pyrrolidinone may be used to incresse the solubdity of Carbohydrates in alcoholic reaction media. However, they cannot be used in the preparation of metal halide adducts, because of their tendency to associate, perhaps by complexing, with metal halide adducts.” Amides are known to form complexes with metal halides. For example, sddium iodide combines with N,Ndimethylformamide~to give NaI.3 N,Ndimethylformamide. Acetamide ctm form NaBr.2 acetamide and Na1.2 acetamide.“ T h e stoichiometry in anhydrous alcoholic media, aa in aqueous media, is variable. The combining ratio for a “stable” adduct prepared at a low concentration of salt may often differ from the ratio obtained at a much higher concentration. The relationship between combining ratio and salt concentration will now be discussed in more detail. 4. Stoichiometry a. Combining Ratioe in Iaolated Adducte.-Information on the stoichiometry of carbohydrate-salt interactions is based largely upon the (66)Y.Gobillon, P.Piret, and M. van MeBresohe, BuU. 8m.Chim. F r a w , MI1 (1962); (67)

206 (1963). Y.Gobillon and P.Piret, A&

Cjyst., 16,

1186 (1962).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

223

cmmpoeition of the complexes iwolated. The most frequently reported conibilling ratios of carbohydrate to salt are 1:l and 2:l. Other ratios are possible for certain carbohydrates, Ratios for adducts thus far prepared in aqueous media have not exceeded 2: 1. The requirements for the formation of a carbohydrate-salt adduct are those for chelation between a metal ion and a multidonor molecule. A group of two,or more, properly oriented hydroxyl groups, or a coqbination of a carbonyl group with one, or more, properly oriented hydroxyl groups is necessary. There has been no indication that the oxygen atom of an ether linkage (or of the glycosidic hemiacetal linkage) can participate as an electron donor and, therefore, serve to bind a cation. That bonding of an alkali metal cation to an alkoxyl group is possible, and may contribute, at least weakly, to chelate stability is shown by the existence of intermolecular bondinp of the type 0-Li. * -0 between molecules of lithium alkoxide in such organic solvents as ether, hexane, and p-dioxane. The greater the number of donor groups in a carbohydrate, the greater is the probability that more than one metal cation can become attached to a single donor molecule to form a po2gcation adduct. Although monosaccharides have not been shown to form polycation adducts, oligosaccharides and polysaccharides associate with two or more cations with great facility. High concentrations of salt favor high ratios of salt to carbohydrate. Ethanolic or aqueous ethanolic media favor high ratios of salt to saccharide in' oligosaccharide adducts. For example, aucmse.2 KOAc can be isolateda by adding ether to an ethanolic solution of sucrose (0.02 M ) and potassium acetate (0.4 M ). Although Mackenzie and Quinsg reported the preparation of sucrose*KCl*HgO,KellyMhas questioned its existence (as an isolable compound) after finding that sucrose and potassium chloride crystallize, separately and simultaneously, from aqueous solution. Gills3was unable to isolate any adducts of sucrose with lithium chloride, lithium iodide, potassium chloride, potassium bromide, or potassium iodide, respectively; with ammonium chloride and ammonium iodide, deliquescent crystals of variable composition were obtained. Much later, however, Wiklundls succeeded in preparing sucrose.KI.2 HaO; and GauthieldO reported the preparation of adducts of sucrose with lithium chloride, lithium bromide, and lithium iodide. Polarimetric studies of the reaction in (homogeneous) aqueous solution have definitely shown that sucrose combines with alkali metal ions in preferred stoichiometric ratio (see Section II,4b, p. 224) Thus, inability to isolate a particular adduct does not prove that the (58) T. V. Talalaeva, G. V. Taareva, A. P. Simonov, and K. A. Kocheshkov, B d . A d . Sci. USSR, DW.Cliem. Sci. (English Trend.), 696 (1964). (59) J. E. Mackenzie and J. P. Quin, J . Chem. Soc., 961 (1929).

224

J. A. RENDLEMAN, JR.

adduct does not exist. Stability, solubility, and ease of crystallization or precipitation of an adduct can vary widely according to the preparative conditions. Senti and WitnaueP have provided the only information yet available on the stoiohiometry of formation of polysacoharide adducts. Their studies of addition compounds of amylose in aqueous ethanolic media showed that the combining ratio of D-glucose residue to sslt is a function of salt concentration, and that the minimum ratios are approached as the sal! concentratidn is i n c r d . Beyond a certain concentration of salt, the ratio becomes almost constant. The anion plays an important role in determining the magnitude of the minimum ratio for an amybse adduct. Potassium bromide and potassium iodide give adducts of minimum ratio 2: 1, whereas potassium acetate and potassium propionate give 1:1 adducts. A study of the composition of the potassium acetate adduct as a function of salt concentration indicated that two, relatively stable adducts axe formed, the 1 : l and the2:l.

b. Stoichiometry in Homogeneous Systems.-The phase rule is not applicable to adduct formation in homogeneous systems. It is useful only for heterogeneous systems containing multiple phases, at least one of which consists of a pure adduct. Failure to observe a phase for an adduct does not n e c e d y signify the nonexistence of the adduct; it could mean that, under the experimental conditions employed, the solubility characteristics of the adduct permit the existence of a metastable solution. This could lead to a solubility curve whose shape would indicate the absence of complex-formation. For homogeneous systems, combining ratios are difficult to determine. The exceptionally low stability of adducts in dilute solution and the rapidly reversible nature of carbohydrate-salt interactions have, thus far, precluded succeeaful electrometric analyses. WiklwxV8 and Ramaiah and Vishnu,dO by applying the method of Job" to solutions containing sucrose and alkali metal salts, have determined the favored combining ratios for various carbohydratesalt adducts in (homogeneous) aqueous solution. The principle of this method is aa follows. If equimolar solutions of two complexing solutes are mixed in different proportione, the concentration of the complex is generally a function of the proportion in which the solutions have been mixed. The Concentration of the oomplex is maximal when the solutions are mixed in the same proportion as that in which the simple componpnts are present in the complex. The position of the maximum is a function of the molar ratio of salt to complexing agent, and is independent (60) N.A. Ramsiah and Viehnu, 19hrkma, 9,3 (1959). (61) P.Job, Ann. Chim. (Pd),0, 113 (10%).

ALKALI AND ALKALINE-EANTH METAL COMPLEXES

225

of both thc! combined concentration and the concentration of the original dutione (prior to mixing). To make use of the above principle, the complex should possess a measurable property that is unlike the corresponding property of its individual components. This is not always feasible, since, frequently, one of the components has properties very similar to those of the complex. Wiklund13 and Ramaiah and VishnusDmeasured optical rotation. Actually, they measured the combined activity of free sucrose and complexed sucrose in the presence of alkali metal salt. The rotations of these two forms were assumed to be additive. The experimental method consisted simply of mixing equimolar solutions in different proportions, and then measuring the optical rotation a for each mixture. The difference Aci = a - aro (where q,is the rotation for the same solution containing no salt) is largest when the solutions are mixed in the proportion in which the individual components are found in the actual complex. Fig. 3 shows plots of Aa against molar ratio of salt, for various sucrose-salt solutions, each of which has a combined concentration of 1 mole per liter of solution. Other studies

3 Mole fraction of sucrose

FIG. 3.-Effect of Salts on the Optical Rotrttion of Sucrose in Aqueous Solution." (Combined coucentrstion of sucrose and salt is 1 M.)

a26

J. A. RENDLEMAN, JR.

showed that the position of maximum Aa changes very little, and in some c w s not at all, when the combined concentration is raised to 2 moles per liter. The magnitude of the maximum Aa is an approximately linear function of the product of salt concentration and sucrose concentration, up to a combined concentration of 4 moles per liter. Although some of the curves are not symmetrical, their maxima lie between 0.5 and 0.6 molar ratio. The date thus provided good evidence for the existence of preponderanily 1:1 sucrowalkali metal salt adduct in aqueous solutions of low to moderate concentration of salt. Similar studies of D-fructose with alkali metal salts have given identical resu1ts.m In anhydrous ethanolic media,@potassium acetate and methyl BDglucopyranoside have been shown polarimetrically to combine in the ratio of 1:1.This ratio is ale0 found in the adduct isolated. Application of Job's principlew to aqueous solutions of sucrose and Itlkalineearth metal salts (magnesium sulfate, calcium acetate, and barium chloride), whose combined concentration waa 1 M, gave curves of Aa against concentration that exhibited two maxima, one at 0.5 M sucrose and the other at 0.66 M sucrose. These observations suggest that both 1:1 and 2: 1 carbohydrat-alt adducts can exist in solution. 5. Solvation

Adducts prepared in aqueous media generdy possess one or more molecules of water of hydration per molecule, the number being a function of cation, anion, and the combining ratio of carbbhydrate to salt. Available data on complexes of simple carbohydrates indicate that three molecules of water per molecule may be the maximum for adducts of alkali metal salts; w many as seven have been reported for those of the alkaline-earth metal salts. Most complexes, however, possese only one or two molecules per molecule. Generally, the higher the combining ratio, the smaller is the number of water molecules that can be accommodated by a molecule of the adduct. In the crystalline complex, solvent molecules can be bonded not only to the cation, but also to the anion end the carbohydrate. A detailed x-ray study has mown that, in sucrose-NaBr.2 each BF ion has bonds to one water molecule, one Nae ion, and four carbohydrate hydmxyl groups.'J2 The ability of a complex to possess solvent of crystallization, even when the free individual components themselves are incapable of doing so under the same conditioriu of temporature, is exemplified by the formation of sucrose~NaCl*2HP at mom temperature. SucroRe and sodium chloride crystallizc from their aqueourJ soliitiotiR in the nonhydmtd form.

Ha,

(62)

W.Cochm, Nature, 167, 87'2 (1V46); C. A. Beover8 and W.Coahran, PTOC. Rog. Soc, (London), Ber. A , 100, 267 (1947).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

227

Adducts of alkali metd salts prepared in anhydrous alcoholic media generally retain very little alcohol of solvation after being dried under vacuum at mom temperature (see Table I). The unusual ability of adducts of Dglucitol to retain alcohol is probably due largely to the great ability of Dglucitol itself to retain solvent. Adducts of alkalineearth metal salts, however, are more strongly solvated by alcohol than adducts of alkali metal salts. For example,28lactose*CaCla*4MeOH is relatively stable at 60" at atmospheric pressure; under vacuum ( < 19 mm. of Hg) , a molecule releases only two of the four molecules of methanol. From aqueous alcoholic media, adducts of alkaline-earth metal salts tend to crystallixe as hydrates.

6. Influence of Size of the Cation on Stability of the Complex Studies of carbohydrate complexes have not yet provided sufficient information to permit the determination of reliable relative reactivities of carbohydrates toward alkali metal ions and alkaline-earth metal ions. Relative electrophoretic mobilities of carbohydrates are a crude, and perhaps unreliable, index of relative reactivity, because the assumption must be made that the mobility is proportional only to the stability of the complex, which is not strictly true. Ease of adduct isolation and the ebulliometric behdvior 'of solutions containing both a carbohydrate and a salt are two other criteria that may be wed to judge reactivity. However, inability to effect precipitation of a complex need not be due entirely to instability of the complex. The order of decreasing ease of isolation (precipitation) is generally Na@> K@> LP. The same order is obtained for the decreasing effectiveness of these ions to promote electrophoretic migration of a carbohydrate. Ebulliometric studieaae on ethanolic solutions of carbohydrates and alkali metal salts have indicated that Na@and Ke ions have very similar reactivities, and that, at salt concentrations of 0.1 M and greater, the percentage of a carbohydrate in the complex form can be quite high. For example, in an ethanolic solution that is 0.0639 molal with respect to D-glucose and 0.1489 molal with respect to potassium iodide, the apparent fraction of mglucose in the complex form is 42%. Only carbohydrates having two or more hydroxyl groups in close proximity to each other show any tendency to complex. The Lie ion was found to possess an immeasurably small reactivity in boiling ethanolic solution. In aqueous solution, the order of decreasing effectiveness in promoting electrophoretic migration of carbohydrateseais (Ca*, Sr@,Ba*@)> Mg2@> Na@> K@> Lie > NHP. No migration occurs in the presence of tetramethylammonium ion. In methanol,sethe order is Caz@> Na@> K@> (63) J. A. Mills, Riockm. Biophys. Res. Commun., 6, 418 (1961/1962).

228

J. A. RENDLEMAN, JR.

PiH$ > Li*.In both solvents, the ions of alkaline-earth metals appear to be more reactive than those of the alkali metals. The ineffectivenesswith which Lie ions complex with carbohydrates can possibly be attributed to the tightly bound sphere of solvation surrounding the ion. The degree of solvation of alkali metal ions in solution docroltsed4 However, the apparently with increasing atomic weight: Lie > NrP > P. greater ability of Na*, relative to K*, to complex with carbohydrates in aqueous media requires a different explanation. Here,the shorter ionic radius of Nae is probably a more important factor than the sphere of solvation. In general, the strength of a bond between a donor molecule and a metal ion is greater, the shorter the radius and the greater the charge on the ion. The apparently greater stability of alkalineearth.metal complexes, relative to alkali metal complexes, is probably due largely to the double charge on alkaline-earth metal ions. The M g a ion differs appreciably from the other alkaline-earth metal ions in electrophoretic behavior, possibly for +b r e m n offered above for the behavior of Lie. In alcoholic solution, carbohydrates possibly complex with undissociated molecules (ion pairs) of salt, as well &B with free cations. Data from optical rotation experiments1ssuggest that, even in aqueous solution, undiasociated molecules of salt may be associated with the carbohydrate (see Section 11,7, p. 230), On the other hand,free aniona do not appear to complex with carbohydrates in solution (see Section 11, 8, p. 234). 7. Effect of Complexing on Optical Rotation

The ability of a dissolved salt to alter the optical rotation of a carbohydrate is now generally believed to be due to complex-formation. However, rotational phenomena have been largely ignored because of the many, often insurmountable, difficulties that can be encountered in their study. Among the factors that require consideration are multiplicity of donor groups on a polyhydroxy ligand, variability of combining ratio, stereochemical conformation and configuration, and the nature of both the mion and the cation. Because the weakness of the chelate bond has, thus far, rendered impractical a quantitative study of complex stability and equilibria in solution, there ie as yet no obvious means of relating a change in optical rotation to tho extent of complex-formation. Vavrineezmhas shown that the optical rotation of tmcrose depends upon the concentration of both tho Rugar and the salt. Empirical relationships for the chlorides, bromides, iodides, mid scetates of sodium arid potassium were determined. (64)R.Flatt and F. Benguerel, Helu. Chim. Ach, 46, 1777 (1882). (65) G. Vsvrineoe, 2.Zwkm'd., 12,261 (1962).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

229

Ramaiah and VishnuMhave, perhaps, come closest to determining the rotation of a pure complex. Working with aqueous solutions of sucrose and of D-fructose, they found that, at a high concentration of alkali metal salt and at a high ratio of salt to sugar (3.5 moles of salt to 40 mmoles of sugar per liter of solution), the addition of more salt to the solutions caused no further change in rotation. This observation was taken as evidence that all of the sugar had been complexed. The rotation was dependeut upon both the cation and the anion. The effect of the cation upon the rotation decreased in the order of Nae > Ke > Li@;that of the anion decreased in the order of Ie > Bre M Cle. The specific rotations for sucrose and TABLE I1 Specific Rotations of Suoroee and of D-Fructoee in 4 M Solutionsof Alkali Metal Salts in Watep

Salt

None Lithium chloride Sodium chloride Sodium bromide Sodium iodide hdium acetate Potsssium chloride Potassium bromide Potsasium iodide Potassium acetate a

From data of RBmaiah and Vishnu."

+66.5

-87.7

+63.6

-97.8

+65 .o

+61.7 +68.4 +61.5 +63.9 +62.1 +58.8 +62.1 b

-

-97.1 -98.5 -99.9

-

Concentration of sugar, 0.04 M.

D-fructose in 4 M solutions of alkali metal salts are given in Table 11. Unfortunately, a quantitative interpretation of these data is impossible without knowledge of the combining ratios and of the structures of the adducts. It is quite possible that more than one ion or ion pair is attached to a sugar moleaule at the high concentrations of salt employed. The effect of salt, ooncentratiom above 5 M on the optical rotation of sucrose was not Rtudied by llsmuinh a i d Vixhnu.RoTherefore, although the sperific rotation R i it~vttriant~ over the range of 3.5 to about 5 A4 salt concentration, it may not be illvariant at higher concentrations. Bigelow and Geschwind67 (66) N. A. Ramaiah and Vishnu, Sharkara, 2, 56 (1959). (67) C. C. Bigelow arid I. I. Geschwind, Compt. Rend. Trav. Lab. Carlaberg, 19, 89 (1961).

230

J. A. RENDLEUN, JR.

have reported that the specific rotation of sucrose in 7.5 M lithium bromide solution is much lower than that in 5.1 Y lithium bromide solution. It is clearly evident from the work of Wiklund” (see Fig. 3) and of Ramdah and Vishnua* that, even at low concentrations of salt, both the cation and the anion affect the optical rotation. Wiklundl*found, at low salt concentration, essentially the same order of decreasing effectiveqess that Ramaiah and VishnuM found at high concentration, namely: Na* > Ke; and I* > BB > OAce > Cle. The pronounced effect of the anions strongly suggests” an important role for anions in the formation of complexes, Because electrophoretic studies in both water and alcohol (see Section II,8, p. 234) offer evidence that free univalent anions do not complex significantly, if at all, with carbohydrates in solution, the effect of the anion on the optical rotation may well stem from an interaction between the carbohydrate and undiseociated salt molecules. Ramaiah and Vishnu66 offered a different explanation for the anionic effect. They suggested that this difference between the anions is due to the difference in their ability to alter the refractive index of water. However, this hypothesis fails to explain why, for example, lead nitratew has no effect on the optical rotation of sucrose. The refractivity of an aqueous solution of lead nitrate is greater than that of an equimolar solution of sodium br0mide.a Salts of alkalineearth metals do not generally differ greatly from salts of alkali metals in their effect upon the specific rotation of carbohydrates. The variation in the rotation of sucmse,(~~6@ D-gZpm-D-gdo-heptose:6 Cr-D-gulose,’& and methyl D-gulopyranosides,” as a function of calcium chloride concentration in aqueous solution, has been mathematically expressed in empirical equations. Aqueous mixtures of sucrose with magneeium sulfate, calcium acetate, barium chloride, and lead nitrate, respectively, have been studied polarimetrically.’O Lead nitrate differs from the other salts in having no apparent effect upon the rotation of sucrose. However, this salt does affect the rotation of Dfructose. The equilibrium rotation of ~guloeeAl. and of D-gZycero-D-gulo-heptosedb v d e s considerably with the concentration of calcium chloride (see Fig. 4). On the other hand, that of pglucose is not greatly influenced. Frush and Isbell” have adequately shown that the influence of calcium chloride is predominantly caused by an adduct formation that leads to a marked shift in the equilibrium between the a- and p-D-pyranose modifications. The higher the concentration of calcium chloride, the greater is the proportion of the u-D modification. The presence of ethanol in the system affects the position of the equilibrium rotation in such a way aa to suggest (68) “International Critical Tablee,” MoGraw-Hi11 Book Co.,Inc., New York, 1930, (69)

VOl. VII. D. 63 ff * If,. F. Ja&son and C. Id. Gillis, Bur. Sbndarda Sci. Pamra. No. 376. 126 (1920)

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

231

+ 10 0

8 2 -10 9

- 20 0

2

4

6

CaClp, g/IOO ml of

8

10

solution

FIG.4.-Effect of Calcium Chloride on the Equilibrium Rotation of ~glycerct~-guloI3eptose.Q (Conaentration of sugar ia 4 g./lOO ml. of solution.)

an increme in the proportion of the CX-Danomer. The mutarotation coefficients for pure D-gulose and wgtycero-wgulo-heptose in aqueous solution are in accord with those of the corresponding calcium chloride adducts. Interpretation of the rotational data on reducing sugars in salt solution must necessarily take into account the possibility that changes in specific rotation of the sugar can be caused, at least partially, by carbohydrat-lt interactions that have no effect upon the proportion of the CY-(D or L) modification. Solutions containing both a salt and a reducing sugar contain two classes of substance: (1) free, uncomplexed sugar consisting of CY and fl (D or L) anomera in equilibrium, and (2) complex4 sugar. The latter class is possibly composed both of charged and uncharged species, formed by the interaction of the sugar with free cations and with undissociated molecules of the salt, respectively. The optical rotation of the solution is, thus, equal to the s u m of the rotations of the different complex species plus the rotation of the free anomers of the sugar. The possibility that both the pyranose and the furanose structures contribute to the overall rotation introduces additional complications. Polarimetric studies have shown that dilution of a solution of a sugar and a salt leads to a lowering in the percentage of sugar in the complex form. For example,@.in a 0.03 M solutionof cu-D-gulose*CaCl2.H2O in water, the percentage of Dgulose in the complex form, estimated from rotatiQnal measurements, was reported to be 0.9%, whereas, at a concentration of 0.34 M ,the percentage indicated waR 15%. 8. Electrophoresis

Electrophoretic migration of carbohydrates in solutions of alkali metal salts or alkaline-earth metal salts demonstrates the ability of carbohydrates

232

J. A. BENDLEMAN, JR.

to unite with free ions; however, it cannot detect the union of carbohydrate molecules with undissociated molecules of salt. The rate of migration is a function of many variables, such as stability of the complex, the stoichiometry, the concentration of salt, the cationic radius, the favored coordination geometry of the cation, and the size, configuration, and confiorniation of the polyhydroxy compound. Obviously, the electrophoretic data done would not permit the determination of the relative complexing abilities of carbohydrates; however, they do permit qualitative information to be obtained concerning the relative complexing abilities of alkali and alkalineearth metal ions (see Section 11,6,p. 227). a. Aqueous Mdia.--Mill@ has used cellulose-paper electrophoresis to provide evidence for the existence, in dilute aqueous mlution, of complexes of polyhydroxy compounds with cations of alkali metals and alkalineearth metals. The relative effectiveness of the different cations in promoting migration toward the cathode has already been given in Section 11,6 (see p. 227). No evidence was found for complexing between the carbohydrate and the anions acetate, nitrate, and perchlorate; however, the sulfate ion appeared to have some complexing ability. Of all the polyhydroxy compounds studied in aqueous solution,"* cisinositol exhibits the greatest mobility. The epi- and allo-inositol and cis-quercitol also show considerable movement, but to a lower degree; other cyclitols are less mobile. Reducing sugars and alditols generally show very little or no movement in the presence of Mg*@and alkali metal ions; all move in the presence of Caa, Sr@, and Baa, but the rates are only moderate. Table I11 gives the relative mobilities of several polyhydroxy compounds in aqueous solutions of various metal ions.

Turn I11 Relative Mobilitles. of Polyhydmxy Compound8 in Aquaour S O ~ U ~ ~ofO X I B Metallic Ionra Compound

Bag

Md'

Na (s

K '

&Inoaitol 6pi-Inodtol GIditol Allitol ~-Talom

82 26

20 2

13

10 3

6 3

5

1 1

1s

0

1 1 2

1 2

1

4 Cstionio movements are given as peroentagsa of the anionio movement (about 10 cm.)of p-tnitropenienmlfonic acid on the mme strip, with 2,3,Btri-O-methyl-~-glucose MI the marker for Bero migration. The eleotrolyte was a 0.1 M solution of the metal acetate in 0.2 A4 Boetio acid. Electrophoreds wae performed for 1 hr. at a potential gradient of about 20 v./om. on Whatman No.4 paper under a uniform preseure of 0.4 atm., with oooling by tep water. Compounds were applied as 0.1 M aqueous solutione.

ALKALI AND ALKALINBEARTH METAL COMPLEXES

233

OH I

FIG.6.4-Inoaitol.

Mills6*attributed the outstanding complexing power of cis-inositol to the presence of three axial hydroxyl groups in a chair conformation (see Fig. 5 ) ; these groups are suitably oriented for close approach to a cation. Arrangements of three hydroxyl groups that are close enough together to be associated with a cation can be discerned in certain of the conformations of the other cyclitols that show catiohc migration. Charley and Saltmane studied the migration of radioactive Cate in the presence and absence of lactose in aqueous solution at pH 7.0. The solutions were buffered with sodium hydrogen carbonate. The inability of calcium to migrate in the presence of lactose indicated that Cat” had reacted with the sugar to form a soluble, uncharged complex.

b. Alcoholic Media.-Studies of the interaction of polyhydroxy compounds with alkali metal salts in alcoholic solutiona6have shown that electrophoretic migration is much faster in methanolic and ethanolic solution than in aqueous solution. This observation indicates that the stability of a complex is much greater in alcoholic than in aqueous systems. Even 2,3,4 ,6-tetra-0-methyl-D-glucose migrates at a small, but measurable, rate. Glass-fiber paper, instead of cellulose paper, was employed in the studies, in order to eliminate the possibility of errors that could arise from the formation of complexes between cellulose and metal ions. The effectiveness with which the solvent promotes migration decreases in the order: methanol > ethanol >> water. The extremely low stability of complexes in water can be explained by the relatively great tendency of metal ions to associate with water molecules.70The difference between the rate in ethanol and that in methanol can be attributed, at least partly, to the fact that salts we more highly dissociated into free ions in methanoP; a higher concentration of free cations would permit a higher concentration of positively charged carbohydrate species. Relative rates of carbohydrate migration are spread over a wider range of values in methanol than in ethanol: this can be attributed to a lower E. Martell and M. Calvin, “Chemistry of the Metal Chelate Compounds,” PrenticA3aI1, New York, 1962, p. 239. (71) N. A. I z d o v , E. I. Vd,and N. N. Salstnikov. Uch.Zap. Kb’kouak. Qos. Univ. Tr. Khim. Fak. i Nauchn.-Isaled. Znut. Khim., 71, No. 14, 29 (1956).

(70) A.

J. A. RENDLEMAN, JR.

234

stability of complexes in methuriolic media. Stability of chelates is known to bo greater in solvents of lower than in those of high The relative rates in an alcoholic medium are virtually independent of the nature of the anion (see Table IV) ; this constitutes a strong indication that free anions do not complex with carbohydrates to a n y significaiit extent. On the other hand, anions do play an important role in determining absolute TABLIC IV Rater of Eleatmphoreticr MigratJona of ~ - X y l ~ rand e D - G I U O Oin ~ Methanolic %duthnr of Varloua Electmly te#

Salt

M Temp., Migration "C.' rate,

Maib

mm./hr.

NaI NaBr NaCl NsOAo

Licl

NI4C1 CaCl, M s 1 ~ Ha0 6

0.03 0.16 0.16 0.03 0.16

0.30 0.30 0.16 0.16 0.16

38

-

-

0.61

38 34

7.2 3.8 6.7 4.7 0.1 3.0 14.0

0.57 0.68 0.66

0.8

0.2

34 38 43 37 39

44

4.0

-

0.60

0.06 0.67 0.56

Temp., Migration "CSb rate, mm./hr.

M~lb

-

-

-

47 38

10.1 8.2

0.64

-

0.66

34

6.6 4.3

-

-

0.64 0.40

39 44

10.2 1.7

0.40

38 -

-

-

0.41

a Zone electrophoresis WM on glass-fiber paper at a potentiel gradient of 16.7 v./in.; referenae (nonmigrating) oompouad waa ohrysene, whioh ie visible under ultraviolet lieht when dry, but not when wet; oompounds were applied aa alooholio solutions (0 .O& 0.08 M,solubility permitting); all carbohydrates migrated towerd the oathode; Mart. rate of migration relative to that of ~-ribose.'Maximum temperature to whioh the system rose.

-

rates of migration. In promoting migration, metal halides are more effective than the corresponding acetates. In methanol, magnesium acetate causes no migration, whereas magnesium chloride (hexahydrate) effects a memurable movement that is roughly comparable to that of ammonium chloride and lithium chloride. This differencein the ability of different salts (poseessing a common cation) to promote migration is possibly due to a difference in the degree of ionic dissociation, and not to complexing between the carbohydrate and the free anion. (72) A. Brtlndstr6m, Arkiu Kemi, 7, 81 (1964).

235

ALKALI APSD ALKALINE-EARTH METAL COMPLEXES

Table V contains a list of the absolute rates and relative rates of niigration for various carbohydrates in methanolic solutions of potassium acet'ate and sodium acetate. Table I V shows the effect of different salts on the absolute rates and relatlve rates of wxylose and wglucose in methanol. The ability TABLEV Rate8 of Electrophoretic Migrationa of Polyhydroxy Compounds in 0.3 M Solution8 of Potaadum Acetate and Sodium Acetate in Methanol= KOAc Compound

Temp.? Migration "C. rate, mm./hr.

D-Xylose

D-Lyxose D-Arabinose a-D-Glucose fructose Sucrose Maltose Ibffinose Meleaitoee 1,6-Anhydro-p-D-g1uc0pyranose Methyl a-D-glucopyranoside B momer Methyl a - ~ msnnopy ranoside D-Glucitol Erythritol Q

See Table IV, footnote a.

42 48 42 48

48 48 42 42 42 42

7.3 10 .a 4.6 6 .O 7.4 9.1 4.8 10.2 4.1 4.8

NaOAc MRib

1.oo 1.oo

0.60

37 38 38

-

-

6 .O

.oo

38 38 38 a7

0.58 0.73 0.90 0.64 1 0 -68 0.64

-

-

42 42 42

4.6 4.4 6.7

0.63 0.62 0.94

42 42

6.2 4 .O

0.69

-

-

Temp.? Migration "C. rate, mm./hr.

0.66

9.4 9.3 4.7

7.8 4.3 7.9

1.oo 1.oo

0.60

-

0.65 0.83

0.46 0.84

-

-

-

37 37 37

4.3 3.4 12.3

0.36 1.31

-

-

-

-

-

-

-

-

MRib

0.4

* Maximum temperatwe to which the system rose.

of a cation to promote migration in methanolic salt solution decreases in the order: Ca2e > Na" > Ke > NH4@> Lie, There is no clear correlation between the electrophoretic data available and the geometry of polyhydroxy compounds. The unusually high, relative rate of migration of 1,6anhydro-~-wglucopyrrtnosemay be due to the presence of the two axially oriented cis hydroxyl groups on G 2 and C-4 of a boat conformation. Such an orientation in a rigid molecule would be

236

J. A. RBINDLDMAN,

JR.

uxpoc:tcd to givo u uo,omplex of stability higher than average, aimilar to that of cia-inoeitol in aqueous media, Electrophoresis in nonaqueous media may be an effective means of separating polyhydroxy compounds from mixtures which would otherwise be difficult to resolve. Subsequent separation of salt from carbohydrate could then be agcon@ished by means of ion-exchange techniquee.

9. Structure of the Complex The true structure of a carbohydrate-salt adduct can be determined only from detailed x-ray diffraction studies, a few of which have been made. Such studies enabled Beevers and Cochrad2 to determine the complete structure of suprose-NaBr.2 HSO. Each Na@ion was found to have sixfold coordination, with almost regular octahedral symmetry. The Bre ion also has sixfold coordination, but the coordination group has no regular shape. Each Na@ion is close to one Bre ion, two water molecules, and three carbohydrate hydroxyl groups. There are only two direct intermolecular bonds between hydroxyl groups themselves; the remaining hydroxyl groups are linked through Na@and Bre ions and the water molecules. The hydroxyl groups of both the D-glucose sjld the D-fructose moiety participate in the bonding with oation and anion. The separation between Na@and B e in the complex (2.94 A.) is actually maller than that in pure, crystalline sodium bromide (2.98 A,). Senti and WitnauerSO made similar x-ray studies of potassium salt adducts of amylose. Adducts having a 2:l ratio of D-glucose residue to salt (iodide, bromide, formate, acetate, and bicarbonate) were found to have tetragonal lattices with fourfold screw symmetry; 1 : l adducts (acetate and propionate) were orthorhombic. The structure of the tetragonal adducts is determined, not by amylose-amylose contacts, but lsrgely by amylose-cation contacts. Amylose-anion contacts appear to be of minor importance, for the unit-cell dimensions are relatively insensitive to the size of the anion in an isomorphous series of potassium salt adducts. The elements of symmetry in these complexes indicate that the D-glucose residues in the amylose chain, or, at least, those chains in the cry-stalline portions (of the complex) that are responsible for the discrete diffraction patterns, are equivalent. The conformation of the &glucose residues was not determined. By means of three-dimensional, x-ray diffraction data, Hybl, Rundle, and W i l l i w solved the crystal and molecular structure of the potassium acetate adduct of cyclohexsamylose, a Schardinger dextrin. Cyclohexaamylose is a macro-ring consisting of six D-glucopyranose rings connected by a - ~(-1-4) -glucosidic linkages. Because there are six D-glucopyranose

ALKALI AND ALKALINHbIMRTH METAL COMPLEXEB

237

residuw in each turn of the helical structure of amylose, cyclohexaamylose is an ideal model compound on which to base a study of both the conformation of the D-glucopyranose rwiduea and the geometry of the CY-D( 1 4 ) -gluoosidic linkage in amylose. The analysis of the complex 2 cyclohexaamylose*3.08KOAc-19.4 HtO showed a translational stacking of the cyclohexaamylose molecules, to yield a cylindrical, carbohydrate canal structure. The a-n-glucose residues are all in the pyranose form, and this i s in,the C1 (D) cgnfonnation (la283e4e5e). Each molecule of cyclohexaamylose has six pocket positions along its surface that are occupied by water molecules and potaasium ions. The potassium ions are in distorted octahedral environments, outside the carbohydrate channels. Two of the three acetate ions in each unit cell are at highly anisotropic, disordered sites inside the cyclohexaamylose macro-rings. The third acetate ion has not yet been accounted for. There are intermolecular hydrogen bonds between the hydroxyl groups at C-2 and C 3 of each pair of contiguous P-glucose residues.

111. COMPLEXES FROM THE INTERACTION OF CAF~BOHYDRATES WITHMETALBASES The interaction of strong bases with polyhydroxy compounds, a1though extensively studied, has not yet been fully clarified. The available evidence indicates that the removal of a proton by a basic anion, to give an alcoholate (reaction 1 ), and the formation of an adduct (reaction 2 ) can both occur. In alcoholic media, reaction 1 has been definitely shown to occur. However, in aqueous media, differentiation between reactions 1 and S has not yet been possible. ROH + MB ROM + HB (11 ROH

+ MB

ROH*MB

0)

where M = a metal ion; and B = OH*, CNe, or an alkoxide ion. Alcoholates of polyhydroxy compounds will be included in the category of complexes, because of the probability that most, if not all, of them are stabilized by inner chelation of the metal ion with neighboring hydroxyl groups, similar to that illustrated in Fig. 6, Adducts, a h , should be stabilized by chelation.

FIQ.&-Chelate Structure of

the Sodium Alcohblste of a 1 ,ZDiol.

238

J. A. RENDLEMAN, JR.

There is considerable supporting evidence for the existence of undissociated inner chelates in aqueous solution. Potentiometric pH meaaurementsn and nuclear magnetic resonance studies74.76 of tiqueous solutions of a- and phydroxy carboxylic acid salta indicate an appreciable association between the alkali metal cation and the organic anion; alkaliie-earth metal ions associate even more strongly than do alkali metal ions. The stability of the complex increases a,s the radius of the cation decreases." All alkali metal cations form complexes with malate ion. The observation that the tetramethylammonium ion is much less strongly bound than an alkali metal ion is understandable, in view of the fact that quaternary ammonium ions are known to resist being solvated, even by water. The constants of formation of various metal kojates, including that of calcium, have been determined by potentiometric titration?' Possibly, the &membered, chelate ring of the metal kojate (see Fig. 7) contributes significantly to ita stability. Chsistepenn has suggested that chelation between alkali metals and

FIQ. 7.--Chehte Structure of Calcium Kojate.

pyridoxal plays a role in biologic4 transport; he studied both the alkali metals and the alkaline-earth metals. The formation of complexes of wgluconate ion with alkaline-earth metal ions has been studied." Supporting the concept of the existence of alkali metal hydroxide adducts is the isolation of highly crystalline, 1:1 molar adducts of potassium hydroxide with certain tertiary acetylenic carbinole and glycols.~~ However, these complexes cannot be strictly compared to alkali metal hydroxidecarbohydrate adducts, because of the probable involvement of the r shell of the carbon-carbon triple bond. Weizmann,@on the other hand, has reported the formation of potassium hydroxide complexes of acetala and of (73) L. E. Erickmn and J. A. Denbo, J . Phyu. C b m . , 67, 707 (1983). (74) 0. Jsrdetriky and J. E. We&, J . Am. Chem. Soc., 84, 318 (1880). (76) L. E. Erickson and R. A. Alberty, J . Phy8. Chem., 66, 1702 (1062). (76) B. E. Bryant and W. C. Fernelius, J . Am. Chem. Soc., 76, 6361 (1964). (77) H. N. ChristenMn, dlcimcc,ll,1087 (1066). (78) R. K. Canand A. Kibrick, J . Am. Chem. Soc., 60, 2314 (1038). (70) R. J. Tedeschi, M. F. Wilson,J. Scanlon, M. Pawlak, and V. Cunicella, J . &g. C h . ,48, 2480 (1963). (80)C. Weimmann, British Pat. 682,191 (1948); C h .AMroatb, 41, a630 (1047).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

239

dialkyl ethers of glycols; this indicates that alkoxyl groups call act 89 electron donors, in the same capacity as hydroxyl and keto groups. However, the electrondonating ability of an alkoxyl group would undoubtedly be much less than that of a hydroxyl or keto group. Both the pH and the conductivity of an aqueous solution of sodium hydroxide are markedly decreased by the presence of polyhydroxy compounds. By assuming that the reactions are due entirely to the removal of one or more hydroxylic protons, several inveatigabrs have calculated ionization constants for a number of simple carbohydrates.a1-’J4A tabulation of most of these constants (as p&) is availab1e.w Hirsch and Schlage showed that the reactions are reversible, and that the extraordinarily high “acidity” of reducing sugars is not due to enolization of the aldehydo form. An aldehydo or keto group is not essential for high reactivity toward alkali metal hydroxide, as shown by a comparison of selected p& (18’) values for D-glucose (12.43), sucrose (-12.6) , and ~-glucitol (13.57). The lesults of calculations in which these values were used indicate that, for aqueous solutions that are 0.16 M in carbohydrate and 0.16 M in sodium hydroxide, the extents to which D - ~ ~ U C O Ssucrose, ~, and wglucitol react to give alcoholate and water (reaction 1 ) are, roughly, 70%, So%, and 20010, respectively. These percentages must, of course, be viewed with some skepticism, since their calculation was based on the assumption that the contribution of reaction 8 is negligible. Apparent ionization constants wheat starchJWand alginate.” have also been determined for In Fig. 8, tha number of hydroxide ions consumed per molecule of carbohydrate, in an aqueous solution that is 0.25 M in sodium hydroxide, has been plotted against the initial concentration of carbohydrste. The ease with which a single molecule of disaccharide can effect the disappearance of more than one hydroxide ion is readily apparent. Starch and dextrin are unable to consume more than 0.5 hydroxide ion per wglucose residue. Should the &values for oligosaccharidea be expressed as molecules of sodium hydroxide consumed per monosaccharide residue, it would at once be apparent that the difference between oligosaccharideand plysaccharide is not very large. Furthermore, on this basis, pure *glucose is more reactive than any of the oligosaccharides studied. Smoleiiski and Porejko8astudied the pH of aqueous solutions containing (81)L. Michaelie and P. Ilona, Biocham. Z., 4@,232 (1913). (82)J. Thamsen, Ada C b m . S m d . 6, 270 (1952).

(83)P. Hirech and R. Sohlags, 1.Phyeilc. Chern. (Leipzig), A, 141, 387 (1929). (84)P.Souchay and R. &had, BuU. Soc. Chim. Fronts, 819 (19M)). (85) B.Capon and W. G. Overend, Advan. Ca&oh&ate Chsm., 16,32 (1960). (80) 8.M. Neale, J . Teztile Inst., 20, 373T (1929). (87)8.P.Sari0 and R. K. Schofield, Proc. Roy. Soc. (London), Ssd.A, 186, 431 (1948). (88)K.Smolehki and 8. Porejko, Roczniki Chem., 16,281 (1936).

J. A. RIJNDLEMAN, JR.

240

8FIO.8.--Relation between Consumption of Sodium Hydroxide and Conoentration of Carbohydrrh in Aqueous Solution." (Concentration of polymcaharidea h a x p r e g e e d in mole of p.plucose &due per liter of solution. Initial conaentration of sodium hydroxide is 0.26 M.e Imole of sodium hydroxide oonsumed psr mole of carbohydrate. Carbohydratea: D-glucoee 0,D-fruotOrw 0, BUDIOBB X, leotoee 0,meltose A, staroh @, and dextrin A.)

both calcium hydroxide and sucro~e.Their reaults were similar to those obtained with sodium hydroxide and ~umom,in that sucrose behaves a~ a weak acid. Measurementsof the pH of alcoholic solutions containing alksli metal hydroxide and carbohydrate have not yet been reported. A pH meter is capable of giving reproducible e.m.f. values in alcohol or alcoholwater solvents,m but the pH numbers read from the inetrument ere subject to no simple, clear interpretation in term of chemical equilibrium. There isJ at this time, no universal scale of acidity for mlventa differing in w a t e ~ alcohol ratio. Studies of the heats of reaction of sucrose with sodium hydroxide, and of sucrose with barium hydtoxide, in aqueous solution have shown that neither reaction ie as simple aa that of an wid-base neutralismtion; the experimental reeults indicated the possible involvement of hydrogen bonding.88. After devoting considerable thought to the problem of the chemical structure of alkali cellulose, B l e a M I and Lositsksyaw have suggested (89) R.Gi. Bab, M.Pesbo, and R. A. Robiaeon, J . Phya. Chenr., 67, 1838 (1983). (SQa) E.Calvet, H.Thibon, and P. Leydet, Bull. Soc. Chim. Fmncs, 2187 (1%). (90)8. V, BleehinskiI and 8. F. Lositateya, Tr. I&. Khim. A W . Not& Kim. 88R. I , 73 (1961).

ALJCALI AND ALKALINE-EARTH METAL COMPLEXES

241

that alkali cellulose exists as a mixture of metal hydroxide adduct and metal alcoholate, the adduct preponderating. They hypothesized that the ratio of adduct to alcoholate vanes with’thesize of the metal cation; the stronger the ion-dipole bonding between a cation and a carbohydrate hydroxyl group, the greater should be the proportion of the adduct. However, there is no unequivocal evidence to confirm their hypothesis. The same attractive force between cation and hydroxyl group that would stabilize an adduct might just as readily stabilize an alcoholate. The metal cation of a carbohydrate alcoholate is, very probably, chelated to neighboring hydroxyl groups. 1. Reactions in Aqueous Media

In homogeneous, aqueous solution, alkali metal hydroxides react with carbohydrates to produce negatively charged carbohydrate species. Although the general feeling among chemists is that these species are free alcoholate anions, the possibility that they are composed, at least partially, of carbohydratehydroxide ion adducts cannot be dismissed. There is electrophoretic evidence that the S0,S ion is capable of weak bonding with polyhydroxy compounds.” If this is true, perhaps the hydroxide ion and certain other oxyanions are likewise capable of weak bonding. Detailed x-ray studies of crystalline lithium hydroxide monohydratee’ and of sodium hydroxide tetrahydratee*have shown that hydrogen bonds can form between water molecules and hydroxide ions. Because contact of a metal ion with a hydroxide ion can increase the dipole moment of a hydroxide ion, and enhance its ability to form hydrogen bonds with hydroxyl groups,g* it is conceivable that undissociated, metal hydroxide molecules should form hydrogen bonds with carbohydrate hydmxyl groups more readily than can free hydroxide ions. In either situation, bonding would be expected to be stronger, the greater the partial positive character of the hydrogen atom on the hydroxyl group of the carbohydrate. Evidence for the existence of OHe(ROH)s, where ROH is an alkyl alcohol, in solutions of alcohols in benzene and in nitrobenzene, has been obtained by Agarwal and Diamond.” Their studies, which involved the use of various quaternary ammonium hydroxides, indicated that the hydroxide ion is capable of associating with three alcohol molecules, regardless of whether it IR in the free form or nmciated with a cation. (91) R. Pepinsky, 2.Krist., la, 119 (1940). (92) G. Beurskena and G. A. Jeffrey, J . Chem. Phge., 41, 924 (1904). (Q3) A. F. Wells,“Structural Inorganic Chemistry,” University Press, Oxford, 1947, p.

360.

(94) B. R. Agarwal and R. M. Diamond, J . Phys. Chem., 67, 2785 (1963).

242

J. A. RBNDLEMAN, JR.

Makolking6studied the rate of exchange of l80between labeled water and alkali cellulose, and between labeled water and the trisodium alcoholate of cellulose, and concluded that merceriration proceeds by reaction 8 ; that is, alkali cellulose is an adduct. He b d his conchpion on the fact that theN is no measurable exchange between water and the alooholate, whereas there is measurable exchange between alkali cellulose and water. His conclusion, which is based upon difference of rate, is, however, not necessarily valid. The trisodium alcoholate may not possess a structure that is as accessible to water aa is that of alkali cellulose. The complex 2 sucrose-NaOH, prepared in aqueous alcoholic media, loses the elements of water at 110"under vacuum, to give the corresponding alcoho1ate.m Treatment of the alcoholate with glacial acetic acid permits B U C ~ Y to ) ~ be ~ recovered in 90% yield. Ahali metal complexes may be analyred for their metal content by simple acidimetric titration. Analysis for adduct (hydroxide) content is more involved, and entails the aseumption that there can be no water of hydration attached to an alcoholate anion. The methodm involves: first, dissolving the complex in anhydrous methanol, and then, treating the resulting solution with an appropriate anhydrous acid, such as tartaric acid. The acid servea to convert any hydroxide ion into water (reaction S),

which can then be quantitatively determined by titration with the Karl Fischer reagent. The amount of water thereby measured is assumed to equal the amount of hydroxide originally preaent in the adduct. By similar means, it has been shown@@. that, when alkali cellulose (prepared in an aqueous sodium hydroxide medium) is dried under vacuum at 6 5 O , the reaulting material consists of both alcoholate and hydroxide adduct. There is the poseibility that complexesieolated from aqueous (or aqueous alcoholic) media are not hydroxide adducts, but, instead, hydrated alcoholatea. The above adaptation of the Karl Fisoher analysis does not, unfortunately, distinguish between a hydroxide ion and a water molecule. Such a distinction could posaibly be made by detailed x-ray analysis; however, neither alcoholates nor adducts have, as yet, been obtained in a form suitable for such a study. Tablo VI contains a comprehensive list of known alkali metal hydroxide ndducts. Complexm prepared by the interaction of carbohydrates or naotiitcn of ctirhohydmtewwith nlkdi motd hydroxide in anh!/drrgusalcoholic (95) I. A. Mskolkin, Zh. Ob8hch. Khim. 12, 306 (1042). (96)J. A. Nendleinn, Jr., J . Org. C h . ,81, 1845 (1966). (!IOU) E.(ifligerand H.Noh, Helu. Chim. A&, 40, 660 (1987).

TABLE VI Adducta of Carbohydrates with Alkali Metal Hydroxides Carbohydrate &and)

Amylose

MOH

CsOH LiOH KOH

Molar r ~ t i o , ~ ligand-MOH

References

Solvent of solvation, molecules/cation

Solvent medium

3: 1 3:l 1:l 3:l

-

H&EtOH H&EtOH H&-EtOH H&EtOH

97 97

-

s

U

k

1:l

-

H&EtOH

96

cellobiose

KOH

1:l 1:2

-

H&EtOH HaEtOH

99 100

Celluld

CsOH

3: 1 1:l 2: 1 2: 1 3:2 4:3 3: 1 1:l 1:l 1:l 2: 1 2:l 3:l

-

Hto H&EtOH HeEtOH HZO

RbOH

NaOH

-

3 HZO HZO

-

3 HtO

f:2

-

4:3

-

HaEtOH H&EtOH HZO H@-EtOH H+EtOH H&EtOH H&EtOH

El20

HZO-EtOH HeEtOH HeEtOH

*

z

97

NaOH

-

c (

98

~Arabiiose

KOH

Tr

101 9% 102

102 101 102 102 101 103

103 104 104 101 103 104 104

*

ET ss

i ! b c1

0

5 Fi

L

h3

8

Tmuc VI (Continued) Adducts of Gubohyhtca w i t h Alkali Metal H y M d e s Carbohydrate

(ligand)

MOH

Molar mttio,ligand:MOH

Solvent of &ation,

Solvent medium

Refere-

molec&/~tion

D-GlUcoSe

Starch

SUCroSe

NaOH

1.S:l 1:l 1:1*2 2.0:l

KOH

(2-3) KOH

KOH NaOH

1:3 1:l

HJO-EtOH H&-EtQH

NaOH

1:1

H&-EtOH

KOH NaOH

1:1 1:l

H&-EtOH H&WH

106 106

NaOH

1:l 1:2

H&EtOH H&-EtOH

107 107

NaOH

1:1 1 :2 2:1

Hto HZO

Hso

108, 109 108,109

2:1 1:l 1:2 1:3

H&EtOH HeEtOH H&EtOH H&EtQH

96 110,111 110 110

LiOH KOH

CsOH KOH

no &O

-

0.2 &o 0.1 H a , 0.08 EtOH

H&-EtQH H&WH H&EtOH H&WH

96 99 96 96

H&EtOH

108,109

Q

-r

NaOH

1:l 1.7:l 1:1.8 1:2.5 1:5.1

0.3 HzO

-

no HtO 0.22 HsO, 0.04EtOH

HeEtOH HeEtOH HeEtOH H&EtOH HtO-EtOH

111 96 96 96

96

r

The Jigand in a polysaccharide adduct is the D-glucose residue. In other adducts, it is the entire carbohydrate molecule. b For adducts of cellulose with lithium hydroxide, 6ee the discussion in Section III, la (p. 250).

r

6

(9i)F.R. Senti and L. P. Witnauer, J. Am. Chern. Soc., 70, 1438 (1948). (98)W. J. Heddle and E. G. V. Percival, J. Chin. Soc., 1690 (1938). (99)E. G.V.Percival, J. C h .Soc., 1160 (1934). (100) E.G.V. Percival and G. G. Richie, J. Chem. Soc., 1765 (1936). (101) E. Heuser and R. Bartunek, CeUu2oseehemie, 6,19 (1925). (102) K. G.Ashar, J . Cltim. Phys., 48, 583 (1951). (103) I. Sakurada and S. Okamura, Kouoid-Z., 81, 199 (1937). (104) G.Champtier and J. Nhl, BuU. Soc. Chim. FMW, 930 (1949). (105) A. Herzfeld, Ann., aa0,206 (1883). (106) A. Bau, Z.Vet. Deoct. Zzccker-lnd., M,481 (1904). (107)K.Beythien and B. Tollens, Ann., 266, 195 (1889). (108) G.Champtier and 0.Yovanovitch, J. Chim. Phys., 48,587 (1951J. (109) 0. Yovanovitch, Compt. Rend..,292, 1833 (1951). (110)E.G.V. Percival, J. Chem. Soc., 648 (1935). (111) E. Soubeiran, Ann., 43, 223 (1842).

E

-.

U

>

E

E 3r

J. A. RENDLEMAN, Jll.

246

TABLEm1 Complexea of Carbohydrates with Alkaline-earth Metal Hydroxides Carbohydrate (ligand) Hydroxide

drabhose p.F~ctose D-G~UCOW

Lactose Maltose

Manninotrioso RaffiIlOee

Be Sr Ca BE Ca Ca Ba Ca Sr Blb Ba Ca

Stachyose

Sr Be

Sucrose

Ba

Sr

C€+

Sr Q

,P-Trehalose

D-XJ’hi?

CR

BE Sr

Molar ratio, ligandiaation

2:1 2:1 1:l 2:1 1.1

1:l 1:1 1:l 1:1

1:l 1:l 1:2 1:l 1:3 1:2 2:3 1 :2 1:6

1:1 1:l 3:l 1:1 1:l 1:2 1:3 1:l 1:2 2:3 2:1 2: 1

Solvent

References

medium HeEtOH HgO-EtOH HlO HZO-EtOH HQ-EtOH HsO Ha-EtOH Ha-EtOH Ha-EtOK HaO-EtOH H&EtOH HiO-EtOH HsO-EtOH HlO HiO-EtOH HaEtOH Hi0 HlO HiO-EtOH

HlO HlO Ha0 HgO-EtOH H&-EtOE HlO Hi0 HqO HsO-EtOH H&EtOH H&EtOH

(112) E. Poligot, Compt. Rand.,90, 163 (1880). (113) H.Wintw, Ann., 144, 296 (1888). (114) H. Will, Arch. Phrni., 816, 812 (1887). (116) C.Tanret, Bull. Soc. Chim. Frame, 87, 947 (1802). (118) L.Lindet, J . Fabr. S w e , 81, 19 (1880). (117) G. Tanret, Compt. Rend.,166, 1620 (1912). (118)P. Horsin-DOon, Bull. doc. Chim. Frunce, 17, 166 (1872). (119) I. Schukow, 2. Ver. Dad. ZuckerZnd., 50,818 (1800).

4

4 69,112,113 114 59 69 105 59, 105 105 115 107 107 116 107 107 115 117 117 59 19 19 69 69 59, 118 69 19,59 19,69 119 4 4

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

247

rneclia*'WJ'O are not listed, b e c a w of the probability that they are, preponderantly ,alcoholates. With the exception of magnesium hydroxide, alkaline-earth metal hydroxides are similar to alkali metal hydroxides in that both types are strongly basic and highly dissociated in aqueous solution. Interaction of an alkaline-earth metal hydroxide or oxide with a carbohydrate results in an increased solubility of the hydroxide or oxide, apparently through the formation of either an alcoholate, a carbohydratemetal hydroxide adduct, or a carbohydrate-metal oxide adduct. Mackenaie and Quin"' have suggested that, in the compounds of reducing sugars with calcium hydroxide, the calcium is united with the hydroxyl group at C-1, possibly in the form 0

/ \

-CH

Ca

or

\ / 0

\

HC-O-Ca-OH,

/

and that, in the compounds of nonreducing sugars, the calcium is present simply aa calcium hydroxide bound to the carbohydrate in a manner similar to that of a salt in a salt-carbohydrate adduct. Because there has been little experimental work with either alkaline-earth metal hydroxides or oxides since that of Mackenzie and Quin:Q any attempt to draw conclusions concerning the composition and structure of the complexes must await further experimentation. For convenience, therefore, all complexes formed from hydroxides or oxides will be called adducts of alkaline-earth metal hydroxides, and they are listed aa such in Table VII. Water of hydration is omitted from Table VII for several reasons. (1) Many investigators did not consider water of hydration in determining the formula for a complex. (2) In all instances where water was determined, it was determined indirectly. The difference between the weight of a complex before and after the complex had been subjected to dehydrating conditions (for example, under vacuum at 100') was often assumed to equal the weight of water of hydration. (3) Dehydration of a complex could involve either the removal of water of hydration or the removal of water formed by chemical reaction, or both. And (4) , there is considerable question concerning the chemical structure of the complex. There has been no reported isolation of a magnesium hydroxide complex. Attempts by BenedikP to prepare such a complex of sucrose were unsuccessful. A barium hydroxide adduct of amylose was prepared by Senti and WitnaueP7 by means of an exchange of barium hydroxide for potassium hydroxide in a potassium hydroxide-amylose adduct; however, the exact (120) R.Benedikt, Ber., 6, 413 (1873).

248

J. A. RENDLEMAN, JH.

composition of the adduct was not reported. Dextran, also, reacts with hydroxides of calcium, strontium, and barium to give complexes; these complexes have pravidg a method of fractionating dextran for molecular size,'21 the process being based upon the fact that fractional acidification of an aqueous suspension of the complex results first in the dissolution of the component having the highest molecular weight. Dialysis studies'" of d u l o s e and dextran in aqueous solutions of barium hydroxide, sodium hydroxide, and "cadoxene" (cadmium hydroxide in ethylenediamine) showed no difference in complexing ability between the two polysaccharides. Furthermore, in solutions having equal base normality, Ba2@,Nae, qnd Cd(e$hylenedismine)P had loughly equal complexing abilities. a. Stoichiometry of Alkali-Metal Hydroxide Reactions.-The combining ratio for alkali metal hydroxide adducts is variable, as with that for alkali metal salt adducts. The ratio is dependent on the concentration both of hydroxide ion and carbohydrate, and on the size both of alkali metal ion and carbohydrate. At low concentrations (<0.1 M ) of hydroxide, there is a tendency for simple carbohydrates to form adducts having a 2: 1 ratio of carbohydrate to metal. Thii ratio is also found in many alkdi metal salt adducts prepared at low concentrationsof salt (seeSection 11,4, p. 222) and in most alcoholate complexes prepared at low concentrationsof base (see Section 1142, p. 259). With increasing concentration of hydroxide, particularly where the ratio of carbohydrate to hydroxide in the reaction mixture is small, oligosaccharide8 tend to form adducts having one or more molecules of metal hydfoxide per molecule of carbohydrate.wnsgThe greater the hydroxide concentration, the greater is the hydroxide content of the resulting adduct. One molecule of sucrose appears able to support as many as five molecules of sodium hydroxide." One molecule of cellobiose can accommodate at least two molecules of potassium hydroxide; maltose can accommodate three.8gLactose is intermediato, forming an adduct possessing between two and three molecules of potassium hydroxide per molecule." Hydroxide adducts of nonsugars and of sugar derivatives have been given but little attention. Heddle and E. G. V Percival" reported the preparation of 1:l adducts of potassium hydroxide with methyl &-Dglucopyranoside and methyl 8-Sglucopyranosidein alcohol-ether containing only a few percent of water. However, Rendleman has repeated the preparation of the 8-D-glucopyranosidecomplex, using Percival's procedure, and has found that the product actually consists of 29% of adduct and (121) E.L. Wimmer, U.8.Pat. 2,686,679; Chem. Abefmcte, 4% 7878 (1066). (122) H. Vink, Maktomol. Chem., 76, 86 (1904).

ALKALI AXD ALKALINE-EARTH METAL COMPLEXES

249

71% of alcoho1ate.m The reportm of the preparation of the p o t m i m i hydroxide adduct of methyl &D-glucopyranosidein an anhydrous ethanolic system is also largely incorrect. Konaqueous systems, &s will be discussed later, favor proton removal and, therefore, alcoholate formation (see , Section 111,2, p. 255). Senti and Witnauerg’ treated amylose with lithium hydroxide, potassium hydroxide, and cesium hydroxide, respectively, in 25% aqueous ethanol, to obtain products having a 1:3 ratio of hydroxide to D-glucose residue. However, adducts having this ratio are stable, in the preparative medium, over only a limited range of hydroxide concentration. The stoichiometry is variable, just, as it is with simple oligosaccharides. Adducts of amylose with sodium hydroxide and with ammonium hydroxide were also prepared. The latter could be formed only by exchange with the alkali metal hydroxide-amylose adduct in ethanolic ammonia solution. Heddle and Percivals reported that they had obtained a 1:1 potassium hydroxideamylose adduct by treating amylose acetate with ethanolic potassium hydroxide. In view of the fact that monosaccharides react with potassium hydroxide in nonaqueous media to give mainly alcoholate, the alleged composition of this amylose complex is questionable. Champetier and Yovanovitchlw treated corn starch with aqueous sodium hydroxide solutions of various concentrations, and obtained three adducts having 1:2, 1 :1, and 2: 1 ratios of sodium hydroxide to D-glucose residue. No 2:3 or 3:4 adducts, such as those reported for cellulose, could be obtained. As the hydroxide concentration is changed, the transition from one addition compound to another is abrupt, thus showing an absence of topochemical fixation. Champetier and NBelIo4also studied cellulose in aqueous sodium hydroxide solutions, and found the following ratios of sodium hydroxide to D-glucoseresidue: 1:2,2:3,3:4, and 1 : l . In theabsenceof metal hydroxide, natural cellulose and mercerized cellulose are composed of units of 2 C~H10O5*H2O and C&Ilo05*H20,respectively. Champetier and NBel suggested that the formation of a sodium hydroxide-cellulose adduct occurs through tl stepwise displacement of water from cellulose hydrate by sodium hydroxide molecules. On the other hand, Chuin, Petitpas, and Marsaudon12ahave explained the phenomenon as a displacement of the water molecules of sodium hydroxide hydrates (NaOH .m H2O) by hydroxyl groups of cellulose, to give NaOH (H20)m-,,( HOcellulose)n. At concentrations of less than 15% of sodium hydroxide, accessibility of the hydroxyl groups of native cellulose to sodium hydroxide is less than that of mercerized cellulose; but, a t concentrations greater than about 15% (the threshold (123) J. ChBdin, G. Petitpas, and A. Marmudon, Mem. Serv. Chim. Eta1 (Paris), 40, 811 (1955).

250

J. A. RENDLEMAN, JR.

concentration for complete mercerization) , the accessibility is the same for both types. Complete merceriration occurs when 85% of the D-glucose residues have each fixed one molecule of sodium hydroxide, equilibrium .’~~ believed that, being reached in less than 48 h o ~ r ~Abadie-Maumert126 when cellulose is immersed in 18% sodium hydroxide, only half of the sodium hydroxide in the complex is combined with the cellulose. He postulated that the other half is present in the intermicellar solution trapped within the adduct. The formula given for the complex was 2 C ~ I O O(NaOH)aombimed* S. (NaOH).oh*ll(H:O),,t.

Ashar’02treated cellulose with aqueous potassium hydroxide, and ob.served the formation of adducts having the same ratios of hydroxide to D-glucose residue as those observed by Champetier and N & P for sodium hydroxide and cellulose, namely: 1:2, 2:3, 3:4, and 1:l. Bleached cotton linters were shaken with aqueous potassium hydroxide solutions of different concentrations at 20°, and the composition of the resultant solids was plotted against the hydroxide concentration. Plateaus corresponding to compounds possessing the aforementioned ratios were obtained. Heddle and PercivaP had also reported the preparation of a 1 :1 potassium hydroxide adduct. The reaction of cellulose with aqueous lithium hydroxide is a continuous function of the hydroxide concentration, and gives no adducts of greatly favored combining ratio. Heuser and Bartunek’Ol isolated an adduct having a 1:2 ratio of lithium hydroxide to D-glucose residue. At the high concentration of 5 M lithium hydroxide,‘* the ratio is 0.75:l. Less attention has been paid to the reaction of cellulose with rubidium hydroxide and with cesium hydroxide. Heuser and Bartunek’O’ isolated adducts of rubidium hydroxide and of cesium hydroxide that had the general formula MOH-3 CsHloOa. Their studies showed that the concentration, in weight percent, of alkali metal hydroxide required for forming a “stable” adduct of the lowest alkali content increases with incresse in the atomic weight of the metal: Li < Na < K < Rb < Cs. However, on a molar basis, this relationship does not hold. No simple relationship exists between the size of cation and the concentration of hydroxide necessary for the formation of a “stable” adduct. Treatment of an amylose-alkali metal hydroxide adduct with a concentrated solution of an inorganic salt (such EMpotassium iodide or potassium acetate) in aqueous ethanol can result in the total displacement of hydroxide This displacement indicates the existence of an equilibrium (124) A. Webei, K.G. h h r , and G . Champetier, Compt. Rend., 288, 1318 (1054). (126) F.A. Abadie-Maumert, Compt. Rend., PI, 1957 (1961). (120) K.G.Aahar, Compl. W .262, , 734 (1961).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

251

bctwecp ail alkali mctal hydroxide tdduct and a salt adduct. Displacement of the rnettll cation by another cation can alRo bccur." In aquem alcoholic solution, the combining ratio for sodium hydroxide cellulose is a function of the kind of alcohol, the concentration of the sodium hydroxide, and the concentration of In aqueous ethanol, maximum consumption of sodium hydroxide occurs at 18% of water, to give R compound containing 1.15 molecules of sodium hydroxide per glucose residue. A smaller percentage of water results in a lower consumption of sodium hydroxide. Higher alcohols are similar to ethanol in behavior; however, the limited miscibility of most of the higher alcohols with water prevents the occurrence of a maximum. In contrast to the relatively high consumption of sodium hydroxide in aqueous ethanol and higher alcohols, consumption in aqueous methanol is very low. Nicoll , Cox,and Conawayln have critically reviewed the different methods generally employed for determining the alkali content in cellulosealkali metal hydrqxide,adducts.

b. Stoichiometry of Alkaline-earth Metal Hydroxide Reactions.The only information available on combining ratios for alkaline-earth metal hydroxide adducts is derived from studies of mono- and oligosaccharides. Various attemptssBto isolate adducts of methyl glycosides have failed. Polysaccharide adducts have thus far been almost entirely neglected. PrzyleckiU8 has prepared a chloride-free calcium compound of starch by treating an aqueous, alkaline solution of starch with calcium chloride. The composition of the product was reportedly constant. It is known with reasonable certainty that monosaccharides combine with alkaline-earth metal hydroxides in 2 :1, 1 :1, and , perhaps, 1:2 ratios of monosaccharideto metal. With oligosaccharides, only adducts containing one or more molecules of metal hydroxide per carbohydrate ligand are known. The ease with which more than one molecule of metal hydroxide becomes fixed to a molecule of oligosaccharide incrertses with increase in atomic weight of the alkaline-earth metal: Ca < Sr < Ba. The maximum number of molecules of calcium hydroxide accommodated by a molecule of sucrose is probably only threepg although certain investigators have reported as many as four1%and six."* The tetrasaccharide stachyose reportedly accommodatessix molecules of strontium hydroxide per m0lecule.~~7 D.Nicoll, N. L. Cox, and R. F. Conaway, in "Cellulose and Cellulose Derivatives," M. W. Gra&, E. Ott, and H. M. Spurlin, eds., Interecience Publishers, Inc., New York, N. Y., 1964, p. 825 ff. (128) 8. J. Przylecki, Sprawozdania Posisdzen Towarz. Nu&. Warmw. W&id ZV, Naxk Biol., SD, 186 (1936); Chem. Zen.&., 109, I, 1992 (1938). (129) W. Wolters, N. 2.Rhuclee7-Znd., 10, 287 (1883).

(127) W.

252

J. A. RENDLEMAN, JR.

c. Electrophoresis.-Although there have been no electrophoretic studies on aqueous solutions of alkaline-earth metal hydroxides, the behavior of a large number of polyhydroxy comy wds in 0.1 M sodium hydroxide wlution has been studied. Frahn a d Mills1aofound definite movemznt toward the anode (positive electrode) of nearly all compounds having more than two hydroxyl groups. This movement can be attributed either to the ionization of hydroxyl groups (alcoholste formation) or to the formation of adducts with free hydroxide ions, or both. Both species are negatively charged and would move toward the anode. In no case was there migration toward the cathode (negative electrode). With certain exceptions, the general order of decreasing rate of migration is: aldoses, ketoses 2 reducing oligosaccharides > nonreducing oligosaccharides > polyhydric alcohols and methyl glycosides. The mobilities of tetra-0methyh-glucopyranose and 2,3,6- and 2 ,4 ,6-tri-O-methyl-~-glucopyrmose are only slightly less than that of unsubstituted D-glucose. Among the monosaccharides, there is a trend toward the highest rates for the conformationally least stable sugars. However, the relatively high rates for D-glucose and D-mannose, both of which occur in the stable C1 (D) conformation, show that conformational instability is not the decisive factor operative. The acidic, angmeric hydroxyl group of reducing sugars probably contributes much to their overall high mobility. The rates for methyl glycosides are very low compared to those for the corresponding sugars. Diols do not migrate, indicating little or no reaction with hydroxide. The higher polyhydric alcohols, except for glycerol, do exhibit migration. In fact, certain heptitols behave as if, in acidity, they are comparable to reducing sugars. The fact that tetra-0-methyl-D-glucopyranoseis very similar to D-glucose in mobility may mean, in some instances, that simple ionization of a hydroxyl group, without concomitant formation of a cyclic complex, is responsible for the migration. Furthermore, cis-inositol, which is well known for its outstanding ability to form complexes with borate ion1" and with the ions of alkali metals and alkaline-earth metals,'Jahas B relatively low mobility, in aqueous sodium hydroxide] that is almost identical to that of D-glucitol. However, the possibility that the cyclitol preferentially forms uncharged complexes, composed of un-ionized polyhydric alcohol and undissociated sodium hydroxide, must not be overlooked. Such electrically neutral entities, should they exist, would be electrophoretically undetectable. Also, the fact that free, alkali metal ions can interact with polyhydroxy compounds in aqueous solution to give positively charged species6*cannot be disregarded in interpreting electrophoretic data on (130) J. L.Frabn and J. A. Mills, Awlrdhn J . C h . ,12, 66 (1959).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

253

hydroxide solutions. Migration of these positively charged species toward the cathode would decrease the overall migration rate of the carbohydrate toward the anode. d. Optical Rotation.-Reeves arid Blouinlg’ measured the rotation of various glycosides in 1 M sodium hydroxide, hoping to gain information that would lead to an understanding of the effect of h l i metal hydroxide upon the optical rotation of amylose.1a2 The optical rotation of many glycosides in neutral aqueous solution is essentially the same as that in 1 M sodium hydroxide. Some glycosides, however, show relatively large, reversible changes in optical rotation at this concentration of hydroxide. To explain the changes, Reeves and Blouinls1suggested that alkali-sensitive glycosides undergo a change in conformation. The removal of a proton from a glycosidic hydroxyl group was postulated to give a charged species more highly solvated than the corresponding, uncharged molecule. This (highly solvated) species would conceivably adopt a conformation in which as many aa possible of the ring hydroxyl groups would assume equatorial positions, where there is less steric hindrance than is afforded by axial positions. Thomsen,laa Smolehski and Kozlowski,’% and Reeves and B l ~ u i n ~ ob*~ served that sodium hydroxide has a relatively large effect on the optical rotation of sucrose. a,a-TrehaloseIu1 on the other hand, is affected only slightly. T h o m m P and Reeves and Blouinl*lmade no attempt to interpret the unusual behavior of sucrose; Smolexkki and Kozlowski,la however, assumed that the reaction was that of alcoholate formation, and they calculated dissociation constants for sucrose. The possibility that either chelation of sodium hydroxide (or OHe ion) with glycosides or inner chelation of undissociated sodium alcoholates is responsible for part or all of the abovediscussed changes in rotation had not been considered. Support for a hypothesis based on chelation in which at least two neighboring hydroxyl groups are involved is provided by the work of Lindberg and Swan.la6These investigators studied the changes in optical rotatiop of various glycosides effected by “cadoxene,” a solution of cadmium hydroxide in 28% aqueous ethylenediamine; they concluded that complexes we formed between cadmium and adjacent hydroxyl groups in a molecule of the glycosides. Methyl /3+glucopyranoside shows no electrophoretic mobility in “cadoxene” and lessens the conductivity of “cadoxene,” which proves that the complexes are uncharged. Except for the magnitude (131) 132) 133) (134) (135)

t

R. E. Reeves and F. A. Blouin, J . Am. Chem. Soc., 79, 2261 (1957). R. E. Reeves, J . Am. Chem. Soc., 76, 4595 (1954). T. Thom’n, Ber., 14, 1647 (1881). K. Smolehski and W. Koslowski, Roczniki Chem., 16, 270 (1936). B. Lindberg and B. Swan, Acta Chem. Scad., 17, 913 (1963).

254

J. A. RINDLEMAN, JR.

of the rotational changes, the results of the polarimetric studies in “cadoxene” are identical with those obtained by Reeves1” in his studiea of glycosides in cuprammonium hydroxide solution. The complexes formed with “cadoxene” are, probably, very similar to those formed with cuprammonium hydroxide: that is, 5-membered, cyclic complexes involving adjacent hydroxyl groups. It has not been shown whether the “cadoxene” complex is an alcoholate or a hydroxide adduct. e. Preparation of Adducts. (1) Alkali Metal Hydroxide Adducts. (a) Mono- and Oligo-saccharides.-The usual method of preparation

is to dissolve the sugar in a small amount of water, dilute with ethanol, and then, with rapid stirring, add either ethanolic or aqueous ethanolic alkali metal hydroxide. Addition of a dilute solution of the hydroxide gives low ratios of hydroxide to carbohydrate; addition of concentrated hydroxide favors high ratios. Fornation of adducts of saccharides having a low molecular weight may require the addition of ether to the reaction mixture to effect their precipitation. Adducts having a very high alkali content may be prepared by dissolving the sugar in a concentrated solution of the alkali metal hydroxide (aqueous), and then adding ethanol to bring about precipitation. In all cases, the water content of the final mixture should be sufficient (10% or more) to prevent the formation of alcoholate. The precipitated adduct ia quickly separated from its mother liquor by the most convenient method (filtration, centrifugation, or decantation) , washed with ethanol (or a mixture of ethanol and ether) ,and then, perhaps, with ether, and dried under vacuum. Care must be taken to protect the adduct from air (which contains carbon dioxide, oxygen, and water vapor, all of which can attack the adduct). Adducts of nonreducing sugars may be stored under nitrogen for many days. However, adducts of reducing sugars are not very stable, and should be used soon after preparation. The adducts are colorless and amorphous. Mtindy and Vavrineczsl reported that they obtained crystalline needles of sucrose*NaOH by slowly evaporating an aqueous solution containing equimolar proportions of the components. However, there has been no repetition of this experiment to confirm the report. Adducts prepared from reducing sugars are exceptionally sensitive to heat, and, even at room temperature, gradually turn yellow, possibly from deep-seated reactions involving enoliration.

(b) Poly8accharides.-Water-insoluble carbohydrates, such aa cellulose, must be equilibrated with aqueous, or aqueous alcoholic, hydroxide solutions. The effects of the concentration of hydroxide and of alcohol on the combining ratio have already been disauased. When equilibrium has (136)

R.E.Reevee, Aduan. Carbohyd*ds Chcna., 6, 107 (1961).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

255

been attained, the product is either pressed free of adhering solution or wiped dry with absorbent tissue. Adsorbed alcohol, which is difficult to remove by vacuum drying, can be removed by humidification over water in a vacuum desiccator. Use of a high concentration of sodium hydroxide’” in water (about 5 M ) permits the formation of a 1:l adduct of cellulose. Lower concentrations lead to adducts having a lower content of alkali. Acetates can be used instead of the free polysaccharide for preparing polysaccharide adducts. For example,” at concentrationa of 0.1 M alkali metal (lithium, sodium, and potassium) hydroxide in 25y0aqueous ethanol, amylose triacetate is deacetylated to form adducts containing approximately one molecule of alkali metal hydroxide per three D-glucose residues. Partially crystalline amylose adducts can be obtained by immersing stretched filaments of amylose triacetate in the deacetylating solution. (2) Alkaline-earth Metal Hydroxide Adducts.-Precipitation of an adduct from an aqueous solution containing both a sugar and a hydroxide is usually effected at room temperature by treating the solution with ethacol. The greater the concentration of the hydroxide with respect to that of tho sugar, the greater is the ease of fixing more than one metal atom upon a molecule of the sugar. High temperatures favor a high ratio of metal to sugar. Prolonged treatment with aqueous ethanol of a complex having a low metal content is known to favor conversion of the complex into one having a greater content of the metal. The isolated complex is washed with aqueous ethanol and dried, either between filter papers or in a vacuum. Amorphous and crystalline complexes have both been prepared by this general method. A review by Mackeneie and Q ~ i non , ~complexes ~ of alkaline-earth metal hydroxides, is recommended to those who desire a more thorough description of the methods of preparation. 2. Reactions in Anhydrous, Alcoholic Media

In anhydrous, alcoholic media at 25”, both the hydroxide and the cyanide of alkali metals react with “nonacidic” carbohydrates, to give colorless, amorphous, hygroscopic precipitates that are preponderantly mono- (alkali metal) alcoh01ate.g~Under the proper conditions of concentration, most of the metal alcoholates combine with an additional molecule of carbohydrate per molecule, to give products whose molar ratios of carbohydrate to alkali metal are greater than 1:l. A small proportion of the hydroxide adduct or the cyanide adduct accompanies the alcoholate as the latter is (137)

E. G . V. Percival, A. C. Cuthbertson, and H. Hibbert, J . Am. Chem. Soe., 6a, 3267 (1930).

256

J. A. RENDLEMAN, JR.

precipitated from aolution. The hydroxide adduct may, actually, be a hydrate of the alcoholatebut no meaw for distinguishing between a hydroxide addyct and an alcoholate hydrate has yet been devised. (For the sake of simplicity, the term adduct is employed throughout this review.) Table VIII gives the composition of a number of complexes formed by the interaction of &ali metal hydroxide with various carbohydrates in TAB- VIII Composition of Variour Alcoholate Complexes Precipitated from Anhydrour Ethanolia Mdiaw Ligand

Metal Molar ratio, Cornpodtion of complep Ethanol of so1vation.b cation ligand/cation % Alcohol- % Hydrox- molecules ate ide adduct per cation

n-Arabinose

Na

1.18

87

13

0.2

D-Glucose

Na

K

2 .o 1.9

90 8s

10 15

0.3 0.3

D-MUUIW

Na

1.18

88

12

0.06

Maltola

X

2 .o

97

3

0 -07

Methyl a+ glucopyranoside 3, anomer

Na

1.8

88

12

0.4

Na

2.3

70

30

undet.

D-Glucitol

Na

1.6 1 .o

88

82

18 12

1.6 1.4

80

20 23

0.2 0.2

Li Sucrose~

Na

2.1 1.4

77

0 The aemmption ie made that the combining ratio for the dcoholate is identical to that for the hydroxide adduct. Percenteges are in mole percent. Solvent remaining after vacuum treatment at 26" fox 10 hr. 0 3-Hydroxu-zmethyl~-pyran~ne. d Uae of methanol inatead of ethanol gave a 1.8: 1 oomplex that consisted of 70% of alcoholate and 30% of adduct.

anhydropa ethanolic medium. The alcoholate content was determined by the modified Karl Fisoher analysis discussed in Section IIIJ (see p. 242). It is interesting that the percentage of alcoholate in the &glucose complex is approximately the same as that in the D-glucitol complex, even though ~gluco~m because , of its free reducing group, is more acidic than D-glucitol. Ordinarily, it would have been expected that this difference in acidity would have a much greater effect on the relative proportions of alcoholate

A L W L I AND ALKALINE-EARTH METAL COMPLEXES

257

in the two complexes. One explanation for the Similarity in alcoholate content is that alcoholates are, possibly, less soluble than adducts and are, therefore, the first to be precipitated. Another explanation is that inner chelation is such an exceptionally strong driving force in the formation of alcoholates that any difference in carbohydrate acidity is of minor importance. Methanol is rarely used as a solvent in the preparation of alcoholates. A monopotassio derivative of 1,2-anhydro-a-~-glucopyranose,lasand a sodio derivative of sucrose (see Table VIII), have been isolated from methanolic media. The facile'removal of hydroxylic protons from 2olyhydroxy compounds was first demonstrated by Sugihara and WolfrornJy8@ who treated cellulose with sodium hydroxide in boiling 1-butanol, and removed water of formation by concurrent azeotropic distillation. Later, Wolfrom and El-Taraboulsi'" similarly treated methyl a-D-ghcopyranoside, and obtained the monosodium alcoholate (reaction 4). Subsequent investigations by

+

GHMOS NaOH

* GHISOiNa + HzO

(4)

RendlemanDB have shown that the monosodium alcoholate of methyl CY-Dglucopyranoside can also be obtained by treatment of the ~-glucosidewith sodium 1-butoxide in boiling 1-butanol (reaction 6). Heat is probably not GHuOS

+ NaOBu * C&O~Na + BuOH

(6)

essential for effecting the formation of the alcoholate from sodium hydroxide in 1-butanol, in view of the ease with which alcoholates are formed in ethanolic sodium hydroxide solution at room temperature. However, to ensure complete removal of hydroxide ion and any possible water of hydration from the isolated complex, azeotropic distillation is a necessary operation. In a refluxing solution of sodium hydroxide and methyl a-D-glucOpyranoside in 1-butanol, possibly both the hydroxide ion and the butoxide ion react directly with the D-glucoside to produce an alcoholate. Sodium 1-butoxide is formed readily, in quantitative yield, by refluxing a solution of sodium hydroxide in an excess of 1-butanol (reaction 6) under dry NaOH

+ BuOH

NaOBu

+ HtO

(6)

nitrogen while water of formation is being removed by azeotropic distillation?' The product isolated from the reaction of methyl a-D-glucopyranoside with either sodium hydroxide or sodium 1-butoxide in refluxing 1-butanol (138) A. Pictet arid P. Castan, Helu. Chim. Ada, 4, 319 (1921). (139) J. M. Sugihttra and M. L. Wolfrom, J . Am. Chem. Soc., 71, 3509 (1949). (140) M. L. Wolfrom and M. A. El-Taraboulsi, J . Am. Chem. Soc., 76, 5350 (1953).

258

J. A. RENDLEIIMN, JB.

contains a small proportion of butoxyl p u p , a which suggests that reaction (7), the formation of a butoxide adduct, contributes significantly to the

+

C1HlrO~ NaOBu

ClrH1dOvNeOBu

(7)

overall reaction. Reducing sugars are decomposed by sodium hydroxide in boiling butanol. Treatment of reducing sugars with alcoholic alkali metal hydroxide must, therefore, bo conducted at room temperature, in order to avoid this decomposition of the ctirbohydrate. There have been few investigations of the reactions of polyhydroxy compounds with alkali metal &oxides in nonaqumus media at room temperature. Percivalw reported the preperstion of ogluoose*NaOEt and D-glucose*NaOMeby treating Dglucoee with sodium ethoxide, and penta0-acetyl-D-glucopyranosewith sodium methoxide, respectively The adducts were dried under vacuum at 00” for 24 hours, a treatment which would make the presence of any significant proportion of alcohol of solvation unlikely. Any traces of moisture in the preparative medium preclude the formation of an alkoxide adduct. Early reports that D-glucose,* ~-fructose,”~and lactosdu react with sodium ethoxide in anhydrous ethanol at room temperature to give alcoholates axe questionable, because the products were not analy5ed for their ethoxyl content. hmpl6n and Kunz”* treated *glucose with sodium in absolute ethanol to obtain a compound that they believed to be a 1:l D-glucose-sodium ethoxide adduct; and Pringaheim and DernikosIu treated the monoaoetate of a datrin with sodium ethoxide, and obtained a compound having one sodium atom per wglucose d u e . However, the analyses performed were insufficientto permit identificationof the products. Alekhinelu treated turanose with sodium ethoxide in ethanol, and obtained a product containing one sodium atom per turanose moiety; however, he did not specify that he used anhydrous conditions, and he did not record an ethoxyl analysis. Pacsu and coworkersla have reported the preparation of the sodium alcoholatc of cellulose by treating either cotton or viscose rayon with either sodium methoxide or sodium l-butoxide in anhydrous alcoholic media, at temperatures ranging from 25 to 120’. No proof of formation of alcoholate waa offered. However, there is little reason to doubt its occurrence at the higher reaction temperatures. The ratio of sodium to *glucose (141) M.H b i g and M. Roeenfeld, Bet., 14, 45 (1879).

.

(143) 0. Zemplcln and A. KUM, Bet., MI, 1705 (leas). (143) € Pllingsheim I. and D. Dernikoa, Ber., 66, 1433 (lea). (144) A. Alekhine, Ann. Chirn. (Paria), l8, 632 (1889). (146) R. F.Bohwenker, T. Kinwhite, K. B e u r h , and E. Pamu, J . Polyma Sci., 51, lSti:(lesl).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

259

residue varied with the time of reaction, the extractant (alcohol or acetone) used in extracting residual sodium alkoxide from the reaction product, the length of time for the extraction, and, to a small extent, the temperature. Sodium 3-methyl-l-butoxide, dispersed in xylene, has dao been used in preparing sodio derivatives of cellulose.1a In summary, the reaction between an alkali metal &oxide and a polyhydroxy compound in hot alcoholic media produces an alcoholate and, possibly, a small proportion of alkoxide adduct; however, the conditions governing the ratio of alcoholate to adduct have not yet been well defined. Reactions with alkali metal hydroxides and cyanides produce mixtures (of alcoholate and adduct) that consist mainly of alcoholate. Occurrence of reactions between alkaline-earth metal hydroxides and polyhydroxy compounds in anhydrous alcoholic media has not been reported. a. Stoichiometry of Alcoholate Formation.-The ratio of carbohydrate to metal in an alcoholate complex is a function of several factors: the cation radius, the concentration of metal hydroxide in the preparative medium, and the configuration of the carbohydrate.w With monosaccharides, oligosaccharides, and many of their derivatives, at hydroxide concentrations of about 0.05 M or less, and with the carbohydrate present in either a stoichiometric proportion or an excess, the ratios approximate to the maximum values (see Table IX); these are, usually, 2:l for alkali metals other than lithium, although there are exceptions where the maximum ratios are 1:1 and 3: 1. Lithium alcoholate complexes do not show ratios greater than 1.5: 1; many me 1:1. High concentrations of hydroxide l e d to smaller ratios. At about 2 M concentration, the ratios generally approach a minimum of 1:1 for monosaccharides and their derivatives. For oligosaccharides, smaller ratios are possible. However, a large fraction of the metal in these low-ratio complexes is probably present as alkali metal hydroxide. Studies of sucrose in the presence of a high concentration of potassium hydroxideg6 indicate that, although the resulting complex contains more than one atom of potassium per carbohydrate moiety, no more than 1 hydroxylic proton is removed from the sugar molecule. Ethanolic potassium hydroxide (20%) reacts with sucrose and with sucrose octaacetate to give complexes having a carbohydrate-to-metal ratio of 1:2. For each complex, a modified Karl Fischer analysis indicated that slightly less than 1 proton had been given up by each carbohydrate moiety. The complex obtained from pure sucrose had the approximate composition CIZH~O-(OH *KOH)1.8(OK)0.7.Conceivably, large molecules having reaction sites (hydroxyl groups) separated (146) V. A. Derevithya, M. Prokof’eva, and 2.A. Rogovin, Zh. Obshch. Khim., 28, 1368 (1958).

z

Tmm IX Maximum combining Ratios of Alcoholate cOmploteso Isolated from Anhydrous Ethanolic MediaMaximum combining ratio, ligand/cation

Li'

Alcoholate

Na " 1.2 (0.2EtOH)

-

1.0 (1.4EtOH) 1.5 1.1 1.5 (no EtOH)

1.4 1.4 1.4

1.5 2.3 1.5 (1.6 EtOH) 2.2 1.9 (0.4EtOH) 2.0 (0.3 EtOH) 2.0 1.2 (0.1EtOH) 2.2

-

2.1 (0.2EtOH) 1.9

K"

1.9 (0.5EtOH) 2.3b 1.9 2.3 1.9e 3.8 (0.3 EtOH) 2.2 2.0 (0.3EtOH) 2.3 2.0 2.1 (0.4EtOH) 2.0 2.0 2.1 1.9 (0.3EtOH)

G 3 '

2.3 2.2 2.6

-

2.0 (0.1 EtOH)

-

2.0 1.9

-

2.1

a A minor, but siepificsnt, fraction of each isolated complex i an alkali metal hydroxide adduct. All complexes were treated 86 pure alcoholate in the calculations of the combining ratio. Except where indicated by superscript c, the ratios are corrected, for ethanol content, only in those casee where the solvent content is known (ethanol of solvation, if known, is reported as molecules per cation in parentheam next to the combining ratio). b Based on the dimeric form of the aldose. The complex is actually composed of approximately four aldose residues. c Estimated on the basis of a b b l e molecule of solvation (ethanol) Der cation.

t..

*

E

Z

E

-Z

P

ALKALX AND ALKALINE-EARTH METAL COMPLEXES

261

by relatively great distances should function as polybasic acids that release 3 or more protons. Gaver’” claimed to have prepared sodium “starchate” by treating starch with alcoholic sodium hydroxide solution; a polysodium alcoholate was presumed to have been formed by the release of a proton from each D-glucose residue. No conclusive proof that each sodium atom is associated with a carbohydrate oxyanion was, however, given. Although the stoichiometry varies with the concentration of hydroxide, favored combining ratios do exist. Certain ratios predominate, over a wide range of concentration. The ability of a metal alcoholate to accommodate an additional molecule of carbohydrate increases with increasing ionic radius:gBLi < Na < K < Cs. The difference in stoichiometry between lithium and sodium is much greater than that between either sodium and potassium, or potassium and cesium. The coordination number of an alkali metal is known to increase with increasing ionic radius. Brewerla reported that the maximum number of donor groups oriented about an alkali metal cation is four for lithium, and as many as six for sodium, potassium, rubidium, or cesium. A greater surface area would allow accommodation of more than one carbohydrate moiety; but, in addition, solvent molecules are more strongly attachpd to cations of smaller radius, and these may not be readily displaced by carbohydrate molecules. The importance of carbohydrate configuration in the formation of complexes is exemplifiede6 by the behavior of certain isomeric sugars. For example, each of the four D-aldopentoses (D-arabinose, D-xylose, D - I ~ X O S ~ , and D-ribose) reacts with potassium hydroxide or cesium hydroxide to form a complex having a carbohydrate-to-metal ratio of 2:l. However, with sodium hydroxide, only D-xylose, D-lyxose, and D-ribose form 2:l complexes; n-arabinose gives a 1:1 complex. Similarly, D-mannose reacts with sodium hydroxide to form a complex having a maximum ratio of 1: 1, whereas both D-glucose and D-galactose form 2 :1 complexes. With potassium hydroxide, all three of these hexoses give 2: l complexes, A 3: l complex is formed by the reaction of levoglucosan (lt6-anhydmj3-Dglucopyranose) with potassium hydroxide. Weber, Ashar, and Champetier*24treated cellulose (dried cotton linters) with 1 M sodium hydroxide in various anhydrous alcohols at room temperature. Although no analyses were performed to determine whether the products were alcoholates or adducts, our present knowledge concerning simple carbohydrates in anhydrous media would strongly suggest that the products contained a large, if not preponderant, proportion of alcoholate. When equilibrium wm established in alcoholic systems that were 1 M in (147) K.M. Gaver, U.S. Pat. 2,397,732 (1946); Chem. Abstracts, 40, 3620 (1946). (148) F.M. Brewer, J . Chem. Soe., 361 (1931).

262

J. A. RENDLEMAN, JR.

sodium hydroxide, the following frtlctions of a mole of sodium hydroxide were found to have reacted per mole of D-glucose residue: in methanol, 0.124; in ethanol, 0.26; in l-propanol, 0.58; and in l-butanol, 0.78. In water, only 0.07 mole of sodium hydroxide reacted. Equilibrium was established very rapidly in methanol ( <24 hours). In the higher alcohols, a period of more than 400 days was required. reacts with butanolic sodium hydroxide The e m with which at the reflux temperature (with concurrent azeotropic distillation to remove water of formation) to give alcoholates containing one metal ion per ~-glucose residue suggests that similar treatment of nonreducing oligosaccharides would, likewise, give alcoholates containing one metal ion per monosaccharide residue. No such reaction with oligosaccharides has, as yet, been reported. The formation of adducts of polyhydroxy compounds either with alkali metal alkoxides or with cyanides has not been sufliciently studied to permit any generalizations to be made regarding its stoichiometry. Table IX (see p. 260) lists many of the known alcoholate complexes prepared from alkali metal hydroxides in snhydroua ethanolic media. For additional complexes from ethanolic media, the reader is referred to Table VIII (p. 256) and to Rsf. 96.

b. Electrophoresis.-Many fmtors other than alcoholate formation might influence the rate and direction of electrophoretic migration: (1) the degree of dissociation of the metal alcoholate into free ions, (2) the size of the carbohydrate anion, (3) the degree to which the metal cations combine with the carbohydrate to give free, positively charged, carbohydrate species, and (4) the carbohydrate-to-metalcombining ratio. The competition between hydroxide ion and metal ion for carbohydrate has been demonstratedMin a combination of electrophoretic experiments involving Dglucom, D-xylose, sucrose, and D-glucitol. In methanolic lithium h y d r m ' h solution, all movements were toward the anode. In methanolic sodium hydToxide solution, movement of D-xylose and D-glucosewas toward the anode, whereas movement of sucrose and D-glucitol was in the oppoeite direction. A reasonable interpretation of these phenomena rests solely on the assumption that a competition exists between the metal cation and the hydroxide ion for the carbohydrate. Free alcoholate anions, as well as any free carbohydrate-hydroxide ion species, would migrate toward the anode. Cationic species, produced by the chelation of a free, metal cation,with one or more carbohydrate molecules, would migrate toward the cathode. Negative and positive species should both exist; however, the mobility of each species and the extent to which each is formed determine the rate and

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

263

direction of carbohydrate migration actually observed. I n alcoholic sodium hydroxide solution, the two opposing movements almost cancel each other, and migration is slow. The somewhat acidic sugars, D-xylose and D-glucose, migrate slowly as an anion, but the much less acidic carbohydrates, sucrose and D-glucitol, which do not have an acidic, hemiacetal hydroxyl group, maintain a net positive charge and migrate toward the cathode. In methanolic lithium hydroxide solution, all carbohydrates migrate toward the anode, because. of the extremely weak ability of lithium cations to form positively charged carbohydrate species (see Section 11,6, p. 227). Electrophoresis does not show the presence of uncharged species, such as undissociated metal alcoholate or carbohydrate-metal hydroxide sdducts. These species are probably present in alcoholic solutions, but their concentration has not yet been ascertained. Their presence is suggested by the relatively low mobility of carbohydrates in alcoholic solutions of alkali metal hydroxide. In aqueous media, where greater dissociation of ion pairs should occur, the mobility is extremely high. The possible existence of free carbohydratehydroxide ion species cannot be disregarded, because of the hydrogen-bonding properties of the hydroxide ion. c. Preparation of Alcoho1ates.-Only a brief description of the general procedure for preparing alcoholates will be given. The solubility of both the carbohydrate and the alcoholate often determines the proportion of solvent necessary. To a solution of carbohydrate in either ethanol or N-methyl-2-pyrrolidinone is rapidly added dropwise, with stirring, an ethanolic solution of alkali metal hydroxide. If the product tends to remain in solution under these conditions, ether is added, either before or after addition of the hydroxide, in order to facilitate precipitation. The concentration of the hydroxide solution being added is usually a.very important factor in determining the combining ratio of the alcoholate (see Section III,2a, p. 259). After the product has been separated from its mother liquor, it is quickly washed with ethanol or with a mixture of ethanol and ether, and then dried under vacuum. Alcoholates of carbohydrates are colorless, amorphous, and, like the corresponding alkali metal hydroxide adducts, very hygroscopic. Alcoholates of nonreducing saccharides (soluble or insoluble) and of saccharide derivatives may also be prepared by the method of Wolfrom and coworkers.g2." A mixture of the carbohydrate and sodium hydroxide in 1-butanol is refluxed, preferably under a nitrogen atmosphere, Pnd all of the water of formation is removed by concurrent azeotropic distillation. This method is preferred when no trace of hydroxide adduct or water is desired in the final product. Butoxide ion is a possible contaminant, how-

264

J. A. RBNDLBMAN, JR.

ever. The use of a 1:1 molar ratio of sodium hydroxide to monosaccharide residue in the remtion medium leads mainly to the monosodium alcoholate ( b d on the monosaccharide residue). The method of GaveP7 for nonreducing carbohydrates (refluxing the carbohydrate in alooholic alkali metal hydroxide solution without concomitant amtropic distillation) night give results similar to thoae obtained by the Wolfrom method.m*" However, the presence of water and hydroxide ion in the final reaction mixture would probably cause a. small fraction of the product to be the hydroxide adduct. 3. Reaction# in Nonhydroxylic Solvents

Media other than water and alcohol have thue far been virtually ignored. However, ethylenediamiaehas received some attention, apparently because of its ability to facilitate the release of protons from compounds whose acidity is ordinarily considered to be very weak. Derevitskaya, Smirnova, and bgovin;4@ in a study of the degree of dissociation of the hydroxyl groups of Dglucose, methyl cu-D-glucopyranoside,methyl @-D-glucopyranoaide, cellobiose, and maltose, electrometrically titrated 0.001 M solutions of these compounds in e t h y l e n d i e with 0.1 M potassium hydroxide (alcoholic), The volume of the titrant was plotted against the derivative of the current with respect to the volume of the titrant. The resulting curves contained a number of peaks that were interpreted as being the equivalence points of the different hydroxyl groups. The first peak on the curves for D-~~UCOBB,maltose, and cellobiose was the highest, and it differed greatly in height from one carbohydrate to another. If the assumption is made that the heights of these first peaks characterize the degree of dissociation of the respective anomeric hydroxyl group, these compounds c m be arranged in the following order of decreasing acidity of the anomeric hydroxyl group: D-glucose > cellobiose > maltose. A comparison of the curves of the two ~glucopyranosidesindicates that the @-D Bnomer is More acidic than the WD anomer. Five peak8 were obtained for pglucose, and four for each of the two D-glucopyranasides. There is some question, however, as to whether each of these peaks corresponds to the titwtion of a different hydroxyl group. Rendlema,n160has treated D-glucose with potassium hydroxide and with sodium hydroxide in ethylenediamine under the conditions employed by Derevitskaya and ooworkers,14@and has found that (1) addition of either potassium hydroxide or sodium hydroxide to the Dglucose solution results in the precipitation of a product (an almost (140)

V. A. Derevitakaya, G. S. Bmirnova, and 2;. A. Rogovin, Proc. A d . 815.USSR,

Chnn. Sect. (Englkh Trend.),U6,1254 (1981). (160) J. A. Rendleman, Jr,, unpubliehed observations.

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

265

invisible gel) before half of the stoichiometric proportion of the hydroxide (the amount reqgred for reaction with five hydroxyl groups per molecule) has been dded, and (2) the product that is isolated after a stoichiometric proportion of hydroxide has been added contains essentially all of the sugar originally present in solution, but no more than two atoms of alkali metal per D-glucose moiety. Complete identification of the derivative has not yet been attempted. Treatment of D-mannose with lithium hydroxide, and with potassium hydroxide, in liquid ammonia161gives a crystalline di-lithio derivative and a crystalline monopotassio derivative, respectively. The structures of these products have not been determined. The use of metal alkyls, metal aryls, metal hydrrdes, and metal carbides for preparing alcoholates of carbohydrates in inert, aprotic solvents has not yet been reported. 4. Structure of Alcoholates and Adducts

The general structure of alkali metal alcoholates of polyhydroxy compounds is probably very similar to those proposed by Martell and CalvixP for the alkali metal chelates of o-salicylaldehyde (see Figs. 9 and 10).laJmJM Unfortunately, because of the highly amorphous nature of nearly aJl pf the alcoholates and adducts formed by the interaction of metal hydroxides with carbohydrates, x-ray diffraction studies have failed to furnish information regarding the precise location of the metal in them complexes. The ease of formation of alcoholates and adducts can be related to both the acidity and the geometry of the polyhydroxy compound. The geometry is important, in that the chelate ring must possess a minimum of strain in order to allow the complex to possess the maximum stability. Reaction of a cyclic 1,2diol with a metal hydroxide is physically impossible if the hydroxyl groups are oriented in directions that are exactly opposite (180') to each other. Cyclic 1,&diols can form chelates if both of the hydroxyl groups are cis and axial. An example of this is the reaction of 1,&anhydro-

Fro. %-Lithium Alcoholate of Salicylaldehyde. (151) (162) (153) (154)

K. Shimo and R. Tada, Sci. Rept. Re8. Inst. Toholcu Uniu., Ssr. A , 7, 235 (1955). Ref. 70, p. 242. N. V. Sidgwick and F. M. Brewer, J . Chem. Soc., 127, 2379 (1925). 0. L. Brad\ and W. H. Bodger, J . Chem. Soc., 952 (1932).

266

J. A. RENDLEMAN, JR.

Fro. lO.-Sodium Alcoholate of Salicylsldehyde.

PD-glucopyranose with potassium hydroxide in ethanol to give an alcoholate, whose structure is possibly that shown in Fig. 11. The location of the metal cation (or cations) in alcoholates has been investigated by the method of substitutive methylation for the sodium derivatives of methyl a-wglucopyranoside, amylose, and cellulose. The method assumes that methylation by either dimethyl sulfate or methyl iodide occurs only at an anionic oxygen stom. Lendu methylated the monosodium alcoholstes (prepmed in boiling, butanolic sodium hydroxide by the

FIU.Il.-PoeSible glucopyrenoee. (165)

Structure of the Potsesiurn Alcoholate of l,BAnhydro-&n-

R.W.Lena, J . Am. C h .Soc., 89, 182 (1960).

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

267

method of Sugihara and Wolfram*@) of methyl a-o-glucopynmoside and cellulose with methyl iodide, and obtained producta that he subsequently hydrolyzed and analyzed by quantitative, paper chromatography. The hydrolyzates contained mono-, di-, and tri-0-methyl-D-glucose and unsubstituted ~glucose.The mono-O-methyl fraction contained the principal isorsers, of which 2-O-methyl-~-glucosewas the ether present in the highest percentage. From the distribution of the methoxyl groups in the monosubstituted wglucosea, the relative rate constants for substitution at the hydroxyl groups on C-2, (2-3, and C-6 were calculated to be 5, 1, and 2.5, respktively. It should be pointed out that the failure to observe monosubstitution at the hydroxyl group on C-4 of methyl eD-glucopyranoside could have been due to a lack of separation of 3-O-methyl-~-glucosefrom the 4-methyl ether by paper chromatography. Thus, the relative rate for substitution calculated for the hydroxyl group on C-3 is, possibly, actually equal to the combined relative rates for substitution at the hydroxyl groups on G 3 and (2-4. To explain the formation of the di- and tri-methyl ethers, Lena1" suggested two possible mechanisms: either (1) methylation occurs on' un-ionized hydroxyl groups, or (2) the distribution of ionized hydroxyl groups (anionic oxygen atoms) changes continually during the course of the reaction. Bines and WhelaxP methylated the monosodium "alcoholate" of amylose (prepared by heating amylose with butanolic d i u m hydroxide for 2 hours in a closed v-1 at 85-87") by means of a method16' which involves heating a mixture of the alcoholate and a methyl halide in toluene at 100" in a sealed reaction vessel for 4 hours. Hydrolysis of the product gave mono-0methyl-D-glucoses (36.4%), di-0-methyl-D-glucoses (14.6%), unsubstituted D-glucose (43.573, and 5.5% of remaining material that had been incompletely hydrolyzed. The 2-, 3-, and 6-O-methyl-wglucoses were present in the proportions of 2.6: 1:1.2. Doane and coworkers1@methylated starch by the method of Hodge, Karjala, and Hilbertl" (sodium metal-liquid ammonia-methyl iodide) to degrees of substitution (D.S.) of 0.34 and 0.90 methoxyl per D-glucose residue. Hydrolysis of the products gave D-glucose, and mono-, di-, and tri-0-methyl-*glucose in the approximate molar ratios of 36: 6 :2 :1 and 6:3:2:i1, for the D.S.0.34 and 0.90 products, respectively. Resolution of the monomethyl ethers into their isomers showed a 1:1ratio of 2-O-methylD-glucose to BO-methyl-~-glucosein the D.S.0.90 hydrolyzate, and a 3:2 (150) B. J. Bmea and W. J. Whelm, J . Chenl. Soc., 4222 (1982). (157) K. M. Gaver, E. P. Lasure, and D. V. Timen, U. 8. Pat. 2,871,779 (1964); f%sm. Abeirmt8, 48, 8589 (1954). (158) W.M.Doane, N.L.Smith, C. R. Rumll, and C. E. Rist, Staerke, 17,225 (1966). (159) J. E.Hodge, 8.A. Karjala, and G.E.Hilbert, J . Am. Chem. Soc., 78, 3312 (1951).

268

J. 4. J t W D L E W ,

JR.

ratio in the D.S.0.34 hydrolyzate. Significantly, there wae no 3-O-methylD-glucose in either hydrolyoate. The sodium alcoholate of cellulose prepared from a sodium alkoxide is probably similar to, if not identical with, that prepared from sodium hydroxide. Manomethylationof the alcoholate prepared with either sodium occura preferentially a t the hydroxyl methoxide or sodium l-b~toxidel4~ groups on C-2 and C-6. Very little methylation occurs a t the hydroxyl group on C-3. In adducts of alkali metal hydroxides, the hydroxide ion is probably chelated to two or more hydroxyl groups and attached most strongly to the hydroxyl group of highest acidity. Percival@-lwJ1O methylated a number of potassium hydroxide adducts in an attempt to determine the positions of the potassium hydroxide residues. His method waa based on the likely assumption that dimethyl sulfate reacts only with those hydroxyl groups to which OHe ion b attached. The results of his studies of product isolation employing simple carbohydrates (D-glucose,” sucrose,11ocellobiose,”JW maltose,”>” and lactosem) indicated that, for D-glucose, the potassium hydroxide is located preferentially, if not exclusively, a t the anomeric hydroxyl group; for sucrose, the potassium hydroxide is attached mainly to the primary hydroxyl groups; for cellobiose, the attachments are at the free anomeric hydroxyl group and at the hydroxyl groups on C-2 and (2-3; for lactose, the attachments are at the anomeria hydroxyl group of the ,D-glucopyranoseresidue and at the hydroxyl groups at C-2 and C-4 of the D-galactopyranose residue; and for maltose, the attachments are at the anomeric hydroxyl group and at the hydroxyl groups at C-2 and C-6 of one of the D-glucopyranose residues. the 1:1 potassium hydroxide adducts According to Heddle and Percival,gB of amylose and cellulose are methylated, by dimethyl sulfate, preferentially at the hydroxyl group on (2-2; no methylation of the hydroxyl group at C-6 was detected. Croon and coworkers,1m however, using more advanced analytical techniQues, later found that sodium hydroxide adducts of amylose and cellulose (which should not differ significantly from the potassium hydroxide adducts) can be methylated with dimethyl sulfate at all three of these hydroxyl groups. The relative rates for the methylation of cellulose at the hydroxyl groups on C-2, C-3, and C-6 are 3.5,1, and 2, respectively; for amylose, they are 6, 1, and 7, respectively. It was suggestedlm that intrachain hydrogen-bonds play an important role in the reactivity of the hydroxyl groups in both celluloBe and amylose. (160)

I. Croon and B. Lindberg, Suenek hpperstid., 6B, 794 (1958); 60, 843 (1957); I. Croon, ibkd., 61, 919 (1968); I. Croon and E. Flsrnm, ibid., 61, 863 (1958); I. Cronu, Ada Chem. Scad., la, 1235 (1969).

ALKALI AND ALKALINE-EARTH METAL COMPLEXEB

269

Htwch can he vinylated with acetylene in the presence of potassium hydroxide in an W~UCOUHtctrahydrofuran medium.16' The mechanism poecJibly involven the addition of the potassio derivative of starch across the carbon-carbon triple bond of acetylene, with subsequent hydrolysis of the organometallic intermediate to give the vinyl ether. Such a mechanism has been postulated for the formation of vinyl ethers from monohydric alcohols and acetylene, in the presence of an alkali metal base as catalyst.162The vinylation of amylose is very similar to the vinylation of amylopectin, except for the relative ratio of mono- to di-substitution.'6" With amylopectin, the proportion of disubstitution is greater. In both starches, the hydroxyl group on C-2 is slightly more reactive than the hydroxyl group on (2-6; there is little substitution at the hydroxyl group on C-3. The fact that a carbohydrate can be vinylated in the presence of a base catalyst in ,an aqueous system is a good argument for the existepce of carbohydrate alcoholates in aqueous media. However, successful vinylation does not imply that the concentration of the alcoholate is necessarily high. Because the interaction between the carbohydrate and the hydroxide ion is rapidly reversible, the concentration of the alcoholate may not have to be large for vinylation to proceed at a moderate rate. There have been no studies of adducts of alkalineearth metal hydroxides with a view to determining the position, or positions, of attachment of alkaline-earth metal hydroxide on carbohydrates.

OF

IV. ALCOHOLATES FROM REACTIONB, IN LIQUID AMMONIA, CARBOHYDRATES WITH ALKALIMETALS,ALKALINE-EARTH METALS, AND ALKALIMETALAMIDES

Chablay16a* treated D-mannitol with sodium and with potassium in anhydrous liquid ammonia, and obtained the respective monoalkali metal alcoholates. Later, Schmid and coworkers1" reported the preparation of monosodium alcoholates of ethylene glycol, glycarol, wmannitol, methyl a-wglycopyranoeidq, starch, inulin, lichenin, glycogen, chitin, ~-glucose, and *fructose by treatment of the respective carbohydrate with sodium in liquid ammonia; similarly, starch, inulin, lichenin, D-mannitol, and (161) J. W. Berry, H. Tucker, and A. J. Deutsahm, Jr., Z n d . Eng. Chum., Procese Dcsign Develop., I,318 (1983). (162) W.Reppe, Ann., 601, 81 (1966). (163) J. W. Berry, A. J. Deutmhmen, Jr., and J. P. Evans, J . Otg. Chsm., I S , 2819 (1964). (1638) E.Chtlthy, Compt. Rend., 140, 1396 (1905). (164) L. Bnhrnid and U. Becker, Ber., 58, 1966 (1925); L. Sahmid, A. Wsschlcau, and E.Ludwig, Monubh., 4S, 107 (1928).

270

J. A. RENDLEMAN, JR.

methyl a-Pglucopyranoside reacted with potassium to give the respective monopotassium alcoholates. Muskatla' improved the procedure, and prepared potaslilium alcoholat- of various highly substituted monosaccharides. The report on the preparation of the alcoholatea of D-glucose and D-fructose is probably incorrect. Liquid ammonia itself reacts with either a free or an acylated anomeric hydroxyl group,laa affording the corresponding glycosylamine, without affecting acyloxy groups attached to other carbon atoms. However, alkali metals remove acyl group of O-acylated glycosylamines to give alkali metal derivatives. Lithium, sodium, and potctssium in liquid ammonia have been used107 t o prepare the respective monoalkali metal derivatives of sucrose. Prey and Grundschobe+ used similar means to prepare the monosodio derivative. Di-, tri-, tetra-, and pentasodio derivatives have also been prepared. ~6~ ~ 7 0Ammonia of solvation, which is strongly held by the alcoholates, can be almost entirely removed by extraction with toluene.'@ Amagasa and Onikura171 treated sucrose with potassium in liquid ammonia under preasure at room temperature, and obtained a dipotassio derivative. Further addition of potassium to the reaction mixture gave a pyrophoric substance that appeared to be a mixture of hexa- and hepta-potaseio derivatives. D-Mannitol, likewise capable of undergoing polysubstitution, can form a di- and a tri-lithium alcoholate when it is treated with lithium in liquid ammonia; sodium and potassium give disubstitution products; and calcium gives a monocalcium alcoholate.lS1Treatment of cellulose with aodium17* can lead to the trisodio derivative. The first atom of sodium enters rapidly; the second and third, slowly. Sodium in either pyridine or morpholine reacts with sucrose to give ti substance that, reportedly, does not have the properties of an al~oholate.~~7 Further work with these solvent media is necessary before any definite conclusions regarding the reaction products can be made. Sodamide in liquid ammoniala7reacts with sucroae much faster than does (165) I. E. Muekat, J . Am. Chcm. Soc., 66, 693, 2449 (1934); P. A. Levene and I. E. Mudcat, J . BioZ. Chcm., 106, 431 (1934). (166)R.8.Tipeon, MeuIods Carbohvdde C h . ,9, 160 (1903). (167) P. C. Arni, W. A. P. Black, E. T.Dewar, J. C. Petenron, and D. Rutherford, J . A w l . Clam. (London), 8, 180 (1959). (168) V. Prey and F. Crundschober, Mondsh., 81, 1186 (1960). (169) W. A. P. Black, E.T.Dewar, and D. Ruthexford, J . C h . Soc., 3073 (1959). (170) W. A. P. Black, E. T. &war, J. C. Patereon, and D. Rutherford, J . Appl. C h . (London), 8, 286 (1959). (171) M. Amagaea and N. Onikurs, Kogyo Kagaku Zasshi, 69, 2 (1949). (172) P. C. Scherer and R. E. Hussey, J . Am. Chem.Soe., 68, 2344 (1931); P.Shorigin and N. Makarowva-SemljanRkaja,Rer., 88, 1713 (1936).

ALKALI AXD ALKALINE-EARTH METAL COMPLEXES

271

sodium to give sodium alcoholates. The use of sodamide haa permitted the isolation of a heptasodio derivative. An octasodio compound that has pyrophoric properties has also been reported. Potassium amide reacts with o-msnnito1161 in liquid ammonia to produce a dipotado derivative. Tipson'" has described, in detail, two general procedures,both involving the use of liquid ammonia and alkali metal, for the methylstion of nonreducing carbohydrcttes and their derivatives. He offered procedures for t4e preparation of 1,2: 5 ,6di-O-isopropylidene-3-O-potassio-cu-D-glucofurmose, methyl tetra-0-potassio-a-o-mannopyranoside, and tri-0-sodiocellulose; and for the conversion of these alkali metal derivatives into 1,2 :5 ,&li-Oisopropylidene-3-0-methyl-~~-~-glu~ofuranose, methyl tetraO-methyl-a-~-mannopyranoside, and tri-0-methylamylose, respectively. Factors that dictate the choice of the alkali metal in the preparation of methyl ethers are: (1) the solubility of the metal, (2) the solubility of the alcoholate. (3) the reactivity of the alcoholate, and (4) the solubility of the metal halide formed during reaction of the alcoholate with methyl halide.

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SYNTHETIC CARDENOLIDES BYW. WERNERZORBACHAND K. VENKATRAMANA BHAT Deparimeni oj C b i a t r y , Oeorgeioum Universiiy, Waahingbn,D. C.

I. Introduction.. ....................................................... 273 11. General Methodology Employed.. ...................................... 275 1. Koenigs-Knorr Method.. ........................................... 275 2. Meystre-Miescher Modification. ..................................... 277 3. Mercuric Cyanide Procedure.. ....................................... 278 4. Limitations Imposed by the Sensitive Nature of the Aglycon. ........... 279 111. Synthesia of Individual Glycosidee. ..................................... 281 1. Cardenolides Containing Sugars Having a Hydroxyl Group a t C-2 ....... 281 2. Cardenolides Containing 2-Deoxy Sugars.. ............................ 297 IV. Contribution of the Carbohydrate Component to Physiological Activity. .... 311 1. Assay Procedure, .................................................. 311 2. Some Correlations.. ................................................ 311 V. Table of Synthetic Cardenolides. ....................................... 318

I. INTRODUCTION Cardenolides, otherwise known as cardiac glycosides (to include, also, bufadienolides) are of plant origin, and comprise an outstanding class of drugs. They exert a specific action on the heart, and are of great clinical value in caws involving decompensation, where the heart is beating in a deficient condition. Auricular fibrillation, a serious cardiac disorder, responds in a dramatic manner to therapy with the drugs, and daily doses of 0.05-0.1 mg. of digitoxin per 0s generally suffice to maintain “normal” heart function. Qualitatively, the cardiotonic activity of the glycosides resides in the aglycon portion: which is steroidal in nature. The most widely studied aglycons are digitoxigenin [38, 14@dihydroxy-5fl-cd-20(22) -enolide ( 1) 3, (1) J. H. Hoch, “A Survey of Cardiac Glycosides and Genins,” University of South Carolina Press, Columbia, 5. C., 1961. (2) (a) R. Tschesche, in “ h r Sterine, Gallensffuren, und verwandte Naturstoffe,”

H. Lettr6, H.H. Inhoffen, and R. Tschesche, eds., Ferdinand Enke Verlag, Stuttgart, Ger., 1964, Vol. 1, p. 287. (h) C. W.S h o p p , “Chemistry of the Steroide,”

Butterworths Scientific Publications, London, Engl., 1968, p. 228. (0) L. FI Fieaer and M. Fieser, “Steroids,” Reinhold Publishing Corp., New York, N. Y.,1959, p. 728. 273

274

W. W. ZOIWACH AND K. VENKATRAMANA BHAT

obtained from Digitalis spp., and strophanthidin [38,58,14@-trihydroxy19-oxocard-20(22)-enolide (2)], occurring as a glycoside obtained from

HO

H

HO (a)

(1)

seeds of Strophanthus , kmnbd. Digitoxigenin (1) may be considered to possess the minimum structural requirements for cardiotonic activity; its structure is unique, and differs totally from that of other classes of saturated steroids, in that both the A/B and the C/D ring fusion we cis. Be cause of this sitdtion, ‘the 8 or “upward” hydroxyl group at C-3 is axially oriented. The (2-14 hydroxyl group has, likewise, the p, or “upward,” configuration. A change in configuration at G5 of ddtoxigenin (1) leads to the aglycon uzarigenin, in which the A/B fusion is traw; this small change results in a sharp diminution in cardiotonic activity.’ Epimerization at C3 to give 3-epidigitoxigenin,Blose of the C-14 hydroxyl jpoup, or saturation of the unsaturated lactone ring attached at C-17 of digitoxigenin ( l ) , leads to a loss in activity. The a,b-unsaturated carbonyl group in the lactone ring is a powerful chromophore, absorbing strongly in the ultraviolet region at 217-218 mp, and having an extinction coefficient in the neighborhood of 16,000. The absorption maximum and extinction coefficient for a given aglycon are influenced little, if any, by glycoside formation at the C-3 hydroxyl group. Knowing in advance, therefore, the extinction coefficient (6) for a particular aglycon, it is possible to calculate the molecular weight ( M )of glycosides prepared therefrom by a simple rearrangement (to give equation l ) of the equation used for calculating the extinction coefficient (E) from the

M

= egV!l

(1)

absorbance (q), where g is the number of grams of sample per liter and 1 is the width of the cell in om. This technique is used routinely by the authors and their coworkers for making a rough estimate of the molecular weighte of products obtained in the partial syntheses of cardenolides, and serves a~ a quick method for ascertaining whether glycoside formation has (8) (a) W. Schmid, € P. I. Mehlinger, C. Tamm, and T. Reichetein, Helv. Chim. Acla, 44, 72 (1959). (b) T.Golah, H.Jaeger, and T. Reichstein, ibid., 48, 2035 (1960).

SYYI'HETIC CARDESOLIDES

275

taken place. Although the error in this procedure amounts to &-I the %, estimateR are valid, owing to the substantial increase in molecular weight when the sugar residue is joined to the aglycon. The sugars found in the naturally occurring glycosides are, for the most part, rare hexoses of the deoxy type, and have been adequately covered in this Series in Chapters by Elderfield4 and Reichstein and Weiss.6 In addition, a review of 2deoxy sugars has been provided.' Although the sugars glycosidically bound in cardenolides have no physiological activity of their own, they enhance (often markedly) the cardiotonic activity which resides in the aglycon. It appears that the sugar residues influence the absorption and distribution of the drugs, and it is suggested that the carbohydrate component contributes to an adsorptive process that promotes the affinity of cardiac tissue for the cardenolide. In view of the important clinical position held by cardenolides, and because of the vital role of the glycosidically bound sugar(s) in promoting cardiotonic activity, the partial synthesis of cardenolides is of paramount importance. The end achieved by partial synthesis may be: (1) confirmation of the proof of structure of a naturally occurring cardenolide, (2) provision of a basis for structure-activity relationships in terms of the carbohydrate component, or (3) securing of cardenolides either more potent than those occurring in Nature or capable of bringing about digitalization more rapidly while having a longer duration of action.

11. GENERALMETHODOLOGY EMPLOYED

1. Kaenigs-Know Method The number of glycosides that have been prepared by the KoenigsKnorr synthesis7 is legion. Two reviews8J' dealing with the synthesis have appeared earlier in thiR Series, as well as a review of 0-acylglycosyl halidedo and one covering mechanisms of replacement reactions.ll The reaction (4) R. C. Elderfield, Aduan. Carbohydrafe Cham., 1, 147 (1945).

(5) T.Reichstein and E. Weiaa, Aduan. Carbohydrate Cham., 17, 65 (1962). W.G.Overend and M. Stacey,Advan. Carbohydrate Chem.,8, 45 (lap3). See also, 8. Hanesaian, This Volume, p . 143. (7) (a) W.Koeniga and E. Knorr, Sikber. Math-naturw. Abt. Bayer. A M . Wi88. Mtinehen, SO, 103 (1900);(b) Ber., 84, 957 (1901). (8) W . L. Evann, D. D. Reynolda, and E. A. Talley, Advan. Carbohydrate C h m . , 6, 27 (1951). (9) J. Conchie, 0. A. J m ~ v y ,and C. A. Marah, Aduan. Carbohydrate Chem.,19, 157 ( 1957). (10) L. J. HayneR and F. Newth, Aduan. Carbohydrate Chem., 10, 207 (1955). (11) R. U. Ikmieux, Aduan. Carbohydrate Chem., 9, 1 (1954). (6)

276

W. W. ZORBACH AND K. VENKATRAMANA BHA'I'

consists, in theory, of allowing an 0-acylglycosyl halide to react with an alcohol, giving a substituted glycoside plus the hydrogen halide. The -0

RCb il 0

presence of the liberated hydrogen halide is deleterious because it (1) tends to reverse glycoside formation, and (2) may effect partial deacylation of the substituted glycoside, thus introducing a source of c o n t a b tion. To obviate the effects, an acid acceptor, commonly silver carbonate: is employed to remove the acid ;and a second reaction takea place, resulting in the formation of silver halide, carbon dioxide, and water. When the 2 HX + AgaCOa -+ 2 AgX + Con + Ha0 Koeniga-Knorr reaction is carried out using simple, liquid aglycons (methanol or ethanol, for example), the rtlcohol also serves aa the solvent for the halide, and is frequently present in large excess. In such cases, the halide is rapidly converted into glycoside, and the water formed in the secondaq reaction between the liberated hydrogen halide and silver carbonate is seldom cause for concern. The synthesis of glycosides of alcohols that are solid at room temperature (especially costly ones) is a different matter. Obviously, the alcohol will not usually play the role of solvent; therefore, the halide and aglycon are brought together in an inert solvent in which they are mutually soluble. Inherent in such a procedure is a dilution effect which militatea agdnst good yields, especially with the less reactive, secondary alcohols, such aa the cardiac aglycons. In the synthesis of steroid glycosides which contain readily available sugtlrs, the steroid aglycon is nearly always the dearer of the two reagents, and, to compensate for the adverse effect of dilution when steroid and halide are permitted to interact in solution, D molar excess of the halide is used. Molar excesses of the halide from 2 to 3,5 times that of the aglycon have been reportedu-16 in the synthesis of steroid glycosides. Because the secondary hydroxyl groups attached to the ring skeleton of steroids react sluggishly with O-acylglyoosyl halides, completion of the (12) F. C. Uhle and R. C. Eldertield, J . Org. Chcm., 8, 102 (1943). (13) R. C. Elderfield, F. C. Uhle, and J. Fried,J . Am. C h . Boc., 69, 2235 (1947). (14) C. Meystre and K. Miescher, Heb.Chim. Acb, 17,231 (1944). (15) K. Reyle, K. Meyer, and T. Reichstein, Helv. Chim. Acfa, 33, 1541 (1950).

SYNTHETIC CARDENOLIDES

277

reaction may require several hours; consequently, water is being generated within the reaction medium long before the halide is completely converted into'glycoside (or otherwise consumed). It is, therefore, of paramount importance to have an efficient means for removing the water as it is being formed, to prevent thc hydrolysis of unconsumed halide, which would not only lower the efficiency of the operation, but would introduce another source of contamination. To remove the water! a drying agent is added to the reaction mixture before the halide is introduced. To this end, anhydrous calcium chloridels or Drierite" have been employed. The latter is a particularly efficient drying agent, and is generally considered to have a power of dehydration superseded only by phosphorus pentaoxide and by sulfuric acid. Consequently, Drierite is an unsuitable desiccant for the synthesis of cardiac glycosides, owing to the eaae of elimination of elements of water from the tertiary hydroxyl group at (2-14 and a neighboring hydrogen atom of the aglycon [see structures (1) and (2)]. In their synthesis of strophanthidin (2) glycosides, Uhle and Elderfield notedu that the use of Drierite led to some decomposition of the aglycon. For this reman, Elderfield and coworkersl*J*employed anhydrous magnesium sulfate aa the dehydrating agent, and obtained yields of between 8 and 31% of the O-acylated intermediate. 2, Meystre-Miescher Modification

In 1944, Meystre and Miescher reported" on a significant improvement in the Koenigs-Knorr synthesis. They noted, in the preparation of steroid glycosides, that the introduction of drying agents did not necessarily lead to good yields of the protected intermediates, especially when halides of disaccharides were employed. They suggested that the drying agent might not be performing efficiently in such caaes, and,l to improve on the relatively low yields, they introduced a technique whereby the water could be removed azeotropically. The steroid aglycon and silver carbonate were added to anhydrous benzene, and the solvent waa brought to boiling and c a d to distil. A solution of the halide in anhydrous benzene waa slowly added to the boiling mixture and, by maintaining the distillation during the addition, the water was removed azeotropically as it was formed. Meystre and Miescher re-investigated the preparation of five previously reported steroid glycosides, using their new method, and in all cases obtained yields approximately double those obtained by the clwical procedure. (16) B. Helferich, E. Bohn, and S. Winkler, Ber., 68,989 (1930). (17) D. D. Reynolds and W. L. Evans, J . Am. Chem. Soc., 60,2559 (1938).

278

W. W. ZORBACH AND K. VENKATRAMANA BHAT

In their synthesis of convallatoxin, Reichstein and coworkers16made some minor modifications in the Meystre-Mieacher procedure. Boctluse of the low solubility of strophanthidiri (2) in bonriene, the anhydrous tglycon wm dimolved iii tb n n i d vdumo of ihmlutn pdioxcmc. I’uro, dry nilver carbonate wtw iabroduaod cud , uiidw crfficioiit stirririg, LL solutioii of the halide in benzene was d d e d over a period of three hours, while distillation was maintained at a rate equal to that of the addition, thus keeping the volume of the solution in the flask at a constant (and small) volume. After addition of the halide was complete, a small volume of absolute benzene was added dropwise over a period of one hour, with distillation maiqtained as in the foregoing. By this procedure, the yield of tri-O-acetylcopvalbtoxin amounted to 44%. Other variations in the Meystre-Miescher procedure relate primarily to the solvent employed, and will be mentioned summarily in Section 111. 3. Mercuric Cyanide Procedure

A significant improvement in the Koenigs-Knorr synthesis was made by Helferich and Wedemeyer’* who, in 1944, introduced mercuric cyanide as an acid acceptor. Subsequently, Helferich and coworkers,1gemploying nitromethane as the solvent, showed that the use of mercuric cyanide led to excellent yields in the synthesis of glycosides from solid aglycons and, further, that it W&B not necessary to use a large excess of the aglycon. A potential side-reaction is the condensation of mercuric cyanide with the 0-acylglycosyl halide, as shown by Helferich and Bettin,@who allowed an 0-aaylglycosyl halide and mercuric cyanide to react in nitromethane in the absence of an aglycon. The latter reaction was re-inyeatigated by Coxon and Fletcher,U who showed that the condensation product is a mixture of the O-acylglycosyl cyanide and a 2-cyano-2-methyl-1 ,3dioxolane derivative. In searching for a.n improved method for the preparation of cardenolides, the mercuric cyanide procedure was investigated by Zorbach and coworkers. On the basis of somewhat limited experimental results, it is suggested that the procedure may not be generally applicable, being dependent on the particular aglymn or halide, or both. The coupling of methyl 2,3,4tri-O-acetyl-l-bromo-1-deoxy-a-D-glucuronate with digitoxigenin ( 1)az.28 (18) B. Helferioh and K. F. Wedemeyer, Ann., 6M,139 (1949). (19) (a) B. Helferich and K. Wth, Chem. Bm., 89, 314 (1966); (b) B. Helferich and R.Steinpreis, ibid., 81, 1794 (1968). (20) B. Helferich and K. L. Bettin, C h . Bm., B4, 1159 (1981). (21) B. Coxon and H. Q. Fletcher, Jr., J . Am. Chsm.Soc., 86, 2637 (1983). (22) W. W. Zorbach, D. V. Kaahelikar, and W. BQhler,Unpublished nreulte.

(23) W. W. Zorbaoh, G. D. Valiaveedan, and D. V. Kaehelikar, J . otg. C h . , 17, 1788 (1962).

279

SYNTHETIC CARDESOLIDES

and of 2,3 ,4-tn-O-benmy~-6-deoxy-a-crnannosy~ bromide with strophanthidin ( 2 ) a gave disappointing results. I n contrast, the coupling of 2 , 3 , 4 tri-0-benmyl-6-deoxy-a-cmannosylbromide with digitoxigenin ( 1) ,under essentially the same conditions, gavesp 3-0- (a-crhamnopyranosy1)digitoxigenin in 44% yield ! Nevertheless, application of the mercuric cyanide procedure to the partial synthesis of cardenolides deserves further investigation. 4. Limitations Imposed by the Sensitive Character of the Aglycon

There are two aspects of the chemistry of cardiac aglycons which further compound the difficulties in synthesizing cardenolides. The C-14 hydroxyl group, present in all cardiac aglycons, is tertiary and easily removed, especially by mineral acids. For example, when digitoxigenin (1) is heated with sulfuric acid (5% in aqueous ethanol), quantitative removal of the C-14 hydroxyl group is resulting in a mixture of two isomeric “anhydrodigitoxigenins,” 3@-hydroxy-5@-carda-8 (14) ,20(22) dienolide (3) and 3fl-hydroxy-5&carda-l4,20(22)dienolide (4). The secondary hydroxyl group at C-3 is unaffected by this procedure. During the Koenigs-Knorr

(3)

(4)

synthesis of cardenolides, removal of the C-14 hydroxyl group has also been observed. In an effort to establish conditions for the synthesis of convallatoxin using the Meystre-Miescher modification, Reichstein and coworkers1s re-investigatedl2 the preparation of the 3-&~-glucoside of strophanthidin (2), but obtained only the 8-D-glucoside of an “anhydrostrophanthidin,” in which the position of the new double bond WLndt specified. Tamm and baselet,% attempting to synthesize the naturally occurring evomonoside [3@-0-(6-deoxy-a-cmannopyranosyl)digitoxigenin], treated digitoxigenin (1) with 2,3 ,Ptri-@acetyl-6deoxy-a-~mannosyl bromide, but obtained the a-crhamnopyranoside of “14( 15)anhydrodigitoxigenin” (4) instead. Coupling of 2deoxy-3,4,6-tri-O-p nitrobenzoyl-a-wzrabimhexosyl bromide with digitoxigenin (1) , in the (24) 8.Smith, J . Chem. Soc., 1050 (1935). (25) C. Tamm and J. P. Rosselet, Helu. Chim. Acta, 86, 1309 (1953).

W. W. ZORBACH AND K. VENKATlMMANA UHAT

280

absence of an acid acceptor, led to rcmoval of the (2-14 hydroxyl group by the liberated hydrogen bromide, giving, after removal of the pnitrobemyl -5&cmd&c&14,20(22)groups, 38-0- (2deoxy-a-~-arabino-hexopyranosyl) dienolide.gb Brief treatment of a cardiac aglycon with methanolic sodium hydroxide leads to an “isogenin” (5). Elderfield and coworkersol interpreted the

If= OH

I

OH

Dfgitoxigenin (1)

transformation of the aglycon (1) aa proceeding by a shift of the double bond in the unsaturated lactone ring from the a,&. to the @,yposition, followed by intramolecular addition of the C-14 hydroxyl group to the double bond. The transformation is irreversible, and the original aglycon cannot be regenerated ; consequently, cardiotonic activity is lost. Because of this transformation, cardiac aglycons and cardenolides should not be exposed to alkalinity greater than that provided by aqueous bicarbonate solution. The use of the Zempldn procedure%for the saponific& tion of O-acylated cardenolides is likewise hazardous, although Elderfield and coworkers12J* were successful in deacetylating their synthetic cardenolides with barium methoxide in methanol. Because a substantial excess of the halide is used in the Koenigs-Knorr aynthesis of cardenolides, it is usually difficult, if not impossible, to secure the acylated intermediate in crystalline form. Hence, Reichstein qnd qoworkers1s introduced a procedure whereby the whole of the pioducts arising from the coupling reaction are saponified, employing a solution of potassium hydrogen carbonate in aqueous methanol. This modified transesterification procedure renders all extraneous carbohydrate materials water-soluble; the methanol is removed by evaporation, leaving an aqueous solution from which the (deacylated) cardenolide may be obtained by (26) W. W. Zorbach arid G. Pietsch, Ann., 666, 26 (1962). (27) W. 1). Pakt, E. R. Blout, F. C. Uhle, and R. C. Elderfield, J . Org. Chern., 6, 273 (1941). (28) (a) G . 7dmplfn and A. Kune, Ber., 66, 1706 (1923); (h) G. Zemplbn, A. Gerecs, and I. H e d w y , iLid., 69, 1827 (1WW).

281

SYNTHETIC CARDSNOLIDES

extraction with organic solvents. A disadvantage to the procedure is that the transeaterification may require from one to three weeks, depending on the nature of the acyl group; nevertheleas, it is at present the method of choice for the isolation of synthetic cardenolides.

111. SYNTHESIS OF INDIVIDUAL GLYCOSIDES 1. Cardenolides Containing Sugare Having a Hydroxyl Group at C-2 The first synthetic cardenolides were reportedI2 in 1943 by Uhle and Eldiefield. Availing themselves of four readily obtainable monosaccharides, they were successful in coupling the respective O-acetylglycosyl bromides with strophanthidin (2) in p-dioxane at room temperature, using silver carbonate as the acid acceptor and magnesium sulfate as the drying agent. In each case, the O-acetylated intermediate was isolated in crystalline form. Saponification of the latter, using barium methoxide in methanol, supposedly led to the @-pyranosides(61, (da), and (6b);however, from

OAc (7)

tetra-O-acetyl-~-~gdwtosylstrophanthidin (7), it was not possible to secure the unsubstituted glycoside as crystalline material. The results of these experiments showed that neither the C-5 nor the E l 4 tertiary hydroxyl group of the aglycon was involved in glycoside formation; this was fortuitous, because C-3 was the desired point of attachment, being the same 88 that which obtains in cardenolides of natural origin. The most significant aspect of the work is that the role of the sugar in promoting cardiotonic activity was clearly demonstrated for the first time. At the time, strophanthidin (2) cardenolides of natural origin containing n-xylose, D-arabinose, or D-glucose, joined directly to the aglycon, were unknown and, in retrospect, the new synthetic cardenolides ( 6 ) )

282

W. W. ZOELBACH AND K. VENKATHAMANA BHAT

(da), and (6b) might be looked upon as “artificial.” Neverthelees, the latter were shown to be highly potent when aiiumyed intravenously in cats, and they compared favorably with severd known strophanthidin (2) hexosides of natural origin.’Jg In a later paper, Elderfield and coworkers,l*employing 2,3,4 ,&tetra-Oacetyl-a-D-glucosyl bromide, prepared the @-bglucopyranosidea ( 8 ) , (8a), and (8b) of digitoxigenin (1) , digoxigenin [3@,12@ ,14@-trihydroxy-58card-20(22)-enolide] ,and periplogenin [3/3 ,5@,14&trihydroxycard-20( 22) enolide], respectively. As with the strophanthidin (2) cardenolides discussed in the foregoing, coupling was carried out at room temperature in pdioxane with magnesium sulfate aa the desiccant, but employing silver

-

HO€I,C

HO

-- AH

(8) RL= CH,, R* H, Ra = H [Digitoxigenin, (l)] (84 R1 = CH,, R* H, Ra OH [Digoxigenin]

(8b) R1

Ch,R* = OH,Ra H

[Periplogenin]

oxide instead of silver carbonate aa the acid acceptor. Whereas silver carbonate and silver oxide are used alternatively in the Koenigs-Knorr synthesis: the use of silver oxide, because of its oxidizing properties, is contraindicated in the synthesis of glycosidea from aglycons containing easily oxidizable groups. Strophanthidin (2), with its C-10 ddehyde group, is vulnerable to oxidation, and there is little doubt that, in Elderfield’s synthesis of strophanthidin (2) cardenolidea, the choice of silver carbonate was dictated by this property. Following this work, Reichstein and coworkers1@effected the first partial synthesis of a naturaIly occurring cardenolide, convallatoxin. ConVallatoxin c3P-O- (0deoxy-a-cmannopyranosyl)strophanthidin (9) 3 is considered to be the most potent of all the known, naturally occurring cardenolides, and is obtained from the blossoms of the Iily-of-thevalley (Conua2la~iamajalis). The coupling of strophanthidin (2) with 2,3,4triO-acotyl-Bdeoxy-a-tmannosyl bromide waa performed using silver car-

SYNTHETIC CAIZDENOLIDEB

283

honate as the acid acceptor. The water was removed azeotropically, employing R modified Meystre-Miescher technique,14and the reaction products were saponified directly, without isolation of the acetylated intermediate, giving convallatoxiri ( 9 ) in 44% yield. When the silver carbonate was replaced by silver oxide, only 11%of (9) was obtained.

I

HO

I

OH (9)

Klyne?P hag noted that, with various steroidal glycosides, the molecular rotation is approximately the s u m of the moIecuIar rotations of the steroidal aglycon and of the methyl a- or @-glycosidecorresponding configurationally to the suzar residue in the glycoside. On this basis, he has shown that cardenolides of natural origin containing D-sugars are B-D anomers (lo), whereas those which contain Gsugars have the same absolute, or LY-L, anomeric configuration (lOa). Moreover, save for two exceptions which

will be discussed subsequently, natural cardenolides are generally 1,2truns isomers, &R shown in the structures (10) and (lOa) . On theoretical grounds, then, it would never be expected that cardenolides uontaining either BGglucoRe or a-D-mannose would be found in Nature. Later in this (29) W. Klyne, Biochm. J., 47, xli (1060).

W. W. ZORBACH AND K. VENKATRAMANA BHAT

284

article, it will be demonstrated that cardenolidea which contain “unnatural,” or (Y-D,linkages show very low potencies in comparison with hexosides of natural origin and, because of this property, it is important to be able to synthesize cardeiiolides having a “natural” (a-L or /%D) linkage. The shble form of the 0-acylglycosyl halides of the enantiomorphous aldohexopyranoses is the (Y-(Dor L) anomer.” In the Koenigs-Knorr reaction, using silver carbonate as the acceptor, when there is a 1,2-cis relationship between the halogen atom at C-1 and the acyloxy group at C-2, replacement occurs at C-1 with inversion of configuration,lO and it is generally agreed that the silver carbonate assists in promoting the inversion. When a trans situation exists between the halogen atom at C-1 and the 20-acyl group, the 0-acyl group may attack the back side of C-1 as the halogen departs, forming an orthoester carbonium ion.” Under the slightly basic conditions (silver carbonate) of the reaction, an alkoxy radical can then attack this carbonium ion, to form a stable orthoeater. When, however, solvolysis is carried out in the absence of an acid acceptor (and, therefore, in acid medium), orthoester formation stops, and simple replacement occurs, with net relention of codguration81 as in (11)d(lla) Under the same conditions, 1,PCis halides undergo solvolysis with inversion of configurationa1as in (12)+( 12a) ; therefore, irrespective of whether a 1,2-trans or a 1,2-cis halide is the starting material, the result

.

R

R

R’CO II

R’CO

(12)

(lad

0

4

(30) 0.Hmeel and B. Ottar, Actu Chem. &and., 1,929 (1947). (31) Compare, R. S.Tipson, J . Biol. Chem., 180, 55 (1939).

285

SYNTHETIC CARDENOLIDES

will be” a 1,2-lruns glycoside, (lla) or (12a). Thus, it is not possible to synthesize 1 ,2-Cis glycosides by conventional methods. In Reichstein’s clynthesisl6 of convallatoxin (9), 2,3,4tri-O-acetyl-6deoxy-a-cmannoRyl bromide (13) (a 1,2-trum halide) waa employed. In contrast to the preparation of simple glycosides, the use of silver carbonate did not lead to orthoester formation; instead, glycoside formation took A

c

Ac

O

P

OAc (13)

+@I

-

H

O

HO

W

OH

(9) R = Strophanthidin (2)

place with net retention of configuration, giving a 1,2-truns glycoside having the “natural” or a-L configuration. It appears, therefore, that the stereochemical course of the reaction in cardenolide syntheses is inde pendent of the silver carbonate, and proceeds as if no acceptor were present at all.J1a In light of the foregoing, assignment of the D-L configuration to the I.arabinopyranosidel2(6a) of strophanthidin (2) must be incorrect. Furthermore, 6a shows a high order of cardiotonic activity, which is suggestive of a “natural,” a-I.-glycosidic linkage. The original assignment is understandable, however, for it waa made prior to the study on the methanolysis of benzojdated halide@* and the partial synthesis of convallatoxin,16both of which demonstrated that 1 ,2-truns halides may give rise to 1,2-trun.s glycosides. illso, it was not possible to specify anomeric configuration by calculation, as Klyne’s ruleZewas not formulated until seven years later. As may be seen, by application of the rule, (6a)is, in fact,the crcarabinopyranoside of strophanthidin (2). “DerJglucocheirotoxin” is a strophanthidin (2) pyranoside of natural origin,@and was originally presumed to contain a B-D-lyxose residue, as shown in structure (14). On the basis of the presumed structure, (14) containu a “natural” (B-D)glycosidic linkage; at the same time, it is a 1,2-cis glycoside, which is an exception to the rule that natural cardenolides are 1 ,2-lru% isomers. In order to provide firm proof of its structure, Reyle and Reichsteinaaset about to synthesize (14) partially. When they treated strophanthidin (2) with amorphous 2 ,3,4-tri-O-acetyl-D-lyxosylbromide under essentially the same conditions, including the isolation procedure, (31a) H. G. Fletaher, Jr., and C. S. Hudson, J . Am. Chem. Soc., 72, 4173 (1950). (32) N. M. Shah, K. Meyer, and T. Reichstein, Phum. Ach. Helv., 24, 113 (1049). (33) K. Reyle and T. Reichetein, Helv. Chim. A&, 86, 98 (1952).

W. W. ZORBACH AND K. VENKATRAMANA BHAT

286

aa those employed in the synthesis16 of convallatoxin (9), 3p-O-(cr-w lyxosyl)strophanthidin ( 14a) WIW obtained, the anomeric configuration of which ww deduced through Klyne’s rule of molecular rotational additivities.’g Other procedures, involving variations of the Koenigs-Knorr synthesis, were investigated, but in no C&BB waa even a partial conversion into the 8-D-lyxoside (14) realized.= Subsequently, Reichstein and coworkers*a*re-investigated the constitution of “deaglucocheirotoxin” and found that the glycoside does not, in fact, contain a ~-1yxoseregidue at all; instead, it has a &deoxy-&D-gulosyl residue! “Desglucocheirotoxin” is, therefore, a 1,%trans glycoside.

WAH Q H

HO

O

f H

)

W

li0

(144

(14)

Honghelin (15) and neriifolin (1Sa) constitute a unique pair of isomeric cardenolides, in that both contain a thevetose (a Bdeoxy-3-Omethylglucose) retidue IW the carbohydrate component. Honghelii (15) wm shown to consist of a digitoxigenin (1) residue and 8 D-thevetose residue; therefore, according to Klyne,” it must have the B-D anomeric configuration. Neriifolin (151) also contains a digitoxigenin (1) residue,= but is a

OH

OCH,

Hongheltn (16)

(33s) J. A . Moore, C. Tamm, and T. Reichstein, €Zulu. Chim. Acfu, 87, 765 (1964). (34) M. Frhejacque, Compl. Rcnd., 480, 127 (1950). (36) H.Helfenberger and T. Reichstein, Helu. Chim.Acta, 81, 1470 (1948).

SY STlI LTIC CARDEICOIJDES

287

derivative of tthevetosa" and has, therefore, the a-Lanomeric configuration." Whereu honghcliri (IS) has a "normal" (trans) relationship b e tween the substituents at C-1 and C-2 of the sugar, neriifolin (15a) is a 1,%-cisglycoside. The synthesis of honghelin (15)was accomplished by Reyle and Reichstein," who treated 'a solution of digitoxigenin (1) in pdioxane with a solubromide tion of amorphous 2,4di-O-acetyl-6deoxy-3-0-methyl-mglucosyl (16)in carbon tetrachloride. Freshly prepared silver carbonate was employed, and the water was removed azeotropically. Saponification of the reaction products using aqueous methanolic potassium hydrogen carbonate, followed by extraction, yielded amorphous material. Chromatography of the latter disclosed that the saponification had been incomplete; the material was, therefore, reacetylated and rechromatographed, giving di-0-

QBr

AcO

OAc (16)

OR (15) R = H (17) R = Ac

acetylhonghelin (17) in 28y0yield. Complete saponification of the acetate ( 17) was finally accomplished by employing methanolic barium methoxide, giving the desired honghelin [38-0- (6-deoxy-3-O-methyl-8-~-glucopyranosyl) digitoxigenin ( 15)1. In an ef!ort to synthesize neriifolin ( 15a), digitoxigenin (1) waa treated bromide* with amorphous 2 ,4-di-O-acetyl-6deoxy-3-O-methyl-~-glucosyl in a manner similar to that for the synthesis of honghelin (15). The reaction proved to be unsatisfactory, however, and they obtained, in low yield only, a mixture of some unreacted aglycon (1) , "14( 15)-anhydrodigitoxigenin" (4), and another substance having, possibly, the structure of a 8-bthevetoside of "14( 15)-anhydrodigitoxigeninJ' (4). Again, the possibility of synthesizing a 1,2458 cardenolide waa ruled out. In 1957, Reichatein and coworkersa re-investigated, for a second time. (36) M. Frhrejncque and V. Hasenfratz, Compt. W., 222,815 (1946). (37) K. lteyle and T. bichstein, Helv. Chim. Acfa, 81, 196 (1052). (38) R. Mtluli, C. Tamm, and T. ReichRtein, Helv. Chiwi. Acla, 40,284 (1957).

W. W. ZORBACH AND K. VENKATRAUNA BRAT

288

the partial synthesis of the ,9+glucopyranoside'* (6b) of strophanthidin (2) under various conditions, and employing alternatively silver carbonate or mercuric cyanide as the acid acceptor. The best results were obtained with silver carbonate under conditions where the water waa removed azaotropically, giving the 0-acetylated derivative of (db) in 35% yield. When mercuric cyanide was employed under essentially the same conditions, the yield was only 17%. The 0-acetylated intermediate waa saponified, giving the unsubstituted glycoside (6b). Treatment of (6b) with sodium borohydride effected the reduction of the aldehyde group at (2-10 of the steroid moiety, without attack on the (2-17 butenolide ring, giving the hitherto unknown strophanthidol 3-/3+gluco&de [3&0- (,9-D-glucopyranosyl)-5fl,l4,9,19-trihydroxycard-20 (22)-enolide]. Its cardiotonic activity, as measured in cats, was approximately the same as that of convallatoxin (9). Evomcinoside (19) is a cardenolide which may be isolated directlyaO from the seeds of Euonymus europaea L., or from the ensymic (strophanthobiase) hydrolysis of evonoside,1° obtained from the same source. The structure of evomonoside (19) was elucidated by Tamm and RosseletP who showed

=O

How i

HO

OH

(18)

(19)

H.Hauenatein, A. Hunger, end T.Reichetein, If&. Chim. A h , 86, 87 (1963). (40)F. Santavy and T.Reichetein, Helu. Chim. Acfa, 81, 1665 (1948). (39)

289

SYNTHETIC CARDENOLIDES

that it is an a-crhamnoside (6deoxy-a-cmnnoside) of digitoxigenin (1). To afford final proof of structure, they attempted its synthesis by coupling 2,3,4-€ri-O-acetyl-fjdeoxy-a-cmannosyl bromide" ( 13) with digitoxigenin in the presence of silver carbonate. Instead of the desired product, they obtained 3B-0- (u-crhamnopyranosyl) -5fl-cardtt44,20 (22)dienolide (18). Being aware that 0-benzoylglycosyl halides are less reactive than the corresponding ctcetylated halides, Zorbach and re-investigated the synthesis of (19) under conditions which would permit glycoside formation without removal of the hydroxyl group at C-14 of the aglycon (1). When digitoxigenin (1) was coupled with the known 2,3,4tri-Obenzoyl-6deoxy-a-cmannosyl bromideu ( 13a), wing silver carbonate and a modified Meystre-Miescher procedure, 6% of evomonoside (19) was obtained after saponification of the reaction products. In an effort to improve the yield, coupling of the bromide (13a) with (1) was carried out in 1,2dichloroethane with mercuric cyanidel9 as the acceptor; after saponification, evonionoside (19) waa obtained in 44% yield. As part of this the synthesis of a digitoxigenin (1) cardenolide containing D-rhamnose [G-deoxy-~-mannose, (24) ] waa considered. Because of the configuration of the hydroxyl group at C-2 in D-rhamnose (24), there would be formed a 1,2-truns glycoside, having the a - ~ or , "unnatural," configuration. DRhamnose (24) has not been reported to occur naturally, hut it has been synthesized, in five steps, starting with methyl a+mannopyranoside.'B In search of a shorter route, a new synthesisu was developed, starting with Dmannose (20). Conversion of (20) into the known4sdimethyl dithioacetal (21), and ptoluenesulfonation of the latter gave (22). Lithium aluminum hydride reduction of (22) gave 6deoxyD-mannose dimethyl dithioacetal (23), and demercaptalation of (23) gave the expected D-rhamnose (24). Because of technical difficulties in I

t!=0 I

t

--

HOCH Ho& H~OH

H~OH dH,OH

CH,S SCH, HOCH I HO~H R$!OH HCOH

&%OH

01) where Te = ptolyleulfonyl. (20)

7

H I

-

2II,SCSCH, I

HYH

HfH

HOCH +OH HCOH

H;OTa

@a)

H

c=o I

HOCH HO~H H&OH HtOH CII,

0s)

04)

(41) E. Fischer, M. Bergmann, and A. Rabe, Ber., M), 2362 (1920). (42) R. K. Neee, H G . Fletcher, Jr., and C. 8. Hudsoq, J . Am. Chem. Soc., 79, 296 (1961). (43) W. T. Haskins, R. M. Hann, and C. S.Hudson,J . Am. Chem. Soc., 68, 628 (1946), (44) W. W. Zorbach and C. 0. Tio, J . Org. Chem., IS, 3543 (1961). (46) H. Zinner, Chem. Ber., 84, 780 (1951).

290

W. W. ZORBACH AND K. VENKATRAMANA BHAT

carrying out the synthesis, and because the yields were not outstanding, another route to an O-acylglycosylhalide of n-rhamnose (24) waa explored.48 An intermediate in Hudson’s synthesis4 of Drhamnose (24) is methyl 2 , 3 , Ctri-O-benaoyl-Bdeoxy-u-D-manno&de (25). Frequently, direct replacement of the methoxyl group at C-1 of O-acylated hexopyranosides by halide proceeds with diiEculty (or not at dl);however, treatment of (25) with hydrogen bromide in acetic acid resulted in the slow replacement of the methoxyl group by bromide ion, to give the desired 2,3,4-tri-O-

( h &

06)

(25)

-

bBr

B%0

BzO

where Be

~

bensoyl.

(27)

benaoyl-G-deoxy-cu-b-mannosyl bromideU (26). Because of the costly procedure in obtaining (26) , and because the mercuric cyanide procedure does not give uniform results in cardenolide syntheses, (26) way coupled with digitoxigenin (1) in 1,2-dichloroethaneJ using silver carbonate and employing an areotropic distillatioil procedure.* Saponification of the reaction producte, followed by extraction procedures, gave 23% of 38-0-(a?-~rhamnopyranosyl) -148-hydroxy-5Bcard-20( 22)-enolide (27). The potency of (27), as measured in cats, was substantially less than that of its aglycon, digitoxigenin (1) ! Coupling of the bromide (26) with strophanthidin (2) under essentially the same conditions as those used in the synthesis of (27) gave 38-0- ( a-u-D-rhamnopyranosyl)-58 ,148dihydroxy-19-oxocard-20 (22)enolide in 53% yield.46 With its “unnatural” (LY-D)glycosidic linkage, the new cardenolide showed a very low order of cardiotonic activity. (46)W. W. Zorbach and Y. h k i , N a t i ~ ~ ‘ m e ~ & f t e50, n , 93 (1063); W. W. Zorbach 6.Swki, and W. BUhler, J . Med. Chem., 8, 298 (1963).

291

SYNTHETIC CARDENOLIDES

Owing to the vital role of D-glucuronic acid in the body aa a detoxifying agent,a the preparation of a Dglucosiduronic 'acid derivative of digitoxiEmploying, alternatively, silver carbonate, genin ( 1) waa silver oxide, and mercuric cyanide, it was found that optimum yields were secured when methyl 2,3 ,4-tri-O-acetyl-l-bromo-1de~y-ce~glucuronate~~ was coupled with digitoxigenin (1) in 1,2dichloroethane, using silver oxide. The coupling was carried out under exclusion of light at mom temperature for 3 days, resulting in a 42% yield of crystalline methyl (digitoxigenin-3&yl 2 ,3 ,4-tri-O-acetyl-&~-glucosid)uronate. All known methods of saponificationfailed to give the free, unsubstituted Dglucosiduronic acid; refluxing of the coupling product with moderately concentrated ,methanolic sodium methoxide resulted largely in regenerated starting material plus smaller amounts of products of illdefined composition. As the result of studies which are discussed in the following Section, evidence has been accumulated to show that deoxygenation at a carbon atom in the carbohydrate component of a cardenolide (excluding those which contain CY-Dor B-L linkages) leads to a loss in cardiotonic potency. With this in mind, Zorbach and Saeki" considered means for changing the o-methyl group to a hydroxymethyl group in the carbohydrate component of the potent convallatoxin [a-crhamnopyranoside (9) of strophanthidin (2)]. Although, in the jntroduction (see p. 279) , the implication is clear regarding the Rensitive nature of the aglycon, it should be pointed out that it is not generally feasible to effect transformations on the sugar residue when it is glycosidically bound to the aglycon. For this reason, it is necessary to work with the sugars themselves to obtain the desired structural features, and then, to bring about coupling to give the desired glycoside. In order to iiitroduce the terminal hydroxyl group in convallatoxin (9), it was necessary to work with the non-naturally occurring bmannose (28) , which may be synthesized according to Sowden and Fischer." Acetylation of (28) under the usual conditions gave 1,2 ,3 ,4 ,&pent&0-acetyl-&c H

C&OH O G

.

A

cChOAc O G

AcOQ ChOAc

OAc

no

OH (28)

AcO

OAc (2 9)

AcO

Ac

(30)

mniinom (29). Trs:ttmcnt of (29) with hydrogen bromide in acetic acid gave the desired 2 , R ,4,&tetra-0-acetyl-a-L-mannosyl bromide (30). (4fin) R. S.Tengue, Ailvan. Carboh@ate Chni., 9, 185 (1964). (47) W.F. (ioebel srid F. 1%.Babem, J . Biol. C h . ,111, 347 (1935). (48) J. C. Sowden and H.0.L. Fiecher, J . Am. Chem. Soc., 68, 1963 (1917).

W. W. ZORBACH AND K. VENXATEWBMNA B U T

292

Because of the expensive nature of the bromide (30), “pilot” experiments were carried out by using the known@2,3,4,6-tetrad-acetyl-a-Dmannosyl bromide (31) in order to establish optimum conditions for the coupling of (30) with (2). Coupling of the acetylated Dmannosyl bromide (31) with strophanthidin (2) was performed in 1,!Michloroethane with exclusion of light, using freshly prepared silver carbonate. The water was removed szeotropically in the usual manner, and the reaction products

(32)

(31)

were sapnified in aqueous methanolic potassium bicarbonate, giving the (32) of strophanthidin (2) , which showed an a-~-manfiopyranoside~~ extremely low order of potency. Coupling the cmannosyl bromide (30) with (2) under essentially the same conditions gave 33% of 6hydroxyconvallatoxin [3&0- (a-cmannopyranosyl) -54 ,14@-dihydroxy-19-oxocard20(22)-enolide, (33)], having a molar potency 12% greater than that of convallatoxin4~(9). The Gmannoside (33) is, therefore, the most potent of all known cardenolides.

AcOQ CH,OAc

Ac6

6Ac (SO)

(2,

~

HOQ CH,OH

HO

Ho

OH (33)

k-strophanthosidd is a glycoside of a trisaccharide and is present in the seeds of Slrophanthus kombd; it may be schematically represented as 0-4(49)

E.A. “alley, D. D. Reynolds, and W. 11. Evans, J . Am. Cham. Soc., 66,577 (1013).

SYNTHETIC CARDENOLIDEB

293

u-~lu~clyl-0-~-D-glut:onyl-~-cymltro~yl~tro~h~nthidin. Hydrolysis of kntrophanthoside with p-DgIucosidase cleaves the terminal Dglucose residue, leaving k-strophanthin-p (35), a glycoside of a disaccharide. Hydrolysis of the latter with Fjtrophanthohiase cleaves the second D-glucose residue, resulting in the hexoside cymarin [3&0-(2 ,6-dideoxy-3-0methyl-&~-ribo-hexopyranosyl)-5/3,14~-dihydroxy-19-oxocard-20(22) - enolide, (34)]. Inspection of the structure of (34) shows that the only un-

OCH, Cymarin (34)

protected position on the pyranoid ring is at C-4. Recognizing this feature, Kochetkov and coworkersMaccomplished the partial synthesis of k-strophanthin-8 (35) , albeit with considerable difficulty. Employing silver (60) N. K. Kochetkov, A. Ya. Khoriin, and A. F. Bochkov, Dokl. A W . Nauk SSSR, 186, 813 (1Sel).

294

W.

W. ZORBACH AND K. VENKATRAMANA BHAT

carbonate arid a coiivcritional aseotropic distillation procedure, they were unable to couple 2,3,4 ,6tetra-O-acety~-u-~-glucosy~ bromide with cymarin (34). In order to effect the coupling, they alternately added portions of the bromide in benzene solution and the silver carbonate in portioiw, to a solution of cymarin (34) in refluxing benrene-p-dioxane over a period of 8.5 hours. The reaction was run in a modified Soxhlet apparatus in which the refluxing solvent wm passed over sodium held in the thimble. The yield of k-strophanthin-8 (35) was approximately 3%. Chernobai61 has reported on the preparation of two interesting strophanthidol (3jl,58,148,19-tetrahydroxycard-20(22)-enolide) glycosides. In a manner analogous to that employed by Reichstein and coworkers= for the synthesis of the 3-B~-glucopyranosideof strophanthidol, strophanthidin (2) WM coupled with 2 ,3,4-tri-O-acetyl-a-r,-rhamnosyl bromide (13) , giving the tri-0-acetyl derivative of oonvallatoxin ( 9 ) - Reduction of the 0x0 group at C-19 with sodium borohydride, followed by a second coupling with (13) , gave 38,19di-O-(a-trhamnopyranosyl)58,148dihydroxycard-20(22)-enolide6' (36) , after saponification of the reaction producta. In a second paper," strophanthidin (2) waa converted into its 3-0-acetyl derivative as a protective measure, and the latter was reduced with sodium borohydride to give 3-0-acetylstrophanthidol.Coupling of the reduction product with 2,3,4-tri-O-acetyl-a-crhamnosyl bromide (13) ,followed by saponificationof the 0-acetylated intermediate, gave 19-0-(a-crhamnopyranosyl)-38,5jl, 14&trihydroxycard-20(22) -enolide (37). HO

nd

OH

on (38)

(51) (62)

V. T. Chernobai, Zh. ObeM. Khim., 84, 1690 (1964).

V. T.Chernobai, Zh. Obeheh. Khim., 84, 3862 (1964).

(37)

295

SYNTHETIC CARDENOLIDES

With a view to providing ndditionul anomeric pairs of cardenolides containing &sugars for a direct comparison of cardiotonic activities, Zorbach and GilliganU investigated anew the possibility of synthesizing 1 , 2 4 5 cardenolides. In a paper by Gorin and Perlin,Mit had been shown that 6Pglucosidea are the preponderant products from the reactions of 4,6di-0acetyl-2,3-O-carbonyl-or-~mannosyl bromide with (a) 1,2,3,Ptetra~Oacetyl-&D-glucose and (b) 1,2,3,4-tetr~O-acetyl-&~-mannose.Only trace amounts of the a-Pglucosides were formed during the reaction. Because the inverted products were formed almost exclusively, the use of glycosyl halides containing the “non-participating” 2,3-O-carbonyl group could, presumably, also lead to the synthesis of 1,Zcis cardenolides. On this basis, the synthesis of the p-D-rhamnosides (44)and (46) of digitoxigenin (1) and strophanthidin (2) was undertaken. Without isolation of the intermediates, methyl a-D-mannopyranoside (38) waa treated in succession with ptoluenesulfonyl chloride, carbonyl chloride, ttnd benzoyl chloride, giving methyl 4-O-benzoyl-2,3-O-carbonyl-

__f

(39)

(38)

BzO

0’ ‘0 (43)

-

(40)

-

0

BZ

(42)

OCH,

(41)

6-O-ptolylsulfonyl-a-~-mannoside (39) in 37% yield. Treatment of (39)

with sodium iodide in acetone gave the 6-iodo derivative (40), which underwent reduction with hydrogen in the presence of a “nickel boride” catalystssto give methyl PO-benzoyl-2,3-O-carbonyl-6deoxy-a-~-mar1noside (41) in 95% yield. Reaction of (41) with hydrogen bromide in acetic acid effected replacement of the methoxyl group at C-1, affording crystalline (53) W.W.Zorbach and W. H. Gilligan, Curbohydrak Res., 1, 274 (1985). (64) P. A. J. Gorin and A. S. Perlin, Can. J . C h . ,89, 2474 (1961).8ee also, G. M. Tener, R. 8.Wright, and H.G. Khorana, J . Am. Chem. &., 78, 606 (1956). (66) R.Paul, P. Biuason, n d N. Joaeph, Znd. Eng. Chem.,44, 1006 (1962).

296

W. W. ZORBACH AND K. VENKATRAMANA B U T

4- 0-benzoyl-2 ,8-O-carbonyl-Bdeoxy-or-D-n~ai~noclyl bromide (42). The bromide (42) is extremely reactive, and can be stored for only short periods in a desiccator in the dark, at -78'. It readily undergoes meth-

anolysis in the presence of silver carbonate to give, by inversion, methyl 4-Obenzoyl-2 ,3-O-carbonyl-6-deoxy-~Dmannoside(a), and these results are consistent with those reported by Gorin and P e r h M However, coupling of digitoxigenin (1) with the bromide (42) in 1,1,2trichlomethme in the presence of silver carbonate, followed by saponification of the reaction mixture, gave 48% of the previously described LY-DrhmmosidtP (27) , but only 13% of the desired &D anomer (44).Coupling

1

of (42) with strophanthidin (2), under easentidy the -me conditions, gave 8% of the known ara-rhamnoside4(b)(45) and 6% of the &D anomer

SYNTHETIC CARDENOLIDES

2c37

(46). In both cases, failure to yield inverted products exclusively is most probably due to a steric situation in which the steroid aglycon attacks the initially formed carbonium ion from the side of the plane of the ring that is opposite to the 5-methyl group, to give the CY-D anomer aa the major product.

2. Cardenolides Containing 2-Deoxy Sugars

The fiist crystalline O-acylglycosyl halide of a 2deoxy sugar was reported by Bergmann and coworkers,66 who prepared 3 ,4 ,6-tri-O-benzoyl2deoxy-ar-~-arabino-hexosylbromide. The bromide is unstable and cannot be stared; consequently, it waa added to methanol containing silver carbonate, giving methyl 3,4 ,6-tri-O-bensoyl-2deoxy-/3-~-arabino-hexoside. Save for this work,a there were no reports of crystalline acyl-2deoxyglycosyl halides prior to 1958 and, moreover, little work had been carried out on the synthesis of 2deoxyglycosides. Also, although the partial synthesis of cardenolides containing sugars having a hydroxyl group at C-2 was becoming commonplace, no cmdenolidea containing 2-deoxy sugars had been prepared. Because of the importance of digitoxose as the carbohydrate component of cardenolides obtained from LXgitalis spp., Zorbach and Payneb7began an investigation with a view to obtaining a stable halide of the sugar for coupling reactions. Whereas 2deoxy sugars are considerably more reactive with respect to replacement reactions at the anomeric center than are the corresponding sugars, the 2 ,6-dideoxy sugars tire even more reactive, behaving like 2deoxypentoses. The greater reactivity of deoxy sugars may be due to the absence of “shielding” at the unsubstituted carbon atom(s),” electronic effects, or a combination of the two. Acetylation of digitoxose [2 ,6-dideoxy-p-n-dm-hexo8e (47)] under the usual conditions gave the triacetate (48) as crystalline material. However, attempts to prepare the bromide of (48) , even under carefully controlled conditions, resulted only in sirups which evolved hydrogen bromide, presumably either by hydrolysis or by elimination. Benzoylation of (47) yielded a crystalline tribenzoate (48a) ,and the results of treating the latter with hydrogen bromide were essentially the same aa those observed with the triacetate (48). In contrast, p-nitrobensoylation of (47) rpulted in a high-melting tris-pnitrobensoate (48b)which, when treated with a little more than one equivalent of hydrogen bromide in dichloromethane,” (56) (57) (58) (60)

M. Bergmann, H. Schotte, snd W. Leschineky, Be?., 68, 1052 (1923). W. W. Zorhach and T. A. Payne, J . Am. Chem. Soc., 80,5564 (1958). F. IT. Newth arid G. 0.Phillips, J . Chem. Soc., 2904 (1953). R. K. Nese and 11. G . Fletcher, Jr., J. Am. Chem. Soc., 78, 1663 (1954).

W. W. ZORBACH AND K. VENKhTRAMANA BHAT

298

0

HO

OH

bR

0

(47)

0-

0

(48b) H

p -O,NCJ&

4

PNBZO

OPNBz

(4s) X

Br

PNBZO m J-( I OPNBz (50)

PNBs

0 1

OPNBz (61)

where PNBs = pnitrobenzoyl.

yielded67 qdalline 2,&dideoxy3 ,4-di-O-pnitrobenzoyl-~D-rib~htxosyl bromide (49).The choice of dichloromethane waa fortuitous; owing to its low solubility in this solvent, the liberated pnitrobenzoic acid separated in dmo& quantitative yield. Filtration left a solution containing the desired bromide (49) , which crystallized readily from ether-dichloromethane. The crystalline (49) was extremely reactive; consequently, the less reactive (49a), likewise crystalline, waa prepared in an analogous manner. Treatment of (49a)with silver pnitrobenzonte gave the original tris-p-nitrobenzoate (48b); both (49)and (49a) reacted with water, in acetone, to yield" the same product, 2,6-dideoxy-3,4-di-~pnitrobenzoyl-D-ribohexose (50). Preliminary experiments on the coupling of the chloride (4Oa) with

BYNTHIEI?C CARDENOLJDES

299

digitoxigenin (l), in the presence of silver carbonate, led to gross decomposition of the halide, and it waa suspected that, under the conditions of the experiments, the silver carbonate was causing elimination of hydrogen chloride. Subsequently, in attempts to couple (49a) with mercury derivatives of pyrimidine bases,dO a carbohydrate product was isolated which had a composition agreeing with that for 1,2,&trideoxy-3,4-di-O-p n i troben~ yl-D-ri~~h~-l~nopyranose (51). In a separate elimination of hydrogen chloride from (49a) was provoked by warming it in pure pyridine only, giving 45% based on (48b)I of the pnitrobenzoylated digitoxal (51). A decision was made, therefore, to carry out a coupling reaction in the absence of an acid acceptor?' Treatment of one millimole of digitoxigenin (1)with an excess of (49a) in a small volume of dichloromethane at room temperature, followed by saponification of the reaction products, yielded an amorphous residue. Partition chromatography*2of the latter gave 2% of a material whose composition agreed with that of a digitoxigenin digitoxopyranoside; however, its physical constants differed from those of the naturally occurring evatromonoside" [36-0- (2,6dideoxy-&~-ribo-hexopyranosyl)-14p-hydroxy-5B-card-20 (22)-cnolide (53)3. A second experiment was carried out,(14involving a two-fold increase in the amounts of the reactants at the same concentration; this gave 3 8 0 (2,6dideoxy-a-~-m'bo-hexopyranosyl) -14B-hydroxy-5&card-20(22)-enolide (52) in 44% yield, without resorting to chromatography! The preparation of (52) constitutes, simultaneously, the first synthesis of a 2deoxycardenolide and the first recorded instance in which a biologically important 2deoxyglycoside WM synthesized, using a crystalline 2deoxy-0-acylglycosyl halide. The digitoxosides (52) and (53) are the first known anomeric pair of cardenolides; when assayed intravenously in cats, synthetic (52) showed a potency only two-fifths that of the anomeric form (53). The possibility of synthesizing the pdigitoxoside (53) by a direct method has been investigated." Digitoxigenin (1) and digitoxose (47) were dissolved in a relatively small volume of pdioxane, and the solution was treated with a small proportion of hydrogen chloride in dichloromethane. The acid was neutralized, and the crude, solid product was chromatogrltphed, giving an approximately 1:1 mixture of the anomeric digitoxogides (52) and (53) in a combined yield of 10% based on (1). (0) W. W. Zorbach and G. J. 1)urr, J . Org. Chem., 17, 1474 (1962). W. W. Lorbech nnd T.A. Peyne, J . Am. Chem. Soc., 81, 1619 (1959). F. Kaiser, E. H a c k , and H. Ypingler, Ann., 608, 75 (1957). R. Tscheache, 9. Wirtz, and G. Snatzke, Chem. Ber., 88, 1619 (1955). W. W. Zorbach and T. A. Payne, J . Am. Chem. SOC.,82, 4979 (1960). W. W. Zorbach, N. Henderson, and S. Saeki, J . Org. C b . ,29, 2016 (1964).

(61) (62) (&3) (64) (05)

300

W. W. ZORBACH AND K. VENKATRAMMA B U T

OH (52)

OH

OH

(47)

(53)

The formation of an a-D-glycoside (52) may be satisfactorily accounted for in terms of the “anomeric” particularly applicable with 2deoxy sugars, in which the absence of substitution at C-2 eliminates fact.org

I

6- I 0

(54)

which would normally govern the stereochemical course of glycoside formation. The effect allows for an attraction between the axially oriented CU-D glycosidic! oxygen atom and C-5, which crtrrieR a partial positive charge. (66) (a) R. U. Ledeux and P. Chu, Ab8lract.a Papm.9 Am. C h . Boo. Meding, 138, 3 1 (1968); ~ (b) J. T. Edward, P. E. Morand, and I. Pudcm, Can. J . Chem., 3% 2089 (1961) suggest, for hexopyrsnoeer not deoxygenated et G6,that through an induative effect, both the G 4 and CB hydroxyl grouprr BBNB to augment the positive charge on C-5.

SYNTHETIC CARDENOLIDES

301

Such an attraction would overcome, at least in part, the conformational instability imposed by the erected oxygen atom. For pyranosides which are not deoxygenated a t (2-6, the added electron-withdrawing power of the hydroxymethyl (or acyloxymethyl) group a t C-5 should increase the charge on C-5, thus favoring cY-D-glycoside formation even more.ee(b) In direct coupling of digitoxose (47) [see (54),R=H] with digitoxigenin (1), this attraction is not strong enough to offset to a large degree the conformational instability, with the result that substantial proportions of the conformstionally more stable 15-digitoxoside (53) were formed. When, chloride however, 2 ,6dideoxy-3 ,4di-0-p-nitrobenzoyl-~-~-ribo-hexosyl (49a) was coupled with (1) in the absence of an acid acceptors’ (therefore, under equilibrating conditions) , the a-D-glycoside (52) was formed exclusively. It is therefore suggested that the powerful electron-withdrawing

0

II

capacity of the pnitrobenzoyl group at C-4 [(54), R = p-OsNCsH&] was sufficient to offset conformational instability to a point where @-D-glycoside formation was excluded. In an extension of this direct method for preparing 2deoxycardenolides using crystalline, pnitrobeneoylated halides, the synthesis of cardenolides containing 2deoxy-~-arabino-hexose (55) was investigated for pharmacological comparison studies.“ With a view to correctly assigning anomeric configurations to the p-nitrobenzoylated intermediates, commercial “2deoxy-D-glucose” [2deoxy-&~-arabinohexose,(55) ] was subjected to a partial anomerization procedure described by Bergmann and coworkers.m The solid material obtained by this procedure is tt mixture of the anomeric forms (56) and (55) ;low-temperature p-nitrobenzoylation of the mixture in pyridine resulted in a mixture of crystalline, anomeric tetrakis-pnitrobenzoates (57) and (58) in a ratio of approximately 1:1. The two had a large difference of solubility in dichloromethane, and a complete separation of (57) from (58) was effected by fractional recrystallization from this solvent. Treatment of either of the anomeric p-nitrobenmates (57) or (58) with an excess of hydrogen chloride in dichloromethane, or with hydrogen bromide in dichloromethane, resulted in 2-deoxy-3,4 ,6-tri-0-pnitrobenzoyl-u-D-arabexosyl chloride (59) , and the corresponding bromide (59a) , respectively. Both httlidcs are crystalline and high melting, and have a stdilitfywhich appears tfo exceed that of 0-acetylglycosyl halides of normal hexoses: oonsequcntly, they could be handled and stored without undue precautions. Treatment of the bromide (S9a) in dichloromethane with silver pnitrobeneoate regenerated the Ftetrakis-pnitrobenzoate (SI).The bromido (5%) reacted also with methanol to give, after neutralieation of tho liberated hydrogen bromide with silver carbonate, methyl

302

W. W. ZORBACH AND K. V E " F U M A N h

BHAT

2-deoxy-3,4,6-tri-O-p -nitrobenaoyl-&~-a~ubino-hexoside(60).Methoxidecatalyaed saponification of the latter resulted in the known66 methyl 2aeoxy-8-Darabino-hexopyran~ide(604. HOH,C

H&OH (55)

PNBeO€i+

w

PNBzO

R (6Q) X 5 C1 (5Qa) X = Br

-

(60)R = PNBz (604 R

H

Further testimony of this stability w a obtained in an attempt to couple the chloride (59) with digitoxigenin (1) in the absence of an acid acceptoP in a manner analogom to that describedMfor the preparation of the adigitoxoside (52). Even on prolonged standing, no reaction whatever took place, and (1) and (59) were recovered unchanged. In order to effect coupling, the more reactive bromide (59a) w&s employed under essentially the same conditiona. Although glycoside formation took place, the product was shown to be 380- (2-deoxy-cu-~-urubinc~-hexopyranosyl) -5pcarda8(14) ,20(22)dierolide (61), in which removal of the (2-14 hydroxyl group from the aglycon moiety was brought about by the liberated hydrogen

SYNTHETIC CARDENOLIDES

303

bromide. This removal was not observed, however, during the coupling of 2 6-dideoxy-3,4-di-O-p-nitrobenzoyl-@-~-ribo-hexosyl chloride with (1) , in which hydrogen chloride was the by-product in the reaction. Inasmuch aa the coupling of (1) with (S9a) was carried out in the absence of an acid acceptor (equilibrating conditions), the formation of an a-u-Pglycosidemay be aacribed to the “anomeric” effect.66 Because of the stability of (59)and (59a) (unprecedented for 2-deoxy0-acylglycosyl halides), it waa considered that coupling couId be accom-

PNBzOH,$!

(5B)

x = c1

(58s) X - B r

304

W. W. ZORBACH AND K. VENKATRAMANA BHAT

plished in the conventional manner, wing the less reactive chloride (59). Accordingly, digitoxigenin (1) was treated with a 1,a-dichloroethane solution of (59) in the presence of ailvor carhonatc, employing a MeystreMieschcr type of unootropic distillatioa procodurc.28Tho reaction products were sapoiiificd (without isolation of the acylated intermediates) giving, in 35% yield, an approximately 1:l mixture of both anomem of the 2deoxy-D-arabino-hexopyranosides[(62) and (63)] of digitoxigenin (1). Despite the use of silver carbonate, the course of the reaction appears to have been governed in part by the “anomeric” effect. As a further extension of this work, the synthesis of cardenolides con(68)] was inve~tigated.~~ taining 2-deoxy-D-rhhexoae ~‘2dooxy-~-allose’’ 2-Deoxy-n-?%bo-hexose(68) has not been reported to occur naturally, but it may be synthesized in five steps, starting with methyl cr-D-glucopyranoside. The conversion of the latter into methyl 2,3-anhydro-4,60-benzylidenen-D-alloside was best accomplished according to Richtmyer,a and, by substituting absolute tetrahydrofuran for ether in the lithium aluminum hydride reduction of the anhydroalloside,6Dan 80% yield of methyl 4 , W benzylidene2deoxy-a-~-ribo-hexosideww obtained. Complete hydrolysis of the latter, in one o p e r a t i ~ ngave , ~ ~ the desired sugar (68). Although this sequence of reactions for synthesizing (68) waa attractive because the yields were good in each stage, an alternative approach waa considered. This second approach70 waa based on the nitromethane synthesis,” and involved, as the key intermediate, 1-C-nitro-1-hexenewrik? ,4 ,5 ,6tetrol tetraacetate (66) , previously reported by Sowden and Fischer.72In restudying their conversion of &ribose (64) into the hexenetetrol (66), it was possible70to secure 2,3,4,5 ,6-penta-O-acety1-1deoxy-l-nitro-D-altritol (65) as crystalline material. When (65) was subjected to the SchmidtH c=o I HCOH I HCOH I HCOH I

CH,NO,, I AcOFH H OAc HCOAc I HCOAc

- - F

CbOH

&&O , Ac

(64)

(65)

(67) (68) (69) (70) (71) (72)

CHNO, I1 CH

YWOa

P

HAOAC

HCOH

HAOAC

HCOH I HCOH --+

H b c

CH,OAc (66)

I I

CHaOH

H c=o I ?Ha HCOH

I

HCOH I HCOH I CHpOH

(67)

W. W. Zorbaoh and W. B W , Ann., 670, 116 (1963). N. K,Richtmyer, Methook Carbohgdrale Chem., 1, 107 (1902). D.A. Prim, J . Am. Chum. h., 70, 3958 (1948). W. W. l;orbsch and A. P. Ollapally, J . Org. Chum.,28, 1790 (1964). J. C. Elowden, Advan. Carbohgdrale C h . ,6, 291 (1961). J. C. Sowden and H. 0. L. Fiecher, J . Am. C h .rSoc., 68, 1048 (1947).

(68)

305

SYNTHETIC CARDENOLIDES

Rutz reaction,'a the hcxenetetrol (66) was obtained in GO% yield. Reduction of the double bond of (66) gave the acetylated deoxynitro alcohol as sirupy material. At this point, an innovation was introduced in which the reduction product was hiydrolyzccl using aqueow hydrochloric acid. This procedure did not bring about changes other than removal of the acetyl groups, resulting in 1 ,2dideoxy- l-nitro-~-rh-hexitol(67) tw crystalline material. The latter readily underwent the Nef reaction14to give 2deoxyD-ribo-hexose (68). Unfortunately, this procedure offered no advantages over the alternative one described in the foregoing. Low-temperature p-nitrobenzoylation of 2-deoxy-~-n'bo-hexose (68) in pyridine gave an anomerically pure tetrakis-pnitrobemoate (69) , presumably having the p - ~configuration at C-1. The reaction of (69) in dichloromethane with hydrogen chloride or with hydrogen bromide gave crystalline 2deoxy-3,4 ,6-tri-O-p-nitrobenzoyl-ar-D-ribo-hexosylchloride (70) and the corresponding, crystalline bromide (70a) , respecti~ely.~~

OH

OPNBZ

OPNBZ (70) X = C1 (70a) X = Br

Both appeared to have a stability rivalling that of the corresponding halides, (59) and (59a) , prepared from 2deoxy-~-urabino-hexose (55). Because the bromide (70a) was chosen for the coupling experiments to be discussed subsequently (see p. 306), a decision was made to perform an elementary analysis on the material, thereby obviating the necessity for further constitutional studies. Without undue precautions to avoid hydrolysis during handling, the bromide (70a) gave excellent values for carbon, hydrogen, and bromine! Initially, the coupling of digitoxigenin (1) with (70a) in dry 1,2-dichloroethane was accomplished67 by employing an azeotropic distillation technique'to remove the water. The silver carbonate used in the reaction had been prepared several days previously, and had been stored under anhydrous conditions in the dark at 0".After completion of the reaction, the products were saponified, followed by extraction, giving solid material which consisted of two components as disclosed by paper chromatography. (73) E.Schmidt and G.Ruts, Bet., 61, 2142 (1928). (74) J. U. Nef, Ann., 260, 263 (1894).

306

W.

W. ZORBACH AND K. VB1NKATRAMANA BHAT

OH (72)

(71)

A resolution of the mixture was accomplished through column partitionchromatography; this gave a small quantity of the desired 3&0-(2deoxy@-~-ribo-hexopyranoayl) -14&hydroxy-5Bcard-20 (22)-enolide (71) plus dig(22) -enolide (72) ], The formaitoxigenone [14/3-hydroxy-3-oxo-5~-c~rd-20 tion of the latter is unprecedented in cdenolide syntheses, and there is little doubt that, in spite of the careful manner in which the silver carbonate had been stored, some conversion into silver oxide must have taken place. It is reasonable to conclude that, under the conditions of prolonged contact at an ele'vated temperature, the oxidation of the secondary hydroxyl group at C-3 of the aglymn (1) was brought about by silver oxide, for this phenomenon haa never been observed when frecllrly prepared silver carbonate haa been used. A second experimentaTW M conducted in an malogous maaner, using freahly prepared silver carbonate. Under these conditions, the 2-deoxy-pM.i'bo-hexopyranpide (71) waa obtained in 46% yield, without recourse to chromatographic procedures. The cardiotonia activity of (71) waa a p proximately the srtme aa that of the corresponding %deoxy-&D-urubhhexopyranoside (62), and it appears that, in this regard, a change in configuration at C-3 of the carbohydrate component may be an unimportant one.

SYNTHETIC CARDENOLIDES

307

The preparation of (71) is given in the following experiment, which servea to typify the synthesis of a 2deoxycardenolide, starting with a !2deoxy sugar. The experiment also embodies a Meystre-Miescher type of azeotropic distillation technique for removing the water, and saponific% tion catalyzed by potassium hydrogen carbonate for isolation of the unsubstitutd cardenolide. A warm solutiop of 5.57 g. (30 mmoles) of pnitrobewoyl chloride in 50 ml. of dry pyridine ia mled'to 0" under stirring, and to the stirred suspension is added, in small portions over a period of 15 min., 0.98 g. (6 mmoles) of finely divided Zdeoxy-& hexose. Stirring ia maintained at 0" for 1.5 hr., and the mixture i a set aaide in a refrigerator for 4 days. The excess p-nitrobenzoyl chloride is neutralized by the careful addition of 60 ml. of saturated, aqueous sodium hydrogen carbonate, and the mixture ia added to 1 1. of icewater. The precipitate ia removed by filtration, washed with water, and dried in the open at room temperature and then in a desiccator over phosphorus pentaoxide. Most of the water of crystalliation is removed by heating the material at llO"/O.l mm. for 15 hr. The dry product is crystallized from nitromethane (decolorh d ) giving 3.46 g. (76%) of 2 & 0 ~ ~ - 1 , 3 , 4B-tetra-O-pnitrobOyl-~'~hexose, , m.p. u)1-202", of satisfactory quality for the following conversion. Its specific rotation is +1M" in chloroform. To 72 ml. of a solution of anhydrous hydrogen bromide in dichloromethane (containing about 0.25 meq. of hydrogen bromide per mi.) ia added 2.28 g. (3 mmoles) of the tetrakia-p-nitrobenzoate. The supension is stirred magnetically for 2 hr. in a stoppered flask, and the separated pnitrobenzoic acid ia removed by filtration through a sintered-glw funnel. The filtrate is evaporated at room temperature under diminished prewure to a volume of about 5 ml., and 10 ml. of dry ether is added. The solution ia kept overnight at room temperature, and the separated material ia filtered by suction, waahed with a little ether, and dried in a vacuum desiccator over phosphorus pentaoxide. The yield of 2deoxy-3,4, B-tri-O-pnitrobe~oyl-P-Mibo-hex6sylbromide h 1.93 g. (90%). It has m. p. 11Q0(dec.)and [ a ] ~198' (in dichloromethane). Into a 50-ml., %necked flask, fitted with a dropping funnel, condenaer, and magnetic stirrer, h introduced a solution of 374 mg. of digitoxigenin in 40 ml. of 1,2dichloroethane followed by 700 mg. of freahly prepared silver aarbonate. The stirred suspension is heated in an oil bath at 106", and 20 ml. of the solvent is distilled off at a moderate rate. Under efficient stirring, a solution of 1.35 g. (2 mmolea) of the bromide in 110 ml. of 1,Zdichloroethane-carbon tetrachloride (5:6) is added from the dropping funnel over a period of 2 hr., during which time, distillation of the solvent from the reaction flask is maintained at a rate equal to that of the addition of the solution of bromide. An additionel 100 ml. of anhydrous 1,Zdichloroethane is added over a period of 2 hr., under the same conditiona aa those for the addition of the solution of the bromide. To the mixture is added 20 ml. of acetone, and the silver salts are removed by filtration and wmhed thoroughly with acetone. The filtrate h evaporated to a small volume, and the solution is added slowly to a solution of 2.5 g. of potsssium hydrogen carbanate in 90 ml. of water and 250 ml. of methanol. The suspension which forms is stirred at room temperature for 12 days, and the resulting opalescent solution ia evaporated under diminished precisure at 30" to about 90 ml. The resulting solution is extracted four times with 100-ml. portions of chloroform, and the combined extracts are waahed with water, dried with Rodium sulfate, and evaporated under diminished pressure. The residue is regvaporatad with a little chloroform, digested with 10 ml. of ether to remove the

+

308

W. W. ZORBACH AND IC, VBNKATRAMANA B U T

methyl p-nitrobenmate, the mpnsion is filtered, and the insoluble material is wmhed with ether and dried. Decolorization snd recry~talliaationfrom a e m d volume of abeolute ethanol gives 240 mg. (40%) of the Zdeoxy-&-~'~hexopytibo-hexopyrsnoeide of digitadgenin, m. p. 223-226', [CXB 27" in ethanol, hg??,jj 217 my (loge 4.2).

+

To lend support to the above conclusion, the preparation of digitoxigenin (1) and strophanthidin (2) cardenolides containing 2,6dideoxy-D-arubino-

hexopyranosewaa considered. A direct comparison could be made, thereby, (53) of digiwith evatromonoside [2, Gaideoxy-8-D~bo-hexopyran~ide toxigenin (l)] and helveti~oside7~.[Z ,Bdideoxy-8-om'bo-hexopyranoside of stmphanthidin (2)], respectively, in which the sole difference in corresponding pairs would be a reversal of the configuration at C-3of the sugar moiety. During the course of the investigation7*to synthesize the hitherto unknown 2,6dideoxy-warubino-hexoae(76), Meyer and coworkers reported76 on its isolation from the hydrolyzate of a cardenolide obtained from ~ i g i t a ~canariensis is L. ~ & g piecedent from the more recently pubH

7'0

HO$H HCOH I HCOH I CH,OH (55)

- HO@

HCOH I HYOH C&OH (73)

HO~H +OH HYOH

CH,OTe (74)

(744 W. Nagata, C. Tamm,and T. Reichatein, Hetu. Chim. Actu, 40,41 (1957). (76) W. W. Zorbach and J. P. Ciaudelli, J . 01-8.C h . ,80, 461 (1965). (76) P. Studer, 8. K, Payanaram, G. R. Gavilanea, H. Linde, and K. Meyer, Helu, Chim. Acb,48,23 (1983).

SYNTHETIC CARDENOLIDES

309

lishedu synthesis of D-rhamnose (24), the readily available “2deoxy-w glucose” (55) waa converted into its knownn diethyl dithioacetal (73). p Toluenesulfonationof (73) gave the crystalline Gptoluenesulfonate (74), which underwent reduction with lithium aluminum hydride to yield 2,G dideoxy-warabinehexose diethyl dithioacetal (75). Demercaptalation of (75) gave the desired 2,kiideoxyhexose (76) as sirupy material, which only became completely crystalline after standing in a desiccator for 6 months; but, on removal from the desiccator, the crystalline mass changed to a sirup within 5 minutes. Because of this property, coupled with the technical difficulties involved in its synthesis, further studies with the sugar (76) were deferred. Natural cardenolides are invariably pyranosides, and the synthetic, non-naturally occurring ones discussed in this article are likewise pyranosides. It was, therefore, of interest to investigate the preparation of furanoid cardenolides;such glycosides might, conceivably, display unexpected biological activity. In 1965, Bhat and Zorbach rep~rted’~ on the preparation of the first stable, crystalline 0-acylglycofuranosyl halide of a 2deoxyhexose, thus paving the way for the synthesis of Zdeoxyhexofuranosyl cardenolides. 2-Deoxy-~-arubino-hexose (55) was treated in methanol with hydrogen chloride according to a pro~edure’~ which was purported to yield almost exclusively a mixture of the anomers of methyl 2deoxy-~-arabino-hexofuranoside. It has now been shown78 that the latter consists, in fact, of an anomerically pure methyl 2deoxy-~-arabim-hexofuranoside(77), plus substantial proportions of an approximately 1:l mixture of methyl 2deoxy-a- and 8-D-arabino-hexopyranosides (78) and (79), and unreacted 2deoxy-~-araZrino-hexose(55). pNitrobenzoylation of the sirupy, quadripartite mixture led to a mixture of pnitrobenzoic esters from which, by fractional recrystallization, there was obtained in pure form a methyl 2deoxy-3,5,6-tri-O-pnitrobenzoyl-~-urubino-hexoside (80). Attempts to replace, directly, the severely hindered methoxyl group at Gl of (80)by halide failed. Methoxidcwmtalyzed saponification of (80) gave a crystalline methyl 2deoxy-~-arubin~-hexofuranoside (77). On conformational grounds, and because of its strongly positive specific rotation (+117O), the a-Danomeric configuration is provisionally assigned to (77), as well as to (80). Treatment of (77) with carbonyl chloride gave the 5,60-carbonyl derivative (81), and pnitrobenzoylation of the latter gave methyl 5,60-carbonyl-2deoxy-3-0-p~trobenzoyl-a-~-arubino-heside (82) , Replacement of the methoxyl group at C-1 of (82) by hydrogen bromide in dichloromethane (77) H.R. Bolliger, Helu. Chim. Acia, 34, 889 (1951). (78) I(.V. Bhat and W.W.Zorbach, Curbohydrutc Rcs., 1, 93 (1965). (79) I. W.Hughes, W.G.Overend and M. Stacey, J . Chem. Soc., 2846 (1949).

W. W. ZORBACH AND K. VENKATRAhfANA B U T

310

(79)

(78)

(55)

HOCH.

-

L

(80)

o=c.

(04)

,OC%

I

1 (89)

was facile, yielding crystalline 5, GO-carbonyl-2deoxy-3-O-p-nitmbensoylD-urabino-hexosyl bromide (83). The anomeric configurationof the bromide (83) has not yet been determined, but is presumed to be c r - ~ ,in which the bromine atom occupies a position trans to the substituted side-chain at C-4 of the furanoid ring. The utility of the bromide (83) in replacement

SYNTHETIC CARDENOLIDES

311

rcactiorin wm demomtrated, in part, by the ease with which it reacted with silver pnitrobenzoote, giving 5,6-0-carbonyl-2deoxy-1,3di-0-pnitrobenzoyl-D-arqbim-hexose (84), the anomeric configuration of which has not yet been determined. Through the latter reaction, indirect support for the structure of the bromide (83) waa provided.

IV. CONTRIBUTION OF THE CARBOHYDRATE COMPONENT TO PHYSIOLOGICAL ACTIVITY 1. Assay Procedure New glycosides are not standardized directly in terms of therapeutic effectiveness in man, but are assayed for toxicity to animals. Because the cat heart behaves toward the drugs in much the same manner as does the human heart, the cat assay has for some time been accepted as the standard. A particularly reliable assay has been developed by K. K. Chen, formerly of the Lilly Research Laboratories, and currently of the Indiana University Medical School. In this method, a dilute solution of the glycoside is injected intravenously, a t a standard rate, into a cat (that has been anesthetized with ether) until death occurs by ventricular fibrillation. Each compound is assayed in a group of adult cats of average body-weight) and a result is expressed a~ the geometric mean of the cat lethal dose (LD). The values thus obtained are inversely proportional to the potencies. A linear relationship obtains through the use of LD per mg., an expression inaugurated by Chen, which is obtained simply by taking the reciprocal of the LD. Practically all of the known cardenolides have been assayed by Chen and his coworkers; the data were obtained in one laboratoqf under conditions as nearly identical as possible. It is for this reason that meaningful results can best be obtained from this method, particularly for comparison studies in terms of structure-activity relationships. Also, it is important to note that the potency figures for cats are applicable to man, when the drugs are administered intravenously. The data cited in Table I are all Chen assay values; those obtained prior to 1961 may be found in an excellent compilation by Hoch.' Cardenolides, for which assay figures have since been given by Chen, will be referenced accordingly. 2. Some Correlations

Of the sixteen isomeric nldohexoses, only D-glucose has been found as a carbohydrate component of cardenolides. It generally occurs in combination with other sugars, and rarely are cardenolides found in which a Dglucose residue is joined directly to the aglycon moiety? Prior to the work

312

W. W. ZORBACH AND

K, VBNKATRAMANA BHAT

of Elderfield and coworkers,uJs the &D-glucosides (6b) and ( 8 ) of strophenthidin (2) end digitoxigenin (1) ,respectively, were unknowneo;nevertheless, both showed remarkably high potenoies when compared with corresponding hexosjdea of natural origh1J3 Indeed, (bb) and ( 8 ) were the only fully hydroxylated hexosidea of (2) and (1) known and, in light of their high potencies, the suggestion was made that a direct relationship between the degree of hydroxylation on the pyranoee component of a cardenolide and the cardiotonic activity of the cardenolide might exist. To this end, a study waa initiated at Georgetown University in 1956 with a view to obtaining a series of cardenolides having minimal structural variations in, the sugar component, in the hope of clarifying the relation between the structure of the carbohydrate residue and the activity of the cardenolide. This work also had as a leading objective the preparation of a series of glycosides of digitoxigenin (1) and of strophanthidin (2), all s adverse effect, containing hexoses or 2deoxyhexoses, in order to ~ s e s the if any, of (1) changing a primary grouping (-CH*OH) to a methyl group, or a secondary grouping (-CHOH) to a methylene group, or (2) changing the configuration of an asymmetric carbon atom at a single position in the pyranoid component. The startling discovery that the synthetic digitoxigenin a-digitoxoside (52) shows a potency substantially less than that of its aglycon (1) invited the preparation of additional hexosides which would contain “unnatural” glycosidic linkages, in order to confinn the observation that such loss in potency is a function of this structural feature. In spite of the substantial progress made, the list of known hexosides of digitoxigenin (1) and of strophanthidin (2) is still small. The limitations on the preparation of a new cardenolide are often severe, especially with one which contains a rare sugar residue. A partial synthesis of the sugar must be accomplished from more readily available carbohydrate starting materials, the sugar must be converted into a relatively stable O-acylglycosy1 halide and, finally, a coupling must be carried out. The time required for the synthesis and the characterization of such a cardenolide, together with the securing of sufficient material (usually 100 mg.) for w a y , may be six to eight months. Because of the limited data, it would be imprudent to attempt at this time to draw sweeping conclusions regarding the role of the sugar in enhancing or otherwise modifying cardiotonic activity. In the following discussion, some trends may be noted, and the correlations drawn therefrom are, essentially, a reflection of observations published previously by Chen and (80)Fourteen y e m later, R. Mauli and C. Temm,[Helu. Chim. Ada, 40,299 (1067)l isolated strophanthidin 8-Pglucoside (Bb) from Periploca nigre8una. (81)K.K. Chen, Proc. Infern. Phu-1. Meeling, let, Sfockholm, 1061, 8, 27 (1982). (82)F.G.Henderson snd K. I(.Chen, J . Med. Chem., 8,677(1966).

SYNTHETIC CARDENOLIDES

313

For each of the aglycons (1) m d (2), the correspoiidiiig glyrosides listed in Table I are arranged iii the older of increasing “deoxygenation” in the carbohydrate component and, for the purpose of the comparisons which follow, the potency values givcn are molar potencies. It may be seen that there is a progressive, although by no means linear, diminution of cardiotonic activity. For the glycosides of digitoxigenin ( I ) , the most active appear to have that conformation in which the hydroxyl groups a t (2-2, C-3, and C-4 of the pyranoid ring are equatorially disposed, as evidenced by the high potency of the /3-D-glucoside ( 8 ) . Also, the &deoxy-@-mglucosidea has a potency somewhat greater than that of the ~ - ~ ~ o x Y - & D mannoside (44).Inspection of the value for the 2deoxy-&~-arabinohexosides” (62) suggests that deoxygenation a t C-2 diminishes the potency less than does a reversal of configuration of this carbon atom. Unfortunately, the b-D-mannoside is not yet available and, therefore, a direct comparison has not been made. As shown in Table I, deoxygenation either at C-2 or at C-6 leads to a loss in potency, yet the 2 ,6-dideoxy-i3-&ribohexoside (53) has a potency only slightly less than that of either (62) or (71), suggesting that the losses in activity through deoxygenation are not necessarily cumulative. For the glycosides of strophanthidin (2), the synthetic a-u-cmannoside (33) has an outstanding potency, this being the highest yet recorded for any cardenolide. A tendency for the highest potency to be displayed by cardenolides in which the sugar residue has an axially oriented hydroxyl group at C-2 is suggested by the observations that (a) the 6-deoxy-8-Dmannoside (46) has a potency only a little lower than that of the fully ~ (6a) hydroxylated p-D-glucoside (6b), and (b) the a r - arabinoside [C-2 OH,a] is considerably more potent than the 8-D-xyloside (6) [C-2 OH,e]. An exception to this is provided, however, by the 6-deoxy-pD-dosidd6; although having approximately the same potency as that of the Gdeoxy-,9-~-mannoside, it differs from the latter by a reversal of the configuration at both C-2 and C-3 of the pyranoid residue. Perhaps the strongest support for the argument, that enhancement of cardiotonic activity by the carbohydrate component is a function of hydroxylation, may be gained by inspection of the potencies of the completely deoxygenated 3-tetrahydropyranyl derivatives@ of digitoxigenin (1) and of strophanthidin (2) (see Table I). In each case, the value ob(83) F,Kaiser, E. Haack, and H. Spingler, Naful.loissenschqfkn,49, 1969 (1962). The Chen value waa communicated privately to the authors by Dr. F. Kaiser, C. F. Boeringer u. Soehne, G.m.b.H., Mannheim-Waldhof, Germany. (84) F. G. Hendemon and K. K. Chen, J . Med. Phann. C h . ,6,988 (1962). (86) (a) P. Mlihlradt, E. Weirs, and T. Reichstein, Heb. Chim. Acfu, 47, 2164 (1964). (b) K. K. Chen and F. G. Henderson, J . Pharmucot. EzptZ. Therap., 160,53 (1965). (88)W. W. Zorbach, W. Blihler, and 8. Saeki, C h . Pharm. Bull. (Tokyo), 18, 735 ( 1965).

314

W. W.

ZORBACH AND K. VENKATRAMANA BHAT

TABLEI Cardlotonic Activities of Some Digitoxigenin (1) and Strophanthidin (2) Pyranoaidea ~

~~

Pyranoayl moiety

Formulp of conlonolids

LD/pmole Referen-

Digitoxigenin (1)o GlucoW, &D-‘ Gaeoxy-, @-DJ 6deoxy-3-0-methyl-, a - t b &deoxy-%O-methyl-,& D - ~ MBN~OBB, 6deoxy-,

BdeoTY-, &D-b adeoxy-, a-D arabiwHexose, 2deoxy-, &D-* tibo-Hexose, 2deoxy-, &DJ 2 I Bdidmxy-, &D-’ 2,6dideoxy-, a - ~ gEymo-Peqtose, 2,3,4-tridoxxy-, =(or G)

4.3

1

2.3 2.7 2.6 1.9 1.5 0.8 2.7 2.7 2.3 0.9 0.2

83

6.3 8.2 2.2 7 .O 6.6

4.0

1 82 82 1 82 82

6.6 6.2 8.4 3.8 0 .Q

1 1 1 82

1 1 1

82 81,82 84 82 1

81 82

[3-tetmhydropyranyl ether] Strophanthidin (2p

Glucose, &pb Mannose, ebb a-D

Bdeoxy-, a-LJ Meoxy-, && 6deoxy-, a - ~ A l l ~ Bde~xy-, , &DJ Arsbinoge, a-bb Xylose, BL9 Lyxme, a-D &erePentose, 2,3,4trideoxy-, -(or G) C3-tetrshydropyranyl ether]

* LD/pmole = 0.8. Nsturel linkage. * LD/mole

-

86

1.2.

tained is lower than that of the corresponding aglycon. It may be reasoned, therefore, that the pyranoid ring makes no contrjbution of its own, but merely acts as a vghicle for carrying hydroxyl functions, and it is suggested that the uneubstituted tetrahydropyranyl ring serves only to “dilute” the cardiotonic activity of the aglycon. In Table I, three p a h of WL-and #I-D isomers are available for direct comparison of activities. These are ( 1 ) the 6deoxy-3-O-methylglucosides

SYNTHETIC CARDENOLIDES

315

(15)arid ( 15a), ( 2 ) the Gaeoxymannosides (19)and (44)of digitoxigeilin (1), and (3)the 6-deoxymannosidw (9)arid (46) of strophanthidin (2). Although the value8 for each pair fall within the same range, it is interesting to riotc that the a-Lisomer is alwlty8 slightly more potent. In contrast, the glymsidw (27), (52), (32),arid ( 4 9 , all of which contain the ~ show very low potencies as compared with the “unnatural,” a - linkage, corresponding &D or a-L isomers, or both. With the digitoxigenin (1) glycosides (27) and (52), it is interesting to note that the values obtained are the same as that for the aglycon (l),and it therefore appears that, in these cases, the sugar makes no contribution whatever. The a-D-hexosides (32) and (45) of strophanthidin (2), although showing low potencies as compared with the corresponding 8-Dor a-Lisomers, are somewhat more active than the parent aglycon (2). The a-D-lyxoside (14a) likewise has a low order of activity as compared with the a-L-arabinoside (6a) and the bwxyloside (6). Particularly surprising is the fact that the &deoxy-a-~mannoside (45) is more potent than the fully hydroxylated a-D-mannoside (32). Whether other such “anomalies” will appear, especially with glycosides having “naturd” linkages, cannot be predicted. Perhaps the most puzzling aspect concerning the role of the sugar in modifying cardiotonic activity is the nature of the glycosidic linkage in natural cardenolides. As shown in the foregoing by three a-L and &D isomeric pairs, the potencies for corresponding pairs, although not identical, fall within the same range, in which the potency of the a-L isomer is a little greater in each case. This fact would seem difficult to interpret, because, considered in the usual terms of the conformational rules for carbohydrates;a-~ and 8-Dimmediately connote the conformers (85) and (86) for ex,and /3-D-cardenolides, respectively.

From a consideration of these two structures, it is reaaonable to 4uggest that, for all cases, the physical properties (to include solubility) of such (87) The symbols CA and CE, sppended to the names of the etructuree shown, follow an improved eyetern for indicating the principd conformations of pyranoid sugars and derivatives [R. S. Isbell and R. S. Tipaon, J . Rss. Natl. Bur. SM., MA, I71 (ISSO)]. Through the UBB of eymbob, a precise specification of all of the principal pyranoid conformera can be made.

316

W. W. ZORBACH AND K. VENKATFUMANA BHAT

pairs would bo widely divorgent. Accordingly, the physiological “distributive” effects of the two should differ markedly, resulting in a differential in cardiotonic activity grertt,er than that observed. A duo to u possibly corract rwcwrnant of the mnformations of a-L and of /3-D-cardenolides is provided by the infrared absorption spectrazsof the a-crhamnoside [evomonoside ( 19) ] and the synthetic a-D-rhamnoside (27) of digitoxigenin (1). The two spectra are so similar that only on very close scrutiny can minor differences be observed; indeed, they are almost superposable. This fact suggested that the vibrational characteristics of the two molecules are nearly identical. Their structures might, then, be represented a8 shown, in which both have equiualeat conformations. It is

\

OH (Y

-L-Rhamnoeide- CA (19)

to be noted that, with either (19) or (27), there are, in the vicinity of the pyranoside ring, no groups present in the steroid portion which could give rise to a different set of interactions. It is well known that the infrared absorption spectra of enantiomorphs are identical, and it is for these reasons that (19) and (27) should have nearly identical vibrational characteristics. On this basis, a naive, although not entirely illogical, conclusion might be drawn, to the effect that the two cardenolides should possess the same “distributive” characteristics and , therefore, should show approximately the same potency. However, this conclusion is not supported by the facts, for the “unnatural” a-r>-rh&mnoside(27) has a potency less than one half that of evomonoside (19) ,and is less active even than its aglycon, digitoxigenin (1). It is now suggested that, with glycosidea having bulky aglycon residues, the usual rules regarding conformation for carbohydratesme not applicable. It is generally considered that, with certain exceptions (such as idose), aldohexopyranoses (or aldohexopyranose residues) assume that chair form

317

SYNTHETIC CARDENOLIDEB

which accommodates the hydroxymethyl (or methyl) group at C-5 as an equatorial substituent, regardess of the nature of the substituent at C-1. It is, however, unreasonable to consider that, when bulky aglycons are involved, the pyranoside component would maintain a conformation which would permit an axial disposition of the aglycon moiety at C-1 in the case of a-glycosidcs. This would be especially difficult to attain with cardenolides, with their huge steroidal residues. It is further suggested, therefore, that, irrespective of the configuration at C-1 of the pywnoside component, all carqholides have conjommtions in which the qlycon residue is an eqw torial substiluent, thereby overriding all conformational eff ecta imposed by substituents on the pyranoside ring. The a-crhamnoside (19) and the a-D isomer (27) would, accordingly, have the following structures, in which the aglycon moiety is equatorially disposed. Whether the two rhamnosides do, in fact, exist aa the conformers shown makes no difference

I

I HO

HO a-?,-Rhamnoside-CE (1s)

a-D-Rhamnoside-CE (27)

aa to their vibrational characteristics, for they have the same equivalence as when the pyranose components are shown in the alternative chair conformation. Nevertheless, this difference may have a profound influence on the absorption of the drugs in animals, and it may be significant in this context that each of the four “unnatural” a-D glycosides (27), (52) , (32), and (IS)has an unusually low solubility in any given solvent aa compared with that of its a - or ~ @D isomer. In the application of this conformational rationale simultaneously to the structures of a - b and 8-D-cardenolides, the a-c and the 8-D-rhamnosides (19) and (44) serve to illustrate the hypothesis that a structural corrwpondence can be obtained, which permits a satisfactory explanation for the fact that a-Land B-D isomers have p9tencies which are, approximately, the same. On the ba& of the structures shown, the aglycon component is equatorially disposed, both pyranose rings have the same chair form, and the order of substituents on the ring is the same. The only difference between (19) and (44) is a reversal of configuration of substituents on the pyranoid ring, which is tantamount merely to a (‘vertical”displacement of

318

W. W. ZORBACH AND K. VENKATRAMANA BHAT

substituents on the pyranoid ring of one with reapect to the other. It is suggested that, insofat twsolubility and physiologioal absorption are concerned] this difference is slight compared with that noted with the &I.and cu-p.rhamnosidea (19) and (27) in the foregoing. Some justification for thir, belief may be taken from the fact that the potencies of digitoxigenin “2-deoxy-@-DglucOside” [(62) C-3 OH, a] and digitoxigenin “2deoxy-&D-alloside” [(71) C-3 OH, e] are substantially the same. Alm, because such a small difference does exist between the structures (19) and (44)I it is reaeonable to ascribe the slightly greater potencies of a - t cardenolidee, aa compared with those of their /?-D isomers, to such structural variation.

V. TABLE OF SYNTHETICCARDENOLIDEB In Table I1 are listed the synthetic cardenolides reviewed in this article, aa well aa the corresponding 0-acylated intermediatea when these have been isolated in crystalline form. In addition to the melting point, specific rotation, and yield, the Table lish the acid acceptor, desiccant, and solvent employed in the coupling reaction for each. The new gIyoosides we grouped according to the aglycon moiety, and we divided into (a) digitoxigenin glycosidoa, (bl strophanthidin glycosides, and (cl miscellaneous glycosides.

TABLE I1 Synthetic Cardenolides

Glymeyl moiety

Formula number

Melting point,OC.

C.3, Acid Desiccante Solventd Reported References degree" aeceptor* pidd, %

a Digitongenin glycusides

Glucose, &Dtetra-o-acetylGlucusiduronate, &D-, tri-0acetyl-, methyl

242-246

163-168 226-228

-4.9, A -8.6, A +2.1, B

otgbirrsHexose

ribo-Hexase zd-Y+= 2,6didmxy+~ @ 8nomer

-=,

223-225 251-255 204-209

6de€lxy-Q!-D@ anomer Bde0Xy-a-c

glgmv-Pentase, 2,3,4trideoxy-D-(or t) [3tetrahydmpyranyl ether]

sc

-

-

PD DCE

15 42

13 13 23

AZ

AZ

DCE DCE

15 19

26 26

+27, A +85.1, B -7.1, B

sc

AZ

DCE DCM PD

46

67

+53.4,B

sc

DCE TCE DCE

23 13 44

63

DCM

66

86

-12.4, B -28.7, B

161-165

so

M M

SC

+67.6, B +11 .8, B

@ Bllomer

so

+31.9, C:

-

8c MC

-

-

AZ CS

-

A2

4.5 4.5'

64 65

23 23

b. Stmphanthidin glycosides Arabinose, a-I.tri-0-acetyl-

210(dec.) #)o (dec.)

-

12 12

212-2#)

+82.1, B; +57.1, E

PD-BZ PD-CT

152-158; !BZ-!236

(dec.)

+46, c

1s2-151 (dec.) 24o-m (dec.)

+7,A +1oJ c +IS, C +21, D +11 .s, c +78.7, F +6.3, F

236-237

(dec.)

234-236 (dec.)

240-260 (dec.)

259-263 (dec.)

285r289 128-133

261-263 228-231 238241

DCM

-

+%.% B 4-15.3, B +l.O, A -9.5,

268 ( k . 1 194-199

-1.4, A -3.3, A

27

-

PD PD

-

PD-BZCT DCE DCE

-

DCE DCM PD-BZ

-

F

240-244

42

-

+56.6, B

179-182

32

PD

7.5 31

-

35

m

30

-

53 7

44 11

-

22

33 33

86 12 12 12 38 38 45 46 46 46 53

15 15

13 13

.

Key: A, ethanol; B, methanol; C, chloroform; D, water; E, pyridine; F, methmol-water (9:l) b Key: SC, silver carbonate; 80, silver oxide; MC, mercuric cyanide. Key: AZ,seeotropic distillation; M, magnesium sulfate; CS,calcium sulfate; .i Key: PD, pdioxane, BZ,benzene; CT,carbon tetrachloride; DCM, dichloromethane; DCE, 1,!Michloroethme; TCE, 1,1,2-trichloroethane.

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BYA. R. ARCHIBALD AND J. BADDILEY

I. Introduction ....., . . . . .......... . , . ......... .. .... . . . . . , , .. . . . . . . . . . 11. Surface Structures of Gram-positive Bacteria. . . . . . , . . . . . . . . . . . . . . . . . . . 111. Discovery of the Teichoic Acida. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Hydrolyeis of Estere of Phosphoric Acid.. . . . . . . , . . . , . . . . . . . , , . . . . . V. Membrane Teichoic Acids. . , . . .. . . . . . . . , . , , . . . . . . . . . . . . . . . . . . . . . . . . . 1. LcrcfubaciUusarabinmua 17-5. . . , . . . . . . , . , . . . . . . . , . . . . . . . . , . . . . . . . . 2. Laetabacillus mei 7469.. . . . . . . . , . . . . * . . . . . . , , . . . . . . . . . . * . . . . . . . 3. Staphybeoccue hctie 7944.. . . . . , . . . . . . . . . . . . . . . . . . . . ’. . . . . . . . . . . 4. Stuphylococms aurm H . . . . . .... ... . . . . , .. . , . . , , . . . . . . . , 5. Group D Streptococci.. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . , . . VI. Wall Teichoic Acids. . . . . , . . .. . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . .. . 1. Glycerol Teiclioic Acids.. . . . . . . . . . , . . . . . . ., . . . . . . . . . . . . . . . . . . . . . , 2. Ribitol Teichoic Acida.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Teichoic Acids of Actinomycetes. , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. The Locstion of Teichoic Acids in Relation to Cell Structure.. , .. . . . . . . . . . 1. W i t i o n with the Membrane. , . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 2. Association with the Wall.. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . IX. Biosynthesis.. .. . .. . . . . .. .. . . . . . . . . . . . . . ... . . . . . .. . .. .. .. . . ... . . ... .

.

.

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

.

.

.

323 324 326 328 332 334 341 342 342 344 346 346 354 363 365 365 368 372

I. INTRODUCTION Teichoic acids’ are natural polymers which occur in Gram-positive bacteria and which possess several novel structural features. Details of their structure vary according to the source, but in general they are composed of residues of glycerol phosphate or ribitol phosphate, Dalanine, and a sugar or 2-acetamide2deoxy sugar. They occur in two distinct locations in the cell-in the wall, and also between the wall and the underlying protoplast membrane,2J probably in association with the latter structure.’ Membrane or “intracellular” teichoic acids are very widely distributed and have been found in almost all of the Gram-positive bacteria (1) (2) (3) (4)

J. Baddiley, Proc. Chem. Soc., 177 (1959). J. Baddiley, J. Roy. Inst. Chem., 86,366 (1962). J. B. Hay, A. J. Wicken, and J. Baddiley, Bioehim. Biophys. Ada, 71, 188 (1963). G. D. Shockman and H. D. Slade, J. Gen. Microbial., 87, 297 (1964). 323

324

A. R. ARCHIBALD AND J. BADDILlY

examined: they are invariably polymers of glycerol phosphate, and it is likely that they are present in all Gram-positive bacteria. Wall teichoic acids are less widely distributed, but have been foundbe in many strains of staphylococci, streptococci, bacilli, laotobaailli, and streptomyces. They are polymers of either a glycerol phosphate or a ribitol phosphate to which sugar residues and D-alanine residues are attached, and, in many cases, they occur in relatively large proportions (up to SO% of the dry weight of the wall). The early postulate has been confirmed”J-1sthat P-cytidine ~ ( P - D glycerol l-pyrophosphate) (“cytidine diphosphate glycerol”) and PIcytidine 5-(P-cribitol l-pyrophosphate) (“cytidine diphosphate ribitol”) are biological precursors of the teichoic acids. Both wall and membrane teichoic acids are serologically active (compare Ref. 14), but nothing is yet known about their function in the living cell. Nevertheless, it has been suggested that they may control the passage of ions across the wallmembrane region,16 or may be concerned in the formation of the rigid, wall polymers.16 The present discussion will be concerned mainly with the chemistry of these polymers. For reaaons of space, and because of the rapid rate of development of the subject, the equally important topic of serological behavior and significance of teichoic acids is not discussed in detail. 11. SURFACE STRUCTURES OF GRAM-POSITIVID BACTERIA Although it would be out of place to give here an extensive account of the\ surface anatomy of bacteria, current interest in the exact location of compounds in the anatomical components of cells, for example, in membranes, granules, mitochondria, and ribosomes, requires a reasonably precise description of the location of teichoic acids. In its turn, this requires an understanding of the main features of the outer regions of bacteria. Gram-positive bacteria possess a rigid cell-wall which is responsible for (5)J. Baddiley and A. L. Davison, J . Gbn. Microbial., 94,296 (1962). (6) A. L.Davisoa and J. Baddiley, J . Gem. Microbial., 89,H l (1903). (7)A. L.Dsvison, J. Baddiley, T. Rofstad, N. Losnegsrd, and P. Oeding, Ndure, 309, 872 (1964);A. L.Davison and J. Baddiley, iW.109,874 , (1904). (8) M.E.Shape, A. L. Davison, and J. Baddiley, J . Urn. Mirrobid., 84,333 (1964). (9) A. C.Raird-Parker, J . Urn. Mirrobiol., 88,363 (1965). (10) M. Burger and 11. G l w r , Biochim.Biophys. A&, 64,675 (1982). (11) M. Burger and L. Qlaaer, J . B+l C h . ,P9,SlSS (1964). (12) L. Glaser, BiocAim. Biophys. A&, 71, 237 (1863). (13) L. Glaser, J . Biol. Cham., ISB, 3178 (1984). (14)M.McCarty and J. I. M o m , Aduan. Immunol.,4,249 (1964). (15)A. R. Arohibald, J. ‘J. Annatrong, J. Baddiley, and J. B. Hay, Nature, 191, 670 (1961). (10)H. R.Perkins, Bacte7iol. Rev., ill, 18 (1963).

THE TEICHOIC ACIDS

325

the shape and integrity of the cell>’*18The wall comprises 10-40~~ of the dry weight of the intact organism, and may vary in thickneas from 100 A. to a,s much a8 800 A. Examination of bacteria by electron microsoopy clearly reveals the prwnce of such walls, which can usually be differentiated from such surfaoe appendages as flagella, from the capsular and other extracellular substances which coat many bacteria, and from the underlying protoplasm with its surrounding membrane. The production of capsular material is subject to genetic and environmental control, and it is often possible to remove capsules from encapsulated organisms without affecting the morphological integrity and viability of the cell.l+*1 Dissolution of the wall of certain organism may be effected with muramidase (formerly known aa lysozyme)e.B; this results in the production of fragile, spherical protoplasts which are readily ruptured by osmotic pressure. The shape and integrity of the cell is thus principally due to the cell wall. The component common to all bacterial cell-walls, and primarily responsible for their strength and rigidity, is a glycosaminopeptide (mucgpeptide) composed of 2-acetamido-2deoxy-~-glucose,Nacetylmuramic acid18~’8~24~26 [Zacetamido-%deoxy- (3-O-lactoy1-~-glucose)l, and four or five amino acids (typically, alanine, glutamic acid, glycine, and either lysine or 2 ,6diaminoheptanedioic acid), some of which have the D configuration. The other components usually found in cell walls are proteins (in small proportion), polysaccharides, and teichoic acids. Complete removal of these soluble components can often be achieved without disintegration of the wall, and their function is, therefore, presumably not structural.I6 The protoplast membrane differs markedly in properties and composition from the wall, and is characterized by a high content of lipid and by the virtual absencflv” of the characteristic components of wall glycosamino(17) I. C. Gunsalus and R. Y. Stanier, “The Bacteria,” Academic Press Inc., New York, N.Y.,1960, Vol. 1.

(18) M. R. J. Salton, “The Bacterial Cell Wall,” Elsevier Publishing Co., Amsterdam, 1984. (19) 0. T. Avery and R. Dubos, J . EzpU. Med., 64, 73 (1931). (20) M.Tod, Med. J . Osaka Unw.,6,726 (1955). (21) M. H. Adams and H. R. Park, Vitology, 2,719 (1956). (22) C. Weibull, Ann. Rev. MicrobioZ., 12, 1 (1968). (23) M. R. J. Salton, Bacterial. h., 21,82 (1967). (24) H. J. Rogers, in “Menibranes and Surfaces of Cells,” Biochem. SOC. Symp. (Cnmbridge, Engl.), 24, 55 (1962). (26) H. J. Rogers, in “Function and Structure in Micrc-organisms,” Symp. SOC.Gen. Mirrobiol., 16, 186 (1965). (26) C. Wiebull and L. Bergstrom, Biochim. Biophys. Ada, 80,340 (1968). (27) G. D. Shockman, J. J. Kolb, B. Bakay, M. J. Conover, and G. Toenniea, J . B p e f d l . , 86, 168 (1963).

A. R. ARCHIBALD AND J. BADDILEY

326

peptide. The teichoic acids formerly described w “intracellular” appear to be closely sssociated with the membrane,’ as are at least some of the enzymes concerned with the biosynthd of both “intracellular” and wall teichoic w i d s . % ~ ~ * ~ ~ 111. DISCOVERY OF THE TEICHOIC ACIDS The application of electron microscopy to the study of fractionation of disrupted bacteria, and the subsequent isolation of homogeneous preparations of wall fractions,a have led to an increasing interest in their chemistry. This interest has been further stimulated by the recognition of the metabolic importance of the wall, and the discovery that the lethal action of such antibiotic substances as the penicillins is principally due to inhibition of wall synthesis (compare Ref. 33). The discovery of teichoic acid aa a major component of the walls of several bacteria was not a direct consequence of thia interest in cell w d s , but followed from the discovery of two new nucleotides, first detected in extracts of LactobadUue arabinoeueJ- but now known to be widely distributed. By degradative studies and by chemical synthesis these were shown to be Pl-cytidine b(P-D-glycerol l-pyrophosphate) (1)*“ and Pl-cytidine Ci-(P-cribitol l-pyrophosphate) (2).@,@*&The con!iguration@b of the glycerol phosphate residue in Phytidine 5-(P%-glycerol l-pyre phosphate) is that of the naturally occurring wglycerol l-phosphate (“cglycerol3-phosphate”),and it seemed likely that this nucleotide would (28) L. Gleser and M. Burger, J . BW. C h . , 989,3187 (1964). (29) M. Burger, Biochim. Biophga. A&, 71,496 (1963). (30) S. G. Nathenson and J. L. Stmminger, J . Biol. C h . , 487, ~63839(1962). (31) S. G. Netheneon and J. L. Strominger, J . Bid. C k . ,oS8, 3161 (1903). (32) M. R. J. Salton and R. W. Home, Biyhim. Biophys. Ada, 7,177 (1951). (33) E.P.Abraham, Endeauour, 18,212 (1969). (34) J. Baddiley end A. P. Mathiaa, J . Chbm. &., 2723 (1964). (36) J. Baddiley, J. 0.Buchanan, B, Cam, A. P. Mathias, and A. R. Sanderson, Biochem. J., 64,699 (1956). (36) J. Baddiley, h. G . Buchanen, A. P. Math-, end A. R. Sanderaon, J . C h . Soc., 4188 (1966). (37) J. Baddiley, J. G. Buchanan, and B. CWM, J . C h . Isloc., 1869 (1967). (38) J. Baddiley, J. G. Buchanan, and A. R. Senderaon, J . C h .Soc., 3107 (1968). (39) J. Baddiley, J. G. Buohanan, B. Cam, and A. P. Ma&, J . C h . Soc., 4683 (1966). (40) J. Baddiley, J. G . Buchanan, and C. P. Fawcett, J . C h . Soc., 2192 (1969). (404 6ee 6.F. Neufeld and W. 2.Hamid, Aduan. CarbolLyh Chum., 18,309 (1983).

(40b) In the original publications on teichoio wide, the numbering of dditob doee not conform to the Rules. The names and formulaa used in the present article are in conformity with the Rules of Cwbohydrate Nomenclature, J . Org. Chem., 98, 281 (1963).

THE TEICHOIC ACIDS

327

be involved in the biosynthesis of known glycerol phosphate derivatives.

No ribitol phosphate was, however, known in Nature, and it was considered that (2) might function either in transformations or in transfer processes leading to the formation of polymeric substances containing ribitol or ribitol phosphate residues. Thus, the cytidine derivatives would be analogous to nucleotides containing either 2-acet.amido-2-deoxy-~-glucoseor N-acetylmuramic acid peptides, which are recognized intermediates in the biosynthesis of the glycosaminopeptide component of bacterial walls.

The correctness of this reasoning has been established by the isolation of polymers cantaining residues of ribitol phosphate or glycerol phosp h a t e t h e teichoic acids-and by the direct demonstration of the participation of the nucleotides in their biosynthesis in cell-free systems. The existence of the polymers was demonstrated by addition of ethanol to trichloroacetic acid extracts of whole cells of Lactobacillus arabinow, whereupon a polymeric material was precipitated which contained derivatives both of glycerol phosphate and of ribitol phosphate." Examination of the isolated walls of Lactobacillus arabinosus and of Bacillus subtilis showed that these contain substantial proportions of phosphorus, which is present as a ribitol phosphate polymer'$; D-glucose and alanine are also compoThe alanine has the D configuration, and is nents of the new sub~tances.~~ attached to the polyribitol plioflphnte through unusually labile, ester linkagc~Similar cmmpound~wcre dctected in the walls of other bacteria, (41) J. Birddilcy, J. C.Buahanan, and G. R. Greenberg, Biochem. J., 66, 5 1 (1957). ~ (42) J. h d d i l ( y , J. (;. Ruchnntm, and B. Cam, Biochim. Biophys A c b , 27,220 (1958). (43) J. J. AriiraLroiig, J. Bnddiley, J. G. BuoBnnnn, and B. Cars, Nature, 181, 1092 (1968).

328

A. R. ARCHIBALD AND J. BADDILUY

and such compounds were called teichoic acids.U Most teichoic acids conform to the general structure (3), Jthough variations on this structure have been noted. The glycerol phosphate polymere isolated from whole cells also contain sugar residues and walanine residues, and are related in structure to the polymere in the cell walls. Although the “intracellular” polymers are now believed to be associated with the cell membrane, they, too, are cdled teichoic acids.

p~

sugar I

HO-P-0-alditol-0 I1 I 0 D-alanine

fs. 87

P-0-aalditol-0 0 D-alanhe

j-~

sugar I P-0-alditol I .o D-alanine

(3)

It is apparent from the literature that teichoic acids had been observed on earlier oacaaions, although their nature had not been elucidated. Thus, in 1935, Julianelle and WieghmP isolated a carbohydrate antigen from staphylocooci which they called plysaccharide A; this has now been shown to be identical in serological properties with the teichoic acid extracted from the walls of SlCsphy2ococcus aureu8.18Similarly, in 1951, Mitchell and Moyle47e@detected a phosphate in the walls and intracellular fractions of Staphylococcus aureus which appeared to be mainly a derivative of glycerol phosphate: it now seems likely that this was a mixture of glycerol teichoic acid and ribitol teichoic acid. McCarty’D haa independently isolated an antigenic material from several bacteria, and shown that it is a polymer of glycerol phoephate. No sugar or D-alanine residues were present, but it is likely that the conditions used during extraction would remove D danine residues, and might remove sugar residues if only small proportions were present originally. IV. THIIHYDROIAXIIS OF EWERS OF PHOSPHORIC ACID The elucidation of the detailed structure of the teichoic acids has been greatly facilitated by advances in the chemistry of phosphate eatersadvanceg which have been stimulated by work on nucleic acids and phospholipids. The development of greatest significance to studies on the (44)J. J. Armstrong, J. Baddiley, J. Q. Buohanan, B. Cam, and Q. R. Oreenberg, J . Chcm. SOC.,4344 (1958). (45) L. A. Jdianelle and C. W. Wieghard, J . EzpU. Med., 6 2 , l l (1935).

(46)G . Hauksnes, D. C. Ellwood,J. Baddiley, and P. Oediig, Biochim. Biophvu. Ada, MI, 425 (1961). (47) P. Mitcheil and J. Moyle, J . Urn. Microbiot., 6,986 (1961). (48) P. Mitchell and J. Moyle, J . Urn. Miwobiol., 6,981 (1951). (49) M. McCarty, J . Exptl. Med., 109,381 (lQ59).

THE TEICHOIC ACIDS

329

structure of naturally occurring esters of phosphoric acid has been the elucidation of the manner in which they undergo hydrolytic cleavage (compare Refs. 50 and 51). Information obtained from the study of model compounds has been applied with great succeas to structural work on naturally occurring phosphates, and a brief summary of the relevant aspects is given here. Simple phosphomonoesters are stable to alkali, but are readily hydrolyzed at pH 4, when the species undergoing hydrolysis is the mono-anion. In more midic solution, the rate decreases, but, at pH values below about 0.5, the rate again increases due to hydrolysis of the protonated phosphate group. In molecules having a hydroxyl group adjacent to the phosphate group, the pattern of hydrolysis is similar; but at low pH values the phosphate grmp can migrate reversibly to the neighboring hydroxyl group, so that, for example, on heating D-glycerol l-phosphate in acid solution, a racemic mixture of 1- and %phosphates is formed." Simple phosphodiesters (such as dimethyl phosphate) are stable under alkaline conditions and a t pH 4, but are hydrolyzed a t low pH values. The presence of a hydroxyl group adjacent to the phosphate group markedly increases the eaae of hydrolyais of phosphodiesters, both in alkali and in acid, the hydroxyl group participating, in each case to effect hydrolysis through an intermediate, five-membered, cyclic phosphate which is readily hydrolyzed to a mixture of isomeric monoesters." Thus, Brown and Todd and their collaborators (see Ref. 50) found that acid- or alkali-catalyzed hydrolysis of alkyl esters of 2- and 3-monoribonucleotides follows the path outlined in Fig. 1. Examination of the products of incomplete hydrolysis of cytidine 3-(benzyl hydrogen phosphate) (4, R = CHGHb) has shown significant difference between the two pathways. Under alkaline conditions, no migration of the alkyl phosphate group in unhydrolyzed material occurs, whereas, in acid, significant migration, (4)+(8), is found. These results suggest that the step (4)+(5) in alkali involves an unsymmetrical transitionstate which may be represented aa (11) and, in acid, involves the formap tion of protonated species derived from intermediates of types (9) and (101, which can then undergo P-O fission to give either ( 6 ) or (7). Triesters of phosphoric acid are readily hydrolyzed under mildly acidic or alkaline conditions. (50) C. A. Vernon, C h . Soc. (London) Spec. Publ., 8,17 (1957). (51) D. M. Brown, Advan. Org. Chem., 8, 75 (y962). (52) P. W. C.Barnard, C. A. Burton, D. R. Llewellyn,K. G . Oldham, B. L. Silver, and C. A. Vernon, Chem. Znd. (London), 760 (1955). (53) D. Y. Brown and A. R. Todd, J . C h . Soc., 52 (1952). (54) D. M. Brown, D. I. Magrath, A. M. Niehn, and A. R. Todd, Nature, 177, 1124 (1956).

330

A.

n. ARCHIBALD

(a)

(8 )

FIQ.1-Participation Phosphodieetsr.

AND J. BADDILEY

(11)

(10)

of an Adjaaent Hydroxyl Group in the Hydrolysis of a

During structural studies on (2), it waa found that inorganic phosphate is readily produced when the nucelotide is heated at 100' in N hydrochloric acid. Synthetic L-ribitol 1-phosphate behaves similarly, whereas glycerol phosphates and wmannitol 1-phosphatebehave normally, and do not give signifiaant amounts of inorganic phosphate under these conditions. A study of the hydrolysis of L-ribitol 1-phosphate (wribitol bphwphate)n*M showed that, in addition to the expected racemisation due to the reversible migration of the phosphate group, large proportions both of inorganic phosphate and 1,$-anhydroribit01 (13) are produced. The mechanism suggested includes protonation of the ester oxygen atom followed by the electronic displacements shown in Fig. 2; the hydroxyl group at C 4 of the D-ribitol ester (12) is, presumably, in a sterically favorable position for the intramolecular,

HOGC

+ HSPO, HO

OH (12)

(13)

FIO. 2.-Formation of Anhydroribitol from Ribitol &phosphate. (66) J. Baddiley, J. G. Buchanan, and B. Car=, J . C h m . Soc., 4058 (1957).

331

THE TEICHOIC ACID8

nucloophilia whtitutiori. liibitol is converted into 1,Panhydroribitol in the presence of dilute acid, presumably by a mechanism which is similar but in which a primary hydroxyl group is protonated; however, this conversion occcrs more slowly than for ribitol phosphate. Traces of an optically active anhydroribitol and its phosphates are proNo anhydroduced when some teichoic acids are hydrolyzed with alkaJi.w*67 ribitol is formed by similar treatment of ribitol, its 1-, 2-, or 3-phosphates, or ribitol 1,9-diphosphate." However, small proportions of anhydroribitol and its phosphates are produced by the action of alkali on a synthetic poly(ribito1 phosphate) prepared by the action of diphenyl phosphorochloridate oil 3,4-O-isopropylideneribitol1-phosphate and 2-pho~phate.~ This observation suggests that 1,Canhydroribitol (13) or its derivatives (IS) are produced by fission of a phosphodiester, for example (14), in the manner indicated in Fig. 3, and that this reaction occurs together

ROH,C

/0 0

vG OH

HO

(14)

00

I H,~O-POR~ II 0

-

+ R'OPOS& OH

HO (15)

FIQ.3.-Formation of Anhydroribitol from a Ribitol Phosphodiester.

with the normal, cyclic phosphate sequence. Hydrolysis of certain phosphodiesters by C-0 fission, resulting from nucleophilic attack by a vicinal hydroxyl group, has been described by Brown and UsherJS0and the intermediate formation of a 3-membered oxide ring haa been demonstrated. However, in the case of a ribitol 1,5-di(phosphodiester), in which the linkages are between positions 1 and 5 of adjacent ribitol residues, it is likely that hydroxyl groups at C-4 of the ribitol are suitably oriented for direct attack on C-1, thereby eliminating phosphate and yielding 1,4anhydroribitol. Supporting this conclusion is the observation6O that a &membered epoxide is probably not an intermediate in the formation of 1,4anhydroerythritol when 1-0-p-tolylsulfonyl-DerythritoI is treated with alkali. (66)J. J. Armstrong, J. Baddiley, and J. G. Buchanan, Biochsm. J., 80, 264 (1961). (67) A. R. Amhibald, J. Baddiley, and J. G. Buchanan, Bioehem. J., 81,124 (1961). (58) D. A. Applegarth, J. (3. Buchanan, and J. Beddiley, J . Chem. Soc., 1213 (1966). (69) D. M. Brown and D. A. Usher, Proc. Chem. Soc., 309 (1963). (80)F. C. Hartman and R. Barker, J . Org. Cham., Y, 1004 (1983).

332

A. R. ARCHIBALD AND J. BADDILEY

The proportioq of 1,4-anhydroribitol formed by treatment of teichoic acids and synthetic poly(ribito1 phosphate) with alkali is small, and the major hydrolytic pathway involves the cyclic phosphate sequence. No 1,4anhydroribitol glycosides have been observed in the alkaline hydrolyzates of teichoic acids; possibly, tho prwnco of a glycosyl substituent makos the reaction stericttlly l a s favorablethan when such substituerits are absent.

V. MEMBRANE TEICHOIC ACIDS As the compounds described hitherto as “intracellular” teichoic acids are now known to occur, at least in those cases studied so far, in the region between the wall and membrane, and are probably attached to the membrane, the term “intracellular” is correct but misleading. It is now proposed to call these compounds membrane teichoic acids. All of those so far examined are polymers of glycerol phosphate in which glycerol residues are joined together through phosphodiester groups at the 1-and 3-positions. The %-positionsof the glycerol residua bear glycosyl or D-alanyl substituents, aa shown in structure (16). Membrane teichoic acids have been prepared by extraction of whole cells with trichloroacetic acida1 or by similar extraction of the cell contents (which contain fragments of membranes) obtained by the removal of walls from suspensions of disrupted bacteria.62-66 In addition to the teichoic acid, such extracts contain large proportions of polysaccharides and polynucleotides; small proportions of ribitol teichoic acid are frequently preaent in acid extracts of cell contents of organisms which have a ribitol teichoic acid in the wall. The significance of the presence of some (ribitol) wall teichoic acid in the cell contents fraction is at present uncertain, but its presence may be a consequence of release during the disintegration of the cells. The particulate fraction obtained by high-speed centrifugation of the cell contents (twp. 366) contains most of the membrane teichoic acid, and extracts of this fraction contain very little of the wall teichoic acid. HO

-

-Lo-,-% II A

CH,- o ~ ~ c l - w - ~ , c ~ - o f ~ o - Hf C &WE a

(16)

where R ~-0lYlinsor glycoryl

(61) (62) (63) (64) (66)

M. V. Kelemen and J. Baddiley, Biochsm. J., 80,246 (1961). P. Critchley, A. R. Archibald, and J. Baddiley, Biochar. J., 85,420 (1962). A. J. Wicken and J. Baddiley, Biochern. J., 87,64 (1883). A. J. Wicken, 8. D. Elliott, and J. Baddiley, J . h. Microbhl., 81,231 (1963). U. L. RRjBhandsry and J. Baddiley, Biochar. J., 87,429 (1965).

THE TEICHOIC ACIDS

333

Essentially pure saniples of teichoic acid have been obtained from the several types of extract by procedures involving fractional precipitation, gel filtration, and ion-exchange chromatography. Such preparations contain equal proportions of glycerol and phosphate, a somewhat smaller proportion of D-ulanine, and a variable proportion of sugar. The alanine from several teichoic acids has been isolated as its hydrochloride and shown by the positive action of Damino acid oxidase to have the D configuration. The alanine residues are attached to the rest of the chain through ester linkageswhich are readily hydrolyzed by ammonia or dilute hydroxylamine, to give alanine and its amide or hydroxamate. Kinetic studies of the reaction with neutral hydroxylamine have shown that the alanine ester groups in the membrane teichoic acids from L. c u a e i S 1 and L. arabinosuSa2exhibit the characteristic, high reactivity of those in the ribitol teichoic acids.66 These ester groups are much more labile than alanine methyl ester toward hydroxylamine. The remarkable lability of the alanine ester residues is characteriRtic of most of the teichoic acids studied, and is comparable to that of the amino acid ester residues in the aminoacyl nucleic acids which are concerned in protein biosynthesis.B7The high reactivity of the nucleic acid derivatives was originally attributed to the presence of a hydroxyl group on the carbon atom adjacent to that carrying the ester linkage.*M The lability of the alanine in the ribitol teichoic acids could also be explained on this basis, but the comparable lability in the glycerol teichoic acids clearly could not be, since in this caae there is no adjacent hydroxyl group. It appears from this conclusion that a neighboring phosphate group might also confer high reactivity on the ester linkages. Accordingly, ~~-2-(alanyloxy) ethanol and its dihydrogen phosphate were prepared and their rates of reaction with neutral hydroxylamine were compared with that of Dkalanine methyl ester." Both of the synthetic model compounds were hydrolyzed at a rate which was similar to that for the teichoic acids and much greater than those for simple, amino acid esters. Activation of the ester linkage may therefore be effected either by vicinal hydroxyl or phosphate groups. Either or both of such groups may be responsible for the lability of the alanine in the ribitol teichoic acids, whereas in the glycerol polymers, the phosphate ester groups are sufficient to cause the observed reactivity. It is of interest that the alanine ester groups in the membrane teichoic (68)J. Baddiley and F. C. Neuhaus, Biochem. J., IS, 579 (1960). (67) P. Berg and E. J. Ofengand, Proc. Natl. Acad. Sn'. U.S., 44, 78 (1958). (68) T. Wieland, J. Merz, and G . Pfieiderer, C h a . Ber., 98, 1816 (1960). (09) H. G. Zachau, Chem. Ber., 98,1822 (1960). (70) H. G. Zachau and W. Ksrau, C h m . Bet., 98,1830 (1960). (71) 2.A. Shabarova, N. A. Hughes, and J. Baddiley, Bioehem. J., 88,216 (1962).

334

A. R. ARCHIBALD AND J. BADDILEY

acids of group D streptococci are appreciably more stable toward alkali than are those of the other teichoic acids examined.a The alanine in the streptococcal compounds is attached to D-glucose reaidues, and c o w quently its stability may be ascribed to the absence either of phosphate or hydroxyl groups in sterically suitable proximity. Membrane teichoic acids have now been detected in a large number of bacteria, including almost all of the Gram-positive organisms examined. Neverthelesa, the proportion present is sometimes small, and separation of the acids from other macromoleculq cell-components, such as nucleic acids, peptides, and polysaccharides, is difEcult. Consequently, few have been obtaiaed in an amount and of a purity adequate for detailed chemical study. Even from the limited studies so far made, it is clear that structural details differ considerably from case to case, and it is convenient to classify these teichoic acids according to the organisms from which they have been isolated. 1. Lactobacillus arablnosus 17-5s" The glycerol teichoic acid obtained by fractionation of cold dilute trichloroacetic acid extracts of the "cell contents" fraction of L.aTabinosua contains 24% of a ribitol teichoic acid, presumably that which is present in the wall of this organism. Ion-exchange chromatography of the mixture, after removal of the alanine ester groups with dilute ammonia, gave a pure preparation of glycerol teichoic acid; it contained glycerol phosphate and glucose in the molar ratio of 1:0.11. Acid hydrolysis gave glycerol (characterized as its tribemoate) , D-glucose (characterbed with &glucose oxidase), glycerol mono- and di-phosphates, and a trace of inorganic phosphate. The formation of these products indicates that the polymer is composed of a chain of glycerol phosphate residues in which glycerol residues are attached to each other through a phosphate group. Acid hydrolysis can occur at either side of the phosphodieater linkages, and will thus give glycerol and its diphosphlltesin addition to the monophosphates. This is illubtrated for an unsubstituted polymer (17) in Fig. 4. The presence of phosphodiester groups was confirmed by potentiometric titration. On treatment with hot alkali, most of the phosphodiester groups were

~.o--f.~-o-i"i-Y-fc~~o{E"

.*....-

-0-EI&

{

-c&-o-

1

O l g C ~ ~

monopbo8phater

I

glycerol

(17)

-......

i

I

I

p,

I

glgcerol dipborph.tr.

FIQ.4.-Hydrolysis of a 1,%LinkedPolymer of Glycerol Phoaphab by Acid.

335

THE TEICHOIC ACIDS

H&H

1

HO,

,o-CH

CH20ap*0 \

A

I C&O<

?i
A

y+o-Y

C q O q *O A

OH I

CqO-P-OH II

0

glycerol monophosphates

glycerol

+ glycerol monophosphatem + glycerol diphosphates

FIG.5.-Hydroly&

of a 1,%Linked Polymer of Glycerol Phosphate by Alkali.

hydrolyzed, and the main products were monophosphates of glycerol, together with some glycerol and its diphosphates. Both 1,2-linked (see Fig. 5) and 1 3-linked (see Fig. 4) chains of a glycerol phosphate polymer have free hydroxyl groups adjacent to the phosphodiester groups, and both types of molecule will, therefore, be labile toward alkali. From a consideration of the mechanism of hydrolysis, it may be seen that different products will be formed from the two kinds of polymer. A 1,2-linked chain can be hydrolyzed by fission at the points A in Fig. 5, to give glycerol and the cyclic phosphates as shown. The cyclic phosphates would then be hydrolyzed to a mixture of glycerol 1- and 2-phosphates, and the cyclic phosphate from the terminal residue would give a mixture of glycerol diphosphates. An alternative, presumably simultaneous, mechanism would involve the stepwise removal of glycerol phosphate residues from the left side of the structure. Again, these would first give cyclic phosphates, which would then be hydrolyzed to a mixture of glycerol 1- and 2-phosphates. The proportions of the various products would depend on the relative rates of the two types of hydrolysis and on the chain length of the polymer. A glycerol phosphate polymer containing 1,3-linkages could be hydrolyzed in alkali by fission at either side of each phosphodiester linkage, to give glycerol and its mono- and di-phosphates in proportions which would

336

A. R. ARCHIBALD AND J. BADDILEY

probably differ from those given by the 1,2-1inked polymer (18). Hydrolysis in both directions from points dong the chain could yield a product that is unique for a l,&phosphodiester structure. Thus, an intermediate cyclic phosphate (19) would be formed from the 1,%polymer by hydrolysis at points A and B (see Fig. 6). Further action of alkali on this product would give the bia(glycerol 2-phosphate) 1,1’- (hydrogen phosphate) (“diglycerol triphosphate’,) (20), together with ieomers in which one or both of the phosphomonoester residues occupy the terminal positions. These isomsrs would be unstable toward alkali, and so would be hydrolyzed further to IL mixture of glycerol mono- and di-phosphates. In compound (20) , however, there are no free hydroxyl groups adjacent to the diester, and corsequently thia would be atable toward W.The formation of a

I

B

A OHo (A B !haion)

Ho-

OH@ orregular ;baton)

$-

-0

k

R

Po,%

k 0 E (20)

phorphrnr

t

m-, =o

L

HbH

&rqOH

p

01)

glycerol t glycerol monqhapla8tem

FIG.B.-Fomation and Struotural Determination of an Alkali-stable.Phosphodieeter in the Hydrolysis of a Glycerol Phosphate Polymer.

337

THE TEICHOIC ACIDS

glycerol phosphates

glycerol phosphates

where G = glycosyl. FIG.7.-Hydrolyis of Phosphodieeter Groups Attached to an O-Glycosylglycerol.

stable diestcr of this type would be diagnostic for the presence of 1,3linkrtgea ia the original polymer. These conclusionsremain unaffected if glycosidic substituents are present in the chain. Thus, glycosidic substitution on glycerol residues in a 1,2linked polymer might influence the course of hydrolysis, but could not give rise to compound (20). A 1,3-1inked polymer having 8 glycosidic substituent on each glycerol residue would be resistant to alkali. With only a small number of glycosyl residues, however, the molecule would be hydrolyzed m d could again give rise to the stable (20). Most of the glycosylglycerol residues would be converted into phosphate free fragments (see Fig. 7). A substituted glycerol residue at the phosphate terminal end of the chain would give glycosylglyceml monophosphate (see Fig. 8), and

PH

HO-P- O-€I&-

::

Y+ c-CH,-O-P A

OH I

-0 -H,C

II

0

7

-cH,o..

....

If

where 0 = glycosyl. Fro. 8.-Formation of nn O-Glycoeylglycerol Phosphate from the P-Terminal End of B Toichoic Acid.

338

A. R. ARCHIUALD AND J. BADDILEY

(25)

FIQ. 9-Alkaliatable with Alkali.

Phoaphodieater Produced by Hydrolysis of Teichoio Acids

small proportions of such compounds aa (22)-(25) (R = glycosyl) might be produwd by a mechttriisni I L I I ~ O ~ O UtoN thtlt in Fig. 9 from Rubstituted glycmol rcniduo~oc!curring withiu the chain. Titratioii of the products of alkaline hydrolysis of the teichoic acid from L.arabinoms showed that 7-8% of the phosphate groups remain aa phoa

THE TEICHOIC ACIDS

339

phodiesters. Four distinct phosphate fractions were obtained on ion-exchange ckmmatography; the two major fractions contained, respectively, glycerol monophosphates and glycerol diphospbtes; the smallest fraction contained a glycosylglycerol phosphate. The fourth fraction contained the characteristic (20); this gave glycerol mono- and di-phosphates on acid hydrolysis. The ratio of total phosphate:inorganic phosphate produced by a phosphomonoegterase waa 3:2, and the resulting diglycerol phosphate gave 2 molar proportions of formaldehyde on oxidation with periodate. Alkaline hydrolysis of the “diglycerol phosphate” gave equal amounts of glycerol ard its monophosphates (see Fig. 6). These observations established both the structure (21) and the presence of 1,3-phosphodiester linkages between the glycerol residues in the teichoic acid. The neutral products from an alkali hydrolyzate were glycerol and 2-0a-D-glucopyranosylglycerol(26), [ a ] ~ 120°,which was not hydrolyzed

+

by a &wglucosidase. Compound (26) reacts slowly with the periodate Schiff spray reagents on paper, indicating that formaldehyde is not produced on oxidation with periodate. This conclusion was verified in a quantitative study when 2 molar proportions of periodate were reduced by (26), and no formaldehyde waa formed. Acid hydrolysis of the oxidized material gave glycerol. These observations show that the neutral product and this conclusion was confirmed by is 2-O-a-~-glucopyranosylglycerol, comparison of its crystalline hexakis-p-nitrobenzoate with that of synthetic Most of the glycosylglycerol (26) in the polymer is liberated as such through the action of alkali; the comparatively small yield of glucosylglycerol phosphate indicates the absence of pIeferentia1 glycosidic substitution of the terminal glycerol residues. This result is in accord with a structure in which the substituted glycerol residues are distributed more or less randomly along the chain. The isolated “diglycerol (72) A. J. Chsrleon, P. A. J. Gorin, and A. S. Perlin, Can. J . Chem., 86, 365 (1957). (73) P. W.Auetin, F. E. Hardy, J. G . Buchansn, and J. Baddiley, J . Chem. Soel, 2128 (1fw.

w

b

OH OH I EO-P-O-~C-C-C&-O-~-O-&C I II H 0

F

::

I I Y - - - p" P-0-V 4x4-0-P-O-Fl$-$!-C&OE It II I

10,@

OH

OH

H

0

0- * *

0

OH OH w4 + 2 HCHO + O=C-C&-O-P-O-€I$-C-C~-O--.-.. H I I

(l)*-e+

(2)

OH I -7-C&H

II

0

I

H

H

OH OH I I H f;-o-~c--c-cE.j-o-P-o-~c-c=o 0

FIG.lO.--StepwiSe Degradstiorr of a Glycerol Teichoic Acid.

I

€I

II

0

X

2

sU

THE TEICHOIC ACIDS

341

triphosphate” accounted for almost all of the phosphodiester detected (by titration) in an alkali hydrolyeate, and thus little, if any, of such compounds aa (22)-(25) were formed. From biosynthetic considerations, glycerol teichoic acid chains should terminate with a phosphornonoester residue at one end and a glycol residue a t the other. The presence of a phosphomonoester at one end of the chain waa demonstmted by titration and by the action of a phosphatase. The ratio of primary to secondary acidic groups waa 17:1, and the ratio of total phosphorus to inorganic phosphate formed by the enzyme was 16.2:1. Periodate oxidation of the polymer gave a formaldehyde:total phosphorus ratio of 1:18.9, showing the presence at the other end of the molecule of a terminal glycerol residue. If the glucosylglycerol residues are randomly distributed along the chain, only 89% of the phosphate-free terminal groups would yield formaldehyde on oxidation with periodate; when this is taken into account, the chain length calculated from the formaldehyde value is 16.8,a value which agrees well with the rmults from potentiometric titration and enzymic hydrolysis. Further evidence for structure (16) was obtained by treating the teichoic acid with phosphatase, and oxidizing the product with periodate (see Fig. 10);the ratio of phosphorus:formaldehyde produced waa 18 :2, indicating the presence of a glycol group at both ends of the chain. It follows that both of the terminal glycerol residues are joined to the polymer by 1,3-phosphodiester linkages. Degradation of the oxidation product with phenylhydrazine (compareRef. 74) gave a modified polymer containing two glycerol residues fewer. Incubation with phosphatase showed the presence of a phosphomonoester group at each end of thia modified polymer (see Fig. 10). The teichoic acid which had not been subjected to ion-exchange chromatography contained D-alanine in characteristic, labile, ester linkage. It contained insufficient glucose to accommodate all of the alanine ester residues (alanine:phosphorus ratio, 0.89: l), and the kinetics of the reaction with hydroxylamine indicated that all of the alanine is attached to the available hydroxyl groups at C-2 of glycerol residues. The structure of the teichoic acid is depicted in (16) , where n = 16 and 1% 0-CrD-glucopyranosyl or D-alanyl, Approximately one sugar residue is prosent to 9 glycerol residues.

-

2. Lactobacillue casei 1%Y’

This W&R the first membrane teichoic acid to be studied and, although the walls of this organism do not contain a teichoic acid, difficulties were experienced in the purification of the material isolated from extracts of (74) D. M.Brown, G . E.Hall, and R. Letters, J . Chem. Soc., 3647 (1959).

342

A. R. ARCHIBALD AND J. BADDILBIY

whole cells. Several aspects of the structure were, nevertheless, elucidated. The beat preparations contained phosphorus, glycerol, amino acids, and reducing sugar in the proportions 1:0.93:0.63:0.17. Acid hydrolysis gave glycerol, its mono- and di-pholsphates, D-alanine, and small proportiona of several sugars. Hydrolysis by alkali gave glyoerol, its mono- and di-phosphates, and the characteristic “diglycerol triphosphate” (20). No trace of a glycoeylglycerol was detected, and it w a concluded that the sugars present in t4e preparation came from contaminating polysaccharidea. The major amino acid found was D-alanine; this is preeent in characteristic, labile, eater linkage. It follows that the teichoic acid is a 1,&linked chain of glycerol phosphate residues having Palanine ester residues attached at C-2 of most of the glycerol residues. Unambiguoue demonstration of the total abseuce of glycosidic substituents was precluded by the presence of impurities which also interfered with attempts to determine the chain length. 3. Staphylococcus kcti8 1944”

The purified material obtained from the particulate fraction of disrupted cells of this organism contains glycerol, phosphate, alanine, and a small proportion or” 2-acetamido-2-dtmxy-D-gahctose (phosphorus:m i n o sugar, 1:0.02). Degradation with acid and alkdi gave products characteristic of a lJ3-linked glycerol phosphate polymer, and the alanine residues were found to be in the usual, labile, ester linkage. The wall of this organism contains a glycerol feichoic acid in which many of the glycerol residues bear Zacetamido-2deoxy-wgalactosyl substituents, and it is possible that the amino sugar in the sample of the membrane material is derived from contaminating wall teichoic acid. The membrane teichoic acid thus resembles that from the strain of L. caaei, and hae few or no glycosidic substituents. 4. S ~ ~ ~ ~ Y Z O Caureua O C C UHSa

This was also shown to be a 1,a-linked glycerol phosphate polymer having D-alanine ester residues attached to C-2 of most of the glycerol residues. Small proportions of glucose and &aoetamido-&deoxy-wglucoeewere also present in acid hydrolyzates. On hydrolysis by alkali, gentiobiosylglycerol and 2-amino-2deoxy-~-g~ucosy~g~ycero~ were produced in small proporis resistant to acid hydrolysis, tions. The 2-umino-2deoxy-~-gluco~ylglycerol but is readily degraded by nitrous acid“ to glycerol and chitose (2,Sanhy(76) D. C. E:lwood, Ph. D. Thesie, University of Newoastle upon Tyne (formerly King’s College, University of Durham), 1862. (76) A. B. Foater, E. F.Martlew, and M. Stecey, Cbm. I&. (London), 825 (1963).

THE TEICHOIC ACIDS

343

dro-smannow) . The reaction with the periodate-Schiff reagentan*” indicated that no formaldehyde is produced on oxidation with periodate; the glycerol (27). I d glycoside is, thus, 2-0- (2-amino-2deoxy-~-glucosyl) cient material was available for determination of the configuration of the on rtnomeric linkage. The rapid production of 2-amino-2-deoxy-~-glucose acid hydrolysis of the teichoic acid contrasts with the stability of the isolated 2-amino-2deoxy-~-glucoside, and i n d i c a h ~that ~ ~ the 2-amino-2deoxy-D-ghosyl residues in the teichoic acid bear N-acyl substituents. Hydrolyais of one molar proportion of the gentiobiosylglycerol with acid (or with 8-wglucosidrtse) gave 2 molar proportions of wglucose to 1 of glycerol. Under milder conditions, the products wore glycerol, a small proportion of rrglucoee, and gentiobiose. Thus, the glycoside must have structure $8). The presence of gentiobiosyl and 2-acylamido-2-deoxy-wglucosyl residues in the teichoic acid is consistent with its behavior toward periodate after removal of w a h i n e : all of the 2-amino-2-deoxy-~-glucose and D-glucose were destroyed, and reduction of the oxidized material with sodium borohydride, followed by acid hydrolysis, gave glycerol but no tetritol.

The D-alanine ester groups are attached to the C-2 hydroxyl group of most of the glycerol residues that are unsubstituted by sugar residues. The proportion of formaldehyde produced by oxidation with periodate indicates that the chain contains 14 units, whereas results of the action of phosphomonoestcme suggest a chain of 35 units. This discrepancy may be due either to loss of phosphate during purification or to steric inhibition of the enzymc, a phenomenon which has often been encountered in this field. Analysis shows that approximately one in every thirty glycerol residues posaesaes a gentiobiosyl Nubstituent, and a similar number carry 2-acylamido-~-clt~xy-i)-gl~icosyl suI)stituc*ntn. (77) J. Baadiley, J. (f. Buohanan, R. E. Handschumacher, and J. F. Prewott, J . Chem. Soc., 2818 (1956). (78) F. E.Hardy arid J. G. Buchanan, J . Chem. Soc., 5881 (1903). (79) A. B. Foeter, D. Horton, and M. Stscey, J . C h .Sm.,81 (1957).

344

A.

R. ARCHIBALD AND J. BADDILEY

5. GroupDStreptococci Most streptococci of intestinal origin belong to a single serological group, Lancefield’s group D. The group D antigen has been identified as the membrane teichoic acid. Preparations of such teichoic acids contain Dglucose, the proportion of which differs in various strains. This difference in structure is associated with small quantitative differences in serologiwl activity. A detailed examination6*of the membrane teichoic acids from strains 8191 and 36 shows that both am composed of a glycerol phosphate, D glucose, and Palanine; small proportions of Glysine (10% of the total amino acid) are also present in the material from strain 8191. The ratio of wglucose:phosphorus in the teichoic acid from strain 8191 is 2.8:1, and, from strain 39, it is 1.5: 1, Acid hydrolysis gave the expected products, but, apart from the removal of D-alanine, neither teichoic acid was detectably degraded by alkali. The resistance to alkqli is consistent with the high content of sugar, and indicates that essentially all of the glycerol residues have glycosidic substituents : thus, no unsubstituted hydroxyl groups occur adjacent to phosphodieater linkages. The glycerol glycosides were not isolated, but their structures were deduced from a study of the products of partial hydrolysis, by acid, of the teichoic acids. Thw, the teichoic acid from strain 39 gave, in addition to glycerol and its phosphates, D-glucose and kojibiose (29), identified by chromatographic comparison with authentic wmples. wGlucose, kojibiose, and kojitriose (30) were the only reducing sugara produced from strain 8191. Kojitriose was identified by partial hydrolysis with acid, when *glucose imd kojibiase were produced; after reduction of the trisaccharide with sodium borohydride, partial h y w y s i s by acid gave D-glucose, D-glucitol, kojibiose, and kojibiitol. Structure (16; R = cu-kojibiosyl) was proposed for the teichoic acid from strain 39, and (16; R = a-kojitriosyl) for that from strain 8191. These structures are supported by the results of studies on the oxidation of the teichoic acicis (D-alanino removed) with periodate, when the amounts of reagent roducled and formic acid produced were those expected from the structures given. The optical rotation of the teichoic acid from strain 8191, [O]D 130°,is consistent with the presence of three ar-rnglucosidiclinkages. The optical rotation of that from strain 38 is also positive, although accurate determination was precluded by the small amounts of material available. The analytical data do not exclude the possible presence of some tetra.saccharide residues in the polymer from strain 8191, or of trisaccharide residues in that from strain 39, although no traces of these were detected in partial, aaid hydrolyBates. It does seem likely from analyea, however,

+

345

TEE TEICROIC ACIDS

that mme kojibiosyl, and possibly Dglucosyl, residues occur in the teichoic acid from strain 8191, and that some mono-0-D-glucosylglyceml residues and possibly a few unsubstituted glycerol residues are present in that from strain 39. The presence of these smaller substituenta may be a consequence of hydrolysis during isolation and purification.

(as)

mw Qoa

p

m?& *QHO H

p (30)

Although the stability of the phosphodiester linkages toward alkali preventcl the direct demonstration of a structure containing 1,3-linked glycerol phosphate residues having the sugar substituenta attached to C-2 on each glycerol residue [structure (16)], such a structure seems likely by analogy with other teichoic acids. Moreover, the alternative, regular Structure which would conform to the pattern of a polymer of a glycerol monophosphate is shown in structure (31), where R = kojibiosyl or kojitriosyl. However, structure (31) is untenable for either of the teichoic acids, sirxe it would be hydrolyzed (see Fig. 11) by alkali through stepwise loss of 0-glycoeylglycerol phosphate residues by a mechanism discussed previously (see p. 329). Potentiometric titration of the teichoic acid from strain 8191 reveals the presence of a terminal phosphornonoester group, and indicates a chain length of 20-22 residues. This phosphomonoester residue is resistant to the action of phosphomonoestemse,presumably because of the neighboring, bulky, sugar substituents.

346

A. R. ARCHIBALD AND J. BADDILEP

giycoeylg1ycsroll-and2-phosphotem

FIG.ll.-HydrolyRiR hy Alkali of diceter Linkages.

B

Glycerol Teichoic Acid Containing 1 ,ZPhosph*

On treatment with ammonia, both teichoic acids give D-alanine and its amide; in addition, that from strain 8191 yields traces of Irlysine and t lysimx-nide. The amino acid ester reddues must clearly be attached to sugar residues, since very few or no glycerol hydroxyl groups are available in these polymers. This difference in structure] aa compared with those of the teichoic acids previously disoueaed, is reflected in their behavior toward ammonia; hydrolysis occum slowly, at a rate comparable with that for alanine methyl ester, showing the absence of suitably situated phosphate or hydroxyl groups. The presence of the amino acid eater groups prevents the oxidatiou, by periodate, of some of the glucose residues, thereby indicating that the 3- or 4hydroxyl groups of some of the Dgluoose residues are involved in the ester linkages.

VI. WALLTEICHOIC ACIDB The walls of Gram-positive bacteria] unlike the membranes, sometimes do not contain teichoic acids. However, there are numerous examples of walls which do contain these compounds in substantial proportions, although in relatively few C ~ M E has detailed structural work been carried out. The purification of wall teichoic acids is often much more readily achieved than for the membrane polymers. Addition of ethanol to trichloroaaetic acid extracts of w& gives a precipitate which is usually pure teichoic acid. Extraction is effected at low temperatures, and for a r888onably short time, in order to avoid extensive degradation of the polymers under the acidic conditions. Conssquently, although walls may contain 20-50% by weight of teichoic acid, practical yields are generally rsther low.

1. Glycerol Teiahoio A d d s Glycerol teichoic acids have been detected in the walls of a number of bacteria. By chromatographic observation of the producta formed on

T H E TEICHOIC ACIDS

347

hydrolynk with acid and alkali, it was shown that almost all of these teichoic acids are structurally similar to those'from membranes; two representatives of this type have been examined in [email protected]' 1 On the other hand, during a study of the relationship between the taxonomy, the chemical structure, and the serology of the teichoic acids in staphylococci, a strain of Staphylococcus lactis waa freshly isolated from an infected chicken." The walls of this organism contain a glycerol teichoic acid which, in its hydrolytic behavior, shows great differences from the conventional type. Further studies have revealed that this polymer has a novel type of structure in which amino sugar phosphate residues form an integral part of the polymer chain.8BPreliminary observations suggest that a similar teichoic acid is present in the walls of Micrococcus hgicus, and it is possible that other representatives may occur in other bacteria. This type of structure has not so far been found in a membrane teichoic acid. a. Staphylococcus lcrctis 794&Bo--The teichoic acid from the walls of this organism contains D-alanine, Z-acetamido-2-deoxy-D-galactose,and glycerol phosphate in the proportions 0.69:0.34: 1. The pattern of acid hydrolysis indicates that the polymer resembles the membrane teichoic acids in being composed of a chain of glycerol residues joined through phosphodiester linkages. Hydrolysis by alkali gave glycerol, Z-O-(Z-amino-%deoxywgalactosy1)glycerol, and a little Z-O-(Z-acetamido-Z-deoxy-wgalactosyl)glycerol, together with a complex mixture of organic phosphates. The phosphates were hydrolyzed with a phosphomonoesterase, and inorganic phosphate, glycerol, and a mixture of phosphodiesters were obtained. The identification of (21) , originating from (20), established the presence of 1,3-phosphodieater liiages between the glycerol residues. By hydrolysis with alkali and subsequent degradation with a phosphtase or with nitrous acid, the other phosphodiesters were identified &s originatingfrom the alkalistable compounds (22) or (23),and (24; R = 2-amino-2-deoxy-Dact~yl). Appreciable proportions of these products would be expected from a polymer in which a reasonably large proportion (up to about 30%) of the glycerol residues have amino sugar substituents. The results of analysis indicated that about every third glycerol residue in the chain is substituted in this way. As the diester (25) was not observed, it is probable that amino sugar substituents do not occur on any adjacent glycerol residues, and thus the polymer is regularly substituted throughout its length. (80)D. C. Ellwood, M. V. Kelemen, and J. Baddiley, Biocha. J., 88,213 (1963). (81) N. Shaw and J. Baddiley, Biochsm. J., 98,317 (1964). (82) A. L. Davieon, Ph. D. Thesis, University of Newoeetle upon Tyne (formerly King's College, University of Durham), 1982. (83) A. R. Arohibald, D. Button, and J. Baddiley, Biochem. J., 96,& (1966).

348

A, R. ARCHIBALD AND J. BADDILEY

Mild, acid hydrolysis of the teichoic acid gave, in addition to some free amino sugar, an acylamidohexose which waa indistinguishable from 2acetamido-2deoxy-~-galactose.Further evidence for the identity of the

amino sugar wa,s obtained by degradation with lJ2,3-indantrione monohydrate (niniiydrin)*4;B lyxom WRR produced, thereby establishing tho configuration of C-3 to C X as that of a 2-unliim-2deoxygalactase. Whaii the hichoic acid was oxidised with sodium periodate, the acetamido sugar residues,were oxidized (see Fig. 12) ; the resulting aldehydic polymer (32) was reduced with sodium borohydride, and acid hydrolysis followed by oxidation with aqueous bromine gave D-serine (33). This showed that or 2-amino-Meoxythe amino sugar is either 2-amino-2deoxy-~-galact.ose ttalose. Studies on the 2-0- ( 2 . a c e t a m i d o - 2 d ~ x y - ~ g a lglycerol ~~~l) showed that the amino sugar has the D configuration and must, therefore, be 2-a,mino-2-deoxy-~-galactose. HOH& B

?

o=c B?- o=c

q AcNH

AcNH

PH

....*.O~~-~-CHa-O-P...... 0 I It B

CQaH

0

(1)

H@

A

-14kHc;o*

p

%OH

(33)

(2) Br,,-W

d

(W

MHaC AcNEl

P

H

......

OH

0 w...O&C-c-cHp-# I

......

'f;P"......

H 0 FIQ. 12.-Degradetion to Eetablbh the Bttmwhemicsl Series of ZArnino-!&deoxyD-gahtose Reaiduee by Converting E.1 to G3 into Beriae.

The 2-0-(2-amino-2-deoxy-~-galtlctosyl)glycerol (34, R = H) was smoothly degraded by nitrous acid t o glycerol and 2,6-anhydro-~-talose (35), and the N-acetyl derivative (35, R = Ac) reduced 1 molar proportion of periodate, giving neither formaldehyde nor formic acid; during (84) P. J. Stoffp and R. W. Jeenloa, Arch. Biochem. Biophys., 68,373 (1964).

349

THE TBICHOIC ACIDS

I

HO%C -y-C€&OH

H

kIaOIi (37)

FIG.13.-Formation of m Anhydro Sugar and of ~-GlycericAcid from %Amino-% deoxy-D-gslactose Residues in s Teiahoic Acid.

this oxidation, the acetamido sugar residue was destroyed, but the glycerol

was not attacked. These observations establish the attachment of the 2-eoetamido-2-deoxy-Dgalactose at the 2-hydroxyl group of glycerol. The configuration of the amino sugar was established by further oxidation of the product from the periodate oxidation (36) with aqueous broxpine, followed by acid hydrolysis, when Dglyceric acid (37) was produced. Neither the 2-0-(2-amino-2-deoxy-~-galactosyl)glycerolnor its N-acetyl derivative crystallized, 80 that direct compmiaon with authentic com-

350

A.

R. ARCWIRALD AND J. BADDILEY

pounds was not possible. However, thc optical rotation of the 2-042acetamido-2-deoxy-~-galactosyl)glycerol,[U]D = 4- 112', shows that most of the glycosidic linkages have the U-D configuration, but the presence of some &D linkages is not precluded. The action of a specific 8-Nacetylgalactosaminidaae (from pig epididymis) on both the 2-acetamido-2deoxy-D-galactoside and the intaat teichoic acid indicated that a small number of O-D linkages, is, indeed, present. Titration shows the presence of a terminal phosphornonoester residue, and the average chain length is about 18. The D-alanine residues have the expected reactivity toward ammonia, and, from a study of the reaction with hydroxylamine, it is concluded that all of these residues are attached to available 2-hydroxyl groups of glycerol, instead of to the 2-acetamido2deoxy-~-galactosyl residues.

b. hctobudlus buchneri NCIB 8001.*1-The usual procedures -tabl i h that the wall teichoic acid of this organism is a normal, 1,3-linked glycerol phosphate polymer having D-alanine residues and a-D-glucopyranosyl residues attached at the C-2 hydroxyl group of glycerol residues. The proportions of D-alanine: D-glucose:phosphorus are 0.45:0.26: 1, and the chain length of the extracted material is about 14. The teichoic acid mainly differs from the membrane polymer from L. urubimm in having a greater number of D-glucose residues, and this is reflected in the isolation and identification, in alkaline hydrolymtes, of the phosphodiesters (22) or (23), (24), and (25; R = Dglucosyl). Only small proportions of 0glycosylglycerol monophosphate (see Fig. 8, p. 337) were found, and it wm concluded that the sugar residues are distributed more or less randomly along the chain. c. Bucillu8 subtilis NCTC 3610.-Walls from this strain differ from those of the strain discussed later (see p. 3M), in that they contain a glycerol teichoic acid. This has been extracted in the usual manner, and found to contain D-glucose, glycerol, and phosphate (1:l:l).The extracted material usually contains less than 0.1 molecular proportion of amino group (preaumably, of alanine) to eaah glycerol residue: this may be due to the conditions under which the walls were prepared, including digeetion with trypsin, ribonuclease, and deoxyribonucleam The teichoic acid is stable toward hydrolysis by alkali, a property consistent with a struoture in which all of the glyoerol residues carry glycosyl substituente. Treatment with 60% hydrogen fluoride at 0' cleaved the phosphate ester groups without significumt hydrolysis of glymsidio linkages, thereby giving an 0-wglycosylglyoeml. AB this was hydrolymd by an cw-D-glucosidaseand gave no formaldehyde on oxidation with periodate, it must have the structure (27), and the teichoic mid is therefore a fully

THE T E I ~ H O I CACIDS

35 1

D-glucosyls.tcdpolymer of glycerol phosphate residues joined through positions 1 and 3. The method of d2gradation with hydrogen fluoride is interesting and promises to be of value in the examination of other teichoic acids, such as those from the membrane of streptococci in group D (see p. 344).

d. Staphylococcue hctis (fresh isolate).@-The teichoic acid in the walls of this organism contains malanine, 2-acetamido-2-deoxy-~-glucose, glycerol, and phosphate, the proportions of these in the purified polymer, which contains 12% of phosphorus, being 1:1:1:1.8.The ratio of alditol: phosphorus in all other tcichoic acids examined is 1:1?and the unusual composition of the compound from Staphylococcus lactis is reflected in its behavior on hydrolysis. Thus, with acid, the major products were danine, 2-amino-2-d~oxy-mglucose,and glycerol diphosphates ;no glycerol and only a small proportion of glycerol monophosphates were produced, together with some 2-amino-2-deoxy-~-glucosephosphate. With alkali, only a small proportion of glycerol monophosphate and no glycerol or 0-(2-amino-2deoxy-wglycosy1)glycerol were produced, the major product of hydrolysis being a mixture of organic phosphates of chromatographic mobility similar to that of a glycerol diphosphate; an appreciable proportion of a saccharinic acid ww also formed. Work on this teichoic acid is incomplete, but structure (38) has been phosphate is a part proposed, in which 2-acetamido-2-deoxy-~-glucosyl of the polymer chain. Such a structure accounts for the observed composition of the teichoic acid and for ita behavior on hydrolysis. Thus, in dilute acid, the very labile sugal-phosphate linkages in 2-acetamido-Zdeoxy-Dglucosyl phosphate residues are hydrolyzed unidmtionally (hydrolysis at A , A in Fig. 14), whereas the other phosphodiester linkage can be hydrolyzed in either direction, giving 2-acetamido-2-deoxy-~-glucoseand glycerol diphosphates (A ,A ,C) or a 2-acetamido-2-deoxy-~-glucosephosphate and B glycerol monophosphate (A,A,B); the observed products indicate that the favored path is (A ,A, C) , involving participation of the free hydroxyl group a t C-2 of the glycerol residues. Some of the phosphodiester linkages and 2-amino-2-deoxy-~-glucose residues are stable to alkali, and fractionation of the resulting phosphates gave, in addition to glycerol diphosphates and a small proportion of a monophosphate, two compounds which, on the basis of the observations described below, are identified as (39) and (40). Hydrolysis of these products with acid under miId conditions gave gIycerol diphosphates together with 2-acetamido-2-deoxy-~-glucoseand 2-amino-2-deoxy-Dglucose, respectively. The acidic compound (39) contains 2-acetamido-2deoxy-D-glucose, glycerol, and phosphate in the proportions 1:1:2, 50%

362

oj

$ &I

I

8-po

i.

Ip-0-0

I

0

9

of the Teichoic Acid from S~phylococevslactis by alkali.

A. R. ARCHIBALD AND J. BADDILEY

I

m-y-d

9I &&O

FIG.14.-Hydroly&

THE TEICHOIC ACIDS

353

of the phosphate being labile to phosphomonoesterase. The internal salt (40)reduced 1 molar proportion of periodate giving no formaldehyde, wheress, after eneymic removal of the phosphomonmter, the product (41) reduced 2 molar proportions of periodate and gave 1 of formaldehyde. Treatment of the salt (40)with nitrous acid, followed by reduction with sodium borohydride, gave 2,5-anhydro-~-mnnitol and a glycerol diphosphate; moreover, with phosphomonoesteme, half of its phosphate content wns removed, and the product (41) gave, with alkali, saccharinic acid and glycerol monophosphates, and, with nitrous acid, an anhydro-m mannose and a glycerol l-phosphate. Purified samples of this glycerol phosphate were unaffected by glycerol phosphate dehydrogenase: hence, it is probably L-glycerol l-phosphate. It follows that the stereochemistry of the glycerol residues in this teichoic acid is as shown in formula (38). The formation of (39) and (40) establishes the presence of Zacetamido2deoxy-~-glucosylphosphate residues in the teichoic acid. The 2-aceb amido-2deoxy-~-glucose residues were not oxidized when the teichoic acid (alanine removed) was treated with periodate, and so they must be substituted at their C-3 or C-4 hydroxyl groups. This observation, together with the formation of a small proportion of a 2-amino-2deoxyD-glucose phosphate on acid hydrolysis of the polymer, is consistent with structure (38) ; the configuration of the glycosyl phosphate linkage follows from studies on optical rotation. This structure also accounts for the linkages toward alkali. Glycolability of the 2-amino-2-deoxy-~-glucosyl sidic and glycosyl phosphate linkages are normally stable toward alkali, but participation of the free hydroxyl group of the glycerol residue in (38), leading to hydrolysis of phosphodiesters through cyclic phosphate intermediates, will result in fission of the glycosyl phosphate linkages. The resulting, substituted 2-amino-2deoxy-~-glucose reaidues have free reducing groups, and will undergo further degradation to a saccharinic acid (see Fig. 14, hydrolysis a t A). An alternative, hydrolytic path, involving the free hydroxyl p u p on C-2 of the glycerol residue, is also shown in Fig. 14 (hydrolysis at C). The cyclic phosphate intermediate can be hydrolyzed to give the monoesters (39) and (40), both of which contain phosphodiester linkages which are stable to alkali; however, the phosphodiester residue in (42) can be hydrolyzed through the usual, cyclic mechanism, giving glycerol diphosphates and a saccharinic acid. The 2-amino-2deoxy-~-glucosyl phosphate linkage is very labile to acid; consequently, the teichoic acid is hydrolyzed under mild conditions to a fragment which is apparent19 (43) or (44). Study of this compound should establish which hydroxyl group of the 2-acetamido-2-deoxy-~glucose residue bears the phosphodiester group. The alanine ester residues (844 UnpubMed work of the authors with Mr. D. Button.

354

A. I?. ARCHIBALD AND J. BADDILEY

are relatively stable to alkali and, with neutral hydroxylamine, a h i n e hydroxamate is formed at a rate similar to that observed with alanine ethyl eater. Comparable rates of hydroxamate formation occurred with the membmne teichoio acids of group D streptococci, and it is likely that, in the Staphy2ococcwr lactis polymer, too, the D-alanine is attached to amino sugar residues instead of to glycerol residues. The mwt likely position for the alanine ester residues is the hydroxyl group at C-6 of 2-acetamido-D-deoxy-D-ghcose, as this does not have a neighboring phaephate group.

Y 8

Bh

*lQoH -

m-P-0-H&-c-CHj-0-

Ho-f;-o-H&-y-cHi-o7 0 P"

08

(43)

ACNH (44)

2. Ribitol Teichoic Acids

All of the ribitol teichoic acids so far examined are composed of chains of ribitol residues joined through phosphodiester groups at Gl and G5. Each chain is terminated by a phosphomonoester residue, and the ribitol residues bear glycosyl and wdanine ester substituents. Detailed structures have been proposed for the polymers from Bacillus subiilis and Lactobacillus arabanosus, and from two strains of Staphylococcus a u r w . The structure of the teichoic acid from Bacillus subfilis was the first to be established in detail; the other polymers differ mainly in the nature of the glycosyl substituents. a. Bacillus subtills.M*"-The teichoic acid (45; R = BD-glucopyranosyl) extracted from walls of this organism contains wglucose, D-alanine, and ribitol phosphate (o-glucose:D-alanine:phosphate, 0.93:0.46: 1). The content of D-alanine is different in different preparations, and is always somewhat leas than that required for the completely regulan substitution shown in the formula; this deficiency has been ascribed to occurrence of hydrolysis duriag purification or during preparation of the walls.

(85)J. J. Amutrong, J. Buddiluy, sud J. G.Buuhanan, Biuchm. J., 76,010 (1900).

THE TEICHOIC ACIDS

355

Acid hydrolytk gave inorganic phosphate, saltwine, Dglumse, l,C anhydroribitol, and ribitol mono- and &-phosphates, together with 1,4anhydroribitol phosphates and 0-mglucoeylribitol mono- and di-phosphates. The formation of ribitol diphosphates indicates the presence of ribitol residues joined to each other through phosphate, and shows that the structure of the teichoic acid differs from those of the pneumococcal polysaccharides that contain ribitol phosphate; in the latter substances, 0-glycosylribitol residues are joined through phosphodiester residueg between a ribitol residue and a sugar residue, the sequencebeing phosphatesugars-ribitol-phosphate-~ugars.~~~~~ Hot alkali completely hydrolyzes the phosphodiester linkages, the main products being n-alanine and a mixture of 0-wglucosylribitol monophosphates. Enzymic dephosphorylation of the (46) in high hydrolysis mixture gave a crystalline 2-O-~-glucosyl-~-ribitol~~~ overall yield; this waa obtained as the anhydrous compound and as the hemihydrate. It has [ a ) ~- 28.6' and is presumably a BD-glucopyranoside. Acid hydrolysis of the D-glucoside (46) gave D-glucose, 1 ,&anhydroribitol, and ribitol. A @-D-glucosidasereadily hydrolyzed it to mglucose and ribitol, confirming the presence of a &D-glucosidic linkage. The position of substitution on the ribitol moiety was determined by oxidation with periodate, when 4 molar proportions of this oxidant were reduced and 2 molar proportions of formic acid and 1 of formaldehyde were produced. Reduction of the oxidized material with borohydride, followed by acid hydrolysis, gave glycerol and no ethylene glycol. The D-glucoside is, thus, ~-O-&Dglucopyranosyl-cribitol (46) or Z-O-&oglucopyranosyl-oribitol (49). In fact, the compound WM shown to be 2-O-&~-glucopyranosy1-~-ribitol (46) by examination of the trialdehyde produced on oxidation with periodate (see Fig. 15). Both trialdehydea (47) and (50) have two glyceraldehyde residues, and, in each, that which arises from C-4 to C-6 of the original D-glucose residue has the D configuration. The other glyceraldehyde residue has the D configuration in structure (47) and the L configuration in structure (50) ;it follows that, whereas the trialdehyde (47) is optically active, that with structure (50) is a meso compound. In fact, the oxidation mixture exhibited pronounced optical activity, and, since all products of oxidation other than the trialdehydes are optically inactive, the trialdehyde produced is thus (47). This was confirmed by further oxidation with aqueous bromine and hydrolysis of the resulting tricarboxylic acid, (48) or (51) , to glyceric acid; as this also exhibited a marked positive rotation, it must have arisen from (46)-(48) , since the glyceric acid from (49)-(51) would be racemic. The ribitol D-glucoside must be 2-O-&~glucopyranosyl-~-ribitol(%) , and this conclusion has been confirmedby chemical synthe&.a Similar degrada(86) P. A. Rebera and M. Heidelberger, J . Am. Chem. Soc., 83,3056 (1961). (87) E.V. Rao, W. K. Roberta, J. G. Buchanan, and J. Baddiley, Proc. Inten. Cong. Biochcm., 6th New Ymlc, 80, vi, 90 (1964). (88)J. Baddiley, J. G. Buchanan, and F. E. Hardy, J . Chem. 8oc., 2180 (1961).

A. R. ARCHIBALD AND J. BADDILEY

356

H

H

H

H

C=O

HoHac*cH.OHH H

Fio. I &-The Degradation of 2-0-8-~-Glucopyrsnosyl-~ and -n-ribitol with Periodate, Bromine, and Acid.

THE TEICHOIC ACIDS

357

tive sequences were followed with the 0-mglucosylribitol from the teichoic acid from the walls of Lactobanl ' lus arabinosw and the (2-amino-2-deoxyaureus?9 C U ~ In mglucosy1)ribitol from that in the walls of S ~ Z ~ ~ & X O C these instances, however, insufficient material was available for polarimetric studies, and so the configuration of the glyceric acid was determined enzymically. A small proportion of 0-D-glucosylribitol was produced directly by hydrolysis of the teichoic acid with alkali (see Fig. 16) ; this product is identical with that obtained by dephosphorylation of the hydrolysis mixture. The major products of such a hydrolysis with alkali were the isomeric monophosphates (58) and (59), in which R = 8-D-glucopyranosyl, both of which gave the 0-D-glucosylribitol on enzymic dephosphorylation. The isomer (58) reduced 3 molar proportions of periodate, and the ribitol residue was oxidized, whereas the isomer (59) reduced 2 molar proportions of periodate, the ribitol residue being resistant to oxidation. Small proportions of the diphosphates (56) and (57) were also produced. Oxidation of the diphosphate (57) with periodate, followed by treatment with alkali to remove the aldehydic residues, gave a ribitol diphosphate. The course of hydrolysis of the teichoic acid in alkali is illustrated in Fig. 16, where, for convenience, only three residues are shown. Hydrolysis involves cyclic phosphate intennedmtes, and, therefore, must proceed in a unidirectioml manner, as each ribitol residue bears a D-glucosyl substituent on a hydroxyl group adjacent to each phosphodiester group. O-~-Glu~o~ylribitol and its diphosphates are formed only from the residues a t the ends of the chain; and so,from a polymer comprising several residues, the major products would be the isomeric monophosphates (58) and (59). Since only the 1- and 2-phosphates were produced, it follows that the inter-ribitol linkages must be at C-1 and C-5 on each residue. This conclusion is also reached by oxidation of the teichoic acid with periodate after removal of the D-alanine; all of the ribitol residues were destroyed, and 80 they must have contained unsubstituted a-glycol groupings. The ratio of the formaldehyde, produced on complete oxidation, to the phosphorus in the polymer was less than 0.12:l. This result shows that the phosphodiester linkages are a t C-1 and (2-5 of each ribitol residue, since a 3,5-linkage would give a ratio of 1:1. The results of potentiometric titration indicated the presence of one primary acid group for each atom of phosphorus; the content of secondary acid group^, one for every nine atoms of phosphorus, is a measure of the terminal phosphomonoester group, and indicates a chain length of xhie units. The ratio of formaldehyde (produced on oxidation (89) J. Baddiley, J. C. Buchanm, U. L. RajBhsndary, and A..R. Sanderson, Biochem.

J., 89,438 (1962).

A. N. ARCHIBALD AND J. BADDILEY

358 OH

OH

P B P

RO OHOH (62)

F

HO-P-O-&C-f I1 0

H

H

+

B B r

~ - ~ - ~ €HOtqC-C-Ct ,

R HO

O ,

o+p~oH

R d d

tTib

+

BPB

HOtqC-C-C-C-CHpH Rd dHdH

H o ~ p ' o

FIG.16.-The Action of Alkali on a Ftibitol Teichoia Acid. with periodate) :total phosphorus (1:8.8) and the ratio of formaldehyde produced :periodate reduced (1:26) are also consistent with a polymer comprising nine units. The teichoic acid shows an infrared absorption band at 1751 crn.-l, characteristic of carboxylic ester groups, which is not observed in samples from which the D-alanine residues have been removed. Removal of the Dalanine was readily effected with ammo& or hydroxylarnine, when D-alaninamide or D-alanine hydroxamate were formed. The kinetics of the reaction with hydroxylamine reveal the high reactivity of its D-alanine ester linkages, which, like those in most other teichoic acids, are activated by the presence of a neighboring phosphate group. That the D-alanine residue is attached directly to the ribitol residues, instead of to the Dglucosyl substituenh, was also shown by oxidation with periodate under controlled conditions of pH, when it was found that the walanine residues protect the ribitol residues from oxidation. Under the same conditions, all of the ribitol residues were oxidized in a sample of teichoic acid from which the u-alanine had been removed, and it is concluded that the eater groups are attached to C-2 or C-3 of the ribitol reaidues.

THE TEICHOIC ACIDS

359

b. Staphylococcus aureua H.-'-This teichoic acid contains ribitol, phosphate, D-alanine, and 2-acetamido-2-deoxy-~-glucose(D-alanine:phosphorus, 0.66: 1). Acid hydrolysis gave products analogous to those obtained from the polymer from Bacillus subtilie, and, in this case also, all of the phosphodiester linkages were hydrolyzed by alkali. Enzymic d e phosphorylation of the products of hydrolysis by alkali gave an 0-(2amino-2-deoxy-~-glucosyl) ribitol (60) which readily yielded a crystalline

octaacekte. The 0-(2-amino-2-deoxy-~-glucosyl)ribitol is stable toward acid hydrolysis under reasonably vigorous conditions, but reacts readily with nitrous acid, to give ribitol and 2,5-anhydm~-mannose.Less vigorous hydrolysis with qlkali, followed by enzymic dephosphorylation, gave an 0-(2-acetamido-2-deoxy-~-glucosyl) ribitol, the octaacetate of which was identical with that obtained by acetylation of the 2-amino-2-deoxy-~glucoside (60). Degradative studies similar to those described for the Dglucoside (46) from the Bacillus subtilit3 teichoic acid showed that the 2-acetamido-2-deoxy-~-glucosylsubstituent is attached to the C-2 (L) hydroxyl group of ribitol. This has been confirmed by The optical rotation of the crystalline octartcetate is consistent with a 8-D configuration for the glycosidic linkage, and this was confirmed by comparison with synthetic 2-amino-2-deoxy-a- and Bwglucosides. However, studies on the rotation of the mother liquors, the unpurified 2acetamido-2-deoxy-~-glucoside,and the teichoic acid itself indicate that a-D- as well as 8-~-glycosidiclipkages are present. Moreover, pure 0(2-acetamido-2-deoxy-~-glucosyl) ribitol obtained by deacetylation of the crystalline octaacetate was completely hydrolysed by a specific &DN-acetylglycosaminidase, but the unpurified 2-acetamido-2-deoxy-~-glucoside and that obtaincd by dc-0-acetylation of the material recovered from (90)J. Baddiley, J. G. Buchanrtn, F. E. Hardy, R. 0. Martin, U. L. RajBhnndary, and A. R. Sanderson, Biochim. Biophyu. Acta, 62,406 (1961). (91) J. Baddiley, J. G. Buchanan, R. 0. Martin, and U. L. RajBhandary, Biockm. J., 86,49 (1962). (92) F. E. Hardy, J. G . Buchanan, and J. Raddiley, J . Chem. Soc., 3380 (1963).

360

A. If. ARCHIBALD AND J. BADDILEY

the mother liquors after crystallization of the @-D compound were incompletely hydmlysed by the enzyme. The unhydrolyaed material was the a-Danomer. As a consequence of the presence of both anomeric 2-amino-2-deoxy-~glucosyl lin'hges in the teichoic acid, four monophosphates are formed on hydrolysis with alkali. These were separated and characterized as the ribitol 1- and 2-phosphates anomers of the 0- (2-amino-2-deoxy-~-glucosyl) (58) and (59) (R = 2-amino-2-deoxy-~-glucosyl) ; &8 no 0-(2-amino-2deoxy-D-glucosyl)ribitol 3-phosphate was found,' the attachment of the dicsters must be a t the hydroxyl groups at C-1 and C-5. Small proportions of diphosphates were produced (see Fig. 16) which, on treatment with nitrous acid, gave ribitol diphosphates, thereby establishing the presence of doubly-linked ribitol, as required by structure (45) (R = 2-acetamido-2deoxy-D-glucosyl). Structure (45) is also supported by studies on the oxidation of the polymer with periodate, when 2.12 molecules of oxidant were reduced for each phosphorus atom. One molecule of formaldehyde was produced for every 8 atoms of phosphorus, and titration of a sample of the teichoic aaid after removal of the D-alanine residues showed the presence of eight primary acid groups and one secondary acid group for every eight phosphate groups. The D-alanine, which is attached to the polymer through the characteristic, labile, ester linkages, affords protection to the ribitol during oxidation with periodate and is, therefore, attached directly to the C-2 or C-3 hydroxyl groups of the ribitol residues. The proportions of CY-D- and BD-glycosidic linkages differed in samples of teichoic acid prepared from different batches of wall. Thus, the optical rotations of four different samples were t40.3, +6, -6, and -15.6'. Acid hydrolysis of each of these showed that they contain the same components, namely, ribitol, phosphate, odanine, and 2-acetarnido-2-deoxy-~-glucose, whereas hydrolysis with alkali revealed that the differences are related to differences in the ratio of WD to 8-Dlinkages. Thus, no WD linkages were detected in the phosphates produced on alkaline degradation of the sample having [ a ] ~- 15.6'. Serological evidence has been presentedg*showing that the 2-acetamido-2deoxy-ar- and -fl-D-glucosyl substituents occur in different chains, and that there are, therefore, two distinct types of teichoic acid in the wall. The variations in the proportions of these in Staphylococcus a u r w H walls are at p r w n t unexplained and do not appear to be due to mutation during cultivation of the organisms. c. Staphylococcus aureus C ~ p e n h a g e n . ~ . ~ ~ - T hteichoic is acid is (93) M. Torii, E. A. Kahat, and A. E. Bezer, J . Ezzptl. Med., 120, 13 (1984). (94) A. R. Sanderson, W.6. JuergenN, and J . L. Strominger, Ba'ochem. Ba'ophye. Res. Cornmula., 6,472 (1961). R. Ssnderson, J. L. Strominger, and S. G . Natheneon, J . BWZ. Chem., 287, 3003 (1882).

(06) A.

THE TEICHOIC ACIDS

361

similar in structure to that from strain H. 2-Acetamido-2deoxy-~-glucosyl groups are attached to the C-2 hydroxyl group of each Gribitol residue, and both a-D- and p-D-glycosidic linkages are present. Incubation of the teichoic acid with a specific BD-N-acetylglucosaminidase resulted in the whereas a mixed hydrolysis of 85% of the 2-acetamido-2deoxy-~-glucose, a- and pD-N-acetylglycosadnidase removed all of the amino sugar residues. After such treatment, the ribitol phosphate is still polymeric, and the product from the action of the p-D-N-acetylglucosamhidinidase contains all of the original walanine, together with the 2-acetamido-2deoxy-aDglucosyl residues (phosphorus :2-acetamido-2-deoxy-sglucose:walanine, 1:0.11:0.44). The D-alanine ester residues are thus attached directly to ribitol residues (not to 2-acetamido-2dleoxy-wglucoseresidues) . Methods similar to those used with other teichoic acids were employed to demonstrate that this polymer closely resembles that from strain H. Measurement of the formaldehyde produced on oxidation, and of the inorganic phosphate formed on incubation with phosphomonoesterase, indicated a chain length of about 14 units. residues (15% of the The number of 2-acetamido-2deoxy-cu-~-glucose total) is somewhat greater than that in the strain H teichoic acid, but is less than that found in material from a third strain (3528)) in which essentially all of the amino sugar is in a-D-glycosidic linkage. These differences in the ratio of a-D to p-D linkages have been correlated with differences in immunological specificity, and strains have been found which possess entirely a - ~or, entirely /3-D, linkages? d. Lactobacillus arabinosus 17-5."-The teichoic acid from the walls of this organism contains D-glucose, D-alanine, ribitol, and phosphate (phosphorus:wglucose: walanine, 1:1.06:0.62). By methods similar to those already described, it was shown to have the usual chain structure of ribitol phosphate residues joined through positions 1 and 5 ; also, there are 7-8 units in each chain. D-Alanine is attached to the ribitol residues in labile, ester linkages, and the wglucose is also attached to the ribitol residues (as a-sglucopyranosyl groups). The pattern of glycosidic substitution is, however, somewhat more complex than in the ribitol polymers described previously. Hydrolysis with alkali, followed by enzymic dephosphorylation, gave a mixture of neutral products, together with some di-0-D-glucosylribitl phosphates. Chromatographic fractionation of the neutral products gave several pure components. Appreciable proportions of free ribitol were produced, together with a much smaller proportion of 1,4anhydroribitol. The formation of 1 ,Panhydriribitol under alkaline conditions has been discussed earlier (see p. 331): it k, apparently, a characteristic product of ribitol phosphate polymers containing unsubstituted ribitol residues. It is of interest that the infrared spectrum of the 1,Canhydroribitol is

362

A. R. AIWHIRALD AND J. BADDILEY

identical with that of optically active 1,4anhydroribitol and is different from that of the racemate. It thus appears that the production of the anhydroribitol is stereoselective, but, unfortunately, the small amount of mtttorisl nv:ailiihlo preoludd twigiininiit of (!onfiguration. 2- O-a-iA ~livni~yr~iiior~yl-~~ril,ibl (61), [u]ii 1W0,WIW c i y ~ t d l i ~ ~ ! d from tho friwtioiiiLtcui liydrolyrute; it8 xtructuru w u dulttriiiiiiod by methods similar to those used for the &D-glucopyranosidefrom Bocillzra crubtilis, and hns been confirmed b y ~ y n t h e s i s . A ~ , small ~ ~ amount of an isomeric 0-D-glucosylribitol was also obtained; this is stable to &D-glucosidase and has a high, positive rotation. On oxidation with periodate in acidic solution, two molar proportions of formaldehyde were formed and about 5 of oxidant were reduced; in alkaline solution, "over-oxidation"f@occurred, and 3 molar proportions of formaldehyde were formed. Theee results, in conjunction with the studies on the di-0-D-glucosykbitl (discussed below) , established that this compound is 3-O-cu-~-glucopyranosylribitol(62) . 2,3-Di-0-~~-~-glucopyranosyl-~ribitol (63), [a]D 4- 137", was obtained,

+

0-CH HO'H !%OH

(62)

:LH its hexahydrate, from one of the fractions. The glycoaide waa unaffected by ~-D-glucosidase,and the presence of two arD-glucosidic linkages is consistent with its optical rotation. Acid hydrolysis gave &glucose, ribitol, (96) J. Baddiley, J. G. Buchanan, P. W. Auetin, and F. E. Hardy, J . Chem. Sm.,2128

(1984). (97) L. J. Sargent, J. 0.Buchanan, and J. Baddiley, J . Cham. Soc. 21% (1962). (98) L. Hough and M. B. Perry, Cham. Id.(London), 788 (1966).

THE TEICHOIC ACIDS

363

and some anhydrorihitol, but, under milder conditions, the products were wglucose and a mixture of the two isomeric ribitol mglucosidee, (61) and (62), together with unchanged “digluc&de.” One molar proportion reduced 5 of periodate, and gave 2 of formic acid and one of formaldehyde: reduction of the oxidized product with borohydride, followed by acid hydrolysis, gave glycerol and erythritol. It follows that its structure is that given in formula (63). Approximately equimolar proportions of unsubstituted, monosubstituted, and disubstituted ribitol residues were present in the teichoic acid examined. However, the proportions differed in other samples, and there is no apparent, regular sequence along the chain. In fact, it is not yet known whether all three types of unit occur in the same polymer molecules, or whether the teichoic acid is a mixture of different molecular species. The production of ribitol diphosphates on acid hydrolysis, and also on alkaline hydrolysis, of the product obtained after oxidation of the teichoic acid with periodate establishes the presence of ribitol phosphate residues joined to each other through phosphodiester groups. On oxidation of the polymer (palanine removed) with periodate, many of the ribitol residues were oxidized, and the quantitative data obtained support a structure in which there are 7-8 units in the chain. From the lability of the Palanine residues to alkali, it follows that these are attached directly to the ribitol residues. Demonstration of this linkage, by oxidation of the intact polymer with periodate, is precluded by the presence of the di-0-D-glucosylribitol residues in which the ribitol is, in any case, resistant to oxidation.

VII. TBICHOIC ACIDSOF ACTINOMYCETES Actinomycetes are mold-like organisms, some of which have properties in common with the Gram-positive Eubacteriales; thus, muramic acid have been found in isolated walls of several and 2-amino-2deoxy-~-glucose of these organisms, and many of them are sensitive to muramidase,m which is specific for the hydrolysis of the glycosaminopeptide in bacterial walls. has rqported the presence, in whole cells and in isolated walls NaumovalOO of such organisms, of alditol phosphate derivatives which resemble the teichoic acids. The degradative procedurea used to determine their structure follow the general pattern developed for the teichoic acids from bacteria. (99) H.H.Romano end W.J. Niokerson, J . Bmbviol., 79,478 (1956). (100) I. B. NBU~OVB, Dokl. A M . NDuk &WR, 6, 199 (1964).

364

A. R. ARCHIBALD AND J. BADDILEY

The polyiner extracted from whole cells of Actinomyces streptmnycini contains 7.3% of phosphorus and 7% of glucose.lol Acid hydrolysis gave products similar to those formed from ribitol teichoic acids, and alkali gave a mixture of phoaphomonoestern, ctiaymic dephosphorylation of which gave ribitol and a ribitol glueoside. The ribitol polymer extracted from the walls of this orgsnism is more heavily sub8tituted with glycosyl residues than the material extracted from whole cells, and, on hydrolysis with alkali, followed by eneymic dephosphorylation, it gave an O-glucosylribitol and an O-(2-amino-2-deoxyglucosyl)ribitol. These have not yet been fully characterized, but qualitative observations suggest that the glucoside has structure (46) and the 2-amino-2-deoxyglucoeide has structure (60). An interesting feature of this polymer is the absence of Dalanine and the presence of succinate.'02 Treatment of the polymer with hydroxylamine gave the monohydroxamate of succinic acid: with ammonia, the monoamide was formed. Structure (64)was proposed for this polymer.

.. 8"-0-bCI...-

8

- - -clii-o-

R d IIiiH

YH

--H

$"

8 8 Y

PH d

777

c- -c-c-cli#-o--p-o-H*c-c--cc-c~~*~'-

*

R J bHbH \

R'b

m&-cHj~c~-co,-

bHbH I

(W

A ribitol phosphate polymer has also been extracted from Aclinomyces vioZuceus.10* The major product of hydrolysis with alkali is an O-glucosylribitol phosphate, and, although D-alanine is apparently absent, acetate groups are believed to be present. The extract of whole cells also contained a glycerol phosphate derivative, and it is concluded that the organism contains a glycerol teichoic acid. Glycerol teichoic acids have been found in straina of Actinomyces r i m o m and A. antibioticus.lo4On acid hydrolysis, both gave galactose, glycerol, and glycerol phosphates. The phosphates produced by alkaline hydrolysis of the polymer from A. rimoswr include glycerol phosphates and a more complex phosphate containing a stable, phosphodiester residue; the properties of this phosphate are consistent with structure (24; R = galaatosyl). No 0-galactoeylglycerol phosphates were produced, and it is suggested that the polymer is a glycerol teichoic acid containing the usual phosphodiester linkagea between the C-1 and (101) I. B. Newnova, A. N. Belozemky, and P. A. Shsfikova, Dokl. Akad. Nauk SSHR, 143,730 (1962). (102) I. B. Newova, 2. A. Shabarova, and A. N. Belozersky, Dokl. Akad. Nauk SSSR, 162, 1471 (1963). (103) I. B.Neumove and M.Z. Zaretakeye, Dokl. Akad. Nauk SSSR, 166, 1464 (1964). (104) I. B. Naumove and M. Z. Zaretakaye, Dokl. Akad. Nauk SSSR,167,207 (1W).

THE TEICHOIC ACIDS

363

C-3 hydroxyl groups of glycerol and having galwtosyl substituents on the C-2 hydroxyl group of the glycerol regidurn. The polymor frorii Aclinomycex untz'biolicusis uiiwud in that appreciable pliosphatc arc produced on hydrolproportions of a11 f-galtta~~~ylglyoerol ysis by alkali. Such a phonphatc could not arise as a major product of hydrolysis of a glycerol phosphate polymer cmtaiuing 1,3-linkagesJ and it has been suggested that glycerol phosphate residues are attached to the hydroxyl groups at C-1 and C-2 of glycerol, a galactosyl residue being attached to the"C-3 hydroxyl group. Such a structure (31; R = galactosyl) would be hydrolyzed by alkali, to give O-galactosylglycerol 1- and 2phosphates, but the evidence does not unambiguously establish this structure. It is of interest, however, that an enzyme systemm from Bacillus lichenifomis ATCC 9945 synthesizes a Dglucosylated glycerol phosphate polymer which, on hydrolysis by alkali, gives an 0-D-glucosylglycerol phosphate as the major D-glucose-containing product. The D-glucose is attached to one of the primary hydroxyl groups of the glycerol, and, here too, it is probable that 1,2-phosphodiester linkages are present. Moreover, 1,2linkages occur between glycerol residues in a teichoic acid from Bacillus sfeurothmophilus, in which D-glucosyl substitutes the C-1 hydroxyl group.'& Strains of Streptmnyces gm'seus and S. niveus contain teichoic acidslMb:these are composed of glycerol or ribitol, phosphate, and glucose, but walanine has not been detected.

VIII. THELOCATION OF TEICHOIC ACIDSIN RELATION TO CELLSTRUCTURE 1. Association with the Membrane

The widespread and probably universal occurrence or" these polymers in Gram-positive bactkria suggests that they may be important in cell metabolism. Although quantitative studies on the proportion in organisms have not been made, data obtained during the usual extraction and purification procedures indicate that the membrane teichoic acid may represent up to 1-2% of the dry weight of the cell. Appreciably less is present in some organisms, and the actual proportion may depend on the age of the cell; thus, it has been shown that much more groupD antigen (membrane (104s) A. J. Wicken, personal communication.

unpublished observations. (104b) P. Critchley and B. BBWS,

360

A. R. ARCHIBALD AND J. BADDILEY

kirhoic soid) in pronorit in resting aclls of f8reptococcue f m l & 3 than in exponentidly growing cello.’o6JW High-speed centrifugation (100,OOOy) of the “cell contents” of many Gram-positive bacteria results in the sedimentation of a particulate fraction consisting of ribosomes and fragments of membrane; this contains nearly all of the membrane teichoic acid.@*This material is not removed during p d c a t i o n of the ribosomal fraction, and it haa been suggested that teichoic acids might be common contaminants in such ribosomal Extraction from the particulate fraction re&ired prolonged treatment with trichloroacetic acid, and a part of the extracted teichoic acid wa8 in close association with polynucleotides. It is possible that such complexes do not occur in the intact cell, but are artefacts arising during the agitation of soluble and particulate material in the disrupted cell. This view is in accord with studies on the location of “intracellular” teichoic acids; these studies indicate that these polymers are in, or external to, the protoplast membrane.uJw-!m Thus, cells of Bacillus ntegaterium* were treated with muramidase in the presence of sucrose; the resulting protoplasts were separated from the wall hydrolyzate and disrupted osmotically, and the cytoplasmic contents and membrane were separated. Analysis of the proportion of teichoic acid present in each fraction showed that the wall hydrolyzate contained about 85% of the total,most of the remainder being present in the membrane fraction. Similar results were obtained with a streptococcus in group D, and it was concluded that “intracellular” teichoic acids are located between the wall and the protoplast membrane, possibly in association with the membrane. Only small proportions of nucleic acid were found in the wall hydrolyzate; consequently, the protoplast membrane was still an effective osmotic barrier, and so it is unlikely that the teichoic acid in the wall hydrolyzate had arisen through diffusion through the membrane after preparation of the protoplasts. The location of the “intracellular” teichoic acid in streptococci of group D has also been studied by using immunological techniques. Shattock and SmithlWJm exmined several strains by digestion of the walls with murami(106) H.D. Slade and G. D. Shockmsn, Iowa Stde J . Sci., 8 8 , s (1963). (106) P. M. F. Shattock and D. G. Smith, J . Urn. Mkobiol., 81, iv (1883). (107) D. G. Smith and P. M. F. 8hattoak, J . Urn. Mirrobiol., 84,165 (1064).

THE TEICHOIC ACIDS

367

d w arid with a phago3ssoci:ited lysin; they, too, concluded that most of the group antigen (teichoic acid) is external to the protoplast menibrane. The location of antigen between the wall and membrane agrees with the observation that, under certain conditions, antigen is synthesized by G foriiis of stroptocowi nnd is excretcd into tho culture niedium. A detailed ~ Jhas ~ been investigation on straiii 9790 has now been carried o ~ t . It clearly demonstrated that the antigen is associated with the protoplast membrane, and membrane preparations containing much of the antigen originally present in the cell have been prepared. Antigen can be removed from such preparations by washing with salts or, especially, water; even after extensive washing, however, a large proportion remains associated with the membrane. Washing causes fragmentation of the membranes, and it is suggested that the effect of washing might not be to dissociate the teichoic acid from the membrane so much as to disrupt the membrane into fragments which are not sedimented at low speeds. This might explain the release of antigen into the wall hydrolyzates in the earlier studies, since membranes prepared from certain, exponential-phase cultures are much more fragile andaJ06J08susceptible to auto1ysis108than are those prepared from stationary-phase cultures. All three investigations strongly suggest that the glycerol teichoic acid antigen is located in or on the external surface of the protoplast membrane. This conclusion is consistent with the observation that at least some of the enzymes concerned in the biosynthesis of the teichoic acid occur in the protoplast membrane. The nature of the association between membrane and teichoic acid is unknown, and it is possible that these teichoic acids are chemically attached to other components of the cell. Samples obtained by extraction with phenol appear to have appreciably higher moleculhr weight than has the purified teichoic acid obtained by extraction with trichloroacetic acid, and it is likely that the prolonged, acid treatment used in earlier work may have caused hydrolysis of some of the phosphodiester linkages. It is noteworthy that this comment on earlier studies does not apply to ribitol teichoic acids. Detailed examination of preparations of membrane teichoic acid obtained by less drastic conditions is highly desirable, in order to confirm the supposed she of the naturally occurring polymers, as well aa (108)

G. D.Shockman, M. J. Conover, J. J. Kolb, P. M. Phillips, L. 8. Riley, and G. Toenniee, J . Backriol., 81,36 (1961).

308

A. R. AllCHIUALD AND J. BADDILEY

t,o cst,tthlish thc chcmicul hi& of thcir aseoaiation with the membrancs.

Such studictj should wit&in deciding the furictioii of them compounds (sec p. 371). 2. Association with the Wall

Teichoic acid accounts for up to 50% of the weight of the wall in several organisms, and it is thus a major component of the cell, comprising some 5-10q0 of the total dry-weight. Complete removal from the walls of several organisms has been effected by extraction with acid under forcing conditions, and the residual, insoluble gIycosaminopeptide has the same shape as the original cell-wal116;thus, the glycosaminopeptide is responsible for the shape and integrity of the wall, and teichoic acids are apparently of little importance as structural components. The difficulty in completely extracting teichoic acids led to the suggestiodw that they might be held in the wall by covalent linkage to glycosaminopeptide. However, such attachment could not involve the amino groups of the D-alanine ester residues, since these are readily (2,4-dinitrophenyl)ated with l-fluoro2,4-dinitrobenzene. Moreover, dilute alkali hydrolyzes all of the D-alanine ester linkages in walls, giving the free amino acid without removing significant amounts of the teichoic acid.16J10Attachment involving the inter-unit phosphodiester groups of the teichoic acid was considered unlikely,’6 as, not only would the resulting phosphotriesters be extremely labile toward acid , but the teichoic acid released by their hydrolysis would contain 2 ,5 (as well as 1,5) inter-unit phosphodiester linkages; moreover, some of the chains in isolated material might be expected to have phosphomonoester residues at C-1 and C-2 of terminal ribitol residues. These consequences follow from the accepted mechanism of hydrolysis of phosphotriesters, and the absence of such structural features in the isolated ribitol teichoic acids indicates that linkages of this kind do not occur in the intact wall. The observation that moat of the phosphorus in walls containing ribitol teichoic acid is converted into water-soluble material when the walls are oxidized with sodium metaperiodate indicates also that multiple linkages between teichoic acid and “glycosaminopeptide” do not 0ccur.11~ Evidence for covalent linkage between these two wall-components followed from the re~ognition‘~ of wall teichoic acid m a major component (109) J. Mandelstam and J. L. Strominger, Biocham. Biophys. Res. Cmmun., 6 , 466 (1961). (110) G. D. Shockman, Nalure, 198, 997 (1963). (111) H.J. Rogers and A. J. Garrett, Biochsm. J., 8 8 , 6 ~(1963).

‘rm

TEICHOIC ACIDS

369

of preparations of “polysacchsride A.” This designation was first given to an important, serologically active component of staphylococci in 1935, and more recent pieparations have been studied by Oeding and his colleagues, Material has been obtained by prolonged extraction of disrupted and it is likely that such treatment causes partial cells in various buffers8,112 autolysis of the cell wall.118The resultant, antigenic material contains teichoic acid associated with smaller proportions of amino acids and amino sugars derived from the cell wall.114Characteristic components of glycosaminopeptide could not be removed by electrophoresis or ionexchange chromatography, and this finding suggests that they are covalently attached to the teichoic acid. A similer conclusion has been reached by Strominger and his collaborators, who studied the hydrolysis of walls of Staphylococcus aureus by enzymes prepared from Streptmyces albus and from C h a l a r o p s i ~ . ~Again,Im ~ ~ J : ~ soluble preparations were obtained in which teichoic acid is covalently attached to fragments of glycosaminopeptide. The properties and composition of such preparations have been studied in some detail,116and it has been shown that no phosphomonoester group is present. Since teichoic acid obtained by extraction with acid contains a terminal phosphomonoester residue, it was suggested”d that a covalent linkage between the two polymers might involve the terminal rnonoester; alternatively, such monoeaters might not occur in “native” teichoic acid, but would arise by hydrolysis of inter-unit phosphodiesters during extraction under the acidic conditions. However, the rate of such of the hydrolysis is low under normal conditions, and a teichoic acid extracted from walls of Staphylococcus aureus, Bacillus subtilis, and Lactobm‘llus arabinoswr has shown that little degradation of the ribitol phosphate polymer chains occurs under the conditions usually employed for the preparation of pure samples of these substances. Cons4 (112) G. Haukenes, N. Loanegard, and P. Oeding, Acta Pathol. Mirrobwl. Scad., 68, 84 (1961). (113) G. D. Haukenes, Ada Patiwl. Mierobiol. Scad., 66,463 (1962). (114) G. D. Haukenee, Ada PaucOZ. Microbwl. Scud., 66,450 (1962). (115) G. D. Haukenee, Acta Palhol. Mirrobial. Sccmd., 66, 110, 117 (1962). (118) J. M. Ghuyaen, D. J. Tipper, and J. L. Strominger, BaOchmi8tsyl 4, 474 (1965). (117) J. M. Ghuyaen, M. Leyh-Bouille, and L. Dierickx, Biochim. Biophy8. Ada,.B8, 286,297 (1982). (118) J. H. Hash, Arch. Biochem. Biophye., la,379 (1963). (119) D. J. Tipper, J. L. Strominger, and J. M. Ghuysen, Science, 146, 781 (1984). (120) J. M. Ghuysen and J. L. Strominger, Biochemietsy, 2,1110 (1963). (121) J. B. Hay, N. B. Davey, A. R. Archibald, and J. Baddiley, Biochem. J., H, 7c (1965).

370

A . 1t. AItCIIlIIALD A N D J. UADDILEY

quently, tho monoester groups in thcse preparations do not arise through hydrolysis of inter-ribitol diesters, and their absence in the enzymically prepared teichoic acid-glycosaminopeptide complex does, indeed, indicate covalent attachment of teichoic acid through its terminal phosphate group. It has been statedII7JB that a teichoic acid in the wall of Bacillus megalmium KM is released, as a soluble complex with fragments of glycosaminopeptide, by treatment with N-acetylmuramidase. Similarly, in a preliminary report it haa been statedU8 that the teichoic acid released on autolysis of isolated walls of Bacillus subtilis remains wociated with other components Moreover, the of the wall, in particular with 2-amino-Zdeoxy-~-galactose. teichoic acid complex contains no phosphomonoester residues, and it appears that, in this caae also, the linkage must involve the terminal phosphate &rOUP* Evidence for the chemical nature of the teichoic acid-glycosaminopeptide linkage is still inadequate, but chemical considerations based on the structure of isolated teichoic acids and on studies of fragments formed from walls by chemical degradation have led to the suggestions that the linkage may be a phosphoramidate linkage involving one of the amino groups of the glycosaminopeptide or, alternatively, a pyrophosphate linkage. Tho bond is readily hydrolyzed by acid, giving teichoic acid having a free phosphomonoester group on the C-5 hydroxyl group of a terminal ribitol residue. Since the C-4 hydroxyl group of this ribitol residue bears a glycosyl residue, hydrolysis could not occur through the usual, cyclic phosphate intermediates, and the acid lability of the linkage must have a different explanation. Teichoic acid is released only slowly when walls are treated with dilute alkali; however, under vigorous conditions, the inter unit phosphodiester groups are hydrolyzed, and, in experiments on walls of Staphylococcus aureus, 0-(2-amino-2deoxy-~-glucosyl)ribitol, and its mono- and di-phosphates are produced in the proportions expected from consideration of the chain length of the teichoic acid in these walls.124 The formation of the theoretical amount of 0-(2~amino-2deoxy-~glucosyl)ribitol diphosphates, in which a phosphomonoester residue occurs adjacent to the glycosidic substituent, again cannot be due to hydrolysis of a simple phosphodiester involving cyclic phosphate intermediates. These observations are consistent with the presence of a phosphoramidate linkage involving one of the amino groups of the amino sugar chain of the RlycoRrtmiiiopcpt,id~,for cxrtmplc (65). Ruth R linkage would he labile to (122) J. M. Ghuyscn, Biochim. Biophys. Ada, 89, 132 (1964). (123) F. E. Young, I). J . Tipper, and J. L. St,rnminger,J . Biol. Chem., 238, ~ ~ 3 6 0 0 (1!)64). (124)

J. B. Hey, A. 11. Arrhilxdd, niitl J. B:ddiley, Biochem. J., 97, 723 (1965).

371

THE TEICHOIC ACIDS Horqc

$.-

GlycocwialnopePtlde---O

B Y ? *b

?H

RN-P-O-H,C-C-F-C-C%-O-P--II 0 Gl& 0

bl

trlchoic WLd

Alp

where I? = H or CHaCO, and Gly = a sugar realdue. (65)

acid, but would probably be rather more stable to alkali. Degradation of the sugar chain by alkali could, however, lead to the elimination of the phosphoramidate group and, subsequently, to the formation of the 0(2-amino-2deoxy-~-glucosyl)ribitol diphosphstes. However, in view of our relatively inadequate knowledge of the reactions of comparably substituted phosphoramidates, the alternative in which the phosphorus atom is attached to a nitrogen atom in a peptide group of glycosaminopeptide cannot be excluded. Linkage through a pyrophosphate group [see formula (M)] to a hydroxyl group of an amino sugar residue in the glycosaminoOH

GlycoramInapeptide-0

BH

-'b -0-P-

4 8

O-H,C-C-&

-

T H -C-CqY

Gld

AA

0

---

ALP

(66)

peptide would also be consistent with the lability toward acid and alkali. Hydrolysis by alkali would proceed through either a &membered cyclic phosphate involving the hydroxyl group at C-3 of the terminal ribitol residue or a 5- or &membered cyclic phosphate on the glycosaminopeptide. In considering the nature of the linkage between the two polymers, the ready removal of teichoic acid from walls by dilute solutions of phenylhydrazine or 1,l-dimethylhydrazineat pH 7 is probably significant.u6For structure (65), this could be explained by the formation of an osazone on the reducing end, but insufficient is known about the behavior of the other suggested structures toward hydrazines to permit a decision between the different possibilities. The function of teichoic acids is still unsettled. The presence in them of highly reactive groupings, for example, aminoacyl ester, might be consistmt, witchn function related to cell-energy relationships. However, as thc rrac4ivc cstcr linkages arc confined almost exclusively to walanine, and in view of the restricted location of the polymers in the cell, such a function seems rather unlikely. Similarly, there is little reason to associate their serological properties with function. It has been suggested (125) A. R. Archibald and J. Baddiley, Biockm. J., 96, 1% (1965).

372

A. H. AHCHIUALD AND J. BADDILEY

that they may participate, in an unspecified manner, in the formation of the wall atructure itself. The conclusion that they occur in association with the membrane is consistent with this suggestion, because it is in this region of the cell that other wall components, such as glycosaminopeptides, are synthesized. It is possible that teichoic acid attached to the outer surface of the membrane, and extending to the inner surface of the wall, presents a region of spatially highly oriented anionic and cationic centers which could direct the course of wall formation. In this connection, also, the region of high charge-orientation would markedly influence the passage of ions through the wall-membrane area, and the function of teichoic acids may be, at least in part, that of ion regulators. It is perhaps noteworthy that organisms containing substantial proportions of teichoic acid in their walls are particularly able to withstand relatively high concentrations of salt in the surrounding medium.lb The function of these compounds will, however, be better understood when more is known about their general metabolism; alresdy it appears that the proportion of teichoic acid in the wall of an organism depends upon its age,146but detailed quantitative information is not yet available.

IX. BIOSYNTHESIS Many Gram-positive bacteria contain enzymes which catalyze the formation of (1) from mglycerol l-phosphate and cytidine triphosphoric acid, and of (2) from L-ribitol l-phosphate (D-ribitol 5-phosphate) and cytidine triphosphoric acid,12’ the L-ribitol l-phosphate arising through the reduction of D-erpthro-pentdose 5-phosphate with reduced adenine-nicotinamide dinucleotide.lS The early suggestion that (1) and (2) would be biological precursors of polymeric substances (teichoic acids) has been demonstrated by the synthesis in vitro both of ribitol phosphate and glycerol phosphate polymers similar to those present in teichoic acids. Poly (glycerol phosphate) synthetaae haa been demonstrated in Bacillus liCht??&ifO9-?niS ATCC 9945, BaciZZus wbtilis ATCC 6051, and Baci~lussubtilis NCTC 3610;incubation of enzyme preparations from these organisms with (1) yields a glycerol phosphate polymer in which the phosphodiester linkages are attached to the C-1 and C-3 hydroxyl groups of adjacent glycerol residues.lOJ1This synthesis shows a marked similarity to the synthesis128 of phosphatidylglycerol phosphate, a prohahlc! precursor of cardiolipin in which three glycerol residues are joiiid through phosphodiester residues. Cardiolipin is structurally related to t,eichoic acids. (126) Unpublished work by the authors with Mrs. N. B. Davey. (127) D. R. D. Shaw, Biochem. J., 81,297 (1982). (128) L.GItww, Rimhim. Biophys. Acta, 07, 528 (1003). (121)) E. P.Kennedy, Federation Proc., 10,934 (1961).

THE TEICHOIC ACIDS

373

The bacterial, poly(glycem1phosphate) synthetase activity is ssaociated with the particulate fraction obtained by ultracentrifugation of sonically disintegrated cells. Attempts to solubilize this enzyme, or to remove the membrane teichoic acid, have been unsuccessful, and it is uncertain whether primer molecules, such as teichoic acid in the enzyme preparation, are required for the synthesis. Membranes obtained through the action of muramidase on whole cells contain all of the synthetase of the cell, and this is sufficient to account for the calculated rate of synthesis of teichoic acid in the living cells. Membranes from Baeillus subtilis NCTC 3610 also contain an enzyme which catalyzes the transfer of a-D-glucopyranosyl residues from uridine 5- (D-glycopyranosylpyrophosphate) to enzymically prepared glycerol phosphate polymer or to natural, membrane teichoic acid from this organism. The D-glucose residues me attached to the C-2 hydroxyl group of each glycerol residue, and the product is indistinguishable from the wall teichoic acid of this organism. Poly (ribitol phosphate) synthetase has been found in particulate fractions from $taphylococcus aureus H, and Lactobacillus plantarum.'2JaJ~ The bulk of the activity in Lactobacillus phntarum was in crude, cell-wall preparations, and the enzyme is apparently located in the membrane, although intimate association with the wall itself has been suggested. Unlike the natural teichoic acid, the enzymically synthesized ribitol phosphate polymer was readily extracted with phenol; hydrolysis by acid and by alkali gave the expected products, and oxidation with periodate indicated a chain length of 5-9 units, a value which compares well with that of 8 units for the natural polymer in the walls of this organism. The particulate fraction from Staphylococcus aurm H contains enzymes groups from uridine 5- (2which transfer 2-acetamido-2-deoxy-~-glucosy~ acetamido-2-deoxy-~-glucopyranosyl pyrophosphate) to a ribitol phosphate polymer acceptorem*" The acceptor was prepared by the action of a ~ D - N acetylglucosaminidw on the teichoic acid from the walls of Staphylococczls aureu~H,and contained the 2-acetarnido-2-deoxy-a~-glucosylgroups present in the original teichoic acid. In view of the more recent discovery that a-Dand B-D linkages occur in separate polymers,gait must be assumed that the acceptor preparation was a mixture of a polymer of ribitol phosphate and a polymer in which 2-acet~mido-2-deoxy-cr-D-glucosylsubstituents are attached to all of the ribitol residues in the chain. Both CY-D and 8-D linkages are formed in proportions similar to those in the original teichoic acid. Enzyme preparatioiis from other strailis of Staphylococcus uz(~eusbring about the incorporation of glycosyl substituents in which the proportions of anomers closely correspond to those of the native (130) N. Ishimoto and J. L. Strominger, Federation Proc., 22,465 (1963).

374

A. R. ARCHIBALD AND J. BADDILBY

teichoic acid of these strains. Acceptor polymers containing alanine ester residues are only half as active as those from which these residues have been removed, and transfer of sugar residues to intact teichoic acid, L-ribitol l-phosphate, or (2) apparently does not occur. It has not been poesible to replace, in the cell-frea system, all of the Zwetamido-2-deoxy-Dglucosyl groups removed by the glucosaminidaae, and it is poesible that, in the living cell, a transfer of ribitol phosphate during lengthening of the polymer chain and the transfer of 2-acetamido-2-deoxy-~-glucosylgroups to ribitol residues might occur sequentially. This possibility could also explain the low activity in vitro of poly(ribito1 phosphate) synthetase, although this may be a consequence of the absence of simultaneoussynthesis of glycosaminopeptide. Synthetic ribitol phosphate polymer, unlike teichoic acid in a wall, is readily extracted from the paxticulate enzyme or membrane preparation by treatment with phenolla Similarly, teichoic acid synthesized by intact and cells in the presence of penicillin is only loosely attached to the it may be significant that, in each case, synthesis of teichoic acid has occurred without the simultaneous synthesis of glycosaminopeptide. It is now known that, in the normal wall, teichoic acid and glycosaminopeptide are attached to each other, and it has been suggested that the low activity of cell-free synthetase is due to the absence of suitable acceptor molecules of glycosaminopeptide. This possibility could account for the erne of removal of teichoic wid formed when simultaneous synthesis of glycosaminopeptide was not possible. The mechanism of biosynthesis of the teichoic acid from the walls of the strain of Staphylococcue hf& in3which , sugar residues form a part of the polymer chain,a has not yet been elucidated. This mechanism must differ considerably from that proposed for the more conventional teichoic acids, as sugar residues could not be attached after the formation of the polymer chain. If it is assumed that (1) is a precursor of this polymer, the evidence from degradation (see p. 351) shows that the phosphorus attached to C-1 of the Z-acetamido-2-deoxy-~-glucoseresidues is not that attached to glycerol residues in the nucleotide. The stereochemical evidence from structural studies would suggest that, at an unspecified stage in the biosynthesis, a +glycerol l-phosphate residue from (1) is transferred to C-3or C-4 of a 2-acetamido-2-deoxy-n-g~ucose residue. The incorporation of the n-alanine ester groups is, presumably, the last &age in tho hiogynthesis of teichoic acids. Several organisms possess oxizymw which activate D-alaninc,that is, which form a walanyl-adenosine Fdphosphate-enzyme complex; but, 90 far, there has been no demonstration of incorporation, in cell-free systems, of walanine into teichoic acid or any

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of its degradation products or precursors.66 It is, perhaps, worth noting that sequential incorporation of the amino acid, ribitol phosphate, and a sugar residue is not excluded. The poly (ribitol phosphate) synthetase w d poly (glycerol phosphate) synthetase are inhibited by vancomycin, novobiocin, and Crystal Violet. Other antibiotic substances which interfere with cell-wall synthesis (such as bacitracin, ristocetin, and streptomycin) are almost without effect on the isolated synthetases, and penicillin is inhibitory at high concentrations only. Moreover, penicillin, vancomycin, and bacitracin do not markedly inhibit synthesis of cell-wall glycosaminopeptide in vitro, although the synthetical activity of extracts of cells which have been pretreated with these antibiotios is lowered.l81J@Convincing evidence that the primary site of inhibition by antibiotics is the biosynthesis of cell-wall material has been obtained only for the penicillins and cycloserine, and it appears that the action of even those antibiotics may be more complex than was originally SUPPA. (131) A. N. Chetterjee and J. T. Park, Proc. Nadl. A d . Sci. U.S., 61, 9 (1964). (132) P. M. Meadow, J. 5. Anderson, and J. L. Strominger, Biochsm. Biophys. Rea. Commun., 14,382 (1964).

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THE EFFECTS OF PLANT-GROWTH SUBSTANCES ON CARBOHYDWTE SYSTEMS

BY H. W. HILTON Ezperiment Station,Hawaiian Sugar Planters’ Association, Hmrolulu, Hawaii

I. Introduction.. .....................................

.

11. Indole-3-acetic Acid and 1-NaphthaleneaceticAcid. 1. Effects on the Cell-wall Polysaccharides. ........ 2. Effecta on Stmch and on Soluble Carbohydrates... . . . . . . . .

3. Auxin Activity, Respiration, and Metabolism. . . . 111. Plant-growth Substances Used as Herbicides.. . . . . . . 1. (2,4-Dichlorc1phenoxy)eoeticAcid and Related Compounds. . . . . 2. 1 , % D i h y M,&pyridazhedione. . . . . . 3. 3-Amino-a-trianole . .............................. 4. . . . . . . . . . . . 402 4. Chlorinated ChlorinatedAliphatic Aliphatic Acide. Acids.... . . . . . . . . 5. 5. Carbamates Carbamatesand and Carbenilates. Carbenilates..................... ....................

6. 6. Substituted Substituted Uress.. Uress. ........................... ............................. . . . . . . . . . . . . . . 404 7.7. Substituted Substituted8-Triazines. s-Triazines........................... ..... ........... ............. 8. Substituted Benzoic Acids.. ........................................ 407 IV. Glycosides and Other Carbohydrate Derivatives Plantgrowth Substances.. 408 1. NaturalGlycosidw....................... . . . . . . . . . . . . . . . . . 408 . . . . . . . I . .

2. SyntheticGlycosidea, Other Carbohydrates, and Carbohydrate Derivatives. 413 V. Gibberellins and Kinins. .............................................. 416 VI. The Effects of Plantgrowth Bubstances on Sugarcane.. .............. 421 VII. Abscission and Ripening. ............................................. 429

I. INTRODUCTION The identification of indole-&acetic acid (1) as the primary, natural regulator of growth in plants, made by Kogl and coworkers1in 1934, and its

(1)

quantitative estimation in trace quantities? set off an intensive search for other natural and synthetic organic compounds having similar ability to (1) (2)

F.K6g1, A. J. Haagen-Smit, and H. Erxleben, 2.Phgeiol. C h . ,PN, 90 (1934). F.W . Went, Rec. Trav. Botan, Need.,16, 1 (1928). 377

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inodify the growth and dcvelopment of plants. Two other groups of substances, the natural gibberellins and, possibly, the kinins, are now recognized, each having unique patterns of growth activity. Many other substances of known or unknown chcniical structure have been iwlt~tdfmni plant tissucs :uid liwc bwii sliowir lo :rtTcvl tho :uiiouat or durtrt8ioirof ~ o i r t c biological process. Ethylene is probably 8 phbnl horniotic iitvolvtut i i i t isNuo aging and senescence. These chemicals of natural origin, mid several hundred synthetic compounds, have been used in extensive studies of normal growth-regulation by the internal (endogenous) substances, and of growth alteration with externally applied (exogenous) chemicals. Many of the synthetic compounds are widely used commercially for profoundly altering normal growth in higher plants, or MItherapeutic agents against plant diseases. It is the author’s intent to discuss the contributions to carbohydrate chemistry from plant physiology, agricultural chemistry, botany, and agronomy. In common with the usual convention in these fields, the D or L configuration will not be signified for natural products, unless it is needed for clarity or was included in the original work. The mass of detail has necessitated certain reatrictions in scope: plant-growth substances are defined as, and limited to, “organic compounds which, at low concentrations, promote, inhibit, or qualitatively modify growth. Their effect does not depend on their caloric value or their content of essential elements.”* Discussion will chiefly be restricted to the effects of externally applied chemicals. The effects of light, altitude, temperature, irradiation, oxygen, carbon dioxide, hydrogen sulfide, and other products of anaerobic metabolism, of most enzyme poisons (such as cyanide, arsenate, and fluoride), and of the macro- and micro-nutrients will not be considered except as they may influence the effects of a growth substance. Only the naturally produced regulators will be considered to be hormones. The usual procedure is to class those compounds which behave like indole-3-acetic acid in the standard biological tests (extension-growth of shoot cells, or pea-stem bending) as natural or synthetic auxins, and to consider all other active chemicals as planbgrowth substances. At first, the synthetic auxins included other indole caxboxylic acids, such as indole-3-propionic acid ;then, 1-naphthalene- and 2-naphthaleneacetic acid, and phenylacetic acid were added‘; later, 2-naphthy1o;xyacetic acid6 and the ring-chlorinated phenoxyacetic acid and benzoic acid series were included.’ The great (3) P. k n , Phnt Physioz., 80, 190 (1966). (4) P. W. Zimmennan, A. E. Hitchcock, and F. Wilooxon, Contrib. Boyes Thompson Inel., 8, 106 (1936). (6) V. C. Irvihe, Uniu. Cob. Stwlice, 1, 69 (1938). (6) P. W. Zimmerman and A. E. Hitchcock, Contrh Boycs Thompson Inst., 12, 321 (1942).

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pract.ical importance, in agriculture and hort icdkirt:, of t lir :wxiii substances for the control of weeds and for the regulation of such diverse phenomena as rooting, sexdetermination, flowering, fruit set and thinning, leaf and fruit abscission, fruit ripening, tuber storage ?nd control of sprouting (bud arid aecd dormancy), relatioilship to light, avoidaiicc of heat and cold injury, and control of water trauspiration, led to the alniost exclusive study of their properties and a search for useful derivatives. Gradually, other compounds lacking auxin activity have assumed importance. One of the major properties of auxins is that they stimulate growth and synthesis at low concentrations (generally 10-lo to M ) and inhibit these (and, perhaps, other) processes at higher levels, after passing through a maximum which depends on the chemical applied and on the plant. Positive responses with plant-root tissue are obtained only at the lowest concentrations of auxin, but leaves and stems, or intact plants, tolerate higher concentrations. Plant responses to auxins are not generally specific. Thimann’ has generalized that “there is no aspect of plant growth and development in which auxin does not play an important role . ,” and he proposed that ( 1 ) in any specific process modified with an externally applied growth-substance, internal (endogenous) substances are normally interacting; and (I)the observed responses in organs or tiasues are far removed from the actual molecular processes catalyzed by each class of growth substance. The mode of action of the natural and synthetic auxins, and, indeed, of most growth-substances, is not known. As a consequence, response has been measured as an observed effect on some organ or tissue, without regard for, or a knowledge of, the mechanism responsible for the effect. Most of the time, even the concentration necessary to produce an effect is unknown, since little is known of the absorption, translocation, interaction, accumulation, and metabolic fate of the auxin, the concentration of which in the external solution may be precisely measured. It has been suggested8 that natural auxins are predisposing agents: that is, the nature of the change produced may be determined by other internal, chemical regulators, once the predisposing auxin has been synthesieed and transported to the site of action. In the past 15 years, an ever-widening array of organic chemicals having profound effects on plants has been found, mainly through empirical screening. Most of these chemicals are not active in the auxin tests, and show increased growth-inhibition with increasing concentration. Again, the agricultural and horticultural usefulness aa herbicides and for plant protection, as fungicides or bactericides, has tended to dominate sppliedresearch efforts. These chemicals will be referred to simply as plant-growth (7) K.V . Thimann, Ann. Reu. Plant Physiol., 14, 1 (1963). (8) N. P.Keffordand P.L.Goldacre, Am. J . Botany, 48, 643 (1961).

..

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nubntmwon, whntcvor tho diroatiori of the roRpotiRc!. Tha term “rcgulutor,” uiitl “ltritiauxiii” will tiol b0 wd for ~ynthr!tic:growth-suh“hort~ioti~~,JJ stancen. A number of general reference-works on plant-growth substances have been consulted.+14 In addition, sections from the 31x4 4th, and 5th International Conferenceson Plant Growth Regulation have been useful, mainly in relation to work with natural and synthetic auxins.1+17 Carbohydrate analyses have been carried out by a variety of standard methods. Soluble substances have usually been extracted from finely divided tissue with cold water, buffer solutions, or boiling 80% ethanol. The aqueous solutions are often treated with lead acetate 01: anion-exchange resins, to remove non-carbohydrate, reducing substances and plant substances which react with carbohydrates. Analytical methods may be found in Paech and Traceyl*and other standard reference works. Starch has been isolated from the materials insolublein ethanol, and determined as D-glucose. Polysaccharides-the “cell-wall substances”-are isolated as a series of heterogeneous fractions by extraation with hot water, dilute acid, and alkali. They are characterized by uronic acid content, furfural formation from pentosans, and reducing substances liberated after hydrolysis of various fraction~.’~J~ The final residue is presumed to be cellulose and is determined by difference. Much of the recent work, and nearly all of the carbon-14 labeled assays, has employed chromatographic or ion-exchange elution techniques. (9) L. J. AuduB, “Plant Growth Substances,” Interscience Publishers, Inc., New York, N. Y.,and Leonard Hill, Ltd.,London, 2nd Edition, 1969. (10) “Encyclopedia of Plant Physiology,” W. Ruhland, ed., “Formation, Storage,

Mobilisation, and Transformation of Carbohydrates,” Springer-Verlag, Berlin, GBttingen, and Heidelberg, 1968, Vol. 6. (11) “Encyclopedia of Plant Phyaiology,” W. Ruhland, ed., “Growth and Growth Substances,” Springer-Verlag, Berlin, GBttingen, and Heidelberg, 1961, Vol. 14. (12) “The Physiology and Biochemistry of Herbicides,” L. J. Audus, ed., Academic Press Inc., New York, N. Y., 1984. (13) A. S. Crafts, “The Chemistry and Mode of Action of Herbicidea,” Interscience Publishers, Inc , New York, N. Y.,1981. (14)’ P. EI. Pilet, “Les Phytohormones de Croieaance,” Maseon et Cie., Paris, 1961. (16) “The Chemistry and Mode of Action of Plant Growth Substances,” R. L. Wain and F. Wightman, eds., Academic Press Inc., New York, N. Y., and Butterworthe Scientific Publications, London, 1966. (16) “Plant Growth bguletion,” R.M. Klein, ed., Boyce Thompson Institute, Yonkers, N. Y.,1969, Iowa State Univ. Press, Amea, Iowa,1961. (17) “Regulateurs Natureh de la C r o h n c e Vegetele,” J. P.Nitsoh, ed.,Gif-sur-Yvette, France, 1983; Centre National de la Recherche Scientifique, Paris, 1904. (18) “Modern Methods of Plant Analysis,” K. Paech and M.V. Traoey, ed., SpringerVerlag, Berlin, GBttingen, and Heidelberg, 1986, Vol. 2. (10) J. F. Bonner, “Plant Biochemistry," Academic Press Ino., New York, N. Y., 1960, p. 71.

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381

Thc niajor pointh to hc einpbzwized licrc are tlxit tlic quuitit:itiw dTw*tx of growth wuhstancw are relative and are ronipared to uatmbkd mntmls. The effcctw arc oftcti highly dapendent 011 tho experinioiital conditions, and aro gcticraliy rcwcrsiblo uiilom thc! cffcct in 190 noverc or prolorlgcd a# to (:awe death of tho plant. Much of tho work in this field is open to criticiem, because conclusions have often been drawn from a single, often excessive, upplication of a chemical; such an application is likely to produce abnormal, disordered, and perhaps herbicidal effects. Controlled release of optimal quantities to bring about slow orderly changes, even if the changes ultimately result in herbicidal injury, is more difficylt to achieve but more promising of meaningful research results.

11. INDOLE-3-ACETIC ACID AND 1-NAPYTHALENEACETIC ACID 1. Effects on the Cell-wall Polysaccharides A t a range of concentration in external solutions (generally 10-8 to 10-4 M , where the auxins stimulate plant-tissue growth by cell enlargement), the initial effect is apparent in the increased uptake of water and in the increased plasticity of the cell walls. The synthetic auxin has the same superficial effect as the natural one, but the former is more stable to oxidation and the effects are of longer duration. The auxins bring about an increase in the rate of plant-cell elongation; an accessory solute, usually D-glucose or sucrose, is required in order to maintain the growth rate by a proportional increase in cell-wall synthesis. The elongation will occur with a solute such as mannitol to maintain osmotic concentration, but the increase in cell substances is a secondary effect, dependent on carbohydrate concentration, K@, and nitrogen, and may or may not be auxinregulated.** HeynZ4is given credit for demonstrating that indole-3-acetic acid increases the cell-wall plasticity. It was later suggestedlap2ethat the changes might occur as a result of the auxin-induced, longitudinal swelling of the intermicellar substances, especially of the pectin-like polysaccharides. Cellulose was presumed to provide tangential strengthening, without being otherwise affected. No change was found in the hemicellulose fraction. Morerecent work has implicated lignin, but its role in young tissue has (20) J. Bonner, in “Plant Growth Regulation,” R. M. Klein, ed., Iowg State Univ. Press, Ames, Iowa,1961, p. 307. (21) H. Buratdm, in “Encyclopedia of Plant Physiology,” W. Ruhland, ed., SpringerVerlag, Berlin, 1961, Vol. 14, p. 285. (‘22) J. P. Nitsch rpd C. Nitsch, Am. J . Botany, 4, 839 (1956). (23) I. Ilan and L. Reihold, Nature, 801, 726 (1964). (24) A. N. J. Heyn, Rec. Trav. Botan. Neerl., 28, 113 (1931). (25) U. Ruge, Biochsrn. Z., 296, 29 (1937). (26) J. van Overbeek, Botan. Rev., 6, 665 (1939).

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generally been overlooked.n Support for softening of pectin came from tho fact that Ca*@decreases the deformability, whereas K@does not. The exchange capacity of the tiasue for CaS could be accounted for aa free, pectic, carboxyl groups.m The extensive examination of the auxin-induced changes in the cell-wdl cwbohydratcs, w p i d l y of tho polyg~ym~idumnir acids, has been reviewed niany tinlcs ::*-a4 the IYWILIM~ whi& follow will sumniarize only the nlore recent findings. The early hypot,hew! have been modified many times, and the pectin-methylation hypothesis as an explanation of wall plasticity has been abandoned by at least one of its early proponents.a1 The “cell wall” in higher plants is an exceedingly complex and highly organized structure composed of glucans, pentosans, polyglycosiduronic acids, lignin, fats, waxes, and inorganic ions.**Plant tissue for analysis is generally divided into a water-soluble fraction and into “cutin,” wax,ash, cellulose, lignin, hemicellulose, and pectin fractions. Other terms, such as “protopectin,” “polyuronidehemicelluloseJJJ and “water-soluble polyuronide,” reveal the lack of homogeneity of the fractions, and the lack of specificity of the extraction procedures. Starch is often ignored entirely, unless, as in the potato, it accounts for 7040% of the material insoluble in cold water (dry weight). Two procedures are common: that of Bonner,lg and extraction with 80% boiling ethanol. Bonner and his group extract the plant tissue with ice-cold, acetate buffer, and then fractionate the insoluble portion with hot water (“cIwica1 pectin”), cold alkali (protopectin and hemicellulose, with lignin, if any), and hot, dilute acid (additional hemicellulose) ; cellulose is the residue, and constitutes 25 to 40% of the cell wall (dry weight). There is no strict agreement concerning the fractional separation, and ClelandWhas observed that the hot, dilute-acid extract of the cell wall has been called “pectins,” “protopectins,” and “hemicelluloses’Jin different references. Starch is also extracted by acids. Since auxin-treated fractions are compared to untreated controls, the fractionation method employed should be less vital than its exact repetition (27) A, W. Gelston and W. K. Purves, Ann. Rev. Plant Physiol., 11, 239 (1960). (28) T. A. Bennet-Clerk, in “The Chemistry and Mode of Action of Plant Growth Substances,” R. L. Wain and F. Wightman, eds., Acedemic Press Inc., New York, N. Y., and Ruttarworths Scientific Publications, Lolidon, 1956,’p. 284. (29) R.Pohl, in “Encyclopedia of Plant Physiology,” W. Ruhland, ed., Springer-Verlag, Berlin, 1961, Vol. 14, p. 703. (30) R. E. Cleland, in “Encyclopedia of Plant Physiology,” W. Ruhland, ed , SpringerVerlag, Berlin, 1961, Vol. 14, p. 754. (31) See T. A. Bennet-Clark, discussion in “Plant Growth Regulation,” R. M. Klein, ed., Iowa State Univ., Ames, Iowa, 1961, p. 328. (32) A. Frey-Wyasling. “Macromolecules in Cell Structure,” Harvard Univ. Pwm, Cambridge, Maea., 1967.

PLINT-GROWTH SUBSTANCES

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for each sample. I'nfortunatcly. little or nothing is known of the effwt of the treatment on the chain length of the polymers, hr:biiching, inolcwltbr structure and configurtition of the components, niicelle organization or orientation, or arrangement or dcgrcc of the bonding, d l of which might influencd the solubility. No change has been noted in the rate of synthesis of cellulose during auxin-induced growth with indole-3-acetic acid or l-naphthaleneacetic acid (compared to the controls), except at severely inhibiting concentrat~ons.s~83-~ The orientation of the microfibrils of oat coleoptiles under stress wm decreased considerably by treatment with indole-3-acetic acid, and this was presumed to be the result of changas in the pectin "glue" through which the microfibrils were forced to move.2oAttempts to measure the rate of deposition of carbon-14 in cellulose have shown an apparent increase from acetate-14C and lo-' M indole-3-acetic acid in wheat-root celIs,a6 but not in oat-stem segments with labeled acetate, sucro~e,~7 glucose,@and galactose.sgGluc0se-6-~~C labeled the cell-wall fractions rapidly and, in time, entered the more insoluble portions; that is, the turnover of labeled carbon was most rapid in the water-soluble materials and then in the acid-soluble materials. Carbon-14 finally entered the cellulose, which showed no turnover. Indole-&acetic acid increased the rate of turnover in the hexoses of the fractions soluble in water and in 0.05 N hydrochloric acid, but there was no change in the turnover in the alkali-soluble fractions or in the cellulose.'0 The cuticular fraction ( 10-1270) of the cell walls has not received much attention, nor has the part (55-70%) which is usually called hemicellulose. Carlier and Buffel" found nearly 50% more pentosan (measured as furfural) in potato tuber treated with l-naphthaleneacetic acid at 10 mg. per liter in agar; pectin increased in almost the same proportion, and in proportion to the increased intake of water; starch decreased by 36%, apparently to provide the substrate for increased respiration. Instances of parallel responses in growth and synthesis have been found,^^^ but other studies have shown no synthesis response.*J3 Indole%acetic acid increased the incorporation of acetate-W and sucrose-14C in the concentrated, alkali-soluble portion of oat coleoptiles,a but this result could not be confirmed with labeled glucosea or galactose.8eNo increased (33) H. Burstrom, Kgl. Fysiograf. Sdlskap. Lund, F&h., 28, 53 (1958). (34) A. Cerlier and K. Buffel, A& Bohn. Neerl., 4, 661 (1955). (35) 8.T. Bayley and G . Setterfield, Ann. Botany (London), 21, 633 (1957). (30) I. B. Perlix and J. F. Nance, Plant Physwl., 31, 451 (1956). (37) H. Boroughs and J. Bonner, Arch. Biochem. Biophys., 46, 279 (1953). (38) L. Ordin, R. Cleland, and J. Bonner, Proc. NaU. A d . Sci. U.S., 41, 1023 (1955). (30) L. Ordin and J. Bonner, Plant Physiol., 32, 212 (1957). (40) L. Ordm and M. Katr, Plant Phyeiol, 39, Suppl., iii (1964).

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incorporutioii of carbon-14 from 1iLt)olod motatc, su(mo~c,glucom, or galactose WBR found in the fractions soluble in hot water, dilute acid, or dilute alkali. Nance“ found that indole-3-acetic acid inhibits acetate-l*C incorporation into pea-stem pectin , “polyuronide hemicelldose,” lipides, and water-soluble material, and stimulatesorganic acids (especially succinic acid) and evolution of carbon dioxide; this is evidence of a shift in tho pathways of acetate utilization. Pretreatment of the tissue with Ca- and K@accentuated the effects, illustrating the difficulty of interpreting data from various sources if the experimental details differ slightly. The “polyuronide hemicellulose” was the fraction soluble in 4% sodium hydroxide, obtained from the residue after extraction with boiling ethanol, water, and hot 0.05 N hydrochloric acid. Pectin was isolated from the acid extract by precipitation with ethanol. Jansen and coworkers42 showed that the hemicellulose fraction soluble in cold alkali (after pectin removal) is composed of glucose, galactose, xylose, arabinose, perhaps ribose, galacturonic acid, and several ultraviolebabsorbing and fluorescing constituents. The whole cell-wall fraction contained about 5% of galacturonic acid residues, measured by evolution of carbon dioxide and by the pectinase-carbazole colorimetric method.” About 5% of the total of this uronic acid constituent was found, by alcohol precipitation, in the fraction soluble in cold water and “not associated with the cell wall” and about 78% of the “cell wall” uronic acid material could not be removed as pectin soluble in hot water. Thus, the “classical pectin” fraction contained only 20-25% of the total uronic acid material in 3.5% of the total dry-weight of the wall material. This fraction was highly esterified, averaging 90% esterification,whereas the portion soluble in cold water averaged about SO%, and the substances insoluble in hot water, about 31% in degree of esterification. Xylose and arabinose were the major remakng constituents of the hot-water extract. Most of the attention has centered on the auxin-induced changes in the methyl esterification of the carboxyl groups of the uronic acid residues. I t had been theorized that increased methyl esterification would account for the increased wall-plasticity, in contrast to the stiffening effect of hydrogen bodding of the free acid groups and especially of the cross-linking of divalent calcium and magnesium ions. The WHI from Gmethionine was more rapidly incorporated as a methyl substituent,@l‘ but it has not been poqsibla@to show a corresponding decrease or turnover of W a ; moreover, the net ester content did not change, nor waa the distribution of methyl (41) J. F. Nance, Plant Phyeiol., 83, 93 (1958); 84, Suppl., iii (1984).

(42)E. F. Jamen, R. J a g , P. Albenheim, and J. Bonner, Plant PhyaioZ., 85,87 (1960). (43) R. M. McCready and E. A. McComb, A d . C h . ,B4, 1630, 1986 (1952). (44) L. Ordin, R. Cleland, and J. Bonner, Plant PhpabZ., SO, 216 (1957). (45) R. Cleland, Plant PhyuioZ., 85, 681 (1980).

PLANT-OROWTH SUBSTANCES

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group altered among forms of different solubility.’* Jansen and coworkers r.nnclucicxl that inddle3-acetic acid influences only the rate of formation (metabolic turnover) of tho pectin methyl ester groups, increasing it to the extcnt of up to 70%. The greatest effect was on the fraction soluble in cold water. DLEthionhe at 0.05 M blocked the auxin-sensitive, methyl ester transfer, yet did not interfere with induced elongationa; thus, pectin methyl esterificationand cell elongation were independent. Clelanda further explained the apparent anomaly between studies of response to auxin and those showing no change, finding that the auxin-induced synthesis of pectin depends on the supply of soluble carbohydrate to the tissues: 2% of sucrose increased the uronic acid content 20% over the controls not containing auxin. In previous work?71~-~2 where no synthesis of polyglycosiduronic acid was found, or where the incorporation of labeled substrates was low or absent, the levels of available carbohydrate were inadequate; where net synthesis of polyglycosiduro~cacid occurred, adequate carbohydrate was ~ u p p l i e d . * ~The ~ * ~auxin-induced ~~7 uptake of Ke and inhibition of NHP has also been found to be inversely dependent on the presence of sucrose.2a Mannitol has often been used as an osmotic regulator in the external solutions, and has been presumed to be inert. It was found to be a respiratory substrate in 15 of 26 species representing 17 families of higher plants, some of which were capable of utilization of mannitol that was equal to that of D-glucose and D-fructose. Oat ( A v m sativa), most often used for the cell-wall studies, showed only a slight output of carbon dioxide from labeled mannitol. About 10% of the carbon in the mannitol was converted, with time, into the hemicellulose and cellulose fractions. Only the glucose, and, perhaps, the cellobiose, was 1abeled.a No firm conclusions are yet possible as to whether auxins bring about direct incorporation of new cell-wall substances or whether this occurs merely as a result of the auxin-induced cell-expansion. Efforts are being made to clarify the relationships of auxins with metal ions present in plant sy~tems.*’J-~~ This topic is irrelevant here, except for noting that it is considered that the auxins are transported to centers of high metabolic activity where they may act as mobilization centers for the metallic ions. Whether or not this view will hold is still not clear; however, it has been founda that carbohydrates and nitrogeneous substances are strongly attracted to these centers of induced metabolism. R. Clelend, Plant Physiol., 88, 12 (1963). P. Albersheim and J. Bonner, J . Biol. Chem., 284, 3105 (1959). P. Trip, G. Krotkov, and C. D. Nelson, Am. J . Botany, 61, 828 (1964). J. W. Mitchell, BuU. Tmey Botan. Club,88, 299 (1961). H.Buratdm, ‘Advan.Botan. Res., 1, 73 (1963). K. V. Thimann and N. Takahashi, in “Plant Growth Regulation,” R. M. Klein, cd., Iowa State Univ. Press, Am-, Iowa, 1961, p. 383. (52) N. W.Stuart, Bohn. ohr., 100, 298 (1938). (46) (47) (48) (49) (50) (51)

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2. Effect@on Starch and on Soluble Carbohydrates

As noted in the previous Section, one general effect of auxins on higher plants is the hydrolysis of carbohydrate reserves, particularly of starch. Starch conversion takes place principally in the leaves; the extent to which it occurs in other storage organs (such aa the stem, tubers, seeds, fruit, or roots) depends on the environmental conditions. The relationship between plant-storage reserve8 and the component monosaccharides has been the subject of considerable applied research with such storage plants as potatoes, sugarbeets, corn, sugarcane (see Section VI), and bananas and other fruits (see Section VII). No consistent, direct mechanism for the hydrolytic activity has been shown. Among those mechanisms which have been advanced, the most attention has been focused on increased enzyme activity of amylase or phosphorylase, osmotic regulation due to increased uptake of water, metabolic pathways altered to produce increased amounts of organic acids, amino acids, or proteins, increased translocation from areas of storage to areaa of expansion, increased photosynthesis, or combinations of these. The auxin-induced depletion of starch reserves in tomato leaves was first noted by Borthwick and coworkers,0*and has been confirmed with many other plants.l* The increase in soluble carbohydrate is only temporary; depletion takes place with time, especially at higher auxin concentrations.Mv66 Fructose is decreased ; and sucrose has shown incrcases, decreases, and no significant change, depending on the plant and the experimental condition^.^^ It is not easy to generalize from the available data, but it appears that plants having starch as a normal reserve show decreased polysaccharide from auxin-induced increases in respiration, with little or no change in the disaccharide content, whereas plants containing littlo or no starch utilize their normal, reserve sucrose or other substances. Attempts have been made to increw the yield of root crops by auxin dips of seed, or auxin sprays on the plant leaves. Claims have been made for 100% increases of potatoes and increased size of the starch grain,”e but these results could not be repeated on another variety”; prolonged dormancy and delayed germination were said to cause a decreased yield in the latter study. Such conflicting reports as this, complete with photomicrographs of the starch grains, emphasize how little is actually known of t’he rrgulatory merhanism, or of tho amount3of auxin prcRent in the (53) (54) (55) (56) (57)

H. A. Borthwick, K. C. Hamner. and M. W. Parker, Botan. Gaz., 98, 491 (1937). D. J. Wort, Plant Phyeiol., 96, 60 (1951). F. G. Smith, C. 11. Hsmner, and A. F. Carlmn, Plant P h p o l . , 14, 68 (1947). M. Ziks, Planla, SO, 151 (1939). R. T Whittanbergor and G. C. Nutting, Pliant Phyeiol., 94, 278 (1949).

PLAST-GROWTII SUBSTANCES

387

tissue. The considerable body of literature on sweet potatoes, sugarbeets, and sugarcane cjhowr;lmany such conflicts. Sections of non-photosynthetic oabtimue fed with radioactive substrates (acetate and Hucrose) incorporated the label into the soluble carbohydrates and lipiden, with no apparent differences between auxin-treated segments and untreated contro1s.n The tissue waa apparently grown with an inadequate substrate of carbohydrate, which may explain why indole-3-acetic acid at 1 mg. per liter increamd the utilization of sucrose, lipides, and organic acids by pea-stem segments in other work.6*None of the ayailable investigations on cellular carbohydrates have provided very much promising information concerning the nature of the metabolic functions directly affected by the auxins. 3. Auxin Activity, Respiration, and Metabolism

Leaves kept in the dark lose their response to auxin at a rate parallel to the loss of carbohydrate used in respiration.'lgSuch metabolic inhibitors as cyanide (which represses both growth and respiration) and iodoacetate (which represses growth without affecting respiration) suppress the auxininduced growth-response60"l; auxins generally enhance the respiration of intact plants, but may act as inhibitors to isolated tissue or enzyme systems. Most of the evidence suggests that auxins act on, or through, the plant enzyme-systems in the living plant. It has often been shown that enzyme systems are affected; an example is the increased activity of amylase, leading to starch degradation (mentioned in the previous Section). However, the cffects'are indirect, as the auxins have no effect on purified amylase The auxin-induced activity of many incubated with pure starch in vif~0.l~ isolated cnzyme-preparations has rarely had any relationship to the effects observed in intact plants.me62There has been no evidence of direct chemical or physical attachment of the auxins, either to the cell wall or to individual enzymes.6a Evidence has been acquired of complexes of indole-3-acetic acid with pyridine n~c!leotides6~; these complexes inhibited certain enzymes, such as malic dehydrogenasc, requiring the nucleotides. There is also some (58) G. S. Christianson and K. V. Thimann, Arch. Biochenz., 20, 230 (1950). (59) F. G. Gregory and B. Samantarai, J. Expfl. Boiany, 1, 159 (1950). (60)J. Bonner, J . Gen.,Physiol., 17, 63 (1933). (61) B. Commoner and K. V. Thimann, J. Gen.Physiol., 24, 279 (1941). (62) W. B. Neely, C. D. Ball, C. L. Hamner, and H.M. Sell, Plant Physiol., 26, 525 (1950).

(63) A. W. Galmn and R. Kaur, in "Plant Growth Regulation," R. M. Klein, ed., Iowa State Univ. Press, Ames, Iowa, 1961, p. 355. (64) Reviewed hy R. T. Wedding and M. K. Black, Plant Physiol., 99, 799 (1964).

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evidence that protein, nucleic acids, and ribonuclease are increased in quantity,”6e66 probably at the expense of same of the carbohydrate. Auxins induce growth, but only under conditions of active, oxidative metabolism. The role of Carbohydrate, necessary for the transport of the auxin, appears to be metabolic, either as a growth substrate or for energy. Light and cwbon dioxide are also noaesssnry for the auxin trunsport.a’ Auxinic growth is stimulated by metabolic aarbohydrattos, adenosine monophosphate, arginine, methionine, L-ascorbic acid, and other organic acids. Reviews of the metabolism and respiratory effects of, and enzyme activation and deactivation by, natural and synthetic auxins have been published~*”Jg; these cover the literature to about 1962. It seems superfluous to repeat much of this work here. What follows is a brief discussion of the methods used to study auxinic metabolism and respiration changes, and of the effects of such changes. With sufficient substrate, auxin-treated plants show an initial increase in oxygen uptakc, with an increase in metabolism, output of respiratory carbon dioxide, and enzyme activities. The content of dry matter often decreases, with a trend for hydrolysis of storage reserves to simpler forms. The stimulatory effects may be short-lived, however, and enzyme activity often falls to normal or even to less than the levels in the control. Some of the major effects are on amylase and phosphorylase systems, as might be expected, although many enzymes are affected. At herbicidal levels, especially of some synthetic auxins, increases in respiration of 50 to 100% have been reported, without increased dry weight, although synthesis of protein and nucleic acid continued to increase. At higher concentrations, oxygen uptake may be depressed, in mitochondria a t least, and the enzymes of phosphorylation may be more involved than those of oxidation. The effects reported are similar to those of the metabolic poisons 2,4dinitrophenol and 2,4dichlorophenol, which permit carbohydrate metabolism without utilization of inorganic phosphate?O Transpiration waa diminished by closing of the stomata?‘ The respiratory quotient (RQ) (of evolution of carbon dioxide to uptake of oxygen) has been found to be near 1.0 for plant tissues and for metabolizcd carbohydrates. FatH cvolvc less carbon dioxide and have an RQ (66) J. Silberger and F. Skoog, Scisncc, 118, 434 (1953). (66) J. C . Shannon, J. R.Haneon, and C. M. Wilson,Planl Phyniol., 99, 804 (1964). (67) J. R. Hiry and K . V. Thimcmn, Plant Phydal., 91, 446 (1956). ((is) n.J . Wort, in “l!hyrlopcdiib of Plrint PhyNioiogy,” W. Ruhlrmi, ed., SpringerVerlug, Bclrlin, 1061, Vol. 14, p. 1110. (69) D. J. Wort, in “The Physiology and Biochemistry of Herbicides,” L. J. Audus, ed., Academic Press Inc., London, and New York, N. Y.,1964, p 291. (70) C. M. Switzer, Plant Physiol., 93, 42 (1957). (71) D. Bradbury and W. B. Enriis, Am. J . Bolany, 99, 324 (1952).

PLANT-QROWTH BUBBTANCES

389

of less than unity, whereas organic acids have an RQ of greater t’hanunity. Auxin treatments of many plants have shifted the RQ slightly in both directions, but it is doubtful that the shifts reflect iniportant changes in metabolism. The energy required for cell expansion would presumably be derived from the catabolism of D-glucose to pyruvate. Two pathways are possible. In the Embden-Meyerhof-Parnas or “glycolytic” pathway, g l u c o ~ e I - ~ ~ C and gluc0se-6-~~Cgive triose phosphate only, which leads to pyruvate, and, finally to W02,labeled equally from either substrate. For each mole of triose phosphate produced, the pentose phosphate pathway leads to the reduction of 6 moles of adenine nicotinamide dinucleotide phosphate, release of 3 moles of 14C02from C-1, and regeneration of 1 mole of hexose. The two pathways are interconnected, with some enzymes common to both. The pentose phosphate pathway yields more energy, and it has been theorized that auxin might shift the C-6/C-1 ratio of labeled carbon dioxide evolved to a lower value as the result of greater utilization of glu~ose-l-~~C. Gibbs and coworkers have discussed the general principles and techniques.72-75 Labeled glucose fed to oat, pea, and corn tissue which had been treated with indole-3-acetic acid or (2,4-dichlorophenoxy)acetic acid resulted in an M unchanged C-6/C-1 ratio, except at inhibitory concentrations of auxin. The latter concentration lowered the ratio.’2 The increased participation of the pentose phosphate pathway at inhibitory levels of auxin was ~ o n f i r r n e d ~glucose ~ , ~ ; uptake by corn roots or peas was decreased by 50% and output of carbon dioxide increased 100%. Bourke and coworkers~J9 do not agree that the pentose pathway is affected, and argue that both pathways are inhibited at concentrations greater than M , with the C-6/C-1 ratio decreasing with increased concentration because of a greater inhibition of the glycolytic pathway. The ratios had no correlation with the herbicidal activity of several phytotoxic and nonphytotoxic ring-chlorinated phcnoxyacetic acids, indole-3-acetic acid, or 1- or 2-naphthaleneacetic acids. Only gibberellic acid did not inhibit uptake of glucose. The lack of agreement is difficult to resolve from a study of the available literature, especially aince an increase in the C-6/C-1 ratio with 1-naphthaleneacetic acid has (72) M.Gibbs, Ann. Rev.Plant Phyaiol., 10, 329 (1959). (75) M.cihbn and H. BwvcrA, Plant Phyaiol., 30, 343 (1955). ~

(74) T.Ap IIWX !t11(1 H. k W ( ! P H , I’lanl Ph~aiol.,36, 870, 830 (1960). 30, p. 7(14, rrfotw inrorrwtly to the “CI/C6nttio.” (75) It~!lt~t-(!nc.c~ (7(i) T. E. Huinphrey~and W. M . Ihgger, Jr., Plant P h ~ e i d .32, , 136, 530 (1957); 34, 112, 580 (1959). (77) C. C. Black, Jr., and T. E. Humphreys, Plant Phy&Z., 87, 66 (1962). (78) J. B. Bourke, J. 5. Butts, and S. C. Fang, Plan6 Physiol., 37, 233 (1982). (79) J. B. Bourke, J. S. Butts, and S. C. Fang, Weeds, 14, 272 (1964).

390

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also been reported with bean-stem tissue." Black and Humphreys found increased activity from (2,Pdichlorophenoxy)aceticacid for several enzymes of the pentose phosphate pathway , including glucose-&phosphate dehydrogenase and Bphosphogluconate dehydmgenase. Scott and coworkersS1 could not confirm this result with indole-3-acetic acid in tobacco tissuecultures. In particular, with increasing auxin, glucose-&phosphate dehydrogenase decreased to undetectable levels, and the &phosphogluconate dehydrogenase was also diminished. The plant species, auxin species and concentration, and time period were different. Since the same effects occurred with the non-herbicidal (2,6dichlorophenoxy) acetic acid as with toxic compounds, glucose metabolic changes are not the important singular effects of auxin herbicides. Indole-3-acetic acid is rather readily oxidized by peroxidases and is, in fact, probably not present in the plant in the free form to any appreciable extent. The nature of the complexing groups is not clear. The inherent instability of the compound in living tissue has made experimental observations difficult, and (the more stable) 1-naphthaleneacetic acid has often been used instead, although it is by no means certain that the biological activities are comparable. One view held is that auxin herbicides are effective either because they do not readily form conjugate systems, or because the conjugate retains the phytotoxic properties. 1-0-(Indole-3-acetyl)-&D-glucopyranose (2) ww identified from its

spectra and from the hydrolytic fragments resulting from the action of ,B-glucosidase, although it was not obtained crystalline>+" It was biologicdly active. Two major, metabolio conjugates were detected, the second apparently being the aspartate. A second glucose ester, possibly the 6-esterJ was not split by the enzyme, and there was also evidence for the presence (SO) C. A. Carlier and C. Van Horne, Nature, 901, 677 (1964). (81) K.J. Scott, J. Daly, and H. H. Smith, Plant PhysioZ., 89, 709 (1964). (82) H. D. Kllmbt, Pkanta, 66, 618 (1981); 67, 339, 391 (1961). (83) M. H. Zenk, Nature, lftl, 493 (1961). (84)M. H. Zenk, Plan&, 66,688 (1902).

PLANT-GHOWTH SUBSTASCES

391

of the &wglucoside of 2-hydroxyindole-3-aceticacid, which was biologically inactive.*W Animals excrete the indole auxin as the l-ter of BDglucuronic acid.” Zenk has suggestedeethat the natural growth-regulator indole3-acetic acid forms stable compounds with =glucose or Gaspartic acid, competitively, depending on the plant species. The glucose conjugate was believed to be the more primitive, as it appears in all plants, including bacteria and fungi. Aspartate competition occurs in higher plants only: 33 of 38 monocotyledonous and 72 of 75 dicotyledonous species form the aspartate, compared to 25 and 48 which form the glucoae ester, respectively. The glucose ester is labile to ammonium ion, ethanol, or amino acids, but Zenk has presented evidence that the glucoside is not an intermediate in aspartate formation. The glucoside is formed rapidly, reaching a maximum in 4-8 hours after the addition of label@ auxin, followed by slow hydrolysis, whereas the formation of aspartate occurs very slowly, but continually, after 6-8 hours. Free, unbound auxin remained at a more-or-less constant, low level of 0.04 pM per g. in St. John’s wort (Hyperium hircinum) leaf. Since there are plants which form only the aspartate or the glucoside, “it is unlikely that either conjugate is an essential part of the growth induction mechanism. We therefore assume that both conjugates are true detoxication products.”8oAlthough the auxin was added at 5 X 1W6M , and was therefore present in considerable excess in the external medium, i t seems curious that the natural, planegrowth regulator requires a “detoxication mechanism,” unless this conjugate formation is a means of regulating the amount of the free, more bioactive, acid. 1-0-(Indole-3-propionyl)-B.~-glucosewas found , by similar means, in incubation cultures of BaciUus megatheriumsO and 1-0-(indolp3-acetyl) arabinose was, from the results of paper-chromatographic separation, believed present in hydrolyzed, immature corn kernels?’ The presence of carbohydrate esters in plants is not unusual; there is good evidence for 1-estem of glucose and gentiobiose, even of esters of phenolic acids having free hydroxyl groups, such as caffeic, ferulic, and pcoumaric acid.@* >Ascorbic acid, considered to be a growth regulator (although whether (86) H.I). Klilmbt, Nalu~un’s8en8chafkn,46, 649 (1959). (86) H.I). Kliirgbt, I:lanb, 66, 309 (1961). (87) H.1). KlfiniI)t, in “Rrguhteim Nutiirrls de In CroiRRltnce Vegetale,” J. P. Nitsch, rd., C o i i l ~ Nirt ~ ion:rl tlr IHi t d i c w h c Sririitifiqur, PILI-~R, 1964, p. 235. (88)J. 13. Jtppwoii, Hiurhsnr. J . , 69, 82 (1958). (89) M. H. Zenk, in “Iiegulateura Naturds de la Croisaance Vegetale,” J. P. Nitsch, ed., Centre National de la Recherche Scientifique, Paris, 1964, p. 241. (90)J. Tabone and D. Tabone, Cmipl. Rend., 297, 943 (1953). (91) E. M. Shsntz arid F. C. Steward, Plunf Physiol., S2, Suppl. viii (1957). (92) I,. Birkofer, C. Kaiser, and H. Kosmol, Nalum*ssmcha&n, 47, 409 (1960).

392

H. W. HILTON

it is inhibiting or promoting depends on the source of information), forms, in Brassica species, an inactive compound with indol&acetic acid. The Czech literature on this compound, known aa aacorbigen, has been r e viewed by Bentley.”~~‘ The goitrogenic “bramica factors” from various species of cabbrrgo (Bramica) seem to inhibit uptake of iodine by the thymid. The inhibition has been attributed to SCNe, which appears as one of the hydrolysis products of mustard oil glycosidea, such m glucobrassicin (3).

(3 1

5( 8-p.Glucopyranosyl)-3-indolylthioacetylhydroximyl0-sulfate Compound (3) was obtained as a crystalline salt, in yields of up to 3% (based on the dry weight of cabbage). The N-methylindole analog, neoglucobrassicin, from the same species, has similir properties!6-@‘ 1-Naphthaleneacetic acid-I4C formed an emulsin-hydrolyzed D-glucose 1-ester in wheat, together with a Pglucoside of 8-hydroxy-1-naphthaleneacetic acid.82-m** Neither waa crystallired. 2-Naphthyloxyacetic acid formed both the 6- and 8-hydroxylated glucosides. The naphthalene auxins show considerably more stability and are converted less rapidly into the COIG jugate form. 111. PLANT-GROWTH SUBSTANCES USEDAS HERBICIDE~S

1. (2,4=Dichforophenoxy)acetioAcid and Related Compounds (2 ,.Q-Dichlorophenoxy)acetic acid, together with the 2-methyl4chloro and the 2,4, &trichloro analogs, 2 4 2,4,5-trichlorophenoxy)propionic acid , and many other related compounds, constitute one of the major classes of herbicides, selective primarily to dicotyledonous plants. Monocotyledons, (93)J. A. Bentley, Ann. Rsu. Pkrnt Phu&ol., 8, 47 (1968). (94) J. A. Bentley, in “Enoyclopedia of Plant Physiology,” W.Ruhland, ed., SpringerVerlsg, Berlin, 1961, Vol. 14, p. f309. (96) R. Gmelin and A. I. Virtannen, Ada Chem. Soond.,14, 607 (1960); 18,1378 (1962). (96) R. Omelin and A. I. Virtannen, Ann. Amd. 815.F m n h , 8er. A . IZ, lW,3 (1961). (97) A. I. V i n n e n , Pkyf0ch3mh3CryJ4, 207 (1966). (98) M. H. Zenk, Plank, MI, 76 (1962).

PLANTGROWTH SUBSTANCES

393

\KY‘:\UW of poor. absorption by the leaf and poor trsllsiocation within the plant. Abnornlal stem-twisting, leaf fusion and curling (epinasty), and rooting at nodes and leaf sxils are characteristic of these auxinic herbicides. The principal differences from the auxins previously discussed are the persistence and the high degree of phytotoxic effect of the phenoxy compounds. The various compounds are similar in their responses to the biological-growth tests; for each compound, the herbicidal properties appear at a critical concentration for the individual species and stage of growth. In common with the natural (and less phytotoxic) auxins, (2,4dichlorophenoxy) acetic acid in low concentrations decreases the reserve carbohydrate, temporarily increases the reducing substances, and increases the content of protein and amino acid, especially of the roots and stems. Although the exhaustive synthesis of nucleic acids and protein was suggested as a mechanism for herbicidal activity,a the depletion of reserves is not a sin,ple starvation process. For example, (2,4-dichlorophenoxy)acetic acid at a toxic concentration of lo00 mg./l. depletes sucrose only in the leaves and roots of kidney bean in 6 days, without altering the reducing substances, starch, acid-hydrolyzable polysaccharides, crude fiber, ash, ether extxact, uhsaponifiable materials, and fatty acids; and yet the reducing and nonreducing carbohydrates are depleted, and starch, crude fiber, and hydrolyzed polysaccharides are diminished in the stems.”Jm Soluble carbohydrate in buckwheat stems and leaves treated with 50, 100, 500, hnd lo00 mg. per liter increased the first day and decreased to 48% of that in the controls in 8 days. Starch in the stems declined immediately, increased above the controls in 12 hours, and then was depleted in 8 days at the highest concentrations. Root-starch levels fell contin~ously.~~ Starch formation wag prevented in tomato leaves, although sucrose increased continuously with low levels of (2-methyl-4-chlorophenoxy ) acetic acid.lO’ Many of the same conclusions may be drawn from results with synthetic auxins as from indole-3-acetic acid: water intake increased, there was a lessening of the downward translocation of photosynthate with temporary increases of soluble carbohydrates in the leaves, and alterations appeared in the metabolic rate and direction and in enzyme activity. However, the direct B i t e of action is not known, and many of the effects are puzzling. &Fructose oligosaccharides in artichoke and chicory storage-tissue were diminished by 70y0 (calculated on the content of dry matter) in 6 days

apwiaUy the grasses, an! not very nmvptible, ptwtly

(99) H. M. Sell, R. W. Leucke, B. M. Taylor, and C. L. Hamner, Phnt Phyehl., 24, 295 (I 949). (100) L. E. Weller, R. W. Leucke, C. L. Hamner, and H. M. Sell, Phnd Phyewl., 25, 289 (1950). (101) A. Rhociees, J . Ezptl. Botany, 2, 129 (1952).

394

H. W. HILTON

with M (2,4-dit:hlorophcnoxy):~cctiaacid, and oiily by 25% with the less toxic ( 3 , bdichlorophenoxy) acetic acid." Increases in hemicellulose, cellulom, sucrose, and total rarhohydrate, and decreases in starch arid of ( 2 , &dic*hlor.ophmrcducing subrstaticw were notd :It nd)lctohttlIcvc~l~ cwd xoybssii"M Iccwcw. Tho diniiiiut8ioiii i i tho oxy)ncetic*wid iii c*otloii*o" ratmeof downward transport of photosyiithete is put icwltrrly soveie with the herbicidal auxins, arid there is interference with photmynthwis.lMThe ripening of detached bananas by (2,4dichlorophenoxy)acetic acid and similar compounds by the more rapid conversion of starch into soluble carbohydrate could only have come about through a direct or indirect effect on the enzyme systems.IwThe ripening of fruit by means of chemicals is discuseed in Section VII (see p. 429). Uptake of carbon-l'C dioxide by tomato leaves was greatly increased with (2 ,.l-dichlorophenoxy)acetic acid, although the pattern of utilization was altered from leaves to fruiLIO"This contrasts with the assumption that auxin diminishes the rate of photosynthesis.lm The incorporation of carbon from sucrosd4C into ylheat and pea-stem segment cellulose and hemicellulose was "greatly enhanced,"'" and yet the comprehensive studies by Stevens and coworkerslw and Bourke and c0workers7~with ~-glucos&~C on pea-root tissue did not confirm this work. The two latter studies, with gluco~e-l-~~C, glu~ose-&~~C, g1ucoseJ4C6, acetate-l-14C,and acetate-PC, and a group of eight chlorinated phonoxyacetic and phenoxypropionic acids at lG-*M, showed that there wm no correlation of phytotoxioity with metabolic changes. All of the compounds inhibited uptake of acetate and increased respiration (as the ratio of carbon dioxide to residue), but did not alter the.distribution of radioactivity in respiratory WOt (from C-1 or C-6 of gluhose, or C-1 or C-2 of acetate), or in the ethanol-soluble or in ethanol-insoluble fractions. Glucose uptake was inhibited by all of the compounds, with little effect on the C-6/C-1ratio at lo-" M.At lo-&M, the C-6/C-1 ratio increased; this wm interpreted as indicating a stimulation of glycolysis. At lo-* M, the C-6/C-1 ratio was lowered, probably by inhibition of glycolysis. Increased radioactivity wm found in the ethanol(102) R. L. Wain, P. P. Rutherford, E. W. Weaton, and C. M. Griffiths, Nature, 208, 504 (1904). (103) D. R. Ergle and A. A. Dunlap, T e r n Agr. Ezpt. 8tu.Bull., 718, 18 pp. (1949). (104) D. E. Wolf, G. Vermillion, A. W a k e , and G. H. Ahlgren, Botan. Um., 112, 188 (1960). (105) A. J. Loustalot and T. J. Muaik, Botan. Uar., 111, 60 (1963). (106) 8.R.Freiberg, Botan. Urn., 117, 113 (1965) (107) N. I. Yakushkina, Fiziol. R a t . , 8, 111 (1982). (108) F. Wightmah and A. C. Noiah, Proe. Iniern. Botan. Congr., 9th Montreal, 1869, p. 430. (100) V L. Stevens, J. S.Butte, and 9. C. Fang, Plan4 P h y h l . , S7, 215 (1962).

PLAXT-QBOWTH SUBSTAHCES

395

dwreafed incopration w w irotd in the itdublc, ''cell-wall" fraction. The apparent alteration, by auxin, of the carbohydrate reserves, especially cjterch, in plants has had two consequences in essentially opposite directionn. First, there have been attempts to correlate the effectiveness of herbicide treatments with the natural variation in the root reserves of various species of weed. Although there has been some evidence of succm with Canadian thistle (Cirsium arvense)', in which the root reserves (fructan) are normally lowest, and most affected by herbicide, just before flowering,'1° the results have in general been disappointing. It is more likely that effective translocation of the foliar-applied herbicide throughoat the plant and into the root system depends on the downward movement of food reserves from the leaves, which in turn is related to the synthetic activity of the plant as a whole. This possibility agrees with a number of observations that treatment with a herbicide is most effective during active, vegetgtive growth.lllJ1*There have been few attempts to determine how much of the applied herbicide enters the phloem system of the plant, or the degree of translocation to various organs of the plant in relation to the effects observed. It also seems unfortunate that some of the published work has considered only the variations in the soluble carbohydrates m a function of the treatment.111,11a,114 The total volume of the root or the root weight will also determine whether recovery will take place.116 Second, attempts have been made to increase the storage reserves both of root crops and stem crops, or to decrease the reducing carbohydrate without altering the reserve of starch or sucrose (see also, Section VI). The accumulation of soluble carbohydrates in the leaves hais been attributed to poor translocation in the stem without immediate cessation of photosynthesis,llBwhich may or may not influence the reserve. Potato plants sprayed with (2,4-dichlorophenoxy) acetic acid at low concentrations, a t eight ounces per acre in August, showed no change in tuber sucrose in October, but had an increased proportion of reducing carbohydrate, whereas a July treatment decreased the reducing carbohydrate and increased the sucrose.117Increased starch and decreased reducing sub-

?dJhltdf2 extract, aid

B. Granstdim, Kgl. Liaabnclce-hg8kOl. Ann., 11, 281 (1954). H. P. Cords and A. A. Bandiei, Weeds, 12, 299 (1964). A. F. W i w and H. E. Rea, Weeds, 10, 58 (1962). H. M.LeBaron, Univ.Mimfilma 6!2-149,213 pp.Dis~erlationAb8tr.~ 12,2542 (1982). C. G. McWhohr, Weed, 9, 563 (1961). D. L. Linscott and M. K. McCarty, Weeds, 10, 298 (1962) L. G. Ganyushkina, uch. z a p . Karehk. Ped. Inat., 6, 135 (1958); Chem. Absh.acte, 68, 20307 (1959). (117) M. G. Payne and J. L. Fults, Am. Po@to J., 81, 144 (1955). (110) (111) (112) (113) (114) (115) (116)

H. W. HILTON

890

cwe*18;in anothcr, there were no differences”*; third showcd 3274 less of tliv rc!ducing substances, but no difference in suc’rosc or nitrogen in riincb i w w s . I w Variety, treatment, climate, and thc agc of thc trctttd plnnts huvc d l vwicd, and t hc cttrbohydrntnc.diffcrewes observed lruvc iwt been 1ttr.p)ailid liavc vwicd with thc oltbpxtd tinic? dtor treutnieiit. Phosphorus, in sll fornis rxcvpt in conibinnt ion in iwlcic acids, wns diminished in several plnrit species treated69 with aP. The particularly large diminution in organic phosphates, coupled with large decreases in incorporation of phosphorus into $,he high-energy adenosine di- and tri-phosphates suggests a major dislocation of the phosphorylase systems. Examination of isolated phosphorylation enzymes, however, shows no consistent alteration of activity. (2,4-Dichlorophengxy)acetic acid-I’C is metabolized by plant tissue to inactive, wt?,ter-soluble substances, some of which regenerate the parent acid on hydrolysis. The rate of metabolism differs with different species, but is usually low in comparison with the indole or naphthalene auxins. One of the earlier studies described a single major metabolite in corn, wheat, peas, and tomatoes,lZ1the identity of which is still uncertain. In another study of differenceR between species which were susceptible and those which were resistant to the herbicide, the resistant red currant metabolized 50% of the carboxyl-labeled and 20% of the methylene-labeled (2,4dichlorophenoxy)acetic acid to “COP in seven days, whereas the susceptible black currant alteredln only 2%. Similar effects were found with apple varieties. Strawberry and lilac decarboxylated the herbicide readily; however, 16 other species, of various susceptibilities to herbicide, were unable to decarboxylate the acid to any significant extent. In both varieties of currant, 5 to 10% of the herbicide was converted into watersoluble derivatives of the parent acid, and 10 to 30% was bound in the leaf tissue in an unextractable form. Two essentially different views of the metabolism of (2 ,4-dichlorophenoxy)acetic acid have been presented. The firstlP8Ja4was based on a study with bean stems, which converted 42% of (2,4dichlorophenoxy)acetic acidJ4C into acidic, water-soluble substances. Two ether-soluble metabolites were formed, and the same two metabolites could be recovered, (118) M. (i.Psync, J. L. Fults, R. J. Hay, and C. H. Livingston, Am. Potato J., 80, stuticw was indic:cttd in OIIC

aid

8

(119) (120) (121) (122) (123) (124)

46 (1!)53). TI. It. l’rrtcrpon, ZX8sertufion Abatr., 18, 147 (1953); 47, 7149 (1953). D. J. Wort, World Rev. Pest Conirol, 1, 6 (1962). 8.C. Fang and J. S.Butts, Plant Physiol., 19, 56 (1954). 5. C. Luckwill and C. P. Lloyd-Jonee, Ann. Appl. B i d , 48, 613, 626 (1980). M. K. Bach, Plant Physiol., 88, 668 (1961). M. K ‘Bach and J. Fellig, in “Plant Growth Regulation,” R. M. Klein, ed., Iowa State Univ. Press, Amee, Iowa, 1981, p. 273.

PLANT-GROWTH SUBBTAXCES

397

after hydmlysis of the water-soluble fmction, ‘‘mi~tainiihatvdwith sugars arid arrrino at-ids.” Spit her was tlir origiirrl wid or 2,~ ~ ~ Hydrogellation, and rc:tc*tion wit11 hydriorlic- wid, p;:t\lr? only pmdwts having intact ring~,bclievcd to t-ontttin Icngthcncd sidc-chains; this is

similar to formation of fatty acid by the addition of tcctyl groups. This view does not agree with earlier observations that the phenoxyalkanoic acids having an even number of carbon atoms in the side chain are converted by &oxidation into the phenoxyacetic acid. Neither does it agree with the conclusion derived from other studies, in which the major metabolites were believed to be either the glucose eater or a reaction product Mineral acids, takadiastase, or emulsin with aspartic acid, or both.126J28 regeperated the parent acid. Radioactive, chromatographic artefacts were found when leaves were dried before extraction with ethanol. 2 ,4-Dichloroanisole, but not 2 ,4-dichlorophenol, appeared to be a likely minor product in the latter work and in that of Crosby, who investigated two major, water-so!uble metabolites.127He believed one to be the glucose ester described by Klambt,B7and the other to be a structurally altered molecule, probably aJm present as a glucose derivative. Kliimbt had suggested the slow formation of (2 ,4dichloro-3-hydroxyphenoxy)acetic acid, but a more definitive study’m with oat tissue failed to reveal its presence. Phenoxyacetic acids, such as the 2-chloro and 2,&dichloro derivatives, with the C-4 unsubstituted, formed the 4-hydroxyl derivative and accumulated as the 4-/3-~-glucoside.The 4-substituted acids, such as (Pchlorophenoxy)acetic acid arid (2,4dichlorophenoxy)acetic acid, were not hydroxylated to any appreciable extent, but formed the glucose ester at equimolar ratios. The infrared epectrum of the acetylated ester isolated was identical with that of synthetic 2 ,3,4,6-tetra-O-acetyl-l-O- (2 ,Michlorophenoxy) acetyl-&r+ glucopyranose. (2,GDichlorophenoxy)acetic acid formed both the glucoside of the Phydroxy derivative and the glucose ester of the parent acid, acetic acid was exceptional in that it formed but (2,4,6-trichlorophenoxy) a glucoside of the 3-hydroxyl derivative. All derivatives could be hydrolyzed with acid or with &glucosidase. Other herbicidal phenoxyacetic and phenoxypropionic acids have some properties Rimilar to those of (2,4dichlorophenoxy) acetic acid, but often have quite different species selectivity. Some of the differences can be explained on thc basis of molecular stability, persistence, or mobility in the toxic form, as well as on the basis of differences in solubility and in absorption through leaves or roots. (126) (120) (127) (128)

R. W. Holley, F. P. Boyle, and D. B. Hand, Arch. Biochem., 87, 143 (1950). E. G. Jaworski and J. S.Butts, Arch. Biochem. Biophys., 88, 207 (1952). D. G. Croeby, J . Agr. Food Chem., 10, 3 (1964). E. W. Thomas, B. C. Laughman, and R. G. Powell, N u b e , 204,286 (1964).

~

398

H. W. HILTON

Of the thrcc monochlorophenoxyacotic acidR, only the 4-chloro derivative is very active and is used to retard abscission of fruit. A study of the effects of the isomers on the carbohydrates of the bean plant showed that the alterations of the carbohydrates were not related to the growth-inhibiting mechanism.’” Reducing substances, starch, mid s u c m decserwed with dl treatments, and the 2-chloro compound had the least effect; polyssccharides other than starch were not much altered. It is of interest that Aspergillus niger detoxified the (2-chlorophenoxy)acetic and (4-chlorophenoxy)acetic acid8 by hydroxylation of the ring, with remctval of the chlorine atom.’” 2. 1,2-Dihydro-3,6-.pyridazinedione

Maleic hydrazide (4) is not active in the Avena auxin test, and is inhibitory to growth, especially of grasses, at all measurable concentrations. 0

One of its curious manifestations is the inhibition of terminal growth without major inhibition of photosynthesis. It has been used to inhibit the sprouting of stored potatoes and to repress the growth of tobacco suckers. Starch content and “quality” of potatoes, and the sucrose in sugarbeets, treated with mrtleic hydrazide, did not change appreciably with storage.1s1Ja2 no effe~t,’**J*~ The content of reducing substances varied :dimin~tion,~~7J~’ or an being noted, depending on the elapsed time of treatment and the iength of storage. The sucrose content either remained unchanged or increased.lg7Plants sprayed with solutions of maleic hydrazide have shown moderate to large incremes in the amount of sucrose in the shoots

H.M. Sell, C. L, Hamner, T. L. Rebsfock, and L. E. Weller, Mich. State Uniu. Agt. E x P ~Sla. . Q’WTt. Bull., 40,306 (1957). (130) J. K.Fadkner and D. Woodcock, J . C k m . Soc., 5397 (1961). (131) D. R. Pateraon, S. H. Wittwer, L. E. Weller, and H. M. Sell, Plant Physiol., 27, (129)

135 (1952). (132) 8.H. Wittwer and C. M. Hansen, PTW. Am. SOC.Sugar Beet Techmlogiets, 6, 90 (1Qb;o). (133) M. E. Highlands, J. J. Licciardello, and C. E. Cunningham, Am. Pdoto J., 29, 226 (1952). (134) E. J. Kennedy and 0. Smith, Proc. Am. Soc. Hwt. Sd.,61, 395 (1953).

PLAATSROWTH SUBSTASCGS

399

c ~ r n , l and ~ J ~of surmse auld shrc.h itr r.I)tt8ir,1* wheprt,'a and bean.'" In t h e latter work, only the frnctan rwervc in barley innremed. Tho gluvoxe content was cithcr iinttltered or w w lower than that of thc aoiit,roln i n thtrxc irivcnt,ilr;utioiin. Three di ffcretit workom have dcwribcd oornlmted niuiiily of (IU(:MSB -from barlcyllm c o r ~ i , and ~~J~ leaf exudi&+q wheat.*a Synthesis or metabolism of starch does tiot appear to have been altered,140at least in tomato or bean plants. The apparent reason for the altered distribution of carbohydrate is that the sucrose or starch photorjynthate accumulates in the leaves, while transport and utilization are hindered. Sugarbeets and sugarcane (see Section VI) treated with foliar sprays of maleic hydrazide have not shown significant increases in reserve sucrose, although minor increases have been found for short, initial periods after treatment.14'-143 N o changes in oxidative phosphorylation, acetate uptake or metabolism, or pathways of carbohydrate metabolism were evident with this compound. The &D-glucoside of maleic hydrazide has been postulated as being one of two metabolites in wheat leaves and in apple, willow, tobacco, and other plants. These are formed144when the plants are supplied with maleic14Chydrazide or with inactive maleic hydrazide and glucoseJ4C. As rather large proportions of the parent compound are needed in order to produce plant responses, it is possible that the glucosidation is a detoxifying mechanism, as the authors suggest, although the activity level of the glucoside has not been determined, and other, alternative mechanisms of erratic performance, such as poor absorption by the leaf and poor translocation, seem equally likely.

and letves cf

3. 3-Amino-s-triazole

The most obvious effect of 3-amino-8-triazole (amitrole) (5) in plants a bleached appearance and diminished photosynthesis, although an effect on the synthesis of protein is probably involved as a more fundamental mechanism of herbicidal activity

is the lack of chloroplwta, resulting in

.

A. W. Naylor and E. A. Davis, Boban. Uaz., 112, 112 (1950). A. W. Naylor, Arch. Bdochem. Bzophys., 98, 340 (1951). W. J. McIlrath, Am. J . Bobany, 37, 816 (1950). D. J. Samborski and M. Shsw, Can. J . Botany, 36, 457 (1957). 'H. B. Currier, B. E. Day, and A. 5. Crafts, Botan. Uaz., 112, 272 (1951). V. A. Greulaoh, Botan. Gaz., 114, 480 (1953). M. Stout, PTOC. Am. Soc. Sugar Beet Technologiste, 8, 95 (1950). D. Ride, D. 8. Mikkelson, and R. S. Bsskett, Proc. Am. Soc. Sugar Beet T e c h t w b ~ t a7, , 88 (1952). (143) F. H. Peto, w.G.Smith, and F. R. Law,PTOC. Am. Soc. Sugar Beet !/'&-fa,

(135) (136) (137) (138) (139) (140) (141) (142)

7, 101 (1962). (144) G . H N. Towers, A. Hutchinson, and W. H. Andreae, Nature, 181, 1536 (1958).

400

H. W. HILTON

Amitrole had a drastic effect on the fixation of WOr by illuminated chloroplasts of ChZoreZZu pyrenoidosa, lowering the incorporation into sucrose by 95% at a concentration of 500 mg. per liter; it did pot affect phosphorylated compounds.146Carbohydrates hydrolynable by acids were higher (510j0) in treated, chlorotic corn leavea than in controls (32%), and this resdt was interpreted aa due to increased metabolism of proteins and fats. The respiratory quotient of 0.8 to 0.88, compared to controls near 1.O, supported this interpretation.146

I

H

Thera is considerable evidence that amitrole does not remain in the plant system as the free amine. For a perit$ of about 12 hours, amitrole does not move readily out of a leaf to which it is applied, although it is eventually quite mobile throughout the leaves, stems, and roots. The lag period has been interpreted as being connected with the rate of formation of one or more bioactive, translocation substances. Sorting out the various metabolic fragments has been fairly unrewarding and has been complicated by the presence of many minor metabolites. Most of the evidence seems to point to two major metabolites: one formed fairly rapidly and translocated easily in the plant; the second formed more slowly, possibly consisting of a transformation product of the first into a more stable form, and translocated with more difficulty. The evidence for transformation of the first compound into the second, as opposed to competitive formation at different rates, is not very clear. Disagreement exists aa to whether the metabolites are compounds formed with glucose, or with glycine or serine; each theory has proponents, and some scientists favor competitive ,or intermedigte formation of both metabolites. From all the evidence, the triazole ring appears to remain intact, even when incorporated into protein. Amitiwle-6-W formed 13 labeled, water-soluble metabolitea, with 50 to 90% transformation, in a variety of resistant and susceptible plant species. The principal compound wm formed with glycine or Gserine; it was ninhydrin-positive, and extremely resistant to hydrolysis (16 hours with 6 N hydrochloric acid in an autoclave sufficed to regenerate amitrole, but (146) a. Gu&in-Dumartrait, C m p t . Rend., 969,1837 (1961). (146) C.G.MoWhorter and W.II. Porter, Physid. Plontcmrm, 18,444 (1880).

PLANT-GROWTH SUBSTANCES

401

not the amino acid)>*--leTwo major metabolites were found in Canadian thistle, but only one in soybeanl". RogerS'Qproposed that a glucose adduct was the more labile compound in thistle; Herrett and Lincklm claimed that this v~aaformed exclusively if D-glucose was fed, with amitrole, to the plants; apparently, it is formed by reaction of amitrole with ~-glucosyl phosphate.-lS1Carter and Naylor149 could not incorporate ~-glucose--l~Ce into their major compound, whereas glycine-l4C entered readily. The Dg1ucow--l4C6 in the presence of amitrole increased the labeling of citric and malic acids, with no change in sucrose or phosphorylated compounds. Later, Naylorla demonstrated some increase in phosphorylated compounds and D-fructose, with inhibition of sucrose incorporation; he believed that the glucose adduct was questionable as a limiting factor in metabolism. Re-examination of three metabolites in Canadian thistle1" showed two to be inactive; a third was more bioactive than the parent compound, although it was different from the synthetic D-glucoside. Translocation of the herbicide required light, which may have brought about reaction to the active, transport form. Neither the synthetic Pglucoside nor reaction mixtures of amitrole with D-mannose, carabinose, D-xylose, or =ribose seemed to be involved in the two major metabolites in cotton.'M Massini166Jm first synthesized 3- (3-amino-s-triazolyl-l-)-Galanine, and identified it as being identical with one plant metabolite in tomato, presumably formed by the reaction of amitrole with serine. He investigated only two m e tabolites, which occurred in the diazotizable fractions: the alanine compound, and a second substance which may be the corresponding glycine compound. The substituted alanine was optically active, [ah -43' (water) , crystalline, and biologically inactive; it was translocated poorly. It appeared to be the most stable metabolite in tomato, increasing in quantity with time. Its similarity to histidine has been noted as a r e w n for its inccrporation into protein.U' Dospite the rather preponderant odds in favor of amino acid reaction, (147) (148) (149) (150) (151) (182) (163) (164) (156) (156) (157)

B. J. Rogers, Weeds, 6, 5 (1967). M. C. Carter and A. W. Naylor, Bdan. olaz., 122, 138 (1960). M. C . Carter and A. W. Naylor, PhysioZ. Plankarum, 14, 20, 62 (1961). R. A. Herrett and A. J. Linck, Physiol. Plankarum, 14,767 (1961). R. B. Shimsbukuro and A. J. Linck, Phyeiol. Planlarum, 17, 100 (1964). A. W. Neylor, J . dgr. Food C h . , 12, 21 (1964). R. A. Hemtt and W. P. Begley, J . Agr. Food Chem., 12, 17 (1964). C. 8.Miller and W. C. Hall, J . Agr. Food Chem., 9, 210 (1961). P. Msseini, Biochim. Biophys. A&, 86, 648 (1969). P. Messini, Acta Bolan. Need., 12, 64 (1963). A. K. William, 5. T. Cox, and R. G. Eagon, Bioehem. Biophy.9. Ree. Commun., 18, 260 (1966).

402

€1. W. HIWON

amitrole does react with carbohydrates to form stable products, but this reaction has not been unequivocally demonstrated in intact, plant systems. However, amitrole and D-glucoselhlQ and D-glucosyl phosphate161J62were condensed in rritro to form crystalline compound, m.p. 215” (dec.) , believed to be 3-(Boglucopyrsnosylamino)-s-tria~le(6) , on the basis of

OH (6)

its spectral properties in the ultraviolet and infrared, its molecular weight, and its slow phosphorylation with yeast hexokinase. It is biologically inactive, nonreducing, ninhydrin-negative, and not diazotized,which would mean that it would not have been investigated by Massini. A compound (presumably the same) referred to aa “triazole glucosazone” waa biologically inactive to wheat roots.Ioa 4. Chlorinated Aliphatic Acids

Two aliphatic acids possess, for grasses, many of the growth-distortion and toxicity effects associated with the synthetic auxins on dicotyledonous plants. Trichloroacetic acid and 2,2-dichloropropionic acid (dalapon), as although the sodium salts, have been called grass “hormones” or “auxinsJJJ Wilkinson’64 could find no growth stimulation at low concentrations, and described dalapon as an “antiauxin” from its interference with indole-3acetic acid effects. The herbicidal properties of trichloroacetate do not depend on ito proteindenaturing ability, and those of 2,2dichloropropionic acid involve, at least indirectly, the synthesis of pantothenic acid. Treatment of various plants with the compounds, at least up to herbicidal levels, has riot shown much alteration in the proportion of soluble carbohydrate, but ha8 shown rather wide variation in the distribution among the forms of the carbohydrates. flugarbeet seedlings increased in (168) A. C. Gentile and J. F. Frcdrick, Phyeiol. Plantarum, 19,862 (1969). (169) J. I?’. Fredrick m d A. C. Gentile, Arch. Biochem. Biophys., 86, 30 (1960). (100) J. F. Fredrick and A. C. Gentile, Arch. Biochem. Biophys., BP, 366 (1961). (161) J. F. Fredrick and A. C. Gentile, Physiol. Planbrum, 18, 761 (1960). (162) J. F. Fredrick and A. C. Gentile, Phytun (Buenos Aires), 16, 1 (1960). (163) E. E. Bchweiaer and B. J. Rogers, weeds, 12, 7 (1984). (164) I%. E. Wilkinson, Weeds, 10, 276 (1962).

PLANT-GROWTH SUBSTANCES

403

reducing substances, imparting freeze resistance to treated plants.1a Sucrose increased in Johnson grass at the expense of reducing substances,114and trichioroacetate lowered sucrose to a quarter of that in controls in stems and shoots of wheat, and increased starch and other root polyaaccharides.la Organic acids remained relatively unchanged in dalapon-treated wheatelm Acid-soluble and acid-insoluble phosphates in ChZoreUa vulgaris increased 2 to 4 fold, depending on the concentration of trichloroacetate, with the soluble phosphorus most affected. However, neither respiration nor the carbohydrate-metabolizing enzymes were particularly sensitive. Little change occurred in the hydrolyzable polysaccharides.l@ Dalapon is hydrolyzed in plants to pyruvic acid.169

5. Carbamates and Carbanilates Several herbicides for monocotyledonous plants are carbamates, as are numerous insecticides and fungicides. Two of the principal herbicides, represeatative of the class, are isopropyl 3-chlorocarbanilate (CIPC) (7) , and S-ehhyl N ,Ndipropylthiocarbamate (EPTC) ( 8 ) .Although the herbi-

0)

(8)

cide activity appears to be associated with interference of cell mitosis, increases occurred in reducing carbohydrates and sucrose in corn and soybeans at herbicidal rates of application of isopropyl 3-chlorocarbanilateJ resulting in an overall increase of 90% in soluble carbohydrate in leaves and ~tems.l7~ The typical, dark blue-green color of the young plants, especially of the cotyledons, suggested a greater content of chlorophyll, but no increase in photosynthesis was S-Ethyl N ,Ndipropylthiocarbamate only moderately inhibited uptake of 14C02 at 1 0 - 2 M in red kidney-bean. No alteration appeared in the distribution of ~ucrose,amino (165) S. R. Miller and W. G. corn^, Cun. J . Botany, 96, 5 (1957). (168) T.L.Rebstock, C. L.Hamer, R. W.Luecke, and H. M. Sell, Plant Phpiol., 28, 437 (1953). (167) C. Oyolu and R. C. Huffaker, Crop Csci., 4, 95 (1964). (168) K. A. H m l l , Physiol. P&darum, 14, 140 (1961). (169) J. K. Lessure, J . A@. Food Chem., 12, 40 (1964). (170) J. A. M ede and A. 0. Kuhn, Wee&, 4 4 3 (1956); 6,68 (1958). (171) F. M Ashton, Wee&, 11, 295 (1963).

404

H. W. HILTON

acids, or organic acids; respiration of bean embryos was unchanged. Phosphorylation was more pronounced, but was not enough to be the cause of the altered growth. Inhibition of fl-amylrtse activity by a series of ringsubstituted carbanilates,with CFa, Br, and C1 substituents, was associated with electmnegativity and solubility, a strong argument for a physical adsorption on enzymes or other actively changing surfaces.ln Diminished carbohydrate reserves resulted in correspondingly greater reducing substances in another study.*TaAmylase, phosphatase, and invertase activity increased with isopropyl carbanilate (IPC) , but reducing carbohydrate did not increase proportionately. Sucrose decreased in proportion to invertase activity.lT4Zinc N ,Ndimethyldithiocarbamate, a fungicide-herbicide, at two to eight pounds per acre, increased glutamic acid about 100% but did not affect yield of sucrose in sugarbeets.lTSVarious carbamates inhibited the Hill reaction (see Section 111,6, p. 405), but not efficiently (as compared with the substituted ureas). No really satisfactory hypothesis explaining the various observed eff ects has yet been offered. The l-thio-r3-P.glucopyranosideof N ,Ndimethyldithiocarbamic acid was isolated in crystalline form, by countercurrent extraction, from potatoes treated with the sodium salt of the fungicide. No data are given in the reference,"a but the compound was said to have been compared with an authentic sample prepared from tetra-0-acetyl-D-glucopyranosyl bromide. The fl-D configuration was inferred from the synthesis, and the compound was a weak fungicide. Another carbanilate herbicide, 4-chloro-2-butynyl 3-chlorocarbanilate (bwban) , formed a water-soluble metabolic product in various plants. The metabolite could not be identified as any simple reaction-product, and it was tentatively proposed that the original herbicide might be sssociated with a plant component such as a flrtvonoid. 3-Chloroaniline could be distilled out after hydrolysis, and several hydrolytic fractions contained carbohydrates but no nitrogen.In 6. Substituted Ureas A group of substituted ureas used as herbicides has a profound inhibiting effect on the photosynthetic mechanism in plants. The relatively advanced (172) D. E. Moreland and J. C. Davis,Proc. sbuthmn Weed Cmf., 9, 150 (1956). (173) V. H. Freed, J . Agr. Food Chem., 1, 47 (1953). (174) C. Tomisirwn arid H. Koikc, Nogyo Gijuku KenkyLsho Hokoku, Byon' Konchu, 4, 26 (1954). (175) J. L.Fults, M. G . Payne, J. 0.Grmkill, L. R. Hao, and A. C. Walker, Botan. Gaz., 113, 207 (1951). (176) J. Ksslander, K. Kaars-Sijpesteijn, and G.J. M. van der Kerk, Biochim. Biophva. Acla, 62, 396 (1961). (177) J. R. Riden and T.R. Hopkins, J . Agr. Food Chem., 10, 455 (1962).

PLANT-OROWTH SUBSTANCES

405

state of photosynthetic research has made possible the study of the inhibition in some detail. The most active compound, which severely inhibits or kills seedlings by root uptake of 1 X 10-7 M solutions, is 3-(3,4dichloropheny1)-1,1-dimethylurea (diuron) (9). A dozen or so other am0

II

c

1

N- C-N, pA1

,cH, CH,

c1 (9)

logs are herbicidal, with 3- (pchloropheny1)-1,1-dimethylurea (monuron) being the most common. Their activity apparently lies in their ability to inhibit photosynthetic phosphcrylation; probably, the photoreduction of adenine nicotinamide dinucleotido or its phosphate is blocked, leading to the lack of an oxygen receptor from the photolysis of water. The effect is meaaured aa an inhibition of the Hill reaction, in which the photoreduction of ferricyanide or pbeneoquinone added to iIluminated suspensionsof chlorophyll is measured with and without the chemical.l*lB1 Diuron inhibition of the Hill reaction was found to be 2500 times that of phenylurethan; if it is not too severe, the inhibition can be partly reversed by ~-gIucose,sucrose, or flavine mononucleotide.182Ja The last compound is a catalyst in the reaction which involves production and reutilization of oxygen from water. There is also apparent interference with the hexokimsecatalyzed photophosphorylation of D-glucose with adenosine triphosphate to give 0-glucose 6-phosphateJ demonstrated1” with *,P,Herbicidal members of many groups of compounds-the ureas, carbanilates, s-triazines, acylamides, and uracils-aJl have essentially the same mode of action, with differences of degree and species selectivity. The mechanism for oxidation of water to oxygen appears to be primarily affected in each case. The urea herbicides do not inhibit seed-germination to any extent, or the growth of nonphotosynthetic tissue, nor do they interfere with darkfixation reactions. Monuron-treated bean-leaves which were fed 14C02in the light, however, decreased in sucrose content (as percent of ethanol-soluble materials) from 70 to 2% in 72 hours.186Most of the carbon-14 label (178) The Hill reaction has been reviewed by I(. A. Clendenning, Ann. Rey. P2cmt Phlpwl., 8, 137 (1957). (179)A. It. Conkc, North Cmtral Weed Cbntrol Cmf., Ree. Rept., 12, 181 (1955). (180) J. 8.C. W e w h and R. van der Veen, Biochim. Biophyu. A h , 19, 548 (1956). (181) B.Exer, Weed &8. 1, 233 (1961). (182)M.J. Geoghegan, New Phytolopiat, 66, 71 (1957). (183) W.A. Gentner and J. L. Hilton, Weeds, 8, 413 (1960). (184) N. E.Good, Plan4 PhfpiOl., 36, 788 (1961). (185) B’. M.Ashton, F. G . Uribe, and G . Zweig, Wee& 91676 (l9Gl).

406

H. W. HILTON

appeared in aspartic acid (58.60j0). Although neither the total percentage of amino acids nor that of organic acids changed much, the distribution ratios changed considerably. All carbohydrates decreased, as measured by dry weight, as "water-insoluble" carbohydrates presumed to be starch, and as soluble substances.188 Amylase activit,y decreased ; within the series of chlorosubstituted ureas, decreased activity varied directly with water solubility, suggesting an adsorptive effect on the cnxyme surface. The enzyme effects were not, considered to be major causes of growth inhibiti~n."~ An inac.tive metabolite formed from monuron in bean plants has noC yet been idcntificd.I87 7. SubBtituted n-Triazines

Members of an extensive group of sym-triazine herbicidos, usually having one or two secondary amine substituents, block the Hill reaction and inhibit photosynthesis in a manner quite similar to that of the urea herbicides. The most widely used, 2-chloro-4-(ethylamino) -6-(isopropy1amino)s-triazine (atraxine), (lo), is one of several hundred herbicidal analogs 'N

R

N

(10)

differing in the three substituents attached to the ring-carbon atoms. An interesting difference in species selcctivity involves the ability of certain plants (and soil) to render the molecule biologically inactive by hydrolytic removal of one or more of the attached groups. Ring fission may also occur. The photosynthetic block precedes sucrose synthesis, and sucrose partially revwscs the inhibition caused by the lack of fixation of carbon dioxide. D-Fructose disappears first, after treatment, followed by sucrose, and then r~-glucose.lssIt is likely that other metabolic systems are involved as well, since the amino acid distributions were not identical to the I4CO2 dark-fixation products.1se Uptake of s u ~ r o s e - ~iricreased ~C some acids (as(186) W. H. Minshall, Can.J. Botany, 88, 201 (1960). (187) S. C. Fang, V. H. Freed, R. B. Johnson, and D. R. Coffee, J . A p . Food Chem., 8, 400 (1955). (188) S. M. Mashtakov and R. A. Prochornik, Dokl. Akad. Nauk Bellmask. SSR, 6,1367 (1962). (18'3) P.M Ashton, G . Zwcig, mid C;. W. Mason, Weeda, 8, 448 (1960).

PLANT-GROWTH SUBSTANCES

407

partic and glutamio) at the expense of others (seine, alsnine, glyceric acid) in the light, but not in the dark. Serine-I4Cdid not form any sucrose; and an increase in glyceric acid suggested a block in the glycolytic scheme wherein the acid required energy from adenosine triphosphate for conversion into glyceric acid 3-phosphate. Acetate-2-14Cor -8-l*C uptake and utilization were decreased by simazine, the bis(ethy1amino) analog.1oo Starch production in leaves cemed after one to six days, depending on the species; su~rose-~~C could be substituted m a source of substrate for starch.'@JCorn (maize) tolerates many of the triazine herbicides, but its isolated chloroplasts do not. Treatment did not influence catalase activity or respiration,Ig1 but stopped oxygen output immediately.102 Simazine and atrazine have little effect on corn, sorghum, sugarcane, and some other grasses, apparently because of the ability of the plant to metabolize the parent molecule. Corn and wheat gave a cyclic hydroxamate, namely, the 2-~-glucosideof 2,4dihydroxy-7-methoxy-l,Pbenzoxazin-3-one ( l l ) , m.p. 168-70",aglycoii m.p. 1587" (dec.), believed to

have replaced the chlorine atom catalytically by a hydroxyl group.lg&l" The campound is not known in other tolerant plants, and it is likely that enzymic metabolism is also involved. 8. Substituted Benzoic Acids

There is little published information of interest to carbohydrate chemists concerning the benzoic acid herbicides. The predominant member, 2,3,6trichlorobenzoic acid, inhibited uptake and utilizationlWof acetate-W. It shows some auxin-like properties, whereas 2 , 3 ,5triiodobenzoic acid appears to block the transport of natural and synthetic auxins, and has often been termed an "antiauxin." The triiodo compound inhibited oxygen (86% at (190) (191) (192) (193) (194) (195) (106)

A. Gast, Ezperidia, 14, 134 (1958). B.Exer, Ezperidia, 14, 136 (1958). W. Roth, Ea~perientiu,14, 137 (1958). R. H. Hamilton and D. E. Moreland, Science, 181,373 (1962). W. Roth and E. Knusli, Ezperientia, 11, 312 (1961). R. H. Hamilton, J . Agr. Food Chem., 12, 14 (1984). 0. WulilrooB iirid A. I. Virtannen, Actu Chem. Sand., 18, 1908 (1959).

408

H. W. HILTON

2 X 1 P M) and phosphorus uptake:o

and the uptake and utilization of acetate.1w Benmic wid, the three monohydroxybenmic acids, and the three monochlorobenzoia acidH decreased carbohydratm in barley leaves, d retarded respiration, although none are particularly phytotoxic. Added sucrose increased the output of carbon dioxide, but did not increw the carbohydrate content in the plants. Plant sucrose was depletd.’@’J@ Many other herbicides and compounds having growth-alteration properties are known, and new ones appear a n n d y from commercial screening. It has been quite evident so far that no correlation has been demonstrated between chemical structure or physical properties and biological activity. The empirical search continues for new structures having biological propertieg, arid bmic research studies continue with newer instrumental techniques for investigating the mechanisms of activity.

IV. GLYCOSIDES AND OTHERCARBOHYDRATE DERIVATIVES AS ~ T - G R O W T HSUBSTANCES 1. Natural Glycosides

In ddition to the naturally occurring indole and gibberellin growthregulators, mrtny “growth factors,” accessory factors, and inhibitory substances have boon either isolated or at least suggested to cover unexplained results. One area of lively academic and practical interest involves the natural inhibitors of growth and of germination of seeds and axillasy buds. Many plantdrgan and seed extracts are inhibitory in a gross sense to other plants, and many seeds will not germinate until some inhibitory fraction has been extracted. Three proposed mechanisms for the activity of the inhibitors are: (1) antagonism, competitive or otherwise, with essential auxins, vit,amins, and so forth; (2) direct inhibition of some growth or development process at sites other than those of direct, hormone activity; and (3) indirect inhibition of growth, affecting enzymes, nutrient uptake, or utilization. There is evidence for all three. This discussion will not cover the direct enzyme and metabolic inhibitora or poisons, which have been so comprehensively reviewed by Webb.1Bg The characterization of many of the growth inhibitors is not definitive, and it would be extremely premature at this point to try to assess the physiological or chemical importance of those that are known. However, (197) M. I. Naguib, Ccn. J . Bdcmy, 41, 939 (1W). (198) M. I. Naguib, Plonla, 04, 20 (1006). (109) J. 1,. Webb, “Enzyme and Metabolic Inhibitors,” Academic Press Ino., New York,

N. Y.,and London, 1083.

PWNT-GROWTH SUBSTANCES

409

so many belong to the flavonoid and relatcd groups, or arc dcrivtitivos of coumarin, that it is believed worthwhile to review them. Most of them exist in the plant as glycosides of phenolic or enolic groups, or as carbohydrate esters. Glycoside formation has generally been considered to be a detoxification, the “more or less universal reaction (together with destructive breakdown) of all organisms to the presence of foreign compounds,” and anthocyanin pigments have been considered to be growthinactive end-products. It is likely, however, that the glycosides and the aglycons should either be treated as individual entities, or that the glycoside formation should be considered to be a regulatory mechanism for controlling the amount of active substance. It seems strange that the plant should treat those compounds which it produces as “foreign substances” requiring detoxification, unless glycoside formation provides some such regulatory mechanism. The statement of Gibbsm is applicable: “Many of the substances, such as the alkaloids, the cyanogenetic glycosides, the phenolics, and so on, that seem to be so useful as taxonomic guides have no obvious usefulness to the plant.. . When we know more of them we may think diffelently.” So far, relatively few glycosides are known to have growth-alteration properties. The earlier work (to about 1958) has been reviewed by Hembergml and Mayer and P~ljakoff-Mayber.~~ Many of the simple phenolic acids are inhibitory, including coumarin, trans- (but not cis-) cinnamic acid, salicylic acid, and 0- and p-coumaric acids, among others. Precursors of the various coumarin derivatives appear to be substituted cinnamic acids. o-Coumaric acid (truns-o-hydroxycinnamic acid) , as the &wglucoside in sweet-clover leaves, formed a n equilibrium mixture containing 89% of the Cis isomer in normal sunlight, with subsequent ring closure to coumarin.*m6 It has been suggested that the glycoside is formed first, and is involved in the Cis-trana isomerization and in the biosynthesis of the coumarin derivatives which are predominantly present as g1ycosides.m Precursors of the flavones and flavanones are probably hydroxylated benzoic acids. Light decreased the auxin response of etiolated peas without affecting the auxin level; if light activates or produces an inhibitor which interacts

.

(200) R. D. Gibbs, in “Chemical Plant Taxonomy,” T. Swain, ed., Academic P m Inc., New York, N. Y., and London, 1963, p. 80. (201) T. Hemberg, in “Encyolopedia of Plant Physiology,” W. Ruhland, ed., SpringerVerlag, Berlin, 1961, Vol. 14, p. 1162. (202) A. M. Mayer and A. Poljakoff-Mayber, in “Plant Growth Ragulation,” R. M. mein, ed., Iowa State Univ. h, Am-, Iowa, 1961, p. 735. (203) 8. A. Brown, G. H. N. Towers, and D. Wright, Cun. J . Biochem., 88, 143 (1960). (204) T. Komge and E. E. Conn, J . BWZ. Chtnn., U,2133 (1969). (206) F. A. Haskins, L. 0. William, and H. J. Gors, P & d Phyeiol., 89, 777 (1964). (208) D. J. Austin and M. B. Meyera, Phyfoehemishy, 4, 256 (1965).

H. W. HILTON

410

with the auxin, it might be poasible to explain the resu1ts.m Galstoil and his group believed that they had characterized the natural inhibiting substances in peas aa being derivatives of the two flavonols: quercetin and kaempferol (12), which exist as mixtures of the 3-O-"tri-r+glucosyl"

GR-

HO

-

R H in kaampferol derivatives

R = OH in quercetin derivatives (la)

pcoumarate (shown),and the 3-O-"tri-~-glucoside~~ forms.208 The structure of the "tri-&glucose" portion is not known. Other derivatives of quercetin had earlier been implicated as inhibitors of pollen germinationm : rutin [quercetin 3- (0-0-/3+rhamnopyranosyl) -Dglucopyranoside], and quercetrin (3-O-8-crhannopyranosylquercetin) Naringenin, (13), isolated m a glycoside from peach buds, inhibited

.

I,7,4'-TrihydroxYfhWne (13)

seed and peach-bud g e r m i n a t i o ~ i ? possibly ~ ~ * ~ ~ by interaction with gibberellins. Since naringenin is structurally similar to kaempferol, it may be t r d o r m e d into the latter as the active form. Other related compounds, such as the flavone apigenin 'I-~-~ucoside, had similar growth-activity. Ten other flavanones, flavones, and isoflavones were found to stimulate (207) A. W. Galston and M. E. Hand, Am. J . Bdany, 86, 86 (1949). (208)M. Furuya and A. W. Galston, Ph@ochmiu@, 4,285 (1966). (209)F. Moewua, Aqpsw. Chm., 63,486 (1960). (210) C.H. Henderehott and D. R. Walker) Bn'ancs, 180, 798 (1959). (211) C.H. Hendersbott and D. R. Wfilker, Proc. Am. Boc. Hort. Bci., 74, 121 (1959). (212) I. D.J. Phillips, Nature, 1011 240 (1961). (213) I. D.J. Phillips, J . Ezpdl. Botany, 18, 213 (1962). (214) C. F.Eagles and P. F. Wareing, PhysME. Plontarum, 17, 697 (1864).

PLANT-QROWTH SUBSTANCES

411

wheat rookgrowth at 1od to l(r M ,but were inhibitory at higher levels. Stenlid216 used the aglycons in the study, but noted that they existed mainly aa glycosides. He suggested that they affected enzymic oxidation of phenols and L-ascorbic acid, The compounds reversed the mobinhibiting properties of indole-%acetic acid, (2,4dichlorophenoxy) acetic acid, D-mannose, D-galactose, and 2-deoxy-~-arabino-hexoseto some extent. Quercetin was not too effective as an antagonist for (2,4dichlorophenoxy)acetic acid (4 X 10-7 M) ,indole-3-acetic acid (4 X lo-*M ),D-mannose (7 X 1 P M ), or 2deoxy-~-urubino-hexose(3 X M), and it failed to show auxinic activity. The uptakes of phosphate, chloride, and nitrate were inhibited, which led to the conclusion that quercetin was growth specific, since n-glucose can reverse D-mannose inhibition, but does not depress ion uptake. No complete parallel existed between the effects of the externally applied substances and the endogenous growth mechanism.216 Work with the kaempferol- and quercetin-“triglucose” esters of p hydroxycinnamic acid has established their structures by spectral data, carbon-hydrogen analysis, and chromatography; the esters examined were not crystdliie. The 1:3 molar ratio of aglycon to n-glucose, and the attachment of the cinnamic acid to the D-glucose chain were determined.*17e218 The present view is that, in etiolated peas, and possibly in all green plants, kaempferol exists as a mixture of the “triglucoside” and “triglucosyl’’ p-coumarate forms, with only traces of the quercetin derivatives, whereas the latter (again as the same kind of mixture) are predominant in green plants in the light.m The kaempferol-3-“triglucosyl” p-coumarate waa found only in the leaves, kaempferol-3-“triglucoside” occurred in leaves and stems; quercetin-3-“triglucosyl~’pcoumarate occurred throughout the plants (except in the roots), and the triglucoside wm abaent from roots and stems. Each organ appeared to have its own pattern of flavonoid synthesis, which could be modified by light. Far-red and red light altered leaf-growth production of kaempferol-3-“triglucosyl” p-coumarate. These effects of light were most likely indirect, ultimately changing the flavonoid synthesis.21eKaempferol “hexaglucoside” has been reported in pea seedlings treated with red light.= The physiological effects of the kaempferol and quercetin derivatives are uncertain: they inhibited indole-3-acetic acid oxidase in vitro; they may induce dormancy, uncouple oxidative phosphorylation, stimulate plant (215) (216) (217) (218) (219) (220)

G. Stenlid, Physiol, Phnfamm, 14,659 (1961); 16,698 (1962). G. Stenlid, Physiot. Phntumrn, 10, 807, 924 (1957); 12, 199, 218 (1959). F. E. Mumford, D. H. Smith, and J. E. Castle, Plutzt Phyeiol., 86, 752 (1961). M.Fwuya, A. W. Galston, and B. B. Stowe, Nuture, 198, 456 (1962). M. Furuya and R. G . Thomas, Plant PhysioZ., 89, 634 (1964) F. E.Mumford, D. H. Smith, and P. G. Heytler, B i o c h . J., 91, 517 (1964).

412

H.

W. HILTON

growlh, or iiihibit flowering. 15sglcx aiid WareingZi4proposed that they be called growthdormancy regulators, to distinguish them from the synthetic growth-inhihitors. FIeterN of chismio acids are more common than glyconides of phenols. Tho moiiocstmv of D-g1uco8el rutiiiosc, or goritiobiow with tho four naturally occurriiig cinnniuic acid8 (cinnamic, ferulic, caffeic, and sinltyic: mid) have been recognized as iiaturttl products--some, as complex products containing more than one cirinamoyl Both kaempferol and quercetin exist as 3-glucosides1rhamnosides, galactosides, arabinosides, gentiobiosides, and more complex forms in numerous p1ants.m Kaempferol 3-~-glucoside (astraglin) and kaempferol 3-~glucosylpcoumarate (tiliroside) increased indole-3-acetic acid activity by inhibiting the oxida~e.**S*~ An auxin (1-naphthaleneacetic acid), 2,3 ,&triiodobenzoic acid, and a gibberellin interfered with formation of anthocyanin pigment in Impatiens balsamina. The first compound increased, and the second and third decreased, the anthocyanin formation. This interaction shifted the genetically controlled pattern of quantitative distribution of anthocyanin toward the base of the stem segments.n6 A series of 36 plants showed only two that form no glucosides when sucrose is injected into the leaves. In the two exceptions, the sucrose was converted into nonsugars. Phosphorylation uncouplers (2,4dinitrophenol, iodoacetate, and the like) did not affect Dglucoside formation.m Plant-growth factors producing positive responses of rapid, controlled cell-growth and celldivision have been found in many places; undoubtedly, some are mixtures of vitamins, carbohydrates and amino acids with the natural growth-regulators. None have proved to be more interesting, more useful, or more difficult to characterize than those from coconut milk or from the corresponding, immature, liquid endosperm of various plants, such as horae-chestnut ( Aeeeulw woerlilzenais) and corn (Zea mays). Fractionstion of the active substances, reviewed by Steward and Shantz,n7 has so far produced only 1,3diphenylurea, a possible indole-3-acetic acid eater of arp,binose, and another substance, 20 mg. of which waa saidm to (221) J. J. Corner, J. B. Hsrborne, 8.G. Humphriea, and W. D. Ollie, Phytochmbfty, 1, 73 (1902). (222) See, for example, J. B. Hsrborne, Arch. Ba&m. Btbphys., 96, 171 (1962). (223) J. B. Harborne, Phybockmbtty, 1, 161 (1964). (224) J. P. Niteoh and R. Parip, BuU. Soc. Botan. Frame, 109, 241 (1902). (228) A. W. Arnold and L.8. Albert, Plant Phyeiol., 19, 307 (1964). (226) D.I. Lisitayn, Uglcvody a Uglcvodnyi Obmsn v Z h i v o t m i Rastilsl'nom Organ&mukh, M M a y KW.,Moeeow, 1868, (Publ. 1969); C h m . Abstraete, 66, 7665 (1961). (227) F. C. Steward and E. M. Shantz, Ann. Rev. Plant Phyeiol., 10, 379 (1959). (228) E. M.Shantz and F. 0.Steward, Plant Physiol., 80, xxxv (1866).

PWNT-GROWTH SUBSTANCES

413

equal the activity of coconut milk at 10%. It w a proposed that the product might be the mono-wglucoside of either a leucoanthocyaninm (14) or a ledcocyanidinm ’(15). No other report of its biological activity has been published.

Leucoanthocyanin hm been found in buckwheat, and is formed both in light and darkness, whereas the anthocyanin pigments are formed independently and only in the light.281 The aglycon had little activity. It is not clear whether these compounds are responsible for the major activity of the endosperm source or for only a d part of it. The constituents of the endosperm are diEcult to isolate and purify. The phenolic constituents should be investigated in greater detail. The 5-&~-glucopyranosideof 2 ,5dihydroxybenzoic acid (gentisic acid) waa identified as an accumulation product in borondeficient sunflower.2n.2ss A number of glycosylamine derivatives are growth factors for fungi and bacteria, notably derivatives of riboflavine, coenzyme A, vitamin Bu, myo-inositol, N-glucosylglycine, Zacetamido-Zdeoxy-0-(D-galactosyl)-Dglucose, and others. These will not be discumd here’ePr,2as 2. Synthetic Glycosides, Other Carbohydrates, and Carbohydrate Derivgtives Glycosides are formed by higher planta from a variety of externally applied substances, regardless of whether or not the substance is toxic or growth active. The herbicidal chemicals have been discussed in other Sections, and this Section will deal with glycoside formation of some other compounds, and with some carbohydrates and derivatives that have shown growth activity. Nearly all plants, excepting the lower plants, algae, and fungi, form (229) G. M. Robirison and R. Robinson, Biochcm. J., 27, 206 (1933). (230) T. Swain, Chem.In&. (London), 1144 (1954). (231) J. R. Troyer, Phyfochniehy,8, 638 (1964). (232) R. Watmabe, W. Chorney, J. Skok, and 8.Wender, Phyiochemiu6q 8,391 (1964). (233) -4. Zaue and 8.H. Wender, C h m . I d . (London), 1836 (1964). (234) Reviewed by N. Fries, in “Encyclopedia of Plant Physiology,” W. Ruhland, ed., Springer-Verlag, Berlin, 1961. Vol. 14, D. 332. (235) W.8.McNutt, C h n . Org. Naturs&$e, 9, 401 (1952).

414

H. W. HIL'TON

mono-&D-glucosides with either quinol (hydroquinone) or resorcinol as ail externd phenol. This phenol glycosylation reaction hss been reviewed.2aa Phloroglucinol-14Cand D-glucose formed the corresponding j3+glucoside, phlorin, in many Those species which seemed to have no glycosylatioq mechanism converted the D-glucose into sucmse or, occasionally, into p-fructose or D-glucitol. MilleP-w has studied the glycosides produced (in proportions aa great aa 13% of the dry weight) when chloral hydrate, 2,2 ,2-trichloroethanol, ethylene chlorohydrin, and o-chlorophenol were added to plants. He originally investigated these compounds as tuberdormancy regulators, and found them to have a low degree of phytotoxicity, except at high concentrations. Seven species formed the gentiobioside instead of the D-glucoside, although gentiqbiose was not known to be present in the plants. The derivatives were compared with authentic compounds. A third, unauthenticated carbohydrate material may have been present aa a primeveroside. The carbohydrate moiety present depended on the aglycon applied. The glycosides did not move readily, were not found in sprouts, and, therefore, were considered to be detoxication products. Compounds isolated and synthesized are listed in Table I. Apart from the utiliestion of the common mono- and di-saccharides as metabolic substrates for growth and energy, there is little information on the growth effects of externally applied carbohydrates. Some, like D-mannose, *galactose, and Zdeoxy-D-urabino-hexose, are inhibitory to root growth,*lapossibly because they provide a high osmotic concentration in solution, from which they are only indifferently utilized, without being actively phytotoxic. J. B. Pridhsm, Phyfochsmistry, 8, 493 (1864). A. Hutahinson, C. Roy, and 0.H. N.Towers, Nature, 181, 841 (1968). L. P. Miller, Confrib. Boyce Thompson Znet., 0, 426 (1938). L. P. Miller, Csontrib. Boyw Thumpson Znef., 10, 139 (1939). L. P. Miller, Codrib. Boyce Thornpuson Znet., 11, 28 (1939). L. P. Miller, Am. J . Botany, 96, 14a (1939). L. P. Miller, Conkib. Boyw Thompon Zneb., 11, 271 (1940). L. P. Miller, Seisnce, 91, 42 (1940). L. P. Miller, Codrib, Boy@Thumpuson Znet., 11, 387 (1941). L. P. Miller, Codrib. Bww Thumpuson Znet., 19, 16 (1041). L. P. Miller, Cdrdb. Boyce Thompm Znet., 19, 26 (1941). L. P. Miller, C d r i b . Boyce Thompson Znat.,14, 29 (1941). L.P. Miller, Contnb. Boycs T h p s o n Zteet., 19, 163 (1941). L. P. Miller, Codrib. Boyw T h p o n Inst., 19, 167 (1941). (250)L. P. Miller, J . Am. C h .SOL, 68,3342 (1941). (251) L. P. Miller, Con&&. Boyes Thampsson Znet., 18,369 (1942). (262) L. P. Miller, Contrib. Boyce Thpson Zmt., 19, 466 (1943). (263) L. P. Miller, Confrdb. Bqce Thompson Znuf., 18, 186 (1943). (254) L. P. Miller, Contra%.Boyce Thompson Znef., 18, 113 (1967).

(236) (237) (238) (239) (240) (241) (242) (243) (244) (246) (240) (247) (248) (249)

TABLE I P k n t Glycosidea from Chloral Hydrate, Ethylene Chlorohydrin, and 0-Chlorophenol

Free

Glyeoside

Peracetate

presence

Refennees

in plant@

glycoside

8-D-Glucopyranoside 2,2,2trichlorOethyl

2-chlomethyl whlorophenylb

153

-

-40.5

172

-65.3

205 (dec.)

-41.2

+ +

249,251-253 238,239, slci

+-

245,247,

-49.5

+

250 242-244,Ws

-47.2 -39.9

+-

250,252 252,254

145 118 150

-29 -13.4 -44.6

184 168 208

-28.5 -m.2

170 176

240

8-Gentiobioeide 2 ,2 ,2-trichloroethyl ZChlmOethyP o-chloropheny1

-

-

amorph.

&Primevenmide 2,2,2trichlor0ethyl~ ZChloroethyl* a

*

+

-

Key: means present; - means absent. Synthetic. Glycoside structure uncertain, but may be a primeveroside.

9 s

H. W. HIYMN

416

The cyclitol, myo-inositol, is a growth promoter for lower plants, and the group of cyclibls is widely distributed in higher plants. Their physiological function has not yet been fuUy established, but they are known to be involved in the storage of phosphate, as phytin, and in the biosynthesis of aromatic rings from carbohydrates. Spruce tissue-cultures were maintained with myo-inositol or sequoyitol (a monomethyl ether) at 50 to 100 mg. per liter. (+)-Pinitol, Anositol, and ecyllo-inositol had slight activity, whereas quebrachitol, and L-, epi-, mwo-, and neo-inositol were all inactive.n-manno-Heptulose inhibited phosphorylation of glucose by inhibiting hexokinase?" and the aldono-1,&lactones inhibited carbohydraaes having the same configuration." R. Brown and coworkersm~360 observed that seeds of Striga, known as witchweed, a semi-parasitic plant of gnaws and corn, germinated only in the presence of root exudates from the living host. They isolated the sirupy exudate, [a33 20' (water), resemblinga pentose, but did not specifically identify the active substance. A study of many carbohydrates, mostly pentoses, showed that only wthreo-pentulose, -32' (water), had the required activity; cthreo-pentulose, [a& 35" (water) ,was not active. Two groups of sucrose derivatives of herbicidal acids have been reported. The herbicidal properties of the sucrose estera of (2 ,P-dichlorophenoxy)acetic acid and other analogs differed somewhat from the salts of the free herbicide acids. This could be accounted for as being due to differences in solubility and penetration, since it is unlikely, by analogy to the fatty acid esters, that the sucrose esters would remain intact in the plant.m The second set of sucrose esters were water-soluble sirups, having surfactant properties, prepared from reaction products of hydroxyethyl .ethers of sucro~eor diglycidyl ethers of poly(oxyethy1ene glycol) with (2,4,5-trichlorophenoxy) acetic acid or other herbicidd acids.Ml

+

+

[aso

V. GIBBERELLINS AND KININS Gibberellic acid and related compounds, referred to generically as gibberellins, form another class of naturally occurring, plant-growth regulators (265) C. Steinhart, L.Anderson, and F. Skoog, P h i Phgeiol., 81, 60 (1962). (266) H. G . Coore and P. J. Randle, Biochcm. J., Q1,bB(1964).

(267) G. A. Lewy, A. J. Hay, and J. Conchie, Biochsnr. J., 89, 102~) 103~(1964);Ql, 378 (l9M). (258) R.Brown and M. Edwarda, Ann. Botany (London), 8, 131 (1944). (269) R.Brown, A.W. J o h n , E. Robinson, and A. R. Todd, Proc. Roy. 8oc. (London), SW. B, i a ~ 1, (1949). (260) H.Domaneka and 1;. Eckstein, Romiki Nauk RolniugcA Ser. A, 88, 69 (1963); &gar Id.Abetr., 48, 671 (1964). (261) A. W.Andenson, U.S.Pat. 2,927,919(DowChemical Go.), imed March 8, 1960; Chem. Abelractu, 66, 14143 (1980).

PUNT-OROWTH SUBSTANCE8

417

or hormones. They produce cell, leaf, and stem extension, especially of dwarf mutants, induce flowering in longday plants during shorbday growth, and break dormancy in seeds and buds. Gibberellic acid salts are URed commercidly to induce flower-induction, to enlarge fruitrsize (e+ pecially in grapes), and to accelerate barley-mlting. The chemical structure

a

Gibberellic Acid (m. p. 232-235’ (dec.), [ u ] z +Q2.0’) (16)

according to Cross and co~orkers,2d8-*~~ namely (16) , as revised, has been derived from that of terpene precursors. The most obvious external effect of gibberellic acid on flowering plants is the rapid increase in shoot length. The total (green) weight of the plant may, however, increase, remain unchanged, or decrease, depending on the genotype, on the amount of applied chemical, and on the growth environment. The gibberellins are not particularly phytotoxic, even at excessive dosages, producing only minor rookinhibition and leaf chlorosis. The chemistry and growth responses have been extensively r e ~ e ~ e d . ~ l ~ ~ The effect of gibberellic acid on amylase in barley was first noted by Hayashi.*” The grains softened, releasing soluble carbohydrates into the medium, and increasing the quantity of extractable amylase without affecting the enzyme activity. The symptoms were analogous to those of the breaking of dormancy just prior to germination; maltose was formed from the starch reserve in the endosperm. If the embryo portion was cut away and discarded, gibberellic acid could replace the natural substances (from the embryo) which diffused into the endosperm under conditions (202) B. E. Crow, J. F. Grove, J. F. MscMillan, and T. P. C. Mulholland, Chem. I d . (London), 954 (1950).

(203)

B. E. Cross, J. F. Grove, P. McCloakey, and T. P. C. Mulholland, C h . Id.

(London), 1345 (1959). (204) G. Stork and H. Newman, J. Am. Chem. Soc., 81, 5618 (1959). (265) F. H. Stodola, “Source Book on Gibberellins (1928-1967),” Agr. Res. Service, U. S. Dept. of Agr., Peoria,Illinois, 1958. (266) B. B. Stowe and T. Yamaki, Ann. Reu. Plant Phyaiol., 8, 181 (1957). (267) T. Hayashi, Bull. Agt. Chem. Sw. Japan, 18, 531 (1940).

418

H. W. HILTON

favorable to germinstion. Gibberellic acid concentrations of from 2 X 10-4 pg to 2 X 104 bg promoted hydrolysis of starch; maltose accounted for 15%, n-glucose for So%, and D-fructose, sucroee~,and, probably, maltotriose and maltotetraose, made up the remainder of the reducing substances.268 With sterile conditions, oxygen uptake remained constant, and gibberellic acid activity WM due entirely to induced production of enzymes capable of hydrolyzing starch. The enzyme resembled amylase, rather than Bamylase, as measured by the shift in the wavelength maximum of the iodine reaction and by the heat stability of the enzyme from potato starch. At the optimum concentration of gibberellic acid (5.8 X 1od M ) at 30°, barley endosperms lost 50% of their dry weight in three days, and this loss was attributed to hydrolysis of starch, protein, and part of the cell-wall constituents. Eighty percent of the soluble portion could be accounted for as D-~IUCOSBor protein nitrogen. Iodoacetate and other enzyme poisons inhibited starch conversion by reacting with thiol groups of the enzyme,28Q and oxygen,270 Case, and n1K@ promoted enzyme activity. The effects suggested participation by several enzymes, although a-amylase is currently considered to be the major product.*s4 Evidence of cell-wall breakdown rests on the identification,in treated malt after seven days, of small amounts of arabinose and xylose which are not present in untreated malt.n6 The carbohydrates, separated chromatographically on carbon by gradient elution, inoreased (in the treated malt) in maltotriose (188%) ,maltose (33%) , isomaltose (13%) , D-glucose (820/,), wfructose (13%) , sucrose (16%), “glucodifructose” (5773, and unidentified di-, tri-, and tetrtcaaccharides. The stimulating effect on ketoses could not be entirely explained by a-amylase production. It is now supposed that gibberellic acid stimulates tho hormonal production of a-amylase (and, probably, of &amylase and others) in the aleurone layer surrounding the endosperm starch-reserve. Gibberellic acid released 400 mg. equivalents of wglucose per g. in 90 hours in wheat grains, with a maximum hydrolytic activity after 60 hours. The effect on pamylase was thought to be a release (to the extent of 85%) from the protein-bound, insoluble form.fl6 (268) L. C;. Paleg, Plant Phyeiol., 86, 293, 902 (1960); 86, 829 (1961). (269) J. E.Varner, Plant Physid., 89, 413 (1964). (270) D.E.Brigge, J . Iml. Brewing, 68, I3 (1963). (271) (272) (273) (274) (276) (276)

J. Yomo and H. Iinumlt, Agr. I3ioZ. Chem (Tokyo), 27, 70 (1963). L. (4. Paleg, D.H. B. Sparrow, snd A. Jenninw, Plant Physiol., 37, 679 (1962). L. G. Paleg, B. G. Coombe, and M. 9. Butt-, Phnf Phyttiol., 87, 798 (1962). L. G. Pdeg and B. Hyde, Plant Phy.iol., SB, 673 (1864). B. Drewa, H. Specht, and H. J. Pieper, Branntweinwirtschuft, 109, 377 (1963). E. V. Rowsell and L. J. Goad, Biochem. J . , 90, 1 1 ~ 1, 2 (1964). ~

PIJANT-QROWTH SUBSTANCE8

419

One paper (not seen by the present author in the original) suggested that gibberellic acid increased the growth in starch-containing plants (such aa taro, water chestnut, canna, and rice) by increasing amylase activity to make the reserve polysaccharide available. Plants lacking reserve starch (garlic, onion, and narcissus) did not respond.” Whether this increased hydrolysis would l e d to decreased (fresh or dry) weights fromthe loss of reserve, or to increases by a shift to other plysaccharides or proteins, fats, and 80 forth, is not apparent from the ab8tract.m Perhaps, favorable growth-conditions, temperature, day length, and other variables would influence the ultimate yield more than the available carbohydrate. Stem elongation induced by gibberellic acid is generally accompanied by a reduction in stem diameter, and by reduced and altered leaf shape and size. It seems unlikely that increases in dry weight occur with any regularity, except perhaps in the dwarfed genotypes. Effects on the starch content from applications to starch-storage crops have been variable. Soluble carbohydrates in other plants (such as sugarbeetsnaBnoand cabbagem) have been claimed to be increased, those in corn were increased”’ or decreaaed,m and they decreased in other p1ants.m In cotton, gibberellic acid so reduced the reserve polysaccharide that the cell-wall and protoplasm substances must have been metabolized to provide energy for celldivision activity. The reduction in dry weight accompanying elongation must have resulted in “diluted” or thinned cell-walls. Inhibition of cell division with indole-3-acetic acid had the opposite effect, of increasing starch and dry weight.” Reduction in starch and increased soluble carbohydrate coincided with a reduced uptake (40%) of mPin corn, in which duration of the effect depended on the concentration.% Photosynthetic activity, measured aa uptake of carbon dioxide, depended on the leaf area. Gibberellic acid did not influence photosynthesis per unit (277) S. Lo and H. Wang, Shih Yen S h g Wu Hmeh Poo, 8, (3-4), 576 (1963); C h . Abekoete, 60, 16433 (1964). (278) G. A. Evtuehenko, Akad. Nauk SSSR, Inst. Fizwl. Rast., 110 (1963); C h m . Abatr&, 60, 16067 (1964). (279) N. I. Yakushkina and E. K. Artemova, Akud. Nauk SflSR, Inst. Fiziol. f i s t . , 121 (1963); C h . Abstracfs, 80, 16067 (1964). (280) E. Ye. Ermolaeva, N. A. Koslova, and A. F. Bel’denkova, A&&. Nu& SSSR, Imt. Fizkd. h l . , 149 (1963); Chem.Ab8fr&s, 60, 16066 (1964). (281) I. L. Zakhar’yants and A. 8.Ioneaova, A M . Nauk SSSR, Inst. Fitiol. Raet., 161 (1963); C h m . Ab8trmt8, 60, 16066 (1964). (282) S. Iatatkov, lev. Inat. po Biol.MetodiiPopov, Bulgar. A W . Nauk., 18, 121 (1983). (283) L. A. Lebedenko, Acta Botan. A d . Sci. Hung., 9 (1-2), 86 (1963). (284) L. 8.Dure and W. A. Jensen, Botan. Om.,118,264 (1956). (286) Yu. P. Starchenkov, Vim. 8il18’kog~dar.Nuuki, 8 (7), 36 (1960); Chem. Ahtracte, 66, 1826 (1961).

420

H. W. HIWON

of leaf area, although gibberellic acid indirectly affected photosynthesis by altering the leaf area: treated rice did not change in leaf area, but decreased in its content of starch, whereas tomato increaaed its leaf area, but showed little change in the ratio of starch to reducing oarb0hydrate.m Gibberellic acid and (2 ,Uichlorophenoxy)acetic’ acid increased the glycoside content of Ormithogalurn umbeZEatum, as meeeured by a biological assay of extracts. The active principle, strophanthidin, is one of the cardiac g1ycosides.m There is little reference in the literature to this type of work, but the hydrolytic effects of gibberellic mid on starch may shift the equilibrium toward phenyl glycoside formation in some plant species. Various gibberellins exist as glycosides in the plant system. The 0-w glucoside of gibberellic acid waa prepared, M a noncrystalline substance, in cucumber leaves in vitro, as establiied by Bwglucosidase hydrolysis and by chromatography.mo”o A presumed P-glucoside has also been isolated from sugarcane as a gummy substance sohble in water.m Another compound, isolated originally from autoclaved or aged deoxyribonucleic acid, and identified as 6-(2-furfurylamino)purine (kinetin), (17) has a powerful initiating effect on cell division. The adenine moiety is

necessary; other replacements (such as phenyl, benzyl, and amyl) give products that are known generically as kinins or kinetinoids. Not one has been conclusively demonstrated in plants, but they are of considerable interest, as their activity resembles the action of red At an optimum concentration of lo-‘ M , kinetin acted similarly to gibberellic (286) T.Hayaahi, in “Plant Growth Regulation,” R. M. Klein, ed.,Iowa State Univ. Press, Amea, Iowa, 1901, p. 679. (287) P. Soresuchart, J. A. Smith, and G. R. Patarson, Can. Pharm. J., Sci Sect., 96, 496 (1962); C h .Abatmctu, 68, 4979 (1863). (288) G. Sembdner,G. Sohneider,J. Weiland, and EL.Sahreiber, Ezperientia, 10,89 (1964). (289) Y. Murakami, Botan. Mag. (Tokyo),74, 424 (1981). (280) B. H. Moat and A. Hughes, Ann. Rapt., Tat6 and Lyk Centml Agr. Rss. Sia., Trinidad, Weat Idkf,lW, p. 279. (291) H. Buratdim, in “Encyclopedia of Plant Phyaiology,”W. Ruhland, ed., Springer-

Verlag, Berlin, 1961, Vol. 14, p. 1166. (292) J. A. Zwar, M. I. Bruce, W. Bottomley, and N. P. Kefford, in “Regulateurs Nature18 de la Croiasanoe Vegetale,” J. P. Nitach, ad., Centre National de la

Recherche Scientifique, Paris, 1964, p. 123.

PWNTGROWTE SUBSTANCES

421

acid at 1W6M in stimulating amylase activity or production in wheat, without affecting phosphorylation. The 42% increased elongation of stem segments corresponded to that produced by D-glucose at 0.1 M ,suggesting that liberation of D-glucose from starch brought about growth as an indirect effect.2g8There is a conflicting view that kinetin stimulates the formation of polysaccharides in pea epicotyl at the expense of mono- and di-saccharides. The polysaccharide formation proceeded first in cellulose and othor cell-wall constituents, an8 in starch; considerable phosphorylase stimulation occurred in this case.294

VI. THEEFFECTS OF PUNT-GROWTH SUBSTANCES ON SUQARCANE Sugarcane is an intensively cultivated, perennial-grass crop grown throughout the world in a belt lying between about 30"N.and 30%. latitude. Its ability to store sucrose in the stalk over long periods of time (2 years or more), without a definite maturity or death, makes it one of the most efficient converters of the energy of sunlight into food. The rate of growth is not uniform throughout the plant's life, because of competition for nutrient and sunlight; and practical considerations make necessary the harvesting and processing of the crop at somewhat less than ultimate yield. Excess nitrogen and reducing carbohydrates are brought to a minimum at harvesting, because they are detrimental to processing, especially because of color formation due to heat, or interfelrencewith sucrose crystallization. Green leaves and apical stem tissue are undesirable, as they contribute a considerable amount of reducing carbohydrate, colored pigments, amino acids, and other organic substances detrimental to factory recovery of sucrose. Finally, the water content of the stalk should be lowered before harvesting, both for greater sucrose concentration in the stalk juice and for lowered D-glucose and D-fructose contents. The many aspects of sugarcane culture have been discussed by Burr and coworkers,@6and the physiological aspects of production of sucrose in the leaves, and its transport and storage in the stalk, have been detailed.2w-2ge (293) D. Boothby and 8.T. C. Wright,Ndute, 198, 389 (1962). (294) W. Maciejeweke-Potapcaykowa and H. Lukasiak, Ada Soc. Botan. Polon., 28, 96 (19S9). (296) G. 0. Burr, C. E. Hartt, H. W. Brodie, T. Tanimoto, H. P. Kortschak, D. Takahashi, F. M. Ashtan, and R. E. Coleman, Ann. Rev. Plant Physdol., 8, 275 (1957). (!!96) c. E. Hartt, Huwnaiian P&a&r8' &cord, 41, 33 (1937); 4 , 8 9 (1940); 47,113,166, ,223 (1943); 48, 31 (1944). (297) C. E.'Hartt.,H. P. Kortschak, A. J. ForbeB, and G . 0. Burr, Phn6 Physiol., 88, 306 (1963); (298) H. P. Kortschak, C. E. Hartt, and Q. 0. Burr, Plant Physiol., 40, 209 (1966). (299) K. T. Glasriou, Plant Phyuiol., 86, 896 (1960).

422

H. W. HIWON

Sucrose storage is an active process in which the stored sucrose in the stem cells is in equilibrium with small proportions of *glucose and D-fructose. Growth-promoting mechanisms (chemical or otherwise) are more apt to stimulate hydrolysis of sucrose, a d utilization of the monosaccharides in respiration and formation of new tissue, than to promote storage. In spite of this somewhat obvious conclusion, numerous attempts have been and are being d e to increase sucrose production both in sugarbeet and sugarcane by the use 6f growth-promoting, chemical treatments, Three areas have received the m?st attention: control of flowering in sugarcane to increase vegetative growth, desiccation of green leaves at harvest to lower the water content and the extraneous leaf trash, and control either of sucrose production or recovery by the use of chemical treatments, usually applied just prior to the harvesting period. The use of chemicttls for weed and insect control in sugarcane is common, but there is little information that shows that sucrose or yield is affected, unless injury is Revere and prolonged. Effects of externdlly applied chemicals on the living, intact plant are usually reversible, at least up to the point of extreme phytotoxicity. Chemical effects on (a) sucrose production, (b) equilibrium of sucrose with reducing carbohydrates, and (c) conversion into new tissue or respiration energy are alao likely to be reversible with time. The direction and amount of an effect will depend, as in other plants, on concentrations of the chemical, the stage of plant growth, the variety of plant, and environmental factors. The rate of growth determines the ultimate crop yield, but the rate of sucrose production may or may not follow the total-yield curve. This suggests that chemical treatment may aim either for increased total yield or for increased sucrose recoverable from the same yield. The latter might be accomplished by decreasing water, nitrogen, or reducing carbohydrates, ILS well as by increasing the percentage of sucrose per unit of dry matter. It seems obvious that there would be an osmotic limit to the sucrose concentration in the cell sap, and that more energy would be required to accumulatesucrose against an increasing concentration gradient. Sugarcane flower initiation is dependent on day length, temperature, age, moisture, and variety, and can be prevented by chemical applications at, or very close to, the date of floral initiation. The effective chemicals have been of two types: (a) photosynthetic inhibitors, such as 3- (p-chloropheny1)1, ldimethylurea (monuron), or (b) led-burning, contact chemicals. The very effective bipyridylium herbicides: 6,7dihydrodipyrido[l ,2-a:2’ ,1’clpyrazidinium dibromide (diquat) and 1 ,l’dimethyl-4,4’-bipyridinium bis(methy1 sulfate) (paraquat) combine the two properties, although the

PIA?N“+ROWI”T BUBSTANCES

423

mechanism for flower prevention is unknown. The herbicidal effect is temporary, and newly emerging leaves show no burn or chlorosis. Vegetative growth continues, resulting in higher ultimate yield than in flowering plants. The bipyridylium compounds have also been dsed experimentally to desiccate sugarcane leaves, and are more effective for this purpose than substituted phenols, oils, and chlorates. The yield of treated cane remained more or less constant for several weeks, and then increased again as new leaves formed. The yield of sucrose decreased as a result of treatment, partly because of the temporary reduction of photosynthesis, and partly as a result of increased stalk-moisture.800 Sugarcane can be artificially “ripened” near the harvesting period to increase tlucrose at the expense of water, nitrogen, and reducing substances. The choice of the term is unfortunate, for a plant which has no physiological ripening processes comparable to those of semnal plants. The st&moisture level decreases with age, from a maximum of 80-8501, to a minimum of about 70% in “ripened” cane. Sucrose formation is almost a mirror image of moisture content, varying from less than 10 to more than 45% of the dry weight. The older view has been that all factors affecting maturity or “ripening” do so by affecting the water content: increased water content results in decreased sucrose content.w1A more recent view is that nitrogen is 12180 intimately involved, and that “ripening” is essentially a reversible reaction which can be altered by water, nitrogen, growth substances, enzymes, decreased respiration, and photosynthetic a1teration.m Ideally, during “ripening,” photosynthesisshould continue while respiration decreases; however, photosynthesis is more sensitive to alteration than is respiration, and respiratory inhibition is likely to affect photosynthesis. Auxins stimulate both proceEses at low concentrations, but repress both at higher levels, although nothing is yet known as to the critical amounts present at the active site. Inhibitors of growth are likely to be more promising than stimulants for sugarcane “ripening.” Burr and coworkersm*observed that many factors have been found to decrease the formation of sucrose in sugarcane, but, up to that time (1956), the only factors which were known consistently to increase the formation Were a r i s i i temperature, improved aeration, and the addition of malic acid. (300) Reviewed by L. G. Davidson, Proc. Intern. Soc. Sugadane Techmlogiekl, 11, 319 (1962) (Publ. 1963). (301) S. V. Parthaeartithy and M. V. Rams Rao, Proc. Bien. Conj. Sugarcane Res. Workers Indkn Union, 1, Pt. 11, P-2, 5 (1951). (302) R. A. Yates and J. F. Bates, Proc. Brit. West Indies Sugar Technologists, 174 (1957).

424

H. W. HILTON

The auxins indole-&acetic acid and indole3-butyric acid, injected into sugarcane at the leaf base, stimulated adventitious rootdevelopment.aOa The same auxins plus l-naphthaleneacetic acid and 2-(o-chlorophenoxy) propionic acid, as soaking treatments for cuttings, lowered the reducing substances and increased the sucrose, perhaps by inhibition of sucrose phosphorylase. High concentrationsof the stme auxins reversed the carbohydrate effects." Invertase activation or synthesis by l-naphthaleneacetic acid, proposed by Sacher and Glaaaiou,806is reminiscent of the effect of gibberellic acid on a-amylase in starch-storage plants. However, indole-3acetic acid had the opposite effect on amylase in cotton.ou (2,4-Dichlorophenoxy)acetic acid and, to a lesser extent, the 2,4,5trichloro and other analogs have been tested and used on sugarcane more widely than any other chemicals. They are among the most common herbicides for dicotyledonous weeds in sugarcane, as they have very little effect on grasses. Beauchamp" in Cuba claimed to have found increases in sucrose averaging 0.5% on cane weight, from sprays of 1.25 lb. per acre applied to the soil 48 days prior to sampling. Five Ib. per acre decreased the yield of total carbohydrate by about the same proportion.amFoliar sprays or dusts, at even lower rates of application, all gave increases in sucrose after 20 and 40 days (over the untreated checks). Expanded testing, however, produced variable results; of 18 field tests, seven showed sucrose improvement, five showed decreases, and six were either not significant or inconclusive. Drought-stressed and mature sugarcane ftiiled to improve.m Beauchamp blamed over-mature cane and excessive applications of (2,Cdichlorophenoxy)acetic acid (about 1.5 lb. per acre) for the poor results in Puerto Rico reported by Loustalot and coworkers.MaBrazilian work in general confirmed the Cuban results, with increased sucrose, purities, and juice solids content up to ten days after foliar application of (2,4dichlorophenoxy)acetic acid salts and powders at low rates (of about 10 to 30 g. per acre). The discussion described a need for many precise experiments to provide rigorous, statistical analysis, but there is no published evidence of further testing either in Cuba or Indian work has tended to support the claims of increased sugar production, approximating 0.5% of sucrose on cane tonnage, from applications of (2,4dichlorophenoxy)acetic acid of from 0.025 lb. per acre to 3 lb. or more per acre, on different varieties, autumn- and spring-planted cane, (Ms) Anon., Prm. Htawaiian S W T Plantcre' A88OC., Rspf., 68, 30 (1938) (Publ. 1939). (304) W. C. Hall and M. A. Kahn,Bohn. Gtu., 116, 274 (1966). (306) J. A. Sacher and K. T. Glanziou, Biochdm. Biophp. Rss. Commun., 8, 280 (1962). (306) 9. E. Beauchamp, PTOC. A8m. Tec. A-T. Cubu, 48, 69 (1949); 24, 147 (1950). ($07) C. E.Bsauohamp, Sugar J., 13 (51, 57 (Oat. 19aO); 13 (61,20 (Nov. 1950). (308) A. J. Loustalot,, H. J. Cruzado, and T. J. Muzik, S U ~J., T 13 (5), 78 (Oct. 1950). (309) Anon., B t a ~ i Acucarciro, l 87, 328 (1951).

PLANT-OROWTH SUBSTANCES

425

irrigated and nori-irrigated, and from two to about 30 days after treatment.a’* *’) I t is difficult to arrive at a definite conclusion from this work. The major criticism which has been made of this and similar testing is that at least 15 samples are required in order to provide statistical significance at 0.5 70 sucrose based on cane yield. Variations in the yield from field blocks are commonly as high as lo%, and often more. Some of the demonstrated “increases” in the percentage of sucrose in the sugarcane have been accompanied by overall decreases in sugarcane yield per upit area. Other experiments have shown no effects on yield. In Jamaica,a no effect wm found from (2,4-dichlorophenoxy)acetic acid, but positive responses were obtained from low rates of application (less than 0.009 lb. per acre) of (2,4,5-trichlorophenoxy)acetic acid asd 2- (2,4,5trichlorophenoxy) propionic acid. Louisiana sugarcane failed to respond to the low rates of (2,Cdichloro- or (2,4,5-trichloro-phenoxy)acetic acid, but (2,4dichlorophenoxy) acetic acid at one and three lb. per acre increased the sugar yield significantly at eight days, although not at 25 or 39 days.S16 No significant differences in sucrose a t rates of 0.25, 0.5, 1, 2, 10, 20, and 30 lb. per acre were reported from Puerto Rico,117and from experiments in Australiaal*nl and in Hawaii,a22 where sucrose decreased at higher rates. Maleic hydrazide has been applied to sugarcane in numerous tests, because it was thought to slow terminal-growth without affecting photosynthetiis. Only one positive report has been madeaz8of 0.86% sucrose (310)S. C. Varma, Proc. B i a . Cmcf. Sugarcane Res. Deuel. Workers Indian Union, 2, Pt. 11, 626 (1954). (311) P.S. Mathur, Proc. Bien. Conf. Sugarcam Res. Devel. Workers Indian Union, 2, Pt. XI, 637 (1954). (312) A. 8. Chacravarti, D. P. Srivasteva, and K. L. Khanna, Sugar J . , 18 (6), 23 (Nov. 1955). (313)A. S. Chacravarti, D. P. Srivastava, and K. L. Khanna, Current Sci. (India), fu, 316 (1955);26, 302 (1956).Chm. Abstracts, 60, 17299 (1956). (314) A. S.Chacravarti, D. P. Srivastava, and K. L.Khanna, Proc. Inlcn. Soc. SugarCane T e c h n o ~ b9,, (1956),Val. 1, 355 (Publ. 1957). (316) A. 9. Chacravarti, D. P. Srivastava, R. D. Sahi, and K. L. Khanna, Proc. Zndiun Acad. Sci., Sect. B, 46, 9 (1957). (316) R. E.Coleman and L.P. Hebert, Sugar Bull., 86, 389 (1957). (317) M. A. Lugo-Upez and R. Grant, J . Agr. Uniu. P w ! o Rico, 86, 187 (1952). (318) H.C. Haskew, Cane &mers’ Quart. BuU., 17,52 (1953). (319) H. C. Haskew, Sugar J., 17 (l), 34 (1964). (320) L.G.Vallance, Ann. Rept. Bur. Sugar Exp. 8b.,Brdsbane, 66, 21 (1955). (321) Anon., AuakalQanSugar J., 47.37 (1956). (322) Anon., Hawaiian Sugar Planbe’ Assoc. Ezp. Sta. C m m . Rcpt., 67 (1956). (323) M.8.Subba Rm, M. H. Haque, R. B. Prasad, and K. L. Khanna, Current Sn’. (India), 26, 116 (1967).

426

H. W. HIXA'ON

increase per unit weight of cane on four varieties, using 100 mg. per liter spray solutions. No significant differences have been found in Puerto Rico:Z4 Louisiana?"JJamaica,8o2Hawaii,aB and Australia,8*6although in the ~ found at 49 days in young cane, laat country, small gains in R U O ~ Rwero but not in niore mature cane. l'ctmporrwy uxuewcs of BUOIUIJOiir the leaves and upper portions of the treated stalk call be accounted for aa being the result of depressed growth or poor trtmnslocation. Varietal, seamnal, and age effects cannot be ruled out, but it is obvious that little information of general applicability has been gained from this type of experiment. Such effects as have been noted appeared during younger, more vigorous growth of cane, not at the more mature, harvatabie age when the increases are most desirable. AlexandeP has tried to assess the effect of indole-3-acetic acid, (2 ,4 dichlorophenoxy)acetic acid , and maleic hydrazide on the soluble carbohydrates and enzyme systems in sugarcane leaves. All of the chemical treatments increased sucrose, total reducing value, wfructose, and D-glucose in leaves (compared with the controls), with a maximum at nine days after applying about two g. per plant. The indole auxin increased sucrose most, followed by the phenoxy compound and the hydrazide; D-glucose increased in the reverse order. Poor translocation from the leaves may have caused the temporary increase in leaf photmynthate. Alterations in the enzyme activities 811 a result of the chemical treatment are difficult to interpret, partly aince so little is known about their relative importance, and partly because the activity in the controls varied by as much tm 100% from one sampling period to the next. Many other compounds have been included in studies on sucrose response. Moat of these have been herbicides or ensyme poisons. None of the common herbicides had any positive effect on sucrose at rates up to that causing severe foliar injury. Earlier reports of response from 2-(2,4,5trichlorophenoxy) propionic acid and 2 ,2dichloropropionic acid could not be substantiated in British Guiana and &ueensland.Pn Some compounds, such as 3- (pchlorophenyl) -1,ldimethylurea (monuron), (2 ,Cdichlorophenoxy) acetic acid in soil, ethylenediaminetetrucetic acid, and leaf desiccants decreased aucroae and juice solids content.8s Field trials with several chemicals in Trinidad showed enhanced sucrose at 14 to 28 days before harvest resulting from the application of 8 and 12 lb. (per acre) of (324) M. A. Lugo-Lbpez, 0. Samuels, and R. Grant, J . Agr. Univ. Puerto Rico, 57, 44 (1983). (326) J. C. Skinner, Tech. Cmmun., Bur. Sugar Exp. Sh.,Briebaw, No. 1 (1960). (320) A. G. Alexander, J . Agr. Univ. P w d o R h , 49, 1 (19sa). (327) R.A. Yatee, Tropical A@. (London), 41, 2% (1964). (328) H. Evans and J. F. Batee, Proc. Intern. Soc. Sugat4aw Technobgiata, 11, (1962) 298 (1883).

PLANT-QROWTH SUBSTANCES

427

2,3,6trichlorobenmic acid or its mixture with (2-methyl-4-chlorophenoxy) acetic acid. One application by air, at four lb. of the mixture per acre, significantly increased sucrose at the 0.01 significant level at 28 days, but not at 9, 14, or 20 days. Lawrie believed that the trichlorobenmic acid was the main cause of the improved quality.aa No further report was made in 1963 or 1964, but this is the only report of positive response which k not been tested and reported elsewhere. Enzyme poisons have been studied mainly in attempts to understand the normal physiological processes. As would be expected, synthesis and accumulation of sucrose have often been drastically lowered. In particular, the inhibition of the formation of D-fructose diphosphate and the conversion of D-glucose into =fructose inhibited the formation of sucrose.290~a90 3-(3,CDichlorophenyl)-1 ,1-dimethylurea (diuron) treatment of a plant crop of sugarcane has been proposed as the cause of severely reduced crop yields and altered enzyme activities in the ratoon crop 25 months after the treatment, but without determining the presence of diuron residue. The sucrose level in cane was not significantly alter+, although the enzyme activities were considered in relation to their effect on sucrose synthesis. In particular, the amylase activity decreased while maltase increased."' The conclusion that, in sugarcane leaves, starch constitutes a primary source of &glucose for sucrose synthesis is not supported by the results of radiocarbon work~Ja2Jsa in which only a trawglycosylase could be demonstrated to synthesize sucrose by transfer of D - ~ ~ U C OtSo~ D-fructose from uridine 5-(~-glucosylpyrophosphate) The glucose is formed in sugarcane by direct photosynthesis. Gibberellic acid applied to stem cuttings before planting, to the leaves, or to the apex of growing plants affects elongation markedly, produ~in@*~*

.

(329) I. D.Lawrie, Ann. Rept. T a b and Lyla Central Agr. a s . Sta., Trinidad, Wed Indim, 96 (1961-2). (330) R. L. Bieleski, Ausbalian J . Biol. A%., 18, 221 (1960). (331) A. G.Alexander and J. G. Ibtifiez, J . Agr. Univ. Puarb Rko, 48,284 (1964). (332) C. E.Cardini, L. F. Leloir, and J. Chiriboga, J . Bid. Chem.,284, 149 (1966). (333) R.B.Frydman and W. Z. Hamid, Nature, 199, 382 (1963). (334)C. E.Chardon, Ann. Meeting Aseoc. Sugar TcchnicMIM, Puerto R h , 1966. (336) Anon.,HawaiMn Sugar Planted Aseoc.Ezp.Sfa. Comm.R@., 11 (1956);19 (1967). (336) R.E.Coleman, Sugar Bull., 86, 24 (1967). (337),R. E. Coleman, Sugar J., 20 (ll), 23 (April 1968). (338) R.E.Coleman, E. H. Todd,I. E. Stokes, and 0. H. Coleman, Proc. Infern. Soc. Sugar-Cane T e c h m ~ b10, , 688 (1969,h b l . 1980). (339) R.E.Coleman, E. H. Todd, I. E. Stokes, and 0. H. Coleman, Sugar J., 2S (3), 11 (Aug. 1980). (340) H. Chang and R. Lm, Rept. Taiwan Sugar Expt. Sta. (Taiwan), 28, 121 (1962), having an English summary and tables. (341) H.P.Varma and S. A. Ali, Indian S u p , 12, 636 (1983). (342)T. A. Bull, Awrtrdian J . Agr. Res., 16, 77 (1984).

TABLE II The ~

e t rof , G;bbenllr' c

Treatment

Cuttings soaked, or spray Apex, weekly treatdnents Spray, 3 treatments Cuttings soaked, leaf w y , soil drench a

Key:

Rderences

J3ffectsnon

Length

Apex, 4 treatme& at %week btervah

Acid on Sugarcane

+

+ (early1; +

nf4

(-1

+ (early);nf4 (finsl) + (early); (final) 119

Fnehwt.

Sucrose or

Per plant

quality increase

-

+

-

341

-

11s

336,337

-

+

+

9

342

2:

119

ns

119

335,340

L

119

ns

338,339

Girth

+, increase over control; -, decrease over control; ns, no aignificant difference.

T:

a

z

PIAVI"TR0WTH SUBSTANCES

429

increases in length of 30 to 100%. Girth and overall platit weight measuremcritn have hccn variable, as Ttiblc I1 shows. Variety, age, temperature, arid timing of applications produced differences. Multiple treatments at thrcc- to four-week iiitervals were needed in order to maintain significant differences for proloiiged periods. Treatments reduced tillering (suckering) and germination of buds, but did not affect flowering. Repeated sprayings produced length increases of up to 30 inches (over controls), but did not affect final yield, juice solids content, sucrose, or purity. Higher concentrations (loo0 mg. per liter) lowered the yields. Gibberellic acid responses appeared to be greater where growth conditions were least favorable and normal growth was suppressed.

VII. ABSCISSION AND RIPENING Defoliation and fruit- and flowerdrop are responses to auxin changes. A number of synthetic compounds, such rn l-naphthaleneacetic acid or its amide, l-naphthyl N-methylcarbamate, (2,4 ,Btrichlorophenoxy)acetic acid, and other phenoxyacetic and phenoxypropionic acids, are used for promoting or inhibiting fruit- and flower-set or -drop, for regulating fruit size and maturity, and for defoliating such plants as cotton or potatoes.'8-a"6 The auxins generally retard abscission, probably by stimulating growthprocesses rather than by a direct effect?4aThe carbamate compound promotes fruit drop, and higher concentrations of the auxins have a similar result. At optimal concentrations, the synthetic auxins apparently make up for a deficit of natural auxin, which is, perhaps, one of the effects of aging, leading to a breakdown of the cell-wall membranes, possibly by polygalacturonase or pectic methylesterase. From the carbohydrate point of view, the main point is that the cell wall (including the cellulose) finally dissolves, probably by enzymic hydrolysis; whether this 'occurs as the result of an auxin gradient across the petiole has been argued."'JU Nitrogen, sulfur, magnesium, and zinc deficiencies, and 'presence of oxygen, ethylene, and alanine stimulated abscission; carbohydrate applied distally (on the leaf side) delayed it. 2,3 ,bTriiodobenzoic acid produced effects opposite to those of auxin addition. Gibberellic acid promoted abscission at all concentrations, possibly by acceleration of protein hydrolysis and polysaccharide hydrolysis; kinetin (343) P. C Marth, W. V. Audia, and J. W. Mitchell, J . Agr. Food C h . ,7, 122 (1959). (344) F. Addicott, in "Encyclopedia of Plant Physiology," W. Ruhland, ed., SpringerVerlag, Berlin, 1901, Vol. 14, p. 829. (346) A. C. Leopold, Ann. Rev. Plant Phyeiol., B, 281 (1958). (346) W. P. Jacobs, M. P. Kaushik, and P. G. Rochmis, Am. J . Botany, 51,893 (1984). (347) B. Rubenstein and A. C. Leopold, PZud Phyeiol., 88, 262 (1963). (348) S. K. Chatterjee and A. C. Leopold, Plunf Phyeiol., 88, 268 (1963).

430

H. W. HILTON

had little effect, except to delay abscissionw at relatively high mncentrations of M. A relationship was found between the awin present and ethylene evolution, and it has been suggested that aoceleration of abscission may be due to ethylene.am The concentration of ethylene increaaes in fruit during ripening, and it is considered to be the natural ripening hormone.”ldM It is formed from G 5 and G6 of D-glucose, presumably through a triose, and BurgF-W believes it to be present throughout the life of the plant, but to be triggered to greater production by some regulatory mechdsm. The conversion of starch to soluble carbohydrate, associated with ripening of many fruits, is probably a result of the overall aging processes rather than a direct effect of the ethylene. Yang and How proposed that phosphorylase, not amylase, hydrolyzes starch to Dglucosyl phosphate, which then forms sucrose. The soluble pectin fraction increases. The concentration gradient across the fruit skin wm related by Bur@a2 to the rate of ethylene production by a factor of 2 ppm/pl./kg./hr., and the response was related to the log of the internal content of ethylene. Application of ethylene-“(7 to plants resulted in only a 2.4% conversion into soluble carbohydrates, 11% into ether-soluble materials, S.9yo into phytol, 31.7% into cellulose and lignin, and 9.6% into soluble protein and non-protein material, mainly ph0sphates.m Treatment of detached fruit (such ae apples, bananas, peaches, figs, and pears) with synthetic auxins, especially (2 ,4,5-trichlorophenoxy)acetic acid, speeded up ripening, aa indicated by color, taste, softness, and starch breakdown.” Other fruits have been similarly ripened,’oBe-861 and the treatments are effective both on climacteric and non-climacteric fruit. (349) (360) (361) (362) (363) (364) (366) (368) (357)

(368) (889) (360) (381) (382)

8.K. Chatterjee and A. C. Leopold, P h d P h W . , 80, 334 (1984). F. B. Abelea and B. Rubenetain, Phnt Phw&Z., 80, 903 (1984). 9. Y. Burg, Ann. Rev. Plant Phyaiol., 18, 286 (1982). 8.P. Burg and E. A. Burg, Phnt Phybiol., 87, 179 (1982). S. P. Burg and E.A. Burg, Science,l#, 1190 (1986). S.P. Burg, in “Ri3gulaburs Naturela de la Crohanae Vegetale,” J. P.Nitsch, ed., Centre National de la Reohemhe hientifique, Paria, 1984, p. 719. 8.Yang and H. Ho,J . Chiwe Chsm. Soc. (Taiwan), 6,71 (1968). W. C. Hall, C. 8. Miller, and F. A. H e m , in “Plant Growth Regulation,” R. M. Klein, ed., Iowa State Univ. Preea, Am-, Iowa, 1981 p. 761. J. W. Mitahell and P. C. Msrth, B&n. Ow., 106, 199 (1944). J. R. Blske and C. D. Steveneon, Qumdund J . A p . Sci., 18, 87 (1969). W. 8.Kemp and D. W. W h n , New Zsakrnd J . Ap., 01, 106,607 (1956). P. C. Marth and J. W. Mitahell, Bobn. Qaa., 110,614 (1949). P. 0.Marth, W. V. Audia, and J. W. Mitahell, B&n. Ght., 118, 108 (1958). W.B. Data and P. B. Mathur, Food Sci. (Mysore), 9, 248 (1960).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES* BYI. J. GOLDSTEIN AND T. L. HULLAR Department of Biologiml Chemistr& Univweily of Mi&gan, Ann Arbor, Michigan, a d Departmen4 of M e d i c i d Chmiatw, S h l of P h i a m , State University of New York at Bug&, Buflalo, New York

Dedicated to the late Professor Fred Smith, who taught ua our carbohydrate chemistry. I. Introduction. ........................................... ............. 431 1. s c o p e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .433 ....... 11. Condensation Polymerization.. . . . . . . . . . . . . . .. .. .. . . . . . . . . . . .. .. . 434 1. General.. . . . . . . . .. . . . . ...... . . ... .. . . , .. . . .. .. . . .. . 434 2. Polymerization in a Solvent. , . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 441 3. Polymerization in the Solid State.... . . . . . . . . . . . . .. .. . . . . . . . .. . . . . . 461 111. Addition Polymerisation.. . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . , . . . . . . 477 1. General ..... ....... .. . . .. ........ .,.... .. .. . .. .... . . .. .. .. .. ..... 477 2. Anhydro sugam.. ...* . . .. . . . . . . . . . . . .. . . . . . . . . * .. . . . .. ., * . 478 3. other............................................................. 491 IV. Methods of Study. ... . . .... . . . . . , . .. . . . . . . . . .. . . .. . . . .. . .. .. . . . 491 1. Ieole~on.......................................................... 492 2. F ~ d i o ~ t i ...................................................... on 492 3. Meeaurement of Homogeneity. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 494 4. Structural Analysis. . . . . . . . . . . . .. .... ... . . . . . . . . . . . . . .. . . . 494 6. Use of Eneymea ,... . . . . . . . . . ... . . . . ... .. . . . . . . . . . . . . . . . . . . . 502 6. Use of Immunochemistry., . . . . . . . .. . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . 504 V. Applications of Synthetic Polysaccharidea.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

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

.. ..

..

.... .. . . .. . .. . ... . . . I

.

.

.

.

I. INTRODUCTION Over the pmt century, organic chemists attempting the chemical syntheRia of specific polysaccharides have invariably obtained polymers having indefinite composition. However, a polysaccharide has now been synthesized which apparently possesses properties similar to that of naturally occurring cellulose.1 Synthesis has also been achieved1*of an apparently linear polysaccharide containing only (1 -+ 6)-linked a-D-glucopyranosyl

* The preparation of ‘thisreview waa supported, in pert, by a grant (GM 12992201) from the National h t i t u t e a of Health, U.S. Public Health SeMce. (1) E. Hueemenn and 0. J. M. MWer, MakrmZ. C h . , 91, 212 (1986);privsta communication from E. Husemann. (la)E.R. Ruckel and C. Schuerch, J. Am. C h m . h., 88,2606 (1968). 431

432

I. J. QOLDSTEIN AND T. L. HULLAR

residues. If confirmed, these would represent the first successful, stereospecific syntheses of carbohydrate polymers. This subject has been of continuing interest for several reasons. First, the present concepts of the chemical constitution of such important biopolymers tw cellulose, amylose, and chitin can be confirmed by their adequate chemicd synthesis. Second, synthetic polysaccharides of defined structure can be used to study the action pattern of enzymes, the induction and reaction of antibodies, and the effect of structure on biological activity in the interaction of proteins, nucleic acids, and lipides with polyhydroxylic macromolecules. Third,it is anticipated that synthetic polysaccharides of known structure and molecular size will provide ideal systems for the correlation of chemical and physical properties with chemical constitution and macromolecular conformation. Fihally, synthetic polysaccharides and their derivatives should furnish a large variety of potentially useful materials whose properties can be widely varied; these substances may find new applications in biology, medicine, and industry. For some purposes, it is sufficient to obtain polymers (of high molecular weight) whose structural features are highly varied and generally undetermined. Considerable success has been achieved in synthe&i this type of polymer. For other purposes, however, it is highly desirable to obtain polymers which possess a defined structure. Chemical synthesis of such a structure requires that there be rigorous control of the position and configuration of the glycosidic bond being formed, the ring size of the incorporated sugar residues, the extent of branching (if desired) , and the molecular size and distribution of the polymers. Adequate control of these several variables requires that suitably protected sugars enter into a mild, facile method of polymerization which insures stereospecific synthesis of the glycosidic bond and formation of the desired ring size. Only very limited efforts toward this more demanding objective have thus far been made. In the biological synthesis of carbohydrate polymers, there is complete control of the factors noted above. Enzymes (transglycosyhes) effect the serial transfer of glycosyl groups, in a highly stereospecific manner, to the ends of a growing polymer chain, all transfers being under strict physiological conditions of pH and temperature? It is the duplication of these syntheses, md,’furthermow,the preparation of new, highly ordered carbohydrate polymers that will occupy the carbohydrate-polymer chemist in the foreseeable future. Because the chemical synthesis of defined polysaccharides is in a crucial stage of its development, and because these synthetic polymers and their derivatives show definite signs of having application in the biological (2) P. Bernfeld, in “Biogeneeis of Natural Compounde,” P. Bernfeld, ed., Pergamon P m ,The Maomillan Company, New York, N. Y.,1963,p. 233.

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

433

sciences, it was considered appropriate to review the subject at this time and to assem the prospects for future progrm. Thia topic will be divided into condensation and addition polymerizations, aa defined in the subsequent discussion. Polysaccharides will arbitrarily be defined as polymers of ten or more eimple augar residues united by glycosidic bonds. Polymers containing less than ten such units will be considered to be oligosaccharides. In keeping with current usage, synthetic polysaccharides will be called “poIyglycoBe8,” for example, polyglucose, to d i a t i i h them from the naturally occurring polysaccharides.

1. scope Only syntheses which involve the formation of new glycosidic linkages will be considered in this article. This restriction excludes many interesting examples of copolymerization in which only one of the monomers is a carbohydrate (or carbohydrate derivative),Ithe polymerization of carbohydrate derivatives which contain a polymerizable group‘ (such aa acrylate) , and the polymerization of sugar 1actoncsP Many of these topics have already been discussed in reviews.6-8Ale0 outside the scope of this article is the chemical modification of naturally occurring polysaccharides~; thus, we have excluded the industrially important process of dextrinization,“Jexcept as it may pertain to acid condensation processes. The radiation-catalyzed polymerization and modification of carbohydrate poly(3) W, for example, J. M. van Bemmelen, J. Pmkt. C h . ,[l] 69, 84 (1856);M. L. Wolfrom, M. 8. Toy, and A. Chaney, J. Am. Chem. h c . , 80, 6328 (1958);M. L. Wolfrom, J. 0. WehrmWer, E. P.Swan, and A. Chaney, J. Org. Chum., 9, 1556 (1958);W. N.Haworth, R. L. Heath, and L. F. Wiggins, J. Chem. Soc., 155 (1944). (4) See, for example, P. L. Nichole and E. Yanovsky, J. Am. Chum. SOC.,66, 1626 (1944);R. H.Treadway and E. Yanovsky, ibid., 67, 1038 (1945);W. N. Haworth, H. Gregory, and L. F. Wiggins, J. C h . Soc., 488 (1946);T. P. Bird, W. A. P. Black, E. T. Dewar, and D. Rutherford, C h .I d . (London), 1331 (1960);8. Kimum apd M, Imqoto, Maktomd. Chum., 60, 155 (1961);R. L. Whistler, H.P. Pwer, and H. J. Roberta, J. Org. C h . ,26,1683 (1961); R.S. Theobald, J . C h . Soc., 6359,5365(1961);W.T.Bird, W. A. P.Blaak, J. A. Colquhoun, E. T. Dewar, and D. Rutherford, C b m . I d . (London), 1073 (1965). (6) H.D. K. Drew and W. N. Haworth, J . Chem. Soc., 776 (1927). (6)Yu. L. Pogoeov and Z. A. Rogovin, Rw8. Chum. Reu. (English Transl.), SO, 645 (1961). ’ (7) J. Klar, ChumiAw ZQ., 87, 731 (1963). (8)J. K.N.Jonerr, Pure A w l . Chum., 4,469 (1962). (9) B. J. Binee and W. J. Whelan, Chum. Id.(London), 997 (1960);B. J. Bines, Z. H. Gunja, and W. J. Whelan, ibid., 1358 (1960);R. L. Whistler and S. Hiram, J . Org. Chum.,26,4800 (1961);E. Husemano and M. Reinhardt, M a k r m l . Chum., 57,109,129(1962);M.L.Wolfrom, M. I. Taha, and D. Horton, J. Org. Chum., 28, 3563 (1963). (10)See, for example, “The Chemistry and Industry of Staroh,” R. W.Kerr, ed., Academic Press Inc., New York, N. Y., 2nd Edition, 1950,pp. 345,351;J. R.Kate, Rsc. Trw. Chim., MI, 565 (1934);B.Brimhall, Znd. Eng. Chum.,86,72 (1944).

434

I. J. GOLDIJTEIN AND T. L. HULLAR

mere is under active investigation, and has been surveyed in reviews.11 Three reviewe of the chemical synthesis of polysaccharideshave appeared.“* The historical aspects of the chemical synthesia of polysaccharideawill be considered in some detail. However, the main emphasis is on progress made during the paat 26 years. Selected examples of the synthesis of higher oliosaccharides are also included, since the methods used and the results obtained bear on the synthesis of polysaccharides. Discussion of modern analytical tools and techniques available for studying the nature of the synthetic products is included and, where possible, application of these techniques to the study of synthetic polysaccharides is described. 11.

CONDENBATION POLYMERIZATION

1. General Condensation polymerization has been defined by Floryl2 as a “polymerilitition process which proceeds by a reaction between pairs of functional groups with the formation of a type of interunit functional group not present in the monomer.” In this review, condemalion polynerization of carbohydrates is defined as a polymerization of eugm or their derivatives to give a polymer of sugar residuea joined together by glycoddic bonds and a by-product composed of the conjugate acid of the substituent originally present on the anomeric carbon atom.’* Thie definition may be illustrated by equation 1 , where HO is an (the) alcoholic hydroxyl group of a sugar moiety, X is the substituent on the anomeric carbon atom, G is the remainder of the sugar moiety, and G-4-G is the newly formed glycosidic 12 HO+X

HW-(O--G-),+-X

+ ( n - 1) HX

(1)

linkage. In the majority of the condensation polymerizatiom of sugars or sugar derivatives, X haa been a hydroxyl group; lesa frequently, it haa been a halogen atom or a carboxylic acid group. Before discussing the various methods used to achieve condensation polymerization of sugars and sugar derivatives, several factors will be (11) [Joe, for example, J. B. Snell, J . Pol-

Sci., Pt. A, 8, 2591 (19615);F. A. Bovey, “The Effects of Ionizing Radiation on Natural and Synthetic High Polymers,” Intemcience Publiuhers, Ino., New York, N. Y., 1958; a. 0. Phillip, Aduan. Ca&ohydv& C h . ,16, 13 (1881). (12) P. J. Flory, “Principles of Polymer Chemiwtry,” Cornell Univereity Prees, Ithaca, N. Y., 10153, p. 39. (13) For oonvenience, “polycondenaation” will be used as a synonym for oondeneation polymerbation, and “polycondeneate” will be ueed aa a eynonym for the product of a condewtion polymerbation.

CHEMICAL 8YNTHESI8 OF POLYSACCHARIDES

435

considered which iduence the course of the polycondenaation and t.he structures of the products. First, condensation polymerization of sugars and their derivatives is an equilibrium process (see, for example, equation W) , a nearly stoichiometric a C&O6

S

+ (a - 1) HsO

C~l,O,(C,H,,O,),,C3llOI

(8)

proportion of a by-product (for example, water) being liberated. Zechmeister14 suggested that the polycondensation of sugars is a reversible process; this was confirmed by Frahm,16 who determined the reducing values of solutions of D-glucose and cellulose in 40.8% hydrochloric acid, and found that both solutions reached the same equilibrium. To support further the idea of revenibility, Frahm" found that a 57% solution of D-glucose in,40.8% hydrochloric acid gave a reducing value 35y0 that of wglucose. Dilution of this solution to 15% of D-glucose in 40.8% hydrochloric acid gave, at equilibrium, the same reducing value (68% that of D-glucose) a8 wa8 obtained when a solution of 15% of D-glucose in 40.8% hydrochloric acid was allowed to reach equilibrium. This increase in reducing value upon addition of water corresponds to a decreaae in molecular size, and is the expected result if equations 1 and W are governed by the law of maas action. The position of equilibriumwas examined further by Silin and Srtpegina,l'J They found that both R 20% solution of wglucose and its polymeric equivalent, an 18% solution of starch, in 0.5 N hydrochloric acid, gave at equilibrium a reducing value 91.8% that of D-~~UCOSELFrom a solution of D - ~ ~ U C O in S ~ 75% sulfuric acid, a mixture of oligomers OF' p,, 5 waa obtained.11) Efficient removal of the by-product, HX, of a polycondenaation will shift the equilibrium of the polymerbation so as to provide high yields of material of high molecular weight. The efforts directed to this goal will be discussed in Section II,2 and 3 (see pp. 441 and 461). Second, the positions of the linkages between sugar residues of a polymer from a polycondensation are dependent on the number and reactivity of the hydroxyl groups which can enter into glycoside formation, (14) L. zechmeieter, 2.Physik. C h . ,108,316 (1922). (15)H.Frahm, Ber., 74, 622 (1941). (16) P. M. Silin and E. A. Sapegina, Tr. Voronashsk. Khirn.-Teknol. Inst., 8-4, 79 (1939);Clrtnr. Abstrads, 86, 8338 (1941). (17)Abbdationn employed throughout are: D.8., average degree of substitution; number average molecular weight; aW, weight average molecular weight; and p,, average degree of polymerbation. (18) P. N. Odincova and A. I. Preobraehenskii, Latvdjoa P8R Zinalnu A M . V C S l i S , lB66,No. 2,73;Chem. Ahlmds, 60, 15107 (1966).

1

a,,

-'

436

I. J. QOLDBTEIN AN9 T.

?J.HULLAR

An uneubstituted hexose, such m D-glucopyranw ( l ) , conbins four alcoholic hydroxyl groups and one hemiacetal hydroxyl group. Such a polyfunctionallg monomer can polymeriae to give a multibranched polymer, such 88 (2). A trisubstituted hexose, such as 2,3,&tri-O-(N-phenylcontains only one alcoholic hydroxyl carbamoyl)-D-glucopyranose (3)

OH

-

OH (11

OH

OH

(2) (after Erlander and Frencha1)

group and one hemiacetal hydroxyl group. This bifunctional monomer can give rise only to a linear polymer (4). However, even with a monomer such as (3), the synthesis of polymer (4) ia not assured, since it must be assumed that, a t lesst at the outeet, no (141) (trehaloee type) linkages are formed and that no isomeriaation or removal of the protecting groups occurred during the reaction. The actual position of the linkagea in a branched polymer, such as (2), will depend on the relative reactivities of the hydroxyl groups. These reactivities are known to be different both in monomeric and polymeric carbohydrates.a* In a studya of the condenaation polymerization of un(lQ)W . 11. Carothers, Tram. Faraday ~ o c . ,82, 39 (1936); C h . Rev., 8, 353 (1931); Ilef. 12, pp. 31-32. (20) E. Huuemann and G . J. M. Mllller, Makromol. CAem., 4@,238 (1961). (21) 8.Erlander and I). French, J . Polyiner Sci., SO, 7 (1956). (22) For a genersl review of the relative wactivitiea of the hydroxyl groups of carbohydrates, aee J. M. Sugihara, Aduan. Carbohydrate C h . ,8, 1 (1953). (23) H. Frahm, Ann., 666, 187 (1944).

CHEMICAL SYNTHElsIS OF POhYSACCHARIDES

437

OR 0

II (S), R = -CNHC,H,

I

(4), R =

-8CNHC,H,

substituted and partially methylated D-glucose in 40.8% hydrochloric acid, the reactivity of the C-6 hydroxyl group was found to be approximately four times that of the C-2, C-3, or C-4 hydroxyl groups. The predominance of ( 1+6) -1inkagea in polyglycoses haa been repeatedly observed. The lower reactivity of the C-3hydroxyl group, relative to the C-2 and C-4 hydroxyl ~ the reactivity of the several hygroups, has been n ~ t e d . * ~In. *addition, droxyl groups may ohange during the course of polycondenaation. Methylation studiee of polysaccharidea under alkaline conditions ahowedl6 that alkylation of one hydroxyl group altered the reactivity of the adjacent hydroxyl group. Such altered reactivity may also prevail in polycondensations of free augara. For example, the reactivity of the G 3 hydmxyl group of a reeidue such as (5) may be different from that of the C-3 hydroxyl group of a rssidue such as (6).The relative reactivities of these hydmxyl groups wiIl depend on their location on the sugar residues,* the substituents on the adjacent carbon atoms, and their location within the polymeric matrix. Thus, any statistical treatment211wof the polyconden(24) A. Bhattacherya and C. Schuerch, J . Org. Chem., 36, 3101 (1961). (25) G. G. 8.Dutton and A. M. Unrau, Can. J . Chem., 41, 2439 (1963). (26) I. Croon, S u m k PaPpe?’.sttid.,88, 247 (1960). (27) P.J. Flory, J . Am. Chsm. h c . , 74,2718 (1952); Ref. 12, pp. 102-103, Chaptar IX.

438

I. J. QOLDSTEIN AND T. L. HULLAR

sation of polyfunctional sugar monomers must allow for the differing reactivityll of the hydroxyl groups instead of considering them to be of equal reactivity,* even though the differenoes in reaativity may be constant throughout the polymerimtion.

0

HO

0

HO

I

OH

0(5 1

(6)

Third, the ring size of the constituent glycosyl residues of a polyglycose will depend on the condition5 used for the polymerization. Thus,mixtures of 5- and 6membered ring-forms are formed, and transformed into one anotherJo)under conditions of acid-catalyzed, glycoside formation. It is, therefore, not surprising that both furanosyl and pyranosyl residues have been found in the acid-catalyzed, vacuum polycondensation of ~glucoses (see Section IIJ3,d).Even a protected sugar [such as (3), which possesses a C-4 hydmxyl group] a n exist and be polymerized aa a mixture of ring forms (equation S), the pyranoid form probably preponderating. Acyclic RO&C

Q;

HO

OR

sugar residues can also be incorporated.*'*" This isomerization of ring forms can often be avoided by utilizing monomers whose ring size is fixed

(28) C. T.Bishop and F.P. Cooper, Can. J . Chem.,40,224 (1982); 41, 2743 (1963). (29) G. G. S. Dutton and A. M.Unrau, Can. J . Chm., 40, 1198 (1982).

CHEMICAL SYNTHE8IS OF WLYSACCWARIDES

439

by the p m n c e of a suitable substituent at C-1. Thus, formation of the glycoaidic bond by direct displacement (equation 4) of the C-1 substituent or by effecting displacement a t C-1 through neighboring-group participation,’OJ1followed by attack with hydroxyl (equation 6 ) resulta in retention of the ring form present in the monomer, since, in neither caae (equations 4 and 6), would the expected transition state allow interconversion of ring forms.*2

mr;

I

1

Fourth, the configuration of the glycosidic bonds in the polyglycose will depend upon the polymerization conditions. In an acid-catalyzed polycondensation of an unsubstituted sugar, the intermediate, a stabilized carbonium ion (7), reacts with a hydroxyl group to give the thermodynamically controlled mixtun9 of anomeric glycosides (equation 6 ) .

A more stereoselective formation of the glycosidic bond, assuming nonequilibrium conditions for the polycondensation, involves the use of a large counter-ion (Ae).a4 The intermediate carbonium ion (7) would be stabilized preferentially from the least hindered side of the ring tw in (8) and, comequently, would furnish one glycoside anomer such as (9) (see Section (30) B. R. Baker,J. P. Joseph, R. E. Schrtub, and J. H. Williams, J. Org. C h . ,19,1786 ( 1954). (31) For a recent, general review of this important mbject, see B. Capon, @art. Rev. (London), 18, 45 (1964). (32) Compare with C. G. Swain, J. Am. Chem. Boc., 14, 4578 (1950); C. G. Swain and J. F.Brown, ibid., 74,2534 (1952). (33) R. U. Lemieux, in “Molecular ftearrangements,” P. de Mayo, ed., Interscience Publishers, New York, N. Y.,1964, Vol. 2, p. 735. (34) J. Kope and C. Schuerch, Proc. Cellulose COY$.,6th, Syacuse, (1965); J . Polymer rsn’., Pt. C., 11, 119 (1966).

I. J. QOLDSTEIN AND T. L. HULLAR

440

1142,b). Stabilization of the carbonium ion can also be achieved by neighboring-group participation.a1 Thie waa probably the principal factor operative in the condensation of (3) to form a largely, possibly completely, B-D-lhked polymer' (equation 7). Incidentally, if the conversion of (3) into (3a) occurred by protonation of the C-1hydroxyl group followed by

OH -

ROH$C

n

H

n

OR

[.@) OR

n

neighboring-group displacement of water by the N-phenylcarbamoyl group. (as seems likely), the pyranoid form would be retained, as in (10). Defined stereochomistry a t the anomeric carbon atom is also possible of achievement by means of displacement reactions at C-1 (see equations 4 arid 6 ) . Only oligomere containing &D-( l-S)-linkages were obtained when

441

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

2,3,4-tri-O-acetyl-a-~glucopyranmyl bromide (11) was subjected to selfcondensation under Koenigs-Knorr conditions, giving (12).'O

QBr-:H0W

HOH$

RO

OR

(ll), R = Ac

OR

(12),

R = AC

In all successful syntheses of polysaccharides, it is essential that the equilibrium be so shifted as to afford good yields of material of high molecular weight. The extent to which the other factors must be controlled will depend upon the type of polymer deaired. 2. Polymerization in a Solvent a. Aqueous Solution.-Polycondeneation of wglucoee was probably' obtained first by Berthelot" by heating D-glUCOSe with sirupy phosphoric acid. M u s c ~ I u found s ~ ~ ~that ~ treatment of wglucose (20g.) with concen[.ID 131 trated sulfuric acid gave a white, hygroscopic product (10g.) ( to 134") possessing only slight reducing power. Only 0.54% of this product dialyzed through parchment paper in 24 hr., being similar in this respect to the so-called ydextrin which Musculus obtained by the action of diastase on starch. The material was hydrolyzed to wglucose by dilute acid, but misted fermentation by yeast. Similar products were obtained by the action of gaseous hydrogen chloridea and of heat (170°)'* on D-glucose. It was found" that treatment of cellulose, starch, or =glucose with concentrated sulfuric acid a t 30-35' gave similar products having optical rotations of +llO" to +138". Chemical evidence for the condensation of Dglucose units under strongly acidic conditions was obtained when FischeP isolated

+

(36) G. ZemplOn and A. Gerece, Ber., 64,1646 (1931). (36) S. Haq and W.J. Whelan, J . Chm. SOC.,4643 (1966); W.J. Whelan and S. Haq, Chem. I d . (London), 800 (1966). (37) M. Berthelot, Ann. chim. et phyu., M, 74 (1868). (38) M. Muaculw, Bull. SOC.Chim. (France), [2] 18, 66 (1872). and A. Meyer, Bull. SOC.Chim. (France), [2] 96,368 (1881); Ber., 14, (39) M.MUECU~UE 850 (1881): (40)A. Gautier, Bull. SOC.Chim. (France), [2] 22, 145 (1874); Ref. 7, footnote 36. (41) See Ref. 7, footnote 67. (42) M. H.H6nig and H. 8. Sahubert, Monatsh., 7,466 (1886). (43) E.Fischer, Ber., 09,3687 (1890); 18,3024 (1896).

442

I. J. QOLDBTEIN AND T. L. HULlrhR

a disaccharide44 (as its osazone), together with dextrinous material, dter D-glucose was treated with concentrated hydrochloric acid. This result was confirmed by Szheibler and Mittelmeier?‘ who employed hot, dilute sulfuric actid to obtain tho same disac!aharido from ~gluc!ostt. Crimaux and I~fevrc‘’ obtaiiiod UII cllaohol-pwoipilablc iiiutcrial wlieii a solution of n-gluco& in five per cent hydrochloric acid waa evaporated under diminished pressure on a waterbath. The properties of this material were simiIar to those of the dextrins obtained from the hydrolysis of starch. WohP extendcd these observations by studying the effect of dilute acid on D-fructose, D-glucose, sucrose, and starch. He observed that the hydrolysis of polysaccharides is not a firstcorder reaction, since the monosaccharide units condensed in acid to form polymeric materials. Moel~yn-Hughes~~ drew similar conclusions, and obtained optical rotatory evidence for the self-condensatim of D-glucose in N hydrochloric acid. WohP wea the first to refer to the formation of higher saccharides by the action of acids on monosaccharides as “reversion.” This process, the acidcatalyzed formation of glycosidic bonds between sugar residues, constitutes the most rudimentary form of condensation polymerization. Reversion of oligosaccharides has also been observed.@Jl The relationehips between the hydrolysis of polysaccharides and the reversion of mono- and oligo-saccharides is illustrated for amylose in Fig. 1. Since these early studies, the reversion of carbohydrates has proved to be of considerable importance in the hydrolysis of starch:*@ in the elucidation of polysaccharide structure,w*61*- and in the synthesis of oligonamed iaomaltoee by Fischer.u Later studies showed46 that the dieanbride waa more likely to have been gentiobiose, not iaomaltose. (46) H. Berlin, J . Am. C h . Soc., 48, 1107, 2627 (1926); G. H. Coleman, M. A. Buchanan, and P. T. Paul, ibid., 67,1119 (1936). For a summary of the hietory of the structural elucidation of hmaltoee, we M. L. Wolfrom, L. W. Georgee., and I. L. Miller, iW.,71, 126 (1940). (48) C. Scheiblor and H. Mittelmeier, Ber., 44, 301 (1891). (47) E. ctrimaux and T. Lefevre, Cmnpt. Rend., 108, 146 (1886). (48)A. Wohl, Ber., UI, 2084 (1890). (49) E. A. Mdwyn-Hughee, Trans. Fcrroday Soc., 46, 603 (1928). (60)D. J. Manners, G. A. Mercer, and J. J. M. %we, J . Chem. Soc., 2150 (1866). (61) K. Taufol, H. Iwainsky, and H. Rutloff,J . Pmkt. Chum., 4, 89 (1956). (62) W. R. Fetzer, E. K. Croaby, C. E. Engel, and L. C. Kirst, Ind. Eng. Chem., 46, (44) The diaaccbaride was

1076 (1963).

(63) G, Graefe, Lsbrke, 4, 27 (1960). (64) A. Thornpeon, K. Anno, M. L. Wolfrom, and M. Inahme, J . Am. C h m . SOC.,76, 1309 (T954). (66)A. Thompson, M. L. Wolfrom, and E. J. Quinn, J . Am. Chcm. SOC.,76,3003 (1953). (66) 8.Peat, W.J. Whelm, T. E. Edwards, and 0. Owen, J . Chem. Soc., 586 (1968).

CHEMICAL 8YNTHESI8 OF POLYBACCHARIDES

Hydrolysis

Reversion

Amylose

Polysac,$Kuides

Maltodextrlns

FIG.1.-Relationship

-

443

it

Higher Ollgoraccharides

Between Hydrolysis and Reveraion of Some Carbohydrates.62

saccharides.” The extensive literature that haa arisen because of this interest puts a complete discussion of reversion beyond the scope of this article.68 Consequently, only a few selected, synthetic applications of the reversion reaction i ~ i lbe l considered. As mer;tioned above, a disaccharide, probably gentiobiose, was obtained by FiecheP and Scheibler and Mittelmeiefl when D-glucose was treated with concentrated hydrochloric acid or dilute sulfuric acid. An oligosaccharide fraction, characterized as a tetramer, was obtained by Myrback and eovorkerso2when D-glucose was treated with 3740% hydrochloric acid. Methylation analysis suggests that the fraction had a branched structure. By using chromatographic methods, some of the disaccharidea obtained by treatlnent OP D- and mwabinose,‘.86 wxyloseJw ~galactose,” and D-mannose&with concentrated acids have been characterized (see Table I). Virtually all of the possible disaccharides of D-glucose have been isolated from the reversion products of D-~~UCOR?*** In addition to lower oligosaccharides, condensation products having high molecular weight were also obtained by early workers. Thus, by subjecting (67) J. Stan&, M. Cerng, J. Kocourek, and J. PacBk, “The Monosaccharidea,” Academic Prm Inc., New York, N. Y., 1963, pp. 110-111. (58) For reviqwa of reversion, aee Refs. 8, M)-67,6981; and K. MUer and K. Taufel, 2.?~bmem.UnteraZlch.-F~~ch., 100, 437 (1966). (59) J. C. Sowden and A. S. Sprigga, J . Am. C h . SOC.,18,2603 (1966). (60)P.S. O’Colla, E. E. Lee, and D. McGrath, J . C h . &c., 2730 (1962). (61) H. 13. Schlubach and E. Lilhra, Ann., 647, 73 (1941). (62) K. Myrbbck, M. Hammarstrand, and H. Gelinder, Atkiu Kemi, 1, 235 (1949). (63) F. A. H. Rice, J . Am. Chem. Soc., 78, 6167 (1956). (04) L. Hough and J. B. Pridham, C h . Id.(London), 1178 (1967). (65)J. K. N. Jones and W. H. Nicholson, J . C h .Soc., 27 (1958). (06) D. H. Ball and J. K. N. Jones, J . Chsm.rsoC., 33 (1968). (67) C. N. Turton, A. Beddington, 8.Dixon, and E. Pacau, J . Am. C h . Soc., 77,2506 (1955).

444

1. J. QOLDllTlilIN AND

T. L. HULLAR

TABLE I Preparation of Dinaooharidesby Reversion of Monosaccharidee

Monosamharide

Disamharides identified

References

~-Arabinosea

&D-Arap(l 4 3)-D-Ara

63

i.-Arsbinoeeb

p t A r a p ( l + 3)-cAra p t A r a p ( 1 4)-~Araa &x.-Arap-( 1 + l)-StArapc

64,66

D-Xyloseb

D-Galactose“

67

a-~-Manp-(1 + @&Man & ~ - M a n p1( -+ @+Man i3+Msnp-( 1 -+ 3)-~-Man & ~ - M a n p1 ( 4)-~-Mana

D-Mmnd

-

65

a I n 20 N sulfuric acid. * In 6 N hydrochloric acid. 0 Tentatively identified. d In 11.7 N hydrochloric acid.

D-glucose to the action of fuming hydrochloric acid, Ost@obtained a preparation which had [ a ] ~ 124’ and a reducing value 11.3% of that of maltose. The so-called “isomaltoee” of Georg and Pictetegwaa prepared in the same way. This preparation waa later stated by Zempldn and Bruckner’o to be a mixture of a disaccharide and higher saccharides; the higher saccharides were subsequently considered61 to be similar to the polyglucose prepared by the action of hydrogen chloride gas on p.glucose.el Reversion of D-glucose in 75% sulfuric acid at 20” gave polymeric material which, after fractionation, showed i?, ranging from 2 to 18 and1*averaging 5. In 75% sulfuric acid, reversion waa slower than in 41% hydrochloric acid; bowever, products of higher polymeriaation were obtained in the sulfuric acid system.71

+

(68) H. Oat, Chemiker Ztg., 19, 1506 (1895);2. A n g ~ C. h . ,17, 1663 (1904); H. Oat and T. Broedkorb, Chcmiksr Ztg., 86, 11% (1911). (69) A. aeorg and A. Pictet, H&. CAim. Ado, 9,612 (1926). (70) 0. Zemplbn and Z.BrucLner, Ber., 64, 1862 (1831). (71) P. N. Odinoova and A. I. Preobrashenskii, Ldvijccr P8R Zinatnu Akad. V d 8 ,

1966, No. 2 (Whole No. 91), 43; Chem. Abtrads, 49, 16393 (1968); ibid., 1966, No. 11,M; C h . A W o d s , 60,14329 (1966).

CHEMICAL BYNTHESIS OF POLYBACCHARIDES

445

A three-step procedure7*has been employed by Merck and Company to obtain a plyglucose suitable for use as a plasma volume-expander. A 50% D-glupose solution (w/v) containing 1 mole yo of phosphoric acid (based on D-glucose) was hcatctl for a period of approximately 20 hours at 150160"/5-10 mm. pressure. The partially water-soluble, crude polymer was then degraded in an unspecified manner, to give a yellow powder which was fractiomted by precipitation from aqueous alcohol. The polyglucose contained 0.244% of phosphorus which appeared to be in the bound form. The polymers were undoubtedly of high molecular weight, and this, perhaps, accounted in some measure for their limited solubility in water. As pointed out in Section II,l, polycondensation of sugars in aqueous acid is a reversible process made inefficient by the presence of water. Polycondensation can be conducted more efficientlyin anhydrous solvents, with the removal of the evolved water. Alternatively, it may be carried out by using derivatives which possess a t C-1 nonhydroxylic substituenta that, on displacement, may be conveniently removed from the reaction system. These methods will be discussed in turn.

b. Non-aqueous Solution-Some polycondensations conducted in partially aqueous media fit more conveniently into this Section and will be discussed here. A short review of acid-catalyzed synthesis of oligo- and poly-saccharides has appeared.7a Liquid hydrogen fluoride was found to convert filter paper into a pulpy m.ma.74 Larger proportions of anhydrous hydrogen fluoride dissolved the filter paper completely within a few seconds76to give D-glucopyranosyl fluoride.76 The polymeric products derived from treatment of cellulose with hydrogen fluoride were studied by Helferich and Btittger." In the presence of anhydrous hydrogen fluoride a t 30", cellulose is transformed into a polymeric product, called celIan.n This substance is water-soluble, strongly dextrorotatory ( [.ID 143'), completely converted i n k D-glucose on refluxing with dilute mineral acid, and only faintly reducing. Molecular-weight determinations (by cryoscopy) on cellan acetate ( [ a ] ~ 128') and on the regenerated polymer indicated it to have a p , of 14. When treated with hydrogen fluoride, D-glucose was converted, in 90% yield, into 9 polymeric material," similar to cellan in its solubility and

+

+

(72) A. J. Zambito, Merck and Co.,Inc., Private oommunication. (73) S. Suauki, Tobku Yakka Daigaku Kiyo, 6, 1 (1958); Chem. Abstracts, 63, 10053 (1969;. (74) J. Gore, J . Chem. h e . , 22, 396 (1809). (76) K. Fredenhagen and G.Cadenbach, 2.Anorg. Allgem. C h . ,178, 289 (1929). (76) K. Fredenhagen and G.Cadenbach, Angeur. C h . ,46, 113 (1933). (77) B. Helfeiich and A. BBttger, Ann., 476, 150 (1929).

446

I. J. GOLDSTEIN AND T. L. HULLAR

optical rotatory properties. However, the molecular weight of the acetate and regenerated polymer showed the P a to be approximately 7. The action of anhydrous hydrogen fluoride on cellobiose gave a polymer similar to that obtained from D - ~ ~ U C O S ~ . In an amlogous fashion, starch in hydrogen fluoride at -20' gave a polymer ([.ID 145O), called amylan," which was similar to cellan. However, amylan differa from cellan in the higher rotation ([a]D 142') of its acetyl derivative and in ita precipitability from aqueous soIution by ethanol. Under similar treatment with hydrogen fluoride, maltose gave a polymer, apparently identical with amylan. The similarity in properties of these several preparations suggeets a similarity in structure. The high, positive rotation is indicative of a preponderanc2of a-D-glucosidic linkages, and the isolation of a large proportion of 2,3,4,6-tetra-O-methy1-~-glucose from the hydrolytic fragments of methylated cellan" showed the polymem to be highly branched. The formation of polymera of Iow molecular weight derived from P-glucose and cellobiose may be due to the greater proportion of water released during condensation, preventing formation of higher polymers. Alternatively, the higher P, of cellan may be due to ita being derived largely through traneglucoeidati~n.~~~~~ Russian workeram found that cellulose and glucose furnish ensantially identical products on treatment with 93% hydrogen fluoride. Analysis of the fractions showed that 7% of the product had a P, of approximately 10 (by iodine and copper numbers) and [a]~ 84'; 20% had a P n of approximately 5, [a]~ 141'; and the remainder had a P n of 2, [a]D 152'. An a-D-(l-b6)-gluaosidic linkage was considered responsible for the high, positive rotation of this "biose" fraction. Polycondensrttion of D-glucose (10-20% concentration) in 45%870 hydrogen fluoride at 10-30" furnished a product consisting of 3-5 reaiduea at equilibrium.** Early studies by mu soul us^ indicated that concentrated sulfuric acid effecta polymerizatiou of ~glucose.Nabmuram found that optimal conditions for this polycondmtion involve hating of D-glucose (20 g.) with concentrated sulfuric acid (1 ml.) at 95' for 10-20 min. Carbonization was conspicuous when 4 ml. of sulfuric acid was used. Under these optimal

+

+

+

+

+

(78) B. Helferioh, A. Stllrker, end 0. Peters, Ann., 489, 183 (1930). (79) B. Helferih and 0. Petem, Ann., 44, 101 (1832). (80)Z. A. Rogovin and Yu. L. Pogoeov, Nauchn. Dokl. VywM Shhbly, Khim. i Khim. Tekhn~l.,19691 NO.2, 868; C h . Abstm~ls,6 8 8 22912 (1959); V. I. ShWkOV md M. G. Smirnova, Zh. Prikl. Khim., 87, 976 (1954); C h . Absfrade, 49, 10869 (1956); J . Appl. C h . USSR. (Englieh Trmsl.), 17, 911 (1954). (81) Yu. L. Pogoeov and Z. A. Rogovin, Uabskak. Khim. Zh., 1980, No. 3, 68; C h . Akfracls,66, 24100 (1981). (82) T. Ne,kamura,Kogyo Kagah Zasshi, 68, 1789 (1960).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

447

coudition8, polymers of P, 9-18 (by iodine methods) were obtained in 20-3OOJoyield following fractionation, by alcohol, of the dialyzed reaction products. (The Pn,based on reducing value, before fractionation was approximately 4.) The polymers gave wglucom on acid hydrolysis, but were unaffected by tt crude, d i v a r y amylase preparation. Methylation analysis of a polyglucose fraction having P n 17 suggested that ( 1 4 ) linkages preponderate, with (l+b)-linkages the next most common. The presence of n few branch points at both C-4 and C-6 was also shown, but a relatively low proportion of nonreducing, terminal glycosyl p u p s were indicated. This rather linear structure containing a sizable proportion of (1+4)-bonds is in contrast to the highly branched structures containing (14)-linkages obtained in the presence of other acid catalysts (such as thionyl chloride or hydrogen chloride). Polycondensation of D-glucose also followed when a mixture of D-glucose (20g.) and water (2 ml.) was heated at 95' with phosphorus trichloride (13), phosphorus pentachloride ( 14), or phosphorus pentsoxide (15) .s2 The addition of water waa essential, since these three compounds apparently failed to catalyze polycondeiisation under anhydrous conditions. Under optimum conditions, polymers of P n 11-13 were obtained in 10-15% yield after 20-30 min. reaction times, when (13) and (14) were used for catalysis. With (ls), polymers of P , 8-11 were obtained in 10-30% yield, but such polycondensations required 1-2 hr., instead of a few minutes (as for 13 and 14). In no case did polymerization appear to be as efficient as when sulfuric acid waa used. Kentm capitalized on the waction between thionyl chloride and water (to form sulfur dioxide and hydrogen chloride) for removing the water of condenaation and generating a gaseous by-product to aid in driving the polymerization to completion. In support of this role of thionyl chloride is the observation that polymerization did not proceed under strictly anhydrous conditions. A small proportion of water facilitated the polymerization, probably by permitting initial formation of hydrogen chloride. The optimum conditions for this type of polymerkationWinvoIved heating a solution of D-glucose monohydrate (18 g.) in thionyl chloride (3 ml.) a t 100' for 45-60 minutes. In this manner, a polymer preparation of P, 12 was obtained in 75% yield. The P. could be increased to approximately 30, if the first polymer preparation was re-treated with thionyl chloride under similar conditions. The optical rotation ( [ a ] ~ 100') suggested that a-Sglucosidic linkages were present. Limited analysis, by periodate oxidation, of the structure of a polyglucose of Pn20 prspared by this thionyl chloride procedure showed 58% of the

+

(83)P.W.Kent, Bwchm. J., 66, 361 (1956).

448

I. J. QOLDSTEIN AND T. L. HULLAR

residues to be either (l+6)-linked or terminal.a4Graded hydrolysis, with acid, of the polymer permitted isolation of isomaltose, thereby demonstrating the presence of a--( 1-+6)-glucosidic linkages. Infrared analysis (see Section IV,4,b) showed the presence of a-w(1+6)-linkagesJ and also suggested the presence of a few WD-( 1-*3)-1inkagea. The thionyl chloride method has also been used@for the condensation polymeriza+ionof mgalactoee, &mannose, and lactose, and for the copolymerizaticn of mixtures of &glucose and D-galactose, wglucose and crhamThe p,, ranged from 4.5 nose, and D-glucose and ~-glucurono-6,3-lactone. (for the product from wglucose and wglucuronolactone) to 12 (for the product from D-wnnose, and from lactose). No structural studies, or efforts to repolymerize thew products to material of higher molecular weight, have been reported. Polyphosphoric acid este196 has been employed for activating carboxyl groups for peptide synthesis," and hrts also been used to activate the hemiacetal hydroxyl group of sugars for glycoside synthesis.a Schramm and coworkers reporteda that a solution of polyphosphoric acid ester and wglucose k~f o m m i d e furnished, after dialysis and deionization, a phosphate-free polymeric material of molecular weight approximately 50,OOO (in 10-20(r0 overall yield). By similar procedures, wribose and D-fructose were found to give polymers having molecular weighte of approximateIy

40,Ooo.

+

The optical rotation ( [ a ] ~ 16") of the polyglucose, together with periodate-oxidation data (one mole of periodate consumed per mole equivalent of sugar residue) was taken as evidence for &n-( 14)-glucosidic linkages (see, however, Section IV,4,c). The high viscosity, [ q ] = 100, of a polyglucose containing aome phosphate ester groups, prepared by using N,N-dimethylformamide as the solvent,@ was stated to indicate linear, unbranched molecules. These data prompted the interpretationa that the polyglucosa contained &D- ( 1 4 )-glycosidic linkages almost exclusively. Similar arkuments permitted the conclusion that the polyribose was chiefly a-w(1-+5)-Iinked.a The high degree of stereoselectivity claimed is surprising. In the system used, no directive effects causing exclusive reaction of the (3-4hydroxyl group of D-glucose are apparent. Indeed, in an independent study using this (84) E. Loudon, R. 8. Theobald, and 0. D. Twigg, Chsm. Id.(London), 1060 (1955). (86) W. Pollmann and G . Schramm, Bwchim. Biophgu. A&, 80, 1 (1964). (86) G.Schramm and H. Wiaamann, C h . Ber., 81, 1073 (1968). (87) G.Schramm, H. Gr8tsch, and W. Pollmann, Angew. C h . , 78, 619 (1961). (88)G.Schramm, H. GrWch, and W. Pollmann, Angew. C h . ,74,53 (1962); Angm. Chem. Inlem. Ed. Engl., 1, 1 (1962). (89) Formamide ia eaidu to retard phwphorylation by the polyphoRphoric acid ester.

CHEMICAL SYNTHESIS OF POLYSACCWRIDES

449

method of polycondensation of wglucose, it was concluded that the syntht+ sis proceeds in a nonspecific mnner, to give branched polyglucosee.w This result is to be expected, since, under conditions of acid-catalyzed polycondensation, the C-2, C-3, and G 4 hydroxyl groups are of approximately equal reactivity, but are less reactive than the C-6 hydroxyl Furthermore, the selective formation of the 8-wglucoeidic linkage usually requires neighboring-group participation by a substituent at C-2.” No such group is evident in this system. Consequently, it seems that a mixture of CY-D and p-D anomeric configurations was, most probably, obtained, the proportions of each being dependent on the position of the thermodynamic equilibrium in the acidic medium employed. A (1+5)-linkage for the polyribose is expected to be a preponderant linkage, based on the relative reactivities of primary and secondary hydroxyl groupsp2and on the tendencyQ1of &ribose to adopt the furanoae form. However, the anomeric configuration of the polyribose is, most ~ since a mixture of D-ribose and probably, a mixture of O-D and p - linkages, adenine, under the influence of polyphosphoric acid esters,= gave, among other products, approximately equal proportions of adenosine and its CY-Disomer.g2 Even though this method of polycondensation probably gives polymers containing several different types of glycosidic linkage, the mildness of the method and the high molecular weights of the polyglycoses obtained suggest that the method itself, or modifications of it, when applied to appropriately substituted sugars, may well constitute a convenient synthesis of structurally defined polyglycoses of high molecular weight. Methyl sulfoxide has been used, chiefly by Micheel and his coworkers, as an effective solvent for the acid-catalyzed polycondensation of s ~ g a r s . ~ In * - view ~ ~ ~of the good yields of material of high molecular weight, the methods developed offered considerable promise. Unfortunately, it was subsequently that, in acidic media, methyl sulfoxide undergoes a (90)E.Husemann, Private communication of unpublished reaulta. (91) See,for example, S. Haneasian and T. H. Hmkell, J . Org. Chem., 28, 2604 (I=),

but compare with Ref. 28. (92) J. A. Carbon, Chem. I d . (London), 529 (1963). (93)F. Micheel and W. Greaser, C h . Ber., B l , 1214 (1958). (94) F. Micheel, W. Neilinger, and F. Zerhueen, Tetrahedron Letters, 1205 (1963). (95) F. Micheel, A. Bbckmann, and W. Mecketroth, M a k r m l . Chem., 48, 1 (1961); F. Micheel and A. Btickmann, Angew. Chem., 72, 2096 (1960). (96) F. Micheel and D. Mempel, Makromol. Chem., 48, 24 (1961). (97) F. Micheel and A. Btickmann, M a k r m l . Chem.,61, 97 (1962). (98) F. Micheel and H. Alfes, Makroml. Chem., 48, 33 (1961). (99) F. Micheel and R. Puchta, Malcroml. Chem., 48, 17 (1961). (100) F. Micheel and A. Bockmann, M a k r m l . Chem., 61, 102 (1982). (101) E.Hueemann and J. Klar, Methods Carbohydrate Chem., 6, 180 (1965).

I. J. QOLDBTIPIN AND T. L HULLAR

450

slow disproportioiiation to form methanethiol and formaldehyde. In an analogous reaction, thionyl chloride is known to react with an excem of methyl sulfoxide, to give, among other products, formaldehyde and hydrogen chloride.lm In the presence of acid, formaldehyde is known to undergo polymerization*oato poly (oxymethylene) and to form methylene acetalP with alcohols. Analysis of the polyglycoees for oxymethylene groups (derived from formaldehyde) showed that the polyglycosea contained between 0.5% and 15% of formaldehyde. The presence of polp (oxymethy!ene) as a contaminating polymer was excluded on the basis of infrared spectral analysis and solubility properties. The presence of cyclic, methylene acetals was considered unlikely, since amylose or starch under the reaction conditions employed for polycondensation did not contain cyclic acehl groupings. In order to study the manner in which oxymethylene unita are bound to sugar residua, methyl 2,4 ,6tri-O-methyl-/3-~glucsoside( 16) and 2,3,4,6-

OR

-

(le), R -CH,

(102) F.G. Bordwell and B. M. Pitt, J . Am. C h .rsoC., 77,672 (1966). (103) J. F. Walker, “Formaldehyde,” Reinhold Book Division, New York, N. Y., 3rd Edition, 1864, p. 160. (104) Ref. 103, pp. 264-276; T. G. Bonner, Methode Carbohydrde Chent., 2, 433 (1963).

CHEMICAL SYNTHEBIB OF POLYBACCHhRIDES

451

tetra-O-methyl-~glucoae (17)-neither of which can undergo polymerization-were treated with methyl sulfoxide and acid under the conditions uaed for the polymerizations of sugars. From the reaction of (16), a aeries of compounds, (18), was obtained, in which two residues of (16) were united by oxymethylene units of various chain lengths. In a similar manner, compound (17) gave a mixture of homologous, oxymethylene compounds, (19), of which the first member, n = 1, was chamcterized, and higher members, n = 2, 3, , were identified chromatographically. Compound (17) also furnished the octa-O-methyl-a,cr- and a,Btrehalose~(20) and (21), respectively. It is poasible that oxymethylene units can also form bridges between nonanorneric and anomeric hydroxyl groups, tw in (22). The observation that hydrolysis by 0.1 N aqueous acid causes ti large decrease in molecular weight of the synthetic polymers constitutes further support for the presence of cxymethylene groups between sugar residues.

I t is clear, &herefore,that polymers synthesized by polycondensation of sugars in acidic methyl sulfoxide are, actually, copolymers of sugars and formaldehyde in which some of the glycosyl residues are joined by glycoeidic bonds, and others are linked by “bridged’ of oxymethylene units. Nonetheleas, because polycondensation of sugars can be effected in methyl sulfoxide, and because the methods developed for the polycondenaation may be the extensive studies reported by applicable to other solvent Micheel and his coworkers will be discussed. The general methodg6developed by Micheel and coworkers consists of allowing a 20% solution of sugar in methyl sulfoxide to undergo condensation in the presence of an acid catalyst for two to eight days, usually at 40’. The acid cahlysts used were (a) hydrogen chloride,06(b) hydrogen chloride together with small proportions of hydrogen bromide:& (c) a mixture of hydrogen chloride, hydrogen bromide, and phosphorus pentaoxide,” and (d) thionyl chloride.06 The weight ratio of catalyst to monomer was 0.3-0.5 to 1.0. For polymerizations catalyzed by hydrogen chloride and (106) Other solventa, such aa dimethyl sulfone, tetramethylene sulfone, or acetic acid, have been stated*4 to give polyglycoaea containing no oxymethylene ~ O U P R .

452

I. J. OOLDBTEIN AND T. L, HULLAR

hydrogen bromide, the water of condensation was removed by azeotropic distiIlation with benrene at 14 Tom at 40".Thionyl chloride and phosphorus pentaoxide served as internal, water scavengers, as well as catalysts. Methyl sulfoxide waa removed by rapid distillation, and the polymer was precipitated by the addition of methanol. Methanol-insoluble polymer was diaIyzed to remove the material of low molecular weight. The sugars used in these polycondensations, and some of the oharacteristics of the methanolinsoluble, dialyzed polymers are summarized in Table 11. Effective polymerization occurred only when the water of condensation was removed by one of the methods mentioned above. The maximal yield of material of high molecular weight waa obtained after a reaction time of 5-12 days (see Table 11, Nos. 2 and 3). The molecular weight and yield of the products depended on the concentration of monomer in solution (see Table 11, Nos. 4 and 5 ) and on the catalyst used. For the neutral sugars, hydrogen chloride furnished preparations of the highest molecular weight in the highest yield; the addition of hydrogen bromide resulted in a 50% diminution in the molecular weight and in a 20% diminution in the yield.06 For 2-acet~mid6-2-deoxy-~-glucose , however, the hydrogen chloridehydrogen bromide system proved to be a catalyst superior to hydrogen chloride alone. From wglticose and from 2-acetamido-2-deoxy-~-glucose, thionyl chloride and hydrogen chloride gave similar yields of polymers, but those obtained by using thionyl chloride were of lower molecular weight. Poly (2-acetamido2-deoxy-D-glucose) prepared by means of thionyl chloride was also reported to contain more furanosyl residues than the polymer prepared by using hydrogen chloride. Phosphorus pentaoxide in combination with hydrogen chloride and hydrogen bromide proved to be an effective catalyst for the polymerization of 2 ,3 ,6 - t r ~ - ~ - m e t h y ~ - ~ - ~ uHowever, c o s e . ~ this polymer may have contained a considerable proportion of oxymethylene bridges, since it has been observeds" that polymers prepared in the presence of phosphoric acid are richer in oxymethylene groups than those prepared by using hydrogen chloride-hydrogen bromide. Calculations based on the formaldehyde content of the polyglycoses indicated that, on the average, blocks of four glycosyl residues are distributed randomly. In view of the ease with which formaldehyde undergoes polymerization, it is more probable that blocks of oxymethylene residues are interposed betwecn blocks of glycosyl residua. Complete hydrolysis of the polyglycoses gave the starting monomer. Hydrolysb with dilute acid gave an initial rise in optical rotation which was attributed to the hydrolysis of furanosidic bonds. The positive rotations suggest the presence of CY-Daa well as of &D-glycosidic linkages. The isolation

TABLE I1 Acid-catalyzed Polycondensationof Sugars in Methyl Sulfoxide

No.

-

Monomer

1 2 3 4 5 6 7 8 9 10

c-(=lucose D-Glucose D-Glucose D-Ghd D-Glumseb D-Glucosec D-GdaCtoSe D-Mannose D-xylQSe 2-Acetsmido-Zdeoxy-D-

11

2-Acetsmid~zdeox~-~ glum& 2,3,6T~i-@rne%hyl-~-

glUCOSe

12

glUCOSe*

13

D-Glucose (6.67 g.) plus Dglucuronic acid (3.33 g.)

14 15 16 17

Maltose

18

Cellobioae

Lsctod

Lactoset Polymaltose (10 g.) plus wglucose (25 9.)

Catalyst (g./10 g. of monomer/!%ml. Time of methyl sulfoxide) (days)

Temper- Yield,o ture ("C.) (%)

40 40

Mae.

References

+97

11 ,900 6,200

-1

3,200

96

+I24

5,600

97

23

+75

12,000

98

2

40 40 40 40

49 36 44 33

+86 +67 +76 +79

%ooo

36,300 10,800 6,300

99 99 99

?i

40

60

+90

32,100

4 5 12

6.60 g. SOCI:' 0.73 g. HCl 0.10 g. HBr 1.50 g. Pa' 4.66 g. HCI 0.133 g. HBr 4.80 g. HCl 4.25 g. HC1 5.1 g. HCl 5.7 g. HCl 0 . 2 g. HBr 4.0 g. HCl 1.0 g. HBr

6

2

30

6

45

31

4

40

5.5 5 5 5 4.5

5 5 6 7 4 6

watep)

95 95 95 95 95 95 95 95 95 96

4.M g. HCI 3.08 g. HC1 0.10 g. HBr 3.18 g. RCl 0.08 g. HBr 6.60 g. SOCls' 4.40 g. HCl 3.60 g. HCl 3.20 g. HCl 3.50 g. HCl 0.60 g. HBr

3

Cab

(degrees, in

40 40 40 20 40 40 40 40

46 40 48 47 46 57 45 53 19 16

4-90 +89 +89 +95 +94 +88

+79

+I03 +96

24,000 12,400

13,800 7,300 7,300 12,500 24, loo

24,400

-3

X

p:

6

0

G m

4

$ c) ! ? I

m

2!

8

*5

zE

U

99 99

For methanol-insoluble, nondialysable-mterial. * Used 20 g. of wglucose/50 ml. of methyl sulfoxide. Used 10 g. of Dgiuoose in 20 d In 20 ml. of methyl sulfoxide. Used 6 g. of monomer in 10 ml. of methyl sulfoxide, with the catalyst mixture given. f Used 13.3 g. of lactose/50 ml. of methyl sulfoxide. a

ml. of methyl sulfoxide.

W

454

I. J. GOLDSTEIN AND T. L. HULLAR

of high proportions of 2 ,3,4 ,6-tetra-0-methylhexoses and of 2 ,3 ,4-tri-Omethyl-D-xyloae from the hydrolygateo of the methylsted products indicated highly branched po1ymers.06A high degree of branching is also suggested by the difficulty06lB6of methylation, relative to acetylation. Periodattwxidation data reveal that, for D-glucoseat lertst, the hydrogen chloride-hydrogen bromide catalyst system gives a polymer having a considerably greater proportion of terminal or ( l 4 ) - l i n k e d residues than does that produced by hydrogen chloride alone. The periodate-oxidation &fa further show that, for polymers derived from D-glucose and wgahctose (using hydrogen chloride as catalyst) , a considerable proportion (30-4070) of the glycosyl residues are substituted on the C-2 and C-4, or on the C-3, hydroxyl groups, as shown by their resistance to oxidation; some are substituted on the (2-2 or C-4 hydroxyl groups, as indicated by periodateconsumption data,whereas others are unsubstituted at the hydroxyl groups on C-2, C.3, and (2-4, aa shown by the formation of formic acid. Radioisotope-incorporation studiesn indicate that the polymers are products of an equilibrium process (see Section I1,1, p. 435). In summary, the acid-catalyzed condensation polymerization of sugars in methyl sulfoxide results in the formation of copolymers of the sugars with formaldehyde. The glycosyl residues probably occur in blocks, instead of being evenly separated by methylene bridges. The polymers are highly branched, and the glycosyl residues appear to be substituted in a random fashion. Altholigh the use of methyl sulfoxide as a solvent for the polycondensation of sugars did not, in them studies, lead to the synthesis of homopolymers, it did provide the basis%for an elegant synthesis of an apparently linear, @ - ~ ( 1 4 ) - l i n k e dpolymer of D-glucose (see equation ‘7).lm To direct polyglycoside formation between the C-1 and C-4 hydroxyl groups, thereby preveqting synthesis of branched polymers, the hydroxyl groups at C-2, C-3, and C-6 were protected by a substituent which was relatively stclble to acid but could be removed by alkali. The N-phenylcarbamoyl group fuIiiLs these requirements.lw To prepare the desired intermediate, celluloee was carbanilatedl*wJO’J@ with phenyl isocyanate, the cellulose derivative was depolymerized in Zmethoxyethanol to the D-glucopyranoside corresponding to 2 ,3,&tri-0- (N-phenylcarbamoyl) -D-glucopyranose (3), which was then hydrolyzed to afford (3). It was found, by study1 of reaction conditions, that polycondensation of (3) to form polymeric products is most satisfactory when a solution of (3) (1 g.) and phosphorus pentaoxide (1 g.) in methyl sulfoxide (1 ml.) and (106) (107)

E.Husamnn and G. J. M. MUer, Angm. Cham.,IS, 377 (1963). H. 0. Eouveng, Ada C h .Smnd., 16, 87 (1961); W.M. Hearon, Methoda Carbo-

h y d w C h . , I,239 (1963). (108) J. N. BeMiller, Methods Carbohydrate Chem., 5 , 400 (1906); 4, 301 (1964).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

455

chloroform (9 ml.) is stirred for 2-14 daysma at 30".Under these conditions, the methyl sulfoxide did not disproportionate to formaldehyde; increase in the proportion of methyl sulfoxide gave traces of formaldehyde. A higher proportion of phosphorus pentaoxide (2g./lO ml.) gave a product containing 0.08% of phosphorus. Phosphorus penlaoxide in methyl sulfoxide is also known to oxidize secondary hydroxyl groups of carbohydrate In the absence of methyl sulfoxide, polymerization in derivatives. chloroform gave products of1low (4-8)P,. Under the optimal conditions, polymers were obtained in 7540% yield, and showed [ q ] of 22-78 and Bwaa high tw 335,000 (by light-scattering) , corresponding to a P, of 640. Protecting groups were removed by treatment of the polymer with sodium methoxide in pdioxane, followed by tetraethylammonium hydroxide. Although nitrogen-free polymers were obtained by this procedure, the polymers had suffered a 16-20'3, decrease in p,,. The resulting polymers were insoluble in water, sodium hydroxide, N ,N-dimethylformamide, and methyl sulfoxide, but were soluble in tetraethylammonium hydroxide and in Schweizer reagent. Total hydrolysis of the polymers gave D-glucose only. Water-soluble derivatkes (ethyl or carboxymethyl ethers) of the polymers were unaffected by a-amylase, but were partially hydrolyzed by a cellulase preparation from Acetobacter xylinum. The optical rotations of several preparations of this polyglucose and of cellulose ( P a 1150) in tetraethylammonium hydroxide were all O", thereby strongly suggesting that the polyglucoses are @-wlinked."'Q On the ba& of the data, it was concluded' that the synthetic polyglucoses obtained by this method are linear, Bn-( 1 4 )-linked gIucans which possess properties similar to those of cellulose. It is especially significant that this polymerization proceeded with apparent stereospecificity, to form only the @-D anomeric linbge. The mild conditions under which the polycondensation occurs probably contribute to this selectivity of reaction. (lo&) A second report of conducting polycondenaationa in methyl sulfoxide and phoaphorua penwxide haa appeared [K.Onodera, 8.Himno, and N.Kaahimura, J . Am. Chem. Soc., 87, 4651 (1966)], but experimental reeults are not yet available. (109) This view is further supported by the following observations. Acid-catalyzed aolvolysia of 2,3,6tri-O-(N-phenylcarbemoyl)celluloae by Zmethoxyethanol allows isolation of a ~-glucoeide, Zmethoxyethyl 2,3,6tri-O-(N-phenylcarbamoyl!-D-glucopyranoside, which shows [PI# - 88" (in pdioxane).' The struo turally AimilrLr a-n-glncmide, methyl 2,3,4,~~t,ra~-(N-phenylcarbamoyl)-cr-Dglitcopyranoaido, lrliows [a]# 73" (iii acelone).'IO The diaparity of these two rotstions in solvent8 of aimilar properties suggeeb that the compound of [a]# -88' ie the &D anomer. Since the glucoside formation and the polycondensation are both carried out under conditions that are undoubtedly equilibrium conditions, the glycoaide formation to afford a polymeric derivative may be expected to p r o d analogously to the glycoside formation to give a &-linked monomeric derivative. Hence, the polymer ahould also have the &slinkage.

+

I. J. GOLDSTEIN AND T. L. HULLAR

456

c. Methods Based on Glycoeide Syntheees.-Condensation polymerization of eugam which possess at C-1 such ncmhydroxylic groups as a halogen atom or an acyloxy group hm been studied. The simplest examplea of such condensetion reactions are the KoenigsKnorr synthews of glycoside8.111 In principle, any of the modifications of this general reaction are available for purposes of polycondensation. Thus, Helferich and Gootz,11*synthesized gentiotetraoae in a stepwise manner by condensing 2,3,4,2’, 3‘ ,4‘, 6’-hepta-O-acetyl-a-~-gentiobiosylbromide (23)118with 1,2,3,Ctetra-O-acetyl-ar-mglucopyranose(24)114 in the presence of silver carbonate, to give the protected trisaccharide (25a). Bromination of (23aj at C-1 (by hydrogen bromide in acetic acid) to give (25b), Ac

-

%

Ac

Ac

A

c

O

Ac*QAC

AC r

Ac

AQ--jQ-Q-j$&

Ac

OAC

C

AcO

OAc

C

(26)

(110) (111)

M.L. Wolfmm and D.E.Pletaher, J . Am. Chcm. Csoc., 63, 1161 (1940). W.L.Ev&1111,D. D. Rsynolde, and E. A. Tdey, Adoon. Carbohydrate C h . ,6,

n

(1951). (112) B. Hnlierich and R. GOO~II, Bsr., 04, 109 (1931). (113) Q. ZempIBn, Bsr., 67, 702 (1924). (114) B. Helferiah and W.Klein, Ann., 460,219 (1926).

457

CHEMICAL BYNTHEBIB OF POLYBACCHARIDEB

followed by condensation of (25b) with (24), gave the blocked tetraectccharide (26) in a 5% overall yield from (23). It is clear that the stepwke synthesis of polyglycoses by such a procedure is laborious and gives low yields. As an alternative approach to the synthesis of polyglycoaes, a monomer possessing both a free hydroxyl group and a 1-bromo substituent should, n HO-M-Br

+

(--O-M-),,

+

HBr

under suitable conditions, self-condense to give a polymer. To this end, Whelan and Haqw treated 2,3,4-tri-O-acetyl-a-D-glucopyranosylbromide (11)a with silver oxide and, after deacetylation and carbon-column chromatography, isolated a series of gentiodextrins (27). The di-, tri-, tetra-, and penta-saccharides [(27), n = 0, 1, 2,and 3,respectively] were characterized &B their crystalline acetatee, and the hexasaccharide [(27),

HOH&

kO\

I

OH

on

n = 41 by its 1"JleM~and1" R M values. Reasonable yields of oligosaccharides wer8 obtained: dimer, 14%; trimer, 22%; tetmmer, 5%; pentamer, 2.3%; and hexamer, 1%. 1 ,6-Anhydro-&~-glucopyranose derived from intramolecular condensation constituted only 25% of the condensation products. The low yield of the higher oligosaccharides, together with the isolation of wglucom, are believed due to reaction of the water (released when the liberated hydrogen bromids reacts with the silver oxide) with the unreacted 1-bromide. The stereospecific formation of the 8-D anomeric configuration is due to the (116) K. Freudenberg and G. Blomqviet, Ber., 68, 2070 (1936). (116) W. J. Wheld, J. M. Bailey, and P. J. P. Roberta, J . Chem. Soc., 1293 (1963); B. Lindberg and J. McPheraon, Adcr C h .Scud., 8,986 (1954). (117) D. French and G. M. Wild, J . Am. Chem. Soc., 76, 2612 (1963).

458

I. J. OOLDSTEIN AND T. L. HULLAR

influence of tho neighborbig C-2 hydroxyl group.w If m a n s are found to remove the water of reaction (whioh results in consumption of monomer) , a method of polycondensation brteed upon the Koeniga-Knorr reaction may be of consiclerphle promise for tho stercospccific syntheees of other polyglycoses. Condensation polymerbation of u-D-mannopyranoeyl fluoride (28) haa been attempted.ll* Reaction of (28) with sodium methoxide gave methyl a-D-mannopyranoside (30) in 75% yield. Like other derivatives posseasing a fluoro group tram to the C-2 substituent (hydroxylllg or ptolylsulfonamidom), the stereospecifio formation of (30) undoubtedly proceeds by way of a 1,2-epoxide intermediate (29) which then undergoes facile ringopening at C-1, to give (30). In support of this mechanism is the obser-

vation that Z-O-methyl-a-P~Mopyranosylfluoridell* (31) reacts with (32), sodium methoxide to give methyl 2-O-methyl-&.~mannopyranoside the product expected by direct S32 displacement of the fluorine atom in the absence of participation by the C-2 methoxyl group.1g1-126 In an effort to extend theae etereospecific reactions to the formation of polymers, (28) waa treated with 50% sodium hydroxide. (See Section 111,2,c,p. 400 for the addition polyrnerizatiorP of an analogous compound, (118) F. Michoel and D. Borrmann, C h . Ber., B3, 1143 (1960). (119) F. Micheel and A. Klemer, Chem. Bsr., 85,187 (1962); F. Micheel, A. Klemer, and 0. b u m , tW.,88, 476 (1965). (120) F. Micheel and H. WuW, Chsm. Bar., 89,1621 (1966); F. Micheel and E. Michaelis, W.,91, 188 (1968). (121) C. M. McCloskey and G. H. Coleman, J . Org. Chem.,10, 184 (1946). (122) M. P. nardolph and 0. H. Coleman, J . Org. Chem.,16, 169 (1960). (123) R. U. Lemieux and 0. Brice,Can. J . Cham., SO, 296 (19Sb). (124) A. Dyfvennan and B. Wndberg, Ada chsnr. Scad., 4,878 (1958). (1248) R. C. Gesman and D. C. Johneon, J . Or#. Chum., 81, 1830 (1968). (126) F. Mi-I end A. Khmer, Char. Bss., Bl, 104,683 (1968). (126) However, the methoxyl group ie known" to lresiet in the dieplacement, by silver aoetab, of a traw, vicinal bromide atom attaohed to s cyclohmme ring. (127) 8. Winntein and R. B. Hendemon, J . Am. Char. Soc., 66, 2196 (1943). (128) 8. Hsq md W. J. Whelan, Nature, 198, 1222 (1966).

459

CHEMICAL SYNTHESIS OF POLYBACCHARIDEB

HOSy:

HOHa?

Brigl’s anhydride, 3,4,6-tri-O-acetyl-l , 2-anhydro-~-glucopyrnose.)Two dieaccbarides.[(34) and (35)] of the trehalose type were isolated in 5.3% yield each, and a trisaccharide (36) was isola.ted in 8.5’% yield. Higher

&C----d

t

Q

HO

HOH,C

0

HO (34)

(35)

oligomccharides were isolated in 7.8% yield, but were not obtained in the pure state. The disaccharides (34) and (35) arose by stereospecificreaction of the 1,%anhydro intermediate (29) with the anomers of wmannose (33), the latter wising from the reaction of (29) with the water or hydroxide ion of the medium. The trisaccharide (36) reaulted from reaotion of the disaccharide (34) with (29). It is clear that the reaction conditions used in this study do not allow formation of polyglycoses from (28). The facility of the 1,2kpoxide formation and of the subsequent ringspening do suggest, however, that different reaction conditions should permit ready synthesis of higher oligo-

460

I. J. QOLDSTEIN AND T. L. HULLAR

wtccharides, aid, poesibly, of polywtcciuirides. lt'or example, reaction of a suitable 3,4di-O-substituted wmannosyl fluoride with an appropriate base should furnish oligomers and polymers aomposed largely of a+( 146)linked wmannopyranosyl residues. The polymerbation of several uilaubetitutud aldosyl fluorides's under mildly acidic conditions has been briefly described.1w Treatment of the aldosyl fluorides with pyridinium hydrochloride or hydrofluoride under diminished pressure gave good yields (see Table 111) of nondialymble, polymeric material. TABU I11 PolyglyocMee from Aldopyranmyl Fluoriddm

Polyaaccharide Fluoride

PD-GIUCORY~ 8-p-GluWsyl

arD-Xyloeyl fl-D-Arabinosyl

Yield, %

72 67 81 66

[ o h m Odegreea ,

+66

+68 +32

- 121

No structural studies of these products have been reported. However, from the greater reactivity of the C-6 hydroxyl groupmunder conditions of acid-catalyaed condensation, it may be expected that (1-+6)-linked wglucosy1 residues preponderate. The positive, optical rotations of the polymers from wglucose and D-xylose indicate the presence of some a-D-glycosidic linkages; the levorotation of the poly(D-arabhose) suggests it to be mainly FD-linked. These anomeric configurations, together with the observation that the a- and pwglucopyranosyl fluorides furnish polymers having similar rotations, suggest thst the polymerbation proceeds by way of a carbonium ion intermediate, to give a thermodynamically controlled mixture of a-3 and P-D linkages. The high yields of polymer obtained suggest that further development of this procedure would be desirable, particularly if partially substituted aldosyl fluorides were used. Acid-catalyzed condensation polymerhation of p-D-glucopyranosyl met+ itoate (37) has also been explored. This method takes advantage of the acid-catalyaed, stereospecific displacement of the mesitoyl group of &Dgluaopyranoeyl mesitoat@ by alcohols to give, with methanol,18amethyl (laS)For B review of glyaoayl fluorides, nw F. Mioheel and A. Klemer, Adrxm. Carbohydrats C h . ,16, 86 (1981). (180) F. Miaheel and 0.Hallerman, Telralledron h & a , 19 (1962). (131)F. Miaheel and Q. Baum,Chsm. Ber., 88,2020 (1966);H.B. Wood, Jr., and H. G. FIetaher, Jr., J . Am. C h m . &oc., 78, !207, 2849 (1966). (132)B. Helfeich and D. V. Haahelika,Chem. Ber., 90,2084 (1957).

461

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

(u-i,-14Iucol,yranoside (38). Thus, self-condensation of (37) in pdioxane, using ptoluenssulfonic acid aa the catalyst, gave, in 88% yield, a identified by molecular-weight studies as being a tetrasaccharide. The optical rotation ([aID 92') indimtcd the prescncc of a-D-linkages; methylation analysis gave 2 ,Y,4,&tetra-0-methyl-%glucose and unidentified di-0- and tri-0-methyl-mglucos, indicating the materiil to be branched. This method may also have synthetic utility, particularly if reaction should be confined to specific hydroxyl groups. In a newly developed, elegant method of a-D-glucoside synthesis,'u the ezo isomer of a 3,4,6-tri-O-acetyl-a-D-glucopyranose1,2-alkyl orthoacetatel" (39) undergoes acid-catalyzed rearrangement, to give the corresponding alkyl2 ,3 ,4, 6-tetra-O-acetyl-a-~-glucopyranoside (40). It r e m b

+

H@ AcO

-Ac

OR OAc

to be seen whether this facile reaction can be extended to the synthesis of oligo- and poly-saccharides possessing glycosidic linkages a.8 to the C-2 hydroxyl groups. In a related reaction,'". 3,4 ,Btri-0-acetyl-cu-Pglucopyranose 1,%(ethyl orthoacetate) reacts in nitromethane containing cholesterol, mercuric bromide, and a trace of ptoluenesulfonic acid to give the transglucoside, cholesteryl 2 ,3 ,4 ,6-tetra-O-acetyl-~-~-glucopyranoside. This method has been applied to the synthesis of polysaccharides. Thus, barabinofuranose 1 $ 2,&orthobenzoate has been polymerized in nitromethane containing mercuric bromide, to furnish'sb a poly(carabinose) containing about 70y0of (1 + 51-linkages. 3. Polymerization in the Solid State

Some of the investigations discussed here do not conform strictly to this (133) B. Helferich, H.G . Germscheid, W. Pid,and W. Oat, Chem.Ber., 91,1354 (1962). (134)R.U. Lemieux, Abetrada Papers Am. Chem.Soc. Mectillg, 148,lO D (1964);R.U. Lemieux and A. R. Morgan, Can. J . Chem.,To be published. (135) R. U. Lemieux and A. R. Morgan, Can. J . C h . , 48,2199 (1906). (135s) P!. K.Kochetkov, A. J. Khorlii, and A. F. Bochkov, Tetrahedron Leusrs, 289 (1884). (135b) N. I<. Kochetkov, A. J. Khorlin, A. F. Bochkov, and I. G . Yaelovetaky, Carbohvdrate Ree., I, 84 (1886).

462

I. J. QOLDSTEIN AND T. L, HULLAR

classification. Thus, although ion-exchange resins have been used for effecting polycondensation in solution, many of the reactions catalyzed by such resins have been conducted in the solid phase. Consequently, for convenience, the use of resins is considered in one place. a. Catdyab by Hydrogen Chloride and Other Acide,--Schlubach and coworkerseLJMinvestigated the action of dry hydrogen chloride gas (at 43 atmospheres) on anhydrous wglucose at mom temperature. A chlorine-free product ( [ a ] ~ 105 to 108") was obtained, which showed a reducing value 15-1870 of that of D-glucose. Repeated fractionation with D I . [ 124") having a reducing value only 0.8a/, ethanol gave a fraction ( of that of D - ~ ~ U C Oand S ~ a b, of 70 (by solution osmometry). A fraction having the same properties was obtained by fractionation of the acetate of the reaction mixture. Product analysis of the reaction mixture was said to indicate 60% of polyglucose, 30% of a trisaccharide, and 10% of unchanged wglucoee. Haworth and Purdie methylations of the polyglucose gave a completely 125.8" in chloroform) which, on hydrolysis, methylated product ([alg gave subJtantia1 yields of crystalline 2,3,4 ,&tetrs,O-methyl-D-glucose, a material that appesred to be crystalline 2,3 ,&tri-0-methyl-wglucose, and a di-0-methyl-wglucose. The authors concluded that the synthetic polyglucose occurred in the form of a branched, ring structure; the low viscosity of its aqueous solution was cited in support of this structure. In all probability, this polyglucose is similar to the typical, highly branched, spherical molecule prepared and studied by other investigators. However, it is D I . [ 124"), somewhat unusual in its relatively high, specific rotation ( indicating a rather high content of a-wglucosidic linkages. The action of dry hydrogen chloride on D-fructose gave di-p-D-fructofwnose 1,2': 2,l'-dianhydride as the product isolated.1" In a seriec of communications, Ricketta and coworkerslm-lmhave described the condensation polymerbation of several sugars, using hydrogen chloride. These polymers were obtained by two procedures: (1) allowing crystalline, anhydrous wglucose to stand at room temperature for seven days in an atmosphere containing water vapor and gaseous hydrogen chloridelmJmg; and (2) passing hydrogen chloride gas into a rotary mixer containing D-glucose monohydrate, to give a plastic mass which was rotated for one to five days.1a D-Galactose, maltose, and lactose were polymerized

+ +

+

+

(136) H. H. Schlubach, H.Elmer, and V. Prochownick, Angslu. Chm., 48, 245 (1932). (137) H. H. Schlubech and C. Behre, Ann., M)8, 16 (1934). (138) C. R. Ricketta, J. C h . &c., 4031 (1954). (139) C. R. Rioketts, Brit. Pat. 824,161 (1969); Chem. Abstracts, 64, 7192 (1980). (140) C. R. Ricketta and C. E.Rowe, J . Chem. Soc., 3809 (1966).

CHEMICAL SYNTHESIS OF POLYSACCEfARIDES

463

by the first method, and a mixture of wglucose and ~ - g a h t m ewas condensed by the second method. The polyglucow prepared by the first procedure1=was fractionated by means of ethanol into seven fractions, only the last three of which contained saccharides mobilc on paper chromatograms. A nondialyzable fraction precipitated with 44.4% ethanol was studied further. Complete hydrolysis by acid gave &glucose only; partial hydrolysis ww reported to give a sugar having the s6me chromatographic and electrophoretic mobility as isomaltow. The specific rotation ([ah 106") suggested the presence of a+glucosidic linkages. On the basis of infrared analysis, it was concluded that the polyglucose contained principally a-~-(1+6)-glucosidic linkages; there were no indications of (1+3) bonds or of 3,6-anhydro rings. The polyglucose produced by the second procedurela gave a 45% yield of a nondialyzable material having properties similar to tho= of the polymer described above. Periodate-oxidation studies showed the consumption of 1.60 molecular proportions of periodate, with the formation of 0.72 molecular proportion of formic acid. Partial hydrolysis gave rise to component8 migrating with the chromatographic mobility of =glucose, isomaltose, isomaltotriose, and isomaltotetraose. =Glucose and isoaaltose were identified as their crystalline acetates. Attemptda to polymerize D-xylose, wfructose, and sucrose failed, because of rapid decomposition of the pentose and ketose (see below, however). Maltme gave a polymer whose P n and behavior with periodate were similar to those of the synthetic glucan. However, the polymaltose could be differentiated from the polyglucose on the basis of its somewhat higher specific rotation ([a13 133") and its more vigorous reaction with Type XI1 pneur,iococcal antiserum. Paper-chromatographic examhtion of polymaltose hydrolyzatesshowed spots having R, values corresponding to those of isomaltose and isomaltotriose, indicating that some rearrangement of glucosidic bonds had occurred during the polymerization. Bishop,141 interested in the relative reactivity of secondary hydroxyl groups without the complication of the very reactive primary hydmxyl group, condensed wxylose under the conditions established by Ricketta and coworkers. A nondialyzable polyxylose having P n 10-15 (by hypoiodite oxidation) wtw isolated in 5.6% yield. Methylation and hydrolysis 55 f 2" gave 2,3 ,Ctri-O-methylof a mole of 4 f q t i o n having [a]# D-xylose (3 moles), 2,3-di-O-methyl-D-xylose(7moles), 2,Pdi-O-methylwxylose (1 role), 3-O-methy1-D-xylose (5 moles), and wxylose (1mole). If, w WM assumed, the ID-xylose residues are in the pyranoid form, the bonds is 6.5:3:1. rstioof (14):(1+2):(1+3)

+

+

+

(141) C.

T.Bishop, Can. J . Chem.,81, 1256 (1966).

464

I. J. QOLDSTEIN AND T. L. HULLAR

Exposure of 2-amino-2-deoxy-~-glucoaeto moist hydrogen chloride vapor for extended periods failed to furnish polymers.1" Nor was this sugar incorporated into the oligosaccharides produced when a mixture of ~ - g a lactose and 2-amino-2deoxy-~-glucosewas treated under the same conditions. This resistance to polymerization wm attributed to electrostatic shielding of the glycosidic center by the ammonium group. A series of oligosacoharides waa obtained, however, when 2-acetmnido-2deoxy-~-glucose WRB treated as above for four [email protected] disaccharides, namely, 0-(2-acetamido-2-deoxy-a- and &D-glucoppnOeyl)- ( 1 4 )-2-amino-2deo ~ ~ - D - ~ ~ uwere c o ~ isolated E, and characterized.

b. Catalysis by Ion-Exchange Resins.-Cation-exchange resins in the hydrogen ion form have been used for the inversion of spcrose,lU the hydrolysis of such water-soluble plysaccharides as starch,ldQand the s y n t h i s of glycoSides.luJM The use of such resins for the synthesis of ol+ and poly-saccharides wm first reported in 1954 by Zempl6n end Kisfaludy.I47 They heated a solution of D-glucose with a phenolsulfonic acid resin at 70" for 72 hours. A complex mixture of oligo- and poly-saccharides was formed, from which gentiobiose was isolated. Laminaribiose was produced by the action of a curtion-exchange resin on D-glucose.la In a series of papers,~J4°-1pO'Colla and coworkers reported their investigations on the synthesis of oligo- and poly-saccharides, using ionexchange resins. A disaccharide corresponding to isomaltose, and other oligosaccharides, were obtainedlQgwhen a mixture of equal weights of Zeo-Karb 215 and ~gluoosemonohydrate was heated rtt 100" for 15 min. Longer heating at 100" gave higher saccharides that were immobile on ( [CY]D 81") paper chromatograms. A non-dialyzable poly~rtccharide~~g waa isolated by heating a 1:1 mixture of D-glucose monohydrate and the cation-exchangeresin Amberlite IRlU) (He) at 1OOo/20 Torr for 4 hours, and then at 146" for 2 hours. Partial hydrolysis of the polyglucoae gave

+

(142) A, B. Foster and D. Horton, J. Chem,A%., 1890 (1958). (143) 8.Kaiohi, B d . C h .Soc. Japan, 80, 844 (1857). (144) 0. Bodaner and R. Kunin, Znd. Eng. C h . ,48, 1082 (1951). (146) W. H.Wadman, J . C h . Soc., 3061 (1952). (146) J. E.Cadotta, F. Smith, end D. Spriestambach,J . Am. C h .Soc., 74,1601 (1962). (147) 0. Zemplh and L. Kiafaludy, Ada Chim. A d . Sci. Hung., 76, 2221 (1964). (148) K. Anno, N. &no, E. Nakamure, H.Srtito, and R. Hoshi, Bull. Agr. C h . Soc. Japan, 9 , 6 7 (1969). (149) P. S. O'Colla and E. Lee, Chmr.Znd. (London), 522 (1956). (150) P. S. O'Colla and E. Lee, J . Chsm.Soc., 2361 (1984). (151) P. S. O'Colle, E. Lee, and D. McGrath, Chsm. Znd. (London), 178 (1962). (162) P. 8. O'Colla and E. Lee, Abetr. Intern. 8ymp. Carbohydrak C h . , Mdnder, 40 (1084).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

465

maltose as the preponderant disaccharide. Oxidation with periodate (1.7 moles of periodate consumed and 0.6 mole of formic acid evolved per molar proportion of mglucose residue) indicated a content of 60% of ( 1 4 ) - l i k e

linkrtges.

A further studye0 of the disaccharide fraction formed by the action of Amberlite IR-120 (He) on molten D-glucose revealed the pmence of isOmalto3e, gentiobiose, laminaribiose, cellobiose, a trehalose, and, possibly, 0-D-glucopyranosyl-( 1-5) -wglucose. The first three oligosaccharides were isolated and characterized aa their crystalline octrtacetates. The high incidence of (1-3)- and (1+6)-1inked disaccharides, and the absence of maltose, is in contrast t o the products isolated from the reversion of mglucose in aqueous mineral acid. O’Colla and coworkers suggestedm that the cation-exchangeresin may exert some steric control under the relatively rapid polymerizing conditions (M contraated with conditions of thermodynamic equilibrium). Polyglucose preparations were obtainedlS0in yields of 13-36% on polymcation-exeriaing Dglucose in the presence of Amberlite IR-120 (He) change resin at 100 to 150’. These polymers had a P, ranging from 18 to 50 (based on reducing power) and DI.[ 64 to + 8 5 O (water). One of these nondialyzable glucans consumed 1.42 moles of periodate and liberated 0.47 mole of formic acid per mole. About 6.2% of D-glucose survived periodate oxidation, aa determined by the Barry degradation. This glucan could not be methylated to completion (found: OCH,, 38.3%; calcd., 45.6%) and, when the incompletely methylated polymer was hydrolyzed, material identical with the resin used in its preparation was isolated, thereby indicating possible chemical combination. Tetra-, tri-, di-, and monomethyl ethers were obtaiped in the ratio of 5:1:4:2. The only qualitative deduction that can be niade from these data supporta a highlybranched structure for the polyglucose. O’Colla and col18&gu881s1have investigated the condensation of partially acetylated sugar derivative. Thus, when a solution of 1,2 ,3,4tetra-0acetyl-&D-glucose and ptoluenesulfonic acid in acetone waa concentrated to remove solvent, the residue heated at 100°/20 Torr for 1.5 hours, and the product deacetylated, chromatographic examination revealed the preeence of D-gliicose and at leaat mven oligosaccharides. Levoglucosan, gentiobiose, gentiotriose, and gentiotetraose were isolated and identified. On using zinc chloride aa the catalyst, the same tetm-O-acetyl-pD-glucose gave a polysaccharide which, after‘deacetylation, had [&ID - 2.9’ (in water) , indicating a preponderance of p-D-linkages. The polymer consumed 2.1 moles of periodate per mole and yielded 1.1molecules of formic acid per D-glucosyl midue, suggesting a (1*6)-lhked polymer. Hydrolysis of the methylated polyglucose afforded 2,3,4-tri- and 2,3,4,6-tetra-O-methyl-

+

466

I. J. QOLDM'EIN AND T. L. HULLAR

D-glucose in the ratio of 8.3: 1 and only a trace of a di-0-methyl-*glucose, further confirming that it waa a (1+6)-linked polymer. The disaccharide fraction from the zinc chloride polymerkation of 1,3 ,4 ,&tetra-O-acetyl-w glucose gave sophorose and kojibiose in the ratio of 3: 1,whereas 1,2,3,6tetra-0-acetybglucose gave cellobiose and maltoae in the same ratio. The melt polymerizations of 1,2,3,4-tetra-O-acsetyl-/3-wmannose and -wgalactose in the presence of zinc chloride have been described.'" Chemical investigation of the structure of the polymsnnose suggested that it is mainly an a+( l4)-linked polymer having a p . of 10. In the same way, 1 , 2 ,3 ,Ctetra-O-acetyl-PgaIactose was polymerized. c. Catalysie by Boric Acid.-A patent by Leuck'" described the polymerhation of pure u- or /~-D-~~UCOIW in the solid shte. The catalyst used wns boric acid or ita anhydride, with sulfur dioxide or hydrogen chloride serving &B a possible secondary catalyst. After heat treatment, the products were light in color, with p,, 5 8. No information waa given on their chemid structure. D-Xylose, maltose, and sucrose were also reportedlM to polymerize when heated above their melting points in the presence of an acid or of a water-soluble ealt, such as barium perchlorate or magnesium sulfate.lMJu This work was re-investigated by Tipson and coworker^.^^^ Heat treatment at 140 to 150°, over several time-intervals, of a dry blend of anhydrous crystalline a- glucose in the presence of metaboric acid ( 5 parts per 100 pa& of D-gluoose) gave rise to a series of polyglucoses. The crystalline appeawce of the starting material W&B retained, but x-ray diffraction patterns revealed that the product waa actually amorphous.168JM The use of metaboric acid was advantageous, since it effected but little decomposition of the D-glucose, and it waa readily removed by extraction of the product with cold methanol. Molecular-weight determinations, using both cryoscopic and reducing methods, led to discrepancies which were partially explained on the basis of the presence of some terminal 1 anhy hydro-@-^glucopyranoee residues. The apparent p,, by cryoscopy ranged from 6 to 17; the [u]B 60' (average) indicated a mixture of a- and j3-D-glucosidic linkages. Limited hydrolysie with acid gave D-glucose and oligoeaccharides, one of which had the mobility of gentiobiose or isomaltose on paper chromat~grams.The kinetics of hydrolysis suggested that the polymers were

+

(163) 0. J. buck, U. 8. Pat. 2,375,684 (1946); Chem. Abalrada, SO, 4608 (1946). (164) Q. J. Jaiick, U. 8. Pat. 2,387,276 (1946); C k m . Abetructe, 40, 7569 (1946); 9. Pat. 2,400,423 (1946); Cheni. Abelruels, 40,4904 (1946); U. 8. Pat. 2,436,967 (1848); C h . Abstmcta, 42,3602 (1948). (165) Corn Product8 Refining Co., Brit. Pat. 689,024 (1947); Chem. Ahtracls, 41, 6426 (1947); Brit. Pat. 688,988 (1947); Chem. Abatracb, 41,6426 (1947). (166) H. W. Durnnd, M. F. Dull, arid R. 8. Tipson, J . Am. Chern. Soc., 80,3691 (1958).

u.

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

467

composed chiefly of wglucopyranwidic residuee, with some non- (1 4 ) linkagee; the initial, rapid rate of hydrolysis by 0.1 N hydrochloric acid was attributed to possible hydrolysis of tenninal 1 , 6 - a n h y b p w g l u ~ pyranoee residuee. Methylation (found OCHa, 44.2%; calcd. 45.6%) followed by qualitative analysis of the O-methyl sugars revealed the preeenoe of high concentrations of 2,3,4,6-tetra-O-methyI-~-glucose and 2,3,4-tri-O-methyl-~-glucose, with lower proportions of 2,3,6tri-O-methyl-D-glucose,2,3-di-O-methylD-gIucose, and other sugars. It waa concluded t b t this polymer is a hifly branched polyglucose having large proportions of ( 1 4 ) -wglucosidic linkages, and some D-glucopyranosyl residues joined by both ( 1 4 ) - and ( 1 4 )-bonds. d. Polymerizations Conducted under Diminished Pressure.-The behavior of solid reducing sugars on being heated under diminished pressure in the abmzce of a catalyst haa beer1 examined by several in~estigators~67-~~~ who have observed a loss of water and the formation of compoundsbelieved to be 1 ,Zanhydro ~ugars~~7--l~ and 0ligosaocharidea.~~7 -IQ Hurd and Edward~~ refuted ( ~ earlier claims that 1,2-anhydro sugars are formed, and noted that materials of higher molecular weight are produced, The anhydro sugar which Pictet and EganlWobtained by heating lactose for 10-12 hours at 185'/4-6 Tom polymerizedlm when heated with zinc chloride. This anhydro sugar could have been 1 ,2-anhydrolactose,-l" T w o substituted sugars, namely 2,3-O-isopropylidene-~-lyxofuranose1~ and 2 , 3 ,5tri-O-methyl-D-lyxofuranose,16*b undergo polymerization during slow distillation under diminished pressure; the latter formed a nonreducing dimer of the trehalose type. A major undertaking was initiated in 1950 by Pacsu and Mora,IMand later continued by Mora and colleagues, to establish optimal conditions for the condensation polymerization of simple sugars, both mono- and disaccharides. These studies also involved the preparation of derivatives of polyglucose [containing -COIH, -OSOsH, -0NO3, -O-CH2CH2NHlr, (157) A. Pictet and P. Castan, Helv. Chim. Ada, S, 645 (1920). A. Pictet and H. Vernet, Helu. Chdm. Ada, 6, 444 (1922). (159) M.Cramer and E. H. Cox, Helv. Chim. A&, 6, 884 (1922). (160) A. Pictet and M. M.Egan, Heh. Chim. Ada, 7,296 (1924). (161)C. D.Hurd and 0. E. Edwarda, J . Org. Chem., 14, 680 (1949). (162) I. E. Puddington, Can. J . Rss., B26,415 (1948). (163) J. R. Clamp, L. Hough, J. L. Hickson, and R. L. Whistler, A h n . Carb0hydm.h Chem., 16, 198 (1961). (163a) P. A. Levene and R. S. Tipson, J . BWZ. Chem., 116, 731 (1936). (l63b)H. G.Bott, E. L. Hirat, and J. A. B. Smith, J . Chem.SOC.,658 (1930). (184) E.Pacsu and P. T. Mora, J . Am. Chem. SOC.,72, 1045 (1950).

(1e)

468

I. J. OOLDSTEIN AND ‘I‘ L. . HULWR

-O--CH2-CH2N

(CHICHI)13 which were subsequently used in biological studies (see Section V), Commencing in 1961,a series of papers by Dutton and Unrau2’.10 have reported structural studies on the synthetic polymers prepared hy Mora and coworkers. These independent investigations represent a major advance in our knowledge of the chemical structure of this highly complex and potentially useful group of eynthetic polymers. conThe initial method,l*-lw first employed by Grimaux and Lefe~re,~’ sisted in evaporating under greatly diminished pressure, at 0 to 45”,a concentrated solution of the sugar in approximately 5% hydrochloric acid. After dialysis against running tap water for several days, the polymers were isolated in 15 to 20% yield by precipitation with methanol. A poly108’ (in water) and p,, 42 glucose prepared in this manner had [a38 (by solution osmometry), and waa barely reducing to Fehling solution. The reportw that the product was attacked by “both salivary enzyme and hemicellulase” has been attributed to the presence of oligosaccharides of low molecular wcight.lfl Mora and Wood1aasubsequently reported three methods for the polymerization of anhydrous a-D-glucoee: (1) a melt polymerization, with infrared lamps &B the heat source; (2) a melt polymerization employing tetramethylene eulfone as an inert solvent; and (3) a two-shge, melt polymerization in which the product from the first stage was isolated, powdered, and reheated in the second stage. A high degree of polymerization was favored by a high concentration of *glucose, the presence of an acid catalyst at a concentration which would not cause side reactions, a high temperature, an effective removal of the water produced during the polymerization, and the absence of free oxygen. All condensations were conductod a t 0.01-1600 X lo-* Torr over the temperature range of 140-170O in the presence of 0.164% of phosphorous acid. These conditions kept such side reactions as decomposition to 5- (hydroxymethyl)-2-furaldehyde and levulinic acid to the minimum. Control was most difficult in the “infrared method,” probably because of uneven heating. In the absence of an acid catalyst, conversion of Dglucose into nohdialyzable polymer was extremely low (14.8% yield, as400) as compared to a conversion of 69 to 82% of nondialyzable product 3,250-28,800) in the presence of acid.’dg

+

(a,,

(165) P. T. Mora and E. Pacau, U. S. Pet. 2,719,179 (1965); Chem. Abetrade, 60, 0823 (1956). (166) E. Pacsu and P. T. Mom, Can. Pat. 530,070 (1066). (187) P. T. Mora, Private communication. (168) P. T. Mora and J. W. Wood, J. Am. Chem. Soc., 80, 686 (1958). (160) A eerieR of polyglucoeea, prepared by DuPont for pilot studies, waa also aynthesized by these methods.

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

469

Depending on the conditions, the products obtainedla were light amber to dark tan, hygroscopic resins which could be hydrolyzed completely (96-1000/o) to Pglucose by boiling 1 N hydrochloric acid. Insoluble gels, attributed to the use of high temperatures, were formed in a few cases. The 58 to +80.5" (in 1 N hydrochloric polymer preparationsla had [u]3 acid). In general, the specific rotations rose as the temperature of polymerization was elevated. were determined by a reducing Number-average molecular weights end-residue method, the assumption being made that each polymer chain actually has a reducing end-residue. This assumption is supported by the results of a study170in which the molecular weight of a polyglucose, prepared by a procesb similarlB7to stage one of the two-stage process,1@was determined by copper171 and radioactive cyanide1T2procedures and by solution osmometry.17a Equivalent results were obtained by each method. However, comparisons have not been performed for the polyglucoses prepared by the prepared by three methods described1@above or for other polyglyco~es~~~ these methods. Thus, it is possible that some of the preparations de~ 7 ~ , 1 7 do 6 not contain one reducing end-residue per polymeric scribed1@ chain, but may, instead, possess nonreducing structures, such as terminal 1,6-anhydro-/3-~-glucopyranoseresidues and trehalose-type linkages. Conversion of reducing end-residues of polyeaccharides into 1,Sanhydro units is known to occur when polysaccharides are heated.l76 Trehalose-type linkages cannot be excluded on the basis of energetic reasons, since the activation energy of hydrolysis for a trehalose is 40.2 kcal./mole and for other dmccharidea is 30.0-38.6 kcal./mole'g; trehdose-type linkages have been found to occur in acid-catalyzed polycondenscLtions.Mlw The intrinsic viscosity of the polyglucoses wm about one-tenth that of dextran,ln indicating spherical molecules. This conclusion supports the highly branched structure deducted by chemical studies. The theory of condensation polymerization of aldoses was considered in

+

(an)

(170) P. T. Mom, J . P01um.t~Sci., 28, Part 1, 345 (1957). (171) H. 8. Isbell, W. W. Pigman, and H.L. Frush, J . Res. Natl. Bur. SM.,24,241 (1940). (172) J. D. Moyer and H. S. Isbell, A n d . C h . ,80, 1976 (1958); H. S. Isbell, Methode Carbohydrde C h . ,6, 249 (1965). (173) R. U. Bonnar, M. Dimbat, and F. H. Stross, "Number-Average Molecular Weight," Interscience Publishera, Inc., New York, N. Y., 1958. (174) P. T. Mom, J. W. Wood, and V. W. McFarland, J . Am. C h . Soc., 82, 3418 (1960). (176) P. T. Mom, J. W. Wood, P. Msury, and B. G. Young, J. Am. Cham.Soc., 80,693 (1958). (176) A. Thornpaon and M. L. Wolfrom, J. Am. C h . Soc., 80, 6618 (1988). (177) A. Jeanes, W. C. Haynea, C. A. Wilham, J. C. Rankin, E. H. Melvin, M. J.

Autin, J. E. Cluskey, B. E. Fisher, H. M. Tsuchiya, and C. E. Rist, J . Am. C h .Soc., 76,5041 (1954).

470

I. J. QOLDSTEIN AND T. L. HULLAR

some detail by Mora and ~ o w o r k e r s , ' ~ ~ ~using " . ' ~ 6the ~ ~ model ~ originated by Flory'"J79 and modified by Erlander and French.9' In this treatment, a monomer may be represented as A-RB,-1, where A is the glycosidic hydroxyl group; B, the nonglycosidic hydroxyl groups; and f, the total functionality of the monomer. In the w e of wglucose, one hemiacetal hydroxyl group and four alcoholic hydroxyl groups gave f = 5. The usual assumption that the B groups are equally reactive does not hold here.%t2" The presence of gel in the product from the infrared procedure is ascribed to B-B units (true ether linkages). However, these linkages are relatively resistant to hydrolysis, as shown by the stability of a "disaccharide" of the true ethec type to prolonged boiling in hydrochloric acid.Im The fact that Mom and Wood'" recovered SS-lOO% of the reducing power of the Pglucose in the polysaccharide suggeste the absence of these linkages, at least in the polymers which they examined. In addition, Mom excluded internal linkages of the A-B type, such as those of 1,&anhydro-D-glucose residues.'*' However, the finding of 1,&anhydr@-D-glucopyranose among the products of reversionK4and in a partial hydrolyzate (by acid) of a pyrode~trin'7~suggests the possibility that thew. residues may exist in polyglucosea prepared by condensation polymerization. Because of the greater reactivity of primary relative to secondary hydroxyI groups*2*" (see Section II,l), it is to be expected that (1-6)linkages will preponderate in these polycondensates. As the temperature is raised, however, this selectivity will be somewhat diminished, and the relative proportion of other types of linkage will be increased. In addition, the degree of branching due to multiple condensations at individual D-glucose residues will also be inorwed. Condensation polymerization at clevated temperatures, then, should lead to heterogeneous polyglucose preparations of broad distribution of molecular weight in which all variations of linkage type, branching, and ring size will be found. The variation of any of the rertction conditions should thus have profound effects on the course of the polymerization and on the nature of the products. As expected, the temperature of polymerization was inversely related to the consumption of periodate and the liberation of formic acid.176 ~ 7 Also, * Mora and Wood'@ found that high temperatures generally produce polyglucose fractions having higher molecular weights and higher intrinsic viscosities. However, fractionation with ethanolmJ7'j was extremely complex: the fractions which were first precipitated appeared to have the higher molecular weights and degree of branching (see Section IV,4,c), (178) P. T. Mora, in "The Origin of Prebiologid 8y&m and of their Molecular Matrim," S. Fox, ed., Academic Prese Inc., New York, N. Y., 1965, p. 281. (179) Ref. 12, Chapter IX. (180) V. E. Gilbert, F. Smith, and M. Stacey, J . Chem. Soc., 822 (1946). (181) However, see h f . 12, p. M.

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

471

although some exceptions were found. The solution properties of polyglucose and its fractionation, as studied by Mom, have been discussed in detail. 188,17036 An intensive study of the chemical constitution of one of Mora's polyglucosea was conducted by Dutton and Unrau.26-m*'82 These workers noted the rapid liberation of formaldehyde*Vduring periodate oxidation of the polymer. Reduction of the polyglucose by sodium borohydride, prior to periodate oxidation, resulted in the liberation of a greater proportion of formaldehyde. A P, of about 165 (in good agreement with that reported by Mora and c o w ~ r k e r s ~was ~ J ~calculated ~) on the assumption that the excess formaldehyde originated from a D-glucitol end-group. A portion of the formaldehyde was believed to stem from D-glucofuranosyl residues unsubstituted at C-5 and C-6, a possibility which Mora and coworkers had also indicated on the same basis.'" A relatively large initial increase in specific optical rotation was observed when polyglucose fractions were subjected to hydrolysis with dilute mineral acid,lTf,perhaps indicating cleavage of some of these labile, furanosidically linked residues. Support for the existence of furanosyl residues waa obtained by subjecting the pekodate-oxidked, reduced polyglucose (polyglucose "polyalcohol") to complete hydrolysis.28The following were obtained (approximate molar ratios given in parentheses) : glyceritol (105), erythritol (15), D-xylose (1), and D-glucose (5).The glyceritol arose from terminal, nonreducing, ( 1 4 ) linked D-glucopyranosyl residues and from terminal, nonreducing D-glucofuranosyl residues (assuming no overoxidation) ;the erythritol, from ( 1 4 ) and (1-+4,6tl) -linked D-glucopyranosyl residues1u; and the D-xylose, from internal, substituted D-glucofuranosyl residues in which the C-5 and C-6 hydroxyl groups were unsubstituted. The periodate-resistant D-glucose originated from those residues which carried substituents a t C-3, or substituenta at both C-2 and (3-4, of wglucopyranosyl residues, or at both C-2 and C-6 of wglucofuranosyl residues. Evidence for (1-+2)-linkages waa also obtained*Oby oxidizing the polyglucose polyaldehyde with bromine, to give the corresponding polycarboxylic acid. Hydrolysis, separation of the glyceric acid by means of ion-exchange resins, lactonization, and subsequent reduction, gave glyceritol. The polyglucose polyalcoho1182was also subjected to a Smith degradation'" (see Section IV,4,d). From the products of partial hydrolysis by acid, both anomers of 0-wglucopyranosyl- (l+l)-~-glyceritol were isolated (182) G. G. S. Duttori and A. M. Unrau, Can. J . Chmn., 42, 2048 (1964). (183) P. T. Mora, E. Merler, and P. Maury,J. Am. Chem.Soc., 81,5449 (1959). (184) G. W. Hay, B. A. Lewis, and F. Smith, Methods CuraOhydrale Chem.,6, 377 (1966)

(and references therein).

(185) I. J. C.oldstein,G. W. Hay, B. A. Lewis, and F. Smith, Meulods Carbohydrate Chem., 6, 361 (1965) (and references therein).

I. J. GOLDSTEIN AND T. L. RULLAR

472

and identified, providing experimental proof for the existence of a- and fl-D- (1 4 6 )-linkages; O-~-xylofuranosyl-( 1 4 2 )-D-erythritol was also obtained, which further confirmed the existence of internal, D-glucofuranosyl residues in the synthetic polysaccharide. Other, more complex substances were isolated, some containing up to eight intact sugar residues. Complete methylation" of the polyglucose d s auhieved with difficulty, to give a product showing the unusual property of being soluble in petroleum ether (30-60") containing only 5% of ohlorofom. Hydrolysis of the methylated polyglucose gave fractions of tetra-, tri-, di-, and mono-0methyl-D-glucose9 in the ratio of 4.9:5.0:2.9:1,in addition to a small proportion of D-glucose. The tetra-0-methyl-P-glucose fraction contained this compound represents about 20% of 2,3,5,6tetra-O-methyl-~glucose; terminal, nonreducing, D-glucofuranosyl residues. Nearly all of the possible pyranose and furanose tri-0-methyl-Dglucoses were found and identified, the presence of which including 3,5,6-and 2,3,5-tri-O-methyl-~-glucose, demonstrates the existence of internal P-glucofuranosyl residues. A highly complex mixture of di-0-methyl-D-glucoses and all possible mono-O-methylD-glucoses was obtained; the great proportion of the 3-methyl ether suggested a relatively unreactive C-3hydroxyl group, at least of the tri-0-Dglucosylated residues (see p. 437). In summary, Dutton and Unraul**found Mora's synthetic glucan to be a highly branched, randomly linked polysaccharide containing every conceivable type of wglucosidic linkage, with the ( 1 4 ) - l i n h g e preponderating. n-Glucofuranosyl residues were shown to occur both as terminal, nonreducing residues and aa internal residues. Using conditions similar to those employed for the preparation of the polyglucoees, Mora and coworker~l'~also polymerized other aldohexoses (D-galactose and mann nose) , two deoxy sugars (Zdeoxy-D-araln'no-hexose and &deoxy-cmannose), aldopentosea (Garabinose, D-xyloBe, and D-ribose) , and a disaccharide (maltose). Milder conditions were required for the deoxy sugars and the pentom. The properties of these polymers are presented in Table IV. The polymer from 2-deoxy-~arabano-hexosewas found to be fairly soluble in alcohol and very sensitive to hydrolysis by acid. There was no significant amount of formic acid liberated on periodate oxidation, since internal residuee do not contain three adjacent hydroxyl groups; the formic acid obtained arose, probably, from overoxidation at the reducing end of the polymer molecule. A positive precipitation reactionIBawith concanavalin A (see Section W,6) suggests the presence of at least a few terminal, residues. nonreducing 2-dooxy-cr-~-arabino-hexopyranosyl (186) I. J. Goldstein, C. E.Hollerman, and J. M. Merrick, Bwchim. BiOphy8. Ada, 97, 68 (1965).

TABLE IV Characterization of Polysaccharides [age,

Polymer of

degrees (inH&)

+42.4

+&.l +75.0 +2i. 1 +53.5 -8.6 -40.4 +105.5 Assuming one reducing end-group.

N HC1 for 1 hr.

~osson

dialysis,

% 17 64 11

76 26 83 31

Reducing

[?I, dl./g.

0.09 0.04 0.03 0.07 0.05 0.06 0.04 0.06

0

m 104-

consumed

molecule per sugar residue

power,*

tsa % 18,700 17,900 7,100 6,300 3,300 5,700 1,500 8,300

81 93 99"

97 93 93 103 104

HCOZH p r o d u d

48hr.

72hr.

48hr.

72hr.

1.17 1.58 0.36 0.67 0.87 0.70 1.08 1.11

1.34 1.79 0.49 0.69 0.89 0.79 1.11 1.12

0.34 0.75 0.04 0.13 0.39 0.22 0.35 0.32

0.37 0.81 0.04 0.13 0.34 0.22 0.38 0.40

Reducing power of sugar recovered after hydrolysis in N HC1 for 1 hr.

Hydrolysis by 0.1

; PF

9 W

8

$ g ii 3!

474

I. J. QOLDSTEIN AND T. L. HULLAR

In contrast to the relatively simple polyxylose of Bishop,14L the polyxylose prepared by Mora Tnd examined by Dutton and Unrau1a7-1aswas extremely complex. Its facile depolymeri~ation~~7 during the early stages of hydrolysis with 0.1 N hydrochloric acid suggested the Iwesctica of D-xylofuraiiosyl residuw. Pcrioctate oxidation msulled in the ootwumption of 0.91 inolc of oxidant pel*niolecular equivalent of sugar residuu, iudiuating the presence of periodate-resistant residues. lcormaldehyde ww also liberated; this was believed to arise from acyclic D-xylosc residues unsubstituted a t C-4 and C-5.

Application of the Smith degradstion'aJ88 gave ethylene glycol (from the nonreducing, D-xylopyranose end-groups) , glyceritol (from internal D-xylose residues and nonreducing D-xyiofuranose end-residues), D-xylose (from readily hydrolyzable D-xylofuranosyl residues) , and a series of nonreducing oligosaccharides. The identification of two of these substances, namely, 0-D-xylofuranosyl-( 141) -cglyceritol and O-n-xylopyranosyl(l+l)-bglyceritol, established the presence of the sequences Xylf-( 1 4 5 ) Xylf and Xylp- ( 1 k 5 )-Xylf. As with most of the synthetic polysaccharides, complete methylationl89 of the polyxylose ww difficult. The fully methylated polymer was soluble in petroleum ether (30-60') containing 5% of chloroform. Hydrolysis of the methylated xylan gave tri-, di-, and mono-0-methyl-mxyloses, together with D-xylose, in the molar ratio of 31 :33:19:5. The tri-0-methyl-D-xylose fraction contained 38% of 2,3, btri-0-methyl-mxylose, the remainder being the 2,3,4-trimethyl ether. The dimethyl ethers included the 2 ,5-, 3,5-, 2,3-, 2,4-, and 3,4-di-O-methyl-D-xyloaee. Evidently, the polyxylose is a very complex, highly branched polysaccharide which contains a high proportion of furanosyl residues (some of which are at nonreducing chain ends) and, possibly, some acyclic residues. The polymabinose preparation ([a18A 27.1 ') of Mora and coworkers17' waa subjected to methylation analysis by Dutton and Unrau.'@ In common with other synthetic polysaccharides prepared by this method, the polyarabinoee had a highly ramified structure; 16% of the nonreducing endp u p s of the polysaccharide were furanoid, as indicated by the isolation of 2,3,6-tri-O-methyl-carabinose. Among the di-0-methyl sugars isolated were 2 ,A- and 3 ,.5-di-O-methyl-L-arbinosp, indicating the presence of internal furanosyl residues in the parent arabinan. All of the possible monomethyl ethers were isolated. The polyrhamnose ww found by methylation analysis'@lto contain 35%

+

(187) (188) 1180) 1190) (191)

G. G. 8.Dutton and A. M. Unrau, Con. J . C h . ,40, 1479 (1962). G. G. 8.Dutton and A. M.Unrau, Con. J . C h . ,40,2101 (1962). G. G. 5. Dutton and A. M. Unrau, Con. J . C h . ,40, 2105 (1962). G. 0 . 8 . Dutton and A. M.Unrau, Can. J . C h . ,43,934 (1965). G. G. S. Dutton and A. M. Unrau, Con. J . Chem., 43, 1738 (1965).

475

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

of the ternliual, noureducing residues as fuirriioid gioup. Of the dimethyl ether fraction, 2,3-di-O-rnethyl+rhamnwe coxlstituted 75%, indicating that ( 1 4 )-linkages preponderate. Isolation of the three Smith-degradation products [namely, 0-a-crhamnopyranosyl- ( 143)-l-deoxy-~-erythrit, the corresponding &L anomer, and 0-&L-rhanmopyraiiwyl- ( 1 4 2 )-1deoxy-perythritol, the &L linkages being inferred from the specific rotatione] is evidence for the sequences O-I,-rhamnopyranoayl- ( 1 - 4 ) -L-rhamnose, both a- and pclinked, and O-&L-rhamnopyranosyl- ( 1-+5)-crhamnofuranose, respectively, in the synthetic rhamnan. The polymaltose prepared by Mora and coworkersl" exhibited a higher 105.5') and lower consumption of periodate specific rotation ([a18 (and lower liberation of formic acid) than the polyglucose ( [ c Y ] ~ 65') prepared by the same method.ld* Significantly, the polymaltose showed a vigorous reaction with lS2 concanavalin A (see Section IV,6) as compared to the po1yglucose.102These results suggest that the polymaltose contains a higher proportion of a-D-glucosidic linkages and more a-D-linked, nonreducing, terminal residues than does the polyglucose. A mechanism for the polycondensation of D-glucose (41) in acidic media was set forth by Mora and Wood.'" They postulated protonation of the ring-oxygen atom followed by ring opening to afford an open-chain, C-1 carbonium ion (42). Reaction of (42) with a hydroxyl group of a second which cyclises to give a &glucose molecule gives the hemiacetal (a), disaccharide, the first product to be expected in such a polymerhation.

+

HOH,?

+

HOHaF

HOHaC

___t

OH (41)

OH (42)

OH. (43), R = glycosyl

Intensive investigation on the mechanism of acid hydrolysis of glycosides haa led to the conclusion that it is the glycosidic oxygen atom which is protonated.la* In view of these findings and of the highly unstable nature (192) I. J. Qsldatein, Unpublished results. (193) For studiea on the mechaniam of hydrolysia of glycoaidw, me C.A. Bunton, T.A. Lewis, D. R. Llewellyn, and C. A. Vernon, J . C h . Soc., 4419 (1966); B. E. C. Banks, Y. Meinwald, A. J. Rhind-Tutt, I. Sheft, and C.A. Vernon, ibid., 3240 (1901); C.h o u r , C.A. Bunton, S.Patai, L. H. Selmm, and C. A. Vernon, sW., 412 (1901); C. Bamford, B. Capon,and W.G.Overend, ibid.,6138 (1902); R. L. Whistler and T. Van ES,J . Org. C h . ,18,2303 (1963); R. L. Whistler and R. M. &well, ibia., as, 3290 (1964).

476

I. J. OOLDSTEIN AND T. L. HULLAR

of (42) , Llic IJL'CYUII~rtutliorw prcfw iJ1ufollowing meohatikm. Protonation of the aoetstl hydroxyl group, followed by loas of water from (44) gives a carbonium ion (45) capable of resonance stabilimtion. Nucleophilic attack by a hydroxyl group from a second D-glucose molecule leads to tho formation of a glucosidic linkage, (46) atid (47). The aiiomeric coilfiguration of the )

carbon atom bearing the newly formed bond will depend on the environment about the carbonium ion at C-1 and on the meny faators deecribed in Section II,1. Since sugar solutions and melts also contain furanoid forms, the same mechanism accounts for the existence of furanmyl residues in synthetic polysaccharides.

e. Thermal Polymerization.-The thermal condensation of methyl Slow distillation of 2-deoxyglycofuranosides has been inve~tigated.'@~-~~7 methyl 2-deoxy-a ,p-L-eylthro-pentofuranoside led to polymeric material whenever superheating oc~urred.194,~9~ The water-soluble polymer was nonreducing to Fehling solution, had a P, of 5-7 (by the Rast method) , and could be converted into methyl 2-deoxy-~-~-erytpentofuranosidewhen treated with 1% methanolic hydrogen Similar observations were made for methyl 2-deoxy-a! BD- lo-hexofuranoside." When methyl 2de)

(194)

R. E. Deriaa, W. G . Overend, M. 2836 ( 1849)*

Stacey, and L. F. Wiggins, J .

i3hsm.

C h . Soc.,

(195) W. 0. Overend, F. Shafhadeh, and M. Stacey, J . Soc., 994 (1951). (196) I. W. Hughee, W. G. Overend, and M. Stacey, J , Chsm. Soc., 2848 (1949). (197) W. G. Overend, F. Bhafieadeh, and M. Stacey, J . C h . Sot., 671 (1950).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

477

oxy-a ,Bu-1yzc~hexol)yrano8idewas heated 'at 230" (12 Torr) for seveid hours, methanol waa evolved, and a brittle, water-soluble glass of P, about 4 wm obtained.lP6J" Ready hydrolysislQsof the polymer to 2deoxyD-lyxo-hexose suggcstcd retcntiou of the furanosidic ring structure during polymerization. Methylation analysis1s gave the 3,5 ,&trimethyl and an undefined dimethyl ether of Zdeoxy-D-lyxo-hexme. On the basis of the experimental data, a ( 1 4 6 )-linked polymer of 2-deoxy-~-Zyzo-hexofuranosyl residues was postulated. 111. ADDITIONPOLYMERIZATION 1. General

In the present article, addition polymerization of carbohydrates is defined aa the polymerization of sugar derivatives to give a glycosidically linked polymer in which there is no evolution of a by-product.'" Polymers obtained by this process are composed of structural units which possess a molecular formula identical with that of the monomeric units. Sugar derivatives possessing anhydro rings (internal ethers) ,such aa 1,&anhydro8-D-glucopyranose'w (levoglucosan), have generally been used in this type of polymerization (see equation 8). Although untried, lJ2-unsaturated sugars (glycals) , also may be suitable.

Addition polymerization of anhydro sugars may be regarded att being a reversible process, for levoglucosan is conveniently prepared by the pyrolysis of starch and cellulose.zOO~zo~ Since there is no by-product to be removed in polyaddition, the methods are experimentally simpler than those used for polycondensation. The general considerations developed in Section I1,1 concerning (a) the reactivity of hydroxyl groups in polyfunctional monomers and (b) the position of linkages in the resulting polymer apply also to addition polymerization of unsubstituted anhydro sugars. The ring size of the monomer incorporated and the configuration of the glycosidic linkage formed depend on the nature of the monomer, the catalyst employed, and the conditions of the plymerimtion. (188)For convenience, "polysddition" will be used aa a aynonym for addition polymerization. (199) C. Tanret, Bull. 8oc. Chim. (France), 8, 11,949 (1894). (200) A. Pictet and J. Saruin, Heb. Chim. A&, 1, 87 (1918). (201) R. B. Ward, Methoda Carbohydrate C h . , I, 394 (1963).

478

I. J. GOLDSTEIN AND T. L. HULLAR

2. Anhydro Sugars a. 1,6-Anhydro Sugars.-The

polymerization of 1,&anhydro sugars

has principally been studied with levoglucosan. The first investigations were conducted by Pictet.a- By heating levoglucossn at 240', a brown, viscous sirup was 0btained.a A product, isolated from this sirup by precipi-

+

tation with ethanol, was a weakly reducing, colorless powder ( [ a ] ~ 109") which gave no color with iodine. The material waa hydrolyzed to D-glucose by boiling, dilute, mineral acid, but w a not fermented by yeast. On the basis of its low molecular weight (677, by cryoscopy), Pictet considered that this material waa a tetramer. Platinum blackm was found to catalyze the polymerization, but its effect was not reproducib1F.m Anhydrous zinc chloride was subsequently used as catalyst. Investigation of the polymerization of levoglucosan a t various temperaturea and pressures led to the conclusion that dimers, tetramers, hemmers, and octamers are formed. One preparation was shown to contain approximately 2% of a nondialyzable fraction.g0d The products of the polymerization of levoglucosan were subsequently studied by several investigators. Pringaheim and Schmalzm repeated Pictet's work, using zinc chloride as the catalyst, and subjected the product to methylation. From the hydrolyzed, methylated material, they isolated one part of a tetra-0-methyl-D-glucose,and one part of a di-0-methyl-Dglucose which did not give an osazone. Irvine and Oldhamm polymerized levoglucossn by heating it at 250' in the presence of zinc dust. The product waa separated, by alcohol precipitation, into three fractions, later designated, on the basis of molecularweight determination of their methyl ethers, as triglucosan ([.ID 83.9'), tetraglucoean ( [.ID 60,7'), and heptaglucosan ([.ID 29.9'). The hydrolyzate of each methylated fraction wm composed of tetra-, tri-, di-, and mono-0-methyl-wgluaosea ;the presence of mono-0-methyl-D-glucose8 waa ascribed to incomplete methylation. From the mixture of methylated sugars, crystalline 2,3,4, gtetra-O-methyl-D-glucose (incorrectly designated as 2,3, Ti, &tetm-O-methyl-~glucose)waa obtained in a yield of 47.5% (by weight). A tri-0-methyl sugar ( D I . [ 77.5'), isolated in more smaller proportion, wm stated to be 2,3,5-tri-O-methyl-~-glucose; 70.5') or the probably, this sugar was the 2,3,4trimethyl ([a]*

+

+

+

+

(202) A. Pictat, Helv. Chim. Ada, 1, 226 (1918).

(203) A. Pidet and J. Pictet, H&. Chim. Ado, 4,788 (1921). (204)A. Pictet and J. Piatat, Cvmpl. Rand., 178, 168 (1921). (206) A. Pictet and J. H, Roea, C m p t . Rend., 174, 1113 (1922). (208)A. Pictat and J. H. Rase, Helv. Chim. Ada, 5,876 (1922). (207) J. C. Irvine and J. W. Oldhsm, J . C h .~ o c . ,147, 2903 (1926). (208) H.Pringaheim and K Schmslz, Bet., 66, 3001 (1922).

+

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

479

+

2,3,6-trimethyl ether ( [ a ] D 70"). With great insight, Irviiie and Oldhamm noted that "In marked contrast to natural polysaccharides, the synthetic dextrins afford striking examples of compounds constituted on an entirely different model in that the hydroxyl groups are not attached uniformly to thc individual C-6 unita." Sincc these workers observed that tri-0-methyllevoglucosan was recovered unchauged after it had been heated a t 250" for 10 hours in the presence of zinc dust, they concluded that the presence of free hydroxyl groups was necessary for polymerization to occur. Later studies have, however, shown that this sugar derivative can be polymeriied by using boron trifluoride etherate.m-210 Wolfrom and coworkers211j212studied the thermal polymerization of levoglucosan, in a re-investigation of Pictet's original study.2mThe anhydro sugar was heated in an open vessel a t 235-240" for 15 min., in the absence of water and acid, to give a product (yield, 40%) isolated by precipitation from aqueous solution with ethanol. This ethanol-insoluble product was subjected to partial hydrolysie with 0.15 N sulfuric acid. Separation of the producta of hydrolysis, using both carbon-column chromatography of the free sugars and Magnesol-Celite column chromatography of the sugar acetates, permitted identification of gentiobiose, isomaltose, maltose, cellobiose, sophorose [O-0-D-glucopyranosyl- ( 1-2) -~-glucose], and levoglucosan. This result established the presence of a- and /3-~-(1*6)-, a- and &~-(1+4)-, and 8-~-(1-+2)-linkages,and also suggested that a t least some of the chains contain 1,6-anhydro-&~-glucopyranoseend-residues. The conditions of hydrolysis and the yields of disaccharides obtained were such as to exclude formation of dkccharides by reversion and trans glycosidation p r o ~ e s s e s . ~ * ~ ~ * Examination of the ethanol-soluble portion212 from the thermal polymerization of lewglucosan revealed the presence of 1,6-anhydromaltose, 1,6anhydrocellobiose, 1,6-anhydrokojibiose, and l16-anhydrosophorose-a11 obtained crystalline-in addition to three unidentified anhydrotrisaccharides. Great interest is to be attached to the isolation of these anhydrodiand anhydmtri-saccharides, for they must represent addition products formed during the early stages of the polymerization of levoglucosan. At the high temperatures employed, direct displacement of the C-6 ether (200) V. V. Korshak, 0.P. Golova, V. A. Sergeev, N. M. Media, and R. Ya. Schneer, Vyeokomolehl. Soedin., 8, 477 (1961); Cham. Aastmdp, 68,583 (1962). (210) V. V. Korehak, V. A. Sergeyev, Ya. A. Surna, and R. Ya. Pernikis, Polyter Sn'. USSR (Engliah transl.), 6, 697 (1964); V~pokomOZekuZ.Soedin., 6, 1693 (1963); Chem. Abutraels, 61,328S (1964). (211) M. L. WoKrom, A. Thornpaon, and R. B. Ward, J . Am. Cham. Soc., 81, 4623 (19rib). (212) M. L. Wolfrom, A. Thompson, R. B. Ward, D. Horton, and R. H. Moore, J . Org. Cham., IS, 4617 (1961).

I. J. QOLDSTEIN AND T. L. HULLAR

480

bridge of (48) by a secoiidary hydroxyl group of another unit of (48) (see equation 9 ) may occur, to give an a-D-hked disaccharide, such as 1,0-anhydromaltose (49). Alternatively, heterolysis1'8 of the 1,6-ether linkage of (48) may take place to give a stabilized mrbonium ion (SO), which interacts with a secondary hydroxyl group of a second unit of (48) to give a mixture of a- and &blinked disaccharides, such as 1,&anhydromaItose (49) and l,&anhydrocellobiose (51) (see equation 10). It is interesting that no (1*3)-linked disaccharides were isolated, once again demon&i.rttingthe relative unreactivity of the C-3 hydroxyl group.

+ HO

(48)

-

(49)

+

In a seriea of papers commencing in 1959, Schuerch and coworkers have reported their extensive examination of the polymerization of levoglucosan*1*,214 and several of its derivatives,216 and of 1,B-anhydro-p-~galactopyranoseP4 (Pgalactosan) and ite 2-methyl ether." Preliminary experiments on the polymerization of levoglucosan were conducted under diminished pressure over a temperature range of 1W13Oo, using several acidic catalysts-formic, acetic, monochloroacetic, phosphoric, and hydrochloric acids,'*#and ainc chloride-in various mole ratios of catalyst to (213)J. da 8.Carvalho, W. Prins, and C. Bchuerch, J . Org. Chem,, 81, 4054 (1969). (214) H.Abe and W. Prins, Makromol. Chem., 42, 216 (1961). (215)A. Jabbar Mien, E. J. Quinn, and C. Schuerch, J . &g. Chem., 27, 1895 (1962). (2J6)The polymerhation of levoglucosan at 120-200O in the prerrence of hydrochloric acid in reportad in Ref. 7, p. 734; we, however, F. Hoffman-Labche & Co. A.-G., Ger, Pat. 613,126 (1928);Chem.Abstracts, 26, 1266 (1931).

CHEMICAL SYNTHESIS OF POLYSACCHAHIDE8

481

monomer, in the presence or absence of methyl sulfoxide or tetramethylene sulfone.**sThe most satisfactory polymerizations occurred at 115-120’ in an evacuated tube in the absence of solvent, using monochloroacetic acid as the catalyst in the ratio of 0.02 mole of catalyst per mole of monomer. Brittle, amber-colored glaases, completely soluble in water and free from gel, were obtained. The products were isolated by precipitation with 85% alcohol. The results of these studiee, are presented in Table V. Since the molecular weights recorded in Table V were determined by light-ecattering, TABLE V Condition6 of Preparation, and Re6ult8, for Selected Polymersa1* Catalyst ClCH,CO,H, mmole/ mole of levoPolymer glucoaan

Time Hr.

Min.

Temp., “C.

Yield by Precipitation in EtOH

(as%), 96

Po ~~

65

28 36 37

10.2 10.2 21.4 21.4 21.4

40

21.4

44

20A 32

21.4 10.7

27 29

a Polymer

40

5

20 12

30 10

123.5 123.8 123.8 124 115 125-121 121 121-126 110 110-115-120 123.8 116.5 123.8

52

4,235 8,000 9,950 43,300 24,300

67

49,200

60 71

309,000 29,400

M a

28 44 57.5

27 did not give any appreciableprecipitate with alcohol. b By light-scattering.

they are abnormally high because of the presence of “microgel,” and, hence, they are not the true molecular weights (see the later discussion) .*l4 Periodateoxidation studies conducted on polyglucose 40 (see Table V) showed that it consumed, on the average, 1.42 moles of periodate and liberated 0.50 mole of formic acid per molar equivalent of D-glucosyl residue. Formaldehyde was not determined. These results correspond, in a very approximate fashion, to 50% of (l+)-Iike linkages, 40% of ( 1 4 ) - or {1+2)-like linkages, and 10% of (1-*3)-like linkages. The (1-+6)-like linkages may be due to nonreducing, terminal Dglucopyranosyl groups or to ( 1-+6)-liiiked D-glucopyranosyl residues; the periodate oxidation

482

I. J. OOLDSTEIN AND T. L. HULLAR

data, considered alone, permit of no decision (see Section IV,4,c). However, the low viscosity of the polymers in aqueous solution, and the polyfunctionality of the monomer, taken together with the periodate-oxidation results and the high solubility in water of the polymerbed product suggest highly branched, spherical molecules, 91' f 5") led The specific optical rotation of theae polymers ([a]: Schuerch and coworkers*la to suggest that they contain a minor preponderance of a-D-glucosidic linkages. Only pyranoid forms were assumed to be present. In view of the finding of furanoid residues in other synthetic polysaccharides,ls~2~~ this supposition is no longer valid. Additionid experimeiital data, such the results of methylation studies, must be acquired before the chemical structure of these complicated polymers can be formulated. A study of the molecular-weight distribution of polyglucose addition polymers prepared by the method of Schuerch21* was undertaken by Abe by thermoelectric, vapor-phase osarid Prins.21' They determined mometry*17;aw and the z-average molecular weight by ultracentrifu) light-scattering. gation ; and weighbaverage molecular weight (~ L S by Measurement of a series of preparations showed ~ @ L Sto be consistently and considerably higher than &; this was attributed to a side reaction responsible for the formation of a few weight per cent of substances, termed microgel, of very high moleoular weight. Abe and PrinsZl4 suggested that the first step in the polymerizatioii reaction of levoglucosan coneista of a dimerization of all of the levoglucosan units. This involves the opening of all 1, h n h y d r o rings, followed by the formation of a reactive, (143)-linked intermediate (52), which polymerizes

+

an

(a,)

dH further. However, tho investigatioas by Wolfrom arid aoworkers211J12 do not support this theory. It will be recalled. that l,&anhydronlaltose, 1,Oanhydrocellobiow, 1,Ganhydrokojibiose, and 1,6-anhydrosophorose were cbractedsed aa being present in the ethanol-soluble fraction from the polymerhation of levoglucosan, thus showing that a- and &D-( 1-4)- and (217) J. van Dam and W.Wins, M e W Carbohydrds

Chem.,6,253 (1965).

CHEMICAL BYNTHESIS OF POLYSACCHARIDES

483

a- and &D-( 1-+2)-glucosidiclinkages are early products; no (1-+6)-linked disaccharide WM found in this fraction by experimental procedures which would have detected even minute quantities of isomaltose or gentiobiose. Furthermore, the periodate-oxidation data2l*do not support such an interpretation. If (52) were formed, the C-6 hydroxyl group would be the most reactive, and would be expected to enter into condensation with a second dimer." This would result, ultimately, in a polymer containing about 70% of (l-tti)-like linkages. The periodate-oxidation data, however, indicate only 50-55010 of (1+6)-like linbgea. Korshak and coworkeram polymerized levoglucoaan in p-diomne a t 80-90', using, w the catalyst, benzenesulfonic acid or such Lewis acids as boron trifluoride, ferric chloride, or aluminum chloride. Highly branched, amorphous products having I@w 38,ooO-g8,000(by light-scattering) were obtained. DGalactosan was polymerized2' a t 110" for 15 hr. in the presence of monochloroacetic acid in the ratio of 0.02 mole of catalyst per mole of mopomer-conditions essentially identical to those used for polymerizing levoglucosan. The product, obtained in 76% yield by precipitation with 85% ethanol, had I@,,1800 (by vapor-phe osmometry) and M w 22,500 (by ultracentrifugation) , demonstrating the wide molecular-weight distriThe specific, bution which is to be expected for this type of polymeri~ation.~~ optical rotation ([a]g 82') suggested the presence of both a- and @-Dgalactosidic linkages. Periodate oxidation studies revealed that this polymer is structurally similar to the polyglucose prepared in the same way; the C-3hydroxyl group is, however, even less substituted in this polygalactose than in the polyglucose. by means of monoPolymerization of 1,6-anhydro-@-~-mannopyranose has chloroacetic acid, using the conditions of Schuerch and cow0rkera,~~*~1~ also been accomplished,21*to give a polymannose in 25% yield. This polymer appears to be highly ramified and to contain a high proportion of terminal, nonreducing a-D-mannopyranosyl groups as determined by the precipitation reaction with conaanavalin A (see Section IV,6). Goldstein and Lindberg2'@ copolymerized levoglucosan and Dgalactosan, using monochloroacetic acid as the catalyst, to obtain a polygalactoglucose ([a]g 94'). A nondialyzable fraction of P,,approximately 90 (by 80lution osmometry) waa selected for study. Acid hydrolysis yielded only wglucose (52%) and n-galactose (48%). Partial hydrolysis by acid gave melibiose (among other cleavage products), indicating the starting material to be a true heteropolymer. The product consumed 1.3 moles of periodate and liberated 0.55 mole of formic acid per mole of hexosyl residue,

+

+

(218) R. de Souas and I. J. Goldstein, Unpublished results. (219) I. J . Goldstein and B. Iindberg, A& Chem. Soand., 16, 387 (1982).

484

I. J. QOLDSTEIN AND T. L. HULLAR

being similar in this mpect to the polyglucosdU and polygalactose2‘ prepared by analogous methods. The periodate-oxidkd polysaccharide waa reduced with sodium borohydride, and the product was hydrolyzed, to afford glyceritol, erythritol, ~-glucose,~-&ctoee, and small proportions of arabinom and xylose. The unoxidLed glucose (12%) and %galactose (10%) indicated the presence of (1+3)-linkage or of residues which are multi-linked, containing, for example, (1-2)- and (14)-linkages. Erythritol arose from those wglucosyl residues which were linked (1-4) or ( 1 4 ) and ( 1 4 6 ) ; the apparent absence of threitol suggested that D-galactosyl residues are not similarly linked. The high proportion of glyceritol isolated from the hydrolyzed poIyalcoho1 is considered to derive, most probably, from a highly branched structure containing a high content of (l-t6)-liilced residues. The presence of arabinom and xylose nray have arisen, reopectively, from galacto- and gluco-furanosyl units substituted at C-2 or C-3 but unsubstituted at C-5 and C-6. Evidence for such residues in the polyglucose obtained by polycondensation in uacw) has been obtained (see p. 471).26~*0~* In an effort to study the mechanism of polymerization of 1,6-anhydrohexoses, Schuerch a d his coworkers attempted the polymerization of several substituted 1,6-anhydrohexosea. Polymerization of 1,6-anhydro2-0-methyl-/?-~-galactopyranof~? failed,” a large proportion of the starting material being recovered. Similar resulta were obtained when attempta were , d P to polymerize derivatives of levoglucosan, namely the 2,3,4trinitrate, the 2,3,4trimethaneaulfonate,the 2,3,4-tri-p-tolue~esulfonate, the 2,3, &trinethy1 ether, and the 2,3 ,4-triacetateJ using one of several different catalysts. The Ruesian workersm also failed in their attempt to polymerize tri-0-metyllevoglucosan. In all cmm, decomposition occurred, or the monomer was recovered unchanged. The unrmtivity of these substances was interpreted by Schuerch m evidence for the neceasity of a free, C-2 hydroxyl group for occurrence of this type of polymerization. The formation of “some intermediate related structurally to 1,Zanhydroglucopyranose” wm considered to be involved in the addition polymerization of 1,Banhydro sugam.llaThe reverse proceas, namely the conversion of 3,4,6tri-0-acetyl-1 ,2-anhydro-~-g~ucopyranoee into 1,&anhydro-&D-glucopyra.nose under alkaline conditions is known to O C C U ~ . ~ * ~ - - U ~ * However, the present authors aro unaware of any instance in which a 1,6-anhydro sugar is transformad into R 1,%anhydro derivative. Bredereck and HutteiP have reported the polymerization of tri-0ac:etyllevoglucosaii in the preserice of tetra-0-acetyl-a-D-glucopyranosyl bromide, acetyl chloride, and silver perchlorate, to give polymeric products, the P, of one fraction reachingm 49. The same authors claimed succw in (n0)H.Bredereck and U. Hutten, Unpublished; cited in Ref. 7, p. 737.

CHEMICAL SYNTHESIS OF POLYSACCHARIDEB

485

polymerization of the trimethyl and tribenzyl ethers of levoglucosan, 88 well as of Brigl's anhydride. As mentioned above, tri-0-methyllevoglucoeandoes not undergo polymerization in the presence of zinc dust at 250" for 10 hours,m or in the presence of any of several acidic or basic catalysts ats16temperatures up to 180".However, Korshak and his coworkersm have found that tri-O-methyllevoglucosan can be polymerized in the presence of boron trifluoride etherate in toluene at room temperature, to give products of V . ~ / C 0.22 to 0.30 and molecular weights of 280,000to 394,000(by light-scattering) . These results were confirmed and extended by Tu and Schuerchel*; they obtained etherinsoluble products in approximately 90% yield, but having p , (by vaporphase osmometry) no higher than 25. The discrepancy in molecular-weight data between the two studieq is most probably attributable to the presence of microgel in the preparations; its presence results in abnormally high molecular weights, aa determined by light-s~attering3~4(see p. 496). The yields of polymer were relatively insensitive to the presence of water. A study of the conditions of polymerisation afforded results permitting the possessing P,,200-300. By x-ray diffraction synthesis of studies, the Russian investigators demonstrated the crystallinity of their poly (tri-O-methyl-D-glucose), but they did not report the optical rotations. Tu and SchuerchZ2'obtained rotations of 197-204",a truly astonish"an overwhelming predominance of a-glucoing finding which suggests2z1.222 sidic linkages." No structural studies on these polymers have been reported, but interesting developments may be expected during the next few years. Korshak and his colleagum reportedZl0that polymerization of tri-0methyllevoglucosan with boron trifluoride etherate in dichloromethane gives products having the highest reduced viscosity. The yield of polymer increased with temperature, and the reduced viscosity reached a maximum at -20". In contrast to tri-0-methylcellulose, poly (tri-0-methybglucoses) prepared in this way are insoluble in water, acetone, or ether, but are soluble in chloroform and a cresol. These workers concluded that the products are crystalline and unbranched. In an effort to prepare a stereospecifically (1+6)-linked glucan, the polymerizationi of 1,6-anhydro-2,3,4-tri-O-benzy1-&~-glucopyranose(triO-benzyllevoglucosan) has been studied. Polymerization of this monomer in dichloromethane at -78", using the Lewis acid catalyst, phosphorus (221) (a) C. Tu and C. Rchuerch, J . Poliimer Sn'., Pt. B, 1, 163 (1963); (b) E. R. Ruckel

+

trritl C. Svhuemh, Privstc! c:oiiiinunic!at,ion;J . Org. C % ~ L 81, , 2233 (1966); (c) The dutwnzylstian hrw I)M!II raportcd (see Ref. lu). (222) (u) The optical rotationll of methylatsd &myloseand methylated dextran B-512-F are +214" (in chloroform)m(b) and +215" (in 1,1,2,2-tBtrach10rOethane),~(0) renpectively; (b) J. E. Hodge, 8. A. Karjals, and G . E. Hilbert, J . Am. Chem. Soc., 73, 3312 (1951); ( 0 ) J. W. Vancleve, W. C. Schaefer, and C. E. Rist, &id., 78, 4436 (1956).

486

I. J. QOLDSTEIN AND T. L. HULLAR

pentafluoride, gave polymeric products of p,, 97 to 178 (by vapor-phase osmometry) in 90-95% yield.nl(b)The high molecular rotations (+472 to +489O) of them products suggests that they are predominantly, if not exclusively, cr-p.linked. Consequently, removal of the benvl groups from them poly(tri-ebe~l-D-glucos)will allow chemical synthesis of an ac-~--(l+6)-linkedglucan.n(*O) Tri-0-ben~yllevoglucosanfailed to polymeriee in the presence of boric anhydride.'" In the presence of such acid catalpate as monochloroacetic acid2L8 or a Lewis md,m a 1,B-anhydro sugar, such as levoglucosan (a), is undoubtedly protonated (or conjugated) on the 1,ðer bridge228 (53). H

(53)

(50)

This specis then undergoes ring opening, probably by assistance (of an to give the carbonium undefined kind) from the C-2 hydroxyl group,24~2~J16

..

(12'

q

(223) Protonation of the 1,5 bridge in (i) in posllible, but, on ring opening, this would

lead to the unstable, eeptanose ringsyetem (ii)?

w-

NO

0

-Q HO

HOCH HAOH I

HCOH OH

(11

ROCHOR I HTOH

(W

H OH (W

The ion (ii) would either return to (i), or would react with alcoholic p u p s to give, probably, an acyclic D-glucom derivative (iii).

Lc

z

g1

Y

6

A

In

h

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

P

d I

0

487

I. J. GIOLDSTEIN AND T. L. H U U R

488

ion (50) (see equation If). The ion(S0) can then undergo self-condensation; it can react with the secondary hydroxyl groups of another molecule of (a),as previously described for thermal polymerization of (48) (see equation 10) ; or, it can react with the 1,ðer oxygen atom of a second molecule of (481, to give (by equation 1%) a direct regeneration of a r e active carbonium ion (54). The last possibility (see equation 1%)is analogous to,the trialkyloxonium ion mechanism which is usually postulated for the polymerization of cyclic ethersmen1,n4 (see later). In all three of these possibilities, an approximately equal distribution of a- and &Dlinkagea would be expected, since no major directive elements (see Section II,1) are present. Polymerization of tri-0-methyllevoglucosan (55) , by means of boron trifluoride, to give a polymer of high stereoregularity may be explained by a mechanism based on the formation of trialkyloxonium salts.m.a11n4Coordination of the 1,Bether oxygen atom 9f (55) with boron trauoride gives the salt (56). Reaction of (56) with the 1,Bether oxygen atom of (55) gives (57), which can react with (55) to give an a-D-linked, growing polymer. Addition of isopropyl alcohol would cause an increase in the size of the counter ion (eBFsOR') and, hence, would increase the stereospecificity of the subsequent, nucleophilic attack on (57). This effect has been observed.211

b, 1,4=AnhydroSugars.-The synthesisn6and polymerizations4of two 1 4-anhydro sugars,n*namely, 1,Canhydro-2,3,&tri-O-methyl-D-galactopykanose (58) and lJ4-anhydro-2 ,3-di-O-methyl-~-arabinopyranose (59) containing 16% of 1 Panhydro-2,3-di-O-methyl-~-xylopyranose, were described by Kops and Schuerch. Treatment of these anhydro sugars with

(58),

R = -CH,

(5Q), R

-CH,

the Lewis-acid catalysts, phosphorus pentafluoride or boron trifluoride etherate, under high vacuum (lo-" Torr) gave ctmorphous polymers of (224) J. Furukawa and T. Saegusa, "Polymerization of Aldehydes and Oxides," Interscience Publiehers, Inc., New York, N. Y., 1983, Chapters 111-V. (226) J. Kopa and C. Schuerch, J . Org. Chum., SO, 3951 (1968). (226) For R syntherrh of 1,4-anhydro-2,3,Btri-O-methyl-a-~glucopyranose, see E. Husfmann and J. Kim, M a k r m l . C k a . , 68, 223 (leS2).

489

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

P, up to 90 (by vapor-phase osmometry) . The structures of the polymers, as indicated by their optical rotation, were strongly influenced by changes in the reaction conditions (especlally by the temperature and polarity of the solvent), Lower temperatures (-78') gave rise to higher yields and greater P,,. No epimerization occurred during polymerbation, other than that at the anomeric carbon atom. Highly levorotatory polymers were obtained when (58) waa polymerized a t either --28' or -9O", but no polymerization occurred at -97'. By comparison of the rotations ([CYID -70 to -95' in chloroform) of the polymers formed from (58) with those of the four unmethylated, anomeric methyl D-galactosides, it was concluded that 6-D-galactofuranosyl residues were the preponderant structural units of these polymers. The results of polarimetric measurements during acid hydrolysis of the polymer indicated that pyranosyl residues, also, were probabIy present. Isolation and characterization of the products from a graded hydrolysis with acid would confirm this contention. From similar considerations, it was concluded that a polymer ( [CYJD - 102' in chloroform) formed from (59) at -28" contained a preponderance of a-L-arabinofuranosyl residues, whereas a polymer ( [ a ] ~-13' in chloroform) formed from (59) at -78" contained a significant proportion of 6-L-arabinofuranosyl residues. This latter polymer contained, in addition, the expected ct-L-arabinofurano8yl and a- and 8-carabinopyranosyl re&dues. A carbonium-ion mechanism was advanced" to account for the postulated structures of the polymers and for the rather stereoselective incorporation of the monomeric units; the mechanism proposed may be ilIustrated for the incorporation of furanosyl units. On steric grounds, the bulky counter ion ( ePFsA-) should occupy the less hindered, B-D side of (60). Consequently, 0 PF~A-

-

polymer

OR I

(60) would be expected to undergo reaction with monomer (58) from the CY-Dside

(path b). However, only 6-D-galactofuranosy1 residues appear to

490

1. J. QOLDSTEIN AND T.

L. HULLAR

be present in the polymer. l'hesterie bulk of theexocyclic (3-5, C-6 functions is apparently sufiicient to offset this expected effect of the counter ion. Consequently, only the thermodynamically more stable O-D-glycoeide is formed. The couiiter ion of Hcorporated ctrabinofurunosyl rosidues (61) should also occupy the lees hindered, a-L side of (61), thereby directing the reaotion to proceed, at least partly, by path b, to give &carabinofuranosyl reaidues. At low temperatures (-78') of polymerization, path b does appear to be operative, since both a- and Bmirabinofuranosyl residues seem to be formedamHowever, at higher temperaturee, where the electro-

static interaction between the ion pair is decreased (and, consequently, the effective steric size of the counter ian is decreased) , the attack by (59) on (61) appears to be by path u, to give the thermodynamicaIly more stable a-tarabinofuranoeyl residues." These polymerizations hold great interest, because of the pronounced tendency for sterio control of glycoside formation. c. I,%-Anhydro Sugars.-Haq and Whelan*= have polymerized Brigl's anhydride by heating it in a sealed tube at 118' for one week. Deacetyhtion and fractionation of the product afforded a ,a-trehalose, kojibiose, and material believed to be a homologous series of (l-+2)-linked oligosaccharides. By conducting this reaction in the presence of basic catalysts in a solvent-free system at 6 5 O , Schuerch and his coworkers convertedaZ8Brigl's anhydride into a polymer in which the presence of P-D-linkages was indicated. The highest molecular weights, and a polymer containing fewer tf-D-Iinkages, were obtained by using an aluminum isopropoxide-zinc chloride (1:11 catalyst in tetratiydmfuran solution. Only D-glucose W M obtained on acid hydrolysis of thee. polymers. (227) Path b in, undoubtedly, possible for (el), since the exocyclic C-4 subetituent of (61) is sterically lea8 restrictive than the bulkier C-4 subetituent of (do). (228) E. Quinn. R. 9. Nevin, C. Schuerch, and K. Elarkanen, Abutracta Papers Am. Chsm. SOC. MeSling, 188, 4D ( 1w).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

49 1

In a related study, a novel type of epoxide polymerization was found to occur when 5,6anhydro-l ,2-0-kopropylidene-3-O-methyl-~-glucofuranose waa kept2* with such strong basea as potaasium hydroxide or cesium hydroxide. The factors affecting this proceas were studied in detail, and a supposed mechanism for the polymerization waa described. The polymers so obtained (P,11-25) were treated with dilute sulfuric acid to cleave the isopropylidene acetals, and the resulting acetone was removed as its (2 ,Cdinitrophenyl) hydrazone. A molecular-weight determination on the hydrolyzed poIymer revealed that the polymer itself had not been degraded. Hypoiodite oxidation of the aldehydic function afforded the corresponding aldonic acid derivative. Inasmuch aa the products do not possess glycosidic linkages, a requisite for the compounds discussed in this article, this interesting polymerization will be accorded no further consideration. 3. Other

The facile, acid-catalyzed formation of gIycosides by the addition of alcohols to glycals may be a potential method for preparing polyglycosea. Reaction of D-gluca1,230D-galactal,2*1+2az and 3,4,6tri-O-acetyl-~-galact~~*~ with methanol containing hydrogen chloride gave the corresponding methyl 2-deoxy-~-glycopyranosides. Reaction of 3,4 ,6tri-O-acetyl-D-galactal with phenol in the presence of p-toluenesulfonic acid gavgScthe corresponding 2-deoxy-cr-~-lyxo-hexoside in 63% yield. Extensive studies by Lemieux and c o ~ o r k e r s 2 ~have ' ~ ~ demonstrated ~~ that 3,4,6tri-O-acetyl-D-glUcal reacts with alcohols in the presence of salts of (positive) halogen ions to give glycosides possessing the a-D-manno and 8-D-gluCo configurations. Consequently, it seems probable that a partially substituted Dglucal could be polymerized with an acidic catalyst to give a poly (2-deoxy-~-arabinohexose) . Thus, 3 ,Fdi-O-methyl-D-glucal would be expected to be polymerized in acid solution, to give (l-&)-linked poly(2-deoxy-3 ,Cdi-Omethyl-D-arabino-hexose). IV. METHODS OF STUDY Many of the methods used in the isolation, fractionation, and measure ment of the homogeneity of polysaccharides have been discussed in recent (229) R. S. Nevin, K. Sarkaneii, and C. Schuerch, J . Org. C h . ,84,78 (1962). (230) I. W. Hughes, W. G. Overend, and M. Strtcey, J . C h . SOC.,2846 (1949). (231) W. G. Overend, F. Shafizadeh, and M. Stacey, J . C h . Soc., 992 (1961). (232) A. B. Foster, W. G. Overend, and M. Stacey, J . C h . rSoc., 974 (1961). (233) K. Wallenfele and J. Lehmann, Ann., 686, 166 (1960). (234) R. U. Lemieux and S. Levine, Can. J . Chm., 42, 1473 (1964). (235) R. U. Lemieux and A. R. Morgan, Can. J . C h . ,48,2190 (1966).

492

I. J. GOLDSTEIN AND T. L. HULLAR

reviews.zPB-2as Consequently, only topics of special pertinence to synthetic polysaccharidea will be discussed here.

1. Isolation The physical state of the crude, polymerized product determines to a large extent the isolation procedures to be employed. If the polymerization has beer_conducted in a solvent, the addition of a leas polar solvent usually causes precipitation of the crude product; alternatively, the solution may be dialyzed directIy. A solid melt may be dissolved in water, and the product precipitated by addition of an organic solvent. Monomer, oligosaccharides of low molecular weight, and residual catalyst may be removed by dialysis of the crude product. Thus, polygIucosea synthesized by Micheel and his co~orkers8~@-~~ were isolated in 8842% yield by precipitation, from methyl sulfoxide, with methanol. Dialysis of these preparations resulted in final yields of 40-57%, due most probably to loss of oligosaccharides in the dialysis step. Removal of the gel which sometimes occurs is readily achieved by centrifugation.l@J76*zl4 The usual problems encountered in the isolation of natural polysaccharides, such ria removal of proteinaceous matter or lignin, are absent in the isolation of synthetic polysaccharides. Consequently, the problem of degradation during isolation2m-aais not serious. 2. Fractionation

Fractionation of mixtures of naturally occurring polysaccharidea commonly utilizes some unique structural feature, such as the presence of a particular sugar or a special functional grouping, such as the cis-diol grouping of ( l d ) - l i n k e d D- or L-mannose residues. Alternatively, a more extensive structural oharacteristic, such as branching or linearity, may be utilized, Polyglycoses prepared under equilibrium conditions generally possess similar structural features in proportions dependent on the conditions of polymerization. Unsubstituted glycoses usually have led to a broad molecular-weight distribution of highly branched, randoinly linked polymers. Protected, bifunctional glycoses have generally furnished broad molecular-weight distributions of specificalIy linked polymers. Consequently, the aim in fractionating polyglycoses is usually to demonstrate that a prepyatipn contains a range of molecules which possess a particular (236) W.Banks and C . T. Greenwood, Advan. Carbohydrate Chem.,18, 357 (1963). (237) H.0.Bouveng and B. Lindberg, Advan. Carbohydm& Chem.,15, 54 (1960). (238) B. Lindberg, Pure Appl. Chem., 5, 67 (1962). (239)9. A. Om11 and E. Bueding, J . Biol. C h . , 288, 4021 (1964).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

493

property tc various degrees, and then to isolate as uniforni a fraction as possible for structurai study. Precipitation with solvent is comnioiily used for fractionating according to molecular size, but fractionation by branching or linkage type may also be achieved. Thus, a polyglucose, prepared by the “solution (melt)” method of polycondensation under diminished pressure, was fractionated The subtle effect of structure by ethanol according to size and bran~hing.”~ on fractionation is evident in the fractionation of polyglucoses prepared by the “infrared” and ‘‘two-stage” methods of polycondensation under diminished pressure. For these preparations, no separation by molecular weight was achieved, but some separation by linkage type resulted.l76 The similarity of these two preparations, and their difference from the product from the solution (melt) preparation, was shown by their different patterns of precipitation. The theoretical and practical complexities involved in simultaneous fractionation according to size and branching have, thus far, been relatively unexplored.168 Other solvents may also be used with similar effects. Isopropyl alcohol has been used for fractionating a p01ygIucose.~~ Examination of the fractions revealed that fractionation according to viscosity and immunological reactivity had occurred.240The excellent soIvent properties of methyl sulfoxide,2“ tetramethylene sulfone, pyridine, N ,N-dimethylformamide, and formamide may also prove useful. Solvents differ in their capacity to effect fractionation. Thus, in a study of the polyaddition of levogIucosan, 83-90% ethanol gave a 7% yield of a fraction of J?fw22,750; and addition of one volume of acetone to the supernatant liquor gave a 7% yield of a fraction having2i3Mw 38,650. Such differences in solvents may be useful, for example, in separating branched from unbranched polymers within a particular molecuiar-weight range. Fractional extraction24*(dissolution) may also be employed, as was demonstrated for a polyxylose, using batchwise extraction with aqueous cthan01.l~’A n-iore convenient procedure would involve adsorption of the polyglycose onto an inert, soIid support (such as cellulose), followed by extraction using a procedure already described.2a The continuous nature of this process, the opportunity for solvent to reach all particles of the polymer, and the possibility of using gradient elution make this method attractive, particularly if the examination of a range of polymeric distributions is desired. (240) M. Heidelberger and A. C. Aisenberg, Proc. Natl. Acad. Sci. U.S., 39,453 (1953). (241) F. A. Abadie-Maumert, Papeterie, 79, 519 (1957); Chem. Abstracts, 63, 12397 (1958). (242) Ref. 12, pp. 341-342. (243) S. Gaidell, Methods Carbohydrate Chem., 6 , 9 (1965).

494

I. J. GOLDSTEIN AND T. L. HULLAR

Ultrafiltration has been used in fractionating de~trans.24~ By use of specially prepared membranes,a46it should be possible to extend this simple, gentle method to effect fractionation of polyglycoses and polysaccharides in general. Gel filtration also permits passage of macromolecules on the basis of their molecular size, and this method has proved useful in the fractionation of dextrans*a and dextrins.?" The ease and capacity of this method should make it valuable in the fractionation of polyglycoses. Other methods of fractionation which utilize the presence of ionizable groups, either natural (such as carboxyl or amino groups), or induced (such as borate complexes), may be useful. Such methods have been disc u e d in reviews.*"v*n Complexation (for example, with Fehling solution) was found useful in the study of a polymannose.a18An insoluble complex waa formed, aa with natural mannans.

3. Measurement of Homogeneity Before conducting physical and chemical studies, the homogeneity of a separated polyglycose should be Since polyglycoses formed from unsubstituted sugars probably always consist of a broad distribution of molecular sizes and linkage types, the chief aim of homogeneity measurements will usually be to assure that a narrow range of these distributions is present. As emphasis on the controlled synthesis of polysaccharides continues, homogeneity measurements should prove useful in detecting the presence of small proportions of contaminating, polymeric species. Homogeneity is best assessed by using several different techniques, some of which have been discussed.2" By ultracentrifugation, the polymers synthesized from unsubstituted sugars in methyl sulfoxide solutionea~sb were shown to be homogeneous. Little effort has yet been made to aase8s the homogeneity of other polyglycose preparations. 4. Structural Analysis

a. Molecular Weight.-Apart from methylation analysis, chemical methods for the determination of molecular weight normally require the presence of one terminal, reducing sugar rwidue per polymer chain. As a consequence, the use of these methods for analysis of synthetic polysaccharides is limited by two conditions. First, certain synthetic poly(244) K. C. B. Wilkie, J. K. N. Jones, B. J. Excell, and R. E. Semple, Can. J . Chem., 86, 796 (1967). (246) L. C. Craig and T. P. King, Methods Biodrcna. Anal., 10, 176 (1962). (248) K.Granath, Methods carbohhydratc f%??n., 6, 20 (1966). (247) P. Nordin, Arch. Biochefil. Biophhys., 99, 101 (1962).

495

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

glycoses do not possess a free reducing group. Second, the reducing group must undergo reaction with the chemical reagent ( 8 ) employed. Many of the methods of polymerization either form or incorporate l16-anhydro sugar residues a t the "reducing" end of the polymer. The presence of trehalose-type linkages in either liiicur or cyclic form caiiiiot be excluded.4f'.l" The applicability of a chemical method to a certain type of polyglycose must, therefore, be affirmed by comparing it with other methods, such as physical procedures, which do not depend upon the presence of a reducing group. Moral" found, for example, that the molecular weight of a polyglucose (prepared by a method similar167to that of stage one of the " two-stage" process of polycondensation under diminished pressure'") was about the same when measured either by a copper reducing method or by solution osmometry. The multiplicity of linkages present in some polyglycoses may be sufficient to interfere with or prevent the necessary chemical reactivity of the reducing residue.%*A procedure which depends on measurement of the formaldehyde liberated when the borohydride-reduced polymer is oxidized with periodate24@requires an unsubstituted, C-2 hydroxyl group. This method, however, measures all of the formaldehyde liberated, and care must be taken2@ to account for formaldehyde released from hexofuranosyl residues unsubstituted on the C-5 and C-6 hydroxyl groups. Other chemical methods can also give abnormal molecular weights.*" Consequently, it is desirable to compare values obtained by a t least two different chemical methodk. For a polyglucose,1~J70 the values obtained by copper reduction,'7' radioactive cyanide,17*and borohydride reduction-periodate oxidation2'g were all e q ~ i v a l e n t . ~ ~ J ~ J 7 ~ J ~ ~ Physical methods of molecular-weight determination,238in contrast to the chemical methods, require no specific chemical grouping. Numberaverage molecular weights, #",may be obtained by solution ~ s m o m e t r y ~ ~ ~ ~ less than but this method is not generally applicable to polymers of 30,000-a range often encountered for synthetic polysaccharidea-because of permeation of solute through the membrane.%"A more satisfactory method is that of vapor-phase ~srnometry*~~; this method was used for and studying the polymerization products of 1,6-anhydro-~-hexoses,2"~~~~~~ possesses the distinct advantage of application in both aqueous and nonaqueous solvents. The rapidity and convenience of this method make it of great potential in the determination of the M n of polyglycoses, particularly

an

(248) For a discussion of several chemical methods for measurement of P,, see H. F. Launer and Y. Tomimatau, Anal. Chem., 33, 79 (1961). (249) A. M. Unrau and F. Smith, Chem. Znd. (London), 330 (1957); G. W. Hay, B. A. Lewis,F. Smith, and A. M. Unrau, Me&& Carbohydrate Chem.,6, 251 (1965).

496

I. J. GOLDRTEIN AND T. L. HULLAR

since the results aocruing from chemical methods may be equivocal. Isothermal distillation has also been advocated for the determination*” of fin. Weightraverage methods also have utility in this field. Ultracentrifugal measurements have been used for studying the polymerization of levoof free sugars.08.a-80This technique g l u ~ o s a nand ~ ~ ~the ~ *polymerization ~ also gives information about the distribution of molecular weighta.86 It is, however, too laborious to be suitable for such routine purposea as following the progress of a polymerization. Light-scattering has also been used in thehstudy of synthetic polysaccharide~,%~8J14 but it is time-consuming and, like ultracentrifugation, requires specialized equipment. It is, therefore, not suitable for routine analyses. Of greater consequence, however, is the high sensitivity of this method to small amounts of material of high molecular weight. Thus, samples of polyglucose prepared from levoglucosan were found to have much higher molecular weights according to measurements by lightscattering than those given by ultracentrifugation techniques.a14This discrepancy was attributed to the presence of “micro-gel” in the material. The presence of gel in material undergoing polycondensations under diminished pressure1@would preclude the meaningful use qf light-scattering. Gel filtration has been used to measure the molecular weights of proteins and c a r b ~ h y d r a t e sand ,~~~ it should also be applicable to polyglycoses.

b. Configuration of the Anomeric Linkages.-The specific, optical rotation hm often been used to indicate the main configuration of the glycosidic linkages in polysaccharides. Considerable care must, however, be exercised, since linkage types and their sequence and branching may have “anomalous” effects on the optical rotation. For example, nigeran [a glucan -linkages] has an unpossessing alternating a-D- ( 1 4 ) - and a - ~(1-4 usually high rotations1 ( [.ID 254”) compared to that of amy1oseZs2 ( [ a ] 4~ 200”), an a-~-(1+4)-hked glucan. Thus, if a reasonable proportion of alternating a-D- ( 1-3) - and a-D- (1 4 )-linkages were present in a polyglucose, the positive rotation obtained would probably not be a true reflection Qf the proportion of a- and @+linkages present. The change in optics1 rotation of a polysaccharide during hydrolysis has also been used as an indication of the configuration of the glycosidic bonds, but this method is subject to the same limitations as have been discussed above. Nonetheless, the sign and magnitude of the specific rotation has been used in analyaing the linkages present in the polyglycoses obtained

+

(250) C. T.Greenwood, Methods Carbohydrate C h . , 6,261 (1865). (251)8.A. Barker and T.R.Carrington, J . C h . SOC.,3538 (1853). (262)W.B. Neely, J . Org. Chem., 38, 3015 (lQ6l).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

497

from 1,4anhydro-2,3-di-O-rnethyl-~-arabinopyranose and lJ4-anhydro2,3,6-tri-O-methyl-~-galactopyranose (see p. 489) .w Analysis of anomeric configuration by infrared absorption2s*has allegedly become of increasing importance. Its usefulness in the study of synthetic polysaccharides is not yet clear, however. In polyglucoses prepared by using hydrogen chloride gas'% and thionyl chloride:' CY-D-(1-6) -linkages were detected by infrared analysis. Polyglucoses derived from boric acid catalysisLs6and by polymerization under diminished pressure,'@ gave illdefined absorption in the region of 730-960 cm.-' that did not permit of specific assignments. In view of the difficulties in interpretation, and the multitude oi linkages which may be present, it is mandatory that the presence of linkages suggested by infrared studies be corroborated by other methods, for example, by chemical studies. Isolation of hydrolytic fragments of known structure under conditions which minimize epimerieation and transglycosylation is still the best absolute procedure for determining the presence of a particular anomeric configuration. Thus, the a - ~(1+6)-linkage in a polyglucose (prepared by using thionyl chloride as the catalyst) was suggested by infrared analysis and waa proved by the isolation of isomaltose." The presence of the a-D(1+6)-linkage in a polygalactoglucosewas proved by isolation of melibiose from the partial hydrolyzate by acid.a1eThe differences in solvolytic action between aqueous acid hydrolysis and a ~ e t o l y s i may s ~ ~ be ~ useful in isolating fragments containing specific anomeric configurations. The Smith degradation (see Section IVJ4,d) has also been used to obtain fragments which demonstrate the presence of a- and P-(D or L)-linkages.182 Enzymic hydrolysis by a- or P- (D or L) -glycosidases could also be used to demonstrate the presence of a particular anomeric configuration. However, this method will probably be successful for polymers which possess little (or moderate) complexity, for enzymic digestion of highly ramified polyglucoses has been possible in only a few special cases (see Section IVJ5). c. Degree of Branching.-Methylation analysis is still the best way of determining the extent of branching of any polysaccharide, natural or (253) For reviews on the application of infrared spectroscopy to carbohydrates, see H. Spedding, Advan. Carbohydrab Chem., 19, 23 (1965), and W. B. Neely, &id., 12, 13 (1957). (254) Acids hydrolyze (1 + 4)-linkages faster than (1 + 6)-linkrtges,l66 whereas acetolysis cleaves (1 + 6)-linkages faster than non-(1 6)-linkages.86-266 (255) M. L. Wolfrom, E. J. Lassettre, and A. N. O'Neill, J . Am. Chem. Soc., 78, 595 (1951). (256) P. A. J. Gorin and A. S. Perlin, Can. J . Chem., 34, 1796 (1956); K. Matsuda, H. Watanabe, K. Fujimoto, and K. Aso, Nature, 191, 278 (1961); I. J. Goldstein and W. J. Whelm, J . Chem. Soc., 170 (1962).

498

I. J. GOLDBTEIN AND T. L. HULLAR

synthetic. Although exhaustive methylation of highly branched polyglycoses has proved difficult,”*m it hae been accomplished.26J*The newer methods of methylationw7 should prove more effective than the classical procedures.w,w The classes of the constituent methylated sugars (for example, the tetra-, tri-J di-, and mono-0-methylhexoses) can be readily separated and their amounts quantitatively determined by gas-chromatographic devicea.lm In contrast to methylation analysis, periodate oxidation is of only limited value in determining the degree of branching. The production of formic acid is indicative of (1-+6)-linked hexosyl residues, terminal hexopyranosyl groups, or terminal pentopyranosyl groups. Lack of oxidation of hexopyranosyl residues is indicative of 3-, 2 ,3-, 2 ,4-, 3 ,6-, 3,4-, 2 ,3 ,4, 2 , 3 ,6-, 2 4, ti-, or 2 ,3 4,6substituted residues. The ambiguities involved in the interpretation of periodate consumption and formic acid liberation preclude any real decision about the branched character of a polyglycose. The major value of such data is in the comparison of different fractions of a polyglycose.~76 The branched nature of polyglucoses prepared from free, unsubstituted sugars has been indicated qualitatively by the pronounced solubility of the and by the necessity for four methylated derivatives in petroleum successive sequences of periodate oxidation and borohydride reduction in order that complete oxidation of all of the sugar residues may be achieved.lS2 A decrease in the intrinsic viscosity has also been used as an indication of an increase in branching.176Quantitative data are not, however, derivable from such observations. d. Position of Glycosidic Linkages.-Methylation a n a l y s i ~ ~and ~-~~ methods based on periodate oxidationJ186,z61 and partial hydrolysis (see p. 497) are the major chemical methods by which the structure of a polysaccharide can be elucidated. Reviews on and descriptive statementa about these methods are available. The present discussion will emphasize the usefulness of these methods in the study of polyglycoses. The utility of these methods, illustrated by the elaborate structural studies carried out (267) For a review, BBB K. Wallenfells, G. Bechtler, R. Kuhn, H. Tnschmann, and H. Egge, Angew. Chem.. I n h . Ed. En& I, 516 (1883); see also, 8. Hakamori, J . Riochem (Tokyo), 66, 205 (1oA4) ibnd P. A. Bandford and H. E. Conrad, Riocheminty, 6, 1508 (1966). (268) F. Smith and R. Montgomery, Methods Biorhem. Anal., 3, 153 (1956). (269) E.L. Hirst and E. Percival, Methode Carbohydrate Chena., 6 , 287 (1965). (260) For a review of the application of gas-liquid chromatography to carbohydrates, we C. T. Bishop, Adwan. Catbohydrate Chem., 19, 95 (1985). (261) G . W. Hay, B. A. Lewis, and F. Smith, Melhodu Carbohydrate Chem., 6,357 (1965),

and references therein.

CHEMICAL SYhTHESIS OF POLYSACCHARIDES

499

by Dutton and Unraus-2D*1sZon the polyglucose synthesized by polycondensation under diminished pressure, can be discussed in terms of the hypothetical struhture (62) and the products which can be derived from it (see Table VI) .

OH 5

OH 6

Periodate oxidation of (62) gives a polyaldehyde. Determination of the consumption of periodate affords the number (8) of vicinal diol groups; the moles of compound formed per mole of (62) will be given in parentheses. The amount of formic acid liberated gives the number of vicinal trio1 groups (2). The quantity of formaldehyde evolved gives the number of D-glucofuranose residues unsubstituted on the C-5 and C-6 hydroxyl groups ( 2 ) . Reduction, by borohydride, of the polyaldehyde to the polyalcohol, followed by complete hydrolysis by acid gives glycolaldehyde (4),glyceritol (3) , erythritol (1), D-XYIOS~( 1 ) , and D-glucose (1). These results are in agreement with the previous data, and permit several deductions respecting the number and substitution patterns of the sugar residues; they do not, however, indicate the number of residues linked to the C-6 hydroxyl groups, since glyceritol, erythritol, and D-glucose can all derive from residues having free or substituted C-6 hydroxyl groups. Furthermore, the results obtained from the periodate oxidation do not establish the position of linkages on residues 2, 3, and 4. Methylation analysis of (62), or of the polyalcohol derived from it, settles both of these points by defining the hydroxyl groups which were unsubstituted in (62).

Q TABLEVI Products Expected From Chemical Operation Periodate oxidation

NdO, %Glucose

Hydrolysis of plyalcohol

consump

HCOZH

tion

evolution

residue

Methylation

1

2,3,4triiI-methyl-~glucose 2,4-di-O-methyl-~-glu~

2

1

glyceritol; glycolaldehyde

0

0

D-glucose

2,3,6tri-@methyl-~glum 2,5,6-tri-O-methyl-~-

1

0

glycolaldehyde;erythritol

1

CP

D-xylose

2

1

glyceritol;glycolaldehyde

2

0n.b

2

3 4

Complete

g~ucose

5

2,3,4,6tetm-O-methyl-~glu-

6

a

2,3,5,6-tetra-O-methyl-~glucose

Formaldehyde liberated.

Possible overoxidation.

glyceritol (if no overoxidation) ; glycolaldehyde

Graded

1-

i

0-D-glucopyranosyl(1 -+ l)-L-glYCeritol 0-,9-D-xylofuranOsyl(1 -+ IL)-D-erythritOl glyceritol glyceritol

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

501

T-he assembled data define the substitution pattern of the monomeric residues, but give no indication as to their sequential arrangement. To accomplish this determination, the polyalcohol is subjected to controlled fragmentation in dilute, aqueous acid-the Smith degradation.186This procedure results in the hydrolysis of all of the true acetal bonds while the glycosidic linkages are preserved. From this hydrolyzate can be isolated 0-&D-glucopyranosyl-( 1-1 )-Lglyceritol (1), O-&D-xylofuranosyl- (1-4) D-erythritol (1), and glyceritol ( 2 ) . Isolation of these compounds signifies that reaidue 2 is linked P-D-glucosidically to residue 1 , and that residue $. forms a &D-glucosidic linkage with residue 3. The union of residues 3 and 6 to residue 2, and of residue 5 to residue 4, must be designated arbitrarily. Thus, even with the most advanced techniques yet devised, the arrangement of monomeric residues in a complex polysaccharide is still plagued with considerable uncertainty. These analyses are laborious and require good quantitation. As mentioned in Section IV,4,c, the methylation analysis can be facilitated by use of the newer methods of methylation and by using gas-liquid chromatography for separation and quantitation. Gas-liquid chromatography has also been used for separating the trimethylsilyl ethers of the oligosaccharide glycosides derived from the Smith degradation.'*?This application is particularly noteworthy, and offers an attractive method for expediting the separation of the products resulting from the Smith degradation. e. Other.-Nuclear magnetic resonance spectroscopy has found considerable application in the study of carbohydrates.2s2In a study of dextrans, it wag found that ( 1-6) -linkages could be distinguished from non- (1-6) linkages.263This finding may prove useful in development of a rapid, instrumental analysis of polyglycoses. The configuration of the glycosidic linkages has been studied by nuclear magnetic re80nance,262.264 and this method may have additional application in studying the anomeric configuration of the products from the Smith degradation. Nuclear magnetic resonance has also been used for showing that D-glucosyl residues in Schardinger dextrins2s4a-264b and in several D-glucose-containing polysaccharides have the C1 ( D ) conformation; it may find similar utility in conformational studies of polyglycoses. Analysis by x-rqy diffraction patterns can be used to insure identity of compounds and to measure the degree of Tipson and co(262) For a recent review, see L. 1). Hall, Aduan. Carbohydrute Chem., 19, 51 (1965). (263) W. M. Pssika and L. H. Cragg, Can. J . Chem., 41, 293 (1963). (264) Y.-C. Lad and C. E. Ballou, Biochemislry, 4, 257 (1965). (2644 V. S. R. Rso and J. F. Foster, J . Phys. Chem., 67, 951 (1963). (264b) C . A. Glass, Can. J . Chem., 43,2652 f1965). (265) For a brief discuwiozi, 8ee J. Marin, Methods Carbohydrate Chem., 3, 114 (1963).

502

I. J. GOLDSTEIN AND T. L. HULLAR

workers used this technique to show that a polyglucose prepared by polymerization in the presence of boric acid was amorphous, even though it appeared crystalline to the eye."' Furthermore, the poly(2,3,4-tri-Omethyl-D-glucose) prepared by polymerkation of 1,6-anhydro-tri-Omethyllevoglucosanwith boron trifluoride etheratem was proved crystalline by this method. One of the criteria which may be employed in confirming the chemical synthesis of such a biopolymer as cellulose is to show the identity of the x-ray diffraction patterns. Maas spectrometry of carbohydrates has become of interest, and offers potential for the identification of naturally occurring carbohydratea.*@j This method may also be applicable in analyzing fragmentation products of polyglycoses. 5. Use of Enzymes ' The use of carbohydrases for the elucidation of the structure of naturally occurring polysaccharides has been reviewed.2mThe present discussion will be limited to their use in analyzing the structures of synthetic polysaccharide. Since the hydrolytic activity, toward polysaccharides, of many glycosidases requires that the substrate possess a sequence of glycosidic linkages of defined position and configuration, it is not surprising that many of the highly branched, randomly linked polyglycosea prepared from unsubstituted sugars am unaffected by various carbohydrases. Thus, even though emulsins attack simple, reversion products and oligosaccharides of low molecular weight:' they are without effect on a polyglucose prepared by means of thionyl ~hloride.~' Another polyglucose prepared by the same method waa unaffected by salivary a-amylase, yeast invertase, and potato phosphorylase.88 Synthetic dextrina were not attacked by salivary a-amylase,B*malt diastaee,**W preaeed yeast,62or yeast from b e e r . ~ ~ Q In ~ Mfact, 2 stability toward enzymic hydrolysis may be regarded as one of the truly remarkable properties of complex, synthetic polysaccharides. The possibility of induced enzymeformation in the presence of synthetic polymers has not yet been examined. The action of amyloglucosidase from Aspergillus niger on various synthetic polyglucoses has been examined.460This enzyme is an exoglucosidase

(200) I). C. DeJongh and 8.Iiarim8ian, J . Am. Chem. Soc., 81, 3744 (1965), and refer-

e n m quoted therein. 9ee N. K. Kochetkov and 0. s. Chizhov, This Volume, p. 39. (267) P. Bernfeld, in "Comparative Biochemietry," M. Florkin and € 6.I. Mason, eds., Academic P w Inc., New York, N. Y.,1962, Vol. 3, p. 368. (268) P. A. Levene, J . Bio2. Cham.,64,476 (1926). (269) I. J. Goldeteiu and R. Porets, Unpublished results.

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

503

which splits a-D-glucopyranosyl groups from the nonreducing ends of amylaceous polysaccharidea. The a - D - ( l d ) bond iS cleaved about a hundred timea more readily than other ar-p.glucosidic linkages.n0 Synthetic polyglucosesl@and a p01ymaltose~~~ (prepared by condensation polymerization) and polyglucoses,21aa p ~ l y m a l t o s eand , ~ a polycellobio&w (prepared by addition polymerization) were investigated. The polymalto~e'~~ released the greatest proportion of D-glucose, the proportion being more than twice that liberated by the polyglucosesl@ prepared by the same procedure, This result indicates that a t least some of the a - ~ - ( 1 4 ) glucosidic linkages of-maltose survive the polymerization conditions and occupy positions a t nonreducing chain-ends in the polymaltose. The synthetic glucan prepared by the addition polymerization of 116-anhydromaltose26gliberated more D-glucose than the one prepared from 1,6anhydrocellobiose, indicating the presence of a higher content of a - ~ (l+)-linkages in the polymaltose. More D-glucose was liberated from polyglucoses prepared by addition polymerization218than from those prepared by condensation polymerization.1B8The latter glucans, which are known to contain approximately 20% of their nonreducing terminal reaidues as furanosyl reaidues,26probably contain fewer linkages susceptible to attack by the enzyme. Amyloglucosidase may thus prove to be of great va h e in the examination of the configuration of D-glucose residues situated at chain ends of synthetic polysaccharides. Enzymes may also be useful in demonstrating the identity of a synthetic glycan with a naturally occurring polymer. If the structures of the two are identical, an enzyme which is known to attack the natural polysaccharide should attack the polyglycose in an identical manner. Consequently, the kinetics and products of hydrolysis should be the same in both cases. Husemann and Miiller have reported the effect of a-amylase and cellulase on a synthetih, cellulose-like polysaccharide' (see p. 455). Since this polysaccharide is water-insoluble, it was converted into a water-soluble carboxymethyl derivative of very low degree of substitution. Employing an w a y dependent on a fall in viscosity, they showed that a-amylase is without action on the polymer, whereas a preparation of cellulase from Acetobacter xylinum brought about a definite fall in viscosity. Cellodextrins were employed as controls, and behaved in a manner similar to that of the synthetic polymer. These findings demonstrated that sequences of @D- ( 14)-glucosidic! liiiknges exist i i i the synthetic polymer. Comparison270* of the action of a dextranase from Penicillium funz'culosum on a synthetic CU-D-(1+6)-linked polyglucose1a~221(b) and a natural dextran

T.Ando, J . Biol. Chem., 256, 297 (1980); M. Abdullah, I. D. Fleming, P. M. Taylor, and W. J. Whelan, Biochem. J.. 89, 3 5 (1963). ~ (270a) E. T.Reese and F. W. Parriah, Biopolymeru, in press. (270) J. H. Paaur and

504

I. J. GOLDSTEIN AND T. L. HULLAR

(clinical dextran, containing about 5 per cent of branch points) indicated a basic similarity of the eynthetic and natural dextrana. Both poIysacch* rides yielded D-glucose, isomaltose, and isomaltotriose, in addition to several higher oligosaccharides (11.4%, 57.4%, 5.6%, and 25.6%, respectively in the case of the natural dextran, and 11,7%, 72.8%, 7.7%, and 7.7%, respectively, in the c a e of the synthetic dextran). The higher oligosaccharides (p, > 3) produced by the action of the dextranrtse on the clinical dextran have been shown to be linear and to contain one a-D(1-*3)-glucosidic linkage. The lower proportion of such oligmaccharides produced from the synthetic dextran indicates a product more linear than the natural polymer. It waa shown that the higher oligosaccharides from the synthetic dextran contain only a-D-glucosidic bonds (digestion by glucamylase and a-glucosidase) and it was suggested that approximately 2 per cent of linkages other than the a-w(1+6) occur in the synthetic product. The difficulties attendant on the isolation of pure ensymes of known specificity is a major barrier to their routine use for the structural analysis of polysaccharidea. As the specificities are separated and rts the action patterns of carbohydrases become better defined, these enzymes may be expected to play aq important and vital role in the investigation of the structure of synthetic polysaccharides containing ordered sequences of sugar residues. Conversely, it may be anticipated that synthetic carbohydrate polymers of known structure will aid in studies of the specificity requirements of purified enzymes. 6. Uee of Immunochemistry

The use of immunochemical methods to study homogeneity and purity, and to elucidate polysaccharide structure, has been discussed.nl The following discussion will deal with the application of immunochemical methods to polyglycosea. As ti part of their investigations on the antigenicity of dextran in man, Kabat and Berg272 examined the capacity of two synthetic polyglucosea to crowreact with antidextran antibodies. They found that one polyglucose (Merck 2947-53) reacted with preimmunization sera from three human individuals, showing the presence of preexisting antibodies containing sites complementary to certain chemical features of the polyglucose. (271) P. Z. Allen, Melhoda Carbohydrate Chem., 6,232 (1905); M. Heidelberger, Forkchr. C h . Org. Naturtdofle, 18, 503 (1900); E. A. Kabat and M. M. Mayer, “Experimental Impunochemistry,” Charles C. Thomas, Springfield, Ill., 2nd Edition, 1961, pp. 381-483; M. Heidelberger, “Lectures in Immunochemistry,” Academic Preae Inc., New York, N. Y., 1966, pp. 13, 14, 82-92. (272) E. A. Kabat and D.Berg, J . Zmmunol., 70, 614 (1963).

505

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

However, the proportion of antibody precipitated by the Merck preparation waa significantly greater after immunization of each of the three individuals with a different dextran. This finding suggests that the three dextrans and the Merck polyglucose possessed structural similarities, such ct8 CY-D-(1+6) -glucosidic linkages. In sharp contrast to the Merck polyglucose was a DuPont preparation (P218-56), which showed no reaction with preimmunization sera, and precipitated only negligible quantities of antidextran antibody. Thus, there appears to be a fundamental difference of some kind between these two polyglucoses. Perhaps, the degradation step in the Merck process involves preferential cleavage of certain D-glucosidic bonds, resulting in an enrichment of other linkages, such as the a- and S-D-(1-6) linkages. The antigenicity of the Merck (2947-53) and DuPont (218-56) polyglucoses in man was studied by Allen and Kabat,2” who found that neither gave evidence of any antibody formation. However, as before, it wct8 shown that the Merck polymer reacted to some extent with preimmunization sera, whereas the DuPont polymer was unreactive. Heidelberger and Aisenberg*” studied the crowreaction of the Merck and DuPont polyglucoses with antibodies to pneumoeoccal C-substance and to type-specific polysaccharides. One of the n-glucose polymers (Merck 52R611) was separated into a series of fractions on the basis of fractionation with alcohol (isopropyl alcohol and ethanol) and with glacial acetic acid, the most insoluble fraction being called A, and the most soluble, E. The yield, analyses, and reactivity of these fractions with Types IX, XII, XX, and XXII antipneumococcal horse sera are presented in Table VII. 4

TABLE^ VII

FractionationPM of 10 g. of D-G~UCOS~ Polymer 52R611

Yield, Fraction

A A’

B D E

g.

0.19 0.14 1.16 1.45 1.S

d6’ r)dim*so 1.182 1.075 1.001 1.030 1.027

3.0 1.25 1.0 0.5 0.46

Maximum antibody N, 48, pptd. from antiserum type

Reducing sugar, as glucose, unhyddyzed, %

Ix

XI1

XX

XXII

4.6 5.0 4.3 4.3 4.0

35 21 19 3 0.1

50 35 30 15 5

50 35 25

35 25 20 4 0.3

0

3

0 Solution (0.6%) in 0.85% sodium chloride solution; “rel” and “dim” refer to relative and diminished viscosities, respectively. * ALSO per 10 g of polyglucose.

(273)P.Z.Ailen shd E. A. Kabat, J . Exptl. Med., 106, 383 (1957).

506

I. J. OOLDSTEIN AND T. L. HULLAR

It is readily apparent that some of these fractions differ extensively in their immunological reactivity, and the possibility exists that the original polymer could have been fractionated on the basis of their reactivity with these sera, Just what role (if any) molecular size may play in this immunochemical interaction is not yet known. Several Merck polyglucoses, one DuPont polyglucose, and three dextrans and it were tested against a series of 17 antipneumococcd horse waa shown that the polyglucosee gave precipitates with certain of these sera (especially antisera of Types 11,VII, IX, XII, XVIII, XX, and XXII). It is interesting that the three samples of dextran were also most reactive with these same antisera. The DuPont polymer behaved quite differently from the Merck polyglucoses, giving a good reaction only with Type XI1 antiserum and a slight reaction with Type XX antiserum. Heidelberger and coworkers demonstrated that g l y ~ o g e n P ~from -~ several murces gave specific precipitates with practically the same antipneumococcal sera with which the synthetic polyglucoses (especially, fractions A and A' of the Merck polymer) react. In many instances, these fractions precipitated more antibody nitrogen than the naturally occurring polyglucoses. A polymaltose prepared by Ricketts and Rowel" reacted with pneumococcus Type XI1 antiserum more extensively than did a polyglucose prepared by the same authors, but both polysaccharides left behind some antibody reactive with glycogen. Neither polymer reacted with Type I1 antiserum. Polygalactose gave a slight precipitate with Type IV antiserum, the capsular polysaccharide of which is known to contain D-galactose. London and coworkerss" observed that their polyglucose gave negative precipitin reactions with three rabbibantisera displaying high, native antidextran titers. However, two of the three antisera gave positive results when the more sensitive, complemenbfixation technique was used. It is generally agreed that antibodies to Types 11, IX, XII, XX, and XXII pneumococcal polysaccharides are, at least partially, directed against a-Bglucopyranosyl residues which are linked (1-6) 12n-279 ( 1 4 ) ,217-*7@,280 ( 1 - , 6 , 4 ~ 1 ,)z 4 0 m a 7 6 m (l-+3),wg1aao and, to a lesser extent, (1-2)?le (274) M. Heidelberger, A. C. Aiaenberg, and W. 2. Hassid, J . Exptl. Med., 89, 343 (1964). (276) M. Heidelberger, H. Jahrmilrker, B. Bjthklund, and J. Adams, J . Zmmuml., 78, 419 (1967). (276) M. Heidelberger, H. Jahrmilrker, and F. Cordoba, J . Zmmunol., 78, 427 (1967). (277) M,Heidelberger, B. Bjthklund, and J. L m e r , J . Zmmunol., 78, 431 (1957). (278) J. W. Goodman and E. A. Kabat, J . Zmmuwl., 84,333 (1960). (279) J. W.Goodman and E. A. Kabat, J . Zmmunol., 84, 347 (1960). (280) S. F. Schlossman, M. L. Zarnita, E. A. Kabat, G. Keilich, and K. Wallenfele, J . Zmmunol., 91, 60 (1983). (281) K. Butler and M. Stacey, J . Chcm. Soc., 1637 (1955).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

507

with the (l+G)-bond playing a dominant role. Thus, the synthetic glucans probably possess various proportions of these residues 88 part of their gross structure. Conclusions that are more specific wdl require much additional study of the structural features which affect these antigenantibody reactions. Concanavalin A is an antibody-like protein isolated from the jack bean.= It was shown to form a precipitate with glycogen and with yeast mannan, and was later also used to differentiate between glycogen-like polysaccharides from various source^.*^^,^^^ It has now been shown that concanavalin A forms a precipitate only with branched polysaccharides which contain terminal, nonreducing a-D-glucopyranosyl or a-D-mannopyranosyl groups.L86The combining sites of the protein appear to be directed against the 2-deoxy-a-~-arabino-hexopyranosyl system.286 The interaction between synthetic polysaccharides and concanavalin A was studied by means of the Ouchterlony, agar-gel, diffusion technique.2sD*28s Polyglucoses, polymaltoses, polymannoses, polyribose, polyxylose, polygalactose, poly (2-deoxy-~-arabino-hexose), polygalactoglucose, and a polycellobiose were examined. The polymannoses, polymal toses, and poly(2deoxy-~-arabino-hexose)gave pronounced lines of interaction with the reagent, signifying the presence of multiple, terminal, nonreducing, a-D-glycopyranosyl groups. The polyribose, polygalactose, polyxylose, and polygalactoglucose were, aa waa to be expected, without action. Most of the polyglucoses were unreactive, whereas a few gave very faint lines of precipitation. The Merck polyglucose, however, gave a good reaction with the concanavalin A reagent, illustrating structural differences between the Merck polyglucose and the other polyglucoses. The polycellobiose gave a weak interaction, suggesting that some anomerization had occurred during polymerization, giving rise to some a-D-glucopyranosyl end-units.28s These results suggest that concanavalin A may find increased appIication as a reagent for the detection and preliminary characterization of certain types of a-D-glycosyl groups a t the nonreducing chain-ends of branched polysaccharides.

V. APPLICATIONSOF SYNTHETIC POLYSACCHARIDES The exploitation of synthetic polysaccharides and their derivatives for biological, medical, and industrial purposes is in the early stages of development. The principal emphasis to date has been directed to the biological, rather than to the industrial, application of these materials. In (282) (283) (284) (285)

J. B. Sumner and S. F. Howell, J . Bid. C h m . , 116, 583 (1936). J. A. Cifonelli, R.Montgomery, and F. Smith, J . Am. Chem. Soc., 78,2485 (1956). R. Montgomery, Arch. Biochem. Biophye., 67, 378 (1957). I. J. Goldatein, C. E. Hollerman, and E. E. Smith, Biochsmiatiy, 4, 876 (1965).

508

1. J. OOLDSTEIN AND 1. L. HULLAR

these biological systems, the polyglycoses have been used mainly as their cationic and anionic derivatives. The derivatives studied include poly(glucose sulfate) ,287 prepared by sulfation with chlorosulfonic acid; "polyprepared by periodate oxidation of polyglucose to (glycose carboxylate) afford the polyaldehyde, followed by oxidation with chlorous acidasg;poly(glucose nitrate),280prepared by nitration with a mixture of sulfuric and nitric acids; and basic derivatives,B' such as the (diethylaminoethyl) and (diethylaminohydroxypropyl) ethers of polyglucose, prepared by treatment of polyglucose with 1-(diethy1amino)S ,3-epoxypropane in the presence of sodium carbonate. Interest in the search for heparin substitutes has helped stimulate the development of synthetic polysaccharides. Although blood anticoagulant activity is exhibited by the sulfate esters of many polymers, especially ~ y l a n , chitin,m*p2@4 ~' dextranJB6 those of such polysaccharides as cellulose,2ga and chondroitinsulfuric acid,m#m4most of these products have also been reported to have toxic properties. However, preparations of dextran sulfate of low molecular weight have been developedm6which are nontoxic and are suitable substitutes for heparin. A polyglucose sulfate exhibited met& chromatic and anticoagulant activity similar to that of dextran sulfate.9o7 A polyglucose of P, aboute4 20, sulfated to a degree of substitution of 1.76, had anticoagulant activity of 6-7 international units/mg., and an acute toxicity in the range of that of heparin (L.D.m lo00 mg./kg., intravenous, in mice), Similarly, Wood and Morazw found that polyglucose sulfates (degree of substitution, 2.6-3.0) have approximately the same activity as clinical preparations of heparin. These workers and their colleagues also suggeated2W that sulfated polyglucosea may be useful in other aspects of blood-coagulation studies. (Diethylaminohydroxypropyl)ethers of polyglucose and polygalactose were shown to be potent, cationic, hemagglutinating agents.n2The hemagglutinating activity increased with increased substitution, a polygaIactose of degree of substitution 1.1still being active a t a concentration of 1pg./ml. Interest has also been expressed in the use of polyglucosea as plasma(287) J. W. Wood and P. T. Mora, J . Am. C h . Soc., 80,3700 (1958). 0

2

~

(288) P. T. Mom, E. Merler, and P. Maury, J . Am. Chem.Soc., 81, 5449 (1959). (289) B. T. Hofreiter, I. A. Wolff, and C. L. Mehltretter, J . Am. Chem. Soc., 79, 6457 (1967). (290) J. W. Wood and P. T. Mora, J . Org. C h . , 27, 658 (1962). (281) J. W. Wood and P. T. Mom, J . Org. C h . ,27, 2115 (1982). (292) J. W. Wood and P. T. Mom, J . P01ym.w Sci., Pt. A , 1, 3511 (1963). (293) P. Karrer, H. Koenig, and E.Uateri, Helu. Chim. Ada, 96, 1296 (1943). (294) K.H. Meyer, R. P. Pirue, and M. E.Odier, Helu. Chim. Ado, 86, 574 (1952). (295) A. Gr6uwaU, B. Ingelman, and H. Mosimen, Ada Soc. Med. Upaaliensie, 60, 397 (1946); Chem. Abetrads, 41, 5213 (1947). (296) C. R. Ricketts and K. W. W d t o ~B , d . J . Phannaml., 8,'476 (1953). (297) K. W. Wdton and C. R. Ricketta, B d . J . ExpU. Pafhol.,86, 227 (1954).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

509

volume ex pa rider^.^^^^^ In extensive animal studies, solutions of a Merck polyglucose72 ([TI 0.20-0.23) exhibited good plasmavolume expander properties, with retention times better than that of dextrans. However, the fitiding that some of these polymers interact (to form precipitates) with the sera of normal individualdT2S2?"atid with certain piieumococcal antisera?10,274-2n such as those of Types 11, IX, XII, XX, and XXII, casts some doubt on their use in human beings. On the other hand, a DuPopt polyglucose appeared to be without action on preimmunization sera.2n,na A rather extensive program designed to investigate the interaction of synthetic polyglycoses and their derivatives with components of biological systems was begun by Mora and coworkers in 1958. The availability of polyglucose fractions of various molecular sizes, together with some knowledge of their structure and of the techniques for introducing both cationic and anionic substituents, made it possible to study these systems with a view to correlating their macromolecular structure and charge density with their biological activity. In general, interaction of charged derivatives of polyglycoses in biological systems depends not only on their charge density, but, to a considerable degree, on the macromolecular structure (which serves as a framework for the various charged groups). In almost all experiments in which the anionic and cationic polyglycoses were shown to be biologically active, simple charged molecules (such as 6-glucose tetrasulfate) either were without action or displayed properties unlike those of the macro-ions. By using these charged macromolecules, it was possible to investigate coulombic interactions in biological systems in a systematic way. Polyglucose sulfates can be used to titrate basic protein.m-2w Neutralization was noted when approximately stoichiometric proportions had reacted, and precipitation of the neutralized products often occurred. Such proteins as protamine sulfate, lysozyme, ribonuclease, adrenocorticotropic hormone, thymus peptides, and cytochrome C all gave precipitates which could be solubilized by suitable alteration of the pH and the ionic strength. The soluble interaction-products migrated as a single component during electrophoresis at pH 3,7,and 10. In contrast, no precipitates were obtained with sulfuric acid, sodium sulfate, or D-glucose tetrasulfate, and the soluble products dissociated during eIectrophoresis. Cationic and anionic derivatives of synthetic polysaccharides may be useful for the fractionation of other charged macromolecules, such as nucleic acids, proteins, arid sulfated polysaccharides, in a manner similar to the use of dextran sulfate.m (298) P. T. Mora and B. G. Young, Ndure, 181, 1402 (1958). (299) P. T. Mora and B. G. Young, Arch. Biochem. Bbphye., 82, 6 (1959). (300) J. L. Oncley, K. W. Walton, and D. G . Cornwell, J . Am. C h . Soc., lS, 4666 (1957).

510

I. J. GOLDSTEIN AND T. L. HULLAR

Mortt ttnd coworkersaolstudied thc in uiuo interaction, in mice, of toxic levels of cationic macromolecules with the carboxyl and sulfate derivatives of polyglucose. Toxic levels of polymyxin, protamine, streptomycin, and neomycin could be counteracted, if the synthetic macroanions were injected five to ten minutes after administration of the proteins. Subcutaneous administration of tho uaionic polyglucoses prior to intraperitoneal injection of toxic levels of basic proteins afforded protection to the mice, and also demonstrated that the animals could survive, even when the two injections were by different routes. Several basic proteins (enzymes) could be reversibly inhibited by polyglucose sulfate.280Thus, lysozyme (60 pg.) could be completely inhibited by polyglucose sulfate (48 Ng.). The most highly sulfated polyglucose fractions exhibited maximum inhibitory activity, whereas the polyglucose carboxylate displayed a somewhat lower inhibitory power. This inhibition of lysozyme by polyglucose sulfate is about 100 times more effective than the inhibition by glutamyl polypeptides or by deoxyribonucleic acid, two of the most potent inhibitors previously known.”* Ribonuclease and hyaluronidase were completely inhibited by polyglucose sulfate, but not by D-glucose tetrasulfate.200 The subsequent addition of basic proteins could reetore enzymic activity to systems inactivated with polyglucose sulfate. For example, the depolymerizing activity of ribonucleaae was restored on addition to the system of lysozyme or hyaluronidaae, both of which are basic proteins. The latter complexes with the anionic polyglucose derivative, thus liberating an active enzyme. A series of carboxyl derivatized polyglucoses were studied aa inhibitors of ribonuclease activity, in an attempt to relate charge density to inhibitory activity.”’ In comparison with other factors, it was concluded that coulombic forces probably play a major role in complex-formation between enzyme and substrate, and between enzyme and inhibitor. However, other specific, nonelectrostatic forces were shown to participate in the binding of bovine pancreatic ribonucleaee to ribonucleic acid.a In a study of the inhibition of components of the cytochrome systems by macromolecular poly-ions , Person and FinemvRMshowed that such polycations as prottlmine sulfate, histone, and ribonuclease inhibit the cytochrome oxidare activity of various systems. The inhibition could be ravcrsed by the addition of poly-ions (and, especially, by polyglucose sulfate) ;the latter waa also shown to inhibit the oxidase system reversibly. (301)P. T.Mora, B. G . Young, and M. J. Shear,Ndure, 184, 431 (1969). (302)R. C.Skarnes and D. W. Watson, J . Budsriol., TO, 110 (1966). (303)P. T.Mora, B. G . Young, and M. J. Shear, M u k ~ m ZChem., . 88, 212 (1960). (304)P.T. Mom, J . Biol. Chem.,NT, 3210 (1902). (306)P. Pereon and A. Fine, Science, 192, 43 (1980). (306)P. Person and A. Fine, Arch. Biodwn. Biophye., 94, 392 (1961).

511

CHEMICAL BYXTHESIS OF POLYSACCHARIDES

The fact thak increase in thc ionic strength of the mediuni causes a progressive weakening of the inhihitiou suggested that electrostatic forces are involved in complex-formation between the oxidase and the polyanion. Polyglucose sulfate, a t conccntrations lower than those necessary for inhibition, activated cytochroiiie oxidatie preparations, ~.lb:well as reviving aged preparations of the oxidaae which had lost some of their activity. I n an extension of the above study, Mora and coworkersm examined the effect, on the cytochrome oxidase system, of a series of synthetic, negatively and positively charged, polyglucoses having graded chargedensity properties. Polyglucose sulfate and polyglucose (diethylaminohydroxypropyl) ether of different degrees of substitLtion were employed as macro-ions. At the level of 1 pg./ml., the macro-ions inhibited the cytochrome system in direct proportion to the charge density of the polyglucose derivatives. Thus, 0-(diethylaminohydroxypropyl)polyglucose of degrees of substitution of 0.2, 0.5, 1.0, and 1.6 inhibited cytochrome oxidase activity to the extent of 0, 51, 65, and 82%, respectively. Coulombic forces were believed to play a central role in the interaction. It was shown that polyglucose sqlfate could reverse the inhibition, by poIymyxin or ribonuclease, of the tumor-damaging effect of endotoxins from Serratia marcescens.aOsIt was also demonstrated that polyglucose sulfate abolishes the ability of lysozyme to lower the fever-producing activity of the bacterial polysaccharide from the same organism. Mora and coworkersm established that the endotoxic polysaccharide from Serratia marcescens is inactivated in vitro by extracts of intact, rabbit-liver cells, the inactivation activity being associated with the baaicprotein ftaction. Addition of polyglucose sulfate reversed the inactivating effect and restored the original, tumor-damaging activity to the endotoxin. This demonstrates the reversible character of the macromolecular interaction of the anionic polysaccharide with the cationic, liver-cell protein, and also of the cationic liver-cell protein with the more highly anionic, polyglucose sulfate. In the l’olyanions were also shown to inactivate ba~teriophage.*~”~’3 presence of polyglucose sulfate of high molectilar weight approx. 16,OOO; degree of substitution, 3.0), bacteriophages TI through T7 were rendered the eflect being more pronounced at room temperature. The inactivation was irreversible. Combination of bacteriophage

(a,,,

(307) (308) (309) (310) (311) (312) (313)

P. Parson, P. T. Mora, and A. S. Fine, J . Bwl. Chem., 238, 4103 (1963). P. T. Mora and B. G. Young, J . @en.Microbiol., 26, 81 (1961). S. I. Orooselan, P. T. Mora, and M. J. Shear, Biochem. Phamcot., 12, 1131 (1983). B. G. Young and P. T. Mora, Virobgy, 12, 493 (1960). B. G. Young and P. T. Mora, Bwchim. Biophya. Ada, 47, 202 (1961). P. T. Mora, B. G . Young, and S. Rievi, J . Biol. Chem., 287, 157 (1962). S. Rievi and P. T. Mom, Bwchim. Biophys. A&, 72, 630 (1963).

,512

I. J. GOLDSTEIN AND T. L. HULLAR

T2with the polyglucose sulfate wa8 demonstrated by thc use of &S-labeled No loss in viability occurred when the phage was incubated inhibitor.810JLZ with anions of low molecular weight. Carboxyl derivatives of polyglucose behaved similarly, but higher concentrations of these less acidic polyanions were required in order to achieve similar effects.81a Irreversible inactivation of Tt bacteriophage also occurred when the phage waa incubated with periodate-oxidieed po1yglucosea1' at pH 8.510.0 in the presenoe of 0.1 M borate buffer. Interpretation of the data is difficult, in view of the known lability and modification of polysacespecially since phage inactivacharide polyaldehydes in alkaline tion occurs only at elevated pH values in the presence of polyaldehyde and 0.1 M borate buffer. Mora and Young"1sshowed that antiserum to bacteriophage T2 can be blocked reversibly with polyglucose sulfate. Polycations also inhibit this interaction, but not so effectively, If such polycations as spermidine and cadaverine are added to the polyanionic polyglucose-Te phage antiserum complex, the polyglucose sulfate is preferentially bound to the stronger polyanion, thereby liberating an active antiserum which inactivates bacteriophage Ts.These workers suggested that electrostatic forces are involved in the polyglucose sulfate-antiserum binding and may also be involved in the antibody-antigen system in general. Solutions of synthetic polyglucoses have also been used for densitygradient centrifugation of viruses.a16 These substances are especially suited to this purpose, since they can be dissolved in water or buffer to yield solutions of high concentration and low intrinsic viscosity and a density similar to that of sucrose solutions. Because of their low rate of diffusion, polyglucoses form gradients that are more stable. A polyglucose nitrate powessed explosive properties, similar to those of starch nitrates and cellulose nitrates.*w is currently used as an adhesive, the Although a synthetic polyglucoseLS6 probability of utilization of most of the synthetic polysaccharides for industrial purposes seems uncertain. The availability of vast stores of starch and other natural gums, and of the knowledge and technology necessary to modify these substances into useful products, make it unlikely that much commercial interest will become focused on synthetic polysaccharides. The preparation of man-made polysaccharides requires the prior preparation of the starting monomer, followed by its repolymerization to a polyglycose. Such an operation would not seem commercially feasible, unless the end product exhibits some unusual or highly valuable properties. (314) R. D. Juthrie, Aduan. Carbohydrate C h . ,16, 153 (1961). (316) P. T . Mom and B. G.Young, J . Bwl. Chrn., 197, 1870 (1962). (316) 8.I. Orosslan, S. Rizvi, T. E. O'Connor, and P.T. Mora, Nature, 102,780 (1964).

AUTHOR INDEX FOR VOLUME 21 Numbers in parentheses are footnote numbers. They are inserted to indicate the citation of an author’s work when his name is not mentioned on the page.

A

Ando, T., 503 Andreae, W. H., 399 Abadie-Maumert, F. A., 250, 493 Anet, E. F. L. J., 160 Abd Elhafez, F. A., 180 Anno, K., 31, 116, 142(73), 178, 442, Abderhalden, B., 38 443(54), 464, 469(54), 470(54), Abdullah, M., 503 479(54) Abe, H., 480,481(214), 482(214), 485(214), Antonakis, K., 153, 158, 190 Applegarth, D. A., 331 492 (214), 495(214), 496 (214) Abeles, F. B., 430 Ap Rees, T., 389 Abraham, E. P., 326 Arohibald, A. R., 324 325(15), 332, Aoton, E. M., 141, 169, 179, 181, 193(142, 333(62), 334(62), 347, 351(83), 242), 200(142), 201(233, 242), 361(57), 366(02), 368(15), 369, 202 (142), 203(142). 370(124), 371, 374(83) Adams, J., 506,509(275) Argoudelis, A. D., 191 Adams, M. H., 325 Arison, B., 158 Armour, C., 475 Addicott, F.,429 Agarwal, B. R., 241 Armstrong, J. J., 324, 325(15), 327, 328, Ahlgren, G. H., 394 331, 354(56), 368(15) Ahmed, M. D., 145 Arni, P. C.,270 Aisenberg, A. C., 493, 505, 506(240), Arnold, A. W., 412 509(240, 274) Artemova, E. K., 419 Akita, E., 172, 202(161). Arzoumanian, H., 141, 169, 179, 181, Albersheim, P., 384, 385(42) 193(142,242),200(42), 201 (233,242), Albert, L. S., 412 202(142), 203(142) Alberty, R. A., 238 Asahi, Y., 184, 185(255),187(255) Alekhine, A., 258 Ashar, K. G., 243(102), 245, 250, 261 Alexander, A. G., 426,427 Ashton, F.M., 403,405,406,421,423(295) Alfea, A., 449,453(98), 492(98), 4116(98) Ashwell, G., 189 Ali, S. A., 427, 428(341) Aso, K., 497 Allen, P. J., 127 Aspinall, G. O., 139, 174, 201 Allen, P. Z., 504, 505, 509(273) Audia, W.V., 429, 430 Allerton, R., 146, 152, 154(23), 162, 194 Audus, L. J., 380 Allpress, C. F., 102 Augestad, I., 103, 104, 105(29, 31, 48), Amagasa, M., 213, 219(20), 270 128(48), 129(48), 140(29, 31, 48), Anderson, A. W., 416 141(48) Andemon, C. B., 62, 204 Austin, D. J., 409 Anderson, C. D., 151, l58(57), 159(.57) Austin, M. J., 469 Anderson, J. S., 373, 375(132) AuRtin, P. 104, 105(44), 138(44), 339, Andemon, L., 416 362 513

w.,

514

AUTHOIl INDEX, VOLUME

Austin, W. C., 216(30), 220 Avery, 0. T., 325 Avigad, G.,126

21

Bau, A,, 244(106), 245 Baum, G., 458,460 b x t e r , J. N., 104, 105(40), 132(40), 133(40), 140(40)

Bayley, 8.T., 383,385(35) B Beauohamp, C. E., 424 Bechtler, G.,498 Babers, F. II., 201 hcker, B., 269 Bach, M. K., 396 Beddington, A,, 443, 444(67) Bacon, J. S. D., 126, 127, 140 Beevers, C. A., 226, 236 Baddiley, J., 104, 105(44), 124, 133(110), Beevers, H., 389 138, 323, 324, 328(15), 326, 327, 328, Behre, C., 462 330(37), 331, 332, 333(61, 62, 63), Bel’denkova, A. F., 419 334(62, 63), 339, 341(61), 342(66), Bell, D. J., 127, 140 344(63), 347, 350(81), 351(83), Belozemky, A. N., 364 354(56), 355, 357, 359(89), 361 (57), BeMiller, J. N., 454 362, 366(2, 3, 62, 63), 368(15, 46), Benedikt, R., 247 369, 370(124), 371, 374(83), 375(66) Benguerel, F., 228 Benitez, A., 158 Baer, E., 13, 175 Baer, H., 216(34), 220 Benn, M. H.,124 BennetClruk, T. A., 382, 383(20) Bagley, W. P., 401 Bent, H. A., 97 Bailey, J. M., 457 Bentley, K. R., 157, 158(94) Baird-Parker, A. C., 324 Baker, B. R., 115, 123, 141(98), 142(65), Bentley, J. A., 392 151, 158(57), 159(57), 184, 169, 176, Beresoovekii, V. M., 124 177, 178, 179, 439, 449(30), 458(30) Berg, D., 504, 508(272), 509(272) Bakey, B., 325, 367(27) Berg, P., 333 Berger, L., 123 Ball, C. D., 387 Ball, D. H., 443, 444(66) Bergmann, M., 38, 149, 193, 197(307), 289, 297, 301(56), 302(56) Ball, D. J., 193, 194(306), 196(306) Ballou, C. E., 103, 127, 501 Bergetrom, L., 325 Berlin, H., 442 Bamford, C., 475 Berlin, Y. A., 184, 185(251), 186 Bandiei, A. A., 395 Banks, B. E. C., 475 Berner, E., 103, 104, 105(!29, 31, a), Banks, W., 492, 494(326), 495(236), 128(48), 129(48), 133, 140(29,31,48), 141(48) 406 (236) Bernfeld, P., 432, 502 Bannikova, A. A., 212, 217(14) Barber, 0.A., 126 Bernstein, H. J., 201 Bardolph, M. P., 458, 484(122) Berry, J. W., 269 Barker, G . It., 103, 1&5(33), 122, 136, Berthelot, M., 441 180(1)a), 140(33) Bettin, K. L., 278 Bltrkor, It., 104, 105(41), 122, 140(41), 331 Beurling, K., 258, 268(145) Barker, H. A., 80, 127, 496 Beurskene, G., 241, 263(92), 264(92) Burnard, P.W. C., 3211 Bey, S., 210, 246(4) nrrrriei.t,,I., 175, i’in(184) Beyer, E., 182 Rarry, J. A,, 214 Beynon, J. H., 39, 44(1) Bartiiimk, It., 243(101), 245, 250 Beythien, K., 244(107), 245, 246(107) Bltrte, Q.R.,173, 17U(188), 191, 195 Beeer, A. E., 360, 362(93), 373(93) Btwkett, R. S.,389 Bhacca, N. S.,184,185(255,256), 187(255, Bat8es,J. H , 423, 425(302), 426(302) 2M), 203(256) Bates, R. G., 240 Bhat, K. V., 148, 309

AUTHOR INDEX, VOLUME

Bhattacharya, A. K., 122, 140(87), 437, 480(24), 483(24), &4(24), -(24), 495(24),496(24) Bieleski, R. L., 427 Biemann, K., 39,40,44(4),46, 53, 54(18), 56(18), 59(16), 61(7, 16, 18), 62(16), 63(16), 66(16), 71(16), 74,75, 77(34), 90,204 Bigelow, C. C., 229 Binea, B. J., 174,267, 433 Bird, T.P.,433 Bird, W.T.,433 Birkofer, L.,391,412(92) Bishop, C. T., 98, 99(15), 104(15), 105(15, 42), 107, 108(15), 109(15), 110(15), 111(15), 136, 140(15), 201, 438, 449(28),463, 474, 493(141), 498 Biusson, P., 295 Bjorklund, B., 506, 509(275,277) Black, C.C.,Jr., 389 Black, M.K.,387 Black, W.A. P., 270,433 Blair, M.G.,146 Blake, J. R.,430 Blanchard, P.H.,127 Bleshinskil, S.V., 240 Blomqvist, G.,457 Blouin, F. A., 253 Blout, E.R.,280 Blumbergs, P., 194, 195, 196, 197(325), 199(325),200(325) Bobbitt, J. M., 132 Bochkov, A. F.,293,461 Bodaner, G.,464 Bodea, C.,213,214 Bodger, W. H.,26q Bockmann, A., 449, 451(95), 452(97), 453(95,97),454(95, loo), 492(95,97, loo), 494(95),400(95,97) Biieseken, J., 8 Boettcher, A., 177 BBttger, A., 445 Bohn, E.,277 Bolliger, H.R., 157, 159, 160, 186, 309 Bolton, C.H.,191 Bonnar, R. U.,469, 495(173) Bonner, J. F., 380, 381, 382(19, 20), 383(20), 3X4(3X), 385(37, 38, 42), 387(37) Bonner, T. C., 450 Bonner, W.A., 146, 156

21

515

Boothby, D., 421 Borders, D.B., 196 Bordwell, F. G.,450 Borjeaon, H., 121, 138(82), 141(82) Boroughs, H.,383,385(37), 387(37) Borrmann, D.,458 Borthwick, H. A., 386 Bott, H.G.,102, 138,467 Bottomley, W.,420 Bourke, J. B., 389, W(78) Bourne, E.J., 80, 127 Bouveng, H.O.,454,492,494(237) Bovey, F.A., 434 Bowlea, W.A.,149 Bows, B., 365 Boyle, F. P.,397 Bradbury, D.,388 Bradinskaya, M.S.,126 Brady, 0.L.,265 Briindstrom, A., 234 Brauns, F., 163 Brazhnikova, E.M.C., 184, 185(251) Bredereck, H.,484 Brewer, F. M.,261, 265(148) Brewster, J. H.,97 Brice, C.,458, 484(123) Briggs, D.E.,418 Brigl, P.,157 Brimacornbe, J. S.,173, 179,185, 187(257, 258) Brimhall, B., 433 Brink, N. G.,139 Broadkorb, T.,444 Brockmann, H.,184, 195,198(324) Brodie, H.W.,421, 423(295) Brown, D.M.,135, 166,329,331, 341 Brown, H.C.,97,125, 168, 169(138) Brown, J. F.,439 Brown, R.,416 Brown, S. A., 409 Bruce, M. I., 420 Bruckner, Z., 444 Bryant, B. E.,238 Buchanan, J. G., 104, 105(44), 124, 133(110), 138(44), 161, 166(110), 170, 326, 327, 328, 330(37), 331(37), 339, 343, 354(56), 355, 357, 359(89), 361(57),362,363 Ruchanan, M. A., 442 Budzikiewice, €I., 39, 40, 41(3), 44(10), 47,63(10), 201

AUTHOR INDEX, VOLUME 21

516

Bueding, E., 492 Buehler, W., 146, 278, 279(22), 290, 292(48), 296(460), 304, 305(67), 306(67), 313, 319(67, M), 320(46,

88)

Buffel, K., 383, 385(34) Bull, T. A., 427, 428(342) Bunton, C. A., 475 Burg, E. A., 430 Burg, S. P., 430 Burger, M., 324, 326(10, ll), 350(28), 365(29), 372(10, 11)

Burk, D., 145 Burr, G. O., 421, 423(295), 427(298) B u r s t r h , H., 381, 383, 385, 420 Burton, C. A., 329 Buss, D., 164 Butler, 0.C., 150 Butler, K., 124, 135(104), 193, 508 Button, D., 347, 351(83), 352, 374033) Buttrose, M. S., 418 Butta, J. S., 389, 394(78), 396, 397, 407(109), 408(109) C

Cabib, E., 200 Cadenbach, G.,445 Cadotte, J. E., 104, 464 Cahill, J. J., 139 Cairnmas, I. M., 139 Calvet, E., 213, 240 Calvin, M., 233, 265(70) Cannsn, R. K., 238 Canvin, D. T., 147 Capon, B., 100, 110, 111(49), 120, 129, 131(49a), 239, 439, 440(31), 475

Carbon, J. A., 449 Cardini, C. E., 427 Carlier, A., 383, 385(34), 390 Carlson, A. F., 386 Caron, E. L., 195 Carothem, W. II., 436 Carrington, T. It., 127, 4% Cam, B., 326, 327, 328, 330(37) Carter, M. C., 401 Carvalho, J. d t ~S., 480,481(213),482(213), 483(213), 484(213), p86(213), 493(213), 496(213), 503(213) Casini, G., 151, 159(56), 202(56) Castah, P., 257,487

Ceatle, J. E., 411 bf, M., 164, 166, 171, 197(329), 198(133), 443

Cervernf, J., 175 Chablay, E., 269 Chacravarti, A. S., 425 Champetier, G., 243(104), 244(108), 245, 249, 250, 261

Chandler, L. B., 124 Chasey, A., 433 Chang, H., 427, 428(340) Chang, P., 176 Chanley, J. D., 173 Charslambow, G., 180, lQO(235) Chardon, C. E., 427 Charley, P. J., 210, 212, 213, 233 Charleon, A. J., 174, 339 Chastain, B. K., 161, 166(113) Chattttrjee, A. N., 373, 375(131) Chattttrjee, 8. K., 429,430 C h a n , J., 249 Chen, K. K., 312, 313, 314(81, 82, 84, 85) Chernobai, V. T., 294, 321(51, 52) Chiriboga, J., 427 Chiahov, 0. S., 40, 46, 47(6, 15), 48(15), 52(17), 56(13), 57(13), 59(13, 15), 61(6, 13, 15), 64(15), 67, 69, 71(13, 15, 17, 27, 29, 30), 72(15, 17, 28, 29, 30), 74(32), 79, 80(35), 81, 84, 89, 93,201,204,502 Chorney, W., 413 Christeleit, W., 18 Chrietensen, H. N., 238 Christensen, J. C., 194 Christensen, J. E., 136 Christianson, G. S., 387 Chu, P., 300, 303(66) Chus, J., 147 Ciaudelli, J. B., 186, 308 Cifonelli, J. A., 136, 507 Cifonelli, M., 136 Clamp,J. R., 467 Cleland, R. E., 382, 383, 384(38), 385(38), 387(30), 388(30), 389(30) Clendenning, K. A., 405 Cluekey, J. E., 469 Clutterbuck, P. W., 138 Coahran, W., 219(52), 220, 226, 236

Coffee, D. R., 406 Coleman, G.H., 442,458,484(121,122)

AUTHOR INDEX, VOLUME

Coleman, 0. H., 427, 428(338, 339) Coleman, R. E., 421, 423(295), =425, 426(316), 427,428(336,337,338,339) Collins, P. M., 181 Colquhoun, J. A., 433 Commoner, B., 387 Compton, J., 176, 178(188) Conaway, R. F., 251 Conchie, J., 96, 275, 416 Conn, E. E., 409 Conover, M. J., 325, 367(27) Conrad, H. E., 498 Cooke, A. R., 405 Cooinbe, B. G., 418 Cooper, F., 98, 99(15), 104(15), 105(15, 42), 107, 108(15), 109(15), 110(15), 111(15), 136, 140(15), 438, 449(28)

Coore, H. G., 416 Cordoba, F., 506, 509(276) Cords, H. P., 395 Corner, J. J., 412 Corns, W.G., 403 Cornwell, D. G., 509 Cory, J. G., 158 Court, A. A., 111, 131(496) Cox, E. H., 467 Cox, N. L., 121 Cox, S. T., 401 Coxon, B., 278 Crafts, A. S., 380, 399 Cragg, L. H., 501 Craig, L. C., 494 Cram, D. J., 180 Cramer, M., 467 Creighton, A. M., 193 Cretcher, L. H., 193 Critchley, P., 332, 333(62), 334(62), 365, 366 (62).

Croon, I., 268, 437, 438(26) Crohy, D. G., 397 Crosby, E. K.,442, 443(52) Crown, B., 197 Crosn, B. E., 417 Cruzado, I f . J., 424 Cumminn, C. S.,173 Cunicella, V., 238 Ciiiiiiirighani, C. E., 398 Cuiiningham, K. Cf., 1.57, 158(94) Currier, H. B., 399 Cuthbertson, A. C., 255

517

21

Cutler, W. O., 18 Cynkin, M. A., 189

D Dahlgard, M., 158, 161, 166(113) Dale, J. K., 216(31), 218(47), 219(31), 220 Daly, J., 390 Danilov, S., 175 DaRooge, M. A., 147 Date, W. B., 430 Davey, N. B., 369,372 Davidson, E. .4,, 172 Davidson, L. G., 423 Davies, D. A. L., 187, 188(266), 189 Davis, E. A., 399 Davis, J. C., 404,406(172) Davison, A. L., 324, 347 Davoll, J., 122 Day, B. E., 399 Dean, G. R., 104 Deferrari, J. O., 124 De Jongh, D. C., 46, 53, 54(18, 56(18), 59(16), 61(16, 18), 62(16), 63(16), 66(16), 71(16), 74, 75, 77(34), 85, 87, 88, 93, 158, 199(356), 201, 204(351), 205(356), 206(354c, 356), 207, 502 de Lederkremer, R. M., 200 Denbo, J. A., 238 Derache, R., 154 Derevitskaya, V. A., 259, 264 Derias, R. E., 153, 476 Dernikos, D., 258 de Souza, R., 483, 494(218) Deulofeu, V., 124, 172 Deutschman, A. J., Jr., 269 Dewar, E. T., 270,433 Diamond, R. M., 241 Dick, W. E., 136 Dickhauser, E., 168, 193(139) Diehl, H. W., 121, 133, 140(144), 146, 153 Dierickx, L., 369, 370(117) Diets, A., 192 Dillon, R. T., 102, 103(21), 106(21) Dimbat, M., 469, 495(173) Diori, IT. W.,173, 179(168), 191, 203(290) Dixon, S., 443, 444(67) Djerassi, C., 39, 40, 41(3), 44(10), 47, 63(10), 201 Dmitriev, B. A., 194

518

AUTHOIt INDEX, VOLUME

I)oane, W.M., 267 Domanska, H.,410 Domovs, K. B., 215, 217(28), 218(28), 227(28) Dowgiallo, A., 153 Drew, H.D.K.,102,433 Drewos, B.,418 Dubos, R.,325 Dubourg, J., 213 D u r n , W.M.,Jr., 389 Dull, M. F., 486, 497(186), 502(158), 512(150) Dunlap, A. A,, 394 Dupuis, Y.,210 Durand, H.W.,466,497(156), 602(156), 512(150) Dure, L. S.,419,424(284) Durr, 0. J., 299 Duachimky, R.,123, 146 Dutton, G.G.S., 138,437, 438, 468, 471, 472(25), 474, 482(25, 26, 182), 484(25, 29, 182), 495(29), 497(182), 498(25), 499, 501(182), 503(26) Dyfverman, A., 458, 484(124)

21

Elsner, H.,462 El-?'s~~b~~lai, M. A., 257 Engel, C.E., 442,443(52) English, J., Jr., 193 Ennis, W.B.,388 Ergle, D. R., 394 Erickson, L.E.,238 Erlander, S., 436,437(21), 438(21), 470 Ermolaeve, E.Ye.,419 Ernst, B;;I90 Erxleben, N.,377 Esipav, S.E., 184,185(251),186 Evans, H.,426 Evans, J. P.,209 Evans, W.L.,275,277(8),282(8), 292,456 Evtushenko, 0.A., 419 Excell, B.J., 494 Exer, B.,405,407

F

Fan, Y.,176 Fang, 6. C., 389, 394(78), 396, 406, 407(109), 408(lOQ) Farmer, V. C.,215 E Faulkner, J. K.,398 Fawoett, C. P.,326 Eagles, C. F., 410,412(214) Feather, M.S.,130 Eagon, R. G.,401 Feingold, D. B., 126 Eckstein, Z.,416 Fellig, J., 396 Fenner, G.D.,135 Edelmrtn, J., 126 Fernandee-Bolafioos, J., 170 Edward, J. T.,300 301(68b),303(66) Ferneliua, W.C.,238 Edwards, M.,410 Ferrante, C.R.,103 Edwards, 0.E., 467 Edwards, T. E., 138, 442, 443(66), Ferrier, R.J., 40, 194,201 Fetzer, W.R., 442,443(52) 479(56) Efremova, L. N.,171 Fiaser, L. F., 273, 292(2) E p n , M.M.,467 Fieser, M.,273,292(2) Egge, H.,498 Finan, P.A., 39,54,81(20),84(20),90,127 Eiah, IT., 176 Firre, A., 510, 511 Elbein, A. D., 174 Finkelstein, H.,193 Elderfield, It. C., 144, 171(1), 183(1), Fucher, E., 8, 38, 95, 101, 122, 148, 149, 275, 276, 277, 278(12), 280(12, 13), 173, 175, 177, 178, 289, 441, 442,443 281, 282(12, IS), 285(12), 288(12), Fischer, H. 0. L., 13, 103, 127, 149, 158, 312, 819(12, 13)' 320(12, 13), 321(13) 175, 178(84), 204(84), 291, 304 Elliott, 8. D., 332 Fischer, W.,199 Ellis, C.P.,122, 125 Fisher, B.E., 469 Ellwood, I). C.,328,342, 347,368(* Flamm, E., 208 El Sawi, N. N., 105 Flatt, It., 228

519

AUTHOH INDEX, VOLUME 21 Fleming, I. D., 50.7 Fletcher, H. G.,Jr., 104, 105(41), 121, 122, 123, 125, 133(85), 140(41, 87, 144), 141(85, 114), 146, 150(25), 153, 154(25), 164, 278, 285, 289, 297, 460 Flood, A. E., 193, 194(306), 196(306) Flory, P. J., 434, 437, 438(27), 470(12), 493(12) Fiynn, E. H., 195 Folkem, K., 139, 158, 176 Forbes, A. J., 421 Forster, N. O., 38 Foster, A. B., 114, 136, 156, 169(88), 191, 192(291), 194, 197(310), 200, 342, 343, 464, 491 Foster, J. F., 501 Fouquey, C., 188, 189(273, 276), 190(273), 198(277), 199(273, 277) Fournier, P., 210 Fox, J. J., 122, 123, 139(93), 146 Frahm, H., 435, 436, 437(23), 449(23), 470(23), 483(23) Frahn, J. L., 252 Fredenhagen, K., 445 Frederick, J. F., 402 Frederiksen, S., 158 Freed, V. H., 404, 406 Frege, C., 213 Freiberg, S. R., 394, 430(106) French, D., 436, 437(21), 438(21), 457, 470 Frhejacque, M., 286, 287 Freudenberg, K., 8, 10, 17, 26, 38, 163, 164, 173, 176, 457 Freund, E. H., 215, 217(28), 218(28), 227(28) Frey-Wyssling, A., 382 Fried, J., 117, 282(13) Fries, N., 413 Frohardt, R. D., 195 Fromme, I., 187, 188, 189(270) Frush, H. L., 217(43a), 218(43a, 45), 220, 230(45), 231(45), 469, 495(171) Frydman, R. B., 427 FurNt,, A., 160 Fiijinioto, K., 4!47 Flllt,n, J. Id., 395, 396, 3!)8(117), 404 Furukawtt, J., 4HR Fitriiytr, M., 410, 41 l(308) Fusari, S. A.. I!b5

G

(falmarini, 0. L., 172 Galston, A. W., 382, 387, 410, 411(208) Ganyushkina, L. G., 395 Gardell, S., 493 Gardiner, J. G., 104, 105(47), 141(47) Garner, H. R., 174 Garrett, A. J., 368, 374(111) Gaskill, J. O., 404 Gasman, R. C., 458, 484(124a) Gasaer, R. J., 195 Gast, A., 407 Gaiimann, E., 190 Gauhe, A., 216(34), 220 Cause, G. F., 184 Gauthier, D.,218(50), 219(26,50), 220,223 Gautier, A., 441, 502(40) Gaves, K. M., 261, 264, 267 Gavilaneu, G. R., 308 Gee, M., 126 Geerdes, J. D., 113, 151(56) Geiger, E., 242 Geisel, F., 173 Gelinder, H., 443, 502(62) Gentile, A. C., 402 Gentner, W. A., 405 Geoghegan, M. J., 405 Georg, A., 210, 444 Georges, L. W., 442 Gerecs, A., 280, 441, 457(35) Germscheid, H. G., 461 Geachwind, I. I., 229 Ghuysen, J. M., 369, 370(116, 117) Gibbs, M., 389 Gibbs, R. D., 409 Gilbert, V. E., 470 Gill, C. H., 219(53), 220, 223 Gilligan, W. H., 295, 319(53), 320(53) Gillis, C. L., 230 Glaser, L., 144, 324, 326(10, 11, 12, 131, 350(28), 372(10, ll), 373(12, 13), 374(13) Glass, C. A., 501 Glaszioii, K. T., 421, 424 Glaxkova, E. L., 212, 217(14) Gmelin, It., 392 Goad, L. J., 418 Gobillon, Y.,222 Godefroi, E. F., 149

520

AUTHOR INDEX, VOLUME 21

Goebel, W. F., 291 Golab, T., 274 Goldacre, P. L., 379 Goldberg, M. W.,172 Goldstein, I. J., 471, 472, 474(185), 475, 483, 486(192), 494(218), 497(219), 498(185), 501(185), 502, 503(269), 507(186, 269) Golova, 0. P., 479, 483(209), 484(209), 485(209), 486(209), 488(209), 502(209) Golovkina, L. S., 58, 79, 80(35), 84, 89 Good, N.E., 405 Goodban, A. E., 139 Goodman, J. W., 506 Goodman L., 115, 136, 141, 142(65), 151, 158(57), 159(56, 57), 169, 178, 179, 181, 193(142, 242), 194, 200(142), 201(233, 242), 202(56, 142), 203(142, 316) Goodyear, E. H., 102 Gootz, R., 456 Gore, J., 445 Gorin, P. A. J., 139, 163, 166, 175, 181, 193(239), 198(132, 185, 239), 295, 296, 339, 497 Gorman, M., 173

Gora, H. J., 409 Grade, G., 44!2,443(53) Gramera, R. E., 170 Granath, K., 494, 496(246) Granstrtim, B., 395 Grant, &, 425, 426 Green, J. W., 86, 112, 113(3), 115(50), 117(62), 118(52), 127, 128(60), 130(50, 52), 141(50, 52, 53) Greenberg, G. R., 327, 328 Greenwood, T., 492,494(236), 495(236), 496(238) Gregory, F. G., 387 Gregory, H., 433 Gresser, W., 449, 402(93), 494(93), 486(93) Greulach, V. A,, 399 Griffith, C. F., 151, 152(53), 186(53) Griffith, C. M., 394 Grimaux, E., 442, 468 Grob, C. A,, 148 Groebke, W., 123

c.

Grtinwall, A,, 608 GrBtrJch, H., 448

Gross, D., 127 Grove, J. F., 417 Grundschober, F., 270 Gueffroy, D. E., 136 Gukrin-Dumartrait, &, 400 Gunja, Z.H., 433 Gunner, S. W., 201 Gunsalue, I. C., 325 Gut, M., 159, 176 Guthrie, R.D., 612 Guahavina, V., 221 Guaman de Fernandea-Bolaflos, R., 176

H Haack, E., 299, 313, 314(83) Haagen-Smit, A. J., 377 Haas, H. J., 164 Hac, L. R., 404 Hachihama, Y., 213, 246(19) Hadacsy, I., 280 Haines, A. H., 194 Hakamori, S., 498 Hall, G. E., 341 Hall, L. D., 97,99,168, 193(139), 201,501 Hall, W.C., 401, 424, 430 Hallerman, G., 460 Halsey, G. D., 214 Hamada, Y., 184 Hamilton, R. H.,407 Hammerstrand, M., 443, 502(62) Hammer, K. C., 386 Harmer, C. L., 388, 387, 393, 398, 403 Hand, D. B., 397 Hand, M. E., 410 Handschumacher, R.E., 343 Hanessian, S., 87, 93, 156, 158, 172, 177, 180,181 (236), 182,183,193(236), 195, 199(356), 200(337), 201, 203(321), 204(351), 205(358), 208(209, 356), 207(249a), 275, 449, 502 Hann, R. M., 176, 217(35), 220, 289, 2W43) Hamen, C. M., 398 Hanson, J. B., 388, 393 Hanae, A. R., 117 Hag, S., 441, 457, 458 Hague, M. H., 425 Harborne, J. R.,214 Hardegger, E., 155, 176 Hardy, F. E., 104, 105(44), 138(44), 339, 343, 355, 359, 362

AUTHOR INDEX, VOLUME

lIarriw, C., 38 Harris, H., 173 harrison, R., 194, 197(310) Hart, P. A., 115, 142(65) Hartigan, J., 127 Hartman, F. C., 331 Hartt, C. E., 421, 423(295), 427(296, 298) Hasenfratz, V., 287 Hash, J. H., 369 Haskell, T. H., 172, 195, 203(321), 449 Haskew, H. C., 425 Haskins, F. A., 409 Haskins, W. T., 176, 289, 290(43) Hassall, K. A., 403 Hassel, O., 284 Hassid, W. Z., 326, 427, 506, 509(274) Hauenstein, H., 288 Haukenes, G., 328, 368(46) Haworth, W. N., 18, 95, 102, 126, 127, 128, 130(23), 136, 139, 140(23, 24), 141(2), 433 Hay, A. J., 416 Hay, G. W., 199, 471, 474(185), 495, 498(185), 501 (185) Hay, J. B., 323, 324, 325(15), 366(3), 368(15), 369, 370(124), 371 Hay, J. R., 388, 396 Hayashi, T., 417, 420 Hayneu, L.J., 116, 275, 284(10) Haynes, W. C., 469 Hearon, W. M., 454 Heath, R. L., 433 Hebert, L. P., 425, 426(316) Heddle, W. J., 243(98), 244(98), 245, 247(98), 248(98), 249(98), 250(98), 268(98) I-ledgley, E. J., 160, 161, 162(107), 163(107), 164(107), 166(107, 11I), l68(lll), 169, 192(107), 198(107), 200(107), 201(111) Heidelberger, M., 355, 493, 504, 505, 506(240), 509(240, 274, 275, 276, 277) Heidt, L. J., 128, 129(134), 130 Heinatz, R., 154, 163(77) Helderman, W. D., 211 Helfenberger, H., 286 Helferich, B., 38, 122, 147, 176, 182, 277, 278, 289(19), 445, 446,456, 460,461, 498(79) Hemberg, T., 409 Hendershott, C. H., 410

21

521

Henderson, F. C., 312,313,314(82, 84,85) Henderson, N., 299, 319(65) Henderson, R. B., 458 Herborn, H., 175 Herrero, F. A., 430 Herrett, R. A., 401 Herrington, B. L., 218(46), 220 Herrfeld, A., 244(105), 245, 246(105) Hestrin, S., 126 Heuser, E., 243(101), 245, 250 Heuwr, H., 144, 145(3), 171(3), 183(3) Hewson, K., 170 Heyn, A. N. J., 381 Heyns, K., 46, 48(14), 52(14), 54(14), 60, 61(19, 25), 69(19), 93, 125 Heytler, P. G., 411 Hibbert, H., 255 Hickson, J. L., 467 Highlands, M. E., 398 Hilbert, G. E., 267, 485 Hill, J., 167, 192(134) Hilton, J. L., 405 Himmelspach, K., 187, 188, 189(271) Himmen, E., 176 Hinman, J. W., 195 Hirano, S., 164, 455 Hirase, S., 433 Hirsch, P.,239, 240(83) Hirst,E. L., 96,102,130,138,174,467,498 Hitchcock, A. E., 378 Hitomi, H., 184 Hixon, R. M., 218(49), 220 Ho, H., 430 Hobbs, K. C., 104, 105(35), 141(35) Hoch, J. H., 273, 274(1), 282(1), 311, 312(1), 314(1) Hockett, R. C., 124, 133, 134(143), 210, 216(3), 217(3), 219(3) Hodge, J. E., 267, 485 Hodges, R., 124, 133(110) Hoeksema, H., 139, 174, 191 Honig, M., 217(39), 220, 258(37), 441 Hoesch, K., 38 Hoffer, M., 123, 146 Hofreiter, B. T., 508 Hofstad, T., 324 Hollerman, C. E., 472, 507(180) Holley, R. W., 397 Holly, F. W., 139 Holneaa, N.J., 56 Honeyman, J., 122, 125

522

AUTHOR INDEX, VOLUME

Ilopkiiix, T. It., 404 Ilortio, 11. W., XLB llorth-l)eori, P., 246, 'JRl(1IH) Ilortoii, D., {Hi, 137, 1!)4, 343, 493, 464, 479,482C211) Ilorwitz, J. P., 147 Hoshi, R.,464 Hough, L., 103, 117, 156, 186, 187, 168, 175, 178, 181, 192(134), 193(139, 239), 198(132, 18h, 239), 382, 443, e44(64), 467 Howard, G. A., 124 Howell, S. F.,507 Huber, H., 159 Hudson, C. S.,8, 14, 102, 103, 125, 126, 127, 129(26, 113, 133), 130(27, 1131, 132(126), 133(126), 135(27), 140(28, 27), 141(113),151, 152(54), 178, 178, 182(198), 188(54),210, 216(3), 217(3, 35), 218(44), 219(3), 220, 285, 288, 290(43) Hiitter, R., 190 Huffaker, R. C.,403 Hughes, A., 420 Hughes, I. W.,138, 309, 478, 491 Hughea, N.A,, 333 Hullar, T.L., 133 Hulyalkar, R. K.,178 Humphreys, T.E.,389 Hunger, A.,288 Hurd, C.D.,487 Hwmann, E., 431, 433, 436(1), 449, 454(1,20), 455(1), 488, 503(1) Huclsey, R. E., 270 Hutchison, A., 399,414 Hubon, D. H.,96, 137 Hutten, U.,484 Hybl, A,, 216(32), 220, 238 Hyde, B.,418

21

lllgl~H,(;, If.., 149,2(H) Iiikntor, J. J., 102

Iimeuiva, A. M., 410 Iilririti, J. C.,478,479,485(807) Ii'vhu, V. C.,378 Irving, J. T.,310 Isbell, 11. 8.,215, 217(42, 42a, 43, 43a), 218(43a, 46), 220, 230(42a, 43, 45), 231(42a, 46), 315, 469, 495(171, 172) Iselin, B., 148, 186 Isherwood, F. A,, 199 Ishidate, M.,121,138(81) Ishimoto, N.,373 Ishirawa, K.,128 Istatkov, S.,419 Iwedare, K.,175, 177 Iwainsky, H., 442,443(51),446(51) Izma'Ilov, N.A,, 233

J

Jabbar Mian, A., 480, 484(215), 485(215), 488(215) Jachymczyk, W.,158 Jackson, A,, 149 Jackson, E. L., 127, 132(128), 133(128), 178 Jackson, R. F., 230 Jacobs, W.P.,429 Jiiger, H., 197, 198(331),199(331),274 Jahrmfirker, H.,506, 509(275, 276) Jakubowski, 2.L., 195 Jarnieson, R. 9.P., 139 Jang, R.,384,385(4!2) Jansen, E.F.,384, 385(42) Jansen, J., 137 Jardetzky, C.D.,97 Jardetrky, O.,238 Jaworski, E.G.,397 Jeanes, A., 469 I Jeanloz, R. W., 148,160,348 Ibaez, J. G., 427 Jeffrey, G.A., 97, 241, 283(92), 264(92) Iinuma, H., 418 Jennings, A., 418 Ilan, I., 381, 385(23) Jenninga, H.,182 Immoto, M.,433 Jensen, W.A., 418, 424(284) Inatome, M.,31, 442, 443(54), 469(54), Jeppson, J. B.,391 470(64), 479(54) Jerkeman, P., 121, 134(83), 138(82), Inch, T. D., 192 141(82,83) Ingelman, B.,508 Jermyn, M.A., 199 Ingle, T.R.,136, 170 Jim, V., 184 Inglw, D. I,., 138 Jirgensons, B. R.,18

AUTHOR INDEX, VOLUME 21

523

Kefford, N. P.,379, 420 Job, P., 224 Keilich, G., 506 Johnson, A. L., 191 Johnson, A. W., 416 Kelemen, M. V., 332,333(61), 341(61), 347 Johmon, D. C., 458, 484(124tl) Keller-Schierlein, W., 190, 191 Johnnon, L. F., 191, 2Wd(290) Kelly, F. 11. C., 219(54), 220, 223 Kelly, F. J., 214 Johruori, It. TT., 406 Joncich, M. J., 182 Kemp, W. S., 430 Jones, A. S., 124 Kempf, W., 217(40), 220 Jones, G. H., 166 Kennedy, E. J., 398 Jones, H. G., 201 Kennedy, E. P.,372 Jones, J. K. N., 103, 153, 164, 166, 168, Kenner, G. W., 124 175, 170, 181, 182, 193(139, 239), Kenner, J., 154, 155, 156(81) 194(306), 196(306), 198(132, 185, Kent, P. W.,113, 158, 162(98), 166, 239), 201, 210, 433, 434(8), 443(8), 198(131), 447, 44€4(83), B02(83) 444(65, 66), 494, 497(65) Khaligue, M. A., 145 Khanna, K. L., 425 Jones, W. J. G., 135, 141(149) Joseph, J. P., 439, 449(30), 458(30) Khorana, H. G., 295 Joseph, N., 187, 295 Khorlin, A. J., 461 Juergens, W. G., 360 Khorlin, A. Ya., 293 Kibrick, A,, 238 Julianelle, L. A., 328 Kiliani, H., 153 Juliano, B. O., 116, 142(69) Kimura, S., 433 K King, T. P., 494 Kinoshita, T., 258, 268(145) Kaars-Sijpesteijn, K., 404 Kabat, E. A., 360, 362(93), 373(93), 504, Kirschenlohr, W., 124 505, 506, 508(272), 509(272, 273) Kirst, L. C., 442, 443(52) Kisfaludy, L., 454 Kackzka, E. A., 158 Kahn, M. A., 424 Kiss, J., 125 Kaichi, S., 464 Kissman, H. M., 176 Kaiser, C., 391, 412(92) Kjoelberg, O., 104, 105(43), 128(43), Kaiser, F., 299, 313, 314(83) 129(43), 133, 134 Kaiser, S., 172 Klilmbt, H. D., 390, 391, 392(82, 87), 397 Kakisawa, H., 184, 185(255), 187(255) Klsr, J., 433, 437(7), 441(7), 449, 480(7), Kaltenbach, U.,199 488 Klein, W., 182, 456 Karan, W., 333 Karjala, S. A., 267, 485 Klemer, A., 458,460 Karrer, P., 152, 161, 177(59), 508 Klundt, I. L., 147 Kashelikar, D. V., 278, 279(22, 23), Klyne, W., 283, 285, 286, 287(29) 289(23), 290(23), 291(23), 2Q6(23), Knoevenagel, C., 176 316(23), 319(23), 460 Knorr, E., 275, 276(7) Knusli, E., 407 Kashimura, N., 164, 455 Kaslander, J., 404 Kocheshkov, K. A., 223 Katz, J. R., 433 Kochetkov, N. K., 40, 46, 47(6, 15), Katz, M., 383 48(15), 52(17), 56(13), 57(13), 59(13, Kaufmann, E., 158 151, el(& 13, 151, 64(15), 67, 69, Kaufmann, H., 173 71(13, 15, 17, 29, 30), 72(15, 17, 28, Kaur, R., 387 29, 30), 74(32), 81, 93, 165, 167(130), Kaushik, M. P., 429 181, 192(130), 194, 201, 204, 293, Kawamatsu, Y., 184, 185(256, 256), 461, 502 187(255, 256), 203(256) Kocourek, J., 171, 197(329), 443 Kawashima, K., 185 Kogl, F., 377

524

AUTHOR INDEX, VOLUME

Koeiiig, XI., 508 Koenigs, W.,275, 276(7) Koffer, H., 174 Kohn, P., 125 Koike, €I., 404 Koine, A., 115, 116(66), 142(66) Kolb, J. J., 325, 367(27) KOIOSOV, M. N., 184, 185(251), 186 Komitsky, F.,Jr., 168, 170(137), 198(137), 201(137), 202(137)

Konstantinova, N. V., 184 Kopecky, K. R., 180 Kops, J., 439, 486(34), 488(34), 489(34), 490(34), 497(34)

479, 483, 484(209), 485(209, 2101, 486(209), 488(209), 502 (209) Kortschak, H. P., 421, 423(295), 427(298) Koemol, H., 391, 412(92) Kosuge, T., 409 Kovsharova, I. N., 184 Kowalewski, Z.,197 Kowkabmy, G. N., 197, 198(328) 199(328) Koslova, N. A., 419 Koslowski, W., 253 Krauss, M. T., 197, 198(331), 199(331) Krauz, C., 175, 178, 189(226) Kreienbring, F., 190 Kristen, H., 136, 177 Krotkov, G., 385 Kruglyak, E. B., 184 Ku, T., 177 KuEerenko, V., 172 Kiihii, A. O., 403 Kiihn, It., 124, 164, 216(34), 220, 498 Kuhn, W.,8 Kullnig, R. K., 201 Kunin, R., 464 Kunstmann, M. P.,173, lQO(l69a) Kuntz, A., 280 Kuns, A., 258 Kurdynkova, V. A., 124 Kuszmnn, J., 149

Korshak, V. V.,

L Lake, H. W.G,, 18 Laland, S., 124, 135(104), 193, 194, 196(309), 197(309)

21

Laiiduu, B., 145 Landauur, 8.R., 165, 181(11'3) Landt, E., 213,214 hrtier, J., 506,609(277) Lamen, P., 378 k t t r e , E. J., 497 Lasure, E. P., 267 Laszlo, J., 145 Launer, H. F.,495 Lavigne, J. B., 176 Law, F. R., 388 Lawrie, I. D., 427 Leasure, J. K.,4@3 LeBaron, H. M.,395 Lebedenko, L. A., 419 Lebedev, N. V., 212, 217(14) Ledeen, R., 173 Lederberg, J., 44 Lederer, E., 188, 189(273, 276), 190(273), 198(227), 199(273, 277)

Lee, E. E., 443,464(60), 465(60, 150, 151), 466(152)

Lee, J. B., 165, 181 Lee, W.W.,158, 194 Lee, Y.-C., 501

Lefevre, T., 442,468 Lehman, J., 114, 192, 194, 197(310), 491 Leloir, L. F., 427 Lernieux, R. U., 18, 97, 98, 100(16), 201, 275, 284(11), 300, 303(66), 439, 461(135), 458, 461, 484(123), 491

LeMinor, L., 189 Lenz, J., 125 Lenz, R. W.,266, 267 Leopold, A. C., 429, 430 Lerner, L. M., 125 Lea, J., 123 Leachinsky, W.,297,301(56), 302(56) Letters, R., 341 Leube, H., 157 Leuck, G. J., 468 Leucke, R. W.,393, 403 Levene, P. A., 102, 103(21), 106, 136, 153, 176, 178(188), 270, 467, 502

Levi, I., 139 Levhe, S., 491 Levvy, G. A., 96, 275, 416

Levy, H. B., 188, 189(271), 194(271) Levy, M. F., 193 Lewak, S., 154

AUTHOR INDEX, VOLUME

I4owiri, I,., 3% Lcwk, 13. A., 1 1 3 , 141(56), I!N, 471, 474(185), 496, 498(105), 501(185) Lewis, L. It., 149 Lewis, T. A., 475 Leydet, P., 240 Leyh-Bouille, M., 369, 370(117) Licciardello, J. J., 398 Lichtin, N. N., 149 Lieberrnann, C., 173 Lin, R., 427, 428(340) Lin, W., 177 Linck, A. J., 401 Lindberg, B., 121, 134(83), 137, 138(82), 141(82, 83), 164, 253, 268, 457, 458, 483,484(124), 492,494(237), 497(219) Linde, H., 308 Lmdet, I,., 246 Linscott, D. L., 395 Lipkin, D., 146 Lisitsyn, D. I., 412 Liu, Y. T., 176 Livingaton, C. H., 396 Llewellyn, I). R., 329, 475 Lloyd-Jones, C. P., 396 Lo, S., 419 London, E., 448, 497(84), 502(84), 506, 508(84) Long, F. A., 130, 131(136) Losnegard, N., 324, 369 Loughman, B. C., 397 Loustalot, A. J., 394, 424 Loveday, G. W., 110, 111(49) Lowy, B. A., 122 Lozitskaya, S. F., 240 Luckwill, L. C., 396 Ludwig, E., 269 Liideritz, O., 144, 187(8, 9), 188, 189(8, 9, 270, 276), 199(8, 9) Liihrs, E., 443,444(61), 462(61), 498(61) Lugo-Upez, M. A., 425, 426 Lukasiak, H., 421 Lutz, O., 18 Lythgoe, B., 124 Lyubetskaya, M. N.,212,217(14) Lyubin, B. O., 212, 217(14)

M McCarty, M., 324, 328 McCart,y, M. K., 395

21

525

1McClmkey, c. M., 458, 484(121) McCloakey, J. A,, 40, 61(7), 90,158 McClowkey, P., 417 McComb, E. A., 384 McCready, 11. M., 126, 138(116), 384 MacDoriald, 1). L., 146, 150(25), 154(25), 156, 178(84), 204(84)

McDonald, E. J., 139 Macek, K., 197, 198(328), 199(328) McFarland, V. W., 469, 472(174), 474(174), 475(174), 503(174)

McGrath, D. I., 135, 329, 443, 464(60), 465(60, 151)

Maciejewska-Potapczykowa, W., 421 McIlrath, W. J., 399 Mackenzie, J. E., 223, 246(159), 247, 251(59), 255

McKinnell, J. P., 172 McLaren, L., 149 MacLennan, A. P., 171, 173(151), 197 MacMillan, J. F., 417 McNally, S., 192 McNutt, W. S., 413 McPherson, J., 457 McSweeney, G. P., 192 McWain, P., 116, 123(70) McWhorter, C. G., 395, 400 Madudina, N. F., 64, 71(27) Ma&, K., 172, 202(161) Makarowa-Semlijanskaja, N., 270 Makolkin, I. A., 242 Malawista, I., 172 Malling, H., 158 Mandelstam, J., 368 Mhndy, T., 219(51), 220, 254 Mann, J., 501 Manners, D. J., 442, 443(50), 446(50), 429(50)

Manolopoulos, P., 149 Markovitz, A., 172, 173(152) Marsaudon, A., 249 Marsh, C. A,, 96, 275 Martell, A. E., 233, 265(70) Marth, P. C., 429, 430 Martin, R. O., 359 Martlew, E. F.,342 Mawell, E. N., 182 Mashtakov, S. M., 406 Mason, D. J., 192

826 Miwoii, G . W.,406 Mewiiii, l’., 401

AUTHOII INDEX, VOLUME

Mathian, A. P., 326 Mathur, P.S.,425,430 Matsuda, K., 168, 170(137), 198(137), 201(137), 202(137), 497 Matsui, M., 121, 138(81) Matsuura, S.,211,217(11) Mauli, R., 287, 294(38), 312, 320(38), 321(38) Maury, P., 138, 469, 470(175), 471(175), 492(175), 493(175), 495(175), 498(175),508 Mayer, A. M., 409 Mayer, M. M., 504 Meade, J. A., 403 Meadow, P.M., 373,375(132) Meckstroth, W.,449, 451(95), 453(95), 454(95), 492(95), 494(95), 496(Q5) Mednick, M., 149 Mehlinger, H. P.,274 Mehltretter, C.L.,508 Meinwald, Y.,475 Meisenheimer, K.,121 Melvin, E.H.,469 Mempel, D., 449, 463(96), %4(98), 492(96), 496(96) Menager, L., 158 Mercer, G. A., 442, 443(60), 448(60), 479(50) Meraz, O., 161, 166(111), 168(111), 201 (111) Merler, E.,471,508 Merlis, N. M., 479, 483(208), 484(209), 485(209), W(2091, 488(208), 502(209) Merrick, J. M.,472, 507(185) Merz, J., 333 Meyer, A., 441,502(39) Meyer, A. S., 176, 180 Meyer, G.M., 102 Meyer, K.,276,278(15), 279(15),280(15), 282(15), 286(15), 286(15), 308, 320(15) Meyer, K. H., 508 Meyers, M. B.,408 Meystre, C.,276, 277,a83 Michaelis, E.,458 Michaelis, L.,239 Michaleik, E.,136

21

Miohml, F., 177, 180, 446, 451(94, 95), 452(94, 07), 453(95, 96, 97, 98, W), 464(05, 96, loo), 468, 460, 469(94), 492(93, 95, 96, 97, 98, 99, loo), 494(93,95),496(93,95,96,97,98,99) Miescher, K., 276, 277, 283 Mikeeh, L. A., 153 Mikhailova, J. A., 171 Mikkebon, D.S.,399 Miksic, J., 172 Miller, C.S.,401,430 Miller, E.J., 102 Miller, I. A., 442 Miller, L. P., 414, 415(238, 239, 240, 241, 242,243,244,246,246,247,248,249, 250,251, 252, 253, 254) Miller, 8. R., 403 Mills, J. A.,227,232,233,241(63),252(63) Mills, R.,214 Minshall, W.H.,406 Mitchell, J. W., 385,429, 430 Mitchell, P.,328 Mitra, A. K.,134 Mitschev, L. A., 173, 190(169a) Mittag, R.,176 Mittelmeier, H.,442,443 Miwa, T.,126 Miyake, M., 184 Miyamoto, M., 184, 185(255, 256), 187(255,2M), 203(256) Mizuno, K., 184 Moelwyn-Hiighea, E. A., 442, 469(49), 495(49) Moewus, F.,410 Momber, F.,17 Momose, A., 115, 142(67) Montavon, R.M., 176 Montgomery, E. M., 103,130(27),135(27), 140(27),218(44), 220 Moptgomery, J. A., 122, 123, 139(94),170 Montgomery, R.,113, 136, 141(56),498, 507 Moore, J. A., 286 Moore, R.H.,479,482(212) Mora, P.T.,138, 467, 468(164),469(167, 168),470(170,175),471,472,474,475, 492(168,175), 493(168,175),495(167, 168, 175), 496(168), 497(168), 498(175), 503(168, 174), 508, 509, 510(299), 511,512(290, 310,311,312)

AUTHOR INDEX, VOLUME 21

Morutid, P.E., 3)O, 301 (66h), 303(66) Morelsiid, 1). E., 404,406(172), 407 Morgsri, A. It., 461, 491 Morgan, W. T. J., 176 Rlori, T., 153 M6rics, M., 161 Moriii, I<. B., 173 Morse, J. I., 324 Mosiman, H., 508 Most, B. H., 420 Mowery, I).F., Jr., 103, 104, 106(45, 46), 106,110 Moyer, J. D., 469, 495(172) Moyle, J., 328 Miihlradt, P., 173, 313, 314(85) Muhlschlegel, H., 157 Muller, A,, 161 Miiller, D., 46, 48(14), 52(14), 54(14), 93 Muller, G. J. M., 431, 436(1), 454(1, 20), 455(1), 503(1) Muller, H., 176, 178 Muller, K., 443 Mukherjee, S., 158, 159 Mukherjee, T. K., 195 Mulholland, T. P. C., 417 Mumford, F. E., 411 Murakami, Y., 420 Murray, D. H., 150, 164 Murray, J. K., 168, 169(138) Murray, L. J., 168, 169(138) Murray, R. K., 136 Musculus, M., 441, 446,502(38, 39) Muskat, I. E., 270 Miuik, T. J., 394, 424 Myrbiick, K., 443, 502(62)

N Naegeli, H., 153 Naffa, P., 213 Nagarajan, K., 195 Nagarajan, It., 98, lOO(l6) Nagase, K., 125 Nagata, W., 308 Nagazawa, K., 146, 154(24) Naguib, M. I., 408 Nakadaira, Y., 184, 185(255, 187(255, 256), 203(256) Nakamura, E., 464

256)

527

Nakamura, T., 446,447(82), 502(82) Naksiiishi, K., 184, 1$5(255, 256), 187(255,256),203(256) Nskazswa, K.,184 J. F., 3S3, 384 NRIICB, NU~~IUIIMOII, S.G., 386, XU,373(30, 31) Naumova, I. B., 363, 364, 365 Naylor, A. W.,399, 401 Neale, S. M., 239 Nid, J., 243(104), 245, 249, 250 Neely, W. B., 387, 496, 497 Nef, J. U., 153, 305 Neilinger, W., 449, 451(94), 452(94), 469(94) Neipp, L., 190 Neish, A. C., 394 Nelson, C. D., 385 Ness, R. K., 122, 123, 125, 133, 140(87, 144), 141(114), 146, 289, 297 Neufeld, E. F., 326 Neuhaus, F. C., 333, 375(66) Neukom, H., 187, 202(135) Nevin, R. S., 490, 491 Newman, H., 417 Newth, F. H., 116, 136, 150, 153, 162, 275, 284(10), 297 Nicholas, S. D., 136 Nichols, P. L., 433 Nicholson, W. H., 179, 443, 444(65), 497(65) Nickerson, M. H., 133, 134(143) Nickerson, W. J., 363 Nicoll, W. D., 251 Nicolson, A., 139 Nielsen, N. A., 194, 19.5 Nielsgn, A. M., 329 Nigrelli, R. F., 173 Nimz, H., 115, 116(66), 142(66), 146 Nishizawa, IS., 213, 219(20), 246(19) Nitsch, C., 381 Nitsch, J. P., 381, 412 Nitta, Y.,115, 142(67) Nobs, H., 242 Noel, M., 147 Nolan, T. J., 181 Nordin, P., 494 Novak, J. J. K., 145 Nowotny, A,, 187 Nutting, G. C., 386 Nys, M., 113, 140(51)

528

AUTHOR INDEX, VOLUME 0

Oakes, E. M., 170 O’Colla, P. S., 443, 464(60), 465(60, 150, 151),466(152) O’Connor, T.E.,612 Odier, M.E.,508 Odincovs, P. N.,435,444(18) Oeding, P., 324, 328,368(48) Ofengand, E.J., 333 Ohle, H.,168, 193(139) Okamura, S.,243(103),245 Oldham, J. W., 478,479,485(207) Oldham, K. G.,329 O h , S. M.,18,116, 142(68) Ollapally, A. P., 147, 304 Oncley, J. L.,509 O’Neill, A. N.,497 Onikura, N., 270 Onodera, K., 164,456 Oparin, A. I., 126 Ordin, L.,383,384(38), 385(38) Oroszlan, €3. I., 511, 512 Orrell, 8.A.,492 Osmn, E.M.,104, 105(35), 141(35) Ost, H.,444 ost, 461 Ottar, B.,284 Overend, W. G., 110, 111(49), 114, 124, 129, 130(60), 131(135), 135(60, 104), 136, 138, 144, 145(6, 7), 146(7), 147(6), 149, 150, 162, 163, 164(23), 160, 161, 162(107), 163(107), 164(107), 160(107, lll), 168(111), 181, 192(107), 193, 194, 106(309), 197(309), 198(107), 200(107), 201(111), 239, 275, 309, 475, 476, 477(195, 197),491 Owen, L. N.,135, 136,141(149), 193 Owen, O.,138,442,443(66), 479(66) Owens, H.S.,139 Oyolu, c.,403

w.,

P Paabo, M., 240 Pact&, J., 164, 166, 171, 197(329), 198(133), 443 Pamu, E., 96, 112, 113(3), 115(50), 117(62), 118(52), 127, 128, 130(60, 62,64), 140(&4), 141(50,62, 53, 54), 142(64, 258, 288(146), 443, 444(67), 467,488(164)

21

Pagnucco, R., 116,123(70) Pa&, W.D.,280 Paleg, L. G.,418 Panzer, H.P., 433 Parikh, V. M.,164 Paris, R.,412 Park, H.R.,325 Park, J. T.,373,375(131) Parker, M.W.,386 Parrish, F. W.,503 Parthasarathy, 8.V., 423 Pasika, W.M.,501 Patai, S.,475 Paterson, D.R., 396,398 Pateraon, G.R.,420 Pateraon, J. C.,270 Patin, D.L.,200 Paul, P.T.,442 Paul, R., 295 Paulsen, H., 91 Pavanaram, S. K.,308 Pawl&, M.,238 Payne, M.G.,395,396,398(117),404 Payne, T.A. J., 123,146,297,299,301(61), 302(64),304(57), 319(64) Pazur, J. H., 503 Peat, S., 18, 135, 138, 141(149), 442 443(56), 479(56) Pedemn, C., 121, 122, 146 Peel, E.W.,139 Peligot, E.,248 Pepinsky, R.,241 Percival, E. E.,96,104, 105(47), 141(47), 172,180,190(235), 498 Percival, E. G. V., 243(98, 99, 100), 244(98, 99, 100, 110), 245, 247(98, 99, 100, 110), 248(98, 99, loo), 249(98), 250(98>,255, 258, 288 Perkine, H. R., 324, 325(16) Perlin, A. S., 104, 106(40), 122, 132(40), 133(40),134,140(40),295,296,339,497 Perlis, I. B., 383 Pernikis, R. Ya., 479,485(210) Perry, M.B.,176,201,362 Person, P.,510,511 Petens, O., 445,498(79) Petitpaa, G.,249 Petro, F.H.,399 Pfeiffer, P., 19 Pfleiderer, G., 333 Phillips, D.D.,102, 105(22), 125, 141(22)

AUTHOR INDEX, VOLUME 21

Phillips, G. O.,297, 434 Phillips, I. D.J., 410 Phillips, P. M.,367 Pictet, A., 257, 444, 467, 477, 478, 479, 502(202) Pictet, J., 478 Piel, W., 461 Pieper, H.J., 418 Pietsch, G., 146, 280, 301(26), 302(26), 304(26),319(26) Pigman, W. W., 217(42), 220, 469, 495(171) Pilet, P. E., 380,386(14), 387(14) Piloty, O.,175,178 Pm, R., 200 Piret, P., 222 Pirue, R. P., 508 Pitt, B. M.,450 Plattner, P. A., 150 Pletcher, D.E.,455(110),456 Pogosov, Yu. L.,433, 434(6),446 Pohl, R.,382 Polglase, W.J., 116, 142(68) Poliakova, L. A., 69 Poljakoff-Mayber, A., 409 Pollmann, W.,448, 449(85) Polonsky, J., 188,189(213,276),190(273), 198(277),199(273,277) Pon, G.,187 Porejko, S., 239 Porete, R., 502, 503(269),507(269) Portar, C. R., 95, 102, 126, 127, 128(2), 130(23), 140(23,24), 141(2) Porter, W. K., 400 Portsmouth, D.,173, 185,187(257,258) Posternak, T.,164 Powell, R. G.,397 Prawd, R. B.,425 Pratt, J. W.,159,160, 167(106) Prelog, V., 190 Preobraehenskii, A. I., 435, M(18) Pmcott, J. F., 124, 133(110), 343 Prey, V., 270 Pridham, J. B.,414, 443, 444(64) Pringsheim, H.,268, 478,498(208) Prins, D. A., 148, 150, 152, 159(36), 160(36, 58), 176, 304 Prins, W., 480, 481(213, 214), 48!!(213, 4141, 4x3 (213), 484(213), 485(214), 480(213), 492(214), 493(213), 496(214,217),496(213,214),503(213)

529

Pritchard, J. G., 130, 131(136) Pritchard, R. A., 168,193(139) Prochornik, R. A., 406 Prochownick, V., 462 Prokof’eva, M.,259 Prokop, J., 164 Proshlyakova, V. V.,184 Pryanishnikov, A. A., 171 Przylecki, S. J., 251 Puchta, R., 449,453(99),492(99), 496(99) Puddington, I. E.,467 Purves, C. B., 102, 125, 126, 128, 129, 130(113, l32), 139, 140(28), 141(113) Purves, W.K.,382 P u s h , I., 300,301(66b), 303(66) Pyle, R. E.,104

Q Quin, J. P., 223,246(59), 247,251(59), 255 Quinn, E. J., 442, 443(55), 479(55), 480, 484(215), 485(215), 486(215), 490

R Rabe, A., 289 Raistrick, H.,139 RajBhandary, U. L., 332, 342(65), 355, 357, 359(89) Ramaiah, N. A., 224, 225, 226(60), 229, 230(60) Rama Rao, M.V., 423 Randell, H.M.,197 Randle, P. J., 416 Rankin, J. C.,469 Rao, E.V., 355 Rao, V. S. R., 501 Raphael, R. A., 157 Reschig, K.,17, 173,176 Raymond, A. L.,102, 103(21), 106(21) Rea, H.E.,395 Rebers, P. A.,355 Rebstock, T. L.,398, 403 Recondo, E.,156 Reed, R. I., 39, 40, 54, 81(20), 84(20), 90,Ql(8) Reeder, W.H.,111, 133, 134(143) Rees, C . W.,130,131(135) Rsese, C. B.,187 Iieeue, E. T.,503 Reeves, R. E., 125, 130, 171, 181(147), 253,254

530

AUTflOlt INDEX, VOLUME

Regna, P., 168 Reichstein, T., 144, 145(2, 4), 148, 160(2), 151(2), 159, 171 (2, 4), 172(2), 173(2), 175, 176(184), 178, 180, 183(2, 4), 188(2), 197(2), 198(331), 199(331), 274, 275, 276, 278, 279(15), 280(15), 282(15), 28.5(15), 286(15, 33), 287, 288, 294(38), 308, 311(5), 313, 314(85), 320(15, 33, 38)'321(38) Reid, W. K., 40, 91(8) Reinhardt, M., 433 Geinhold, L., 381, 385(23) Reist, E. J., 115, 136, 142(65), 169, 177, 178, 179 Rembarz, G., 153, 160, 190 Rendleman, J. A., Jr., 217(36), 218(36), 219(36), 220, 222(36), 223(36), 226(36), 227(36), 230(36), 233(36), 234(36), 235(36), 242, 243(96), 244(96), 245(96), 248(96), 249(96), 255(96), 250(96), 257, 258(96), 159(96), 260(96), 261(96), 262(96), 263(96), 264(96) Rennie, R. A. C., 160, 161, 162(107), 163(107), 164(107), 166(107, lll), 168(111), 192(107), 198(107), 200(107), 201(111) Reppe, W., 269 Reyle, K., 276, 278(15), 279(15), 280(16), 282(15), 285(15), 286(15, 33), 287, 320(15, 33) Reynolds, D. D., 275, 277(8), zS2(8), 292, 456 Rhind-Tutt, A. J., 475 Ithodes, A,, 393 Itice, F. A. H., 443,444(63) Richards, C. N., 153, 164, 166, 156(81), 162 Hichardmn, A. C., 167, 191, 192(134) Richie, G. G., 243(100), 244(100), 246, 247(100), 248(100), 268(100) Richtmyer, N. K., 151, 152(54), 159, 160, 167(106), 176, 178, 182(198), 186(54), 304 Ricketts, c. I<., 462, 463(138, 140), 41)7(138),506, 508 Riden, J. R., 404 RiechetA.! 1Ht2 Higaud, L.,h71 Riggs, G. M., 194 Riley, L. S., 367

21

Rinderknecht, H., 156 Rinehart, K. L., Jr., 196 Ride, IX, 399 Riet, C. E., 267, 469, 485 Rizvi, 8., 511, 612(312) Roberts, H. J., 433 Roberts, P. J. P., 457 Roberts, W. K., 356 Robertson, H. J., 151, 152(53), 186(53) Robins, M. J., 194 Robins, R;K., 149, 194 Robinson, E., 416 Robinson, G. M., 413 Robinson, R., 413 Robinson, R. A., 240 Itochmis, P. G., 429 Rodionova, E. P., 124 Rogers, B. J., 401, 402 Rogers, H. J., 325, 368, 374(111) Rogovin, Z. A., 269, 284, 433, 434(6), 446

Romano, H. H.,363 Rona, P., 239 Roamri, G., 191 Rosanoff, M. A., 10 Rosenfeld, D. A., 176, 182(198) Itosenfeld, M., 217(39), 220, 258(39) Row, J. H., 478 Rosselet, J. P., 279, 288 Roth, W., 407 Rowe, C. E., 402, 463(140), 506 Rowe, J. J. M., 442, 443(60), 446(50), 479(50) Rowell, R. M., 136, 475 Rowmll, E. V., 418 Roxborgh, C. M., 157 Roy, C., 414 Rubenwtein, B., 429, 430 Ruckel, E. R., 431, &(la), 486(221b), 488(2!21), 495(221), 503(la) Rude, E., 190 Ruell, D. A., 126 Ruff, O., 153, 177(67) Ruge, U., 381 Rii-Jen Lee Han, 161, lSe(113) Riindle, R. E., 216(32), 220, 236 RuHsell, C. R., 267 Rutherford, D., 270, 433 Rutherford, P. P., 394 Rutloff, H., 442,443(51), 446(51) Rutz, G., 306

AUTHOIi INDEX, VOLUME

21

531

Ryan, K. J., 141, 169, 179, 193(142), Schmid, H., 152, 177(59) 200(142), 201(233), 202(14!2), Schmid, L.,269 Schmid, M.D.,157 203(142) Schmid, W.,274 Ryder, A., 195 Schmidt, E.,305 Rydon, H.N.,165, lSl(129) Schmidt, H.W.H., 167,202(135) s Schmits, E.,182 Schmuts, J., 175, 181(179) Sacher, J. A., 424 Schneer, R. Ta.,479, 483(209),484(209), Saegusa, T.,488 485(209), 486(209), 488(209), Saeki, S., 290, 291, 292(46), 296(466), 502(209) 299,313, 319(65,86),320(46,86) Schneider, G.,420 Sahi, R. D., 425 Schneider, W.. 114,115(61), 128, 201 Saito, H.,464 Schnoes, H.K.,40, 46, 59(16), 61(7, 16), Sakurada, I., 243(103),245 62(16), 63(16), 66(16), 71(16), 204 Salatnikov, N. N.,233 Schofield, R. K., 239 Saltman, P.,210,212, 213,233 Schotte, H.,149,297,301(56),302(56) Salton, M.R.J., 171,325,326 Schramm, G.,448, 449(85) Samantarai, B.,387 Schreiber, K.,420 Samaritano, R. H.,125 Schroeder, W.,139 Samborski, D.J., 399 Schubert, H.S., 441 Samueh, G.,426 Sanderson, A. R.,326,355, 357, 359(89), Schuerch, C., 431, 437, 439, 480(24), 481(213), 482(213), 483(24, 213), 360 484(24, 213, 215), 485(1a, 215), Sandford, P. A., 498 486(24, 34, 213, 215, 221b), 488(34, Sapegina, E.A., 435 221), 489(34), 490(34),491,493(213), Sarasin, J., 477 495(24, 221), 496(24, 213), 497(34), Sargent, L.J., 362 503(la, 213) Saric, S. P.,239 Schukow, I., 246 Sarkanen, K.,490, 491 Schwarz, J. C.P., 149 Sarkar, B., 213 Schweizer, E.E.,402 Scanlon, J., 238 Scattergood, A., 113, 128(54), 140(54), Schwenker, R. F., 258,268(145) Scott, K.J., 390 141(54) Seeliger, A., 164 Schaal, R.,239 Seligman, A. M.,121, 138(80), 141(80) Schade, W.,182 Selix, M.,213 Schlifer, W.,i82 Sell, H.M.,387, 393, 398, 403 Schaefer, W.C.,485 Scharmann, H.,40, 54, 60, 61(19, 25), Selman, L. H., 475 Sembdner, G.,420 69(19), 93 Schaub, R. E.,123, 141(98),439,449(30), Semple, R.E.,494 Seno, N.,464 458(30) Senti, F. R.,216(29), 220, 221, 222(29), Scheibler, C.,442,443 224, 236, 243(97), 246, 249, 250(29, Schellenbaum, M.,155 97), 251(97), 255(97) Scherer, P. C.,270 Schindler, O.,197, 198(331), 199(331) Sepp, J., 114,115(61), 128 Sequeira, J. S., 130, 131(135) Schinle, R.,157 Sergeev, V. A., 479, 483(209), 484(209), Schlags, R.,239,240(83) 485(209, 210), 486(209), 488(209), Schlossman, S.F.,506 Schlubach, H.H.,212, 182, 443, 444(61), +502(209) Sergeyev, V. A., 479,485(210) 462(61),498(61) Setterfield, G.,383, 385(35) Schmalz, K.,478,498(208)

532

AUTHOR INDEX, VOLUME

Shabarova, Z. A,, 333, 364 Shafikova, P. h., 364 Shafiradeh, F., 98, 97, 107, 119, 128, 135(5), 138, 476, 477(195, 197), 491 Shah, N. M., 285 Shannon, J. C.,388,393 Shantarovitch, P.,175 Shantr, E.M.,391, 412 Sharkov, V. I., 221,448 Sharpe, M.E.,324 Shasha, B., 199 Shattock, P. M. F., 366, 367(106), 368(106) Shaw, D.R. D., 372 Shaw, G,, 125 Shaw, M.,399 Shaw, N.,347, 350(81) Shear, M.J., 510,511 Shecter, H.,97 Sheft, I., 475 Sheinker, V. Sh., 64, 71 (29),72(29) Shemyakin, M.M.,184, 185(251), 186 Shibata, M.,184 Shimabukuro, R. H.,401 Shimidate, T,,121 Shimo,K.,265,270(161),271(151) Shinohara, M., 184, 185(255, 256), 187(255,256), 203(256) Shockman, G.D.,323, 32S,326(4), 366(4), 367(4,27, lO5), 368 Shoppea, C.W.,273, 292(2) Shorigin, P.,270 Shunk, C.H.,139,176 Sidgwick, N.V., 265 Silberger, J., 388 Silin, P.M.,435 Silver, B. L.,329 Sirnonov, A. P.,223 Sinclair, H.B.,182 Skarnes, R.C.,510 Skinner, J. C.,426 Skok, J., 413 Skoog, F.,388,416 Blade, IT. D., 323, 326(4), 366(4), 367(4, 105)~S B H ( I ~ T , ) Slomp, G.,174 flmirnova, G.S.,264 Sminiova, M.G.,446 Smith, I). C. C., 108, 1nr,(33), 140(83), 154,155

21

Smith, D. G., 366,367(106),368(106) Smith, D.H.,411 Smith, D.W.,197 Smith, E.E.,507 Smith, F., 104, 113, 133, 136, 141(56), 199, 484, 470, 471, 474(185), 495, 498(185),501(185) Smith, F. G., 386 Smith, G.,139 Smith, H.H.,390 Smith, J. A., 420 Smith, J. A. B., 102,138, 467 Smith, N.L.,267 Smith, O.,398 Smith, R. H.,216(33), 220 Smith, R. M.,192 Smith, S.,279 Smith, W.G.,399 Smolehaki, K.,239,253 Snatzke, G.,299 Snedden, W.,39, 54, 81(20),84(20) Snell, J. B., 434 Snover, J. A., 168, 169(138) So,L.L.,507 Sobotka, H.,173 Sorasuchart, P.,820 Sorkin, E.,151 brrn, F., 145 Soubeiran, E.,244(111),245 Souchay, P.,239 Sowa, W.,164 Sowden, J. C.,146, 147, 149(30), 150(30), 153,291, 304,443 Sparrow, D. H. B., 418 Specht, H.,418 Spedding, H.,497 Spencer, J. F.T., 139 Spencer, M.,97 Spencer, R. R.,169, 177, 178 Spiegelberg, H.,125 Spingler, H.,299,313,314(83) Sporrs, J. W.,136 Spriwterbach, D.,104,464 Spriggu, A. S., 443 Spring, F. G., 157, 158(94) Hpringer, G.F.,172 Hrivastavrt, I). P., 425 Stacey, M., 114, 124, 130(60), 135(60, 104), 136, 138, 139, 144, 145(6, 7), 146(7), 147(6), 149, 153, 154(23),

AUTHOR INDEX, VOLUME

158, 162(98), 179, 185, 187(25), 191, 192(291), 193, 194, 196(309), 197(309), 275,309,342,343,470,476, 477(195, 197), 491, 508 Starker, A., 446 Stafford, F. E., 130, 131(136) Stahl, E., 199 Sbngk, J., 114, 130(60), 135(60), 149, 166, 171, 172(146), 176, 183(146), 197(329), 198(133), 443 Stanier, R. Y., 325 Starchenkov, Yu. P., 419 Stark, K.-H., 136 Staub, A. M., 187, 188, 189 Steel, B. J., 214 Steinhart, C., 416 Steinpreis, R., 278, 289(19) Stenhouse, J., 217(37), 220 Stenlid, G., 411, 414(216) Sternbach, L. H., 172 Stevens, C. L., 194, 195, 196, 197(325), 199(325), 200(325) Stevens, V. L., 394, 407(109), 408(109) Stevenson, C. D., 430 Steward, F. C., 391-412 Stierlin, H., 188, 189(270) Stiller, E. T., 136 Stirm, S., 188, 189(272) Stitt, C. F., 213 Stodola, F. H., 417 Stoffyn, P. J., 348 Stokes, I. E., 427, 428(338, 339) Stokes, J. M., 214 Stokes,R. H., 214 Stork, G., 417 Stout, M., 399 Stowe, B. B., 411, 417 Std’chunas, L. I., 124 Strobach, D. R., 146 Strominger, J. L., 326, 360, 368, 369, 370(116), 373(30, 31), 375(132) Stross, F. H., 469, 495(173) Stuart, N. W., 385, 386(52) Studer, P., 308 Subba R+o, B. C., 168, 169(138) Subba Rao, M. S., 425 Hugihara, J. M., 257, 262(139), 267(139) 436, 449(23), 470(22) Suhadolnik, R. J., 158 Sultori, J. E., 187

533

21

Sumner, J. B., 507 Sundaralingam, M., 99, lOO(17) Surna, Ya.A., 479, 485(210) Susbielle, H., 210 Suzuki, s., 133, 44s Sveahnikova, M. A., 184 Swain, C. G., 439 Swain, T., 413 Swan, B., 263 Swan, E. P., 433 Switzer, C. M., 388, 408(70) Szab6, L., 153, 154, 158 Szab6, P., 158

T Tabone, D., 391 Tabone, J., 391 T d a , R., 265, 270(151), 271(151) Taha, M. I., 117, 433 Tajmr, L., 176 Takahashi, D., 421, 423(295) Takahashi, N., 385 Talalaeva, T. V., 223 Talley, E. A., 275,277(8), 282(8), 292, 456 Tamm, C., 144, 145(5), 171(5), 183(5), 197, 198(328), 199(328), 274, 279, 286, 187, 288, 294(38), 308, 312, 320(38), 321(38). Tanabe, K., 184 Tanimoto, T., 421, 423(295) Tanret, C., 246, 251(117), 477 Taube, C., 175 Taufel, K., 442,443(51), 446(51) Taylor, B. M., 393 Taylor, E. C., 149 Taylor, I(.G., 195 Taylor, N. F., 194 Taylor, P. M., 503 Taylor, T. J., 156, 178 Teague, R. S., 291 Tedemhi, R. J., 238 Teem, E. G., 153 Tegge, G., 211, 212,217(12, 40),220 Tener, G. M., 295 Thacker, D., 100, 111, 120, 131(49a) Thansen, J., 239 Theander, O., 127, 164 Theobald, R. S., 433,448,497(84), 502(84), 506(84), 508(84)

534

AUTHOIi INDEX, VOLUME

Thibon, H., 213, 240 Thimann, K. V., 379, 386,387,388 Thomas, E. W., 397 Thomaa, G . H. S., 164 Thomas, H. J., 122, 123, 139(94) Thomas, R. G., 411 Thompson, A., 31, 115, 116, 123(70), 442, 443(54, 55), 469(54), 470(54, 176), 479(54, 55), 480(176), 482(211, 212) Thompson, J. L., 168, 1[)3(139) Thomen, T., 253 Thurkauf, M., 186 Tieszen, 13. V., 267 Tinelli, R., 187, 189 Tio, C. O., 177, 289, 209(44) Tipper, D. J., 369, 370(116) Tipson, R. S., 122, 139(91), 152, 177(60), 193(60), 195, 215, 270, 271, 284, 285(31), 315, 486, 467, 497(156), 502(150), 512(156) Tjelveit, 0. J., 104, 105(43), 128(43), 129(43) Toda, T., 197 Todd, A. R., 124, 135, 168, 159, 329, 416 Todd, E. H., 427, 428(338, 339) Toenniea, G.,325, 387(27) Tollens, B., 216(33), 220, 244(107), 245, 240(107) Tomimatau, Y., 495 Tomilrawa, C., 404 Tong, G. L., 194 Torii, M.,32S, 360, 362(93), 373(93) Towers, G.H. N., 399, 409,414 Toy, M. S., 433 Traube, H., 217(38), 220 Treadway, R. H., 433 Trendelenberg, E., 38 Trenner, N. R., 158 Trip, P., 385 Trischmann, Ii., 408 Trister, 8.M.,113 Troyer, J. R., 413 Teareva, G. V., 223 Tschesche, R.,273,292(2), 299 TROU, K.-C., 121, 138(80), 141(80) Tmchiya, H. M., 46R Tu, C., 485, 488(221), 495(221) Tucker, H., 269 Tucker, L. C. N., 179

21

Turner, W. N., 194 Turton, C. N., 443,444(67) Twigg, G. D., 448, 497(84), 502(84), 5 w w , 608(84)

U Ugo, R., 213 Uhle, F. C., 276, 277,279(12), 280(12, 13), 281, 28a(ia, la), 2 ~ 4 1 2 )2~~ ( 1 2 ) , 312(12, 13), 319(12, 13), 320(12, 13), 321(13) Ukholina, R.S., 184 Ukita, T., 146, 154(24) Ulrich, P., 186 Umezawa, K., 172, 202(161) Unrau, A. M., 138, 147, 437, 438, 468, 471, 472(25), 474, 482(25, 29, 182), 484(25, 29, 1821, 495(29), 497(182), 498(25), 499, 601(182), 503(25) Urbaa, B., 136 Uribe, F. G., 405 Usher, D. A., 331 Usov, A. I., 74, 92(33), 165, 167(130), 181(130), 192(130) Usteri, E., 508 Utkin, L. M.,218(48), 220

V Vail, E. I., 233 Valentin, F., 172, 175 Valiavwdan, G. D., 278, 279(23), 289(23), 290(23), 291(23), 296(23), 316(23), 319(23) Vallance, L. G., 425 van Bemmelen, J. M., 433 VanCleve, J. W., 485 van Dam,J., 482, 495(217) van der Kerk, G. J. M., 404 van der Vean, R., 405 Van Es, T., 475 Van Home, C., 390 van Meemche, M.,222 van Overbeek, J., 381 van Tamelen, E., 174 Vargha, L., 149, 181 Varma, H. P., 427, 428(341) Varma, 8.C., 425

AUTHOR INDEX, VOLUME

l-ariier, J. E., 418 Vaughan, G., 136, 150 Vavrinecz, G., 219(51), 220,228, 254 Venner, H., 146, 154, 198(74) Venugopalan, M,, 62, 204 Venus-Danilova, E.,175 Verheijden, J. P., 113, 140(51) Vermillion, G., 394 Verner, G., 161 Vernet, H., 467 Venion, C. A., 329, 475 Vink, H., 248 Virtannen, A. I., 392, 407 Vis, E., 121, 133(85), 141(85), 161 Vischer, E., 176 Vishnu, 224, 225, 226(60), 229, 230(60) Vogel, H., 210 von Bebenburg, W., 157 von Harnack, A., 38 von Lippmann, E. O., 210 von Weinberg, A., 38 Votohk, E., 172, 175, 177

W Wadman, W. H., 103, 104, 464 Wachneldt, T., 184, 195, 198(324) Wagenitr, E., 182 Wahlroos, O., 407 Wain, R. L., 394 Waine, A. C., 102, 130(23), 140(23) Waisbrot, S. W., 115 Walker, A. C., 404 Walker, D. R., 410 Walker, H. G., Jr., 126, 138(116) Walker, J. F., 450 Walker, R. W., 158 Wall, R. A., 210 Wallace, A., 394 Wallenfels, K., 401, 498, 506 Walsh, J. P., 216(30), 220 Walston, W. E., 104, 105(35), 141(35) Waiton, K. W., 508, 509 Walz, D. E., 117 Wang, IT., 419 Wmg, Y.,177 Ward, P. F. V., 166, 198(131) Ward, R. B., 477, 479, 482(211, 212) Wareing, P. F., 410, 412(214) Warren, C. I)., 127

21

535

Warrener, R..N., 125 Waschkau, A,, 269 Watanabe, H.,497 Watanabe, R., 413 Watkins, W.M., 103, 141(34) Watson, D. W.,510 Watters, A. J., 210, 216(3), 217(3), 219(3) Wax, J., 173 Webb, J. I., 102 Webb, J. L., 408 Webb, J. M., 188, 189(271), 194(271) Webber, J. M., 191, 192(291), 194, 197(310)

Weber, A., 250, 261 Wedding, R. T., 387 Wedemeyer, K. F., 278 Weeks, I. A., 214 Wehrli, W., 173 Wehrmiiller, J. O., 433 Weibull, C., 325 Weidemann, G., 199 Weigel, H., 127 Weigher, E., 103, 105(29), 140(29) Weiland, J., 420 Weis, K., 278, 289(19) Weisblat, D. I., 115, 117 Weiss, E., 144, 145(2), 150(2), 151(2), 171(2), 172(2), 173(2), 183(2), 186(2), 197(2), 275, 311(5), 313, 314(85) Weirmann, C., 238 Weller, L. E., 393, 398 Wells, A. F., 241 Wempen, I., 122, 139(93) Wender, S. H., 413 Went, F. W., 377 Wertz, J. E., 238 Weswls, J. S. C., 405 Wessley, K., 177 Westphal, O., 144, 187(8), 188, 189(8, 270, 271, 272, 276), 190, 199(8) Westphal, W., 176 Westgarth, G. C., 126 Weston, E. Vy., 394 Westwdod, J. H., 191, 192(291) Weygand, F., 123, 135, 156, 157, 163 Whelaa, W. J., 138, 174, 267, 433, 441, 442, 443(56), 457, 458, 479(56), 497, 503 Whistler, R. L., 136, 170, 199, 218(49), 220, 433, 467, 475

536

AUTHOIt INDEX, VOLUME

Whitehouse, M. W., 113 Whitely, T. E.,168, 169(137), 170(137), 198(137), 201(137), 202(137) Whittenberger, 11. T.,386 Wicken, A. J., 323,931, 384(63),344(08), 385,366(3, 63) Wieghard, C. W., 328 Wieland, T., 333 Wieae, A. F.,395 Wigert, H.,194 Wiggim, L. F., 138, 153, 158, 162(98), 192,433,476 Wightman, F., 394 Wiklund, O., 211, 212(13), 219(13), 223, 224,225, 228(13), 230 Wilcoxon, F., 378 Wild, G.M.,457 Wiley, P.F., 191 Wilham, C.A.,469 Wilkie, K. C. B., 494 Wilkinson, J. F.,139 Wilkinson, R.E.,402 Will, H.,246 William, A. K.,401 William, D.E.,216(32), 220,238 Williams, D. H.,39, 40, 41(3), 44(10), 47,63(10), 201 William, J. H., 123,141(98),439,449(30), 458(30) Williams, L. G., 409 Williams, N. R.,40, 201 Williamson, P.,172 Wilson, C.M.,388,393 Wilson, D.W.,430 Wilson, E. J., Jr., 115, 130(84), 142(84) Wilson, J. M.,40, 64,81(20), el@) Wilson, M.F.,238 Wimmer, E.L.,248 Winkler, S.,277 WinHtein, S.,58, 458 Winter, H.,246 Wirta, S.,299 Wiwmann, II., 448 Witnauer, L. P., 216(29), 220, 221, 222(29), 224, 236, 243(97), 245, 248, 249, 250(!!9, 07), 251 (97),255(07) Wittenburg, E., 146 Wittwer, 8. H.,398 Wohl, A., 10, 17, 177,442 Wolf, A., 104

21

Wolf, D.E.,394 WoM, I. A., 508 Wolfrom, M. L., 18, 31, 116, 116, 117, 123(70), 128, 143(68, 69, 71, 72, 73), 156, 157, 168,169(88, 137), 170(137), 177, 178, 180, 181(236), 193(236), 198(137), 200, 201(137), 202(137), 206(2W), 257, 262(139), 267(139), 433, 442, 443(54, 55), 455(110), 456, 469(84), 470(54, 176), 479(54, 55), 480(176), 482(211, 212), 497 Wolters, W., 251 Wols, H., 135, 156, 163 Woo, P. W. K., 173, 179(168), 191, 203(290) Wood, D. L., 196, 197(325), 199(325), 200(325) Wood, H.B.,Jr., 121,164, 480 Wood, J. W., 138,488,469(168), 470(175), 471(168, 175), 472(174), 474(174), 475(174), 492(168, 175), 493(168, 175), 495(168, 175), 496(168), 497(168), 498(175), 603(168, 174), 508, 512(290) Woodoock, D., 398 Woolf, D.O.,Jr., 195 Wort, D. J., 388, 388,393, 396(69) Wright, D.,409 Wright, K., 145 Wright, R.S.,295 Wright, 8.T.C.,421 Wlilfing, J. A., 217(41), 220 Wulf, G.,154, 163(77) wulff, H.,458 Wulfson, N. S., 40, 46, 47(6), 56(13), 57(13), 59(13), 61(6, 13), 71(13), 79, 80(35),84,89 Wyler, R., 155 Y

Yakushkina, N. I., 394, 419 Yamaki, T.,417 Yang, S.,430 Yanovsky, E.,433 Yatea, R.A., 423, 425(302),426(302) Yazlovetsky, I. G., 461 Yi, W., 177 Yomo,J., 418 Ywiaawa, Z., 116, 142(69, 71,72)

21

537

%PllIISOII, b’.,

449, 451 (!Id), 4&2(94),

AUTHOR INDEX, VOLUME

Yoiiiig, B. G . , 138,469,470(175), 471(175), 492( 175), 493( 1751, 495 (175), 498(175), 509,510(299), 511,512(310, 311, 312) Young, F. E., 370 Young, R. J., 174 Yovanovitch, O., 244(108, 109), 245, 249 Yung, N., 123, 146 Yungsten, H., 174 2

46’3(94)

Ziemann, H., 123 Zika, M., 386 Zimmermaii, P. W., 378 Zinner, H., 115, 116(66), 136, 142(66), 146, 154, 163(77), 177, 190, 194, 289

Zissis, E., 178 Zolotarev, B. M., 40, 46, 47(6), 48,52(17), 56(13), 57(13), 58, 59(13), 61(6, 13), 64, 67, 71(13), 17, 29, 30), 72(17, 28, 29, 30), 81, 204

Zach, K., 175 Zachau, H. G., 333 Zahner, H., 190 Zakhar’yants, I. L.,419 Zambito, A. J., 445, 493(72), 509(72) Zane, A,, 413 Zaretskaya, M. Z., 364 Zarnitz, M. L., 506 Zechmeister, L., 435 Zemplh, G., 258, 280, 441, 444, 456, 457(35), 464

Zenk, M. H., 390, 391, 392

Zorbach, W. W., 123, 146, 147, 177, 186, 278, 279(22, 23), 280, 289(23), 290(23), 291(23), 292(46), 295 296(23, 466), 297, 299, 301(26, 61), 302(26, 64), 304(26, 57), 305(67), 306(67), 308, 309(44), 313, 316(23), 319(23, 26, 53, 64, 65, 67, 86), 320(46, 53, 86) Zust, A., 155 Zwar, J. A., 420 Zweifel, G., 125, 168, 169(138) Zweig, G., 405, 406

SUBJECT INDEX FOR VOLUME 21 Act,iiio~pc:loac,1I) 1 Acyl migration, 24 Abequose, 187, 189, 190 Adenine, 9-(6aeoxy-p-~-eryfhr~he%-~nAbscission, 429 ulofuranosy1)-, 174 Acetals -, 9-(2-deoxy-~-arabinoexopyranosyl)-, aldofuranonide formation from, 111 .in cancer chemotherapy, 145 wdeoxy sugttrs by catalytic reduckion -, 6-(2-furfurylamino)-, as plant-growth of, 176 substance, 420 of monosaccharides, mam epectra of, 74 -, I)-glUCOSyl-,23 of polyhydric alcohols, nxw spectra of, Adonitol, 17 79 Agricultural chemistry, carbohydrate Acetic acid chemistry and, 378 -, chloro-, IW catalyst for polymerisation Agronomy, carbohydrate chemistry and, of 1 ,&anhydro sugars, 486 378 -, (4chloro-2-methylphenoxy)Alanine, 3-(3-amino-s-trial;olyl)-,~,401 effect on sugarcsne, 427 Alcoholatw as herbicide, 392 carbohydratselkali metal complexes, -, (2-chlorophenoxy)-, as plant-growth 258 substance, 398 csrbohydratea, preparation of, 263 -, (3-chlorophenoxy)-, aa plant-growth Alcohols, polyhydric, m&89 spectra and substance. 398 stereochemistry of, 79 -, (4chlorophenoxy)-, aa pbnt-growth Alditols substance, 398 acetates, ma88 speatra of, 89 -, (2,rklichlorophenoxy)-, compounds with acetone, 19 effect on sugarcane, 424 with benraldehyde, 19 as herbicide, 392 dideoxy, 189 metabolism by plant tiwue, 396 electrophoreais in metal salt solutions, -, (3,5dichlorophanoxy)-, ~ F I herbicide, 232 394 Aldofuranosidea -, t,rii!hloro-, nn plant-growth subwtance, from furanose esters and halides, 121 402 hydrolysis of, 129 -, (2,4,&trichlorophenoxy)by alkali, 137 effect on abscission and ripening, 429 mechanism of formation of, 117 on Rugarcane, 424 phenyl, melting points and specific opa8 herbicide, 392 tical rotations of, 141 Acetic acid-’%’, (2, Qaichlorophenoxy)-, rate of formation of, 106 metabolism by plant tissue, 396 synthesis of, 96 Acetone, compounds with sugars and Aldohexofuranoaides alditols, 19 conformation of, 100 a-Acritol, see DkMannito1 oxidation of glycol groups in, 133 a-Acrose, Bee Fructose Aldonic acids, epimerisation of, 11 PAcrose, 8ee Sorbose Aldopentofuranmidea Actinomycetes, teichoic aoid from, 363 formation of, 109 638 A

lf

SUBJECT INDEX, VOLUME

methyl, cwnformations of, 99 oxidation of glycol groups in, 133 Aldopyranosides, hydrolysis by alkali, 137 Aldopyranotiyl fluorides, polyglycoses from, 460 AldoseLl condensation polymerization of, theory of, 468 D series, 9, 11 mass spectra of, 66 reaction with thiols, 19 Aldono-1 ,Slactones, as plant-growth substances, 416 Aldotetrofuranoaides, oxidation of glycol groups in, 132 Alkali, effect on aldosides, 137 Alkali metal alcoholates carbohydrate complexes with, 260 of carbohydrates, structure of, 265 Alkali metal alkoxides, reactions with carbonydrates, 258 Alkali metal amidea, reaction with carbohydrates in liquid ammonia, 269 Alkali metal-carbohydrate complexes, 209-27 1 electrophoresis, 231 preparation of, 216-220 solvation of, 226 stability of, 227 stoichiometry of, 222 structure of, 236 Alkali metal hydroxides, carbohydrate compledes with, 238, 243, 244, 245, 256 Alkali metah, reaction with carbohydrates in liquid ammonia, 269 Alkali met,al salts, effect on specific rotations of Rucrose atid D-fructose, 229 Alkaliiie-earth metal-carbohydrate complexw, 209-271 electrophoresis, 231 preparation of, 216-220 solvation of, 226 stability of, 227 stoichiometry of, 222 structure of, 236 Alkaline-earth metal hydroxides, carbohydra@ compiexes, 246, 255

21

539

Alkaliiir-curth metnls, ioiwt i o i i with carbtr hydratm in liquid aniwonia, 269 Alkaliiie-earth metal salts, effect 011 specific rotations of rctrbohydrates, 230 Allofuranoaide, nietliyl 6-deoxy-2,3-0-isopropyhdene-D-, 178 - , 178, 180 Allose, ~ ~ ~ O X Y - D173, -, 6-deoxy-2,3-di-O-methyl-~-, 173, 179 -, 6-deoxy-2-0-methyl-~-, 173 -, 6-deoxyy-%0-methy1-D-,173 2,3,4,5,6-penta-O-acetyl-lAltritol, deoxy-1-nitro+-, 304 Altrose, 2-amino-2deoxy-~-, 18 -, ~ - ~ W X Y - D173, - , 176 -, 6deoxy-3-O-methyl-r.-, 173 Amicetin, 195 Amicetose, 195, 197 Amecetoside, methyl, 195 Amino sugars, 17 maas spectra of, 67, 93 Amitrole, aa plant-growth substance, 399 Ammonia, liquid, reactions of carbohydrates with alkali metals, alkali metal amides, and alkaline-earth metals, 269 Amygdalin, 22 Amylan, 446 Amylose, B-deoxy-, 174 Analysis, by mass spectrometry, 44 Angustmycin A, 174 Angustom, 174-176 Anhydro sugars 1,2-, 467 addition polymerization of, 477 2-deoxy sugars from, 150 3deoxy sugars from, 159 mass spectra of, 81, 93 reduction of, to d e o x y sugars, 176 Antigens, 3,6-dideoxyhexoses in artificial, 189 Apigenin 7-~-glucoside, as plant-growth substance, 410 Arabinofuranoaide, ethyl 2-acetamido-2deoxy-l-thio--&r.-, 116 -, ethyl l-thio-b-D-, 116 -, methyl WD-, 103 conformation of, 96 -, methyl FD-, conformation of, 98

540

-,

BUBJECT INDEX, VOLUME

21

methyl a-b, conformation of, 100 -, 2 , 3,&trichloromethyl tri-O-methyla,&L-, mats speceffect on sugarcane, 427 trum of, 63 as herbicide, 407 Arabinopyranose, 1 ,4-auhydro-2, .Mi-0- 1,CBenzoxazin3-one, 2,44ihydroxy-7methyl-r,-, synthesis and polymmethoxy-, ZD-glucwide, 407 erization of, 488 ’ Bioohemistry, stereochemistry and, 34 Arabinopyranoside, methyl tri-&methylBiological activity, of herbicides, chemP-D, ma8 spectrum of, 60 ical structure, physical proper-, methyl 2,3,Ctri-O-rnethyl-p-t, mass ties and, 408 Npectrum of, 48 Biosynthesis, of teichoic acids, 372 Arabiriom 4,4’-Bipyridinium bis(methy1 sulfate, D-, diethyl dithioacetal tetraacetate, 1 , l‘dmethyl-, effect on sugarmass spectrum of, 85 cane, 422 Boivinose, 186 -, 5-deoxy-w, 177 -, ~ - ~ ~ o x Y - L -176-178 , Boric acid, catalysis of polymerization by, -, 1,2:3 ,Cdi-0-isopropylidene-PI mass 466 spectrum of, 76 Boron compounds, in synthesis of deoxy -, l-O-(indole-3-acetyl)-, 391 &ugars,168, 169, 181 Ascaryloee, 188, 190 Botany, carbohydrate chemistry and, 378 Ascorbic acid, L-, as growth regulator, 391 Brigl’s anhydride, polymerization‘of, 490 Ascorbigen, 392 Bufadienolides, synthetic, 273-321 Astraglin, as plant-growth substance, 412 1-Butanol, reactions with carbohydrates Atrazine, as herbicide, 406 and sodium hydroxide, 257 Auxins, 378 C effect on respiration and metabolism in plants, 387 Calcium chloride on starch in plants, 386 complex with lsctose and methanol, 215 on sugarcane, 424 effect on equilibrium rotation of D glyce?0-n-guIo-heptJ231 Canaroee, L-, 186 B Cancrer chemotherapy, deoxy sugars in, BaciUua aubtilia, teichoic acid from, 350, 146,188 354 Carbamatea, as herbicides, 403 Bacteria, surfac! structures of Gram- Carbamic acid, N , N-dimethyldithiopositive, 324 1-thio-Bwglucopyranoside,404 Barban, as herbicide, 404 zinc salt, fungicide-herbicide, 404 Barium oxide, complex with sucrose, 213 -, N ,N-dipropylthio-, ðyl ester, as Benzaldehyde, compounds with alditols, herbicide, 403 19 -, N-methyl-, 1-naphthyl eater, effect on Benroic acid absciasion and ripening, 4% as herbicide, 408 Carbanilatea, as herbicides, 403 -, 2-0hlomJ aa herbicide, 408 Carbanilic acid -, 3-chloro-, as herbicide, 408 isopropyl eeter, t~ herbicide, 404 -, Cchloro-, ad herbicide, 408 -, 3-chloro-, %hydmxy-, me Salicylic acid Cchloro-2-butynyl ester, as herbicide, -, 3-hydroxy-, as lierbicide, 408 404 -, 4hydroxy-, aa herbicide, 408 isopropyl ester, as herbicides, 403 -, 2 ,3 ,6-triiodoCarbohydrate-alkali metal alcoholate comeffect on abscission and ripening, 429 plexes, 260 aa herbicide, 407 Carbohydrate-metal base complexes, 237

-,

SUBJECT INDEX, VOLUME

21

541

-, 38,,5,14p, 1%telr:rliy\.cirosy-,sre Carbohydrate-meta1 halt complexes Stmphanthidol electrophoresis, 231 -, 3@,5,1.1p-trihydrosy-, w c Periplooptical rotations of, 228 genin preparation of, 216-220 -, 3fl, 12@,14fl-trihydroxy-, 8ee I)iproof of exiateiice of, 21 1 goxigeiiin solvalioik of, 226 -, 3@,5,1@-trihydroxy-ls-Oxo-, see stability of, 217 Strophanthidin stoichiometry of, 222 Cardenolides structure of, 236 furanoid, 309 Carbohydrates mercuric cyanide in synthesis of, 278 complexes with alkali metals and molecular weight determination, 274 alkaline-earth metals, 209-271 physiologicd activity of, 311, 314 effect of plant-growth substances on, synthetic, 273-321 377-430 properties of, 319-321 electrophoretic migration of, 234, 235 Carboxylic acids, a-substituted, configura- Cardiac glycosides, synthetic, 273-321 Cardiotonic activity, 274 tion of, 33 of cardenolides, 311, 314 58-Carda-8(14), 20(22)dienolide, 38-0(2-deoxy-ct-~-arabhexopyran- Catalysis, of polymerization of sugars by boric acid, 466 08yl)-, 280, 302 by hydrogen chloride, 462 -, 3&hydroxy-, 279 by ion-exchange resins, 464 5&Carda-l4,20(22)-dienolide, 38-hyCatalysts droxy-, 279 for glycofuranoside formation, 104 -, 3&0-(cu-~rhamnopyranosyl)-,289 for polymerization of sugars, 452 5@-Card-20(22)-enolide, 3&0-(2deoxyj3-D-ribo-hexopyranosy1)-, 148- Cellan, 445 Cellobiose, 30 hydroxy-, 306 -, 3@-(2, 6-dideoxy-a-D-ribo-hexopyran- Cellulose polymerization in hydrogen fluoride, 445 osyl)-14&hydroxy-, 299, 301, 312 reaction with alkali metal alkoxides, -, 3&0-(2,6dideoxy-&~-n'bhexopyran257, 258 0~~1)-14@-hydr0~~-, 301,299, 308 -, 3&0-(2,6-dideoxy-3-0-methyl-j3-~- Cell wall bacterial, teichoic acids in, 324 tibo-hexopyranosyl)-5 ,14g-dihyplant, effect of plant-powth substances drOXy-ls-OXo-,293 -, 3@,14g-dihydroxy-,273 on, 381 Cell-wall polysaccharides, effect of auxins -, 3p, I Odi-O-(a+rhamnopyranosyl)5,14&dihydroxy-, 294 on, 381 --, :~jj-O-(p-~-glucopyranosyl)-5,14p, 1% Chalcomycin, 173, 179 trihydroxy-, 288 Chalcose, 191, 192 -, 14p-hydroxy-3-oxo-, 306 Chartreusin, D-fucoue from, 172 Chebulinic acid, 27 -, 3j3-O-(cr1.-mannopyranosyl)-5,148dihydroxy-19-oxo-, 292 Chelation, in carbohydrate-metal base complexes, 237 -, 3p-O-(~~-rhrtmnopyranosyyl)-5,148dihydroxy-19-oxo-, 290 Chinovin, 6-deoxy-~-g~ucose from, 173 -, 3,%O-(a-~-rhamnopyranosyl)-14&hy- Chinovose, 17 Chromatography, of deoxy sugars, 197-199 dwxy-, 290, 296 -, 3&0-(&~-r~mnopyranosy1)-148-h~- Chromomycin, 184, 185 Chromose A, 184, 185, 187 droxy-, 296 Chromose B, 185 -, 194-(a-1.-rhamnopyranosyl)-38, 5, l@trihydroxy-, 294 Chromose C, 186, 186, 308

542

SUBJECT INDEX, VOLUME

Chromom D, 185, 187 Cinnamic acid, tram-, EM plant-growth substance, 409 Colitose, 188,190 Complex compounds carbohydm%ahli metal, 209-271 carbohydrate-dkaline-earthmetal, 209271

Concanavalin A, 507 Conductivity (electrical), effect of carbohydratecomplex salt formation on, 213 Configuration of monoses, 8, 13, 15 of mubstitiited carboxylic acids, 33 of tartaric acids, 32 Conformation of aldopentofuranosides,99 of glycofuranoddes, 96 Convallatoxin Byntheais of, 278, 282, 285 -, &hydroxy-, 292 Convolvulin, D-fucose from, 172 Corchaularose, 106 Cordycepin, 157 Cordycepose, 157,159 o--cOumrtric acid, as plant-growth substance, 409 pCoumaric acid, as plant-growth substance, 409 Coumsrin and derivatives, BB plant-growth substances, 409 Cram’s rule, 180 Curacoae, D-, 172 Curamycin, D-fucose derivative from, 172 Cyclitols electrophoresis in metal salt solutioncl, 232 as plant-growth substances, 416 Cymarin, 293 Cytidine, S(benEy1hydrogen phwpbte), hydrolysis of, 329

D Dalapon, &B plant-growth substance, 402 l’)eaoynine, 174 Defoliation, 429 Degradation of 2-deoxv sumrs. 147

21

of higher sugars to deoxy sugars, 153 of teichoic acid, 340 Deoxy sugars, 143-207 2-

esters, 146 synthesis of, 147 3-, synthesis of, 157 4,synthesis of, 166 5-, synthesis of, 167 in cardenolides, 275 chromatography Of, 197-199 2, Mideoxy, 193 2 ,Mideoxy, 183 3,6-dideoxy, 187 4,8-dideoxyJ190 5, &dideoxy, 192

from higher sugars by degradation, 153 maea spectra of, 64,204 nomenalature of, 14 nuclear magnetic resonance of, 202, 203 omega occurrence of, 171 syntheaia of, 176 synthesia of, 18 trideoxyhexoeeg, 195 Deeglucocheicotoxin, 285 Deuterium, labeled methylated monosac cheridea, maw spectra of, 71 Dextrans, antigenicity of, 504 Dideoxy sugars, 866 Deoxy sugars Di-&D-frudofuranoae 1,2’:2, I’dianhydride, 402 m-Digallic acid, 26 Digitoxal, 3,rkli-0-nitrobenaoyl-, 299 Digitoxigenin, 273 -, anhydro-, 279 -, ~ ( 2 ~ e o x y ~ w s a b i n o - b e x o p ~ n ~ sy1)-, 304 -, 3-(2-deoxy-pwrabino-hexopyranoBYl)-, 304, 308 -, 3-(2-deoxy-&~hexopyranosyl)-,

-, -, -,

308

3-0-(6deoxy-a-cmsnnopyrsnosyl)-, 279, 288

~(sdeoxy-3.0-rnethyl-~D-glucopyranosy1)-, 287 SO-pD-glucopyranosyl)-, 282, 312 -, 3-U-(cr-crhamnopyrenaayl)-, 279 Digitoxigenin digitoxwide, 299, 312

SUBJECT ISDEX, VOLUME

1hgi toxigwi i 11 glycositicn cardiotonic activitiw of, 314 properties of, 313, 319 Digitoxigenone, 306 Digitoxme, 186, 297 Digitoxosidw, 299 Digoxigenin, 3-&D-glucopyranosyl-, 282 Dipyrido[l ,k : 2 ’ ,l’-c]pyrazidinium dibromide, 1,l’dimethyl-, effect on sugarcane, 422 Diquat, effect on sugarcane, 422 Dieaccharidea derived, 30 furanoid, 126 mas8 spectra of, 69 by reversion of monosaccharides, 443,

444 synthetic, 31 Dithioacetals, 8ee Thioacetals Diuron effect on sugarcane, 427 as herbicide, 405 Drierite, in cardiac glycosidesynthesis, 277

E Electrophoresis in carbohydrate-alkaline-earth hydroxide reactions, 252 of carbohydratea in alcoholic alkali metal hydroxide solutions, 262 in solutions of metal salts, 231, 232, 234,235 Ellagic acid, 26 Enzyme poisons, effect on sugarcane, 427 Enzymes, in structure analysis of polyeacchari&s, 502 3-EpidigitoxigeninJ274 E p i g l u d n e , 18 Epimeriration, 178 of aldonic acids, 11 of ladones, 176 Epimycarose, acetyl-, 185 Epoxides, sugar, deoxy sugars from, 152, 159 Ethanol, complexes with carbohydrates and alkali metal hydroxides, 256 Ethylene effect on fruit ripening, 430 88 Dlant hormone. 378

543

21

Ethylenediamine, as solvent for reaction of carbohydrates and alkali metal hydroxides, 264 Et,hyleiiedinitrilott~aceticacid, effect 011 sugarcane, 426 Evatromonoside, 299, 301, 308 Evomonoside, 279, 288 Evonoside, 288

F Ficoll, conductivity and viscosity of solutions of, 214 Fischer, Emil biography, 2 carbohydrate chemistry and, 1-38 Fischer-Sowden reaction, synthesis of zdeoxy sugars, 149 Flavanones, as plant-growth substances, 410 Flavonea, w plant-growth substances, 410 Flavonoids, as plant-growth substances, 408

Flower drop, 429 Formaldehyde, copolymers with sugars, 450 Fragmentation, of pyranose and furanose rings, 47 Fructofuranose, 1’,2-anhydro-l-O-(o-Dfructofuranosyl)-,%D-, 100 Fructofuranoside, benzyl a-D-,125, 126 -, ethyl &D-, 126 -, methyl D-, conformation of, 101 -, methyl c r ~102, , 103, 126 -, -, methyl &D-, 103, 126 Fructopyranose, 1 , 2:4,5di-O-isopropylidene-D-, masa spectrum of, 76 -, 2 ,3 :4,5-di-O-isopropylidene-D,mass spectrum of, 76 Fructose D

optical rotations in solutions of alkali metals, 22s polymerization by hydrogen chloride, 452 DL-, 12, 13 phenylosazone, 11 -, I-amino-l-deoxy-, 18 -, 0-galloyl-D-, 27

544

SUBJECT INDEX, VOLUME

Friiit, drop, 420 E'ucopyrariotm, 1,2:3,4-cli-0-iwiiproi)yliderie-D-, m w llpectriim of, 7fJ Fucopyranoside, methyl tri-o-rnethyl-pD-, maas Rpectrum of, 64 Fucose D-, 172, 177 I.-, 172 -, 2 ,edi-O-methyl-D-, 172 -, 4-0-methyl-D-, 172 Fucoside, methyl CY-D-, 182 Functional groups, determination by mass spectrometry, 44 Furaldehyde, tetrahydro-, dkcetate, mass spectrum of, 62 Furanose polymers, 138 Furanose residues, natural occurrenca of, 139 Furanose ring fragmentation of, 47 maaa spectrum of, 61 stability of, 135 0

Galactofuranose, 1,2 :5 ,6di-O-ieopropylidene-w, maae spectrum of, 77 Galaotofuranoside, ethyl &D,117, 121 hydrolysis of, 131 -, ethyl Zacetamido-2-deoxy-1-thioa(and B)-D-, 116 -, ethyl l-thio-a-D-, 116 -, methyl CX-D-, 103 -, methyl &D-, 103 conformation, 100 Oalactopyranhse, 1,4anhydro-2,3 ,6-tri0-methyl-w, synthesis and polymerization of, 488 -, 1,2 :3,4-di-O-iipropylidene D-, ~ & B R spectrum of, 74 tetra-0Galactopyranoside, methyl methyl-crD-, maas spectrum of, 61 Galsctosan, D-, polymerization of, 483 G&ctoM

configuration of, 15 relation to rhamnose, 16 -, &leOXy-~-, 176 -, &deoxy-t, aee tFucose -, O-gahctmyl , 31 -, O-glUaOByl-, 31

21

Ciullo(sLtiiiinn, 26 (iuiitiobiose, 29 Gentiodextrins, 467 Gentiotetraose, 456 Gibberellic acid abscission promotion by, 429 effect on sugarcane, 427,428 as plantgrowth substance, 416 Gibberellins, 378, 416 Glucal, 18 Glucans, 455, 472, 485 Glucobrsssicin, 392 Glucitol D-, hexaacetate, rruw spectrum of, 89 -, 1,4 :3 ,6-anhydro-D-, msas spectrum of, 84 Glucofuranose, 6-brorno-l,2:3,5di-O-isopropylidene-a-D-, 165 -, 3deoxy-biodo-l , 2 :5,Mi-O-isopropylidene-D-, 166 -, 5,&dideoxy-a-D-, 193 -, 1,2 :6,6di-O-isopropylidentw-D-,mass spectrum of, 77 Glucofuranoside, benzyl I-thio-WD-, 115, 118 -, 6erkbutyl l-thio-&nd &D-, 121 -, ethyl a-D-,95, 102 -, ethyl &D-, 95, 102, 125 -, ethyl Zacetamido-2deoxy-l-thioa(and @)-D-,116 -, ethyl l-thiO-a-D-, 114, 118 -, ethyl l-thio-j3-D-, 115 -, isopropyl =(and &D-, 125 -, methyl D,21 -, methyl WD-,102,125 -, methyl &D-, 117, 125 acidic methanol action on, 110, 111 conformation of, 100 -, methyl 3,6-anhydro-a-~-, r n w spectrum of, 81 -, methyl 2 ,&di-O-methyl-en-, 125 -, methyl 1-thio-a-n-, 115 -, Pl'OpYl I-thiO-a-D-,115 Glucofuranosiduronic acid, ethyl l-thioe ~sodium , salt, 115 -, methyl l-thio-eD-, sodium salt, 116 -, propyl I-thio-a-D-, sodium salt, 115 Glucofuranosiduronolactone, 2-naphthyl &D-, 121 -, phenyl &D-, 121

SUBJECT INDEX, VOLUME

21

545

in hydrogen fluoride, 445 Glucopyranose D-, polymerization of, 436 by ionexchange resins, 464 -, 1 ,&anhydro-@+ in solid state by boric acid, 468 in sulfuric acid, 446 mass spectrum of, 81 potassium alcoholate, structure of, 266 synthesis of, 12 -, 5,6-anhydro-l, 2-0-isopropylidene-3system with sodium chloride, 211 0-methyl-D-, polymerization of, -, 2-acetamido-2-deoxy-~-, polymeriza491 tion by hydrogen chloride, 464 -, l-O-(indole-3-acetyl)-@D-, 390 -, 0-(2-acetamido-2-deoxya(and @)-, 3,4,6-tri-O-acetyl-l, flanhydro-D-, D-glucopyranosy1)-(1-6)-2polymerization of (Brigl’s anamino-2-deoxy-D-, 464 -, acetobromo-a+-, 20 hydride), 490 Glucopyranoside, ethyl Zacetamido- -, acetodibronio-, 17 3,4,6-tri-O-aaetyl-2deoxy-l-thio-, l-O-acetyl-2,3,4,6-tetra-O-methyla(and @)-D-, 116 BD-,mass spectrum of, 56 -, methyl 3,6-anhydro-a-~-,mass spectra -, 2-amino-2-deoxy-~-,18 polymerization by hydrogen chloride, of, 81 -, methyl tetra-0-methyla-D-, mass resistance to, 464 -, &amino-6-deoxy-, 18 spectrum of, 61, 73 -, phenyl tetra-0-methyl-a(and B)-D-, -, 2deoxy-~-,19 mass spectra of, 56 -, 6-dmxy-D-, 173, 175, 177 Glucopyranosyl bromide, 2,3,4-tri-O- -, ~ ~ ~ O X Y - C Y - L177 , 173 acetyl-a+-, polymerization of, -, 6-deoxy-2,3-di-O-methyl-~-, 441 -, 0-galactosyl-, 31 Glucopyranosyl mesitoate, BD-,poly- -, 3-O-galloyl-D-, 27 merization of, 460 -, l-O-(indole-3-propionyl)-&D-, 391 S(~~-Glucopyranosyl)-3-indolylthioace- -, 2,3,4, B-tetra-O-methyl-~-, reaction tylhydroximyl 0-sulfate, as with methyl sulfoxide, 451 growth regulator, 392 Glucoside, methyl a(and @)-D-, 21 S-(pD-Glucopyranosyl)-N-methyl-3-indo- -, methyl 2-acetamido-2-deoxy-3,4,6lylthioacetylhydroximyl O-sultri-0-methyl-D-, m s spectrum fate, as growth regulator, 392 of, 67 Clucosaminide, methyl N-acetyltri-0- -, methyl 4,60-benzy1idene-3-deoxyy-3methyh-, mass spectrum of, 67 iodo-a+, 162 Gliicofie -, methyl 6-bromo-6-deoxy-a-~-,182 u-, methyl 6-chloro-6-deoxy-a-~-,182 complex with potassium bromide, 215 -, methyl 2-deoxy-, mass spectrum of, 64 with potassium iodide, 215 -, methyl 3deoxy-, mass spectrum of, 64 with sodium ethoxide, 258 -, methyl 4-deoxy-, mass spectrum of, 64 with sodium methoxide, 258 -, methyl 2,3,4,6-tetra-O-methyla(and component of cardenolides, 311 B)-D-, mas8 Spectra Of, 56 configuration of, 13 -, methyl 2,4,6-tri-O-methyl-&~-, reaceffect of calcium chloride on specific tion with methyl sulfoxide, 450 rotation of, 230 -, phenyl 1-thio-, 22 electrophoretic migration of, 234 Glucosiduronic acid, digitoxigenin-3&yl periodate oxidation of methyl deriva2,3, Ctri-O-acetyl-&~-, methyl tives, 500 ester, 291 phenylosazone, 11, 12 Glucosyl bromide, 2,3,4tri-O-acetyl-6polymerization of, 437, 441, 468, 475 bromo-6-deoxy-D-, 17 by hydrogen chloride, 462 Glycals, in synthesis of Pdeoxy siigarx, 148

546

SUBJECP INDEX, VOLUME

Glyceraldehyde, D-(+)-, synthesis of, 17 Glyeerose, 12 Glycofuranoseg, occurrence in Nature, 139 Glycofuranosidee,95-142 conformation of, 96 formation in acidic methanol, 101, 105 hydrolysis by acids, 128, 130, 131 kinetics of formation of, 106, 108 melting points and specific optical rotations of, 140, 141 oxidation of glycol groupe in, 132 polymerization of, 138 preparation from dithioacetala, 112, 113 ntructure of, 127 -, methyl 2deoxy-, thermal polymerisation of, 476 Glycofuranoeylanines, 122 Glycol groups, oxidation of, in glycofuranosides, 132 Gly copyranosidea hydrolysis of, 130, 131, 137 kinetics of formation of, 106, 108 Glycosides Koenige-Knorr synthesis of, 275 mercuric cyanide in syntheaie of, 278 Meystre-Miescher modification in synthesis of, 277 molecular weight determination, 274 as plant-growth gubetimces, 408,413 rate of formation of, 108 synthesis of, 21, 491 synthetic, as plantgrowth substances, 413, 415 (IlycoRylamines, as plantgrowth subHtances, 413 Giilopyranowide, methyl D-, effeot of calaium chloride on specific rotation of, 230 Oiiltw, WD-, effect of slkaline-earth metal salts on specific rotation of, 230 -, bdWxy-D-, 173, 178, 181 -, B - d e ~ ~ y - ~178 e,

H Ilalogeiiat,ion,of 6-deoxyhoxow, 182 Helveticodde, 308 Heparin substitutes, polyglycose Aulfaterr as, 608

Heptom, ~~Zycero-n-gulo-, effect of calcium chlorode on equilibrium rotation of, 230, 231

-,

21

7deoxy-~glycero-ylala~, 178 Heptulose, m n w , as pbt-growth substance, 416 Herbicides 3-amino+triarole, 399 benzoic acid derivatives, 407 carbematea and carbanilatat, 403 chlorinated aliphatic acids, 402 maleio hydraride, 398 phenoxyacetic acids, 392 plant-growth substances an, 392 for sugarc&ne,422 a-triazines, 406 urea derivatives, 404 l-€Iexene~-n'b63,4 ,5 I &tetrol, I-C-nitro-, tetraacetate, 304 liex-3-enofuranose, 3-deoxy-l,2 :5,6-di-0hpropylidensn-erythro-, 163 Hex-l-enopyranose, 1,2,6-trideoxy-3,4di-O-pnitrobenzoyl-D-n'bo-, 299 Hexitol, 1,2-dideoxy-l-nitro-wrin'bo-,305 Hexofuranose, 5deoxy-~-zyL,169, 171 -, cnleaxy-1, 20-isopropylidene-~yb1 168 Hexofuranoside, methyl 2deoxy-~ambino-, 114,309 Hexopyranoae, 1 ,6-anhydro-3-deoxy-b w r a b i w , 160 -, 2-deoxy-a-wmbino-, tetraacetate, 146 -, %deoXy-D(Or &urabino-, tetraacetate, mses spectrum of, 62 Hexopyranoside, ethyl 2,hiideoxy-a-Demlhto-, 193 -, ethyl 2, 3-dideoxy-a-D-UIreo-, 194 -, methyl 4 , ~ b e x i z y l i d e n s 3 d e o x y - ~ wmbino-, 160 -, methyl Zdeoxy-&Darabino, 149, 302 -, methyl 2deoxy-a-D-n'bJ 160 -, methyl 3-deoxy-&D-ribo-, 164 Hexopyranosiduronic acid, methyl 4 deoXy-&LaTabim, methyl ester, 167 Hexos-2,Sdiulose, 4,6-dideoxy-glycero-, 101 Tlexose, 3-0-acctyl-2, Cilideoxy-D-lym-, 184-187 -, 4-O-acetyl-2,6-dideoxy-3-C-methyC ~ r a b i n o -185 , -, 5,6-0-rarbony1-2-deoxy-l,3di-O-p nitrobenzoyl-nsrab, 311 -, 2deoxy-~-,polymer, 472

SUBJECT INDEX, VOLUME

21

547

-, methyl 3-deoxy-&~xylo-, 161 2deoxy-wrabino-, 149, 157 3-deoxy-xwrabino-, 159,164 -, methyl rMeoxy-&~-zylo-, 161 -, methyl zdeoxy-3,4,6-tri-O-pnitro4-deoxy-D-arabino-, 166 benzoyl-&Da7abim-, 302 -, ~ - ~ ~ o x ~ - D &159 zo-, -, 2de0~y-D-ribo-,304 -, methyl zdeoxy-3,5,6-tri-O-pnitrobenzoyl-mzrabino-, 309 in cancer chemotherapy, 145 -, methyl 3,4,6-tri-O-benzoyl-2-deoxy-& -, 3 - d e o ~ y - ~ - r i b o159, - , 163 D-arabino-, 297 6-pho8phab1 158' ~Iexos-Brilose,3deoxy-~-erylhro-,164 -, 3-deoXy*D-n'bO-, 160, 164 Hexos-5-rilose, 6-deoxy-D-arabino-, 174 -, 5deOXy-DdO-, 169 -, Zdeoxy-D-zylo-, in carwer chemo- Hexosyl bromide, 5,6-O-carbonyl-2deoxy3-O-pni trobenzoyl-n-arabino-, therapy, 145 310 -, ~ ~ ~ O X Y - D - Z Y ~ O159, - , 161, 163 -, 4-deoxy-~-xyZo-,166 -, 2-deoxy-3,4,6-tri-O-p-nitrobenzoylD-arabinu-, 301 -, li-deoxy-D-Zylo-, 168 -, 2deoxy-3,4,6-tri-O-pnitrobenzoyI-, 2-deoxy-l,3,4, &tetra-0-p-nitrobena-~-n'bo-,305,307 zoyl-Drab, 307 -, 2,6-dideoxy-Darabino-, 185, 186, 308 -, 2,6-dideoxy-3, Mi-0-pnitrobenzoylp-~-n'bo-, 298 -, 2,6-dideoxy-~-arabino-, 186 -, 3,6-dideoxy-oarabino-, 188, 189, 190 -, 3 ,4 ,6-tri-O-benzoyl-2deoxy~~-arabino-, 297 -, 3,6-dideoxy-mrabino-, 189, 190 Hexosyl chloride, 2deoxy-3,4,6-tri-O-p-, 5,6dideoxy-~-arabino-, 193 nitrobenzoyl-a-D-arabino-,301 -, 2,6-dideoxy-~-lyxo-,184, 186 -, 3 ,&dideoxy-D-(and L)-lyxO-, 190 -, 2deoxy-3 ,4 ,6-tri-0-pnitrobenzoyl~Y-D-n'bo-, 305 -, 2,6dideoxy-p-~-ribo-,see Digitoxose -, 3, 6dideoxy-Mibo-, 188, 189 -, 2,6dideoxy-3,Mi-0-pnitrobenzoyl-, 3,6-dideoxy-L-ribo-, 190 &D-n'bo-, 298 Hexulose, 6-deoxy-D-arabiwl 176 -, 2,6-dideoxy-~-xylo-, 186 -, 3 ,6dideoxy-~-zylo-, 188-1 90 -, Cdeoxy-D-ergthro-, 166 -, 3 ,6-dideoxy-rrxylo-, 188, 190 -, .jdeoxy-n-etythre-, 168 -, 6deoxy-~-lyzo-, 176 -, 4, 6-dideoxy-D-zylo-, 192 -, 5,6-dideoxy-D-zybI 193 -, ~ ~ ~ o x Y - c x ~ Z O - , 174-176 -, 5,6-dideoxy-l, 2-0-isopropylidene-~- -, 5 ,6-dideoxy-~-threo-,193 Hill reaction, inhibition of, 405 x y b , 169 -, 2 ,6-dideoxy-40-methyl-~-lyzo-, 184, Holoruthin A, 6deoxy-D-glucose from, 173 Honghelin, 286 185, 187 -, 4, B-dideoxy-3-0-methyl-~-xyZo-,191, Hydrazine, 1-benzyl-1-phenyl-, 147 -, (pbromopheny1)-, in sugar chemistry, 192 -, 2 ,3 ,6trideoxy-n-erylhro-, 195, 197 11 --, 1 ,ldiphenyl-, in sugar chemistry, 11 -, 2 ,3 , 6 - t ~ d s o x y - ~ ~ - e ~ t h197 ro-, -, 2,3,6-trideoxy-~-threo-, 197 -, 1-methyl-1-phenyl-, in sugar chemistry, -, 2 ,3 ,6-trideoxy-Dtlhreo--,197 11 -, 2 ,3,&trideoxy-x.-threo-, 195 -, phenyl-, derivatives, discovery by Fischer, 3, 10 Hexoside, methyl 4,6-O-benaylidene2Ilydroboration reaction, 168, 169, 181 deoxy-a-D-ribo-, 304 -, methyl 4,6-0-benzylidene-3-deoxy-u- Hydrogen bromide, as catalyst in polymD+fbo-, 161, 162 erization of sugars, 452 -, methyl 4,6-0-benzylidene-2,3dide- Hydrogen chloride, catalysis of polymOxy-~-D-W&'O-, 162 erization by, 452, 462 -, methyl 5,6-O-carbonyl-2-deoxy-3-0- Hydrogen flrioride, polymerization in priitroberizoyl-a-D-arabi7lo-, 309 liquid, 445 -, -, -,

548

SUBJE'CT INDEX, VOLUME

Hydrolysis acid, of glycofuranosides, 128 alkaline, of methyl aldosides, 137 of phosphate estem, 328 of teichoic acids, 331, 345, 340,352 Hygromycin, 174

I Iditol, D-, 17 Idofuranose, 3-O-beiizyl-&deaxy-l,2-0isopropylidene-&Ir, 180 Idose, Weoxy-Ir, 176, 180 Immunochemishry, in structure analytis of polymccharides, 504 Indole-3-acetic acid, 377 effect on cell-wall polysaccharides, 381 on augarcane, 424 Indole-3-but yric acid, effect 011 sugarcaiie, 424

Inositols electrophoresis in metal salt solutione, 232

maw spectra of, 91 as plant-growth substances, 416 Inversion, in w-deoxy sugar synthesis, 178 Isodigitoxigenin, 280 Isoflavonts, as plant-growth substances,

410

Isolactose, 31 Isomaltose, Fmcher'n, 31, 444, 464 Isomtation, furanoid ring detection by, 127 Isotrehalose, 32

J

21

Kojic acid, caloium salt, chelate structure of, 238 Kojitriose, 344

Labilomycin, 172 Labilose, 172 Ladobacillus arabinosus, teichoic acid from, 334, 361 Ladobaccilus buehnen', teichoic acid from, 350

Lactokcillus cuaei, teichoic acid from, 341 Lactones, epimerization of sugar, 175 Lactose, 28 complex with calcium chloride and methanol, 215 Laminaran, permethyl-, mass spectrum of, 90

Laminaribioae, 464 Lankavose, 190 Levoglucosan maw spectrum of, 81 polymerization of, 478 Linamerin, 23 Lithium bromide, complex with sucrose, 223

Lithium chloride, complex with sucrose, 223

Lithium iodide, complex with sucrose, 223 Locust-bean gum, conductivity of solutions of, 214 Lyxofuranoside, methyl WD-, 122 conformation of, 98 methyl B-D-, conformation of, 98 Lyxopyranoside, methyl tri-0-methyl-a (and &D-, mass spectra of, 60 LYXOW,~ - ~ W X Y - D177 -, -, Bdeoxy-tr, 176, 177 -J

Javose, 173

K Knempferol, derivative, as plant-growth substance, 410 Ketoses, maw Rpectra of, 66 Kinetics, of glycofuranoside and glycopyranoside formation, 106, 108 Kinstin effect on ahNciRtlion and ripening, 429 a8 pht-growth substance, 420 Kinins, 378 as plant-growth substances, 416 KoenigR-Knorr reartion, 20, 276, 441, 456 Kojibiitol, 344 Kojibiose, 344

M MacDonald-Fischer degradation, deoxy sugani by, 166, 178 Maltose, 30 polymerization by hydrogen chloride, 465 in hydrogen fluoride, 446 Maleic hydraaide effect on sugarcane, 425 as plant-growth substance, 398

SUBJECT INDEX, VOLUME

21

549

Manriitrrl, DL-, 12, 13 Metabolism Maiinoruranone, P , 3 :5,6-di-O-isopropyliof auxins, 396 dene-D-, mats spectrum of, 77 in plants, auxin activity an, 387 Mannofuranoside, methyl a-D, 102, 122, Methyl sulfoxide, polymerization of sugars 126 in, 449, 453, 454 kinetics of formation of, 110 Meystre-Miescher modification, of Koe-, methyl 8-D-,conformation of, 100 nigs-Knorr synthesis, 277 Mannoppnoside, ethyl l-thio-fl-w, 117 Molecular weight -, methyl WD-, 458 determination by mass spectrometry, 4 -, methyl tetra-0-methyl*-D-, mass determination of, of glycosides and spectrum of, 61 cardenolides, 274 Mannopyranosyl fluoride, a-D-, polymof polysaccharides, 494 erization of, 458 hlonosaccharidep Mannose, and phenylhydrazone, 10 alkali metal hydroxide adducts, 254 -, 6-deoxy+, see Rhamnose mass spectra of, 46 Mannoaide, met>hyl 4-0-benroyl-2,3-0reversion of, to disaccharides, 444 carbonyl-6-deoxy-a-n-, 295 Monoses, configuration of, 8 -, methyl 4-O-benzoyl-2,3-O-carbonyl-6- Monuron deoxy-pD-, 296 effect on sugarcane, 422, 426 -, methyl 44-benzoyl-2,3-O-carbonyl- as herbicide, 405 6-O-p-tolylsulfonyl-~-~,295 Mycinose, 173, 179 Mannosyl bromide, 4-0-benzoyl-2 ,Scarbonyl-B-deoxy-WD-, 296 N -, 2,3,4,&tetra-O-acetyl-a-I, 291 -, 2,3,4tri-O-benzoyl-&deoxy-a-u-,290 1-Naphthaleneacetamide, effect on abscission and ripening, 429 Ma.. spectra 1-Naphthaleneacetic acid of acetals, 74, 70 effect on abscission and ripening, 429 of aldom and ketoses, 66 on cell-wall polyeaccharides, 381 of amino sugars, 67, 93 on sugarcane, 424 ~urw of anhydro sugars, 93 Naringenin, as plant-growth substance, of carbohydrate derivatives, 46 410 of deoxy sugars, 204 Nef reaction, synthesis of 2aeoxy sugars of dimcchaddes, 69 by, 150 effect of substituents on, 54 Neoglucobrassicin, 392 of nucleosides, 90 Neriifolin, 286 of oligosaccharides, 93 Neutramycin, 173 principles of interpretation of, 43, 45 Nomenclature stereochemistry and, 79, 92 of deoxy sugars, 1 4 of stereoisomers, 59 of tartaric acid, 34 of thioacetals, 93, 205, 206 Nuclear magnetic resonance, 191, 195 Mass spectrnmeterx, 40 of chromoses, 185 Mam Rpectromeiry conformation of glyoofiiranosides and, of carbohydrate derivatives, 39-93 97 wrope aiid limitiitioiis of, 43 of deoxy Rugars, 202, 203 Melibione, 313 of polywarcharidex, 5131 Meri*trptalr,of aldosen, I!, Nuc.leoAlefi, 28 Mewsptrrris, react ioir with nldmes, 19 mmr hpertra of, 90 Mcwciric- cyaiiide, in cwdenolido q n pyrimidine, rorltaining 2,3-dideoxy suthesis, 278 gar, 194

550

SUBJECT INDEX, VOLUME

Nucleotidea, 24 Numbering, of carbon atom in sugars by Fischer, 10

-, -,

21

zdeOXy-DLc@h-, 157 Zdeoxy-D-threa-, 151, 153 -, Sde~~y-wryUltb, 157, 159 5-phosphateJ 158 0 -, 3-deoxy-catyihro-, 158 -, 4-deoxy-cerylhro-, 166 Oligosaccharides, 27 -, 2,3-dideoxy-cglycei-o-, 194 complexes with alkali metd hydroxides, -, 2, Mideoxy-n-ctythro-, 194 254 Pentase-l-le, 2deoxy-~-ery&ro-,150 with metal nalts, 221 Pentasidq methyl 3deoxy-Lerythro-, 159 dofinition of, 4d3 Pentulose, wihreo-, as plant-growth subfuranoid, 126 stance, 416 mass spectra of, 46, 93 -, 1-amino-1 ,&anhydro-ldeoxy-D-threo-, Ryntheais of, 457 ma88 spectrum of, 91 Olioee, acetyl-, 184, 186 -, Saeoxy-D-threo-, 175 Olivomose, 184 Periplogenin, 3-@-glucopyranosyl-, 282 Olivomycin, 184, 186 Perseitol, 17 Olivomycose, 184 Phosphatm (estcm), hydrolysis of, 328 Olivose, 184 Phosphorus pentachloride, polycondensaOptical rotation tion of Dglucoee in aqueous, 447 effect of carbohydrate-complex ealt Phosphorus pentaoxide, &a catalyst for formation on, 213 polycondensation of sugars, 447, of complexing on, of carbohydrates, 452,454 228 Phosphorus trichloride, polycondensation of metal hydroxides on, of carboof mglucose in aqueous, 447 hydrates, 253 Physical properties, of herbicides, biologof glycofuranosides, 140, 141 ical activity and, 408 of sucrose, effect of salts on, 225 Pinitol, as plant-growth substance, 416 of teichoic acids, 344 Plant-growth substances of 1-thioaldofuranosides, 142 effect on carbohydrate systems, 377-430 Organic compounds, m w spectrometry on sugarcane, 421 and structure of, 39 flavonoids, 409 Orthoacetates, of sugars, 20 glycosidea and other carbohydrate deOsaaones, diecovery of, 10 rivatives as, 408, 413 OsoneR, 11 aa herbicides, 392 Oxidation, of glycol groups in glycofuranoPlant physiology, carbohydrate chemistry sides, 132 and, 378 Oxymethylene groups, in polyglycoses, 450 Pollen germination, inhibitors Of, 410 Polyarabinose, 474 P Polygalactoglucose, 483 Paraquat, effect on sugarcane, 422 Polygalactose, 483 Paratose, 188, 189 in immunochemistry, 506 Pentitol, 1,2dideoxy-~-threo-,157 Polyglucose, 462, 463, 465, 467, 469, 471, Pentofuranoside, methyl 2,Mideoxy-o-D481 erythro-, 194 fractionation of, 505 -, methyl 3,5-dideoxy-o-n-erythro-,194 glycosidia linkages in, 448 Pentofuranosyl chloride, !2deoxy-~in immunochemistry, 504 wthro-, esters, 146 Polyglycoses, 433 Pentose, 2deoxy-~-e@hro-, 149, 151, 153, from aldopyranosyl fluorides, 460 156 nuclear magnetic raqoriance smctrosesters, 146 copy of, 501

SUBJECT IMDEX, VOLUME

21

551

preparation of, 491 Potassium chloride, complex with sucrose, 223 sulfates, as heparin substitutes, 508 x-ray diffraction of, 501 Potassium iodide, complex with s u m e , Polymaltose, 463, 475 215, 223 in immunochemistry, 506 Propionic acid, Z(o-chlorophenoxy)-, efPolymannose, 466,483 fect on sugarcane, 424 -, 2,2dichloroPolymerization effect on sugarcane, 426 addition, 477 as plant-growth substance, 402 catalysis by boric acid, 466 -, 2-(2,4,5-trichlorophenoxy)by hydrogen chloride, 462 by ion-exchange resins, 464 effect on sugarcane, 425 as herbicide, 392 catalysts for, of sugars, 447, 452 Peicofuranine, 174 condensation, of carbohydrates, 434 Purine, 4(2,3-dideoxy-19-Dglycer~pentoeffect of pressure on, 467 epoxide, 491 furanosy1)-, 194 Pyran, 2, Wiacetoxytetrahydro-, m&89 of glycofuranosides, 138 spectra of cis- and trans-, 62 in methyl sulfoxide, 449, 453 Pyranose ring in solid state, 461 thermal, 476 fragmentation of, 47 Polymers, furanose, 138 maea spectrum of, 61 Polyphosphoric acid esters, in glycoside stability of, 136 3,6-Pyrazinedione, 1,2dihydro-, see Masynthesis, 448 Polyrhamnose, 474 leic hydraside Purines, N-glycosyl derivatives, 23 Polyribose, glycosidic linkages in, 448 Polysaccharides, 27 adducts with alkali metal hydroxides, Q 254 branching, determination of degree of, Quebrachitol, as plant-growth substance, 496 416 complexes with alkali-metal and alka- Quercetin, 3-O-&crhamnopyranosyl-, as line-earth-metal salts, 221, 224 plant-growth substance, 410 cell-wall, effect of indole-3-acetic acid -, 3-(&O-~~rhamnopyranosyl-~-glucoand of 1-naphthaleneacetic acid pyranosy1)-, &B plantrgmwth subon, 381 stance, 410 characteriration of, 473 Quercetin derivatives, as plant-growth configurrrtion of anomeric linkages, 496 substances, 410 fraotioriation of, 493 Quercetrin, as plant-growth substance, glycosidic linkages in, 498 410 homogeneity of, 494 isolation of, 402 R mass spectra of, 46 structural analysis of, 494 Itaffinose, 30 structure of, enzymes in analysis of, 502 Resins immiirioc*hemixtryin study of, 504 catalysis of polymerixat,ion by ionexsynthe& of, 431-512 change, 4G4 w s of synthetic, 507 ion-exchange, aa catalysts iri glycohiranPolyxylose, 463, 474 oside formation, 104 Potassium acetate, complex with sucrose, ReBpiration, in plants, effeat of aiixiiw 0 1 1 , 223 387 Potamium broinide, romplex with xucrose, 'Iteversion, of saccharide#, 442 215 Rhamnofuranoside, ethyl a(arid &L-, 112

552

SUBJECT INDEX, VOLUME

Rhamnose configuration of, 17 D-

occmnca in Nature, 172 synthesis of, 177, 289 I-

1 ,2-(methyl orthoamtab), 20 occurrence in Nature, 171 relation to galactose, 16 Rhodinw, 195 Rhodomycin, 195 Ribitol. U)-&D-glucopyranosyl-n(andL)degradation of, 356 Ftibofuranoside, benrsyl &D-, 125 -, pchlorophenyl PD-, 121 -, ethyl l-thio-a-D-, 115, 116 -, isopropyl 1-thio-arD-, 115 -, methyl WD,122 conformation of, 98 -, methyl &D-, 121 conformation of, 98 -, methyl 2,3-anhydro-a(and a)-~-,conformation of, 99 -, methyl l-thio-crD-, 115 -, phenyl &D-, 121 -, propyl 1-thio-a-m, 115 Ribopyranoside, methyl tri-0-methyl-B-D-, m&88 spectrum of, 60 Ribose, D-, 17 -, 5db00xy-~-, 176,177, 179 Rings furanoid, stability of, 135 pyranoid, stability of, 136 Ripening, 429 Ruff degradation, of higher sugars to deoxy sugars, 153, 177 Rutin, BB plantgrowth substance, 410 I

S

Salicylaldehydc, alkali metal chelates, 265 Salicylic acid BB herbicide, 408 as plant-growth substance, 409 Sambunigrin, 23 Bequoyitol, as plant-growth substance, 416 Silicon compounds, trimethylsiiyl ethers of oligoaaccharidea, mass Rpectra and, 93 Simazine, BX herbiaide, 407

21

Sodium bromide, complex with sucrose, Structure of, 236 Sodium carbonate, complex with sucrose, 213 Sodium chloride, system with D-glucoee, 211 Sodium ethoxide, complex with wglucose, 258 Sodium iodide camplex with aucrose, 215 systems with sucrose, 211 Sodium methoxide, complex with D glucorw, 258 Solvation, of carbohydrate-metal salt complexw, 226 Sorbose, DL-, 12, 13 Stability of carbohydrabmetal salt complexes, 227 of furanoid and pyranoid rings, 135 Staphylomcua a u m Copenhagen, teichoic acid from, 360 H,teichoic acid from, 359 teichoic acid from, 342 8 t a p h ~ ~ c o c wIrrdis, u teichoic acid from, 342,347,350 Starch effect of auxins on, 386 polymerination in hydrogen fluoride, 446 Stereochemistry biochemistry and, 34 mass spectrometry and, 79, 92 Stereoieomsre, mars spectra of, 59 Stoichiometry of cerbohydrahlkali metal dcoholate formation, 259 of carbohydrate-alkali metal hydroxide complexes, 248 of carbohydrate-alkaline-earth metal complexes, 251 of carbohydratemetal salt complexes, 222 StreproooeCi, teichoic acid from group D, 344 Streptolydigin, 186 Strontium oxide, complex with sucrose, 213 Strophanthidin, 274 -, 3rr-carabinopymnosyl-, 281, 285 -, 3-(6-deoxy-&D-gulosyl)-, 286

kUl3JECT INDEX, VOLUME

,

21

553

fhibntituenb :!-~-(~-t~~!~JXy-~-lrlIlJt?lll~JpyrtLn~lHyl)-,

determination by nuss spectrometry, 44 nee Corrvalltitoxiri 3-(2,6-dideoxy-&~-n'bo-hexopyraneffect on ma^^ spectra, 54 0syl)-, 308 Sulfuric acid, polymerization of Dglucose -, 3-j3-~-glucopyranosy1-, 281, 288, 312 in, 446 -, o-j3-D-glucosyl-o-~-D-glucosyl-&cyT ~ ~ ~ r o s y293 l-, -, 3-o-(a-D-lyXOSyl)-, 286 Talose, ~ - ~ B ~ x Y - D -173 , -, 3-~-~-mannopyranosyl-,292 -, 6-deo~y-1.~~ 173, 179, 181 -, 3a-~-rhamnopyranosyl-,296 Tartaric acid, configuration of, 32 -, 3-&~-rhamnopyranosyl-,296, 313 nomenclature of, 34 -, 3-fi-~-xylopyranosyl-,281 Teichoic acids, 323-375 Strophanthidin glycosides from actincmycetes, 363 cardiotonic activities of, 314 from Bacillus subtilie, 350, 354 biosynthesis of, 372 properties of, 313, 319 degradation of, 340 Strophanthidol, 3,19-di-O-(a-brhamnopydiscovery of, 326 ranosy1)-, 294 -, 3-&D-glucopyranosyl-, 288 function of, 371 -, 1W-a-~rhamnopyranosyls294 glycerol, 334, 346 Ic-Strophanthin-fi, 293 hydrolysis of, 331, 345, 346,352 from Laclobacillus aralvinosus, 334,361 k-strophanthoside, 292 from Laclobacillus buchneri, 350 Structure from Laclobacillus casei, 341 of carbohydrates, mass spectrometry location in relation to cell structure, 365 and, 39 (Jf herbicides, biological activity and, 408 membrane, 332 Sucrose, 29 ribitol, 354 alkali action on, 358 complexos with alkali metals, 223 from Staphylococcus arabinosus 17-5, 363 with sodium bromide, structure of, 236 from Stuphylococcwr aureua, 343 with sodium carbonate, 213 with sodium iodide, 215 from Staphylococcus aureus Copenhagen, esters, herbicidal properties of, 416 360 hydrolysis of, 131 from Staphylococcus aurewr H, 359 optical rotation of, effect of salts on, 225 from Stuphylococcwr laclie, 342, 347, 350 from Sikeptowcei, 344 in solution of metal salts, 229, 230 structure of, 128 wall, 346 system with barium oxide, 213 Tetrose, 2-deoxy-~-glycero-, 154 with sodium iodide, 211 Theophylline, N-glucosyl-, 24 with strontium oxide, 213 Thevetose, cardenolides containing, 286 Sugarcane, effect of plant-growth sub- Thioacetala stances on, 421 aldofuranosides and thioaldofuranoSugars sides from, mechanism of, 117 acetohalogens, 20 glycofuranoside preparation from di-, amino, 17 112, 113 mass spectra of, 67, 93 mass spectra of, 85, 93 anhydro, see Anhydro sugars of deoxy sugars, 205, 206 deoxy, see Deoxy sugars Thioaldofuranosides, 114 epoxide, deoxy sugam from, 152, 159 ethyl, melting points and specific optical polycondensation in methyl sulfoxide, rotations of, 142 449, 453 mechanism of formation of, 117 uneaturated, synthesis of, 194 Thiols, reaction with aldoseu, 19

-,

554

SUBJECT INDEX, VOLUME

Thioiiyl chloritlo, polymcristltion of augam by, 447 34 Threaric acid, L,-(+)-, Tilironide, aa plant-growth aubatanm, 412 Trelialow, 29 cl-Tritlrilie, 8diloro-4,6bib.(uthylaiiliiio)-, aa herbicide, 407 -, 2-chloro-4(ethylamino)-B(isopropylamino)-, ae herbicide, 408 e-Triaaole, 3-amino-, aa plantgrowth substance, 399 -, 3-(&D-glucopJrranosylamino)-, 402 Trideoxyhexoses, 195 Trideuteriomethyl group, in identification of methylated monosaccharides by II~&BBspectra, 71 Trieaccharides, 28 Turanoae, 30 Tylosin, 173 Tyvelom, 187,189, 190

U

Urea, %@-chlorophenyl)-l ,l-dimethyleffect on sugarcane, 422,426 aa herbicide, 406 -, 343,1Michlorophenyl)-l,l-dimethyleffect on sugarcane, 427 aa herbicide, 405 -, D - ~ u c o s ~24 ~-, -, glucosylthio-, 24 Uearigenin, 274

21

V Vecciniiti, 24, 26 Vallarose, 173 Vieconity, eKec:h of cerbohydratecomplex salt formation on, 213 Volemitol, 17

W Walden inversion, 20 Wohl degradation, 5-deoxy pentosea by, 177

X Xanthatea, deoxy sugara from, 163 X-ray diffraction conformationof glycofuranosidesand, 97 of polyglycOses, 501 Xylitol, 1-acetamidotri-0-acetyl-1 ,5-anhydro-ldeoxy-, maas spectrum of, 91 Xylofuranoae, 1,2 :4 ,5di-o-iaopropylidencm-, maw spectrum of, 78 Xylofuranoaide, ethyl Zacetamido-2deoxy-1-thio-on-, 116 -, msthyl a(and P)-D-, conformation of, 98

Xylopyranoside, methyl tri-Gmethyl-pD-, maaa spectrum of, 60 Xylose D-

-,

electrophoretic migration of, 234 polymerization by hydrogen chloride, 463 Saeoxy-~-,synthesis of, 177

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-21 A

ADAMS,MILDRED.See Caldwell, Mary L. ANDERSON,ERNEST, and SANDS,LILA,A Discussion of Methods of Value in Research on Plant Polyuronides, 1, 329-344

ANDER~ON, LAURENS. See Angyal, S. J. ANET, E. F. L. J., 3-Deoxyglycosuloses (BDeoxyglycosones) and the Degradation of Carbohydrates, 19, 181-218 ANQYAL,S. J., and ANDERSON, LAWNS, The Cyclitols, 14, 135-212 ARCHIBALD,A. R., and BADDILEY,J., The Teichoic Acids, 21, 323-375 ASPINALL, G. O., The Methyl Ethers of Hexuronic Acids, 9, 131-148 ASPINALL, G. O., The Methyl Ethers of n-Mannose, 8, 217-230 ASPINALL,G. O., Structural Chemistry of the Hemicelluloses, 14, 429-4458

B

BEBLIK, ANDREW, Kojic Acid, 11, 145-183 BELL, D. J., The Methyl Ethers of DGalactose, 6, 11-25 BEMILLER,J. N. See Whistler, Roy L. See Zorbach, BHAT,K. VENKATRAMANA. W. Werner. BINKLEY,W. W., Column Chromatography of Sugars and Their Derivatives, 10, 55-94 BINKLEY,W. W., and WOLFROM, M. L., Composition of Cane Juice and Cane Final Molasses, 8,291-314 BIRCH, GORDONG., Trehaloses, 18, 201-225

BISHOP,C. T., Gas-liquid Chromatography of Carbohydrate Derivativea, 19, 95-147

BLAIR, MARYGRACE,The ZHydroxyglycals, 9, 97-129 BOBBITT,J. M., Periodate Oxidation of Carbohydrates, 11, 1 4 1 BOESEKEN, J., The Use of Boric Acid for the Determination of the Configuration of Carbohydrates, 4, 189-210 BONNER, T. G., Applications of Trifluoroacetic Anhydride in Carbohydrate Chemistry, 16, 59-84 BONNER, WILLIAM A., Friede1-Craft.a and Grignard Processes in the Carbohydrate Series, 6, 251-289 BOURNE,E. J., and PEAT,STANLEY, The Methyl Ethers of DGlucose, 6,

BADDILEY, J. See Archibald, A. R. BAILEY, R. W., and PRIDHAM, J. B., Oligosaccharides, 17, 121-167 BALLOU,CLINTONE., Alkali-sensitive Glycosides, 9, 59-95 BANKS,W., and GREENWOOD, C. T., Physical Properties of Solutions of Polysaccharides, 18, 357-398 BARKER,G. R., Nucleic Acids, 11,285-333 145-190 BARKER, S. A., and BOURNE,E. J., E. J. See also, Barker, S. A. Aoetals and Ketals of the Tetritols, BOURNE, BOUVENQ,H. O., and LINDBERG,B., Pentitols and Hexitols, 7, 137-207 Methods in Structural Polyeaccharide BAR~TT,ELLIOTT, P., Trends in the Chemistry, 16, 53-89 Development of Granular AdsorbBRAY, H. G., n-Glucuronic Acid in ents for Sugar Refining, 6, 205-230 Metabolism, 8, 251-275 BARRY,C. P., and HONEYMAN, JOHN, Fructose and its Derivatives, 7, 53-98 BRAY, H. G., and STACEY,M., Blood Group Polysaccharides, 4,37-55 BAYNE,S., and F E W S ~J. R ,A., The BRIMACOMBE], J. 8.See How,M. J. Osones, 11, 43-96 5515

5%

CUMULATIVE AUTHOR INDEX FOR VOLS.

C

CAESAR,GEORCIE V., Starch Nitrate, 18, 331-345

1-21

~IIMLER, R. J., 1 ,6-Anhydrohexofura-

noses, A New Clam of Hexosana, 7, 37-52

M. See IIitmid, W. Z. CALDWELL, MARYL., and ADAMB,MIL- I)OUDOROFI, I ~ R A CP. HS , ee Mehta, N. C. DRED, Action of Certain Alpha I ~ T C H E RJAMEB , D., Clieniid,ry of the Aniylwtw, 6, 2 2 ! ! 8 8 Amino Sugars Derived from AntiCANTOR,SIDNEY M., [Obituary of] John biotic Subshncas, 18, 259-308 C. Sowden, 20, 1-10 CANTOR,SIDNEYM. See also, Miller, Robert Ellsworth. E CAPON,B., and OVEREND, W. G., Constitution and PhysicochemicalProper- ELDERFIELD,ROBERTC., The Carboties of Carbohydratea, 16, 11-51 hydrate Components of the Cardiac CARR, C. JELLEFF, and KRANTZ,JOHN Glycosidea, 1, 147-173 C., JR., Metabolism of the Sugar EL KHADEM, HASBAN, Chemistry of OsaAlcohols and Their Derivativw, 1, zones, 20, 139-181 175-192 E L KHADEM HASSAN,Chemistry of OsoCHIZHOV, 0.9.See Kochetkov, N. K. thaoles, 18, 99-121 CLAMP,JOHN R., HOIJQH,L., HICKSON, ELLIS,G. P., The Maillard Reaction, 14, JOHN L., and WHISTLER,ROY L., 63-134 Lactose, 16, 159-206 ELLIS, 0. P., and HONEYMAN, JOHN, COMPTON, JACK, The Molecular ConstituGlycosylaminea, 10, 95-168 tion of Cellulose, 8, 185-228 EVANS,TAYLORH., and HIBBERT, CONCHIE,J., LEVVY,G. A., and MARSH, HAROLD,Bacterial Polysaccharides, C. A., Methyl and Phenyl Glyco2, 203-233 sides of the Common Sugars, 12, EVANS,W. L., REYNOLDS, D. D., and 157-187 TALLEY, E. A., The Synthesis of COURTOIS,JEANEMILE, [Obituary of] Oligosaccharides, 6, 27-81 Emile Bourquelot, 18, 1-8 CRUM,JAMES D., The Four-carbon SacF charinic Acids, 18, 169-188

D

FERRIER, R. J., Unsaturated Sugars, 20, 67-137 I ~ V I E BD. , A. L., Polysaccharides of J. A. See Bayne, 8. Gram-negative Bacteria, 16, 271-340 FEWSTER, HEWITTG., JR., The ChemDEAN,G . H., mid GOTTFRIED, J. B., The FLETCHER, istry and Configuration of the CycliCommercial Production of Cryetalline tole, 3, 45-77 Dextrose, 6, 127-143 HEWITT G., JR., and RICHTDIo BELD~ER, A. N., Cyclic Amtala of the FLETCHER, Aldonun and AldoRides, 20, 219-302 MYER, NELSON K., Applications in the Carbohydrate Field of Reductive DEITZ,VICTORR. See Liggett, R.W. Deaulfurization by Raney Nickel, 6, DEWEL,1%.See Mehta, N. C. 1-28 DEUEL,HARRY J., JR.,and MOREHOUSE, MARGARET G., The Interrelation of FLETCHER, HEWITT G., JR. See alao, Carbonydrate and Fat Metabolism, Jeanloe, Roger W. FORDYCE, CHARLESR., Cellulose Eaters I, 119-160 of Organic Acids, 1, 309-327 DEULOFRW,VENANCIO,The Acylated Nitrilea of Aldonic Acids and Their FOSTER, A. B., Zone Electrophoresis of Degradation, 4, 119-151 Carbohydrates, 12, 81-115

CUMULATIVE AUTHOR IYDEX FOR VOLS.

FOL~TER, A. I%, arid ~ ~ O R T I)., O H Asperts , of i tic Chemistry of t,lrc Amino Siigarli, 14, 2l:%-2Xl

I ~ + T E R ,A. J3., arid I ~ ~ J I W AA.R J., I ) ,‘l%e Ctioniihlry c i f I lcparin, 10, :$:$5-;168 FOSTER, A. 13., a i d STACEY,M., The Chemitllry of the %Amino Sugars (2-Amino-2-deoxy-sugars),7, 247-288 FOSTER, A. B., and WEBBER,J. M., Chitin, 16, 371-393 Fox, J. J., and WEMPEN,I., Pyrimidine Nucleosides, 14, 283-380 FRENCH, DEXTER,The Raffinose Family of Oligosaccharides, 9, 149-184 FRENCH,DEXTER,The Schardinger Dextrins, 12, 189-260 FREUDENBERG, KARL, Emil Fischer and his Contribution to Carbohydrate Chemistry, 21, 1-38 G

GARCfA, GONZALEZ,F., Reactions of Monosaccharides with beta-Ketonic Esters and Related Subst.ances, 11, 97-143 GARL‘fA <;ONZ6LEZ, CHEZ,

P.,arld G6MEZ 86N-

A., R.eaut,ionsof Amino Sirgars

with beh-Dicarbonyl Compounds, 20, 303-355

GOEPP, RUDOLPHMAXIMILIAN, JR. See Lohmar, blland. GOLDSTEIN, I. J., and HULLAR,T. L., Chemical Synthesis of Polysaccharides, 21, 431-512 GOMEE, SANCHEZ, A. See Garcia GonxMes, F. GOODMAN, IRVINQ, Glycosyl Ureides, 13, 215-236

GOTTFRIED, J. B. See Dean, G. R. GOITSCHALK, ALFRED, Principles Underlying Enzyme Specificity in the Domain of Carbohydrates, 6, 49-78 GREEN,JOHN W., The Glycofuranosides, 21,95-142.

GREEN,JOHN W., The Halogen Oxidation of Simple Carbohydrates, Excluding the AcLion of Periodic Acid,~.3. 123-184

1-21

557

GREENWOOD, C. T., Aspects of the Physical Chemistry of Starch, 11, 335-385 GREENWOOD, C. T., The Size and Shape of Some Pcilyslmcharide Molecules, 7, Px!)-:$:9’; 11, 385-3‘33 GREENWOOD, C. T. See also, Banks, W. GURIN,SAMUEL, Isotopic Tmcem iii the Study of Carbohydrate Metabolism, 3, 229-250

GUTHRIE, R. D., The “Dialdehydes” from t)he Periodate Oxidation of Carbohydrates, 16, 105-158

H HALL,L. D., Nuclear Magnetic Resonance, 19, 51-93 HANESSIAN, STEPHEN, Deoxy Sugars, 21, 143-207

HARRIS, ELWINE., Wood Saccharification, 4, 153-188 HASKINS,JOSEPH F., Cellulose Ethers of Industrial Significance, 2, 279-294 HASSID,W. Z., and DOUDOROFF, M., Enzymatic Synthesis of Sucrose and Other Disaccharide, 6, 29-48 IIASSID,W. 2. See also, Neufeld, Elizabeth F. HAYNES,L. J., Naturally Occurring CGlycosyl Compoiinds, 18, 227-258; 20, 357-369

HAYNES,L. J., and NEWTH,F. H., The Glycosyl Halides and Their Derivatives, 10, 207-256 HEHRE, EDWARD J., The Substitutedsucrose Structure of Melezitose, 8, 277-290

HELFERICH,BURCKHARDT, The Glycals, 7, 2W245 HELFERICH, BURCKHARDT, Trityl Ethers of Carbohydrates, 3, 79-111 HEYNS, K., and PAULSEN, H., Selective Catalytic Oxidation of Carbohydrates, Employing Platinum Catalysts, 17, 169-221 HIBBERT,HAROLD. See Evans, Taylor H. JOHN L. See Clamp, John R. HICKSON, HILTON,H. W., The Effects of Plantgrowth Substances on Carbohydrate Systems, 21, 377-430

558

CUMULATIVE AUTHOR INDEX FOR VOLS.

I IINUEHT, MARJURIID. ,gee KWdJiilOB, J. v. IInwT, 15. L., [Obituary of] Jamw colqiihoiin Irvine, 8, xi-xvii EIIRBT,EL L., [Obituary of] Walter Norman Haworth, 6, 1-9 HIRBT,E. L., and JONES, J. K. N., The Chemistry of Pectic Materials, 2, 235-251

HIRST,E. L., and Ross, A. G., [Obituary of] Edmund George Vincent Percival, 10, xiii-xx HODGE,JOHNE., The Amadori Rearrangement, 10, 169-205 HONEYMAN, JOHN, and MORGAN, J. W. W., Sugar Nitrates, 12, 117-135 HONEYMAN, JOHN. See also, Barry, C. P. HONEYMAN, JOHN. See also, Ellis, G. P. HORTON, D., [Obituary of] Alva Thompson, 19, 1-6 HORTON,D., Tables of Properties of 2Amino-2deoxy Sugars and Their Derivatives, 16, 159-200 HORTON,D., and HUTSON,D. H., Developments in the Chemistry of Thio Sugars, 18, 123-199 HORTON, D. See aho, Foster, A. B. HOUOH,L., and JONES, J. K, N., The Biwynthwis of the Monosaccharides,

1-21

IIULLAR, T. L. See Coldstuin, I. J. IIUTBON, D. H. See Horton, D.

J JEANLOZ, ROGERW., [Obituary of] Kurt Heinrich Meyer, 11, xiii-xviii JEANLOZ, ROOPRW., The Methyl Ethers of ZAmino-2deoxy Sugars, 13, 189214

JEANLOZ, ROGER W., and FLETCHER, HEWITTG., JR., The Chemistry of Ribose, 6, 135-174 JEFFREY, G. A,, and ROBENSTEIN, R. D., Crystal-structure Analysis in Carbohydrate Chemistry, 19, 7-22 JONXIS, DAVIDM., Structure and Some Reactions of Cellulose, 19, 219-246 JONES, J. K. N., and SMITE, F., Plant Gums and Mucilages, 4,243-291 JONES, J. K. N. See also, Hirst, E. L. JONES, J. K. N. See also, Hough, L. JONSEN, J., and LALAND,S., Bacterial Nucleosidea and Nucleotides, 16, 201-234

K

11, 185-262 KARABINOB, J. V., Psicose, Sorbose and HOUQH,L., PRIDDLE, J. E., and THEOTagatom, 1, 99-136 BALD, R. S., The Carbonates and KARABINOS, J. V., and HINDERT,MARThiocarbonates of Carbohydrates, 18, JORIP, Carboxymethylcellulose, @, 91-158

HOUGH,L. See aieo, Clamp, John R. How, M. J., BRIMACOMBE, J. S., and STACEY,M., The PneumococcalPolysaccharides, 19, 303-367 HUDSON, C. S., Apiose and the Glycosidea of the Parsley Plant, 4, 57-74 HUDSON, C. S., The Fischer Cyanohydrin Synthesis and the Configurations of Higher-carbon S u g m and Alcohols, 1, 1-36 HUDSON, C. S., Historid Aspects of Emil Fischer’s Fundamental Conventions for Writing Stemformuha in a Plane, 8, 1-22 HUDSON, C. S., Melezitose and Turanoae, 2, 1-36

HuaQARD, A. J. See Foster, A. B.

285-302

KENT,P. W. See Stacey, M. KERTESZ,2. I., and MCCOLMCH,R. J., Enzymes Acting on Pectic Substances, 6,79-102 KLEMER, ALMUTH.See Micheal, Fritz. KOCHETKOV, N. K., and CHIZHOV,0. R., MW Spectrometry of Carbohydrate Derivatives, 21, 39-93 KOWKABANY, GEORQEN., Paper Chromatography of Carbohydrates and Related Compounds, 9,303-353 KRANTZ, JOHN C., JR.See Carr, C. Jeleff.

L LAIDWW,R. A., and PERCIVAL, E. G. V., The Methyl Ethers of the Aldopen-

CUMULATIVE AUTHOR ISDEX FOR VOLS.

1-21

559

MCCL~SKEY, CHESTER &I., Benzyl Ethers ic w s rid of Ithamnosc and FIICOSC, of Sugars, 12, 137-156 7, 1 -30 XI(:COLI,OCH, R. J. See Kertesz, Z. I. I,ALANI), S.See Jonrieti, d. EMMA J., The Polyfructosttiis I,EDEREH,E., Glywlipids (Jf Acid-fast MCDONALD, and Difriictose Anhydrides, 2, 25313ac:teria, 16, 207-238 2i7 LEMIEUX, R. U., Some Iriiplical,imri in Carbohydrate Chemistry of Theories Relating to the Mechanisms of Replacement Reactions, 9, 1-57 LEMIEUX,R. U., and WOLFROM, M. L., The Chemistry of Streptomycin, 3, 337-384

MEHLTRETTER, C. L., The Chemiwl Synt,hesis of D-Glucuronic Acid, 8, 231-249

MEHTA,N. C., DUBACH, P., and DEUEL, H., Carbohydrates in the Soil, 18, 335-355

LESPI~AU, R,., Synthesis of Hexitols and MESTER,L., The Formazan Reaction in Carbohydrate Research, 13, 105-167 Pentitols from Unsaturated PolyMESTER,L., [Obituary of] G6za Zemplhn, hydric Alcohols, 2, 107-118 LEVI,IRVING, and PURVES, CLIFFORD B., 14, 1-S and KLEMER,ALMUTH, The Structure and Configuration of MICHEEL,FRITZ, Glycosyl Fluorides and Azides, 16, Sucrose (alpha-D-Glucopyranosyl beta85-103 n-Fructofuranoside), 4, 1-35 and CANLEVVY, G. A., and MARSH,C. A., Prepara- MILLER,ROBERTELLSWORTH, TOR, SIDNEY M., Aconitic Acid, a tion and Properties of &GlucuroniBy-product in the Manufacture of dase, 14, 381428 Sugar, 6, 231-249 LEVVY, G. A. See also, Conchie, J. LIGGETT,R. W., and DEITZ,VICTORR., MILLS, J. A., The Stereochemistry of Color and Turbidity of Sugar ProdCyclic Derivatives of Carbohydrates, ucts, 9, 247-284 10, 1-53 LINDBERO, B. See Bouveng, H. 0. JOHN A., A N D THOMAS, H. MONTGOMERY, LOHMAR, ROLLAND, and GOEPP,RUDOLPH JEANETTE, Purine Nucleosides, 17, MAXIMILIAN, JR.,The Hexitols and 301-369 MOODY,G. J., The Action of Hydrogen Some of Their Derivatives, 4,211-241 Peroxide on Carbohydrates and ReM lated Compounds, l B , 149-179 MAHER,GEORGE G., The Methyl Ethers MOREHOUSE, MARGARET G. See Deuel, of the Aldopentoses and of Rhamnose Harry J., Jr. and Fucose, 10, 257-272 MORGAN, J. W. W. See Honeyman, Joliri. MAHER,GEORGE G., The Methyl Ethers MORI, T., Seaweed Polysaccharides, 8, 315-350 of n-Galactose, 10,273-282 MALHOTRA, OMPRAKASH. See Wallenfels, MUETGEERT,J., The Fractionation of Kurt. Starch, 16, 299-333 MANNERS, D. J., Enzymic Synthesis and MYRB~CK, KARL,Products of the EuDegradation of Starch and Glycogen, zymic Degradation of Starch and 17, 371-430 Glycogen, 3, 251-310 MANNERS, D. J., The Molecular Structure N of Glycogens, 12, 261-298 MARSH,C. A. See Conchie, J. NEELY,W. BROCK,Dextran: Structure and Synthesis, 16, 341-369 MARSH,C. A. See Levvy, G. A. MCCABLAND, G. E., Chemical and Phys- NEELY,W. BROCK,Infrared Spectra of ical Studies of Cyclitols Containing Carbohydrates, 12, 13-33 Four or Five Hydroxyl Groups, 20, NEUBERQ, CARL,Biochemical Reductions 11-65 a t the Expense of Sugars, 4, 75-117

500

CITMI~LATIVE AUTHOR IXDEX FOB VOLS.

l ’ ~ I d I X A R I T I I Id’., t t t d I~AHHID, W. %., BiosynLhedn of Sltccharideri from Ulycopyranonyl Eatern of Nucleotides (“Sugar Nucleotides”), 18, 309356 NEWTH,F. H., The Formation of Fiimii Compounds from Hexoses, 6, 83-106 NEWTH,F. H. See also, Haynes, L. J. NICKEREON, R. F., The Relative Cryatallinity of Celluloses, 6, 103-126 NORD,F. F., [Obituary of] Carl Neuberg, 1SI 1-7

N&tica1.b,

0 OLSON,E. J. See Whwtler, Roy L. OVEREND, W. G., and STACEY, M., The Chemistry of the ZDesoxy-sugars, 8, 45-105 OVEREND, W. G. See also, Capon, B.

P

1-21

IZBEVEH, I ~ I O H A H U IC., CiiprammoniumGlycoclide Complexes, 6, 107-134 I~EICHSTEIN, T., and WEISS, EKKEHARD, The Sugam of the Cardiac Glycosides, 17, 65-120 I ~ N D L E M AJ.N ,A,, JR., Complexen of Alkali Metals and Alkaline-earth Metals with Carbohydrates, 21, 209271 REYNOLDS, D. D. See Evans, W. L. R.ICHTMYER, NELSONK., The Altrose Group of Substances, 1,37-76 RICHTMYER,NBIMONK., The 2-(aZdoPolyhydroxyalkyl)benzimidazoles, 6, 175-203 RICHTMYER, NELSONK. See also, Fletcher, Hewitt G., Jr. ROBENSTEIN, R. D. See Jeffrey, G. A. Ross, A. G. See Hirst, E. L. S

SANDS, LILA.See Anderson, Ernest. SATTLER, LOUIS, Glutose and the Unfermentable Reducing Substances in Cane Molasses, 8, 113-128 SCHOCH, THOMAS JOHN, The Fractionation of Starch, 1, 247-277 SHAFIZADEH, F., Branched-chaii Sugars of Natural Occurrence, 11, 263-283 SHAFIZADEH, F., Formation and Cleavage of the Oxygen Ring inSugars, 13,9-61 SMITH,F., Analogs of Ascorbic Acid, 2, 79-106 SMITH,F. See also, Jones, J. K. N. SOWDEN, JOHN C., The Nitromethane and 2-Nitroethanol Syntheses, 6, 291-318 JOHN C., [Obituary of] Hermann SOWDEN, Otto Laurenz Fischer, 17, 1-14 SOWDEN, JOHN C., The Saccharinic Acids, 12, 35-79 SPECK, JOHNC., JR., The Lobry de Bruyn-Alberda van Ekenstein Transformation, 13, 63-103 SPIDDINCI, H., Infrared Spectroscopy and Carbohydrate Chemistry, 10, 23-49 SPRINSON, D. B., The Biosynthesis of b m a t i c Compounds from D-clucone, 16, 235-270 B M., The Chemistry of MucoSTACIOY, RAYMOND, ALBERTL., Thio- and Selenopolysaccharides and Mucoproteins, Rugam, 1, 129-145 2, 161-201

PACSU,EUQENE, Carbohydrate OrthoeHteM, 1, 77-127 PAULSEN, H. See Heyns, K. PEAT,STANLEY, The Chemiatry of Anhydro Sugars, 2, 37-77 PEAT,QTANIBY. See also, Bourne, E. J. PERCIVAL, E. G. V., The Structure and Reactivity of the Hydrazone and Osazone Derivatives of the Sugars, 3, 23-4.4 PERCIVAL, E. G. V. See also, Laidlaw, R. A. PERLIN,A. S., Action of Lead Tetraacetate on the Sugars, 14, 9-61 PHILLIPS, G. O., Photochemistry of Carbohydrateg, 1899-59 PHILLIPS, G. O., Radiation Chemistry of Carbohydrates, 16, 13-58 POLOLASE, W. J., Polysaccharides h o ciated with Wood Cellulose, 10, 283-333 PRIDDLE, J. E. See Hough, L. PRIDHAM, J. B., Phenol-Carbohydrate Derivatives in Higher Planta, 20, 371-408 PRIDHAM, J. B. See also, Bailey, R. W. PURVER, CLIFFORD B. See h v i , Irving.

CUMULATIVE AUTHOR INDEX FOR VOLS.

STACEY, M., and KENT,P. W., The PolyRaccharides of Mycobaderium tubercu~OS~S 3,, 311-336 STACEY, M. See also, Bray, H. G. STACEY, M. See also, Foster, A. B. STACEY, M. See also, How, M. J. STACEY, M. See also, Overend, W. G. STOLOFF, LEONARD,Polysaccharide Hydrocolloids of Commerce, 13,265-287 SUGIHARA, JAMES M., Relative Reactivities of Hydroxyl Groups of Carbohydrates, 8, 1-44

T TALLEY, E. A. See Evans, W. L. TEAWE,ROBERTS., The Conjugates of DGlucuronic Acid of Animal Origin, 9,185-246

THEANDER, OLOF, Dicarbonyl Carbohydrates, 17, 223-299 THEOBALD, R. S. See Hough, L. THOMAS, H. JEANETTE. See Montgomery, John A. TIMELL, T. E., Wood Hemicelluloses: Part I, 19, 247-302; Part 11, 20, 409-483 TIPBON, R. STUART, The Chemistry of the Nucleic Acids, 1, 193-245 TIPSON,R. STUART,[Obituary of] Harold Ribbert, 16, 1-11 TIPRON, R.. STIJART, [Obituary of] Phoebus Aaron Theodore Levene, 12, 1-12 TIPSON, R. STUART,Sulfonic Esters of Carbohydrates, 8, 107-215 TURVEY, J. R., Sulfates of the Simple Sugars, 20, 183-218

1-21

561

WEBBER,J. M., Higher-carbon Sugars, 17, 1583

WEBBER,J. M. See also, Foster, A. B. WEIGEL, H., Paper Electrophoresis of Carbohydrates, 18,61-97 WEISS, EKKEAARD. See Reichstein, T. WEMPEN,I. See Fox, J. J. WHISTLER, ROY L., Preparation and Properties of Starch Esters, 1, 279-307

WHISTLER,ROYL., Xylan, 6, 269-290 WHISTLER, ROYL., and BEMILLER,J. N., Alkaline Degradation of Polysaccharides, 13, 289-329 WHISTLER,ROY L., and OLSON,E. J., The Biosynthesis of Hyaluronic Acid, 12, 299-319

WHISTLER,ROY L. See uko, Clamp, 3ohn R. WHITEHOUBE, M. W. See Zilliken, F. WIGGINS, L. F., Anhydrides of the Pentitols and Hexitols, 6, 191-228 WIGQINS,L. F., The Utilization of Sucrose, 4, 293-336 WISE, LOUIS E., [Obituary of] Emil Heuser, 16, 1-9 WOLFROM, M. L., [Obituary of] Claude Silbert Hudson, 9, xiii-xviii WOLFROM, M. L., [Obituary of] Rudolph Maximilian Goepp, Jr., 3, xv-xxiii WOLFROM, M. L. See also, Binkley, W. W. M. L. See also, Lemieux, R. U. WOLFROM, Z

ZILLIKEN,F., and WAITEEOUSE,M. W., The Nonulosaminic Acids-Neuramink Acids and Related Compounds (Sialic Acids), 13, 237-263 W ZORBACH, W. WERNER,and BHAT,K. WALLENFELS, KURT,and MALHOTRA, OM VENKATRAMANA, Synthetic CardePRAKASH, Galactosidases, 10,239-298 nolides, 21, 273-321

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-21 A

Acetals, cyclic, of the aldom atid aldmidw, 20, 219-302

of hexitols, perititrh, and tetritols, 7, 137-207

Acetic acid, trifluoro-, anhydride, applications of, in carbohydrate chemistry, 16, 59-84 Aconitic acid, 6, 231-249 Adsorbents, granular, for sugar refining, 6, 205-230 Alcohols, higher-carbon sugar, configurations of, I,, 1-36 unsaturated polyhydric, 2, 107-118 Aldonic acids, acylated nitriles of, 4, 119-151 Aldopentoses, methyl ethers of, 7, 1-36; 10, 257-272 Aldoses and aldosidea, cyclic acetals of, 20, 219302 Alkaline degradation, of polysaccharides, 18, 289-329 Altrose, group of compounds related to, 1,37-76 Amadori rearrangement, 10, 169-205 Amino sugars. See Sugars, 2-amino-2deoxy. Amylases, certain alpha, 6, 229-268 Analysin, of myth1 striicture, in carbohydrate chemintry, 19, 7-22 Anhydrides, difrur!iom, 2, 253-277 of hexitok, 6, 1!)1-228 of peiibil.olw, 6, Il)l-T28 Auhydm ~ugaw.the !hga[LpR, anhydrn. AriimalH, roiijiigates of n-glururonic acid originating in, 9, 186-240

Antibiotic substances, chemistry of the amino sugars derived from, 18, 259-308 Apiose, 457-74 Ascorbic acid, analogs of, 2, 70-106 Aromatic compounds, biosynthesis of, from D-glUCWe, 16, 235-270

B Bacteria, glycolipidea of acid-faat, 16, 207-238 nucleoaida and nucleotidea of, 16, 201-234

polyseccharides from, 2, 203-233; 8, 311-338

polysaccharides of Gram-negative, 16, 271-340

Benrimidaeolea, 2-(aldo-poyhydroxydkyl)-,6, 175-203 Benzyl ethers, of sugars, 12, 137-156 Biochemical reductions, a t the expense of sugaw, 4, 75-117 Biosynthesis, of aromatic compounds from D-glucose, 16,235-270

of hyaluronic acid, 12, 299-319 of the monosacoharides, 11, 185-262 of saccharides, from glycopyranosyl eaters of nucleotidea, 18,309-356 Blood groups, polyseccharides of, 4,37-55 Boric acid, for determining configuration of wrbohydrah, 4, 180-210 Bourquelot, Emile, obituary of, 18, 1-8 Branched-chain sugars. See Sugan, branched-chain.

562

CUMULATIVE SUUJEC” INDEX FOR VOLS.

C

Cane juioe, composition of, 8, 291-314 Cane molassa. See Molasses, cane. Carbohydrates, action of hydrogen peroxide on, 19, 149-179

application of reductive desulfuriratip by Raney nickel, in the field of, 6, 1-28

application of trifluoroacetic anhydride in chemistry of, 16, 59-84 as components of cardiac glycosides, 1, 147-173

carbonates of, 16, 91-158 chemistry of, Emil Fischer and his contribution to, 21, 1-38 complexev of, with alkali metals and alkaline-earth metals, 21, 209-271 constitution of, 16, 11-51 crystal-structure analysis of, 19, 7-22 degradation of, 19, 181-218 determination of configuration of, with boric acid, 4, 189-210 dicarhonyl, 17, 223-299 enzyme specificity in the domain of, 6, 49-78

formazan reaction, in research on, 13, 105-167

Friedel-Crafts and Grignard processes applied to, 6, 251-289 gas-liquid chromatography of derivatives of, 19, 95-147 halogen oxidation of simple, 3, 129-184 infrared spectra of, 12, 13-33 infrared spectromopy of, 19, 23-49 masa spectrometry of derivatives of, 21,39-93

mechanisms of replacement reactions in chemistry of, 9, 1-57 metabolism of, 2, 119-160; 3,229-250 orthoesters of, 1, 77-127 paper electrophoresis of, 18, 61-97 periodute oxidation of, 11, 141 t.he “tlialdehydes” from, 16, 10.5-158 phenol rlerivetives, in higher plants, 20, 37 1-408

photmhemistry of, 18, 9-59 ~)hynii.oc.lic-Iiiiclll properlien of, 16, 11-51

563

1-21

radiation chemistry of, 16, 13-58 and related compounds, action of hydrogen peroxide on, 19, 149-179 paper chromatography of, 9,303353 relative reactivities of hydroxyl groups of, 8, 1-44 selective catalytic oxidation of, employing platinum catalysts, 17, 169-221 in the soil, 16, 335-355 stereochemistry of cyclic derivatives of, 10, 1-53

sulfonic esters of, 8, 107-215 systems, effects of planegrowth substances on, 21,377-430 thiocarbonates of, 16, 91-158 trityl ethers of, 3, 79-111 zone electrophoresis of, 12, 81-115 Carbonates, of carbohydrates, 16, 91-158 Carboxymethyl ether, of cellulose, 9, 285-302 Cardenolides, synthetic, 21, 273-321 Catalysts, platinum, in selective catalytic oxidation of carbohydrates, 17, 169-221 Cellulose, carboxymethyl-, 9,285-302 esters of, with organic acids, 1, 309-327 ethers of, 2, 279-294 molecular constitution of, 3, 185-228 of wood, polysaccharides associated with, 10, 283-333 Celluloses, relative crystallinity of, 6, 103-126 some reactions of, 19, 219-246 structure of, 19, 219-246 Chemistry, of the amino sugars, 14, 213-281 of the 2-amino sugars, 7, 247-288 of anhydro sugars, 2, 37-77 of carbohydrates, applications of trifluoroacetic anhydride in, 16, 59-84 Emil Fischer and his contribution to, 21, 1-38

crystal-structure

analysis

in,

19,

7-22

infrared spectroscopy and, 19, 23-49 some implications of theories relating to the mechanisms of replacement, react.ions in, 9, 1-57

564

CUMULATIVE SUBJECT INDEX FOR VOLE.

Chemistry-Continued of the cyclitols, 8, 45-77 of cyclitols containing four or five hydroxyl group, 20, 11-65 of t,he 2deoxy twgars, 8, 45-105 of heparin, 10, 335-368 of mucopolysaccharides and mucoproteins, 2, 161-201 of the nucleic acids, 1, 193-245 of osazones, 20, 139-181 of osotriazoles, 18, 99-121 of pectic materials, 2, 235-251 of ribose, 6, 135-174 of streptomycin, 8, 337-384 of t h o sugars, 18, 123-199 physical, of carbohydrates, 16, 11-51 of starah, 11, 335-385 radiation, of carbohydrates, 16, 13-58 stereo-, of cyclic derivstives of carbohydrates, 10, 1-53 structural, of the hemicellulosm, 14, 429-468 of polysaccharides, 15, 58-89 Chitin, 16, 371-393 Chromatography, column. See Column chromatography. gas-liquid. See Gas-liquid chromatography. paper. See Paper chromatography. Color, of sugar products, 9, 247-284 Column chromatography, of sugars and their derivativea, 10,55-94 Complexes of carbohydrates, with alkali metals and alkaline-earth metals, 21, 209-271 cuprammonium-glycoside, 6, 107-134 Configuration, of carbohydrates, determination of, 4, 189-210 of cyclitols, 8, 45-77 of higher-carhi RiiRRr ahhnlx, 1, 1-86 of ~ucrn~n, 4, 1-36 Corijirgulu, of n-glu(!umriic! wid, 9, 18&2M Coiistitiition, of carbohydraten, 16, 11-51 Crystallinity, relative, of cellulolies, 6, 103-126 Cuprammoniiim-glyooside complexes, 6, 107-134

1-21

Cyanohydrin synthesia, Fischer, 1, 1-36 Cyclic acetals, of the aldoses and aldosidm, 20, 219-302 Cyclic derivativee, of carbohydrata, Rtereochemiatry of, 10, 1-63 Cyclitols, 14, 135-212 chemistry and configuration of, 8, 45-77 containing four or five hydroxyl groups, chemical and physical studies of, 20, 11-65

D Degradation, of acylated nitriles of aldonic acids, 4, 119-151 of carbohydrates, 19, 181-218 enzymic, of glycogen and atarch, 3, 251-310; 17, 407-430 3-Deoxyglycosnnes, See Glycosuloses, 3deoxy-.

3-Deoxyglycosulmes. See Glycosuloses, 3-deoxy-. Deoxy sugars. See Sugam, deoxy. Desulfurization, reductive, by Raney nickel, 6, 1-28 Dextran, structure and synthesis of, 16, 341369 Dextrins, the Schardinger, 12, 189-260 Dextrose, commercial production of crystalline, 6, 127-143 “Dialdehydes,” from the periodate oxidation of carbohydrates, 16, 105-158 Dicarbonyl derivatives, of carbohydrates, 17, 223-299 IXfructcxce, anhydrides, 4, 253-277 I)isaccharides, enzymic RynthesiR of, 6, 2%4R trehalose, 18, 201-225

E Elect,mphoresis,of carbohydrates, paper, 18, 61-97 zone, 12, 81-115

CUMULATIVE SUBJECT INDEX FOR VOLS.

Eneymes. See also, Amylases, Galactosidasea, pGlucuronidese. acting on pectic substances, 6, 79-102 degradation by, of starch and glycogen, 3, 251-310; 17, 407-430 specificity of, in the domain of carbohydrates, 6, 49-78 synthesis by, of glycogen and starch, 17, 371-407

of sucrose and other disaccharides, 6, 2

M

Esters, of cellulose, with organic acids, 1, 309-327

glycopyranosyl, of

nucleotides, 18,

309-356

beta-ketonic (and related substances), reactions with monosaccharides, 11,

1, 279-307

sulfonic, of carbohydrates, 8, 107-215 Ethanol, %nitro-, syntheses with, 6, 291-318 Ethers, benzyl, of sugars, 12, 137-156 carboxyrnethyl, of cellulose, 9, 285-302 of cellulose, 2, 279-294 methyl, of the aldopentoses, 7, 1-36; 10, 257-272

of %amino-2deoxy sugars, 13, 189214

565

Formulas, stereo-, writing of, in a plane, 3, 1-22 Fractionation, of starch, 1, 247-277; 16, 299-333 Friedel-Crafts proc~ss, in the carbohydrate Heries, 6, 251-289 Fructana, 2, 253-277 Fructofuranoside, wD-glucopyranosyl p-m, 4, 1-35 Fructosans, poly-. See Fructans. Fructose, and its derivatives, 7, 53-98 di-, anhydridea, 2, 253-277 Fucose, methyl ethers of, 7, 1-36; 10, 257-272 Furan compounds, formation from hexoses, 6, 83-106

97-143

nitrate, of starch, 13, 331-345 of starch, preparation and properties of,

1-21

G

Galactose, methyl ethers of D-, 6, 11-25; 10, 273-282

Galactosidaaea, 16, 239-298 Gas-liquid chromatography, of carbohydrate derivatives, 19, 95-147 Glucose. See aleo, Dextrose. biosynthesis of aromatic compounds from D-, 16, 235-270 methyl ethers of D-, 6, 145-190 Glucuronic acid, D-, chemical synthesis of, 8, 231-249 conjugates of, of animal origin, 9, 185-246

of of of of

fucose, 7, 1-36; 10,257-272 D-galactose, 6, 11-25; 10, 273-282 D-glUCose, 6, 145-190 hexuronic acids, 9, 131-148 of D-mannose, 8, 217-230 of rhamnose, 7, 1-36; 10, 257-272 trilyl, of carbohydratw, 3, 70-111

F Fat, metabolism of, 2, 1l(9-100 Fincher, Emil, and his coritributiori t o carbohydrate chemistry, 21, 1-38 Fischer, Hermann Ot,to Laurenz, obituary of, 17, 1-14 Formazan reaction, in carbohydrate reclearch, 13,~105-167

in metabolism, 8, 251-275 8-Glucuronidaae, preparation and properties of, 14, 381-428

Glutose, 3, 113-128 Glycals, 7, 209-245 -, 2-hydroxy-, 9, 97-129 Glycofuranosides, 21, 95-142 Glycogens, enzymic degradation of, 3, 251-310; 17, 407-430

enzymic synthesis of, 17, 371-407 molecular structure of, 12, 261-298 Glycolipides, of acid-fast bacteria, 16, 207-238 Glycoside-cuprammonium complexes, 0, 107-134

1-21

CUMULATIVE SUUJECT INDEX FOR VOW.

,560

Glycosides, alkali-ae~itive,9, 50-06 cardiac, 1, 147-173 the sugsrs of, 17, 6b120 methyl, of the common sugars, 10, 157-187

of the parsley plant, 4, 67-74 phenyl, of the common sugars, 12, 167-187

GlycoBiduronic acids, of animals, 9, 186-246 poly-, of plants, 1, 3294344 Glycosones, Meoxy-. See C3lyctmulom, 3d~xy-. Glycosuloaes, 3-deoxy-, and the degradation of carbohydrates, 19, 181-218 Glycosylamines, 10, 95-188 Glycosyl asides, 16, 85-103 C-Glycolryl compounds, naturally occurring, 18, 227-268; 00, 357-369

Glycosyl fluorides, 16, 86-103 Glycosyl halides, and their derivatives, 10, 207-256 Geopp, Rudolph Maximilian, Jr., obituary of, 8, xv-xxiii Grignard process, in the carbohydrate series, 6, 251-289 Gums. See also, Hydrocolloida. commercial, 18, 285-287 of plants, 4, 243-291

H Halogen oxidation. Isee Oxidation, halogen. Haworth, Walter Norman, obituary of, 6, 1-9 Hemidulows, nt.ructara1 chemistry of, 14, 428468 of wood, 19, 247-302; 90, 400-483 I feparin, cheminlry of, 10, 836-3813 Ileiimr, Emil, ohit8iiaryof, 16, 1-9 llexit OIN, acetals of, 7, 137-207 anhydrides of, 6, 191-228 and some of their derivatives, 4 2 1 1-241 synthesis of, I,107-114 Hexofuranoses, 1,&anhydro-, 7, 37-52

Hexoaans, 7 , 3 7 4 2 Hexoses. SSe also, Hexofurenosas. formation of furan compounds from, 6, 83-106

'

Hexuronic eoids, methyl ethers of, 9, 131-148 Hibbert, Hamld, obituary of, 16, 1-11 Hiidwn, Claude Silbert, obituary of, 0, xiii-xviii Il yaluronic acid, biosynthwis of, 12, 200-319 I l ydrasonea, Of RUp&W, 8, 23-44 Hydrocolloids, commercial, polysaccharidic, 18, 265287

Hydrogen peroxide, action, on carbohydrates and related compounde, 19, 140-179 JWmxyl ~ ~ O U P , relative reactivities of, 8, 1-44 I

Infrared spectra, of carbohydrates, ll, 13-33 Infrared rcpectroscopy, and carbohydrate chenlistry, 19, 23-49 Irvine, James Colquhoun, obituary of, 8, xi-xvii Isotopic t r a m . Isee Tracers, isotopic.

K Ketale. See Acetals. Kojic acid, 11, 145-183

L Lactose, 16, 159-206 Lead tetraacetate, action of, on the sugars, 14, 9-61 Levene, Phoebub Aaron Theodor, obituary of, 12, 1-12 Lobry de Bruyn-Alberda van Ekenstein tratisformation, 18, 83-103

M Maillard reaction, 14, 83-134 Mannose, methyl ethem of D-, 8, 217-230

CUMULATIVE SUBJECT INDEX FOR VOLS.

Maee spectrometry,

of carbohydrate derivatives, 21, 39-93 Mechanism, of replacement ref3CtiOnS in carbohydrab chemistry, 9,147 Melmitose, 2,138 structure of, 8,277-290 Metabolism, of carbohydrates, 2, 119-160 use of isotopic tracers in studying, 3, 229-260 of fat, 2, 119-160 of the sugar alcohols and their derive tivea, 1, 175-192 n-glucuronic acid in, 8, 251-275 Methane, nitro-, syntheses with, 6, 291-318 Methods, in structural polysaccharide chemistry, 16, 53-89 Methyl ethers. See Ethers, methyl. Meyer, Kurt Heinrich, obituary of, 11, xiii-xviii Moleseps, cane, 8, 113-128 cane find, composition of, 8, 291-314 Molecular structure, of glycogens, 12,261-298 Monoeaccharides, biosynthesis of, 11, 185-262 reactions of, with beta-ketonic estara and related substances, 11, 97-143 Mucilages. 8~ also, Hydrocolloids. commercial, 13,265-287 of plants, 4,243-291 Mucopolyaaccharidea. See Polyaaccharides, mum-. Mucoproteins. See Proteins,muco-. Mycobacttmurn tubernclos4e, polyaacchsrides Of, 8, 311-336

N Nenberg, Carl, obituary of, 13, 1-7 Neuraminic acids, arid related compounds, 13,237-263 Nickel, Raney. See h n e y nickel. Nitrates, of atmch, 13, 331-345 of sugars, 12, 117-135

1-21

567

Nitrilea, acylated, of aldonic acids, 4, 119-151 Nonuloesminic acids, 18, 237-263 Nuclear magnetic mnance, 19, 51-93 Nucleic acids, 1, 193-245; 11, 285-333 Nucleosidea, baderial, 16,201-234 purine, 17, 301-369 pyrimidine, 14, 283-380 Nucleotides, bacterial, 16, 201-234 glycopyranosyl esters of, 18,309-356 0

Obituary, of Emile Bourquelot, 18, 1-8 of Hermann Otto Laurene Fischer, 17, 1-14 of Rudolph Maximilian Goepp, Jr., 3, xv-di of Walter Norman Haworth, 6,14 of Emil Heuser, 16,l-Q of Harold Hibbert, 14, 1-11 of Claude Silbert Hudson, 9, xiii-xviii of James Colquhoun Irvine, 8, xi-xvii of Phoebua Aaron Theodor Levene, la, 1-12 of Kurt Heinrich Meyer, 11, xiii-xviii of Carl Neuberg, 13, 1-7 of Edmund George Vincent Percival, 10, xiii-xx of John Clinton Sowden, 10, 1-10 of Alva Thompson, 19,ld of G6sa Zempl6n, 14, 1-8 OIigosaccharides, 17, 121-167 the raffinose family of, 9, 149-184 synthesis of, 6, 27-81 Orthoesters, of carbohydratea, 1, 77-127 Osasones, chemistry of, 20, 139-181 of sugars, s,23-44 Osones, 11, 43-96 Osotriasolw, chemistry of, 18, 99-121 Oxidations, halogen, of simple carbohydrates, 3, 129-148 lead tetraacetate, of sugars, 14, 9-61

568

CUMULATIVE SUBJECT INDEX FOR VOLS.

Oxidntions-Continusd periodate, of wbohydrata, 11, 1-41 the “dialdehydee” from, 16, 105-168 selective catalytic, of carbohydrates, employing platinum ostalyete, 17, 169-221

Oxygen ring, formation and cleavage of, in sugars,13, 9-61

P Paper chromatography, of carbohydrates and related compounds, e, 303-363 Paper electrophoresis, of carbohydrates, 18,61-97 Parsley, glycosides of the plant, 4, 67-74 Pectic materiala, chemistry of, 2, 236251 enaymes acting on, 6, 79-102 Pentitoh, acetala of, 7, 137-207 anhydrides of, 6, 191-228 Byntheais of, 2, 107-118 Percival, Edmund George Vincent, obituary of, 10, xiii-xx periodah oxidation. Scs Oxidation, periodate. Phenol-casbohydrate derivatives, in higher plants, 20,371-408 Photochemistry, of carbohydrates, 18,9-59 Physical chemistry, of Carbohydrates, 16, 11-51 $ starch, 11,335-385 Physical properties, of solutions of polysaccherides, 18, 367-398

Physical studies, of cyclitola containing four or five hydroxyl groups, 20, 11-66 Plantgrowth substanma, effect on carbohydrate system, 21,377430

Plants, glycosides of parsley, 4, 67-74 of, 4,243-291 mucilages of, 4, 243-291 polyuronides of, 1, 329-344

1-21

Platinum. See Catalysts. Pneumococoal polysaccharides,19,303-367 Polyfruchmm. See Fructsns. Polyglycosiduronic acids. Sea Glycosiduronic acidB, poly-. Polysaccharidea. See aleo, Carbohydrates, Cellulose, Dextragp, Dextrins, Fructans, Glycogen, Glycosidumnic acids (poly-), Pectin materials, Starch, and xylan. alkaline degradation of, 13, 289-329 aasociated with wood cellulose, 10, 283-333

bacterial, I, 203-333; 16,271440 blood group, 4,37-55 chemical synthesis of, 21, 431-512 hydrocolloidal, l3, 266-287 methods in structural chemistry of, 16, 63-89 muco-, chemistry of, 2, 161-201 of Gram-negative bacteria, 16, 271-340 of Mgmbaclerium tuberculosis, 8,311-336 of seaweeds,8, 315-350 physical properties of solutions of, 18, 357-398

pneumocoud, 19, 303-357 shape and size of molecules of, 7, 289332; 11, 385-393 Poly uronides, of plants, 1, 329-344 Preparation, of esters of starch, 1, 279-307 of pglucuronidase, 14, 381-428 Properties, of 2-amino-2-deoxy sugars and their derivatives, 16, 159-200 of esters of starch, 1, 279-307 of &glucuronidaae, 14, 381-428 physical, of solutions of polysaccharides, 18, 357-398

physicochemical, of carbohydrates, 16, 11-51

Proteins, mucQ., chemistry of, 3, 161-201 Psicose, 7, QQ 13-6 Purinea, nucleosidea of, 17, 301-369 Pyrimidines, nucleosides of, 14, 283-380

CUMULATIVE SUBJECT INDEX FOR VOLS.

R Radiation, chemistry of carbohydrates, 16, 13-58

1-21

569

Sialic acids, 13, 237-263

si,

of some polysaccharide molecules, 7,

289-332; 11, 385-393 Soil, family of oligosaccharides, 9, 149-184 carbohydrates in, 16,335-355 h n e y nickel, Solutions, reductive desulfurization by, 6, 1-28 of polyeaccharides, physical properties Resction, of, 18, 357-398 the formasan, in carbohydrate research, Sorbose, 7, 99-136 18, 105-167 Sowden, John Clinton, the Maillard, 14, 63-134 obituary of, 20,1-10 Reactions, Specificity, of amino sugars with beta-dicarbonyl of enzymes, in the domain of carbocompounds, 20, 303-355 hydrates, 6,49-78 of oBllulose, 19, 219-246 of monosaccharides with beia-ketonic Spectra, infrared, of carbohydrates, 12, 13-33 esters and related substances, 11, Spectrometry, m m , 97-143 of carbohydrate derivatives, 21, 39-93 Reactivities, relative, of hydroxyl groups of carbo- Spectroscopy, infrared, and carbohydrate chemistry, 19, 23-49 hydrates, 8, 1-44 Starch, Rearrangement, enzymic degradation of, 9, 251-310; 17, the Amadori, 10, 169-205 407-430 Reductions, enzymic synthesis of, 17, 3 7 1 4 7 biochemical, a t the expense of sugars, 4, fractionmtion of, 1,247-277; 16,299-333 75-1 17 nitrates of, IS, 331-345 Replacement reactions, physical chemistry of, 11, 335-385 mechanisms of, in carbohydrate chempreparation and properties of esters of, ktw, 9, 1-57 1, 279-307 Rhamnose, Stereochemistry, methyl ethers of, 7, 1-36; 10, 257-272 of cyclic derivatives of carbohydrates, Ribose, 10, 1-53 chemistry of, 6, 135-174 formulas, writing of, in a plane, 3, 1-23 Streptomycin, S chemistry of, S, 337-384 Saccharides, Structural chemistry, biosynthwis of, from glycopyranosyl of the hemicelluloses, 14, 429-488 eaters of nucleotides, 18, 308-356 Structure, molecular, Saccharification, of cellulose, 19,219-246 of wood, 4,153-188 of dextran, 16, 341-369 Saccharinic acids, 12, 35-79 of glyCOgens, 12, 261-298 four-carbon, 18, 169-188 of sucrose, 4, 1-35 Schardinger dextrins, 12, 1891260 Sucrose. See alao, Sugar. Seaweeds, enzymic synthesis of, 6 , 2 W polyaaccharides of, 8, 315-350 structure and configuration of, 4, 1-35 Seleno sugars. Sec Sugars, seleno. utilization of, 4, 293-336 Shape, sugrrr, of some polysaccharide molecules, 7, aconitic acid as by-product in manutso 289-332; 11, 385.393 tm Of, 6, 231-249

570

CUMULATIVE SUBJECT INDEX FOR VOW.

Sugar alcohols, higher-carbon, configurations of, 1, 1-36 and their derivatives, metabolism of, 1,

175-192 “Sugar uucleotidw.” See Nuuleotidtw, glycopyranosyl eetere of.

Sugar products, color and turbidity of, 9, 247-284 Sugar refining, granular adwrbents for, 6,206-230 sugam, action of lead tetraacetate on, 14, 9 8 1 amino, aspects of the chemistry of, 14,

213-281

derived from antibiotic substances,

18,268308

methyl ethers of, 18, 188-214 properties of, 16, 158-200 mactione with bskr-dicerbonyl wmp~unda,40,303366 2-amino. See Sugars, ZSmino-2deoxy. %amin0-2deoxy,7,247-288 d Y b , chemistry of, P, 37-77 benzyl etbera of, lP, 137-166 biochemical reductions at the expense of, 4, 75-117 branched-chain, of natural occurrence,

11,263-283

of the cardiac glycosidea, 17, 86120 d w v i 31, zdeoxy, 8, 45-106 higher-carbon, 17,15-83 configurations of, 1, 1-38 hydrarones of, 8, 23-44 methyl glycosidee of the common, 19,

167-187 nitrates of, 19, 117-136 omzonw of, 8, 23-44 oxygen ring in, formation and aleavage of, 18,9 4 1 phenyl glymidea of the common, 19, 157-187 and their derivetivee, column chrcmatography Of$ 10,M-94 &td t o dt-, 1837-76 seleno. 1. 144-145 sulfa& of the simple, 10,183-218

1-21

thio, 1,129-144 developments in the uhemistry of,

188 123-198 87-137

UtWtlMtd, w),

Sulfatmr, of the uimple sugw, 90, 183-218 Sulfonic eaters, of CsrbOhyhW, 8, 107-215 Synthesis, biochemical, of monosaccharides, 11,

186-282

of cardenolides, 91,273-321 chemical, of u-glucuronic acid, 8, 231-

240

pOlySeCcharides, 91,431-512 of dextran, 16, 341-369 enzymio, of glywgem and starch, 17, 371-407 of sucrose eJld other dmccharidw, 6, Of

29-48

T Tagatoee, 7,QQ-136 Teichoic acide, 91,323-375 Tetritole, scat& of, 7,137-207 ThioCarbonah, of Carbohydrates, 16,91-158 Thio sugars. See Bugare, thio. Thornpeon, Alva, obituary of, 19, 1-8 Tram, isotopic, 8, 229-250 Tradomtion, the Lobry de Bruyn-Alberda van

Ekenetsin, 18,63-103 Trehaloeee, 18,201-225 Trityl ethers, of carbohydrates, 8, 78-111 Tunmoee, 3,1-36 Turbidity, Of sw prOdu~te>9, 247-284

U Unseturated sugars. Sea Sugars, unsaturated. Umidee, & ~ y l ,18, 215-236

CUMULATIVE SUBJECT INDEX FOR VOW.

W wood, hemicelluloses of, 19, 247-302; 20, 409-483 polysaccharidea associated with cellulose of, 10, 283-333 saccherification of, 4, 153-188

1-21

X Xylen, 6, 269-2110 Z Zernplh, GBm, obituary of, 14, 1-8 Zone electrophoresis, of carbohydrate*, 12, 81-1 15

571

ERRATA VOLUME19 Page 20,Table IV, column 4,line 2 of entries. Insert “0-3.” Page 21, Table IV, column 5,line 2 of entries. Delete I ‘ , 0-3.” Page 21, Table IV, column 8, line 2 of entries. For “6” read “8.”

VOLUMB20 Page 10,column 2,line 4 up. For “Strobmk” read “Strobach.” Page 71,line 9. For “glycofuranoaly” read “glycofuranosyl.” Page 245, Ref. 194. For “(1944)” read “(lW).” Page 265,line 3 up. For “ p =dioxane” read “pdioxane.” Page 267, line 2 up. For “WL” read “@-L.” Page 269, line 8 up, For “8-L’’ read “a-L.” Page 271, line 9. For “WL” read ‘(B-L.” Page 282,line 1. For “6-0-tolylsulfonyl” read “6-0-ptolylsulfonyl.” Page 295,line 8 up. For “&L” read “u-L.” Page 300, line 13 up. For “&L” read “a-L.” Page 376, Ref. 11, line 2. For “480” read “408.”

672