Advances in Carbohydrate Chemistry and Biochemistry, Volume 25

Advances in Carbohydrate Chemistry and Biochemistry Volume 25

1902-1969

Advances in Carbohydrate Chemistry and Biochemistry Editor R. STUART TIPSON Assistant Editor DEREK HORTON

Board of Advisors W. W. PIGMAN

S . ROSEMAN

WILLIAMJ. WHELAN

ROY L. WHISTLER

Board of Advisors for the British Commonwealth A. B. FOSTER

SIR EDMUNDHIRST

J. K. N.

JONES

MAURICESTACEY

Volume 25

ACADEMIC PRESS

New York and London

1970

COPYRIGHT 6 1970, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD.

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LIBRARY OF CONGRESS CATALOG CARD

NUMBER: 45

PRINTED IN THE UNITED STATES OF AMERICA

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1135 1

CONTENTS LIST OF CONTRIBUTORS .................... PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

ix xi

....

1

Stanley Peat (1902-1969) J . R. TURVEY

Text

......................................................... Gel Chromatography of Carbohydrates SHIRLEYC. CHURMS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Types of G e l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Theory of Gel Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IV. Application to Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Value of the Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13 14 16 31 51

Crystal-structure Data for Simple Carbohydrates and Their Derivatives GERALDSTRAHS Introduction . . . . . . . . . . . . . . . ........................ 53 hydrates , . . General Features of the Crysta Monosaccharides and Derivatives .......................... Disaccharides . . . . . . . ........................ 75 Oligosaccharides .................................. . . . . . . . . . . . . . 77 Antibiotic Substances ........................ 80 Nucleosides and Nucleotides . . . . . . . . . . . . . . . . . . . . . . Miscellaneous Carbohydrates: tamins, and Hydrazones . . . . . 90 Enzyme-Substrate Complexes ............................ 93 .................... 98 Conclusions . . . . . . . . . . . . . . . XI. Addenda. ................................................. 107

I. 11. 111. IV. V. VI. VII. VIII. IX. X.

Oxirane Derivatives of Aldoses NEIL R. WILLIAMS Introduction.. ....................................................... Synthesis.. .......................................................... Reactions ............................................................ Characterization ..................................................... V. Tables of Aldose Oxiranes ............................................

I. 11. 111. IV.

V

109 110 120 170 172

CONTENTS

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2. 5.Anhydrides of Sugars and Related Compounds J . DEFAYE 1. Introduction

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

I1. Methods of Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

III . heactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Table of Properties of 2,5-Anhydrides of Sugars, Alditols, and Aldonic Acids .............................................

181 183 210 215 219

Alditol Anhydrides S. SOLTZRERG

.

......................................................... 229 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 236 Physical Properties ................................................... 250 Reactions ........................................................... 256 Uses ................................................................. 267 Tables of Properties of the Anhydrides and Their Derivatives . . . . . . . . . . 271

1 Introduction

I1 . 111. IV . V VI .

.

The Sugars of Honey

I . R . SIDDIQUI I . Introduction .......................................................... I1. Honey Monosaccharides .............................................. I11 . Honey Oligosaccharides .............................................. IV. Honey Polysaccharides ............................................... V . Honeydew ...........................................................

285 289 295 306 307

Reactions of Free Sugars with Aqueous Ammonia

M . J . KORT I . Introduction ......................................................... I1. Products Obtained ................................................... I11. Isolation of Products. and Proportions Obtained ....................... IV. Mechanism .......................................................... V. Applications .........................................................

311 314 328 332 349

Synthesis of Nitrogen Heterocycles from Saccharide Derivatives HASSANEL KHADEM I . Introduction

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351

I1. Formation of Three-membered Nitrogen Heterocycles . . . . . . . . . . . . . . . . . . .352 I11. Formation of Five-membered Nitrogen Heterocycles ..................... 357

. .

IV Formation of Six-membered Nitrogen Heterocycles ...................... 394 V Formation of Higher-membered Nitrogen Heterocycles ................... 404

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Aspects of the Structure and Metabolism of Glycoproteins R . D . MARSHALLAND A . NEUBERGER I . The Nature and Occurrence of Clycoproteins .......................... I1. Carbohydrate-protein Linkages ........................................ I11. Polypeptide Chains Carrying More than One Type of Carbohydrate-peptide Linkage ....................................... IV. Heterogeneity in Glycoproteins ....................................... V. The Size of the Carbohydrate Moieties in Glycoproteins . . . . . . . . . . . . . . . . VI . Features of the Structure of the Carbohydrate Moieties of Some Clycoproteins ............................................. VII . The Biosynthesis of Glycoproteins .................................... VIII . Some Genetically Determined Diseases in Which Clycoproteins Are Implicated ........................................ IX. Concluding Remarks .................................................

AUTHOR INDEXFOR VOLUME 25 .......................................... SUBJECT INDEX FOR VOLUME 25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CUMULATIVE AUTHOR INDEX FOR VOLUMES 1-25 ......................... CUMULATIVE SUBJECT INDEX FOR VOLUMES 1-25 .......................... ERRATA...................................................................

407 417 439 443 447 452 467 472 477 479 507 525 533 544

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

SHIRLEYC. CHURMS, Council for Scientific and lndustrial Research Carbohydrate Research Unit, Department of Chemistry, University of Cape Town, South Africa (13)

J. DEFAYE,' Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientijique, 91 Gif-sur-Yuette, France (181) HASSANEL K H A D E M , ~ Institut de Chimie des Suhstances Naturelles, Centre National de la Recherche Scientijique, Paris, France (351)

M . J. KORT, Department of Chemistry, Uniaersity of Natal, Pietermaritzburg, and Sugar Milling Research Institute, Durlian, South Africa (311) R. D. MARSHALL,Department of Chemical Pathology, St. Mary's Hospital Medical School, London, W.2, England (407) A. NEUBERGER,Department of Chemical Pathology, St. Mary's Hospital Medical School, London, W.2,England (407)

I . R. SIDDIQUI,Food Research Institute, Canada Department of Agriculture, Ottawa, Ontario, Canada (285) S. SOLTZBERG, Atlas Chemical Industries, lnc., Wilmington, Delaware (229) GERALDSTRAIIS, Biochemistry Department, New York Medical College, New York, New York (53)

J. R. TURVEY, Department of Chemistry, Unioersity College of North Wales, Bangor, Caernarvonshire, Great Britain (1) NEIL R. WILLIAMS,Chemistry Department, Rirkbeck College, University of London, Malet Street, London W.C.1, England (109)

Present address: Centre de Recherche sur les Macromolecules VBgbtales, C.N.R.S., Domaine Universitaire de Grenoble, 38 St. Martin d'HAres, France. t Permanent address: Faculty of Science, Alexandria University, Alexandria, Egypt.

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PREFACE

With this twenty-fifth Volume, Advances in Carbohydrate Chemist r y and Biochemistry has completed its first quarter-century. This serial publication was initiated with the dual objective of presenting definitive accounts of the status of matured fields and of providing, for areas of high activity, critical evaluations that would serve as guidelines for future research. The past 25 years have seen an acceleration of research unprecedented in the history of science, and the extent to which Advances has usefully fulfilled a need, and yet provided flexibility in accommodating to change, may be judged by the frequency with which many of the older articles are still cited. Over its lifetime, Advances has developed into a permanent source of reference in organized form for practically all of the major subdivisions of knowledge in the field of carbohydrates. It has also stimulated, by means of timely articles on active and controversial areas of research, the exploration of important fields that might otherwise have been neglected or investigated in a more haphazard fashion. The breadth of coverage, as originally conceived and subsequently maintained, has allowed the discussion of carbohydrates from the viewpoints of many specializations. Structural and synthetic organic chemistry have certainly been highly influential, but biochemistry and physical chemistry have been no less important, and the techniques and ideas of agricultural chemistry, analytical chemistry, industrial chemistry and technology, microbiology, pharmacology, and many other disciplines have brought the full breadth of scientific inquiry to bear on this, the largest, class of natural products. In the present era, the idea of interdisciplinary research has become much in vogue, and it is therefore interesting to observe that the original Advisory Board for Advances, in setting a policy of studying a single major class of natural products by a broad spectrum of many classical disciplines, was instrumental in the achievement, over the years, of that very type of cooperation between specialists of different persuasions that is only now becoming properly recognized as an important trend in the future development of science. Bearing in mind the not infrequent asseverations in the past by certain classical specialists (especially, organic chemists) that the study of carboxi

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hydrates is a narrow specialization, the tenacity of the Editors and the Advisory Board in adhering to the original interdisciplinary concept is a fitting testimony to the soundness of their ideas in the face of changing opinion. The present volume was in the planning stage at the time of the death of Professor Melville L. Wolfrom, but it is one of the few volumes in the history of this Series not to have received his editorial attention. His influence always reflected the precision and accuracy that he applied in his own writings, and the present Editors will endeavor to meet these criteria. This volume includes an obituary, contributed by J. R. Turvey, of the late Professor Stanley Peat, F.R.S., who was for many years a member of the Board of Advisors of Advances, and who served as Associate Editor for the British Isles for a number of years. The separation of macromolecules by molecular-sieve techniques constitutes a major technical advance, especially for biochemists. The principles of the method and applications in the carbohydrate field are surveyed by S. C. Churms (Rondebosch). The field of X-ray crystal-structure analysis is undergoing rapid evolution because of major advances in methodology; automatic diffractometers and computerized systems for data reduction have advanced the technique to the point where the solution of many simple structures is almost routine, and the chapter by G. Strahs (New York) surveys developments since the article by Jeffrey and Rosenstein in Volume 9 of Advances. The chapter marks a transition between the era of the classical crystallographer, who determined a structure for its own sake, and that of the newer generation of crystallographers concerned with the broader implications of a coordinated plan of attack, where crystallography provides the tool rather than the objective for the study of fine points of the molecular structure of carbohydrates in relation to their conformations and biological roles. The wideranging subject of anhydrides of sugars and their derivatives is treated from the organic chemical viewpoint in three separate chapters in this volume. Because of the extensive literature on sugar anhydrides of various types, it was found impossible to treat developments in this whole area within the confines of one chapter, or even in the three chapters here presented; other aspects remain to be treated in future issues. Oxiranes (epoxides) are discussed by N. R. Williams (London), and ring-forming reactions, of aldoses, that result in the formation of 2,banhydro rings are delineated in the chapter by J. Defaye (Gif-sur-Yvette). The anhydrides of alditols are considered separately by S. Soltzberg (Delaware).

PREFACE

xiii

Modern methods of separation continue to reveal the complexity of mixtures of even simple carbohydrates in various natural sources, as demonstrated in the chapter by 1. R. Siddiqui (Ottawa) on the sugars present in honey. The reactions of sugars with ammonia and amines, a field related to important problems in the food industry, constitute a subject of continuing interest, and are treated by M. J. Kort (Pietermaritzburg). The polyfunctional nature of sugars can be exploited in the synthesis of a multitude of types of heterocyclic derivative, but the uninitiated reader may find the literature confusing and disorganized because of the plethora of structural types possible, even from simple reactions; H. El Khadem (Alexandria) has performed a valuable service by organizing the facts, fictions, and paradoxes in this domain. In the final chapter, R. D. Marshall and A. Neuberger (London) explore the recent developments in our understanding of the structure and metabolism of glycoproteins, an area at the broad frontier of much advancing knowledge in modern biochemistry. The Subject Index was compiled by Dr. L. T. Capell. A well-assorted, international representation of authorship is evident in recent volumes of Advances; the original British-American liaison on which the publication was founded has been substantially expanded to the international level. The present volume includes, in addition to contributions from North America and Great Britain, articles from continental Europe and, coincidentally, three separate chapters by authors based at different points on the African continent.

Kensington, Maryland Columbus, Ohio November, 1970

R. STUART TIPSON DEREKHORTON

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STANLEY PEAT 1902- 1969

On the 21st of February, 1969, Professor Stanley Peat, D. Sc., F.R.S., suffered a stroke at his home in Bangor, Wale;, and he died 36 hours later without regaining consciousness. Thus ended the career of a scientist who, even as a child, displayed an unquenchable thirst for knowledge and who, all his life, regarded it as a duty to impart that knowledge to others. His parents, Ada and John H. Peat, lived at Bolden, County Durham, but Mrs. Peat was staying with her sister Alice in South Shields when their first child, Stanley, was born in 1902. John Peat was a mining engineer, but, despite his status, the family was rather poor and the care of the infant boy was handed over to his Aunt Alice and her husband, James Gibson, who were childless. Unfortunately, despite their care and attention, the child developed bovine tuberculosis when only a few months old, and had to receive hospital treatment for some time. Possibly as a result of this illness, the child was left with a permanent curvature of the spine. This disability was to have a profound effect on the development of Stanley Peat, since, denied an outlet for his energies in active sports and games, he was thrown back onto his mental resources for amusement and interest. The first consequence of this childhood illness was that Stanley was a rather frail child, requiring much attention from his aunt; this she gave unstintingly, often at the expense of her husband. Both Gibsons were keen members of the Salvation Army, the husband being a musician in the Army Band. The child was thus raised in a household where both Christianity and music were an important part of everyday life, and both were to influence him for the rest of his life. His frailty also resulted in a delayed start to his schooling; he did not attend a formal school until he was eight years old. Fortunately, he was blessed with an inquiring mind and this, coupled with the enforced idleness, resulted in his learning to read and write before he went to school. He became an avid reader, and the local library became an important point of call whenever he went out. From the library he was able to borrow books on travel, science, and a wide 1

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variety of other subjects. Two books in particular fired his imagination, one on chemistry and the other on photography. At the age of eight, he started a chemistry set with which to experiment, and, by the age of ten, he was developing and printing his own photographs in an improvized dark-room. This love for experimental chemistry and photography persisted throughout his schooldays and into College, when he still retained his private laboratory and dark-room at home. The lack of formal schooling in his early days had, however, left large gaps in his education which, in spite of his thirst for knowledge, he had been unable to fill by reading alone. Mathematics was a difficult subject to him and was to remain so for the rest of his life. Once at school, Stanley Peat rapidly showed that he was a gifted pupil. He was never happy until he felt that he really understood a subject and, unable to follow active outdoor pursuits, he tended to devote much of his spare time to study and general reading. Not that he was completely debarred from all childish games; he used to play with the local children, and indulged in the usual boyish pranks. At the age of ten, he had returned to live with his parents at Station Road, Walker-on-Tyne, a mining community, and, as he subsequently used to relate with great glee, he was chased by the local policeman for “scrumping” apples from a nearby orchard. One other incident from this period is worth recording, as it throws some light on the boy’s character. His maternal grandfather, then aged 60, was only semi-literate, and young Stanley took it upon himself to help his grandfather to write. Armed with a penny exercise book, a present from Stanley, the grandfather practised his writing under the watchful eye of his grandson. He made such rapid progress, that, within a short time, he was attempting to write an adventure story. This exercise book, with its spidery writing and frequent misspellings, was treasured by Stanley Peat all his life. From Tyne Dock Grammar School, which he attended for some years, Stanley won a Scholarship in 1915 to the Rutherford College Boys’ School in Newcastle. This school had a long tradition of excellence in its teaching of science subjects, and possessed some extremely able and inspiring science masters. Among these was William Carr, the chemistry master, who came to regard Stanley as one of his favorite and most able pupils. Certainly, the young boy spent nearly all his spare time studying, and he was regarded by his schoolfellows as a pleasant but extremely serious young fellow. In 1917, further hospital treatment for his back caused him some pain, but did not prevent him from winning the Form Prize at school. He obtained his School Certificate in 1919, and proceeded to the Sixth Form, where

OBITUARY -STANLEY PEAT

3

he specialized in Chemistry and Physics. In 1921, he was awarded the Higher School Certificate, with distinctions in chemistry and physics; at the same time he won a State Exhibition, an Entrance Exhibition, and an Earl Grey Memorial Scholarship to study at Armstrong College (now the University of Newcastle-upon-Tyne). It is interesting that fellow pupils of Peat at Rutherford College included Professor E. E. Aynsley, W. Charlton, R. Chirnside, and Sir James Taylor. At Armstrong College, Peat read for Honours in Chemistry, with physics and mathematics as ancillary subjects. Once again, he proved to b e an outstanding student, becoming Senior Pemberton Scholar in 1922 and Saville Shaw Medalist in 1923, and winning the Friere-Marreco Medal and Prize in 1924, when he graduated with First Class Honours in Chemistry. In addition to his normal studies at Armstrong College, Peat also found time to pursue a course of lectures and practical work in physiology, a fact that was to influence his subsequent career profoundly. The Professor of Chemistry at that time was W. N. (later, Sir Norman) Haworth who, with E. L. (later, Sir Edmund) Hirst, was carrying on pioneer research work, originated with Sir James Irvine at St. Andrews, on the use of methylation in the study of the ring structures of sugars. Both Peat and Charlton, who had graduated with him, were invited to conduct postgraduate work in Haworth’s research school. While continuing to live with his aunt in South Shields, Peat joined this enthusiastic group of research workers, and rapidly immersed himself in the work on methylation and in the controversies surrounding it. An insight into his character comes from Professor R. Spence, then a junior student and a fellow commuter on the train from South Shields to Newcastle: “ H e was rather senior to me, but h e was completely unpatronizing in his friendship. This readiness to help a younger man was, I am sure, strongly characteristic. He offered his collection of chemicals and apparatus to me when he left Newcastle . . . . Although a loyal co-worker, Stanley could, on occasion, make a pungent comment on the polemics in which Haworth was involved.” In 1925, Haworth was invited to the Chair of Organic Chemistry at Birmingham University, and h e took with him Stanley Peat (as his research assistant), J. Avery, W. Charlton, E. Goodyear, A. Learner, and V. Nicholson. Peat, Charlton, and Learner were in the same lodgings in Birmingham until they were all awarded the Ph. D. degree in 1928. Stanley (or, as h e was more generally known, “Sammy”) Peat did not have many interests outside his work, not that there was much free time after an average 12-hour day in the laboratory. An

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occasional hand at bridge and listening to classical music were his chief amusements outside the laboratory. However, while waiting for experiments to “come to the boil,” Peat would indulge in lengthy and fierce arguments on every possible subject with Learner and anyone else who cared to participate. It was usually Peat who took the orthodox viewpoint, which he would argue both logically and forcefully, while Learner upheld the revolutionary view. It is a measure of their characters that, though they argued vociferously they never quarrelled. They found common ground in reading the whole of Ibsen’s and Shaw’s plays, and in inviting lecturers on Psychoanalysis or Comparative Religion to address them on Sundays. It is difficult to assess Peat’s attitude towards religion. The Salvation Army influence of his childhood had been modified in his schooldays by regular attendance at a Methodist chapel with his mother. He had an exceptional knowledge of the Bible, being able to recite long passages from memory and, when older, he took pains to study most of the world’s major religions. In spite of this knowledge, he never became an ardent member of any particular faith; he was too full of intellectual curiosity to accept blindly any dogma or principle that he could not subject to a scientific test. In his later dealings with colleagues and students, however, he was to display many Christian attributes, tolerance and understanding of their viewpoint, and a willingness to temper the wind to the shorn lambs among his undergraduates. In those early days, Stanley Peat’s contribution to carbohydrate chemistry was allied completely with Haworth’s pioneering work on the constitution and ring structures of sugars. His first paper (with Haworth) in 1926 was a revision of the structural formula of D-glucose, in which it was established that the known methyl P-D-glucoside, and, hence, probably D-glucose itself, existed in the pyranoid ring-form. In 1926 also, his name appeared on two papers dealing with the structure of maltose, in which it was unequivocally shown that the linkage was (1-4) between two D-glucose residues, both of which were in the pyranoid ring-form. This was the “classical” type of research which was fast making Haworth’s school the leading center of carbohydrate chemistry in Europe. In yet another paper, the importance of the synthetic approach to the structure of sugar derivatives was underlined by the preparation of D-glucono- and D-mannono1,5-lactones from D-arabinose, by using a cyanohydrin synthesis. Following the award of his Ph. D. degree in 1928, Peat was offered the post of lecturer in Biochemistry in the Physiology Department of the Medical School at Birmingham University. This department

OBITUARY -STANLEY PEAT

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wished to-expand its teaching on the biochemistry side, and Professor de Burgh Daly had appealed to Haworth to find him a suitable lecturer. Knowing of Peat’s interest in physiology and of his potential as a teacher, Haworth nominated him. Peat threw himself with enthusiasm into the task of gathering material for his lectures to the medical students and of preparing experiments suitable for large classes in very limited laboratory space. To this problem of overcrowding, Peat brought an orderliness and precision that ensured that each student was able to carry out his allotted experiments without hindrance. A few well-chosen words from Peat were enough to quell the most mischievous prankster, thus ensuring the efficient working of the class. Accounts of such incidents were often related with obvious humor by Peat to his friends. To those who did not know him well, he appeared a shy and very earnest young man. Only with his colleagues and the many friends he made in the Founders’ Room at the Edmund Street branch of the University did he show his tremendous sense of humor, his penchant for intellectual argument, and his deep humanity. During this period, h e also taught himself German, and he subsequently made frequent trips to Austria and Germany. On these trips, h e was able to indulge to the full a growing passion for grand opera and classical music. In Birmingham, he started playing golf, using specially shortened clubs, and was frequently to b e seen practising on the University playing fields by the Bristol Road. His greatest enjoyment, however, came from the concerts given by the Birmingham Symphony Orchestra, from the plays at the local “Rep” theatre, and from learning to play the mandolin. Although a heavy teaching-load curtailed his research activities, in 1931 he collaborated in an investigation of a case of phenol poisoning and, with MacGregor, began a series of investigations on the physiological role of histamine in the animal body, subsequently publishing three papers on aspects of this topic. This experience with biological systems and the metabolism of compounds did much to influence his later work. In 1934, Haworth invited Peat to return to the Chemistry Department as a lecturer. Peat thus joined a team which, during the next few years, was to include such eminent workers as E. L. Hirst, J. K. N. Jones, E. G. V. Percival, Fred Smith, M. Stacey, and L. F. Wiggins. I n 1936, Haworth divided the direction of his rapidly growing research school among Peat (plant polysaccharides and amino sugars), Smith (plant gums), and Stacey (polysaccharides of microorganisms); these “compartments” were not, however, “water-tight,” and fruitful collaboration between the groups frequently occurred

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and often led to joint publications. Under Haworth’s guidance, Peat began to develop two main lines of research. The first was a continuation and extension of the methylation method for investigating the structure of polysaccharides. Over the next few years, there appeared a steady stream of papers based on the use of this technique that described the constitution of agar, a-amylodextrin, cellulose, dextran, and xylan. Cellulose was the polysaccharide to which he frequently returned; its end-group assay by methylation, the importance of excluding oxygen during its methylation, and the properties of oxycellulose and hydrocellulose were among the aspects that he investigated. The second line of research was a detailed study of the formation and reactions of anhydro sugars. In 1938, with Wiggins, he described the base-catalyzed elimination of the p-tolylsulfonyloxy group from with consequent methyl 3-O-p-toly~sulfonyl-fl-~-glucopyranoside, formation of various methyl anhydrohexosides. The fact that Walden inversion frequently accompanied this type of elimination was stressed, and the opening of the anhydro ring by alkaline reagents, again with inversion, was described. Other p-toluenesulfonic esters were then investigated, and the opening of the anhydro ring by methanolic ammonia to produce aminodeoxy sugars was reported. Peat readily appreciated the importance of these reactions in the synthesis of the rarer sugars, and, particularly, in the preparation of amino sugars. The synthesis of “chitosamine,” and the proof that this sugar is 2-amino-2-deoxy-~-glucose,followed from this work. Also of importance was the recognition, by Peat, Hands, and W. G. M. Jones, that the labile sugar present in agar is 3,6-anhydro-~-galactose. Much of this work and later contributions were elegantly summarized by Peat in an article in Volume 2 of Advances in Carbohydrate Chemistry. During this period, he was also a regular contributor to the Annual Reports of the Chemical Society. At this time, Haworth asked Peat to take over from him the teaching course to first-year students, a course which both regarded as the most important of the syllabus. Peat was desirous of introducing into the teaching the new concepts of “electronic mechanisms” as a help to students in understanding why chemical reactions occur. He designed such a course and, in his meticulously careful way, delivered it with obvious enthusiasm. The students responded to this new approach, and all who attended the course agreed that it was outstanding in its clarity and mode of presentation. This avenue to the teaching of elementary organic chemistry was, with suitable modification, continued by Peat for nearly twenty years in Birmingham and Bangor.

OBITUARY- STANLEY PEAT

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In the teaching laboratory, he strove to instil into students the importance of neatness, accuracy, and precise observation. An accomplished experimentalist himself, and second only to Fred Smith in his ability to produce crystals from syrups, he would not tolerate untidy or careless work. His private life, which had pursued an even tenor for several years, was also due for a change. During his days in the Medical School at Birmingham University, he had gone to lodge in Station Road, Harborne, with a Mr. and Mrs. Barnes. Mr. Barnes, a dental surgeon, had been forced by ill health to curtail his practice and, in 1937, to retire completely. Stanley Peat bought a house in Edgbaston, and the Barnes family moved in with him. Their daughter Elsie, several years younger than Stanley, would sometimes accompany him on his frequent visits to concerts or the theatre. In the spring of 1939, following a sharp bout of influenza during which Elsie was his faithful nurse, he proposed to her, and they were married in the summer. Peat threw himself wholeheartedly into the business of being a married man; and he planted a garden, but his early efforts were not an outstanding success. To his great joy, a daughter, Gillian, was born to them in 1940, and another daughter, Wendy, in 1942. He derived tremendous pleasure from the company of these children. Unfortunately, the Second World War had brought an end to the happy state of the Chemistry Department. Haworth had agreed to turn much of the research potential in his department over to a study of uranium compounds, a project contributing in no small measure to the production of the atomic bomb. After six months of research on uranium carbonyls, Peat found that he could generate little enthusiasm for the work itself, and none at all for the project to which it was contributing. He persuaded Haworth that he could contribute more to the war effort by carrying out selected research in carbohydrates, with a view to increasing food production. With Haworth’s approval, he took over from M. Stacey a project aimed at producing D-glucose directly from potatoes, and saw this process through to the stage of commercial production. His knowledge of cellulose and its derivatives was called for as a member of the Cellulose and Cordite Panel of the Ministry of Supply, and his interest in agar was used on the Committee charged with finding from among the British seaweeds a suitable substitute for Japanese agar. The eventual collection of seaweeds and their processing to afford a product acceptable to microbiologists, even if somewhat inferior to agar, was a direct result of the work of this Committee. In addition, members of his research team undertook work on Asdic recordings for the Admiralty.

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J. R. TURVEY

With all this war work progressing, a small group was still able to carry on with the normal carbohydrate research. Papers on starch, D-ghCUrOnOlaCtOne, bacterial levans (with Stacey), and the polysaccharide associated with beta-amylase, all appeared in the Journals. It was in 1940 that a series of papers by C. S. Hanes appeared that were to change completely the ultimate direction of Peat’s research. These papers dealt with the synthesis from D-glucosyl phosphate of a sparingly soluble “starch” by a plant enzyme, phosphorylase. Hanes prepared a batch of this starch and sent it to Haworth for a chemical investigation. With Peat and Heath, Haworth showed that the starch was a short-chain amylose, and that it did not contain any of the branched compound, amylopectin. From that time on, Peat devoted more and more of his time to a study of starch, and particularly to its enzymic synthesis and degradation. A study of the amylolytic degradation of starch was followed in 1944 by the first synthesis of amylopectin, which led directly to the discovery of the Q-enzyme. This work, in which E. J. Bourne played a significant part, added impetus to the search for new methods that would separate whole starch into amylose and amylopectin fractions. One such method, involving the use of thymol or cyclohexanol, was the subject of a patent taken out by Haworth and Peat. A survey of the starches of many plant species followed, and this led to the recognition that certain varieties of pea produce a starch containing an abnormally high proportion of amylose. Many starch derivatives were also prepared, and one in particular, sodium starch glycolate, has since assumed importance as an indicator for iodimetric analyses. In 1946, Peat was made an Associate Editor of a new periodical, Advances in Carbohydrate Chemistry, serving in this capacity until 1954, and he continued as a member of the Board of Advisors for the British Isles until his death. The end of the war brought some changes to the University of Birmingham. Peat became a Reader in Organic Chemistry, and was awarded the D. Sc. degree. The quality of his researches was also recognized in 1948 by his election to Fellowship in the Royal Society, and, shordy afterwards, by his appointment to the Chair of Chemistry at the University College of North Wales, Bangor. He chose two young lecturers to accompany him to Bangor, W. J. Whelan and F. H. Newth, together with two research students. With the enthusiastic co-operation of Whelan and Newth, he soon established a thriving research school despite crowded conditions and lack of finances. With Whelan, further research on starch-metabolizing enzymes was instigated, while Newth concentrated his efforts on anhydro sugars and reaction

OBITUARY - STANLEY PEAT

9

mechanisms of the acetylated glycosyl bromides. The purification and mode of action of several enzymes were studied, and the notable discoveries of R-enzyme, Z-enzyme, and D-enzyme followed. In 1950, Whistler and Durso described the separation of mono-, di-, and tri-saccharides from each other by chromatography on charcoal (activated carbon). This method was immediately tested by Peat and Whelan, and was shown to be outstanding for the resolution of oligosaccharide mixtures, such as are obtained b y enzymic or partial acid hydrolysis of polysaccharides. By 1952, carbon columns were in operation throughout the Bangor chemical laboratories, and the purified oligosaccharides so obtained were being used to elucidate the action patterns of the enzymes. A series of papers on the enzymic synthesis and degradation of starch, the first appearing in 1945 and the last (Part 24) in 1959, represented a major contribution to this field of study and rightly won Peat world renown. Of equal importance, however, was the realization by Peat and Whelan that partial hydrolysis of a polysaccharide with acid, followed by separation on carbon columns and identification of the products, is a supporting method to methylation for investigating polysaccharide structures. The linkages present in the polysaccharide are usually retained intact in the derived oligosaccharides, and, unlike the methylation procedure, the method can give valuable information as to the sequence of linkages in the polysaccharide chain. The method was applied with outstanding success to laminaran, to the glycogen, glucan, and mannan of yeast, and to lichenan, isolichenan, and Aoridean starch. By 1955, Peat was extremely busy, not only with research and teaching but also with the administration of a vigorous department. Wishing to take his share of the burden of other duties, he served for a period as Chairman of the Carbohydrate Nomenclature Committee of The Chemical Society (having already served on the Council and Publications Committee of this Society); also, he had been Dean of the Faculty of Science at Bangor, and was on the College Council. He was a consultant to the Brewing Industries Research Association, and to the Bakers and Flour Millers Research Association. He was also a member of the Scientific Advisory Committee of the Institute of Seaweed Research. When Whelan left Bangor in 1956, it was decided that much of the work on the starch enzymes should be continued by him at the Lister Institute. With J. R. Turvey and P. F. Lloyd (who replaced Whelan), Peat decided to branch into a line of research that he had touched upon previously but now wished to develop more fulIy. The structure of

10

J. R. TURVEY

agar, and, more particularly, the role of sulfuric hemi-esters in certain algal polysaccharides had always intrigued him. With Turvey, a systematic study of the preparation, properties and reactions of sugar sulfates was commenced. This work was coupled with an examination of the galactan sulfates of red algae. Lloyd, who had brought to Bangor an interest in amino sugars and the sulfated polysaccharides of animal tissues, commenced work on some aspects of amino sugar chemistry and on the sulfatase systems present in marine molluscs. The isolation of L-galactose 6-sulfate from an algal polysaccharide, and (with D. A. Rees), from a seaweed of an enzyme system which (as was later established by Rees) could convert galactose 6-sulfate residues in a polysaccharide into 3,6-anhydrogalactose residues, constituted verification of ideas put forward by Peat 15 years previously. In 1959,The Chemical Society recognized Peat’s contribution to the field of investigation of carbohydrate structure and metabolism by inviting him to give the Hugo Miiller Lecture, an invitation that he regarded as a signal honor. He made plans for this lecture, but a heart attack in September put him into hospital. His recovery was very slow, and it was almost two years before he was again able to visit his department regularly. Throughout his convalescence, he encouraged visits to his home in Penrhos by all his staff and research students. Being forbidden to exert himself physically, he took great delight in conversation. His wide knowledge and love of music, of literature (especially Dickens, selected volumes of whose works he read every Christmas Season), and of the history of ancient civilizations provided a constant source of topics for discussion. Once installed in the study of his large house with its beautiful garden, the visitor found it difficult to leave, and hours of delightful conversation ensued. It was during these conversations that he displayed to the full his wonderful sense of humor, his deep interest in his fellow man and, although he claimed to be an agnostic, his essentially Christian outlook on life. When he was again able to return to the department, uncertain health forced him to give up lecturing and to play a less active part in research, both of which he did reluctantly. The supervision of his research students was left entirely to Turvey and Lloyd, but he was always ready to give advice and encouragement whenever these were sought. This fatherly interest in the research work of his department extended to all sections, and he also continued to play an active part in the design of suitable teaching courses for the undergraduates. He tended to view with some caution the new physical methods that were being used by research groups in his department.

OBITUARY - STANLEY PEAT

11

While conceding that much information of a preliminary nature could be obtained by “knob-twiddling” -as he jokingly called it -he stoutly maintained that results obtained by such methods should be treated with caution until reinforced by more tangible evidence. Over the last few years, although still retaining his authority as Head of the Department, he came to rely more and more on the willing co-operation of his colleagues in ensuring the smooth running of the department. Since coming to Bangor he had striven to obtain funds for a new Chemistry Building, and these were granted in 1961. He then gave up all interests outside the department, and devoted much of his energies to the planning and designing of the new building, which came into operation in 1965 and is now one of the showplaces of the College. The research work in his group continued; the investigations on algal polysaccharides and of their degradation by bacterial enzymes were of particular interest to him. Further work on sugar sulfates, an investigation of heparin, and the unravelling of the complexities of molluscan sulfatases were also topics in progress at the time of his death. At home, he preferred relaxing with his books, or selectively viewing television, which provided some consolation because of his love of music and drama. The advent, over the years 1962 to 1964, of four grandchildren was a source of much pride to him, and he derived great pleasure from their company in the ensuing years. He was fascinated by the processes involved in learning in the very young, and would often comment on some observation he had made while watching his grandchildren. In September, 1968, he suffered another heart attack, from which he was still convalescing when he had his fatal illness. Preparations for a Special Issue of the journal Carbohydrate Research in his honor had been made early in 1968, and the issue was in the printers’ hands at the time of his death. Equally tragic was the fact that a Meeting of the Carbohydrate Discussion Group of The Chemical Society had been arranged in his honor in Bangor for Easter, 1969. It was unfortunate that his premature death, a few months before he was due to retire, should have deprived him of the pride and pleasure that these two events would undoubtedly have afforded him. In his address as Dean of the Faculty of Science to the Court of Governors of the College in 1953,Stanley Peat said: “My four predecessors in the Chair of Chemistry-Dobbie, Orton, Simonsen, and E. D. Hughes - were each in their turn elected to the Royal Society, a sufficient indication of the value of their research work and, because of that, of their qualification as university teachers. The influence of

12

J.

R. TURVEY

these men has extended far beyond the boundaries of our College.” The name of Stanley Peat can now, with complete justification, be added to this list of distinguished chemists.

J. R. TURVEY The help of the following people is gratefully acknowledged: Mrs. E. Peat, Dr. J. Avery, Professor E. E. Aynsley, Professor E. J. Bourne, Dr. A. Learner, Professor R. G. S. MacGregor, Mrs. G. Shepherd, Professor R. Spence, Professor M. Stacey, Dr. D. H. Strangeways, and Sir James Taylor.

GEL CHROMATOGRAPHY OF CARBOHYDRATES*

BY SHIRLEYC. CHURMS Council for Scientijic and Industrial Research Carbohydrate Research Unit, Department of Chemistry, University of Cape Town, South Africa I. Introduction.. ........................................................ 11. Types ofGel ........................................................... 111. Theory of Gel Chromatography ......................................... 1. Principles and Definitions ............................................ 2. Separation by Gel Chromatography. .................................. 3. Determination of Molecular Weight. ................................. IV. Application to Carbohydrates ........................................... 1. Sugars ............................................................. 2. Polysaccharides .................................................... 3. Miscellaneous Carbohydrates ........................................ V. Value of the Technique ................................................

13 14 16 16 17 21 31 31 35 47 51

I . INTRODUCTION Gel chromatography (also known as gel filtration, gel-permeation chromatography, or molecular-sieve chromatography) is based on the decreasing permeability of the three-dimensional network of a swollen gel to molecules of increasing size. If a solution containing a mixture of solutes of different molecular sizes is passed through a column packed with a suitable gel, the smaller molecules penetrate farther into the gel pores than do the larger, and they are therefore retained for a longer time on the column. The solutes are thus eluted in the order of decreasing molecular size. Consequently, this procedure affords a rapid, relatively simple method for separating substances that differ in molecular size, or for fractionating polymers, such as polysaccharides, having broad molecular-weight distributions. Because mild conditions are used, the technique is particularly useful for labile biological materials. Also, as the order of elution of a series of similar substances from a gel column is governed largely by mo*This review owes much to the guidance and encouragement of Professor A. M. Stephen, to whom the author expresses her gratitude. The financial support of the South African Council for Scientific and Industrial Research is also gratefully acknowledged.

13

14

SHIRLEY C. CHURMS

lecular weight, gel chromatography provides a means of determining molecular weights of polymers. The technique has been the subject of a detailed and extensive general review.’

11. TYPESOF GEL Gel chromatography originated in 1956 with the work of Lathe and Ruthven,2 who achieved some degree of separation of tri-, di-, and mono-saccharides on a column of potato starch. Owing to the disadvantages attendant on the use of starch in gel chromatography, namely, its high resistance to flow, its instability, and its ill-defined composition, starch was soon superseded by artificially cross-linked dextran gels, the use of which was first reported in 1959 by Porath and F10din.~Dextran gels, commercially available under the name S e p h a d e ~are , ~ ~now widely used in gel chromatography; these gels are hydrophilic. A lipophilic gel, namely, Sephadex LH-20, prepared by alkylation of most of the hydroxyl groups in dextran, is also available. The use of agar in gel chromatography was first reported, in 1961, by Polson.‘ Owing to their more open structure as compared with dextran gels, agar gels are capable of fractionating much larger molecules. However, agar has the disadvantages of ill-defined composition and a strong tendency to adsorb basic substances, owing to the presence of charged groups in the agaropectin component. The agarose component, which is a neutral, linear polymer consisting of alternate residues of D-galactose and 3,6-anhydro-~-galactose,~ has been found superior to native agar as a medium for gel chromatographyBsAgarose gels are now commercially available under the names Sepharose- and Sagarose.’ Another hydrophilic gel that has been successfully used in gel chromatography is the poly(acry1amide) type, first introduced in (1) L. Fischer, in “Laboratory Technique in Biochemistry and Molecular Biology,” P. S. Work and E. Work, eds., Wiley-Interscience, New York, N. Y., 1969,Vol. 1, Part 11, pp. 157-391. (2) G. H. Lathe and C. R. J. Ruthven, Biochem.], 62,665(1956). (3)J. Porath and P. Flodin, Nature, 183,1657(1959). (3a) Pharmacia Fine Chemicals, Uppsala, Sweden. (4)A. Polson, Biochirn. Biophys. Acta, 50,565(1961). (5)C . Araki, Bull. Chem. Soc.Jap.,29,543(1956). (6)S.Hjerten, Biochim. Biophys. Acta, 53,514(1961). (7)Seravac Laboratories, Cape Town, South Africa, and Maidenhead, Berkshire, England.

GEL CHROMATOGRAPHY O F CARBOHYDRATES

15

1962 by Lea and Sehon8 and simultaneously by Hjerten and Mosb a ~ h These .~ gels, prepared by polymerization of acrylamide in the presence of N,N'-methylenebisacrylamide, are available commercially (Bio-Gel P series).ln Gels that swell in organic solvents (for example, polystyrene gels) are required for gel chromatography of hydrophobic macromolecules. Such gels have found wide application in the fractionation of synthetic polymers.11*12 Cross-linked polystyrene containing hydrophilic groups (Aquapakl3) has now become available, permitting the fractionation of water-soluble materials, such as dextrans, on the polystyrene medium.I4 The use of ion-exchange resins to fractionate oligosaccharides15 is possibly another example of molecular-sieve chromatography of hydrophilic solutes on a polystyrene matrix, although, in this instance, other factors may also be involved in the separation. The use of porous glass as a support for molecular-sieve chromatography was suggested by H a l l e P in 1965, and this material, now available in bead form (Bio-Glas'O), has proved useful in the fractionation of solutes of high molecular weight,"*18particularly at high temperat u r e at ~ which ~ ~ organic gels are not suitable. The rigidity and resistance to thermal, chemical, or bacteriological degradation, which are advantages of the use of porous glass as a medium for gel chromatography, are also characteristics of porous silica beads.20 These beads, which have become commercially available ( P o r a ~ i l ' ~are ) , now finding increasing application in gel chromat ~ g r a p h y . ~ ~Certain -*~ polymers [for example, poly(viny1 alcohol)] (8) D . J. Lea and A. H. Sehon, Can.J.Chem., 40,159 (1962). (9) S. Hjerten and R. Mosbach, Anal. Biochem., 3,109 (1962). (10) Bio-Rad Laboratories, Richmond, California, U. S. A. (11) M. F. Vaughan, Nature, 188,55 (1960). (12) J. C. Moore, J . Polym. Sci., Part A, 2,835 (1964). (13) Waters Associates, Framingham, Massachusetts, U. S. A. (14) K. J. Bombaugh, W. A. Dark, and R. N. King,J. Polym. Sci., Part C,21, 131 (1968). (15) S. A. Barker, B. W. Hatt, J. F. Kennedy, and P. J. Somers, Carbohyd. Res., 9, 327 (1969). (16) W. Haller, Nature, 206,693 (1965). (17) S. A. Barker, S. J. Crews, and J. B. Marsters, Symp. Biochem. Eye, Schloss Tutzing (1966). (18) G . Meyerhoff, Angew. Makromol. Chem., 415,268 (1968). (19) J. H. Ross and M. E. Casto, J. Polym. Sci., Part C , 21, 143 (1968). (20) A. J. de Vries, M. LePage, R. Beau, and C. L. Guillemin, Anal. Chem., 39, 935 (1967). (21) M. LePage, R. Beau, and A. J. de Vries,J. Polym. Sci., Part C , 21,119 (1968). (22) S . A. Barker, B. W. Hatt, J. B. Marsters, and P. J. Somers, Carbohyd. Res., 9, 373 (1969). (23) S. A. Barker, B. W. Hatt, and P. J. Somers, Carbohyd. Res., 11,355 (1969).

16

SHIRLEY C. CHURMS

tend to be irreversibly adsorbed on porous silica, but a deactivated form of this support has been developed for use in such worksz4 The most suitable media for gel chromatography of carbohydrates are poly(acry1amide) gels, hydrophilized polystyrene, and porous glass or silica beads. Dextran and agarose gels have the disadvantage of being themselves carbohydrates, so that the effluent solution is liable to contamination with carbohydrate material from the gel. Sephadex and agarose have, nevertheless, been extensively used in this field, but are now gradually being superseded by the newer, non-carbohydrate media. 111. THEORY OF GEL CHROMATOGRAPHY

The theory of gel chromatography has been comprehensively reviewed by DetermannZ5and Altgelt.zsIn the present article, emphasis is laid upon the fundamental aspects most likely to arise in carbohydrate chemistry. 1. Principles and Definitions

In a column packed with a swollen gel, two solvent phases may be distinguished, one within the gel (the stationary phase), and the other outside (the mobile phase). The volume of solvent in the gel is known as the internal volume, designated V,, and the volume of solvent outside the gel particles is the void volume of the column, designated V,. A solute will distribute itself between the two phases to an extent measured by the distribution coefficient Kd, a constant determined by the nature of the solute, the solvent, and the gel, but independent of column ge~metry.~’ The volume of solvent required for eluting the solute from the column in maximum concentration is called the elution volume and is designated Ve; it is equal to the sum of the void volume and the volume of the stationary phase available to the solute, given by & Vi. Hence,

ve= v, + K d - v,,

(1)

from which may be derived the relationship

Kd = w e - V0)lVt (24) K. J. Bombaugh, W. A. Dark, and J. N. Little, Anal. Chem., 41,1337 (1969). (25) H. Determann, “Gel Chromatography” (English Edition), Springer-Verlag New York Inc., New York, N.Y., 1968. (26) K. H. Altgelt, Adoan. Chromatogr., 7,3 (1968). (27) P. Flodin,]. Chromatogr., 5,103 (1961).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

17

If the solute molecules are so large that they are completely excluded from the gel, Kd = 0. In this case, the solute passes through the column entirely in the mobile phase, and its elution volume equals the void volume V,. Small solute molecules that can freely penetrate the gel pores will have a Kd value of 1; here, V, = V, Vi. Between these two extremes lie all solutes that can enter the gel phase to various, limited extents; these will have Kd values lying between 0 and 1, and elution volumes between V, and V, Vi, with both Kd and V, increasing as the molecular size of the solute decreases. The value of Kd cannot exceed unity if the molecular-sieve effect is the sole factor involved, but higher Kd values are found where adsorption effects are superimposed upon the molecular-sieve mechanism. For example, basic substances are adsorbed by ionexchange on gels having ionizable groups (for example, Sephadex ~ ~ , aromatic ~~ and and agar) in solvents of low ionic ~ t r e n g t h , and ~ ~a .mechanism ~~ believed3' heterocyclic compounds are a d s ~ r b e dby to involve the delocalized welectrons present in such compounds.

+

+

2. Separation by Gel Chromatography

The degree of separation of two solutes on any gel depends upon their Kd values on that gel. If one solute has a distribution coefficient K d ' , and the other, K d t ' , their elution volumes on a given column will respectively. The two solutes will, be (v, K d rVi) and (V, K d t t Vi), therefore, emerge from the column separated by a volume V i( K d fthis indicates that separation is improved by increasing Vi, so a column of large volume is desirable. The critical factor is, however, the difference in Kd between the two solutes. This difference depends on the size of the gel pores, which must be of such a size as to differentiate between the appropriate molecular sizes to the greatest possible extent. Optimal separation, on a gel column, of substances having small differences in molecular size is obtained if the gel pores are of a size comparable with that of the substances A gel of given porosity is, therefore, effecbeing chromat~graphed.~~ tive in separating substances within a certain range of molecular size only, the sizes involved decreasing with decreasing size of the gel pores; this range of molecular sizes is known as the fractionation range for the gel.

+

+

&'I);

(28) B. Gelotte,]. Chromatogr., 3,330 (1960). (29) P. Andrews, Nature, 196,36 (1962). (30) K. Sun and A. H. Sehon, C a n . ] . Chem., 43,969 (1965). (31) J.-C. Jamon,]. Chromatog.,28,12(1967). (32) J. Porath, Biochim. E i o p h y s . Acta, 39,193 (1960).

SHIRLEY C. CHURMS

18

Fractionation ranges are conventionally given in terms of molecular weights. The ranges given by manufacturers usually apply to globular proteins. A polysaccharide having a relatively open-chain (random-coiled) structure, such as a dextran, will be larger than a globular protein of the same molecular weight, and, consequently, will be excluded to a greater extent from a gel of a given porosity. The gel will, therefore, be effective in separating dextrans of somewhat lower molecular weight as compared with proteins.33 The fractionation ranges of Sephadex gels, both for protein^^^.^^ and d e x t r a n ~ ? ~ ~~ are well established (see Table I), as are those for S e p h a r o ~ e(see TABLEI Fractionation Ranges of Sephadex Gels Fractionation range (mol. wt.)

Type of gel

Globular p r o t e i n ~ ~ ~ ~ ~ ~ Dextrans36

G-10 G-15 G-25 (2-50 (2-75 G-100 G-150 G-200

700

=s703

s 1,500

< 1,500

1,000-5,000 1,500-30,000 3,000-70,000 4,000- 150,000 5,000-400,000 5,000-800,000

100-5,000 500-10,000 1,000-50,000 1,000-100,000 1,000-150,000 1,000-200,000

Table 11), but, for other gels, only the ranges for proteins have been published by the manufacturers; the ranges for polysaccharides and other substances have to be determined by experiment. For carboTABLEI1 Fractionation Ranges of Sepharose Gels3fi Type of gel -

Agarose (%)

6B 4B 2B

4

6

2

Fractionation range (mol. wt.) Globular proteins

Dextrans

1 X lo4-4 X lo6 4 x 105-20 x 106 3 X 106-40X lo6

1 x 104-1 x 106 3 X 10’-3 X lo6 2 X 106-25X 10‘

(33) P. Andrews, Biochern.J.,91,222 (1964). (34) P. Andrews, Biochern.].,96,595 (1965). (35) K. A. Granath and P. Flodin, Makrornol. Chem., 48,160 (1961). (36) Manufacturers’ literature (1969).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

19

hydrates, the fractionation ranges of the poly(acry1amide) gels BioGel P-300, P-10, and P-4 have been published (see Table III).37 TABLE111 Fractionation Ranges of Some Poly(acry1amide)Gels Type of gel Bio-Gel P-4 P-10 P-300

Fractionation range (mol. wt.) Globular proteins’O 500-4,000 5,000- 17,000

100.000-400.000

Dextrans3’

< 4,000 250-15,000 5.000- 100.000

Values of Kd in gel chromatography are not affected to any great extent by the experimental conditions. Temperature has a very small effect; Selby and Maitland3snoted that a sharp decrease in room temperature caused a slight decrease in the Kd values of certain enzymes on a Sephadex gel, and a corresponding small increase in Kd with increase in temperature from 9 to 60” was observed by Obrink and coworkers39 for fractions of the synthetic “polysucrose” Ficoll on Sephadex. The increase in Kd with increasing temperature has been ascribed39to a decrease in V i that results from shrinking of the gel at higher temperatures; this is possibly due to increased coiling of the dextran chains as a result of changes in the degree of interaction with the solvent. The temperature effect has so far been observed only with dextran gels; whether or not it is universal remains to be ascertained. In some cases, but not in others, the concentration of the sample has been found to have a small effect on the elution volume, and, consequently, on K d . Winzor and Nicho140 noted a slight increase in the elution volumes of certain proteins on Sephadex G-100 as the sample concentration was increased from 1 to 12 mg per ml, but no (weight-average such effect was observed with a dextran of molecular weight) 500,000. The effect observed with the proteins was ascribed to dependence of their migration rates on the concentration. 500,000 with An increase in the elution volume of the dextran of

aw

a,

(37) S. C. Churms and A. M. Stephen, S.Afr. Med.J . , 43,124 (1969). (38) K. Selby and C. C. Maitland, Biochem.]., 94,578 (1965). (39) B. Obrink, T. C. Laurent, and R. Rigler,J. Chromotogr., 31,48 (1967). (40) D. J. Winzor and L. W. Nichol, Biochim. Biophys. Acto, 104,1(1965).

SHIRLEY C. CHURMS

20

increasing concentration (5-20 mg. ml-l) was observed41 in studies using Sephadex G-200. This was attributed to increased void volume, due to shrinkage of the gel resulting from the higher osmotic pressure that accompanies increasing concentration of the non-penetrating solute in the mobile phase. Increase in elution volume with increasing concentration of the sample has also been noted in the chromatogthe dependence raphy of synthetic polymers on polystyrene ge1s,42,43 on concentration being greatest with samples having a broad distribution of molecular weight.42 The elution volumes of dextrans on porous silica (Porasil-D) have also been found to vary with the molecular-weight distribution at high concentrations of the sample;23this has been ascribed to mutual interaction of the solute molecules at high concentrations. The concentration dependence of the elution 10,000, and even of D-glucose, on the volumes of a dextran of poly(acry1amide) gel Bio-Gel P-10 (see Table IV), observed in this

am

TABLEIV Effect of Concentration of Sample on V, and K d of Carbohydrates on Poly(acry1amide) Gels4'

Solute

Gel

Column dimensions (cm)

D-Glucose

Bio-Gel P-10

60 X 1.2

Bio-Gel P-300

60 X 1.2

Bio-Gel P-10

60 X 1.2

Bio-Gel P-300

9OX

Dextran -

M, 10,000 1.5

Sample concentration (mg. m1-I)

1.6 16.5 1.9 19.6 1.6 10.3 18.7 3.8 8.5 19.9

62.7 64.1 70.1 70.1 30.5 31.9 33.7 100.0 100.4 100.6

0.23 0.26 0.30 0.64 0.64 0.64

laboratory,44 can probably be similarly explained. N o such effect was noted with the (more porous) Bio-Gel P-300; less interaction may be expected in gels having large pores. (41) E. Edmond, S. Farquhar, J. R. Dunstone, and A. G. Ogston, Biochem. J., 108, 755 (1968). (42) M. J. R. Cantow, R. S . Porter, and J. F. Johnson, J . Polymer Sci., Part B , 4, 707 ( 1966). (43) K. A. Boni, F. A. Sliemers, and P. B. Stickney, Amer. Chem. SOC., Dio. Polym. Chem., Preprints, 8,446 (1967). (44) S. C. Churms, A. M. Stephen, and P. van der Bij1,J. Chzomatogr.,47,97 (1970).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

21

The degree of separation, or resolution, obtained in any chromatographic process depends not only on the aforementioned factors, which influence the positions of the solute peaks in the resulting chromatograms, but also on the peak widths, which depend on the efficiency of the column. Although Kd values are largely independent of experimental conditions, the column efficiency, which must be high if the narrow peaks necessary for good resolution are to be obtained, is governed by several operating variables, and, therefore, attention to these is as essential in gel chromatography as in other chromatographic processes. The effect of operating conditions on the efficiency of gel chromatography has been investigated by several w ~ r k e r s , ~ ~and , ~ ”it ~has ~ been found that the greatest efficiency is obtained with long columns having a small diameter, gels having small particle size, particles uniformly packed, small volumes of sample, and low rates of flow of the solution. With regard to the last factor, however, it should be noted that an equation of the Van Deemter type48 has been to apply to gel chromatography. The use of very low rates of flow will, therefore, result in a certain amount of peak broadening, due to the effect of longitudinal diffusion of the solute in the mobile phase, although this factor is bel i e ~ e dto~be ~ less significant in gel chromatography than in other chromatographic processes. A n d r e w ~has ~ ~found flow rates in the range of 3 to 10 ml per hr per cm* to be optimal for gel chromatography. The efficiency of gel chromatography is independent of the concentration of the sample,27except where the viscosity of the solution increases rapidly with the concentration; resolution then deteriorates with increasing concentration of the sample, owing to the peak broadening that results from the irregular flow-pattern caused b y the greater viscosity of the s o l ~ t i o n . ~ ~ * ~ ~ 3. Determination of Molecular Weight There is at present no general agreement as to the best method of relating the elution volumes and Kd values in gel chromatography (45) (46) (47) (48)

J. C. Giddings and K. L. Malik, Anal. Chem., 38,997 (1966). W. Heitz and J. CoupekJ. Chrornntogr.,36,290 (1968). J. C. Giddings, Anal. Chem., 40,2143 (1968). J. J. van Deemter, F. J. Zuiderweg, and A. Klinkenberg, Chem. Eng. Sci., 5,

271 (1956). (49) P. Andrews, Brit. Med. Bull., 22,109(1966). (50) J.L. Waters, Amer. Chem. Soc., Dlo. Polym. Chem., Preprints, 6, 1061 (1965).

22

SHIRLEY C. CHURMS

to the molecular weights of the substances being chromatographed; several different relationships have been proposed. An early worker in this field was P ~ r a t h , who, ~ l on the assumption that the gel pores are conical, derived the equation

where k is a constant, characteristic of the gel and the solute, r is the average radius of the gel pores, and a is the effective radius of the solute. For flexible polymers consisting of identical segments, the effective (gyration) radius in solution has been found52to be proportional to the square root of the molecular weight, M. Such a relationship holds, to a good approximation, for d e x t r a n ~ .Here, ~ ~ equation 3 implies a linear relationship between Kd113 and M112for solutes of this type on a given gel. This relationship was observed by Porath, who, to test the equation, used the data of Granath and F 1 0 d i n ~for ~ dextran fractions on Sephadex gels. By using a model of the gel phase in which the gel matrix was assumed to consist of straight, rigid rods that were infinitely long and randomly distributed, Laurent and Killander54derived the equation &, = exp 1 - d (rr + ~ 2 1 , (4) where Z&, is the fraction of the total gel-volume available to the solute, given by (V, - V,)/(V, - V,) [where V, is the total volume of the gel phase (including that of the gel matrix), L is the concentration of the rods in the gel, expressed as cm of rod per cm3, rr is the radius of the rods, and r, is the Stokes radius of the solute]. This equation is based on an equation derived by Ogston55 for the volume available to spherical particles in a system of this type. Assuming a value of rr of 70 nm for dextran gels (a value somewhat larger than the value accepted for the radius of linear dextran chains, since branching and crosslinking effectively thicken the chains), Laurent and Killandef14 determined L for various Sephadex gels by plotting the &, values of a series of solutes of known Stokes radius against r,, and fitting equation 4 to the experimental curves. The constants having been established, the equation was applied to experimental data from the (51)J . Porath, Pure Appl.Chem.,6,233(1963). (52)B.H.Zimm and W. H. Stockmeyer,]. Chem. Phys., 17,1301(1949). (53)K.A.Granath,]. ColZoidSci.,13,308(1958). (54)T.C.Laurent and J . Killander,]. Chromatogr., 14,317(1964). 54,1754(1958). (55)A.G.Ogston, Trans. Faraday SOC.,

GEL CHROMATOGRAPHY OF CARBOHYDRATES

23

literature on the gel chromatography both of proteins and dextrans; it was found to fit, to a good approximation, all of these data. Equation 4 relates K,, to the Stokes radius of the solute molecules, not to their molecular weight. Laurent and Granath56calculated the molecular weights of dextran and of Ficoll fractions from their K,, values on a Sephadex gel by using this equation to calculate a radius equivalent to the Stokes radius for each polysaccharide fraction, and, thence, an apparent diffusion constant for each. The sedimentation constant of each fraction was determined by ultracentrifugation, and this value and the calculated diffusion constant were substituted into the equation of Svedberg and Pedersens7 in order to obtain an apparent molecular weight for the polysaccharide fraction. Siegel and Montys8also recommended this procedure. If, however, the Stokes radius of the solute bears some simple relationship to its molecular weight, the latter may be determined directly from gel chromatography by means of the Laurent-Killander equation.s4 Sims and FolkesS9have pointed out that equation 4 may be more simply expressed in the form (-ln K,,)1/2 = a(p

+

T,)

where a and p are constants characteristic of the gel. Equation 5 predicts a linear relationship between (-ln K,u)1’2and T,, and this has been confirmed experimentally for proteins on various Sagarose gels.60 If the solute molecule is considered to be spherical, as is the case for globular proteins, r, is proportional to the cube root of the molecular weight, M. The linear relationship between (-ln Ka,)112and r, here implies that (-ln K,,)1/2 will also vary linearly with and this has been observed for proteins on Sagarose gels.61 as For dextrans, rs may be regarded as being proportional to already mentioned; therefore, a linear relationship should be observed between (-ln K,,)1’2 and M112.Data obtained by Churms and Stephens2 for the chromatography of dextran fractions on a poly(acry1amide) gel (Bio-Gel P-300) have demonstrated the validity of this relationship. (56) T. C. Laurent and K. A. Granath, Biochim. Biophys. Acta, 136,191 (1967). (57) T. Svedberg and K. 0. Pedersen, “The Ultracentrifuge,” Clarendon Press, Oxford, 1940, p. 5. (58) L. M. Siegel and K. J. Monty, Biochim. Biophys. Acta, 112,346 (1966). (59) A. P. Sims and B. F. Folkes, unpublished work, cited in Sagarose manufacturers’ literature (1966). (60) A. P. Sims, A. H. Bussey, and B. F. Folkes, Ref. 59. (61) Manufacturers’ literature (1966). (62) S. C. Churms and A. M. Stephen, unpublished work.

24

SHIRLEY C . CHURMS

Squires3 used a model of the gel phase in which the volume elements available to solvent within the gel were regarded as a combination of cones, cylinders, and crevices, and derived expressions for the volumes available to a solute of Stokes radius u in these three types of pore. Certain arbitrary assumptions regarding the distribution of solute among the different types of pore gave the following equation:

which simplifies to

where g is a constant characteristic of the gel and the nature of the solute, and T is the average radius of the gel pores. For spherical molecules, a is proportional to M113,and equation 6 may be written as

where C is a constant corresponding to the molecular weight of the smallest spherical solute that is completely excluded from the gel pores. Equation 7 predicts a linear relationship between (V,/V,)113and M113,and this was confirmed by means of experimental data taken from the literature. Squires3 claimed that equation 7 is applicable to dextrans, as well as to proteins. However, in view of the observed proportionality of a and M112(not M113)for dextrans, it is the opinion of the author that, when applied to dextrans, the Squire equation should be modified to

where C represents the molecular weight of the smallest dextran molecule that is completely excluded from the gel. A linear relationship between (Ve/V,)'I3 and M1I2 is predicted by equation 8, and this has been founds2to hold for dextran fractions on the poIy(acry1amide)gel Bio-Gel P-300. The various, theoretically derived relationships between gelchromatographic elution-parameters of macromolecules and their molecular weights are given in Table V. (63) P. G . Squire, Arch. Biochem. Biophys., 107,471 (1964).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

25

TABLEV Theoretically Derived Relationships between Elution Parameters and Molecular Weight Molecular type

Relations hipa

Dextrans Globular proteins Dextrans Globular proteins Dextrans

K d I l 3 = k 1 - k2 MI12 (-ln K,,)1i2= k l k , M1I3 (-ln = kl + k, ML12 (Ve/Vo)113= k , - k, (Ve/V0)"3=k,- k, M'/'

+

References

51 54,59-61 62 63 62

"k, and k, are constants, defined differently in each equation.

A ~ k e r shas ~ ~ interpreted gel chromatography in terms of steric and frictional resistance to the diffusion of the solute in the gel pores, and, on this basis, he has used an equation originally proposed by R e n k i ~ Pfor deriving the following relationship between Kd and the Stokes radius, a, of the solute:

where r is again the effective radius of the gel pores, here assumed to be cylindrical. Values of Kd computed from this equation for solutes of known a on gels of known T were found by A c k e d 4 to agree well with experimental values. An advantage of the Ackers equation is that no arbitrary constants are involved; the only constant is I , which is a property of the gel. The value of T for a given gel can be determined experimentally by measuring the Kd values of solutes of known Stokes radius and substituting the appropriate values of Kd and a into equation 9. With the value of T known, the equation can then be used for determining the Stokes radii of other solutes from their Kd values on the gel. Here, however, no simple correlation with molecular weight is obtainable; in order that the Ackers equation may be used for determining molecular weights, it is necessary that ultracentrifugation data be combined with the data obtained by gel chrornat~graphy.~~ In addition to the various relationships based on theoretical principles, several empirical correlations of gel chromatography data with molecular weights have been reported (see Table VI). A linear correlation between V, and IogM, holding over a molecular-weight (64) G. K. Ackers, Biochemistry, 3,723 (1964). (65) E.M. Renkin, J . Gen. Physiol., 38,225 (1955).

26

SHIRLEY C.

CHURMS

TABLEVI Empirical Correlations between Elution Parameters and Molecular Weight Molecular type

Relationshipa

Proteins, polysaccharides, synthetic polymers Proteins Pol ysaccharides Proteins Dextrans Peptide hormones

V,= k,- k2 IogM

29,33,66-68

& =kl-

30 69 70-72 35 73

k2

IogM

K,, = k,- k, logM VJV, = k,- k, 1ogM V,lV,= k , - k, logM Kd=(kl/logM)- k2

References

"k,and k , are constants that are different in each case. '

range coinciding with the fractionation range for the gel, has been observed in many systems when series of similar solutes have been chromatographed on the same gel co1umn.29,33*66-68 This correlation, which is now widely applied in the determination of molecular weights by gel chromatography, has been observed for protein^,^^*^"^^ polysaccharides,M and synthetic polymed8 on S e p h a d e ~ agar,29 ,~~ p o l y ( a ~ r y l a m i d e ) ,and ~ ~ *po1ystyrene'ja ~~ gels, and may therefore be regarded as universally applicable. A linear correlation between & (or &), and logM under the same conditions has also been observed with several different solute-gel systems, for example, proteins on poly(acry1amide) gels30 and dextran fractions on S e p h a d e ~This .~~ correlation, too, is widely used in molecular-weight determinations. Several ~ o r k e r s have ~ ~ -reported ~~ a linear correlation between VJV, and logM for proteins on Sephadex gels, and Granath and F 1 0 d i n ~ ~ noted a linear relationship between VJV, and log%,, (number-average molecular weight) for dextran fractions on these gels. All of these correlations are with logM; in contrast, Sanfellipo and Surak13found that, on Sephadex G-50, & varies linearly with (logM)-' for the hormonally active proteins and peptides of the anterior pituitary gland. In an attempt to account for these empirical correlations on the (66)D. M.W.Anderson, I.C. M. Dea, S. Rahman, and J. F. Stoddart, Chem. Commun., 145 (1965). (67)A. M.del C.Batlle,]. Chromatogr.,28,82(1967). (68)S.Yamada, S. Kitahara, and Y. Hattori, Kobunshi Kagaku, 23,683(1966). (69)K.A. Granath and B. E. Kvist,]. Chromatogr., 28,69(1967). (70)J. R.Whitaker, Anal. Chem.,35,1950(1963). (71)A. A. Leach and P. C. O'Shea,]. Chromatogr., 17,245(1965). (72)H.Determann and W. Michel,]. Chromatogr., 25,303(1966). (73)P. M.Sanfellipo and J. G . Surak,]. Chromatogr., 13,148(1964).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

27

basis of the various theoretical treatments of gel chromatography, Anderson and S t ~ d d a r t plotted '~ the Kd values computed by A c k e d 4 (by means of equation 9) against the logarithms of the corresponding values of aIr. The Porath treatment"l was also used for calculating values of & for known values of a h ; a value of 1.64, applicable to dextran fractions on Sephadex gels, was used for the constant k of equation 3, and the Kd values thus obtained were also plotted against log a h . Both plots gave sigmoid curves, having extended linear portions, which could be expressed as . .

where k, and k, are constants. For any solute, a is proportional to some fractional power of the molecular weight M, a power that depends on the shape of the molecule but will be the same for any series of similar molecules. If a = M" for a certain type of molecule, then, for any series of solutes of this type on a gel of pore radius r, equation 10 may be written as

=-

k, x logM + k, logr+ k2

=- b logM

+

(11)

C,

where b and c are constants. Equation 11 predicts a linear relationship between & and logM for a series of similar solutes on a given gel over the range of molecular size for which equation 10 holds; this gives theoretical justification to the empirical correlation r e p ~ r t e d . ~ ~ . ~ ~ Anderson and St0dda1-t'~have also justified the correlation between V, and logM by this treatment. From equations 2 and 11,

+ Vi logM + c Vi + V,.

(V, - V,)lVi = -b logM

:. V,

= -b

C.

For a given column, V, and V, are constants, so that this simplifies to V,=-b'logM

+ c',

(12)

where b' and c' are constants. Equation 12 predicts the observed linear relationship between V, and logM. (74) D. M. W. Anderson and J. F. Stoddart, Anal. Chim. Acta, 34,401 (1966).

SHIRLEY C. CHURMS

28

The two other correlations, between logM and V,lV, or VJV,, can also be deduced in this way. The only reported correlation that is not accounted for by this treatment is that observed by Sanfellipo and Kd and (logM)-', for which no theoretical justificaS ~ r a between k~~ tion has as yet been obtained. The work of Anderson and S t ~ d d a r t has ' ~ thus given a sound theoretical basis to the correlations, formerly empirical, of gel-chromatography data with molecular weights, data which have been used with considerable success in the determination of the molecular weights of macromolecules. Of these correlations, the one most universally employed is the simplest; namely, the linear correlation between V, and logM for a series of similar solutes on a given gelcolumn. A number of substances of known molecular weight, or polymer fractions of narrow molecular-weight distribution, with or g,,,are chromatographed, under identical conknown values of ditions, on a column packed with a gel of appropriate fractionationrange, and the elution volumes observed are plotted against logM to give the calibration curve for the column; this should be linear over a range of molecular weights coinciding with the fractionation range of the gel. The substances under examination are then chromatographed on the same column under the same conditions as were used for the calibrating solutes, and their molecular weights are found from the observed elution volumes by reference to the calibration curve. The substances used for calibration must be structurally similar to those being examined, as the relationship between the Stokes radius and the molecular weight should be similar if the calibration is to be valid. For example, if the substances under study are globular proteins, the column must be calibrated with globular proteins; other types, such as glycoproteins, are not suitable for the calibration owing to their different structure.34Well-characterized fractions of dextran are used for caIibration in determinations of the molecular weight of p o l y s a c c h a r i d e ~ . ~ ~ . ~ ~ Values of molecular weight obtained by this method have been found to agree within 5-10% with values determined by classical methods. For example, from chromatography of fractions of the polysaccharide gum from Acacia senegal on Bio-Gel P-300, Anderson and coworkerP obtained Gnvalues of 99,000 and 35,000, respectively; the corresponding values obtained by osmometry were 105,000 and 37,000. Churms and S t e ~ h e n , ~on ' chromatographing the polysaccharide gum from Acacia podalyriaefolia on the same gel, obtained

al,.

GEL CHROMATOGRAPHY OF CARBOHYDRATES

29

a value of 31,500 for M,, the value from sedimentation and diffusion measurement^'^ being 33,500. In order to compare molecular weights determined by use of the simple correlation of V, and logM with the values given by the various theoretical equations, the author has calculated the molecular ~ the elution pattern weights corresponding to 3 peaks ~ b s e r v e d 'in of the gum from Acacia elata on Bio-Gel P-300, and has used, in addition to the V, versus logM correlation, the relationships arising from the P ~ r a t h ,La~rent-Killander,~~ ~~ and Squire63 treatments of gel chromatography. The validity of the linear relationships between M112and the functions Kd113,(- In K,J1/2, and (Ve/V0)1/3, predicted by the Porath equation and the modified versions of the Laurent-Killander and Squire equations, respectively, was tested with data obranging tained by chromatography of dextran fractions of known from 10,000 to 70,000, on the same column (90 X 1.5 cm) packed with the same gel. Plots of these functions against the square root of Mw were found to be linear in all cases. The values of the three functions corresponding to the elution volumes at the peaks in the elution pattern of the A. elata gum were then calculated, and the M , values of the appropriate polysaccharide fractions were estimated by interpolation of these linear plots. These molecular weights were also determined by reference to a V, versus log%, plot for the column, obtained with the same dextran fractions. The results given by the different treatments are compared in Table VII, from which it may be seen that agreement is fairly good, no result deviating from the mean by more than 10%.

zw,

TABLEVII Comparison of Values of Molecular Weight Obtained by Different Treatments M W

V, (ml)

PorathJ'

LaurentKilIandeP

Squirea3

V, versus log7iiw correlation

Mean

82 93 98

31,700 21,900 19,000

32,400 22,500 19,300

28,600 20,200 15,900

29,900 20,000 16,800

30,600 21,200 17,800

(75)G . R. Woolard, Ph.D. Thesis, University of Cape Town (1968). (76)P. I. Bekker, S. C. Chums, A. M. Stephen, and G. R. Woolard, Tetrahedron, 25, 3359 (1969).

30

SHIRLEY C. CHURMS

It may, therefore, be concluded that all of these approaches are valid for galactans of the type i n v e ~ t i g a t e d(and, ~ ~ perhaps, for polysaccharides in general) on poly(acry1amide) gels. The V, OerSus logM correlation has the advantage of simplicity, and it is, therefore, possibly the best method of determining molecular weights by gel chromatography. In the case of polymers having a distribution of molecular weights, such as polysaccharides, any determination of molecular weight can give only an average. The type of average obtained depends upon the experimental method used. Thus, a method that effectively counts the number of molecules present (for example, any method involving colligative properties, such as osmotic pressure) will give the numberaverage molecular weight Be, whereas a weight-average molecular weight is obtained from such methods as light-scattering that depend on the weight of the molecules instead of on their number. Gel chromatography is, however, not an absolute but a relative method of molecular-weight determination; all of the equations purporting to relate gel-chromatographic data to molecular weights or sizes contain constants that can be determined only by calibration of the gel column with similar solutes of known molecular weight or Stokes radius. Therefore, molecular weights obtained in this way depend upon those used in calibration of the column; if M, values are used, the results should be regarded as 2, values, whereas calibration in terms of G,, values gives results that will be closer to 2, for the polymer under examination. That both alternatives are permissible is demonstrated by the linearity, over a wide range of molecular weight, of plots of &” against both GWand %, calculated from results obtained by Granath and KvisP on chromatography of dextran fractions on Sephadex. The fact that the G, values of dextran fractions (from light-~cattering~~) were used by the author in establishing the validity of the Porath equation5I and the modified Laurent-Killandel.s4 and Squires3 equations for dextrans, whereas P ~ r a t h , ~ ~ Laurent and Killander,54 and Squirea3themselves used the results of Granath and F10din~~ (who gave ii?, values) to confirm the applicability of their equations to dextrans, also indicates that either type of average fits these treatments. Thus, gel chromatography may be used to determine either Gwor of a polymolecular substance.

an

(77) Pharmacia, Uppsala, Sweden, technical literature on dextrans (1966).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

31

Iv. APPLICATIONTO CARBOHYDRATES 1. Sugars

a. Monosaccharides. -The behavior of aldoses in gel chromatogby using dexraphy has been thoroughly investigated b y Mar~den,'~ tran gels having a high degree of cross-linking and, consequently, very small pores capable of differentiating between such small solute molecules. Marsden found that the Kd values of these sugars did not decrease monotonically with increasing molecular weight. The value (0.64) found for an aldotetrose, namely, D-erythrose, was slightly higher than that for D-glyceraldehyde (0.61);the latter value may have been lowered as a result of dimerization of the D-glyceraldehyde in solution. The aldopentoses were found to have K d values higher than that of D-erythrose; Marsden attributed this property to the greater flexibility of the pyranose ring of the aldopentoses, as compared with that of the furanose ring in D-erythrose, the rigidity of which might be expected to hinder the entry of D-erythrose molecules into the gel pores. All of the aldohexoses examined exhibited Kd values lower than those of the corresponding aldopentoses, molecular weight being the main factor involved, as both classes tend to adopt the pyranose ring structure. Variations in Kd within the aldopentose and aldohexose series were observed, the Kd values of the aldopentoses increasing from 0.646 for D-arabinose to 0.710 for D-ribose, whereas those of the aldohexoses varied from 0.573 for D-glucose and D-galactose to 0.676 for D-talOSe. This result was attributed to differences in the degree of steric hindrance, resulting from the different spatial arrangements of the hydroxyl groups, to the entry of the various sugar molecules n ~ ~ that, within each series of sugars, into the gel phase. M a r ~ d e noted Kd decreased with decreasing instability rating (Kelly scale79)of the chair conformers of the sugars, that is, with increasing proportion of forms more liable than other conformations to steric hindrance in the gel phase. That this variation in Kd among isomeric sugars is related to conformation is further demonstrated by the absence of such differences in Kd within the corresponding alditol series; all pentitols were found to have a Kd of 0.576, and all hexitols, of 0.547. Despite the presence of anomers, differing in instability rating of their chair conformations, the elution profiles obtained on gel (78) N. V. B. Marsden, Ann. N. Y. Acad. Sci., 125, 428 (1965). (79) R. B. Kelly, Can. /. Chem., 35, 149 (1957).

32

SHIRLEY C. CHURMS

chromatography showed a single, sharp peak for every sugar studied; each sugar was eluted as if it were a single compound. Since mutarotation usually proceeds much more rapidly than elution, the & values determined by Marsden'* are those corresponding to the equilibrium mixtures of the anomers of the various sugars. The enantiomorphs of a given sugar were found to exhibit no appreciable difference in &; for example, the values for D- and L-mannose were 0.626and 0.627,respectively. It appears, therefore, that stereospecific adsorption to the gel, which could possibly result from the presence of the dissymmetric residues of D-glucopyranose in the dextran matrix, is not a significant factor. The & values found for methyl glycosides were lower than those of the corresponding sugars; for example, the values for D-glUCOSe and methyl a-D-glucopyranoside were 0.573 and 0.496,respectively. This behavior may be ascribed to the added bulk of the methyl group. M a r ~ d e n 'did ~ not attempt to separate mixtures of monosaccharides by gel chromatography; the small differences in K d among the different sugars indicate that separation could be achieved only on very large columns by use of the eluants used by this author, namely, deionized water and buffers of ionic strength 0.05(pH 7-8)composed (Tris) and hydrochloric of 2-amino-2-(hydroxymethyl)-1,3-propanediol or acetic acid. A good separation of L-rhamnose, 2-acetamido-2-deoxy-~-glucose, D-glucose, and 2-amino-2-deoxy-D-glucose, eluted in that order, was obtained by ZeleznickRoon a small column packed with the highly cross-linked, dextran gel Sephadex G-25,with 62:15:25 (v/v) butyl alcohol-it4 acetic acid-water as the eluant; however, the gel functioned more as a support for partition chromatography than as a molecular sieve. An almost complete separation of D-glucose from D-ribose on the poly(acry1amide) gel Bio-Gel P-2,with water as the eluant, has been reported by John and coworkers.81A water-jacketed column (127X 1.5 cm) at a temperature of 65" and gel of very fine particle-size (400 mesh) were used. Only under such conditions, where resolution is optimal, does good separation of monosaccharides by gel chromatography become possible.

b. Oligosaccharides. - Mixtures of oligosaccharides differing in (80) L. D. Zeleznick, J . Chromatogr., 14, 139 (1964). (81) M. John, G. TrBnel, and H. Dellweg, J . Chromatogr., 42, 476 (1969).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

33

molecular weight are readily separated by gel chromatography, so that the technique is useful in the isolation of the oligosaccharides produced in degradative studies of polysaccharides. The value of the method was first demonstrated by Flodin and Aspberg,s2who used gel chromatography on Sephadex G-25 in distilled water to separate the sugars produced by acetolysis of cellulose followed by deacetylation of the resulting acetate^.^^,*^ An almost complete separation of the products, from D-glucose up to cellohexaose, was achieved. Similar gel chromatography of the oligosaccharides produced by acetolysis of yeast m a n n a n ~ has ~ ~ *been ~ ~ found to yield elution patterns characteristic of the yeast strains involved.86 For example, whereas the elution pattern given by the degradation products from Saccharomyces cerevisiae mannan revealed the presence of D-mannopentaose, -hexaose, and -heptaose, which, on further acetolysis, yielded di-, tri-, and tetra-saccharide~,~~ the pattern observed for acetolyzates of mannans from Candida strains was indicative of a high proportion of D-mannose, together with acetolysis-stable pentaand hexa-saccharides.86 In this way, the position in the mannan of ,~~ the (1 + 6)-linkages, the ones most susceptible to a c e t o l y s i ~ was indicated. Stewart and Ballous6have suggested the use of this technique in the classification of yeasts. John and coworkersa’ have reported the successful use of the highly cross-linked poly(acry1amide) gel Bio-Gel P-2 to separate the series -( oligosaccharides synthesized by the action of of a - ~ 1-+4)-linked Escherichia coli ML 30 on maltose. D-Glucose and oligosaccharides containing up to 13 D-glucose residues were resolved when the conditions used for separating D-glucose from D-ribose, already described, were employed. On this column, these workers also resolved maltose and isomaltose, and maltotriose and isomaltotriose, the (1+6)linked isomer being eluted first. Isomaltose and isomaltotriose were shown to be absent from the product obtained on incubation, with beta-amylase, of the oligosaccharide mixture formed by the action of amylomaltase in E . coli on maltose; the elution pattern on Bio-Gel P-2 showed that, after incubation for 2 hours, maltotriose, maltose, (82) P. Flodin and K. Aspberg, “Biological Structure and Function,” Academic Press, Inc., New York, N. Y., 1961, Vol. 1, p. 345. (83) E. E. Dickey and M . L. Wolfrom, /. Amer. Chem. Soc., 71, 825 (1949). (84) M . L. Wolfrom and J . C. Dacons,]. Amer. Chem. Soc., 74, 5331 (1952). (85) T. S. Stewart, P. B. Mendershausen, and C. E. Ballou, Biochemistry, 7, 1843 (1968). (86) T. S. Stewart and C. E. Ballou, Biochemistry, 7, 1855 (1968). (87) M . L. Wolfrom, A. Thompson, and C. E. Timberlake, Cereal Chem., 40,82 (1963).

34

SHIRLEY C. CHURMS

and D-glucose were the only degradation products. This further demonstrated the exclusive formation of a-D-( l+.Q)-linkages by action of amylomaltase on maltose.88 The oligosaccharides produced in degradative studies of glycosaminoglycans can also be separated by gel chromatography. For example, Flodin and coworkersss used Sephadex G-25for separating the lower members of the series of oligosaccharides produced by digestion of hyaluronic acid and chondroitin 4-sulfate with testicular hyaluronidase. These workersss succeeded in isolating the di-, tetra-, hexa-, and octa-saccharides by this procedure. The use, instead of distilled water, of an eluant containing sodium chloride in fairly high concentration was found to improve resolution, the best results being achieved with 0.1 M sodium chloride for the digest of hyaluronic acid, and M sodium chloride for the digest of chondroitin 4-sulfate. Eluants of high ionic strength, which have the effect of eliminating any form of electrostatic interaction (such as hydrogen bonding) of the solute molecules with the gel or with one another, are to be recommended in the gel chromatography of carbohydrates in general. In the course of an investigation of the chondroitin 6-sulfate-protein linkage, Helting and Rod&nsofractionated on Sephadex G-25 the oligosaccharides obtained on acid hydrolysis of chondroitin 6sulfate from umbilical cord, with 9:l (v/v) water-ethanol as the eluant. The separation of these oligosaccharides permitted their subsequent identification (by paper chromatography) as 3-O-P-D-galactosyl-4-O-P-D-galactosyl-D-xy~ose, 3-O-~-D-ga~actosyl-D-ga~actose, 4-0~-D-ga~actosyl-D-xy~ose, and 3-O-(~-D-glucosyluronicacid)-D-galactose. with 0.01 M Dietrichsl used gel chromatography on Sephadex G-25, acetic acid as the eluant, to fractionate the products obtained on degradation of heparin by enzymes of adapted Flavobacterium heparinium. The fractions from the gel column were subsequently purified by paper chromatography, and the components were idenditified as the 6-sulfuric ester of 2-deoxy-2-(sulfoamino)-~-glucose, --4)-linked ( to D-glucusaccharides consisting of this molecule a - ~ 1 ronic acid or its 3-sulfate, and tetra- and hexa-saccharides composed of these disaccharide units. Raftery and coworkerss2 have reported the fractionation of the (88) S.A. Barker and E. J. Bourne,]. Chem. Soc., 209 (1952). (89) P. Flodin, J. D . Gregory, and L. RodBn, Anal. Biochem., 8, 424 (1964). (90) T. Helting and L. RodBn, Biochim. Biophys. Acta, 170,301 (1968). (91) C. P. Dietrich, Biochem. I., 108, 647 (1968). (92) M. A. Raftery, T. Rand-Meir, F. W. Dahlquist, S. M. Parsons, C. L. Borders, Jr., R. G. Wolcott, W. Beranek, Jr., and L. Jao, Anal. Biochem., 30,427 (1969).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

35

oligosaccharides obtained on acid hydrolysis of chitin by chromatography on Bio-Gel P-2, with water as the eluant. A good separation of the sugars from 2-acetamido-2-deoxy-~-glucose up to chitohexaose was obtained. Gel chromatography has also been used for fractionating derivatives of these sugars; for example, N-(trifluoroacety1)ated derivatives can be separated on Bio-Gel P-2 in water, and p-nitrophenyl glycosides on the same gel, with 0.1 M sodium chloride as the eluant. Peracetylated derivatives have been separated on Sephadex LH-20 with methanol as the eluant. Dahlquist and Rafteryg3have also used gel chromatography in investigating the action of lysozyme on chitotriose. Chromatography of the product (incubated during 6 hours at 40" at pH 5.0) on Bio-Gel P-2, followed by paper chromatography of the fractions obtained, led to identification of the components present as 2-acetamido-2-deoxy-~glucose and chito-biose, -triose, and -tetraose. It was concluded that transglycosylation (resulting in the formation of the tetraose) occurred, as well as hydrolysis to chitobiose and 2-acetamido-2-deoxy-~-glucose. Naturally occurring oligosaccharides have also been isolated by gel chromatography. Ohman and HygstedP have reported the isolation, from colostrum, of sialic acid oligosaccharides, including diO-sialoyl-lactose [ O-N-acetylneuraminoy1-(2 + 8)-O-(N-acetylneuraminoy1)-(2+ 3)-O-P-D-galactopyranosyl-( 1 + 4)-a-~-glucopyranose], by a procedure involving gel chromatography on Sephadex G-25 with 99: 1 water-butyl alcohol. The lower members (containing 1-10 Dfructose residues) of the fructan series present in dahlia tubers have been separated by Pontisss on Bio-Gel P-2, with distilled water as the eluant. After the fractions corresponding to each peak in the resulting elution curve had been pooled and freeze-dried, paper chromatography showed each product to be a different, pure compound; the Dfructose oligosaccharides were thus completely separated by this method. 2. Polysaccharides a. Dextrans. -The fractionation of dextrans by gel chromatography has been much investigated. Following the earlier work of Flodin and coworker^,^*^^ which demonstrated the feasibility of fractionating dextrans according to molecular weight on Sephadex gels, and established the fractionation ranges of these gels for dextrans, Laurent (93) F. W. Dahlquist and M. A. Raftery, Nature, 213, 625 (1967). (94) R. Ohman and 0. Hygstedt, Anal. Biochem., 23, 391 (1968). (95) H. G. Pontis, Anal. Biochern., 23, 331 (1968).

36

SHIRLEY C. CHURMS

TABLEVIII Separation of Sugars and Derivatives by Gel Chromatography Gel Sephadex G-25

Eluant water

99: 1(vlv) water-butyl alcohol

9: 1(vlv) waterethanol 0.01 M acetic acid 0.1 M sodium chloride

M sodium chloride 62: 15:25 (vlv)butyl alcohol44 acetic acid-water Bio-Gel P-2

water

0.1 M sodium chloride Sephadex LH-20

methanol

Sugars separated

References

oligosaccharides from acetolysis of cellulose oligosaccharides from acetolysis of yeast mannans sialic acid-containing oligosaccharides from colostrum oligosaccharides from acid hydrolysis of chondroitin 6-sulfate oligosaccharides from enzymic degradation of heparin oligosaccharides from hyaluronidase degradation of hyaluronic acid oligosaccharides from hyaluronidase degradation of chondroitin 4-sulfate D-g1ucOSe, L-rhamnose, 2-amino-2-deoxy-D-glucose, 2-acetamido-2-deoxy-~glucose D-ghCOSe from D-ribose; maltose from isomaltose; maltotriose from isomaltotriose; oligosaccharides formed from maltose by amylomaltase fructans (1-10 fructose residues) from dahlia tubers 2-acetamido-2-deoxy-~glucose, chito-biose, -triose, 4etraose 2-acetamido-2-deoxy-Dglucose and oligosaccharides from acid hydrolysis of chitin; N-(trifluoroacety1)ated derivatives of these p-nitrophenyl glycosides of chitin oligosaccharides peracetylated derivatives of chitin olieosaccharides

82 85,86 94

90

91

89

89

80

81

95 93

92

92 92

GEL CHROMATOGRAPHY OF CARBOHYDRATES

37

and Granaths6 fractionated a dextran having a broad distribution of weight into relatively homogenous fractions, ranging in M , from 15,000 to 63,000, by gel chromatography on Sephadex G-200 with 0.2 M sodium chloride as the eluant. Hummel and D. C. Smithy6 investigated the fractionation of dextrans in the molecular-weight range of 30,000 to 500,000 on Sephadex, agar, agarose, and poly(acry1amide) gels, and found that the best fractionation of the dextrans of high molecular weight was achieved on a 7% agar gel, with a 0.2 M Tris hydrochloride buffer (pH 8.0) as the eluant. A 4% agarose gel, with distilled water as the eluant, also proved effective. Sephadex and poly(acry1amide) gels are unsuitable for the fractionation of dextrans of molecular weight above -100,000. Granath and KvisP9 have described a method of determining, by gel chromatography, the molecular-weight distributions of dextrans in the range of 10,000 to 100,000. A column packed with a mixture of two Sephadex gels, G-200 and G-100, in the dry-weight ratio of 1:2 (so that the two occupied equal volumes when swollen) was used by these workers; the eluant was 0.3% sodium chloride. After the column had been calibrated with 17 dextran fractions of known M , and M,, determined by independent methods (light-scattering and end-group analysis, respectively), the dextrans under examination were chromatographed under the same conditions, and the molecular weights corresponding to the observed K,, values were read from the calibration curve. The technique has proved useful in clinical investigations involving the use of d e x t r a n ~ . ~ ~ Dextrans of low molecular weight have also been fractionated by gel chromatography. Bremner and coworkers,97 in preparing (by alkaline degradation) dextrans of low molecular weight suitable for incorporation in the clinically important iron-dextran complex, fractionated their product on Sephadex G-50, with 0J. M sodium chloride as the eluant. Separate fractions, ranging in M , (as determined by osmometry) from 1510 to 4860, were obtained in this way. The newer supports for gel chromatography, such as lyophilized p o l y ~ t y r e n e 'and ~ porous s i l i ~ a , have ~ ~ - ~been ~ successfully applied to the fractionation of dextrans over a wide range of molecular weight. A lipophilic gel, namely, Sephadex LH-20, has found application in studies of partially acetylated dextrans. In describing a modified procedure for the replacement of the 0-acetyl groups in such dextrans by 0-methyl groups prior to acid hydrolysis and identification of the fragments (which indicates the distribution of the substituents in molecular

(96) B. C. W. Hummel and D. C. Smith,J. Chromatogr., 8, 491 (1962). (97) I. Breniner, J. S. G. Cox, and G. F. Moss, Carbohyd. Res., 11, 77 (1969).

38

SHIRLEY C. CHURMS

the dextrans), de Belder and Norrmanesrecommended the use of this gel, with methanol and acetone as eluants, in the isolation of the products obtained after the various steps. In this way, the substituted dextrans are readily separated from reagents and products of low molecular weight.

b. Galactans. - Except for a preliminary investigation by Andrews and Roberts,ss who, in 1962, reported (in a note on the use of agar in gel chromatography) that a larch arabinogalactan and a Strychnos galactan are eluted from an agar column in that order, and both eluted after starch and a galactomannan from clover seed, interest in the gel chromatography of galactans commenced in 1965, when Anderson and coworkers66 described the use of a poly(acry1amide) gel, Bio-Gel P-300, in the determination of the molecular weights of fractions of the arabinogalactan gum from Acacia senegal. On calibration of the gel column with dextran fractions of known the correlation between V, and log il?, was found to be linear over the il?, range of 5,000 to 125,000. Values of Enwithin this range could, therefore, be estimated from elution volumes by reference to this calibration curve.66JooSodium chloride (M)was used as the eluant. This method has been successfully applied by Anderson and coworkers in strucand Araucarido7 gums. Detertural studies of various Acacia100-106 mination of the %, values of the products of hydrolysis and Smith degradation of the gums has permitted certain conclusions to be drawn regarding the distribution of (1+3)- and (1+6)-linkages in the arabinogalactans. The present author and coworkers have used the method of Anderson and coworkers to determine the molecular-weight distributions of the constituents of a number of Acacia and other plant gums, and their degradation product^.^^*^*^^^ The degree of resolution achieved by this procedure is illustrated

En,

(98) A. N. de Belder and B. Norrman, Carbohyd. Res., 8, 1 (1968). ~ (99) P. Andrews and G . P. Roberts, Biochem.J.,84, 1 1 (1962). (100) D. M. W. Anderson and J. F. Stoddart, Carbohyd. Res., 2, 104 (1966). (101) D. M. W. Anderson, Sir Edmund Hirst, and J. F. Stoddart, J . C h e n . Soc. ( C ) , 1959 (1966). (102) D. M. W. Anderson and I. C. M. Dea, Carbohyd. Res., 6, 104 (1968). (103) D. M . W. Anderson, I. C. M. Dea, and R. N. Smith, Carbohyd. Res., 7,320 (1968). (104) D. M. W. Anderson and I. C. M. Dea, Carbohyd. Res., 8,440 (1968). (105) D. M. W. Anderson and I. C. M. Dea, Carbohyd. Res., 8,448 (1968). (106) D. M. W. Anderson, I. C. M . Dea, and Sir Edmund Hirst, Carbohyd. Res., 8,460 (1968). (107) D. M. W. Anderson and A. C. Munro, Carbohyd. Res., 11, 43 (1969).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

39

in Fig. 1, and Fig. 2 demonstrates the reproducibility of the elution patterns obtained. Chromatography of two specimens of A. elata gum collected at different times from the same tree, and of a specimen collected from a different tree in the same locality, gave elution patterns of striking similarity, revealed by comparison of Figs. 2 and 3. These results indicated that the elution pattern given on gel chromatography of a plant gum may be characteristic of the species involved and, therefore, highly significant in chemotaxonomy Products of a Smith degradation of A. elata gum have been fractionated on a preparative scale by gel chromatography on Bio-Gel P-10, with water as the eIuant.62 Anderson and coworkers'08 have reported the successful applica-

.

Volume Lmll

FIG. 1.-Elution Pattern of Material Precipitated by Ethanol from the Mild-Acid Hydrolyzate of Acacia elata Cum. [Bio-Gel P-300, 90 X 1.5 cm column, M sodium chloride as the eluant; flow rate, 3 ml/hr; sample, 2 mg in 1 ml of M sodium chloride.]

(108) D. M . W. Anderson, I. C. M. Dea, and A. C. Munro, Carbohyd. Res., 9,363 (1969).

40

SHIRLEY C. CHURMS

Volume (rnl)

FIG.2.-Elution Pattern of Ethanol-Precipitated A. elata Gum, Collected from Tree 1 in January, 1967. [Bio-Gel P-300,90 x 1.5 cm column, M sodium chloride as the eluant; flow rate, 4 mllhr; samples: (1) 5 mg in 1ml of M sodium chloride, and (2) 3 mg in 1 ml of M sodium chloride.]

tion of an agarose gel, Sepharose 4B, in the fractionation of Acacia gum polysaccharides of molecular weight above the upper limit of in the fractionation range of Bio-Gel P-300. Polysaccharides of the range of -3 x lo5 to 3 x lo6, including many occurring in plant gums, can be fractionated on this gel. Porous glass has also been used in the chromatography of plant gums of high molecular weight. Stoddart and Jonestogestimated the M , for Citrus Zimonia gum by comparison of its elution pattern on Bio-Glas 500 with those given by the gums from Acacia senegal and A. arabica, for which was known. Porous silica is also applicable to the molecular-sieve chromatography of plant-gum polysaccharides. 110

a,

a,

(109) J. F. Stoddart and J. K. N. Jones, Carbohvd. Res., 8, 29 (1968). (110) D. M. W. Anderson, A. Hendrie, and A. C. Munro,]. Chromatogr.,44,178 (1969).

GEL CHROMATOGRAPHY O F CARBOHYDRATES

41

The arabinogalactans occurring in larch wood have been widely investigated by gel chr~matography.~~'-"~ The Sephadex elutionpatterns demonstrate the presence of two components, A and B, in all heartwoods examined. Arabinogalactan A -75,000) preponderates, the proportion of B (M,-14,000) increasing with the age of the wood.112Component B (low molecular weight) is believed to be a product of slow, acid-catalyzed hydrolysis of the material of high molecular weight. Gel chromatography has shown that both A and B are polymolecular, althmgh each has a relatively narrow molecular-weight distribution.'12

(M,

-

M~

I

I00

40

20

10

5

'

I

I

I

I

I

40

1

80

I20

Volume I m l )

FIG. 3.-Elution Pattern of Ethanol-Precipitated A. elata Gum. [Bio-Gel P-300, 90 x 1.5 em column, M sodium chloride as the eluant; flow rate, 4 ml/hr; samples: (1) gum collected from tree 1 in February, 1969 (5 mg in 1 ml of M sodium chloride), (2) gum collected from tree 2 in February, 1969 (3 mg in 1 ml of M sodium chloride.)]

(111) B. V. Ettling and M. F. Adams, T a p p i , 51, 116 (1968). (112) B. W.Simson, W. A. Cote, Jr., and T. E. Timell, Suensk Papperstidn., 71,699 (1968). (113) H. A. Swenson, H. M. Kaustinen, J. J. Bachhuber, and J. A. Carlson, Macromolecules, 2, 142 (1969).

42

SHIRLEY C. CHURMS

c. Other Polysaccharides. -The fractionation of a number of starch dextrins on Sephadex G-75 in distilled water was reported in 1962 by Nordin.l14 The dextrins produced on hydrolysis of glycogens from various sources, and of amylopectin, by pancreatic alpha-amylase, were fractionated b y Heller and Schramm1I5by using Sephadex G-50 with water as the eluant. For the digest obtained from shellfish glycogen, rechromatography of the dextrin fractions first eluted from the column led to the isolation of previously unsuspected dextrins of high molecular weight, having a degree of polymerization (from reducing end-group analysis) ranging from 150 to 330. Such dextrins were not found in the digests from rabbit-liver glycogen, phytoglycogen, and amylopectin. The molecular-size distribution of the dextrins produced on digestion with the enzyme was found to vary considerably from one glycogen to another as a result of the different distributions of branch points in glycogens from different sources, concerning which much information can be obtained from experiments of this type. The molecular-weight distribution of a sample of a glucan, pulM a n , isolated from cultures of the fungus Pullularia pullulans grown in sucrose solutions,'Ifi was determined by Granath and KvisPg by the method developed by these workers for use with dextrans. Here, too, the column was calibrated with dextran fractions, as the shape of the pullulan molecule was believed to be sufficiently similar to that of a dextran to permit use of the same correlation of KUL,with molecular weight for both polysaccharides. However, in determining the molecular-weight distribution of a sample of a fructan, inulin, Granath and Kvistfi9calibrated the column instead of with dextran with fractions of inulin of known Ewand fractions. A different correlation of K,, with molecular weight was observed, indicating that the (1+2)-~-fructofuranosidiclinkages of the inulin chain"' give rise to a relationship between molecular weight and size that differs from that holding for dextrans. Inulin appears to have the more compact structure. Fractionation of the synthetic "polysucrose" Ficoll (manufactured by Pharmacia AB, Uppsala, Sweden) was achieved by Laurent and Granath56by the method already described for dextran. From material having a broad distribution of molecular weight, several fairly homo-

En,

(114) P. Nordin, Arch. Biochem. Biophys., 99, 101 (1962). (115) J. Heller and M . Schramm, Biochim. Biophys. Actn, 81, 96 (1964). (116) H. 0. Bouveng, H. Kiessling, B. Lindberg, and J. McKay, Acta Chem. Scnnd., 16, 615 (1962). (117) E. L. Hirst, D. J. McCilary, and E. C . V. Percival, J. Chem. Sac., 1297 (1950).

GEL CHROMATOGRAPHY O F CARBOHYDRATES

43

geneous fractions, ranging i n m , from 7,000 to 95,000, were obtained. The hemicelluloses that are precipitated on addition of methanol to the neutralized, 10% sodium hydroxide extract of powdered, Norwegian-spruce wood were fractionated by Kringstad and Ellefsen118by gel chromatography on Sephadex. After preliminary fractionation on Sephadex G-100, with 0.5 M sodium sulfate as the eluant, the components of low molecular weight were further separated by rechromatography on Sephadex G-25 in distilled water. The fractions of high molecular weight were subjected both to acid and alkaline hydrolysis, and the hydrolyzates obtained after different periods of time were chromatographed on Sephadex G-100 in 0.05 M sodium sulfate. From the elution patterns obtained, conclusions could be drawn regarding both the structure of the hemicelluloses and the mechanism of the hydrolysis. In an investigation of the polysaccharides present in soil, Stacey and used chromatography on Sephadex G-100 in distilled water to separate these polysaccharides from other components of the soil extracts. The polysaccharides were eluted much earlier than the colored substances present in the soil. Further fractionation of these polysaccharides was achieved by ion-exchange chromatography. Their isolation by this means permitted preliminary characterization; paper chromatography of the acid hydrolyzates revealed the presence of D-glucose, D-galactose, D-mannose, D-xylose, L-arabinose, L-rhamnose, L-fucose, and D-glucuronic acid r e s i d ~ e s . " ~ Barker and coworkers have applied gel chromatography in studies of pneumococcal polysaccharides."' Purification of the type-specific polysaccharide of Pneumococcus Type I1 was effected b y chromatography on Sephadex G-200 in M sodium chloride; in this way, the ribonucleic acid, a persistent impurity in preparations of this polysaccharide, was almost completely removed. The complex formed between the polysaccharide and the nucleic acid is largely dissociated in M sodium chloride, so that the two are free in this solvent and may be separated on the basis of their differing molecular size. Cell-wall lipopolysaccharides of Gram-negative bacteria (Shigetla strains) have also been purified by gel chromatography.122These (118)K. Kringstad and 0. Ellefsen, Papier, 18, 583 (1964). (119)S. A. Barker, P. Finch, M . H . B. Hayes, R. G. Simmonds, and M . Stacey, Nature, 205,68 (1965). (120)S. A. Barker, M. H . B. Hayes, R. G. Simmonds, and M. Stacey, Carbohyd. Res., 5, 13 (1967). (121)S. A. Barker, S. M. Bick, J . S. Brimacombe, and P. J. Somers, Carbohyd. Res., 1, 393 (1966). (122)E.Romanowska, Anal. Biochem., 33,383(1970).

44

SHIRLEY C. CHURMS

polysaccharides are readily separated from smaller contaminants (ribonucleic acids and nonspecific polysaccharides) on the agarose gels Sepharose 2B or 4B. Water has been used as the eluant, but 0.01 M ammonium hydrogen carbonate has proved more effective. After a preliminary, ion-exchange fractionation, *the C polysaccharide from pneumococci was fractionated by Gotschlich and Liu123 by gel chromatography on Sephadex G-200 in M acetic acid. From the elution volumes corresponding to peaks in the elution patterns obtained, the molecular weights of the components were estimated; these were found to range from 65,000 to >200,000. Analysis of the acid hydrolyzates of the fractions revealed the presence of %amino2-deoxy-~-glucose,2-amino-2-deoxy-D-galactose 6-phosphate, muraand its mic acid [2-amino-3-0-(~-l-carboxyethyl)-2-deoxy-~-glucose] 6-phosphate, D-glUtamiC acid, D- and L-alanine, and L-lysine, the proportions differing from one fraction to another. On this basis, it was suggested that the C polysaccharide is a cross-linked aggregate of two or more different types of polymeric chain, variation in the relative proportions of these chains giving rise to the heterogeneity observed. Sialoglycosaminoglycans extracted from rat brain have been purified by Brunngraber and Brown124by a procedure involving gel chromatography on Sephadex G-200 in distilled water; this resulted in the separation of these polysaccharides from u.v.-absorbing impurities. Elution of the sialoglycosaminoglycans commenced at the void volume of the column, whereas most of the impurities emerged much later. The sialoglycosaminoglycans give an elution pattern consisting of a broad, unsymmetrical peak that indicates considerable heterogeneity. Analysis of the fractions of high molecular weight, eluted before the peak, and of the fractions eluted after the peak, revealed that the former contained a higher proportion of sialic acid and a lower proportion of L-fucose than the latter. It was, therefore, suggested that the heterogeneity of the sialoglycosaminoglycans arises from the presence of variable proportions of sialic acid and L-fucose linked to a hexosamine-hexose chain that is common to all of them. The estimation of the molecular weights of glycosaminoglycans by gel chromatography is often complicated by a strong tendency to aggregation, and by changes in molecular shape and size caused by variation in solute-solvent interaction. Brunngraber and BrownlZ4 found that the use of water as the eluant in the gel chromatography (123) E. C. Gotschlich and T.-Y. Liu, J . B i d . Chern., 242, 463 (1967). (124) E. G. Brunngraber and B. D. Brown, Biochern.J.,103, 65 (1967).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

45

of sialoglycosaminoglycans resulted in early elution of these solutes, owing to the formation of aggregates. If, instead of water, a 0.05 M Tris hydrochloride buffer (pH 7.5) that was 0.1 M in potassium chloride was used for elution, the peak elution-volume increased to a value corresponding to a molecular weight of just under 10,000, a value much lower than the apparent molecular weight in water. However, the u.v.-absorbing impurities were not removed, so that, for the main purpose of the experiment, water was the better eluant. The gel-chromatographic behavior of the glycosaminoglycan heparin has also been found to b e markedly dependent on the ionic strength of the eluant. Chromatography of heparin samples on Sephadex G-100 and G-200, with a series of phosphate buffers (pH 6.8) of different concentrations as eluants, revealed a tendency for the elution volume to increase and the peak to broaden with in~ ~the effect persisted at creasing ionic strength of the e 1 ~ a n t . IAs ionic strengths high enough to eliminate any possibility of exclusion of heparin from the gel by reason of repulsion between ionized acidic groups and similar groups in the gel, it was concluded that the lower elution-volumes observed at low ionic strengths could not be entirely due to this factor. The dependence of many properties of heparin (such as viscosity and sedimentation constant) on the ionic strength of the medium suggests that the shape, size, and degree of solvation of the molecules change with the ionic strength. This effect is probably attributable to accompanying changes in solute-solvent interaction, a factor particularly important for heparin, as it contains a large number of sulfate groups per molecule. This behavior makes determination of its molecular weight very difficult; wide discrepancies have been observed between the values of molecular weight estimated from gel chromatographyAz6and those given by other methods. By gel chromatography on Sephadex G-200, with an eluant consisting of 0.15 M sodium chloride mixed with 0.12 M sodium phosphate buffer solution, pH 7.4 (9:1, v/v), heparin of porcine mucosal origin has been shown to be highly polymolecular.A27N o increase in blood anticoagulant activity with increasing molecular size, indicated by the results of an earlier study,'28 was observed when the activity of fractions selected from different parts of the elution curve was determined. The anticoagulant potency of the sample studied was, (125) M. Skalka,]. Chromatogr., 33, 456 (1968). (126) G. B. Sumyk and C. F. Yocum, J. Chromatogr., 35, 101 (1968). (127) M. Hall, C. R. Ricketts, and S. E. Michael, /. Pharm. Pharmacol., 21,626 (1969). (128) C. R. Ricketts, P. L. Walton, and D. R. Bangham, Brit../. Haematol., 12,310 (1966).

46

SHIRLEY C. CHURMS

however, relatively high, and it was suggested that, in a less potent sample, various degrees of alteration of chemical structure may occur. In this case, potency may well vary with molecular weight. Hyaluronic acid from human synovial fluid was isolated by Barker and Young129by gel chromatography on Sephadex G-200 at 4", with M sodium chloride as the eluant. Chromatography was preceded by sterile digestion of the synovial fluid with the proteolytic enzyme pronase, in a Tris hydrochloride buffer (pH 7.9) for 8 hours at 37". The hyaluronic acid, largely excluded from the gel, was eluted in the early fractions, and was well separated from the protein debris (low molecular weight) remaining after the pronase digestion. The same authors130used gel chromatography on agarose for isolating the undegraded hyaluronic acid-protein complex from human synovial fluid. A study by How and Long131of the molecular-weight distribution of hyaluronic acid from normal synovial fluid and of that of patients having rheumatoid diseases has afforded some interesting results. The polymolecularity and average degree of polymerization of the samples of hyaluronic acid were determined by chromatography on agarose gels (1%and 2%) at 2", with 0.01 M phosphate buffer (pH 7.3) in 0.2 M saline as the eluant. The hyaluronic acid in fluid from diseased joints was found to have a lower average degree of polymerization than that in normal synovial fluid. The authors suggested that this may be due either to deficient biosynthesis, or to depolymerization (of the hyaluronic acid) resulting from the action of reducing substances or enzymes in the fluid from patients having rheumatoid disease. The latter possibility is favored by the detection and hyaluronidase activity of 2-acetamido-2-deoxy-/3-~-glucosidase in fluid from arthritic joints. How and Long131used the same method for investigating the effect of drug therapy on the molecular-weight distribution of hyaluronic acid in the synovial fluid of patients having rheumatoid disease. It was found that clinical improvement following the administration of certain drugs was always accompanied by an increase in the average molecular size of the hyaluronic acid, indicating that the symptoms of this group of diseases are related to the degradation of the hyaluronic acid in the synovial fluid. The presence of chondroitin sulfate in the synovial fluid of arthritic (129)S. A. Barker and N. M. Young, Carbohyd. Res., 2, 49 (1966). (130)S. A. Barker and N. M. Young, Carbohyd. Res., 2, 363 (1966). (131)M.J. How and V. J. W. Long, Clin. Chim. Acta, 23, 251 (1969).

47

GEL CHROMATOGRAPHY OF CARBOHYDRATES

patients was demonstrated by Barker and coworkers132by gel chromatography on Sephadex G-200. The glycosaminoglycans in the synovial fluid were isolated in this way after a preliminary ion-exchange fractionation had removed all free protein from the protein-polysaccharide complexes. The use of gel chromatography to determine the molecular-weight distribution of chondroitin sulfate, on a micro scale, has been reported by W a ~ t e s o n .Columns '~~ (60-100 cm x 0.8-3.0mm id.; volume 0.5-7 ml) were packed with Sephadex G-200; a special technique involving the use of vibration was used in packing. Samples containing 100 pg in 50-100 pI were chromatographed on these columns, with M sodium chloride as the eluant. In this way, chondroitin 4-sulfate from and of the various bovine nasal septa was fractionated, the fractions being determined by sedimentation and osmometry, respectively. Linear relationships between K,, and the logarithms of and Gnwere observed over the molecular-weight range both covered (11,500-41,300). The calibration curves thus obtained were used for determining and of another sample of chondroitin 4-sulfate, chromatographed under the same conditions. The values obtained agreed well with those determined by other methods. The molecular-weight distribution of acidic glycosaminoglycans in normal urine, and that of patients having Hurler's syndrome (gargoylism), has been determined by Constantopoulos and coworkers 134 by chromatography on Sephadex G-200, with 0.025 M sodium chloride were as the eluant. Fractions of chondroitin 4-sulfate, of known used for calibration. The high concentrations of heparitin sulfate and chondroitin sulfate B (dermatan sulfate) characteristic of Hurler's syndrome were clearly reflected in the elution patterns and molecular weight averages of the urinary glycosaminoglycans from these patients, which differed significantly from those of normal subjects.

aw an

aw

aw an

aw,

3. Miscellaneous Carbohydrates The phenolic D-glucosides salicin and tremuloidin (2-0-benzoylsalicin) occurring in the bark of Populus tremula have been separated from each other, and from the other carbohydrates present, by gel

(132) S. A. Barker, C. F. Hawkins, and M . Hewins, Ann. Rheumatic Diseuses, 25,209 (1966). (133) A. Wasteson, Biochim. Biophys. Acta, 177, 152 (1969). (134) G. Constantopoulos, A. S. Dekaban, and W. R. Carroll, Anel. Biochem., 3 1 59 ( 1969).

SHIRLEY C. CHURMS

48

TABLEIX Gel Chromatography of Polysaccharides Packing Sephadex G-25 Sephadex G-50

Sephadex G-75

Eluant water water

water

Sephadex G-100 water 0.1 M ammonium hydrogen carbonate 0.5 M sodium sulfate Sephadex G-100 0.3% sodium and G-200 (2:1, chloride dry-weight) Sephadex G-200 water

25 mM sodium chloride 0.15 M sodium chloride-0.12 M phosphate buffer, pH 7.4 (9:1, vlv) 0.2 M sodium chloride M sodium chloride

M acetic acid Bio-Gel P-300

M sodium chloride

A p p1i cation

fractionation of dextrans, M, <5,000 fractionation of dextrans, M . 1,000-7,000 fractionation of dextrins from enzymic hydrolysis of glycogens fractionation of dextrins from hydrolysis of starch fractionation of arabinogalactans from larch wood fractionation of arabinogalactans from larch wood isolation of soil polysaccharides fractionation of arabinogalactans from larch wood fractionation of hemicelluloses from spruce wood determination of molecular-weight distribution ofdextrans (M,1x 10'-1 x lo5),inulin, pullulan isolation of sialoglycosaminoglycans from rat brain determination of molecularweight distribution of urinary glycosaminoglycans fractionation of heparin

fractionation of polymolecular dextran and Ficoll isolation of hyaluronic acid from synovial fluid determination of molecular-weight distribution of chondroitin 4-sulfate isolation of pneumococcal pol ysaccharide fractionation of pneumococcal pol ysaccharide determination of molecular-weight distribution of arabinogalactans from plant gums, and degradation products of these

References 35

35 115

114

111 112 119,120 111 118 69 124 134 127

56 129 133

121 123 37,66,76, 100-107

49

GEL CHROMATOGRAPHY OF CARBOHYDRATES TABLEIX (Continued) Packing Bio-Gel P-10

4% Agarose gel (Sepharose 4B)

4% and 2% agarose gels (Sepharose 4B and 2B) Agarose gels (1% and 2%)

Porous glass (Bio-Glas 500) Porous silica (Porasil-C and D) Porous silica (Porasil-E) Hydrophilized polystyrene (Aquapak) Sephadex LH-20

Eluant

Application

water

preparative-scale fractionation of degradation products of gum polysaccharide M sodium fractionation of plant gum chloride arabinogalactans of high molecular weight (Gw 3 x 1053 X lo6) water or isolation of bacterial 0.01 M ammonium lipopolysaccharides hydrogen carbonate

References 62

108

122

isolation of hyaluronic acidprotein complex from synovial fluid

130

determination of average molecular size of hyaluronic acid from synovial fluid

131

estimation of average molecular weight of Citrus limoniu gum fractionation of dextrans

109

M sodium chloride-0.01 M phosphate buffer (PH 7.0) 0.01 M phosphate buffer (pH 7.3)in 0.2 M sodium chloride 0.25 M ammonium formate water 0.2 M sodium chloride water

fractionation of hyaluronic acid fractionation of dextrans

22

methanol, acetone

isolation of substituted dextrans

98

22,23

14

chromatography, with distilled water,135of an extract of the bark on Sephadex G-25. A trisaccharide, sucrose, D-glucose, salicin, and tremuloidin were eluted, in that order. Salicin was eluted after D-glucose, despite its higher molecular weight, because of the presence, in the salicin molecule, of an aromatic ring which causes adsorption to the gel.2*The elution of tremuloidin after salicin was attributed to the additional aromatic ring in the former; the effect of aromatic adsorption here outweighs that of molecular size. Several other phenolic D-glucosides, from different sources, have (135) A. Repa6 and B. Nikolin, /. Chromatogr., 35, 99 (1968).

50

SHIRLEY C. CHURMS

also been separated by gel chr~matography.'~~ On Sephadex G-25, with water as the eluant, salicin, populin, and salireposide were eluted in that order. Fragilin was eluted together with salicin, and tremuloidin with populin. Better separations were obtained with 9:l (vlv) water-methanol (or ethanol) as the eluant. Under these conditions, populin was separated from tremuloidin; a complete separation, from each other, of salicin, populin, tremuloidin, salireposide, and grandidentatin, eluted in that order, was achieved. A separation of salicin, populin, tremuloidin, and salireposide from each other was also obtained on Sephadex LH-20 in ethanol. All of these separations depend upon differences in degree of adsorption to the gel, occasioned by differences in aromaticity and in the number of hydroxyl groups, and not on molecular sieving. In an investigation of the iron-carbohydrate complexes used in the treatment of iron-deficiency anemia, Ricketts and coworkers137.138 used gel chromatography on various Sephadex gels to isolate the complexes and estimate their molecular weights. It was concluded that the iron-D-glucitol-citrate complexes138 are smaller, on the average, than the iron-dextran c o r n p l e x e ~ , a' ~factor ~ that may cause differences in the distribution of these complexes in the body after injection. TABLEX Gel Chromatography of Miscellaneous Carbohydrates Application Separation of phenolic D-glucosides

Structural studies of irondextran complex

Structural studies of ironD-glucitol-citrate complex

Gel Sephadex G-25

Sephadex LH-20 Sephadex (2-75, G-200 Sephadex G-75 Sephadex G-15, (2-25, (2-50, G-75 Sephadex G-75

Eluant

References

water water-methanol (9:1, vlv) water-ethanol (9:1, v/v) ethanol 0.9%sodium chloride M sodium sulfate 0.9%sodium chloride

135,136 136

M sodium sulfate

138

136 136 137 137 138

(136) A. Repa:, B. Nikolin, and K. Dursum,J. Chromatogr.,44,184 (1969). (137) C. R. Ricketts, J . S . G. Cox, C. Fitzmaurice, and G. F. Moss, Nature, 208, 237 (1965). (138) M . Hall and C. R. Ricketts,J. Pharm. Phormacol., 20,662 (1968).

GEL CHROMATOGRAPHY OF CARBOHYDRATES

51

V. VALUEOF THE TECHNIQUE

The work discussed in this article serves to illustrate the applicability of gel chromatography to a wide variety of carbohydrates. The technique can be used not only as a means of fractionation but also, provided that the relationship between molecular weight and such other molecular parameters as size and shape remains constant, as a method of estimating the molecular weights of polysaccharides. Often, information derived from gel chromatography has proved useful in structural elucidation.

This Page Intentionally Left Blank

CRYSTAL-STRUCTURE DATA FOR SIMPLE CARBOHYDRATES AND THEIR DERIVATIVES* BY GERALD STRAHS Biochemistry Department, New York Medical College, New York, New York I. Introduction

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

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

11. General Features of the Crystal Structure of Carbohydrates . . . . . . . . . . . . .

53 56

Monosaccharides and Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Disaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Oligosaccharides . . . . . . . ...... Antibiotic Substances .................... Nucleosides and Nucleotides . . . . . . . . . . . Miscellaneous Carbohydrates: Glycosides, Vitamins, and Hydrazones . . . 90 Enzyme-Substrate Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 1. Hydrogen Bonds.. . . . . . ....................................... 98 .......... .. 101 2. Conformations ........................... 3. Nonbonded Interactions (Repulsions) . . . . . . . ......... .. 104 XI. Addenda ............................................................. 107

111. IV. V. VI. VII. VIII. IX. X.

I. INTRODUCTION This Chapter covers the subject of carbohydrate crystallography from the time of the previous review' in 1964 to the present. It is meant to be reasonably complete, but oversights will undoubtedly have occurred. Crystallography of the polysaccharides is not included, as the subject was discussed in an article in this Series2; polysaccharides do not form single crystals, and may be regarded as being polycrystalline. Although the survey of the literature was completed in January, 1969, some later papers are discussed. Two groups of scientists are now maintaining computerized listings of carbohydrate 'The author acknowledges the assistance of Dr. Anthony Herp in the preparation of this article, which was supported by Grant AM-04619 and, in part, by Grant FR-05398 from the National Institutes of Health, U. S. Public Health Service. The author also thanks Dr. Ward Pigman for encouragement, and Dr. R. S . Tipson for constructive criticism.

(1) G. A. Jeffrey and R. D. Rosenstein, Adoan. Carbohyd. Chern., 19,7 (1964). ( 2 ) R. H. Marchessault and A. Sarko, Advan. Carbohiyd. Chem., 22,421 (1967).

53

54

GERALD STRAHS

structures that have been solved by crystallographic means, and further information may be obtained from D. G . Watson, Cambridge University, England, and R. D. Rosenstein, University of Pittsburgh, Pennsylvania, U. S . A. Such efforts should be encouraged, because, if these listings are widely distributed, not only may laborious search of the literature be shortened or avoided, but there is also the potential advantage of discouraging duplication. Some of the older crystallographic work can obviously be improved by application of modem techniques. Crystallography is recognized as the preferred method for deducing the complete details of molecular geometry in the solid state? Other physical and chemical methods generally give only partial structural information. However, for crystallography to utilize its fullest potential, it should be used in conjunction with other methods (or data) in order to correlate the known information about a molecule, its behavior, and its properties. The first requirement for studying a crystal structure is to obtain a good single crystal (about 0.3 mm in each dimension). Some compromises in the size of the crystal used are possible, but this dimensional requirement has prevented many studies from proceeding beyond the initial stage. The crystal is placed in an X-ray diffraction apparatus (camera or difiactometer) where the X-ray pattern is recorded photographically or by measuring the intensity of the X-rays electronically. The resulting intensity values are used to obtain the “observed structure factors” which constitute the fundamental experimental data from which the crystal and molecular structures are derived. The structure derived is used for calculating “structure factors” that are compared with the experimental “structure factors” during the period when the derived structure is being modified to fit the experimental data. Unfortunately, the experimental data cannot be correlated directly with the crystal structure, because some of the information needed is lost in recording the X-ray intensity pattern. Indeed, the diffracted X-ray beam is a vector, and it has both an intensity and a phase angle. The information concerning phase angle is lost during measurement of the X-ray intensity. The phase angle could, perhaps, be recorded by a holographic method, but X-ray lasers do not as yet exist. Various methods have been used to circumvent “the phase problem.” The earliest method was based on trial-and-error procedure and works well for relatively simple molecules (diatomic and triatomic). The most successful method has been the “heavy atom” method, wherein an electron-dense atom (for example, bromine or

CRYSTAL STRUCTURE OF CARBOHYDRATES

55

bromide having 35 or 36 electrons) is introduced into the molecule or the crystal. The position of the heavy atom can be determined, and then used for finding the positions of the other atoms in the crystal. More recently, statistical or “direct” methods have achieved a spectacular success. The basis of the method is that the lost information on the phase angle can be derived from the X-ray intensities. Two large structures thus solved by statistical methods are the dichloromethane-anthracene-tetracyanoethylene~ o m p l e x ,namely, ~ (8 Cz,H,,N4-2CHzC1,-C6N,),and 8,14-anhydrodigito~igenin.~ After the crystal structure of the compound has been solved, or deduced, from the X-ray data, the initial parameters (atomic positions, bond lengths, and bond angles) are only approximate and have to be improved. The usual method employed is that of least-squares refinement, although electron-density difference-maps and trial-and-error procedures are also used. Electron-density difference-maps give the approximate difference between the actual structure and the trial structure. One disadvantage of structures derived from X-ray data is that the configuration obtained is a relative configuration. All conformations are correct relative to each other, but the absolute configuration cannot be determined except when certain heavy atoms are introduced into the molecule to yield an “anomalous” X-ray picture. Such anomalous scattering was used to deduce absolute Configuration. However, as the heavy atom in the molecule diffracts the X-rays more than do the lighter atoms, the positions of “light” atoms could not be determined as accurately as in the absence of the heavy atom. The anomalous scattering of oxygen has been used for (+)-tartaric acid6 (configuration 2R,3R) and cellobiose.’ As the anomalous scattering of oxygen is small (about 0.04 electron, as compared to the total scattering of 8 electrons), extremely accurate intensity data are needed. The absolute configuration of a-D-glucopyranose monohydrate has also been confirmed directly b y the anomalous scattering of the oxygen atoms.7aAfter the atomic positions had been refined, the structure factors were calculated by using the oxygen anomalous scatter(3) Structure, in this article, refers to both the structure of the molecule and the relationship or geometry between the molecules in the crystal. (4) I. L. Karle and A. V. Fratini, Actu Crystallogr., B26,596 (1970). (5) R. D. Gilardi and I. L. Karle, Actu Crystullogr., B26,207 (1970). (6) H. Hope and U . de la Camp, Nature, 221,54 (1969). (7) J. W. Moncrief and S. P. Sims, Actu Crystullogr., A25, S74 (1969). (7a) S. Neidle and D. Rogers, Nature, 225,376 (1970).

56

GERALD STRAHS

ing for both D and L configurations. The D configuration was confirmed at a 98.5% confidence level, based on all the data. When Neidle and Rogers used a more sensitive set of data (247 of the 912 X-ray reflections), the D configuration was confirmed at a >99.5% confidence level.

11. GENERALFEATURESOF THE CRYSTAL OF CARBOHYDRATES STRUCTURE The occurrence of hydroxyl groups in carbohydrate molecules gives several potential sites for hydrogen bonding, the hydroxyl groups being capable of acting both as donors and as acceptors of hydrogen atoms in hydrogen-bond formation. The actual number of hydrogen bonds formed in crystals is (or is close to) the maximum number possible. For example, a-D-glucopyranose monohydrate has seven of the seven hydrogen bonds possible,8 a-D-glucopyranose has five of the five hydrogen bonds p o ~ s i b l eand , ~ fl-D-glucopyranose contains four of the five hydrogen bonds possible.’O The bond lengths and bond angles differ considerably from one sugar to another, and in no carbohydrate are their values either ideal or average. For a simple sugar, the molecule may become slightly distorted in forming hydrogen bonds, whereas a sugar derivative that cannot form hydrogen bonds will be distorted because of such steric factors as nonbonded repulsion or interaction. The most common distortion of an ideal chair form of a pyranoid compound is a slight flattening; further puckering does not occur, presumably because it would increase the nonbonded interactions. Furanoid rings are not planar; if they were, all of the non-ring atoms would eclipse each other. The most common distortions affect one carbon atom, usually C-2 or C-3, which is about 0.5A out of the plane of the four other atoms of the ring, giving an “envelope” conformation. Some mean bond-lengths are listed in Table I, with standard deviation and spread. These values are in good agreement with the C-C single-bond length of 1.54A and the C - 0 single-bond length of 1.43A. The C-H and 0 - H bond distances are not nearly so accurately determined as those for the heavier atoms, and the lengths determined with X-rays are about 0.lA shorter than those obtained by neutron (8) R. C. G. Killean, W. G. Ferrier, and D. W. Young, Acta Crystallogr., 15, 911 ( 1962). (9) G . M. Brown and H. A. Levy, Science, 147,1038 (1965). (10)S . S. C. Chu and G . A. Jeffrey, Acta Crystallogr., B24,830(1968).

CRYSTAL STRUCTURE OF CARBOHYDRATES

57

TABLEI Mean Bond-lengths" (in A) in CarbohydratesIz

c-0

c-c Pyranose rings (a) C-1-0-1 equatorial 1.520k0.003 (0.023) (b)C-1-0-1 axial 1.524 3 . 0 0 4 (0.021) Furanose rings 1.520LO.007 (0.017)

1.420 3 0 . 0 0 4 b (0.016)

*

1.425 0.0046 (0.016) 1.436f l . 0 1 5 (0.023)

"The values given are average bond-lengths with standard deviation; the values for spread are given in parentheses. *Excluding C-1-0-1, C-1-0-5, and C-5-0-5.

diffraction. One reason for the difference in the positions of the hydrogen atom as measured by neutron diffraction and by X-rays is that neutrons are diffracted by the nucIeus, whereas X-rays are scattered by electrons. The single electron of the hydrogen atom does not surround the hydrogen atom symmetrically, but is polarized toward the bonding atom (either a carbon atom or an oxygen atom, in carbohydrates). For L-ascorbic acid, HvosleP' compared C-H and O-H bond lengths, as measured by both methods, and found the following average values: O-H (X-rays) 0.83, C-H (X-rays) 1.017, O-H (neutrons) 0.980, and C-H (neutrons) 1.1008,. The O-H bond Iengths correlated with the 0-0 distances in the hydrogen bonds, and the O-H I - * 0 angles of the hydrogen bonds correlated with the 0-0 distances. The anomeric bond in aldoses (C-1-0-1) is shorter than the other C-OH bonds, indicating that it possesses more double-bond character. For an equatorial anomeric bond,12the shortening is about 0.0388,, and for an axial anomeric bond it is 0.024A. The corresponding changes in C-1-0-5 may not be significant (M.008Aequatorial, and -0.0048, axial), and the changes in C-5-0-5 are smaller than the anomeric changes (M.012A equatorial, M.0158, axial). (11) J . Hvoslef, Acta Crystallogr., B24,1431 (1968). (12) H. M. Berman, S . S. C. Chu, and G . A. Jeffrey, Science, 157,1576 (1967).

58

GERALD STRAHS

111. MONOSACCHARIDESAND DERIVATIVES

The tetroses (four-carbon sugars) have not yet been studied crystallographically, undoubtedly due to the lack of suitable crystals. Handbooks list D- or L-erythrose as a colorless syrup, and D- or L-threose as very hygroscopic, colorless, microscopic needles. The X-ray difiaction patterns for erythritol13 and for several derivatives of D- or Ltartaric acid have been solved. In contrast, much information is available about five-carbon sugars. The most widely studied D-pentoses, D-ribose and 2-deoxy-~-erythropentose, are discussed in Section VII (see p. 86). The aldopentoses can form either furanose or pyranose rings, and both structures are known, although not in the same crystal. Crystallographic data have not been obtained for either of the 2-pentuloses D-erythm- and Dthreo-2-pentulose (“D-ribulose” and “D-XylUlOSe”), as they are syrupy. The crystal structure of methyl a-D-lyxofuranoside was solved by a computer,14and 65,536 (i.e.,21s)sign combinations were tested. This statistical method led to the correct structure in projection; the third coordinate was obtained, from an assumed model, by varying its position in the third dimension and observing the agreement between calculated and observed X-ray data, the actual enantiomorph being assumed to be D. One goal was to obtain detailed data on a furanoid ring; it was found that this ring is puckered, with C-3 endo, C-3 being displaced by 0.7A from the plane of the four other atoms in the furanoid ring. The anomeric C-1-0-1 bond is markedly shortened (1.38A as against 1.41A for the other carbon to (hydroxyl) oxygen bond distances). All four of the hydroxyl oxygen atoms participate in hydrogen bonds, to give a total of 6 hydrogen bonds per molecule (although 3 are equivalent to 3 others, by symmetry). These hydrogen bonds link each sugar molecule with four other sugar molecules in a twodimensional network. Many papers fail to describe how the crystals were obtained; many of the crystals are donated to crystallographers, and the crystallization details are not obtained or are unavailable. Methyl 1-thio-/I-D-xylopyranoside was synthesized from D-xylopyranose tetraacetate and methanethiol, and crystallized by sublimation or by evaporation of an ethanol ~olution.’~ The initial structure was obtained by recognizing (13) A. Shimada, Actu Crystallogr., 11,748 (1958);Chern. Abstracts, 53, 16639 (1959). (14) P. Groth and H. Hammer, Actu Chem. Scand., 22,2059 (1968). (15) A. McL. Mathieson and B. J . Poppleton, Actu Crystallogr., 21,72 (1966).

CRYSTAL STRUCTURE OF CARBOHYDRATES

59

that the strong (Patterson) peaks at 2.5A were caused by atoms p to each other. The D-xylopyranose derivative was found to have the C1 (D) conformation, in which all substituents are equatorially attached. In the crystal are two independent molecules having almost identical molecular dimensions. The anomeric bond is not a C - 0 bond but a C-S bond, and its length is 1.80A, which is appreciably shorter than the mean of 1.83A for carbon-sulfur single bonds. The three hydroxyl groups are intermolecularly bonded to each other, forming helices. Neither the sulfur atom nor the ring-oxygen atom is involved in hydrogen bonding. One ring-angle, namely, C-2-C-3-C-4 is significantly larger than a tetrahedral angle, and this distortion may be due to the hydrogen bonding of 0-2, 0-3, and 0-4. The crystal study of p-D-and /3-L-arabinopyranose'6gave the first determination of structure of a free D- and L-sugar. The advantage in dealing with a crystal having a center of symmetry is that a more straightforward determination of its structure results, with a better accuracy, as the estimated standard deviations of 0.004A in bond lengths may be considered good. The molecule exists in that chair conformation in which the hydroxyl groups are la2e3e4a. As expected, the anomeric bond C-1-0-1 is shorter by about 0.03A than the other C - 0 bonds. All five of the oxygen atoms are involved in hydrogen bonding, to give a total of eight hydrogen bonds linking one molecule to five other molecules. The enantiomorphs have the same conformation, namely, 1C (D) and C1 (L). DL-ArabinitoI was examined.16aThe five carbon atoms are coplanar, and the oxygen atoms lie above and below this plane. Each molecule is surrounded by, and hydrogen-bonded to, six other arabinitol molecules, four of which are of the same chirality, and two of the opposite chirality. Each of the five oxygen atoms accepts one hydrogen bond and donates its hydrogen atom to form another hydrogen bond, giving a total of ten hydrogen-bonds per molecule. This hydrogenbonding scheme affords four helices per unit cell; two are lefthanded and two are right-handed. Ribitol is meso, and has no optical activity. The five carbon atoms might be expected to be coplanar, as in 1. In the crystal of ribitol, however, the first four carbon atoms (C-1 to C-4) are coplanar" with 0-4, but not with C-5, as in 2. Atom 0 - 4 is in the plane and minimizes (16) H. S. Kim and C . A. Jeffrey, Actu Crystallogr., 22,537 (1967). (16a) F. D. Hunter and R . D. Rosenstein, Acta Crystallogr., in press. (17) H. S. Kim, G . A. Jeffrey, and R. D. Rosenstein, Act0 Crystallogr., B25, 2223 (1969).

60

GERALD STRAHS

Ribitol (planar, zigzag conformation) (1)

Ribitol (sickle conformation) (2)

nonbonded interactions; were C-5 co-planar, there would be a close approach (2.55A) of 0 - 2 to 0-4, which is less than the Van der Waals contact of 2.8k This reasoning, as originally pointed out for such molecules in solution,ls also applies to (D- and L-) glucitol, iditol, talitol, and allitol. The last four compounds have not yet been studied by X-ray crystallography. The hydrogen bonding of these alditols is similar to that of the arabinitols, in which each hydroxyl group is the donor and the acceptor of one hydrogen bond. The molecules are linked in helices and zigzag chains in" a "very regular and elegant pattern which utilizes their full potential for hydrogen bond formation . , as [in] . , . other polyol structures which have been studied." Crystallographic data have not yet been reported for one of the four hexuloses, namely, D- or L-psicose, not yet crystallized. Preliminary results are available for ~-D-fructopyranOSe.'*aAs expected, the sugar has the 1C (D) conformation. The hemiacetal group on C-2 is not bound to a hydrogen atom, whereas 0-6 is linked to two hydrogen atoms. a-D-Tagatose also crystallizes as a pyranose in the C1 (D) confor-

.

(18)H. S. El Khadem, D. Horton, and T. F. Page, 1. Org. Chem., 33, 734 (1968); D. Horton and J. D. Wander, Carbohyd. Res., 10, 279 (1969);13, 33 (1970); 15,271(1970). (18a) R. D. Rosenstein, Amer. Crystallogr. Ass. Abstracts (1968).

CRYSTAL STRUCTURE OF CARBOHYDRATES

61

mation.lg The hydroxymethyl group on C-2 is equatorially attached, and the hydroxyl groups are 2a3a4e5e. T h e two axial hydroxyl groups are, of course, on opposite sides of the pyranose ring. D-Sorbose (D-xyb-hexulose) has a similar environment at C-2 and C-6, as it has the same configuration as D-fructose (D-arabino-hexulose) except at C-5. Crystalline a-L-sorbose was studied.20The pyranose ring has the chair conformation in which the hydroxyl substituents are 2a3e4e5e (C-1 is equatorially attached). The intermolecular hydrogen bonding is extensive, with every hydroxyl group except that on C-1 acting as both a donor and acceptor, and the ring oxygen atom (0-6) as an acceptor. The 1-hydroxyl group is disordered over two (almost equivalent) positions in which it donates the hydrogen to either a normal hydrogen bond or, possibly, to a bifurcated hydrogen bond. The aldohexoses have not been extensively studied by X-ray crystallographers. Crystals of a- and P-D-galactose were examined by Sheldrick,21 who gave directions for crystallizing both anomers. In one preparation, the crystals of a-D-galactose were small plates; large prismatic crystals of P-D-galactose were also obtained. The crystal structure of a-Dand a-L-mannose has been solved,22 but full details are not yet available. With calcium chloride, D-mannose forms a crystalline compound (C,H,,O, CaC12 * 4H@) which appears to contain the furanose ring.23The crystal structure is of interest because of the hranose ring and the nature of the presumed complex. In the complex are three heavy atoms (one calcium and two chloride ions) which could help in solving the structure. The structures of crystalline a- and P-D-glucopyranose are known. Many studies of D-glucose derivatives are available for comparison, because one or more D-glucose residues form part of the structure of many compounds, such as the D-ghcosides, including cellobiose, maltose, sucrose, amylose, and cellulose. Neutron diffraction was used for a precise determination of the molecular structure of a - ~ - g l u c o s e . ~ The crystal used weighed 31.7 mg, which is enormous b y X-ray crystallographic standards, where a typical crystal might weigh 0.04 mg. The sugar is present as the pyranose form in that chair conforma-

-

(19) S. Takagi and R. D. Rosenstein, Carbohyd. Res., 11,156 (1969). (20) H. S. Kim and R. D. Rosenstein, Acta CrystaZEogr.,22,648 (1967). (21) B. Sheldrick,]. Chem. SOC., 3157 (1961). (22) F. Planisek and R. D. Rosenstein, Amer. Crystollogr. Ass. Abstracts (1967). (23) (a) J. K. Dale, J . Amer. Chem. SOC., 51, 2788 (1929);(b) H. S. Isbell, ibid., 55, 2166 (1933); ( c ) H. S. Isbell and W. W. Pigman, J . Res. N a t . Bur. Stand., 18, 141 (1937).

GERALD STRAHS

62

tion (3)having the anomeric hydroxyl group axially and the other sub-

OH ff -D-Glucopyranose-

CI

(3)

stituents equatorially attached. The C-H and 0 - H bond lengths have mean values of 1.098 and 0.968& respectively, and the axial anomeric bond is significantly shorter than the other C - 0 bonds. There are five unique hydrogen bonds per molecule, with every hydroxyl oxygen atom acting as a donor, and the ring-oxygen atom and every hydroxyl oxygen atom, except that on C-1, acting as an acceptor. The crystal structure is shown in Fig. 1. Each a-D-glucopyranose molecule appears to have 9 hydrogen bonds. A tenth hydrogen bond is located at 0-4. The 10 hydrogen bonds result from each of the 5 hydrogen atoms that connect two oxygen atoms, the donor and the acceptor I

y.0

I

y=b

FIG.I.-The Crystal Structure of a-D-Glucopyranose,Viewed Along the [OOi] Axis. (There are two hydrogen bonds at each 0-4, but one has been omitted for clarity. Diagram kindly provided by Dr. George M. Brown, Oak Ridge National Laboratory, Oak Ridge, Tennessee.)

CRYSTAL STRUCTURE OF CARBOHYDRATES

63

atom, through covalent and electrostatic forces, respectively. The structure of P-D-ghcopyranose’o (4) is similar, the major

0-D-Clucopyranose- CI (4)

difference being that the C-1-0-1 bond is equatorial and is shorter by about 0.04A than the other C - 0 bonds. Per molecule, there is one hydrogen bond fewer than for a-D-glucopyranose. D-Ghcopyranose crystallizes in an “anomalous” fashion that is possibly explained by these crystal structures. Although aqueous solutions of D-glucose contain about 63% of the p-D anomer, the ~ when e v a p ~ r a t e d The . ~ ~ /3-D anomer solutions give the a - anomer has the greater These facts can be correlated with the crystal structures. It would be expected that the structure having the larger number of intermolecular hydrogen bonds (the a anomer) would be the more stable, have a lower solubility, and crystallize the more readily. A thermodynamic argument is applicable, because, as the intramolecular bonding in the crystals of the two sugars is almost identical, the energy difference must lie in the energy of intermolecular (hydrogen) bonding. The structures of a- and /3-D-glucopyranose may be compared with that of methyl a-D-ghcopyranoside.26In the latter, the C-1-0-1 bond is shorter than a C - 0 single bond, but not as short as in a-D-glucopyranose. Part of this shortening may affect the C - 0 bonds of the ring carbon atoms, as the C-1-0-5 bond is slightly shorter than the C-50 - 5 bond. Sedoheptulosan monohydrate, the only C, sugar derivative whose crystal structure has been published, has two fused rings.27 One of these is a pyranoid ring in a chair conformation involving -C-2-C-3C-4-C-5-C-6-0-6-, in which C-1, 0-3, and 0-4 are equatorially (24) J. C. Sowden, in “The Carbohydrates,” W. Pigman, ed., Academic Press, Inc., New York, N. Y., 1957, p. 92. (25) Ref. 24, p. 93. (26) H. M. Berman and H. S. Kim, Acta Crystallogr., B24,897 (1968). (27) G . M . Brown and W. E. Thiessen, Acta Crystallogr., A25, S195 (1969).

GERALD STFUHS

64

attached and 0-5, C-7, and 0 - 2 are axially attached, as shown in 5.

OH HO

3

Sedoheptulosan-IC

(5)

The last two atoms are bonded to each other as part of a nonplanar 1,3dioxolane ring involving -C-7-0-2-C-2-0-6-C-6-. A third ring, involving -0-2-C-2-C-3-C-4-C-5-C-6-C-7-, may be envisaged that includes both of the previous rings except for 0-6; it has a boat conformation having C-4 at the “prow” and C-7 and 0 - 2 at the “stern.” The ring closure introduces strain, and so the molecule is distorted from the idealized shapes, The chair conformation of the pyranoid ring is flattened in the region of C-4, and the C-0-C angle at 0 - 6 is 10”less than that of an ideal chair conformation. The crystals are extremely hard, and this is an indication of extensive hydrogen bonding; six different hydrogen bonds per molecule are present, namely, four linked with hydroxyl hydrogen atoms and two linked with hydrogen atoms of the water present. Crystal structures of two hexitols, galactitol and D-mannitol, have been published. Galactitol is meso, but the permissible intramolecular center of symmetry is not utilized in the crystal.28The molecules crystallize as enantiomorphic pairs that, in conformation, are almost centrosymmetric molecules; the difference therefrom is of the same order of magnitude as the thermal motion of the atoms. The carbon atoms and terminal oxygen atoms form an approximately planar chain. All of the oxygen atoms are both donors and acceptors for an intricate network of hydrogen bonding. D-Mannitol crystallizes in three (or more) crystal forms, two of which (B and K) have been solved in three dimension^,^^^^^ and one

(28)H.M.Berman and R. D. Rosenstein, Acta Crystdogr., 824,435(1968). (29)H.M. Berman, G. A. Jeffrey, and R. D. Rosenstein. Acta Crgstafbgr., B24,442 (1968). (30)H. S. Kim, G. A. Jeffrey, and R. D. Rosenstein, Acta Crystallogr., B24, 1449 ( 1968).

65

CRYSTAL STRUCTURE OF CARBOHYDRATES

(A') in two dimension^.^^ The molecule has a symmetry-required, twofold axis in the A' form, but has only an approximate two-fold axis for the non-hydrogen atoms of the B and K forms. The carbon chain is slightly bowed from planarity in the I3 and K forms. The intermolecular hydrogen-bonding scheme for the B form has each oxygen atom as a donor and as an acceptor. In the K form, too, each oxygen atom again is both a donor and an acceptor, but the hydrogen bonding is quite different. The lactones may be regarded as cyclic, internal esters. An example of a simple molecular structure is D-galactono-1,4-lact0ne,~' which contains a single furanoid ring system. The two C - 0 bonds of the lactone ring are C-1-0-4 and C-4-0-4; the carbonyl group is at C-1 and the lengths of these two bonds differ by about 0.1A. As the lactone group is planar, the shortness of the shorter bond, namely, C-1-0-4, may be attributable to resonance of the type:

-

-c-c=o-c-0

-c-c-o-c0 I1

do

The furanoid ring is nonplanar. All of the oxygen atoms, except 0-1 and 0 - 4 of the lactone group, participate in hydrogen bonding (see formula 6). 0-3 . . .. ... ........ I

" '

OH

n 0

:

:

:

0

on. . . . . . . . . . . . . . . _ .

n

o n . . ,' . _ ' . . .... .. H

0 - 5 0-6 D- Galactono- 1,4- lactone

(6)

A more complex ring-system occurs in ~-D-g~ucofuranurono-6,3lactone,32which contains a furanose ring and a furanoid lactone ring, and may be envisaged as having an eight-membered ring (see formu(31) G. A. Jeffrey, R. D. Rosenstein, and M . Vlasse, Acta C r y s t d l o g r . , 22, 725 (1967). (32) H. S. Kim, G . A. Jeffrey, R. D. Rosenstein, and P. W. R. Corfield, Acta Crystcdlogr., 22,733 (1967).

66

GERALD STRAHS

la 7). The dihedral angle between the two five-membered rings was

P-D-Glucofuranurono6, 3-lactone (7)

given as 111.3’; however, there is an uncertainty of several degrees, as the rings are not planar, and slightly different planes may be chosen as the “plane” of each ring. In the furanoid ring, C-1 is exoplanar by 0.5& it is endo, that is, displaced toward the lactone ring. In the lactone ring, the carboxyl group -C-5-C-6-0-3- is planar,

It

0-6 and is 10.7A away from the plane of the lactone ring. Atom C-5 is 0.25A out of the plane of the lactone ring; it is exo, that is, away from the hranose plane. The two C - 0 bonds in the lactone ring differ in length by 0.135A, showing that there is a contribution from a resonance form, as in ~-galactono-1,4-lactone.The a-D anomer would have both 0 - 1 and 0-2 exo with respect to the furanose ring, at a distance of about 2.6A (the Van der Waals radius of oxygen is 1.4&, and this too-close contact may explain its behavior during mutarotation. The crystals of a-D-glucopyranosyl (dipotassium phosphate) dihydrate are fairly dense and quite hard, because of elaborate intermolecular bonding between the glycosyl group, the water, the potassium ions, and the phosphate The C-1-0-1 bond is slightly shorter than the normal length, and the phosphate bond (0-1-P) is longer than normal. Early results suggested that one of the P-0 bonds had some double-bond character, but Rosenstein’s refinemenP4 (33) C. A. Beevers and G . H. Maconochie, Acta Crystallogr., 18,232 (1965). (34) R. D. Rosenstein, personal communication.

CRYSTAL STRUCTURE OF CARBOHYDRATES

67

shows that the three bonds are of essentially the same length (1.50, 1.51, and 1.52A). Oxidation of D-glucose at C-1 and C-5 gives ~-ryb5-hexulosonic acid, which is an effective chelating agent for calcium. The structure dihydrate (8) was reported by of calcium (D-xy2o-5-he~ulosonate)~

HO

Calcium ( 0 - x y l o - 5 hexulosonate), - 2 H,O (8)

Balchin and C a r l i ~ l e .The ~ ~ acid anion contains an almost planar furanose ring formed by cyclization of the ketone group at C-5 with the hydroxyl group on C-2. The asymmetric center thus formed at C-5 has the hydroxyl group cis to the 4-hydroxyl group, giving a cis-diol. The exoplanar ring atom is C-2. Each calcium ion is situated on a two-fold rotation axis, so that each ion is identically chelated by two sugar-acid molecules. The oxygen atoms chelating the calcium ions are 0-la, 0-2, 0-6, and the oxygen atom of water. As the bond lengths in the carboxyl group are C-1-0-la = 1.23 +0.03A and C1-0-lb = 1.28 -+0.03A there is difficulty in choosing between two forms of the carboxyl group, namely, O-la=C-O-lb-' and 0-la-''2 LC O-lb-*'*,because the difference in bond lengths is less than two standard deviations. The normal C - 0 and C=O bond distances are 1.41 and 1.21A, and thus the carboxyl group is probably a resonance form between the two structures mentioned. Except for the ring-oxygen atom (0-2), all of the oxygen atoms participate in hydrogen bonding. (35)A. A. Balchin and C. H. Carlisle, Acta Crystallogr., 19, 103 (1965).

68

GERALD

STRAHS

The calcium (9) and strontium salts of “a”-~-g~ucoisosaccharinic

Calcium [S-deoxy-2-C- (hydroxyrnethyl)-D- erythro -pentonate], (9)

acid are an isomorphous ~ a i r , 3 ~and * ~ ’these compounds can be discussed together, because the main difference between them is that the metal-oxygen bond lengths are longer for strontium than for calcium. Each metal ion, located on a two-fold axis, is chelated to four 3-deoxy-2-C-(hydroxymethyl)-~-erythro-pentonate ions b y one carboxyl oxygen atom, and 0-2, 0-4, and 0-5. The anion is acyclic, with 0-2’, C-2’, C-2, C-3, and C-4 forming an almost planar, zigzag chain. A short contact (2.60A) between 0 - 0 and 0 - 2 is found in the metal coordination polyhedron. Extended Huckel calculations were made for the acid. An interesting result was that the four hydroxyl protons were estimated to have a net charge of +a64 (or 0.36 electron), whereas the seven methine protons have a net charge of M.ll to M.14 (or 0.86-0.89 electron). The methine protons having 0.86-0.89 electron per proton were readily located in the electron-density difference map, but the hydroxyl protons were not found because they have a much lower electron density, calculated to be only 0.36 electron per proton. The preceding three structures were obtained by the use of alkaline-earth cations, and the following structures were solved with the help of halogen atoms. Iodine is the heavy atom in l,e-O-(amino(36)R. Norrestam, P. E. Werner, and M . von Glehn, Acta Chem. S c a d , 22, 1395 (1968).

(37)P.E.Werner, R. Norrestam, and 0.Ronnquist, Acta Crystallogr., B25,714(1969).

CRYSTAL STRUCTURE OF CARBOHYDRATES

69

isopropy1idene)-a-D-glucopyranose h y d r i ~ d i d e . ~The ~ pyranoid ring assumes a flattened chair-conformation intermediate between the C1 (D) and 4H5(D)conformations (10a and lob), not a boat form,

1,2-O-(Aminoisopropylidene)a-D-glucopyranose hydriodide

(lea)

(lob)

as had been suggested. The five-membered, 1,3-dioxolane ring is almost planar, C-7 being only 0.36A out of the plane, and is directed toward the D-glucopyranose ring. The aminomethylene substituent is in an almost equatorial position, and the methyl group has an almost axial position on the 1,3-dioxolane ring. The iodide ion is hydrogen-bonded to hydroxyl groups, and every “active” hydrogen atom (partially charged positively) in the molecule participates in hydrogen bonds. A sugar derivative in which the iodine is covalently bound is methyl (methyl 4,5-di-0-benzoyl-3,7-dideoxy-7-iodo-a-~-urubinoheptulopyranosid)~nate.~~ The pyranoid ring assumes that chair conformatior. in which the anomeric methoxyl group is axially attached and the (bulky) benzoyl groups are equatorially attached. The structure of 2-amino-2-deoxy-a-~-glucose hydrochloride and hydrobromide has been determined in p r o j e ~ t i o n ,and ~ ~ in three dimension^.^' Jeffrey, in repeating and extending his thesis work (completed 25 years earlier) with modern methods, used computer techniques. For both structures, the assignment of the position of the primary alcohol group was changed. The sugar is a pyranose in that chair conformation having all substituents equatorially attached, except for the anomeric hydroxyl group. The primary amino group is hydrogen-bonded to the halide ion and to (38) J. Trotter and J. K. Fawcett, Actu Crystullogr., 21,366 (1966). (39) C. C. Chu, Ph. D. thesis, Columbia University (1967); Dissertation Abstracts, 28,469B (1967). (40) R. Chandrasekaran and M. Mallikarjunan, Current Sci., 33, 4 (1964); Chem. Abstracts, 60,753h (1964). (41) S. S. C. Chu and G . A. Jeffrey, Proc. Roy. Soc., Ser. A, 285,470 (1965).

GERALD STRAHS

70

three oxygen atoms. One way in which three protons can form four hydrogen bonds is by involvement of a bifurcated hydrogen bond; this bifurcated bond appears to consist of two unequal hydrogen bonds having N - 0 distances of 2.75 and 2.95& respectively. The halide ion has four hydrogen bonds, to three hydroxyl oxygen atoms and the amino nitrogen atom. The conformation of the molecule was determined from the “anomalous” dispersion of the X-ray pattern produced by the chlorine atom.42 The crystal structure of 2-amino-2-deoxy-a-~-glucose is still unwas solved43 determined, but that of 2-acetamido-2-deoxy-a-~-glucose as part of a project on the study of an enzyme, lysozyme. The pyranose assumes that chair conformation in which 0-1is axially attached and the other substituents are equatorially attached, 0-1 and N-2 being cis to each other. All but four of the hydrogen atoms were located; three belong to the methyl group, and the fourth is on 0-4 and is not hydrogen-bonded. Four “active” hydrogen atoms, on 0-1,0-3, 0-6, and N-2 are involved in hydrogen bonding, as in formula 11. I

OH-----0-4

-03

OH,

‘.

‘0- 5

2-Ace tarnido- 2-deoxy - (Y - D glucopyranoee-C1 (11)

The surprising feature of such a structure is the possibility of the occurrence of the p-D anomer in the crystal. The electron density can be explained by the presence of 22% as the p-D anomer; it is indicated by the height of an unexplained peak near C-1 at the correct distance and angle for the p-D structure. When once-recrystallized 2-acetamido-2-deoxy-cr-~-glucopyranose is dissolved, its initial ~ -21.5” rotation is [ale +56.5”, as compared to +82” for the a - and (42) G. N. Ramachandran, R. Chandrasekaran, and K. S . Chandrasekaran, Biochim. Biophys. Acta, 148,317 (1967).

CRYSTAL STRUCTURE OF CARBOHYDRATES

71

for the p-D form; this indicates that 22.5% of the p-D form exists in More remarkable recrystallized 2-acetamido-2-deoxy-~-glucose. than the agreement between the electron-density measurement and the optical rotation is that the anomeric oxygen atom of the p anomer has no short contacts and can even accept a hydrogen bond from 0-6. Johnson43 proposed this as a hypothesis, but the presence of 20-25% of the p-D form seems to be the simplest and perhaps the only explanation; it could be tested by determining whether the p-D form has the same space-group as the CY-Danomer, although, should the two forms not have the same space group, the cocrystallization of the anomers would not be disproved. The occurrence of cocrystallization of both anomers of a monosaccharide is the only one to have been demonstrated crystallographically, and shows the close relationship between the anomers. The structures of four sulfur derivatives of sugars have thus far been solved.* Two are sulfides, the third is a C-sulfonate, and the fourth is a sulfoxide. Sinigrin, from the seeds of black mustard,

,NOSOP

M@

S C \ ~ ~ ,CH, ~ ~ =

Sinigrin

was crystallized as potassium myronate monohydrate (C,,H,,KNO,S, H20).44The myronate ion contains a 1-thio-p-D-glucopyranosyl group in that chair conformation in which the hydrogen atoms are axially attached and the substituents are equatorially attached. The sulfur atom of the thiol group is part of a five-atom plane, namely, -CH,-C = N-0-. The other sulfur atom is part of a sulfonate group that is syn-related to the 1-thio-D-glucopyranosidering. The second thioglycoside is ethyl l-thio-~~-~-glucofuranoside,~~

OH

Ethyl l-thio-cuo-glucofuranoside *A fifth structure was discussed on page 58. (43) L. N. Johnson,Acta Crystallogr., 21,885 (1966). (44) J. Waser and W. H. Watson, Nature, 198,1297 (1963). (45) R. Parthasarathy and R. E. Davis, Acta Crystallogr., 23,1049 (1967).

72

GERALD STRAHS

a compound originally believed to be ethyl p-D-glucofuranoside dihydrate. The sulfur atom was used as the heavy atom in order to solve the crystal structure, and as an “anomalous,” scattering atom in order to confirm the absolute conformation. All C-C bonds in the furanoid ring appear to be shorter (average length, 1.507A) than a single C-C bond (1.54A). The anomeric C-1-S bond is 0.039A shorter than the C,H,-S bond. The furanose ring is puckered, presumably because of nonbonded interactions, and the puckering, with C-3 exoplanar, relieves the strain to give an envelope conformation. All of the oxygen atoms are involved in hydrogen bonding. The sugar sulfonic acid is l-O-(6-deoxy-6-sulfo-a-~-glucopyranosyl)glycerol isolated from a plant ~ u l f o l i p i d .The ~ ~ plant sulfolipid

H,COH

Sulfonic acid from plant sulfolipid

is the only sugar sulfonic acid derivative yet known to occur in Nature, but its ubiquity and abundance imply that it plays a role in plant physiology perhaps as important as that of sugar phosphates. The conformation of the D-glucopyranosyl part is a chair. The sulfonate ion, which is on the same side of the sugar ring as most of the glycerol, and four oxygen atoms (two from the sugar, and one each from the sulfonate group and from the glycerol) form a group that is comparable to the four oxygen atoms of P-D-galactopyranose; this relationship explains the action of P-D-galactopyranosidase on some D-glucosides of this sulfonic acid. The sulfoxide that has been studied was obtained by the oxidation of a sulfide, namely, (2S,6R)-6-(hydroxymethyl)-2-methoxy1,4-0xathiane,~’synthesized from methyl 6-0-trityl-a-D-glucopyrano-

(2 s, 6 ~)-6-(Hydroxymethyl)2-methoxy- 1,4-oxathiane S-oxide

(46) Y. Okaya, Acta Crystrtllogr., 17,1276 (1964). (47) K. W. Buck, T.A . Hamor, and D. J . Watkin, Chem. Commun., 759 (1966).

CRYSTAL STRUCTURE OF CARBOHYDRATES

73

side; C-2and C-6have the same configuration. The chair conformation is flattened, resulting in a further separation of the oxygen atom of the methoxyl group from the syn-axial hydrogen atom on C-6, perhaps d u e to repulsion of nonbonded atoms. Several halogenated carbohydrates have been studied. One exBromine ample is methyl 6-bromo-6-deoxy-a-~-galactopyranoside.~~

Methyl 6-bromo-6-deoxy~-~-galactopyranoside

was used as the heavy atom in solving the structure. The pyranoside adopts that chair form in which all substituents are equatorially attached, except for 0-1and 0-4. The anomeric C-1-0-1 bond is not short, perhaps because of the presence of a methyl group on 0-1. In the crystal, the molecules are packed in an interesting way, the bromine atoms from different molecules being arranged in layers that become cleavage planes because there are no bonds across these planes, and the rest of the crystal is held together by intermolecular hydrogen-bonding. In methyl 4,6-dichloro-4,6-dideoxy-cr-~-glucopyranoside,~~’ 0-1

Methyl 4,6-dichloro-4,6dideoxy- a-D-glucopyranoside

is part of the methoxyl group, as in the previously discussed sugar, and the anomeric bond is a normal C-0 single bond. The chlorine atoms were used in deducing both the crystal structure and the conformation. The compound adopts a chair conformation; it possesses one normal, intermolecular, hydrogen bond (2.70A long) and a long 0-0 distance (3.0SA) that might b e a weak hydrogenbond. (48) J. H. Robertson and B. Sheldrick, Acta Crystallogr., 19,820,(1965). (49) R. Hoge and J. Trotter,]. Chem. Soc. (A), 267 (1968).

74

GERALD STRAHS

3,4,6-Tri-O-acetyl-2-bromo-2-deoxy-a-~-mannopyranosyl flu~ride,~o adopts the expected CI (D) conformation. The intermolecular bonding was not described. A correlation with three other bromodeoxyglycosyl fluorides and with a deoxyiodoglycosyl fluoride was obtained by using I9F nuclear magnetic resonance spectroscopy and measuring the H-F coupling-constants. Three of the compounds were shown to have the C1 (D) conformation, and 2-bromo-2-deoxy-~-~-arabinopyranosyl fluoride has the 1C (D) conformation. Addition of dichloromethylene to 3-deoxy-l,2:5,6-di-O-isopropylidene-a-~-erythro-hex-3-enofuranose gives 3-deoxy-3,4-C-(dichloromethylene)-1,2:5,6-di-O-isopropylidene-a-~-galacto~ranose.~~ [The

3-Deoxy-3,4-C-(dichlorornethylene)-l,2 : 5 , 6 - d l - 0 isopropy lidene - (Y - D galactofuranose

-

X-ray work was hampered by radiation damage to the crystal, which also hindered the study of a caldariomycin derivative in which there are also two chlorine atoms on one carbon atom.52]All of the fivemembered rings are nonplanar, although the furanoid ring is almost planar. The initial formulation of structure was based on the concept that dichloromethylene would react with the double bond on the accessible, exo side of the trioxabicyclo[3.3.0]octene ring-system, and the structure found confirmed this hypothesis. Thus, the two rings attached to the furanoid ring are trans (or anti)to each other. A similar situation is found in 6-azido-5,6-dideoxy-5-iodo-1,2-0isopropylidene-P-L-idohranose,prepared by the action of iodine azide (IN,; a pseudohalogen) on 5,6-dideoxy-l,2-O-isopropylidenea-D-xy~o-hex-5-en0franose.~~ The addition of iodine azide to the (50) J. C. Campbell, R. A. Dwek, P. W. Kent, and C. K. Prout, Carbohyd. Res., 10,

71 (1969). (51)J. S. Brimacombe, P. A. Gent, and T. A. Hamor, J . Chem. SOC. (B), 1566 (1968). (52)S. M.Johnson, I. C. Paul, K. L. Rinehart, Jr., and R. Srinivasan, J . Amer. Chem. SOC., 90,136(1968). (53)J. S . Brimacombe, J. G. H. Bryan, T. A. Hamor, and L. C. N. Tucker, Chem. Commun., 1401 (1968).

CRYSTAL STRUCTURE OF CARBOHYDRATES

75

double bond is anti-Markownikov, possibly for steric reasons. The 5-membered rings are nonplanar, as expected. The heaviest atom thus far used in structure determination of sugar derivatives is mercury in methyl 2-(chloromercuri)-2-deoxy-a-~t a l ~ p y r a n o s i d eA. ~chair ~ form of the pyranoside was the only structure that would fit the observed electron density in one projection. Several other conformations were rejected as they did not fit the results.

IV. DISACCHARIDES The crystal structure of cellobiose [O-P-D-glucopyranosy1-( D-g~ucopyranose](12)has been determined for the third time.’O The

p-Cellobiose

(1 2)

bond lengths and angles are all in the expected ranges, except for the equatorial (2-1-0-1 bond, which is shorter than a C-0 single bond. There is one intramolecular hydrogen-bond, from the hydrogen atom on 0-3’ of the D-glucopyranose residue to the ring-oxygen atom (0-5)of the D-glucopyranosyl group in 12.There is a small twist in the “backbone” of the molecule that brings the hydrogen-bonded atoms 0-3’and 0-5close together. Methyl P-maltoside monohydrate ( 13) contains a D-glucopyranosyl

Methyl p-maltoside monohydrate (13)

(54) J. Bain and M.M.HardingJ. Chem. SOC., 4025 (1965).

76

GERALD STRAHS

group linked a - ~ 1+4) - ( to a D-glucopyranose residue, whereas cellobiose (12)has a @-D-glucopyranosidiclinkage. Both of the D-glucose moieties adopt the C1 (D) conformation. The molecule of water is hydrogen-bonded to both of the D-glucopyranose residues in 13 and may be considered to be part of the maltose A small twist in the “backbone” of molecule 13 moves the donor atoms 0-6 and 0-6’ apart, to make room for the molecule of water, and 0-2 and 0-3’, which participate in an intramolecular hydrogen-bond, are brought closer together. The water molecule is the acceptor for both of the “intramolecular” hydrogen bonds, and the two protons of the water are donors in intermolecular hydrogen-bonds. Four more intermolecular hydrogen-bonds afford a total of nine hydrogen-bonds per molecule. The hydrogen bonds link the maltose molecuIes in helical chains (both 3-unit, left-handed helices and 4-unit, right-handed helices). It is of interest that @-maltose crystallizes as the monohydrate. However, there is no reason to assume that the water molecule does or does not form hydrogen bonds to maltose in either a similar or identical manner. In fact, the water molecule forms four tetrahedrally arranged hydrogen-bonds to four different maltose molecule^.^^ The single intramolecular hydrogen-bond is the 0-2-0-3’ bond. All of the hydroxyl groups participate in hydrogen bonding. The (highly distorted) structure of sucrose (14) is shown for com-

Sucrose

(14)

parison of the intramolecular hydrogen bonds.57Thus, it is complementary to cellobiose (12)and methyl @-maltopyranoside(13),being the only disaccharide thus far studied that has two direct intramolecular hydrogen-bonds . (55)S.S . C.Chu and G. A. Jeffrey,Acta Crystallogr., 23, 1038 (1967). (56)G. J. Quigley, A. Sarko, and R. H. Marchessault, Acta Crystatlogr., A25, S196 (1969). (57)G.M.Brown and H. A. Levy, Science, 141,921(1963).

CRYSTAL STRUCTURE O F CARBOHYDRATES

77

It is unfortunate that structural data have not yet been obtained for lactose, as a comparison would be interesting. Buma and W i e g e r P pointed out that the cell dimensions of p-lactose and p-cellobiose monohydrate are very similar. As the two disaccharides have the same space-groups, their crystal structures may be very similar. Buma and Wiegers gave X-ray powder data for anhydrous a-lactose, anhydrous “stable” a-lactose prepared b y boiling a-lactose hydrate crystals with methanol for one hour, a-lactose hydrate, and p-lactose. An investigation of the crystal forms of lactose in milk powders9 demonstrated the occurrence of an amorphous form, a-lactose monohydrate, and a crystal form containing anhydrous a-lactose and anhydrous p-lactose in the ratio of 5:3 that might be formed by true cocrystallization of the two anomers. The authorss9 also reported powder diffraction data for the four modifications of lactose previously mentioned, as well as for the unusual crystal form, although they mistakenly identified the anhydrous forms as “anhydrides.” V. OLIGOSACCHARIDES

The only trisaccharide for which a crystal structure has thus far been reported60 is raffinose [a-D-galactopyranosyl-( 1 + 6 ) - 0 - a - ~ glucopyranosyl p-~-fructofuranoside]pentahydrate. The bond angles and distances for the individual monosaccharide moieties are in good agreement with average values. All of the hydroxyl groups are donors and acceptors of hydrogen bonds, but crystalline raffinose contains no intramolecular hydrogen-bonds and differs in this respect from the disaccharides shown in 12, 13, and 14, including sucrose, a residue of which constitutes a part of the raffinose molecule. Each raffinose molecule is partly helical, and, in the crystal, raffinose molecules are alternately arranged in helices parallel to a crystal axis. One crystalline tetrasaccharide, namely, p-cellotetraose (15), was

I

H

H

0- Cellotetraose (1 5)

(58) T. J. Buma and G. A. Wiegers, Neth. Milk DairyJ., 21,208 (1967). (59) J. H. Bushill, W. B. Bright, C. H. F. Fuller, and A. V. Bell, J . Sci. Food Agr., 16,622, (1965). (60) H. M. Berman, Acta Crystallogr., B26,290 (1970).

GERALD STRAHS

78

studied6' in an attempt to throw light on the structures of cellulose I and cellulose 11. From the results, Poppleton and MathiesonGIwere able to propose new and improved models for cellulose, although their task was made quite difficult because the cellotetraose crystals available were very thin (0.01 mm). The tiny crystals necessitated long X-ray exposures (up to 900 hours), and only a few X-ray reflections could be measured, so that use of the customary least-squares refinement technique was precluded. The analysis confirmed the presence of (1+4)-linkages of the ~-D-glucopyranosylresidues in cellotetraose and cellulose, and the similarity of these residues to those in cellobiose. As in cellobiose, the four D-glUCOpyranOSe residues are bent by approximately 25" relative to one another; they are also twisted with respect to the other residues. Also, three similar, intramolecular, hydrogen bonds exist between 0-3 and the ring-oxygen atom ( 0 - 5 ) of adjacent residues. The arrangement of the intermolecular hydrogen-bonding is not yet clear, as there are alternative possibilities for arrangement of the hydroxyl group on C-6 of each residue, so that the hydrogen bond could be either interchain or intrachain (to the 0-2); perhaps, this is the key to the solution of the structure of cellotetraose and cellulose. Were there disorder at C-6, much improvement on the results of Poppleton and Mathieson could not be expected, because the results for cellotetraose would be so similar to those for cellulose, or, more precisely, cellulose I1 (regenerated or mercerized cellglose). Quite a different result was found by Hybl and coworker?2 for the cyclohexaamylose-potassium acetate complex (16). They were able 0-3

H:o

0-3

0-3

I

H*O Half of cyclohexaarnylose-potassium acetate (16)

(61) B. J. Poppleton and A. McL. Mathieson, Nature, 219,1046 (1968). (62) A. Hybl, R. E. Rundle, and D. E. Williams,J. Amer. Chem. Soc., 8'7, 2779 (1965).

CRYSTAL STRUCTURE OF CARBOHYDRATES

79

to collect two sets of crystallographic data, even though the crystal was decomposing during the experiment. The contents of potassium acetate and water were found not to bear a simple relationship to the content of cyclohexaamylose. A similarity was noticed between the X-ray diffraction pattern of cyclohexaamylose and of benzene, each D-glucopyranose residue of cyclohexaamylose being analogous to each carbon atom of benzene. The initial refinement was concerned with the location and the orientation of three D-glucopyranose residues, because the other three D-glucopyranose residues were found to be related by symmetry. Later, the position of each atom and the thermal motion were refined. The a-D-glucopyranose residues are present in the C1 (D) conformation (la2e3e4e5e),and an intrachain hydrogen bond connects 0-2 and 0-3 of adjacent D-ghcopyranose residues. A direct, interchain hydrogen-bond connects 0-2and 0-6,and 0-2and 0-5have a water molecule lying between them to which both are hydrogen-bonded. However, this water molecule is randomly replaced by a potassium ion. The hydrogen bonds in the cyclohexaamylose structure are similar to those in the model that Hybl and coworkers elaborated for V amylose. The model consists of a helix of 6 D-ghcopyranose residues per turn, with a hydrogen bond between 0-2and 0-3of contiguous Dglucopyranose residues, and a hydrogen bond between 0-6and 0-2 of D-glucopyranose residues that are one helix turn apart. When the vapor pressure of the water exceeds 26 torr (100% relative humidity at 26.5"),a water molecule can add to the exterior of the helix, forming additional hydrogen bonds between 0-2and 0 - 5 of D-glucopyranose residues that are one helix turn apart. The potassium acetate-cyclohexaamylose complex may be regarded as a channel structure in which the rings are stacked atop one another. The structures of two other complexes, both of which are tetrahydrates, have been solved; one contains iodine, and the other, propyl The cyclohexaamylose rings are arranged in a fishbone manner, the side of a ring being adjacent to the top of the next ring. Iodine as the guest is coaxial with the ring, whereas propyl alcohol as the guest is mainly coaxial but is, statistically, disordered. The cyclohexaamylose molecules are held together by hydrogen bonds between each other and water molecules. The next higher analog is cycl~heptaamylose,~~ which forms a series (63) R. K. McMullan, I. P. Fayos, D. Mootz, and W. Saenger, personal communication. (64) J. A. Hamilton, L. K. Steinrauf, and R. L. Etten, Actu Crystullogr., B24, 1560 (1968).

GERALD STRAHS

80

of crystalline inclusion complexes. Five of these (including those with rn-iodobenzoic acid and 2,5-diiodobenzoic acid) constitute an isomorphous series. A structural analysis is in progress to determine (a) the stereochemistry of fit of the guest molecule in the cycloamylose and (b) the variability of the water of hydration.

VI. ANTIBIOTIC SUBSTANCES The application of crystallography to antibiotic substances that contain carbohydrate residues has been limited by the lack of availability of suitable crystals. The first suitable crystalline derivative of streptomycin that became available was streptomycin oxime sesquiselenate t e t r a h ~ d r a t e . ~ ~ Streptomycin contains residues of the dibasic streptidine and of two NH

HO

H,NCHN

OH

HO

Streptomycin

sugars, namely, 2-deoxy-2-(methylamino)-~-glucose and L-streptose (5-deoxy-3-C-formyl-~-lyxofuranose). The residues of streptidine and 2-deoxy-2-(methylamino)-~-glucose are respectively linked glycosidically to C-l and C-2 of L-streptose, and these residues are trans to each other. The selenate ion was used as the heavy atom, in order to solve the structure by the heavy-atom method. In addition, the “anomalous” scattering of the CuKa X-radiation by the selenium atom was used to confirm the absolute configuration of each of the asymmetric atoms in streptomycin. The uniqueness of this crystal form, in terms of forming large crystals, is attributed to an elaborate system of hydrogen bonds. (65) S. Neidle, D. Rogers, and M. B. Hursthouse, Tetrahedron Lett., 46,4725 (1968).

CRYSTAL STRUCTURE OF CARBOHYDRATES

81

The antibiotic kanamycin is chemically related to streptomycin, as it is 4-0-(6-amino-6-deoxy-a-D-glucopyranosyl)-6-0-(3-amino-3deoxy-cu-~-glucopyranosyl)-2-deoxystreptamine. Two isomorphous crystalline compounds, namely, kanamycin monosulfate monohydrate and kanamycin monoselenate monohydrate, were used in elucidating

Kanamycin A

the structure.fi6The technique employed was the method of single, isomorphous replacement; this requires, in addition to one crystal form, an isomorphous crystal form containing one (or more) heavy atoms, and is an approximation to the method of multiple, isornorphous replacement, for which three or more isomorphous crystals containing heavy atoms are needed. As the method of single, isomorphous replacement is approximate, the uncertainty about the phase angle of the X-ray reflection was resolved6” by use of the “anomalous” scattering of the MoKa X-radiation by the selenium atom. “Anomalous” scattering also gives two phase angles when it is used to solve an unknown structure, but the two methods can be combined to find the correct phase angle, because the two ambiguities differ; the ambiguity of the phase angle is about 0” for the isomorphous-replacement method, and 90” for the “anomalous” scattering. For example, phase angles of +60 and -60” may be obtained by the first method, and 60 and 120” by the second method, and so 60” is the correct value of the phase angle. The results confirmed the relative and absolute configurations of kanamycin as determined by chemical methods, and gave its conformation. All three of the six-membered rings have a

(66) G . Koyama and Y. Iitaka, Tetrahedron Lett., 15,875 (1968).

82

GERALD STRAHS

chair conformation in which all of the substituents are equatorially attached, except for the two anomeric oxygen atoms. The crystal structure of the hydrochloride and hydrobromide of isoquinocycline A was elucidated b y the method of single, isomorphous r e p l a ~ e m e n t .The ~ ~ glycosyl group, which constitutes about 20% of the molecule, is that of a branched-chain, eight-carbon sugar, namely, 2,6-dideoxy-4-(~-gZycero-l-hydroxyethyl)-~-~-~~Z~hexopyranose-lC (17). OR

2,6- Dideoxy - 4 - (L-glYcero -

1-hydroxyethy1)-a-L- XYlOhexopyranose-1C (R = H) (17)

Erythromycin A, a widely used, macrolide, antibiotic substance, was crystallized as the hydriodide dihydrate. It would be expected that the iodine atom would be used as the heavy atom in solving the structure, and, indeed, the authors tried the heavy-atom method first.68 Unfortunately, the iodine atom lies on a crystallographic mirror-plane, and so this method failed. Because erythromycin con-

Me

Erythromycin A hydriodide

(67)A.J. Tulinsky,J. Amer. Chem. SOC.,86,5368(1964). (68)D.R. Harris, S . 6.McGeachin, and H. H. Mills, Tetrahedron Lett., 11,679 (1965).

CRYSTAL STRUCTURE OF CARBOHYDRATES

83

tains asymmetric carbon atoms, there can be no mirror planes in the crystal; correspondingly, there can be no mirror planes in the correct space-grouping. However, as the y coordinate of the iodine atom is zero, it lies on a mirror plane of another space group, of higher symmetry than the true space-grouping. When the iodine atom was used as the heavy atom, the pseudo-space group prevailed; the correct structure and its mirror image overlapped and could not be resolved from each other. The authors then solved the structure by using the “anomalous”-dispersion effect of the iodine atom. The sugar originally thought to be P-cladinose was later identified as a-cladinose (2,6dideoxy-3-C-methyl-3-O-methyl-~-ribo-hexose); the other sugar is p-desosamine [3,4,6-trideoxy-3-(dimethylamino)-~-~-xyZo-hexose]. The chemical identification of the anomeric configuration of the cladinose moiety was repeated, and found to be a-L,in agreement with the crystallographic results. The antibiotic substances formycin and formycin B are C-glycosyl

c2

HOCH, 0

HO

OH

Formycin

HO

OH Formycin B

compounds having a nucleoside-like structure, namely, 7-amino-3~-~-ribofuranosylpyrazolo-[4,3-d]pyrimidine and 3-/3-~-ribofuranosylpyrazolo-[3,4-d]-6(H)-7-pyrimidinone, respectively. Formycin B is a deaminated product of formycin. For formycin, the crystal form used for determination of the structure was formycin hydrobromide monohydrate,@‘and the bromide ion served as the heavy atom. Although the first trial-structure contained both formycin and its mirror image, the authors were able to reject the “ghost peaks,” refine the correct structure, and use the “anomalous” scattering to determine the absolute configuration. (“Ghost” peaks are peaks that do not correspond to atoms, but appear on electron-density maps.) The success in rejecting ghost peaks in the study of formycin, but not in that of erythromycin, may be due to two factors: ( a ) formycin has the lower molecular weight, and ( b ) bromine has fewer electrons than iodine (69) G . Koyama, K. Maeda, and H. Umezawa, Tetrahedron Lett., 6,597 (1966).

GERALD STRAHS

84

and is not so heavy. Instead of the carbon-nitrogen bond present in nucleosides, formycin contains a carbon-carbon single bond. I n nucleoside terminology, the torsion angle between the sugar and the base is 148", indicating the normal, syn orientation. The D-ribofuranose ring adopts a twist conformation, C-2' and C-3' being displaced (on opposite sides of the ring) by 0.33 and 0.38& corresponding to (2-2'-endo-C3'-exo. The structure of showdomycin (3-~-~-ribofuranosylmaleimide)

"

HOCH,

HO

0

OH

Showdornycin

was inferred from the crystal structure of the 2,3-isopropylidene acetal of N-methylbis(deoxycy1oshowdomycin) hydr~bromide.'~The bromine atom was used in solving the structure by the heavy-atom method. The bromide monohydrate was also used" in establishing the crystal structure of blasticidin S. The dimensions of the unit cell ( a =

Me

Blasticidin S

20.39, b = 21.34, and c = 4.81A) indicate that the molecules are almost flat and are essentially perpendicular to the short cell-edge. In contrast, all three axes of the crystal lattice of sodium chloride are 5.63A long, or about 15% longer than the short axis of blasticidin S monohydrate. Consequently, the structure could be solved in projection on the xy plane. However, the flatness (planarity) of the molecule or its components cannot be verified, as the structure is two-dimensional. The carbohydrate moiety is a residue of 1,2,3,4-tetradeoxy-P-~erythro-hex-2-enopyranuronicacid. (70) Y. Tsukuda, Y. Nakagawa, T. Kano, T. Sato, M . Shiro, and H. Koyama, Chem. Commun., 975 (1967). (71) S.Onuma, U. Nawata, and Y. Saito, Bull. Chem. Soc.Jap.,39,1091 (1966).

CRYSTAL STRUCTURE OF CARBOHYDRATES

85

The crystallographic data for other antibiotic substances that have been solved, but not yet published, include 7(S)-chloro-7-deoxyl i n c o m y ~ i nand ~ ~ synthetic methyl ~~-kasugarninide.’~ Me

Methyl kasugaminide

OH 7(S)-Chloro-?deoxylincomycin

Lincomycin consists of an amino acid (a substituted hygric acid) Me I

OH

Lincomycin

linked by an amide group to a monosaccharide. The hydrochloride of lincomycin was used in solving the structure; the sugar residue was identified as that of 6-amino-6,8-dideoxy-l-thio-~-er~thro-~-guZuctooct~pyranoside.~~ Aristeromycin contains no carbohydrate residues, but it is included because of its resemblance to adenosine; it is (l’R,2‘S,3%,4’R)-9[P-2a,3a -dihydroxy-4P - (hydroxymethyl)cyclopenty1I adenine. 75 The antibiotic activity may be due to replacement of a furanose moiety (72) D. Duchamp, personal communication. (73) G. Koyama, personal communication. (74) R. E. Davis and R. Parthasarathy,Actu Crystallogr.,21, A109 (1966). (75) T. Kishi, M. Muroi, T. Kusaka, M . Nishikawa, K. Kamiyo, and K. Mizuno, Chem. Commun., 852 (1967).

86

GERALD STRAHS

by a cyclopentane moiety. The crystal structure of the hydrobromide was solved.75

HO

OH

Aristerornycin

VII. NUCLEOSIDES AND NUCLEOTIDES The nucleosides and nucleotides are components of nucleic acids; they contain D-ribofuranose and 2-deoxy-~-erythro-pentofuranose residues. The large number of structures solved reflect the current interest in genetic material. The crystal structures of constituents of nucleic acids have been reviewed by Sundaralingam and coworker^,^^ nucleotide conformations by Arnott and H ~ k i n s and , ~ ~ furanoid . ~ ~ nucleic acid components adopt structures b y S ~ n d a r a l i n g a mThe many different conformations in the crystal. However, the configurational requirements imposed upon D-ribose and 2-deoxy-D-erythropentose by helices of the nucleic acids are quite restrictive. The most stringent restrictions apply to ribonucleic acid (RNA), in which the D-ribofuranose residues have the C-3-end0 conformation (that is C-3 and C-5 are on the same side of the furanose ring), because only an axially attached hydroxyf group will not interfere with the backbone.79The observed forms of RNA are A, A‘, and A“, which correspond to the A form of 2‘-deoxyribonucleic acid (DNA). T h e helical hybrid of DNA with RNA has the constraint already mentioned, namely, C-3 endo. Only DNA exists in the B form,. which is the familiar model for DNA having C-2 endo. Structures containing D-ribofuranose and 2-deoxy-~-erythro-pentofuranose residues are listed in Tables I1 and 111. In general, the furanoid rings have four atoms in, essen(76) M. Sundaralingam and L. H. Jensen, 1, Mol. Biot. 13, 930 (1965); S. T. Rao and M . Sundaralingam, in “Synthetic Procedures in Nucleic Acid Chemistry,” W. W. Zorbach and R. S. Tipson, eds., John Wiley and Sons, Inc., New York, N.Y., Vol. 2, in press. (77) S. Arnott and D. W. L. Hukins, Nature, 224,886 (1969). (78) M. Sundaralingam, J. Amer. Chem. SOC., 87, 599 (1965). (79) S. Arnott, W. Fuller, A. Hodgson, and I. Prutton, Nature, 229,561 (1968).

CRYSTAL STRUCTURE O F CARBOHYDRATES

87

TABLEI1 Derivatives of D-Ribofuranose Compound

Description

Cytidine c-3 5-Methyluridine c-3endo 5-Bromouridine methyl C-2 elldo sulfoxide 4-Thiouridine C-3 endo ~-Ribofiiranosyl-6-~1,urine- C-2 endo thin1 Inosine 4-Thiouridylate disulfide Adenosine 3’-phosphate dih ydrate Cytidine 3‘-phosphate Guanosine 5’-phosphate trihydrate Guanosine 5’-phosphate Barium inosine 5’-phosphate Disodium inosine 5‘-yhosphate Sodium inosine 5’-phosphate hexahydrate H ydroyen-bonded complex of adenosine and 5-bromouridine 5-Bromouridine Adenosine 2’-(uridine 5’-phosphoric acid) Adenosine 3’:5’-cyclic phosphate Triethylammonium uridine 3’:5’-cyclic phosphate Triethylammonium uridine-2‘:3‘-0,0cyclothiophosphate

Displacement from plane (A)

References

0.58 0.60 0.60

80 81 82

83 84

C-3 endo

0.605 0.612,0.632 (two independent molecules) 0.63 0.54,0.58 (twohalf-molecules) 0.562

87

c - 2 elkdo C-3 endo

0.609 0.515

88 89

C-3 endo C-2 endo

0.515

89

C-2 endo

90

C-2 endo

(two independent molecules) 0.56

C-3 endo C-3 endo

0.0620 0.591

92

c-2 C-2 enclo C-3 endo C-4 e m ( ? )

0.5183 0.62 0.60 at least 0.6 in two independent molecules 0.64, 0.58 (two independent moleculesu) 0.15

82 93

C-3 endo C-3 endo

C-3 endo

c-3

85 86

91

94 95

96

“This may have C-4 out of plane, as in adenosine 3’:5’-cyclicphosphate. (80) S. Furberg, C. S . Petersen, and C. Romming, Acta Crystallogr., 18,313 (1965). (81) D. J. Hunt and E. Subramanian, Actn Crystnllogr., in press. (82) J. Iball, C. H. Morgan, and H. R. Wilson, Proc. Roy. Soc., Ser. A , 302,225 (1968). (83) W. Saenger and K. H. Scheit, personal communication. (84) E. Shefter, I . Phnrrn. Sci., 57, 1157 (1968). (85) P. Tollin and A. R. I. Munns, personal communication. (86) E. Shefter and T. I. Kalman, Biochern. Biophys. Res. Cornrnun., 32,878 (1968).

GERALD STRAHS

88

TABLE111 Derivatives of 2-Deoxy-~erythro-pentofuranose Compound ~

Displacement from plane (A)

References

~

2’-Deoxyadenosine monohydrate 2‘-Deoxycytidine hydrochloride Thymidine 2’-Deoxy-5-fluorouridine [ 1-(2-Deoxy-ao-ribofuranosy1)uracil-5-vll disulfide Hydrogen-bonded complex of 2’-deoxyguanosine and 5-bromo-2’-deoxycytidine S-Bromo-S’-deoxyuridine I

Description C-3 exo C-3 endo C-2 exo C-3 exo

0.552 0.361 0.245 0.57

c-2 C-2 endo C-3 exo c-3 c-4

0.592

C-2 endo C-2 endo

0.454 0.540

102

C-2 endo

0.5917

103

97 98 99 100 101

-

tially, a plane, C-2 or C-3 lying -0.5-0.6Aabove or below the plane of the ring, that is, in an envelope conformation. As the four “coplanar” atoms are usually not quite coplanar (within the standard deviation) and the twist conformations rarely have their two exoplanar atoms symmetrically displaced, the conformation lies somewhere between the envelope and the twist. In the envelope conformation, there is no doubt as to location of the exoplanar atom, because one plane fits the (87) M. Sundaralingam, Acta Crystallogr., 21,495 (1966). (88) M. Sundaralingam and L. H. Jensen,J. Mol. Biol., 13,914 (1965). (89) W. Murayama, N. Nayashima, and Y. Shimuzu, Acta Crystallogr., in press. (90) N. Nagashima and Y. Iitaka, Acta Crystallogr., B24, 1136 (1968). (91) S. T. Rao and M. Sundaralingam, Chem. Commun., 995 (1968). (92) A. E. V. Haschemeyer and H. M. Sobell, Acta Crystallogr., 18,525 (1965). (93) E. Shefter, M. Barlow, R. Sparks, and K. N. Trueblood, Acta Crystallogr., B25, 895 (1969). (94) K. Watenpaugh, J. Dow, L. H. Jensen, and S. Furberg, Science, 159,206 (1968). (95) C. L. Coulter, Science, 159,888 (1968). (96) W. Saenger and F. Eckstein, personal communication. (97) D. G. Watson, D. J. Sutor, and P. Tollin, Acta Crystallogr., 19,111 (1965). (98) E. Subramanian and D. J. Hunt, Actu Crystallogr., B26,303 (1970). (99) (a) P. Tollin, H. R. Wilson, and D. W. Young, Nature, 217, 1148 (1968);(b) D. W. Young, P. Tollin, and H. R. Wilson, Actu Crystallogr., B25,1423 (1969). (100) D. R. Harris and W. M. Macintyre, Biophys.j., 4,203 (1964). (101) E. Shefter, M. P. Kotick, and T. J. Bardos,]. Pharm. Sci., 56,1293 (1967). (102) A. E. V. Haschemeyer and H. M. Sobell, Actu Crystallogr., 19,125 (1965). (103) J. Isbell, C. H. Morgan, and H. R. Wilson, Proc. Roy. SOC.,Ser. A, 295,320 (1966).

CRYSTAL STRUCTURE OF CARBOHYDRATES

89

observations much better than do others (see Tables I1 and 111).With the twist conformations, an arbitrary choice must be made, because any three atoms can form one plane; it is customary to choose the plane encompassing 0-4, C-1, and C-4. The wide variety of conformations observed indicates that the energy of the various forms must be similar, and that different forms are assumed as the result of the forces of crystal packing, including hydrogen bonding. Consequently, some of the compounds can crystallize in more than one crystal form. In order to minimize repulsion of nonbonded atoms, the furanoid ring is puckered, not planar. The substituents, staggered around the puckered ring, are quasi-axially or quasi-equatorially attached thereto. Future work in this area will probably include studies of larger molecules, such as adenosine 5'-pyrophosphate, adenosine 5'-triphosphate (only small crystals of which have as yet been obtained104), dinucleotides, trinucleotides, oligonucleotides, and dinucleotides joined by complementary base-pairing. Well-equipped laboratories can conduct a study of these structures of higher molecular weight, because of advances in data collection and in computers. However, problems may be encountered in obtaining suitable crystals. There is a large gap in molecular weight between a dinucleotide, or nucleotide base-pair, having a molecular weight of about 600, and a transfer-RNA having a molecular weight of about 30,000. Single crystals of transferRNA have been obtained in several laboratories,'05 and it may be assumed that its structure will be determined. The problems likely to be encountered with transfer-RNA will probably be comparable to those obtaining in the structural study of a protein, a problem much more difficult than determination of structure of compounds of low molecular weight. The molecular structure of transfer-RNA may be assumed to contain both helical and nonhelical regions, as certain portions of the molecular sequence always consist of bases complementary to another section of the transfer-RNA molecule, and these would form RNA double helices. Other portions of the molecule cannot form helices. For the helical regions, the restriction already mentioned must apply, that is, C-3 will be endo, and the region will correspond to the A form of DNA. However, there is no such restriction on the conformation of the D-ribofuranose residues. Various con(104) M . Zeppezauer, E. Zeppezauer, and C. I. Briinden, Actci Chem. Scund., 22, 1036 (1968). (105) (a) S. H. Kim and A. Rich, Science, 162, 1381 (1968);(b)A. Hampel, M. Labananansak, P. C . Connors, L. Kirkegard, U. L. Rajbhandany, P. B. Sigler, and R. M . Bock, ibid., 162, 1384 (1968); (c) J. R. Fresco, R. D. Blake, and R. Langridge, Nature, 220,1285 (1968);(d)W. Saenger, personal communication.

GERALD STRAHS

90

formations of the D-ribofuranose residues may even be expected in the nonhelical regions.

VIII. MISCELLANEOUSCARBOHYDRATES: GLYCOSIDES, VITAMINS, AND HYDRAZONES Although many crystalline glycosides are available for X-ray study, very few have thus far been so examined, for reasons that are not obvious. A possible reason is their molecular weight; for example, if a crystallographer is interested solely in the aglycon, the hexosyloxy group (C,H,,O,) would, in its hexoside, add 12 more (non-hydrogen) atoms to the problem. Similarly, the aglycon in a glycoside adds atoms to the carbohydrate structure. One D-glucoside, the rubidium salt of monotropein (C,,H,,O, ,Fib * C0,W

IG;

HO

OH Monotropein, rubidium salt

2H20) has been studied.'O, The conformation of P-D-glucopyranose was used in establishing the conformation of monotropein and four other monoterpene D-glucopyranosides. Another D-glucopyranoside whose structure has been solved is the p-iodobenzene~ulfonate~~~ of fusicoccin (C42H591014S). The a-D-glucopyranosyl group has an Me

HO

Fusicoccin A

(106)N. Masaki, M. Hirabayashi, K. Fuji, K. Osaki, and H. Inouye, Tetrahedron Lett., 25,2367(1967). (107)M. Brufani, S. Cerrini, E. Fedeli, and A. Vaciago, Acta Crystallogr., A25, S202 (1969).

CRYSTAL STRUCTURE OF CARBOHYDRATES

91

acetyl group on 0-3, and a 1,1-dimethyl-2-propenyl group on 0-6. The structure of the aglycon had been solved chemically,'08 but prior determination of the crystal structure might have saved organic chemists much work, because the compound contains 13 acentric carbon atoms. The structure of the normal crystal of L-ascorbic acid (L-threo-hex2-enono-1,4-lactone) has been established by X-ray studies,lo9and that of the partially deuterated crystal by neutron-diffraction experiments." The C-H and 0 - H bonds are discussed on p. 57. The furanoid ring and the three oxygen atoms are almost planar, whereas the enediol group is exactly planar. The bond lengths are different throughout the molecule. It is noteworthy that the length of the C-3-0-3 bond is 1.326 A, which is shorter than a normal single-bond, but longer than a double bond. Consequently, 0-3 must be the source of the acidic proton, and structural studies of sodium L-ascorbate are in progress to check this assignment." In the hranoid ring, the C-1-0-4 bond is shorter than the C-4-0-4 bond, owing to resonance and to partial double-bond character (see formula 18). The neutron0-3 \

H I

0-1

L-Ascorbic acid (18)

diffraction study of normal and deuterated L-ascorbic acid (in addition to the X-ray study) is an extremely ambitious undertaking. Unfortunately, because of ( a ) the large, incoherent scattering of neutrons by the hydrogen atoms, and ( b )the partial deuteration (about 42%), three completely independent sets of data could not be obtained. (108) E. Hough, M. B. Hursthouse, S. Neidle, and D. Rogers, Chem. Commun., 1197 (1968). (109) J. Hvoslef, Actu Crystallogr., B24,23 (1968).

92

GERALD STRAHS

Riboflavine hydrobromide monohydrate1l0 contains a ribitol resiC H,OH I

HOCH I

HOCH I HOCH

Riboflavine

due, which is in a sickleI8conformation. Vitamin B12 is a D-ribofuranose derivative, and the structure of vitamin BI25'-phosphate, a precursor"' of vitamin BIZ,has been determined. The a-D-glycosidic bond lies 16' (0.51 & out of the plane of the 5,6-dimethylbenzimidazole ring, because of steric or nonbonded interactions. The D-ribofuranose moiety has that envelope conformation having C-2 exo (0.77 A exoplanar); C-3 and C-5 bear phosphate groups. The structures of two (pbromopheny1)hydrazones have been determined from projection data. The first was that of D-ribose,'I2 in which the D-ribose is acyclic, in a sickle18conformation, and C-1, C-2, C-3, and C-4 are coplanar. Rotation of C-5 out of this plane reIieves the 0-2, 0-4interaction that would be present in the extended conformation and brings 0-5 close to 0-2, so that an intramolecular hydrogen-bond can be formed; this is the first (and, thus far, the only) case of an intramolecular hydrogen bond in a crystal of a monosaccharide derivative. The hydroxyl groups on C-2, C-3, and C-4 form three intermolecular hydrogen-bonds. p-D-Glucose (p-brom~phenyl)hydrazone"~is intramolecularly cyclized by addition of the 5-OH group across the C-1-N double bond, and crystallizes in that chair conformation in which all of the substituents are equatorially attached. It contains five unique hydro(110) N. Tanako, T. Ashida, Y. Sasada, and M. Kakudo, Bull. Chem. .Sot. Jup., 40, 1739 (1967). (111) C. L. Coulter, S. W. Hawkinson, and H . C. Friedman, Biochirn. Biophys. Acta, in press. (112) K. Bjamer, S. Furberg, and C. S. Petersen, Acta Chem. Scand., 18,587 (1964). (113) T. Dukefos and A. Mostad, Actu Chem. Scand., 19,685 (1965).

CRYSTAL STRUCTURE OF CARBOHYDRATES

93

gen-bonds that link each molecule to six other molecules in the crystal. IX. ENZYME-SUBSTRATE COMPLEXES Lysozyme is an enzyme that hydrolyzes some bacterial cell-walls, the bacterium used for the assay being Micrococcus lysodeikticus. Lysozyme is found in a wide variety of species and locations, including bacteriophages, blood, egg white, gastric secretions, milk, nasal mucus, papaya, sputum, and tears. The outstanding achievement in this field has been the elucidation of the crystal structures of some of the lysozyme-substrate complexes. The successful application of X-ray crystallography of proteins to solution of the structure of lysozyme has been spectacular, but is beyond the scope of this article. However, the enzyme-substrate complexes are of considerable interest, because these substrates and the (structurally similar) inhibitors are carbohydrates. Inhibitor molecules were added to the protein crystals b y cocrystallization, or by adding the inhibitor to the mother liquor containing lysozyme crystals and permitting diffusion of the inhibitors into the crystals."l Seven different inhibitors were studied b y low-resolution crystallography: (a) 2-acetamido-2-deoxy-~-glucose,( b ) Nacetylmuramic acid, ( c ) methyl 2-acetamido-2,6-dideoxy-6-iodo-a-~glucopyranoside, (d) N-acetylbenzylmuramic acid, ( e ) di-N-acetylchitobiose, u)N-acetyl-6-0-(2-acetamido-2-deoxy-~-~-g~ucosy~)n~uramic acid, and (g) tri-N-acetyl-P-chitotriose (19). In general, these Ring A

Ring B

Ring C

Tri- N-acetyl-P- chitotriose

(19)

inhibitors were found in the cleft of the lysozyme molecule. Of these inhibitors, only 2-acetamido-2-deoxy-~-glucoseand tri-N-acetylchitotriose had been studied by high-resolution crystallography (as of 1967), because the high-resolution structures ( 2 A for high resolution (114)C . C. F. Blake, L. N . Johnson, G. A. Mair, A. C. T. North, D. C. Phillips, and V. R. Samia, Proc. Roy. Svc. (London), Ser. B, 167,378 (1967).

94

GERALD STRAHS

as compared with 6 8, for low resolution) require 27 times as many data (-10,000 X-ray reflections as against 400). At a resolution of 2 A, 2-acetamido-2-deoxy-~-glucopyranose binds to lysozyme in two distinct, but related, ways, depending on whether the (Y or p anomer of the sugar is added. In aqueous solution, the anomers are rapidly interconverted, but only the p-Danomer occurs as lysozymal substrate, and this was the anomer used as oligosaccharide inhibitor for X-ray studies. Both anomers of the acetamido sugar form the same type of hydrogen bond through the N-acetyl group, namely, between the oxygen atom and the amino group of residue No. 59 (L-asparagine) and between the nitrogen atom and the carboxyl group of residue No. 107 (L-alanine). 2-Acetamido-2-deoxy-p-~glucose binds as shown in residue C in Fig. 2, 0-3 being directed toward the N H group of residue No. 63 (L-tryptophan), and 0-6being linked to the ring N H of the L-tryptophan residue in position No. 62. The 2-acetamido-2-deoxy-a-~-g~ucose residue is linked in a different orientation to 0-1,which is hydrogen-bonded to the amino group of residue No. 109 (L-valine). The nonpolar (hydrophobic) interactions also contribute to stabilization of the enzyme-substrate complex. The enzyme undergoes an induced fit, amino acid residue No. 62 moving 0.758,; this narrows the cleft (the active site) in the crystal, resulting in better bonding of the lysozyme molecule to the 2-acetamido-2deoxy-D-glucose molecule. These high-resolution results explain some data obtained by low-resolution crystallography. 2-Acetamido-2-deoxy-~-glucose had previously been found to possess a large, diffuse binding-site; this is now interpreted as consisting of the two binding sites for the a-and p-D anomers. Methyl 2-acetamido-2,6-dideoxy-6-iodo-~-glucoside binds only as the p-D anomer, because of steric hindrance between the methyl group and the a - site. ~ Several of the solution results obtained with other inhibitors are now interpreted in a similar way. 2-Acetamido-2-deoxy-~-mannose, a lysozymal inhibitor, can also bind at this site, because the N-acetyl group forms two of the four substratehydrogen bonds to the lysozyme molecule. The most important result, however, is the binding of the trisaccharide tri-N-acetylchitotriose, as the model proposed for substrate binding and enzymic hydrolysis is based on this compound. The three substituted p-D-glucopyranose residues are labeled A, B, and C in Fig. 2. Two observations are the same as for 2-acetamido-2-deoxy-Dglucose: residue C has the same binding as the /3-D anomer, and the resulting shift of amino acid residue 62 (L-tryptophan) is 0.75A. The hydrogen bonds between lysozyme and carbohydrates A and B are shown in Fig. 2 and listed in Table IV. Residue A, which is located

CRYSTAL STRUCTURE OF CARBOHYDRATES

95

FIG. 2. -Atomic Arraiigement in the Lysozyme Molecule in the Neighborhood of the Cleft with a Hexa-N-acetylchitohexaose Molecule Shown Bound to the Enzyme. (The main polypeptide chain is shown speckled, and N H and C O are indicated b y line and full shading, respectively. Sugar residues A, B, and C are as observed in the binding of tri-N-acetylchitotriose. Residues D, E, and F occupy positions inferred from model building. It is suggested that the linkage hydrolyzed by the action of the enzyme is between residues D and E.) [Reprinted from Brookhaven Symp. Biol., 21, 120 ( 1968).I

GERALD STRAHS

96

TABLEIV Binding of Hexasaccharide to L y s ~ z y m e " ~ Saccharide residue A B C

D E

F

Polar interactions NH-Asp 101-0 0-6-Asp 101-0 0-6-Try 62-N 0 - 3 - T v 63-N NH-CO-107 (Ala) CO-NH-59 (Asn) 0-6-CO-57 (Gln) 0-1-Glu 3 5 - 0 0-3-Gln 57 NH-CO-35 (Glu) CO-Asn 44 0-6-CO-34 (Phe) 0-6-Asn 37 0-5-Arg 114-N 0-1-Arg 144-N

Number of non-polar interactions" 7 11 30

35 45

13

'Van der Waals interactions of less than 4di.

at the top of the cleft in the crystal, is hydrogen-bonded to the enzyme (NH and the side chain C0,- of L-aspartic acid No. 101) and hydrogenbonded to saccharide B (0-5 of A and 0-3 of B), and it also has several nonpolar contacts with the lysozyme. Carbohydrate B forms a hydrogen bond with lysozyme (0-6 of the sugar and the oxygen atom of L-aspartic acid, No. 101, as in A) and two intrachain hydrogen-bonds (linking OH-3 of B to 0-5 of A, and 0-5 of B to 0-3 of C). In addition, there are several nonpolar interactions. The binding site of the substrate appears to be a hexasaccharide or a hexasaccharidic part of a polysaccharide. Smaller substrate molecules occupy only part of the active site. Hence, model building was used for adding three more saccharides (D, E, and F in Fig. 2) to the active site; these are listed in Table IV. Crystallographers can work only with crystals of inhibited enzymes and substrates, or with crystalline enzyme and inhibitors; active enzymes cannot be used with the substrate, as the hydrolysis of the substrate is too rapid. Model binding thus bridges a gap between the inhibitor and the substrate complexes of enzyme crystals. The carbohydrate marked D in Fig. 2 was added to the reducing end of the trisaccharide at C, but there were unlikely close contacts by C-6 and 0 - 6 to lysozyme. This overcrowding was relieved by distortion of the chair conformation to a half-chair conformation, with the bond to C-6 axial, as shown in Fig. 2. Carbohydrates

CRYSTAL STRUCTURE OF CARBOHYDRATES

97

E and F were then added to the substrate; they formed hydrogen bonds without distorting their rings. However, E cannot form an intrachain hydrogen-bond to D because of the conformational distortion of the latter. Three of these saccharides have to be N-acetylmuramic acid, and are either A, C, and E, or B, D, and F. The choice is easy, because the hydroxyl group on C-3 of carbohydrate C points toward the cleft and is hydrogen-bonded to the ring-nitrogen atom of L-tryptophan at position NO. 63 of the protein core; hence, there is no space for the lactoyl side-chain of N-acetylmuramic acid. Consequently, B, D, and F are N-acetylmuramic acid residues, and this identification is consistent with the low-resolution results. T h e lactoyl side-chains point out of the cleft, so that there is room for a peptide "tail," such as occurs in the cross-linking of the cell walls of Micrococcus ZysodeiktiCUS.''5

The substrate is known to be hydrolyzed at the /3-0-(1+4) glycosidic link between N-acetylmuramic acid and the oxygen atom remaining on C-4. The bond broken does not connect carbohydrates B and C, as shown both by crystallographic and chemical evidence; therefore, the hydrolysis must occur between carbohydrates D and E. The glycosidic oxygen atom is near to the side-chain carboxyl groups of L-glutamic acid No. 35 and L-aspartic acid No. 52. T h e mechanism proposed for the hydrolysis involves the concept that the L-aspartic acid residue No. 52 is negative and promotes and stabilizes a carbonium ion (CH+)at C-1 of carbohydrate D. The half-chair conformation of D is stabilized by the sharing of the positive charge between C-1 and 0-5, and this weakens the (2-1-0 bond. Then L-glutamic acid (No. 35) acts as a proton donor for the ring-oxygen atom, and the disaccharide EF is released. The hydrolysis can be completed by addition, from a molecule of water, of a hydroxyl group to the carbonium ion, and diffusion of the tetrasaccharide ABCD away from the enzyme. This theory is, indeed, a commencement, as it marks the end of one stage of research, namely, the proposing of a mechanism of hydrolysis, and it should stimulate much more research on all aspects of this mechanism. A preliminary description of the sequence of the amino acids in human lysozyme has been published, and it may be compared with the sequence in hen egg-white lysozyme."fi The human enzyme, which is 2.7 to 3.0times as active as the hen's enzyme, was obtained (115) L. N. Johnson, D. C. Phillips, and J. A. Rupley, Brookhaoen Symp. B i d . , 21,

lZO(1968). (116) R. E. Canfield, Brookhauen Symp. Biol., 21,136 (1968).

98

GERALD

STRAHS

from the urine of a leukemia patient who secreted almost 100 g in one year. In general, the essential amino acids in both lysozymes are, with few exceptions, the same. The structure of human lysozyme as determined by low-resolution experiments resembles that of hen egg-white lysozyme. 11' Bovine a-lactalbumin is one of the two enzymes in lactose synthetase, and its amino acid sequence shows striking similarities to that of lysozyrne.ll8 A model based on the lysozyme model has been built, and the side-chain interactions found are convincing, showing that the model is essentially correct. The active cleft in the crystal is, however, shorter than that in the model, and is consistent with a mono- or di-saccharide as the substrate. Thus, the lysozyme structure may serve as a model for some enzymes that synthesize and hydrolyze carbohydrates. The crystallography of enzyme-substrate (or enzyme-inhibitor) complexes is rapidly expanding, and will probably constitute the subject of a Chapter within a few years.

X. CONCLUSIONS 1. Hydrogen Bonds

Intramolecular hydrogen-bonding between two oxygen atoms in the same monosaccharide is of minor importance in a crystal lattice. Only one example is at present known, namely, in D-ribose (p-bromophenyl)hydrazone.112 Hydrogen bonding between monosaccharide molecules or between the monosaccharide residues in an oligosaccharide molecule is prevalent in their crystal structures. Intermolecular hydrogen-bonding is the primary directing effect in the formation of crystals of simple carbohydrates. In a sugar molecule, the geometry of the carbon and oxygen atoms is fixed or almost fixed, but the hydrogen atom of each hydroxyl group has freedom of motion, and its rotation around the C - 0 bond facilitates occurrence of hydrogen bonding. Formation of hydrogen bonds can also be considered from the thermodynamic point of view. The free energy (G) is defined as G = H - T S , where H is the enthalpy, T is the absolute temperature, and S is the entropy. The enthalpy decreases by hydrogen-bond formation; concomitantly, the entropy is lowered as a result of the ordering by position (arrange(117) D. C. Phillips, personal communication. (118) W. J. Browne, A. C. T. North, D . C. Phillips, K. Brew Vanaman, and R. L. Hill, 1. M o l . Biol., 42,65 (1969).

99

CRYSTAL STRUCTURE OF CARBOHYDRATES

ment) of the hydrogen atoms in the crystal state, provided that no other changes occur; the decrease in change in enthalpy must be greater than that of the entropy term (TAS) for crystals to form. Thus, it is apparent from the free-energy changes (AG) that crystallization of a sugar is favored at lower temperatures, and that, normally, crystals become more soluble in water as the temperature is increased. A notable feature of carbohydrate crystals is the absence (or small proportion) of water of hydration. As monosaccharides are hydrophilic and water-soluble, considerable deposition of water (association) in a sugar crystal may be expected. The absence of water from the crystal lattice of a sugar must, therefore, be explained by supposing that the sugar is able to form sufficiently strong and numerous hydrogen bonds without the involvement of molecules of water; this has been observed, although there are some exceptions. This lack of water of hydration may also be observed when the structural arrangement of the individual hydroxyl groups in hydrogen bonding is considered. In many crystal structures, each hydroxyl group may have four nearest neighbors, arranged tetrahedrally, with a covalent bond directed to the carbon atom and sharing a hydrogen atom in a hydrogen bond; simultaneously, it accepts two hydrogen bonds from two hydroxyl groups nearby. In the absence of water of crystallization, a typical hydroxyl group in a carbohydrate molecule has only three nearest neighbors, and, consequently, it can form only one hydrogen bond in the crystal. The length of a hydrogen bond is usually defined as the distance between the center of the oxygen donor and the center of the oxygen acceptor atoms, because of the uncertainty as to the location of hydrogen atoms as measured by X-ray analysis. Some idea as to the length of hydrogen bonds and the variability in their length may be obtained by considering three crystal structures, namely, those of a-D-glucopyran~se,~ P-D-glucopyranose,*O and cellobiose.1° These three structures will be used, because they have been measured precisely. The lengths and variability of some typical hydrogenbonds are listed in Table V. TABLEV Hydrogen Bonds in Three Carbohydrates Sugar

Number of hydrogen bonds

Range (A)

Average (A)

References

a-D-Clucopyranose P-D-Chcopyranose Cellobiose All three

5 4 8 17

2.71-2.85 2.67-2.77 2.71-2.81 2.67-2.85

2.76 2.71 2.77 2.76

9 10 10

GERALD STRAHS

100

Some of the oxygen atoms in a sugar molecule do not accept hydrogen bonds; for example, none of the glycosidic oxygen atoms in cellobiose,’* methyl @-maltopyrano~ide,~~ and raffinose60 accept hydrogen bonds. No examples are known for hydrogen-bond formation involving the ring-oxygen atoms of glycofuranosides and methyl g l y c o p y r a n ~ s i d e s Zhdanov .~~~ and coworkerslZ0calculated that the ring-oxygen atom in an aldopentofuranose has a charge of -0.2642 electron (erroneously given the plus sign in the reference cited); the ring-oxygen atom in an aldopentofuranose actually has a charge of -0.2631 electron, and each hydroxyl oxygen atom has a charge of -0.47 electron (ranging from -0.463 to -0.475 electron). The charge of -0.26 electron cannot by itself explain the lack of hydrogenbond formation to the ring-oxygen atom of glycofuranosides, especially as the ring-oxygen atom in pyranoses, having the same charge, can accept a hydrogen bond. However, the ring-oxygen atom of pyranoses usually accepts equatorial hydrogen-bonds only. The results of quantum-mechanical calculations apply only to pentoses; they are in the right direction for, but not strictly applicable to, pentose derivatives or higher sugars. Crystallographic results indicate a decreased negative charge for the anomeric oxygen atom, and for the ring-oxygen atom in glycofuranosides and methyl glycopyranosides. Two calculations of the net charge on individual atoms in carbohydrates are tabulated in Table VI. The two methods of calculation TABLEVI Charge (in Electrons) on Atoms in Carbohydrates Carbohydrate

Methine proton

“ru”-D-Glucoisosaccharinic“ acid36 +0.11 to-tO.14 Aldopentopyranose’*” +0.05 to +0.06

Hydroxyl proton $0.64

+0.32

Carbon

Oxygen

-0.27 t0-tl.71 -1.02 to-1.25 +0.03 to +0.19 -0.46 to -0.47’

“3-Deoxy-2-C-(hydroxymethyl)-~-erythro-pentonic acid. ’Pyranose ring-oxygen atom, -0.26.

are respectively based on the extended Huckel theory36and on the LCAO-MO (linear combination of atomic orbitals-molecular orbital) method.’2aThe protons and oxygen atoms have positive and negative charges, respectively. One of the six carbon atoms (C-3) of “ a ” - ~ (119) M . Sundaralingam, Biopolymers, 6, 189 (1968). (120) Y. A. Zhdanov, V. I. Minkin, Y. A. Ostroumov, and C . N. Dorofeenko, Carbohyd. Res., 7, 156 (1968).

CRYSTAL STRUCTURE OF CARBOHYDRATES

101

glucoisosaccharinic acid lacks a hydroxyl group, and it is the only negatively charged carbon atom therein. In aldopentopyranoses, C-5 has a similar environment to C-3 in that acid, and it has only a small positive charge (+0.04electron). The only large positive charge for a carbon atom (+1.71) is that on the carboxyl group. When these three carbon atoms are omitted, the eight other carbon atoms have charges ranging from +0.10 to +0.63 electron. That the equatorial orientation is favored over the axial in the formation of hydrogen bonds to the oxygen atom of the pyranose ring cannot be well explained by a lack of sufficient negative charge or by simple steric considerations. However, the charge can be considered to be distributed over the four tetrahedral, hybrid orbitals around the oxygen atom, two of which are directed along the C - 0 bonds, the other two orbitals being respectively axial and equatorial to the pyranose ring. The equatorial orbital has a greater electrondensity than the axial orbital, explaining the bonding to the ringoxygen atom b y equatorial hydrogen-bonds. Steric factors also contribute to an explanation of equatorial hydrogen-bond formation, and they may explain the difference in electron density. When a hydrogen atom approaches the ring-oxygen atom of a pyranoside, there seems to be free access to the oxygen atom from either the axial or the equatorial direction. As axially attached substituents are usually positively charged protons (+0.05 electron), an incoming proton is repelled. 2. Conformations All of the crystalline pyranoses thus far examined adopt a chair conformation. A boat conformation has not yet been found for crystalline monocyclic compounds of sugars. Fused-ring systems seem to be required for part of the molecule to adopt a boat form, as in sedoheptulosan ( 5 ) (where a chair form is also a part of a boat form (fused to the boat form)27and 1,6-anhydro-/3-~-glucopyranose. A half-chair conformation of a crystalline monosaccharide has not been observed. A half-chair conformation for the fourth 2-acetamido2-deoxy-P-D-glucopyranosylresidue (residue D) in the lysozyme substrate has not been detected, although, on the basis of model fitting, its presence has been suggested (see p. 96). 2-Acetamido-2-deoxyp-D-glucosyl groups were added to a molecular model constructed by use of data obtained from the nature of the enzyme-trisaccharide complex; it was implicit that the lifetime of the half-chair conformation would he quite short.

102

GERALD STRAHS

On the other hand, flattened chair conformations have been found more often than not in crystals of sugar derivatives. The degree of flattening can be obtained by consideration of the dihedral angles. In an ideal chair conformation, the dihedral angles are 60 and 180" respectively, whereas a flat chair or planar ring has dihedral angles of 0 and 120". In 1,2-O-(aminoisopropylidene)-a-D-glucopyranose hydriodide3* (1Oa and lob), the non-hydrogen dihedral angles are 16, 12, 90, 24, 69, 50, 55, and 66" (all *3"), whereas 60" is the angle for an ideal chair conformation; the pyranoid ring is constrained by the cyclic acetal group. Information about the conformation of a pyranoid compound may also be obtained by crystallography. The usual form, that is, CI (D), 1C (L), has been observed for the structures of most crystaIIine sugars, but the alternative form, that is, 1C (D), C1 (L), has also been observed, although rarely. Crystallography is used for determining, not for predicting, the conformation of a carbohydrate. Predictions can, however, be made by considering the conformational free-energy as being the sum of the interaction energies between nonbonded atoms; the results of such estimations are in agreement with crystallographic results. A question arises as to the relationship between the structure of a compound in solution and that existing in the crystals. The structure in the crystal has always been found to agree with the structure of one of the components in solution (as deduced by n.m.r. spectroscopy, for example). A fundamental difference exists between the structure of a reducing sugar in the crystal state and in solution, in that the solution contains an equilibrium mixture of several tautomers and conformers, whereas the solid usually contains one form. Mixtures of anomers, C1 and 1 C conformers, and pyranose, furanose, acyclic, and anhydride forms are known to coexist, although some of these forms are present in insignificant proportions.121Of the various forms present, the process of crystallization favors one form, and this form is not necessarily the form that preponderates in solution; for a discussion of D-glucose, see p. 63. A few examples of the C1 (L), that is, the 1C (D) conformation have been described. /3-D-Arabinopyranose and its enantiomorph , ' ~illustrated in formula crystallize in this conformation ( l u 2 e 3 e 4 ~ )as 20 for the / 3 - compound. ~ The C1 (D) conformer is shown, for comparison, in formula 21. As may be seen, neither form has any large nonbonded interactions. Both forms have o n e axial hydroxyl group on (121) For background information on mutarotation of carbohydrates in solution, see W. Pigman and H. S. Isbell, Aducin. Curbohud. Chem., 23, 11 (1968), and H. S. Isbell and W. Pigman, ibid., 24, 13 (1969).

CRYSTAL STRUCTURE OF CARBOHYDRATES

P-D-Arabinopyranose-lc

P-D-Arabinopyranose-Cl

( l a 2e 3e 4 a ) (20)

(le2a3a4e)

103

(21)

each side of the pyranose ring and two equatorial hydroxyl groups. Angya1'22 has calculated that there is a difference of only 0.5 kcal per mole between the 1C (D) and C1 (D) forms, the 1C (D) form being the more stable. For a-D-arabinopyranose, the 1C (D) conformer is (by 1.15 kcal per mole) more stable than the C1 (D) form. In the C1 (D) form, 0-1and 0 - 3 are both attached axially on the same side of the ring and have a large, nonbonded, interaction energy. Another compound that crystallizes in the 1C (D) form is the 1-(hydroxymethyl) derivative of P-D-arabinopyranose, namely, ~-D-fructopyranose.lsThe 1C (D) form 22 has the bulky hydroxymethyl group equatorial, and the axial hydroxyl groups, on C-2 and C-5, are on opposite sides of the ring. The C1 (D) form (23) has a

(3 -D - Fructopy ranose- IC

( l e 2 a 3e4e 5 0 ) (22)

p-D-

Fructopyranose-C1 (laZe3a4o5a) (23)

large, nonbonded, interaction energy between the primary alcohol group on C-2 and the hydroxyl group on C-4. P-D-Psicopyranose, not yet crystallized, would be the only other 2-ketohexose that would be expected to crystallize in the 1C (D) form. The pyranoid part of sedoheptulosan monohydrate ( 5 ) has the C1 (L) conformation, and of 1,6-anhydro-/3-~-glucopyranose,the 1 C (D) conformation. The predictions for the conformations of pyranoses agree surprisingly well with the conformations actually existing in their crystals, despite the fact that the pyranoid rings are always somewhat distorted in the crystal. Perhaps the structure in solution is closer to the ideal (122) S. J. Angya1,Aust.J. Chern., 21,2737 (1968).

GERALD STRAHS

104

shape, or appears to be ideal, because of the apparent averaging of several similar conformers over a finite time-interval. The pyranoid ring is quite flexible and can be distorted considerably. The conformation that a furanoid compound will adopt in the crystal is not readily predicted. A furanoid compound can adopt one of four envelope conformations in which C-2 or C-3 is endo or exo, namely, 2E, E2, ,E, and E,; less commonly, it may adopt a twist conformation. The energy difference between the four common envelope conformations may be so slight that other effects predominate in determining the conformation that the compound will adopt, both in the crystal and in solution. Such effects include those caused by hydrogen bonding, solvation, and close contacts with an aglycon.

3. Nonbonded Interactions (Repulsions) As has already been pointed out (see p. 59), the carbon chain in ribitol’? is nonplanar, and the molecule adopts a “sickle” conformation.18 This conformation is just one of the conformational changes that can be directly attributed to interaction (repulsion) between nonbonded atoms. Were ribitol planar, the contact between 0 - 2 and 0 - 4 would be close, as shown in Fig. 3 . The C - 0 bonds would be 0-2

0-4 I

FIG. 3.-Distance Between 0 - 2 and 0 - 4 in the Planar, Zigzag Conformation of Ribitol.

parallel and coplanar, making the 0-2-0-4 distance identical with the C-2-C-4 distance. As calculated from the trigonometric formula (1.532 1.532- 2(1.53)(1 . 5 3 ) ~ 0 ~ 1 1 3 ”the )*~~ latter , distance equals 2.55 A; this value is quite important, because it is applicable to other atoms (such as hydrogen, nitrogen, oxygen, and halogen), and will vary little with small changes in the bond lengths and angles. PaulingIz3 gives the Van der Waals radius for the hydrogen atom as 1.2 A; for nitrogen, 1.5 A, for oxygen, 1.4 A; and for a methyl group, 2.0 A. These radii are, however, only approximations or guidelines;

+

(123) L. Pauling, “The Nature of the Chemical Bond,” Cornell University Press, Ithaca, N. Y., 3rd Edition, 1960, p. 260.

CRYSTAL STRUCTURE OF CARBOHYDRATES

105

thus the only “good’ contact between hydrogen atom and hydrogen atom occurs where the sum of the Van der Waals radii is 2.4 A. A hydrogen-oxygen contact of 2.6 A could be accommodated by small changes in any or all of three angles. Jeffrey and Kim have reviewed the conformation of a l d i t ~ l s , ’and ~ ~ predicted the conformation adopted in the crystals of 3 pentitols, 6 hexitols, and several octitols. They formulated a rule according to which the carbon chain of the alditol adopts a planar, zigzag conformation when all pairs of alternate, acentric carbon atoms have different configurations, but is nonplanar if the configuration is the same for any one pair of alternate acentric carbon atoms. As for ribitol formulas 1 and 2 (see p. 60),one way to relieve the strain can be achieved specifically by rotation about the C-3C-4 bond. A similar, symmetrically related rotation about the C-2-C-3 bond, which would move 0 - 2 out of the plane of interaction, gives the mirror image of the ribitol molecule as shown in formula 2a. The OH

H‘..,,* I

I‘

*. ,’,H

Hoe

I

’\

‘H

\

H,’ ‘

OH

HO‘ Ribitol (sickle conformation)

Ribitol (sickle conformation)

(2)

@a)

crystal of ribitol actually contains both of these conformers (2 and 2a). A most interesting example is D-glucitol, for which two distinct ways are conceivable for relieving this strain, as shown in Fig. 4. A rotation of 120”about the C - 2 4 - 3 bond relieves the strain, as shown in Fig. 4(b). The contact between oxygen atoms can also be eliminated by a rotation of 120” about the C-3-C-4 bond and b y a second rotation about the C-4-C-5 bond (see Fig. 4c). These conformations have lower energy states than the planar form, and should be the most abundant species in solution, with the terminal hydroxymethyl groups rotated to avoid close contacts. This rule for alditol conformations can be extended to acyclic sugars, or acyclic portions of cyclic sugars.I8 However, in the crystalline state, the D-gluconate ion is considered to have an extended chain of carbon atoms.*25 (124) G . A. Jeffrey and H . S. Kim, C ~ r b o h y dRes., . 14, 207 (1970). (125) c.D. Littleton, Acto C r y s t d o g r . , 6,775 (1953).

106

GERALD STRAHS

HO

HOh

H

, , i HO H

FIG.4. -Conformations of D-GIUCitOl. (a, Planar; b, nonplanar, after rotation about C-2-(2-3; c, nonplanar, after rotation about C-3-C-4 and C-4-C-5.)

The consequences of a nonbonded oxygen repulsion are clear in P-D-glucopyranose, which has all substituents equatorial in the C1 (D) conformation (formula 4, p. 63). The 1C (D) conformation (24) has CH,OH

I

OH

OH ,3- 0-Glucopyranose- I C (24)

all substituents axially attached, with 0-1, 0-3, and the hydroxymethyl group above the pyranose ring, and 0-2 and 0-4below the ring; thus, it has 4 pairs of too-close contacts. Angya1lZ2estimated an unfavorable energy difference of 5.95 kcal/mole for the change from C1 (D) to 1C (D) for P-D-glUCOpyranOSe. As expected, this corresponds to one of the most unfavorable C1 (D) to 1C (D) transitions (for the Daldopentopyranoses and o-aldohexopyranoses), precisely because of the interaction between nonbonded atoms on alternate carbon atoms.

CRYSTAL STRUCTURE O F CARBOHYDRATES

107

Jeffrey and Kim'24 gave 3.30 A as the distance between the axially attached 0 - 2 and 0 - 4 in 1,6-anhydro-/3-~-glucopyranose.This increased separation occurs, however, not only because nonbonded repulsion flattens the ring but also because of the formation of the anhydro ring. The earlier article' on the crystallography of carbohydrates described its infancy, and the present one covers its adolescence. The lack of maturity is attributable to the scarcity of interdisciplinary studies and correlations. The coming of age of carbohydrate crystallography will occur when widespread use is made of conformational studies, reaction mechanisms, and the charge distribution, the geometry and the electron density being determined crystallographically.

XI. ADDENDA

A half-chair conformation (H:) has been found for crystalline Dglucono-1,5-lactone,'26 and the conformation of crystalline 1,2:4,5-diO-isopropy~idene-/3-~-fructopyranose~~*~~~ is intermediate between 1C and H i . A mixture of the anomers (a:p= 2: 1)occurs in crystalline methyl 2-chloro-2-deoxy-a- and /3-D-galactopyranosides.'28 Presence of a seven-membered ring has been verified in crystalline 5 - 0 (chloroacety1)-1,2:3,4-di-O-isopropylidene-a-~-glucoseptanose.~~~ The crystal structures of two additional seven-carbon sugars have been published, namely, those of a-coriofuranose ( ~ - n l t r o - 3 - h e p t u l o s e ) ~ ~ ~ and D-rnanno-3-heptulose m ~ n o h y d r a t e . ' ~The ' crystal structures of three disaccharide derivatives have been elucidated, namely, those of a-lactose m ~ n o h y d r a t e ,methyl '~~ P - c e l l ~ b i o s i d e , and ' ~ ~ a new trianhydrosucrose, 3,6-anhydro-a-~-glucopyranosy~1,4:3,6-dianhydro-p-D-fructofuranoside. 134 (126) M . L. Hackertand R. A. Jacobson, Chern. Commun., 1179 (1969). 7 (127) S . Takagi and R . D. Rosenstein, Acta Crystaltogr., A25 [S3], ~ 1 9 11969). (128) R. Hoge and J . Trotter,]. Chem. Soc. ( A ) ,2170 (1969). (129) J . Jackobs and M . Sundaralingam, Chem. Commrrn., 157 (1970). (130) T. Okuda, K. Osaki, and T. Taga, Chem. Corrzmur~.,851 (1969);T. Taga, K. Osaki, and T. Okuda, Acta Crystullogr., B26,991(1970). (131) T. Taga and K. Osaki, Tetrohedron Lett., 4434 (1969). (132) D. Fries, S.T. Rao, and M . Sundaralingam, Amer. Crystatfogr.Ass. Abstract,&0-6 (1970). (133) J . T . Ham, Ph.D. Thesis, Institute of Paper Chemistry, Appleton, Wisconsin (1969);Dissertatiori Abstracts, 30B, 1068 (1969). (134) N. W. Isaacs, C. H. L. Kennard, G . W. O'Donnell, and G. N. Richards,]. Chem. SOC.( D ) ,360 (1970); Chem. Abstracts, 72, 126,077k (1970).

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OXIRANE DERIVATIVES OF ALDOSES BY NEIL R. WILLIAMS Chemistry Depcirtment, Birklxdi College, Unioersit!y of Loiidorl, Malet Street, London W. C. 1 , Englond I. Introduction . . . . . . . 11. Synthesis

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

1. General Methods. . 2. Other Methods. . . . . . . . . . . . 111. Reactions . . . . . . . . . ..................... 1. Stability of the Oxi 2. Stereochemical Features. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. 1,6:2,3- and 1,6:3,4-Dianhydrohexopyranoses. 6. 2,3-Anhydro-4,6-0-benzylidenehexopy 7. 2,3- and 3,4-Anhydrohexopyranosides.

.....

109

120

. . . . . . . . . . . 131 . . . . . . . . . 134 . . . . . . . . . 141

10. 5,6-Anhydrohexofuranoses. . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Miscellaneous Oxiranes. ................................ IV. Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Spectroscopic Methods. . . . . . . . . V. Tables of Oxirane Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

163

172

I. INTRODUCTION Oxirane derivatives of aldoses were last reviewed in this Series by Peat' in 1946, as part of a chapter on anhydro sugars. It is no longer possible to cover such a broad field within the confines of a single article, and this review is concerned only with the oxirane derivatives of aldoses, excluding ketoses, alditols, and cyclitols. Even within this narrow field, little attention will be given to 1,2-aiihydro compounds, typified by the Brig1 anhydride, as they behave differently from other aldose oxiranes, and little new work has been done on their chemistry beyond their use in the synthesis of glycosides. (1) S. Peat, Adoan. Carhohyd. C h e m . , 2, 38 (1946).

109

110

NEIL R. WILLIAMS

Carbohydrate oxiranes are also commonly called epoxides, and, although the latter is a less specific term, both terms will be used in this article. The nomenclature system is based on the anhydride principle, the oxirane being considered as derived from the aldose of corresponding configuration by removal of the elements of water, but, in order to avoid confusion with carbohydrates containing larger ether rings, the general term anhydride or anhydro sugar will not be used. The great interest shown in aldose oxiranes stems from their value as intermediates in the synthesis of a variety of modified sugars, including deoxy, amino, thio, and branched-chain sugars, and the rarer simple sugars. The stereochemistry of their reactions has posed problems of continuing interest from the theoretical viewpoint, and attention will be focused on this aspect of their chemistry. The methods available for their synthesis, with special reference to their ease of formation, will be discussed, together with their reactions (considered generally, and then in more detail under the various classes of aldose oxirane that have been studied) and the methods available for their characterization. The extensive investigations that have been made since Peat’s article’ have been discussed in several review papers and notably in one by N e ~ t hOther . ~ comprehensive articles on oxiranes have paid some attention to carbohydrate representative^.^,^

I I . SYNTHESIS 1. General Methods The standard method for the formation of the oxirane ring in carbohydrate compounds is the treatment with alkali of sulfonic esters having vicinal, trans-hydroxyl groups that are free or esterified. Methanesulfonic or p-toluenesulfonic esters are commonly used, the adjacent hydroxyl group being unsubstituted, or acetylated, benzoylated, or sulfonylated. The mechanism involves displacement of the sulfonyloxy group by rearside attack of the adjacent oxide anion, an s N 2 process requiring the anti-periplanar orientation for the sulfo(2) S. J. Angyal, Chem. Ind. (London), 1230 (1954); W. G. Overend and G. Vaughan, ihid., 995 (1955); G. Huber and 0. Schier, Helo. Chim. Acta, 43, 129 (1960). (3) F. H . Newth, QuaTt. Reu. (London), 13, 30 (1959). (4) R. E. Parker and N. S. Isaacs, Chem. Reo., 59, 737 (1959). (5) A. Rosowsky, in “The Chemistry of Heterocyclic Compounds,” A. Weissberger, ed., Interscience, New York, N . Y., 1964, Vol. 19, Part I, p. 1.

OXIRANE DERIVATIVES OF ALDOSES

111

nyloxy group and the oxide anion, and leading to inversion of configuration at the substitution enter.^

I

YR'

-c-cFtSO(

A '

-

I

@O

-c-cI

@O

-,,C-c\, : \ /

0

-,c-c

,I \

/ \

t

rS0io

where R ' = H, RCO, o r WO,

I

W0,O

.

RSOP

In a preliminary step, adjacent ester groups undergo initial hydrolysis or alcoholysis, by the base, to the oxide anion. This step is reasonable for carboxylic esters, which are rapidly hydrolyzed under the conditions usually employed, but is rather more surprising for sulfonic esters, which are usually hydrolyzed only slowly by the nucleophilic attack on sulfur required for this reaction6 With these disulfonic esters, the ease of hydrolysis has been explained by the inductive effect of the adjacent sulfonyloxy group.'

No problems arise where either group is primary, but, where both are secondary and attached to a furanoid or pyranoid ring, a trcms configuration is essential, and a conformation must be available that allows the two groups to approach an cinti-periplanar orientation to each other. The rather wide range of conditions needed for satisfactory yields of epoxide from different pyranoid sulfonic esters may be rationalized by considering the ease with which the required conformation can b e adopted, together with the influence of polar effects that affect the amount of energy required for reaching the transition state. Sodium methoxide in methanol, often with chloroform as cosolvent, has customarily been the basic reagent employed. Less frequently, particularly with water-soluble esters, sodium or potassium hydroxide in aqueous solution has been used. Generally, an excess of the basic reagent is taken, except where the possibility of epoxide migration arises (see p. 127). In the latter situation, only a limited excess of reagent is used, at low temperature, or, alternatively, the (6) R. S. Tipson, Advan. Cafbohyd. Chern., 8, 207 (1953). (7) S. J. Angyal and P. T. Gilman,]. Cheni. SOC., 3691 (1957).

NEIL R. WILLIAMS

112

reaction mixture is titrated with the base in the presence of an alkali indicator, such as phenolphthalein.8-10 In conformationally mobile systems in which the required antiperiplanar arrangement is readily achieved (even though this may not be the favored conformation), the reaction is usually complete within hours at 0-25", or after a few minutes at the reflux temperature. Thus, 1,2-O-isopropy~idene-6-O-p-to~y~su~fony~-a-~-g~ucofuranose ( 1) affords 5,6-anhydro-l,2-O-isopropy~idene-a-~-glucofuranose ( 2 ) within TsOCH,

HOCH I

,CH2 Oh!H

0

0

qy - Q? 0,CMe2

0,CMe2

(21

(1)

minutes at room temperature." Similarly, 6-O-benzoyl-1,2-O-isopropy~idene-5-O-p-to~y~sulfonyl-a-~-glucofuranose yields12 5,6-anhydro-1,2-O-isopropylidene-p-~-idofuranose within 2.5 hours at 0". 1,6-Anhydro sugar derivatives that have the two reacting groups held in the required anti-periplanar (diaxial) orientation afford oxiranes very readily. Thus, 1,6-anhydro-2-O-p-toly~su~fonyl-~-D-g~ucopyranose (3) yields 1,6:2,3-dianhydro-p-~-mannopyranose (4) within

OTs (3)

10 minutes at room temperature with M sodium n i e t h 0 ~ i d e .The l ~ ease with which this reaction occurs suggests, incidentally, that the inductive effect of the acetal group at C-1 does not greatly retard substitution at C-2. However, a comparative study of the rates of reaction of 3 and its 3- and 4-0-p-tolylsulfonyl analogs with 5 mM sodium hydroxide gavei4 the ratios of 1:180:23, which indicates that some retar(8) G. Charalamlious and E. Percival, J . Chem. Soc., 2443 (1954). (9) J. G. Buchaiian and J. C. P. Schwarz, J . Chem. Soc., 4770 (1962). (10) J. Jar$ and K. Capek, Collect. Czech. Chem. Commun., 31, 315 (1966). (11) H . Ohle and L. Vargha, Rer., 62,2435 (1929). (12) A. S. Meyer and T. Reichstein, Helu. Chim. Acta, 29, 152 (1946). (13) M . Cern$, J . Padk, and J. Stankk, Collect. Czech. Chem. Commun., 30, 1151 (1965). (14) M. Cern9, J. StanSk, and J. Pacik, Collect. Czech. Claem. Commun., 34,849 (1969).

OXIRANE DERIVATIVES OF ALDOSES

113

dation may be attributed to this effect, although a polar interaction between the oxide anion at (2-3 and the anhydro-bridge oxygen atom seems a more likely explanation for the slowness of the reaction of 3. A similar interaction between the sulfonyloxy group at C-3 and the bridge oxygen atom, aiding the expulsion of the ester group, would account for the speed of the reaction of the 3-0-p-tolylsulfonyl isomer, which gives a 7:3 mixture of 1,6:3,4- and lY6:2,3-dianhydroD-allopyranose. If the reacting groups are diequatorially arranged in a 1,6-anhydro sugar, much more vigorous conditions become necessary. 1,6-Anhydro-3-O-p-to~ylsulfonyl-@-D-altropyranose ( 5 ) requires reflux temperatures for several hours in order to afford 1,6:2,3-dianhydro-P-~mannopyranose (4), which rearranges under these conditions to give

h0 V

HO (5)

1,6:3,4-dianhydro-@-~-altropyranose (see p. 127). Here, an unstable, skew conformation ( S ; ) must first be adopted before reaction can O C C U ~ . ~The ~ , ~ 2-0-p-tolylsulfonyl ~ isomer of 5 fails to react, even under these condition^,'^ and this behavior may be attributed to an even more severe interaction between the bridge oxygen atom and the 2-sulfonyloxy group of the skew conformation. The i n e r t n e ~ s ' ~ of the 3,4-di-O-~1-tolylsulfonylanalog of 5 has been attributed by Newth3 to an additional, passing interaction between the two sulfonyloxy groups on changing from the chair to a boat conformation. However, this behavior is better understood as due to the partial eclipsing of these groups in the resulting skew form, an interaction, both steric and polar, which becomes emphasized in the transition state leading to the 4-0-p-tolylsulfonyl analog of 4. Skew conformations must also be involved in the reaction of 4,6-0-benzylidene-~-glucopyranoside derivatives, in which the trails ring junction again prevents the alternative chair conformation ( lC4) from being adopted. Reaction is easier than with the 1,6-anhydro-~altrose derivatives already discussed, but, again, significant differences in rate are found between displacement of sulfonyloxy groups on C-2 and C-3. Whereas methyl 4,6-0-benzylidene-2-O-p-tolylsulfonyl-a-D-glucopyranoside ( 6 ) appears to require reflux tempera(15) F. H. Newth,/. Chem. SOC.,441 (1956).

NEIL R. WILLIAMS

114

-

* o ,

*;p h f

OMe

/c Ts

00

TsO

OM e

/

tures,I6 leading to the D - ~ U W O epoxide (7), the 2,3-di-O-p-tolylsulfonyl-D-glucoside (8) reactsI7 at 0" to give the a-~-uZloepoxide (9). The skew conformation required for the D - ~ U T L ~epoxide W possesses an interaction between the methoxyl group on C-1 and the p-tolylsulfonyloxy group on C-2 that will be emphasized in the transition state as atoms C-1 to C-4 move towards coplanarity, eclipsing these groups; such an interaction is absent from the transition state for the (16) G . J. Robertson and C. F. Griffith,/. Chern. SOC.,1193 (1935). (17)N. K. Richtmyer and C. S. Hudson, J. Amer. Chern. SOC., 63,1727 (1941).

OXLRANE DERIVATIVES OF ALDOSES

115

D-UZZO epoxide. The same unfavorable interaction could also account for ( a ) the much lower rate of reaction of methyl 6-deoxy-2-0-p-tolylsulfonyl-a-D-ghcopyranoside as compared with that of its 4-0-p-tolylsulfonyl isomer,'O as a similar situation will arise in the transition state, even though the alternative chair form ('CJ may initially be adopted, instead of the skew form, and ( b )the much greater reactivity of methyl 4,6-0-benzylidene-2-O-p-tolylsulfonyl-~-~-galactopyranoside (10 minutes at 00) as compared to that of its anomer (no reaction after 24 hours).la Newth3 has interpreted these differences in terms of passing interactions, between the sulfonic ester group and the methoxyl group, that raise the energy barrier to the interconversion of the conformers. However, differences in the rate of reaction more properly reflect differences in energy of the transition states, and the different rates observed are better understood in terms of the presence or absence of interactions in the transition state for reaction. The interaction may be both steric and polar in origin (if, indeed, these effects can be clearly separated from each other), and R i c h a r d ~ o n has ' ~ stressed the importance of polar interactions from adjacent, axial, polar groups in the transition state for sN2 displacement of sulfonic esters by azide ions, where eclipsing interactions arise either with the incoming nucleophile or the outgoing sulfonic ester group. A similar effect can occur in epoxide-ring formation, where the sulfonic ester group is cis to an adjacent methoxyl or other polar group.

+

'OTs

It is of interest that methyl 4,6-0-benzylidene-2,3-di-O-p-tolylsulfonyl-a-D-galactopyranoside, epimeric with 8 at C-4, is unreactive at room temperature, in contrast to 8; this behavior may be attributed to the 3-OTs-4-0 interaction arising in the transition state. On refluxing, displacement of the 3-sulfonyloxy group occurs, to give the D(18) L. F. Wiggins, J . Chem. SOC., 522 (1944). (19) A. C. Richardson, Carbohyd. Res., 10, 395 (1969).

116

NEIL R. WILLIAMS

gulo epoxide.20*21 Although, in principle, this compound has the of the opportunity to adopt the alternative chair conformation (T4) pyranoid part, because the ring junction is cis, this possibility does not appear to offer any advantage; this might suggest that the skew form is still adopted. Of course, in the alternative chair form (C4), the benzylidene ring will also be inverted, making the phenyl group syn-axial with C-3, a highly unfavorable arrangement. For furanoid derivatives, 2,3-epoxides are readily formed from trans-related groups. Other stereochemical features do not appear to be important, and this reflects the mobility of the orientations on such five-membered rings. Here, eclipsing polar interactions between cis sulfonate and methoxyl groups are not likely to alter much on passing to the transition state; and, accordingly, both anomers of form the 2,3-anhydromethyl 2-O-methy~sulfonyl-~-xy~ofuranoside D-lyxofuranoside on treatmentz2with sodium methoxide at 0". One further effect would seem to permit rationalization of a number of apparently anomalous results observed in the reaction of disulfonate compounds to give epoxides. In the preliminary hydrolytic step, a sulfonate group flanked by two equatorial groups is less readily hydrolyzed than one flanked by at least one axial group. This behavior may be regarded as a steric inhibition of hydrolysis; although this explanation was rejected by Dick and Jonesz3as an adequate way of interpreting their results with a series of 4-azido-4-deoxy-2,3-di-Omethylsulfonyl-a-U-pentopyranosides, it does seem to be plausible (from a study of models), and does explain their results. The explanation would account for the fact that, whereas methyl 4,6-0-benzylidene-2,3-di-O-p-tolylsulfonyl-a-~-galactopyranoside (10) gives mainO (ll),its anomer (12) gives the ~ - t &epoxide ly the D - ~ U ~epoxide (13),20and also for the fact that the anomer of the D-glucoside derivative 8 is quite unreactive at room temperatureaz4Dick and Jonesz3 explained their results by considering the relative stabilities of the two possible transition states that would lead to epoxides, depending on which sulfonic ester group is hydrolyzed first, but, for this stability to govern the ratio of epoxides formed, either ( u ) transesterification must occur, in order to maintain the balance of unhydrolyzed di-ester, (20) E. Sorkiri and T. Reichstein, Helti. Cliim. Acta, 28, 1 (1945). (21) H . Huller and T. Reichstein, Helti. Chim. Actci, 31, 1645 (1948). (22) B. R. Baker, R. E. Schaub, and J. H. Williams,./. Amer. Chem. S O C . , 77.7 (1955). (23) A. J. Dick and J. K. N . Jones, Can. J. C h e n . , 44, 79 (1966). (24) E. J. Hedgley, R. A. C. Rennie, and W. G. Overend, ./. Claeni. SOC.,4701 (1963).

OXIRANE DERIVATIVES OF ALDOSES

TsO

117

TsO

OMe

a possibility that they were unable to substantiate, or ( b )the hydrolysis step and the epoxide formation must be concerted, a possibility that seems unlikely, because partial hydrolysis of the di-0-p-tolylsulfonyl-D-glucoside (8) gives a mixture of the D-UZZO epoxide and the 3-O-p-to~y~sulfony~-D-g~ucoside, as expected for a stepwise mechanism involving preliminary hydrolysis of the 2-p-toluenesulfonic ester group.25 Methyl 4,6-0-ethylidene-2,3-di-O-p-tolylsulfonyl-~-~-glucopyranoside affords the D-UZZO epoxide as readily as the benzylidene analog,26 contrary to an earlier report of its stability at room temperat~re.~' Nitric esters also behave similarly to sulfonic esters in leading to epoxide~,~' but the reactions are preparatively less satisfactory, and have not been exploited.

(25) J. Honeyman and J. W. W. Morgan, J . Chem. SOC., 3660 (1955). (26) P. M. Collins, unpublished observations. (27) E. G . Ansell and J. Honeyman, J . Chem. SOC., 2778 (1952).

118

NEIL R. WILLIAMS

2. Other Methods a. From Deoxyhalo Sugars. -With alkali, the bromo-, chloro-, and iododeoxy sugars react analogously to sulfonic ester derivatives, with the same stereochemical limitation, readily affording epoxides in good yield.2B-30The reaction is of little preparative importance, because the deoxyhalo sugar is usually derived from the epoxide, but it has been used in establishing the trans relationship of adjacent halogen and hydroxyl groups. Thus, the 4-chloro-4-deoxypentoside obtained from methyl D- or L-arabinopyranoside with sulfuryl chloride must be the D- or L - X ~ ~ isomer, O because, with alkali, it affords an epoxide which, on acid hydrolysis, yieIds a Iyxose and a x y l o ~ e . ~ ‘

b. From Amino Sugars.-The deamination of amino sugars with nitrous acid can also yield oxirane derivatives if the amino group and adjacent hydroxyl groups are in, or can very readily achieve, an antiperiplanar orientation; otherwise, simple hydrolysis or other rearrangements preferentially occur. Thus, whereas methyl 2-amino4,6-0-benzylidene-2-deoxy-a-~-altropyranoside (14) gives the D-UZZO epoxide (9)with nitrous acid, the isomeric D-glucoside (15) undergoes rearrangement to yield the 2,5-anhydro-~-mannosederivative (16) instead of the ~ - m a n epoxide n~ (7),the ring-oxygen atom being untiperiplanar to the amino group in this case.32

(16)

(28) F. H . Newth, W. G. Overend, and L. F. Wiggins, J . Chem. SOC., 10 (1947). (29) G. N. Richards and L. F. Wiggins,]. Chem. SOC., 2442 (1953). (30) J. G. Buchanan, J . Chem. SOC., 955 (1958). (31) J. K. N. Jones, M. B. Perry, and J. C. Turner, Can. J . Chem., 38, 1122 (1960). (32) L. F. Wiggins, Nature, 157, 300 (1946); V. G. Bashford and L. F. Wiggins, ibid., 165, 566 (1950); S . Akiya and T. Osawa, Chem. Pharm. Bull. (Tokyo), 7, 277 (1959).

OXIRANE DERIVATIVES OF ALDOSES

119

Elimination of the amino group can also be achieved by the Hofmann procedure, namely, by heating the quaternary ammonium hydroxide.33

c. From Unsaturated Sugars.-The epoxidation of unsaturated compounds with peroxy acids or hydrogen peroxide has been utilized for the synthesis of a number of carbohydrate oxiranes. However, in most instances, racemic non-carbohydrate precursors have been used, affording racemic mixtures of epoxides that have not been resolved, and this limits the usefulness of the products. A further limitation is that mixtures of cis and trans forms are usually obtained, and these need to be ~ e p a r a t e d . ~ Although ~ - ~ ~ the reaction of glycal derivatives with peroxy acids has been known for forty years,4o the resulting 1,2-anhydro sugars being hydrolyzed under the reaction conditions to the simple sugars, only recently have other unsaturated carbohydrates been investigated. Defaye4* has prepared the 5,6epoxide 17 (formally, a derivative of a pyranosid-5-ulose) by treating methyl 2,3,4-tri-O-acetyl-a-~-xylo-hex-5-enopyranoside with p-nitroperoxybenzoic acid, and Ferrier and P r a ~ a d have ~ ~ reported the synthesis of 2,3-anhydro-cllo- and -manno-pyranoside epoxides by treating the corresponding glyc-2-enosides with hydrogen peroxide and benzonitrile. The general pattern is that the oxygen atom is added to the double bond so that it is trans to allylic alkoxyl groups present, but cis to hydroxyl groups. Thus, trans-5,6-dihydro-2-methoxy-6(methoxymethyl)-2H-pyran (18) yielded >95% of methyl 2,3-anhydro4-deoxy-6-O-methyl-au-~~-lyxo-hexopyranoside ( 19) with m-chloroperoxybenzoic and methyl 4,6-di-O-acetyl-2,3-dideoxy-a-Derythro-hex-2-enopyranoside(20) gave a 3:2 ratio of D - ~ U W Z O to Dallo epoxide, whereas, for the unacetylated analog, the ratio was421:3. The overall stereochemistry of addition may be reversed by utilizing the alternative route for epoxidation, namely, first forming the (33) A. B. Foster, M . Stacey, J . M . Webber, and J . H . Westwood, Proc. Chern. Soc., 279 ( 1963). (34) (a) T. Iwashige, Chern. Pharrn. Bull. (Tokyo), 9, 492 (1961); (b) T. Iwashige, M . Asai, and I. Iwai, ibid.,11, 1569 (1963). (35) F. Korte, V. Claussen, and G. Snatzke, Tetrahedron, 20, 1477 (1964). (36) J. Jar$ and K. Kefurt, Collect. Czech. Chern. Cornnun., 31,2059 (1966). (37) (a) F. Sweet and R. K. Brown, C a n . ] . Chern., 46, 707 (1968); (b) 46, 2283 (1968). (38) V. B. Mochalin, Y. N. Porshnev, and G. I. Samokhvalov, Zh. Obshch. Khim., 38, 85 (1968). (39) H. Newman,J. Org. Chern., 29, 1461 (1964). (40) P. A. Levene and A. L. Raymond, ]. B i d . Chern., 88, 513 (1930); P. A. Levene and R. S. Tipson, ibid.,93,631 (1931). (41) J . Defaye, Compt. Rend., 255, 794 (1962). (42) R. J. Ferrier and N. Prasad, J . Chem. SOC. (C), 575 (1969).

NEIL R. WILLIAMS

120

AcOO

O

M

e

halohydrin from the unsaturated compound and then subjecting this to alkaline hydrolysis. Thus, 2-ethoxy-5-(tetrahydropyran-2-yloxymethyl)-2,5-dihydrofuran (21) (mainly the DL-CIS form), which fails to react with peroxybenzoic acid, with calcium hypochlorite, followed by potassium hydroxide, to yield the D-~ZJXO (22) and D-&O (23)epoxides in the ratio of 3 :1.

where R

=

0 111. REACTIONS

1. Stability of the Oxirane Ring Oxiranes react with a wide variety of nucleophilic reagents, under neutral or base- or acid-catalyzed conditions, and this property has led to their extensive exploitation in synthesis. They also undergo hydrogenation in the presence of Raney nickel catalyst, which,

OXIRANE DERIVATIVES OF ALDOSES

121

for convenience, will be considered to be a nucleophilic reagent (although its true mechanism of reaction is not yet clear). However, these reactions usually require somewhat elevated temperatures for several hours, and, by using milder conditions, it is often possible to leave the oxirane ring intact while carrying out a reaction elsewhere in the molecule. 4,6-Benzylidene acetal groups may be selectively removed from 2,3-anhydro sugars by treatment with 5 mM sulfuric acid in aqueous methanol,43or with aqueous oxalic Alternatively, they may be removed by catalytic hydrogenation in the presence of palladium.45 It is interesting that hydrogenation of methyl 2,3-anhydro-4,6-0benzylidene-a-D-talopyranoside in the presence of Raney nickel under vigorous conditions causes preferential opening of the epoxide ring to yield methyl 4,6-0-benzyIidene-3-deoxy-cr-~-Eyxo-hexopyranoside, together with some of the 4,6-O-cyclohexylidene-3-deoxy sugar.21The D-gulo epoxide behaves similarly. Hydrogenation over Raney nickel may be so controlled as to allow the preferential reduction of iodomethyl to methyl groups,46 and lithium aluminum hydride has been used for preferentially reducing a primary sulfonic ester group to a methyl group without affecting a sugar epoxide ring.47 Trityl ether groups can be selectively hydrolyzed with 80% aqueous acetic acid.48 The relative stability of the oxiranes to bases at room temperature allows the selective ammonolysis of carboxylic ester groups with ammonia in methanol,4yand, conversely, free hydroxyl groups may be esterified with acid chlorides or anhydrides in pyridine without affecting the oxirane ring. Hydroxyl groups may also be converted into alkyl ethers by using either the Haworth50 or the Purdie-Irvine pr~cedure.~*-~* Catalytic oxidation of a terminal hydroxymethyl group in a 3,4(43) M. Gut and D . A. Prins, Helu. Chim.Acto, 30, 1223 (1947). (44) W. H. Myers and G . J. Robertson, J . Amer. Chem. Soc., 65, 8 (1943). (45) S. Peat and L. F. Wiggins, 1. Chem. Soc., 1088 (1938). (46) (a) P. Chang and Y . Liu, Hua Hsueh Hsueh Pao, 23, 67 (1957); (b) ibid., 23, 175 (1957);Chem. Abstracts, 52, 12875, 16220. (47) H . R. Bolliger and M . Thiirkauf, Helu. Chim.Acta, 35, 1426 (1952). (48) J . Davoll, B . Lythgoe, and S . Trippett,]. Chem. Soc., 2230 (1951). (49) J. M. Hunter (to Upjohn Co.), U. S. Pat. 3,288,781 (1966); Chem. Abstracts, 66, 85993 (1967). (50) D . S . Mathers and G . J. Robertson,J. Chem. Soc., 1076 (1933). (51) A. Miiller, Ber., 67, 421 (1934). (52) P. J. Garegg, Acta Chem. Scand., 14, 957 (1960).

122

NEIL R. WILLIAMS

anhydropyranoside to give the corresponding 3,4-anhydro-uronic acid has also been achieved.53Potassium permanganate may also be used in this way without affecting the epoxide ring (see Ref. 125a). A p-tolylsulfonyl group can be removed photochemicallysa from a sugar epoxide p-toluenesulfonate to give the corresponding derivative having a free hydroxyl group without affecting the oxirane ring,sb thus providing a potentially valuable method for the interconversion of oxirane derivatives. 2. Stereochemical Features The great majority of the reactions of oxiranes lead to products formed by cleavage of the epoxide ring, the nucleophile becoming attached to one carbon atom, and a hydroxyl group to the other. In a few cases, elimination of the oxygen atom occurs, notably with potassium e t h y l ~ a n t h a t eand ~ ~ potassium ~ e l e n o c y a n a t eleading ,~~ to unsaturated compounds; and with t h i ~ u r e a ~or ~ , potassium ~' thio~ y a n a t e leading ,~~ to episulfides. Here, however, substitution in the usual way probably occurs as a first step, and this is followed by further reaction of the a-hydroxy sulfur or selenium derivative formed; this explains the inversion of configuration at both of the carbon atoms that is observed on converting an epoxide into an episulfide. The general mechanism for the cleavage of epoxides to give ahydroxy derivatives4 appears to be always followed, that is, by an sN2 substitution at one of the carbon atoms, with inversion at that center, leading to a trans a-hydroxy product. Under neutral or basic conditions, an oxide anion is generated, and this is subsequently protonated. Under acid-catalyzed conditions, proton addition to the oxygen atom occurs first, to generate an oxonium ion, which assists cleavage of the carbon-oxygen bond, and this is followed by nucleophilic attack at the carbon atom. In this event, the possibility arises of a borderline sN2 mechanism in which bond breaking is relatively far advanced in the transition state. In both mechanisms, two products (53) G. D. Shyrock and H. K. Zimmerman, Chem. Commun., 263 (1966). (53a) S. Zen, S. Tashima, and S. Koto, Bull. Chem. Soc. l a p . , 41, 3025 (1968). (53b) A. D. Barford, A. B. Foster, and J. H. Westwood, Carbohyd. Res., 13,189 (1970). (54)D. Horton and W. N. Turner, Tetrahedron Lett., 2531 (1964); E. L. Albano, D. Horton, and T. Tsuchiya, Carbohyd. Res., 2,349 (1966). (55) T. van Es, Carbohyd. Res., 5,282 (1967). (56) L. D. Hall, L. Hough,and R. A. Pritchard,]. Chem. Soc., 1537 (1961). (57) R. D. Guthrie and D. Murphy,]. Chem. Soc., 6666 (1965).

OXIRANE DERIVATIVES OF ALDOSES

123

are possible, depending on which carbon atom is attacked by the nucleophile. The ratio of the two products depends upon the structure of the epoxide, the reagent, and the conditions, and the rationalization of the ratios observed provides a subject of enduring interest. The transition state for the substitution step requires that, for maximum stability, the incoming nucleophile be coplanar with the three-membered oxide ring, and, for an epoxide on a pyranose ring (for which a half-chair conformation is expected, by analogy with

H

cyclohexene oxides8),this leads to preponderance of a product having, in the related chair conformation, a trans-diaxial orientation of the two groups. This diaxial rule was first propounded by Furst and Plattners9 for steroid epoxides, and was first suggested for (rigid) carbohydrate epoxides by Mills.6oThese ideas were quickly adopted by other worker^,^^^^^^ and they form the basis for all subsequent discussions of the stereochemistry of cleavage of epoxide rings.

x

X

(58) B. Ottar, Acta Chem. Scand., 1, 283 (1947). (59) A. Furst and P. A. Plattner, Abstr. Pupers Znt. Congr. Pure A p p l . Chem., 12th, New York, 1951, p. 409. (60) J. A. Mills, cited by F. H. Newth and R. F. Homer in J . Chem. Soc., 989 (1953). (61) R. C. Cookson, Chetn. Ind. (London), 223,1512 (1954).

124

NEIL R. WILLIAMS

The concept serves to rationalize most of the observed reactions of aldose oxiranes, and, in the following discussion, attention will be focused on the apparent exceptions to the Furst-Plattner rule. The formation of products derived from the alternative mode of ring opening, which will be called the abnormal mode, leading to diequatorial groups, might reflect the relative stability of a transition state that leads directly to equatorial disposition of the two groups in the related chair conformation, but, because the incoming nucleophile and the departing oxygen atom must then move in quite different directions, such a transition state would be expected to be much more strained, and of higher energy, than the transition state for diaxial opening, as pointed out by Cookson.61This theory is borne out by the fact that epoxides having more rigidly held half-chair conformations (for example, steroid and 1,6-anhydro sugar oxiranes) show little tendency to give diequatorial products. It seems more probable that abnormal products arise either by change of the conformation of the epoxide first to its alternative, less stable, half-chair conformation before reaction (in which case, the transition state will then reflect the alternative (unfavored) chair form of the product having the groups diaxially arranged), or the transition state may reflect and lead to one of the skew conformations of the product, when, again, the colinear arrangement of the nucleophile and the departing oxygen atom at the substituted carbon atom, required for sN2 substitution, can be preserved. In both mechanisms, these alternative chair or skew conformations can then rapidly revert to the more stable chair form, in which the two groups are equatorial. Where the alternative half-chair conformation is not possible of formation by the epoxide, reaction must involve the skew form. Even where the alternative half-chair form is possible, the skew alternative may still provide a reaction pathway energetically more favorable. X

J

OXIRANE DERIVATIVES OF ALDOSES

125

3. Classification of Reagents The following summary indicates the range of groups that have been introduced (X groups in the formulas), with the reagents given in parentheses. For individual references, see later Charts. a. Hydrogen (LiAIH,, H,/Raney Ni); b. Oxygen nucleophiles: OH (H+/H,O or OH-/H,O), OMe, OCH,Ph, OAc, OBz, OPO(OH),, OPO(OCH,Ph), (generally as sodium or potassium salts); c. Sulfur nucleophiles: SMe, SEt, SBu, SCH,Ph, SPh, SCN, SBz (as sodium or potassium salts); d. Nitrogen nucleophiles: NH2, NHR, NR2, NHC(NH,)=NH (as corresponding amine), N3 (NaN,); e. Halogen nucleophiles: I (Mg12,LiI, NaI, RMgI), Br (HBr, MgBr,, LiBr, RMgBr), C1 (HCI, MgCI,, RMgCI), F (HF, KHF,); and f. Carbon nucleophiles: M e (MgMe,, LiMe), Et (MgEt,), Ph (MgPh,), CH=CHPh, CH=CMe2, C=CPh (as lithium alkyl), CN (NaCN, HCN/AIEt,), CH,OH [CO/H,/NaCo(CO),I, CH2C0,Et [via CH,(CO,Et),, sodium salt]. General Comments. The formation of deoxy sugars by hydrogenation over Raney nickel often leads to the abnormal isomer (namely, that formed by diequatorial opening of the oxirane ring) as the major product, in contrast to the product afforded by lithium aluminum hydride; this suggests that a different mechanism is involved in the nickel-catalyzed reaction. Hydrolysis may be effected under either acid or alkaline conditions, the latter being generally preferred in order to avoid concomitant hydrolysis of acetal or glycosidic groups present. On the other hand, Buchanan and coworkers have found that, in the hydrolysis with aqueous acetic acid of acetylated epoxy sugars having an acetoxyl group trans to the epoxide ring, advantage can be taken of neighboring-group effects to control the stereochemistry of the cleavage of the epoxide ring (incidentally, with a greatly increased rate of r e a ~ t i o n ) . ~Thus, , ~ ~ whereas methyl 3,4-anhydro-6-0-trityl-a-~altropyranoside (24) (or its 2-benzyl ether) in 80% aqueous acetic acid is only partially consumed after 3 hours at loo", to give a mixture of D-mannoside (25) and D-idoside (26, R = H), the 2-0-acetyl derivative (27)reacts within 30 minutes, to give mainly the D-mannoside, and the 6-0-acetyl-2-0-benzyl analog (28) as readily gives mainly the idoside (26, R = CH,Ph). In each example, formation of an acetoxonium ion is envisaged (involving a 5- or 6-membered ring) which undergoes cis hydrolytic cleavage. The initial products are a mixture of acetates, which are readily deacetylated. (62) J . G. Buchanan and R. M. Saunders,]. Chem. SOC., 1791 (1964).

NEIL R. WILLIAMS

126 TrOCH,

TrOCH,

0&)Me

o

b

O

AcOCH,

M

e

o&OMe

where Bzl = benzyl.

H I

qoy c-c-c d\/(0 I

Me

--c

,c-c-c

OF.> I Me

OH I

OH I_t

c-c--c

I

I

0

I

0

H,Ac

Although sodium methoxide is also generally used for epoxide formation, it cleaves the epoxide ring only after heating at reflux temperatures for several hours (to give methyl ether derivatives), and no difficulty is experienced in controlling these separate reactions. Simple Grignard reagents effect halogen substitution instead of carbon substitution, Magnesium, lithium, or sodium iodides and bromides are simpler reagents for this purpose. Hydrogen chloride and bromide, in more or less anhydrous media, have been used for preparing bromo- or chloro-deoxy sugars (see p. 137). (Chloromethylene)dimethyliminium chloride has been recommended as a reagent for the preparation of chlorodeoxy sugars.628 For carbon substitution, magnesium dialkyls (and biphenyl) and lithium alkyls are effective in the absence of metal halides. Conditions have also been found for cyanide substitution and for hydroformylation. As mentioned on p. 122, sulfur nucleophiles can also lead to elimi(6%) S. Hanessian and N. R. Plessas, J . Org. Chem., 34,2163(1969).

OXIRANE DERIVATIVES OF ALDOSES

127

nation, or to episulfide formation with inversion of configuration at both of the carbon atoms involved. 4. Anhydro-ring Isomerizations

a. Epoxide Migration. - Oxiranes that possess a hydroxyl group vicinal and trans to the epoxide ring may undergo an intramolecular, nucleophilic substitution by the hydroxyl group, especially under basic conditions, to generate an isomeric a-hydroxy epoxide, with inversion at the middle carbon atom. This process is clearly reversible, the position of the equilibrium being dependent on the relative OH

\

@O

\ ,I / I

,c\-?-c, 0

0 / \ /

\ / \ / - ,

-/c,-pc,

0

,,C-C-C, \ /I

OO

0

/ \

,c-c-c, c' I 1 no

, ,

stabilities of the two epoxides. The unsuspected occurrence of such epoxide migration confused the interpretation of the results obtained by Oldham and Robertson":' and Labaton and Newthe4in their studies on methyl 3,4-anhydro-a-~-galactopyranoside. Buchanan has since shown"0*6sthat the syrupy product, supposedly the epoxide, was probably a mixture of methyl 3,4-anhydro-a-~-galactopyranoside with methyl 2,3-anhydro-a-~-gulopyranoside (see Chart I, Series I, R = CH,OH) by preparing the pure crystalline epoxide, and demonstrating that the products obtained by the earlier workers arise only from a mixture; h e identified one of the products as a 2-substituted D-idoside that can only result from the 2,3-anhydro-~-guloside. A number of studies have now been made, notably by Buchanan and coworkers, with the object of determining the position of the equilibrium between the various pairs of interconvertible epoxides; the findings are summarized in Chart I. For the dianhydro compounds, the epoxides that contain the free hydroxyl group quasiaxial, and that also have the possibility of a polar interaction between the epoxide ring and the 1,6-anhydro bridge, are clearly less stable than those in which it is quasi-equatorial, although the relative importance of these two factors is uncertain. The situation is less clear for the monocyclic epoxides. The half-chair conformations indicated are considered to be favored on the basis that the alkyl (63) J. W. H. Oldham and G. J. Robertson, J . Chern. SOC., 685 (1935). (64) V. Y. Labaton and F. H. Newth, J . Chem. Sac., 992 (1953). (65) J . G. Buchanan,J. Chern. SOC.,2511 (1958).

NEIL R. WILLIAMS

128

CHART I Equilibria Between Interconvertible Epoxides Series I

where R = H (Ref. 68), Me (Ref. lo), CH,OCH,Ph (Ref. 67), or CH,OTr (Ref. 67)

Series I1

rati when DR>9:lH,;

ek+$koH

=

Y "+ g

0.4:l

where R = Me (Ref. 69), CH,OH (Ref. S ) , or CH,OTr (Ref. 9)

ratio 4 : l (Ref. 70)

7

ratio 1:> 20

H

' 0

(Ref. 13)

OMe

OXIRANE DERIVATIVES OF ALDOSES

129

substituent on 0-5 would then be equatorial or quasi-equatorial, and this group is the most important in determining conformation of simple pyranosides.66 However, that this is so for the 3,4-anhydro compounds is by no means certain, because the group on C-5 may adopt a quasi-axial orientation without marked interaction with the axial substituent on C-1. A quasi-axial hydroxyl group may cause some destabilization, but polar interactions between the oxygen atom of the oxirane ring and either the oxygen atom of the pyranose ring or the anomeric oxygen atom seem to be more important than effects produced by a quasi-axial hydroxyl group. The following sequence of interactions seems to be discernible. This sequence can be justified

by the results of consideration of the stereochemistry of the interacting, oxygen lone-pairs. However, this theory does not fully explain the reversal of the equilibrium in Series 11 (see Chart I) when R = H, because, even in the alternative half-chair conformation (lH0),the 3,4epoxide might be expected to be less stable on the basis of the anomeric effect, and this should favor a quasi-axial orientation for the anomeric methoxyl group. The possibility of occurrence of this kind of epoxide migration should be borne in mind in essaying the preparation and considering the reactions of compounds of this kind in basic media, particularly at elevated temperatures.

b. Other Isomerizations. - Hydroxyl groups present elsewhere in the sugar may similarly participate in an intramoIecuIar substitution reaction that leads to larger anhydro rings. It has long been recognized that 3,6-anhydro-~-glucofuranosederivatives may arise from the alkaline treatment of the corresponding 5,6-anhydro-~-glucofuranose,71 and, similarly, 3,6-anhydrohexopyranosides can result from 2,3-anhydrohexopyranosidesin which a free hydroxyl group on C-6 (66) E. L. Eliel, N. L. Allinger, S. J. Angyal, and G . A. Morrison, “Conformational Analysis,” Interscience, New York, N. Y., 1965, pp. 42, 356, and 371. (67) J . G . Buchanan and R. Fletcher,]. Chem. SOC., 6316 (1965). (68) J. G . Buch%nanand R. Fletcher,]. Chem. SOC. (C), 1926 (1966). (69) J. Jar;, K. Capek, and J. Kova:, Collect. Czech. Chem. Commun., 29, 930 (1964). (70) M. Cernj., I. Buben, and J. Pachk, Collect. Czech. Chem. Commun., 28, 1659 (1963). (71) E. Seebeck, A. Meyer, and T. Reichstein, Helu. Chim. Acta, 27, 1142 (1944).

NEIL R. WILLIAMS

130

is suitably located to attack the rearside of the epoxide ring. Thus, (29) yields methyl 3,6-anhymethyl 2,3-anhydro-a-~-allopyranoside dro-a-D-glucopyranoside (30) with sodium hydro~ide.'~ Methyl 3,4anhydro-a-D-galactopyranosidealso reacts with alkali, to afford the epoxide migration occurs first, isomeric 3,6-anhydro-~-galactoside;~~

OMe-

HOCH,

no

OMe

(30)

(29)

to give the 2,3-anhydro-~-gulopyranoside, which then reacts analogously to the D-UZZO epoxide. These isomerizations may also be acidcatalyzed. For example, methyl 2,3-anhydro-4,6-0-benzylidene-a-~gulopyranoside also yields methyl 3,6-anhydro-a-~-galactopyranoside on hydrolysis with 0.05M sulfuric acid,73hydrolysis of the benzylidene group occurring in a preliminary step that liberates a free hydroxyl group at C-6. Buchanan and O a k e have ~ ~ ~demonstrated the existence of a reversible equilibrium between 5,6-anhydro-1,2-0-isopropylidene-P-~HzF\o HC'

11 (32)

(72) A. B. Foster, M. Stacey, and S. V. Vardheim, Acta Chem. Scand., 12,1819 (1958). (73)J. G. Buchanan, Chem. Ind. (London), 654 (1958);J. G.Buchanan and J. Conn, J . Chem. SOC., 201 (1965). (74) J. G . Buchanan and E. M. Oakes, Tetrahedron Lett., 2013 (1964):

OXIRANE DERIVATIVES OF ALDOSES

131

idofuranose (31)and the oxetane 3,5-anhydro-l,Z-O-isopropylidenea-D-glucofuranose (32);this exists concomitantly with the irreversible formation of 1,2-O-isopropylidene-p-~-idofuranose and its 3,6-anhydro derivative. T h e isomeric 5,6-anhydro-~-glucofranosebehaves similarly, affording the 3,5-anhydro-p-~-idofuranose oxetane. As with the oxirane ring, the oxetane ring may undergo nucleophilic substitution with inversion of configuration at either carbon atom attached to the oxygen atom. The intermediacy of an oxetane serves also to rationalize the products obtained by the alkaline hydrolysis of methyl 2,3-anhydro-p-~-ribofuranoside (33). With sodium hydroxide (or cyanide), the 3,5-anhydro derivative 34 may be isolated; and, with sodium methoxide, besides the expected products from the trans opening of the epoxide ring, the major product is methyl 5 - 0 methyl-P-D-xylofuranoside (35),which must result from substitution at C-5 in the intermediate o ~ e t a n e . 'By ~ contrast, only the normal 2- and 3-0-methyl products result from similar treatment of the a

anomer. This difference has been attributed to the fact that, in the

p-D anomer, the oxirane ring is relatively hindered as regards substitution resulting from attack by external reagents; this encourages the intramolecular reaction leading to the oxetane, whereas, for the a anomer, reagents can readily attack C-2, which is more a c ~ e s s i b l e ' ~ (see p. 155).

5. 1,6:2,3-and 1,6:3,4-Dianhydrohexopyranoses In this and the following Sections, the reactions of the various classes of aldose oxirane are summarized in a series of Charts. Unless otherwise indicated, reactions classified as normal give the diaxial product indicated as the preponderant or the only product (or as the only product isolated, although, in some studies, the yield of product isolated was <50%). Conversely, abnormal reactions give the diequa(75) P. W. Austin, J. G . Buchanan, and E. M . Oakes, Chem. Commun.,374 (1965);P. W. Austin, J. G . Buchanan, and R. M. Saunders, J . Chem. SOC. ( C ) ,372 (1967).

132

NEIL R. WILLIAMS

torial isomer, with substitution at the alternative carbon atom, as the major product or in amounts comparable to that of the diaxial product. Reactions in which the simple substitution product is not obtained are listed as “other reactions.” Chart I1 summarizes the reactions of these epoxides with nucleophilic reagents. In all but one reaction, the product is very preponderantly, if not exclusively, that derived by diaxial opening of the epoxide ring. The absence of the alternative, diequatorial isomer argues against the likelihood, in the general case, of a mechanism CHART I1

2, 3-Anhydro-D-tala

%-Subs. D -g U lU C tO ( R ’ = OH, R = H)

Normal: X = NHz,’6~77 OMe,78OH,7nHSza(R’ = OMe, R = H), X = NH,,’O 2,3-anhydro-~-manno 2-subs. D-ghco (R = OH, R’ = H) Normal: R = OMe, X = NHZ8O(R = OCH,Ph, R’ = H), X = F,S1 H,8” OMe.’” 2-subs. D-xylo-hex0(R’ = R = H) 2,3-anhydro-4-deoxy-~-Zyxo-hexo Abnormal: (3-subs. D-arabtno-hexo) X = OHnZ(1:l ratio, 2- to 3-subs.)

OH

2, 3-Anhydro-~-gulo

3-subs. o-galacto (R‘= OH, R = H)

(76) S. P. James, F. Smith, M. Stacey, and L. F. Wiggins, J. Chem. SOC.,625 (1946). (77) R. W. Jeanloz, J . Amer. Chem. SOC., 81, 1956 (1959). (78) R. B. Duff,J. Chem. SOC.,1597 (1949). (79) R. W. Jeanloz and P. J. StoEyn, J . Amer. Chem. Soc., 76, 5682 (1954). (80) L. J. Carlson, J . Org. Chem, 30, 3953 (1965). (81) J. P+k, Z. Tocik, and M. Cem);, Chem. Commun., 77 (1969). (82) M. Cern); and J. Pacik, Collect. Czech. Chem. Commun., 27,94 (1962). (8%) P. A. SeibJ. Chem. Soc. (C), 2552 (1969).

OXIRANE DERIVATIVES OF ALDOSES Normal: X=OH'O((R'=OMe, R = H ) , X = O H 7 " 2,3-an hydro-4-deoxy-~-ribo-hexo

3-subs. 4-deoxy-~-xylo-hexo ( R ' = R = H)

Normal: X = OHS2 2,3-anhydro-~-allo Normal: X = OMeSZa

3,4-Anhydro-~-talo

133

3-subs. D-glUC0 (R = OH, R'

=

H)

4-6ubs. ~ - m a n t ~ (R' = OH, R = H)

Normal: X = NH2,76OH,s3SCH2Phe4 3,4-anhydro-~-galacto 4-subs. D - ~ ~ U C(R O = OH, R' = H) Normal: X = OH,70F53a(R = OTs, R' = H), X = OH,'3 OMe,*O H,S2 OCH2ph,85 F.53a NaOH gives a mixture of epoxides70(R = OMe, R' = H), X = OH.70

3,4-Anhydro-D-dtro

3-sub6. D - manno (R' = OH, R = H)

Normal: X = (R' = OMe, R = H), X = OH70 3,4-anhydro-~-ah Normal: X = OMe.8Za

3-subs. D-glUC0 (R = OH, R'

= H)

involving a transition state leading directly to the diequatorial isomer. The one apparent exception is the alkaline hydrolysis of 1,6:2,3dianhydro-4-deoxy-P-~-lyxo-hexopyranose, which gives an approximateIy 1:1 mixture ofthe I ,6-anhydro-4-deoxy-P-~-xyZoand -amhino-

(83) R. M. Hann and C. S. Hudson, J. Amer. Chem. Soc., 64, 925 (1942). (84) L. N; Owen and P. L. Ragg, J. Chem. SOC.(C), 1291 (1966). (85) M. CernJ, L. Kalvoda, and J. PacAk, Collect. Czech. Chem. Commun., 33, 1143 (1968).

134

NEIL R. WILLIAMS

hexopyranoses. Here, however, polar efforts could be very significant; the oxygen atom in the pyranose ring would be expected to hinder the approach of the hydroxide ion to C-2 more than it does to (2-3, by a polar-field effect; and C-2, adjacent to the acetal carbon atom, should be markedly less reactive than C-3, which is adjacent to a deoxy position, because electron-withdrawing groups appear to deactivate an adjacent, oxirane ring to substitution at the nearer carbon atom.86 Furthermore, the absence of a substituent on C-4 facilitates the change from a half-chair to a skew conformation that is required in order to give the abnormal product. These factors, taken together, could offset the usual tendency for diaxial opening of the epoxide ring from a half-chair conformation.

6 . 2,3-Anhydro-4,6-0-benzylidenehexopyranosides Chart I11 summarizes the reactions of these sugars. The d o and manno epoxides have, between the pyranose and benzylidene rings, a trans junction that prevents adoption of the alternative half-chair conformation ('HJ, although the conformation is more flexible than for the dianhydro compounds already considered. In the gulo and tuZo epoxides, the alternative half-chair form is, in principle, possible; nevertheless, it is very unlikely, because, were it adopted, not only would the C-6 group be axially oriented on the sugar ring, but the phenyl group would also become axial on the benzylidene ring. Although the cis ring-junction does confer greater flexibility, it is interesting that these epoxides are, in fact, less prone to give abnormal products than is the allo epoxide. With the allo and gulo epoxides, abnormal products arise either from hydrogenation over Raney nickel catalyst, so that all four of the isomeric 2,3-epoxides give mainly 3-deoxy sugar derivatives with this reagent, or from reactions catalyzed by acids or Lewis acids. Hydrogenation may well proceed by a special mechanism for which poIar effects are much more important than steric effects. In the acidcatalyzed reactions, a borderline sN2 mechanism may be involved, in which the transition state is achieved when bond breaking is well advanced and bond making is only just beginning. Here, a considerable positive charge may develop on the substituted carbon atom, making it more sensitive to polar effects; also, the steric requirements (86) M . Mousseron, G. Manon, and G . Combes, Bull. SOC.Chim. Fr., 396 (1949); M. Mousseron, R. Jacquier, M . Mousseron-Canet,and R. Zagdoun, ibid., 1042 (1952); R. U . Lemieux, R. K. Kullnig, and R. Y. Moir,J. Amer. Chem. SOC.,80,2237 (1958).

OXIRANE DERIVATIVES OF ALDOSES

135

CHART 111 H

no 2, 3-Anhydro-D-all0

i

R'

2-subs. D - f f l f 7 0

(R' = OMe, R" = H). Normal: X = H,8' OH,I7OMe,lBOPO(OCH2Ph)z,88 SMe,89SEt,SO SCHzPh,g' SPh,gla SBz,'O NH2,44.45,93 NHMe ,93 NHC(NH,) = NH,e3 N,,94 Cl,95Br,2g.95.96 I,29*9s-97CHzSOMe,g8CHzCOzEt,gg Me,99aSCSNME,,99bC, Cls.9" Abnormal (3-subs. D-gluco): H,24,8T OH z8 C1 28,93.95.1w Br, ** 12g*90~998 Other reactions: KCS,OEt + eliminations9 or sulfide,lO' CS(NH2), + episulfide5'; ' ~ ~ ; POCH2C0,Et NH,SCN, KSCN + sulfide101;MeLi + elimination (see t e ~ t ) ~ " .(EtO), + cyclopropane derivative1O3;Me,SO 4 diuloselo4 p (R" = OMe, R' = H). Normal: X = OMe105 p (R" = theophylline, R' = H). Abnormal: X = OPO(OCH,Ph),88 ci

,

I

(87) D. A. Prins, ]. Amer. Chem. Soc., 70,3955 (1948). (88) W. E. Harvey, J. J. Michalski, and A. R. Todd, ]. Chem. Soc., 2271 (1951). (89) R. W. Jeanloz, D. A. Prins, and T. Reichstein, Helo. Chim. Acta, 29, 371 (1946). (90) F. H. Newth, G. N. Richards, and L. F. Wiggins,]. Chem. Soc., 2536 (1950). (91) N. C. Jamieson and R. K. Brown, Can. J. Chem., 39, 1765 (1961). (91a) S. Hanessian and N. R. Plessas, Chem. Commun., 706 (1968). (92) J . Kocourek, Carbohyd. Res., 3, 502 (1967). (93) S. N. Danilov and I. S. Lyshanskii, Zh. Obshch. Khim., 25,2106 (1955). (94) R. D. Guthrie and D. Murphy, ]. Chem. Soc., 5288 (1963). (95) G. N. Richards, L. F. Wiggins, and W. S. Wise,]. Chem. Soc., 496 (1956). (96) M. Sharma and R. K. Brown, Can.]. Chem., 44,2825 (1966). (97) R. U. Lemieux, E. Fraga, and K. A. Watanabe, Can.]. Chem., 46,61(1968). (98) M. Sharma and R. K. Brown, Can.]. Chem., 46,757 (1968). (99) L. I. Kudryashov, M. A. Chlenov, and N. K. Kochetkov, Izu. Akud. Nauk S S S R , Ser. Khim., 75 (1965). T. D. Inch and G. J. Lewis, Carbohyd. Res., 15, 1 (1970). S. Ishiguro and S. Tejima, Chem. Phnrm. Bull. (Tokyo), 16,1567 (1968). J. B. Lee and B. Scanlon,]. Chem. Soc. (D), 955 (1969). S. Mukhejee and H. C. Srivastava, Proc. Zndian Acad. Sci.Sect. A , 35,178 (1952). M. Kajima, M. Watanabe, and T. Taguchi, Tetrahedron Lett., 839 (1968). A. A. J. Feast, W. C . Overend, and N. R. Williams, J. Chem. Soc., 7378 (1965). W. Meyer zu Reckendorf and U. Kamprath-Scholtz, Angew. Chem. Intern. Ed. Engl., 7, 142 (1968). G. Henseke and G. Hanisch, Chem. Ber., 101, 4170 (1968). S. Peat and L. F. Wiggins, J. Chem. Soc., 1810 (1938).

NEIL R. WILLIAMS

136

4 -

'0'

r

OMe

2,S-Anhydro-a - D - manno

Normal: X = H,87J08OH,16 OMe,16 SMe,'" SCH,Ph?' SCN,lo7NH2,44J08J09 N 3,O4 C1 110 Me,lo2Et,", CH2SOMe,88CN,IL3Me,00aEt.g88 Abnormal (2-subs. D-glUc0):C1,'O0Br,lo0(see text), Phil4 Other reactions: KSCN, CS(NH,), + episulfideS7;KSeCN + eliminations5 The 4,6-O-ethylidene analog reacts normally with NaSCH2Ph115 The phenyl glycoside analog reacts normally with lithium aluminum hydride 1

Lo R"

,

2,3-Anhydro-n-gulo

R' HO 2 subs. ~ - i h

(R'=OMe, R" = H). Normal: X = H,llEOH,20OMe,ZoSMe,lI7NH 2,44~118C130 Abnormal (3-subs. D-golacto):X = H21 Other reactions: H2S04aq. + 3,6-anhydrogalactoseT3 OMe20 p ( R ' = OMe, R' = H). Normal: X = H,'l8 (I

(106) H. R. Bolliger and D. A. Prins, Heh. Chim. Acta, 29, 1061 (1946). (107) J. E. Christensen and L. Goodman,]. Amer. Chem. SOC., 83,3827 (1961). (108) L. F. Wiggins,]. Chem. Soc., 18 (1947). (109) B. R.Baker and R. E. Schaub,J. Org. Chem. 19,646 (1954). (110) F. H. Newth and R. F. Homer, ]. Chem. Soc., 989 (1953). (111) G. N. Richards,]. Chem. Soc., 4511 (1954). (112) A. B. Foster, W. G. Overend, M. Stacey, and G. Vaughan, J . Chem. SOC., 3308 (1953). (113) B. E. Davidson, R. D. Guthrie, and A. T. McPhail, Chem. Commun., 1273 (1968). (114) G. N. Richards,]. Chem. SOC., 2013 (1955). (115) L. Goodman and J. E. Christensen, ]. Org. Chem., 28, 158 (1963). (115a) S. Svensson, Acta Chem. Scand., 22, 2737 (1968). (116) H. Hauenstein and T. Reichstein, Helu. Chim. Acta, 32, 22 (1949). (117) A. C. Maehly and T. Reichstein, Helu. Chim.Acta, 30, 496 (1947). (118) J. G. Buchanan and K. J. Miller,]. Chem. SOC.,3392 (1960). (119) T. Golab and T. Reichstein, Helu. Chim. Acta, 44, 616 (1961).

OXIRANE DERIVATIVES OF ALDOSES

2, 8-AnhydrO-D-talo

137

3-subs. o-id0

a (R’ = OMe, R” =H) Normal: X = H,Z1OH,’%OMe,2O NH2,ll6N3’*’

Other reactions: H 2 S 0 4aq. + 3,6-anh~droidose’~

p (R” = OMe, R’ = H). Normal: X=OH,20OMe,18*20 SMe,’**NH2I8

for the entering and leaving groups in the transition state will be less rigorous. It is, perhaps, significant that most of the abnormal products arise with the allo epoxide, when 2-substitution is expected. Proton acid catalysts lead mainly to 3-substituted gluco products, with concomitant debenzylidenation which, as it occurs first, allows the alternative half-chair conformation (‘HJ to be adopted for the reaction. Grignard reagents, magnesium and lithium halides, and methyllithium all give normal 2-substitution mainly, except for methylmagnesium iodide, which yields the 3-deoxy-3-iodo-~-glucoside, as does methylzinc iodide. This behavior is at present very difficult to explain, especially as both ethylmagnesium iodide and magnesium iodide behave “normally,” even allowing for the complex structure of Grignard reagents in solution; possibly, only the molecule of methylmagnesium iodide is small enough to complex effectively with the epoxide oxygen atom, so as to encourage operation of the borderline, s N 2 mechanism. The abnormal substitution of 7-(2,3anhydro-4,6-O-benzylidene -/?-D-allopyranosy1)theophyllinewith dibenzyl hydrogen phosphate, to give the D-glucose 3-phosphate derivative, probably results from the large steric effect of the purine ring, which would inhibit substitution at C-2. The manno epoxide, by contrast, unambiguously exhibits abnormal behavior with diphenylmagnesium only. Here, a steric effect may again be discerned, as normal substitution at C-3 would lead to (120) M. Gyr and T. Reichstein, Helt.. Chim.Acta, 28, 226 (1945). (121) S. Hanessian and T. H. Haskell,]. Org. Chem., 30, 1080 (1965). (122) M. Gut, D. A. Prins and T. Reichstein, Helu. Chim. Acta, 30, 743 (1947).

138

NEIL R. WILLIAMS

syn-axial phenyl and anomeric methoxyl groups, a severely overcrowded situation. In this series, ethylmagnesium iodide also acts as a reducing agent (a well known characteristic of alkyl Grignard reagents having a hydrogen atom /3 to the magnesium atom) and leads to methyl 4,6-0-benzylidene-3-deoxy-a-~-a~abino-hexopyranoside as a "byproduct" in up to 50% yield."' An investigation on the reaction of the allo- and manno-epoxides with Grignard reagents has shown that the use of alkylmagnesium chlorides does afford the deoxy, branched-chain sugars expected by alkyl substitution and diaxial opening of the epoxide ring, the chloride ion being less nucleophiIic than bromide or iodide, which generally give rise to deoxyhalo sugars, or unsaturated or deoxy products derived therefrom, Also, the interesting observation was made that, whereas the all0 epoxide reacts normally with freshly prepared methyl- or ethyl-magnesium iodide in tetrahydrofuran to give unsaturated or deoxy sugars derived from the 2-deoxy-2-iodoaltroside, an aged solution of the Grignard reagent in an excess of tetrahydrofuran gives high yields of the 3-deoxy-3-iodoglucoside, the product derived by abnormal opening of the epoxide ni@ g r.=' Conflicting results have been reported for the reaction of the manno epoxide with hydrochloric acid in acetone. Mukherjee and Srivastavalooclaimed that approximately equal amounts of the normal and abnormal products are formed, whereas Newth and Horner'lO reported that 3-substitution (ratio 92:8) mainly occurs, as would be expected. Although the former workers claimed similar results for hydrobromic acid, their anomalous results need re-investigation.

OXIRANE DERIVATIVES OF ALDOSES

139

The “unusual” behavior of the d o epoxide with methyllithiumIo2 has been clarified by Sharma and B r o ~ n . Methyllithium ~ , ~ ~ that contains lithium iodide (because it was prepared from methyl iodide) Ieads’O’ to 4,6-O-benzylidene-~-allal (36), whereas methyllithium free from iodide yields,% instead, the 2-C-methyl analog of this unsaturated sugar (37). In both reactions, the epoxide reacts normally with the iodide and methyl nucleophiles, respectively, but the initial products then undergo elimination catalyzed by methyllithium, a strong base. In the one reaction, the iodide undergoes nucleophilic attack, with trans elimination of iodide and methoxyl groups, to give 36. In the other, the proton at C-2 is removed, followed by p-elimination of the methoxyl group, to yield 37. At present, the reaction of the allo- and manno-epoxides with sulfur nucleophiles presents rather a confusing picture. Although the reaction of alkanethioxides is unexceptional, other reagents give products that appear to depend on the conditions employed. Thiourea gives mainly the episulfide, with double inversion; thus, the d o epoxide gives methyl 4,6-O-benzylidene-2,3-dideoxy-2,3-epithioa-D-mannopyranoside (38) in 63% yield,57 together with 20% of methyl 4,6-0-benzylidene-2,3-dideoxy-a-~-erythro-hex-2-enopyranoside (39). Ammonium thiocyanate in 2-methoxyethanol also yields

these two compounds in low yield, but the principal product islO1the bis(2-deoxy-altrosid-2-yl)disulfide (40). However, potassium thiocyanate under the same conditions, yields the bis(2-deoxyaltrosid2-yl) monosulfide (41), as does potassium ethylxanthate in methanol,Io1the latter reagent also giving some of the thiol 42. On the other

NEIL R. WILLIAMS

140

hand, potassium ethylxanthate in refluxing butanol gives5?principally the unsaturated sugar 39. A different pattern emerges with the manno epoxide. The a110 episulfide, isomeric with 38, results with thiourea, but in poor yield,s7whereas ammonium thiocyanate gives the simple 3-deoxy-3-(thiocyanato)-~-mannoside (43), but no episulfide. Io7 The latter is produced in poor yield by use of the potassium salt, especially in the presence of ammonium ~hloride.~' Finally, potassium ethyl-

xanthate does produce the episulfide in ethanol, but the latter reacts further in butanol, to give5?the unsaturated sugar 39. Presumably, the reactions are each initiated by normal, nucleophilic substitution in the epoxide, but the resulting diaxial products generally react further by a number of pathways that depend not only on the precise conditions but also on the stereochemistry of the given intermediate. The reaction of the gulo and talo epoxides in dilute sulfuric acid, to give7:l 3,6-anhydro-D-galactose and 3,6-anhydro-~-idose,respectively, clearly represents the reaction of the debenzylidenated epoxides, and selective removal of the benzylidene group can be achieved synthetically. The products then arise by nucleophilic attack at C-3 by the 6-hydroxyl group. For the talo epoxide (44), it is noteworthy that preliminary glycosidic hydrolysis must also occur, before the 6-hydroxyl group is sterically capable of attacking the rearside of C-3, an attack required by the configuration of the product and expected from the usual mechanism, either through the acyclic form of the sugar or through a furanose form. Generally, acid-catalyzed cleavage

n I c=o

I HOCH

OH-

I

HCO HCOH

(44)

H,COH

OXIRANE DERIVATIVES OF ALDOSES

141

of the epoxide ring in pyranoside oxiranes can be achieved selectively, before glycosidic hydrolysis occurs. The d o epoxide 9 has been shown to undergo an unusual reaction with diethyl (ethoxycarbonylmethy1)phosphonate and sodium hydride, affording1O3the cyclopropane derivative 45. This reaction presumably proceeds

by an attack at C-2 by the phosphorus ylid that generates a betaine, which undergoes decomposition in a way similar to that of the intermediate in the standard Wittig reaction. Both the d o and manno epoxides (9)or (7), or their debenzylidenated analogs, undergo oxidation with methyl sulfoxide and boron trifluoride,l0' but, unlike 5,6-anhydrohexofuranose derivatives (see p. 163), they do not yield a simple a-hydroxy carbonyl compound, as would be expected by analogy with simple oxiranes; instead, the rearranged diulose (46) is obtained in low yield, possibly by elimination of methanol from the intermediate (47).

7. 2,3- and 3,4-Anhydrohexopyranosides Chart IV summarizes the reactions of these 2,3- and 3,4-anhydrohexopyranosides. The half-chair conformations shown are those anticipated on the assumption that the alkyl substituent on C-5 governs the conformation assumed, as it usually does with the chair confonna-

NEIL R. WILLIAMS

142

CHART IV

2, 3-Anhydro-n-allo

2-subs. n-altro

(R'= OMe, R" = H). Normal: (Y = OMe) X = OH,I" OMe,Iz3FIz4;(Y = OAc) X = FIz4; (Y = OTs) X = H.iz5Methyl (methyl 2,3-anhydro-4-azido-4-deoxy-a-~-allopyranosid)uronate with iodide gives 2:l ratio, 2:3 subs.125a Abnormal (3-subs. D-gluco): (Y = OMe) X = O',I NHMe, gives 1:l ratio, 2,3 subs.Iz6 (Y =H) NHMe,gives l : l r a t i o , 2 , 3 s ~ b s . ~ -4=OCH,Ph,Y ~(Y -6=OTr)I"*' Other Reactions: NaOH --i 3,6-anhydro-~-glucopyranoside~~ p (R" OMe, R' = H). Normal: (Y = OMe) X = OMe.'05 Abnormal: Ethyl or benzyl 2,3-anhydro-4-deoxy-P-~~-ribo-hexopyranosides gives 3-subs. DL-xylo-hexopyranosides with NH3 or NHMeZ126b Q

R' X

2,3-Anhydro-~-ninnm

3-subs. n-nEtro

Q (R' = OMe, R" = H). Normal: (Y = 2 = OH) X = OH,' NHz44;(Y = OH, 2 = OTr) X = OH'; (Y = OAc, 2 = OTr) X = (Y =OH, 2 = H) X = NH2,Iz8NMeZiz8;(Y = 2 = H) X = NMe,.3s P (R"= OMe, R' = H). Abnormal: (Y = 2 = OMe) X = OMe.lZ9

(123) G. J . Robertson and H. G . Dunlop,/. Cliem. Sac., 472 (1938). (124) I. Johansson and B. Lindberg, Carbohyd, Res., 1, 467 (1966). (125) H. R. Bolliger and P. Ulrich, Helu. Chirn. Actn, 35, 93 (1952). (125a) R. S. Goody, K. A. Watanabe, and J. J. Fox, Tetruhedron Lett., 293 (1970). (126) A. B. Foster, T. D. Inch, J. Lehmann, M. Stacey, and J. M. Webber, /. Chem. Soc., 2116 (1962). (126a) S. Dimitriyevich and N. F. Taylor, Carbohyd. Res., 11,531 (1969). (126b) V. B. Mochalin, Y. N. Porshnev, and G . I. Samokhvalov, Zh. Obshch. Khim., 39, 109, 681, 701 (1969); V. B. Mochalin, Y. N. Porshnev, G. I. Samokhvalov, and M. T. Yanotovskii, ibid., 39,116 (1969). (127) J. G. Buchanan and R. M. Saunders, 1. C h e n . Sac., 1796 (1964). (128) J. JaG, J. KovLi, and K. Capek, Collect. Czech. Chem. Cornmun., 30, 1144 (1965). (129) W. H. G. Lake and S. Peat,J. Chem. Soc., 1417 (1938).

OXIRANE DERIVATIVES OF ALDOSES Y

143

/?

Normal: (Y = OH, Z = OTs) and (Y = Z = OTs) X = H.Iz9 Abnormal: (Y = OAc, Z = OTr) and (Y = OAc, Z = OCH,Ph) X

= OH.s7

Y

a (R' = OMe, R" = H). Normal: (Y = O H , Z = H)" X = H,'* OMeI8; (Y = OH, Z = Nil) X = NS1"; (Y = OH, Z = NHAc) X = NH2,"'o~'3' Abnormal (2-subs. D-gahcto): (Y = OMe, Z = HI* X = OMe.18 p (R" = OMe, R' = H). Normal: (Y = Z = O H ) X = NHp18;( Y = Z = OMe) X = OMe.18

X

3, 4-Anhydro-D-a110

4-subs.D - ~ u ~ o

(R' = OMe, R" = H). Abnormal (3-subs. D - ~ Z U C O ) : (Y = OH) X = OMe,45 NH2.105 (Y = H)" X = P (R" = OMe, R' = H). Abnormal: (Y = OH) X = NHqlo5;(Y = OMe) X = NH,.45 a

(130) H . R. Bolliger and T. Reichstein, Helu. Chim. Acta, 36, 302 (1953). (131) (a) H. Weidmann, K. B. Hendriks, and K. Balda, Tetrahedron Lett., 903 (1965); (b) H. Weidmann and N. Wolf, Ann., 687,250 (1965). (132) A. ZobP6ovP and J. Jar?, Collect. Czech. Chem. Commun., 31, 2282 (1966). (133) J. P. Marsh, C. W. Mosher, E. M . Acton, and L. Goodman, Chem. Comrnun., 973 (1967).

NEIL R. WILLIAMS

144

4-subs. 0-id0

3,4-Anhydro-o-altVO

(Y (R' = OMe, R" = H) Normal: (Y = OH, Z = OTr) X = 0H.O; (Y = OCHZPh,Z = OAc) X = OH.@*

Abnormal (3 subs. D-manno): (Y = OAc, Z = OTr) X = OH9; (Y OCHtPh, Z = OTr) X = OH:* ( 1 : l ratio, 3:4 subs.) p (R" = OMe, R' = H). Normal: (Y = 2 = OMe) X = OMeI3'

= Z = OAc) and

(Y =

HO

3,4-Anhydro-0-galacto

3-subs.D - g U l O

a (R'= OMe, R" = H). Normal: (Y= Z = OH) X = H,24OH,B5SMe,14Cl,65Br2'; (Y =OH, 2 = OTr) X = OHB5;(Y= OAc, Z = OTr) X = OH65*B7; (Y = Z = OAc) X = OH,65 CN113; (Y = OAc, Z = OCHzPh)13sa X = OHe7;(Y = OH, Z = H) X = H,I35*l35(IBr.135 Abnormal (4-subs. D-ido); (Y = Z = OH) X = H,24(1:l ratio, 3:4 subs.) (Y = OH, Z = H)

X = H,135S B U , ' N3,136 ~ ~ (1:l ratio, 3:4 subs.) Other Reactions: (Y = Z = OH) NaOH+3,6-anhydrogalactopyranoside; R' = OCH,Ph, R" = H, Y = NHCOzCH,Ph, Z =OH; H O A ~ o x a ~ o l i d i n o n e . ~ ~ ~ p (R" = OMe, R' = H) Normal: (Y = Z = OH) X = H,29J38SMe,I3O Sr0;(Y = OH, 2 = H)

X=OH,'41OMe.";(R''=OCH2Ph,R'=H,Y=OH,Z=OTs)X=NH,131 Abnormal: (Y = Z = OH) X =

(1:l ratio, 3:4 subs.)

(134) W. H. G. Lake and S. Peat,J. Chem. SOC., 1069 (1938). (135) K. Capek, J. NGmec, and J. Jary', Collect. Czech. Chem. Commun., 33,1758 (1968). (135a) G.qSiewert and 0.Westphal, Ann., 720, 171 (1968). (136) K Capek and J. J a 6 , Collect. Czech. Chem. Commun., 31, 2558 (1966). (137) P. H. Gross, J. Brendel, and H. K. Zimmerman, Ann., 680,155 (1964). (138) M . Dahlgard, B. H. Chastain, and R. L. Han, J. Org. Chem., 27, 929 (1962). (139) M. Dahlgard, B. H. Chastain, and R . L. Han, f. Urg. Chem., 27, 932 (1962). (140) M . Dahlgard,J. Org. Chem., 30, 4352 (1965). (141) H. Kaufmann, Helo. Chim. Acta, 48, 769 (1965).

OXIRANE DERIVATIVES OF ALDOSES

145

HO

3,4-Anhydro-n- falo

3-subs.

id^

Normal: (R = OH) X = H,B' OMe.8' NH,,'42' NMe,,'420N:,,'43(R = OMe) X = H.8" Abnormal: ( R = O H ) X = O H 8 " ; (R=OMe)X=OMeB";( R = O B Z ) X = N : $ . ' ~ ~ 'L Series actually studied.

tions of simple pyranosides.66 Buchanan and coworkers have shown'44 that methyl 2,3-anhydro-a-~-guloand -manno-pyranosides and their 6-deoxy analogs, and also methyl 3,4-anhydro-a-~-altroand -galactopyranosides all adopt the half-chair conformation, depicted in Chart IV, that has the alkyl group on C-5 attached equatorially or quasiequatorially, and the anomeric methoxyl group attached axially or quasi-axially. In all of these examples, the anomeric effect will support the tendency of the substituent on C-5 to adopt an equatorial position. The favored conformation of the p-Danomers, where these two effects are in conflict, has not yet been investigated. However, it should be recognized that with 3,4-anhydro compounds, where the alkyl group on C-5 is attached either quasi-equatorially or axially, the stereochemical difference between these positions is likely to be smaller than the equatorial-axial difference in chair conformations; and, in particular, the quasi-axial position will be less sterically crowded. The 3,4-anhydro compounds are more prone to abnormal reactions, and this could be due to a smaller en( rgy difference between the two possible half-chair conformations of the epoxide, which would be reflected in the transition states for the two pathways. In any event, the epoxides of monocyclic aldopyranoses can all adopt the alternative half-chair conformation without difficulty, so that either form may be involved in a given reaction, and it might be anticipated that the ratio of normal to abnormal products will be more dependent on (142) J. ]a$. K. Capek, and J. Kovai, Collect. Czech. Chem. Con~mun.,28,2171 (1963). (143) C. L. Stevens, S. J. Gupta, R . P. Glinski, K. G. Taylor, P. Blurnbergs, C. P. Schaffner, and C. H. Lee, Carbohyd. Res., 7, 502 (1968). (144) J. G. Buchanan, R. Fletcher, K. Parry, and W. A. Thomas, J . Chem. SOC. ( B ) , 377 (1969).

146

NEIL R. WILLIAMS

reagents and conditions than with the more rigid epoxides so far considered. Methyl 2,3-anhydro-4,6-di-O-methyl-a-~-allopyranoside exhibits abnormal behavior with methylmagnesium iodide, as does the 4,60-benzylidene derivative, whereas magnesium iodide gives the normal 2-deoxy-2-iodo-~-altroside with the latter, but the 3-deoxy3-iodo-D-glucoside with the former.g0These results could mean that, for both reagents, a borderline s N 2 mechanism that favors 3-substitution is operative, but that, with the 4,6-O-benzylidene derivative, for which the alternative half-chair conformation is not available, magnesium iodide is sterically hindered from following this pathway, and, instead, takes the normal course, resulting in 2-substitution. with Methyl 2,3-anhydro-4-0-benzyl-6-O-trityl-a-~-allopyranoside methylmagnesium iodide also yields the 3-deoxy-3-iodo-~-glucoside.12Ba Dimethylamine yields comparable proportions of 2- and 3-substituted products with the all0 epoxide,'26perhaps reflecting the combination of a greater sensitivity to polar effects of a reagent more weakly nucleophilic than the other reagents tested, together with the effects of the high temperature used, which would tend to equalize the ratio of the two reacting conformations and the energies of the resulting transition states. A very similar behavior is shown by the 4,6-dideoxy analog, namely, ethyl 2,3-anhydro-4,6-dideoxy-m,&~~ribo-hexopyran~side,~~ which gives a 3:2 ratio of the 3-(dimethylHere, an amino)- to the 2-(dimethylamino)-trideoxyhexopyranoside. additional factor in favor of 3-substitution is the presence of the deoxy (methylene group) at C-4, which will enhance the polar effect shown by the allo epoxide. It is not surprising that the isomeric ethyl 2,3anhydro-4,6-dideoxy-cY,P-~~-lyxo-hexopyranosidereacts entirely at C-3, because, here, polar and steric effects combine to favor substitution at this atom. The importance of the polar factor is borne out by the claim that a number of C-6-substituted ethyl or benzyl 2,3anhydro-4-deoxy-~-~~-ribo-hexopyranosides react with ammonia or secondary amines to give 3-amino-3-deoxy-xylo-hexose derivatives. lab With sodium hydroxide, methyl 2,3-anhydro-a-~-allopyranoside yields methyl 3,6-anhydro-c~-~-glucopyranoside by intramolecular attack on the epoxide ring by the 6-hydroxyl group." Methyl 2,3anhydro-a-D-gulopyranosidebehaves similarly, and its intermediacy accounts for the conversion of methyl 3,4-anhydro-m-~-galactopyranoside into methyl 3,6-anhydro-a-~-galactopyranoside on treatment with alkali.67

OXIRANE DERIVATIVES OF A L D O S E S

147

Methyl 2,3-anhydro-c~-~-mannopyranoside behaves normally, but the 4,6-dimethyl ether of its anomer gives, with sodium methoxide, methyl 2,4,6-tri-O-methyl-p-~-glucopyranoside as the only crystalline compound isolated (less than 50% of the total product, h o ~ e v e r ) . " ~ Here, an anomeric effect may help to stabilize the alternative halfchair conformation (5H,);but it should be noted that this is not the only instance in which abnormal products result from the treatment, with sodium methoxide in methanol, of a sugar epoxide possessing a methoxyl group adjacent and cis to the oxirane ring. Methyl 2,3-anhydro6-deoxy-4-0-methyI-c~-~-talopyranoside (48) also behaves abnormally, in giving the 2,4-di-O-methyl-~-galactosederivative, although the unmethylated epoxide 49 gives the expected 3-substituted-~-idoside, and methyl 3,4-anhydro-6-deoxy-2-O-methyl-ar-~-talopyranoside gives whereas the unmethylthe abnormal 2,4-di-O-methyl-~-mannoside, ated epoxide yields the expected 3-0-methyl-idoside." The results of comparison of these examples suggest that the polar interaction between the epoxide-ring oxygen atom and the pyranose-ring oxygen atom may be important in determining the half-chair conformation favored, thus increasing the tendency to assume the alternative halfchair form having an axially attached substituent at C-5, for example, 48, an interaction that a cis quasi-axial hydroxyl group can offset by intramolecular hydrogen-bonding to the oxirane oxygen atom or to the pyranose-ring oxygen atom, or both, but that a methoxyl group is unable to do, restoring the balance in favor of the conformation having an equatorially attached group, at C-5, such as compound 49. The possibility that hydrogen bonding stabilizes a half-chair form (OHs), as in 49, was suggested by Newth,3 but, in methanolic sodium meth-

(48)

(49)

oxide solution, the hydroxyl group would be expected to be extensively ionized. Another possibility is that the cis-hydroxyl group, when attached quasi-axially, assists the protonation of the oxygen atom of the epoxide (which it cannot do when quasi-equatorially attached), facilitating the reaction in this conformation. The abnormal behavior of methyl 3,4-anhydro-2-O-benzoyl-6-deoxy-a-~-talopyranoside with azide ion, to give the 4-azido-4,6-dideoxy-~-mannoside as

148

NEIL R. WILLIAMS

the principal product, may be similarly explained, because the unbenzoylated epoxide reacts normally, giving the 3-azido-3,6-dideoxyD-idoside as the major p r 0 d ~ c t . Perhaps, l~~ the abnormal behavior of the unsubstituted 3,4-anhydro-talo epoxide with barium hydroxide, giving mainly the 6-deoxymanno~ide,~ may be explained by assuming that intramolecular hydrogen-bonding fails to occur in the aqueous solution used, so that the importance of oxygen-oxygen polar repulsions is reasserted. On this basis, the apparently normal behavior of methyl 3,4-anhydro-6-deoxy-2-O-methyl-a-~-talopyranoside with lithium aluminum hydride in ether, to give methyl 3,6-dideoxy-2-0methyl-a-D-Zyxo-hexopyranoside, becomese “anomalous,” but, as the product was isolated in only 17% yield, this may not have been the major product formed. Such compounds deserve further exploration. Neighboring-group participation by the vicinal, trans-acetoxyl group (see p. 125)serves to explain the “abnormal” behavior of methyl 4-0-acetyl-2,3-anhydro-6-O-benzylor -trityl-cr-~-gulopyranosidewith hydrogen chloride in acetone, or with 80% aqueous acetic acid, which give D-galactose, instead of the D-idose, derivative^.^' In the same way, 2-0-acetyl-3,4-anhydro-~-altropyranosides yield D-mannosides, not ~ - i d o s i d e s . ~ ~ ~p.~ 125). (see Methyl 3,4-anhydro-a-(or P-)D-allopyranoside derivatives have not received much study, but, in every example studied, abnormal products have been isolated, although in poor yield and from impure epoxides. This would suggest that the alternative half-chair conformation (‘Ho) for this epoxide has a stability comparable to that of the expected form (OH,), and, in fact, affords an easier pathway for substitution. With the a - anomer, ~ the interaction previously suggested between the epoxide-ring oxygen atom and the anomeric oxygen atom would destabilize 50, and intramolecular hydrogen-bonding would stabilize 51. However, methyl 3,4-anhydro-2,6-di-O-methyl-

P-D-allopyranoside lacks both of these features, and yet it reacts with ammonia, apparently, through the alternative half-chair form (‘H,,; 52), to give, primarily, the 3-amino-3-deoxy-~-idoside.~~ The alternative half-chair conformation (‘H0)is here stabilized by a pronounced

OXIRANE DERIVATIVES OF ALDOSES

149

anomeric effect, the quasi-axially attached methoxyl group on C-2 enhancing the tendency for the anomeric methoxyl group to be axial. The common feature linking these epoxides is that substitution occurs OMe

at the oxirane carbon atom farther from the alkyl group on C-5. Perhaps, the relatively small steric effect that this group would be expected to exert when attached quasi-equatorially is, nevertheless, sufficient to dominate the relative stabilities of the two transition states, but this series merits further study before more definite conclusions are drawn. 3,4-Anhydro-D-galactose derivatives, by contrast, have been extensively studied, although interpretation of the earlier work was confused by the unsuspected presence of 2,3-anhydro-~-gulose analogs as impurities, formed by epoxide migration under the alkaline conditions employed in the synthesis. Most reagents behave normally, but, again, the results of hydrogenation over Raney nickel are anomalous,” as comparable amounts of normal and abnormal products are given by methyl 3,4-anhydro-a(and P)-D-galactopyranoside,24, I :is and primarily the abnormal methyl 4,6-dideoxy-a-~-xylohexopyranoside is given by methyl 3,4-anhydro-6-deoxy-a-~-galactopyranoside.l:’s No marked inductive effects would be anticipated that would affect substitution in 3,4-anhydro sugars, because the adjacent carbon atoms are similarly substituted. In the expected OH, conformation 53, some hindrance to substitution at C-3 by the anomeric group at C-1 might be expected, especially with the a-D anomer, whereas, in the alternative ‘H, form 54, C-4 could only be subject to possible polar effects of the pyranose-ring oxygen atom, and, to some extent, of the quasi-axial group on C-2. T h e ratio of the “

H,OR

NEIL R. WILLIAMS

150

products may, therefore, well depend on the precise steric requirements and polar effects of the reagents concerned. This hypothesis may account for the fact that, with methyl 3,4-anhydro-a-~-galactopyranoside, sodium methanethiolate in ethanol reacts n ~ r m a l l y , ' ~ but the 6-deoxy analog with sodium butanethiolate in butanethiol reacts a b n ~ r m a l l y ;perhaps, '~~ this behavior reflects different solventeffects on the nucleophiles and on the resulting transition-states. A comparable stability of the two half-chair forms (53 and 54) of this 6-deoxy epoxide is also suggested by the fact that sodium azide with ammonium chloride in aqueous 2-methoxyethanol gives a 1:1 mixture of 3- and 4-azido-4-deoxy derivatives.136 Benzyl 3,4-anhydro-2-(benzyloxycarbonylamino)-2-deoxy-a-~-galactopyranoside undergoes solvolysis in 50% aqueous acetic acid to give the oxazolidone derivative 55 by neighboring-group participation from the adjacent amide group. Subsequent alkaline hydroderivative, and this lysis leads to the 2-amino-2-deoxy-~-gu~ose sequence represents overall, normal hydrolysis of the epoxide ring.'"'

t

where 2 is CO-OCH,Ph.

8. 2,3- and 3,4-Anhydropentopyranosides Chart V summarizes the reactions of 2,3- and 3,4-anhydropentopyranosides. Buchanan and coworkers have shown144that the methyl 2,3-anhydro-a(and P)-lyxosides, and 3,4-anhydro-@-~-arabinoside adopt that half-chair conformation (5H0)in which the anomeric methoxyl group is axially attached; this indicates the importance of the ano-

OXIRANE DERIVATIVES OF ALDOSES

151

CHART V

OH 3-subs. D-xylo

a(R' = OMe, R" = H): ( Y = OH) X = SMe.145'(2:l ratio, 3:2 subs.) = OMe, R' = H): (Y = OH) X = H,'45."14fi SMe,'450NH,,109"C1,'46 Br,148 CN,113'.149 Ph,102 CH = CMe,,Io2 C = C-Ph,'"Z CH = CHPh'02; (Y = OMe) X = OH,'50 OMe,'J";(Y = OCHLPh)X = I,'5I0 F1s2'.'53., (Y = NJ X = OH,154.1550 OMe.23 P(R" = OCH,Ph, R' = H): (Y = OH) X = F.IS6;(Y = OMe) X = OH.5' Abnormal: a (R' = OMe, R" = H) (Y = NJ X = O H ( D and L series), (1:1 ratio, 2:3

p (R"

(R' = OMe, R" = H) (Y = OAc) X = OHfi7(3-subs.); (Y = N,) X = OH154( D and L series) (3-SllbS.) p (R" = OMe, R' = H) (Y = OAc) X = OH6' (3-subs); (Y = N J X = OH.'54(2-subs.) 01

(145) S. Mukhejee and A. R. Todd,]. Chern. Soc., 969 (1947). (146) R. Allerton and W. G. Overend,]. Chern. Soc., 1480 (1951). (147) J . Honeyman,j. C h e m Soc., 990 (1946). (148) P. W. Kent, M. Stacey, and L. F. Wiggins,/. Chern. SOC.,1231 (1949). (149) N. R. Williams, Chem. Comrnun., 1032 (1967). (150) L. Hough and J. K. N. Jones, ]. Chem. Soc., 4349 (1952). (151) (a) N . F. Taylor and G . M. Riggs, Clzem. Ind. (London), 209 (1963);(b)]. Chern. Soc., 5600 (1963). (152) N. F. Taylor, €3. F. Childs, and R. V. Brunt, Chern. I n d . (London), 928 (1964). (153) J. A. Wright and N. F. Taylor, Corbohyd. Res., 3, 333 (1966-67). (154) A. J . Dick and J. K. N. Jones, Can. J. Chem., 45, 2879 (1967). (155) A. J. Dick and J. K. N. Jones, Can. ]. Chern., 43, 977 (1965). (156) S. Cohen, D. Levy, and E. D. Bergmann, Chem. Ind. (London), 1802 (1964).

152

NEIL R. WILLIAMS

3,4-Anhydro-p-~-ribo

(R = OMe) X

=

4-subs. O-lyXO

NH2,ls7NHMe,lS7 NMep,lS7NHPh,lS7 Br.’58 (R = OEt) X = OMe,159

SCH2PhISm

3, 4-Anhydro-D- avabino

a!

3-subs. D-lYXO

(R’= OMe, R” = H) X=OH.67

p (R” = OMe, R’ =H) X = OH.s7 *L Series actually

studied.

meric effect in determining the conformation. Methyl 3,4-anhydroa-D-arabinopyranoside (56), however, favors a half-chair conforma-

tion in which the methoxyl group is equatorially attached; this can be explained by the presence of a marked interaction between the epoxide-ring oxygen atom and the anomeric methoxyl group in the alternative form. For 2,3-anhydro-p-~-ribopyranosides, the normal mode of cleavage leads to 3-substitution, giving D-X& derivatives. This result implies (157) W. G. Overend, A. C. White, and N. R. Williams, Chem. Ind. (London), 1840 (1963). (158) P. W. Kent and P. F. V. Ward, 1.Chem. SOC., 416 (1953). (159) J. Piotrovsky, J. P. H. Verheyden, and P. J. Stofin, Bull. SOC. Chim. Belges, 73, 969 (1964). (159a) J. P. H. Verheyden and J. G. Moffatt, J . Org. Chem., 34,2643 (1969).

OXIRANE DERIVATIVES OF ALDOSES

153

that the transition state develops from the diaxial opening of the oxirane ring in the alternative half-chair form of the epoxide (57). This result is most readily rationalized on the basis of the simple steric effects of the substituents at C-1 and C-4, the anomeric group trans to the oxirane ring hindering the approach of the nucleophile to C-2, whereas the substituent on C-4 is cis related and offers no resistance to substitution on C-3. Accordingly, in the two reactions investigated

/

in the a-D series, in which both adjacent substituents are cis related to the oxirane ring, the extents of substitution at C-2 and C-3 are comparable. Polar effects would be expected to support substitution at C-3, but the results in the a - ( or ~ L) series suggest that these effects are not dominant. Conformational effects seem also to be unimportant, as, in the a-series, transition states are equally readily achieved from either half-chair form; and in the @-series,the two halfchair forms would be expected to be of comparable stability, besides being readily interconvertible. 2,3-Anhydrolyxopyranosides have received little attention. Buchanan and R. FletcherG8have shown that the hydrolysis of the oxirane ring in the anomers of methyl 4-0-acetyl-2,3-anhydro-~lyxopyranoside in 80% aqueous acetic acid takes place with participation from the acetoxyl group, to give D-arabinose derivatives. Alkaline hydrolysis of methyl 2,3-anhydro-4-azido-4-deoxy-a-~-(or L)-lyxopyranoside proceeds with substitution at C-3, to give the corresponding arabinose derivative, whereas the @ anomers undergo substitution at C-2, to give the xylose derivatives. Again, a simple steric effect of the trans-azido group can account for favored substitution at C-2, the less hindered position, in the @ anomers, and

154

NEIL R. WILLIAMS

substitution at C-3 in the a anomers could result from a dominant polar effect, as both substituents adjacent to the oxirane ring are trans, making comparable the steric effects at the two positions. H ~ n e y m a n ' ~reported ' the synthesis of methyl 2,3-anhydro-p-~lyxopyranoside, and claimed that, on alkaline hydrolysis, this gives a 2: 1 ratio of L-xy1oside:L-arabinoside, but these results could not be substantiated by Buchanan and R. Fletcher,fiRwho recorded different constants for the epoxide and for the disulfonic ester claimed'47to be the starting material for its synthesis. The same epoxide has been synthesized by Reist and coworkers,IfiOand its properties are in agreement with Buchanan and Fletcher's results. Ethyl or methyl 3,4-anhydro-p-~-ribopyranoside undergoes substitution at C-4 in all the reactions thus far studied. Neither half-chair conformation would seem to be clearly favored, but the specificity observed can be rationalized by considering the steric and polar interactions that may arise in the transition state; substitution at C-3 in conformation 58 would involve marked interactions between the nucleophile and

OMe

i/

(58)

HO

the axially attached anomeric alkoxyl group, which would be avoided in the alternative half-chair form ('HJ; in this conformation, the pyranose-ring oxygen atom would be expected to offer less hindrance to the nucleophile. In the 3,4-anhydro-arabinose series, only the hydrolysis of the anomers of methyl 2-0-acetyl-3,4-anhydro-~-arabinopyranoside with aqueous acetic acid appears to have been studied;fiw both compounds undergo substitution at C-3, with participation of the acetoxyl group, leading to the corresponding D-lyxose derivatives. (160) E.J . Reist, L. V. Fisher, and D. E. Gueffroy, J . Org. Chem., 31,226 (1966).

OXIRANE DERIVATIVES OF ALDOSES

155

9. 2,3-Anhydropentofuranosides Chart VI summarizes the reactions of the 2,3-anhydropentofuranosides. With these compounds, the oxirane ring constrains the four carbon atoms in the furanose ring to be coplanar, forcing the molecule to assume an envelope conformation in which the ring-oxygen atom is above or below the plane of the carbon atoms. No conformational difference therefore exists between C-2 and C-3 in the ground state, and conformational effects, if of any significance at all, will only become apparent in the transition state. The direction of cleavage of the epoxide ring appears to b e governed by a combination of the steric and polar effects of the groups adjacent to the epoxide ring. Groups that are trans-related hinder substitution at the adjacent carbon atom. Polar effects appear to favor substitution at C-3, as for pyranosides. Both effects combine to direct a-1yxo epoxides to h u b stituted arubino products, and no exceptions have as yet been found for this stereochemical rule. On the other hand, the two effects oppose each other with the a - r i h epoxides, and either 2- or 3substitution occurs. 2-Substitution seems to be favored in most cases, suggesting the predominance of the steric factor, but, whereas ammonia attacks C-2 in the 5-deoxy epoxide, it is reported to attack CHART VI

x 2 , 3 - A n h y d r o - a - ~ -lyxo

3-subs. ( Y - D - arabino

(R' = OMe): (Y= OH) X = OMe,'" SCH2Ph,IV2NH2**~161~'fi3; (Y= OAc) X OMe) X = OMe,'61NHylK:';(Y= OCH,Ph) X = F.'":' (R' = theophylline) (Y= OH) X = SEt.48

= B P 4 ; (Y =

(161)E.E.Percival and R. Zobrist, \ . Cltem. Soc., 564 (1953). (162)J . E. Christensen and L. Goodman, J. Org. Chern., 28, 2995 (1963). (163)(a) J . M . Ariderson and E. Percival, .J. Clzent. Soc., 1042 (1955);(I>) ibid., 819 (1956). (164) E. J. Reist and S. L. Holton, Curboh!/d.Res., 2, 181 (1966).

156

WR'

YCH,

0

NEIL R. WILLIAMS

+

=YCL;;"

X 2,3-Anhydro-P-D- lyxo

YC.cz)"

X 3-subs. p - ~ - ~ a b i ? ~ ~

2-subs. p-~-xylo

(R' = OMe): (Y = OH) X = NH222,163 (3 subs.), SCH,PhlE5(3:2)*, SCN1B6(3:7)"; (Y = OAc) X = Brl@ (2:l)'; (Y = OTr) X = SBzlE7(1:l)'; (Y = OMe) X = NH,IB3 (3 subs); (3:4)' (Y = OCHzPh)X = (R'= OEt): (Y = OH) X = NHZ3' (3 subs.); (Y = tetrahydropyran-2-yloxy)X = NHz34 (3 subs.). (R' =adenine): (Y = O H ) X = O B Z '(3 ~ ~subs.), S E P 9 (3 subs.), SCH,Ph'68 (1:5)', N:%I7O (3 subs.); (Y = H) X = OBz"' (1:2)', SCHzPh172 (l:l)', N31T2(1:3)' (R' =6-(dimethylamino)-2-(methylthio)purine:(Y = O H ) X=NH2173(3 subs.) (R'= uracil): (Y = OH) X = OH174(3:17)', NHz175(3 subs.), Br175R (3 subs.); (Y = OMS) X =OH174(1:4)0,0CHzPh174(3subs., followed by 2,5,-anhydride formation)

YCH, D

0 O

YCH, M

e

-

" V O M e HO

2, 3-Anhydro-a-D-ribo

2-subs. a-o-arabino

+

0

QOMe OH

3-subs.(Y-D-XYIO

(165) G. Casini and L. Goodman,J. Amer. Chem. SOC.,85,2357 (1963);86,1427 (1964). (166) L. Goodman, Chem. Commun., 219 (1968). (167) K. J . Ryan, E. M. Acton, and L. Goodman, J . Org. Chem., 33, 3727 (1968). (167a) J. A. Wright and J. J. Fox, Carbohyd. Res., 13, 297 (1970). (168) W. W. Lee, A. Benitez, L. Goodman, and B. R. Baker J. Amer. Chem. SOC., 82, 2648 (1960). (169) A. P. Martinez, W. W. Lee, and L. Goodman, J . Org. Chem., 31, 3262 (1966). (170) W. W. Lee and A. P. Martinez, unpublished results, cited in Ref. 171. (171) E. J . Reist, D. F. Calkins, and L. Goodman, J. Org. Chem., 32, 2538 (1967). (172) E . J. Reist, V. J. Bartuska, D. F. Calkins, and L. Goodman, J . Org. Chem., 30, 3401 (1965). (173) B. R. Baker and R. E. Schaub, J. Amer. Chem. SOC.,77,5900 (1955). (174) I. L. Doerr, J. F. Codington, and J. J. Fox, J . Org. Chem., 30, 467 (1965). (175) J. F. Codington, R. Fecher, and J. J . Fox, J . Org. Chem., 27, 163 (1962). (175a) M. Mirata, T. Naito, and Y.Nakai, Japan Pat. 6,813,214 (1968);Chem. Abstracts, 70,38051 (1969).

OXIRANE DERIVATIVES OF ALDOSES

157

(Y = OH) X = OMe,75(2 subs.), NH21769177 (3 subs.); NH4SCN+3,5-thietane1"; KCN+ 3,5-1act0ne~~; (Y=OAc) X=BrI7'(5:2)"; (Y =OTs)X=H1"** (2 subs.), SCH,Ph17Hon (2 subs.); (Y = H) X=NH21R"(Zsubs.);(Y =OCH,Ph) X=F1"'

(R' = OMe): (Y= OH) X = H,I6*SEt,'*' NH2176.177; NH,SCN+3,5-thietane17s; (Y= OAc) X = Brl79; (y OTs) X = H,17BooSEtlD8"'., (Y = OCH,Ph) X = F'"; (Y = H) X = NH,.''" (R' = adenine) (Y = OH) X = S E P ; (Y = H) X = SCH2Ph,172 N:
C-3 in the unsubstituted ~ o m p o u n d . ' ~ However, ~~'~' at the time of these experiments, only C-3 substitution had been observed with 2,3-anhydropentofuranosides,and the results of investigation of this reaction suggest that the amino sugar substituted at C-2 is the major product.184 With thiocyanate ion, substitution also occurs a t C-3, leading to the thietane derivative, namely, methyl 3,5-anhydro-3-thioa-D-xylofuranoside (59); but, as this compound was obtained in only 25% yield, the major pathway may involve 2-substitution, leading to products not ~ h a r a c t e r i z e d . ' ~ ~

(176) R. E. Schaub and M. J. Weiss,J. Amer. Chem. SOC.,80,4683 (1958). (177) C. D. Anderson, L. Goodman, and B. R. Baker, J . Amer. Chem. SOC., 80, 5247 ( 1958). (178) L. Goodman,J. Amet. Chem. SOC., 86, 4167 (1964). (179) E. J. Reist and S. L. Holton, Carbohyd. Res., 9, 71 (1969). (180) H. Kuzuhara and S. Emoto, Agr. Biol. Chem. (Tokyo), 27, 689 (1963); 28, 184 (1964). (180a) J. A. Wright, N. F. Taylor, and J. J. Fox, J . Org. Chem., 34,2632 (1969). (181) C. D. Anderson, L. Goodman, and B. R. Baker, J . Amer. Chem. SOC., 81, 898 (1959). (182) J . A. Wright and N. F. Taylor, Carbohyd. Res., 6,347 (1968). (183) C. D. Anderson, L. Goodman, and B. R. Baker, J. Amer. Chem. SOC., 81, 3967 (1959). (184) J. G. Buchanan and D. R. Black, unpublished observations.

158

NEIL R. WILLIAMS

OH (59)

OH (60)

It is interesting that, apparently, cyanide ion does substitute75 mainly at C-3, the cyano group being hydrolyzed under the conditions of the reaction with assistance from the 5-hydroxymethyl group, to give the lactone 60. Here, the reaction may be rationalized if the initial addition of cyanide is reversible, C-3- substituted product being irreversibly removed by h y d r o l y ~ i s . ~ ~ For the p anomers of these epoxides, where the substituents at C-1 and C-4 are either both cis or both trans to the oxirane ring, the steric effects of the two groups might be expected to be comparable, so that the direction of cleavage of the epoxide ring becomes controlled by polar effects. Accordingly, favored 3-substitution is exhibited by both the p-lyxo and p-ribo epoxides, particularly in the latter series, where no exceptions to the rule have been found. Epoxides having the ribo configuration have both substituents trans to the oxirane ring, and these groups hinder the approach of the incoming nucleophile; consequently, the greater stereoselectivity in this series may be explained if the grouping at C-1 offers more hindrance than that at C-4. This is a reasonable explanation where the group at C-1 is a residue of a purine derivative, but it is more surprising for a methoxyl group, as compared to the 4-(hydroxymethyl) substituent, in view of the relative sizes of these groups that are suggested by con formational free-energy differences for cyclohexane compounds.185 However, an electronic repulsion of the nucleophile by the lone pairs of the anomeric oxygen atom could significantly increase the energy of the transition state for 2-substitution, and Reist and H 0 1 t o n ' ~have ~ suggested this explanation for the fact that, with magnesium bromide, the LY anomer of methyl 5-O-acetyl-2,3-anhydro-~-ribofranoside reacts much faster than the p anomer, the former reacting at C-2 (unhindered position), and the latter at C-3 (the less-hindered position). The steric effects of the adjacent trans substituents seem also to be responsible for the unreactivity of the oxirane ring in methyl 2,3anhydro-5-O-trityl-~-~-ribofuranoside with ammonia,180and for the similar inertness of 9-(2,3-anhydro-p-~-ribofuranosyl)-6-(dimethyl(185)Ref. 66, p. 44.

OXIRANE DERIVATIVES OF ALDOSES

159

amino)-2-(methylthio)purine and its 5-trityl ether,'*6 in which one (or both) of the substituents adjacent to the oxirane ring is particularly bulky. However, the ethanethiolate anion, a more powerful nucleophile, reacts with (2,3-anhydro-5-0-trityl-~-~-ribofuranosyl)theophylline to give the 3-ethylthio d e r i v a t i ~ e9-( . ~2,3-Anhydro-5-deoxy-P-~~ ribofuranosy1)adenine reacts with sodium benzoate in N,N-dimethylformamide to give a product for which the anhydro-nucleoside structure 61 was suggested on the basis of ultraviolet spectral evidence, but the compound was not characteri~ed.'~'

OH

For the p-1yxo epoxides, steric effects would be expected to be minimal, because both of the groups adjacent to the oxirane ring are on the side of the furanose ring opposite to the approaching nucleophile, so that polar effects should control the stereochemistry of the reaction. A general tendency to 3-substitution is observed, but methyl 2,3-anhydro-P-~-~yxofuranoside with sodium m-tol~enethioxide,'~~ and the 5-acetate of this epoxide with magnesium bromide,Ifi4both give, primarily, 2-substituted products, and, in most of their reactions, both isomers possible can be detected. Clearly, polar influences here are not very strong, and inductive effects are readily modified by more subtle differences in the reagents and in the conditions employed. Nitrogen nucleophiles show a greater tendency than the more nucleophilic sulfur reagents to give 3-substituted products, but it is, at present, difficult to rationalize the precise pattern observed. Thus, sodium a-toluenethioxide in methanol shows a slight tendency to favor 2-substitution with methyl 2,3-anhydro-/3-~-lyxofuranoside,'g~ but the 3-substituted product mainly results from similar treatment of the glycosylamine derived from adenine,lfiSwhereas the 5-deoxy analog of the latter gives approximately equal proportions of the 2- and (186) R. E. Schaub, M. J. Weiss, and B.R. Baker,]. Arner. Chern. SOC., 80,4692 (1958).

NEIL R. WILLIAMS

160

3-substituted products.172These particular observations might be rationalized by assuming that, in addition to inductive effects, the electronic interaction between the developing oxide anion and adjacent polar groups is important in the transition state, a more polar substituent encouraging substitution at the carbon atom nearer to it, so that the oxirane oxygen atom moves away from the polar group.

10. 5,6-Anhydrohexofuranoses Chart VII summarizes the oxirane reactions of 5,6anhydrohexofuranoses. With these oxiranes, polar and steric effects combine to favor substitution at the (terminal) primary carbon atom, and in every instance, 6-substitution predominates. However, hydrogenation of 5,6-anhydro-1,2-0-isopropylidene-a-~-glucofuranose in methanol, in the presence of Raney nickel and ofan acid resin to remove residual traces of alkali, gives'89 a mixture of deoxy sugars containing up to in ad33% of 5-deoxy-1,2-O-isopropylidene-a-~-xylo-hexofuranose, dition to the major product, namely, 6-deoxy-1,2-O-isopropylidene-cwD-ghcofuranose, This result re-emphasizes the distinctive character of hydrogenation catalyzed by Raney nickel as a method for the cleavage of epoxides which is relatively insensitive to the polar and steric effects that clearly govern the stereochemistry of related reactions with other reagents. It is interesting that, prepared in the usual way without neutralization of the alkali, Raney nickel affords the expected 6-deoxy sugar ~ n l y . ~ ~ ~ J ~ ~ Hydrolysis of 5,6-anhydro-1,2-0-isopropylidene-a-~-glucofuranose with sodium hydroxide under controlled conditions can lead7'*74to and 3,6both 3,5-anhydro-l,2-O-isopropylidene-~-~-idofuranose anhydro-1,2-O-isopropylidene-a-~-glucofuranose, in addition to 1,2-O-isopropylidene-a-D-glucofuranose (see p. 130), whereas mild hydrolysis with 5 mM sulfuric acid can give up to 25% of 2,5-anhydro' ~ ~ reaction must inL-idose (62),besides the expected D - g l u ~ o s e .This CHO I

HCOH

I HOCH

I

HYOH

HOH,CQH0

HC, H,kO

HO

OXIRANE DERIVATIVES OF ALDOSES

161

CHART VII XCH, I

I

I

z '0

i.-.~

5.6-Anhydro-o! - 0 - g Z ~ c o

I

6-subs. LY

I

2..

-D-&K~

(Z = H)

(Y = OH, Z = H) X = H,ls7-18yOH," OP=O(OH),,LSO OP=-O(OCH,Ph),,8S S B Z ,NH,,lS1 ~~ NHCHRC0,H,'Y2 CHO,IY3 CO,Me,l"; NaOH+3,6-anhydro-D-gl~cose~'~~'; 5 mM H2S0,-t2,5-anhydro-~-idose'95; CS2+dithiothionocarbonate1s6*'y7; (NH,),CS-episulfide56;KSeCN-hex-5-eno~e~~; Me,SO/BF,,~dialdose.'ya (Y= OMe, Z = H) X = OMe,Ig9OCH,PhLoo;KOH+polymer.zol (Y = OCH,Ph, Z = H ) X = H," OH,', OCHPPh,202Ph.,03; (NH2),CS-episulfide'OZ; MeLi+he~-5-enose.~~~ 5,6-Anhydro-~-~-allo+6-subs. ff-D-do (Y=H, Z=NHAC)X=NH,.'~'

(187) K. Freudenberg, H. Eich, C. Knoevenagel, and N. Westphal, Ber., 73,441 (1940); E. J. Reist, R. R. Spencer, and B. R. Baker,]. Org. Chem., 23,1753 (1958). (188) E. Vischer and T. Reichstein, Helv. Chim.Acta, 27, 1332 (1944). (189) E. J . Hedgley, 0. MerBsz, and W. G. Overend, J. Chem. Soc. ( C ) ,888 (1967). (190) G. P. Lampson and H. A. Hardy,]. B i d . Chem., 181, 693 (1949). (191) J . Jary', V. HeiniAnkovii, and K. KovAi., Collect. Czech. Chem. Commun., 31: 2048 (1966). (192) K. Gluzman and V. I. Kovalenko,]. Gen. Chem. USSR (Engl. Transl.),23,283,287 (1953); Sb. Statei Ohshch. Khim., Akucl. Nauk SSSR, 1, 457, 462, 465, 469, 482 (1953);Chem. Abstracts, 49, 878, 879, 881 (1955). (193) A. Rosenthal and G. Kan, Tetrahedron Lett., 477 (1967). (194) A. Rosenthal and J. N. C. Whyte, C o n . J . Chem., 46,2239 (1968). (195) C. A. Dekker and T. Hashizume, Arch. Biocheni. Biophys., 78, 348 (1958). (196) G. P. McSweeney and L. F. Wiggins, Nuture, 168, 874 (1951). (197) A. M. Creighton and L. N. Owen, 1.Chem. Soc., 1024 (1960). (198) G. Henseke and G. Hanisch, Cltem. Ber.., 101, 2074 (1968). (199) R. B. Duff and E. G. V. Percival,]. Chem. Soc., 1675 (1947). (200) J. Kenner and C . N. Richards, ]. Chem. Soc., 3277 (1954). (201) R. S. Nevin, K. Sarkanen, and C. Schuerch, ./. Amer. Cheni. Soc., 84, 78 (1962). (202) U. G. Nayak and R. L. Whistler,]. Org. Chem., 34, 97 (1969). (203) J. English, Jr., and M. F. Levy, J. Amer. Chem. Soc., 78, 2846 (1956). (204) U. G. Nayak and R. L. Whistler, Chem. Commun., 434 (1969).

162

NEIL R. WILLIAMS

5,6-Anhydro- p-L- id0

~ - s u ~ sf i.- ~ - i &

(Y = OH) X = H,12 OH',; NaOH+3,5-0xetane'~; (NH,),CS+epis~lfide~~; KSeCN+ hex-5-eno~e.~~ ( Y = OMS) X = NH,.205 (Y= OCH,Ph) X = H,'* N3206;M e L i j h e x - 5 - e n o ~ e . ~ ~ ~ 5,6-Anhydro-3-deoxy-P-~-lyxo-hexo+6-subs. 3-deoxy-P-~-lyro-hexo(Y = H) X = OCHzPh.m' 5,6-Anhydro-3-deoxy-l,2-O-isopropylidene-c~-~-xylo-hexofuranose~3-deoxy-~-xylohexose 6-pho~phate.'~'

volve preliminary hydrolysis of the isopropylidene acetal, because the sugar must achieve an acyclic form before formation of the anhydro ring becomes sterically possible. It is interesting that the 2,5-anhydro ring is favored over the 2,6or the 3,6-anhydro ring, even though its formation involves substitution at C-5 (a secondary position). This behavior may be rationalized on the basis of the well known greater ease of formation of 5- over 6membered rings; the 2,5- is formed more readily than the 3,6-anhydro ring, as the 2-hydroxyl group has more conformational freedom to approach the rearside of C-5 than the 3-hydroxyl group has to approach C-6, the oxirane ring meanwhile holding C-4, C-5, and C-6 in a rigid structure. Reaction of 5,6-anhydro-1,2-0-isopropylidene-a-~-glucofuranose (2) with thiourea yieldss6 5,6-dideoxy-5,6-epithio-1,2-O-isopropylidene-p-L-idofuranose (63), and its 3-benzyl ether behaves similarly.202 Conversely, the ~ - i d oepoxide gives the D-gluco episulfide.56Reacbion of 2 with carbon disulfide and potassium hydroxide 5,6-thioleadsIg7 to 1,2-O-isopropylidene-5,6-dithio-~-~-idofnose nocarbonate (64), originally incorrectly assigned the a-gluco configuration.'% Potassium selenocyanate with 2 (or the ~ - i d oisomer) y i e l d P 1,2-0-isopropylidene-a-~-xyEo-hex-5-enofuranose (65) by elimination of the oxirane oxygen atom. A similar elimination had earlier been (205) J. K o d i and J. ]a$, Collect. Czech. Chern. Commun., 33, 549 (1968). (206) H. Saeki, T. Iwashige, and E. Ohki, Chern. Pharrn. Bull. (Tokyo), 16,188 (1968). (207) K. Antonakis, A. Dowgiallo, and L. Szabb, Bull. SOC. Chirn. Fr., 1355 (1962).

OXIRANE DERIVATIVES OF ALDOSES

163

observed in the reaction of the 3-benzyl ether of 2 or its ~ - i d oisomer with methyllithium;'O" methyl substitution at C-6 had been expected. This behavior contrasts with that shown by the 2,3-anhydro-allo and -manno epoxides discussed previously (see p. 134), although similar eliminations are known for the reaction of simple oxiranes with Grignard reagents. Oxidation of 2 with methyl sulfoxide and boron trifluoride proceeds by conversion of the oxirane ring into an a-hydroxy aldehyde group, just as for simple oxiranes, g i ~ i n g ' the ~ " dialdose derivative 66.

11. Miscellaneous Oxiranes a. 1,2-Anhydrohexopyranoses.- Carbohydrate oxiranes in which the anomeric carbon atom is included in the oxirane ring are much more reactive than members of any other class of sugar oxiranes; they always react by substitution at the anomeric carbon atom, because any positive charge that develops on this carbon atom during the substitution reaction can readily be delocalized to the adjacent ring-oxygen atom, thus lowering the energy of the required transition state. In contrast to the behavior of other oxiranes, substitution is not necessarily accompanied by inversion of configuration; this may be attributed to encouragement of s N 1 substitution by the relative stability of the glycosidic carbonium ion, together with the possibility of neighbouring-group participation of the 6-acetoxyl group (which is present in all compounds in which retention of configuration has been observed).

NEIL R. WILLIAMS

164

Attention has largely been given to 3,4,6-tri-O-acetyl-l,2-anhydroa-D-glucopyranose (67);this is commonly called the Brigl anhydride, as Brigl first reported its synthesis and reactions.208Compound 67 may be prepared by first treating penta-0-acetyl-D-glucopyranose with phosphorus pentachloride to yield the acetylated 2-O-(trichloroaCetr.l)-D-gIUCOpyrdnoSyl chloride (68); on reaction with ammonia, 68 loses the trichloroacetyl group to give a mixture (69) of triacetate AcOYH,

AcOYH,

AcO

OAc

AcOYH,

AcOYH,

(67)

chlorides, the p anomer of which reacts further with ammonia to give the acetylated 1,2-anhydro s ~ g a r . ” ~The ~ ’ ~6-deoxy ~ analog of the Brigl anhydride may be similarly prepared.*’O With titanium tetrachloride, the Brigl anhydride gives”’ the CY anomer of the chloride 69. The value of such epoxides in synthesis lies in their ready reaction with alcohols to give glycosides. The Brigl anhydride reacts readily with methanol or ethanol at room temperature to yield the corresponding P-D-glycosides by inversion of configuration at the anomeric carbon atom. Although higher primary alcohols also give p-D-gly-

(208) P. Brigl, Z . Physiol. Chem., 122, 245 (1922). (209) Y. Inoue, K. Onodera, I. Karasawa, and Y. Nishisawa, Nippon Nogei Kngaklt Kaishi, 24,362 (1950-51). (210) E. Hardegger and R. M. Montavon, Helr;. Chim. Acta, 30, 632 (1947). (211) Z. Csiiriis, G. Dehk, and M . Haraszthy, Acta. Chim.Acad. Sci. Hung., 21, 181 (1959);Chem. Abstracts, 54,16389 (1960).

OXIRANE DERIVATIVES OF ALDOSES

165

cosides,212the claim by Hardegger and de PascuaP3 that, stereospecifically, isopropyl, tert-butyl, and benzyl alcohol react similarly, confirming an earlier observation with isopropyl alcohol and menthol by Hickinbottom,2" has been contested b y Veibel2lSon the basis of the optical rotation of the products. Veibel determined the values for the pure p-D anomers prepared by the Koenigs-Knorr method, and checked their purity by hydrolysis with emulsin. These bulky alcohols apparently give ar,p mixtures ,215 as does chole~tanol,"~ whereas cholesterol gives a low yield of a D-glucoside that, apparently, has the a - ~ The reactions with these bulky alcohols require elevated temperatures. The Brigl anhydride reacts with carbohydrate derivatives to give a-D-glucosides, and this fact has been utilized by Lemieux and coworkers in the synthesis of maltose,216trehalose,"' and sucrose."" Phenol also gives the a-Dglucoside."4 Lemieux and Huber2'" have explained the tendency of the Brigl anhydride to give a-D-glucosides with the bulkier alcohols on the basis of the steric effect of the 5-(acetoxymethyl) group on the pyranose ring; in the reactive, iH4 conformation 70, which allows diaxial opening of the oxirane ring, this substituent is axially attached and can hinder the approach of the reagent alcohol, thus preventing the formation of the P-D-glucoside by the normal sN2 mechanism. If this occurs, the 6-acetoxyl group can participate in the reaction, giving an acetoxonium ion (71) which may then react with the alcohol to give the a-D-ghcoside. It would be interesting to learn whether

(212) I. Karawasa and R. Onishi, DoshfshaJoshiDaigaku, 9 , 5 (1958);Chern. Abstracts, 54,3215 (1960). (213) E . Hardegger and J. de Pascual Teresa, Helw. Chlrn. Acta, 31, 281 (1948). (213a) C. L. Stevens and R. E. Harmon, Carbohyd. Res., 11, 99 (1969). (214) W. J. Hickinbottom,]. Chem. Soc.,3140(1928). (215) S. Veibel, Helo. C h i n . Acta, 31,736 (1948). (216) R. U. Lemieux, Can..\. Chem., 31,949 (1953). (217) R. U.Lemieux and H . F. Bauer, C a n . ] .Chem., 32,340 (1954). (218) R. U.Lemieux and G . Huber,/. Amer. Chern. Soc., 78,4117 (1956).

166

NEIL R. WILLIAMS

the 6-deoxy analog of the Brigl anhydride shows a similar stereochemical behavior, because, for this compound, the acetoxonium ion (70) is not possible. Lemieux has since stated that substitution is not completely specific for a-D-glucosides, and that it is not necessary to invoke participation of the acetoxyl group in order to account for predominant a-substitution,2'Y because the open, stabilized carbonium ion (72) that can be formed on heating the epoxide will tend to undergo diaxial addition to the unsaturated group, to give the

CH,OAc

(72)

a-D-glucoside from the depicted conformation (TI3 anticipated ) to be favored, Other a-D-linked disaccharides have been synthesized from the Brigl anhydride,219a*21gb which gives both a- and P-D-glycosides with b e n ~ e n e t h i o lWith . ~ ~ ~Lewis-acid ~ catalysts, the Brigl anhydride readily polymerizes to give oligomers having a degree of polymerization of 3 to 5, the ratio of a to p linkages depending on the conditions, with a preponderance of the a anomer suggested by optical rotation studies. Open carbonium ions are postulated to account for the cr-D-linkages formed.219d Where the alcohol cannot be used in excess, yields of D-glucoside are generally poor, but the addition of a small proportion of an acid catalyst ensures much improved yields,22othe yield for cholesterol being raised from 0.5 to 58%. 1,2-Anhydro sugars react similarly with amines; substituted anilines lead to N-aryl-P-D-glycosylamine derivatives.221 With acetic acid at room temperature, the Brigl anhydride rein high arranges, to give 1,3,4,6-tetra- O-acety~-P-D-g~ucopyranose yield."'" With dibenzyl hydrogen phosphate, dibenzyl 3,4,6-tri-0acety~-~-D-glucopyranosy~ phosphate is formed.213a (219) R. U. Lemieux, Methods Carbohyd. Chem.,6 (1971), in press. (219a) J. Kiss, Tetrahedron Lett., 1983 (1970). (219b) G . Maghuin-Rogister, Bull. SOC. Chim. Belges, 77, 575 (1968). (2194 H . Frenzel, P. Nuhn, and G . Wagner, Arch. Pharm. (Weinheim), 302,62 (1969). (21%) J. Zachoval and C. Schuerch,]. Polym. Sci., 28, 187 (1969). (220) J. de Pascual Teresa, An. Real S O C . Espan. F k . Quim., Ser. B , 50, 79 (1954); Chem. Abstracts, 49,3027 (1955). (221) Y. Inoue, K. Onodera, and I. Karawasa, N i p p o n Nogei Kagaku Kaishi, 28, 193 ( 1954).

OXIRANE DERIVATIVES OF ALDOSES

167

On treatment with aqueous alkali, the Brig1 anhydride yields 1,6-anhydro-~-glucopyranose by nucleophilic substitution at C-1 by the oxide anion generated at C-6 on hydrolysis,'22 and this reaction substantiates the hypothesis that 1,2-anhydro-~-glucoseis the intermediate in the alkaline hydrolysis of phenyl p-D-glucopyis obtained. ranoside, from which 1,6-anhydro-~-glucopyranose

b. Oxiranes of Branched-chain Sugars. -A number of oxirane derivatives of branched-chain sugars have been synthesized by the ~ methylsulfox-~~~ treatment of aldosuloses with d i a z ~ m e t h a n e , ~ 'di onium methylide,"7*2'8 or dimethylsulfonium methylide."" Spiro epoxides are formed, and these are valuable intermediates for the synthesis of a variety of branched-chain sugar derivatives. The ratio of the two isomers obtained depends on the reagent, and, with diazomethane, higher homologs are also possible. Thus, with diazometh(73) ane, methyl 3,4-O-isopropylidene-~-~-ert~thro-pentosidulose yields22:3 a 3:1 mixture of the ribo and arabino epoxides (74) and (75). Dimethylsulfoxonium methylide yields the same compounds, but in (76, inverse ratio.'" 1,2-O-Isopropy~idene-c~-~-g~z~cero-tetros-3-u~ose R = H) with diazomethane gives the D-apio-D-furanose epoxide (77, R = H) as the preponderant isomer,22fialso obtained in poor yield with dimethylsulfonium methylide,22Hwhereas the D-apio-L-furanose epoxide (78, R = H) preponderates in the ratio of 6:l with dimethylsulfoxonium methylide.2'8 5-O-Benzoyl-l,2-O-isopropylidene-a-~erythro-pentofuranos-3-ulose(76, R = CH'OBz) gives only the x y l o isomer (78), R = CH,OBz) with diazomethane in ether, but the 4-deoxyhexose epoxide (79) is also formed, as the major product, in methanol-ether,22spresumably by way of an initial ring-expansion of the pentosulose by reaction with diazomethane to give the hexosulose, which then reacts with more diazomethane to give the spiro-epoxide. Here, again, dimethylsulfoxonium rnethylide gives the isomeric ribo (222) M . P. Bardolph and G. H. Coleman,]. Org. Chem., 15, 169 (1950);A. Dyfverman and B. Lindberg, Acta Chem. Scand., 4, 878 (1950). (223) W. G. qverend and N. R. Williams,]. Chem. Soc., 3446 (1965); J. J. K. Novkk and F. Sorm, Collect. Czech. Chem. Commun., 30, 3303 (1965); D. Horton and J . S. Jewel], Carbohyd. Res., 2,251 (1966). (224) B. Flaherty, W. G. Overend, and N . R. Williams, J . Chem. Soc., 398 (1966); Chem. Commun.,434 (1966). (225) S . Nahar, W. G. Overend, and N. R. Williams, Chem. Ind. (London), 2114 (1967). (226) A. D. Ezekiel, W. G. Overend, and N. R . Williams, Tetrahedron Lett., 1635 (1969). (227) R. D. King, W. G. Overend, J . Wells, and N. R. Williams, Chem. Commun., 726 (1967). (228) D. H. Ball, F. A. Carey, J . L. Klundt, and L. Long, Jr., Carbohyd. Res., 10, 121 ( 1969).

168

NEIL R. WILLIAMS

OMe

'

epoxide (77, R = CH,OBz) as the major p r o d u ~ t . "Similarly, ~ methyl 4,6-0-benzylidene-2-deoxy-a-~-erythro-hexopyranosid-3-ulose with dimethylsulfoxonium methylide yieldszz7the 2-deoxy-~-ribo-hexose

0

(79)

spiro-epoxide (BO), and diazomethane in methanol-ether givesri4 the epoxide (81), together with ring-expanded 2,3-dideoxy-~-ribo-heptose a small proportion of the 2-deoxy-~-xylo-hexoseisomer of 80. As

OXIFMNE DERIVATIVES OF ALDOSES

169

would be expected on both steric and polar grounds, these epoxides undergo substitution exclusively at the primary, branch carbon atom, thus preserving the stereochemistry of the spiro carbon atom.

The racemic form of methyl 3,4-anhydro-P-mycaroside (82) has been s y n t h e ~ i z e dit; ~is~an epoxide of a branched-chain sugar having the oxirane ring fused to the pyranose ring. Hydrolysis of the oxirane ring with hydrochloric acid in aqueous p-dioxane occurs with substitution at C-4, the (more accessible) secondary position, as would be expected from its favored OH, conformation (82) anticipated, derivabecause it is an analog of the 3,4-anhydro-~-allopyranoside tives considered previously (see p. 148).

(229) R. J . Ferrier, W. G. Overend, G . A. Raffetty, H. M . Wall, and N . R. Williams, .I.Chem. Sac. (C), 1091 (1968).

170

NEIL R. WILLIAMS

IV. CHARACTERIZATION 1. Chemical Methods a. Sodium Thiosulfate-Phenolphthalein(Ross Test).*”’-A solution ( 1 m M ) of the sugar epoxide in water, aqueous acetone, or aqueous p-dioxane is treated with neutralized 0.2 M sodium thiosulfate containing phenolphthalein. Development of a pink coloration, occurring slowly at room temperature or more rapidly on warming, indicates the presence of an epoxide. The reaction can be used for quantitative estimation of the amount of epoxide present by titration of a weighed sample against 0.2 M acetic acid, so as to discharge the pink color as it forms, until the end point is reached, usually within 20 minutes. The test depends on the rapid, nucleophilic substitution of the oxirane ring by the thiosulfate, which generates a strongly alkaline alkoxide group.

b. Methyl Iodide-Methyl Red.-A 5% solution of sodium iodide in butanol containing 0.01% of Methyl Red has been proposed by Buchanan and Schwarzy as a spray reagent for detecting epoxides on paper chromatograms. After being sprayed, the paper is heated for a few minutes at 140”;epoxides present produce yellow spots on a red badkground. A mechanism similar to that for the Ross test is involved. c. Hydrochloric Acid-Magnesium Chloride. - The ready reaction of sugar oxiranes with 0.5 M hydrochloric acid saturated with magnesium chloride, with monitoring of the consumption of acid by titration with alkali, has been proposed as a test for epoxides b y Wiggins and coworkers.Y5The reaction is usually complete within 15 minutes. However, the test has not been generally adopted, the Ross test providing a simpler method, especially as a qualitative test.

2. Spectroscopic Methods a. Infrared Spectroscopy. - The oxirane ring does not provide clearly diagnostic frequencies in infrared spectra, because the bands that it gives occur in regions where carbohydrate compounds, in general, tend to show absorption bands. However, the presence of a band at -860 cm-I (11.1-11.6 pm), in the region expected for a cis-oxirane derivative, has often been advanced as evidence for the presence of the epoxide structure in sugar derivatives. A variable band at -1250 cm-I (8 pm) has also been utilized, but this band can overlap carbon-oxygen and hydroxyl group-frequencies. (230) W. C. J. Ross,]. Cheni. Soc., 2257 (1950).

OXIRANE DERIVATIVES OF ALDOSES

171

b. Nuclear Magnetic Resonance Spectroscopy. - In chloroform, the oxirane protons of 2,3-anhydroaldopyranosides appear”’*2”2 somewhat upfield of other protons on the carbohydrate chain, in the region r 6.4-6.9, and J2,3 4.0 Hz. These protons show no coupling to adjacent protons on C-1 or C-4 that are trans related (as in the a-manno and p-riho epoxides), and only weak coupling, J 2.5-4.5 Hz, where they are cis-related (as in the a-allo epoxide); this renders difficult the assignment of peaks to H-2 and H-3. Sweet and have demonstrated that, in fact, H-3 is upfield of H-2 (with a chemicalshift difference of 0.3 ppm); they studied a number of 2-alkoxy3,4-epoxytetrahydropyrans (2,3-anhydro-4-deoxypentopyranosides) in the spectra of which the signals for the H-4 protons on the methylene (“deoxy”) carbon atom appear well upfield of the oxirane protons. The results of decoupling experiments then permit confirmation of the assignment of the signals for H-3. The conformations of some methyl 2,3- and 3,4-anhydroglycopyranosides have been determined’44from their n.m.r. spectra at 100 MHz, by utilizing not only vicinal-proton coupling-constants, but also long-range coupling-constants over four bonds and differences in the chemical shifts of equatorially and axially attached protons. For 2,3-anhydro compounds,J,,, orJ4,5,provides the clearest indication of the half-chair conformation favored where the two protons under consideration are trans, and, therefore, either diaxial or diequatorial. 3,4-Anhydro compounds can similarly be analyzed from J1,2 values. The chemical shift of H-1 appears to be the most generally useful parameter, as it lies in the range r 4.95-5.43 when the proton is equatorial or quasi-equatorial, and has a value of 7 5.82 for H - l in methyl 3,4-anhydro-cr-~-arabinopyranoside (83), the only compound thus

&* 0’ 0

OMe (83)

far examined that is considered to have an equatorially attached anomeric group when it assumes its favored conformation (11,26.81 Hz). That this half-chair form is favored in this instance has been attributed to the strong polar interaction between the oxygen atom of (231) D. H. Buss, L. Hough, L. D. Hall, and J. F. Manville, Tetrahedron, 21,69 (1965). (232) F. Sweet and R. K. Brown, Can.J. Chem.,46,1481 (1968).

172

NEIL R. WILLIAMS

the methoxyl group and the oxirane oxygen atom in the alternative form, which offsets the usual, stabilizing, anomeric effect of an axially attached anomeric group (see p. 129). In the spectra of 2,3-anhydroaldofuranose derivatives, the peaks for H-2 and H-3 overlap those for the other protons on the carbohydrate chain, and, somewhat surprisingly, H-2 in methyl or ethyl glycosides generally shows no coupling to H-1, no matter whether the latter is cis- or truns-related.2g3Some differentiation is possible on the basis of chemical-shift differences, as H-1 in a-ribo epoxides appears in the region T 4.73-4.80, whereas, for p-ribo and a- or P-lyro epoxides, it appears upfield, at T -4.89-5.01, but, for these compounds, the technique does not have its usual value for the assignment of configuration. With the spiro-epoxides of branched-chain sugars, the oxirane protons appear somewhat farther upfield, between T 6.8 and 7.5, the two protons showing a variable difference in chemical shift.'224,227 '

v. TABLESOF

ALDOSEOXIRANES

The following Tables list the aldose oxirane derivatives that have been prepared. The solvents used in determination of the specific rotation are given by A, acetone; chloroform; E, Ethanol; E.A., ethyl acetate; M, methanol; P, pyridine; and W, water.

c,

TABLEI Derivatives of 1,6-Anhydro-fi-~-hexopyranoses 1,6-Anhydro-P-n compound Allopyranose, 2,3-anhydro4-0-benzyl4-0-11-tolylsulfonylAllopyranose, 3,4-anhydro2- O-methyl2-O-p-tolylsulfon yl-

M.p.9 degrees

Lab, degrees

94-96 93-96 74-76 146-147 102- 103 104- 106 83-85 116-1 17

41 55 127 52 -125 -134 -174 -95

Rotation solvent

A W C C A W C C

References 82a 233a 233a 233a 82a 233a 233a 233a

(continued) (233) L. D. Hall, Chern. Znd. (London), 950 (1963); T. Hiraoka, T. Iwashige, and I. Iwai,vChem. P h o n n . Bull. (Tokyo),13, 285 (1965). (233a) M. Cern;, T. Trnka, P. Beran, and J. PacPk, Collect. Czech. Chem. Commun., 34,3377 (1969).

OXIFUNE DERIVATIVES OF ALDOSES

173

TABLEI (continued) 1,6-Anhydro-p-~ compound

[a]D,

M.p.7 degrees

Altropyranose, 3,4-anhydro2-O-p-tol y lsulfonyl-

Galactopyranose, 3,4-anhydro2-O-meth yl2-O-),-tolylsulfonylGulopyranase, 2,3-anhydro4-0-acetyl4-0-methyl4-0-p-tolylsulfonylHexopyranase, 2,3-anhydro-4-cleoxy-I!/xo2,3-anhydro-4-deoxy-rihoMannopyranose, 2,3-anhydra4-0-l)enzyl4-O-meth yl4-0-11-tolylsulfonyl4-0-tritylTalopyranose, 2,3-anhydro4-0-methyl3,4-anhvdro-

degrees

158 161 104-106 102-103 67-69 9 1-93 150- 151 148-150 135-137 83-85 36-38 69-70 66-67 68-70 63-65 52-58 137-139 138-140

-108 -124 -76 -70 -80 -77 -37 -40 30 47 4.4 26 -36 30 -35 -34 -40 -37 -17

132 51 73-74

-88 -104 -49.5

Rotation solvent

References

A A

C M C C

15 70 13 70 70 70 80,234 235 70 70 70 70 236 82 13 81 80 13 13

W M W

76 79 83

Rotation solvent

References

M C

123 126a 125a 1251 126a 16 6221 43 27.45

C C W

C C C W

c c C

W W W

TABLE11 Derivatives of 2,3-Anhydrohexopyrai1osides Methyl 2,3-anhydro compound

M.P., degrees

a-D-Allopyranoside 4,6-di-O-acetyl4-azido-4-deoxy 4-azido-4-deoxy-6-0-trityl4-0-benzy l-6-0-trityl4,6-O-benzylidene6-chloro-6-deoxy6-deoxy -4-O-p-toly lsulfony l4.6-0-ethvlidene-

105- I07 67-68 102-103 162 200 93-94 117-1 18 128

[fflD,

degrees 153 160 236 122 91.5 140 155 157 100

c C C C C C C

(contintled)

(234) H. W,. Jeanloz and A. M. C. Rapin,./. Org. Chem.,28,2978 (1963). (235) M . Cerhy, V. Gut, and J . Pacik, Collect. Czech. Client. Cotnttiun., 26, 2542 (1961). (236) M . Cerny, J . Pacik, and J. Stan&k,Chent. Ind. (London),945(1961).

NEIL R. WILLIAMS

174

Methyl 2,3-anhydro compound

4,6-di-O-methyl4,6-di-O-(methylsulfonyl)4,6-O-propylidene6-0-tritylP-D-Allopyranoside 4,6-O-benzylidene4,6-di-O-methyla-D-Allopyranosiduronio acid 4-azido-4-deoxymethyl ester a-D-Gulopyranoside 4-0-acetyl-6-O-benzy l4-0-acetyl-6-deoxy6-O-benzy l4,6-O-benzylidene6-O-p-tol ylsiilfon yl4,6-di-O-p-tolylsuIfonyl6-0-trityl4-0-acetylP-D-Gulopyranoside 4,6-O-benzylidenea-D-Mannopyranoside 4-O-acetyl-6-0-trityl4,6-O-benzylidene6-deox y4-0-acetyl6-iodo-4-O-p-tol ylsul fony l4-O-p-tol ylsul fonyl4,6-O-ethylidene4,6-di-O-p-tolylsulfonyl6-0-trityla-D-Mannopyranoside, phenyl 4,6-0-benzyIidene/3-D-Mannopyranoside 4,6-O-benzylidene4,6-di-O-methyla-D-Talopyranoside 6-acetamido-6-deoxy6-azido-6-deoxy4,6-O-benzylidene-

M.P., degrees

[1ylIA

degrees

Rotation solvent

References

63 138 131- 132 184 61 138 51

189 139.5 120.9 96.5 -6.1 -15.6 35.3

C C C C EA C C

50,124 126a 27 126a 45 45 45

136-138 66-67

230 232

C C

125a 125a

4.8 20 38.1 -7.4 24.7 24 21.8 -28

C C C C C C C C

67 10 67 20 130 130 67 67

-1 19 108 50.4 107.4 116 128.5 82.2 118.6 108 71.5 15.6

C C C C C C C C

20 9,237 9 16 69 69 69 69 115 69 9

200 -25 -40 -30.7 24 40

C W EA C W EA

55 97.4

W M

77-78 83.5-84.5 178-179 60-62 150-151 174-175 104-105 147 82-83 140-141 147 63.5-65.5 92.5-94 80.5-82 100 160-161 160-161 182-184

183 69

204 208-209 65-67 242

-40

C C

115a 238 45 238 129 131 132 121 20

C

(continued)

(237) R. W. Jeanloz and D. A. Jeanloz,]. Amer. C h e k Soc., 80,5692 (1958). (238) W. N. Haworth, E. L. Hirst,and L. Panizzon,J. Chem. Soc., 154 (1934).

OXIRANE DERIVATIVES OF ALDOSES

175

TABLEI1 (continued) Methyl 2,3-anhydro compound P-D-Talopyranoside 4,6-O-benzylidene4,6-di-O-methyla-L-Talopyranoside 6-deoxy4-0-methyl-

References

Lab, degrees

Rotation solvent

103-104 248-249 72-73

-86.1 -142.5 -148.4

C P C

18 20 18

95-97 108-110

-88 0

W CAW

8 8

M.P., degrees

TABLE111 Derivatives of 3,4-Anhydrohexopyranosides 3,4-Anhydro compound

M.P., degrees

P-D-Allopyranoside,benzyl 2(benzyloxycarbonylamino)-2-deoxy- 108-110 6-O-(methylsulfonyl)134-135(d) P-D-Allopyranoside,methyl 2,6-di-O-methyl46 a-D-Altropyranoside,methyl 6-0-acetyl-20-benzyl6-0-trityl135-136 2-0-acetyl143 2-0-benzyl94-95 0-D-Altropyranoside,methyl 2,6-di-O-methyla-D-Galactopyranoside,benzyl 2-(benzyloxycarbonylamino)-2-deoxy158 6-O-(niethylsulfonyl)124 P-D-Galactopyranoside,benzyl 6-0-p-tolylsulfonyl66-68 a-D-Galactopyranoside,methyl 118.5-119-5 2-O-acetyl-6-O-benzyl2-0-acety l-6-deoxy112.5-114 2,6-di-O-acetyl6-0-benzyl41-42 6-deoxy65.5-66.5 2-0-p-tolylsulfonyl87-88 6-0-trityl144.5 2-O-acety l119-120 P-D-Galactopyranoside,methyl 158 6-deoxy2-O-acetyl-6-O-trityl-

114-115 181

[ a ] ~ , Rotation

degrees

solvent

References

-142 -134

P P

137 137

-144.5 97.3 23.6 22 22 13.7

C W C C C C

45 9 62 9 9 62

-21

W

239

71 -64

P P

137 137

-117 67.5 58.9 131.5 57.4 9.3 70 60.5 8.7 31 -118 -121.8 -64.6 -91.8

C W C C C C W C C C W W M C

131(b) 65 67 10 65 67 10 10 65 65 240 138 141 240 (continued)

(239) W. H. G . Lake and S. Peat,]. Chem. SOC., 1069 (1939). (240) B. Helferich and A. Miiller, Ber., 63,2142 (1930).

NEIL R. WILLIAMS

176

TABLE111 (continued) 3,4-Anhydro compound 2,6-di-O-acetyl2,6-di-O-benzoyl2,6-di-O-methyl6-0-methylP-D-Galactopyranoside, phenyl 2,6-di-O-acetylHexopyranoside, methyl P-DL-Tibo2,6-dideoxy-3-C-methyla-D-Tdopyranoside, methyl 6-deoxy2-0-benzoyla-L-Talopyranoside, methyl 6-deoxy-

M.P., degrees

[a]~, degrees

Rotation solvent

118 133 84 121 150 123 76

-114 -80 -148.2 -141.6 -161 -104.5

C C C W W C

240 240 51 51 241 24 1 35

65-66 72-73 68 65

2-0-methyl-

References

143 143 -1 10 -1 16 -140

W W M

8 142 8

TABLEIV Anhydro Derivatives of Pentopyranosides ~

Compound a-D-Arabinopyranoside, methyl 3,4-anhydro2-0-acetylP-L-Arabinopyranoside, methyl 3,4-anhydro2-0-acetyla-D-Lyxopyranoside, methyl 2,3-anhydro4-0-acetyl4-azido-4-deox y0-D-Lyxopyranoside, methyl 2,3-anhydro4-azido-4-deoxya-L-Lyxopyranoside, methyl 2,3-anhydro4-azido-4-deox yP-L-Lyxopyranoside, methyl 2,3-anhydro4-0-acetyla-D-Ribopyranoside, methyl 2,3-anhydro4-azido-4-deoxyP-D-Ribopyranoside, benzyl2,3-anhydro4-0-methylP-D-Ribopyranoside, methyl 2,3-anhydro4-0-acetyl-

M.p., degrees 95-96 107-108 32-33 49-5 1 62-63

65-66 70-70.5

43-44 76-77 98-100 46 73

[a]D,

degrees

Rotation solvent

References

65.6 52.1 133 170 111 88.6 105

W C W C W C C

68 68 68 68 68 68 23

-54

C

23

C

-104 59.4

w

81.7

C

23 68 160 68

-67 -19 -35.8 22.4

C C C C

23 52 52 148 146 (continued)

(241) B. Helferich and F. Straws,]. Prakt. Chem., 142, 13(1953).

OXIRANE DERIVATIVES OF ALDOSES

177

TABLEIV (continued) M.p., degrees

Compound

52-52.5 49-50 51-52

P-L-Ribopyranoside, methyl 2,3-anhydro4-0-acetyl-

82-83 70-71 44-45 151

4-0-benzyl4-O-(p-nitrophenylsuIfonyl)P-L-Ribopyranoside, methyl 3,4-anhydro-

Rotation solvent

References

0 25.4 9.1 -7 24.6 -25.5 -145 -252

C C E W C C C C

148

M

35.7 73.6 22.6 -12

C C C C

98.6

A

23 146 102 150 146 146 145 23 159 159a 109 145,147 147 151(b) 151(b) 151(b) 158

[a]D,

64-64.5 106 42-43 75-77 159-160 89 73

4-azido-4-deoxy4-0-benzoyl4-O-benzy l4-O-methyl4 - 0 4p-nitrobenzoy1)4-0-p-tolylsulfonyla-L-Ribopyranoside, methyl 2,3-anhydro4-azido-4-deoxyP-L-Ribopyranoside, ethyl 3,4-anhydro-

degrees

TABLEV 2,3-Anhydrofuranosides 2,3-Anhydro compound

M.p.9

[ah

degrees

degrees

p-D-Allofuranoside, methyl 6-0-benzoyl-5-0-p-tolylsulfonyl- 11 1 5,6-di-O-benzoyl5,6-di-O-p-tolylsulfonyl116 a-D-Lyxofuranoside, ethyl 58-59 5-0-acetyl5 - 0 4tetrahydropyran-2-y1)a-D-Lyxofuranoside, methyl 81 80-82 5-0-acetyl72-73.5 5-0-benzyl5-deoxy5-deoxy-5-iodo77-78 5-0-methyl43 5-O-~~-tolylsulfonyl80-81 P-D-Lyxofuranoside, ethyl 5-0-acetyl5-O-(tetrahydropyran-2-yl)-

-45 -96.2 -26.3 40.3 44 21 57 67 65 23.4 66.9 70 60 24.5 -77 4 7 -79

Rotation solvent

References

C E C W C C W W C C

242 242 242 34b) 34(b) 34(b) 161 22 164 153 46(1>) 46(a) 161 46(a) 34(b) 34(b) 34(b)

M W C C

(contintred)

(242) H. Ohle and H. Wilcke, Ber., 71,2316 (1938).

178

NEIL R. WILLIAMS

2,3-Anhydro compound P-D-Lyxofuranoside, methyl 5-0-acetyl5-0-benzyl5-deoxy5-iodo5-0-methyl5-0-p-tolylsulfonyl5-0-trityla-D-Ribofuranoside, ethyl 5-0-acetyl5-0-(tetrahydropyran-2-yl)a-D-Ribofuranoside, methyl 5-0-acetyl5-0-benzyl5-deoxy5-0-(p-nitrobenzoyl)5-O-p-tolylsulfony lp-D-Ribofuranoside, ethyl 5-0-acety15-04tetrahydropyran-2-y1)P-D-Ribofuranoside, methyl 5-0-acetyl5-0-benzyl5-deoxy5-O-(p-nitrobenzoyl)5-0-p-tolylsulfonyl5-0-trityl-

TABLEV (continued) tab, M.P., degrees degrees 74-75

62-64 14-15 76-77 156-157

21-23 134-136 93-95

2-3 98-99 66.5-67 127

-102 -80 -67 -28 -113 -88 -89 -76 13.1 -18.4 -14.3 13 -2.1 -18.1 26 -26

7 -89.5 -108 -99 -109 -112 -90.8 -153.4 -95 -80 -59

Rotation solvent

References

W C E

22 164 167a 46(4 4%) 163(a) 46(4 167 34(W 3403) 34b) 177 177 180a 180 177 178 34b) 34W 3403) 177 177 182 180 177 178 176

W

C C C C W C E M C C W C C W C C M C C C

g-(P-D-PentofuranosyI)adenineDerivatives

Lyxofuranosyl 5-deoxyRibofuranosyl 5-deoxy-

205-206 208-209d 200-203 190d

-23 -3 42

168 171 183 171

W WIP C

6-(Dimethylamino)-2-(methylthio)-9-(p-~-pentofuranosyl)punne Derivatives

Lyxofuranosy 1 Ribofuranosyl 5-0-trityl-

211-212 172-173 213

P

68.7

C

173 186 186

-53.5 23.6

P C

48 48

-43

7-(~-Pentofuranosyl)theophylline Derivatives

a-Lyxofuranosy 1 p-Ribofuranosyl, 5-0-trityl-

204-205 202-203

(continued)

OXIRANE DERIVATIVES O F ALDOSES

179

TABLEV (continued) 2,S-Anhydro compound

M.P., degrees

[ffID, degrees

Rotation solvent

34 3 26

W C W D W

References

l-(p-D-PentofuranosyI)uraci~ Derivatives

Lyxofuranosyl 5-0-benzoyl5-deoxy5-iOdO5-O-(methylsulfonyl)-

139-140 187-190 146-146.5 218-220 177-177.5

-4

16

175 175 175 175 175

TABLEVI 5,6-Anhydrohexofuranoses

5,B-Anhydro compound a-D-Glucofuranose, 1,2-O-isopropylidene3-acetamido-3-deoxy3-0-benzyl3-O-meth yl3-04methylsulfonyl)3-O-p-tol ylsulfonyla-D-Glucofuranose, 1,2-0-(trichloroethy1idene)8-L-lyxo-Hexofuranose, d-deoxy1,2-O-isopropylideneLu-D-xylo-Hexofuranose,3-deoxy1,2-O-isopropylidenep-L-Idofuranose, 1.2-0-isopropylidene3-0-benzyl3-O-(methylsulfonyl)-

M.p.,

degrees 133.5 142-143

80-81 145

27 73-75 96.5-98.5

[ a ] ~ , Rotation

References

degrees

solvent

-26.5 67.5 -51.2 44.8 430 -50.9 -29.8

W C C A C C P

11,243 191 12 188 191 191 244

-27.6

C

204

-15 -25.2 -78.7 -7.4

D C C C

207 12 12 205

(243) K. Freudenberg, H. Toepffer, and C. C. Andersen, Ber., 61,1750 (1928). (244) R. Grewe and G. Rockstroh, Chem. Ber., 86,536 (1953).

This Page Intentionally Left Blank

2,S-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS”

BY J. D E F A Y E ~ ” lnstitut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91 Cif-stir-Yvette, France

I. Introduction,. .......................................................... 11. Methods of Formation., ................................................. 1. Deamination of Aminoaldoses with Nitrous Acid ........................ 2. Halogenolysis of Halogenated Derivatives .............................. 3. Intramolecular Displacement of Sulfonates by the Action of a Base . . . . . . . . 4. Solvolysis of Sulfonates ............................................... 5. Miscellaneous Methods. ..................... ................. 111. Reactions. .............................................................. 1. Stability of the Oxolane Ring. .......................................... 2. Reactions Involving a Carbonyl Group .................................. IV. Utilization.. ............................................................ V. Table of Properties of 2,s-Anhydro Derivatives of Sugars, Alditols, and Aldonic Acids ................................

181 183 183 194 198 203 209 210 210 212 215

219

I. INTRODUCTION Almost a century has elapsed since Ledderhose,’ seeking to establish the structure of an amino sugar (“glycosamin”) obtained by hydrolysis of chitin with acid, subjected “glycosamin” to deamination with nitrous acid. He obtained an amorphous, tasteless, nonfermentable, strongly reducing product that he found exceedingly disconcerting, as all his attempts to characterize it led to extensive decomposition. The structure of “glycosamin” (2-amino-2-deoxy-D-glucose) was not established2 until half a century later. As regards the product of deamination with nitrous acid, namely, 2,5-anhydro-~-mannose (chitose), it was not until 1956 that the polemics concerning its struc*Translated from the French by D. Horton. **Presentaddress: Centre de Recherche sur les Macromolecules Vegetales, C.N.R.S., Domaine Universitaire de Grenoble, 38 St. Martin d’HL.res, France.

(1) G . Ledderhose, Z . Physiol. Chem., 4,139 (1880). (2) W. N. Haworth, W. H. G. Lake, and S. Peat,j. Chem. Soc., 271 (1939).

181

182

J. DEFAYE

ture came to an end. Nevertheless, the way was opened for a whole series of brilliant studies that, from Tiemann3+ to Levene7-19by way of Fischer, 6,zo enabled sugar chemistry to contribute to fundamental chemical knowledge of general interest. The chemistry of the 2,5-anhydrides of aldoses subsequently entered a prolonged lull, and Peat's reviewz1of 1946 in this Series does not report on any work later than 1925. The experimental basis of the deamination of amino sugars with nitrous acid was, nevertheless, established. The progress afterwards made in the conformational analysis of sugars made it possible for Shafizadehzzto draw a parallel with the nitrous acid deamination of the aminocyclohexanols, and to rationalize the whole of these results. During the same period of time, the use of chromatography, together with developments in physical methods for the determination of structure, permitted reaction mixtures to be studied more thoroughly. Many investigations revealed the formation of 2,5-anhydrides of aldoses during reactions where such anhydrides were not anticipaied. It is noteworthy that, although the five-membered carbocyclic rings are more strained and possess a higher fi-ee-energy than the sixmembered analogs, the oxolane ring (five-membered, oxygencontaining heterocycles) of the 2,5- or 3,6-anhydrides are common in the aldose and alditol series. The theoretical aspects of ring closure in the sugars has been the subject of two articles in this S e r i e ~ , ~ ~ , ~ ~ and will not be treated further here. (3) F. Tiemann, Ber., 17, 241 (1884). (4) F. Tiemann and R. Haarmann, Ber., 19, 1257 (1886). (5) F. Tiemann, Ber., 27,118 (1894). (6) E. Fischer and F. Tiemann, Ber., 27,138 (1894). (7) P. A. Levene and F. B. LaForge,]. Biol. Chem., 20,433 (1915). ( 8 ) P. A. Levene and F. B. LaForge,]. Biol. Chem., 21,345 (1915). (9) P. A. Leveneand F. B. LaForge,]. Biol. Chem.,21,351(1915). (10) P. A. Levene and F. B. LaForge,]. Biol. Chem.,22,331 (1915). (11) P. A. Levene and G . M. Meyer,J. Biol.Chem.,26,355 (1916). (12) P. A. Levene,]. Biol. Chem., 31,609 (1917). (13) P. A. Levene,]. Biol. Chem., 36,73 (1918). (14) P. A. Levene,]. Biol. Chem., 36,89 (1918). (15) P. A. Levene,]. Biol. Chem., 39,69 (1919). (16) P. A. Levene and E. P. ClarkJ. Biol. Chem., 4 6 , l Q(1921). (17) P. A. Levene, Biochem. Z., 124,36 (1921). (18) P. A. Levene,]. B i d . Chem., 59,135 (1924). (19) P. A. Levene and R. Ulpts,]. Biol. Chem.,64,475 (1925). (20) E. Fischer and E. Andreae, Ber., 36,2587 (1903). (21) S. Peat, Aduan. Carbohyd. Chem., 2,37 (1946). (22) F. Shafizadeh,Advan. Carbohyd. Chem.,13,9 (1958). (23) J. A. Mills, Aduan. Carbohyd. Chem.,10,1(1955).

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

183

The attention focused on facile methods for obtaining heterocycles of this type has led to recent utilization of some of them as intermediates in synthesis, inasmuch as certain ones, such as m u s ~ a r i n e , ~ ~ have been obtained from natural sources, and various others have been shown to have interesting biological proper tie^.^^ The purpose of this article is to attempt a rationalization among the numerous methods for access to 2,5-anhydrides of aldoses; and, from the mass of scattered, and frequently fragmentary, information in the literature, to single out sugar derivatives in this class from the viewpoint of their properties and their utilization. 11. METHODS O F FORMATION

1. Deamination of Aminoaldoses with Nitrous Acid Deamination of aminoaldohexoses with nitrous acid constitutes the oldest route of access to the 2,5-anhydroaldohexoses (see the Introduction). It is only in recent years, however, that the results obtained by this method could be rationalized on a theoretical basis as a consequence of concomitant developments in certain other areas of organic chemistry. The action of nitrous acid on a primary, aliphatic amine is still a complex process, inasmuch as it is necessary to postulate certain steps that are imperfectly understood, but it can nevertheless be considered that the process leads ultimately to a carbonium i ~ n .The ~ ~ , ~ ~ carbonium ion thus formed is termed “hot,” that is to say, it is nonsolvated and chemically activated; the nature of the products resulting from its decay is essentially determined by the conformation of the initial state; the electronic factors in effect at the time of the transition state exert little influen~e.~’ A route through a diazonium-ion intermediate has long been postulated’* (see Scheme 1, path A) from the fact that primary, aromatic amines react with nitrous acid in an acid medium to give diazonium saltsz9that are sufficiently stable to be isolable. However, more-recent (24) S. Wilkinson, Quart. Reo. (London), 15,153 (1961). (25) J. Defaye, P. P. Slonimski, G. Perrodin, and E. Lederer, Compt. Rend., 251, 817(1960). (26) F. C. Whitmore and D. P. Langlois,]. Amer. Chem. Soc., 54,3441 (1932). (27) J. H. Ridd, Quast. Reo. (London), 15,418 (1961). (28) P. Brewster, F. Hiron, E. D. Hughes, C. K. Ingold, and P. A. D. S. Rao, Nature, 166,179 (1950). (29) H. Zollinger, “Azo and Diazo Chemistry,” Interscience Publishers, Inc., New York, N. Y., 1961.

J. DEFAYE

184

work has suggested that, in the aliphatic series, the reaction proceeds by direct decomposition of an intermediate diazohydroxide having the anti configuration30(see Scheme 1, path B).

R-N=N-OH

.. ..

-

..

p.0

R-N=N-O-H

ad..

-

0

R-NEN

..,H + O,H

path A R-NH,

.. ..

H N o z t ~ - NI - ~ = ~ :

-

H

’

.. @

2 0 .

’

+

N,

Ro+

- - t R, .N=N, ’.

HR. *Q-N@

CB.

0

R-N=N-0: I * - .* H

H,O

*.

R ,.N q=N,@

OH

*

p H 2

Scheme 1. Action of nitrous acid on a primary, aliphatic amine

Undoubtedly, the vicinal groups play a fundamental role in the outcome of this reaction, especially with the sugars, where the favored conformation of the molecule at equilibrium is controlled at the outset by groups that determine whether the molecule exists as a cyclic or acyclic structure. The deamination of cyclic and acyclic amino sugar derivatives by nitrous acid will be considered in turn. a. Cyclic Amino Derivatives. (i) 2-Amino-2-deoxyaldohexopyranoses. - The deamination of these compounds by nitrous acid was reviewed in 1958 by Shafizadeh,*‘ who compared the results with the behavior of aminocyclohexanols on deamination with nitrous acid. Various papers devoted to this s u b j e ~ t ~ O show - ~ ~ that the outcome of the deamination of these compounds by nitrous acid depends essentially on the group antiparallel to the amino group. Thus, the deamination by nitrous acid of the isomeric 2-aminocyclohexanols, for which the favored conformation is rigidly fixed by the presence of a bulky substituent at C-4, is highly stereoselective, and M . ChBrest, H. Felkin, J. Sicher, F. SipoS, and M. Tichy, J . Chem. Soc., 2513 (1965). G . E.McCasland,J. Amer. Chen. SOC., 73,2293(1951). W.Klyne, Progr. Stereochern., 1,72(1954). D.Y. Curtin and S. Schmukler,]. Amer. Chem. Soc., 77,1105(1955). W.G.Dauben and K. S. Pitzer, in “Steric Effects in Organic Chemistry,” M. S . Newman, ed., JohnWiley and Sons, Inc., New York, N. Y., 1956,p. 3.

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

185

leads uniquely to the product of rearrangement resulting from attack on the carbonium ion at C-2 by the group trans and antiparallel to the amino group30 (see Scheme 2).

Scheme 2. Deamination by nitrous acid of the epimeric 2-amino-4-tert-butylcyclohexanols (accordingto Ref. 30)

It may, therefore, be expected that the deamination with nitrous acid of those 2-amino-2-deoxyaldopyranoses in which the amino group is oriented equatorially in the most stable conformation will lead to a 2,5-anhydroaldose, with inversion of the configuration at C-2. For example, the deamination of 2-amino-2-deoxy-~-glucose(1) with nitrous acid proceeds principally to 2,5-anhydro-~-mannose'*'~-'~ (2), and the methyl 2-amino-2-deoxy-a- and -P-D-glucopyranosides behave similarly.38The low rate of deamination of the a-Danomer has been attributed to steric hindrance to the approach of the reagent; This difference in rate has been proposed as a method for establishing anomeric configurations in glycosaminoglycans. The structure of 2,5anhydro-D-mannose (for many years known as chitose) was contro-

(35) S. Akiya and T. Osawa, Yakugaku Zasshi, 74,1259 (1954). (36) A. B. Grant, New Zealand/. Sci. Technol., B37,509 (1956). (37) 8.C. Bera, A. B. Foster, and M . Stacey,J. Chem. SOC.,4531 (1956). (38) A. B. Foster, E. F. Martlew, and M. Stacey, Chem. Znd. (London),825 (1953).

J. DEFAYE

186

versial for a long time.39-42In the same way, the deamination of 2amino-2-deoxy-~-ga~actose (3)with nitrous acid Ieads to 2,5-anhydroD - t a l o ~ e ' " ~(4). ~'~~

0

where -Xo = -NP or -N,OH,

The key sequence in the determination of the structures of 2,5anhydro-D-mannose (2) and -D-talose (4) was their reduction to the corresponding 2,5-anhydroalditols (5 and 6) and identification of the asymmetric "dialdehyde" (7) resulting from oxidation of the anhydroalditols with p e r i ~ d a t e . ~ ' . ~ ~ HOCH,

0 HOH,C,H

I(C%OH HO

HOH,C

,c-0-c,

H,CH,OH CHzOH

f

OMe

(39) C. Neuberg, H. Wolff, and W. Neimann, Ber., 35,4009 (1902). (40) W. Armbrecht, Biochern. Z., 95, 108 (1919). (41) L. Zechmeister and G . T6th, Ber., 66, 522 (1933). (42) P. Schorigin and N. N. Makarowa-Semljanskaja,Ber., 68,965 (1935). (43) J. Defaye, Bull. Soc. Chirn. Fr.,999 (1964). (44) E. Vankata Rao, J. G . Buchanan, and J. Baddiley, Biochern.]., 100,801 (1966).

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

187

The nitrous acid deamination of 2-amino-2-deoxy-~-mannose(8),in the favored conformation, the amino group ofwhich is axially attached, leads, in contrast, uniquely to D - g l u ~ o s e ,characterized, '~ after oxidation by nitric acid, as D-glucaric acid (9).This result has also been verified by direct crystallization of the D-glucose and b y assay with Dglucose o x i d a ~ e . ~ ~ ~

t HNO,

CO,H I

HCOH i HOCH I HCOH I

HCOH I CO,H (81

0

(91

where -X* = - N P or -N20H,

It is remarkable that the only compound isolated from this reaction results from attack by solvent on the carbonium ion at C-2. By analogy with the deamination of the 2-aminocyclohexanols by nitrous acid, it might have been expected that a 3-0x0 derivative would have been formed as a result of hydride migration, because the C-3 proton is antiparallel to the leaving group. LeveneI5 reported that heating of 2-amino-2-deoxy-D-mannose (8) in the presence of silver oxide leads to a crystalline, nitrogen-free compound to which he attributed the structure of 2,5-anhydro-~glucose on the basis of its elemental analysis. The possibility of interconversion between the two chair forms C1 (D) + 1C (D), which would bring the amino group at C-2 into equatorial orientation, has been postulated.22Without excluding this possibility, it remains to be proved that the deamination by silver oxide does, indeed, proceed by (44a)D.Horton and K. D. Philips, unpublished results.

188

J. DEFAYE

way of a carbonium ion. The physical properties of this 2,s-anhydride'* are, moreover, unusual (see p. 214). As might be expected, the nitrous acid deamination of methyl 2amino-4,6-O-benzylidene-2-deoxy-a-~-altropyranoside ( 10) hydrochloride leads45,46 uniquely to methyl 2,3-anhydro-4,6-0-benzylidenea-D-allopyranoside (11). The benzylidene group does not play an

essential role in this reaction, unless it is required for stabilization of the conformation of the aminohexose. Although an initial report indicated4' that deamination with nitrous acid of the hydrochloride of ( 12) methyl 2-amino-4,6-0-benzylidene-2-deoxy-~-~-glucopyranoside led to 4,6-O-benzylidene-~-mannopyranose, the results of subsequent work corrected this result, and showed46that the sole product obtained under these conditions is 2,5-anhydro-4,6-0-benzylidene-~mannose (13). The same reaction performed on methyl 2-amino-2deoxy-4,6-O-ethylidene-3-O-methyl-~-glucopyranoside led to a similar r e ~ u l t . ~ "

(45) L. F. Wiggins, Nature, 157,300 (1946). (46) S. Akiya and T. Osawa, Chem. Pharm. Bull. (Tokyo), 7,277 (1959). (47) J. C. Irvine and A. Hynd,]. Chem. SOC., 105,698 (1914). (48) S. Akiya and T. Osawa, Yakugaku Zasshi, 76,1276 (1956).

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

189

(ii) 2-Amino-2-deoxyaldonolactones. -The deamination of Z-amino2-deoxyaldonolactones by nitrous acid was investigated by Leveneg,l4 with promising results, but the theoretical basis for interpreting his observations was not available at that time. Levene found that the nitrous acid deamination of 2-amino-2-deoxyD-mannonolactone (14) gives 2,5-anhydro-~-mannonicacid (16),a result identical with that obtained by similar deamination of the corresponding acid (15). On the other hand, the same reaction performed

H OH H,N o c o

Hop / HCOH I

(15)

HCOH I CqOH

J . DEFAYE

190

on 2-amino-2-deoxy-~-idonolactone (17) led, after oxidation with nitric acid, to 2,5-anhydro-~-gularicacid (18). The inversion of con-

OH

(18) where -Xo

0

= - N P o r -N,OH,

figuration at C-2 evident in the latter example can be explained, assuming a five-membered-ring structure for these lactones, by the fact that the hydroxyl group at C-5 in 17 is, sterically, well placed for attacking a carbonium ion developing at C-2. After deamination with nitrous acid followed by oxidation, 2-amino-2-deoxy-~-idonic acid (19) gives 2,5-anhydro-~-idaricacid (20) (see p. 191). COJi I H,NCH I HYOH HOCH I HCOH I

ChOH

m%

1

OH

b. Acyclic Amino Derivatives. -The outcome of deamination of these compounds by nitrous acid is essentially dependent on the nature of the participating group at C-1. A wide variety of compounds has been obtained to date, and the nature of these is not, as the initial results on the nitrous acid deamination of 2-amino-2-deoxyaldonic acids had previously led investigators to suppose, limited to the class of 2,5-anhydrides of sugars.

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

191

(i) 2-Amino-2-deoxyaldonic Acids. - Deamination of the eight 2-amino-2-deoxy-~-aldohexonic acids with nitrous acid was investigated by Levene and coworkers in an outstanding series of paper^.'."'^,'^ A discussion of these results has already been presented in a Volume in this Series.22 In all known instances, the deamination of 2-amino-2-deoxyaldohexonic acids by nitrous acid takes place with formation of a 2,5anhydro ring and with retention of the configuration at C-2, as illustrated in the sequence 21+22-23.

*H0Cv”’

HCNH, I

HOYH

HNO, +

HOCH I

HCOH

HCOH

HCOH

HCOH

I

I

CH,OH

0

1 0

I

I

CH,OH

HO

(22)

where

The mechanism proposed by Foster49for this deamination postulates the formation of an unstable, intermediate a-lactone (22a),which subsequently undergoes attack by the hydroxyl group on C-5; this process would explain, by a double inversion, the net retention of configuration that is observed. (ii) 2-Amino-2-deoxyalditols. - By analogy with the nitrous acid deamination of 2-amino-2-deoxyaldonic acids, it might be expected that an oxolane ring would result from the deamination of 2-amino-2deoxyalditols with nitrous acid. Although the formation of such a compound had once been envisaged,50 no confirmation of the preliminary claim has been forthcoming. In fact, even though the deamination of l-amino-l-deoxyalditols with nitrous acid certainly leads in the case of the D-mannitols” and D-glucitol derivatives5] to the corresponding 1,4-anhydrides, the same reaction5’ applied to 2-amino-2-deoxy-D-glucitol (24) gives 2-deoxy-~-arabino-hexose (26).Under the same conditions, a mixture (49) A. B. Foster, Clrern. Ind. (London),627 (1955). (50) V. G. Bashford and L. F. Wiggins, Nature, 165,566 (1950). (51) V. G. Bashford and L. F. Wiggins,J. Chern. Soc., 299 (1948). (52) Y. Matsushima, Bull. Chem. Soc.Jnp.,24,144 (1951).

J . DEFAYE

192

CH,OH

HC=O

I

I

HFNH, HOCH I

HYOH

-

HCOH I

CH,OH

HNO,

HO&@ 1

-

1

I HOCH

HCOH

HCOH

HCOH

HCOH

I

I

I

I

c&or

CH,OH

-

1

7% HOCH HCOH I

HCOH I C&OH

(251

(24) where -X@

=

Q 0 -N2 or -",OH,

of 2-amino-2-deoxy-~-arabinitol and 2-amino-2-deoxy-~-ribitol, obtained by reductive cleavage of the (o-nitropheny1)hydrazone of Derythro-pentulose, underwent d e a m i n a t i ~ n ~ to~ give 2-deoxy-Derythro-pentose. A mechanism involving a hydride migration (25) has been proposed by Foster.49 (iii) Dithioacetals of 2-Amino-2-deoxyaldoses. - Bivalent sulfur possesses an electronic structure analogous to that of oxygen. On the other hand, being more polarizable than the oxygen atom, it is more apt to stabilize a carbonium ion by formation of a three-membered ring. In numerous examples, the possible intervention of an episul. ~ ~ participation fonium ion has been pointed out in the l i t e r a t ~ r eThis ~ ~ - ~ ~ is frequently accompanied by migration of the sulfur g r o ~ p to the carbon atom originally carrying the group displaced, and this factor makes prediction of the outcome of the reaction uncertain. In attempting to obtain 2,5-anhydro-~-glucosediethyl dithioacetal, Defaye5*performed the nitrous acid deamination of 2-amino-2-deoxyD-glucose diethyl dithioacetal (27) under weakly acidic conditions (acetic acid and sodium nitrite), and obtained a principal product later shown5' to be ethyl 2-S-ethyl-l,2-dithio-cr-~-mannofuranoside (28). Shortly before, Horton and coworkers60 had reported the forma-

(53) Y. Matsushinia and Y. Imanaga, Bull. Chem. Soc.Jap.,26,506 (1953). (54) For a review, see L. Goodman, Aduati. Carbohyd. Chem., 22, 109 (1967). (55) N. A. Hughes and R. Robson,./. Chem. Soc. ( C ) ,2366 (1966). (56) N . A. Hughes, R. Robson, and S. A. Saeed, Chem. Commun., 1381 (1968). (57) N. A. Hughes and R. Robson, Chem. Commun., 1383 (1968). (58) J. Defaye, Bull. Soc. Chim. Fr., 1101 (1967). (59) J. Defaye, T. Nakamura, D. Horton, and K. D. Philips, Carbohyd. Res., 16, 133 (1971). (60) D. Horton, L. G . Magbanua, and J . M. J. Tronchet, Chem. Ind. (London), 1718 (1966); A. E. El Ashmawy, D . Horton, L. G. Magbanua, and J. M . J. Tronchet, Carhohyd. Res., 6,299 (1968).

2,5-ANHYDRIDES OF SUGARS A N D RELATED COMPOUNDS

193

-

HOCH,

-

I

HOCH

I

HCOH I

-

W

CH,OH

0 S

E

t

(28)

pH 5 . 6

EtS,H,SEt C I HCNHz I HOCH I

HCOH

HNO,

I

H SEt 0,C’ EtS I ‘CH I

HOCH I

HCOH I

HCOH

HCOH

I

I

CH,OH

CH,OH

(27 )

HOCH,

H

O

W

O

H

(29)

tion of 2-S-ethyl-2-thio-~-glucose(29) as the major product from the same dithioacetal (27)when the deamination was performed with aqueous nitrous acid at a more acidic pH; a minor product was identical with compound 28. In both instances, the participation of an ethylthio group in initial stabilization of the carbonium ion is evident. The subsequent course of the reaction nevertheless appears to be strongly influenced by the nature of the medium. In acetic acid solution, there is attack at C-1 by the hydroxyl group on C-4, whereas, in the more-acidic, aqueous medium, it is the solvent that attacks at the same (least hindered) carbon atom, with formation of a hemithioacetal that is rapidly hydrolyzed to give the aldehyde group. To account for

J. DEFAYE

194

the net retention of configuration in the reaction leading to 29, it was ) , which atpostulated that the initial episulfonium ion ( D - ~ u w ~ o in tack by solvent at C-1 would be hindered by the hydroxyl group on C-3, equilibrates with a second episulfonium ion (D-gluco), and the latter is rapidly attacked by solvent at the unhindered, C-1 position.60 Although 2-S-ethyl-2-thio-~-mannoseis readily epimerized to 29 in the presence of base,60asuch epimerization does not occur at the pH levels ~ s e din ~the~deamination * ~ ~ of 27. 2. Halogenolysis of Halogenated Derivatives a. Brominolysis of 2-Deoxy-2-iodo Derivatives in the Pyranose Series.-The action of bromine in the presence of silver acetate on (30)in acetic methyl tri-O-acetyl-2-deoxy-2-iodo-~-~-glucopyranoside acid containing potassium acetate leads6*to 173,4,6-tetra-O-acety1-2,5anhydro-D-mannose methyl hemiacetal (31), obtained as a mixture of the C-1 epimers. The structure of this compound was established by ( a ) conversion into the corresponding dimethyl acetal (32) by acidcatalyzed methanolysis, and (b) obtaining 2,5-anhydro-~-rnannitol (33) upon treatment of the hemiacetal 31 with sodium borohydridg.

Br,, AgOAc

t

KOAc, AcOH

ACO-

A

c

O

W

HC(0Me) (OAc)

OMe

AcO

How

i

MeOH-HC1

J

CH,OH

(33)

AcO

(60a)B. Berrang and D. Horton, Chem. Commun., 1038 (1970). (61) R. U. Lemieux and B. Fraser-Reid, Can. J . Chem., 42, 547 (1964).

(32)

195

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

This reaction has been compared61with the nitrous acid deamination of 2-amino-2-deoxy-~-glucose;attack by a bromonium ion could lead to an intermediate, alkyl-iodonium cation (2-C-I-Br)O, which would decompose through nucleophilic attack by 0-5,which is antiparallel to the leaving group at C-2. This hypothesis would seem justifieds2 by the fact that the same reaction performed on the epimeric halide (manno configuration) (34)leads to a mixture containing 60% of the 3,3,4,6-tetraacetate of methyl 2-bromo-2-deoxy-a-~-urubino-hexopyranosid-~u1ose 3-hydrate (38) and 20% of 1,3,4,6-tetra-O-ace~l-2-O-methyl-~-~-glucopyranose

~ ; c p o h r y A c ; m

OAc

Me0 Me (35) A

c

O

m

OMe (34)

/

(36) (20%)

Br, , AgOAc, KOAc, AcOH

\

(60%)

(36). The mechanism postulated62 for this reaction involves two parallel pathways leading from a common precursor, a carbonium ion at C-2. Stabilization of the carbonium ion by participation of the axial methoxyl group at C-1, to give the intermediate methoxonium ion (35),is followed by attack by solvent at the anomeric position to give the ether (36) obtained. In the alternative route, the initial carbonium ion could subsequently undergo elimination to give an intermediate enol acetate (37), which is then brominated stereoselectively at C-2, to give compound 38 as the major product of the reaction.

b. Action of Lead Tetrafluoride on Pyranoid Glycals.-3,4-Di-Oacetyl-1,5-anhydro-2-deoxy-~(39) and -L-erythro-pent-l-en-itol [D(62) R. U. Lemieux and B. Fraser-Reid, C a n . ] . Chem.,42,539 (1964).

1. DEFAYE

196

and L-arabinal (ribal) diacetates] react63.64with hydrofluoric acid in the presence of lead tetraacetate to give, after deacetylation, 2,5anhydro-l-deoxy-1,l-difluoro-D(40) and -L-ribitol, respectively. The same reagents acting upon 3,4,6-tri-O-acetyl-l,5-anhydro-2-deoxyD-arabino-hex-l-enitol (D-glucal triacetate) (41) a ringcontraction of the same type, to give 2,5-anhydro-l-deoxy-l,l-difluoroD-mannitol (43). Compound 43 is likewise obtained65 by the action of the hydrofluoric acid-lead tetraacetate reagent on ethyl 4,6-diO-acetyl-2,3-dideoxy-a-~-erythro-hex-2-enopyranoside (42), with subsequent deacetylation.

AcO

HO

OH (40)

(39) CH,OAc

2. NaOMe, MeOH HO

f

(43)

?H,OAc

It is known66 that the hydrofluoric acid-lead tetraacetate reagent leads, with unsaturate?. steroids, to cis additions of fluorine. This fact suggests that a cis-difluoro adduct (39b, 42b) is the starting point for the ring contractions observed in the preceding examples. (63) P. W. Kent, J. E. G. Barnett, and K. R. Wood, Tetrahedroii Lett., 1345(1963). (64) P. W. Kent and J. E . G. Barnett, Tetrohedron, S u p p l . 7,69 (1966). (65) K. R. Wood and P. W. Kent,]. Clzein. SOC.(C), 2422 (1967). (66) A. Bowers, P. G. Holton, E. Denot, M. C. Loza, and R. Urquiza, ]. Anier. Chem. Soc., 84, 1050 (1962).This reagent [A. L. Henne and T. P. Waalkes,J. Anier. Chem. Soc., 67, 1639 (1945)l supposedly leads, i n situ, to lead tetrafluoride, an unstable compound that is difficult to isolate.

197

2.5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

Such an intermediate is quite plausible for the D-glucal derivative (41a), which leads to a derivative (43a) of 2,5-anhydro-~-mannitol. The process could be very similar to that already discussed for the brominolysis of 2-deoxy-2-iodo derivatives of D-glucopyranose. The similar rearrangement observed with the "pseudo-glucal" diacetate (42a) could be due to an attack at C-3 by an acetoxyl group, leading to migration of the double bond, with displacement of the ethoxyl group at C-1. From such a reaction scheme, it would, however, be expected that a derivative (44) of 2,5-anhydro-~-arabinitolwould be obtained 1-enit01 from 3,4-di -0-acetyl- 1,5-anhydro-2-deoxy-~-erythro-pent(39a), and not the T i h product observed.

Ace"\; AcO

\

MeONa, MeOH

(39b)

MeOH

"

V

C I HO

H

F

2 (44)

198

J. DEFAYE

3. Intramolecular Displacement of Sulfonates by the Action of a Base

The sulfonic esters of sugars are fundamental intermediates in synthesis, as they permit facile access, through displacement by nucleophiles, to amino sugars, deoxy sugars, halogeno sugars, thio sugars, and anhydro sugars.66aIn the last example, the nucleophilic agent is a hydroxyl group of the same molecule. The reactions of sulfonates are normally of the s N 2 type and proceed with configurational inversion at the carbon atom that originally bore the sulfonyloxy group. Inasmuch as the factors leading to closure of a new oxygen-containing ring are strongly influenced by any ring already present, it is evident that the displacement of sulfonate groups in acyclic derivatives may follow a course quite different from that observed for cyclic derivatives. a. Sulfonie Esters of Acyclic Structures.-The action of one molar equivalent of p-toluenesulfonyl chloride on various dialkyl dithio~ ~ ,D-lyxose,68 ~~ in pyridine solution acetals of D-ribose, D - ~ y l o s e ,and at a temperature below -5”, leads to the corresponding dithioacetals (45, 48, and 51) of 2,5-anhydro-~-ribose,-D-xyIose, and -D-lyXOSe, respectively. The nature of the substituent on sulfur playss7 no particular role in this reaction. The structures of the compounds obtained have been proved by oxidation with lead tetraacetate and, for the D-ribose and D-xylose derivatives, by the isolations7 of a crystalline (p-nitropheny1)hydrazone after cleavage of the dithioacetal group by bromine. Cleavage of the dithioacetal groups from the products, followed by reduction of the resultant carbonyl derivatives (46, 49, 52) with sodium borohydride leads,68with the three compounds (45, 48, and 51), to 1,4-anhydro-~-ribitol(2,5-anhydro-~-ribitol)(47), 1,CanhydroL-xylitol (2,5-anhydro-~-xylitol) (50), and 1,4-anhydro-~-arabinitol (2,5-anhydro-~-lyxitol) (53), identified by comparison with their 1,4-anhydro-~-xylitol,~~ and enantiomorphs, 1,4-anhydro-~-ribitol,~~ 1,4-anhydro-~-arabinitol.’~ (66a)R.S. Tipson, Adoan. Carbohyd. Chem., 8,107 (1953);D . H. Ball and F. W. Parrish, ibid. 23, 233 (1968); 24, 139 (1969). (67) H. Zinner, H. Brandhoff, H. Schmandke, H . Kristen, and R. Haun, Chem. Ber., 92, 3151 (1959). (68) J. Defaye, Bull. Soc. Chim. Fr., 2686 (1964). (69) R. Kuhn and G. Wendt, Chem. Ber., 81, 553 (1948). (70) E. J. Hedgley and H. G. Fletcher, Jr., J. Amer. Chem. Soc., 86, 1576 (1964). (71) R. Barker and H. G. Fletcher, Jr., J . Org. Chem., 26, 4605 (1961).

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

199

HO

HO

(52)

(53)

In sharp contrast to the behavior observed when the three dithioacetals aforementioned are treated with p-toluenesulfonyl chloride in pyridine, dialkyl dithioacetals of D-arabinose, treated under the same conditions, are converted into the corresponding 5-p-toluenesulfonates, generally isolable crystalline in high yielde7' This remarkable difference has been interpreted7*/ on conformational grounds; the D-arabinose dithioacetals are stable in the extended, planar zigzag conformation, whereas the other three examples experience some destabilization in the extended form, because of parallel 1,3-interaction~.~~* Furthermore, the transition state for closure of the 2,s-anhydro ring would be quite strained in the Darabinose series, but not in the other three.?*/ For the reaction leading to the 2,8anhydride, it has been postulated that initial esterification to give the 5-sulfonic ester is followed by intramolecuIar displacement of the 5-substituent by 0-2, either directly or during processing of the reaction mixture in an aqueous medium.67 Another hypothesis, which supposes a dehydration without passage through an intermediate sulfonic ester, has also been advanced.73 (72) H . Zinner, K. Wessely, and H. Kristen, Chem. Ber., 92, 1618 (1959). (72a)J.Defaye and D. Horton, Carbohyd. Res., 14, 128 (1970). (72b)D. Horton and J . D. Wander, Carbohyd. Res., 10,279 (1969). (73) H. Zinner, K. H. Stark, E. Michalzik, and H. Kristen, Chem. Ber., 95, 1391 (1962).

J. DEFAYE

200

b. Sulfonic Esters of Cyclic Structures-The action of sodium methoxide on methyl or ethyl 5-O-p-tolylsulfonyl-a-~-arabinofuranoside (54) leads'4 to the corresponding aIkyl 2,5-anhydro-cu-~-arabinofuranosides (55), the structures of which have been demonstrated by conversion, by way of the aldehyde 56, into 2,5-anhydro-~-arabinitol(57) and comparison of the latter with its enantiomorph, namely, 2,5-anhydro-~-arabinitol.'~

\

H2, Ni

(56)

GHzoH

(57) where R = M e o r Et

Compound 55 is one of the rare examples of a 2,5-anhydro sugar that also possesses a furanoid ring; it has various interesting properties as a result of ring strain in the bicyclo[2.2.1] system, and these are discussed later (see p. 212). A glycosylamine analog in the D series (59), a derivative of uracil, has been p r e ~ a r e d 'by ~ the action of sodium benzyloxide on 1-[2,3anhydro-5-0-( methylsulfonyl)-~-~-lyxofuranosyl]uracil(58). (74) M. Cifonelli, J. A. Cifonelli, R. Montgomery, and F. Smith,]. Arner. Chern. SOC., 77, 121 (1955). (75) A. B. Foster and W. G . Overend, J. Chem. SOC., 680 (1951). (76) I. L. Doerr, J. F. Codington, and J. J. Fox, J. Org. Chern., 30, 467 (1965).

2,S-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

201

0

PhC4ONa PhC&OH

PhCH,O

0

I

(59)

I

PhCbO

Compound 59 is substantially more stable toward acid hydrolysis than the alkyl anhydroarabinoside 54, because boiling for three hours under reflux in 0.2 M sulfuric acid is needed in order to liberate the uracil. It should be noted, however, that the acid hydrolysis of pyrimidine nucleosides requires77 conditions considerably more severe than those needed for cleaving the anhydronucleoside 59. The action of sodium methoxide on 1-[5-0-(methylsulfony~)-/3-Dlyxofuranosyl]uracil (60) leads78to 1-(2,5-anhydro-/3-~-lyxofuranosyl)uracil (61); the 3,5-oxetane ring that might equally well have been

M8"cm OH

%

qc&(61)

(77) R. S. Tipson, Advan. Carbohyd. Chem., 1, 193 (1945); J. J. Fox and I. Wempen, ibid., 14, 283 (1959). (77a)B. Capon, Chem. Reu., 69,407 (1969). (78) J . F. Codington, I. L. Doerr, and J. J . Fox, J . Org. Chem., 30, 476 (1965).

202

J. DEFAYE

formed in this reaction is not observed, confirming that formation of an oxolane ring is favored when there are the two possibilities. The conditions for acid hydrolysis of this nucleoside derivative are more or less the same as those for the D-arahinose derivative 59. 2,5-Anhydro-D-lyxose could not, however, he isolated from the hydrolyzate thereof. Inspection of molecular models reveals that 2,5-anhydro-arahinose and -1yxose are the only 2,5-anhydroaldopentoses wherein the simultaneous presence of a 1,li-furanoid ring is sterically possible. c. Displacement of Halide Ions.-The displacement of a halide ion by an anionic atom of oxygen is quite analogous to the displacement of a sulfonate under the same conditions, and two examples of this type of reaction are, accordingly, presented in this Section. The action of (p-nitropheny1)hydrazine on 3,4-di-O-acetyl-2-hromoZdeoxy-D-xylopyranose (62)leads79to two (p-nitrophenyl)hydrazones, namely, 3,4-di-O-acety1-2,5-anhydro-~-xylose (p-nitropheny1)hydra(p-nitropheny1)zone (63) and 3,4-di-O-acety1-2,5-anhydro-~-lyxose hydrazone (64). Simultaneous formation of the two compounds, unex-

(79) A. Gerecs, Magy. Kern. Foly., 68, 211 (1962).

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

203

pected at the outset, can nevertheless be explained if it is considered that displacement of the bromine atom at C-2 by the oxygen atom of the heterocycle 62a leads in the first stage to 3,4-di-O-acetyl-2,5anhydro-aldehydo-D-lyxose(62b) which, under the influence of the basic medium, undergoes epimerization at C-2, by way of the intermediate 62c, before undergoing conversion into the corresponding hydrazones 63 and 64. The 2,5-anhydrides of 2-ketoses constitute a special case, in which the hemiacetalic carbon atom is involved in the anhydride bridge. Such anhydrides more closely resemble the 1,6-anhydroaldoses (glycosans) than the 2,5-anhydroaldoses. An example of a 2,5-anhydride of a 2-hexulose is obtained by the action of base on a D-fructosyl fluoride derivative; treatment of l-O-methyl-/3-D-fructopyranosyl fluoride at 90" with concentrated, aqueous sodium hydroxide leads,Eo in poor yield, to 2,5-anhydro-l-O-methyl-P-D-fructopyranose.

4. Solvolysis of Sulfonates The solvolysis of sulfonic esters of sugars in acid media does not seem to have been greatly exploited. The examples that can be given indicate, nevertheless, that the method has great potentialities for the interconversion of ring systems. From the mechanistic point of view, the reaction can be interpreted, in general, in terms of protonation on an oxygen atom of the ester group, which leads to a displacement of charges on the carbon atom of the carbonyl group thereof, and proceeds further to the formation of a carbonium ion. The action of 1% methanolic hydrogen chloride on 1,2-O-isopropylidene-3,5-di-O-p-tolylsulfonyl-~-xylofuranose (65) leads,E' after boiling for three hours under reflux, to 2,5-anhydro-3-0-ptolylsulfonyl-D-xylose dimethyl acetal (66). The structure of 66 was demonstrated b y conversion into the disulfonate 67; this was prepared independently from a 2,5-anhydro-~-xylosedialkyl dithioa~etal'~ (48) by p-toluenesulfonylation followed by exchange of the acetal group in methanol in the presence of mercury salts. A reaction identical from the standpoint of mechanism is the conversion*' of 3-O-benzyl-1,2-O-isopropylidene-5,6-di-O-p-tolylsulfonyl-D-glucofuranose (68) into 2,5-anhydro-3-0-benzyl-6-O-ptolylsulfonyl-L-idose dimethyl acetal (70) by boiling a solution of 68 in methanol containing 2% of concentrated hydrochloric acid for 40 hours under reflux. These two reactions involve initial removal (80) F. Micheel and E. A. Kleinheidt, Chem. Ber., 98, 1668 (1965). (81) J. Defaye and J. Hildesheim, Tetrahedron Lett., 313 (1968).

J. DEFAYE

204 TsO

-

MeOH, HCl,l%_ 0

Me

MeOH, HC1,2%

1 hour HO

OMe

2.5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

205

of the O-isopropylidene group, with formation, in the case of the D-glucofuranose derivative, of the corresponding methyl D-glucoside (69), which can be isolated as an intermediate after one hour of refluxing. The formation of the dimethyl acetal takes place only as a consequence of the second step, subsequent to, or simultaneous with, the closure of the 2,5-anhydro ring. This solvolysis resembles a substitution of the sN2 type, because, for the D-glucofuranose derivative,R2there is an inversion of configuration at the carbon atom carrying the group displaced. It should be noted that, if the carbonium ion is formed at C-5, the leaving group is sufficiently close to the molecule that the attack can take place only from the opposite side. Apparently related to the two preceding reactions is the action of 2% methanolic hydrogen chloride on 1,2-O-isopropylidene-5-selenoa-D-xylofuranose (72) (obtained from the benzylseleno derivative, 71), which affords83 a mixture of 2,5-anhydro-5-seleno-~-xylose(and -Dlyxose) dimethyl acetal (73). Similarly, the action of the same reagent

Na-NH,

*

O+Me Me

on 1,2-O-isopropylidene-5-thio-D-xylofuranose (74) furnishesM42,5anhydro-5-thio-~-xylose(and -D-lyxose) dimethyl acetal (75).

(82) J. Defaye and V. Ratovelomanana, Curbohyd. Res., in press (1971). (83) T. Van Es and R. L. Whistler, Tetrtihedron, 23, 2849 (1967). (84) B. Nestadt and T. Van Es, Tetrahedron, 24, 1973 (1968).

J. DEFAYE

206

Tq0

MeOH-HCI (2%)

HO

OLOMe

Me

(74)

(75)

A mechanism involving nucleophilic displacement of the hydroxyl group on C-2 by the selenium atom at C-5 has been proposed for the formation of the acetal 73. This mechanism does not, however, account for the presence of two products, epimeric at C-2, in the reaction mixture. It should be noted that 2,5-anhydro-aZdehydo-~-idose (79) had already been obtained85 by the action of aqueous sulfuric acid on 5,6-anhydro-1,2-0-isopropylidene-1~-~-glucofuranose (76); anhydride 79 is formed i n 25% yield, together with D-glucose (80), which is the major product (60%) in the reaction.

L

f

(77)

(85) C . A. Dekker and T.Hashizume, Arch. Biochem. Biophys., 78,348 (1958).

2,8ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

207

The competition between attack by solvent a t C-6 (intermediate 78) and attack at C-5 by the hydroxyl group on C-2 (intermediate 77) explains these results, which show the extent to which closure of an oxolane ring is favored. In principle, a third (unobserved) possibility exists, namely, closure of a six-membered, oxygen-containing heterocycle as a result of opening of the epoxide at C-6 by the hydroxyl group on C-2. Other examples of the solvolysis of sulfonates to give oxolanes have been reported. Thus, when 2,3-O-benzylidene-tri-O-(methylsulfonyl)D-arabinitol, in a mixture of 9 M acetic acid and 10 M hydrochloric acid is heated for 30 minutes at loo", it gives 2,5-anhydro-l,4-di-O(methylsulfonyl)-D-arabinitol.86 Additional instances of the formation of internal anhydrides (not always well characterized) by acid hydrolysis of various sulfonic esters of alditols have been given by the same authors.86 The formation in low yield of 3,6-anhydro-4,5-0-isopropylidene-~allose dimethyl acetal (82), together with methyl 3-O-p-tolylsulfonylD-glucopyranoside (83) as the main product, by the action of boiling 2% methanolic hydrochloric acid (under reflux for 27 hours) on 1,2:5,6di-O-isopropylidene-3-O-p-tolylsulfonyl-~-~-glucof~~ose (81) has been reported.87

4C-0

Me

TsO "'$-o

Me

HI X

MeOH-HCl(28)

0 Me

(86) S. S. Brown and (7. M. Timmis, J . Chem. SOC., 3656 (1961). (87) R. Ahluwahlia, S. J. Angyal, and M. H. Randall, Carbohyd. Res., 4, 478 (1967).

208

J. DEFAYE

In the cyclitol series, Gorings obtained 2',5-anhydroquinicol (83b) by the action of 50% acetic acid (for 6 hours at 57") on 5-0-p-tolyl-

sulfonyl-epi-quinicol(83a).

P H

o

~

HOAco I&O

~

*" (834

l

O

O

H

(83b)

In another example, the formations9 of racemic 1,rianhydroribitol by the action of dilute hydrochloric acid for 20 hours at 100" on Dribitol 1-phosphate has likewise been assumed to take place by protonation of the oxygen atom of the ester function, followed by an intramolecular substitution by the hydroxyl group on C-4. Formation of this protonated phosphoric ester likewise explains the racemization observed, caused by partial migration of the ester group at C-1 to C-5. Another example of solvolysis of a sulfonate has been described by Buchanan and coworkers;90 it occurs without acid, but utilizes a highly polarizable sulfonic ester group. Solvolysis of methyl 2-0(p-nitrophenylsulfony1)-a-D-glucopyranoside (84) in water in the presence of sodium acetate for 6 hours at 100" leads, after reduction of the reaction mixture by means of sodium borohydride, to a fraction identified as 2,5-anhydro-~-mannitol (85). This reaction, which

where N s

= p-02NC,H4S0,-

(85)

(88)P. A. J . Gorin, Can.]. Chem., 41, 2417 (1963). (89) J. Baddiley, J. G. Buchanan, and B. Carss,]. Chem. SOC., 4058 (1957). (90)P. W. Austin, J. G. Buchanan, and R. M. Saunders,]. Chem. SOC. (C), 372 (1967).

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

209

proceeds by a rearrangement of the Wagner-Meenvein type, resembles the nitrous acid deamination of 2-amino-2-deoxy-D-glucose, which also leads (after reduction of the product) to 85 (see p. 186).

5. Miscellaneous Methods It is conceivable that, starting from a pre-formed, five-membered heterocycle of the tetrahydrofuranol type, or even a furanosyl derivative, a contraction or extension of a side chain, according to the situation, would permit synthesis of a 2,5-anhydride of a sugar. This method has been used effectively on several occasions. a. Oxidative Cleavage of the Side Chain of a 3,8Anhydrohexose. 3,6-Anhydro-~-mannitolis readily obtaineds1 by the action of concentrated hydrochloric acid on D-mannitol. Protection of the hydroxyl groups on C-4 and C-5 as the isopropylidene acetal (86), followed by oxidation with lead tetraacetate, gives a quantitative yield of 2,5-anhydro-3,4-0-isopropylidene-~-arabinose (87).

2,5-Anhydro-~-xylose, characterized as the benzimidazole derivative formed from the corresponding 2,5-anhydro-~-xylonicacid and o-phenylenediamine, has likewise been preparede2 by the action of one molar equivalent of periodic acid on 1,4-anhydro-~-glucitol.

b. Attachment of a Side Chain to a G1ycofuranose.-The action of mercuric cyanide on 2,3,5-tri-O-benzoyl-~-ribofuranosyl bromide (88) in nitromethane affords,s3 in 88% yield, the corresponding nitrile (89) having the p-D configuration; hydrolysis of 89 with hydrochloric acid gives 2,5-anhydro-3,4,6-tri-O-benzoyl-~-allonic acid (90).This elegant synthesis also permits,s4 b y reduction of the nitrile 89, the synthesis of l-amino-2,5-anhydro-1-deoxy-~-al~itol (91), (91) A. B. Foster and W. G . Overend, J. Chem. SOC., 680 (1951). (92) C. F. Huebner and K. P. Link,]. Biol. Chem., 186, 387 (1950). (93) M. Bobek and J. Farkai, Collect. Czech. Chem. Commun., 34, 247 (1969). (94) M . Bobek and J. Farkai, Collect. Czech. Chem. Commun., 34, 1684 (1969).

J. DEFAYE

210

BzOCH,

0

BzO

Hg(CN), MeNO,

(90)

OBz (88)

(89)

characterized as its crystalline salicylidene Schiff base; compound 91 may be used directly as a precursor in the synthesis of analogs of nucleosides (see p. 218). 111. REACTIONS

The reactivity of the 2,5-anhydrides of aldoses is determined by two essential structural features that do not exist in the sugars, namely, the presence of an oxolane ring and of a carbonyl group (most frequently, free) a to the ring-oxygen atom. These two characteristics make the 2,5-anhydroaldoses closer to tetrahydro-2-furaldehyde than to the aldoses, where only in exceptional cases is the carbonyl group not masked by the formation of an intramolecular, five- or sixmembered, hemiacetal ring. 1. Stability of the Oxolane Ring The stability of cyclic ethers toward hydrolytic agents is largely a function of the size of the ring and, hence of the internal strain. Thus, whereas the oxiranes (epoxides) are particularly sensitive to these reagent^,^^.^^ the oxetanes are relatively more table.^' Oxolanes of the tetrahydrofuranol type seem, in particular, little affected by (95)R. E. Parker and N. S. Isaacs, Chem. Rev., 59, 737 (1959). (96)F. H.Newth, Quart. Rev. (London), 13, 30 (1959). (97)N. R. Williams, Adoan. Carbohyd. Chem. Biochem., 25, 109 (1970).

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

211

these reagents; opening of the ring generally takes place only after a step leading to formation of an unsaturated system. This aspect involves consideration of the stability of furan systems to acid; such heterocycles are readily opened by acid.gs Certain authors have presumed that the presence of a carbonyl group (Y to the oxygen atom of the heterocycle renders the ring more sensitive to acid hydrolysis. In fact, however, the action of M hydrochloric acid for three hours at 100" on 2,5-anhydro-3,4-0-isopropylidene-D-arabinose leads75only to hydrolysis of the acetal protectinggroup, without affecting the oxolane ring. There is, however, a certain amount of decomposition under these conditions, because, after the action of methanolic hydrogen chloride on this hydrolyzate, the yield of 2,5-anhydro-~-arabinosedimethyl acetal is only -35%; c01ored, unidentified products constitute the major proportion of the hydrolyzate. F. Smith and coworkers74observed the formation of 2-furaldehyde by the action of 0.1 N acid on 2,5-anhydro-~-arabinose,an observation that has been confirmed by other author^.^^,^^ The formation of furan derivatives by acid-catalyzed dehydration of a 2,s-anhydroaldose can be ascribedg9 to the elimination of a group p to the carbonyl group. It is known that electron-attracting groups strongly reinforce the acidic character of the proton removed by nucleophilic agents in the course of this type of reaction. In contrast, the corresponding anhydroalditols are not affected under the same conditions of treatment.99 Furthermore, 2,5-anhydro-3-deoxy-~-erythro-pentose, which lacks a hydroxyl group p to the carbonyl group, is recoveredloo unchanged after treatment with boiling 0.05 M sulfuric acid for 2.5 hours under reflux. It is probable that a tetrahydrofuranol ring formed between two secondary alcohol groups is more stable toward nucleophilic agents than a similar type of heterocycle where at least one of the original alcohol groups is primary. This property is illustrated by the hydrolysis of 2,5:3,6-dianhydro-~-glucitol in concentrated hydrochloric acid for 8 hours at 100";this treatment cleaves the 3,6-anhydride ring, and 1eads'O' to 2,5-anhydro-6-chloro-6-deoxy-~-glucitol, isolated as its 173,4-triacetate. To date, the only reagent found capable of opening the ring of a (98) F. H. Newth, Adoan. Carbohyd. Chem., 6,83 (1951). (99) J. Defaye and S. D . CBro, Bull. Soc. Chim. Biol., 47, 1767 (1965). (100) J. Defaye and J. Hildesheim, unpublished results. (101) L. Vargha and J. Kuszmann, Carbohyd. Res., 8, 157 (1968).

J. DEFAYE

212

2,5-anhydroaldose is phenyIhydra~ine.~~ This fact has been the cause of a long polemic argument on the structure of 2,5-anhydro-~-mannose, because treatment thereof with phenylhydrazine gives Darabino-hexosulose phenylo~azone,~' not the anticipated phenylhydrazone of 2,5-anhydro-~-mannose. 2. Reactions Involving a Carbonyl Group a. Addition Compounds. - In contrast to the 3,6-anhydroaldohexopyranoses, where, despite a certain amount of strain between the two cyclic systems, it is somewhat unusual for the carbonyl group not to be masked by the formation of an intramolecular hemiacetal, the 2,5-anhydrides of aldoses are only in exceptional cases encountered as hemiacetals of this type. The alkyl 2,5-anhydro-p-~-and -CU-Larabinofuran~sides'~.~~ and l-(2,5-anhydro-~-~-lyxofuranosyl)uraci1~~ represent the sole examples yet known. It is noteworthy that ethyl 2,5-anhydro-a-~-arabinofuranoside in water at room temperature is hydrolyzed completely in 96 hours.74 The action of boiling 1.4% methanolic hydrogen chloride for two (92) hours under reflux on ethyl 2,5-anhydro-a-~-arabinofuranoside affords74a quantitative yield of the corresponding dimethyl acetal(93);

OEt

MeOH-HCI (1.4%) reflux, 2 hours

the reaction well illustrates the high degree of strain that exists in this bicyclic system. The same reagent applied to 2,5-anhydro-~the corresponding dimethyl acetal (95), only. mannose (94) gives36.99

HOCH,

0

MeOH-HC1 (1%)

OC=. reflux, 1 hour

HO

HO

2,5,-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

213

N o formation of a methyl glycoside under these conditions has been detected. Inspection of molecular models of the 2,5-anhydroaldohexoses reveals that, when the carbonyl group and the primary alcohol group on C-6 are cis-disposed, as in 2,5-anhydro-aZdehydo-~-glucose (96) for example, the formation of a 1,6-hemiacetal (97) is possible without

HOCH

0

HC=O

HO 0

much strain. This factor is of particular relevance to the “mutarotational” behavior of the 2,s-anhydrides of aldehydo-aldoses. With the exception of 2,5-anhydro-~-glucose,’~,~~ “mutarotation” has been observed for all 2,5-anhydrides of aldehyde-aldoses reported to date (see Table I). Grant36 has attributed the “mutarotation” of 2,s-anhydro-D-mannose to the formation of a 1,4-hemiacetal, although he has shown that the action of methanolic hydrogen chloride on this comTABLEI Mutarotation of Various Free or Partially Substituted 2,5-Anhydro-aZdehydo-aldoses 2,5-Anhydride of D-Mannose

TemperConcen- ature ReferEquilibrium Solvent tration (degrees) ences

[ a l (degrees) ~ Initial +70.8 +33.6 +33.9 -55.8 +8.3 +15.3

1 hour

+21.9 +22.2 +27 -43 +16.6 12.5

D-Talose D-Ribose D-XylOSe D-LyXOSe 3,4,6-Tri-O2.5 hours 14.8 methyl-D-mannoSe +24 w 3,4-0-1~0propylidene-n73 hours -176 arabinose -126 3,4-O-Isopropylidene-D-ribose - 46.8 12 hours - 64.7

+ +

-

HzO H,O H,O H,O H,O H,O

2.6 2.2 0.44 0.86 0.60 3.2

20

25 25 25 25 25

43 68 68 68

CHCI,

2.5

25

99

20

74

27

117

CHCI, ~

35 99

J. DEFAYE

214

pound affords only the corresponding dimethyl acetal. On the other shows hand, 2,5-anhydro-3,4,6-tri-O-rnethyl-aldehydo-~-mannose “mutarotation” in certain solvents (see Table I), and 2,5-anhydro-3,40-isopropylidene-aZdehydo-~-arabinose behaves ~imilarly‘~;these products cannot form internal hemiacetals, 1n.view of these results, the apparent absence of “mutarotation” for 2,5-anhydro-aldehydo-~-glucose, to the impossibility of its forming an internal hemiacetal, remains to be confirmed. Instead of necessarily being the consequence of the formation of intramolecular hemiacetals, it seems likely that the “mutarotation” observed with the 2,5-anhydroaldoses could also be attributable to the formation of hydrates or of unstable hemiacetals with hydroxylated solvents, or even to dimerization. Known examples in related series lend support to these suppositions. It is known, for example, that 1,2-O-isopropylidene-5-~Zdehydo-a-~-xyZo-pentodialdo-1,4-furanose (98) readily forms102a dimer (99). Working with related com-

O=CH

QT

0

O+Me Me O+Me Me (99 )

pounds, Rosenthal and coworkers103have shown that 4,s-di-0-acetyl-

2,6-anhydro-3-deoxy-aldehydo-~-xyloand -D-lyxo-hexose (which, because of the absence of free hydroxyl groups, cannot form dimers of type 99) exist in solution partially in the hydrated form. Horton and coworkerslM have likewise shown that 1,2:3,4-di-Oisopropylidene -a-D-galacto-hexodialdo- 1,s-pyranose exists exclusively as the hydrate in deuterium oxide, and that this compound remains partially hydrated in moist chloroform. As early as 1931, W ~ l f r o mhad ~ ~shown ~ that the formation of hemiacetals from aldehydo sugars in hydroxylic solvents is a common occurrence. (102) R. Schder and H. S . Isbell, J . Amer. Chern. SOC., 79, 3864 (1957). (103) A. Rosenthal, D.Abson, T . D. Field, H. J. Koch, and R. E. J. Mitchell, Can. J . Chern., 45, 1525 (1967). (104) D.Horton, M. Nakadate, and J. M. J. Tronchet, Carbohyd. Res., 7, 56 (1968); D.Horton and J. D. Wander, ibid., 16, in press (1971). (105) M. L.Wolfrom,J.Amer. Chern. SOC., 52,2464 (1930);53,2275 (1931).

2.5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

215

Also relevant to this matter is the fact that the n.m.r. spectra of the in deuterium oxide rarely show106 free 2,5-anhydro-aZdehydo-aldoses the low-field signal characteristic of the free aldehyde group. Such a signal is generally visible when the spectrum (in chloroform-d) of compounds that are partially substituted is recorded, but its intensity is often weak. In 1925, Levene and Ulptsis proposed the possibility that polymerization was responsible for some of the unusual properties of the 2,5-anhydroaldoses. The formation of dimers, hydrates, and hemiacetals with the solvent can likewise be held responsible for the characteristic migration behavior observed for the 2,5-anhydroaldoses with aqueous alcoholic solvents on paper chromatograms.68

b. Dehydration. - The mechanism that leads to rapid transformation, in the presence of acid, of 2,5-anhydro sugars into furan derivatives has been discussed in the previous Section. Although the formation, in low yield, of an oligosaccharide that gives 2,5-anhydroL-idose and D-glucose on hydrolysis with acid had already been noted'*' as a result of the action of an acid on D-glucose or on starch, it seems unlikely that passage through such intermediates constitutes a fundamental pathway in the acid-catalyzed degradation of hexoses and pentoses. For example, the yield of 5-(hydroxymethy1)2-furaldehyde from 2,5-anhydro-~-mannoseis only 12%, whereas, under the same conditions, D-fructose gives 20-25% of the same furan derivative, and the aldohexoses afford the furan derivative in 1% yield.lo8 From present evidence,lo9 it seems that several pathways in each, of which the common stem is a 1,2-enediol precursor, are involved. It is, however, a plausible supposition that one of the minor pathways to 5-(hydroxymethyl)-2-furaldehydeinvolves direct passage by way of a 2,5-anhydro sugar. IV. UTILIZATION Although 2,5-anhydro sugars had, for a long time, been poorly understood, they have now found useful application as intermediates in synthesis, and even as derivatives for the characterization of certain amino sugars. For example, a useful method for the analytical deter(106) J. Defaye, unpublished results. (107) P. Slonimski, J . Defaye, J . Asselineau, and E. Lederer, Compt. Rend., 249, 192 (1959). (108) W. Alberda van Ekenstein and J. J. Blanksma, Ber., 43, 2355 (1910). (109) E. F. L. J. Anet, Aduan. Carbohyd. Chem., 19, 181 (1964).

J. DEFAYE

216

mination of 2-amino-2-deoxy-~-glucose and -D-galactose involves initial deamination with nitrous acid followed by treatment of the resulting 2,5-anhydroaldohexose with indole in an acid medium, to give a colored product having a maximum absorption at 492 nm.Ilo This color reaction is applicable to all of the known 2,s-anhydroaldoses, and appears to be relatively ~ p e c i f i c ; ~ "however, ~" it has been statede5that 2-deoxy-~-erythro-pentosegives a positive reaction in this test. As regards the use of2,5-anhydro sugar derivatives as intermediates in synthesis, the work of Hardegger"' on the synthesis of nonnuscarine (104) is particularly noteworthy. Treatment of the methyl ester (100) of 2-amino-2-deoxy-~-gluconicacid with nitrous acid gave the methyl ester (101) of 2,5-anhydro-~-gluconicacid; the diniethylamide (102) of this acid was readily converted into the corresponding tris(p-toluenesulfonate) (103) which, on reduction with lithium aluminum hydride, afforded normuscarine (104). 0, C '

,OMe I

H,NCH I

HCOH I

HOCH I

HOCH

J HO

TsO

Starting from the appropriate sulfonylated acetals 105, 106, and 107 (from 2,5-anhydro-~-ribose,-D-xylose, and -D-lyXOSe, respectively), the application of a method for elimination of vicinal, secondary disulfonates by reaction with sodium iodide in N,N-dimethylformamide in the presence of zinc112 has permitted113 the preparation of the (110)Z.Dische and E. Borenfreund,]. Biol. C h e n . , 184, 517 (1950). (111)E. Hardegger and F. Lohse, Helo. C h i n . Acto, 40,2383 (1957). (112)R. S . Tipson and A. Cohen, Carbohyd. Res., 1, 338 (1966). (113)J. Defaye and J. Hildesheim, Bull. SOC. Chim. Fr., 940 (1967).

2,5-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

217

enantiomorphic R (112) and S (113) forms of tetrahydro-2-furaldehyde114 by way of the corresponding acetals (108 and 109) of the dihydro-4H-2-furaldehydes and those (110 and 111) of the tetrahydro-2-furaldehydes.

TsO

OTs

TsO (107)

NaI, Zn, HCONMe,

0

HC(OR),

0

(114)

(114) J. Defaye, Bull. SOC. Chim. Fr., 2099 (1968).

J . DEFAYE

218

The 2(R)-tetrahydro-2-furaldehyde (112) is reduced1l5 by sodium borohydride to the corresponding alcohol (114). 2,5-Dihydrofurfuryl alcohol can be prepared, starting from 2,5-anhydro-l-O-benzoyl-3,4di-0-p-tolylsulfonyl-D-ribitol, by using the Tipson-Cohen112 method for introducing the 3,4-double bond, with subsequent saponification of the ester."' Finally, the use of 2,5-anhydroalditols in the synthesis of nucleoside analogs that are modified in the base-sugar linkage may be cited. It is known that many synthetic analogs of nucleosides, having structural modifications either in the base or in the sugar, are readily inactivated in vivo by the action of glycosidases. The introduction of a methylene group into the base-sugar linkage, with retention of a furanoid ring, has been p r o p o ~ e d " ~asJ ~a~method of avoiding this enzymic destruction. A new series of nucleoside analogs of such types starting as 115, 116, 117, and 118 have thus been prepared,94J15*117**18 from derivatives of 2,5-anhydroalditols.

(117)

where R = OH, R' = H R = OH; R' = Me R = NH,; R' = H

(118)

where R = OH or NH,

(115) J . Defaye, M. Naumberg, and T. Reyners,]. Heterocycl. Chem., 6,229 (1969). (116) J. CIBophax, J. Hildesheim, and S. D. Gkro, Bull. SOC. Chim. Fr., 4111 (1967). (117) J. Defaye and T. Reyners, Bull. SOC. Chim. Biol., 50, 1625 (1968). (118) J. Defaye and T. Reyners, to be published.

2,B-ANHYDRIDES OF SUGARS AND RELATED COMPOUNDS

219

v. TABLEOF

PROPERTIES OF 2,5-ANHYDEUDES OF SUGARS, ALDITOLS, AND ALDONIC ACIDS

In Table I1 are given the melting point, boiling point, and specific rotation of the 2,5-anhydrides of sugars, alditols, and aldonic acids that have thus far been studied.

E3

TABLEI1

E3 0

Properties of 2,s-Anhydrides of Sugars, Alditols, and Aldonic Acids [ffltl,

Derivative

M.p., "C.

2,5-Anhydro-n-allo-pentitol 181.5-2.5 1-(6-azauracil-5-y1)3,4O-isopropylidenesyrup 119.5-21 1-[2,3,4,5-tetrahydro-3-thioxo-as-triazin-5-2(~-one-~yl 12,5-Anhydro-~-altro-pentitol 182-3.5 1-(6-azauracil-5-yl)240-1.5 3,4-0-isopropylidene233-5 1-[2,3,4,5-tetrahydro-3-thioxo-a~-tri~in-5-(2~)-one-6-y~]2,5-.4nhydroallaric acid 2,5-Anhydroallitol l-amino-l-deoxysyrup 3,4,6-tri-O-benzyl-l-(4-chloro-l,2-dihydropyrimidin-2-one-l-yl)-l-deoxyarnorph. amorph. 3,4,6-tri-O-benzyl-l-deoxy-l-(uracil-l-yl)88.5-9.5 3,4,6-tri-O-henzyl-l-deoxy-l-ureido213-15 1-(cytosin-1-y1)-1-deoxy191-2.5 l-deoxy3,4-O-isopropylidene-l-( uracil-1-yl)118-9 ldeoxy-1-(salicy1ideneamino)146-7 1-deoxy-(1-uracil-1-y1)amorph. l-deoxy-l-ureido2,5-Anhydro-~-allonicacid SYNP 215-7 4,6-diamino-5-aminopyrimidineester arnorph. 3,4,6-tri-O-benzoyl203-4 2,5-Anhydrogalactaric acid 2,5-Anhydro-~-galactonic acid 244 brucine salt

B.p., "C./torr

degrees +48.8

+ 109.2 -122.9 -183.4 0

Rotation solvent

Reference 129 129 129 129 129 129 16 94

-7.6 -1 +16.7 -52.5

94 94 94 94

+9.9 +55.2 +31.3 0

94 94 94 94 93 93 93 10

-9.4

12

-18.1 -42.9 +3.8

* v

5B

2,5-Anhydro-~-glucaricacid 2,5-Anhydro-~-glucaricacid 2,5-Anhydro-~-glucitoI 1,3,4-bi-O-acety1-6-chloro-6-deoxy1,6-di-O-(phenylsulfonyl)1,6-di-O-benzoyl2,5-Anhydro-~-gluconicacid 2,5-Anhydro-~-gluconicacid dimethylamide 3,4,6-tri-O-p-tolylsulfonylmethyl ester 2,5-Anhydro-aZdehydo-~-glucose (?) B,J-Anhydro-~-idaricacid 2,5-Anhydro-~-iditol

1,3,4,6-tetra-O-acetyl1,3:4,6-di-O-benzylidene1,6-dideoxy-1,6-diiodo1-0-p-tolylsulfonyl4,6-O-isopropylidene6-0-trityl1,6-di-O-p-tolylsulfonyl-

2,5-Anhydro-aldehydo-~-idose 3-O-benzyl-6-O-p-tolylsulfonyl-, dimethyl acetal 1-(2,2-diphenylhydrazone)

163 163 56-9 130-1 115-2OiO.25 109-9.5 137-8 135-7 amorph. 144 172 141-2 d. 175 d. 240 226 117-8 111-3 116.5-8.0

-19.1 -29

124-40/0.003 147-8 110-1 109-10 146 98- 100 syrup 146 148-9.2 151 amorph. SYNP 154-6

+38.53 -38.79 +24.4 -53 -4.4 +21 +3.2 +1.2 +70.3

-96 -93.3 t-9.7 + 12.6 +6.2 -13.2 +32.3 +97.1 + 105.7 +3.8 +8.1 + 16.3 +6.6 -6.6 -9 +11 20.4 +20

9 9 120 121 101 121 122 101 18 111 111 111 111

15 9.14

124 123 120 123 124 126 82 82,125 106 125 124,126 85 82 85 82 85 (continued)

N

E3

TABLEI1 (continued)

KJ

to bID1

Derivative

2,5:4,6-Dianhydro-~-idose &O-benzyl-, dimethyl acetal 2,SAnhydro-~mannaricacid 2,5hhydro-~-mannitol 1,3,4,6-tetra-O-acetylldeoxy- 1,l-difluoro6deoxy-6-iodo-3,4-di-O-p-tolylsulfonyl3,4,6t1i-O-methyl3,4,6-t~i-O-p-tolylsulfonyl1,3:4,6-di-O-methylene1,6-di-O-p-tolylsulfonyl1,6-di-O-trityl2,5-Anhydro-~mannonicacid

ethyl ester methyl ester 3,4,6-tri-O-methylmethyl ester 2,5-Anhydro-mmannose

1,3,4,6-tetra-O-acetyl-, (methyl “a”-hemiacetal) 1,3,4,6-tetra-O-acetyl-, (methyl “B”-hemiacetal) 4,6-O-benzylidene1-(2-benzy1-2-phenylhydrazone) diethyl dithioacetal dimethyl acetal

M.p., “C. 52-3 184-5 100-01 syrup syrup 85

B.p., “C./torr 132-5/0.1

degrees +41 +46.1 +58.2 +27.3; +26.4 +31.6

60-64/0.02 95 120 133.5 149 amorph. 106 syrup SYNP amorph. amorph. amorph. SYNP syrup

145-7 144-5 85 SYNP SYNP

+16 -59.1 r+ 6 +9.6 +60.7 +44.5 +38.3 +46.7

125/0.1 100/0.1

+69.9 +55.3 +33&1 +70.8++21.9 +33.6++22.2 +49.4 +46.3 -22.5 -22.4 +50.2 +60.4 +30.9

Rotation solvent

Reference

CHCI, HzO HzO CHCl, HzO

82 5,6,15 37,120 61 65 65 65 CHCl, 65 p-dioxane 127 35 C&N C&N 35 HzO 6 5%HCI in H,O 18 36 EtOH 36 HzO 36 HzO 36 H,O 19 H,O 35 H,O 99 CHC1, 61 CHCl, 61 Me,CO 46 Me2C0 47 36 EtOH Me&O 36 HzO 36

?

*

-I -4

M

3,4,6-tri-O-acetyl3,4,6-tri-O-methyl3,4,6-tri-O-methylsemicarbazone (p-nitropheny1)hydrazone

(2,4-dinitrophenyl)hydrazone 1-(2,2-diphenylhydrazone)

syrup 148 185 175 144-5

90/0.1 l2OlO.l

144-5 2,5-Anhydro-~talitol tetra-0-acetyl2,5-Anhydro-~-talonicacid, brucine s a l t 2,5-Anhydro-~-talose

112-3

t37.5 +7.1 +49.8 +30 +28.3 +44.5

MeOH EtOH MeOH

+7.4 +72

+ 193 +15.2 +121 +115 85-100/0.001 65-7510.135

-81.7 - 167 - 141

137 SYNP amorph. syrup syrup syrup

CHC13 HzO

-12.4 +20 +33.9++27

218 amorph. amorph.

(2,5-Anhydro-l,3,6-trideoxy-~-ribo-hexitol-l-yl)-t~imethyl179-80 ammonium chloride [L-(+)-Muscarinechloride] 2,5-Anhydro-P-~-arabinofuranose 194-5 l-deoxy-1-(5,6-dihydrouracil-l-yl)d. 260-2 1-deoxy-1-(uracil-1-y1)249-50 3-0-benzoyl241-3 3-0-benzyld. 196-8 3-O-(methylsulfonyl)2,5-Anhydro-a-~-arabinofuranoside ethyl methyl %carbanilate 3-0-methyl2,5-Anhydro-~-arabinose dimethyl acetal 3,4-O-isopropylidene-

110-15/0.005 160-5/0.3

+41.7 +39.8

115-20/0.01 118-24/0.03

-114 -22 -44.4 -131.5 - 126-+- 176 12

+

HzO HzO Hz0

61 36 36 36 36 36 35 42 43 43 12 19 43

H20

111

EtOH MeOH HZO

H20 HzO MeZCO p-dioxane Me,CO

76 76 76 76 76

0.1 M NaOH in HzO HzO Me&O

74

Hz0 HzO MeOH C.5& C6H6

H@

74 74 74 91 91 91 74 74

(continued)

Ls

tQ w

TABLEI1 (continued) [CIID,

Derivative

1-(2-benzyl-2-phenylhydrazone) dimethyl a c e d 2,s-Anh ydro-~arabinitol 3-O-acetyl-1,4-di-O-(methylsulfonyl)1,3,4-tri-O-benzoyl1,4-di-O-(methylsulfonyl)1,3,4-tri-O-p-tolylsulfonyl2,5-Anhydro-~-arabini to1

1,3,4-triazido-l,3,4-trideoxy1,3,4-tri-O-p-tolylsulfonyl2,5-Anhydro-~-arabinonic acid, methyl ester 2,5-Anhydro-~lyxitol 1,3,4-tri-O-benzoyI1,3,4-tri-O-(p-nitrobenzoy1)2,5-Anhydro-~-lyxitoI 4-azido-l-O-benzoyl-4-deoxy-3-O-p-tolylsulfonyl1,4-diazido-l,4-dideoxy-3-O-p-tolylsulfonyl1,3,4-tri-O-(p-nitrobenzoyl)1-(2,5-Anhydro-p-~-lyxofuranosyl)uracil 3-04methy1sulfonyl)2,S-Anh ydro-D-lyxose diisobutyl dithioacetal 3,4-di-O-p-tolylsulfonyldibutyl acetal 3,4-di-O-p-tolylsulfonyl2,5-Anhydro-~-ribitol 1-amino-1-deoxy-, hydrochloride

M.p., "C.

B.p., 'C./torr

degrees

Rotation solvent

115-20/0.005 125-35/0.24 syrup 75-6 117-8 153-4 128-9 115-25/0.09 120-2510.05 128 syrup 14S/0.0006 syrup 80- 1

Reference

+30.5 - 1.4 20.5 -0.5

MeOH HjO H,O

-36.1 +21 +27.4 +0.2 +34.7 -27.4

CHCI, CiHiN CHCl , H,O CHCI, CHCl,

74 74 74 128 86 128 86 74 74 130 74

t25.3 +77.5 -8.5

MeOH CH,CI, CHCl

74 68 128 68

-38

CHCl,

129-30

67-8 liquid 80-2 220-3 d. 190-1 amorph. 100-01 98

+85.1 +234 + 163 +15.3++12.5 +46.8 +35

syrup 98-9 164-8

-67 -79

CHCI, H,O p-dioxane H20 CHC1, CHCl

130 130 71 78 78 68 68 114

H,O H,O

114 68 119

w

z

?i

P

.e

m

l-azido-l-deoxy-3,4-O-isopropylidene62-510.005 l-azido-l-deoxy-3,4-di-O-p-tolylsulfonyl69-70 l-O-benzoyl-3,4-di-O-p-tolylsulfonyl98-9 l-O-benzyl-3,4-O-isopropylidene125-710.02 l-O-benzyl-3,4-di-O-p-tolylsulfonyl87-88 1-(6-arninopurin-9-y~)-l-deoxy-, hydrochloride 205-206 l-(cytosin-l-yl)-l-deoxy263-265 1-deoxy-1,l-difluoro75 l-deoxy-l-(2,4-dinitrophenyl)amino-3,4-O-isopropylidene-144,5-145,5 l-deoxy-l-(2,4-dini trophenyl)amino-3,4-di-O-p-tolylsulfonyl- 75 l-deoxy-l-iodo-3,4-O-isopropylidene6510.03 l-(N-acetylcytosin-l-yl)-l-deoxy-3,4-O-isopropylidene203-205 1-(5-amino-4-chloropyrimidin-6-yl)-l-deoxy-3,4-0-

isopropylidene1-(6-aminopurin-9-y1)l-deoxy-3,4-O-isopropylidene1-deoxy-1-( purin-6-ylamino)-3,4-O-isopropylidenel-deoxy-3,4-O-isopropylidene-l-(uracil-I-yl)l-deoxy-l-(purin-6-ylamino)1-deoxy-1-(thymin-1-yl)1-deoxy-1-(uracil-1-yl)-

3,4-0-isopropylidene1-0-p-tolylsulfon yl3,4-di-O-p-tolylsulfonyl1,3,4-tri-O-j~-tolylsulfonyl-

2,5-Anhydro-~-ribitoI 1,3,4-tri-O-benzoyl1-deoxy-1, l-difluoro2,5-Anhydro-~-ribose dibutyl acetal,3,4-di-O-p-tolylsulfonyl diisobutyl dithioacetal 3,4-O-isopropylidene-

145-146 185-187 244-245 179,5-180 230 178-180 198-199

-46 - 104 - 74 -28.6 -38 -51 -78.4 -26 -63.6 -82 -23.1 - 134 -98 -98

-85.3 -78

-67.5 -58.5 -69.6

60-510.005 62-63 118 125-126 99-100 73-75 73-4 amorph. amorph.

-40.1 -21.3 -53.5 -45.6 +66.7 +lo7 +25 -55.8+-43 -95.4

CHCI, CHCI, CHCI, CHCI, CHCl, H,O

KO

MeOH CHCI, CHCI, CHCI, MeOH

CHCI, MeOH MeOH CHCls H,O H,O HzO

MeOH CHCI, CHCI, CHCI:, H,O

CHCI, MeOH HzO

H,O

syrup

89-90 120-125/0.02

-85.6 -51.9

MeOH

CHCI,

119 130 116 116 116 117 118 64 119 130 118 118 117 117 119 118 119 118 118 119 119 116 116 69,71 128 64 68 67 114 67 117 (continued)

!2

TABLEI1 (continued) Derivative 3,4-di-O-p-tolylsulfonyl3,4-O-isopropylidene(p-nitropheny1)hydrazone dipropyl dithioacetal 2,5-Anhydro-~xylitol 1,3,4-tri-O-acetyl-

3,4-di-O-acetyl-1-0-trityl2,5-Anhydro-~-xylitol 1,3,4-tri-O-acetyl2,5-Anhydro-~xylose 3,4-di-O-acetyl-,dimethyl acetal dibenzyl dithioacetal 3,4-bis(phenylurethan) diisobutyl dithioacetal 3,4-di-O-p-tolylsulfonyldimethyl acetal, 3-0-p-tolylsulfonyl3,4-di-O-p-tolylsulfonyl(p-nitropheny1)hydrazone 2,5-Anhydro-3-deoxy-~-erythro-pentitol 1,4-di-O-acetyl2,5-Anhydro-3-deoxy-~-erythro-pentose diethyl dithioacetal 2,5-Anhydro-4-deoxy-~-erythro-pentitoI 1,3-di-O-benzoyl1,3-di-O-(p-chlorobenzoy1)-

M.p., "C.

90- 1 140 67-70

153

amorph. amorph. 98 168 94 76-8 76-8 86-7 196

amorph.

47-8 60-61

B.p., 'C./torr

blu, degrees

-50 -46.8+-64.7 50-5lO.02 +6.5++3.2 -91.4 10 150-55/0.2 -45.5 90/0.02 -39.3 90/0.02 +6 -11.2 k1 160-70/0.02 90-98/0.04 +31.5 k1.5 +9.0 +8.3++16.6 110-15/0.15 - 16 +159.6 +9.3 f11.5 +23.5 +53.7 ffi4.5 +145-++93.3 +1 *I 8W0.02 -40 55-60/0.02 +13.5++15 -t+8 - 15 90/0.01 105- 15/0.02 t48.1 -59.4 +68.1

+

Rotation solvent

CHCI, CHCI, MeOH MeOH HzO CHCI, CHzCIz CsH, HzO CH,CI, HzO HzO CHCI, CHCI, C,H,N CHCl, CHCln CHCI, CHCI, MeOH MeOH CHCI,

HzO CHCI, H20 CHzCI, CH,CI,

Reference 113 117 67 67 68 68 68 68 70 70 67 68 131 67 67 67 113 81 81,113 67 100 100 100

100 128 128 128

? ?1

t;M

1,3di-O-( p-methylbenzoy1)1,3-di-O-(p-nitrobenzoyl)Optically active di- and tetra-hydrofurfury1 derivatives formed from 2,5-anhydropentoses (2R)-2,5-Dihydro-2-fancarboxaldehyde dibutyl acetal diisobutyl dithioacetal (2s)-2,5-Dihydro-2-furancarboxaldehyde dibutyl acetal diisobutyl dithioacetal (2R)-2,5-Dihydro-2-frylmethanol O-benzoylO-benzyl(2R)-Tetrahydro-2-furancarboxaldehyde dibutyl acetal (2S)-Tetrahydro-2-furancarboxaldehyde dibutyl acetal (2R)-Tetrahydro-2-furylmethan01 O-benzoylldeoxy-l-iodoldeoxy-1-(uracil-1-y1)1-0-p-tolylsulfonylDianhydrides 2,5:3,6-Dianhydro-~-glucitol 4-0-acetyl-1-0-tritylldeoxyl-deoxy-l-iodo4-0-(rnethylsulfony1)ldeoxy-1-iodo-4-O-p-tol ylsulfonyll-deoxy-4-O-(methylsuIfonyl)1-deoxy4-0-p-tolylsulfonyl-

1,4-di-O-(methylsulfony1)1,4-di-O-p-tolylsulfonyl-

CH,Clz CHzClz

128 128

t97.8 +95.5

CHCl, CHCI, CHCI,

114 113 116

118 syrup

-123.3 -95.3 170 f101.2 +67 -3.4 +9.1 f4.2 - 10.7 -14.8 -23 19 -52.5 -15.5

CHCl, CHCl, MeOH CHCI, CHCl, MeOH CHCI, MeOH CHC1, CHCl, CHCI, CHCl, CHCl, CHCl,

114 114 116 116 116 114 114 114 114 115,132 116 115 115 115

117-9 143-5 81-2 111-3 113-4 92-3 85-6 123-5 123-4

+94.4 +30.7 107.9 +61.1 +46.7 +69.3 f60.1 +50.2 +37.7

HzO CHCl, Hz0 CHCl, CHCl, CHCI, CHCl, CHCl, CHCl,

101 101 101 101 101 101 101 101 101

+63.5 +65

63-5 137-8

90-95/0.1 90-95/0.02 103-05/0.01 90-95/0.05 90-95/0.005 120/40 78-80/0.01 80-8210.01 75-80/0.02 60-5/0.05 75-80/0.02 60-6510.005 85/18 80-8210.05 35-40/0.05

+123.5

+

+

+

rA

c Q

k v)

?-

z

U

E3

El

4

228

J. DEFAYE

(119) J. CIBophax, J. Defaye, and S. D. CBro, Bull. Soc. Chim. Fr., 104 (1967). (120) J. W. LeMaistre, personal communication (1965). (121) J. M. Sugihara and D. L. Schmidt,/. Org. Chem., 26, 4612 (1961). (122) R. C. Hockett, M. Zief, and R. M. Goepp, Jr., J. Amer. Chem. Soc., 68,935 (1946). (123) L. Vargha, T. Puskas, and E. Nagy, /. Amer. Chem. SOC., 70, 261 (1948). (124) L. Vargha and E. Kasztreiner, Chem. Ber., 93, 1608 (1960). (125) L. Vargha, Ber., 68, 1377 (1935). (126) L. Vargha and T. Puskcis, Ber., 76, 859 (1943). (127) A. K. Mitra and P. Karrer, Helo. Chim. Actu, 38, 1 (1955). (128) A. K. Bhattacharya, R. K. Ness, and H. G . Fletcher, Jr., /. Org. Chem., 28, 428 ( 1963). (129) M. Bobek, J. Farkag, and F. Sorm, Collect. Czech. Chem. Commnn., 34, 1673 (1969). (130) J. ClBophax, J. Hildesheim, R. E. Williams, and S. D. GCro, Bull. S O C . Chim. Fr., 1415 (1968). (131) J . Defaye and M. Naumberg, unpublished results. (132) The enantiomorphic tetrahydro-2-furylmethanols and certain of their derivatives can also be obtained by resolution of the racemic forms, or by cyclization of a 1,2,5-pentanetriol derivative. For relevant references, see Ref. 115.

ALDITOL ANHYDRIDES BY S. SOLTZBERG Atlos Chemical Industries, Inc., Wilmington, Delaware

I. Introduction.. . . .

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

229

...........

1. Industrial.. .......................................................... 267 268 2. Biological.. .......................................................... VI. Tables of Properties of the Anhydrides and Their Derivatives. ............... 270

I. INTRODUCTION

An article on the anhydrides of polyhydric alcohols appeared in Volume 5 of this Series,' and it is the purpose of the present article to bring the subject up to date. Wiggins' article' was limited to the anhydrides of the pentitols and hexitols; the present article will include the anhydrides of tetritols and of alditols higher than the hexitols. However, anhydrides having the three-membered (oxirane) ring will not be considered, as they are discussed elsewhere in this Volume.la 2,5-Anhydrides of aldoses are also treated in this Volume.lb (1) L. F. Wiggins, Aduan. Carbohyd. Chem., 5, 191 (1950). (la) N. R. Williams, This Volume, p. 109. (lb) J. Defaye, This Volume, p. 181.

229

230

S. SOLTZBERG

In a broad sense, little that is new has been added to the procedures for synthesizing the anhydroalditols and their derivatives. However, three reactions, which will be discussed at the appropriate places, are of general interest. These are ( a )the oxidation of isolated hydroxyl groups to ketone groups in the presence of platinum oxide, (b) the isomerization of 1,4:3,6-dianhydrohexitols,and (c) the synthesis of C-aryl and -alkyl derivatives of anhydroalditols (C-glycosyl compounds, the so-called “C-glycosides”). A fourth very interesting reaction, of limited application, is formation, from 1,4:3,6-dianhydro-~glucitol (“isosorbide”) sulfonates, of 1,4:2,5:3,6-trianhydro-~-mannitol. As many derivatives as possible of the anhydroalditols are listed in Tables, with references to their synthesis.

11. SYNTHESIS

1. Anhydrotetritols 1,4-Anhydroerythritol (cis-3,4-tetrahydrofurandiol) has been obtained in 94.2% yield by dehydration of erythritol in the presence of an anionic resin.2 A mixture containing mainly 1,4-anhydro-o~threitol (truns-3,4-tetrahydrofurandiol),with some 1,4-anhydroerythritol, was formed by refluxing 2-butene-174-diol for 20 hours with hydrogen peroxide in the presence of such oxidizing acids as nitric or peroxysulfuric acid and salts of arsenic, bismuth, mercury, or tin;3 the yield of 174-anhydrothreitolranged from 60 to 84%, depending on the cation. Similarly, 2,5-dihydroxytetrahydrofuranwas converted by hydrogen peroxide in 85% formic acid into 3,4-tetrahydrofurandiol in high yield,4 but no mention was made of the distribution of isomers. Hartman and R. Barkefl synthesized 174-anhydroerythritol(3)by (1).The reaction the saponification of 1-0-p-tolylsulfonyl-D-erythritol apparently proceeded by direct reaction of the 4-hydroxyl group with the 1-p-tolylsulfonoxy group, and not through the intermediate formation of the l,e-anhydride, which would have afforded the epimer, namely, 1,4-anhydro-~-threitol(2). Similarly, 1-O-p-tolylsulfonyl-Dthreitol gave 1,4-anhydro-~-threitol(2). (2)F.H.Otey and C. L.Mehltretter,J. Org. Chem., 26,1673(1961). (3)Badische Anilin und Soda-Fabrik Akt.-Ges., Ger. Pat. 833,963 (Mar. 13, 1952); Chem. Abstracts, 52,10201(1958). (4)Badische Anilin und Soda-Fabrik Akt.-Ges., Ger. Pat. 855,861(Nov. 17,1952); Chem. Abstracts, 52,9215 (1958). (5)F.C.Hartman and R. Barker, J. Org. Chern., 28, 1004 (1963).

ALDITOL ANHYDRIDES H.,COTs I HCOH I HCOH I H&OH

231

4 p HO (2)

HO

OH (3)

However, saponification of 2-O-p-tolylsulfonyl-~-erythritol or of 2-O-p-tolylsulfonyl-D-threitol did not furnish any 1,4-anhydro compounds, but only the respective tetritol. Erythritol and L-threitol have also been dehydrated by means of 50% sulfuric acid at 120" to the corresponding 1,4-anh~drides.~ On heating erythritol in the presence of a small proportion of a xylenesulfonic acid at 130- 150" under vacuum, an 89%yield of 1,4-anhydroDL-erythritol was obtained.' 2. Anhydropentitols a. 1,4-Anhydropentitols. - The synthesis of anhydropentitols by acid-catalyzed dehydration of pentitols was discussed by Wiggins,' but the only pentitol mentioned was xylitol. A solution of ribitol in 2 M hydrochloric acid was heated for 24 hours at 100" to yield an by analogy with anhydroribitol assumed to be 1,4-anhydro-~~-ribitol~ the anhydridation of xylit01.~ It was also shown that, under the conditions applied to ribitol, L-arabinitol and D-xylitol afford negligible proportions of anhydrides.8 That the anhydro product from ribitol is a derivative of 1,4-tetrahydrofuran was established'O from the results of periodate oxidation (consumption of 1 mole of periodate per mole, with no formation of (6) H. Klosterman and F. Smith, J . Amer. Chem. SOC.,74, 5336 (1952). (7) C. M . Himel and L. 0. Edmonds, U. S. Pat. 2,572,566 (1951); Chem. Abstructs, 46, 6157 (1952). (8) J. Baddiley, J. G . Buchanan, B. Carss, and A. P. Mathias,]. Chem. SOC.,4583 (1956). (9) J . F. Carson and W. D . Maclay, J . Amer. Chem. SOC., 67, 1808 (1945). (10) J. Baddiley, J . G . Buchanan, and B. Carss,]. Chem. SOC..4058 (1957).

S. SOLTZBERG

232

formic acid) and the correspondence of the infrared (i.r.) spectrum (with minor variations) to that of an authentic sample of 1,4-anhydroD-ribitol. MacDonald and coworkers" corroborated the fact that L-arabinitol is resistant to anhydridation under conditions that, applied to ribitol, gave an almost quantitative yield of 1,4-anhydro-~~-ribitol. The resistance of L-arabinitol and D-xylitol to anhydride formation under relatively mild conditions may be due to greater nonbonded interactions than with ribitol. Although this situation is not readily apparent from the stereo structures shown, inspection of models reveals that the relatively bulky hydroxymethyl group on C-5 is sterically unfavorable to the 3-hydroxyl group of 6 (eclipsed arrangement); and for D-arabinitol(5), the hydroxymethyl and the 2-hydroxyl group are opposed transannularly. It would, therefore, appear that the nonbonded interaction of the cis-hydroxyl groups of 4 is weaker than those of 5 and 6, thus permitting more facile anhydride formation.12

OH

HO

HO

OH

Ribitol

D-Arabinitol

Xylitol

(41

(5)

(6)

In contrast to ribitol and xylitol (which form a DL mixture on anhydride formation because of their molecular symmetry), Darabinitol may, in principle, on heating with acid, give two different anhydrides, namely, a 1,4-anhydroarabinitol or a 2,5-anhydroarabinitol (1,4-anhydro-lyxitol), as the following reaction sequence illustrates.

(1 1) D. L. MacDonald, J. D. Crum, and R. Barker, J . Amer. Chem. SOC., 80,3379 (1958). (12) Baddiley and coworkers* speculated that the threitols would be more resistant to anhydride formation than erythritol, because their secondary hydroxyl groups are trans-disposed. It is of interest that Klosterman and Smith: using somewhat more vigorous conditions, had found earlier, that, for anhydride formation, a lower temperature and shorter heating period could be used for erythritol than for threitol, thus lending support to this speculation.

ALDITOL ANHYDRIDES

233

However, if the reasons already given for the differences in ease of (8) anhydridation of the pentitols are valid, 1,4-anhydro-~-arabinitol should be the chief product, because, in 7, not only would there be a transannular, nonbonded interaction between the hydroxymethyl group and the 2-hydroxyl group, but the (bulky) hydroxymethyl group and the 3-hydroxyl group would be in eclipse. H,YOH HOCH I

HYOH H,COH (7)

Baddiley and coworker^'^ further found Lat, under the same conditions (loo0 and 2 M hydrochloric acid), allitol (9) and D-altritol (D-talitol) (10) are converted into anhydrides in high yield, as shown CH,OH I HCOH I

ym HOCH I

HCOH I HCOH

HCOH I HCOH

HCOH

HCOH

I

I

C&OH (9)

I

I

CH,OH

(10)

by paper chromatography of the products. These hexitols, like ribitol, possess three contiguous, cis-hydroxyl groups. The hexitol anhydrides were not isolated, however, and their structures were not established, but it was supposed that they were the 174-anhydrides.DGlucitol, D-mannitol, and L-iditol were also stated to yield anhydrides to various extent^.'^ l74-Anhydropentito1s have also been synthesized by the reductive desulfurization of l-thioglycofuranosides.l Syrupy 1,4-anhydro-~arabinitol, characterized as the tris( p-nitrobenzoate), and crystalline 1,4-anhydro-~-ribitol'~ have been obtained by reduction of the cor-

(13) J . Baddiley, J. G. Buchanan, and B. Carss,J. Chem. Soc., 4138 (1957). (14) (a) R. Barker and H. G, Fletcher, Jr.,J.Org. Chenr., 26,4605 (1961).(b) F. Weyyand and F. Wirth, Chem. Ber., 85, 1000 (1952).

S . SOLTZBERG

234

responding acylated pentofuranosyl bromides with lithium aluminum hydride, a procedure introduced by Hudson and coworkers15during the synthesis of 1,5-anhydroalditols. On heating pentitols with acid under more drastic conditions, derivatives of 1,4-anhydrides may be produced. Thus, on treating molten xylitol with dry hydrogen chloride at loo", or in concentrated hydrochloric acid at 106-108",a moderate yield of syrupy 1,Panhydro5-chloro-5-deoxy-~~-xylitol was obtained.16 Several 2,5-anhydropentitols have been synthesized from suitably protected pentoses by standard procedures (see also, Ref. lb). Thus, ethyl 5-O-p-tolylsulfonyl-c~-~-arabinofuranoside (11) was converted" into the corresponding 2,5-anhydride ( 12) by treatment with methanolic sodium methoxide. The product was hydrolyzed, and the resulting 2,5-anhydro-~-arabinosewas hydrogenated in the presence of Raney nickel to give 2,5-anhydro-~-arabinitol(13) as a syrup.

("9"

uoEt

OH HO H,COTs (11)

OH (12)

(13)

The enantiomorph, also a syrup, was synthesized from 3,6-anhydro4,5-O-isopropylidene-~-mannitol'~ by periodate oxidation to the corresponding D-arabinose derivative, followed by reduction in the presence of Raney nicke1.l' 2,5-Anhydro-~-xylitol,2,5-anhydro-~ribitol, and 2,5-anhydro-~-lyxitol(1,4-anhydro-~-arabinitol)'~*~~ were (15) R. K. Ness, H. G. Fletcher, Jr., and C. S. Hudson,]. Amer. Chem. SOC.,72, 4547 (1950). (16) S. N. Danilov and V. F. Kazimirova, Sb. Statei Obshch. Khim., 2, 1646 (1953); Chem. Abstracts, 49, 6840 (1955). (17) M. Cifonelli, J. A. Cifonelli, R. Montgomery, and F. Smith,]. Amer. Chem. SOC., 77, 121 (1955). (18) A. B. Foster and W. G. Overend,]. Chem. SOC., 680 (1951). (19) J. Defaye, Bull. SOC.Chim. Fr., 2686 (1964). (20) DefayelS called attention to the fact that the 2,5-anhydro-tri-O-(p-nitrobenzoyl)-~lyxitol that he prepared is the enantiomorph of Barker and Fletcher's 1,4-anhydrotri-O-(p-nitrobenzoyl)-~-arabinitol.'~'~' This is one of the relatively few instances on record where alditol anhydrides derived, without Walden inversion, from two configurationally different sugars have been shown experimentally to possess the same, although enantiomorphic, configuration.

235

ALDITOL ANHYDRIDES

obtained by reduction of the corresponding 2,5-anhydro-~-pentoses with sodium borohydride. The xylitol and arabinitol derivatives were syrups. The anhydro-D-arabinitol was characterized as the 3,4-diO-acetyl-l-O-trityl derivative. 2,5-Anhydro-l-deoxy-l,l-difluoro-~-ribitol~~ (15) was, by an unusual ring-contraction, obtained from di-O-acetyl-D-arabinal (3,4-di-0acetyl-1,5-anhydro-~-en~th~o-pent-l-enitol) (14) by way of the following reaction sequence.

AcO

AcO

(14)

OAc

HO

OH (15)

Also, di-O-acetyl-L-arabinal was converted into the corresponding anhydro-L-ribitol derivative (see also, pp. 195- 197). The configuration of the products was established by periodate oxidation of the D compound, and reduction of the resulting dialdehyde to an optically active diol whose sign and magnitude of optical t2.5"in water) correspond to that of the diol ( [a]iO t-6.8" rotation ( [a]iO in water) obtained by similar treatment of methyl a-L-arabinopyranoside. The bis(p-nitrobenzoates) of the two diols exhibited similar relationships. Hence, it appears likely that the configuration at C-2 of the anhydride is the same as that at C-1 of the L-arabinoside. The cis relationship of the hydroxyl groups was retained from the arabinal started with. The formation of oxetane (1,3-anhydro) rings has received but was oblittle attention. 1,3-Anhydro-2,4-0-methylene-~~-xylitol tained'l from 2,4-0-methylene-l-O-p-to~y~su~fony~-D~-xy~ito~. The 1,3-ring was established by elimination of the possibility of other products, through test reactions and formation of derivatives.22 No work on the synthesis of 1,5-anhydropentitols appears to have been reported since the earlier article.' Only two dianhydropentitols have been reported. One was ob(21) (a) P. W. Kent, J . E. G. Bamett, and K. R. Wood, Tetrahedron Lett., 1345 (1963). (b) K. R. Wood and P. W. Kent,J. Chem. Soc. (C), 2422 (1967). (22) R. M. Hann, N . K. Richtmyer, H. W. Diehl, and C. S. Hudson, J. Amer. Chem. Soc., 72, 561 (1950).

236

S. SOLTZBERG

tained, as a syrup, by refluxing a solution of 174-anhydro-5-chloro-5deoxy-DL-xylitol'6 in dry acetone with dry sodium hydroxide, or by the action of sodium methoxide in acetone at room temperature; it was supposed that the product was 1,4:2,5-dianhydro-~~-xylitol (16). Wiggins suggested that a dianhydro-xylitol obtained by GrandelZZa by heating xylitol with methanedisulfonic acid was the 1,4:2,5dianhydride (16). The second dianhydro-xylitol was obtainedz3by methylenation of 1,4-anhydro-~~-xylito1, with paraformaldehyde and concentrated hydrochloric acid, to give the 3,5-methylene acetal; this was methylated to the 2-methyl ether, which was hydrolyzed, and the product mono-p-toluenesulfonylated to the 5-p-toluenesulfonic ester. On treatment with aqueous sodium hydroxide at 40-50", the last gave syrupy 1,4:3,5-dianhydro-2-O-methyl-~~-xylitol (17, D form).

The crucial methylenation step in this synthesis undoubtedly gave the 3,5-methylene acetal, not the 2,3-acetal (or the highly improbable 2,5-methylene acetal), because, otherwise, a 6-O-methyl-2(or 3)-0p-tolylsulfonyl compound would have been obtained which could only have formed an epoxide (oxirane). T h e oxirane ring might have been opened under the conditions of the saponification; the product would not then have been a dianhydride. 3. Anhydrohexitols a. Anhydrohexitols. - Synthesis of 1,4-anhydrohexitols by new procedures has not been reported. However, 1,4-anhydro-~-mannitol (224 F. Crandel, U. S. Pat. 2,375,915 (1945), characterized, by boiling point only, the dianhydro-xylitol that he prepared (b.p. 170"/5 torr). As xylitol is symmetrical, an anhydride ring would b e formed first at C-l,C-4 (= C-5,C-2). Examination of Fisher-Hirschfelder models reveals that a second tetrahydrofuran ring would b e extremely difficult to form unless inversion had occurred at some point. This is a possibility (see Ref. 82). T h e same steiic considerations apply to the compound mentioned in Ref. 16. (23) G. E. Ustyuzhanin, E. M. Kogan, N. S. Tikhomirova-Sidorova, and S. N. Danilov,

Zh. Obshch. Khim., 33,3622 (1963); Chem. Abstracts, 59, 5246 (1963).

ALDITOL ANHYDRIDES

237

was isolatedz4 as one of the products formed on heating D-mannitol with sodium pyrosulfite solution at 130-180". The reduction of per-0-acylaldopyranosyl halides has been shown to constitute a definitive synthesis of 1,5-anhydrohexitols. Thus, Hudson and coworkers'5 reduced tetra-O-acety~-a!-D-glucopyranosyl bromide, and tri-0-acetyl-a-L-rhamnopyranosyl bromide with lithium aluminum hydride; deacetylation of the respective product gave 1,5-anhydro-~-glucitol (polygalitol) and 1,5-anhydro-~-rhamnitol. cox or^^^ used this method for synthesizing 1,5-anhydro-~-allitolfrom D-allOSe. He prepared the enantiomorph (18) by reduction of 2,3,4tri-0-benzoyl-D-ribopyranosyl cyanide, followed by saponification, reduction of the product with lithium aluminum hydride (with simultaneous deacetylation), and deamination.25 2,6-Anhydro-~-aIlitol (19) is 1,5-anhydro-~-allitol(IS).

HO

OH

Because of an analogous relationship, 1,9anhydro-~-mannitol (styracitol) was obtained by hydrogenation of 2,6-anhydro-~-mannose.26Also, 1,5-anhydro-~-gulitol,obtained by reduction of tetra-0acetyl-D-gulopyranosyl bromide with lithium aluminum hydride, and its enantiomorph, obtained2' by similar reduction of tetra-o-benzoylP-D-fructopyranosyl bromide are 2,6-anhydro-~- and -D-glUCitOl, respectively. It should be noted that reduction of the tetra-o-acylketohexopyranosyl halide results in inversion at C-2; hence, the D-glucitol derivative is formed (the 1,5-anhydro-~-mannitol derivative was obtained in very low yield). Because a solution of the bromide in ether was dropped into a solution of the reductant, the aluminum bromide or aluminum bromide etherate formed in the initial reduction may have anomerized the succeeding portions of the D-fructose derivative. With per-0-acylaldopyranosyl halides, epimerization at C-1 does not affect the configuration of the resulting anhydroalditol. (24) B. Lindberg and 0. Theander, Suensk Papperstidn., 65, 509 (1962); Chem. Abstracts, 58, 9773 (1963). (25) B. Coxon, Tetrahedron, 22, 2281 (1966). (26) F. Micheel, W. Neier, and T. Riedel, Chem. Ber., 100, 2401 (1967). (27) R. K. Ness and C. S. Hudson, J . Amer. Chem. Soc., 75, 2619 (1953).

238

S. SOLTZBERG

1,5-Anhydro-D-altritol was obtained2s in low yield by reduction of tetra-0-benzoyl-D-altropyranosyl bromide with lithium aluminum hydride, in contrast to the high yields of anhydroalditols generally obtained by this technique. It is possible that most of the bromide was decomposed prior to reduction, as a very low yield of crude intermediate was obtained at this stage. A different approach,28starting with 1,5-anhydro-~-glucitol,afforded 1,5-anhydro-~-altritolin satisfactory overall yield. 1,5-Anhydr0-4,6-O-benzylidene-2,3-di-Op-tolylsulfonyl-D-glucitol was transformed in methanolic sodium methoxide into the corresponding 2,3-anhydride whose configuration was not established; this was treated with hot, aqueous alkali to give 1,5-anhydro-4,6-0-benzylidene-~-altritol, which was hydrolyzed to the anhydroalditol. The physical constants of the product differed from those of 1,5-anhydro-~-glucitolor -D-mannitol, and hence, it had the D-altritol configuration. A different reductive approach was employed by Zervas and Zioudrou,29 who catalytically reduced tetra-0-acetyl-a-D-glucopyranosyl bromide in the presence of palladium to obtain 1,5-anhydroD-glucitol. It would be of interest to determine whether this or a similar reduction of tetra-0-benzoyl-P-D-fructopyranosyl bromide would give the D-mannitol anhydride. Synthesis of 1,5-anhydroalditols by use of a compound already having a pyranoid ring was accomplished by Rice and Inatome.so They reduced tetra-0-acetyl-a-D-glucopyranosyl nitrate and tri-0acetyl-P-L-arabinopyranosyl nitrate with sodium borohydride, and obtained the corresponding 1,Sanhydroalditol acetates; saponification afforded the corresponding 1,5-anhydroalditols. As the acetylated glycosyl halides had been used for preparing the nitrates, the procedure has no advantage over reduction of the halide. Inasmuch as it was shown that sodium borohydride does not reduce nitric esters of primary or secondary alcohol groups, nitrated sugars are not suitable starting materials. In a different synthesis of 1,5-anhydrohexitols from a compound having a pyranoid ring, Lehmann and Friebolinsl treated 1,Sanhydro2-deoxy-~-arabino-hex-l-enitol (D-glucal) (20) with a-toluenethiol in the presence of light, and obtained 1,5-anhydro-2-S-benzyl-2-thio-~mannitol (21) and the epimeric anhydro-D-glucitol in equal amounts. (28) E. Zissis and N. K. Richtmyer, I . Amer. Chem. SOC., 77, 5154 (1955). (29) L. Zervas and C. Zioudrou, J. Chem. Soc., 214 (1956). (30) F. A. H. Rice and M. Inatome, J. Amer. Chem. SOC., 80, 4709 (1958). (31) (a) J. Lehmann, Carbohyd. Res., 2, 486 (1966); (b) J. Lehmann and H. Friebolin, ibid., 2, 499 (1966).

239

ALDITOL ANHYDRIDES

(21)

(20)

where Bzl = PhCH,.

The configuration at C-2 was established by comparison with the optical activities of 1,5-anhydro-~-mannitoland -D-ghcitOl and the proton magnetic resonance (p.m.r.) spectra of their acetate^.^"^' This reaction is useful for the preparation of C-2 substituted derivatives, but the configuration at C-2 of the products must then be established. T r i - O - a c e t y l - 1 , 5 - a n h y d r o - 2 - d e o x y - ~ - a r a b i o (tri-0l acetyl-D-glucal) was the starting point for the synthesis of 1,5-anhydro-2-chloro-2-deoxy-~-glucitol or -D-mannitol by way of the 1,2dichloride (22), which was reduced:j2b y means of lithium aluminum hydride to the 2-chloro-2-deoxy derivative (23).That the chlorine AcOCH, AcO

AcOCH,

-

..

A

c

O

Y

o

\

A

C T “ c1

atom was situated on C-2 was inferred by analogy with the comparable reduction of peracetylated glycosyl halides (see p. 237) and from its low reactivity toward sodium hydrogen carbonate or silver nitrate, as compared with that expected for an a-chloro ether. Because the distilled product, obtained in good yield, was a viscous syrup, it may have consisted of a mixture of the C-2 epimers. Anhydroalditol compounds of a rather unusual type are the Cglycosyl compounds (erroneously called “C-glycosides”). As pointed out by Treibs,”%these are not glycosides, but they are anhydroof approaches has been employed for synthesis of a l d i t o l ~A. ~variety ~ (32) C. D. Hurd and H. Jenkins, Carbohyd. Res., 2, 240 (1966). (33) W. Treibs, Nuturwissenschuften, 48, 378 (1961); Chem. Abstracts, 55, 24705 (1961). (34) See Ref. 37, footnote 2. It is now suggested that these compounds be named similarly to the alditols higher than the hexitols, because an asymmetric carbon atom has been introduced at C-1 of the anhydride.

240

S. SOLTZBERG

this class of compound. Hurd and Holysz35treated tetra-O-acety1-aD-glucopyranosyl chloride or tetra-0-acetyl-a-D-mannopyranosyl bromide with such metallo-organic compounds as phenyllithium and butyllithium. The reaction between the former glycopyranosyl chloride and phenyllithium gave three products, which were separated by chromatography on alumina. Two of the compounds were shown to be the tetraacetates of the known36P-D-glucopyranosyl- (24) and a-D-glucopyranosyl-benzene (25). The third product has been in(26). ferred to be 1,5-anhydro-2-C-phenyl-~-glucito1~~

HO HO

HO

HO

HO

Ph

The formation of the tetraacetate of the a-D anomer (25a) (no inversion) has been postulated to take place by way of a four-center transition state:

It was presumed that the third product (26) results from elimination of the elements of hydrogen chloride, in addition to deacetylation, by the strong base, to give a 2-hydroxy-~-glucal (1,5-anhydro-~arabino-hex-1-enitol) anion (27) which then adds phenyllithium

(27)

(35)C.D.Hurd and R. P. Holysz, ]. Amer. Chem. SOC., 72, 1735 (1950). (36)W.A. Bonner and J . M. Craig,J. Amer. Chem. SOC., 72, 3480 (1950). (37)C.D.Hurd and H. T. Miles,]. Org. Chem., 29,2976(1964).

ALDITOL ANHYDRIDES

241

across the double bond. As proof of this theory, 2-hydroxy-~-glucal tetraacetate was treated with phenyllithium, and the mixture was processed as for the reaction of tetra-O-aCetyl-cu-D-glUCOpyranOSyl chloride. It was demonstrated, both by paper chromatography of the deacetylated, partially purified, crude material and by gas-liquid chromatography of the (fully acetylated) material, that the principal component of the mixture corresponded in behavior to pure 1,5anhydro-2-C-phenyl-~-glucitol. This configuration,,instead of the D-manno form, is based on the assumption that the tertiary hydroxyl group of the hypothetical Dmanno derivative, being in a favorable, trans-coplanar arrangement with the axial hydrogen atom on C-1, should be dehydrated when it is acetylated under forcing conditions. On the other hand, the tertiary hydroxyl group of the D-gluco configuration is not trans-coplanar with any neighboring C-H bond. Inasmuch as dehydration did not occur on forced acetylation (acetic anhydride and sodium acetate at 140"), it was decided the compound had the D-ghco configuration. Mild acetylation at loo", or in acetic anhydride-pyridine at 25", yielded only a triacetate (not a tetraa~etate).3~ The 1,s-anhydride, namely, P-D-glucopyranosylbenzene (24), was formed by the nucleophilic-displacement type of reaction expected. The reaction of tetra-O-acetyl-a-D-mannopyranosylbromide with phenyllithium likewise gave a mixture; from it, only tetra-O-acety1-aD-mannopyran~sylbenzene~~ was isolated crystalline. The two other products have, apparently, not been further investigated. A mixture of products was obtained from the reaction of butyllithium with tetra0-acetyl-a-D-ghcopyranosyl bromide; from it, a crystalline tetra-0acetyl-D-glucopyranosylbutanewas isolated, and identified as the a anomer, because of its identity (by mixed melting point) with the broproduct from the reaction of tetra-O-acetyl-a-D-glucopyranosyl mide with the butyl Grignard reagent. The use of the Grignard reaction to synthesize 1-C-substituted carbohydrate derivatives from peracetylated glycosyl halides was . ~ ~reaction gives more introduced in 1945 b y Hurd and B ~ n n e rThis homogenr-ms products in much better yields than the organolithium reaction. However, the product isomeric at C-1 is generally produced simultaneously to a greater or lesser extent. Only sporadic use was made of the reaction until the late 1950's, when Zhdanov and coworkers undertook the synthesis of numerous (38)C.D.Hurd and R. P. Holysz,J. Amer. Chem. SOC.,72, 1732 (1950). (39)C.D.Hurd and W. A. Bonner,]. Amer. Chem. Soc., 67, 1972 (1945).

242

S. SOLTZBERG

l-C derivatives embracing a variety of substituted phenyl compounds and such unsaturated acyclic groups as ally1 and vinyl.40 That it is not necessary to employ substituted aryl compounds in order to obtain a substituted product was demonstrated by Gerecs and Windholz,4' who nitrated tetra-O-acety~-~-D-glucopyranosylbenzene with cuprous nitrate in acetic anhydride to give a mixture (-4: 1)of the corresponding o- and p-nitro derivatives. D-Glucopyranosylation of an acetylene compound was achieved by treating tetra-0-acetyl-a-D-glucopyranosyl bromide with 2-phenylethynylmagnesium bromide.42 The product, namely, tetra-o-acetylp-~-glucopyranosyl-( 2-phenylethyne), was reduced to the corresponding phenethyl derivative, which was obtained directly by use of phenethylmagnesium bromide. The p-D configuration was inferred from the levorotation of the crystalline products. The mother liquors were dextrorotatory, and were assumed to contain the a - anomers. ~ The tetra-O-acetyl-(phenylethyne) derivative could be crystallized in both the anhydrous and the monohydrated form, depending on the solvent used. From 95% ethanol containing ethyl ether, the deacetylated product was also obtained as a solvate, but it was not determined whether it contained water or alcohol of solvation. An attempt was made to extend the reaction to sodium acetylide, but only a small amount of an unidentified, crystalline, carbohydrate derivative (40) (a) Yu. A. Zhdanov and G. N. Dorofeenko, Dokl. Akad. Nauk SSSR, 112,433(1957); (b) ibid., 113, 601 (1957); Chem. Abstracts, 51, 13767, 14561 (1957). (c) Yu. A. Zhdanov, G. A. Korol'chenko, and S. I. Uvarova, ibid., 122, 811 (1958); Chem. Abstracts, 53, 4183 (1959). (d) Yu. A. Zhdanov, G. A. Korol'chenko, and L. A. Kubasskaya, ibid., 128, 1185 (1959); Chem. Abstracts, 54,8644 (1960). (e) Yu. A.

Zhdanov, G. A. Korol'chenko, L. A. Kubasskaya, and R. M. Krivoruchko, ibid., 129, 1049 (1959); Chem. Abstracts, 54, 8641 (1960). (f) Yu. A. Zhdanov, G. N. Dorofeenko, and L. E. Zhivoglazova, ibid., 117, 990 (1957); Chem. Abstracts, 52, 8056 (1958). (9)Yu. A. Zhdanov, G. A. Korol'chenko, G. N. Dorofeenko, and G. V. Bogdanova, ibid., 152, 102 (1963); Chem. Abstracts, 59, 15373 (1963). (h) V. I. Komilov, S. S. Doroshenko, V. I. Tikhonov, and Yu. A. Zhdanov, Materialy Vses. Konf. Probl. Khim Obmen Uglevodou, 3rd, Moscow, 1963,85 (1965). (i) Yu. A. Zhdanov and V. I. Komilov, Izo. Vysshikh Uchebn. Zavedenii Khim. Khim. Teknol., 9, 65 (1966); Chem. Abstracts, 65, 13806 (1966). fj)Yu. A. Zhdanov and G. A. Korol'chenko, Dokl. Akad. Nauk SSSR, 139, 1363 (1961); Chem. Abstracts, 56, 531 (1962). (k) Yu. A. Zhdanov, G . A. Korol'chenko, G. N. Dorofeenko, and G. V. Bogdanova, ibid., 152, 102 (1963); Chem. Abstracts, 59,15373 (1963). (1) Yu. A. Zhdanov, Uglevody i Uglevodn. Obmen v Zhiootn. i Rust. Organizmakh Materialy Konf., Moscow, 1958,53 (1959); Chem. Abstracts, 54, 19503 (1960). (41) A. Gerecs and M . Windholz, Acta Chim. Acad. Sci. Hung., 13, 231 (1957); Chem. Abstracts, 52, 11778 (1958). (42) R. Zelinski and R. E. Meyer, J . Org. Chem., 23, 810 (1958).

ALDITOL ANHYDRIDES

243

having an analysis corresponding to the composition calculated for C16H2009 was obtained. A reaction that appears to be of fairly broad application, and that yields a mixture of compounds containing some 1-C-substituted anhydroalditol, consists in treating a free reducing sugar, an oligomer, or a polysaccharide with an aromatic compound in the presence of liquid hydrogen fluoride. The procedure was first reported by Linn.43However, the structure of the anhydride obtained was not fully elucidated; nor were the structures of the compounds obtained from the acidcatalyzed dehydration of l-deoxy-l,l-bis(3,4-dimethylphenyl)-~g l u ~ i t o l , whose ~~ configuration was not rigorously established. It has been found that numerous plants contain 1-C-substituted anhydroalditols. For example, barbaloin, a major constituent of commercial aloin, is l0-(a-~-glucopyranosyl)-1,8-dihydroxy-3-(hydroxymeth~1)anthrone.~~ Numerous other 1-C-substituted anhydroalditols have been isolated from a variety of plant sources by various investigators. The antibiotic substances ~ h o w d o m y c i n ,formycin ~~ and formycin B,47348and l a u r u ~ i n *have ~ been shown to be 1,4-anhydroribitol-l-y1 compounds. It was proved that laurusin and formycin B are the same compound.48 A new synthesis of 2,5-anhydro-~-iditolwas achieved49by reduction of 2,5-anhydro-~-idosewith sodium borohydride. The product was identified as its crystalline 1,6-bis(p-toluenesulfonate), which was already known.' The synthesis of an alditol having a 4-membered (oxetane) ring was first reported by Ustyuzhanin and coworkers,50who prepared 1,3anhydro-5,6-di-O-methyl-2,4-0-methylene-~-glucitol by saponification of the 1-p-toluenesulfonate of the corresponding derivative of D(43)C.B. Linn, Am. Chem. Soc., Div. Petrol. Chem., Preprints, 2, No. 3, 173 (1957). (44)J. Heerema, G.N. Bollenback, and C. B. Linn, Am. Chem. SOC. Div. Petrol Chem., Preprints, 2, No.4,185 (1957);1.Amer. Chem. SOC., 80, 5555 (1958). (45)(a) J. E. Hay and L. J. Haynes,]. Chem. SOC., 3141 (1956).(b) R.A. Barnes and W. Holfeld, Chem. Znd. (London), 873 (1956). (46)K. R. Darnall, L. B. Townsend, and R. K. Robins, Proc. Nut. Acad. Sci. U . S . , 57, 548 (1967). (47)G . Koyama, K. Maeda, and H. Umezawa, Tetrahedron Lett., 597 (1966). (48)R. K. Robins, L. B. Townsend, F. Cassidy, J. F. Gerster, A. F. Lewis, and R. L. Miller, J . HeterocycL Chem., 3, 110 (1966). (49)C. A. Dekker and T. Hasizume, Arch. Biochem. Biophys., 78, 348 (1958). (50)G. E. Ustyuzhanin, N. S. Tikhomirova-Sidorova, and S. N. Danilov, Zh. Obshch. Khim., 33, 453 (1963);Chem. Abstracts, 59,5247 (1963).

S. SOLTZBERG

244

glucitol. Later, Haslam and R a d f ~ r dwhile , ~ ~ investigating the synthesis of 1,5-anhydro-2,4-0-benzylidene-~-glucitol, prepared 5,6-anhydro-2,4-O-benzylidene-l-O-p-tolylsulfonyl-~-glucitol. On saponification, instead of the 5,6-anhydro-2,4-0-benzylidene-~-glucitol expected, de-p-toluenesulfonyloxylation occurred, affording a crystalline anhydride (28) that did not agree in physical properties with the monoanhydromonobenzylidenehexitols known. The possibility that it was a 1,5-anhydro-~-iditolcompound was excluded on the basis of the following evidence. The nuclear magnetic resonance (n.m.r.) spectrum of the ethers obtained by treatment of the 5,6-anhydride with an alcohol and a base, and of their monoacetates, indicated that the free hydroxyl group in the ether was attached to a methine carbon atom (not to a methylene group); this showed that the 5,6-anhydro ring had opened normally. Hence, the reactions must have proceeded as shown. &COTS I HCO I \ HOCH CHPh I / HCO I

r (I F \

0 HCO

NaOH_LCH I

/

CHPh

HCO I HCOH I H,COH

lX;i;:,

@ ,:

AcSO

OCH CHPh I /

HCO I HCOAc I

GCOAc

(28)

E: I

\

-

CKOTs I

HCO I

\

OCH CHPh ROHsbase HOCH CHPh I / I / HCO HCO

I HCOH I H,COR

HkOH

I H,COTs

where R = Me, Et, or PhC&.

It is probable that the two 1,Sanhydro compounds just described are anhydrides of the same alditol. The compound of Ustyuzhanin and coworkerss0 is unequivocally a 1,3-anhydro-~-glucitolderivative, because the hydroxyl groups at C-2,4,5, and 6 were protected prior to the ring formation. Positive identity of the two would have been achieved by converting the monomethyl ether obtained by Haslam (51)(a) E.Haslam and T. Radford, Chem. Commun., 631 (1965).(b) E.Haslam and T. Radford, Carbohyd. Res., 2, 301 (1966).

ALDITOL ANHYDRIDES

245

and Radford5' into the diether. Moreover, periodate oxidation of 28 could have provided incontrovertible chemical proof, through the formation of formaldehyde, that ring closure could only have taken place between C-1 and C-3. Oxepane (seven-membered) rings (1,6-anhydrohe~itols)~* have been prepared from the 3,4-isopropylidene acetals of D-mannitol, Dglucitol, and L-iditol, by way of alkaline hydrolysis of the corresponding 1,2:5,6-dianhydrides. The ring structures of the products were established through periodate oxidation and lead tetraacetate oxidation; the requisite amount of formic acid was produced, and 3 equivalents of lead tetraacetate were consumed. No inversions at any of the asymmetric centers were involved in the reactions conducted, so the oxepanes had retained the configurations of the starting hexitols. In the synthesis of 1,6-anhydro-~-ghcitol,at least, a 2,6-anhydride is a b y p r ~ d u c tIts .~~ structure was assigned on the basis of its i.r. spectrum, in comparison with those of the 1,6-and 1,5-anhydro-~-glucitols, and the fact that no diglycolic acid was obtained after periodate oxidation followed by oxidation with hypobromite. b. Dianhydrohexitols. -A dianhydro-D-glucitol having 1,5 and 3,6 rings was synthesized by S. B. Baker;54he saponified 2,4-0-methylene-1-0-p-tolylsulfonyl-D-glucitol with dilute aqueous alkali, to form a monoanhydride that did not consume lead tetraacetate. This result eliminated the possibility of a 1,3-anhydro ring, but left open the question whether the compound was a 1,5-or 176-anhydride. Presumptive evidence that the compound contained a primary hydroxyl group was presented by the formation of a trityl ether. The compound was then p-toluenesulfonylated to a mono-p-toluenesulfonate, and this was saponified to the 2,4-O-methylene derivative of the postulated dianhydro-D-glucitol. The structure was conclusively established by starting with 1,5-anhydro-~-glucitol (polygalitol), mono-p-toluenesulfonylating, acetylating, and then saponifying the diester. A dianhydride was obtained that afforded a methylene acetal identical with that obtained on starting with 2,4-0-methylene-1-0-ptolylsulfonyl-D-glucitol. It is noteworthy that saponification of Baker's first compound afforded a 1,5-anhydride, whereas saponification of the corresponding 5,6-anhydro-2,4-0-benzylidene derivative furnished 1,3-anhydro-2,4O-benzylidene-~-glucitol.~' (52) L. Vargha and E. Kasztreiner, Chem. Ber., 93, 1608 (1960). (53) P. Sohar, L. Vargha, and E. Kasztreiner, Tetmhedron, 20,647 (1964). (54)S . B. Baker, Can.J.Chem., 32, 628 (1954).

246

S. SOLTZBERG

Vargha and KuszmannJ5 obtained the compound long known as “p-mannide”5g together with a small proportion of material believed (from its chromatographic behavior) to be 1,4:3,6-dianhydro-~-mannito1 (isomannide) on treating either 1,6-dichloro-l,6-dideoxy-~-mannito1 or 1,6-di-O-(methy~sulfony~)-~-mannito~ with methanolic sodium methoxide. A 2,5:3,6-dianhydro structure was suggested for “p-mannide” because, on tritylation, it gave a monotrityl derivative (indicative of the presence of one primary hydroxyI group); also, its bis(ptoluenesulfonate) gave only a monoiodo product on treatment with sodium iodide in acetone, indicative of the presence of one primary p-tolylsulfonyloxy group. No periodate was consumed by the “pmannide,” suggesting that contiguous hydroxyl groups were absent. The absence of oxirane and oxetane rings was indicated by the resistance of the compound to hot dilute acid or to alkali, There remained, therefore, only the 2,5:3,6-dianhydro structure that satisfied the evidence. The compound is the same as that obtained if ring closure is 1,4:2,5. It was shown to possess the D-gluco configuration as follows: treatment with fuming hydrochloric acid at 100” opened the 3,6 (1,4) ring, because displacement of the chlorine in the resulting monochloromonodeoxy anhydride by benzoyloxy, followed by deacylation and conversion into a dibenzoate, yielded a compound identical with 2,5-anhydro-l,6-di-O-benzoyl-~-glucitol. Of all the anhydrohexitols currently known, the structure of two dianhydrohexitols (reported by Wiggins5’) remain unresolved. The two dianhydrides were obtained on heating D-mannitol with hydrochloric acid; one had m.p. 118” and [a]D-34, and the second was isolated only as its dimethanesulfonate, m.p. 113-114”. The apparent enantiomorph of the first was formed on treating 1,6-dichloro-1,6dideoxy-D-mannitolwith sodium methoxide. Wiggins5Tureported that the physical constants of a product that he had obtained from 1,2:5,6-di-0-isopropylidene-3,4-di-O-p-tolylsulfonyl-D-mannitol by deacetalation with 70% acetic acid followed by acetylation and saponification with sodium methoxide were those of 1,4:3,6-dianhydro-~-iditol. Tipson and CohenJ7*have presented evi(55) L. Vargha and J. Kuszmann, Corbohvd. Res., 8, 157 (1968). (56) A. Siwoloboff, Ann., 233, 368 (1886). (57) L. F. Wiggins, Nature, 164,672 (1949). (57a) L. F. WigginsJ. Chem. SOC., 1403 (1947). (5%) R. S. Tipson and A. Cohen, Corbohyd. Res., 7,232 (1968).

ALDITOL ANHYDRIDES

247

dence, based on a study of the saponification of the 3,4-dimethanesulfonate and 3,4-bis(p-toluenesulfonate)of D-mannitol, that Wiggins’ product must have been grossly impure. They likewise pointed out that the physical properties reported by Wiggins, as compared to those of the authentic enantiomorph, and the yields of ester derivatives, also showed that the product had been a mixture.

c. Trianhydrohexitols. -Only one example of a trianhydroalditol is thus far known. Cope and Shen5*obtained 1,4:2,5:3,6-trianhydroD-mannitOl on boiling a solution of 1,4:3,6-dianhydro-2,5-di-O-ptolylsulfonyl-D-glucitol in ethanol containing sodium ethoxide under reflux. As the endo-p-tolylsulfonyl group (on 0-5) is protected by the neighboring ring, they postulated a trans-p-toluenesulfonylation resulting from an sN2s displacement of the exo-p-tolylsulfonyl group, with transfer of the endo-p-tolylsulfonyl group to the e m position. The resulting endo-alkoxide group could then bring about a normal sN2 displacement of p-toluenesulfonate. The reasonableness of this postulated mechanism was demonstrated by the fact that 2-0-acetyl1,4:3,6-dianhydro-5-O-p-tolylsulfonyl-~-glucitol~~ (erroneously assumed by them to be the 5-0-acetyl-2-0-p-tolylsulfonyl isomer) gave the same product. Hence, trans-p-toluenesulfonylation and the same intermediate were involved. Lemieux and M c I n n e ~ pointed ~ ~ ~ ) out the error in the assumption, and, by means of i.r. spectroscopy, unequivocally established the structure of the mono-p-toluenesulfonic and mixed esters, and verified conversion of the 2-O-acetyl-5-O-ptolylsulfonyl compound into 1,4:2,5:3,6-trianhydro-~-mannitol. Jackson and H a y ~ a r dhad ~ ~previously ~) pointed out that Cope and Shen’s assumption that the exo-hydroxyl group in 174:3,6-dianhydroD-glucitol is preferentially sulfonylated prior to conversion into the mixed ester is incorrect. They based their conclusions on studies of the rate of replacement of p-tolylsulfonyloxy group by iodide in the three dianhydro stereoisomers. However, their proof of structure was questioned by Lemieux and M c I n n e ~as~ being ~ ~ ) equivocal, because of possible formation of an acetoxonium intermediate prior to attack by the iodide ion.

(58) A. C. Cope and T. Y. Shen,]. Amer. Chem. SOC., 78,6912 (1956). (59) (a) R. U. Lemieux and A. G . McInnes, C a n . ] .Chem., 38,136 (1960).(b) M. Jackson and L. D. Hayward, ibid., 37, 1048 (1959).

248

S. SOLTZBERG

4. Anhydrides of Higher Alditols

a. Anhydroheptitols. - Sowden and FischeFI obtained a 2,6-anhydro-5,7-O-benzylidene-l-deoxy-l-nitroheptitol as a byproduct in the condensation of 4,6-O-benzylidene-~-glucopyranosewith nitromethane; this was hydroIyzed to the free anhydroheptitol. The configuration at C-2 (C-1of the original D-glucopyranose) has not yet been established. By analogy to the cyclization of 1-deoxy-1-nitro-D-mannit01,'~~ it would be expected that the compound should possess the D-glycero-D-ido configuration. Rosenthal and coworkers have applied the hydroformylation technique to D-glucal t r i a ~ e t a t e D-galactal ,~~~ t r i a ~ e t a t e 2-hydroxy,~~ D-glucal tetracetate, and 2-hydroxy-~-galactalt e t r a ~ e t a t e(Hydroxy.~~ methy1)ation occurred at C-1 in all cases. [After deacetylation, there were obtained 2,6-anhydro-3-deoxy-~-gZuco-heptitol and 2,6-anhydro3-deoxy-~-manno-heptitol,~~ 2,6-anhydro-3-deoxy-~-gaZacto-heptitol and 2,6-anhydro-3-deoxy-~-tuZo-heptitol(2,6-anhydro-S-deoxy-~~Ztro-heptitol),~~ and 2,6-anhydro-~-gZycero-~-guZo-heptitol as the principal product from 2-hydroxy-~-glucal,and 2,6-anhydro-~-gZyceruL-manno-heptitol as the principal product from 2-hydroxy-~-galactal.5w The latter compounds were shown to be identical with those described by Coxon and F l e t ~ h e r , ~which ~ ~ * ~ were ~' prepared by h y d r ~ g e n a t i o nof ~ ~2,3,4,6-tetra-O-acetyl-/3-~-glucopyranosy1~~ and -P-D-galactopyranosyl cyanides, respectively, and subsequent deamination by nitrous acid. Hough and Shute5* heated 1-deoxy-1-nitro-D-glycero-L-mannoheptitol in water, and obtained the corresponding 2,6-anhydride (28a) as the chief product; this behavior parallels that of l-deoxy-lnitro-D-mannit01.'~~Hough and Shute5* also obtained evidence for the probable presence of 2,6-anhydro-l-deoxy-l-nitro-~-gZyceru-~gZuco-heptitol and 2,5-anhydro-l-deoxy-l-nitro-~-gZycero-~-munnoand -L-gluco-heptitol. Sowden and Oftedah1132have suggested that such anhydridation occurs by way of an intermediate l-nitroald-l(59a)J. C. Sowden and H. 0. L. Fischer, U.S. Pat. 2,480,785(1949);Chem. Abstracts, 44,656 (1950). (59b)A. Rosenthal and H. J. Koch, Can.J.Chem., 43,1375(1965). (5%) A. Rosenthal and D. Abson, Can.]. Chem.,43,1985(1965). (59d) A. Rosenthal, Carbohyd. Res., 3, 112 (1966). (59e)B.Coxon and H. G. Fletcher, JrJ. Amer. Chem.Soc., 85,2637(1963). (590 B.Coxon and H. G . Fletcher, Jr.,J. Amer. Chem. Soc., 86,922(1964). (59g)L. Hough and S. H. Shute, 1. Chem. Soc., 4633 (1962). (59h)A. Camerman, H.J. Koch, A. Rosenthal, and J. Trotter, Can. J . Chem., 42,2630 (1964).

ALDITOL ANHYDRIDES

249

enitol, as shown in the following reaction sequence (according to Hough and Shute). HCNO,

%?NO, HCOH I HCOH I HOCH

HOCH

HOCH

HOCH

II

HO

HC I

HCOH I

I

I

C%NO2

I

I

HCOH

HCOH I H,COH

I

H,COH

2,B-Anhydro-1-deoxy- l-nitroD-,qlycero -L- manno-heptitol

b. Anhydrooctitols. - 1,5-Anhydro-~-erythro-~-ga~acto-octitols appear to be the only members of this group thus far reported. These anhydro-octitols have been prepared by the Raney nickel desulfurization of derivatives of the lincomycin group of antibiotics. Thus, 6acetamido- 1,5-anhydro-6,8-dideoxy-3,4-O-isopropylidene-~-eryt~roD-gazacto-octitol (28b)59iJ(“N-acetyl-3,4-O-isopropylidenelincosaminol”) was obtained from the corresponding 1-thiol, 1,5-anhydro-6,8dideoxy-l\r-(4-propyl-~-hygroyl)-~-eryth~o-~-gaZacto-octitol~~~ (anhydrolincomycitol) (2&) from the corresponding 1-S-methyl-1-thio Me I

pHrQ Me

HOCH I AcNHCH

Me

I

Q

‘CNHCH

Pr

OH

(28b)

OH (28~)

derivative, and the 7-0-methyl derivatives9’of 28b (“N-acetyl-3,4-0isopropylidenecelestoraminol”) from a celesticetin degradation product. (59i) H. Hoeksema, US. Pat. 3,255,175(1966);Chem. Abstracts, 65, 17039 (1966). (59j) W. Schroeder, B. Bannister, and H. Hoeksema, J . Arner. Chem. SOC., 89, 2448 (1967).

(59k)R. R. Herr and G. Slomp,J. Amer. Chem. SOC., 89,2444 (1967). (591) H. Hoeksema, J. Arner. Chern. SOC., 90, 755 (1968).

250

S. SOLTZBERG

111. PHYSICAL PROPERTIES 1. Infrared Spectra

ani -2-OThe infrared spectra of 1,4-anhydro-3,5-0-methy~nemethyl-DL-xylitol have been studied.s0 The 2-methyl ether was obtained by converting 1,4-anhydro-3,5-0-methylene-~~-xylitol into its monomethyl ether, and then hydrolyzing off the methylene group. A methyl ether prepared from the known 1,4-anhydro-3,5-0-isopropylidene-2-O-methyl-~~-xylitol proved to be identical with this compound, thus establishing at the same time that the methylene group in the known acetal is attached to 0-3 and 0-5 of 1,4-anhydro-~~xylitol. The methylene group, having a 1,3-dioxolane structure, was characterized by an absorption band at about 2800 cm-’. It was e ~ t a b l i s h e dthat ~ ~ the ring structures of anhydroalditols of unknown structure could be inferred solely on the basis of the multiplicity of the C-H stretching-vibration frequencies, without interpreting the bands below 1600 cm-’. The anhydroalditols examined were 1,5-, 1,6-, and 2,6-anhydro-~-glucitoland 1,6-anhydro-~iditol, and partially deuterated forms of the first two and the last. The following wavenumbers between 3000 and 2960 cm-’ (LiF) were assigned to the C-OH bonds on the ring: for 1,5-anhydro-~-g~ucitol, a strong band at 2985 cm-’ was attributed to the two axial C-OH bonds “above” the plane of the ring, and a weaker band at 2998 cm-’, to the single axial C-OH “below” the plane. It was claimed that the hydroxymethyl group in this compound was responsible for the bands at 2940 and 2885 cm-’. In 1,6-anhydro-~-glucitol,three of the four axial C-OH groups are spatially similar, and a strong absorption at 2975 cm-’ was assigned to them, the fourth (in the opposite direction) was assumed to cause the band at 2990 cm-’. 1,6-Anhydro-~-iditolhas four axial groups in 2 pairs, with absorptions of equal intensities; the bands at 2998 cm-I and 2960 cm-I were attributed to them, the former being assigned to the pair “above” the plane. The band due to the C-H part of the CH(0H) grouping is very weak in all of the compounds. For 2,6anhydro-D-glucitol, the assignments were 2992 cm-’ for the axial hydroxyl group “above” the plane, 2980 cm-’ for the equatorial hydroxyl group “above” the plane, and 2970 cm-’ for the equatorial hydroxyl group “below” the plane. All of the bands were of medium intensity. The asymmetrical and symmetrical stretching-frequencies of the (60) A. N. Anikeeva, E. I. Pokrovskii, L. G . Revel’skaya, E. F. Fedorova, and S. N. Danilov,J. Gen. Chem. USSR (Engl. Transl.), 36, 203 (1966).

ALDITOL ANHYDRIDES

251

-CH-(0-) grouping were weak, and ranged from 2920 to 2928 cm-I for the former, and 2860 to 2862 cm-' for the latter. The assignment of structure to 2,6-anhydro-D-glucitol (previously unknown) through its i.r. spectrum was based on the following reasoning. ( a ) There are four strong bands for CH2groups: -CH,-(OH), at 2938 and 2880 cm-'; -CH2-(O-),at 2920 and 2860 cm-'. As may be seen, the compound has a primary hydroxyl group as well as a primary \ ether group. (b) There are three ,CH-(OH) bands, thus excluding the presence of a ring of less than six members. ( c ) There is a weak shoulder on the band at 2938 cm-', presumably due to coalescence of the weak secondary ether grouping with the strong absorption band of the hydroxymethyl group at the same wavenumber. ( d ) The spectrum is not identical with that of 1,5-anhydro-~-glucitol.(e) A molecular model of the compound shows that it has one axial and two quasi\ equatorial ,CH-(OH) groups. (Stretching and out-of-plane deformation vibrations for OH and OD were also given.) Barker and Stephens6' studied the i.r. spectra of 3,6-anhydro-~and glucitol, 1,4-anhydro-~-mannitol,1,4:3,6-dianhydro-~-glucitol, 1,4:3,6-dianhydro-2,5-di-O-methyl-~-mannitol, and grouped the absorption bands into four characteristic types: Type A, a symmetrical, ring-breathing frequency of the single hydrofuranol ring (924 -1-13cm-', strong); Type B, a rocking vibration of the methylene group in the ring (879 &7 cm-'); Type C, at 858 +7 cm-I; and Type D, at 799 217 cm-' (tentatively assigned to the C-H deformation mode of a hydrogen atom attached to the ether carbon-atom of the hydrofuranol ring). The frequencies observed (cm-I) are summarized in Table I. TABLEI Frequencies (cm-') of Absorption Bands of 3,6-Anhydro-D-glucitol (l), 1,4-Anhydro-~-mannitol(2),1,4;3,6-Dianhydro-2,5-di-O-methyl-~-mannitol (3), and 1,4:3,6-Dianhydro-~-glucitol (4) Compound

A

B

C

D

1 2

933s 929s

886s 880s

781m 782s

3

923s 915s

880vs

867s 868s 860s 842m 846s

922m

884m

4

865111

816vs 814m 802m 830s 733s

"C-0stretching mode of methyl ethers. (61) S. A. Barker and R. Stephens, J . Chem. SOC., 4550 (1954).

Other bands

789m 753m

981s 995vs 949vs 966s 981vs 9440 976s 951m

252

S. SOLTZBERG

For dianhydroalditols having two hydrofuranol rings fused together, Type A and D absorptions appear as doublets, in conformity with the spectra of other sugar derivatives having fused hydrofuranol rings. However, for the dianhydro-D-mannitol, the bands at 923 and 816 cm-’ are single peaks, because of the identical nature of the anhydro rings. The i.r. spectrum of 1,4:3,6-dianhydro-~-glucitol dinitrate (isosorbide dinitrate) was recorded by Sammul and coworkers.62Hayward and coworkers6Yfound that the symmetrical stretching frequencies (us) for the NO, bands were characteristic for the endo (1282 *1 cm-’) and exo (1274 -+Icm-*) configurations in the mono- and di-nitric esters of the 1,4:3,6-dianhydrides (isohexides) of D-glucitol, D-mannitol, and L-iditol. The us (NO,) (1642-1647 cm-’) was not significantly affected by the configuration at the carbon atom to which the nitrate group was attached. Likewise, v(0N) (841-846 cm-I) was relatively constant. On the other hand, u(0H) showed configurational influence, the band for the exo-hydroxyl group appearing at 3640 cm-I, whereas the band for endo attachment was located at 3595 cm-’ for the dianhydro-Dmannitol and at 3565 cm-’ for the dianhydro-D-glucitol (OH-5). The assignments for the endo (hydrogen-bonded to the ring-oxygen atom of the second ring) and exo (free OH) hydroxyl groups are in agreement with the findings of Brimacombe and coworker^,^^ who found the OH stretching frequency to be at 3540 cm-’ for 1,4:3,6dianhydro-D-mannitol (endo OH) and at 3624 cm-’ (exo OH) and 3540 cm-’ (endo OH) for 1,4:3,6-dianhydro-~-glucitol. Included in the study were the i.r. spectra of 1,4-anhydro-2-deoxy-~-threoand -L-erythro-pentitols and 1,4-anhydro-~-threitoland -erythritol. The i.r. bands observed were consistently lower for the hydrogen-bonded OH as compared to the non-bonded or “free” OH. 2. Nuclear Magnetic Resonance Spectra

Proton coupling-constants were determined65 for the tri-O-acetyl derivatives of bis(ethylsulfony1)-a-D-lyxopyranosylmethane [2,6anhydro-l,l-bis(ethylsulfonyl)-ldeoxy-~-galactitol] (29) and bis(eth(62) 0. R. Sammul, W. L. Brannon, and A. L. Hayden, J . Ass. Ofic.Agr. Chem., 47, 918 (1964). (63) L. D. Hayward, D. J. Livingstone, M. Jackson, and V. M. Csizmadia, Can. J . Chem., 45, 2191 (1967). (64) J. S. Brimacombe, A. B. Foster, M. Stacey, and D. H. Whiffen, Tetrahedron, 4, 351 (1958). (65) L. D. Hall, L. Hough, K. A. McLauchlan, and K. Pachler, Chern. Znd. (London), 1465 (1962).

ALDITOL ANHYDRIDES

253

yIsu1fonyl)-a-D-arabinopyranosylmethane[2,6-anhydro-I,l-bis(ethylsu~fony~)-l-deoxy-D-glucitol] (30). Based on the coupling constants found (see Table 11), the angles calculated (by using the equation TAE~LE I1 Proton Coupling-constants (Hz) for Compound 29"

Jm.,

Anele (decrees)

11.2

1.7

~~

10.4 180 180

Estimated Ideal geometry

13.4

12.3

~~

3.0 54 60

3.3 52 60

"In chloroform, 3 mole percent.

] =J,cos28- 0.28 Hz and values of J,, = 9.26 Hz for angles between 0

and 90" andJ, = 10.35Hz for angles between 90 and 180")were almost in agreement with those required for the paired hydrogen atoms of a chair conformation, namely, 1C (D). 5

6

4

3

OAc

AcO

a

0

The acetoxyl-proton resonances were also in accordance with the (D) conformation, namely, one equatorial and two axial acetoxyl resonances for 29, and the converse for 30 (see Table 111), since methyl groups of axially oriented acetoxyl substituents of a pyranoid ring generally resonate at lower field than those of similar, equatorially attached groups.

1C

TABLEI11 Proton Chemical-shifts" (7-values) Compound

29 30

Acetoxyl-proton resonances

7.98 8.46

7.84 8.62

7.84 7.88

'In chloroform, 3 mole percent.

Proton magnetic resonance spectra of the three 1,4:3,6-dianhydrohexitols and their diacetates and dimethanesulfonates were used in

254

S. SOLTZBERG

1 , 4 :3,6-DianhydroL- iditol

1 , 4 :3,B-DianhydroD-glUCitOl

1 , 4 :3,B-DianhydroD-mannibl

determining the shapes of the oxolane ringss6 Proton couplings throughout the series were found to be constant, thus indicating that one conformation was common to all three compounds. The intramolecular hydrogen-bonding with the ether oxygen atom of the other and 174:3,6-dianring that occurs in 1,4:3,6-dianhydro-~-mannitol hydro-D-glucitol as a result of the presence of endo hydroxyl groups has little or no effect on the shape of the ring. Long-range couplings were identified, and it was noted that the coupling of the endohydroxyl proton to the proton on the same carbon atom was almost twice that for compounds having an exo-hydroxyl group. The n.m.r. spectrums7 of dianhydro-xylitolZ3and its methyl ether confirmed the chemical evidence that it has a 4-membered and a 5membered ring, and hence is 1,4:3,5-dianhydro-xylitol. This conclusion was further supported by the i.r. spectrum of the methyl ether, which has bands at 980 cm-' (oxetane ring) and at 1050 cm-I (oxolane ring).67

3. Circular Dichroism Spectra Circular dichroism of the nitrato (ONO) chromophore of the mono- and di-nitric esters of 174:3,6-dianhydrohexitolswas found to consist of two dichroic bands, one weak and positive, at about 265 nm, and a stronger band at -228 nm, which was positive for the endo-(R)-nitrato chromophore and negative for the em-(S)-nitrato dinitrate, which group.88 However, for 1,4:3,6-dianhydro-~-glucitol has both an endo and an exo nitrato group, both bands were positive, (+ 7,260) for the band at 225 nm was about half of the but [t$J,, algebraic sum of this band for the di-endo (+ 18,400) and di-exo (-4,460) compounds. (66)F. J. Hopton and G. H. S . Thomas, Can.J. Chem., 47, 2395 (1969). (67) G . E. Ustyuzhanin, A. I. Kol'tsov, N. S. Tikhomirova-Sidorova, and S. N. Danilov, Zh. Obschch. Khirn., 34, 3805 (1964); Chem. Abstracts, 62, 11892 (1965). (68) L. D. Hayward and S. Claesson, Chem. Commun., 302 (1967).

ALDITOL ANHYDRIDES

255

4. Crystal S h c t u r e The crystal and molecular structures of 1,4:3,6-dianhydro-2-0(p-bromophenylsulfony1)-(em)-D-glucitol 5-nitrate(endo) were reported by Camerman, Camerman, and Trotter.6gThe bond distances and valency angles were found to be normal, with an intramolecular apparent attraction between an oxygen atom of the NO, and the ring-oxygen atom of the second ring; the distance was found to be 0.29 nm.

5. Surface Activity By using light-scattering measurements, Becher'O observed a configurational effect influencing the micellar behavior of the four isohexide monostearates dissolved in benzene. He found that micelles are formed only when the molecule contains an endo stearic ester group (see Table IV). TABLEIV Micellar Behavior of Monostearates of Dianhydrohexitols in Benzene Solution Monostearate of

Critical micelle conc. (gldl)

Micellar molecular weight

Aggregation number

none

-

-

0.51

14,300

35

0.35

9,000

22

1,4:3,6-Dianhydro-~-glucitol (em) 1,4:3,6-Dianhydro-~-glucitol (endo) 1,4:3,6-Dianhydro-~-mannitol (endo) 1,4:3,6-Dianhydro-~-iditol (ex01

none

-

-

It is possible that a fused, oxygen-containing ring-system can behave as the center for micelle formation. For the dianhydro-D-mannito1 monostearate (endo), the stearate group is out of the way, and cannot interfere with agglomeration of the isohexide moieties. On the other hand, for the dianhydro-D-iditol monostearate ( e m ) , the stearate group can cover up the isohexide portion, and thus inhibit micelle formation. Hence, for dianhydro-D-glucitol monostearate, (69)A. Camerman, N. Camerman, and J. Trotter, Acta Cwstallogr., 19,449 (1965); Chem. Abstracts, 63, 10793 (1965). (70)P.Becher, 1.Phys. Chem., 64, 1221 (1960).

256

S. SOLTZBERG

the 2-ester ( e m ) does not form micelles, whereas the 5-ester (endo) does. Surface activity and micellar behavior in aqueous solutions of a number of D-glucopyranosyl alkyl-substituted benzenes and alkanes were s t ~ d i e d . 'It ~ was found that D-glucopyranosylto~uenedoes not form micelles, although it is surface-active. The higher alkyl derivatives (ethyl and butyl) show micelle formation. Moreover, D-glucopyranosylhexane does not form micelles, whereas the higher Dglucopyranosylalkanes (for example, the derivatives of octane and dodecane) form micelles at regularly decreasing concentration; these occur in the same order as with the D-glucopyranosyl derivatives of the alkylbenzenes. T o k i ~ a 'investigated ~ the surface tensions of a homologous series of D-glycopyranosyl derivatives of alkylbenzenes, and developed a relationship between the surface tension and the concentration. Likewise, an equation of state was evolved for the monolayers at relatively high pressures. It was observed that the free energy of absorption changes by about 650-700 cal/mole for each additional methylene group in the chain, except for methyl to ethyl, where the change was 380 cal. mole-'.

IV. REACTIONS 1. Ring Opening a. Anhydrohexitols.-Ring opening of an anhydroalditol was and was discussed first described for 1,4:3,6-dianhydro-~-mannitol by Wiggins.' Foster and coworker^'^ treated 1,4:3,6-dianhydro-~glucitol with an excess of boron trichloride in the presence of dichloromethane at room temperature, and, after removal of borate and unreacted boron trichloride, obtained a mixture of products that was treated with benzaldehyde. The major portion of the crude, solid benzylidene acetal produced proved to be 2,4-O-benzylidene-l,6dichloro-1,6-dideoxy-~-glucitol, and there was a small proportion of the corresponding 2,4:3,5-dibenzylidene acetal. Also, a small proportion of other (unidentified)benzylidene-containing substances was isolated. The yield of benzylidene derivatives of 1,6-dichloro-1,6(71) E. Hutchinson, V. E. SheaEer, and F. Tokiwa, J . Phys. Chem., 68, 2818 (1964). (72) F. Tokiwa, Bull. Chem. Soc.Jap., 38,751 (1965); Chem. Abstracts, 63,3646 (1965). (73) M. A. Bukhari, A. B. Foster, and J. M. Webber, Carbohyd. Res., 1, 474 (1966).

ALDITOL ANHYDRIDES

257

dideoxy-D-glucitol was high, compared to that obtained74by heating the starting dianhydride with fuming hydrochloric acid in a sealed tube at 110". Institoris and coworkers, in a patent issued to Chinoin from Gyogys~er,'~prepared 1,6-dibromo-l,6-dideoxy-~-mannitol 1,4:3,6-dianhydro-~-mannitol, reportedly in 91% yield, by using a methanolic solution of concentrated aqueous hydrobromic acid as the reagent. The ring opening of 1,4:3,6-dianhydro-~-iditol does not appear to have been attempted, although it should occur analogously to that of the other dianhydrides mentioned. Cope and Shen58 observed that, when a solution of 1,4:2,5:3,6trianhydro-D-mannitol in concentrated hydrochloric acid is heated on a steam-bath, the 1,4- and 3,6-rings are opened, affording 2,5-anhydrol,6-dichloro-l,6-dideoxy-~-mannito1 in good yield. It had earlier been mentioned55that treatment of 2,5:3,6-dianhydroD-glucitol with fuming hydrochloric acid at 100" causes opening of the 3,6-oxolane ring. The marked tendency for ring opening between the oxygen atom and the methylene (terminal) carbon atom is consistent with the known behavior of ethers and of oxolane (tetrahydrofuran) derivatives.

b. Anhydropentitols. - 1,4-Anhydro-5-deoxy-5-fluoro-~~-xylito~ was obtained in about 30 % yield by heating 1,4:3,5-dianhydro-~~-xylitol with potassium fluoride in aqueous diethylene glycol.7sThe product was identical with that obtained by heating 1,4-anhydro-5-chloro-5deoxy-DL-xylitol with powdered potassium fluoride in diethylene glycol. On heating 1,4-anhydro-xylitol with dry hydrogen chloride77 at 105", a crystalline "xylityl chloride," presumably l-chloro-l-deoxyxylitol, was formed, but, on the basis of results of derivatization, it probably retained the anhydride structure. Moreover, a subsequent report7* indicated that, under apparently the same conditions, 1,4anhydro-xylitol affords 1,4-anhydro-5-chloroS-deoxy-~~-xylitol. (74) R. Montgomery and L. F. Wiggins, J. Chem. Soc.. 237 (1948). (75) Chinoin Gyogyszer es Vegyeszeti Termeker Gyara Rt., Hung. Pat. 149,870 (1962); Chem. Abstracts, 60, 10776 (1963). (76) S. N. Danilov and E. Ya. Afanas' eva, Zh. Obshch. Khim., 36, 1406 (1%); Chem. Abstracts, 66, 18827 (1967). (77) S. N. Danilov, A. N. Anikeeva, N. S. Tikhomirova-Sidorova,and A. N. Shirshova, Zhur. Obshch. Khim., 27, 2434 (1957); Chem. Abstracts, 52, 7162 (1958). (78) S. N. Danilov, N. S. Sidorova, A. N. Anikeeva, Yu. A. Bol'shukhina, G. M. Zarubinskii, T. I. Orlova, and G . E. Ustyuzhanin, Sintez i Suoistoa Monomerou, Akad. Nauk S S S R , 1962,247 (1964); Chem. Abstracts, 62,9218 (1965).

258

S. SOLTZBERG

Danilov and coworker^'^ treated 1,4:3,5-dianhydro-~~-xylitol with a number of primary and secondary amines. The 3,s-anhydro ring was opened, to give the corresponding 5-amino-5-deoxy compounds. Hedgley and FletcheflO found that 1,4-anhydro-n-ribitol and 1,4anhydroerythitol are comparatively stable to liquid hydrogen fluoride at 18". 2. Isomerization

a. Acid-catalyzed Isomerization. - In an extensive investigation of the behavior of acylated inositols and 1,4- and 1,s-anhydroalditols in liquid hydrogen fluoride, Hedgley and Fletcher have showneothat a feature common to all instances in which inversions occur is the presence of a cis-trans sequence of three ester groups; inversion occurs at the middle carbon atom of the sequence. A mechanism involving intermediates of the charged, cyclic ortho-ester type was postulated. Hence, 1,4-anhydroerythritol diacetate does not isomerize to 1,4anhydrothreitol,80 although ring opening with formation of threitol and erythritol occurs. On the other hand, 1,4-anhydro-~-ribitol tribenzoate yields a complex mixture containing lY4-anhydro-ribitol, 1,4-anhydro-lyxitol (2,5-anhydro-arabinitol), and, possibly, 1,4-anhydro-arabinitol (as shown by the results of paper electrophoresis) and at least one pentitol, namely, DL-arabinitol. 1,4-Anhydro-~arabinitol triacetate afforded a mixture which, by paper electrophoresis, was found to contain 1,4-anhydro-lyxitol, 1,4-anhydro-ribitol, and 1,4-anhydro-arabinitol in one fraction, and arabinitol, ribitol, and xylitol in the second fraction. On the other hand, 1,4-anhydroD-XylitOl triacetate gave, solely, 1,4-anhydro-~-ribitol. Although these anhydropentitols lack the cis-trans triple-ester arrangement, the following respective intermediates from 1,Canhydrowere tri-0-benzoyl-D-ribitol and tri-O-acetyl-l,4-anhydro-~-xylitol proposed, in order to account for the epimerizations that occur.

(79)(a) S. N. Danilov, N. S. Tikhornirova-Sidorova, G. E. Ustyuzhanin, and G. A. Efirnova, Zh. Obshch. Khim., 32, 3614 (1962);Chem. Abstracts, 59,5246 (1963). (b) ibid., 32, 3617 (1962);Chem. Abstracts, 59, 5246 (1963).(c) G.A. Efimova, G . E. Ustyuzhanin, N. S. Tikhomirova-Sidorova, and S. N. Danilov, ibid., 33, 1429 (1963);Chem. Abstracts, 59, 12893 (1963).(d) S. N. Danilov, N. S. Tikhomirova-Sidorova. G. E. Ustyuzhanin, and C. A. Efirnova, USSR Pat. 162,522(1964); Chem. Abstracts, 61,9574(1964). (80) E. J. Hedgley and H. G. Fletcher, Jr., /. Amer. Chem. SOC.,86, 1576 (1964).

ALDITOL ANHYDRIDES

259

-0-C-Ph

HO,

,Me I

For 1,4-anhydro-xylitol, ring opening was postulated to occur as follows: H,COH I HCOAc

I

ACOCH,

I

OAc

HCO

I

H,COAc

(31)

Intermediate 31 can then rearrange to the isomeric pentitol carbonium intermediates, through attack by a neighboring acetyl group. These intermediates then cyclize to the epimeric 1,4anhydropentitols as a result of attack by the 1-hydroxyl group, or are hydrolyzed to the corresponding pentitols. An analogous ring-opening was proposed for tri-0-benzoyl-D-ribitol, in order to account for the 2,5-anhydroarabinitol and DL-arabinitol formed. 1,4-Anhydro-~-glucitoltetraacetate was envisaged as opening to form the intermediate 32 which, through a series of rearrangements and cyclizations, yields 1,4-anhydro-~-glucitol,1,4-anhydro-~-man-

260

S. SOLTZBERC H&OH 1

HCOAc

nitol, an unidentified product that is neither 1,4-anhydro-~-iditol nor 1,4-anhydrogalactitol, and galactitol, D-glucitol, L-iditol, and Dmannitol. In contrast, the derivatives of galactitol and D-mannitol that contain the requisite cis-trans arrangement within the ring gave only simple mixtures of the unchanged anhydride and one epimer, namely, 1,5-anhydro-~-gulitol (2,5-anhydro-~-glucitol) and 1,5-anhydro-~altritol, respectively, in accordance with predictions.81 On the other and tri-O-acetyl-1,5-anhyhand, tetra-O-acetyl-1,5-anhydro-~-glucitol dro-D-arabinitol, which lack the cis-trans arrangement are not epimerized.sl Although allitol, galactitol, and talitol, which lack a trans arrangement of the 3- and 4-hydroxyl groups, are difficult to anhydridize to the 1,4:3,6-dianhydrides, Hartmanns2 found that, on heating these hexitols or their 1,4-anhydrides at 110 to -185" with a catalytic amount of an acid (such as sulfuric acid or p-toluenesulfonic acid), 1,4:3,6dianhydrides were formed that were not dianhydrides of the starting hexitol. In this way, galactitol was transformed into 1,4:3,6-dianhydroDL-gluCitO1, inversion of configuration occurring at either C-3 or C-4.

b. Isomerization by Hydrogenation Catalysts.- The isomerization of a 1,4:3,6-dianhydrohexitolwas first reported by Fletcher and or -D-manG ~ e p pwho , ~ ~treated either 1,4:3,6-dianhydro-~-glucitol nitol with Raney nickel at 140" in the absence of added hydrogen, and then at 190-200" in the presence of hydrogen. From both compounds, the product was found to contain 1,4:3,6-dianhydro-~-iditol, isolated as the dibenzoate. Wright and Brandners4 have shown the reversible interconversion of aqueous solutions of these three dianhydrides in the presence of a nickel-on-kieselguhr catalyst under hydrogenating conditions. At the steady state, the product contains 57% of 1,4:3,6-dianhydro-~(81) E. J. Hedgley and H. G . Fletcher, Jr., J . Amer. Chem. Soc., 85, 1615 (1963). (82) L.A. Hartmann (to Atlas Chemical Industries, Inc.), US. Pat. 3,454,603 (1969). (83) H. C. Fletcher, Jr., and R. M. Coepp, Jr., J. Amer. Chem. Soc., 68, 939 (1946). (84) L. W. Wright and J. D. Brandner,]. Org. Chem., 29,2979 (1964).

ALDITOL ANHYDRIDES

26 1

iditol, 36% of the corresponding D-glUcitOl dianhydride, and 7% of the D-mannitol dianhydride. In the presence of 3% copper-promoted, nickel-on-kieselguhr catalyst, the respective yields weres5 58.9, 35.5, and 5.7%.

3. Selective Behavior a. Catalytic Oxidation. -When aqueous solutions of the three 1,4:3,6-dianhydrohexitolswere treated, at or above room temperature, with Adams’ catalyst and oxygen, only the endo-hydroxyl groups gave 174:3,6-dianhydrowere oxidized; 1,4:3,6-dianhydro-~-mannitol D-fructose and 1,4:3,6-dianhydro-~-threo-2,5-hexodiulose, 1,4:3,6dianhydro-D-glucitol gave 1,4:3,6-dianhydro-~-sorbose, and 1,4:3,6dianhydro-L-iditol was u n ~ h a n g e d . ~ ~ . ~ ~ For 174-anhydridesof the hexitols, oxidation of hydroxyl groups on C-2 or C-3 is determined by whether they are cis or trans, and whether the side chain is cis or trans to the 3-hydroxyl group on the oxolane ring and can form a hemiacetal ring on oxidation of the primary hydroxyl group.88 The final oxidation products from 1,4-anhydro-~glucitol (33) were 3,6-anhydro-~-gulono-1,4-lactone (34) and the hydrated form (35) of 3,6-anhydro-xyZo-~-hexulosono-1,4-lactone. Similarly, 1,4-anhydro-~-iditol (36) gave 3,6-anhydro-~-idono-1,4lactone (37) and 35. 1,4-Anhydro-~~-galactitol (38) gave 3,6-anhydro-

HoLQ H,COH

(33)

-4 (34)

OH

0

OH

H,COH I

DL-galactonic acid (39). 1,4-Anhydro-~~-talitol(3,6-anhydro-~~altritol) (40) gave 2-O-(carboxymethyl)-D~-threaric acid (41) and 1,4anhydro-D-mannitol(42)gave 20-(carboxymethy1)erythraricacid (43). (85) L. W. Wrightand J. D. Brandner, U.S. Pat. 3,023,233 (1962). (86) K. Heyns, W. P. Trautwein, and H. Paulsen, Chem. Ber., 96,3195 (1963). (87) Atlas Chemical Industries, Inc., French Pat. 1,425,204 (Jan. 28, 1966). (88) E. Alpers, Dissertation, University of Hamburg (1967).

262

S . SOLTZBERG H,COH

CO,H

HO

HO

I

(38)

H,COH I HCOH 0

(39)

CO,H 1

COzH

I

HCOCH,CO,H HO,C

H,COH

CO,H

C0,H

I

CO,H

CO,H I

HCOCH$O,H HO,C (42)

CO,H

I CO,H

(43)

The lY4-anhydrotetritolswere oxidized to diglycolic acid. However, 1,4-anhydrothreitol, in which the hydroxyl groups are trans, was much more resistant than 174-anhydroerythritolto attack. The following order of oxidizability or selectivity was formulated: primary OH = cis OH on the oxolane ring > trans OH. If the resulting anhydroaldonic acid can lactonize, the secondary hydroxyl group on the lactonic ring is oxidized.

b. Selective Acylation.-Of the anhydroalditols, lY4:3,6-dianhydroD-glUCitOl provides an interesting instance of selective acylation. (The two other dianhydrohexitol isomers either have both hydroxyl groups endo, or both exo, so that differentiation would not be expected.) In the D-glucitol dianhydride, the 2-hydroxyl group is exo, and the 5-hydroxyl group is endo and, apparently, blocked sterically by the second ring. Therefore, it had been assumed by Cope and Shen5*that the 2-hydroxyl group would be the more readily esterified. However, Jackson and Hayward5O'*)showed, by comparison of the rates of reaction of the mono-p-toluenesulfonates of the isomers with iodide, that the 5-hydroxyl group is probably more readily p-toluenesulfonylated. That this is correct was firmly established by Lemieux and M c I n n e ~ , ~who ~ ( ~proved, ) by i.r. spectroscopy, that the

ALDITOL ANHYDRIDES

263

mono-p-toluenesulfonate obtained in 11.7%yield had an intramolecular hydrogen bond, whereas, the mono-p-toluenesulfonate obtained in 45.4% yield showed no evidence of hydrogen bonding. Because only the endo hydroxyl group is favorably situated for hydrogen bonding, the first ester must be the 2-ester, and the second, the 5-ester. Selective acetylation of the 5-hydroxyl group was observed by Buck and who also showed that the presence of hydrogen chloride strongly influences the yields of isomers. At room temperature, on acetylation with acetic anhydride-pyridine, the 2-acetate was obtained in somewhat higher yield (27.6-28.6%) than the 5-acetate (16.9-17.1%). However, when acetylation was performed in the presence of pyridine hydrochloride, the yield of the 5-acetate was 42.9%, and that of the 2-acetate was 12%. These yields are in remarkable agreement with those of Lemieux and M c I n n e ~ ~for ~ ' ~the ' monosulfonylation reaction. As the latter reaction was conducted with ptoluenesulfonyl chloride, pyridine hydrochloride was formed, and it seems likely that, in some as-yet-unexplained manner, the presence of a strong acid augments the hydrogen-bonding. This effect is further illustrated in the partial esterificationgOof the dianhydro-Dglucitol with p-phenylazobenzoyl chloride in pyridine at 37". A 36% yield of the 5-ester and 12% of the 2-ester were obtained. A less obvious instance is the acylation of 1,5-anhydro-4,6-0benzylidene-D-glucitol. The 1,5-anhydride has a chair conformation, and the 2- and 3-hydroxyl groups are equatorially attached trans to each other, and are, apparently, essentially equivalentg1However, the 2-benzoate and 2-p-toluenesulfonate were obtained almost exclusively on using the acyl chlorides in pyridine, although in the former acylation, an appreciable proportion of the dibenzoate was formed. In order to explain his observations on the conversion into the corresponding 2,3-anhydride7 Newthsl suggested a conformational shift to a boat conformation. However, should a conformational shift occur early during the esterification (as a result of the formation of a small proportion of hydrogen chloride), the hydroxyl groups could become trans-axial, and the 2-hydroxyl group would be in a position to form a hydrogen bond with one of the acetal oxygen atoms, almost analogously to the situation with 1,4:3,6-dianhydro-~-glucitol. (89) K. W. Buck, J. M. Duxbury, A. B. Foster, A. R. Perry, and J. M. Webher, Carbohyd. Res., 2,122 (1966). (90)K. W. Buck, A. B. Foster, A. R. Perry, and J. M. Webber,]. Chern. Soc.,4171(1963). (91) F. H. Newth, ]. Chern. SOC.,2717 (1959).

264

S. SOLTZBERG

c. Reaction of Sulfonyloxy Groups with Nucleophiles. - It is well known that isolated, secondary sulfonyloxy groups on sugars react with difficulty with nucleophilic reagents.92Hence, it was incorrectly assumed that the same would apply to alditols, and that the sulfonic esters of the 1,4:3,6-dianhydrohexitolswould be resistant to displacement. However, it has been found that a considerable difference in the ease of reaction exists between the endo- and em-sulfonic esters of the 1,4:3,6-dianhydrohexitol isomers; this was discussed by Wiggins.' In the early work, no attention was paid to the fact that Walden inversion occurs, and the compounds formed were incorrectly named. Cope and Sheng3called attention to this serious error, and corrected the configurations that had been given for many of the compounds. Those corrected by Cope and Shen,g3 and some additional compounds, are listed in Table V. TABLEV Revised Configurations of Derivatives of Anhydroalditols

New structure

Old configuration

References

1,4:3,6-Dianhydro-5chlaro-5-deoxy-

D-glucitol

16,93

L-iditol 1,4:3,6-Dianhydro-5-deoxy-5-iodo-2-O-ptolylsulfonyl-L-iditol

D-glucitol

93

1,4:3,6-Dianhydro-2,5-dichloro-2,5dideoxy-L-iditol 1,4:3,6-Dianhydr0-2-chloro-2-deoxy-5-0-

93

(methylsulfonyl)-D-glucitol

93

1,4: 3,6-Dianhydro-2-chloro-2-deoxy-5-O(phenylcarbamoy1)-D-glucitol

D-mannitol

93

D-mannitol

93

D-mannitol D-mannitol

93 93

xylitol

79b

xylitol

79b

xylitol

79b

1,4:3,6-Dianhydro-2,5-dideoxy-2,5diiode L-iditol

2,5-Diamino-l,4:3,6-dianhydro-2,5dideoxy-L-iditol

1,4:3,6-Dianhydro-2,5-dithio-~-iditol 1,4:3,5-Dianhydro-2-anilino2-deoxy-~~-arabinitol?

1,4-Anhydro-2,5-dideoxy-2,5dipiperidino-DL-arabinitol?

1,4-Anhydro-2,5dideoxy-2,5-bis(diethylamino)-DL-arabinitol?

1,4-Anhydro-2-(butylamino)-2-deoxy3,5-O-methylene-~~-arabinitol? 1,4-Anhydro-2-anilino-2-deoxy3,5-O-methylene-DL-arabinitol?

xylitol

145

xylitol

145

(92) R. S. Tipson, Adoan. Carbohyd. Chen., 8, 107 (1953). (93) A. C. Cope and T. Y. Shen,]. Amer. Chem. SOC., 78,3177 (1956).

ALDITOL ANHYDRIDES

265

The three 1,4:3,6-dianhydrohexitolsshow the shielding effect of the cis-fused oxolane ring on their di-0-sulfonyl derivatives. As C-2 and C-5 of 1,4:3,6-dianhydro-~-mannitol 2,5-di-p-toluenesulfonate and 2,5-dimethanesulfonate are open to attack from the exo direction, they react readily with such nucleophilic reagents as benacetate in acetone,93t h i ~ a c e t a t e , ~ ~ zoate in N,N-dimethylf~rmamide,~~ and iodide in acetic anhyphthalimide in N,N-dimethyIf~rmamide,~~ dride.59(b1 Inversion occurs at these positions and the L-iditol configuration results, as pointed out in the several examples in Table V. However, hydroxide ion may merely saponify,92with retention of c~nfiguration.~~ 2,5di-p-toluenesulfonate, generFor 1,4:3,6-dianhydro-~-glucitol ally only the 5-p-tolylsulfonyloxy group (endo) is displaced, with formation of the L-ido onf figuration;^^.^ but it may lose both sulfonyloxy groups under more vigorous conditions, affording 1,4:2,5:3,6trianhydro-~-mannitol~* as already mentioned. as The 2,5-bis(p-toluenesulfonate) of 1,4:3,6-dianhydro-~-iditol, would be expected, is quite resistant to nucleophilic attack and can be recovered unchanged in good yield.93,96,97 However, reaction occurs to some e ~ t e n t , ~ ~but ( * ) the , ~ ~resulting product or products have not as yet been isolated. Under very vigorous conditions, the 2-p-tolylsulfonyloxy group reacts, and of 1,4:3,6-dianhydro-2,5-di-O-p-tolylsulfonyl-~-glucitol unsaturated products r e s ~ l t . ~ " ~ ~ d. Formation and Behavior of Acetals. -The benzylidenation of 1,4-anhydroerythritol has been monitored by n.m.r. s p e c t r o s c ~ p y . ~ ~ The initial product was postulated to have the phenyl group endo to the fused-ring system. It was found that, at equilibrium, the endo and exo forms are present in approximately equal amounts. For 1,4anhydro-3,5-O-benzylidene-~-mannitol in N,N-dimethylformamide containing p-toluenesulfonic acid, the low-field benzyl proton signal (T 4.35) changed to the high-field signal (T 4.54) of the known endo-2,3-O-benzylidene isomer.gRHowever, on prolonged treatment, a low-field signal again appeared, but, this time, it was that of the exo-2,3-O-benzylidene isomer. The latter was isolated by column chromatography on silica gel; it had m.p. 111-112", [aID-40" (in (94) D. H. Buss, L. D. H9I1, and L. Hough, I . Chem. Soc., 1616 (1965). (95) P. Bladon and L. N. Owen, J . Chem. SOC.,585 (1950). (96) N. K. Matheson and S. J. Angya1,J. Chern. Soc., 1133 (1952). (97) L. F. Wiggins and D. J. C. Wood, J . Chern. Soc., 180 (1951). (98) F. S. Al-Jeboury, N. Baggett, A. B. Foster, and J. M. Webber, Chern. Comrnun., 222 (1965).

266

S. SOLTZBERG

water), as compared with m.p. 94-96", [a],, -88" (in water) for the 2,30-benzylidene isomer previously known. As the zeta1 group in 2,5-O-methylene-~-mannitol is relatively resistant to hydrolysis, S. B. Bakerss studied the stabilities of 1,4:3,6dianhydro-2,5-O-methylene-~-mannitol and -D-iditol relative to that of the D-mannitol acetal. He synthesized 1,4:3,6-dianhydro-2,5-0methylene-D-mannitol by cyclizing 2,5-O-methylene-l,6-di-O-p-tolylsulfonyl-D-mannitol or by methylenation of 1,4:3,6dianhydro-~-mannitol. The D-iditol isomer was prepared by cyclizing 1,6-di-O-benzoyl2,5-0-methylene-3,4-di-O-p-tolylsulfonyl-~-mannito~. 2,5-O-Methylene-~-mannitolis quite stable to hydrolysis by dilute acids, or to acetolysis by acetic anhydride-sulfuric acid. It was found that its 1,4:3,6dianhydride could be hydrolyzed merely by treatment with hot water. The dianhydro-D-iditol derivative is stable to hot water and hot mM hydrochloric acid, but not to hot 10 mM acid. Inspection of molecular models fails to reveal any reason to expect the behavior observed for these two dianhydro-0-methylene isomers, as it would appear that a greater strain should be present in the bonds of the methyIene bridge in the exo (D-iditol) isomer than in the endo (D-mannitol)isomer. It is possible that the oxygen atoms of the oxolane rings strongly repel the acetal oxygen atoms of the dianhydro-0methylene-D-mannitol, favoring rupture of the methylene bonds; or, because of the proximity of the oxygen atoms to each other, a proton could have a longer residence-time in the vicinity, thus destabilizing the methylene group. Indeed, it is probably more noteworthy that 1,4:3,6-dianhydro-2,5-O-methylene-~-iditol is incapable of existence at all, unless a considerable adjustment of the fused oxolane rings should occur.

e. Miscellaneous.- On treatment of 1,4:3,6-dianhydro-2,5-dideoxy2,5-diiodo-~-iditolwith silver nitrate in dry acetonitrile, racemization occurs, with formation of the corresponding 2,5-dinitrates of 1,4:3,6dianhydro-D-glucitol, -D-mannitol, and -L-iditol in poor yield (although 70% of the theoretical yield of silver iodide was isolated). The low yield was not attributable to destruction of nitric ester, because 1,4:3,6-dianhydro-~-glucitol dinitrate was recovered in almost 90% yield when it was subjected to the same conditions.loOThis test also served to demonstrate that racemization had occurred during the formation of the nitric ester, and, hence, a carbonium ion intermediate was postulated. Different reactivities of the dinitrates of the three dianhydro(99)S. B. Baker, Can. /. Chem., 31, 821 (1953). (100)L. D.Hayward, M. Jackson, and I. G . Csizmadia, Can./. Chem. 43, 1656 (1965).

ALDITOL ANHYDRIDES

267

hexitols toward hot pyridine was observed.'"' Arrhenius activation energies of 21,24, and 41 kcal.mole-' were found for the dianhydro-Liditol, -D-ghcitol, and -D-mannitol dinitrates, respectively, indicating that the exo nitrato group is the more readily affected. 1,4:3,6-Dianhydro-~-mannitol was directly converted,lo2 in quantitative yield, into 1,4:3,6-dianhydro-2,5-dideoxy-2,5-diiodo-~-iditol on treatment with triphenyl phosphite methiodide in dry benzene at 20".

v. USES

1. Industrial As mentioned by Wiggins,' the principal industrial use of the hexitol anhydrides (prepared almost exclusively from D-glucitol and D-mannitol) is in the manufacture of nonionic surfactants. The anhydrides are generally formed in situ during esterification with a variety of saturated and unsaturated fatty acids. However, the anhydrides, frequently in the form of poly(oxythy1ene) adducts, are mentioned in a wide range of patents, of which the following selection is only a small sample. On monoacylation with lauric acid and treatment of the product with ethylene oxide, anhydro-D-glucitols afford an antistatic agent for photographic film.ln3 1,4:3,6-Dianhydro-~-glucitol was one of a number of diols used in preparing polymeric phosphites (esters) useful as flame-proofing agents.lo4 Water-soluble glycidyl with ethers were prepared by treating 1,4:3,6-dianhydro-~-glucitol epichlorohydrin and 50%, aqueous sodium hydroxide solution.1o5 Poly(oxypropy1ene) ethers of 1,4-anhydro-~-glucito1or -D-mannitol have been used in the preparation of nonfriable polyurethan foams.lM 1,4:3,6-Dianhydro-~-glucitol, 1,4-anhydro-D-glucito~,or 1,5-anhydroD-mannitol are used in modifying thermosetting melamine resins.lo7 (101) M . Jackson and L. D. Hayward, Can.]. Chem., 38,496 (1960). (102) (a) N. K. Kochetkov and A. I. Usov, Tetrahedron, 19,973 (1963).(b) N. K. Kochetkov and A. I . Usov, lzv. Akad. Nauk S S S R , Otd. Khim. Nauk, 1042 (1962);Chem. Abstracts, 57,15213 (1962). (103) Adox Fotowerke, Ger. Pat. 1,134,586 (Aug. 9, 1962); Chem. Abstracts, 57, 12006 (1962). (104) L. Friedman and H. Could, U.S. Pat. 3,053,878(Sept. 11,1962);Chem.Abstracts, 58, 3354 (1963). (105) J . G. Morrison, U.S. Pat. 3,041,300 (June 26, 1962); Chem. Abstructs, 57, 8739 (1962). (106) (a) Takeda Chemical Industries, Ltd., Brit. Pat. 989,144 (April 14, 1965); Chem. Abstracts, 63, 1963 (1965). (b) Takeda Chemical Industries, Japan Pat. 16,198 (Sept. 12, 1966); Chem. Abstracts, 66, 18605 (1967). (107) (a) J. D. Larkin and G. M. Grudus, U.S. Pat. 3,194,719 (July 13, 1965). (b) G. M. Grudus and J. D. Larkin, U.S. Pat. 3,194,720. (c) G. M. Grudus and J. D. Larkin, U.S. Pat. 3,194,723.

268

S. SOLTZBERG

Clear lacquers that dry to tack-free films in four hours were obtained by use of unsaturated polyesters of D-glucitol anhydrides having two or more free hydroxyl groups.l0*Linear polymers were obtained from the acetate and the acetals of l74-anhydro-xy1itoI monomethacryits diethers, and its diesters may late.loS1,4:3,6-Dianhydro-~-glucitol, be used for stabilizing polypropylene against oxidation and discoloraand -di-0-ethtion by sunlight.'1° 1,4:3,6-Dianhydro-2,5-di-O-methylyl-D-glucitol are useful stabilizers for solutions of tetracycline and related antibiotic substances."' A so-called "liquid, universal shortening composition" is prepared from a mixture of fatty acid esters of 1,4:3,6-dianhydro- and 1,4-anhydro-~-glucitoland their poly(oxythylene) derivatives with vegetable oils.112 2. Biological 1,4:3,6-Dianhydro-~-glucitol dinitrate has found important medicinal use as a coronary vasodilator, and a considerable volume of literature has developed from studies thereof; of this, the references given here constitute only a part.'13 In one investigation, the pharmacological behavior of the three isomeric dianhydrohexitol dinitrates was c ~ m p a r e d . " ~ 'It~ ' was shown that, although glycerol trinitrate dinihas a slightly more rapid action, 1,4:3,6-dianhydro-~-glucitol trate has a longer-lasting effe~t."~(~'-'~' (108) Howards of Ilford, Ltd., Brit. Pat. 927.786 (June 6, 1963); Chem. Abstracts, 59, 6534 (1963). (109) A. N. Anikeeva and S. N. Danilov, U.S.S.R. Pat. 175,660 (Oct. 9, 1965); Chem. Abstracts, 64,6786 (1966). (110) P. E. Oberdorfer, Jr., U.S. Pat. 2,967,169 (Jan. 3, 1961). (111) R. A. Nash and B. E. Haeger, U.S. Pat. 3,219,529 (Nov. 23, 1965). (112) A. S. Geisler, U.S. Pat. 3,184,575 (May 25, 1965). (113) (a) J. E. Halliday and S. C. Clark,]. Pharm. Pharmacol., 17,309 (1965).(b) W. H. Bunn, Jr., and A. N. Cremos, Angiology, 14, 48 (1963); Chem. Abstracts 58, 11876 (1963). (c) A. L. Smith, Angiology, 13, 425 (1962); Chem. Abstracts, 58, 2768 (1963). (d) D. A. Sherber and I. J. Gelb, Angiology, 12, 244 (1961); Chem. Abstracts, 55, 18991 (1961). (e) J. P. Buckley, M. D. G. Aceto, and W. J. Kinnard, Angiology, 12, 259 (1961); Chem. Abstracts, 55, 18991 (1961). (f) A. J . Dietz, Jr., Biochem. Pharmacol., 16, 2447 (1967); (g) V. N. Dzyak, L. T. Furs, and B. N. Bezborod'ko, Farmakol. i Toksikol., 26, 47 (1963); Chem. Abstracts, 59, 4439 (1963). (h) J. C. Krantz, Jr., G. G. Lu, F. K. Bell, and H. F. Cascorbi, Biochem. Pharmacol., 11, 1095 (1962).(i) E. Kimura, K. Ushiyama, T. Yamazaki, K. Yoshida, N. Kojima, and T. Kanie, Proc. Asian-Pacific Congr. Cardiol. 3rd Kyoto, 1, 745 (1964); Chem. Abstracts, 65, 1265 (1966).(j) V. V. Buyanov, Famakol. i Toksikol., 30, 30 (1967); Chem. Abstracts, 66, 84327 (1967). (k) H. P. Cjuchta and R. F. Gautieri, J . Pharm. Sci., 52, 974 (1963). (1) A. Skoda, G. G. Rowe, W. C. Lowe, and C. W. Crumpton, Amer. J. Med. Sci., 246, 584 (1963). (m) K. Ogawa and S. Gudbjarnason, Arch. Int. Pharrnacodyn. Ther., 172, 172 (1968); Chem. Abstracts, 68, 113158 (1968).

ALDITOL ANHYDRIDES

269

Interest has developed in the use of 1,4:3,6-dianhydro-~-glucitol as an orally administered, osmotic diuretic. It has been examined in the lab~ratory,"~ and ~linically"~ in patients having cirrhosis of the liver. It was found to be similar in effectiveness to intravenous Dmannit01."~Its use in diuretic compositions has been patented.ll6 The dianhydro-D-glucitol lessens cerebrospinal fluid pressure and brain mass when administered orally to dogs;"' this effect is accompanied by osmotic diuresis. Intravenous injection likewise produces a drop in the cerebrospinal fluid pressure, but this is followed b y a greater "rebound." have shown that orally administered 1,4: Becker and 3,6-dianhydro-~-glucitol, at doses of 1.5 to 2 g/kg, effectively lessens the intraocular pressure in patients having cataracts, and at doses of 0.5 to 2 g/kg for glaucoma. Only minimal side-effects were noted in two of nineteen patients. were 1,5-Anhydro-~-glucitol and 1,5-anhydro-6-deoxy-~-glucitol used in a study of the mechanism of the active transport of sugars.118 1,5-Anhydro-~-glucitol,D-glucose, D-galactose, and 6-deoxy-~-glucose were found mutually to inhibit the active transport of each of the other compounds, indicating that a single transport-mechanism is involved for all of In connection with the transport of sugars, the Michaelis constant, the relative maximum rate with respect to D-glucose, and the phosphorylation coefficient were found to respectively, for the hexokinase in rat-epidibe 50, 0.5, and 3 x dymal, adipose-tissue homogenate~."~ The competitive inhibition of aldolase by a number of structural analogs of D-frUCtOSe 176-diphosphate has been investigated. Among these analogs were 1,4-anhydro-~~-ribitol 5-phosphate7 1,4anhydro-DL-xylitol 5-phosphate, 1,4-anhydro-~-arabinitol5-phosphate, 2,5-anhydro-~-mannitol 176-diphosphate, and 2,5-anhydroD-glUCitOl l,6-diphosphate. Their respective, enzyme-inhibitor, (114)(a) J. F.Treon, L. E. Gongwer, and W. H. C. Rueggeberg, Proc. Soc. Erp. Biol. Med., 119, 39 (1965).(b) J. H.Shinaberger, J. W. Coburn, R. C. Reba, K. G. Barry, and L. C. Clayton,]. Phormacol. Erp. Ther., 158,460(1967). (115)0.Gagnon, P. M. Gertman, and F. L. Iber, Amer. ]. Med. Sci., 254, 284 (1967). (116)Atlas Chemical Industries, Inc., Brit. Pat. 1,067,298(May 3,1967). (117)B. L. Wise, J . L. Mathis, and J. H. Wright,j. Neumsurg., 25,183 (1966). (117a)B.Becker, A. E. Kolker, and T. Krupin, Arch. Ophthalmol., 78, 147 (1967). (118)(a)R. K. Crane and P. Mandelstam, Biochim. Biophys. Acta, 45, 460 (1960). (b)R. K. Crane, ibid., 45,477(1960).(c)I. Bihler, K. A. Hawkins, and R. K. Crane, ibid., 59,94 (1963).(d) R. A. Ferrari, P. Mandelstam, and R. K. Crane, Arch. Biochem. Biophys., 80,372(1959). 86,166 , (1963). (119)A. Hernandez and A. Sois, Biochem.I. (120)F.C. Hartman and R. Barker, Biochemistry, 4, 1068 (1965).

S. SOLTZBERG

270

(K,)were found to be 4.6 X 3.8 X 1.3 x 3.0 x and 1.3 x respectively. It was concluded that the binding to the enzyme is due principally to the phosphate dissociation constants

groups. The contribution of the hydroxyl groups is not significant. Constituents of certain nonionic surfactants (SpanlZoQ 40, Span 60, Span 65, and TweenlZ0“65) were found to cause a flocculation reaction with serums containing C-reactive protein.121‘Q’ These surfactants are partial esters of fatty acids with a mixture of anhydro-D-glucitols. 1,4:3,6-Dianhydro-~-glucitol and 1,4-anhydro-~-glucitolmonostearates were found to be highly The presence of a free hydroxyl group was found to be a requisite for binding. The relatively low toxicity of the partial esters of the hexitol anhydrides makes them desirable emulsifiers for injectable medications. “D-Mannide” mono-oleate was used in a patented, single-injection, vaccine compositionlzZand in an adjuvant vaccine.Iz3 1,4:3,6-Dianhydro-~-mannitol, its 2,5-dimethyl ether, and 1,4:3,6dianhydro-2,5-dichloro-2,5-dideoxy-~-iditol were found to be inactive as adjuvants for pyrethrins in fly sprays.12*

VI. TABLES OF PROPERTIES OF THE ANHYDRIDES AND THEIRDERIVATIVES Tables VI-XI show the physical properties of the alditol anhydrides and certain of their derivatives.

(12Oa)Registeredby Atlas Chemical Industries, Inc. (121) (a) I. M . Tuomioia, P. Kajanne, and R. Junnila, Ann. Med. E x p . Biol. Fenniae (Helsinki), 39,29 (1961). (b) ibid., 39,35 (1961). (122) Wright-Fleming Institute of Microbiology, St. Mary’s, Brit. Pat. 1,081,796 (Aug. 31,1967);Chem. Abstracts, 68,35362 (1968). (123) A. F. Woodhour and T. B. Stim, U.S. Pat. 3,149,036 (Sept. 15, 1964); Chem. Abstracts, 61,13136 (1964). (124) R. W. Ken, Aust. Commonwealth Sci. Znd. Res. Organization, Bull. No. 261, 32 (1951); Chem. Abstracts, 46,2227 (1952).

TABLEVI Anhydrotehitols and Their Derivatives

M.P., Compound 1,4Anhydro-D-threitol 2,3-di-O~p-nitrobenzoyl)1,4Anhydro-~-threitol

2,3-di-O-(p-nitrobenzoyl)-

degrees 191-2 60-1 63-4 191-2

160-5"/0.17tom 144/2-3 torr

1,4AnhydIO-DL~rythntOl 2,3-sulfite 2,3-O-benzylidene2,3-di-O-(p-nitrobenzoyl)-

B.P., degrees

106-8

160-5a/0.05tom

173-4

"Bath temperature. Temperature, 24".Temperature, 20".

[alo, degrees

Rotation solvent

-115.0 -5 -4

CHCl, HzO HzO

nD

References 5 6 64 6

1.437V 1.4767'

6 64 133 134 6

$

z

$ > z

2U

EFl

10

TABLE VII

-4 10

Anhydropentitols and Their Derivatives" Compound

M.P., degrees

B.P., degrees 14516mtorr

2,3,5-hi-O-(p-nitrobenzoyl)2,3,5-tri-O-benzyl1,4Anhydro-~-arabinitol 2,3,5-tri-O-p-nitrobenzoyl-

1,4-Anhydro-~~-arabinitol* tri-0-acetyltri-0-benzoyl1,4-Anhydro-~~-ribitol 2,3,5-tri-O-benzoyl-

1,4-Anhydro-~-ribitoI 1,4-Anhydro-D~-xylitol 3,5-0-methylene-2-0methacryloyl3,5-0-benzylidene-2-0methacryloyl3,5-O-isopropylidene-2-0methacryloyl

80-1 S Y W

80-2 115-16 122-3 144-5 74-5 76.5-7 113-14 116-17 100-1 99 98-9

la],, degrees

Rotation solvent

25.3

MeOH

-85 +0.6

CHCI, CHCl,

85.1

CHCl,

nD

References

1.4917 1.4868

19 19 19 142 14a 146 146 146 10 11 10 11 14a 14b 19

77

126

138

126

57

126

v,

E 4

N W

M

T1

2,3-di-O-benzoyl-5-chloro114-15 5-deoxy3,5-0ethylidene-2-0-p86 tolylsulfonyl5-chIoro-S-deox y3,5-Oethylidene96-7 3,s-0-methylene82-3 2-0-methyl-3,5-0-methylene- 48 1,4-Anhydro-~-xylito1 1,4-Anhydro-~-xylitol (2,5-anhydro-~-xylitol) 2,3-di-O-acetyl-5-0-trityL 153 104-5 2,4-O-methylene2,4-0-methylene-5-082-3 p-tolylsulfonyl5-deox) 5-iodo-2,4-097-8 methylene5-deoxy-2,4-0-methylene64-5 3,5-Anhydro-~-xylitoI l-deoxy-2,4-O-methylene84-5 2,5-Anhydro-~-arabinitol ( 1,4-anhydro-~-lyxitol) 75-6 3,4-O-isopropylidene1,5-Anhydro-~-arabinitol 50-2 2,3,4-tri-O-acetyll,S-Anhydro-%deoxv-D-threo-pentitol 68 3,4-di-O-acetyl1,5-Anhydro-2-deoxy-~erythro-pentitol 3,4-di-O-acetyl-

16 125 16 125 23 23 80

160-1/3 tom

160-70/0.02 tom 150-5/0.2 ton

-11.2k1 10

HzO

1.4957

19 19 22 22 22 22

H20

127

-40.5

H20

17

73.6 -29.6 -38

CHCI, HzO CHCI,

30 64 64

H D Hi0

64

28.6

102/0.5 torr

l20/0.2-0.3 tom 86-9010.2 tom

64 75

64 ~~

(continued)

to

TMLE VII (continued)

M.P., Compound

degrees

1,5-Anhydro-2-deoxy-~erythro-pentitol 42-4 3,4-di-O-benzoyl89-91 3,4di-O-acetyl3,4-di-O-(p-nitrobenzoyl)117-19 1,s-Anhydro-DL-ribitol tri-0-acetyl133 1,4:3,5-Dianhydro-D~-xylitol 2-0-methyl2-0-methyl-3-0-p-tolylsulfonyl- 77

B.P., degrees

4

A

degrees

Rotation solvent

-51.4 -65.1 -47.3 -97.1

H# CHCl, CHC13 CHCl,

[ab

nD

References 128 128 128 128 136

5112 tom

1.4542

23 23

“For additional compounds, see Ref. 1 and J. Defaye, This Volume, p. 181. q h i s appears to be a case of isomerization of xylitol. The original report was unavailable, and so was not discussed under “Isomerization.”

vl v)

n

F 4 N

TABLEVIII Monoanhydrohexitols and Their Derivatives" Compound 1,5-Anhydro-D-allitol(2,6-anhydro-~-allitol) 1,5-Anhydro-~-allitol 6-amino-6-deoxy-, hydrochloride

M.P., degrees

B.P., degrees

+ 34.0

151-2 151-2 67-9

- 33.4 - 23 - 18.4

6-acetamido-tri-0-acety l-6-deoxy-

3,6-Anhydro-~-altritol( 1,4-anhydro-~-talitol) 1,2:4,5-di-O-isopropylidene4,5-O-isopropylidene1,5-Anhydro-~altritoI(2,6-anhydro-~talitol) 4,6-O-benzylidene2,3,4,6-tetra-O-benzoyl1,4-Anhydro-~-galactitol

tetra-0-benzoyl3,6Anhydro-~-galactitol I-deoxy1-deoxy-tri-0-p-tolyIsulfonyl1,5-Anhydro-~galactitol 4,6-benzeneboronate 1,5-Anhydro-2-deoxy-~-lyro-hexitol 4,6-benzeneboronate 2,6Anhydro-~-glucitol( 1,5-anhydro-~-gulitol) tetra-0-acetyl4,5-di-O-benzoyl-1,3-0-benzylidene1,3-O-ben~ylidene-~

72-610.1 tom 77-9 127-9 125-6 176-7 95-6

-37 -43

28.4 - 22.7 - 6.8

- 35.2 - 18.0

99-1Olb 87-9Zb

41.7

92.5-3 73

Rotation solvent

69

114-15 amorph. 115-8 156-SlO.03 tom

n,

References

HzO HZO H*O CHCI,

25 25 25 25

CHC1, CHCl, H@ CHCl, CHCl, EtOH H20 CHC1,

135 135 28 28 28 138 138 138 138

140 140

32.2

141-2

177-80 173-6 164-7

[&,

degrees

p-dioxane

139

60 pdioxane 7.8 20.2 EtOH 15.4 H*O 20.38 CHCI, 3.6 50.2 CHC1, -6.8 k0.3 EK3H

139 27 53 53 27 27 (continued)

c 2

? $

2 e E

2.t

4 u1

TABLE VIII (continued)

Compound

4,5-di-O-benzoyl-l,3-O-benzylidene2,6-Anhydro-~~-glucitol l,%O-benzylidene4,Mi-0-benzoyl- 1,3-O-benzylidene1,5-Anhydro-L-glucitoI (2,6-anhydro-~-gulitoI) 6-deoxy-&nitro1,5-hhydro-D-glucito1(2,6-anhydro-~-gulitol; polygalitol) hepta-O-acetyl-4-O-~-D-glucopyrnnosyl-

M.P.,

B.P.,

degrees

degrees

amorph. 168-9 176-8 177-80 136-41 164-5 141-2 115-6; 135-6O

194 172 Z-S-benzyl-2-thi0-~ 167-8 104-5 3,4,6-tri-O-acetyl-ZS-benzyl-Zthio134.5-5.5 2-C-phenyl161.5-2 3,4,6-tri-O-acetyl-2-C-phenyl142-3 tetra-0-acetyl-2-C-phenyl3,4-di-0-acetyl-2-C-phenyl-6-O-p-tolylsulfonyl- 164-5 (dec.) 176.5-7.5 tri-O-acetyl-2-C-phenyl-6-O-p-tolylsulfonyl177-8 3,4di-O-acetyl-6-deoxy-6-iodo-2-C-phenyl154-5 SO-benzyl101-2 hi-0-acetyl-3-0-benzyl144-5 tri-O-acetyl8-O-p-tolyIs~IfonyI143.5-4.5 135-5.5 2,4-O-methylene176-7 4,6-benzeneboronate 4-O-p-D-glUCopyEi1IOSyl-

N

2 degrees

Rotation solvent

-7.920.2 5.8 k0.4

HzO EtOH

27 27

-3.1 40.2

CHCI,

27

- 15.2

EtOH CHCI, HZO HzO

27 27 132 132

4.6 29.5 30.5 2% 13.9 49.3 - 2.3 58.8 21.7 48.6 34.6 15.0

CHCI, HzO MeOH CHCI, EtOH CHCl, CHCI, CHCI, CHCI, CHC1, p-dioxane pdioxane

29 29 31a 31b 37 38 38 37 37 37 51b 51b 51b

62.2 - 24.2 -80"

CHCl, H& p-dioxane

54

[a],,

e0.0 -0.0 -40.2

n,

References

54 131

v)

E 4 N W

m

8

Weoxytri-0-acetyl-6-deoxy1,3-Anhydro-~-glucitoI 5,6-di-O-methyl-2,4-0-methylene2,40-benzylidene5,6-di-O-acety1-2,4-O-benzylidene1.6-Anhydro-sglucitol ~3,4,5-di-O-benzyIidene-d l,&Anhydro-L-iditol 3,4O-isopropylidenetetra-0-acetyl1,S-Anhydm-D-mannitol(styracitol) tetra-o-methyl3,Pdi-O-meth y l3,kli-O-methyl-2,6di-O-( p-nitrobenzoy1)2-S-benzyl-2-thio-c 3,4,6-tri-O-acetyI-2-S-benzy1-2-thio1,6Anhydro-~-rnannitol 2,&Anhydro-~mannitol(1,5-anhydro-~-mannitoI) ldeoxy-l-nitrotri-0-acetyl- ldeoxy- 1 - n i b 1-amino-1-deoxy-,oxalate I-amino-ldeoxy-, oxalate, monohydrate 2,5-Anhydro-~mannitoI 1,tidichloro-1,tidideoxy1,6-dichloro-1,6-dideoxy-3,4di-O-(methyIsulfonyl)3,4di-O-acetyl-1,6-dichloro-l,6dideoxy3,4di-O-acetyl-l,6-dideoxy-

149-50 119-21 9819 109-10 121-3 81-2 137-8 163-4 129-31 124-5 69-70

29 7.7 - 1 - 55.9

MeOH CHCl,

11.0 - 20.0 - 62.0 49.1 52.5 44.1

p-dioxane H,O CHCI, H*O H2O CHCI,

80-80.5 146.5-7.5 151-1.5 79-80

- 40.2 - 16.3 - 37.6 - 12.9 14.5 34

136-7

-51.1

EtOH CHCI, CHCI, MeOH MeOH CHCI, HZO

loo c/2 tom

170-1 77-8 124-5 128-31 100-01 87.6-88 98.2-99.2

137 137 51 50 51b 51b 52 52 52 52 52 1.4521

- 52.5 -69 - 42.6 -39.5 +58.2 14.2

15.2/0.2 tom 1lo/12 torr

1.4750

15.1

MeOH

141 141 141 31a 31b 31b 52 132 132 132 132 14% 58 58 58

58 (continued)

N

TABLEVIII (continued) M.P., Compound

degrees

B.P., degrees

41

[a],,,

degrees

Rotation solvent

nD

References

3,6-Anhydro-~-mannitol(1,4-anhydro-~-mannitol) 4,5-O-isopropylidene83-4 143 2-0-acetyl-4,5-0-isopropylidene-l-O-p-tolylsulfonyl- 95-6 -23.2 MeOH 143 l-bromo-l-deoxy-4,5-O-isopropylidene115-2oC/0.01torr -42.1 MeOH 1.4970 143 l-deoxy-4,5-O-isopropylidene78-82c/0.01ton -62.9 MeOH 1.4554 143 l-deoxy-4,5-O-isopropylidene-2-O-p-tolylsulfonyl70-1 -70.1 CHCI, 143 l-deoxy-l-iodo4,5-U-isopropylidene75-6 -44.6 CHCI, 143 l-amino-l-deoxy-4,5-O-isopropylidene-36.9 EtOH 1.4610 143 1,5-Anhydro-6-deoxy-~-mannitol (1,5-anhydro-~-rhamnitol) 123-4 83.8 HZO 15 tri-0-acetyl61-2 48.1 CHCl, 15 hi-0-benzoyl169-70 279 CHCl, 15 "For additional compounds, see Ref. 1 and J. Defaye, This Volume, p. 181. *Dimorphic. =Corrected configuration; see Ref. 31(b). dRing positions not determined. Bath temperature.

?

g cl

N

m

P

Q

TABLEIX Di- and Tri-anhydrohexitols and Their Derivatives"

Compound 1,5:3,6-Dianhydro-~-glucitol 2,4-O-methylene2,5:3,6-Dianhydro-~-glucitol (p-mannide) 1,4:3,6-Dianhydro-~-glucitol 2-0-acetyl-5-O-p-toly lsulfonyl-

5-0-acetyl-2-0-p-tolylsulfonyl5-nitrate 5-nitrate, 2-O-p-toly lsulfonyl2-O-(p-bromophenylsulfonyl)-, 5-nitrate di-0-nicotinoyldi-0-benzyl-

M.p.9 degrees 150-2 79.5-80 117-19 118-19

oil

52.3 74.5-5.5 75.0-6.0 143-5 155-60/0.01tom 200-10/0.01 tom 195-200/0.01ton

I

91.5-2 54.5-6 44-4.5 72.5-3.0 55.5-7 83.5-4.5

[a],,

degrees

Rotation solvent

n,

4.1 - 44.9 94.4 94

64-5 65.5-6 64-5

bis-0-(dipropylphosphinite) bis-0-(dipropylphosphonite) bis-0-(dipropylthiophosphonite)

2-0-stearoyl5-0-stearoyl1,4:3$-Dianhydro-~-iditol 2-0-acetyl-5-deoxy-5-iodo2-O-nitro-5-O-p-tolylsulfonyl2-0-nitro2(5)-O-stearoyl1.4:3.6-Dianhvdro-~-mannitol Mononicotinate

B.P.,

degrees

50.5 79.2 77.9 83.6

29.8 75.7 26.9 47.6 64.3 35.12 50.2 100.5

References 54 54 55 1

CHCI, CHCI, CHCl, CHCI,

CHCl, CHC1,

CHCI, CHCl, CHCl,

CHCl,

1.5544 1.4910 1.4750 1.5225

58 59a 59b 59a 63 63 63 129 130 144 144 144 147 147

9

z

r 0 9

z

213 $

59b 63 63 147

_

113.5- 14.5

110

CHCI,

129 (continued)

r; (D

tQ 0

TABLEIX (continued)

Compound dinicotinab

%nitrate 2-0-nitro-90-p-tolylsulfonylbis-0-(dipropylphosphinite)

m.p., degrees

bis-0-(dipropylthiophosphonite)

2(5)-O-stearoyl1,4:2,5:3,6-Trianhydro-~mannitol

b, degrees

[ah

57.5

131-2 69.0-70.5 119-20 I55-60/0.01 tom 190-5/0.01 tom

bis-0-(dipropylphosphonite)

W

64-5 57-9 66.5-7.2 68-8.6

"For additional compounds, see Ref. 1 and J. Defaye, This Volume, p. 181.

Rotation solvent CHC1,

1.4910 1.4780

42.5 79.8 101.3 128.4

n,

HA)

References 129 63 63 144 144 144 147 58 59a

(" vl

0 r 4

N

m !a

m

Q

ALDITOL ANHYDRIDES

281

TABLEX Anhydroheptitols ~

Compound 2,6-Anhydroheptito1 l-deoxy-l-nitro5,7-O-benzylidene2,6-Anhydro-~-glyceroL-manno-heptitol penta-0-acetyll-amino-l-deoxy1-amino-1-deoxy-, hydrochloride l-acetamido-3,4,5,7tetra-0-acetylI-deoxyI-amino-1-deoxy-, p-toluenesulfonate l-deoxy-l-nitro3,4,5,7-tetra-0acetyl-l-deoxyl-nitro2,6-Anhydro-~-glyceroD-gulo-heptitol penta-0-acetyll-amino-l-deoxyl-acetamido-3,4,5,7tetra-0-acety ll-deoxy2,6-Anhydro-3-deoxy-~mnnno-heptitol tetra-o-acetyl4,5,7-tri-O-acetyI1-0-p-tolylsulfonyl2,6-Anhydro-3-deoxy-~gluco-heptitol tetra-0-acety l4,5,7-tri-O-acetyIl-0-p-tolylsulfonyl4,5,7-tri-O-acetyl1-0-( p-bromopheny1)sulfonyl-* 2,6-Anhydro-3-deoxy-~galacto-heptitol tetra-0-(p-nitrobenzoy1)-

M.P., degrees 177-7.5 211-12

[&lo, degrees

Rotation solvent

8.2 -35

HZO MeOH

59a 59a

References

121-22" 55-57 189-91(dec.)

32.6" 14.2 30.0

HzO CHCI, Hz0

59d,f 59f 59f

21 1

31.3

HzO

59f

CHCI,

59f

153-54 141-43 198.5-9.5

102-3 204-5 203-5 89 91-2 164-65

1.3 19.7 36.5

18.2 0.0 20.3 optically inactive 0.0 -0.2

59g 59g CHCI, HzO

59g 59e 59d

CHCl,

-6.7

HzO

59e 59d 59e

120

4.5

CHCll

59e

152-153 123-124

60 28

HzO Me,CO

59b 59b

30

CHCI,

59b

137-138 79-80

-1 2

Hz0 Me,CO

59b 59b

117-118

-5

CHCI,

59b

104

-10

CHCl,

59h

158-159 210-211

24 -23

H1 0 CHCla

59c 59c

oil

(continued)

282

S. SOLTZBERG TABLEX (continued) M.p.9

Compound 2,6-Anhydro-3-deoxy-~talo-heptitol 4,5-O-isopropylidene2,5-Anhydro-~-glyceroL-gluco-heptitol l-deoxy-l-nitro-

[a]D,

degrees

degrees

168 104-105

68 12

155-157

-0.5

Rotation solvent

References

HzO CHCI,

59c 59c

59g

“Hemihydrate. bCrystal structure has been determined by X-ray analysis.5* TABLE XI Anhydro-octitol ~~~~

~

~

degrees

Rotation solvent

References

220-35

70

5070EtOH

59ij

206-10”

not measurable

H,O

59k

198- 205

71

50% EtOH

59k

149-50

65

5070EtOH

59k

M.P.,

Compound 1,5-Anhydro-~-erythroD-gulacto-octitol 6-acetamido-6,8dideoxy-3,4-0isopropylidene6-amino-6,8-dideoxyN-(propylhygroyl)-, hydrochloride 6-acetamido-6,8dideoxy-3,4-0isopropylidene-7-0methyl6-acetamido-6,8dideoxy-3,4-0isopropylidene-2,7-di-Omethyl~~

“Hemihydrate.

degrees

[a]D,

ALDITOL ANHYDRIDES

283

(125)A. N. Anikeeva and S. N. Danilov, Zh. Obshch. Khim., 34,2532 (1964);Chem. Abstracts, 61,16139(1964). (126)A. N. Anikeeva and S. N. Danilov, Zh. Obshch. Kham., 34, 1063 (1964);Chem. Abstracts, 61,1929 (1964). (127)E.Zissis and N. K. Richtmyer,]. Amer. Chem. So&;75,129 (1953). (128)A. K. Bhatracharya, R. K. Ness, and H. G. Fletcher, Jr., J . Org. Chem., 28,428 (1963). (129)Aspro-Nicholas, Ltd., French Pat. M2318 (Mar. 2, 1964);Chem. Abstracts, 61, 715 (1964). (130)R. Allerton and H. G. Fletcher, Jr.,]. Amer. Chem. Soc., 76,1757(1954). (131)R.J. Ferrier,]. Chem. Soc., 2325 (1961). (132)J . C.Sowden and M.L. Oftedahl, 1.Org. Chem., 26,1974(1961). (133)J. S. Brimacombe, A. B. Foster, E. B. Hancock, W. G. Overend, and M. Stacey, J. Chem. Soc., 201 (1960). (134)N. Baggett, K.W. Buck, A. B. Foster, and J. M.Webber,]. Chem. Soc., 3401 (1965). (135)J. S. Brimacombe, M. E. Evans, A. B. Foster, and J. M.Webber, J . Chem. SOC., 2735 (1964). (136)S. Tejima, T. Maki, and M. Akagi, Chem. Pharm. Bull. (Tokyo), 12,528(1964). (137)M. Akagi, S. Tejima, and M. Haga, Chem. Pharm. Bull. (Tokyo), 11, 58 (1963). (138)R. K. Ness, H. G. Fletcher, Jr., and C. S. Hudson,]. Amer. Chem. Soc., 73,3742 (1951). (139)R.J. Ferrier, A. J. Hannaford, W. G. Overend, and B. C. Smith, Carbohyd. Res., 1,38(1965). (140)S. Akiya and A. Hamada, Yakugaku Zasshi, 78, 119 (1958);Chem. Abstracts, 52, 10892(1958). (141)E.D. M.Eades, D. H. Ball, and L. Long, Jr.,J. Org. Chem., 30,3949(1965). (142)Y. Rabinsohn and H. G. Fletcher, Jr.,]. Org. Chem., 32,3452(1967). (142a)B. C.Bera, A. B. Foster, and M.Stacey,J. Chern. Soc., 4531 (1956). (143)A. B. Foster and W. G. Overend,]. Chem. Soc., 1132 (1951). (144)K. A. Petrov, E. E. Nifant’ev, A. A. Shchegolev, and N. A. Knudyntsev, Zh. Obshch. Khim., 32,3074(1962);Chem. Abstracts, 58,11456(1963). (145)A. N. Anikeeva, T. L. Orlova, and S. N. Danilov, Zh. Obshch. Khim.,31, 3544 (1961);Chem. Abstracts, 57,9934(1962). (146)K. Anno, Nippon Nogei Kagaku Kaishi, 22, 145 (1949);Chem. Abstracts, 46, 3501 (1952). (147)L.Hartmann, Atlas Chemical Industries, Inc., unpublished data.

This Page Intentionally Left Blank

THE SUGARS OF HONEY BY I. R. SIDDIQUI Food Research Institute, Canada Department of Agriculture, O t t a w a , Ontario, Canada

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

285

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 ...................... ................................ 293 2. Granulation . . . . . . . . . . . . . . . . . . . . 111. Honey Oligosaccharides . . . . . . .............. . . . . . . . . . .295 1. Composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 2. Origin . . . . . . . .......................... 298 IV. Honey Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 V. Honeydew . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

I. INTRODUCTION “And thy Lord inspired the bee, saying: choose thou habitations in the hills and in the trees and in that which they thatch; then eat of all fruits, and follow the ways of thy Lord, made smooth (for thee). There cometh forth from their bellies a drink of diverse hues, wherein is healing for mankind.”’ This is by no means the oldest account of honey, but it is undoubtedly an authentic one, put forth 1,400 years ago. The oldest evidence of the association of man with honey dates back to the Stone Age, about 15,000 years ago, by the discovery of a prehistoric, Spanish rock-painting. From prehistoric days to the dawn of recorded history, as honey was the only concentrated sweetener available to man to satisfy his many needs, it was destined to occupy an exalted position in his art, legend, literature, medicine, mythology, and religion. The latest treatise on the biology of the honeybee2 has an entire volume devoted to history, ethnology, and folklore. It is full of amusing and little-known details, and is undoubtedly a very

(1) M. M . Pickthall, “The Meaning of the Glorious Koran,” Allen and Unwin, London, 1953, p. 199. (2) “Treatise on the Biology of the Honeybee,” R. Chauvin, ed., Masson and Co., Pans, 1968. 285

286

I. R. SIDDIQUI

interesting extension of the classical works of B e ~ s l e rB, ~~ d e n h e i m e r , ~ F r a ~ e rand , ~ Ransome.6 If the ancients regarded honey as an elixir of life, the folk medicine7-*of our time has proclaimed it to be a universal cure. Medical science has substantiated some of these claims, and has enumerated The - ~ aforementioned ~ treatise on other medicinal uses of h ~ n e y . ~ the biology of the honeybee2 has a considerable portion of Volume 3 devoted to the therapeutic aspects of hive products, including honey. The use of honey in athletic nutrition is also documented. Lloyd Percival of Sports College in Canada, from the results of a four-year study, has no hesitation in recommending honey for athletes and those interested in maintaining a high level of energy.29White lists a number of other sports30wherein honey has been used with beneficial results. There are constant reports in the bee literature advo(3) J. G. Bessler, “Geschichte der Bienenzucht,” Kohlhammer, Stuttgart, 1886. (4)F. S. Bodenheimer, “Materialen zur Geschichte der Entomologie bis Linne,” Junk, La Haye, 1928. (5)J. G. Frazer, “Totemism and Exogamy,” Macmillan, London, 1910. (6)H. M. Ransome, “The Sacred Bee,” Allen and Unwin, London, 1937. (7) D. C. Jarvis, “Folk Medicine,” Fawcett Publications, Inc., Greenwich, Conn., 1958. (8) D. C. Jarvis, “Arthritis and Folk Medicine,” Fawcett Publications, Inc., Greenwich, Conn., 1960. (9)R. Moreaux, Rev. Pathol. Comparke H y g . Gkn.,51,59(1951). (10)F. J. Pothmann, Z. Hyg. Znfektionskrankh., 130,468(1950). (11)A.C. Mikhailov, Pchelooodstoo, 2,117(1950). (12)W.Blechschmidt, Med. Monatsschr., 4,506(1950). (13)P. Beckmann, Deut. Med. Wochschr.,75,426(1950). (14)E.Hiller, Med. Monatsschr., 5,626(1951). (15)R. Chauvin, Reu. Fr. Apicult., 11,619(1951). (16)H. Stadler, Deut. Z. Verdaungs. Stoffwechselkrankh.,12,108(1952). (17)G. K.Osaulko, Vestn.Oftalmol., 32,25(1953). (18)K.Fochem, Strahlentherapie, 93,466(1954). (19)0. Martensen-Larsen, Brit. Med.].,4885,468(1954). (20)M.Pestel, Proc. Med. Gaz. Apicult., 56,63(1955). (21)M.W.Bulman, Middlesex Hosp.].,55,63(1955). (22) N.P. Ioirish, Pchelouodstuo, 33,50(1956). (23) K.Von Am, Praxis (Bern), 46,717 (1957). (24)N. Rubin, A. R. Gennaro, C. N. Sideri, and A. Osol, Amer. J. Pharm., 131, 246 ( 1959). (25)F. Alison and R. Narbouton, Nutr. Dieta, 3,33(1959). (26)V. I. Maksimenko, Pchelouodstuo, 37,49(1960). (27)F. Baungarten, Deut. Bienenwirtsch., 11,137(1960). (28)R. Lyonnet and R. Berthelier, Lyon Med., 38,505(1961). (29)L. Percival, Amer. BeeJ.,95,390(1955). (30) J. W.White, Jr., in “The Hive and the Honey Bee,” R. A. Grout, ed., Dadant and Sons, Inc., Hamilton, Illinois, 1963,p. 369.

THE SUGARS OF HONEY

287

cating honey for diabetics; such claims are unfounded. It is, however, possible that honey is a lesser evil to the diabetic than table sugar (sucrose), but surely, because on the average its content of D-glucose is -3070,the use of honey by diabetics cannot be freely advocated. The U. S. Federal Food and Drug Act of 1906 defined honey as “The nectar and saccharine exudation of plants, gathered, modified and stored in the comb by honey bees (Apis mellifera and Apis dorsata) is levorotatory; contains not more than 25% water, not more than 0.25% ash and not more than 8% sucrose.” According to White30 and Feinberg,31 these figures are unrealistic; the values for moisture and sucrose are too high, and the recommended values for ash are far too low. The Canadian Food and Drug Act and Regulations state that honey should be derived entirely from the nectar of flowers and other sweet exudations of plants by the work of the bees, and should not contain more than 20% of moisture, 8% of sucrose, and 0.25% of ash, and should contain not less than 60% of invert sugar. In most European countries, honey is defined in similar terms. However, certain quality factors considered by Europeans, especially the Germans, of importance in the marketing of honey are the levels of the enzymes invertase and diastase, and of 5-(hydroxymethyl)-2furaldehyde. White has discussed these requirements in relation to suggested standards for the Codex A l i m e n t a r i u ~ The . ~ ~ German insistence on these requirements is outlined in a volume of A p i a ~ t a . ~ ~ The Codex Alimentarius Commission, in its Draft Provisional Standard34 (following definition and description, and subsidiary definitions and designations) has laid down certain compositional criteria. It is obvious that all definitions of honey so far discussed deal with two chemically and physically distinct commodities: namely, nectar honey and honeydew honey. A comparison between the two shows that honeydew honey is lower in D-fruCtOSe and D-glucose and higher in pH, oligosaccharides, acidic components, ash, and nitrogen than nectar honey. A distinct feature of honeydew honey is the trisaccharide melezitose, which has been identified in the exudate manna of the Douglas fir; the European larch, and the North American Jack or scrub pine. According to the late Professor C. S. Hudson, Turkestan manna contains 20-38% of melezitose, and Douglas-fir manna, 5070 of melezitose. At one time, melezitose formed 20-30% of the total (31) B. Feinberg, Amer. BeeJ.,91,471 (1951). (32) J. W. White, Jr., Amer. Bee]., 107, 374 (1967). (33) H. Duisberg, Apiactn, 3,26 (1967). (34) Joint FAO/WHO Food Standards Program, Codex Alimentarius Commission Draft Provisional Standardfor Honey, March, 1968.

288

I. R. SIDDIQUI

sugars of Maryland and Pennsylvania honeys that had been collected during a period of drought when nectar was not readily a ~ a i l a b l e . ~ ~ . ~ Regarding the origin of melezitose, it has been stated that the sugar arises by the action of an aphid enzyme on plant-sap su~rose.~’ It is therefore obvious that bees could not be regarded as the sole producers of honeydew honey. For this reason, and because nectar honey is a commodity that is traded internationally, it will be advisable to treat it as an entity separate from honeydew honey. Further distinctions between floral or nectar honeys, as set forth in the Codex Standard, are based on mixed floral and unifloral sources of nectars, and are too sophisticated to be practical. Such limits as 65 5% for the invert sugar and similar limits for other variables, to embrace all or most nectar honeys, would constitute better criteria. The terms “reducing sugar as invert sugar” and “apparent sucrose,” used in the Codex draft, are ambiguous and misleading. The characteristic to be given should be either (a) total reducing sugar, which includes D-glucose, D-fructose, and the reducing oligosaccharides, or (b) invert sugar, which represents merely D-glucose and D-fruCtOSe. Similarly, “apparent sucrose” should be modified to “nonreducing oligosaccharides,” in view of the fact that the Codex methods currently in use measure (a) total reducing sugar and (b) nonreducing oligosaccharides (by difference, after hydrolysis). If such values as percentage of invert sugar, true sucrose, and other reducing and nonreducing oligosaccharides, some of which are present in honey in proportions greater than s u c r o ~ e ,are ~ ~ to * ~be ~ measured, resort will have to be made to chromatographic methods. In this connection, adoption of a paper-chromatographic procedure, followed by quantitative elution and colorimetric determination of the components, will have a better chance of wide acceptance than a more sophisticated, elaborate, and expensive gas-phase chromatographic procedure. Most of us know that bees are busy little creatures, but how busy and persistent they are is apparent from the estimate that a worker bee gathers, from one flower, a minimum of 10 micrograms of nectar and brings back after one flight a total of 50 milligrams, yielding approximately 20 milligrams of honey; to produce a kilogram, the bees would have to fly approximately 250,000 miles, or the equivalent _+

(35) C. S. Hudson and S. F. Sherwood,]. Amer. Chem. SOC., 40,1456 (1918). (36) C . S. Hudson and S. F. Sherwood,j. Amer. Chem. SOC., 42,116 (1920). (37) J. S. D. Bacon and B. Dickinson, Biochem.J.,66,289 (1957). (38) I. R. Siddiqui and B. Furgala,.!. Apicult. Res., 6, 139 (1967). (39) I. R. Siddiqui and B. Furgala,J.ApicuEt. Res., 7,51(1968).

THE SUGARS OF HONEY

289

of a flight to the It is also estimated that the present worldproduction of honey is 500,000 tons, produced by about 5 million beekeepers from 40-50 million colonies comprised of 1.5 billion bees .41 11. HONEYMONOSACCHARIDES

1. Composition and Analysis In the production of honey, the foraging bees carry the nectar in their honey sac, mix it with enzyme-rich secretions from their hypopharyngeal glands, carry it to the hive, and pass it over to the house bees. The house bees, in turn, transmit it among themselves, and carry out the process of ripening by mixing it with further amounts of glandular secretions and by removing water. The process of ripening is continued until the raw material loses about 50% of its water content; subsequent ripening takes place automatically in the cells of the comb, in the stream of dry air that constitutes the ventilation system of the hive. When a moisture content of 20% is reached, the cells containing the honey are capped by the bees.42a43 During the whole process of ripening, and afterwards in storage, honey undergoes very intricate enzymic reactions about which very little is known. However, two fundamental steps in the manufacture of honey are the removal of water and the inversion of the sucrose contained in the nectar. Honey, therefore, consists of a concentrated solution of two monosaccharides, D-glUCOSe and D-fructose, and it is correct to state that these sugars constitute, in the free and the combined form, -95% of the honey solids. The ratio of D-glucose to D-fructose is characteristic of certain types of honey. In mixed, floral honeys, they are present in almost equal proportions. On the other hand, unifloral honeys contain appreciably more D-fructose than D-glucose. Examples of honeys rich in D-fructose are Robinia, Salvia, Tupelo, and Sweet chestnut; honeys rich in D-glucose are rarer, examples being those from dandeA monosaccharide derivative that occurs in honey to lion and rape.44*45 (40) I. Khalifman, “Bees,” Foreign Languages Publishing House, Moscow (1953). (41) E. Crane, in Ref. 30, p. 18. (42) 0. W. Park,]. Econ. Entomol., 26, 188 (1933). (43) J . F. Reinhardt,J. Econ. Entomol., 32,654 (1939). (44) A. Maurizio, Bee World,40,275 (1959). (45) J . W. White, Jr., M . L. Riethof, M. H. Subers, and I. Kushnir, US.Dept. Agr. Tech. Bull. No. 1261, (1962).

290

I. R. SIDDIQUI

an appreciable extent is D-gluconic acid; inorganic and organic acids, including amino acids, are also present, and D-gluconic acid con~ . ~formation ' in honey is stitutes 70-90% of the organic a ~ i d s . ~ Its ascribed to the presence of a D-gluCOSe oxidase that produces Dgluconic acid and hydrogen peroxide from D-glUCOSe.4s Inhibine, the antibacterial agent reported in honey, is now recognized to be hydrogen peroxide. The presence of traces of D-galaCtOSe was reported in one instance,49but, in the author's opinion, this was merely an attempt to justify the frequent reports of the occurrence of raffinose in honey. Analysis of honey on this continent dates back to 1892, when 500 commercial samples of honey were analyzed.50 The analytical methods used were, with certain modifications, employed over the years by several worker^,^'-^^ until a critical study of the methods of sugar analysis was made by White and coworkers.55 Following the development of carbon-Celite column chromatogr a p h ~ and , ~ ~its application to sugar analysis of honey by White and collaborators, a new procedure for the analysis of honey emerged and was termed the selective-adsorption method. Application of this technique resulted in the recognition of the presence in honey of reducing disaccharides and higher oligosaccharides, and more accurate values for the content of D-gluCOSe and D-fructose were ~ b t a i n e d . ~ ~This + l method was also used in Canada,6z Chile,63and (46)E. E.Stinson, M. H. Subers, J. Petty, and J. W. White, Jr., Arch. Biochem. Biophys., 89,6(1960). (47)S. Maeda, A. Mukai, N. Kosugi, and Y. Okada, J . Food Sci. Technol. (Japan), 9, 270 (1962). (48)J. W. White, Jr., M. H. Subers, and A. I. Schepartz, Biochim. Biophys. Acta, 73, 57 (1963). (49)S. Goldschmidt and H. Burkert, Z. Physiol. Chem., 300,188(1955). (50)H.W. Wiley, U . S. Diu. Chem. Bull., 13,pt. 6,744(1892). (51)C.A. Browne, U . S . Bur. Chem. Bull., llO(1908). (52)R. E.Lothrope and R. L. Holmes, lnd. Eng. Chem.,Anal. Ed., 3,334(1931). (53)J. E. Eckert and H. W. Allinger, Caltf.Agr. E x p . Sta. Bull., 631 (1939). (54)J. A. Elegood and L. Fischer, Food Res., 5,559(1940). (55)J. W. White, Jr., C. Ricciuti, and J. Maher, J . Assoc. Ofic.Agr. Chemists, 35, 859 (1952). (56)R.L.Whistler and D. F. Durso,J. Amer. Chem. SOC.,72,677(1950). (57)J.W. White, Jr., and J. Maher,]. Assoc. Ofic. Agr. Chemists, 37,466(1954). (58)J. W. White, Jr., and J. Maher,]. Assoc. Ofic. Agr. Chemists, 37,478(1954). (59)J. W. White, Jr.,J. Assoc. Ofic. Agr. Chemists, 40,326(1957). (60)J. W. White, Jr.,J.Assoc.Ofic. Agr. Chemists, 42,341(1959). (61)R. A. Osborn, M. Oakley, and K. L. Milstead, J . Assoc. Ofic. Agr. Chemists, 42, 26 (1959). (62)G. H. Austin, Proc. Intern. Congr. Entomol. IOth, Montreal, 1956,4,1001(1958). (63)D.A. Bravar, An. Tac. Quim. Farmac., 9,149(1958).

THE SUGARS OF HONEY

291

South The average composition of 490 samples of honey, with respect to sugar constituents and moisture and their range of values, as determined by White and collaborator^,^^ is given in Table I. The values given in Table I for maltose and sucrose do not TABLEI Average Composition of Honey from 490 Samples45 Component

Percentage

Standard deviation

Range

Moisture D-Fructose D-Glucose Sucrose Maltose Higher sugars Undetermined

17.2 38.19 31.28 1.31 7.31 1.5 3.1

1.46 2.07 3.03 0.95 2.09 1.03 1.97

13.4 -22.9 27.25-44.26 22.03-40.75 0.25- 7.57 2.74-15.98 0.13-13.2 0.0 -13.2

represent the true content of these sugars. Sucrose values are interfered with by other ketose disaccharides, including turanose, and maltose values are subject to interference by other reducing disaccharides, such as kojibiose and n i g e r o ~ e . ~ ~ In recent years, it has become fashionable to determine the composition of honey, and in the current literature are many reports dealing with the compositional aspects of honey from various parts of the w ~ r l d . ~ It ~ -is, ' ~however, regrettable that application of paper chromatography, which has played an outstanding role in the development of carbohydrate chemistry, has not yet been fully explored in (64) R. H. Anderson and L. S. Perold, S. Afr.1. Agr. Sci., 365 (1964). (65) C. Pelimon and H. Baculinschi, An. Inst. Cercet. Zooteh. (Bucharest), 13, 621 (1955). (66) A. K. Mallick, J . Indian Inst. Chem., 30, 171 (1958). (67) Y. Arai, K. Akiyama, S. Sakai, M. Doguchi, N. Suzuki, and S. Ogawa, l a p . B e e l . , 13, 101 (1960). (68) T. Watanabe, Y. Motomura, and K. Aso, TohokuJ.Agr. Res., 12,187 (1961). (69) J. Pourtallier, Bull. Apic. Inform. Doc. Sci. Tech.,5,138 (1962). (70) C. H. W. Flechtmann, C. F. Caldas Felho, E. Amaral, and J. D. P. Arzolla, Bol. Ind. Animal. (Sao Paulo), 21,65 (1963). (71) V. G. Chudakov, Pchelooodstoo, 40,18 (1963). (72) S. Aoyagi, K. Fudeya, and S. Takeshima, Tamagawa Daigaku Nogakutsu Kenkyu Hokoku, 8, No. 7,181 (1968). (73) Y. Okimoto, K. Toshida, M . Yamazaki, and H. Fujioka, Tamagawa Daigaku Nogakutsu Kenkyu Hokoku, 8, No. 7,184 (1968).

292

I. R. SIDDIQUI

connection with the quantitative analysis of sugars of honey. In this laboratory, the analysis of a large number of honey samples from various parts of Canada was undertaken, in order to standardize Canadian honey composition.73aThe sugars were separated by paper chromatography, quantitatively eluted, and determined spectrophotometrically by the Nelson-Somogyi m e t h ~ d . ? ~The - ~ ~average composition of 95 Canadian honeys is given in Table 11. TABLEI1 Average Composition of Honey from 95 Samples73o Component Moisture D-Fructose D-Glucose Oligosaccharides Undetermined

Percentage

Range

17.9 37.1 33.7 7.4 3.9

15.0-21.8 3 1.1-4 1.4 28.5-40.7 2.2-15.2 0.0-10.8

Of the 95 samples examined, 4.2% had monosaccharides below 65%; 32.6%, between 65 and 70%; and 63.296, above 70%. It is not intended to imply that this method of analysis is the only one worthy

of consideration; other colorimetric methods in conjunction with paper chromatography will be as useful. What is implied is that simple, paper-chromatographic separations, followed by colorimetric determinations, provide results equally as good as, if not better than, those afforded by other available methods. Moreover, the chromatographic methods are easy to handle and less time-consuming, and do not require complicated instrumentation. However, paper chromatography has limitations; for example, its lack of resolution between oligosaccharides (such as sucrose from turanose, maltose from other disaccharides, and melezitose from other trisaccharides) does not permit determination of important individual components. Such problems can be resolved by the application of paper electrophoresis, thin-layer chromatography, or gas-liquid chromatography. The use of thin-layer chromatography for the determination of sugars in honey has been p r ~ p o s e d , and ~ ' the potential value of gas(73a)I. R. Siddiqui and associates, unpublished results. (74) N. Nelson,]. B i d . Chem., 153,375 (1944). (75) M . Somogyi,]. Biol.Chem., 160,61(1945). (76) M . Somogyi,]. Biol.Chem., 195,19(1952). (77) J. Pourtallier, Bull. Apic. Inform. Doc. Sci. Tech., 7,197 (1964).

THE SUGARS OF HONEY

293

liquid chromatography in the quantitative analysis of the sugars of honey has been demonstrated. Experiments testing the reproducibility of the results were conducted with one sample of honeydew honey from a coniferous tree. In four independent determinations, the following results were obtained: D-fructose, 36.7; 37.0; 36.8; and 37.2%; and D-glucose, 27.8, 28.0, 27.5, and 28.0%. The sugars of honey were transformed into their volatile per(trimethylsily1) derivatives, and these were injected into a gas chromatograph. The chromatography was conducted with a column (10 ft. X 0.125 in.) of 4% SE 52 on Chromosorb W (100-120 mesh) at a nitrogen flow-rate of 30 ml/min, with temperature programming. Good separations of D-glucose from D-fructose were obtained. The retention times for the D-fructose and P-D-glucose derivatives, with reference to that of a-D-glUCOSe, were 0.73 and 1.51, re~pectively.'~ The resolution of oligosaccharides is, however, far from optimal and is, indeed, a challenging problem because of their diversity and complexity. Perhaps, further experimentation, with different column packings and manipulation of conditions of analysis, would lead to a solution of the problem. The present challenge is to determine, quantitatively, most, if not all, of the sugars in honeys, and not merely to determine the content of D-glucose and D-frUCtOSe b y yet another method.

2. Granulation Granulation of honey has been a problem to the honey producer for a long time. A fully crystalline pack is acceptable, but a clear, liquid pack is more appealing to the eye of the consumer and lends itself more readily to packaging in the little containers that have become popular in recent years. Partially crystalline packs containing much larger crystals constitute a real problem; such packs sometimes have to be redissolved and repacked, resulting in additional costs. The quest for methods for keeping honey in the liquid state for reasonable periods of time has demanded the attention of honey specialists for a long time. Several indexes have been proposed for describing the tendency of honey to granulate, in order to provide a basis for blending honeys for packing in the liquid form. Among these, the L/D ratio (where L denotes levulose and D, dextrose) was, for a long time, considered a criterion of the crystallization (78) J. Pourtallier, Z . Bienenforsch.,9,217(1968).

294

I . R. SIDDIQUI

potential of honey; high values are indicative of liquid, or slowgranulating, honey. From a study of the solubility of pure D-glucose in the presence of D-fructose, sucrose, and a combination of the two, Jackson and Silsbee79proposed two indices, namely, the supersaturation coefficients and the (D-W)/Lratio, where W denotes water. Later, on the basis of a comparison of L/D ratios and supersaturation coefficients, Austin62 concluded that the supersaturation coefficient is a more logical index, but that it is not so good as the D/W ratio; he pointed out that the latter was simpler to determine, since it could be calculated from only two variables. He also suggested that, when honeys are compared by this method, their composition should be calculated on the basis of equivalent moisture-content. White and have shown that DlW ratios, not adjusted to a common moisture-content, show the most closely related relationship to the granulating tendency than does any other index. DlW ratios of 1.7 or lower indicate nongranulating honey, and values of 2.1 and higher predict occurrence of rapid granulation. In the writer’s laboratory, results obtained from the analysis of a large number of samples of Canadian honey by paper chromatography were expressed in these ratios; namely, L/D, (D-W)/L, and DlW, and these were compared with the granulation rate observed for each sample during 6 months by the method of White and coworkers.45 For the Canadian honeys thus examined, there was no discernible relationship between the rate of granulation and any of the indexes used. The purpose of a granulating index is to relate the composition of honey to its granulating tendency, in order that such behavior may be predicted; such predictions were, of course, not possible, because the factor actually involved is the presence or absence of appropriate crystal nuclei. In conformity with this conclusion are the results on the crystallization of honey published in a recent bookeofrom the Ministry of Agriculture in Greece. In relating crystallization to chemical composition, the authpr proposed yet another index. This was, however, a nongranulating index B-D/D, where B represents degrees Brix and D represents the content of D-glucose. As could have been predicted, the indexes L/D, (D-W)/L, and D/W were not useful in this connection. (79) R. F. Jackson and C. G. Silsbee, Nut. Bur. Stand. (U.S . ) Technol. Pap., No. 259, 18,277 (1924). (80) M. I. Kodoyne, “The Crystallization of Honey,” Ministry of Agriculture, Athens, Greece (1962).

THE SUGARS OF HONEY

295

111. HONEYOLIGOSACCHARIDES 1. Composition

For a long time, honey was considered to be a mixture of D-glucose, D-fructose, and sucrose. Although the presence of maltose in some honeys was recognized 45 years ago,81it remained for Van VoorstR2 and Hurd and coworkersR3to show that it is probably a component of all honeys. Recognition of the presence of other oligosaccharides (for systematic names, see Table 111) was only a matter of time, and was made possible by the development of paper chromatography and its application to sugar analysis. By using paper chromatography, KeupS4demonstrated the presence of as many as 15 components, corresponding, among others, to maltose, turanose, isomaltose, erlose, kestose, raffinose, and melibiose. After fractionation of honey by carbon-column chromatography and paper chromatography, Aso and coworkersE5detected 22 components, 15 of which were classified as ketoses. Other reports dealing with the paper-chromatographic identification of honey oligosaccharides are those of Goldschmidt and B ~ r k e r t , 4 ~ Po~rtallier,~ Flechtmann ~ and coworkers,70 and Curylo.H6Attempts to identify the oligosaccharides of honey by methods other than chromatography were limited by the difficulties encountered in the isolation of these compounds in the pure form. An attempt to isolate and identify some of the disaccharides and their octaacetates by infrared spectroscopy was made by White and HobanR7by comparison of the spectra with those of authentic samples of disaccharides and their derived octaacetates. Fractionation of honey by carbon-Celite chromatography, and analysis by gas-liquid chromatography of the per(trimethylsilyl) derivatives of the fractions eluted with water, and 2.5%, 5%, and 10% aqueous ethanol, resulted in the detection of a glucose, a fructose, sucrose, turanose, maltulose, leucrose, kojibiose, nigerose, maltose, isomaltose, melezitose, and two undetermined components.s8 However, (81) E. Elser, Mitt.Geb. Lebensmittelunters. Hyg., 25, 92 (1924). (82) F. T. Van Voorst, Chem. Weekbl.,38,538 (1941). (83) C. D. Hurd, D. T. Englis, W. A. Bonner, and M . A. Rogers, J . Amer. Chem. SOC., 66,2015 (1944). (84) N. Keup, Inst. Grand-Ducal Luxembourg, Sect. Sci. Nut. Phys. Math. Arch., 24, 91 (1957). (85) K. Aso, T. Watanabe, and K. Yamau, Hakko Kogaku Zasshi, 36,39 (1958). (86) J . Curylo, Pszczel. Zeszyty Nauk., 6,1(1962). (87) J. W. White, Jr., and N . Hoban, Arch. Biochem. Biophys., 80,386 (1959). (88) J . Matsuyama, G. Fudeya, T. Ishida, and T. Echigo, Tamagawa Daigaku Nogakubu Kenkyu Hokoku, 8, No. 7, 188 (1968).

I. R. SIDDIQUI

296

TABLEI11 Glossary of Relevant Oligosaccharides Trivial name Centose Erlose Gentiobiose Isomaltose Isomaltotriose Isomaltotetraose

Isomaltopentaose

Isomaltulose Isopanose I-Kestose Kojibiose Laminarabiose Leucrose Maltose Maltotriose Maltulose Melibiose Nigerose Panose Raffinose Sucrose Theanderose a$-Trehalose Turanose (I

n

a

‘No recognized trivial name.

Systematic name O-a-D-GlUCOpyranOSyl-(1+4)-0-[a-~glucopyranosyl-(1+2)1-D-glucopyranose O-CZ-D-GI ucopyranosyl-( 1+ 4)-a-~-glucopyranosyl j3-D-fructofuranoside O-P-D-Glucopyranosyl-(1+ 6)-D-glucopyranose O-a-D-Clucopyranosyl-(1+ 6)-D-glucopyranose 0-a-D-Glucopyranosyl-(1+ 6)-O-a-D-glucopyranosy~(1+6)-D-glucopyranose O-a-D-Glucopyranosyl-(1+ 6)-O-a-D-glucopyranosyl(1 +6)-O-a-~-glucopyranosyl-( 1 6)-~glucopyranose O-a-D-Ghcopyranosyl-(1+ 6)-O-a-D-glucopyranosyl(1+6)-O-a-~-glucopyranosyl-(1 4 6 ) - 0 - a - ~ gIucopyranosyl-(1-+6)-~-glucopyranose O-a-D-Glucopyranosyl-(1+ 6)-D-fmctofuranose 0-a-D-Glucopyranosyl-(1+ 4)-O-a-D-glucopyranosyl(1+6)-D-glucopyranose O-cY-D-Glucopyranosy1-(1+2)-j3-D-fructofuranosyl(1+2) j3-D-fructofuranoside O-a-D-Glucopyranosyl-(1+ 2)-D-glucopyranose O-j3-D-Glucopyranosyl-(1+ 3)-D-glucopyranose O-a-D-Glucopyranosyl-(1+5)-D-fructopyranose O-a-D-Ghcopyranosyl-(1+4)-~-glucopyranose O-a-D-Glucopyranosyl-(1+ 4)-O-a-D-glucopyranosyl(1+4)-D-glucopyranose 0-a-D-Glucopyranosyl-(1+4)-D-fructose 0-a-D-Galactopyranosyl-( 1+ 6)-D-glucopyranose O-a-D-GlucopyranosyI-(1+ 3)-D-glUCOpyrWOSe 0-a-D-Glucopyranosyl-(1+ 6)-O-a-D-glucopyranosyl(1-+4)-D-glucopyranose 0-a-D-Galaclopyranosyl-(1+6)-0-a-~glucopyranosyl-(1+2) j3-D-fructofuranoside a-D-Glucopyranosyl j3-D-fructofuranoside 0-a-D-Glucopyranosyl-(1+ 6)-a-D-glucopyranosyl j3-D-fructofuranoside a-D-Glucopyranosyl P-D-glucopyranoside O-a-D-Glucopyranosyl-(1 3)-D-fructose O-a-D-Glucopyranosyl-(1+ 6)-O-a-~-glucopyranosyl(1-B 8)-D-glucopyranose O-B-D-Glucopyranosyl-(1+ 6)-O-a-D-glucopyranosyl(1+4)-D-g~ucopyranose 1-O-a-bglucopyranosyl-D-fructose -+

THE SUGARS OF HONEY

297

the first unequivocal identification of four disaccharides, namely, kojibiose, nigerose, maltose, and isomaltose, in honey -as their crys~ ~ work talline P-octaacetates -was made by Watanabe and A s o . This was made possible by the development of Magnesol-Celite column chromatography by Wolfrom and coworkers.90 Following these reports, a large-scale fractionation was undertaken by Siddiqui and F ~ r g a l a ~on * , a~ carbon-Celite ~ column; they used an enriched oligosaccharide fraction, isolated from honey by using a batch operation employing carbon-Celite as the adsorbent. The fractions eluted with 2.515% aqueous ethanol were further separated by paper chromatography, occasionally by paper electroph~resis,~' and, frequently, by thin-layer c h r o m a t ~ g r a p h y after , ~ ~ acetylation. These fractionation procedures resulted in the characterization of at least 24 oligosaccharides. The oligosaccharides maltose, kojibiose, isomaltose, nigerose, a$-trehalose, gentiobiose, laminarabiose, melezitose, O-a-D-glucopyranosyl-( l+6)-O-a-D-g~ucopyranosyl-(1-4)D-glucopyranose, and maltotriose were identified as crystalline P-octa- and hendeca-acetates. Sucrose, turanose, l-kestose, and panose were identified as the crystalline sugars, maltulose as the crystalline phenylosazone, and isomaltotriose as its crystalline P-hendecabenzoate. Erlose and theanderose were identified by the isolation and characterization of the disaccharides derived by partial hydrolysis with acid or an enzyme. Isopanose, isomaltotetraose, and isomaltopentaose were identified b y their specific optical rotations and behavior on hydrolysis with acid or an enzyme. The disaccharides isomahlose and l-O-a-D-glucOpyranOSyl-D-fructose were tentatively identified. Evidence was also presented for the presence in honey of two new trisaccharides, namely centoseS3{O-a-D-glucopyranosyl(1+4)-O-[a-D-glucopyranosyl-( 1-+2)]-D-glucopyranose} and 0-p-Dglucopyranosyl-(1+6)-O-a-~-glucopyranosyl-(i ~ ~ ) - D - g ~ u c o p y r a n o s e . A synthesis of centose s3a was presented during the preparation of this review. The approximate proportions of these sugars in the oligosaccharide fraction (3.65%) of the honey were: maltose, 29.4; kojibiose, 8.2; turanose, 4.7; isomaltose, 4.4; sucrose, 3.9; ketose band (mixture (89) T. Watanabe and K. Aso, TohokuJ.Agr. Res., 11,109 (1960). (90) W. H. McNeely, W. W. Binkley, and M. L. Wolfrom,]. Amer. Chem. Soc., 67,527 (1945). (91) A. €3. Foster,]. Chem. SOC., 982 (1953). (92) M . E. Tate and C. T. Bishop, Can.].Chem.,40,1043 (1962). (93) I. R. Siddiqui and B. Furgala, Carbohyd. Res., 6,250 (1968). (93a)B. H. Koeppen, Carhohyd. Res., 13,417 (1970).

298

I. R. SIDDIQUI

of at least three ketoses, including maltulose and isomaltulose), 3.1; nigerose, 1.7; a,p-trehalose, 1.1;gentiobiose, 0.4; laminarabiose, 0.09; erlose, 4.5; theanderose, 2.7; panose, 2.5; maltotriose, 1.9; 1-kestose, 0.9; isomaltotriose, 0.6; isomaltotetraose, 0.33; melezitose, 0.3; isopanose, 0.24; isomaltopentaose, 0.16; centose, 0.05%; O - ~ - D glucopyranosyl-(1+6)-O-a-D-glucopyranosyl-( 1+3)-D-glucopyranose, minute proportion; and O-p-D-glucopyranosyl-(1+6)-O-a-D-glucopyranosyl-(1+4)-D-glucopyranose, minute proportion. This extensive analysis also revealed certain features of composition that were at variance with the results of other workers. Although Watanabe and Asos9 detected leucrose in their sample of honey by paper electrophoresis, its presence could not be confirmed by Siddiqui and F ~ r g a l a . On ~ ” ~the ~ other hand, although the former workers searched for trehalose, they failed to detect its presence. Another point of difference from the results in the literature was uncovered in the reported presence of raffinose in a variety of honeydew and nectar honeys.49.70.77.84*H6 It was found that 0-a-D-glucopyran0syl-(1+6)-a-D-glucopyranosyl p-D-fructofuranoside, assigned the trivial name theanderose, had been confused with raffinose owing to the identical behavior of the two sugars in paper chromatography and toward ketose spray-reagents. This clarification further demonstrated that paper-chromatographic methods alone are insufficient to warrant such conclusions. The confusion would not have arisen had an attempt been made to confirm the supposed presence of raffinose by paper electrophoresis, since the M G values (0.18 for theanderose and 0.31 for raffinose) are sufficiently different to have clarified the situation. 2. Origin

The answer to the biochemical origin of honey oligosaccharides lies in recent developments in the field of sugar and enzyme chemistry. It is obvious that, in the formation of these saccharides, both trans-D-glucosylation and trans-D-fructosylation occur. In the former process, D-glucopyranosyl groups are transferred from a D-glucopyranosyl donor to an acceptor molecule, which may be a mono-, oligo-, or poIy-saccharide. In the latter process, D-fructofuranosyl groups are transferred by a D-fructofuranosyl-transferring enzyme to other sugars, giving D-fructose-containing oligosaccharides. These transfer reactions probably occur through the formation of a Dfructofuranosyl-enzyme or D-g~ucopyranosy~-enzyme complex, but there is at present no direct evidence for the formation of such

299

THE SUGARS OF HONEY

complexes. A simple scheme for trans-D-fructosylation has been postulated as follows.94 D-Fruf-D-Gp

+E

+

~-Frnf-E D-Fruf-D-Gp D-Fruf-E

Fruf-E

+ D-Gp

D-Fruf-Fruf-D-Gp

+E

+ D-Fruf-D-Fruf-D-Gp

+

D-Fruf-D-Fruf-D-Fruf-D-Gp E, and so on,

where D-Fruf= D-fructofuranosyl, E =the enzyme, and D-Gp= D-glucopyranosyl. Trans-D-glucosylation can similarly be depicted by the following scheme:

+

D-GP-D-G~ E e D-Gp+ D-GP-E

+ D-GP-D-GP-D-G~ + D-GP-E * D-GP-D-GP-D-GP-D-G~+ E, and so on. +

E~ D-GP-D-G~ D-GP-E e D - G ~ - D - G ~ - D - G

Trans-D-glucosylation in the monosaccharide series has been known for a long time,95 but, in the oligosaccharide series, it was first described by Pigman and Blair.96.97The ability of carbohydrases to catalyze both the hydrolysis and the synthesis of oligosaccharides is now well established. It has been reported that preferential trans-D-glucosylation to primary alcoholic groups on aldose-containing substrates is brought about by many a-D-glucosidases from molds.98 Although maltose is reported to be present in some nectars,99 it is unlikely that the sugar occurs in proportions sufficient to account for the percentage found in honey. In conformity with this conclusion, certain evidence indicates that it is also formed in the bee stomach by tranS-D-ghCOsylation. White and Maherloo have demonstrated the presence in “honey invertase” of a trans-D-glucosylase capable of synthesizing maltose and isomaltose from D-glucose. Studies have been reported on the trans-D-glucosylating action of the tropical yeast Schizo(94) E. H. Fischer, J. Kohtes, and J . Fellig, Helo. Chim. Acta, 34, 1132 (1951). (95) J. Rabate, Compt. Rend., 204,153 (1937). (96) W. W. Pigman,]. Res.Not. Bur. Stand.,33, lOS(1944). (97) M. C. Blair and W. W. Pigman, Arch. Biochem. Biophys., 48,17 (1954). (98) J. Edelman,Aduan. Enzymol., 17,189 (1956). (99) C . R. Wykes, New Phytologist, 51,210 (1952). (100) J. W. White, Jr., and J. Maher, Arch. Biochem. Biophys., 42,360 (1953).

300

I. R. SIDDIQUI

saccharomyces pombe on a variety of substrates, including maltose,10'.'02 ~ - g l u c o s e , 'and ~ ~ a starch-syrup substrate.lM From these investigations, it follows that D-glucose and several members of the a - ~ 1+4) -( homologous series give rise to such oligosaccharides as kojibiose, nigerose, isomaltose, panose, and isomaltotriose. From the work of Peat and coworkers,lo5it is known that small proportions of nigerose are produced by the action of an enzyme preparation of Aspergillus niger on concentrated solutions of D-glucose. However, Aspergillus oryzae enzyme yields a substantially greater proportion of nigerose from a mixture of D-glucose and maltose.'06 Isomaltose, panose, and isomaltotriose have also been synthesized from maltose by various enzyme preparations from mold^.'^^-'^^ However, panose was first crystallized by Pan and coworkersl'O as a product of trans-D-fructosylation of maltose b y a culture filtrate of Aspergillis niger. Yasumura"' found maltulose and turanose in the mixture obtained by action of a brewers' yeast extract on sucrose; these were obviously formed by a transfer of &-D-glUCOSyl groups to 0 - 3 and 0-4of D-fructose. Avigad'I2 has reported the isolation of a number of oligosaccharides, including ~-O-a-D-g~ucopyranosy~-Dfructose, isomaltulose, and maltulose, as products of trans-D-glucosylation of D-fructose moieties by a hybrid-yeast a-D-glucosidase. A trehalose is an important constituent of the blood sugar of ins e c t ~ , " ~ , "including ~ the honey bee,'lS where it ha.s been reported to occur as the a,a form. By paper chromatography, it was shown'ls that the blood of pollen-collecting bees is very rich in a trehalose, and that the level of D-glucose and D-fructose is almost nil. On the other hand, the blood of the nectar-collecting bees is comparatively rich in D-glucose and D-fructose. However, Maurizio116has found (101)K.Shibasaki and K. Aso, TohokuJ.Agr.Res., 5,131(1954). (102)K.Shibasaki, TohokuJ.Agr. Res., 6,47(1955). (103)K.Shibasaki, Tohoku]. Agr. Res., 6,171(1955). (104)K.Shibasaki, Mem. Publ. Fac. Agr., Tohoku Uniu., 26 (1958). (105)S.Peat, W. J. Whelan, and K. S. Hinson, Chem. lnd. (London), 385 (1955). (106)J. H. Pazur, T. Budovich, and C. L. Tipton,]. Amer. Chem. Soc., 79,625(1957). (107)J . H.Pazur and D. French,]. Biol. Chem., 196,265(1952). (108)S.A. Barker and T. R. Carrington,]. Chem. Soc., 3588 (1953). (109) K. V. Grii, A. Nagabhushanam, V. K. Nigam, and B. Bellavadi, Science, 121, 898 (1955). (110)S.C. Pan, L. W. Nicholson, and P. Kolacho,J. Amer. Chem. Soc., 73,2547(1951). (111)A. Yasumura, Seikagaku, 26,200(1954). (112)G. Avigad, Biochem.J.,73,587(1959). (113) G. R.Wyatt and G. F. Kalf,J. Gen. Physiol., 40,833(1957). ., (1959). (114)R.Geigg, M. Huber, D . Weismann, and G. R. Wyatt, Acta T T O ~16,255 (115)D.R.Evans and V. G. Dethier,]. Inst. Physiol., 1,3(1957). (116)A. Maurizio,]. Inst. Physiol., 11,745(1965).

THE SUGARS OF HONEY

301

that a trehalose is a constituent of the blood sugar of all three types of bee, namely, workers, drones, and queens. a#-Trehalose was known only as a synthetic preparation until its presence, together ~ with the a,a isomer, was detected in saki and in koji e ~ t r a c t . ”These two sugars have also been reported to occur in Royal Jelly.”* a,aTrehalose has been synthesized enzymically by Peat and and P,P-trehalose has been isolated from an almond-emulsin digest of D-glUCOSe after incubation for 5 weeks. Theanderose, together with panose and other oligosaccharides, has been synthesized by the action of a cell-free extract of Aspergillus niger 152 on a mixture of sucrose and maltose. The analog erlose, namely 0-a-D-glucopyranosyl-(1+4)-a-~-glucopyranosyl p-D-fructofuranoside, was obtained by White and Maher1Ig by treating sucrose with honey invertase. Of the honey trisaccharides, 1-kestose [O-a-D-glucopyranosy1(1+2)-O-P-D-fructofuranosyl-( 1+ 2) p-~-fructofuranoside] has been the most widely investigated. It has been synthesized (sometimes accompanied by other kestoses) by the action of trans-D-fructosylases from various sources on sucrose: by the action of takadiastase,lZ0 yeast invertase120J22 and various fungal enzyme^.'^^-'^^ Barker and were the first to obtain it crystalline. The oligosaccharide has also been prepared by the action of enzymes from sugar-beet leaves and from other higher plants.lZ6 It has been identified as a component of the soft xylem of aspen and of the oligosaccharides of maple sap.lZ8 The absence of appreciable proportions of melezitose from nectar honeys suggests that it is not formed during elaboration of the nectar in plants or in the ripening of honey by the bees; hence, it is logical to assume that the small proportions of this sugar present in floral honey may have originated as a result of interference by aphids. On the other hand, honeydew honeys contain a high proportion of this sugar, but

(117) K. Matsuda, TohokuJ . Agr. Res., 6,271 (1956). (118) I. R. Siddiqui and B. Furgala,]. Apicult. Res., 4,89 (1965). (119) J. W. White, Jr.. and J. Maher,].Amer. Chem. SOC.,75,1259 (1953). (120) J. S . D. Bacon and D. J . Bell,]. Chem. Soc., 2528 (1953). (121) J. S. D. Bacon, Biochem.]., 57,320 (1954). (122) D. Gross, P. H. Blanchard, and D. J. Bell,]. Chem. SOC.,1727 (1954). (123) J. H. Pazur,j. Biol. Chem., 199,217 (1952). (124) S. A. Barker and T. R. Carrington,]. Chem. SOC., 3588 (1953). (125) S. A. Barker, E. J. Bourne, and T. R. Carrington,]. Chem. SOC.,2125 (1954). (126) P. J. Allen and J. S. D. Bacon, Biochem.]., 63,200 (1956). (127) J. B. Pridham, Biochem. J . , 76, 13 (1960). (128) S. Haq and G. A. Adams, Can.]. Chem., 39,1165 (1961).

302

I. R. SIDDIQUI

there is a divergence of views as to its origin. Hudson129considered that melezitose is a constituent of the sap of various species of plants, whereas others believe that insects produce it. Bacon and D i ~ k i n s o n ~ ~ have discussed, in considerable detail, the origin of melezitose and erlose in honeydew honeys, and have shown that these sugars originate by the interaction of certain enzymes from aphids with plantsap sucrose. The trisaccharide O-cY-D-glucopyranosy1-(1+6)-O-a-D-glucopyranoSyl-(1- 3)-D-glUCOpyranOSehas been obtained by the action of potato T-enzyme on nigerose. The same trisaccharide is probably also produced, among others, by the transfer of D-glucopyranosyl groups from maltose to D-glucose in the presence of enzymes from Aspergillus oryzae.lW Other examples and explanations of such reactions are to be found ~ ~ Jother ~ ~ examples of the biosynin review^^^^^^^' and b o ~ k s . ’ Still thesis of some of these oligosaccharides, including isopanose, are those brought about by bacterial and algal As regards the occurrence of p-D-linked disaccharides in the absence of a p-D-linked substrate, one is tempted to conclude that these oligosaccharides are synthesized by the enzymic reversion of D-glucose by a P-D-glucosidase, As White and Maherloohave found that their honey-invertase preparation had no /3-D-glucosidase activity, it would appear that these sugars are carried into the hive as constituents of nectar. In vitro syntheses with enzymes almost invariably result in poor yields, but use of such syntheses is essential when chemical syntheses have not yet proved feasible. Another advantage of enzymic processes lies in their similarity to the processes occurring in Nature. It is implicit that similar enzymes or similar enzymic reactions are operative in the formation of honey, but, unfortunately, our knowl(129) C. S. Hudson, Adoan. Carbohyd. Chem.,2,1(1946). (130) S . A. Barker and E. J . Bourne, Quart. Reo. (London), 1,56 (1953). (131) R. A. Dedonder, Ann. Reo. Biochem., 30,347 (1961). (132) R. W. Bailey, “Oligosaccharides,” Macmillan, New York, N.Y., 1965. (133) J. Stanek, M. Cerny, and J. Paclk, “The Oligosaccharides,” Academic Press Inc., New York, N.Y., 1965. (134) M . Killey, R. Dimler, and J . E. Huskey,]. Amer. Chem. Soc., 77,3315 (1955). (135) S. A. Barker, E. J . Bourne, P. M. Grant, and M . Stacey, Nature, 178,1221 (1956). (136) D. S. Feingold, G. Avigad, and S. Hestrin, Biochem.]., 64,351 (1956). (137) K. Aso, K. Shibasaki, and M. Nakamura, Nature, 182,1303 (1958). (138) E. J . Bourne, D. H. Hutson, and H. Weigel, Biochem.].,79,549(1961). (139) J. H. Pazur and T. AndoJ. Biol. Chem.,235,297 (1960). (140) W. A. M. Duncan and D. J. Manners, Biochem.]., 69,343 (1958).

THE SUGARS OF HONEY

303

edge of the enzyme chemistry of honey is still inadequate. Honey has been shown to contain an acid phosphatase and a phosphorylase141J42; and an enzyme (D-glucose oxidase) that produces acid has been reported. However, the most important enzymes in honey are invertase and diastase. Invertase brings about the inversion of sucrose in nectar, and has been recognized to be an a-D-glucosidase: the end products it produces from sucrose differ from those formed by yeast invertase. Enzymic synthesis from action of invertase on SUcrose gives rise to six oligosaccharides, five of which differ from those produced by yeast invertase; the structure of only one of these has This invertase preparation appears to be as yet been a mixture of enzymes that contains a little D-fructosylase and maltase activity. Under the circumstances, the most favored reaction with honey invertase would be resynthesis of oligosaccharides from sucrose by transfer of D-glucosyl and D-fructosyl groups. In this connection, it should be emphasized that, although the honey invertase would be expected to be specific with regard to the donor molecule and the resulting glycosidic linkage, its specificity towards the acceptor molecule will vary considerably; hence, further experimentation, with other acceptor oligosaccharides, should lead to very interesting results. It is highly probable that theanderose would be the product of one such reaction. Further fractionation of crude a-D-glucosidase of honey by ionexchange chromatography, gel filtration, and starch-gel electrophoresis has shown that it contains 7-18 components (isozymes) having143 a molecular weight of -51,000. The origin and function of diastase are little underin honey, supposedly arising chiefly from the bee,144 stood. The same is true of phosphorylase. Phosphorylases, which transfer the D-glucopyranosyl group from D-glucopyranosyl phosphate to a monosaccharide acceptor (and thus give rise to a disaccharide), have been found in many bacteria, and several disaccharides have The mode of synthesis of oligobeen synthesized by their use.145-148 saccharides in honey is subject to further complications in view of the presence of osmophilic (sugar-tolerant) yeasts, said to become active (141) W. Zalewski, Pszczel. Zeszyty Nauk., 9, l(1965). (142) F. Gunther and 0.Burckhart, Deut. Lebensm. Rundschau, 63,41(1967). (143) J. W. White, Jr., and I . Kushnir,]. Apicult. Res., 6,69 (1967). (144) R. Ammon, Biochem. Z., 319,295 (1949). (145) W. Z. Hassid and M. Doudoroff, Aduan. Enzymol., 10,123 (1950). (146) C. J. Sih, N. M . Nelson, and R. H. McBee, Science, 126,1116 (1957). (147) E. W. Putman, C. Fithing-Litt, and W. Z. Hassid,]. Amer. Chem. Soc., 77,4351 ( 1955). (148) Z. Selinger and M. Schramm,/. Biol. Chem., 236,2183 (1961).

304

I. R. SIDDIQUI

with an increase in the moisture content and the granulation of the honey. White has discussed these aspects, and has made recommendations for arresting or minimizing these effects. Of the osmophilic yeasts, four have been shown to be Schwanniomyces occidentilis, Saccharomyces torulosus, Saccharomyces bisporus, and Zygosaccharomyces japonicus; Nematospora ashbya gossypii has been tentatively identified, and a new species is reported that needs further i d e n t i f i ~ a t i o n . ' ~ ~ Logical questions in connection with the origin of oligosaccharides in honey are the composition of the raw material (or nectar) with respect to these constituents, and the extent of alterations in the composition caused by the nectar enzymes and those incorporated by the bees. Occurrence of both trans-D-glucosylation and trans-Dfructosylation has been established, the former in the nectar from flowers of Robinia pseudoacacia, and the latter, in the extra-floral nectar of Impatiens but very little is known concerning the nature and composition of sugars present in nectars, and the work has so far been of a qualitative or routine quantitative nature.100*'s2-15S The principal reason for this deficiency is the difficulty encountered in obtaining sufficient quantities of the material. For this reason, information is only available on the three major sugar constituents of nectars, namely, D-glucose, D-fructose, and sucrose (and their relative proportion, in some sources). Indications have also been obtained that small proportions of other sugars are present, but these have not yet been properly identified. Maurizio has reviewed the results of some of these investigations in It is apparent that our knowledge of the oligosaccharide composition of nectars is elementary, and the same is true of the changes caused by the bee enzymes. Some information regarding these effects is based on paper-chromatographic analysis of mixtures of sugars obtained from in vitro experiments (149) S. Aoyagi and C. Oryu, Tamagawa Daigaku Nogakubu Kenkyu Hokoku, 8, No. 7, 203 ( 1968). (150) M. H. Zimmermann, Ber. Schweiz. Botan. Ges., 63,402 (1953). (151) M. H. Zimmermann, Experientia, 10,145 (1954). (152) R. Beutler, Z. Vergleich. Physiol., 12,72 (1930). (153) A. Maurizio, Ann. Abeille, 4,291 (1959). (154) M. S. Percival, New Phytologist, 60,235 (1961). (155) B. Furgala, T. A. Gochnauer, and F. G. Holdaway, Bee World, 39,203 (1958). (156) D. Wanic and L. Mostowska, Zesz. Nauk. Wyzszej Szkoly Rolniczej Olsztynie, 17,54311964). (157) E. A. Mikhailova, Zzu. Tomsk. Otd. Vses. Botan. Obshchestua, 5,121 (1864). (158) A. G. R. Nair, S. Nagarajan, and S. S. Subramanian, Cum. Sci., 33, 401 (1964). (159) A. Maurizio, Bee World, 43,66 (1962).

THE SUGARS OF HONEY

305

using crude extracts from the hypopharyngeal glands and the mid-gut of the honey bee; these are the organs where sugar-inverting enzymes have been found.160The effects of these preparations on sucrose, maltose, a,a-trehalose, and other disaccharides have been examined and discussed by M a ~ r i z i o . ' ~ It ~ -is, ' ~ however, ~ difficult to draw any definite conclusions, except that enzymic changes occur and that these sugars are not only hydrolyzed but also give rise to higher oligosaccharides. It was found that the enzymes from the two sources, the hypopharyngeal gland and the mid-gut, differ in their speed of action on these substrates, and produce different end-products. Differences in the ease with which summer and winter bees inverted sugars were noted, and their age, nutrition, physiological condition, and race were found to affect the efficiency of the enzymes. Goldschmidt and BurkerP have attempted to explain the origin of a number of the sugars in the various honeys from the results obtained by paper chromatography of nectar honey, honeydew honey, and the honey derived by feeding sucrose syrup to bees. It was concluded that such sugars as 1-kestose and melezitose, which occur in honeydew honey in considerable proportions (but not to an appreciable extent in nectar honey), are apparently carried to the hive from the plant. On the other hand, such sugars as maltose, erlose, isomaltose, and isomaltotriose are formed as enzymic, secondary products formed by trans-D-glucosylation in the body of the bee. The complexity of the operations involved in the formation of honey is indicated by the fact that those processes that start in the nectaries (the plant glands that secrete nectar) continue in the secreted nectar, are modified in the bee stomach, and continue in the hive during ripening and then in storage. White and coworkers164have shown that chemical changes in composition and biochemical activity occur, even when honey is stored at 26 k3". During two years of such storage, there was a conversion of about 9% per year of monosaccharides into oligosaccharides. Free D-glucose disappeared more rapidly than free D-fructose, so the D-fructose/D-glucose ratio increased markedly. It may therefore be concluded that the biochemical origin of oligosaccharides in honey is still at the speculative stage. Thorough knowledge of nectar composition, including sugar components and enzymes involved, and in vitro studies using nectar, bee, and honey (160) E. Kralky, 2. Wiss. Zool. Abt. A, 139,120 (1931). (161) A. Maurizio, Ann. Abeille, 5,215 (1962). (162) A. Maurizio, Ann. Abeille, 8,113 (1965). (163) A. Maurizio, Ann. Abeille, 8,167 (1965). (164) J. W. White, Jr., M. L.Riethof, and I. Kushner, J . Food Sci., 26, 63 (1961).

306

I. R. SIDDIQUI

enzymes under properly controlled conditions are needed for explaining on a more realistic basis the enzymic build-up of oligosaccharides.

IV. HONEYPOLYSACCHARIDES Despite the voluminous literature on honey, its colloidal constituents have remained essentially uninvestigated. The gummy material from the filters of honey-processing plants contains a large proportion of a polymer associated with undesirable turbidity, caramelization, and crystallization tendencies of honey. The presence of such constituents has been recognized for many years; they have been reported to consist of gummy and colloidal aggregates of proteins, waxes, and polysaccharides, and have been thought to affect the properties of honey rather adversely. Their removal has been shown to result in the elimination of turbidity and the production of brilliant clarity in honey. After removal of these constituents, which amount to 0.2-1% of light to dark honeys, the caramelization and crystallization tendency of honey is also greatly l e ~ s e n e d . ' ~ A ~-'~~ more definitive report on the nature of these colloidal constituents appeared'68 in 1953. On the basis of electrophoretic and sedimentation analysis of buckwheat honey, the presence of three polymeric constituents was claimed; the two major components were proteins, and the minor component appeared to be a polysaccharide. was made, by the Following these reports, a detailed author, of the polysaccharide isolated from honey collected at the Central Experimental Farm, Ottawa. The crude polymer constituted 0.2% of the honey, and provided two polysaccharide fractions that amounted to only about 0.002%of the honey. On hydrolysis, the impure fraction gave six monosaccharide components, and, on this basis, it appeared to be a mixture of polysaccharides. However, the purified fraction yielded only three sugars, namely, D-mannose, Larabinose, and D-galactose (ratios 1.0:2.04:4.04) and appeared to be homogeneous on the basis of boundary electrophoresis and sedimentation analysis. Methylation and hydrolysis of the methylated product afforded seven methylated fragments which were fully characterized. From the methylation data, it was concluded that an average unit of the polysaccharide is made up of 22-23 sugar residues consisting of (165)R.E.Lothrope and H. S. Paine, Amer. BeeJ.,71,28 (1931). (166)H.E.Lothrope and H. S. Paine, Amer. Bee]., 72,444 (1932). (167)H.S.Paine and R. E. Lothrope, Amer. Bee]., 73,53(1933). (168)T.C.Helvey, Food Res., 18,197(1953). (169)I. R.Siddiqui, Can.J.Chem., 43,421(1965).

THE SUGARS OF HONEY

307

6 terminal, nonreducing end-groups comprising 3 residues of Dmannose, 2 of D-galactose, and 1 of L-arabinose; 5 to 6 residues of Dgalactose are involved in branching at 0-3 and 0-6. The remaining 11nonterminal units consist of six (1+6)-linked D-galactose residues, and one (1+3)-linked and four (1-5)- or (1+4)-linked (or both) Larabinose residues. Although the molecular weight of the polysaccharide was not determined, its rapid rate of diffusion on ultracentrifugation, and the fact that synthetic boundary-cells had to be used, indicated that the molecular weight was less than 10,000. The molecular weight of buckwheat-honey polysaccharide is 9,000. It was not possible to formulate a unique structure for the polysaccharide from the data obtained. Further studies, including fragmentation analysis, are needed for determination of its detailed structure and its triheteropolymer nature. Other polysaccharides were undoubtedly present, but their isolation and fractionation in quantities sufficient to permit detailed studies have been major obstacles to their structural elucidation. Regarding the origin of these polysaccharides, no information is available. However, there are several ways in which these compounds could originate. They could, for example, be components of the pollen grains present in honey, or, alternatively, could result from the fermentation of honey by sugar-tolerant yeasts. An interesting speculation is that these polysaccharides may be products of the detoxifying agents or enzymes in the bee, since Dgalactose, D-mannose, and L-arabinose are reputed to be bee poisons. V. HONEYDEW The importance of honeydew as raw material for honey is increasing in many European countries as a consequence of changed agricultural practices which have imposed restrictions on bee pastures. Both kinds of raw material, nectar and honeydew, have a common origin in the phloem saps of plants, but the two reach the bees through different routes; whereas nectar is drawn from flower nectaries, honeydew passes through insects, and, in the process, undergoes characteristic and noteworthy changes that result in the production of the so called honeydew honey or forest honey. It is appropriate to mention that, in certain regions of central Europe, honeydew honey is more highly prized than floral honey, although, on the North American continent, it is, from the esthetic point of view, considered inferior to floral honey. The insects associated with the formation of honeydew honey are the homopterous insects, such as

I. R. SIDDIQUI

308

plant lice (aphids) and scale insects that thrive on various parts of the host plant. In the past, there has been a divergence of opinion concerning the origin of honeydews (and mannas); however, the prevalent view is that insects are always responsible for formation of h ~ n e y d e w . "Such ~ insects pierce the plant tissues and suck the sap into their alimentary tract and, after modification by aphid enzymes, the unwanted constituents of the sap are excreted in the form of droplets that collect on the leaves and other parts of the plants. The plants associated with these deposits are such trees as ash, beech, cedar, elm, fir, hickory, linden, maple, oak, poplar, spruce, tulip, willow, and fruit trees.30 Bees collect the mannas or honeydews from these sources when nectar is not readily available, and process it to a product which, as already mentioned, is darker in color, lower in D-glucose and D-fructose, and higher in pH, oligosaccharides, total free acid, ash, and nitrogen than the original honeydew. The average composition of honeydew honey with respect to carbohydrates and moisture, based on 14 samples, is given in Table IV. TABLEIV Average Composition of Honeydew Honeys and Range of Values for 14 Samples, According to White30 Component, % Moisture D-Fructose DGlucose Sucrose Maltose Higher sugars Undetermined

Average

Standard deviation

16.3 31.80 26.08 0.80 8.80 4.70 10.1

1.74 4.16 3.04 0.22 2.51 1.01 4.91

Range

12.2 -18.2 23.91-38.12 19.23-31.86 0.44- 1.14 5.11-12.48 1.28-11.5 2.7 -22.4

A characteristic feature of honeydew honey is the presence of the trisaccharides rnelezitose and erlose; the presence of the former was recognized35 as early as 1918, and the latter was found"' in 1954. The mode of formation of these trisaccharides, as pointed out earlier (see p. 302), has been discussed at length by Bacon and Di~kinson.~' However, for the present, it suffices to state that, based on the fore(170)G. Tanret, Bull. SOC. Chim. Fr., 27,56(1920). (171)H.E.Gray and G.Frenkel, Science, 118,304(1954).

THE SUGARS OF HONEY

309

going evidence, White30 believed that there are at least two types of honeydew honey; namely, the melezitose type, which may granulate rapidly (frequently in the comb itself), and the erlose type, which does not granulate. To this list must be added yet a different type of honeydew honey; this supposedly arises from the phloem sap secreted from wounds on a plant, without passage through the body of insects; according to some beekeepers, bees often collect such sap.

This Page Intentionally Left Blank

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA* BY M. J. KORT Deparfment of Chemistry, University of Natal, Pietermaritzburg, and Sugar Milling Research Institute, Durban, South Africa

I. Introduction.. ..........................................................

11. Products Obtained.. .................................................... 111. Isolation of Products, and Proportions Obtained. .........................

IV. Mechanism.. ........................................................... 1. Lobry de Bruyn-Alberda van Ekenstein Transformation ................. 2. Formation of Glycosylamines and Aminodeoxy Sugars . . . . . . . . . . . . . . . . . . 3. Fragmentation Mechanism ............................................ 4. Saccharinic Acids. ........................... ..................... 5. Reaction of Dicarbonyl Compounds with Ammo ..................... 6. Fission of Reducing Sugars in Alkaline Solution.. ...................... 7. Imidazole Formation.. ............................................... 8. Recombination of Sugar Fractions.. ................................... V. Applications ................................... .....................

311 312 328 332 332 333 340 341 344 345 347 348 349

I. INTRODUCTION It is well known that sugars react with aqueous ammonia to produce heterocyclic compounds in low yield. The products of the reaction of the mono- and di-saccharides with concentrated, aqueous solutions of ammonia are dependent on three factors: (I) the length of time during which the reaction proceeds, (2) the temperature of the reaction, and (3) the catalyst used. This matter has been briefly but comprehensively reviewed.' The present article is more detailed; it covers the literature to the end of July 1970, and has been prepared for the use of chemists who may not be specialists in the carbohydrate field, as well as of those who are. * The author thanks Professor D. A. Sutton for suggesting that this subject be re-

viewed. (1) M. R. Grimmett, Reti. Pure Appl. Chern., 15,101(1965).

311

TABLEI Reaction of Ammonia with Various Sugars at Low Temperature (Short Reaction Time, with No Catalyst) Reaction

Sugar

D-GluCOSe ( 11

Temp. (depees)

Time (hours)

37

-

D-arabinose (2)

37

48

-

-

Dpsicose (5) di-mglucopyranosylamine (6) 2-amino-2deoxy-m glucose (8)

38

mFructose (3)

38

48

48

References

Products dark, high polymer

D-fructose (3)

imidazoles

Dfructose (3)

Dglucopy8 imidazole ranosylamine (7) compounds

D-mannose (4)

2

mmannose (4)

3 4

5 5

2-amino-2deoxy-D-

glucose (8) Lactose (4-0-8-Dgalactopyranosy l-D glucopyranose)

37

48

=galactose (9)

lactulose

Dlyxose (10)

resinous matter

s

?

Dtagatose (11)

3

R

g

4

TABLEI (continued) Maltose

37

48

D-arabinose (2)

D-fructose (3)

(4-0-a-DglucopyranOSyl-D-glUCOpyranose)

maltulose (4-0-a-nglucopyran-

4-0-a-~glucopyranOSYl-Dpsicose

Dglucose (1)

imidazoles

3

>

n

2

D-mannose (4)

5

OSyl-D-

Melibiose (6-0-w~galactopyranosyl-Dglucopyranose)

37

48

fructose) 6-O-cx-~galactopyranosyl-p-Dmannopyranose pentosecontaining disaccharide

w

m

-l

w Dgalactose (9)

imidazoles

Dtagatose (11)

melibiulose (6-0-a-~galactopyranosyl-Dfructose)

4(5)-methylimidazole

6

m m

2Q

* ZI

3 X

>

0 C

m

0

5

(2) L. Hough, J. K. N. Jones, and E. L. Richards, Chem. Ind. (London),545 (1954). (3)L. Hough, J. K. N. Jones, and E. L. Richards,J. Chem. SOC.,2005 (1953). (4) M. KGmoto, Nippon Nogei Kagaku Kaishi, 36, 305, 310 (1962);Chem. Abstracts, 59,15361d,f (1963). (5) K. Heyns and W. Koch, 2. Natulforsch., B , 7,486 (1952). (6) L. Hough, J. K. N. Jones, and E. L. Richards,/. Chem. SOC.,295 (1954).

?i 5

M. J. KORT

314

11. PRODUCTSOBTAINED When sugars are treated with aqueous ammonia for a short time at low temperature in the absence of a catalyst, the reaction is arrested before heterocyclic compounds can be formed in appreciable proportion, and the products are mainly epimerization products of the sugars, probably formed by way of their 2,3-enediols. These epimerization products are summarized in Table I which shows the reactions of D-glUCOSe, D-fructose, lactose, maltose, and melibiose with aqueous ammonia for a short time at low temperature. A dark-colored, high polymer is also formed in some instances (the browning reaction). In the ammoniacal solution, the monosaccharides are epimerized; the disaccharides are epimerized and, in addition, may be hydrolyzed to monosaccharides that can also be epimerized; hence, the variety of products obtained may be considerable. With a prolonged reaction-time, the reaction is more complex and passes beyond the epimerization stage. Many products are formed; from a single sugar, Hough and coworkers' obtained at least 15 components, and K6moto8 separated 8 compounds by paper chromatography. Many substituted imidazoles and pyrazines have been isolated and identified. Compounds isolated by various workers are shown in Table I1 (which includes compounds 12 through 58). The products H

c=o

I HCOH

I HOC H I HCOH I HCOH I C &OH (1)

H

c=o HOCH I

HOCH I

HCOH I HCOH I C &OH (4)

H

c=o I

HOCH I HCOH I

HCOH I C H,OH (2)

CH,OH I

CH,OH

I

c=o I

HOC H I

HCOH I HCOH I CH,OH (3)

7

c=o I

HCOH I HCOH

HCOH

HCOH

HCO

I

I

CH,OH

(5)

(6)

(7) L. Hough, J. K.N. Jones, and E. L. RichardsJ. Chem. Sac., 3854 (1952). (8) M. KiSmoto, Nippon Nogei Kagaku Kaishi, 36, 407 (1962); Chem. Abstracts, 61, 12227g(1964).

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA H

H

c=o

c=o

I

I

HCNH, I HOCH

HOCH I

HCOH I

HOCH

I

HCOH

I

HCOH I HCOH

I

HCO I

315

HOCH I HCOH I

I

CH,OH

CH,OH

CQOH

(7)

(8)

(9)

C€&.OH I

H

c=o

c=o

I HOCH I HOCH I HCOH I CH,OH

I

HOCH I HOCH I HCOH I CH,OH 111)

(10)

TABLE I1

Compounds Isolated from the Reaction of Free Sugars with Aqueous Ammonia Cornpound number

Compound name Miscellaneous "Galactosimine," C,HlaNO,.C,H ,N,O,.4 H,O-Zn(OH), Nitrogen-containing disaccharide, CiJL&"io(V Oxalic acid D-Xylosylamine

12

13 14 15

Imidazoles

16 (continued)

M. J. KORT

316

TABLEI1 (continued) Compound number

Compound name SubstituentsO at

17 18

C-2 CHSCOH

c-4 Me R

c-5 H 2-acetyl-4(5)-methylH 4(5)-(~-erythro-2,3-dihydroxybuty1)-

OH -CHzC-CH,OH H H Me H Me Me Me H H H Et H H Et Et Me H -CHzCHzOH H H H H -CH,OH -CH,OH H H Me H -CHzOH R' H -CH,OH H

4(5)-(~-glycero-2,3-dihydroxy-

30 31 32 33 34 35 36 37

H H H Me H H H H

H -COzH -CONH, H Me R" R' R"'

H H H H H H H H

Pr0PYl)2,4(5)-dimethyl4,5-dimethyl2-ethyl4(5)-ethyl4(5)-ethyl-5(4)-methyl4(5)-(2-hydroxyethyl)2-(hydroxymethy1)4(5)-(hydroxymethyl)2-(hydroxymethyl)-4(5)-methyl2-(hydroxymethyl-4(5)-(D-lyxotetrahydroxybuty1)4(5)-imidazole -4(5)-carboxylic acid -4(5)-formamide 2-methyl4(5)-methyl4(5)-(~-arabino-tetrahydroxybutyl)4(5)-(~-lyxo-tetrahydroxybutyl)4(5)-(~-erythro-2,3,4-trihydroxy-

38 39 40 41

H H H Me

R"" R""' R""" Me

H H H Me

19

20 21 22 23 24

25 26 27 28 29

buty1)-

4(5)-(D-erytho-trihydroxypropyl)4(5)-(~-erythro-trihydroxypropyl)4(5)-(~-threo-trihydroxypropyl)2,4,5-trimethyl-

Pyrazines Substituentsaat c-2

42 43 44 45

R" Me Me Me

C-3 H Me H H

C-5

R" H Me H

C-6 H H H Me

2,5-bis(~-arabino-tetrahydroxybutyl)2,S-dimethyl2,5-dimethyl2,6-dimethyl(continued)

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

317

TABLEI1 (continued) Compound number

Compound name

46 47 48 49

-CH,OH -CH,OH Me Me

H H H H

H H R”

H H H H

50

Me

H

H

R“

51

Me

R“”

R”

H

52 53

H R”

H H

H R”’

H H

Me

2-(hydroxymethy1)2-(hydroxymethyl)-5-methyl2-methyl2-methyl-5-(~-arubino-tetrahydroxybuty1)2-methy~-6-(~-arabino-tetrahydroxybuty1)2-methyl-5-(~-arabino-tetrahydroxybutyl)3(D-erythro-trihydroxypropyl) pyrazine 2-(~-arabino-tetrahydroxybuty~)-5-(D-

erythro-2,3,4-trihydroxybutyl)-

$x H

H

Piperazines

Substituents at

c-2 54

Me

C-3 H

C-5 Me

C-6 H 2,5-dimethyl-

Pyridines

55 56 57

Pyridine 2-Methylpyridine (a-picoline) C,H,NO probably a (hydroxymethy1)pyridine C,H,,NO, probably 4-acetyl-5,lidihydro-3-methyl-2-pyridinol

58

H H

H H OH

H OHOH

“where R = -CH,-C-C-CH,; R’ = -C-C-C--CH,OH; R“ =-C-C-C--CH,OH;

OHH H OHOH OHOHH OHOH OHOH H H R”‘ = -CH,-C--C-CH,OH; R”“ = -C-C-CH,OH; R””‘ = -C-C-CH,OH; and H H H H OHOH H OH R””” = -C-C--CH,OH. OHH

M. J. KORT

318

obtained on prolonged reaction of sugars with aqueous ammonia at low temperature in the absence of a catalyst are summarized in Table 111. TABLE111 Reaction of Ammonia with Various Sugars at Low Temperature (Long Reaction Time with No Catalyst) Reaction sugar

Temp. Time Products Refer(degrees) (days) 1 6 7 8 13 15 27 30 34 35 42 49 50 51 NH, ences

2-Amino-2-deoxy- room D-glucose D-Fructose 20-23 room D-Fructose" room D-Glucose 37 20 20-23 D-Glucose* 20

0 0

9

0 0 0 0 0 0 0

10 11 11

0

180 42 60

0 0

0 0

60 14 16 42 7

0

7

0 0

12 10 13

00

0 0

0 0 0 0 0 0

0

0

0 0 0 (+ epimerization

products) D-Glucosylamine' D-Xylose

20 20

10 0 0 7

0 0

0

(+unidentified heterocycles)

12 13 ~

"Plus oxygen. bVapors of 25% aqueous ammonia. cWater present, but no ammonia.

At elevated temperatures, sugars react more quickly than at lower temperatures with aqueous ammonia, to give, mainly, substituted imidazoles and pyrazines, summarized in Table IV, and a catalyst is not needed. At the higher temperatures, there is an increase in the methyl-pyrazine and -imidazole fractions and a decrease in the (hydroxymethy1)imidazole and (tetrahydroxybuty1)pyrazinefractions, indicating occurrence of thermal cleavage of the side chains.1° (9) M. I. Taha,J . Chem. SOC.,2468 (1961). (10) I. Jezo and I. LuzAk, Chem. Zuesti, 20, 586 (1966); Chem. Abstracts, 65, 1866913 (1966). (11) j. Parrod, Ann. Chim. (Paris), 19, 205 (1933); Bull. Soc. Chim. Fr., 51, 1424 (1932); 53,196 (1933). (12) M. KGmoto, Nippon Nogei Kagaku Kaishi, 36, 403 (1962); Chem. Abstracts, 61, 12227d (1964). (13) M. S. Dudkin, N. G . Shkantova, and A. F. Yatsuk, Zh. Prikl. Khim., 41,385 (1968); Chem. Abstracts, 69,19441r (1968).

TABLEIV Reaction of Ammonia with Various Sugars at High Temperature with No Catalyst Reaction sugar D-Glucose

D-Ghcosylamine" Invert sugar Molassesb

Temp. Time Products (degrees) (hours) 1 3 6 7 8 13 20 21 27 34 35 42 44 45 46 47 48 49 50 51 52 54 55 56 NH3 References

100 100 100 100 120 120 120

Molasses' Sucrose

1 35 40 1 2 2

000 0 0 0 0

-

140 220 120-260 110

16 16 18 >5

0

0

000

0

0 0

2

-

0 0

0 0 0

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0

0 0 0 (+lOimidazole spots) (+substituted pyrazines and imidazoles) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

12 14 14,15 12 16 17.18 19 19a

10 10

20 21

"Water present, but no ammonia. 60 lb. in.?. 'Molasses itself, no treatment with aqueous ammonia; the necessary ammonia must have arisen during the processing of the sugar beet (or cane). (14) P. Brandes and C. Stoehr,]. Prakt. Chem., 121,54,481(1896). (15)C . Tanret, Compt. Rend., 100, 1540 (1885);Bull. SOC. Chirn. Fr., 44, 102 (1885). (16)B. K.Davison and L. F. Wiggins, Chem. Ind. (London), 982 (1956). (17)L. F. Wiggins, Proc. Congr. Intern. SOC. Sugar-Cane Technologists, gth, India, 2,525(1956). (18)L.F.Wiggins and W. S . Wise, Intern. Sugar]., 57,435(1955). (19)L. F.Wiggins and W. S . Wise, Chem. Ind. (London), 656 (1955). (19a)1. Jezo, Listy Cukrooar., 82,300(1966);Chem.Abstracts, 66,66989~ (1967). (20)I. Jezo,Chem. Zuesti, 17,126(1963);Chem. Abstracts, 60,4139b(1964). (21)M. R.Grimmett, R. Hodges, and E. L. Richards, Aust.]. Chem., 21,505(1968).

9

0 C

m

0

c,

M. J. KORT

320

Use of a catalyst in the reaction of sugars with ammonia does not appear to alter the reaction qualitatively. The reaction still requires a longer time at room temperature (see Table V) than at higher temperatures (see Table VI), and the products of the reaction at low temperature and long reaction-time (see Table V) do not appear to TABLEV Reaction of Ammonia with Various Sugars for a Long Time, at Room Temperature in the Presence of a Catalyst Reaction Sugar

L-Arabinose D-Fructose

D-Galactose D-Glucose D-Glucose" Lactose Maltose D-Mannose L-Rhamnose* D-Sorbose Sucrose L-Xylose

Time (months)

Catalyst

Products Refer12 14 20 24 27 28 30 34 35 57 58 ences

1.5 4 1 2

2 2 4 1.5 1.5 1.5 4 3 6,18 6,18 4 6 12 4 24 4

0 0 0

0

0

0

0

? 11 0 11 0 23 0 23 0 22 0 24,25 0 23 0 0 26 0 23 0 23 0 23 0 0 23 0 0 0 0 0 27,28 0 23 no imidazole compound 23 0 23

0

0

22 23 11 11

OHOHH H H "Plus acetaldehyde. *L-Rhamnose = H , C - C - C - - C - C - - C = O . H H OHOH

(22) K. Inouye, Ber., 40,1890 (1907). (23) A. Windaus, Ber., 40,799 (1907). (24) A. Windaus and F. Knoop, Ber., 38,1166 (1905). (25) K. K. Koessler and M. T. HankeJ. Biol. Chem., 39,497 (1919). (26) A. Windaus, Ber., 39,3886 (1906). (27) A. Windaus and A. Ullrich, 2. Physiol. Chem.,92,276 (1914). (28) A. Windaus and W. Langenbeck, Ber., 55,3706 (1922).

TABLEVI Reaction of Ammonia with Various Sugars for a Short Time in the Presence of a Catalyst

sugar

Temp. Time (degrees) (hours) Catalyst

D-Arabinose” 95 0.5 L-Arabinosea 95 0.5 Cane and beet 200-275 18 molasses Cellobiose 200-275 18 Cellulose 200-275 18 “Dialdehyde” 200-275 18 of starch D-Fructose 60 4 1 D-Fructose“ 100 D-Galactose room 96 “Galactosimine” (12) 100 2 D-Glucose 40 %,48 42 120 60 6 100 4 D-Glucose” 40 24 60 1 plus 70 5 D-GlUCOSe‘ 40 24 Hydrolyzed starch 200-275 18 60 6 Invert sugar Lactose 200-275 18 L-Rhamnosed 44-45 130

Products

Refer-

12 18 19 20 21 23 24 25 27 28 30 34 37 38 39 40 43 44 45 48 54 ences

0

Cu(OAc), Cu(OAc), (NH&PO,

0

(NH&PO, (NH&PO, (NHJSPO,

0 0

0

0

0 0 0 0

0 0 0 0

0 0

0 0

0 0 0 0 0 0 0 0

0

0

29 29 30 30 30 30

v,

C

Q

Zn(OH), Cu(OAc), Zn(OH), 0

0

-

Zn(OH), Zn(OH), Zn(OH), Zn(OH), Zn(OH), ZnC03 Zn(OH), (NH,),PO, Zn(OH), (NHJ3P04 Zn(OH),

31 32 23 23 31 8,33-36 31 31 31 37

0

P

O

0

0

0 0 0 0 0 0

(+2unidentified)

0 0

0 0 0

0

0

0 0 0

0

0 0

0 0 0 0 0

0 0 0 0

0 0 0 0 (+ 2 unidentified)

31 30 31 30 38,39

(continued)

b

w

E

TABLEVI (continued)

0

E

Reaction Sugar

D-Ribose" Starch of various origins Sucrose D-XyIOSe"

Temp. Time (degrees) (hours) Catalyst

Products Refer12 18 19 20 21 23 24 25 27 28 30 34 37 38 39 40 43 44 45 48 54 ences

95 200-275

0.5 18

CU(OAC)~ (NH,),PO,

120-160

18

(NHJaPO, ZnO

160 95

6,183 0.5 CU(OAC)~

0 0

0

0

0 0 0 0 0

29 30

0 0

0

0

0 0 0 0

20

0 0

0

0

0 0 0 0

20 29

0

was obtained. "Plus acetaldehyde. aPlus formaldehyde. *A racemic mixture, namely 4(5)-(~~-glycero-2,3-dihydroxypropyl)imidazole, OHOHH H H Paper chromatograms of the products obtained in the reaction of L-rhamnose and L-fucose dL-Rhamnose= H3CC-C-C-C-C=O. H H OHOH (6-deoxy-~-galactose)with aqueous ammonia are identi~al.~* e(NH4)2Mo04,(NH4)3P04,NH,VO,, (NH,),WO,, Co,O,, Cu(NH,),SO,, CuO, NiO, Na2A102+ SOz,ZnC12,and ZnO. B. N. Ames, H. K. Mitchell, and M. B. Mitchel1,J. Amer. Chem. SOC., 75, 1015 (1953). I. Jeio and I. LuGk, C k m . Zuesti, 17,255 (1963);Chem. Abstracts, 60,4139e (1964). K. Bemhauer, Z . Physiol. Chem., 183,67 (1929). R. Weidenhagen, R. Hemnann, and H. Wegner, Ber., 70, 570 (1937). M. Emoto, Nippon Nogei Kagaku Kaishi, 36, 461 (1962); Chem. Abstracts, 60, 2332d (1964). M. Gmoto, Nippon Nogei Kagaku Kaishi, 36,464, 546 (1962); Chem. Abstracts, SO, 2332qh (1964). F. Fujii and M. Ksmoto, Nippon Nogei Kagaku Kaishi, 39, 114 (1965);Chem. Abstracts, 63, 14953e (1965);M. KGmoto, ibid., 36, 541 (1962); Chem. Abstracts, 60,2332g (1964); M. Gmoto, S. Fujii, and H. Tsuchida, Hyogo Noka Daigaku Kenkyu Hokoku, 5, 124 (1962); Chem. Abstracts, 60, 2249d (1964). S. Fujii, H. Tsuchida, and M. Gmoto, Agr. Biol. Chem. (Tokyo), 30, 73 (1966). R. W. Liggett and H. L. Hoffman, Jr. (to Atlas Chemical Industries, Inc.), U.S. Pat. 3,030,376 (1962); Chem. Abstracts, 57, 9859g (1962). H. Tsuchida and M. KGmoto, Agr. Biol. Chem. (Tokyo), 31, 185 (1967). M. KGmoto and H. Tsuchida, Agr. Biol. Chem. (Tokyo), 32,983 (1968).

5 ?

3

REACTIONS O F FREE SUGARS WITH AQUEOUS AMMONIA

323

TABLEVII Reaction of Ammonia with Various Sugars with Oxygen Bubbled Through the Solution at Room Temperature in the Presence of a Catalyst Reaction Sugar

L-Arabinose D-Fructose

Time (days) 30 15 30 30

60 60 D-Fructose" b b

D-Galactose D-Glucose

20 60 15 C

Invert sugaf' D-Mannose L-Rhamnose L-Xylose

15 30 30

Catalyst Cu(OH)* Cu(OH)* MethyleneBlue NH,HCO, + Cu(OH)* (=CuCO, + NH,) Fe2(S04)3, MnS04, FeSO, Ca(OH)* CUCO, cuco3 Cu(OAc), Cu(OH)* Cu(OH)* CU(OH)~ Cu(0H)z CUCO$ CU(OH)~ CU(OH)~ CU(OH)~

Products Refer14 27 29 30 31 32 33 34 35 36 38 ences 0 0 0

0 0 0

0 0 0

11 11,40,41 11,42 11,43

0 0

0

11 0

0

?(+at least 1 unidentified)

0 0 0 0 0 0

0 0 0 0 0

0 0 0

0 0

0 0 0

0 0 0

0 0

11 44

45 45,46 1 1,47 11 11,40 48 49 11,50 11 11

uPlus formaldehyde. *2.5hr at 100"."3years (at room temperature). d2hr on hot-water bath, plus 6-8hr at room temperature.

(40)J. Parrod, Compt. Rend., 192,1136 (1931);P. Girard and J. Parrod, Ann. Physiol. Physicochim. Biol.,7,295(1931). (41)P. Girard and J. Parrod, Compt. Rend., 190,328(1930). (42)J.Parrod, Compt.Rend., 195,285(1932). (43)J. Parrod and Y.Garreau, Compt. Rend., 195,1110(1932). (44)W.J. Darby, H. B. Lewis, and J. R. Totter,J. Amer. Chem. SOC., 64,463(1942). (45)J. R.Totter and W. J. Darby, Org. Syn., 24,64(1944). (46)R. I. Meltzer, A. D. Lewis, F. H. McMillan, J. D. Genzer, F. Leonard, and J. A. King,]. Amer. Pharm. Assoc., Sci. Ed., 42,594(1953). (47)Y.Garreau and J. Parrod, Compt. Rend., 194,657(1932). (48)A. Windaus and A. Ullrich, Z. Physiol. Chem.,90,366(1914). (49)L. P. Kulev and R. N. Gireva, Zh. Prikl. Khim., 30, 811 (1957);J . Appl. Chem. USSR, 30,858(1957). (50)J. Parrod and Y. Garreau, Compt. Rend., 193,890(1931).

w

TABLEVIII

to

A

Reaction of a-Dicarbonyl or a-Hydroxycarbonyl Compounds with Ammonia in the Presence of Formaldehyde Reaction Time

Temp. Reactant 2,SButanedione (CHa-CO-CO-CH,) 3-Deoxy-D-glyceropentosulose (59) 3,6-Dideoxy-~-erythrohexosulose (60) 1,4-Dihydroxy-2-butanone (CH,OH-CO-CH,-CH,OH) 1,3-Dihydroxy-2-propanone (CH ,OH-CO-C H 2 0 H ) Glycolaldehyde (CHXOH-CHO) Glyoxal (CHO-CHO) D-aruhino-Hexosulose (61) 2-Oxobutanal (CH:$-CH 2-C0-CHO) Pyruvaldehyde (CH:,-CO-CHO)

Products

(degrees) (hours)(months)

Catalyst

0 120 room

12 8 24

-

room

30

-

55 ?

0.5 ?

room 40 95 100

short ?

? 0 room

? 12 24

room room 40

100

6 2.4

6

24 2

23

Zn(OH), Zn(0Hh Zn(OH)2 Zn(OH).L

25

27 30 34

35 41

0 0

0 0

0

References 51 51,52 36 38

0

CU(OAC)~ cuco, Zn(OH), Zn(OH), C u (0Ac) Cu(OAc), Zn(OH),!

immediate

18 19 21

0 0

53 0 0

54

31 55 55

0 0

0

56

0 0

0 0 0 0

g

33

11,40 39 54 31 31 31

g

REACTIONS O F FREE SUGARS WITH AQUEOUS AMMONIA

325

H

H

c=o I c=o I FHP HCOH

I

CH,OH (59)

H

c=o I c=o

c=o c=o I

7H2 HOCH I HOCH I c H3 (60)

H

c=o I c=o

I HOCH I HCOH

HCOH

HCOH

HCOH

I

I

CH,OH

(61)

I

y

2

I I

C H,OH

(62)

differ significantly from those under similar conditions in the absence of a catalyst (see Table 111). Similarly, the products of the reaction at higher temperature and shorter reaction-times (see Table VI) do not differ significantly from those under the same conditions in the absence of a catalyst (see Table IV). In Table VII are listed further reactions of sugars with aqueous ammonia in the presence of a catalyst at room temperature, but, in these cases, oxygen was passed through the solutions. The imidazoles formed in the reaction of aqueous ammonia with other a-hydroxycarbonyl compounds, for example, the triose DLglyceraldehyde, and such a-dicarbonyl compounds as 3-deoxy-~glycero-pentosulose (59), and the 3,6-dideoxy-~-erythro-,D-arahino-, and 3-deoxy-~-erythro-hexosu~oses (60, 61, and 62), respectively, are summarized in Table VIII for reactions in which formaldehyde was added, and in Table IX for reactions in which it was not added. (51) R. G. Fargherand F. L. Pyman,]. Chem. Soc., 115,217 (1919). (52) A. Windaus, Eer., 42,758 (1909). (53) C. F. Huebner,J.Amer. Chem. Soc., 73,4667 (195 1). (54) B. J. Sjnllema and A. J. H. Kam, Rec. Trac. Chim., 36, 180 (1916). (55) R. Weidenhagen and R. Herrmann, Ber., 68,1953 (1935). (56) R. Behrend and J. Schmitz, Ann., 277,310 (1893).

TABLEIX

Reaction of a-Dicarbonyl or a-Hydroxycarbonyl Compounds with Ammonia in the Absence of Formaldehyde Reaction Time

Temp.

Catalyst Products

Reactant 2,3Butanedione

3-Deoxy-~glyceraldehyde OH CH&--CHO H 3Deoxy-D-eythrohexosulose (62) 1,3-Dihydroxy-2propanone DL-Glyceraldehyde (CHZOH-CHOH-CHO) Glycolaldehyde

(degrees) (hours)(weeks) andreagent cold plus 95 100

Refer14 16 17 20 22 23 24 25 26 27 28 30 33 34 37 41 ences

-

0

57

z

.

8

3

4

1 1

-

room

21

-

room 40

24

2

CU(OH)~,O~ 0 Zn(OH), +CH3CH0

37

8

-

room

8

-

0

0

0

0

39

0 0

0

0 0

0

0

11 31

Ob

58

‘I

0

0

21

59

Glyoxal

60-70 room 0 ?

Hydroxypymvaldehyde (CH 2 0 H-CO-CHO) Pymvaldehyde

room room

-

48

48

Zn(OH)*+ CH,CHO NH,OAc HOAc (no NH,) + CHZOH-CHO

+

CHZOH-CHO

60 61 62 63

0 0

0

-

5

room

0 0

CH,CH,CHO

6 ?

0 0

CH,CHO (noNH,) CH,CHO.NH,

12

19 40

95

-

short ? 24 1

0 0

63 59

0 0

0 0

6 4 31

0

8

0

g +c,

2 3 g

’II

!a

m

m vl

c

’3 +0

0

58

“Possibly not found because of the small quantities used. bAlso identified: 1,3-dihydroxy-%propanone,DL-gluCOSe, DL-fructose, mannose, DL-arabinose, DL-lyxose, and DL-XylOSe (and, possibly, DL-ribose).

DL-

3:

s+ m 0 C vl

(57)H. von Pechmann, Ber., 21,1411(1888). (58)M.R. Grimmett and E. L. Richards, Aust.]. Chem., 17,1379(1964). (59) M. R. Grimmett and E. L. Richards, Aust.]. Chem., 18,1855(1965). (60) H. Debus, Ann., 107,199(1858). (61)G . Wyss, Ber., 10,1365(1877). (62)B. Radziszewski, Ber., 15,2706(1882). (63) B. Radziszewski, Ber., 16,487(1883). (64)M.R.Grimmettand E. L. Richards,]. Chem. SOC., 3751 (1965).

Ez

0

$ 0 E3

4

328

M. J . KORT

111. ISOLATION OF PRODUCTS, AND PROPORTIONS OBTAINED From the Tables of the products obtained from the reaction of various sugars with ammonia, it may be seen that the results often appear to be irreproducible from one worker to another; this is because, in some experiments, only certain products were being investigated or specifically synthesized. Often, other compounds were pres~ ' ~spraying -~~*~~*~~*~~ ent, as shown by paper c h r o m a t ~ g r a p h y , ~ - ~ ~ ~with with diazotized sulfanilic acid (the Pauly reagent),s5 with or without ammoniacal silver nitrate,66but all of them were not identified, and some could not be obtained crystalline." In the earlier work, too, the yields might have been too low for presence of the compounds to have been observed (before the advent of paper and thin-layer and thin-layer21.59*64 chrochromatography). Papel.8-'0.21.33-36,38,39,58.59,64 matography have been used as an aid in identifying the various products formed. Chromatography4 on ion-exchange resins applied to the reaction mixture, and cellulose-column chromatography of the brown syrup or after obtained after concentrating the reaction mixture,s*7,g,21.36*38,39 passing the reaction mixture through a column of an ion-exchange resin and then c o n ~ e n t r a t i n g ,have ~ ~ * ~been used for separating the components of reaction mixtures. Column chromatography on alumina . ~ ~ , ~the~ brown syrup was extracted has also been ~ ~ e dAlternatively, with ethefl*20*30 or ethanol,6' and, after removal of the solvent, the residue was distilled to yield the imidazoles8-6'or a pyrazine fraction and an imidazole f r a c t i ~ n . The ~~.~ still ~ residue8 could be separated into the constituent imidazoles by fractional recrystallization of their picrates. Fractional recrystallization of the picrates has also been pyrazine~,"-'~ -'~*~~*~~ used by other workers to isolate i m i d a ~ o l e s , ~ ~ ' ~ the reaction mixtures. The percentages of the and p i c ~ l i n e 'from ~ pyrazine and imidazole fractions obtained at various temperatures and with various proportions of catalyst^,^^*^^ and with different catalysts and times of reaction,20 have been given in detail. With sucrose,Po the overall yield of both fractions increased with time, indefinitely, 18 hours being the most convenient length of time. In the absence of a catalyst, 220" was the optimal temperature (16.8% of pyrazine fraction and 8.3% of imidazole fraction). The most effective catalysts were ammonium phosphate (0.625%in the reaction mixture gave the highest yields) and zinc oxide, with yields at the (65) H. Pauly,2. Physiol. Chem., 42,508(1904);44,159(1905). (66)S.M.Parbidge,l3iochem.J..42,238(1948).

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

329

optimal temperature (200") of 12.60 and 10.20% of pyrazine and 7.20 and 2.20% of imidazole fractions, respectively. With wheat starch,305% was the optimal concentration of the ammonium phosphate catalyst; and the optimal temperature for beet and cane molasses, lactose, cellobiose, starch syrup, dextrin, and the "dialdehyde" of starch was 225", and for cellulose and wheat, corn, and potato starch, it was 250". The percentages of the individual imidazoles and pyrazines obtained from the imidazole and pyrazine fractions from the various carbohydrate^^^.^^ were given in detail. Only one example, sucrose, is shown in Table X, in which the quantities of the products obtained in some of the reactions detailed in Tables I and 111-IX are summarized. The yields of some of the products obtained are given in Table XI for those experiments in which the amounts of the insoluble copper or zinc salts of the imidazoles were determined. Brandes and StoehrI4 (see Table IV) added potassium hydroxide to each reaction mixture before distilling it, and added sodium hydroxide to the distillate, liberating an oil which was separated and purified, and from which pyridine and four pyrazines were separated by means of their mercury and gold salts. In the various experiments in which a zinc hydroxide catalyst was used (see Tables V, VI, VIII, and IX), the zinc salts of the imidazoles precipitated from the reaction mixtures, and, after they had been or in filtered off, they were suspended either in warm waters*1'*24,36--39 and the zinc was precipitated as sulfide by bubacetic bling hydrogen sulfide through the suspension. The zinc sulfide was filtered off, the clear filtrate was evaporated to a syrup, and the base was either (a) extracted directly into chloroform and the product distilled after removal of the chl0roform,3~or (b) extracted into acetone,2s into ether,22 or into ether after addition of potassium Removal of the ether yielded an oil from which the various imidazoles were separated by fractional recrystallization of their oxalates23-27~31*54 or p i ~ r a t e s . ~ Cellulose-column ~,~~,~' chromatography of the ether extract also separated the constituent imidazo1es.36,38,39 The free imidazoles could be liberated from their oxalates24,25*54 by adding potassium carbonate, extracting with ether, and evaporating the extract; and from their picrateP by adding sulfuric acid, filtering off the picric acid (which was completely removed from the solution by washing with ether), concentrating the solution, adding potassium carbonate, extracting with ether, evaporating the extract, and distilling the residue to give a pure product. Similarly, where copper salts were used as the catalyst (see Tables -27331s4

0 0 0

TABLEX Yields of the Products Obtained Reactant

16

%Amino-Zdeoxy&glucose L-Arabinose 2,3-Butanedione D-Fructose

20

21

27

28

Weight (g) of product, based on 100 g of reactant 30 31 34 39 41 42 44 45 48 3.0

49

50

51

54

55

1.05 3.67

9

5

29 13.2

19.6 25.528.2

D-Glucose

0.05 6.71

0.12

0.2 0.1

1.o

0.2

4 Glyoxal Invert sugar

References

1.5

2.75 16.423.7"

7

8

8

s

14 48 61 49

Pyruvaldehyde

75.8b

54 64

Sucrose

12.4 4.71

"Assulfate.bAs oxalate.

0.38 0.15 0.31

2.65 0.88 9.95

0.81

m

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

331

TABLEXI

Yields of Products Obtained, Where the Amount of the Copper or Zinc Salt Was Determined Reactant L-Arabinose

Cu"

ReferWeight (9)of product, based on 100 g of reactant Zn" 1 4 20 27 29 30 33 34 35 36 ences

105

4.4

11 22 23 11

31

12.5 21 1,3-Dihydroxy- 150 2-propanone 1,3-Dihydroxy2-propanone* 155 D-Fructose

0.1

36

0.1

12

8 3

15

31 11

2.5

26 20 D-Galactose

38.9

77.8 180

0.4 15

5

0.5

10

2.8

24.2 D-Glucose

1.5

145

24

1

26 20 31

10 9.4c

40

D-Ghcose* Invert sugar Lactose Maltose D-Mannose

Pyruvaldeh ydeb L-Rhamnose 10 D-Sorbose L-Xylose

31

38.7 24 16 0.8,1.4 4.8,7.9 80

8

4

10

5

3

26 33.3 0.1 I .5

3

1.7 21

8.5 9.6c

1.9

33 32 26

142

11 23 31 32 11 22 23 11 23 24 26

7.3

37 31 31 23 23 11 23 31 11 23 27 23 11 23

"Cu = Insoluble copper salt of the imidazoles; Zn = insoluble zinc salt of the imidazoles. *Plusacetaldehyde. 'As oxalate.

V to IX), the copper complexes of the imidazoles also precipitated from the reaction mixture, and were filtered off. Oxalic acid and 4(5)-imidazole (30)were determined in the f~ltrate.".~~*~' The complex was suspended in hot water,11~29~32~40*4'~43~47*50~53~55 dilute sulfuric a ~ i d , ~or * ,dilute ~ ~ hydrochloric the copper was removed as the sulfide, with hydrogen sulfide or sodium sulfide,49and the excess of hydrogen sulfide was removed with lead a ~ e t a t e . " * ~The ~ * clear ~'~~~

332

M.J. KORT

solution obtained after filtering off the precipitated copper sulfide was further purified with decolorizing carbon32*49*53 and evaporated in oucuo to give the i m i d a z ~ l e , ~or~the * ~product ~ * ~ ~ in the residue was then extracted into c h l o r o f ~ r m ,or ~ ~the residue was distilled55 to afford the pure imidazole. In addition, ion-exchange chromatography has been used for isolating the i m i d a z ~ l e s .Alternatively, ~~ the imidazoles were isolated from the copper-free solution as the picrates,11~40~41*44~47*so which were separated ( a ) by fractional recrystallization, (b) as the p h o s p h o t u n g ~ t a t e ~ (after ~ * ~ ~removal , ~ ~ * ~ ~of ammonia by extraction with ether48),or (c) by concentration of the solution in the presence of barium hydroxide and removal of the barium ions with sulfuric The free base was liberated from the picrate by adding sulfuric acid, removing the picric acid liberated by filtration and ether extraction, neutralizing the sulfuric acid with barium carbonate, filtering, and concentrating the liquid to a small volume, whereupon the free base crystallized out on Alternatively, for isolating the free base, a mixture of a solution of the picrate with potassium carbonate was evaporated to dryness, and the free base was extracted into hot acetone, from which it crystallized on To obtain the free base from the phosphotungstate, the salt was suspended in water, barium hydroxide solution was added, and the suspension was filtered. After addition of carbon dioxide to remove the barium, and filtration, the filtrate was evaporated, and the residue was recrystallized from a l ~ o h o l " , [for ~ ~ ,4(5)~~ (hydroxymethy1)imidazole (27)] or from aqueous acetone4s [for imidazole-4(5)-carboxylicacid (31)l. Use has been made by Grimmett and R i c h a r d ~of~ a~ quantitative, ,~~ colorimetric method for determining imidazole derivative^.^^ IV. MECHANISM

1. Lobry de Bruyn- Alberda van Ekenstein Transformation

The products obtained by the reaction of sugars with aqueous ammonia for a short time at low temperature, in the absence of catalysts (see Table I) are simply those obtained by the action of alkali on the sugars. The reaction is known as the Lobry de BruynAlberda van Ekenstein transformations7 after the chemists who dis(67)c.A. Lobry de Bruyn, Rec. Trao. Chim., 14, 150 (1895);c.A. Lobry de Bruyn and W. Alberda van Ekenstein, ibid., 14, 195 (1895);15,92 (1896);16,241,245, 256, 264 (1897);18, 147 (1899);J. C. Speck, Jr., Aduan. Casbohyd. Chern., 13, 63 (1958).

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

333

covered this rearrangement, which occurs when a reducing sugar is dissolved in water containing an alkaline catalyst; the transformation is illustrated in Scheme 1 for D-glucose. H C=Q

H cCOH

rl

11

H-COH

0

C-0-H I

I

HOCH

HOCH I

I

R

-

R

ChOH I

n

C=O 17 HOC-H I R

CH,OH I

COH II

HOC I

R

o-Fructose

o-Glucose

(3)

(1)

H CH,OH

C=O

I

c=o

HOCH

I HCOH I R

I

HOCH I

R

o-Peicose

D-Mannose

(5)

(4)

I HCOH I

where R = HCOH I

CH,OH

. Scheme I

The reaction of the sugars with ammonia was stopped before condensation with ammonia could occu13 and only the epimeric aldoses and the corresponding ketose were ~ b t a i n e dHowever, .~ on prolonged reaction, i m i d a z o l e ~ ~or- ~other ~ ~ condensation products4a5[namely, di-D-glucopyranosylamine (6), D-glucopyranosylamine (7), and 2amino-2-deoxy-D-glucose (8)] were also isolated (see Table I). 2. Formation of Glycosylamines and Aminodeoxy Sugars There are two theories as to the mechanism of the formation of imidazole and pyrazine compounds, namely, ( a ) the prior formation of glycosylamines and aminodeoxy sugars, and ( b )the fragmentation mechanism. The similarity of the products obtained from the mono- and the di-saccharides supports the hypothesis of Je5020*68 and Jeio and Luihk30 that the di- and poly-saccharides are first hydrolyzed to the monosaccharides. Although most glycosidic linkages are stable to (68)I.Jeio,Listy Cukroun~.,82,259(1966);Chem.Abstracts, 67,3201k(1967).

M. J. KORT

334 hydrolysis Sucrose

__ __t

+

o-Glucose

o-Fructose (3)

HO H NH, I l l R-C-C-C-H I

H

l

HO R-C-

l

OHOH D-

Amadori

H

I I

OH

Fructosylamine Heyns

(see Schemes 4 and 5)

HO

I I

NH, C-CH,OH

I

H

0-Glucosylamine

R-C-

I

HO I

H

H

l

l

R- C -C- C =O

C -CH,NH, I1 O

I

H

1 -Amino- l-deoxy-

1

NH,

2- Amino- 2- deoxy~-glucoiie

D- f ructose

HO I R-C-C=O

HN

‘CH-C-R

I

-+

2-Amino- 2-deoxy D-glucose

+

2 -Amino- 2-deoxy D- glucose

1- Amino- 1-deoxy o-fructose

+

2. dehydration

..

A

m

e

H

r ization

- H,O

-

F

H P OH

where R = -c-L-CH,OH. I I H H

Formation of Pyrazines

Scheme 2

I I OH

2- Amino- 2-deoxy o- glucose

the double bond

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

335

alkali at 25- lOO", Lindberg and coworkers69investigated the alkaline hydrolysis of some glycosides at 170". Hydrolysis certainly occurred, although often to only a small extent. The reaction of sucrose with ammonia was conducted at a high temperature, and even should it only be hydrolyzed to a small extent, this would be sufficient to support the mechanism of J e i o and Lui&ka3O The hypothesisz0,68for the next step is the formation of 2-amino-2-deoxy-D-glucose from Dfructose and ammonia, without prior fragmentation of the sugar molecules.I0 Condensation of this product with a second molecule or with a molecule of l-amino-l-deoxyof 2-amino-2-deoxy-~-glucose D-fructose (similarly formed from the D-glucose produced and ammonia) would give the substituted pyrazines,20,68as illustrated in Scheme 2, and the substituted imidazoles,68as shown in Scheme 3. The Amadori rearrangemenPo of D-glucopyranosylamine to 1amino-1-deoxy-D-fructopyranose, and the Heyns rearrangement7' of D-fructopyranosylamine to 2-amino-2-deoxy-D-glucopyranose,are shown in Scheme 4. These mechanisms for the Amadori and Heyns rearrangements are, in effect, two alternatives for the same type of transformation, even though the former involves protonation of the nitrogen atom at C-1, and the latter, protonation of the oxygen atom at C-6. Indeed, the mechanism of the Amadori rearrangement has been explained on the basis of N - p r o t o n a t i ~ nand ~ ~ of O-protonat i ~ n Hodge70 . ~ ~ prefers the former. Although the mechanisms shown in Scheme 4 are acid-catalyzed, the Amadori rearrangement occurs also in alkaline media. The mechanisms proposed for the Amadori and Heyns rearrangements in alkaline solution are shown in Scheme 5. Jeio further proposedz0 that the methylpyrazines are formed by thermal detachment of the side chains (see Scheme 6); these hagments could then combine with ammonia to form the imidazoles. From Scheme 2, it may be seen that D-glucosylamine and D-fructosylamine are also considered to be intermediates in the formation of heterocyclic compounds. This intermediary formation of l-amino1-deoxy-D-fructose and D-glucosylamine from D-glucose and am-

(69)B. Lindberg, Suensk Papperstidn., 59, 531 (1956);E. Dryselius, B. Lindberg, and 0. Theander, Acta Chem. Scand., 11, 663 (1957);12, 340 (1958);J. Janson 14,2051(1960). and B. Lindberg, ibid., 13,138(1959); (70)J . E. Hodge, Adoan. Carbohyd. Chem., 10,169(1955). (71)K. Heyns, H. Paulsen, R. Eichstedt, and M. Rolle, Chem. Ber., 90, 2039 (1957); K. Heyns and K.-H. Meinecke, ibid., 86,1453(1953). (72)R. Kuhn and F.Weygand, Ber., 70,769(1937);F.Weygand, ibid., 73, 1259 (1940); H.S.Isbell, Ann. Reu. Biochem., 12,205(1943). (73)A. Gottschalk, Biochemj.,52,455(1952).

M. J. KORT

336

Sucrose

I

hydrolysis

INH,

D-Glucose

t

1

R----T

D-

2- Amino- 2-deoxy- D- glucose

H

H0 H H OH H/NHe\ I I LCH-C-C-R

I

n HC=O U .

H

I

I

Cf,:

I-\/> HI OH I cOH R-CH ,CH-C-C-R

1

OHH

HocH,-A-iiH I Ll HO H

HN? ' H

H

I

where R = -C-C-CH,OH 1 1 HO OH

Fructosylamine

i

rearrangement

1- Amino-1-deoxy-D-fructose

I

(see Scheme 2)

+

D-Glucosylamine

H

D-Fructose

,

Formation of Imidazoles Scheme 3

I

I

HO

H

"7 Hi[g

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

337

Amadori Rearrangement

0

cfi".

CH H-COH f-1 I

+Ho

HCOH I

HOCH

HOCH

~

I

HCOH I HCO

-

HCOH

1

HCOH I HCOH

H&Oq I

I

CH,OH

1

CH,OH

CH,OH

a-D - Glucopyranosylamine

I

HYOH

HFOH

HToH J

HCOH/

I

HOCH I

HYOH HCOH

.. CH,Od-H I

C%O

1-Amino- 1-deoxy

-

I

CH,OH

- (Y

D-fructopyranose

Heyns Rearrangement

-

HO~H I I

I CH,O (Y-D-

CH,-0

i

HCOH I

HCOH I

CH,OH

H

/lH@

Fructopyranosylamine

HCNH, I HOYH

"PO

HCO I

CH20H

Z-Amino-2-deoxy-ao-glucopyranose

Scheme 4

HCO-H 10

CH,OH

M. J. KORT

338

"1

Amadorl Rearrangement

HOCH

r-

HCNH, H-C-0 /I f-El I alkali c. HOCH I

HCOH

HCOH

HCO

HCO I CH,OH

I

I

CH,OH

CH,NH, I

c=o

-

I HOCH

1

HCOH

n

H&O@ I CH,OH

a-D-Glucopyranosylamlne

CH,NH, I

c=o I

-

1

HCOH

HOCH HCOH I HCOH

I

C H,O

C H,OH

1-Amino- 1 -deoxy- a-0-f ructopyranose

Heyns Rearrangement H

c=o

H ~ - C 1H- J -- C N ~

HCOH HCOH

-

I HOCH I HCOH I HCOH I f CH,O

alkali

CH,O

I

HCNH, I

HOCH I

__c

HCOH I HCOH &,O@

a-u-Fructopyranosylamlne H

c=o

HCOH

-

HCO I

CH,OH

I HCNH, I HOCH I

HCOH I HCOH I

CH,OH

2-Amino-2-deoxy- a-D-glucopyranose

Possible Mechanisms of Amadori and Heyns Rearrangements in Alkali Scheme 5

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA H O*C-

+

I

H

339

OH

CI -CH,OH 1

H

Scheme 6

monia, and of 2-amino-2-deoxy-~-glucoseand D-fructosylamine from D-fructose and ammonia, has some experimental support. (a) The isolation of 2-amino-2-deoxy-~-glucose from the reaction of D-glucose and D-fructose with a m m ~ n i aat ~ low ~ ’ ~temperature (see Tables I and 111). ( b ) The isolation of D-glucosylamine from the reaction of Dglucose with ammonia at low temperature4*I2(see Tables I and 111) and at high temperature’* (see Table IV). ( c ) 2-Amino-S-deoxy-~glucose and ammonia at low temperature for 6 months yieldedg three of the substituted pyrazines (42, 50, and 51) (see Table 111) obtained from sucrose and ammonia at high temperaturelo for a shorter time (see Table IV). The high temperature would decrease the time of the reaction with sucrose, and would be necessary in order that the sucrose would first be hydrolyzed. ( d )2,5-Bis(~-arabino-tetrahydroxybuty1)pyrazine (42) has been synthesized by self-condensation of two molecular proportions of 1-amino-l-deoxy-~-fructose~~ or of 2-amino-2-deoxy-~-glucose.~ The pyrazines 50 (Ref. 9), 51 (Ref. 9) (see Table III), and 53 (Ref. 74) were also obtained from the selfIn addition, a similar condensation of 2-amino-2-deoxy-~-g~ucose. disubstituted pyrazine, namely, 2,5-bis(~-erythro-2,3,4-trihydroxybutyl)pyrazine, has been synthesized from two molecular proportions Indeed, the formation of 2,5of 3-deoxy-~-ribo-hexosylamine.~~ bis(D-arabino-tetrahydroxybuty1)pyrazine (42) (D-frUCtOSaZine)from 1-amino-1-deoxy-D-fructose(“isog~ucosamine”),76from 2-amino-2deoxy-D-glucose (“D-g~u~osamine”),~~ and from D-fructose plus ammonia78 (which form 2-amino-2-deoxy-~-glucose)~has been known for a long time. Although the early workers could not prove the formation of 2-amino-2-deoxy-~-g~ucose from the reaction of D-fruCtOSe with a m m ~ n i a , Lobry ~ ~ - ~ de ~ Bruyn assumed its presence. Heyns and Koch5 have, however, since obtained 2-amino-2-deoxy-~glucose from D-fructose plus ammonia, and have proposed a mecha(74) R. Kuhn, G. Kruger, H. J . Haas, and A. Seeliger, Ann., 644,122 (1961). (75) F. Micheel, S. Degener, and I. Dijong, Ann., 701, 233 (1967). (76) K. Maurer and B. Schiedt, Ber., 68,2187 (1935). (77) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. Truu. Chim., 18, 77 (1898); C. A. Lobry de Bruyn, Ber., 28, 3082 (1895); 31, 2476 (1898); R. Breuer, ibid., 31, 2193 (1898); K. Stolte, Beitr. Chem. PhysioE., 11, 19 (1908). (78) C. A. Lobry de Bruyn, Rec. Trau. Chim., 18, 72 (1898).

340

M. J. KORT

nism for the reaction. By analogy, it is therefore possible that l-aminol-deoxy-D-fructose might be formed in the reaction of D-glucose with ammonia. In contradiction to this intermediary formation of the amines, K?5moto'2 found that D-glucosylamine partially decomposes at 20", and at 100", to D-glUCOSe and ammonia (see Tables I11 and IV). He maintained that D-glucosylamine, the condensation product of Dglucose with ammonia, does not, therefore, appear to be the primary intermediate product in the browning reaction between D-glucose and aqueous ammonia. However, the D-glucosylamine was only partially decomposed, and K6moto estimated the velocity of browning by measuring the absorption at 400 and 580 nm. Hence, his evidence does not negate the possible presence of D-glucosylamine as an intermediate in the formation of the pyrazines and imidazoles. 3. Fragmentation Mechanism

For imidazole formation, Windaus and Kn0opz4proposed that a molecule of D-glucose decomposes to give two D-glyceraldehyde molecules, and that the D-glyceraldehyde could either decompose further to formaldehyde, or form pyruvaldehyde by dehydration followed by migration of a hydrogen atom. The fragmentation products from L-rhamnose were also d i s c ~ s s e d . ~P~a, ~r r*~ d " 'extended ~~ this hypothesis to D-fructose, which would fragment to D-glyceraldehyde plus 1,3-dihydroxy-2-propanone. He then proposed occurrence of oxidation of the free sugars to a-ketoaldehydes; for example, of D-glucose (l),D-mannose (4, and D-fructose (3)to D-arabino-hexosulose (61),D-galactose (9) to D-lyxo-hexosulose, and D-glyceraldehyde and 173-dihydroxy-2-propanone to hydroxypyruvaldehyde. D-Glyceraldehyde could further fragment to formaldehyde and glyoxal, which could be oxidized to the oxalic acid that he isolated in his reactions. KGmoto also proposed the conversion of decomposition products of D-glucose into unstable a-diketones and aldehyde^.^,^^,^^ The aketoaldehydes could then condense with ammonia and the formaldehyde to give the various imidazoles shown in Scheme 7. In support of this mechanism, imidazoles are commonly prepared from adicarbonyl compounds (a-diketones and a-ketoaldehydes) and ahydroxycarbonyl compounds, with or without a catalyst, some examples of which are listed in Tables VIII and IX. The results of further investigations strongly support this formation of a-diketones and aldehydes. Before this work is considered, it is pertinent to discuss the action of alkali on sugars. As has already

REACTIONS O F FREE SUGARS WITH AQUEOUS AMMONIA

341

been noted, the action of ammonia on reducing sugars initially parallels the action of alkalis, which cause epimerization (the Lobry de Bruyn-Alberda van Ekenstein transformation).

44

H-N

.
%=CR

-

H-NH

HN tS\H yCR

L E 'R

c/

HO

H-NH, L A

-

HN.

\H R'C=O CR I n I R"C=O :NH, LJ

where (A) = an aldehyde, (B)= an a-diketone or a-ketoaldehyde, and (C) = a substituted 4(5)-imidazole.

Scheme 7

4. Saccharinic Acids Among the products of the action of alkalis on substituted hexoses are saccharinic acids; their nature depends on the position of substiand l-O-substituted-~-fructoses~~~~~ tution of the hexose. D-Fruct~se'~ yield some saccharinates together with high yields of lactic acid;81 3-0-substituted s ~ g a r s ~and * , ~D ~ - g l u c o ~ egive ~ ~ metasaccharinates, The and 4-0-substituted sugars produce i s o s a c c h a r i n a t e ~ . ~ ~ . ~most ~ widely accepted mechanism for formation of saccharinic acids is that suggested by Isbell,86which involves ( a ) formation and ionization of an enediol; (b) the p-elimination of a hydroxyl or an alkoxyl group; (c) rearrangement to an a-dicarbonyl intermediate; and ( d ) a benzilic acid type of rearrangement to the saccharinic acid (see Scheme 8). (79) A. Ishizu, B. Lindberg, and 0. Theander, Acta Chem. Scand., 21, 424 (1967). (80) A. A. J. Feast, B. Lindberg, and 0. Theander, Acta Chem. Scand., 19, 1127 (1965). (81) J. Kenner and G. N. Richards, J . Chem. Soc., 1784 (1954). (82) W. M. Corbett and J. Kenner, J. Chem. SOC.,3274 (1954). (83) J. Kenner and G. N. Richards, J . Chem. Soc., 278 (1954). (84) H. Tsuchida, S. Fujii, and M. Komoto, Hyogo Noka Daigaku Kenkyu Hokoku, 6, 73 (1964); Chem. Abstracts, 64, 5280c (1966). (85) J. Kenner and G. N. Richards, J . Chem. Soc., 1810 (1955). (86) H. S. Isbel1,J. Res. Nut. Bur. Stand., 32,45 (1944); J. C. Sowden,Aduan. Carbohyd. Chem., 12, 66 (1957).

M. J. KORT

342

H

H r c-0

C=%

H

0

c=o

@OH

H ~ O H I ROCH (a)

01 RO-CH

HLOH I

HAOH I

HCOH I CqOH

A-f-H CkH -

1

HCOH I

HCOH

HCOH I CGOH

H-C=O

CO8H I CHOH

LAG

(c)

I

I ?Ha HCOH I HCOH

I

+H@&

I

CH,OH

CHaOH

chon

Glucometasaccharinic acid

(62)

u-Glucose (R = H) or 3-0-substituted u-glucose (R = alkylf

HCOH I I

+OH@ HCOH

(65)

H

c=o

CH,OH

c=o lY(

HCOH I I

HOCH I

HYOR HCOH I CbOH

*

HOC-H 1 7 I

HYOR

HCOH I CH,OH

4 - 0 -Substituted D-glUCOM

(R = alkyl)

Isosaccharink acid (66)

Scheme 8

CH,OH

('1

;rO@ (!OH HC-OR fl I

HCOH I

CH,OH

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

c=o I

HOCH H~OH I

- *

HCOH I CH,OH

H-COH I C=O I 0

H~OH I HCOH

-

343

c4 I

HCOH I HCOH I C&OH

I

C&OH

1 -0-Substituted

D-fructose (R = alkyl)

CO,H I H,CCOH (4 I HCOH r: I +H@ HCOH I

ChOH Saccharinic acid

+OHa

6-C-H

(&3

c=o I

HCOH I

HCOH I

C&OH

A=O I w HCOH e (c)

I

HCOH

(!&OH

(64)

(6 7)

where

*

is the Lobry de Bruyn-Alberda van Ekenstein transformation.

Scheme 8

Each of the reactions in Scheme 8 requires the formation of a dicarbonyl intermediate (62,63, and 64).Two of these, namely, 3-deoxy~-erythro-hexosulose~'(62)and 4-deoxy-~-gZycero-2,3-hexodiulose~~ (63),have been isolated from the action of alkali on substituted sugars (3-0-benzyl- and 4-O-substituted-~-glucose,respectively). In addition, the first intermediateE9(62)and the third intermediate, namely, l-deoxy-~-erythro-2,3-hexodiulose~ (64),have been synthesized. On treatment with lime-water, these three dicarbonyl compounds, 62, 63, and 64,yielded the expected saccharinic acids, D-glucometasaccharinic acid (65)fr0m8738962,D-glucoisosaccharinic acid (66)frome8 63,and D-glucosaccharinic acid (67)fromg064.Similarly, D - u ~ u ~ ~ o hexosulose (61) has been found to rearrange to D-mannonic acid in alkaline solution.g1 (87) G. Machell and G. N. Richards, J . Chem. SOC., 1938 (1960). (88) R. L. Whistler and J. N. BeMiller, j . Amet. Chem. SOC.,82,3705 (1960);G. Machell and G. N. Richards, J . Chem. SOC., 1924, 1932 (1960). (89) E. F. L. J. Anet, J . Amer. Chem. SOC., 82, 1502 (1960);Aust. J . Chem., 14, 295 (1961). (90)A. Ishizu, B. Lindberg, and 0. Theander, Carbohyd. Res., 5, 329 (1967). (91) B. Lindberg and 0. Theander, Acta Chem. Scand., 22, 1782 (1968).

M. J. KORT

344

5. Reaction of Dicarbonyl Compounds with Ammonia This benzilic acid type of rearrangement is the result of the action of alkali on the dicarbonyl compound, and is accelerated by calcium ions. The formation of saccharinic acids by the action of aqueous alkali on sugars is very well k n ~ ~ nhowever, ; ~ ~ if ~ammonia ~ ~ *is ~ ~ present, very little6 or no production of saccharinic acid has been reported. The reaction of the intermediate carbonyl compounds with ammonia is faster than the benzilic acid type of rearrangement to give saccharinic acid, and, hence, substituted imidazoles are formed, as illustrated in Scheme 9. 3-0- Substituted glucose

H

c=o I c=o

H

I

7%

alkaline flssion

1

H&=o

'

HCOH I HCOH 1 ChOH

X

n

(37)

(62)

Scheme 9

Again, supporting evidence that the hexosulose (62) is an intermediate in the formation of imidazoles in the reaction of D-glucose with ammonia was provided by Grimmett and coworkers.21 They treated hexosulose 62 with ammonia, and obtained the imidazole 37 (see Table IX). The same imidazole was obtained from 3-0-methylon D-glucose and from turanose (3-O-c~-~-glucopyranosyl-~-hctose) treatment with ammonia (60 hours and 2 weeks, respectively, at room temperature).21 Sophorose (2-O-~-D-g~ucopyranosy~-D-g~ucopyranose) yielded no imidazoles with ammonia ( llOo, 5 hours),21in agreement with the fact that 2-0-substituted sugars do not form saccharinic acids with aqueous alkaLg3This treatment was obviously too short for hydrolysis of the disaccharide to occur. Indeed, after prolonged heating, traces of 4(5)-methylimidazole (34) could be (92)W.M.Corbett and J. Kenner, J . Chem. SOC., 2245 (1953);1789,3281(1954);1431 (1955). (93)R. L.Whistler and W. M. Corbett, J . Amer. Chem. SOC., 77,3822,6328 (1955).

REACTIONS OF FREE SUGARS W I T H AQUEOUS AMMONIA

345

detected in a mixture of sucrose and ammonia.92Other imidazoles have also been synthesized from glycosuloses: 4(5)-(~-erythro-2,3and 4(5)-(Ddihydroxybuty1)-, 4(5)-(~-gZycero-2,3-dihydroxypropyl)-, arabino-tetrahydroxybuty1)-imidazole (18, 19, and 35), from formaldehyde, ammonia, and 3,6-dideoxy-~-erythro-hexosulose~~ (60), 3-deoxy-D-glycero-pentosulose36(59), and D-arabino-hexosulose11~40 (61),respectively (see Table VIII).

6. Fission of Reducing Sugars in Alkaline Solution It has been known for more than sixty years that formaldehyde, glycolaldehyde, glyceraldehyde, 1,3-dihydroxy-2-propanone7 pyruvaldehyde, and l-hydroxy-2-propanone result from sugars treated under alkaline conditions, through reversed aldol reactions, internal oxidations and reductions, dehydrations, and rearrangement reactions.94 Acids representative of lower-carbon fragments were also obtained by the action of caustic alkali on hex0ses,9~and four-, five-, and sixcarbon acids were detected in a study of the saccharinic acids obtained from D-xylose and D-fructose in alkaline medium.51 It was therefore concluded that isomerization of the sugars to the saccharinic acids involves fragmentations and recombination^.^^ The modes of breakdown of glyceraldehyde,58 hydroxypyruvaldeh ~ d e and , ~ reducing ~ sugars1*11,33*34 have been discussed. KiYmoto3S*34 (62) from postulated the formation of 3-deoxy-~-erythro-hexosulose D-gluCOSe (1). Compound 62 could subsequently break down to pyruvaldehyde (68) and D-glyceraldehyde (69) by a reverse aldol reaction, and the D-glyceraldehyde [or 173-dihydroxy-2-propanone (70)] could break down to glycolaldehyde (71) and formaldehyde (another reverse aldol reaction) (see Scheme 10). Kcmoto detected lactic acid in the mixture from reaction of D-glucose with a m r n ~ n i aand , ~ presumed that it was produced from pyruvaldehyde formed by decomposition of D-glucose. Lactic acid has, indeed, been found as a product of the action of alkali (lime-water) on substituted D-glucose and substituted ~ - f r u c t o s e , * and ~ * ~the ~ * mech~ anism of its formation involves the reversible aldol reaction, followed by formation of pyruvaldehyde, and the benzilic acid rearrangement already described for saccharinic acid; this is i l l u ~ t r a t e d *in~ Scheme *~ 11. (94)J . U. Nef, Ann., 357,214 (1907);376,1 (1910);403,204 (1913). (95)J. U. Nef, Ann., 335, 191 (1904). (96)J.Kenner and G. N. Richards, J . Chem. Soc., 2916 (1956).

M. J. KORT

346

H

H

c=o I c=o I

H

c H3

c=o

c=o

I I C=O HCoH I (as in I HOCH Scheme 7) cYH, I L HCOH H-C~-H I I HCOH HCOH I I CH,OH CH,OH 3-Deoxy-DD-(;luCOSe erythro hexosulose

H

c=o I

+

H,C=O

CH,OH

(68) - t

,=a/f

H

H~AOH

I

CH,OH

(71)

H-COH ‘k-w-H

\

CH,OH

~ = o

I

I C %OH

CH,OH (70)

(69)

(62)

(1)

Scheme 10

H o H

c=o I

HCOH I

HOCH

n HC-0-H I HCOH I CH,OMe

c=o

f-1

H-COH I CH,OH

L

+

-H

H o

c=o /I H-COH

I

CH,OMe Lactic acld

Scheme 11

In the presence of ammonia, as already mentioned for 3-deoxy-Derythro-hexosulose (see Scheme 9), the pyruvaldehyde will condense with ammonia (to form imidazoles) faster than the competitive reaction of formation of lactic acid (the benzilic acid rearrangement). Indeed, by “trapping,” with (2,4-dinitrophenyl)hydrazine, any ketones formed, Tsuchida and coworkers84were able to demonstrate the formation of pyruvaldehyde, formaldehyde, glycolaldehyde, and 3-deoxy-~-g~ycero-pentosulose, as well as 3-deoxy-~-erythro-hexosulose, from the reaction of D-glucose with lime-water, although glucometasaccharinic acid, lactic acid, and four other carboxylic acids were still obtained.

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

347

Strong support for the hypothesis that ammonia reacts with the dicarbonyl intermediate pyruvaldehyde is given by the fact that 4(5)-methylimidazole is the main imidazole produced when 1,6linked disaccharides react with ammonia.6 In addition, 4(5)-methylimidazole, sometimes only in traces, found in the products of reaction of all reducing disaccharides with ammonia, can be attributed to some reverse aldolization of the carbohydrates to form pyruvaldehyde and f ~ m a l d e h y d e The . ~ ~ intermediary formation of pyruvaldehyde from D-ghCOSe in ammonia has been demon~trated~l by its isolation as its phenylosazone, (p-nitropheny1)osazone and its bis(semicarbazone). In hexose-ammonia systems, formaldehyde is undoubtedly formed by direct degradation of the hexose molecule,54and Grimmett and RichardP4have demonstrated the formation of formaldehyde from pyruvaldehyde in aqueous, ammoniacal solution, although previous ~ o r k e r s ~had l . ~not ~ observed this.

7. Imidazole Formation

One-carbon to six-carbon carbonyl compounds are, thus, clearly obtained by the alkaline degradation of sugars, and K O ~ O ~ O , ~ * ~ ~ ~ S ~ ~ , probably ~ ~ * ~ ~ Parrod,’ l s 4 0 and Windaus and C O W O ~ ~ ~ therefore, correctly, postulated the formation of the different imidazole derivatives they obtained from the various breakdown products of the sugars. To give a few examples, in Scheme 7 (see p. 341),firstly, with R = R’ = H (A = formaldehyde) and R“ = CH3, B = pyruvaldehyde, and C = 4(5)-methylimidazole (34);if R” = -CH2CH20H, B = 3deoxytetrosulose, and C = 4(5)-(2-hydroxyethyl)imidazole (25);if R” H OHOH = -C--C--C--CH,OH, B = D-arabino-hexosulose, and C = 4(5)-(DOHH H arabino-tetrahydroxybuty1)imidazole (35) (only obtainable from D-glucose, D-mannose, D-fructose, or D-arabino-hexosulose); secondly, with R = R” = CH3 and R’ = H, A = acetaldehyde, B = pyruvaldehyde, and C = 2,4(5)-dimethylimidazole (20); and finally, with R = H, R’ = CH3, and R“ = CH3CH2, A = formaldehyde, B = 2,3-pentanedione, and C = 4(5)-ethyl-5(4)-methylimidazole (24). The formation of this last compound from L-rhamnose was unexpected .27*28 The almost exclusive formation of 4(5)-(hydroxymethyl)imidazole (27) from D-fructose, 1,3-dihydroxy-2-propanone,and D-fructosecontaining compounds (for example, sucrose) is to be expected

348

M. J. KORT

because the required intermediate, hydroxypyruvaldehyde, would be readily obtained by cleavage of the D-fructose to 1,3-dihydroxy-2propanone and oxidation of the latter. However, where this imidazole was obtained from ~ - g l u c o s e(see ’ ~ Table 111),epimerization products of D-glucose were also observed, and D-fruCtOSe might have been one of them. Similarly, 2-methylimidazole was only obtained from Lrhamnose, as only this 6-deoxy sugar could give rise to acetaldehyde on fragmentation. The hypothesis of Windaus and U l l r i ~ that h ~ ~imidazole-4(5)-carboxylic acid (31) is formed after oxidation of D-glucose to 2-oxomalonaldehydic acid, OCH-CO-C02H, needs revision. As the reaction mixture was kept for three years, it is far more probable that a 4(5)(hydroxyalky1)imidazole was first formed, and that this was subsequently oxidized by the copper hydroxide in the reaction mixture to imidazole-4(5)-carboxylicacid.

8. Recombination of Sugar Fractions As Fujii and coworkers3s isolated 4(5)-(~~-glycero-2,3-dihydroxypropyl)imidazole, not the D form (19), in the reaction of D-glucose with aqueous ammonia, they expressed the view that pentose may be formed in the reaction mixture not by direct fission of the C - 1 4 - 2 or C-5-C-6 bond of D-glucose but by recombination of fragments from D-glucose, for example, of a triose and glycolaldehyde. Indeed, Grimmett and Richards5* identified 1,3-dihydroxy-2-propanone7 DL-glucose, DL-fructose, DL-mannose, DL-arabinose, DL-lyxose, DL-xylose, and, possibly, DL-ribose in the mixture when DL-glyceraldehyde was kept at 37”with ammonia (see Table IX). In addition, the isolation of 4(5)-ethylimidazole (23)from the reactions of L-rhamnose (see Table VI), L-fucose (6-deoxy-~-galactose), and 3-deoxy-~-glyceraldehyde(see Table IX) with aqueous ammonia, led Kzmoto and T ~ u c h i d to a ~believe ~ that the 2-oxobutanal forming the 4(5)-ethylimidazole (23) arose from an aldoI reaction of the 3-deoxy-~-glyceraldehyde (formed by dealdolization in the cases of L-rhamnose and L-fucose) with formaldehyde, with subsequent loss of water from the 4-deoxytetrose so formed. In their investigations of aspects of the chemistry of the browning reaction of reducing sugars, Fleming and coworkerss7 interpreted (97) M. Fleming, K. J. Parker, and J. C. Williams, “Aspects in the Chemistry of the Browning Reaction of Reducing Sugars.” Paper delivered at the 13th Congr. Intern. SOC.Sugar-Cane Technologists, Taiwan, Formosa, March, 1968.

REACTIONS OF FREE SUGARS WITH AQUEOUS AMMONIA

349

their results as evidence in support of a glycodiulose intermediate (l-deoxy-~-erythro-2,3-or 2,5-hexodiulose), derived either from the reducing sugar directly, or by resynthesis from pyruvaldehyde and 1,3-dihydroxy-2-propanone.

V. APPLICATIONS An application of the formation of imidazoles from sugars plus ammonia that might prove extremely useful is the microdetermination of the position of the linkage in (1 + 2)-, (1 + 3)-, (1--* 4)-, and (1+ 6)-linked disaccharides of hexoses. It has been showna8 that these hexose disaccharides react with ammonia to produce different proportions of imidazoles. Another structure-elucidative technique is used to differentiate between reducing sugars containing a terminal methyl group and other reducing sugars. 4(5)-Ethylimidazole (23) is only obtained in the reaction of aqueous ammonia with reducing sugars containing a terminal methyl group, for example, L-rhamnose and L-fucose (see Table VI).39 The imidazole obtained from the reaction of a periodate-oxidized polysaccharide with ammoniass is specific to the linkage in the parent polysaccharide, and provides another basis for a micro-method for linkage analysis: (1 + 3)-linkage (laminaran) gives only traces of imidazoles; (1 + 4)-linkage (amylose) gives 4(5)-imidazole (30) and 4(5)-(2-hydroxyethyl)imidazole(25); and (1+ 6)-linkage (dextran) results in 4(5)-imidazole (30)and 4(5)-methylimidazole (34). In addition, sugars can be used as starting materials for the synthesis of substituted imidazoles, for example, 4(5)-(hydroxymethy1)imidazole (27)from ~ - f r u c t o s e . ~ ~ The formation of the imidazoles and pyrazines in the ammoniation of m o l a s ~ e s ' ~ -increases '~ the nitrogen content of the molasses, and makes it more valuable as animal fodder, a d e ammoniated molasses is then a cheap source of protein and not merely a source of energy. Although imidazoles and pyrazines exhibit certain toxicities to animals, these can be o v e r c ~ m e . ' ~

(98) M. R. Grimmett, R. W. Bailey, and E. L. Richards, Chem. Znd. (London), 651 (1965). (99) E. L. Richards,Aust.J. Chem., 23,1033(1970).

This Page Intentionally Left Blank

SYNTHESIS OF NITROGEN HETEROCYCLES FROM SACCHARIDE DERIVATIVES

BY HASSANEL KHADEM lnstitut de Chimie des Substances Naturelles, Centre National de la Recherche Scientijique, Paris* I. Introduction ........................................................... 351 11. Formation of Three-membered Nitrogen Heterocycles . . . . . . . . . . . . . . . .352 1. Saccharides Containing Aziridine Rings (Epimino Sugars) . . . . . . . . . . . . . .352 111. Formation of Five-membered Nitrogen Heterocycles . . . . . . . . . . . . . . . . . . , .357 1. Pyrrolidines, Pyrrolines, and Pyrroles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,357 2. Pyrazolines and Pyrazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .364 3. Imidazolidines, Imidazolines, and Imidazoles . . . . . . . . . . . . . . . . .. . . .367 4. Oxazolidines, Oxazolines, and Oxazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . .375 5. Thiazolidines and Thiazolines . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. .. .. . .385 6. 1,2,3-Triazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . .387 7. Thiadiazolines . . . . . . . . . . . . . . . . . . . . . . . .. . .. .. . . ,. . . . . . . . .. . . . . . . . .393 8. Tetrazoles .......................................................... 393 IV. Formation of Six-membered Nitrogen Heterocycles . . . . . . . . . . . . . . . . . . . . ,394 1. Piperidines . . . . . . . . . . . . . . . . . . . . . . ... ... ................. ...... ... . 394 2. Diazines ........................................................... 396 3. Oxazines and Thiazines . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,400 V. Formation of Higher-membered Nitrogen Heterocycles . . . . . . . . . . . . . . .404

.

.

.

.

..

.

.

.

.

.

.

.

..

.

. .

. .. .

. . . . . .

.

.

.

..

.

.

.

I. INTRODUCTION The synthesis of nitrogen heterocycles from saccharide derivatives can be traced back to the early days of organic chemistry, when it was shown (1860) that pyrrole could be obtained by the pyrolysis of the ammonium salt of galactaric acid. Since then, a large number of heterocyclic compounds has been prepared from saccharide derivatives, with the aim of finding uses for abundant and relatively inexpensive sugars, or of studying the chemistry and biological activity of the heterocyclic compounds prepared and of using them as intermediates in the synthesis of amino sugar derivatives. 'Permanent address: Faculty of Science, Alexandria University, Alexandria, Egypt.

351

352

HASSAN EL KHADEM

This Chapter will discuss the formation of heterocycles that contain nitrogen and that involve atoms of the original sugar, and not the combination of existing heterocycles with saccharide derivatives. Thus, for example, the synthesis of nucleosides will not be included, but the formation of anhydronucleosides, in which a new heterocyclic ring has been introduced into the molecule, will be treated. The heterocycles are classified into three-, five-, six-, and highermembered rings, and are subdivided into the different types of monocyclic heterocycle, irrespective of other rings present in the molecule. This procedure has the advantage of limiting the number of classes of heterocycle, and permits the discussion of a certain kind of heterocyclic ring in a single Section, irrespective of whether it is attached to a furanose or a pyranose ring or to an acyclic sugar residue. When more than one ring is formed, the heterocycle will be classified under the simplest type, namely, the one having the smallest ring or the smallest number of hetero atoms.

11. FORMATION OF THREE-MEMBERED NITROGEN HETEROCYCLES 1. Saccharides Containing Aziridine Rings' (Epimino Sugars) The first saccharide derivative having an aziridine ring was prepared in 1960 by Christensen and Goodman,2 who treated a D-altrose derivative (1) with sodium methoxide, and obtained, by nitrogen participation, 4,6-0-benzylidene-2,3-dideoxy-2,3-epimino-~-~-allopyranoside (2). The formation of an aziridine ring is often accompanied by the production of a five-membered ring, and the thiazoline 3 was obtained by participation through ~ u l f u rThe . ~ relative yields of the three- and five-membered ring compounds vary considerably with the type of cyclizing agent used and the nature and stereochemistry of the reacting groups.

(1)These compounds were examined in a Chapter on neighboring-group participation, L. Goodman, Adoan. Carbohyd. Chem., 22,109(1967). (2) J. E. Christensen and L. Goodman, J . Amer. Chem. SOC., 82,4738 (1960). (3)L. Goodman and J. E. Christensen, J . Amer. Chem. SOC., 83,3823 (1961).

SYNTHESIS OF NITROGEN HETEROCYCLES

353

N Y S SMMe

(3)

The aziridine ring of epimino sugars is formed by interaction of a nitrogen-containing, participating group with a suitable, neighboring leaving-group, such as the methylsulfonyl or p-tolylsulfonyl group or the halogen atom of a 2-amino-2-deoxyglycosyl halide derivative. The participating group and the leaving group must be trans-disposed to one another, and, preferably, trunsdiaxial. Several aziridines have been prepared from amino sugars by protecting the amino group, introducing the leaving group, and cyclizing with, for example, sodium methoxide or potassium cyanide in N,Ndimethylformamide. Examples of protecting groups (R) attached to such amino groups in compounds (4) that have been cyclized to R-substituted aziridines (5) are the acety14 and benzoyl groups,4-s

(4) D. H. Buss, L. Hough, and A. C. Richardson, J . Chern. Soc., 5295 (1963). (5)R. D.Guthrie, D. Murphy, D. H. Buss, L. Hough, and A. C. Richardson, Proc. Chern. Soc., 84 (1963). (6) W. Meyer zu Reckendorf, Chern. Ber., 97,325(1964).

(7) C. F. Gibbs, L. Hough, and A. C. Richardson, Carbohyd. Res., 1, 290 (1965). (8)W.Meyer zu Reckendorf, Chern. Bet., 98,93 (1965). (9)A. D.Barford and A. C. Richardson, Carbohyd. Res., 4,408(1967).

HASSAN EL KHADEM

354

the methylsulfonyl1° and p-tolylsulfonyl" groups, and the 2,4-dinitrophenyl group.1z In the same way, such groups as CN (Ref. 13), NHzCO (Ref. 13),NHzC=NN02 (Ref. 14), NH,C=NNOH (Ref. 15), and NH,C=S (Refs. 16-18) were attached to the nitrogen atom of an amino sugar, and the product was converted into an aziridine having these respective substituents attached to the hetero atom. Sugar azides are also important starting-materials for the synthesis of epimino sugars; an azido group adjacent to a p-tolylsulfonyloxy group, as in 6 or 8, may be simultaneously reduced and cyclized with Raney nickel to form an aziridine ring, as in 7, especially if the two groups are in the trans-diaxial p o ~ i t i o n . ' ~ - ~ ~

An amino aziridine (10) was synthesizedz3 by direct interaction between hydrazine and a 5,6-dimethanesulfonate (9). MsOCH,

I MsOCH

(10) B. R. Baker and T. L. Hullar, /. Org. Chem., 30, 4053 (1965). (11) B. R. Baker and T. L. Hullar, /. Org. Chem., 30, 4045 (1965). (12) P. F. Lloyd and G . P. Roberts,]. Chem. Soc., 2962 (1963). (13) B. R. Baker and T. Neilson, /. Org. Chem., 29, 1057 (1964). (14) B. R. Baker and T. Neilson,/. Org. Chent., 29, 1047 (1964). (15) B. R. Baker and T. Neilson, J. Org. Chem., 29, 1063 (1964). (16) B. R. Baker and T. Neilson,]. Org. Chem., 29, 1051 (1964). (17) B. R. Baker and T. L. Hullar, /. Org. Chem., 30, 4049 (1965). (18) B. R. Baker and T. L. Hullar, 1.Org. Chem., 30, 4038 (1965). (19) J. CIBophax, S . D. GBro, and R. D. Guthrie, Tetrahedron Lett., No. 6, 567 (1967). (20) J. CICophax, J. Hildesheim, A.-M. Skpulchre, and S. D. G&o, Compt. Rend. (C), 266, 720 (1968). (21) J. Clkophax, S. D. GBro, and J. Hildesheim, Chem. Commun., 94 (1968). (21a)J. Clbophax, S. D. GBro, J. Hildesheim, R. D. Guthrie, and C . W. Smith, Chem. Commun.,784 (1969). (22) R. D. Guthrie and I). Murphy, /. Chem. Soc., 5288 (1963). (23) H. Paulsen and D. Stoye, Chem. Ber., 102,820 (1969).

SYNTHESIS OF NITROGEN HETEROCYCLES

355

In the foregoing examples, the imino nitrogen atom, no matter whether already present in the molecule or formed during the reaction, participates in the formation of an aziridinium ion (12 from 11, or 15 from 14), which facilitates the removal of the leaving group, and leads to the neutral, aziridine ring-compound (13 or 16). AS a result, the original configuration at the carbon atom bearing the imino nitrogen atom is retained in the product, and the configuration at the carbon atom bearing the leaving group is reversed.

w-w-v HN

I R

H’

1 R

I

R

R

The aziridine ring may be opened by a variety of reagents, including hydrochloric acid and hydriodic acid, sodium azide, potassium acetate, and potassium thioacetate. A complex mixture of products is usually obtained that can be separated by thin-layer chromatography. The mixture usually contains the isomeric amino sugars formed by addition, and the rearrangement products formed by ring expansion. Their ratios depend to a large extent on the reagent used and on the configuration of the epimino sugar studied. As a rule, trans-diaxial amino sugars are formed, but, from epimino allopyranosides, transdiequatorial products are obtained. Thus, whereas the reaction of methyl 2,3-(benzoylepimino)-4,6-O-benzylidene-2,3-dideoxy-a-~mannopyranoside (17) with sodium azide in N,N-dimethylformamide yields a trans-diaxial amino sugar, namely, methyl 3-azido-2-benzamido-4,6-0-benzylidene-2,3-dideoxy-a!-~-altroside (18), and an oxazoline (19), the a210 isomer, methyl 2,3-(benzoylepimino)-4,6-O-benzylidene-2,3-dideoxy-a-~-allopyranoside (20), yields8*24,25 the transdiequatorial amino sugar azide, namely, methyl 2,3-(benzoy1epimino)(24) €3. D. Cuthrie and D. Murphy, J . Chem. SOC., 3828 (1965). (25)D.H.Buss, L. Hough, and A. C . Richardson,J.Chem. Soc., 2736 (1965).

HASSAN EL KHADEM

356

ph
. c

P

h

< 0

O

OMe

N I Bz

m

(20)

(21)

I Bz

4,6-0-benzylidene-2,3-dideoxy-cr-~-glucopyranoside (21). A study of epimino lyxofuranosides revealed that, here too, trans-diaxial ringopening occurs.z6 Isomerization of methyl S-benzamido-2,3-(benzoylepimino)-2,3,5-trideoxy-/3-~-lyxofuranoside (22) with sodium iodide in acetonitrile yields a mixture of oxazolines, the isomer having the nitrogen atom attached to C-2, namely, 23,preponderating; the isomer (24) having the oxygen atom on (2-2 was also isolated, but in a lower yield.27When an epimino sugar that is unsubstituted on the B

z

H

N

C

W

B

z

H

N

C

U

A

__c

N’

Major product

Minor product

(23)

(24)

nitrogen atom, such as methyl 4,6-0-benzylidene-2,3-dideoxy-2,3epimino-a-D-allopyranoside(25), is treated with nitrous acid, it first undergoes nitrosation; thus, 25 gives 26, and 26 is then converted28 into an alkene (27). This reaction may be regarded as an example of ring contraction. (26) J. Hildesheim, J. ClBophax, A. M. SBpulchre, and S. D . GBro, Cnrbohyd. Res., 9, 315 (1969). (27) J. Hildesheim, E. Walczak, and S. D . GBro, Cornpt. Rend. (C), 267, 980 (1968). (28) R. D. Guthrie and D. King, Carbohyd. Res., 3, 128 (1966).

SYNTHESIS OF NITROGEN HETEROCYCLES

p

h

<

o

-

OMe HNO,

b

p

h

< 0

N H

357

OMe

o

N I NO

(25)

(26)

J

ph
0 (27)

111. FORMATION OF FIVE-MEMBERED NITROGENHETEROCYCLES 1. Pyrrolidines, Pyrrolines, and Pyrroles a. Pyrrolidines - Pyrrolidines are obtained from amino aldosesZeain particular, from the 4-amino and 3-amino derivatives (whichcan form imino bridges between C-4 and C-1 and between C-3 and C-6). The first type of cyclization has been effected with tetroses, pentoses, and hexoses, and the parent pyrrolidine of this series, namely, 29, was (28) by cyclizasynthesized from 4-acetamido-4-deoxy-~-erythrose tion with methanolic hydrogen chloride.29 When 28 was replaced by the corresponding D-erythronic acid derivative, ~-erythro-3,4-dihydroxy-2-pyrrolidinone (30) was obtained.29a The cyclization of 4amino-4-deoxyaldopentoses is exemplified b y the periodate oxidation of 2-acetamido-2-deoxy-3,4,6-tri-O-benzyl-~-talitol (31) to the 4-acetH

c=o

I HOCH I HOCH I H,CNHAc

Ac

H

(28a)The chemistry of five-membered heterocycles obtained from amino sugars has been reviewed in detail by D. Horton, in “The Amino Sugars,” R. W. Jeanloz, ed., Academic Press, Inc., New York, N.Y., 1969, p. 114. (29) W. A. Szarek and J. K. N. Jones, Con. J . Chem., 42,20 (1964). (29a)S. Hanessian and T. H. Haskell, J . HeterocycZ. Chem., 1, 57 (1964).

358

HASSAN EL KHADEM

amido-4-deoxy-~-ribosederivative, which is directly cyclized30 to the pyrrolidine (32). CH,OH I HCOH I

I0,O

BzlOCH I BzlOCH I AcHNCH I CH,OBzl

&H,oH

BzlOC HZ

(31)

(32)

where Bzl = Benzyl.

Periodate oxidation of 2-acetamido-1,2-dideoxy-3,4-0-isopropylideneD-glucitol (33) affords 4-acetamido-4,5-dideoxy-2,3-O-isopropylidenealdehydo-L-xylose, which, on hydrolysis with acetic acid, g i v e P the pyrrolidine 34.The same series of reactions was performed31aon the H

FHS

HCNHAc

c=o I

I

OCH +,CMe,

Ac

10,” __c

nco

HCO’ I AcHNCH I

I HCOH I

CH,OH

HO

CH3

(34)

(33)

enantiomeric D-xylose derivative. The presence of an aldehydic group at C-1 is by no means essential; methyl 4-acetamido-4-deoxy-a-~ribopyranoside (35) is ~ o n v e r t e d by ~ ~acetic , ~ ~ acid into the pyrrolidine 36. For the hexose derivatives, an ingenious method was used.

A c o c i

QOMe

H,OH

AcHN I

HO

1

OH (35)

I

AcO

1

OAc (36)

(30) J. Gigg and R . Gigg, J . Chem. SOC. (C), 1876 (1966). (31) A. E. El-Ashmawy and D. Horton, Carbohyd. Res., 1, 164 (1965). (31a)J.S. Brimacombe and J. G. H. Bryan,]. Chem. SOC. ( C ) , 1724 (1966). (32) E. J. Reist, D. E. Gueffroy, and L. Goodman, J . Arner. Chem. SOC., 87,677 (1965). (33) E. J. Reist, D. E. GueEroy, R.W. Blackford, and L. Goodman, J. Org. Chem., 31, 4025 (1966).

SYNTHESIS OF NITROGEN HETEROCYCLES

359

2,3:5,6-Di-O-isopropylidene-uZdehydo-~-glucose dimethyl acetal was p-toluenesulfonylated at 0-4, and the ester was converted, with methyl sulfoxide and acetic anhydride, into the 4-ketose derivative; with hydroxylamine, this gave the oxime, which was reduced to a mixture of 4-amino-4-deoxy-D-gluco and -D-gulucto derivatives. These were converted34b y sulfur dioxide into the corresponding pyrazolidines, namely, 4-amino-4-deoxy-D-g~ucofuranosylsulfite (37) and 4-amino4-deoxy-~-galactofuranosylsulfite (38).

HOCH,

I OH

AH

HCOH I CH,OH

(38)

(37)

Nuclear magnetic resonance (n.m,r.) studies have revealed that 4a m i n o - 4 - d e o x y - ~ - x y l o s e , ~4-amino-4,5-dideoxy-~-xylose,~~~ ~*~~~ and 4-amin0-4-deoxy-D-glucose~~ (40)exist as equilibrium mixtures; for example, of the pyrrolidine 40, the pyrroline 39 formed by its dehydration, and a dimer (41), the equilibrium lying strongly towards the last. Similar studies have shown that the hydrochlorides of 4-aminoCH,OH I HOCH CH,OH I HOCH

Q (39)OH

CH,OH I HOCH QH,oH

( 40)OH

-

L

qN HCOH q

O (41)

(34) H. Paulsen, K. Propp, and K. Heyns, Tetrahedron Lett., 683 (1969). (35) A. E. El-Ashmawy and D. Horton, Carbohyd. Res., 8, 191 (1966). (35a)H. Paulsen, J. Briining, and K. Heyns, Chem. Bet., 102,459 (1969). (35b)H. Paulsen, K. Propp, and J. Briining, Chem. Ber., 102, 469 (1969).

k&OH

HASSAN EL KHADEM

360

4-deoxy-D-gl~cose~~ and 4-amino-4,6-dideoxy-~-glucose~~ exist preponderantly in the pyranose form; thus, the latter consists, in solution, mainly of 42, with very little of 43.

The conversion of 3-amino-3-deoxy sugars into pyrrolidines requires the presence of a leaving group, such as a p-tolylsulfonyloxy group on C-6; this is exemplified by the conversion of 3-azido-3deoxy-1,2-O-isopropylidene-6-O-p-tolylsulfonyl-(~ -D-glucofuranose (44) and methyl 3-azido-2-benzamido-2,3-dideoxy-6-O-p-toly~sulfonyl-P-D-glucopyranoside (46) into the respective pyrrolidines, 45 and 47, by reduction of the azido group, followed by ~ y c l i z a t i o n . ~ ~

iWBz

TsM: H, I HOCH

q

O,-CMe,

1 . reduction 2 . Bz,O

-

(44)

O,CMe,

(45)

rYH

TsOCH,

2. cyclization I

NHBz (46)

NHBz (47)

(36) C. L. Stevens, P. Blumbergs, F. A. Daniher, D. H. Otterbach, and K. G . Taylor, J. Org. Chem., 31, 2822 (1966). (37) W. Meyer z u Reckendorf, Chem. Ber., 97, 1275 (1964); 101, 3802 (1968).

SYNTHESIS OF NITROGEN HETEROCYCLES

361

Cyclization between a I-amino group and C-4 of an anhydropentito1 has been achieved: 2,5-anhydro-l-azido-l-deoxy-3,4-di-O-p-tolylsu~fony~-D-xylito~ (48) was hydrogenated to the l-amino compound and then this was N-p-toluenesulfonylated, giving 49; the product was cyclized by use of sodium methoxide t ~the bicyclic ~ ~ compound * ~ ~ (50).

b. Pyrrolines. - In the previous Section (see p. 359), it was shown that 4-amino-4-deoxy-~-glucoseexists in an equilibrium mixture of the pyrrolidine form (40), its dimer (41), and the pyrroline 39 formed by its dehydration. The presence of the last was demonstrated by its reduction to a p y r r ~ l i d i n e .The ~ ~ same kind of reduction was performed on 4-amino-4-deoxy-~-xylose(51a) and on 4-arnin0-4~5-deoxyL-xylose (51b),which gave35pyrrolidines (52a) and (52b),respectively. H

HO

HO

(51)

a. R = CH,OH b . R = Me

(52)

c. Pyrro1es.-In the Introduction, it was mentioned that the preparation of pyrrole from galactaric acid by pyrolysis of its ammonium salt40dates from the early days of organic chemistry. It is remarkable that this method is still in use today, and that it is recommended, with some modifications, in Organic S y n t h e ~ e s . ~By ' using primary amine (38) J. Clkophax, S . D. Gem, and A. M. Sepulchre, Carbohyd. Res., 7, 505 (1968). (39) J. Clkophax, J . Hildesheim, A. M . Skpulchre, and S. D. Cero, Bull. SOC. Chirn. Fr., 153 (1969). (40) H. Schwanert, Ann., 116, 278 (1860). (41) S. M. McElvain and K. M. Bolliger, Org. Syn., Coll. Val., 1, 473 (1941).

362

HASSAN EL KHADEM

salts of galactaric acid, instead of the ammonium salt, it is possible to prepare N-alkyl- and N-aryl-pyrroles. Thus, N-meth~1-,4**~~ N-eth~1-,4~,~~ N - b ~ t y l - and , ~ ~ N - i ~ o p e n t y -pyrrole l~~ have been prepared by the pyrolysis of the corresponding diamino salts of galactaric acid. The same kind of synthesis was performed with arylamine salts of galactaric and -p-tolyl-pyrr0le,4~'~~ N-m-, -0-, acid, giving N - ~ h e n y l -N-m-, , ~ ~ -0-, and -p-chl~ro-pyrrole,~~ N-o-nitr~phenylpyrrole,~~ and N-1- and -2n a p h t h y l p y r r ~ l e .It ~ ~should be noted that, with liquid and solid amines, it is not necessary to isolate the amine salt of galactaric acid; instead, the calculated weights of the amine and galactaric acid are simply ground together, and the mixture is heated in a pyrolysis f i a ~ k The . ~ ~ disadvantage of this method is that the yields of substituted pyrroles decrease considerably with increase in their molecular weight; being less volatile, the compounds of higher molecular weight decompose instead of distilling, Another class of pyrrole derivative may be obtained by the interaction of 1-amino-1-deoxy-%ketoses or 2-amino-2-deoxyaldoses with a p-dicarbonyl compound. Unlike the previous type (which is N substituted), these pyrrole derivatives have a tetrahydroxybutyl group in the a- or P-position with respect to the nitrogen atom of the ring, in addition to other groups arising from the dicarbonyl compound used in the condensation. The formation and reactions of this type of pyrrole derivative have been discussed in detail in two articles in this ~ e r i e s ~ *they , ~ ~ will, ; therefore, only be treated briefly. l-Amino-1deoxy-D-fructose (53)reacts with 2,4-pentanedione to give50 pyrrole derivative 54a; similar pyrroles were obtained with ethyl acetoacetate,50*51 which yields 54b.

(42) C. A. Bell, Ber., 10, 1861 (1877). (43) A. Pictet, Ber., 37, 2792 (1904). (44) C. A. Bell and E. Lapper, Rer., 10, 1962 (1877). (45) T. Reichstein, Helu. Chim. Acta, 10, 389 (1927). (46) L. Lichtenstein, Ber., 14, 933 (1881). (47) H . El Khadem, M. M. A. Abdel Rahman, and S. El Sadany,]. Chem. Soc., in press (1970). (48) F. Garcia Conzalez, Aduun. Curbohyd. Chem., 11, 97 (1956). (49) F. Garcia Gonzilez and A. G6mez Sanchez, Aduan. Curbohyd. Chem., 20, 303 (1965). (SO) F. Garcia Gonzalez, A. Gbmez Sanchez, and J. Gasch G6mez, An. Fis. Quim. (Madrid), 54B,513 (1958). (51) A. Gbmez Sinchez, L. Rey Romero, and F. Garcia Gonzalez, An. Fis. Quim. (Madrid), 60B,505 (1964).

SYNTHESIS OF NITROGEN HETEROCYCLES

363

R

O=C H,CNH, I c=o

R O=C

I HOCH I

Me

\

H2C-C

HCOH I HCOH I CH20H

(53)

/

/

Me

\

c=c\/

I

fJH

C=CH

I

HOCH

'b

I

HCOH

I I

HCOH CH,OH a. R = Me b. R = OEt

(54)

Much more studied is the reaction of p-dicarbonyl compounds with 2-amino-2-deoxyaldoses; in particular, with 2-amino-2-deoxy-~-glucose (55), both in neutral and alkaline medium. In neutral methanol or aqueous acetone, 2-amino-2-deoxy-~-glucosereacts with 2,4-pentanedione to give52-543-acetyl-2-methyl-5-(~-arabino-tetrahydroxybuty1)pyrrole (56a), and, with ethyl a ~ e t o a c e t a t e the , ~ ~ pyrrole 56b. Similar (tetrahydroxybuty1)pyrrole.s have been prepared from other p-keto esters, such as ethyl 3-oxohexanoate, ethyl thiolacetoacetate, and diethyl 3-0xopentanedioate.~~*~~*~~~ R O=C,

Me

c=c\

,

H

c=o I

HCNHHCNH,

I HOCH Ho II

HCOH

R

/

'=$ Ozc

n"m /Me

\

H2C-C

b'

I

HCOH

I

CH20H

(55)

I

I

hH

HC=C

I HOCH I HCOH I HCOH I

CH20H

a. R = Me b. R = OEt

(56)

(52)R. Boyer and 0. Furth, Biochem. Z . , 282,242 (1935). (53)A. Ollero and R. de Castro, An. Fis. Quim. (Madrid), 41,868 (1945). (54)F. Garcia ConzPlez and R. de Castro Brzezicki, An. Fis. Quim. (Madrid), 46B, 68 (1950). (55)F. Garcia Conzalez, An. Fis. Quim. (Madrid), 32, 815 (1934). (56)A. Ollero Gomez and J. Femindez Jimenez, An. Fis. Quim. (Madrid), 41, 1165 (1945). (56a)A. G6me.z Sanchez, H. Tena Aldave, J. Valesco del Pino, and U. Scheidegger, Carbohyd. Res., 10,19 (1968).

HASSAN EL KHADEM

364

The condensation of amino sugars with p-keto esters in an alkaline medium results in considerable degradation of the (tetrahydroxybuty1)pyrrole produced, giving rise to small yields of S-methylpyrr ~ l e , ~ which ' . ~ ~ seems to be the main chromophore in the ElsonMorgan reaction. Under these conditions, 3-amino-3-deoxyhexoses yield 2-methylpyrrole-4-carboxylic acid,59which is, in part, responsible for the coloration produced with p-(dimethy1amino)benzaldehyde.

2. Pyrazolines and Pyrazoles The saccharide derivatives best suited for cyclization to pyrazolines and pyrazoles are the hydrazones and osazones, which possess the two adjacent nitrogen atoms required. a. Pyrazolines. - The periodate oxidation of a saccharide osazone, such as 57, to mesoxalaldehyde bis(pheny1hydrazone) (60) is effected under mild conditions,60 as, under more vigorous conditions, overoxidation to the acid 61 occurs, followed by cyc1ization6' to l-phenyl4-phenylazo-5-pyrazolinone(58). The latter is also formed when a hexulose or pentulose phenylosazone is refluxed in methanol containing some hydroxylamine hydrochloride,61phenylhydrazine hydrochloride,62or semicarbazide hydrochloride.62 In an alkaline medium, compound 58 is converted into the enol form (59), and this can be O-benzoylated.62 HC= NNHPh

I

C=NNHPh

I HOCH I

HCOH

I

-

PhHNN

$!zL PhN=N

HFOH CH,OH

Ph

(58)

(57)

I

HC =NNHPh

I

C=NNHPh I

c=o H

HO

Ph

(59)

A

(60)

HC=NNHPh

I

C=NNHPh I 'OZH (61)

(57)J. W.Cornforth and M. E. Firth, J. Chem. Soc., 1091 (1958). (58)C. Cessi and F. Serafini-Cessi, Biochem. J., 88, 132 (1963). (59)S. Hanessian andT. H. Haskell,]. Org. Chem., 30,1080 (1965). (60)E.Chargaff and B. Magasanik,J . Amer. Chem. SOC.,69,1459(1947). (61)0.Diels, R. Meyer, and 0.Onnen, Ann., 525,94 (1936). (62)H.El Khadem and M. M. A. Abdel Rahman, Carbohyd. Res., 3, 25 (1966).

365

SYNTHESIS OF NITROGEN HETEROCYCLES

T h e trihydroxypropyl derivative 63 was prepared from dehydroL-ascorbic acid phenylosazone (62) by opening the lactone ring with warm alkali and acidifying the mixture after a few minutes, whereupon the pyrazolinone 63 separated i r n m e d i a t e l ~The . ~ ~ structure of this compound was established by d e g r a d a t i ~ n and , ~ ~ confirmed by a study of its n.m.r. spectrum.64 The p-tolyl-, p-(bromopheny1)-, and p-(iodopheny1)-osazones of dehydro-L-ascorbic acid were also converted into the corresponding l-ary1-4-phenylazo-3-(trihydroxypropyl)-5-pyrazolinone, and their acetylation and benzoylation products were prepared.64 CH,OH

I

Ph I

HCOH

CH,OH (63)

(62)

Another 2,3-bis(phenylhydrazone),namely, 1,5:4,6-dianhydro-2,3hexodiulose bis(pheny1hydrazone) (62a), was also found to cyclize to a pyrazoline (63a) by the action of copper(I1) sulfate or benzaldehyde, which usually convert osazones into aldosuloses or osotriaz 0 1 e s . ~Reduction ~ of this bicyclic compound (63a) led to the pyrazolidine 64, and periodate oxidation of 63a gave the expected aldehyde 65. 07

P h - N q - P h CHOH 1

CH,OH

‘N-Ph

(63) H. Ohle, Ber., 67, 1750 (1934). (64) H. El Khadem and S. H. El Ashy, J . Chem. SOC. (C), 2248 (1968). (65) G. Hanisch and G. Henseke, Chem. Ber., 101,4170 (1968).

CHOH I

CH,OH

HASSAN EL KHADEM

366

The flavazoles, an interesting type of pyrazoline derivative obtained

by interacting sugar quinoxalines with phenylhydrazine, will be discussed in the next Section (see p. 398). b. Pyrazo1es.-It has been shown66that degradation of sugar hydrazones (66) with alkali results in the fission of the C-3-C-4 bond and cyclization to 1-phenylpyrazole (67). The 4-phenylazo derivative of this compound is obtained from the osazone 68 by periodate oxidation followed by cyclization of the mesoxalaldehyde bis(pheny1hydx-azone) (69) produced with acidic reagents.62Under these conditions, mesoxalaldehyde tris(pheny1hydrazone) also yields 1-phenyl-4-phenylazopyrazole (70), the unchelated hydrazone residue on C-3 being hydrolyzed before cyclization occurs. HC=NNHPh I

HCOH I

HOCH I HOCH

2% KOH

____c

C N - P h

I

HCOH I CH,OH

(67)

(66)

HC=NNHPh

HC=NNHPh I

-

I

C=NNHPh

H@

___c

CHO

C N - P h

Another type of substituted pyrazole is obtained by refluxing osazones with acetic anhydride; during the acetylation, two molecules of water per molecule are removed. The structure of the resulting 5-(~-gZycero-diacetoxyethyl)-3-formyl1-phenylpyrazole 2-acetyl-2phenylhydrazone (71) was determined by deacetylation followed by oxidation with (a) periodate to a pyrazolaldehyde (72),and (b) peracid (73), manganate to the known l-phenyl-pyrazole-3,5-dicarboxylic as well as by a study of its n.m.r. spectrum, which confirmed the pres(66) H. Simon and W. Moldenhauer, Chem. Ber., 100,3121 (1967).

SYNTHESIS OF NITROGEN HETEROCYCLES

367

ence of the 12-diacetoxyethyl side-chain and of the N-acetyl g r o ~ p . ~ ~ - ~ ~ This reaction seems to be quite general; it was successfully applied 6-deoxyhexoses, and to substituted p h e n y l o s a ~ o n e s ~of ~ *pentoses, ~’ s ~ ~of disaccharides having hexoses, as well as to those of h e p t o ~ e and (1+ 6 ) - l i n k a g e ~It. ~is~useful for determining the D or L configuration of a hexose, as the sign of the optical rotation of the D isomer is positive, and that of the L isomer is negative.73

HC//NN(Ac)Ph

/flN(Ac)Ph

(68)

___c

“ P - P h HCOAc I CH,OAc

<

9

-

P

h

CHO (72)

(71)

3. Imidazolidines, Imidazolines, and Imidazoles a. Irnida~olidines~~ (Tetrahydroimidazoles).-The 5-amino-5-deoxypentoses are cyclized to piperidine derivatives (see p. 394). It is, therefore, not surprising that 5,6-diamino-5,6-dideoxyhexoses cyclize to bicyclic compounds having, in addition to the piperidine ring formed between C-1 and the nitrogen atom on C-5, a five-membered imid(67) H. El Khadem and M. M. Mohammed-Ali, J . Chem. Soc., 4929 (1963). (68) H. El Khadem, Z. M. El-Shafei, and M. M. Mohammed-Ali,J. Org. Chem., 29, 1565 (1964). (69) H. El Khadem, j . Org. Chem., 29,3072(1964). (70)H. El Khadem, Z. M. El-Shafei, and M. M. A. Abdel Rahman, Carbohyd. Res., 1, 31 (1965). (71) H.El Khadem and M. M. A. Abdel Rahman, Carbohyd. Res., 6,470 (1968). (72) H. El Khadem, M. M. A. Abdel h h m a n , and M. A. E. Sallam, J . Chem. SOC. (C), 2411 (1968). (73) H. El Khadem and Z. M. El-Shafei, Tetrahedron Lett., 1887 (1963). (74) These compounds were discussed in an article by H. Paulsen and K. Todt, Aduon. Carbohyd. Chem., 23, 115 (1968).

368

HASSAN EL KHADEM

azolidine ring. The reaction is illustrated by the conversion of 5,6diamino-5,6-dideoxy-l,2-0-isopropylidene-~-~-idofuranose (74) into the acyclic sulfite 75, and bicyclization thereof to 76 by the reaction of barium h y d r o ~ i d e . ~Compound ~,'~~ 76 can be acetylated to the di-Nacetyl derivative 77.

K.. \

OAc

Unlike the free base 76, which exists in equilibrium with the acyclic form, the di-N-acetyl derivative 77 exists only in the bicyclic form, the presence of four rotamers of which has been revealed by n.m.r. studies.76 Other tetrahydroimidazoles have been obtained from the reaction of 2-amino-2-deoxyhexoses with isocyanates and with thiocyanates. Thus, 2-amino-2-deoxy-~-g~ucopyranose hydrochloride (78) reacts readily with phenyl isocyanate to yield7' the corresponding 4,s-Dglucopyrano-l-phenyl-4-imidazolidin-2-one(79). Alternatively, the tetraacetate 80 was converted into the urea derivative 81, the prod-

(75) H. Paulsen and K. Todt, Angew Chem., 77, 589 (1965);Angew Chem. Intern. Ed. Engl., 4, 592 (1965). (75a)H. Paulsen and K. Todt, Chem. Ber., 99, 3450 (1966). (76) H. Paulsen and K. Todt, Chem. Ber., 100, 3397 (1967). (77) F. MicheeI and W. Lengsfeld, Chem. Ber., 89, 1246 (1965).

SYNTHESIS OF NITROGEN HETEROCYCLES CH,OH

Q f

PhNCO

HOQ

369

HO

O NH,C1 H

HN

(78)

CH,OAc

TH,OH

Lo\

TH,OH

n A n

uct was deacetylated to give compound 82, and the resulting product was cyclized with 20% acetic acid.7s A similar reaction with a Darabinofuranoside (83) has been reported,1° the cyclization being effected with methanolic sodium methoxide to give the imidazolinone 84. Substituted guanidines can cyclize in an analogous way, to give

M

s

W

V

HNCONHPh

-

MsocR HNYN-p

(83)

0

(84)

iminoimidazolines; thus, on treating cyanamide (85) with boiling ethanolic benzylamine under reflux, the intermediate guanidine derivative (86) was formed, and this was converted18 into the tricyclic 4-imidazolidin -2 -one, namely, 1-benzyl -4,5-(methyl 4,6-O-benzylidene-a-~-mannopyranosid)-3-p -tolylsulfonyl-4-imidazolidin -2-one (87). The condensation with thiocyanates had earlier been a c h i e ~ e d ' ~ (78) C. J . Morel, H e h . Chirn. Acta, 44, 403 (1961).

HASSAN EL KHADEM

370

J

by using both the alkyl and aryl d e r i v a t i v e ~ ~ ~however, -~l; the products were formulated in the acyclic, imidazole form. This structure was soon discarded in favor of the bicyclic, 4,5-(D-glucopyrano)imidazolidine-2-thione structure 88 in the light of the results of periodate oxidation of the products obtained by desulfurization with Raney

CH OH

(78)

-

( 1 0

(79) C. Neuberg and H. Wolff, Ber., 34, 3840 (1901). (80) F. Garcia Gonzrilez and J. Femrindez-Bolafios, An. Fis. Quim. (Madrid), 44B, 233 (1948). (81) F. Garcia GonzPlez and J. Fernrindez-Bolaiios, An. Fis. Quim. (Madrid), 45B, 1527 (1949).

SYNTHESIS OF NITROGEN HETEROCYCLES

371

n i ~ k e l , ' ~and . ~ ~the structure was confirmed by X-ray crystallographic s t ~ d i e s . ~As * - with ~ ~ aryl isocyanates, the formation of imidazolidine2-thiones can be achieved with the hexosamine acetates78(see p. 369). On being heated with acetic acid, imidazolidine-2-thiones of the type of 88 undergo rearrangement to the corresponding monocyclic 4-(tetrahydroxybutyl)imidazoline-2-thione~s7~8s (89);these will be discussed in the next Section.

R

(88)-

HOCH I HCOH I HCOH I CH,OH (89)

-

b. Imidazolines. The 4-(D-arabino-tetrahydroxybuty1)imidazolin2-one (90) and -2-thione (92) are obtained from the reaction of substituted l-amino-l-deoxy-D-fructose (91)with isocyanate and thiocyanate ions, respectively?O The l-alkyl-2-thiones (92) are also obtained by

(82) F. Garcia Gonzalez, J. Femandez-Bolaiios, and J . Ruiz Cruz, An. Fis. Quim. (Madrid), 478,299 (1951). (83) F. Garcia Gonzilez, J. Femandez-Bolaiios, and A. Paneque Guerrero, An. Fis. Quim. (Madrid), 57B,379 (1961). (83a)GeigyA.-G., French Pat. 5403 (1967). (84) M. Gubero, L. Roldan, and A. PBrez Puente, An. Fis. Qui'm. (Madrid), 53A, 126 (1957). (85) L. Bru and M. PBrez Rodriguez, An. Fis. Quim. (Madrid), 53A, 149 (1957); 54A, 31 (1958). (86) P. Hunoz ConzQlez, An. Uniu. Hisp. (Sevilla, Spain), 20, 47 (1960). (87) F. Kruger and H. Rudy, Ann., 669, 146 (1963). (88) F. Garcia Gonzalez, J. Femandez-Bolaiios, and A. Heredia Moreno, An. Fis. Quim. (Madrid),62B,999 (1966). (89) F. Garcia Gonzilez, M. MenBdez Gallego, F. Ariza Toro, and C. Alvarez Gonzfdez, An. Fis. Quim. (Madrid), 64B,407 (1968). (90)G. Huber, 0. Schier, and J. Dmey, Helo. Chirn. Acta, 43, 713, 1787 (1960).

HASSAN EL KHADEM

372

HCOH I

CqOH

(90)

HCOH I ChOH (91)

HCOH I C&OH (92)

the rearrangement of the bicyclic imidazolidine-2-thiones already discussed (see p. 371), and the unsubstituted derivatives are obtained from the reaction of 2-amino-2-deoxy-D-glucose hydrochloride with potassium t h i o ~ y a n a t e . ~The ' ~ ~ ~structure of these compounds was established by periodate oxidation of the product obtained by desulfurization with Raney nickel or by alkylation of the tautomeric imidazole-2-thi01.~~-~~ On the other hand, acetylation does not involve the 2-thione group, but only the tetrahydroxybutyl side-chain.M Like many of the nitrogen heterocycles possessing tetrahydroxybutyl side-chains (which tend to form anhydrides), the unsubstituted imidazoline-2-thione 92 readily loses water on heating its aqueous solution under pres~ul-e,~' to give 93, and as with other l-substituted 2-thiones, it is converted into the l-aryl-4-(D-arabino-tetrahydroxylbuty1)imidazole (94) by desulfurization followed by oxidation of the product with oxygen in the presence of a platinum catalyst.08

(91)H.Pauly and E. Ludwig, Z. Physiol. Chem., 121,170 (1922). (92) K. Ishifuku, Yakugaku Zasshi, 48,584 (1928). (93)F. Garcia GonzAlez and J. FemLndez-Bolaiios, An. Fis. Q d m . (Madrid), 45B, 1531 (1949). (94) F. Garcia GonzLlez, J. Femlndez-Bolarios, and M. Menbndez Gallego, An. Fis. Quim. (Madrid), 60B,653 (1964); 62B,1061 (1966). (95)J. Femhdez-Bolaiios and M. Menbndez Gallego, An. Fis. Quim. (Madrid),62B, 1005 (1966). (96)J. Femhndez-Bolafios, F. Garcia GonzLlez, J. Gasch Gbmez, and M. Menbndez Gallego, Tetrahedron, 19, 1883 (1963). (97) M. Repetto, J. Femandez-Bolaiios, and M. J. Martin, An. Fis. Quim. (Madrid), 64B,1013 (1968). (98) J . FemLndez-Bolaiios, M. Martin Lomas, D. Martinez Ruiz, and M. A. Pradera, An. Fis. Quim. (Madrid),64B,203 (1968).

SYNTHESIS OF NITROGEN HETEROCYCLES

(92)

I

__c

HOCH I HCOH

I

o+oH

HCOH I OH

(93)

373

CH,OH (94)

c. 1rnida~oles.~~Imidazole (94), discussed in the previous Section, may be considered the parent compound of this series. More important and better studied are the benzimidazoles. These compounds are obtained by condensing o-diamines with aldonic acids and aldaric acids. In the early the benzimidazoles were obtained in low yield from aldoses by a combined oxidation and condensation reaction with o-phenylenediamine. The main reaction product is the corresponding quinoxaline (see p. 396). When, instead of using an aldose, the reaction is conducted with an aldonic acid (such as 95, which gives 96), the yield rises1wJ05to about 80%. The high yield and excellent crystallizing properties of the benzimidazoles make them useful for the characterization of aldonic a ~ i d s . ~ ' J + ~ ~ ~ (99) The chemistry of 2-(aldo-polyhydroxyalkyl)benzimidazoles was reviewed by N. K. Richtmyer, Aduan. Carbohyd. Chem., 6, 175 (1951). (100)P. Griess and G. Harrow, Ber., 20,2205,3111 (1887). (101)0.Hinsberg and F. Funcke, Ber., 26, 3092 (1893). (102)B. Schilling, Ber., 34,902(1901). (103)H. Ohle, Ber., 67,155(1934). (104)W.T. Haskins and C . S . Hudson,]. Amer. Chem. SOC., 61, 1266 (1939). (105)S. Moore and K. P. Link, J . B i d . Chem., 133,293 (1940);J . Org. Chem., 5,637 (1940). (106)N. K.Richtmyer and C . S. Hudson, J . Amer. Chem. SOC., 64, 1609 (1942). (107)A. T. Memll, R. M. Hann, and C. S. Hudson,]. Amer. Chem. SOC., 65,994(1943). (108)G. R. Barker, K. R. Cooke, and J. M. Gulland, J. Chem. SOC., 339 (1944). (109)R. M. Hann, A. T. Memll, and C. S . HudsonJ. Amer. Chem. SOC.,66,1912(1944). (110)R. J. Dimler and K. P. Link, ]. Biol. Chem., 150,345 (1943);C . F.Huebner, R. Lohmar, R. J. Dimler, S. Moore, and K. P. Link, ibid., 159,503 (1945). (111)E. Zissis, N. K. Richtmyer, and C. S . Hudson, J. Amer. Chem. Soc., 72, 3882 ( 1950). (112)D.A. Rosenfeld, N. K. Richtmyer, and C. S . Hudson, J . Amer. Chem. SOC., 73, 4907 (1951);D.A. Rosenfeld, J. W. Pratt, N. K. Richtmyer, and C. S. Hudson, ibid., 73, 5907 (1951). (113)E. Zissis, D. R. Strobach, and N. K. Richtmyer, J . Org. Chem., 30, 79 (1965).

374

HASSAN EL KHADEM

HO I

" N y N

c=o

I HCOH

HCOH I HOCH I HCOH I HCOH L&OH

I I

HOCH

HCOH I HCOH 1 C4OH (95)

(96)

With the aldaric acids, condensation occurs on both ends of the molecule, yielding products having two benzimidazole rings separated by a polyhydroxyalkylidene group, as in 97. These products are well adapted for the characterization of uronic acids and their polymersl14-l 16 and of the hydrolysis products of nucleic acids."'

HCOH I

HOCH

HCOH

-

HCOH I HOCH HCOH I

I

c=o I

HO

Q Like many tetrahydroxybutyl derivatives of heterocycles, a molecule of the benzimidazole 98 readily loses a molecule of water to (114) R. Lohmar, R. J. Dimler, S. Moore, and K. P. Link,]. B i d . Chem., 143,551(1942). (115) G. A. Levvy, Btochem. J., 42, 2 (1948). (116) J. K. Grant and G . F. Manian, Btochem.], 47, 1 (1950). (117) G. R. Barker, K. R. Farrar, and J. M. Gulland, 1.Chem. SOC., 21 (1947).

SYNTHESIS OF NITROGEN HETEROCYCLES

375

gi"elos.llo.l18 the anhydro derivative 99. When boiled with acetic anhydride, 99 loses two molecules of water, giving 2-(2-furyl)benzimidazole (100); the structure of both anhydro compounds was established by

HCOH I HOCH I HSOH

f

t

s y n t h e s i ~ . "Also ~ in common with other heterocycles having sugarlike side-chains, the configuration of the carbon atom adjacent to the ring determines the sign of rotation of the benzimidazole: when the hydroxyl group on C-2 of the aldonic acid precursor is to the right in the Fischer projection formula, the rotation is positive, and vice uersu.120 The configuration of this carbon atom also determines the sign of the Cotton effect in the optical rotatory dispersion curves121of these compounds. 4. Oxazolidines, Oxazolines, and Oxazoles a. Oxazolidines.'4-Tw~ isomeric oxazolidines, 103 and 105, have been obtained by cyclization of 5-aminod-deoxy-l,2-0-isopropylidene-L-idofuranose (101) by way of the sulfite 102 or the 6-amino-6deoxy-L-idosyl sulfite derivative (104).74,75,75a On hydrogenation, a (118)R. C. Hockett, M. H. Nickerson, and W. H. Reeder, ]. Amer. Chem. SOC., 66, 472 (1944). (119)C. F.Huebner and K. P. Link,]. Biol. Chem., 186, 387 (1950). (120)N. K. Richtmyer and C. S. Hudson,]. Amer. Chem. SOC., 64, 1612 (1942). (121)W. S.Chilton and R. C. Krahn,]. Amer. Chem. SOC., 89,4129(1967).

376

HASSAN EL KHADEM

compound of the first type (103) is converted into piperidine (106), whereas, a compound of the second type (105) undergoes ring expansion to the seven-membered nitrogen heterocycle 107; in each instance, the oxygen bridge is ruptured.'22 HO,

,SO,H C I HCOH

CH,OH I

I

I

HCOH I H,NCH I CH,OH

HO

Another type of oxazolidine (109) was obtained from a substituted 1-deoxy-1-p-toluidino-D-fructose (108) by cyclization with benzalde?

f

H

a

M

e

P~CHO ____c

HOG

H

-

Ph

HO

HO (108)

(122) H. Paulsen and K. Todt, Chem. Ber., 100, 512 (1967).

(109)

SYNTHESIS OF NITROGEN HETEROCYCLES

377

h ~ d e . An ' ~ anhydronucleoside ~ (111)was obtainedlZ4by participation of oxygen from a dimethanesulfonic ester (110) of guanosine, the ring thus formed with C-2 being an oxazolidine ring. Similar five-membered rings, such as 113, have been obtained125from such uracil nucleosides as 112, having leaving groups on C-2.

t

Oxazolidinones have been prepared by participation of an Nbenzyloxycarbonyl group with a leaving group on'26-'3' C-1 or C-3 such as 3,4,6-tri-O-acetyl-2-(benzylof a 2-amino-2-deoxyhexose,13z-'34 (123) R. Kuhn and G. Kriiger, Ann., 618, 82 (1958). (124) M. Ikehara and K. Muneyama, J . Org. Chem., 32, 3039 (1967). (125) K. A. Watanabe and J. J. Fox,J. Org. Chem., 31, 211 (1966). (126) S. Konstas, I. Photaki, and L. Zervas, Cheni. Ber., 92, 1288 (1959). (127) S. Umezawa, S. Koto, and Y. Ito, Bull. Chem. SOC.J a p . , 36, 183 (1963). (128) T. Suami, S. Ogawa, and S. Umezawa,BuZZ. Cherri. Soc.Jap.,36,459 (1963). (129) K. Heyns, R. Harrison, and H. Paulsen, Chem. Ber., 100,271 (1967). (130) S. R. Kulkarni and H. K. Zimmerman, Jr., Ann., 684, 223 (1965). (131) P. H. Gross, K. Brendel, and H. K. Zimmerman, Jr., Ann., 680, 159 (1964). (132) P. H. Gross, K. Brendel, and H. K. Zimmerman, Jr., Ann., 681, 225 (1965). (133) K. Brendel, P. H. Gross, and H. K. Zimmerman, Jr., Ann., 691, 192 (1966). (134) K. Heyns, K. Propp, R. Harrison, and H. Paulsen, Chem. Ber., 100, 2655 (1967).

HASSAN EL KHADEM

378

oxycarbonylamino)-2-deoxy-a-~-glucopyranosyl chloride (114), benzyl 3,4-anhydro-2-(benzyloxycarbonylamino)-2-deoxy-a-~-galactopyranoside (116), and 3,4,5,6-tetra-O-aceyl-2-(benzyloxycarbonylamino)-2-deoxy-~-glucosediethyl dithioacetal(118), which yield 115, 117, and 119, respectively. On cyclization, methyl 4,6-O-benzylidene3-deoxy-2-O-(methylsuIfonyl)-3-ureido-a-~-altroside (120) yielded a mixture of imino-oxazolidine 121 and oxazolidinone 122; the N-benzyl derivative (124) of the latter was obtainedI3 from the N-benzylcyanamido derivative 123.

FH,OAc

FH,OAc

-

AcOQCi

QA0

AcO

N H

HN-YO

CH,OH

CH,OH

Q O C H , P h -

" Q O C H , P h

OKNH

OC/NH OCH,Ph I

0

(117)

(116) H EtSCSEt I HF-NH-CO-0-CH,Ph RO~H

H E tSC SEt I

HYN,H -c

I

HFOR

HYOR

HCOR (118)

Ph

HCOR I

I CH,OR

(119)

-(b 0

HN\

(120)

*\_,

MsO OMe -ph-(ob

CH,OR

OMe + p h - ( o o

OMe

co I

NH,

(121)" " YNH O

(122)

0

SYNTHESIS OF NITROGEN HETEROCYCLES

(123)

379

(124)

Cyclization with the primary hydroxyl group of a 2-amino-2-deoxyD-glucitol (125) has been reported35 to give 1.26. Direct interaction H,C-0 CH,OH I HYNH,. HCI ROCH

I

HFOR H~OR

I

CH,OR

(125)

1

COCI,

\ /c=o

HC-NH I

ROCH

I

HFOR

HCOR I

CH,OR

(126)

between D-glucopyranosylamine (127) and phosgene also affords an oxazolidinone, the nitrogen atom being attached135to C-1, as in 128.

Another type of heterocycle accessible from sugars by a one-step reaction was investigated by Zempl6n after a German patent reported that D-glucose reacts with thiocyanate to give a compound thought to be D-gluconothi~lactone.~~~ He soon established that the product (135) P. R. Steyemark, J. Org. Chem., 27, 1058 (1962). (136) Kali-Chemie A.-G., German Pat. 590,580; Chem. Abstracts, 28, 3187 (1934).

380

HASSAN E L KHADEM

contains an oxazolidine ring,137J38and similar heterocyclic derivatives were obtained from other sugars.'39 That their exact structure is that exemplified by 129 was, however, determined later, both by degradation studies and the results of periodate o ~ i d a t i o n . ' ~ ~ - ' ~ ~

b. 0xazolines.'-It has been shown (see p. 352) that, during the preparation of epimines from acylamido sugars, oxazolines are formed by participation of the oxygen atom of the acyl group (instead of participation by nitrogen, which leads to an epimine). The formation of an oxazoline requires the presence of an acylamido group adjacent to a trans-leaving group, such as the methyl- or p-tolyl-sulfonyloxy group, or the halogen atom of a glycosyl halide. Here, as with epimines, the trans-diaxial orientation is more favorable than the trans-diequatorial. Opening of the oxazoline ring leads to a 1,2-cis-acylamido alcohol (instead of the original, trans arrangement present before ring formation). This method therefore constitutes a very useful means of effecting configurational inversion, and is at present the major synthetic use for sugar oxazolines. Several reactions involving inversions on the carbon atoms adjacent to R

R

I

X

R

i

(137) G. Zemplkn, A. Gerecs, and M. Rados, Ber., 69,748 (1936). (138) C . ZemplCn, A. Gerecs, and E. Illes, Ber., 71, 590 (1938). (139) W. Brornund and R. M. Herbst,J. Org. Chem., 10, 267 (1945). (140) J. T. Edward and E. F. Martlew, Chem. Ind. (London), 1034 (1952). (141) J . C. P. Schwarz, J. Chem. Soc., 2644 (1954). (142) A. WickstrBm and J . K. Wold, Acta Chem. Scand., 13, 1129 (1959). (143) J . C. Jochims, A. Seeliger, and T. Taigel, Chem. Ber., 100, 845 (1967).

SYNTHESIS OF NITROGEN HETEROCYCLES

381

acylamido groups have been attributed to the intermediate formation of o x a z o l i n e ~ . ' ~ ~Most - ' ~ ~of the sugar oxazolines thus far isolated have been obtained from the readily available 2-amino-2-deoxy-Dglucose by cyclizing an N-acetyl or N-benozyl group with C-1. For 2-methyloxazolines, the reaction is so facile that it does not require method a leaving group of the type just d e s ~ r i b e d . ' ~A~ convenient -'~~ of preparation consists in treating a 2-acylamido-2-deoxyaldose such as 130 with acetic anhydride and zinc chloride to 131, and, for hranosides, the thioglycoside (132) is treated with chlorine,157 to give 133. YH,OH

CH,OAc I

$!H,OH

FH,OAc

(144)B. R. Baker and R. E. Schaub, J. Amer. Chem. SOC.,75,3864 (1953);77,5900 ( 1955). (145)B. R. Baker and R. E. Schauh,J. Org. Chem., 19,646 (1954). (146)B. R. Baker, R. E. Schauh, J. P. Joseph, and J. H. Williams,J. Anier. Chenz. Soc., 76,4044 (1954). (147)E.J. Reist, R. R. Spencer, and B. R. Baker, J . Org. Chern., 24, 1618 (1959). (148)R. W. Jeanloz,J. Amer. Chem. S O C . , 79,2591 (1957). (149)R. W. Jeanloz and D. A. Jeanloz,]. Org. Chenz., 26, 537 (1961). (150)R. W. Jeanloz, Z. T. Glazer, and D. A. Jeanloz,]. Org. Chem., 26, 532 (1961). (151)A. C.Richardson and K. A. McLauch1an.J. Cheni. SOC.,2499 (1962). (152)Earlier reports of an oxazoline [see T. White, J . Chem. Soc., 428 (1940)] were subsequently shown to be incorrect; D. H. Leaback and P. G. Walker, Chent. Ind. (London), 1012 (1957);F.-P. van d e Kamp and F. Micheel, Cheni. Ber., 90,2054(1957). (153)F. Micheel, F.-P. van de Kamp, and H. Wulff, Chem. Ber., 88,2011 (1955). (154)A. Yu. Khorlin, M. L. Shulman, S. E. Zurabyan, I. M. Privalova, and Yu. L. Kopaevich, Izv. Akad. Nauk S S S R Ser. Khim., 227 (1968). (155)N. Pravdii., T.D. Inch, and H. G. Fletcher, Jr., J. Org. Chern., 32,1815 (1967). (156)W.L.Salo and H. G. Fletcher, Jr.,J. Org. Chem., 33,3585(1968);in press (1970). (157)M. L. Wolfrom and M. W. Winkley,J. Org. Chern., 31,3711 (1966).

HASSAN EL KHADEM

382

For the 2-phenyloxazolines, the reaction is performed, for example, by treating tri-O-acetyl-2-benzamido-2-deoxy-a-~-glucopyranosyl bromide (134)with hydrobromic w~to give 135.Alternatively, the unacetylated 2-acylamido-2-deoxy-~-glucose is treated with hydrochloric acid in acetone, and the 5,6-isopropylidene acetal (136)is obtained.Ie5 Compound 136 has proved a valuable intermediate in the synthesis of several amino sugars.1w-172 FH,OAc

FH,OAc

In addition to the cyclization of 2-acylamido groups with C-1, the 2-acylamido group can form an oxazoline with C-3. The 2-methyloxazolines, which are readily hydrolyzed, have not been isolated, but their probable presence is indicated by the inversion observed.'44 For a 2-benzamido group adjacent to a suitable leaving-group on C-3, the oxazolines formed are quite stable and can be isolated. The reaction has been conducted on pyranoside d e r i v a t i v e ~ , ~ . ~175 ~ Jsuch 7~as methyl 2-benzamido-4,6-O-benzylidene-2-deoxy-3-O-(methylsulfonyl)-P-D-glucopyranoside(137) to give 138, furanoside deriva(158)F. Micheel and H. Kochling, Chem. Ber., 90,1597(1957). (159)F. Micheel, F.-P. van d e Kamp, and H. Petersen, Chem. Ber., 90,521 (1957). (160)F.Micheel and E. Drescher, Chem. Ber., 91,670 (1958). (161)F.Micheel and H. Kiichling, Chem. Ber., 91,673(1958). (162)F.Micheel and H. Petersen, Chem. Ber., 92,298 (1959). (163)F. Micheel, H. Petersen, and H. Kochling, Chem. Ber., 93,1 (1960). (164)F. Micheel and H. Kiichling, Chem. Ber., 93,2372 (1960). (165)S. Konstas, T. Photaki, and L. Zervas, Chem. Ber., 92,1288 (1959). (166)T. Osawa, Chem. Phnrm. Bull. (Tokyo), 8,597(1960). (167)R. Gigg and P. M. Carroll, Nature, 191,495 (1961). (168)R. Gigg, P. M. Carroll, and C. D. Warren, J . Chem. Sac., 2975 (1965). (169)R. Gigg and C. D. Warren, J . Chem. SOC. (C),295 (1969). (170)W.Meyer zu Reckendorf and W. A. Bonner, Chem. Ber., 95,996 (1962). (171)W.Meyer zu Reckendorf and J. Feldkampf, Chem. Ber., 101,2289(1968). (172)W.Meyer zu Reckendorf, N. Wassiliadou-Micheli, and D. Delevallk, Chem. Ber., 102,1076 (1969). (173)W.Meyer zu Reckendorfand W. A. Bonner, Chem. Ber., 95,1917 (1962). (174)A. C.Richardson, J. Chem. Sac., 5464 (1964). (175)Y. Ali and A. C. Richardson, J. Chem. SOC. (C), 320 (1969).

SYNTHESIS OF NITROGEN HETEROCYCLES

383

tives, 176-1711 such as methyl 2-benzamido-2-deoxy-5,6-O-isopropylidene-3-O-(methylsulfonyl)-~-~-glucofuranoside (139) to give 140, and'79on the substituted 2-benzamido-2-deoxy-3-O-(methylsulfonyl)L-iditol (141) to give 142. Similar oxazolines have been obtained

CH,OR I HC-NH-CO-Ph I MsOCH I HCOR I ROCH I CH,OBz (141)

CH,OR I HC-N

I

HC-0

' F h

I

___t

HCOR I

ROCH I CH,OBz (142)

where R = CH,Ph

(176) R. Gigg and C. D. Warren,J. Chem. Soc., 1351 (1965). (177) R. Gigg, C. D. Warren, and J. Cunningham, Tetrahedron Lett., 1303 (1965). (178) J. Gigg, R. Gigg, and C. D. Warren, J . Chem. SOC. ( C ) , 1872, 1882 (1966). (179) R. Giggand C. D. Warren,./. Chem. SOC. (C),2661 (1968).

HASSAN E L KHADEM

384

from epimino sugars by ring expansion (see p. 356). Oxazolines have also been prepared by cyclization of the 3-benzamido group with C-4 of 3-benzamido-3-deoxy-1,2:5,6-di-O-isopropylidene-4-O-(methylsulfonyl)-D-altrose180 (143) to give 144, as well as between the 4-benzamido group and C-3of methyl 4-benzamido-2-0-benzoyl-4-deoxy-3-0p-tolylsulfonyl-a-D-xylopyranoside'81(145) to give 146, and between the 6-benzamido group and C-5 of 6-benzamido-6-deoxy-1,2-O-isopropylidene-5-O-p-tolylsulfonyl-a-~-glucofuranose'~~ (147) to give 148.

HC-N Hb-NHCOPh I HFOMs HkO, I ,CMe,

H,CO

___c

I

HC-0

' b h

I

HCO, I ,CMe,

H,CO

(180) B. R. Baker and A. H. HainesJ. Ow. Chem..28,442 (1963). (181) W. Meyer zu Reckendorf and Ny Wassiliadou-Micheli,'Chem. Ber., 101, 2293 (1968).

SYNTHESIS OF NITROGEN HETEROCYCLES

385

c. Oxazoles. -The preparation of sugar oxazoles has been reported. Treatment of oxazoline 149 with potassium tert-butoxide in methyl sulfoxide causes the removal of the proton on C-4 of the oxazoline ring and the rupture of the 0 - C bond of the furanoid ring, giving riselB2to the oxazole derivative 150. Ph

I

ROCH

I

HCOH

I

HCO,

I

H,CO'

CMe,

5. Thiazolidines and Thiazolines

a. Thiazolidines. -Thiazolidines are obtained by the direct interproducts are probably action of aldoses with ~ - c y s t e i n e . ' ~lB5~ These derivatives of the aldehydo form, and are similar to the thiazolidines obtained from L-cysteine and simple aldehydes. D-Glucose gives the thiazolidine 151. Thiazolidines have also been obtained from Larabinose, D-xylose, D-mannose, D-galactose, and lactose. The same

I

HCOH

I

HOCH I HCOH

I

HCOH I CH,OH

(151)

(182) R. Gigg and C. D. Warren, /. Chem. SOC. ( C ) , 1903 (1968). (183)M.P.Schubert,]. Biol. Chem., 130,601(1939). (184)I. Vadopalaite and J. V. Karabinos,Trans. Illinois State Acad. Sci., 46,266(1953). (185)G.Weitzel, J. Engelmann, and A.-M. FretzdoB, 2. Physiol. Chem., 315, 236 (1959).

HASSAN EL KHADEM

386

kind of reaction has been conducted with 2-aminoethanethio1, to give a thiazolidine.186The structure of the D-glucitol derivative 152 was determined by acetylating, opening the sulfur ring in the acetate (153)with Raney nickel, and identifying the product as 2,3,4,5,6penta-O-acetyl-l-deoxy-l-(N-ethylacetamido)-D-glucitol(l54).

n

n

HNYS..,YS HCOH I HOCH I HCOH I HCOH I C H,OH

-

HCOAc

A~O&H I

HCOAC

I

HCOAc

-

I

AcOCH

I

CH,OAc

(152)

/Et Hz(fNLAc HCOAc HLOA~ I

HCOAc I CH,OAc (154)

(1 53)

b. Thiazolines. -As with oxazolines, the thiazolines are formed by the participation of sulfur, but the thioureido and the dithiocarbamoyl groups are involved. Thus, a 2-(dithiocarbamoyl) group may cyclize with either C-1 or C-3 of an aldose, according to the nature of the leaving group and the stereochemistry of the molecule. The conversion of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-(dithiocarbamoyl)-~-~glucopyranose (155)into 156 illustrates the first type of c y ~ l i z a t i o n ~ ~ ~ ; FH20Ac

CH,OAc I

HN-qS

SMe

I

SMe

(155)

(1 56)

there are several examples of the second type,3,16,18,'88*189 such as the formation of 158 from 157. Examples are also known11*16.1eo of sulfur participation between a 3-(dithiocarbamoyl) or 3-(thioureido) group and C-2 to afford such thiazolines as 160 from 159, and 3 from 1. (186) W. A. Bonner and W. Meyer zu Reckendorf, Chem. Ber., 94,225 (1961). (187) T. Ito, Con. J . Chem., 44, 91 (1966). (188) W. Meyer zu Reckendorfand W. A. Bonner, Proc. Chem. Soc., 429 (1961). (189) W. Meyer zu Reckendorf and W. A. Bonner, Tetrahedron, 19, 1721 (1963). (190) L.Goodman and J. E. Christensen,J. Org. Chem., 28, 2610 (1963).

SYNTHESIS OF NITROGEN HETEROCYCLES

387

0 7

ph-Q

sc

0

I /NH

s y 5 / N

I

R

R

(158) R = SMe o r NH,

(157)

0

0

WOMe - R:e /I MeOCOCH,

II MeOCOCH,

0

(1) -(3)

HM N \eCSI S

”“---(

SMe

(159)

(160)

6. 1,2,3-Triazoles Three types of saccharide triazole are known at present. In type I, the glycosyl residue is attached to N-1 or N-3 of the triazole ring; in type 11, the saccharide residue i s attached to C-4 of the triazole ring, which bears an acyl or aryl group on N-1; and in type I11 (the best known), the saccharide residue i s on C-4, and an aryl group is on N-2.

R

R zN

7 Sacch.

Type

HASSAN EL KHADEM

388

a. Type I. Triazoles Having a Sugar Residue Attached to a Nitrogen Atom.-The first member (161) of this series was prepared by Micheel and Baum”’ from phenylacetylene and tetra-O-aCetyl-P-D-glUCOpyranosy1 azide (162). The same azide was later cyclized with esters of acetylenedicarboxylic acid.lS2The D-XylOSe analog of 161 has also been prepared.isg The formation of triazoles 161 and 163 from 162 follows RO,C

AcOCH,

OAc

(161)

(162)

OAc (163)

a general method of synthesis of triazoles from the reaction of azides with acetylenic compounds; this method is known to give rise to 1substituted triazoles. Furthermore, the structure of the compounds prepared was established by hydrolysis to D-glUCOSe and to known, substituted triazoles. Triazoles of this type are glycosylamines and, consequently, they lack the stability of triazoles of other types. However, acid-stable triazoles of this type are obtained when primary or secondary azides of sugars are treated with phenylacetylene. Is3

b. Type 11. 1-Substituted Triazoles Having a Saccharide Residue Attached to C-4.-It has been possible to cyclize acyl osazones of sugars with iodine and mercuric oxide to triazoles having the acylamido group at N-1 and the saccharide residue attached to C-4 of the triazole ring.is4*’s5 Thus, 164 gives 165. A similar reaction is known

(191) F. Micheel and G . Baum, Chem. Ber., 90, 1595 (1957). (192) J. Baddiley, J, G . Buchanan, and G. 0.Osbome,J. Chem. SOC.,1651,3606 (1958). (193) H. El Khadem, D . Horton, and M . H. Meshreki, Carbohyd. Res., in press (1971). (194) H. El Khadem and M. A. E. ShabanJ. Chem. SOC.( C ) ,519 (1967); H. El Khadem, M . A. M. Nasr, and M. A. E. Shaban, ibid., 1465 (1968). (195) H. El Khadem, M. A. E. Shaban, and M. A. M. Nasr, J. Chem. SOC. (C), 1416 (1969).

SYNTHESIS OF NITROGEN HETEROCYCLES

389

to occur with simple bis(benzoy1hydrazones) of 1,2-dicarbonyl compounds; the products were originally believed to be tetrazines,'% but a more detailed study has revealed that they are 1-substituted tri-

p?

/C=N BzO

BzYH

\N-N

HC=N-NH-Bz I

C=N-NH-Bz ACOCH

I

HYOAc HCOAC i CH,OAc

oxidation

ACOCH I HFOAc HCOAC 1 CH,OAc

NH,OH

I HFOH

HCOH 1

CH,OH

a ~ o l e s . ' ~The ' structure of l-benzamido-4-(~-arabino-tetrahydroxybutyl)-1,2,3-triazole (166) was determined by periodate oxidation to l-benzamido-4-formyl-1,2,3-triazole(167) and 168. The benzoylhydrazone of the isomeric 5-formyl derivative was obtained from the trishydrazone 169 by c y c l i z a t i ~ nto ' ~l7Oand ~ 171.

1-N-Aryl analogs have been synthesizedIg3 by cycloaddition of terminal-acetylenic sugar derivatives to azides, as exemplified by the conversion of the pentyne derivative 171a into l-phenyl-4-(D-threo(196) R. Stolli., Ber., 59, 1742 (1926). (197) D. Y. Curtin and N. E. Alexandrou, Tetrahedron, 19, 1697 (1963).

HASSAN EL KHADEM

390

CH

Ph

trihydroxypropyl)-l,2,3-triazole(171b).These compounds are positional isomers of the arylosotriazoles (Type 111).

c. Type 111. Aryltriazoles Having a Saccharide ResidueIs8Attached type of triazole has been by far the most extensively studied; its members are commonly designated simply as sugar triazoles, or saccharide o s o t r i a z ~ l e s The . ~ ~ ~first member of this series was prepared by Hann and Hudson1ss by treating osazones with boiling, aqueous copper(I1) sulfate under reflux; for example, 172 gave 173. A large number of osotriazoles has since been prepared; they are useful in characterization of the osazones from monosaccharides200-209 and disaccharides,210-216 and of anhydro-osaz~nes.~~~-~~~ to C-4.-This

(198) The chemistry of osotriazoles was reviewed by H. El Khadem, Advan. Carbohyd. Chem., 18,99 (1963). (199) R. M. Hann and C. S. Hudson,]. Amer. Chem. SOC., 66, 735 (1944). (200) W. T. Haskins, R. M. Hann, and C. S. Hudson,J. Amer. Chem. Soc., 67,939 (1945); 68, 1766 (1946);69, 1050, 1461 (1947);70, 2288 (1948). (201) D. A. Rosenfeld, N. K. Richtmyer, and C. S. Hudson, J . Amer. Chem. SOC., 73, 4907 (1951). (202) L. C. Stewart, N. K. Richtmyer, and C. S. Hudson,J. Amer. Chem. SOC., 74,2206 (1962). (203) J. W. Pratt, N. K. Richtmyer, and C. S. Hudson, J . Amer. Chem. Soc., 74, 2210 (1952). (204) J. V. Karabinos, R. M. Hann, and C. S. Hudson, J . Amer. Chem. Soc., 75, 4320 (1953). (205) N. K. Richtmyer and T. S. BodenheimerJ. Org. Chem.,27,1892 (1962). (206) P. P. Regna, J.Amer.Chem. SOC., 69,246(1947). (207) V. Ettel and J. Liebster, Collect. Czech. Chem. Commun., 14,80 (1949). (208) E. Hardegger and H. El Khadem, Helv. Chim. Acto, 30,900,1478 (1947). (209) E. Hardegger, H. El Khadem, and E. Schrier, Helo. Chim. Acto, 34, 253 (1951). (210) J. F. Carson, J. Arner. Chem. SOC., 77, 1881 (1955). (211) W. Z. Hassid, M. Doudoroff, and H. A. Barker, Arch. Biochem., 14, 29 (1947). (212) W. Z. Hassid, M. Doudoroff, A. L. Potter, and H. A. Barker, J. Amer. Chem. Soc., 70, 306 (1948). (213) F. H. Stodola, H. J. Koepsel, and E. S. Sharpe, J . Amer. Chem. Soc., 74, 3202 (1952). (214) A. Thompson and M. L. Wolfrom, J . Amer. Chem. SOC., 76, 5173 (1954).

SYNTHESIS OF NITROGEN HETEROCYCLES

391

Ph HC=N-NHPh

I

CZN-NHPh I HOCH cuso,

I HCOH I HCOH I

CH,OH (172)

9

HOCH I HCOH

-t PhNHa

HAOH

1

CH,OH

(173)

The bishydrazones of the 1,e-diketones from inositols have also of arylosazones been converted into t r i a ~ o l e s . The ~ ~ ~conversion ,~~~ into the corresponding osotriazoles requires the presence of an oxidant, and it is obvious that simple removal of aniline from the osazone, as suggested by the equation, is not involved. In addition to copper(I1) sulfate, the reagent most commonly used, other oxidizing heavy-metal salts, such as ferric sulfate and ~ h l o r i d e , ~and ~4 mercuric acetate,223have been used, as well as halogenszz5and nitrosulfonates.226The osazone acetates are converted into osotriazoles by nitrous which decomposes the unacetylated osazones to the aldosulosesZz8;and the osazone formazans are cyclized with warm

(215) F. H. Stodola, E. S. Sharpe, and H. J. Koepsel, J. Amer. Chem. Soc., 78, 2514 (1956). (216) D. Rutherford and N. K. Richtmyer, Carbohyd. Res., in press (1969). (217) E. Hardegger and E. Schreier, Hell;. Cham. Acta, 35, 623 (1952). (218) H. El Khadem, E. Schreier, G. Stohr, and E. Hardegger, Helu. Chirn. Acta, 35, 993 (1952). (219) S. Bayne, J. Chem. Soc., 4993 (1952). (220) E. Schreier, G . Stohr, and E. Hardegger, Helu. Cham. Acta, 37, 574 (1954). (221) G. Hanisch and G. Henseke, Chem. Ber., 100,3225 (1967); 101,2074 (1968). (222) L. Anderson and J. N. Aronson, J . Org. Chern., 24, 1812 (1959). (223) A. J . Fatiadi, Chem. Znd. (London), 617 (1969). (224) H. El Khadem and 2. M. El-Shafei,J. Chem. Soc., 3117 (1958); 1655 (1959). (225) H. El Khadem, Z. M. El-Shafei, and M. H. Meshreki,J. Chern. SOC., 2957 (1961); 1524 (1965); H. El Khadem, Z. M. El-Shafei, and Y. S. Mohammed, ibid.,3993 (1960); H. El Khadem, A. M. Kolkaila, and M. H. Meshreki, ibid., 2531 (1963). (226) H. J. Tauber and G. Jellinek, Chem. Ber., 85, 95 (1952). (227) M. L. Wolfrom, H. El Khadem, and H. Alfes, J. Org. Chem., 29,2072 (1964). (228) G. Henseke and M. Winter, Chern. Ber., 93,45 (1960).

392

HASSAN EL KHADEM

I

HCOH 1 HCOH I CkOH

acetic acidzz9to phenylazotriazoles, such as 174. The saccharide osotriazoles are very stable compounds; they can withstand nitration with concentrated nitric-sulfuric and are unaffected by alkali. Like other heterocycles attached to hydroxyalkyl chains (see pp. 373, 375), they undergo dehydration, affording 3,6-anhydro comp o u n d ~ ~ 'such * * ~as~ 175. ~ In addition to its ability to form 3,6-anhydro Ph

I

(173)

HO

OH 1175)

rings, C-3 of the parent sugar (C-1 of the side-chain) exhibits a marked influence both on the groups attached to it and on the optical properties of the compound. Thus, the methine proton attached to C-3 is more deshielded than any of the protons of the side chain,23'and the configuration of C-3 determines the sign of the optical r o t a t i ~ n ' ~ * ~ ~ ~ and of the Cotton effect in the o.r.d.lz1and c.d.233spectra of saccharide osotriazoles. (229) L. Mester and F. Weygand, Bull. SOC. Chim. Fr., 350 (1960);L. Mester, ibid., 381 (1962). (230) (a) H. El Khadem and M. H. Meshreki, Nature, 194,373 (1963);(b) H. El Khadem, M . H. Meshreki, and G. H. Labib,J. Chem. SOC., 2306 (1964). (231) H. El Khadem, D. Horton, and T. F. Page, J . Org. Chem., 33, 734 (1968). (232) H. El Khadem, I. Org. Chem., 28, 2478 (1963); J. A. Mills, Aust. J. Chem., 17, 227 (1964). (233) G. G. Lyle and M. J. Piazza, J . Org. Chem., 33, 2478 (1968).

393

SYNTHESIS OF NITROGEN HETEROCYCLES

7. Thiadiazolines The thioaldonic phenylhydrazides obtained by reduction of saccharide formazans condense with benzaldehyde to give 1,3,4-thiadiazolines, characterized by their high optical rotatory p o ~ e r . ~ ~ ~ . ~ 5

I HCOH I HOCH H o b I HCOH I CH,OH

H2S

1 HCOH

I

HOCH I HOCH I HCOH I CH,OH

PhCHO ____c

I HCOH I HOCH

I I HCOH I

HOCH

CH,OH

8. Tetraz~les”~ Acyclic sugar hydrazones react with benzenediazonium chloride to give red formazans. When oxidized with N-bromosuccinimide (or, after acetylation, with lead tetraacetate), these yield tetrazolium salts. On reduction,237-239 the formazans are regenerated.

HcoR I

ROCH

I

ROCH

oxidation P

reduction

I

HCOR I C H,OR

HCOR I ROCH

I I

ROCH HCOR I

CH,OR where R = H o r Ac

(234) G . Zemplen, L. Mestes, and A. Messmer, Chem. Ber., 86, 697 (1953). (235) A. Messmer and L. Mester, Chem. Ind. (London), 423 (1957); L. Mester and A. Messmer, Methods CarhohrJd. Chem., 2, 126 (1963). (236) See L. Mester, Adoan. Carbohyd. Chem., 13,105 (1958); “Derivks Hydraziniques des Glucides,” Hermann, Paris, 1967, p. 54. (237) G. Zemplen, L. Mester, and E. Eckhart, Chem. Ber., 86, 472 (1953). (238) L. Mester and A. Messmer, 1. Chem. Soc., 3802 (1957). (239) L. Mester and A. Messmer, Methods Curbohyd. Chern., 2, 123 (1963).

394

HASSAN EL KHADEM

Iv. FORMATION OF

SIX-MEMBERED

NITROGENHETEROCYCLES

1. Piperidines Piperidines are obtained from 5-aminod-deoxy sugars by cyclization with C-1; their preparation therefore follows the general methods of preparation of such amino sugars. The 5-0-p-tolylsulfonyl- or 5-O-(methylsulfonyl)-pentoses constitute excellent starting-materials, as these sulfonyloxy groups can be replaced by azide, and the azides can be reduced to the 5-amino derivatives, which are capable of cyclization.*40-251 The formation of piperidine 178 from 5-0-p-tolylsulfonyl-L-arabinose diethyl dithioacetalZ4O(176) and 177, and of 181 from benzyl 2,3-O-isopropylidene-5-O-(methylsulfonyl)-a-~1yxofuranoside”O (179) and 180, are examples of this reaction sequence. H

H

EtSCSEt

EtSCSEt

I

HCoH I H°CH I

-Ac

I

1 . NaN, 2

s

HCOH I H°CH I

CdCO,

Ac __c

(240) S. Hanessian and T. H. Haskel1,J. Org. Chem., 28, 2604 (1963). (241) S. Hanessian, Chem. Commun., 1296 (1965);796 (1966). (242) S. Hanessian, 1.Org. Chem., 32, 163 (1967). (243) H. Paulsen, Angew. Chem., 74, 901 (1962);Angew. Chem. Intern. Ed. Engl., 5 , 495 (1966). (244) H. Paulsen, K. Todt, and F. Leupold, Tetrahedron Lett., 597 (1965). (245) H. Paulsen, F. Leupold, and K. Todt, Ann., 692, 200 (1966). (246) H. Paulsen, I. Sangster, and K. Heyns, Chem. Ber., 100, 802 (1967). (247) H. Paulsen and G . Steinert, Chem. Ber., 100, 2467 (1967). (248) J. K. N. Jones and W. A. Szarek, Can.1.Chem., 41, 636 (1963). (249) D. L. Ingles, Chem. Znd. (London), 927 (1964);Tetrahedron Lett., 1317 (1965). (250) J. S . Brimacombe, F. Hunedy, and M. Stacey, J . Chem. SOC. (C), 1811 (1968). (251) A. Zobacovi and J. Jar$, Collect. Czech. Chem. Commun.,29,2042 (1964).

SYNTHESIS OF NITROGEN HETEROCYCLES

395

A similar series of reactions was performed on a 6-azido-6-deoxy-2ketose.246,2s2 In most of the foregoing syntheses, the piperidine formed is accompanied by the tautomeric furanose. The tautomers can, however, be separated without occurrence of equilibration (which can be induced by changing the p H of the medium). For the ketose derivacompound 184 and a tive 183, the equilibrium mixture dehydration product (182). The conformation and anomeric effect in these piperidines have been studied by n.m.r. s p e c t r o s ~ o p yand ,~~~ their fragmentation pathways have been determined by mass spectrometry. 254 H o o c H 2 0 H

=Hop Hp oH,cH,oH

HO

H,NC

HO (182)

oH,cH,oH

OH

(183)

(184)

Piperidones of two types have been prepared from saccharide derivatives. Those of one type are lactams, exemplified by 187, ob5-azido-5-deoxy-2,3-O-isopropyidene-/3-~-ribotained by furanose (185) to 186, reducing the lactone 186 to the amine, and cyclizing this to 187. The other type comprises the 4-piperidones such as 189;this is obtained256by oxidizing methyl a-D-arabinopyranoside (188) with periodate, and treating the product with methylamine and 3-oxopentanedioic acid; the bicyclic compound obtained from the a-glycoside differs from that from the /3 anomer.

r;B" -

N,CH,

0

CrO,

(185)

0, o , C Me,

N

3

c

0, (186)

~

0

H,

/o

(188)

OH

(187)

(y) OMe

+ HO

0

HO

C Me,

OMe

CH

HoQ

0-0MeNH,

MeN

0

HO,C

(189)

(252)S. Hanessian, Chern. Znd. (London), 2126 (1966). (253)H.Paulsen, K.Todt, and H. Ripperger, Chern. Ber., 101, 3365 (1968). (254)S. Hanessian, 1.Org. Chem., 32, 163 (1967). (255)S. Hanessian and T. H. Haskell, J. Heterocycl. Chern., 1, 55 (1964). (256)R. D.Guthrie and J. F. Mecarthy, J . Chem. SOC. ( C ) , 62 (1967).

HASSAN EL KHADEM

396

2. Diazines

-

a. 1,BDiazines. Substituted hydrazones usually cyclize to fivemembered rings; however, in some cases, diazines (six-membered rings) are formed. Examples are the formation of (a) 3-(D-arabinotetrahydroxybuty1)cinoline (190) as a byproduct in the formation of the phenylosazone from D-glucose by reaction with phenylhydrazine and (b) the bicyclic diazine in the presence of hydrochloric 192 obtained from dehydro-L-ascorbic acid phenyIosazone 6-ptoluenesulfonate (191) by nitrogen participati0n.~5~

H,YOTs

0 -

I

HOCH

I HCOH I HCOH I

PhHNN

NNHPh

CH,OH

(190)

b. 1,4-Diazines.- It has long been known100-103that reducing sugars react with o-diamines to give mainly quinoxalines, together with imidazoles (see p. 373).The reaction bears some resemblance to osazone formation, because, at one stage, a dehydrogenation takes place, caused either by the effect of atmospheric oxygen or by hydrazine hydrate added to the reaction mixture. This oxidation is not necessary if the glycosulose or its l-hydrazone is used as the starting material. Thus, both D-glucose (193)and D-urabino-hexosulose (195) give 2-(~-arubino-tetrahydroxybutyl)quinoxaline (194). Quinoxalines have been prepared from various mono- and d i - s a ~ c h a r i d e s , ' ~ ~ - ' ~ ~ ~ 259-262 2-amin0-2-deoxyhexoses,~~~ Amadori compounds [ l-(ary1amino)(257) H. J. Haas and A. Seeliger, Chem. Ber., 96, 2427 (1963). (258) H. El Khadem and S . H. El Ashry, Carbohyd. Res., 13,57 (1970). (259) H. Ohle and M. Hielscher, Ber., 74, 13 (1941). (260) H. Ohle and R. Liebig, Ber., 75,1536 (1942). (261) H. Ohle and J. J. Kruyff, Ber., 77, 507 (1944). (262) K. Maurer and B. Schiedt, Ber., 67, 1980 (1934). (263) R. Lohmar and K. P. Link, J . B i d . Chem., 150, 351 (1943).

SYNTHESIS OF NITROGEN HETEROCYCLES

397

l-deo~y-2-ketoses],~~267 glycosuloses,26s-271 and 1-substituted glyco-

HO,

,CHO

H 4 HOYH

[ 01

-

I

HOCH I HFOH

HCOH

C,

o=c

HOFH

HCOH

I I

HCOH

I

I

HCOH

HCOH

CHzOH

I

(194)

(193) CH,OH

HO

I

(195) CH@H

sulose hydra zone^.^^*^^* - 275 The 2-quinoxalinols are obtained from L-ascorbic acid, or hexulosonic acids (“2-ketoaldonic acids”) such as 196, by condensation with o - d i a r n i n e ~ ; ~thus, ~ ~ - 196 ~ ~ ~gives 197,

9

Q

NHZ

HZN

f

o=c , C O P I

HOCH

I

HCOH

I I

-

N+

OH

HOCH

I

HCOH

I

HCOH I CH,OH

HCOH (196) CH,OH

Q-

(1 97)

-

.Go

HOCH

I I HCOH I HCOH

CH,OH

(198)

(264) F. Weygand, Ber., 73, 1284 (1940). (265) F. Weygand and A. Bergmann, Chem. Ber., 80,255 (1947). (266) G. Henseke and M. Winter, Chem. Ber., 89,956 (1956). (267) S. Kitaoka and K. Onodera, J . Org. Chem., 28, 231 (1963). (268) E. Fischer, Ber., 22, 87 (1889). (269) C. R. Bont, E. C. Knight, and T. K. Walker, Biochem. J., 31, 1033 (1937). (270) D. M. Hall and E. E. Turner,J. Chem. Soc., 694 (1945). (271) G. Henseke and K. J. Bahner, Chem. Ber., 91, 1605 (1958). (272) G. Henseke and E. Brose, Chem. Ber., 91,2273 (1958). (273) G. Henseke and C. Bauer, Chem. Ber., 92,501 (1959). (274) G. Henseke and W. Lemke, Chem. Ber., 91, 101 (1958). (275) G. Henseke and R. Jakobi, Ann., 684, 146 (1965). (276) H. Erlbach and H. Ohle, Ber., 67, 555 (1934). (277) H. Ohle, W. Gross, and A. Wolter, Ber., 70, 2148 (1937). (278) H. Ohle, M. Hielscher, and G. Noetzel, Ber., 76, 1051 (1943). (279) H. Hasselquist, Ark. Kemi, 4, 369 (1952). (280) G. Henseke and K. Dittrich, Chem. Ber., 92, 1550 (1959). (281) S. Ogawa, Yakugaku Zasshi, 73,309 (1953). (282) G. Henseke, D. Lehmann, and K. Dittrich, Chem. Ber., 94, 1743 (1961). (283) H. Dahn and H. Moll, Helu. Chim. Acta, 47, 1860 (1964). (284) H. Zellner and G. Zellner, Chimia, 19, 587 (1965).

HASSAN EL KHADEM

398

which is in equilibrium with 198. The condensation has also been conducted with phenyl ~-arabino-hexosidulose,~~ 1,5-anhydro-~erythr0-2,3-hexodiulose,~~~ and l-O-benzoyl-6-deoxy-2,5-O-methylene-~-threo-3,4-hexodiulose (199), which affordedz8' 200.

The side chain of 24~-arabino-tetrahydroxybuty1)quinoxaline tends to assume a planar, zigzag conformation, as revealed by n.m.r. It can be oxidized by periodate to a quinoxalinaldehyde capable of undergoing the Cannizzaro reaction and the benzoin condensation, and of reacting with nitrogenous compounds to afford compounds containing additional heterocyclic rings.28s c. Flavazoles. - Quinoxalines react with phenylhydrazine in the presence of acetic acid to give the pyrazoloquinoxalines that are commonly designated flavazoles. In this reaction, C-1 of the side chain (that is, C-3 of the parent sugar) reacts with the hydrazine in a way resembling the reactions that occur during osazone formation, with the formation, in both reactions, of a hydrazone residue on a carbon atom bearing a hydroxyl group. The intermediate hydrazone cyclizes to the flavazole. The reaction has been applied to several

HCOH

HAOH I CH,OH (201)

HCOH I HCOH

HCOH

I C&OH

I

CqOH (202)

(285) H. Ohle and M. Hielscher, Be?., 74, 18 (1941). (286) 0. Theander, Acta Chern. Scand., 12, 1887 (1958). (287) J. W. Bird and J. K. N. Jones, Can. J . Chern., 41, 1887 (1963). (288) D. Horton and M. J. Miller, J . Org. Chem., 30, 2457 (1965). (289) For references, see G . Henseke, 2.Chern., 6,329 (1966).

(203)

SYNTHESIS O F NITROGEN HETEROCYCLES

399

q u i n o ~ a l i n e s *292 ~ ~and ~ ~2~- q~~~i n~o~x a- l i n o l s Thus, . ~ ~ ~ 201 gives 202, and 202 cyclizes to 203. Flavazole formation has been applied to monosaccharides, d i s a c ~ h a r i d e s , ~ ~and ~ , ~ ~higher ~ - ~ ~ oligosaccharides. 293-299

d. Pterins.-If, instead of condensing a reducing sugar with an aromatic diamine, a heterocyclic diamine is used, a 1,4-diazine fused to a heterocyclic ring is formed. Thus, aldoses and glycosuloses react with 2,4,5-triamino-6-pyrimidinolto give pteridine compounds. In view of their biological interest, these “sugar pterins” have been extensively s t ~ d i e d . For ~ ~ example, ~ - ~ ~ ~D-glucose gives 204, which is in equilibrium with 205.

(290) H. Ohle and G. A. Melkonian, Ber., 74, 279, 398 (1941). (291) H. Ohle and A. Itgan, Ber., 75, 1 (1943). (292) G. Henseke and W. Lemke, Chem. Ber., 91, 113 (1958). (293) G. Neumuller, ArkfuKemi, Mineral. Geol., 21A, No. 19 (1946). (294) U. Rosenquist, G. Neumiiller, and K. Myrback, Arkiu Kemi. Mineral. Geol., 24A, No. 14 (1946). (295) J. H. Pazur and D. French, J. B i d . Chem., 196,265 (1952). (296) T. Kobayashi, T. Haneishi, and M. Saito, Nippon Nogei Kagaku Kaishi, 36, 189 (1962). (297) R. L. Whistler and J. L. Hickson, J . Amer. Chem. SOC., 76, 1671 (1954). (298) D. French, G. M. Wild, and W. J. James, J . Amer. Chem. SOC., 75, 3664 (1953). (299) J. D. Dutcher, J. Reid, and 0.Wintersteiner, 1. Org. Chem., 28, 999 (1963). (300) P. Karrer, R. Schwyzer, B. Erden, and A. Seigwart, Helu. Chim. Acta, 30, 1031 (1947). (301) P. Karrer and R. Schwyzer, Helo. Chim. Acta, 31, 777, 782 (1948); 32, 423, 1041 (1949). (302) H. G. Petering and D. J. Weisblat, J . Amer. Chem. Soc., 69, 2567 (1947). (303) H. G. Petering and J’. A. Schmitt, J . Amer. Chem. Soc., 71, 3977 (1949). (304) R. B. Angier, C. W. Waller, J. H. Boothe, J. H. Moyat, J. Semb, B.L. Hutching, E.L. R. Stokstad, and Y. Subharow, J . Amer. Chem. SOC., 70,3029 (1948). (305) H. S. Forrest and J. Walker, Nature, 161, 308 (1948); J. Chem. SOC., 7 , 2077 (1949). (306) H. S. Forrest and K. Mitchell, 1.Amer. Chem. SOC., 77,4865 (1955). (307) F. Weygand, A. Wacker, and V. Schmied-Kowarzik, Experientia, 4, 427 Chem. Ber., 82, 25 (1949). (308) F. Weygand, H. Simon, K. D. Keil, and H. Millauer, Chem. Ber., 97, 1002 1964). (309)G. Henseke and H.-G. Patzwaldt, Chem. Ber., 89,2904 (1956). (310) H. Rembold and H. Metzger, Chem. Bet., 96, 1395 (1963).

HASSAN EL KHADEM

400

I

HCOH

I I CH,OH

HCOH

(205)

A similar condensation has been reported that involves the reaction of the glycosulose monohydrazone 206 with 4,5-diamino-&phenyl1,2,3-triazo1e3"to give 207.

Ph I Ph H,N

NH,

+

___4L

O=C-C=N-N(Me)Ph I

HOCH I HCOH I

HCOH I

CH,OH (206)

HOCH I HCOH I HCOH

I

CH,OH (207)

3. Oxazines and Thiazines a. l,%Oxazines. -Several 1,3-oxazines have been prepared by reactions involving oxygen participation; especially, derivatives of uracil nucleosides and of thymine nucleosides that contain a leaving group are thus converted into anhydronucleosides having such

(311) H.-J. Binte, Symp. Heterocycl. Compounds, Reinhardsbrunn, East Germany (1967).

SYNTHESIS OF NITROGEN HETEROCYCLES

401

rings.312-319 For example, 208 gives 209, and 210 gives the isomeric oxazolidine 211. An oxazine has been obtained320by oxygen participation between

w- Q

ROCH,

MsO

ROCH, .O

0

OH

(208)

the benzamido group on C-6 and the leaving group on C-4 of methyl 2,6-bis(benzamido)-2,6-dideoxy-3-0-methyl-4-O-(methylsulfonyl)-~D-glucopyranoside (212) to give 213. The oxidation product (214) of 1,2-O-isopropylidene-c~-~-glucofuranose was condensed321with benzylamine to give 215. The formation of the ret than^^^ 217 from 216, (312) For earlier references, see A. M. Michelson, “The Chemistry of Nucleosides and Nucleotides,” Academic Press, New York, N. Y., 1963, p. 68. (313) J. J. Fox, N. Young, and A. Bendich, J . Amer. Chem. SOC., 79, 2775 (1957). (314) I . L. Doerr, J . F. Codington, and J. J. Fox,J. Org. Chem., 30, 467 (1965). (315) K. A. Watanabe and J . J . Fox,J. Org. Chem., 31, 211 (1966). (316) R. Letters and A. M. Michelson, J . Chem. SOC., 1410 (1961). (317) Y. Mizuno, T. Sasaki, T. Kanai, and H. Igarashi,]. Org. Chem., 30, 1533 (1965). (318) W. V. Ruyle, T. Y. Shen, and A. A. Patchett,J. Org. Chem., 30, 4353 (1965). (319) J. P. Horowitz, J. Chua, M. Noel, and J. T. Donatti,J. Org. Chem., 32,817 (1967). (320) W. Meyer zu Reckendorf, Chert&.Ber., 96, 2019 (1963). (321) H. Paulsen and K. Todt, Chem. Ber., 101, 3358 (1968). (322) F. Lichtenthaler and H. K. Yahya, Chem. Ber., 101,908 (1968).

HASSAN EL KHADEM

402

and of the l a ~ t a m 219 ~~ from 218, are other examples of the formation of l,&oxazines.

'0- "0 H N i

HN Ph-CO

Ms

HNBz

HNBZ

(213)

(212)

Bzl

I

b

O

H

H,NHNCO

HOFH,

Q

HO

(323) H. H. Baer and F. Kienzle, J . Org. Chem., 32, 3169 1967).

SYNTHESIS OF NITROGEN HETEROCYCLES

403

b. 1,4-Oxazines (Morpholines).- This class of heterocycle is obtained from the reaction of periodate-oxidized glycosides (that possess the oxygen atom required), after reduction to the diols and conversion into disulfonic esters, with ammonia or amines. The morpholine324220 is an example of such a product. Similarly, oxidation of 221,reduction of the product, sulfonylation to give 222,and reaction of 222 with methylamines25gives 223.

c. Thiazines. -Aldopentoses and aldohexoses react with 3-aminopropanethiol hydrochloride to give3w tetrahydro-(2-hydroxyalkyl)thiazines. Thus, D-glucose gives 2-(~-gZuco-pentahydroxypentyl)thiazoline (224).On acetylation, compound 224 undergoes both N- and 0acetylation to give 225,and action of Raney nickel on 225 ruptures the ring, affording the 1-amino-1-deoxyhexitol226.

(324) K. W. Buck, F. A. Fahim, A. B. Foster, A. R. Perry, M. H. Qadir, and J. M . Webber, Carbohyd. Res., 2,14(1966). (325) J . X. Khym, Biochemistry, 2, 344 (1963);D. M. Brown and A. P. Read, 1.Chem. Soc., 5072 (1965). (326) R. Mani, W. Meyer zu Reckendorf, and W. A. Bonner, Chem. Ber., 95,1000 (1962).

HASSAN EL KHADEM

404

n

n

HNY D-Glucose

__c

HCOH I HOCH

I HCOH I HCOH I

CH,OH

(224)

v. FORMATIONOF

Ac/NyS

-

HCOAc 1 AcOCH

I HCOAc I HCOAc I

CH20Ac

(225)

-

Ac I CH2-N-Pr

+

H OAc AcOCH I HCOAc

I

HCOAc I CH20Ac (226)

HIGHER-MEMBERED NITROGENHETEROCYCLES

Seven-membered heterocycles containing one nitrogen atom may be obtained either from 105 (see p. 376) or 6-amino-6-deoxy-~-glucose (227) by reductionlZ2to 228, or from 6-azido-6-deoxy-~-glucopyranose (229) by oxidation to 230 followed by reduction of 230 to 231. A

lactam is formed327from 231. Large rings that contain two nitrogen atoms and an oxygen atom are formed by interaction of phenylhydrazine with periodate-oxidized pyranosides and polysaccharides that have the 4-hydroxyl group protected.32s Thus, 232 gives 233, and this gives 234. (327) S. Hanessian, J . Org. Chem., 34, 675 (1969). (328) R. D. Cuthrie and L. F. Johnson, J . Chem. Soc., 4116 (1961);see also, R. D. Guthrie, Aduan. Carbohyd. Chem., 16, 105 (1961).

SYNTHESIS OF NITROGEN HETEROCYCLES

405

Anhydronucleosides in which an oxygen or nitrogen atom of the base is involved in ring formation with C-5 constitute examples of formation of large heterocyclic rings.312*329-333 Thus, 235 gives 236, and 237 gives 238.

AgOAc

RO

RO

(236)

I

heat ___t

(237)

(238)

(329) E. J. Reist, D. F. Calkins, and L. Goodman, J . Org. Chern., 32, 169 (1967). (330) A. Hampton and A. W. Nichol, J . Org. Chern., 32, 1688 (1967). (331) M. Ikehara and K. Muneyama, 1. Org. Chern., 32, 3042 (1967). (332) R. F. Nutt, M. J. Dickinson, F. W. Holly, and E. Walton,]. Org. Chern., 33, 1789 (1968). (333) I. L. Doerr, R. J. Cushley, and J. J. Fox, J . Org. Chern., 33, 1592 (1968).

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ASPECTS OF THE STRUCTURE AND METABOLISM OF GLYCOPROTEINS BY R. D. MARSHALL AND A. NEUBERGER Department of Chemical Pathology, S t . Mary’s Hospital Medical School, London, W. 2, England

I. The Nature and Occu oproteins . . . . . . . . . . . . . . . . . . .407 11. Carbohydrate-protein ................ 1. 2-Acetamido-1-N-/3 eOXy-/3-D-ghCopyran as a Linking Moiety, .......................... 2. Linkages in which 2-Acetamido-2-deoxy-~-galactose is . . . . . . . . . . . .42s to L-Serine or L-Threonine Residues ............. 3. 0-/3-D-Xylosyl-L-serine as a Carbohydrate-peptide Li 4. 5-O-/3-D-Ga~actopyranosyloxy-L-lysine as a Carbohydrate-peptide Linking Moiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .436 111. Polypeptide Chains Carrying More than One Type of Carbohydrate-peptide Linkage . . . . . . . . . . . . . . . . . . . . . . . . ............ .439 IV. Heterogeneity in Glycoproteins ..................... V. The Size of the Carbohydrate Moieties in Glycoproteins . . . . . . VI. Features of the Structure of the Carbohydrate Moieties of Some Glycoproteins ........................ . . . . . . . . . . . . 452 . 1. Blood-group Substances from Ovarian Cysts ......................... ,452 2. Pig Submaxillary-gland Glycoprotein . . . . . . . . . . . . . . . . . . . . . . . .458 3. Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .459 ............................. 461

VIII. Some Genetically Determined Diseases in which Glycoproteins are Implicated. ......................................................... 472 . . . . . . . . . .477 IX. Concluding Remarks ....................

I. THE NATURE AND OCCURRENCE OF GLYCOPROTEINS Many proteins consist of one or more polypeptide chains that are composed solely of amino acids for which codons exist in the genome. Others contain modified amino acids for which, so far as is known, 407

408

R. D. MARSHALL AND A. NEUBERGER

there is neither a specific activating enzyme nor a specific transferribonucleic acid (tRNA) molecule. Examples include pyroglutamic acid, which blocks the N-terminus of a number of proteins’*2and which may occur through enzymic conversion3 of L-glutaminyl-tRNA or possibly at a later stage of protein ~ y n t h e s i s .fi-Hydroxy-~-lysine ~ and hydroxy-L-proline residues are formed, at least mainly, from L-lysine and L-proline moieties, respectively, after formation of the polypeptide chain^.^ Other groups include acetyl groups at the N-termini of a number of protein chains, L-serine and L-threonine phosphates, and 6-N-mono- and di-methyl-L-lysines. In all of these examples, the macromolecule formed consists of an aprotein to which certain prosthetic groups are attached by covalent bonds. By analogous considerations, glycoproteins may be described as macromolecules composed of one or more polypeptide chains that are formed by the normal processes of translation at the polysomal level and to which sugar molecules are attached through covalent bonds. There are a number of definitions of glycoproteins, and these have been fully discussed.6 The definition employed here is wider than those adopted b y many workers in the past, as it has a number of advantages in the present discussion. The class of glycoproteins contains a very large number of compounds; they occur as such biologically active materials as enzymes, hormones, and immunoglobulins. Glycoproteins are components of the structure of blood vessels and skin, and are commonly found in epithelial secretions. Most of the serum proteins contain carbohydrate, as do many of the proteins present in milk and egg white. The gelatinous fluids of certain tumors are rich in glycoproteins, as are many cell membranes. Often, the isolation of a glycoprotein has been achieved by the use of techniques that do not involve cleavage of covalent bonds; glycoproteins of this type are in their native state, apart, possibly, from the degree of aggregation, to which reference will later be made. Examples of these glycoproteins are described in Table I, together

(1) R. F. Doolittle and R. W. Armentrout, Biochemistry, 7,516 (1968). (2) I. J . O’Donnell, Aust.J. B i d . Sci., 21,1327 (1968). ( 3 ) M. B. Bemfield and L. Mester, Biochim. Biophys. Res. Commun., 33, 843 (1968). (4) M. Messer and M. Ottesen, Biochim. Biophys. Actu, 92, 409 (1964). (5) S. Udenfriend, Science, 152, 1335 (1966). (6) A. Gottschalk, in “Glycoproteins,” A. Gottschalk, Ed., Elsevier Publishing Co., Amsterdam, 1966, Chapter 2.

TABLE1 The Carbohydrate Components of Some Glycoproteins ~~~~~

~

~

~~~~~

~

~

No. of residues per molecule of protein," or percentages

No.

Class

Protein

Source

1 Enzymes ribonuclease Bb ox pancreas deoxyribonu- ox pancreas 2 clease Be 3 prnteinase bd snakevenom 4 NADase' Neurospora

Mol.wt. Man

14,700 31,000

Gal

Fuc

G

5 5

Total neutral suears

5 5

GN

2 2

95,000 10 36,500 68

30

16,300 4

1

1

-

6

5

30,000 2.3

2.0

3.0

-

7.3

n.d.

2.9

1.o

-

10

-

10 28 270 5.8

66

tf

-

-

32

42 136

34 n.d.

GalN -

n.d.

Total hemsamine

Sialic acid

2 2

-

Total sugar Referresidues ences

7 7

7 10

-

85 140

12 14

7

0.25

13

15

6.2

n.d.

13.5

16

8

1.1

19

17,18

1 9 59 0.7

20 58 54 1 10

21 22 23 24

34 4

9

crassa

5 Hormones luteinizing

6

folliclestimulating

7 Immunoglobulins 8 9 10 11

rG'

12 13

MOPC70 MOPC 195

YG YA YM MOPC 46h

14 MOPC 172 15 Structural aorta GP I proteins 16 collagen' 17

basement membrane'd

sheep pituitary gland pig pituitary gland rabbit serum

140,000

5.8

human serum 140,000 5 human serum 140,000 14 + human serum 1,000,000 mouse urine 23,000 2 (Bence-Jones) 23,OOo n.d. + mouse serum 140,000 (yG-Be2) 140,000 + ox aorta 60,000

+

dog Achilles tendon ox kidney

14

2.0 2 49

3

LT

-

n.d.

n.d.

3

+

n.d.

+ + +

+

+

-

1.8

1.1 0.4%

-

0.28%

-

0.77%

3.05%

0.22% 2.47%

8

2

n.d

+

-

9 n.d. n.d. 3.6

n.d. n.d.

0.85 11.2

n.d. n.d.

n.d. n.d.

1.9 17

n.d. 11

n.d.

0.68%

n.d.

n.d.

&5%,

1.5%

-

-

0.22%

9 21 212 3.6 0.75 7.1

0.1

3.6

0

11

0

3.6

1.7 18 6 33

26 26 26 27.28

0.04%

0.0670

0.8%

29

1.7%

12%

9.4%

30

(continued)

TABLEI (continued) No. of residues per molecule of protein,' or percentages Total neutral

No.

Class

Protein

Source

Mol.wt. Man

Gal

~~

18 Mucins 19 20

21 Plant 22

Fuc

G

~

1,100

-

-

-

~~~

980

-

49

-

11.0% 28

-

2,070 27.1%

W

2.77% 5

Sialic acid

Total sugar Referresidues ences

72

4,120

~~~

1,090

51.4 70

17.3% 24

2,050

GalN

GN

sugars

~~~~~~

ovarian 1,0oO,OOO - 950 cyst-fluid gastric GP' human gastric juice 17 submaxillary sheepsub1,0oO,OOO 8 GP maxillary gland glycoproteinkI kidney beans 7.8% Ahemagsoybeans 110,OOO 28 glutinin A-substance

Total hexosamine

800

-

800

2.77% 5

~

2.5%

800

-

~~

83.6% 1,600

14% 33

"-, absent; a d . , not determined. bRibonucleases C and D were also prepared from ox pancreatic juice. They contain mgalactose, L-fucose, and sialic acid, in addition to Dmannose and 2-amino-2-deoxy-~glucose.~ Pig pancreatic ribonuclease contains very much more carbohydrate, and is highly heterogeneous owing to variations in the carbohydrate ~ o n t e n tcA . ~ closely related enzyme, namely, deoxyribonuclease A, was found to contain two moles of Dmannose and 2-amino-%deoxy-~glucoseper mole." dThis proteinase is from the venom of Agkistrodon halys blomhofi, and other glycoprotein pro' similar glycoprotein enzyme, in which both %amino-Sdeoxy-D-glucose and Samino-Zdeoxy-Dgalactose are teinases are also present.I3 % = trace. A present, occurs in Bacillus subtilis. A specific inhibitor for this enzyme is also a glycoprotein." OThe values given are average values: various protein fractions having total hexosamine contents ranging from 8.2 to 23.4 moles per mole of protein have been isolated.'g Some of the molecules contain %amino2-deoxy-D-galactose.20hAsimilar protein produced by another transplant line of the same tumor had somewhat different sugar contents.% 'Sugar contents *Small are expressed as percentages. 'There are at least two glycoproteins that form the basement membrane, as shown with preparations from the proportions of L-fucose, Dxylose, and Larabinose were also present in these preparati~ns.~'

31

32

33

34 35

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

411

with their sugar analyses; certain glycoproteins that occur in plants are included. This list is far from complete, and other compilations are a ~ a i l a b l e , ~ Bbut - ~ ~ the examples shown demonstrate certain

(7) T. H. Plummer and C. H. W. Hirs,]. Biol. Chem., 238, 1396 (1963). (8) T. H. Plummer,]. Biol. Chem., 243,5961 (1968). (9) V. N. Reinhold, F. T. Dunne, J. C. Wriston, M. Schwartz, L. Sarda, and C. H. W. Hirs,J. Biol.Chem., 243,6482 (1968). (10) B. J. Catley, S. Moore, and W. H. Stein,]. Biol. Chem., 244,933 (1969). (11) P. A. Price, T.-Y. Liu, W. H. Stein, and S. Moore,J. Biol. Chem., 244,917 (1969). (12) G. Oshima, S. Iwanaga, and T. Suzuki,]. Biochem. (Tokyo), 64,215 (1968). (13) C . Oshima, Y. Matsuo, S. Iwanaga, and T. Suzuki, ]. Biochem. (Tokyo), 64, 227 ( 1968). (14) J. Everse and N. 0. Kaplan,]. Biol.Chem., 243,6072 (1968). (15) E. F. Walborg and D. N. Ward, Biochim. Biophys. Ac~Q, 78,304 (1963). (16) A. Segaloff and S. L. Steelman, Recent Progr. Hormone Res., 15,115(1959). (17) E. R. B. Graham and A. Neuberger, Biochem.]., 106,593,(1968). (18) E. R. B. Graham and A. Neuberger, Biochem.]., 109,645(1968). (19) R. S. Blacklow and R. D. Marshall, Biochim. Biophys. Acta, 165,179 (1968). (20) D. G. Smyth and S. Utsumi, Nature, 216,332 (1967). (21) J. W. Rosevear and E. L. Smith,]. Biol. Chem., 236,425 (1961). (22) N. Heimburger, K. Heide, H. Haupt, and H. E. Schultze, Clin. Chim. Acta, 10, 293 (1964). (23) H. E. Schultze, H. Haupt, K. Heide, G. Mbschlin, R. Schmidtburger, and G. Schwick, 2. Naturforsch., B, 17,313 (1962). (24) T. J. Coleman, R. D. Marshall, and M. Potter, Biochim. Biophys. Acta, 147, 396 (1967). (25) F. Melchers, E. S. Lennox, and M. Facon, Biochem. Biophys. Res. Commun., 24, 244 (1966). (26) R. D. Marshall and M. Potter, Abstr. 3rd Meeting Fed. Europ. Biochem. Soc., Warsaw, 1965,258. (27) B. Radhakrishnamurthy, A. F. Fishkin, G. J. Hubbell, and G. S. Berenson, Arch. Biochem. Biophys., 104,19 (1964). (28) B. Radhakrishnamurthy and G. S. Berenson,]. B i d . Chern., 241,2106 (1966). (29) N. A. Kefalides and R. J. Winder, Biochemistry, 5,702 (1966). (30) R. G. Spiro,J. Bid. Clzem.,242,1915(1967). (31) D. Aminoff, W. T. J. Morgan, and W. M. Watkins, Bioclaem.]., 46,426 (1950). (32) R. Martin, A. Berard, A. Jovenceaux, and R. Lambert, Compt. Rend. Soc. Biol., 161,848(1967). (33) W. Pigman and A. Gottschalk, in Ref. 6, Chapter 11, Section 5. (34) A. Pusztai, Biochem.]., 101,379 (1966). (35) H. Lis, N. Sharon, and E. Katchalski,]. Biol. Chem., 241,684 (1966). (36) R. G. Spiro, N e w E n g l a n d j . Med., 269,566,616 (1963);281,991, 1043 (1969). (37) E. H. Eylar, j . Thew. Biol., 10,89 (1965). (38) R. D. Marshall and A. Neuberger, in “Carbohydrate Metabolism and its Disorders,” F. Dickens, P. J . Randle, and W. J. Whelm, Eds., Academic Press, Inc., New York, N.Y., 1968, Chapter 7.

TABLEI1 Sugar Contents of Glycoproteins Obtained from Proteolytic Digests of Various Materials Moles per mole of protein, or percentages

Source of material

1 Carcinoma of human stomach 2 Humanstomach-wallQ 3 Dog fundic stomach mucosa* 4 Sheep colonic epitheliumC 5 Ratcolonicmucosad 6 Breast colloid carcinoma 7 Human red-cells’

Total neutral

Total hexosamine

Mo1.W.

Sulfate

Gal

Fuc

sugars

GN

GalN

270,000 164,OOO

50 tr

483 340

123 113

606 453

183 142

134 78

317 220

100,000

16

143

57

200

78

78

212,000 250,000

46 80

197 300

52 175

249 475

304 225

-

22 n.d.

22 19%

-

n.d.

n.d.

20,000e

n.d.

-

Sialic acid

Total sugars

References

77 11

1000 684

39 39

156

29

385

41

122 75

426 300

82 63

755 838

42 43

37 n.d.

37 21%

1 35%

60 75%

44 45

“Other fractions, having a higher content of sulfate, were isolated from the gastric wall of the dog.40 *The analyses are those described for fraction IIIA, and are based on a molecular weight of 100,000. Other closely related fractions, that differed in their contents of sulfate, L-fucose, and sialic acid, were also described. L-Threonine and L-serine constituted 58%of the amino acid residues. “The sedimentation rate of this protein is strongly dependent on its concentration; the value used in determining the molecular weight is that at zero concentration of protein. T h e values given (for fraction F 3) are based on a molecular weight of 250,000. Other, closely related, fractions were also isolated. In all materials, the protein core consisted of about 100 residues of L-threonine, 20 of L-serine, and 30 of Lproline. Other amino acids were present in much smaller proportions. “The S,,, value for this material at 1%concentration is 2.35. The shape of the molecule is unknown, and a molecular weight has been assumed. Of the amino acid residues, 45%are L-threonine, with but little L-serine present. ’In this example, the values are reported as percentages.

F

P

9

z

U

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

413

features. In the first place, the carbohydrate content of a glycoprotein may vary from quite low values, as in collagen (item 16 of Table I) or in yG globulin (items 7 and 8), to over 80% in the blood-group substances from ovarian cysts (item 18). Sugars that often occur are Dmannose, D-galactose, and L-fucose, and the amino sugars 2-amino-2deoxy-D-glucose and 2-amino-2-deoxy-~-galactose, and sialic acid. The amino sugars are almost invariably N-acylated, the hexosamines with acetyl groups, and sialic acid with acetyl or glycoloyl groups. D-Glucose occurs less frequently (items 4 and 15-17), as well as Dxylose and, occasionally, L-arabinose. Uronic acids occur in glycoproteins of a type that has sometimes been excluded by other writers; these are discussed later (see Table IV, p. 416). It may also be noted that certain Bence-Jones proteins contain carbohydrate (items 11 and 12), although, most frequently, these proteins are devoid of sugars. Furthermore, myeloma proteins antigenically of the same subclass may differ in their carbohydrate contents (items 13 and 14). Finally, it may be seen that a given glycoprotein may be homogeneous with regard to its amino acid sequence, but heterogeneous with respect to carbohydrate components (items 1, 2, 7, and ll), and this kind of variation is probably a very common feature. In addition to these clearly defined glycoproteins, other materials have been isolated after proteolysis, with the use of one or more purified enzymes, of less well characterized substances (see Table 11). Whether the materials isolated occur as such in cells, or are degradation products of other glycoproteins, is as yet unknown. These materials frequently contain sulfuric ester linkages, and are usually free from D-mannose. The Lowry method45agives a gross underestimate of the content of protein component, and, because their content of aromatic amino acids is low, this is to be expected. Procedures based on that described by are often employed

(39) E. Kimoto, T. Kuranari, H. Masuda, and M . Takeuchi, J . Biochem. (Tokyo), 63, 542 (1968). (40) I. Hakkinen, K. Hartiala,and T. Terho, Acta Chem. Scand., 19,800 (1965). (41) T. Pamer, G . B. J. Glass, and M. I. Horowitz, Biochemistry, 7,3821 (1968). (42) P. W. Kent, J. P. Ackers, and J. C. Marsden, Biochem.]., 105,24~(1967). (43) S . Inoue and Z. Yosizawa, Arch. Biochem. Biophys., 117,257 (1966). (44) J. B. Adams, BiochemJ.,94,368 (1965). (45) S. Okhuma and T. Furuhata, Proc.Jap.Acad. Sci., 45,417 (1969). (45a) 0.H. Lowry, N. J. Rosebrough, A. L. Fan, and R. J. Randall,]. B i d . Chem., 193, 265 (1951). (46) J . E. Scott, Methods Biochem. Anal., 8, 145 (1960).

TABLEI11

e

The Carbohydrate Content of Some Glycopeptides and of the Tamm-Horsfall Glycoprotein Found in Normal Urine

rp

Moles per mole of substance

Fraction

Total 2-Amino-2- 2-Amino-2- Total neutral deoxy-D- deoxy-D- hexo- Sialic Total Referglucose galactose samhe acid sugar ences Mol. wt. D-Mannose D-Galactose L-Fucose D-Glucose sugar

9,500 B3an 5,800 Ah S-5D-Iab 4,500 RS-1' 17,000 UF-4A-5d 1,200 C 4.6" Tamm-Horsfall protein' 100,000

+

+H-

-

16

-t+t ft

+I+t

n.d.

n.d.

ft

+I+

32

32

+t

10 8 ++(3)

8 1.2

-

-

+ -

n.d.

+(1.4%)

+

5

-

28 24

9 8

13

+t+

25 4

11 n.d.

31%

-l-kk

69

45

1 2

+

3 n.d.

++ 5

10 10 5 14 2 9%

1 0.3 2

50

15

-

0.1 2%

39 35 20 39 6

50 51 52 53

P

42%

54

$ r

50

134 55,s

"Several other, closely related, fractions were also obtained. It has been suggested47athat this material and also the next one in this Table, A,b, are derived from blood-group substances. Although this is not impossible, the ratio of the contents of 2-amino-2-deoxy-~ glucose to -galactose, and the presence of D-mannose in one of them ( B g ) , are not in keeping with what is thus far known of the sugar composition of the soluble, blood-group substances. bAbouttwenty related fractions, soluble in 50% ethanol, that differed markedly in the contents of the various sugars were obtained by chromatographic methods. The molecular weight of 4,500 was deduced from the S,, value of 0.85, and is used so as to give an order of magnitude. T h i s fraction and a somewhat smaller one, RS-IC, both exhibited hemagglutinin inhibitory activity. T h i s is probably a group of chemically similar mucoids in which the proportions of sialic acid and L-fucose are inversely related. 'This glycopeptide contains 4-hydroxy-~-proline;several other similar glycopeptides, having somewhat different compositions, occur. In solution, they all pass through Sephadex G-50. Another glycopeptide that contains 4-hydroxy-~-proline and sarcosine was described by Cherian and Rama~handran.~~*~* 'The monomeric form of this glycoprotein appears to have a molecular weight of the order of 100,000, as determined in dissociating solvents.55*"The data citedJJfor the sugar composition of the protein are closely similar to those in another report," but both sets differ considerably from earlier results. Thus, the aldohexose values quoted in an earlier review47aare rather lower and, in particular, the ratio of -galactose to D-mannose is quite different; but the hydrolysis conditions used in these earlier experiments in order to release these sugars were probably not adequate to liberate the monosaccharides quantitatively. Earlier values for hexosamine were derived from measurements made under conditions of hydrolysis that are applicable to proteins generally, but that lead to extensive decomposition of the 2-amino sugars. The proportion of sialic acid in the protein differs from one preparation to a n ~ t h e r : ~but . ~ ~the considerably higher values reported earliel.4'" are probably due, at least in part, to the use of assay procedures that are not completely specific.

?¶

Eg r

*

z

?

M S

STRUCTURE AND METABOLISM OF CLYCOPROTEINS

415

in the isolation of these charged compounds. Proteolysis of defatted, rat-brain tissue yields a large number of glycoproteins, many of which are associated with synaptosomes, microsomes, and ax on^.^^ Urine contains a number of glycoprotein~~'~ that are identical with, or closely related to, the serum proteins. Tamm-Horsfall protein48 also occurs therein, and it may well have originally been part of the renal-tubule cells.49This protein has a relatively large proportion of carbohydrate as part of its structure (see Table 111). Also, a number of glycopeptides isolated from normal urine have sugar compositions similar to those of mammalian glycoproteins in general (see Table 111). Sometimes, a uronic acid is a component of such g l y ~ o p e p t i d e s . ~ ~ Of considerable interest is a glycopeptide that contains a uronic acid, hydroxy-L-proline, and ~ - s a r c o s i n e . ~ A ' * ~glycopeptide ~ has been isolated from urine in crystalline form; on hydrolysis, it yields L-alanine, L-aspartic acid, L-glutamic acid, glycine, L-serine, 2-aminoD-glu2-deoxy-D-galactose, D-galactose, 2-amino-2-deoxy-~-g~ucose, curonic acid, and D-xylose .59 These urinary glycopeptides may arise from the catabolism of glycoproteins. The occurrence of 0-D-glucosylO-D-galaCtOSyl-(1 + 5)-5-hydroxy-~-lysine and of 0-D-galactosyl(1-+ 5)-5-hydroxy-~-lysinein normal urine suggests (see p. 444) that they arise from turnover of collagen.60 All of the glycoproteins and glycopeptides thus far mentioned probably contain their carbohydrate as moieties whose size does not exceed -15 sugar residues; this point will be discussed later (see p. 447). Other glycoproteins are known in which at least some of

(47) E. G. Brunngraber, V. Aguilar, and A. Aro, Arch. Biochem. Biophys., 129, 131 (1969). (47a)E.H. F. McGale, Aduan. Carbohyd. Chem. Biochem., 24,435 (1969). (48) I. Tamm and F. L. Horsfall, J. Erp. Med., 95, 71 (1952). (49) T. Friedmann, Experientia, 22,624 (1966). (50) A. Lundblad, Biochim. Biophys. Acta, 101,46 (1965). (51) R. Bourillon, P. Cornillot, and R. Got, Clin. Chim. Acta, 7,506 (1962). (52) J. S. King, M. L. Fielden, H. 0. Goodman, and W. H. Boyce, Arch. Biochem. Biophys., 95,310 (1961). (53) J. S. King, M. L. Fielden, and W. H. Boyce, Arch. Biochem. Biophys., 90,12 (1960). (54) R. Bourillon and J. L. Vernay, Biochim. Biophys. Acta, 117,319 (1966). (55) A. P. Fletcher, A. Neuberger, and W. A. Ratcliffe, Biochem. J., in press (1970). (56) F. Stevenson and P. W. Kent, Biochem.J.,116,791 (1970). (57) M. G. Cherian and A. N. Radhakrishnan, Biochim. Biophys. Acta, 101,241 (1965). (58) M. G. Cherian and A. N. Radhakrishnam, IndianJ.Biochem., 3,101 (1966). (59) D. Basu, Biochem.J.,112,379 (1969). (60) L. W. Cunningham, J. D. Ford, and J. P. Segrest,]. Biol. Chem., 242,2570 (1967).

TABLEIV The Sulfate and Sugar Contents of Some of the Glycosaminoglycan-proteinComplexes Content (%) of

Substance

Source

Total neutral Sulfate D-Mannose D-Galactose L-Fucose sugar

Keratan sulfate 1) ox cornea 17.7 human-rib (KS 11) cartilage 18.9 Host fact09 chick dlantoic(HF-A) sac fluid 6.9 (Norway) 12 Horitin sulfuric Charonia lampas acidc liver 24 Chondroitin ox nasal septa 12.5 sulfate-protein pigtrachea 14.5

+ +

Total hexo- Sialic Uronic Total ReferGN GalN samine acid acid sugar ences

+ti+

Trace

35.3

25.1

1.1

26.2

-+cH

2.7

28.7

21.4

2.7

24.1

3.4

-

+ti+

-

27

1.0 4.3

26.0 31.3

12.0 24

8.6 11

20.6 35

13.0 n.d.

4

8

8 n.d.

26 8

6 6 3.4 20.2

11.8 23.6

2.1 n.d.

20.0

39.9 66 52 67.68

unde- unde- 27.5 tected tected

27.5

0.5

ftt

69,70

Trace (3.4)’

F

u

5

Ei5

61.5

64

55.2

64

(3.7)o 59.6 66.3

64

0

65

?

(2.4)’

r

z z

tr.d

undetected

i-tk

undetected

undetected

-

M m

8 w

~

~~~

“The color given in the carbazole reaction was different from that given by uronic acids. T h e Lowry procedure gives a large underestimate of the quantity of protein present. cThe analyses reported are for one of the several fractions described. The substance also contains -4% Of D-glUCOSe residues, and has%a molecular weight of -5,000. %aces of D-glucose were also present.

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

417

the carbohydrate moieties are much larger. Macromolecules of the type found in wheat flour, in which it is reported that both an arabinoxylan and an arabinogalactan are linked to a polypeptide chain, are in this group,61and it is possible that, in its native state in the cuticle of insects and crustaceaes2 or in the cell walls of such molds as Actinornucar e l e g ~ n s , 6chitin ~ occurs as part of a glycoprotein. Of more immediate interest are such compounds as the chondroitin 4-and 6-sulfates, dermatan sulfate, and the various keratan sulfates, the structures of which consist, at least in part, of well defined repeatingsequences of alternating sugar-residues. The compositions of some of these compounds are described in Table IV. The isolation procedures frequently involve a proteolytic digestion, so that interpretation of the results in terms of the native structure of the materials from which the compound was derived must be cautious. Differences in the analyses reported for the “host factor” (resembling keratan sulfate) obtained by separate groups of workers may partly stem from the fact that, in one case (HF-A), proteolysis preceded the separation methods, but not in the other (Norway).

LINKAGES 11. CARBOHYDRATE-PROTEIN In this Section, the nature of the linkages between the protein chain and the carbohydrate moieties in glycoproteins will b e considered, and some indication of their occurrence will be given. It will become clear that, in a number of examples, a given polypeptide chain is glycosidically substituted at more than one point. Sometimes, the amino acid residue at two such points is of a different type, so that more than one type of carbohydrate-peptide bond occurs in the same protein. It is likely that more examples of this kind of structure will be found as more features of the constitution of glycoproteins involved in membranes and connective tissue are determined. (61) W. Kundig and N. Neukom, Helo. Chim. Acta, 46,1423 (1963). (62) R. H. Hackman, Aust.J. B i d . Sci., 13,568 (1960). (63) N. Wai, Bull. Inst. Chem. Acud. Sinica, 15,1(1968). (64) K. Meyer, V. P. Bhavanandan, D. Yung, L. T. Lee, and C. Howe, Proc. N u t . Acad. Sci. U . S., 58, 1655 (1967). (65) G. Haukanes, A. Harboe, and K. Mortensson-Egnund, Acta Pathol. Microbiol. Scand., 66,510 (1966). (66) S . Inoue, Biochim. B i o p h y s . Acta, 101, 16 (1965). (67) S. M. Partridge, H. F. Davies, and G . S. Adair, Biochem. J., 79,15 (1961). (68) S . M. Partridge and D. F. Elsden, Biochem.J.,79,26 (1961). (69) A. J . Anderson, Biochem.J.,78,399 (1961). (70) H. Muir, Biochem. J., 69,195 (1958).

R. D. MARSHALL AND A. NEUBERGER

418

Of the dozen or so amino acid residues whose side chains are of such a nature that glycosylation might be possible, only asparagine, serine, threonine, and 5-hydroxy-~-lysinehave been unequivocally demonstrated to be commonly involved in such linkages, although, in certain glycoproteins that form part of the cell wall of some plants, ',~~ 4-hydroxy-~-prolineconstitutes part of a bond of this t y ~ e . ~Thus, treatment of cell walls of plants with 0.44 M barium hydroxide for 6-8 hours at 90-105" was followed by chromatography; this led to the isolation of L-arabinose-containing fractions that were found also to contain 4-hydroxy-~-proline having free imino and carboxyl groups. Oxidation with sodium hypobromite and subsequent reactions with the Ehrlich reagent afforded the major evidence for this deduction; other amino acids were not present. A type of linkage in which L-glutamic acid is bound to a sugar has, among others, been conbut direct evidence for this sidered by Katsura and David~on,'~ structure is lacking. y-L-Glutamyl derivatives of D-gluc~sylamine~~ are available as and of 2-acetamido-2-deoxy-~-glucosylamine~~ reference compounds. The number of types of sugar involved in such linkages is, apparently, also strictly limited (see Table V). A few other sugars, such as TABLEV The Amino Acid and Sugar Residues that are Generally Engaged in Carbohydrate-peptide Linkages in Glycoproteins Amino acid L-Asparagine L-Threonine L-Serine 5-Hydroxy-L-1ysine

Corresponding sugar 2-acetamido-2-deoxy-~-glucose 2-acetamido-2-deoxy-~-gdactose 2-acetamido-2-deoxy-~-ga~actose or D-XylOSe g galactose

L-arabinose, have also been reported to be in direct linkage with a protein. L-Arabinose may also be linked to L-serine and L-threonine in a hyaluronic acid-polypeptide complex from ox v i t r e o ~ s - h u m o r . ~ ~ ~ D-Mannose is reported to be glycosidically linked in a glucoamylase [( 1+4)-a-D-glucan glucohydrolase, EC 3.2.1.31 from Aspergillus (71) D.T.A. Lamport, Nature, 216,1322(1967). (72) D.T. A. Lamport, Biochemistry, 8,1155(1969). (73) N.Katsura and E. A. Davidson, Biochim. Biophys. Acta, 121,128(1966). (74) C.Coutsogeorgopoulos and L. Zervas,]. Amer. Chem. Soc., 84,1885(1961). (75) A. Yamamoto, C. Miyashita, and H. Tsukamoto, Chem. Pharm. Bull. (Tokyo), 13, 1041 (1965). (75a)A. H. Wardi, W. S. Allen, D. L. Turner, and 2. Stary, Biochim. Biophys. Acta, 192,151(1969).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

419

n i g e ~ ’ and ~ in yeast invertase (EC 3.2.1.26; p-D-fructofuranoside fmctohydrola~e),~~ but the evidence was based on the results obtained from experiments involving the use of alkali (see pp. 427 and 456).Suggestions have also been made that 2-amino-2-deoxy-~-glucose is involved in carbohydrate-peptide linkages in yeast i n v e r t a ~ e . ~ ~ n-Galactose may be directly linked to L-threonine in the collagen from earthworm-cuticle; the experiments from which this was deduced involved the use of alkaline conditions to effect cleavage.7s It has been suggested that, in a number of proteins, the carbohydrate is bound to protein by glycosyl ester bonds involving Laspartic and L-glutamic acid residues, although it was later demonstrated that the Hestrin hydroxylamine reaction79a gives negative results with these proteins.80 The earlier deductions were originally drawn from the fact that the linkages are split under relatively mild conditions of alkalinity,H1*82 but it is now clear that glycosidic linkages that involve L-serine and L-threonine, where these amino acids are themselves bound in a peptide chain, are split by p-elimination. More-decisive evidence appeared to have been provided by the observation that reduction of certain modified glycoproteins with lithium borohydride in tetrahydrofuran led to the formation of 2amino-4-hydroxy-~-butyricacid and 2-amino-5-hydroxy-~-pentanoic acid r e ~ i d u e s . However, ~ ~ . ~ ~ Gottschalk and KOnigs5consider that the formation of these o-hydroxy acids occurs directly, by reduction of the free carboxyl groups of L-aspartic and L-glutamic acid residues within the protein. Decomposition of the excess of lithium borohydride with an anhydrous solution of hydrogen chloride in methanol, the usual technique employed in earlier studies on the proteins, appears to lead to the production of diborane and, probably, of dimethoxyborane,*s and these reagents effectively reduce carboxylic

(76) D. R. Lineback, Carbohyd. Res., 7,106 (1968). (77) H. Greiling, P. Vogele, R. Kisters, and H.-D. Ohlenbusch, Z. PhysioZ. Chem., 350,517 (1969). (78) S. Gascon, N. P. Neumann, and J. 0.Lampen,]. B i d . Chem., 243,1573 (1968). (79) Y. C. Lee and D. Lang,]. B i d . Chem.,243,677 (1968). (79a)S.Hestrin,J. BioZ. Chem., 180,249 (1949). (80) M. Bertolini and W. Pigman,]. BioZ. Chem.,242,3776 (1967). (81) J . H. Pazur, K. Kleppe, and E. M. Ball, Arch. Biochem. Biophys., 103,515 (1963). (82) E. R. B. Graham, W. H. Murphy, and A. Gottschalk, Biochim. Biophys. Acta, 74, 222 (1963). (83) W. H. Murphy and A. Gottschalk, Biochim. Biophys. Acta, 52,349 (1961). (84) S. M. Bose, “Aspects of Protein Structure,” G. N. Ramachandran, ed., Academic Press, Inc., New York, N.Y., 1963, p. 357. (85) A. Gottschalk and W. Konig, Biochim. Biophys. Acta, 158,358 (1968).

420

R. D. MARSHALL AND A. NEUBERGER

a ~ i d s . ~ Lithium ~,~' borohydride reduces carboxylic acids, but, in general, only slowly.E8The preparation and some properties of a number of l-O-(2-acetamidoacy~)-2,3,4,6-tetra-0-acety~-~-~-glucopyranoses have been described.E8" It has been suggested that one of the major components of normal, human-adult hemoglobin (fraction IAc)is a glycoprotein in which at least one of the &chains has the amino group of the terminal Lvaline residue engaged in Schiff-base formation. Reduction of the protein with tritium-labeled sodium borohydride, followed by acid hydrolysis, esterification, and acetylation, led to formation of a compound having a mass spectrum similar to, but not identical with, that of N-l-(penta-O-acetyl-l-deoxy-D-galactitol-l-yl)-L-valine ethyl ester. The structure of the P-chain at the N-terminal end was, therefore, suggested to be that of a sugar combined as a Schiff base with Lvaline.8YThe linkage is particularly unstable at neutral and alkaline pH, so reduction with sodium borohydride is most successfully achieved at pH 3.5.The compound gives no reaction in the anthrone or orcinol tests for sugars,g0and its precise nature is as yet unknown. Three types of covalent linkage of carbohydrate to protein are widely distributed. 1. 2-Acetamido-l-N-~-~-aspartyl-2-deoxy-~-~-glucopyranosylamine as a Linking Moiety In this compound, the anomeric carbon atom of the 2-acetamido2-deoxy-~-glucosyl group is P-D-glucosidically linked to the amide group of L-asparagine. Many glycoproteins are now known in which this type of linkage (1) exists; included among these are the

H

nH N-C-CH,-C 0 I1

O

yI -CO,H z

H

Ac (1)

2- Acetamido- 1 -N-P-L-aspartyl2-deoxy-P-D-glucopyranosylamine

egg-white proteins, namely, albumin, ovomucoid, and avidin, a number of serum proteins, such as ceruloplasmin, fetuin, fibrinogen, (86) H. C. Brown and W. Korytnyk,J . Amer. Chem. Soc., 82,3966 (1960). (87) A. F. Rosenthal and M. Z. Atassi, Biochim. Biophys. Acta, 147,410 (1967). (88) R. F. Nystrom, S. W. Chaikin, and W. G . Brown, J . Amer. Chem. Soc., 71, 3245 ( 1949). (88a)A. Kornhauser and D. Keglevid, Carbohyd. Res., 11,407 (1969). (89) R. M . Bookchin and P. M. Gallop, Biochem. Biophys. Res. Commun., 32,86 (1968). (90) W. R. Holmquist and W. A. Schroeder, Biochemistry, 5,2489 (1966).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

421

transferrin, and orosomucoid, and the immunoglobulins. Pancreatic ribonucleases of ox and pig, and some plant glycoproteins, contain this linking moiety. Much of the evidence for these findings, and many of the properties of this type of compound, have been summari~ed.~] This type of linkage is relatively stable to acids, and is split mainly by two pathways, as in the following scheme. H,OH

+

NH,

+

(jl

I

1

NH,O

NH I

Ac

Asp-OH

h

(7

+

NH,

+

Asp-OH

NHQ Asp

0 I1

"1'?

where A s p = -C-CH,-C-CO,H I

H

The rate constants k, and k, have values of 20 x and 22 x min-', respectively, in 2 M hydrochloric acid at loo", and the overall rate of decomposition of 2-acetamido-l-N-P-~-aspartyl-2-deoxy-~-Dglucosylamine occurs with a rate constant of 42 x min-', that is, a half-life of the order of 17 minutes.s2 L-Aspartic acid and ammonia are released at the same rate.s2-s5 This type of linkage is likewise relatively stable under alkaline conditions, ammonia being released by 0.2 M sodium hydroxide at loo", with a half-life of about 100 minute^.^^*^*^' (91) R. D. Marshall and A. Neuberger, in press (1970). (92) R. D. Marshall, Biochim. Biophys. Actu, 192,381 (1969). ~ (93) G . S. Marks, R. D. Marshall, and A. Neuberger, Biochern.J.,85, 1 5 (1963). (94) I. Yamashimaand M . Makino,J. Biochem. (Tokyo),51,359 (1962). (95) J . Yoshimura and H. Hashimoto, Curbohyd. Res., 4,435 (1967). (96) A. Neuberger, Biochem.J.,32, 1435 (1938). (97) G. S. Marks, R. D. Marshall, and A. Neuberger, Biochem. J., 87, 274 (1963).

TABLEVI Amino Acid Sequences in the Region of the Glycosylated L-Asparagine Residues of Some Glycoproteins ~~~~

Protein Human orosomucoidb

Rat Yoshida ascites tumor a,-acid glycoprotein Human Ba-a,-glycoprotein Ox fibrinogenc Human yG globulin Human yG myeloma protein (Eu) Rabbit yG globulin Mouse Bence-Jones K-chain (MOPC 46)d Human transferrin

Sequence'

References

ASN.Pro.Lys Thr.ASN.Ala ASN .Gly .Thr. Thr.ASN.Lys ASN.Thr(Ser,Ala)Gly Glu(Ser.)ASN.Thr(Gly) Thr.Pro.ASN.Lys Pro.Thr.ASN.(Tyr,Leu) Glu.Thr.Gly.ASN Thr.Glu.ASN ASN(Ala.Thr.Pro.Val) ASN.Lys.Thr.Ser Val.Gly .Glu.ASN.Arg

98

G1u.Glu.Asn.Tyr.Gln.ASN.Ser.Thr Lys.Pro.Arg.Glu.Glu.Gln.Phe.ASN.(Ser,Thr) Pro.Leu.Arg.Glu.Gln.Gln.Phe.ASN.Ser.Thr.IIe.Arg.Val.Val.Ser Ala.Ser.Gln.ASN.1le.Ser.Asn.Asn.Leu Phe.Gly.Ser.ASN.Val.Thr.

21,109,110 112,113 114 24,115 117,117a

100,101 101

100 102 102 106-108

Ala.Glu.Asn.Tyr.ASN.Lys.Ser. Human thyroglobulin Hen egg-white albumin Hen ovomucoide

Ala.Leu.Glu.ASN.Ala.Thr.Arg Glu.Glu.Lys.Tyr.ASN.Leu.Thr.Ser.Va1.Leu ASN.Thr.Asp Thr.ASN Phe.Pro.Ala.ASN Ala.ASN .Thr

1I8 119-122 123 124 125

Hen egg-white avidin Hen ovoglycoprotein

Hen ovotransferrin'

G1y.Lys.Try.Thr.Asx.Asx.Leu.Gly.Ser.ASN.Met ASN.Thr.Ser ASN.Gly Thr.ASN ASN.Ser

Cly.Leu.1le.His.ASN.Arg.Thr.Gly Ala.ASN.Leu.Thr.Gly Cys.Met.ASN.Asp.Ser.Phe

126 123

127,127a

127a Hen egg-yolk vitellin ASN.Thr.Ser(Ala,Gly,Val)Ile 128 Ox pancreatic ribonuclease' Asn.Gln.Met.Met.Lys.Ser.Arg.ASN.Leu.Thr.Lys.Asp.Arg.Cys.Lys 129,130 Arg.Arg.ASN.Met.Thr.Gln.Gly.Arg 131 Pig pancreatic ribonuclease* Ox pancreatic deoxyribonucleases Ser.ASN.Ala.Thr 133 Ala.Phe.Gly.ASN.Cly 28 Ox aorta glycoprotein Pineapple-stem bromelain Ala.Arg.Va1.Pro.Arg.Asn.ASN.Glu.Ser.Ser.Met 134 alpha-Amylase (Aspergillus oryzae) Ser.ASN 135 "ASN represents glycosylated asparagiue. *This protein probably contains eight carbohydrate moieties per molecule.99Both Satakes8 and Yamauchi'O' and their coworkers worked with serum orosomucoid, whereas Bourillon and coworkers'@"studied pleuromucoid. fibrinogen contains six or seven carbohydrate moieties104"05per molecule. dCertain human A-type Bence-Jones proteins are also glycosylated, but the type of linkage has not as yet been unambiguously determined."' 'Hen ovomucoid probably contains four carbohydrate moieties per molecule.123The sequences reported by Montgomery and WulZ4and Tanaka125were obtained with ovomucoid that may also have contained ovoglycoprotein. 'The first, and, possibly, the second, of these two amino acid sequences, where carbo~ ~position * ~ ~ ~ number 34. hydrate is found, also appears in hen-serum tran~ferrin.'~'T h e glycosylated L-asparagine residue O C C U ~ S ' at hPig ribonuclease has L-asparagine residue number 34 glycosylated. I n addition, other L-aspamgine residues within the polypeptide chain are covalently bonded to sugar, namely, one in a sequence (Asn,Ser6)at positions 17-23, and another in the sequence Tyr(Asn, Glx,Thr,Ser2,Met) at132positions 73-79.

: p

C

0 4

s

M

*z

U

zM

+I

>

0" g

8 0

sE 5

424

R. D. MARSHALL AND A. NEUBERGER

(98) M. Satake, T. Okuyama, K. Ishihara, and K. Schmid, Biochem. J., 95,749 (1965). (99) R. W. Jeanloz, in Ref. 6, Chapter 11, Section 4B. (100) R. Bourillon, R. Got, and D. Meyer, Biochim. Biophys. Acta, 83,178 (1964). (101) T. Yamauchi, M. Makino, and I. Yamashima, J . Biochem. (Tokyo), 64,683 (1968). (102) A. Caputo, A. Floridi, and M. L. Marcante, Biochim. Biophys. Acta, 181, 446 (1969). (103) K. Ishihara and K. Schmid, Biochemistry, 6,112 (1967). (104) L. Mester, E. Moczar, G. Medgyesi, and K. Laki, Compt. Rend., 256,3210(1963). (105) M. A. Cynkin and R. H. Haschmeyer, Fed. Proc., 23,273 (1964). (106) R. H. Haschmeyer, M. A. Cynkin, L.-C. Han, and M. Trindle, Biochemistry, 5 , 3443 (1966). (107) L. Mester, E. Moczar, and L. Szabo, Compt. Rend., 265,877 (1967). (108) S. Iwanaga, B. Blomback, N. J. Grondahl, B. Hessel, and P. Wallen, Biochim. Biophys. Acta, 160,280(1968). (109) J. R. Clampand F. W. Putnam,J. B i d . Chem., 239,3233(1964). (110) N. Duquesne, M. Monsigny, and J. Montreuil, Compt. Rend., 262,2536(1966). (111) G. M. Edelman, B. A. Cunningham, W. E. Gall, P. D. Gottlieb, U. Rutishauser, and M. J. Waxdal, Pmc. Nut. Acad. Sci. U.S., 63, 78 (1969). (112) C. Nolan and E. L. Smith,J. Biol. Chem.,237,446(1962). (113) N. Duquesne, M. Monsigny, and J. Montreuil, Compt. Rend., Ser. D, 261, 1430 (1965). (114) R. L. Hill, R. Delaney, R. E. Fellows, and H. E. Lebovitz, Proc. Nat. Acad. Sci. U.S., 56,1762 (1966). (115) F. Melchers, Biochemistry, 8,938 (1969). (116) A. B. Edmundson, F. A. Sheber, K. R. Ely, N. B. Simonds, N. K. Hutson,and J. L. Rossiter, Arch. Biochem. Biophys., 127,725(1968). (117) G. Spik and J. Montreuil, Intern. Symp. IV. Chromatographie EZectrophorese, Presses Acad. Europbenes, Bruxelles, 1968,p. 385. (117a)J.Williams and I. Graham, personal communication. (118) A. B. Rawitch, T. Liao, and J. G. Pierce, Biochim. Biophys. Acta, 160,360(1968). (119) P. G. Johansen, R. D. Marshall, and A. Neuberger, Biochem.J.,78,518 (1961). (120) Y. C. Lee and R. Montgomery, Arch. Biochem. Biophys., 97,9 (1962). (121) L. W. Cunningham, R. W. Clouse, and J. D. Ford, Biochim. Biophys. Acta, 78, 379 (1963). (122) E. D. Kaverzneva, F. V. Shmakova, and A. P. Andrejeva, Biokhimiya, 32, 964 (1967). (123) M. Monsigny, A. Adam-Chosson, and J. Montreuil, Bull. Soc. Chim. Biol.,50, 857 (1968). (124) R. Montgomery and Y . C . Wu, J . B i d . Chem., 238,3547(1963). (125) M. Tanaka, Yakuguku Zasshi, 81,1470(1961). (126) R. J. DeLange, Fed. Proc., 28,343 (1969). (127) J. Williams, Biochem.]., 108,57 (1968). (127a)T.C. Elleman and J. Williams, Biochem.]., 116,515(1970). (128) J. Z. Angustyniak and W. G. Martin, Cun.J.Biochem., 46,983 (1968). (129) T. H.Plummer and C. H.W. HirsJ. B i d . Chem., 239,2530 (1964). (130) T. H. Plummer, A. Tarentino, and F. Maley,]. Biol.Chem., 243,5158(1968). (131) R. L. Jackson, V. N. Reinhold, and C. H. W. Hirs, Fed. Proc., 27,529 (1968). (132) C. H. W. Hirs, personal communication. (133) B. J. Catley, S. Moore, and W. H. Stein,]. B i d . Chem., 244,933(1969). (134) Y. Yasuda, N. Takahashi, and T . Murachi, Biochemistry, 9,25 (1970). (135) M. Anai, T. Ikenaka, and Y. Matsushima, J . Biochem. (Tokyo), 59,57 (1966).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

425

Studies on the sequence of amino acids in the neighborhood of that L-asparagine residue to which the 2-acetamido-2-deoxy-~-glucosy1 moiety is attached (see Table VI) has revealed that no single, favored, primary structure exists in this region, with an important exception: in the great majority of compounds thus far studied, X-Q-YThr (or Ser) occurs, where Q represents the glycosylated residue, and X and Y are amino acid residues. I t was suggested that the sequence in the apoprotein may be a necessary condition for glycosylation to occur,136but the finding of sequences of this type in proteins not glycosylated suggests that this is not a sufficient condition. Thus, the overall stereochemistry and other factors must also be considered. Examples of such non-glycosylated proteins that contain a sequence of this type include human137calcitonin M, a penicillinase from Staphylococcus aureus in which it occurs twice (residues 186-188 and 241-243),13"nucleases from the same bacteria (residues 118-120),139 and guinea-pig immunoglobulin K-chain, close to the C-terminal end.140It may be mentioned that an earlier report to the effect that a sequence of this type occurs as residues number 102-104 of ox chymotrypsin was erroneous, and that residue 102 is not L-asparagine, but L-aspartic acid.14' Other non-glycosylated sequences of this type have been de~cribed.'~'" It has been suggested'42 that the nature of the side chain of residue Y may influence the size of the carbohydrate moiety found at that point, but further data are needed before this hypothesis can be considered firmly established.

(136) A. Neuberger and R. D . Marshall, in "Symposium on Foods-Carbohydrates and their Roles," H. W. Schultze, R. F. Cain, and R. W. Wrotstad, eds., Avi Publishing Co.,Westport, Connecticut, 1969, p. 115. (137) P. Sieber, M. Brugger, B. Kamber, B. Riniker, and W. Rittel, Helu. Chim. Acta, 51,2057 (1968). (138) R. P. Ambler and R. J. Meadway, Nature, 222,24 (1969). (139) C. L. Cusumano, H. Taniuchi, and C. B. Anfinsen, J . B i d . Chern., 243, 4769 (1968). (140) M. E. Lamm and B. Lisowska-Bernstein, Nature, 220,712 (1968). (141a)L.T. Hunt and M . 0. Dayhoff, Biochern. Biophys. Res. Cornmun., 39,757 (1970). (141) D. M. Blow, J. J. Birktoft, and B. S. Hartley, Nature, 221,337 (1969). (142) C. H. W. Hirs, R. L. Jackson, and I. Kabasawa, Abstracts Papers Amer. Chern. Soc. Meeting, 158, CARB 048 (1969).

426

R. D. MARSHALL AND A. NEUBERGER

2. Linkages in which O-2-Acetamido-2-deoxy-a-~-galactopyranosyl is Linked to L-Serine or L-Threonine Residues Structures of this type (2) occur in a number of glycoproteins. HO

H O I

1

HO,C -C-C-R I I H,N H 0 -@-Acetamido- 2-deoxy - (Y -Dgalactopyranosy1)-L-serine (R = H) or -L-threonine (R = Me)

(2)

Through studies with purified enzymes, it has been established that this type of linkage is of the a-Danomeric c o n f i g ~ r a t i o n . ' ~ ~In -'~~" this Section are included certain glycoproteins for which identification of the sugar residue involved in the carbohydrate-peptide linkage has not yet been unambiguously made. Recognition of the amino acid, and, more particularly, of the sugar involved in the linkage, would clearly be accomplished most satisfactorily were the sugar-amino acid compound constituting this part of the structure to be isolated from various proteins and characterized by the techniques of organic chemistry. Sometimes, this approach is difficult, because of the inability of proteolytic enzymes to hydrolyze the glycoprotein; this is particularly encountered with those molecules in which the extent of substitution by sugars is very high, as in the blood-group substances from ovarian cysts,145in which every second amino acid residue is an 0-glycosylated a-amino-phydroxy acid. With other glycoproteins, this problem does not arise, and it may be noted that the half-life of the glycosidic bond in O-aD-glucosyl-L-serine in 0.4 M hydrochloric acid at 100" is146about 6 hours; this indicates that the glycoside has a stability to acid some(143) B. Weissmann and D. F. Hinrichsen, Biochemistry, 8,2034 (1969). (144) E. Buddecke, H. Schauer, E. Werries, and A. Gottschalk, Biockem. B i o p h y s . Res. Cotnmrin., 34, 517 (1969). (144a)A. S. R. Donald, J . M. Creeth, W. T. J. Morgan, and W. M . Watkins, Biochem. J., 115,125 (1969). (145) W. M. Watkins, i n Ref. 6, Chapter 11, Section 7. (146) V. A. Derevitskaya, M. G. Vatina, and N. K. Kochetkov, Cnrbohyd. Res., 3, 377 (1967).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

427

what greater than the P-aspartylglycosylamine linkage (see p. 421). Moreover, if the sugar involved in the linkage is an N-acetylhexosamine, it might b e expected that, during hydrolysis with acid, a relatively much more stable 0-(aminodeoxyhexosy1)-L-serinewould be produced to some extent. Thus, when 0-(2-acetamido-2-deoxy@-D-glucosy1)-L-serinewas treated with 1.5 M hydrochloric acid for 90 minutes at loo", a compound having the electrophoretic mobility expected for the deacetylated derivative was formed. Similar results were obtained with the corresponding derivative of ~ - t h r e o n i n e . ' ~It' has been customary to design experiments based on the effects of alkali, in order that the amino acid and the sugar involved in the linkage might be recognized. Usually, the aim is to bring about a @-eliminationof the carbohydrate moiety. a. The P-Elimination Reaction. -One of the highly characteristic features of a compound having a structure of the type depicted in 2 is the facility with which it may undergo @elimination under alkaline conditions, and this type of reaction can be considered in general mechanistic t e r r n ~ . ' T~h~e ~reactions '~~ of glycoproteins here considered require the presence of a base, indicating that the reaction is a bimolecular elimination and that it may be represented as follows.

Transition state

where X is the 0-( 2-acetamido-2-deoxy-~-galactosyl)group, R is H or CH, (for L-serine or L-threonine, respectively), and R' and R" are the -CO- and -NH- parts of the peptide groups involved in bonding to the remainder of the chain. It should be emphasized that, in the notation generally used to , carbon atoms are the reverse describe this type of reaction, the LY and B of those usually employed in amino acid chemistry. The effects, in general, of the groups X, R, R', and R" were con(147) M. Monsigny, M . Buchet, and J. Montreuil, Intern. Syrnp. ZV ChrornatogruphieElectrophorese, Presses Acad. EuropCenes, Bmxelles, 1968, p. 361. (148) W. Hanhart and C. K. Ingold, j . Chern. SOC.,997 (1927). (149) M. L. Dhar, E. D. Hughes, C. K. Ingold, A. M. N. Mandour, G. A. Maw, and L. I. Woolf,J. Chem. SOC., 2093 (1948).

428

R. D. MARSHALL AND A. NEUBERGER

sidered in detail by Ingold and his colleagues.lS0The group X must always be a strongly electron-attracting group. Reaction is facilitated through operation of the inductive effect if R’ and R” are strongly electron-withdrawing, and because -NHR’ and -COR” are not alkyl groups, they cannot affect the reaction through hyperconjugation. The presence of the methyl group (R) in derivatives of L-threonine may, through hyperconjugation, have a stabilizing effect on the transition state, but the extent to which this is operative cannot in general be predicted.ls1 b. Side Reactions that may Occur When a Glycoprotein or Glycopeptide is Subjected to Conditions Suitable for &Elimination. When the conditions that result in occurrence of this reaction are applied to glycoproteins, a number of other reactions of relevance may occur. In the first place, N-deacetylation of the 2-acetamido-2deoxy-D-galactosyl group (X) may occur to some extent, and the resultant group, X’,has a lessened electron-withdrawing ability as compared with that of X. The effect of this change in structure on the rate of p-elimination has not yet been investigated. The effect of splitting the peptide bond -COR” will be replacement of a powerful, electron-attracting group by a powerfully electron-donating, negatively charged carboxyl ion, so that reaction will then occur much more slowly. Splitting of the peptide bond -NHR’- will, likewise, be expected to result in a diminution in the rate of reaction, but the effect of splitting this peptide bond would be expected to be less than in the aforementioned case, because the uncharged amino group thereby produced is still slightly electron-withdrawing.1s2 Moreover, the uncharged amino group may stabilize the transition state through the operation of a mesomeric effect. The extent to which these two opposing effects affect the rate of reaction cannot be predicted. c. The Effects of Alkali on Model Compounds of This Type. - The effects of substituents on the rate of p-elimination of model compounds have been examined q~antitative1y.l~~ During 24 hours at 37”, at pH 11, N-( benzyloxycarbony1)-0-P-D-glucopyranosyl-L-serine methylamide reacts to the extent of 95%, whereas, under the same (150) C. K. Ingold, “Structure and Mechanism in Organic Chemistry,” G. Bell & Sons, Ltd., London, 1963, Chapter 8; 2nd. Edition, 1969, Chapter 9. (151) J. W. Baker, “Hyperconjugation,” Oxford University Press, Oxford, 1952, Chapter 6 . (152) M. J. S. Dewar, “Electronic Theory of Organic Chemistry,” Oxford University Press, Oxford, 1949, p. 52.

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

429

conditions, the corresponding free acid, namely, 0-P-D-glucopyranosyl-L-serine is completely stable. Similarly, O-P-D-glUCOpyranOSylL-serine methylamide is undegraded under these conditions. Somewhat surprising is the finding that, under the same conditions, 0P-D-glucopyranosyl-N-glycyl-L-serine methylamide is stable. Moreover, it was found that the free acid derivative already mentioned is not degraded by treatment with 0.1 M sodium hydroxide during 8 days at 37". Similar results were obtained with the corresponding derivatives of D-galactose. Under somewhat different conditions, namely, 0.1 M sodium hydroxide plus 0.3 M sodium borohydride during 24 hours at 20°, the 0-(2-acetamido-2-deoxy-~-glucosyl) derivatives of L-serine and L-threonine are stable, whereas their amides are almost completely ruptured by occurrence of 8-elimination.'53 Synthetic compounds in which the hydroxyl group of L-serine is glycosidically coupled with derivatives of D-glUCOSe,'46'154,155*155a D-gala~tose,'~ 2~ - amino , ~ ~ ~- 2 , ~- deoxy ~ ~ ~ - D - glucose,155bJ56*157 D - xy1ose,'55~158~159 lactose, and c e l l ~ b i o s e , 'as ~ ~well ~ as ones in which L-threonin? is linked to D-xylose,160D-glucose,'61or 2-amino-2-deoxyD-glUCOSe,'62have been described. d. Recognition of the Amino Acid Involved in the Carbohydratepeptide Bond. -Treatment with alkali of glycoproteins that contain glycosidic linkages involving L-serine or L-threonine, or both, leads to the formation, from 0-glycosylated L-serine and L-threonine residues, respectively, of 2-aminoacrylic and 2-aminocrotonic acid residues within the chain. The difference in the number of residues of L-serine or L-threonine, or both, in acid hydrolyzates of the glycoprotein, both before and after treatment with alkali, is, in general, (153) J. Montreuil, M . Monsigny, and M. Buchet, Compt. Rend., Ser. D , 264, 2068 (1967). (154) J. K. N. Jones, M. B. Perry, B. Shelton, and D. Walton, C a n . ] . Chem., 39, 1005 (1961). (155) F. Kum and S. Roseman, Biochemistry, 5,3061 (1966). (155a)K.Kum, Carbohyd. Res., 11,269 (1969). (155b)E.Rude and M. Meyer-Delius, Carbohyd. Res., 8,219 (1968). (156) F. Micheel and H. Kochling, Chem. Ber., 93,2372 (1960). (157) J . R. Vercellotti and A. E. Luetzow,]. Org. Chem.,31,825 (1966). (158) B. Lindberg and B. Silvander, Acta Chem. Scand., 19,530 (1965). (159) K. Brendel and E. A. Davidson, Carbohyd. Res., 2,42 (1966). ~ (160) M. Higham, P. W. Kent, and P. Fisher, Biochem.]., 1 0 8 , 4 7 (1968). (161) V. A. Derevitskaya, E. M. Klinov, and N. K. Kochetkov, Carbohyd. Res., 7, 7 (1968). (162) J. R. Vercellotti, R. Femandez, and C. J. Chang, Carbohyd. Res., 5,97 (1967).

430

R. D. MARSHALL AND A. NEUBERGER

approximately equal to the number of carbohydrate moieties that were formerly linked through these residues and that are released from the polypeptide chains. However, this reaction may not always be sufficiently specific for glycosylated L-serine or L-threonine moieties. It has been found that, under conditions often employed, namely, 0.5 M sodium hydroxide for 96 hours at 4", there is often little or no decomposition of nonglycosylated L-serine or L-threonine residues, as judged from the results of control experiments in which edestinIti3 or orosomucoid1"J65 were employed. However, it has been reported that P-hydroxy a-amino acid residues in the protein derived from sheep submaxillary-gland glycoprotein by removal of the sugar residues are unstable under alkaline conditions.'66 Treatment of lysozyme at pH 12.8 (ionic strength 1.6)for 45 minutes at 70" led to a loss of somewhat less than 15%of the L-serine and L-threonine contents of the protein.'"' We believe that data concerned with alkaline conversion of L-serine and L-threonine residues into the corresponding unsaturated derivatives must be interpreted with caution. It would not be altogether surprising were certain unsubstituted P-hydroxy a-amino acid residues, in their protein environment, to undergo this reaction with facility, Decrease in the proportion of L-serine or L-threonine, or both, when pig gastric blood-group substance,lti4 casein,16Eor a mucin derived from colloid carcinoma of the breast44 were subjected to alkaline conditions may suggest that they all contain linkages of this type. The increase found in the proportion of glycine in the lastmentioned glycoprotein when it was treated with alkali and the product was hydrolyzed with acid was interpreted as being due to a second possible pathway for breakdown of 2-aminocrotonic acid residues, namely a retroaldol reaction.1Bg Peptides that contain 2-aminoacrylic acid and 2-aminocrotonic acid groups absorb in the ultraviolet region, the former at a wavelength maximum of 240 nm, and the latter with a more generalized absorption. At 240 nm, the molar absorptivity is the same (4,200) for the two (163)B. Anderson, P.Hoffman, and K. Meyer, Biochim. Biophys. Acta, 74,309(1963). (164)B. Anderson, N. Seno, P. Sampson, J. G . Riley, P. Hoffman, and K. Meyer,]. Biol.Chem., 239,PC 2716 (1964). (165)B. Anderson, P.Hoffman, and K. Meyer,]. Blol. Chem.,240,156(1965). (166)J. E. McGuire and S. Roseman,]. Biol. Chem., 242,3745(1967). (167)S. Harbon, G. Herman, B. Rossignol, P. JollBs, and H. Clauser, Biochem. Biophys. Res. Commun., 17,37(1964). (168)F.H. Malpress and M. Seid-Akhaven, Biochem.]., 101,764(1966). (169)J. B. Adams, Biochem.]., 97,345(1965).

TABLEVII The Effects on the Contents of Serine and Threonine of Some Glycopeptides and Glycoproteins on Treatment with Alkali in Presence of Borohydridea Alkaline conditions Type of material

Molarity Molarity Glycoprotein

Glycopeptide yA myeloma protein rabbit yC globulin lactotxansfemn yA lactoglobulin Glycoprotein ox submaxillarygland glycoprotein pig submaxillarygland glycoprotein earthworm-cuticle collagen

Temp.

Time

of OH@ of NaBH, (degrees) (hr.) 0.1

0.3

0.5

-

0.5 0.1 0.1 0.1 0.1 0.5 0.1 0.1 0.05

0.3 0.3 0.3 0.3 0.3

-

0.3 0.3 0.15

Reduced further with

Decrease inb Increase inb

Refer-

Ser

ences

Thr

270

-

2

20

-

1.0

20

I70 70 70 216 216 96 6 6 24

-

1.2 3.9 3.5 218 170 243 394 373 162

4

20 20 5 5 4 45 45 30

0.15 2.9 sodium-liquid ammonia 2.3 357 NaBH,-palladiumchloride 317 H2-Adams’catalyst 462 NaBH,-palladiumblack 683 NaBH,-palladium chloride 553 51

Ala

But

0.3

172 20

0.13 1.75 0.63 3.4 2.9 311 32 147 311 92 90 192 567 483 258

117 173 171 174 164 175 79 ~~

“The 2-aminoacrylic and 2-aminocrotonic acid residues initially produced by alkali are reduced to L-alanine and 2-aminobutyric acid (But) residues, respectively. Use of a further reducing agent is indicated. T h e results are expressed as decrease or increase in moles of amino acid for the glycopeptides, and as pmoles per g for the glycoproteins.

432

R. D. MARSHALL AND A. NEUBERGER

types of derivative,I7O so that, in some instances, quantitative measurements may be used for determining the number of unsaturated residues formed. L-Cysteine and L-cystine residues might further complicate the interpretation of the data, as could also the presence of phosphorylated p-hydroxy a-amino acid residues. Addition reactions across the double bonds of the 2-aminoacrylic and 2-aminocrotonic acid residues formed often permit comparisons of the amounts of L-serine or L-threonine, or both, that are decomposed by alkaline conditions, and estimates of the quantities of the addition products to be made. Pigman and coworkers171introduced the use of sodium borohydride to reduce the double bonds formed during alkaline treatment, and this reagent is highly effective in converting 2-aminoacryloyl into L-alanyl residues within the modified polypeptide chain (see Table VII). However, the corresponding derivative of L-threonine is less readily reduced by this reagent, and further reduction with sodium borohydride in alkali containing palladium chloride leads to higher, but still not quantitative, yields of 2-aminobutyric acid moieties (see Table VII). Related studies with the A and B substances from ovarian cysts, as well as with pig gastric-mucin (A H) substance showed that over 80% of the Lserine and L-threonine was decomposed on treatment with sodium borohydride (0.26 M ) in alkali (0.2 M ) during one week at room temperature. The extent of formation of 2-aminobutyric acid was small. It was further shown that alkaline decomposition of L-serine and Lthreonine residues proceeds to a greater extent in the presence than in the absence of sodium b~rohydride."~."~ L-Cysteic acid residues were produced within the peptide chain when submaxillary-gland glycoprotein from sheep was treated with alkali (pH 9.0), during 24 hours at room temperature in the presence of sulfite (0.1 M).The yield was -55% on the basis of the L-serine decomposed, and the corresponding derivative from L-threonine was not detected.IB7

+

(170)V. E.Price and J. P. Greenstein, Arch. Biochem. Biophys., 18,383(1948). (171)K. Tanaka, M.Bertolini, and W. Pigman, Biochem. Biophys. Res. Commun., 16, 404 (1964). (172)G . Dawson and J. R. Clamp, Blochem../.,107,341(1968). (173)J. Descamps, M.Monsigny, and J. Montreuil, Conpt. Rend., Ser. D, 266, 1775 (1968). (174)K.Tanaka and W. Pigman,./. Blot. Chem.,240, PC 1487(1965). (175)N.Payza, S. Rizvi, and W. Pigman, Arch. Btochem. Btophys., 129,68(1969). (176)E. A. Kabat, E. W. Bassett, K. Pryzwansky, K. 0. Lloyd, M. E. Kaplan, and E. J. Layug, Biocherndstry,4,1632(1965). (177)D. M. CarlsonJ. Blol. Chem.,243,616(lQ68).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

433

e. Recognition of the Sugar Involved in the Carbohydrate-peptide Bond.-It has been shown that alkaline conditions, namely 0.5 M sodium hydroxide, during 48 hours at 22", cause elimination of 2acetamido-2-deoxy-~-galactose from glycopeptides prepared from submaxillary-gland glycoprotein of sheep. The glycopeptides used residues as the contained single 2-acetamido-2-deoxy-~-galactose carbohydrate moieties, and the number of these residues (which behaved colorimetrically as free 2-acetamido-2-deoxy-~-galactose after the reaction) was equal to 90% of the number of L-serine and L-threonine residues decomposed b y the treatment. The difference (10%)was found to be equivalent to the amount of free 2-acetamido-2deoxy-D-galactose which, in control experiments, was converted into under compounds not estimatable as 2-acetamido-2-deoxy-~-galactose these condition^.'^^ However, in general, when the carbohydrate attached to the glycopeptide or glycoprotein under examination has a more complicated structure, very considerable decomposition of the sugar moiety originally involved in the carbohydrate-peptide linkage may occur under the conditions used to split the latter. Thus, when blood-group substances from ovarian cysts were treated with alkaline borohydride or alkaline borodeuteride under the conditions frequently employed to cause P-elimination from glycoproteins, namely, 0.2 M alkali with 0.26 M sodium borohydride or sodium borohydride-d4, the elimination reaction was succeeded by extensive alkaline degradation of the oligosaccharide chains concomitant with reduction, to the corresponding alditols, of the reducing-sugar moieties thereby e x p o ~ e d . ' ' ~ Similar losses were observed when pig submaxillary-gland glycoprotein and the M- and N-active substances from human erythrocytes were treatedIs0 under similar conditions, namely, 0.02 M sodium hydroxide plus 0.4 M sodium borohydride for 16 hours at 25", or 0.2 M sodium hydroxide plus 0.2 M sodium borohydride for 48 hours at 25". Alkaline degradation of an oligosaccharide leads to loss of the sugar originally in the reducing position, because the reaction may be expected to proceed by p-elimination. If the vicinal sugar is linked to 0 - 3 of the reducing-sugar residue, the reaction occurs with much greater facility than when 0-4or 0-6is involved. A linkage involving 0 - 2 is stable under alkaline conditions. Studies have been made with oligosaccharides of the type encountered in glycoproteins.'s' For the blood(178) R. Carubelli, V. P. Bhavanandan, and A. Gottschafk, Biochim. Biophys. Acta, 101,67 (1965). (179) K. 0. Lloyd, E. A. Kabat, and E. Liciero, Biochemistry, 7,2976 (1968). ( 1 8 0 ) P. Weberand R. J. Winzler, Arch. Biockem. Biophys., 129,534 (1969). (181) A. Neuberger and R. D. Marshall, in Ref. 6, p. 262.

434

R. D. MARSHALL AND A. NEUBERGER

group substances already mentioned, it is considered that 2-acetamido2-deoxy-D-galactose residues are linked to L-serine and L-threonine residues within the polypeptide chain, and that some of these sugar residues are substituted at 0 - 3 (see p. 452). It is not at present possible to interpret unambiguously the finding that part of the 2-amino-2deoxy-D-glucose originally present in the material resembling keratan sulfate that is a constituent of chick allantoic fluid is decomposed when the material is treated with alkali.64 The occurrence of 2-acetamido-2-deoxy-D-galactose as part of the carbohydrate-peptide linkage in several proteins is based on acceptable evidence. In the submaxillary-gland glycoprotein from sheep and ox, the carbohydrate moieties consist of 2-acetamido-2deoxy-6-O-sialyl-~-galactosylresidues. The action of neuraminidase results in the production of modified glycoproteins in which 2-acetamido-2-deoxy-~-galactoseis by far the major sugar component. This was also the only amino sugar in a glycopeptide isolated from rabbit y G globulin.20 Treatment of blood-group B substance from ovariancyst fluid with a solution of 25 mM sulfuric acid in acetic acid at 60" for 24 hours led to the production of a modified glycoprotein that had an amino acid composition closely similar to that of the parent material, but from which sugars other than 2-acetamido-2-deoxy-~-galactose had been largely eliminated.'44n

3. 0-P-D-Xylopyranosyl-L-serine as a Carbohydrate-peptide Linking Moiety Linkages of this type (see 3) that occur within glycoproteins may

0-CH,-C-CO,H

I

H

0-$-o-Xylopyranosyl-L- serine (3)

also undergo p-elimination under alkaline conditions. In acid, however, the rate of cleavage may be expected to be of the same order as that for 0-P-D-glucopyranosyl-L-serine, which is reported'46 to be quite stable in solution at p H 1.5 during 24 hours at 100". O-P-DXylopyranosyl-L-serine has been isolated from acid hydrolyzates (pH 1.55), after 3 hours at loo", of commercial heparin containing L-serine as the only amino acid present in significant proportion. The

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

435

preparations of heparin used were presumably from heparin proteoglycans that had been subjected to proteolysis prior to isolation. The nature of the compound isolated was established by hydrolysis with 4 M hydrochloric acid during 3 hours at loo", followed by chromatographic identification of both a xylose and a serine. That the sugar was D-xylose was proved with the use of D-xylose isomerase (Dxylose aldo-keto-isomerase, E.C. 5.3.1.5).183The quantity of material obtained accounted for 13% of the original L-serine, in addition to a further 135%of an 0-D-galactosyl-D-xylosyl-L-serine and 17% of the free amino acid. O-D-Xy1osyl-L-serine was isolated from acid hydrolyzates (pH 1.5) obtained, after 4 hours at loo", from the serine-oligosaccharides obtained from enzymic hydrolyzates of "chondromucoprotein," consisting mainly of chondroitin 4-sulfate, from nasal septa.184 Isolation methods have been employed for demonstrating that, in umbilical cord, chondroitin 6-sulfate is linked to a protein chain by the same type of bond.IK5It had earlier been established that chondroitin 6-sulfate from shark cartilage undergoes a considerable loss in L-serine (35 pmoles per gram) when it is treated with 0.5 M sodium hydroxide for 19 hours at 0 or 24". About half of this L-serine is converted into L-alanine by hydrogenation in the presence of Adams' catalyst. le5 Moreover, D-xylose occurs in glycopeptides isolated from enzymic digests of chondroitin 6-sulfate-protein complexes from a variety of sources.Is6 Comparable experiments were performed with chondroitin 4-sulfate, with similar results. Xylitol was identifiedIx7 by gas-liquid chromatography after chondroitin 4-sulfate from pig costal-cartilage had been subjected to the action of 0.2 M potassium hydroxide and sodium borohydride during 20 hours at 4".Xylitol was also identifiedIs8 as its pentaacetate after treatment with 0.5 M sodium hydroxide and 0.25 M sodium borohydride during 75 minutes at room temperature. O-@-D-Xylosyl-L-serinewas also isolated from the cartilage,Is8as well as from chick-embryo ~ a r t i 1 a g e . l ~ ~ (182) E. R. B. Graham and A. Cottschalk, Biochim. Biophys. Acta, 38,513 (1960). (183) U. Lindahl and L. Rod&]. Biol. Chem., 240,2821 (1965). (184) L. Rod& and U. Lindahl,]. Biol. Chem., 240,606 (1965). (185) T. Helting and L. Rod&, Biochim. Biophys. Acta, 170,301 (1968). (186) M. Schmidt, A. Dmochowski, and B. Wierzbowska, Biochim. Biophys. Acta, 117,258 (1966). (187) N. Katsuraand E. A. Davidson, Biochim. Biophys. Acta, 121,120 (1966). (188) E. E. Grebner, C. W. Hall, and E. F. Neufeld, Arch. Biochem. Biophys., 116, 39 1 (1966). (189) H. C. Robinson, A. Telser, and A. Dorfinan, Proc. Nut. Acad. Sci. U . S., 56, 1859 (1966).

436

€3. D. MARSHALL AND A. NEUBERGER

Comparison of the optical rotatory dispersion exhibited by 0-p-Dxylopyranosyl-L-serine isolated from glycosaminoglycans with those of the two synthetic anomers revealed that the natural material has Moreover, the O-D-XylOSyl-L-Serine the p-D anomeric configurati~n.'~~ present in normal urine also has the p-Dconfiguration, as had earlier been suggested.190The origin of the urinary compound is unknown, but it may be a catabolite of certain glycoproteins. Although this type of linkage is frequently labile under alkaline conditions, it is known that extensive digestion with papain of "chondromucoprotein" may lead to the formation of chondroitin 4-sulfate in which L-serine is the major if not the only amino acid present. Not surprisingly, under the circumstances, the carbohydrate-amino acid bond is not readily split'O by 0.15 M sodium hydroxide during 96 hours at 20" or1"' by 0.2 M potassium hydroxide during 20 hours at 4". At one linkage of this type in the chondroitin 4-sulfate from pig c~stal-cartilage,'~~ the sequence of amino acids is reported to be -Glu-Gly-Ser-Gly-, where Ser is the glycosylated residue. including The presence of D-XylOSe in several other glycoprotein~,~~ an alpha-amylase (see p. 442) and pineapple-stem b r ~ m e l a i n has '~~ been reported, but its mode of attachment is not yet known. 4. 5-O-/3-D-Galactopyranosyloxy-L-lysine as a Carbohydrate-peptide Linking Moiety This structural entity (see 4) occurs at the junction of the oligosacHO

H

H

5- 0 - p - D - Galactopyranosyloxy-L-lysine (4)

charide units to the polypeptide chain in both the citrate-soluble and citrate-insoluble collagen'g1 from guinea-pig skin, in the lens capsule (190) F. Tominaga, K. Oka, and H. Yoshida,]. Riochem. (Tokyo), 57,717 (1965). (191) L. W. Cunningham and J. D. Ford,]. B i d Chem.,243,2390 (1968).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

437

of the eye,lgla in the soluble stroma of calf cornea,lg2 and in the basement membrane of ox-kidney g l o m e r u l ~ s . ' Compounds ~~ in which a disaccharide is linked through the hydroxyl group of 5hydroxy-L-lysine have been isolated from the hydrolysis, with 2 M sodium hydroxide during 16-20 hours at QO-lOF, of glycopeptides isolated from these proteins, or, for the basement membrane, from the protein itself. Not unexpectedly, this type of linkage is stable under alkaline conditions. The material obtained from basement membrane was further degraded by 0.05 M sulfuric acid during 28 hours at 100" and 5-O-~-~-galactopyranosyloxy-~-lysine was isolated.lg3 The occurrence of the positively charged amino group in the aglycon under acidic conditions has the effect of stabilizing the glycosidic linkage to a considerable degree. lg3 This factor has been investigated quantitatively by comparing the rate of acid hydrolysis of ethyl P-D-glucopyranoside with that of 2-aminoethyl p-D-glucop y r a n 0 ~ i d e . The l ~ ~ presence, in the aglycon, of the positively charged group attached to the carbon atom in the P position to the anomeric oxygen atom lowers the rate of hydrolysis of the glycosidic bond by a factor much less than that for glycosides of 2-amino-2-deoxy-D-glucose (see Table VIII). The most probable explanation for this difference TABLE VIII The Rates of Hydrolysis of Some Glycopyranosides in 2 M Hydrochloric Acid at 80" Glycopyranoside Ethyl P-D-glucopyranoside 2-Aminoethyl P-D-glucopyranoside Methyl 2-amino-2-deoxy-a-D-glucopyranoside"

k (min-') 19.2X 4.3 x lo-:' 0.09 x lO+J

References

195 194 196

"In 2.5M hydrochloric acid.

lies in the fact that the doubly charged, positive, oxonium-ion intermediate produced in the hydrolysis of 2-aminoethyl P-D-glucopyranoside undergoes cleavage in such a way as to cause separation of the two positive charges (see Fig. la). On the other hand, the oxonium (191a)R.G. Spiro and S. Fukushi,]. B i d . Chem.,244,2049(1969). (192)E. Moczar, L.Robert, and M. Moczar, Eur0p.J. Biochem., 6,213(1968). (193)R.G. Spiro,]. Biol. Chem., 242,4813(1967). (194)E. R. B. Graham and A. Neuberger,]. Chem. SOC. ( C ) ,1638(1968). (195)W.G. Overend, C. W. Rees, and J. S. Sequeira,]. Chem. SOC.,3429 (1962). (196)R. G . C. Moggridge and A. Neuberger,J. Chem. SOC.,745 (1938).

438

R. D. MARSHALL AND A. NEUBERCER

FIG.1.-Reaction Mechanism for the Acid Hydrolysis oE(a) 2-Aminoethyl p-~-Glucopyranoside and (b) Methyl 2-Amino-2-deoxy-~-~-g~ucopyranoside.

ion produced when methyl 2-amino-2-deoxy-~-glucopyranoside is protonated is likely to undergo conversion into the carbonium ion with difficulty, because this reaction involves a still closer approach of two positive charges to each other (see Fig. lb). An alternative explanation is based on the assumption that the protonation required as the preliminary step in cleavage of the glycosidic bond occurs on the ring-oxygen atom.197If, in 2-aminoethyl P-D-glucopyranoside, a di-cation having positive charges on the amino group and on the ring-oxygen atom is cleaved, repulsion between the two charges will be greatly lessened. In contrast, with the a shift of the proton from ethyl 2-amino-Zdeoxy-~-gIucopyranosides, the anomeric oxygen atom to the ring-oxygen atom would not cause the distances between the two positive charges to be altered to the same extent. The two explanations are not mutually exclusive. were Isomeric forms of 5-O-~-D-galactopyranosyloxy-L-lysine isolated after hydrolysis, with 2 M sodium hydroxide during 16 hours at go", of skin collagen,198and it was suggested that racemization of the amino acid moiety had occurred during the hydrolysis. In the natural compound, the anomeric configuration is believed to be p-D, because a-D-galactosidase is without effect on the 5-O-P-D-galaC(197)C . A. Bunton, T. A. Lewis, D. R. Llewellyn, and C. A. Vernon, J. Chem. SOC., 4419 (1955). (198) L. W. Cunningham, J. D. Ford, and J. P. Segrest, J. B i d . Chem., 242,2570 (1967).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

439

topyranosyloxy-L-lysine isolated from basement membrane, either before or after N-acetylation. Moreover, although the compound is not split by P-D-galactosidase, its N-acetylated derivative is, albeit very slowly.1s3 The sequence of amino acids in the vicinity of at least some of the glycosylated 5-hydroxy-~-lysineresidues in the collagen from guineapig skin was shown1ss to be the following, relatively rare structure,

-Gly-Met-Hyl-Gly-His-Arg-, where Hyl represents the amino acid that is glycosylated. This sequence is not the only, or even the necessary, sequence for glycosylation, because glycopeptides from the basement membrane do not contain this sequence. It has been shown that one molecule of these glycopeptides contains about two molecular proportions of glycine and one of L-glutamic acid, in addition to 5-hydroxy-L-lysine.*OOBoth the citrate-soluble and the citrate-insoluble collagen from guinea-pig skin contain -19 5-hydroxy-~-lysineresidues per 3 X lo5daltons, and about one-third of these are g l y c o ~ y l a t e d . ' ~ ~

111. POLYPEPTIDECHAINSCARRYING MORE THANONE TYPEOF CARBOHYDRATE-PEPTIDE LINKAGE

The possibility of categorizing glycoproteins according to the nature of the carbohydrate-peptide bond contained has been sugg e ~ t e dTo . ~ some ~ extent, this method is possible, but, as was pointed out, it does not result in a rigid classification, for examples are known in which more than one type of carbohydrate moiety is attached to a given polypeptide chain. In studies conducted by Partridge and Elsden,68 it was found that " chondromucoprotein" prepared from ox nasal-septa without the use of alkali or proteolytic enzymes gave rise, on treatment with 0.5 M sodium hydroxide during 24 hours at 25", to protein-free chondroitin sulfate and a heterogeneous, glycoprotein fraction whose carbohydrate composition suggested that it was keratan sulfate. The carbohydrate-peptide linkage binding the latter to protein appears to be more resistant to alkali than that joining chondroitin sulfates to the polypeptide chain (see p. 435). It is, therefore, likely that chondroitin sulfate and keratan sulfate may be attached to the same polypeptide chain by different types of linkage, as indicated also by the (199) W. T. Butler and L. W. Cunningham,J. Biol. Chem.,241,3882 (1966). (200) R. C . Spiro, j . B i d . Chem., 242,1923 (1967).

440

R. D . MARSHALL AND A. NEUBERGER

work of Gregory and R o d h Z o 1Human knee-joint cartilage also contains a complex composed of protein attached to keratan sulfate and chondroitin sulfate; it has been reported that Li-hydroxy-~-proline occurs in the glycopeptides obtained from papain digests of this material.z0zIt should be emphasized, however, that this compound is not always found, for chondroitin 4-sulfate-protein complexes extracted from pig laryngeal-cartilage are free from keratan sulfate.203 The absence of the latter from these glycoprotein preparations might result from the polypeptide chain's not containing the necessary marker-sequence (see p. 425) that may well be required so that keratan sulfate can become attached to the protein. The polypeptide chain to which chondroitin sulfate is attached is not always the same.70~165~zo3--205 There is probably a variety of polypeptide chains to which chondroitin sulfates, keratan sulfate, and smaller oligosaccharides may be attached. In some complexes, the nature of the recipient chain for the sugar moieties, or the distribution of the relevant sugar transferases, is such that more than one type of carbohydrate moiety becomes attached. 2-Acetamido-2-deoxy-~-galactose residues are attached to the polypeptide chain that is part of the structure of the keratan sulfate prepared from Pan-Protease digests of old human-rib cartilage, and these residues are linked to P-hydroxyl groups of L-serine and Lthreonine residues. Some, at least, of the 2-acetamido-Z-deoxy-~galactose residues form branch points, probablyzo5with substituents at 0-3 and 0-6. Other types of keratan sulfate have been shown also to contain alkali-labile linkages in which L-serine and L-threonine p a r t i ~ i p a t e . It ' ~ is, ~ however, possible that the disaccharide repeatingunit (which is that structure comprising the major component usually identified as keratan sulfate) is not attached to this 2-acetamido-2deoxy-D-galactose, and L-serine or L-threonine residues (or both) either directly or indirectly, because the major carbohydrate component is not split from the keratan sulfate isolated from chick allantoic-fluid when this material is treated with 0.5 M sodium hydroxide for 48 hours at room temperature. All of the samples of keratan sulfate used contained various proportions of sialic acid and L(201)J. D.Gregory and L. Rodbn, Biochem. Biophys. Res. Commun.,5,430(1961). (202)H.Greiling and H. W. Stuhlsatz, 2.Physiol. Chem.,350,449(1969). (203)H.Muir and S. Jacobs, Biochem.]., 103,367(1967). (204)A. A. Castellani, B. Bonferoni, S . Ronchi, G. Ferri, and M. Malcovati, Ital. J . Biochem., 11,187(1962). (205)C. P.Tsiganos and H. Muir, Biochem.J,113,885(1969). (206)B.A.Bray, R. Lieberman, and K. MeyerJ. BioE. Chem.,242,3373(1967).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

441

f u c o ~ e . These ~ ~ ‘ constituents may occur as part of carbohydrate moieties that involve at least some of the 2-acetamido-2-deoxy-~-galactose residues in structures analogous to those present in blood-group substances. D-Mannose, also, was found in keratan sulfate isolated from human rib208 and kneezo9 cartilage. Some preparations from rib cartilage have chondroitin 6-sulfate linked, by way of D-xylose, to the same polypeptide hai in.^^^,^^^ By the use of somewhat different techniques, a keratan sulfate that is largely free from L-fucose and sialic acid may be isolated from human costal cartilage, and the protein and carbohydrate entities of such preparations cannot be cleaved”’ with 0.5 M potassium hydroxide during 48 hours at 25”. Ox-corneal keratan sulfate is also attached to the protein core by linkages stable under alkaline conditions207 and the linkage is believed212 to involve L-asparagine and 2-acetamido-2-deoxy-~-glucose, although definite proof is still lacking. It is, therefore, reasonable to infer that cartilage contains glycoproteins in which there are residues of ( a ) L-asparagine that are substituted with 2-acetamido-2-deoxy-~-glucoseand that, to some extent, may be part of the keratan sulfate side-chain(s),( b )L-serine substituted by D-XylOSe attached to chondroitin sulfate polysaccharide moieties, and (c) L-serine and L-threonine to which are attached 2-acetamido-2deoxy-D-galactose residues that form a part of small oligosaccharide moieties; the antigenic cross-reactivity of keratan sulfate and bloodgroup substances may be relevant here.213More work is required before a general hypothesis can be put forward with confidence. In interpreting some of the earlier data, there are difficulties partly attributable to the fact that proteolytic digestion had preceded isolation of the material; in these studies, different proteolytic enzymes were used. The finding that, when the protein-polysaccharide complex (mainly chondroitin 4-sulfate) from pig rib-cartilage was treated with 0.2 M potassium hydroxide for 20 hours at 4”,only about 80% of the carbohydrate moieties were removed by &elimination, was interpreted as (207) N. Seno, K. Meyer, B. Anderson, and P. Hoffman,J. Biol. Chem., 240,1005 (1965). (208) V. P. Bhavanandan and K. Meyer (1967), quoted in Reference 206. (209) H. Greiling, in “Aktuelle Probleme des Rheumatismus,” H. Riissler and R. Heister, eds., F. K. Schattauer-Verlag, Stuttgart and New York, 1969, p. 53. (210) K. Meyer, P. Hoffman, and A. Linker, Science, 128,896 (1958). (211) M. B. Mathews and J. A. Cifonelli,J. Biol. Chem., 240,4140 (1965). (212) H. Greiling, H. W. Stuhlsatz, R. Kisters, and L. Plagemann, Ahstr. 5th Meeting Fed. Europ. Biochem. Soc., Prague, 1968, p. 155. (213) 0.Rosen, P. Hoffman, and K. Meyer, Fed. Proc., 19,147 (1960).

442

R. D. MARSHALL AND A. NEUBERGER

evidence that the remainder were bound to the polypeptide chain by linkages of a type different from the glycosidic ones involving Lserine and L-threonine. The inability to detect D-XylOSe or D-galactose in the fraction stable to alkali supports the view that a second type of linkage may be present.73 Several other examples of more clearly defined glycoproteins are known in which there is more than one type of carbohydrate-peptide bond. In rabbit yG globulin, an L-asparagine residue in the Fc region of the heavy chains is substituted by 2-acetamido-2-deoxy-~-glucose (see Table VI), and an L-threonine residue is glycosylated with 2-acetamido-2-deoxy-~-galactose in the hinge region of about 35% of the total number of heavy chains.20These two types of linkage occur also in yA (Bra) myeloma protein172and in human, chorionic gonadot r ~ p i n , ~but, " in these examples, L-serine provides the site of attachment of 2-acetamido-2-deoxy-~-galactose.A "heavy-chain disease" protein (Cra),215l a ~ t o t r a n s f e r r i n ,yA ~~~ l a c t ~ g l o b u l i n ,and ~ ~ ~an oxaorta glycoprotein216are yet further compounds in which these two types of linkage are present. alpha-Amylase from Aspergillus oryzae (EC 3.1.1.1)was found to have an amino acid sequence around a carbohydrate moiety

-Ser-Glu-Asp-Gly-(Ala,Thr)-, and to the L-serine residue was attached an oligosaccharide that contained 8 molecules of D-mannose and one of D-XylOSe per molecule.217 Although the whole molecule contained about two molecules of hexosamine, the amino sugar(s) was (were) not present in the glycopeptides isolated. Later studies with other preparations of alphaamylase revealed the presence of another linkage, involving 2acetamido-2-deoxy-~-glucose and ~ - a s p a r a g i n e . 'Interpretation ~~ of these data is complicated by the realization that the carbohydrate composition of preparations of enzyme probably varies from one batch to another.217u Collagen and basement membrane may contain several types of carbohydrate-peptide linkage. They clearly contain linkages in which 5-hydroxy-~-lysine is involved, as already discussed (see p. 436). From the preparations studied were also isolated glycopeplinking tides in which an asparagine-2-acetamido-2-deoxy-~-glucose moiety was p r e ~ e n t . ' ~ ' * ~ ~ (214) 0.P. Bahl,]. B i d . Chem., 244,575 (1969). (215) J. R. Clamp, G. Dawson, and E. C. Franklin, Biochem.]., 110,385 (1968). (216) B. Radhakrishnamurthy and G. S. Berenson,]. Biol. Chem., 241,2106 (1966). (217) A. Tsugita and S. Akabori,J.Biochem. (Tokyo),46,695 (1959). (217a)J. F. McKelvy and Y. C. Lee, Arch. Biochem. Biophys., 132,W (1969).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

443

Whether, in these examples, both types of linkage occur in one polypeptide chain is at present undecided, but the finding, in normal urine, of glycopeptides that, on acid hydrolysis, give rise to 4-hydroxyL-proline and other amino acids, as well as to D-galactose, 2-amino-2deoxy-D-glucose, 2-amino-2-deoxy-~-galactose, and smaller proportions of L-fucose,218might suggest that collagen contains types of carbohydrate-peptide linkage other than that involving 5-hydroxy-Llysine, assuming that these glycopeptides are catabolites of collagen or basement membranes, or both.

IV. HETEROGENEITY IN GLYCOPROTEINS Much of the early work was performed on glycopeptides that, as is now realized, were heterogeneous with regard to their carbohydrate moieties. Such heterogeneity is not surprising in view of the mechanism of biosynthesis of the prosthetic groups, and there is the further complication that our knowledge of the catabolism of these substances is still scanty. Heterogeneity of this kind is probably widespread, and it is likely that the separation of glycopeptides containing carbohydrate moieties that are structurally very closely related may require highly refined techniques. Differences in the structure of glycoproteins can manifest themselves in a number of ways. Perhaps the first point to be emphasized is that the nature of the polypeptide chain to which oligosaccharide moieties become attached is not always the same. This is a situation that we have already discussed in connection with certain of the glycosaminoglycans (see p. 440). Differences between proteins occur with respect to the attachment of carbohydrate; a given amino acid may be present either unsubstituted or glycosylated. Ribonucleases A and B appear to differ solely by virtue of the latter having an oligosaccharide moiety attached through L-asparagine residue number 34 from the N-terminal end.lZ9 Only about 35% of the heavy chains of rabbit yG immunoglobulin occur as a form in which an L-threonine residue is substituted by a glycosy1 group,2oSuch examples are not likely to be the only ones. Analogous situations are known wherein a potential amino acid acceptor for entities other than carbohydrate in a protein does not appear to be present, in at least some molecules, in a modified form. Thus, 5hydroxy-L-lysine and L-lysine residues occupy equivalent positions in the amino acid sequence219in the a, chains of collagen, as do L(218)A.Bourillon and J. L. Vernay, Biochim. Biophys. Acta, 117,319(1966). (219)E.J. Miller, J. M. Lane, and K. A. Piez, Biochemistry, 8,30(1969).

444

R. D. MARSHALL AND A. NEUBERGER

proline and hydroxy-L-proline.220The same applies to L-lysine and 6-N-acetyl-~-lysinein calf-thymus histoneZz1and L-serine and 0phosphono-L-serine in egg albumin and other proteins.222The finding that collagens can be glycosylated, by uridine 5’-(D-galaCtOpyranOSyl pyrophosphate) and an enzyme isolated from rat kidney, may well be an indication that a similar situation exists with regard to the glycosylation of the 5-hydroxy-~-lysineresidues of collagen.223The significance of these findings, either in chemical or biological terms, is not as yet understood. Another type of variation is, perhaps, best illustrated by the results of studies with various types of collagen.224The carbohydrate moiety occurs, in part, as 2-O-a-D-glUCOpyranOSyl-o-~-D-galaCtOpyranosyl and, in part, as O-P-D-galaCtOpyranOSyl groups, that is, both with and without addition of the terminal D-glucopyranosyl groups. Similar variations in structure occur in corneal g l y c o p r o t e i n ~ . ~ ~ ~ It is not unreasonable to suppose that, at least in part, these two (closely related) types of carbohydrate moiety are attached to 5hydroxy-L-lysine residues that occur in the same relative position in the peptide chain. In other glycoproteins, a given carbohydrate moiety may, in some molecules, be present in molecules in which one or more terminal sialic acid residues are present, and, in others, in which no sialic acid occurs at all. As examples of this situation , ~ ~ ~46, ~~ and a may be mentioned a mouse Bence-Jones p r ~ t e i n MOPC yA myeloma ~ r 0 t e i n . lIt~ ~is not unlikely that ox pancreatic ribonucleasess B, C, and D are further examples. Variations of this type in the structure of glycoproteins have been described as peripheral heterogeneity.172 Analogous variations in the structure of various oligosaccharides in other glycoproteins almost certainly occur; these include f e t ~ i n transferrin,226 ,~~~ o r o s o r n ~ c o i d ,/3,-glycoprotein,22s ~~~ and zinc az-glycoprotein.229The extents of their heterogeneity, as revealed by electrophoresis in poly(acry1amide) gels, were in all (220) P. Bomstein, J. Biol. Chem., 242,2572 (1967). (221) R. J. DeLange, D. M . Famborough, E. L. Smith, and J. Bonner, J. Biol. Chem., 244,319 (1969). (222) G. E. Perlmann, “Phosphorus Metabolism,” W. D. McElroy and B. Glass, eds., Johns Hopkins Press, Baltimore, Md., 1952, Vol. 2, p. 167. (223) R. G. Spiro and M. J. Spiro, Fed. Proc., 27,345 (1968). (224) R. G. Spiro,]. Bid. Chem.,244,602 (1969). (225) Y. Oshiro and E. H. Eylar, Arch. Biochem. Biophys., 127,476 (1968). (226) S.-H.Chen and H. E. Eldon, Genetics, 56,425 (1967). (227) K. Schmid, J. P. Binette, S. Kamiyama, V. Pfister, and S. Takahashi, Biochemistry, 1,959 (1962). (228) H. E. Schultze, K. Heide, and H. Haupt, Naturwissenschu~fer1,48,719(1961). (229) K. Schmid and S. Takahashi,Nature, 203,407 (1962).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

445

instances markedly decreased after removal, by treatment with neuraminidase, of their sialic acid residues. A similar structural variation may be present in human-serum alkaline p h o s p h a t a ~ e . ~ ~ ~ Related examples in which oligosaccharides are present in glycoproteins, either with or without terminal glycosyl groups (other than the charged sialic acid residues), are known. Differences of this kind occur in the blood-group substances (see p. 452). The presence of oligosaccharide moieties, attached at a specific position on the polypeptide chain of a yA myeloma protein, has been described; some contained nonreducing, terminal 2-acetamido-2-deoxy-~-glucoseand L-fucose residues, and some did There are conflicting reports concerning the structure of the oligosaccharide prosthetic group of ox residues ribonuclease B. None of the 2-acetamido-2-deoxy-~-g~ucose in a glycopeptide isolated from the protein could be oxidized with periodate,130 although other workersz3’ reported that 2-acetamido-2deoxy-D-glucose can be removed from a nonreducing, terminal position of other preparations of ribonuclease B by use of a highly (2-acetamido-2-deoxypurified 2-acetamido-2-deoxy-~-~-glucosidase @-D-ghcoside2-acetamido-2-deoxy-~-~-glucohydrolase, E.C. 3.2.1.30) from Phaseolus vulgaris. It seems possible that the preparations of ribonuclease B differed with respect to the absence or presence of a nonreducing, terminal 2-acetamido-2-deoxy-~-glucoseresidue. In addition to heterogeneity of this type, which is primarily concerned with the nonreducing, terminal sugar residue(s), there may be a more complicated variation in the structure of the carbohydrate moieties of glycoproteins. This is best illustrated by considering the single oligosaccharide unit that occurs in hen’s-egg albumin. Although a number of physical tests failed to reveal any heterogeneity in the carbohydrate moiety of glycopeptide isolated from this protein,z3zthe glycopeptide could be fractionated by chromatography on DowexdO X2 in buffers of low ionic strength. The fractions thus obtained had various contents of D-mannOSe and 2-amino-2-deoxy-~glucose.z33-z3sIt was further shown that the differences found were (230) J. C. Robinson and J. E. Pierce, Nature, 204,472 (1964). (231) 0. P. Bahl and K. M. L. Agrawal,]. BioE. Chem.,243,98 (1969). (232) A. Neuberger and R. D. Marshall, in Ref. 6, p. 306. (233) L. W. Cunningham, J. D. Ford, and J. M. Rainey, Biochim. Biophys. Acta, 101, 233 (1965). (234) V. D. Bhoyroo and R. D. Marshall, Biochem.].,9 7 , l l p (1965). (235) L. W. Cunningham, in “Biochemistry of Glycoproteins and Related Substances,” E. Rossi and E. Stoll, eds., S. Karger, New York, 1968, Part 11, p. 141. (236) C. C. Huang and R. Montgomery, Biochem. Biophys. Res. Commun., 37, 94 (1969).

446

R. D. MARSHALL AND A. NEUBERGER

unlikely to be due to genetic variation.z35Treatment of similar material with a molecular sieve, Sephadex G-25, also gave rise to a number of glycopeptide fractions containing differing proportions of sugars.237 A number of other proteins are now known to exhibit heterogeneity with respect to their carbohydrate content; these include pig panyG immunoglobulin, creatic ribonuclea~e,~ rabbit19 and human109*238 and the c e r u l o p l a ~ r n i n ,2-acetamido-2-deoxy-j?-~-glucosidase,~~~ ~~~ blood-group substances from ovarian ~ y s t s . ' ~ ~ * ~ ~ ' In summary, a polypeptide chain may have one or more oligosaccharide units attached to it, although, in some, only a portion of an amino acid residue that is a potential acceptor-site for a sugar actually becomes glycosylated. The size of the prosthetic group attached at different points along the polypeptide chain may differ radically, as in calf thyroglob~lin,2~~ which is reported to contain nine carbohydrate units consisting of five residues of D-mannose and one of 2-amino-2deoxy-D-glucose, and 14 very much larger units, per molecule of protein (1 mole = 670,000 g). Structural features of this type have also been shown to occur in ox-aorta glycoprotein218and o v o m ~ c o i d . ~ ~ ~ This type of structural feature was described as central heterogeneity,172a term that might better be reserved for such situations as that already described for egg albumin, in which structural variations occur within a carbohydrate moiety attached at a specific position of the polypeptide chain. It is not unreasonable to suppose that the last-mentioned form of variation in the structure of glycoproteins may be widespread, and, as already discussed, more-sensitive fractionation procedures must probably be applied to these materials. It was found that glycoprotein prepared from sheep submaxillarygland182,244 contains, as prosthetic groups, almost exclusively a large number of disaccharide units, namely, 2-acetamido-2-deoxy-6-0sia~y~-a-D-ga~actosy1 groups. This result might suggest that substitution by sialic acid residues is, in all cases, complete in z)iz)o,but it is possible that the properties of those molecules in which this reaction is incomplete would be sufficiently different as to lead to their removal during purification procedures. Other variations in structure may also (237) G. A. Levvy, J. Conchie, and A. J. Hay, Biochim. Biophys. Acta, 130,150 (1966). (238) J. L. Fahey and A. P. Horbett, J. Biol. Chem., 234,2645 (1959). (239) G. A. Jamieson,J. Biol. Chem.,240,2019(1965). (240) D. Robinson and J. L. Stirling, Biochem.J.,107,321 (1968). (241) W. M. Watkins, Science, 152,172 (1966). (242) R. G . Spiro,J. Biol. Chern.,240,1603 (1965). (243) R. Montgomery and Y.-C. Wu,J. Biol. Chem., 238,3547 (1963). (244) V. L. N. Murty and M. I. Horowitz, Carbohyd. Res., 6,266 (1968).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

441

occur, in view of the presence of other types of sugars, even in those molecules in which the disaccharide units are all complete, and may be responsible, at least in part, for the heterogeneity found by immunological methods in similar protein preparations from cattle.245 The analogous protein from pig has a more complicated structure, the oligosaccharide groups varying from a single 2-acetamido-2-deoxy-~galactose residue to a pentasaccharide. Products, obtained by reduction in alkali, that are believed to correspond with the carbohydrate moieties existing in the protein will be described (see p. 458).

V. THESIZE OF

THE

CARBOHYDRATE MOIETIES IN GLYCOPROTEINS

A number of measurements have been made of the molecular weights of glycopeptides isolated from glycoproteins. Often, the determinations may have been performed on material that was not homogeneous. To illustrate the sort of problems that may arise, the studies of Clamp and Putnam1O9may be considered. The sedimenting patterns obtained with glycopeptides isolated from human yG globulin appeared to indicate that the substance was, from the point of view of size, fairly homogeneous. However, application of the mathematical methods of Trautman and C r a m p t ~ to n ~the ~ ~data led to the deduction that t w o species, having molecular weights of 1170 and 3150, respectively, were present. On the other hand, the related procedure of Klainer and K e g e l e ~appeared ~~~ to indicate that a species of only one size was present and that this had a molecular weight of 2,200. More-varied analytical procedures for sedimentation data obtained with other glycopeptides might indicate wider heterogeneity with regard to size than has thus far been reported. Despite all the problems of heterogeneity with respect to the carbohydrate moieties mentioned, certain statements about structural features can be made. At a given position in the polypeptide chain, the prosthetic group may differ widely from one such group to another. It may consist of a structure as simple as a single sugar residue, as in pig submaxillary-gland glycoprotein, or a disaccharide, as in submaxillary-gland glycoproteins or collagen, or the moiety may consist of -12 to 15 sugar residues. Some of the relevant data are summarized in Table IX, where the methods often employed for de(245)M. I. Horowitz, L. Martinez, and V. L. N. Murty, Biochirn. Biophys. Acta, 83, 305 ( 1964). (246) R. Trautman and C. F. Crampton,j. Amer. Chem. SOC., 81,4036 (1959). (247) S . M. Klainer and G. Kegeles, j . Phys. Chern., 59,952 (1955).

TABLEIX Molecular Weights of Glycopeptides Isolated from Various Glycoproteins Glycomotein" Fetuin (3) Orosomucoid (11)*=

Guinea-pig a,-acid glycoprotein

Human yG globulin

yA Myeloma protein (4) & Globulin Ceruloplasmin (10) Sheep ICSH Human chorionic gonadotropin (2) Calf thyroglobulin (23)

Mol.wt. 4300 4500 2800 2540 2840 2600 1800 4120 4260 1805 2100 3070 2700 2450 3Ooob 22w 28W 3305 1950 3160 3300 4300 1250 4100

Method employed sediment. equil. sediment. rate and viscosity sediment. rate vapor phase osmometry trinitrobenzenesulfony1 subst. sediment. equil. sediment. equil.

Relevant data uW

(determined) 0.664 ml/g

[qlup (determined) 5.83 ml/g

(assumed) 0.664 ml/g u (calculated)0.614 ml/g -

u (assumed) u (assumed) 0.644 mllg

)sediment. equil. -

sediment. equil. gel filtration '(Sephadex G 25) sediment. equil. sediment. rate Archibald method freezing-pt. osmometry sediment. equil. Archibald method dinitrophenyl substitution gel filtration (Sephadex G 50) sediment. rate

u (assumed) 0.664 ml/g

-

u (assumed) 0.7 ml/g

Total amino acid Referpresent (moles) ences contains 77% of carbohydrate

248

2.0 2.1 2.1 1.07 1.04 6.8 6.5 n.d. 1.4 3.7 1.4 1.4 5 3.16 1

249 250 251 252

)2% 21 109 255

-

u (assumed) 0.664 ml/g

v (determined) 0.78 ml/g

-

u (calculated)

0.634 ml/g

n.d. 3.22 9 9.7 13.1 2.2

253 239 257 214 242

“Egg white” (mainly ovomucoid) Ovomucoid (3)

Egg albumin (1)

Urinary T-H protein Ox aorta glycoprotein Deoxycholate soluble fraction of rat-liver microsomes Earthworm-cuticle collagen Soybean hemagglutinin (1) Chondroitin 4sulfate’* (ox nasal-septa)

Chondroitin 4-sulfate (pig laryngeal cartilage)

-2000

diffusion rate

258 -

2500 2840 2580 3000 2800 1880 1580 1570 1500 1560 -4000 2300 1500 2950 2360

di- and tri-Dgalactosides 4600 1500 15300 18300 21700 29800 41300 150008*h.‘

sediment. equil.

v

(assumed) 0.664 ml/g

gel filtration (Sephadex G 25) phenylthiohydantoin subst. dinitrophenyl substitution gel filtration (Sephadex G 25) electrothermal micromethod benzyloxycarbonyl substitution isopiestic method thermoelectric osmometry analysis and rate of dialysis

1.09 1.09 2 1 1 1 1

analytical data analytical data gel filtration (Biogel P-2) -

u (calculated)0.615 ml/g

sedimentation rate and sedimentation equil.

254 243 254 259 260 261 262 263

n.d. 6.5 5.7 7.1

28

nonee

265

264

v, 4

c: 5e n

2 P

$ U

g R

4

9m

8 E ~

1

0

analytical data, based on Dxylose/2-amino-2-deoxy-Dgalactose ratio and on the (continued)

$ a

TABLEI X (continued) ~~~

Glycoprotein" Chondroitin 4-sulfate (pig costalcartilage) Dermatan sulfate (pig skin)

Mol.wt.

Method employed

1430OgAJ

pelimination reaction sedimentation equilib.

27WhJ

sedimentation equilib.

Relevant data (assumed) 0.57 ml/gz69

~

~

Total amino acid Referpresent (moles) ences

0

205 268

-

u (determined) 0.57 ml/g

6% of amino acids 269

"The number of carbohydrate residues per molecule of glycoprotein is indicated in parentheses. bThe material used in this study was heterogeneous with respect to size, and only an order of magnitude is indicated. "See the comments on p. 447. dGel-filtration on Sephadex G-25 and G-50 showed that glycopeptides from a yA myeloma protein had molecular weights of 1,900 and 2,600, respectively. Glycopeptides from a yM globulin had= molecular weights of 1500 and 1730. 'The materials isolated were prepared by treating the protein with aIkaline (0.05M sodium hydroxide) borohydride (0.15 M) for 24 hours at 30", but the oligosaccharides are likely to exist in the protein in units of about the size given.28sThe saccharides are composed of o l - ~ - ( l +2)-linked D-galactose residueszGa'The values are for various fractions isolated by Scott's procedure.46 The unfractionated material hadzwM , 25,300 and M . 20,800. Weightaverage molecular weights of 21,700 and 21,100 were found for materials prepared from digests with (0.5 M sodium hydroxide for 19 hours at 4") and papain, respectively.267'As used here, the term chondroitin sulfate refers to the carbohydrate chain only (not to its complex with protein). "The carbohydrate chains are heterogeneous with respect to molecular size. 'A number-average value. 'A weightaverage value.

?

z

m

C

STRUCTURE AND METABOLISM OF CLYCOPROTEINS

451

termining the molecular weights are also described. At present, it is difficult to set a precise upper limit to the size of a carbohydrate unit, but, in general, the molecular weight would appear not to exceed -3,000 in those moieties in which there is not, so far as we know, a repeating pattern. However, in soybean hemagglutinin, the value would appear3s to be of the order of -4,500,and the single carbohydrate moiety in this glycoprotein consists of approximately 25 residues of a mannose and 5 of 2-amino-2-deoxy-~-g~ucose. The carbohydrate chains of the chondroitin sulfate type would seem to have rather larger molecular weights, up to values of the order of 40,000 daltons. Here again, there is considerable heterogeneity with respect to size (see Table IX). The size of the carbohydrate chains of keratan sulfate, having repeating units of (1+3)-0fi-D-galactopyranosyl-( 1+ 4)-2-acetamido-2-deoxy-fi-~-glucopyranosyl 6-sulfate (see p. 459),is unknown. Ox-corneal keratan sulfate was separatedZ7Ointo a number of fractions having molecular weights of 4,100 (F = 0.55 ml/g), 8,800(0.53ml/g), 10,000 (0.51ml/g), 12,500 (248) R. G. Spiro, J. Biol. Chem.,237,646 (1962). (249) N. Ui and 0.Tarutami,J. Biochem. (Tokyo), 51,370 (1962). (250) P. V. Wagh, I. Bomstein, and R. J. Winzler,]. Biol. Chem.,244,658 (1969). (251) S. Kamiyama and K. Schmid, Biochim. Biophys. Acta, 58,80 (1962). (252) E. H. Eylar, Biochem. Biophys. Aes. Commun., 8,195 (1962). (253) W. E. Marshall and J.Porath,J. Biol.Chem.,240,209 (1965). (254) W. L. Cunningham and J. L. Simkin, Biochem. J., 99,434 (1966). (255) G. Dawson and J. R. Clamp, Biochem. Biophys. Res. Commun., 26,349 (1967). (256) T. Bhatti and J . R. Clamp, Biochim. Biophys. Acta, 170,206 (1968). (257) H. Papkoff, Biochim. Biophys. Acta, 78,384 (1963). (258) P. A. Levene and A. Rothen,J. Biol.Chem., 84,63 (1929). (259) E. D. Kaverzneva and V. P. Bogdanov, Biokhimiya, 27,273 (1962). (260) A. P. Fletcher, R. D. Marshall, and A. Neuberger, Biochim. Biophys. Acta, 71, 505 (1963). (261) P. G. Johansen, R. D. Marshall, and A. Neuberger, Biochem.J.,78,518 (1961). ~ (262) Y. C. Lee, Y.-C. Wu, and R. Montgomery, Blochem.]., 9 1 , 9 (1964). (263) A. Gottschalk, in “Chemistry and Biology of Mucopolysaccharides,” G. E. W. Wolstenholme and M. O’Connor, eds., J. & A. Churchill, Ltd., London, 1958, p. 287. (264) Y.-T. Li, S.-C. Li, and M. R.Shetlar,]. Biol. Chem.,243,656 (1968). (265) Y. C. Lee and D. LangJ. Biol.Chem., 243,677 (1968). (265a)L.Muir and Y. C. LeeJ. Biol. Chem., 244,2343 (1969). (266) A. Wasteson, Biochim. Biophys. Acta, 177, 152 (1969). (267) M. Luscombe and C. F. Phelps, Biochem. J . , 103,103 (1967). (268) E. Marler and E. A. Davidson, Proc. Nut. Acad. Sci. U . S., 54,648 (1965). (269) C. Tanford, E. Marler, E. Jury, and E. A. Davidson, /. Biol. Chem., 239, 3034 (1964). (270) T. C. Laurent and A. Anseth, Exp. Eye Res., 1,99 (1961).

452

R. D. MARSHALL AND A. NEUBERGER

(0.49 ml/g), 13,700 (0.52 ml/g), 17,300 (0.49 ml/g) and 19,100 daltons (0.47 ml/g). Some or all of these fractions may have contained more than one carbohydrate chain linked together by short lengths of peptide, but it is clear that the smallest chain of keratan sulfate has a molecular weight that does not exceed -4,000. Keratan sulfates from other sources have number-average molecular weights of 11,500 (from bull-shark cartilage), 10,000 (human cartilage), 8,900 (calf cornea), and 9,700 (chicken cornea).211 The factors responsible for limiting the size of the carbohydrate moieties are as yet unknown. Speculations may be made as to whether, in the prosthetic group itself, certain structural features, such as terminal fucosyl, sialyl, a-D-galactosyl or 2-acetamido-2-deoxy-cu-~-galactosyl residues may, in appropriate cases, play a role here. However, kinetic factors, and the distribution and specificities of the various activated sugar transferases during passage of the nascent protein through the cysternae of the endoplasmic reticulum, may be of prime importance.

VI. FEATURES OF THE STRUCTURE OF THE CARBOHYDRATE MOIETIES OF SOME GLYCOPROTEINS Many methods are available for determination of the structure of carbohydrate moieties. One method involves periodate oxidation, succeeded by reduction of the hemialdal groups formed. The resulting polyalcohols are hydrolyzed by acid, and, from the nature of the products, it may be possible to deduce a structure for the carbohydrate under considerati~n.~'~ However, problems of interpretation are likely to arise if the prosthetic group is markedly heterogeneous. It is possible that, so far, the most valuable data concerned with the structure of the carbohydrate moieties (including the anomeric configuration of glycosyl groups) of many glycoproteins have been obtained by techniques involving partial hydrolysis.

1. Blood-group Substances from Ovarian Cysts Some of the results determined in this way with blood-group substances are described in Table X. A number of other oligosaccharides

(271) M. B. Mathews and J . A. Cifonelli,]. Biol.Clzem., 240,4140 (1965). (272) M. Abdel-Akher, J. K. Hamilton, R. Montgomery, and F. Smith, J . Amer. Chem. SOC.,74,4970 (1952).

T A ~ LX E Oligosaccharides Isolated from Partial Hydrolyzates of Certain Blood-group Substances Oligosaccharide isolated 1" P-D-Gd-( 1

-+

Source ovarian cyst Leo substance

3)-D-GNAc 4

t

2

p-D-Gd-( 1

3

~-L-Fuc-(1

4

-+

-+

Hydrolysis conditions triethylamine (2.5%) in 50% aqueous methanol; 60"; 18hr

References 273

2 2 C

n 4

C

1 a-L-Fuc 4)-D-GNAc-

2

m triethylamine (2.5%) in 50% aqueous methanol; 60"; 18hr triethylamine (2.5%) in 50% ovarian cyst H substance aqueous methanol; 60"; 18 hr ovarian cyst A and B substances triethylamine (2.5%) in 50% aqueous methanol; 60"; 18hr 0.2 M NaOH; 0.25 M NaBH,; ovarian cyst A,B and H room temp.; 7 days substances triethylamine (2.5%) in 50% ovarian cyst H substance aqueous methanol; 60"; 18hr ovarian cyst A and B substances triethylamine (2.5%) in 50% aqueous methanol; 60"; 18hr triethylamine (2.5%) in 50% ovarian cyst A substance aqueous methanol; 60"; 18hr ovarian cyst Lea substance

2)-D-Gal

~ - L - F u c - -+ ( ~ 2)-~-Gal-(1 4)-D-GNAc -+

1-+ 4)-D-CNAc 5b a-D-CalNAc-(1 -+ 3)-p-~-Gd-(

2

T

a-L-FUC 6C.d a-D-Gd-( 1-+ 3)-P-D-Gd-(1 4)-D-GNAc 2 -+

t

ovarian cyst B substance

triethylamine (2.5%) in 50% aqueous methanol; 60";18 hr

273 274 275 276 274 275 275

275

1

~-L-Fuc (continued)

TABLEX (continued) Source

Oligosaccharide isolated 7

1 + 3)-D-Gd p-~-Gal-(l-+~)-P-D-GNAc-( 2 4

t

8

ovarian cyst Lebsubstance

T

1 a-L-Fuc a-L-Fuc P-~-Gal-(l-+ 3)+3-~-GNAc-(l + 6)-~-GalacitolNAc ovarian cyst Lea substance 3

Hydrolysis conditions water-soluble, alkaline resin, pH 8.5; 100"; 20 min

+

References 277

0.2 M NaOD 0.25 M NaBD, in D,O; room temp.; 7 days

179

0.2 M NaOD +0.25 M NaBD, in D,O; room temp.; 7 days

179

t

1 p-~-Gal 9

p-~-Gal-(l+ ~)-P-D-GNAc 1

ovarian cyst Lea substance

.1

6 Galactitol 3

?

t

C

z

m

1 10

p-~-Gal-(l-+ 3)-P-D-GNAc D-GdacitolNAc

11 p-~-Gal-(1 --* 4)-P-D-GNAc-(1 + 3)-/3-D-Gd3

ovarian cyst Lea substance ovarian cyst Lea substance

0.2 M NaOD -k 0.25 M NaBD, in D20;room temp.; 7 days water-soluble, alkaline resin; pR 8.6; loo"

278

t

1 a-L-Fuc 12

p-~-Gal-(1-+ ~)-P-D-GNAC-( 1+ 6)-p-D-Gal

ovarian cyst Lea substance

water-soluble, alkaline resin; pH 8.6; 100"

278

15

a-~-GalNAc-(l+ 3)-P-~-Gal-(1+ 3)-&GNAc a-D-GalNAc-(1+ 3)-P-~-Gal-(1 + 4)-D-GNAc a-D-Gal-(1 + 3)-/3-D-Gal-(1 + 3)-D-GNAc

ovarian cyst A substance ovarian cyst A substance ovarian cyst B substance

16

a-D-Gal-(1 + 3)-p-D-Gal-(1+ ~)-D-GNAc

ovarian cyst B substance

17

p-D-Gal-(l

13 14

18

19 20

+ 3)-@-D-CNAc-(l+ 3)-D-Gd

ovarian cyst A,B,H, and Lea substances p-D-Gd-( 1 + 4)-P-D-GNAc-(1 + 3)-D-Gal ovarian cyst A,B,H,Lea substances 1 -+ 3)-p-D-Gal-(1 + 3)-~-GalNAc ovarian cyst A,B,H,Lea ~-D-G-NAc-( substances a - ~ - G a l N A c1(-+ 3)-P-D-Gal-(1 3)-P-~-GaladitolNAcpig submaxillary gland 2 6 glycoprotein -+

t

21

+

t

2 CX-L-FUC ff -D-sidyl 1+ 2)-P-D-Gal-(1-+4)-P-D-GalactitolNAc CY-L-FUC-( 1

0.1 M HCl; 100"; 30 min 279,280 0.1 M HCI; 100"; 30min 200 0.04 N poly(styrenesu1fonic acid); 281 87" 0.04N poly(styrenesu1fonic acid); 281 87" 0.04 N poly(styrenesu1fonic acid); 282 90";4 hr 0.02 N (polystyrenesulfonicacid); 282 90"; 4 hr 0.02 N (polystyrenesulfonicacid); 282 90"; 4 hr 177 0.05 M KOH 1.0 M NaBH,; 45"; 15h r

v)

2

5

2 n z

* U

5

*m 4

? pig submaxillary gland glycoprotein

0.1 M NaOH +0.3 M NaBH,; 28"; 120 hr

283

"A related trisaccharide probably also occurs at the nonreducing terminal of some of the oligosaccharide side-chains of Lea substance, in which the positions of the two terminal sugar residues are interchanged; this structure is almost devoid of activity as a determinant bOligosaccharides of a clearly g r ~ u p . ' ~This g compound may be related to the tetramer isolated by Marr and coworkers (see item 11).278 related type have been isolated from hydrolyzates of A substance (0.2 M NaOH at room temperature for 7 days) in the presence of 0.25 M NaBH,: they were blocked at their reducing end by a hex-3-ene-1,2,5,6-tetrol. Both mono-L-fucosyl and di-L-fucosyl oligosaccharides were described, the latter having a second L-fucose residue attached at the 2-acetamido-2-deoxy-D-glucose residue, but not276at 04. COligosaccharides analogous to those described in footnote b were isolated from B substance.276dOligosaccharides analogous to those described in footnote b were isolated from H substance,*" but there was no nonreducing, terminal a-D-galactosyl group.

-

F 30

5fl z

v)

456

R. D. MARSHALL AND A. NEUBERGER

that could be related biogenetically to those described therein have also been isolated. Thus, such structures as P-D-GNAc-(1 --* 6)-~-Gal 3

t

P-D-GNAc-~ or even simpler related ones, had been isolated some years earlier by Y o ~ i z a w a ~from * ~ hydrazinolyzates of blood-group substance A obtained from pig gastric mucus; they may have arisen from a region similar to that from which the oligosaccharide shown as item 9 in Table X was derived. Alkaline hydrolysis is particularly valuable in obtaining oligosaccharides, because the compounds isolated must have arisen from preliminary cleavage of the carbohydrate-peptide bond, followed by breakdown, to a greater or lesser extent, from the reducing, terminal end thereby formed. The oligosaccharides isolated must, therefore, have occurred at nonreducing, terminal positions in the carbohydrate moieties of the glycoprotein or glycopeptide. The oligosaccharide isolated may, in some instances, have a modified, reducing terminus, because of the action of the alkali. Some of the work with blood-group substances is of particular interest in this regard. First, oligosaccharide 1 (see Table X) represents that structure shown by serological tests285to be largely responsible for Lea antigenic activity. Both this structure and the disaccharide N-acetyl-

(273) V. T. Rege, T. J. Painter, W. M. Watkins, and W. T. J. Morgan, Nature, 204, 740 (1964). (274) V. T. Rege, T. J. Painter, W. M. Watkins, and W. T. J. Morgan, Nature, 203, 360 (1964). (275) T. J. Painter, W. M. Watkins, and W. T. J. Morgan, Nature, 206,594 (1965). (276) K. 0.Lloyd, E. A. Kabat, E. J. Layug, and F. Gruezo, Biochemistry, 5,1489 (1966). (277) A. M. S. Marr, A. S . R. Donald, W. M. Watkins, and W. T. J. Morgan, Nature, 215, 1345 (1967). (278) A. M. S. Marr, A. S. R. Donald, and W. T. J. Morgan, Biochem.]., 110,789 (1968). (279) G. SchifFman, E. A. Kabat, and S. Leskowitz, J. Amer. Chem. Soc., 84.73 (1962). (280) I. A. F. L. Cheese and W. T. J. Morgan, Nature, 191,149 (1961). (281) T. J. Painter, W. T. J. Morgan, and W. M. Watkins, Nature, 199,282 (1963). (282) V. P. Rege, T. J. Painter,-W. M. Watkins, and W. T. J. Morgan, Nature, 200,532 11963). (283) k. L. katzman and E. H.Eylar, Biochem. Biophys. Res. Commun., 23,769 (1966). (284) Z. YosizawaJ. Blochem. (Tokyo), 51,145 (1962). (285) W. T. Watkins and W. T. J. Morgan, Nature, 180,1038 (1967).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

457

lactosamine (item 2) occur in nonreducing, terminal positions in the various carbohydrate moieties of Lea substance. Clearly, the carbohydrate moieties are independently formed, and neither of them is likely to be the precursor of the other. The second of these structures is believed to be the antigenic determinant responsible for interaction of the macromolecule with antibody produced in the horse against the capsular polysaccharide of Type XIV pneumococcus.285,286 Probably, the N-acetyl-lactosamine is usually linked to D-galactose, and this sugar may have different degrees of substitution and different positions of the substituents. Substituents on 0-4of D-galactose may occur, but direct evidence for such substitution is 1 a ~ k i n g .Other l~~ structures are also situated at the nonreducing, terminal ends of some of the carbohydrate side-chains (see, for example, items 8-12, footnote a of Table X). One of these (item 8) has been suggested as being derived from the region through which linkage to the polypeptide chain occurs, although decisive evidence is not available.179 It has been found that some of the carbohydrate moieties of A and B substances do not contain the 2-acetamido-2-deoxy-a-~-galactose or a-D-galactose residues, respectively, that are essential features of these antigenically determinant sites (see items 3 and 4). Furthermore, it is clear that, unless a very high degree of branching occurs, the antigenic determinants for A and B activity (items 5 and 6) cannot be present on the same carbohydrate moieties; both activities are probably present in the same molecule. The Leb antigenic determinant (item 7) has structural features in common with both H (items 3 and 4)and Lea determinants. The juxtaposition of the two L-fucose residues in the Leb-active carbohydrate moieties sufficiently affects the general stereochemistry of the group to make it a completely new antigenic determinant, quite distinct from those for either H or Lea substance. The Leb characteristic of blood-group substance seems always to occur with one or more other such activities as A, B, and H,2x7so that it is likely that there is considerable variation in the structure of various oligosaccharides in the same molecule. Other oligosaccharides obtained from ovarian-cyst blood-group substances indicate that these macromolecules have many features in common (items 17-19) between the various types; but, as they have been isolated after partial hydrolysis with acid, it is, in general, not possible, from this information alone, to assign a position to them within the carbohydrate moieties. Those items represented by 13, (286) P. Z. Allen and E. A. Kabat,J. Imtnunol., 82,340(1959). (287) W. T. Watkins, Science, 152, 172 (1966).

458

R. D . MARSHALL A N D A. NEUBERCER

14 (from A substance) and those by 15,16 (from B substance) are likely to arise from reducing, terminal regions of carbohydrate chains of A and B substances, respectively: numbers 13 and 15 are of the structural type designated type I, in which the 2-acetamido-2-deoxyD-glucose, as the reducing terminal sugar, is linked by 0-3, and numbers 14 and 16 are of type 11, in which 0 - 4 of this corresponding sugar is substituted.282Structural features of this type may form in the macromolecular part of such branched moieties as that represented by item 9. Also, it is not yet known whether such structures as those represented by 13-16 exist as such, as part of the macromolecule (that is, without any terminal L-fucose residues). Oligosaccharides of the type represented by items 5 and 6 would be expected to lose L-fucose residues relatively easily under acidic conditions.288 Serological and enzymic techniques involving various glycosidases have been used for determining certain structural features of the secreted blood-group substances. Reviews of these data are available,241*285 and so these aspects will not be discussed. This very brief survey of some of the extensive and elegant studies made over many years, mainly by Morgan, Watkins, Kabat, and Yosizawa and their colleagues, has emphasized certain features of the chemistry of the secreted blood-group substances. The carbohydrate moieties in a given substance may differ quite markedly from one another, both in the nature of the nonreducing terminal residues and in the structures of the “internal” parts of these prosthetic groups. The sizes of the moieties probably differ, possibly quite extensively. If an attempt is made to construct a model in which are incorporated all of the various oligosaccharides listed in Table X, the resultant prosthetic groups would have to consist of more than 20 sugar residues, a somewhat unlikely possibility. It is more reasonable to suppose that these glycoproteins have a number of types of somewhat smaller carbohydrate moieties, in which certain variations of structure occur. It would be desirable to examine homogeneous preparations of glycopeptides from these sources, but the resistance of the glycoproteins to proteolytic digestion”’ makes this a difficult task at present. 2. Pig Submaxillary-gland Glycoprotein

A protein obtained from the submaxillary gland of pigs and having blood-group A activity was found to contain, as part of its carbo(288) A. Neubergerand R. D. Marshall, in Ref. 6, Chapter8

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

459

hydrate moieties, the structure whose reduction product is given as item 20 in Table X. Closely related, but reduced, “incomplete” oligosaccharides in which (a)the terminal 2-acetamido-2-deoxy-cr-Dgalactosyl group and ( b )the nonreducing, terminal trisaccharide were missing from this structure have been found, as well as all of the analogous substances lacking a N-glycoloylneuraminic acid residue. In addition, a similar type of reduced oligosaccharide was isolated from the same source by Katzman and EylarZs3;in it, the @-D-galaCtOSe residue was linked to 0-4 of 2-acetamido-2-deoxy-~-galactitol(item 21). Some prosthetic groups consisted of monomeric 2-acetamido-2deoxy-D-galactose residue^.'^' The structures of these oligosaccharides seem to be simpler than some of those that occur in bloodgroup substances from ovarian cysts.

3. Glycosaminoglycans

The structures of the carbohydrate chains of the various glycosaminoglycans have been described in considerable detail,289-291a and therefore only a few limited aspects concerned with these glycoproteins will be discussed. Keratan sulfate has usually been obtained by treatment of extracts of cornea and of cartilage with proteolytic enzymes, followed by treatment with hyaluronidase and application of various fractionation procedures. The resulting material usually contains up to 5-1070of amino acids; these are probably liberated by hydrolysis of a polypeptide backbone to which carbohydrate branches may be linked. The term keratan sulfate is often applied to the carbohydrate moiety composed of a disaccharide repeating unit, namely, (1-3)-O-p-D-galactopyranosyl-(1~4)-2-acetamido-2-deoxy-~-~-glucopyranose, in some of which, both sugars are sulfated at C-6 (but far from completely in each type of sugar moiety). Other sugars are also often found in the substance isolated from various sources; these include sialic acid, L-fucose, and 2-amino-2-deoxy-~-galactosein relatively small proportions.207From methylation studies, it seems likely that L-fucose, as usual, occurs in a nonreducing, terminal position and, moreover, that, in keratan sul(289) H. Muir, Intern. Reo. Connect. Tissue Res., 4,lO (1964). (290) J. S. Brimacombe and J. M. Webber, “Mucopolysaccharides,” Elsevier Publishing Co., Amsterdam, 1964. (291) S. Schiller, Ann. Rev. Physiol., 28,137 (1966). (291a)C. Quintarelli, “Chemical Physiology of Mucopolysaccharides,”Little, Brown & Co., Boston (1968).

460

R. D . MARSHALL AND A. NEUBERGER

fate from rib cartilage, some D-galactose is likewise in a terminal, nonreducing p o s i t i ~ n .Keratan ~ ~ ~ * sulfate ~ ~ ~ from the latter source is relaand the extent to tively richer in 2-amin0-2-deoxy-D-galactose,~"~ which this sugar, as well as L-fucose and some of the D-galactose, occurs as carbohydrate moieties independent of the repeating disaccharide unit that makes up other prosthetic groups has not yet been fully established. As mentioned earlier (see p. 440), such units could be responsible for the cross-reactions with anti-blood-group sera that have been described.213The recognition of D-mannose (which is not released from keratan sulfate by treatment with alkali) indicates that there are further, unknown, structural features.292It has been suggested209that this sugar occurs in such a position that the side chains of keratan sulfate of human, knee-joint cartilage have the general structure [Gal- GNAc In- Man- GalNAc- Ser, but further studies on this aspect are desirable (see p. 440). The antigenic activity of the keratan sulfate-like, protein complex from chick allantoic fluid results mainly from the presence of relatively large proportions of prosthetic groups (hexasaccharides) that differ greatly from that usually described as keratan sulfate.293This complex is immunologically identical with a component of influenzavirus envelope.294 Chondroitin sulfate, heparin, and heparitin sulfate chains are linked to the polypeptide backbone by the following linear sequence of sugars:

1--* 3)-/3-D-Ga1-(1 + 3)-/3-D-Gal-(1+-4)-/3-~-Xyl-L-Ser, @-D-GUA-( where the anomeric carbon atom of D-xylose is involved in the carbohydrate-peptide bond. This structure was demonstrated for chondroitin 6-sulfate prepared from umbilical cord,'85 for chondroitin 4-sulfate from ox nasal septa,295for dermatan sulfate from pig skin2Mand for heparin from pig intestinal m u c ~ s a . ~The ~ ' sugar sequence in this It is * *of~interest ~~ region is probably identical in heparitin ~ u l f a t e . ~ @ (292) V. P. Bhavanandan and K. Meyer, J . Biol. Chem., 243,1052 (1968). (293) K. Meyer, N. Seno, and B. Anderson, Rheumatismus (Darmstadt),36,13 (1965). (294) W. G. Laver and R. G. Wehster, ViroEogy,30,104 (1966). (295) L. Roden and G. Armand,]. B i d . Chem., 244,65 (1966). (296) L. A. Fransson, Biochim. Biophys. Acta, 156,311 (1968). (297) U. Lindahl, Biochim. Biophys. Acta, 130,368 (1966). (298) J. Knecht, J. A. Cifonelli, and A. Dorfman,J. Biol. Chem., 242,4652 (1967). (1963). (299) S. Jacobs and H. Muir, Biochem.J.,87,37~

STRUCTURE AND METABOLISM O F GLYCOPROTEINS

461

TABLEXI The Main Structural Features of the Carbohydrate Chains of Glycosaminogl ycans Carbohydrate chain

Main disaccharide repeating unit

References

4404

I

Dermatan sulfate"

a-( 1 + 4)-p-~-IdoUA-( 1 + J)-D-GalNAc-

289- 29 l a

4-SO4

I Chondroitin 4-sulfate*

+-(l

+ ~)-P-D-GUA-(~ + 3)-~-GalNAc-

289-291a

640,

I

-p-(1 4 ~)-/~-D-GUA-( 1 + 3)-~-GalNAc-

289-291a

Heparind

-p-(1+ 4)-P-D-GUA-(l -+ 4)-D-GNSO,

297

Heparitin sulfate'

D-GUA-D-GNSO, D-GUA-D-GNAc

298

6-sulfatec

aIn dermatan sulfate from shark skin, there is about a 40% excess of sulfate groups over 2-acetamido-2-deoxy-~-galactose residues."o The small proportions of D-glucuronic acid (D-GUA) present in dermatan sulfate occur in regions of the carbohydrate chains both near to, and far from, the carbohydrate-peptide linkage region. The isolation of the tetrasaccharide so4

so4

I

I

GUA-CalNAc-IdoUA-GalNAc from dermatan sulfate from pig skin reveals that the carbohydrate chains are heterogeneous in at least some preparation^.^"' "Cartilage of squid and of horse-shoe crab contains chondroitin 4-sulfate, sulfated at 0 - 4 of 2acetamido-2-deoxy-D-galactoseresidues to the extent of about 75% only. Overall, there are about 1.5 molar equivalents of sulfate per mole of 2-acetamido-2-deoxyD-galactose."u' 'From shark skin may be obtained various fractions containing 1.0to 1.3 molar equivalents of sulfate per mole of 2-acetamido-2-deoxy-~-galactose.~~~ dThe 2amino-2-deoxy-D-glucose residues (in the repeating sequence of this sugar containing uronic acid in the region of the linkage region) is N-acetylated, not N - ~ u l f a t e d . ~ ~ ~ Small proportions of L-iduronic acid are also reported to be present,28' as well as some unsubstituted hexosamine residues.3a3As well as the N-sulfate groups, there are, on average, a further 1.5 sulfate groups per disaccharide repeating nit;^"^,'^ these are mainly on 0 - 2 of about one-third of the D-glucuronic acid residues and on 0-6 of 2-amin0-2-deoxy-D-glucose.~" Probably, branch points also occur in heparin from lungs.3os 'An alternating sequence of 2-acetamido-2-deoxy-D-g~ucose-uronic acid and N-sulfated 2-amino-2-deoxy-D-glucose-uronicacid moieties is not present; long sequences of alternate D-glucuronic acid and 2-acetamido-2-deoxy-~glucoseresidues are present.*% The material probably comprises a family of closely related compounds, and may be separated into fractions having various proportions of total sulfate, Nsulfate, and acetyl groups.3q

462

R. D. MARSHALL AND A. NEUBERGER

that, even in dermatan sulfate, in which the preponderant uronic acid is L-iduronic acid, it is D-glucuronic acid that occurs near the carbohydrate-peptide linkage region. Furthermore, the sugar attached by its anomeric carbon atom to the D-glucuronic acid residue in the linkage region of heparin is not the N-sulfate of 2-amino-2-deoxy-DThus, the main strucglucose, but 2-acetamido-2-deoxy-~-glucose.~~~ tural features of the carbohydrate chains of the glycosaminoglycans (see Table XI) do not extend to the carbohydrate-protein linkage area. The occurrence of heterogeneity in them, as exemplified by the small proportions of D-glucuronic acid (instead of L-iduronic acid) in dermatan sulfate, is a further aspect of the heterogeneity of glycoproteins . The carbohydrate chains of chondroitin sulfate are not antigenic, although antibodies can be raised in rabbits and guinea pigs against pig chondromucoprotein.307The nature of the site(s) against which antibodies are produced is as yet unknown.

4. Some Uses of Glycosidases

A large number of studies have been concerned with the structure of the carbohydrate moieties of glycoproteins, but these will not be listed, partly because of limitations of space. Also, there are discrepancies in some of the data obtained by different workers and in their interpretation. Such discrepancies may, at least in part, arise from problems of heterogeneity, as discussed in various parts of this Chapter. There are, however, a number of characteristics common to a large number of these prosthetic groups. Firstly, many of them contain p-D-galactose attached to 0 - 4 of 2-acetamido-2-deoxy-~-glucose: N-acetyl-lactosamine is a structural feature of blood-group substances

(300)N. Seno and K. Meyer, Biochim. Eiophys. Acta, 78,258(1963). (301)L.A. Fransson and L. Rod&, J . Biol. Chen., 242,4170(1967). (302)M. B. Mathews, J. Duh, and D. Person, Nature, 193,378(1962). (303)G.J. Durant, H. R. Hendrickson, and R. Montgomery, Arch. Eiochem. Eiophys., 99,418(1962). (304) I. Danishefsky, H. Steiner, A. Bella, and A. Friedlander, J . Biol. Chem., 244, 1741 (1969). (305)J. A.Cifonelli, Fed. Proc., 24,354(1965). (306) J. A.Cifonelli and A. Dorfman,]. Eiol. Chern.,235,3283(1960).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

463

(see item 2, Table X), and of keratan sulfate (see p. 459), and it has been isolated after partial, acid hydrolysis of a number of other glycoproteins (see Ref. 181). Secondly, frequently branched structures are present in the carbohydrate entities (see Ref. 315).Finally, the effect of purified glycosidases indicates that there are a number of common features. When present, sialic acid is always in a nonreducing, terminal position. In certain other natural products, such as colominic sialic acid has been found to be further substituted, but only by sialic acid, and, in view of their occurrence in oligosaccharides found in colostrum,30git would not be surprising should such structures occur in some glycoproteins. The use of neuraminidase purified from a number of sources leads to the release from many glycoproteins of the major part of the sialic acid, without any changes in the protein (see Table XII). However,

TABLEXI1 Release of Sialic Acid from Certain Glycoproteins by Use of Neuraminidase

G1ycoprotein

Mol. Wt.

Source of neuraminidase

Sialic acid (moleslmole of protein) Total

Released

References

1 Human, chorionic

27,000

Vibrio cholerae

8

8

310

gonadotropin 2 Fetuin 3 a,-Acidglycoprotein

45,000 40,000

Vibrio cholerae Diplococcus pneumoniae Vibrio cholerae Vibrio cholerae

13 14

13 14

248 311

16.9%

680

312 182

76

61

39

6

4

313

4 Lensubstance 5 Submaxillary-gland 1,000,000 glycoprotein (sheep) 6 Stomach-carcinoma glycoprotein 7 a,-Acute-phase globulins

270,000 45,000

Clostridium pelfringens Vibrio cholerae

17.9% 800

(307) G . Loewi and H. Muir, Immunology, 9,119(1965). (308)G.T.Barry and W. F. Goebel, Nature, 179,206(1957). (309)R. Kuhn and A. Gauhe, Chem. Ber. 98,395(1965). (310)0.P.Bahl,]. Biol. Chem., 244,567(1969). (311)R.C. Hughes and R. W. Jeanloz, Biochemistry, 3,1535(1964). (312)A. Pusztai and W. T. J. Morgan, Biochem.]., 78,135(1961). (313)A. H. Gordonand L. N. Louis, Biochem.J.,113,481(1969).

464

R. D. MARSHALL AND A. NEUBERGER

structures may occur in which, although sialic acid is in a nonreducing, terminal position, it is nevertheless not split off by neuraminidase. An example is sialylganglio-N-biose 11: P-D-GalNAc-(1+ 4)-D-Gal 3

t

2 a-D-sialyl The 2-acetamido-2-deoxy-~-galactose residue has to be removed from this compound before the sialyl linkage can become susceptible to n e ~ r a m i n i d a s e Sialic . ~ ~ ~ acid may also be released from glycoproteins under conditions of very mild acidity, very few other changes occurring in the glycoproteins, apart from the loss of small proportions of ~-fucose.~~~ Sialic acid occurs naturally as both the N-acetylated and the N glycoloylated form, the proportion in a given environment being different from one species to anotherS3lsOxidation of the acetyl group to the glycoloyl group occurs after the formation of 2-acetamido-2deoxy-~-glucose.~~~ In those proteins containing L-fucose, it would seem that this sugar is, probably always, also in nonreducing terminal positions.315This may not, however, be a general rule, because it would appear that only -75% of the L-fucose in A and B substances from ovarian cysts is released318after prolonged heating in M acetic acid at 100”.This result could indicate that L-fucose is not in a nonreducing, terminal position, so that cleavage of a second glycosidic linkage would be necessary in order that free L-fucose might be released, but this is not the only explanation possible for these findings. Thus, if, in some carbohydrate moieties, L-fucose were glycosidically attached to 0-3 of an N-acetylhexosamine residue, degradation of the moiety would probably involve, in part, a deacetylation of this “aglycon.” The L-fucosidic bond of this product would be more resistant to hydrolysis than that in the original entity, because of the presence of the positively charged

(314)R. Kuhn and Wiegandt, 2.Naturforsch., B , 18,541(1963). (315)See Ref. 6. (316)E. Martensson, A. Raol, and L. Svennerholm, Btochlm. Biophys. Acta, 30, 124 (1956). (317)R. Shauer, H.J. Schoop, and H. Faillard, Z. Phystol. Chem., 349.645 (1968);350, 155 (IeSQ). (318)R. A. Gibbons and W. T. J. Morgan, Blochem.J., 57,283(1954).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

465

amino group on the 2-amino-2-deoxyhexosyl group. The finding that, either before or after release of sialic acid, the single molecule of L-fucose is removed from a molecule of human, chorionic gonadotropin by use of the a-L-fucosidase from Aspergillus nigeP9 shows that this sugar residue is a nonreducing terminus. D-Galactose tends to be the next innermost sugar in nonreducing, terminal positions. Thus, about 70-80% of the D-galactose residues of sialic acid-free f e t ~ i n ,or~ of~ a~ glycopeptide , ~ ~ ~ isolated from it,248 were released by the action of P-D-galactosidase. Similar results were TABLEXI11 Release of 2-Acetamido-2-deoxy-~-glucose from Glycoproteins and Glycopeptides (GP) by Use of 2-Acetamido-2-deoxy-~-~-glucosidase 2-Acetamido-2-deoxyD-gluCOSe (moles/ mole of protein) Glycoprotein or GP

M.W.

Source of enzyme

1 Egg albumin GP 1,500 pig epididymis 2 Human IgG-GP 2,300 pig epididymis 3 Human, chorionic 27,000* Proteus vulgaris gonadotropin Proteus vulgaris 4a HCG-GP, 4,300 b 5a HCG-GP, 3,300 Proteus uulgaris b 6 al-Acid 40,000 Diptococcus pneumoniae Glycoprotein 7 Fetuin-GP 4,300 pig epididymis 8 Ovomucoid 28,000 Turbo cornutus

Total Released

References

1" 3.1 8'

319-321 109 214

24

O.gd 4.0" 1.7d 3.8' 6.2"

214 214 214 214 311

15 24

9.3d 3

323 324

3 7.7 11

5.6 4.9

"2-Acetamido-2-deoxy-~-glucosidase released about 1.5 moles of 2-acetamido-2deoxy-D-glucose from an amount of an egg-albumin glycopeptide containing 4 moles of this s ~ g a yResults . ~ ~ ~ obtained by Huang and Montgomery by use of enzymesz3' d o not agree with' those of other They rep0I-P" that all residues of 2-acetamido-2-deoxy-~-glucose but one are released by 2-acetamido-2-deoxy-~-glucosidase. T h i s is the molecular weight of the reduced, alkylated "The substance was free from sialic acid; it had been pretreated with P-D-galactosidase. No 2-acetamido-2deoxy-D-glucose was released, unless the sialic acid was first removed. dBy direct action on the glycopeptide (which was free from sialic acid).

(319) 0. P. Bahl and K. M. L. Agrawal,]. B i d . Chem., 244,2970 (1969). (320) H. H. Kaufman and R. D. Marshall, Abstr. 6th Intern. Congr. Biochem., New York, 2,92 (1964). (321) J. R. Clamp and L. Hough, Biochem.]., 94,502 (1965). (322) J. Conchie, A. J. Hay, I. Strachan, and G. A. Levvy, Biochern.]., 115, 717 (1969). (323) R. G. Spiro, Methods Enzymol., 8,26 (1966). (324) T. Muramatsu,]. Biochem. (Tokyo), 64,521 (1968).

R. D. MARSHALL AND A. NEUBERGER

466

obtained with orosomucoid311*319 and human, chorionic gonadotropin.319D-Galactose is not released at all, unless sialic acid has previously been removed. 2-Acetamido-2-deoxy-~-~-glucosidase acts directly on a number of glycoproteins and glycopeptides (see items 1,2,and 7 of Table XIII). Sequential use of enzymes may give results of considerable value. was allowed to act Thus, if 2-acetamido-2-deoxy-~-~-glucosidase directly on two glycopeptides prepared from human, chorionic gonadotropin (HCG) from which sialic acid had been removed, amounts of 0.9 mole (HCG-GP,, item 4a) and 1.7 moles (HCG-GP,, item 5a) of 2-acetamido-2-deoxy-~-g~ucose,respectively, were released per mole. However, if this reaction was preceded by the action of P-D-galactosidase (P-D-galactosidegalactohydrolase, E.C. 3.2.1.23), considerably larger amounts were released (see items 4b and Sb, respectively). Glycoproteins or glycopeptides are frequently treated with a - ~ mannosidases; (a-D-mannoside mannohydrolases, E.C. 3.2.1.24); D-mannose is often released (see Table XIV). Prior treatment with other enzymes is often found necessary (see items 5 and 6). TABLEXIV Release of D-Mannose from Glycoproteins and Glycopeptides (GP) by Use of a-D-Mannosidase D-Mannose (moles/ mole of protein) ReferGlycoprotein or GP

1 Eggalbumin 2 Egg albumin GP

3 Ovomucoid 4 Human, chorionic gonadotropin 5 HCG-GP, 6 HCG-GP, 7 a,-Acid glycoprotein

M. W.

45,000 1,500

Source of enzyme

Jack-bean meal Jack-bean meal Charonia lampas liver Turbo cortunus liver 28,000 Jack-bean meal Aspergillus 27,000b niger 4,300 Proteus vulgaris 3,300 Proteus vulgaris 40,000 Jack-bean meal

Total 5 5 5

Released 1.1" 3.3 1.3

ences 325 325 326

5

3.3

327

7.3

0.7

325

8.0 5.1

1.8' 4.1c,d 4 . 9 ~ 2.gC

214 214 ~214 325

5.4 12

"Another group of workers has reported that 2.5 moles of D-mannose per mole are split off by use of this enzyme,3" and another reported that all of the D-mannose is removed. bThis is the molecular weight of the reduced, (carboxymethy1)ated proteir~.~'O CAfterremoval of sialic acid, followed by treatment with p-D-galactosidase and 2-acetamido-2-deoxy-~-~-glucosidase. dD-Mannose was not released unless the substrate had been pretreated with p-Drgalactosidase and 2-acetamido-2-deoxy-p-~glucosidase.

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

467

The glycosidic linkages in which 2-acetamido-2-deoxy-~-galactose participates are frequently a - ~ These . linkages occur in the nonreducing, terminal residues of A substance (see Table X) and in the carbohydrate-peptide linkage of at least some g l y c ~ p r o t e i n s . ' ~ ~ * ' ~ ~ Although D-galactose is usually @-linked, it is a-D-linked in B substance (see Table X). Full interpretation of data of this type must await further understanding of the specificities of the enzymes involved, of the nature of the residual oligosaccharide, and of the problem of possible heterogeneity discussed earlier. An advantage of enzymic methods is that the anomeric configuration may be revealed.

VII. THE BIOSYNTHESIS

OF

GLYCOPROTEINS

1. The Carbohydrate-peptide Linkages Certain parts of ihe structure of glycoproteins are rigidly controlled; it may therefore be deduced that a fairly high degree of specificity is involved in their biosynthesis. It would appear that an L-asparagine moiety in a polypeptide chain may be an acceptor of only one type of sugar, namely, 2-acetamido-2-deoxy-~-glucose, provided, in general, that the acetamido sugar is followed, in the p position on the C-terminal side, by a P-hydroxy-a-amino acid residue (see p. 425). As the acquisition of the sugar residue appears to depend on the presence of an amino acid (L-serine or L-threonine) that is incorporated into the peptide chain after the L-asparagine moiety, it is reasonable to suppose that the sugar residue concerned is added, as a post-ribosomal event, to a predetermined sequence of amino acids in the polypeptide chain. Direct experimental verification of this inference is at present lacking. On the other hand, there is evidence for direct, enzymic addition of 2-acetamido-2-deoxy-D-galactoseresidues to L-serine and Lthreonine residues of the polypeptide chains that are probable precursors of the submaxillary-gland glycoproteins. T h e enzymes, and sheep,16fihave a largely particulate in the glands from high degree of specificity, not absolute with regard to the receptor, as discussed later (see p. 468). The activated 2-acetamido-2-deoxy-

(325) Y.-T.Li,]. Biol. Chern.,241, lOlO(1966). (326) T. Murarnatsu,]. Biochem. (Tokyo),62,487 (1967). (327) T. Muraniatsu and F. Egami,]. Biochem. (Tokyo),62,700 (1967). (328) A. Hagopian and E. H. Eylar, Arch. Biochem. Biophys., 129,515 (1969)

468

R. D. MARSHALL A N D A. NEUBERGER

D-galactose is specific, and is not transferred, by the enzyme, from pyrophosphate) uridine 5'-(2-acetamido-2-deoxy-~-galactopyranosyl to other sugar molecules, regardless of whether they are monosaccharides or exist as integral parts of glycoproteins. Known receptors are the ox and sheep submaxillary-gland glycoproteins from which the disaccharide prosthetic groups have been removed. Peptides derived from pronase digests of these modified proteins do not function as receptors, and treatment of these proteins with trypsin leads to products that can be glycosylated, although much less readily. Most of the natural proteins do not function as acceptors, although at least one is known to, namely, a basic protein, encephalitogen, having a molecular weight of 16,400, isolated from the myelin of spinal cord; it is normally devoid of carbohydrate, but, presumably, some of the L-threonine residues have the environment requisite to occurrence of g l y c o ~ y l a t i o n .Enzymes ~~~ of this type presumably operate first on those polypeptide chains that are destined to become glycoproteins. Probably, enzymes that add 2-acetamido-2deoxy-D-galactose residues to existing carbohydrate moieties belong to a different class. linkage of collagen is formed The ~-galactose-5-hydroxy-~-lysine after production of the polypeptide chain, and, indeed, as a step subsequent to the conversion of L-lysine into 5-hydroxy-~-lysine residues.330 An enzyme involved in the biosynthesis of this type of linkage was identified in skin of embryonic guinea-pig and was purified 160-fold. The nature of the receptor is highly specific, only 5-hydroxy-~-lysineacting in this capacity, preferably when this amino acid is combined in collagen.331 Enzymic transfer of D-XylOSe from uridine 5'-(D-xylopyranosyl-'"C pyrophosphate) to L-serine residues of endogenous protein acceptors from ( a ) a cell tumor of the mouse188and ( b )chick-embryo cartilagelS9 occurs in cell-free extracts of both of these tissues, in the absence of biosynthesis of protein. The enzyme preparations employed were from the supernatant liquor, although activity was also present in the insoluble fractions. In these two types of tissue, the acceptors are heparin and chondroitin sulfate, respectively, but the presence of other D-xylose-containing glycoproteins in ascites fluid from (329) A. Hagopian and E. H . Eylar, Arch. Biochem. Biophys., 126,785 (1968). (330) J. Rosenbloom, N . Blumenkrantz, and D. J . Prokop, Biochem. Biophys. Res. Conmuti., 31,792 (1968). (331) H. B. Bosmann and E. H. Eylar, Biochem. Biophys. Res. Commun., 33, 340 (1968).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

469

cancer patients332 may indicate that other acceptors for D-xylose also exist. 2. Addition of Other Sugar Residues

The formation of the carbohydrate moieties of glycoproteins occurs by enzymic transfer of single sugar residues from glycosyl esters of nucleotides to nonreducing, terminal positions of the growing prosthetic groups. Certain oligosaccharide esters of nucleotides occur n a t ~ r a l l y , and ~ ~ ,it~ is ~ ~not impossible that these may be intermediates in the formation of glycoproteins; however, there is, at present, no proof of this hypothesis. The possibility must also be considered that the branch points that occur in the oligosaccharide moieties of a number of glycoproteins are formed by enzymes having activities analogous to that of amylo-( 1 -+ 4)+( 1 + 6)-trans-D-glucosidase (EC 2.4.1.18). Activated sugar-transferases prepared from the particulate fraction of chick embryo were used in a study of the formation of the linkage region (see p. 460) of chondroitin s ~ l f a t e . T~h~e ~ enzyme ,~~~ preparation transfers D-galactose from uridine 5’-(D-galactopyranosyl pyrophosphate) to D-xylose or u-xylosides, 0-D-xylosyl-L-serine, or endogenous acceptor, so that, in all of these reactions, D-galactose becomes attached to 0 - 4 of D-xylose. In the second stage of the reaction, D-galactose becomes attached to such substrates as DGal-( 1-+4)-D-Xyl and D-Gal-(1+4)-D-Xyl-L-Ser, as well as to endogenous acceptors; this step of the reaction appears to have fairly exacting requirements as regards the structure of the acceptor molec ~ l e The . ~ ~ enzyme ~ involved in attaching the D-glucuronic acid residue to 0 - 3 of the D-galactose in the neighborhood of the carbohydrate-peptide linking moiety of chondroitin 4- and 6-sulfate is probably different from that involved in the formation of later stages of the carbohydrate chains.335 A large number of glycosyl ester nucleotide transferases are known to exist; these include those involving cytidylic acid-sialic acid336*337 (332) K. Sugimoto, Tohoku J. E x p . Med., 64,271 (1964). (333) Y. Nakanishi, S. Shimizu, N. Takahashi, M. Sugiyama, and S. Suzuki, 1. H i d . C h e m . ,242, 967 (1967). (334) T. Helting and L. RodCn, ./. B i d . Cheni., 244, 2790 (1969). (335) T. Helting and L. RodCn, J . B i d . C h i n . , 244, 2799 (1969). (336) S. Roseman, “Biochemistry of Glycoproteins and Related Substances,” E. Rossi and E. Stoll, eds., S. Karger, Basel and New York, 1968, Part 11, p. 244. (337) M . J . Spiro and R. G. Spire,]. B i d . Chem., 243,6520 (1968).

470

R. D. MARSHALL AND A. NEUBERGER

as well as uridine 5‘-(D-glucopyranosyl pyrophosphate) for formation of the carbohydrate moieties of collagen.”8 Transferases acting upon uridine 5’-(D-galactopyranosyl pyrophosphate) are also known; some of these may result in the formation of P-D-galactosyl groups,339 and others, in the production of a-D-gahCtoSyl groups”O in carbohydrate moieties. Enzymes that catalyze the transfer of 2-acetamido-2deoxy-D-galactose from uridine 5’-(2-acetamido-2-deoxy-~-galactopyranosyl pyrophosphate) to precursors of the blood-group A type of carbohydrate moieties (see Table X) occur in milkJ4‘ and submaxillary glands::’4zof A and AB individuals, as well as in pig gastric mucosa.““:’ Transferase activities acting with guanosine 5’-(~-fucosyl pyrophosphate) occur in human milk, that forming the ( 1 + Z)-Iinkage to D-galactose being absent from that of those individuals lacking A, B, or H blood-group, specific antigens,“44and that forming the (1+ 4)-linkage to 2-acetamido-2-deoxy-D-g~ucosebeing absent from that of those lacking Lewis determinant^."^ All of these enzymes have, with regard to the acceptor molecules, fairly rigid requirements as to specificity.

3. Incorporation of Sulfate into Carbohydrate Moieties The incorporation of sulfate into the carbohydrate moieties of glycoproteins occurs after incorporation of the sugar moieties, and thus, here again, is a post-ribosomal event. The process is, therefore, not directly under control of the genome. Sulfation of the sugar moieties of chondroitins occurs by a sequence of steps, the first of which involves formation of 3‘-O-phosphono5’-adenylyl hydrogen sulfate, the sulfate d ~ n o r . ” ~This J ~ ~enzyme is present in a number of the tissues in which these glycoproteins (338) H. B. Bosmann and E. H. Eylar, Biochem. Biophys. Res. Commun.,30,89 (1968). (339) M. J. Spiro and R. G . Spiro,}. Biol. Chem., 243,6529 (1968). (340) A. Kobata, E. F. Grollman, and V. Ginsburg, Biochem. Biophys. Res. Commun., 32,273 (1968). (341) A. Kobata, E. F. Grollman, and V. Ginsburg, Arch. Biochem. Biophys., 124, 609 (1968). (342) V. M. Hearn, Z. G . Smith, and W. M. Watkins, Biochem.}., 109,315 (1968). (343) H . Tuppy and W. L. Staudenbauer, Nature, 210,316 (1966). (344) L. Shen, E. Grollman, and V. Ginsburg, Proc. Nat. Acad. Sci. U . S., 59,224 (1968). (345) E. Grollman, A. Kobata, and V. Ginsburg, Fed. Proc., 27,345 (1968). (346) P. W. Robbins and F. Lipmann,J. Amer. Chem. Soc., 78,2652 (1956). (347) S. Suzuki and J. L. Strominger,}. Biol. Chem., 235,257 (1960).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

471

are sulfated, including chick-embryo ~ a r t i l a g e ~ ~and ~ - ~the ~ Oisthmus of the hen As already mentioned (see p. 470),it is probable that sulfation of the carbohydrate chains occurs, to a considerable extent at least (if not wholly), after the polymerization has occurred. This probability was suggested by Meyer and c o ~ o r k e r s , ~because ~ ’ , ~ ~ ~of their finding of chondroitin in ox cornea that was free from, or very low (<2%) in, sulfate; this substance was later shown to be enzymically sulfated by extracts of the isthmus of hen oviduct at a higher rate than all other substances assayed.353 Chondroitin prepared by chemical desulfation procedures3s4 from chondroitin sulfate isolated either from pig-costal cartilage35s or ox trachea3s6was not sulfated by extracts of chick-embryo cartilage hydrogen sulfate. in the presence of 3’-O-phosphono-5’-adenylyl As substrate, the enzyme may require a chondroitin-protein complex; this, probably, was largely destroyed during the alkaline conditions (0.1 M sodium hydroxide at room temperature for 2 days) used during the preparation of the chondroitin. However, chondroitin prepared in a similar way functions as a sulfate acceptor when extracts of oviduct isthmus are It was demonstrated that the rate of sulfation, by extracts of isthmus, of carbohydrate chains of the type found in chondroitin or chondroitin sulfate A increases with the length of the acceptor chain with two notable exceptions: the non-sulfated trisaccharide (GalNAcGA-GalNAc) and pentasaccharide (GalNAc-GA-GalNAc-GAGalNAc) undergo esterification with sulfate at rates about 15 to 20 times greater than might be expected, and that are, indeed, about 50 to 60%of the value at which chondroitin itself is ~ u l f a t e d . ~ ~ ~ ~ : ~ ~ ~ residues do not become Some 2-acetamido-2-deoxy-~-galactose sulfated, and some of them accept two sulfate groups. Although a 2acetamido-2-deoxy-~-galactosesulfate can be isolated in relatively it is unlikely to be a direct prelarge amounts from hen (348) F. D’Abrarnoand F. Lipmann, Biochim. Biophys. Acta, 25,211 (1957). (349) J. B. Adams, Biochem.].,76,520 (1960). (350) S. D e Luca and J. E. Silbert,]. Biol. Chem., 243,2725 (1968). (351) E. A. Davidson and K. Meyer,J. B i d . Chem., 211,605 (1954). (352) K. Meyer, A. Linker, and P. Hoffman, Biochim. Biophys. Acta, 21,506 (1956). (353) S. Suzuki and J. L. StromingerJ. Biol. Chem.,253,267 (1960). (354) T. G. Kantor and M. Schubert,J. Amer. Chem. Soc., 79,152 (1957). (355) E. Meezan and E. A. Davidson,J. B i d . Chem., 242,1685 (1967). (356) J . B. Adams, Biochirn. Biophys. Acta, 32,559 (1969). (357) S. Suzuki and J. L. Strominger,]. B i d . Chem. 235,274 (1960). (358) J. L. Strominger, Biochirn. Biophys. Acta, 17,283 (1955).

472

R. D.MARSHALL AND A. NEUBERGER

cursor of chondroitin sulfate, because its rate of formation in the system already discussed is extremely low.353 From observations with the electron microscope, it was suggested that sulfation of the carbohydrate moieties occurs on the endoplasmic reticulum or in the Golgi a p p a r a t u ~ . The ~ ~ ~finding , ~ ~ ~ of sulfotransferase (E.C. 2.8.2) activity in microsomal fractions from both chickembryo e p i p h y ~ e and s ~ ~mouse, ~ mast-cell tumoP1 may be supporting evidence. Several sulfotransferases probably occur in a given t i s s ~ e . ~ ~ ~ * ~ Presumably, sulfation of the glycoproteins found in the digestive tract (see Table 11, p. 412) occurs by a similar mechanism, in which 3’-O-phosphono-5’-adenylyl hydrogen sulfate is the donor. Neither the origin of, nor the acceptor site for, the sulfate group reported to occur in human Tamm-Horsfall protein is k n o ~ n . ~ ~ ~ , ~ ~ ~

VIII. SOMEGENETICALLY DETERMINEDDISEASES IN WHICH GLYCOPROTEINS ARE IMPLICATED Many pathological states are associated with marked changes in the level of protein-bound carbohydrate in serum.365*366 However, there are difficulties in interpreting the experimental results obtained,367and these aspects will not be considered here. Of more immediate relevance are a number of examples in which a specific defect in the structure or metabolism of one or more glycoproteins is associated with a pathological state. Inherited, abnormal forms of fibrinogen are known in a number of families. In one of these types, fibrinogen Detroit, there are, in addition to replacement of the L-arginine residue at position 19 of the a(A)-chains by a neutral (359)M. Peterson and C. P. Leblond,]. Cell Biol., 21,143(1964). (360)N. Lane, L. Caro, L. R. Otero-Vilardebo, and G. C. Goodman,]. Cell. Biol.,21, 339 (1964). (361)J. E. Silbert,]. Biol. Chem., 242,5146(1967). (362)S. Suzuki, R. H. Thremm, and J . L. Strominger, Biochim. Biophys. Acta, 50, 169 (1961). (363)L.Odin, Nature, 170,663(1952). (364)W.H. Boyce and M. Swanson,]. Clin. Inuest., 34,1581(1955). (365)J . P. Greenstein, “The Biochemistry of Cancer,” Academic Press, Inc., New York, N.Y., 1954,pp. 551-557. (366)R. J. Winder, “The Plasma Proteins,” F. W. Putnam, ed., Academic Press, Inc., New York, N.Y., 1960,Vol.1, pp. 309-310. (367)C. L. Heiskell, C. M. Carpenter, H. E. Weiner, and S. Nakagawa, Ann. N . Y. Acad. Sci., 94,183(1961).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

473

amino acid,3s8unusually small proportions of sialic acid, hexosamine, and neutral sugars.369The rate of conversion of this fibrinogen into fibrin is abnormally low. Another abnormal molecular form of fibrinogen (Paris) has been shown to contain little or no D-galactose and twice the usual proportion of sialic acid. The polymerization reaction of the fibrin monomers arising from this fibrinogen is abnormally slow. If a difference in the structures of the oligosaccharide moieties of this molecule, compared with those of fibrinogen in normal individuals, is confirmed as being the only abnormality, the participation of these prosthetic groups in clotting will be fully i r n p l i ~ a t e d . ~ ~ ~ - ~ ~ ~ It is not impossible that fibrocystic disease of the pancreas373is primarily an expression of an abnormal metabolism or structure (or both) of glycoproteins. This disease is a hereditary condition, with recessive character, showing secretions of highly viscous glycoproteins in the bronchi, the pancreatic ducts, and the intestinal tract. It has been suggested that the glycoproteins of secretions from the duodenum have an abnormal structure.374J o h a n ~ e n ~examined ’~ certain highly insoluble glycoproteins occurring in plugs formed in the digestive tracts of two very young children having the disease. In each, the major constituents were a closely related family of glycoproteins. The composition of the two samples was similar, although some differences were noted in their sugar compositions, particularly with respect to L-fucose. The structure and metabolism of the various glycoproteins in this disease need further extensive study, but it cannot be assumed that the metabolic defect is not one that affects these macromolecules indirectly.373 A description has been given of the presence, in patients having diabetes mellitus, of an unusually high concentration of a hemoglobin having many properties in common with hemoglobin IAc,to which reference has already been made (see p. 420). This compound differs (368) M . Blomback, B. Blomback, E. F. Mammen, and A. S. Prasad, Nature, 218, 134 (1968). (369) E. F. Marnmen, A. S . Prasad, M. I. Barnhart, C. C. Au, andV. Schwandt,J.Clin. Inoest., 48,235 (1969). (370) J. Soria, J. Coupier, M . Samama, P. Tixier, and G. Bilski-Pasquier, Abstr. 12th Intern. Cong. Haematol., New York, 1968, p. 180. (371) L. Mester and L. Szabados, C o n p t . Rend., Ser. D, 266,34 (1968). (372) L. Mester and L. Szabados, Bull. SOC.Chim. Biol., 51,635 (1969). (373) P. A. Di Sant’Agnese, in “Cystic Fibrosis,” E. Rossi and E. Stoll, eds., S. Karger, Base1 and New York, 1967, Part 1, p. 10. (374) Z. Dische, Ann. N. Y. Acad. Sci., 106,259 (1963). (375) P. G. Johansen, Biochem.], 87,63 (1963).

474

R. D . MARSHALL AND A. NEUBERGER

from the latter in that, on acid hydrolysis, it yields a compound that reacts with ninhydrin and that has the chromatographic behavior of an amino sugar, whereas I,, does not yield this compound. It has been tentatively suggested that this hemoglobin from diabetic patients is a glycoprotein that contains a h e x o ~ a m i n e . ~ ~ ~ A most interesting metabolic defect in a brother and sister, both of whom were mentally retarded, has been described. It would appear that there is a deficiency377of the enzymic activity responsible for splitting 2-acetamido-l-N-~-~-aspartyl-2-deoxy-~-~-g~ucopyranosy~amine. Lysosomal enzymes that split this type of linkage occur in a wide variety of tissues, including liver, kidney, spleen, testis,378,380,383 intestine, seminal vesicle,378 pancreas,379lung and heart,378*37s and skeletal muscle.379It occurs in seminal plasma, and in small proportions in the serum of a number of species.379~380*384~38s The enzyme is also present in hen in the visceral hump of the limpet,380and in a It is a lysosomal enzyme,378,379 and it functions as an amidase, so that the products of the enzymic cleavage of 2-acetamido-l-N-P-~-aspartyl-2-deoxy-~-Dglucopyranosylamine are, initially, L-aspartic acid and Z-acetamido2 - d e o x y - ~ - D - g ~ ~ c o s y l a r n iThe n e . specificity ~ ~ ~ ~ ~ ~ ~ requirements ~~~~~~~~ of the enzyme are such that the P - ~ a s p a r t ygroup l of the compound cannot be in peptide linkage either at its amino group or its carboxyl residue may be g r o ~ p s , but ~ ~ the ~ - 2-acetamido-2-deoxy-~-glucose ~ ~ ~ linked to other sugars .37s,384-386 In the mentally retarded adults already mentioned, relatively ~ s2-acetamido-l-N-P-~-aspartyl~ large amounts (about 300 r n g / d a ~ ) of 2-deoxy-~-D-glucopyranosylamine, as well as closely related gly(376) S. Rahbar, 0.Blumenfeld, and H. M . Ramney, Biochem. Biophys. Res. Commun., 36,838 (1969). (377) R. J. Pollitt, F. A. Jenner, and H. Merskey, Lancet, ii, 253 (1968). (378) S. Mahadevan and A. L. Tappel, 1.B i d . Chem., 242,4568 (1967). (379) T. Ohgushi and I. Yamashima, Biochim. Biophys.Acta, 156,417 (1968). (380) J . Conchie and 1. Strachan, Bioclzem.].,115,709(1970). (381) M. Murakami and E. H. Eylar,]. BioZ. Chem.,240, PC 556 (1965). (382) J . R. Clamp, C . Dawson, L. Hough, and M . Y. Khan, Carbohyd. Res., 3,255 (1966). (383) C. P. J . Roston, J . C. Caygill, and F. R. Jevons, Biochern.].,9 7 , 4 3 (1965). ~ (384) M . Makino, T. Kojima, and I. Yamashima, Biochem. Biophys. Res. Commun., 24,961 (1966). (385) M . Makino, T. Kojima, T. Ohgushi, and I. Yamashima, J. Biochem. (Tokyo), 63, 186 (1968). (386) A. L. Tarentino and F. Maley, Arch. Biochem. Biophys., 130,295 (1969). (387) E. D. Kaverzneva, Khimia, 130 (1966). ~ (388) F. A. Jenner and R. J. Pollitt, Biochern.].,1 0 3 , 4 8 (1967).

STRUCTURE AND METABOLISM O F CLYCOPROTEINS

475

copeptides, were present in the urine.377 The enzyme responsible for splitting this type of linkage was absent from the seminal fluid of the brother, and it has been suggested that there may have been a generalized deficiency of the enzyme in both patients.377 The “mucopolysaccharidoses” have attracted considerable attention”$ during the past fifty years, since they were first described clinically by HunteFO and Hurler.“$’ At least six classified types of disease are now In types I (Hurler syndrome), I1 (Hunter syndrome), and I11 (Sanfilippo syndrome), specific enzyme defects have been reported, and, because of this, some aspects of these types of disease will be briefly discussed. An excessive storage of brain g a n g l i o s i d e ~ dermatan ,~~~ sulfate, and heparitin sulfate occurs in a number of tissues. In normal individuals, -3 to 15mg of glycosaminoglycans is passed in the urine every 24 h0urs,3$~the greater part being chondroitin 4-sulfate, with smaller amounts of dermatan sulfate and heparitin sulfate.394Large amounts (10to 40 times normal) of dermatan sulfate and heparitin sulfate are excreted in the urine of patients having the Hurler and Hunter syndromes, and of heparitin sulfate only in those having the Sanfilippo ~ y n d r o r n e . ~ ~ $ , ~ $ ~ * ~ ~ The molecular size of the glycosaminoglycans isolated from tissues and from the urine of patients is smaller than usual; the compounds contain relatively few amino acids, apart from ~ - s e r i n e ~and ~ ~may ,~$~ well be partially degraded fragments of the original glycoproteins. The rate of degradation of the glycosaminoglycans is lower than normal, as was shown by studies conducted with fibroblasts derived from the skin of patients having the Hunter and Hurler syndromes.398 Many tissues of patients having types 1-111 disease have been reported to be deficient in a specific, lysosomal j3-D-galactosidase. The

(389) V. A. McKusick, “Heritable Disorders of Connective Tissue,” C. V. Mosby Co., Saint Louis, 3rd Edition, 1966, p. 325. (390) C. H. Hunter, Proc. Roy. Soc. Med., 10,104 (1917). (391) G . Hurler, Z . Kinderheilk., 24,220 (1919). (392) R. Leeden, K. Salsman, J. Gomatas, and A. Taghavy,]. Neuropathol. E x p . Neurol., 24,341 (1965). (393) C . Rich, N. DiFerrante, and R. M. Archibald,]. Lab. Clin. Med., 50, 686 (1957). (394) A. Linker and K. D. Terry, Proc. Soc. E r p . Biol. Med., 113,743 (1963). (395) K. Meyer, M . B. Brurnbach, A. Linker, and P. Hoffman, Proc. Soc. E x p . Biol. Med., 97,275 (1958). (396) A. Dorfman and A. E . Lorincz, Proc. Nut. Acad. Sci. U . S., 43,443 (1957). (397) A. Dorfman, Biophys.J., 4, 155 (1964). (398) J. C. Fratantoni, C. W. Hall, and E. F. Neufeld, Proc. Nut. Acad. Sci. U. S . , 60, 699 (1968).

476

R. D. MARSHALL AND A. NEUBERGER

tissues involved include liver,399-402skin,403 kidney,401.402brain, and spleen.401Usually, the activities of other acid hydrolases are greatly increased, but an undiscriminated hyperactivity of the lysosoma1 system is not found.399In five other patients examined, there at appeared to be a complete absence of @ - ~ - g a l a c t o s i d a s eIt. ~is~ ~ present difficult to interpret fully the relationship between the enzymic deficiency and the accumulation of glycosaminoglycans, and it is not clear whether the glycosidases and the accumulated glycosaminoglycans may interact to form enzymes of lowered activities. The finding of increased proportions of a keratan sulfate-like substance in the liver and spleen of patients having GM,-gangli~sidosis~~~ would, perhaps, be more easily understandable were a low @-Dgalactosidase activity present. Related types of mucopolysaccharidosis are known that cannot easily be fitted into any of the categories described by M c K ~ s i c k . ~ * ~ Three cases have been reported in which there was a complete absence of a-L-fucosidase activity in liver, brain, lung, and kidney, although other glycosidase activities were increased.39sProblems arise in the interpretation of these data similar to those encountered in the enzyme deficiencies in the mucopolysaccharidoses just mentioned. A disease, related in its clinical signs and symptoms, that appears to be the result of an abnormal storage, in liver and brain cortex, of oligosaccharides containing D-mannose and 2-amino-2-deoxy-D-g~ucose has been described. The level of a-D-mannosidase in the liver and brain of the patients having the disease was quite low, although other glycosidases had raised activity compared with the norma1.405-408 The name mannosidosis has been suggested for the disease. The Morquio syndrome (type IV mucopolysaccharidosis) is associated with the excretion of large amounts of keratan sulfate in the urine.389The patients appear to have a normal pattern of enzymic activities in the liver l y s o s ~ m e sTypes . ~ ~ ~ V (Scheie syndrome) and (399) F. Van Hoof and H. G . Hers, Europ.]. Biochern., 7,34 (1968). (400) P.-A. Ockermann, Scund. J. Clin. Lab. Inuest., 22, 142 (1968). (401) M. C. MacBrinn, S. Okada, M. Woollacott, V. Pattel, A. L. Tappel, and J. S . OBrien, New EnglandJ. Med., 281,331 (1969). (402) M. W. Ho and J. S. O'Brien, Science, 165,611 (1965). (403) P. -A. Ockermann, Clin. Chirn.Acta, 20,1(1968). (404) K. Suzuki, Science, 159, 1471 (1968). (405) P. -A. Ockermann, Lancet, ii, 239 (1967). (406) P. -A. Ockermann, Lancet, i, 734 (1969). (407) P. -A. Ockermann,]. Puediatrics, 75, 124 (1969). (408) B. Kjellman, I. Gamstorp, A. Brun, P. Ockermann, and B. Palmgren, J. Paediatrics, 75,133 (1969).

STRUCTURE AND METABOLISM OF GLYCOPROTEINS

477

VI (Maroteaux-Lamy syndrome) are very rare, and both are associated with urinary excretion of large amounts of dermatan sulfate, although the diseases differ in their clinical manifestation^.^^^ Other forms of this disease that are, at present, unclassified are known, in which chondroitin 4-sulfate is excreted in excessive amounts in the ~ r i n e . It~ is ~ reasonable ~ * ~ ~ ~ to suppose that other glycosidase deficiencies may be found to underlie other genetically acquired diseases that involve glycoproteins.

REMARKS IX. CONCLUDING We have not attempted to give a complete review of the various aspects of glycoproteins, as this would be impossible in the space available. Instead, we have selected a number of topics intended to give an indication of the general status of the subject at the present time, in the hope of providing a stimulus to further studies. Glycoproteins are widespread, and they can be classified in such a way as to include glycosaminoglycans; indeed, we feel that not to include them precludes a consideration of systems in which both long-chain polysaccharides, such as are found in keratan sulfate, and smaller oligosaccharides occur attached to the polypeptide chain. Moreover, such diseases as the mucopolysaccharidoses (see p. 475) may result from deficiencies of various glycosidases which could affect the catabolism of many glycoproteins, although numerous observations point to a primary defect in the metabolism of some of the glycosaminoglycans. There are still many unanswered questions here. Four general types of carbohydrate-peptide linkage are now known, each requiring a specific enzymic pathway for its biosynthesis. Incorporation of this “first” sugar residue occurs, at least to a large extent, as an event subsequent to the ribosomal-translation reaction of messenger ribonucleic acid to polypeptide chain. More than one type of carbohydrate-peptide bond may be formed on the same polypeptide chain. The remainder of the sugar residues are usually added as a series of steps involving the addition of one sugar residue at a time, so that “finishing” of the carbohydrate moiety may not always occur in all molecules; this situation is possibly attributable to ( a ) lack of absolute specificity of the various glycosidases, and ( b ) the particular way in which these enzymes are arranged in the endoplasmic reticulum. This hypothesis might account for the hetero(409) M. M . Robins, H. F. Stevens, and A. Linker,]. Paediatrics, 62,881 (1963). (410) M . Philippart and G. I. Sugarman,Lancet, ii, 854 (1969).

478

R. D. MARSHALL AND A. NEUBERGER

geneity that appears to be common to the carbohydrate moieties of glycoproteins. However, partial catabolism may also be responsible for this type of variation in the structure. A related problem concerns the way in which the sizes of the carbohydrate moieties are limited. The classical studies of the C ~ r i s , ~which ll demonstrated for the first time a relationship between a deficiency in a single enzyme activity and the resultant, genetically determined, metabolic abnormality in von Gierke's disease, led to an understanding of analogous, enzymic defects in other diseases. That various storagediseases involving glycoproteins should occur is not surprising. It will clearly be of interest to determine whether the deficiency that has been d e s ~ r i b e d ~ of ' ~ one ,~~~ of the 2-acetamid0-2-deoxy-P-~hexosidase isoenzyme activities in extracts of a number of tissues in patients having Tay-Sachs disease results in an abnormal catabolism of certain glycoproteins, as well as of gangliosides. Urinary casts, often excreted by patients having renal disease, possess414antigenic determinants in common with the Tamm-Horsfall protein (see p. 414), and, indeed, this glycoprotein is the major component of the However, the reason for its aggregation is as yet unknown. Other pathological states may well be found to b e associated with abnormalities in the structure or metabolism, or both, of glycoproteins.

(411) G. T. Cori and C. F. Cori,]. Biol. Chem., 199,661 (1952). (412) S. Okada and J . S. O'Brien, Science, 165,698 (1969). (413) K. Sandhoff, FEBS Lett., 4,351 (1969). (414) E. G . McQueen,]. Clin. Puthol., 15,367(1962). (415) E. G. McQueen and G. B. Engel,]. Clin. Puthol., 19,392 (1966). (416) A. P. Fletcher, J . E. McLaughlin, W. Smith, and D. A. Woods, Biochim. Biophys. Acta, 214, 299 (1970).

AUTHOR INDEX FOR VOLUME 25 Numbers in parentheses are reference numbers and indicate that an author’s work is referred to although his name is not cited in the text.

A Abdel-Akher, M., 452 Abdel Rahman, M. M. A., 362, 364, 366 (62), 367 4bson, D., 214, 248, 281(59c), 282(59c) Aceto, M. D. G., 268 Ackers, G. K., 25, 27 Ackers, J. P., 412(42), 413 Acton, E. M., 143, 156, 178(167) 4dair, G. S., 416(67) Adams, G. A., 301 Adams, J. B., 412(44), 413, 430, 470 Adams, M. F., 41, 48(111) Adam-Chosson, A., 422 ( 123), 423 (123), 424 Afanas’eva, E. Ya, 257 Agrawal, K. M. L., 445, 465, 466(319) Aguilar, V., 415 Ahluwahlia, R., 207 Akabori, S., 442 Akagi, M., 274( 136), 277 (1371, 283 Akiya, S., 118, 185, 188, 213(35), 222(35, 46). 223(35), 275(140), 283 Akiyama, K., 291 Albano. E. L., 122, 14Q(54) Alberda van Ekenstein, W., 215. 332, 339 Alexandrou, N. E., 389 Glfes, H., 391 Ali, Y., 382 4lison, F., 286 GI-Jeboury, F. S., 265 Gllen, P. J., 301 Allen, P. Z., 457 Allen, W. S., 418 Allerton, R., 151, 176(146), 177( 1461, 279 (130), 283 Minger, H. W., 290 Allinger, N. L., 129, 145(66), 158(185) Alpers, E., 261 Mtgelt, K. H., 16 Alvarex Gonzilez, C., 371 4maral. E., 291, 295(70). 298(70) 479

Ambler, R. P.,425 Ames, B. N., 321(29), 322, 330(29), 331 (29), 332(29) Aminoff, D., 410(31), 411 Ammon, R., 303 Anai, M., 423(135), 424, 442(135) Andersen, C. C., 179 Anderson, A. J., 416 (69), 417 Anderson, B., 430, 431(164), 435(165), M ( 1 6 4 , 165), Ml, 459(207), %O (207) Anderson, C. D., 157, 178(177, 183) Anderson, D. M. W., 26, 27, 28(66), 38, 39, 4Q, 48(66, 100, 101, 102, 103, 104, 105, 106, 107), 49(108) Anderson, J. M., 155, 156 (1631, 178(163a) Anderson, L., 391 Anderson, R. H., 291 Ando, T., 302 Andreae, E., 182 Andrejeva, A. P., 422(122), 424 Andrews, P., 17, 18, 21, 26(29, 33), 28 (34), 38 Anet, E. F. L. J., 215, 343 Anfinsen, C. B., 425 Angier, R. B., 399 Angustyniak, J. Z., 423(128), 424 Angyal, S. J., 103, 106, 110, 111, 123(2), 129, 145(66), 158(185), 207, 265 Anikeeva, A. N., 250, 257, 268, 272(126), 273 (125). 283 Anno, K., 272(1%), 283 Ansell, E. G., 117, 173(27), 174(27) Anseth, A., 451 Antonakis, K., 162, 179(207) Aoyagi, S., 291, 304 Arai, Y., 291 Araki, C., 14 Archibald, R. M., 475 Ariza Toro, F., 371 Armand, G., %O Armbrecht, W., 186 Arnott, S., 86

AUTHOR INDEX, VOLUME 25

480

Aro, A., 415 Aronson, J. N., 391 Anolla, J. D. P., 291, 295(70), 298(70) Asai, M., 119, 120(34b), 156(34), 177 (34b). 178(34b) Ashida, T., 92 Aso, K., 291, 295, 297, 298, 300, 302 Aspberg, K., 33, 36(82) Asselineau, J., 215 Atassi, M. Z., 420 Au, C. C., 473 Austin, G. H., 290, 294 Austin, P. W., 131, 157(75), 158(75), 208 Avigad, G., 300, 302

B Bachhuber, J. J., 41 Bacon, J. S. D., 288, 301, 302, 308 Baculinschi, H., 291 Baddiley, J., 186, 208, 231, 232, 233, 272 (lo), 388 Baer, H. H., 402 Baggett, N., 265, 271 (134), 283 Bahl, 0. P., 442, 445, 448(214), 463, 465 (214, 310), %6(214, 310, 319) Bahner, K. J., 397 Bailey, R. W., 302, 349 Bain, J., 75 Baker, B. R., 116, 136, 151(109), 155(22), 156(22), 157, 159, 161, 177(22, log), 178(22, 168, 173, 177, 183, 186), 354, 369(10, la), 378(13), 381, 384, 386, (11, 16, 18) Baker, J. W., 428 Baker, S. B., a 5 , 266, 276(54), 279(54) Balchin, A. A., 67 Balda, K., 143, 144(131), 174(131) Ball, D. H., 167, 169(228), 277(141!, 283, 198 Ball, E. M., 419 Ballou, C. E., 33, 36(85, 86) Bangham, D. R., 45 Bannister, B., 249, 282 (59j) Bardolph, M. P., 167 Bardos, T. J., 88 Barford, A. D., 122, 353 Barker, G. R., 373, 374 Barker, R., 198, 224(71), 225(71), 230, 232, 233, 234(14a), 269, 271(5), 272(11, 14a)

Barker, H. A., 390 Barker, S. A., 15, 20(23), 34, 37(22, 23), 43, %, 47, 48(119, 120, 121, 129), 49(22, 23, 1301, 251, 300, 301, 302 Barlow, M., 87(93), 88 Barnes, R. A., 243 Bamett, J. E. G., 196, 225(64), 235 Barnhart, M. I., 473 Barry, G. T., 463 Barry, K. G., 269 Bartuska, V. J., 156, 157(172), 160(172) Bashford, V. G., 118, 191 Bassett, E. W., 432 Basu, D., 415 Batlle, A. M. del C., 26 Bauer, C., 397, 399(293) Bauer, H. F., 165 Baum, G., 388 Baumgarten, F.: 286 Bayne, S., 390(219), 391 Beau, R., 15, 37(21) Becher, P., 255 Becker, B., 269 Beckmann, P., 286 Beevers, C. A., 66 Bekker, P. I., 29, 30(76), 38(76), 48(76) Bell, A. V., 77 Bell, C. A., 362 Bell, D. J., 301 Bell, F. K., 268 Bella, A., 461(304), 462 Bellavadi, B., 300 BeMiller, J. N., 343 Bendich, A., 401 Benitez, A., 156, 178(168) Bera, B. C., 185, 186(37), 222(37), 277(142a), 283 Beran, P., 172 Beranek, W., Jr., 34, 36(92) Berard, A,, 410(32), 411 Bereneon, G. S., 409(27, 281, 423(28), 442, 446(216), 449(28) Bergmann, A., 397 Bergmann, E. D., 151 Berman, H. M., 57, 63, 64, 65(29), 77, 180(60) Bernfield, M. B., 408 Bernhauer, K., 321(31), 322, 324(31, 56), 325, 326(31), 327(31), 329(31), 331 (31), 347(31)

AUTHOR INDEX, VOLUME 25 Berrang, B., 194 Berthelier, R., 286 Bertolini, M., 419, 431 (171), 432 Bessler, J. G., 286 Beutler, R., 304 Bezborod’ko, B. N., 268 Bhattacharya, A. K., 224(128), 225(128), 226( 128), 227 (128), 228, 274(128), 283 Bhatti, T., 450(256), 451 Bhavanandan, V. P., 416(64), 417, 433, 434(64), 440(64), 441, 460(208) Bhoyroo, V. D., 445 Bick, S. M., 43, 48(121) Bihler, I., 269 Bilski-Pasquier, G., 473 Binette, J. P., 444 Binkley, W. W., 295 Binte, H. J., 400 Bird, J. W., 398 Birktoft, J. J., 425 Bishop, C. T., 297 Bjamer, K., 92, 98(112) Black, D. R., 157 Blackford, R. W., 358 Blacklow, R. S., 410(19), 411, 446(19) Bladon, P., 265 Blair. M. G., 299 Hlake, C. C. F., 93 Blake, R. D., 89 Blanchard, P. H., 301 Blanksma, J. J., 215 Blechschmidt, W., 286 Blomback, B., 422(108), 424, 473 Blomback, M., 473 Blow, D. M., 425 Blumbergs, P., 145, 148(143), 176(143), 360 Blumenfeld, O., 474 Hlumenkrantz, N., 468 Bobek, M., 209, 220(93, 94, 129), 228 Bock, R. M., 89 Bodenheimer, F. S., 286 Bodenheimer, T. S., 390 Rogdanov, V. P., 449(251), 451 Bogdanova, G. V., 242 Hollenback, G. N., 243 Bolliger, H. R., 121, 136, 142, 143, 174( 130)

481

Bollinger, K. M., 361 Bolshukhina, Yu. A., 257 Bombaugh, K. T., 15, 16, 37(14), 49(14) Bonferoni, B., 440 Boni, K. A., 20 Bonner, J., 444 Bonner, W. A., 244 241, 295, 382, 386, 403 Bont, C. R., 397 Bookchin, R. M., 420 Boothe, J. H., 399 Borders, C. L., Jr., 34, 36(92) Borenfreund, E., 216 Bornstein, 444, W ( 2 5 0 ) , 451 Bose, S. M., 419 Bosmann, H. B., 468, 470 Bourillon, R., 414(51, 541, 415, 422(100), 423, 424, 443 Bourne, E. J., 34, 301, 302 Bouveng, H. O., 42 Bowers, A., 196 Boyce, W. H., 414(52, 531, 415, 473 Boyer, R., 363 Branden, C. I., 89 Brandes, P., 319, 329, 330(14) Brandhoff, H., 198, 199(67), 225(67), 226 (67) Brandner, J. D., 260, 261 Brannon, W. L., 252 Bravar, D. A., 290 Bray, B. A., 440 Bremner, I., 37 Brendel, J., 144, 150(137), 175(137) Brendel, K., 377, 429, 436(159) Breuer, R., 339 Brewster, P., 183 Brew Vanaman, K., 98 Bright, W. B., 77 Brigl, P., 164 Brimacombe, J. S., 43, 48(121), 74, 252, 271(64, 133), 273(M), 275(135), 283, 358, 394, 459, %1(290) Bromund, W., 380 Brose, E., 397 Brown, B. D., 44,48(124) .Brown, D. M., 4Q3 Brown, G. M., 56, 61(9), 63, 76, 99(9), 101(27)

482

AUTHOR INDEX, VOLUME 25

Camerman, N., 255 Campbell, J. C., 74 Canfield, R. E., 97 Cantow, M. J. R., 20 Capek, K., 112, 115(10), 128(10, 691, 129, 142, 144, 145, 149(135), 150(135, 136), 174(10, 691, 175(10), 176(142), Capon, B., 200 Caputo, A., 422(102), 424 Carey, F. A., 167, 169(228) Carlisle, C. H., 67 Carlson, D. M., 432, 455(177) Carlson, J. A., 41 Carlson, L. J., 132, 133(80), 173(80) Caro, L., 472 Carpenter, C. M., 472 Carrington, T. R., 300, 301 Carroll, P. M., 382 Carroll, W. R., 47, 48(134) Carson, J. F., 231, 390 Carss, B., 208, 231, 232(8), 233, 272(10) Carubelli, R., 433 Cascorbi, H. F., 268 Casini, G., 156, 159(165) Cassidy, F., 243 Castellani, A. A., 440 Casto, M. E., 15 Catley, B. J., # ( l o ) , 411, 423(133), 424 Caygill, J. C., 474 Cernf, M., 112, 128(13, 701, 129, 132, 133(13, 70, 82), 172, 173(13, 70, 81, 82), 302 Cerrini, S., 90 Cessi, C., 364 Chaikin, S. W., 420 Chandrasekaran, K. S., 70 Chandrasekaran. R., 69, 70 Chang, C. J., 429 Chang, P., 121, 177(%a, %b), 178(%a) Charalambous, G., 112, 145 (81, 147 (81, 148(8), 175(8), 176(8) Chargaff, E., 3 a Chastain, B. H., 144, 149(138), 175(138) Chauvin, R., 285, 286(2) Cheese, I. A. F. L., 455(280), 456 C Chen, S.-H., 444 ChCrest, M., 184, 185(30) Caldas Feiho, C. F., 291, 295(70), 298(70) Calkins, D. F., 156, 157(171, 1721, Cherian, M. G., 414(57, 58), 415 159(171), 160(172), 178(171), 405 Childs, R. F., 151 Chilton, W. S., 375, 392(121) Camerman, A., 248, 255, 281(59h3

Brown, H. C., 420 Brown, R. K., 119, 135, 136(91, 98), 139, 169(232), 171 Brown, S. S., 207, 224(86) Brown, W. G., 420 Browne, C. A., 290 Browne, W. J., 98 Bru, L,321 Briining, J., 359 Brufani, M., 90 Brugger, M., 425 Brumbach, M. B., 475 Brun, A., 476 Brunngraber, E. G., 44, 48t124.1, 415 Brunt, R. V., 151 Bryan, J. G. H., 7 4 358 Buben, I., 128(70), 129, 133(70), 173(70) Buchanan, J. G., 112, 118, 125, 127(30), 128(9, 67, 681, 129, 130(67), 131, 136(30,73), 137(73), 140(73), 142(9), 143(67), 144(9, 62, 65, 67), 145, 1%(67), 148(9, 62, 67), 150(1@), 152(67), 153, 154, 157 (75), 158(75), 161 (741, 162(74), 170, 174(9, 67), 175(9, 62, 65, 67), 176(68), 186, 208, 231, 232(8), 233, 272(10), 388 Buchari, M. A., 256 Buchet, M., 429 Buck, K. W., 72, 271(134), 263, 283, 403 Buckley, J. P., 268 Buddecke, E., 426 Budovich, T., 300, 302(106) Bulman, M. W., 286 Buma, T. J., 77 Bunn, W. H., Jr., 268 Bunton, C. A., 438 Burckhart, O., 303 Burkert, H., 290, 295, 298(49), 305 Bushill, J. H., 77 Buss, D. H., 169(231), 171, 265, 353, 355 Bussey, A. H., 23 Butler, W. T., 439 Buyanov, V. V., 269

AUTHOR INDEX, VOLUME 25 Chlenov, M. A., 135 Christensen, J. E.. 136, 14001107), 155, 174(115), 352, 386(3) Chu, S. S. C., 56, 57, 63(10), 69, 75(10), 76, 99(10), 100(10, 55) Chua, J., 401 Chudakov, V. G., 291 Churms, S. C., 19, 20, 23, 24(62), 25(62), 28, 29, 30(76), 38(37, 62, 761, 39(62), 48(37, 76), 49(62) Cifonelli, J. A., 200, 211(74), 212(74), 213(74), 214(74), 223(74), 224(74), 234, 273(17), 441, 452f211), 460, 461 (298, 305, 3061, 462, 475(298) Cifonelli, M., 200, 211(74), 212(74), 213(74), 214(74), 223(74), 224f741, 234, 273(17) Cjuchta, H. P., 268 Claesson, S., 254 Clamp, J. R., 422(109), 424, 431(172), 432, 442(172), 444(172), 445(172), 4%(109, 172), 447, 448(108, 255), 450(256), 451, 4 5 f 1 0 9 ) , 474 Clark, E. P., 182, 191(161, 220(16) Clark, S. C., 268 Clauser, H., 430, 431(167) Claussen, V., 119, 169(35), 176(35) Clayton, L. C., 269 ClCophax, J., 218, 224(119, 1301, 225(116, 119, 1301, 227(116), 228, 354, 356, 361 Clouse, R. W., 422(121), 424 Coburn, J. W., 269 Codington, J. F., 156, 179(1?5), 200, 201, 211(76), 212(76, 781, 223(76). 224 (78), 401 Cohen, A., 216, 218, 246 Cohen, S., 151 Coleman, G. H., 167 Coleman, T. J., 41)9(24), 411, 422(24) Collins, P. M., 117 Combes, G., 134 Conchie, J., 446, %5, %6(322), 474 Conn, J., 130, 136(73), 137(73), 140(73) Connors, P. G., 89 Constantopoulos, G., 47, 48 (134) Cooke, K. R., 373 Cookson, R. C., 123, 124 Cope, A. C., 247, 257, 262, 264, 265(58, 93), 277(58), 279f581, 280(58)

43

Corbett, W. M., 341, 344(82), 345(92) Cori, C. F., 478 Cori, G. T., 478 Cornfield, P. W. R., 65 Cornforth, J. W., 364 Cornillot, P., 414(51), 415 Cote, W. A., Jr., 41, 48(112) Coulter, C. L., 87(95), 88, 92 Coupek, J., 21 Coupier, J., 473 Coutsogeorgopoulos, C., 418 Cox, J. S. G., 37, 50 Coxon, B., 237, 248, 275(25), 281(59e, 5%) Craig, J. M., 240 Crampton, C. F., 447 Crane, E., 289 Crane, R. K., 269 Creeth, J. M., 426, 434( 144a), 467( 144a) Creighton, A. M., 161, 162(197) Cremos, S. C., 268 Crews, S. J., 15 Crum, J. D., 232, 272(11) Crumpton, C. W., 268 Csizmadia, I. G., 266 Csizmadia, V. M., 252, 279(63), 280(63) Csiiros, Z., 164 Cunningham, B. A., 424 Cunningham, J., 383 Cunningham, L. W., 415, 422(121), 424, 436,438,439, 442(191), 445, 446 (2351, 4848 (254), 449(254, 451) Curtin, D. Y., 184, 389 Curylo, J., 295, 298 (86) Cushley, R. J., 4Q5 Cusumano, C. L., 425 Cynkin. M. A., 422(106), 423(105), 424

D D’Abramo, F., 471 Dacons, J. C., 33 Dahlgard, M., 144, 149(138), 175(138) Dahlquist, F. W., 34, 35, 36(92, 93) Dahn, H., 397 Dale, J. K., 61 Daniher, F. A., 360 Danilov, S. N., 135, 234, 236(16), 243, 250, 257, 258, 268, 272(126), 273(16, 23, 125), 274(23), 277(50), 283 Danishefsky, I., 461 (3041, 462

484

AUTHOR INDEX, VOLUME 25

Darby, W. J., 323, 330(44), 331(44), 332 (44), 349 (45) Dark, W. A., 15, 16, 37(14), 49(14) Darnell, K. R., 243 Dauben, W. G., 184 Davidson, B. E., 136, 144(113), 151(113) Davidson, E. A., 418, 429, 435, 436(159, 187), 442(73), 450(268, 2-59), 451, 47 1 Davies, H. F., 416(67), 417 Davis, R. E., 71, 85 Davison, B. K., 319 Davoll, J., 121, 155(48), 157(48), 159(48), 178(48) Dawson, G., 431(172), 432, 4+42(172), 444(172), 445(172), 4%(172), 448 (255), 451, 474 Dayhoff, M. O., 425 Dea, I. C. M., 26, 28(66), 38(66), 39, M(66, 102, 103, 104, 105, 106), 49 (108) Deik, G., 164 de Belder, A. N., 38, 49(98) Debus, H., 327 de Castro, R., 363 de Castro Bnezicki, R., 363 Dedonder, R. A., 302 Defaye, J., 119, 183, 186, 192, 194(59), 198, 199, 203, 205, 211, 212(99), 213(43, 68, 99), 215(68), 216(43, 68), 217, 218, 221(82, 106), 222(82, 991, 223(43), 224(68, 114, 1191, 225(68, 114, 117, 118, 119), 226(68, 81, 100, 113, 117, 131), 227(113), 228(115), 229, 234, 235(20), 272(19), 273(19) Degener, S., 339 Dekaban, A. S., 47, 48(134) Dekker, C. A., 160(195), 161, 206, 211 (85), 216(85), 221(85), 243 de la Camp, U., 55 Delaney, R., 422(114), 424 DeLange, R. J., 423(126), 424, 444 Delevallt, D., 382 Dellweg, H.,32, 33(81), 36(81) De Luca, S., 471, 472(350) Denot, E., 196 de Pascual Teresa, J., 165, 166(213) Derevitskaya, V. A., 426, 428(1%), 429 (1%), 429, 434(1%) Descamps, J., 431(173), 432, 4+42(173)

Determann, H., 16, 26 Dethier, V. G., 300 de Vries, A. J., 15, 37(21) Dewar, M. J. S., 428 Dhar, M. L., 427 Dick, A. J., 116, 151(23), 176(23), 177 (23) Dickey, E. E., 33 Dickinson, B., 288, 302, 308 Dickinson, M. J., 405 Diehl, H. W., 235, 273(22) Diels, O., 364 Dietrich, C. P., 34, 36(91) Dietz, A. J., Jr., 268 DiFerrante, N., 475 Dijong, I., 339 Dimitriyevich, S., 142, 146(126a), 173 (126a), 174(126a) Dimler, R. J., 302, 373, 374, 375(110) DiSant'Agnese, P. A., 473 Dische, Z., 216, 473 Dittrich, K., 397 Dmochowski, A., 435 Doerr, 1. L., 156, 200, 201, 211(76), 212(76, 78), 223(76), 224(78), 401, 405 Uoguchi, M., 291 Donald, A. S. R., 426, 434(144a), 454 (277, 278), 455(278), 456, %7(14+4a) Donatti, J. T., 401 Doolittle, R. F., 408 Dorfman, A., 435, 460, 461(298, 3061, 462, %8(189), 475(298) Dorofeenko, G. N., 100, 242 Doroshenko, S. S., 242 Doudoroff, M., 303, 390 Dow, J., 87(94), 88 Dowgiallo, A., 162, 179(209) Drescher, E., 382 Druey, J., 371 Dryselius, E., 335 Duchamp, D., 85 Dudkin, M. S., 318, 348(13) Duff, R. B., 132, 161 Duh, J., 461 (302), 462 Duisberg, H., 287 Dukefos, T., 92 Duncan, W. A. M., 302 Dunlop, H. G., 142, 173(123)

485

AUTHOR INDEX, VOLUME 25 Dunne, F. T., 410(9), 411, 446(9) Dunstone, J. R., 20 Duquesne, N., 422(110, 113), 424 Durant, G. J., %1(303), 462 Durso, D. F., 290 Dursum, K., 50 Dutcher, J. D., 399 Duxbury, J. M., 263 Dwek, R. A., 74 Dyfverman, A., 167 Dzyak, V. N., 268

E Eades, E. D. M., 277(141), 283 Echigo, T.,295 Eckert, J. E., 290 Eckhart, E., 393 Eckstein, F., 87(%), 88 Edelman, G. M., 424 Edelman, J., 299 Edmond, E., 20 Edmonds, L. O., 231 Edmundson, A. B., 423(116), 424 Edward, J. T., 380 Efimova, G. A,, 258 Egami, F., 466(327), 467 Eich, H.,161 Eichstedt, R., 335 El Ashmawy, A. E., 192, 194(60), 358, 359, 361(35), 379(35) El Ashry, S. H., 365, 3% Eldon, H. E., 444 Elegood, J. A., 290 Eliel, E. L., 129, 145(66), 158(185) El Khadem, H. S., 60, 92(18), 103(18), 104(18), 362, 364, 365, 366(62), 367, 388, 389(193), 390(218), 391, 392(73, 218), 396 Ellefsen, 6, 43, 48(118) Elleman, T. C., 423(127a), 424 El Sadany, S., 362 Elsden, D. F., 416(68), 417, 439 Elser, E., 295 El-Shafei, Z. M., 367, 391, 392(73) Ely, K. R., 423(116), 424 Emoto, S., 157, 158(180), 178(180) Engel, G . B., 478 Engelmann, J., 385 Englis, D. T.,295

English, J., Jr., 161, 163(203) Erden, B., 399 Erlbach, H.,397 Ettel, V., 390 Etten, R. L., 79 Ettling, B. V., 41, 48(111) Evans, D. R., 300 Evans, M. E., 275(135), 283 Everse, J., 4Q9(14), 411 Eylar, E. H., 411, 444, 448(252), 451, 455(283), 4.56,459, %7, 468, 470, 474 Ezekiel, A. D., 167

F Facon, M., 410(25), 411 Fahey, J. L., 446 Fahim, F. A., 403 Faillard, H., 464 Famborough, D. M., 444 Fargher, R. G., 324(51), 325, 328(51), 330(51), 345(51) Farkai, J., 209, 220(93, 94, 1291, 228 Farquhar, S., 20 Farr, A. L,413 Farrar, K. R., 374 Fatiadi, A. J., 391 Fawcett, J. K., 69, 102(38) Fayos, I. P., 79 Feast, A. A. J., 135, 136(102), 139(102), 151(102), 177(102), 34J Fecher, R., 156, 179(175) Fedeli, E., 90 Fedorova, E. F., 250 Feinberg, B., 287 Feingold, D. S., 302 Feldkampf, J., 382 Felkin, H., 184, 185(30) Fellig, J., 299 Fellows, R. E., 422(114), 424 Fernandez, R., 429 Ferdndez-Bolaiioa, J., 370, 371, 372 Fernhdez Jimenez, J., 363 Ferrari, R. A., 269 Ferri, G., 480 Ferrier, R. J., 119, 169, 275(139), 276 (1311,283 Ferrier, W. G., 56 Field, T. D., 214 Fielden, M. L., 414(52, 53). 415 Finch, P., 43, 48(119)

486

AUTHOR INDEX, VOLUME 25

Firth, M. E., 364 Fischer, E., 182, 185(6), 222(6), 397 Fischer, E. H., 299 Fischer, H. 0. L., 248, 279(59a), 280(59a), 281 (59a) Fischer, L., 14, 290 Fisher, L. V., 154, 176(160) Fisher, P., 429 Fishkin, A. F., 409(27), 411 Fithing-Litt, C., 303 Fitzmaurice, C., 50 Flaherty, B., 167, 168(224), 172(224) Flechtmann, C. H. W., 291, 295, 298(70) Fleming, M., 348 Fletcher, A. P., 414(55), 415, 449(260), 451, 478 Fletcher, H. G., Jr., 198, 224(71, 128), 225(71, 128), 226(70, 128), 227(128), 228, 233, 234(14a), 237(15), 248, 258, 260, 272(14a, 1421, 273(80), 274 ( 128), 275 ( 138), 278 ( 151, 279 (1301, 281(59e, 59f), 283, 381 Fletcher, R., 128(67, 68), 129, 130(67), 143(67), 144(67), 145, 1%667), 148 (67). l50(144), 152(67), 153, 154, 174(67), 175(67), 176(68) Flodin, P., 14, 16, 18, 21(27), 22, 26(35), 30, 33, 34, 35(3, 35), 36(82, 891, 48(35) Floridi, A., 42!2(102), 424 Fochem, K.,286 Folkes, B. F., 23, 25(59) Ford, J. D., 415, 422(121), 424, 4 6 , 438, 439, 442(191), 445 Forrest, H. S., 399 Foster, A. B., 119, 122, 130, 136, 142(72), 1%(72, 126), 185, 186(37), 191(49), 192, 200, 209, 211(75), 222(37), 223 (91), 234, 252, 256, 263, 265, 271(64, 133, 134), 273(64), 275(135), 277 (142a), 278(143), 283, 297, 403 Fox, I. J., 142, 156, 157, 173(125a), 174 (125a), 178(167a, 18Oa), 179(175), 200, 201, 211(76), 212(76, 78), 223 (76), 224(78), 377, 401, 405 Fraga, E., 135 Franklin, E. C., 442 Fransson, L. A., 460, 461(301), %2 Fraser-Reid. B., 194, 195(61), 222(61), 223(61)

Fratantoni, J. C., 475 Fratini, A. V., 55 Frazer, J. G., 286 French, D., 300, 399 Frenkel, G., 308 Frenzel, H., 166 Fresco, J. R., 89 Fretzdorff, A.-M., 385 Freudenberg, K., 161, 179 Friebolin, H., 238, 239(31b), 276(31b), 277(31b), 278(31b) Friedlander, A., 461 (3041, 462 Friedman, H. C., 92 Friedman, L., 267 Friedmann, T., 415 Fries, D., 107 Fudeya, G., 295 Fudeya, K., 291 Fiirst, A., 123, 135(59) Fuji, K., 90 Fujii, S., 321(35, 36), 322, 324(36), 328 (35, 36), 329(36), 341, 344(82), 345(36), 346(84), 348 Fujioka, H., 291 Fukushi, S., 437 Fuller, C. H. F., 77 Fuller, W., 86 Funcke, F., 373, 3%(101) Furberg, S., 87(94), 88, 92, 98(112) Furgala, B., 288, 291(38), 297, 298, 301, 304 Furs, L. T., 268 Furth, O., 363 Furuhata, T., 412(45), 413

C Gagnon, O., 269 Gall, W. E., 424 Gallop, P. M., 420 Gamstorp, I., 476 Garcia Gonzilez, F., 362, 363, 370, 371, 372 Garegg, P. J., 121, 151(52), 176(52) Garreau, Y., 323, 331(43, 47, SO), 332(43, 47, 50) Gasch G&rnez, J., 362, 372 Gascon, S., 419 Gauhe, A., 463 Gautier. R. F., 268 Geigg, R., 300

AUTHOR INDEX, VOLUME 25

M7

Gottschalk, A., 335, 408, 410(33), 411, 419, 426, 433, 435, 446(182), 449 (2631, 451, 463(182), %4(6). Gould, H., 267 Graham, E. R. B., 409(17, 18), 411, 419, 435, 437, 446( 182), 463(182) Graham, I., 422 (117a), 424 Granath, K. A., 18, 22, 23, 26(35), 27(69), 28(69), 30, 35(35), 37, 42, 48(35, 56, 69) Grandel, F., 236 Grant, A. B., 185, 212(36), 213, 222(36), 223(36) Grant, J. K., 374 Grant, P. M., 302 Gray, H. E., 308 Grebner, E. E., 435, 468(188) Greenstein, J. P., 432, 472 Gregory, J. D., 34, 36(89), 440 Greiling, H., 419, 440, 441, 460(209) Grewe, R., 179 Griess, P., 373, 396(100) (40) Griffith, C. F., 114, 135(16), 136(16), Gireva, R. N., 323, 330(49), 331(49), 173(16), 174(16) 332 (49) Grii, K. V., 300 Glass, G. B. J., 412(41), 413 Grimmett, M. R., 311, 319, 326(21), 326 Glazer, Z. T., 381 (58, 59), 327, 328(21, 58, 59, 64), Glinski, R. P., 145, 1481443). 176(143) 330(64), 332, 344, 345(1, 58, 59), Gluzman, K., 161 347(64), 348, 349 Gochnauer, T. A., 304 Grollman, E. F., 470 Goebel, W. F., 463 Grondahl, N. J., 422(108), 424 Goepp, R. M., Jr., 221(122), 228, 260 Gross, D., 301 Golab, T., 136 Gross, P. H., 144, 150(137), 175(137), 377 Goldschmidt, S., 290, 295, 298(49), 305 Gross, W., 397 Gomatas, J., 475 Groth, P., 58 Gbmez Sinchez, A., 362 Grudus, G. M., 267 Gongwer, L. E., 269 Gruezo, F., 455(276), 456 Goodman, G. C., 472 Gubero, M., 371 Goodman, H. O., 414(52), 415 Gudbjarnason, S., 268 Goodman, L., 136, 140(107), 143, 155, Gueffroy, D. E., 154, 176(160), 358 156, 157(171, 172), 159(165, 169, Guillemin, C. L., 15 171), 160(172), 174(115), 178(167, Gulland, J. M., 373, 374 168, 171, 177, 178, 183, 192, 352, 358, Gunther, F., 303 380(1), 386(3), 405 Gupta, S. J., 145, 1488(143), 176(143) Goody, R. S., 142, 173(125a), 1741125a) Gut, M., 121, 137, 173(4.3) Gordon, A. H., 463 Guthrie, R. D., 122, 135(57), 136(57, 94). Gorin, P. A. J., 208 139(57), 14Q(57), 144(113), 151(113), Got, R., 414(51), 415, 422, 423(100), 424 353, 354, 355, 356, 395, 4Q4 Gotschlich, E. C., 44, 48(123) Gyr, M., 137 Gottlieb, P. D., 424

Geisler, A. S., 268 Gelb, I. J., 268 Gelotte, B., 17 Gennaro, A. R., 286 Gent, P. A., 74 Genzer, J. D., 323 Gerecs, A., 202, 242, 380 Gdro, S. D., 211, 212(99), 213(99), 218, 222(99), 224(119, 130), 225(116, 119, 130), 227(116), 228, 354, 356, 361 Gerster, J. F., 243 Gertman, P. M., 269 Gibbons, R. A., %4 Gibbs, C. F., 353 Giddings, J. C., 21 Gigg, J., 358, 383 Gigg, R., 358, 382, 383, 385 Gilardi, R. D., 55 Gilman, P. T., 111 Ginsburg, V., 470 Girard, P., 323, 324(4Q), 331(40, 411, 332(4Q, 41). 340(40), 345(40), 347

AUTHOR INDEX, VOLUME 25

488

H Haarmann, R., 182 Haas, H. J., 339, 3% Hackert, M. L., 107 Hackman, R. H., 417 Haeger, B. E., 268 Haklcinen, I., 412(40), 413 Haga, M., 277 (137), 283 Hagopian, A., 467, 468 Haines, A. H., 384 Hall, C. W., 435, %8(188), 475 Hall, D. M., 3!?7 Hall, L. D., 122, 161(56), 162(56), 169 (231), 171, 172, 252, 265 Hall, M., 45, 48(127), 50 Haller, W., 15 Halliday, J. E., 268 Ham, J. T., 107 Hamada, A., 275(140), 283 Hamilton, J. A., 79 Hamilton, J. K., 452 Hammer, H., 58 Hamor, T. A., 72, 74 Hampel, A., 89 Hampton, A., 405 Han, L. C., 422(106), 424 Han, R. L., 144, 149(138), 175(138) Hancock, E. B., 271(133), 283 Haneishi, T., 399 Hanessian, S., 126, 135, 137, 143(121), 173(62a), 174(121), 357, 364, 394 395, 4Q4 Hanhart, W., 427 Hanisch, G., 135, 141(lOQ), 157 (198), 161, 163(198), 364, 390(221), 391 Hanke, M. T., 320, 329(25), 332(25) Hann, R. M., 133, 173(83), 235, 273(22), 373, 390 Hannaford, A. J., 275 (139), 283 Haq, S., 301 Haraszthy, M., 164 Harboe, A., 416(65), 417 Harbon, S., 430, 432(167) Hardegger, E., 164, 165, 166(213), 216, 221(111), 223(111), 390(217, 218, 2201, 391, 392(218) Harding, M. M., 75 Hardy, H. A., 161 Harmon, R. E., 165, 166(213a) Harris, D. R., 82, 88

Harrison, R., 377 Harrow, G., 373, 3%(100) Hartiala, K., 412(4Q), 413 Hartley, B. S., 425 Hartman, F. C.,230, 269, 271(5) Hartmann, L A., 236(82), 260, 279(147), 283 Harvey, W. E., 135, 161(88) Haschemeyer, A. E. V., 87(92), 88 Haschmeyer, R. H., 422(106), 423(105), 424 Hashimoto, H., 421 Hashizume, T., 160(195), 161, 206, 211 (85), 216(85), 221(85), 243 Haskell, T. H., 137, 143(121), 174(121), 357, 36% 39% 395 Haskins, W. T., 373, 390 Haslam, E., 244, 245(51), 276(51b), 277 (51) Hasselquist, H., 397 Hassid, W. Z, 303, 390 Hatt, B. W., 15. 20(237), 37(22, 231, 49(22, 23) Hattori, Y., 26 Hauenstein, H., 136, 137(116) Haukanes, G., 416 (65). 417 Haun, R., 198, 199(67), 225(67), 225(67) Haupt, H., W(22,231, 411, 4-44 Hawkins, C. F., 47 Hawkins, K. A., 269 Hawkinson, S. W., 92 Haworth, W. N., 174, 181 Hay, A. J., 446, 465, %6(322) Hay, J. E., 243 Hayden, A. L., 252 Hayes, M. H. B., 43, 48(119, 120) Haynes, L. J., 243 Hayward, L. D., 247, 252, 254, 262, 265 (59b), 266, 267, 279(63), 280(63) Hearn, V. M., 470 Hedgley, E. J., 116, 135(24), 1444241, 149(24), 150(24), 160(189), 161, 198, 226(70), 258, 260, 273(80) Heerema, J., 243 Heide, K., 4Q9(22, 23),411, 444 Heimburger, N., 409(22), 411 Heiskell, C. L., 472 Heitz, W., 21 Helferich, B., 175, 176(24QO) Heller, J., 42, 48(115)

AUTHOR INDEX, VOLUME 25 Helting, T., 34, 36(90), 435, 460(185),

%9 Helvey, T. C., 306 Hendrickson, H. R., 461(303), %2 Hendrie, A., 40 Hendriks, K. B., 143, 144(131), 174(131) Henne, A. L., 196 Henseke, G.,135, 141(104), 157(198), 161,

163 (198), 364, 390(221), 391, 397, 398, 399(273, 289) Herbst, R. M., 380 Heredia Moreno, A., 371 Herman, G.,430, 432(167) HeIminkovL, V., 161, 179(191) Hernandez, A., 269 Herr, R. R., 249, 282(59k) Herrmann, R., 321 (32), 322, 324(55), 325, 331(32, 551, 332(32, 55) Hers, H. G., 476 Hessel, B., 422(108),424 Hestrin, S., 302, 419 Hewins, M.,47 Heyns, K., 261, 312(5), 313, 333(5), 335, 339(5), 359, 360(34), 361(34), 377, 394, 395 (246) Hickinbottom, W. J., 165 Hickson, J. L., 399 Hielscher, M., 396, 397, 398, 399(278, 285) Higharn, M., 429 Hildesheim, J., 203, 211, 216, 218, 224 (130), 225(116, 130), 226(81, 100, 113), 227(113, 114, 115, 116), 228, 354, 356, 361 Hill, R. L., 98, 422(114),424 Hiller, E.,286 Himel, C. M., 231 Hinrichsen, D. F., 426, 467(143) Hinsberg, O., 373, 396(101) Hinson, K. S., 300, 301(105) Hirabayasbi, M., 90 Hiraoka, T., 172 Hiron, F., 183 Hirs, C. H. W., 409(7), 410(9), 411, 423 1129, 131, 1321, 424, 425, 448(129), M(9) Hirst, E. L., 38, 42, 48(101, lM),174 Hjerten, S., 14, 15 Ho, M. W.,476 Hockett, R. C., 22(122), 228, 375 Hodge, J. E., 335

489

Hodges, R., 319, 326(21), 328(21) Hodgson, A., 86 Hoeksema, H., 249, 282(59i, 59j) Hoffman, H. L., Jr., 321(37), 322, 329

(37),331 (37) Hoffman, P.,

430, 431(164), 435(165), 440(164, 165), 441, 459(207), 460 (207,213), 471, 475 Hoge, R., 73, 107 Holdaway, F. G., 304 Holfeld, W.,243 Holly, F. W.,405 Holmes, R. L., 290 Holmquist, W. R., 420 Holton, P. G.,1% Holton, S. L., 155, 156(164), 157(164),

158, 159( 164), 177( 164), 178( 164) Holysz, R. P., 240, 241(35), 276(38) Homer, R. F., 136 Honeyman, J., 117, 151, 154, 173(27), 174

(27),177(147) Hope, H., 55 Hopton, F. J., 254 Horbett, A. P.,446 Horowitz, J. P., 401 Horowitz, M. I., 412(41), 413, 446, 447 Horsfall, F. L., 415 Horton, D.,60, 92(18), 103(18), 104(18),

122, 140(54), 167: 187, 192, 194G9, 60), 199, 214 357, 358, 359, 361(353, 379(35), 388, 392, 398 Hough, E., 91 Hough, L., 122, 151, 161(56), 162(56), 169(231), 171, 177(150), 248, 252, 265, 281(59g), 282(59g), 312(2, 3), 313, 314, 318(7), 328(2, 3, 6, 71, 330(7), 333(2, 3, 6), 344(6), 347(6), 353, 355, 465, 474 How, M. J., 45, 49(131) Howe, C., 416(64), 417, 434(64), 440(64) Huang, C. C., 445, 465(236), 466(236) Hubbell, G. J., 4Q9(27), 411 Huber, G.,110, 165, 371 Huber, H., 116, 121(21), 136(21), 137 (21) Huber, M., 300 Hudson, C. S., 114, 133, 135(17), 173(83), 234, 235, 237 ( 151, 273(22). 275 (27, 138). 276(27), 278(15), 283, 288, 302, 308(35), 373, 375, 390

AUTHOR INDEX. VOLUME 25

490

Huebner, C. F., 209, 324(53), 325, 331 !53), 332 (53), 373, 375 (110) Hughes, E. D., 183, 427 Hughes, N. A., 192 Hughes, R. C., 463, %6(311) Hukins, D. W. L., 86 Hullar, T. L., 354, 369(10, 18), 386(11, 18) Hummel, B. C. W.,37 Hunedy, F., 394 Hunoz Gonzilez, P., 371 Hunt, D. J., 87, 88 Hunt, L. T., 425 Hunter, C. H., 475 Hunter, F. D., 59 Hunter, J. M., 121 Hurd, C. D., 239(37), 2444 241(35), 276 (37, 38), 295 Hurler, G., 475 Hursthouse, M. B., 80, 91 Hutching, B. L., 399 Hutchinson, E., 256 Hutson, D. H., 302 Hutson, N. K., 423(116), 424 Hvoslef, J., 57, 91(11) Hybl, A., 78 Hygstedt, O., 35, 36(94) Hynd, A., 188, 222(47)

I Iball, J., 87 Iber, F. L., 269 Igarashi, H., 401 Iitaka, Y., 81, 87(90), 88 Ikehara, M., 377, 4Q5 Ikenaka, T., 423 (135), 424, 442(135) Illes, E., 380 Imanaga, Y., 192 Inatome, M., 238, 273(30) Inch, T. D., 135, 136(99a), 138(99a), 142, 146(126), 381 IngIes, D. L,394 Ingold, C. K., 183, 427, 428 Inoue, S., 412(43), 413, 416(66), 417 Inoue, Y., 164, 166 Inouye, H., 90 Inouye, K., 320, 329(22), 331(22) Ioirish, N. P., 286 Irvine, J. C., 188, 222(47) Isaacs, N. S., 110, 111(4), 122(4), 210,

218(95) Isaacs, N. W.,107 Isbell, H. S., 61, 102, 214, 335 Isbell, J., 88 Ishida, T., 295 Ishifuku, K., 372 Ishiguro, S., 135 Ishihara, K., 422(98), 423(98, 1031, 424 Ishizu, A., 341, 343 Itgan, A., 399 Ito, T., 386 Ito, Y., 377 Iwai, I., 119, 120(34b), 156(34), 172, 177 (34b), 178(34b) Iwanaga, S., 409(12), 410(13), 411, 422 (108), 424 Iwashige, T., 119, 120(34b), 156(34), 162, 172, 177(34b), 178(34b)

J Jackobs, J., 107 Jackson, M., 247, 252, 262, 265(59b), 266, 267, 279(63), 280(63) Jackson, R. F., 294 Jackson, R. L., 423(131), 424, 425 Jacobs, S., MU, 460 Jacobson, R. A., 107 Jacquier, R., 134 Jakobi, R., 397 James, S. P., 132, 133(76), I73(76) James, W. J., 399 Jamieson, G. A., 4.16, M ( 2 3 9 ) Jamieson, N. C., 135, 136(91) Janson, J., 335 Jao, L., 34, 36(92) Jarvis, D. C., 286 J a ~ J., , 112, 115(10), 119, 128(10, 69), 129, 142, 143, 144, 145, 149(135), 150 (135, 1361, 161, 162, 174(10, 69, 1321, 175(10), 176(142), 179(191, 205), 394 Jeanloz, D. A., 174, 381 Jeanloz, R. W., 132, 135, 169(234), 173 (791, 174, 381, 423(99), 424, 463, 466(311) Jeffrey, G. A., 53, 56, 57, 58(16), 59, 60 (17), 63(10), 64, 65(29), 69, 75(10), 76, 99(10), lOO(10, 551, 102(16), 1@4(17), 105, 107 Jellinek, G., 391 Jenkins, H., 239

AUTHOR INDEX, VOLUME 25 Jenner, F. A., 474, 475(377) Jensen, L. H., 86, 87(87, 94), 88 Jevons, F. R., 474 Jewell, J. S., 167 JeZo, I., 318, 319(10), 321(30), 322, 328 (10, 20, 30). 329(20, 30), 330(20), 333, 335(10, 20, 68),339(10) Jochims, J. C., 380 Johansen, P. G., 422(119), 424, 449(261), 4S1, 473 Johansson, I., 142, 174( 124) John, M., 32, 33, 36(81) Johnson, J. F., 20 Johnson, L. F., 404 Johnson, L. N., 71, 93, 96(115), 97 Johnson, S. M., 74 Jollts, P., 430, U I ( 1 6 7 ) Jones, J. K. N., 40, 49(109), 116, 118, 151 (23), 176(23), 177(23, 150). 312(2, 3), 313, 314, 318(7), 328(2, 3, 6, 7), 330(7), 333(2, 3, 6), 344(6), 347(6), 357, 394, 398, 429 Joseph, J. P., 381 Jovenceaux, A., 410(32), 411 Junnila, R., 270 Jury, E., 450(269), 451

K Kabasawa, I., 425 Kabat, E. A., 432, 433, 454(179), 455 (276, 279), 456, 457(179) Kajanne, P., 270 Kajima, M., 135, 139(101) Kakudo, M., 92 Kalf, G. F., 300 Kalman, T. I., 87 Kalvoda, L., 133 Kam, A. J. H., 324(54), 325, 329(54), 330(54), 347(54) Kamber, B., 425 Kamiyama, S., 444, 448(251), 451 Karniyo, K., 85, 86(75) Kamprath-Scholtz, U., 135. 140(103) Kan, G., 161 Kanai, T., 401 Kanie, T., 268 Kano, T., 84 Kantor, T. G., 471 Kaplan, M. E., 432 Kaplan, N. O., 409(14), 411

491

Karabinos, J. V., 385, 390 Karasawa, I., 164, 165, 166 Karle, I. L., 55 Karrer, P., 222(127), 228, 399 Kasztreiner, E., 221(124), 228, 245, 250 (531, 275(53), 277(52) Katchalski, E., 410(35), 411, 449(35), 461 (35) Katsura, N., 418, 435, 436(187), 442(73) Katzman, R. L., 455(283), 456, 459 Kaufrnan, H. H., 465 Kaufmann, H., 144, 175(141) Kaustinen, H. M., 41 Kavelzneva, E. D., 422(122), 424, 449 (259), 451, 474 Kazimirova, V. F., 234, 236(16), 272(16) Kefalides, N. A., 409(29), 411 Kefurt, K., 119 Kegeles, G., 447 Keglevic, D., 420 Keil, K. D., 399 Kelly, R. B., 31 Kennard, C. H. L., 107 Kennedy, J. F., 15 Kenner, J., 161, 341, 344(82), 3465(81, 83, 92 ) Kent, P. W., 74, 151, 152, 176(148), 177 (158), 196, 222(65), 225(64), 235, 412(42), 413, 414(56), 415, 429 Kerr, R. W., 270 Keup, N., 295, 298(84) Khalifrnan, I., 289 Khan, M. Y., 474 Khorlin, A. Yu., 381 Khym, J. X., 403 Kienzle, F., 402 Kiessling, H., 42 Killander, J., 22, 23, 25(M), 29, 30 Killean, R. C. G., 56 Killey, M., 302 Kim, H. S., 58(16), 59, 60(17), 61, 63, 64, 65, 89, 102(16), 1@4(17), 105, 107 Kirnoto, E., 412(39), 413, 463(39) Kimura, E., 268 King, D., 356 King, J. A,, 323 King, J. S., 414(52, 53), 415 King, R. D., 167, 168(227), 172(227) King, R. N., 15, 37(14), 49(14) Kinnard, W. J., 268

A U T H O R INDEX, VOLUME 25

492

Kirkegard, L., 89 Kishi, T.,85, 86(75) Kiss, J., 166 Kisters, R.,419, 441 Kitahara, S., 26 Kitaoka, S., 397 Kjellman, B., 476 Klainer, S. M.,447 Kleinheidt, E. A., 203 Kleppe, K., 419 Klinkenberg, A., 21 Klinov, E. M., 429 Klosterman, H.,231, 232, 271(6) Klundt, J. L., 167, 169(228) Kluskey, J. E., 302 Klyne, W., 184 Knecht, J., 460, 461 (298),475(298) Knight, E. C., 397 Knoevenagel, C., 161 Knoop, F., 320, 329(24), 331(24), 340,

347 (24) Knudyntsev, N. A., 279(144), 283 Kobata, A., 470 Kobayashi, T., 399 Koch, H. J., 214, 248, 279(59b), 281(59b,

(84), 345(4, 33, 34, 36, 38), 3%(84), 347, 348(36), 349(39) Konstas, S., 377, 382 Kopaevich, Yu. L., 381 Kornhauser, A., 420 Kornilov, V. I., !N2 Korol’chenko, G. A., 242 Korte, F., 119, 169(35), 176(35) Korytnyk, W.,420 Kosugi, N., 290 Kotick, M. P., 88 Koto, S., 122, 133(53a), 377 Kovalenko, V. I., 161 KovaF, J., 128(69), 129, 142, 145, 161, 162,

174(69), 176(142), 179(191, 205) Koyama, G., 81, 83, 64, 85, 243 Krahn, R. C., 375, 392(121) Kralky, E., 305 Krantz, J. C., Jr., 268 Kringstad, K., 43, 48(118) Kristen, H.,198, 199(67), 225(67), 226

(67)

Krivoruchko, R. M., 242 Kriiger, C.,339, 377 Kruger, F.,371 Krupin, T., 269 59h ) Kruyff, J. J., 396 Koch, W., 312(5), 313, 333(5), 339(5) Kochetkov, N. K.,135, 267, 426, 428(146), Kubasskaya, L. A., 242 Kudryashov, L. I., 135 429(146), 434(1%) Kuhn, R., 198, 22!5(69), 335, 339, 377, 463, Kocourek, J., 135, 161(92) 464 Kodoyne, M. I., 294 Kulev, L. P., 323, 330(49), 331(49), 332 Kochling, H.,382, 429 (49) Kijnjg, W.,419 Kulkarni, S. R., 377 Koeppen, B. H.,297 Kullnig, R. K., 134 Koepsel, H. J., 390(215), 391 Kum, F., 429 Koessler, K. K., 320, 329(25), 332(25) Kundig, W.,417 Kogan, E. M.,236, 273(23), 274(23) Kuranari, T., 412( 39), 413, 463( 39) Kohtes, J., 299 Kusaka, T.,85, 86(75) Kojima, N.,268 Kushnir, I., 289, 291(45), 294(45), 303, 305 Kojima, T.,474 Kuszmann, J., 211, 221( 101) , 227( 101), Kolacho, P., 300 246, 257(55), 279(55) Kolkaila, A. M.,391 Kuzuhara, H.,157, 158(160), 178(180) Kolker, A. E., 269 Kvist, B. E., 26, 27(69), 28(69), 30, 37, Kol’tsov, A. I., 254 42, 48(69) Komoto, M., 312(4), 312, 314. 318, 319

(12), 321(8, 33, 34, 35, 36, 38, 391, 322, 324(33, 36, 38, 39), 326(39), 327 (8), 328(4, 8, 33, 34, 35, 36, 38, 391, 329(8, 36, 38, 39), 330(8), 333(4), 339(4, 12), 340(4, 33, 34), 341, 364

L Labananansak, M., 89 Labaton, V. Y., 127 Labib, G. H.,392

AUTHOR INDEX, VOLUME 25 LaForge, F. B., 182, 189(9), 191(7, 91, 220(10), 221(9) Lake, W. H. G., 142, 143(129), 144, 147 (129), 174(129), 175, 181 Laki, K., 423 (104), 424 Lambert, R., 410(32), 411 Lamm, M. E., 425 Lampen, J. O., 419 Lamport, D. T. A., 418 Lampson, G. P., 161 Lane, J. M., 443 Lane, N., 472 Lang, D., 419, 431(79), 449(265), 450 (265), 451 Langenbeck, W., 320, 347(28) Langlois, D. P., 183 Langridge, R., 89 Lapper, E., 362 Larkin, J. D., 267 Lathe, G. H., 14 Laurent, T. C., 19, 22, 23, 25(54), 29, 30, 37, 42, 48(56),451 Laver, W. G., 460 Layug, E. J., 432, 455(276), 456 Lea, D. J., 14, 15 Leaback, D. H., 381 Leach, A. A., 26 Leblond, C. P., 472 Lebovitz, H. E., 422(114), 424 Ledderhose, G., 181 Lederer, E., 183, 215 Lee, C. H., 145, 148(143), 176(143) Lee, J. B., 135 Lee, L. T., 416(64), 417, 434(64), 440 (64) Lee, W. W., 156, 159(169), 178(168) Lee, Y. C., 419, 422(112), 424, 431(79), 442, 449(262, 265), 450(265, 265a), 451 Leeden, R., 475 Lehmann, D., 397 Lehmann, J., 142, 1%6(126), 238, 239 (31b), 276(31a, 31b), 277(31a, 31b), 278 (31b ) LeMaistre, J. W., 221 (120), 222 (120), 228 Lemieux, R. U., 134, 135, 165, 166, 194, 195(61), 222(61), 223(61), 247, 262, 263 Lemke, W., 397. 399 Lengsfeld, W., 368

493

Lennox, E. S., 410(25), 411 Leonard, F., 323, 330(44) LePage, M., 15, 37(21) Leskowitz, S., 455(279), 456 Letters, R., 401 Leupold, F., 394 Levene, P. A., 119, 182, 186(12), 187(15), 188(18), 189, 191(7, 9, 14, 16), 213 (15, 18), 214(18), 215, 220(10, 12, 16), 221(9, 14, 15, 181, 222(15, 18, 19), 223(12, 19), 449(259), 451 Levvy, G. A., 373, 4%, 465, %6(322) Levy, D., 151 Levy, H. A., 56, 61(9), 76, 99(9) Levy, M. F., 161, 163(203) Lewis, A. D., 323 Lewis, A. F., 243 Lewis, G. J., 135, 136(99a), 138(99a) Lewis, H. B., 323, 330(44), 331(44), 332

(44) Lewis, T. A., 438 Li, S.-C., 449(264) Li, Y.-T., 449(264), 451, %6(325), 467 Liao, T., 422(118), 424 Lichtenstein, L., 362 Lichtenthaler, F., 401 Liciero. E., 433, 454(179), 455(179), 457 (179) Lieberman, R., 4 4 Liebig, R., 396 Liebster, J., 390 Liggett, R. W., 321(37), 322, 329(37), 331 (37) Lindahl, U., 435, 460, %1(297), %2(297) Lindberg, B., 42, 142, 167, 174(124), 237, 335, 341, 343, 429 Linehack, D. R., 419 Link, K. P., 209, 373, 374, 375(105, 110), 396 Linker, A., 441, 471, 475, 477 Linn, C. B., 243 Lipmann, 7..470, 471 Lis, H., 410(35), 411, 449(35), 451(35) Lisowska-Bernstein, B., 425 Little, J. N., 16, 37(14), 49(14) Littleton, C. D., 105 Liu, T.-Y., 44,410(11), 411 Liu, Y., 121, 177(%a, %b), 178(46a) Livingstone, D. J., 252, 279(63), 280(63) Llewellyn, D. R., 438

494

AUTHOR INDEX, VOLUME 25

Lloyd, K. O., 432, 433, 454( 179), 455 (276, 179). 456, 457(179) Lloyd, P. F., 354 Lohry de Bruyn, C. A., 332, 339 Loewi, G., 462(307), %3 Lohmar, R., 373, 374, 375(110), 396 Lohse, F., 216, 221(111), 223(111) Long, L., Jr., 167, 169(228), 277(141), 283 Long, V. J. W., 46, 49(131) Lorincz, A. E., 475 Lothrope, R. E., 290, 306 Louis, L. N., 463 Lowe, W. C., 268 Lowry, 0. H., 413 Loza, E., 196 Lu, G. G., 268 Ludwig, E., 372 Luetzow, A. E., 429 Lundblad, A., 414(50), 415 Luscombe, M., 450(267), 451 Lui& I., 318, 319(10), 321(30), 322, 328 (10, 30), 329(30), 333, 335(10), 339 (10) Lyle, G. G., 392 Lyonnet, R., 286 Lyshanskii, I. S., 135 Lythgoe, B., 121, 155(48), 157(48), 159 (48), 178(48)

M McBee, R. H., 303 MacBrinn, M. C., 476 McCarthy, J. F., 395 McCasland, G. E., 164 MacDonald, D. L., 232, 272(11) McElvain, S. M., 361 McGale, E. H. F., 414(47a), 415 McGeachin, S. G., 82 McGilary, D. J., 42 McGuire, J. E., 430, 467(166) McInnes, A. G., 247, 262, 263 Macintyre, W. M., 88 McKay, J., 42 McKelvy, J. F., 442 McKusick, V. A., 475, 476(389), 477(389) McLauchlan, K. A., 252, 381 McLaughlin, J. E., 478 Machell, G., 343 Maclay, W. D., 231 McMillan, F. H.,323

McMullan, R. K., 79 McNeely, W. H., 297 Maconochie, G. H., 66 McPhail, A. T., 136, 144(113), 151(113) McQueen, E. G., 478 McSweeney, G. P., 161, 162(196) Maeda, K., 83, 243 Maeda, S., 290 Maehly, A. C., 136 Magasanik, B., 364 Maghanua, L. G., 192, 194(60) Maghuin-Roqister, G., 166 Mahadevan, S., 474 Maher, J., 290, 299, 301, 302, 303(119), 304 ( 100) Mair, G. A., 93 Maitland, C. C., 19 Makarowa-Semljanskaja, N. N., 186, 223 (42.2) Maki, T., 274(136), 283 Makino, M., 421, 422(101), 424, 474 Maksimenko, V. I., 286 Malcovati, M., 440 Maley, F., 423 (130), 424, 445 (130), 474 Malik, K. L,21 Mallick, A. K., 291 Mallikarjunan, M., 69 Malpress, F. H., 430 Mammen, E. F., 473 Mandelstam, P., 269 Mandour, A. M. N., 427 Mani, R., 403 Manners, D. J., 302 Manon, G., 134 Manville, J. F., 169(231), 171 Marcante, M. L,422(102), 424 Marchessault, R. H., 76 Marks, G. S., 421 Marler, E., 450(268, 269), 451 Marr, A. M. S., 454(277, 278), 455(278), 456 Marrian, G. F., 374 Marsden, J. C., 412(42), 413 Marsden, N. V. B., 31, 32 Marsh, J. P., 143 Marshall, R. D., 409(24, 26), 410(19), 411, 421, 422(24, 119), 424, 425, 433, 436(38), 439(38), 445, 4%(19), 449 (260, 261), 451, 458, 465, %9(38) Marshall, W. E., 448(253), 451

AUTHOR INDEX, VOLUME 25 Marsters, J. B., 15, 37(22), 499(22) Martensen-Larsen, O., 286 Martensson, E., 464 Martin, R., 410(32), 411 Martin, W. G., 423(128), 424 Martinez, A. P., 156, 159(169) Martinez, D., 372 Martinez, L., 447 Martlew, E. F., 185, 380 Masaki, N., 90 Masuda, H., 412(39), 413, 463(39) Mathers, D. S., 121, 174(50) Matheson, N. K., 265 Mathews, M. B., 441, 452(211), 461(302), 4 2 Mathias, A. P., 231, 232(8) Mathieson, A. McL, 59, 78 Mathis, J. L., 269 Matsuda, K., 301 Matsuo, Y., 410(13), 411 Matsushima, Y., 191, 192, 423(135), 424, 442 ( 135) Matsuyama, J., 295 Maurer, K., 339, 396 Maurizio, A., 289. 300, 304, 305 Maw, G. A., 427 Meadway, R. J., 425 Medgyesi, G., 423 (104), 424 Meezan, E., 471 Mehltretter, C. L., 230 Meinecke, K.-H., 335 Melchers, F., 410(25), 411, 422(115) Melkonian, G. A., 399 Meltzer, R. I., 323 Mendershausen, P. B., 33, 36(85) MenCdez Gallego, M., 371, 372 MCrCsz, O., 160(189), 161 Merrill, A. T., 373 Merskey, H., 474, 475(377) Meshreki, M. H., 388, 391, 392 Messer, M., 4Q8 Meesmer, A., 393 Mester, L., 392, 393, 422(107), 423(104), 424, 473 Metzger, H., 399 Meyer, A. S., 112, 129, 161(12, 71), 162 (12), 179(12) Meyer, D., 422 ( l o o ) , 423 ( l o o ) , 424 Meyer, G. M., 182 Meyer, K., 416(64), 417, 430, 431(164),

495

434(64), 435(165), 440(64, 164, 1651, 441, 459(207), 460(207, 208), 461 (300), 462, 471, 475 Meyer, R., 364 Meyer, R. E., 242 Meyer, W., 135, 141(103) Meyer-Delius, M., 429 Meyerhoff, G., 15 Meyer zu Reckendorf, W., 135, 141(103), 353, 355(8), 360, 382(6, 37), 384 386, 401, 4Q3 Michael, S. E., 45, 48(127) Michalski, J. J., 135, 161(88) Michalzik, E., 199 Micheel, F., 203, 237, 339, 368, 381, 382. 388, 429 Michel, W., 26 Michelson, A. M., 401, 4Q51312) Mikhailov, A. C., 286 Mikhailova, E. A., 304 Miles, H. T., 239(37), 240, 276(37) Millauer, H., 399 Miller, E. J., 443 Miller, K. J., 136 Miller, M. J., 398 Miller, R. L., 243 Mills, H. H., 82 Mills, J. A., 123, 182, 392 Milstead, K. L., 290 Minkin, V. I., 100 Mirata, M., 156 Mitchell, H. K., 321 (29), 322, 330(29), 331 (291, 332(29) Mitchell, K., 399 Mitchell, M. B., 321(29), 322. 330(29), 331(29), 332(29) Mitchell, R. E. J., 214 Mitra, A. K., 222(127), 228 Miyashita, C., 418 Mizuno, K., 85, 86(75) Mizuno, Y., 401 Mochalin, V. B., 119, 142, 146(126b) Moczar, E., 423(104), 422(107), 424, 437 Moczar, M., 437 Moschlin, G., 409(23), 411 Moffatt, J. G., 152, 177(159a) Moggridge, R. G. C., 437 Mohammed, Y. S., 391 Mohammed-Ali, M. M., 367 Moir, R. Y., 134

496

A U T H O R INDEX, VOLUME 25

Moldenhauer, W., 366 Moll, H.,397 Moncrief, J. W.,55 Monsigny, M., 422(110, 113, 123), 423

(123), 424, 427, 429, 431(173), 432, 442( 173) Montavon, R. M., 164 Montgomery, R., 200, 211 (74), 212(74), 213(74), 214(74), 223(74), 224(74), 234,257, 273(17), 422(120, 123), 423, 424, 445, 446, 449(243, 262), 451, 452, %1(303), 462, 465(236), 466(236) Montreuil, J., 422(110, 113, 117, 1231, 423 (123),424, 427, 429, 431(117, 1731, 432, 442(117, 173) Monty, K. J., 23, 25(58) Moore, J. C., 15 Moore, S., 373, 374, 375(105, 1101, 409 (101, 410(11), 411, 423(133), 424 Mootz, D., 79 Moreaux, R., 286 Morel, C. J., 369, 371(78) Morgan, C. H.,87, 88 Morgan, J. W. W., 117 Morgan, W. T. J., 410(31), 411, 426, 434 (144a), 453(273, 274, 275), 454 (277, 278), 455(278, 280, 281, 282), 456, 457(285), 458(282, 285), 463, 464, 467 (144i3) Morrison, G. A., 129, 145(66), 158(185) Morrison, J. G.,267 Mortensson-Eznund, K.,416(65), 417 Mosher, C. W.,143 Moss, G. F., 37, 50 Mostad, A., 92 Mostowska, L., 304 Motomura, Y., 291 Mousseron, M., 134 Mousseron-Canet, M., 134 Moyat, J. H.,399 Miiller, A., 121, 175, 176(51, 240) Muir, H.,416(70), 417, 436(70), 440(70), 450(205), 459, 460, 461(289), %2 (307),463 Muir, L,450(265a), 451 Mukai, A,, 290 Mukherjee, S., 135, 136(100), 151, 177 (145) Muneyama, K., 377, 405 Munns, A. R. I., 87

Munro, A. C., 38, 39, 40,48(1071, 49(108) Murachi, T., 423(134), 424, 436(134) Murakami, M.,474 Muramatsu, T.,465, 466(326, 3271, 467 Murayama, W.,87(89), 88 Muroi, M., 85, 86(75) Murphy, D., 122, 135(57), 136(57, 941,

139(57), 140(57), 353, 354, 355 Murphy, W. H.,419 Murty, V. L. N., 446, 447 Myers, W. H.,121, 135(44), 136(44), 142

(44) Myrback, K., 399

N Nagabhushanam, A., 300 Nagarajan, S., 3 W Nagashima, N., 87(89, 90), 88 Nagy, E., 221(1231, 228 Nahar, S., 167 Nair, A. G. R., 304 Naito, T.,156 Nakadate, M., 214 Nakagawa, S., 472 Nakagawa, Y.,84 Nakai, Y., 156 Nakamura, M., 302 Nakamura, T., 192, 194(59) Nakanishi, Y.,469 Narbouton, R., 286 Nash, R. A,, 268 Nasr, M. A. M., 388, 389(193, 195) Naumberg, M., 218, 226(131), 227(1151,

228( 115) Nawata, U., 84 Nayak, U. G., 161, 179(204) Nef, J. U., 345 Neidle, S., 55, 80, 91 Neier, W.,237 Neilson, T.,354, 378(13), 386(16) Neimann, W.,186 Nelson, N., 292 Nelson, N. M., 303 Nkmec, J., 144, 149(135), 150(135) Ness, R. K.,224(128), 225(128), 226(128),

227(128), 228,234,237(15), 274(128), 275(27, 1381, 276(27), 278(15), 283 Nestadt, B., 205 Neuberg, C., 186, 370

AUTHOR INDEX, VOLUME 25 Neuberger, A., 409(17, 18, 411). 414(55),

415, 421, 422(119) 424, 425, 433, 436 !38), 437, 439(38), 445, 449(260, 261), 451, 458, 469(38) Neufeld, E. F., 435, %8(188), 475 Neukom, N., 417 Neumann, N. P.,419 Neumuller, G., 399 Nevin, R. S., 161 Newrnan, H., 119, 142(39), 1%(39) Newth, F. H.,110, 113, 115, 118, 123(3), 127, 133(15), 135(28), 136, 142(90), 1%(90), 147, 173(15), 210, 211, 263 Nichol, A. W., 405 Nichol, L. W., 19 Nicholson, L. W.,300 Nickerson, M. H.,375 Nifant'ev, E. E., 279(1U), 283 Nigam, V. K.,300 Nikolin, B., 49, 50(135) Nishikawa, M., 85, 86(75) Nishisawa, Y., 164 Noel, M.,401 Noetzel, G., 397, 399(278) Nolan, C.,422(112), 424 Nordin, P.,42, 48(114) Norrestam, R., 68, 100(36) Norman, B., 38, 49(98) North, A. C. T., 93, 98 NovU, J. J. K., 167 Nuhn, P., 166 Nutt, R. F., 405 Nystrom, R. F., 420

0 Oakes, E. M., 130, 131, 157(75), 158(75),

161 (74), 162(74) Oakley, M., 290 Oberdorfer, P. E., Jr., 268 O'Brien, J. S., 476, 478 Odin, L., 472 O'Donnell, G. W.,107 O'Donnell, I. J., 408 Obrink, B., 19 Ockerniann, P.-A., 476 Of~edahl,M. L., 24dl(132), 276(132), 277

(1321,283 Ogawa, K.,268 Ogawa, S.,291, 377, 397 Ogston, A. G., 20, 22

497

Ohgushi, T., 474 Ohki, E., 162 Ohle, H., 112, 161(11), 177, 179(11), 365,

373, 396(103), 397, 398, 399(278, 285) Ohlenbusch, H.-D., 419 Ohman, R., 35, 36(94) Oka, K.,436 Okada, S., 476, 478 Okaya, Y.,72, 290 Okhurna, S., 412(45), 413 Okimoto, Y.,291 Okuda, T., 107 Okuyarna, T.,442(98), 423(98), 424 Oldham, J. W. H., 127 Ollero, A., 363 Ollero Gbmez, A., 363 Onishi, R., 165 Onnen, O., 364 Onodera, K.,164, 166, 397 Onuma, S., 84 Orlova, T. I., 257, 283 Oryu,

C., 304

Osaki, K.,90, 107 Osaulko, G. K.,286 Osawa, T. 118, 185, 188, 213(35), 222135,

46), 223(35), 382 Osborn, R. A., 290 Osborne, G. O., 388 O'Shea, P. C.,26 Oshirna, G., 409(12), 410(13), 411 Oshiro, Y., 444 Osol, A., 286 Ostroumov, Y. A., 100 Otero-Vilardebo, L. R., 472 Otey, F. H., 230 Ottar, B., 123 Otterbach, D. H., 360 Ottesen, M.,408 Overend, W. G., 110, 116, 118, 123(2),

135(24, 28), 136(102), 139(102), 144 (24), 149(24), 150(24), 151(102), 152, 160(189), 161, 167, 168(224, 227), 169, 172(224, 227), 176(1%), 177(102, 146). 200, 209, 211(75), 223 (91). 234, 271(133), 275(139), 278 (143),283, 437 Owen, L. N., 133, 161, 162(197), 265

498

AUTHOR INDEX, VOLUME 25

(13, 70, 82), 172, 173(13, 70, 81, 82), 302 Pachler, K., 252 Page, T. F., 60, 92(18), 103(18), 104(18), 392 Paine, H. S., 306 Painter, T. J., 453(273, 274, 275), 455 (281, 282), 456, 458(282) Palmgren, B., 476 Pamer, T., 412(41), 413 Pan, S. C., 300 Paneque Guerrero, A., 371 Panizzon, L., 174 Papkoff, H., 448(257), 451 Park, 0. W., 289 Parker, K. J., 348 Parker, R. E., 110, 111(4), 122(4), 210, 218(95) Parrish, F. W., 198 Parrod, J., 318, 320(11), 323(11), 324(11, 40), 326(11), 328(11), 329(11), 331 (11, 40, 41, 43, 47, 50), 332(11, 40, 41, 42, 43, 47, 50), 340(11), 345(11, a),347 Parry, K., 145, 150(144) Parsons, S. M., 34, 36(92) Parthasarathy, R., 71, 85 Partridge, S. M., 328, 416(67, 68), 417, 439 Patchett, A. A., 401 Pattel, V., 476 Patzwaldt, H.-G., 399 Paul, I. C., 74 Pauling, L., 104 Paulsen, H., 261, 335, 354, 359, 360(34), 361(34), 367, 368, 375(74, 75, 75a), 376, 377, 394, 395(246), 401, 404(122) Pauly, H., 328, 372 Payza, N., 431 (175), 432 Pazur, J. H., 300, 301, 302(106), 399, 419 Peat, S., 109, 121, 135(45), 142(105), 143 (45, 105, 129), 144, 147(129), 148 (45), 173(45), 174(45, 129), 175(45), 181, 182, 300, 301 Pedersen, K. O., 23 Pelimon, C., 291 Percival, E., 112, 145(8), 147(8), 148(8), 155, 156(163), 175(8), 176(6), 177 (1611, 178(163a) Percival, E. G. V., 42, 161

Percival, L., 286 Percival, M. S., 304 Pkrez Puente, A., 371 Pkrez Rodriguez, M., 371 Perlmann, G. E., 444 Perold, L. S., 291 Perrodin, G., 183 Perry, A. R., 263, 403 Perry, M. B., 118, 429 Person, D., 461(302), 462 Pestel, M., 286 Petering, H. G., 399 Petersen, C. S., 87, 92, 98(112) Petersen, H., 382 Peterson, M., 472 Petrov, K. A., 279(144), 283 Petty, J., 290 Pfister, V., 444 Phelps, C. F., 450(267), 451 Philippart, M., 477 Philips, K. D., 187, 192, 194(59) Phillips, D. C., 93, %(115), 97, 98 Photaki, I., 377 Photaki, T., 382 Piazza, M. J., 392 Pickthall, M. M., 285 Pictet, A., 362 Pierce, J. E., 445 Pierce, J . G., 422(118), 424 Piez, K. A., 443 Pigman, W. W., 61, 102, 299, 410(33), 411, 419, 431(171, 174, 175), 432 Piotrovsky, J., 152, 177(159) Pitzer, K. S., 184 Plagemann, L., 441 Planisek, F., 61 Plattner, P. A., 122, 135(59) Plessas, N. R., 126, 135, 173(62a) Plummer, T. H., 409(7), 411, 423(129, 130), 424, 443(129), 444(8), 445(130) Pokrovskii, E. I., 250 Pollitt, R. J., 474, 475(377) Polson, A., 14 Pontis, H. G., 35, 36(95) Poppleton, B. J., 59, 78 Porath, J., 14, 22, 25(51), 27, 29, 30, 35(3), 448(253), 451 Porshnev, Y. N., 119, 142, 146(126b) Porter, R. S., 20 Pothmann, F. J., 286

AUTHOR INDEX, VOLUME 25 Potter, A. L., 390 Potter, M., W ( 2 4 , 26), 411, 422(24) Pourtallier, J., 291, 292, 293, 295: 298(77) Pradera, M. A., 372 Prasad, A. S., 473 Prasad, N., 119 Pratt, J. W., 373, 390 PravdiE, N., 381 Price, P. A., 410(11), 411 Price, V. E., 432 Pridham, J. B., 301 Prins, D. A., 121, 135, 136(87), 137, 173 (43) Pritchard, R. A,, 122, 161(56), 162(56) Privalova, I. M., 381 Prokop, D. J., 468 Propp, K., 359, 360(34), 361(34), 377 Prout, C. K., 74 Prutton, I., 86 Pryzwansky, K., 432 Puskai, T., 221(123, 126), 228 Pusztai, A., 410(34), 411, 463 Putman, E. W., 303 Putnam, F. W., 422(109), 424, 446(109), 447, 448(109), 465(109) Pyman, F. L., 324(51), 325, 328(51), 330 (511, 345(51)

Q Qadir, M. H., 403 Quigley, G. J., 76 Quintarelli, C., 459. 461(291a)

R Kabate, J., 299 Rabinsohn, Y., 272 (142), 283 Radford, T., 244, 245, 276(51b), 277(51) Radhakrishnarnurthy, B., 409(27, 281, 411, 423(28), 442, 446(216), 449(28) Radhakrishnan, A. N., 414(57, 58), 415 Rados, M., 380 Radziszewski, B., 327 Rafferty, C. A., 169 Raftery, M. A., 34, 35, 36(92, 93) Ragg, P. L., 133 Rahbar, S., 474 Rahman, S., 26, 28(66), 38(661, 48(66) Rainey, J. M., 445 Rajhhandany, U. L., 89

4%

Raniachandran, G. N., 70 Ramney, H. M., 474 Randall, M. H., 207 Randall, R. J., 413 Rand-Meir, T., 34, 36(92) Ransome, H. M., 286 Rao, P. A. D., 183 Rao, S. T., 86, 87(91), 88, 107 Raol, A., 464 Rapin, A, M. C., 169(234), 171 Ratcliffe, W. A., 414(55), 415 Ratovelomanana, V., 205, 221(82), 222 (82) Rawitch, A. B., 422(118), 424 Raymond, A. L., 119 Read, A. P., 403 Reba, R. C., 269 Reeder, W. H., 375 Rees, C. W., 437 Rege, V. T., 453(273, 2741, 455(282), 456, 458 (282) Kegna, P. P., 390 Reichstein, T., 112, 116, 121(21), 129, 135, 136(20, 21), 137(20, 21, 116), 143, 160(188), 161(12, 71), 162 (12), 174 (20, 1301, 175 (20), 179 ( 12, I%), 362 Reid, J., 399 Reinhardt, J. F., 289 Reinhold, V. N., 410(9), 411, 423(131), 424, 446(9) Reist, E. J., 154, 155, 156(164), 157(164, 171, 172), 158, 159(164, 171), 160 (172), 161, 176(160), 177(164), 178 (164, 1711, 358, 381, 405 Reinbold, H., 399 Renkin, E. M., 25 Rennie, R. A. C., 116, 135(24), 144(24), 149(24), 150(24) RepaS, A., 49, 50(135) Repetto, M., 372 RcvelSkaya. L. G., 250 Rey Romero, L., 362 Reyners, T., 218, 225(117, 118), 226(117), 227 ( 1151, 228 ( 115) Ricciuti, C., 290 Rice, F. A. H., 238, 273(30) Rich, A., 89 Rich, C., 475 Richards, E. L., 312(2, 3), 313, 314, 318 (7), 319, 326(21, 58, 591, 327, 328(2,

500

AUTHOR JNDEX, VOLUME 25

3, 6, 7, 21, 58, 59, 64), 330(7, 641, 332, 333(2, 3, 61, 344(6, 21), 345(58, 59), 347(6, 64),348, 349 Richards, G. N., 107, 118, 135(29, 95), 136, 138(111), 142(90), 1%(90), 161, 170 (95), 341, 343, 345(81, 83) Richardson, A. C., 115, 353, 355, 381, 382 Richtmyer, N. K., 114, 135(17), 235, 238, 273(22, 127), 275(28), 283, 373, 375, 390(216), 391 Ricketts, C. R., 45, 48(127), 50 Ridd, J. H., 183 Riedel, T., 237 Riethof, M. L., 289, 291(45), 294(45), 305 Riggs, G. M., 151, 177(151b) Rigler, R., 19 Riley, J. G., 430, 431(164), 440(164) Rinehart, K. L., Jr., 74 Riniker, B., 425 Ripperger, H., 395 Rittel, W., 425 Rizvi, S., 431(175), 432 Robbins, P. W., 470 Robert, L., 437 Roberts, G. P., 38, 354 Robertson, G. J., 114, 121, 127, 135(16, 441, 136(16, 441, 142(44), 173(16, 1231, 174(16, 50) Robertson, J. H., 73 Robins, M. M., 477 Robins, R. K., 243 Robinson, D., 446 Robinson, H. C., 435, 468(189) Robinson, J. C., 445 Robson, R., 192 Rockstroh, G., 179 Rod& L., 34, 36(89, 90), 435, 440, %O (185). 461(301), 462, %9 Rogers, D., 55, 80, 91 Rogers, M. A., 295 Roldan, L., 371 Rolle, M., 335 Romanowska, E., 43, 49(122) Romming, C., 87 Ronchi, S., 44-0 Ronnquist, O., 68 Rosebrough, N. J,, 413 Roseman, S., 429, 430, 467(166), 469 Rosen, O., 441, 460(213) Rosenbloom, J., 468

Rosenfeld, D. A., 373, 390 Rosenquist, U., 399 Rosenstein, R. D., 53, 59, 60(17), 61, 64, 65(29), 66, 104(17), 107 Rosenthal, A., 161, 214, 248, 279(59bf, 281(59b, 59c, 59d, 59h), 282(59c) Rosenthal, A. F., 420 Rosevear, J. W., 409(21), 411, 422(21), 448(21) Rosowsky, A., 110 Ross, J. H.,15 Ross, W. C. J., 169(230), 170 Rossignol, B., 430, 431(167) Rossiter, J. L,423(116), 424 Roston, C. P. J., 474 Rothen, A., 449(258), 451 Rowe, G. G., 268 Rubin, N., 286 Rudy, H., 2.71 Rude, K., 429 Rueggeberg, W. H. C., 269 Ruiz Cruz. I., 371 Rundle, R. E., 78 Rupley, J. A., 96(115), 97 Rutherford, D., 390(216), 391 Ruthven, C. R. J., 14 Rutishauser, U., 424 Ruyle, W. V., 401 Ryan, K. J., 156, 178(167)

S Saeed, S. A., 192 Saeki, H., 162 Saenger, W., 79, 87(76), 88 Saito, M., 399 Saito, Y., 84 Sakai, S., 291 Sallam, M. A. E., 367 Salo, W. L,381 Salsman, K., 475 Samama, M., 473 Sammul, 0. R., 252 Samokhvalov, G. I., 119, 142, 1%(126b) Sampson, P., 430, 431(164), 44Q(164) Sandhoff, K., 478 Sanfellipo, P. M., 26, 28 Sangster, I., 394, 395(2%) Sarda, L., 410(9), 411, 446(9) Sarkanen, K., 161 Sarko, A., 76

AUTHOR INDEX, VOLUME 25 Sarma, V. R., 93 Sasada, Y.,92 Sasaki, T., 4Q1 Satake, M., 422(98), 423, 424 Sanders, R. M., 125, 131, 142, 144(62), 148(62), 157(75), 158(75), 175(62), 208 Scanlon, B., 135 Schaffner, C. P., 145, 148(143), 176(143) Schafter, R., 214 Schaub, R. E., 116, 136, 151(109), 155 (22), 156(22), 157, 159, 177(22, 109), 178(22, 173, 176, 186), 381 Schauer, H., 426 Scheidegger, U., 363 Scheit, K. H., 87 Schepartz, A. I., 290 Schiedt, B., 339. 596 Schier, 0..110, 371 Schiffman, G., 455(279), 456 Schiller, S., 459, 461 (291) Schilling, B., 373, 3% (102) Schmandke, H., 198, 199(67), 225(67), 226(67) Schmid, K., 422(98), 423(98, 103), 424, 444, 44$(251), 451 Schmidt, D. L., 221 (121), 228 Schmidt, M., 435 Schmidtburger, R., 409(23), 411 Schmied-Kowanik, V., 399 Schmitt, J. A., 399 Schrnitz, J., 324(56), 325 Schmukler, S., 184 Schoop, H. J., 464 Schorigin, P., 186, 223(42) Schramm, M., 42, 48(115), 303 Schrier, E., 390(217, 218, 220), 391, 392 (218) Schroeder, W.,249, 282(59j) Schroeder, W. A., 420 Schubert, M., 471 Schubert, M. P., 385 Schuerch, C., 161, 166 Schultze, H. E., 409(22, 23), 411, 444 Schwandt, V., 473 Schwanert, H., 361 Schwartz, M., 410(9), 411, M ( 9 ) Schwan, J. C. P., 112, 125(9), 128(9), 142(9), 144(9), 148(9), 170, 174(9), 175(9), 380 Schwick, G., 4Q9(23), 411

501

Schwyzer, R., 399 Scott, J. E., 413, 450 Seebeck, E., 129, 161(71) Seeliger, A., 339, 380, 3% Segaloff, A., 409(16), 411 Segrest, J. P., 415, 438 Sehon, A. H., 14, 15, 17, 26(30), 27(30) Seib, P. A., 132, 133(82a), 172(82a) Seid-Akhaven, M., 430 Seigwart, A., 399 Selhy, K., 19 Selinger, Z., 303 Semb, J., 399 Seno, N., 430, 431(164), 440(164), 441, 459(207), %0(207), 461(300), %2 Sepulchre, A. M., 356, 361 Sequeria, J. S., 437 Serafini-Cessi, F., 364 Shaban, M. A. E., 388, 389(193, 195) Shafizadeh, F., 182, 184, 187(22), 191(22), 214(22) Sharma, M., 135, 136(98), 139 Sharon, N., 410(35), 411, 449(35), 451 (35) Sharpe, E. S., 390(215), 391 Shauer, R., 464 Shchegolev, A. A., 279 (144), 283 Sheaffer, V. E., 256 Sheber, F. A., 423(116), 424 Shefter, E., 87(93), 88 Sheldrick, B., 61, 73 Shelton, B., 429 Shen, L., 470 Shen. T. Y., 247, 257, 262, 264, 265(58, 93), 277(58), 279(58), 280(58). 401 Sherher, D. A., 268 Sherwood, S. F., 288, 308(35) Shetlar, M. R., 449(264), 451 Shibasaki, K., 300, 302 Shimada, A., 58 Shimizu, S., 469 Shimuzu, Y., 87(89), 88 Shinaberger, J. H., 269 Shiro, M., 84 Shirshova, A. N., 257 Shkantova, N. G., 318, 348(13) Shmakova, F. V., 422(122), 424 Shulman, M. L., 381 Shute, S. H., 248, 281(59g), 282(59g) Shyrock, G. D., 122 Sicher, J., 184, 185(30)

502

AUTHOR INDEX, VOLUME 25

Siddiqui, I. R., 288, 291(38), 292, 297, Sorkin, E., 116, 136(20), 137(20), 174 (!ZOO), 175(20) 298, 301, 306 Sorm, F., 167, 220(129), 228 Sideri, C. N., 286 Sowden, J. C., 63, 248, 276(132), 277(132), Sidorova, N. S., 257 279(59a), 280(59a), 281(59a), 283,341 Sieber, P., 425 Sparks, R., 87(93), 88 Siegel, L. M., 23, 25(58) Spencer, R. R., 161, 381 Siewert, G., 144 Spik, G., 422(117), 424, 431(117), 442 Sigler, P. B., 89 (117) Sih, C. J., 303 Spiro, M. J., 444, 469, 470 Silbert, J. E., 471, 472(350) Spiro, R. G., 409(30), 411, 437, 439(193), Silsbee, C. G., 294 442(200), 444, 4%, 448(242, 248), Silvander, B., 429 451, %3(248), %5(248), 469, 470 Simkin, J . L,448(254), 449(254), 451 Squire, P. G., 24, 25(63), 29, 30 Simmonds, R. G., 43, 48(119, 120) Srinivason, R., 74 Simon, H., 366, 399 Srivastava, H. C., 135, 136(100) Simonds, N. B., 423(116), 424 Stacey, M., 43, 48(119, 1201, 119, 130, Sims, A. P., 23, 25(59) 132, 133(76), 136, 142(72), 1%(72, Sims, S. P., 55 1261, 151, 173(76), 176(148), 185, Simson, B. W., 41, 48(112) 186(37), 222(37), 252, 271(64, 133), Sipoi, F.. 184, 185(30) 273(64), 277(142a), 283, 302, 394 Siwoloboff, A., 2% Sjollema, B. J., 324(54), 325, 329(54), Stadler, H., 286 StanBk, J., 112, 128(13), 133(13), 173 330(54), 347(54) (13), 302 Skalka, M., 45 Stark, K. H., 199 Skoda, A., 268 Stary, Z., 418 Sliemers, F. A., 20 Staudenbauer, W. L,470 Slomp, G., 249, 282(59k) Steelman, S. L., 409(16), 411 Slonimski, P. P., 183, 215 Stein, W. H., 409(10), 411(11), 411, 423 Smith, A. L., 268 (133), 424 Smith, B. C., 275(139), 283 Steiner, H., %1(304), %2 Smith, C. W., 354 Steinert, G., 394 Smith, D. C., 37 Smith, E. L,409(21), 411, 422(21, 112), Steinrauf, L K., 79 Stephen, A. M., 19, 20,23, 24(62), 25(62), 424, 444,448(21) 28, 29, 30(76), 38(37,62, 76), 39(62), Smith, F., 132, 133(76), 173(76), 200, 211, 48(37, 761, 49(62) 212(74), 213(74), 214(74), 223(74), 224(74), 231, 232, 234, 271(6), 273 Stephens, R., 251 Stevens, C. L., 145, 148(143), 165, 166 (17), 452 (213a), 176(143), 360 Smith, R. N., 38, 48(103) Stevens, H. F., 477 Smith, W., 478 Stevenson, F., 414(56), 415 Smith, Z. G., 470 Smyth, D. G., 410(20), 411, 431(20), 434 Stewart, L. C., 390 Stewart, T. S., 33, 36(85, 86) (20), 442(20), 443(20) Steyermark, P. R., 379 Snatzke, G., 119, 169(35), 176(35) Stickney, P. B., 20 Sobell, H. M., 87(92), 88 Stim, T. B., 270 Sohar, P., 245, 250(53), 275(53) Stineon, E. E., 290 Sois, A., 269 Somers, P. J., 15, 20(23), 37(22, 231, 4, Stirling, J. L., 4% Stockmeyer, W. H., 22 48(121), 49(22, 23) Stoddart, J. F., 26, 27, 28(66), 38(66), Somogyi, M., 292 40, 48(66, loo, 101), O(l09) Soria, J., 473

AUTHOR INDEX, VOLUME 25 Stodola, F. H., 390(215), 391 Stoehr, C.,319, 329, 330(14) Stoffyn, P. J., 132, 152, 173(79), 177(159) Stohr, G.,390(220), 391, 392(218) Stokstad, E. L. R., 399 Stolle, R., 389 Stolte, K.,339 Strachan, I., 465, %6(322), 474 Strauss, F., 176 Strobach, D. R., 373 Strominger, J. L.,470, 471 (347),472 (353) Stuhlsatz, H. W.,440, 441 Suami, T., 377 Subbarow, Y., 399 Subers, M. H.,289, 290, 291(45),294(45) Subramanian, E.,87 Subramanian, S. S., 304 Sugarman, G. I., 477 Sugihara, J. M.,221(121),228 Sugimoto, K.,469 Sugiyama, M.,469 Sumyk, G. B., 45 Sun, K.,17, 26(30), 27(30) Sundaralingam, M.,86, 87(87, 88, 91). 88,

100, 107 Surak, J. G.,26, 28 Sutor, D. J., 88 Suzuki, K.,476 Suzuki, N.,‘ 291 Suzuki, S., 469, 470, 471(347), 472(353) Suzuki, T., 409(12),4143(13),411 Svedberg, T., 23 Svennerholm, L., 464 Svensson, S., 136, 174(115a) Swanson, M.,472 Sweet, F., 119, 169(232), 171 Swenson, H. A., 41 Szabados, L., 473 Szabb, L., 162, 179(207), 422(107), 424 Szsrek, W. A., 357. 394

T l a g a , T., 107 Taghavy, A., 475 Taguchi, T., 135, 139(101) Taha, M. I., 318, 328(9), 330(9), 339(9) Taigel, T., 380 Takagi, S., 61, 107 Takahashi, N., 423(134), 424, 436(134),

469

503

Takahashi, S., 444 Takenchi, M., 412(39), 413, 463(39) Takeshima, S., 291 Tamm, I., 415 Tanaka, K.,431 (171,1741, 432 Tanaka, M.,422(125), 423, 424 Tanako, N., 92 Tanford, C., 450(269), 451 Taniuchi, H.,425 Tanret, C., 319 Tanret, G., 308 Tappel, A. L., 474, 476 Tarentino, A., 423 (130), 424, 445 (130),

474 Tarutami, O., 448(249), 451 Tashima, S., 122, 133(53a) Tate, M. E., 297 Tauber, H. J., 391 Taylor, K. G., 145, 148(143), 176(143),

360 Taylor, N. F., 142, 146(126a), 151, 155

(153),157, 173(126a), 174(126a), 177 151b, 153), 178(180a, 182) Tejima, S., 135, 274(136), 277(137), 283 Telser, A., 435, 468(189) Tena Aldave, H.,363 Terho, T.,412(40), 413 Terry, K. D.,475 Theander, O., 237, 335, 341, 343, 398 Thiessen, W. E., 63, lOl(27) Thomas, G. H. S., 254 Thomas, W. A., 145, lSO(144) Thompson, A., 33, 390 Thremm, R. H.,472 Thiirkauf, M., 121 Tichj, M., 184, 185(30) Tiemann, F., 182, 185(6), 222(5, 6) Tikhomirova-Sidorova, N. S., 236, 243, 254, 257, 258, 273 (23), 274(23), 277 (50) Tikhonov, V. I., 242 Timberlake, C. El, 33 Timell, T. E., 41, 48(112) Timmis, G. M.,207, 224(86) Tipson, R. S., 111, 119, 198, 201, 216, 218, 246, 264, 265(92) Tipton, C. L., 300, 302(106) Tixier, P., 473 Tocik, Z., 132, 173(81) Todd, A. R., 135, 151, 161(88), 177(145) Todt, K., 367, 368, 375(74, 75, 75a), 376, 394, 395, 401, M(122)

AUTHOR INDEX, VOLUME 25

504

Toepffer, H., 179 Tokiwa, F., 256 Tollin, P., 87, 88 Tominaga, F., 436 Toshida, K., 291 T6th, G., 186, 212(41) Totter, J. R., 323, 330(44), 331(44), 332

(441, 349945) Townsend, L. B., 243 Trautman, R., 447 Trautwein, W. P., 261 Treibs, W., 239 TrCnel, G., 32, 33(81), 36(81) Treon, J. F., 269 Trindle, M.,422(106), 424 Trippett, S., 121, 155(48), 157(48), 159 (481,178(48) Trnka, T., 172 Tronchet, J. M. J., 192, 194(60), 214 Trotter, J., 69, 73, 102(38), 107, 248, 255, 281(59h) Trueblood, K. N., 87 (93),88 Tsiganos, C. P.,W , 450(205) Tsuchida, H., 321 (35, 36, 38, 39), 322, 324(36, 38, 39), 326(39), 328(35, 36, 38, 39), 329(36, 38, 39), 341, 344 (84), 345(36, 38), 346, 348(36), 349 (39) Tsuchiya, T., 122, 140(54) Tsugita, A., 442 Tsukamoto, H.,418 Tsukuda, Y.,84 Tucker, L. C. N., 74 Tulinsky, A. J., 82 Tuomioia, I. M.,270 Tuppy, H.,470 Turner, D. L., 418 Turner, E. E., 397 Turner, J. C., 118 Turner, W. N., 122, 140(54) U Udenfriend, S., 408 Ui, N., 448(249), 451 Ullrich, A., 320, 323, 329(27), 330(48),

331 (27, 48), 332(48), 340(27), 347 (27), 348 Ulpts, R., 182, 215, 222(19), 223(19) Ulrich, P., 142 Umezawa, H., 83, 2.23, 377

Urquiza, R., 196 Ushiyama, K., 268 Usov, A. I., 267 Ustyuzhanin, G. E., 236, 243, 244(50), 254,

257, 258, 273(23), 274(23), 277(50) IJtsumi, S., 410(20), 411, 431(20), 434

(20),442(20),443(20) Uvarova, S. I., 242

V Vaciago, A., 90 Vadopalaite, I., 385 Valesco del Pino, J., 363 van Deemter, J. J., 21 van de Kamp, F.-P., 381, 382 vander Bijl, P., 20 van Es, T., 122, 136(55), 161(55), 162

(55),203 Van Hoof, F., 476 Vankata Rao, E., 186 Van Vnorst, F. T., 295 Vardheim, S. V., 130, 142(72), 146(72) Vargha, L., 112, 161(11), 179(11), 211,

221(101,123,124, 125, 1261,227(101), 228, 245, 2%, 250(53), 257(55), 275 (53),277(52), 278(55) Vatina, M. G., 426, 428(146), 429(146), 434(146) Vaughan, G., 110, 123(2), 136 Vaughan, M. F., 15 Veibel, S., 165 Vercellotti, J. R., 429 Verheyden, J. P. H., 152, 177(159, 159a) Vernay, J. L., 414(54). 415, 444 Vernon, C. A., 438 Vischer, E., 160(188), 161, 179(188) Vlasse, M.,65 Vogele, P.,419 Von Arx, K.,286 von Glehn, M.,68, lOO(36) von Pechmann, H.,326(57), 327

W Waalkes, T. P., 196 Wacker, A., 399 Wagh, P. V., 448(250), 451 Wagner, G.,166 Wai, N., 417 'WdbOrg, E. F., #9(15), 411

AUTHOR INDEX, VOLUME 25 Walczak, E., 356 Walker, J., 399 Walker, P. G., 381 Walker, T. K., 397 Wall, H! M., 169 Wallen, P., 422(108), 424 Waller, C. W., 399 Walton, D., 429 Walton, E., 4Q5 Walton, P. L., 45 Wander, J. D., 60, 92(18), 103(18), 104 (la), 199, 214 Wanic, D., 304 Ward, D. N., 409(15), 411 Ward, P. F. V., 152, 177(158) Wardi, A. H., 418 Warren, C. D., 382, 383, 385 Waser, J., 71 Wassiliadou-Micheli, N., 382, 384 Wasteson, A., 47, 48(133), 449(266), 450 (266), 451 Watanabe, K. A., 135, 139(101), 142, 173 (125a), 174(125a), 377, 401 Watarabe, T., 291, 295, 297, 298 Watenpaugh, K., 87(94), 88 Waters, J. L., 21 Watkin, D. J., 72 Watkins, W. M., 410(31), 411, 426, 434 (144a), 446(145), 453(273, 274, 275), 454(278), 455(281, 282), 456, 457 (285), 458(241, 282, 285), %7(144a), 470 Watson, D. G., 88 Watson, W. H., 71 Waxdal, M. J., 424 Webber, J. M., 119, 142, 1%(126), 256, 263, 265, 271 ( 1341, 275 ( 135), 283, 403, 459, %1(290) Weber, P., 433 Webster, R. G., 460 Wegner, H., 321(32), 322, 331(32), 333 (32) Weidenhagen, R., 321 (32), 322, 324(55), 325, 331(32, 55), 332(32, 55) Weidmann, H., 143, 144(131), 174(131), 175(131b) Weigel, H., 302 Weiner, H. E., 472 Weisblat, D. J., 399 Weismann, D., 300 Weiss, M. J., 157, 159, 178(176, 186)

505

Weissmann, B., 426, 467 (143) Weitzel, G., 385 Wells, J., 167, 168(227), 172(227) Wempen, I., 201 Wendt, G., 198, 225(69) Werner, P. E., 68, lOO(36) Werries, E., 426 Wessely, K., 199 Westphal, N., 161 Westphal, O., 144 Westwood, J. H., 119, 122 Weygand, F., 233, 272(14b), 335, 391, 397, 399 Whelan, W. J., 300, 301(105) Whiffen, D. H., 252, 271(64), 273(64) Whistler, R. L., 161, 162(202), 179(204), 205, 290, 343, 344, 399 Whitaker, J. R., 26 White, A. C., 152 White, J. W., Jr., 286, 287, 289, 290, 291, 294, 295, 299, 301, 302, 303(119), 304 (100).305, 308(30) White, T., 381 Whitmore, F. C., 183 Whyte, N. C., 161 Wickstrom, A., 380 Wiegandt, Z., 464 Wiegers, G. A., 77 Wienbowska, B., 435 Wiggins, L. F., 115, 118, 121, 132, 133 (76), 135(28, 29, 45), 136(95), 137 ( l a ) , 142(90, 105), 143(18, 45, 1051, 1%(90), 148(45), 151, 161, 162 (1961, 170(95), 173(45, 76), 174(45), 175 (18, 45), 176(148), 188, 191, 229, 233 (1). 235(1), 243(1), 2%, 256, 257, 264, 265, 267, 279(1), 319, 328(17, 18, 19), 349(17, 18, 19) Wilcke, H., 177 Wild, G. M., 399 Wiley, H. ,W., 290 Wilkinson, S., 183 Williams, D. E., 78 Williams, J., 422 (117a), 423 (127, 127a), 424 Williams, J. C., 348 Williams, J. H., 116, 155(22), 156(22), 177 (221, 178(221, 381 Williams, N. R., 135, 136(102), 139(102), 151(102), 152, 167, 166(224, 2211, 169, 172(224, 227), 177(102), 210, 229

AUTHOR lNDEX, VOLUME 25

506

Williams, R. E., 224(130), 225(130), 228 Wilson, H. R., 87, 88 Windaus, A., 320, 321(23), 323, 324(52), 325, 329(23, 24, 26, 27), 330(48), 331 (23, 24, 26, 27, 48), 332(48), 3400(27, 28), 347, 348 Windholz, M., 242 Winkley, M. W., 381 Winter, M., 391, 397 Wintersteiner, O., 399 Winder, R. J., 409(29), 411, 433, 448 (250), 451, 472 Winzor, D. J., 19 Wirth, F., 233, 272(14b) Wise, B. L., 269 Wise, W. S., 135, 136(95), 170(95), 319, 328(18, 19), 349(18, 19) Wolcott, R. G., 34, 36(92) Wold, J. K., 380 Wolf, N., 143, 144(131), 174(131), 175 (131b) Wolff, H., 186, 370 Wolfrom, M. L., 33, 214, 297, 381, 390, 391 Wolter, A., 397 Wood, D. J. C., 265 Wood, K. R., 196, 222(65), 235 Woodhour, A. F., 270 Woods, D. A., 478 Woolard, G. R., 29, 30(76), 38(76), 48 (76) Woolf, L. I., 427 Woollacott, M., 476 Wright, J. A., 151, 155(153), 156, 157, 177(153), 178(167a, 180a, 182) Wright, J. H., 269 Wright, L. W., 260, 261 Wriston, J. C., 410(9), 411, 446(9) Wu, Y.-C., 422(124), 423, 424, 446, 449 (243, 262), 451 Wulff, H., 381 Wyatt, G. R., 300 Wykes, G. R., 299 Wyss, G., 327, 328(61), 330(61)

Y Yahya, H. K., 403 Yamada, S., 26 Yamamoto, A., 418 Yamashima, I., 421, 422(101), 424, 474

Yaniau, K., 295 Yamauchi, T., 422(101), 423, 424 Yamazaki, M., 291 Yamazaki, T., 268 Yanotovskii, M. T., 142, 146(126b) Yasuda, Y., 423(134), 424, 436(134). 442 (1) Yasumura, A., 300 Yatsuk, A. F., 318, 348(13) Yocum, C. F., 45 Yoshida, H., 436 Yoshida, K., 268 Yoshimura, J., 421 Yosizawa, Z., 412(43), 413, 456 Young, D. W., 56, 88 Young, N., 401 Young, N. M., 46, 48(129), 49(130) Ynng, D., 416(64), 417, 434(64), 440(64) Z

Zachoval, J., 166 Zagdoun, R., 134 Zalewski, W., 303 Zarubinskif, G. M., 257 Zechmeister, L., 186, 212 (41) Zeleznick, L. D., 32, 36(80) Zelinski, R., 242 Zellner, G., 397 Zellner, H., 397 Zemplen, G., 380, 393 Zen, S., 122, 133(53a) Zeppezauer, E., 89 Zeppezauer, M., 89 Zervas, L., 238, 276(29), 377, 382, 418 Zhdanov, Y. A., 100, 242 Zhivoglazova, L. E., 242 Zief, M., 221(122), 228 Zimm, B. H., 22 Zimmerman, H. K., Jr., 122, 144, 150(137), 175(137), 377 Zimmermann, M. H., 304 Zinner, H., 198, 199(67), 225(67), 226(67) Zioudrou, C., 238, 27609) Zissis, E., 238, 273(127), 275(28), 283, 373 ZobiEovi, A., 143, 174(132), 394 Zobrist, R., 155, 177(161) Zollinger, H., 183 Zuiderweg, F. J., 21 Zurabyan, S. E., 381

SUBJECT INDEX FOR VOLUME 25 Aldonic acids, characterization of, 373 -, 2-amino-2-deoxy-, deamination of, 191 Acetals, of anhydroalditols, formation and -, 2,5-anhydro-, properties of, 220-229 behavior of, 265 -, dithio-, of 2-amino-2-deoxyaldoses, de- Aldonolactones, 2-amino-2-deoxy-, deamination of, 189 amination of, 192 Aldopentopyranoses, electric charges on Acetylene compounds, D-glucopyranosylaatoms of, 100 tion of, 242 Aldopentoses, gel chromatography of, 31 Ackers equation, 25 Aldoses Acrylamide polymers, gels aldehydo-, 2-5-anhydrides, mutarotation in chromatography, 14 of, 213 fractionation ranges of, 19 gel chromatography of, 31 Acylation, selective, of anhydroalditols, 262 hydrogen bonding in, 57 Agar oxirane derivatives, 109-179 constitution of, 6 -, amino-, deamination of, 183-194 in gel chromatography, 14 -, 2-amino-2-deoxy, djthioacetals, desubstitute for, 7 amination of, 192 Agarose, gels, in chromatography, 14 -, 2,5-anhydro-, 182, 185 Albumin, carbohydrate linkage in, 420, color reaction for, 216 444,445 hemiacetals, 212 Alditols reactivity of, 210 conformations of, 105 Alkali crystallography of, 60 effect on glycoproteins, 428-431 gel chromatography of, 31 reaction with sugars, 341, 345 sulfonic esters, solvolysis of, 207 -, 2-amino-2-deoxy-, deamination of, 191 Allal, 4,6-O-benzylidene.o-, 139 -, 4,6-0-benzylidene.2-C-rnethyl-n-, 139 -, anhydro-, 229-283 Allitol acetals, 265 anhydridation of, 233 configurations (revised) of, 264 crystallography of, 60 infrared spectra of, 250-252 -, l-amino-2,5-anhydro-l-deoxy-~-, 209 isomerization of, 258 -, l,S-anhydro-e-, 237 ring opening of, 256 -, 1,5-anhydro-~-,237 uses, industrial, 267 Allonic acid, 2,5-anhydro-3,4,6-tri-O-ben-, 2,s-anhydroZOyl-D-, 209 in nucleoside synthesis, 218 Allononitrile, 2,5-anhydro-3,4,6-tri-O-benproperties of, 220-229 ZO)’l-il?-D-,209 reactivity of, 211 Aldofuranoses, 2,3-anhydro-, nuclear m a g Allopyranose, 1,6:2,3-dianhydro-n-, 113 -, 1,6:3,4-dianhydro-o-, 113 netic resonance spectra, 172 Aldohexonic acids, 2-arnino-%deoxy-o-, Allopyranoside, methyl 2,3-anhydro-a-n-, derivatives, 146 deamination of, 191 Aldohexopyranoses, 2-amino-2-deoxy-, dea- -, methyl 2-3-anhydro4,6-0-benzylidenea-D-, 114, 117, 118, 141, 188 mination of, 184 -, methyl 4,6-0-benzylidene-2,3-dideoxyAldohexoses, 2,3-epimino-a-D-, 352, 356 crystallography of, 61 Allose, 3,6-anhydro-4,5-0-isopropylidene-~-, gel chromatography of, 31 -, 2,5-anhydro-, 183 dimethyl acetal, 207 Aldolase, inhibition of, anhydroalditols in, Aloin, 243 269 Altritol, D-, anhydridation of, 233

A

507

508

SUBJECT INDEX. VOLUME 25

-, 1,5-anhydro-o-, 2-38 -, 3,6-snhydro-n- and -L-, crystallography

with lead tetrafluoride, 1% Arabinitol DL-, crystallography of, 59 of, 60 L-, anhydridation of, 231 -, 3,6-anhydro-o~-,catalytic oxidation of, -, 2-amino-2-deoxy-o-,deamination of, 192 261 Altropyranose, 1,6:3,4-dianhydro-B-n-, 113 -, 1,4-anhydro-D-, 198, 233 Altropyranoside, methyl 2-O-acetyl-3,4-antriacetate, isomerization of, 258 hydro-6-0-trityl-n-o-,cleavage of oxirane -, 1,4-anhydro-~-,233 ring in, 125 -, 2,5-anhydro-o-, 234 -, methyl 2-amino-4,6-O-benzylidene-2- -, 2,5-anhydro-~-,200, 234 deoxy-a-n., deamination of, 188 -, 2,5-anhydro-1,4-di-0-(methylsulfonyl) D-, 207 -, methyl 3,4-anhydro-n-n., 145 -, methyl 3,4-anhydro-6-0-trityl-n-~-, -, 2,3-O-benzylidenetri-0-(methylsulfonyl) -D-, solvolysis of, 207 cleavage of oxirane ring in, 125 Arabinofuranoside, alkyl 2,5-anhydro-B-nAmadori rearrangement, 335, 337, 338 and -n-L-,hemiacetals, 212 Amino acids -, alkyl 2,5-anhydro-a-~-,200 in glycoproteins, 417 identification of, in glycoproteins, 429 -, ethyl 2,5-anhydro-n-~-, hydrolysis of, sequences in glycoproteins, 422-425 212 Amino sugars, see Sugars Arabinogalactans, gel chromatography of, 41 Ammonia Arabinopyranose reactions of carbonyl compounds with a-D-, conformation of, 103 aqueous, 324-327, 344 of sugars with aqueous, 311-349 P-D-, conformation of, 102 a-Amylase, 436 p-D-and P-L-, crystallography of, 59 carbohydrate-peptide linkage in, 442 Arabinopyranoside, methyl 2-0-acetyl-3,4a-Amylodextrin, constitution of, 6 anhydro-n-, 154 Amylomaltase, action on maltose, 33 -, methyl 3,4-anhydro-a.n-, 152 Amylopectin nuclear magnetic resonance spectrum, gel chromatography of, 42 171 synthesis of, 8 -, methyl 3,4-anhydro-F-~-,150 Amylose, 8 Arabinopyranosyl fluoride, 2-bromo-2-deoxy. Anhydrides P-D-, crystal structure of, 74 of alditols, 229-283 Ara binopyranosylmethane, bis (ethylsulof aldoses, 109-179 fonyl) -n-D-, triacetate, nuclear mag2,5-, of sugars, and related compounds, netic resonance spectrum of, 253 181-228 Arabinose Anhydronucleosides, 405 D-, dialkyl dithioacetal, reaction with pAnhydro sugars, see Sugars toluenesulfonyl chloride, 199 Anthracene, complex with dichloromethane gel chromatography of, 31 and tetracyanoethylene, structure of, L-, in glycoproteins, 413 55 L-, protein linkage with, 418 Anthrone, 10-(a-D-glucopyranosyl) -1,l-dihy- -, 2,5-anhydro-n-, 202 droxy-3-(hydroxymethyl) -,243 dimethyl acetal, 211 Antibiotics, 243 -, 2,5-anhydro-~-,2-furaldehyde from, 211 anhydrooctitols from, 249 -, 2,5-anhydro-n-~-,dimethyl acetal, 212 crystal structures of, 80-86 -, 2,5-anhydr0-3,4-0-isopropylidene-o-, stabilizers for, anhydroalditols for, 268 209 Antistatic agents, anhydrohexitols, 267 hydrolysis of, 211 Aquapak, 15 -, 2,5-anhydro-3,QO-isopropylidene-aldeArabinal, D- and L-, diacetates, reaction hydo-w, mutarotation of, 214

-

SUBJECT INDEX, VOLUME 25

509

Aristeromycin, crystal structure of, and hydrobromide, 85 Ascorbic acid, L-, crystal structures of, and sodiiim salt,

Carbonium ion, 183 Carbonyl compounds, reaction with ammonia, 324-327,344 Carbonyl group, reactions involving, of 91 sugars, 212 L-, hydrogen bonding in, 57 Catalysts, effect on reaction of sugars and Asparagine, L-, in glycoprotein linkages, ammonia, 320-323,328 418, 420 Cataracts, 1,4:3,6-dianhydro-~-glucitolin L-Aspartic acid, in glycoproteins, 419, 421 treatment of, 269 Avidin, carbohydrate linkage in, 420 Cclestoraminol, N-acetyl-3,4-O-isopropylAzepine, sugar derivatives, 404 idene-, 249 Azides, sugar, aziridine sugars from, 354 Cellobiose Aziridine ring, saccharides containing, 352configuration of, 55 356 crystal structure of, 75, 77 hydrogen bonds in, 99 B p-Cellotetraose, crystal structure of, 77 Cellulose Barbaloin, 243 constitution of, 6 Benzene, a-D- and 8-o-glucopyranosyl-, crystal structure of, 80 tetraacetates, 240 Cellulose I, structure of, 80 -, o-mannopyranosyl-, tetraacetate, 241 Cellulose 11, structure of, 78 Benzilic acid rearrangement, 344 Centose, in honey, 297 Benzimidazole, sugar derivatives, 373 Ceruloplasmin Bio-Gel P, 15, 20, 23 carbohydrate linkage in, 420 Bio-Glas, 15 heterogeneity in, 4% Biosynthesis, of glycoproteins, 467-472 Chemotaxonomy, 39 Blasticidin S, crystal structure of, 84 Chitin Bovine a-lactabumin, crystal structure of, gel chromatography of hydrolysis prod98 ucts of, 35 Brig1 anhydride, 164 in glycoproteins, 415 Bromelain, pineapple-stem, 436 Brominolyais, of 2-deoxy-2-iodopyranoses, Chitobiose, di-N-acetyl-, crystallography of,

194

93

Butane, tetra-0-acetyl-a-D-glucopyranosyl-, Chitosamine, synthesis of, 6 Chitose, see Mannose, 2,5-anhydro-~241 Chitotriose, gel chromatography of lysozyme products with, 35 C -, p-tri-N-acetyl-, crystallography of, 93 Calcitonin M, amino-acid sequence in, 425 Chondroitin sulfates, 439 Caldariomycin, derivative, crystal structure carbohydrate chains in, 460.462 of, 74 gel chromatography of, 46 Carbohydrate-peptide linkage, of glycomolecular weights of chains in, 451 proteins, 467-472 4- and 6-,435 gel chromatography of oligosacchaCarbohydrate-protein linkages, 417-439 rides from, 34 Carbohydrates crystal structure of, and derivatives, 53in glycoproteins, 417 107 Chondromucoprotein, 435, 436, 439 gel chromatography of, 13-51 Chromatography, 9 glycoprotein components, 409-414 of 2,5-anbydrides of sugars, 182 Carbon nucleophiles, for oxirane aldoses, gel 125 of carbohydrates, 13-51

510

SUBJECT INDEX, VOLUME 25

principles and definitions, 16 separation by, 17 theory of, 16-30 gels for, 14 of honey, 290-293, 295 of sugar and ammonia reaction products, 328 Cinnoline, 3-(o-nrabino-tetrahydroxybuty1)-, 396 Circular dichroism spectra, of 1,4:3,6-dian. hydrohexitol nitrates, 254 Cladinose, a+, 83 Cocrystallization of anomers, 71, 77 of proteins with inhibitor molecules, 93 Codex Alimentarius Commission Draft, on honey, 287, 288 Collagen carbohydrate-peptide linkage in, 419, 436, 438, 442, 443, 468 heterogeneity in, 447 Colominic acid, %3 Colostrum, sialic oligosaccharides from, gel chromatography of, 35 Concentration, effect on gel chromatog raphy, 19 Configuration, of anhydroalditols, 264 Conformation of carbohydrates, hydrogen bonding effect on, 56 crystallography and, 101-10.2 effect on gel chromatography, 31 on oxirane ring, 111, 123 Coronary vasodilators, dianhydrohexitol dinitrates as, 268 Cotton effect, 375 Crystal structure of carbohydrates and derivatives, 53-107 of dianhydrohenitol nitrates, 255 of monosaccharides, 58-75 Crystallography, molecular structure and, of carbohydrates, 53-56 Cycloheptaamylose, crystal strueture of, 79 Cyclohexaamylose, crystal structure of, 79 Cyclohexaamylose-potassium acetate complex, crystal structure of, 78 Cyclohexanol, 2-amino-, deamination of, 184 -, 2-amino.4-tert-buty1-, deamination of, 185

D Dahlia tubes, fructose oligosaceharides from, gel chromatography of, 35 Desmination of aminoaldoses, 183-194 of aminocyclohexanols, 184 of 2-amino-2-deoxyaIditols, 191 of 2-amino-2-deoxyaldohexoses,183 of 2-amino-2-deoxyaldonic acids, 191 of 2-amino-2-deoxyaldonolactones, 189 of 2-amino-2-deoxyaldose dithioacetals, 192 of sugars, 182 Dermatan sulfate carbohydrate chains in, 460, %l in glycoproteins, 417 Desosamine, p-, 83 Dextran gels, in chromatography, 14 Dextrans constitution of, 6 gel chromatography of, 18, 19, 35 molecular-weight determination by gel chromatography, 22 Dextrins, starch, gel chromatography of, 42 Diabetes mellitus, glycoprotein in, 474 Diastase, in honey, 303 lJ-Diazine, derivatives, from sugar derivatives, 396 1,4-Diazine, derivatives, from sugar derivatives, 396-398 Dichloromethane-anthracene-tetracyanoethylene complex, structure of, 55 Digitoxigenin, 8,14-anhydro-, structure of, 55 Disaccharides, crystal structure of, 75-77 Distribution coefficient, in gel chromatography, 16 Disulfide, bis(2-deoxyaltrosid-2-yl), 139 Dithioacetals, of 2-amino-2-deoxyaldoses, deamination of, 192 Diuretics, 1,4:3,6-dianhydro-~-glucitol as, 269

E Electron density, carbohydrate structure and, 55 Electrophoresis, paper, in honey analysis, 297 Emulsifiers, anhydrohexitol esters as, 270 Encephalitogen, 468

SUBJECT INDEX. VOLUME 25

511

structure in, 473 Ficoll, gel chromatography of, 42 Flame-proofing agents, anhydrohexitols, 267 Flavazoles, 398 Floridean starch, 9 Fly sprays, anhydrohexitols in, 269 Foams, polyurethan, anhydrohexitols for, 267 Formaldehyde, reactions of carbonyl compounds and ammonia in presence of, 324 Formazans, 393 Formycin, 243 crystal structure of, and hydrobromide monohydrate, 83 Formycin B, 243 crystal structure of, 83 Fractionation ranges, in gel chromatog raphy, 18 Fragilin, gel chromatography of, 50 Fragmentation mechanism, of reaction of sugars with ammonia, 340 Fructopyranose, PDconformation of, 103 crystallography of, 60 -, 2,5-anhydro-l-O-methyl-j3-~-, 203 Fructopyranosyl fluoride, I-O-methyl-p-D-, reaction with sodium hydroxide, 203 Fructose, Din honey, 289, 295 in nectars, 304 reaction with aqueous ammonia, 312, 315 -, 1,4:3,6-dianhydro-o-, 261 -, l-0-a-D-glucopyranosyl-D-, in honey, 297, 300 Fucose, Lin glycoproteins, 413 in keratan sulfate, 441 2-Furaldehyde, formation from 2,5*anhydroaldoses, 211 -, 5-(hydroxymethyl)-, 215 -, tetrahydro-, preparation of enantiomorphic, 217 Furanoid compounds, conformation of, 104 F Fiirst-Plattner rule, 123 Fetuin, carbohydrate linkage in, 420, 444, Fusicoccin, crystal structure of, and p %5 iodobenzenesulfonate, 90 Fibrinogen G carbohydrate linkage in, 420 effect of glycoprotein structure on, 472 Galactans Fibrocystic disease, pancreas, glycoprotein gel chromatography of, 38 Enthalpy, of hydrogen bonding, 98 Entropy, of hydrogen bonding, 98 Enzymes D-, 9 deficiency, in certain diseases, 478 as glycoproteins, 408 in honey, 302 inhibition of, anhydroalditols for, 269 lysosomal, 474 Q-, 8 R-, 9 starch-metabolizing, 8 z, 9 Enzyme-substrate complexes, crystal structures of, 93-98 Epimino sugars, 352-356 Epoxides, carbohydrate, 110 Erlose in honey, 295, 301, 302 in honeydew honey, 308 Erythraric acid, 2-0-(carboxymethyl)-, 261 Erythritol, crystallography of, 58 -, 1,4-anhydro-, 230 acetalation of, 265 catalytic oxidation of, 262 diacetate, isomerization of, 258 infrared spectrum of, 252 reaction with hydrogen fluoride, 258 Erythrocytes, human, carbohydrate linkages of, 433 Erythromycin A, crystal structure of, and hydriodide dihydrate, 82 Erythrose D-, gel chromatography of, 31 D- and L-, crystallography of, 58 Esterification, selective, of anhydroalditols, 262 Ethylene, tetracyano-, complex with anthracene and dichloromethane, structure of, 55 Ethyne, tetra-0-aCetyl-B-D-glUCOpyranoSyl2-phenyl-, 242

512

SUBJECT INDEX,VOLUME 25

molecular-weight determination by gel Chromatography, 30 Galactitol, crystallography of, 64 -, 1,4.anhydro.n~-, catalytic oxidation of, 261 -, 2,6-anhydro-l,l-bis(ethylsulfonyl)-l. deoxy-o-, triacetate, nuclear m a g netic resonance spectrum of, 252 Galactofuranose, 3-deoxy-3,4-C-(dichloromethylene )-1,2:5,6-di-O-isopropyIidenea-D-, crystal structure of, 74 Galactonic acid, 3,6-anhydro-o~-,261 D-Galactono-1,4-lactone, crystallography of, 65 Galactopyranoside, methyl 3,4-anhydro-a-o-, 127, 145 methyl 6-bromo-6-deoxy-a-n-, crystal structure of, 73 Galactopyranosyl, 0-2-acetamido-2-deoxy.aD-, linkage with amino acids, 425-434 Galactose

-.

D-

gel chromatography of, 31 in glycoproteins, 413 in honey, 290 protein linkage with, 418 a-D- and P-D-, crystallography of, 61 L-, 6-sulfate, 10 -, 2-acetamido-2-deoxy-oin glycopeptides, 433 protein linkage with, 418 -, 2-amino-2-deoxy-~deamination of, 186 determination of, 216 in glycoproteins, 413 -, 3,6-anhydro-o-, 140 -, 3,6-anhydro-~-,in agar, 6 -, 3-O-is-D-galaCtOSyl-D-,34 -, 3-0-(~n-glucosy~uronic acid)+, 34 Gel chromatography, see Chromatography Gentiobiose, in honey, 297 Glass, porous, in chromatography, 15 Glaucoma, 1,4:3,6-dianhydro-~-glucitolin treatment of, 269 r G Globulin heterogeneity in, 447 rabbit, carbohydrate-peptide linkages in, 442, 443 Glucal, D-, triacetate, reaction with lead tetrafluoride, 196

Glucan, 9 Glucito1 D-

anhydridation of, 233 conformation of, 105 D- and L-, crystallography of, 60 -, 2-amino-2-deoxy-~-,deamination of, 191 -, 1,4-anhydro-ooxidative cleavage of, 209, 261 tetraacetate, ring opening and isomerization of, 259 -, 1,5-anhydro-~-,237, 238 infrared spectrum of, 250 -, 1,6-anhydro-n-, 245 -, 1,6-anhydro-~~-, infrared spectrum of, 250 -, 2,6-anhydro-o-, 237, 245 infrared spectrum of, 250 structure of. 251 2,6-anhydro-~.,237 3,6-anhydro-o-, infrared spectrum of, 251 1,3-anhydro-2,4-0-benzylidene-n-, 244, 245 1,5-anhydro-2,4.0-benzylidene-n-, 244 l,S-anhydro-4,6-0.henzylidene-n-, acylation of, 263 5,6-anhydro-2,4-0-benzylidene-O-p tOlylSUlfOnyl-D-, 244 1,5-anhydro-2-S-benzy1-2-thio-o-, 238 2,6-anhydro-l,l-bis(ethylsulfonyl)-ldeoxy.D-, triacetate, nuclear m a g netic resonance spectrum of, 253 1,5-anhydro-2-chloro-2-deoxy-~-, 239 2,5-anhydr0-6-chloro-6-deoxy-n-, 211 1,3-anhydro-5,6-di-0.methy1-2,4-0methylene-o-, 243 1,5-anhydr0-2-C-phenyl-o-, 240 1,4:3,6-dianhydro-oacylation (selective) of, 262 biological uses of, 268 dinitrate, circular dichroism spectrum of, 254 infrared spectra of, and nitrates, 251,2.52 isomerization of, 260 monostearate, surface activity of, 255 nuclear magnetic resonance spectra of, and derivatives, 254 ring opening of, 256 -, 1,4:3,6-dianhydro-~-,catalytic oxidation of, 261

SUBJECT INDEX, VOLUME 25

-, 2,5:3,6-dianhydro-~-, 246

513

-, 1,6-anhydro-j3-~-,conformation of, 101, hydrolysis of, 211 103, 107 O-@-n-glucopyranosyl-( 14)-8-~-, ring opening of, 257 -, 1,4:3,6-dianhydro-2-0-(p-bromophenylsee Cellobiose sulfony1)-(eZO)-D-, crystal and molec- -, 0-a-o-glucopyranosyl-(1-4)-O-[a-nular structures of, 255 glucopyranosyl-(l-2)1-D-, in honey, 1,5:3,6-dianhydro-2,4O-methylene-o-, 297 245 -, O-a-o-glucopyranosyl-(1-6)-0-a-~-, 1,6-dichloro-1,6-dideoxy-~-, 256 glucopyranosyl-(1-3)-D-, in honey, Glucoamylase, carbohydrate-peptide link297, 302 ages in, 418 -, O-f?-D-glUCOpyIXVIOSyl(16) -0-a-DGlucofuranose, 5,6-anhydro-1,2-0-isopropyliglucopyranosyl-( 1-41 -D-, in honey, dene-a+-, 112, 162 297 solvolysis of, 206 -, 1,3,4,6-tetra-O-acetyl-2-o-methyl-~-~-, -, 3-O-benzyl-1,2-0-isopropylidene-5,6-di195 O-p-tOlylSUlfOnyl.D-, solvolysis of, 203 -, 3,4,6-tri-O-acetyl-1,2-anhydro-a-~-, 164 -, 1,2:5,6-di-O-isopropylidene-3-O-p-tolyl- Glucopyranoside, methyl R-D-, gel chromasulfonyl-a-n-, solvolysis of, 207 tography of, 32 Glucofuranoside, ethyl 1-thio-a-n-, crystal -, methyl 2-acetamido-2,6-dideoxy-6-iodostructure of, 71 Q-D-, crystallography of, 93 -, methyl 3-0-benzyl-5,6-di-O-p-tolylsul- -, me thy1 2-amino-4,6-O-benzylidene-2deoxy-&D-, deamination of, 188 fOnyl-D-, 205 ~-~-Glucofuranurono.6,3-lactone, crystallog -, methyl 2-aminod-deoxy-a-o- and -j3-0-, raphy of, 65 deamination of, 185 Glucoisosaccharinic acid -, methyl 2-amino-2-deoxy-4,6-O-ethylD-, 343 idene9-o-methyl-n-, deamination of, " a -D188 calcium and strontium salts, crystal- -, methyl 4,6-dichloro-4,6-dideoxy-a-~-, lography of, 68 crystal structure of, 73 electric charges on atoms of, 100 -, methyl 2-0-(p-nitrophenylsulfonyl) -aGlucometasaccharinic acid, D-, 343 D-, solvolysis of, 208 Gluconic acid, D-, in honey, 290 -, methyl 3-O-p-tOlySUlfOnyl-D-, 207 D-Glucono-1,5-lactone, preparation of, 4 -, methyl tri-O-acetyl-Z-deoxy-Z-iodo-~-D-, Glucopyranose brominolysis of, 194 a-D-,monohydrate Glucopyranosylamine, Z-acetamido-l-N-@Lconfiguration of, 55 aspartyl-2-deoxy-p-b, protein-linking hydrogen bonding in, 56 with, 420-425 Q-D- and 8-0a-D-Glucopyranosyl (dipotassium phoscrystallography of, 61 phate) dihydrate, crystallography of, hydrogen bonds in, 99 66 POD-Glucopyranosyltoluene, surface activity of, 256 conformation of, 106 Glucosaccharinic acid, D-, 343 hydrogen bonding in, 56 Glucose -, 1-0-(2-acetamidoacyl) -2,3,4,6-tetra-ODaCetyl-P-D-, 420 -, 0-N-acetylneuraminoyl- (28)-0from 2-amino-2-deoxy-D-mannose,187 gel chromatography of, 31, 32 (N-acetylneuraminoyl) -(2-3)-0in glycopiuteins, 413 8-o-galactopyranosyl-(1-4)-a-D-, 35 -, 1,2-0-(aminoisopropylidene) -a+ in honey, 289, 295 in nectars, 304 conformation of, 102 hydriodide, crystallography of, 68 preparation from potatoes, 7

-.

-.

$,

514

SUBJECT INDEX. VOLUME 25

chromatography of, 9 reaction with aqueous ammonia, 312, 315 gel chromatography of, 42 structural formula of, 4 Glycopeptides P-D-, (p.bromopheny1) hydrazone, crystal carbohydrate identification in, 433 structure of, 92 molecular weights of, 447-452 -, 2-acetamido-2-deoxy-~in urine, 414, 415 Glycoproteins crystallography of, 93 gel chromatography of, 32 biosynthesis of, 467-472 protein linkage with, 418 carbohydrate components of, 409.414, 469, 477 -, 2-acetamido-2-deoxy-a-D-,crystal struccarbohydrate-peptide linkages in, 467ture of, 70 472 -, 2-amino-2-deoxy-~, see also Chitosadiseases related to changes in, 472-477 mine @elimination reaction of, 427 deamination of, 185, 195 heterogeneity in, 443-447, 477 determination of, 216 metabolism and structure of, 407-478 diethyl dithioacetal, deamination of, 192 molecular weights of polypeptides from, gel chromatography of, 32 447-452 in glycoproteins, 413 nature and occurrence of, 407-417 “glycosamin,” 181 pig submaxillary-gland, 433, 458 protein linkage with, 419 sheep submaxillary-gland, 446 -, 2-amino-2-deoxy-a-D-,hydrobromide and structure of carbohydrate moieties of. hydrochloride, crystal structures of, 452-467 69, 70 sulfate incorporation into, 470-472 -, 2,5-anhydro-o-, 187 Glycopyranosides, hydrolysis of, 437 diethyl dithioacetal, 192 -, 2,5-anhydro-aZdehydo-D-, 1,6-hemiacetal, “Glycosamin,” deamination of, 181 Glycosidases, uses of, 462-%7 213 Glycosides -, 2-S-ethyl-2-thio-n-, 193 crystal structures of, 90 Glucosidase, O-D-, of honey, 303 synthesis of, 164 --, 2-acetamido-2-deoxy-p~-, 4% -, methyl, gel chromatography of, 32 Glucoside, methyl P-D-, ring structure of, 4 Glucosides, D-, phenolic, gel chromatog “C-Glycosides,” 239 Glycosuloses, reaction with ammonia, 345 raphy of, 49, 50 Glycosylamines L-Glutamic acid, in glycoproteins, 419 formation in reaction of sugars and Glycals, lead tetrafluoride action on pyraammonia, 333 noid, 195 synthesis of, 166 Glycans, glycosamino-, 436, 477 Gonadotropin, 466 configuration determination of, 185 carbohydrate-peptide linkage in, 442 gel chromatography of, 47 Grandidentatin, gel chromatography of, 50 of degradation products of, 34, 44 Granulation, of honey, 293 in mucopolysaccharidoses, 475 Grignard reagents protein complexes, 416 reaction with oxirane aldoses, 126, 137 structure of carbohydrate chains in, 459in synthesis of 1-C-substituted carbo462 hydrates, 241 Glyceraldehyde Gularic acid, 2,5-anhydro-n-, 190 D-, gel chromatography of, 31 Gulitol, 1,5-anhydro-o-, 237 DL-, reaction with ammonia, 325 -, 1,5-anhydro-~-,237, 245, 250, 251 Glycerol, I-~-(6-deoxy-6-su~fo-a-~-glucopyGulono-1,4-lactone, 3,6-anhydro-~-,261 ranosyl) -,crystal structure of, 72 Gulopyranoside, methyl 2,3-anhydro-a-o-. Glycogen 127, 145, 1%

SUBJECT INDEX, VOLUME 25

515

-, methyl 2,3-anhydro-4,6-benzylidene-o-, Hex-1-enitol, 3,4,6.tri-O-acetyl.1,5-anhydro116 Gums, gel chromatography of, 38

2-deoxy-~-arabino-,reaction with lead tetrafluoride, 196 Hex-5-enofuranose, 1,2-O.isopropylidene-aD-, 162 H Hex-2-enopyranoside, ethyl 4,6-di-O-acetylHalogen nucleophiles, for oxirane aldoses, 2,3-dideoxy-a-~-erythro-, reaction with 125 lead tetrafluoride, 196 -, methyl 4,6-0-benzylidene-2,3-dideoxyHalogenolysis, of halogenated deoxy a-D-erythro-, 139 sugars, 194 Halogens, displacement of, in sugar deriv- Hexitols anhydro-, 236-247, 275-278 atives, 202 Hemagglutinin, soyhean, 451 catalytic oxidation of, 261 Hemiacetals ring opening of, 256 of 2,5-anhydroaldoses, 212 uses of aldehydo sugars, 214 biological, 268 industrial, 267 Hemicelluloses, gel chromatography of, 43 1,6.anhydro-, 245 Heparin, 11 amino acid in, 434 conformation of, 105 carbohydrate chains in, 460 gel chromatography of, 31 gel chromatography of, 45 dianhydro-, 245-247 of degradation products of, 34 circular dichroism spectra of, 254 Heparitin sulfate, 47 di- and trianhydro-, and derivatives, 279, carbohydrate chains in, 460 280 Heptitol, 2,6-anhydro-, 248 trianhydro-, 247 -, 2,6-anhydro.o-glycero-~-manno-, 248 Hexodialdo-1,5-pyranose,1,2:3,4-di-O-isopro-, 2,6-anhydro-5,7-0-benylidene-l-deoxypylidene-a-o-galacto-, and hydrate, 214 1-nitro-, 248 2,3-Hexodiulose, I-deoxy-o-erythro., 343 -, 2,6-anhydro-3-deoxy-o-galacto-, 248 -, 8-deoxy-n-glycero-, 343 2,6-anhydro-3-deoxy-~-gluco-, 248 2,5-Hexodiulose, 1,4:3,6-dianhydro-o-threo-, -, 2,6-anhydro-3-deoxy-o-manno-, 248 261 .-, 2,6-anhydro-3-deoxy-o-talo-, 248 Hexofuranoses, 5,6-anhydro-, 160-163, 179 -, 2,6-anhydro-5-deoxy-o-altro-, 248 Hexofuranosides, 2,3-anhydro-, 177 -, 2,5-anhydr0-1-deoxy-l-nitro-n-g~ycero- Hexokinase, 269 L-gluco-, 248 Hexopyranoses, 1,2-anhydro-, 163-167 -, 2,5-anhydro-l-deoxy-l-nitra-o-glycero-, 1,6-anhydro-p-~-,172, 173 L-manno-, 248 -, 1,6:2,3-dianhydro-, 131-134 -, 2,6-anhydro-l-deoxy-l-nitro-o-gZycero- -, 1,6:3,4-dianhydro-, 131-134 L-gluco-, 248 Hexopyranoside, ethyl 2,3-anhydro-4,6.di-, 2,6-anhydro-l-deoxy-l-nitro-~-glycerodeoxy-a,t3-DL-~yxo-,146 L-manno-, 248 -, ethyl 2,3-anhydro-4,6-dideoxy-o~-ribo-, Heptitols, anhydro-, and derivatives, 248, 146 281, 282 -, methyl 2,3-anhydro-4-deoxy-6-O-methylHeptulopyranosidonic acid, methyl 4,s-di-0a-DL-lyxo-, 120 benzoyl-3,7-dideoxy-7-iodo-a-~-arabino-, -, methyl 4,6-~-benzylidene.3.deoxy-a-omethyl ester, crystal structure of, 69 lyxo-, 121 Heterocyclic compounds Hexopyranosides, 2,3-anhydro-, 141-150, 171 nitrogen, synthesis from saccharide de- -, 3,4-anhydro-, 141-150, 171, 175, 176 rivatives, 351-405 -, 2,3-anhydro-4,6-0-benzylidene-, 134-141 from sugars and aqueous ammonia, 311- -, methyl 2,3-anhydro-, 173-175 349 Hexopyranosid-3-ulose, methyl 2-bromo-2-

-.

516

SUBJECT INDEX, VOLUME 25

deoxy-a-n-arabino-, 3-hydrate 3,3,4,6tetraacetate, 195 Hexose, 2-deoxy-n-arabino-, 191 -, 4,5-di-O-acetyl-2,6-anhydr0-3-deoxyaldehydo-n-lyzo-, 214

-, 4,5-di.O-acetyl-2,6-anhydr03-deoxyaldehydo-o-xylo-, 214 Hexosulose, o-arabinomannonic acid from, 343 phenylosazone, 212 reaction with ammonia, 325 -, 3-deoxy-o-erythro-, 343 reaction with ammonia, 325 -, 3,6-dideoxy-~-erythro-, reaction with ammonia, 325 Hexuloses, crystallography of, 60 5-Hexulosonic acid, D-ZYZO-, calcium salt dihydrate, crystallography of, 67 Hexulosono-1,4-lactone, 3,6-anhydro-t-xylo-, 26 1 Heyns rearrangement, 335, 337, 338 Histone, calf-thymus, 444 Honey chromatography of, 290-293, 295 composition and analysis of, 289-293 definition of, 287 enzymes in, 303 folklore, 286 granulation of, 293 history, 285 honeydew, 287, 307-309 monosaccharides, 289-294 nectar, 286 oligosaccharides composition of, 295-298 origin of, 298-306 polysaccharides in, 306 sugars of, 285-309 uses of, 286 Honeydew, honey- from, 287, 307-309 Hormones, as glycoproteins, 408 Hyaluronic acid, gel chromatography of, 34, % Hydrazine, phenyl-, reaction with 2,5.anhydroaldoses, 212 Hydrocellulose, 6 Hydrochloric acid-magnesium chloride test, for sugar oxiranes, 170 Hydrogen bonding in carbohydrates, 56, 57

in crystal structures, 98-101

I Jdaric acid, 2,5-anhydro-n-, 190 Iditol crystallography of, 60 I.-, anhydridation of, 233 -, 1,4-anhydro-~-,catalytic oxidation of, 261 -, 1,6-anhydro-~-,infrared spectrum of,

250

-,

2,5-anhydro-~-,243 -, 1,4:3,6-dianhydro-o-, 2% monostearate, surface activity of, 255 ring opening of, 257 --, 1,4:3,6-dianhydro-~-, 260 catalytic oxidation of, 261 infrared spectra of, and nitrates, 252 nuclear magnetic resonance spectra of, and derivatives, 254 Idofuranose, 5,6-anhydro-1,2-0-isopropylidene-p-L-, 112 -, 6-azido-5,6-dideoxy-5-iodo-1,2-O-isopropylidene-p-L-, crystal structure of, 74 -, 5,6-dideoxy-5,6-epithio-1,2-O-isopropylidene-P-L-, 162 -, 1,2-O-isopropylidene-5,6-dithio-p-~-, 5,6-thionocarbonate, 162 Idonic acid, 2-amino-2-deoxy-o-, deamination of, 190 Idonolactone, %amino-2-deoxy-D-, deamination of, 190 Idono-1,4-lactone, 3,6-anhydro-~-,261 Jdose, 3,6-anhydro-n-, 140 -, 2,5-anhydro-aZdehydo-~-, 206 -, 2,5-anhydro-3-0-benzyl-6-O-p-tolylsulfonyl-L-, dimethyl acetal, 203 Idoside, methyl D-, 125 -, methyl 2.0-benzyl-~-,125 Imidazole, sugar derivatives, 315, 316, 318, 328, 336, 344, 347, 349, 373 -, 4- (n-arabino-tetrahydroxybutyl) -,derivatives, 372, 373 Imidazolidine, sugar derivatives, 367-371 Imidazoline, sugar derivatives, 371 Iminium chloride, (chloromethy1ene)dimethyl-, reaction with aldose oxiranes, 126 r G Immunoglobulin, 4% Jmmunoglobulins

SUBJECT INDEX. VOLUME 25 carbohydrate linkage in, 421, 425 as glycoproteins, 4Q8 Infrared spectra of anhydroalditols, 250-252 of sugar oxiranes, 170 Inhibine, in honey, 290 Inulin, gel chromatography of, 42 Tnvertase in honey, 303 yeast, carbohydrate-peptide linkages in, 419 Iron-dextran-citrate complexes, gel chromatography of, 50 Iron-o-glucitol-citrate complexes, gel chromatography of, 50 Isolichenan, 9 Isomaltopentaose, in honey, 297 Isomaltose gel chromatography of, 33 in honey, 295, 300 Isomaltotetraose, in honey, 297 Isomaltotriose gel chromatography of, 33 in honey, 297, 300 Isomaltulose, in honey, 297, 300 Isomannide, 2% Isomerization of anhydroalditols, 258-261 anhydro-ring, 127-131 Isopanose, in honey, 297, 302 Isoquinocycline A, crystal structure of hydrobromide and hydrochloride, 82 Isosaccharinates, 341 Isosorbide, infrared spectra of, and nitrates. 252

K Kanamycin, crystal structure of, 81 Kanamycin monoselenate monohydrate, crystal structure of, 81 Kanamycin monosulfate monohydrate, crystal structure of, 81 Kasugaminide, methyl DL-, crystal structure of, 85 Keratan sulfate, 439 carbohydrate chains in, 459-462 in glycoproteins, 417 molecular weights of chains in, 451 1-Kestose in honey, 295 synthesis of, 301

517

Ketoses, in honey, 295 Kojibiose, in honey, 295, 300

L Lacquers, anhydroalditols for, 268 Lactic acid, from D-glUCOSe and ammonia, 345 Lactoglobulin, carbohydrate-peptide linkage in, 442 Lactose crystal structure of, 77 reaction with aqueous ammonia, 312, 315 -, di-0-sialoyl-, 35 Lactose synthetase, 98 Lactotransferrin, carbohydrate-peptide linkage in, 442 Laminarabiose, in honey, 297 Laminaran, 9 Laurusin, 243 Lead tetrafluoride, reaction with pyranoid glycals, 195 Leucrose, in honey, 295 Lichenan, 9 Lincomycin anhydrooctitols from, 249 crystal structure of, and hydrochloride,

85 -,

7(S)-chloro-7-deoxy-, crystal structure of, 85 Lincomycitol, anhydro-, 249 Lincosaminol, N-acetyl-3,4-O-isopropylidene-, 249 Lipopolysaccharides, gel chromatography of, 43 Lithium compounds, organic, in synthesis of 1-C-substituted carbohydrates, 240 Lobry de Bruyn-Alberda van Ekenstein transformation, 332 Lysine, 5-O-@-o-galactopyranosyloxy-L-, linkage in glycopeptides, 436 -, S-hydroxy-~-,in glycoprotein linkages, 418 Lysozyme action on chitotriose, gel chromatography of products, 35 crystal structures of, and substrate complexes, 93-98 Lyxitol, 2,5-anhydro-n-, 234 Lyxofuranoside, ethyl 2,3-anhydro-5-0-(tetrahydropyran-Z-yl)-o-, 120 -, methyl a+, crystallography of, 58

518

SUBJECT INDEX, VOLUME 25

-, methyl 2,3-anhydro-n-, 116 Lyxopyranoside, methyl 2,3-anhydro-n-, 153 -, methyl 2,3-anhydro-j3-~-,154 Lyxopyranosylmethane, bis(ethylsulfony1) a-D-, triacetate, nuclear magnetic resonance spectrum of, 252 Lyxose, D-, dialkyl dkhioacetal, reaction with p-toluenesulfonyl chloride. 198 -, 2,5-anhydro-o-, 202 dialkyl dithioacetal, 198 -, 2,5-anhydro-5-seleno-o-, dimethyl acetal, 205 -, 2,5-anhydro-5-thio-~-,dimethyl acetal, 205 -, 3,4-di-0-acetyl-Z,5-anhydro-o-, ( pnitrophenyl) hydrazone, 202 Lyxoside, methyl 2,3-anhydro-a-~-and -p D-, 150

-

M Maltopyranoside, methyl p-, hydrogen bonding in, 100 Maltose j3-, crystal structure of, 76 gel chromatography of, 33 in honey, 295, 299 reaction with aqueous ammonia, 313, 315 structure of, 4 synthesis of, 165 Maltoside, methyl p, monohydrate, crystal structure of, 75 Maltotriose gel chromatography of, 33 in honey, 297 Maltulose, in honey, 295, 300 Manna, 287, 308 Mannan, yeast, 9 o-Mannide, monooleate, as emulsifier in vaccine, 270 “p-Mannide,” 2% Mannitol, Danhydridation of, 233 crystallography of, 64 1,4.anhyd10-D-, 236 catalytic oxidation of, 261 infrared spectrum of, 251 -, 1,5-anhydro-~-,237 -, 2,5-anhydro-o-, 194, 208 -, 3.6-anhydro-w, oxidative cleavage of, 209 -9

1,4-anhydr0-2,3- and -3,5-O-benzylideneD-, 265 -, 1,5-anhydro-2-S-benzyl-2-thio-~-, 238 -, 1,5-anhydro-2-chloro-2-deoxy.n., 239 -, 2,5-anhydro-l-deoxy-l,l-difluoro-o-, 196 -, 2,5-anhydro-1,6~dichloro-1,6-dideoxy-~-, 257 -, 1,4:3,6-dianhydm-o-, 246 catalytic oxidation of, 261 infrared spectra of, and nitrates, 252 isomerization of, 260 monostearate, surface activity of, 255 nuclear magnetic resonance spectra of, and derivatives, 254 ring opening of, 256 -, 1,4:3,6-dianhydro-2,5-di-O-methyl-o-, infrared spectrum of, 251 -, 1,6-dibromo-1,6-dideoxy-~-, 257 -, 1,4:2,5:3,6-trianhydro-o-,247 ring opening of, 257 Mannofuranoside, ethyl 2-S-ethyl-1,2-dithioa-D-, 192 Mannonic acid, D-, 343 -, 2,5-anhydro-n-, 189 Mannonolactone, 2-amino-%deoxy-n-, deamination of, 189 o-Mannono-1,5-~actone,preparation of, 4 Mannopyranose, 1,6:2,3-dianhydro-p-n-, 112, 113 Mannopyranoside, methyl 2,3-anhydro.a-D-, 145, 147 methyl 2,3-anhydro.4,6-0-benzylideneD-, 114, 141 -, methyl 4,6-0-benzylidene-2,3-dideoxy2,3-epithio-a-~-,139 Mannopyranosyl fluoride, 3,4,6-tri.O-acetyl2-bromo-2-deoxy-a-n-, crystal structure of, 74 Mannose -,

-.

D-

in glycoproteins, 413 in keratan sulfate, 441 protein linkage with, 418 D- and L-, gel chromatography of, 32 LI-D- and P-O-, crystallography of, 61 -, 2-amino-2-deoxy-o-,deamination of, 187 -, 2,5-anhydro-D-, 181, 185 dimethyl acetal and, 212 mutarotation of, 213 reaction with phenylhydrazine, 212

SUBJECT INDEX, VOLUME 25 2,5-anhydro-4,6-0-benzylidene-~-, 118, 188 2,5-anhydro-3,4,6-tri-O-me thyl-aldehydoD-, mutarotation of, 214 P-S-ethyl-2-thio-o-, 194 1,3,4,6-tetra-0-acety1-2,5-anhydro-o. dimethyl acetal, 194 methyl hemiacetal, 194 Mannosidase, Q-D-, 466 Mannoside, methyl D-, 125 -, methyl 4.64-benzylidene-3-deoxy-3(thiocyanato)-n-, 139 Melamine resins, anhydrohexitols for, 267 Melibiose in honey, 295 reaction with aqueous ammonia, 313, 315 Melizitose in honey, 295, 301 of honeydew honey, 286, 308 Mental retardation, lysosomal enzyme and glycopeptides in, 474 Mercury compound, crystal structure of methyl 2- (chloromercuri)-2-deoxy-a-otalopyranoside, 75 Metabolism, of glycoproteins, 4Q7-478 Metasaccharinates, 341 Methane, dichloro-, complex with anthra. cene and tetracyanoethylene, structure of, 55 Methylation, of polysaccharides, 6 Methyl iodide-Methyl Red test, for sugar oxiranes, 170 Methyllithium, reaction with aldose oxirane, 139 Molecular weights determination by gel chromatography, 21-30 of glycopeptides from glycoproteins, 447452 of polymers, determination of, 14, 22 Monosaccharides crystal structure of, 58-75 gel chromatography of, 31 honey, 289-294 Monosulfide, bis (2-deoxyaltrosid-2-yl), 139 Monotropein, rubidium salt, crystal structure of, 90 Morpholine, sugar derivatives, 4003 Mucopolysaccharidoses, glycosaminoglycans in, 475-477

519

Muramic acid, N-acetyl-, crystallography of, 93 -, N-acetyl-6-0-(2-acetamido-2-deoxy-Poglucosyl) -, crystallography of, 93 -, N-acetylbenzyl-, crystallography of, 93 Mutarotation, of 2,5.anhydro-aZdehydo. aldoses, 213 Mycaroside, methyl 3,4-anhydro-P, 169 yA Myeloma protein, carbohydrate-peptide linkage in, 4 2 , 445

N Nectars, sugars in, 304 Neuraminidase, use of, 463 Nigerose, in honey, 295, 300 Nitrogen heterocycles, synthesis from saccharide derivatives, 311-349, 351-405 Nitrogen nucleophiles, for oxirane aldoses, 125 Nitrous acid, deamination of amino sugars by, 181-194 Nomenclature, of aldose oxiranes, 110 Normuscarine, synthesis of, 216 Nuclear magnetic resonance spectra of anhydroalditols, 252 of sugar oxiranes, 171 Nucleic acids characterization of hydrolysis products of, 374 nucleosides and nucleotides, crystal structures of, 86-90 Nucleophiles reaction with oxirane-containing aldoses, 120, 125 with sulfonyloxy groups of alditols, 264 Nucleosides anhydro-, 405 crystal structure of, 86-90 synthesis of, 218 Nucleotides, crystal structure of, 86-90

0 Octitol, 6-acetamido-1,5-anhydro-6,8-dideoxy-3,4-0-isopropylidene-~-ery.throo-galacto-, 249

-, 6-acetamido-1,5-anhydro-6,8-dideoxy. 3,4-O-isopropylidene-7-O-methyl-~erythro-D-galacto-,249 --, 1,5-anhydro-~-erythro-~-galacto-, 249 -, 1,5-anhydro.6,8-dideoxy-N(4-propyl-~.

520

SUBJECT INDEX,VOLUME 25

hygroyl) .o-erythro-D-ga&cto-, 249 Octitols anhydro-, 249, 282 conformation of, 105 Oligosaccharides chromatography of, 9 crystal structure of, 77-80 gel chromatography of, 32-35 honey composition of, 295-298 origin of, 298-306 of ovarian-cyst blood-group substances, 452-458 sialic acid, from colostrum, gel chromatography of, 35 Orosomucoid, carbohydrate linkage in, 421, 444, %6 Ovarian cysts, blood-group substances, 426, 433, 446,452-458 Ovomucoid carbohydrate linkage in, 420 heterogeneity in, 446 1,4-0xathiane, (2S, 6R) -6-(hydroxymethyl) 2-methoxy-, S-oxide, crystal structure of, 72 1,3-0xazine, sugar derivatives, 400-402 l,COxazine, sugar derivatives, 403 Oxazole, sugar derivatives, 385 Oxazolidine, sugar derivatives, 150, 375380 Oxazoline, derivatives, from sugar derivatives, 356, 380-384 Oxepane ring, anhydrohexitols containing, 245 Oxetane ring alditols Containing, 235, 243 by isomerization of oxirane ring, 129 stability of, 210 Oxidation, catalytic, of anhydrohexitols, 261 Oxirane ring aldoses containing, 109.179 in branched-chain sugars, 167-169 characterization of, 170-172 cleavage of, 125 formation in aldoses, 110-120 migration of, 127 stability of, 120, 210 Oxolane ring opening of, in alditols, 257 stability of, 210

-

Oxycellulose, 6 Oxygen nucleophiles, for oxirane aldoees, 125

P Pancreatic ribonucleases, carbohydrate linkage in, 421 Panose, in honey, 297, 300 Pent-1-enitol, 3,4.di-O-acetyl-l,5-anhydro-2deoxy-o- and -L-erythro-, reaction with lead tetrafluoride, 195 Pentitol, 1,4-anhydro-2-deoxy-~-erythro-, infrared spectrum of, 252 -, 1,4-anhydro-2-deoxy-~-threo-, infrared spectrum of, 252 Pentitols conformation of, 105 gel chromatography of, 31 -, anhydro-, 231-236, 212-274 ring opening of, 257 -, 1,5-anhydro-, 235 -, dianhydro-, 235 Pentodialdo-1,4-furanose,l,2-0-isopropylidene-5-aldehydo-a-o-xylo-, and dimer, 214 Pentofuranose, 2-deoxy-n-erythro-, nucleo. sides and nucleotides, crystal structures of, 86-90 Pentofuranosides, 2,3-anhydro-, 155.160, 177-179 Pentopyranosides, 2-3-anhydro-, 150.154, 171, 176, 177 -, 3,4anhydro-, 150.154, 176, 177 Pentose, 2,5-anhydro-3-deoxy-~-erythro-, reactivity of, 211 -, 2-deoxy-D-erythro-, 192 crystallography of, 58 Pentoses, crystallography of, 58 Pentosulose, 3-deoxy-o-glycero-, reaction with ammonia, 325 2.Pentuloses, crystallography of, 58 Phosphatase, in honey, 303 Phosphorylase, 8 in honey, 303 Picoline, from sugars and ammonia, 328 Pig submaxillary-gland, glycoprotein of, 433, 458 Piperazine, derivatives from sugars and ammonia, 317 Piperidine, derivatives, from sugar derivatives, 394

SUBJECT INDEX, VOLUME 25 Pneumococcal polysaccharides, gel chromatography of, 43, 44 Polyacrylamide, gels in chromatography, 14 fractionation ranges of, 19 Polygalitol, 237, 238 Polymers, molecular-weight determination by gel chromatography, 14, 22 Polysaccharides gel chromatography of, 35-49 in honey, 306 methylation of, 6 molecular-weight determinations of, 28 pneumococcal, gel chromatography of,

6,44 soil, chromatography of, 43 Polystyrene, gels, in chromatography, 15 Polysucrose, gel chromatography of, 42 Polyurethan foams, anhydrohexitols tor, 267 Populin, gel chromatography of, 50 Porasil, 15 Potatoes, o-glucose from, 7 Proline, 4-hydroxy-~-,in glyeoprotein linkages, 418 Protein-carbohydrate linkages, 417-439 Proteins chromatography (gel) of globular, 18 crystallography of, 93 eggwhite, carbohydrate linkages in, 420, 445 molecular-weight determination by gel chromatography, 23 serum, carbohydrate linkages in, 420 Psicopyranose, P-D-, conformation of, 103 Psicose, 60 Pterins, sugar, 399 Pullulan, gel chromatography of, 42 Pyranoid compounds, conformation of, 102, 104 Pyrazine, derivatives formation in ammoniation of molasses, 349 from sugars and ammonia, 316-318, 328, 335 Pyrazole, derivatives, from sugar hydrazones, 366 Pyrazoline, derivatives, from sugar derivatives, 364

Pyridine, derivatives, from sugar and ammonia, 317, 328, 329

521

Pyrrole, and derivatives, from sugar derivatives, 361-364 Pyrrolidine, derivatives, from amino aldoses, 357-361 Pyrroline, derivatives, from sugars, 361 Pyruvaldehyde, reaction with ammonia, 347

Q Quinicol, 2',5-anhydro-, 208 -, 5-O-p-tolylsulfonyl-epi-, solvolysis of, 208 Quinoxaline, derivatives, from sugar derivatives, 373, 396-399 -, 2-(~-arabino-tetrahydroxybutyl)-, 396, 398

R Rafinose crystal structure of, 77 in honey, 290, 295 hydrogen bonding in, 100 Repulsion, by nonbonded atoms in conformational changes, 1@4 Resins, melamine, anhydrohexitols for, 267 Rhamnitol, 1,5-anhydro-~-,237 Rhamnose, L-, gel chromatography of, 32 Ribitol conformation of, 104 crystal structure of, 59 1-phosphate, solvolysis of, 208 -, %arnino-2-deoxy-D-, deamination of, 192 -, 1,4-anhydro-n-, 233 reaction with hydrogen fluoride, 258 tribenzoate, isomerization of, 258 -, 1,4-anhydro-~~-, 208, 231 -, 1,4-anhydro-~-,198 -, 2,5-anhydro-o-, 234 -, 2,5-anhydro-l-deoxy-l,l-difluoro-~-, 235 -, 2,5-anhydro-l-deoxy-l,l-difluoro-~and -L-, 1% Riboflavine, hydrobromide monohydrate, crystal structure of, 92 Ribofuranose, D-, nucleosides and nucleotides, crystal structures of, 86-90 Ribofuranoside, ethyl 2,3-anhydro-5. (tetrahydropyran-2-yl)-D., 120 Ribonuclease, 446 Ribopyranoside, ethyl 3,4-anhydro-P-~-,1% -, methyl 2,3-anhydro-p-~-,152

522

SUBJECT INDEX. VOLUME 25

Ribose, D(p-bromophenyl)hydrazone crystal structure of, 92 hydrogen bonding in, 98 crystallography of, 58 dialkyl dithioacetal, reaction with p toluenesulfonyl chloride, 198 gel chromatography of, 31, 32 -, 2,5-anhydro-~-, dialkyl dithioacetal, 198 Rings, opening of, in anhydroalditols, 256 Ross test, for sugar oxiranes, 170

Sodium thiosulfate-phenolphthalein test, for sugar oxiranes, 170 Soil polysaccharides, chromatography of, 43 Solvolysis, of sulfonates of sugars, 203-209 Sorbose, D- and a+, crystallography of, 61 -, 1,4:3,6-dianhydro-~-,261 Spectroscopy, infrared, of sugar oxiranes, 170 Stability, of aldose oxiranes, 120 Starch enzymic synthesis and degradation of, 9 floridean, 9 sodium glycolate, 8 S synthesis, 8 Saccharides, nitrogen heterocycles from, Stereochemistry, of oxirane derivatives of 351-405 aldoses, 122 Saccharinic acids, 341 Steric hindrance, effect on gel chrornaSagarose, 14, 23 tography, 31 Salicin, gel chromatography of, 47 Streptomycin, crystal structure of, 80 Salireposide, gel chromatography of, 50 Streptomycin oxime sesquiselenate tetraSedoheptulosan hydrate, crystal structure of, 80 conformation of, 101, 103 Structure crystaliography of, and monohydrate, 63 crystallography and, of carbohydrates, 53-56 Sephadex, 14 of glycoproteins, 407-478 fractionation ranges of, 18 Styracitol, 237 Sepharose, 14 Styrene polymers, gels, in chromatography, fractionation ranges of, 18 15 Serine, L-, in glycoprotein linkages, 418, Sucrose 425 -, 0-(2-acetamid0-2-deoxy-a-~-galactopy- crystal structure of, 76 in honey, 291, 295 ranosylh-, glycoprotein linkage, 426 in nectars, 304 -, O.B-D-xylopyranosyl-L-, glycoprotein synthesis of, 165 linkage, 434 Sugars Sheep submaxillary gland, glycoproteins, amino, 6 446 aziridine sugars from, 353 Shortening composition, anhydroglucitols deamination of, 182 for, 268 intermediate for synthesis of, 382 Showdomycin, 243 oxirane sugar derivatives from, 118 crystal structure of, 84 aminodeoxy, 6 Sialic acid formation in reaction of sugars and in glycoproteins, 413, %3 ammonia, 333 in keratan sulfate, 440 anhydro, 6 oligosaccharides, from colostrum, gel 2,5-anhydro chromatography of, 35 properties of, 220-229 Sialoglycosaminoglycans, gel chromatog related compounds and, 181-228 raphy of, 44 utilization of, 215-218 Silica heads, porous, in chromatography, azides, aziridine sugars from, 354 15 aziridine derivatives, 352 branched-chain, oxiranes of, 167 Sinigrin, crystal structure of, 71

SUBJECT INDEX, VOLUME 25

523

methyl 2- (chlorornercuri)-Z-deoxy-a-n-, crystal structure of, 75 Talose, D-, gel chromatography of, 31 -, 2,5-anhydro-o-, 186 Tamm-Horsfall protein, 472, 478 in urine, 414, 415 Tartaric acid configuration of, 55 D- and L-, derivatives, crystallography of, 58 Tay-Sachs disease, 478 Temperature, effect on gel chromatography, 19 Tetrazole, derivatives, from sugar derivatives, 393 Tetritols, anhydro-, 230 derivatives and, 271 Tetroses, crystallography of, 58 Theanderose, in honey, 297, 301, 303 Thiadiazoline, derivatives, from sugar derivatives, 393 Thiazine, derivatives, from sugar derivatives, 403 Thiazolidine, derivatives, from sugar derivatives, 385 Thiazoline, derivatives, from sugar derivatives, 352, 386 Thietane ring, sugar derivative, 157 (carboxymethy1)-nL-, Threaric acid, 2-0261 Threitol, 1,4-anhydro-, 230 catalytic oxidation of, 262 -, 1,4-anhydro-~-,infrared spectrum of, 252 Threonine, L-, in glycoprotein linkages, 418, 425 T -, 0-(2-acetamido-2-deoxy-a-~-galactoTagatose, a-D-, crystallography of, 60 pyranosyl)-L-, glycoprotein linkage, Talitol 426 D-, anhydridation of, 233 Threose, D- and L-, crystallography of, 58 D- and L-, crystallography of, 60 Thyroglobulin, 446 -, 1,4-anhydro-o~-,catalytic oxidation of, Transferases, sugar and glycosyl ester 261 nucleotide, 469 Talopyranoside, methyl 3,4-anhydro-2-0- Transferrin, carbohydrate linkage in, 421, benzoy1-6-deoxy-a-n-, 147 444 -, methyl 2,3-anhydro-4,6-0-benylidene- Transfructosylation, of honey oligosacchaD-, 116, 140 rides, 298, 304 -, methyl 2,3-anhydro-6-deoxy.4-0-methyl- Transglucosylation, in honey oligosacchaa-L-, 147 rides, 298, 304 -, methyl 3,4-anhydro-6-deoxy-2-O-methyl- Transglycosylation, 35 a-L-, 147 Trehalose, synthesis of, 165 constitution and ring structure of, 4 deoxyhalo, 194 oxirane aldoses from, 118 preparation of, 126 epimino, 352-356 of honey, 285-309 nitrogen heterocycles from, 351-405 reactions with alkali, 341, 345 with aqueous ammonia, 311-349 reducing, fission in alkaline solution, 345 separation by gel chromatography, 36 sulfates, 10 sulfonic esters displacement of, 198-202 solvolysis of, 203-209 transport of, anhydroalditols in, 269 unsaturated, oxirane sugar derivatives from, 119, 167 Sulfation, of glycoproteins, 470-472 Sulfolipid, plant, 72 Sulfonic esters, of sugars displacement of, 198-202 solvolysis of, 203-209 Suifonyloxy group displacement of, in aldoses, 110-117 reaction with nucleophiles, 264 Sulfotransferases, in glycoprotein biosynthesis, 472 Sulfur nucleophiles, for oxirane aldoses, 125, 139 Surface activity, of anhydroalditols, 255 Surfactants anhydroalditols, 270 anhydrohexi tols, 267

-,

524

SUBJECT INDEX, VOLUME 25

a,a-Trehalose, 301 a#-Trehalose, in honey, 297, 300 p,p-Trehalose, 301 Tremuloidin, gel chromatography of, 47 1,2,3-Triazole, derivatives, from sugar derivatives, 387-392 Turanose, in honey, 295, 300

-, 1,4-anhydro-5-deoxy-5-fluoro-o~-, 257 -,

1,4-anhydro-2-0-methyl-o~-, infrared spectrum of, 250 -, 1,3-anhydr0-2,4-O-methylene-o~-, 235 -, 1,4-anhydr0-3,5-O-methylene-o~-, infrared spectrum of, 250 -, 1,4:2,5-dianhydro-o~-,236 -, 1,4:3,5-dianhydroU nuclear magnetic resonance spectrum of, 254 IJracil, 1-(2,5-anhydro-3-O-benzyl-@-~-araring opening of, 257, 258 binofuranosyl) -, 200 -, 1,4:3,5-dianhydro-2-O-methyl-n~-, 236 -, 1-(2,5-anhydro-~-~-lyrofuranosyl) -,201 nuclear magnetic resonance spectrum hemiacetal, 212 of, 254 Urine, glycopeptides and glycoproteins of, Xylofuranose, 1,2-O-isopropylidene-3,5-di-O414, 415 p-tolylsulfonyl-o-,solvolysis of, 203 Uronic acids characterization of, 374 -, 1,2-O-isopropylidene-5-seleno-o-, solvoin glycoproteins, 413, 415 lysis of, 205 -, 1,2-O-isopropylidene-5-thio-~-, solvolysis of, 205 V Xylofuranoside, methyl 3,5-anhydro-3.thioVaccines, anhydrohexitols as emulsifiers a-D-, 157 for, 269 Xylopyranose, 3,4-di-O-acetyl-Z-bromo-2-deValine, L-, protein linkage with, 420 OXYO-, reaction with (p-nitrophenyl) Van Deemter equation, 21 hydrazine, 202 Viscosity, effect on gel chromatography, 21 Xylopyranoside, methyl 1-thio-p-D-, synVitamins, crystal structures of, 90-92 thesis and crystal structure of, 58 von Gierke’s disease, 478 Xylose, Ddialkyl dithioacetal, reaction with pW toluenesulfonyl chloride, 198 in glycoproteins, 413 Wagner-Meenvein rearrangement, 209 protein linkage with, 418 Walden inversion, 6, 264 Water of hydration, in carbohydrate crys- -, 2,5-anhydro-n-, dialkyl dithioacetal, 198 tals, 99 -, 2,5-anhydro-~-,209 , 2,5-anhydro-5-seleno.n-,dimethyl acetal, X

-

205

X-Ray diffraction, of carbohydrate crystals, 54 Xylan, constitution of, 6 Xylitol, D-, anhydridation of, 231 -, 1,4-anhydroreaction with dry hydrogen chloride, 257 ring opening and isomerization of, 259 -, 1,4-anhydro-o-, triacetate, isomerization of, 258 -, 1,4-anhydro-i-, 198 -, 2,5-anhydro-n-, 234 -, l,Canhydro-5-chIoro-5deoxy-~~-, 234, 257

-,

-,

2,5-anhydro-5-thio-D-,dimethyl acetal, 205 2,5-anhydro-3-0-p-tolylsulfonyl-n-, dimethyl acetal, 203 3,4-di-O-acetyl-2,5-anhydro-~-, (p-nitrophenyl) hydrazone, 202 4-O-p-D-galactosyl-D-,34

-,

3-~-~-D-galaCtOSyl-4-O-~-D-galaCtOSy~-D-,

-, -,

34

Y Yeasts, osmophilic, in honey, 303

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-25 and Hexitols, 7, 137-207 BARNETT,J. E. G., Halogenated CarboADAMS,MILDRED. See Caldwell, Mary L. hydrates, 22, 177-227 ANDERSON, ERNEST,and SANDS,LILA, A BARRETT,ELLIOTT, P., Trends in the Discussion of Methods of Value in Development of Granular Adsorbents Research on Plant Polyuronides, 1, for Sugar Refining, 6, 205-230 329-344 BARRY,C. P., and HONEYMAN, JOHN, ANDERSON, LAURENS. See Angyal, S. J. Fructose and its Derivatives, 7, 53-98 ANET, E. F. L. J., 3-Deoxyglycosuloses BAYNE,S., and FEWSTER,J. A., The (3-Deoxyglycosones~ and the DegraOsones, 11, 43-96 dation of Carbohydrates, 19, 181-218 BEELIK,ANDREW, Kojic Acid, 11, 145-183 ANGYAL,S. J., and ANDERSON, LAURENS, BELL, D. J., The Methyl Ethers of DThe Cyclitols, 14, 135-212 Galactose, 6, 11-25 ARCHIBALD, A. R., and BADDILEY, J., The BEMILLER,J. N., Acid-catalyzed HydroTeichoic Acids, 21, 323-375 lysis of Glycosides, 22, 25-108 ASPINALL,G . O., Gums and Mucilages, BEMILLER, J. N. See also, Whistler, Roy L. 24, 333-379 BHAT, K. VENKATRAMANA. See Zorbach, ASPINALL, G. O., The Methyl Ethers of W. Werner. Hexuronic Acids, 9, 131-148 BINKLEY,W. W. Column Chromatography ASPINALL, G. O., The Methyl Ethers of of Sugars and Their Derivatives, 10, o-Mannose, 8, 217-230 55-94 ASPINALL, G. O., Structural Chemistry of BINKLEY,W. W., and WOLFROM,M. L., the Hemicelluloses, 14, 429-4668 Composition of Cane Juice and Cane Final Molasses, 8, 291-314 BIRCH, GORDONG., Trehaloses, 18, 201B 225 BISHOP,C. T., Gas-liquid Chromatography BADDILEY, J., See Archibald, A. R. of Carbohydrate Derivatives, 19, 95BAER, HANS H., The Nitro Sugars, 24, 14'7 67-138 BLAIR, MARY GRACE, The 2-HydroxyBAER, HANS H., [Obituary of] Richard glycals, 9, 97-129 Kuhn, 24, 1-12 J. B., Oligo- BOBBITT,J. M., Periodate Oxidation of BAILEY,R. W., and PRIDHAM, Carbohydrates, 11, 1-41 saccharides, 17, 121-167 J., The Use of Boric Acid for BALL,D. H., and PARRISH, F. W., Sulfonic BOESEKEN, the Determination of the Configuration Esters of Carbohydrates: of Carbohydrates, 4, 189-210 Part I, 23, 233-280 BONNER,T. G., Applications of TrifluoroPart 11, 24, 139-197 acetic Anhydride in Carbohydrate BALLOU,CLINTONE., Alkali-sensitive GlyChemistry, 16, 59-84 cosides, 9, 59-95 BONNER, WILLIAMA., Friedel-Crafts and BANKS, W., and GREENWOOD, C. T., Grignard Processes in the CarboPhysical Properties of Solutions of hydrate Series, 6, 251-289 Polysaccharides, 18, 357-398 The BARKER, G. R., Nucleic Acids, 11, 285- BOURNE,E. J., and PEAT,STANLEY, Methyl Ethers of o-Glucose, 5, 145. 333 190 BARKER,S. A., and BOURNE,E. J., Acetals BOURNE, E. J. See also, Barker, S. A. and Ketals of the Tetritols, Pentitols

A

525

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-25

526

BOUVENG, H. O., and LINDBERG, B., Methods in Structural Polysaccharide Chemistry, 15, 53-89 BRAY, H. G., D-Glucuronic Acid in Metabolism, 8, 251-275 BRAY, H. G., and STACEY, M., Blood Group Polysaccharides, 4, 37-55 BRIMACOMBE, J. S. See How, M. J.

C CAESAR,GEORGEV., Starch Nitrate, 13, 331-345

CALDWELL, MARY L., and ADAMS, MILDRED, Action of Certain Alpha Amylases, 5, 229-268 CANTOR,SIDNEY M., [Obituary of] John C. Sowden, 20, 1-10 CANTOR,SIDNEYM. See also, Miller, Robert Ellsworth. CAPON,B., and OVEREND, W. G., Constitution and Physicochemical Properties of Carbohydrates, 15, 11-51 CARR,C. JELLEFF, and KRANTZ,JOHN C., JR., Metabolism of the Sugar Alcohols and Their Derivatives, 1, 175-192 CHIZHOV,0. S. See Kochetkov, N. K. CHURMS,SHIRLEY C., Gel Chromatography of Carbohydrates, 25, 13-51 CLAMP,JOHN R., HOUGH, L., HICKSON, JOHN L., and WHISTLER, ROY L., Lactose, 16, 159-206 COMPTON,JACK,The Molecular Constitution of Cellulose, 3, 185-228 CONCHIE,J., LEVVY,G. A., and MARSH, C. A., Methyl and Phenyl Glycosides of the Common Sugars, 12, 157-187 COURTOIS,JEAN EMILE, [Obituary of] Emile Bourquelot, 18, 1-8 CRUM, JAMESD., The Four-carbon Saccharinic Acids, 13, 169-188

DEFAYE,J., 2,5-Anhydrides of Sugars and Related Compounds, 25, 181-228 DEITZ,VICTORR. See Liggett, R. W. DEUEL,H. See Mehta, N. C. DEUEL, HARRYJ., JR., and MOREHOUSE, MARGARET G., The Interrelation of Carbohydrate and Fat Metabolism, 2, 119-160

DEULOFEU,VENANCIO,The Acylated Nitriles of Aldonie Acids and Their Degradation, 4, 119-151 DIMLER,R. J., 1,6-Anhydrohexofuranoses, A New Class of Hexosans, 7, 37-52 DOUDOROFF, M. See Hassid, W. 2. DIJBACH, P. See Mehta, N. C. DUTCHER,JAMES D., Chemistry of the Amino Sugars Derived from Antibiotic Substances, 18, 259-308

E ELDERFIELD, ROBERTC., The Carbohydrate Components of the Cardiac Glycosides, 1, 147-173

EL KHADEM,HASSAN,Chemistry of Osazones, 20, 139-181 EL KHADEM,HASSAN,Chemistry of Osotriazoles, 18, 99-121 EL KHADEM,HASSAN,Synthesis of Nitrogen Heterocycles from Saccharide Derivatives, 25, 351-4Q5 ELLIS,G. P., The Maillard Reaction, 14, 63-134

ELLIS, G. P., and HONEYMAN,JOHN, Glycosylamines, 10, 95-168 EVANS,TAYLOR H., and HIBBERT,HAROLD, Bacterial Polysaccharides, 2, 203-233 EVANS,W. L., REYNOLDS,D. D., and TALLEY,E. A,, The Synthesis of Oligosaccharides, 6, 27.81

D

F

DAVIES,D. A. L., Polysaccharides of Gramnegative Bacteria, 15, 271-340 DEAN,G. R., and GOTTFRIED, J. B., The Commercial Production of Crystalline Dextrose, 5, 127-143 DE BELDER,A. N., Cyclic Acetals of the Aldoses and Aldosides, 20, 219-302

FERRIER, R. J., Unsaturated Sugars, 20, 67-137; 24, 199-266 FEWSTER, J. A. See Bayne, S. FLETCHER, HEWITTG., JR., The Chemistry and Configuration of the Cyclitols, 3, 45-77

FLETCHER, HEWITT G., JR.,

and RICHT-

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-25 MYER,NELSONK., Applications in the Carbohydrate Field of Reductive Desulfurization by Raney Nickel, 5, 1-28 FLETCHER, HEWITT G., JR., See also, Jeanloz, Roger W. FORDYCE, CHARLESR., Cellulose Esters of Organic Acids, 1, 309-327 FOSTER, A. B., Zone Electrophoresis of Carbohydrates, 12, 81-115 FOSTER, A. B., and HORTON,D., Aspects of the Chemistry of the Amino Sugars, 14,213-281 FOSTER, A. B., and HUGGARD, A. J., The Chemistry of Heparin, 10, 335-368 FOSTER,A. B., and STACEY,M., The Chemistry of the 2-Amino Sugars (2-Amin0-2-deoxy-sugars)~ 7, 247-288 FOSTER,A. B., and WEBBER,J. M., Chitin, 15,371-393 FOX,J. J., and WEMPEN,I., Pyrimidine Nucleosides, 14, 283-380 Fox, JACKJ. See also, Ueda, Tohru. 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 GARCIA GONZALEZ,F., Reactions of Monosaccharides with beta-Ketonic Esters and Related Substances, 11, 97-143 GARCIA GONZALEZ, F., and GOMEZ SANCHEZ, A., Reactions of Amino Sugars with beta-Dicarbonyl Compounds, 20, 303-355 GOEPP, RUDOLPH MAXIMILIAN,JR. See Lohmar, Rolland. GOLDSTEIN,I. J., and HULLAR,T. L., Chemical Synthesis of Polysaccharides. 21, 431-512 A. See Garcia Gonzllez, G ~ M ESANCHEZ, Z

F. IRVING, Glycosyl Ureides, 13, GOODMAN, 215-236 GOODMAN,LEON, Neighboringgroup Par-

527

ticipation in Sugars, 22, 109.175 GOAIN, P. A. J., and SPENCER, J. F. T., Structural Chemistry of Fungal Polysaccharides, 23, 367-417 GOTTFRIED, J. B. See Dean, G. R. GOTTSCHALK, ALFRED,Principles Underlying Enzyme Specificity in the Domain of Carbohydrates, 5, 49-78 GREEN,JOHN W., The Glycofuranosides, 21, 95-142 GREEN,J O H N W., The Halogen Oxidation of Simple Carbohydrates, Excluding the Action of Periodic Acid, 3, 129184 GREENWOOD, C. T., Aspects of the Physical Chemistry of Starch, 11, 335-385 GREENWOOD, C. T., The Size and Shape of Some Polysaccharide Molecules, 7, 289-332; 11, 385-393 GREENWOOD, C. T., The Thermal Degradation of Starch, 22, 483-515 GREENWOOD, C. T., and MILNE, E. A., Starch Degrading and Synthesizing Enzymes: A Discussion of Their Properties and Action Pattern, 23, 281-366 GR~ENWOO C.DT. , See also, Banks, W. GURIN, SAMUEL,Isotopic Tracers in the Study of Carbohydrate Metabolism, 3, 229-250 GUTHRIE,R. D., The “Dialdehydes” from the Periodate Oxidation of Carbohydrates, 16, 105-158 GUTHRIE,R. D., and MCCARTHY,J. F., Acetolysis, 22, 11-23

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., Enzymgtic Synthesis of Sucrose and Other Disaccharides, 5, 29-48

528

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-25

HASSID,W. Z. See also, Neufeld, Elizabeth F. HAYNES,L. J., Naturally Occurring CGlycosyl Compounds, 18,227-258; 20, 357-369 HAYNES,L. J., and NEWTH,F. H., The Glycosyl Halides and Their Derivatives, 10,207-256 HEHRE, EDWARDJ., The Substitutedsucrose Structure of Melezitose, 8, 277-290 HELFERICH, BURCKHARDT, The Glycals, 7, 209-245 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. HICKSON,JOHN L. See Clamp, John R. HILTON. H. W., The Effects of Plantgrowth Substances on Carbohydrate Systems, 21, 377-430 HINDERT,MARJORIE.See Karabinos, J. V. HIRST, E. L., [Obituary of] James Colquhoun Irvine, 8, xi-xvii HIRST,E. L., [Obituary of] Walter Norman Haworth, 6, 1-9 HIRST, 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, JOHN, E., The Amadori Rearrangement, 10, 169-205 HONEYMAN, JOHN, and MORGAN, J. W. W., Sugar Nitrates, 12, 117-135 JOHN.See also, Barry, C. P. HONEYMAN, HONEYMAN, JOHN. See also, Ellis, G. P. HORTON,D., [Obituary of] Alva Thompson, 19, 1-6 HORTON,D., Tables of Properties of 2Amino-2-deoxy Sugars and Their Derivatives, 15, 159-200 HORTON,D., and HUTSON,D. H., Develop ments in the Chemistry of Thio Sugars, 18, 123-199 HORTON,D. See also, Foster, A. B.

J. K. N., The HOUGH, L., and JONES, Biosynthesis of the Monosaccharides, 11, 185-262 HOUGH,L., PRIDDLE, J. E., and THEOBALD, R. S., The Carbonates and Thiocarbonates of Carbohydrates, 15, 91-158 HOUGH,L. See also, Clamp, John R. How, M. J., BRIMACOMBE, J. S., and STACEY,M., The Pneumococcal Polysaccharides, 19, 303-357 HUDSON,C. S., Apiose and the Glycosides of the Parsley Plant, 4, 57-74 HUDSON,C. S., The Fischer Cyanohydrin Synthesis and the Configurations of Higher-carbon Sugars and Alcohols, 1, 1-36 HIJDSON,C. S., Historical Aspects of Emil Fischer’s Fundamental Conventions for Writing Stereo-formulas in a Plane, 3, 1-22 HUDSON,C. S., Melezitose and Turanose, 2, 1-36 HUGGARD, A. J. See Foster, A. B. HULLAR,T. L. See Goldstein, I. J. H~JTSON, D. H. See Horton, D. I ISBELL,HORACES., and PIGMAN, WARD, Mutarotation of Sugars in Solution: Part 11, Catalytic Processes, Isotope Effects, Reaction Mechanisms, and Biochemical Aspects, 24, 13-65 ISBELL,HORACE S. See also, Pigman, Ward.

J ROGERW., [Obituary of] Kurt Heinrich Meyer, 11, xiii-xviii JEANLOZ, ROGERW., The Methyl Ethers of 2-Amino-2-deoxy Sugars, 13, 189214 JEANLOZ, ROGER W., and FLETCHER, HEWITT G., JR., The Chemistry of Ribose, 6, 135-174 JEFFREY, G. A., and ROSENSTEIN. R. D., Crystal-structure Analysis in Carbohydrate Chemistry, 19, 7-22 JONES, DAVID M., Structure and Some Reactions of Cellulose, 19, 219.2% JEANLOZ,

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-25

J. K. N., and SMITH,F., Plant Gums and Mucilages, 4, 243-291 JONES, J. K. N. See aZso, Hirst, E. L. JONES, J. K. N. See also, Hough, L JONSEN, J., and LALAND,S., Bacterial Nucleosides and Nucleotides, 15, 201234 JONES,

529

LEWY, G. A., and MARSH,C. A.. Preparation and Properties of p-Glucuronidase, 14, 381-428 LEWY, G. A. See also. Conchie, J. LIGGETT,R. W., and DEITZ, VICTOR R., Color and Turbidity of Sugar Products, 9,247-284 LINDBERG, B. See Bouveng, H. 0. LOHMAR.ROLLAND,and GOEPP, RUDOLPH MAXIMILIAN,JR., The Hexitols and Some of Their Derivatives, 4, 211-241

K KARABINOS, J. V., Psicose, Sorbose and Tagatose, 7, 99-136 KARABINOS, J. v., and HINDERT, MARJORIE, Carboxymethylcellulose, 9, 285-302 KENT, P. W. See Stacey, M. M KERTESZ,Z. I., and MCCOLLOCH.R. J., Enzymes Acting on Pectic Substances, MAHER,GEORGEG., The Methyl Ethers of the Aldopentoses and of Rhamnose 5, 79-102 # and Fucose, 10, 257-272 KISS, J., Glycosphingolipids (SugarSphingosine Conjugates), 24, 381-433 MAHER,GEORGEG., The Methyl Ethers of o-Galactose, 10, 273-282 KLEMER,ALMUTH.See Micheel, Fritz. See Wallenfels, N. K., and CHIZHOV,0. S., MALHOTRA,OM PRAKASH. KOCHETKOV, Kurt. Mass Spectrometry of Carbohydrate MANNERS,D. J., Enzymic Synthesis and Derivatives, 21, 39-93 Degradation of Starch and Glycogen, KORT, M. J., Reactions of Free Sugars 17, 371-430 with Aqueous Ammonia, 25, 311-349 D. J., The Molecular Structure KOWKABANY, GEORGEN., Paper Chroma- MANNERS, of Glycogens, 12, 261-298 tography of Carbohydrates and ReMARCHESSAULT, R. H., and SARKO,A., lated Compounds, 9, 303-353 X-Ray Structure of Polysaccharides, KRANTZ,JOHN C.,JR. See Cam, C. Jelleff. 22, 421-482 L MARSH,C. A. See Conchie, J. LAIDLAW,R. A., and PERCIVAL, E. G. V., MARSH,C. A. See Levvy, G. A. The Methyl Ethers of the Aldopentoses MARSHALL,R. D., and NEUBERGER,A., and of Rhamnose and Fucose, 7, 1-36 Aspects of the Structure and MetabLALAND, S. See Jonsen, J. olism of Glycoproteins, 25, 47-478 LEDERER, E., Glycolipids of Acid-fast MCCARTHY, J. F. See Guthrie, R. D. Bacteria, 16, 207-238 MCCASLAND, G. E., Chemical and Physical LEMIEUX,R. U., Some Implications in Studies of Cyclitols Containing Four Carbohydrate Chemistry of Theories or Five Hydroxyl Groups, 20, 11-65 Relating to the Mechanisms of Re- MCCLOSKEY,CHESTERM., Benzyl Ethers placement Reactions, 9,1-57 of Sugars, 12, 137-156 LEMIEUX,R. U., and WOLFROM,M. L., MCCOLLOCH, R. J. See Kertesz, Z. I. The Chemistry of Streptomycin, 3, MCDONALD, EMMAJ., The Polyfructosans 337-384 and Difructose Anhydrides, 2, 253-277 LESPIEAU,R., Synthesis of Hexitols and MCGALE,E. H. F., Protein-Carbohydrate Pentitols from Unsaturated PolyCompounds in Human Urine, 24, 435hydric Alcohols, 2, 107-118 452 LEVI, IRVING, and PURVES, CLIFFORDB., MEHLTRETTER, C. L, The Chemical SynThe Structure and Configuration of thesis of o-Glucuronic Acid, 8,231-249 Sucrose (alpha-n-Glucopyranosyl beta- MEHTA,N. C., DUBACH.P., and DEUEL.H., D-Fructofuranoside), 4, 1-35 Carbohydrates in the Soil, 16, 335-355

530

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-25

R. F., The Relative CrystalMESTER,L., The Formazan Reaction in NICKERSON, linity of Celluloses, 5, 103-126 Carbohydrate Research, 13, 105-167 MESTER,L., [Obituary of] GCza ZemplCn, NORD,F. F., [Obituary of] Carl Neuberg, 13, 1-7 14, 1-8 MICHEEL,FRITZ,and KLEMER,ALMUTH, 0 Glycosyl Fluorides and Azides, 16, OLSON,E. J. See Whistler, Roy L. 85.103 OVEREND,W. G., and STACEY, M., The MILLER,ROBERTELLSWORTH, and CANTOR, Chemistry of the 2-Desoxy-sugars, 8, SIDNEY M., Aconitic Acid, a By45-105 product in the Manufacture of Sugar, OVEREND, W. G. See also, Capon, B. 6, 231-249 P MILLS,J. A., The Stereochemistry of Cyclic Derivatives of Carbohydrates, 10, 1-53 PACSU, EUGENE, Carbohydrate OrthoMILNE, E. A. See Greenwood, C. T. esters, 1, 77-127 MONTGOMERY, JOHN A., and THOMAS, H. PARRISH,F. W. See Ball, D. H. H., and TODT, K., Cyclic MonoJEANETTE,Purine Nucleosides, 17, PAULSEN, saccharides Having Nitrogen or Sulfur 301-369 in the Ring, 23, 115-232 MONTGOMERY, REX, [Obituary of] Fred PAULSEN, H. See also, Heyns, K. Smith, 22, 1-10 The Chemistry of AnMOODY,G. J., The Action of Hydrogen PEAT,STANLEY, hydro Sugars, 2, 37.77 Peroxide on Carbohydrates and RePEAT,STANLEY. See also, Bourne, E. J. lated Compounds, 19,, 149-179 MOREHOUSE,MARGARETG. See Deuel, PERCIVAL,E. G. V., The Structure and Reactivity of the Hydrazone and OsaHarry J., Jr. zone Derivatives of the Sugars, 3, 23MORGAN, J. W. W. See Honeyman, John. 44 MORI, T., Seaweed Polysaccharides, 8, PERCIVAL, E. G. V. See also, Laidlaw, R. 315-350 A. MUETGEERT, J., The Fractionation of Starch, PERLIN, A. S., Action of Lead Tetraacetate 16, 299-333 on the Sugars, 14, 9-61 MYRBACK, KARL,Products of the Enzymic Degradation of Starch and Glycogen, PERLIN,A. S., [Obituary of] Clifford Burrough Purves, 23, 1-10 3,251-310 PHILLIPS, G. O., Photochemistry of Carbohydrates, 18, 9-59 N PHILLIPS,G. O., Radiation Chemistry of Carbohydrates, 16, 13-58 NEELY,W. BROCK,Dextran: Structure and PIGMAN, WARD,and ISBELL, HORACES., Synthesis, 15, 341-369 Mutarotation of Sugars in Solution: NEELY, W. BROCK,Infrared Spectra of Part I. History, Basic Kinetics, and Carbohydrates, 12, 13-33 Composition of Sugar Solutions, 23, NEUBERG,CARL, Biochemical Reductions 11-57 at the Expense of Sugars, 4, 75-117 PIGMAN, WARD.See also, Isbell, Horace S. NEUBERGER, A. See Marshall, R. D. POLGLASE, W. J., Polysaccharides AssociNEUFELD,ELIZABETH F., and HASSID,W. ated with Wood Cellulose, 10,283-333 Z., Biosynthesis of Saccharides from PRIDDLE,J. E. See Hough, L. J. B., Phenol-Carbohydrate Glycopyranosyl Esters of Nucleotides PRIDHAM, Derivatives in Higher Plants, 20, (“Sugar Nucleotides”) , 18, 309-356 371-408 NEWTH, F. H., The Formation of Furan PRIDHAM, J. B. See also, Bailey, R. W. Compounds from Hexoses, 6, 83-106 PURVES, CLIFFORDB. See Levi, Irving. NEWTH,F. H. See also, Haynes, L. J.

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-25

R RAYMOND, ALBERT L., Thio- and Selenosugars, 1, 129-145 REES, D. A,, Structure, Conformation, and Mechanism in the Formation of Polysaccharide Gels and Networks, 24, 267-332

531

SOLTZBERG, S., Alditol Anhydrides, 25, 229-283

SOWDEN, JOHN C., The Nitromethane and 2-Nitroethanol Syntheses, 6, 291-318 SOWDEN, JOHNC., [Obituary of] Hermann Otto Laurenz Fischer, 17, 1-14 SOWDEN,JOHN C., The Saccharinic Acids,

12, 35-79 REEVES,RICHARD E., Cuprammonium-GlySPECK, JOHNC., JR., The Lobry de Bruyncoside Complexes, 6, 107-134 Alberda van Ekenstein TransformaREICHSTEIN,T., and WEISS, EKKEHARD, tion, 13,63-103 The Sugars of the Cardiac Glycosides, SPEDDING, H., Infrared Spectroscopy and 17,65-120 Carbohydrate Chemistry, 19, 23-49 RENDLEMAN, J. A., JR., Complexes of SPENCER, J. F. T. See Gorin, P. A. J. Alkali Metals and Alkaline-earth D. B., The Biosynthesis of AroMetals with Carbohydrates, 21, 209- SPRINSON, matic Compounds from D-Glucose, 271 REYNOLDS, D. D. See Evans, W. L. RICHTMYER,NELSON K., The Altrose Group of Substances, 1, 37-76 RICHTMYER,NELSON K., The 2-(aldo. Polyhydroxyalkyl) benzimidazoles, 6, 175-203

RICHTMYER, NELSONK. See also, Fletcher, Hewitt G., Jr. ROSENSTEIN, R. D. See Jeffrey, G. A. ROSENTHAL, ALEX, Application of the 0x0 Reaction to Some Carbohydrate Derivatives, 23, 59-114 Ross, A. G. See Hirst, E. L.

S SANDS, LILA. See Anderson, Ernest. SARKO, A. See Marchessault, R. H. SATTLER,Lours, Glutose and the Unferrnentable Reducing Substances in Cane Molasses, 3, 113-128 SCHOCH, THOMAS JOHN,The Fractionation of Starch, 1, 247-277 SHAFIZADEH, F., Branched-chain Sugars of Natural Occurrence, 11, 263-283 SHAFIZADEH, F., Formation and Cleavage of the Oxygen Ring in Sugars, 13, 9-61 SHAFIZADEH, F., Pyrolysis and Combustion of Cellulosic Materials, 23, 419-474 SIDDIQUI, I. R., The Sugars of Honey, 25, 285-309 SMITH,F., Analogs of Ascorbic Acid, 2, 79-106 SMITH,F. See also, Jones, J. K. N.

15, 235-270

STACEY,M., The Chemistry of Mucopolysaccharides and Mucoproteins, 2, 161-201 STACEY, M., and KENT, P. W., The Polysaccharides of Mycobacterium tuberculosis, 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 STRAHS,GERALD, Crystal-structure Data for Simple Carbohydrates and their Derivatives, 25, 53-107 SUCIHARA, JAMES M., Relative Reactivities of Hydroxyl Groups of Carbohydrates, 8, 1-44

T TALLEY, E. A. See Evans, W. L. TEACUE, ROBERTS., The Conjugates of D-Glucuronic Acid of Animal Origin, 9, 185-246 THEANDER,OLOF, Dicarhonyl Carbohydrates, 17, 223-299 THEOEALD, R. S. See Hough, L. THOMAS, H. JEANNETTE. See Montgomery, John A. TIMELL,T. E., Wood Hemicelluloses: Part I, 19, 247-302 Part 11, 20, 4119-483

CUMULATfVE AUTHOR INDEX

532

TIPSON, R. STUART,The Chemistry of the Nucleic Acids, 1, 193-245 TIPSON,R. STUART,[Obituary of] Harold Hibbert, 16, 1.11 TIPSON,R. STUART,[Obituary of] Phoebus Aaron Theodor Levene, 22, 1-12 TIPSON, R. STUART,Sulfonic Esters of Carbohydratee, 8, 107-215 TODT,K. See Paulsen, H. TURVEY, J. R., [Obituary of] Stanley Peat, 25, 1-12 TURVEY,J. R., Sulfates of the Simple Sugars, 20, 183-218

U UEDA, TOHRU, and Fox, JACK J., The Mononucleotides, 22, 307-419

V VERSTHAETEN, L. M. J., o-Fructose and itts Derivatives, 22, 229-305

W WALLENFELS, KURT, and MALHOTRA, OM PRAKASH, Galactosidases, 16, 239-298 WEBBER,J. M., Higher-carbon Sugars, 17,

15-63 WEBBER,J. M. See also, Foster, A. B. WEICEL, H., Paper Electrophoresis of Carbohydrates, 18, 61-97 WEISS,EKKEHARD. See Reichstein, T. WEMPEN,I. See Fox, J. J. WHISTLER, ROY L., Preparation and

FOR VOLS. 1-25

Properties of Starch Esters, I, 279-

307 WHISTLER,ROY L, Xylan, 5, 269-290 WHISTLER,ROY L., 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 also, Clamp, John R. WHITEHOUSE, M. W. See Zilliken, F. WIGGINS,L. F., Anhydrides of the Pentitole and Hexitols, 5, 191-228 WIGGINS,L. F., The Utilization of Sucrose,

4,293-336 WILLIAMS,NEIL R., Oxirane Derivatives of Aldoses, 25, 109-179 WISE, Lours E., [Obituary of] Emil Heuser, 15, 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, 2

ZILLIKEN, F., and WHITEHOUSE,M. W., The Nonulosaminic Acids-Neuraminic Acids and Related Compounds (Sialic Acids), 13, 237-263 ZORBACH,W. WERNER,and BHAT, K. VENKATRAMANA, Synthetic Cardenolides, 21, 273-321

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25 A

of aldoses, 25, 109-179 of hexitols, 5, 191-228 of pentitols, 5, 191-228 Anhydro sugars. See Sugars, anhydro. Animals, conjugates of D-glUCUrOniC acid originating in, 9, 185-246 Antibiotic substances, chemistry of the amino sugars derived from, 18, 259-308 Apiose, 4, 57-74 Ascorbic acid, analogs of, 2, 79-106 Aromatic compounds, biosynthesis of, from D-glucose, 15, 235-

Acetals, cyclic, of the aldoses and aldosides, 20, 219-302 of hexitols, pentitols, and tetritols, 7, 137-207 Acetic acid, trifluoro-, anhydride, applications of, in carbohydrate chemistry, 16, 59-84 Acetolysis, 22, 11-23 Aconitic acid, 6, 231-249 Action pattern, of starch degrading and synthesizing enzymes, 23, 281-366 Adsorbents, 270 granular, for sugar refining, 6, 205-230 Alcohols, higher-carbon sugar, configurations of, B 1, 1-36 Bacteria, unsaturated polyhydric, 2, 107-118 glycolipides of acid-fast, 16, 207-238 Alditols, anhydrides, of, 25, 229-283 nucleosides and nucleotides of, 15, 201Aldonic acids, acylated nitriles of, 4, 119-151 234 Aldopentoses, polysaccharides from, 2,203-233; 3, 311methyl ethers of, 7, 1-36; 10, 257-272 336 Aldoses, oxirane derivatives of, 25, 109-179 polysaccharides of Gram-negative, 15, Aldoses and aldosides, 271-340 cyclic acetals of, 20, 219-302 Benzimidazoles, Alkaline degradation, 2-(aldo-polyhydroxyaIkyl)-,6, 175-203 of polysaccharides, 13, 289-329 Benzyl ethers, Altrose, of sugars, 12, 137-156 group of compounds related to, 1, 37-76 Biochemical aspects, Amadori rearrangement, 10, 169-205 of mutarotation of sugars in solution, Amino sugars. See Sugars, 2-amino-2-deoxy. 24, 13-65 Ammonia, aqueous, reactions with free Biochemical reductions, sugars, 25, 311.349 at the expense of sugars, 4, 75-117 Amylases, Biosynthesis, certain alpha, 5, 229-268 of aromatic compounds from D-ghlcOSe, Analysis, 15, 235-270 of crystal structure, in carbohydrate of hyaluronic acid, 12, 299-319 chemistry, 19, 7-22 of the monosaccharides, 11, 185-262 Anhydrides, of saccharides, from glycopyranosyl es2,5-, of sugars, 25, 181-228 ters of nucleotides, 18, 309-356 difructose, 2, 253-277 Blood groups, of alditols, 25, 229-283 polysaccharides of, 4,37-55

533

534

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25

Boric acid, for determining configuration of carbohydrates, 4, 189-210 Bourquelol, Emile, obituary of, 18, 1-8 Branched-chain sugars. S e e S u g a r s, branched-chain. C

Cane juice, composition of, 8,291-314 Cane molasses. See Molasses, cane. Carbohydrates, action of hydrogen peroxide on, 19, 149-179 application of reductive desulfurization by Raney nickel, in the field of, 5, 1-28 application of trifluoroacetic anhydride in chemistry of, 16,59.84 application of the 0x0 reaction to some derivatives of, 23, 59-114 as components of cardiac glycosides, 1, 147-173 carbonates of, 15, 91-158 chemistry of, Emil Fischer and his contribution to, 21, 1-38 complexes of, with alkali metals and alkaline-earth metals, 21, 209-271 compounds with proteins, in human urine, 24, 435-452 constitution of, 15, 11-51 crystal-structure analysis of, 19, 7-22 crystal-structure data for, 25, 53-107 degradation of, 19, 181-218 determination of configuration of, with boric acid, 4,189-210 dicarbonyl, 17,223-299 enzyme specificity in the domain of, 5, 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 gel chromatography of, 25, 13-51 halogen oxidation of simple, 3, 129.184 halogenated, 22, 177-227

infrared spectra of, 12, 13-33 infrared spectroscopy of, 19,23-49 mass 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 periodate oxidation of, 11, 1-41 the “dialdehydes” from, 16, 105-158 phenol derivatives, in higher plants, 20, 371-408 photochemistry of, 18,9-59 physicochemical properties of, 15, 11-51 radiation chemistry of, 16,13-58 and related compounds, action of hydrogen peroxide on, 19,149-179 paper chromatography of, 9, 303-353 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; 23, 233280; 24, 139-197 systems, effects of plant-growth substances on, 21, 377-430 thiocarbonates of, 15, 91-158 trityl ethers of, 3, 79-111 zone electrophoresis of, 12,81-115 Carbonates, of carbohydrates, 15, 91-158 Carboxymethyl ether, of cellulose, 9,285-302 Cardenolides. See also, Glycosides, cardiac. synthetic, 21, 273-321 Catalysts, effects of, in mutarotation of sugars in solution, 24, 13-65 platinum, in selective catalytic oxidation of carbohydrates, 17, 169-221 Cellulose, carboxymethyl-, 9,285-302 esters of, with organic acids, 1, 309-321 ethers of, 2, 279-294 molecular constitution of, 3, 185-228

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25 of wood, polysaccharides associated with, 10, 283-333 Celluloses, relative crystallinity of, 5, 103-126 some reactions of, 19,219-246 structure of, 19,219-246 Cellulosic materials, combustion and pyrolysis of, 23, 419474 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 reactions in, 9, 1-57 of the cyclitols, 3, 45-77 of cyclitols containing four or five hydroxyl groups, 20, 11-65 of the 2-deoxy sugars, 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, 3, 337-384 of thio sugars, 18, 123-199 physical, of carbohydrates, 15, 11-51 of starch, 11, 335-385 radiation, of carbohydrates, 16, 13-58 stereo-, of cyclic derivatives of carbohydrates, 10, 1-53 structural, of fungal polysaccharides, 23, 367-417 of the hemicelluloses, 14,429-468 of polysaccharides, 15, 53-89 Chitin, 15, 371-393 Chromatography, column. See Column chromatography. See Gas-liquid chromatog gas-liquid. raphy.

535

gel. See Gel chromatography. paper. See Paper chromatography. Color, of sugar products, 9, 247-284 Column chromatography, of sugars and their derivatives, 10, 5594 Combustion, of cellulosic materials, 23, 419-474 Complexes, of carbohydrates, with alkali metals and alkaline-earth metals, 21, 209271 cuprammonium-glycoside, 6, 107-134 Composition, of sugar solutions, 23, 11-57 Configuration, of carbohydrates, determination of, 4, 189-210 of cyclitols, 3, 45-77 of higher-carbon sugar alcohols, 1, 1-36 of sucrose, 4, 1-35 Conformation, in formation of polysaccharide gels and networks, 24, 267-332 Conjugates, of D-glucuronic acid, 9, 185-246 of sugars with sphingosines, 24, 381-433 Constitution, of carbohydrates, 15, 11-51 Crystallinity, relative, of celluloses, 5, 103-126 Crystal-structure, analysis, in carbohydrate chemistry, 19, 7-22 data, for simple carbohydrates and their derivatives, 25, 53-107 Cuprammonium-glycoside complexes, 6, 107.134 Cyanohydrin synthesis, Fischer, 1, 1-36 Cyclic acetals, of the aldoses and aldosides, 20, 219302 of hexitols, pentitols, and tetritols, 7, 137-207 Cyclic derivatives, of carbohydrates, stereochemistry of, 10, 1-53

536

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25

Cyclic monosaccharides, having nitrogen or sulfur in the ring, 23, 115-232 Cyclitols, 14, 135-212 chemistry and configuration of, 3, 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 starch, 3, 251-310; 17, 407-430 thermal, of starch, 22,483-515 3-Deoxyglycosones. See Glycosuloses, 3deoxy-. 3-Deoxyglycosuloses. See Glycosuloses, 3deoxy-. Deoxy sugars. See Sugars, deoxy. Desulfurization, reductive, by Raney nickel, 5, 1-28 Dextran, structure and synthesis of, 15, 341-369 Dextrins, the Schardinger, 12, 189-260 Dextrose, commercial production of crystalline, 5, 127-143 “Dialdehydes,” from the periodate oxidation of carbohydrates, 16, 105-158 Dicarbonyl derivatives, of Carbohydrates, 17,223-299 Difructose, anhydrides, 2, 253-277 Disaccharides, enzymic synthesis of, 5,29-48 trehalose, 18, 201-225

degradation by, of starch and glycogen, 3, 251-310; 17, 4Q7-430 specificity of, in the domain of carbohydrates, 5,499-78 starch degrading and synthesizing, 23, 281-366 synthesis by, of glycogen and starch, 17,371-407 of sucrose and other disaccharides, 5, 29-48 Esters, of cellulose, with organic acids, 1, 309327 glycopyranosyl, of nucleotides, 18, 309356 beta-ketonic (and related substances), reactions with monosaccharides, 11, 97.143 nitric, of starch, 13, 331-345 of starch, preparation and properties of, 1, 279-307 sulfonic, of carbohydrates, 8, 107-215; 23, 233-280; 24, 139-197 Ethanol, 2-nitro-, syntheses with, 6,291-318 Ethers, benzyl, of sugars, 12, 137-156 carboxymethyl, of cellulose, 9, 285-302 of cellulose, 2, 279-294 methyl, of the aldopentoses, 7, 1-36; 10, 257-272 of 2-amino-2-deoxy sugars, 13, 189214 of fucose, 7, 1-36; 10, 257-272 of o-galactose, 6, 11-25; 10, 273-282 of D-glucose, 5, 145-190 of hexuronic acids, 9, 131-148 of o-mannose, 8, 217-230 of rhamnose, 7, 1-36; 10, 257-272 trityl, of carbohydrates, 3, 79-111

E

F

Electrophoresis, of carbohydrates, paper, 18,61-97 zone, 12,81-115 Enzymes. See also, Amylases, Galactosidases, 8-Glucuronidase. acting on pectic substances, 5, 79-102

Fat, metabolism of, 2, 119-160 Fischer, Emil, and his contribution to carbohydrate chemistry, 21, 1-38 Fischer, Hermann Otto Laurenz, obituary of, 17, 1-14

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25 Formazan reaction, in carbohydrate research, 13, 105-167 Formulas, stereo-, writing of, in a plane, 3, 1-22 Fractionation, of starch, 1, 247-277; 16,299-333 Friedel-Crafts process, in the carbohydrate series, 6,251-289 Fructans, 2, 253-277 Fructofuranoside, a-D-glucopyranosyl P-D-, 4, 1-35 Fructosans, poly-. See Fructans. Fructose, and its derivatives, 7, 53-98; 22,229-305 di-, anhydrides, 2, 253-277 Fucose, methyl ethers of, 7, 1-36; 10, 257-272 Fungal polysaccharides, structural chemistry of, 23, 367-417 Furan compounds, formation from hexoses, 6,83-106

G Galactose, methyl ethers of D-, 6,11-25; 10, 273282 Galactosidases, 16,239-298 Gas-liquid chromatography, of carbohydrate derivatives, 19, 95-147 Gel chromatography, of carbohydrates, 25, 13-51 Gels, polysaccharide, 24,267-332 Glucose. See also, Dextrose. biosynthesis of aromatic compounds from D-, 15,235-270 methyl ethers of D-, 5, 145-190 Glucuronic acid, D-, chemical synthesis of, 8, 231-249 conjugates of, of animal origin, 9, 185246 in metabolism, 8,251-275 p-Glucuronidase, preparation and properties of, 14, 381428 Glutose, 3, 113-128 Glycals, 7, 209-245 -, 2-hydroxy-, 9,97-129 Glycofuranosides, 21, 95-142

537

Glycogens, enzymic degradation of, 3, 251-310; 17, 4Q7-430 enzymic synthesis of, 17,371-407 molecular structure of, 12, 261-298 Glycolipides, of acid-fast bacteria, 16,207-238 Glycoproteins. See Proteins, glyco-. Glycoside-cuprammoniurn complexes, 6, 107-134 G1ycosides, acid-catalyzed hydrolysis of, 22, 25-108 alkali-sensitive, 9, 59-95 cardiac, 1, 147-173 the sugars of, 17,65-120 methyl, of the common sugars, 12, 157187 of the parsley plant, 4, 57-74 phenyl, of the common sugars, 12, 157187 C-Glycosides. See C-Glycosyl compounds. Glycosiduronic acids, of animals, 9, 185-246 poly-, of plants, 1, 329-344 Glycosones, 3-deoxy-. See Glycosuloses, 3deoxy-. Glycosphingolipids, 24, 381-433 Glycosuloses, 3-deoxy-, and the degradation of carbohydrates, 19, 181-218 Glycosylamines, 10,95-168 Glycosyl azides, 16,85-103 C-Glycosyl compounds, naturally occurring, 18, 227-258; 20, 357-369 Glycosyl fluorides, 16,85-103 Glycosyl halides, and their derivatives, 10,207-256 Goepp, Rudolph Maximilian, Jr., obituary of, 3, xv-xxiii Grignard process, in the carbohydrate series, 6,251-289 Gums (see also, Hydrocolloids), 24, 333379 commercial, 13,265-287 of plants, 4,243-291

H Halogen oxidation. See Oxidation, halogen. Halogenated carbohydrates, 22, 177-227

538

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25

Haworth, Walter Norman, obituary of, 6, 1-9 Hemicelluloses, structural chemistry of, 14, 429-468 of wood, 19, 247-302; 20, 409-4.83 Heparin, chemistry of, 10, 335-368 Heuser. Emil, obituary of, 15, 1-9 Hexitols, acetals of, 7, 137-207 anhydrides of, 5, 191-228 and some of their derivatives, 4, 211-241 synthesis of, 2, 107-114 Hexofuranoses, 1,6-anhydro-, 7, 37-52 Hexosans, 7, 37-52 Hexoses. See also, Hexofuranoses. formation of furan compounds from, 6, 83-106 Hexuronic acids, methyl ethers of, 9, 131-148 Hibbert, Harold, obituary of, 16, 1-11 History, of mutarotation, 23, 11-57 Honey, the sugars of, 25, 285-309 Hudson, Claude Silbert, obituary of, 9, xiii-xviii Hyaluronic acid, biosynthesis of, 12, 299-319 Hydrazones, of sugars, 3, 23-44 Hy drocolloids, commercial, polysaccharidic, 13, 265287 Hydrogen peroxide, action on carbohydrates and related compounds, 19, 149.179 Hydrolysis, acid-catalyzed, of glycosides, 22, 25-108 Hydroxyl groups, relative reactivities of, 8, 1-44

I Infrared spectra, of Carbohydrates, 12, 13-33 Infrared spectroscopy, and carbohydrate chemistry, 19, 23-49

Irvine, James Colquhoun, obituary of, 8, xi-xvii Isotopes, effects of, in mutarotation of sugars in solution, 24, 13-65 Isotopic tracers. See Tracers, isotopic.

K Ketals. See Acetals. Kinetics, basic, of mutarotation, 23, 11-57 Kojic acid, 11, 145-183 Kuhn, Richard, obituary of, 24, 1-12

L Lactose, 16, 159-206 Lead tetraacetate, action of, on the sugars, 14, 9-61 Levene, Phoebus Aaron Theodor, obituary of, 12, 1-12 Lipids, glyco-. See Glycolipids. glycosphingo-. See Glycosphingolipids. Lobry de Bruyn-Alberda van Ekenstein transformation, 13, 63-103

M Maillard reaction, 14, 63-134 Mannose, methyl ethers of D-, 8, 217-230 Mass spectrometry, of carbohydrate derivatives, 21, 39.93 Materials, cellulosic, combustion and pyrolysis of, 23, 419.474 Mechanism, in the formation of polysaccharide gels and networks, 24, 267-332 of replacement reactions in carbohydrate chemistry, 9, 1-57 Melezitose, 2, 1-36 structure of, 8, 277-290 Metabolism, of carbohydrates, 2, 119-160 use of isotopic tracers in studying, 3, 229.250 of fat, 2, 119-160 of glycoproteins, 25, 407-478

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25 of the sugar alcohols and their deriva tives, 1, 175-192 u-glucuronic acid in, 8, 251-275 Methane, nitro-, syntheses with, 6, 291-318 Methods, in structural polysaccharide chemistry, 15,53-89 Methyl ethers. See Ethers, methyl. Meyer, Kurt Heinrich, obituary of, 11, xiii-xviii )Molasses, cane, 3, 113-128 cane final, composition of, 8, 291-314 Molecular structure, of glycogens, 12, 261-298 Mononucleotides, 22, 307-419 Monosaccharides, biosynthesis of, 11, 185-262 cyclic, having nitrogen or sulfur in the ring, 23, 115-232 reactions of, with beta-ketonic esters and related substances, 11, 97-143 Mucilages (see also, Hydrocolloids), 24, 333-379 commercial, 13, 265-287 of plants, 4, 243-291 Mucopolysaccharides. See PoIysaccharides, mnco-. Mucoproteins. See Proteins, muco-. Mu tarotation, of sugars in solution: Part I. History, basic kinetics, and composition of sugar solutions, 23, 11-57 Part 11. Catalytic processes, isotope effects, reaction mechanisms, and biochemical aspects, 24, 13-65 Mycobacterium tuberculosis, polysaccharides of, 3, 311-336

N Neighboringgroup participation, in sugars, 22, 109-175 Networks, polysaccharide, 24, 267.332 Neuberg, Carl, obituary of, 13, 1-7 Neuraminic acids, and related compounds, 13, 237-263

539

NickeI, Raney. See Raney nickel. Nitrates, of starch, 13, 331-345 of sugars, 12, 117-135 Nitriles, acylated, of aldonic acids, 4, 119-151 Nitrogen heterocycles, synthesis from saccharide derivatives, 25, 351-405 Nitro sugars. See Sugars, nitro. Nonulosaminic acids, 13, 237-263 Nuclear magnetic resonance, 19, 51-93 Nucleic acids, 1, 193-245; 11, 285-333 Nucleosides, bacterial, 15, 201-234 purine, 17, 301-369 pyrimidine, 14,283-380 Nucleotides, bacterial, 15,201-234 glycopyranosyl esters of, 18, 309-356 mono-, 22, 307-419

0 Obituary, of Emile Bourquelot, 18,1-8 of Emil Fischer, 21, 1-38 of Hermann Otto Laurenz Fischer, 17, 1-14 of Rudolph Maximilian Goepp, Jr., 3, xv-xxiii of Walter Norman Haworth, 6, 1-9 of Emil Heuser, 15, 1-9 of Harold Hibbert, 16, 1-11 of Claude Silbert Hudson, 9, xiii-xviii of James Colquhoun Irvine, 8, xi-xvii of Richard Kuhn, 24, 1-12 of Phoebus Aaron Theodor Levene, 12, 1-12 of Kurt Heinrich Meyer, 11, xiii-xviii of Carl Neuberg, 13, 1-7 of Stanley Peat, 25, 1-12 of Edmund George Vincent Percival, 10, xiii-xx of Clifford Burrough Purves, 23, 1-10 of Fred Smith, 22, 1-10 of John Clinton Sowden, 20, 1-10 of Alva Thompson, 19, 1-6 of G b a Zemplkn, 14, 1-8 Oligosaccharides, 17, 121-167 the raffinose family of, 9, 149-184

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25

540

synthesis of, 6, 27-81 Orthoesters, of carbohydrates, 1, 77-127 Osazones, Chemistry of, 20, 139-181 of sugars, 3, 23-44 Osones, 11, 43-96 Osotriazoles, chemistry of, 18, 99-121 Oxidation, halogen, of simple carbohydrates, 3,

129-148 lead tetraacetate, of sugars, 14, 9-61 periodate, of carbohydrates, 11, 1-41 the “dialdehydes” from, 16, 105-158 selective catalytic, of carbohydrates, employing platinum catalysts, 17,

169-221 Oxirane derivatives, of aldoses, 25,109-179 0 x 0 reaction, application to some carbohydrate derivatives, 23,59-114 Oxygen ring, formation and cleavage of, in sugars,

13. 9-61 P Paper chromatography, of carbohydrates and related compounds, 9, 303-353 Paper electrophoresis, of carbohydrates, 18, 61-97 Parsley, glycosides of the plant, 4,57-74 Participation, neighboringgroup, in sugars, 22, 109.

175 Peat, Stanley, obituary of, 25, 1-12 Pectic materials, chemistry of, 2, 235-251 enzymes acting on, 5, 79-102 Pentitols, acetals of, 7, 137-207 anhydrides of, 5, 191-228 synthesis of, 2, 107-118 Percival, Edmund George Vincent, obituary of, 10, xiii-xx Periodate oxidation. See Oxidation, periodate.

Phenol-carbohydrate derivatives, in higher plants, 20, 371-408 Photochemistry, of carbohydrates, 18, 9-59 Physical chemistry, of Carbohydrates, 15, 11-51 of starch, 11, 335-385 Physical properties, of solutions of polysaccharides, 18, 357-

398 Physical studies, of cyclitols containing four or five hydroxyl groups, 20, 11-65 Plant-growth substances, effect on carbohydrate systems, 21, 377-

430 Plants, glycosides of parsley, 4,57-74 gums of, 4,243-291 mucilages of, 4,243-291 polyuronides of, 1, 329-344 Platinum. See Catalysts. Pneumococcal polysaccharides, 19, 303-

357 Polyfructosans. See Fructans. Polyglycosiduronic acids. See Glycosiduronic acids, poly-. Polysaccharides. See also, Carbohydrates, Cellulose, Dextran, Dextrins, Fructans, Glycogen, Glycosiduronic acids (poly-), Pectic materials, Starch, and Xylan. alkaline degradation of, 13, 289-329 associated with wood cellulose, 10, 283-

333 bacterial, 2, 203-233;15, 271-340 blood-group, 4, 37-55 chemical synthesis of, 21, 431-512 fungal, structural chemistry of, 23, 367-

417 gels and networks, role of structure, conformation, and mechanism, 24,

267-332 hydrocolloidal, 13, 265-287 methods in structural chemistry of, 15,

53-89 muco., chemistry of, 2, 161.201 of Gram-negative bacteria, 15, 271-340 of Mycobacterium tuberculosis, 3, 311-

336 of seaweeds, 8, 315-350

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25

541

physical properties of solutions of, 18, Reactions, 357-398 mechanisms of, in mutarotation of sugars pneumococcal, 19,303-357 in solution, 24, 13-65 shape and size of molecules of, 7, 289of amino sugars with beta-dicarhonyl 332; 11, 385-393 compounds, 20, 303-355 x-ray structure of, 22, 421-482 of cellulose, 19,219-246 Polyuronides, of free sugars with aqueous ammonia, of plants, 1, 329-344 25, 311-349 Preparation, of monosaccharides with beta-ketonic of esters of starch, 1, 279-307 esters and related substances, 11, of p-glucuronidase, 14,391-428 97-143 Properties, Reactivities, relative, of hydroxyl groups of carboof 2-amino-2-deoxy sugars and their derivatives, 15, 159-200 hydrates, 8, 1-44 of esters of starch, 1, 279-307 Rearrangement, the Amadori, 10, 169-205 of @-glucuronidase, 14,381-428 Reductions, physical, of solutions of polysaccharides, 18, biochemical, at the expense of sugars, 357-398 4,75-117 physicochemical, of carbohydrates, 15, Replacement reactions, 11-51 mechanisms of, in carbohydrate chemProteins, istry, 9, 1-57 compounds with carbohydrates, in hu- Rhamnose, man urine, 24, 435-452 methyl ethers of, 7, 1-36; 10, 257-272 Ribose, glyco-, aspects of the structure and chemistry of, 6, 135-174 metabolism of, 25, 407-88 muco-, chemistry of, 2, 161-201 S Psicose, 7, 99-136 Purines, Saccharides, nucleosides of, 17, 301-369 biosynthesis of, from glycopyranosyl Purves, Clifford Burrough, esters of nucleotides, 18, 309-356 obituary of, 23, 1-10 synthesis of nitrogen heterocycles from, Pyrimidines, 25, 3 5 1 4 5 nucleosides of, 14,283-380 Saccharification, Pyrolysis, of cellulosic materials, 23, 419of wood, 4, 153-188 474 Saccharinic acids, 12, 35-79 four-carbon, 13, 169-188 R Schardinger dextrins, 12, 189-260 Radiation, Seaweeds, chemistry of carbohydrates, 16, 13-58 polysaccharides of, 8, 315-350 Raffinose, Seleno sugars. See Sugars, seleno. family of oligosaccharides, 9, 149-184 Shape, Raney nickel, of some polysaccharide molecules, 7, reductive desulfurization by, 5, 1-28 289.332; 11, 385-393 Reaction, Sialic acids, 13, 237-263 the formazan, in carbohydrate research, Size, 13, 105-167 of some polysaccharide molecules, 7. the Maillard, 14,63-134 289-332; 11, 385-393 the 0x0, application to some carbo- Smith, Fred, obituary of, 22, 1-10 hydrate derivatives, 23, 59-114

542

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25

Soil, carbohydrates in, 16, 335-355 Solutions, of polysaccharides, physical properties of, 18, 357-398 of sugars, mutarotation of, 23, 11-57; 24, 13-65 Sorbose, 7,99-136 Sowden, John Clinton, obituary of, 20, 1-10 Specificity, of enzymes, in the domain of carbohydrates, 5, 49-78 Spectra, infrared, of carbohydrates, 12, 13-33 Spectrometry, mass, of carbohydrate derivatives, 21, 39-93 Spectroscopy, infrared, and carbohydrate chemistry, 19, 23-49 nuclear magnetic resonance, 19, 51-93 Sphingosines, conjugates with sugars, 24, 381-433 Starch, degrading and synthesizing enzymes, 23, 28 1-366 enzymic degradation of, 3, 251.310; 17, 407-430 enzymic synthesis of, 17, 371-407 fractionation of, 1, 247-277; 16,299.333 nitrates of, 13, 331-345 physical chemistry of, 11, 335-385 preparation and properties of esters of, 1, 279.307 thermal degradation of, 22, 483-515 Stereochemistry, of cyclic derivatives of carbohydrates, 10, 1-53 formulas, writing of, in a plane, 3, 1-22 Streptomycin, chemistry of, 3, 337-384 Structural chemistry, of fungal polysaccharides, 23, 367-417 of the hemicelluloses, 14,429-468 Structure, molecular, of cellulose, 19, 219-246 of dextran, 15, 341-369 of glycogens, 12, 261-298 of glycoproteins, 25, 4Q7-478 of polysaccharide gels and networks, 24,

267*332 of sucrose, 4, 1-35 x-ray, of polysaccharides, 22, 421-482 Sucrose. See also, Sugar. enzymic synthesis of, 5, 29-48 structure and configuration of, 4, 1-35 utilization of, 4, 293-336 Sugar, aconitic acid as by-product in manufacture of, 6, 231-249 Sugar alcohols, higher-carbon, configurations of, 1, 1-36 and their derivatives, metabolism of, 1, 175-192 “Sugar nucleotides.” See Nucleotides, glycopyranosyl esters of. Sugar products, color and turbidity of, 9, 247-284 Sugar refining, granular adsorbents for, 6, 205-230 Sugars, action of lead tetraacetate on, 14, 9-61 amino, aspects of the chemistry of, 14, 213281 derived from antibiotic substances, 18,259-308 methyl ethers of, 13, 189-214 properties of, 15, 159-200 reactions with beta-dicarbonyl compounds, 20, 303-355 2-amino. See Sugars, 2-amino-2-deoxy. 2-amino-2-deoxy, 7, 247-288 2,5-anhydrides of, 25, 181-228 anhydro, chemistry of, 2, 37-77 benzyl ethers of, 12, 137-156 biochemical reductions at the expense of, 4,75-117 branched-chain, of natural occurrence, 11, 263-283 of the cardiac glycosides, 17, 65-120 conjugates, with sphingosines, 24, 381433 deoxy, 21, 143.207 2-deoxy, 8, 45-105 free, reactions with aqueous ammonia, 25,311-349 higher-carbon, 17, 15-63 configurations of, 1, 1-36

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-25 of honey, 25, 285-309 hydrazones of, 3, 23-44 methyl glycosides of the common, 12, 157-187 neighboringgroup participation in, 22, 109-175 nitrates of, 12, 117-135 nitro, 24, 67-138 osazones of, 3, 23-44 oxygen ring in, formation and cleavage of, 13, 9-61 phenyl glycosides of the common, 12, 157-187 related to altrose, 1, 37-76 seleno, 1, 144-145 solutions of, mutarotation of, 23, 11.57; 24, 13-65 sulfates of the simple, 20, 183-218 and their derivatives, column chroma. tography of, 10, 55-94 thio, 1, 129-144 developments in the chemistry of, 18, 123-159 unsaturated, 20, 67-137; 24, 199-266 Sulfates, of the simple sugars, 20, 183-218 Sulfonic esters, of carbohydrates, 8, 107-215; 23, 233. 280; 24, 139-197 Synthesis, biochemical, of monosaccharides, 11, 185-262 of cardenolides, 21, 273-321 chemical, of D-g~UCUrOnic acid, 8, 231. 249 of polysaccharides, 21, 431-512 of dextran, 15, 341-369 enzymic, of glycogen and starch, 17, 371-407 of sucrose and other disaccharides, 5, 29-48 of nitrogen heterocycles from saccharide derivatives, 25, 351-405

T Tagatose, 7, 99-136 Teichoie acids, 21, 323-375 Tetritols, acetals of, 7, 137-207

543

Thiocarbonates, of carbohydrates, 15, 91-158 Thio sugars. See Sugars, thio. Thompson, Alva, obituary of, 19, 1-6 Tracers, isotopic, 3, 229-250 Transformation, the Lobry de Bruyn-Alberda van Ekenstein, 13, 63-103 Trehaloses, 18, 201-225 Trityl ethers, of carbohydrates, 3, 79-111 Turanose, 2, 1-36 Turbidity, of sugar products, 9, 247-284

U Unsaturated sugars. See Sugars, unsaturated. Ureides, glycosyl, 13, 215-236 Urine, human, protein-carbohydrate compounds in, 24, 435-452

W Wood, hemicelluloses of, 19, 247-302; 20, 409483 polysaccharides associated with cellulose of, 10, 283-333 saccharification of, 4, 153-188

X X-Rays, crystal-structure analysis by, 19, 7-22 Xylan, 5, 269-290

Z Zemplin, Giza, obituary of, 14, 1-8 Zone electrophoresis, of carbohydrates, 12, 81-115

ERRATA VOLUME 22 Page 32, Table 11, column 2, line 3. For “0.679” read “0.295.” Page 32, Ref. 46. For “17, No. 6 (1940)” read “19,No. 1 (1941).” Page 49, Table l X , column 1 , entry 5. For “2-Butyl” read “Isobutyl.” Page 49, Table IX, entry 6, columns 5 and 6. For “36.2” and “f22.9” tead “329” and “+ 12.4” Page 49, last paragraph, sentence 2. Delete rest of sentence after “constant.” Page 57, lines 2 and 3. Delete “or C-3 each.” Page 57, line 4. Insert “1 or 11,ooO” before “[(I91 compared with ( Z O ) ] ” ; after this, insert “; a hydroxyl group at C-3 diminishes the rate constant by a factor of about 3.4 “22) compared with (ZO)].” Page 64, line 3. Before “ionized” insert “un-.” Page 64, line 4. For “~-D-glucopyranosiduron~te” read “B-o-glucopyranosiduronicacid.” Page 64, Table XVI, cohmn 3. For “190” read “19.0”; for “62” read “6.2.” Page 65, lines 14-16. Delete sentence, and insert “From the data in Table XVIIA, they concluded that the rate of hydrolysis increases only slightly as the electronegativity of the aglycon is increased.” Page 66, Table XVllB, heading. For “121a” read “120a” Page 67, Table XYIIC, heading. For “121a” read “120a.” Page 67, Table XVllC, entry 4, columns 5 and 6. For “36.2” and “t22.9” read “32.9” and “+ 12.4.” Page 49, Table I X; Page 74, Table X X I I ; and Page 75, Table XXIII. All of the rate constants listed are too high by a factor of In 10. Page 1*04, Table VI, column, 2, line 10. For “24.2” read “24.2-27.0”; delete entry in column 3.

544