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Advances in Carbohydrate Chemistry and Biochemistry

Volume 41

This Page Intentionally Left Blank

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

Board of Advisors BENGTLINDBERG HANSPAULSEN NATHAN SHARON MAURICE STACEY ROY L. WHISTLER

LAURENS ANDERSON J. ANGYAL STEPHEN CLINTON E. ~ i L L o u GUYG . S. DUTTON ALLANB. FOSTER

Volume 41

1983

ACADEMIC PRESS A Subsidiary of Harcourt Brace Iovanovich, Publishers

Paris

San Diego

New York London S%oPaulo Sydney Tokyo Toronto

San Francisco

COPYRIGHT @ 1983, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS,INC.

111 Fifth Avenue, New York, New York 1Mx13

United Kingdom Edition published by ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London NWI

7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 45- 1 135 1 ISBN 0-1 2-007241 -6 PRINTED IN THE UNITED STATES OF AMERICA 83 84 85 86

9 8 7 6 5 4 3 2 1

CONTENTS CONTRIBUTORS . . . PREFACE. . . . .

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

vii ix

John Kenyon Netherton Jones (1912-1977) A . SZAREK. MAURICESTACEY. AND GEORGE W. HAY WALTER

Text . . . Appendix

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

1 11

Carbon-13 Nuclear Magnetic Resonance Spectroscopy of Monosaccharides KLAUSBOCKAND CHRISTIAN PEDERSEN I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Sampling Techniques . . . . . . . . . . . . . . . . . . . . . . . . . 111. Assignment Techniques . . . . . . . . . . . . . . . . . . . . . . . . IV. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27 28 34 39 44

Structural Chemistry of Polysaccharides from Fungi and Lichens ELIANA BARRETO-BERGTER AND PHILIP A . J . GORIN I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . a-D-Linked Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . 111. p-o-Linked Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Glucans from Lichens . . . . . . . . . . . . . . . . . . . . . . . . . V. Mannans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Galactans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . 2-Acetamido-2-deoxy-o-glucuronic Acid Polymer . . . . . . . . . . . . VIII . 2-Amino-2-deoxy-~-galactopyranan . . . . . . . . . . . . . . . . . . . IX. Heteropolysaccharides Based on o-Mannan Main-Chains . . . . . . . . X . Heteropolysaccharides Based on Galactan Main-Chains . . . . . . . . . XI . Miscellaneous Polysaccharides . . . . . . . . . . . . . . . . . . . .

68 68

72 75

77 87 88

88 89 100 101

Biosynthesis of Cellulose DEBORAH P. DELMER

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. A Survey of Organisms Useful for the Study of Cellulose Biosynthesis

111. IV. V. VI . VII .

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

Structural Considerations Relevant to Biosynthesis Cytological Investigations of Cellulose Biosynthesis The Mechanism of Polymerization . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . Addendum . . . . . . . . . . . . . . . . . . . . .

V

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

105 107 110 116 125 150 152

vi

CONTENTS Capsular Poiysaccharides as Human Vaccines HAROLD J . JENNINGS

I . Introduction . . . . . . . . . . . . . . . . . . . . I1. Structures of Capsular Polysaccharides . . . . . . . I11. Other Important Structural and Physical Features of Capsular Polysaccharides . . . . . . . . . . . . . . IV. Immune Response to Bacterial Infection . . . . . . V. Polysaccharide Vaccines and Immunity . . . . . . VI . Bacterial Virulence . . . . . . . . . . . . . . . . .

. . . . . . . . .

155

. . . . . . . . . . 158 . . . . . . . . . 174 . . . . . . . . . . 186

. . . . . . . . . . 191 . . . . . . . . . 202

High.Resolution. 'H-Nuclear Magnetic Resonance Spectroscopy as a Tool in the Structural Analysis of Carbohydrates Related to Glycoproteins JOHANNES

F. G . \'LIEGENTHART. LAMBERTUS DORLAND. A N D HERMAN VAN HALBEEK

I . General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 209 I1 . Hiyh.resolution. 'H-N.m.r. Spectroscopy of Carbohydrates Related to Glycoproteins of the N-Glycosylic Type . . . . . . . . . . . . . . . . 218 111. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . 371 IV. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 AUTHORINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

375 393

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ELIANABARRETO-BERGTER, Departamento de Microbiologia Geal, Universidade Federal do Rio de Janeiro, Brazil (67) KLAUSBOCK,Department of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark (27)

DEBORAH P. DELMER,* MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 (105) LAMBERTUS DORLAND, Department of Bio-Organic Chemistry, University of Utrecht, Utrecht, The Netherlands (209) PHILIP A. J. GORIN,Prairie Regional Laboratory, National Research Council, Saskatoon, Saskatchewan S7N OW9, Canada (67) GEORGE W. HAY,Carbohydrate Research Znstitute and Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada (1) HAROLD J. JENNINGS, Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KIA OR6, Canada (155) CHRISTIAN PEDERSEN, Department of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark (27) MAURICE STACEY, 12 Bryony Road, Weoley Hill, Birmingham B29 4BU, England (1) WALTER A. SZAREK, Carbohydrate Research Znstitute and Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada (1) HERMAN VAN HALBEEK, Department of Bio-Organic Chemistry, University of Utrecht, Utrecht, The Netherlands (209) JOHANNES F. G. VLIEGENTHART, Department of Bio-Organic Chemistry, University of Utrecht, Utrecht, The Netherlands (209)

*Present address: ARC0 Plant Cell Research Institute, 6560 Trinity Ct., Dublin, California 94568. vii


PREFACE In perhaps no other field of biological chemistry has n.m.r. spectroscopy played such an important role as it has in the structural investigation of the carbohydrates. Its use as an investigative tool has had significant implications across the whole range of carbohydrates, from simple sugar derivatives to complex polysaccharides and glycoconjugates. It is therefore fitting that, in Aduances, this technique should constitute a sustained theme of interconnected articles that are devoted to various important groups of carbohydrates and to the implications of rapid advances in instrumental methodology. As well demonstrated by Gorin’s article in Volume 38 of this Series, carbon-13 n.m.r. spectroscopy has proved to be of profound significance in the structural investigation of polysaccharides; it has taken its place in complementing modern versions of such traditional techniques as methylation analysis and periodate oxidation, and may in large measure replace them as our library of reliable reference data for the simple sugar constituents is consolidated. In the present volume, a most significant step in this direction is taken by Klaus Bock and Christian Pedersen (Lyngby, Denmark) in their extensive and careful compilation of carbon-13 data for a wide range of monosaccharides and their derivatives. The data are conveniently arranged in a selection of representative Tables, and the fact that the authors have themselves conducted extensive verification of the data presented offers the user a measure of convenience and confidence that could never be met by the scattered and often conflicting data in the primary literature. In a similar vein, but with reference to proton-n.m.r. spectroscopy, Vliegenthart and coworkers (Utrecht, The Netherlands) have assembled the fruits of their detailed, comparative studies, by state-of-theart, n.m.r. instrumentation, on a large number of carbohydrates related to glycoproteins. Much of this work was conducted with materials isolated in the laboratories of J. Montreuil (Lille, France), who contributed a landmark article on the structure of glycoproteins to Volume 37 of Aduances. The present, complementary article displays the great power of high-field n.m.r. spectroscopy in applications related to glyco-conjugates of considerable complexity. An article by Barreto-Bergter (Rio de Janeiro, Brazil) and Gorin (Saskatoon, Canada) likewise invokes strong emphasis on n.m.r. methods for structure determination, in this instance by use of carbon13 techniques in delineating the structural chemistry of polysaccharides from fungi and lichens. In comparison with the foregoing complex polysaccharides and conjugates, the structure of the world’s most abundant chemical comix

Y

PHE FrlCE

pound, nanwly cellulose, may seem prosaic indeed, and yet it is quite astonishing that. despite a high level of sophistication in our understanding of the mode of hiosynthesis of many rare, complex carbohydrates, we still have remarkably little definitive knowledge of the way in which Nature builds this ubiquitous, plant polysaccharide. In an article that offers challenges to established dogmas and invites fresh thotight, Deborah P. Delmer (now of Dublin, California) challenges the validity of conclusions that have often been taken for granted, and emphiasizes the need for open-minded, new research on the biosynthesis of the cellulose fiber. The dntmatic success of antibiotics for therapeutic control of many microbial infections has tended to overshadow the value of immunochemical approaches. The article by Jennings (Ottawa, Canada) provides an up-to-date discussion of the structures of a variety of bacterial capsular polysaccharides, and sewes to emphasize the important uses that such compounds have as human vaccines. In a voltimc ha\,ing strong emphasis on polysaccharide topics, it is especially appropriate to recognize the life and work of J. K. N. Jones. The article h e r e contributed by Szarek and Hay (Kingston, Ontario, Canada) and Stacey (Birmingham, England) provides a sensitive account of Jones’s work on both sides of the Atlantic, and includes a useful appendix that lists his scientific publications. The Editors note with regret the recent passing of Louis Malaprade, University of Nancy, discoverer of the stoichiometric oxidation of glycols by periodate, a reaction that has had such profound implications in the structural investigation of carbohydrates; and of Karl Freudenberg, Heidelberg, last surviving student of Emil Fischer’s, pioneer of important stereochemical concepts, and a scientist whose extensive contributions to synthesis included the classic, widely used acetone derivatives (isopropylidene acetals) of the monosaccharides. The Subject Index was compiled by Dr. Leonard T. Capell.

R. STUART TIPSON DEREK HORTON

Advances in Carbohydrate Chemistry and Biochemistry

Volume 41

1912 -1977

JOHN KENYON NETHERTON JONES 1912-1977

Many great men have compelled the admiration of their associates, but few have won the respect and the affection of their colleagues and coworkers to the extent achieved by John Kenyon Netherton Jones. Professor Jones was at all times an educator of the highest rank, and an inspiration to a large number of graduate students, from whom he evoked, as a result of his enthusiasm, sincerity, and gentle character, tremendous fealty and dedication. His life belies the popularly accepted quip “nice guys finish last.” On January 28,1912, J. K. N. Jones was born in Birmingham, England, the eldest son of George Edward Netherton Jones and Florence Jones (n6e Goldchild). His father was a shipping agent for the Elder-Dempster line; during the latter part of his life he was in poor health as a consequence of being badly gassed during World War I, and he died in the early 1920’s from tuberculosis. For the next few years, Jones’s mother strove to secure a pension for herself and her seven children, but because a pension was not granted until 1926, shortly before she died from blood poisoning, hardship characterized the early life of the Jones family. The family, now bereft of both parents, was separated, and the six oldest children were made wards of the Ministry of Pensions, and dispersed among five families of relatives. The youngest, Geoffrey David, who had been born after the end of the war in 1918, was not supported by the Ministry of Pensions, and was sent to an orphanage. Ken Jones had a particularly warm affection for his youngest brother, and experienced enormous grief when Geoffrey, a bomber pilot during World War 11, was killed in action in June, 1944. Ken’s school days were happy ones, and although he lived with several aunts and uncles in Birmingham, they afforded him the security and warm affection so necessary to a growing boy. Hereminisced fiequently of the joyous summer days when he was able to cycle out to the home of a paternal uncle, Jack Jones, who, with his wife Lucy, lived in the country near Ross-on-Wye, Herefordshire. He spent his holidays with them, and these visits engendered in him a life-long interest in gardening and an abiding love of plants and flowers. Between 1917 and 1923, Ken attended the local Bordesley Green Council School. He received a scholarship to the Waverley Grammar 1

Copyright 63 1 W by Academic Press,Inc. All nghts of reproduchon in any form reserved. ISBN 0-124x72414

2

W. A. SZAREK, M. STACEY, AND G . W. HAY

School, an institution noted for the excellence of its science and engineering students, many of whom progressed to Birmingham University. At both Bordesley Green and Waverley Schools, Ken immersed himself in reading and studying as a means of forgetting the harsh times of his childhood and enduring the loneliness occasioned by the prolonged separation from his brothers and sisters. Later, these two activities became a habit and, eventually, a pleasure for him. Ken inherited his mother’s athletic talents and developed into a fine athlete. In 1929, he was both the captain of the school’s association football team and an athletics champion. He was a typical ectomorph, above average in height, seemingly impervious to weight gain, and walking with a characteristic gait that made him stand out in any gathering of scientists. In 1930, Ken entered Birmingham University, having won a Polytechnic Bursary and a Kitchener Scholarship. He began his studies with great enthusiasm, finding particular enjoyment in the laboratory work. Ken had been advised to take a degree in metallurgy, and he studied this subject in his first year at Birmingham, together with chemistry and physics. He found physical chemistry difficult, and his whole interest turned to organic chemistry. During the vacations, he assisted Dr. W. J. Hickinbottom with his researches; this experience presumably persuaded him to become an organic chemist. Ken completed his studies towards the B.Sc. degree, with first-class honours, in 1933,and received the Frankland Medal for having attained the highest standing in his year. The financial constraints imposed by the dire economic conditions of the time thwarted his endeavor to secure financial assistance to work on platinum compounds with Dr. William W. Wardlaw. Happily for carbohydrate chemistry, Ken was offered a research scholarship to study for his Ph.D. with Professor W. N. Haworth and Dr. E. L. Hirst. Under the supervision of this illustrious team, Ken became engaged in studies related to L-ascorbic acid, and he was allocated to Maurice Stacey, at that time a Research Fellow. The Chemistry Department was aglow with excitement because the determination of structure and the synthesis of L-ascorbic acid had just been achieved, and Ken was assigned the topic of repeating on a large scale the synthesis of L-ascorbic acid, and elucidating the structures of some of the intermediates. When Maurice Stacey departed for London, in late 1933, Ken began a happy collaboration with the late Fred Smith, under Edmund Hirst’s general direction. The long hours expended in the laboratory by this team resulted in rapid progress towards the development of a process for the production of L-ascorbic acid on a commercial scale. In 1937, the rights to a patent (with W. N. Haworth, E. L. Hirst, and F. Smith) on the nitric acid oxidation of

OBITUARY-JOHN

KENYON NETHERTON JONES

3

L-sorbose to L-ascorbic acid were sold for a return of $100 sterling to each co-author. The year 1937 was important to Ken Jones both professionally and personally, for, in June of that year, he married Marjorie Ingles Noon, Fred Smith being the best man. The couple first met as teenagers through a family association-Ken’s uncle, Tom, with whom he had lived for a time, had married Majorie’s aunt, Elsie. Later, Ken and Marjorie met at school dances and nurtured their relationship through enjoyable walks and tandem cycling together. Marjorie understood Ken’s commitment to laboratory work, and devotedly spent long evenings, Sundays, and holidays with him in the laboratory, eventually learning enough chemical language to proof-read his writings. Marjorie was an only child; her father was for many years a maintenance and electrical engineer at the offices of the Birmingham Post and Mail,Birmingham’s leading newspaper. In 1936, Ken received the Ph.D. degree. In that same year, when E. L. Hirst was appointed to the Chair of Organic Chemistry at Bristol University, Ken accepted Hirst’s invitation to go with him as an assistant lecturer and to be part of the nucleus of his carbohydrate-research group. Thus began a close and productive partnership that lasted until Hirst moved to Edinburgh in 1948. Jones readily acknowledged that Hirst was his inspiration to diligence in research, and Ken regarded him as a model and a father figure. Ken Jones’s first topic of research at Bristol was the elucidation of the structure of damson gum. Maurice Stacey had collected several pounds of the raw material in Shropshire and had donated it to Hirst. The unravelling of the structure of this complex material was a truly formidable task, but Ken was not daunted by this challenge. He rapidly established himself as a leader, building up, with such colleagues as G. T. Young, a powerful carbohydrate-research team, and they extended their interests to other plant gums, alginates, and unusual starches. Regrettably, the deepening gravity of the war demanded that the major research effort on carbohydrates be suspended in 1940, at which time the Bristol University Chemistry Department, under the headship of Professor W. E. Gamer, was asked to house Professor Bennett of King’s College, London, and his Chemistry Department, and also to find accommodation for sections of the Woolwich Arsenal staff. The organic chemists accepted an invitation to do “war work” with Professors Gamer and Cecil Bawn on problems concerning explosives, such as the use of low-grade toluene to make TNT, and to assist with work on the then-supersecret RDX (hexahydro-l,3,5-trinitro-l,3,5-triazine, a high explosive) and on plastic explosives.

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During this period, Ken supervised the laboratory work at the University, thereby enabling Edmund Hirst to attend to Governmental activities. Under Ken Jones’s leadership, the team, over a period of six years, made a very significant contribution to the war effort, but, because of the nature of the work, received very little publicity. Despite the personal cost to himself, Ken adhered rigidly to the terms of the Official Secrets Act. In 1945, Ken, who was now Lecturer at Bristol University, was again invited by Hirst to move with him to Manchester University, this time as Senior Lecturer in Organic Chemistry. Once more, it became necessary for Hirst to concentrate his efforts on University and Government committee work. Ken Jones, therefore, took charge of the carbohydrate-research group, and supervised the completion of the explosives work. During this interval, Ken enjoyed the able collaboration of Dr. T. G. Halsall in studies on the structures of starch, cellulose, and glycogen, and on the oxidation of carbohydrates by periodate. The close association of Ken with Professor Hirst, which continued at Manchester University until 1948, was a tremendously fruitful one; over 50 joint publications resulted from their research on complex polysaccharides. In 1948, Edmund Hirst moved to Edinburgh University, and Ken returned to Bristol University as Reader in Organic Chemistry. At Bristol, Ken rapidly developed his own carbohydrate-research group and, with great foresight, impressed upon his colleagues the need to apply biochemical methods to the study of natural products, a point of view fully shared by his brilliant assistant (later Professor) Leslie Hough. The decision, in 1951, to accept an invitation to spend six months at the institute of Paper Chemistry, in Appleton, Wisconsin, changed irrevocably the course of Ken’s professional career. i n 1953, he moved to the Chown Research Chair of Chemistry at Queen’s University, Kingston, Ontario, Canada. Of this move, Ken wrote in his Royal Society Record “I stayed (at Appleton) from April to September. The people were very kind and helpful and the weather was hot and sunny. The scenery was good and I liked the large open area. When I saw the advert. for the Chown Research Chair in 1953 I put in for it. I have never regretted moving here. Facilities at first were poor but J. A. McRae, Dean Ettinger and the National Research Council gave me funds to buy apparatus and with the assistance of devoted graduate students we have never looked back.” The Chown Research Chair required the expenditure of a minimum amount of time for administration and teaching, and afforded Ken the maximum possible time for research. One early difficulty in Canada was the restriction of his re-

OBITUARY-JOHN


5

search effort occasioned by the lack of available Canadian graduates, but this was soon overcome by Ken’s ability to attract research students and postdoctoral researchers from overseas, particularly from Bristol. His research group grew steadily. In the course of time, he did succeed in attracting Canadian graduates into his research group, amongst them being Walter Szarek. Like E. L. Hirst, Professor Jones throughout his career attracted the close collaboration of a number of University colleagues. Thus, at Bristol University, Dr. (later Professor) L. Hough collaborated with him, and in Canada, at Queen’s University, he found senior collaborators in the persons of Dr. M. B. Perry until 1962, and Dr. (later Professor) W. A. Szarek from 1967 to 1977. Despite Professor Jones’s abiding passion for research in carbohydrate chemistry, his life was much broader than his science alone. It encompassed as well an appreciation of the beauties of Nature in general, and a devotion to family life. Three children, Stephanie Netherton, Stephen Howard, and Jonathan Ingles Netherton were born into their family. These children, from 1953 onwards, rapidly became “real” Canadians, enjoying life to the full. The children chose to follow diverse educational paths. Stephanie graduated as a nurse, Stephen as an engineer from Queen’s University, Kingston, and Jonathan as a biologist from Brock University, St. Catharines, Ontario. The Jones family had a charming home on Treasure Island on the St. Lawrence River near Kingston. From their grounds, they could enjoy swimming, boating, fishing, and partaking of the beautiful scenery of the St. Lawrence River. Ken took much pride and joy in cultivating and displaying his flowers and garden. Together with his wife Marjorie, he had an active interest in the cultural affairs of Kingston, such as the promotion of live theatre and the Symphony Orchestra. He was an experienced and extremely eager traveller. Indeed, his travels took him to countries on five continents. He was on sabbatical leave in Brazil for the period September 1967 to March 1968,in South Africa from March 1968 to June 1968, and again in Brazil from January 1976 to June 1976. Ken’s hobbies, which he could share with the family, were simple -music, photography, foreign stamps, chess, and the collecting of plants. He took a general interest in military affairs. He was quite proud of the active role he played in Bristol, where he was a part-time officer in the Royal Corps of Signals attached to the University Training Corps. At the end of the war, he resigned with the rank of Captain. As Chown Research Professor at Queen’s University, Professor Jones, in addition to the supervision of the research of a large number of graduate students, postdoctoral fellows, and fourth-year undergraduates, taught courses in Natural Products Chemistry and Carbohy-

6


drate Chemistry. These were graduate-level courses, but were open to fourth-year undergraduates. Occasionally, Ken gave a series of lectures on Carbohydrate Chemistry to students at the Royal Military College in Kingston. Although he was loath to accept administrative responsibilities beyond those inherent in his research operation, Ken, nevertheless, did fulfil such tasks on occasion, at both the Departmental and Faculty level, but administration was not his forte. During his time at Queen’s University, he served briefly as a Member of the Department of Chemistry Graduate Committee, and one term as Chairman of that Division of the School of Graduate Studies and Research which encompassed the physical sciences (Division IV).The Chairmen of the various Divisions are Members of the Council of the School of Graduate Studies. As well, Ken was Secretary of the Committee on Scientific Research, a committee that considered applications from faculty members for financial support of research. He contributed to the design of some of the laboratory renovations (completed in 1964) in Gordon Hall. Professor Jones’s participation in professional societies and affairs outside the University were as follows: Rapporteur for the Royal Society of Canada (Chemical Section) in 1971, and Convenor in 1972; Member of the Advisory Committee to the Atlantic Regional Laboratories of the National Research Council, Halifax, Nova Scotia; Member of the Board of Governors of the Ontario Research Foundation; Member of the Board of Advisors for the British Commonwealth for Advances in Carbohydrate Chemistry and Biochemistry; Member of the Editorial Advisory Board of Carbohydrate Research; Chairman of the Fourth International Conference on Carbohydrate Chemistry, which was held in Kingston in 1967; and a Corresponding Member of the Nomenclature Committee of the Division of Carbohydrate Chemistry, American Chemical Society. Professor Jones was a member of The Chemical Society, the Biochemical Society, the Royal Institute of Chemistry (Associate),the Chemical Institute of Canada, the American Chemical Society, and the New York Academy of Sciences. Professor Jones’s outstanding achievements in carbohydrate chemistry were recognized by his receipt of numerous awards and honors. In 1957, he was elected a Fellow of the Royal Society of London, and, in 1959, a Fellow of the Royal Society of Canada and a Fellow of the Chemical Institute of Canada. The Division of Carbohydrate Chemistry of the American Chemical Society presented him with the Claude S. Hudson Award in 1969. He was the 1975 recipient of the Anselme Payen Award from the Cellulose, Paper, and Textile Division. In March 1975, he was awarded the third Sir Norman Haworth Memorial Medal of The Chemical Society (London).

OBITUARY- JOHN KENYON NETHERTON JONES

7

An International Symposium entitled “Perspectives in Carbohydrate Chemistry” was organized in Kingston to honor Professor Jones on the occasion of his 65th birthday and his retirement from the Chair as the Chown Research Professor at Queen’s University. Alas, a few weeks before the Symposium was to commence, he did not survive a second major operation for cancer of the stomach, and the Symposium was held, in May, 1977, as a memorial to him. Over 200 participants attended the Symposium, which was a fitting expression of appreciation of the life and work of a fine scientist and true gentleman. As has already been intimated, J. K.’s earliest publications (with W. N. Haworth and E. L. Hirst) were concerned with L-ascorbic acid (vitamin C) and its analogs. Four papers and one patent resulted from the L-ascorbic acid work at Birmingham. When E. L. Hirst was appointed to the Chair of Organic Chemistry at Bristol University in 1936, he took J. K. with him from Birmingham. At this time, Fred Smith’s work was mainly concerned with the structures of gum arabic and gum tragacanth, topics suggested by Edmund Hirst, and J. K. and Fred agreed to maintain a general collaboration in order to avoid overlap. The early Bristol work indeed owes much to the generous provision by Fred Smith of reference samples of partially methylated sugars. J. K. undertook the major task of elucidating the structures of damson gum and various pectic substances. The next four years was a period of tremendous activity for him, necessitating spending even longer hours than previously in the laboratory, as few research students were available at Bristol. The techniques used for structural determination of the highly complex, acidic carbohydrate polymers were initially those developed by the Birmingham school for the determination of polysaccharide structures : exhaustive methylation of the carbohydrate polymer, or its acid-degraded derivative, followed by partial and complete hydrolysis and then quantitative separation and identification of the constituent methylated mono-, di-, and oligo-saccharides. J. K. realized that new techniques were urgently needed to lessen the enormous amount of labor required for these difficult investigations. J. K. developed new methods of methylation using thallium compounds and methyl iodide, and new oxidative methods for the determination of end groups in saccharide chains. He was quick to perceive the potential of various chromatographictechniques for the separation of complex mixtures of simple saccharides and their methyl ethers. The results were published in a series of papers concerned with the structures of such diverse substances as damson gum, peanut arabinan, pectic acids, cherry gum, slippery-eIm mucilage, and citrus arabinan. The move to Manchester in 1945 made a welcome change, although J. K. had once more to build up a new research team. The cessation of hostilities had

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W. A. SZAREK,

M. STACEY, A N D G. W. HAY

cleared the way for the resumption of his primary interest, namely, polysaccharide chemistry. Salient among the excellent work performed during this period was the elucidation of further major structural features of the complex macromolecules of damson gum, cherry gum, and peach gum. Damson gum is an exudate gathered in the form of resin-like nodules from the bark of the tree Prunus insitia. It is normally isolated in the form of an ash-free, water-soluble, acidic white powder. Hydrolysis by mineral acid afforded D-xylose, L-arabinose, Dgalactose, D-mannose, and D-glucuronic acid. Damson gum differed from gum arabic in its content of D-mannose and D-xylose, and by its lack of L-rhamnose. As in the case of gum arabic, L-arabinose was liberated by autohydrolysis, and was present in the furanoid form. No fewer than 18 methylated saccharide derivatives were isolated from the hydrolyzed, methylated gum and the methylated, degraded gum. The quantitative separation of these constituents and an examination of their modes of union permitted an assignment of structure to a large part of the highly branched molecule. Similar studies were made on cherry gum, to which periodate-oxidation techniques were applied successfully. In the many gum exudates and mucilages studied, striking similarities, and yet often wide differences, in saccharide constituents and their modes of linkage were disclosed. The work was greatly expedited by J. K.’s development of automated fraction-collectors. Column chromatography and paper partition-chromatography were developed into a fine art at Bristol, owing to J. K.’s skill and that of such of his students as A. E. Flood, F. Brown, W. H. Wadman, and L. Hough. Ken’s geographical transition to the New World was accompanied by a concomitant transition in his research emphasis. Although he maintained an interest in polysaccharide chemistry, the publication record from Queen’s University attests to the universality of his interests in carbohydrate chemistry. J. K. made major contributions to synthetic carbohydrate chemistry, stereochemistry , biosynthetic mechanisms, and metabolism of carbohydrates, and the application of such separational techniques as paper and gas-liquid chromatography in the carbohydrate field. The results of his lifetime of research were documented in over 300 scientific publications. Clearly, it would be impractical to review this number of papers individually, and consequently, only a representative sample will be treated. A list of Professor Jones’s publications is appended to this article. During the 1950’s and early 196O’s, J. K. and his coworkers achieved new syntheses of a number of simple sugars. These included D-tag& tose and mpsicose, 5,&dideoxy-~-xylo-and -h-arubino-hexose, derivatives of Dribitol, 5-S-ethyl-5thio-~threo-2-pentulose, wgZycer0-D-

OBITUARY-JOHN


9

rnanno-heptose, l-deoxy-~-arabino-3-hexulose, L-arabinoseS-lC, D apiose, 3-acetamido-3-deoxy-~glucose,L-mycarose, L-cladinose, D glycero-Daltro-, L-glycero-L-galacto-, Dglycero-L-gluco-, and Bglycero-L-galacto-octulose,and 3-hexuloses. The classic problem of disaccharide synthesis also attracted J. K.’s attention. Syntheses of 3O-~-D-galactopyranosy~-D-ga~actose, 3-~-~-D-xylopyranosy~-D-xy~ose,

lactose, 2-0- and 5-O-P-D-glUCOpyranOSyl-D-XylOSe,and 4-O-P-D-galactopyranosyl-Dgalactose were included in his achievements. Two accomplishments of particular significance were the synthesis of sugars in which the ring-oxygen atom had been replaced by nitrogen, and the investigation of the reaction of sulfuryl chloride with sugars and their derivatives. The former development was a consequence of J. K.’s study of the microbiological oxidation of sugar derivatives by Acetobacter suboxyans. A series of papers on the oxidation of terminally substituted, polyhydric alcohols was published. In connection with studies of the oxidation of acetamidodeoxyalditols,5-acetamido-5-deoxy-~-arabinose was prepared and, interestingly, was found to exist in two forms, namely, the normal furanoid form, and a pyranoid form in which the ring heteroatom was nitrogen, not oxygen. A number of examples of this new type of compound were synthesized, including 5-acetamido-5-deoxy-~-xylopyranose, methyl 4-acetamido-4-deoxy-~-erythrofuranoside, and methyl 4-acetamido-4deoxy-D and -L-arabinofuranoside. Concomitant with these developments at Queen’s University, researchers in other countries, particularly the United States and West Germany, were synthesizing a variety of analogous compounds in which the oxygen atom of the ring was replaced by nitrogen, phosphorus, selenium, or sulfur. J. K. and his colleagues extensively studied the reaction of sulfuryl chloride with carbohydrates. This work elucidated the stereochemical principles involved in the various transformations, and made available a convenient and effective procedure for the preparation of chlorodeoxy sugars, derivatives that have been found to be extremely valuable intermediates in the synthesis of a wide variety of rare sugars. The synthesis of chlorodeoxy sugars involves the initial formation of chlorosulfuric esters, followed by bimolecular displacement of certain of the chlorosulfonyloxy groups by chloride ion liberated during the chlorosulfation. It is often possible to predict the reactivity of a chlorosulfonyloxy group by a consideration of the steric and polar factors affecting the formation of the transition state. Thus, it has been found that the presence of a vicinal, axial substituent, or of a P-transaxial substituent, on a pyranoid ring inhibits replacement of a chlorosulfonyloxy group; also, a chlorosulfate group at C-2 has been observed to be deactivated to nucleophilic substitution by chloride ion.

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Some of the rare sugars that have been prepared by way of chlorodeoxy derivatives are 4,6-dideoxy-3- 0-methyl-~-xyZo-hexose(D-chalcose), 3,6-dideoxy-D-ribo-hexose (paratose), 3,6-dideoxy-~urabinohexose (tyvelose), methyl 2,3-dideoxy-~-~-glycero-hex-2-enopyranosid-4-ulose, and certain aminodeoxy sugars. Although the bulk of J. K.’s work at Queen’s University concerned reactions of monosaccharides, he nevertheless did not lose interest in polysaccharides and the chemistry of L-ascorbic acid. The polysaccharides examined during the Canadian period included the hemicelluloses of loblolly pine (Pinus taeda) and aspen (PopuEus trernuloides), linseed mucilage from flax (Linurn usztatissimum), the type VIII Pneurnococcus specific polysaccharide, the hemicelluloses and a-cellulose from a specimen of ancient wood from Cedrus penhallowii, an arabinogalactan from Monterey pine (Pinus radiata ), the “gum asafoetida” polysaccharide, a water-soluble arabinogalactan from mountain larch (Larix lylatli, Parl), the glucomannan of bluebell seed (ScyZla n.onsc7ipta L.), polysaccharides from the seeds of the huacra pona palm (friarteauentricosa), cholla gum (Opuntia fulgida),the capsular polysaccharide of Pneumococcus XII, arabinobioses from Acacia nilotica gum, the type-specific polysaccharide from type XIX Pneumococcus, the mucilage from the bark of Ulmus fulua (slipperyelm mucilage), lemon gum (CitrusZiminia), lipopolysaccharides of Proteus, and the galactan from the albumin glands of the snail (Strophocheilus oblongus). In the course of the preparation of C - and 0benzyl derivatives of L-ascorbic acid, it was observed that the L-ascorbate ion acts as an ambident nucleophile; this was the first example of a carbohydrate structure exhibiting this property. The universality of J. K.’s interests was further manifested by his continuing studies of biosynthetic mechanisms and the metabolism of carbohydrates, and the application of such separational techniques as gas-liquid chromatography in the carbohydrate field. In 1961, the mode of the linkage of sugars to amino acids in glycoproteins and “mucoproteins” was obscure. J. K. and his colleagues attempted to determine the nature of the carbohydrate -peptide bonds occurring in natural glycoproteins. In 1967, Walter Szarek returned to Queen’s University as a faculty member, after a three-year absence in the United States, and was asked to direct J. K.’s entire research group while J. K. was on sabbatical leave in Brazil and, subsequently, South Africa. This marked the beginning of the very close association and collaboration between J. K. and Walter Szarek, a partnership that continued until J. K.’s death. The fruit of this close relationship was recorded in the numerous publications that ensued. These were primarily in the area of synthetic carbohydrate chemistry, and involved such topics as synthesis and


11

chemical modification of carbohydrate antibiotics, design of biologically active nucleosides, development of new routes to those sugars and their derivatives that are of interest to biochemistry and chemotherapy, conformationaland mechanistic studies of carbohydrate reactions, microbiological oxidation of sugars and their derivatives, chemical modification of polysaccharides, photochemistry of carbohydrates, stereochemistry, and heterocyclic conformational analysis. In 1976, the long tradition of excellence in carbohydrate chemistry at Queen’s University led to the formation of the Carbohydrate Research Institute. J. K. was a very strong supporter of such an interface between the University, Government, and Industry and was, with Walter Szarek and G. W. Hay, a Founding Member. Regrettably, his deteriorating health at this period obviated his active involvement in the formation and subsequent development of the Institute, which came into being through the efforts, and under the directorship, of Walter Szarek. Professor Jones died on April 13, 1977, after a 10-month struggle with cancer. His family and friends, his carbohydrate chemistry, and his love and appreciation of the beauties of Nature were, for him, his life. His genuine humility precluded any inclination towards extensive eulogizing or ceremony. He was indeed “one among a thousand” (Job, 33: 23). The character of the man was accurately portrayed by the University Chaplain during the Memorial Service at Queen’s University in the words ‘‘. . . our friend, highly regarded and greatly beloved among us . . . cared for all living and growing things and cherished the beauty and wonder of woods and fields, rocks and water flowing by. His loyalty and thoughtfulness toward others taught us that a faithful friend is the medicine of life.” WALTERA. SZAREK MAURICE STACEY GEORGEW. HAY

APPENDIX

The following is a chronological list of the scientific publications of Professor J. K. N. Jones and his colleagues. “Synthesis of Ascorbic Acid and its Analogues: The Addition of Hydrogen Cyanide to Osones,” W. N. Haworth, E. L. Hirst, J. K. N. Jones, and F. SmithJ. Chem. SOC. (1934) 1192- 1197. “Ascorbic Acid and its Analogs,” W. N. Haworth, E. L. Hirst, J. K. N. Jones, and F. Smith, Br. Pat. 443,901 (1936).

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W. A. SZAREK, M. STACEY, AND G. W. HAY

“Gluco-ascorbic Acid,” W. N. Haworth, E. L. Hirst, and J, K. N. Jones,]. Chem. Soc., (1937)549-556. “Pectic Substances. Part I. The Araban and Pectic Acid of the Peanut,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., (1938)496-505. “Analogues of Ascorbic Acid Containing Six-membered Rings,” W. N. Haworth, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1938) 710-715. “The Constitution of Damson Cum. Part I. Composition of Damson Gum and Structure of an Aldobionic Acid (Clycuronosido-2-mannose)Derived from It,” E. L. Hirst and J. K. N. Jones,-/. Chem. Soc., (1938) 1174-1180. “Methylation of a-Methylglucoside by Thallous Hydroxide and Methyl Iodide,” C. C. Barker, E. L. Hirst, and J. K. N. Jones,J. C h m . Soc., (1938) 1695-1698. “Methyl Ethers of Arab-ascorbic Acid and Their Isomerism,” E. G. E. Hawkins, E. L. Hirst, and J. K. N. Jones,J. Chem. Soc., (1939) 246-248. “Pectic Substances. Part 11. Isolation of an Araban from the Carbohydrate Constituents of the Peanut,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., (1939) 452-453. “Pectic Substances. Part 111. Composition of Apple Pectin and the Molecular Structure of the Araban Component of Apple Pectin,” E. L. Hirst and J. K. N. Jones,J. Chem. SOC.,(1939)454-460. “The Constitution of Cherry Gum. Part I. Composition,” J. K. N. Jones,]. Chem. Soc., (1939) 558-563. “Constitution ofthe Mucilage from the Bark of Ulmusfuloa (Slippery Elm Mucilage). Part I. The Aldobionic Acid Obtained by Hydrolysis of the Mucilage,” R. E. Gill, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., (1939) 1469-1471. “The Constitution of Damson Cum. Part 11. Hydrolysis Products from Methylated Degraded (Arabinose-free) Damson Grim,” E. L. Hirst and J. K. N. JonesJ. Chem. Soc., (1939) 1482- 1490. “Pectic Substances. Part IV. Citrus Araban,” G. H. Beaven, E. L. Hirst, and J. K. N. Jones,]. Chem. S o c . , (1939) 1865-1868. “2:3:4-Trimethyl Mannose,” W. N. Haworth, E. L. Hirst, F. A. Isherwood, and J. K. N. Jones,]. Chem. Soc., (1939) 1878-1880. “The Structure of Alginic Acid. Part I,” E. L. Hirst, J. K. N. Jones, and (Miss) W. 0. Jones,]. Chem. Soc., (1939) 1880-1885. “Structure of Alginic Acid,” E. L. Hirst, J. K. N. Jones, and W. 0.Jones, Nnture, 143 (1939) 857. “Molecular Structure of Pectic Acid,” C. H. Beaven and J. K. N. Jones, Chem. Znd. (London),(1939) 363. “The Constitution of Banana Starch,” E. G. E. Hawkins, J. K. N. Jones, and G. T. Young, J . Chem. Soc., (1940)390-394. “The €-Galactan of Larch Wood,” E . L. Hirst, J. K. N. Jones, and W. G. Campbell, Nature, 147 (1941)25-26. “Separation of Methylated Methylglycosides by Adsorption on Alumina. A New Method for End-group Determinations in Methylated Polysaccharides,” J. K. N. Jones, 1. Chein. Soc., (1944) 333-334. “The Condensation of Glucose and p-Diketones,” J. K. N. Jones,]. Chem. Soc., (1945) 116- 119. “The Quantitative Estimation of Xyiose,” L. J. Breddy and J. K. N. Jones,]. Chem. SOC., (1945)738-739. “Nitrogenous Substances Synthesized by Moulds,” A. H. Campbell, M. E. Foss, E. L. Hirst, and J. K. N. Jones, Nature, 155 (1945) 141. “Application of New Methods of End-group Determination to Structural Problems in the Polysaccharides,” F. Brown, S. Dunstan, T. C. Halsall, E. L. Hirst, and J. K. N. Jones, Nature, 156 (1945) 785-786.


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“The Constitution of Damson Gum. Part 111. Hydrolysis Products from Methylated Damson Gum,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1946) 506-512. “Methylation of B-Methylglucopyranoside and ap-Methylxylopyranosidesby Thallous Hydroxide and Methyl Iodide,” C. C. Barker, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1946) 783-784. “Constitution of the Mucilage from the Bark of Ulmusfulua (Slippery Elm Mucilage). Part 11. The Sugars Formed in the Hydrolysis of the Methylated Mucilage,” R. E. Gill, E. L. Hirst, and J. K. N. Jones, J. Chem. SOC., (1946) 1025-1029. “The Chemistry of Pectic Materials,” E. L. Hirst and J. K. N. Jones,Adu. Carbohydr. Chem., 2 (1946) 235-251. “Structure of Starch and Cellulose,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones, Nature, 159 (1947) 97. “Quantitative Estimation of Mixtures of Sugars by the Paper Chromatogram Method,” A. E. Flood, E. L. Hirst, and J. K. N. Jones, Nature, 160 (1947) 86-87. “The Chemistry of Some Plant Gums and Mucilages,” E. L. Hirst and J. K. N. Jones, J . SOC. Dyers Colour., 63 (1947) 249-254. “The Quantitative Determination of Galactose, Mannose, Arabinose, and Rhamnose,” E. L. Hirst, J. K. N. Jones, and E. A. Woods,]. Chem. SOC., (1947) 1048-1051. “The Constitution of Cherry Gum. Part 11. The Products of Hydrolysis of Methylated Cherry Gum,” J. K. N. Jones, J. Chem. SOC., (1947) 1055-1059. “The Synthesis of 3-Methyl and 3:5-Dimethyl L-Arabinose,” E. L. Hirst, J. K. N. Jones, and (Miss) E. Williams,J. Chem. SOC., (1947) 1062-1064. “The Constitution of Egg-plum Gum. Part I,” E. L. Hint and J. K. N. Jones,]. Chem. SOC., (1947) 1064-1068.

“Pectic Substances. Part V. The Molecular Structure of Strawberry and Apple Pectic Acids,” G. H. Beaven and J. K. N. Jones,]. Chem. SOC., (1947) 1218-1221. “Pectic Substances. Part VI. The Structure of the Araban from Arachis hypogea,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1947) 1221-1225. “Pectic Substances. Part VII. The Constitution of the Galactan from Lupinus albus,” E. L. Hirst, J. K. N. Jones, and (MIS.) W. 0. Walder,]. Chem. SOC., (1947) 1225-1229. “Some Derivatives of DGalacturonic Acid,” J. K. N. Jones and M. Stacey,]. Chem.

SOC., (1947) 1340-1341. “Synthesis of Some Derivatives of D and L-Arabinose,” J. K. N. Jones, P. W. Kent, and M. Stacey,]. Chem. Soc., (1947) 1341-1344. “The Quantitative Separation of Methylated Sugars,” F. Brown and J. K. N. Jones,]. Chem. SOC., (1947) 1344-1347. “The Structure of Glycogen. Ratio of Non-terminal to Terminal Glucose Residues,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1947) 1399-1400. “Oxidation of Carbohydrates by the Periodate Ion,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1947) 1427-1432. “The Galactomannan of the Lucerne Seed,” E. L. Hirst, J. K. N. Jones, and (MIS.)W. 0. Walder, J . Chem. SOC., (1947) 1443-1446. “The Structure of Starch. The Ratio of Non-terminal to Terminal Groups,” F. Brown, T. G. Halsall, E. L. Hirst, and J. K. N. Jones,J. Chem. Soc., (1948) 27-32. “The Structure of Egg-plum Gum. Part 11. The Hydrolysis Products Obtained from the Methylated Degraded Gum,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1948) 120- 128.

“The +Galactan of Larch Wood (Larix decidua),” W. G. Campbell, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., (1948) 774-777. “The Galactomannan of Carob-seed Gum (Gum Gatto),” E. L. Hirst and J. K. N. Jones, J . Chem. SOC., (1948) 1278-1282. “The Structure of Almond-tree Gum. Part I. The Constitution of the Aldobionic Acid

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Derived from the Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., (1948) 1677-1679. “Quantitative Analysis ofMixtures of Sugars by the Method of Partition Chromatography. Part I. Standardisation of Procedure,” A. E. Flood, E. L. Hirst, and J. K. N.Jones,]. Chem. SOC., (1948)1679-1683. “Structure of Acorn Starch,” E. L. Hirst, J. K. N. Jones, and A. J. Roudier,]. Chem. SOC., (1948)1779-1783. “Pectic Substances. Part VIII. The Araban Component of Sugar-beet Pectin,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1948)2311-2313. “Separation and Identification of Methylated Sugars on the Paper Chromatogram,” F. Brown, E. L. Hirst, L. Hough, J. K. N. Jones, and H. Wadman, Nature, 161 (1948)720. “Application of Paper Partition Chromatography to the Separation of Sugars and their Methylated Derivatives on a Column of Powdered Cellulose,” L. Hough, J. K. N. Jones, and W. H. Wadman, Nature, 162 (1948)448. “The Amylose Content of the Starch Present in the Growing Potato Tuber,” T. G. Halsall, E. L. Hirst, J. K. N. Jones, and F. W. Sansome, Biochem.]., 43 (1948)70-72. “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part 11. The Separation and Determination of Methylated Aldoses,” E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chern. SOC., (1949)928-933. “The Polysaccharides ofthe Florideae. Floridean Starch,” V. C. Barry, T. G. Halsall, E. L. Hirst, and J. K. N. Jones,J. Chem. SOC.,(1949)1468-1470. “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part 111. Determination of the Sugars by Oxidation with Sodium Periodate,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., (1949)1659-1662. “The Constitution of Egg-plum Gum. Part 111. The Hydrolysis Products Obtained from the Methylated Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,]. Chen. SOC., (1949)1757-1761. “Cholla Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,], Chem. SOC., (1949)17611766. “Reactions of Nitroparaffins. Part XI. The Reaction of 2-Nitropropane with Formaldehyde and Ammonin,” J. K. N. Jones and T. Urbariski,J. Chem. SOC., (1949)17661767. “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part IV. The Separation of the Sugars and Their Methylated Derivatives on Columns of Powdered Cellulose,” L. Hough, J. K. N. Jones, and W. H. Wadman,J. Chem. SOC., (1949)2511-2516. “Cherry Gum. Part 111. An Examination of the Products of Hydrolysis of Methylated Degraded Cherry Gum, Using the Method of Paper Partition Chromatography,” J. K. N. Jones,J. Chem. Soc., (1949)3141-3145. “The Action ofp-Amylase on Amylopectin and on Glycogen,” T. G. Halsall, E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chern. SOC., (1949)3200-3207. “Pear Cell-wall Cellulose,” E. L. Hirst, F. A. Isherwood, M. A. Jermyn, and J. K. N. Jones,J. Chem. SOC., (1949)s182-sl84. “Composition of the Gum of Sterculia setigera: Occurrence of D-Tagatose in Nature,” E. L. Hirst, L. Hough, and J. K. N. Jones, Nature, 163 (1949)177. “Chromatographic Analysis. The Application of Partition Chromatography to the Separation of the Sugars and their Derivatives,” E. L. Hirst and J. K. N. Jones, Discuss. Faraday SOC., 7 (1949)268-274. “Plant Gums and Mucilages,” J. K. N. Jones and F. Smith,Ado. Carbohydr. Chem., 4 (1949)243-291. “The Structure of Peach Gum. Part I. The Sugars Produced on Hydrolysis of the Gum,” J. K. N. Jones,J. Chem. SOC., (1950)534-537.


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“On the Structure of Knudsen’s Base and of Related Compounds. Part I,” M. E. Foss, E. L. Hirst, J. K. N. Jones, H. D. Springall, A. T. Thomas, and T. Urbairski,]. Chem. SOC.,(1950) 624-628. “The Synthesis of 2:bDimethyl L-Rhamnose; The Action of Sodium Metaperiodate on 2:3- and 3:4-Dimethyl L-Rhamnoses,” F. Brown, L. Hough, and J. K. N. Jones, J. Chem. Soc., (1950) 1125-1127. “The Constitution of Xylan from Esparto Grass (Stipa tenacissima, L.),” S. K. Chanda, E.L. Hirst, J. K. N.Jones, and E. G. V. Percivd,]. Chem. SOC.,(1950) 12891297. “On the Structure of Knudsen’s Base and of Related Compounds. Part 11,”M. E. Foss, E. L. Hirst, J. K. N. Jones, H. D. Springall, A. T. Thomas, and T. Urbairski,]. Chem. SOC.,(1950) 1691-1695. “Grapefruit and Lemon Gums. Part I. The Ratio of Sugars Present in the Gums and Isolated by the Structure of the AIdobionic Acid (4-~-Glucuronosido-~-galactose) Graded Hydrolysis of the Polysaccharides,” J. J. Connell, (Miss) R. M. Hainsworth, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., (1950) 1696-1700. “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part V. Improved Methods for the Separation and Detection of the Sugars and their Methylated Derivatives on the Paper Chromatogram,”L. Hough, J. K. N. Jones, and W. H. Wadman,]. Chem. Soc., (1950) 1702-1706. “Frog-spawn Mucin,” B. F.Folkes, R. A. Grant, and J. K. N. Jones,]. Chem. Soc., (1950) 2136-2140. “The Structure of the Mannan Present in Porphyra umbiliculis,” J. K. N. Jones, J . Chem. SOC.,(1950) 3292-3295. “Composition of Linseed Mucilage,” D. G. Easterby and J. K. N. Jones, Nature, 165 (1950) 614. “Constitution of the Mucilage from the Bark of Ulmu~fuloa (Slippery Elm Mucilage). Part 111. The Isolation of 3-Monomethyl &Galadose from the Products of Hydrolysis,” E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chem.SOC.,(1951) 323-325. “The Synthesis of Sugars from Simpler Substances. Part I. The in oitro Synthesis of the Pentoses,” L. Hough and J. K. N. Jones,]. Chem. Soc., (1951) 1122-1126. “The Synthesis of Sugars from Simpler Substances. Part 11. The Synthesis of DL-Ribose in oitro from D-Glyceraldehyde and Glycollic Aldehyde,” L. Hough and J. K. N. Jones,]. Chem. SOC.,(1951)3191-3192. “Toluene+-sulphonylhydrazones of the Pentose Sugars, with Particular Reference to the Characterisationand Determination of Ribose,” D. G. Easterby, L. Hough, and J. K. N. Jones,]. Chem. SOC.,(1951) 3416-3418. “The Colorimetric Determination of Methylated Sugars: An Improved Micromethod of End-group Assay,” J. K. Bartlett, L. Hough, and J. K. N.Jones, Chem. Ind. (London), (1951) 76. “The Chemical Composition and Properties of Pectins,” J. K. N. Jones, Chem. Ind. (London),(1951) 430-431. “The Origin of the Sugars,” L. Hough and J. K. N. Jones, Nature, 167 (1951) 180. “Some Observations on the Constitution of Gum Myrrh,” L. Hough, J. K. N. Jones, and W. H. Wadman,]. Chem. SOC.,(1952) 796-800. “Methylene Derivatives of &Galactose and &Glucose,” L. Hough, J. K. N. Jones, and M. S. Magson,]. Chem. SOC.,(1952) 1525-1527. “Mannose-containing Polysaccharides. Part 11. The Gdactomannan of Fenugreek Seed (Trigonellafoenurngraecum),” P. Andrews, L. Hough, and J. K. N. Jones,]. Chem. SOC.,(1952) 2744-2750. “The Hemicelluloses Present in Aspen Wood (Populus tremulotdes).Part I,” J. K. N. Jones and L. E. Wise,J. Chem. Soc., (1952) 2750-2756.

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“The Hemicelluloses Present in Aspen Wood (Populus tremuloides). Part 11,” J. K. N. Jones and L. E. Wise,]. Chem. Soc., (1952)3389-3393. “An Investigation of the Polysaccharide Components of Certain Fresh-water Algae,” L. Hough, J. K. N. Jones, and W. H. Wadman,]. Chem. Soc., (1952)3393-3399. “The Reaction of Amino-compounds with Sugars. Part I. The Action of Ammonia on ~ G l u c o s e , ”L. Hough, J. K. N. Jones, and E. L. Richards,]. Chem. Soc., (1952) 38543857. “The Synthesis of Sugars from Simpler Substances. Part 111. Enzymic Synthesis of a Pentose,” L. Hough and J. K. N. Jones,]. Chem. SOC., (1952)4047-4052. “The Synthesis of Sugars from Simpler Substances. Part IV. Enzymic Synthesis of 6-Deoxy-D-fructose and 6-Deoxy-L-sorbose,” L. Hough and J. K. N. Jones, ]. Chem. SOC., (1952)4052-4055. “A Synthesis of 3:4-Dimethyl D-Xylose and 4-Methyl D-Xylose,” L. Hough and J: K. N. Jones,j. Chern. Soc., (1952)4349-4351. “Methylation of Carbohydrate Using Diazomethane,” L. Hough and J. K. N. Jones, Chem. Znd. (London), (1952) 380. “The Enzymatic Synthesis of Methylpentose,” L. Hough and J. K. N. Jones, Chem. Ind. (London), (1952)715. “The Enzymic Synthesis of Heptose Sugars,” L. Hough and J. K. N. Jones, Chem. lnd. (London),(1952)907. “Arabopyranose Residues in Larch E-Galactan,” J. K. N. Jones, Chem. lnd. (London), (1952)954. “Mannose-containing Polysaccharides. Part I. The Galactomannans of Lucerne and Clover Seeds,” L. Hough and J. K. N. Jones,]. Am. Chem. Soc., 74 (1952) 40294032. “Identification of L-Rhamnose in Aspen Wood,” J. K. N. Jones and J. R. Schoettler, Tappi, 35 (1952) 1mA. “Pentahydric Alcohols and their Oxidation Products,’’ J. K. N. Jones, in E. H. Rodd (Ed.), Chemistry of Carbon Compounds, Val. IB, Chap. XIX, Elsevier, Amsterdam, 1952, pp. 1197-1223. “Hexa- and Poly-hydric Alcohols and their Oxidation Products. Carbohydrates and Related Compounds,” J. K. N. Jones, in E. H. Rodd (Ed.), Chemistry of Carbon Compounds, Vol. IB, Chap. XX,Elsevier, Amsterdam, 1952, pp. 1224-1286. “The Synthesis of Sugars from Simpler Substances. Part V. Enzymic Sypthesis of Sedoheptulose,” L. Hough and J. K. N. Jones, j . C h . Soc., (1953) 342-345. “Mannose-containing Polysaccharides. Part 111. The Polysaccharides in the Seeds of In’s achroleuca and I. sibirca,” P. Andrews, L. Hough, and J. K. N. JonesJ. Chem. Soc., (1953) 1186-1192. “The Synthesis of Sugars from Simpler Substances. Part VI. Enzymic Synthesis of D-Idoheptulose,” P. A. J. Gorin and J. K. N. Jones,]. Chem. Soc., (1953) 1537-1538. “The Reaction of Amino-compounds with Sugars. Part 11. The Action of Ammonia on Glucose, Maltose, and Lactose,” L. Hough, J. K. N. Jones, and E. L. Richards,]. Chem. SOC., (1953)2005-2009. “The Synthesis of Sugars from Simpler Substances. Part VII. Enzymic Synthesis of 5-Deoxy-~-xylulose,”P. A. J. Gorin, L. Hough, and J. K. N. Jones,]. Chem. Soc., (1953) 2140-2142. “The Isolation of Oligosaccharides from Gums and Mucilages. Part I,” P. Andrews, D. H. Ball, and J. K. N. Jones,]. Chem.SOC., (1953) 4090-4095. “Structure of the ‘Triuronide’ from Pectic Acid,” J. K.N. Jones, Chem. Ind. (London), (1953)303. “The Galactan of Strychnos nux-uomica Seeds,” P. Andrews, L. Hough, and J. K. N. Jones, ]. Chem. Soc., (1954) 806-810.

OBITUARY-JOHN


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“The Structure of the Oligosaccharides Produced by the Enzymic Breakdown of Pectic Acid. Part I,” J. K. N. Jones and W. W. Reid,]. Chem. SOC., (1954)1361-1365. “The Isolation of Oligosaccharides from Gums and Mucilages. Part 11,” P. Andrews and J. K. N. Jones,]. Chem. SOC., (1954)1724-1726. “The Hemicelluloses of Scots Pine (Pinus syloestris) and Black Spruce (Picea nigru) Woods,” A. R. N. Gorrod and J. K. N. Jones,]. Chem. SOC., (1954)2522-2525. “The Synthesis of Sugars from Smaller Fragments. Part VIII. The Synthesis of D-Idoheptulosan from D-Xylose,” J. K. N. Jones,]. Chem. SOC., (1954)3643-3644. “The Isolation ofOligosaccharides from Gums and Mucilages. Part 111. Golden Apple Gum,” P. Andrews and J. K. N. Jones,]. Chem. Soc., (1954)4134-4138. “A Synthesis of 4-Deoxy-~-erythrohexulose,” P. A. J. Gorin, L. Hough, and J. K. N. Jones,]. Chem. SOC., (1954)4700-4701. “Some Observations on the Browning Reaction Between Glucose and Ammonia,” L. Hough, J. K. N. Jones, and E. L. Richards, Chem. Ind. (London),(1954)545-546. “Colorimetric Estimation of Sugars with Benzidine,” J. K.N. Jones and J. B. Pridham, Biochem. 1..58 (1954) 288-290. “Hemicellulose of Esparto (Stipa tenucissima L.). Part I,” J. K. N. Jones and G. Guzman, An. R. Soc. Esp. Fts. Qutm., Ser. B , 50 (1954)505-516. “An Improved Synthesis of D-Xylose 5-(Barium Phosphate),” P. A. J. Gorin, L. Hough, and J. K. N. JonesJ. Chem. Soc., (1955)582-583. “The Isolation of Oligosaccharides from Gums and Mucilages. Part IV. The Isolation from Lemon Cum,” P. Andrews and J. K. N. of 3-O-~-~-Arabopyranosyl-~-arabinose Jones,]. Chem. SOC., (1955)583-584. “The Synthesis of L-Glycerotetrulose and Related Compounds,” P. A. J. Gorin, L. Hough, and J. K. N. Jones,J. Chem. SOC., (1955)2699-2705. “The Constitution of Gum Myrrh. Part 11,” J. K. N. Jones and J. R. Nunn,J. Chem. Soc., (1955)3001-3004. “The Epimerization of Sugars,” J. K. N. Jones and W. H. Nicholson,]. Chem. Soc., (1955)3050-3053. “The Synthesis of Sugars from Simpler Substances. Part IX.The Enzymic Synthesis P. A. J. Gorin, L. Hough, and J. K. N. Jones,]. Chem. of 5:6-Dideoxy-~threohexulose,” SOC., (1955)3843-3845. “Methylene Derivatives of L-Rhamnose,” P. Andrews, L. Hough, and J. K. N. Jones, J. Am. Chem. Soc., 77 (1955)125-130. “The Structure of Frankincense Gum,” J. K. N. Jones and J. R. Nunn,]. Am. Chem. SOC., 77 (1955)5745-5764. “Chemistry ofthe Carbohydrates,” J. K. N. Jones,Annu. Reo. Biochem., 24 (1955)113 - 134. “A Synthesis of D-Tagatose from D-Galacturonic Acid,” P. A. J. Gorin, J. K. N. Jones, and W. W . Reid, Can.]. Chem., 33 (1955)1116-1118. “Preparation of L-Sorbose from 5-Keto-D-gluconic Acid (L-Sorburonic Acid),” J. K. N. Jones and W . W. Reid, Can. ]. Chem., 33 (1955)1682-1683. “The Analysis of Plant Gums and Mucilages,” J. K. N. Jones and E. L. Hirst, in K. Peach and M. V. Tracey (Eds.), Modern Methods of Plant Anulysis, Vol. 11, SpringerVerlag, Berlin, 1955,p. 275. “Properties of Dextrans Extracted from Plasma and Urine of Dogs,” R. E. Semple, B. J. Excell, and J. K. N. Jones, Fed. Proc., Fed. Am. Soc. Erp. Biol., 14 (1955)443. “The Structure ofthe Oligosaccharides Produced by the Enzymic Breakdown of P e e tic Acid. Part 11,” J. K. N. Jones and W. W. Reid,]. Chem. Soc., (1955)1890-1891. “The Separation ofan Essential Oil and of Methylated Sugars by Thermal Diffusion,” D. H. Ball, R. M. Butler, W. H. Cook, and J. K. N. Jones, Chem. Ind. (London),(1955) 1740-1741.

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“The Synthesis of Sugars from Smaller Fragments. Part X. Synthesis of LClucoheptulose,” J. K. N. Jones and R. B. Kelly, Can. I . Chem., 34 (1956)95-97. “A Synthesis of 5-0-Methyl =Glucose and of2-0-Methyl D-Glyceronamide,” J. K. N. Jones, Can.]. Chem., 34 (1956)310-312. “Fractionation of Polysaccharides,” A. J. Erskine and J. K. N. Jones, Can.].Chem., 34 (1956)821-826. “The Hemicellulose of the Fossilized Wood of Cedrus penhallawii,” J. K. N. Jones and E. Merler, Can. J. Chem., 34 (1956)840. “4-6-OisoPropylidene-rnethyl-a-~-glucoside,” J. K. N. Jones, Can. J . Chern., 34 (1956) 840-842. “The Action of Alkali Containing Metaborates on Wood Cellulose,” J. K. N. Jones, L. E. Wise, and J. P. Jappe, Tappi, 39 (1956)139-141. “The Synthesis of 3-Hexuloses. Part 1.2O-Methyl-~-rylo-3-hexulose,”J. K. N. Jones, J. Am. Chem. SOC., 78 (1956)2855-2857. “The Structure of the Hemicelluloses of Loblolly Pine,” D. H. Ball, J. K. N. Jones, W. H. Nicholson, and T. J. Painter, Tappi, 39 (1956) 438-443. “Reactions of Aliphatic Nitro Compounds. Formation of a Derivative of 1,5-Diazabicyclo(3.3.3)undecane from 1-Nitropropane, Formaldehyde and Ammonia,” J. K. N. Jones, R.Kosinski, H. Piotrowska, and T. Urbariski, Bull. Acad. Pol. Sci. Cl. 3,4 (1956) 509-510. “Reactions of Aliphatic Nitro Compounds. XXVII. On Formation of a Derivative of 1,5-Diazabicyclo(3.3.3}undecane from 1-Nitropropane, Formaldehyde and Ammonia,” J. K. N. Jones, R. Kolinski, €1. Piotrowska, and T. Urbdski, Ron. Chem.,31 (1957) 101109. “The Synthesis ofo-DeoxywS-ethylpoIyols,”J. K. N. Jones and D. L. Mitchell, Can. I. Chem., 36 (1957) 206-211. “The Hemicelluloses of Loblolly Pine (Pinus taeda) Wood. Part I. The Isolation of Five Oligosaccharide Fragments,” J. K. N. Jones and T. J. Painter,]. Chem. Soc., (1957) 669-6’73. “The Hemicelluloses Present in Aspen Wood (Populus tremuloides). Part 111. The Constitution of Pentosan and Hexosan Fractions,” J. K. N. Jones, E. Merler, and L. E. Wise, Can. J . Chem., 35 (1957) 634-645. “The Fractionation of Polysaccharides by the Method of Ultrafiltration,” K. C. B. Wilkie, J. K. N. Jones, B. J. Excell, and R. E. Sernple, Can. l. Ckm.,35 (1957) 7957%. “A Synthesis of 5,6Dideoxy-~-xylohexose(5-Deoxy-X-methyl-D-xylose),”J. K. N. Jones and J. L. Thompson, Can.J . Chem., 35 (1957) 955-959. “The Structure of the Type VIII Pneumococcus Specific Polysaccharide,” J. K. N. Jones and M. B. Perry,J. Am. Chem. SOC., 79 (1957) 2787-2793. ‘‘The Structure of Linseed Mucilage. Part I,” A. J. Erskine and J. K. N. Jones, Can.J. Chem., 35 (1957) 1174-1182. “The Synthesis of Disaccharides,” D. H. Ball and J. K. N. JonesJ. Chem. Soc., (1957) 4871-4873. “Isolation of Disaccharides from Golden Apple Cum,” J. K. N. Jones and B. 0. Lindgren,Acta Chem. S c a d . , 11 (1957) 1365. “The Acidcatalyzed Reversion of L-Arabinose and of DMannose,” J. K. N. Jones and W. H. Nicholson,J. Chem. Soc., (1958) 27-33. “The Acid-catalyzed Reversion of DXylose,” D. H. Ball and J. K. N. Jones,J. Chem. SOC., (1958)33-36. “A Synthesis of3-0-j3-DCalactopyranosyl-~galactose,” D. H. Ball and J. K. N. Jones, 1. Chem. SOC.,(1958)905-907.


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“Carbohydrate Chemistry at Queen’s University,” J. K. N. Jones, Pulp Pap. Mag. Can. Tech. Sect., 59 (1958) 145-147. “The Preparation of Some Derivatives of DRibono-14-lactone and DRibitol,” L. Hough, J. K. N. Jones, and D. L. Mitchell, Can. J. Chem., 36 (1958) 1720-1728. “The Hemicellulosas of Loblolly Pine (Pintts taedu) Wood. Part 11.The Constitution of Hexosan and Pentosan Components,” J. K. N. Jones and T. J. Painter,]. Chem. SOC., (1959) 573-580. “Structural Studies on Clinical Dextrans. Part I. Methylation and Pcriodate Oxidation Studies,” J. K. N. Jones and K. C. B. Wilkie, CanJ. Biochem. Physiol., 37 (1959) 377390. “The Characterization of TriO-tosyl Sucrose,” P. D. Bragg and J. K. N. Jones, Can. J. Chem., 37 (1959)575-578. “The Oxidation of Some Terminal-substituted Polyhydric Alcohols by Acetobacter suboxydans,” L. Hough, J. K. N. Jones, and D. L. Mitchell, Can. J . Chem., 37 (1959) 725-730. “Structure of Some Water-soluble Polysaccharides from Wood,” D. J. Brasch, J. K. N. Jones, T. J. Painter, and P. E. Reid, Proc. Cellul. Con$, 2nd, Syracuse, 1959, 3-15. “Structure of Some Water-soluble Polysaccharides from Wood,” D. J. Brasch, T. J. Painter, P. E. Reid, and J. K. N. Jones, Pulp Pap. Mag. Can., Tech. Sect., 60 (1959)T342T345. “5,6-Dideoxy-~arabino-hexose (S-Deoxy-5C-methyl-L--arabinose),” D. H. Bali, A. E. Flood, and J. K. N. Jones, Can. J. Chem., 37 (1959) 1018-1021. “The Reaction of Sulphuryl Chloride with Glycosides and Sugar Alcohols. Part I,” P. D. Bragg, J.K. N. Jones, and J. C. Turner, Can. J. Chem., 37 (1959) 1412-1416. “The Structure of an Arabogalactan From Monterey Pine (Pinus radiata),” D. J . Brasch and J. K. N. Jones, Can.J. Chem., 37 (1959) 1538-1545. “The Synthesis of S-Deoxy-5S-ethyl-D-threo-pentulose,” J. K. N. Jones and D. L. Mitchell, Can. J. Chem., 37 (1959) 1561-1566. “Separation of Sugars on Ion Exchange Resins,” J. K. N. Jones, R. A. Wall, and A. 0. Pittet, Chem. Ind. (London),(1959) 1196. “Synthesis of Sugars from Smaller Fragments. Part XI. Synthesis of L-Galactoheptulose,” J. K. N. Jones and N. K. Matheson, Can. J. Chem., 37 (1959) 1754-1756. “Investigation of Some Ancient Woods,” D. J. Brasch and J. K. N. Jones, Tappi, 42 (1959) 913-920. “The Reaction of Sodium Metaperiodate with Some Nitrogen Derivatives of Carbohydrates,” M. J. Abercrombie and J. K. N. Jones, Can. J. Chem., 38 (1960)308-309. “Synthesis of Sugars from Smaller Fragments. Part XII. Synthesis of SGlycero-D altro-, cGEycero-Lgalacto-, DClycero-Ggluco-, and DGlycero-cgalacto-odulose,” J . K. N. Jones and H. H. Sephton, Can. J. Chem., 38 (1960) 753-760. “Some Open-chain Derivatives of Glucose and Mannose,” E. J. C. Curtis and J. K. N. Jones, Can. J. Chem., 38 (1980) 890-895. “The Synthesis of 2O-~-~-Glucopyranosyl-~-xylose,” J. K. N. Jones and P. E. Reid, Can.]. Chem., 38 (1960) 944-949. “The Reaction of Sulphuryl Chloride with Glycosides and Sugar Alcohols. Part 11,” J. K. N. Jones, M. B. Peny, and J. C. Turner, Can.]. Chem., 38 (1960) 1122-1129. “The Synthesis of 3O-~-~-Xylopyranosyl-~-xylose and the Recharacterization of Some Benzylidene Derivatives of ~Xylose,”E. J. C.Curtis and J. K. N. Jones, Can.]. Chem., 38 (1960) 1305-1315. “The Polysaccharides of Cryptococcus laurentii (NRRL Y-1401). Part I,” M. J. Abercrombie, J. K. N. Jones, M. V. Lock, M. B. Perry, and R.J. Stoodley, Can.]. Chem., 38 (1960) 1617-1624.

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“A Chemical Procedure for Determination of the C“ Distribution in Some Labelled Carbohydrates,” M. J. Abercrombie and J. K. N. Jones, Can.J . Chem., 38 (1960)1999-

2006. “The Polysaccharides of Cryptococcus laurentii (Y1401).Part 11. Biosynthesis of the Carbohydrates Found in the Acidic Polysaccharide,” M. J. Abercrombie, J. K. N. Jones, and M. B. Perry, Can. J . Chem., 38 (1960)2007-2014. “The Separations of Sugars on Ion-exchange Resins. Part I,” J. K. N. Jones, R. A. Wall, and (in part) A. 0.Pittet, Can. J . Chem., 38 (1960)2285-2289. “The Separations ofSugars on Ion-exchange Resins. Part 11,” J. K. N. Jones and R. A. Wall, Can. J. Chem., 38 (1960)2290-2294. “The Structure of the ‘Gum Asafoetida’ Polysaccharide,” J. K. N. Jones and G. H. S. Thomas, Can. J . Chem., 39 (1961)192-202. “Analysis of Sugar Mixtures by Gas-Liquid Partition Chromatography,” S. W. Gunner, J. K. N. Jones, and M. B. Perry, Chem. Ind. (London), (1961)255-256. “The Demethylation of Sugars with Hydrogen Peroxide,” B. Fraser-Reid, J. K. N. Jones, and M. B. Perry, Can. J . Chem., 39 (1961)555-563. “The Synthesis ofhcetamido-deoxy Ketoses b y Acetobacter suboxydans. Part I,” J. K. N. Jones, M. B. Perry, and J. C. Turner, Can./. Chem., 39 (1961)965-972. “The Carbohydrate-Protein Linkage in Glycoproteins. Part I. The Syntheses of Some Model Substituted Amides and an L-Seryl-Sglucosaminide,” J, K. N. Jones, M. B. Perry, B. Shelton, and D. J. Walton, Can. J . Chem., 39 (1961)1005-1016. “Constitution of a 4-O-Methylglucuronoxylan From the Wood of Trembling Aspen (Populus tremuloides Michx.),” J. K. N. Jones, C. B. Purves, and T. E. Timell, Can. J. Chem., 39 (1961)1059-1066. “The Gas-Liquid Partition Chromatography of Carbohydrate Derivatives. Part I. The Separation of Glycitol and Glycose Acetates,” S. W. Gunner, J. K. N. Jones, and M. B. Perry, C a n . J .Chem., 39 (1961)1892-1899. “The Synthesis of Acetamido-deoxy Ketoses by Acetobacter suboxyduns. Part 11,” J. K. N. Jones, M. B. Perry, and J. C. Turner, Can./. Chem., 39 (1961)2400-2410. “Biogenesis of Carbohydrates in Wood,” J. K. N. Jones, Pure Appl. Chem., 5 (1962)

21 -35. “The Synthesis of Acetamido-deoxy Ketoses by Acetobacter suboxydans. Part 111,” J. K. N. Jones, M. B. Perry, and J. C. Turner, Can.]. Chem., 40 (1962)503-510. “Biosynthesis of Sugars Found in Bacterial Polysaccharides. Part I. Biosynthesis of L-Rhamnose,” J. K. N. Jones, M. B. Perry, and R. J. Stoodley, C a n . ] . Chem., 40 (1962) 856-863. “The Biological and Chemical Synthesis of Polysaccharides,” J. K. N. Jones, Pure Appl. Chem., 5 (1962)469-482. “The Structure of Linseed Mucilage. Part 11,” K. Hunt and J. K. N. Jones, Can. J .

Chem., 40 (1962)1266-L279. “The Reaction of Sulphuryl Chloride with Reducing Sugars. Part I,” H. J. Jennings and J. K. N . Jones, Can. j . Chem., 40 (1962)1408-1414. “The Gas-Liquid Partition Chromatography of Carbohydrate Derivatives. Part 111. The Separation of Amino Glycose Derivatives and of Carbohydrate Acetal and Ketal Derivatives,” H. G. Jones, J. K. N. Jones, and M. B. Perry, Can.]. Chem.,40 (1962)1559 -1563. “Biosynthesis of Sugars Found in Bacterial Polysaccharides. Part 11. Biosynthesis of D-#lycero-D-manno-Heptose,”J. K. N. Jones, M. B. Perry, and R. J. Stoodley, Can. J . Chem., 40 (1962)1798-1804. “The Carbohydrate-Protein Linkage in Glycoproteins. Part 11. The Synthesis ofN-LSeryt-D-glucosamine andN-L-Threonyl-Sglucosamine,” J. K. N. Jones, J. P. Millington, and M. B. Perry, Can. /. Chem., 40 (1962)2229-2233.


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“5-Acetamido-5-deoxyy-~-arabinose: A Sugar Derivative Containing Nitrogen as the Hetero-atom in the Ring,” J. K. N. Jones and J. C. Turner,./. Chem. SOC., (1962)46994703. “Recent Progress in Polysaccharide Chemistry,” J. K. N. Jones, An. Assoc. Bras. Quim., Numero Expec., 21 (1962) 41-55. “Chromatography on Paper,” L. Hough and J. K. N. Jones, Methods Carbohydr. Chem., 1 (1962) 21-31. “Enzymic Methods for Determination of DGlucose. Quantitative Determination of D-Glucose by Oxidation with D-Glucose Aerodehydrogenase,” L. Hough and J. K. N. Jones, Methods Carbohydr. Chern., 1 (1962)400-404. “Determination of isotopic Carbon Distribution in Aldoses. Chemical Oxidation to Carbon Dioxide,” J. K. N. Jones and R. J. Stoodley, Methods Carbohydr. Chem., 2 (1963)489-493. “Structural Studies on the Water-Soluble Arabinogalactans of Mountain and European Larch,” J. K. N. Jones and P. E. Reid,J. Polym. Sci., Part C , (1963) 63-71. “Synthesis of a Sugar Derivative with Nitrogen in the Ring,” J. K. N. Jones and W. A. Szarek, Can.J . Chem., 41 (1963) 636-640. “The Reaction of Chlorosulphate Esters of Sugars with Pyridine,” H. J. Jennings and J. K. N. Jones, Can.J.Chem., 41 (1963) 1151-1159. “The Synthesis of D-glycero-Dmanno-Heptose,”R. K. Hulyalkar. J. K.N. Jones, and M. B. Perry, Can. J. Chem., 41 (1963) 1490-1492. “The Synthesis of 3-Hexuloses. Part 11. Derivatives of 1-Deoxy-Lurabo-3-hexulose (Syn. 6-Deoxy-~-Zyxo4-hexulose),”J. W. Bird and J. K. N. Jones, Can. J . Chem., 41 (1963) 1877-1881. “Synthesis of ~-Arabinose-5-C’~,” R. K. Hulyalkar and J. K. N. Jones, Can. J. Chem., 41 (1963) 1898-1904. “Carbon-Oxygen Fission: Degradation of Polysaccharides,” J. K.N. Jones and M. B. Perry, in K. W. Bentley (Ed.), Elucidation of Structures b y Physical and Chemical Methods, Part 11, Technique of Organic Chemistry, Vol. X I , Interscience Publishers, New York, 1963, pp. 707-750. “The Synthesis of 5#-~-D-Glucopyranosy1-Dxylose and 3,5-Di.0-P-~-glucopyranosyl-D-xylose,” J. K. N. Jones and P. E. Reid, Can. J . Chem., 41 (1963) 2382-2387. “The Occurrence of Dglycero-D.manno-Heptosein the Extracellular Polysaccharide Produced by Azotobacter indicum,” J. K. N. Jones, M. B. Perry, and W. Sowa, Can. J. Chem., 41 (1963)2712-2715. “The Structure of the Extracellular Polysaccharide of Azotobacter indicum,” V. M. Parikh and J. K. N. Jones, Can. J . Chem., 41 (1963)2826-2835. “Synthesis of Methyl 4-Acetamido4-deoxy-~-erythrofuranoside: A Sugar with Nitrogen in a Five-membered Ring,” W. A. Szarek and J. K. N. Jones, Can. /. Chem., 42 (1964)20-24. “The Chemistry of Apiose. Part I,” D. T. Williams and J. K. N. Jones, Can. J . Chem., 42 (1964)69-72. “The Glucomannan of Bluebell Seed (Scylla nonscripta L.),” J. L. Thompson and J. K. N. Jones, Can.]. Chem., 42 (1964) 1088-1091. “Hindered Internal Rotation in Carbohydrates Containing Nitrogen in the Ring,” W. A. Szarek, S. Wolfe, and J. K. N. Jones, Tetrahedron Lett., (1964)2743-2750. “Polysaccharides From the Seeds of the Huacra Pona Palm (Zriartea uentricosa),” W. Sowa and J. K. N. Jones, Can. J . Chem., 42 (1964) 1751-1754. “The L-Ascorbate Ion as an Ambident Nucleophile,” E. Buncel, K.G. A. Jackson, and J. K. N. Jones, Chem. Ind. (London), (1965)89. “Structure of Cholla Gum (Opuntia fulgida),” V. M. Parikh and J. K. N. Jones, J . Polym. Sci., Part C , (1965) 139-148.

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“The C- and 0-Benzylation of L-Ascorbic Acid,” K. G. A. Jackson and J. K. N. Jones, Can. J . Chem., 43 (1965)450-457. “The Oxidation of Sugar Acetals and Thioacetals by Acetobacter suborydans,” D. T. Williams and J. K. N. Jones, Can.]. Chem., 43 (1965)955-959. “Synthesis of 4-Acetamido4-deoxy-sugars,” A. J. Dick and J. K. N. Jones, Can. J.

Chem., 43 (1965)977-982. “The Reaction of Galactose with Hydrazine at Elevated Temperature,” J. K. N. Jones, P. Reid, and J. R. Turvey, Can.]. Chem., 43 (1965)983-985. “The Chemistry of D-Apiose. Part 11. The Configuration of D-Apiose in Apiin,” R. K. Hulydkar, J. K. N. Jones, and M. B. Perry, Can.]. Chem., 43 (1965)2085-2091. “Carbohydrates Containing Nitrogen in a Five-membered Ring and an Attempted Synthesis of a Carbohydrate with Nitrogen in a Seven-membered Ring,” W. A. Szarek and J. K. N. Jones, Can .J. Chem., 43 (1965)2345-2356. “Reactions of Sugar Chlorosulfates. Part V. The Synthesis of Chlorodeoxy Sugars,” H. J. Jennings and J. K. N. Jones, Can.J . Chem., 43 (1965)2372-2386. “Synthesis of 40-~-D-Galactopyranosyl-D-galactose,” E. J. C. Curtis and J. K. N. Jones, Can. J . Chem., 43 (1965)2508-2511. “Reactions of Sugar Chlorosulfates. Part VI. The Structure of Unsaturated Chlorodeoxy Sugars,” H. J. Jennings and J. K. N. Jones, Can.J. Chem., 43 (1965)3018-3025. “Synthesis of 5-Benzamido-5-deoxy-~-xylopyranose,” M. S. Patel and J. K. N. Jones, Cun. J . Chem., 43 (1965)3105-3108. “Direct Displacement of a Primary Tolyl-p-sulfonyloxy Group by the Methoxide Ion: A More Direct Route to 5-0-Methyl-L-arabinose and 3,5-Di4l-methyl-~-arabinose,” S. C. Williams and J. K. N. Jones, Can. J. Chem., 43 (1965)3440-3442. “Oxidation of Sugars with Ruthenium Dioxide-Sodium Periodate: A Simple Method for the Preparation of Substituted Keto Sugars,” V. M. Parikh and J . K. N. Jones, Can.J . Chern., 43 (1965)3452-3453. “Selective Nucleophilic Substitution and Preferential Epoxide Formation,” A. J. Dick and J. K. N. Jones, Can. J . Chem., 44 (1966)79-87. “Cholla Gum. Part I. Structure of the Degraded Cholla Gum,” V. M. Parikh and J. K. N. Jones, Can. J . Chem., 44 (1966)327-333. “The Separation of Aldopentose and Aldohexose Diethyl Dithioacetal Derivatives by Gas-Liquid Partition Chromatography,” D. T. Williams and J. K. N. Jones, Can. J . Chem., 44 (1966) 412-415. “Chlorosulphate as a Leaving Group: The Synthesis of a Methyl Tetrachloro-tetradeoxy-hexoside,” A. G. Cottrell, E. Buncel, and J. K. N. Jones, Chem. Ind. (London), (1%) 552. “Reactions of Sugar Chlorosulfates. Part VII. Some Confonnational Aspects,” A. G. Cottrell, E. Buncel, and J. K. N. Jones, Can.]. C h m . , 44 (1966)1483-1491. “Cholla Gum. Part 11. Structure of the Undegraded Cholla Gum,” V. M. Parikh and J. K. N. Jones, C a n . J .Chem., 44 (1966) 1531-1539. “The Capsular Polysaccharide of Pneumococcus Type XII, SXII,” J. A. Cifonelli, P. Rebers, M. B. Perry, and J. K. N. Jones, Biochemistry, 5 (1966)3066-3072. “A One-step Conversion of Cyclohexene Oxide into cis-l&Dichlorocyclohexane,” J. R. Campbell, J. K. N. Jones, and S. Wolfe, Can.J . Chem., 44 (1966)2339-2342. “A New Synthesis of 3-Acetamido3-deoxy-~glucose,” D. T. Williams and J. K. N. Jones, Cun. J . Chem., 45 (1967)7-9. “A Synthesis of Dihydroxyacetone Phosphate From Dihydroxyacetone,” R. L. Colbran, J. K. N. Jones, N. K. Matheson, and I. Rozema, Carbohydr. Res., 4 (1967)355-358. “The Synthesis, Separation, and Identification of the Methyl Ethers of Arabinose and ‘Their Derivatives,” S. C. Williams and J. K. N. Jones, Can.J.Chem., 45 (1967)275-290.


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“Further Experiments on the Oxidation of Sugar Acetals and Thioacetals by Acetobacter suborydans,” D. T. Williams and J. K. N. Jones, Can.]. Chem., 45 (1967)741744. “Acetonation of D-Xylose Diethyl Dithioacetal,” D. G. Lance and J. K. N. Jones, Can. .J. Chem., 45 (1967)1533-1538. “Reactions of Sugar Chlorosulfates. Part VIII. @Ribose and Its Derivatives,” S. S. Ali, T. J. Mepham, (Miss) I. M. E. Thiel, E. Buncel, and J. K. N. Jones, Carbohydr. Res., 5 (1967)118-125. “Gas Chromatography of Derivatives of the Methyl Ethers of D-Xylose,” D. G. Lance and J. K. N. Jones, Can. 3. Chem., 45 (1967)1995-1998. “The Synthesis of L-Mycarose and L-Cladinose,” G. B. Howarth and J. K. N. Jones, Can. ]. Chem., 45 (1967)2253-2256. “Selective Benzoylation of Benzyl P-L-Arabinopyranoside and Benzyl a-~-Xylopyranoside,” T. Sivakumaran and J. K. N. Jones, Can. 1.Chem., 45 (1967)2493-2500. “Epoxide Ring Opening of Methyl 2,3-Anhydro4-azido4-deoxy-pentopyranosides,” A. J. Dick and J. K. N. Jones, Can.]. Chem., 45 (1967)2879-2885. “The Synthesis of ~-Arcanose,” G. B. Howarth, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1968)62-63. “Isolation of Two kabinobioses From Acacia nilotica Gum,” R. C . Chalk, J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Con.]. Chem., 46 (1968)2311-2313. “Synthesis of 6-Deoxy3-C-methyl-2-0-methyl-@a~lose,” G. B. Howarth, W. A. Szarek, and J. K. N. Jones, Can. 3. Chem., 46 (1968)3375-3379. “Synthesis of 6-Chloro-4(6’-deoxy-3’C-methyl-2‘,3‘,4‘-~-0-methyl-~-@allopyranosy1)purine: A Branched-chain Sugar Nucleoside,” G. B. Howarth, W.A. Szarek, and J. K. N. Jones, Can. J. Chem., 46 (1968)3691-3694. “Photolysis of Carbohydrate Nitro-olefins,” G. B. Howarth, D. G. Lance, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1968)1349. “Branched-chain Sugar Nucleosides. Synthesis of a Purine Nucleoside of 4-0-AcetylL-arcanose,” G. B. Howarth, W. A. Szarek, and J. K. N. Jones,]. Org. Chem., 34 (1969) 476-477. “Syntheses Related to the Carbohydrate Moiety in Lincomycin,” G. B. Howarth, D. G . Lance, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969)75-79. “Photolysis of a Carbohydrate Nibbolefin,” G. B. Howarth, D. G. Lance, W. A. Szarek, and J. K. N. Jones, Can. J. Chem., 47 (1969)81-87. “Some Structural Features of the Mucilage From the Bark of Ulmus fulua (Slippery Elm Mucilage),” R. J. Beveridge, J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 9 (1969)429-439. “An Improved Procedure for Oxidation of Carbohydrate Derivatives with Ruthenium Tetraoxide,” B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 10 (1969) 456-458. “Synthesis of 8-Deoxy-D+rythro-D-galacto-octose.Determination of the Configuration of Two Octenoses,” D. G. Lance, W. A. Szarek, J. K. N. Jones, and G. B. Howarth, Can.]. Chem., 47 (1969)2871-2874. “Some 0-Isopropylidene Derivatives of @Ribose Diethyl Dithioacetal,” D. G. Lance, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969)2889-2891. “Synthesis of DChalcose,” B. T. Lawton, D. J. Ward, W. A. Szarek, and J. K. N. Jones, Can. ]. Chem., 47 (1969)2899-2901. “Large Heterocyclic Rings From Carbohydrate Precursors,” J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969)3213-3215. “A Simple Synthesis of Azidodeoxy-sugars uia Chlorodeoxy-sugars,” B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1969)787-788.

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“Reachon of Methyl 4,6C)-Benzy~idene3C-methyl-2-O-p-tolylsulfonyl-a-~-allopyranoside with Sodium Methoxide in Methyl Sulfoxide: Synthesis of 6-Deoxy-34methyl3-O-methyl-aallose (2-Hydroxy-~-cladinose),”G. B. Howarth, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 11 (1969)257-262. “The Synthesis of N-Acetyl-lincosamine (6-Acetamido-6,8-dideoxy-D+rythro-~-galacto-octose), a Derivative of the Free Carbohydrate Moiety in Lincomycin,” G. B. How&, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1969) 1339-1340. “Synthesis of 3-Hexuloses,” K. G. A. Jackson and J. K. N. Jones, Can.J . Chem., 47 (1969)2498-2501. “Separation and Identification of Methyl Ethers of DGlucose and D-Glucitol by GasLiquid Chromatography,” H. G. Jones and J. K. N. Jones, Can.]. Chem., 47 (1969)3269 -3271. E. H. Wi1“Synthesis of Olivomycose (2,6-Dideoxy3C-methyl-~-arabino-hexose),” liams, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47 (1969) 4467-4471. “Addition of Pseudohalogens to Unsaturated Carbohydrates. Part 111. Synthesis of 3DeoxySE-nitromethyl-&allose, a Branchedchain Nitro Sugar,” W. A. Szarek, J. S. Jewell, 1. Szczerek, and J. K. N. Jones, Can.J. Chem., 47 (1969)4473-4476. “Carbohydrate Fluorosulfates,” E. Buncel, H. J. Jennings, J. K. N. Jones, and I. M. E. Thiel, Carbohydr. Res., 10 (1969)331-332. “Structural Feature of Pneumococcus Type XIX Specific Polysaccharide,” T. Miyazaki and J. K. N. Jones, Chem. Pham. Bull., 17 (1969) 1531-1533. “The Isolation and Properties of the Skin-reactive Substance in Aedes aegypti Oral Secretion,” W. H. Newsome, J. K. N. Jones, F. E. French, and A. S. West, Can.J. Biochem., 47 (1969) 1129-1136. ‘?V-(4,6-O-Benzylidene-l-O-methyl-3-oximino-a-~-ribohexopyranos-2-y1)pyridinium p-Toluenesulfonate. A Novel Versatile Carbohydrate Substrate,” W. A. Szarek, B. T. Lawton, and J. K. N. Jones, Tetrahedron Lett., (1970) 4867-4870. “A Facile Synthesis of 4,6-Dideoxy-~-xylo-hexose,” B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 14 (1970) 255-258. “The Synthesis of Lincomycin,” G. B. Howarth, W. A. Szarek, and J. K. N. Jones, 1. C h . SOC., C, (1970) 2218-2224. “Synthesis of Deoxy and Aminodeoxy Sugars by Way of Chlorodeoxy Sugars,” B. T. Lawton, W. A. Szarek, and J. K. N.Jones, Carbohydr. Res., 15 (1970) 397-402. “Polysaccharides of Type XIX Pneumococcus. Part I. Isolation of Type Specific Polysaccharide,” T. Miyazaki,T. Yadomae, and J. K. N. Jones,]. Biochem. (Tokyo), 68 (1970) 755-758. “Synthesis of Paratose (3,6-Dideoxy-D.ribo-hexose)and Tyvelose (3,6-Dideoxy-~arabino-hexose),” E. H. Williams, W. A. Szarek, and J. K. N. Jones, Can. ]. Chem., 49 (1971) 796-799. “Some Structural Studies on the Galactan from the Albumen Glands of‘the Snail, Strophocheilus oblongus,” J. H. Duarte and J. K. N. Jones, Carbohydr. Res., 16 (1971) 327-335. “Reaction of Methyl 4,6-Dichloro4,6-dideoxy-a-D-galactopyranoside2,3-Di(chlorosulfate) with Sodium Azide, and with Sodium Bromide, in N,N-Dimethylformamide,” H. Parolis, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 19 (1975) 97-105. “Isolation of Three Oligosaccharides from the Mucilage from the Bark of Ulmusfulua (Slippery-Elm Mucilage). Synthesis of O-(30-Methyl-~-~-galactopyranosyl)-(14)-~rhaanose,” R. J. Beveridge, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 19 (1971) 107-1 16. “Reactions of‘Carbohydrate a-Keto Toluene*-sulphonates. Reaction of Methyl 4,6O-Benzylidene-20-toluenep-suIphonyl-a-D-ribo-hexop~~nosid~-ulose with Triethyl-

OBlTUARY- JOHN KENYON NETHERTON JONES

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amine-Methanol,” A. Dmytraczenko, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1971) 1220-1222.

“Preparation of Unsaturated Carbohydrates from Methyl 4,W-Benzylidene-3chIoro-3-deoxy-pDallopyranoside,and Their Utility in the Synthesis of Sugars of Biological Importance,” E. H. Williams, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 20 (1971) 49-57. ‘‘Structure of Slippery Elm Mucilage (Ulmusfulua),” R. J. Beveridge, J. K. N. Jones, R. W.Lowe, and W . A. Szarek,]. PoZym. Sci., Part C,36 (1971) 461-466. “Studies on Lipopolysaccharides of Proteus,” B. A. Dmitriev, N. A. Hinton, R. W. Lowe, and J. K. N. Jones, Can.]. MicrobioZ., 17 (1971) 1385-1394. “An Evaluation of Methods for the Preparation of 1,2:3,4-Di-O-isopropylidene-a-~galacto-hexodialdo-1,Spyranose. Oxidation of 1,2:3,4DiO-isopropylidene-a-~galactopyranose with Lead Tetraacetate-Pyridine,” D. J. Ward, W.A. Szarek, and J. K. N. Jones, Carbohydr. Res., 21 (1972) 305-308. “Addition of Pseudohalogens to Unsaturated Carbohydrates. Part V. Addition of Iodine Trifluororoacetate,” R. G. S. Ritchie and W. A. Szarek, Can. J. Chem., 50 (1972)

507-511. “Some Reactions of Unsaturated Carbohydrates in the Presence of Iodine,” I. Szczerek, J. S. Jewell, R. G. S. Ritchie, W.A. Szarek, and J. K. N. Jones, Carbohydr. Res., 22 (1972) 163-172. “Amination of Sugar Derivatives with a Mixture of Phthalimide, Triphenylphosphine, and Diethyl Azodicarboxylate,” A. Zamojski, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 23 (1972) 460-462. “Selective Oxidation of a Diol with Methyl Sulfoxide-Acetic Anhydride,” T. B. Grindley, J. W. Bird, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 24 (1972) 212215. “Synthesis of Carbohydrate Furoxan Derivatives,” C. S. Wu, W. A. Szarek, and J. K. N. Jones, Chem. Commun., (1972) 1117-1118. “Ethers of Sugars,” J. K. N. Jones and G. W. Hay, in W. Pigman and D. Horton (Eds.), The Carbohydrates, Vol. IA, Academic Press, New York, 1972, pp. 403-422. “Reaction of Some Die-isopropylidenehexoseswith Cyanuric Chloride,” A. Zamojski, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 26 (1973) 208-214. “Reaction of Methyl 2,3-O-lsopropylidene-6~~-tolylsulfonyl-a-DEyxo-hexofuranosid-5-ulose with Triethylamine-Methanol,” A. Dmytraczenko, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 26 (1973) 297-303. “Reductive Cleavage of Carbohydrate p-Toluenesulfonates with Sodium Naphthalene,” H. C. Jarrell, R. G. S. Ritchie, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 51 (1973) 1767-1770. “Conversion of 2-Hexuloses into 3-Heptuloses: Synthesis of D-manno3-Heptulose,” R. W. Lowe, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 28 (1973) 281-293. “The Total Synthesis of Carbohydrates,” W. A. Szarek and J. K. N. Jones, in J. W. ApSimon (Ed.), The Total Synthesis of Natural Products, Wiley-Interscience, New York, 1973, pp. 1-80. “Lipopolysaccharides of Proteus,” J. K. N. Jones, in Mbthodologie de la Structure et du Mbtabolisme des Glycoconjuguk, Colloques Internationaux du Centre National de la Recherche Scientifique, No. 221, June 20-27, 1973, Villeneuve d’Ascq, Vol. 1, pp. 533-543. “A Reinvestigation of the Reaction of Methyl 8-DGlucopyranoside with Sulfuryl Chloride,” D. M. Dean, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 33 (1974) 383-386. “Reaction of Hexopyranoside a-Keto Toluene-p-sulfonates with Triethylamine-

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Methanol,” W. A. Szarek, A. Dmytraczenko, and J . K. N. Jones, Carbohydr. Res., 35 (1974)203-219. “Reaction o f Methyl Yentofuranosides with Sulfiiryl Chloride,” B. Achmatowicz, W. A. Szarek, J. K. N. Jones, and E. H. Williams, Carbohydr. Res., 36 (1974) c14-c16. “Arthur Charles Neish, 1916-1973,” J. K. N. Jones, Biogr. Mem. Fellows R . Soc., 20 (1974)294-315. “Synthesis of Nucleosides by Direct Replacement of the Anomeric Hydroxy-group,” W. A. Szarek, C. Depew, H.C. Jarrell, and J. K. N. Jones,]. Chem. Soc., Chem. Commum, (1975)648-649. “Syntheses Related to Dendroketose,” H. C. Jarrell, W. A. Szarek, J. K. N. Jones, A. Dmytraczenko, and E. B. Rathbone, Carbohydr. Res., 45 (1975) 151-159. “Decarhonylation of Aldehydo Sugar Derivatives with Chlorotris(methyldipheny1phosphine)rhodium(I),” D. J. Ward, W. A. Szarek, and J. K. N. Jones, Chem. Ind. (London), (1976) 162-163. “Syntheses Towards the Carbohydrate Moiety of Lincomycin,” G. R. Woolard, E. B. Rathbone, W. A. Szarek, and J. K. N. Jones,/. Chem. SOC. Perkin Trans. 1 , (1976)950954.

“Synthesis of Carbohydrate-Saccharin Conjugates,” W. A. Szarek, C. Depew, and J. K. N. Jones,/. Heterocycl. Chem., 13 (1976) 1131-1133. “Selective, Reductive Dechlorination of Chlorodeoxy Sugars. Structural Determination of Chlorodeoxy and Deoxy Sugars by I3C Nuclear Magnetic Resonance Spectroscopy,” W. A. Szarek, A. Zamojski, A. R. Gibson, D. M. Vyas, and J. K. N. Jones, Can./. Clnem., 54 (1976)3783-3793. “Oxidation of a Branched-chain Alditol by Acetobocter suboxydans: a Stereospecific Synthesis of L-Dendroketose,” W. A. Szarek, G. W. Schnarr, H. C. Jarrell, and J. K. N. Jones,Carbohydr. Res., 53 (1977)101-108. “Preparation and Activity of Immobilized Acetobacter suboxydans Cells,” G . W. Schnarr, W. it. Szarek, and J. K. N. Jones,AppL Enoiron. Microbiol., 33 (1977)732-734. “Synthesis of Glymsides: Reactions of the Anomeric Hydroxyl Group with NitrogenPhosphorus Betaines,” W. A. Szarek, H. C. Jarrell, and J. K. N. Jones, Carbohydr. Res., 57 (1977) c13-cI6. “Stereospecific Chemical Synthesis of L-Dendroketose Derivatives,” H. C. Jarrell, W. A. Szarek, and J. K. N. Jones, Carbohydr. Res., 64 (1978)283-288.

ADVANCES IN C N O H Y D R A T E CHEMISTRY AND BIOCHEMISTRY. VOL. 41

CARBON-13 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY OF MONOSACCHARIDES BY KLAUS BOCKAND CHRISTIANPEDERSEN Department of Organic Chemistry. The Technical University of Denmark. DK-2800 Lyngby. Denmark I . Introduction ................................................. I1. Sampling Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Conditions for Optimal Signal-to-Noise Ratio ...................... 3. Referencing of Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Resolution Enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Assignment Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Comparison with Model Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Isotopic Substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Correlation with Proton Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . RelaxationRates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Paramagnetic Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Protonation Shifts ........................................... IV . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Identity of Monosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Structure Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Conformational Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Relaxation Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Complexation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27 28 29 30 31 32 33 34 34 35 36 37 38 39 39 39 40 43 43 43 44

I . INTRODUCTION

The first two reports on carbon-13 nuclear magnetic resonance ( 13Cn.m.r.) spectra of carbohydrates appeared1. in 1968 and 1969; since then. 13C-n.m.r. spectroscopy has become increasingly important as a tool for the characterization and structural elucidation of sugars and their derivatives . Although 13C-n.m.r. is closely related to 'H-n.m.r. spectroscopy, especially when both types of spectra are recorded with

.

(1) F. J . Weighert. M Jautelat. and J. D. Roberts. Proc. Natl .Acad . Sci . USA. 60 (1968) 1152-1155 . (2) A . S . Perlin and B . Casu. Tetrahedron Lett., (1969) 2921-2924 .

27

Copyright @ 1983 by Academic Press. Inc All nghts of reproduction in any form reserved ISBN 0-12-007261-6

28

KLAUS BOCK AND CHRISTIAN PEDERSEN

Fourier-transform instruments, the two techniques are sufficiently different to be valuable complements to each other. In many cases, in particular when dealing with complex molecules, such as polysaccharides, the amount of information obtainable from 'H-n.m.r. spectra is limited, compared to that revealed3 by I3C-n.m.r. spectra. Monosaccharides may also yield 'H-n.m.r. spectra that are poorly resolved, even at high field, and that contain little information. On the other hand, proton-decoupled, I3C-n.m.r. spectra are well resolved and, even if the signals are not assigned, a spectrum will provide an almost unambiguous identification of a compound. The application of I3C-n.m.r. spectroscopy to carbohydrates has already been reviewed many times,4-" and has been discussed in two monograph~.'~.'~ In each of those reviews, limited numbers of chemical-shift data for carbohydrates were given. As, for identification purposes, it is useful to have convenient access to an extensive list of chemical-shift data, the main purpose of the present article is to provide an almost complete collection of W-n.m.r. chemical-shifts of monosaccharides, their methyl glycosides, and acetates; see Tables IV. In addition, examples of shift data for as many different types of monosaccharide derivative as possible will be given; see Tables VIXXI. Nucleosides and nucleotides are not included, but data on compounds of these types have been reported, for example in Refs. 8, 12, and 14. The literature covered in the present article includes most of that published in 1980, together with a few subsequent papers. 11. SAMPLINGTECHNIQUES The fundamental principles of Fourier-transform, n.m.r. spectroscopy have been described in books and reviews.'"-" (3) P. A. J. Gorin,Adc;. Carbohydr. Chem. Biochem., 38 (1980) 13-104. (4) B. Coxon, Dec;. Food Carbohydr., 2 (1980)351-390. (5) A. S. Perlin, M T P Znt. Reo. Sci., Org. Chem. Ser. One., 7 (1976) 1-34. (6) S. N. Rosenthal and J. H. Fendler, Ada. Phys. Org. Chem., 13 (1976)292-424. (7) .4. S. Shashkov and 0. S. Chizhov, Bioorg. Khim., 2 (1976) 437-497. (8) F. W. Wehrli and T. Nishida, Fortsclrr. Chem. Org. Nuturst., 36 (1979) 1-229. (9) R. Barker and T. E. Walker, Methods Carbohydr. Chem., 8 (19130) 151-165. (10) T. D. Inch, Annu. Rep. N M R Spectrosc., 5A (1972)305-352. (11) G. Kotowycz and R. U. Lemieux, Chem. Rev., 73 (1973)669-698. (12) J. B. Stothers, Carbon-13 NMR Spectroscopy, Academic Press, New York, 1972, pp. 458-468. (13) E. Breitmaier, G. Jung, and W. Voelter,Angew. Chem., 83 (1971) 659-672. (14) M.-T. Chenon, R. J. Pugmire, D. M. Grant, R. P. Panzica, and L. B. Townsend, J . Am. Chem. SOC., 97 (1975) 4627-4636. (15) F. W. Wehrli and T. Wixthlin, lntefpretation of Carbon-13 NMR Spectra, Heyden, London. 1976.

13C-N.M.R.SPECTROSCOPY OF MONOSACCHARIDES

29

1. Sample Preparation The solvents most frequently used for the measurement of 13Cn.m.r. spectra are deuterium oxide (D20) and deuteriochloroform (CDCl,). Deuterated dimethyl sulfoxide (Me2SO-d,) is frequently used, especially for oligo- and poly-saccharides,, and a range of other solvents, including pyridine-d, , have also been employed. The 13Cn.m.r. chemical-shifts of carbohydrates cover a range of -200 p.p.m., and, as solvent-induced shifts are usually less than 1 p.p.m., the choice of solvent does not have a large effect on proton-decoupled, 13C-n.m.r.spectra. Exceptions to this are, however, spectra of basic or acidic carbohydrates (amino sugars, and aldonic and uronic acids), which are strongly pH-dependent. Proton-coupled, 13C-n.m.r. spectra may also be affected by a change in solvents owing to their profound effect on the IH-n.m.r. spectra. The concentration of the sample in a particular solvent has little effect on chemical-shift values and, because of the inherently low sensitivity of I3C-n.m.r. spectroscopy, it is advantageous to use as concentrated solutions as possible when measuring these spectra. However, increased concentration, and consequently increased viscosity, causes line broadening due to decreased, spin-lattice relaxation-times (TI values),'* and thus, poorer resolution. Certain solvents that tend to give viscous solutions (for example, Me2SO-d6) may also give decreased resolution. The temperature of the sample solution has a profound effect on the viscosity and, hence, on the resolution; that is, a higher temperature results in better resolution, because of lower viscosity (larger T, values). The most important aspect of temperature changes in the sample is, however, its effect on chemical-shift values. Thus, a series of I3C-n.m.r. spectra recorded for methyl a-aglucopyranoside in D 2 0 solution showedl9 linear changes in chemical shifts of up to 0.015 p.p.m./degree. Hence, when data have to be compared accurately, I3Cn.m.r. spectra should be recorded at the same temperature, and for samples that have reached temperature equilibrium in the probe. It is obvious that the best resolution is obtained from samples that contain no insoluble impurities, and no paramagnetic materials. The line broadening caused by soluble paramagnetic impurities" may be

(16) E. Breitmaier and W. Voelter, 1 3 4 N M R Spectroscopy, Verlag Chemie, Weinheim, 1974. (17) M. L. Martin, J.-J. Delpuech, and G . J. Martin, Practical NMR Spectroscopy, Heyden, London, 1980. (18) K. Bock, L. D. Hall, and C. Pedersen, Can. J . Chern., 58 (1980) 1916-1922. (19) K. Bock, B. Meyer, and M. R. Vignon,J. Magn. Reson., 38 (1980) 545-551.

30


diminished20 by treatment with an ion-exchange resin or by addition of small amounts of (ethylenedinitri1o)tetraacetate(EDTA).Dissolved oxygen also causes some line broadening; it may be removed sufficiently by boiling the solution in the sample tube for 1 minute.

-

2. Conditions for Optimal Signal-to-Noise Ratio The signal-to-noise ratio (s/n) obtained when a l3C-n.m.r. spectrum is recorded for a given sample solution depends, of course, on the type of instrument used, and it is obvious that a high-field instrument, quadrature detection, and large sample tubes are factors that all result in increased s/n in a given time. Increased concentration of the sample results in a larger s/n, but only to a certain extent, as too high a concentration will lead to line broadening, which will, in turn, have an adverse effect on the s/n. The pulse width is an important factor in the measurement of pulsed spectra. The optimal pulse-width may be estimated21from the equation cos a = exp(- T 1 / T ) ,in which a is the pulse width (in degrees), TI the spin-lattice relaxation-time (in s), and T the pulse-repetition time (in s). For monosaccharides in 20% aqueous solution, TI values of the protonated carbon atoms are22 1 s at 30". Using 8 k of computer memory for the acquisition, and a sweep width of 5-6 kHz, T becomes 0.6-0.8 s, and the equation gives an optimum pulse-width of -60". In Fig. 1 is shown a series of spectra measured at different pulse-widths, all other variables being kept constant. The best s/n is seen to correspond to a 63" pulse. If '%-n.m.r. spectra are recorded for very concentrated solutions, or impure samples, the TI values may become small, and, in such cases, a 90"sample pulse will be optimal. The s/n is, of course, directly proportional to the amount of sample present in the sample tube (more correctly, in the volume defined by the receiver coil); hence, a better s/n is obtained when a large sampletube is used. If, however, a limited amount of compound is available, it may be advantageous to use a smaller probe-insert, because this gives a better coupling between the receiver coil and the nuclei in the sample. The increased s/n resulting from measuring the same amount of compound in a 5-mm sample tube rather than in a 10-mm tube is illustrated in Fig. 2. It may be seen that the s/n ratio in the 5-mm insert is -3 times that in the 10-mm. Consequently, with the 5-mm tube, a

-

(20) M.Cohn and T. R. Hughes, Jr.,]. B i d . Chem., 237 (1962) 176-181. (21) R. R. Emst and W. A. Anderson, Rev. Sci. Instrum., 37 (1966) 93-102. (22) K. Bock and L. D. Hall, Carbohydr. Res., 40 (1975) c3-C5.

13C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

31

FIG. 1.-22.63-MHz, 13C-N.m.r. Spectrum of Methyl 8-D-Xylopyranoside in D,O (0.5 M) at 274 K. [All spectra were obtained under the same experimental conditions, but with different pulse-widths:A, 90";B, 63";C, 45"; and D, 27". Obviously, the optimal signal-to-noise ratio is obtained with a 63" pulse-width.The numerals in A indicate the signals of carbon atoms 1-5.1

sln corresponding to that shown in Fig. 2A could have been achieved with only 20,000/9 = -2000 scans, because the s/n is proportional to the square root of the number of scans.

3. Referencing of Signals Carbon-13 chemical-shifts are defined relative to the carbon signal of internal tetramethylsilane (Me,Si); hence, when measuring spectra in organic solvents, Me4Si should be added to the sample solution as the internal reference-~tandard.2~ However, any homo- or hetero-nuclear signal of the solvent, or of an added reference compound, may be used to calculate the 13C-chemical shifts, provided that its shift rela(23) Pure A p p l . Chem., 45 (1976) 217-219.

32


10mm

3

1

2

4

6

5 mm

B

s/n = 24.4/1

FIG.2.-22.63-MHz, %-N.m.r. Spectrum of Methyl /3-D-Xylopyranoside (10 mg) in 40. [A. Measured in a 10-mm sample-tube in 0.9 mL of DzO. B. Measured in a 5-mm sample-tube i n 0.3 mL of 40. Experimental conditions for the acquisition of the two spectra were exactly identical, and both spectra were obtained with 20,000 scans. In A, the numerals 1-5 indicate the signals of corresponding carbon atoms, and 6 indicates the signal of the 0-methyl group.]

tive to Me,Si is known. In aqueous solution, in which MF4Si is insoluble, it is necessary to use a water-soluble reference-compound unless external Me,Si (in a capillary tube) is employed. In most cases, internal 1,4-dioxane (67.4), acetone (30.5),or methanol (49.6 p.p.m.) are used as references when D,O is the solvent. For routine purposes, when accurate chemical-shifts are not important, it may be convenient to use the deuterium signal of the solvent as a heteronuclear reference, thus avoiding addition of any reference compound. 4. Quantitative Analysis It is often assumed that quantitative data cannot be satisfactorily obtained from integrated, I3C-n.m.r. spectra, because of saturation phenomena and nuclear Overhauser effects. However, if spectra are measured under suitable conditions, and if integrals (or peak heights) of

I3C-N.M.R.SPECTROSCOPY OF MONOSACCHARIDES

33

signals from carbon atoms carrying the same number of hydrogen atoms are compared, it is possible to obtain rather accurate information (f5%; that is, comparable to integrals obtained from 'H-n.m.r. spectra) about the relative amounts of components in a mixture. This has been discussed both from a general point of viewz4and, more specifically, with regard to carbohydrate^.^*'^-^^^^^^ It may be concluded that one of the most important conditions for correct integrals is a good s/n, which is, of course most, readily obtained for concentrated solutions. In such samples, the TI values are small, and hence, saturation is less likely. Furthermore, a sufficient digital resolution (at least 5 points per line) is necessary, in order to define the lines in a spectrum. This may be achieved by narrowing the sweep width, by using a sufficiently large computer memory, or by multiplying the free induction decay (f.i.d.) by a sensitivity-enhancement factor corresponding to a line broadening of 2-3 Hz. Obviously, integration can only be performed on signals that are completely separated; hence, high-field instruments are better suited for this purpose. Integrated, 13C-n.m.r. spectra have been used extensively to study mutarotational equilibria of monosaccharides, especially of ketoses, which do not have well-resolved, 'H-n.m.r. ~ p e c t r a , 2 ~ and - ~ also ~ have been used to determine the composition of crude reaction-mixture~.~~

5. Resolution Enhancement Better separation of poorly resolved signals can obviously be achieved by measuring a spectrum at higher field. However, because increased relaxation-times result in sharper lines,18the resolution can also be improved by using a low concentration, a high temperature, and a nonviscous solvent (for example, acetone). Besides, the use of a (24) S. Gillet and J.-J. Delpuech,]. Magn. Reson., 38 (1980)433-445. (25) J. W. Blunt and M. H. G. Munro, Aust. J . Chem., 29 (1976) 975-986. (2%) D. Horton and Z. Wataszek, Carbohydr. Res., 105 (1982) 145-153. (26) D. Doddrell and A. Allerhand,J. Am. Chem. SOC.,93 (1971)2779-2781. (27) L. Que and G. R. Gray, Biochemistry, 13 (1974) 146-153. (28) D. J. Wilbur, C . Williams, and A. Allerhand,J. Am. Chem. SOC.,99 (1977) 54505452. (29) C. Williams and A. Allerhand, Carbohydr. Res., 56 (1977) 173-179. (30) A. S. Perlin, P. C. M. H. du Penhoat, and H. S. Isbel1,Adu. Chem. Ser., 117 (1973) 39-50. (31) S. J. Angyal and G. S. Bethell, Aust. J . Chem., 29 (1976) 1249-1265. (32) W. Funcke, C. von Sonntag, and C. Triantaphylides,Carbohydr. Res., 75 (1979) 305 -309. (33) P. C. M. H. du Penhoat and A. S. Perlin, Carbohydr. Res., 36 (1974) 111-120. (34) K. Bock, C. Pedersen, and H. Thegersen, Acta Chem. Scand., Ser. B , 35 (1981) 441-449.

34

KLAUS BOCK A N D CHRISTIAN PEDERSEN

5-mm insert and sample tube, instead of the usual 10-mm tubes, will, with most instruments, result in sharper lines, in addition to the increased sensitivity mentioned earlier (see Fig. 2). Alternatively, the resolution of a spectrum may be improved by various mathematical methods, readily performed with a computer and normally described in the instruction manuals for the various n.m.r. instruments. A detailed discussion of data processing in Fourier-transform, n.m.r. spectroscopy was given in Reference 35. It should be mentioned that any mathematical improvement of resolution inevitably leads to a loss of s/n. Resolution enhancement is usually not important in proton-decoupled, W-n.m.r. spectra of monosaccharides. However, in the much more complex, proton-coupled, carbon spectra, this technique is useful if the rather small, two- or three-bond, C-H couplings have to be measured. 111. ASSIGNMENT TECHNIQUES The assignment of signals to specific carbon atoms is a necessary prerequisite to the application of 13C-n.m.r. spectroscopy in structural investigations. As assignment techniques have been described in numerous reviews and book~,3*~~'~*'"-" this area will be treated relatively briefly in the present article.

1. Comparison with Model Compounds In earlier publications, the assignment of signals in 13C-n.m.r. spectra of monosaccharides relied mostly on comparison with those of model compounds3697;this approach led to a number of simple, general rules, summarized as follows. ( a ) The anomeric carbon atoms in pyranoses and furanoses, and in their derivatives, resonate at lowest field (90-110 p.p.m.), except in 1-thioglycosides (see Table VI). (b) Carbon atoms carrying primary hydroxyl groups are found at 60-64 p.p.m. (c) Carbon atoms bearing secondary hydroxyl groups, in pyranoses and furanoses, give signals at 65-85 p.p.m. Signals of alkoxylated carbon atoms, including C-5 in pentopyranoses and C-4 in furanoses, are shifted 5-10 p.p.m. to lower field when compared with the corresponding, hydroxy-substituted carbon atoms. (35) J. C. Lindon and A. G . Femge, Prog. Nucl. Magn. Reson. Spectrosc., 14 (1980)2766. (36)A. S. Perlin, B. Caw, and H. J. Koch, Can. /. Chem., 48 (1970)2596-2606. (37) D.E. Dorman and J. D. Roberts,/.Am. Chem. Sac., 92 (1970)1355-1361.


35

A number of more complicated rules on the influence of axial or equatorial substituents on the chemical shifts of a-,p-, or y-carbon atoms may be safely applied to simple, alicyclic m o l e ~ u l e s . 3 ~In~ ~ - ~ ~ the authors’ opinion, however, such rules are generally of limited value for pyranoses or hranoses, because these contain several, mutually interacting substituents, and use of these rules has, in several instances, led to erroneous assignments. 2. Isotopic Substitution

If a compound in which carbon atoms at known positions are substituted with deuterium or carbon-13 is available, the assignment of its l3C-n.m.r. spectrum is greatly facilitated. Substitution with carbon-13 results in a much stronger signal from the enriched carbon atom, and hence, in its unambiguous assignment. In addition, l3C-I3C couplings may be visible in the spectra of I3C-enriched compounds, and these, together with isotope-induced shifts, may assist in the assignment of carbon atoms in positions a or p to the enriched carbon atom.9940-44 In the I3C-n.m.r. spectra of C-deuterated compounds, the deuterium-carrying carbon atom usually gives no signal, due to coupling to deuterium, longer spin-lattice relaxation-time, and quadrupolar broadening of the signal. Furthermore, the p-carbon atoms may be assigned because of the small, deuterium-induced, upfield shift~.4~-~* A convenient procedure for the preparation of glycosides labelled with deuterium at the hydroxyl-bearing carbon atoms has been deve10ped.4~-~~ Introduction of such magnetic nuclei as I9F or 31Pleads to spin-spin (38)D.K. Dalling and D. M. Grant,]. Am. Chem. Soc., 89 (1967)6612-6622. (39)D. E. Doman and J. D. Roberts,]. Am. Chem. Soc., 93 (1971)4463-4472. (40)T.E. Walker, R. E. London, T. W. Whaley, R. Barker, and N. A. Matwiyoff,]. Am. Chem. SOC., 98 (1976)5807-5813. (41)T. E. Walker, R. E. London, R. Barker, and N. A. Matwiyoff, Carbohydr. Res., 60 (1978)9-18. (42)T.E.Walker and R. Barker, Carbohydr. Res., 64 (1978)266-270. (43)A. S. Serianni, E. L. Clark, and R. Barker, Carbohydr. Res., 72 (1979)79-91. (44)G . Excoffier, D. Y. Gagnaire, and F. R. Taravel, Carbohydr. Res., 56 (1977)229238. (45)P. A. J. Gorin, Can. ]. Chem., 52 (1974)458-461. (46)P. A. J. Gorin and M. Mazurek, Can.]. Chem., 53 (1975)1212-1223. (47)H. J. Koch and A. S. Perlin, Carbohydr. Res., 15 (1970)403-410. (48)E.Breitmaier and U. Hollstein, Org. Magn. Reson., 8 (1976)573-575. (49)H. J. Koch and R. S. Stuart, Carbohydr. Res., 67 (1978)341-348. (50) S.-C. Ho, H. J. Koch, and R. S. Stuart,Carbohydr. Res., 64 (1978)251-256. (51)F.Balza, N.Cyr, G. K. Hamer, A. S. Perlin, H. J. Koch, and R. S. Stuart, Carbohydr. Res., 59 (1977)c7-cll.

36


coupling with neighboring carbon atoms, and their I3C-signals may therefore be readily identified.4"s"-"6 Whereas introduction of I3C or deuterium onto carbon atoms requires more-or-less laborious syntheses, 0-deuteration of hydroxyl groups or N-deuteration of amino groups is readily achieved by exchange of protons by deuterons with D,O. In the deuterated carbohydrates thus obtained, only small isotopic-shifts are observed in the '"C-n.m.r. spectra; however, when measured under appropriate conditions, these shifts are very useful for the assignment of 13C-signa1s.50,S7-6i

3. Correlation with Proton Spectra An assignment technique that requires no chemical modification of

the compound studied involves the use of proton-coupled, or off-resonance-decoupled, 'W-n.m.r. spectra. A proton-coupled spectrum, usually measured by the "gated decoupling" technique,'"'' contains information about the I3C-'H coupling-constants, but, as these are large, the 13Cmultiplets may overlap. In an off-resonance-decoupled ~pectrurn,~"-" the C-H couplings are lessened and, hence, overlap of signals is less likely. Both types of spectra show unambiguously how many protons are attached to each I3C nucleus. In addition to the large, one-bond, I3C-H couplings,2,62-65 fully proton-coupled spectra having good resolution will show two- or three-bond, 13C-H COUplings that may be useful for the assignment of signals to certain car(52) K. Bock and C. Pedersen, Acta Chem. Scand., Ser. B , 29 (1975)682-686. (53) V. Wray, J. Chem. Soc., Perkin Trans. 2, (1976) 1598-1605. (54) G . Adiwadjaja, B. Meyer, H. Paulsen, and J. Thiem, Tetrahedron, 35 (1979)3733%.

(55)J. V. O'Conner, H. A. Nunez, and R. Barker, Biochemistry, 18 (1979) 500-507. (56) T. A. W. Koerner, Jr., R. J. Voll, L. W. Cary, and E. S. Younathan, Biochemistry, 19 (1980) 2795-2801. (57) D. Y. Gagnaire and M. Vincendon,J. Chem. Soc., Chem. Commun., (1977) 509510. (58) D. Y. Gagnaire, D. Mancier, and M. Vincendon,Org. Magn. Reson., 11 (1978)344349. (59) P. E. Pfeffer, K. M. Valentine, and F. W. Parrish,J. Am. Chem. SOC., 101 (1979) 1265- 1274. (60) P. E. Pfeffer, F. W. Panish, and J. Unruh, Carbohydr. Res., 84 (1980) 13-23. (61) K. Bock, D. Y. Gagnaire, and M. R. Vignon, C . R . Acad. Sci., Ses. C , 289 (1979) 345-348. (62) K. Bock and C. Pedersen,]. Chem. Soc., Perkin Trans. 2, (1974)293-297. (63)K. Bock and C. Pedersen, Acta Chem. Scand., Ser. B , 29 (1975) 258-264. (64) J. A. Schwarcz and A. S. Perlin, Can. J . Chem., 50 (1972)3667-3676. (65) H. Paulsen, V. Sinnwell, and W. Greve, Carbohydr. Res., 49 (1976)27-35.


37

bon atoms. Two- and three-bond, l3C-H couplings have been discussed in several a r t i ~ l e s , 4 O . ~and * ~ ~in- ~a ~review.72 The most straightforward way of assigning 13Csignals is through selective, proton decoupling. By this technique, one proton is irradiated at its resonance frequency with a low-power, single frequency, causing the signal of the carbon atom to which it is bound to appear as a singlet in the l3C-n.m.r. spectrum, whereas all of the other carbon atoms are coupled to protons, and hence give off-resonance, decoupled multiplets. This is clearly illustrated in Fig. 3. This technique, however, requires a fully assigned, 'H-n.m.r. spectrum having well-dispersed proton-signals (separated by at least 10 Hz), and is therefore best conducted with high-field instruments and for acylated carbohydrates, which afford better-separated proton-signals. With modem, pulsed Fourier-transform instruments, series of selective proton-decouplings may be performed automatically, provided that the correct, decoupling frequencies have been measured.15 Correlation between proton and carbon chemical-shifts and coupling-constants may also be obtained through heteronuclear, twodimensional, n.m.r. ex~eriments.733~~ 4. Relaxation rate^^,^"^

Carbon-13 relaxation-rates of monosaccharides are dominated by dipolar-relaxation mechanisms,18,22 and primarily give information ahor:t molecular m ~ t i o n , in ~ ~addition , ~ ~ to the somewhat trivial distinction between C, CH, CH, , and CH, groups. However, by measuring spectra with a suitable pulse-sequence, the differences in spin-lattice relaxation-rates can be used for the assignment of signals from overlapping C H and CH, groups.77

(66)R. U. Lemieux, T. L. Nagabhushan, and B. Paul, Can.]. Chem., 50 (1972)773-776. (67) A. S. Perlin, N. Cyr, R. G. S. Ritchie, and A. Parfondry, Carbohydr. Res., 37 (1974) cl-c4. (68)J. A. Schwarcz, N. Cyr, and A. S. Perlin, Can.]. Chem., 53 (1975) 1872-1875. (69) R. G. S. Ritchie, N. Cyr, and A. S. Perlin, Can.J . Chem., 54 (1976)2301-2309. (70) N. Cyr and A. S. Perlin, Can.J . Chem., 57 (1979)2504-2511. (71) R. U. Lemieux, Ann. N . Y. Acad. Sci., (1973) 915-934. (72) P. E. Hansen, Prog. Nucl. Magn. Reson. Spectrosc., 14 (1981) 175-296. (73) R. Freeman and G. A. Morris,]. Chem. SOC., Chem. Commun., (1978) 684-686. (74) L. D. Hall and G. A. Moms, Carbohydr. Res., 82 (1980) 175-184. (75) M. F. Czarniecki and E. R. Thomton,]. Am. Chem. Soc., 99 (1977)8279-8282. (76) J. M. Berry, L. D. Hall, and K. F. Wong, Carbohydr. Res., 56 (1977)C16-~20. Lallemand,]. Chem. SOC., Chem. Commun., (1981) 150-152; (77) C. LeCoco and J.-Y. D. M. DoddreIl and D. T. Pegg,]. Am. Chem. SOC., 102 (1980)6388-6390.

86


38

I;,

H-1

~ - n.4 3 n-2

A

14.ti 65

OMe

90 MHz

i

I

C

D

/I

t.

.

I I I

FIG.3.--90-MWz, 'H-N.m.r. Spectrum in Deuteriochloroform (0.1M ) and 22.63-MHz, *T-N.m.r. Spectra of Methyl TetraO-acetyl-a-D-glucopyranosidein Deuteriochloroform (1 M). [A. The W M H z , 'H-n.m.r. spectrum, with the assignment ofthe signals given above the resonances. 8.The 22.63-MHz, 13C-n.m.r., proton-noise-decoupled, lacn.m.r. spectrum, with the assignment of the signals indicated below the resonances. C, D, E, and F show the results of a series of selective, proton decouplings, applied at the frequencies indicated in A, at positions C to F.]

5. Paramagnetic Reagents It is well known from 'H-n.m.r. spectroscopy that the addition of soluble, paramagnetic reagents (notably europium, gadolinium, and cupric complexes) causes large changes in chemical shifts and line widths. Similarly induced changes are observed in 13C-n.m.r.spectra, and their use for assignment of carbon signals have been discussed in ,'~ shift-reagents have general terms by several a u t h o r ~ . ' ~Paramagnetic


39

also been applied in the study of I3C-n.rn.r. spectra of carbohydrate~.~~-~~

6. Protonation Shifts The chemical shifts observed in the I3C-n.m.r. spectra of aminodeoxy sugars are strongly dependent on the pH of the sample solution, and the spectra of such compounds should, therefore, be measured with control of the pH. Comparison of I3C-n.m.r. spectra, measured at low or high pH, that is, for compounds having protonated or free amino groups, may be used for the assignment of carbons a and p to the amino groups.11J6,81,82 Similar, but smaller, effects are-observed in the spectra of other ionizable compounds, such as aldonic or uronic acid^.^^,^^

IV . APPLICATIONS

1. Identity of Monosaccharides The most important, practical application of 13C-n.m.r.spectroscopy is probably the simple characterization and identification of organic compounds. Because of the simplicity of proton-decoupled carbon spectra, and the sensitivity of carbon-13 chemical-shifts towards structural changes, carbon spectra are extremely well suited for this purpose (see, for example, Ref. a), and it is for this reason that the emphasis of the present article has been placed on presenting chemical-shift data of monosaccharides and their derivatives. Such data are also important for structural studies of oligo- and poly-saccharides? and for the investigation of such mixtures as those arising from r n u t a r o t a t i ~ n ~(see ~ - ~Section ~ II,4) or from other reactionsa3* (78) B. Caw, G. Gatti, N. Cyr, and A. S. Perlin, Carbohydr. Res., 41 (1975) d - C 8 . (79) S. Hanessian and G . Patil, Tetrahedron Lett., (1978) 1031-1034. (80) P. McArdle, J. 0.Wood, E. E. Lee, and M. J. Conneely, Carbohydr. Res., 69 (1979) 39-46. (81) K. F. Koch, J. A. Rhoades, E. W. Hagaman, and E. Wenkert,J.Am. Chem. SOC., 96 (1974) 3300-3305. (82) R. U. Lemieux, K. Bock, L. T. J. Delbaere, S. Koto, and V. S. Rao, Can.].Chem., 58 (1980) 631-653. (83) K . Bock and C. Pedersen, unpublished results. (84) R. C. Beier, B. P. Mundy, and G. A. Strobel, Can.J . Chem., 58 (1980) 2800-2804. (85) W. Voelter, E. Breitmaier, and G. Jung,Angew. Chem., 83 (1971) 1011-1012. (86) S. J. Angyal, G. S. Bethell, D. E. Cowley, and V. A. Pickles, Aust. J . Chem., 29 (1976) 1239-1247. (87) C. F. Midelfort, R. K. Gupta, and H. P. Meloche,]. Biol. Chem., 252 (1977) 34863492.


40

When studying the course of reactions, %-n.m.r. spectra may be used to monitor the progress of a reaction,% or to detect intermediates. The latter was achieved in a study of the Kiliani-Fischer reaction?* 2. Structure Determination The sensitivity of carbon-13 chemical-shifts towards changes in substitution renders W-n.m.r. spectroscopy very useful for the determination of the structures of unknown compounds. This is clearly seen from the large changes in carbon-13 chemical-shifts encountered when deoxy, aminodeoxy, deoxyhalogeno, thio, or unsaturated h n c tions are introduced into monosaccharides (see Tables X-XII, and XIV) and it reflects the influence of electronegativity and polarizability on the chemical shifts. It may be noted that whereas a chlorine and bromine atom situated on C-1 of aldose derivatives causes upfield shifts of 2 and 5 p.p.m., respectively (see Table VI), a much larger effect is observed when substitution takes place at other carbon atoms of pyranoses or furanoses. Thus, replacement of oxygen by chlorine at C 4 or C-6 of galactopyranose causes upfield shifts of 7 and 19 p.p.m., respectively; the corresponding shifts for bromine are -20 and -28 p.p.m., respectively. Similar, carbon-13 chemical-shifts are found in deoxy sugars; but deoxy and deoxyhalogeno carbon atoms can be readily differentiated through the multiplicities of their protoncoupled, W-n.m .r . spectra. A change of ring size is also accompanied by a change of chemical shifts; thus, furanoses and other five-membered rings have chemical shifts downfield from those of the configurationally related, six-membered (see Tables 1-111). Similar relationships are found for five- and six-membered lactonesSR(see Table XX). Acyclic derivatives show chemical shifts at higher field than those of the corresponding cyclic compounds (see Tables XV and XVI). In five-menibered, isopropylidene derivatives that are monocyclic, or fused to a pyranoid ring, the chemical shifts for the quaternary carbon atoms are 108.5111.4 p.p.m., whereas values of 111.4-115.7 p.p.m. are found when they are fused to a furanose ring. Six- and seven-membered, isopropylidene derivatives show the quaternary carbon atoms at 97.1-99.5 and 101- 102 p.p.m., Similar data have been educed from 13C-n.m.r.spectra of benzylidene derivativesw The chemical shifts of the methyl groups of isopropylidene derivatives may also give information concerning the ring size.89The two carbon atoms engaged in

-

-

(88) R. M. Blazer and T. W. Whaley,J. Am. Chem. Soc., 102 (1980)5082-5085. (89) J. G. Buchanan, M. E. Chac6n-Fuertes, A. R. Edgar, S. J. Moorehouse, D. I. Rawson, and R. H. Wightman, Tetrahedron k t t . , (1980)1793-1796. (90)T. B. Grindley and V. Culasekharam, Carbohydc Res., 74 (1979)7-30.

W-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

41

an epoxide give carbon signals at higher field than those of five- and six-membered rings (see Table XIII). Furthermore, the signals of epoxide carbon atoms may be assigned from their large (180-190 Hz), one-bond, C-H coupling-~onstants.~~ Although many pairs of anomers give quite different signals for the anomeric carbon atoms, it has not been found possible to discover a general relationship between the anomeric configuration and the chemical shifts. However, for those furanoses in which the substituents at C-1 and C-2 are trans-oriented, the signals of the anomeric carbon atoms are always found at lower field than in those of the corresponding cis isomers.92For pyranoses, this relationship does not hold, but the anomeric structure of pyranoses can always be determined2s4. 62,83,64,74 from the one-bond coupling-constants, namely, JC--I,H--l. The corresponding coupling-constants of furanoses cannot be used to determine anomeric structures. Alkylation of oxygen leads to a rather large, downfield shift of the a-carbon atom (see Section II1,l and Table VIII), as discussed in reviews3s5Jand in several papers.36,37,93-97 Similarly, formation of cyclic acetals in downfield shifts of the furanose or pyranose car(91) K. S. Kim, D. M. Vyas, and W. A. Szarek, Carbohydr. Res., 72 (1979) 25-33. (92)R. G. S. Ritchie, N. Cyr, B. Korsch, H. J. Koch, and A. S. Perlin, Can.J . Chem., 53 (1975) 1424-1433. (93)P. A. J. Gorin and M. Mazurek, Carbohydr. Res., 48 (1976)171-186. (94) J. Haverkamp, J. P. C. M. van Dongen, and J. F. G. Vliegenthart, Tetrahedron, 29 (1973)3431-3439. (95) J. Haverkamp, J. P. C. M. van Dongen, and J. F. G. Vliegenthart, Carbohydr. Res., 33 (1974)319-327. (96) J. Haverkamp, M. J. A. De Bie, and J. F. G. Vliegenthart, Carbohydr. Res., 39 (1975)201-211. (97) R. Usui, N. Yamaoka, K. Matsuda, K. Tuzimura, H. Sugiyama, and S. Seto, J . Chem. Soc.,Perkin Trans. 1, (1973) 2425-2432. (98) W. Voelter, E. Breitmaier, E. B. Rathbone, and A. M. Stephen, Tetrahedron, 29 (1973)3845-3848. (99) W. A. Szarek, A. Zamojski, A. R. Gibson, D. M. Vyas, and J. K. N. Jones, Can. J . C h m . , 54 (1976)3783-3793. (100) A. S. Shashkov, A. I. Shienok, M. Islomov, A. F. Sviridov, and 0. S. Chizhov, Bioorg. Khim., 3 (1977)1021-1027. (101) A. Lip&, P. Nhhsi, A. Neszmklyi, and H. Wagner, Carbohydr. Res., 86 (1980) 133-136. (102) E. Conway, R. D. Guthrie, S. D. Gero, G. Lukacs, and A.-M. Sepulchre,J. Chem. Soc., Perkin Trans. 2, (1974) 542-546. (103) A. Lip&, P. Fugedi, P. Nhnhsi, and A. NeszmBlyi, Tetrahedron, 35 (1979)11111119. (104) A. NeszmBlyi. A. LipKk, and P. Nbhsi, Carbohydr. Res., 58 (1977) ~ 7 - m . (105) P. J. Garegg, B. Lindberg, and I. Kvamstrom, Carbohydr. Res., 77 (1979)71-78. (106) P. J. Garegg, P.-E. Jansson, B. Lindberg, F. Lindh, J. Lonngren, I. Kvarnstrom, and W. Nimmich, Carbohydr. Res., 78 (1980) 127-132.

42


bon atoms (see Table IX).Introduction of an acyl group onto oxygen causes a smaller (1.5-4p.p.m.), downfield shift of the a-carbon atom than that of an alkyl group. However, as 0-acylation causes the signal of the @-carbonatom to shift upfield (1-5 p.p.m.), the cumulative effect of several acyl groups may be difficult to predict. Acylation effects on simple alcohols have been d i ~ c u s s e d , ~and ~ *systematic '~~ studies of I3C-n.m.r. spectra of carbohydrates selectively 0-acylated in different positions have been reported by several a ~ t h o r s . ' ~ ~ - ~ ~ ~ Just as introduction of a magnetic nucleus into a known position may help in assigning the signals in a 13C-n.m.r.spectrum (see Section III,Z), the placement of an isotope in an unknown position may be determined from isotope shifts or from, for e ~ a m p l e , ' ~ C - *coupling ~C constants, or both. In most cases, the stereochemistry of the quaternary carbon atom in branched-chain carbohydrates cannot be elucidated from 'H-n.m.r. spectra, but 13C-chemical shifts, or long-range, I3C-lH coupling-constants, may often yield valuable inf~rmation."*-"~Likewise, the stereochemistry of acetal carbon atoms of benzylidene derivatives,103* l w and of acetals derived from pyruvic acid,105*10g may be determined from I3C-chemical shifts. Finally, from the 13C-chemicalshifts of glycopyranosides, it is possible to obtain information about the stereochemistry of chiral aglycons. I?'

(107) Y. Terui, K. Tori, and N. Tsuji, Tetrahedron Lett., (1976) 621-622. (108) M. R. Vignon and P. J. A. Vottero, Tetrahedron Lett., (1976) 2445-2448. (109) M. R. Vignon and P. J. A. Vottero, Carbohydr. Res., 53 (1977) 197-207. (110) K. Yoshimoto, Y. Itatani, and Y. Tsuda, Chem. Pharm. Bull., 28 (1980)2065-2076. (111) K. Yoshimoto, Y. Itatani, K. Shibata, and Y. Tsuda, Chem. Pharm. Bull., 28 (1980) 208-219. (112) H. Komura, A. Matsuno, Y. Ishido, K. Kushida, and K. Aoki, Carbohydr. Res., 65 (1978) 271-277. (113) P. E. Pfeffer, K. M. Valentine, B. G. Moyer, and D. L. Gustine, Carbohydr. Res., 73 (1979) 1-8. (114) P. M. Collins and V. R. N. Munasinghe, Carbohydr. Res., 62 (1978) 19-26. (115) J.-C. Depezay, A. Dukault, and M. Saniere, Carbohydr.Res., 83 (1980)273-286. (116) M. MiljkoviC, M. GligorijeviC, T. Satoh, D. GliSin, and R. G. Pitcher, J . Org. Chern., 39 (1974) 3847-3850. (117) K. Sato, M. Matsuzawa, K. Ajisaka, and J. Yoshimura, Bull. Chem. SOC. Jpn., 53 (1980) 189-191. (118) A.-M. Sepulchre, B. Septe, G. Lukacs, S. D. Gero, W. Voelter, and E. Breitmaier, Tetrabdron, 30 (1974) 905-915. (119) S. Seo, Y. Tomita, K.Ton, and J. Yoshimura,J. Am. Chem. Soc., 100 (1978)33313339.

W-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

43

3. Conformational Analysis Relatively little use has yet been made of I3C-n.m.r. spectroscopy in conformational analysis. The most extensive studies have been conducted on furanoses, the conformational equilibria of which may be studied by consideration both of carbon-13 chemical-shifts and of twoand three-bond, C-H coupling-constants.70The conformation of pentopyranoses has been investigated through one-bond, C-H couplingconstantsF3 Other applications of two- and three-bond, C-H couplings are described in Refs. 120 and 121. An experimental method for the determination of long-range, C -H coupling-constants has been describedIe2;this technique can conveniently be used with modem, n.m.r. instruments having full computer-control of the decoupling channels.

4. Relaxation Rates Carbon-13, spin-lattice relaxation-rates may be readily measured with pulsed, Fourier-transform instruments, and they primarily provide information about the molecular motion in s o l ~ t i o n . ~ , ~ ~ ~ ~ , Carbon-13 relaxation-rates have mostly been used to obtain structural information on polysa~charides.~

5. Complexation Carbon-13 chemical-shifts have been used to study the interaction of monosaccharides with such complexing agents as b o r a t e ~ ' ~ *and J~~ calcium ion^.'^^,'^^ Paramagnetic complexing-agents are mentioned in Section III,5.

(120) D. Y. Gagnaire, R. Nardin, F. R. Taravel, and M. R. Vignon, Nouo. J . Chim., 1 (1977) 423-430. (121) R. U. Lemieux and S. Koto, Tetrahedron, 30 (1974) 1933-1944. (122) K. Bock and C. Pedersen,J. Magn. Reson., 25 (1977) 227-230. (123) A. Neszmelyi, K. Ton, and G . Lukacs,J. Chem. SOC.,Chem. Commun., (1977)613 -614. (124) P. A. J. Gorin and M. Mazurek, Carbohydr. Res., 27 (1973) 325-339; Can. J . Chem., 51 (1973)3277-3286. (125) W. Voelter, C. Biirvenich, and E. Breitmaier,Angew. Chem., 84 (1972) 589-590. (126) M. F. Czamiecki and E. R. Thornton, Biochem. Biophys. Res. Commun., 74 (1977) 553-558. (127) L. W. Jaques, J. B. Macaskill, andW. Weltner, ]I.,]. Chem. Phys., 83 (1979) 14121421.

44


V. TABLES* In the following Tables are presented I3C-n.m.r. chemical-shifts of a variety of monosaccharides and their derivatives. As far as possible, complete sets of shift values are given for all of the pentoses, hexoses, methyl glycosides, alditols, and aldonic acids. In addition, the chemical shifts of a selection of the most common types of derivatives of monosaccharides are given. For many compounds, especially free sugars or methyl glycosides, carbon-13 chemical-shifts have been published several times for the same compound. In such cases, references are not necessarily given to all relevant articles, but primarily to those that give a complete assignment. When more than one reference is given to the same compound, the chemical-shift data have been taken from the reference marked with an asterisk in the Table. The majority of the spectra given in the Tables have been unambiguously assigned. Those which are not assigned (indicated with a superscript a ) are included because each constitutes a valuable identification of the compound. For many carbohydrate derivatives, only a few examples of spectra are given. Those references that contain a considerable number of additional data on similar derivatives are marked, or mentioned in footnotes to the Tables. The chemical shifts given in the Tables are, unless otherwise stated, from spectra recorded for solutions in D,O or in deuteriochloroform. Carbon-13 chemical-shifts published for a particular compound may differ considerably (by 1 to 2 p.p.m.), depending on the concentration, the temperature, and the reference standard used. Apart from changes caused by temperature,IYthe variations are generally the same for all of the carbon atoms in a compound, causing a parallel shift of signals. Because of these variations, the values in the Tables have been rounded off to one figure after the decimal point.

* The authors are grateful to Professor S . J. Angyal for a number of suggestions regarding, and corrections to, the data in the Tables. Data on heptoses, heptuloses, and heptitols will be published by S. J. Angyal and coworkers.

I3C-N.M.R.SPECTROSCOPY OF MONOSACCHARIDES

45

TABLE I 13C-N.m.r.Data for Aldoses Compound

C-1

D-Hexopyranoses a-All 93.7 P943 U-Alt 94.7 P92.6 a-Gal 93.2 P97.3 (Y-GlC 92.9 P96.7 CY-GUl 93.6 P94.6 a-Ido 93.2 P93.9 a-Man 95.0 P94.6 a-Tal 95.5 P95.0 D-Pentopyranoses a-Ara 97.6 P93.4 a-Lyx 94.9 P95.0 a-Rib 94.3 P94.7 a-Xyl 93.1 P97.5 D-Hexofuranoses a-All 96.8 P101.6 a-Alt 102.2 P96.2 ff-Gal 95.8 P101.8 P-ClC 103.8

c-2

c-3

C-4

c-5

C-6

References

67.9 72.2 71.2 71.6 69.4 72.9 72.5 75.1 65.5 69.9 73.6" 71.1" 71.7 72.3 71.7' 72.5"

72.0 66.9 72.0 67.7 71.1 66.0 71.3 65.2 70.2 70.3 73.8 69.7 73.8 70.6 76.7 70.6 71.6 70.2 72.0 70.2 72.7" 70.6" 68.8c 70.6' 71.3 68.0 74.1 67.8 70.6c 66.0 69.6" 69.4

37,83,*128" 67.7 61.6 62.1 36,37,63* 74.4 61.6 129 72 .O 129 75.0 62.5 36,37,59,*98 71.4 62.2 36,37,59,*98 76.0 62.0 72.3 61.6 29,36,37,40.46,59,*85,130" 29,36,37,40,46,59,*1306 76.8 61.7 67.2 61.7 83 83 74.6 61.8 73.6" 59.4 83 75.6" 62.1 83 73.4 62.1 28,36,37,40,46,*130,b131 77.2 62.1 28,36,37,46,*1306 72 .O 62.4 59,*131 59,*131 76.5 62.2

72.9 69.5 71.0 70.9 70.8 71.8 72.5 75.1

73.5 69.5 71.4 73.5 70.1 69.7 73.9 76.8

69.6 69.5 68.4 67.4 68.1 68.2 70.4 70.2

67.2 63.4 63.9 65.0 63.8 63.8 61.9 66.1

72.4 76.1 82.4 77.5 77.1 82.2 81.8"

d

73.3 76.9 76.0 75.1 76.6

84.3 83.0 84.3 82.1 81.6 82.8 82.1'

70.2 71.7 72.5 73.4

d

36,37,46,59,*131,132* 36,37,46,59,*131,132* 36,37,83* 36,37,83* 36,48,83* 36,37,48,63,*131 36,37,46,59,*131,133 36,37,46,59,*131,133 83 83 129 129

71.5

63.1 63.3 63.3 63.3 63.3 63.6

d

d

29

d

83 83

(continued)

(128) W. A. Szarek, D. M. Vyas, S. D. Gero, and G. Lukacs, Can. J . Chem., 52 (1974) 3394 -3400. (129) K. Bock and M. Beck Sommer, Acta Chem. Scand., Ser. B , 34 (1980) 389. (130) R. Kasai, M. Okihara, J. Asakawa, K. Mizutani, and 0. Tanaka, Tetrahedron, 35 (1979) 1427- 1432. (131) W. Voelter and E. Breitmaier, Org. Magn. Reson., 5 (1973) 311-319. (132) K. Mizutani, R. Kasai, and 0. Tanaka, Carbohydr. Res., 87 (1980) 19-26. (133) J.-P. Utille and P. J. A. Vottero, Carbohydr. Res., 85 (1980) 289-297.

KLAUS BOCK A N D CHRISTIAN PEDERSEN

46

TABLEI (continued) Compound ~~

C-2

C-1 ~

C-3

C-4

C-5

C-6

References

~

97.3 101.4 a-ldo 102.5 B96.3 a-Tal 101.8 P97.3 DPentofuranoses a-Ara 101.9 896.0 a-Lyx 101.5 &-Rib 97.1 8101.7 m-Erythrose a-Furanose 96.8 P-Furanose 102.4 Hydrate 90.8 DL-Threose a-Furanose 103.4 P-Furanose 97.9 Hydrate 91.1 DL-Glyceraldehyde Hydrate 91.2 Glycolaldehyde Hydrate 91.2 Formaldehyde Hydrate 83.3 CV-GUI

P-

d

d

78.1 78.6 77 .O 76.1 71.6

d

83 83

75.6‘ 75.9 72.7 72.0

80.4 80.3 82.2 81.6 82.7 83.3

62.6 63.2 70.3‘ 63.4 71.7c 63.4 71.6 63.7 63.8

82.3 77.1 77.8 71.7 76 .O

76.5 75.1 71.9 70.8 71.2

83.8 82.2 80.7 83.8 83.3

62.0 62.0 61.9 62.1 63.3

72.4 77.7 74.9

70.6 71.7 73.0

72.9 72.4 64 .O

43 43 43

82.0 77.5 74.6

76.4 76.2 72.2

74.3 71.8 64.4

43 43 43

75.5

63.4

83 83 59,*131 59.*131 83 83 83 48,83* 48,83*

43 43

66.0

43

In dimethyl sulfoxide-d,. Not resolved.

Assignment may have to be reversed.

In pyridine-d,.

TABLE I1

W-N.m.r. Data for Methyl Aldosides ~~

~

~~

~

~

C-2

C-3

C-4

C-5

D-Hexopyranosides a-All 100.0 68.3 P101.9 72.2 a-Alt 101.1 70.0 P100.4 70.7 a-Gal 100.1 69.2 P104.5 71.7 a-Glc 100.0 72.2

72.1 71.4 70.0 70.2 70.5 73.8 74.1

68.0 68.0 64.8 65.6 70.2 69.7 70.6

67.3 74.8 70.0 75.6 71.6 76.0 72.5

Compound

P-

C-1

104.0 74.1 76.8

70.6 76.8

C-6 @Me

References

83 83 55.4 36 83 57.7 36,59,*131 56.0 36,59,*131 58.1 55.9 36,37,40,46,49,59,*60,99,” 130,”134 61.8 58.1 36,37,40,46,49,59,*60,99,” 130,b134

61.7 62.2 61.3 61.7 62.2 62.0 61.6

56.3

58.0

I3C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

47

TABLEI1 (continued) Compound

C-1

100.4 102.6 a-Ido 101.5 101.9 a-Man p101.3 102.2 a-Tal D-Pentopyranosides 105.1 a-Ara p101.0 (Y-LYX 102.0 100.4 a-Rib 103.1 6100.6 a-Xyl p105.1 D-Hexofuranosides a-All 103.8 p109.0 a-Gal 103.8 p109.9 a-Glc 104.0 p110.0 109.7 a-Man p103.6 D-Pentofuranosides 109.2 a-Ara P103.1 109.2 a-Lyx P103.3 103.1 a-Rib 108.0 p103.0 a-Xyl p109.7 D-Tetrofuranosides a-Ery 103.6 p109.6 a-Thr 109.4 p103.8 a-Gul

p-

C-2

C-3

C-4

C-5

65.5 69.1 70.9 71.2 70.6 70.7

71.4 72.3 71.8 71.8 73.3 66.2

70.4 70.5 70.3 68.0 67.1 70.3

67.3 74.9 70.8 73.7 76.6 72.1

71.8 69.4 70.4 69.2 71.0 72.3 74.0

73.4 69.9 71.6 70.4 68.6 74.3 76.9

69.4 70.0 67.7 67.4 68.6 70.4 70.4

67.3 63.8 63.3 60.8 63.9 62.0 66.3

72.3 75.6 78.2 81.3 77.7 80.6 77.9 73.1

69.9 72.7 76.2 78.4 76.6 75.8 72.5 712d

85.9 83.4 83.1 84.7 78.8 82.3 80.5 80.7

72.7 73.8 74.5 71.7 70.7 70.7 70.6 71.W

81.8 77.4 77.0 73.2 71.1 74.3 77.8 81.0

77.5 75.7 72.2 71.0 69.8 70.9 76.2 76.0

84.9 82.9 81.4 82.1 84.6 83.0 79.3 83.6

62.4 62.4 61.5 62.7 61.9 62.9 61.6 62.2

72.8 76.4 80.5 77.4

69.9 71.4 76.4 75.8

73.6 72.6 73.7 72.0

C-6 0-Me

62.0 62.1 60.2 62.1 61.4 62.3

56.3 58.1 55.8 55.9 56.9 55.6

58.1 56.3 55.9 56.7 57.0 56.0 58.3 63.5 63.9 64.1 63.6 64.2 64.7 64.5 64.4

References

135 136 36 36,46,* 13CP 36,*130" 83 46,59,*131 , 1 3 2 , " ~ 46,59,*1 3 1 , 1 3 2 , " ~ 63 63 63,*131,134 36,46,*131,134 36,46,*131,134

56.6 56.4 57.2 55.6 57.0 56.3 57.2 56.8

92 92 92

56.0 56.3 56.9 56.7 55.5 55.3 56.7 56.4

46,92,*137 46,92,*137 92 92 46,92,*137 46,92,*137 92,*138 92,*138

56.7 56.6 55.5 56.2

92 92 92 92

92 92 92 92 92

~

a In dimethyl sulfoxided,. may have to be reversed.

* In pyridined,.

Contain additional data. Assignment

(134) E. Breitmaier, W. Voelter, G. Jung, and C. Tanzer, Chem. Ber., 104 (1971) 11471154. (135) H. Naganawa, Y. Muraoka, T. Takita, and H. Umezawa, J . Antibiot., Ses. A, 30 (1977) 388-396. (136) S. Jacobsen and 0. Mols, Acta Chem. Scand., Ser. B , 35 (1981)163-168. (137) E.Breitmaier, G . Jung, and W. Voelter, Chimia, 26 (1972) 136-139. (138) P. W. K. Woo and R. D. Westland, Carbohydr. Res., 31 (1973) 27-36.


48

TABLE111 'T-N.m.r. Data for Ketoses and Their Methyl Glycosides

Compound

C-1

DHexopyranoses a-Fni 65.9 B64.7 a-Psi 64 .O

B-

64.8

64.5 64.8 864.4 DHexofuranoses a-Fm 63.8 863.6 a-Psi 64.2 B63.3 a-Sor 64.3 a-Tag P63.5 D-Hexopyranosides B-FN 61.8 a-Psi 61.1 B57.7 a-Sor 61.2 &-Tag 61.0 B61.7 D-Hexofuranosides a-Fni 58.7 B60.0 a-Sor 60.7 B57.7 a-Tag 58.8 P60.3 a-Sor a-Tag

C-2

C-3

G4

C-5

C-6

99.1 98.4 992 98.5 99.0 99.1

70.9 68.4 66.4 71.2 71.4 70.7 64.6

71.3 70.5 72.6 65.9 74.8 71.8 70.7

70.0 66.7 69.8 70.3 672 70.1

64.1 58.8 65.0 62.7 63.1 61.0

105.5 102.6 104.0 106.4 102.5 105.7 103.3

82.9 76.4 71.2 75.5 77.0 77.6 71.7

77.0 75.4 71.2 71.8 76.2 71.9 71.8

82.2 81.6 83.6 83.6 78.6 80.0 80.9

61.9 63.2 62.2 63.7 61.6

101.4 100.7 102.6 100.9 102.4 101.4

69.3 67.3 69.7 72.0 69.6 65.5

70.5 72.1 65.7 74.5 71.7 71.5"

70.0 66.7 69.9 70.1 66.8 70.4a

64.7 58.9 65.4 63.0 63.4 61.1

49.3 49.1 48.7 49.2 48.5 49.3

31 31 31 31 31 31

109.1 104.7 1042 109.9 108.7 105.3

81.0 77.7 80.0 80.3 75.2 73.4

78.2 75.9 76.5 772 71.9 71.7

84.0 82.1 78.8 83.4 80.6 82.0

62.1 63.6 61.6 62.1 60.8 61.9

49.1 49.8 49.9 49.3 49.6 49.8

31 31 31 31 31 31

C-OMe

References 31 26,27,31* 27,30,31,*33 27,30,31,*33 31,*27 31,*27 31,*27 26,27,31,*32 26,27,31,*32 27,30,31,*33 27,30,3 1,*33 31,*27 31,*27 31,*27

61.9

Assignments may have to be reversed. For further data, see p. 66.

TABLEIV W-N.m.r. Data for Glycosides of Aromatic Aglycons C-2

C-3

C4

C-5

C-6

References

Phenyl D-glucopyranosides a 97.9 72.0 B 103.1 75.8 a p-NO, 100.5 74.1 B 102.7 76.0 B m-NO, 103.6 76.1 p &NO* 103.3 76.0

73.3 79.5 76.9 80.1 80.0

70.2 72.4 72.5 72.4 72.6 72.4

73.9 79.3 75.8 79.3 79.2 79.5

61.1 63.6 63.5 63.5 63.6 63.5

83 134 134 134 134 134

Compound

C-1

80.1

49


TABLEIV (continued)

Compound

C-1

C-2

Phenyl D-gdactopyranosides 104.0 73.6 P 75.5 a p-NOI 100.8 103.4 73.2 P p m-NO, 101.2 73.3 73.2 fi o-NOZ 104.1 Phenyl Dmannopyrawsides 73.4 a p-NO, 101.5

C-4

C-5

C-6

References

76.4 712 76.1 76.1 76.3

71.3 70.5 71.1 71.1 71.1

78.3 72.1 78.6 78.7 78.7

63.7 63.0 63.5 63.5

134 134 134 134

63.5

134

72.5

69.5

78.0

63.8

134

C-3

TABLEV 13C-N.m.r. Data for Peracetylated Pyranoses and Furanoses ~

Compound

C1

D-Hexopyranoses P-All 90.1 a-Alt 90.2 ff-Gal 89.5 P91.8 a-Glc 89.2 P91.8 p-Gul 89.7 a-Ido 90.4 a-Man 90.4 ff-Tal 91.4 D-Pentopyranoses a-Ara 92.2 B90.4 a-Lyx 90.7 88.7 a-Rib P90.7 a-Xyl 88.9 P91.7 D-Pentofuranoses a-Ara 99.4 P93.7 a-Lyx 98.0 P93.2 a-Rib 94.1 P98.1 a-Xyl 92.8 P98.9 a

C-2

C-3

C-4

C-5

C-6

References

68.2 68.2 67.2 67.8 69.3 70.5 67.3" 65.9 68.6 65.2"

68.2 66.4 67.2 70.6 69.9 72.8 67.1" 66.2 68.2 66.3"

65.8 66.4 66.2 66.8 68.O 68.1 67.1" 65.9 65.4 65.3"

71.2 64.4 68.5 71.5 69.9 72.8 71.1 66.2 70.5 68.8"

61.9 62.1 61.0 61.0 61.6 61.7 61.3 61.8 62.0 61.5

63 63 83 63 108 108,109* 83 63 62 63

68.2 67.3 68.2 67.1 67.1 69.2 69.3

69.9 68.7 68.2 65.6 66.0 69.2 70.8

67.3 66.9 66.6 66.5 66.0 68.8 68.1

63.8 62.9 61.9 59.3 62.5 60.5 62.5

80.6 75.4 75.0 70.5 70.0 74.1 75.3 79.4

76.9 74.8 70.6 68.5 69.8 70.5 73.8 74.3

82.4 79.7 77.0 77.7 81.6 79.2 75.4 79.9

63.1 64.5 62.4 62.8 63.3 63.6 61.6 62.3

63 63 63 63 63 63+*133* 63,*133b 139 139 139 139 139 139 139 139

Assignments may have to be reversed. * Contains additional data.

(139) B.L. Kam,J.-L. Barascut, and J.-L. Imbach, Carbohydr..Res., 69 (1979) 135-142.

.50

KLAUS BOCK AND CHRlSTlAN PEDERSEN

TABLEVI '"C-N.rn.r.Data for Tetra-0-acetyl-(benmy1)-Dglycopyranos yl Derivatives" ~~

Compound

C-1

C-2

C-3

86.1 87.3 86.5 89.5 87.1 66.8 103.5 105.7 96.3 101.1 94.3 98.8 80.1

69.7 70.3 70.4b 70.2b 72.4 69.4 69.9 70.6 70.4 70.9 70.5 71.1 65.8 70.4 70.8* 69.6b 71.W 68.76 69.4

69.8 72.2 72.P 70.3b 73.0 73.3 69.1 71.4 69.7 72.5 70.1 71.8 71.0 72.1 70.6 73.7b 70.7b 73.56 70.3

68.9 64.6b 67.6 68.5 69.1 69.3

68.2 66.6b 67.6 70.2 68.8 70.4 69.3 68.2 67.4 66.0 69.1 71.0

C-4

C-5

C-6

Me

-

References

~ g l u c oderivatives a-Azide

Pa-Bromide a-Chloride

P@-Cyanide a-Fluoride

Pa-Methoxy

Pa-Phenoxy

Pa-Phenylamino

P-

84 .O

81.8 83.2 a-Methylthio 83.0 P82.3 a-Methoxy, benzoate 96.8 Methyl mglycopyranosides ,&All 99.3 a-Alt 98.2 a-Gal 96.5 P101.5 a-Man 98.1 a-Ara 101.9 P97.6 a-Lyx 98.4 a-Rib 97.5 a-Ethylthio

P-

Pa-Xyl

P-

68.4

69.3 67.5 99.4 68.3 96.4 70.5 101.0 70.2

70.1 73.6 70.W 68.8b 74.9 77.3 69.6 71.5 66.8 71.4 68.1 72.5 72.1 72.8 67.6b 75.6b 68.96 67.76 68.v 75.5b 67.5 71.8 68.1

67.6 67.W 66.8b 67.2 67.8 67.1 67.0 68.2 68.1 68.4 68.2 68.5 68.7 68.7 68.2b

66.1 64.1b 67.0 66.8 65.8 67.9 67.2 66.6 66.1 66.9 68.8 68.3

70.0 68.96 65.7 70.6 68.0 63.2 60.3 59.4 57.9 61.1 57.7 61.3

61.7 61.4 60.8 60.4 61.2 61.8 61.0 61.3 61.5 61.6 61.7 61.8 61.7 62.0 62.0 61.9 62.1 61.8 62.9 62.1 62.2 61.2 61.0 62.1

114.5

55.6 56.6

12.4 55.4

140 62 62 62 62 141 52,62* 52,62* 62,63,*142 62,63,*142 83 62 62 62 143 62 143 62 62

56.0 144 55.0 62 54.8 62,63,*142 56.6 62,*142 54.9 62,63* 63,*142 56.6 55.4 63,*142 54.9 63 56.2 63 55.7 63 54.7 63,*133,142 55.8 63,*133,142

Additional data for related compounds are given in Refs. 145-148. may have to be reversed.

Assignments

(140) T. Takeda, Y. Sugiura, Y. Ogihara, and S. Shibata, Can.1.Chem., 58 (1980)26002603. (141) B. Coxon, Ann. N . Y. Acad. Sci., (1973) 952-970. (142) A. 1. Kalinovskii and E. V. Evtushenko, Khim. Prir. Soedin., 1(1979) 6-8. (143) B. S. Petersen, Ph.D. Thesis, Danrnarks Tekniske Haiskole, Lynehy, 1978. (1M) K. Bock, S. R. Jensen, B. J. Nielsen, and V. Nom, Phytochemistry, 17 (1978) 753757. (145) H. Pauisen, A. Richter, V. Sinnwell, and W. Stenzel, Carbohydr. Res., 64 (1978) 339-364.

i3C-N.M.R. SPECTROSCOPY O F MONOSACCHARIDES

51

TABLEVII 13C-N.m.r.Data for Anhydropyranose Derivatives" Compound

C-1

C-2

C-3 C-4

C-5

C-6 0 - M e C-7

1,6Anhydro-fl-~-hexopyranoses All 101.5 70.2 63.5 70.1 76.8 65.4 101.9 72.9 69.9 70.3 77.6 66.0 Alt Gal 101.3 71.9 70.8 64.9 74.9 64.1 Glc 102.1 70.9 73.3 71.6 76.9 65.8 Gul 101.7 70.5 70.5 69.9 74.9 63.8 101.9 74.7 74.7 71.4 75.8 65.4 Ido Man 101.9 66.6 70.9 72.2 76.4 65.3 1022 69.1 69.2 67.1 74.8 65.1 Tal Per-0-acetylated 1,6anhydr0-/3-~-hexopyranoses 99.0 68.0 62.4 67.7 74.0 64.8 All Alt 99.2 71.8 67.0 69.2 74.7 65.6 Gal 98.7 70.9 67.3 64.6 71.9 64.3 99.5 70.1 69.9 71.0 74.0 65.5 Glc Gul 98.9 68.9 66.7 68.6 71.8 63.9 98.7 72.3 70.1b 70.W 73.5 65.2 Id0 Man 99.2 67.0 67.6 71.8 73.8 65.2 99.0 68.6 66.4 66.0 72.1 65.6 Tal Methyl 3,6anhydro-~hexopyranosides gal 98.6 69.8 77.7 70.5 81.5 69.5 p103.4 72.7 78.4 70.5 81.2 70.9 a-Glc 99.5 71.8 72.0 70.4 76.4 69.8 p104.1 72.5 72.8 71.8 75.3 70.2 2,7-Anhydro-8-n-heptulopyranoses 60.4 107.9 72.8 70.7 70.7 78.3 Alt Gal 61.1 107.1 71.7 71.7 64.9 76.3 Glc 61.4 107.2 71.W 74.4 70.6b 78.3 60.8 107.9 70.4 70.2b 69.9 76.1 Gul Ido 60.5 108.1 74.8 75.4 71.7 76.6 Man 60.9 107.7 66.5 71.3 72.7 78.6 ~

~

References

65,69* 65,69* 65,69* 65,69,*124,149,150 65,69* 65,69* 65,69* 65.69; 69 69 69 69 69 69 69 69 83,*151 83,*151 124 124

58.0 56.2 58.5 56.5 67.0 65.2 66.7 65.1 66.5 66.5 ~~~~

69 69 69 69 69 69

~

Additional data for related compounds are given in Refs. 152 and 153. Assignments may have to be reversed. a

(146) V. Pozsgay and A. Neszmblyi, Carbohydr. Res., 80 (1980) 196-202. (147) B. L. Kam and N. J. Oppenheimer, Carbohydr. Res., 77 (1979)275-280. (148) C. L a t e , A. M.N. Phuoc Du, F. Winternitz, R. Wylde, and F. Pratviel-Sosa, Carhohydr. Res., 67 (1978) 105-115. (149) Y. Halpern, R. Riffer, and A. Broido,J. Org. Chem., 38 (1973) 204-209. (150) N. Gullyev, A. Ya. Shmyrina, A. F. Sviridov, A. S. Shashkov, and 0. S. Chizhov, Bioorg. Khim., 3 (1977) 50-54. (151) A. S. Shashkov, A. I. Usov, and S. V. Yarotskii, Bioorg. Khim., 3 (1977) 46-49. (152) C. Subero, L. Fillol, and M. Marth-Lomas, Carbohydr. Res., 86 (1980)27-32. (153)T. Trnka, M. cerng, A. Ya. Shmyrina, A. S. Shashkov, A. F. Sviridov, and 0.S. Chizhov, Carbohydr. Res., 76 (1979) 39-44.


52

TABLEVIII

'W-N.m.r. Data'' for 0-Substituted Monosaccharide Derivatives Compound

C-1

C-2

C-3

C-4

C-5

C-6

OMe

References

72.W 76.1b 72.8 77.3 71.7 76.1 71.4 75.8

61.4 61.5 62.3 62.3 62.1 62.1 72.6 72.6

58.4 60.9 61.3 61.3 61.6 61.6 60.3 60.3

62,*97 62,*97 37,97,*108 37,97,*108 97 97 97,*154 97,*154

71.0 75.4

72.4 72.4

94' 94'

72.1 76.3 70.5 74.7

61.5 61.5

155 155 155 155

74.1 77.9 73.1 77.2 75.0 78.2 71.3 75.6 72.6 76.1 70.1

61.9 62.4 62.7 62.9 62.4 62.7 64.8 64.8 63.7 63.7 64.4

55 55 55 55 55 55 83 83 156 156

83.0 81.3 81.4 80.8

62.6 63.3 64.5 65.4

56

0-Methyl-D-glucopyranose

90.1 81.3 72.8* 70.5 84.4 76.6h 70.5 96.5 84.1 70.6 93.4 72.6 a 375.1 86.7 70.4 P 97.2 a 493.2 73.0 73.9 80.5 75.8 76.7 80.5 97.1 P 93.3 73.0 74.3 71.4 671.4 75.8 77.2 P 97.3 Methyl tetra-0-methyl-D-glucopyranoside a 93.2 82.6 84.3 80.6 P 105.0 84.6 87.2 80.5 D-GlUCOpyranOSe sulfate a 392.9 71.1 83.1 68.3 73.8 85.2 68.3 P 96.5 70.2 93.1 72.3 73.6 a 675.0 76.5 70.2 P 96.9 Phosphate D-PyranOSeS a Clc 196.3 72.9 74.3 70.9 75.3 76.9 71.2 P 98.9 a Gal 196.5 69.7 70.7 70.7 73.9 70.3 73.0 99.5 P 68.1 72.1 71.6 a Man 197.3 72.6 74.2 68.2 P 96.7 69.9 93.0 72.2 73.3 a G l t 669.9 74.8 76.3 96.7 P 67.1h 94.8 71.3 70.6 CI Man 666.7" 94.4 71.9 73.3 P P Fni 167.4 99.0 69.0 70.4 D-Furanoses a Fni 183.0 77.0 P 66.0 77.4 75.2 a Fru 663.8 105.3 82.6 76.9 P 63.8 102.4 76.2 75.4 a 2-

P

68.1 68.1

56

56

56 56 (continued)

1154) R. Colson, K. N. Slessor, H. J. Jennings, and I. C. P. Smith, Can. ]. Chem., 53

(1975) 1030-1037.

(155) S. Honda, H. Yuki, and K. Tahiura, Carbohydr. Res., 28 (1973) 150-153. (156)P. A. J. Gorin, Can. ]. Chem., 51 (1973) 2105-2109. (157) A. S. Serianni, J. Pierce, and R. Barker, Biochemistry, 18 (1979) 1192-1199. (158) S. A. Abbas, A. H. Haines, and A. G. Wells,]. Chem. SOC., Perkin Trans. I , (1976) 1351- 1357.

I3C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES

53

TABLEVIII (continued) Compound

C-1

C-2

C-3

71.9 71.3 97.5 71.7 76.4 102.4 76.7 102.2 82.2 a Ara 575.1 77.0 P 96.3 n-Glyceraldehyde &phosphate, hydrate 91.3 74.9 66.0 n-Glycolaldehyde phosphate, hydrate 90.7 68.2 a Rib 5-

P

C-4

C-5

83.6 82.5 83.1 81.1

65.8 66.6 65.1 66.2

C-6

OMe

References

157 157 157 157 157 157

a Additional data for related compounds are given in Refs. 93, 95, 96, and 158. Assignments may have to be reversed. Contain additional data.

TABLEM W-N.m.r. Dataa for Isopropylidene and Benzylidene Derivatives* Compound

C-1

C-2

C-3

C-4

C-5

C-6

1,2:5,6-Di-O-isopropylidene-a-~-~ucofuranose 3-substituted derivatives OH 105.4 85.2 75.0 81.3 73.3 67.7 105.0 83.3 76.0 79.6 72.4 67.0 OAc OBz 105.2 83.4 76.7 80.0 72.7 67.3 105.3 82.6 81.7 81.3 72.5 67.4 OBn OMe 105.1 83.6 81.9 81.0 72.3 67.1 105.3 83.8 82.8 79.9 72.2 67.6 OMS 105.1 83.4' 82.1" 79.9 71.8 67.1 OTs Cld 104.3 85.4 62.2 79.7 73.2 66.4 104.8 82.3 93.5 80.4 71.7 66.9 F 105.0 79.8 34.7 77.9 76.2 66.1 Deoxyd 1,2 :5,6-DiO-isopropylidene-a-n-allofuranose 103.9 79.7' 75.6 79.1c 72.4 65.8 2,3 :5,6-Di-O-isopropylidene-a-~-mannofuranose 101.1 80.1' 79.7' 85.6 73.4 66.5 l,2 :3,4-DiO-isopropylidene-cr-~-galactopyranose 96.3 70.8" 70.6' 68.3" 71.5 62.1 Methyl 4,6Q-benzylidene-hexopyranosides a-All 100.2 67.9 68.8 78.1 56.9 68.8 a-Alt 101.6 69.6 68.8 76.0 57.8 68.8 99.4 70.8 68.6 76.5 62.8 68.6 8a-Gal 100.8 69.2' 69.5' 76.5 63.0 69.3 104.2 72.8" 71.2' 76.0 66.8 69.3 P99.9 72.4 70.5 80.8 62.0 68.5 a-Glc 104.2 74.2 72.9 80.3 65.9 68.3 8101.7 70.6 68.0 78.5 62.9 68.4 a-Man

C-7

0-Me

References

83,*100,101 83,*100 83 83 83 83 83,*100,101 99,*100 83

99,*100 83,*100 83 83 101.5 101.8 101.8 101.4 101.5 101.5 101.5 101.7

55.7 55.0 56.4 55.7 57.2 54.9 56.8 54.4

102,*128d 102 102 83 83 102 102 102

Additional data for related compounds are given in Refs. 90 and 103-106. For assignment of dependence of chemical shifts on ring size, see Refs. 89 and 90. Assignments may have to be reversed. In dimethyl sulfoxide-d,.


54

TABLEX

13C-N.m.r.Data for Aminodeoxy-, Deoxyhalo- and Thio-substituted Derivatives ~

Compound

~~

C-1

Aminodeoxy-D-pyraw se" o-Glucose a 2-, HCI 89.9 93.5 P Me a 2-,base 99.7 98.7 Me a 3-, base base 99.0 Me (Y 6, a 1-N-Acetyl 79.1 81.8 B a 2-N-Acetyl 92.1 96.2 P Me a 2-N-acetyl 98.6 102.3 P Mannose u 2-, HCI 91.1 91.8 P a 2-iV-A~etyl 94.3 91.3 P Galactose a 2-N-Acetyl 92.2 96.5 P 99.1 M e a 2-N-acetyl Thio-D-pyranoses' B 1-thio-Clc 85.1 73.9 B Sthio-Glc 64.8 a 6-thio-Fru 66.4 P Deoxyhalo-n-pyranoses (I 6-Cl-Gk 93.4 P 97.1

~

~~

C3

C4

C-5

55.3 57.8 54.9 71.7 71.6 71.9 74.3 55.3

70.5 72.8 74.1 54.1 73.1 75.6 80.0 72.0 58.0 75.2 54.3 71.9 56.1 74.6

70.5 70.5 69.7 69.6 71.2 71.9 71.8 71.4 712 70.4 70.9

72.4 76.9 71.7 71.3 712 75.2 79.0 72.8 77.2 72.2 76.3

61.3 61.3 60.6 60.6 41.4 63.1 63.1 61.9 62.0 61.4 61.5

55.3 67.7 56.4 70.3 54.4 70.1 55.3 73.2

67.1 67.0 68.0 67.8

72.8 76.9 73.2 77.5

61.2 61.2 61.7 61.7

51.4 68.6 54.9 72.3 50.8 68.7

69.7 69.0 69.4

71.6 76.3 71.6

62.4 622 62.1

71.4 76.0 73.3* 71.9"

80.6 43.9 68.W 71.7d

62.3 61.0 27.1 30.4

163 83 164 164

71.3 712

71.4 75.6

45.6 45.1

154 154

C-2

79.6 74.4 84.4 85.3

77.9 74.4 72.7d 70.1

72.5 73.6 752 76.5

C-6 OMelNAc References

23.3 23.5 55.6 57.2

42,62* 42,62* 81 81 81 159 159 159,160* 159,160* 161b 82,161b*

23.2 23.2

42,162; 42,162* 160 160

54.9 54.5 54.8

23.2 23.4

160 160 81

(continued)

(159) S . Shibata and H. Nakanishi, Carbohydr. Res., 86 (1980)316-320. (160) D. R. Bundle, H. J. Jennings, and I. C . P. Smith, Can.]. Chem., 51 (1973) 38123819. (161) A. S. Shashkov, A. Yu. Evstigneev, and V. A. Derevitskaya, Carbohydr. Res., 72 (1979) 215-217. (162) T. Yadomae, N. Ohno, and T. Miyazaki, Carbohydr. Res., 75 (1979) 191-198. (163) P. Friis, P. 0.Larsen, and C. E. Olsen,]. Chem. SOC., Perkin Trans. 1 , (1977)661665. (164) M. Chmielewski and R. L.Whistler, Carbohydr. Res., 69 (1979) 259-263.

55

13C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES TABLEX (continued)

Compound

C-1

a 2,6-Br2-Glc

93.1 96.6 101.5 Me B 2-Cl-Glc' Me a 2,6-Br2-Man 94.1 92.3 P 98.9 Me a 4-Cl-Gal' Me a 4,6-C1,-Gale 99.1 103.5 B"

P

C-2

C-3

C-4

C-5

C-6 OMe/NAc References

53.5 56.3 62.3 56.3 60.8 67.1 66.8 68.9

70.9 74.9 75.8 70.1d 71.8d 67.1 66.8 70.3

73.3 73.O 69.9 68.8 69.7 63.9 63.9 62.7

73.6 76.7 75.8 72.1d 75.7d 68.5 68.4 71.8

34.4 33.6 59.7 34.6 33.8 60.0 43.4 42.7

165 165 128 83 83 128 128 128

55.1 53.6 53.9 55.5

" Additional data for related compounds are given in Refs. 145 and 166. * Contains additional data. Additional data for related compounds are given in Refs. 167 and 168. Assignments may have to be reversed. In dimethyl sulfoxide-do. TABLEXI

13C-N.m.r.Data for Some Deoxy Sugars Compound

C-1

C-2

ZDeoxy-Whro-pentose 92.5 34.5 a-Pyranose 35.9 P94.6 98.9 41.9 a-Furanose" 98.9 41.8 P-" Deoxy-D-hexopyranoses 92.1 38.3 a-2-, arabino 94.1 40.5 P91.8 67.4 a&, ribo 69.7 P98.8 a4-,xylo 93.6 74.1" 76.9" P97.1 69.2 a-6-, Galacto 93.3 72.8 P97.3 a-6-,Gluco 93.1 72.9 75.6 P96.8 a&-, Manno 95.0 71.9 94.6 72.4 PDeoxy-whexofuranoses 97.4 73.9 a 3 - , ribo 102.6 76.5 P96.5 77.0 a-5, rylo '77.0 P102.5 a

C-3

C-4

C-5

65.1" 67.3" 71.7 72.0

68.1" 68.3" 86.1 86.6

66.8 63.6 62.3 62.3

68.8 71.4 34.7 39.3 69.3" 73.2" 70.4 74 .O 73.6 76.6 71.1 73.8

72.0 71.7 653 65.3 35.1 35.1 73.0 72.5 76.4 76.1 73.3 72.9

72.8 76.8 73.1 82.8 67.8" 71.3' 67.4 71.9 68.6 73.0 69.4 73.1

61.6 61.9 61.6 61.9 64.6 64.5 16.7 16.7 18.0 18.0 18.0 18.0

62 62 60 60 83 83 37,40,46* 37,40,46* 46 46 37,46.*130,"148 37,46,*130,"148

31.9 33.7 77.0 76.1

77.6 78.O 81.6 79.8

71.6 73.9 31.9 32.6

63.5 63.8 59.5 59.5

60 60 83

C-6

References

83,*137 83,*137 83,*137 83,*137

Assignments may be reversed. In pyridined,.

(165) K. Bock, I. Lundt, and C. Pedersen, Carbohydr. Res., 90 (1981)7-16. (166) B. Paul and W. Korytnyk, Carbohydr. Res., 67 (1978)457-468. (167) J. E. N. Shin and A. S. Perlin, Carbohydr. Res., 84 (1gSO) 315-327. (168) J. E. N. Shin and A. S. Perlin, Carbohydr. Res., 76 (1979) 165-176.

83


S6

TABLEXI1 %-N.rn.r. Data for Methyl Deoxypyranosides Compound DPentopyranosides a-2-Deoxy+m~thro

P j3-2-Deoxy-threo P-3-Deoxy-erythro a-2,3-Dideoxy-glycero

P a3,4-Dideoxy-glycero

P DHexopyranosides a-2-Deoxyurabino

C-1

C-2

C-3

C-4

101.3 99.6 101.5 104.7 99.8 100.3 102.3 104.9

34.6 33.1 372 67.8 27.9 26.0 70.1 68.8

67.9" 65.0" 70.8 36.4 27.4 26.0 28.5" 29.0"

67.4" 689" 702 65.0

65.3 63.6 64.8 68.0 652 66.2 65.1 66.0 25.7" 62.5 23.3" 64.8

100.8 103.2 98.9 106.1 101.4 108.3 103.1 102.4 104.5 100.3 104.3 101.9

39.1 40.7 67.1 68.5 65.8 68.3 67.4 75.6 75.8 72.6 74.5 71.0

70.8 72.9 35.3 39.1 35.7 39.7 33.3 71.1 71.2 73.9 76.7 71.3

73.6 73.6

C-5

OMe

References

56.8 55.6 57.0 57.0 55.7 55.9 57.8 56.9

169 169 169 169 169 169 169 169

56.9 169 59.1 169 a3-Deoxy-n'bo 65.0 55.6 169 B 652 57.7 169 a3-Deoxy-xylo 68.5 57.5 169 P 68.0 59.3 169 u3-Deoxyf yxo 682 57.3 169 a-4-Deoxy-xy lo 36.6 57.5 169 B 35.1 57.9 169 a-6-Deoxy-gluco 76.2 56.2 46 B 76.2 58.3 46 a-6-Deoxy-manno 73.1 55.8 46,*130,b133, 134,145, 147,169,170 B 102.0 71.2 73.0 73.0 73.6 17.6 57.6 130,b169,* 170 a-6-Deoxy-galacto 100.5 69.0 70.6 72.9 67.5 16.5 56.3 46,*82,171 P 104.8 71.5 74.1 72.4 71.9 16.5 58.3 46,*171 a-6-Deoxy-altm 101.3 70.9" 70.9" 70.7" 66.9 17.2 56.3 83 a-2$-Dideoxyerythro 98.1 29.0 26.9 66.0 74.3 61.8 54.9 169 103.5 30.3 30.3 66.1 80.6 62.2 57.0 169 P a-2,3-Dideoxy-threo 98.9 25.4" 23.7" 64.9 71.9 62.9 55.1 169 a3,4-Dideoxy-erythro 100.0 69.8 26.0 26.3 68.5 64.8 55.6 169 106.5 69.9 30.2 26.7 77.4 64.7 57.5 169 B

P

74.6 78.6 73.2 80.5 73.1 80.9 74.0 69.7 73.3 68.7 73.0 69.4

C-6

63.3 63.6 61.5 61.8 64.0 63.8 64.4 66.2 64.5 17.6 17.8 17.7

(continued)

(169) L. Wiebe, Ph.D. Thesis, Danmarks Tekniske Hq~jskole,Lyngby, 1976. (170) L. V. Backinowsky, N. F. Balan, A. S. Shashkov, and N. K. Kochetkov, Cnrbohydr. Res., 84 (1980) 225-235. (171) J.-H. Tsai and E. J. Behrman, Carbohydr. Res., 64 (1978) 297-301. (172) V. Pozsgay and A. Neszrnelyi, Carbohydr. Res., 85 (1980) 143-150. (173) 6. Monneret, C. Conreur, and Q. Khuong-Huu, Carbohydr. Res., 65 (1978)3545. (174) D. R. Bundle,J. Chem. SOC., Perkin Trans. 1 , (1979) 2751-2755. (175) D. R. Bundle and S. J. Josephson, Can. J . Chem., 56 (1978) 2686-2690.


57

TABLEXI1 (continued) Compound

C-1

C-2

C-3

C-4

102.9 67.3 105.0 67.7 a-2,6-Dideoxy-arabim 98.9 37.7

26.7 30.1

22.8 23.1 77.4

a3,4-Dideoxy-threo

P P

a3,6-Dideoxy-ribo

P a3,6-Dideoxy-xylo

P a3,6-Dideoxyarabim a4,6-Dideoxy-xylo

P a4,6-Dideoxy-ribo

P

a4,6-Dideoxy-lyxo

103.2 99.1 106.2 99.9 106.7 100.8 102.6 106.1 100.8 102.1 101.9

68.7

79.0 72.7 35.4 70.6 39.1 70.5 33.9 69.2 38.1 69.1 34.6 70.7 69.9 42.6 73.1 42.4 68.1 39.1 68.6 39.5 66.0 36.2

41.0 67.4 68.8 63.9 66.4 68.4 75.7 77.5 68.5 71.7 68.8

a Assignments may be reversed. data.

C-5

C-6 OMe

72.2 67.0 79.2 66.8 68.7 17.8

57.1 58.7 55.3

74.5 69.2 76.6 67.0 74.9 68.0 67.3 71.2 60.7 68.1 64.3

59.1 55.6 57.8 55.8 57.6 55.7 57.7 59.7 56.3 57.7 54.7

19.4 17.3 17.6 16.2 16.5 17.9 22.5 22.4 20.4 20.7 21.1

In dimethyl sulfoxided,.

References 169 169 165,*172, 173' 169 169 99,b169,*174 83,*175 83 172,175* 169 169 169 169 172

Contains additional

TABLEXI11 13C-N.m.r.Dataa for Methyl Anhydro-D-glycosides

Compound D-Pentopyranosides a-2,3-Anhydro-lyxo P-2,3-Anhydro-ribo D-Pentofuranosides a-2,3-Anhydro-ribob

C-1

C-2

C-4

C-5

96.4 51.1 56.8 96.3 52.7 52.4

61.4 62.3

59.9 61.9

56.4 56.6

83 83

56.0 55.4 55.6 55.1

78.5 78.6 76.6 76.4

61.5 61.7 59.6 59.6

55.3 54.8 54.6 55.7

91 91 91 91

52.7 54.7 52.9 54.1

76.8 76.4 73.8 73.7

59.9 60.5 61.5 67.8

67.8 68.0 68.3 68.3

54.9 55.8 55.1 56.3

91 91 91 91

67.1 62.1 67.7 61.9

56.3 56.1

83 83

101.1 55.1 101.7 54.8 a-2,3-Anhydro-lyxob 101.5 53.7 Pb 101.8 54.4 4,6-O-Benzylidene-hexopyranosides a-2,3-Anhydro-alld 94.6 49.9 Pb 97.1 50.6 a-29-Anhydro-mannob 96.0 49.8 Pb 99.1 50.1 D-Hexopy ranosides a3,4-Anhydro-galado 97.0 64.4 a3,4-AnhydK1-t& 100.5 65.4

Bb

OMe References

C-3

54.0" 51.8" 52.6" 52.0"

C-6

Additional data for related compounds are given in Ref. 176. In dimethyl sulfoxided,. Assignments may be reversed. (176) M.Chmielewski, J. Mieczkowski, W. Priebe, A. Zamojski, and H. Adamowich, Tetrahedron, 34 (1978) 3325-3330.

58

KLAUS BOCK AND CHRlSTIAN PEDERSEN TABLEXIV

W-N.m.r. Data” for Unsaturated Carbohydrate Derivatives Compound

C-1 ~~

C-2

C-3

C-4

C-5

C-6

OMe

References

~ _ _ _ _

mGlycals 146.6 101.4 63.7’ 68.4’ 65.8 Xylal GDeoxygfucal 144.6 104.4 6 9 9 74.ab 75.7 17.2 Allal 146.2 101.3 62.5 67.0 74.8 61.3 Calactal 144.8 103.4 64.8 65.5 77.9 62.1 Glucal 144.6 103.8 69.7’ 69.2* 79.1 61.0 Methyl 4,6-O-benzylidene-~-hex-%enopyranoses aerythro 96.3 130.9 126.9 75.5 64.2 69.6 56.0 P 97.8 130.1’ 126.8’ 73.6 69.0 76.6 53.3 Methyl 40-acetyi-mpent-Zenopyranosides a-glycero 95.1 129.P 128.7b 64.9 60.1 55.7 P 94.2 130.9 125.1’ 63.4 61.3 55.6 Methyl 4,6-di-O-acetyl-~-hex-%enopyranosides 95.3 129.1’ 127.8’ 65.3 66.9 63.0 55.6 aerythro 63.9 63.0 54.9 72.4 95.6 129.8 125.9 P 62.6 55.2 94.8 130.4 125.0 66.6 62.6 a-threo Methyl U)-acetyl-3,4-dideoxy-~-pent-3-enopyranosides a-glycero 96.1 66.5 121.8 129.3 602 56.1 P 98.9 66.0 120.4 1312 59.4 55.9 Methyl 2,6di-O-acety13,4dideoxy-~-hex-3-enopyranosides aerythro 96.0 66.5‘ 124.3 127.9 66.8’ 65.3 56.0 56.1 71.6 65.9 B 100.2 67.3 124.7 1292 66.3 65.4@ 56.0 98.9 65.3* 122.4 130.8 a-threo 71.8 65.8’ 56.6 98.0 65.1’ 124.2 130.0 P 6-Deoxy-l,2 :3,4di-O-isopropylidene-~-~-am~no-hex-5-enopyranose 97.3 73.2 70.9 72.V 152.4 100.4 Methyl 5,6dideoxy-2~-isopropylidene-a-~-Z~-hex-5-enofuranoside 1072 81.6b 81.P 85.4 132.4 119.1

83 83 177 83 177 83 83 83 83 83 83 83 178 178 178 178 178 178 179 180

” Additional data for related compounds are given in Refs. 176,181,and 182. * Assignments may be reversed.

(177) A. I. R. Burfitt, R. D. Guthrie, and R. W. Irvine,Aust.J. Chem., 30 (1977) 10371043. (178) M.Chmielewski, A. Banaszek, A. Zamojski, and H. Adamowicz, Carbohydr. Res., 83 (1980)3-7. (179) B. Coxon and R. C. Reynolds, Carbohydr. Res., 78 (1980) 1-16. (180) K. Bock and C. Pedersen, Acta Chem. S c a d . , Ser. B, 31 (1977) 248-250. (181) R. D. Guthrie and R. W. Irvine, Carbohydr. Res., 82 (1980)207-224. (182) R. D. Guthrie and R. W. Irvine, Carbohydr. Res., 82 (1980) 225-236. (183)W. Funcke and C. von Sonntag, Carbohydr. Res., 69 (1979)247-251. (184) G. W. Schnarr, D. M. Vyas, and W. A. Szarek, J . Chem. SOC., Perkin Trans. 1 , (1979)496-503. (185) P. Finch and Z. M. Merchant, Carbohydr. Res., 76 (1979)225-232.

13C-N.M.R. SPECTROSCOPY O F MONOSACCHARIDES

59

TABLEXV '3C-N.m.r. Dataa for Some Acyclic Monosaccharide Derivatives Compound

C-1

C-2

C-4

C-3

C-5

C-6

OMe

References

75.46 74.8 72.6b 73.1b 73.7b 72.9 70.7 67.6

73.6b 64.5 73.0 64.6 72.3b 71.8b 64.2 64.2 71.7b 71Zb 64.8 64.8 73.4b 72.6 64.5 73.2b 72.3b 64.5

61.6 61.9 61.3 61.8 61.8 61.7 61.9 61.9

183 183 183 183 183 183 183 183

70.5 70.8 69.7* 69.8 69.8b

71.4b 72.6 69.4b 72.1 69.4b

63.6 62.8 70.0 71.4 71.4

63.2 63.4 63.8

67.6

72.5

71.3

63.2

~~

D-, 0-Methyloximes syn, Rib 152.2

71.4 67.7 anti, Rib 153.4 151.5 71.4 syn, Glc anti, Glc 153.5 67.5 69.8 syn,Gal 153.0 anti,Gal 155.2 66.0 syn, Fru 56.1 161.9 anti, FN 61.6 162.6 D-,Diethyl dithioacetals 54.5 71.6b Arac 54.4 74.2 xyl" 54.7 71.6 GalC 54.1 75.3 Glc" 55.0 73.8 ManC D-, Dimethyl acetals Glc" 104.1 73.6

184 184 184 184 184 53.1, 54.5

184

Additional data for related compounds are given in Ref. 185. Assignments may be reversed. In dimethyl sulfoxided&.

TABLEXVI W-N.m.r. Data for Alditols and Their Acetates Compounds Hexitols Allitol Altritol Galactitol Glucitol Iditol Mannitol Pentitols Arabinitol Ribitol Xylitol

C-1

C-2

C-3

C-4

C-5

C-6

References

63.7 64.4 64.5 63.8 64.1 64.6

73.5 71.8 71.5 74.3 73.1 72.2

73.7 72.2 70.7 71.0 72.5 70.7

73.7 73.0 70.7 72.6 72.5 70.7

73.5 74.0 71.5 72.5 73.1 72.2

63.7 63.4 64.5 64.2 64.1 64.6

184,"186* 184,"186* 184,a186,*187 154,184,"186,*188 184,"186* 184," 186,*187

64.4 63.8 63.9

71.6 73.5 73.2

71.9 73.6 72.0

72.3 73.5 73.2

64.3 63.8 63.9

184,"186,*187 154,184,a186,*187 184,a186,*187 (continued)

(186) S. J. Angyal and R. Le Fur, Carbohydr. Res., 84 (1980) 201-209. (187) W. Voelter, E. Breitmaier, G. Jung, T.Keller, and D. Hiss, Angew. Chem., 82 (1970) 812-813. (188) A. P. G. Kieboom, A. Sinnema, J. M. van Der Tom, and H. von Bekkum, Red. Trau. Chim. PUYS-BUS,96 (1977) 35-37.


60

TABLEXVI (continued) ~

Compounds

C-1

Tetritols Erythritol 64.0 Threitol 63.9 Other alcohols Glycerol 64.0 Ethylene glycol 63.8 Hexitol acetates Allitol 61.8 Altritol 62.1 Galactitol 62.3 Clucitol 62.0 Iditol 61.8 Mannitol 62.0 Pentitol acetates Arabinitol 62.1 Ribitol 61.8 Xylitol 62.0 Tetritol acetates Erythritol 61.9 Threitol 62.0 Other alcohol acetates G lycero 1 62.4 Ethylene glycol 62.4 'I

~~

C-2

C-3

C-4

C-5

73.3 72.9

73.3 72.9

64.0 63.9

73.5 63.8

64.0

69.7 68.4 67.8 69.6 69.3 68.1

69.4 69.1 67.7 68.7 68.9 67.7

69.4 68.7 67.7 69.0 68.9 67.7

69.7 70.0 67.8 68.9 69.3 68.1

68.3 69.6 69.4

68.6 69.4 69.3

68.3 69.6 69.4

61.9 61.8 62.0

69.4 69.4

69.4 69.4

61.9 62.0

69.4 62.4

62.4

C-6

References

184,"186,*187 184,"186* 184:186,*187 186,*187 61.8 61.7 62.3 61.6 61.8 62.0

186 186 186 186 186 186 186 186 186 186 186 186 186

In dimethyl sulfoxided,. TABLEXVII

13C-N.m.r. Data'' for Anhydroalditols _ _ _ _ ~ ~ Compound C-1 c-2 C-3 C-4 C-5

C-6

References

1,4Anhydrohexitols Allitol 72.9 Altritol 74.1 Galactitol 73.7 Glucitol 74.3 Gulitol 72.2 iditol 72.9 Mannitol 71.9 Talttol 73.7

63.4 64.0 63.9 64.5 63.8 63.3 64.O 64.3

27,189* 189 189 189 27,189* 189 27,189* 189

72.1 78.3 77.9 77.3 72.3 77.2 72.3 72.5

72.9 79.1 79.3 76.8 71.3 76.5 71.2 73.3

82.9 86.5 85.8 80.8 81.3 80.9 81.1 82.1

72.5 72.7 72.3 70.3 71.6 71.3 70.3 72.2

(continued ) (189) H. Thggersen, Ph.D. Thesis, Danmarks Tekniske Hgjskole, Lyngby, 1980. (190) B. Matsuhiro and A. B. Zanlungo, Carbohydr. Res., 63 (1978) 297-300. (191) J. C. Goodwin, J. E. Hodge, and D. Weisleder, Carbohydr. Res., 79 (1980) 133141.

61

I3C-N.M.R. SPECTROSCOPY OF MONOSACCHARIDES TABLEXVII (continued) Compound

C-1

174-Anhydropentitols 74.1 Arabinitol Lyxitol 72.1 Ribitol 73.1 Xylitol 73.6 1,CAnhydrotetritols 72.3 Erythritol Threitol 73.8 2,!j-Anhydrohexitols 62.9 Allitol Altritol 61.3 Galactitol 60.5 Glucitol 61.1 Iditol 61.0 Mannitol 61.9 1,SAnhydrohexitols 66.1 Allitol Altritol 67.1 Galactitol 70.1 Glucitol 69.8 Gulitol 65.8 Iditol 68.2 Mannitol 70.8 Talitol 71.6

C-2

C-3

C-4

C-5

C-6

References

77.8 72.1 71.9 77.2

79.0 71.4 72.5 77.6

86.5 81.7 82.7 81.8

62.5 61.3 62.3 60.8

71.8 77.2

71.8 77.2

72.3 73.8

84.4 81.5 79.9 81.9 81.3 83.2

72.4 72.5 71.6 77.9 77.6 77.3

72.4 72.7 71.6 79.0 77.6 77.3

84.4 82.1 79.9 85.7 81.3 83.2

62.9 62.5 60.5 62.4 61.0 61.9

189 189 189 27,189* 27,189* 27,189*

66.8 70.4 67.4 70.4 66.6 70.1 70.0 70.1

70.0 70.4 75.1 78.4 71.0 70.8 74.5 70.6

67.1 65.8 70.1 70.7 71.0 70.6 68.2 69.2

77.0 77.2 80.4 81.3 76.6 77.4 81.5 80.4

62.2 62.3 62.3 61.5 62.4 62.0 62.1 63.4

27,189* 27,189* 27,189* 27,189* 189 189 27,189,*190 27,189*

27,189* 27,189* 27,189* 27,189* 27,189* 27,189*

Additional data for related compounds are given in Ref. 191.

TABLEXVIII I3C-N.m.r. Data for Aminodeoxydditols and Aminoanhydrodeoxydditols18g Compound

c-1

c-2

c-3

1-Amino-1-deoxy-D-hexitol hydrochloride 43.6 67.2 71.2 Galactitol Mannitol 43.1 67.6 71.2 1-Amino-1-deoxy-wpentitol hydrochloride 43.2 66.9 71.7 Arabinitol Lyxitol 43.0 67.7 72.3 Ribitol 41.6 68.2 73.1 Xylitol 42.6 68.3 72.1 5-Amino-l,Panhydro-5-deoxy-~-pentitol hydrochloride 74.6 77.5 80.0 Arabinitol Lyxitol 72.0 71.5 71.1 Ribitol 74.5 72.1 73.7 Xylitol 74.1 76.9 78.1

C-4

c-5

C-6

70.0 69.3

70.7 71.0

64.O 63.5

71.1 70.4 72.1 72.1

63.3 63.4 62.8 62.9

82.4 75.9 78.3 77.9

42.4 39.8 42.7 40.4

KLAUS BOCK AND CHRlSTIAN PEDERSEN

62

TABLEXIX 'T-N.m.r. Data for Uronic Acids or Uronolactones Compound

C-2

C-1

C-4

C-3

C-5

C-6

DGlucopyranumnic acid 72.4 71.4 172.9 93.2 72.0 73.4 75.4 173.8 72.2 13 96.9 74.7 76.3 76.9 176.9 73.0 a (pH 7.8) 92.9 72.2 73.5 72.6 177.6 72.7 96.7 75.0 76.5 P Methyl D-ghcopyranosiduronic acid and methyl ester 100.7 71.9 73.8 72.5 71.9 &-Acid a-Ester 100.8 71.9 73.7 72.4 71.9 @-Acid 104.3 73.8 76.5 72.3 75.6 " P-Ester 104.6 73.7 76.3 72.4 75.7 'I D-Glucofuranurono-6,3-lactone ff 99.1 74.8 85.6 76.7 70.4 177.8 P 103.7 74.8 85.6 78.4 70.1 177.9 D-Gdactopyranuronic acid a 93.2 68.7 69.5 70.9 70.5 172.6 B 97.0 72.1 73.1 70.9 74.8 173.5 Methyl (methyl a-mannopyranosid)uronate a 102.3 70.4 71.1 69.2 72.9 a Methyl 2-hexulosonate a-D-urubino 170.6 97.0 70.6' 71.7b 65.7* 63.5 P 170.6 97.0 69.3 69.5 69.1 64.7 a-L-xy lo 170.5 96.6 72.8 73.7 69.5 62.8

0-Me

59 59 59 59

a (pH 1.8)

_

_

-~ ~

~

References

56.7 56.8,54.2 58.5 58.7,56.2

46 46,*192 46 46 59 59 83,*127 83,*127

56.534.1

46

53.5 53.9 53.9

193 193 193

('Not resolved. * Assignments may b e reversed. (192) A. S. Shashkov, A. F. Sviridov, 0. S. Chizhov, and P. KovaC, Carbohydr. Res., 62 (1978) 11-17. (193) T. C. Crawford, G. C. Andrews, H. Faubl, and G. N. Chmumy,]. Am. Chem. SOC., 102 (1980) 2220-2225. (194) H. S. Isbell and M. A. Salam, Carbohydr. Res., 90 (1981)123-126. (195)W. Kondo, F. Nakazawa, and T. Ito, Carbohydr. Res., 83 (1980) 129-134. (196) M. Chmielewski, Tetrahedron, 36 (1980) 2345-2352. (197) S. Berger, Tetrahedron, 33 (1977) 1587-1589. (198) T. Ogawa, J. Wzawa, and M. Matsui, Carbohydr. Res., 59 (1977) c32-c35. (199) G . Schilling and A. Keller,]ustus Liebigs Ann. Chem., (1977) 1475-1479. (200) D. M. Vyas, H. C. Jarrell, and W. A. Szarek, Can.J. Chem., 53 (1975) 2748-2754. (201) A. K. Bhattacharjee, H. J. Jennings, and C. P. Kenny, Biochemistry, 17 (1978) 645 -651. (202) R. Cherniak, R. G. Jones, and D. S. Gupta, Carbohydr. Res., 75 (1979)39-49. (203) V. Eschenfelder, R. Brossmer, and H. Friebolin, Tetrahedron Lett., (1975)30693072. (204) H. J, Jennings and A. K. Bhattacharjee, Curbohydr. Res., 55 (1977) 105-112. (205) L. W. Jaques, B. F. Riesco, and W. Weltner, Jr., Carbohydr. Res., 83 (1980)21-32. (206) M. F. Czamiecki and E. R. Thomton,]. Am. Chem. SOC., 99 (1977) 8273-8279. (207) J. M. Beau, P. Sinay, J. P. Kamerling, and J. F. G. Vliegenthart, Carbohydr. Res., 67 (1978) 65-77.

63


TABLEXX 13C-N.m.r. Datan for Salts of Aldonic Acids and for Aldonolactones Compound

C-1

C-2

C-3

C4

D-Aldonic acid salts (pH 14) Allonic 179.5 74.7b 74.6b 73.6b Altronic 180.5 74.W 73.9 72.7b Galactonic 180.6 72.4b 72.4b 71.1b Gluconic 179.8 75.2 72.4 73.8 Gulonic 180.4 75.6b 73.9 73.6b Idonic 179.5 73.8 73.2b 72.5b Mannonic 180.3 75.4b 72.2b 72Jb Talonic 180.7 75.3b 74.0" 72.6b Arabinonic 180.5 73.4b 72.6b 72.3b Lyxonic 180.0 75.1b 72.9 72.3b Ribonic 179.9 759 74.6b 73.W Xylonic 180.4 74.2b 74.V 73.6 Erythronic 179.6 74.4b 74.2b 62.8 Threonic 179.4 73.V 73.1b 63.1 Glyceric 179.2 74.4 64.9 D-Hexono-,. .pentono-, and tetrono-1.4lactones Allono 178.7 86.9 70.9 69.5b 81.3 Altrono 176.8 74.8b 73.3b 80.9 74.5b 73.7b Galactono 176.7 Glucono 177.9 73,4b 73.8b 80.5 71.7b 71.V Gulono 178.8 822 Idono 176.5 74.W 71.9 79.9 Mannono 178.8 79.3 71.5b 70.2b Talono 179.3 71.2b 71.V 86.9 Arabinono 176.9 82.0 74.6b 73.2b 82.4 71.3b 70.3b Lyxono 179.0 Ribono 179.3 70.3b 69.8b 87.5 73.9 72.9 Xylono 177.9 812 Erythrono 179.3 73.7 70.5 69.7 Threono 178.0 74.0 73.1 70.4 D-Glucono-1,S-lactone 174.5 82.3b 73.4b 71.7b n-Gluconic acid (pH-3) 176.5 73.1b 72.3b 71.9

C-5

C-6

References

72.7b 72.6b 70.7b 72.0 71.7 71.9 71.6b 72.0 64.1 64.0 63.9 63.6

63.4 63.3 64.3 63.6 63.6 63.9 64 .O 64.4

83 83 83 83,*194 83 83 83 83 83 83 83 83 83,*43 83,*43 195,*43c

69.W 71.2b 69.8b 71.2b 70.4b 68.8b 68.5b 69.4b 60.1 60.6 61.4 59.9

62.8 62.3 62.9 63.2 62.4 62.9 63.4 62.7

83 83 83 83 83 83 83 83 83 83 83 83 83 83

67.9

60.8

83

71.4b

63.5

83

Additional data for related compounds are given in Ref. 196. Assignments may be reversed. Data for acid at pH -3. For further data, see p. 66. (I

(208) Y. Terui, K. Ton, K. Nagashima, and N. Tsuji, Tetrahedron Lett., (1975) 25832586. (209) J. Boivin, M. Pai's, and C. Monneret, Carbohydr. Res., 64 (1978) 271-278. (210) K.-I. Harada, S. Ito, and M.Suzuki, Carbohydr. Res., 75 (1979) ~17-c20. (211) K. Olsson, 0. Theander, and P. h a n , Carbohydr. Res.. 58 (1977) 1-8. (212) S. Mizsak, G. Slomp, A. NeszmBIyi, S. D. Gem, and G. Lukacs, Tetrahedron Lett., (1977) 721-724.

C

CA

TABLEXXI

0" R

W-N.m.r. Data for Some Biologically Significant Monosaccharides Compound

c-1

c-2

c-3

c-4

c-5

C-6

c-7

C-8

c-9

References

r2

U

L-Ascorbic acid

174.2

118.9

156.6

77.3

60.0

63.4

197,*198

97.8 101.5 94.8 95.3

78.3 81.2 75.3 75.7

70.6 71.6 76.9 66.6

81.6 82.5 68.9 68.6

62.9 63.6 65.6 63.6

61.3 62.9 61.1 63.4

199 199 199 199

g

106.1 84.3 82.2 3-Deoxy-D-manno-octulosonicacid, sodium salt a-Pyranose 177.9 97.6 34.8 Me a-Pyranoside 176.5 102.5 35.2 P 174.8 102.4 35.5 A'-Acetyl-D-neuraminic acid, methyl pyranoside 41.0 174.1 101.6 a Acid 40.8 176.1 101.4 P 39.7 170.7 100.1 a Me ester 40.1 171.2 100.1 P

62.3

73.2

200

m

67.8" 67.4" 68.6"

67.4" 67.1" 66.5"

72.4 72.5 74.6

70.5 70.5 70.3

64.2 64.2 65.2

51.9 52.9

201,*202 201 201

69.0 67.1 69.0 67.2

52.9 53.1 52.8 52.6

73.4" 71.1" 73.8" 71.5"

69.2" 69.5" 69.2" 69.0"

72.6" 71.1" 71.5" 70.8"

63.6 64.5 64.0 64.3

203*-206 202,*205-207 203,*204,206 203,*204,206

D-Hamamelose a-Furanose

B a-Pyranose

B

2

3 U

1,u)-Isopropylidene-a-apiose

z

n

Methyl 2,6-dideoxy-3-C-methyl-3-O-methyl-~-ribo-hexopyranoside~ a 98.8 37.8 74.9 78.0 70.8 18.2 P 97.5 35.2 73.0 78.0 64.5 17.9 Methyl 2-deoxy-3C-methyl-a-D-n'bo-hexopyranoside 98.2 40.5 69.4" 71.2 69.7" 62.9 Methyl 4,~-benzylidene-2-deoxy-2C-methyl-~-methyl-~-mannopyranoside a 104.2 37.6 76.6 79.1 63.8 69.1 P 103.8 38.1 79.5 78.9 67.6 68.9 Methyl 3-amino-2,3,6trideoxy-fl-~qlo-hexopyranoside 99.1 34.6 49.7 72.2 69.3 16.5 Methyl 3-acetamido-2,3,6trideoxyhexopyranoside a-tmrabino 97.6 35.0 48.1 74.2 68.6 17.0 a-dyxo 98.2 30.2 45.5 69.9 65.9 16.8 P-L-ribo 99.1 33.5 46.3 72.2" 71.8" 18.6 Methyl 3,4,6-trideoxy-3-(dimethylamino)-~qb-hexopyranoside a 99.6 68.7 60.3 29.3 64.8 21.2 P 104.9 69.9 65.4 28.8 69.5 21.2 Thioglycosides 82.5 80.9 78.1 73.0 70.1 61.7 Ally1 glucosinolatec Lincomycind 89.2 68.8 71.4 69.5 70.0 54.9 N-acetyl-" 88.2 68.8 71.3 69.4 69.5 53.8

21.1 21.9

208 208

25.6

118

54.7 56.9

11.0 5.7

116 116 209

56.0

6

2

+ v)

54.6 54.8 55.9

209,210* 209 209

55.0 56.5

39.9 40.3

67.4 65.7

17.2 20.6

208 208 14.2 13.3

163 212 212

" Assignments may have to be reversed. Additional data for related compounds are given in Refs. 115 and 117. Additional data for related compounds are given in Ref. 211. The carbon chemical-shifts for the pyrrole ring of lincomycin hydrochloride are given in Ref. 212. Pyrrole ring substituted with an N-acetyl group.

2

3

a8 cc

%

5

n Ec

$ i U E

66

KLAUS BOCK AND CHRISTIAN PEDERSEN ADDENDUM

For 'SC-n.m.r. data (Table 111) on B-Sorp and B-Solf, see Ref. 213. For IS-n.m.r. data on D-xylono- and D-mannono-1,5-lactone, and on the four Daldopentonic acids at pH 1-3 (Table XX), see Refs. 214-217.

(213) G.-J. Wolff and E. Breitmaier, Chem. Ztg., 103 (1979) 232-233. (214) A. S . Serianni, H. A. Nunez, and R. Barker,]. Org. Chem., 45 (1980)3329-3341. (215) D. Horton and Z . Waiaszek, Cnrbohydr. Res., 105 (1982) 95-109; 111-129. (216) Z. Wdaszek and D. Horton, Carbohgdr. Res., 105 (1982) 131-143. (217) 2.Wdaszek, D. Horton, and I. Ekiel, Cnrbohydr. Res., 193-201.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 41

STRUCTURAL CHEMISTRY OF POLYSACCHARIDES FROM FUNGI AND LICHENS

BY ELIANABARRETO-BERGTER Departamento de Microbiologia Geral, Uniuersidade Federal do Rio de Janeiro, Brazil

AND

PHILIPA. J. GORIN

Prairie Regional kboratory, National Research Council, Saskatoon, Saskatchewan S7N OW9, Canada

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. a-&Linked Glucans . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . 1. Amylose . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 2. Glycogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pseudonigeran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Glucans Containing a - ~ - ( 1 + 3 )and cw~(1-A)Linkages . . . . . . . . . . . . 5. Pullulan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. P-D-Linked Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Linear /3-D-Glucans 3. Branched-chain P-DIV. Glucans from Lichens

.. .

.. . .. . . . .

.

. .. .

1. Linear Mannans

68

. 68 68 69 69 70 72 72 72

.... . . . . . . ..... ,. .. . .

VI. Galactans . . . VII. 2-Acetamido-2 VIII. 2-Amino-2-deo 1. Rhamnomannans . . . ....................... 89 2. Glucomannans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 3. Galactomannans . . . . . . . . . . . 92 4. Miscellaneous Heteropolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 X. Heteropolysaccharides Based on Galactan Main-Chains . . . . . . . . . . . . . . . 100 1. Glucogalactans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 100 2. Fuco(manno)galactans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Miscellaneous Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 67

Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12007241-6

68

E. BARHETO-BERGTER AND PHILIP A. J. GORIN

I. INTRODUCTION In this Chapter, the authors discuss the chemical structures of the polysaccharides of fungi and lichens investigated from 1967 to the middle of 1980. Work conducted on fungal polysaccharides before 1967 was covered earlier by Gorin and Spencer.' That article and the present one consider the same classes of fungi, except for lichens. Herein, it is convenient to group the polysaccharides in terms of their chemical structures, according to the nature of the component sugars, the predominant linkage and configuration, and, in the case of heteropolymers, the nature of the main chain. Investigations that provided few structural details are not included, except when an unusual structural feature is concerned. Also not discussed are biosynthesis, industrial utilization, and morphological location when related to the immunology of polysaccharides. The chemistry and biochemistry o f fungal polysaccharides have been periodically reviewed.2 Other reviews have appeared on the cell-wall chemistry, morphogenesis, and taxonomy of fungi,3 fungal cell-wall glycoproteins and peptido-polysaccharides,4 and the chemical composition, organization of polymer, and biosynthesis in the cell wall.5 Summaries have also appeared on the use of proton-n.1n.r. spectra of yeast polysaccharides in identification and classification of yeasts," and on 13C-n.m.r. spectroscopy of fungal polysaccharides, as an aid in monitoring the formation of polysaccharides and in their structural identificati~n.~ 11. CY-D-LINKED GLUCANS

1. Amylose A11 reports on starchlike polymers cannot be covered herein. However, the cell walls of Fusicoccum amygdali are stained blue with iodine and attacked by alpha amylase.x Extracts of Sporothrix schenckii

(1) P. A. J. Gorin and J. F. T. Spencer, Ado. Carbohydr. Chem., 23 (1968)367-417. (2) Carbohydr. Chem., Spec. Period. Rep., 3 (1970)236-243; 5 (1972)266-281; 6 (1973) 261-273; 7 (1975)282-293; 8 (1976)254-261; 11 (1979)299-308. (3) S. Bartnicki-Garcia,Annu. Rev. Microbiol., 22 (1968)87-108. (4) J. E. Gander,Annu. Reo. Microbiol. 28 (1974) 103-119. (5) R. F. Rosenberger, in J. E. Smith and D. R. Deny (Eds.),The Filamentous Fungi, Vol. 2, Wiley, New York, 1976, pp. 328-344. (6) P. A. J. Gorin and J. F. T. Spencer, Ado. Appl. Microbiol., 13 (1970)25-89. (7) P. A. J. Gorin, ACS Symp. Ser., 126 (1979) 159-182. (8) K. W. Buck and M. A. Obaidah, Biochem. I., 125 (1971)461-471.

POLYSACCHARIDES FROM FUNGI AND LICHENS

69

and Ceratocystis stenoceras contain 4-0-substituted D-glucopyranosyl units, and the solutions give a blue color with i ~ d i n e . ~

2. Glycogen Glycogens have been isolated from Candida albicans,tOBlastocladiella emersonii," Neurospora crassa,12Allomycesrnacrogynu~,'~ Rhizophydium sphaerotheca, and Monoblepharella elongata.l4 They have p-amylolysis values of 4 5 4 5 % and average chain-length values of 11-14 D-glucosyl units. A c.l. decrease of 15 to 9 corresponded to the different developmental stages of B . emersonii during the 12-20-h culture age-period of resistant, sporangial plants." Polyp o n s circinatus glycogen from young cells hast5a B-amylolysis value of 34%, which decreases with age to 23% (with a x value of 6).

(x)

3. Pseudonigeran Pseudonigeran, isolated following alkaline extraction of cell walls ofAspergillus niger, is a (1-+3)-linked a-D-glucopyranan, as shown by methylation, periodate, and partial-hydrolysis studies. The hot-waterinsoluble polymer contains only 1% of ( 1 - 4 linkages, which are present in the polymer, or are from contaminating material, such as hotwater-soluble nigeran16;this is much less than the 4 to 10%previously reported."J8 Pseudonigeran is present in the cell walls ofAspergilZus nidulans, where it is a reserve material serving for growth of cleistothecia,lS and in Phytophthora infestans,20Cryptococcus, and Schizosaccharomyces spp.*l Many other fungi contain alkali-soluble, (1+3)-linked a-D-glucopyranans; these are summarized in Table I. In Histoplasma capsulatum, the cell wall contains a glucan, whereas the mycelial form con(9)J. 0.Previato, P. A. J. Gorin, R. H. Haskins, and L. R. Travassos, E x p . Mycol., 3 (1979)92-105. (10)H. Yamaguchi, Y.Kanda, and K. Iwata,J. Bacteriol., 120 (1974)441-449. (11)J. Nomnan, G. Wober, and E. C. Cantino, MoZ. Cell. Biochem., 9 (1975)141-148. (12)G.Takahara and K. Matsuda, Agric. BioZ. Chem., 40 (1976)1699-1703. (13)D.B. Coulter and J. M. Aronson, E x p . Mycol., 1 (1977)183-193. (14)D. B. Coulter and J. M.Aronson,Arch. Microbiol., 115 (1977)317-322. (15)J. D.Fontana and G. T. Zanch,J. Bacteriol., 129 (1977)141-148. (16)M. Horisberger, B. A. Lewis, and F. Smith, Carbohydr. Res., 23 (1972)183-188. (17)I. R. Johnston, Biochem. J., 96 (1965)651-658;659-664. (18)S.Hasegawa, J. H. Nordin, and S. Kirkwood,]. B i d . Chem., 244 (1969)5460-5470. (19)B. J. M. Zonneveld, Biochim. Biophys. Acta, 273 (1972)174-187. (20)T.Miyazaki, M. Yamada, and T. Ohno, Chem. Pharm. Bull., 22 (1974)1666-1669. (21)J. S. D.Bacon, D. Jones, V. C. Farmer, and D. M. Webley, Biochim. Biophys. Acta, 158 (1968)313-315.

70

E, RARRETO-BERGTER AND PHILIP A. J. GORIN

TABLEI Fungi Whose Cell Walls Contain Alkali-Soluble, (1+3)-Linked a-D-GlUCOpy~ananS Fungus

Methods used for characterization

References

Tremelln mesentericu Aspergillus nidulons (mycelia) Schizophyllum commune Histoplusma capsulutum Histoplusmu farcinosum Purucoccidioides brasiliensis Blastomyces dermutiditis

X-ray diffraction [a],,,periodate resistance x-ray diffraction, [a],,methylation methylation, [alD,i.r. methylation, i.r., enzymolysis methylation, [ale [a],,, i.r., enzymolysis

24 27 28-30 22 23 31 24

b i n s none.22 Yeast forms preferentially contain the glucan in Histoplusma f a r ~ i n o s u mBlastomyces ,~~ d e r m a t i d i t i ~and , ~ ~ Paracoccid ioides hrusil iEnsi.~.25

4. Glucans Containing a-D-( 1+3) and a-D-( 1 - 4 Linkages

Few reports exist on nigeran, except that it occupies an inaccessible position in the cell wall ofAspergihs niger, and is resistant to enzymic attack.32 An a-D-glucopyranan from fruit bodies of Lentinus edodes is slightly branched, with (1-3) and ( 1 4 4 ) linkages in the ratio of 5.3 : 1. It is partially degraded by amylolytic enzymes, and it was concluded that the (1-4) linkages are concentrated in regions not far from nonreducing ends:{" An a-D-glucopyranan containing (143) and (1-4) linkages in the ratio of 1:2 was obtained on alkaline extraction of the cell walls of Cladosporium herbarum, but the distribution of the linkages thi-oughout (22) F. Kanetsuna, L. M. Carbonell, F. Gil, and I. Azuma, Mycopathol. Mycol. Appl., 54 (1974) 1-13. (23) G. San-Blas and L. M.Carbonel1,J. Bucteriol., 119 (1974) 602-611. (24) F. Kanetsuna and L. M. Carbonel1,J. Bacteriol., 106 (1971) 946-948. (25) F. Kanetsuna and L. M. Carbonell,]. Bocteriol., 101 (1970) 675-680. (26) I. D. Reid and S. Bartnicki-Garcia,]. Gen. Microbiol., 96 (1976) 35-50. (27) B. J. M. Zonneveld, Biochim. Biophys. Acto, 249 (1971) 506-514. (28) J. G. H. Wessels, D. R. Kreger, R. Marchan5 B. A. Regensburg, and 0. M. H. d e Vries, Biochim. Biophys. Acto, 273 (1972) 346-358. (29) D. Siehr, Con. ]. Biochem., 54 (1976) 130-136. (30)J. H. Sietsrna and J. G. H. Wessels, Biochim. Biophys. Acta, 496 (1977) 225-239. (31) F. Kanetsuna, L. M. Carbonell, I. Azuma, and Y. Yamamura,J. Bucteriol., 110 (1972)208-218. (32)K. K. Tung and J. H. Nordin, Biochem. Biophys. Res. Commun., 28 (1967) 519524. (33) M. Shida, T. Uchida, and IL Matsuda, Carbohydr. Res., 60 (1978) 117-127.

71


the polymer was not determined.34Elsinan, isolated from the culture filtrate of Elsinoe leucospila, is a predominantly h e a r ff--0-ghcopyranan having 4-0-and 3-0-substituted units in the ratio of 2.5: 1. It consists mainly of the alternating, repeating sequence 1, although 3 -cr-D-GlCp-(l-4)-cr-D-GlCp-(l-4)-cu-D-Gl~p

-(1-4)-

1

consecutive, (1+4) linkages were evidenced by formation of a small proportion of maltotetraose on partial h y d r ~ l y s i s . Such ~ ~ , ~a~polysaccharide may be present in the hyphal wall of Coprinus mucrorhizus var. m i c r ~ s p o r u sPart . ~ ~ of the cell wall of Neurosporu crussa consists of a glucan having (1-4) and (1+3) linkages and, perhaps, the LY-D configurati~n.~~

1

1

I10

100

I 90

I 00

I

I

70

60

p. p. m. FIG. l.-W-N.m.r.

Spectrum of Pullulan from Tremella mesenterica, at pD 7.0.

(34) T. Miyazaki and Y. Naoi, Chem. Pharm. Bull., 22 (1974) 2058-2063. (35) Y. Tsumuraya, A. Misaki, S. Takaya, and M. Toni, Carbohydr. Res., 66 (1978) 5365. (36) A. Misaki, Y. Tsumuraya, and S. Takaya,Agric. Biol. Chem., 42 (1978) 491-493. (37) C. B. Bottom and D. J. Siehr, Carbohydr. Res., 77 (1979) 169-181. (38) L. Cardimil and G. Pincheira,J. Baetetiol., 137 (1979) 1067-107'2.

72

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

5. Pullulan Pullulan is present in the culture filtrate of Tremella mesentericu NRRL-6158, as shown by methylation, partial hydrolysis,Rgand Wn.m.r.-spectraleoevidence. Its l3C-n.rn.r. spectrum (see Fig. 1)is identical to that of pullulan. The three-unit repeating-sequence 2 was confirmed, because, although it only gives 14 signals in its W-n.ni.r. spectrum, these may be interpreted in terms of 18 signals, some of which overlap. A gliican from Cyttariu harioti Fischer, having (1-6)and (1+4)-linked a-D-glucopyranosyl units in the ratio of I :2.4, also resembles pullulan.41Although pullulans have the overall repeatingunit 2, that of Aureobasidium (Pullularia)pullulans contains a minor sub-unit having 3 consecutive, (1-+4) linkages.42 - a -D - GlC P - ( 1 - 4 ) -

(I-D-

GlCP - (1-

4)-

(2

-D-Glc p- (1-6)

-

2

111. PD-LINKEDGLUCANS 1. Cellulose both cellulose and chitin occur According to infrared (i.r.) in the cell walls of Cerutocystis oliuacea. Based on cytochemical and X-ray data, cellulose occurs in the cell walls of 31 of 47 species of Cerutocystis, and in 4 species of E ~ r o p h i u mConvincing .~~ i.r. evidence was obtained for the presence of cellulose in the cell walls of Europhium ~ u r e u malready , ~ ~ examined by the X-ray technique.

2. Linear I&D-GIuc~~s &D-Giucans are of interest because of their potential, antitumor act i ~ i t yAs . ~they ~ are present in virtually all fungi, the number of investigations conducted on them is considerable, and too great to be covered here. Thus, only those glucans whose structures have been determined in some detail are considered. (39) C. G. Fraser and H. J. Jennings,Can. J . Chem., 49 (1971) 1804-1807. (40) H. J. Jennings and I. C. P. Smith,J. Am. Chem. SOC., 95 (1973) 606-608. (41) N. Waksman, R. M. de Lederkremer, and A. S. Cerezo, Carbohydr. Res., 59 (1977) 505-515. (42) B. J. Catley and W. J. Whelan,Arch. Biochem. Biophys., 143 (1971) 138-142. (43) A. J. Michell and G. Scurfield, Trans. Br. Mycol. SOC., 55 (1970) 488-491. (44) T. R. Jewell, Mycologiu, 66 (1974) 139-146. (45) A. C. M . Weijman, Antonie uan Leeuwenhoek,J . Microbiol. Serol., 42 (1976) 315324. (46) R. L. Whistler, A. A. Bushway, P. P. Singh, W. Nakahara, and R. Tokuzen, Adv. Carbohydr. Chem. Biochem., 32 (1976) 235-275.


73

Pachyman, the major constituent of the cell wall ofPoria COCOS Wolf consists of (l+3)-linked ED-glucopyranosyl units, with only a few (1+6) links in the side chains and one in the main chain. Its numberaverage degree of polymeri~ation~~ is 690, which is higher than previously determined. A (1+3)-linked p-glucan is a major constituent of the conidial wall of Neurospora c r a s ~ aand , ~ ~is similar to that of the mycelial cell-wall. The fruit bodies of Pleurotus ostreatus (Fr.) QuCl contain a glucan having 3-0-substituted p-D-ghCOpyranOSyl, and minor 4-0-substituted a-D-glucopyranosyl, units.49A similar combination of linkages occurs in the cell-wall glucan of Coprinus macrorhizus var. microsporus.50A soluble glucan consisting of 77%of (1-+3)linked @D-ghcopyranosyl units was isolated from the mycelia of Phytophthora cinnamoni.51 The /+D-ghcopyranan from the mycelial form of Paracoccidioides brasiliensis contains 90% of (1-3) linkages.31Although one of the glucans of Saccharomyces cerevisiae is almost linear, it is, for convenience, dealt with in the following section (3). A linear cell-wall glucan(s) from Sporothrix schenckii contains 3 0 , 6 0 , and 4-0-substituted @D-glucopyranosy1 units, the @D-glycosidic configuration being determined by 13C-n.m.r. spectrosc~py,~ by virtue of the C-1 signals, at relatively low field, of 6, 103.8 and 104.8. 3. Branched-chain p-~-Glucans

Cell walls of Saccharomyces cerevisiae were found to contain a ( 1+6)-linked ED-glucopyranan; this was isolated, and identified by i.r. s p e c t r o s ~ o p yand ~ ~ chemical-analysis techniques." The alkali-insoluble glucan from S. cerevisiae contains this and a (1+3)-linked p-Dglucopyranan in the ratio" of 1:5.7. The former, of mol. wt. 2 x lo5,

has 6-0- and 3-0-substituted units in the ratio of 4.4: 1, and contains 14% of 3,6-di-O-substituted units.55(A similar heterogeneity occurred (47) G. C. H o h a n n , B. W. Simson, and T. E. Timell, Carbohydr. Res., 20 (1971)185188. (48) P. R. Mahadevan and U.R. Mahadkar, lndiun J . Exp. BioE., 8 (1970)207-210. (49) H. Sait6, T. Ohki, Y. Yoshioka, and F. Fukuoka, FEBS Lett., 68 (1976) 15-18. (50) C. B. Bottom and D. J. Siehr, Can.J . Biochem., 58 (1980) 147-153. (51) L. P. T. M. Zevenhuizen and S. Bartnicki-Garcia,]. Gen. Microbiol., 61 (1970) 183188. (52) J. S. D. Bacon, V. C. Farmer, D. Jones, and I. F. Taylor, Biochem. J., 114 (1969) 557-567. (53) D. J. Manners and A. J. Masson, FEBS Lett., 4 (1969) 122-124. (54) D. J. Manners, A. J. Masson, and J. C. Patterson,]. Gen. Microbiol., 80 (1974)411417. (55) D. J. Manners, A. J. Masson, J. C. Patterson, H. Bjorndal, and B. Lindberg, Biochem. j., 135 (1973) 31-36.

74

E. BARRETO-BEHGTER A N D PHlLIP A. J. GORIN

in the polysaccharides in the walls of Kloeckera apiculata, Schizosaechurorriyces pombe, Saccharomyces fragilis, and Saccharomyces fermentati.) The latter polysaccharide, of mol. wt. 2.4 x lo5, is less branched, havings6 (143) (85%)and interchain, (1+6) linkages (3%). Smith degradations of total glucans gave glucosylglycero1,j6~5'laminardbiosyiglycerol, and larninaratriosylglycer01.~~ The alkali-soluble P-D-giucopyranan contains 3-0- (80-85%), 6 0 (8- 12%),and 3,6-di0-substituted (3-4%) units'." 'Chc pu-glucopyranans of Candida albicatis serotype B and Candicfu yarupsilosis are mainly linear, with only 10%of branch points, and contain, principally, ( 1 4 6 ) linkages (67 and 63%, respectively).59

-

TABLEI1 Degree of Side-chain Substitution in Glucans Having the General Structure 3 ~

Source of glucan

Sclerotinia glucanicum, exocellulaP1 Pythircm ncunthicum, hyphal wall" Sclerotinia libertinna, exocellulaP Monolinia fructigena, exocellular (mol. wt. 7 x l(r)&la Schizophyllum communewD Claciceps fusiformus (Loveless), exocelluIaP Aweobusidium (Pulluluria) pullulnns, cell wallm Pin'culuria oryzue, cell wall6'

~

~~

~~

Value of n in structure 3

2 2 2 1 2 3 8 5

(56) D. J. Manners, A . J. Masson, and J. C. Patterson, Biochem. I. 135 ,(1973) 19-30. (57) A. Misaki, J . Johnson, Jr., S. Kirkwood, J. V. Scaletti, and F. Smith, Carbohydr. Has.. 6 (1968) 150-164. (58) G . H. Fleet and D. J. Manners,J. Gen. Microbiol., 94 (1976) 180-192. (59) R. J. Yti, C . T. Bishop, F. P. Cooper, F. Blank, and H. F. Hasenclever, Can. J. Cherii., 45 (1967) 2264-2267. (60)K. Buck, A. LV. Cheti, A. G. Dickerson, and E. B. Chain, J. Gen. Microbiol., 51 (1968)337-352. (61) I . j. Goldstein, G. W. Hay, B. A. Lewis, and F. Smith,Abstr. Pap. Am. Chem. SOC. 3fcrt.. 135 (19591 3D. (62) J. H. Sietsma, J. J. Child, L. R. Nesbitt, and R. H. Haskins,]. Cen. Microbiol., 86 (1975) 29-38. (63) Y. Ueno, Y. Hachisuka, H. Esaki, R. Yamauchi, and K. Kato,Agric. B i d . Chem.,44 (1980) 353-359. (Wa) F. Santamaria, F. Reyes, and R. Lahoz,]. Cen. Microbiol., 109 (1978) 287-293. (644h)S. Kikumoto, T. Miyajima, K. Kimura, S. Okubo, and K. Kumatsu, Nippon Nogei Kupaku Kaishi, 45 (1970) 162-168. (65) S . Komatsu, S. Ohkubo, S.Kikumoto, G. Saito, and S. Sakai, Gunn, 60 (1969) 137144. (66) R. G. Brown and B. Lindherg,Acto Chem. Scand., 21 (1967) 2379-2382. (67) T. Nakajima, K. Tamari, K. Matsuda, H. Tanaka, and N. Ogasawara, Agric. Biol. Chem., 36 (1972) 11- 17.


75

A number of P-D-glUCanS have been reported to contain a (143)linked, ~-D-glUCOpyranOSyl main-chain, partially substituted at 0-6 by (single unit) PD-glUCOpyranOSyl groups, as, for example, in repeatingunit 3. The degree of substitution varies with the fungus (see Table p-~-Glcp

I

6

-[p-D-Glcp -(1-t3)]n-@-D-GlCp -(1-+3)3

11). With Claviceps fusiformis, the exocellular glucan has a degree of branching that increases to a maximum of 1, in every third, main-chain residue, after 14 days, and then slowly declines.60The glucans from Aureobasidium (Pullularia) pullulans and Pirikularia oryxae cellwalls contain little branching, and, in the case of the latter glucan, the main chain has a few (1-6) linkages. Lentinan, from Lentinus edodes, appears to have a main chain consisting largely of (1-+3) [and some (1+6)] linkages, together with (143) and (146) links in the side chains.68 The fruit bodies of Auricularia auricula-judae contain two branched FD-glucopyranans having (143) and (1+6) linkages. The soluble one could have a structure similar to that of 3, but the other, which is insoluble in alkali, contains 6-0-substituted units.69

IV. GLUCANSFROM LJCHENS A partially acetylated, (1-+6)-linked P-D-glucopyranan has been extracted from Gyrophora esculenta Miyoshi. The degree and position(s) of substitution have not yet been determined. A similar glucan has been found in Lasallia papulosa (Ach.) Llano.70The unacetylated glucan gives a 13C-n.m.r. spectrum7l similar to that of pustulan from Umbilicaria p ~ s t u l a t a . ~ ~ Common glucans from lichens are lichenan, a p-D-glucan having (143) and (1-4) linkages in the ratio of 3: 7 (approximate repeatingunit 4), and isolichenan, an a-D-glucan having (1-+3) and ( 1 4 4 ) linkages in the ratio of 11 :9. Such components are present in Cetraria ri(68) T. Sasaki and N. Taksuka, Carbohydr. Res., 47 (1976) 99-104. (69) Y. Sone, K. Kakuta, and A. Misaki, Agric. Biol. Chem., 42 (1978)417-425. (70) S. Shibata, Y. Nishikawa, T. Takeda, M. Tanaka, F. Fukuoka, and M. Nakanishi, Chem. Pham. Bull., 16 (1968) 1639-1641. (71) H. Sait6, T. Ohki, N. Takasuka, and T. Sasaki, Carbohydr. Res., 58 (1977)295-305. (72) D. Bassieux, D. (Y.) Gagnaire, and M. (R.) Vignon, Carbohydr. Res., 56 (1977) 19-33.

76

E. BARRETO-BERGTER AND PHILIP A. J. GORIN

churdsonii Hook,73Alectoria sulcata (Lev.) Nyl., and Alectoria sarmentosa. 74 -S-D-GlCP -(1-4)-p-D-Glcp-(1-4)-~-D-Glcp-(l~3)4

Purnieliu caperuta (L.)Ach. provided a cold-water-insoluble amglucopyranan having (1-3)- and (1-+4)-linked residues in equimolar proportions. As there were no successive, (1-+3) and (1-+4)linkages, the glucan is structurally similar to nigeran (repeating unit 5).75 - 0 - D-

GlC p- (1-

3)- (1-D-Glcp - ( 1 - 4 ) 5

From chemical the glucan from Stereocaulon japonicum apparently had a-(1-3) and a-(1-4) linkages in the ratio of 3 : 1, but This technique the ratio was shown to be 2: 1 by a 'T-n.m.r. was useful in showing that a-Dglucans from Sphaerophorus glohosus and Acroscyphus sphaerophoroides had (1-+3) and (1-4) linkages in the ratio of 2 :3, with 30,000; when the radioactive material isolated was re-incubated with the membranes, a small fraction of the radioactivity was transferred to the cellulosic fraction. However, as values were given as the percentage of the total radioactivity supplied, it is difficult to assess the statistical significance of this result. Although of some potential interest, further characterization of this borate-soluble material has not yet been reported. In later s t ~ d i e s , ’ Colvin ~ ~ J ~ and ~ coworkers isolated a fraction, insoluble in 60% ethanol but soluble in water, from the medium in which A . zylinum cells had been grown. Methylation analysis indicated that the material contained a glucan having p-(1-+4) linkages with single glucosyl groups as branches at 0-2 of every third glucosyl residue, on the average. They suggested that this water-soluble glucan was a precursor to cellulose, but gave no proof of a precursor-product relationship. Furthermore, the significance of this polymer with respect to the synthesis of cellulose has since been questioned by Colvin and cow o r k e r ~ , ’and ~ ~ the possible relationship between this polymer and the borate-soluble polymer previously studied is also not clear. Benziman and c o w ~ r k e r soffered ~ ~ ’ ~ additional evidence of a different sort for high-molecular-weight precursors to cellulose in A. xylinum. These workers monitored the kinetics of labeling of various cellular fiactions during incubation of “resting cells” (lacking an external source of nitrogen) in radioactive glucose. Following extraction of the labeled cells with chloroform-methanol, they were able to extract labeled, non-dialyzable, water- and alkali-soluble polymeric materials. During a subsequent chase with unlabeled glucose, radioactivity declined in these fractions, and a corresponding increase in radioactivity (143) G. C . S. King and J. R. Colvin, A w l . Polym. Symp., 28 (1976)623-636. (144) J. R. Colvin, L. Chene, L. C. Sowden, and M. Takai, Can.]. Biockm., 55 (1977) 1057- 1063. (145) L. C. Sowden and J. R. Colvin, Can. 1. Microbiol., 24 (1978) 772-777. (146) J. R. Colvin, L. C. Sowden, V. Daoust, and M. B. Perry, Can. J . Biochem., 57 (1979) 1284-1288.

BIOSYNTHESIS OF CELLULOSE

137

Time ( m i d

FIG.3.-Pulse-chase Experiment with Acetobacter ryZinum.1° {Incorporation of D ['4C]glucose (3,300 c.p.m. per nmol) into the water- and alkali-soluble fractions, and its subsequent transfer from these fractions into cellulose. In the pulse, cells were incuo s0" e in buffer at pH 6.0; at the time of the chase, cells bated in 3 mM ~ [ ~ ~ C l g l u c at were diluted in cold buffer, centrifuged, and re-incubated at 30" in buffer either conor lacking &glucose (--).} taining 40 mM unlabeled Dglucose (-)

in cellulose was observed (see Fig. 3).The kinetics of chase suggested that the two fractions (water-soluble and alkali-soluble) were distinct, and that the alkali-soluble polymer(s)served as precursor to the watersoluble material which, in turn, served as precursor to cellulose. The fractions were rendered dialyzable by digestion with crude cellulase, and partially dialyzable by treatment with pronase. Thus, the authors suggested that these polymers might be glucoproteins that serve as precursors to cellulose. Subsequent methylation analysis of these fractions indicated that the radioactivity is mainly located in (1+4)-glucosyl residues, with lesser, and variable, proportions of (1-*2)-glucosyl residues.147Thus, although these fractions have not yet been purified, or completely characterized structurally, they seem to possess properties consistent with a role as precursors to cellulose. b. Protothecu zopfiL-Studies by Hopp and coworkers,17described earlier in connection with the alga Prototheca xopfii, indicated that a radioactive, water-soluble polymer is produced when membrane fractions are incubated with radioactive UDP-glucose. As -40% of the polymer was "hydrolyzed" by pronase treatment, the authors sug(147) U. Rothschild, M. Benziman, and D. P. Delmer, unpublished results.

138

DEBORAH P. DELMER

gested that the polymer is a glucoprotein. The existence of p-D-(1+4)glucosyl residues in the polymer was reasonably well established. However, as in the work with A. xylinnm, rigorous proof that the polymer was a glucoprotein is still lacking; and a role for this polymer as preciirsor to cellulose is still not definitely established. The Prototlzecci \ystem does, however, appear to be a promising one for in vitro studies, and it is to be hoped that future work will clarify the nature of this water-soluble polymer.

c. Higher Plants.- Some circumstantial evidence for a high-moleciilar-weight precursor to cellulose was also educed from studies with higher plants. MortimeP" found that radioactive a-cellulose, formed in barley leaves and sugar beet by labeling with radioactive carbon dioxide, contained a distinct fraction of glucan, of higher specific activity, that could be released by extraction of the a-cellulose with hot, dilute trichloroacetic acid. Following a chase with unlabeled CO, , this radioactivity was transferred to the portion of the a-cellulose fraction that was insoluble in this acid. An intermediate role was suggested for this glucan; an alternative interpretation is, however, that the glucosyl residues most recently incorporated were the most accessible to partial hydrolysis with acid, resulting in the release, by trichloroacetic acid, of glucan fragments having low molecular weights. Satoh and coworkers149observed a somewhat comparable phenomenon during i n cioo labeling of mung-bean hypocotyl-segments in radioactive glucose. However, in this instance, a glucan fraction of high specific activity, that could be hydrolyzed by an impure cellulase to yield glucose and (probably) cellobiose, was found associated with membranes and not with the cell-wall fraction, as was the case in Mortimer's study."* Some turnover of the radioactivity in this fraction could be observed during an in oioo, pulse-chase experiment; furthermore, the synthesis ofthe "cytoplasmic" glucan could be inhibited by coumarin, a relatively specific inhibitor of cellulose synthesis (see Section V,4). These studies appear to be of potential importance; nevertheless, definitive, structural characterization of this fraction as (~+4)-~-D-glucan is required, and, as yet, no further analysis of this fraction has been published. In 1975, FranzI5Oreported that incubation of mung-bean membranepreparations with radioactive UDP-glucose resulted in the production of a variety of polymeric products, one of which was identified as a glucoprotein. The radioactivity associated with this presumed glucoprotein could be solubilized by treatment of the alkali-insoluble prod(148)D. C. Mortimer, C a n . / . Bot., 41 (1963) 995-1004. (149) S. Satoh, K. Matsuda, and K. Tamari, Plant Cell Physiol., 17 (1976) 1213-1254. (150) C . Franz, A p p l . Polym. Symp., 28 (1976) 611-621.


139

ucts with pronase, or by hydrazinolysis. The released material contained all of its radioactivity in glucose residues; upon digestion with an impure cellulase, compounds migrating coincident with cellobiose and cellotriose standards, during paper chromatography, were released. Some turnover of the “glucoprotein” fraction was indicated in pulse-chase experiments. These results appeared quite promising, but surprisingly have not been pursued further by Franz. As with the results of Satoh and coworkers,149the characterization of the glucan moiety was not complete; crude cellulases were employed and, in both studies, it is uncertain whether the chromatographic techniques employed really allowed resolution of di-, tri-, and tetra-saccharides having different linkages. Quite a different sort of possible, high-molecular-weight precursor to cellulose, namely, ( 1+3)-p-D-g1ucan7has also been considered for higher plants. This suggestion has arisen primarily from studies with developing cotton-fibers by the groups of Delmer, Meier, and Waterkeyn. Meinert and DelmeP first observed the appearance of substantial quantities of (1+3)-glucosyl residues in cell walls of cotton fibers during the early stages of secondary-wall formation. Subsequently, Maltby and coworkers151and Huwyler and coworker^^^*'^^ definitely characterized this material as (1+3)-/%D-glUCan.Some peculiarities in the pattern of labeling of the (1+3)-p-D-glucan in viuo, using cultured cotton-fibers, led Maltby and coworkers151to consider whether this glucan might exhibit turnover and, perhaps, serve as a precursor to cellulose; thus, they observed that, in short-term, labeling experiments, the rate of incorporation of label into (1+3)-p-D-glucan exceeded that expected on the basis of chemical analyses of levels of accumulation of (1+3)-P-D-glUCan in the cell wall at this stage of development. However, in repeated, pulse-chase experiments, significant turnover of this glucan fraction could not be demonstrated (see Fig. 4A). also observed, during short-term labeling Pillonel and of fibers, using intact plants, that (1+3)-p-D-glucan is synthesized at a rate that appears to exceed the accumulation observed in the walls, and they, also, suggested that turnover must occur. In subsequent studies, Meier and coworker^^^^,^^^ were able to demonstrate a slow turnover of the (1+3)-p-D-glUCan fraction in vivo (see Fig. 4B).Tech(151) D. Maltby, N. C. Carpita, D. Montezinos, and D. P. Delmer, Plant Physiol., 63 (1979) 1158-1164. (152) H. R. Huwyler, G . Franz, and H. Meier, Plant Sci. Lett., 12 (1978) 55-62. (153) C. Pillonel, A. J. Buchala, and H. Meier, Planta, 149 (1980)306-312. (154) H. Meier, L. Buchs, A. J. Buchala, and T. Homewood, Nature, 289 (1981) 821822. (155) H. Meier, in Ref. 27, pp. 75-83.

m-s 3 - A -

a 0

c 0

0

60

rnin

120

Ieo

Days after beginning of 14C02 pulse FIG.4.-A. Pulse-chase Experiment using Cotton Fibers (Gossypium hirsuturn) Cultured in oitro; Kinetics of Labeling of Cellulose and (1+3)-/3-~-Glucan.'~~ {Fibers, with (20 mM; 0.08 pCi per pmol). their associated ovules, were incubated in ~-['~C]glucose At the time of the chase, half of the remaining ovules with fibers were briefly rinsed and then incubated in 100 mM unlabeled r>-glucose. Cellulose and (1+3)-p-~-glucan were pulse; (---) chase; (0)cellulose; (0)water-soluanalyzed as d e ~ c r i b e d . ' ~Key: ' (-) ble (1+3)-p-~-glucan; (A) water-insoluble (1-+3)-/3-~-glucan.) B. Pulse-chase Feeding1Mof W02 to Branches of Intact Plants of Gossypium arhoreirm L. ['*C02 (7.4 MBq; 2.0 FCi) was fed in the morning in bright sunlight to branches, each with three or four leaves and one capsule, at 35-40 days post-anthesis, inside poly(ethy1ene)bags. After 30 minutes, -80% of the original radioactivity in the bags had been taken up by the branches which were, however, left inside the bags for another 90 minutes. The latter were then removed, and the plants were kept under normal day and night conditions, when photosynthesis could occur normally, until the capsules were harvested. The radioactivity was determined in the fractions 80%soluble in methanol ( O ) ,and (1+3)-@-~-glucan(callose) (O),and (1-*4)-p-D-glucan (cellulose) (a) fractions of the fibers. Numbers in parentheses are the percent radioactivity in the callose and cellulose type of Dglucans, respectively.]


141

nical difficulties with obtaining a clear chase in vivo using intact plants made it hard to decide from these studies whether a quantitative conversion of radioactivity from (1+3)-p-D-ghcan into cellulose had occurred. In the results of Maltby and coworkers,151the chase was effective, as evidenced by a cessation of incorporation of label into cellulose, and in those experiments no turnover was observed. Other cytochemical studies of developing cotton-fibers by Waterk e ~ n indicated '~~ that (1+3)-P-~-glucanis always localized, independent of the age of the fiber, in the innermost wall-layer. The basis of identification of the (1+3)-j%~-glucan was that it showed a specific fluorescence after staining with Aniline Blue, a procedure that is not always specific for (1+3)-P-D-gl~can.'~~ However, Waterkeyn is experienced with the use of this dye, and, assuming that his identification was correct, such a localization for the (1+3)-P-~-glucancould be interpreted to indicate a precursor function. Alternatively, Waterkeyn also offered the suggestion that the glucan could play a role in providing a matrix wherein microfibrils undergo "maturation" and orientation. By chemical analyses of levels of (1+3)-p-~-glucan, Maltby and coworker^'^' found that the maximum level of accumulation occurred during a time when an overlap takes place between the phases of fiber elongation and secondary-wall synthesis, and they similarly suggested that the role of the (1+3)-j3-~-glucancould be in modulating the extensibility of the wall, rather than as a precursor to cellulose. Nevertheless, there are peculiarities in the pattern of labeling of this glucan in vivo that are not readily explained, except by invoking turnover; but, even if such turnover occurs, a specific conversion into cellulose has by no means been proved as yet. A curious analogy in A. rylinurn to the whole (1+3)-P-~-glucanpuzzle in higher plants may also be worth mentioning. In addition to catalyzing the synthesis of (1+3)-/?-D-glucan from UDP-glucose, A. xyZinum membrane-preparations are also active in catalyzing the synthesis from UDP-glucose of water-soluble ( 1 + 2 ) - ~ - ~ - g l u c a n . " ~ * ~ ~ ~ Such a glucan has not been described as a structural component of this organism, although it has been reported to exist in other Grsm-negative bacteria (see references cited in Ref. 112). It has also been observed that some synthesis of this glucan is found in labeling studies in v i ~ dhowever, ~ ~ ; no data as yet exist to support, or refute, an intermediate role for this polymer in cellulose biosynthesis. In summary, much incomplete evidence has been given that offers

(156) L. Waterkeyn, Protoplasma, 106 (1981) 49-67. (157) M. M. Smith and M. E. McCully, Protoplasma, 95 (1978) 229-254.

142

DEBORAH P. DELMEH

suggestions for a role for high-molecular-weight precursor(s) to cellulose. Taken in sum, the evidence is not yet fully convincing, nor does it preclude such a possibility. It is, perhaps, useful to consider the implications for biosynthesis that the existence of such a polymer would have. Based on current concepts of mechanisms of polysaccharide synthesis, it is difficult to envisage a mechanism of microfibril synthesis involving such polymers. If, as Colvin' believes, such precursors associate without the aid of enzymes, to afford crystalline cellulose, it is exceedingly difficult to explain three things: (1 ) as cellulose I1 has a structure much more stable than that of cellulose I, it would be expected that spontaneous crystallization of a soluble form would yield cellulose 11, and yet we know that the native cellulose is cellulose I; ( 2 )the d.p.ofthe cellulose would be expected to be low, and we know that it is not; Colvin' proposed the possible existence of chain ligases, but no evidence exists for such enzymes as of this writing; and ( 3 ) whatever polar group exists that must confer solubility to such an intermediate glucan must be removed, a process that would surely require some kind of enzyme. If, on the other hand, transfer of glucan chains from a (protein?) carrier is mediated by an enzyme, it is difficult to envisage why a process of elongation involving carrier-mediated transfer of oligosaccharides should require such long oligosaccharides, instead of D-glucose or cellobiose. Perhaps, a more likely possibility is that such polymers are primers that are subsequently elongated by a different mechanism, to afford the final D-glucan chains in cellulose. With respect to the possibility that (1+3)-P-D-glucan might serve as an intermediate in cellulose synthesis in higher plants, transfer of Dgliicosyl groups to cellulose by trans-D-glucosylation could 6e envisaged; the spontaneous crystallization of the cellulose might serve to drive this reaction in the appropriate direction. Exoglucanases having transglucosylase activity are known to exist, but the new linkages generated are often random. Meier's group'j5 demonstrated the existence in cotton fibers of a wall-bound exoglucanase that has a preference for (1--d)-~-D-giucansas substrate, but they have yet to demonstrate any trans-D-gliicosylase activity for this enzyme. One attractive argument for the hypothesis that this glucan is a precursor to cellulose is that it could nicely explain the rapid production of (l+d)-P-D-glucan that occurs on wounding of plant cells; if the final conversion of this glucan into cellulose is catalyzed by a very labile enzyme-system, it might be expected that, on cellular damage, the precursor would rapidly accumulate. However, much more study is needed before such a possibility can be considered to be more than just speculation.


143

4. Genetic Mutations, and Chemical Inhibitors of

Cellulose Biosynthesis The elucidation of metabolic pathways has often been aided by the use of genetic mutations, or specific, chemical inhibitors that result in the blockage of a specific, metabolic reaction along the pathway; such inhibition can sometimes lead to the accumulation of prior intermediates, and thus facilitate their isolation and identification. Mutants of A. xylinum that are incapable of, or impaired in their, ability to synthesize cellulose have been isolated. One such mutant was partially characterized by Swissa and coworker^.^ Although this mutant lacked the capacity to synthesize cellulose, it possessed capability for the normal metabolism of hexose phosphates and UDP-glucose; of considerable interest was the observation that, during in vivo labeling with radioactive D-glucose, it showed enhanced accumulation of label in chloroform-, water-, and alkali-soluble material (as compared to the wild type). Such a finding supports a possibility proposed by these workers, namely, that the water- and alkali-soluble fractions may constitute precursors to cellulose. However, a chemical characterization of these fractions in the mutant strain has yet to be reported. They also found that the mutant is still capable of catalyzing the synthesis, from UDP-glucose, of D-glucans in vitro; this is in contrast to results with another mutant studied by Cooper and M a n l e ~ , ' ~ ~ who found their mutant strain incapable of D-glucan synthesis from UDP-glucose. Incorporation, in whole cells, of label from labeled D glucose into the lipid fraction was also lessened in this mutant. Garcia and also mentioned that they observed a lower capacity for glucolipid synthesis from UDP-glucose with another cellulose-less mutant. Unfortunately, the lack of a good genetic system for A. xylinum has made genetic analyses of these mutants impossible to date; in none of the foregoing was it known how many mutations were involved, or where the specific blocks occurred in these metabolic pathways. To the best of the present author's knowledge, there are, unfortunately, in the algae or higher plants, no known mutants available that are specifically blocked in cellulose synthesis. However, several, relatively specific, chemical inhibitors of the process have been characterized. One of these, coumarin, has been reported to inhibit cellulose synthesis in A. x y Z i n ~ m , ' ~as - 'well ~ ~ as in higher plant^?^,'^^ Relatively (158) D. Cooper and R. S. J. Manley, Biochim. Biophys. Acta, 381 (1975) 109-119. (159) S. Satoh, M. Takahama, and K. Matsuda,Plant Cell Physiol., 17 (1976) 1077-1080. (160) J. Burgess and P. J. Linstead, Planta, 133 (1977)267-273.

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high concentrations (in the millimolar range) are required in order to obtain a substantial inhibition; this appears to be relatively specific for cellulose synthesis, at least in comparison to its effects on the synthesis of other cell-wall polymers. Coumarin has, however, been reported to have some other side effects in plants, as discussed by Montezinos and Delmer.85Nevertheless, it is of some interest that it was effective in inhibiting the synthesis of proposed intermediates in cellulose synt h e ~ i s , ' ~as~ discussed ,'~~ in Section V,3. Some indication that Mg2+may be an important ion for cellulose synthesis comes from studies by Montezinos and as discussed earlier (in Section IVJ). Also of interest is the observation by Quader and RobinsonBdthat calcium ionophores and the cryptates 211 and 212 were potent inhibitors of cellulose synthesis in Oocystis, although their mode of action is at present not understood. One of the most promising inhibitors studied to date is 2,6-dichlorohenzonitrile (DCB), which has been marketed as a herbicide under the names of Casoron and Dichlobenil. Hogetsu and coworkers'61first provided an indication that the mode of action of DCB as a herbicide could be due to its effect on cellulose biosynthesis in plants. Subsequently, Montezinos and DelmeP showed it to be a specific and reversible inhibitor of cellulose synthesis, effective in low concentrations (1-10 pM)in cotton fibers. Meyer and Herthls2also found DCB to be an effective and reversible inhibitor of cell-wall regeneration in tobacco protoplasts. Aloni and Benziman'O reported that DCB also inhibits cellulose synthesis in A. xylinum. Further studies, with cotton fibers and soybean cells,lm indicated that DCB does not inhibit mglucose uptake, or the synthesis of hexose phosphates or UDP-glucose, nor does it affect ATP levels. However, attempts to demonstrate a DCB-induced accumulation of any intermediates beyond the level of UDP-glucose were not successful. Montezinos and Delmef15pointed out that use of this inhibitor for studies of cellulose synthesis should be confined to short-term experiments, as some indication exists that DCB can be metabolized to a derivative that can affect oxidative phosphorylation.lM The documented, herbicidal activity of DCB offers some promise that inhibition of the process of cellulose synthesis could be further exploited as a safe and effective target of herbicide action. (161) T. Hogetsu, H. Shibaoka, and H. Shimo-Koriyama, Plant Cell Physiol., 15 (1974) 389-393. (162) Y. Meyer and W. He&, Plantn, 142 (1978) 253-262. (163) N. C. Carpita, A. Klein, and D. P. Delmer, unpublished results. (164) D. E. Moreland. G . G . Hussey, and F. S. Fanner, Pestic. Bwchem., Physiol., 4 (1974) 356-364.


145

5. Possible Factors Affecting the Lability of the Polymerizing System

From the preceding discussions, it is evident that, in all systems studied, and, in particular, in higher plants, attempts to synthesize cellulose in vitro have met with only limited success; this therefore leads to the conclusion that, for poorly understood reasons, the cellulose synthetase complex is a highly labile system. As a conclusion to this article, it may prove useful for future research to discuss possible reasons for this apparent lability. a. Effect of Proteases.-One obvious possibility is that the complex is highly susceptible to proteolytic attack. Chao and Ma~lachlan'~~ reported that, present in extracts of pea seedlings was an endogenous factor, suggested to be a protease, that caused partial inactivation of UDP-glucose:(l4)-/3-D-glucan synthetase activity. (As discussed earlier, it is not certain whether this enzyme functions in synthesis of cellulose or of xyloglucan.) Nevertheless, attempts by these workers to prevent the inactivation by the addition of protease inhibitors or high concentrations of nonspecific protein were unsuccessful. Through conversations with colleagues in the field, as well as personal experience, it is clear that numerous attempts to inhibit protease activity have not resulted in a substantial enhancement of UDP-glucose:(1+4)-p-~glucansynthetase activity. Likewise, the present author knows of no successful attempts to stimulate activity by limited protease treatment, a procedure used with great success for chitin ~ynthetase'~*'~' (EC 2.4.1.16). b. Effect of Poly(ethy1ene Glycol).-It has observed that inclusion of 0.06 molal poly(ethy1ene glycol)-4O00 (PEG-4000) in the isolation medium results in a considerable enhancement of UDP-g1ucose:glucan synthetase activities in membrane preparations derived from cotton fiber~.l@J~~ Polymerization of both &( 1+3)- and p-( 14)-glucosyl residues is enhanced; whereas, in previous work,3l it was possible to detect synthesis only of (1+3)-p-~-glucan,from UDP-glucose in the (165) H.-Y. Chao and G. A. Maclachlan, Plant Physwl., 61 (1978) 943-948. (166) E. Cabib, R. Ulane, and B. Bowers, in B. L. Horecker and E. R. Stadtman (Eds.), Current Topics in Cellular Regulation, Vol. 8, Academic Press, New York, 1974, pp. 1-32. (167) J. Ruiz-Herrera, E. Lopez-Romero, and S. Bartnicki-Garcia,J . Biol. Chem., 252 (1977) 3338-3343. (168) D. P. Delmer, M. Benziman, A. Klein, A. Bacic, B. Mitchell, H. Weinhouse, Y. Aloni, and T. Callaghan,]. Appl. Polym. Sci., in press. (168)A. Bacic and D. P. Delmer, Planta, 152 (1981)346-351.

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DEBORAH P. DELMER

presence of PEG4000, it is now routinely observed that -25% of the glucan products contain p-( 14)-glucosyl residues.169Poly(ethy1ene glycols) of lower molecular weight were less effective. A similar enhancement of activity (- 10-fold) by PEG4000 has also been observed for the A. xylinum UDP-glucose:( 1+4)-p-~-glucansynthetase."jRMaclachlan and coworkers170also observed some enhancement of UDPglucose:( 1+4)-P-~-glucan synthetase from pea tissue on using PEG400. Inclusion of PEG in isolation buffers may, therefore, prove to be of considerable help in stabilizing such enzymes. Poly(ethy1ene glycol)~ of high molecular weight are known to promote protein-protein and this could be the mechanism whereby these substances stabilize the activity of a multi-subunit enzyme-complex. It should also be noted, however, that inclusion of PEG4000 in the isolation buffer leads to the production of abnormally large, membrane vesicles that sediment at low centrifugal forces, presumably due to the known ability of PEG to promote membrane fusions. At present, it is not known whether these vesicles contain components only from the plasma membrane, or represent mixed-membrane fusions. c. Attempts to Assay Solubilized Glucan Synthetases-Some attempts to solubilize and purify a UDP-glucose:( 1+4)-p-D-glucan synthetase activity have been made. Tsai and H a ~ s i dwere ' ~ ~ able to solubilize p-( b 3 ) - and p-( l-A)-glucan synthetase activities from membranes of oat seedlings by use of high concentrations of digitonin; they were also able to resolve these activities by chromatography in a column ofhydroxylapatite; however, the solubilized enzymes were quite unstable, and further purification was not attempted. Larsen and BnLmmond'i4 also succeeded in solubilizing, with digitonin, these activities from membranes of Lupinus albus; however, no purification was attempted. Klein16H-1i5 achieved good solubilization of these activities from membranes derived from cultured soybean cells; her soliibil ization procedure involved treatment of the membranes for 15 minutes at 0" with 30 mM cholate at pH 7.8. The solubilized enzymes were quite labile, but could be both stimulated and stabilized by high concentrations of glycerol. Advances in solubilization and re(170) G. Maclachlan, M. Durr, and Y. Raymond,Methodol. Sum. (B)Biochem., 9 (1979) 147-153. (171) L. A. Halper and P. A. Srere, Arch. Biochem. Biophys., 184 (1977) 529-534. (172) J. C. Lee and L. L. Y. Lee,J. Biol. Cliem., 256 (1981)625-631. (173) C. M. Tsai and W. Z . Hassid, Plont Physiol., 47 (1971) 740-744. (174) G. L. Larsen and D. 0. Brurnrnond, Phytochemistry, 13 (1974)361-365. (175) .4. S. Klein, Ph.D. Thesis, Michigan State University, 1981.


147

constitution of membrane-bound proteins into artificial lipid vesicles suggest that it would be profitable to pursue these preliminary studies farther. However, to date, no dramatic increases in activity for UDPglucose:( 1+4)-P-D-glucan synthetase have been reported as a result of working with a solubilized form of the enzyme. d. Requirement for an Intact Cell for Activity; Possible Modulation of Activity by a Transmembrane, Electrical Potential.-Assay of cel-

lulose synthetase activity in intact cells has been attempted in a number of laboratories. In higher plants, incubation of pea-epicotyl slices in radioactive UDP-glucose results in production of P-glucan, but the product is mainly ( 1 + 3 ) - P - D - g l u ~ a n . ~Anderson * ~ ~ , ~ ~ ~ and Ray115concluded that this activity occurred primarily at the cut edges of the tissue. Similarly, Delmer and coworkers1lsobserved that intact cottonfibers do not utilize UDP-glucose, but substantial activity for synthesis of (1-*3)-P-D-glUCan is obtained when the cells are damaged; Brett176found a similar phenomenon with suspension-cultured, soybean cells. Klein and Delmel.26*175 observed that preparations of “intact,” soybean protoplasts could utilize UDP-glucose for synthesis of (1+3)-P-D-glucan, but activity was enhanced at least 10-fold when the protoplasts were lysed; therefore, it was difficult to exclude the possibility that the “intact” protoplast-preparation contained a low percentage of damaged protoplasts. The main conclusions from all of these experiments are that (a) intact plant-cells utilize UDP-glucose poorly, or not at all; and (b) when damaged (or even just rendered permeable by treatment with a detergent or dimethyl sulfoxide, or by cold shock”’), they can utilize the substrate for synthesis of (1+3)-P-D-glucan, but not for synthesis of cellulose. This, in turn, leads to the conclusion that the cellulose synthetase complex can only accept UDPglucose from the inner face of the plasma membrane and, unfortunately, loss of cellular integrity results in inactivation of the complex. Carpita and Delmer177*178 proposed that one feature of an intact cell, that is, a transmembrane, electrical potential (A$) may be an important factor for maintaining an active, cellulose-synthetase complex. This hypothesis evolved from observations of another effect of PEG-4000 on cellulose synthesis in cotton fibers; it was found that cutting of intact fibers (just once) with scissors resulted in an essentially total cessation of the synthesis of radioactive cellulose from radioactive D-ghCOSe supplied. [The synthesis of (1+3)-p-D-glucan, was not, however, substantially lessened, suggesting that energy-generating systems neces(176) C. T. Brett, Plant Physiol., 62 (1978) 377-382. (177) N. C. Carpita and D. P. Delmer, Plant Physiol., 66 (1980)911-916, (178) N. C. Carpita, in Ref. 10, pp. 225-242.

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DEBORAH P. DELMER

n-n- L n :ontrol

K - MES

4L

K-l

SIVAL

(+)

BTP/N03 (-)

Frc. 5.-Stimulation of UDP-g1ucose:glucan Synthetase Activities by Conditions that Lead to Induction of a Transmembrane, Electrical P0tentia1.l~~ {The experiment was performed by using membrane vesicles prepared from developing cotton-fibers;

into total P-D-glUCanS was meaincorporation of radioactivity from UDP-~-[~~C]glucose sured. Anion and cation concentrations were 50 mM; valinomycin (VAL) was present at 5 p M ;and UDP-glucose at 0.1 mM; 1 pCi per pmol.}

sary for synthesizing activated Dglucose substrates were not seriously impaired by this procedure.] However, if the fibers were cut in the presence of 0.06 molal PEG-4000, the rate of cellulose synthesis observed was 50% of that of uncut fibers. A variety of observations led to the conclusion that this protective effect of PEG is mainly due to a promotion of resealing of the cut fiber-membranes, which thereby results in restoration of an “intact” cell. Thus, it was reasoned that some feature of an intact cell was essential for maintaining active synthesis of cellulose. It was possible to rule out turgor pressure, and to propose, instead, that a transmembrane, electrical potential could be the critical factor required. Results of later studies by Delmer and coworker^^^^*^^^ provided some support for this hypothesis. Thus, it was shown that re-establishment of a transmembrane, electrical potential (positive-inside) across vesicles isolated from cotton fibers resulted in a 4-12-fold stimulation of p-D-glucan synthesis from UDP-glucose (see Fig. 5). Such a potential was established by the addition of K+ in the presence of an imper-

-

(179) D. P. Delmer, N. C. Carpita, A. Bacic, and D. Montezinos, Proc. Ekman-Days Int. Symp. Wood Pulp. Chern., 3 (1981) 25-27.


149

meant anion, such as 2-(4-morpholino)ethanesulfonicacid (MES), to membrane vesicles incubated in the presence of valinomycin (VAL). VAL is a K+-specific ionophore that allows the free movement of K+ down its concentration gradient, thereby establishing a net, positive-inside potential. Conditions that should lead to a negative-inside potential, such as the addition of an impermeant cation, for example, 1,3-bis[tris(hydroxymethyl)methylamino]propane (“bistrispropane”; BTP) together with a permeant anion such as NO3-, resulted in no substantial stimulation. Other experiments provided further evidence that the effect was truly due to a creation of A$, and not just to stimulation by K+; it was also shown that creation of a ApH across the vesicles did not lead to stimulation; only creation of A$ (positive-inside) was successful. It is of interest that only a positive-inside potential was effective. As UDP-glucose can presumably only be accepted as substrate from the inner face of the plasma membrane, it is presumed that only inverted vesicles are active; therefore, a positive-inside potential in an inverted vesicle mimics the in vivo situation, which is negative-inside. Analyses of the linkages found in the products of the stimulated reaction revealed that polymerization of both p-( 1+3)- and p-( 14)-glucosyl residues was enhanced. Once again, it is necessary to point out that no proof exists at present that this UDP-glucose:(14)-p-&glucan synthetase activity is related to a true, cellulose-synthesizing reaction. Results of Delmer and coworkers,’80with A. xylinum also indicated that the existence of a A$ across the cell membrane may be a crucial factor for maintaining active synthesis of cellulose. In these studies, it was shown that dissipation of A$, by addition of K+ and VAL to EDTA-treated, A. xylinum cells in the presence of the impermeant anion S042-, resulted in an essentially complete inhibition of cellulose synthesis from supplied D-glucose. When the experiments were performed under conditions where energy metabolism could still be driven18’ by a transmembrane ApH (that is, at pH 4.5;ApH -1.3 or 76 mV), the effect of dissipating A$ was specific for cellulose synthesis; it was also reversible, because, when cells were transferred from a high-K+ medium to a high-Na+ medium, cellulose synthesis resumed. Several possible mechanisms can be envisaged to explain how A$ may modulate the activity of the cellulose-synthetase complex. The effects of membrane fluidity on membrane-bound enzymes is well (180) D. P. Delmer, M. Benziman, and E. Padav, Proc. Natl. Acad. Sci USA, 79 (1982) 5282-5286. (181) E. D. Padan, D. Zilberstein, and H. Rottenberg,Eur. J . Biochern., 63 (1976) 533541.

1*50

DEBORAH P. DELMER

documented.’”2 Lelkes’s:’ has shown that changes in AJ, markedly influence the fluidity (and, almost certainly, also, the orientation of lipids) in phospholipid vesicles. Thus, it is quite conceivable that changes in the lipid environment surrounding a highly organized, enzyme complex could result in conformational changes in the complex. Another possibility is that changes in A+ could influence the movement of substrates (UDP-glucose, lipid intermediates, or proteinlinked carriers) within the membrane. In any case, the loss of A+ upon cellular disruption could well be one factor responsible for the loss of enzyme activity in citru. VI. CONCLUSIONS Many difficulties have been encountered in the study of the biosynthesis of cellulose, chief among them being the apparent lability of the cellulose-synthetase system. Based on evidence accumulated to date, a current model of cellulose synthesis184can be envisaged as indicated in Fig. 6. Polymerization of the mglucan chains occurs by way of a multi-subunit, enzyme complex embedded in the plasma membrane; an almost simultaneous association, by means of hydrogen bonds, of the newly formed chains results in formation of partially crystalline microfibrils. This mechanism of polymerization and crystallization results in the creation of microfibrils whose chains are oriented parallel (cellulose I). In A. xylinurn, the complex is apparently immobile, but, in cells in which cellulose is deposited as a cell-wall constituent, it seems probable that the force generated by polymerization of the relatively rigid microfibrils propels the complex through the fluid-mosaic membrane. The direction of motion may be guided through the influence of microtubules. Much controversy has surrounded the question as to the nature of the active form(s) of D-glucose that serve(s) as precursor to cellulose. Current evidence strongly favors a role for UDP-glucose; much suggestive, but by no means conclusive, evidence indicates that lipid- or protein-linked intermediates, or both, may also be involved. Much of the difficulty in studying cellulose biosynthesis may be at(182) H. K. Kimeiberg, Cell Surf. Rev., 3 (1977)205-293. (183)P. I. Lelkes, Biochem. Biophys. Res. Commun., 90 (1979)656-662. (184)D.P. Delnier, in C. C. Black and A. Mitsui (Eds.), CRC Handbook Series ofBiosolar Resources, Vol. I : Basic Principles, CRC Press, Boca Raton, Florida, 1982, pp. 351-355.


Cell wall

1

u\

Plasma membrane

151

Cytoplasm

Mobile cellulose svnthetase

r

D- gtucose 6 - P

\I

0-

-

glucose I P

Individual D-glucan chain

.UDP

w

fructose Growing microfibril

microtubules?) involved in the orienting movement

sucrose

FIG.6.-Hypothetical Model for the Biosynthesis of [Numbers refer to reactions catalyzed by the following enzymes: 1, invertase (EC 3.2.1.26); 2, sucrose synthetase; 3, hexokinase (EC 2.7.1.1); 4, phosphoglucomutase (EC 2.7.5.1); 5, UDPglucose pyrophosphorylase; and 6, 7, and 8, hypothetical reactions in the pathway to cellulose.]

tributed to the apparent lability of the synthesizing complex. Such compounds as glycerol or PEG4000 have been found to offer some protection of UDP-glucose:( ld)-p-D-ghcan synthetase activity. A hypothesis that the activity may be modulated by the transmembrane, electrical potential may offer another clue to the lability of the complex in vitro. Nevertheless, it is clear that many questions concerning the nature of the complex, and of the process in general, remain unanswered. The writing of such an article as this is a tedious process; but, if even just one imaginative young scientist is stimulated to join the quest as a result of reading it, the effort will have been well worth while. The study of cellulose biosynthesis requires both a combination of careful scientific analyses and imagination, and the field can only profit from the entry of more scientists possessing these qualities. An invitation is extended to you to join in.

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DEBORAH P. DELMER

VII. ADDENDUM Since this article was sent to press, several noteworthy findings have been reported in the literature. The first concerns the structural identification of the radioactive compounds which appeared to serve as precursors to cellulose inA. xylinum (see Fig. 3). In a note added in proof to Ref. 168, Delmer and coworkers have concluded that the radioactive compounds present in the water- and alkali-soluble fractions analyzed by Swissa and coworkerss and Aloni and Benziman’O consist of a mixture of sugar phosphates (Dfructose 1,6-bisphosphate7 Dglucose 6-phosphate7and Dfructose &phosphate) and fine fibrils of cellulose. During a chase with unlabeled Dglucose, labeled carbon from the sugar phosphates is rapidly converted into cellulose, concomitant with a much slower, apparent “chase” of the fine fibrils of cellulose into the mat of larger aggregates of cellulose produced upon prolonged incubation in high concentrations of Dglucose. Thus, Delmer and coworkers168concluded that, as a result of a quite extensive analysis of the chloroform-methanol-soluble, water-, and alkali-soluble fractions, no positive evidence exists for intermediates beyond the level of UDP-glucose in A. xylinum. The second finding concerns reports by Aloni and coworkers185and Benziman and coworkersla6 of success in achieving high rates of in vitro synthesis of (1+4)-&~glucanfrom UDP-glucose by using membrane preparations derived from A. xylinum. The key to this success lay in the discovery that the A. xylinum enzyme-system can be activated by GTP. Activation by GTP requires the presence of an additional, protein factor; this factor tends to dissociate from the enzyme, but enzyme-factor association can be promoted by PEG-4000 or by Ca*+.Under optimal conditions, that is, in the presence of GTP, factor, and PEG4000 (or Caz+),initial rates of synthesis of (l-;.4)-P-~-glucan that are 200 times higher than any previously reported can be achieved; such rates exceed 40% of the in viuo rate of synthesis of cellulose in A. xylinum. The enzyme system has also been successfully solubilized by using digitonin, and the solubilized enzyme possesses high activity and displays all of the regulatory properties observed for the membrane-bound enzyrne.l8’ These findings offer new hope that future, in uitro studies can lead to a detailed understanding of the (185) Y. Aloni, D. P. Delmer, and M. Benziman, Proc. Natl. Acad. Sci. USA, (1982) in press. (186) M. Benziman, Y. Aloni, and D. P. Delmer, J . A p p l . Polym. Sci., (1982)in press. (187) Y. Aloni, M. Benziman, and D. P. Delmer,J. BWL. Chem., (1983) in press.


153

mechanism and regulation of the synthesis of cellulose in A. xylinum. It will certainly also be of interest to know if the activation by poly(ethy1ene glyco1)s of plant D-ghcan synthetases (see Section V, 5,b) relates to a similar, regulatory mechanism for the synthesis of cellulose in higher plants.


.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY VOL. 41

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES BY HAROLDJ . JENNINGS Division of Biological Sciences. National Research Council of Canada. Ottawa. Ontario KIA OR6. Canada

I . Introduction ............................. . . . . . . . . . . . . . . . . .155 I1. Structures of Capsular Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . .158 158 1. Neisseria meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 2 . Haemophilus influenzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 3 . Group B Streptococcus ....................................... 170 4 . Streptococcus pneumoniae .................................... 111. Other Important Structural and Physical Features of Capsular Polysaccharides ...................................... 174 . . . . . . . . . . . . 174 1. Structural Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . .175 2. Determinants and Immunological Specifi 3. Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 4 . Molecular Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 183 5 . Location .................................................. IV . Immune Response to Bacterial Infection ............................ 186 187 1. Phagocytosis ............................................... 2 . Role of Complement ......................................... 187 189 3 . Humoral Antibodies to Polysaccharide Vaccines .................... V. Polysaccharide Vaccines and Immunity ............................. 191 1. Streptococcus pneumoniae .................................... 191 ............................ 193 2 . Neisseria meningitidis . . . . . . . 3 . Haemophilus injluenzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 4 . Group B Streptococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 197 5 . Polysaccharide-Protein Conjugates ............................. 6 . Natural Immunity, and Polysaccharide Serological Cross-reactions . . . . . .200 VI . Bacterial Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 1. Role of the Capsular Polysaccharide ............................. 202 206 2. Polysaccharide Structure and Pathogenicity .......................

I . INTRODUCTION Vaccination has proved to be one of the most useful scientific developments in the control and eradication of human disease . Early vaccines were based on whole-organism preparations. or on protein toxins isolated from different bacteria and. although these methods 155

Copynght @ 1983 by Academic Press. Inc. All nghts of reproductlon in any form reserved. ISBN 0-12-007241-6

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HAROLD J. JENNINGS

are still effective, the discovery of a “specific soluble substance” secreted by pneumococcal organisms during growth,’ and the immunogenicity of these substances (capsular polysaccharides),2 opened the door to a new and important development in vaccine technology. In 1923, Heidelberger and Avery3 demonstrated that this substance was, in fact, a type-specific, polysaccharide antigen that was able to precipitate: quantitatively, antibodies produced in animals by injection of the homologous, whole organisms. Heidelberger5 has given an interesting account of these significant, early discoveries of the immunogenicity of polysaccharides. In subsequent, pioneering work, he and his associates6 demonstrated that, when used as human vaccines, these purified, pneumococcal polysaccharides provide type-specific protection against pneumococcal infections. However, at that critical stage of development, the phenomenal success of the newly discovered antibiotics in treating bacterial diseases overshadowed the early promise of polysaccharide vaccines. Since then, the prophylaxis of bacterial disease has been the subject of renewed, intensified research,’ due in large part to the expanding incidence of antibiotic-resistant, bacterial strains6 Also, clinical and epidemiological studies have demonstrated that the antibiotic treatment of infectious diseases caused by encapsulated bacteria does not always prevent their morbidity and m ~ r t a l i t y Thus, .~ “cured” H. inJuenzue type b rneningitidis is the leading cause of acquired mental retardation,9 and epidemiological statistics indicate that deaths due to pneumococcal pneumonia occur at the same rate as in the pre-antibiotic era.’oCurrent interest in the capsular polysaccharides has evolved simultaneously with this resurgence of interest in the prophylaxis of human, bacterial disease, because of their potential as good immunogens in providing protection against bacterial infections. The concept of using a purified polysaccharide immunogen devoid of its accom(1) A. R. Dochez and 0. T. Avery, J . E r p . Med., 26 (1917)477-493. (2) T. Francis, Jr., and W. S. Tillet,]. E r p . Med., 52 (1930) 573-585. (3) M. Heidelberger and 0. T. Avery,]. E x p . Med., 38 (1923) 73-79. (4) M. Heidelberger and F. E. Kendall,]. E x p . Med., 61 (1935) 563-591. (5) M.Heidelberger, Annu. Reo. Microhiol., 31 (1977) 1-12. (6) C. M . McLeod, R. G. Hodges, M. Heidelberger, and W. G. Bernhard,J. E x p . Med., 82 (1945) 445-465. (7) J. B. Rohbins, Zmmunochemistry, 15 (1978) 839-854. (8) M. Finland, Rec;. Infect. Dis., 1 (1979) 4-21. (9) H. W. Sell, R. E. Merril, 0. E. Doyne, and E. P. Zimsky, Pediatrics, 49 (1972)206211. (10) R. Austrian, in R. F. Beers, Jr., and E. G. Bassett (Eds.),The Role of Immunological Factors in Infectious, Allergic and Autoimmune Processes, Raven Press, New York, 1976, pp. 79-89.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

157

panying, complex, bacterial mass is technically elegant. Besides their demonstrated immunogenicity in man, these materials are nontoxic, thus avoiding unpleasant (for example, pyrogenic) and, possibly, other deleterious effects associated with whole-cell vaccines. Another important feature of these purified, polysaccharide immunogens is that they can be chemically and physically defined: criteria that add a greater measure of control over their efficacy as vaccines than can be attained by using whole-cell vaccines. As a measure of the success of these vaccines, up to 1978, 130 million individuals had been immunized with capsular polysaccharides, resulting in a high degree of protection and no fatalities or significant adverse effects.' The purpose of this Chapter is to outline the development of bacterial-polysaccharide vaccines, and also to relate the structures of these capsular polysaccharides to their many roles in the immune response to bacterial infection. Because bacterial disease is host-related, this article will be concerned only with bacterial polysaccharides associated with human disease, particularly those capsular polysaccharides currently being used as human vaccines, or those having some immediate potential as human vaccines. The requirements that mediate this decision include clinical importance, the presence of meaningful epidemiological studies, and the identification of a stable, polysaccharide immunogen. Although Klebsiella pneumoniae" and Staphylococcus aureus'* possess defined, capsular polysaccharides, they have not yet satisfied the first two requirements, and will thus be referred to only briefly. The genus Klebsiella is largely restricted to hospital infections, and, because it has 72 serotypes, more-comprehensive epidemiological studies will be needed before the design of a capsularpolysaccharide vaccine is p ~ s s i b l e . ' ~ In a number of pathogenic bacteria (for example, Salmonella and Shigella), the capsular polysaccharide is replaced by the 0-chain polysaccharide of their lipopolysaccharides. Although these 0-chains have been demonstrated to be the immunological equivalent of the capsular poly~accharides,~~ they will not be covered in this Chapter, because they are unique, in that they can only be isolated from bacteria in their high-molecular-weight form, attached to a highly toxic, and physiologically active, lipid A moiety. This circumstance has generally discouraged the development of lipopolysaccharide vaccines,

-

(11) W. Nimmich, Z. Med. Mikrobiol. Zmmunol., 154 (1968) 117-131. (12) W. W. Karakawa and A. J. Kane,]. Zmmunol., 115 (1975) 564-568. (13) J. Z. Montgomerie, Reo. Infect. Dis., 1 (1979) 736-748. (14) 0.Liideritz, 0.Westphal, A. M. Staub, and H. Nikaido, in G . Weinbaum, S. Kadis, and S. J. Ajl (Eds.),Microbial Torins, Vol. IV, Academic Press, New York, 1971, pp. 145-233.

158

HAROLD J. JENNINGS

as the removal of lipid A results in the non-immunogenicity of the resultant O-chain. However, the conjugation of these 0-chains to nontoxic, protein carriers has obvious significance in the future development of 0-chain A previous review of microbial p~lysaccharides'~ has been updated by reviews on the structure'8 and immunological r e ~ p o n s e of ' ~ polysaccharides; a pertinent and comprehensive review of vaccines for the prevention of encapsulated bacterial diseases has also been published.' The term polysaccharide has been used throughout this Chapter, although, strictly speaking, some of the phosphorylated, capsular antigens bear a close, structural resemblance to teichoic acids.

11. STRUCTURESOF CAPSULARPOLYSACCHARIDES I. Neisseria meningitidis Neisserin meningitidis is a Gram-negative organism that has been classified serologically20into groups A, B, C, 29-e, W-135,X, Y, and Z. This grouping system depends on the presence of capsular polysaccharides that, although identified some time ago in the case of groups A (Ref. 21) and C (Ref. 22), were not compositionally defined until later.'""'" In these studies, it was established that the group A polysaccharide is a partially 0-acetylated, (1+6)-linked homopolymer of 2acetamido-2-deoxy-D-mannopyranosyl and that groups B and C pol ysaccharides are homopolymers of sialic

(15)S. R. Svenson and .4. A. Lindberg,]. Immunol., 120 (1978)1750-1757. (16)H. J. Jorbeck,S. B. Svenson, and A. A. Lindberg, Infect. Immun., 32 (1981)497502. (17)K. Jann and 0. Westphal, in M. Sela (Ed.), The Antigens, Vol. 111, Academic Press, New York, 1975,pp. 1-125. (18) L. Kenne and B. Lindberg, in G. 0. Aspinall (Ed.), The Polysaccharides, Vol. 2 , Academic Press, New York, in press. (19)C. T.Bishop and H. J. Jennings, in Ref. 18,Vol. 1, 1982,pp. 291-330. (20) E. C . Gotschlich, T.-Y. Lui, and M. S. Artenstein,J. Exp. Med., 129 (1969)13491365. (21) E. A. Kabat, H. Kaiser, and H. Sikorski,]. E x p . Med., 80 (1944)299-307. (22)R. G. Watson, G. V. Marinetti, and H. W. Scherp,]. Immunol., 81 (1958) 337-344. (23)T.-Y. Lui, E. C. Gotschlich, E. K. Jonssen, and J. R. Wysocki,]. B i d . Chem., 246 (1971)2849-2858. (24) T.-Y. Lui, E. C. Gotschlich, F. T. Dunne, and E. K. Jonssen,]. Biol. Chem., 246 (1971)4703-4712.


159

3-Deoxy-Dmanno-2-octu~osonicacid (KDO) has also been identified as a component of the group 29-e polysaccharidez5Sz6 and the Escherichia coli K6 capsular poly~accharide.~~ Because of the presence of complex, 3-deoxy-2-glyculosonic acid components and phosphoric diester linkages in these polysaccharides, plus their great fragility, particularly to acid treatment, problems were encountered in the application of more-conventional, chemical techniques to their structural elucidation. This situation prompted a search for new techniques with which to tackle these problems, and one that has been used with great success is l3C-nuclear magnetic resonance (l3C-n.m.r.) spectroscopy. The potential of this technique as applied to polysaccharides had been demonstrated in studies on amyloseZ8and the more-complex polysaccharide heparin.29Subsequent studies on the polysaccharides of N. meningitidis and other bacterial polysaccharides (see later) served to consolidate the method as a powerful technique in structural and conformational investigations of polysaccharides. Since the earlier studies, reports of the application of this technique to other bacterial polysaccharides, and to polysaccharides in general, have been prodigious; these, beyond the scope of this article, have been discussed in a previous Volume of this Series.3oMore-pertinent reviews on the application of 13C-n.m.r. spectroscopy to polysaccharides of human pathogenic bacteria have also been p u b l i ~ h e d . ~ ~ * ~ ~ The structures of the repeating units of these capsular polysaccharides of N. meningitidis are shown in Table I. Although all of the polysaccharides are linear and acidic, and contain acetamido groups, they can be divided into two categories, based on their acidic components: those containing phosphoric diester bonds, and those containing 3deoxy-2-glyculosylonic acid residues. Interestingly, except for the group A polysaccharide, on which some structural information was al-

(25) A. K. Bhattachaqjee, H. J. Jennings,and C. P. Kenny, Biochem. Biophys. Res. Commun., 61 (1974)439-443. (26) A. K. Bhattacharjee, H. J. Jennings,and C. P. Kenny, Biochemistry, 17 (1978)645651. (27) F. M. Unger, Ado. Carbohydl-. Chem. Biochem., 38 (1981)323-388. (28) D. E. Dorman and J. D. Roberts,]. Am. Chem. Soc., 92 (1970) 1355-1361. (29) A. S. Perlin, N. M. K. Ng Ying Kin, S. Bhattacharjee, and L. F. Johnson, Can. J . Chem., 50 (1972)2437-2441. (30) P. A. J. Gorin,Adu. Carbohydr. Chem. Biochem., 38 (1981) 13-104. (31) H. J. Jennings,A. K. BhattacharJee,D. R. Bundle,C. P. Kenny, A. Martin, and I. C. P. Smith,]. Infect. Dis., Suppl., 136 (1977) s78-s83. (32) W. Egan, in J. S. Cohen (Ed.),Magnetic Resonance in Biology, Vol. 1, Wiley, New York, 1980, pp. 197-258.

HAROLD J. JENNINGS

160

TABLEI Structures of the Capsular Polysaccharides of Neisseria meningitidis Group

structure

References

0

1I

A

+

6)-a~-ManpNAc-l-O-P-O-

9

33

I OH

I

OAc + 8)aD-NeupAc(2+ + 9)aD-NeupAc(2+ 718

B C

34 34

I

I

OAc + 3)-a~-CalpNAc(l + 7)p~-KDOp(2 + 4(5

29e

26

I

OAc -+ 6)-a~-Galp( 1+ 4)a~-NeupAc(2+ 0

W-135

35

II

X

+~)~D-G~c~NAc-~-O-P-O-

33

I

Y

+

OH fi)-a~-Glcp(l-+4)a~-NeupAc(2-+ (contains OAc groups) 0

35

II Z

+

3)-a&CalpNAc(l

+

l)glycerol-3-O-P-O-

36

I

OH

ready available,23all of the other structures were deduced entirely by I3C-n.m. r. s p e c t r o s ~ o p y . ~ ~ ~ ~ ~ * ~ ~ - ~ ~ Some of the fundamental principles involved in these structural The lacanalyses are outlined here for the group A poly~accharide.~~ n.m.r. spectrum thereof is shown in Fig. 1; although complex, due to the presence of 0-acetyl substituents, it is considerably simplified, to an eight-resonance spectrum (carbonyl signal at 175.8 p.p.m. not (33) D. R. Bundle, I. C. P. Smith, and H. J. Jennings,]. Biol. Chern., 249 (1974) 22752281.

(34) A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and 1. C. P. Smith, J . B i d . Chern.,250 (1975) 1926-1932. (35) A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and 1. C . P. Smith, Can. 1. Biochem., 54 (1976)1-8. (36) H. J. Jennings, K.G. Rosell, and C. P. Kenny, Can.]. Chern., 57 (1979)2902-2907.


161

p.p.m.

FIG. 1. -Fourier-transformed, %-N.m.r. Spectrum of the Native Polysaccharide Antigen of Croup A Neisseria meningitidis. [Upper, containing ( a )0-acetylated and ( b ) unacetylated residues, and lower, its fully O-deacetylated form.]

shown), on removal of the 0-acetyl groups. This simplicity indicated that the polysaccharide consists of a linear arrangement of a 2-acetamido-mannopyranosyl phosphate repeating-unit. The individual signals in the spectrum of the 0-deacetylated, group A polysaccharide were assigned by using the generally applicable, empirical methodology of comparing the chemical shifts of the signals of the polysaccharide with the corresponding chemical shifts of the anomers of its monomeric or oligomeric constituents. Experience has indicated that the chemical shifts of the monosaccharides are similar to those of the monosaccharide residues within the polysaccharide, except for substituent These effects, caused by the attachment of any substituent to a sugar moiety, cause an increase in the chemical shift of the carbon atom directly involved in the linkage; this increase is usually accompanied by a deckease of smaller magnitude (or, sometimes, an increase) in the chemical shifts (37) H. J. Jennings and I. C. P. Smith, Methods Enzymol., 5OC (1978)39-50. (38) H. J. Jennings and I. C. P. Smith, Methods Carbohydr. Chem., 8 (1980)97-105.

162

HAROLD J . JENNINGS

of the neighboring P-carbon atoms. Thus, these chemical-shift differences serve to determine the position of linkages; similarities in chemical shifts, especially those involving carbon atoms known to be sensitive to change in anomeric configuration, can be employed to determine the configuration of linkages. By using this approach, the l-+6)-linked.33 ( The linkgroup A polysaccharide was shown to be a - ~ age position was also confirmed by the pattern of the two- and threebondi11P-13Ccoupling manifest in the spectrum of the O-deacetylated polysaccharide. Chemical-shift differences between the signals of the native, and the O-deacetylated, group A polysaccharide (see Fig. 1)indicated that the O-acetyl substituents were linked to 0-3 of the 2-acetarnido-2-deoxy-~-mannopyranosyl residues; a comparison of the intensities of' the characteristic methyl signals of the O-acetyl and N-acetyl groups indicated that 70% of these residues were substituted in this way. A similar analysis indicated that the analogous, gronp X polysaccharide is composed of a repeating unit of a-~-(l+4)-linked2acetarnido-2-deoxy-D-glucopyranosyl phosphate.33 The group Z polysaccharide can be included in the same category as the aforementioned polysaccharides, as it also contains phosphoric diesters.36 The structure was shown to be a repeating unit of 10-(2-acetamido-2deoxy-a-D-ga1actopyranosyl)glyceroljoined through phosphoric diester groups at 03 of glycerol and 03 of the 2-amino-2-deoxy-D-galactose residue. However, in this case, the phosphoric diester is not glycosidically linked to the 2-amino-2-deoxy-~-galactoseresidue, and the structure closely resembles that of a teichoic acid. In the second category of meningococcal polysaccharides, those containing 3-deoxy-2-glyculoses, the group B and C polysaccharides are the simplest in structure34;this is illustrated in the 13C-n.m.r.spectrum of the O-deacetylated, group C polysaccharide, shown in Fig. 2. The simple, eleven-resonance spectrum indicated that the group C polysaccharide is a linear polymer of sialic acid. The group B polysaccharide gives a similar, simple, eleven-resonance spectrum. By using the methyl a- and p-D-ketosides of sialic acid as model compounds, and comparing the chemical-shift differences between some of their carbon atoms with those of the sialic residues in the polysaccharides, it was indicated that the group B polysaccharide is (2-+8)-linked, whereas the group C polysaccharide is (2+9)-linked.34 Similarities in the chemical shifts of the configurationally sensitive signals (C-1, C 4 , and C-6) of the polysaccharides with those of the methyl a-D-ketoside, permitted the a-Dconfiguration to be assigned to both polysaccharides. The carboxylate signal (C-1) proved to be extremely useful in these configurational determinations, as it is readily discernible and undergoes a significant, chemical-shift displacement (- 2 p.p.m.) with


r

163

I4m7

5

P.P.m FIG.2. -Fourier-transformed, W-N.m.r. Spectrum of the Native Polysaccharide Antigen of Group C Neisseria meningitidis (upper),and its 0-Deacetylated Form (Iower).

change in anomeric config~ration.~~ This characteristic displacement is dependent on the orientation of the carboxylate group (axial or equatorial), and could prove to be generally applicable to 3-deoxy-2glyculose r e s i d ~ e s . ~ ~ , ~ ~ , ~ ~ The 0-acetyl substituents of the native, group C polysaccharide were located by comparing its 13C-n.m.r. spectrum with that of the 0deacetylated, group C polysaccharide (see Fig. 2). The appearance of characteristic muhiplets in some of the signals of the native polysaccharide showed that the 0-acetyl groups are distributed exclusively between 0-7 and 0-8of its sialic acid residues. The group Y and W-135 capsular polysaccharides also contain sialic acid. However, unlike the groups B and C polysaccharides, they are not homopolymers, as they also contain, respectively, D-glucosyl and D-galactosyl residues.35 Structural studies indicated that the group Y (39) H. J. Jennings and A. K. Bhattacharjee, Carbohydr. Aes., 55 (1977) 105-112. (40) H. J . Jennings, K.-G. Rosell, and K. G. Johnson, Carbohydr. Res., 105 (1982)4556.

164

HAROLD J . JENNINGS

polysaccharide has a -+6)-a-D-Glcp-(14)-a-~-NeupAc-(2+repeating unit, whereas that of group W-135 has a -6)-a-D-Galp-(l+4)-a-DNeupAc-(2+ repeating unit.35Interestingly, these two, serologically distinguishable, polysaccharides differ only in the configuration of one hydroxyl group in their respective, disaccharide repeating-units. The group Y polysaccharide contains 0-acetyl groups (1.3mol per sialic acid residue), but the locations of these have not yet been established. The group 29-e polysaccharide is composed of an alternating sequence of 3-deoxy-p-~-manno -2-octulosylonic acid and S-acetamido2-deoxy-a-~-glucopyranosyl residues; linkage is to 0-7 of the former and to 03 of the latter. 0-Acetyl substituents were also located on both 04 and 0-5 of the KDO residues.26It is of interest that, whereas sialic acid has only been found in bacterial polysaccharides, and elsewhere in Nature, as its a-Danomer, there is strong evidence to suggest that KDO probably exists in bacterial polysaccharides in both of its anomeric f ~ r m ~ . ~ ~ , ~ ~ 2. Haemophilus influenzae The Haemophilus influenzae are Gram-negative organisms that can be serologically classified into six types (a through f) on the basis of their type-specific, capsular polysaccharides. Analytical studies indicated that those of types a, b, c, and f a r e poly(sugar phosphates):’ whereas that of type e contains a “hexosamine-uronic acid” component,’2 since characterized as 2-acetamido-2-deoxy-~-mannuronic acid.43*,44 This component sugar is also a constituent of the type d polys a c ~ h a r i d e . Thus, ~ ~ . ~ ~like the meningococcal polysaccharides, the type-specific polysaccharides of H. influenzae may be divided into two groups on the basis of their acidic components, 2-acetamido-2deoxy-D-mannuronic acid replacing the 2-glyculosonic acids of the former. The structures of the type-specific polysaccharides are shown in Table 11, and, except for one particular of type e, are composed of linear arrangements of disaccharide repeating-units. The clinically (41) E. Rosenberg, G . Leidy, J. Jaff, and S. Zamenoff,]. Biol. Chern., 236 (1961) 28412844. (42) A. R. Williamson and S. Zamenoff, J . Biol. Chem., 238 (1963)2255-2258. (43) P. Branefors-Helander, L. Kenne, B. Lindberg, K. Petersson, and P. Unger, Carbohydr. Res., 88 (1981)77-84. (44) F.-Y.Tsui, R. Schneerson, and W. Egan, Carbohydr. Res., 88 (1981)85-92. (45) P. Branefors-Helander,L. Kenne, B. Lindberg, K. Petersson, and P. Unger, Carbohydr. Res., 97 (1981)285-291. (46) F.-P. Tsui, R. Schneerson, R. A. Boykins, A. B. Karpas, and W. Egan, Carbohydr. Res., 97 (1981)293-306.

165

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES TABLEI1 Structures of the Capsular Polysaccharides of Haemopkilus influenzae

Type

References

Structure

0 a

ll

+ 4)p~-Glcp(l+4)D-ribito&O-P-O-

48

I

OH 0

II

b

-+

3)p~-Ribf(l-+ l)~-ribito1(5-O-P-O-

47,s

I

OH 0

II C

d e

4)pD-GIcpNAc(l+ 3fc~D-Gdp(l-O-P-O3 I t OH OAc + 4)p~-GlcpNAc( 1+ 3)pD-ManpANAc(1-+ -+ 3)p~-GlcpNAc( 1+ 4)p~-ManpANAc( 1+ 3 -+

49,50

45,46 43,44

t

2 p~-Fr~p 0

f

1I

.+ 3)p~-GalpNAc( 1+ 4)a~-GalpNAc( 1-O-P-O-

3

t

51,52

I

OH

OAc

important, type b polysaccharide was the first to have its structure determined4'; I3C-n.m.r. spectroscopy played a prominent role in this early investigation. This technique was also extensively used in subsequent structural determinations on the other H. influenzae, typespecific p o l y ~ a c c h a r i d e s . ~ ~ - ~ ~ ~ ~ ~ - ~ ~ (47) R. M. Crisel, R. S. Baker, and D. E. DormanJ. Biol. Chem., 250 (1975)4926-4930. (48) P. Branefors-Helander,C. Erbing, L. Kenne, and B. Lindberg, Carbohydr. Res., 56 (1977) 117-122. (49) P. Branefors-Helander, B. Classon, L. Kenne, and B. Lindberg, Carbohydr. Res., 76 (1979) 197-202. (50) W. Egan, F.-P. Tsui, P. A. Climenson, and R. Schneerson, Carbohydr. Res., 80 (1980) 305-316. (51) P. Branefors-Helander, L. Kenne, and B. Lindqvist, Carbohydr. Res., 79 (1980) 308-3 12. (52) W. Egan, F.-P. Tsui, and R. Schneerson, Carbohydr. Res., 79 (1980)271-277.

166

HAROLD J. JENNlNGS

Except for the anomeric configuration of the ribofuranosyl residue,53 the repeating unit (1) of the type b polysaccharide was proposed by

Crisel and coworker^.^' They established that the type b polysaccharide is composed of ribose, ribitol, and phosphate in the molar ratios of 1: 1: 1, and, by periodate oxidation studies, that the ribitol is linked at both of its hydroxymethyl groups. The 13C-n.m.r.spectrum ofthe type b polysaccharide exhibited ten individual, carbon signals, indicative of a simple, linear arrangement of the disaccharide repeating-unit. The presence of 13C-31Pscalar couplings in five of these signals was also consistent with the structure proposed. Later studiess3 established the chirality (D) of the ribitol residue, and the anomeric configuration (p-D)of the ribofuranosyl residue. Additional structural studies%*confirmed the structure of the type b polysaccharide. The remaining N. infEuenzae polysaccharides in the phosphoric diester category (a, c, and f) were structurally elucidated by. using similar procedures, and were shown to have other structural similarities; all of them are composed of linear, disaccharide phosphate repeatingunits. The type a polysaccharide, which contains D-glucose, D-ribitol, and phosphate in the molar ratios of 1:1:1was shown to be composed of 4-O-~-D-ghcopyranosyl-D-ribitolresidues linked by phosphoric . ~ independent ~ diesters between 0-5of ribitol and 03 of D - g l u ~ o s eIn studies, two different groups of researchers proposed identical structures for the respective type c (Refs. 49 and 50) and type f (Refs. 51 and 52) polysaccharides. The type c polysaccharide was reported to conand phosphate in the tain D-galactose, 2-amino-2-deoxy-D-glucose, (53) P . Branefors-Helander, C. Erbing, L. Kenne, and B. Lindberg,Acta Chem. Scund., Ser. B, 30 (1976)276-277. (54) B. A. Fraser, F.-P. Tsui, and W. Egan. Carbohydr. Res., 73 (19'79)59-65.


167

molar ratios of 1:1:1. The structure is based on a 34-(2-acetamido-2deoxy-p-D-glucopyranosyl)-a!-D-galactopyranosyl phosphate repeating-unit, and the phosphate is attached to 0-4of the 2-amino-2-deoxyD-glucose residues in the polysaccharide structure. The type c polysaccharide also contains acetyl substituents situated at 03 of 80% of the 2-amino-2-deoxy-D-glucose residues. The structures of the two remaining 2-acetamido-2-deoxy-~-mannuronic acid-containing, H. injluenzae capsular polysaccharides (d and e) were also elucidated independently by the same two groups of re~earchers.~ Both ~ - ~polysaccharides ~ also contain 2-amino-2-deoxyD-glucose, and the structures of both are based on alternating 2-amino2-deoxy-D-glucose and 2-amino-2-deoxy-D-mannuronic acid residues. The type d p o l y ~ a c c h a r i d eis~ composed ~~~~ of a +4)-/3-D-GlcpNAc(1+3)-/3-~-ManpANAc-( l+ repeating unit, with L-alanine, L-serine, or L-threonine linked to the carboxylate group of 2-acetamido-2deoxy-D-mannuronicacid by an amide bond, whereas the type e polyis composed of a differently linked, -*3)-/3-D-GlcpNAc(1+4)-p-~-ManpANAc-(l+ repeating-unit. Strain-dependent structure-variations have also been demonstrated for the type e polysaccharide.43 One particular strain of the type e organism produced a polysaccharide possessing the foregoing structure but having additional, terminal p-D-fmctofuranosyl groups linked to 03 of all of the 2-acetamido-2-deoxy-D-mannuronic acid residues. 3. Group B Streptococcus

L a n ~ e f i e l d ~characterized ~-~' two polysaccharide antigens obtained from Group B Streptococcus: a group antigen common to all strains, and the type-specific, capsular polysaccharides that distinguish four major serotypes, namely, Ia, Ib, 11, and 111. The type-specific polysaccharides were originally isolated by extraction of the whole, streptococcal organisms with hot hydrochloric acid, and all had identical constituents: galactose, glucose, and 2-acetamido-2-deoxyglucose.57-62 This extraction procedure produces immunologicallyincomplete antigens that form a lower molecular weight core to the complete, native (55) R. C. Lancefield,]. Exp. Med., 57 (1933) 571-582. (56) R. C. Lancefield,]. E r p . Med., 59 (1934)441-458. (57) R. C. Lancefield,]. E r p . Med., 67 (1938)25-40. (58) R. C. Lancefield and E. H. Friemer,]. Hyg., 64 (1966) 191-203. (59) H. Russell and N. L. Norcross,]. lmmutwl., 109 (1972)90-96. (60)J. A. Kane and W. W. Karakawa, Infect. lmmun., 19 (1978)983-991. (61) J. Y. Tai, E. C. Gotschlich, and R. C. Lancefield,]. E r p . Med., 149 (1979) 58-66. (62) D. L. Kasper, C. J. Baker, R. S. Baltimore, J. H. Crabb, G. Sc hihan, and H. J. Jennings,]. E x p . Med., 149 (1979) 327-339.

168

HAROLD J. JENNINGS

The latter antigens, which can be obtained by neutral or buffered (pH 7.0) extraction of whole organisms,61.62,65,67a contain additional, terminal sialic acid residues. The presence of these residues proved to be essential for the immunological expression of the native antigens. The native types Ia (Ref. 68, 69), Ib (Ref. 61, 69), and I11 (Ref. 63) antigens contain D-galactose, D-glucose, 2-acetamido-2-deoxy-~-glucose, and sialic acid in the molar ratios of 2 : 1:1:1, and constitute a group of isomeric polysaccharides, whereas the type I1 (Ref. 70) native antigen has identical component sugars, but in the ratios of 3 :2 : 1:1. The structures of the repeating units of the types Ia, Ib, 11, and I11 polysaccharides are shown in Table 111. These structures were elucidated by first determining the structures of the simpler, core antigens, as describedm for the type I11 polysaccharide. A comparison of the methylation analysis of the core with that of the native polysaccharide permitted the position of linkage of the terminal sialic acid residues in the native antigen to be established. The anomeric configurations of the sugar residues were determined by W-n.m.r. spectroscopy. Structurally, all of the Group B streptococcal antigens constitute an interesting group of polysaccharides, both in their relationships to each other, and to other biologically important molecules (glycoproteins). All of the polysaccharides have in their ~ t r u c t u r e s ~a*com~-~~ mon p-~-GlcpNAc-( l+S)-P-~-Galp-(1+4)-p-~-Glcp trisaccharide which forms the repeating unit of the backbone of the type I11 polysaccharide. This was also presumed, in a previously proposed structure:* to be true of the type Ia polysaccharide; however, the evidence on which this structure was based proved not to be definitive, as it was also compatible with an alternative structure in which the 2-amino-2deoxy-0-glucose residue of the trisaccharide becomes a part of the (63)H. J. Jennings, K.-C. Rosell, and D. L. Kasper, Can. ]. Biochem., 58 (1980) 112120. (64)E. H. Friemer,]. E x p . Med., 125 (1967) 381-392. (65) H. W. Wilkinson, Infect. Zmmun., 11 (1975) 845-852. (66) C. J. Baker and D. L. Kasper, Infect. Immun., 13 (1976) 284-288. (67) J. A. Kane and W. W. KarakawqJ. Immunol., 118 (1977) 2155-2160. (67a) C. J. Baker, D. L. Kasper, and C. E. Davies,]. E r p . Med., 143 (1976) 259-5370. (68) H. J. Jennings, K.-G. Rosell, and D. L. Kasper, Proc. Natl. Acad. Sci. U . S . A., 77 (1980) 2931-2935. (69) H . J . Jennings, E. M. Katzenellenbogen, C. Lugowski, and D. L. Kasper, Biochemistry, in press. (70) H. J. Jennings, K.-G. Rosell, and D. L. Kasper,]. Biol. Chem., in press. (71) H. J. Jennings, E. M. Katzenellenbogen, C. Lugowski, and D. L. Kasper, unpublished results.

TABLEI11 Structures of the Capsular Polysaccharides of Group B Streptococcus Type

Structure

References

Ia

+ 4)-p-D-Gkp-(1 + 4)-p-D-Gdp-(1 +

69

3

t p-~GlcpNAc 4

t

Ib

1 c~-~-NeupNAc-(2 + )-P-DGalp + 4)-p-Dklcp-(l + 4)-p-~-Galp-(l+ 3

69

t

1 p-~ClcpNAc 3

t

I1

1 a-D-NeupNAc-(S+ 3)-P-&alp --* 4)-p-D-GlcpNAc-(l+ 3)-B-D-Galp(l+ 4)-p-D-Glcp-(l -* 3 ) - p - ~ - G l ~ p - ( l 2)-8-~-Galp-( + 1+ 6 3

t

I11

1 p-DGdp +4)-p-~-Gl~p 1 --* ( B)-p-D-GlcpNAc-(1 + 3)+-D-G&-( 1 + 4

t

1 a-~-NeupNAc-(2--* 6)-/3-DGdp

70

f

2 a-D-NeupNAc 62

170

HAROLD J. JENNINGS

branches of the type Ia polysaccharide. This was indicated in subsequent, extensive degradation studies on the types Ia and Ib polysaccharides?” the results of which are consistent only with both having trisaccharide branches as shown in Table 111. All of the incomplete core-structures have branches terminating in P-D-galactopyranosyl residues ,63 ,I%-?1 and the fact that the type 111, core antigen has a structure identical to that of the capsular polysaccharide of type 14 S. pneum ~ n i a e is ? ~of some serological significance.62*63 in the native type Ia (Refs. 68 and 69), Ib (Ref. 69), and I11 (Ref. 63) polysaccharide antigens, the terminal P-D-galactopyranosyi residues of the core antigens are completely masked by sialic acid residues, forming branches that terminate in sialic acid residues. These sialic acid residues are linked to 0-3of the P-D-galactopyranosyl residues of the native type Ia (Refs. 68 and 69) and Ib (Ref. 69) polysaccharides and to 0-6 of the type I11 polysaecharide.63 These branches are of considerable serological importance to the group B streptococcal organism, because of their structiiral homology with some important, human serum-glycoproteins. The terminal 3-0-(N-acetyl-a-o-neuraminy~)-~-D-galactopyranosyl group of the types Ia and Ib antigens is the end group in the human $1 and N blood-group substance^,^^ and the 6-O-(N-acetyl-a-~-neuraminyl)-P-D-galactopyranosylmoiety of the type I11 polysaccharide is also a structural feature of human ~erotransferrin.~~ 4. Streptococcus pneumoniae

Strelitococczis pneumoniae are Gram-positive organisms which, like the group B Streptococcus, have a common, group antigen (Cs ~ b s t a n c e ) ,and ~ ~ .different ~~ type-specific, capsular polysaccharides. Unlike the organisms previously described, S. pneumoniae have been identified in a prolific number of immunologically distinguishable types based on these capsular polysaccharides. To date, there are at and these have been designated least 84 known type-spe~ificities,~.’O types 1-84 in the American system. However, on the basis of serological cross-reactivity among these polysaccharides, the organisms have also been conveniently classified into serologically related groups (see Table IV) in the Danish system. The pneumococcal polysaccharides are particularly important, because early investigations into (72) B. Lindberg, J. Likmgren, and D. A. Powel1,Carbohydr. Res., 58 (1977) 177-186. (73) J . E. Sadler, J. C. Paulson, and R. L. Hill,/. BioE. Chem., 254 (1979)2112-2119. (74) G . Spik, B. Bayard, B. Fournet, G . Streker, S. Bouquelet, and J . Montreuil, F E B S . k t t . , 50 (1975)296-299. (75) D. E. Brundish and J. Baddiley, Biochem.]., 110 (1968) 573-581. (76) H. J. Jennings, C. Lugowski, and N. M. Young, Biochemistry, 19 (1980) 47124719.


171

their immunological and structural properties resulted in the acquisition of knowledge fundamental to the development of human, polysaccharide vaccines. Structural studies on these polysaccharides also provided technical and conceptual contributions to the general problem of polysaccharide structure, and it is a tribute to earlier investigators that these contributions were made without the benefit of modem instrumental methods. The structures of the pneumococcal polysaccharides have been re~iewed.'~,''Because of their number and structural complexity (up to 7 sugar components in their repeating units), they can be dealt with only briefly in this chapter, and only the structures of those used in the current, pneumococcal vaccine (see Section V,1) are listed in Table IV. Two of the pneumococcal polysaccharides [types 14 (Ref. 72) and 37 (Ref. 78)] are neutral; in fact, that of type 14 is a rare example of a neutral-polysaccharide capsule involved in human bacterial The rest of the pneumococcal polysaccharides are acidic, and can be classified according to their common acidic components. Types 1 (Ref. 79), 2 (Ref. 80),3 (Ref. 81), 5 (Ref. 18), 8 (Ref. 82),9A (Ref. 83), 9N (Ref. 84), and 9V (Ref. 85) have D-glucuronic acid residues, whereas types 6A (Ref. 86), 6B (Ref. 87), 11A (Ref. SS), 13 (Ref. 89), 15F (Ref. W),17F (Ref. 91), 19F (Refs. 92 and 93),19A (Ref. 94),23F (Ref. 95), 27 (Refs. 96 and 97), 29 (Ref. 98),and (77) 0. L a m and B. Lindberg, Ado. Carbohydr. Chem. Biochem., 33 (1976) 295-322. (78) J. C. Knecht, G. Schiffman, and R. Austrian,J . Exp. Med., 132 (1979) 475-487. (79) B. Lindberg, B. Lindqvist, J. Lonngren, and D. A. Powell, Carbohydr. Res., 78 (1980) 111-117. (80) L. Kenne, B. Lindberg, and S. Svensson, Carbohydr. Res., 40 (1975) 69-75. (81) R. E. Reeves and W. F. Goebel,]. Biol. Chem., 139 (1941) 511-519. (82) J. K. N. Jones and M. B. Perry,]. Am. Chem. Soc., 79 (1957) 2787-2793. (83) L. G. Bennet and C. T. Bishop, Can. J . Chem., 58 (1980) 2724-2727. (84) H. J. Jennings, K . G . Rosell, and D. J. Carlo, unpublished results. (85) M. B. Perry, V. Daoust, and D. J. Carlo, Can.]. Biochem., 59 (1981) 524-533. (86) P. A. Rebers and M.Heidelberger,J. Biol. Chem., 139 (1941) 511-519. (87) L. Kenne, B. Lindberg, and J. K. Madden, Carbohydr. Res., 73 (1979) 175-182. (88) D. A. Kennedy, J. G. Buchanan, and J. Baddiley, Biochem.]., 115 (1969) 37-45. (89) M. J. Watson, J. M. Tyler, J. G. Buchanan, and J. Baddiley, Biochem.]., 130 (1972) 45-54. (90) M. B. Perry, D. R. Bundle, V. Daoust, and D. J. Carlo, Mol. Immunol., 19 (1982) 235-246. (91) M. B. Perry, personal communication. (92) H. J. Jennings, K . 4 . Rosell, and D. J. Carlo, Can.J. Chem., 58 (1980) 1069-1074. (93) H. Ohno, T. Y. Yadomae, and T. Miyazaki, Carbohydr. Res., 80 (1980) 297-304. (94) C.-J. Lee and B. A. Fraser,J. B i d . Chem., 255 (1980) 6847-6853. (95) M. B. Perry, V. Daoust, and R. Lowe, unpublished results. (96) L. C. Bennet and C. T. Bishop, Can.]. Chem., 55 (1977) 8-16. (97) L. C. Bennet and C. T. Bishop, lmmunochemistry, 14 (1977) 693-696. (98) E. V. Rao, M. J. Watson, J. G. Buchanan, and J. Baddiley, Biochem.]., 111 (1969) 547-556.

TABLEIV Structures of Some of the Capsular Polysaccharides of Streptococcus pneumoniae Contained in the Current, Pneumococcal Vaccine Structureb

Type"

w

1 2

References

79

-+ B)a-Sugp(1 + 4)aD-GalpA(1 S)aD-GalpA(1 + S)a~-Rhap(l S)a~-Rhap(l+ 3)P~-Rhap(l+ 4)aD-GIcp(l -+ 2 -+

-+

4

&a

80

-+

t

3 4

1 a ~ - G l c p A ( l - +6 ) a ~ G l c p + 4)PD-GlCp(l S)pD-GIcpA(1 -+ -+ 4)P~-ManpNAc(l + S)a~-FucpNAc(l-+ S)aD-GalpNAc(l -+

-+

4 ) a ~ - G a l (-+ l

H,C 5

-+

x

81 100

CO,H 18

Z)PD-GkpA(1 -+ B)aL-FucpNAc(1 -+ 4

t -+

2)aD-Gdp(1 -+

a-Sugp(l 3)crD-Glcp(1

1 0 4)P~Glcp II 3)a~-Rhap( 1 -+ 3)-ribitol-(5-O-P-O-

-+

-+

I

OH

86

12F (12)

+ 4)a~-FucpNAc( 1+ 3)p~-GalpNAc(l+ 4)p~-ManpANAc(l+

3

3

t

t

1

1 aDGalp a ~ - G l c p (+ l 2)aDGkp + 4)pD-Gkp(l+ G)pD-GlcpNAc(l+ 3)p~-Galp(l + 4

14

82 a4 101

72

t

1

0

II

19F (19)

2

+ 4)pD-ManpNAc(1+ 4)aD-Gkp(1--* 2)a~-Rhap( 1-O-P-O-

92,93

I

OH 23F (23)

+ 4)p~-Gfcp(l + 4)pD-Galp(l + 4)a~-Rhap(l+

I

2

P

t

1 aL-Rhap a

US. typing system in parentheses. *Sug = 2-Acetamido-l-arnino-2,4,6-trideoxygalactose.

95

174

HAROLD J . JENNINGS

34 (Ref. 99) contain phosphate. Type 27 also contains p y r u ~ a t e ~as ~,~' an additional acid component, and type 4 contains pyruvate as the sole acid component." Type 1 2 F contains 2-acetamido-2-deoxy-~-mannuronic acid as its only acidic component.101 The structure of the type 3 polysaccharide was the first to be established, by Reeves and Goebel,8' and thus this polysaccharide became a model for many immunological investigations. The concept of a repeating unit was also established in this work, and this was elegantly confirmed in later, chemical-degradation studies by Rebers and Heidelberger,H6in which they isolated the tetrasaccharide repeating-unit of the type 6A polysaccharide in crystalline form in 94% yield. Another early study!' in which partial hydrolysis with acid was employed, established the structure of the type 8 polysaccharide as having a tetrasaccharide repeating-unit containing cellobiouronic acid. Other, extremely valuable, early degradative studies were carried out b y Baddiley and coworkers on the types 11A (Ref. 88), 13 (Ref. 89),29 (Ref. 98), and 34 (Ref. 99)polysaccharides; they were able to establish that each was composed of a pentasaccharide phosphate repeating-unit, and to locate the phosphoric diester linkages in these polysaccharides. Finally, the combined use of gas-liquid chromatography and mass spectrometry by Lindberg and coworker^'^^^'^^ has had a profound impact on the structural analysis of polysaccharides. This was demonstrated in work on the type 2 polysaccharide,8° and in subsequent, structural elucidations of other pneumococcal polysaccharides (see Table IV). 111. OTHER IMPORTANT STRUCTURAL AND PHYSICAL OF CAPSULAR POLYSACCHARIDES

FEATURES

1. Structural Heterogeneity There is now abundant structural, spectroscopic, and biosynthetic evidence to suggest that, except for the possibility of minor structural irregularities, the fundamental structures of bacterial polysaccharides consist of fairly small, regular repeating-sequences of from 1to 7 saccharide units. Thus, as the polysaccharides are multivalent antigens, the effect of minor irregularities in their structures would, in general, (99)G. J. F. Chittenden, W. K. Roberts, J. G . Buchanan, and J. Baddiley, Biochem.J., 109 (1968) 597-602. (100) P.-E. Jansson, B. Lindberg, and U. Lindquist,Carbohydr. Res., 95 (1981) 73-80. (101) K. Leontein, B. Lindberg, and J. L(inngren,Can.]. Chem., 59 (1981) 2081-2085. (102) B. Lindberg, Methods Enzymol.,28B (1972) 178-195. (103) B. Lindberg, Methods Enzymol., 5OC (1980) 3-34


175

be unimportant to their immunological specificity. However, minor structural irregularities cannot be ignored in other immunological properties, as it has been demonstrated that the presence of extremely small proportions of attached lipid can have a profound effect on the immunogenicity of polysaccharides (see Section 111,4). A larger degree of structural heterogeneity has been documented for some bacterial, capsular polysaccharides, and this is mostly introduced by the distribution of 0-acetyl substituents on these polysaccharides. Only 70% of the 2-amino-2-deoxy-D-mannose residues of the group A meningococcal polysaccharide have 0-acetyl s~bstituents,3~ and this heterogeneity is also exhibited by the group C meningococcal polysac~ h a r i d e . 3In ~ the latter, an a-D-(2+9)-linked homopolymer of sialic acid, the molar ratio of 0-acetyl to sialic acid is 1.2:l-0. These 0acetyl groups are distributed on the sialic acid residues, in a complex pattern, at 0-7 and 08 (monosubstitution and disubstitution), and some of the sialic acid residues remain unsubstituted (see formulas 25). Interestingly, in the group C polysaccharide, the pattern of O-acetyl substitution is dependent on the conditions used to grow the group C organisms31; this could be important in the production of human polysaccharide vaccines. However, it is unlikely that the extent of this structural variation would be sufficient to impart complete, serological specificity to the variant group C polysaccharide and to preclude its use as an effective, group C meningococcal vaccine, because, even the 0-deacetylated group C polysaccharide can function as an effective, group C meningococcal vaccine in man (see Section V,2).

-

2 R=R'=H 3 R = R' = COCH, 4R=H,R=COCH, 5 R=COCH,,R'=H

The Four Different, N-Acetylneuraminic Acid Residues in the Native Polysaccharide Antigen of Croup C Neisseria meningitidis.

2. Determinants and Immunological Specificity An important step in understanding the immunology of polysaccharides consists in establishing which part of the polysaccharide is re-

176

HAROLD J. JENNINGS

sponsible for its immunological specificity. This part of the polysaccharide is called a determinant, and, from early studies by Goebel,lW it became apparent that determinants constitute only a small part of the large polysaccharide molecule. Antibodies made to the type 3 pneumococcal polysaccharide are strongly inhibited by cellobiouronic acid, the disaccharide repeating-unit of the polysaccharide; conversely, antibodies made to a cellobiouronic acid-protein conjugate cross-react with the type 3 pneumococcal polysaccharide. This procedure of using compounds of low molecular weight that are representative of parts of the polysaccharide structure, in order to inhibit the classical, antibody-antigen (polysaccharide) precipitin reaction of Heidelberger and Kendall? was used extensively by Kabat in ~ t u d i e s ' ~on ~ Jthe ~ linear dextran-antidextran reaction. This model system is probably representative of all linear polysaccharides, with the possible exception of those terminating in nonreducing sialic acid groups (see later); as such, it is pertinent to polysaccharide vaccines, as most of the polysaccharides involved are linear. In these definitive studies, this procedure furnished information on the location and size of determinant groups, and on the heterogeneous nature of antibodies in terms of their specificities. By using a series of oligosaccharides of the isomaltose series with human antidextran sera, it was found that the inhibitory power of the oligosaccharides increases with molecular size until it becomes more or less constant at the hexasaccharide, and this was interpreted as a measure of the optimum size of the combining site of the antibody molecule. From the relative, inhibitory powers of each oligosaccharide, it was possible to calculate the contribution, to the binding energy, of each successive D-glucose residue; it was found that, although the terminal mglucose unit contributed most to this binding energy, each succeeding D-glucose unit also made incrementally smaller contributions. The nonreducing, terminal D-glucosyl groups were called immunodominant, although, in fact, they remain a part of the larger determinant. Another important finding in these studiesIo7was the heterogeneous nature of the antibodies in regard to their serological specificities. The antibodies were adsorbed onto Sephadex, and fractionated by elution with isomalto-oligosaccharidesof' different molecular sizes. Inhibition studies on the different fractions indicated that antibodies having a (104) W. F. Goebel,]. Exp. Med., 68 (1938)469-484. (105)E. A. Kabat,]. Immunoi., 84 (1960) 82-85. (106) E. A. Kabat, Experimental Immunochemistry, 2nd edn., Charles C. Thomas, Springfield, Illinois, 1967, pp. 241-267. (107) J. Gelmer and E. A. Kabat, Immunochemistry, 1 (1964)303-316.


177

specificity both to small (disaccharide)and large (hexasaccharide) determinants were present in the antidextran serum. Branched polysaccharides differ from linear polysaccharides in having many more terminal, glycosyl residues on each polysaccharide molecule. This fact, together with the more exposed, and, thus, more accessible, position of these residues, tends to make them immunodominant, although not exclusively so, as populations of antibodies having specificities to the backbone of the polysaccharide can also be formed. Although the immunodominance of terminal p-D-glucosyluronic acid groups was recognized in early studies,lWthis phenomenon was more definitively resolved in classical studies on the serological determinants of the lipopolysaccharides of SaZmoneZZa; these have been reviewed." As with the capsular polysaccharides, the 0chains of the lipopolysaccharides consist of a linear arrangement of oligosaccharide repeating-units,the majority of which contain unique, terminal 3,6-dideoxyhexosyl groups in each repeating unit. These terminal saccharides are, to a large degree, responsible for the specificity of antibodies made to the Salmonella organisms; however, antibodies having a specificity for the backbone are also detected in these antisera. In serological studies on branched Dmannans, Ballou and coworkerslOgJ1O determined that the participation of the backbone D mannosyl residues in the immunological response to these branched D-mannans is dependent on the length of the branch structure. When the side chains extend to a tetrasaccharide unit, they are able completely to inhibit antibodies made to the homologous Dmannan. Interesting exceptions to the general rule of the immunodominance of branch saccharides have become apparent as regards the capsular polysaccharides of Group B Streptococcus. These are structure-related, and are important in both the human immunologicalresponse to (see Section 111,3),and the virulence of (see Section VI,Z), the Group B streptococcal organisms."' The type I11 polysaccharide has a 6-0(N-acetyl-cu-Dneuraminyl)-P-wgalactopyranosyl branch,= whereas those of types Ia and Ib have terminal 3-0-(N-acetyl-a-Dneuraminy1)p-Dgalactopyranosyl ~ n i t s .Neither ~ ~ . ~ of ~ these terminal-branch disaccharides is immunodominant, and this can probably be attributed to structural homology between the type 111, and the types Ia and Ib polysaccharides and human serum glycoproteins (serotransferrin and (108) M. Heidelberger, Fortschr. Chem. Org. Natumt., 18 (1960) 503-536. (109) C. E. Ballou,]. BioZ. Chem., 245 (1970) 1197-1203. (110) C. E. Ballou, P. N. Lipke, and N. C. Rashke,J. Bacteriol., 117 (1974)461-467. (111) H. J . Jennings, C. Lugowski, and D. L. Kasper, Biochemistry, 20 (1981) 45114518.

178

HAROLD J. JENNINGS

the M and N blood-group substances, respectively)."' The production of antibodies to these determinants would be highly unfavorable, and, consequently, is probably suppressed by the human immune-system. Following this reasoning, it is highly improbable that terminal sialic acid residues can be a part of any determinant responsible for significant amounts of antibody, although these residues exercise confonnational control over these determinants"' (see Section 111,3). This would also imply that nonreducing, terminal sialic acid residues of any linear polysaccharide (for example, groups B and C meningococcal polysaccharides) would not be immunodominant. Many of the bacterial polysaccharides contain small, noncarbohydrate substituents that could be regarded as branches, and that can also be important in the serological reactions of polysaccharides. These substituents have been r e ~ i e w e d , ' ~and , ' ~ the most important, in terms of the formulation of current, polysaccharide vaccines (see Section V,2), is the 0-acetyl substituent. These substituents can be immunodominant, but are not exclusively so; other populations of antibodies having specificities for other sectors of the polysaccharide are usually formed.

3. Conformation It has been established that the primary structures of the capsular polysaccharides are responsible for their serological specificity. Obviously, conformational factors must also piay a role in this specificity. Rees112showed that polysaccharides, like proteins, can have ordered (helical) structures in which interchain and intrachain associations are both involved, and that these polysaccharides undergo temperatureinduced, order-disorder transitions. Rees'I3 also found that the secondary structures are responsible for the physical and biological properties of these polysaccharides. Of special interest to the present discussion is the fact that the ordered conformation of the capsular polysaocharide from the Gram-negative organism Xanthomonus campestris, a plant pathogen, is necessary, in order that the bacteria may bind to the surface of the plant-host cell^."^ A similar dependence of specific binding to antibody molecules on the ordered (helical) structure of capsular polysaccharides has not been established. However, similar, order-disorder transitions have been detected in a number of (112) D. A. Rees, Biochem. I., 126 (1972) 257-273. (113) D. A. Rees, M T P Int. Reu. Sci., Org. Chem., Ser. One, 7 (1973) 251-283; M T P Int. Ret;. Sci., Biochem., Ser. One, 5 (1975) 1-42. (114) E. R. Morris, D. A. Rees, G. Young, M. D. Walkinshaw, and A. Darke,J. MoZ. Biol., 110 (1977) 1-16.


179

the capsular polysaccharides from Klebsiella, which would suggest that these polysaccharides have some helical character in solution at lower temperature^."^ Helical structures for some of these polysaccharides have also been reported from study of their X-ray fiber, diffraction pattern^.'^^,"^ Interestingly, the K8 polysaccharide has a two-step, temperature transition, the first step of which is due to the breaking of interactions between its terminal Dglucosyluronic acid groups and its backbone saccharides that results in a change in the backbone conformation of the poiysaccharide.l15 Although helical structure in solution has not been demonstrated for capsular polysaccharides associated with human vaccines, it has been identified in oriented fibers and films of the capsular polysa~charides."~,~~~ In an X-ray fiber diffraction study, the pneumococcal type 3 polysaccharide was found to exist in an extended, left-hand, helical conformation, hydrogen bonding being involved in maintenance of this secondary structure."* This was confirmed by X-ray studies of oriented films of both the types 3 and 8 pneumococcal poly~accharides."~The former exists as a two-fold helix, and the latter as a three-fold helix. Extended conformations in solution have also been assigned to the groups A (Ref. 33), X (Ref. 33), and Z (Ref. 36) polysaccharides of N. meningitidis on the basis of large, three-bond (31P-13C)coupling-constants in their 13C-n.m.r. spectra, but the presence of any helical content in solutions of these polysaccharides has not been established. Laser light-scattering techniques were applied to the group C meningococcal polysaccharide, and these studies indicated that it behaves like a random coil in solution.lZ0This conclusion is consistent with data obtained from 13Cn.m.r.-relaxation studies on the same polysaccharide that indicated32 that a fair degree of flexibility is exhibited by the group C polysaccharide in solution. This situation could prove to be representative of the majority of capsular polysaccharides in solution, although, as in the case of amylosic chain conformations,121the occurrence of regions of helical content in these polysaccharides is also a distinct possibility. (115)C. Wolf, V. Elsasser-Beile, S. Stirm, G. G . S. Dutton, and W. Burchard, Biopolymers, 17 (1978) 731-748. (1161 D. H. Isaac, K. H. Gardner, E. D. T. Atkins, V. Elsasser-Beile, and S. Stirm, Carbohydr. Res., 66 (1978)43-52. (117) D. H. Isaac, E. D. T. Atkins, H. Niemann, and S. Stirm, I n t . ] . Biol. Macromol., 3 (1981) 135-139. (118) R. H. Marchessault, K. Imada, T. L. Bluhm, and P. R. Sundararajan, Carbohydr. Res., 83 (1980)287-302. (119) W. T. Winter and I. Adelsky, Biopolymers, 20 (1981) 2691-2694. (120) T. Tsunashima, K. Mom, B. Chu, and T.-Y. Lui, Biopolymers, 17 (1978)251-265. (121) R. C. Jordan, D. A. Brant, and A. Cesbo, Biopolymers, 17 (1978) 2617-2632.

180

HAROLD J. JENNINGS

Because interactions between antibodies and polysaccharides are restricted to comparatively small regions of the polysaccharides, the orientation of the glycosidic linkages between the individual saccharide units is a hndamental parameter; this is the linkage orientation,122,123 and is defined by the torsion angles (A4 and A+) between these saccharides. Although Se1alz4has shown that antibodies made to peptide sequences did not recognize the same sequences when they formed part of a protein helical structure, this type of conformational specificity is not the general rule for polysaccharides. In fact, regions of conformational similarity are found in polysaccharides of different structures, and this is manifested in the extensive, serological crossreactivity of polysaccharides (see Section V,6). These regions of conformational similarity have been demonstrated in X-ray studies of oriented films of the cross-reacting types 3 and 8 pneumococcal polysaccharide^."^ The common cellobiouronic acid unit adopts the same conformation in both polysaccharides, despite the fact that it is the repeating unit of the former and is separated by a 4-O-a-~glucopyranosyl-cu-D-galactopyranosyl spacer in the structure of the type 8 polysaccharide (see Fig. 3). Rees and S k e r ~ - e t tshowed '~~ that disaccharides can generally be fitted into polysaccharide structures without significant changes in their torsion angles, and this is the basis of the use of

FIG. 3.-Conformations of the Types 3 (Lower) and 8 (Upper) Polysaccharides of Streptococcus pneumoniae, Showing the Common Disaccharide Unit. (122) D. A. Rees,J. Chem. SOC., B , (1969)217-226. (123) D. A. Rees,j. Chem. SOC., B , (1970)877-884. (124) M. Sela, B. Schecter, I. Schecter, and F. Barek, Cold Spring Harbor Symp. Quant. Biol., 32 (1967) 537-545. (125) D. A. Rees and R. J. Skerrett, Carbohydr. Res., 7 (1968) 334-348.


181

hard-~phere,'~~-'~' and other, more sophisticated, calculations,128in predicting the conformations of homog1ucans,lz6diheteroglycans,126 and even more-complex, saccharide sequence^.^^^^^^^ Not all disaccharides maintain their torsion angles in different structural environments, however, and this results in the type of conformationally controlled, and highly serologically-specific, determinants demonstrated by Jennings and coworkers111J31 in the type 111 capsular polysaccharide of group B Streptococcus. The repeating unit of this capsular polysaccharide is shown in Fig. 4. The conformation of the determinant of this polysaccharide, responsible for the population of antibodies involved in the protection of humans against type I11 group B streptococcal infections, is dependent on its terminal sialic acid groups, even though these are not immunodominant (see Section V,4) I I

I

.'

/O

FIG.4.-Proposed Conformation of the Repeating Unit of the Type I11 Polysaccharide Antigen of Group B Streptococcus. (126) D. A. Rees,]. Chem. SOC., B, (1969) 217-226. (127) D. A. Rees and W. E. Scott,J. Chem. SOC., (1971) 469-479. (128) D. A. Rees and P. J. C. Smith,]. Chem. SOC., Perkin Trans. 2, (1975)836-840. (129) R. U. Lemieux, K. Bock, L. T. Delbaere, S. Koto, and V. S. Rao, Can.]. Chern., 58 (1980) 631-653. (130) K. Bock, S. Josephson, and D. R. Bundle,]. Chem. SOC., Perkin Trans. 2, (1982) 59-70. (131) H. J. Jennings, C. Lugowski, K.-G. Rosell, and D. L. Kasper, in D. A. Brant (Ed.), Solution Properties of Polysaccharides, A.C.S. Symp. Ser., 150, American Chemical Society, Washington, D. C., 1980, pp. 161-172.

182

HAROLD J. JENNINCS

and are, most probably, not even a part of the determinant. By using '"C-n.m.r.-spectroscopicdata on the native, and modified-native, type I11 polysaccharides, it was possible to determine that the terminal sialie acid groups exert conformational control over the torsion angles of the penultimate 2-acetamido-2-deoxy4O-~-D-ga~actopyranosy~-~-Dglucopyranosyl unit of the polysaccharide. It is possible that this control is achieved by interactions (possibly hydrogen bonding) between these terminal sialic acid groups and the backbone of the type I11 polysaccharide. It is also possible, and, indeed, probable, that substituent groups smaller than sialic acid groups, such as 0-acetyl, pyruvate, and phosphate, could also confer immunospecificity in polysaccharide determinants by a similar mechanism. 4. Molecular Size One of the most important physical parameters in the effectiveness of capsular polysaccharides as vaccines is their molecular size. This ' ~ ~ demonstrated that the fact was discovered b y Kabat and B e ~ e r ,who molecular weight of native dextrans that are highly immunogenic in man is of the order of several million. They then proceeded to ascertain the dextran of lowest molecular weight that could still retain its immunogenicity; this was achieved by monitoring the increase in serum-antibody titers following the injection of humans with dextrans having different ranges of molecular weight. They found that, in the molecular weight range of 90,OOO and above, the dextrans remained excellent immunogens, whereas, at values of 50,000and below, they exhibited poor immunogenicity. In subsequent, similar experiments on the pneumococcal, type 3 capsular polysaccharide, Howard and cow o r k e r ~ injected '~~ fractions of different molecular weight of the type 3 polysaccharide into mice; the immunogenicity of each fraction was determined by monitoring the number of plaque-forming cells in the spleens of the mice. It was found that the number of such cells was directly related to the molecular size of the fraction; the native polysaccharide was easily the most effective immunogen. In studies on the capsular polysaccharides of N . meningitidis, Gotschlich and coworkersznwere able to obtain the group A and C polysaccharides in their high-molecular-weight form by precipitation directly from the liquid culture by means of Cetavlon. These highmolecular-weight polysaccharides were highly immunogenic in man. However, the group A polysaccharide isolated from cultures concen(132) E. A. Kabat and A. E. Bezer,Arch. Biochem. Biophys., 78 (1958)306-310. (133) J. G . Howard, H. Zola, G. H. Christie, and B. M. Courtenay,]. Immunol., 21 (1971)535-546.


183

trated by rotary evaporation had lower molecular weights (less than 50,000), and proved to be non-immunogenic in humans. This depolymerization of the group A polysaccharide was attributed to the action of specific enzymes in the culture medium during evaporation. Lui and coworkerslMthen made the interesting and important observation that estimations of the molecular size of the group A polysaccharide by reducing end-group analysis were considerably lower than those obtained by gel filtration; this phenomenon was also reported earlier for the group X polysaccharide of N . m e n i n g i t i d i ~ . 'On ~ ~ the basis of their results, Lui and coworkers postulated134that some form of aggregation was occurring between individual polysaccharide chains, resulting in a macromolecular structure, and that lipid components could be responsible for this aggregation. In subsequent experiments by Gotschlich and coworkers,136aggregation was found to occur in the groups A, B, and C capsular polysaccharides of N. meningitidis and in the type K92 polysaccharide of E. coli. Experiments were then carried out to determine the nature of this aggregation. They showed,136as previously postulated, that a small proportion of lipoidal material was attached to all of these polysaccharides (8040% of di-0-palmitoylglycerol and 10-20% of di-0stearoylglycerol), and that these diO-acylglycerols were glycosylically attached to the reducing eqd of the polysaccharides by phosphoric diester bonds (see Fig. 5). This small proportion of lipid was sufficient to impart micellar behavior to the individual chains of the polysaccharides. These results couid be significant in our perceptions of capsular polysaccharides, in that an apparently minor component can have such a profound effect on their physical (molecular size) and immunological (immunogenicity) properties. In addition, this minor, lipoidal component could be the entity by which these polysaccharides are actually attached to the outer membrane of the bacterium (see Section 111,5).

5. Location

The importance of capsular polysaccharides in the immune response to bacterial infection is due to their location on the outer surface of the bacteria. They are at the interface of the many host-bacte(134) T.-Y. Lui, E. C. Gotschlich, W. Egan, and J. B. Robbins,]. Inject. Dis., Suppl., 136 (1977) S71-S77. (135) D. R. Bundle, H. J. Jennings, and C. P. Kenny,]. Biol. Chem., 249 (1974)47974801. (136) E. C. Gotschlich, B. A. Fraser, 0.Nashimura, J. B. Robbins, and T.-Y.Lui,J. Biol. Chem., 256 (1981) 8915-8921.

r

1

Ac AC

0

FIG.5.-Proposed Structure of the Lipid Functional Group of the Group C Meningococcal Polysaccharide.


185

ria interactions, and constitute the principal antigens in most of the pathogenic, Gram-negative and Gram-positive organisms. However, the outer membranes of encapsulated bacteria are complex, and other antigens, such as protein^^"^^ and lipopolysaccharide~,~~~ can also play a minor, but important, role in the human immune-response to bacterial infection. Physical experiments have established the surface location of capsular polysaccharides on bacteria; from early experiments, previously reviewed,19using the optical microscope to visibilize the interactions of capsules and specific antibody (quellung reaction) with India ink, to improved techniques using electron microscopy to study the latter intera~ti0ns.l~~ Electron microscopy has also been used to study the capsules of bacteria following the incubation of the bacteria with ferritin-conjugated, specific antibody.140When applied to the group B Streptococcus, the surface location of the type Ia, Ib, Ic, 11, and 111 capsules was experimentally confirmed; types Ib and Ic also have additional, surface-protein antigens.141 The definition of a capsular polysaccharide is arbitrary; it is generally described as an envelope of mucilaginous material surrounding the bacterium. A fundamental question concerning these capsules is whether they are actually attached to the bacteria. Although the detection of free, capsular polysaccharide in culture filtrates is indicative of only weak forces of attraction, this conclusion need not necessarily be correct, as the release of capsular polysaccharide from the bacteria could be due to enzymic activity.142Evidence that covalent bonding could be involved in the attachment of some capsular polysaccharides to bacteria was reported by Tai and coworkers61;they had to use muralytic enzymes in order to isolate the type Ib, group B streptococcal polysaccharide. The identification of end-group diO-acylglycerol phosphate moieties on the group A, B, and C meningococcal and type K92 E. coli polysaccharides has also raised the possibility that these esters could be involved in the anchoring of the capsular polysaccharides to the outer membranes of their respective bacteria.13s Endgroup phosphoric esters have also been detected, by 31P-n.m.r. spectroscopy, in the H. injuenzae capsular polysaccharides; these esters

(137) T. M. Buchanan, in M. Inouye (Ed.), Bacterial Outer Membranes, Wiley, New York, 1979, pp. 475-514. (138) H. J. Jennings,A. K. Bhattacharjee, L. Kenne, C. P. Kenny, and G. Calver, Con.J . Biochem., 58 (1980) 128-136. (139) M. E. Bayer and H. Thurow.]. Bacterial., 130 (1977) 911-936. (140) J. Swanson, K. C. Hsu, and E. C. Gotschlich,]. Exp. Med., 130 (1969) 1063-1075. (141) D. L. Kasper and C. J. Baker,]. Infect. Dis., 139 (1979) 147-151. (142) F. A. Troy, Annu. Reu. Microbial., 33 (1979) 519-560.

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could have been part of an original linkage to the outer membrane of the bacteria.143 Iv. IMMUNE RESPONSE TO BACTERIALINFECTION

In order to understand the function of capsular polysaccharides as human vaccines, it is necessary to describe the human immuneresponse to infections caused by encapsulated bacteria. This response consists of a highly complex interplay of different cells and molecular components, and, of necessity, will only be described in an abbreviated and simplified way. More-extensive information on this subject can be obtained in books and review^.'^^-^^^ Once micro-organisms have penetrated subepithelial tissue and have invaded the human circulatory system, the immune mechanism is the last line of defense against proliferation of the bacteria and the eventual establishment of the disease state in the host. The fact that infections are common indicates that the host defenses do not constitute an impenetrable barrier for micro-organisms. In fact, we are all engaged in a constant struggle with invasive bacteria, a struggle in which various strategies employed by micro-organisms play an important role. As surface components of the bacteria, capsular polysaccharides are implicated in the complex, host uerszis micro-organism interactions, and, in particular, are responsible both for the stimulation of the human immune-system against the invading bacteria and for the virulence of the encapsulated bacteria (see Section V1,l). Two excellent reviews have been written on the intriguing subject of bacterial strategy and the human, immunological-defense ~ y s t e m . ~ " The * ' ~ ~process of eliminating the invading, encapsulated bacteria from the circulatory system is based on three factors; phagocytosis, the activation of complement, and the production of humoral antibodies. Cell-mediated immunity, as opposed to humoral immunity, is less important in the critical, acute and early stages of infections due to encapsulated bacteria, but is probably important in long-term immunity. (143)W.Egan, H. Sclineerson, K. E. Werner, and G. ZonJ. Am. Chem. Soc., 104 (1982) 2898-2910. (144) 31. C. Raff; Nature, 242 (1973) 19-23. (145) E. S. Golub, The Cellular Basis of the Immune Response, Sinauer Assocs., Sunderland, Mass., 1977. 1146) W. E. Paul, in J. B. Robbins, R. E. Horton, and E. M. Krause, New Approaches f o r Inducing Natural Immunity to Pyogenic Organisms, Proceedings of a Symposium, DHEW Publ., No. (NIH) 74553. (147) P. J. Baker and B. Prescott, in J. A. Rudbach and P. J. Baker (Eds.),Development in Immunology, Vol. 2, Elsevier-North Holland, New York, 1979, pp. 67-104. (148) P. Densen and G. L. Mandell, Rev. Infect. Dis., 2 (1980)817-838.


187

1. Phagocytosis The engulfment and digestion of micro-organisms, termed phagocytosis, is the function of specialized cells in the human circulatory system.149J50 These cells consist of two major types, the largest and most complex of which are the macrophages. The macrophages constantly monitor the subepithelial tissues and all circulatory fluids, and have an important, immediate function, in that they are able to adhere directly to microbes by some kind of rather primitive, recognition mechanism. They are also able to adhere to microbes by a more specific recognition mechanism involving the prior coating of the bacteria with specific antibody, or the third component (C3) of complement. These molecules function as ligands between the macrophages and the bacteria by adhering to specific, macrophage receptor-sites to the Fc portion of the antibody, or to the C3 component of complement. Macrophages are also important in cell-mediated immunity and in the instigation of the immune response to invading micro-organisms. They achieve this by stimulating antibody-producing cells (see Section IV,3) to produce antibodies having specificities for surface components of micro-organisms to which the macrophages have previousIy adhered. Polymorphs are small, less complex cells that are of extreme importance to the immune system, as they are highly specific and short-lived, and can be rapidly produced by the body and delivered to the tissues by chemotactic response. Like the macrophages, they operate in association with complement and antibody through their respective C3 and Fc receptors. 2. Role of Complement The complement system has been r e v i e ~ e d ' ~ it~ is ~ 'composed ~~; of a series of proteins, Cl-C9, present in normal human serum, that serve as important mediators in the host defense. The terminal components, C3-C9, are involved in the destruction of invading microorganisms, but, in order to achieve this, they have to be activated. This activation process can be divided into two pathways, the alternative pathway and the classical pathway, although both pathways can occur simultaneously in the host defense-mechanism. Surface carbohydrates of micro-organisms are able to activate the al(149)C.A. Mims, The Pathogenesis of Infectious Disease, Academic Press, New York, 1977. (150) M. J. Taussig, Processes in Pathology, Blackwell, Oxford, 1979. (151)H. J. Muller-Eberhard and R. D. Schrieber,Adu. Zmmunol., 29 (1980)1-53. (152)J. A. Winkelstein, Reu. Infect. Dis., 3 (1981)289-298.

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ternative pathway, directly generating C3 convertase activity; those important to human immunity to bacterial infection are the cell-wall mucopeptide of Gram-positive o r g a n i ~ m s , ~in ~ ~which - l ~ ~the teichoic * ' ~ the ~ lipopolysaccharides acid moiety is probably f ~ n c t i o n a l , ' ~and of Gram-negative organisms.'58 The convertase splits C3, to give C3b, which binds to the microbe, and a chemotactic agent that serves to attract polymorphs. The polymorphs then migrate towards the site of infection, and are able to adhere readily to the C3b-coated microbes, because of their surface C3b-receptors. The exact, structural basis of the activation of the alternative pathway is as yet but little understood, but it performs an important, immediate function in the destruction of invading microbes, because of the speed at which this defense mechanism can be deployed as compared to the classical pathway. Although antibody is not required for the activation of the alternative pathway, there is evidence to show that it can participate functionally in this mechanism.15z The classical pathway generally requires the presence of antibody ( 1 s ) having a specificity for a surface component of the bacterium, in order that activation may occur. The antibody adheres to the bacterium, and the resulting, immune complex, through a conformational change in the Fc portion of the antibody molecule, activates the first component of complement (Cl).This, in turn, activates C2 and C4, to yield C3b from C3, in which respect, this pathway now converges with that of the alternative pathway. In fact, the generation of C3b at this stage can lead to the activation of the alternative pathway.152Interestingly, in a few isolated cases, the classical pathway can be activated without the participation of antibody by some molecular structures; this has been reported for the lipid A moiety of lipop~lysaccharides,~~~ for a polysaccharide found in ant venom,'6oand for some synthetic oligosaccharides when linked to an 8-methoxycarbonyloctanol carrier.161 (153) J. A. Winkelstein and A. Tomasz,]. Zmmunol., 118 (1977)451-454. (154) P. H. Quinn, F. J. Crossan, J. A. Winkelstein, and E. R. Moxon, Inject. Zmmun., 16 (1977)400-402. (155) J. W. Tauher, M. J. Polley, and J. B. Zabriskie,J. E r p . Med., 143 (1976) 13521366. (156) J. A. Winkelstein and A. Tomasz,J. Zmmunol., 120 (1978) 174-178. (157) B. A. Fiedel and R. W. Jackson, Infect. Zmmun., 22 (1978)286-287. (158) C. Galanos and 0. Luderitz, Eur. J . Biochem., 65 (1976)403-408. (159) D. C. Morrison and L. F. Kline,]. Zmmunol., 118 (1977)362-368. (160) D. R. Schultz, P. I. Arnold, M . X . Wu, T. M. Lo, J. E. Volkanakis, and M. Loos, Mol. Zmmunol., 16 (1979) 253-264. (161) D. R. SchuIk and P. I. Amold,J. Zmmunol., 126 (1981) 1994-1998.


189

Capsular polysaccharides are actively involved in the mediation of complement, in that they are able to suppress the activation of the immediate, alternative-pathway mechanism, thus forcing the immune system to use the classical pathway; this is an important factor in the virulence of bacteria (see Section VIJ).

3. Humoral Antibodies to Polysaccharide Vaccines

An important distinction must be made between the humoral response to a pure, capsular polysaccharide, and to the same polysaccharide when it is an integral part of the bacterium. Thus, the immunity received on recovery from infection by encapsulated bacteria, in terms of the polysaccharide antigen, differs from that generated by purposeful immunization with purified capsular-polysaccharide vaccines. Fortunately, with the exception of infants, the polysaccharide vaccines still stimulate protective-antibody levels in humans, despite these differences. In infants, due to the immature nature of their immune systems, these polysaccharide vaccines are of only marginal benefit.7 Some insights into the nature of these different responses in humans can be found in studies on the cellular basis of the immune response to polysaccharides. However, for the purposes of this Chapter, it would be inappropriate to provide a lengthy description of this incompletely understood mechanism; in-depth reviews of this burgeoning field of research can be referred t ~ . ~ ~ - ~ ~ ~ , ~ ~ For most antigens, the production of antibody (immunoglobulin) is based on the cooperative interaction of two types of lymphocyte, called T-cells (thymus-derived) and B-cells (bone marrow-derived). The T-cells, preprimed with macrophage-presented antigen, stimulate the B-cells to secrete copious quantities of antibody. However, on the basis of animal studies, such polysaccharide antigens as the type 111 pneumococcal polysaccharide have been considered to be T-cellindependent, as they are capable of triggering B-cells to produce antibody (IgM) in T-cell-deficient mice.16' These studies also indicated (162) P. J. Baker, H. C. Morse, S. C. Gross, P. W. Stashak, and B. PrescottJ. Infect. Dis., Suppl., 136 (1977) s20-~24. (163) D . E. Mosier, N. M. Zidivar, E. Goldings, J. Mond, 1. Sher, and W. E. Paul, J . Infect. Dis., Suppl., 136 (1977) s14-s20. (164) H. Braley-Mullen, Immunology, 40 (1980) 521-527. (165) E. C. Gotschlich, I. M . Goldschneider, M. L. Lepow, and R. Gold, in E. Huber and R. M. Krause (Eds.),Antibodies in Human Diagnosis and Therapy, Raven Press, New York, 1977, pp. 391-402. (166) P. J. Baker, D. F. Amsbaugh, P. W. Stashak, G. Caldes, and B. Prescott, Reu. Infect. Dis., 3 (1981) 332-341. (167) J. G. Howard, G. H. Christie, B. M. Courtenay, E. Leuchars, and A. J. S. Davies, Cell. Immunol., 2 (1971)614-626.

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that the same type of antibody (IgM) is produced in normal mice by the same polysaccharide z ~ n t i g e n . ' ~ ~ - ' ~ ~ The majority of antibodies can be broadly classified into three main categories ( I s , IgM, and I d ) on the basis of their different structure and functions in the immune response, and IgG is the most important type of antibody in promoting the classical pathway of the complement system."O Further experiments in mice demonstrated that, although pol ysaccharides are capable of functioning as T-cell-independent antigens in athymic mice, in normal mice, polysaccharides do stimulate T-cell 'activity, although the T-cell response differs markedly (more restricted) from that generated by the injection of mice with whole b a ~ t e r i a . 'T-Cells ~ ~ , ~ ~can ~ modulate the immune response in mice b y either an effector or a suppressor mechanism, and, unlike the situation for the whole bacteria, polysaccharides cause the supAlso, unlike the response of pressor mechanism to be d~rninant.'~'~''~ whole bacteria in mice, polysaccharide antigens fail to induce a memory (amnestic) re~ponse.'~" If the results of the foregoing experiments in mice were projected to the human situation in general, the use of polysaccharides as efficacious, human vaccines would show little promise. However, the immunological response to polysaccharides is species-dependent, and, in humans, with the exception of young infants, a fuller range of antibody types is produced. Thus, polysaccharides stimulate the production of IgG antibodies in humans, in addition both to IgM and IgA ant i b ~ d i e s , 'but, ~ ~ as in the mouse experiments, they fail to exhibit a sizable, amnestic response to subsequent, booster injection^.^ Although the presence of this effect would be a decided advantage in immunoprophylaxis, it is not detrimental, because efficacious, antibody levels in humans are maintained for up to 8 years follbwing the injection of pneumococcal poiysa~charides.'~~ Human infants have immature, immune systems in relation to polysaccharide vaccines, (168) B. Anderson and €1.Blombren, Cell. Zmmunol., 2 (1971) 411-424. (169) C . F. Mitchell, F. C. Grumet, and H. D. McDevittJ. E x p . Med., 135 (1972) 126135. (170) E. C. Gotschlich, I . M. Coldschneider, and M. S. Artenstein,J. E x p . Med., 129 (1969) 1367- 1384. (171) P. J. Baker, N. D . Reed, P. W. Stashak, D. F. Amsbaugh, and B. Prescott,]. E x p . Med., 137 (1973) 1431-1441. (172) P. J . Baker, T r Q n S p h f .Rer;., 26 (1975) 3-20. (173) A. Basten and J. G. Howard, Contemp. T o p . Zmmunobiol., 2 (1973)265-291. (174) W. J . Yount, M. M. Domer, H. J. Kunkel, and E. A. Kabac]. E x p . Med., 127 (1968) 633-646. [175) M. Heidelberger, M. M. Dilapi, M. Siegel, and A. W. Walter,]. lmmunol., 65 (1950) 535-541.


191

and do not produce the necessary IgG antibodies (see Sections V,5 and 6). This behavior is similar to that exhibited by mice, and, if it is permissible (not yet proved) to use these experiments in mice as models, this immaturity could be attributed to T-suppressor-cell functi~n.’~~

v. POLYSACCHAFUDE VACCINES AND

IMMUNITY

1. Streptococcus pneumoniae

Pneumonia was, and still remains, among the leading causes of death in the United States. It is responsible for lower-respiratory-tract infections in humans, and is also the most common cause of otitis media (a bacterial infection of the middle ear) in children. The high rate of mortality caused by this disease prompted a search for a preventive approach to its control, an investigation in which the pneumococcal, capsular polysaccharides were the first, purified polysaccharides to be used as human vaccine^.^*'^^*'^^ This followed directly from the discovery by Francis and TilleP that the intradermal injection of type 1 and 2 pneumococcal polysaccharides induced serum antibodies in humans. These results led to the demonstration by Heidelberger and his associates: in a large field-trial under epidemic conditions, that a vaccine composed of types 1,2,5,and 7 polysaccharides is efficacious against disease caused by S. pneumoniae. These studies also confirmed the type-specific protection induced in humans by these capsular polysaccharides. Other successful field-trials were subsequently carried out; in one of these, Heidelberger and demonstrated that a hexavalent, polysaccharide vaccine administered in a single injection induced the corresponding, satisfactory, serotypeantibody levels, which persisted for up to 8 years.175This success rapidly led to the commercial licensing of hexavalent, pneumococcalpolysaccharide vaccines. However, interest in the prophylaxis of pneumococcal pneumonia waned at this time, due to the advent of the “sulfa” drugs, and then the unprecedented success of antibiotic therapy. This development engendered such a complacent attitude towards pneumococcal infections that even the accurate serotyping of disease isolates was discontinued in most medical centers. However, subsequent epidemiological studies of pneumococcal pneumonia by conclusively showed that, despite the success of antibi(176) R. Austrian, Reu. Infect. Dis., Suppl., 3 (1981) sl-sl7. (177) M. Heidelberger, C. M. McLeod, and M. M. DilapiJ. E r p . Med., 88 (1948)369372. (178) R. Austrian, Am. J . Med. Sci., 57 (1959) 133-139.

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otic therapy, the disease occurs with the same frequency, and the same mortality rate, as in the pre-antibiotic era. This fact, together with the emergence of antibiotic-resistant strains: led to consideration of reviving the preventative approach to control of the disease; the eventual development and licensing of a pneumococcal-polysaccharide vaccine179gave fruition to the earlier, interrupted research. Because of the diversity of pneumococcal-capsular types (84 have so far been identified), and a reluctance to use them all in a single, multivalent vaccine, the final composition of the vaccine was the result of a compromise in which the number of serotype polysaccharides was limited to fourteen, presumably with minimum loss of effective coverage. This decision was based on epidemiological s t ~ d i e s , ' ~con~,'~~ ducted in the United States, which indicated that 80-9O% of pneumococcal, bacteremic infections are caused by fourteen serotypes (see Table V). However, because of the restricted nature of these epidemiological studies, this vaccine can only be regarded as a core vaccine to which other serotypes may have to be eventually added. This flexibility will probably be required, in order to allow for geographical variations in pneumococcal serotypes, for time-related changes in the prevalence of serotypes in disease isolates, and, also, for the possibility of the tailoring of pneumococcal vaccines in response to agerelated, epidemiological studies (infant ~accine).'.''~ A further factor that enabled the limitation of the number of serotypes used in the vaccine was the occurrence of some structural homology in the pneumococcal polysaccharides, resulting in extensive cross-reactions and cross-immunity in their serological properties. This is exemplified in the Danish serotyping system, which designates capsular types within groups, based on this cross-reactivity. For example, the two types recognized as 6A (Ref. 86) and 6B (Ref. 87), that is, types 6 and 26 in the U. S. system, differ structurally by only one linkage position in a tetrasaccharide phosphate repeating-unit, the a-L-rhamnopyranosyl residues of type 6B being linked to 04 of the ribitol residues instead of to 03 of the same residues, as in the case of type 6A (see Table IV).This structural similarity, accompanied by a degree of conformational retention in the structure, allows for the production of antibodies common to both types. Because of this crossreactivity (see Section V,6), only type 6A is used in the vaccine. Other significant cross-reactions that occur within the polysaccharides used in the vaccine are types 19F (Refs 92 and 93) and 19A (Ref. 94),of which, type 19F is used in the vaccine, and types 9A (Ref. 83), 9V (Ref. (179) R. E. Weibel, P. P. Vella, A. A. McLean, A. F. Woodhour, W. L. Davidson, and M. R. Hilleman, Proc. SOC. E x p . Biol. Med., 156 (1977) 144-150.


193

TABLEV Distribution of Pneumococcal Types Responsible for Bacteremic Infection in the United States During 1968-1973 Pneumococcal type" 1 2 3 4 5 6A (6) 7 8 9N (9) 12 14 18 19F (19) =F (23) All other Total

Number of isolates

Percentage of isolates

293 10 237 320 70 160 213 325 132 182 2.40 159 134 117

9.1 0.3 7.3 9.9 2.2 5.0 6.7 10.1 4.1 5.6 7.4 4.9 4.2 3.6 19.6 100.0

633 3225

American type-designation is given in parentheses.

85), and 9L and 9N (Ref. 84), of which, type 9N is used in the vaccine (see Table V). The choice of these serotypes (6A, 19F, and 9N) was made because, of all the serotypes in each cross-reacting group, these occur more frequently in disease isolates. 2. Neisseria nzeningitidis

Meningococcal disease (purulent meningitis) commonly occurs in children, but is also observed in adults. Without antibiotic treatment, the mortality rate is high (85%), and, even with this treatment, cured patients can suffer serious and permanent neurological deficiencies.ls5 These facts, together with the emergence of antibiotic-resistant strains: prompted the rapid development of a commercial vaccine. This vaccine was developed almost simultaneously with the pneumococcal vaccine. In contrast to the pneumococcal vaccine, however, the composition of the meningococcal vaccine was greatly simplified, due to the fact that fewer polysaccharides were required. Based on their capsular polysaccharides, there are only eight different serogroups of N.meningitidis (A, B, C, 29e, W-135, X,Y,and Z), of which, groups A, B, and

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C account for more than 90% of meningococcal d i s e a ~ e . ' ~The ~-~*~ high-molecular-weight, group A and C polysaccharides raise titers of bactericidal antibody in adults,16j although, in young children, their use has been only marginally (see Section V,5). The group A and C polysaccharide vaccines have also been used in numerous, successful, human field-trials.165-1R' Interestingly, because of the lack of a suitable animal model in which to test the efficacy of these vaccines, the standards for their licensure and release were, for the first time, based purely on physiochemical criteria. However, one major problem in the design of a comprehensive, trivalent pol ysaccharide, meningococcal vaccine inclusive of group B is the poor immunogenicity of the group B polysaccharide in man.lW2 Two major reasons have been proposed to account for this phenomenon. One is that the rr-~-(2-+8)-linkedsialic acid homopolymer is rapidly depolymerized in human tissue, because of the action of neuraminidase; the other is that this structure is recognized as "self" by the human immune-system, and, in consequence, the production of antibody having a specificity for this structure is suppressed. The weight of evidence is in favor of the latter explanation. A neuraminidase-sensitive variant of the group C polysaccharide [an a-D-(2+9)-linked, sialic acid homopolymer] having no 0-acetyl groupsIR3still proved to be highly immunogenic in man.Is4In addition, in experiments using tetanus toxoid conjugates of the group B polysaccharide, it was demonstrated that, when conjugated at the nonreducing sialic acid group (thereby producing a neuraminidase-resistant, group B polymer), its immunogenicity was not enhanced.185Finally, the Escherichia coli K92 capsular polysaccharide contains alternating sequences of CPD( 2 4 ) -and -(2-+9)-linked sialic acid.1*6This polysaccharide proved to be immunogenic, but produced only antibodies that cross-reacted with the group C polysaccharide [a-~-(2+9)-linked]. The immune mechanism avoids the production of antibody having a specificity for the cy-1)-(2+8)linkage.'"*'Rg (180) E. C. Gotschlich, in Ref. 10, pp. 91-101. (181) R. Gold, M. L. Lepow, I. M. Goldschneider. and E. C. Gotschlich,]. Infect. Dis., S U ? I ~ >136 ~ . , (1977) S31-S35. (182) F . A. Wyle, M. S. Artenstein, D. L. Brandt, D. L. Tramont, D. L. Kasper, P. Altieri, S. L. Berman, and J. P. Lowenthal,]. Infect. Dis., 126 (1972)514-522. (183) M . A. Apicellq]. Infect. Dis.,129 (1974) 147-153. (1%) s.1. P. Glode,E. B. Lewin,A. Sutton,C. T. Le,E.C. Gotschlick,and J. B. Robbins, J . In-fect.Dis., 139 (1979)52-59. (185)H. J. Jennings and C. Lugowski,]. lmmunol., 127 (1981) 1011-1018. (186) W. Egan,T.-Y. Lui, D. Dorow, J. S. Cohen, J. D. Robbins, E. C. Gotschlich, and J. B. Robbins, Biochemistry, 16 (1977)3687-3692. .


195

Attempts to surmount this problem have included the use of ( a ) other surface-components of the group B organism (type-specific prot e i n ~ ) , ' ~(b) ~ . 'group ~ ~ B polysaccharide conjugates (see Section V,5), and (c) cross-reacting polysaccharides as alternative vaccines. In the last category, a proposal has been made to use a cross-reacting, E. coli p o l y s a c ~ h a r i d eThe . ~ ~ E. ~ coli K1 polysaccharide is ~ t r u c t u r a l l y 3 ~ ~ ~ ~ and serologically'" identical to the meningococcal group B polysaccharide (see Section V,6), but a form variant (OAc+)of this organism was isolated that produces a polysaccharide randomly 0-acetylated at 0-7 and 0-9 of its sialic acid residues.lgl This form variant (OAc+)of the E. coli K1 organism is more immunogenic in rabbits than the OAcvariant, and produces antibodies having specificities for both the 0acetylated and the nonacetylated polysa~charides.'~~ Human trials using the 0-acetylated K1 polysaccharide as a vaccine are now needed, in order to determine whether this approach to the problem will show any promise.

3. Haemophilus infiuenzae Although there are six capsular types of H. influenzae, the most serious disease is caused'9z by type b. This organism is the cause of meningitis, which occurs exclusively in infants, and even survivors of this disease can suffer severe, and permanent, neurological defect^.^ In consequence, an extensive amount of work has been dedicated to finding a vaccine for this organism, and the capsular polysaccharide was a prime candidate. Heidelberger and coworkers's3 demonstrated that protective antibodies in hyperimmune rabbit antisera could be removed by absorption with the purified H. influenzae type b polysaccharide. Since then, Anderson and coworkers,194and Parke and cow o r k e r ~ , 'were ~ ~ able to induce, in adults, long-lived, complement(187) C. E. Frasch and E. C. Gotschlich,]. E x p . Med., 140 (1974) 87-104. (188) W. D. Zollinger and R. E. Mandrell, Infect. Immun., 18 (1977)424-433. (189) J. B. Robbins, R. Schneerson, J. C. Parke, T.-Y. Lui, Z. T. Handzel, I. Brskov, and F. Brskov, in Ref. 10, pp. 103-120. (190) D. L. Kasper, J. L. Winkelhake, W. D. Zollinger, B. Brandf and M. S. Artenstein, J . Immunol., 110 (1973) 262-268. (191) F. Brskov, A. Sutton, R. Schneerson, L. Wenlu, W. Egan, G . E. Moff, and J. B. Robbins,J. E r p . Med., 149 (1979)669-685. (192) J. C. Parke, R. Schneerson, J. B. Robbins, and J. J. Schlesselman,J. Infect. Dis., S ~ p p l . 136 , (1977) S25-~30. (193) H. E. Alexander, M. Heideiberger, and G . Leidy, Yale 1. Biol. Med., 16 (1944) 425-438. (194) P. Anderson, G . Peter, R. B. Johnston, L. H. Wetterlow, and D. H. SrnithJ. Clin. Inoest., 51 (1972) 39-44.

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mediated, bactericidal antibodies by using the purified type b polysaccharide as a vaccine. However, the development of this pure, polysaccharide vaccine was retarded when it was discovered that the p l y saccharide induced only short-lived immunity in older infants, and little or no protection in younger infants.lS5Current research to obviate this problem has focussed on the use of ( a ) type b polysaccharide-protein conjugates (see Section V,5) and (b) cross-reacting organisms. The latter approach would involve the deliberate colonization of infants with the non-pathogenic, cross-reacting E. coli (see Section V,6).

4. Group B Stseptococcw Group B Streptococcus is a major cause of bacterial meningitis in new-born infant^.'^^'^^ The organisms can be into four distinct serotypes (Ia, Ib, 11, and HI), of which, type I11 is the most important in human disease.Is7 The original isolation of the type 111 capsular polysaccharide was achieved by using acid-extraction proced u r e ~ ~ ~this . ~ resulted '; in the isolation of an immunologically incomplete, core antigen that originated from a native polysaccharide containing labile, terminal sialic acid residuesw The complete, native, type I11 antigen could be isolated by growing the type I11 organism under pH-controlled conditions and isolating the polysaccharide by mild extraction-procedures.62The core antigen is structurally identical to the capsular polysaccharide of type 14 S . pneumoniae,63and, on the basis of serological experiments in animals using the latter organism, it was suggested that antibodies to the core polysaccharide could be functional in the production of protective antibodies against type 111, group B Streptococcus organisms.1s8However, confirmatory evidence for the essential participation ofthe native, type I11 polysaccharide in the development of human immunity to the disease was obtained when it was demonstrated that, in human sera, only antibody directed to the native antigen correlated most highly with opsonic (bactericidal) a ~ t i v i t y . ~ ~ * ' ~ ' I n a disease that is restricted to the newborn, the use of immunoprophylaxis is impractical, due to the time lag before effective levels of (195) I>. H . Smith, G . Peter, D. L. Ungram., A. L. Harding, and P. Anderson, Pediatrics, 52 (1973) 637-645. (1%) T. C . Eickhoff, J . 0. Klein, A. K. Daly, D. Ingall, and M. Finland, New Engl. /. Med., 271 (1964) 1221-1228. (197) C. J. Baker,Ado. Intern. Med., 25 (1930) 475-499. (198) G. W.Fisher, G . M. Lowell, M. H. Cumrine, and J. W. Bass,]. E x p . Med., 148 (1978) 776-786.


197

protective antibodies are produced. Therefore, a different vaccination strategy is envisaged for this disease, one in which the target population for the polysaccharide vaccine would be pregnant mothers deficient in antibodies specific for the native, type 111, group B streptococcal polysaccharide. The type I11 polysaccharide is immunogenic in and the baby could acquire immunity by the placental transfer of antibody ( 1 s ) . It has been demonstrated that babies born of mothers having high levels of type I11 polysaccharide-specific antibody are less liable to infectionse2than others. 5. Polysaccharide-Protein Conjugates

The utilization of the previously described, capsular polysaccharides as human vaccines is only partially successful, due to the fact that they are poor immunogens in young children. This situation is highly undesirable, as this section of the population experiences the highest incidence of disease (particularly meningitis) caused by these pathogenic bacteria.' The immunological basis of this phenomenon, discussed in Section IV,3, is the inability of young children to generate a mature and amnestic response involving the production of IgG antibodies to these purified-polysaccharide antigens. A possible solution would be to enhance the immunogenicity of these pure polysaccharides by converting them into thymus-dependent antigens. One method of achieving this objective would be to conjugate them to a protein carrier. The feasibility of this approach is well established. Fifty years ago, Goebel and AverylWcoupled the type 3 pneumococcal polysaccharide to horse serum-globulin by the diazotization of p aminobenzyl ether substituents on the polysaccharide. They demonstrated that this polysaccharide conjugate,2O0and a similar conjugate made with the oligosaccharide repeating-unit (cellobiouronic acid) of the type 3 pneumococcal polysaccharide,2°0were able to induce polysaccharide-specific antibody in rabbits previously unresponsive to the pure polysaccharide. GoebelzO1*zOz also established that the cellobiouronic acid conjugate was able to confer immunity to pneumococcal infection in mice. Other investigators confirmed these results by using the type 3 pneumococcal polysaccharide covalently linked to proteinzwand to erythrocytes,204and noncovalently linked in ionic as(199) W. F. Goebel and 0. T. Avery, ]. E x p . Med., 54 (1931)431-436. (200) 0. T. Avery and W. F. Goebel,]. E x p . Med., 54 (1931)437-447. (201) W. F. Goebel,]. Em. Med., 72 (1940)33-48. (202) W. F. Goebel,]. E r p . Med., 69 (1939)353-364. (203) W. E. Paul, D. H. Katz, and B. Benacerraf,]. lmmunol., 107 (1971)685-688. (204) H. Braley-Mullen,]. lmmunol., 113 (1974) 1909-1920.

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sociation with methylated bovine serum albumin.205In some of these methods, the coupling techniques employed were far too drastic for the highly sensitive polysaccharides currently used as human vaccines. Also, the carrier proteins and the coupling methods employed in the synthesis of these conjugates resulted in the formation of conjugates having constituents or structural features (for example, aromatic groups) highly undesirable for use in human vaccines. More-comprehensive studies on polysaccharide-protein conjugates directed specifically to their use as human vaccines have now been reported; the development of simple, and efficient, coupling procedureszo6has resulted in the formation of linkages to compounds containing more-innocuous, and more-acceptable, structural features. The H. injuenzue type b polysaccharide was conjugated to a number of proteins by Schneerson and coworkers207by using an adipic dihydrazide spacer between the molecules. These conjugates were relatively nontoxic, and, in contrast to the pure polysaccharide, functioned as thymic-dependent (T-cell-dependent) antigens. They produced polysaccharide-specific, serum antibodies in mice and other animals, and the level of these antibodies could be augmented by reinjection of the conjugate. The group C meningococcal polysaccharide was also successfully converted into a thymic-dependent antigen b y Beauvery and coworkers,2°M who linked it directly through the carboxyl groups of its sialic acid residues to the amino groups of tetanus toxoid (amide linkages), using l-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride. The foregoing conjugation methods employed random activation of the many functional groups of the polysaccharides, and more-specific coupling was obtained in the formation of artificial SaZmoneZZa typhimurium and Pseudomonas aeruginosa vaccines. Svenson i n d Lindberg15*.209 synthesized the former by coupling the smaller molecularsize octa- and dodeca-saccharides, obtained by treatment of the 0chain of the Salmonella typhimurium lipopolysaccharide with phage enzymes, to bovine serum albumin. A carboxyl group, unique to the oligosaccharide, was generated on their reducing (end-group) rham(205) 0. J. Plescia, W. Braun, and N. C. Palczuk, Proc. Natl. Acad. Sci. U . S. A., 52 (1964)279-285. (206) C. P. Stowell and Y. C. Lee, Ado. Carbohydr. Chem. Biochem., 37 (1980) 225281. (207) R. Schneerson, 0.Barrena, A. Sutton, and J. B. Robbins,]. E r p . Med., 152 (1980) 361 -376. (208) E. C. Beauvery, F. Miedema, R. W. Van Delft, and J. Nagel, in J. B. Robbins, J. C. Hill, and J. C. Sadoff (Eds.), Seminars in Infectious Disease. Bacterial Vaccines, Vol. 4, Thieme-Stratton, New York, 1982, pp. 268-274. (209) S. B. Svenson and A. A. Lindberg, J . Immunol. Mkthods, 25 (1979)323-335.


199

nose residues, through which they were linked to the protein by amide linkages by using 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride. The conjugates elicited good antibody responses in rabbit^,'^,^'^ but not in mice,2l0although it was demonstrated that the rabbit antibody was able passively to protect the mice against infection by live, homologous Salmonella organisms.210In subsequent work, Seid and Sadoff prepared a tetanus toxoid conjugate of the (larger molecular size) whole, base-treated lipopolysaccharide of type 5 Pseudomonas aeruginosa by the formation of amide linkages between the carboxyl groups of its few KDO residues and the amino groups of 1,4-diaminobutane spacers pre-attached to the protein. The conjugate proved considerably less toxic than the original lipopolysaccharide, and preliminary, immunological results indicated that IgG antibodies having a specificity for the 0-chain can be readily induced in mice by using this conjugate.211Except for the potentiaI loss of alkali-labile substituents on lipopolysaccharide 0-chains, this method could prove to be applicable to all bacterial lipopolysaccharides, and it has obvious potential in the synthesis of human vaccines. In an attempt to extend the monofunctional-group approach to the conjugation of the meningococcal polysaccharides, and thus to produce conjugates more chemically defined, Jennings and L ~ g o w s k i ' ~ ~ inserted a unique, terminal, free aldehyde group into the groups A, B, and C polysaccharides. This was achieved by controlled, periodate oxidation of the native, group B and C polysaccharides and of the group A polysaccharide premodified by reduction of its terminal, reducing 2-acetamido-2-deoxy-~-mannose residue (see Fig. 6). These monovalent molecules were then specifically coupled to tetanus toxoid by reductive amination, using sodium cyanoborohydride, without activating the other functional groups in the polysaccharide. When used as vaccines in mice and rabbits, the group A and C polysaccharide-tetanus toxoid conjugates produced high-titer antisera having bactericidal activity against the homologous organisms, indicating the potential of these conjugates as human vaccines. In contrast, the group B polysaccharide-tetanus toxoid conjugate failed to elicit detectable, polysaccharide-specific antibodies in these anim a l ~Inhibition . ~ ~ ~ experiments indicated that the antibody produced was not specific for the polysaccharide, but was highly specific for the linkage between the lysine residues of tetanus toxoid and the nonreducing (end-group) heptulosylonic acid group of the oxidized group B polysaccharide. (210) S. B. Svenson and A. A. Lindberg, Infect. lmmun., 32 (1981) 490-496. (211) R. C. Seid, Jr., and J. C. SadofT,/. B i d . Chem., 256 (1981) 7305-7310.

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HAROLD J. JENNINGS

6 R=R'=H IR=R'=COCH, 8 R=H,R'=COCH, 9 R = COCH,, R' = H

1

L R = OCCH, or H

FIG.6.-Shucture ofthe Group C (uppermost),B (middle), and Terminally Reduced Group A (lowest) Polysaccharide Antigens OfNeisseria meningitidis, Depicting the Positions of Cleavage on Oxidation by Periodate.

6. Natural Immunity, and Polysaccharide Serological Cross-reactions Adult-animal sera contain antibodies to a variety of polysaccharides, including those of human pathogenic bacteria, indicating that, to confer immunity, disease is not required. Robbins and were able to detect antibodies having a specificity for Vibrio cholerue (212)J. B.Robbins, R. Schneerson,M. P. Glode, W. Vann,M. S. Schiffer,T.-Y. Lui, J. C . Parke, and C. Huntley,]. Cell. Clin. Zmmunol., 56 (1975)141-151.


201

in animals, as the animals were allowed to mature without the possibility of contact with the homologous organism. In addition, in serological studies on a healthy human population, there is frequently detected the presence of antibodies having a specificity for the capsular polysaccharides of groups A, B, and C N. meningitidis and type b H. influenme that cannot be satisfactorily explained by asymptomatic Similar findings were reported for antibodies to the pneumococcal type 3 polysaccharide in children.213These populations of antibodies result from exposure to cross-reacting antigens among the nonpathogenic bacteria found in the nasopharyngeal or gastrointestinal Serological cross-reactions among polysaccharides are a well documented phenomenon, due in large part to the extensive work conducted in this area by Heidelberger and This phenomenon is due to the ability of polysaccharide antigens to promote the formation of heterogeneous populations of antibodies, and to the special property of polysaccharides to retain domains of structural and conformational similarity despite some structural differences. This is well illustrated by the cross-reactions exhibited between the different pneumococcal polysaccharides (see Sections III,3 and VJ). However, cross-reactions between polysaccharides from different species of other organisms have been used to great advantage in probing polysaccharide structures. Thus, an antiserum of one polysaccharide of known structure becomes a reagent to monitor for similar structural features in other polysaccharides. Heidelberger and coworkers have used this type of analysis extensively, and the results have constituted the subject of several reviews.108*214-219 In addition to its analytical value, this phenomenon is probably the basis of the important mechanism by which humans develop natural imrn~nity.~ In fact, it has also been postulated that exposure to these cross-reacting, T-cell-dependent organisms is the most satisfactory explanation for the eventual maturation of the polysaccharide immuneresponse in infants.165It can be shown, for instance, that there is an age-related increase in natural antibodies to the group A meningococcal polysaccharide in children, even though the group A organism is

(213) M. Finland,]. Infect. Dis., 128 (1973) 76-124. (214) M. Heidelberger, Res. lmmunochem. lmmunobiol., 3 (1973) 1-40. (215) M. Heidelberger, in J. B. G. Kwapinski (Ed.), Research in Zmmunochemistry, Vol. 3, University Park Press, Baltimore, 1973. (216) M. Heidelberger, Annu. Reo. Biochem., 36 (1967) 1-12. (217) J. M. Tyler and M. Heidelberger, Biochemistry, 7 (1968) 1384-1392. (218) M. Heidelberger and W. Nimmich,J. lmmunol., 109 (1972) 1337-1344. (219) M. Heidelberger and W. Nimmich, Immunochemistry, 13 (1976) 67-80.

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rarely isolated in the United States.22oFrequently found in normal human flora are cross-reacting organisms that could be responsible for natural immunity to groups A, B, and C N. meningitidis and type b H. infiuenzae. Robbins and coworker^^*^^^ and Egan and coworkers222 identified some of these important organisms; they are listed in Table VI. Serological studies indicated that the capsular polysaccharides of these organisms are responsible for the cross-reactions, and this is confirmed by the structural similarity of some of these polysaccharides (see Table VI) to those from N. meningitidis (see Table I) and H. influenzcre (see Table 11). Schneerson and rob bin^^^^ clearly demonstrated the feasibility of this mechanism by deliberately feeding nonpathogenic E. coli possessing the K-100 capsule to human-adult volunteers, Colonization readily occurred, and antibodies specific for the H. infiuenzae type b polysaccharide were induced.

VI. BACTERIAL VIRULENCE

1. Role of the Capsular Polysaccharide We are constantly in contact with a wide range of micro-organisms in our external environment, but few of these prove to be virulent. The virulence of bacteria is dependent on their ability to invade the human host and to evade the host's immune system, thus allowing the bacteria to propagate within the host. The varied strategies employed by the bacteria to evade the host's immune system have been re~iewed.':'~.'*~ Although the mechanisms behind these different strategies are incompletely understood, research in this area is encouraging, and suggests that an understanding of the pathogenesis of infectious diseases at the molecular and macromolecular level will eventually be possible. Because of their surface location, capsular polysaccharides are important agents in bacterial pathogenesis, as they interact directly with the host's immune system. The initial event in the pathogenesis of most bacterial infections is the attachment of the bacteria to the mucosal surface. This probably occurs by a receptor mechanism that exhibits a high degree of cellular specificity. Capsular polysaccharides have not been implicated in this (220) I. M. Goldschneider, M. L. Lepow, E. C. Gotschlich, F. T. Mauck, F. Bache, and M. Randolph,]. Infect. Dis., 128 (1973)769-776. (221) J. B Robbins, R. L. Myerowitz, J. K.Whishant, M. Argaman, R. Schneerson, Z.T. Handzel, and E. C. Gotschlich, Infect. Zmmun., 6 (1972)651-656. (222) W. Egan, F.-P. Tsui, and H.Schneerson,]. Biol. Chem., submitted for publication. (223) R. S. Schneerson and J. B. Robbins, New Engl.]. Med., 292 (1975) 1093-1095.

TABLEVI

Polysaccharides of Bacteria, Frequently Found in Human Flora, That Cross-react with the PolysaccharideCapsules of Human Pathogenic Bacteria Pathogen Neisseria meningitidis Group A Croup B Group C

Cross-reacting organism Bacillus pumilis Streptococcus fecalis Escherichia coli K 1 Escherichia coli K92

Structure

References

2-acetamido-2-deoxymannosyl phosphate residues

22 1 7 191 186

+ g)cyD-NeupAc(z+

and its OAc' variant

+ 8)a~-NeupAc(2 + g)aD-NeupAc(2+

Haemophilus influenme

0 Type b

Escherichia coli KlOO

+ 3)p~-Ribf( 1-+

II

2)D-ribitol(5-O-P-

222

I

Staphylococcus aureus Bacillus pumilis Bacillus subtilis Lactobacillus plantarum

OH teichoic acids containing ribitol phosphate

7

204

HAROLD J. JENNINGS

mechanism, which probably involves interactions between the glycose moieties of the surface glycoprotein of human cells and surface ~ ~ , ~ ~ ~polysacchaproteins (pili or fimbriae) of the b a ~ t e r i a . ' Capsular rides, however, are very much involved in the pathogenesis of bacteria following the penetration of these bacteria into body tissue. There is abundant accumulated evidence to demonstrate that capsular polysaccharides are important virulence factors in disease caused by Neisseria meningitidis, Haemophilus influenxae, group B Streptococcus, and Streptococcus pneu~oni~e.7~14E~'s5.225~227 This is also the case for the capsular polysaccharides of other pathogenic bacteria, including those in which the high-molecular-weight 0-chains of their lipopolysaccharides are functionally equivalent to the capsular polysaccharides.17 The importance of capsular polysaccharides in pneumococcal infections was demonstrated fifty years ago, when it was shown that the enzymic depolymerization of the capsular polysaccharide on the surface of type 111 S. pneumoniae organisms considerably decreased the virulence of these organisms in mice.z28The property of the capsular polysaccharide that enhances the virulence of bacteria is its ability to mediate the host's immune system. Except for a few cases of molecular mimicry (see Section VI,2), the major mechanism involved in this mediation is its function as an inhibitor of the fast-acting, alternative pathway of complement-induced phagocytosis, thus forcing the immune system to utilize the slower, classical pathway. The complement system is briefly explained in Section IV,2. Because the classical pathway has a requirement for polysaccharide-specific antibody, and because the process of producing this antibody takes a few days, the host is compromised during the initial, acute stages of bacterial infection, and is liable to die, or to acquire serious morbidity effects. Thus, the rationale behind vaccination with capsular polysaccharides is to maintain a long-lasting, effective level of polysaccharide-specific antibody in the host. The function of the polysaccharide capsule in inhibiting the alternative pathway is most satisfactorily and simply explained by the fact that it masks the underlying, bacterial structures (for example, teichoic acids), which are known to be powerful activators of the alternative p a t h ~ a y . ' ~ " -However, '~ although this mechanism is no doubt

(224)E. H. Beachey,]. Infect. Dis., 143 (1981) 325-345. (225) R. Bortolussi, P. Ferrieri, B. Bjorksten, and P. G. Quie, Infect. Immun., 25 (1979) 293-298. (226) C . M. McLeod and M. R. Krauss,]. E r p . Med., 92 (1950) 1-9. (227) C. J . Howard and A. A. Glynn, Immunology, 20 (1971) 767-777. (228)0.T. Avery and R. Dubos,J. E x p . Med., 54 (1931) 73-89.


205

operative, the concept of the capsule as a simple, physical barrier is inadequate to explain all of the experimental results and observations. For instance, why are not all encapsulated bacteria pathogenic? To understand this mechanism filly, it will first be necessary to comprehend the molecular events leading to the complement-mediated phagocytosis of bacteria. Although most of these events have been elucidated, the critical mechanism whereby the alternative pathway is first activated still remains o ~ s c u ~ However, ~ . ~ ~ from ~ , ~ the~point ~ , of ~ ~ ~ view of the capsular polysacckaride, evidence has been accumulated that helps to reveal some of its functional aspects in the inhibition of complement-mediated phagocytosis of bacteria. Evidence that the capsule creates a simple, physical barrier to the underlying surface of the bacteria can be found in the fact that only encapsulated bacteria are pathogens, and, for any given pathogenic strain of bacteria, its virulence is directly related to the amount of capsu1e.176,226,231 However, the amount of capsule is not the only criterion on which virulence is based, because although heavily encapsulated type 3 pneumococci are extremely virulent in mice, type 37 pneumococci, having the same degree of encapsulation, are In addition, type 12 pneumococci, having very small capsules, are extremely virulent in humans.176On the basis of these results, plus the known species-specificity of bacterial pathogenesis, other physical, compositional, or structural properties must be postulated to account for the role of the polysaccharide capsules in bacterial pathogenesis. Experimentation leading to the delineation of this role is hampered by the complexity of the bacterial surface and by lack of knowledge as to whether the polysaccharide capsules function alone as mediators of the complement system. However, the use of molecular cloning-techniques has considerably simplified the problem, and has demonstrated that not all capsular polysaccharides have the same function in bacterial pathogenesis. Moxon and Vaughn232demonstrated that the polysaccharide capsule of type b H. injluenzue is necessary, and, indeed, sufficient, to perform this function. Type b and d transformants were made from the same, unencapsulated, H. influenme strain, thus giving the transformants DNA homology, except for the regions that determine serotype specificity. As in the clinical situation, the type b (229)D.T.Fearon and K. F. Austen, Proc. Natl. Acad. Sci. U . S . A.,74 (1977)16831687. (230)R. D. Schrieber, M. K. Pangburn,P. H. Lesavre, and H. J. Muller-Ebenhard,Proc. Natl. Acad. Sci. U . S . A., 75 (1978)3948-3952. (231)A. A. Glynn, W. Brumfitt, and C. J. Howard, Lancet, (1977)514-516. (232)E. R. Moxon and K. A. Vaughn,]. Infect. Dis., 143 (1981)517-524.

206

HAROLD J. JENNINGS

transformant proved to be more invasive and virulent in rats than that of type d. This experimental result is also consistent with the structural basis of bacterial pathogenicity. In similar studies by Silver and coworkers,233the cloned genes responsible for synthesis of the polysaccharide capsule of the highly pathogenic E. coZi K 1 organisms were able to synthesize the identical capsule in the non-pathogenic E. coZi K12 organisms. In this case, however, the capsule alone was not able to induce the same virulence properties in the transformed organism nonnally associated with the E. coli K1 bacteria, indicating that the E . coli K1 capsular polysaccharide probably functions in concert with other surface components of the K1 organisms.

2. Polysaccharide Structure and Pathogenicity

That polysaccharide structure could be involved in bacterial pathogenesis can be deduced from the observation that, of all the encapsulated bacteria, only a few are virulent in man, and interestingly, two of these different species of bacteria share a common capsular polysaccharide. Group B N. meningitidis and K1 E . coZi produce the same aD-(2+8)-linked sialic acid homopolymer (see Table V) as their only common surfacecomponent, and both are a major cause of meningitis in children. Some experimental evidence234is also consistent with the structure of polysaccharide capsules’s being implicated in the virulence of bacteria, which stems from their differing abilities to inhibit the activation of complement by way of the alternative pathway. In measuring the survival time of the six serotypes (a, b, c, d, e, and f ) of H . influenme in antibody-free sera containing complement, only the type b organisms were able to survive complement-mediated phagocytosis for any appreciable length of time. Although it has, to date, not been possible to identify any common structural feature among all the polysaccharide capsules of bacteria associated with the most pathogenic human disease, there is one common feature in many of them. The capsular polysaccharide of type I11 group B Streptococcus has terminal sialic acid residues in its struct~ree,6~ asqdo ~ ~ the groups B and C N. rneningitidis and K 1 E . C O Z ~ . ~ ~ ~ ~ ~ The ability of terminal sialic acid residues to inhibit the activation of complement by way of the alternative pathway has been well docu(233)R. P. Silver, C. W. Finn, W. F. Vann, W. Aaronson, R. Schneerson, P. J. Kretschmer, and C. F. Garon, Nature, 289 (1981)696-698. (234) A. Sutton, R. Schneerson, S. Kendail-Morris, and J. B. Robbins, Infect. Immun., 35 (1982)95-104.


207

mented for erythrocyte-membrane ~ u r f a c e s . ~ ~Edwards ~ - ~ ~ ' and cow o r k e r ~also ~ ~observed ~ this phenomenon on bacterial surfaces. Normally, type I11 group B streptococcal organisms are potent inhibitors of the alternative pathway, but, when they are grown in neuraminidase, which removes the terminal sialic acid residues, they are converted into alternative-pathway activators. F e a r ~ described n ~ ~ ~ a similar experiment for the conversion, using neuraminidase, of sheep erythrocytes into activators of the alternative pathway. In an attempt to delineate the structure-function relationship of the terminal sialic acid residues of the type I11 group B streptococcal polysaccharide with this inhibition, Edwards and coworkers238chemically modified the polysaccharide on the surface of the bacteria. Reduction of the carboxylate groups of the sialic acid residues to hydroxymethyl groups also changed the surface of the type I11 organisms to become alternative-pathway activators, thus indicating that removal of these terminal residues in order to expose underlying, structural features is not required for activation to occur. F e a r ~ nalso ~ ~demonstrated ~ that removal of only C-8 and C-9 of the glycerol end-chain of terminal sialic acid residues of sheep erythrocytes is sufficient to convert them into activators; in this experiment, the charge on the sialic acid residues was retained, thus indicating, by extrapolation to the type I11 group B streptococcal polysaccharide, that the charge alone is not responsible for its inhibitory properties. All of these experiments involving the chemical modification of terminal sialic acid residues gave results that are consistent with the hypothesis that any change in the integrity of the sialic acid residue could alter its capacity to modulate the complement system, and this is also consistent with the work of Varki and K ~ r n f e l dwho , ~ ~showed ~ that the extent of 0-acetylation of sialic acid residues is directly related to the capacity of murine erythrocytes to activate the alternative-complement pathway. However, this hypothesis may be oversimplistic, and there still remains the possibility that there are involved more-complex, structural features in which the sialic acid residues could provide tertiary conformation to those surface structures. Certainly, such conformationally controlled determinants (235) D. T. Fearon, Proc. Natl. Acad. Sci. U . S. A., 75 (1978) 1971-1975. (236) M. K. Pangbum and H. J. Muller-Eberhard,Proc. Natl. Acad. Sci. U . S . A., 75 (1978) 2416-2420. (237) M. D. Kazatchkine, D. T. Fearon, and K. F. Austen,J. Immunol., 122 (1979) 7581. (238)M. S. Edwards, D. L. Kasper, H. J. Jennings, C. J. Baker, and A. NicholsonWeller,J. Zmmunol., 128 (1982) 1278-1283. (239) A. Varki and S. Komfeld,J. E r p . Med., 152 (1980)532-544.

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have been identified in the type I11 group B streptococcal polysaccharide.76.131 Another mechanism whereby capsular polysaccharide could mediate the human immune-system is by molecular mimicry. If a bacterium were able to coat itself with molecules having a structure similar to that of those found in the host’s tissue, the production of antibodies having a specificity for these structures would be suppressed, as they would be recognized as part of “self” by the host. Some organisms (schistosomes) are able to acquire human blood-group determinants on their surfaces in order to circumvent the deleterious effects of the host’s immunological response.%O However, known examples of this type of molecular mimicry in bacteria are very few. Although the capsular polysaccharides of both types Ia and Ib, and type I11 group B Streptococcus have a high degree of structural homology with the respective M and N human blood-group s ~ b s t a n c eand s~~ human ~ ~ serotran~ferrin,6~ there is no evidence that this structural homology interferes with the production of polysaccharide-specific antibody in humans.62The best example of molecular mimicry is probably that of the a-D-(2+8)-linked sialic acid homopolymer, which serves as the capsule for both groups B N . meningitidis and K1 E . coli (see Table VI). This polysaccharide is poorly immunogenic in humans,ls2 and there is evidence to suggest that this poor immunogenicity could be attributed to its structural homology with human glycoprotein.lS5Already highly pathogenic, were it not for the production of antibodies against other surface components of both organisms, they would, indeed, be superpathogens.

(240) 0.L. Goldring, J. A. Clegg, S . R. Smithers, and R. J. Teny, Clin. Enp. Zmmunol., 26 (1976)181-187.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 41

HIGH-RESOLUTION, 'H-NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AS A TOOL IN THE STRUCTURAL ANALYSIS OF CARBOHYDRATES RELATED TO GLYCOPROTEINS BY JOHANNES F. G. VLIEGENTHART, LAMBERTUS DORLAND, AND HERMANVAN HALBEEK Department of Bio-Organic Chemistry, Unioersity of Utrecht, Utrecht, The Netherlands

I. General Introduction ........................................... 209 1. High-resolution, 'H-N.m.r. Spectroscopy ......................... .211 2. Literature Data on High-resolution, 'H-N.m.r. Spectroscopy of Carbohydrates Derived from Glycoconjugates ....................214 11. High-resolution, 'H-N.m.r. Spectroscopy of Carbohydrates Related .218 to Glycoproteins of the N-Glycosylic Type ....................... 1. Carbohydrate Chains of the N-AcetylladosamineType .............. .218 2. Carbohydrate Chains of the Oligomannoside Type ................. .343 3. Additivity Rules ........................................... .365 111. Concluding Remarks .......................................... .371 IV. Experimental ................................................. 373

I. GENERALINTRODUCTION Glycoproteins are biopolymers consisting of a polypeptide backbone bearing one or more covalently linked carbohydrate chains. During the past decade, interest in the structure and function of glycoproteins has increased greatly, as it was found that the carbohydrate chains of these polymers are involved in several important biochemical processes. In particular, their roles in recognition phenomena, in immunological events, and in determining the life-span of cells and glycoproteins must be mentioned. For understanding of the biological function of the glycan chains, detailed knowledge of their structures is a prerequisite. The carbohydrate chains of glycoproteins may be classified according to the type of linkage to the polypeptide backbone. N-Glycosylic chains are attached to the amide group of asparagine (Asn), whereas the 0-glycosylic chains are linked to the hydroxyl group of such amino acid residues as serine (Ser), threonine (Thr), and hydroxylysine (Hyl). As a whole, the carbohydrate chains show a large variety in pri209 Copyright @ 1883 by Academic Press, Inc.

All rights of repmduaion in any form reserved. ISBN 0-1%0072418

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mary structure, as has been discussed by Montreuil,1*2the Kornfelds,3 and Sharon and Lis.* The unambiguous determination of the primary structure of carbohydrates is much more cumbersome than for other biopolymers. The large number of glycosylic linkages possible, in conjunction with the occurrence of branching, yields a fantastically large number of theoretically possible isomers, even for a relatively simple oligosaccharide. This demands a high degree of sophistication in methods used for analysis of the structure. The first analytical problem is encountered at the level of the glycoprotein as such. Owing to natural or artificially introduced (micro)heterogeneity in the carbohydrate chains, it is virtually impossible to obtain the polymer in the form of a single molecular species. Secondly, analysis ofthe complete primary structure can so far not be conducted on intact glycoproteins, and degradation to glycopeptides, oligosacckarides, or oligosaccharide-alditols is obligatory. Thirdly, the fractionation of more or less complex mixtures of (closely) related, partial structures is difficult. Reliable checks for the purity of the compound isolated are indispensable, because, if a sample is considered to be homogeneous, but, in fact, consists of a mixture of closely related components, incorrect structures may be deduced. This might be one of the reasons why it is not exceptional that, for one and the same carbohydrate side-chain of a certain glycoprotein, more than one structure has been reported. Over the years, several strategies have been developed for determination of the structure of carbohydrate chains; for concise reviews, see Refs. 3 and 4.In particular, the refinements of methylation a n a l y s i ~ , ~ , ~ ~ and c h e m i c a P and enzymic degradation method^^-'^ have permitted (1) J. Montreuil, Adz-. Curbohydr. Chern. Biochem., 37 (1980) 157-223. (2) J. Montreuil, in A. Neuberger (Ed.), Comprehensive Biochemistry, Vol. 19 B, Part 11, Elsevier, Amsterdam, 1982, pp. 1-188. (3) R. Komfeld and S. Komfeld, in W. J. Lennalz (Ed.),The Biochemistry ofGlycoproteins und Proteoglycans, Plenum, New York, 1980, pp. 1-34. (4)N. Sharon and H. Lis, in H. Neurath and R. L. Hill (Eds.), The Proteins, 3rd edn., Vol. V, .4cademic Press, New York, 1982, pp. 1-144. ( 5 ) B. Lindberg and J. Liinngren, Methods Enzymol., 50 (1978) 3-33. (54 H. R a ~ i ~ a l J, a , Finne, T. Krusius, J. Kirkkainen, and J . Jamefelt,Ada. Carbohydr. Chem. Biochem., 38 (1981) 389-416. (6)B. Lindbery, J. Lonngren, and S. Svensson, Ado. Carbohydr. Chem. Biochern., 31 (1975) 185-240. (7) G. 0. Aspinall, Pure A p p l . Chem., 49 (1977) 1105-1134. (8) G. Strecker, A. Pierce-Cretel, B. Foumet, G. Spik, and J. Montreui1,Anal. Biochem., I 1 1 (1981) 17-26. (9) Y.-T.Li and S.-C. Li, in M. I. Horowifz and W. Pigman (Eds.),The Glycocunjugates, Vol. I, Academic Press, N e w York, 1977, pp. 51-67. (9a) H. M. Flowers and N. Sharon, Ado. Enzymol., 48 (1979) 29-95. (10) A. Kobata, A n d . Biochem., 100 (1979) 1-14.

'H-N.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

21 1

the unraveling of new structures. The enzymic methods were greatly improved by rigorous purification of isolated exo- and endo-glycosidases. However, the classical methods have some inherent limitations, and are time- and material-consuming. In the past few years, we have had the opportunity to introduce high-resolution, 'H-n.m.r. spectroscopy for determination of the structure of glycan chains of glycopr~teins.'~-'~ In close collaboration with J. Montreuil and his colleagues (Lille, France), we have shown that this technique, in conjunction with methylation analysis, is extremely suitable for structural studies on N-, as well as on 0-,glycosylic glycans. The present article contains a discussion of the high-resolution, 'H-n.m.r. spectra of compounds, derived from N-glycosylically linked carbohydrate chains, comprising the N-acetyllactosamine and oligomannoside types of structure.

1. High-resolution, 'H-N.m.r. Spectroscopy 'H-N.m.r. spectroscopy has contributed significantly to extensions of our knowledge on the structure and conformation of biomolecules as well as on intra- and inter-molecular, interaction processes. N.m.r. spectroscopy is, however, an inherently insensitive technique, and the richness of information contained in an n.m.r. spectrum often limits the size and complexity of the molecules that can usefully be studied. Advances in instrument design have now greatly improved the sensitivity of the spectrometers, so that resonances can readily be observed in aqueous solutions of, for instance, complex carbohydrates, at concentrations of the order13 of 0.05 mM. Increase in the strength of magnetic fields (up t014 14 Tesla) has enabled the study of molecules of larger molecular weight and complexity by inducing better spectral-resolution. Moreover, the availability of sophisticated computerfor example, programs allows an artificial resolution-enhan~ement,'~ by Lorentzian to Gaussian transformation; for general reviews, see (11)J, Montreuil and J. F. G. Vliegenthart, in J. D. Gregory and R. W. Jeanloz (Eds.), Glycoconjugate Research, Proc. lnt. Symp. Glycoconjugates, 4th, Vol. I, Academic Press, New York, 1979, pp. 35-78. (12)J. F. G.Vliegenthart, H. van Halbeek, and L. Dorland, in A. Varmavuori (Ed.), IUPAC Int. Congr. Pure A p p l . Chem., 27th, Helsinki, 1979, Pergamon, Oxford, 1980, pp. 253-262. (13)J. F.G. Vliegenthart, H. van Halbeek, and L. Dorland, Pure A p p l . Chem., 53 (1981) 45-77. (14)M. Llinis, A. de Marco, and J. T. J. Lecomte, Biochemistry, 19 (1980)1140-1145; D. G. Davis and B. F. Gisin, FEBS Lett., 133 (1981)247-251; M.L. Hayes, A. S. Serianni, and R. Barker, Carbohydr. Res., 100 (1982)87-101. (15)R. R. Emst, Ado. Magn. Reson., 2 (1966)1-135.

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Refs. 16-20. Already, a partial interpretation of an n.m.r. spectrum can provide detailed information about molecular structure, as will be shown. For interpretation of the ‘H-n.m.r. spectrum of a carbohydrate chain in terms of primary structural assignments, the concept of “structuraEThis means that the chemical reporter groups” was shifts of protons resonating at clearly distinguishable positions in the spectrum, together with their coupling constants and the line widths of their signals, bear the information essential to permit assigning of the primary structure. In high-resohtion, ‘H-n.m.r. spectra of relatively large, N-glycosylic carbohydrate structures, resonances of the following structural-reporter groups can be discerned. (a) Anomeric Protons ( H - l Atoms). Their chemical shift and coupling constant provide information on the kind of sugar residue, as well as on the type and configuration of its glycosylic linkage. (b) Mannose H-2 and H-3 Atoms. The pattern of their signals is, as a whole, indicative of the type of substitution of the common, mannotriose branching-core. ( c )Sialic Acid H-3 Atoms. Their chemical shifts are characteristic for the type and configuration of the glycosylic linkage of the sialic acid residue, and, in some cases, for the location of the residue in the chain. ( d )Fucose H - 5 and CH, Atoms. The chemical shifts of these protons, together with that of H-1 of the residue, are indicative of the type and configuration of its glycosylic linkage, and of the structural environment, in particular of the residue to which the fucose is attached. ( e )Galactose H-3 and H-4 Atoms. Their chemical shifts are, in some cases, useful for characterizing the type and configuration of the glycosylic linkage between galactose and its substituent. cf)Amino Sugar

(2-Acetamido-2-deoxyglucoseand Sialic Acid) N-Acetyl-CH, Protons. Their chemical shifts are sensitive to even small structural variations, making this region of the spectrum highly informative. The line widths of the signals of the structural-reporter groups are influenced by the local mobility of the protons. This will be illustrated, in particular, for the anomeric-proton signals and for the N-acetyl signals. (16) R. A. Dwek, Nuclear Magnetic Resonance (N.M.R.)in Biochemistry: Applications to Enzyme Systems, Clarendon Press, Oxford, 1973. (17) P. F. Knowles, D. Marsh, and H. W. E. Rattle, Magnetic Resonance of Biomolecules, Wiley, New York. 1976. (18) K . Wuthrich, NMR in Biological Research: Peptides and Proteins, North-Holland, Amsterdam, 1976. (19) L. J. Berliner and J. Reuben, Biological Magnetic Resonance, Vols. 1 and 2, Plenum, New York, 1978. (20) R. G. Shulman, Biological Applications of Magnetic Resonance, Academic Press, New York, 1979.

.‘H-N.M.R. SPECTRA OF

GLYCOPROTEIN CARBOHYDRATES

213

Besides the aforementioned n.m.r. parameters [chemical shift (a), coupling constant and line width], spectral integration can give valuable information. The relative intensities of the structural-reporter-group signals in the n.m.r. spectrum can be used as markers for the purity of the compound. Often, from the spectrum, it can be deduced whether or not the sample consists of more than one carbohydrate structure, and in which molar ratios the components of the mixture, and the sugar residues in each of these, occur. In the ‘H-n.m.r. spectrum of a carbohydrate chain, a broad signal is observed, between 6 -3.4 and -4.0, that is derived from the bulk of nonanomeric, sugar-skeleton protons, but, so far, it could not be resolved into contributions of individual protons. In the case of glycopeptides, additional signals, derived from protons of the amino acid residues, are found in the spectrum. It should be stressed that a high-resolution, ‘H-n.m.r. spectrum of a compound provides a measure of the structural identity which, even if the spectrum cannot be completely interpreted, renders possible a comparison with the spectra of compounds obtained from other sources, allowing a decision as to whether or not the compounds are identical. After recording of this “identity card,” the unimpaired compound may be submitted to chemical and enzymic degradation-procedures. In this article, the 500-MHz, or, sometimes, 360-MHz, ‘H-n.m.r. spectra of some seventy N-glycosylic carbohydrate chains will be discussed. The spectra were recorded in D 2 0 at ambient temperature and at p D -7, unless indicated otherwise; for experimental details, see Section IV. First, the outstanding features of the spectra of fundamental elements of N-acetyllactosamine-type structures will be treated in detail (compounds 1-20); this part also covers the characteristics of the intersecting, GlcNAc residue in this type of structure. Secondly, extensions of these chains with differently linked, sialic acid residues (compounds 21-41), and thirdly, with fucose residues (compounds 42-54), will be discussed. Besides the structural-reportergroup signals of the newly introduced residues, the effects of extension on the remaining signals in the spectra will be traced. Next, some unusual, N-acetyllactosamine-type, N-glycosylic carbohydrate structures (compounds SS-SO) containing a virtually abnormal core-region, namely, PGal(1+4)pGlcNAc( l-*N)Asn, will be considered. Finally, the n.m.r. characteristics of oligomannoside-type carbohydrate chains (compounds 61-72) will be presented; in particular, the second branching-point, and the characteristics and influences of a-(1-2)linked mannose residues occurring in these structures, are the subjects of discussion.

u),

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2. Literature Data on High-resolution, 'H-N.m.r. Spectroscopy of Carbohydrates Derived from Glycoconjugates

The fundamental work of Lemieux and coworkers21introduced the successful application of 'H-n.m.r. spectroscopy to structural problems in the carbohydrate field. Since then, numerous studies have been devoted to the (partial) determination of primary structures of carbohydrates and derivatives thereof, as well as to the elucidation of their conformation in solution, by means of 'H-n.m.r. spectroscopy. An exhaustive discussion of all these contributions is beyond the scope of this article. For comprehensive reviews, see Refs. 22-28. Regarding the applicability of 'H-n.m.r. spectroscopy for elucidation of the structure of the carbohydrate moieties of glycoconjugates, several reports have been published. In 1973, one of the first examples of employment of high-resolution, 'H-n.m.r. spectroscopy for structural studies on intact g l y c ~ l i p i d safforded ~~ the 220-MHz, 'Hn.m.r.-spectral data for some peracetylated galactocerebrosides, determined in three different solvents. In 1979, Falk and described 270-MHz, 'H-n.m.r. spectroscopy of non-degraded, permethylated and permethylated-reduced derivatives of (blood-group active) glycosphingolipids (spectra were recorded in chloroform solution, at probe temperatures of -25 and -40"), as a suitable approach for the determination of the configuration of the anomeric linkages in the sugar chains. According to Karlsson and c o ~ o r k e r s , 3 3 -these ~~~ data, at most, supplement the structural fingerprinting of lipid-linked (21) R. U . Lemieux, R. K. Kullnig, H. J. Bemstein, and W. G. SchneiderJ. Am. Chem. SOC., 80 (1958) 6098-6105. (22) L. D. Hall, Adc. Carbohydr. Chem., 19 (1964) 51-93. (23) T. D. Inch, Annu. Aec. N M A Spectrosc., 2 (1969) 35-82. (23a) P. L. Durette and D. Horton,Adr;.Carbohydr. Chem. Biochem., 26 (1971)49-125. (24) B. Coxon, Adr. Curbohydr. Chem. Biochem., 27 (1972) 7-83. (25) G. Kotowycz and R. U. Lernieux, Chem. Rec., 73 (1973) 669-698. (26) L. D. Hall, Adc. Carbohydr. Chem. Biochem., 29 (1974) 11-40. (27) D. B. Davies, Nucl. Magn. Reson., 9 (1980) 182-203. (28) L. D. Hall, in W. Pigrnan and D. Horton (Eds.), The Carbohydrates: Chemistry und Biochemistry, 2nd edn., Vol. IB, Academic Press, New York, 1980, pp. 13001.326. (29) M. Martin-Lornas and D. Chapman, Chem. Phys. Lipids, 10 (1973) 152-164. (30) K.-E. Falk, K.-A. Karlsson, and B. E. Samuelsson, Arch. Biochem. Biophys., 192 (1979) 164-176. (31) K.-E. Falk, K.-A. Karlsson, and B. E. Sarnuelsson, Arch. Biochem. Biophys., 192 (1979) 177- 190. (32) K.-E. Falk, K.-A. Karlsson, and B. E. Sarnuelsson, Arch. Biochem. Biophys., 192 (1979) 191-202. (32a) K.-E. Falk, K.-A. Karlsson, H. Leffler, and B. E. Samuelsson, FEBS Lett., 101 (1979) 273-276.


215

oligosaccharides by mass spectrometry. Since 1980, Dabrowski and coworker^^^-^^^ have published 360MHz, 'H-n.m.r. data for a number of underivatized glycosphingolipids (blood-group active glycosylceramides, as well as more-complex compounds related to the Forssman glycolipid); the spectra were recorded at 65" for solutions in dimethyl sulfoxideds containing a trace of DzO. These authors were able to assign all of the anomeric-proton signals, and a number of nonanomeric-proton resonances. The chemical shifts of these protons were found to be dependent on essential, primary, Most of the chemical shifts of the ring prostructural tons were determined by spin-decoupling, and nuclear Overhauser, difference s p e c t r o s ~ o p y . ~ Concerning ~ , ~ ~ - ~ ~ ~ the potential of J-resolved, two-dimensional, 'H-n.m.r. spectroscopy for this purpose, Yamada and drew attention to the fact that, despite the many useful aspects of this method for carbohydrates (see Refs. 28 and 39), these experiments require a fair amount of time and material. In the field of glycoproteins, the following high-resolution, 'Hn.m.r.-spectral studies for solutions in D,O on underivatized, glycan chains related to, or identical with, those that will be described herein deserve mention. Wolfe and coworkers publishedPO in 1974 and4' 1975, 220-MHz, 'H-n.m.r. spectra of two oligosaccharides related to the N-acetyllactosamine type of N-glycosylic carbohydrate chain. (33) K.-A. Karlsson, in Ref. 12, pp. 171-183. (33a) M. E. Breimer, G . C. Hansson, K.-A. Karlsson, and H. Leffler, Biochim. Biophys. Acta, 617 (1980) 85-96. (33b) G . C. Hansson, K.-A. Karlsson, and J. Thurin, Biochim. Biophys. Acta, 620 (1980) 270-280. (34) K.-E. Falk, K.-A. Karlsson, and B. E. Samuelsson,FEBS Lett., 124 (1981) 173-177. (34a) J. Angstrom, M. E. Breimer, K.-E. Falk, I. Griph, G. C. Hansson, K.-A. Karlsson, and H. LeHer,J. Biochem. (Tokyo), 90 (1981) 909-921. (35) J. Dabrowski, H. Egge, P. Hanfland, and S. Kuhn, in C. C. Sweeley (Ed.), Cell Surface Glycolipids, ACS Symp. Ser. 128, American Chemical Society, Washington D. C., 1980, pp. 55-64. (36) J. Dabrowski, H. Egge, and P. Hanfland, Chem. Phys. Lipids, 26 (1980) 187-196. (37) J. Dabrowski, P. Hanfland, and H. Egge, Biochemistry, 19 (1980) 5652-5658. (37a) J. Dabrowski, P. Hanfland, H. Egge, and U. Dabrowski, Arch. Biochem. Biophys., 210 (1981) 405-411. (37b) P. Hanfland, H. Egge, U. Dabrowski, S. Kuhn, D. Roelcke, and J. Dabrowski, Biochemistry, 20 (1981) 5310-5319. (374 J. Dabrowski, P. Hanfland, and H. Egge, Methods Enzymol., 83 (1982) 69-86. (38) A. Yamada, J. Dabrowski, P. Hanfland, and H. Egge, Biochim. Biophys. Acta, 618 (1980) 473-479. (39) L. D. Hall, G. A. Moms, and S. Sukumar, Carbohydr. Res., 76 (1979) c7-cQ. (40) L. S. Wolfe, R. G. Senior, and N. M. K. Ng Ying KinJ. Biol. Chem., 249 (1974)1828 -1838. (41) N. M. K. Ng Ying Kin and L. S. Wolfe, Biochem. Biophys. Res. Commun., 66 (1975) 123- 130.

J. F. G. VLIEGENTHAHT et uZ.

2 16

These compounds were isolated from the 1ivefO and the urine4' of patients suffering from GM,-gangliosidosis type I. Their structures are identical to those of the di- and tri-antennary, asialo oligosaccharides 7 and 10 ofthis article (see Chart 1). The spectra were recorded at 70°, and the chemical shifts were measured in p.p.m. relative to external tetramethylsilane as the standard. In later s t ~ d i e s , 4 *these , ~ ~ authors arrived at a partial interpretation of the 100-MHz, and 90-MHz, 'Hn.m.r. spectra, recorded at 70 and 60°, respectively, of some oligosaccharides stemming from the liver of a patient who died of GMz-gangliosidosis variant 0 (Sandhoff- Jatzkewitz disease). The structures of the isolated hexa- and hepta-saccharides are the same as those respectively denoted 6 and 14 herein, and as that of the minor constituent present in the heptasaccharide mixture of which compound 14 forms the major part (see legend to Fig. 15).The linear, GMz-gangliosidosis tetrasaccharides described in Ref. 43,namely, /3GlcNAc(1+2)aMan(1+3)BMan( 1+4)GlcNAc and /3GlcNAc(lA)crMan(1+3)/3Man(1+4)GlcNAc, are not incorporated in the series of compounds discussed herein. In addition, the authors described43 the W M H z , 'H-n.m.r. data for a trisaccharide (compound 5) isolated from the urine of mannosidos is patients . In 1977 and 1980, we respectively introduced the application of 360-MHz, and 500-MHz, 'H-n.m.r. spectroscopy for elucidation of the structure of underivatized carbohydrate chains obtained from glycoproteins. Since then, several other research groups have become active in this field. For reasons outlined later, the results of their work will be briefly summarized first. In 1979, Kin and Wolfe" published the 220-MHz, 'H-n.m.r. spectra, recorded at 70°, of a mono-antennary glyco-asparagine possessing the same structure as 46 or 47, and of the corresponding oligosaccharide, h a v i n e GlcNAc-2 as the terminal, reducing-sugar residue. Like 46 and 47, the compounds were isolated from the urine of a hcosidosis patient. published some 360-MHz, 'H-n.m.r. data Schachter and for a series of glycopeptides having N-glycosylically linked carbohydrate chains of the N-acetyllactosamine type, and one having a chain of the oligomannoside type; spectra were recorded at a variety of probe temperatures (20,25,30,70, and 85"). The compounds (42) N. M.K. Ng Ying Kin and L. S. Wolfe, Biochem.Biophys. Res. Commun., 59 (1974) 837-844.

(43)N. M. K. Ng Ying Kin and L. S. Wolfe, Carbohydr. Res., 67 (1978) 522-526. (44) N. M. K. Ng Ying Kin and L. S. Wolfe, Biochem.Biophys. Res. Commun., 88 (1979) 696-705. (44a) For the system of numbering used for the sugar residues in N-glycosylic carbohydrate chains, see the footnote on page 221. (45) S. Narasimhan, N. Harpaz, G. Longmore, J. P. Carver, A. A. Grey, and H. Schachter, I. Biol. Chem., 255 (1980) 4876-4884.


217

a M a n ( l + 3), 'pMan( 1+ 4)pGlcNAc( 1-+ 4)pGlcNAc(1+ N)Asn, / / aMan(1 + 6) a F u c ( 1 + 6) pGlcNAc( 1+ 2)aMan( 1+ 31, aMan(1-+ 6)/

/3Man(1-+ 4)/3GkNAc( 1 + 4)/3GlcNAc( 1 + N)Asn, / aFuc(1 -+ 6)

and pGlcNAc( 1+ 2)aMan( 1-+ 3) \ pGlcNAc( 1+ 4)8Man( 1-+ 4)pGlcNAc(1+ 4)pGlcNAc( 1+ N)Asn, / aMan(1 -+ 6)/ aFuc(1 + 6)

were prepared from a human myeloma IgG; the other two, namely, pGlcNAc( 1 + 2)aMan( 1-+ 3), pGlcNAc( 1 + 4)pMan( 1-+ 4)PGlcNAc( 1+ 4)bGlcNAc( 1-+ N)Asn, pGlcNAc( 1 + 2)aMan(1+ 6f

and aMan(1 + , 1 3 pMan( 1-+ 4)pGlcNAc( 1 -+ 4)pGlcNAc( 1+ N)Asn,

crMan(l-+ 3)

\

aMan( 1+ 6)/

were obtained from hen-egg albumin. Subsequently, Cohen and BallouM exhaustively discussed the 180-MHz7'H-n.m.r. data (40")for a large number of building units (both as oligosaccharides and as glycopeptides) of the oligomannoside type of N-glycosylic carbohydrate chain possessing 1 through 8 mannose residues. Most of the compounds treated in Ref. 46 will also be subject to discussion herein (corresponding to structures 5,62-65, and 68-70). However, some ofthem, for example, pMan( 1 4 ) G l c N A c and aMan( 1-+ 3 ) , pMan( 1+ 4)GlcNAc, aMan( 1+ 6)'

are not included. It should be noted that the pioneering work of Gorin and coworker^^^-^^ on yeast mannans and galactomannans provided, for resonankes characteristic for a series of oligosaccharides, assignments (46) R. E. Cohen and C. E. Ballou, Biochemistry, 19 (1980) 4345-4358. (47) P. A. J. Gorin and J. F. T. Spencer, Can. J. Chem., 46 (1968) 2299-2304. (48) P. A. J. Gorin, M. Mazurek, and J. F. T. Spencer, Can. J. Chem., 46 (1968) 23052310. (49) P. A. J. Gorin, J. F. T. Spencer, and S. S. Bhattachaiee, Can. I . Chem., 47 (1969) 1499-1505.

2 18

J . F. G. VLIEGENTHART et

a1

that proved to be valuable for high-resolution, 'H-n.m.r.-spectral Finally, in addition to 'H-n.m.r. data for structures identical to 62 and 63, Carver and coworkers50 reported the 360-MHq 'H-n.m.r.spectral dsta for two glycopeptides possessing a hybrid type1 of structure, namely, pGlcNAc( 1

2)aMan(1 -+ 3)\ pClcNAc( 1 + 4)@Man(1 4 4)PGlcNAc(1 + 4)PGlcNAc(1 + N)Asn, uMan(1 -+ 3 ) , aMan(1 -+ 6) uhIlan(1 + 6') -+

/

and

4)

pGal( 1 -+ 4)/.3CkNAc(1 + pGlcNAc(1 + 2)aMan(l + 3)\ pGlcNAc( 1 + 4)BMan(1 -+ 4)PGlcNAc(1 -+ 4)@GlcNAc(1 + N)Asn

a.Cfan[l + 3)\ a M ~ n ( -+ 1 6)/

/

aMan(1 + 6)

In this study, n.0.e. difference-spectroscopy was applied to enable the making of some assignments. The spectral data for the compounds, the detailed structures of which have been presented in Section I, 2, are not compiled in the following Sections, because they were obtained from spectra recorded under more or less different experimental conditions (regarding strength of magnetic field, and reference standard, but, most of all, probe temperature) (compare, our experimental conditions, Section IV). This choice was dictated solely by the impossibility of making these data comparable with ours, and is not intended to imply any criticism of the work performed in other laboratories. 11. HIGH-RESOLUTION, IH-N.M.R.SPECTROSCOPY OF CARBOHYDRATES RELATED TO GLYCOPROTEINS OF T H E N-GLYCOSYLIC TYPE

1. Carbohydrate Chains of the N-Acetyllactosamine Type a. Fundamental Elements of Carbohydrate Chains of the N-Acetyllactosamine Type (Compounds 1-20).- Symbols employed for compounds 1-20 are depicted in Chart 1. (50) J. P. Carver, A. A. Grey, F. M.Winnik, J. Hakimi, C. Ceccarini, and P. H. Atkinson, Biochemistry, 20 (1981) 6600-6606.

CHART 1. Symbols Employed for Compounds 1-20 Key to the symbolic notation: 0 = GlcNAc

+ = Man W = Gal

1

0 = c~-NeuAc-(2-*6)

A = c~-NeuAc-(2+3) 0 = Fuc

e A s n

11 2 3

l-

Asn

rAsn 12

4

RAsn

5 13 6 14 7 15 8

16 9

17 10

18

19

20

>y

Thr

220

J. F. G. VLIEGENTHART et al.

The simplest element of the linkage region between an N-glycosylic carbohydrate chain and the polypeptide backbone to which it is attached is pGlcNAc(l+N)Asn (compound 1).Among various sources, 1 may be isolated from the urine of patients with aspartylglucosaminria.^'-% The 360-MHz, 'W-n.m.r. spectrum of compound 1 is presented in Fig. 1.The resonances in this spectrum may be divided into signals from the following groups of protons. The structural-reporter groups resonate at clearly distinguishable positions and provide essential information on the primary structure. The GlcNAc anomeric proton resonates at a lower field (see Table I) than could be expected [the normal range of S for an axial anomeric proton in a free or 0-glycosylically linked GlcNAc residue in the 'Cc,(D) conformation is 4.40-4.75 p.p.m.1. It is reasonable to assume that this effect is due to the electron-withdrawing amide group attached to C-1 (compare with Ref. 54).TheJl3 value (9.8 Hz) is characteristic for a p-glycosylic linkage. The value is relatively large, due to the N-type of glymsylic linkage. The chemical shift of the singlet of the N-acetyl group is typical for GlcNAc linked to Asn. As will be shown later, its position may vary, depending on the nature of the peptide moiety. The nonanomeric protons resonate in the range of 3.4-4.0 p.p.m. For this simple compound, a complete interpretation was achieved, and this was confirmed, and refined, by computer simulation of the spectrum. The n.m.r. data for compound 1 are summarized in Table I. By means of a modified Karplus equation, the 'Cc,(D)conformation of the GlcNAc group was deduced.55 The amino acid protons of Asn resonate apart from the GlcNAc protons. In glycoproteins, substitution at 0-6 of GlcNAc-1 by an (Y-L-FUC group frequently occurs.* Compound 2 can be isolated from the urine of patients suffering from f u ~ o s i d o s i s . ~The ~ , ~360-MHz, *~ 'H-n.m.r. spectrum of 2 is given in Fig. 2. (51) J. N. Isenberg, in E. F. Walborg, Jr. (Ed.), Glycoproteins and Glycolipids in Diseuse Processes, ACS Symp. Ser. 80,American Chemical Society, Washington D. c., 1978, pp. 129-131, and references cited therein. (52) G. Strecker and J. Montreuil, Biochimie, 61 (1979) 1199-1246. (53) In the description of the n.m.r.-spectralfeatures of each compound discussed in this article, only the actual source(s)of the compound used for the n.m.r. investigations is (are)mentioned. For other possible glycoprotein sources of these carbohydrates, the reader is referred to reviews'-'; see also, Section IV. (54) M. Tanaka and I. Yamashina, Carbohydr. Res., 27 (1973) 175-183. (55) L. Dorland, B. L. Schut, J. F. C. Vliegenthart,C. Strecker, B. Fournet, G. Spik, and J. Montreuil, Eur. I . Biochern., 73 (1977) 93-97. (56) G . Strecker, B. Foumet, J. Montreuil, L. Dorland, J. Haverkamp, J. F. G. Vliegenthart, and D. Dubesset, Biochimie, 60 (1978) 725-734.

221


TABLEI 'H Chemical Shifts and Coupling Constants for pGlcNAc(l+N)Asn (Compound 1) and aFuc(l-&)pGlcNAc(l+N)Asn (Compound 2) Coupling constant (Hz)"

Chemical shift (p.p.m.)"

Proton Compound 1 Compound 2

Residue ~

Compound 1 Compound 2

~

GlcNAC-1

H-1 H-2 H-3 H-4 H-5 H-6a H-6b NAc H-1 H-2 H-3 H-4 H-5 CHI H-(Y H-P H-P'

(YFUC( 1+6)

Asn

a

5.072 3.821 3.601 3.472 3.527 3.876 3.739 2.013

9.85 9.85 9.50 9.50 2.10 5.55 -11.40

-

3.75 10.30 3.40 0.60 6.55

-

-

6.30 3.80 - 17.25

3.968 2.866 2.932

Chemical shifts and coupling constants measured at 360 MHz and T = 300 K.

Comparison of the spectral data for 1and 2 shows that the chemical shifts of the GlcNAc structural-reporter groups, that is, H-1 and the N-acetyl-CH,-protons, are slightly, but significantly, influenced, whereas theJ,,2 value is but little affected by the extension of 1with a Fuc group. For the Fuc group, the resonances of its H-1, H-5, and CH3-groupprotons are characteristic. TheJ1,2value (3.75Hz) of Fuc is * Coding of monosaccharides for the carbohydrate chains described in this Chapter: Ny8-7

\ N-6- 5 - 4

\

9-3-2-l- Asn N'- 6 - 5 ' - 4

a-

7'

/

'

for 1-54

b D1-C-4

N -c- b-a

- 1I

F

Asn

for 55-60

&"\ 03- B

'3-2-1/

Asn

for 61-72

J . F. G. VLIEGENTHART et al.

222

0 -GlcNAc - (1--N) -Asn 1

1

NAc

CH3 p o I w

anomeric proton

I

60

-1

50 \

I I

\

\ \

I 1 I GlcNAc

n-i

A

I I I

, \

I

\ \

\

Asn H-*

\

GlcNAc

H-2 H-6a

\

H-5

H-6b H-3

H-4

A m

H - 6 H-P

\

I

I

1

5M 2 SO

FIG.1.- Resolution-enhanced, Overall, 360-MHz, IH-N.m.r. Spectrum of Compound 1 (Upper Trace), Supplied with Assignments in Full Detail, Most of Which are Indicated in the Expanded Regions (Lower Trace).[A small amount of sodium 4,4-dimethyl4-silapentane-I-srilfonate (DSS) had been added to the D,O solution, in order to serve as an internal reference for the chemical shifts (S(CH,),, DSS 0). (In the expanded pattern for the Asn P-CH, protons, the signals marked by asterisks originate from DSS.)]

indicative of an a-L-glycosidic bond between this group and the GlcNAc residue. The signals of the nonanomeric protons form a bulk that could be completely interpreted. The refined n.rn.r. data, obtained after spectral simulation, are given in Table I. For both GlcNAc and Fuc, the normal chair conformations [", (D) and ' C 4 ( ~ )respectively] , were cal-

'H4.M.R. SPECTRA OF GLYCOPROTEIN CARBOHYDRATES

223

0 -GlcNAc - (1-N)-Asn /

a-Fuc-(l-B)

I

'

2 NAC

C H 3 protons

Fuc -CH3 protons

..2

5.068 4.682 -4.77 5.136 4.922 4.606 4.575 4.444 4.549 4.253 4.197 -4.11b

-*> 45

- Asn

,"-

AS"

5.068 4.682 -4.77 5.118 4.940 4.575 4.606 4.545 4.447 4.253 4.190 -4.11b

H 3 of

H-1 of H-5 of CH, of

6 6' aNeuAc(2+6) aNeuAc(2+3) aNeuAc(2+6) aNeuAc'(2+6) aNeuAc(2+3) aFuc(l+6) aFuc(1+6) aFuc(l+6)

NAc of

1

H-3a of H3e of

2 5 5' aNeuAc(2+6) aNeuAc(2+3)

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