Advances in Carbohydrate Chemistry, Volume 12

ADVANCES IN CARBOHYDRATE CHEMISTRY VOLUME 12

Advances in Carbohydrate Chemistry Editor

MELVILLE L. WOLFROM Associate Editor

R. STUART TIPSON Board of Advisors

c. B. PURVES

HERMANN 0. L. FISCHER R. C. HOCEETT W. W. PIGMAN

J. C. SOWDEN ROYL. WHISTLER

Board of Advisors for the British Isles E. L. HIRST

STANLEY PEAT

MAURICE STACEY

Volume 12

1957

ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N. Y.

Copyright,@ 1957, by ACADEMIC PRESS INC. 111 Fifth Avenue New York 3, N. Y. All Rights Reserved

No part of this book may be reproduced in any form, by photostat, microfilm, or any other means without written permission from the publishers. Library of

COngTeS8

Catalog Card Number: 45-11351

PRINTED I N THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME 12

J. CONCHIE, Rowett Research Institute, Bucksburn, Aberdeenshire, Scotland A. B. FOSTER, Chemistry Department, The University of Birmingham, England DEXTER FRENCH, Department of Chemistry, Iowa State College, Ames, Iowa JOHN HONEYMAN, Chemistry Department, King’s College, University of London,England* G. A. LEVVY,Rowett Research Institute, Bucksburn, Aberdeenshire, Scotland CHESTERM . MCCLOSKEY, California Institute of Technology, Pasadena, California D. J. MANNERS,Department of Chemistry, The University of Edinburgh, Scotland C. A. MARSH,Rowett Research Institute, Bucksburn, A berdeenshire, Scotland J.’ W. W. MORGAN,Chemistry Department, King’s College, University of London, England ** W. BROCK NEELY, Research Department, G. D. Searle and Company, Chicago, Illinois*** E. J. OLSON,Department of Biochemistry, Purdue University, Lafayette, Indiana JOHNC. SOWDEN,Department of Chemistry, Washington University, St. Louis, Missouri R. STUART TIPSON, Mellon Institute, Pittsburgh 13, Pennsylvania**** ROY L. WHISTLER,Department of Biochemistry, Purdue University, Lafayette, Indiana * Present Address: The British Cotton Induatrg Reeearch Association, Shirley Imtitute, Mancheater, Enoland

** Preaent Address: British Celanese Ltd., Putteridge Burg, Bedfadahire, England *** Present Address: Biochemistry Department, The Dow Chemical Company, Midland, Michigan **** Present Address: National Bureau of Standurds, Washington 66, D. C.

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PREFACE With this Volume, the Advances in Carbohydrate Chemistry enters upon its twelfth year of publication with chapters shared about equally between British and American writers. Foster (Birmingham) continues our series on modern carbohydrate-separation techniques with a contribution on zone electrophoresis. After lying dormant for many years, the theory and practice of saccharinic acid formation is undergoing a current revival, reported on by Sowden (Washington University). Modern instrumentation has made the infrared absorption region of molecules readily available to the chemist. The complexities found in this spectral area with carbohydrate substances are still largely uninterpretable, but a start has been made which has been summarized by Neely (Dow Chemical Co.). Topics mainly hiochemical in nature are treated by French (Iowa State), Manners (Edinburgh), and by Whistler and Olson (Purdue); these are, respectively, the Schardinger dextrins, the glycogens, and hyaluronic acid. The fundamental organic chemistry of sugars is represented by chapters on sugar nitrates by Honeyman and Morgan (London), benzyl ethers by McCloskey (Pasadena), and simple glycosides by Conchie, Levvy and Marsh (Rowett Research Institute, Scotland). One of the editors (R. S. T.) has contributed a sketch of the life and work of the pioneer biochemist Phoebus A. Levene. Columbus, Ohio

M. L. WOLFROM

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R. STUART TIPSON

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CONTENTS Contributors to Volume 12.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phoebus Aaron Theodor Levene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

vii 1

Infrared Spectra of Carbohydrates BY W. BROCK NEELY,G. D. Searle and Company, Chicago, Illinois ...................................................

13

11. Molecular Spectra.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 111. Infrared Spectra.. . . . . . . . ................. IV. Interpretation of Infrared Spectra of Carbohydrates.. . . . . . . . . . . . . . . . . . . V. Application of Infrared Spectroscopy to Carbohydrates.. . .

The Saccharinic Acids BY JOHN C. SOWDEN, Department of Chemistry, Washington University, Saint Louis, Missouri

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 11. The Individual Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Mechanism of Formation of Saccharinic Acids.. ........................ 62 IV. Table of Properties of Saccharinic Acid Derivatives ......... 76 Zone Electrophoresis of Carbohydrates BYA. B. FoaTER, Chemistry Department, The University of Birmingham, England

I. Introduction., . . . . . . . . . . . ............................... . . . 81 11. Technique of Zone Electrophoresis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 111. Zone Electrophoresis of Carbohydrates in the Presence of Borate. . . . . . . 86 IV. Zone Electrophoresis of Carbohydrates in the Presence of Complexing 106 Agents Other than Borate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Determination of Molecular Size of phoresis ............................. . . . . . . . . . 107 VI. Zone Electrophoresis of Carbohydrates . . . . . . . . . . . . . . . . . 109 VII. Zone Electrophoresis of Polysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 VIII. Separations of Carbohydrates on Ion-exchange Resins.. . Sugar Nitrates BYJOHN HONEYMAN A N D J. W. W. MORGAN, Chemistry Department, King’s College, University of London, England

I. Introduction.. . . . . ix

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CONTENTS

I1. Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I . Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

118 122 123 124 134

Benzyl Ethers of Sugars

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BY CHESTERM MCCLOSKEY, California Institute of Technology and Ofice Naval Research, Pasadena, California I . Introduction . . . . . . . . . . . . ...................................... I1. Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Hydrogenolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV Chemical Properties . . . . . . . . . . . . . . ............................ V Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

of

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137 142 148 150 153

Methyl and Phenyl Glycosides of the Common Sugars

. .

.

BY J . CONCHIE,G A LEVVYAND C . A MARSH,Rowett Research Institute, Bucksburn, Aberdeenshire, Scotland

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

158

I1. Preparation of Sugar Derivatives Employed in Glycoside Synthesis . . . . . . 158 I11. Condensation of Alcohols and Phenols with Sugars and Sugar Derivatives . 163 IV . Deacetylation of Glycoside Acetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Special Methods of Glycoside Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I Description of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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171 174 178

The Schardinger Dextrins

BY DEXTERFRENCH, Department of Chemistry, Iowa State College, Ames, Iowa ................................................... I1. Historical Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Fractionation and Purification of the Schardinger Dextrins . . . . . . . . . . . . . IV . Bacillus macerans Amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Other Biochemical Properties of the Schardinger Dextrins . . . . . . . . . . . . . . V I . Molecular Size of the Schardinger Dextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Molecular Constitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I I I . Complex Formation and Inclusion Compounds . . . . . . . . . . . . . . . . . . . . . . . . . I X . Ring Conformation in the Schardinger Dextrins . . . . . . . . . . . . . . . . . . . . . . . . X . Derivatives of the Schardinger Dextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI . Significance of the Schardinger Dextrins with Respect t o the Constitution and Behavior of Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

190 192 211 219 231 234 243 247 252 254 257

The Molecular Structure of Glycogens

BY D . J . MANNERS,Department of Chemistry, The University of Edinburgh, Scotland

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

262

CONTENTS

I1. Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Structural Analysis by Chemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I V . Structural Analysis by Enzymic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Molecular Structure of Glycogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Biological Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I I . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi 274 280 284 289 296 298

The Biosynthesis of Hyaluronic Acid

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BY ROYL . WHISTLERAND E J . OLSON,Department of Biochemistry, Purdue University, Lafayette, Indiana I Introduction . . . . . . . . ................................. 299 I1. Metabolism of D - G ~ u ................................. 304 I11. Metabolism of D - G ~ ................................. 308 IV . Biosynthesis of Hyaluronic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

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Author Index for Volume 12. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject Index for Volume 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cumulative Author Index for Volumes 1-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cumulative Subject Index for Volumes 1-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Errata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

321 337 357 361 367

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PHOEBUS AARONTHEODOR LEVENE 1869-1940 I n describing the life and work of many a scientist, the article might conveniently be divided into two parts, one discussing his life, the other his work. Such a procedure is here almost impossible. Of Dr. Levene, it can truly be said that “his work was his life, and his life was his work-they were inextricably intertwined.” Phoebus Aaron Theodor Levene, known to his intimates as Fedya, was born a t Sagor in Russia on February 25th, 1869, the second of the eight children of Solom and Etta (Brick) Levene. In 1873, his family moved to St. l’etersburg, where he began his education in private schools; he eventually attended the Classical School (“Gymnasium”) and was graduated in 1886. His study of Latin and Greek, for eight years each, may have contributed to his subsequent linguistic ability. Becoming interested in biology, he decided t o proceed to the study of medicine. So, when the opportunity arose, he applied for entrance to the Imperial Military Medical Academy in the same city, and was one of the few Jewish students admitted. Ivan Pavlov was then a privatdozent in physiology there, and Levene’s Professor of Chemistry was the famous musician, Alexander P. Borodin, composer of the opera “Prince Igor” (which was completed, after his death, by RimskyKorsakov) . Borodin’s son-in-law, Professor Alexander Dianin, who was in charge of organic chemistry, permitted young Levene to work at will in the chemical laboratory. Although in the midst of his medical courses, he somehow managed to find time to carry out his first research in organic chemistry, participating in Dianin’s studies on the condensation of phenols with aldehydes and ketones. Incidentally, one of Dianin’s compounds is still receiving a good deal of attention, since it is capable of forming an apparently unique set of inclusion complexes. During the year 1891-that is, when Levene was twenty-two-the Levene family came to the United States because of the growing religious persecution in Russia. Rather appropriately, they arrived here on July the Fourth. Shortly thereafter, Levene bravely returned to St. Petersburg, completed his examinations, and received the M.D. degree in the autumn of 1891. Early the next year, he came back to New York, passed his examinations for the practice of medicine, and then practised on the lower East Side of New York City for the next four years. Although his general training had been in medicine, he gradually reached 1

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the conclusion that a permanent career as a physician was not for him, and he decided t o turn to related, fundamental research a t the first opportunity. Realizing the growing importance of chemistry to the advancement of medicine as a science, he enrolled as a special student in the Chemistry Department of the School of Mines a t Columbia University, and extended his chemical training while still a medical practitioner. His teachers in organic chemistry included Professors Colbe and Marston T. Bogert. At the same time, Professor John G. Curtis was kind enough to place a t his disposal working-facilities in his own laboratory in the Department of Physiology of the College of Physicians and Surgeons, Columbia University, then on W. 59th Street. Here he conducted research in biological chemistry and, by 1894, he had begun to publish scientific papers. The first two were concerned with the biochemistry of sugars, a fore-shadowing of a life-long interest. This busy life continued for several years and, one summer, he even managed to return to Europe to spend some time in the laboratory of Professor E. Drechsel in Berne. However, although his laboratory associates were astounded a t the amount of work he accomplished, he began to find these arrangements inadequate, since the limited time permitted him for research (when he was actively practising medicine) was insufficient for satisfying his aroused curiosity regarding the chemical nature and interactions of biological substances. It was therefore immensely fortunate to the future of biochemistry that, in 1896, he was appointed Associate in Physiological Chemistry in the new laboratories of the Pathological Institute of the New York State Hospitals, under the direction of Dr. Ira von Giesen. Here, he developed an interest in the nucleic acids and, to his joy, found that he was permitted to devote all his time to his researches. Characteristically, he immediately tackled problems of extreme difficulty, and he threw himself into the work with such unbounded enthusiasm and such unflagging zeal that, by November of the same year, he had contracted tuberculosis. While recuperating for a year a t the sanatorium a t Saranac Lake, N. Y., where he formed lasting friendships with Dr. Trudeau and his medical staff, he decided to devote his life to biochemistry. H e therefore travelled to Europe to study under one of the great masters of the day-Drechsel in Berne-, but was too ill to work there and so went to Davos in Switzerland to recuperate. Later the next year, though still not very strong, he managed to work with Drechsel for a while. After these two years of rest, he returned to New York and resumed his work at the New York State Pathological Institute where, all this time, his post had been held open for him. But soon thereafter, the laboratory was closed for reorganization, and so he proceeded to Marburg to spend some time working in the laboratory of Professor Kossel, then the authority

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on nucleic acids. Simultaneously, he was a student in the electrochemical laboratory of H. Hofer in the Technische Hochschule in Munich. Returning to the U. S., he accepted the position of chemist in the Saranac Laboratory for the Study of Tuberculosis, and, though he was still far from well, he worked on the chemistry of the tubercle bacillus a t Saranac from 1900 to 1902. By 1900, he had already published his first paper on nucleic acids. One wonders if he then realized that his studies of these profoundly interesting cell-materials would become a life work. In addition, he had now acquired an interest in the chemistry of amino acids and proteins, and he was able to spend a summer with Professor Emil Fischer (at the University of Berlin) in a study of the hydrolysis of gelatin; the resulting publication describes one of the first analyses of a protein by Fischer’s ester method for amino acids. He returned to New York City in 1902 to resume his researches in the chemical laboratory of the re-opened New York State Pathological Institute, where he remained until 1905. From 1904 to 1905, he was honored by being asked to present the Herter lectures in Pathological Chemistry a t New York University and Bellevue Medical College; and he was one of the first invited to lecture before the newly formed Harvey Society in 1905. By reason of these multifarious activities and his consuming passion for biochemical research, he had come to be well known. Then, in 1905, came his golden opportunity. Dr. Simon Flexner, Director of the newly formed Rockefeller Institute for Medical Research, invited him to join the small group of eminent scientists then gathering to form the nucleus of that great research institute. His appointment commenced on January 14th, 1905, and continued uninterrupted until his death. Starting as Assistant in Chemistry, with one laboratory helper in temporary facilities (while the main Institute building was being erected a t 66th Street and York Avenue), he initiated his researches with his customary vigor, and his capabilities were speedily recognized. Within two years he had been promoted to the rank of Life Member in charge of the Division of Chemistry, a post which, with gradual growth in equipment and personnel, enabled him to continue the unparalleled series of scientific contributions which were to appear steadily for the rest of his life. As Dr. L. W. Bass, one of his former coworkers, has said: “The Rockefeller Institute provided the ideal environment for the flowering of his genius. The stimulus of an inspiring atmosphere, the unexcelled laboratory facilities, and the opportunity for choice of problems without regard to their difficulty and length, afforded a widened horizon of scientific endeavor to which he devoted his abilities for thirtyfive years.” His facilities, taking up most of the top two floors of the North Building were splendidly equipped and included (besides his private laborat)ory, reception room, and office-library) separate laboratories for his collaborators, preparation rooms, cold room with individual lockers, animal

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rooms, and a microanalytical laboratory. The last-mentioned was the first such laboratory to be set up in the United States in conjunction with research in organic chemistry; it was first manned by Oskar Wintersteiner , from Pregl’s laboratory, and later by A. Elek. Levene was a short, wiry man of slight build; his hair, originally dark, was dark gray in his later years and was worn longer than that of his contemporaries; he had dark-brown eyes, heavy eyebrows, and a small moustache. He was always impeccably but conservatively dressed, his sole eccentricity being the wearing of an extremely battered hat (which his wife was only able t o persuade him to replace a t most infrequent intervals) ; he thus immediately conveyed the impression of student, artist, and scientistall of which, indeed, he was. A man of liberal views, he held several particularly strong opinions, one of which was opposition to capital punishment. He was an unusually good linguist, who (in addition to his native Russian) spoke English, French, and German fluently, and Spanish and Italian adequately. He read the masters of European literature in the original languages; this reading often occupied the hour before breakfast, as his evening hours were largely devoted to keeping abreast of the avalanche of scientific journals. His earlier association with the medical group a t Saranac was maintained through occasional visits, often during vacation, to Dr. Trudeau and his associates; and on one of these trips, in the autumn of 1919, he met there a very wonderful and remarkable woman, Miss Anna M. Erickson of Lewistown, Montana, who became his wife on the following June 9th. Their home, containing discriminately chosen furniture, pictures, and other art objects, became the center for a wide circle of friends-scientific, artistic, and literary-who enjoyed the congenial and delightfully stimulating atmosphere and the hospitality of their versatile and interesting hosts. Levene was particularly appreciative of music and of classical and modern art. His beloved violin brought him pleasant relaxation. During his sojourns in Switzerland and Germany, he became interested in the work of the Renaissance; and a vacation in Spain in 1909 resulted in his closer acquaintance with the art of the Spanish schools. About 1913, the Cubists attracted him, and later he found much of interest in the work of the more modern artists and sculptors. The walls of his office and of his home were almost covered with reproductions of works of art. His ever-growing personal library, of which he was justifiably proud, was exceptional and large; besides containing a surprising number of scientific books and runs of journals, it satisfactorily encompassed literature and art. I n July 1939, Dr. Levene retired and became a Member Emeritus of the Institute, but he still continued his researches in the same laboratory with unabated vigor. He died unexpectedly at his home in New York City on September 6th, 1940, survived by

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Mrs. Levene for several lonely years. It is related that almost his last words were “Thank God!”-when he was told of the transfer of the destroyers from the U. 5. t o the U. K. I n his death, biochemistry and organic chemistry lost a man who had contributed almost beyond measure to their upbuilding. He was one of those who, early in this century, brought to the United States the finest European traditions and helped stimulate the sound development of chemistry over here. I n that burgeoning, Levene’s discoveries played a conspicuous part. An equally important contribution, however, lay in his subtle influence in developing young scientists. Although the Rockefeller Institute was undoubtedly intended primarily to be an institution devoted to research on medical and allied subjects, the provision of post-doctorate research opportunity was deemed almost equally important by its founders. Many productive American investigators became inspired, early in their careers, through their association with Levene’s laboratories; for no one could work with him without being at least partly imbued with his ardent spirit, and a not insignificant part of the growth of biochemistry in America is undeniably attributable t o men trained and motivated in his department. I n addition, numerous foreign students-from Austria, China, England, Germany, Japan, Poland, Russia, Scotland, Serbia, Spain, Sweden, and Switzerlandwere accepted as collaborators; and, excepting those from the Orient, he was able t o converse with them in their native tongues. Thus was his scientific influence carried far and wide. Moreover, besides scientific leadership, he gave his men warm personal interest. His kindly smile and genial disposition, combined with his human understanding, wide experience, and good judgment, made him a trusted counsellor (in both personal and scientific matters) to his coworkers and other associates; thus, all who worked with him hold his memory in affection. No one could be associated with Levene for very long without realizing that he was to be regarded as a genius. More extensive contact revealed that, in combination with a great intellect, the secret of his genius largely lay in his “infinite capacity for taking pains.” Solving riddles of chemistry and biochemistry afforded him much happiness and satisfaction ; and his rather constant successes led to an optimistic approach and supplied a continual driving force. Every day, he made a point of visiting the laboratory of each of his coworkers for stimulating discussions on the progress of the work, usually enlivened by new ideas developed from his chemical reading the previous night. He displayed a truly remarkable capacity for hard work, despite his seemingly rather frail physique. Besides directing the researches of his often numerous coworkers, adniinistering his Department, writing scientific papers, and welcoming American and foreign scientific visitors, he always personally carried out a great deal of delicate experi-

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mental work and, even in his later years, seemed to accomplish as much of this in a day as any of his more youthful coworkers. The sight of this diminutive figure in his hip-length, white lab-coat, surrounded by large pieces of apparatus often towering above him, was one not readily forgotten.’ To record his experimental results, in his minuscule but nonetheless legible handwriting, he stood at a special lectern-desk; since possible patent protection was of no interest to him, these records were kept on 5” x 8” ruled index cards, a most convenient system for the academic researcher. He worked long hours in the laboratory, and was possessed of great personal skill in laboratory technique. He was a veritable artist in isolating salient compounds from apparently hopeless mixtures, and was blessed with an almost uncanny ability to induce such recalcitrant substances as sugar sirups to crystallize. He was a great believer in the test tube as one of the most valuable pieces of laboratory apparatus. As a result of his medical training, he could perform (on animals) operations of which few organic chemists would be capable; for instance, his producing and using (in collaboration with E. S. London) an intestinal fistula in dogs as an apparatus with “built-in” reactant for achieving controlled hydrolysis of deoxyribonucleic acid was a feat that no ordinary chemist would be capable of conceiving, let alone putting into practice. A noteworthy point is that he ever retained a youthful outlook and was always ready to adopt any new theoretical concept or experimental technique which would help to solve the problems he encountered; to this end, he read extensively (in several languages) on every relevant subject. Both in the laboratory and in his private chemical library, Chemistry and its medical implications were a consuming passion. I n his own laboratory work, he received loyal help from a number of devoted “dieners” or “lab. boys” (technicians, often almost his own age), among whom Joseph Lender was outstanding. These helpers, lacking any prior formal training in science or in laboratory skills, nevertheless became conspicuously competent, albeit in restricted ways, under his tutelage and guidance. The writer well remembers the many occasions on which he said to Lender, “Joe, sometime soon, I’d like ten grams of D-ribose [or adenosine, uridine, etc.1”-and Joe’s invariable reply of ‘(Jawohl, Herr Doktor”-followed by the sight of a larger sample of the desired material (perhaps not completely pure, but always satisfactory after a single recrystallieation) reposing on his laboratory bench the very next morning; Joe’s secret lay in his ability to foresee probable demands for certain compounds and to stockpile such materials in advance. Some of these helpers took courses at local Universities and eventually at,tained degrees in Science. (1) The frontispiece is from a completely unposed, “candid-camera” photograph taken by Martin Kuna.

PHOEBUS AARON THEODOR LEVENE

In appraising the work of Dr. Levene, it should always be borne in mind that he was an M.D. and did iiot have a degree in Chemistry. This renders his astounding achievements all the more remarkable, because few, indeed, have possessed such innate aptitude for biochemical research. Essentially, he was a self-made man in this field. In addition, he became a n accomplished organic chemist who could also employ physical chemistry when the need arose. Levene’s publications, largely shared by his collaborators and nearly all describing original, experimental work, numbered over seven hundred and twenty. His few review articles and books were unusually well done, but he begrudged the time, stolen from laboratory time, necessary for producing these to meet his meticulous standards of historical and scientific accuracy. The extremely wide range of fields he explored, often concurrently, in studying the chemistry of tissue components, included the proteins and amino acids, lipids, nucleoproteins, the nucleic acids, glycoproteins, carbohydrates (including amino sugars, sugar phosphates, and uronic acids) ; enzymes and autolysis; an investigation of the stereochemistry of natural products, which developed into an extensive study on stereochemical configuration and the interrelationship of a wide range of simple synthetic compounds; studies on the concentration and isolation of the vitamin B complex; an examination of the mechanism and nature of the Walden inversion, which contributed greatly to the modern concepts thereof, and the results of which were summarized in his Nichols Medal award address; and, during his last few years, the chemistry of the pectins and gums, a project directly stimulated by his interest in the pneumococcal polysaccharides. This immense amount of valuable, versatile work was recognized by his election t o membership in the American Association for the Advancement of Science, the American Chemical Society, the American Philosophical Society, the American Physiological Society, the American Society of Biological Chemists (of which he was a charter member, being present a t its inaugural meeting on December 26th, 1906), the American Society of Naturalists, Bayerische Akademie der Wissenschaften, Deutsche Akademie der Naturforscher (Halle), Deutsche Chemischen Gesellschaft, the Harvey Society, the National Academy of Sciences, the Royal Society of Science (Sweden), Soci6t6 de Chimie Biologique, Soci6t6 Chimique de France, Soci6t6 Royale des Sciences Medicales et Naturelles de Bruxelles, Soci6t6 Suisse de Chimie, the Society of Experimental Biology and Medicine, and t o honorary membership in Phi Lambda Upsilon. I n addition, he was awarded the Willard Gibbs medal of the Chicago Section of the American Chemical Society, in 1931, and the William H. Nichols medal of the New York Section in 1938. Many chemists, faced by the imposing list of Levene’s chemical investigations, have failed to grasp that there was a plan implicit in all of his re-

8

OBITUARY

searches. More than once he was asked why it was that he had worked on so many different topics, whereas most academic investigators will stay with one topic until they feel that they have exhausted its possibilities. The fact of the matter is that this is precisely what he did do in several fields, often simultaneously; and he would reply that he was guided by one primary fundamental interest: namely, the chemistry of life processes with special reference to the chemical basis of individuality. That is to say, starting with cells which appear to be very much alike, growing in a quite similar environment, why do some become brain cells or liver cells or heart cells; or why do some aggregations of cells end u p as yeasts, worms, pigs, monkeys, or human beings? I n analyzing the possible biological significance of the various constituents of tissues, he broadly distinguished three kinds. I n one category are such substances as the conjugated sulfuric acids, the nucleic acids, and the lipids, which apparently exhibit little or no particular specificity or individuality, although they are essential to life processes. Examples of a second group are the enzymes and hormones, which may be identical in various species and yet may or may not be present in different organs or in different species. The third set includes such ubiquitous compounds as the proteins, with precise structures differing from species to species. Apparently, it was his background of medicine and biology which guided him successfully through the morass of intriguing chemical problems, so that he tackled only those most imperative and rewarding in advancing our knowledge of the life processes; yet, only recently has the significance of much of his work become really appreciated by many biochemists. Indeed, it is a remarkable tribute to his vision that, within a decade after his death, all the fields in which he was almost a lone pioneer had become the subjects of intensive research, engaging the activities of many hundreds of investigators. Although his researches encompassed almost all fields of biochemistry, they actually had a coordinated, logical pattern. His first papers, which appeared near the start of this century, presented a preview of the principal topics for his future study. His career in independent research began with a study of the transformation, in animals, of proteins into carbohydrates; his interest in these two important types of biological material was destined to be life-long. I n quick.succession, he proceeded to a diversity of other biochemical problems, including the nucleic acids, mucins, and phospholipids. The interrelationships of this group of interests are as follows. His early work on the proteins mainly involved the isolation, characterization, and identification of individual members. Then he turned to the question of their structure, and from a study of the racemization of synthetic diketopiperazines, came to the conclusion that the structure of the giant molecules of proteins could be explained (in terms of Emil Fischer’s polypeptide chains) by the classical theories of valency, without the neces-

PHOEBUS AARON THEODOIt LEVENE

9

sity of assuming the functioning of mysterious auxiliary valeiicies for uniting an assemblage of relatively small molecules consisting of diketopiperazine rings. Interestingly, his isolation of prolylglycine anhydride from tryptic digests of gelatin, in 1906, had originally constituted a challenge to the peptide-chain theory, but this apparent anomaly was later satisfactorily resolved. Two outstanding results came from this work. One was the development of improved analytical methods for proteins; the other, the isolation of a crystalline intermediate from a protein hydrolyzate. However, the emphasis in his protein studies gradually turned in the direction of conjugated proteins; and, through the years, he essayed a series of pioneering investigations on the characteristic non-protein constituents of the nucleoproteins, the glycoproteins, and the lecithoproteins (together with a little work on certain phosphoproteins and chromoproteins) . Levene’s name is probably most often associated with the nucleic acids, which are essential constituents of all living things. Knowledge of their structure was in a singularly elementary state a t the beginning of the century; all that was known was that they consist of non-nitrogenous and nitrogenous organic compounds combined with phosphoric acid. No information as to the nature of the non-nitrogenous constituents, the quantitative relationships between the different components, or their mode of union, had been adduced. During some four decades, these substances were patiently investigated by Levene, so that practically every detail of their structures is now known. Prior to his work, it had seemed that no two nucleic acid preparations were identical. However, he succeeded in distinguishing two main groups. Beginning, early in the century, with a study of the nuclein materials from varied sources, his perseverance was rewarded a few years later by the isolation of the four nucleosides of one type of nucleic acid and then by the identification of the sugar thereof as D-ribose, a sugar not previously known to occur in Nature. Fortunately, L-ribose had just been synthesized by Alberda van Ekenstein and Blanksma, and the two sugars proved t o be identical in every respect except that they possessed equal but opposite optical rotatory power. (Incidentally, in collaboration with W. A. Jacobs, he then synthesized the new hexoses, D-allose and D-altrose, from D-ribose.) These striking achievements were followed by the isolation of the four corresponding nucleotides, and he was then able to show that the nucleic acids are highly polymerized compounds consisting essentially of four nucleotides, often united in approximately equimolar proportions. He demonstrated that each nucleotide is cornposed of phosphoric acid, a sugar, and a purine or pyrimidine base. Similarly, the socalled animal (“thymus”) nucleic acid which, by 1912, he could with some confidence distinguish from plant (“yeast”) nucleic acid, gave a different set of four nucleotides and four nucleosides. The identification of the sugar

10

OBITUARY

in this nucleic acid, a problem which had baffled all other workers, was finally achieved by him in 1929, some twenty years after the sugar of the other nucleic acid had been identified. It turned out to be 2-deoxy-~-ribose (2-deoxy-~-erythro-pentose),a hitherto unknown sugar. The sequence of union of the units, the ring structures of the sugars, and the positions of the substituent nitrogenous bases and phosphoric acid groups on the sugars were next established. As a result of his work, we now have an exact concept of most features of the architecture of these giant molecules, probably the most complex biological compounds the structures of which had till then been ascertained. In 1931, he published a monograph entitled “Nucleic Acids” in collaboration with Dr. L. W. Bass, a former coworker, then of the Mellon Institute. However, when only part way along in the above project, Levene reached a point where he was forced to transfer his major activities to the carbohydrates and related compounds, because lack of knowledge in this field prevented his presenting a complete formulation for the building units of the nucleic acids. Ways for determining two important features of sugar structure evolved from his researches. For ascertaining the position of u n i o n in disaccharides or substituted monosaccharides, two methods were developed, one involving oxidation and subsequent examination of the rate of lactonization, the other employing catalytic reduction under high pressure. The determination of ring structures of sugars and their derivatives was conducted in various ingenious ways. These methods proved essential in his elucidation of the complicated structures of the nucleic acids. Another reason for his becoming interested in sugars stemmed from his work on the glycoproteins, which he had commenced in 1900. These substances contain high proportions of carbohydrates. From the mucoids he isolated nitrogenous sugars. These hexosamines presented problems of great interest and difficulty as regards their relationship t o the simple sugars. Whereas, in the latter, the relative position in space of every hydrogen atom and hydroxyl group had been determined, that of the nitrogen atom could not, a t that time, be allocated by the methods of classical organic chemistry; and new, indirect procedures had to be devised. Consequently, Levene embarked upon an exhaustive synthetic study of the hexosamines and the corresponding hexoses; and, from the resulting data, indirect evidence suggesting that chondrosamine is 2-amino-2-deoxy-~-galactose and that chitosamine is 2-amino-2-deoxy-~-g~ucose was adduced ; these allocations were later unequivocally proved, by Haworth, Peat, and their coworkers, by use of direct chemical methods. These voluminous investigations were summarized in his monograph entitled “Hexosamines and Mucoproteins” (1925). This work led directly t o his fundamental correlations of chemical struc-

PHOEBUS AARON THEODOR LEVENE

11

ture with optical activity. Furthermore, the work on nitrogenous sugars had involved rases of Walden inversion, which led Levene to more general considerations of ronfiguratiorial relationship. This topic berame, to him, one of his most enthralling. During somewhat more than a decade, a tremendous volume of work on this important phase of stereochemistry came from his laboratory. In 1938, he was awarded the Nichols medal of the New York Section of the American Chemical Society “for his study of the configurational relationships of the simpler optically-active organic compounds.” One of the important results of this work was the announcement of a theory of the mechanism of Walden inversions from the modern viewpoint. The lipids, another group of compounds having complicated formulas, were classified as a result of Levene’s researches in this notoriously laborious and difficult field. The literature was in a chaotic state, but, by devising methods for isolating these substances pure, he was able to replace confusion by scientific order. He found that there are three main groups of lipids, two containing phosphorus and one free from phosphorus. I n the members of two groups, there is a sugar unit in the molecule. As regards the lecithins, his main contribution, arising from a study of their fatty acids, was to show that the pure lecithins isolated from different organs of the body contain different fatty acids ; previously, it had seemed doubtful whether more than one lecithin occurred in Nature. Levene isolated the important phospholipid, sphingomyelin, from a variety of animal organs, showed that the various preparations were identical, and, from a study of its scission products, propounded a formula for it. His pioneer work on the cerebrosides -phrenosin and kerasin-was fruitful. The latter afforded (‘kerasinic acid” which he showed was identical with lignoceric acid. The former gave “ phrenosinic acid” ( ‘ I neurostearic acid” or cerebronic acid) which, on oxidation, afforded the known tetracosanic acid; he was able to show that cerebronic acid is the a-hydroxy derivative of a higher homolog of lignoceric acid. As early as 1900, Levene had worked on mucin. Returning to the mucoproteins, Levene found that the prosthetic group is composed of four components in equimolecular proportions, namely, a hexosamine, sulfuric acid, acetic acid, and D-glucuronic acid. Two types of mucoid were recognized, differing only in the nature of the hexosamine. The mucoproteins from navel cord, vitreous humor, cornea, gastric mucus, and serum mucoid yield mucoitinsulfuric acid which contains 2-amino-2-deoxy-~-glucose,the only amino sugar known prior to Levene’s work in 1916. Those from cartilage, tendons, aorta, and sclera yield chondroitinsulfuric acid, which contains a new sugar that Levene named “chondrosamine,” and which was eventually proved to be 2-amino-2-deoxy-~-galactose. Hydrolysis of chondroitin-

12

OBITUARY

sulfuric acid affords chondrosine, a ‘Ldisaccharide,”which Levene showed is composed of n-glucuronic acid and chondrosamine. One of his last papers (published posthumously) dealt with the applications of the then latest methods to the problem of the structure of chondrosine, although he had suggested a plausible formula for it in 1921 and had, from this, tentatively advanced a formula for chondroitinsulfuric acid. These investigations, together with then-recent studies on pneumococcal polysaccharides, stimulated his interest in the uronic acids and led to his studying, during his last two or three years, the structure of “aldobionic acids” (aldobiouronic acids), pectins, and vegetable gums and mucilages. This work led to the introduction of high-pressure catalytic hydrogenation as a tool in the realm of sugar chemistry. In 1931, Levene was awarded the Willard Gibbs medal of the Chicago Section of the American Chemical Society as “the outstanding American worker in the application of organic chemistry to biological problems.” That citat,ion is one which will go unchallenged for many years to come. R. STUART TIPSON Appendix The following is a list of the 92 scientists who published articles in collaboration with Dr. P. A. Levene., R. H. Aders (Plimmer); C. H. Allen; C. L. Alsberg; E. R. Baldwin; P. D. Bartlett; L. W. Bass; W. A. Beatty; I. Bencowitz; F. W. Bieber; F. J. Birchard; A. Carrel; C. C. Christman; E. P. Clark; J. Compton; F. Cortese; L. H. Cretcher, Jr.; R. T. Dillon; A. Dmochowski; E. Fischer; J. Garcia-Blanco; A. Gratia; H. L. Haller; S. A. Harris; G. W. Heimrod; J. H. Helberger; K. Heymann; D. W. Hill; L. E. Holt; T. Ingvaldsen; W. A. Jacobs; E. Jorpes; P. A. Kober; S. Komatsu; L. C. Kreider; L. Kristeller; M. Kuna; F. B. LaForge; K. Landsteiner; I. Levin; F. A. Lipmann; E. S. London; J. L6pez-SuBrez; J. M. Luck; J. A. Mandel; D. D. Manson; S. Mardashew; R. E. Marker; I. Matsuo; F. Medigreceanu; C. L. Mehltretter; W. G. Melvin; L. B. Mendel; G. M. Meyer; B. Michailowski; L. A. Mikeska; T. Mori; M. Muhlfeld; I. E. Muskat; M. Osaka; G. Ovakimian; K. Passoth; Mimosa H. Pfaltz; E. G. Pickels; A. L. Raymond; Ida P. Rolf; A. Rothen; C. A. Rouiller; J. Scheidegger; G. Schmidt; A. Schormuller; J. K. Senior; H. S. Simms; H. A. Sobotka; R. E. Steiger; P. G. Stevens; E. T. Stiller; L. B. Stookey; J. E. Sweet; F. A. Taylor; R. S. Tipson; R. Ulpts; B. J. C. van der Hoeven; J. van der Scheer; D. D. Van Slyke; G. B. Wallace; A. Walti; Ione Weber; C. 5. West; 0. Wintersteiner; M. L. Wolfrom; M. Yamagawa; and P. S. Yang.

INFRARED SPECTRA OF CARBOHYDRATES

BY W. BROCK NICELY* G. D . Searle and Company, Chicago, Illinois I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 11. Molecular Spectra. ........................... ............... 14 1. Description.. . . . . . . 2 . Spectroscopic Units ............................ 15 111. Infrared Spectra.. .... 1. Origin and Limitations. . . ............................. 15 2. Experimental. ................................. ...................... 21 IV. Interpretation of Infrared Spectra of Carboh 1. Introduction.. ............................ 2 . Tetrahydropyran N 3. Partial Assignment of Frequencies in the Carbohydrate Nucleus. . . . . . . . 23 V. Application of Infrared Spectroscopy to Carbohydrates . . . . . . . . . . . . . . . . . . . 29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 1. Early Work ....................... 2. Hydroxyl Absorption in Cellulose and Related Compounds. . 3. Mucopolysaccharides.. ............ a. Hyaluronic Acid and Chondroiti b. Pneumococcal Polysaccharides . c. Bacterial Dextrans.. . . . . . . . . . . . 32 4. Comparative S t u d y . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION This Chapter will deal with the use of infrared spectra in ascertaining some of the configurational and structural aspects of various carbohydrate molecules. It must be realized at the outset that the use of infrared spectroscopy in this field is a useful tool for the chemist but should not be considered as supplanting the classical chemical methods for determining these particular entities. Many excellent text books’! have been written on the subject of infrared spectroscopy. Accordingly, no attempt will be made to discuss the the-

* Present address : Biochemistry Department, The Dow Chemical Co., Midland, Michigan. (1) G. Herzberg, “Infrared and Raman Spectra of Polyatomic Molecules,” D. Van Nostrand Co., Inc., New York, N. Y., 1945. (2) G. Hersberg, “Spectra of Diatomic Molecules,” D. Van Nostrand Co., Inc., New York, N. Y., 1950. 13

14

W. BROCK NEELY

oretical aspects of molecular spectra, other than to provide sufficient background material for the discussion to follow.

11. MOLECULAR SPECTRA 1. Description

A molecule, like an atom, can exist in a number of energy levels, and the change from one level to another will result in the absorption or emission of a definite quantum of energy. Three categories of molecular spectra have been recognized : electronic, vibrational, and rotational. Each of these will be described briefly. The energy difference between two electronic levels is of the order of 5 E. v., or about 8 X 10-l2 ergs per molecule. Substitution of this value in Planck’s equation e = hv, where h is Planck’s constant and has a value of I

ULTRAVIOLET

INFRARED

I

A:

0.01

Ir:

-

V:

FIG. 1.-A

I

I

1

10

100

1000

0.01

0.1

I

I 1

1,000

RADIO

I

1 ;

105 10

I

106

I 107

100 1000 1000 100 10 Schematic Representation of the Electromagnetic Spectra.

1 10,000

6.6 x erg-sec., shows that the frequency of absorption is about 1.2 x 1OI6 vibrations per sec., or is equivalenta to a wavelength of 2500 8. The electronic spectra will thus appear in the visible and ultraviolet part of the spectra. In each electronic level, a molecule can have a number of vibrational sublevels, which in turn contain a set of rotational sublevels. As yet, the latter, which appear in the far infrared and microwave regions, have had little application in organic chemistry. Whiffen has written a review on this aspect of spectroscopy4 and O’Loane6 has reported on some recent work in this region. The energy differences within the three groups vary by a factor of 10-100. If a change occurred in the vibrational energy only, the energy difference would be about 1.6 x 10-13 ergs per molecule, which would correspond to a wave length of 100,000 8. or 10 p . Actually, however, the (3) The various units are defined in the following Section. (4) D. H. Whiffen, Quart. Revs. (London), 4, 131 (1950). (5) J. K. O’Loane, J . Chem. Phys., 21, 669 (1953).

INFRARED SPECTRA OF CARBOHYDRATES

15

vibrational energy differences are always accompanied by rotational energy differences, thus giving the effect of widening the vibrational line into a band, termed the vibrational-rotation band. The radiation accompanying such a change would lie in the near infrared at 1-25 p, the region in which we are chiefly interested. Fig. 1provides a graphical illustration of a portion of the electromagnetic spectrum. 2. Spectroscopic Units

The position of the various bands may be expressed either as frequencies

(v) or as wavelengths (A). In order to demonstrate the relationship between the two, the following symbols will be defined. X = wavelength (cm., p, c = velocity of light (cm. sec.-l). v = frequency (sec.-l). tt = wave number (cm.-l). E l , E2 = energies (in ergs) of the two levels involved in a spectral transition. h = Planck's constant (erg. sec.). These quantities are related by the following equations.

w.).

c = VX

El - E2 = hv = hc/X = hcs The units for expressing the positions of the various bands are as follows. 1. AngstrBm @.I. 1W. = 10-8 cm. = 10-1 mp, 2. Millimicron (mp). 1 mp = 10-3 p = 10-7 cm. = 10 A. 3. Micron ( p ) . lp = m. = mm. 4. Wavenumber (v). t = l / X cm. = 108/X A. Wavenumbers and wavelengths expressed in microns are convenient units for infrared spectra. The former have the additional advantage of being directly proportional to energy, which is preferred in theoretical considerations.

111. INFRARED SPECTRA 1. Origin and Limitations The requirements for the absorption of energy in the infrared are that the molecule either has a permanent dipole moment or vibrates in such a way as to produce a dipole moment, as in Fig. 2, A, ii.6 An external electric field will tend to orient the permanent dipole-moment, whereas a field that is periodically changing will tend to swing the molecule alternately (6) H. W. Thompson, J . Chem. Soc., 183 (1944).

16

W. BROCK NEELY

in opposite directions. Energy can, therefore, be absorbed to make the molecule rotate. For simple molecules like carbon dioxide and water which possess some degree of symmetry, it becomes possible to describe the geometrical form of these vibrations as shown in Fig. 2. These picturizations allow the use of such terms as “breathing,” “rocking,” “bending,” or ((twisting” vibrations. They also demonstrate the difference between deformation modes, when the nuclei move at right angles to the bonds, and valence- or stretching vibrations, in which the nuclei move in the direction of the bonds, The problem of schematically representing the normal vibrations, as in the above case, becomes increasingly difficult as the number of atoms in the molecule increases. This follows from the fact that a molecule con-

A

B

Fra. 2.-The Normal Vibrations for (A) C02 (a Linear Triatomic Molecule); (B) Ha0 (a Nonlinear Triatomic Molecule). (The arrows represent displacements of the nuclei from their equilibrium positions.)

taining n atoms will have 3n - 6 normal vibrations (3n - 5 for a linear molecule). A normal mode of vibration is defined as a mode in which (a) the center of gravity of the molecule does not move and (b) all the atoms move with the same frequency and in phase. The derivation of 3n - 6 is obtained in the following manner. The motion of an atom is described completely by specifying the three Cartesian coordinates; thus, for n atoms we would need 3n coordinates, and the system would be described as having 3n degrees of freedom. However, the molecule has certain equations of constraint, namely the translation and rotation of the molecule as a rigid body. These two motions may be characterized by the three coordinates of the center of mass and the three Eulerian angles, respectively. The remaining 3n - 6 degrees of freedom must then describe the motions of nuclei relative to each other; that is, they describe vibrational motions. For a linear molecule, only two angles are required to describe rotation, hence the number of vibrational motions will be 3n - 5.

INFRARED SPECTRA OF CARBOHYDRATES

17

To make the situation slightly more complicated, the number of observed vibrations may be greater than the theoretical amount due to a combination of the following. (a) Harmonics or overtones which are approximate multiples of the fundamental frequency, the ratios of the fundamental, first, and second harmonics being7 of the order 1:2 :3. (b) Digerenee tones, which are merely the difference between two frequencies in which the molecule is in one excited state and absorbs enough energy to raise it to another excited state. (c) Combination tones which are the sum of two or more frequencies, where enough energy is absorbed to excite two states simultaneously. (d) Resonance, where the harmonic of one vibration is equal in magnitude to another fundamental, and consequently leads to a pair of new frequencies. To compensate for the above, the number of theoretical normal vibrations may be reduced by two inherent factors of the molecule. Some vibrations may be degenerate. For example, a linear triatomic molecule should, by theory, have four vibrational modes. However, the deformational mode of carbon dioxide (see Fig. 2, A, iii) is not uniquely defined, since the motions could take place either in the plane of the paper or in a plane perpendicular to it. If a molecule is highly symmetrical, it is probable that certain vibrations will not be accompanied by a change in the dipole moment, thus the frequency will be “forbidden in the infrared.”*#9 To illustrate the last point we shall look a t a molecule with a center of symmetry. Carbon dioxide, benzene, and ethylene all have this common property, that is, they have a point such that a line, drawn from one atom to this point and extended an equal length beyond, will contact the twin of the first atom. Water (see Fig. 2, B) and most other molecules do not possess such a center of symmetry. If there is molecular symmetry, a vibration may be either symmetric or antisymmetric. For a symmetric vibration, the displacement vector of one atom will be the mirror image of the displacement vector of the opposite atom (see Fig. 2, A, i). Such a vibration obviously leaves the dipole moment unaltered and is thus forbidden in the infrared. On the other hand, the antisymmetric vibration (see Fig. 2, A, ii) does produce a change in the dipole moment. The moment is zero in the equilibrium position and is some value other than zero at either end of the vibration. This vibration will be active in the infrared. I n addition, there are certain mechanical limitations of the spectropho(7) S: Glasstone, “Textbook of Physical Chemistry,” Macmillan and Co., Ltd., London, 2nd Edition, 1953,p. 567. (8) This theory is discussed by Herzberg, ref. 1, pp. 251 ff. (9) A. G. Meister, F. F. Cleveland and M. J. Murray, Am. J . Phys., 11,239 (1943).

18

W. BROCK NEELY

tometer which tend to diminish the number of vibrations observed. The fundamentals may occur at such low wavenumbers as to fall outside the region of the spectrophotometer. The instrument may be too insensitive to detect certain of the frequencies and, finally, some of the vibrations may be so nearly alike that their resolution is at present impossible. A mathematical treatment of the infrared spectra could, if successful, make possible the unique determination of the structure. Theoretically, such a calculation would be possible provided that the strengths of all the interatomic forces were known. The degree of difficulty of such a calculation, however, would be a linear function of the number of atoms and of the symmetry of their geometrical arrangement. Carbon dioxide and water, both simple molecules, have been subjected to very thorough investigations. On the other hand, most compounds that the organic chemis't encounters, such as the ortho-substituted phenols wherein the symmetry is completely destroyed, require the solution of a thirty-third degree equation. As an alternative method in the attempt to correlate the structure of the molecule with the observed frequencies, attention has been directed to the purely empirical approach. This method is based on the fact that the vibrations for a certain group (such as C-H linkages in a molecule) would, to all intents and purposes, be independent of the rest of the molecule. This assumption has been strengthened by the study of hundreds of molecules containing C-H bonds. These molecules have shown an absorption at 2900 cm.-' (C-H stretching) and another at 1400 cm.-I (C-H bending). This premise has been further substantiated by the use of deuterated compounds. From theory, the C-D frequency should be less than the C-H frequency by a factor of fi.This was borne out experimentally when molecules with the C-D bond showed a stretching frequencylO of 2100 cm.-l. Thus, by comparing the spectra of a large number of compounds having a common group, it is possible to find an absorption band which remains relatively constant, regardless of the rest of the molecule, The band can then safely be assigned to that particular group.

2. Experimental The infrared spectrum covers a range of wavelengths from approximately 1 p-104 p. Between 1 p-25 p (104 cm.-l - 4 x 102 cm.-l), a prism spectrophotometer may be used in which the prism may be calcium or lithium fluoride, or sodium chloride, depending on the particular region under investigation. The region beyond 25 p usually requires a diffraction grating. (10)R . B. Barnes, U. Liddel and V. 2. Williams, Anal. Chern., 16, 659 (1943).

I NF R AR E D SPECTRA O F CARBOHYDRATES

19

Infrared radiation is usually detected by means of a bolometer or a therniocouple. Since emission spectra are often too weak to be detected, we are here primarily interested in absorption spectra. These are obtained by int,erposing the substance between the prism and a source of infrared emission, such as a Neriist glower or any other suitable incandescent solid body a t temperatures of 1000 to 1500°C. Several discussions on this subject are avai1able.l’. l2 Substances may be studied in either the gaseous, liquid, or solid state. Solutions may also be used, but it must be mentioned that great care should be taken to choose a solvent that will not absorb in the region in which one is interested. By preparing solutions in carbon tetrachloride and carbon disulfide, the entire range from 2-15 p can be covered. Torkington and Thompson13have prepared a list of solvents (with their regions of transparency) which may be used in certain parts of the spectrum. Water is a poor solvent for two reasons: it causes dissolution of the sodium chloride plate and i t absorbs throughout much of the near infrared region. A common practice for studying solids involves the “mull” technique, using a purified mineral oil such as Nujol. The mull is made by grinding a sample (1-5 mg.) in a few drops of the oil and placing the suspension between two sodium chloride plates. The grinding is essential in order to obtain a homogeneous mixture of the sample and oil and t o destroy the orientation of the crystal structure which causes strengthening and diminishing of certain bonds.** Since Nujol exhibits only five frequencies from 600 cm.-l to 4000 cm.-l, it is well suited for this type of work. I n order to obtain the spectrum of the sample in these particular regions of absorption, one of the fluorinated hydrocarbons may be used in the place of Nujol. Recently, potassium bromide Jilms of the sample have been used for infrared absorption measurements.16-l6 This technique consists in mixing the material with analytically pure potassium bromide in the ratio of 1:100, both components being ground to pass through a fine-mesh screen, The mixture is then placed in a die, the die is evacuated, and pressure is applied. Certain sugars treated in this manner have shown progressive (11) G. R. Harrison, R . C. Lord and J. R. Loofborrow, “Practical Spectroscopy,” Prentice-Hall, New York, N. Y., 1948. (12) R. B. Barnes, R. C. Gore, U. Liddel and V. 2. Williams, “Infrared Spectroscopy,” Reinhold Publishing Corp., New York, N. Y., 1944. (13) P. Torkington and H. W. Thompson, Trans. Faraday Soc., 41, 184 (1945). (14) F. A. Miller in “Organic Chemistry,” H. A. Gilman, ed., John Wiley and Sons, Inc., New York, N. Y., 2nd Edition, 1953, Vol. 3, p. 139. (15) Miriam M. Stimson and Marie J. O’Donnell, J . Am. Chem. Soc., 74, 1805 (1952). (16) U. Scheidt and H. Reinwein, 2.Naturforsch., 7B, 270 (1952).

20

W. BROCK NEELY

changes in their spectra as the films were stored1' (see Fig. 3). This phenomenon has recently been clarified.'* Films prepared from potassium bromide which had been dried a t (350" C. for 4 hours showed no spectral change when stored. The product resulting from the film prepared from a-D-glucopyranose and moist potassium bromide was shown to be the monohydrate of a-D-glucopyranose. This was established by examining

930

890

850

810

770

Wave numbers, cm.-' Infrared Spectrum of a-~-Glucopyranose~~ in a Potassium Bromide Film Between 930-750 em.?: (A) Initially; (B) After 3 days; (C) After 7 days. (The absorption scales have been displaced, in order to spread out the three spectra.)

FIQ.3.-The

the spectrum of the monohydrate, which was identical with curve C, Fig. 3. Thus, the drying procedure becomes necessary when the compound under examination is prone to hydrate formation. I n comparing spectra of two samples which are thought t o be identical, it becomes imperative that they shall both be in the same physical state. (17) S. A. Barker, E. J. Bourne, W. B. Neely and D. H. Whiffen, Chemistry & Industry, 1418 (1954). (18) S. A. Barker, E. J. Bourne, H. Weigel and D. H. Whiffen, Chemistry & Zndustry, 318 (1958).

INFRARED SPECTRA O F CARBOHYDRATES

21

Crystalline a - ~ - g l u c ~ p y r a n exhibits o ~ e ~ ~ a characteristic peak at 837 cm.-l ~ which is shifted to 849 cm.-’ in a sirupy C U , mixture.2* IV. INTERPRETATION OF INFRARED SPECTRAOF CARBOHYDRATES 1. Introductirm

As stated previously (see p. IS), infrared spectral diagnosis is based upon the empirical study of a large number of compounds. Such study has revealed that certain groups have characteristic absorption bands. Examples of this type of collation are now very numerous and many 21-2a A group in a molecule parreference tables have been taking in such a localized oscillation represents an idealized extreme. I n the actual vibrations, however, the characteristic frequencies will be modified by various factors, such as the influence of neighboring groups, conjugation, ring strain, and the formation of hydrogen bridges. The more complex the molecule, the more will these factors have an influence in modifying the correlation rules (and so lead to difficulty in assigning a frequency to a particular group in the molecule). 2. Tetrahgdropyran Nucleus

The infrared spectra of even the simplest carbohydrates present a very complex picture throughout most of the region (3000-700 em.+). One method of approach which would tend to reduce this complexity, and simultaneously make the task of interpretation easier, would be the investigation of simple compounds. Such a molecule (containing the pyranose ring often found in sugars) presented itself in the form of tetrahydropyran. The first problem was, therefore, the identification of the bands arising from the stretching vibrations of the tetrahydropyran ring. Although these vibrations are not identical in the two molecules, a knowledge of the spectrum of the tetrahydropyran nucleus was of great assistance in the interpretation of the infrared spectra of carbohydrates. With this in mind, Burket and Badger2‘ attempted to assign frequencies to the various vibrations arising from the tetrahydropyran nucleus. They (19) S. A. Barker, E. J. Bourne, M. Stacey and D. H. Whiffen, J. Chem. Soc., 171 (1964). (20) S.A. Barker, E. J. Bourne, M. Stacey and D. H. Whiffen, J . Chem. SOC.,3468 (1954). (21) H.W. Thompson, J . Chem. SOC.,328 (1948). (22) Ref. 14, p. 122. (23) L. Bellamy, “Infrared Spectra of Complex Molecules,” John Wiley and Sons, Inc., New York, N. Y., 1954. (24) S. C. Burket and R. M. Badger, J . Am. Chem. SOC.,72,4397 (1950).

22

W. BROCR NEELY

were successful both in this endeavor and in presenting new spectroscopic arguments in support of the stable chair conformation. The foundations for this study had been thoroughly established by previous work on cycloh e ~ a n e . ~Following ~-~’ a similar method, Burket and Badger deduced that the normal vibrations for tetrahydropyran could he pictured essentially as shown by Ramsay.26 Two of these are depicted in Fig. 4. The intricacies of the discussion, which culminated in the assignments of the frequencies, are of great interest to spectroscopists, since the study was based to a greater extent than usual on a consideration of probable intensities. How-

A B FIG.4.-Two of the Normal Vibrations of TetrahydropyranZ4:(A) Symmetrical Ring Breathing Frequency; (B) Antisymmetrical Ring Bending Frequency. (The arrows represent displacements of the nuclei from their equilibrium positions. Vertically-lined circle = oxygen above xy plane, carbon above zy plane, 0 carbon below xy plane.)

ever, a general account of their results will satisfy the purpose of the present Chapter. The ring breathing frequency of tetrahydropyran (see Fig. 4, A) was derived from the symmetrical mode of cyclohexane (Fig. 4, A, with the oxygen replaced by a methylene group). This particular mode in cyclohexane was forbidden in the infrared region because of the symmetry of the molecule. Tetrahydropyran, on the other hand, should exhibit an absorption due to this vibration. Since the band at 813 cm.? was present for tetrahydropyran and absent for cyclohexane, it was assigned to the ring breathing frequency of tetrahydr0pyranLn.2~ The antisymmetrical C-0-C vibration was closely associated with the ring stretching mode of tetrahydropyran (see Fig. 4, B). The contribution (25)

C.W.Beckett, K. S. Pitzer and R, Spitzer, J . A m . Chem. SOC.,69,2488 (1947). A. Ramsay, Proe. Roy. SOC.(London), A190, 562 (1947).

(26) D.

(27) 0. Hassel and B. Ottar, Aeta Chem. Scand., 1, 929 (1947).

23

INFRARED SPECTRA OR' CARBOHYDRATES

of such an effect should produce an intense band in the infrared. The absorption at 875 cm.-', being the most intense, was assigned to this particular mode of vibration.24

3. Partial Assignment of Frequencies to the Carbohydrate Nucleus The intensive study of infrared spectra of carbohydrates has been confined to the frequency range 730-960 cm.-'. This is the region of the spectra TABLEI Characteristic Infrared Bands Derived from D-Ghcopyranose ~

Frequencies of absorption peaks Compounda oj a

,dry CHiOH

917 f 13

b

844 i 8

766 f 10

H

Ho

I'

H

I

OR OH a-n-glucopyranose 891 i 7

920 f 5

H

774 i 9

OH p-n-glucopyranose

~

~

(1

~~

~

R varies from hydrogen t o polysaccharide chain.

in which the molecule vibrates as a wh01e.l~Consequently, any differences in the stereochemistry of the various molecules should be evident in this region. Beyond 960 cm.-l, C-0 and C-C stretching, as well as C-H deformation and skeletal frequencies, make correlation between band positions and molecular structure difficult. Derivatives of D-glucopyranose, both a and p, were the first compounds to be examined t h o r o ~ g h l y .28~ ~In . the particular regions studied, there (28) S. A. Barker, E. J. Bourne, M. Stacey and D. H. Whiffen, Chemistry & I n dustry, 196 (1953).

24

W. BROCK NEELY

were three principal sets of bands common to all, for which the average values are shown in Table I. The assignment of the type 1 absorption was based on the following considerations18:the ring vibration in tetrahydropyran (see Fig. 4,B) was given the value of 875 cm.-l by Burket and Badger.*4This particular mode included a considerable contribution from the antisymmetrical ring C--0-C stretching and was of considerable intensity in the absorption spectra. Since the type 1absorption at 920 cm.-' in the carbohydrate spectra was of medium strength, it was tentatively assigned to this particular mode. The symmetrical ring breathing vibration (see Fig. 4, A) in tetrahydropyran was correlated with the type 3 vibration.1g The symmetrical nature of this vibration was substantiated by a study of the infrared spectrum of scyllo-inositol (I),a centrosymmetrical inosit01.~~ This molecule, being perfectly symmetrical, will not produce a change in the dipole moment during the ring breathing vibration. Consequently, the type 3 absorption should be forbidden in the infrared, which was found to be the case. The antisymmetrical inositols examined did exhibit this particular absorption. The lowered frequency of the type 3 absorption in sugars as compared to the 813 om.-'of tetrahydropyran was explained on the basis of the extra weight of the constituents involved in the sugar molecule. H

OH

I

Type 1 and type 3 absorption were shown to vary with the type of linkIn the starch class, a-n-( 1 + 4) ages involved in various polysac~harides.~~ linkages, there is a gradual transition from 907 to 930 cm.-l for type 1 and from 778 to 758 f 2 cm.-' for type 3, in going from the disaccharide to ~ 3) and C W - D - (4) ~ +linkages] the polysaccharide. Nigeran [alternate C W - D - (4 exhibits an absorption a t 793 f 2 cm.-l, aa compared to the normal type 3 absorption of the D-glucopyranoses. The dextran class [ a - ~ - + ( l 6 ) linkages], on the other hand, shows fairly constant frequencies of 917 f 2 cm.-I and 768 f 1 cm.-l for the two types, in passing from isomaltose to the higher polymer. This constancy for dextran may be attributable to the fact that the dextran linkages are not intimately involved with the ring. Such an (29) S. A . Barker, E. J. Bourne, R . Stephens and D. H. Whiffen, J . Chem. Soc., 4211 (1954).

INFRARED SPECTRA OF CARBOHYDRATES

25

explanation would tend to substantiate the assignment of types 1 and 3 vibrations to modes involved with the pyranose ring. This use of infrared spectra of polysaccharides is becoming very helpful in characterizing the types of linkages in an unknown polysaccharide (see Section V of this Chapter for a further discussion of this point). The type f? absorption showed only a very small deviation, regardless of whether a simple hexose or a long-chain polysaccharide was being examined. The conclusion from this observation was that the motion was an anomeric C-H deformation mode rather than a motion involving the external oxygen attached to the anomeric carbon atom.lg The a and /? anomers of the sugars examined differed in the fact that the a-D-anomeric hydrogen lies in the equatorial belt, whereas the p-D-anomeric hydrogen ie in the axial position. Reevesao.31 has discussed this point thoroughly and has shown that the most stable chair conformation of the pyranose ring is the one which contains the maximum number of hydrogen atoms in the axial position. From a study of formulas I1 and I11 for a- and 8-D-glucopyranose it was seen that the stable conformations have an axial hydrogen atom on C5. It wm possible, therefore that, during the deformation mode of the anomeric C-H, the axial hydrogen on C1 came into closer contact with the hydrogen atom on C5 than the equatorial hydrogen atom on C1. With such close contact of C1 and C5 hydrogen atoms, van der Waals’ forces would become appreciable, thus leading to an increased frequency, which was exactly the situation found for P-D-glucopyranose (111). This postulation was given confirmation by the work of Burket and Badger,24who found that the axial hydrogen atoms on C1 and C5 are closer together in tetrahydropyran than in cyclohexane. A study of derivatives of a- and j3-D-galactopyranoses and -mannopyranoses gave support to the types 2a, 2 4 and 3 assignments. There has been some recent work on the H

H

6H 11 (30) R. E. Reeves, J . A m . Chem. SOC.,72, 1499 (1950). (31) R. E. Reeves, Advances in Carbohydrate Chem., 6, 107 (1951)

26

W. BROCK NEELY

infrared absorption of a- and @-D-talopyranoseand related derivatives31a; each anomer exhibits the absorptions which have been considered characteristic of the a and p modifications.lgSuch anomaly might be explained on the basis that both chair conformations (as described by Reeves30s31)might exist. If this situation occurs, the a and p forms would contain both equatorial and axial hydrogens on C l . However, regardless of the reason, the study by Isbell and associate~~la does indicate that the absorption in the 2a and 2b regions cannot be used indiscriminately for the assignment of the a and P configuration to all types of pyranose derivatives. Derivatives of D-galactopyranose and D-mannopyranose show an extra absorption (type 2c) at 875 cm.?. These sugars differ from D-glucopyranose on C4 and C2 respectively. Here the C-H bonds are equatorial rather than axial. Since this is the only difference, the new peak must arise from a C-H equatorial deformation other than an anomeric C-H mode. Various reasons for the rise in frequency from 840-875 cm.-l were given.20They included an application of van der Waals' forces and also the suggestion that the low frequency for the anomeric C-H equatorial deformation is due to an altered force-constant (resulting from the attachment of two oxygen atoms to the anomeric carbon atom). Type 2c was given further confirmation by work on the hexahydric inositols and the pentahydric quercitols, respecti~ely.~g The stable chair form, which contains equatorial hydrogen atoms, exhibited a similar absorption peak at approximately 875 cm.-'. From the above discussion, it would appear that axial C-H deformation modes other than the anomeric axial C-H should absorb at frequencies higher than 890 cm.-'. This region, for previously mentioned reasons, is very complicated, and the separation and identification of the various absorption peaks became quite difficult. Consequently, no definite assignment could be made for these axial C-H deformation modes. In assigning absorption peaks to the methylene group of deoxy sugars, the spectra of the inositols and quercitols were examined.29 The quercitols showed an extra peak at 853 cm.-l, as compared to the corresponding inositol. This must have been due to a ring methylene group which was in close agreement to the absorption at 856 cm.-l assigned to the methylene groups in tetrahydr~pyran.~~ The various deoxy sugars, other than the 6-deoxy, showed a new peak at 867 cm.-' which has been assigned to the ring methylene rocking vibration.29 This assignment has been further strengthened by work on the 4,6-O-benzylidene derivatives of a-D-glucopyranose.82An absorption at 877 cm.-l was apparent; this has tentatively been assigned to the ring methylene group formed by C6 of the a-D-gluco(31a) H. S. Isbell, J. E. Stewart, Harriet L. Frush, J. D. Moyer and F. A. Smith, J . Research Natl. Bur. Standards, 67, 179 (1956). (32) W. B. Neely, unpublished results.

27

INFRARED SPECTRA O F CARBOHYDRATES

pyranose fused to the benzylidene group. Complications developed, however, in the compounds containing equatorial hydrogen atoms on carbon atoms other than the anomeric carbon atom. Here, the presence of the type 2c absorption made identification of the methylene group very difficult. The 6-deoxy derivatives of D-galactopyranose and ~ - ma n n o p y ra n o s eex~~ amined showed a peak a t 967 cm.-l. The rigid assignment of such a frequency t o any particular group would, however, be impossible, because of the numerous absorption bands appearing in this region. A preliminary investigation of furanose derivatives was made by Barker and Stephens33 in which tentative assignments were allocated. A ring breathing frequency at 924 f 13 cm.-' was made, along with a C-H deformation mode (799 f 17 cm.-l) where the hydrogen atom was present TABLE I1 Frequencies Characteristic of the Sugar Pyranose Ring Frequency (cm.-')

Antisymmetrical ring vibration Symmetrical ring breathing vibration a-Anomeric C-H deformational vibration 8-Anomeric C-H deformational vibration Equatorial C-H deformational vibration, other than anomeric C-H Ring Inethylene rocking vibration Terminal methyl group rocking vibration

917 770 844 891 880

f 13 f 14 f8 f7 f8

867 f 2 967 f 6

Referesces

19 19 19 19 20 29 29

on the carbon atom directly attached to the ring oxygen atom. Unlike the anomeric carbon atom of the pyranose, where axial and equatorial hydrogen atoms are present, the furanose hydrogen atoms were in equivalent positions, and no differentiation between a- and ,&forms was possible. The results of all this discussion have been summarized briefly in Table I1 and, along with the data of Table 111,form a list of the common frequencies encountered in infrared spectra of carbohydrates. The Tables have been divided in order to emphasize that there is a sharp difference in the certainty of the assignments higher than 1350 cm.-' and of those below. For the higher frequencies, the assignments are fairly definite because the vibrations are well divided and t,here is little likelihood of interactions causing frequency shifts. I n the lower regions, this is not true, as the combination of force constants may give rise to frequencies in the same region or result in interactions causing unpredictable frequency shifts. (33) S. A. Barker and R. Stephens, J . Chem. Soc., 4550 (1954).

28

W. BROCE NEELY

TABLEI11 Group Frequencies of Interest i n Carbohydrates ~

~~~

Group

A. Hydrogen Stretching 1. a. 0-H (free) b. 0-H (bonded) 2. a. N-H (free) b. N-H (bonded) C. N-Hz 3.C-H 4. S-H B. Carbon-Carbon Stretching 1. C-C (beneenoid) 2. c=c 3. c=c C. Double Bond Stretching 1. Carbonyl a. anhydride b. ring carbonyl c. ester d. carboxyl e. aldehyde, ketone f . amide g. carboxyl salt h. ionized carboxyl i. lactone, gamma j . lactone, delta 2. Phenyl ring D. Hydrogen Bending or Deformation 1. a. N-HI b. N-substituted amide 2. a. saturated CHa b. methyl hydrogen 3.0-H E. Sulfur-Oxygen Stretching 1. sulfone 2. sulfonate 3. sulfate F. Other Important Frequencies 1. Nujol 2. Atmospheric COZ 3. Liquid water

Range (cm.-L)

References

2400-3800 3500-3700 3100-3500 3200-3500 3100-3500 3200-3300 2800-2900 2500-2580

34 10, 35 10, 35 10 10 10 36, 37 38

1500-1600 1600-1700 1950-2350 1475-1875 1550-1850 1800-1850 1750-1800 1725-1750 1650-1725 1650-1725 1625-1700 1550-1600 1400-1450 1760-1800 1725-1750 1500 and 1600

39 39 40 34 41 41 41 41 42, 43 44 41 41 41 41 10

1590-1650 1525-1575 1425-1475 1350-1400 1050-1075

10, 39 38 10, 21

1350-1450 1150-1200 1100

21 21 45

23

2918, 2861, 1458, 1378, 720 (weak) 2367, 2336, 721, 667 1600-1650

(34) R. B. Barnes, R. C. Gore, R. W. Stafford and V. Z. Williams, Anal. Chem., 20, 402 (1948).

(35) J. J . Fox and A. E,. Martin, PTOC. Roy. SOC.(London), A162,419 (1937).

INFRARED SPECTRA O F CARBOHYDRATES

29

V. APPLICATION OF INFRARED SPECTROSCOPY TO CARBOHYDRATES 1. Early Work

C ~ b l e n appears z ~ ~ to have been the first person to examine the infrared spectra of carbohydrates. He studied D-fructose and D-glucose in the region 3200-833 cm.‘ (3-12 p ) . Rogers and Williams4’ extended the series to include D- and L-arabinose, D- and L-lyxose, D-galactose, and D-mannose. They used the same region as had Coblenz, and found a few additional peaks which, they claimed, resulted from an improved technique of handling the sample. C o b l e n ~used ~ ~ melted samples, whereas Rogers and Williams used the crystalline material. Barr and C h r i ~ m a n employing ,~~ aqueous solutions, examined the spectra of several other simple sugars. They reported the appearance of new bands at 4630 and 4348 cm.-l (2.16 p and 2.3 p ) . Because of the difference in preparation of the samples, it would be difficult to ascertain if the “new” bands were attributable to the sugars or to the physical state of the materials. 2. Hydroxyl Absorption in Cellulose and Related Compounds

The investigator in the field of cellulose chemistry has been confronted with several problems ideally suited for infrared analysis. The first was concerned with reactions involving substitutions in the cellulose polymer; these were found to proceed more rapidly in the material which was less crystalline. Interest in the second problem arose from the postulation that secondary valence forces associated with the hydroxyl groups are responsible for stabilizing of the cellulose chains in the cell wall. The state of crystallinity and the stabilization forces in cellulose fibers of plants will both cause variations in the degree of hydrogen bonding. Therefore, the hydroxyl stretching frequency around 3300 em.-’ (3 p ) in the cellulose spectra should be influenced. These questions have stimulated a great deal of interest in the infrared I

(36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) (48)

J. J. Fox and A. E. Martin, Proc. Roy. SOC.(London), A167.257 (1938). J. J. Fox and A. E. Martin, Proc. Roy. SOC.(London), A176, 208 (1940). I. F. Trotter and H. W. Thompson, J . Chem. SOC.,481 (1946). N. Sheppard and Delia M. Simpson, Quart. Revs. (London), 7 , 19 (1953). J. H. Wotiz and F. A. Miller, J. A m . Chem. SOC.,71,3441 (1943). R. S. Rasmussen and R. R . Brattain, J . A m . Chem. Soc., 71, 1073 (1949). H. Hartwell, R. E. Richards and H. W. Thompson, J. Chem. SOC.,1436 (1948). H. W. Thompson and P. Torkington. J . Chem. SOC.,640 (1945). R. E. Richards and H. W. Thompson, J. Chem. SOC.,1248 (1947). F. A. Miller and C. H. Wilkins, Anal. Chem., 24, 1253 (1950). W. W. Coblenz, Carnegie Inst. Wash. Publ. N o . 66 (1906). L. H. Rogers and D. Williams, J. Am. Chem. SOC.,60, 2619 (1938). E. S. Barr and C. H. Chrisman, Jr., J . Chem. Phys., 8, 51 (1940).

30

W. BROCK NEELY

analysis of cellulose and related substan~es.~~-63 As the extent of hydrogen bonding increased, the frequency of the hydroxyl band shifted to a slightly longer wavelength. The observations of these and other workers tended to confirm the results of x-ray investigation^^^ which showed that hydrogen bonding tends to stabilize tJhecellulose fibers in plants and that substitution reactions proceed more rapidly in the less crystalline state.e2 These studies were thus able to strengthen some of the theories regarding the structure and reactivity of carbohydrate polymers. The hydroxyl region has been used with some success in the confirmation of the structure of a sugar derivative. One of the earliest examples of such definitive use of infrared spectra was in detectings4~the unacetylatable tertiary hydroxyl group in the streptose portions of the streptomycin molecule. Infrared analysis was used by Clark66in support of his conclusion that 2,3-dideoxy-2-dimethylamino-4-O-methylaldotetrose contains a free hydroxyl group and, possibly, a R3N@Hgroup. WeiglS6studied the exchange reaction between ascorbic acid and heavy water. An examination of the 0-H and C-H stretching frequency region of the normal and the deuterated compound led him to the conclusion that ascorbic acid contains labile hydrogen atoms attached to both carbon and oxygen.

3. Mucopolysaccharides StaceyK7 has written an excellent review on the subject of mucopolysaccharides, which he classified on the basis of their containing both hexosamine and hexuronic acid residues, one or the other of these sugar derivatives, or neither. Hyaluronic acid, chondroitinsulfuric acid, Type I pneumococcal polysaccharides, and heparin are members of the first class. Types 11, 111, and VIII pneumococcal polysaccharides are examples containing hexuronic acid but no hexosamine. Chitin and Types IV and XIV pneumococcal polysaccharides contain hexosamine but no hexuronic acid; and bacterial dextrans, mold polysaccharides, and levans contain neither hexosamine nor hexuronic acid. (49) J. W . Ellis and Jean Bath, J . A m . Chem. Soc., 62, 2859 (1940). (50) J. W. Rowen and E. K. Plyler, J . Research Natl. Bur. Standards, 44,313 (1950). (51) E. Treiber, Kolloid-Z., 130, 39 (1953). (52) L. Brown, P. Holliday and I. F. Trotter, J . Chem. Soc., 1532 (1951). (53) J. W . Ellis and Jean Bath, J . Chem. Phys., 6, 221 (1938). (54) H. Mark, Chem. Revs., 26, 169 (1940). (54a) F. A. Kuehl, Jr., E. H. Flynn, N. G . Brink and K . Folkers. J . A m . Chem. Soc., 68, 2096 (1946); R . U. Lemieux and M. L. Wolfrom, Advances in Carbohydrate Chem., 3 , 359 (1948). (55) R. K. Clark, Jr., Antibiotics & Chemotherapy, 3, 663 (1953). (56) J. W. Weigl, Anal. Chem., 24, 1483 (1952). (57) M. Stacey, Advances in Carbohydrate Chem., 2, 161 (1946).

INFRARED SPECTRA OF CARBOHYDRATES

31

a. Hyaluronic Acid and Chrmdroitinsulfuric Acid.-The examination of the infrared spectra of this class of mucopolysaccharide has led to some 69 was able to demonstrate by a comparison very interesting of the intensity ratio of the bands at 1736 cm.? (due to the carboxylic acid group) and 1560 cm.-l (amino group) that the hexosamine and hexuronic acid moieties occur in a 1:1 ratio in the chondroitinsulfuric acid from trachea. These results were based on the assumption that such a ratio exists in hyaluronic acid. Meyer and associates60-62 have confirmed the validity of this assumption in a series of papers on the chemical constitution of hyaluronic acid. was also able to assign the frequencies of 1240 cm.-l and 820 cm.-l to the sulfate group present in hyaluronic acid and related compounds. The assignment of the 1240 cm.-' band was founded on the interpretation of the spectrum of alkyl sodium sulfate,63where a similar band was shown to be due to the sulfate group. This vibrational mode was undoubtedly analogous to the C=O stretching vibration present in acetates a t 1740 cm.-'. Acetates show an additional absorption at 1240 cm.-', which Thompson and T o r k i n g t ~ nascribed ~~ to a C-0-C system. It would be reasonable, system, at therefore, to expect to find a similar mode within the C-0-S correspondingly lower frequencies. The spectrum of polysulfated hyaluronic acid69disclosed the presence of such a band (at approximately 820 cm.-l) which must have been due to the aforementioned vibration. The exact position of this band a t 820 cm.-l might be correlated with the location of the sulfate group in an equatorial or a polar position on the pyranose ring. This suggestion had a precedent in the study of the 3-acetoxy steroids.64 In this case, the polar-polar or the equatorial-polar relationship of the 3-acetate bond and the 5-hydrogen bond could be differentiated by investigating the nature of the absorption at 1240 cm.-'. With more work on this point, it might be possible to establish the exact location of the sulfate group in this important group of mucopolysaccharides. b. Pneumococcal Po1ysaccharides.-Stevenson and Levines6P 66 compared the spectra of purified pneumococcal polysaccharides. They showed that the use of infrared analysis affords a single, rapid, physical test permitting (58) S.F.D.Orr, R . J. C. Harris and B. SyIvBn, Nature, 169,544 (1952). (59) S. F. D.Orr, Biochim. et Biophys. Acta, 14, 171 (1954). (60) M. M. Rapport, K. Meyer and A. Linker, J . Am . Chem. Sac., 73,2416 (1951). (61) B. Weissmann and K. Meyer, J . A m . Chem. Soc., 76, 1753 (1954). (62) K.H.Meyer, J. Fellig and E. H. Fischer, Helv. Chim. Acta, 34, 939 (1951). (63) I. M. Klotz and D. M. Gruen, J . Phys. & Colloid Chem., 62, 961 (1948). (64) R.N.Jones, P. Humphries, F. Herling and K . Dobriner, J. Am . Chem. Sac., 73, 3215 (1951). (65) H . J. R.Stevenson and S . Levine, Science, 116, 705 (1952). (66) S. Levine, H.J. R. Stevenson and P. W. Kabler, Arch. Biochem. and Biophys., 46, 65 (1953).

32

W. BROCK NEELY

the identification of type-specific polysaccharides. I n addition, the result may also be used as a criterion of purity of the sample. c. Bacterial Dextrans.-Burket and Melvin,67 from the infrared analysis of various dextrans, showed that a marked difference exists in the spectra at approximately 794 cm.-l(12.6 p ) . Other workers have confirmed this and have correlated the increased intensity at 794 cm.-' shown by certain dextrans with the presence of (1 -+ 3)-glucosidic linkages (see Section IV of this Chapter). Additional evidence for this correlation came from the chemical elucidation of the normal, highly branched dextran produced by Betucoccus arabinosaceous.71 This dextran was shown to consist of branch points witha-n-(1 + 3) linkages, and the absorption spectrum exhibited a band a t 794 cm.-'. The dextran produced by the same organism grown in a magnesium-deficient medium is an essentially straight C Y - D - ( ~--+ 6) de~tran.'~ The infrared absorption spectrum showed a peak at 768 cm.-', consistent with (1 + 6) linkages (with no absorption at 794 cm.-' detectable).

4. Comparative Study K ~ h nwas ? ~ the first to make a serious attempt a t correlating differences in sugar molecules with their infrared spectra. He published the absorption curves of a number of sugars and their derivatives, and showed that the anomeric forms of various glycosides are readily distinguished by their infrared curves; he failed, however, to assign any of the observed frequencies to the anomeric carbon atom. The various oligosaccharides were also shown to have different absorption curves. Whistler and House:4 also, have reported that the spectra of the anomers of sugars can be used to differentiate between them. Fletcher and Diehl,'6 in studying the preparation of melibiose from rafEinose by the fermentative hydrolysis of the trisaccharide:6 noticed a new form of the disaccharide. By observing the mutarotation of the new form, and by comparing the infrared spectra with that of an authentic (67) S. C. Burket and E. H. Melvin, Science, 116, 576 (1952). (68) R. Lohmar, J . Am. Chem. SOC.,74, 4974 (1952). (69) Allene Jeanes and C. A. Wilham, J. Am. Chem. Soc., 74, 5339 (1952). (70) S. A. Barker, E. J. Bourne, M. Stacey and D. H. Whiffen, Chemistry & Industry, 1156 (1952). (71) S. A. Barker, E. J. Bourne, G . T. Bruce, W. B. Neely and M. Stacey, J . Chem. SOC.,2395 (1954). (72) S. A. Barker, E. J. Bourne, A. E. James, W. B. Neely and M. Stacey, J. Chem. SOC., 2096 (1955). (73) L. P. Kuhn, Anal. Chem., 22, 276 (1950). (74) R. L. Whistler and L. R. House, Anal. Chem., 26, 1463 (1953). (75) H. G. Fletcher, Jr., and H. W. Diehl, J . Am. Chem. Soc., 74, 5774 (1952). (76) C. 5.Hudson and T. 5. Harding, J . Am. Chem. SOC.,37,2734 (1915).

INFRARED SPECTRA O F CARBOHYDRATES

33

sample of p-melibiose, they concluded that their form must be the LY modification. A trisaccharide produced from sucrose by Aspergillus niger (152) was investigated by Barker, Bourne and C a r r i n g t ~ nDuring . ~ ~ the course of this study they compared the infrared spectra of their trisaccharide and of a known trisaccharide78 produced from sucrose by Takadiastase (a commercial, mold-enzyme preparation), The spectra were identical, and later methylation and hydrolysis studies confirmed the fact that the two trisaccharides were the same. The infrared spectra of a large number of carbohydrates have been published; all of these will be useful for future comparative work. The work of K ~ h n and 7 ~ of Stevenson and Levinea6has already been mentioned. In addi? ~ recently published the spectra of tion, Solms, Denzler and D e ~ e l have several derivatives of poly-D-galacturonic acid. The collection of infrared spectra of the sugar acetates and related compounds also forms a valuable source of data for comparative work.so (77) S. A. Barker, E. J. Bourne and T. R . Carrington, J . Chem. SOC.,2125 (1954). (78) J. S.D. Bacon and D. J. Bell, J . Chem. SOC.,2528 (1953). (79) J. Solms, A. Denzler and H. Deuel, Helv. Chim. Acta, 37, 2153 (1954). (80) H.S. Isbell, F. A. Smith, C. Creitz, J. D. Moyer and Harriet L. Frush, Natl. Bur. Standards (U.S.)Report 1358,NR 055208, Dec. 31, 1951.

This Page Intentionally Left Blank

THE SACCHARINIC ACIDS

BY JOHN C . SOWDEN Department of Chemistry. Washington University. Saint Louis. Missouri

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... I1. The Individual Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. DL-Lactic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. D L - ( ~ ,4-Dihydroxybutyric Acid) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............................. 3. ~~-[2,4-Dihydroxy2-(hydroxymethy1)butyric Acid] . . . . . . . . . . . . . . . . . . . . .............................. .............................. 4 The Five-Carbon Metasaccharinic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

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

b . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. “or”-D-Glucosaccharinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . Configuration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d . Miscellaneous Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . “cy”-D-Isos&ccharinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . Configuration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d . Miscellaneous Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e . “0 ”-D-Isosaccharinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. The D-Galactometasaccharinic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) “a”-D-GalactometasaccharinicAcid . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) “j3”-s-Galactometasaccharinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 The D-Glucometasaccharinic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Structure ................................................. c . Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d . Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11 . Mechanism of Formation of Saccharinic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The Fragment-recombination Mechanism of Kiliani and Windaus . . . . . 2 . The Isomerication Mechanism of Nef .................................

. .

.

.

35

36 38 38 38 38 39

40 40 40 41 41 41 42 43

43 44

46 47 48 48 49 51 52 52 53 54 55 55 56 59 59

60 61

61 62 62 62 63

36

JOHN C. SOWDEN

3. The Ionic Mechanism of Isbell.. ...................................... 4. Saccharinic Acids from Substituted Sugars.. .......................... 5. Fragment Recombination and Saccharinic Acid Formation. . . . . . . . . . . . . 6. Saccharinic Acid Formation by Various Bases.. ....................... IV. Table of Properties of Saccharinic Acid Derivatives.. ....................

66 69

72 75 76

I. INTRODUCTION In a paper presented before the French Academy of Sciences in 1838, Eugene Peligot reported that an acid “trds hmgique” was among the products of the action of barium hydroxide or calcium hydroxide on glucose.’ This observation, that acidic materials may result from the treatment of reducing sugars with aqueous alkalis, marked the beginning of investigations that were to uncover one of the most intriguing, and a t the same time one of the most perplexing, reaction sequences in carbohydrate chemistry. Confusion entered the sugar-alkali reaction picture with the report by Mulder2 in 1840 that, apparently, aqueous acid or aqueous alkali act upon glucose in the same manner, to form an acidic product (“glucinic acid”). A natural consequence of this early work was the conclusion that glucose might be an ester whose hydrolysis by acids or alkalis led to acid and alcohol moieties. However, a clear distinction between the products obtainable from the reducing sugars through the action of acids and alkalis, respectively, was eventually achieved. The hexoses were found to afford, through strenuous treatment with acids, a mixture of levulinic and formic acids: whereas with alkalis the principal products are lactic acid plus a series of six-carbon, deoxyaldonic acids (saccharinic acids) isomeric with the starting sugars. In addition, minor amounts of racemic 3-deoxytetronic acid are formed in the hexose-alkali reaction. COZH

COzH

COzH

CHOH

CHa

CHOH

I CHOH I

CHzOH Saccharinic acid

I CHOH I CHZOH Isosaccharinic acid

I I

CHOH CHzOH Metasaccharinic acid

(1) E. Peligot, Compt. rend., 7, 106 (1838). In this same paper it was also recognized that the crystalline sugar obtainable from grapes, honey, starch hydrolyzates, and diabetic urine is one and the same substance, and the name glucose was proposed for it. (2) G . J. Mulder, J . prakt. Chem., 21, 203 (1840). (3) A. F. von Grote and B. Tollens, Ber., 7, 1375 (1874).

THE SACCHARINIC ACIDS

37

Three structurally isomeric forms have been established for the six-carbon saccharinic acids. In the order of their discovery, these are the saccharinic or 2-C-methylpentonic acids, the isosaccharinic or 3-deoxy-2-C(hydroxymethy1)-pentonic acids, and the metasaccharinic or 3-deoxyhexonic acids. Although none of these six-carbon, deoxyaldonic acids has been crystallized, six are known in the form of crystalline lactones (saccharins). All the possible metasaccharinic acids of less than six-carbon content have been obtained, in the form of crystalline derivatives, by the sugaralkali reaction. Only one example of a branched-chain deoxyaldonic acid (the racemic, five-carbon isosaccharinic acid) of other than six-carbon content has been so obtained. The formation of saccharinic acids containing more than six carbon atoms remains to be explored. Isomeric with the saccharinic acids that arise from the sugar-alkali reaction are the w-deoxy- and 2-deoxy-aldonic acids. Both of these latter types may be obtained by oxidation of the corresponding deoxy sugars, and the 2-deoxyaldonic acids also result from the action of lead oxide on the 1,2dideoxy-1 ,2-dihalogeno-aldoses.* In addition, Glattfeld and coworkers6 have synthesized, mainly from non-sugar starting materials, all the racemic deoxytetronic acids possible. However, the present article deals only with those acids which have been isolated from the sugar-alkali reaction. Mention should be made of the origin of the terms saccharin and saccharinic acid. Peligote isolated the first crystalline lactone (“a”-D-glucosaccharin) of a deoxyaldonic acid produced by the hexose-alkali reaction. A slightly erroneous analysis of this new substance led him to believe that it had the same carbon and hydrogen content as has ordinary cane sugar. In addition, the lactone yielded an initially neutral aqueous solution, and Peligot, after concluding that his substance was simply an isomer of sucrose (saccharose), named it saccharin. Although Scheibler’ soon thereafter recognized the lactonic character of Peligot’s saccharin, he retained the name and expanded it to saccharinic acid for the corresponding free acid. Subsequent workers have perpetuated this nomenclature, and the terms saccharinic acid and saccharin are now used extensively in the generic sense. (4) 8. N. Danilov and A. M. Gakhokidze, Z h w . Obshchei Khim., 6, 704 (1936); Chem. Abstracts, SO, 6333 (1936). (5) J. W. E. Glattfeld and G. E. Miller, J . Am. Chem. SOC.,43, 2314 (1920); J. W. E. Glattfeld and F. V. Sander, ibid., 43, 2675 (1921); J. W. E. Glattfeld and L. P. Sherman, ibid., 47, 1742 (1925); J. W. E. Glattfeld and Sybil Woodruff, ibid., 49,2309 (1927); J. W. E. Glattfeld, Gladys Leavell, G. E. Spieth and D . Hutton, ibid., 63,3164 (1931); J. W. E. Glattfeld and J. W. Chittum, ibid., 66, 3663 (1933); J. W. E. Glattfeld and J. M. Schneider, ibid., 60,415 (1938). (6) E. Peligot, Compt. rend., 89, 918 (1879). (7) C. Scheibler, Ber., 13, 2212 (1880).

38

JOHN C. SOWDEN

11. THEINDIVIDUAL ACIDS 1. DL-Lactic Acid COzH

I I

CHOH CHI 3-Deoxy-~~-glyceronic acid

m-Lactic acid is the metasaccharinic acid related to the triose sugars.

It has been obtained as a product of the action of alkali not only on glycerose (glycera1dehyde)a but also on hexosesg and pentoses,*O whence it arises via cleavage and isomerization. The racemic form of the acid is always obtained from the sugar-alkali reaction since the nonasymmetric enediol related to glycerose is an intermediate in its formation (see Section 111). As a consequence of the biochemical importance of lactic acid, its chemistry has been thoroughly studied and is adequately documented elsewhere. The production of lactic acid by the action of alkalis on the sugars has been reviewed by Montgomery." 2. DL- ( 2 ,,&Dihydroxybutyric Acid) COzH

1 I CHa I

CHOH

CHzOH

DL- (3-Deoxytetronic

acid)

Only the racemic form of this acid is obtained from the sugar-alkali reaction. As in the formation of lactic acid, a non-asymmetric enediol is an intermediate in its production (see Section HI), and hence the racemate is the sole representative of the four-carbon metasaccharinic acid class. a. Preparation.-The conversion of the threoses and erythroses to their related metasaccharinic acid by the action of alkali has apparently not been explored because of the relative inaccessibility of these tetroses. The acid is, however, formed as one of the products of the action of hot, concentrated sodium hydroxide on the pentoses or hexoses.12 Its isolation from these (8) J. U. Nef, Ann., 336, 247 (1904). (9) F. Hoppe-Seyler, Ber., 4,346 (1871). (10) T. Araki, Hoppe-Seyler's 2. physiol. Chem., 19,422 (1894). (11) R. Montgomery, Sugar Research Foundation, N . Y., Sci. Rept. Ser., No. 11 (1949). (12) J. U. Nef, Ann., 376, 1 (1910).

39

THE SACCHARINE ACIDS

sources by Nef was accomplished through a complex and arduous sequence of operations which afforded, at the same time, other saccharinic acids produced in the reactions. Nef's method for resolving these complicated mixtures into their component saccharinic acids is here described in general terms only, and the reader is referred to the original work for details.12Following the isomerization with sodium hydroxide, the reaction mixtures were treated with a very slight excess of hydrochloric acid, concentrated, and the residues heated to effect lactonixation. The saccharinic acids and their lactones were then isolated from the sodium chloride by extraction with organic solvents. The acid-lactone mixture was next acetylated, and the products were separated (by extraction with ether) from colored, gummy byproducts. Following deacetylation, the refined acid-lactone mixtures were repeatedly extracted from their aqueous solution with ether and ethyl acetate, to provide fractions of different degrees of solubility in these organic solvents. Finally, fractional recrystallization of the quinine or brucine salts was employed for separating the individual saccharinic acids. Application of the techniques of chromatography and ion exchange, not known to Nef, should greatly simplify the isolation of individual saccharinic acids from such mixtures. b. Structure.12-Nef observed that oxidation of the four-carbon metasaccharhie acid with nitric acid yields DL-malic acid. Moreover, DL- (3,4-dihydroxybutyric acid), prepared by condensation of l-chloro-l-deoxyglyceritol (glycerol a-chlorohydrin, 3-chloro-1,2-propanediol) with potassium cyanide, followed by hydrolysis, provides a phenylhydrazide (m. p., 99") different from that (m. p., 130-131') obtained from the saccharinic acid. Accordingly, the product from the sugar-alkali reaction is ~~-@-deoxytet, acid)]. ronic acid) [ D L - ( ~4-dihydroxybutyric CH aOH-CH 2-CHOH-CO

zH

DL- (2,4-Dihydroxybutyric

acid)

HN03 A

HO &-CH n-CHOH-CO

2H

DL-Malic acid

(phenylhydrazide, m. p., 130-131")

(m. p. and mixed m. p., 128') THNOs

ClCH2-CHOH-CHzOH 1-Chloro-1-deoxyglyceritol

KCN HzO

___f

HOIC--CH~-CHOH-CH~OH D L - ( 4-Dihydroxybutyric ~, acid) (phenylhydrazide, m. p., 99")

Additional evidence regarding the structure of ~~-(S-deoxytetronic acid) was obtained (by Nef) by resolution of the acid through the brucine salt.

40

JOHN C. SOWDEN

One of the two resulting enantiomorphs gave, on oxidation with nitric acid, the enantiomorph of naturally occurring (- )-malic acid. 3.

D L - [ ~ 4-Dihydroxy-2-(hydroxymethyl)butyric , Acid]

COaH

CH~OH

~~-[3-Deoxy-2-C-(hydroxymethyl) tetronic acid]

This recently discovered, racemic acid of the five-carbon series is only the third example of an isosaccharinic acid to be identified as a product of the sugar-alkali reaction. The other examples are the a- and p-D-isosaccharinic acids of the six-carbon series (see pages 48 and 52). a. Preparation.-The crystalline lactone of D L - [ 4-dihydroxy-2-(hydroxy~, methyl) butyric acid] may be isolated after treatment of xyl~biose'~ (4-0P-D-xylopyranosyl-D-xylose) or the related trisaccharide, xylotrio~e,'~ (both obtained by partial hydrolysis of xylan) with lime-water. b. Structure.-The structure of this isosaccharinic acid is established by its synthesis from 1 ,4-butynediol dia~etate.'~ CHaOH CH-0 COCHI

CHa

CHa

C H ~ O CO C H ~

CHaOCOCHs

I

1,4-Butynediol diacetate

I

I

/OH C

I

CHzOH DL- [ Z , 4-Dihydroxy-P(hydroxymethyl) butyric acid]

The branched-chain structure of the acid is further confirmed through its reduction, with hydriodic acid and red phosphorus, to 2-methylbutyric acid.14 Oxidation of one mole of the isosaccharinic lactone with periodate produces approximately one molecular equivalent of f~rrnaldehyde.'~ Whistler and CorbettI4record barely detectable, positive optical rotations (13) G. 0. Aspinall, Mary E. Carter and M. Los, Chemistry & Industry, 1553 (1955). (14) R. L. Whistler and W. M. Corbett, J . Am. Chem. Soc., 78, 1003 (1956).

41

THE SACCHARINIC ACIDS

for the lactone and calcium salt of the acid. These optical rotations, if real, are probably due to unremoved D-xylose, since the accepted mechanism for the production of this five-carbon isosaccharinic acid in the sugar-alkali reaction would predict a racemic product (see Section 111). 4. The Five-Carbon Metasaccharinic Acids COzH II HCOH

I CHz I HCOH I

CHzOH

3-DeoxyD-erythropent onic acid

COzH II

HOCH

I CHa I HCOH I

CHzOH

3-DeoxyD-threopentonic acid

COzH II

HOCH

I CHz I HOCH I

CHzOH

3-DeoxyL-erythropentonic acid

COIH II

HCOH

I I HOCH I

CHI

CHzOH

3-DeoxyL-threopentonic acid

These four acids comprise all the possible five-carbon metasaccharinic acids; and all were obtained by Nef, in the form of crystalline derivatives, from the pentose-alkali reaction. In reading Nef’s description of these substances and their preparation, it must be borne in mind that the available, naturally occurring D-xylose was a t that time called I-xylose. Moreover, Rosanoff’s convention16 for assigning configurational prefixes was then relatively new and was not utilized by Nef. Accordingly, Nef’s I-xylose and I-arabinose, and d-erythro-, I-threo-, I-ergthro-, and d-threo-1,3,4-trihydroxyvaleric acids are respectively, by modern nomenclature, D-xylose and L-arabinose, and D-erythro-, D-threo-, L-erythro-, and L-threo-1 , 3 ,4-trihydroxyvaleric acids (3-deoxypentonic acids). a. Preparation.-Nef applied his conditions of isomerization (with hot, 8 N sodium hydroxide) to L-arabinose and D-xylose, and was able to obtain from the reaction mixtures, through his complicated system of fractionations (see page 39), all four possible 3-deoxypentonic acids. L-Arabinose yielded 3-deoxy-~-erythro-and-L-threo-pentonic acids, and D-xylose provided the corresponding enantiomorphs. The reader is referred to the original work12for details of the separations involved. b. Structure.12-The structures of the four 3-deoxypentonic acids were established through study of their oxidation with nitric acid to the related 2,4-dihydroxyglutaric acids. The interpretation of the results of these oxidation experiments is intimately related to the prior proof of the structure (15) M. A. Rosanoff, J . Am. Chem. Soc., 28,114 (1906).

42

JOHN C. SOWDEN

of “a”-D-isosaccharinic acid (see page 49). Thus, both 3-deoxy-~-erythropentonic acid (from D-xylose) and 3-deoxy-~-erythro-pentonic acid (from L-arabinose) yielded meso-2,4-dihydroxyglutaricacid on oxidation. The last acid, accompanied by an optically active 2,4-dihydroxyglutaric acid, had been obtained previously by Kiliani and M a t t h e P through oxidation, followed by decarboxylation, of “a”-D-iSosacCharinic acid. Moreover, 3-deoxy-~-threo-pentonic acid (from L-arabinose) gave, on oxidation, the enantiomorph of the optically active 2,4-dihydroxyglutaric acid obtained by Kiliani and Matthes from “a”-D-isosaccharinic acid. COzH I

C OzH

1

COsH

\OH

HNOa

- - coz I

CHz

HOCH

+

CH2

I

I

HCOH

COzH

“~”-D-Isosaccharinic acid

I

CHz

-

I HCOH I CH2 I HOCH I

CHz

CHzOH

3-Deoxy-~-threopentonic acid

I I

HCOH

I

COzH

HNO3

HCOH

1

COiH ~-threo-2,4-Dihydroxy- meso-2,4-Dihydroxyglutaric acid glutaric acid

COzH

I

HNO3

HCOH

I

I

HCoH

I

HCOH

CHzOH

COzH

I

I

I HCOH I , CHz I HOCH I

COzH

~-threo-2,4-Dihydroxyglutaric acid

CHz OH 3-Deoxya-erythropentonic acid

COzH

I

HOCH

I

CHz

I I

HOCH CHzOH 3-Deoxy-~-erythropentonic acid

Finally, a comparisonof the properties of the phenylhydrazides of 3-deoxyD-threo-pentonic acid (from D-xylose) and 3-deoxy-~-threo-pentonic acid (from L-arabinose) showed these two acids to be enantiomorphs. c. Configuration.-Much of the evidence quoted above as proof of the structure of the 3-deoxypentonic acids is also applicable to the establishing of their respective configurations. Nef’s theory of the mechanism of formn(16) H. Kiliani and 0. Matthes, Bey., 40,1238 (1907).

43

THE SACCHARINIC ACIDS

tion of the saccharinic acids led to the conclusion that, in the transformation of a pentose or higher sugar into its related metasaccharinic acid, the configuration would not be disturbed beyond C3. The currently accepted modification of Nef’s theory does not alter this conclusion, and the retention of configuration a t C4 in the formation of metasaccharinic acid has been confirmed experimentally in the hexose series (see page 61). Thus, for example, the two 3-deoxypentonic acids obtained from D-xylose have the D-threo and D-erythro configurations, respectively, and the acid possessing the latter configuration provides meso-2,4-dihydroxyglutaric acid on oxidation. It should be noted that this assignment of configuration t o the 3-deoxypentonic acids, taken in conjunction with the evidence cited in the preceding Section, also confirms the D configuration for the penultimate, secondary carbon atom of L‘Q”-D-isosaccharinicacid. The directions of the optical rotations of the lactones (presumably gamma lactones) and phenylhydrazides of the four 3-deoxypentonic acids are in agreement with those predicted, on the basis of the assigned configurations, by the lactonell and phenylhydrazide18 rules. 5. “~”-D-GZucosaccharin~c Acid COzH

I I HCOH I HCOH I

CHZ-COH

CHZOH

2-C-Methyl-~-ribo(?)-pentonic acid a. Preparation.-The lactone of this saccharinic acid is prepared most conveniently by the action of calcium hydroxide on D-glucose, D-fructose, or invert sugar. Peligotlg noted that D-fructose yields the lactone more readily than does D-glucose, and this result was confirmed by Scheibler.7 For preparative purposes, Kiliani preferred invert sugar, and an abstract of his directions20 based on this starting material follows, A cold solution of 1 kg. of inverted sucrose in 9 liters of water is treated with 100 g. of calcium hydroxide and allowed to stand in a stoppered flask with frequent shaking. Fourteen days later, an additional 400 g. of calcium hydroxide is added. After ~~

~

~

~

-

(17) C. S. Hudson, J. Am. Chem. SOC.,32,338 (1910). (18) P . A. Levene, J . Biol. Chem., 23, 145 (1915); P.A. Levene and G. M. Meyer, ibid., 31,623 (1917) ; C. S. Hudson, J. Am. Chem. SOC.,39,462 (1917). (19) E. Peligot, Compt. rend., 90, 1141 (1880). (20) H. Kiliani, Ber., 16, 2953 (1882).

44

JOHN C. SOWDEN

one to two months, with occasional shaking, the solution reduces Fehling reagent only slightly. The mixture is filtered, the filtrate is saturated with carbon dioxide, and the dissolved calcium ions are then precipitated by the addition of an exactly equivalent amount of oxalic acid. After filtration, the solution is concentrated to a thin sirup and allowed to crystallize in the cold. When the crystallization is complete (several days), the mother liquors, from which no appreciable further amount of the lactone can be obtained, are drained from the crystals, and the latter are recrystallized from water. The yield is approximately 100 g. of pure “oc”-D-glucosaccharinic lact one.

Scheibler’ describes a similar preparation of the lactone, except that the long period of standing a t room temperature is replaced by several hours at 100”. However, KilianiZ0states that the yield obtained by this rapid method is unsatisfactory. b. Structure.-In the belief that “a”-D-glucosaccharhic acid possessed a straight, carbon chain, Scheibler? reduced its lactone with hydriodic acid and red phosphorus in an attempt to obtain n-hexanoic acid. He obtained instead, however, a neutral oil of b. p. 203-204’ which he assumed to be the lactone of a hydroxyhexanoic acid. The presence of a methyl group and of a branched, carbon chain in “a))-D-glucosaccharinicacid was established by Kiliani.2°sI1 Oxidation of “a”-D-glucosaccharinic lactone with silver oxidez2gave a mixture of acids

o=c-

I

COzH

I

I

I/CHaI

CHaCOiH

+ CHaOH

I

C02H

I

/CHa C

I\oH

HNOa ____,

A&O

I

CHOH

CHOH

CHO

CHOH

I

CHz OH “a”-D-GlucostLccharinic lactone

I I

COnH Saccharonic acid

1

AgzO but no

(21) H. Kiliani, Ber., 16, 701 (1882). (22) For a more recent study of the oxidizing action of silver oxide on the sugars, see K. G. A. Bwch, J. W. Clark, L. B. Genung, E. F. Schroeder and W. L. Evans, J . Org. Chern., 1, 1 (1936-37).

45

THE BACCHARINIC ACIDS

including formic, glycolic, and acetic, the last indicating the presence of a methyl group in the original lactone. Oxidation of ‘‘a”-D-glucosaccharinic lactone with nitric acid provided in high yield a crystalline monolactone (saccharon, C6HsO6) of a dibasic acid (saccharonic acid, CaHloO?).Oxidation of this dibasic acid with silver oxide yielded acetic acid but no glycolic acid, indicating that, in the original oxidation with nitric acid (“a”-D-ghcosaccharinic lactone + saccharon), a hydroxymethyl group had been oxidized to a carboxyl group. Thus, it was shown that “Cu”-D-glucosaccharinic acid contains the groups -CHI, -CHzOH, and -C02H, and so must possess a branched, carbon chain. The disposition of the functional groups in “a”-D-glucosacCharinic acid was also established by Kiliani.23Reduction of saccharon with hydriodic acid and red phosphorus gave the known:* crystalline ~~-(2-methylglutaric

o=c

CHI

I

CHa-CH--CH*-CH-PO

I

0

I

C

I CHa-CHz-CH2-CH-COzH DL-(2-Methylvaleric acid)

C H O l

I

CHzOH I < 01 1 9 -D-Glucosaccharinic lactone

C I

~~-(Z-MethyIgIutaric acid)

+

HI P

CHI

CHOH

I I

CHOH

I

HOz C-CH=CH-CH-C OIH ~~-(4-Methylglutaconic acid)

COzH Saccharonic acid ~

(23) H. Kiliani, Ann., 216, 361 (1883). (24) J. Wislicenus and L. Limpach, Ann., 192,128 (1878).

46

JOHN C. SOWDEN

acid). A byproduct of the reduction was a crystalline, unsaturated, dibasic acid which later was recognized as ~~-(4-methylglutaconic acid) .26 Thus, the position of attachment of the methyl group in L‘a”-D-glucosaccharinic acid was restricted to one of the penultimate carbon atoms of a pentonic acid carbon chain. A repetition of Scheibler’s reduction’ of “~”-D-glUCOsaccharinic lactone then showed that the neutral product obtained was similar in its properties to known26~~-(2-methylvalero-l ,4-lactone). Accordingly, L‘a”-D-ghcosaccharinicacid must be a 2-C-methylpentonic acid. Confirmation of this structure for “a”-D-glucosaccharinic acid was obtained by Liebermann and Scheibler27when they demonstrated that the known2* ~~-(2-methylvaleric acid) [DL-(methylpropylacetic acid)] was also formed, in low yield, in the reduction of “a”-D-glucosacchariniclactone to the 2-methylvalero-1,4-lactone. Moreover, under more strenuous conditions of reduction with hydriodic acid and red phosphorus (in a sealed tube a t 200°), the ~~-(2-methylvaleric acid) is the principal product. c. Con$guration.-The coilfiguration of “a”-D-glucosaccharinic acid is not known with certainty, although the D-rib0 arrangement is indicated by the evidence so far accumulated. In his theory of the mechanism of saccharinic acid formation. Nefl2,29 assumed that this acid is produced from D-glucose by changes involving only the first three carbon atoms of the sugar and, hence, that the D-erythro configuration is retained in the two lowest asymmetric carbon atoms of the saccharinic acid. This latter contention has not been changed by subsequent modifications of the Nef mechanism (see Section 111) which are currently used for explaining saccharinic acid formation. Supporting evidence for the D classification of “a”-D-glucosaccharinic acid is found in the optical rotation of its lactone. “a”-D-Glucosaccharinic lactone, in view of its marked stability in water or aqueous acids, is almost certainly a gamma lactone. Thus, as pointed out by VotoEek,3° its positive optical rotation indicates the D configuration for C4 on the basis of the lactone rule.” If the two lowest asymmetric carbon atoms possess the D-erythro configuration, ‘(a”-D-glucosaccharinic acid must be either 2-C-methyl-~arabino-pentonic acid or 2-C-methyl-~-ribo-pentonicacid. The initial choice between these two possibilities was made by Nef,12who chose the D-arabino configuration because of similarities between certain alkaloid salts of the saccharinic acid and the corresponding salts of D-arabinonic acid. However, subsequent developments make the D-rib0 configuration appear the more (25) M. Conrad and M. Gutzheit, Ann., 222,249 (1884); see Ref. 60. (26) R. Fittig and L.Gottstein, Ann., 216, 26 (1883). (27) C . Liebermann and C. Scheibler, BeT., 16, 1821 (1883). (28) A. Saytzeff, Ann., 193,349 (1878). (29) J. U.Nef, Ann., 367, 301 (1907);403, 204 (1914). (30) E. VotoEek, Collection Czechoslov. Chem. Communs., 2 , 158 (1930).

THE SACCHARINIC ACIDS

47

probable. The phenylhydrazide of LLa”-D-ghcosaccharinicacid is strongly dextrorotatory ([a]: +50.3°),12 and the phenylhydrazide rule’s would thus assign the D configuration to the tertiary carbon atom. Obviously, such assignment involves the assumption that replacement of hydrogen by methyl on the a-carbon atom does not invalidate the phenylhydrazide rule. Evidence in support of the presence of a cis arrangement of the hydroxyl groups on C2 and C3 of the ‘La”-D-ghcosacchariniclactone ring is provided , of the lactone for three by its behavior upon a c e t o n a t i ~ n .32~ ~Treatment hours at room temperature with a 2% solution of sulfuric acid in acetone gives a crystalline monoisopropylidene compound [m. p., 62-63’, [a]% -38.4’ (in chloroform)] in 83 % yield. This derivative, after titration with sodium hydroxide to open the lactone ring, consumes one molecular equivalent of sodium metaperiodate, with the production of formaldehyde, and is, lactone. therefore, 2,3-0-isopropylidene-“a”-~-glucosaccharinic The epimer of the known “a”-D-glucosaccharinic acid has not been detected among the products of the D-hexose-alkali reaction, although its concurrent formation is to be expected. d. Miscellaneous Reactions.-Two obviously attractive reactions of ‘(a”D-ghcosaccharinic acid would be its reduction to the corresponding 2-Cmethylaldopentose and its degradation to a 1-deoxy-2-ketopentose. The reduction of “a”-D-glucosaccharinic lactone with sodium amalgam was investigated by S ~ h e i b l e rwho , ~ ~ reported briefly that hydrogen is absorbed by the lactone under these conditions. Subsequently, F i ~ c h e noted r~~ that, if the reduction is carried out at or near neutrality, the product is a reducing sugar. The reduction with sodium amalgam was repeated by VotoEek?O but his attempts to prepare a crystalline hydrazone of the amorphous product with phenylhydrazine, p-bromophenylhydrazine, or 1methyl-1-phenylhydraainewere unsuccessful. However, application of the cyanohydrin synthesis to the sirupy sugar provided what was presumably a mixture of 3-C-methylaldohexoses, from which a crystalline phenylosazone and a crystalline p-bromophenylosazone were obtained. The successful degradation of ‘La”-D-glucosaccharinicacid to a l-deoxypentulose has apparently not been recorded. Experiments in the author’s laboratory have indicated that the oxidation of calcium (‘a”-D-glucoSaccharinate with hydrogen peroxide and ferric acetate (the Ruff degradation) proceeds normally to yield a reducing product. However, no crystalline derivative of the expected deoxypentulose has been obtained as yet. (31) Dorothy J. Kuenne, Ph.D. Dissertation, Washington University, St. Louis, (1953). (32) L. M. Utkin and G. 0. Grabilina, Doklady Akad. Nauk S. 8. S. R., 93, 301 (1953); Chem. Abstracts, 48, 12676 (1954). (33) C. Scheibler, Bes., 18,3010 (1883). (34) E. Fischer, Ber., 22, 2204 (1889).

48

JOHN C. SOWDEN

6. “a”-D-IsoSaccharinic Acid COzH

c I‘oa CHz

I I

HCOH CH2OH 3-Deoxy-2-C-(hydroxymethyl)-(~-erythro or D-threo)-pentonic acid

Shortly after the discovery of Peligot’s “a”-D-glucosaccharin, Dubrunf a u P reported that the calcium salt of a monobasic acid resulted from the action of lime-water on maltose. CuisinieP named the acid isosaccharinic acid, after he had prepared from it a crystalline lactone (CeHloOs) isomeric with Peligot’s (‘a”-D-glucosaccharin.The name was expanded t o (‘a’’-D-isosaccharinic acid after Nef12 obtained evidence of the concurrent formation of its epimer, “p”-D-isosaccharinic acid, in the hexose-alkali reaction. a. Preparatim.--“a”-D-Isosaccharinic lactone is obtained in a 15 to 20 % weight-yield by the action of lime-water on malt0se,~6lactose,8eor cellobiose?’ Somewhat lower yields (4to 12 % by weight) can be obtained from partially degraded cellulose plus lime-water.a8 Relatively little “a”-D-isosaccharinic acid is formed in the reaction of D-glucose or D-galactose with hot 8 N sodium hydroxide.’z The efficacy of the (1+4)-linkeddisaccharides in producing the isosaccharinic acid is discussed in Section 111. Lactose is the most convenient source of “a”-D-kosaccharinic acid and Kiliani’s directionsa@ based on this disaccharide follow. Lactose (1 kg.) in 9 liters of water is treated with 200 g. of calcium oxide (slaked and cooled), and the resulting mixture is maintained in a stoppered flask a t room temperature, with frequent shaking, for 3 days. The solution is then heated in a boiling-water bath for 10 hours, filtered, and evaporated to a volume of 3 liters. The highly insoluble calcium “UJ’-D-isosaccharinate(199g.) crystallizes; i t is accompanied by a small amount (14g.) of calcium carbonate. The salt is separated by filtration and

(35) A. P. Dubrunfaut, Monit. sci. Docteur Quesneville, [3]12, 520 (1882). (36) L. Cuisinier, Monit. en’. Docteur Quesneville, [3]12,521 (1882);Bull. S O C . chim. (France), [2]38, 512 (1882). (37) S. V. Hintikka, Ann. Acad. 81%.Fennicae, Ser. A , ZZ, N o . 9 (1922);Chem. Abstracts, 17, 3486 (1923). (38) 0.von Faber and B. Tollens, Ber., 32, 2589 (1899);J. J. Murumow, J. Sack and B. Tollens, ibid., 34,1427 (1901);C. G.Schwalbe and E. Becker, J . prakt. Chem., [2]100, 19 (1920).J. Palm&, Finska Kemietsamfundets Medd., 38, 108 (1929); Chem. Abstracts, 24, 1625 (1930). (39) 11. Kiliani, Ber., 42, 3903 (1909).

49

THE SACCHARINIC ACIDS

is then heated with a solution of an equivalent amount of oxalic acid. Filtration of the calcium oxalate, followed by concentration of the filtrate to a sirup, affords the readily crystallizable “cr”-D-iaosaccharinic lactone.

b. Structure.-Soon after the discovery of “a”-D-isosacchar~icacid, Kiliani applied to i t 4 0 the same methods he had used previously to establish the structure of “a”-D-glucosaCcharinic acid (see page 44). The reduction of L1a”-D-isosacchariniclactone (CeHloOs) with hydriodic acid and red phosphorus at atmospheric pressure yielded, as had the similar reduction of “a”-D-glucosaccharinic lactone, a 2-methylvalero-1 4-lactone. When the reduction was carried out at higher temperatures in a sealed tube, the prodacid), which also had been obtained similarly uct was ~~-(2-methylvaleric from “a”-D-glucosaccharinic lactone. However, “a”-D-isoSaccharinic lactone, on oxidation with silver oxide, yielded (in contrast to the behavior of “a11-D-glucosaccharinic lactone) no acetic acid. Thus, a methyl group is not present in “a”-D-isosaccharinic acid, and, in view of the formation of the CHs

I

CHa-CH-CHz-CH-C=O

I

HI

AgzO

[no CHsC02Hl

CeHia06 P ‘1 a J 9 -~-Isoaaecharinic

I

2-Methylvalero-l,4-lactone

+ CHa

lactone

CH~-CH~-CH~-~H-CO~H DL- (2-Methylvaleric acid)

2-methylvalero-l , 4-lactone on reduction, it must possess one of two alternate structures (I or 11). COzH I

I/ I

CHzOH

C

COzH I CHzOH C

‘OH

I

CHa

I

CHOH

I

CHzOH I 3-Deoxy-2-C-(hydroxymethy1)pentonic acid

(40)H. Kiliani, Ber., 18,631 (1885).

I

CHOH

I I

CHOH CHzOH

I1 2-Deoxy-2-C-(hydroxymethy1)pentonic acid

50

JOHN C. SOWDEN

In agreement with these postulated alternate structures (I or II), it was observed that oxidation of “cr”-D-isosaccharinic lactone with nitric acid yields a tribasic acid, COHsOs Furthermore, the tribasic acid readily loses a molecule of carbon dioxide when warmed to loo”, a behavior consistent with the presence of two carboxyl groups on a single carbon atom. The product initially isolated by Kiliani41from this decarboxylation was an optically inactive dihydroxyglutaric acid. This latter acid was found to differ in properties from a Zf3-dihydroxyglutaric acid obtained by the successive bromination and hydrolysis of glutaconic acid. Accordingly, Kiliani concluded that the dibasic acid (obtained by oxidation, followed by decarboxyl-

.

COzH

COZH

COSH

COaH

CHOH

CHOH

CH

CHOH

I

CHzOH “a”-o-Isosaccharinic acid

I COzH

I

COzH Dihydroxy- Glutaconic glutaric acid acid (m. p., 106” +)

I

C02H 2,3-Dihydroxyglutaric acid (m. p., 155-156”)

ation, of “Cr”-D-isosaccharinic lactone) must be a 2,4-dihydroxyglutaric acid and, hence, that “a”-D-isosaccharinic acid is a 3-deoxy-2-C-(hydroxymethy1)pentonic acid (I). The above reasoning, based on the dihydroxyglutaric acids, is fallacious, as was recognized subsequently by Kiliani and Herold,42since in no event could the same properties be expected for the dihydroxyglutaric acids obtained, respectively, from “Cr”-D-isosacchariniclactone and from glutaconic acid. Glutaconic acid, on bromination followed by hydrolysis, would yield a mixture of the two possible racemates of 2,3-dihydroxyglutaric acid. In contrast, structure I1 for “cY”-D-isosaccharinic acid would provide, on oxidation and decarboxylation, a single, enantiomorphous 2 , S-dihydroxyglutaric acid. Finally, structure I for “a”-D-isosacchari~cacid would lead to a mixture of diastereoisomeric 2,4-dihydroxyglutaric acids, one of which would be asymmetric and the other meso. The correctness of structure I for “a”-D-isosaccharhic acid was even’ tually confirmed by Kiliani and Matthe@ when they isolated from the oxidation and decarboxylation, not only the previously obtained meso-dihydroxyglutaric acid, but also the accompanying, optically active isomer (41) H. Kiliani, Ber., 18,2514 (1885). (42) H. Kiliani and F. Herold, Ber., 38, 2671 (1905).

[:"

51

THE SACCHARINIC ACIDS

1

COzI[

O

H

O

H

CHa OH CHzOH

,CHIOH ,,CHZOH

I'

COzH

HCOH

C

HOCH

C

CHz

CHOH

CHOH

\

- COZ

CHz

I CHO H I

CHZOH

I (Enantiomorph)

CHz

I

CHOH

I

COzH

CH~

1 I CHOH CHOH I I

COzH

CHzOH

I

CHOH

I

CO,H

2,4I1 2,3Dihydroxyglutaric (Enantiomorph) Dihydroxyglutaric acids (one meso, one acid enantiomorph) (enantiomorph)

of this acid. Meanwhile, had also established the presence of a hydroxyl group on the tertiary carbon atom of “a”-D-isosaccharinic acid, by degrading the latter with hydrogen peroxide and ferric acetate to a deoxypentose (CbH1004). Thus, the structure of “a))-D-iSosaccharinic acid is established beyond question as that of a 3-deoxy-2-C-(hydroxymethyl)pentonic acid. c. Configuration.-The D configuration may be assigned to C4, the penultimate secondary carbon atom of “a))-D-isosaccharink acid, from several considerations. The currently accepted mechanism for the formation of this acid from the (1 -+4)-linked disaccharides (see Section 111) involves no change in configuration at C4 (C5 of the original D-glucose moiety of the disaccharide). Moreover, the configuration of this carbon atom has been experimentally related to that of C4 of the 3-deoxy-~-pentonicacids (see page 42). Finally, the positive optical rotation of “a’)-D-isosaccharinic lactone, presumably a gamma lactone, assigns the D configuration for C4 on the basis of the lactone rule.’’ The configuration of C2, the tertiary carbon atom, of “a))-D-isosaccharinic acid has not been established. Unfortunately, application of qualitative rules of configuration based on optical rotation affords disagreeing conclusions in this instance. The positive optical rotation of the phenylhydrazide would indicate the D configuration for C2 on the basis of the phenylhydraBide rule.’s On the other hand, the reported negative optical rotation of the acid amide44would assign the L configuration to this carbon at,om on the (43) 0.Ruff, Ber., 36, 2360 (1902). (44) R. A. Weerman, Rec. trav. chim., 37, 16 (1917).

52

JOHN C. SOWDEN

basis of the amide The amide is, however, reported to be unstable, and assignment of configuration on the basis of the available data for this compound may be unreliable. The anilide, in contrast to the amide, shows a positive optical rotation. d. Miscellaneous Reactions.-The reduction of “a”-D-isosaccharink lactone with sodium amalgam gives a sirupy product from which a crystalline p-nitrophenylhydrazone of the branched-chain sugar, 3-deoxy-2-C-(hydroxymethy1)-(D-erythroor D-threo)-aldopentose, can be ~btained.~” Acetylation of the sirupy sugar yields a mixture of the crystalline, anomeric triacetates; the tertiary hydroxyl group is presumably inert toward acetylation, as is the similar tertiary hydroxyl group of methyl hamameloside.47 The Ruff d e g r a d a t i ~ nof~ ~L‘a”-D-isosaccharinicacid to a deoxypentose has been mentioned above. It is interesting that the soluble, lead salt of the acid was used for the degradation instead of the more usual calcium salt which, in this instance, is only very slightly soluble in water. Although Ruff was able to obtain the crystalline benzylphenylhydrazone of the pentose, the yield was so low that cleavage of this hydrazone to the pure sugar could not be studied. The reaction invites repetition and improvement in view of the rare nature of the product, a 3-deoxy-2-pentulose (3-deoxy-~-glyceropentulose). e. “P”-D-Isosaccharin~cAcid.-During the recrystallization (from ethanol) of the brucine salt of 2,4-dihydroxybutryic acid, obtained from the hexose - alkali reaction, Nef4*always observed the presence of a small amount of a less-soluble brucine salt. He concluded that this latter product was a mixture of the brucine salts of “a”-D-isosaccharhic acid and its epimer. After fractional recrystallization of the mixed salts (22 g.) to remove brucine “a”-D-isosaccharinate, he isolated a minor amount (0.6 g.) of a sirupy lactone that still, however, contained about 10% of “cY”-Disosaccharinic lactone. The principal constituent of the lactone mixture yielded brucine, quinine, and calcium salts, as well as a phenylhydrazide (no optical rotation for which was given), all of which were quite different in properties from the corresponding derivatives of “a”-D-isosacch&rinic acid. The various derivatives of the sirupy lactone were, however, similar to those of the corresponding derivatives of 3-deoxy-~-erythro-pentonic acid (from D-xylose plus alkali). Accordingly, Nef concluded that he must have in hand a 3-deoxy-2-C-(hydroxymethyl)pentonic acid, the epimer of ( I 1) a -D-isosaccharinic acid. (45) C. S. Hudson, J . Am. Chem. Soc., 40, 813 (1918). (46) P. Schorigin and N. N. Makarowa-Semljanskaja, Ber., 66,387 (1933). (47) 0. T . Schmidt, Ann., 476,250 (1929); see F. Shafizadeh, Advances i n Carbohydrate Chem., 11, 270 (1956). (48) Ref. 12, pp. 56-58 and pp. 64-65.

53

THE SACCHARINIC ACIDS

In support of his contention that “/3”-D-isosaccharinic acid is present in the hexose-alkali reaction mixture, Nef also cited certain observations of Kiliani arid Ei~enlohr,4~* 6o who oxidized (with nitric acid) the residue obtained, after substantial removal of LLa”-D-isosaccharinic acid and the metasaccharinic acids, from the lactose-alkali reaction mixture. Among the products identified was the tribasic acid, (H02C)zC(OH)-CH2-CHOHC0211, previously obtained by a similar oxidation of “a”-D-isosaccharinic acid (see page 50). Nef concluded that the tribasic acid must in this instance have arisen from “/3”-D-isosaccharinic acid. This conclusion ignores, however, the experimental demonstration by Kiliani and Eisenlohr60 that the residue subjected to oxidation had still contained a small proportion of ‘(a”-D-isosaccharinicacid, isolable as the slightly soluble calcium salt. The best evidence for the formation of L‘/3J1-D-isosaccharinic acid in the sugar-alkali reaction is the recent observation61that treatment of lactose, maltose, or 4-O-methyl-~-glucosewith lime-water at room temperature provides initially a mixture of saccharinic acids consisting almost exclusively of “a”-D-isosaccharinic acid plus an acid with the properties of Nef’s “/3’1-D-isosaccharinicacid [brucine salt, m. p. 185 to 210’ (dec.), [a]: -20 to -22O; lactone, [a]%+6 to +8.5O]. An experimental proof that this substance possesses the isosaccharinic acid structure would provide the necessary evidence that it is, indeed, the epimer of “a”-D-isosaccharinic acid.

7. The D-Galactometasaccharinic Acids COZH

I

HCOH

I

COzH

I I

HOCH

CHz

CHz

HOCH

HOCH

I

I I

HCOH CHzOH 3-Deoxy-~-xy~o-hexonic acid (“d-D-galactometasaccharinic acid)

I

I I

HCOH CHeOH 3-Deoxy-~-lyxo-hexonicacid (“8”-D-galactometasaccharinic acid)

3-Deoxy-~-x&1-hexonicacid (“a”-D-galactometasaccharinic acid) was first detected as a product of the prolonged action of lime-water on lactose (49) H. Kiliani, Ber., 41, 2650 (1908). (50) H. Kiliani and F. Eisenlohr, Ber., 42, 2603 (1909). (51) W.M.Corbett and J. Kenner, J . Chem. Sac., 2245 (1953); 1789 (1954); J. Kenner and G. N. Richards, ibid., 1810 (1955).

54

JOHN C. SOWDEN

at room temperature. After having removed the very slightly soluble calcium L‘a’’-D-isosaccharinatefrom one of these reaction mixtures, IGliani52 noted the slow deposition of a second calcium salt. This latter material could be recrystallized from hot water; it yielded, after removal of the calcium, a crystalline lactone with the familiar formula, CsH,,05 , of a sixcarbon saccharin. It was recognized later that the initial action of liinewater on lactose yields ‘L~ll-D-isosaccharinic acid and D-galactose, with ensuing conversion of the hexose to the epimeric D-galactometasaccharinic acids. 3-Deoxy-~-lyxo-hexonic acid (“p”-D-galactometasaccharinic acid) was discovered by Kiliani and Sandas3as a minor product of the D-galactose-alkali reaction. Kiliani believed that this product was a new type of branchedchain saccharinic acid, and referred to it throughout subsequent publications as L‘parasaccharinic”acid. The evidence, provided both by his own work and that of Nef, that Kiliani’s “parasaccharinic” acid contained, in fact, the epimer of “rY1’-D-galaetometasaccharinicacid, is outlined on page 56. a. Preparation.-The epimeric D-galactometasaccharinic acids are produced concurrently, in yields of 15 to 20 %, by the action either of hot, concentrated sodium hydroxide12 or of lime-water a t room temperature 011 D-galactose. The xylo epimer apparently predominates in the mixture; it can be readily isolated in pure form through its slightly soluble calcium salt. The lyxo epimer is, however, extremely difficult to purify by recrystallization because of its tendency to form mixed salts with those of the xylo epimer. Kiliani and Sandals directions63 for the preparation of “cr1’-D-ga1aCtOmetasaccharinic acid follow. A solution of one part of D-galactose in ten parts of water is treated with half a part of freshly prepared calcium hydroxide. The mixture is maintained a t room temperature in a stoppered flask for 4 weeks, with initial frequent shaking. The resulting voluminous precipitate is removed by filtration and the filtrate is heated t o boiling, while being maintained a t constant volume, for 3 hours. The new precipitate (of basic calcium salts) is then removed and the filtrate is saturated with carbon dioxide. The solution is again heated, filtered, and concentrated t o about twice the weight of the original D-galactose. After seeding with calcium “a”-D-galactometasaccharinate, if seeding crystals are available, the crystallization of this salt is completed by storing in the cold for about 10 days. The yield is about 14% of the weight of sugar used initially.

From the mother liquors of preparations similar t o the above, Kiliani and coworkerss3,s 4 , 5s isolated a crystalline barium salt of the mixed, (52) (53) (54) (55)

H. Kiliani, Ber., 16, 2625 (1883). H. Kiliani and H. Sanda, Ber., 26, 1649 (1893). H. Kiliani and P. Loeffler, Ber., 37, 1196 (1904). H. Kiliani and H. Naegell, Ber., 36, 3528 (1902).

55

THE SACCHARINIC ACIDS

epimoric D-galactometasaccharinic acids. Their preparations of the lyxo epimer (“parasaccharin”) were obtained from this mixed salt by conversion to the mixed lactones and removal, through crystallization, of the xylo epimer. In some instanceslb4~ 66 they briefly record the crystallization of the lyxo epimer (“@”-D-galactometasaccharin). Nef b7 also isolated the crystalline ‘(@” epimer from the D-galactose-sodium hydroxide reaction. His directions include “protracted” fractional recrystallization of crude brucine salts, followed by successive fractional recrystallizations of the strychnine and barium salts. It appears certain that chromatographic and ion-exchange methods, not known to Kiliani and Nef, could be used to advantage in future preparations of “@”-D-galactometasaccharinicacid. b. Structure.-(1) “a”-D-GalactometaSacchar~n~c Acid.-Kilianib8 observed that reduction of “a”-D-galactometasaccharink? acid with hydriodic acid and red phosphorus, under reflux at atmospheric pressure, yielded n-hexanoic 1,li-lactone. Further reduction, at higher temperature in a sealed tube, gave a low yield of n-hexanoic acid. Thus a straight-chain structure, with a hydroxyl group gamma to the carboxyl group, was established for the

o=cAI H T I AI H T I

n-Hexanoic 1,4-1actone

COeH

COzH

CHz CHZ I

CHOH

n-Hexanoic acid

“a’l-D-Galactometasaccharinic acid

I

1 I

I IHNOa

C OaH

COzH

I

I

cHoH

I

CHz CHOH

- I

I I

(56) H. Kiliani, Ber., 44, 109 (1911). (57) Ref. 12, pp. 62-66 and 76-77. (58) H. Kiliani, Ber., 18, 642 (1885).

CHz CHI

I I

CHOH

CHz

COzH

COzH

Trihydroxyadipic acid

Adipic acid

56

JOHN C. SOWDEN

metasaccharinic acid. Oxidation of the latter with nitric 58 led to a crystalline trihydroxyadipic acid which, on reduction with hydriodic acid and red phosphorus, was converted to the known, crystalline adipic acid. These latter observations established a non-terminal position for the deoxy function in “a”-D-galactometasaccharinic acid. A Ruff degradation, with hydrogen peroxide and ferric acetate, of the calciumss or barium60 salts of “a”-D-gdactometasaccharinic acid provided a crystalline deoxypentose (C5HI004, Limetasaccharopentose”)which failed t o give an osazone on treatment with phenylhydrazine. Oxidation of the deoxypentose with bromine yielded a trihydroxyvaleric acid which, upon lactonization and then reduction with hydriodic acid and red phosphorus,60 gave a n-valero-1 ,$-lactone. The silver salt of the corresponding acid was found to be crystallographically identical with the known silver 4-hydroxyn-valerate. COzH

I

CHO

CHOH

1

CHz

I I CHOH I CHOH

CHzOH I t (z

H2 0

2

-F e w

-D-

Galactometasaccharinic acid

I CHz I

CHOH

I I

o=c-

COzH

I

--Brz -+

CHOH CHzOH 2-Deoxypentose

I I CHOH I

CHOH

CHzOH

Trihydroxyvaleric acid

I I CH2 I CHOI CH2

CH2

HI

CHa

n-Valero-l,4lactone

Considered together, the above observations provided evidence for the presence of hydroxyl groups on C2, C4, C5, and C6 of “a”-D-galactometasaccharinic acid, and, hence, for its formulation as a 3-deoxyhexonic acid. (2) “p”-D-GuZactometusucchur~n~c Acid.-Kiliani and Sanda53 reduced their “parasaccharinic acid” in the usual manner with hydriodic acid and red phosphorus. The product was a hexanoic lactone whose boiling point (217.5’) was precisely intermediate between that (220’) of the n-hexanoic 1,4-1actoneobtained by a similar reduction of “a”-D-galactornetasaccharinic acid and that (215”) reported6I for 2-ethylbutyro-l , 4-lactone. Kiliani, however, chose the latter structure for his lactone, since the corresponding acid, acid) and unlike ejther the enantiomorlike ~~-(2-ethyl-4-hydroxybutyric (59) H. Kiliani, Ber., 18, 1555 (1885). (60) H. Kiliani and P. Loeffler, Ber., 38, 2667 (1905). (61) M. B. Chanlaroff, Ann., 226,340 (1884).

57

THE SACCEIARINIC ACIDS

phous62or racemic 4-hydroxy-n-hexanoic gave a readily crystalline barium salt. On the basis of this identification, “parasaccharinic acid’’ was assigned one of the three structures I, 11, or 111. CHa

CHzOH

CH2 OH

CHOH

CHOH

CH2

I

I

I

I

I

CHOH

I

CHzOH I

CHOH

I

CHZOH I1

I

CH2 OH I11 (“Parasaccharinic acid”)

Structure I was quickly ruled out by the observation that the saccharinic acid contains no methyl group, since it gives no acetic acid on oAdation with silver oxide. Moreover, a Ruff degradationK4of the “parasaccharinic acid” gave a crystalline “parasaccharopentose” (CsHlo04) and, hence, it was concluded that structure 111 shows the correct disposition of the functional groups. As a further observation in support of the branchedchain structure, Kiliani and LoeffleP4 reported that oxidation of the saccharinic acid by nitric acid yields a tribasic acid (presumably a hydroxycitric acid) accompanied by the crystalline monolactone (CeHaOs) of a dibasic acid. Evidence that “parasaccharinic acid” probably contains “p”-D-galactometasaccharinic acid (or its unremoved “a” epimer) was soon forthcoming from Kiliani’s own laboratory. Crystallographic comparison of “parasaccharopentose” with “metasaccharopentose,” of their respective crystalline oximes, and of the phenylhydrazides of their derived deoxypentonic acids, showed the two sugars to be identical.6STo bring this observation into conformity with his proposed branched-chain structure for “parasaccharinic acid,” Kiliani suggested that the latter gives the expected 2-deoxy-3pentulose in the Ruff degradation but that this deoxypentulose structure is unstable and rearranges spontaneously to the 2-deoxypentose, “metasaccharopentose” (IV + V). (62) H. Kiliani and S. Kleemann, Ber., 17,1296 (1884). (63) R. Fittig and E. Hjelt, Ann., 208, 67 (1881). (64) H. Kiliani and P. Loeffler, Ber., 37, 3612 (1904). (65) H. Kiliani and A. Sautermeister, Bey., 40, 4294 (1907); H. Kiliani, ibid., 41, 120 (1908).

58

JOHN C. SOWDEN

CHO

CH~OH’

CHOH

I

CHzOH “Parasaccharinic acid”

I CHz I c=o I CHOH I

CH2OH.

I I CHOH I CHOH I CHz

+

CHzOH

v IV Further serious doubt was cast on the branched-chain structure by NefG6 when he observed that “cr”-D-galactometasaccharink acid can be readily isomerized into a product that very closely resembles Kiliani’s “parasaccharinic acid.” On heating the pure “a” epimer in a sealed tube at 200°, with or without pyridine, it was partly converted to “P”-D-galactometasaccharinic acid, whose brucine salt showed properties in excellent agreement with th‘ose of Kiliani’s “brucine parasaccharinate.” The isomerization product was further characterized, through its strychnine salt and phenylhydrazide, as Nef’s “/3”-D-galactometasaccharinic acid, obtainable directly by the action of alkali on D-galactose. At this stage, the possible presence of a branched-chain saccharinic acid in Kiliani’s preparation was supported only by (a) the properties of the barium salt of the hydroxyhexanoic acid obtained from it on reduction and (b) the reported oxidation of “parasaccharinic acid” with nitric acid t o a tribasic acid. The latter evidence was retracted by Kiliani in his final report on the matter,66when he stated that the previous identification of “hydroxycitric acid” was in error and that this “tribasic acid” is, in fact, (-)-tartaric acid. In addition, he now observed that oxidation of “parasaccharinic acid” with nitric acid, followed by reduction with hydriodic acid and red phosphorus, gives a low yield of adipic acid. The hypothesis of the existence of the branched-chain “parasaccharinic acid” now depended solely on the identity of the reduction product, hydroxyhexanoic acid. To support his previous contention that the barium salt of this acid is, indeed, barium 2-ethyl-4-hydroxybutyrate, Kiliani66 also prepared the calcium salt and found that it, too, closely resembled the corresponding salt of 2-ethyl-4-hydroxybutyric acid. As emphasized by Kiliani, neither the reductions with hydriodic acid and red phosphorus nor the oxidations with nitric acid proceed in good yield to single products. Accordingly, Kiliani remained firm in his conviction that his preparation, although it was apparently a mixture, nevertheless contained the branched(66) Ref. 12, pp. 78-82.

59

THE SACCHARINIC ACIDS

chain “parasaccharinic acid.” The accumulated evidence points overwhelmingly to the presence therein of “/3”-D-galactometasaccharinicacid. Whether or not Kiliani’s “parasaccharinic acid” is also formed in the hexose-alkali reaction is a matter requiring further study. c. Configuration.-The accepted theory of the mechanism of formation of metasaccharinic acids predicts, as mentioned previously, that no change in the configuration of the starting sugar will occur a t carbon atoms below C3. Accordingly, the galactometasaccharinic acids should have the D-threo configuration a t C4 and C5. Thus, “metasaccharopentose,” obtainable from either of the galactometasaccharinic acids by the Ruff degradation, should be 2-deoxy-~-threo-pentose.This sugar, prepared by the glycal rneth0d,~7 shows properties (m. p., and m. p. of the benzylphenylhydrazone) in close agreement with those of “metasaccharopentose.” The product from the glycal synthesis showed a final optical rotation which was slightly negative -2’ (in water)], whereas “metasaccharopentose” was reported64* e6 to be optically inactive. Hence, no decision concerning the D or L classification of the latter is available from these data. The galactometasaccharins are, however, gamma lactones, and both show negative optical rotations, thus permitting assignment of the L configuration to C4 of both on the basis of the lactone rule.’? Thus, “metasaccharopentose” must have the D-threo configuration, and the epimeric galactometasaccharinic acids are the 3-deoxy-~-xylo-and -D-lyxo-hexonic acids. Finally, on the basis of the phenylhydrazide rule,’* the “a” epimer is the 3-deoxy-~-xylo-hexonicacid. It is interesting that Nef assigned the correct configurations to the D-galactometasaccharinic acids, as well as t o the D-glucometasaccharinic acids, on the basis of analogies between the optical rotations of D-tartaric acid, the 2,4-dihydroxyglutaric acids (obtained by oxidation of the fivecarbon metasaccharinic acids), and the 2,3,5-trihydroxyadipic acids (obtained by oxidation of the six-carbon metasaccharinic acids).

8. The D-Glucometasaccharinic Acids COzH

I HCOH I

COzH

I I

HOCH

CH2

CHz

HCOH

HCOH

HCOH

HCOH

I

I

I

CHzOH 3-Deoxy-~-ribo-hexonicacid (“a”-~-glucometasaccharinicacid)

I I

CHzOII 3-~)eoxy-~-arabino-hexonic acid (“B”-n-glucometasaccharinic acid)

(67) P. A. Levene and T. Mori, J . Biol. Chem., 83, 813 (1929).

60

JOHN C. SOWDEN

The epimeric D-glucometasaccharinic acids were first isolated by NefL2 from the interaction of D-glucose and hot, concentrated sodium hydroxide. D-Glucose is isomerized and smoothly degraded under these conditions to a mixture of saccharinic acids, in a yield of over 80 %. a. Preparation.-From the isomerization of 100 g. of D-glucose with hot 8 N sodium hydroxide, Nef reported as products, after careful fractionation, 40 to 45 g. of m-lactic acid, 10 to 15 g. of ~~-(2-hydroxybutyro-l,4lactone), 20 g. of the epimeric D-glucometasaccharinic lactones, and 2 g. of the epimeric D-isosaccharinic lactones. The six-carbon lactones, consisting almost entirely of the D-glueometasaccharhie lactones, were separated with relative ease from the products of lower molecular weight. Accordingly, D-glucose is an attractive source for these metasaccharins. The “/3” epimer (3-deoxy-~-arabino-hexonic acid) is readily isolable in pure form through its calcium salt, which is sparingly soluble in cold water. The “a” epimer (3-deoxy-~-ribo-hexonicacid), however, is relatively difficult to separate from the mixture. Here again, it is probable that chromatographic or ion-exchange methods may serve to good advantage. A recently developed methodE8for preparing the epimeric D-glucometasaccharinic acids is based on the action of lime-water on the seaweed polysaccharide, l a m i n a r i ~ The ~ . ~ ~mixed D-glucometasaccharinic acids are obtainable from this source in practically pure condition, as their calcium salts, after separation from unchanged polysaccharide. The directions for their preparation from “insoluble” laminarin follow. “Insoluble” laminarin (50 g.) is treated with an oxygen-free suspension of calcium hydroxide (509.) in 1 liter of water. After 8 days a t room temperature, the suspension is filtered, and calcium is precipitated by t h e addition of the equivalent amount of oxalic acid. Concentration of the filtrate to a volume of 500 ml. causes precipitation of polysaccharide (21.4 g.). After filtration, and concentration t o a sirup, extraction with ethanol (3 X 100 ml.) leaves further polysaccharide (7.7 g.). Evaporation of the ethanol extract affords a mixture of the sirupy D-glucometasaccharinie lactones (13.8 g.). After their conversion t o t h e calcium salts, and crystallization from water (finally with the gradual addition of ethanol), there is obtained calcium “j3”-D-glucometasaccharinate (5.8 g.), calcium “a”-D-ghcometasaccharinate (0.6 g.), and a residue of the mixed salts (3.7 g., principally “01” epimer). Partial, acid hydrolysis of the recovered polysaccharide, followed by retreatment with lime-water, yields an additional amount (11 g.) of the mixed calcium salts.

Similar treatment of “soluble” laminarin with lime-water, but at 100” for 3 hours, gives approximately the same yield of the calcium D-glucometasaccharinates. (68) W.M.Corbett and J. Kenner, J . Chem. SOC.,1431 (1955). (69) V. C. Barry, Sci. Proc. Roy. Dublin SOC., 21, 615 (1938);22, 59 (1939).

61

THE SACCBARINIC ACIDS

b. Structure.-Nef12 established the structural similarity of the D-glucometasaccharinic acids to the D-galactometasaccharinic acids by oxidizing the former to the two corresponding 2,3,5-trihydroxyadipic acids and converting these individually, by dehydration, to the lactone of 3-hydroxymuconic acid. The latter was also obtained, in a precisely similar fashion, from “a”-D-galactometasaccharinic acid. Thus, since Kiliani had previously established the structure of the galactometasaccharinic acid, Nef concluded that the D-glucometasaccharinic acids are also 3-deoxyhexonic acids. C OzH

COzH

CHZ

I I HCOH

I II

CHOH

CH

I

HNOa

A

HCOH

I

O=C-

I

I CHOH I

CHzOH o-Glucometasaccharinic acids

CHz

I HCOH I HCOH I

AcaO

CH

TiF

CO-

CO,H 2,3,5-Trihydroxyadipic acids

I II

CH

I

CO2H 3-Hydroxymuconic lactone

c. Configuration.-On the assumption that the D-erythro configuration had been retained at C4 and C5 in the conversion of D-glucose to the D-glucometasaccharinic acids, Nef assigned the D-rib0 and D-arabino configurations to the latter. Moreover, on the basis of analogies between their optical rotations and those of D-tartaric acid and the five-carbon metasaccharinic acids, he concluded that the “a” epimer is 3-deoxy-~-ribo-hexonicacid and acid. the ((P” epimer is 3-deoxy-~-arabino-hexonic The correctness of Nef’s reasoning has been fully borne out by subsequent observations. Ruff degradation of the D-glucometasaccharinic acids, gives the known 2-deoxy-~-erythro-pentose either mixedloor indi~idually,~~ (“2-deoxy-D-ribose”) . The formulation of the “P” epimer as 3-deoxy-~arabino-hexonic acid is, in view of the negative optical rotation of its phenylhydrazide, in accord with the configurational prediction for C2 by the phenylhydraeide rule.18Finally, the identity of “P”-D-glucometasaccharinic lactone with 3-deoxy-~-arabino-hexonolactone (prepared from authentic by hydrolysis and subsequent oxidamethyl 3-deoxy-~-arabino-hexoside’~ tion) has been e~tablished.~’ (70) J. C. Sowden, J . Am. Chem. Soc., 76, 3541 (1954). (71) G. N. Richards, J . Chem. Soe., 3638 (1954). (73) H. R. Bolliger and D. A. Prins, Helu. Chim. Acta, 29, 1061 (1946).

62

JOHN C. SOWDEN

d. Degradation.-In view of the great biochemical interest in 2-deoxy-~ribose and the many attempts to develop a satisfactory synthesis for this sugar,73it is surprising that the degradation of the D-glucometasaccharinic acids has been investigated only recently.70* 71 The preparation (from D-g1UCOSe by Nef’s method) of the mixed metasaccharinic acids in a state of sufficient purity for the Ruff degradation is readily achieved. The degradation of the calcium metasaccharinates proceeds normally, and the resulting deoxypentose may be isolated as its “anilide” without difficulty. In laboratory scale preparations,70200 g. of D-glucose yields approximately 20 g. of 2-deoxy-N-phenyl-~-ribosylamine. The free, crystalline 2-deoxy-Dribose is obtained from the “anilide” in almost quantitative yield by cleavage with benzaldehyde.

111. MECHANISM OF FORMATION OF SACCHARINIC ACIDS 1. The Fragment-recombination Mechanism of Kiliani and Windaus

Although Kiliani supplied a preponderant amount of the experimental data concerning the preparation and proofs of structure of the saccharinic acids, he theorized but little on the mechanism of their formation. In a footnote74to one of his early articles, he pointed out that glycerose had been reported16 to be one of the products of the action of alkali on D-glUCOSe, and he suggested that glycerose might afford D-glucosaccharinic acid through condensation with the lactic acid also present in the isomerization mixture. HCHO HOz C

\ /

+

CH3

KO2 C

HCOH

+

CHO

I I

CHOH CHzOH

+

CH3

\ / COH I CHOH I CHOH I

CHpOH

n-Glucosaccharinic acid

/

COzH

CHOH

I I CHOH I

--t

7 O Z H COH

I I CHOH I

CHz

CHI

CHpOH

CHzOH

D-Isosacc harinic acid

This idea was expanded by Windaus,7s who suggested that not only (73) See W. G. Overend and M. Stacey, Advances in Carbohydrate Chem., 8 , 45 (1953). (74) Ref. 02, p. 1302. (75) M. Nencki and N. Sieber, J . prakt. Chem., [a] 26, 1 (1882). (76) A. Windaus, Chem. Ztg., 29, 564 (1905).

63

THE SACCHARINIC ACIDS

D-glucosaccharinic acid but also D-isosaccharinic acid and Kiliani’s parasaccharinic acid might be formed by recondensation of appropriate aldehydic fragmentation products with a lower-carbon metasaccharinic acid. He proposed that the unbranched metasaccharinic acids, in contrast, are formed by direct dismutation of the isomeric sugars. CHzOH

I

CHO

CHO

+

CHzOH COzH

/

CHOH

I CH2 I

CHZOH

I

~

CHOH COzH

\ / COH I CH2 I

CHzOH

I CHOH I CHOH I CHOH I CHOH I

COiH

I I . direct. , CH2 dismutation 1

CHzOH

Kiliani’s Parasaccharinic acid

CHOH

CHOH

I I

CHOH CHzOH Metasaccharinic acid

Recently, C14-labeling experiments, discussed in Section 111, 5 have confirmed that fragment recombination is not involved to any significant extent in the conversion of a sugar to the related metasaccharinic acids. Also confirmed by the C14-labelingdata is the fact that fragment recombination is an important feature of the formation of the branched D-glucosaccharinic acid from an unsubstituted D-hexose. However, the specific fragments suggested by Kiliani, and the direct condensation to the final product, D-glUcosaccharinic acid, now seem improbable. 2. The Isomerixation Mechanism of Nef

Nef’s theory of the mechanism of formation of the saccharinic acids is outlined, in its original form, in a paper published in 1907” and, in its final form, in his comprehensive article of 1910.l2The theory proposes that the reaction takes place in two major steps: (a) the isomerization of the sugar, with loss of water, to an a-dicarbonyl compound, and (b) a benzilic acid type of rearrangement of the latter, with hydration, to the saccharinic acid. The second step involves chain rearrangement in the production of the saccharinic, isosaccharinic, and Kiliani’s parasaccharinic acids, but not in the production of the metasaccharinic acids. (77) J. U. Nef, Ann., 367, 214 (1907).

64

JOHN C. SOWDEN

HCO

HCO

HCO

:0

I CHONa + I

7"""

-HC,

CHOH

I

-'&=O I

CH2 I

Ho"

I1

I Aldose

HCO

I

I11

-

IV

COzH

I I CH2 I '

CHOH

Metasaccharinic acid

According to the theory, the initial reaction is the formation of an alkoxide (I) between the base and the sugar hydroxyl group vicinal to the .carbony1 group. A molecule of base is then eliminated, to give the free, methylenic intermediate (11). The latter isomerizes to the epoxy compound (111) and thence to the a-dicarbonyl intermediate (IV). Finally, a benzilic acid type of rearrangement, with hydration and dismutation, gives the saccharinic acid. CH2OH

CH20H

'i=" CHONa-

c=o c,

I CHOH

&OH

I

I

I1

CH2OH

CHzOH I

c=o I

I 3-Ketose

--+

:7

+

c=o I

I I/ o HC I

HC,

I

I 2-Ketose

I CHONa I

CH2OH

CHzOH (C=O I

c=o I

1

d

l c=o I I

I1

CH2

CH2

I11

- 7'"

CO2H

I

Isosaccharinic acid

IV

C+ I 0 HC'

I

COsH

c=o I IV

I11

Saccharinic acid

The successive isomerization of an aldose to a 2-ketose and then to a 3-ketose was explained by assuming the intermediate formation of 1,2- and 2,3-enediols.?sThus, a single aldose, under the influence of alkali, could produce all three types of saccharinic acid. CHO

CHOH

I I

COH

CHOH CHOH

I

Aldose

II I CHOH I 1,a-Enediol

CHzOH

CHzOH

CHZOH

I I

COH

I II

CHOH

C=O CHOH

I

2-Ketose

.--)

COH

I

2,3-Enediol

I

7 c=o I

3-Ketose

(78) The possibility of the presence of enediolic forms in alkaline solutions of the sugars had been discussed previously by E. Fischer, Ber., 28, 1145 (1895) and by A. Wohl and C. Neuberg, ibid., 33,3095 (1900).

85

THE SACCBARlNfC ACIDS

To account for the formation of saccharinic acids of carbon content lower than that of the original sugar, it was proposed that the enediols are subject to cleavage at the double bond, to produce lower-carbon sugars which could then also undergo the saccharinic acid rearrangement. Thus, according t o Nef, a molecule of a hexose 3,4-enediol, after cleavage to two molecules of glycerose, could provide two molecules of lactic acid (3-deoxyglyceronic acid). It is now considered more probable79that cleavage of the sugar chain CHzOH

I I

CHOH COH

II

COH

CHO

I CHOH I

I

-+

2 CHOH

I

CHzOH

CHzOH

Hexose 3,4-enediol

Glycerose

COzH -+

I I

2 CHOH

CHa Lactic acid

under the influence of alkali takes place by a reverse aldolization (V VI) a t the carbon-carbon single bond situated a,@ to the double bond of the enediol. This mechanism involves a 1,2-enediol, instead of a 3,4-enediol, in the cleavage of a hexose t o two triose fragments. An equally plausible mechanism (VII + VI), that utilizes the 2-ketose as the immediate precursor of the triose fragments,sOwould predict the more rapid cleavage of ketoses than of aldoses. .--)

+GHOH COH

HCVH I CHOH I

CHzOH

V

CHzOH

CHzOH

C-OH I

JG

II

CHOH CHO

I

CHOH

I

CHzOH VI

HCOH I CHOH

I

CHzOH VII

I n the original formulation of his theory,77Nef chose the hydroxyl group

p to the carbonyl group as the site of alkoxide formation with the base, in the initial step of the saccharinic acid rearrangement. This was later amended12 to the formulation shown above, in order to accommodate the (79) 0. Schmidt, Chern. Revs., 17, 137 (1935). (80) H. S. Isbell, private communication.

66

JOHN C. SOWDEN

observation that, in the presence of air or other oxidants, the action of alkali on the sugars leads to aldonic acids instead of to saccharinic acids. Nef’s mechanism for the aldonic acid formation is shown in VIII + IX. HCO

I CHOH I CHOH I

HCO +

I CHONa I

HCO +

CHOH

I

,1 C, I

HCO &‘&=O

CHOH

I

I

COzH

I

CHOH

-1

I

CHOH CHOH

I

IX

VIII Aldose

Aldonic acid

The general statement of the isomerization mechanism, as given in the opening paragraph of this Section, is accepted at the present time as a mechanism of saccharinic acid formation. However, Nef’s concept of the mode of isomerization of the original sugar to the intermediate a-dicarbonyl compound has undergone radical revision.

3. The Ionic Mechanism of Isbell The final phase of the Nef mechanism, which involves a benzilic acid type of rearrangement of a-dicarbonyl intermediates to the saccharinic acids, is at present accepted as a feature of saccharinic acid formation. Nef’s concept of the conversion of reducing sugars to the a-dicarbonyl structures required revision, however, when it became evident that the formation, in this step, of the proposed methylenic intermediates is highly improbable. A departure from the methylenic intermediates was suggested in 1926 by Evans and Benoy,s’ who proposed that the a-dicarbonyl intermediates of the Nef mechanism might arise by successive dehydration and rehydration from the enediols. It is now recognized, however, that forma-

CHOH

II C-OH I CHOH I 1,a-Enediol

- HzO A

11;’

HC

C

I I

CHOH

+ &O

- HzO

CHI

I I c=o I

c=o

a-Dicarbonyl intermediate

tion of the unsaturated oxide structures (pictured as resulting from the initial (81) W. L. Evans and Marjorie P. Benoy, cited in W. L. Evans, Rachel H. Edgar and G. P. Hoff, J . Am. Chem. Soc., 48,2665 (1926).

67

THE SACCHARINIC ACIDS

dehydration of the enediols) is also improbable. An acceptable course for the initial isomerization, based on consecutive electron-displacement reactions and in accord with the principal experimental facts of saccharinic acid formation, was eventually developed in 1944 by Isbell.@ As a prolog t o the Isbell ionic mechanism, Shaffer and Friedemann had concluded,a3after studying the kinetics of sugar activation by alkali, that saccharinic acids result from spontaneous rearrangement of the unstable, sugar anions that are formed in alkaline solution. They also pointed out that the sugars may behave in such solutions not only as monobasic but also as dibasic or polybasic acids, thus giving rise to unstable mono-, di-, or poly-valent anions as precursors of the saccharinic acids. An experimental demonstration that the Nef mechanism for the initial conversion of a sugar by alkali to the a-dicarbonyl structure is not acceptable was provided by a study of the action of alkali on 2-hydroxy-3-methoxy-3-phenylpropiophenone.Nicolet observeda4that the products in this case are 2,3-diphenyllactic acid and methanol. This conversion, which is completely analogous to the formation of a saccharinic acid from a reducing sugar, demonstrated clearly that carbon-oxygen cleavage occurs at the p-carbon atom, rather than a t the a-carbon atom, with respect to the carbonyl group. The Isbell ionic mechanism for the formation of the various types of saccharinic acid, as well a8 for Nicolet’s conversion of 2-hydroxy-3-methoxy3-phenylpropiophenone to 2,3-diphenyllactic acid, involves the following successive steps: (1) the formation and ionization of a n enediol; (2) the ,&elimination of a hydroxyl or an alkoxyl group; (3) rearrangement to a n a-dicarbonyl intermediate; and (4) a benzilic acid type of rearrangement to the saccharinic acid. H-C-Q@

H-C=O

I

H-C=O

.-

C-OIH 11-CI-OH +OH. I ------ CHOH

I

I

H-b CHOH

I I

COiH

p,--

I

-

I

I-I-C-H CHOH

I

I I

CHOH II@OH@

- 1

CH2 CHOH

I I

CHOH

CHOH

CHOH

CHOH

CHzOH

CHzOH

CHzOH

CIIzOH

I

I

hletasnccharinic ncid (82) H. S. Isbell, J . Research Null. Bur. Standards, 32,45 (1944). (83) P. A . Shaffer and T. E. Friedemann, J . Biol. Chem., 86,345 (1930). (84) B. H. Nicolet, J . Am. Chem. Soc., 63,4458 (1931).

68

JOHN C. SOWDEN

__-__

H~c<- OH

@s,C-QQ I

-p. -i:::

HZC3

-

COzH

C-O!H III n,---

C=O

1

y=O CHOH

H@OH~

CHOH

CHOH

CHOH

CHOH

CHOH

CHOH

CHzOH

CHzOH

CHzOH

CHzOH

CHOH

I

I I

I

I

I

Saccharinic acid

2,3-Enediol

CHzOH

CHzOH

C-43"

c=o

I

H-C:-OH

I

.-----

YHoH CHzOH

2,3-Enediol

I I

1

I

COzH

-

HCH

I I

CHOH

CHOH

CHOH

CHzOH

CHzOH

CHzOH

I

I

Isosaccharinic acid

2-Hydroxy-3methoxy-3phenylpropiophenone

It is interesting L a t Isbell's extension of L e mechanism to the hexose 3'4-enediol series would explain the formation of Kiliani's parasaccharinic acid from this source. Indirect evidence is, however, available that the hexose 3,4-enediols are, at most, minor constituents of the hexose-alkali reaction mixtures. A 3,4-enediol (or the 3-hexulose with which it is in equilibrium) should give, by chain cleavage, formaldehyde and a pentose

69

THE SACCHARINIC ACIDS

Kiliani’s Parasaccharinic acid

1 ,a-enediol. The latter would be expected to afford, among other products, five-carbon metasaccharinic acids. Despite his painstaking analyses of the hexose-alkali reaction products, Nef was never able to obtain evidence for the presence therein of five-carbon structures. Recently, however, chromatographic evidence has been obtained86 for the production of S-deoxypentonic acids, in low yield, from D-glucose plus alkali. Thus, if hexose 3,4-enediol is the necessary precursor of Kiliani’s parasaccharinic acid, the latter must be formed to only a minor extent. 4. Saccharinic Acids from Substituted Sugars

It has long been known that D-glucose substituted a t C4, as in the disaccharides lactose, cellobiose, and maltose (see page 48), is particularly suitable for the preparation of ‘La’’-D-isosaccharinicacid through treatment with lime-water. Since this acid is only a minor product of the action of alkali on unsubstituted D-glucose, the presence of a substituent a t C4 must preferentially direct the alkaline degradation to the isosaccharinic structure. The role of substitution in determining the course of saccharinic acid formation has been critically examined recently by Kenner and coworkers. They conclude that an 0-glycosyl or 0-alkyl anion is more readily extruded from the sugar enediol anion of the Isbell mechanism than is a hydroxyl ion.86 I n addition, substitution a t certain positions in the sugar molecule may inhibit competing side-reactions. For example, a substituent a t C4 of the hexose molecule inhibits cleavage (by reverse aldolization) into two three-carbon fragments and the resultant formation of lactic acid:? a result that had been demonstrated earlier by the experiments of Evans and his A combination of the two above effects, then, preferentially (85) J. W. Green, J . Am. Chem. SOC.,78, 1894 (1956). (86) J. Kenner and G . N. Richards, J . Chem. SOC.,278 (1954). (87) J. Kenner and G. N. Richards, J . Chem. SOC., 1784 (1954). (87a) W. L. Evans and Marjorie P. Benoy, J . Am. Chem. SOC.,62, 294 (1930); W. L. Evans and R. C. Hockett, ibid., 63,4384 (1931).

70

JOHN C. SOWDEN

channels the reaction of the substituted sugars, to aff ord specific, saccharinic acid structures. On this basis, isosaccharinic acids are to be expected as the principal products from 4-0-substituted hexoses. This expectation has been experimentally confirmed in studies of the action of lime-water on 61 lact~ lo s e ,~ cellobiulose,68 ' maltu61 c e l l ~ b i o s e 68 ,~~~ 4-O-methyl-~-fructose~~~ and lose,61cellotetraose,684-0-methyl-~-glucose,~~ 4, G-O-benzylidene-~-glucose.~~ The 2 3-enediol precursor for the formation of isosaccharinic acid (by the Isbell scheme) is produced from the above ketoses by a single enolization, whereas the aldoses must proceed through reversible isomerizations, by way of the 1 ,2-enediols and ketoses, t o arrive a t the 2 3-enediol structure. Accordingly, the 4-0-substituted ketoses in the preceding list are all degraded somewhat more rapidly with lime-water than are the related aldoses. I n the case of 3-0-substituted hexoses, the 1,2-enediol anion is the reactive species because of the ready elimination of the 0-alkyl or 0-glycosyl residue. Hence, the metasaccharinic acid structures are the principal products. Experimental confirmation of this concept is seen in the action of lime-water on 3-O-methyl-~-glucose,~~ 3-O-methyl-~-fructose,~~ 3-0-(/3-~glucosy1)-D-glucose (laminaribiose),89 3-O-(a-~-glucosyl)-~-fructose (turanose),89 G-O-benzyl-3-O-methyl-~-glucose,~~ 4 6-0-benzylidene-3-0-methylD - ~ ~ u c oand s ~ ,2,3: ~ ~5 G-di-O-isopropylidene-D-mannose.88 In contrast to the behavior of 4-0-substituted hexoses, the 3-0-substituted aldoses are degraded with lime-water a t approximately the same rate as are the related ketoses. This is to be expected since, in this case, a single enolization of either (aldose or ketose) produces the 1,2-enediol precursor for metasaccharinic acid formation. With a 1-0-substituted 2-hexulose, metasaccharinic acid formation is blocked by the stability of the monosubstituted 1,2-enediol anion (I) t o alkali. However, the alternative 1-0-substituted 2 3-enediol anion (11) CHOR

II c-00 I CHOH I I

CHzOR

I II

C-OH C-00

I

I1

can proceed, through elimination of the OR anion at C l , to the saccharinic acid structure. Experimentally, 1-0-methyl-D-fructose provides a higher (88) W. M. Corbett, J. Kenner arid G . N. Richards, J . Chem. SOC.,1709 (1955). (89) W. M. Corbett and J. Kenner, J . Chem. Sac., 3274 (1954). (90) J. Kenner and G . N . Richards, J . Chem. Sac., 3277 (1954).

THE SACCHARINIC ACIDS

71

yield of ‘‘af’-D-g1ucosacchariiiicacid, when treated with lime-water, than does ~ - f r u c t o s e . ~ ~ As would be expected from the above considerations, substitution a t C6 of a hexose has, a t most, only a minor effect on the course of saccharink acid formation. Thus, 6-O-(a-~-galactosyl)-~-glucose (melibiose) plus lime-water gives lactic acid and a mixture of the corresponding metasaccharinic, isosaccharinic, and saccharinic acids.S7a, 91 The qualitative experiments with melibiose and with 6-O-methyl-~-glucose,based mainly on paper chromatography, suggest that the metasaccharinic acids may be the principal products from hexoses substituted at C6. I n their initial publications on the saccharinic acids, Kenner and his associates utilized the Isbell ionic mechanism, as given in the preceding Section, to interpret their results. S u b s e q ~ e n t l yhowever, ,~~ they have suggested that the enediol di-ion is the reactive species in saccharinic acid formation; this conclusion was reached when it was observed that related 3-0-substituted aldo- and keto-hexoses are degraded by lime-water a t approximately equal rates. This observation does not, however, necessitate any modification of the original Isbell scheme. As pointed out above, the comnion 1’2-enediol is reached from either the aldose or the ketose by a one-step process, and ionization at C1 of the enediol can then initiate the metasaccharinic acid rearrangement. More recently, Whistler and Corbettg2 have cited the moderate stability of 2-O-(~-xylopyranosyl)-L-arabinose t o alkali as further evidence for the necessity of di-ion formation in the saccharinic acid rearrangement. The failure of this substance t o form saccharinic acids under mildly alkaline conditions is adequately explained, however, by the following considerations. The elimination of the hydroxyl CHOe

CHO

COR

COR

II I

HOCH

I I11

+

I I1 CH I IV

group from C3 of the mono-ion (111) is undoubtedly slow, as is the formation of saccharinic acids from unsubstituted aldoses a t room temperature with lime-water. Moreover, subsequent steps in the Isbell mechanism leading to saccharinic acids are effectively blocked a t structure IV owing to the inability of the glycosyl residue (R) a t C2 t o ionize. At higher tempera(91) W. M. Corbett and J. Kenner, J . Chem. SOC.,3281 (1954); J. Kenner and G. N. Richards, i b i d . , 2916 (1956). (92) R. L. Whistler and W. M. Corbett, J . Am. Chem. Soc., 77,3822 (1955).

72

JOHN C. SOWDEN

tures, the 2-0-substituted pentose is degraded by lime-water to acidic products, presumably because of chain cleavage. The preceding explanation of the failure of a 2-0-substituted aldose to form saccharinic acids (on treatment with alkali) finds substantiation in the results of a study of the action of lime-water, at room temperature, The presence, in this latter reaction mixon 2,3-di-O-methyl-~-glucose.8~~ ture, of an a,p-unsaturated aldehyde(IV, R = CH3) was established by its further, facile conversion to 5-(hydroxymethyl) -2-furaldehyde upon acidification. The fact that an enediol di-ion is not essential for the initiation of the saccharinic acid rearrangement is further amply demonstrated by the observations of Corbett, Kenner and Richards88 on 2 , 3 :5,6-di-O-isopropylidene-D-mannose. This compound is converted to a mixture of the 5 , 6 - 0 HC-Q’

0-0

( C H ~ ) ~ C+CH < 1~ . III V

-

CHO (CH)C-0-C

I1

CH

I

VI

COzH

I

1

7

CHOH 1

CH2

I

VII

isopropylidene-D-glucometasaccharinicacids (VII) on treatment with limewater at 100’. The authorss8depict the reaction as occurring through the intermediate VI. Obviously, the formation of an enediol di-ion prior to the anionic elimination at C3 is here ruled out, as it is in the case of 2,3-di-0methyl-D-glucose, and the reactive mono-ion (V) of the normal Isbell mechanism is involved. Thus, although enediol di-ions may well exist in alkaline solutions of reducing sugars, and may participate in rearrangements of the latter to saccharinic acids,83 the evidence so far advanced does not seem to necessitate any embellishment of the original mechanism advanced by Isbell. 5 . Fragment Recombination and Saccharinic Acid Formation

Aldol condensations involving glycerose (glyceraldehyde) and “glycerulose” (dihydroxyacetone) are known to provide ketohexoses of both straightchain and branched-chain structure.93Since these three-carbon sugars are known to be also formed through chain cleavage of hexoses by alkali,7sit is clear that the hexose-alkali reaction mixtures must contain products derived from fragment recombination. It is noteworthy, for example, that DL-sorbose,which is one of the major products of the action of dilute alkali (92a) J. Kenner and G . N . Richards, J . Chem. Sac., 2921 (1956). (93) E. Fischer and J. Tafel, Ber., 20, 1088, 2566 (1887); H . 0. L. Fischer and E . Baer, Helv. Chim. Acta, 19, 519 (1936); L. M. Utkin, Doklady Akad. Nauk S . S. S. R . , 67, 301 (1949); Chem. Abstracts, 44, 3910 (1950).

73

THE SACCHARINIC ACIDS

on ~~-glycerose,9~ has also been obtained by the action of alkali on D-fructose and by treatment of D-glucose with a strong-base resin.96 Tracer experimentsgehave revealed that fragment recombination plays only a minor role in the conversion of D-galactose to the (La”-D-galactometasaccharinic acid by lime-water at room temperature. Thus, the metasaccharinic acid prepared from ~-galactose-l-C’~ was found to contain approximately 95% of the original radioactivity* in C1, in accord with the prediction from the Isbell mechanism. The remaining 5 % of the radioactivity was presumably distributed elsewhere in the molecule as a result of intermediary fragmentation of the D-galactose followed by recombination to hexose. *CO$

I

CHO I

CHZ

HCOH

.degradntion

HOCH I

I

HCOH 2-Deoxy-D-xylose”

(cu. 5% of original

radioactivity)

CHZ 1 HOCH 1

o-plicnylene-

diamine; KMn04

’

HFoH CIlzOH

I

CHzOH ‘I

I

Ruff

-

“a”-D-Galactometasaccharinic acid (from D-galactose-I-&4)

heat

N

\;/ I

NH

COzH

Benzimidazole of original radioactivity)

(ca. 95%

In contrast to the above formation of metasaccharinic acid, fragment recombination appears to be a predominant feature in the formation of the branched-chain “a”-D-glucosaccharinic acid from D-mannose-l-CI4 plus g6 In this case, the radioactivity originally present in C1 of lime-~ater.~’’ the hexose was found to have become distributed almost entirely between the methyl carbon atom and the tertiary carbon atom of the saccharinic acid, with the latter atom more heavily labeled than the former. In contrast to these observations, the Isbell mechanism, in the absence of compli(94) E. Schmitz, Ber., 46,2327 (1913). (95) M. L. Wolfrom and J. N. Schumacher, J. Am. Chem. SOC.,77, 3318 (1955); M. Grace Blair and J. C. Sowden, ibid., 77, 3323 (1955). (96) J. C. Sowden and Dorothy J. Kuenne, J. A m . Chem. SOC., 16,2788 (1953).

74

JOHN C. SOWDEN

cations engendered by fragment recombination, predicts the appearance of the radioactivity entirely at the methyl carbon atom. 1

COzH

3 , 2 CH,COH I I 4

CH,CO,H

3,

o-phenylenedinmine

NnIO,

HCOH

I

-

coz

1

HFoH CHzOH

6 “u”-D.Glueoaaccharinic acid (from D-mannose-1-C”)

4+5

HCOzH

6

HCHO

Q-0 oxidation,

N

\&/

NH

I

3

N

NH \z / CH

CH,

2-Rlethylhenzimidazole (ca. 96% of the original radioaitivity)

Benzimidazole of the original radioactivity) (ca. 577,

o-phcnylenediaminc

y/

NH

N

I

2

CO,H

Benaimidazole-’2-cnrboxylic acid (ca. 59%,of the original radioactivity)

Benzimidazole (ca. 2% of the original radioactivity)

The above experimental result has been explained by Kenner and Richards*’ as attributable to fragmentation of the hexose to the trioses, D-glycerose (from C4,C5, and CS) and “glycerulose” (from C1, C2, and C3), followed by aldolization of these same two fragments. This would provide ketohexose labeled equally a t C1 and C3. The latter would then yield, by way of the Isbell mechanism, L‘a”-D-ghcosaccharinicacid labeled equally a t the methyl carbon atom and the tertiary carbon atom. This explanation is attractive by reason of its simplicity, but must be discarded since, although it explains the experimentally observed positions of labeling, it fails to explain the relative extent of labeling in the two principal radioactive atoms of the saccharinic acid. If the acid were formed solely by this route, the methyl and tertiary carbon atoms would each contain 50% of the original radioactivity. Moreover, the formation of uny of the acid without prior fragmentation and recombination would be reflected in a greater extent of labeling at the methyl carbon atom than at the tertiary carbon atom. No combination of the route proposed by Kenner and Richards with the normal Isbell mechanism can explain the appearing of a greater extent of labeling at the tertiary carbon atom than a t the methyl carbon atom. Experimentally, the tertiary carbon atom was found, by the methods depicted above, to contain 57 % of the original radioactivity, whereas the methyl carbon atom contained 39 %. The atoms C l , C4 C 5 ,

+

THE SACCHARINIC ACIDS

75

and CCj of the ‘ L a ~ ’ - ~ - g ~ ~ ~ ~ ~acid a ~ ~contained h a r i n i2,c 2, and 0 %, respectively, of the original radioactivity. From the above considerations, the conclusion may be drawn that, although fragment recombination is implicated in the formation of “ c ~ ” - D glucosaccharinic acid from unsubstituted hexoses, the nature of the fragments involved in the recombination step is not yet known with certainty.

6 . Saccharinic Acid Formation by Various Bases Differences in the details of isomerization of the reducing sugars by different bases were recognized by Lobry de Bruyn and Alberda van Ekenstein.97That these differences extend also into the saccharinic acid rearrangement was apparent from the early work of Kiliani and Nef. Thus, whereas D-glucose plus lime-water at room temperature gives “a”-D-glucosaccharinic acid as the principal six-carbon product (Kiliani) , the same sugar with hot, concentrated sodium hydroxide is reported t o give no trace of this branched-chain saccharinic acid but, instead, the metasaccharinic acids plus a lesser amount of “a”-D-isosaccharinic acid (Nef). Whether all such differences in the saccharinic acid rearrangement are due entirely t o differences in the pH of the reaction mixtures, or whether there is also involved a specific, cationic effect, is not yet known. Corbett and KennerE9 suggest that the difference in behavior of glucose in the above two instances may be due to differences in the ionization behavior of the enediols a t the different pH values. Thus, they propose that, a t low pH, the 2,3enediol di-ion (I) is formed and that a preferential elimination of the hyCH~[OH

I c-08 It c-08 I HCOH I I

CHzOe

I II c-08 I c-08

HCI-OH

I

I1

droxyl group at C l then leads to “a”-D-gIucosaccharinic acid. At higher pH, ionization may also occur a t C l of the 2,3-enediol. Elimination of the hydroxyl group from the resulting 2,3-enediol tri-ion (11) is then restricted to C4, and “a”-D-isosaccharinic acid results. Interesting differences in saccharinic acid formation by different bases (97) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trav. chim., 14, 203 (1895); 16,92 (1896); 16,257, 262, 282 (1897); 18, 147 (1899); 19. 1 (1900). See also, A . Kuzin, Ber., 68, 619, 1494 (1935); 69, 1041 (1936). J. C. Sowden and R . Schaffer, J. A m . Chem. Soc., 74, 499 (1952).

76

JOHN C. SOWDEN

are revealed by tracer experiments. Gibbs98 observed that ~-glucose-l-C'~ plus 3 N potassium hydroxide at 50" provide lactic acid labeled equally (and only) in C l and C3. A similar result was obtained in this laboratoryg9 when ~-glycerose-3-C'~ was isomerized with 1.68 N sodium hydroxide at 25". The radioactivity in the lactic acid thus produced was found to be distributed between C1 and C3, with a negligible amount at C2. With saturated lime-water a t 25", however, D-gly~erose-3-C~~ formed lactic acid labeled at all its carbon atoms, in the percentages shown in the formulas. 44%

COzH

I

CHOH

56%

I CH,

NaOH

CHO

I HCOH I

-

CI~H,OH

COzH

CdOH),

I CHOH I CHI

37% 13% 50%

The following explanation of this result is offered. The reverse isomerization of glycerose with "glycerulose," in the presence of either base, is fast as compared to the rate of saccharinic acid formation. Accordingly, the formation of lactic acid occurs essentially through triose enediol labeled equally at C1 and C3. The a-dicarbonyl intermediate of the Isbell mechanism is in this case pyruvaldehyde, which is subject to migration either of the methyl group (saccharinic mechanism) or of the hydrogen atom (metasaccharinic mechanism) in the rearrangement step. The latter migration is almost exclusive in the presence of sodium hydroxide, and produces lactic acid labeled only at C1 and C3. With lime-water, migration of the hydrogen atom preponderates, but some migration of the methyl group also occurs, t o produce lactic acid labeled at C2 and C3. Any combination of the two mechanisms results in the location of 50 % of the radioactivity in the methyl group. Thus, if methyl-group migration occurs to the extent of 26 %, and hydrogen-atom migration to the extent of the remaining 74 %, the lactic acid obtained will be labeled precisely as is observed experimentally. The possibility of the existence of specific, cationic effects, as opposed to pH effects, in the saccharinic acid rearrangement requires further study. IV. TABLE OF PROPERTIES OF SACCHARINIC ACIDDERIVATIVES The melting points and optical rotations of saccharinic acids and their derivatives are recorded in Table I with the corresponding literature references. (98) M. Gibbs, J . Am. Chem. SOC.,72, 3964 (1950). (99) Eva K. Pohlen, M.A. Thesis, Washington University, St. Louis (1954).

77

THE SACCHARINIC ACIDS

TABLEI Properties of Saccharinic Acid Derivatives Compound

felling point, “C.

Four-carbon Metasaccharinic Acid ~ ~ - ( 3 - D e o x y t e t r o nacid), ic [~~-(2,4dihydroxybutyric acid)] anilide brucine salt phen ylhydrazide Fiue-carbon Zsosaccharinic Acid ~ ~ - [ 3 - D e o x y - 2 -(hydroxymethyl) Ctet,ronic acid], ( ~ ~ - [ 2 , 4 - d i h y d r o xy-2-(hydroxymethyl) butyric acid]) brucine salt lactone Five-carbon Metasaccharinic Acids 3-Deoxy-~-erythro-pentonic acid phenylhydrazide quinine salt 3-Deoxy-~-threo-pentonicacid brucine salt lactone phenylhydrazide quinine salt 3-Deoxy-~-erythro-pentonic acid lactone phenylhydrazide sodium salt 3-Deoxy-~-threo-pentonicacid brucine salt lactone phenylhydrazide quinine salt sodium salt Six-carbon Saccharinic Acid 2-C-Methyl-~-ribo(?)-pentonic acid (“d’-D-glucosaccharinic acid) anhydrobenzimidazole anilide brucine salt lactone phen ylhydrazide quinine salt6

115-116 188 130-131

192-195 95-96

[&”, degrees

-27

References

85 12 12

-27.6

14 13

150 172

- 104

+9.4

12 12

145-150 amorph. 110-112 160-1 62

-34 $42.5 -25 - 119

12 12 12 12

amorph. 150

-45 t o -55 -8.9 -20

12 12 12

160 amorph. 110 172

- 19 -36 t o -40 +26 - 103 +24

12 12 12 12 12

240-241 193-195 152 160-161 167-169 141-142

+58 1-55(95%EtOH

-26 +93.5 +50.3 - 103

96 85 12

7, 12, 19 12, 100 12, 64,101

JOHN C. SOWDEN

TABLE I (continued) COrn~OUfld

Six-carbon Isosaccharinic Acid 3-Deoxy-2-C-(hydroxymethyl) (n-erythro or D-threo)-pentonic acid (“a”-D-isosaccharinic acid) amide anilide brucine salt lactone phenylhydrazide quinine salt strontium saltc Six-carbon Metasaccharinic Acids 3-Deoxy-~-xylo-hexonicacid ( ‘W’-Dgalactometasaccharinic acid) anilide barium salt brucine salt hydrazide lactone phenylhydrazide quinine salt strychnine saltd 3-Deoxy-n-lyzo-hexonic acid ( “ f i ” - ~ galactometasaccharinic acid of Nef; parasaccharinic acid of Kiliani) barium salt brucine salt lactone phenylhydrazide quinine salt strychnine salt 3-Deoxy-~-ribo-hexonicacid ( “ a ” - ~ glucometasaccharinic acid) anilide brucine salt calcium salt lactone phenylhydraeide quinine salt strychnine salt

Welling p o d , “C.

86-89

[aIDa,degrees

References

44

-28 t o -7, 13 days +13 -26 62 +19.6 - 118 -5.8

32, 85, 102 12 12, 36 12 54, 101 32, 50

136, 140 122-1 23 141-142, 144 113-115, 145 134-135, 144 185-195

$57.3 $27.4 -13.2 18 -48.4, -45.3 +34.4 -90 -8.4

32 12 12, 50 103 12, 52 12, 50, 56 12, 54, 101 12

13G137, 137 55-60 85-90 134-135, 142 125-130

-1.3 -27, -25.6 -63 -1.9 -106, -104 -23.5

12 12, 50 12 12 12, 54, 101 12

169-171 164 95-96 120-122 191-192

108-109

89-90 145-150 104 lO(r103 135-140 145-147

+

+

k40 (95%EtOH - 23 -5 +25.3 0 - 101 -19.5

85 12 68 12 12 12 12

79

THE SACCHARINIC ACIDS

TABLEI (continued) Compound

Six-carbon Metasaccharinic Acids 3-Deoxy-~-arabino-hexonic acid (“8”-D-glucometasaccharinic acid) anilide brucine salt calcium salt lactone phenylhy draside quinine salt strychnine salt

Welling point, “C.

[alou, degrees

123-124 ca. 130

-63 (95%EtOH) -33 -23.3 +8.2 -30.7 -113.6 -30.8

92 124-1 26 150-155 180-190

Rejerences

85 12 12 12 12 12 12

The rotation solvent is water unless otherwise noted. copper,20 potassium,7 1 0 and rubidiumIo4salts have also been reported for “a”-D-glucosaccharinic acid. The crystalline calcium saltsbe b2 of “or”-D-isosaccharinic acid (soluble t o t h e extent of 1.19 g. in 100 g. of hot water) was the first known salt of this acid. Crystalline calcium,s* copper,62 lead,62 and strontium64 salts have also been reported for “o”-D-galactometasaccharinic acid.

* Crystalline ammonium,’

(100) (101) (102) (103) (104)

E. Fischer and F. Passmore, Ber., 22, 2728 (1889). H . Kiliani, P. Loeffler and 0. Matthes, Ber., 40, 2999 (1907). B. Sorokin, J. prakt. Chem., [2] 37, 318 (1888). R . A. Weerman, Rec. trau. chim., 37, 52 (1917). E . Rimbach and E . Heiten, Ann., 369, 317 (1908).

This Page Intentionally Left Blank

ZONE ELECTROPHORESIS* OF CARBOHYDRATES

BY A. B. FOSTER Chemistry Department, The University of Birmingham, England

I. Introduction ........................................................... 81 11. Technique of Zone Electrophoresis. ................... 1. Apparatus for Zone Electrophoresis. ............... a. The Suspended-strip Method. .................................... 84 b. The Immersed-strip Method.. ............................... 84 c. The Enclosed-strip Method.. .............................. 111. Zone Electrophoresis of Carbohydrates in the Presence of Borate. ...... 86 1. Neutral Carbohydrates.............................................. 89 a. Monosaccharides.. ................................................ 89 b. Methylated Sugars.. .. ..................................... 92 c. Di- and Oligo-sacchari ..................................... 95 d. Glycopyranosides ..................................... e. Glycofuranosides ..................................... f . Polyhydric Alcohols.. ............................................ 102 g. Flavonoid Glycosides. .......... ............................... 104 2. Acidic and Baaic Carbohydrates and Related Substances.. ..... 104 IV. Zone Electrophoresis of Carbohydrates in the Presence of Comp Agents Other than Borate. ............................................ 106 V. Determination of Molecular Size of Carbohydrates by Zone Electro107 phoresis.. .............................................................. VI. Zone Electrophoresis of Carbohydrates on Glass Paper. ........ VII. Zone Electrophoresis of Polysaccharides. ............................... 110 1. Acidic Polysaccharides.. ........................................ 2. Neutral Polysaccharides.. . . VIII. Separations of Carbohydrates on Ion-exchange Resins. ................. 114

I. INTRODUCTION The scope of paper' and column2chromatography in carbohydrate chemistry is now well defined, and the impact of these techniques has been widely felt. Complementary to chromatography, and at present developing rapidly, is zone electrophoresis. It may be stated that, whilst chromatography has

* Zone electrophoresis is the most precise name for the technique. Other terms in common use are ionophoresis, ionography, electromigration, and electrochromatography. (1) G. N. Kowkabany, Advances i n Carbohydrate Chem., 9,303 (1954). (2) W. W. Binkley, Advances i n Carbohydrate Chem., 10,55 (1955). 81

82

A. B. FOSTER

reached its majority, zone electrophoresis is currently adolescent, although its potentialities have been fully recognized. The general technique of electrophoresis may be classified conveniently into boundary electrophoresis and zone electrophoresis.3 Boundary electrophoresis, which is a well-established analytical method, involves the migration of charged molecules in free solution under the influence of an applied electric field. The migration of the components of a mixture may be observed by means of optical methods that utilize the differences in density at the boundaries between the solution of the migrating substances in the electrolyte and the electrolyte itself. Precautions must be taken to eliminate convection currents in the conducting solution, and, because of diffusion effects, the technique is usually applied to molecules of relatively high molecular weight, especially proteins. Complete separation of the components of a mixture is not normally achieved in boundary electrophoresis. In zone electrophoresis, the conducting solution and the migrating substances are supported on a solid matrix. By means of this device, convection effects are effectively eliminated; diffusion effects are, in general, counterbalanced by the high mobilities of the migrating substances attendant on the application of high voltage gradients; and it is usual for the components of a mixture to be separated into discrete zones. Location of the zones on the supporting matrix may be accomplished by making use of specific chemical reactions or physical properties of the migrating substances. The molecular weight of the migrating substance in zone electrophoresis is not necessarily so critical a factor as often is the case in boundary electrophoresis. I n fact, zone electrophoresis has been successfully applied to mono-, oligo-, and poly-saccharides and to an analogous range of molecules in the protein field. In the realm of carbohydrates, the majority of investigations have been concerned with mono- and oligo-saccharides. A comparative account of the application of boundary and zone electrophoresis to proteins has been given by Tiselius and F10din.~The development and application of zone electrophoresis and allied techniques in the carbohydrate field will be the main concern of this Chapter. Isolated examples of the use of zone electrophoresis have long been on record: but not until 1952 were the potentialities of its application to problems of carbohydrate chemistry realized generally. The development and wide application of zone electrophoresis in the amino acid-peptideprotein (3) A. Tiselius and P. Flodin, Advances i n Protein Chem., 8,461 (1953). (4) See, for example, C. W. Field and 0. Teague, J. E x p t l . Med., 9, 86 (1907); J. Kendall, E. R. Jette and W. West, J . Am. Chem. Soe., 48,3114 (1926); T. B. Coolidge, J . B i d . Chem., 127, 55 (1939). Current interest appears to have stemmed from the work of R. Consden, A . H. Gordon and A. J. P. Martin, Biochem. J. (London), 40, 33 (1946) and subsequent papers.

ZONE ELECTROPHORESIS OF CARBOHYDRATES

83

field3 is, perhaps, riot surprising, since the molecules there encountered possehs a nct charge or may be given one by c*ontrollitigthe pE-1 of their environnient. On the other hand, most members of the carbohydrate family arc elcctrically neutral. This, the major obstacle, was overcome by making use of the long-known reaction between borate ions and carbohydrates in aqueous solution, a reaction which leads to the formation of negatively charged complexes that migrate in an applied electric field. It cannot be over-emphasized that the techniques of chromatography and zone electrophoresis, which are based on very different physicochemical principles, are compkmentary; and, in those cases where both may be applied, a more powerful analytical method obtains than when either technique is used alone. Especially is this so in the field of carbohydrate chemistry.

11. TECHNIQUE OF ZONEELECTROPHORESIS Recently, detailed accounts of the methodology of zone electrophoresis have appeared,3*6 so that only the salient points of the technique need be mentioned here. The supporting matrix most commonly used in zone electrophoresis is filter paper, although such other substances as starch, silica, powdered glass, silica gel, and even single cellulose fibers, have found some applicationlo and may be more advantageous than paper in certain cases (see page 109). I n essence, zone electrophoresis involves the application of an electric field across a strip of filter paper17impregnated with a suitable conducting solution, onto which a suitable amount of the substance (5-150 pg.) under examination has been introduced as an arbitrarily located, compact zone. After a suitable interval of time, the paper strip is processed in much the same manner as in standard paper-chromatographic procedure. The position of the zones may be ascertained by making use of specific physical properties (for example, ultraviolet absorption) or chemical reactivity (for example, reducing power) of the migrated substances. Many of the detecting reagents which have been developed in connection with the chromatography of organic compounds are equally useful in zone electrophoresis. An extensive compilation of these detection reagents has been described by Block, Durrum and Zweig.6(*) (5) (a) R. J. Block, E . L. Durrum and G. Zweig, “A Manual of Paper Chromatography and Paper Electrophoresis,” Academic Press Inc., New York, N. Y., 1955. (b) M. Lederer, “Introduction t o Paper Electrophoresis and Related Methods,” Elsevier, Amsterdam, 1955. (c) H. J. McDonald, “Ionography,” The Year Book Publishers Inc., Chicago, Ill., 1955. (6) I,. F. J. Parker, Analyst, 80, 638 (1955). (7) All subsequent references t o zone electrophoresis imply the use therein of filter-pitper strips, unless otherwise stated.

84

A. B. FOSTER

1. Apparatus for Zone Electrophoresis In view of the fact that filter paper is the supporting matrix by far the most frequently used in zone electrophoresis, only those types of apparatus which employ filter-paper strips will be mentioned. Since, in zone electrophoresis, the impregnated, filter-paper strip is the effective resistance in an electrical circuit, heat will be generated within its structure. Ineffective or incomplete dissipation of this heat will lead consecutively to evaporation of the conducting solution, establishment of a concentration gradient, and, ultimately, the breakdown of the circuit if a dry zone is formed across the paper strip. The various types of apparatus in current use may be classified according to the devices employed to control the development of heat within the paper strip or to facilitate its dissipation. a. The Suspended-strip Method.-This technique involves the simplest, and possibly the least expensive, type of equipment. The impregnated or in an inverted paper strip may be suspended horizontally,8 ~ertically,~ V shapelo with the ends dipping into the electrode chambers. Precautions are usually taken to prevent polarized electrolyte (from the electrode chambers) from reaching the ends of the paper strip. The whole apparatus may be enclosed in a humidified chamber in order to minimize evaporation of liquid from the paper strip. Because of the largely inefficient cooling system (air convection), relatively low potential-gradients only may be applied, especially when conducting solutions of high ionic strength are being used. This restriction is reflected in the considerable intervals of time which may be found necessary in order to achieve satisfactory separations. b. The Immersed-strip Method.l'--In this type of apparatus, the filterpaper strip is immersed in a solvent which is immiscible with the conducting solution. In conjunction with an aqueous conducting solution, carbon tetrachloride or heptane may be used. The cooling is mainly effected by liquid convection and is more effective than in (11, la), thus permitting the use of higher voltage-gradients. c. The Enclosed-strip Method.l-It is the author's opinion that this type of apparatus, originally devised by Kunkel and Tiselius,12(8)is the most versatile of the three noted. Essentially, the apparatus comprises a filter(8) W. Grassmann andK. Hannig, Hoppe-Seyler's Z. physiol. Chem., 2 W , l (1952); see Ref. 5(a), p. 366. (9) H. Michl, Monatsh., 82,489 (1951). (10) E. L. Durrum, J . A m . Chem. SOC.,73,2943 (1950). (11) J. D. Smith and R. Markham, Nature, 168,406 (1952); E. L. Durrum, unpublished method, described in Ref. 5(a), p. 359. (12) (a) H. G . Kunkel and A. Tiselius, J . Gen. Physiol., 36, 89 (1951). (b) A. B. Foster, Chemistry & h&?tr?/, 1050 (1952).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

85

paper strip enclosed between glass plates; the ends of the paper strip project beyond the edges of the glass plates, and dip into the electrode chambers. The plates are clamped together and onto a metal cooling-plate. Cooling is effected by conduction through the glass plates and it is surprisingly efficient in most instances. Enclosing and cooling the paper strip in this manner effectively prevents evaporation and permits the use of relatively high potential-gradients. Cooling may also be effected by immersing the clamped glass plates in a bath of ch10robenzene.l~Alternatively, the employment of low voltage-gradients or of conducting solutions of low ionic strength, or both, may obviate the need for a special cooling-device. With high potential-gradients and with conducting solutions of high ionic strength, the heat generated within the paper strip may be too great for rapid dissipation if glass plates are used. This difficulty may be overcome by substituting polythene sheets for the glass plates and by clamping them between two metal co~ling-blocks.~~ Two advantages may be gained by the use of high potential-gradients in zone electrophoresis. Firstly, and obviously, a rapid separation of the components of a mixture may be permitted and this will be of considerable importance in routine work. Secondly, diffusion effects (which may interfere with the resolution of mixtures of substances having closely similar mobilities) may be eliminated. I n fact, certain resolutions may be effected only under very high potential gradients.16 In practice, in designing equipment for routine use, a balance must be sought between the magnitude of the potential gradient employed and the associated electrical hazard. A convenient procedure12(b)for preparation of the paper strip is as follows. Solutions of the substances under examination are introduced onto the paper strip as compact zones along the origin line (which is a t right angles to the length of t h e strip and arbitrarily located). One end of the paper strip is then immersed in the conducting solution until the liquid front is about 1 cm. from the origin line. The impregnated portion is thoroughly blotted by inserting between two sheets of absorbent paper and strongly compressing with a rubber roller. The second portion of the paper is treated likewise; the two liquid fronts are then equidistant from, and near to, the origin line. By this procedure, t h e zones of the substances to be migrated are compactly retained on the origin line. The paper strip is inserted between the glass plates and the apparatus is assembled. The blotting procedure ensures tha t no liquid will be extruded from the paper strip when i t is compressed between the glass plates. A suitable potential-gradient is applied, and, after a n appropriate interval of time, the paper strip is processed by standard chromatographic methods. (13) H. D. Cremer and A. Tiselius, Biochem. Z . , 320, 273 (1950). (14) D. Gross, Nature, 172,908 (1953). (15) D. Gross, Nature, 176,362 (1955).

86

A. B. FOSTER

Several strips may be used simultaneously if they are arranged in sandwich fashion and are separated by polythene sheets. The number of papers which may be so used will be limited by the amount of heat to be dissipated, which, in turn, will depend upon the thickness of the paper strips, the ionic strength of the conducting solution, and the potential gradient applied. Kunkel and Tiselius12(s)recommend that the glass plates, which enclose the paper strip, should be covered with a film of silicone grease in order to eliminate any anomalous movement at the paper-glass interface. This precaution is unnecessary if the paper strip is prepared as described above. The possibility that some of the substances in the migrating zones might be lost by transference to the glass plates was ruled out when it was shown that, after zone electrophoresis of a nucleic acid hydrolyzate, the only ultraviolet-absorbing material transferred to the glass plates originated from the paper itself and not from the migrating nucleic acid components.16 Thus, the enclosed-strip technique may be used for quantitative work.

111. ZONE ELECTROPHORESIS OF CARBOHYDRATES IN THE PRESENCE OF BORATE which showed that, in In 1952, a number of publications the presence of alkaline borate, a wide variety of neutral carbohydrates migrate toward the anode in zone electrophoresis. It is of interest to note that Coleman and Miller,22ain 1942, had observed the migration of D-glucose and maltose toward the anode when a potential was applied across their solution in aqueous borax. The reaction of polyhydric alcohols with borate ions has long been knownz3and it has been suggestedz4that the following equilibria occur.

(16) A. B. Foster, A. S. Jones and S. B. H. Rizvi, Chemistry & Industry, 914 (1956). (17) R.Consden and Winifred M. Stanier, Nature, 169,783 (1952). (18) Y. Hashimoto, I. Mori and M. Kimura, Nature, 170,975 (1952). (19) H. Michl, Monatsh., 8s, 737 (1952). (20) L. Jaenicke, Naturwissenschaften, 39, 86 (1952). (21) L. Jaenicke and P. Vollbrechthausen, Naturwissenschaften, 39,86 (1952). (22) A. B. Foster, Chemistry & Industry, 828 (1952). (22a) G. H. Coleman and A. Miller, Proc. Iowa Acad. Sci., 49, 257 (1942). (23) See C. A. Zittle, Advances i n Enzymol., 12,493 (1951). (24) J. Boeseken, Rec. trav. chim., 61,82 (1942). See also, H. S. Isbell, J. F. Brewster, Nancy B. Holt and Harriet L. Frush, J . Research Natl. Bur. Standards, 40, 129 (1948). See also, Ref. 40.

ZONE ELECTROPHORESIS OF CARBOHYDRATES

Lo~o.l” + HO

87

HO

OH

\

R &

HO’

HO

[ H o x l > R I

+

I

HO

R, \

~

HO I1

Presumably, it is the ionic species I and I1 which migrate in zone electrophoresis. Since the equilibria (l), (2), and (3) are dynamic, then, over a sufficiently long period of time, all the carbohydrate molecules R(OH), will be associated with a negative charge. The magnitude of this charge will be determined by the position of the equilibria ( l ) , (2), and (3), which will be influenced, among other things, by the stereochemical disposition of the hydroxyl groups in R(0H)z (see p. 98). The concentration of the ionic species I and I1 in aqueous boric acid is low, and an increase in pH would be expected to raise their concentration and, concomitantly, to result in an increased electrophoretic mobility of the polyhydric alcohols. That this is indeed the case is clearly demonstrated by the results of Consden and Stanier.” Figs. 1 and 2 show the mobility-pH relationship for a range of simple carbohydrates. Two important inferences may be drawn from Figs. 1 and 2. (1) The maximum mobilities of the carbohydrates are generally in the pH region 9-10; and, consequently, borate buffers within this range are usually selected for zone electrophoresis. (2) The relative mobilities of certain pairs of carbohydrates may be critically dependent on pH; for example, at pH 9-10, D-glucose has a mobility greater than that of D-fructose, whereas, at pH 7-8, the reverse relationship obtains. Thus, careful selection of an appropriate pH may be of value in facilitating certain separations. Consden and Stanierl7 noted that, at high pH values, the migrated zones were sharp and circular, but that, a t lower pH values, “some were rather elongated, suggesting the existence of more than one species of complex.” A t pII 7 , only the ketoses and n-ribose had any appreciable mobility and, in boric acid (pH 5 ) , the migrated zones of these sugars became elongated. A further significant observation” was that, at pH 8, the mobility of carbohydrates is proportional to the borate content of the buffer, suggesting that, under these conditions, some of the carbohydrates in solution are uncomplexed.

88

A. B. FOSTER

The M a value has been suggested12(b)v 26 as a convenient index of the mobility of a carbohydrate in zone electrophoresis. The M Q value, which bears a formal resemblance to the RQ value employed in chromatography, is given by

MQ=

true distance of migration of a substance true distance of migration of D-glucose

-

-

PH PH FIG. 1.-Relationship Between Mobility and pH, for Glucose (l),Galactose ( 2 ) , Fructose (3), Ribose (4), and Raffinose ( 5 ) . FIG. 2.-Relationship between Mobility and pH, for Sorbose (l),Arabinose ( 2 ) , Mannose (3), Rhamnose (4), and Cellobiose ( 5 ) .

The true distances of migration are obtained by correcting for movement due to electroendosmotic flow. With borate buffers, the electroendosmotic flow is toward the cathode, so that the true distances of migration are usually greater than the apparent distances. Movement occasioned by electroendosmotic flow is determined by referring to the position of substances which do not react with borate ions, for example, 2,3,4,6-tetra-O-methylD-glucose12(b)and trans- 1 ,2-~yclohexanediol.~~ For a given system of zone electrophoresis, the M o values are fairly reproducible, and they will be used (25) A. B. Foster, J . Chem. Soc., 982 (1953). (26) A. B. Foster, unpublished observation.

ZONE ELECTROPHORESIS OF CARBOHYDRATES

89

in this Chapter wherever possible. It has been inferred2'. 28 that there is little or no selective absorption of low molecular-weight carbohydrates (that is, hexasaccharides and smaller oligosaccharides) on paper during zone electrophoresis. Several observations support this inference; thus, di$erent substances which do not complex with borate ions, for example, methyl a-D-xylopyranoside, 2,3,4,6-tetra-O-methyl-~-glucose, and trans1,2-cyclohexanediol, migrate at identical rates under the influence of the electroendosmotic flow. Further, in a glycine buffer at pH 11, members of the oligosaccharide series maltose-maltohexaose, and also the a- and P-Schardinger dextrins, all migrated to the same extent under the influence of the electroendosmotic flow?* Elimination of the observed slight variations in M , values for a given zone-electrophoretic system is experimentally very difficult, since it would demand precise control of (1) the quality and thickness of the paper strips, (2) the buffer content per unit volume of paper, (3)the pressure exerted on the paper strip by the enclosing medium, (4)the amount of carbohydrate in each migrating zone, and (5) the temperature. For most practical purposes, these variations are not very important, since a comparison between known and unknown compounds is performed on a single, paper strip. 1. Neutral Carbohydrates

In the following Sections, the various types of carbohydrate which have been subjected to zone electrophoresis have been classified arbitrarily. I n view of the fact that zone electrophoresis is, as yet, relatively little used in carbohydrate chemistry, an attempt has been made to emphasize and illustrate the applicability of the technique and to compare it, at the appropriate point, with paper chromatography, rather than to compile a catalog of applications. a. Monosaccharides.-Most of the early investigations on the zone electrophoresis of carbohydrate~17-~0 were concerned, in the main, with monosaccharides. A comparative list of M , and R p values of a range of monosaccharides is shown in Table I, from which it may be seen that there are significant differences in the movement of carbohydrates in zone electrophoresis and in paper chromatography (see also, Table 11).For example, D-galactose, D-glucose, and D-mannose may be easily separated by zone electrophoresis, but not readily by paper chromatography. It is important to note that separations achievable by both techniques will, in general, be much more rapidly accomplished by zone electrophoresis. It is also clear from Table I that, in zone electrophoresis, carbohydrates of different (27) E. J. Bourne, A. B. Foster and P. M. Grant, J. Chem. SOC.,4311 (1956). (28) A. B. Foster, Primula A. Newton-Hearn and M. Stacey, J. Chem. SOC.,30 (1956).

90

A. B. FOSTER

“types,” for example, pentoses and hexoses, may have identical or closely similar M , values ; specific examples are the pairs D-xylose-D-glucose and L-arabinose-D-galactose. This observation suggests the manner in which zone electrophoresis and paper chromatography may be used complementarily in the identification of carbohydrates or in substantiating the homogeneity of individual components of a mixture. Separation of a mixture of carbohydrates into fractions of different types may be achieved by paper chromatography or by column chromatography. Elution of the fractions TABLE I Comparative MG and RF Values of Some Monosaccharides R p i n solvent systema Sugar

L-Arabinose D-Ribose D-Xylose L-Fucose L-Rhamnose D-Galactose D-Glucose D -Mannose D-Fructose L-Sorbose L-galacto-Heptulose n-manno-Heptulose

h!

0.96 0.77 1 .oo 0.89 0.52 0.93 1.00 0.72 0.90 0.95 0.89 0.87

1

2

3

4

0.43 0.56 0.50 0.44 0.59 0.34 0.39 0.46 0.42 0.40 -

0.21 0.31 0.28 0.27 0.37 0.16 0.18 0.20 0.23 0.20 -

0.51

-

-

0.12 0.21 0.15 0.21 0.30 0.06 0.082 0.11 0.12 0.10 0.11

-

0.34 0.59 0.56 0.35 0.29 0.35 0.45 0.36 -

a 1. Water saturated with 2,4, 6-collidineBe 2. 1-Butanol-acetic acid.29 3. Phenolacetic acid-water.80 4. l-Butanol-ethanol-water.81

from the paper chromatogram or from the column, and subjecting them to zone electrophoresis, will in many cases indicate homogeneity or identity. This sequence of operations was employed to advantage by Gross32in an investigation of the products of the action of yeast invertase on sucrose. A further example is provided by R i ~ k e t t s , who * ~ observed that the product obtained on treatment of a dextran sulfate with aqueous alkali gave, on acidic hydrolysis, a mixture of reducing sugars which migrated as a single zone in paper chromatography. The Rp value was similar to that of D-glucose, but more than one substance appeared to be present. On treatment (29) S. M. Partridge and R. G. Westhall, Biochem. J. (London), 42,238 (1948). (30) J. N. Counsell, L. Hough and W. H. Wadman, Research (London), 4, 143 (1951). (31) E. L. Hirst and J. K. N. Jones, Discussions Faraday Soc., 7, 268 (1949). (32) D. Gross, Nature, 173,487 (1954). (33) C. R . Ricketts, J . Chem. SOC., 3752 (1956).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

91

by zone electrophoresis, four discrete zones were obtained, with M , values identical to those of D-glucose (1.00), D-gulose (0.84),D-altrose (0.92), and D-mannose (0.72). The rapid separations of carbohydrates by means of zone electrophoresis may be of value in studying the course of a reaction. Thus, Stacey and coworkers34were able to follow the conversion of D-glUCa1 and of D-galactal into 2-deoxy-D-‘(glucose” and Z-deoxy-D-“galactose,” respectively, under the influence of a cation-exchange resin (He form). The reaction was shown to be quite complex. An indication of the rapidity of zone electrophoretic separations is shown by the fact that, using the enclosed-strip technique12(b)with a borate buffer a t pH 10 under a potential gradient of about 20 v./cm., a mixture of maltose and isomaltose may be completely resolved in 20 minutes. Under similar conditions, a mixture of D-glucose and D-galactose would be separated in about 4 hours. Most routine separations may be effected in time periods lying between these two limits. I n the interests of time saving, it should be noted that, by using specially designed equipment, potential gradients of up to 240 v./cm. may be applied to a 50-cm. paper strip.36 Many proteins contain small amounts of bound polysaccharide, and paper-chromatographic analysis of the hydrolyzates of these substances (for carbohydrates) is rendered difficult by the excess of amino acids present. The isolation of carbohydrate-rich fractions may be a n essential preliminary to hydrolysis. These difficulties may be overcome to a large extent by zone electrophoresis, which frequently will permit the separation of sugars from amino acids.” In a borate buffer at pH 8.6, only the “peptides of glutamic acid and aspartic acid will migrate to positions similar to those of the monosaccharides,” but, even so, they are usually present in small proportions. The separated sugars may be examined subsequently by paper chromatography and, if necessary, by zone electrophoresis again. The general movement of sugars in zone electrophoresis is slowed down by the presence of amino acids and peptides. Consden and Stanier” have employed a single paper sheet, first for zone electrophoresis and second for paper chromatography. They used the following general method. The protein polysaccharide is hydrolyzed at 100-110” for 3 hours with N sulfuric acid, neutralized with baryta solution, filtered, and the filtrate concentrated t o small volume. Zone electrophoresis in borate buffer (pH 8.6)is then carried out for a suitable time, and the paper strip is dried and sprayed with ninhydrin30 (to locate the amino acids) and with aniline hydrogen phthalate37 (to detect the reducing sugars).

(34)A. S.Matthews, W . G. Overend, F. Shafizadeh and M. Stacey, J . Chem. Soc., 2511 (1955). (35)D. Gross, Nature, 178, 29 (1956). (36) Ref. 5(a), p. 88. (37) S. M. Partridge, Nature, 164, 443 (1949).

92

A. B. FOSTER

The relative positions of the amino acids and sugars may give useful information. Alternatively, the paper may be subjected t o chromatography in a direction a t right angles to that of the zone electrophoretic separation; in which case, the paper should finally be sprayed first with aniline hydrogen phthalate and then with ninhydrin.

Using this procedure, Consden and Stanierl’ were able to show that a hydrolyzate of Group A hemolytic streptococci contained hexosamine, rhamnose, ribose, and glucose; and, also, that a hydrolyzate of purified, human fibrin contained uronic acid, hexosamine, mannose, and galactose. A similar combination of techniques has been employed by Woodin38in a study of the composition of a corneal mucopolysaccharide. It is of interest that the conditions of acidic hydrolysis necessary for releasing the D-glucosamine moiety from heparin also result in destruction of the concomitantly released D-glucuronic acid.agTraces of the intact uronic acid may be revealed by zone electrophoresis of the hydrolyzates.26 b. Methylated Sugars.-The behavior of methylated sugars under zone electrophoresis was first described by Foster,22who found that mixtures of 2,4- and 3,4-di-O-methyl-~-rhamnose, which are difficult to resolve by paper chromatography, are easily separated by zone e1ect)rophoresis (see Table 11) since only the latter ether can form a complex with borate ions. This behavior was predicted from the work of Boeseken,4O who inferred that, for cyclic carbohydrates, only vicinal cis-hydroxyl groups can form a complex with borate ions in aqueous boric acid. The observation**that 2,3-di-O-methyl-~-glucose, the furanose and pyranose forms of which have no vicinal cis-hydroxyl groups, has an MQ value of 0.12 suggested that, in alkaline media, other types of borate-ion interaction could occur. The M Qvalues listed in Table I1 indicate that this is indeed the case (RQ values are included for purposes of comparison). Several of the derivatives listed, especially those of D-glucose substituted at C2, show an appreciable mobility on zone electrophoresis in alkaline borate, although the furanose and pyranose forms have no vicinal cis-hydroxyl groups. A further important observation is the low MQ value of 4-0-methyl-~-glucose;in this case, the furanose form is precluded, but the a-D-pyranose form has vicinal cishydroxyl groups at C1 and C2. The M Q values of the mono-0-methyl-& glucoses suggest that the hydroxyl groups on C2 and C4 in the parent sugar are very important in complex formation with borate ions. It is possible that the important role of the hydroxyl group at C4 is associated with a reaction of the furanose form with borate ions; however, other results (see page 101) suggest that the furanose form may not be significantly involved (38) A. M.Woodin, Biochem. J . (London), 61,319 (1952). (39) M.L.Wolfrom and J. V. Karabinos, J. A m . Chem. Soc., 67,679 (1945). (40) J. Boeseken, Advances i n Carbohydrate Chem., 4, 189 (1949). (41) A. B.Foster and M. Stacey, J . Appl. Chem. (London), S,l9 (1953).

93

ZONE ELECTROPHORESIS OF CARBOHYDRATES

in complex formation. The sequence of the M Qvalues in Table I1 has been rationalized by postulating that the aldehydo form of the sugars is the principle one involved in complex formation.41aIn this event, the pair of hydroxyl groups sterically most favorable for complex formation are those on C2 and C4. It must be admitted, though, that little or no information is available which would indicate how the equilibria of furanose, pyranose, TABLEI1 MQ and RF V a l u e s of Some Methylated, Acetamidodeoxy and Deoxy Sugars Sugar derivative

D-Glucose 2-0-Methyl-~-glucose ‘‘~-D~OX~-D-~~UCOB~” 2-0-Methyl-~-galactose “2-Deoxy-~-galactose” 2-Acetamido-2-deoxy-~-glucose 2-Acetamido-2-deoxy-~ -galac tose 3-O-Methyl-~-glucose 4-O-Methyl-n-glucose 4-O-Methyl-~-galactose 6-O-Methyl-~-glucose 2,3-Di-O-methyl-~-glueose 2,4-Di-O-methyl-~-ghcose

3,4-Di-O-methyl-~-glucose 2,3,4-Tri-O-methyl-~-glucose 3,5,6-Tri-O-methyl-~-glucose 2,3,4,6-Tetra-O-methyl-~-ghcose 2,3-Di -0-methyl-L-rhamnose 2,4-Di-O-methyl-~-rhamnose 3,4-Di-O-methyl-~-rhamnose

RQ

Mg26.41

1.00 0.23 0.29 0.32 0.37 0.23 0.35 0.80 0.24 0.27 0.80 0.12 <0.05 0.31 0.00 0.71 0.00 <0.05 <0.05 0.36

in solvent“ B

0.09 0.22 0.23 0.25 0.26 -

-

0.27 0.57 0.52 0.85 1.oo 0. 832a 0. 872a 0.892’

0.69*’ 0. 6441 0. 6541

a Solvent A is 1-butanol-ethanol-water.Solvent B is l-butanol-ethanol-waterammonia.

and open-chain forms of reducing sugars in alkaline media are influenced by complex formation with borate ions. A limitation of zone electrophoresis in the field of methylated sugars is the non-reaction of several polymethylated derivatives with borate ions, but, as the results in Table I1 testify, in the mono- and di-0-methyl series there is a much wider variation in Mg values than in Ro values. Bell and Northcote& have reported the mobilities of the 0-methyl-Dfructoses given in Table 111. (41a) See H. Bouveng and B. Lindberg [Acta Chem. Scand., 10,1283 (1956)l for the application of this theory to the mono-0-methyl-D-gdactoses. (42) D. J. Bell and D. H. Northcote, Chemistry & Industry, 1328 (1954).

94

A. B. FOSTER

Zone electrophoresis has found application in the structural investigation of 2,4-di-O-methyl-~-rhamnose,isolated from a methylated polysaccharide from Pneumococcus Type 1143and from okra mucilage,44and of 2,4-di-O-methyl-~-glucoseisolated after methylation and hydrolysis of a dextran elaborated by Betacoccus a r a b i n o s a c e o ~ s The . ~ ~ behavior of several mono- and di-0-methyl ethers of D-ribose in zone electrophoresis has been been ~tudied.~0. 47 It was found47that exhaustive methylation of uridylic acid “b” with the Purdie reagents, followed by hydrolysis, gave, after paper chromatography, a di-0-methyl-D-ribose fraction which was shown, by zone electrophoresis, to contain two components corresponding to 2 , 5 and 3,5-di-O-methyl-~-ribose. Evidently, phosphate migration had ocTABLE I11 Mobilities of O-Methyl-D-fructoses42 D-Fructose deriwalive

Dislance migrated‘ i n cm.

1-0-Methyl 3-0-Methyl3,4-Di-O-methyl4,5-Di-O-methyl1,3,4-Tri-O-methyl1,4,5-Tri-O-rnethyllJ4,6-Tri-0-methyl3,4,6-Tri-O-methyla The mobility of D-fructose itself was not recorded. pH 9.2. c Used a8 a marker.

14.7 14.0 10.8 13.0 0.00” 9.7 12.5 9.3 Borate buffer (0.05 M ) of

curred during the methylation. Application of zone electrophoresis to the product obtained by the action of diethylmagnesium on methyl 2,3-anhydr0-4,6-O-benzylidene-cu-~-mannoside, followed by acidic hydrolysis, helped to substantiate its identity as a mixture of 3-deoxy-3-C-ethyL~altrose and its 1,6-anhydro d e r i ~ a t i v e . ~ ~ The examples selected above illustrate some of the many possible applications of zone electrophoresis. (43) K. Butler, P. F. Lloyd and M. Stacey, Chemistry & Industry, 107 (1954). (44) R. L. Whistler and H. E. Conrad, J . Am. Chem. SOC.,7 6 , 3514 (1954). (45) S. A . Barker, E. J. Bourne, G. T. Bruce, W. B. Neely and M. Stacey, J . Chem. Soc., 2395 (1954). (46) D. M. Brown, G. D. Fasman, D. I. Magrath and A . R. Todd, J . Chem. SOC., 1448 (1954). (47) D. M. Brown, D. I. Magrath and A . R. Todd, J . Chem. Soc., 1442 (1954). (48) A. B. Foster, W. G. Overend, M. Stacey and G. Vaughan, J . Chem. SOC.,3308 (1953).

95

ZONE ELECTROPHORESIS OF CARBOHYDRATES

c. Di- and Oligo-saccharides.-From the zone-electrophoretic behavior of the mono-0-methybglucoses, it might be predicted that the reducing disaccharides of D-glucose (only) with (1 -+ 2) or (1 -+ 4) linkages would have much lower M Qvalues than those containing a (1 + 3) or a (1 -+ 6) linkage. The M , values in Table IV amply confirm this prediction: maltose and cellobiose have much lower mobilities than have isomaltose and gentiobiose. Furthermore, certain pairs of disaccharides which contain linkages at similar positions (but of different configuration) have appreciably different M , values, for example, maltose and cellobiose, and isomaltose and gentiobiose. The migration of di- and oligo-saccharides in paper chromatography with the customary solvent systems is very slow, but special solvent systems have been evolved which result in much higher RQ values.49Al-

TABLEIV MG Values of Some Disaccharidese6 Disaccharide (DGkbCOSyl-D-gllUOSC)

Sophorose Nigerose Laminaribiose Maltose Cellobiose Lactose Isomaltose Gentiobiose

Linkage

P-D-(1

-+

2)

CU-D-(l + 3)

P-D-(l -+ 3) a-D-(l 4) D-D-(1 -+ 4) p-D-(1 + 4) -+

C U - D - ( ~+ 6)

p - ~ - (+ 1 6)

0.24 0.69 0.69 0.32 0.23

0.38 0.69 0.75

ternatively, the di- or oligo-saccharides may be converted, on the paper, to a suitable derivative (for example, the N-benzylglycosylamine60)which will have a greater solubility in the mobile phase and hence a higher RF value. It has been emphasizedzs that a combination of these chromatographic procedures with zone electrophoresis may be of considerable value in the identification of di- and oligo-saccharides. The most important structural feature which governs the magnitude of the M Qvalue of a reducing D-gluco-oligosaccharide (and probably of many other saccharides), is the point of attachment of the “remainder” of the molecule to the reducing moiety. The M Qvalue will be influenced to a much smallcr extent by structural variations in the “remainder” of the molecule. An interesting example of this phenomenon was encountered in structural investigations of the polyglucan elaborated by Aspergillus niger.61The poly(49) Allene Jeanes, C. S. Wise and R . J. Dimler, Anal. Chem., 23, 415 (1951). (50) R . J. Bailey and E. J. Bourne, Nature, 171, 385 (1953). (51) S. A . Barker, E. J. Bourne and M. Stacey, J . Chem. Soc., 3084 (1953).

96

A. B. FOSTER

saccharide contains an alternating sequence of a-~-(l-+ 3) and C X - D4 - ( ~4) links, and graded acidic hydrolysis afforded a trisaccharide fraction (by carbon-Celite column chromatography) which appeared homogeneous by paper chromatography using the benzylamine method,60 but which had an R, value intermediate between that of a (1 + 3) ,(1 4 3) linked trisaccharide and a (1 + 4) ,(1 -+4) linked trisaccharide. Zone electroph~resis~~ of the trisaccharide fraction gave two components with the mobilities expected for (1 -+ 3), (1 -+ 4) and (1 4 4), (1 -+ 3) linked trisaccharides. Use has been made of zone electrophoresis in structural investigations of glycogen. Peat, Whelan and Edwards62isolated, by carbon-Celite column chromatography, a trisaccharide fraction from a partially hydrolyzed glycogen from baker’s yeast. Zone electrophoresis showed two components, of low and high mobility. Extraction of the slow-moving component from the paper, followed by reduction (with NaBH4) and acetylation, gave panitol dodecaacetate, indicating the parent trisaccharide to be panose [an a - ~ - ( l-+ 6) , a - ~ - (-+l 4)-linked trisaccharide of D-glucose (only)]. Thelow mobility of this trisaccharide is not unexpected. It was suggested62 that the component of high mobility might be the a - ~ - ( + l 4) , c x - D - ( ~+ 6)linked trisaccharide. Recently, Wolfrom and Thompson63fractionated a partial hydrolyzate of glycogen by carbon-Celite column chromatography, paper chromatography, and zone electrophoresis, successively. A trisaccharide fraction was obtained which had a mobility much higher than that of panose. Structural investigation showed it to be isomaltotriose, and not the (1 -+ 4) ,(1 + 6)-linked trisaccharide which might have been expected. This significant observation reveals that some of the (1 -+ 6) links in glycogen must be in adjacent positions. The preceding examples elegantly demonstrate the point at which zone electrophoresis may be used to advantage in structural determinations on polysaccharides, namely, in further resolving fractions isolated by carbonCelite column chromatography or paper chromatography, or both. Recovery of sugars from “pherograms”64 (paper electrophoretograms) impregnated with sodium borate involves extraction with water followed by the removal of inorganic material from the extracts. This may be accomplished by removing cations with a suitable ion-exchange resin, followed by evaporation of the solution and the removal of boric acid as the volatile methyl borate by repeatedly distilling added methanol from the residue.66 (52) S.Peat, W.J. Whelan and T. E. Edwards, J . Chem. SOC.,355 (1955). (53) M.L.Wolfrom and A. Thompson, J . Am. Chem. Soc., 78, 4182 (1956); 79, 4214 (1957). (54) T. Biicher, D.Matselt and D. Pette, Klin. Wochschr., 30,325 (1952). (55) Compare L.P.Zill, J. X. Khym and G. M. Cheniae, J . Am. Chem. SOC.,76, 1339 (1953).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

97

I n the case of di- and oligo-saccharides, a simpler method may be employed. The aqueous extract of the pherogram is added to a short carbon-Celite column, inorganic material is eluted with water, and the carbohydrate is subsequently eluted with aqueous ethanol.26This method may be used for any substance which is not readily eluted from a carbon-Celite column by water but which can be eluted with aqueous ethanol. Alternatively, the saccharide extracted from the pherogram may be a c e t ~ l a t e d . ~ ~ Special precautions may be necessary in the recovery of saccharides which include a (1+ 3) link at the reducing end of the molecule. These saccharides are readily decomposed by alkali.66It is interesting to note that laminaribiose and nigerose are stable under the conditions (borate pH 10) of zone electrophore~is,2~ but are decomposed on extraction from the pher~gram.~’ In this case, it may be necessary to render the pherogram neutral before performing the extraction. Isolation, using zone electrophoresis, of a quantity of an oligosaccharide sufficient for structural studies may be a tedious process, and it may be necessary to prepare and then extract many pherograms. An important, column technique has been developed68which will overcome this difficulty in many cases. The method is based on the well-known, carbon-Celite colunin technique of Whistler and D u r s ~ The . ~ ~column, prepared in the usual manner, is impregnated with a borate buffer (pH lo), and the eluant (aqueous ethanol) contains this buffer at the same p H . Saccharides which complex strongly with borate ions are eluted more rapidly from the column than would be the case in the absence of borate. The reason for this behavior lies in the fact that the borate complexes have some of the characteristics of inorganic salts, and it is well known that many salts are not firmly retained b y carbon-Celite columns. Thus, of a pair of isomeric saccharides, the one complexing the more strongly with borate ions will be the more rapidly released from the column. For example, a mixture of melibiose and maltose (with M , values of 0.80 and 0.32, respectively) is incompletely resolved on a normal, carbon-Celite column when a gradient of aqueous ethano160 is applied; the disaccharides emerge when the eluant contains 3.2 % and 5.3 % of alcohol, respectively. Using the borate-impregnated column, resolution is complete, and the melibiose emerges a t 0.8% and the maltose a t 4.6% concentration of alcohol. It should, therefore, be feasible, in many cases, to translate a microscale, zone-electrophoretic (56) Compare W. M. Corbett and J. Kenner, J . Chem. Sac., 3274 (1954). (57) S. A. Barker, E. J. Bourne and M. O’Mant, unpublished observations, cited in Ref. 58. (58) S. A. Barker, E. J. Bourne and 0. Theander, J . Chem. Sac., 4276 (1955). (59) R. L. Whistler and D. F. Durso, J . A m . Chem. Sac., 7 2 , 677 (1950). (60) B. Lindberg and B. Wickberg, Acta Chem. Scand., 8,569 (1954).

98

A.

€3.

FOSTER

separation to a macroscale separation, using a borate-impregnated carbonCelite column. Information on the feasibility of such separations will be obtainable from the relative M u values. TABLE V MG Values of Some Pento- and Hero-pyranosides and Related Compoundse1-B3 Glycoside

Methyl a-D-xylopyranoside j3 anomer lt5-Anhydroxylitol Methyl a-D-arabinopyranoside j3 anomer 1,5-Anhydro-~-arabinitol Methyl a-D-lyxopyranoside p anomer Methyl 8-D-ribopyranoside 1,5-Anhydroribitol Methyl a-D-glucopyranoside fl anomer 1,5-Anhydro-~-glucitol Methyl a-D-galactopyranoside j3 anomer 1,5-Anhydro-~-galactitol Methyl a-n-mannopyranoside 8 anomer 1,5-Anhydro-~-mannitol Methyl a-L-rhamnopyranoside j3 anomer 1,5-Anhydro-~-rhamnitol Met hy1 a m - gu 1opy ranosi de j3 anomer Methyl a-D-fructopyranoside fl anomer Sucrose Trehalose, a,aa,@-

838-

0.00 0.00 0.00 0.38 0.38 0.39 0.45 0.27 0.53 0.53 0.11 0.19 0.20 0.38 0.38 0.38 0.42 0.31 0.40 0.31 0.14 0.31 0.59 0.72 0.71 0.59 0.17 0.19 0.23 0.19

d. G2ycopyranosides.-The zone-electrophoretic behavior of a wide range of glycopyranosides has been studied,26zm3and the M , values are recorded in Table V. The zero M u values of D-xylopyranosides indicate that vicinal, trans-hydroxyl groups do not react with borate ions. This is in accord with the observations of Boeseken using boric acid.40It is of interest to note that , (61) A. B. Foster, E. F. Martlew and M. Stacey, Chemistry & Industry, 825 (1953). (62) A. B. Foster and M. Stacey, J . Chem. Soc., 1778 (1955). 1395 (1957). (63) A. B. Foster, J . Chem. SOC.,

ZONE ELECTROPHORESIS OF CARBOHYDRATES

99

whilst the anomeric methyl D-arabinopyranosides and 1,5-anhydro-~arabinitol have similar affinities for borate ions, this is not the case with the anomeric methyl D-lyxopyranosides. Methyl a-D-lyxopyranoside has a much higher M Qvalue than has the p anomer. I n fact, in all the examples presented in Table V where vicinal, cis-hydroxyl groups are flanked by a cis related methoxyl group (that is, in methyl 8-D-lyxopyranoside, methyl P-L-rhamnopyranoside, methyl p-D-mannopyranoside, and methyl a-D-gulopyranoside), the reaction of the hydroxyl groups with borate ions appears to be hindered. Although a precise explanation of this effect cannot be given a t present, it seems probable that the relative instability of the borate complexes of these glycosides, resulting in low M , values, is due in part t o adverse, non-bonded interactions within the complexes. In this regard, it would be instructive to determine the M , value of methyl a-D-ribopyranoside. The mobilities of methyl a- and 0-D-glucopyranosides in zone electrophoresis are attributable to the formation of a borate complex across the hydroxyl groups at C4 and C6. This has been shown by blocking individual hydroxyl groups in derivatives of these glycosides. An explanation of the lower mobility of methyl a-D-glucopyranoside (O.ll), compared to that of the p anomer (0.19), may be offered in terms of non-bonded interactions. The borate complexes of the a and ,f3 anomers may be represented by 111 and IV, which show the complexes to be rigid, trans fused, bicyclic systems. The glycosidic methoxyl group in the borate complex of methyl p-D-glUCOpyranoside (IV) occupies an equatorial position, where it is relatively free from strong non-bonded interactions. In the complex of the a anomer (111), however, the glycosidic methoxyl group occupies a n axial position and will interact strongly with the axial hydrogen atoms on C3 and C5. This effect will tend to destabilize the borate complex of the a anomer and is reflected in the lower M a value.

OH

OH

I11

1v

Sugihara and P e t e r ~ e nhave ~ ~ shown that metaboric acid will condense with methyl a-D-glucopyranoside under suitable conditions. Benzoylation (64) J. M. Sugihara and J. C.Petersen, J . A m . Chem. Soc., 78, 1760 (1956).

100

A. B. FOBTER

of the condensate, followed by methanolysis of the boric ester groups and acetylation of the liberated hydroxyl groups, yielded a mixture of products in which there predominated the 4,6-diacetate-2,3-dibenzoate and the 3,4-diacetate-2,6dibenzoate of the original glycoside. Since the acetyl groups substituted the hydroxyl groups which had originally carried the boric ester groups, it appears that 4,6- and 3,4-borate esters are formed in the initial condensation. As noted above, a borate-ion interaction at the hydroxyl groups at C4 and C6 has been observed in the zone electrophoresis of methyl a-D-glucopyranoside. No evidence has been obtained which would indicate-the formation of a complex across the hydroxyl groups at C3 and c4. Both of the steric effects mentioned above are operative in the D-mannopyranosides. Thus, in methyl a-D-mannopyranoside, the 4,6-borate complex is hindered, but the 2,3-c0mplex is not. The reverse situation obtains for the 8 anomer. Since the 2,3-c0mplex is formed more readily than the 4,6-complex, the a anomer has the greater M , value. The similar M , values of the anomeric methyl D-galactopyranosides and the anomeric methyl D-arabinopyranosides indicate that their reactions with borate ions are independent of configuration at the glycosidic center, It is the occurrence of borate-ion interactions, similar to those found for methyl a- and 8-D-glucopyranoside, that is responsible for the mobility of sucrose and the trehaloses in zone electrophoresis. The structures allocated on the basis of the reaction of the anomeric methyl D-fructopyranosideswith borate ions (as reflected in the M , values) are consistent with the structures allocated on the basis of their optical rotations.03It is interesting to note that methyl a-D-fructopyranoside (V) and methyl P-D-gulopyranoside (VI), which have a closely related disposition of their hydroxyl groups, have closely similar M , values. CHzOH

H

H

HO V

VI

e. G1ycojuranosides.-From the M , values of the anomeric methyl D-arabinofuranosides and methyl D-xylofuranosides,it may be inferred that, whereas vicinal, cis, hydroxyl and hydroxymethyl groups (as in the, D-xylofuranosides) in these glycosides react strongly with borate ions, a trans arrangement of these groups (as in the D-arabinofuranosides) permits a

101

ZONE ELECTROPHORESIS OF CARBOHYDRATES

very weak interaction. Thus, the structures allocated to the anomeric methyl D-fructofuranosides, on the basis of their M , values, are the same as those allocated on the basis of their optical rotations.aaA knowledge of this type of borate-ion interaction has been of value in structural investigations on 2,5-anhydro-~-mannito1,66and in interpreting the M , values of the adenosine 2-, 3-, and 5-phosphate~.~~, 66 Together with a knowledge of the borate-ion interactions noted in Section 111, Id, the (observed) mobility relation kestose (a-~-Gp-(l+ 2)-P-~-Fruf-(6+ 2)-P-~-Fruf)> neokestose (P-~-Fruf-(24 6)-a-~-Gp-(l4 2)-p-~-Fruf)would have been predicted. The isomeric trisaccharides kestose and neokestose were obtained by the action of yeast invertase on sucrose,67and this investigation provides TABLEV I Me Values of Some Glycofuranosides and a Related Cornpound62*68 Dnivafioe

Methyl a-D-arabinofuranoside B anomer Methyl a-D-xylofuranoside j3 anomer Methyl a-D-fructofuranoside 13 anomer Methyl a-D-glucofuranoside 1,2-O-Isopropy~idene~-~-glucofuranose Methyl a-D-galac tofuranoside j3 anomer

0.035 0.035 0.56 0.33 0.60 0.04 0.73 0.73 0.41 0.31

yet another example of the value of zone electrophoresis in the resolution of mixtures of isomeric saccharides. The high M , value of the D-glucofuranoside derivatives in Table VI is due to complex formation with the hydroxyl groups at C3, C5, and C6, and is of considerable interest in connection with the role of furanose forms in the reaction of reducing derivatives of D-glucose with borate ions (see page 93). The low M o value of 2-O-methyl-~-glucose,in which the hydroxyl groups at C3, C5, and C6 are free would suggest that the furanose form of this sugar is not significantly involved in complex formation. It is not improbable that the hydroxyl groups a t C3, C5, and C6 in the D-glucofuranoside derivatives shown in Table VI may form a “ tridentate complex” of the type suggested for certain inositol derivativesas (see page 104). (65) B.C.Bera, A. B. Foster and M. Stacey, J. Chem. SOC., 4531 (1956). (66) D.C.Burke and A. B. Foster, Chemistry & Industry, 94 (1955). (67) D.Gross, P.H. Blanchard and D. J. Bell, J. Chem. Soe., 1727 (1954). (68) S.J. Angyal and D. J,McHugh, Chemistry & Industry, 1147 (1956).

102

A. B. FOSTER

f. Polyhydric Alcohols.-Using a borate buffer at pH 9.2, Frahn and Millss3have studied the zone-electrophoretic behavior of a number of diols (see Table VII). Thus 1,4-butanediol and 1 ,5-pentanediol were found not to react with borate ions, indicating that borate complexes involving 7- and 8-membered rings have little tendency to be formed. The more ready reaction of threo-2 ,3-butanediol with borate ions, in comparison with the erythro isomer, is not unexpected, since the 5-membered ring in the borate complex of the latter (but not the former) would possess eclipsed methyl groups a t C2 and C3. The relative reactivity of the 2 ,3-butanediols toward borate TABLEVII MG Values of Some Pol?/hydric Alcohols 27.09 Derivative in solvent" A

1,4-Butanediol 1,5-Pentanediol threo-2,3-Butanediol erythro-2,3-Butanediol 2,4-Pentanediol 1,3-Pentanediol Glyceritol Erythritol u-Arabinitol Galactitol u-Glucitol D-Mannitol

in solvenP B

0.00 0.00 0.56 C.14 0.00, 0.35 0.05, 0.19

0.76 0.92 0.83

0.44 0.75 0.90 0.98 0.89 0.90

Solvent A is borate buffer of pH 9.2. Values of M a are calculated from the d a t a of Frahn and Mills.69 Solvent B is borate buffer27 of p H 10.

ions is closely analogous to the reaction of acetone with threo- and erythro1 ,2-di-C-phenyi-l , 2-ethanediol.'o The stereochemical implications involved in the cis cyclization reactions, of which the above are examples, have been discussed by Barton and Cookson.71Although it is understandable that 2,4-pentanediol should yield two components on subjection t o zone electrophoresis, it is difficult to see why 1,3-pentanediol should behave in a similar manner, since the two possible forms of the diol are enantiomorphs and should have a n equal affinity for borate ions. Gross16 has pointed out that, until the advent of zone electrophoresis, (69) J. L. Frahn and J. A. Mills, Chemistry & I n d u s t r y , 578 (1956). (70) P. H. Hermans, 2. physik. Chem., 113, 337 (1924). (71) D. H. R . Barton and R. C. Cookson, Quart. Revs. (London), 10, 44 (1956).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

103

there was no satisfactory micromethod for the separation of the hexitols shown in Table VII. The zone electrophoretic separation of galactitol, D-glucitol, and wmannitol is complicated by the fact that their mobilities lie within a iiarrow range. Attempts to resolve a mixture of the hexitols at low potential-gradients may be complicated by the normal diffusion of the migrating zones (an effect which will tend to cause the zones to overlap). The diffusion effects can be minimized sufficiently to permit a satisfactory resolution of the mixture by operating a t high potential-gradients.15 Gross was also able to separate these three hexitols from D-glucose, D-fructose, D-mannose, and L-sorbose. The mobility sequence of the hexitols may be critically dependent on the conditions of zone electrophoresis, since, a t pH TABLEV I I I Ma Values of Some Inositols72, 73 Inosilol ~

~~

myo-Inositol (+)-Inositol (-)-Inositol scyllo-Inositol epi-Inositol do-Inositol muco-Inositol Quebrachitol Pinitol Bornesitol Sequoyitol

I

MQ

0.53 0.63 0.63 <0.05 0.73 0.88 0.96 0.31 0.66 0.12 0.18

9.2, Frahn and Mills69 observed the mobility sequence of D-glucitol > D-mannitol > galactitol, whereas GrossI6 (using pH 9.2) and Bourne, Foster and Grant2' (using pH 10) observed the sequence galactitol > D-mannitol > D-glucitol. The zone-electrophoretic behavior of a number of inositols has been studied,72p7 3 and the M , values listed in Table VIII indicate a wide range of mobilities within the group. Recently, Angyal and McHugh68have postulated the formation of a novel kind of borate complex with certain inositols. They observed that inositols with cis related hydroxyl groups at C1, C3, and C5 give complexes with borate ions even when cis-1 ,2-glycol groupings are absent. For example, scyllo-quercitol (VII) is postulated to yield the '' tridentate" complex VIII. (72) A. B. Foster and M. Stacey, Chemistry & Industry, 279 (1953). (73) A. B. Foster, Chemistry & Industry, 591 (1953).

104

A. €3. FOSTER

OH

I

aH OH

HO

HO

OH VII

H

VIII

The stability of the tridentate complex and, hence, the position of the equilibrium between the tridentate and the free inositol will be dependent on the stereochemical disposition of the hydroxyl groups not linked to the boron atom in VIII. The greater the number of these hydroxyl groups in axial positions, the less stable will be the complex. This effect is elegantly illustrated by the magnitude of the equilibrium constant in the following quercitol and inositol series (the number of free hydroxyl groups in the tridentate in axial positions is given in parentheses after the cyclitol): scyllo-quercitol (2) 5.0; epi-quercitol (1) 310; cis-quercitol (0) 7.9 x lo3; myo-inositol (2) 25; epi-inositol (1) 7.0 X lo3;and cis-inositol (0) 1.1 X lo6. As the number of axially disposed free hydroxyl groups in the tridentate decreases, the equilibrium constant rises sharply. Zone electrophoresis has been employed in structural investigations of the mono-0-methylinosito1s.~z-74 g. Flavonoid G1ycosides.-Relatively little attention has so far been paid to the zone electrophoresis of flavonoid glycosides. Hashimoto and coworkers1*have reported the migrated distances given in Table IX.

2. Acidic and Basic Carbohydrates and Related Substances The zone-electrophoretic behavior of a few carbohydrate acids has been reported. Foster and Stacey*' demonstrated the mobility sequence of D-glucuronic acid > n-galacturonic acid, in a borate buffer at pH 10. Consden and StanierI7 noted that uronic acids and hexosamines may be separated from neutral sugars in borate buffers of suitable p H . I n the author's opinion there is much scope for the application of zone electrophoresis in structural investigations of polysaccharides containing uronic acids. Gross36has studied the mobility of a range of nonvolatile organic acids in non-borate buf(74) L.Anderson and A. M. Landel, J . Am. Chem. SOC.,76,6130 (1954).

105

ZONE ELECTROPHORESIS O F CARBOHYDRATES

fers. He observed a fairly good correlation between the dissociation constants and the mobilities. A conducting solution at pH 2 (0.75 M formic acid) was found most satisfactory; at higher pH values there was a tendency to form multiple spots. The following mobility sequence was observed by Gross? gluconic acid < 2-ketogluconic acid < a-ketobutyric acid < a-ketoglutaric acid < glucosyl phosphate < glyceritol 3-phosphate < fructose 1,6-diphosphate. The most suitable use of borate buffers will be in the separations of mixtures of isomeric polyhydroxy acids. The behavior of phosphate, triose phosphate, and hexose phosphate under a variety of conditions in nonborate buffers was examined by Neil and Walker:6 who found that the

Migration Distance2 Flauonoid Glycoside

TABLEIX f Some Flavonoid Cflycosidesla 'icinal cis-hydroxyl :roups in the sugar moiety

Vicinal hydroxyl groups in the aglycon

1 0 0

1 1 1 1

0 0 0 0 0 0

1 1 0 0 0 0

Myricitrin Rutin Quercitin Myrecetin Lutedin 7-glucoside Naringin Hesperidin Acacetin Morin Robinin

1

Migrationa distance ( t o cathode)

30 25 16 10 3 3 3 3 2 2

The buffer solution was 2% borax.

hexose monophosphates could not be separated from each other. These authors point out that an advantage of zone electrophoresis over chromatography is that useful fractionation of tissue extracts may be achieved directly. Separations of phosphates of certain isomeric carbohydrates are possible using borate buffers as shown by the mobility sequencedl: D-galactosyl phosphate > D-glucosyl phosphate, D-galactosyluronic acid phosphate > D-glucosyluronic acid phosphate. The speed of separation of acidic carbohydrates by zone electrophoresis is illustrated by the fact that, under suitable conditions,4l D-glucuronic acid may be separated from D-glucosyluroriic acid phosphate in 1.5 hours, whereas 4-6 days are necessary for effecting a resolution by paper chromatography. A considerable amount of attention has been focussed on the zone-electrophoretic behavior of components of nucleic acids, but this field has (75) N. W. Neil and D. G. Walker, Biochem. J . (London), 66, xxvii (1954).

106

A. B. FOSTER

recently been adequately reviewed by SmithT6and need not be further mentioned here. The separation of aminodeoxy sugars from neutral and acidic sugars may be readily achieved26using an acetate buffer at pH 5 (the N-acetyl derivatives of D-glucosamine and D-galactosaminehave characteristic M , value# in borate buffer at pH 10). M. C. Foster and A ~ h t o n ’have ~ found zone electrophoresis to be useful in the separation of streptomycin components. They observed the following mobilities ( M X lop5): streptomycin, 22.5; mannosidostreptomycin, 19.5; streptothricin, 24.0; streptidine, 24.9; and streptarnine, 6.3.

TABLEX Relative Migrations of Some Carbohydrates i n the Presence of Borate and Other Complexing A g e n t P Carbohydrate

D-Glucose D-Mannose D-Fructose D-Mannitol D-Glucitol Galactitol

I

I

Mobilitp Borax (1)

Arrenife (2)

17.0 11.7 15.1 14.1 15.7 12.8

1.0 2.4 5.1 7.5 6.3 9.3

I

Basic lead acetalc ( 3 )

0.7 4.5 2.8 2.8 3.6 4.6

a The figures quoted are the distances migrated (in cm.) under standard conditions. For the significance of solutions ( l ) , (2), and (3), see the text.

Although phenylhydrazones of carbohydrates have not been studied by zone electrophoresis, the behavior of phenylhydrazone derivatives of numerous a-ketoacids has been reported.78

IV. ZONE ELECTROPHORESIS OF CARBOHYDRATES IN THE PRESENCE OF COMPLEXING AGENTS OTHER THAN BORATE The zone electrophoresis of carbohydrates in the presence of complexing agents other than borate is a virtually unexplored field. Frahn and Mills69 have described some preliminary experiments in which the zone-electrophoretic behavior of carbohydrates in the following conducting solutions was compared: (1) borax, pH 9.2; (2) sodium arsenite-arsenious acid, pH (76) J. L). Smith, in “The Nucleic Acids,” E. Chargaff and J. N. Davidson, eds., Academic Press Inc., New York, N. Y., 1955, Vol. 1, p. 267. (77) M. C. Foster and G. C . Ashton, Nature, 172, 958 (1953). (78) W. J. P. Neish, Rec. t7an. chim., 72, 105 (1953); B. Mondori and F. Narazio, G i o ~ nbiochim., . 3, 259 (1954); H. Tauber, Anal. Chein., 27, 287 (1955).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

107

9.6; and (3) basic lead acetate. In solutions (1) and (a), the carbohydrates migrated as anions; they moved as cations in solution (3). The rates of migration in solutions ( 2 ) and (3) were much lower than those in solutiorl (1). The relative migrations are summarized in Table X, from which it may be seen that the sequence of increasing migration distances is not the same for each conducting solution. Of especial interest is the behavior (in the arsenite buffer) of the hexitols, which, in addition to migrating more rapidly than the sugars, are more widely separated from each other than in borax (compare Gross16). I n conducting solutions containing sodium tungstate (pH 6.2) and ammonium molybdate (pH 5.6), D-mannitol, D-glucitol, and galactitol migrated rapidly as anions on subjection to zone electrophoresis, but were not separated. Reducing sugars also migrated, but extensive streaking occurred. The behavior of carbohydrates on carbon-Celite columns69impregnated with molybdate has recently been described,7gand comparisons were made with their behavior on borate-impregnated columns6s. Zone electrophore~is~~ of the hexitols in sodium metavanadate (pH 8.6) revealed the mobility sequence : D-glucitol > D-mannitol > galactitol.

V. DETERMINATION OF MOLECULAR SIZEOF CARBOHYDRATES BY ZONEELECTROPHOEESIS The fact that carbohydrates of different molecular sizes [for example, pentoses and hexoses (see Table I)] may have identical M , values on subjection to zone electrophoresis (in borate buffers) indicates that this system cannot be used for determining the molecular size of a carbohydrate. It has been emphasized a t several points in this Chapter that the most potent use for zone electrophoresis of carbohydrates in borate buffers is after mixtures of carbohydrates have been resolved into groups of similar molecular size by chromatographic procedures. Zone-electrophoretic methods have recently been developed whereby the molecular weight of an aldose may be ascertained. The first method, due to Stacey and coworkers,80involves the conversion of the aldose into the N-benzylglycosylamine on the pherogram (by interaction with benzylamine). Thereafter, the glycosylamine is caused to migrate as the glycosylammonium ion by zone electrophoresis in a formic acid-sodium formate buffer (pH 1.8). The mobilities listed in Table XI are expressed relative to that of N-benzyl-n-glucosylamine. It was shown that the mobility of N-benzylglycosylammonium ions is inversely proportional to the mo (79) S. A. Barker, E. J. Bourne, A . B. Foster and R . B. Ward, Nature, 179, 262 (1957). (80) S. A. Barker, E. J. Bourne, P. M. Grant and M. Stacey, Nature, 177, 1125 (1956).

108

A. B. FOSTER

lecular weight of the ions and is independent of the stereochemistry of the sugar and of the configuration of the linkages in di- and oligo-saccharides. Frahn and Millss1 have shown that zone electrophoresis of aldoses in TABLE XI Relative Mobilities of Aldoses, as N-Benzylglycosylammonium IonsaQ and as B k ite Complexess1

-

Aldose or derivative

liobilily o j Nieneylglycosylmmonwm ton, relative to that zf N-bcneyl-Dglucosylomine

Pentose

1.09-1.15

6-Deoxyhexose Hexose

1.oo

0.91

liol. put.

Aldose or derivative

Mobility of isul&c com$lex, relative to thot of D-glucose

150 150 150 164 180 180 180 206

Heptose tetra-0-methylhexose 4,6-0-benaylidenehexose Hexose disaccharide

0.71-0.78

342 342 342 342 342

Hexose trisaccharide

0.59-0.63

504

Hexose tetrasaccharide Hexose pentasaccharide Hexose hexasaccharide

0.49-0.51

504 666

0.42

828

0.33

990

210 236 268

D-xylose D-ribose D-arabinose L-rhamnose D-glucose D-galactose D-mannose 4,6-O-ethylidene-~-glucose

1.13 1.16 1.15 1.07 1.00 1.02 1.00 0.97

2,3,4,6-tetra-O-rnethyl-~

0.90

glucose 4, 6-0-benzylidene-~-glucose cellobiose maltose lactose melibiose isomaltose maltotriose panose maltotetraose

0.85 0.70 0.71 0.69 0.71 0.70 0.55 0.55 0.45

-

aqueous sodium bisulfite results in a separation according to the molecular weights. The technique involves the introduction of the aldose onto the pherogram, which has already been impregnated with 0.4M aqueous sodium bisulfite, and a suitable interval of time must be allowed in order to permit the formation of the bisulfite complex. During migration, the com(81) J. L. Frahn and J. T. Mills, Chemistry &Industry, 1137 (1956).

ZONE ELECTROPHORESIS O F CARBOHYDRATES

109

plexes undergo slow decomposition, to an extent dependent on the nature of the aldose. Under the conditions of zone electrophoresis described b y Frahn and Mills,s1ketoses did not afford a migrating component. The relative mobilities as determined by each method are listed in Table XI, from which it may be seen that they are related to the molecular weight; this permits the determination of the molecular size of an aldose, a t least up t o a hexasaccharide. The decrease of mobility with increase in molecular weight is not linear. Frahn and Millss1 applied the bisulfite method in a study of the acidreversion products of D-galactose. It was shown chromatographically that, after reversion, a t least five reducing compounds are formed in addition to n-galactose. After elution from the chromatogram, these components could be classified as di-, tri-, or tetra-saccharides by determining their zoneelectrophoretic mobility in aqueous sodium bisulfite.

VI. ZONEELECTROPHORESIS OF CARBOHYDRATES ON GLASSPAPER There are some disadvantages associated with the use of paper strips in the zone electrophoresis of carbohydrates ; for example, numerous nonreducing carbohydrates are difficult to locate after migration, and certain polysaccharides, especially the amylosaccharides, tend to be adsorbed (see page 113). To a large extent, these difficulties may be overcome by using strips fabricated of fiber-gla~s.2~. S2 Fiber-glass sheets of structure and properties suitable for zone electrophoresis are now available commer~ially.8~ Smith and coworkers838have obtained the most satisfactory results with the fiber-glass sheets made by the National Bureau of Standards a s recommended by O’Leary, Hobbs, Missimer and E r ~ i n g . 8 ~ ~ The chemical inertness of the fiber-glass sheets permits the application of vigorous chemical reactions for detecting zones of migrated carbohydrate. For example, 0.5 % potassium permanganate in N sodium hydroxide will detect sugars, methylated sugars, sugar alcohols, lactones, sugar phosphates, and neutral and acidic polysaccharides.s2 The more stable methylated methyl glycosides, methylated polysaccharides, and acetal derivatives may be detected by using 5 % 1-naphthol in 10 N sulfuric acid.s2The spray reagents are applied to the dried pherogram at looo, and may be applied successively to the same fiber-glass sheet. Smith and coworkers82 point out that certain quantitative determinations of carbohydrates may be simplified by the use of fiber-glass, since no contaminant carbohydrate can be (82) D. R. Briggs, E. F. Garner and F. Smith, Nature, 178,154 (1956). (83) Supplied by H. Reeve Angel and Co., Ltd., Bridewell Place, London, England. (83a) Prof. F. Smith, private communication to Prof. M. L. Wolfrom. (83b) M. J. O’Leary, R. B. Hobbs, J. K. Missimer and J. J. Erving, T a p p i , 37, 446 (1954).

110

A. B. FOSTER

extracted from the fiber-glass as may be the case with cellulose sheets. Furthermore, by using a suitable washing treatment, the fiber-glass sheets may be reclaimed for further use. Bourne, Foster and Grantz7have studied the zone-electrophoretic behavior of a wide range of simple carbohydrates on fiber-glass and cellulose sheets under similar conditions. It was observed that, whilst D-glucose has an appreciably lower absolute mobility on cellulose than on fiber-glass, the M , values of the range of compounds studied were only slightly different (in general, < 0.05),and it was concluded that there was negligible selective adsorption on either of these electrolyte supports. The electroendosmotic flow for fiber-glass was toward the cathode, but it was very much higher than with cellulose and the migrating zones tended t o become more diffuse on fiber-glass than on cellulose. As noted earlier, however, diffusion effects may be diminished by operating at high potential-gradients (page 103). It is the author’s opinion that the use of fiber-glass sheets will be of great value in studying the zone-electrophoretic behavior of polysaccharides.*

VII. ZONE ELECTROPHORESIS OF POLYSACCHARIDES 1. Acidic Polysaccharides

One of the earliest investigations of the zone-electrophoretic behavior of carbohydrates was concerned with the separation of mixtures of certain acidic poly~accharides.~~ BlixS6had shown that the boundary-electrophoretic mobilities of hyaluronic acid acd chondroitin hydrogen sulfate are quite different. Gardell, Gordon and Aqvista4examined the zone-electrophoretic behavior of these mucopolysaccharides, using 0.1 M acetate buffer and slabs of powdered silica or diatomaceous earth (Hyflo Supercel) as the electrolyte support. The latter support was found to be the more effective. The mucopolysaccharides were located by cutting the slab into strips, eluting with water, and determining the carbohydrate content of the eluate by a colorimetric method. In this manner, mixtures of chondroitin hydrogen sulfate and hyaluronic acid (20 mg. of each) could be resolved. The observed higher mobility of chondroitin hydrogen sulfate is to be expected, since the mucopolysaccharide contains sulfate groups, whereas the hyaluronic acid does not. The method was subsequentlys4applied to an extract of pig skin, and it was qualitatively demonstrated that two components, with mobilities

* Since this manuscript was completed, the zone-electrophoretic behavior of several neutral polysaccharides (including yeast mannan and snail galactan) on fiberglass strips (and on silk) has been described in detail by K. W. Fuller and D. H. Northcote [Biochem.J . (London), 64, 657 (1956)]. (84) S. Gardell, A . H. Gordon and S. i q v i s t , Acta Chem. Scand., 4,907 (1950). (85) G. Blix, Acta Physiol. Scand., 1, 29 (1940).

111

ZONE ELECTROPHORESIS OF CARBOHYDRATES

similar to those of hyaluronic acid and chondroitin hydrogen sulfate, are present. RienitsX6later studied the zone-electrophoretic behavior of hyaluronic acid, chondroitin hydrogen sulfate, and heparin on paper strips in nonborate buffers; he found that, whereas hyaluronic acid can be separated from chondroitin hydrogen sulfate and heparin, the latter two mucopolysaccharides cannot be separated. The mucopolysaccharides could be, located by the use of a method similar to that of Gardell, Gordon and A q v i ~ t , 8 ~ or by staining the pherogram with Toluidine Blue. The mobility of hyaluTABLEXI1 Mobilities of Some Neutral and Acidic Polusaccharidesas MobililyD

Polysaccharids

(Ir X 10-6 cm.?hec.-l/v.-l) ~~

Heparin N-(2,4-Dinitrophenyl)heparin Chondroitin hydrogen sulfate (from bovine trachea) (from bovine septa) Pneumococcus polysaccharide, Type I Type I1 Type I11 Rhizobium radicicolum polysaccharide Alginic acid Dextr an Amylose

-13.8 -13.5 -10.7 -11.9 -9.1 -3.4 -8.3 -9.9 -12.9 0.0 0.0

Determined in 0.06 M barbiturate buffer of pH 8.5. Detected by Toluidine Blue (acidic polysaccharides)

.

ronic acid was observed to be dependent on its state of polymerization; the more degraded samples had a higher mobility. The highly polymerized preparations of hyaluronic acid may have been adsorbed on the paper t o some extent. I n this connection, it has been reported that the mucopolysaccharides from thyroid follicle tend to be adsorbed on the paper in zone electroph~resis.~~ Rien itP applied his method t o extracts of pig skin, and concluded that several mucopolysaccharides appeared to be present. The mobilities of the polysaccharides in Table XI1 were obtained by Pasternak and Kentes using a 0.06 M barbiturate buffer at pH 8.5. (86) K. G. Rienits, Biochem. J . (London), 63,79 (1953). (87) G. J. Hooghwinkel, G. Smits and D. B . Kroon, Biochim. et Biophys. Acta, 16, 78 (1954). (88) C . A. Pasternak and P. W. Kent, Research (London)] 6,486 (1952).

112

A. B. FOSTER

2. Neutral Polysaccharides

Kent and coworkerssgbriefly reported the detection, after application of zone electrophoresisin barbiturate and other buffers, of a number of neutral polysaccharides, although no details of mobilities were noted. In a borate buffer at pH 8.0, amylopectin was observed to migrate toward the cathode and to be stained red with iodine, whereas amylose is stained blue and remains stationary. Preece and Hobkirkgohave attempted the fractionation of water-soluble, cereal polysaccharides (obtained from rye and oats), using zone electrophoresis on paper in a borate buffer at pH 11 and an acetate buffer at pH 4.The fractions were located on the pherogram by segmenting it, eluting TABLE XI11 Zone-electrophoretic Separation of Cereal PolysaccharideP Cereal

Buffer

Mooemenl

1

I Rye

borate

Oats

borate

Oats

acetate

a Key: tected.

-+ cathode

stationary cathode stationary 4 anode --* cathode stationary + anode --f

I XylosP I Galaclose" I +++ ++ +

GlucosP

-I

f

+++ f

f

+++ -

+-

-

-

+-

-

++ ++ ++ ++ +++ ++ +++

Arabinosc?'

++

++ +++ +++ +++ +++ +++ +++

+++, major component; ++, minor component; +, trace; -, not de-

the segments, hydrolyzing the eluted carbohydrate, and then checking the sugars present by chromatography. The results shown in Table XI11 indicate that some fractionation was obtained; this permitted an interpretation of the composition of the original mixtures. In a detailed study of the zone-electrophoretic behavior of amylosaccharides on paper using a borate buffer (pH lo), Foster, Newton-Hearn and StaceyZ8showed that, whilst the amylopectin (blue value, 0.18) underwent appreciable migration toward the anode with but little adsorption of the polysaccharide in the path of movement, the pattern of movement of amylose (blue value, 1.35) depended on the amount of the polysaccharide put on the paper. Thus, a sufficiently small amount was adsorbed at the origin, but, with larger amounts, migration toward the anode occurred. A mixture of amylose and amylopectin was easily resolved, whereas, with (89) R. M. Greenway, P. W. Kent and M. W. Whitehouse, Research (London) 6 , 6s (1953). (90) I. A. Preece and R. Hobkirk, Chemistry & Industry, 257 (1955).

70NE ELECTROPHORESIS OF CARBOHYDRATES

113

phosphate and glycine buffers, little migration occurred and no separation could be achieved. Thus, it appeared that the migration both of amylose and amylopectin was due to the acquisition of a negative charge by complex formation with borate ions. As is known,62 methyl a- and @-D-glucopyranosides react with borate ions exclusively at the hydroxyl groups at C4 and C6, so that, in the amylosaccharides, which are linked CY-D-(~-i4), only the nonreducing chain-ends may react in this manner with borate ions. Since 4-O-methyl-~-ghcoseis known to react with borate ions (see Table 11),the reducing ends of the amylosaccharide chains may also react with borate ions. The contribution to the net negative charge of the borate complexes of amylose and amylopectin (from the reducing ends) would be expected to be the same, since there is only one reducing D-glucose unit per molecule. However, amylopectin has a much greater number of nonreducing chain-ends per molecule than has amylose, and glycogen has a still greater number. The observed mobility sequence: glycogen > ainylopectin > amylose is therefore to be expected. The probability of occurrence of other types of complex formation with borate ions, involving parts of the amylosaccharide molecules other than the end groups, must be considered. Both the a- and the p-Schardinger dextrin, in which all the hydroxyl groups at C4 are involved in glycosidic linkages, form complexes with borate ions. The Schardinger dextrins are well known for their ability t o form inclusion complexes with a variety of molecules, and it appears that a similar complex is formed with borate ions thereby conferring a negative charge on the cyclodextrins. The helical structure of the chains in amylose and amylopectin may similarly entrap borate ions. Foster, Newton-Hearn and Stacey28 provided examples of the application of zone electrophoresis to amylosaccharides in the presence of borate. thus, i t was possible to demonstrate some significant differences between amylose and amylopectin, on the one hand, and the synthetic amylosaccharides obtained by the action of P- and &-enzymes (from potatoes) on a-D-glucosyl ph~ sp h ate,~on' the other. The zone-electrophoretic behavior of the polysaccharide elaborated by Neisseria perJava was found to be unusual. Chemical investigationsgZhave revealed the polysaccharide to be a glycogen in type, but it has some rather unusual proper tie^.^^ On zone electrophoresis, it was found to be completely absorbed at the origin, whereas glycogens from other sources (ox liver, hog round-worm, bee-drone larvae, bass liver, human liver, sheep tapeworm, dog liver, frog liver, chicken liver, guinea-pig liverS*(b))all migrated toward the anode to a similar extent. (91) S. A. Barker, E. J. Bourne, S. Peat and I. A. Wilkinson, J . Chem. Soc., 3022 (1950); S. A. Barker, E. J. Bourne and I. A. Wilkinson, ibid., 3027 (1950). (92) (a) S. A. Barker, E. J. Bourne and M. Stacey, J . Chem. Soc., 2884 (1950); (b) M. Abdel-Akher and F. Smith, J . A m . Chem. SOC.,73,994 (1951). (93) S. A. Barker, A. Bebbington and E. J. Bourne, J . Chem. Soc., 4051 (1953).

114

A. B. FOSTER

These results emphasize the value of results obtained by the application of zone electrophoresis in the comparison of polysaccharides of supposedly similar chemical structure. An interesting study of the behavior of a range of neutral polysaccharides on subjection t o boundary electrophoresis in the presence of borate has been described by N o r t h c ~ t e . ~ ~ VIII. SEPARATIONS OF CARBOHYDRATES ON ION-EXCHANGE RESINS The technique of the separation of carbohydrates on a borate anion-exchanger was originated by Khym and Zill,96and a review of some of their results has been given by C ~ h nIt. is~ mentioned ~ in concluding this Chapter because of its relationship to zone electrophoresis. Briefly, the technique consists in eluting mixtures of carbohydrates from a column of strong-base resin (Dowex-1) by means of aqueous solutions of boric acid or sodium borate. Those sugars which react strongly with borate ions, thereby acquiring a high negative charge, will be more strongly sorbed by the resin than sugars which complex weakly with borate ions; and they will strongly resist elution from the column. Thus, there should be a relationship between the M , values of sugars (as determined by zone electrophoresis) and their affinity for a borate anion-exchanger: namely, the higher the M , value of a sugar, the more difficult it should be to elute that sugar from the borate anion-exchanger. The following sequences show the relative ease of elution96 and the M , values (in parentheses) for a series of simple sugars: ribose (0.77) >> fructose (0.90) > galactose (0.93) > glucose (1.00); and ribose (0.77) >> arabinose (0.96) > xylose (1.00). The correlation between M , value and affinity for the column is clear. Many separations of carbohydrates on borate anion-exchangers have been described by Khym, Zill, 9 6 * 97 These include the separation of pentoses, hexand their oses, heptoses, di- and tri-saccharides, deoxy sugars, sugar alcohols, and sugar phosphates. Other workerss8have studied sugar phosphates and methylated sugars,99and, in the majority of cases where a comparison may be made, D. H . Northcote, Biochem. J . (London), 68, 353 (1954). J. X . Khym and L. P. Zill, J . Am. Chem. SOC., 73,2399 (1951) ;74,2090 (1952). W. E. Cohn, in Ref. 76, p. 235. G. R. Noggle and L. P. Zill, Arch. Biochem. and Biophys., 41,21 (1952) ;M. A Chambers, L. P. Zill and G. R . Noggle, J . Am. Pharm. Assoc., 41, 691 (1952); J. X. Khym and W. E. Cohn, J . A m . Chem. SOC.,76, 1153 (1953); J. X. Khym, D. G. Doherty and W. E. Cohn, ibid., 76,5523 (1954) ;J. X. Khym and W. E. Cohn, Federation PTOC., 13, 241 (1954). (98) J. 0. Lampen, J . Biol. Chem., 204,999 (1953); M. Goodman, A. A. Benson and M. Calvin, J . A m . Chem. SOC.,77, 4257 (1955). (99) M. V. Lock and G. N. Richards, J . Chem. SOC.,3024 (1955). (94) (95) (96) (97)

ZONE ELECTROPHORESIS O F CARBOHYDRATES

115

the above noted relationship between the M , value and the column affinity obtains. An obvious value which stems from this correlation is that information may be provided (by 41, values) which will be of use in translating a microscale, zone-electrophoretic separation to a macroscale, borate anionexchange separation. It is of interest to note that borate-complex formation increases the affinity of a carbohydrate for a borate anion-exchanger and decreases its affinity for a carbon-Celite column.68

This Page Intentionally Left Blank

SUGAR NITRATES B Y JOHNHONEYMAN* AND J.

w. w. MORGAN?

Chemistry Department, King’s College, University of London, England

......

. . 117 . . . . . . . . . . . . . . . . 118

1. Nitric Acid. . .

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

118

. . . . . . . . . . . . . . . . 118 3. Nitric Acid in Chloroform. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 4. Dinitrogen Pentoxide in Chloroform.. . . . . . . . . . . 5. Nitric Acid in Acetic Anhydride. ..................................... 121 6 , Silver Nitrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . .........

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

V. Reactions.. . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................ 2. Reductive Denitration.. . . . . . . ......... .. 3. R.eactions with Sodium Iodide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Reactions with Sodium N i t r i t e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Reactions with Pyridine. ...................... 6 . Alkaline Hydrolysis. .......................... VI. Uses.. ......................................

123 125 125 127 128 129 130 134

I. INTRODUCTION This account, supplementary to the recent review1 of the chemistry of the nitrates of simple aliphatic alcohols, describes the preparation, properties, and reactions of carbohydrate nitrates, but deals neither with the important physical properties nor with the thermal and explosive decompositions of polysaccharide (especially cellulose) nitrates. Occasionally, such common nitrates as those of cellulose and glycerol are erroneously called ‘(nitro” compounds; throughout this article, the nitrate ester of an alcohol (as in R-O-NO2) is invariably referred to as a nitrate, with (‘nitro” used only when the -NO, group is attached t o a carbon atom, as in nitrobenzene.

* Present address : British Cotton Industry Research Association, Shirley Institute, Didsbury, Manchester 20, England. t Present address: British Celanese Ltd., Putteridge Bury, Near Luton, Bedfordshire, England. (1) R. Boschan, R. T. Merrow and R. W. Van Dolah, Chem. Revs., 66,485 (1955). 117

118

JOHN HONEYMAN AND J. W. W. MORQAN

11. PREPARATION Many of trhe reagents used for nitrating aromatic compounds are also successfully employed for preparing the nitrates of alcohols, including sugars. The esterification reactions are usually rapid, requiring from five minutes t o an hour, and are carried out a t or below room temperature. Care is required to avoid rise in temperature during the preparations, otherwise the exothermic reactions may proceed with extensive decomposition or even explosively. When the desired reaction is complete, the solution is poured into ice and water, sometimes containing sufficient sodium carbonate to prevent excessive acidity, and the nitrate separates and often crystallizes. The majority of the compounds are readily obtained pure by recrystallization, but some require to be first freed from acid by processing of a chloroform solution of the product. 1. Nitric Acid

Nitric acid alone has not been used extensively for making nitrates, although Colley2 prepared crystalline 2,3,4,6-tetra-O-acetyl-a-D-glucosyl nitrate by dissolving the corresponding chloride in fuming nitric acid.

2. Nitric and Sulfuric Acids The old-established, commercial preparation of cellulose nitrates of various degrees of substitution involves treating cellulose with a cold mixture of concentrated nitric and sulfuric acids, the extent of esterification being controlled mainly by the water content of the mixture. I n an investigation3 of the byproducts of this nitration, the mixed acids were used for preparing the nitrates of several sugars. Some of the nitrates reported were impure, including those of D-glucose, sucrose, and raffinose, but these workers obtained pure crystalline samples of the fully esterified derivatives of L-rhamnose, L-arabinose, lactose, maltose, trehalose, methyl a-D-glucopyranoside, and methyl a-D-mannopyranoside, as well as two isomeric anhydro-D-fructose trinitrates3" and both anomers of D-galactopyranose pentanitrate. Later, crystalline sucrose octanitrate was obtained by using a mixture of 100 % nitric and 100 % sulfuric acids? Like most esterifications of this kind, treatment of D-mannitol with the mixed acids gives the fully substituted compound, the hexanitrates 6 ; nevertheless, by employing care3

(2) A. Colley, Compt. rend., 76, 436 (1873). (3) W. Will and F. Lenze, Ber., 31, 68 (1898). (3a) These have now been shown t o be hexanitrates of di-D-fructose dianhydrides; A. Schwager and Y. Leibowitz, Bull. Research Council Israel, 6A. 266 (1956). (4) E. J. Hoffman and V. P. Hawse, J . A m . Chem. Sac., 41, 235 (1919). (5) N. Sokoloff, J . Russ. Phys.-Chem. Soc., 11, 136 (1879). (6) T.S. Patterson and A. R. Todd, J . Chem. Soc., 2876 (1929).

SUGAR NITRATES

119

fully controlled conditions, there has been isolated (in low yield) a pentanitrate,? the subject of further discussion later in this review. Galactitol hexaand penta-nitrates are obtained similarly.

3. Nitric Acid in Chloroform Esterification with mixed acids is often suitable for the preparation of carbohydrate nitrates, but sometimes a less vigorous method is required. For this purpose, a solution of anhydrous nitric acid in chloroform was introduced by Koenigs and Knorra and found suitable for preparing 2,3,4,6-tetra-O-acetyl-~~-~-glucosyl nitrate from the corresponding bromide and from P-D-glucopyranose pentaacetate (but not from the anomeric pentaacetate). Acetyl groups a t positions 2,3,4, and 6 are unaffected b y the reagent, but the bromine atom or the acetoxyl group with the p-Dconfiguration on C l is replaced by nitrate. The stability of the acetoxyl group attached to C l with the a-D configuration has been confirmed b y Behrend and Rothg when nitric acid containing phosphorus pentoxide was employed. They suggested use of this property for separating the a anomer from the mixed D-glucopyranose pentaacetates, since the CY anomer alone remains unreacted and undissolved. This difference in reactivity of the C1 acetoxyl groups toward nitric acid is, doubtless, similar to that obtaining in other replacement reactions (discussed by Lemieux) .lo Many esterifications have been accomplished with a solution of nitric acid in chloroform containing phosphorus pent0xide.l' In addition to reacting with free alcoholic groups, this reagent also opens the anhydro ring in the trimethyl ether and the triacetate of 1,6-anhydro-~-~-glucopyranose, giving finally the 1,6-dinitrates with the C ~ - Dconfiguration. The stability of the glycosidic methyl group to this reagent is apparent from the successful conversion of methyl 2,3-di-O-methyl-P-~-glucopyranoside to its 4,6-dinitrate.I2 I n this and many other nitrations, better yields were obtained by conducting the rapid reactions on a small scale, presumably because of the resulting closer control of time and temperature. Similarly, methyl 2,4-di-O-acetyl-~-~-xylopyranoside gives its crystalline 3-nitrate,I3 and methyl 2,3-di-O-methyl-a-~-galactopyranoside affords its crystalline 4,6-dinitrate.14 (7) (a) J. H. Wigner, Ber., 36,794 (1903). (b) G. G. McKeown and L. D. Hayward, Can. J. Chem., 33, 1392 (1955). (8) W. Koenigs s n d E. Knorr, Ber., 34, 957, 4343 (1901). (9) R . Behrend and P. Roth, Ann., 331, 359 (1904). (10) R. U. Lemieux, Advances in Carbohydrate Chem., 9, 1 (1954). (11) J. W. H. Oldham, J. Chem. Soc., 127, 2840 (1925). (12) J. W. H. Oldham and Jean K. Rutherford, J. A m . Chem. Soc., 64,366 (1932). (13) G. J. Robertson and T. H. Speedie, J. Chem. Soc., 824 (1934). (14) G. J. Robertson a n d R . A. Lamb, J . Chem. Soc., 1321 (1934).

120

JOHN HONEYMAN AND J. W. W. MORGAN

The glycosidic methyl group is stable to nitric acid in chloroform, but a disadvantage of this method of esterification is that the more acid-labile groups are removed and are replaced by nitrate ester groups. For example, methyl 4,6-O-ethylidene-/3-~-glucosideis converted by nitric acid in chloroform solution into methyl P-D-glucoside 2 , 3 , 4 6-tet,ranitrate,l6. and into the methyl 2,6-di-O-methyl-3,4-O-isopropylidene-a-~-galactoside corresponding 3,4-dinitrate.l7 Although methyl ethers are unaffected, the aromatic ring of benzyl ethers is nitrated. Thus, 1 , 2 , 4,G-tetra-0-acetyl3-O-benzyl-p-~-glucoseyields 2,4,6-tri-O-acetyl-3-0-(nitrobenxyl)-~-~-g~ucosy1 nitrate.'a The trityloxy group is also replaced by nitrate by use of this reagent, making convenient the preparation of methyl 2,3,4-tri-0acetyl-0-D-galactoside 6-nitratelg and the analogous a-D-glucosidez0derivative from the respective acetylated 6-trityl ethers. An elegant synthesis of methyl 6-O-acetyl-P-~-glucoside2,3,4-trinitrate involves replacement of an 0-(1-acetoxyethyl) group by nitrate, as follows. Methyl 4,g-O-ethylidene-p-D-glucoside 2,3-dinitrate (prepared by the method described in the following Section), subjected to acetolysis with acetic anhydride containing a trace of sulfuric acid, yields methyl 4-O-(l-acetoxyethyl)-6-0-acetylP-D-glucoside 2,3-dinitrate, which is converted by nitric acid in chloroform into the 6-acetate 2 , 3 ,4-trinitrate.16 4. Dinitrogen Pentoxide in Chloroform Gibsonz1reported that dinitrogen pentoxide is useful for esterifying carbohydrates and similar compounds. By keeping a mixture of tartaric acid and the pentoxide in a vacuum desiccator over sodium hydroxide, tartaric acid dinitrate was obtained. Similarly, galactaric (mucic) acid was converted into its tetranitrate,22 whereas, during treatment with mixed nitric and sulfuric acids, unreacted mucic acid separated from the reaction solution. The use by T . B. Clark of dinitrogen pentoxide in dry chloroform to prepare nitrates of sugar derivatives was reportedz3in 1934, but the method was in common use in the St. Andrews laboratories before then and was really introduced by J. W. H. Oldham. With this reagent, lJ2:4,5-di-0isopropylidene-D-fructose is converted into its 3-nitrate,z3 and methyl (15) (16) (17) (18) (19) (20)

(21) (22) (23)

D. J. Bell and R . L. M. Synge, J. Chem. Soc., 1711 (1937). J. Dewar, G. Fort and N. McArthur, J. Chem. SOC.,499 (1944). D. J. Bell and S. Williamson, J. Chem. Soc., 1196 (1938). I<. Freudenberg and E. Plankenhorn, Ann., 636, 257 (1938). J. W. H . Oldham and I). J. Bell, J . A m . Chem. SOC.,60, 323 (1938). E. K. Gladding and C. B. Purves, J. Am. Chem. SOC.,66. 153 (1944). G. E. Gibson, Proc. Roy. SOC.Edinburgh, A28, 705 (1908). A. C. Brown and G. E. Gibson, Proc. Roy. SOC. Edinburgh, A29, 96 (1909). T. N. Montgomery, J. Am. Chem. SOC., 66,419 (1934).

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121

4, G-0-ethylidene-p-u-glucoside mainly into its 2,3-dinitrate,l6 whereas nitric acid in chloroform yields methyl p-D-glucoside 2 , 3 , 4 , G-tetranitrate. Dinitrogen pentoxide in chloroform nitrates as readily as does a solution of nitric acid, but has the advantage that it does not so readily remove acid-labile groups, although better yields again result by conducting the reactions on a small (2 g.) scale. Nevertheless, some methyl p-D-galactoside 2 , 3 , 4 ,G-tetranitrate was isolated as a byproduct during the conversion, by dinitrogen pentoxide in chloroform, of methyl 3,4-O-isopropylidene-pD-galactoside into its 2,6-dinit1-ate.~~ The cleavage may be caused by nitric acid present originally as an impurity or produced as esterification proceeds. If sodium fluoride is added to the solution, nitric acid is sequestered (as a hydrogen-bonded complex) as fast as it is formed, the dinitrogen pentoxide being left unaffected. This ingenious method, introduced in order to effect complete nitration of starch while avoiding excessive degradation,26 was also successfully used in the preparation of the pentanitrates of D-gluconic and D-galactonic acids26;at the same time, the necessity for using freshly prepared solutions of nitrogen pentoxide in chloroform with sodium fluoride was demonstrated.

5 . Nitric Acid in Acetic Anhydride Anhydrous nitric acid in acetic anhydride plus acetic acid has been used for preparing the crystalline tetranitrates of the methyl D-glucopyranosides and also methyl 0-cellobioside heptanitrate on quite a large s ~ a l e . ~ 7 Nitration of a- and p-D-glucose led to the formation of the crystalline, anomeric pentanitrates.28 Fuming nitric acid in acetic anhydride, well known for effecting aromatic nitration, is convenient for preparing the nitrates of many sugar derivative^,^^ since it has advantages over nitrogen pentoxide in chloroform (including its easier preparation). Fuming nitric acid in acetic anhydride can be used under mild conditions for converting methyl 4,6-O-benzylidene-a-~-glucosideinto its 2 , 3-dinitrate,30 whereas dinitrogen pentoxide in chloroform causes simultaneous nitration of the aromatic gro~p.~1,32 Care must be taken to keep solutions of nitric acid in acetic anhydride a t or below room temperature, as, otherwise, vigorous (24) J. S. D. Bacon and D. J. Bell, J . Chem. Soc., 1869 (1939). (25) G. V . Caesar and M. Goldfrank, J. A m . Chem. Soe., 68, 372 (1946). (26) M. L. Wolfrom and A. Rosenthal, J . A m . Chem. SOC.,76, 3662 (1953). (27) L. Brissaud, MBm. services chim. Btat (Paris), 30, 120 (1943); Chem. Abstracts, 41, 715 (1947). (28) G. Fleury and L. Brissaud, Compt. rend., 222, 1051 (1946). (29) J. Honeyman and J. W. W. Morgan, Chemistry &Industry, 1035 (1953). (30) J. Honeyman and J. W. W . Morgan, J. Chem. SOC.,3660 (1955). (31) J. W. H . Oldham, J . SOC.Chem. Znd. (London), 63, 236T (1934). (32) J. Dewar and G. Fort, J . Chem. Soc., 492 (1944).

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JOHN HONEYMAN AND J. W. W. MORGAN

decomposition may occur; but, by observing this precaution, esterifications on a larger scale (10 8.) have been carried out in excellent yield. Fuming nitric acid in trifluoroacetic anhydride, a nitrating agent for aromatic compounds, has been used for preparing D-mannitol hexanitrate and D-glucitol hexanitrate.33 6. Silver Nitrate The action of silver nitrate on an ethereal solution of 2 ,3 ,4,6-tetra-Oacetyl-a-D-glucosyl chloride gives 2,3,4,6-tetra-O-acety~-@-D-g~ucosy~ nit ~ - a t eOther . ~ ~ preparative methods involving use of polar solvents invariably yield the a-D anomer, into which the @-D anomer is transformed by polar solvents. Indeed, the ethanol present in pharmaceutical (for example, U. S. P.) chloroform is sufficient to effect this anomerization. Silver nitrate, usually in acetonitrile solution, has been widely used for introducing a nitrate group at the primary carbon atom of a sugar derivative, by interaction with the deoxyiodo derivative. The latter is obtained by the preferential substitution of a primary tosyloxy group by an iodine atom, through interaction with sodium iodide in acetone. Methyl 2 ,3-di-O-acetyl-P-~glucopyranoside can be preferentially esterified to give the 6-p-toluenesulfonate, which has been converted to the 6-nitrate by applying the above reaction sequence.I2A similar example, in the pentose series, is the conversion of 1,2,3-tri-O-acetyl-5-deoxy-5-iodo-~-arabinose into the corresponding 5 - n i t ~ a t e .More ~ ~ information concerning the scope of this replacement reaction is contained in Tipson’s re~iew.~6

111. PHYSICAL PROPERTIES Nitrates of sugars and their derivatives are colorless compounds which do not deteriorate on storage at room temperature. Nearly all crystallize readily.37 Their solubility depends partly on any other substituents present in the molecule, but, in general, the introduction of nitrate groups increases the solubility in less polar solvents. Most sugar nitrates, prepared by the methods described in the preceding Section, are isolated by pouring the reaction solution into water. In many cases, the products solidify and are obtained pure by direct recrystallization from the appropriate solvent. Purification by distillation is not recommended because of the danger of expl0sion.~8Chromatography on alumina is outstandingly successful for (33) E. J. Bourne, M. Stacey, J. C. Tatlow and J. M. Tedder, J . Chem. SOC.,1695 (1952). (34) H. H. Schlubach, P. Stadler and Irene Wolf, Ber., 61,287 (1928). (35) P. A. Levene and J. Compton, J . B i d . Chem., 116, 189 (1936). (36) R. S. Tipson, Advances in. Carbohydrate Chem., 8 , 107 (1953). (37) E. G. Ansell and J. Honeyman, J . Chem. Sac., 2778 (1952). (38) J. C. Irvine and Jean K. Rutherford, J . A m . Chem. Sac., 64, 1491 (1932).

SUGAR NITRATES

123

purifying and separating mixtures of D-glucose nitrates, and derivatives thereof.30 37 Although fully nitrated monosaccharides reduce Fehling s ~ l u t i o n pre,~ sumably because of decomposition by the alkaline reagent, there is little evidence suggesting that they mutarotate. No indication of this is found in reports on the physical constants of the D-glucopyranose pentanitrates2* or the D-galactose pentanitrates. Although L-arabinose tetranitrate undergoes a rotational change in ethanol ([.ID -101.3' + -90" in 20 hours), this may well be evidence of slow hydrolysis. Nevertheless, tetra-o-acetylP-D-glucopyranosyl nitrate is converted rapidly into the (Y anomer in polar solvents.34 Mono- and di-nitrates of monosaccharides and their derivatives are sufficiently stable t o heat and shock to be handled without undue risk if caution is exercised, but the compounds become more sensitive as more nitrate groups are introduced. An interesting comparison of the stability of carbohydrate nitrates, made39 by measuring the loss in weight of different compounds a t loo", shows that esters of the hemi-acetal form, such as D-xylopyranose tetranitrate and the u-glucopyranose pentanitrates, are extremely unstable, whereas the nitrates of the corresponding methyl glycosides are more stable. I n compounds of the same chemical type, the stability increases with increase in molecular weight. Increased stability is considered to arise from steric hindrance.

IV. ANALYSIS When a compound containing a nitrate ester group is stirred with cold concentrated sulfuric acid, nitric acid is liberated. This is detectable with each of which gives an indiphenylaminell or N , N'-diphenylben~idine,~~ tense blue color. For the quantitative estimation of the nitrogen content of cellulose nitrates, the Dumas meth0.d has only limited value because of the explosive nature of these materials. Good results are obtained by using the Dupont nitrometer method, especially on the micro scale,(O or with the semi-micro adaptation of the Kjeldahl 42 For sugar nitrates, Oldham31boiled an acetic acid solution of the compound with excess titanous sulfate, and estimated the residual reducing agent with ferric alum. Possibly due to losses of nitrogen oxides during boiling, the calculation of nitrate content had to be based on 7.5 atoms of titanium, instead of the theoretical 8. (39) G. Fleury, L. Brissaud and P. Lhoste, Compt. rend., 224, 1016 (1947). (40) P. J. Elving and W. R. MoElroy, Znd. Eng. Chem. Anal. Ed., 14, 84 (1942). (41) T. E. Time11 and C. B. Purves, Svensk Papperstidn., 64, 303 (1951). (42) M. L. Wolfrom, J. H. Frazer, L. P. Kuhn, E. E. Dickey, S. M. O h , D. 0. Hoffman, R. S. Bower, A. Chaney, Eloise Carpenter and P. McWain, J . A m . Chem. Soc., 77, 6573 (1955).

124

JOHN HONEYMAN AND J. W. W. MORGAN

Good results were obtained from 13 sugar nitrates, including the methyl 4 ,6-0-(nitrobenzylidene)-p-~-glucosidemono- and di-nitrates, in which

the nitro group reacted like a nitrate. This method was found to require unusually careful control and was therefore replaced by a rapid, accurate procedure, based on use of Devarda’s alloy,43in which the nitrate group is reduced and estimated as ammonia. I n this method, the nitro group does not react similarly to the nitrate ester group. On the micro scale, Shepherd44 has successfully used a colorimetric method46 in which the nitrate group is removed as nitric acid, which is then converted into 5-nitro-2,4-xylenol. Determination of the nitrate group attached to a primary carbon atom depends on its unique replacement by iodine on interaction with sodium This method was successfully used for measuring the proportion of substitution a t C6 of partially nitrated cellulose. Anapproximate estimate of the labile nitrate on C1 of aldoses is made42by boiling the compound with barium carbonate in methanol and determining the liberated nitrate ion with the Nitron reagent.

V. REACTIONS The nitrate group is unaffected by most of the reagents used for achieving substitution in carbohydrates. To illustrate this stability, the following examples are cited. Methyl P-D-glucopyranoside 2 ,3-dinitrate is, on the one hand, converted by methyl iodide and silver oxide into its 4 ,6-dimethyl ether,15*47 and, on the other, by successive treatments with trityl chloride and with acetic anhydride in pyridine, into methyl 4-0-acetyl-6-0-tritylp-D-glucoside 2 ,3-dinitrate.48 Esterifications in pyridine proceed without complication, as shown by the ready preparation of the crystalline 2-henzoate and 2-p-toluenesulfonate from methyl 4,6-0-ethylidene-/3-~-glucoside 3-1itrate.~~ The nitrate group is also stable during the condensation of methyl P-D-galactopyranoside 6-nitrate with acetone.Ig On treatment with paraldehyde containing concentrated sulfuric acid, the benzylidene group of methyl 4,6-0-benzylidene-a-~-glucoside 2,3-dinitrate is replaced by e t h ~ l i d e n eA. ~nitrategroup ~ or C l of an aldoseis, however, exceptionally labile. Thus, both anomers of tetra-0-acetyl-D-glucopyranosyl nitrate are converted into 8-D-glucopyranose pentaacetate by heating them with sodium acetate plus acetic anhydride,s- 60 whereas similar treatment of methyl (43) (44) (45) (46) (47) (48) (49) (50)

J. Dewar and Gina W. Brough, J. SOC. Chem. Ind. (London), 66,207T (1936). D . M. Shepherd, J. Chem. SOC., 3635 (1953). H . Yagoda, Ind. Eng. Chem. Anal. E d . , 16, 27 (1943). G . E. Murray and C. B . Purves, J . A m . Chem. SOC., 62,3194 (1940). D. O’Meara and D . M. Shepherd, J . Chem. Soc., 4232 (1955). D . J. Bell and R . L. M. Synge, J. Chem. Soc., 836 (1938). J. Dewar and G. Fort, J. Chem. SOC.,496 (1944). Z. H. Skraup and R . Kremann, Monatsh., 22, 1037 (1901).

SUGAR NITRATES

125

4 ,6-O-benzylidene-cu-~-glucoside3-nitrate yields its 2-acetate1 without replacement of the nitrate group.30 1. Reactions with Acids

Fully nitrated sugars are reported to react with hot, concentrated hydrochloric acid with the evolution of chlorine? With dilute acids, sugar nitrates are generally stable, although a thorough investigation has not yet been made. For example, there is no record as to whether a nitrate group on a glycosidic carbon atom is affected by this reagent. The nitrate group on C3 of n-fructose has been supposed, without evidence, t o be particularly labile23(to explain the poor yield of n-fructose 3-nitrate resulting from acid hydrolysis of the corresponding di-0-isopropylidene acetal) , but, in other compounds, many instances of their stability have been usefully employed. Methyl P-n-galactopyranoside 2 ,6-dinitrate is readily obtained by the acid hydrolysis of its 3 ,4-0-isopropylidene acetal. Although the nitrate groups in methyl 4 ,6-0-ethylidene-P-D-glucoside2 ,3-dinitrate increase the resistance of the ethylidene group to acid h y d r o l y ~ i s49, ~this ~ ~ can still be removed without affecting the nitrate g r o u p ~Alternatively, .~~ the ethylidene group can be preferentially removed by mild acetolysis. With 0.1 % concentrated sulfuric acid in acetic anhydride, at room temperature for five minutes, the above 2 , 3-dinitrate yields methyl 4-O-(l-acetoxyethyl)-6-0acetyl-P-n-glucoside 2 ,3-dinitrate.lK Under more drastic conditions, however, replacements occur. With 10 % absolute sulfuric acid (hydrogen sulfate) in acetic anhydride, for 24 hours at O", the nitrates of several aldoses and polyhydric alcohols have been converted into their acetates in high yield.51 2. Reductive Denitration

Several methods have been used for the reductive removal of nitrate groups from sugars and their derivatives; in every instance, the product is the corresponding sugar alcohol. One early example is the reduction of Dmannitol hexariitrate to D-mannitol by hydriodic A method of wider application, using iron dust in boiling acetic acid, successfully removes the nitrate group from methyl 2 ,3 ,4-tri-O-methyl-~-~-glucoside 6-nitrate." Even better results were obtained by replacing iron dust with a mixture of iron and zinc dusts: this removes the nitrate group from methyl 6-deoxy6-iodo-2,3-di-0-methyl-~-~-glucoside 4-nitrate) but leaves the iodine atom unaff ected.12 Sodium sulfide in aqueous ethanol de-esterifies methyl 4,6-di-O-methyl(51) M. L.Wolfrom, R. S . Bower and G . G . Maher, J. Am. Chem. Soc., 73, 874 (1951). (52) E.J. Mills, J. Chem. SOC.,17, 153 (1864).

126

JOHN HONEYMAN AND J. W. W. MORGAN

P-D-glucoside 2,3-dinitrate.l6 Methyl 3,4-0-isopropylidene-2-O-methyl-PD-galactoside is obtained in almost quantitative yield after its 6-nitrate has been boiled for 20 minutes withl9 “ a solution of sodium hydroxide halfsaturated with hydrogen sulphide.” A mechanistic study of the de-esterification of butyl nitrate by this means has established, among other things, that alkaline hydrolysis is not involved.6a Reduction with sodium amalgam in aqueous ethanol is also successful, provided that alkali-labile groups are absent.23 Quantitative, reductive removal of nitrate groups by high-pressure, catalytic hydrogenation at room temperature is successfully accomplished with palladium-on-calcium carbonate (at 1500 pounds per sq. in.) or on charcoal (at 300 pounds per sq. in.).64 These reductions are believed to proceed just as smoothly at atmospheric pressure. Reduction of p-toluenesulfonate esters with lithium aluminum hydride may give the corresponding alcohol or hydr0carbon.3~In sugar derivatives where the sulfonate ester group is on a primary carbon atom (for example, C6 of aldohexoses), the “hydrocarbon” is obtained (for example, the 6deoxy sugar). The sulfonate ester group on a secondary carbon atom leads usually to the corresponding alcoho1,66 although there is evidence that the “hydrocarbon” may also be produced.66All nitrates so far examined are converted by lithium aluminum hydride into the parent alc0h01.~~~ b7 Reduction of simple aliphatic nitrates with hydrazine occurs smoothly a t room temperature in the presence of a platinum or palladium catalyst.68 Without catalyst, reaction is slow unless concentrated solutions are used. Primary aliphatic nitrates have been shown to undergo substitution (as the principal reaction, leading to alkylated hydraxines), as well as reduction to the alcohol. The substitution reaction is favored by increasing the concentration and by omitting aqueous ethanol as solvent.69 Sugar nitrates are reduced by hydrazine: even in aqueous alcoholic solution, production of the corresponding alcohol is apparently the only reaction. In boiling solution for 90 minutes (without catalyst), a high yield of methyl 4,6-O-benzylidenea-D-glucoside is obtained from the 2,3-dinit~ate,~O whereas the analogous (53) R.T.Merrow, S. J. Cristol and R. W. Van Dolah, J . Am. Chem. SOC.,76, 4259 (1953). (54) L.P. Kuhn, J . Am. Chem. Soc., 68, 1761 (1946). (55) H.Schmid and P . Karrer, Helv. Chim. Acta, 32, 1371 (1949). (56) R.Allerton and W. G. Overend, J. Chem. SOC., 3629 (1954). (57) L.M.Soffer, Elizabeth W . Parrotta and Jewel1 Di Domenico, J . Am. Chem. Soc., 74, 5301 (1952). (58) L.P. Kuhn, J . Am. Chem. SOC.,73,1510 (1951). (59) R.T.Merrow and R. W. Van Dolah, J . Am. Chem. Soc., 76,4522 (1954). (60) K. S. Ennor, T. C . Stening and J.-Honeyman, Chemistry & Industry, 1308 (1956).

127

SUGAR NITRATES

2 ,3-di-p-toluenesulfonate is unchanged. After this treatment, 1 ,2 : 3 ,4-di0-isopropylidene-D-galactoseis obtained from its G-nitrate. I n another reaction, the nitrate is replaced by acetate by carrying out the reduction with zinc dust and anhydrous hydrogen chloride in acetic anhydride.01

3. Reactions with Sodium Iodide I n this Section, the reactions of sugar nitrates are compared with those of sugar sulfonate esters. Sodium iodide in acetone solution (Finkelstein's reagents2) is most commonly used. By means of this reagent, a nitrate or a sulfonate ester group36 on a primary carbon atom is replaced by iodine.", l2

I

CHz--!

I

!

' i-

0-NOz

CHZ--/-O-SO2-CH3

+ NaI

+ NaI

I

I

-+

+ NaN03

CHZI

-+

CHzI

+ NaO-S02CH3

There is a marked difference between nitrate groups and sulfonate groups on secondary carbon atoms. Only a few cases are known where, under prolonged, vigorous reaction conditions, replacement of such a sulfonate group is effected. Methyl 2 ,3-di-0-methyl-4 ,G-di-O-tosyl-~-~-glucoside reacts with Finkelstein's reagent to give a nearly quantitative yield of methyl 6-deoxy-G-iodo-2,3-di-0-methyl-4-0-tosyl-~-~-glucoside.~~ By reaction at 135O for three days, 1 , 2,3 ,6-tetra-0-acetyl-4-0-mesyl-~-glucose gives 1 , 2 , 3 ,G-tetra-0-acetyl-4-deoxy-4-iodo-"~-glucose~~ in 46 % yield.63No proof was given for designating this product as a D-glucose derivative, yet inversion of configuration of C4 during replacement is certainly possible. Such a reaction has not been reported for a nitrate group which may, however, react with sodium iodide (more or less readily according t o its position), so that the nitrate group is replaced by a hydroxyl group but not by a n iodine atom. With sodium iodide in acetone (at 100' for about 20 hours), methyl 2,3-di-O-methyl-P-~-glucoside4,G-dinitrate gives methyl 6-deoxy6-iodo-2 ,3-di-O-methyl-P-~-glucosidetogether with a small proportion of its 4-nitrate.12 Analogous products arise similarly from methyl 3-0-acetyl2-O-methyl-~-~-glucoside 4, G-dinitrate.32 The different reactivities of nitrate groups on different secondary carbon atoms is shown by the reaction of methyl @-D-ghcopyranosidetetranitrate with sodium iodide.16The mixed (61) D. 0. Hoffman, R . 5. Bower andM. L. Wolfrom, J . Am. Chem. Soc., 69,249 (1947). (62) H . Finkelstein, Ber., 43, 1528 (1910). (63) B. Helferich and A. Gnuchtel, Ber., 71, 712 (1938).

128

JOHN HONEYMAN AND J. W. W. MORGAN

reaction products, after treatment with silver nitrate in acetonitrile to replace (by a nitrate group) the iodine atom introduced a t C6, were methyl p-D-glucopyranoside 2,6-dinitrate (isolated as its 3,4-diacetate) and a smaller amount of the 3,6-dinitrate. The nitrate group on C4 appears to react the most readily, and then that on C3. The over-all yield from the reaction was, however, low, and so caution is necessary in interpreting these results. This apparently enhanced reactivity of the group at C3 stands in contrast with the well-substantiated behavior of methyl 4,6-O-ethylideneP-D-glucoside 2,3-dinitrate and its analogs.30-37 With these, reaction with sodium iodide a t 100" for about 20 hours leads to good yields of the 3-nitrates. These reactions can equally well be conducted a t atmospheric pressure in boiling acetone, methanol, or p ~ r i d i n eIn . ~acetic ~ anhydride, methyl 4,6-O-ethylidene-P-~-glucoside2,3-dinitrate is converted by sodium iodide a t 100" into the 2-acetate 3-nitrate and, in the boiling solution, into the 2,3-dia~etate.~~ The mechanism by which a nitrate group on a primary carbon atom is replaced by iodine clearly involves the rupture of the carbon-oxygen bond illustrated earlier in this Section. What happens when a secondary nitrate group is replaced by a hydroxyl group is more obscure, but one suggestion30 is that the oxygen-nitrogen bond breaks as follows.

NOJ

+ NaI

-+

NaNOz

+ Iz

The sodium derivative reacts with solvent, or with water during isolation, to yield the secondary alcohol. This mechanism, although not fully substantiated, accounts for the production of sodium nitrite and iodine and for the invariable retention of the original configuration when a n asymmetric carbon atom is involved in the reaction. In the above cases, the nitrate group on C2 is apparently activated by the strongly electropositive nature of the nitrate group on C3. Similarly, the nitrate group on C3 is rendered labile to sodium iodide by the presence of a mesyloxy or tosyloxy group on3*C2

.

4. Reactions with Sodium Nitrite Sodium nitrite in boiling aqueous ethanoP can be used instead of sodium iodide for removing certain nitrate groups on secondary carbon atoms. I n fact, this solution has certain advantages, especially that the reaction solution does not discolor much. Several hours' treatment in this way of methyl 4 ,6-0-ethylidene- and methyl 4 ,6-O-benzylidene-c~-~-glucoside2 , S-dinitrate gives the corresponding 3-nitrate, The remaining nitrate group on

SUGAR NITRATES

129

C3 is again resistant to reaction unless activated by a sulfonyloxy group on c 2 .6* When the nitrate group is on a primary carbon atom, a replacement reaction analogous to that with sodium iodide does not occur. If reaction does take place, the nitrate is converted into the primary alcohol. After several days' boiling in sodium nitrite solution, 1,2 ,3 ,4-di-0-isopropylidene-D-galactose 6-nitrate was recovered unchanged. The nitrate group in 3 ,5-0-benzylidene-1 ,2-O-isopropy~idene-~-glucose 6-nitrate is more readily removed: after 14 hours' boiling, 34 % had reacted.60 5. Reactions with Pyridine Sugar derivatives containing one or two nitrate groups are stable to cold pyridine, which has, in fact, been used in the esterification of such compounds. Tetranitrates of methyl a- and p-D-glucopyranosides are recovered to the extent of 60% after 16 hours in anhydrous pyridine, although unidentified, highly colored substances are D-Mannitol hexanitrate7(") 86 and galactitol hexanitrate7cb) are, however, converted readily and exothermically by pyridine, or by ammonium carbonate in aqueous acetone,66into 1 ,2 ,3 ,5 ,6-pentanitrates. In the denitration by pyridine, nitric and nitrous oxides and nitrogen are evolved. Moisture is required in the pyridine in order to provide the proton which replaces the nitronium ion (NOz@). The use of rigorously dried pyridine greatly reduced the yield, but, as the water content was increased, the yield roseB7 to a maximum of 75 %. Cellulose nitrate is greatly degraded by pyridine. To avoid this (by converting into the oxime any carbonyl groups produced) , Segall and Purves added hydroxylamine to the reaction mixture.'j* The resulting, partially denitrated cellulose (1.7 nitrate groups for each D-glucose unit) contained only a trace of oxime (0.08 per D-glucose unit). With hydroxylamine hydrochloride, one oxime group per D-glucose unit was introduced. Similar results were obtained with methoxyamine and its hydrochloride. Pyridine or hydroxylamine, separately, have but little effect on methyl P-D-glucopyranoside tetranitrate, but together they initiate a vigorous reaction a t room temperature. From the resulting mixture, methyl P-D-glucopyranoside 2,3,6-trinitrate (28 %) and 3 ,&dinitrate (17 %) were isolated.'j4Here again, the nitrate on C4 is most readily removed. When methyl 4,6-O-benzyli(64) L. D. Hayward and C. B. Purves, Can. J . Chem., 32, 19 (1954). (65) L. D. Hayward, J . Am. Chem. Soc., 73, 1974 (1951). (66) D. E . Elrick, N . S. Marans and R. F. Preckel, J. A m . Chem. Soe., 76, 1373 (1954). (67) J. R . Brown and L. D. Hayward, Can. J . Chem., 33, 1735 (1955). (68) G. H . Segall and C. B. Purves, Can. J. Chem., 30, 860 (1952).

130

JOHN HONEYMAN AND J. W. W. MORGAN

dene-a-D-glucoside 2,3-dinitrate is heated at 100" with hydroxylamine hydrochloride in pyridine, a small amount of the crystalline dioxime of methyl 4,6-O-benzylidene-2,3-dideoxy-2,3-diketo-a-~-erythro-hexoside (I) is isolated.30

6. Alkaline Hydrolysis

In this field, mildly alkaline solutions do not remove nitrate groups. Catalytic quantities of sodium in methanol remove only the acetyl groups from sugar acetate nitrates. In this way, methyl P-D-galactopyranoside 6-nitratelg and methyl P-D-glucoside 3, 4-dinitrateB9are obtained from their acetates. Similarly, a 5 % solution of dimethylamine in ethanol selectively deacetylates methyl 2,3,4-tri-O-acetyl-P-~-ghcoside6-nitrate." Treatment of 6-O-acetyl-l , 2-O-isopropy~idene-~-glucose 3,5-dinitrate with a more concentrated solutioii of dimethylamine removes the nitrate group from C3 , as well as causing deacetylati~ii,~~ whereas, under similar conditions, only the acetyl groups of methyl 2,4-di-O-acetyl-P-~-xyloside3-nitrate are removed.I3 There are three main reactions of simple alkyl nitrates with ethanolic potassium hydroxide; these are as follows. (1) Ether formation: R-CH2-O-NO2 KOH

+

+ C2H60H

-+

R-CH2-O-C2H6 (2) Alcohol formation: R-CH2-O-NO2 (3) Carbonyl formation: R-CHz-O-NO2

+ HzO + KNOa

+ KOH * R-CH20H + KN03 + KOH -+ R-CHO + H20 + KNO2

With most of the compounds studied, the three reactions were considered to proceed simultaneously, although methyl nitrate gave the ether exclusively, and benzyl nitrate was converted almost quantitatively into ben~aldehyde.~~ There is some similarity between nitrates, halides, and sulfonates, although, with nitrates, formation of olefins has minor impor(69) D.J. Bell and R. L. M. Synge, J. Chem. SOC.,833 (1938). (70) D.J. Bell, J . Chem. SOC.,1553 (1936). (71) J. U. Nef, A m . , 309, 126 (1899).

131

SUGAR NITRATES

tance, whereas the reaction leading to a carbonyl compound plus inorganic nitrite is significant. A kinetic study of the hydrolysis of alkyl nitrates72 has led to postulation of the following mechanisms.

Nucleophilic substitution (alcohol formation)

+

OH'

CHzR

I

CHzONOz

+ I

CHzR CHzOH

+ NO$'

&Hydrogen elimination (olejin formation) OHe

+

CHzR

I

CHR

--f

Hz0

CHz ON02

+ 11

CHz

+ NOS@

a-Hydrogen elimination (carbonyl formation) OH'

+

CHzR

I

CHzONOe

CHzR

+

He0

+ I

CHO

+ NOz'

I n this work the organic products were not isolated, and the possibility of ether formation was not considered. With methyl, ethyl, isopropyl, and terl-butyl nitrates, the principal reaction was nucleophilic substitution, but the proportion of the elimination reactions increased with increasing temperature and decreased with increasing polarity of the solvent. I n carbohydrate derivatives having several nitrate groups, the production of inorganic nitrite plus carbonyl compounds is the chief reaction. Because the alkaline reagents attack the carbonyl compounds as they are produced, the isolation and identification of organic reaction products is difficult. Considerable amounts of nitrite are present after the alkaline hydrolysis of the nitrates of ethylene glycol, glyceritol, erythritol, D-mannitol, and galactitol (dulcitol) .73 The action of alcoholic alkali on some alkyl P-D-ghcopyranoside tetranitrates has been found to lead to the formation of three molecular proportions of nitrite ion per molecule of the tetranitrate. The alkali consumed in excess of the theoretical four molecules was assumed to be involved in the further degradation of the initial ketonic products. No organic compound was identified after the reaction, but methyl p-Dglucopyranoside was not present after the hydrolysis44of methyl p-D-glUC0pyranoside tetranitrate. The reactions of compounds containing one nitrate group a t C1, C2, or C6 have been compared with those of the corresponding halides and sulfonate esters.74 Barium carbonate in boiling methanol converts 2 , 3 , 4 , 6 (72) J. W. Baker and Dorothy M. Easty, J. Chem. SOC.,1193, 1208 (1952). (73) L. Vignon and I . Bay, Compt. rend., 136, 507 (1902). (74) E. K. Gladding and C. B. Purves, J . Am. Chem. SOC., 66, 76 (1944).

132

JOHN HONEYMAN AND J. W. W. MORGAN

tetra-0-acetyl-a-D-glucosyl nitrate8 and 2,3,4-tri-O-methyl-a-~-glucose 1,g-dinitratell into the corresponding methyl p-D-glucopyranosides. With sodium methoxide in methanol, 2,3,4,6-tetra-O-acetyl-a-~-glucosyl nitrate gives methyl p-D-ghcopyranoside (28 % yield of product, after re-acetylation), whereas, with sodium hydroxide in aqueous dioxane, 1,6-anhydroP-D-glucopyranoseis obtained (33 % of product, after methylation). Methyl 2,3,4-tri-O-acetyl-a-~-glucoside 6-nitrate reacts in aqueous or alcoholic alkali to give a high yield of methyl 3,6-anhydro-a-~-glucopyranoside. These reactions of the nitrate on C1 or on C6 are accompanied by the formation of only small proportions of nitrite, and lead to the products which are obtained in a similar manner from the corresponding halides and sulfonate esters. I n the case of a 6-nitrate where C3 does not bear a free hydroxyl group after hydrolysis (for example, methyl 2,3,4-tri-O-methyl-a-~-glucoside 6-nitrate) , the alcohol (methyl 2,3,4-tri-O-methyl-a-~-glucoside) is slowly produced (final yield, about 75 %) ,the amount of nitrite being higher at about 20 % yield of the original nitrate. Although hydrolysis of methyl 3,4,6-tri-O-acetyl-a-~-glucoside2-nitrate leads to the formation of only 2 % of nitrite ion, the reaction appears to be complex. No pure product was isolated, but the resulting sirup (from its analysis and its resistance to oxidation by periodate) was considered to be essentially a mixture of methyl anhydro-a-~-hexosides.~~ Cautious interpretation of these results gave reason for believing that the nitrate group on C2 had been removed like a similarly situated sulfonate group. Further t o investigate this similarity between nitrates and sulfonates, methyl 4,6-O-benzylidene-(and -alkylidene)-a-D-glucoside 2,3-dinitrates were treated with sodium methoxide in methanol30'37 under the conditions used for converting the 2,3-ditosylates into the 2,3-anhydro-a-~-allosides. Much degradation occurred, even a t O", and a large proportion of nitrite was formed. The only products isolated were the 2- and 3-nitrates of methyl 4,6-O-benzylidene-a-~-glucoside (in low yield). In boiling methanol, there was an over-all yield of 7.5%, consisting of approximately equal parts of the 3-nitrate, the 2,3-anhydro-a-~-alloside,and methyl 4,6-0-benzylidenea-D-ghcoside. Production of this last compound shows that both nitrate groups are partly removed (with some difficulty) in the manner of hydrolysis of carboxylic esters, without inversion of configuration or anhydro compound formation, by the breaking of the oxygen-nitrogen bond. Formation of the anhydro-a-D-alloside shows, however, that the dinitrate also reacts like the corresponding ditosylate, that is, the group on C2 is removed by oxygen-nitrogen bond fission, whereas that on C3 comes off through carbon-oxygen fission with inversion of configuration. The bonds broken are shown by the arrows in the accompanying formulas.

133

SUGAR NITRATES

1 1 0zN-OCH 1 1

HCO-NO2

I

\

2,3-Dinitrate

HCO-CO 1 1

\+

I

HCOH

I

HOCH CHS

//

I

1 '

CHICO-OCH

I

I 2,3-Diacetate

' 1 I

HCO-NO, 02NO-CH

TI

2,3-Dinitrate

1 1

>+"I>(> HC

HCO-SO2-CaH4CH3-p

I

I

2,3-Anhydro-a-Dalloside

By the use of water containing OIS, the existence of these two modes of heterolytic fission has been confirmed for the alkaline hydrolysis of nit r a t e ~ Extensive .~~ decomposition, accompanied by the formation of inorganic nitrite, also occurred during the alkaline hydrolysis of the anomers of methyl 4 ,6-0-ethylidene-~-glucoside 2,3-dinitrate. The production of carbonyl compounds was confirmed by including o-phenylenediamine in one reaction mixture; a low yield of the quinoxaline derivative of methyl 4,6-0benzylidene-2,3-dideoxy-2 ,3-diketo-a-~-erythro-hexoside (I) was obtainedPO Confirmation for the positions assigned to bond fission during the removal of the nitrate groups on C3 is obtained from the results of the alkaline hydrolysis of methyl 4,6-O-benzylidene-c~-~-glucoside 3-nitrate in boiling methanol. Decomposition was greatly reduced ; the parent D-glucoside derivat,ive (35 %) and the 2,3-anhydro-a-~-alloside(21 %) were obtained. (75) M. Anbar, I. Dostrovsky, D. Samuel and A . D. Yoffe,

(1954).

J. Chem. Soe., 3603

134

J OHN HONEYMAN AND J. W. W. MORGAN

When the hydrolysis was conducted at room temperature, the compounds isolated included the unchanged 3-pitrate (44 %) and the corresponding 2-nitrate (5 %). Hence, in this reaction, some nitrate group migrated30from C3 to C2. This is the only recorded instance of such a movement, and the small extent to which i t occurred justifies the general confidence in the stability of nitrate groups against migration. Alkaline hydrolysis of the 2-nitrate 3-tosylate and of the 3-nitrate 2tosylate of methyl 4 ,6-O-benzylidene-a-~-glucosidegave the products to be expected if initial removal of the C2 substituent is assumed. Like the 3-tosylate1 the 2-nitrate 3-tosylate gave methyl 2,3-anhydro-4 ,6-0-benzylidene-cu-D-alloside, whereas the 3-nitrate 2-tosylate gave the same mixture of products as is obtained from the 3-nitrate. Nevertheless, over-all yields were low in these cases and some nitrite ion was These results of alkaline hydrolysis are summarized by stating that a single nitrate group, attached to a primary or glycosidic carbon atom of a sugar, behaves like a sulfonate group or a halogen atom. When attached to a secondary carbon atom, the nitrate may be hydrolyzed like a carboxylic acid ester or like a sulfonate, but with the additional reaction leading to carbonyl compounds plus inorganic nitrite becoming apparent. In compounds having more than one nitrate group, carbonyl-compound formation predominates.

VI.

USES

Cellulose nitrate is manufactured on a large scale, as the basis for plastics and lacquers. The amount of esterification is controlled a t about 2.2 nitrate groups per D-glUCOSe unit, to give a product which is not explosive and which is soluble in a wide range of inexpensive solvents. The inflammability of the resulting materials has probably limited their further development. Some of the carbohydrate nitrates containing a high proportion of nitrogen have been used as explosives. In addition to the widely used cellulose nitrates and glycerol nitrates, the hexanitrate of D-mannitol is employed in the United States in blasting caps and in explosive rivets. The nitrates of D-glucose, D-mannose, methyl a-D-ghcopyranoside, D-glucitol, and sucrose have all been investigated and considered for commercial exploitation. Starch nitrate has been employed in some types of munitions. The ability of certain nitrates, especially glycerol trinitrate, to lower the blood pressure has been used clinically in the treatment of hypertension and angina pectoris. A correlation was established between the rate of alkaline hydrolysis and the effectiveness of the nitrate in lowering the blood pressure: D-mannitol hexanitrate, hydrolyzed more rapidly than glycerol

SUGAR NITRATES

135

trinitrate, was 50 % more effective.’6 Crystalline 1,4: 3,G-dianhydro-Dglucitol 2,5-dinitrate is especially active physiologically.77 This effect of nitrates has been reviewed.78 The chief chemical use of such nitrates has arisen from opportunities to take advantage of their stability, the easy, selective removal of the nitrate groups from certain positions, and their ready and complete denitration by reductive processes. These properties have permitted preparation of several new methyl ethers of ~-glucose,~*. 49 u - g a l a c t o ~ eand , ~ ~~-fructose.*~ (76) R. F. Herrman, C. D. Leake, A . S. Loevenhart and C. F. Muehlberger, J. Pharnzacol. Exptl. Therap., 27, 259 (1926). (77) L. Goldberg, Acla Physiol. Scand., 16, 173 (1948). (78) W. F. Oettinger, Natl. I d s . Health Bull., NO. 186 (1946).

This Page Intentionally Left Blank

BENZYL ETHERS OF SUGARS

BY CHESTERM. MCCLOSKEY California Institute of Technology* and Office of Naval Research

I. Introduction.. ................ ... . . . . . . 137 . , , , , , , , , . , , , . , 142 11. Preparation . . . . . . . . . . . . d a Beneyl Halide,, . . . . . . . . . . . . . . . . . . . . . . 142 1. With an Alkali Hydro . . . . . . . . . . . 144 a. Preparation of 1,6-Anhydro-2,4-di-O-benzyl-~-~-glucose. b. Preparation of 6-O-Beneyl-3,5-O-benzylidene-l, Z-o-isopropylidene-~. . . . . . . . . . . . . . . . . 145 glucose . . . . . . . . . . . . . . . . . . . . . . . . . 2. From an Alkali Salt of a Sugar and a Beneyl Halide.. . . . . . . . . . . . . . . . . . 145 a. Preparation of 3-0-Benzyl-1 I 2:5,6-di-O-isopropylidene-~-glucose 3. By Opening of an Epoxide Ring.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Hydrogenolysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Catalytic Methods.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Chemical Methods.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 ..................... 150 IV. Chemical Properties. ..................... 1. Action of Alkaline Reagents.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 150 2. Action of Acidic Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 a. Aqueous and Alcoholic Acids.. . . . . . . . . . . . . . . . . . . . . . . b. Acetolyzing Reagents.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 c . Hydrogen Bromide-Glacial Acetic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 d. Mercaptan-Hydrochloric Acid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 e. Nitric A ci d . . . . . . . . . . . . . . . . . . . .......................... 152 152 3. Action of Oxidizing Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 4. Action of Reducing Agents.. . . . . . . . . . . . . . . . . . . . V. Physical Properties. . ................................................ 153 154 Table of Benzyl Ethers of Sugars and Some of Their Derivatives

I. INTRODUCTION The benzyl ethers of sugars comprise one of the important classes of derivatives employed in sugar chemistry. These compounds are of value synthetically because the benzyl group can be readily removed under mild conditions by hydrogenolysis according t,o the following reaction. CaHaCHzOR

+

Hg

---t

+

C O H ~ C H ~HOR

This property, together with the availability of methods for the preparation of certain of these ethers (in high yield) and their relatively inert *Contribution No. 2031 from the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, California.

137

138

CHESTER M. MCCLOSKEY

nature in general, has made them especially useful. A notable example is that in which it is desired to protect a hydroxyl group during a chemical manipulation and to uncover it later without fear of migration or inversion. Of particular value to the sugar chemist is the ability to remove by hydrogenolysis the benzyl group from a benzyl glycoside or sugar ether without reduction of the sugar. The first established preparation of benzyl ethers of sugars was described by Gomberg and Buchlerl in 1921, a contemporary claim2 in the patent literature not being substantiated by revelation of experimental details. This was followed by the synthesis of S-O-benzyl-~-glucosein 1925 by Freudenberg, von Hochstetter and Engels.3 Freudenberg and vom Hove: in 1928, contributed the next significant step, the application of hydrogenolysis to sugar derivatives. They demonstrated that benzyl ethers of sugars can be split chemically by sodium and alcohol or catalytically by hydrogen and platinum in glacial acetic acid. In the same year, Kariyone and Kondo6 (see Richtmyere for a discussion of this work) split aucubin and arbutin by the action of hydrogen and a platinum catalyst. working with non-sugar Following the lead of scattered systems, Fischer" in 1931, and Fischer and BaerI2,l3 in 1932, reported their success in synthesizing derivatives of glycerose by utilizing its benzyl cycloacetals. These acetals were converted to the substituted glycerose by hydrogenolysis in glacial acetic acid employing palladium as the catalyst. RichtmyeP in 1934 clearly demonstrated the excellence of palladium as a catalyst for the hydrogenolysis of benzyl ethers, its superiority over platinum, and its suitability for the hydrogenolysis of benzyl glycosides. He (1) M. Gomberg and C. C. Buchler, J. Am. Chem. Soc., 43, 1904 (1921). (2) L. Lilienfeld, British Pat. 149,320 (1920); Chem. Abstracts, 16,436 (1921). (3) K. Freudenberg, H. von Hochstetter and H . Engels, Ber., 68, 666 (1925). (4) K. Freudenberg, W. Diirr and H . von Hochstetter (with H. vom Hove, W. Jacobi, A. Noe and E. Gartner), Ber., 61,1735 (1928). (5) T. Kariyone and K. Kondo, J . Pharm Soc. Japan, 48, 684 (1928) ; Chem. Abstracts, 23, 393 (1929). (6) N. K. Richtmyer, J. Am. Chem. Soc., 66,1633 (1934). (7) K . W. Rosenmund and F. Zetzsche, Ber., 64, 2038 (1921); 0. Wolfes and W. Krauss, German Pat. 407,487 (1923) ;P. Friedlaender, Fortschr. Teerfarbenfabrikation, 14,421 (1925). (8) T. Kariyone and Y. Kimura, J . Phurm. Soc. Japan, No. 500,746 (1923) ; Chem. Abstracts, 18,386 (1924). (9) W. Krauss, German Pat. 417,926 (1924) ; P. Friedlaender, Fortschr. Teerjurbenfabrikation, 16, 98 (1928). (10) N. M. Carter, Ber., 63, 1684 (1930). (11) H. 0. L. Fischer, 2.angew. Chem., 44, 187 (1931). (12) H. 0. L. Fischer and E. Baer, Ber., 66, 337 (1932). (13) H. 0. L.Fischer and E. Baer, Ber., 66, 345 (1932).

BENZYL ETHERS OF SUGARS

139

also demonstrated the specificity of the catalyst, showing th a t phenyl and 2-phenylethyl ethers are not split by hydrogen with palladium catalysts. The next advance was the introduction of a simple method for the preparation of benzyl ethers in good yield by Zemplh, Csuros and Angyal in 1937.14 Freudenberg and P1ankenhorn,16 in 1938, demonstrated the synthetic value of the benzyl group, employing it in the synthesis of 1 ,2 ,4 ,6 tetra-0-acetyl-P-D-glucose and 2,4,6-tri-O-methyl-~-glucose.I n 1940, numerous papers utilizing the benzyl ethers for the synthesis of various sugar derivatives began to appear. Benzyl ethers have been employed in synthesis of 2 3 and 4-0-methyl-,17-19 2 ,4-,20 2,6-,2l 3,5-,22 4 , 6-,23-26and 3-05,6-di-O-meth~l-i~~ and 2 ,3 , 5-22and 3,4,6-tri-O-methyl-~-ghcose~~; methyl-~-glycerose'~;D-glycerose 3-phosphate12 27 ; L-idose28; 6-deoxy-~idoseZ8;3-O-methyl-~-fucose( d i g i t a l o ~ e ) ~2,3-di-O-methyl-~-rhamnose~~; ~; 2-amino-2-deoxy-6-0-methyl-~-glucose~~ ; and adeno~ine.3~ A typical use of benzyl ethers is the synthesis of n-glycerose 3-phosphate by Ballou and Fi~cher.2~ 1,3: 4,5-Di-O-methylene-~-mannitol(I) was benzylated (to give 11) and converted by selective a ~ e t o l y s i sto~ ~2,5-di0-benzyl-n-mannitol (111). Oxidation with sodium periodate gave 2-Obenzyl-D-glycerose (IV). This was converted, first, to the diethyl dithioaceta1 (V), and then to the dimethyl acetal (VI), which was phosphorylated with diphenyl phosphorochloridate to give VII. Hydrogenolysis of VII, first with a palladium catalyst to remove the benzyl group, followed by a 3

(14) G. ZemplBn, Z. Csiiros and S. Angyal, Ber., 70, 1848 (1937). (15) K. Freudenberg and E. Plankenhorn, Ann., 636, 257 (1938). (16) F. Weygand and 0. Trauth, Chem. Ber., 86,57 (19523. (17) 1). I. McGilvray, J . Chem. SOC.,3648 (1952). (18) J. Kenner and G. N. Richards, J . Chem. SOC. 1810 (1955). in press. (19) C. M. McCloskey and C. G. Niemann, J . Am. Chem. SOC., (20) Mildred H. Adams, R . E. Reeves and W. F. Goebel, J. Biol. Chem., 140, 653 (1941). (21) K. Freudenberg and G. Hull, Ber., 74, 237 (1941). (22) G. H. Coleman, S. Brandt and C. M. McCloskey, J. Org. Chem., in press. (23) D. J. Bell and J. Lorber, J. Chem. SOC.,453 (1940). (24) K. Freudenberg and E. Plankenhorn, Ber., 73, 621 (1940). (25) J. C. Dennison and D. I. McGilvray, J. Chem. Soc., 1616 (1951). (26) R. L. Sundberg, C. M. McCloskey, D. E. Rees and G. H. Coleman, J. A m . Chem. SOC.,67, 1080 (1945). (27) C. E. Ballou and H. 0. L. Fischer, J . A m . Chem. SOC.,77, 3329 (1955). (28) A. S. Meyer and T. Reichstein, Helv. Chim. Acta, 29, 152 (1946). (29) 0. Th. Schmidt and E. Wernicke, Ann., 668, 70 (1947). (30) 0. Th. Schmidt, E. Plankenhorn and F. Kiibler, Ber., 76, 579 (1942). (31) R. W. Jeanloz, J. A m . Chem. SOC.,76, 558 (1954). (32) G, W . Kenner, C. W. Taylor and A. R. Todd, J . Chem. SOC.,1620 (1949). (33) R. Allerton and H. G. Fletcher, Jr., J. A m . Chem. SOC.,76, 1757 (1954).

140

CHESTER M. MCCLOSKEY

platinum catalyst to remove the phenyl groups, gave on hydrolysis D-glycerose 3-phosphate (IX). 0 CH,

1 I

I I OCH I

HOCH

Hz C

HYo--l HCOH

C aHs CHz C1 KOH

I

HzCO T

I

0 CHz

I HzP

I

CsHsCHzOCH

I I

1. AcPO,

OCH

AcOH, HZSO, 2. HCI

H C 0 CHI CeHs HzCO I1

ce

HOCHs II ~ C 6H o ~CH

I I HCOH I

HOCH

NaIO,

EtSH HCI

HC 0 CH2 CeH5

I

HzCOH

III HC(SEt)z I

N HC(OMe)z

HzCOH

H,COH

V

VI

141

BENZYL ETHERS OF SUGARS

HC(OMe12

HC(0Me)a

I

1

HCOCHzCaHa

VII

DA

U

I

HC=O urn

I

-

VIII

Ix

The preparation and hydrogenolysis of benzyl glycosides has been employed in a method f or analyzing methylated polysaccharide~.~~ Splitting by hydrogenolysis is not a property unique to benzyl ethers among the groups often employed in carbohydrate chemistry. The triphenylmethy1’8 36-38 ethers and the cyclic acetals derived from benzaldehyde8e 21, 39-41 are also cleaved by this means. This property of these groups has not yet been fully utilized. Closely related t o the benzyl group are the benzyloxycarbonyl (“carbobenzoxy”) and benzyloxymethyl groups, both of which are subject to hydrogenolysis. The former was employed successfully in the synthesis of polypeptides by Bergmann and Z e r v a ~ and , ~ ~ its use has been extended to hydroxyl compounds by others.43 The benzoxymethyl group was employed effectively in the synthesis of 5,6-di-O-methyl-~-glucose,~~ and an advantage for it is claimed because of the milder preparative conditions involved. A novel reagent whose use has exploited subsequent hydrogenolysis is diphenyl phosphorochloridate.27’ 3 8 , 45 By reaction therewith, suitable (34) E.E . Combs, C. M. McCloskey, R. L. Sundberg and G. H . Coleman, J . A m . Chem. SOC., 71, 276 (1949). (35) F.Micheel, B e r . , 66, 262 (1932). (36) H.Bredereck and W . Greiner, Chem. Ber., 86, 717 (1953). (37) W. Anderson, 1).H. Hayes, A. M. Michelson and A. R. Todd, J . Chem. Soc., 1882 (1954). (38) C. E. Ballou, H. 0. L. Fischer and D. L. McDonald, J . A m . Chem. SOC.,7 7 , 2658 (1955). (39) K. Freudenberg, H. Toepffer and C. C. Anderson, Ber., 61, 1750 (1928);F. Sigmund, Monatsh., 63-64, 607 (1929). (40)M. Bergmann and N. M. Carter, Hoppe-Seyler’s Z . physiol. Chem., 191, 211 (1930). (41) R. Fischer, H.R . Bolliger and T . Reichstein, Helv.’Chim. Acta, 37, 6 (1954). (42) M. Bergmann and L. Zervas, Ber., 86, 1192 (1932). (43) H. 0.L. Fischer andB. Gohlke, HeEv. Chim. Acta, 18,1130 (1933);P. A.Levene and A. L. Raymond, J . Biol. Chem., 102, 327 (1933);107, 75 (1934). (44)M. R. Salmon and G. Powell, J . A m . Chem. SOC.,61, 3507 (1939). (45) K.Zeile and Hildegard Meyer, Hoppe-Seyler’s 2. physiol. Chem., 266, 131 (1938);P.Brig1 and H. Muller, Ber., 72,2121 (1939);H. Bredereck, Eva Berger and JohannaEhrenberg,ibid.,73,269(1940);E.Baer andH.O.L.Fischer,J. Riol.Chem., 160, 213,223 (1943);E.Baer and C. S. McArthur, ibid., 164, 451 (1944);Kathleen R. Farrar, J . Chem. SOL, 3131 (1949);A.B. Foster, W.G. Overend andM. Stacey, ibid., 980 (1951);J. M. Gulland and G. I. Hobday, i b i d . , 746 (1951);J. Lecocq and A. R.

142

CHESTER M. MCCLOSKEY

sugar derivatives afford diphenyl phosphate esters which yield the sugar phosphate on hydrogenolysis with platinum as the catalyst. Also of value in the preparation of sugar phosphates have been the dibenzyl phosphates introduced by Z e r ~ a and s ~ ~exploited largely" by Todd and coworkers3' 48 in nucleotide syntheses. 9

11. PREPARATION Three methods have been utilized successfully in the preparation of benzyl ethers of sugars. 1. With an Alkali Hydroxide and a Benzyl Halide

The first preparation of benzyl ethers of sugars was recorded by Gomberg and Buchlerl in 1921. They employed benzyl chloride and aqueous alkali with an aqueous solution of the sugar at a temperature of 90-95", in a manner analogous to the well known Haworth methylation technique. Incompletely benzylated products were obtained by one treatment with the above reagents, and so repeated treatment was necessary in order to obtain completely benzylated products. The method was successfully employed only with glycosides, non-reducing sugars, or polysaccharides. The results with D-glucose were unsatisfactory. Methyl glycosides were reported to be partially converted to benzyl glycosides. The modification of this method to give the procedure commonly used Todd, ibid., 2381 (1954) ;P. A. J. Gorin, L. Hough and J. K. N. Jones, ibid., 582 (1955) ; J. L. Barclay, A. B. Foster and W. G. Overend, ibid., 2505 (1955); J . L. Barnwell, W. A. Saunders and R. W. Watson, C a n . J. Chem., 33, 711 (1955); J. M. Anderson and Elizabeth E. Percival, J . Chem. Soc., 814 (1956). (46) L. Zervas, Naturwissenschaften, 27, 317 (1939). (47) A . Deutsch and 0. Ferno, N a t u r e , 166, 604 (1945); R . S. Wright and H. G. Khorana, J . Am. Chem. Soc., 77, 3423 (1955). (48) F. R. Atherton, H. T . Openshaw and A. R. Todd, J. Chem. Soc., 382 (1945) ; J. Baddiley and A. R. Todd, ibid., 648 (1947); F. R . Atherton and A. R. Todd, ibid., 674 (1947) ;F. R . Atherton, H. T. Howard and A. R. Todd, ibid., 1106 (1948) ; J. Baddiley, V. M. Clark, J. J. Michalski and A. R. Todd, ibid., 815 (1949) ;A. M. Michelson and A . R. Todd, ibid., 2476,2487 (1949) ;J . Baddiley, A.M. Michelson and A. R. Todd, ibid., 582 (1949); V. M. Clark and A. R. Todd, ibid., 2023, 2030 (1950); D. M. Brown, L. J. Haynes and A. R. Todd, ibid., 3299 (1950) ; H. S. Mason and A. R. Todd, ibid., 2267 (1951); W. E. Harvey, J. J. Michalski and A. R. Todd, ibid., 2271 (1951); D. M. Brown and A. R. Todd, ibid., 44 (1952) ; N . S. Corby, G. W. Kenner and A. R. Todd, ibid., 1234 (1952); N. Anand, V. M. Clark, R. H. Hall and A. R. Todd, ibid., 3665 (1952) ;N . S. Corby, G. W. Kenner and A. R. Todd, ibid., 3669 (1952) ; G. W. Kenner, A . R . Todd and F. J. Weymouth, ibid., 3675 (1952) ;H. G. Khorana and A. R. Todd, ibid., 2257 (1953) ;A. M . Michelson and A. R. Todd, i b i d . , 34 (3954) ;S. M. H. Christie, G. W. Kenner and A. R. Todd, ibid., 46 (1954); D. M. Brown, G. D . Fasman, D. I . Magrath and A. R . Todd, i b i d . , 1448 (1954) ; G. W. Kenner, A. R. Todd, R . F. Webb and F. J. Weymouth, ibid., 2288 (1954).

BENZYL ETHERS OF SUGARS

143

today was developed by ZemplBn, Csuros and Angyal.l* These workers carried out the interaction of benzyl chloride, potassium hydroxide, and the sugar (or sugar derivative) a t 90-100" in the absence of water. By employing a large excess of benzyl chloride, a good yield of benzylated product was obtained. The method is fairly general and yields are consistently good (70-95 %) . As commonly employed, the compound t o be benzylated plus 3 t o 8 times its weight of ' benayl chloride (depending on the number of hydroxyl groups t o he benzylated) is vigorously stirred with 1 t o 2 times its weight of powdered potassium hydroxide or sodium hydroxide a t 90-100"for 4 t o 6 hours. To isolate the product, most of the excess benzyl chloride is removed under diminished pressure, and the residue is steam-distilled. The product is isolated from the residue by filtration or extraction.

The compound to be thus benzylated should not contain groups that are sensitive to alkali. Thus, it is common practice to convert reducing sugars into their glycosides or to other non-reducing derivatives before benzylation. Nevertheless, reducing sugars substituted in a manner to provide solubility in benzyl chloride have been directly benzylated s u ~ c e s s f u lly .~ ~ Esters are readily saponified under the reaction conditions, and so are a s readily benzylated as if the parent substance containing free hydroxyl groups had been used. A nitrogen atmosphere is often employed, but is not usually essential. Toluene or xylene often have been employed as a solvent replacing a portion of the benzyl chloride; there is no particular advantage to this practice except conservation of benzyl chloride, whereas on the other hand, it may result in incomplete benzylation.14, 49 I n large-scale preparations, "practical grade" benzyl chloride has been used advantageously. Where several runs are to be made, the recovered reagent, although probably somewhat contaminated, has been re-used successfully. The early investigators employed potassium hydroxide, but sodium hydroxide gives satisfactory yields as well. Powdered alkali is preferred, but crushed flake can be employed. Vigorous stirring must be employed, to keep the alkali in suspension. Either a steam or water bath is satisfactory as a heat source. The major part of the reaction is usually over within thirty minutes, but an extended reaction period is commonly employed t o ensure complete reaction. In large-scale reactions, or where several hydroxyl groups are to be benzylated, it is often desirable to add the alkali hydroxide and the sugar derivative portionwise in order to moderate the reaction. The reaction mixture sometimes becomes quite pasty for a short period of time after the reaction has started, and although it soon thins out, (49) I. Angyal, Magyar Biol. Kutat&ntBzetMunk&i,10,449 (1938); Chem. Abstracts, 33, 4963 (1939).

144

CHESTER M. MCCLOSKEY

mixing during this period is greatly facilitated by portionwise addition of the reagents. Benzylation under the above conditions appears to be somewhat selective. Zemplh and coworkers14.49 studied the benzylation of 1,6-anhydroP-D-glucopyranose (levoglucosan) and found that a reasonable yield of the 2,4-dibenzyl ether could be obtained (as well as the 2,3,4-tribenzyl ether). By varying the concentration of reagents and diluents, the results given in Table I were obtained. It is possible that other isomers were present, but such were not isolated. TABLEI Eflect'P of Concentration of Reagent on the Benzylation of 1 ,b-Anhydro-B-~glucopyranose Triacetate" Bcneyl chloride, g.

32 25 25 15 10 6 4

Tolrenc, ml.

0 15 25 45 44 40 40

Potassiumg,

9 7.5 9 7 10 10 10

Yield, % '

Time, hr.

7

0.75 1.5 1 1 1 1

Di-0-benzyl

Tri-0-benzyl

17 25 22

55 35 29 63

6 little little 0

68 40

a . Preparation of i16-Anhydro-R,~-di-0-benzyl-@-~-glucose.-In a 1-liter, threenecked flask equipped with a mechanical stirrer, a seal, and a reflux condenser4gaare placed 60 g. (0.38 mole) of 1,6-anhydro-~-~-glucopyranose triacetate, 250 ml. (2.2 moles) of benzyl chloride, and 200 ml. of xylene. The flask is heated t o 90" by means of a n oil bath, and 100 g. (1.75moles) of powdered potassium hydroxide is added in small portions over a period of thirty minutes; the mixture is stirred vigorously during the entire reaction period. The temperature is then raised t o 105" for thirty minutes. The reaction mixture is cooled t o room temperature, 400 ml. of water is added, and the mixture is stirred until the residue dissolves. The aqueous layer is separated, extracted with 40 ml. of benzene, and the benzene extract is combined with the nonaqueous layer. This solution is extracted with 50 ml. of water, and is transferred to a Claisen flask arranged for distillation under diminished pressure. The flask is gradually heated t o 100", a pressure of 5 mm. being maintained. The residue is dissolved in 250 ml. of alcohol, 10 ml. of water is added, and the solution is set aside overnight. (ca. 50 g.) are then reThe crystals of 1,6-anhydr0-2,3,4-tri-0-benzyl-~-~-glucose moved by filtration, and t h e filtrate is steam-distilled. The residue is dissolved in 100 ml. of alcohol, and 10 t o 20 ml. of water is added; after scratching the sides, crys(49a) A vigorous reaction which results in boiling of the xylene is sometimes encountered.

BENZYL ETHERS OF SUGARS

145

tals of 1,6-anhydro-2,4-di-0-benzyl-~-~-glucose form, and, after standing for several hours, are removed (15 g., 21% of the theoretical amount). They are recrystallized by dissolving them in 90 ml. of alcohol and adding 20 ml. of water, t o give 11 g. of product, m. p. 96-97.5". If a purer product is desired [m. p. 103", [CU]~*D- 28.5" (in chloroform)], i t is best purified by conversion t o itb acetate. b. Preparation of 6-O-Benzyl-3,Q-O-benzylidene-1,$-O-isopropylidene-a-D-glucose. -A mixture of 100 g. (0.32 mole) of 3,5-O-benzylidene-1, 2-O-isopropylidene-c-~glucose and 125 g. (2.2 moles) of powdered potassium hydroxide is added in four port i o n t~o ~500 ~ ml. ~ (4.45 moles) of benzyl chloride vigorously stirred in a 1-liter, three-necked flask on a steam bath; the last three additions are made when the reaction mixture has thinned after the previous addition. After 5 hours, the liquid part is poured into a 1-liter Claisen flask, and the major portion of the benzyl chloride is removed by distillation under diminished pressure (10 mm.). The solid residue in the reaction flask is dissolved in a little water and added t o the residue from the distillation. The mixture is steam-distilled until appreciable amounts of insoluble liquid no longer distil over. The residue is then poured into a flask, where i t solidifies on standing overnight. The solid is removed by filtration, washed with water, and dried, to give 128 g. (99% of the theoretical amount) of product. It is recrystallized from ligroine (60-70") or ether (by cooling i n an ice bath), m. p. 83.5"-84", [cY]'~D -1.3" (in chloroform).

2. From an Alkali Salt of a Sugar and a Benzyl Halide

The second general method for the preparation of benzyl ethers of sugars, the reaction of the alkali salt of a sugar derivative with a benzyl halide, was developed by Freudenberg, von Hochstetter and Engelsa in 1925, not long after Gomberg and Buchler's original preparation of carbohydrate benzyl ethers. The former authors reported the synthesis of 3-O-benzylD-glucose by way of 3-O-benzyl-l , 2 :5,6-di-O-isopropylidene-a-~-glucose. They prepared the latter by the action of benzyl bromide on the sodium in ethyl ether. This derivative of 1, 2 :5,6-di-O-isopropylidene-cu-~-glucose is analogous to the procedure used by Freudenberg and Hixon60 for the preparation of the methyl ether with methyl iodide. This general method, consisting of the preparation of a sugar sodium salt of the alkoxide type and its reaction with a benzyl halide, usually in some inert solvent, has been employed many times. A number of methods have been utilized to prepare the sodium derivatives. Sodium metal with ether has been the reaction mixture most commonly employed, although dioxane4' has also been found satisfactory. 62 However, the A solution of sodium in ammonia has been (49b) The reaction mixture becomes very thick, and dia c ult to stir, if the reagents are added in one portion. (50) K. Freudenberg and R. M. Hixon, Ber., 68, 2119 (1923). (51) W. T. Haskins, R. M. Hann and C. S. Hudson, J . Am. Chem. Soc., 84, 132 (1942) . (52) W. T. Haskins, R. M. Hann and C. S. Hudson, J . Am. Chem. SOC.,70, 1290 (1948)

.

146

CHESTER

M. MCCLOSKEY

best yields by this procedure have been obtained where the ammonia was removed before the benayl halide was added. Sowden and F i ~ c h e r64~ reported ~. a very intriguing variation of the general method. They employed the sodium-naphthalene reagent in the dimethyl ether of ethylene glycol. This reagent is deep-green but turns colorless on reaction with an active hydrogen atom, for example, that of a hydroxyl group of a sugar. This property permits ready determination of the completion of the reaction. Since hydrogen and sodium are exchanged in this reaction, reducing conditions are avoided. The method should be especially useful where reducing conditions or the presence of free sodium cannot be tolerated. Both benzyl bromide and benzyl chloride have been successfully employed in the reaction with the sodium derivatives. A tertiary base, often pyridine, is added at the end of the reaction to remove unreacted benzyl halide by quarternary salt formation. The relative reactivity toward sodium of the hydroxyl groups a t the various carbon atoms of a sugar has been exploited in order to prepare selected derivatives. By the addition of one molar equivalent of sodium to 4,5-0isopropylidene-D-fucosedimethyl acetal in ethyl ether, followed by benzyl chloride, Schmidt and WernickeZ9were able to isolate a 42 % yield of 2-0benzyl-4,5-O-isopropylidene-~-fucose dimethyl acetal. Freudenberg and N0e4reacted molar equivalents of 1,2-O-isopropy~idene-a-~-glucofuranose and sodium in boiling dioxane. Subsequent reaction with benzyl chloride, and acetylation, gave a 29 % yield of crystalline 5,6-di-O-acetyl-3-O-benzyl1,2-O-isopropylidene-c-~-glucose. a . Preparationof S-O-Benzyl-l,2:6,6-di-O-isopropylidene-ol-~-glucose.-In a250-ml. flask equipped with a drying tube are placed 45 g. (0.17 mole) of 1,2:5,6-di-O-isopropylidene-a-D-ghcose (m. p. 107-109"; b. p. 126-128/0.2 mm.) and 100ml. of absolute ether. T o this solution is added 9 g. (0.39 mole) of sodium wire, and the mixture is allowed t o stand for 16 hours. The remaining sodium wire is removed, t h e flask is equipped for distillation, and 22 ml. (0.18 mole) of benzyl bromide is added. The ether begins t o distil slowly, and heat is gradually applied until the temperature of the mixture reaches 70". The mixture is maintained a t t ha t temperature for 5 hours. The residue is dissolved in 300 ml. of ligroine (60-70") and is washed 5 times with 200-ml. portions of water. The ligroine is removed by distillation under diminished pressure, and the residue is distilled under high vacuum (b. p. 146-149" a t 0.05 mm.) ;some 38-44 g. (63-73% of the theoretical amount) of product is obtained.

3. By Opening of an Epoxide Ring Benzyl alcohol or sodium benzoxide have been found to add readily to epoxides in the instances reported. Sodium benzoxide opens 5,G-anhydro(53) J. C. Sowden and H. 0. L. Fischer, J . A m . Chem. SOC.,63,3244 (1941). (54) J. C. Sowden and Dorothy J. Kuenne, J . A m . Chern. SOC.,74,686 (1952).

147

BENZYL ETHERS OF SUGARS

TABLE I1 Typical Benzylations of If gars and Related Compounds Contpound bemylaled

Solvent

Reagents"

i " C

?t

:*

Peferences

*

-

W i t h a n A l k a l i Hydroxide and a Benzy, Halide 2,3-O-Isopropylidene-~-rhamnose KOH KOH 1,6-Anhydro-p-~-glucopyranose triacetate KOH 1,2-O-Is0propy~idene~-~-glucofuranosc KOH KOH 3,5-O-benzylidene acetal 6-methyl ether KOH Methyl 4,6-O-benzylidene-ol-~-gluco-KOH side KOH diacetate NaOH Phenyl 4,6-O-bensylidene-p-~-glucosid~ Methyl 2,3-O-isopropylidene-~-ribo- KOH fursnoside 1,3:4,6-Di-O-methylenegalaetitol diace KOH tate A l k a l i Salt of a Sugar and a Benzyl Halide 1,2:5,6-Di -0-isopropylidene -a- D-glu Na Na cose C I OH sNaz 0-Isopropylidenegly ceritol CloHsNaZ Na Na 1,3:4,6-Di-O-benzylidenegalactitol Methyl 2,3-O-isopropylidene-~-ribo- Nn furanoside Nn 1,6-Anhydro-2,3-O-isopropylidene-& n-galactopyranose Na 1 , 2 - O - I s o p r o p y l i d e n e - -glucofuranosc ~~ Nn 4,5-O-Isopropylidene-~-fucose dimethyl acetal a

A A

A A

41 82

30 14

xylene

21 90 99 76 92

14 16 22 21 24 23 19 32

A

A A

A

xylene

A A

xylene

75 94 83

A

toluene

99

64

B B B B A A A

EtzO EtzO MeOCH2) 2 MeOCH2) EtzO C4HsOz NH3

46 77 50 70 66 46 55

3 28 54 53 53 51 32

A

NHsf

89

52

B A

CiHsOe EtzO

29 42

4 29

-

-

A represents benzyl chloride; B represents benzyl bromide. b Yield given is t h a t

of completely benzylated product unless otherwise specified. Yield of crystalline noncrystalline isomers are benzyl 5-0-bensy1-2,3-O-isopropylidene-~-rhainnoside; not included. d Yield of 1,6-anhydro-2,4-di-0-bensyl-p-~-glucose. Yield of crude product. f All of the ammonia was removed before the addition of t h e bensyl chloride. 0 Yield of 5,6-di-O-acetyl-3-0-benzyl-l, 2-O-isopropylidene-a-~-glucose.A Yield of 2-0-benzyl-4,5-O-isopropy~idene-~-fucose.

148

CHESTER M. MCCLOSKEY

1 ,2-O-isopropylidene-a-~-glucoseto form 6-O-benzyl-l , 2-0-isopropylidenea-D-glucofuranose.Ohle and Tessmars6found that the yields obtained from benzyl alcohol were higher (76 %) than those from methanol (70 %), ethanol (44%), or other alcohols. Benzyl alcohol was found to open Brigl's anhydride66 (3 ,4 ,6-tri-0-acetyl-1,2-anhydro-a-~-glucose) to give benzyl 3 ,4 ,6-tri-O-acetyl-p-~-glucoside. Benzyl glycosides may be prepared by the Koenigs-Knorr glycoside synthesis from benzyl alcohol plus a glycosyl halide, or from benzyl alcohol and the sugar in the presence of an acidS34 Typical results obtained from the benzylation reaction are given in Table 11.

111. HYDROGENOLYSIS Both catalytic and chemical methods are effective for the hydrogenolysis of benzyl ethers. Catalytic methods have found much wider use than the chemical methods, presumably because of the milder conditions that prevail. A general survey of the hydrogenolysis of benzyl compounds (benzyl amines, sulfides, and esters are also subject to hydrogenolysis to varying degrees) has recently been made,S7 and the reader is referred to this discussion for a complete treatment of the scope of the reaction. 1. Catalytic Methods

Palladium is the favored catalyst, although Raney nickel has been found satisfactory for the hydrogenolysis of benzyl ethers of sugars. Copper chromite has been successfully employed in other systems.67 Palladium has been utilized in many forms. Palladium black,Bv 16*6 3 , 54 palladium 0xide,2~30 and palladium on charcoal2l'2 6 - 2 7 , 34 are the forms most commonly employed with sugar ethers. Quantitative yields are approached in most cases, and the conditions are mild-high temperatures and pressures not being required. Side reactions are at the minimum and do not interfere with most sugar derivatives. Numerous solvents have been employed with palladium catalysts. Glacial acetic acidl6>2 6 , 34 meth2 9 * 30 64 ethyl acetate,ls, 21 tetrahydrofuran,16 and ethyl anollz4* ether68have been successfully employed. Acetones6was utilized with palladium in the hydrogenolysis of a trityl ether. MozingoS9has ably described the preparation of several palladium catalysts suitable for hydrogenolysis, including the popular palladium-on-charcoal catalyst. The preparation of (55) H . Ohle and K. Tessmar, Ber., 71, 1843 (1938). (56) E. Hardegger and J. de Pascual, Helv. Chim. Acta, 31,281 (1948). (57) W. H. Hartung and R . Simonoff, Org. Reactions, 7 , 263 (1953). (58) C. E. Ballou, S. Roseman and K. P. Link, J . Am. Chem. SOC.,73,1140 (1951). (59) R. Mosingo, Org. Syntheses, 26, 77 (1946).

BENZYL ETHERS O F SUGARS

149

another favorite, palladium black, is described by Tausz and von Putnoky.60 Occasionally, it is necessary to use two successive portions of catalyst in order to obtain a reasonable reaction rate. The presence of small amounts of poisons sometimes contaminates the catalyst, so that the rate of hydrogenolysis becomes very low. In such cases, it is desirable to remove the initial lot of catalyst and add a fresh portion. Platinum should be avoided because of the reduction in yield occasioned by the competing reaction, hydrogenation of the aromatic ringE1;the hexahydrobenzyl ethers are not cleaved by hydrogen. Raney nickel18*62 has been shown to catalyze effectively the hydrogenolysis of benzyl ethers. Recently, Meyer and Reiehstein28utilized Raney nickel to open a 5,6-anhydro ring and remove a beneyl group simultaneously. In this manner, they prepared 6-deoxy-l , 2-~-isopropylidene-ar-~-g~ucose from 5,6-anhydr0-3-0-benzyl-l, 2-O-isopropylidene-~-glucose, and 6-deoxy-1 ,2-O-isopropylidene-~-idosefrom the corresponding idose derivative. Both methanol and ethyl acetate were used as solvents, with 100 atmospheres of hydrogen a t 70". Use of Raney nicke141may result in some (6.8 %) hydrogenation of the aromatic ring and thus reduce the yield. Raney nickel, when boiled with an ethanolic solution of substrate, was found by Kenner, Taylor and ToddS2to split the benzyl ethers and remove simultaneously any RS- groups that were present. The stereospecificity of the hydrogenolysis reaction was demonstrated by Ballou, Roseman and Link.68Utilizing acetylated phenyl glycopyranosides in an ether solution with a palladium catalyst, the rate of hydrogenolysis was measured for these derivatives of D-glucose, D-xylose, and L-arabinose. With D-glucose derivatives, the 0-D-glycoside was split in three minutes, but the a anomer required 8 hours. The difference in rate of hydrogenolysis between the anomers of the pentosides was less than with the Dglucosides; nevertheless, the rate for the 0-D-xyloside was four times that for the CY anomer. With the L-arabinosides, the order was reversed, the a anomer being split eight times faster than the 0. The retarding effect of a cis configuration (on the hydroxyl groups at C1 and C2) strongly suggests steric or neighboring-group effects. 2. Chemical Methods

Metallic sodium plus alcohol constitutes the most commonly used system for chemical hydrogenolysis. One of the first reagents employed: it has found continual use ever since. The benzylated compound is dissolved in the alcohol, and small pieces of sodium are added. After the sodium has (60) J. Tause and N . von Putnoky, Ber., 63, 1576 (1919). (61) L.J. Heidt and C. B. Purves, J . Am. Chem. Soc., 66,1385 (1944). (62) E.M.Van Duzee and H. Adkins, J . Am. Chem. Soc., 67, 147 (1935).

150

CHESTER M. MCCLOSKEY

all dissolved, the product is isolated. Absolute4 and 97 %I7. 25* ethanol have been employed. The method has the advantage of simplicity, and is especially useful where the substrate is contaminated by compounds which would act as catalyst poisons or the substrate contains groups that could act as such poisons. The yields are often not as good as with catalytic methods, and the method cannot be used if alkali-sensitive groups are present.

IV. CHEMICAL PROPERTIES Benzyl ethers have chemical properties that are, in general, similar to those of alkyl ethers. The exceptional property is the ease of hydrogenolysis of the former class. The other differences are found in the degree, but not in the type, of reaction that they undergo. Since benzyl ethers are used primarily t o protect a selected hydroxyl group during chemical manipulation at other positions in the molecule, it is pertinent to consider briefly what can be expected of a benzyl group during such treatments. I. Action of Alkaline Reagents

Renzyl ethers are very stable to alkaline reagents, as is evidenced by their preparation in high yield in the presence of hot (100') alkali. 2. Action of Acidic Reagents

a. Aqueous and Alcoholic Acids.-Aqueous acids do not rapidly split beneyl ethers, and this property attracted the early investigators.4 Benzylidene, isopropylidene, and methylene groups can be removed, and methyl glycosides can be converted to free sugars by acid hydrolysis or alcoholysis, without excessive damage to the benzyloxy group. However, some degree of caution should be exercised when acidic conditions are employed, since benzyl ethers are split (at low, but significant, rates) particularly when the benzyloxy group is attached to a primary carbon atom. SintenisGSreported in 1872 that benzyl phenyl ether is split by hydrochloric acid to give benzyl chloride plus phenol. Benzylidene groups have been removed from 2 ,5-di-O-benzyl-l , 3 :4 ,6di-0-benzylidenegalactitol by refluxing for one hour in a solution in dioxane and N hydrochloric acid (4:1, by volume), to give a 90% yield of 2,5-0ben~ylgalactitol,~~ and the residue contained a monobenzyl derivative. Refluxing phenyl 2,3-di-0-benzyl-4,6-0-benzylidene-/3-~-glucoside in a solution containing acetone, water, and hydrochloric acid (600:50: 1, by volume) for 5.5 hours resulted in a 68% elimination of the benzylidene group; the residue was unchanged starting material.lg The isopropylidene group was removed from the 1 ,2-positions of 3 , 5 ,6tri-O-benzyl-l , 2-O-isopropy~idene-a-~-glucose in 79 % yield16 by refluxing (63) F. Sintenis, Ann., 161, 329 (1872).

BENZYL ETHERS OF SUGARS

151

for four hours in a solution of methanolic hydrogen chloride (0.5%),and from the 3,4-positions (in 79 % yield) of methyl 2-0-benzyl-3,4-o-isopropylidene-D-fucosideby heating on a steam bath with N sulfuric acid.29 The methylene groups were removed from 2,5-di-O-benzyl-l , 3 :4,6di-0-methylenegalactitol in 64% yield by heating at 100" a solution in ethanol, water, and concentrated hydrochloric acid (10: 1.5: 1, by volume) for 12 hours in a pressure bottle. Also, the di-0-methylene derivative of di-0-benzylgalactitol can be prepared in 57% yield by heating the latter (0.5 g.) in a solution of 5 ml. of dioxane, 2.5 ml. of concentrated hydrochloric acid, and 2.5 ml. of 37% formaldehyde on a steam bath for 15 minutes.s4 Methyl 3-0-benzyl-2 ,4, 6-tri-O-methyl-~-glucosidewas hydrolyzed with 20 parts of 5 % hydrochloric acid and 5 parts of methanol at 70" for several hours, to give 3-0-benzyl-2,4 ,6-tri-O-methyl-~-glucose.~~ The hydrolysis of 178 g. of methyl 6-0-benzyl-2 ,3,4-tri-0-methyl-~-glucosidewith a solution of 1.8 liters of water, 1.3 liters of acetic acid, and 356 ml. of hydrochloric acid on a steam bath for 4.5 hours gave, on fractionation, 96 g. of starting material, 72 g. of 6-0-benzyl-2,3,4-tri-O-methyl-~-glucose, and 4 to 5 g. of 2,3,4-tri-O-methy~-~-g~ucose.~~ b. Acetolyzing Reagents.-Debenzylation proceeds much more readily by acetolysis than by hydrolysis. ZemplBn, Csuros and Angyal14 successfully opened the 1,6-anhydro ring of 1,6-anhydro-2,3 ,4-tri-O-benzyl-P-~-glucosewith an acetolyzing reagent (acetic acid plus acetic anhydride plus sulfuric acid) to give 1,6-di-O-acetyl2 ,3 ,4-tri-O-benzyl-~-glucose.Subsequent deacetylation gave 2,3,4-tri-Obenzyl-D-glucose in an over-all yield (from the 1,&anhydride) of 84.5 %. The conditions were, however, relatively mild, involving a low concentration of sulfuric acid (0.22%) as the catalyst and a short reaction time (3 minutes). Allerton and Fletcher,33prompted by the reportaaof the ease of acetolysis of aryl benzyl ethers, reiiivestigated the acetolysis of 1,6-anhydro-2,3,4-tri-O-benzyl-P-~-ghcose and found that, by using 2 % sulfuric acid and a reaction period of 18 hours the product was a-D-glucopyranose pentaacetate. The acetolysis of the benzyl ethers of the hexitols was studied in detai1.33 It was found that the benzyl ethers are readily cleaved by acetolyzing reagents. Primary ethers are split more readily than secondary. Comparing the acetolysis of benzyl ethers and methylene acetals, the primary ethers are split more readily than the secondary-secondary bridges, but the secondary benzyl ethers are acetolyzed more slowly than is a pri(64) R. M. H a m , W. T. Haskins and C. S . Hudson, J . Am. Chem. Soc., 64, 986 (1942) . (65) G. H . Coleman, C. M. McCloskey and C. Dornfeld, unpublished results. (66) H. Burton and P. F. G.Praill, J . Chem. Soc., 522 (1951).

152

CHESTER M. MCCLOSKEY

mary-secondary methylene bridge (see also, Ballou and Fischer*'). In the first instance, a short reaction time permitted recovery of 32 % of the methylene acetal, and in the latter, use of a low concentration of acid (0.09%) permitted recovery of some benzyl ether. In both cases, prolonged acetolysis removed both groups. It is evident that caution should be exercised if retention of a benzyl group is desired and the molecule is to be subjected to acetolyzing conditions. c. Hydrogen Bromide-Glacial Acetic Acid.-A solution of hydrogen bromide in glacial acetic acid, if judiciously used, can be successfully employed to remove a 6-0-trityl group without serious elimination of benzyl groups elsewhere in the molecule. The trityl ethers react at a rate sufficiently higher than that of the benzyl ethers to permit isolation of the latter. However, informationz0 concerning this reaction is so fragmentary that it is not yet poasible to estimate accurately the extent of benzyl elimination. A safer procedure that has proved very satisfactory for the elimination of 6-0-trityl groups from molecules also containing benzyloxy groups is to reflux the sugar derivatives for one hour in an 80% solution of acetic acid.17,l9 d. Mercaptan-Hydrochloric Acid.-Benzylated sugars can be satisfactorily converted to the sugar dithioacetals by means of a thiol plus hydrochloric acid. A short reaction time and low temperatures are, however, necessary, otherwise the bensyl group may be eliminated. Schmidt and W e r n i ~ k efound ~ ~ that concentrated hydrochloric acid and a-toluenethiol, allowed to react with 2-O-bensyl-~-fucoseovernight at room temperature, caused elimination of the benzyl group. It was retained however, when ethanethiol and hydrochloric acid (saturated a t - 15") were employed for 0.5 hour at 0". Kenner, Taylor and Todda2converted 5-O-benzyl-~-riboseinto 5-O-benzyl-~-ribosediethyl dithioacetal by the action of ethanethiol and concentrated hydrochloric acid on the sugar in dioxane at 0" during 25 minutes. Ballou and Fiecherz7obtained an excellent yield of 2-O-benzyl-~-glycerosediethyl dithioacetal from 2-O-benzyl-~-glycerose, ethanethiol, and concentrated hydrochloric acid at 0" during 30 minutes. e. Nitric Acid.-Red fuming nitric acid in chloroform may cause nitration of the aromatic nucleus. Treatment of tetra-0-acetyl-3-0-benzyl-~-glucopyranose with fuming nitric acid in chloroform yielded tri-O-acetyl-3-0(nitrobensy1)-D-glucopyranosyl 3. Action of Oxidizing Agents Sugars with 0-benzyl substituents can be oxidized to the corresponding aldonic acid. The oxidation of 2-O-benzyl-~-fucoseto 2-O-benzyl-~-fuconic (66a) J. Honeyman and J.

W. W. Morgan, this volume, p. 117.

BENZYL ETHERS OF SUGARS

153

acid by the iodine-barium iodide-barium hydroxide method of Goebe16’ was s u c ~ e s s f u lSodium .~~ periodateZ7or lead tetracetate51 do not attack the benzyloxy group.

4.Action of Reducing Agents Several reducing agents can be employed in the presence of benzyl ethers without causing hydrogenolysis of the latter. Lithium aluminum hydride under moderate conditions does not cleave benzyl ethers. Allerton and Fletcher33treated 1 , 4 :3,6-dianhydro-2,5-di-Obenzybmannitol with an excess of lithium aluminum hydride in boiling tetrahydrofuran for six hours, and recovered the starting material in 82 % yield. The stability of the benzyloxy group to this reagent had previously been reported.68 Some caution should be exercised if vigorous conditions are employed. In the presence of cobaltous chloride, benzyl phenyl ether is hydrogenolyzed to a small extent by lithium aluminum h ~ d r i d eZinc .~~ dust-acetic acid is reported as not reacting with benzyl ethers.32 V. PHYSICAL PROPERTIES The benzyl ethers of sugars and their derivatives are often crystalline compounds, and many can be distilled. Table I11 gives some physical properties of some benzyl ethers of sugars and of some of their derivatives. (67) W. F. Goebel, J . B i d . Chem., 73, 809 (1927). (68) K . E. Hamlin and F. E. Fischer, J . Am. Chem. Soc., 73, 5007 (1951); M. E. Speeter, R. 0. Heinrelmann and D. I. Weisblat., ibid., 73, 5514 (1951). (89) P. Karrer and 0. Ruttner, Helv.Chim. Acta, 33, 812 (1950).

154

CHESTER M. McCLOSKEY

TABLX111 Benzyl Ethers of Se irs and Some Com)ound

Their Derivatives

Baiting $oint,

Rotation solvent

"C./mm.

2-0-Benzyl-~-glycerose,diethyl 140-145/0,1 dithioacetal dimethyl acetal 4 100-105/0.1 semicarbazone 132 li-O-Benzyl-~-ribose diethyl dithioacetal 170-180a/10-4 2,3,4-triacetate 1504/10-3 Methyl 5-0-benzyl-2,3-O-isopro- 95-100"/10pylidene-D-riboside 1,6-AnhydroS-O-benzyl-3,4-O-iso84-85 propylidene-8-D-galactose Phenyl 2,3-di-O-benzyl-p-~115 galac toside 4,6-0-benzylidene acetal 185-195 Methyl 2-0-benzyl-3-0-methyl103 8-D-glucopyranoside 4,6-0-benzylidene acetal 147-148 Methyl 2-O-benzyl-3,4,6-tri-O42 125/0.008 methyl-8-D-glucoside 3-O-Benzyl-~-glucose 138-141 136-138 phenylosazone 149-150 tetrakis(ppheny1azobenzoate) 246 tetraacetate (a) 107 tetrabenzoate 203 2,4,6-trimethyl ether 127-128 methyl D-glucoside 149/0.4 137/0.2 124/0.03 6-trityl ether triacetateb 145-200 1,2:5,6-di-O-isopropylidene 146-149/0.05 acetal 165-169/0.2 1,2-0-isopropylidene acetal (furanose) 119.5 5,6-diacetate 119-120 5-acetyl-6-benzoyl ester 95-96 6-benzoyl-5-tosyl ester 68-70 5,6-ditosyl ester 5,g-dimethyl ether 160/0.2 5,6-anhydro derivative 132-13310.07 1I 2-0-benzylidene-5,6-o-iso92-93 propylidene acetal 93.5 6-O-Benzyl-~-glucose

References

16.9

27

25.1 -8.5 9. -4.2 -36.

EtOH CHCla CHCla CHCla

27 51 32 32 32 32

-81.9

CHC13

52

-7

C SHSN 23,52

- 10 -5.8

CHCla Me&O

52 26

-30.3 9.9

CHCla CHCL

26 26

H2 0 HzO

3,20 54 3 24 15 15 15 15

41.9 41.8 -48b -1.2 8.6 54.6 43.5

CHCL CHCls CHCla CHCla EtOH

19.4 50.3

CHCla CHCla

-26.9 EtOH CHCls - 45 - 53 -46.4 -67.1 -2.2 -6 -15.8 -51.2 -10.2

39.3

CzHaCI CHCla CHCla CHCla CHCla Me2C0 CHCla CHCls HzO

20 15,20 28 3 28 4 28 28 28 28 24 28 54 55 __

TABLEI11 (Continued) Compound

1,2-0-isopropylideneacetal (furanose) 3,5-O-benzylidene acetal 3,5-dimethyl ether 2,3,4-trimethyl ether methyl D-glucoside Methyl 2,3-di-0-benzyI-cu-o-glucopyranoside 6-trityl ether 4-methyl ether 4,6-0-benzylidene acetsl 4,6-dirnethyl ether

-

Boilin point, c./g,m.

Melting point, "C.

172/0.16

79

127-128/0. OOt 150-152/0.02 116-117/0.02t

84 81

75-76 79-80 93 99 215-220/0.03 200-210/0.45 122-123

Methyl 2,3-di-O-benzyl-p-~-glucopyranoside 4-methyl ether

4M1 88

6-trityl ether 6-trityl ether 4,6-O-benzylidene acetal Phenyl 2,3-di-o-benayl-/3-~-glucopyranoside 6-benzoate 4,6-dibenzoate 4,g-dirnethyl ether 4-methyl ether 6-benzoate 4,6-O-benzylidene acetsl 2,4-~i-O-benzyl-o-glucose 1,6-anhydro-j3 derivative 3-tosyl ester 3,5-Di-O-benzyl-l,2-0-isopropy- 208-21 1/0.05 lidene-6-0-methyl-cr-~-glucose Methyl 3,5-di-O-benzy1-6-0- 185-192/0.05 methyl-o-glucoside Methyl 3,5-di-O-benzyl-2,6-di-O-203-207/0.01 methyl-o-glucoside Methyl 4,6-di-O-benxyl-2,3-di-O-195-19910.3 methyl-a-D-glucoside 2,3,4-Tri-O-benzyl-~-glucose 1,6-diacetate, ap anorner 1,6-anhydride, /3benzyl j3-D-glucoside 155

119-120 125 137 164 60.5 114-116 92.5 75-79 103 106 3941

90-91 66 63.5 90 116

Rolalion

solsenl

2.0

CHCL

Referencd

22,55

22 22 65 65 18.8 CHCla 23 88.7 MezCO 24 14.5 CHCls 18 13 CHCls 18 31.2 CHCla 23 23.5 MezCO 24 32.9 CHC13 23 97.9 MezCO 24 -13.3 CHCla 25 -1.3 41.6 50.3

CHCls CHCla CHCla

32 42.9 20.4 -12.6 -35.8 3.8

CHCla CHC13 CHC13 CHCla CHCla Me&O

25 17 17 17 25 19

CHCla -41.0 CHC13 6.0 CHCls 1.0 CHCla 2.5 CHCla -43.6 MezCO 25.1 EtOH 28.7 CHCla CHCla -5.7

19 19 19 19 19 19 14 14 14 21

-30.9

CHCla

21

-21

CHCla

21

121.9

MezCO

24

18.6 81.5 17.4 -29.5 2.9

EtOH EtOH CHCls CHCL CHCls

14 14 14 14 14

- 43

156

CHESTER M. MCCLOSKEY

TABLEI11 (Concluded) Compound

Boilin poinl, C.I??l,.

3,5,6-Tri-O-benzyl-1,6-di-O-(pphenylazobenzyl) -D-glucose 3,5,6-Tri-O-benzyl-l,2-O-isopro- 240/0.05 pylidene-a-D-glucose Methyl 3,5,6-tri-O-benzyl-~-glu220-230/0.05 cosidec 2-methyl etherc 230-235/0.02 Benzyl 2,3,4,6-tetra-O-benzyl-~glucoside Methyl 2-acetamido-3,4-di-Obenzyl-2-deoxy-cu-~-glucoside 6-methyl ether 3-0-Benzyl-l , 2-0-isopropylideneL-idofuranose 20-130/0.055 triacetate 137-139/0.05 5,6-anhydro derivative 1,6-Anhydro-4-O-benzyl-2,3-0isopropylidene-8-D-mannose 2-0-Benzyl-~-fucose phenylhydrazone diethyl dithioacetal triacetate dimethyl acetal 142-145/0.05 4,5-O-isopropylidene acetal 125-128/0.04 3-methyl ether 152-157/0.1 Methyl 2-0-benzyl-3-0-methyl-~fucoside c Methyl 2-0-benzyl-3,4-0-isopro-136-138/0.00 pylidene-D-fucoside Benzyl 5-0-benzyl-~-rhamnoside 180/0.01 2,3-O-isopropylidene acetal 160-170/0.1 2,a-dimethyl ether 140/0.02 Methyl 5-0-benzyl-2,3-di-0methyl-L-rhamnoside

Melting poinl, "C.

146-166

[ah

Rofation

solvent

72.5 CHCla

-34.7

CHCla

16

16 16 16 1,14

83.5 195-197 197-198

99-100 165 141 96 62-63 93-94

31 99 -64.5

CHCla CHCla

31 28

-78.7 -13.0

CHCls CHCla

28 28 52

66.3 17.4 -4.6 4.5 11.1 21.1 7.4

H20 MeOH EtOH EtOH Hz0 EtOH MeOH

29 29 29 29 29 29 29 29

97.2

CHCla

29

MezCO MezCO MezCO MezCO

30 30 30 30

48.2 77.5 84,s 104 30.3 119 71.7 93 - 72

-

Bath temperature. b 6252 A. e Mixture of a and 6 anomers. 5780 A. still. Metastable crystal form. a

Refmemces

Molecular

METHYL AND PHENYL GLYCOSIDES OF THE COMMON SUGARS BY J. CONCHIE, G. A. LEVVYAND C. A. MARSH Institute, Bucksburn. Bucksburn, Aberdeenshire. Aberdeenshire, Scotland Rowett Research Institute.

............................................................ Introduction.. .......................................................... 158 II.. Introduction 11.. Preparation of Sugar Derivatives Derivatives Employed in Glycoside Synthesis Synthesis.. I1 . . . . . . . 158 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Halides.. . . . ............................................ 11.. 0-Acylglycosyl Halides 2 . Acetylated Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 161 a . Sodium Acetate as Catalyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161 b. b . Zinc Chloride as Catalyst. Catalyst . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. ............................ 161 c. c . Pyridine as Catalyst.. Catalyst . . ............................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 d . Perchloric Acid as Catalyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 e. e . Sulfuric Acid as Catalyst.. Catalyst . . .............................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 I11. Condensationof Alcohols and Phenols with Sugars and Sugar Derivatives . . 163 1 . With 0-Acylglycosyl Halides .......................................... 163 a . I n Presence of Silver Oxide or Silver Carbonate . . . . . . . . . . . . . . . . . . . . 164 b . In Presence of Alkali in Aqueous Acetone . . . . . . . . . . . . . . . . . . . . . . . . . . 165 c . In Presence of Organic Bases Alone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 d . In Presence of Mercuric Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 2 . Condensation of Phenols with Acetylated Sugars . . . . . . . . . . . . . . . . . . . . . . 168 a. a . Zinc Chloride as Catalyst.. Catalyst . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ...... . . . . ...................................168 b. p-Toluenesulfonic Acid as Catalyst.. Catalyst . . .. .. .. .. .. . . . . . ...... .. .. ....... .. .. ....... .. .. ...... . . . 169 c. c . Other Catalysts .. .. ......... .. .. .. .. ......... .. .. .. .. ......... .. .. .. .. ......... .. .. .. .. ...... . . . . . . . . . . . . . .169 . 3 . Condensation of Simple Alcohols with Free Sugars . . . . . . . . . . . . . . . . . . . . 170 a. a . Hydrochloric Acid as Catalyst. Catalyst . . . . . . ......... . . . . .. ......... . . . . .. ....... .. . . . . .... . . . 170 b. b . Cation-exchange Resins as Catalysts. Catalysts . . . . . ............ . . . . . ............. . . . . . .... . . 171 IV . Dcacetylation Dcacetylation of Glycoside Acetates. Acetates . . . . . . . . . . . . ........ .. .. .. ....... .. .. .. ......... .. .. .. .... . . .171 IV. . 1 .. By Alkali .. .. .. ........... .. .. .. .. ........... .. .. .. .. .. ........... .. .. .. .. .. ........... .. .. .. .. .......... . . . . . . . . . . . .. . .171 2. 2 . By Sodium Methoxide. Methoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 3. 3 . By Methanolic Ammon Ammonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .173 4. 4 . By Barium Methoxide. Methoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 5. 5 . By Potassium Methoxi Methoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .173 6. 6 . By Dimethylamine. Dimethylamine . .. ........... .. .. .. .. ........... .. .. .. .. ........... .. .. .. .. .......... . . . . . . . . . . . . . . . . . .174 V. V . Special Methods of Glyco Glycoside Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 1 . Anomerization.. Anomerization . . ......................... 1, a. a . With Titanium Tetrachloride .. .. ........... .. .. .. .. .. ............. .. .. .. .. ............. . . . . . . . . . . 174 175 b. . b . With Boron Trifluoride.. Trifluoride ............................................. c. c . With Other Catalysts.. Catalysts . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . ...................... . . . . . . . . . . . . . . . . . . . . . 175 2. 2 . Aglycon Exchange. Exchange . .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .175 3 .. Oxidation of Glycosides to Glycosiduronic Acids.. Acids . . ..................... . . . . . . . . . . . . . . . . . . . . . 175 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................... .. .. .. . 178 4 . Miscellaneous.. Miscellaneous . .................................... 4. VI. VI . Description Description of Tables .................................................... ....................................................178 157

158

J. CONCHIE, G. A. LEVVY AND C. A. MARSH

I. INTRODUCTION Although glycosides of unsubstituted sugars have no great importance as reference compounds in carbohydrate chemistry, they are valuable as standard substrates in enzyme chemistry. Moreover, when it comes to the synthesis of natural glycosides, disaccharides, and oligosaccharides, the simple alkyl and aryl glycosides serve as prototypes. The synthesis of many of the simpler glycosides dates back to the nineteenth century, and often no modification of the original preparation is to be found in the literature. There is no doubt, however, that in many instances the synthesis of a given compound could easily be simplified and improved. Several new general methods or modified procedures for glycoside synthesis have been introduced in the last thirty years. Not all of these are widely known to non-specialists. Throughout this article the emphasis is on preparatory methods, as far as possible those of general application, and theoretical considerations will not be discussed. The application of typical reactions to D-glucopyranose is illustrated in Fig. 1. For purposes of illustration, both anomers of the methyl and phenyl glycosides of some of the commoner sugars are quoted in the Tables. Where the methyl or phenyl derivative is unknown, constants and details of preparation are sometimes given for some other representative member of the series. Any selection of the “common sugars” must of course be arbitrary, and our choice is as follows: n-glucopyranose, D-galactopyranose, D -mannopyranose, D -fructopyranose, D -fructofuranose, N-acetyl-D-glucosamine (2-acetamido-2-deoxy-~-glucopyranose), D-xylopyranose, D-ribopyranose, L-arabinopyranose, L-arabinofuranose, D-arabinopyranose, D-arabinofuranose, maltose, cellobiose, lactose, D-glucopyranuronic acid, D-glucofuranuronic acid, D-galactopyranuronic acid, and D-mannopyranuronic acid.

11. PREPARATION OF SUGAR DERIVATIVES EMPLOYED IN GLYCOSIDE SYNTHESIS 1. 0-Acylglycosyl Halides

The 0-acylglycosyl halides, where the acyl group at the potential reducing group in a fully acylated reducing sugar has been replaced by halogen, are the most generally useful intermediates in the synthesis of alkyl and aryl glycosides, and the 0-acetylglycosyl bromides (“acetobromosugars”) are by far the most commonly employed. They are more reactive than the chlorides and more stable than the iodides. In some instances, however, the bromide is so unstable that the chloride is preferred. In the first reported synthesis of a glycoside, phenyl 0-D-glucopyranoside,’ (1) A. Michael, Am. Chem. J., 1 , 305 (1879).

159

GLYCOSIDES OF THE COMMON SUGARS

H

CH20H

OAc

H

-

HQR

OH OH 6

OR H

OH I

HO

H

H

(V3)

OH

HO

H

; QH

i

OH

HO

CHZOH OH

H

OH

H

h

FIG. 1.-Methods for the Synthesis of D-Glucopyranosides and Their Oxidation t o D-Glucopyranosiduronic acids. [ROH = AlkOH or ArOH. Numbers in brackets refer t o Sections and Subsections in the text, describing the general reactions. a , a-D-glucopyranose; b , penta-0-acetyl-a-D-glucopyranose; c , penta-O-acetyl-8-D-glubromide; e , R tetra-0-acetyl-a-Dcopyranose ; d , tetra-0-acetyl-a-D-glucopyranosyl glucopyranoside; f, R tetra-O-acetyl-~-D-glucopyranoside;g, R a-D-glucopyranoside ; h , R 8-D-glucopyranoside ;i, R a-D-glucopyranosiduronic acid; j , R 8-D-glucopyranopiduronic acid.]

160

J. CONCHIE, G. A.

LEVVY AND C. A. MARSH

potassium phenoxide was condensed with tetra-0-acetyl-a-D-glucopyranosyl chloride, made by the action of acetyl chloride on D-glucose? Considerably later, Koenigs and Knorr3 introduced the use of the corresponding bromide, prepared in the same way. A comprehensive review of the preparation and properties of the O-acetylglycosyl halides, together with tables of constants, has already appeared in this Series.4 The chief method of preparation is by treatment of the fully acetylated sugar (either anomer) with the hydrogen halide in acetic acid. However, the method described by Bdrczai-Martos and Korosyb for the preparation of bromides, in which acetylation and bromination are done successively without isolation of the acylated sugar, can be recommended on all grounds. Tetra-0-acetyl-a-D-glucopyranosylbromide was made6 as follows. To a mixture of 400 ml. of acetic anhydride and 2.4 ml. of 60% perchloric acid, 100 g. of o-glucose is added over a period of 30 min. The temperature should not exceed 40" (to avoid caramelization) nor fall below 30" (to maintain steady reaction). After cooling in ice, 30 g. of amorphous phosphorus is introduced. This is followed by the gradual addition of 180 g. of bromine, the temperature being kept below 20". With careful mixing and cooling, 36 ml. of water (90% of the theoretical) is dropped in, over a period of 30 min. The stoppered vessel is kept a t room temperature for 90120 min., and 300 ml. of chloroform is added. After pouring the mixture into 800 ml. of ice-water, the chloroform layer is separated, filtered to remove particles of phosphorus, and washed twice with an equal volume of ice-water; the water is backwashed twice with 30 ml. of chloroform. A final extraction of the chloroform with saturated sodium bicarbonate solution brings the pH to about 6. The yellow solution is rapidly dried (30 min.) and simultaneously decolorized with a mixture of calcium chloride, 5 g. of charcoal, and a pinch of sodium bicarbonate, calcium carbonate, or magnesium carbonate. The residue obtained on evaporating the filtered solution to dryness under diminished pressure a t 60" is dissolved in dry ether, from which i t crystallizes on cooling or is precipitated with petroleum ether; m. p. 84'; yield 85%; m.p. 87" after recrystallization from ether.

Yields of O-acetylglycosyl bromides obtained from other sugars by this method were: lactose 85 %, arabinose 50 %, maltose 60 %, cellobiose 72 %, and galactose 75 %. With cellobiose, some glacial acetic acid has to be added at the start to maintain solution. In general, the products are pure enough to be used without recrystallization in the next stage of glycoside synthesis. The method is not applicable to methyl glucuronate.s Most of the stable O-acetylglycosyl halides have the a-configuration, (2) A. Colley, Ann. chim. et phys., [4] 21, 363 (1870). (3) W. Koenigs and E. Knorr, Ber., 34,957 (1901). (4) L. J. Haynes and F. H. Newth, Advances in Carbohydrate Chem., 10,207 (1955). (5) M. Bgrczai-Martos and F. Korosy, Nature, 166, 369 (1950). (6) G. N. Bollenback, J. W. Long, D. G. Benjamin and J. A . Lindquist, J . A m . Chem. SOC.,77, 3310 (1955).

GLYCOSIDES OF THE COMMON SUQARS

161

notable exceptions being the derivatives of D- and L-arabinopyranose, D-ribopyranose, and D-fructopyranose. Since Walden inversion normally occurs during condensation with alcohols and phenols, the Koenigs-Knorr reaction is useful for the synthesis of glycosides of configuration opposite to that of the stable halide. 0-Benzoylglycosyl bromides, made by the action of hydrogen bromide on fully benzoylated sugars, have been employed for the synthesis of methyl and ethyl glycosides, not necessarily with Walden inversion7. * , (see Section 111, 1). 2. Acetylated Sugars

Apart from their use as starting materials for the preparation of O-acetylglycosyl halides, polyacetylated sugars are employed extensively in glycoside synthesis by the Helferich reaction,1°in which they are condensed directly with low-melting phenols. This reaction has only been applied to alcohols in one or two instances, but there seems to be no reason why it should not be more often used in this way. So far as we are aware, benzoylated sugars have not been employed in the Helferich reaction. Intermediates in the preparation of the 0-benzoylglycosyl halides, they are made by the action of benzoyl chloride on the sugar in the presence of alkali or an organic base, usually pyridine.9 Acetic anhydride is used exclusively in making the poly- 0-acetylated sugars, and a catalyst is essential. The choice of catalyst determines the predominant anomer obtained. Once isolated, the anomers can be interconverted by the use of suitable catalysts.9 The following Subsections deal with the principal catalysts employed in acetylation. a. Sodium Acetate as Catalyst.-This, one of the earliest catalysts used, I I , l 2 i s still of major importance. Heating a sugar with acetic anhydride in the presence of anhydrous sodium acetate gives predominantly the ,&form of the acetyl derivative. The following procedureIs is typical. Heat 2.5 g. of anhydrous sodium acetate on the steam bath with 25 ml. of acetic anhydride until dissolution is nearly complete. Cautiously add 5 g. of n-glucose (7) R. W. Jeanloz, H. G. Fletcher, Jr., and C. S. Hudson, J. Am. Chem. Soc., 70, 4052 (1948). (8) R. W. Jeanlos, H . G. Fletcher, Jr., and C . S. Hudson, J. Am. Chem. Soc., 70, 4055 (1948). (9) R. K. Ness, H. G. Fletcher, Jr., and C. S. Hudson, J. Am. Chem. Soc., 72,2200 (1950). (10) B. Helferich and E. Schmitz-Hillebrecht, Ber., 66, 378 (1933). (11) C. Liebermann and 0. Hormann, B e y . , 11, 1618 (1878). (12) C. Tanret, Bull. 8oc. chim. (France), [3] 13, 261 (1895). (13) F. G. Mann and B. C. Saunders, “Practical Organic Chemistry,” Longmans, Green and Co., London, 2nd Edition, 1938, p. 98.

162

J. CONCHIE, G. A. LEVVY AND C. A. MARSH

(anhydrous or monohydrate) with shaking, and continue heating for 1 hr. Pour slowly into 250 ml. of ice-cold water, with vigorous stirring. The oil which separates crystallizes; the mixture is then filtered and the crystals are washed very thoroughly with water; yield 10.0-10.5 g.; twice recrystallized from 95% ethanol: m. p. 130-131".

b. Zinc Chloride as Catalyst.-This is the first and best known catalyst14 for obtaining the a-acetyl derivatives from sugars. It is also employed for the anomerization of poly-0-acetylated and -benzoylated P-~-sugars.~a 16, l 6 , l7 The reaction with D-glucose is performed as follow^.'^ Heat 1 g. of powdered, dry zinc chloride with 25 ml. of acetic anhydride on the steam bath until most of the salt has dissolved. Add 5 g. of D-glucose and proceed asinsection II,2a; yield, 7.5-8.0 g.; twicerecrystallizedfrom 95% ethanol: m. p. 110111". The same procedure can be employed for anomerization of the fl form. I n the case of L-arabinose, anomerization of the polyacetate by zinc chloride proceeds from a t o 8.18 c. Pyridine as Catalyst.lS-Treatment of a sugar with a mixture of pyridine and acetic anhydride, usually a t O", results in a mixture of a- and @-polyacetates, which can often be separated by fractional recrystallization. This method can be recommended for making penta-0-acetyl-@-D-mannopyranose from commercial D-mannose.20Since mutarotation is slow in its presence, this catalyst is particularly valuable in obtaining from a pure a- or &sugar the acetyl derivative of the same configuration. d . Perchloric Acid as Catalyst.-Like other acid catalysts, this gives predominantly the a-acetyl derivative. Its use was developed by Nicholas and Smith.z1On treatment at room temperature with a mixture of acetic acid and acetic anhydride and a few drops of perchloric acid, crystalline derivatives were obtained from D-glucose, maltose, and some polyhydric alcohols, but not from D-galactose or D-mannose. e. Sulfuric Acid as Catalyst.-This has been used as a catalyst for the acetylation of, for example, D-glucose,22but has no advantage over perchloric acid for the acetylation of free sugars. A more concentrated solution of sulfuric acid in acetic anhydride gives, however, an excellent yield of octa-0-acetyl-a-cellobiose from c e l l ~ l o s eA . ~ mixture ~ of acetic anhydride and glacial acetic acid containing 2 % (v/v) concentrated sulfuric acid has (14) (15) (16) (17) (18) (19) (20) (21) (22) (23)

E. Erwig and W. Koenigs, Ber., 22, 1464 (1889). C. S. Hudson and J. K. Dale, J . A m . Chem. Soc., 37, 1280 (1915). C. S. Hudson and J. M. Johnson, J . A m . Chem. SOC.,37, 2748 (1915). C. S. Hudson and H. 0. Parker, J . A m . Chem. SOC.,37,1589 (1915). C. S. Hudson and J. K. Dale, J . A m . Chem. SOC.,40,992 (1918). R. Behrend and P. Roth, Ann., 331, 359 (1904). J. Conchie, G. A. Levvy and C. A. Marsh, unpublished results. S. D. Nicholas and F. Smith, Nature, 161, 349 (1948). C. E. Redemann and C. Niemann, Org. Syntheses, Coll. Vol. 3, 11 (1955). G. Braun, Org. Syntheses, Coll. Vol. 2 , 124 (1948).

GLYCOSIDES O F T H E COMMON SUGARS

163

been advocated for converting sugar @-acetatesinto the a! forms at room temperat~re.2~

111. CONDENSATION OF ALCOHOLS AND PHENOLS WITH SUGARS AND SUGAR DERIVATIVES 1. With 0-Acylglycosyl Halides

The Koenigs-Knorr r e a ~ t i o n ,in ~ which 0-acetylglycosyl halides are condensed with alcohols or phenols in the presence of a heavy metal or organic base, is dealt with in previous reviews in this Series.4-2 6 , 26 Whilst the bromides are employed almost invariably, there are some instances in which the chloride is to be preferred, for example in the preparation of methyl a!-~-fructopyranoside,2~~ 28 and in the synthesis of N-acetyl-glucosaminides.29~298 Although Koenigs and Knorfl isolated a small yield of methyl /3-D-glucopyranoside (as such) from a solution of tetra-0-acetyl-a-D-glucopyranosyl bromide in methanol that had stood at room temperature for several days, it is customary to add an “acid acceptor” to speed up the reaction and to prevent deacetylation of the product. Silver, in the form of the oxide or a salt, was the first acid acceptor to be employed, and is still the one in most common use. Unless the aglycon is a simple alcohol, it is usual to dissolve the reactants in a solvent, which is often an organic base to act as an additional acid acceptor. Walden inversion at C l is almost the invariable rule when the reaction is done in the presence of silver ion. Under special circumstances, however, both anomeric glycoside acetates may be obtained.30 Walden inversion at C1 is also the rule when alkali is employed as the condensing agent.31 An organic base (nearly always quinoline) may be used in the same way, but, in the absence of silver, a mixture of the a- and p-glycoside acetates results. These are usually easy to separate by fractional (24) Edna M. Montgomery and C. S. Hudson, J . Am. Chem. Soc., 66, 2463 (1934). (25 W. L. Evans, D. D. Reynolds and E. A. Talley, Advances in Carbohydrate Chem., 6, 27 (1951). (26) R. U.Lemieux, Advances in Carbohydrate Chem., 9, 1 (1954). (27) H. H. Schlubach and G. A. Schroter, Ber., 61, 1216 (1928). (28) H. H. Schlubach and G. A. Schroter, Ber., 63, 364 (1930). (29) L. F. Leloir and C. E. Cardini, Biochino. et Biophys. Acta, 20, 33 (1956). (29a) As an alternative to ethereal hydrogen chloride, titanium tetrachloride has been employed to convert N-acetyltetra-0-acetyl-D-glucosamineinto l-chloro-ldeoxy-D-glucosaminetetraacetate, in chloroform solution (B. R. Baker, J. P. Joseph, R . E. Schaub and J. H. Williams, J . Org. Chem., 19, 1786 (1954)). (30) H. S. Isbell and Harriet L. Frush, J . Research Nail. Bur. Standards, 43, 161 (1949). (31) C. Mannich, Ann., 394, 223 (1912).

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recrystallization. Mercuric salts have recently become prominent as acid acceptors. When 0-benzoylglycosyl bromides are used instead of the O-acetylglycosyl bromides, Walden inversion occurs during the reaction in the presence of silver ion. It has now, however, been shown that the O-benzoylglycosyl bromides react rapidly with simple alcohols at room temperature in the absence of any acid acceptor, and without debenzoylation of the product.8, 9 9 3 2 , a3 Under these conditions, the configuration of the product is determined by steric hindrance. As a general rule, “in the absence of an acid acceptor all the benzoylated glycopyranosyl halides . which have a benzoyloxy group a t C2 trans to the halogen, react with methanol without net Walden inversion while those halides having a cis relationship between the groups on C1 and C2 react with inversion at Cl.”32Thus, since the aglycon always takes up a trans position with respect to the benzoyl group at C2, this reaction yields p-D-glucosides, p-D-ribosides, and p-D-xylosides, but a-D-mannosides and a-D-arabinosides. The following Subsections deal in more detail with the condensation of 0-acetylglycosyl halides with alcohols and phenols, in the presence of different acid acceptors. a. I n Presence of Silver Oxide or Silver Carbonate.--In their original experiments, Koenigs and Khorr3 used silver carbonate or concentrated aqueous silver nitrate to remove the hydrogen halide produced in the condensation; silver oxide was subsequently found to be equally effective, and this or the carbonate have come to be used exclusively. Many workers have stressed the importance of using dry reagents and excluding moisture. In an inert solvent, the water produced by the reaction of hydrogen halide with silver oxide or carbonate may reduce the efficiency of the condensation, and improved yields are claimed for the use of an internal desiccant, such as calcium chloride or Drierite.26 For some reason, the velocity of condensation is diminished by the desiccant but the normal speed is restored by iodine?4*36 Except with simple alcohols, the most common procedure is to dissolve the 0-acetylglycosyl bromide in a dry solvent (usually methylene chloride, alcohol-free chloroform, benzene, or quinoline), after adding silver oxide or carbonate (dry, freshly prepared36), and then to add an excess (at least 100%) of the appropriate aglycon. Although the reaction is usually exo-

..

(32) H . G . Fletcher, Jr., and C. S.Hudson, J . A m . C h e w Soc., 73, 4173 (1950). (33) R . K . Ness, H. G . Fletcher, Jr., and C. S.Hudson, J . A m . Chem. SOC., 73, 959 (1951). (34) B. Helferich, E. Bohn and S.Winkler, Ber., 63, 989 (1930). (35) D. D. Reynolds and W. L. Evans, J . A m . Chem. Soc., 60,2559 (1938). (36) C. M. McCloskey and G . H. Coleman, Org. Syntheses, Coll. Vol. 3, 434 (1955).

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thermic, application of heat may be necessary in particular instances. When reaction is complete, the solution is filtered, and, in the case of phenols, washed free from excess aglycon with dilute sodium hydroxide. If evaporation of the dried solution does not result in a crystalline product, the material can usually be crystallized by addition of methanol or ethanol. This reaction has been employed extensively for making the 0-glycosides of phenols and alcohols, including methanol and ethanol. Although not glycosides, aldopyranosyl phosphates can be made similarly, by condensing the O-acetylglycosyl bromides with silver dihydrogen phosphate?’ Whilst Walden inversion at C l is the rule in the presence of silver compounds, other factors may modify the reaction and so affect the final re~ult.~’J Whereas tetra-0-acetyl-a-D-glucopyranosyl bromide (a cis halide) yielded 90-95 % of methyl tetra-0-acetyl-p-D-glucopyranoside under all tried conditions of condensation with methanol in the presence of silver carbonate, the corresponding a-D-mannopyranosyl bromide (a trans halide) gave a variable product. In pure methanol at 20°, the product was 78% of D-mannopyranose 1,2-(methyl orthoacetate) triacetate and 15 % of methyl tetra-0-acetyl-0-D-mannopyranoside; at 50°, 53 % of the methyl orthoacetate, 25 % of the P-D-mannoside acetate, and 8 % of the a-D-mannoside acetate were obtained. In 2.5% methanol in ether, the yields a t 20” were 7 % of the methyl orthoacetate, 23 % of the P-D-mannoside acetate, and 34% of the a-D-mannoside acetate. The use of benzene instead of ether gave a similar result. Because of Walden inversion during the replacement of the halogen, orthoacetate formation (as a competing process to glycoside synthesis) was possible in the case of the trans, but not the cis halide. Subsequent methanolysis of the orthoacetate gave a mixture of anomeric glycosides. that a wide miscellany of metallic Helferich and W e d e m e ~ e r3g~ found ~ oxides and salts and other compounds, including albumin, are efficient “acid acceptors” in the condensation of tetra-0-acetyl-a-D-glucopyranosyl bromide with methanol at room temperature to give methyl tetra-o-acetylp-D-glucopyranoside. b. I n Presence of Alkali in Aqueous Acetone.-This method, applicable only to phenols, proceeds invariably with Walden inversion a t C1. O-Acetylglycosyl halides can be condensed directly with sodium or potassium phenoxide, either by fusion, as in the synthesis of phenyl tri-O-acetylp-~-glucosaminide,~~ or in ethanol solution, as in the synthesis of phenyl 9

(37) 0.Touster and V. H. Reynolds, J . Biol. Chem., 197,863 (1952). (38) €3. Helferich and K.-F. Wedemeyer, Ann., 663, 139 (1949). (39) B.Helferich and K.-F. Wedemeyer, Chem. Ber., 83,538 (1950). (40) B.Helferich, A. Iloff and H. Streeck, Hoppe-Seyler’s Z . physiol. Chem., 226, 258 (1934).

166

J. CONCHIE, G . A. LEVVY AND C. A. MARSH

tri-o-acetyl-@-D-glucopyranosiduronic acid methyl esterls from the corresponding acetyl-a-D-glycosyl bromide. By far the most convenient procedure, where applicable, is to employ a dilute solution of sodium or potassium hydroxide in aqueous acetone as the condensing medium, as in the preparation of o-nitrophenyl tetra-0-acetyl-@-~-glucopyranoside.~~ o-Nitrophenol (3 9.) in a solution of sodium hydroxide (1.2 g.) in 30 ml. of water was added t o a solution of tetra-0-acetyl-a-D-glucopyranosyl bromide (6.3 g.) in 45 ml. of acetone. The mixture was homogeneous and there was no rise in temperature. After 5 hr. a t room temperature, the acetone was removed under diminished pressure. Separation of the glycoside acetate commenced, and was completed by adding 300 ml. of water; recrystallized from ethanol: m. p. 158-159”; yield, 65% in terms of onitrophenol.

This same general procedure is said to give a 37 % yield of phenyl [email protected] c. I n Presence of Organic Bases Alone.-In presence of dry quinoline as the sole acid acceptor, Fischer and his collaborator^^^^ 44 found that mixtures of the a- and @-glycosidetetraacetates are obtained after the condensation of alcohols and phenols with 0-acetylglycosyl bromides. This method has been employed in preparing I-menthyl a-~-glucoside~~ and phenyl a-D-galactoside.46Pyridine, often used in presence of silver compounds, was not recommended as sole acid acceptor,46,47 although pyridine in ether has been successfully employed in the synthesis of methyl tetraO-acetyl-a-~-fructopyranoside.~~ d. I n Presence of Mercuric Salts.-Mercuric bromide, acetate, and cyanide have frequently been used in recent years as “acid acceptors” in the Koenigs-Knorr reaction with alcohols or phenols. Whilst there seems to be a general tendency for the reaction to proceed with Walden inversion, either anomer may be produced, depending upon the experimental conditions. The effect of varying the aglycon concentration-on the condensation of hepta-0-acetyl-a-cellobiosyl bromide with ethanol, in the presence of mercuric acetate-was studied by Zempl6n and Gerecs4*;benzene was added to keep constant the final volume of the solution, and the reaction (41) E. Glaser and W. Wulwek, Biochem. Z . , 146, 514 (1924). (42) J. Stantik and J. Kocourek, Chem. Listy, 47, 697 (1953); Chem. Abstracfs, 49, 190 (1955). (43) E. Fischer and L . von Mechel, Ber., 49, 2813 (1916). (44) E. Fischer and M. Bergmann, Ber., 60, 711 (1917). (45) B. Helferich and H. Bredereck, Ann., 466, 166 (1928). (46) E. Fischer and K. Raske, Ber., 43, 1750 (1910). (47) B. Helferich, A. Doppstadt and A. Gottschlich, Naturwissenschaften, 40, 441 (1953). (48) G. Zemplen and A. Gerecs, Ber., 63, 2720 (1930).

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167

was carried out for 30 min. under reflux. It was found that the optical rotation of the twice-recrystallized product varied according t o the number of moles of ethanol originally present per mole of glycosyl bromide. Thus, with one mole, [a]=was 54", and with two moles, +57" (corresponding to the pure a-glycoside acetate). Above 3 moles of ethanol, [aIDfell, to reach -23" with 50 moles (corresponding to the pure 0-glycoside acetate). The preparation of the a anomer was repeated in slightly better yield, using mercuric bromide along with calcium hydride to remove hydrogen bromidet9; initial formation of a 0-glycoside (that is, normal Walden inversion) with subsequent anomerization was postulated. On the other hand, using mercuric cyanide, the tri-0-acetyl-0-glycosides of N-acetyl-D-glucosamine were obtained when the corresponding glycosyl bromide was condensed in benzene at room temperature with simple aliphatic alcohols present in relatively small excess (2 or 3 moles).K0Up to 85 % yields of methyl tetra-0-acetyl-0-D-glucopyranosidewere claimed when tetra-0-acetyl-a-D-glucopyranosylbromide reacted with methanol (amount unstated) at room temperature in presence of mercuric bromide, acetate, or cyanide, using tertiary bases (2,6-lutidine or 2,4,6-collidine) as solvents.47In presence of a considerable excess of phenol, with mercuric cyanide as catalyst, methyl l-bromo-l-deoxy-tri-O-acetyl-a-D-glucopyranuronate in benzene yielded 45 % of the p-D-glucopyranosiduronate acetateBK1 The complexity of the reaction is illustrated by the yields of methyl tetra-0-acetyl-0-D-glucopyranoside obtained when the appropriate a-glycosy1 bromide was shaken with pure methanol (50 moles) at room temperature in the presence of mercuric cyanide.38 After 30 minutes, the yield was 64 %, rising to 89 % after 40 mins., and falling to 12 % after 100 min. Reacetylation after 150 min. brought the yield up to 80 %, suggesting that there had been deacetylation after condensation. Tetra-0-acetyl-a-D-galactopyranosides of substituted phenols have been preparedK2by fusion of the phenol with the O-acetylglycosyl bromide and mercuric cyanide, in the molar ratio 3 : 1 : 0.5, at 80-100". The a anomer was obtained in good yield after fractional recrystallization. In boiling nitromethane (b. p., 101") in the presence of mercuric cyanide, p-cresol gave a yield of the a-D-galactoside tetraacetate comparable to the yield (43 %) from a melt. No great success attended attempts to make a-D-glucosides by fusion in these experiments. Theuse of O-benzoylglycosylbromides in boiling nitromethane, in the pres-

+

(49) B. Lindberg, Arkiv Kemi, Mineral. Geol., 18B,No.9 1 (1945). (50) R.Kuhn and W. Kirschenlohr, Chem. Ber., 86, 1331 (1953). (51) K. Heyns and C. Kelch, Chem. Ber., 86, 601 (1953). (52) B. Helferich and K.-H. Jung, Ann., 689, 77 (1954).

168

J. CONCHIE, 0. A. LEVVY AND C. A. MARSH

ence of mercuric bromide or cyanide, is recommended for the synthesis of alkyl @-D-glycosides.mAlthough a considerable excess of the alcohol is normally required, equimolar concentrations of all three reagents were employed for benzyl alcohol and I-menthol with excellent results. A solution of 6.6 g. of tetra-0-benzoyl-a-D-glucopyranosyl bromide, 1.6 g. of Z-mentho1 (dried over phosphorus pentoxide), and 2.5 g. of mercuric cyanide in 20 ml. of dry nitromethane was refluxed for 7 hr. with exclusion of moisture. The sirup obtained on evaporation under diminished pressure was dissolved in 20 ml. of hot benzene. On cooling, the mercury salts separated and were filtered. The residue after evaporation was crystallized from methanol (100 ml.); 5.6 g. (75% of the theoretical) ; twice recrystallized from ethanol: m. p. 139-140".

2. Condensation of Phenols with Acetylated Sugars

The use of this reaction has been confined almost entirely to the synthesis of phenolic glycosides, by fusion of the phenol with the 0-acetylated sugar in the presence of anhydrous zinc chloride or p-toluenesulfonic acid as catalyst; the former favors the formation of the a and the latter of the @ anomer.l0 Improved yields result from the removal, under diminished pressure, of the acetic acid produced in the reaction, as well as of any that may be added.64*6 6 a. Zinc Chloride as Catalyst.-Yields of tetra-0-acetyl-a-D-glucosides have been improved by using the a anomer of penta-0-acetyl-D-glucose and by adding the zinc chloride as a solution in acetic acid-acetic anhydride (95 : 5 by vol.), the reaction being performed66under diminished pressure. T o a melt of 25 g. of penta-0-acetyla-D-glucose (0.064 mole) and 24 g. of phenol (0.255 mole) was added 6.3 g. of anhydrous zinc chloride dissolved in 20 ml. of an acetic acid-acetic anhydride mixture. The flask was kept in a bath a t 120-125" for 2 hr. and evacuated with a water pump. After dissolving the resulting red sirup in 300 ml. of ethylene dichloride (or a larger volume of benzene*'), the zinc chloride and phenol were removed by successive washing with water, dilute sodium hydroxide, and water. The solution was dried with calcium chloride and evaporated under diminished pressure, and the product was crystallized from 150 ml. of ethanol. A dense mass of isomeric acetates was obtained, totalling 92% of the theoretical. Slow recrystallization from 350 ml. of ethanol gave the OI-D form. After four recrystallizations, pure phenyl tetra-0-acetyl-a-D-glucopyranoside was obtained; m. p. 115"; [aID 169" (c, 2 in chloroform); yield 64%. The 8 anomerwas obtained in 26% yield from the original mother liquor. With penta-0-acetyl-8-D-glucose as the starting material, a 42% yield of each anomer was obtained.

+

With zinc chloride as catalyst, the 0-glycoside acetate sometimes pre(53) B. Helferich and K. Weis, Chem. Ber., 89, 314 (1956). (54) K. Sisido, J. ~%c. Chem. Id.,Japan, 39, Suppl. p. 217 (1936) ;Chern. Abstracts, 30, 7118 (1936). (55) Edna M. Montgomery, N. K. Richtmyer and C. S. Hudson, J . Am. Chem. Soc., 64, 690 (1942).

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dominates in the condensation product, for example, in the condensation with phenols.6 of methyl tetra-0-acetyl-0-n-glucopyranuronate b. p-Toluenesulfonic Acid as Catalyst.-Good yields of the @-glycoside tetraacetates of D-glucose, D-galactose, D-xylose, D-fructose, and cellobiose were obtained when the poly-0-acetylated P-D-sugars were heated in a melt (usually a t 100") with an excess ( 2 4 moles) of phenol or a substituted phenol, in the presence of p-toluenesulfonic acid (0.7 % of the total weight).lo An 85 % yield of phenyl tetra-0-acetyl-P-D-glucopyranosidewas obtained by carrying out the reaction under diminished pressure,66 and the same procedure has been employed in preparing aryl tri-0-acetyl-P-D-glucopyranosiduronic acid methyl esters in 30-60 % yield from methyl tetra-0acetyl-P-D-glucopyranuronate.EIt would appear that. the optimum pro67 portion of catalyst may vary with the sugar c. Other Catalysts.-A few catalysts other than zinc chloride and p-toluenesulfonic acid have been successfully employed in the Helferich reaction, for example phosphoryl chloride,6*sulfuric and anhydrous stannic chloride,E1 all of which yield the p anomer. Very good yields of aryl tetra-0-acetyl-P-D-glycosides were obtained with anhydrous aluminum chloride in a melt.62Boron trifluoride was used in traces a8 catalyst for the condensation of penta-0-acetyl-P-D-glucose with phenol (four moles) in benzene solution a t room temperatures3; the p-D-glucoside tetraacetate was obtained in 70 % yield after five days. In other solvents, much more boron trifluoride was required. In the first reference to the direct use of an acetylated sugar in glycoside synthesis, the aglycon was an alcoh01.~4On warming octa-0-acetyl-a-cellobiose in chloroform with sublimed ferric chloride, a complex was formed. Removal of chloroform and addition of ethanol gave ethyl hepta-o-acetyla-cellobioside in 40 % yield. Isolation of the corresponding derivative of maltose under somewhat similar conditions was claimed. The synthesis of methyl tetra-0-acetyl-P-D-glucopyranoside in 50-60 % yield was recently accomplisheds1 by condensing equimolar amounts of penta-0-acetyl-P-Dglucose and methanol a t 40' in benzene or chloroform solution, using stannic chloride as catalyst; the 0-acetylglycosyl chloride was isolated as a (56) B. Helferich, Ber., 77, 194 (1944). (57) C. D. Hurd and R. P. Zelinski, J . A m . Chenz. SOC.,69, 243 (1947). (58) T. H. Bernbry and G. Powell, J . Am. Chem. SOC.,64, 2419 (1942). (59) B. Helferich, S. Demant, J. Goerdeler and R . Bosse, Hoppe-Seyler's 2. physiol. Chem., 283, 179 (1948). (60) M. A . Jermyn, Australian J . Chem., 7,202 (1954). (61) R. U. Lemieux and W. P. Shyluk, Can. J. Chem., 31, 528 (1953). (62) C. D. Hurd and W. A. Bonner, J . Org. Chem., 11, 50 (1946). (63) H. Bretschneider and K. Beran, Monatsh., 80,262 (1949). (64) G. ZemplBn, Ber., 62, 985 (1929).

170

J. CONCHIE, G . A. L E V W AND C. A. MARSH

byproduct. When the a anomer of penta-0-acetyl-D-glucose was used in this reaction, it was recovered unchanged.

3. Condensation of Simple Alcohols with Free Sugars The oldest and simplest method of glycoside synthesis is by the Fischer reaction,66 in which the sugar is condensed directly with an alcohol in presence of hydrogen ion. One or other, or both, anomers may be separated from the reaction mixture; some sugars, such as D-mannose, give essentially only one anomer of the glycoside. Unfortunately, the method is applicable only to the lowest aliphatic alcohols and to monosaccharides. It is not applicable to phenols at all, nor, because of alcoholysis, to disaccharides. There is no way of altering the a! : p ratio in the final equilibrium mixture, and mixtures of anomeric glycosides are not as a rule as easy to fractionate as their acetates. It may thus be wellnigh impossible to separate the anomer required, even if it is formed in significant Although the introduction of cation-exchange resins as catalysts in place of hydrochloric acid sometimes yields more-readily crystallizable products, recourse to the Koenigs-Knorr reaction (Section 111, 1) is still often necessary, in order to obtain a specific glycoside. Although the final equilibrium in the Fischer reaction favors the pyranosides, the furanosides appear to be formed first,66and they can sometimes be isolated by performing the reaction under mild conditions and arresting it at an early stage. In the same way, advantage can sometimes be taken, a t the expense of the yield, of the fact that the a! and @ anomers (furanose or pyranose) may be formed a t different rates.66aIf an alcoholic glycoside is refluxed with the alcohol and an acid catalyst, equilibrium between the different forms is re-established. a. Hydrochloric Acid as Catalyst.-The sugar is refluxed in anhydrous alcoholic hydrogen chloride (0.25-3 %, w/v) for several hours, and acid is removed with silver oxide or carbonate, prior to concentration of the solution. In some cases, the preparation can be simplified by adding an inert solvent from which the glycoside separate^.^' By doing the reaction a t (65) E. Fischer, Ber., 26, 2400 (1893). (65a) A. L. Raymond and E. F. Schroeder, J . Am. Chern. SOC.,70, 2785 (1948), found that methyl 8-D-ghcopyranoside can be readily separated, in good yield, as a complex with potassium acetate, after condensing D-glucose with methanol by the Fischer method. (66) P. A. Levene, A. L. Raymond and R . T. Dillon, J. B i d . Chem., 96, 699 (1932). (66a) For analysis of the reaction between methanolic hydrogen chloride and D-galactose, see D. F. Mowery, Jr., and G. R. Ferrante, J . ARL Chern. Soc., 76, 4103 (1954). (67) F. Smith and J. W. Van Cleve, J. Am. Chem. Xoc., 77,3159 (1955).

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room temperature, it is sometimes possible to prepare the furanosides, for and example, the methyl glycosides of CX-D-~*and p-~-fructofuranose,~~ a-D-arabinofuranose?* Of the glycopyranosiduronic acids, only methyl a-D-galactopyranosiduronicacid methyl ester has yet been made by the Fischer reaction.7I Treatment of N-acetyl-tetra-0-acetyl-D-glucosamine with methanolic hydrogen chloride yielded crystalline methyl N-acetyl~-glucosaminide~~; the O-acetyl groups were removed under the conditions of the condensation. The product was comprised of 85 % of a and 15% of p anomer, and the former could only be obtained pure after complete rea~etylation.7~ b. Cation-exchange Resins as Catalysts.-An elegant improvement in technique was made when Cadotte, Smith and Spriestersbach74 introduced cation-exchange resins as catalysts that could simply be filtered off at the end of the reaction. Good yields of the methyl glycosides of a-D-glucopyranose, a-D-mannopyranose,a-L-rhamnopyranose, and p-D-and p-L-arabinopyranose were obtained, whilst D-glucuronolactone gave the a and p anomers of methyl ~-glucofuranosidurono-6,3-lactone in 10 % and 60 % yield, respectively.

IV. DEACETYLATION OF GLYCOSIDE ACETATES With the exception of the Fischer reaction, the general methods employed for the formation of the glycoside bond result in the formation of glycoside acetates (or benxoates) . Deacylation is accomplished by treatment with alkali, to which the glycoside bond is stable, unless the bond approximates to the ester type. The original method of deacetylation was by quantitative saponification with sodium hydroxide, potassium hydroxide, or barium hydroxide. It was noted, however, that, in anhydrous media, much less than the theoretical amount of alkali was required, and the reaction has since been made entirely catalytic, with considerable gain in efficiency and convenience. The same methods have been employed for debenxoylation. 1. B y Alkali

The acetate was shaken at 0" or room temperature with aqueous sodium hydroxide, potassium hydroxide, or barium hydroxide in considerable (68) C. B. Purves and C. S. Hudson, J.Am. Chem. Soc., 66,708 (1934). (69) I. Augestad, E. Berner and Else Weigner, Chemistry & Industry, 376 (1953). (70) Edna M. Montgomery and C. S. Hudson, J. Am. Chem. SOC.,69,992 (1937). (71) J. K. N. Jones and M . Stacey, J . Chem. Soc., 1340 (1947). (72) R. C. G. Moggridge and A. Neuberger, J. Chem. SOC.,745 (1938). (73) R . Kuhn, F. Zilliken and Adeline Gauhe, Chem. Ber., 86, 466 (1953). 74, 1501 (74) J. E. Cadotte, F. Smith and D. Spriestersbach, J. Am. Chem. SOC., (1952).

172

J. CONCHIE, 0. A. LEVVY AND C. A. MARSH

excess for several hours, until complete dissolution was achievedP8 ‘6, l8 Excess alkali was carefully neutralized with sulfuric acid, or alternatively, in the case of barium hydroxide, by passing in carbon dioxide. After concentration, the solution was poured into alcohol, and the precipitated salts were filtered. Last traces of salts were removed by evaporating the filtrate to dryness and extracting the glycoside with alcohol. Sometimes, shaking was obviated by dissolving the acetate in alcohol or acetone before adding the aqueous alkali.”- 78 Recently, deionization of the solution with ion-exchange resins has been intr0duced.7~8o 0

2 . By Sodium Methoxide

The first recorded use of this type of reagent in carbohydrate chemistry was made by Fischer and BergmannS1who found that only 0.2 mole of sodium ethoxide was required for complete deacetylation of a sugar or glycoside acetate in ethanol a t room temperature. This method was modified by Zemplh, mainly in connection with a study of cellobiose derivatives.a2B8 3 , 84 The acetate was dissolved in chloroform, and a solution of sodium methoxide (0.1 to 0.2 molar proportion) in methanol was added. A gelatinous, addition compound separated at 0” and was decomposed by the addition of water. After neutralization, the aqueous layer was processed in the usual way (see Section IV, 1). that a solution or suspension of a glyIt was subsequently coside acetate in absolute methanol can be completely deacetylated in a few minutes on the boiling-water bath by using catalytic quantities of sodium methoxide (about 0.002 molar proportion), with production of methyl acetate. This method is of very wide application. In a typical experiment, 0.1 ml. of 0.1 N sodium methoxide was added to 0.54 g. of methyl tetra-0-acetyl-a-n-mannopyranoside in 5 ml. of cold, absolute methanol. After 2 min. on the boiling-water bath, crystals of methyl a-D-mannopyranoside (75) C. S. Hudson and D. H. Brauns, J. A m . Chem. Soc., 37,1283 (1915). (76) E. Abderhalden and G. ZemplBn, Hoppe-Seyler’s 2. physiol. Chem., 72, 58 (1911). (77) Z. H . Skraup and J. KGnig, Ber., 34, 1115 (1901). (78) B. Helferich and A. Iloff, Hoppe-Seyler’s 2.physiol. Chem., 221, 252 (1933). (79) W. W. Binkley, M. Grace Blair and M. L. Wolfrom, J . A m . Chem. Soc., 67, 1789 (1945). (80) S. Roseman and A. Dorfman, J . B i d . Chem., 191,607 (1951). (81) E. Fischer and M. Bergmann, Ber., 62,829 (1919). (82) G . ZemplBn, Ber., 69, 1254 (1926). (83) G. Zernplbn, Ber., 60, 1555 (1927). (84) E. Pacsu, J . A m . Chem. SOC.,62,2571 (1930). (85) G. ZemplBn and E. Pacsu, Ber., 62, 1613 (1929). 186) G. ZemplBn, A. Gerecs and Ilona H a d h y , Ber., 69, 1827 (1936).

OLYCOSIDES OF THE COMMON SUGARS

173

separated. Sodium methoxide was prepared by adding the metal in small portions to cold, absolute methanol, but a chip of freshly-cut sodium may simply be added to the solution of glycoside acetate.

The period of heating varies with different compounds, and the solution may require concentration before the glycoside separates. In some cases, for example where there is a potential reducing group, it may be preferable to perform the reaction at room temperature for a longer period. Moisture must be rigorously excluded. Other primary alcohols, straight- or branchedchain, can be used instead of methanol to provide the alkoxide, but secondary and tertiary alcohols are much less efficient.87 3. By Methanolic Ammonia This reagent is still used to some extent.60* 7 3 , 88, 89 The solution or suspension of the acetate in absolute methanol at 0" is saturated with dry ammonia, or treated with an equal volume of saturated methanolic ammonia. After several hours a t 0", the solution is evaporated to dryness to give the glycoside. 4. By Barium Methoxide

Barium methoxide, introduced by Weltzien and Singer,90 is often used in catalytic amounts as a somewhat milder alternative to sodium methoxide.oln 92 As a convenient general procedure, the glycoside acetate (1 g.), dissolved or suspended in dry methanol (30-50 ml.) is treated with 0.4 N barium methoxide (1 m1.-prepared from the metal or the oxide), and left at 0" for about 24 hr. Barium may be convertedg1into the sulfate at 0", but the removal of colloidal barium sulfate may present difficulties87; hence, use of carbon dioxide was introduced.92aBarium methoxide has been particularly recommended for debenzoylation.8

5. By Potassium Methoxide 87 Potassium methoxide can be used catalytically in exactly the same way as sodium methoxide, and the added advantage is claimed that potassium ion can be removed by titration with perchloric acid at the end of the reaction ; potassium ethoxide is equally convenient. (87) W.A. Bonner and W. L. Koehler, J . Am. Chem. Soc., 70,314 (1948). (88) B. Helferich, A. Lowa, W. Nippe and H. Riedel, Hoppe-Seyler's 2.physiol. Chem., 128, 141 (1923). (89) K.-C. Tsou and A. M. Seligman, J . Am. Chem. SOC.,74, 3066 (1952). (90) W. Weltsien and R. Singer, Ann., 443, 71 (1925). (91) H. 5. Isbell, Bur. Standards d . Research, 6, 1179 (1930). (92) W. A. Mitchell, J . Am. Chem. SOC., 63, 3534 (1941). (92a) P. A. Levene and R. S. Tipson, J . B i d . Chem., 93,631 (1931).

174

J. CONCHIE, G. A. L E V W AND C. A. MARSH

6. By Dimethylamine When the deacetylation of methyl tetra-0-acetyl-a-D-fructopyranoside with ammonium methoxide was attempted, the produced acetamide formed a stable complex with the fructoside.28A method developed for the deacetylation of octa-0-acetylisosucrose9~was therefore adopted. The fructoside acetate was heated with a 30% solution of dimethylamine in absolute ethanol in a sealed tube at 100" for 3 hr., and amine, alcohol, and N,N-dimethylacetamide were removed under diminished pressure.

V. SPECIAL METHODS OF GLYCOSIDE SYNTHESIS Synthesis of glycosides of the required structure (and configuration) from the appropriate sugars and alcohols or phenols has been discussed in the preceding Sections. In this Section, the transformation of one glycoside into another, whether by anomerization, substitution of one aglycon for another, or alteration of the sugar residue, will mainly be dealt with.

1. Anomerization In practice, anomerization is almost always applied to the glycoside acetates, and in almost all recorded instances the transformation is from 8-D to a-D.Many of the catalysts employed in the synthesis of acetylated sugars or in their condensation with aglycons (for example, zinc chloride or sulfuric acid) have been employed for the anomerization of the glycoside acetates. This list does not, however, include titanium tetrachloride, the best-known anomerizing agent. The theory of anomerization is dealt with in an earlier review in this a. With Titanium Tetrachloride.-Pacsu, who introduced this reagent,84 9 4 , 96 obtained a 25 % yield of methyl hepta-0-acetyl-a-cellobioside after refluxing the B compound with titanium tetrachloride in chloroform. A nearly 70% anomerization was accomplished in the same way with the and methyl 0-D-mannotetraacetates of ethyl /?-~-galactopyranoside~~ pyranoside,g7 but the method was unsuccessful when applied to phenyl tetra-0-acetyl-~-~-g~ucopyranoside.~~ A 50 % yield of the a anomer was produced by titanium tetrachloride from methyl tetra-0-benzoyl-p-D-glucopyranoside in chloroform in 17 min. at room t e m p e r a t ~ r e . ~ ~ (93) (1929). (94) (95) (96) (97) (98) (99)

J. C. Irvine, J. W. H. Oldham and A. F. Skinner, J. Am. Chem. SOC.,61, 1279 E. Pacsu, J. A.m. Chem. Soc., 62, 2563 (1930). E. Pacsu, J. Am. Chem. Soc., 62, 2568 (1930). L. Asp and B. Lindberg, Acta Chem. Scand., 4, 1386 (1950). L. Asp and B. Lindberg, Acta Chem. Scand., 6,947 (1952). R. U. Lemieux and W. P. Shyluk, Can. J . Chem., 33, 120 (1955). R. E. Reeves and L. W. Maesero, J . Am. Chem. SOC., 76, 2219 (1954).

GLYCOSIDES O F THE COMMON SUGARS

175

b. W i t h Boron Tri$uoride.-LindbergloOo lol considers boron trifluoride to be preferable to titanium tetrachloride for the transformation of alkyl 8-D-glucosides into the a anomers. The ease of transformation of the tetraacetates was in the order: is0 Pr > Et > Me 2 ally1 fi benzyl; whereas the isopropyl D-glucoside required only 30 min. a t room temperature, the methyl compound needed 24 hr. for complete reaction. Methyl tetra-0-acetyl-P-n-glucopyranoside(1 g. ; dried over phosphorus pentoxide) in anhydrous chloroform was saturated with gaseous boron trifluoride and the solution was kept for 24 hr. The gelatinous precipitate was decomposed by shaking with sodium bicarbonate solution, and the chloroform layer was washed with water. After drying with calcium chloride, i t yielded 0.98 g. of the 01 anomer; m. p. 97-98O; [aID+121". Recrystallization from ethanol gave material of m. p. 100-101"; [aID +130°. c. With Other Catalysts.-Other catalysts include stannic chloride, considered less efficient than titanium tetrach1oride,l02and sulfuric acid in an acetic acid-acetic anhydride mixture.96 1O3 Undoubtedly, in some instances the catalyst is simply re-establishing equilibrium between the anomeric glycoside acetates. Thus, a 30 % transformation of phenyl tetra-o-acetylp-D-glucopyranoside into the a form was obtained66by fusion with phenol and zinc chloride under diminished pressure for 3 hr. at 120-125'. The reverse transformation, to the extent of 33 %, was produced by p-toluenesulfonic acid.62 2. AgEycon Exchange Methyl p-D-fructopyranoside can be obtained*04in 80% yield from the corresponding benzyl glycoside by treatment with 0.03 N methanolic hydrogen chloride for 40 min. at 20'; benzyl p-D-fructopyranoside was prepared in 30 % yield by condensing D-fructose with benzyl alcohol in the presence of 0.2 N hydrogen chloride for 60 min. at 20". A variation of the Helferich reaction (see Section 111, 2), which might be of practical use under certain circumstances, is illustrated by the preparation of phenyl tetra-0-acetyl-a-D-glucopyranosidefrom the methyl a-D-ghcoside acetate by fusion with phenol and zinc chlorideb6;the yield of the phenyl a anomer was 55 %, and some of the phenyl p anomer (15 %) was also isolated. I

3. Oxidation of Glycosides to Glycosiduronic Acids There are two general methods for the synthesis of glycosiduronic acids. The first is that used for other glycosides, namely, by condensing the (100) (101) (102) (103) (104)

B. Lindberg, Acta Chem. Scand., 2, 426 (1948). B. Lindberg, Acta Chem. Scand., 2,534 (1948). E. Pacsu, Bey., 61, 137 (1928). B. Lindberg, Acta Chem. Scand., 3, 1153 (1949). C. B. Purves and C. S. Hudson, J . Am. Chem. SOC.,69, 1170 (1937).

176

J. CONCHIE, G. A. LEVW AND C. A. MARSH

aglycon with the poly-0-acetylglycuronic acid or with the acetyl-1-bromo1-deoxy-glycuronic acid (see Sections 111, 1 and 2), both in the form of the methyl ester. The second involves the selective oxidation of the primary alcohol group of the corresponding glycoside. The first of these general methods, using the acetyl-a-D-glycosyl bromide, has been used to prepare, amongst other compounds, methyl 6-D-galactopyranosiduronic acidlo6and pregnanediol-3 @-D-ghcopyranosiduronicacid,Iosthe latter being the first of the known urinary glucosiduronicacids to be synthesized.lW D-Glucuronic acid having now become freely available in the form of the lactone, the synthesis of phenyl P-D-glucopyranosiduronic acid and other aryl P-D-glucopyranosiduronic acids by both types of condensation reaction has been intensively studied, and good yields have been obtained.6*61 It is noteworthy that no great success has attended attempts to prepare the a anomers in this way, even in the Helferich reaction with zinc chloride as catalyst (see Section 111,2a) .6 Whereas D-glucuronic acid and its esters and salts are normally pyranose, ~-glucurono-6,%lactone is furanoselOsand can be used in condensation reactions for preparing p-D-glucofuranosidur o n o l a c t ~ n e s110~ ~(compare ~~ Section 111, 3b). Many attempts have been made in the past to find a general reaction for the oxidation of glycosides to glycosiduronic acids. Of purely historical interest is the preparation of I-menthyl a-D-glucopyranosiduronk acid in low yield (3-5 %) by hypobromite oxidation of the D-glucosidelll;the very low solubility of this particular glucosiduronic acid permitted its isolation from the reaction mixture. Anhydrous nitrogen tetroxide was employed to oxidize methyl a-D-galactopyranoside to the glycosiduronic acid in about 50 % yield,l12but this reagent gave poor yields of methyl a- and p-D-glucopyranosiduronic acids (which can only be isolated as the triacetates of the methyl esters) .l13* 11* Nevertheless, the use of nitrogen tetroxide would appear to be the most promising of the non-catalytic methods for the oxidation of glycosides to glycosiduronic acids. Irradiation of hexoses in dilute aqueous solution with 1 MV electrons has been found to yield hexuronic (105) S.Morell, L.Baur and K. P. Link, J . Biol.Chem., 110,719 (1935). (106) C.F.Huebner, R . S. Overman and K. P. Link, J . Biol. Chem.,166,615 (1944). (107) The occurrence of methyl 8-D-glucopyranosiduronicacid in urine is a rather special case; see I. A. Kamil, J. N. Smith and R. T. Williams, Biochem. J . (London), 64, 390 (1953). (108) F. Smith, J . Chem. Soc., 584 (1944). (109) K.-C. Tsou and A. M. Seligman, J . Am. Chem. SOC.,76, 1042 (1953). (110) K.-C. Tsou and A. M. Seligman, J . A m . Chem. SOC.,74,5605 (1952). (111) M. Bergmann and W. W. Wolff, Be?-.,66, 1060 (1923). (112) K. Maurer and G . Drehfahl, Chem. Ber., 80. 94 (1947). (113) E.Hltrdegger and D. Spitz, Helv.Chim. Acta, 83,,2165(1949). (114) E.Hardegger and D. Spits, Helv. Chim. Acta, 33, 337 (1950).

GLYCOSIDES O F THE COMMON SUGARS

177

acids by specific oxidation116at C6, and there would appear to be no difficulty in applying this reaction to the synthesis of glycosiduronic acids. Gaseous oxygen in the presence of a platinum catalyst was found to in neutral or mildly alkaconvert 1 2-O-isopropy~idene-cY-~-glucofuranose line solution into the D-glucuronic acid d e r i v a t i ~ e , ~and ~ ~ -this ~~~ reaction 8 was developed into a general one for the preparation of alkyl glycosiduronic acids from the corresponding alkyl glycosides.*18 )

A powdered platinum catalyst was prepared in aqueous suspension from Adams' platinum oxide catalyst by hydrogenation at atmospheric pressure; i t could be stored under water for about a week before there was any undue loss in activity. A suspension of the catalyst (0.1 9.) in an aqueous solution (20 ml.) of methyl or-o-galactopyranoside monohydrate (1.069.) was placed in a water-bath a t 60" and vigorously stirred. Oxygen was passed into the liquid, and small samples were periodically withdrawn for measurement of pH. Neutrality was maintained by suitable addition of N sodium bicarbonate. Reaction was complete in 5 hr., with the consumption of 4.9 ml. of bicarbonate solution. The catalyst was filtered and the filtrate was made definitely alkaline with ammonia. Excess basic lead acetate was added t o precipitate the lead salt of methyl a-D-galactopyranosiduronicacid. After being washed with water on the centrifuge, the lead salt was decomposed with hydrogen sulfide at 0". Evaporation of the colorless filtrate under diminished pressure gave a gum which crystallized on drying. Two recrystallizations from 95% ethanol yielded the pure glycosiduronic acid as the dihydrate; m. p. 110" (sintering at 105" and decomposing a t 126"); [uID 128" ( c , 2 in water); yield, 42% of the theoretical.

+

Early attempts to prepare phenyl Q- and p-D-glucopyranosiduronic acid by this reaction were not always successfuLs1,118 However, by performing the oxidation a t a slightly higher temperature (about 90") and at pH 8-10, and by using more catalyst, it has been found possible to obtain botb compoundss, l o o ; the use of really fresh catalyst is also important.z0 The following modificationz0of the original procedures for the preparation of the CY anomer has been found to improve the yield nearly four-fold. A solution of phenyl a-D-glucopyranoside monohydrate (2.16 g.) in water (50 ml.) was adjusted t o pH &lo, and catalyst (0.8 9.) was added. Oxygen was passed into the stirred suspension maintained a t 90" and constant pH, and further catalyst (0.2g.) wa8 added after 1 hr. Reaction was complete after a further 90 min., with a total uptake of 15.5 ml. of 0.5 N sodium bicarbonate (99% of the theoretical). The filtered solution was evaporated under diminished pressure t o 10 ml., and 100 ml. of hot ethanol was added. After immediate filtration and cooling, the sodium salt of phenyl (115) G. 0. Phillips, Nature, 173, 1044 (1954). (116) R.Fernhndea-Garcia, L. Amor6s, Hilda Blay, E. Santiago, Hilda SolteroDiaz and A. A. CoMn, El Crisol, 4,40 (1950). (117) C. L. Mehltretter, B. H. Alexander, R. L. Mellies and C. E. Rist, J. A m . Chem. Soc., 73, 2424 (1951). (117a) C.L.Mehltretter, Adnrances in Carbohydrate Chem., 8,231 (1953). (118) C.A. Marsh, J . Chem. Soc., 1578 (1952).

178

J.

CONCHIE,

G . A. L E W Y AND C. A. MARSH

a-D-glucopyranosiduronicacid separated (1.82 g.). The crude sodium salt was dissolved in water (18 ml.) and the pH was adjusted to 2.2 with sulfuric acid. After continuous extraction with ethyl acetate for 2.5 hr., the aqueous layer gave only a feeble Tollens reaction. Concentration of the ethyl acetate layer t o 5 ml. gave the glycosiduronic acid (1.35 g., 62% of the theoretical), m. p. 148-149". Recrystallized from moist ethyl acetate it had m. p. 149-160'; [&II, 150" (c, 2 in water) ; yield, 52% of the theoretical.

+

4. Miscellaneous Until recent years, the sole source of 8-D-ghcopyranosiduronic acids, and indeed of D-glucuronic acid itself, was the urine of animals fed with the appropriate aglycons, and in most instances the chemical synthesis in the laboratory has yet to be achieved. In some cases [for example, phenolphthalein (mono-)p-D-glucopyranosiduronic acid], laboratory synthesis still presents difficulties. The most economical method of preparing certain methyl glycosides is by methanolysis of polysaccharides (for example, the preparation of methyl a-D-mannopyranoside from mannan1Ig). Selective methylation of D-mannose with one molar equivalent of dimethyl sulfate is a recognized way of making methyl /3-D-mannopyranoside.'20 The mixture of anomers can be fractionated only after acetylation, and the deacetylated /3 anomer has so far only been crystallized with one molecule of isopropyl alcohol of crystallization. Diazomethane has also been employed121 for the selective methyl glycosidation of a sugar at C1. The preparation of crystalline methyl a-L-arabinofuranoside was accomplished by treating L-arabinose diethyl thioacetal in methanol with mercuric chloride, mercuric oxide, and DrieritelZ2;ethyl a-lactoside was prepared by a somewhat similar proced~re.1~3 Tri-O-acetyl-l , 2-anhydro-cr-~-glucopyranose (Brigl's anhydride) can be employed for forming a- or 0-D-glucopyranosides, depending on the conditions,4,26 but has not yet found wide application in the field of simple glycosides.

VI. DESCRIPTION OF TABLES Tables I to IV give constants for the known methyl and phenyl a- and p-glycosides of the sugars listed in the Introduction. Rotations have been measured in water unless there is a footnote to the contrary. The column headed Preparation indicates those Sections and Subsections in the text (119) C. S. Hudson, Org. Syntheses, Coll. Vol. 1 , 371 (1948). (120) H . S. Isbell and Harriet L. Frush, J. Research N a t l . Bur. Standards, 24, 125 (1940). (121) R. Kuhn and H. H. Baer, Chem. Ber., 86, 724 (1953). (122) J. W. Green and E. Pacsu, J . A m . Chem. SOC.,60, 2056 (1938). (123) J. Stanek and J. S&da,Collection Czechoslov. Chem. Communs., 14,540 (1949) ; Chsm. Abstracts, 44, 5820 (1950).

GLYCOSIDES O F THE COMMON SUGARS

179

that deal with the general method of synthesis; where there are alternatives, the presumed best of the published methods has been selected. Sometimes, however, the constants refer to a glycoside that was simply isolated as a byproduct in making the anomer; obviously, then, a more appropriate method of synthesis may be worth investigating. In yet other cases, there is no doubt that the use of a more recent method would lead to a considerable improvement in yield. Where the intermediate is an 0-acylglycosyl halide, the 0-acetylglycosyl bromide was always employed, except in the cases of methyl a-D-fructopyranoside (from the 0-acetylglycosyl chloride) and methyl p-D-ribopyranoside (from the 0-benzoylglycosyl bromide). The first reference to the literature deals in each instance with the method of synthesis; other references give further details or better constants. Tables V to VII give constants for the crystalline glycoside acetates that have been isolated in the course of the syntheses listed in Tables I to IV. Optical rotations were observed in chloroform, unless otherwise stated. Since the acylated sugars are of such importance as intermediates in the syntheses of glycosides, constants for the poly-acetates and -benzoates have been collected in Tables VIII to X. Optical rotations are, again, usually given for solutions in chloroform. To the best of our knowledge, there is no recent compilation of constants for these compounds. For the other important class of intermediates in glycoside synthesis, the 0-acylglycosyl halides, the reader is referred to the Tables in a previous review in this Se~ies.~

180

J. CONCHIB, G . A. LEVVY AND C. A. MARSH

TABLE I Phenyl a-Glycosides Y.fi., "C.

D-Glucopyranose& D-Galactopyranoseb D-Mannopyranose N-Acetyl-D -glucosamine D-Xylopyranose L-Arabinopyranose D-Arabinopyranose Maltose Cellobiose D-Glucopyranuronic acid D-Galactopyranuronic acid

173-174 146 132-133 246-247 145 153-155 153-155 sirup 25lC 147-149 192-193

[aln degrees (daler)

Pre$aration"

References

+181 +217 +114 +213 189 +6.0 -5.5 198 124 154 +156

III2a, IV2 I I I l c , IV2 IIISb, IV2 III2a, IV4 1112b, IV4 IIIla, IV2 I I I l a , IV2 III2a, IV2 III2a, IV2 v3 v3

55, 43 45, 124, 59 125 80, 20 55 126 127 128 10, 129 6 20

+

+ +

+

a Refers t o Sections and Subsections i n the text, dealing with general methods used in the preparation. b Monohydrate; m. p. and [aIDfor the anhydrous compound. Uncorrected. M. p. and for hemihydrate.

(124) B. Helferich and H. Appel, Hoppe-Seyler's 2.physiol. Chem., 206,231 (1932). (125) B. Helferich and S. Winkler, Ber., 66, 1556 (1933). (126) B. Helferich, S. Winkler, R. Goota, 0. Peters and E. Gunther, HoppeSeyler's 2.physiol. Chem., 208,91 (1932). (127) B. Helferich, H. Appel and R. Gootz, Hoppe-Seyler's 2. physiol. Chem., 216, 277 (1933). (128) B. Helferich and S. R. Petersen, Ber., 68, 790 (1935). (129) G. Jayme and W. Demmig, Chem. Ber., 88, 434 (1955).

GLYCOSIDES OF THE COMMON SUGARS

181

TABLE11 Phenyl P-Gl ycosides Parenl sugar

M.

)., "C.

!RID,

degrees (waler)

o-Glucopyranoseb o-Galactopyranosec u-Mannopyranose u-Fructopyranose N-Acetyl-u-glucosamine o-Xylopyranose o-Ribopyranosed L-Arabirtopyranose o-Arabinopyranose Maltose Cellobiose Lactose o-Glucopyranuronic acid'

175-176 155-156 175-177 173-174 249-250 178-180 143- 144 176- 179 177-1 79 96 211-213 191-192 161-162

-72 -43c - 72 - 210 -10.3 - 49 - 108" +243 -244 +34 - 60 - 36 -91

n-Glucofuranuronic acidu o-Galactopyranuronic acidi

185-186 173

-115 - 73

Piegaration"

References

I I I W , IV2 IIIBb, IV2 III2a, IV2 IIIQb, IV2 IIIQb, IV4 III2b, IV2 1112b, IV3 III2a, IV2 IIIZa, IV2 IIIBb, IV1 I I I l b , IV4 I I I l b , IV2 V3 (or III2b, IV4) 1112b, IV3h v3

55, 43 56, 130, 50 125 10 80, 20 55, 124 89 127 127 131, 132 133 134 109, 135, 6 110 20

SeeTable I. Dihydrate; m. p. and [a10on arihydrouscompound. Hemihydrate; m. p. on anhydrous compound. 6-Bromo-2-1i~plithylderivative: mono-methanolate. e I n dioxane. f Dihydrate; m. p. and [a111 on anhydrous compound. Lactone of 2naphthyl derivative, monohydrate; m.p. and [a],,(in dioxane) on anhydrous compound. Resnltant amid? decomposed with HNOr . Monohydrate; [@In in methanol. (130) E:. Fischer and l3. F. Armstrong, Ber., 36, 833 (1902). (131) L. Asp and B. Lindberg, Acta Chem. Stand., 6, 941 (1952). (132) E. Fischer and E. F. Armstrong, Ber., 36, 3153 (1902). (133) Edna M. Montgomery, N . I<. Richtmyer and C. S. Hudson, J . A m . Cheni. Soc., 66, 1848 (1943). (134) B. Helferich and R. Griebel, Ann., 644, 191 (1940). (135) G. A. Garton, 1). Robinson and R. T.Williams, Riocheni. J . (London), 46, 65 (1949).

182

J. CONCHIE, G. A. LEVVY AND C. A. MARSH

TABLE111 A4elhyl CY -G1gcosides Parent szrgar

n-Glucopyranose o-Galactopyranose* D-Mannopyranose D-Fructopyranose D -Fruc tof ur anose N-Acet,yl-n -glucosamine n-Xylopyranose n-Ribopyranose L-Arabinopyranose L-Arabinofuranose n-Arabinopyranose o-hrahinof uranose Cellobiose Lactose* n-Glucopyranuronic acidi u-Glucofuranuro~iicacidi o-Galactopyranuronic acidk o-Mannopyranuronic acidG

M. p., "C.

[ a ] o degrees ,

165-166 111-112 193-194 102 69 187-188 90-92 sirup 131

+158 +196 +82 +47c +93 +131 +154 +103" +17 -125 -17 +123 +97 -52 +46 +147 +128 +66

-f -a

65-67 144-145 sirup 141-142 148 112-114 108

Preparation"

III3b III3a III3b IIIlc, IVl I I I 3 a (or IV6) III3bd III3a III3a III3a V4 -0

III3a V l a , IV2 V4 V3 11131) V3 V3

Rejerences

74, 136 137, 138 74, 139 27, 28 68 73 140, 138 141 140 122 142 70 8.1 123 118 74 118, 112, 143 118, 144

a See Table I. Monohydrate; m. p. and [ a ]on~ anhydrous compound. In ethanol, [a]" 93". Freed from anomer by acetylation, fractional recrystallization, and deacetylation. In methanol. Too hygroscopic for m. p. 0 No details given. Ethyl derivative; [ a ]inethanol. ~ Z-Menthyl derivative; in. p. and [a]" (in ethanol) on dihydrate. 7 Lactone.k M. p. anti [ a ]on ~ dihydrate.' M. p. and [aln on monohydrate.

+

(136) T. S. Patterson and J . Robertson, J . Cheni. Sac., 300 (1929). (137) J. K. Dale and C. S. Hudson, J . Am. Chern. Soc.. 6 2 , 2534 (1930). (138) E. Fischer, Be?-., 28. 1145 (1895). (139) F,. Fischer and I,. Beensch, Ber., 29, 2927 (1896). (140) C. S.Hudson, J . A m . Chem. Soc., 47, 265 (1925). (141) G. R. Barker and I). C. C. Smith, 6. Chem. Soc., 2151 (1954). (142) E. L. Jackson and C. S. Hudson, 6. A m . Chem. Soc., 69, 994 (1937). (143) S. Morel1 and I<. P. Link, J . Riol. Chem., 100, 385 (1933). (144) R. G. Ault, W. N. Hnworth and E. I,. Hirst, J. Chem. Soc., 517 (1935).

GLYCOSIDES OF THE COMMON SUGARS

183

TABLE IV Methyl p-Glglrosides Parenl sugar

u-Glucopyranoseh

u -Galactopyranose wMannopyranowr D-Fruct opyranose D -Fruct ofuranose N-Acetyl-u-glucosamiiie L)-Xylopyranose D-Ri bopyranose L-Arabinopyranose o-Arabinopyranose Maltose’ Cellohioseu Lactosch D-Glucopyraiiuronic acid’ D-Glucofuranuronic acid? D-Galactop).ranuronic acid’ D-Mannopyrariuronic acid

M. f l . , “C.

1aIt1, degrees

(waler)

Preparalionn

Rejerences

110 178-180 74-75 119-120 sirup 204-205 157 83-84 172 172 111-113 193 206 75-77 14CL141 163-165 sirup

-32 +0.61 - 53 - 172 - 50 -44 - 66 - 105 246 - 244 +85 - 19 +5.6 - 99 -58 -40 25

11138 III3a v4 v2 1113ad I I I l d , IV3 III3a IIIl,e IV4 III3b III3b I I I l a , IV2 I I I l a , IV3 I I I l a , IV2 v3 III3b v3 v3

65a, 136 137, 138 120 104, 145 69, 146 50 140 7, 8, 147 74, 140 74 148 88 149, 150 118, 151 74, 20 118, 105 118

+

+

See Table I. * Hemihydrate; m. p. and [a]=on anhydrous compound. e M. p’ and [all, on mono-2-propanolate. Separated chromatographically. No added condensing agent or catalyst. f M. p. and [ a ] D on monohydrate. Monohydrate; m. p. and [a]=on anhydrous compound. Monohydrate. l-Menthyl derivative, 1. 5 H20 ([a]= in ethanol) ; varying const,ants are reported for biosynthet,ic preparations. j Lactone. (145) (146) (147) (148) (1942). (149) (150) (151)

C. S. Hudson arid D. H . Brauns, J . Am. Chem. SOC.,38, 1216 (1916). H. H . Schlubach and H . E. Bartels. ilnn., 641, 76 (1939). E. I,. Jackson and C. S. Hudson, J . Am. Chem. SOC.,63, 1229 (1941). T . J. Schoch, E. J. Wilson, Jr., and C. S. Hudson, J . Am. Chent. SOC., 64,2871 F. Smith and J. W. Van Cleve, J . .tni. Chent. SOC.,74, 1912 (1952). R. Ditmar, Ber., 36, 1951 (1902). R. T. Williams, Biorhenz. .J. (London), 33, 1519 (1939).

184

J. CONCHIE, G . A . LEVVY A N D C. A . MARSH

TABLE

v

Polyacetates of Phenul a-Gi "osides

Parent sugar

v-Glucopyranose D-Galactopyranose D-Mannopyranose N - Acetyl-D-glucosamine i~-Xylopyranose L-Arabinopyranose Maltose Cellobiose D-Glucopyranuronic acidu a

M. fi., "C.

[ale, degrees

(CHCla)

References

114-115 131-132 79-80 122-123 64-65 87-89 184-185 228 110-1 12

+I68 +I76 74 138 +135 25 +170 +83 163

10 45 125 80 55 126 128 10 6

+

+

+

+

Methyl ester.

TABLE VI Polyacetates of Phenyl 6-Glycosides Parent sugar

o-Glucopyranose D-Galactopyranose D -Mannopyranose v-Fruc topyranose N-Acetyl-D-glucosamine D-Xylopyranose n-Ribopyranose Maltose Cellobiose Lactose D-Glucopyrariuronic acidd v-Glucofuranuronic acid'

Y.$., "C.

125-126 123-1 24 169-1 70 128-1 31 209-210 148 161-162 157-158 206-208 162 127-128 231-232

[ a ) . , degrees

(CZCls) -23 -26"

- 63h - 146 -21 -51 -54 +42* -36 -23

References

55 130 125 10 80, 20 55 89 131, 132 133 134 6 110

a I n benzene. Solvent unstated. 6-Bromo-2-naphthyl derivative; m. p. after drying. d Methyl ester. Lactone of 2-naphthyl derivative. In pyridine.

TABLE VII Polyacetates of Methyl a- and P-GIycosides M . p., “C.

Parenl sugar

8-D-Mannopyranose or-D-Fructopyranose 8-D-Fructopyranose N-Ace tyl-a-D-glucosamine N-Acet yl-p-D-glucosamine @-Maltose a-Cellobiose p-Cellobiose 8-Lactose Q

,DI.[

degrees (CZIC1a)

159-160 112 75-76 107-108 163-164 128-129 185 187 65-66

-48

+45 - 125 +lo0 - 24= 53 56 -26b +6.4

+ +

Rejerences

120

27 145 73 50 152,148 84 88 150

In methanol. a In acetylene tetrachloride: values in chloroform are similar.

TABLEV I I I Polyacetates of a-Pyranose Sugars Parenl sugar

D-Glucose n-Galactose D-Mannose n-Fructose N-Acetyl-D-glucosamine D-XylOHC n-Ribose L-Arabinose D-Arabinose Maltose Cellobiose Lactose n-Glucopyranuronic acid* D-Galaotopyranuronic acidb a

Y.p . , “C.

112-113 96 74 122-123 139-140 59 sirup 97 99-100 125 230 152 111-1 12 142-143

[.ID,

degrees (CHCla)

+102 +107

f57 47 +94 +89 +5lU 43 -44 +123 43 54 98 143

+ + + + +

+

References

153 17 154,15,155 156 157,158 16 159 18 160 161 161,23 162 163 164

In methanol. * Methyl ester.

(152)J. C. Irvine and I. M. A. Black, J . Cheni. Soc., 862 (1926). (153) C. S.Hudson and J. K. Dale, J . A m . Chem. Soc., 37,1264 (1915). (154) 1’. A . Levene, J . B i d . Cheni., 69, 141 (1924). (155) P. A. Levene and R . S. Tipson, J . Biol. Chem., 90,89 (1931). (156)E.Pacsu and F. €3. Cramer, J . Am. Chem. SOC.,67, 1944 (1935). (157)0. Westphal and H. Holzmann, Ber., 7 6 , 1274 (1912). (158) C. S.Hudson and J. K. Dale, J . A m . Cheni. Soc., 38, 1431 (1916). (1591 H. Zinner, Cheni. Rer., 86, 817 (1953). (160)A. M. Gnkhokidze, Z h w . Obshchei Khim., 16, 539 (1945);Chem. Abstracts, 40, 4674 (1946). (161)C. S.Hudson and J . M. Johnson, J . A m . Chert,. SOC.,37, 1276 (1915). (162)C. S.Hudson and J. M. Johnson, J . A m . Chem. Sor., 37, 1270 (1915). (163)W. F. Goebel and F. H. Babers, J. Biol. Chem., 106, 63 (1934). (164)S.Morel1 and K. P. Link, J . Biol. Cheni., 108, 763 (1935). 185

1%

J. CONCHIE, G. A . LEVVY A N D C. A. MARSH

TABLEI X Polyacetates of P-Pyramse Sug rs and of ~-~-Glucofuranurono-G,S-lacto.ne Parent sugar

n-Glucose D-Galactose D-Mannose o-Fructose N-Acetyl~D-glucosamine D-Xylose D -Ribose L-Arabinose D-Arabinose Maltose Cellobiose Lactose D-Glucopyranuronic acidb D-Galactopyranuronic acidb ~-Glucofuranurono-6,%lactone a

M . 9 . . “C.

[a]” degrees

(CHCla)

References

132 142 117-1 18 108-109 188-189 128 113-114 86

+3.9 1-25 -25 - 121 +1.2 - 25 -58 147 - 147 +63 - 15 -4.4 +9 58 +90

153 17 15, 165 75 166, 158 16, 167 168, 169 18 170 161, 171 161 162 163, 6 172 110

-a

159-160 202 90 178 142-143 193-194

+

+

No m. p. given. Methyl ester.

(165) E. Fischer and R. Oetker, Rer., 46,4029 (1913). (166) M. Bergmann and L. Zervas, Ber., 64,975 (1931). (167) W . E. Stone, Am. Chem. J., 16, 653 (1893). (168) G. €3. Brown, J. Davoll and B. A. Lowy, Biochem. Preparations, 4, 70 (1955). (169) 1’. A. Levene and R . S. Tipson, J. Biol. Chem., 92, 109 (1931). (170) Edna M. Montgomery, R . M. Hann and C . S. Hudson, J . A m . Cheni. Soc , 69, 1124 (1937). (171) I>. H . Brauns, J . ilm. Chem. Soc., 61, 1820 (1929). (172) E. L. Pippen and R . M. McCready, J. Org. Chem., 16, 262 (1951).

187

GLYCOSIDES O F THE COMMON SUGARS

TABLE X Polybenzoates of Furanose and P Parent sugar

a - Glucopyranose ~ 8-D-Glucopyranose u-D-Galactopyranose a-o-Mannopyranose P - D - Mnnopyranose ~ P-D-Friictopyranose'~ o-Frur tofuranoseb a - D - X y lopyranose 8-0-Xy lopyr anose 8-D-Ribopyranose a-L-Arabinopyranose 8-L-Arabinopyranose a-o-Arabinopyranose 8 - D - Arabinopyranose a-D-Galactopyranuronic acid<

.v.p . , "C. 190-191 189-192 158-159 152-153 161-162 179-182 124-125 119-120 177 131 164-165 173-174 164-165 160-161 181-182

anose Sugurs [a], , degrees

(CUCla)

References

+139 +24 +187

9,173 9 174,175

- 10

-82

- 172

-6

4 -14 +I50 -42 - 102 +114 +325 - 114 -323 279

+

9

9 176 177 178 178 7 179 175 179 179 164

a 1,3,4,5-Tetrabenzoate; there is no satisfactory evidence for the existenceof a n y ring-form pentabenzoates.lsO 1,3,4,6-Tetrabenzoate; see a. Methyl ester.

(173) P. A. Levene and G . M. Meyer, J . Biol. Chem., 76,513 (1928). (171)V. Deulofeu and J. 0. Deferrari, J . Org. Chem., 17,1097 (1952). 1175) M.L . Wolfrom and C. C. Christrnan, J . <4na.Chem. Soc., 68, 39 (1936). (176)R.K.Ness and H . C . Flekher, Jr., J . A m . Cheni. Soc., 76,2619 (1953). (177) P. Brig1 and R. Schinle, Ber., 67,127 (1934). (178)H . G. Fletcher, Jr., and C. S. Hudson, J . A m . Cheni. SOC.,69,921 (1947)(179) H. G. Fletcher, Jr., and C. S. Hudson, J . A m . Chem. Soc., 69, 1145 (1947). (180) C. P.Barryand J. Honeyman, Advances in Carbohydrate Cheni., 7,53(1952).

This Page Intentionally Left Blank

THE SCHARDINGER DEXTRINS

BY DEXTER FRENCH Departnienl of Ch.emistry, Iowa State College. Anies. Io.wa

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 I1. IIistorical Review . . . . . . . . . ........................................ 192 1. Discovery of the Schard Dextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 2 . Primitive Concepts as to Chemical Structure and Molecular Size . . . . . 198 3 . The Maturation Period 1935-1950 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Other Members of the Schardinger Dextriu. Family . . . . . . . . . . . . 111. Fractionation and Purification of the Schardinger Dextrins . . . . . . . . . . . . . 211 IV . Bacilliis maceruns Amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 1. Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 220 2 . Measurement of B . inaceruns Amylase Activity . . . . . . . . . . . . . . . . . . . . . . 3 . Substrates for Production of Schardinger Dextrins by B . maceruns Amylase . . . . . . . . . . . . . . . . . . . . . . ..................................... 224 4 . Action Pktttern of I3 . niacer Amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 V . Other Biochemical Properties of the Schardinger Dextrins . . . . . . . . . . . . . . 231 1. Degradat.ion by Amylases . . . . . . . . . . . . . . . . . . . . . . . . 2 . Inhibition of Phosphorylase . . . . . . . . . . . . . . . . . . . . . . 3 . Utilization of Schardinger Dextrins by Organisms . . . . . . . . . . . . . . . . . . . . 232 V I . Molecular Size of the Schurdinger Dextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 1 . Molecular Size from Measurement. s of Colligative Properties . . . . . . . . . 234 2 X-Ray Molecular Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 238 4 . Sedimentation and Diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Partial Hydrolysis by Acid or Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 5 . Paper Chromatography, Optical Rotation, and Miscellaneous Observations Bearing on the Molecular Size of t.he Schardinger Dextrins . . . . . 241 VII . Molecular Constitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 1. The Points of Glycosidic At’tachment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 246 2 . Anomeric Configuration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Complex Formation and Inclusion Compounds . . . . . . . . . . . . . . . . . . . . . . . . . 247 1 . Complexes wit-h Organic Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 2 . Complexes wit.h Iodine arid Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 I X . Ring Conformation in the Schaldinger Dextrins . . . . . . . . . . . . . . . . . . . . . . . . 252 X . Ilerivirtives of the Schardinger Dest.rins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 1 . Acetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 2 . h’itrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 3 . Methyl I
.

189

190

DEXTER FRENCH

I. INTRODUCTION The Schardinger dextriiis are a group of homologous oligosaccharidw, obtained from the breakdown of starth by the artioii of Bacitltts macvrnns amylase. They bear the name Srhardingcr’ in recognition of thc fact that, Schardinger first identified BaciUiis maccrans and first described their preparation and properties ill reliable detail. These curious compounds have a fascination for the carbohydrate chemist? in that, they have properties markedly different from those of other

FIG.l.-Alpha

tlextrin from 60% propanol, hexagonal

carbohydrates (especially other dextrins) in their molecular-weight range. Some of these unique properties are as follows. (1) Brief obituary notices of Franz Schardinger are published in 2. angew. Chem., 33, 396 (1920), Chem.-Ztg., 44, 744 (1920), and Osterr. C h e m - Z t g . , 23, 150 (1920) (in translation) : “Government counsel Franz Schardinger, M.D., I’h.D., chemist, and bacteriologist, retired chief inspect,or of the Untersiichiingsanst:Llt fur Lebensmittel in Vienna, died during the night of Sept,. 27-28, 1920, a t Iririsbruck as the result of a stroke, at the age of 67 years. Schardinger has rendered considerable service t o science through his discovery of: (a) the levolact,ic acid bacillus, (b) the methylene blue reaction for the distinction between raw and pasteurized milk, (c) Bacillus macerans, (d) acetone fermentat.ion by B . niurernn,~,and (e) formation of cryst.alline ‘polysaccharides’ [Schardinger dext,rins] from starch by H. w~acerans. “Schardinger was est,eemed by everyone by virtue of his solid character, open manner and even disposit,ion. TJrifort,iiii:Ltely, he lived to enjoy his well-deserved retirement only it short time. Honor his memory!” The aiit.hor is indebted t.o Dr. Hans Tiippy for providing t,hese references. (2) In 1945, the late C. S. Hudson observed to t,he ituthor t.hnt2hc considered the Schardinger dext,rins and their relationship to st,:Lrch t,o he the most, perplexing prohlem in starch chemist,ry. (3) D. French, M. 1,. Levine, J. H. l’azur and ICthelda Norberg, J . Anc. Chenr. Soc., 71, 353 (1949).

THE SCHARDINGEH DEXTHINS

191

(a) The Schardinger dextrins are homogeiieous cyclic molecules composed of G or more a-D-glucopyranose units linked 1-4 as in amylose. (b) As a coiisequeiice of the cyclic arrangement they have neither a reducing end-group nor a noiircducing end-group. They are not decomposed by hot aqueous alkali.

FIG. 2.-Alpha

destrin f r o m 60% proptnol, blade form.3

FIG.3.--Rett

dextriri from

SOY0 p r ~ p a n o l . ~

(c) They are rather resistant to acid hydrolysis and the ('ommoil starchsplit,tiiig a-amylases (except microbial eiizyines) , arid are completely resistant t o yeast fermentation and to @-amylase. (d) They crystallize well from water arid from aqueous alcaohols (see Figs. 1-4). (e) They form an abundance of crystalline complexes with organic substances, especially with organic liquids of low solubility in water. (f) They form a variety of inorganic coiiiplexes with neutral salts, halo-

192

DEXTER. FRENCH

gens, and bases. The crystalline complexes with iodine-iodide resemble in many respects the starch-iodine complexes. The literature on the Schardinger dextrins has been summarized up to 1932 by Pringsheim4 (in English) and through 1940 by Samec6s (in German). More recent reviews are fragmentary and c*oncerned with special aspects of the Schardinger dextrins or B. macerans amylase. The present article deals with the formation, structure, properties, and reactions of the Schardinger dextrins, together with material on the action of B. macerans amylase. The literature has been covered up through August 1956. It is hoped that the result will be a working guide from a consistent point of view over this particularly fascinating field.

FIG.4.-Gamma

dext.rin from 60% propa~iol.~

11. HISTORICAL REVIEW I. Discovery of the Schardinger Dextrins

Table I will serve as a means of orienting the reader with respect to the historical development of the Schardinger dextrins. The names under which the Schardinger dextrins have been known have undergone many mutations, some of which are listed in Table 11. The early literature contains frequent references to iicqstalline” dex(4) H. Pringsheim, “Chemistry of t.he Saccharides,” McGrnw-Hill, Kew York, 1932, p. 280; H. Pringsheim, in “A Comprehensive Survey of Starch Chemistry,” R . P. Walton, ed., Chemical Cat,alog Co., Inc., New York, Y . Y., 1928, 13. 35. (5) M. Samec and M. Rlinc, Kolloid-Beih., 49, 75 (1939). Sec pp. 211-23. (6) M. Samec arid M . Blinc, “Die neuere Entwicklung der Iiolloidchemie der Starke,” Steinkopff, Dresden and Leipzig, 1941, p. 239.

19.3

THE SCHARDINGER DEXTltINS ‘rA€lI,E

1

Historical Survey IIliLstrating the Metattiorphosis v j the Srhurdinaer Dextrins Rej’erences

Euenl or Quolalion

1891 1903 1904 1911 1912

Isolation, empirical formula of p-dextrin (“cellulosine”) from crude hacterial digest of starch Isolation of a- and p-dextrins Isolation of Bacillus macerans Empirical formula of a-dextrins a-Dextrin “series” : diamylose, tetraamylose, hesaamylose : p-dextrin ‘keries” triamylose, hexaamylose ; a-diamylose :

/ \

-1 CH-(

CH?-CHOH-CH-( 1 0 -

19261930 1934 1935 19361939 1942 1945 1918-

1948-.

Schardingers Schardingerg SchardingerIo I’ringsheim”

07 CHOH)~-CH-CHOH-CHZ

0

1921 1922 1924

Villiers?

CHOH)2--CH I

\ 0 /

Acetolysis of a- and 6-dextrins Existence of 8-triamylose is doubtful Methylation analysis of p-dextrin gives 2,3,6-tri0-methyl-D-glucose (Methylation analysis of maltose and starch) Existenre of a-diamylose is doubtful a-Dextrin is a mixture of chain molecules coritaining 4-5 D-glucose units Fractionation scheme; isolation of y-dextrin; a = 5, p = 6 o-glucose units Cyclic formula for Srhardinger dextrins containing all maltose linkages Discovery of Bacillus niacerans amylase X-ray molecular weights: 01 = 6, p = 7 o-glucose units Transglycosylase mechanism in enzymic synthesis of Schardinger dextrins Molecular size and structure for y-dextrin = 8 o-glucose units joined by maltose linkages Reversible action of B . ntacerans amylase

Irvine16 Haworth17 Miekeley ‘8 Freudenberg’ Freudenberg20 Frei~denberg~’-~~ TildenZG French27 Cori28 MyrbackZ9 Freudenberg3” French3’ French32 N01-bei-g~~

(7) A. Villiers, Compt. rend., 112. 536 (1891).

(8) F. Schardinger, 2. Untersurh. Nahr. u . Gent~ssni.,6, 865 (1903). (9) F. Schardinger, W i e n . klin. Worhschr., 17, 207 (1904). (10) F. Schardinger, Zentr. Rakferiol. Parasitenk., Abt. ZI, 29, 188 (1911). (11) H. Pringsheim and A . La~lghans,Ber., 46, 2533 (1912). (12) P. Karrer and C. Niigeli, H e l v . (‘him. Aclu, 4, 169 (1921).

W

TABLE I1 Nomenclature o j the Schardinger dezliins

Ip

Dextrin a

Villiers7 SchardingerE-*O.

Pringsheimll. 3 5 . 36 and Karrer1a-14

Freudenherg20’37

French27. 3’

crystallized dextrin ,4 crystallized amylose hexagonal A “Schlamm” crystallized dextrin LY (a) -diamylose (a) -tetraamylose a - hexaaniylose (a)-octaamylose a-amylosan a-allo-amylosan a-iso-amylosan a-dextrin cyclohexaglucan (a-1 --t 4) c yclohexaamylose

B

r

6

b-D-GP(1 + 411 7 C V ~ O cellulosine crystallized dextrin B crystallized amylodextrir

crystallized dextrin p (8)-triamylose 8-hexaamylose

p-amylosan B-allo-amylosan 8-iso-amylosan p-dextrin cycloheptaglucan (a-l --t 4)

cy cloheptaamy lose

a r-dextrin cyclooctaglucan (a-1 4) cyclooc taamglose cyclononaamylose --f

cyclodecaamylose

Cramer3* Freudenberg used the terms 6-dextrin and c-dextrin t o refer to higher rotating fractions obtained in separation of Schardinger dextrin mixtures. These fractions most likely contained lower Schardinger dextrins plus starchy residues with little if any of the 9- nr 10-membered rings. Q

THE SCHAHDINGEK DEXTRINR

195

trins obtained from starch, through the action of enzymes, arids, etc. Most of these reports are concerned with sphcroc*rystalliiie dextrins similar to Nageli’s amylodextrin, which is a short htraight-c+haindextrin completely differrnt in constitution and properties from thc Schardiiiger dextrins. The first report of crystalline dextrins of the Schardinger type is that of Villiers’ (1891). Using primitive bacteriological techniques arid probably impure cultures, Villiers obtained a small amount of rrystalline material from digests of Bacillus amylobacter (Clostridium butyricum) on starch. In addition to reducing dextrins formed by hydrolytic reactions, “. . . there is formed in very small amounts (about 3 g. per 1000 g. of starch) a carbohydrate which forms beautiful radiate crystals after a few weeks in the alcohol from which the dextriris were preclipitated. These crystals contain water and alcohol of crystallization. The proportion of the latter is rather small, about 4 %. In air they become opaque; they lose alcohol and absorb water without ally notable change in weight. Upon dissolving in a rather large amount (13) P. Karrer, C . NSigeli, 0. Huraitz nnd A . WSilti, Helo. Ch,ini. Aeta, 4,678 (1921). (14) P. Karrer and Elisabeth Biirklin, tIelit Chin?,.A d a , 6, 181 (1922). (15) J. C. Irvine, H. Pringsheim arid J. Macdonsld, J . Chem. Soc., 126,942 (1924). (16) J. C . Irvine and I. M. A . Black, J . Cheni. Soc., 862 (1926). (17) C. J. A. Cooper, W. N . Haworth and S. Pcat, J . Cheni. Soc., 876 (1926). (18) A. Miekeley, Ber., 63, 1957 (1930). (19) K. Freudenberg, 2. angew. Ch,eni,., 47, 675 (1934); Osterr. Cherri.-Ztg., 37, 162 (1934). (20) K. Freudenberg and It. Jacobi, A n n . , 618, 102 (1935). (21) K . Freudenberg, G. Blomquist, Lisa Ewald and K. Soff, Be,., 69, 1258 (1936). (22) K. Freudenberg and W.Rapp, Ber., 69, 2041 (1936). (23) K. Freudenberg, H. Boppel and Margot, Meyer-Delius, Naturwissenschaften, 26, 123 (1938). (24) K. Freudenberg and Margot Meyer-Delius, Bet-., 71, 1596 (1938). (25) K . Freudenberg, F:. Schaaf, Gert.rtid Dilmpert, and T . I’loetz, Naturwissenschuftsn, 27, 850 (1939). (26) Evelyn B. Tilden and C. S. Hudson, J . A m . (,”hem. Soc., 61, 2900 (1939). (27) D. French and R. E. Itundle, J . A m . Chem. Soc., 64, 1651 (1942). (28) C . F. Cori, Federation Proc., 4. 226 (1945). (29) K. Myrback and L . G. Gjorling, Arkiu Kenti, Mineral. Geol., A 20 (5) 1 (1945). (30) K. Freudenberg and F. Cramer, 2 . Natzrrjorsch., 3b, 464 (1948). (31) D. French, Doris W. Knnpp and J. H. Paxur, J . A m . Chem. Soc., 72, 5150 (1950). (32) D French, J. Pazur, M. I,. 1,evitie and 1’:thrld:t Korberg, J . A m . Cheni. Soc., 70, 3145 (1948). (32u) K. Myrhiirk and k:bL):t Willstaedt., iicltr (“hen/.S c u d . , 3, 91 (1949). (33) Et,helda Norberg arid I). French, J . A n / . (Then!. Soc., 72, 1202 (1950). k . , I I , 22, 98 (1908). (34) k’. Schnrtlinger, Z e n l r . Bakteriol. P u r ~ ~ s ; / e r / Abt. (35) 11. l’ringsheini, A . Wiener and A. Weitlinger, H e r . , 63, 2628 (1930). (36) €1. I’ringsheim, A . Weidiiiyer and P. Ohlmcyer, Ber., 64, 2125 (1931). (37) K. Fretidenberg and F. Cramer, Cheni.H e r . , 83, 296 (1950). (38) F. Cramer :tnd D . Steirile, A n n . , 696, 81 (1955).

196

DEXTER FRENCH

of hot water, and cooling, small brilliant crystals are obtained, remaining unchanged in air and having the composition represented by a multiple of the formula ( C E H I O O ~HzO. ) ~ . ~. . .” This description and the physical constants given by Villiers for his compound agree very well with those of the P-dextrin later described by Schardinger. In Koch’s opinion,39Villiers used impure cultures. As the spores of Bacillus macerans would have survived Villiers’ sterilization procedure, it is quite likely that his digests contained sufficient Bacillus macerans to account for the small amount of crystalline dextrin obtained. Villiers named his crystalline product “cellulosine” because of a fancied resemblance to cellulose (for example, with regard to difficulty of acid hydrolysis). In some cases, Villiers reported that he obta,ined two distinct crystalline “cellulosines,” the second cellulosine probably corresponding to the a-Schardinger dextrin. During a study of food ~poilage,~” Schardinger’s attention had been drawn to various isolated strainsRof bact,eria which survived the cooking process and which were thought to be responsible for certain cases of food poisoning. One of these heat-resistant organisms, which he called strain 11,had considerable starch-fermenting power. When subcultured on starch, strain I1 broke down starch, giving an alcohol-insoluble “soluble starch” together with crystalline dextxins A (fine hexagonal plates) and B (stout prismatic crystals). These dextrins were described as regards their lack of reducing power, hydrolysis to reducing sugar, and, for dextrin B, the water of crystallization, optical rotation, and C and H analysis. Schardinger observed that his crystalline dextrin B was most likely identical with Villiers’ “cellulosine.” It was Schardinger’s intention to continue his study of these crystallized dextrins, with the expectation that they would shed some light on the synthesis and degradation of starch. However, he was unsuccessful in maintaining a culture of Strain I1 which had the characteristic starch-degrading a~tivity.~ Meanwhile, Schardinger had isolatedg a new organism which he found as an accidental contaminant in a nutrient medium which had been “sterilized” by treatment with streaming steam for an hour on each of three successive days, with incubating between steam treatments. This cont,aminating microbe a t first annoyed Schardinger, but on noting that it produced acetone by fermentation of carbohydrate media, he became interested in it. On examining the growth charact,eristics of t,he hacillus on various vegetable nutrients, he observed that in each case t,here was a pronounced disint,egratiiig or “rotting” action, as well as the product,ion of (39) It. Koch, Juhresber. Gtirungsoryunisrne?l, 2, 242 (1801). (40) F. Schardinger, W i e n . klin. Wochschr., 16, 468 (1903).

THE SCHARDINGER DEXTRINS

197

acetone and ethyl alcohol on sugar or starch-containing plant material. Schardinger at first named the organism “Rottebazillus 1.” When the assignment of a Latin name appeared desirable, Schardinger felt that his knowledge of the Latin language was not adequate for expressing “forming acetone and ethyl alcohol” in a single Latin He compromised by naming his microbe Bacillus mace ran^^^ (macerare, to rot), which name is currently accepted in “Bergey’s Manual of Determinative Bacteriology.”42 Schardinger stated that BacilEus macerans was also found in the flax-retting pits, which has led to the misconception that he obtained it originally from this source. Schardinger was so engrossed with the acetone-forming property and other biochemical features43of his new bacillus that it was some time later before he tested its action on starch. On inoculating a starch paste with the culture, he found3*that the starch was broken down; by evaporating, and extracting the solid residue with alcohol, he obtained in the alcoholic extract crystalline dextrins which were identical in nature with those he had previously described. In addition to characterizing these dextrins rather carefully, Schardinger observed that they formed characteristic iodine addition products on the addition of iodine-iodide solution. Schardinger remarked that these crystalline degradation products of starch, which stand midway between maltose and starch, might be of considerable theoretical interest in the study of starch, and inasmuch as they form crystalline iodine addition products they might be used to shed some light on the starch-iodine reaction. He was sufficiently impressed by the analogy between his A and B dextrins and amylose and amylodextrin, especially with respect to their iodine color-reactions, that he proposed the names “crystallized amylose” and “crystallized amylodextrin.” Later Schardinger withdrew the namesla in favor of the noncommittal names “crystallized dextriri a’’and “crystallized dextrin @”; the Greek letter designations have been perpetuated in the scientific literature with occasional modification and with extension of the Greek alphabet to include higher members of the series. The following abridged quotation (in translation) from Schardinger summarizes his views a t the conclusion of his work on the crystalline dextrins.*O (40a) The name “Bacillus acetoethy1icw)i” was subsequently coined by J. H. Northrop, L. H. Ashe and J. K. Senior, J . Biol. Chern., 39, 1 (1919), for an organism found i n decaying potatoes, subsecluently identoifled as R . macerans. (41) F. Schardinger, Zentr. Bakteriol. Parasitenk., Abl. 11, 14, 772 (1905). (42) D. H. Bergey, R. S. Breed, E. C;. D. Murray and A. 1’. Hitachens, “Bergey’s Manual of Determinative Bact.eriology,” 6th Edit,ion, Williams and Wilkins, Bs1t.imore, 1948. (43) F. Schardinger, Zentr. Bakteriol. Parasifenk., A b f . IT, 19, 161 (1907).

198

DEXTER FRENCH

(1) Starch pastes give dextrins under tJhe action of specific microorganisms. ( 2 ) Root starches are converted by Bacillus m.acerans to wat8er-soluble products, but cereal starches less so. (3) About 25-30% of t,he starch is converted t o crystalline dextrins. Amorphous dextrins are also produced. (4) All starches examined gave a- and B-dextrin, a preponderating. ( 5 ) The crystalline dextrins are precipitated from aqueous solution by alcohol as well as by ether, chloroform, or iodine solution. They are nonreducing to copper reagents and non-fermentable by yeast’. (6) The simplest means of distinguishing the a and ,B dextriris is the iodine reaction. Crystalline a-dextrin-iodine complex in thin layers is blue when damp, gray-green when dry; the crystalline B-dextrin-iodine complex is brownish (red-brown) damp or dry. In his closing paragraphs on the crystalline dextrins Schardinger‘O notes: “I realize that there are many questions which have not yet been answered. I must leave the answering of them to the person who, thanks to more favorable circumstances, can engage himself more intensively with the subject.”

2. Primitive Concepts as to Chemical Structure and Molecular

In the 24 years following Schardinger’s final paper, the field of research on the chemistry of the crystalline dextrins was dominated by Pringsheim. This relatively fruitless period saw the development of considerable confusion with regard to the chemical structure and molecular weight of the crystalline dextrins, and later a degeneration into a rather bitter argument between Pringsheim and Karrer. It is unfortunate that little information of lasting value has come to us from this period. The literature is voluminous, but much of it is repetitive, controversial, or based on erroneous coricepts. Pringsheim held the view4 that the Schardinger dextrins arose through the bacterial “depolymerization” of starch to the fundamental units: the amylose fraction being broken down t o the alpha series of dextrins or poly(44) K. Freudenberg, Chenz.-Ztg., 60, 853 (1936) : (in translation) “Regarding the forces which hold a polymer t,ogether, we have had no notions, or a t most only hazy ones. This uncertainty has been transferred, completely erroneously, t,o the rigidly established body of concepts mainly associated with the name of Emil Fischer. Following Fischer’s deat,h it seemed that the gates and doors stood open t o fantasy with regard to the binding forces in polysaccharides. The development, of polysaccharide chemistry i n the German eult,ural community during the years 1919-1927 stands for :dl time as one of t h e most remarkable examples of t,he effect, on the scientific world of the loss of an unerring leader, which came a t u moment when the social, int.eliectnit1 and politjical foiuidations of a people had been shaken t o their deepest levels.”

THE SCHARDINGER I)EXT&INS

199

amyloses containing 2% D-glWOSe units per molecule, arid the amylopectiri fraction being degraded to the beta series i*ontaining3n D-glucose units per molecule. Thus, Pririgsheim considered a t least four distinct alpha polyamyloses: alpha diamylose, alpha tetraamylose, alpha hexaamylose, and alpha octaamylose. In the beta series, a triamylose and hexaamylose were supposedly distinct compounds. The members of any one series were readily intcmonverted by such reactions as were thought to induce polymerization or depolymerieation. A typical reactionlIv 46-60 was acetylation in the presence of an acidic catalyst to ‘‘depolymerize” a-tetraamylose to the acetate of the corresponding diamylose ; P-hexaamylose was similarly converted to 0-triamylose. “Polymerization” of a-diamylose to a-hexaamylose was considered to occur on allowing a concentrated aqueous solution of a-diamylose to stand a t room temperature for an extended period of time. These transformations were not limited to the members of one series, as Pringsheim contended that long standing of a-hexaamylose in water changes it into P-hexaamylose and that long-continued heating in boiling water reverses the reaction. Heating a-tetraamylose in glycerol a t 200” converted a small amount of it into a-hexaamylose and about one fifth into P-triamylose. Some of the transformations reported by Pringsheim are shown in Scheme I. The great weakness in I’ringsheim’s studies lies in the fact that he worked with incompletely separated fractions and that he refused to recognize the inherent unreliability of cryoscopic methods for molecular-weight determiriatioiis on complex and often impure substances of high molecular weight. The crystalline dextrins, as well as their acetates, have a high tendency t o form complexes with organic compounds; these products often depend for their characteristic crystalline form upon the particular “small molecule of solvation.” In the specific cases of complexes with water and (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) (58) (59) (60)

H. H. H. H.

Pririgsheim and F. Eissler, H e r . , 46, 2959 (1913). Pringsheim and F. Eissler, Ber., 47, 2565 (1914). Pringsheim and Stephanie Lichtenstein, Ber., 49, 364 (1916). Pringsheim and W. Persch, Ber., 64, 3162 (1921). €3. Pringsheim and W. Pcrsch, Ber., 66, 1425 (1922). H. Pringsheim and D. Dernikos, Ber., 66, 1433 (1922). H. Pringsheim and K. Goldstein, Ber., 66, 1446 (1922). H. Pringsheim and K. Schmalz, Ber., 66, 3001 (1922). H. Pringsheim and K. Goldstein, Ber., 66, 1520 (1923). H. Pringsheim and J . Leibowitz, Ber., 67, 884 (1924). H. Pringsheim and A Steingroever, Ber., 67, 1579 (1924). J . Leibowitz and S. H. Silmann, Ber., 6B, 1889 (1925). H. Pringsheim and J . Leibowitz, Ber., 69, 2058 (1926). 13. Pringsheim and P. Meyersohn, Her., 60, 1709 (1927). €1. Pringsheim, A. Weidinger and H. Sallentien, Ber , 64, 2117 (1931). H. I’ringsheim and A. Beiser, Rer., 66, 1870 (1932).

200

DEXTER FRENCH

ethanol, i t has been shown that there are a t least ten different modifications61s62 of the a-dextrin which vary in their x-ray crystal patterns as well as in water content arid alcohol content. These complexes are frequently very stable, being broken down neither by prolonged drying a t elevated temperature under high vacuum nor by dissolving in water. Freudenberg has insisted that, to remove the last trace of alcohol or other complexing agent, i t is necessary t o evaporate the aqueous solution to dryness on a water bath three successive times. It is understandable, then, that PringSCHEME I Transformation Scheme of the Polyamyloses According to Pringsheim a-Series

8-Series

-alsoamylosan

0.isoamylosan

-

solution in formarnide acetylation in zinc chloride water

starch digest with Bacillus rnacemns

a4etraamylose 0-hexaarnylose a.Hexaamylose (“Schlamm”)

a.0ctaamylose (“Schlamm”)

-712-KI

hexagonal prisms

red-brown prisms

I,-K1

sheim was frequently confronted with peculiar apparent molecular weights for his preparations, even though these were crystalline. In a paper entitled “On the Questionable Existence of the So-called Alpha Diamylose,” Miekeleyl* showed that the acetylation of a-tetraamylose, whether catalyzed by pyridine or an acid, always gave a-tetraamylose acetate, rather than a-diamylose acetate (as Pringsheim had claimed). On (61) M. Ulmann, C . Trogus and K. Hess, Ber., 66, 682 (1932). (62) K. Hess, C . Trogus and M. Ulmann, 2. physik. C‘hem., B21, 1 (1933). Additional data relating t o the similarity of powder patterns of “a-diamylose” and alcohol-precipitated “V”-starch are given by J. R. Kate and J. C. Derksen, ibid., A168, 337 (1932).

THE SCHARDINGER DEXTRINS

20 1

deacetylation, the product obtained always corresponded to a-tetraamylose. As a result of using carefully recrystallized and presumably purer materials, Miekeley concluded that Pringsheim’s polymerization-depolymerization scheme needed further evidence before it could be considered acce~table.~3 Karrer, who was interested in the Schardinger dextrin~12-~~, 64-70 during the 1920’s, appeared convinced that th ea “series” of dextrins was composed of a t least four distinct members, differing in molecular size according to Pringsheim’s scheme. However, he sharply disputed Pringsheim’s evidence for the subdivision of the ,8 series into triamylose and h e ~ a a m y l o s e . ~ ~ ~ ’ ~ Karrer produced several rather forceful arguments showing that triamylose and beta hexaamylose were identical, including identity of crystalline form, solubility, specific rotation, etc. One of the principle techniques of structure analysis developed during this troubled period was the method of acetolysis by reaction of the saccharide with acetyl bromide or a mixture of hydrogen bromide and acetic anhydride.I2In these ways, Karrer was able to obtain hepta-O-acetylmaltose from the Schardinger dextrins, including “B-triamylose,” in excellent yield-in fact, in as high a yield as from maltose or starch. The high yield of a disaccharide would be very difficult to understand if the starting product were a trisaccharide. Pringsheim remained unconvinced by Karrer’s arguments and, as late as 1932, stated that he believed he had successfully defended his case against Karrer.4 Even the principal conclusion, that maltose linkages occur in the Schardinger dextrins and starch to the extent of at least 50 %, was riot accepted by Pringsheim. A second major structural method applied to the Schardinger dextrins was methylation analysis. This method was fraught with experimental difficulties, and in a few cases it had led to rather embarrassing errors in assigning of structure.” Severe difficulty was experienced in obtaining a completely methylated product, so that 20 or more methylations were necessary. Here again, there was the suspicion that structural alterations might have been occurring during the prolonged methylation period. Even the main result, namely (63) A . Miekeley, Ber., 66, 69 (1932). (64) P. Karrer and C. Nageli, H e l v . chin^. Acta, 4, 263 (1921). (65) P. Karrer, Helv. Chini. Acta, 4, 811 (1921). (66) P. Karrer, M. Staub and A . Walti, H e l v . Chim. A d a , 6, 129 (1922). (67) P. Karrer and A . 1’. Smirnoff, Helv. Chitti. Acla, 6, 187 (1922). (68) P. Karrer, Ber., 66, 2854 (1922). (69) P. Karrer and W. Fiorini, Helv. Chin,. Acta, 6, 396 (1923). (70) P. Karrer, HeZu. Chim. A d a , 6, 402 (1923). (71) In the case of maltose, the original methylation work by W. N . Haworth and Grace C. Leitch, J . Chern. Soc., 116, SO9 (1919), led to an incorrect formula which was not corrected until 1926.16-I 7

202

DEXTER FRENCH

that 2,3,6-tri-O-methyl-~-glucose was the predominant or exclusive hydrolysis product, was interpreted variously as indicating the possibility of alternating a and (3 linkages or alternating pyranose (“amylene oxide”) and furanose (“butylene oxide”) ring forms. Because of the foggy notions as to the true molecular size of either starch or the Schardinger dextrins, these compounds were frequently represented as sterically improbable biose or triose anhydrides. CHzOH 0-

I I

CH

C H p f

CHOH

CHOH

I O CHOH ]

I--0

‘_I

CH

I

CH2 OH “a-Diamylose” (Pringsheim)

In the period 1930-1931, the already complex situation was further complicated by Pringsheim’s an n o u ~ icemen 36 t~ ~, of the production of ‘‘new polyamyloses” by treating the polyamyloses in formamide and precipitating the carbohydrate with alcohol. It was assumed that the formamide had a dispersing effect on the molecular aggregates, reducing them in some cases to C ~ H ~ units. O O ~These “amylosans” were characterized by means of the crystalline iodine complexes (which appeared identical with those of the original polyamyloses) , molecular weight, crystal form, and optical rotation. “Isoamylosans” and “allo-amylosans” were supposedly further transformation products resulting from heating the amylosans with water or formamide. Thus, at the end of a 20-year period following Schardinger’s original work, the principle significant advances which have stood up under more detailed study are as follows: (1) a very rough idea as to the approximate size (of a-tetraamylose and 0-hexaamylose) ; (2) production of 2,3,6-tri-0methyl-D-glucose on methylation and hydrolysis; (3) partial degradation by acetolysis to a maltose derivative; (4) further characterization of the complexes with iodine; complex formation noted with bromine and several organic solvents; (5) prepaiation and properties of crystalline acetates and several other derivatives; ( G ) resistance of the Schardinger dextrins to attack by most ordinary amylases, but their hydrolysis by fungal enzymes.

T H E SCHARDINGER DEXTRINS

208

3 . The Maturation Poriod 19Sc5-1950 I+om 1935 for a period of approximat>ely1 5 years, t,he main contributions t)oward t,he chemistry of tJheSchardingcr dcxtriiis were devcloped by b’reudenberg and his group. At the outset,, Freudenberg dismissed the work of l’ringsheim and his contemporaries as practically valueless,72since most of it was based upon work with dextrin mixhres and upon serious misconceptions relating to the structural principles of high polymers. Freudenberg set out a t once to rectify one of the main sources of confusion during the previous era, namely, the lack of individual Schardinger dextrins of unquestionable purity (in the sense of freedom from contamination by other members of the series). In a highly significant, paper by Freudenberg and Jacobi,2O straightforward means were described for obtaining the a and @ dextrins (see the following Section on fractionation). With these preparations in hand, i t became possible to carry out definitive studies on the constitution and properties of the compounds. Freudenberg originally considered the Schardinger dextrins to be chain molecules~g intermediate between maltose and starch. If chain molecules were involved, it would be necessary t o have a norireducing chain termination, such as D-glUcitOl, D-gluconic acid, or a levoglucosan type of unit. However, enzymic hydrolysisZ2gave no trace of a sugar unit other than D-glucose, and, furthermore, careful methylation studies*?-?*failed to reveal the presence of any D-glucose units which gave a product other than 2,3,6-tri-O-methyl-D-glucose. It has subsequently been amply e~tablished7~ that open-chain compounds in this molecular-size range are easily attacked by the common starch-splitting enzymes, whereas the Schardinger dextrins are remarkably stable to diastatic breakdown, especially by @-amylase. Thus, the preponderance of evidence led t o the conclusion that the Schardinger dextrins must be truly cyclic m0lecules.7~ The nature of the glycosidic bonds was studied by following the changes in optical rotation and reducing power during acid hydrolysis of the Schardinger dextrins arid their fully methylated ethers (see Figs. 5-7). After a brief initial rise in rotation, the Schardinger dextrins gave rotation-time curves closely parallel to those given by starch .21 The initial interpretation (72) “The confusion which has arisen in puhlic:ti.ions concerning these compounds is due to the fact t h a t they form mixtures which are hard to separzite and addition product<swith salt.s, water, alcohol and ot,licr solvents, and like all substances of t,he kind give rise to false values of molecular weight. by the 0rdin:tr-j methods” (translated from Frendenberg and .J:teobiz0). (73) D. French, M. I,. I,evine, ,J. H. 1’:izur :tritI IMel(1~iNorberg, J . A t t t . Chent. Soc:., 72, 1746 (1950). (74) “We must, therefore, with all caut,ion, consider t,he possibility of large rings” [for the Schardinger dextrins]: K. Freudenhrrg.2’

204

DEXTER FRENCH

of the rise in rotation was that it corresponded to a labile @-linkage,perhaps of the levoglucosan or cellobiose type. However, studies of the kinetics of hydrolysis of the Schardinger dextriris together with those on such compounds as starch, rrialtosc, levoglucosan (1 ,6-anhydro-@-wglucopyranose),

FIG.5.-Rotational changes during hydrolysis in 51% sulfuric acid.*l 1, Starch; 2, a-dextrin; 3, p-dext,rin; 4, maltose; 5, 1,6-anhydromaltose; 6, levoglucosan.

FIG.6.-Increase in iodometric reducing value during hydrolysis in 5170 sulfuric acid.” 1, Starch, observed; 2, starch, calculated; 3, a-dextrin; 4, maltose.

205

THE SCHARDINGEH DEXTHINS

maltose anhydride [ 1 ,G-atihydro-.l-O-(a-i,-~lucopyranosyl)-D-glucopyranose], and c~omparisonwith the behavior of cellulose, indicated that there could be 110 ,&linkages present ill the structures. The initial increase in rotation was explained by assuming that ring closure itself lowers the optical rotation; hence, on opening the ring the rotation a t first ineresses. During acetolysis, the Schardinger dextrins were also shown to be more nearly similar t o starch than to compounds of the levoglucosan type (see Fig. 8). Thus, we see that Freudenberg passed through a change in views, from the notion that the Schardinger dextrins were chain molecules with a peculiar thain termination which rendered them non-reducing, to the current

2

FINAL

2.8-

$ 2.62.4-

2 . 2 - l

FIG.7.-Rotational

2

j k

5 6 7 8 MINUTES x I O - ~

9

I0

1‘1

1‘2

3

changes of methylateci Schardinger dextrins in 51% sulfuric

acid.24

picture of truly cyclic symmetrical molecules in which each D-glucose unit is linked to the next by an a - ~(1 - +-l)-glurosidic bond. 4. Other Members of the Schardinyer Dextrin Family

Schardinger recognized only the (Y and /3 dextrins. Freudenberg definitely obtained the y dextrin arid raised the possibility that higher homologs exist, though it is doubtful whether the “6-dextrin” and “edextrin” fractions represented the homologous 9- and 10-membered saccharides. More recently, Akiya and c o ~ o r k e r s have ~ ~ ~ claimed -~ the discovery of (74a) S. Akiya and T. Watanabr, J Pha,n/ S o c . b n p a n , 62, 207 (1942); f‘hctt,. A b stracts, 44, 11138 (1950).

(74b) S. Akiya and T. Watanabe, J . P h a r ~ So(. . . l a p a n , 62, 209 (1942). (74c) Y. Akiya and S Okni, J . I’haruc. Soc. Jupnrr, 71, 865 (1051), (’hen/. rlb~lrarls, 46, 8018 (1952). (74cl) S. Akiya, S. Okui arid 8 . Suxuki, J . Phn, ni S o i . J a p a n , 72,1554 (1952); Chew. Abstracts, 47, 8657 (1953).

206

DEXTEIZ FRENCH

“new” cyclic oligosaccharides similar t o the Schardinger dextrins. A “new” strain of R. macerans is also claimed, hut 110 information is given by which it could be distinguished from previously described organisms. Inasmuch as Tilden arid Hudson have shown that all examined strains of B. macerans were capable of forming Schardinger dextrins, it is most likely that the “new” compounds of Akiya are in fact the a- and p-dextrins. Descriptions

FIG.8.-Rotation:d changes during acetolysis.22 1, Tri-0-acetylstarch; 2, a-o-glucose pentaacetate; 3, a-dextrin triacetate; 4, p-dextrin triacetate; 5, y-dextrin t r i acetate; 6 , hexa-O-acetyl-l,6-anhydromnltose.

and reported properties of the “new” dextrins fail to show any respect in which they differ from the previously known dextrins. In an effort to see how far the Schardinger dextrin series extends, French75 subjected radioactive glycogen t o the prolonged action of B. macerans __ (74e) S. Akiya and T. Watanahe, J . Pharni. Sor. Jnpan, 70. 572 (1950). (74f) S. Akiya imd T. Watanabe, ,I. P h a m . SOC.J a p a n , 70, 576 (1950). (74g) S. Akiya m d T. Watanabe, J . Pharrn. Soc. J a p a n , 70, 579 (1950). (74h) T. Watanabe, .I. Pharnz. Soc. J a p a n , 70, 582 (1950) (75) D. French and J. A . Effenhergcr, unpublished work.

THE SCHARDINGEH DEXTRINS

207

amylase. The resulting mixture, after iiiac.tivatioii of the eiieyrne and treatment of the residue with @-amylase (to destroy traces of linear oligosaccharides) , was subjerted to paper vhromatography and radioautography

FIG.9.--Hadioautograph of paper chromatogram showing radioactive Schardinger dextrins produced from radioactive glyrogen by B. nracer‘ans a m y l a ~ e . ’Small ~ amounts of linear oligosaccharides together with unreacted residues from the glycogen were removed by beta amylase action : ~ n dconverted to maltose and multotriose.

(see Fig. 9). Radioactive areas were seen, corresponding in position to maltose, maltotriose, high molecular-weight residuc, and a-,@- and 7-dextrins. A spot of low intensity appeared about midway between the origin and the 7-dextrin position. There seems little doubt that this spot is, in fact, &dextrin, the next higher homolog of the Schardinger dextrin series. A second unidentified spot, of intensity barely above the background (see

208

DEXTEIL PliENCH

Yig. lo), appears midway between the origin and the "&dextrin" spot. We tentatively ascribe this radioactivity to the e-dextrin. If subsequent work confirms that these radioactive areas are higher members of the Schardinger dextrin family, they would be cyclorioriaamylose and cyclodecaamylose, the 9- and 10-unit members. There is no obvious reason why the series should stop here, but higher members are indiscernible on the radioautograph.

I1

';o'40'$01t30 'Ido'l;o'14-o'I~o DISTANCE ( rnrn.) FIG. 10.-Densitometer tracing of the radioautograph of Fig. 9.76 The 6-dext,rin shows as a clearly resolved peak; the e-dextrin area appears only as a very weak but reproducible peak or shoulder against 6he backgroiind. O0'0

It may be remarked that although the series may extend beyond the 6or e-dextrin on the high molecular-weight side, there is no evidence a t all for a compound of lower size than a-dextrin. This abrupt termination of the series with a-dextrin may well be related to the fact that the smallest cycloamylose ring which can he constructed, using space-filling models, is cyclohexaamylose, provided that the D-glucose units exist in the C l conformation (see Figs. 11-15 and the discussion on ring conformation in Section IX). Rings larger than about 10 D-glucose units would also be strained (strainless super-rings are not possible here, because of the severe restriction of rotation about the glucosidic bond in the amylose chain). On the other hand, with D-glucose units in the JlexibZe (cis or boat) ring conforniation, it is possible to construct essentially strainless cycloamylose

THE SCHAIZDINGER DEXTRINS

209

rings having any number of n-glucose units from 3 to infinity (see Fig. IG).

No radioactive areas in the range between maltotriose arid a-dextrin can be discerned, which most likely means thatt Schardhlger dextriris below the a-dextrin do riot exist?6

FIG.11.-Model of a-dextrin from top. The u-glricose residues are in the C1 conformation. The h:trkgrountl is ruled :it 5 crn. intcrv:tls, corresponding to 5.5 d. in the models.

Fro. l2.-Model

of a-dextrin from the side

(76) However, since the lower members of the Srhnrdinger dextrin family do not fall in n. regular chromatographic pattern, it is impossible t o predict where t o expect the 5-membered compound on n. chromatogram. Conceivably it might be unresolved from the a-dextrin, in which case it would not have been detected even if it were present.

210

DEXTER FRENCH

FIG.I3.-Model

of p-dextrin from the top.

F I ~ ;14.-Model .

of 7-tiextrin from the top.

THE SCHARDINGER DEXTRINS

21 1

FIG.15.--Modcl of 6-tlrsirin from the top.

FIG. 16.-Model of hypothetical “cJrlot,ri:tmJlose” with the u-glucose residues in the flexible (“boat”) corifomnt.ioii.

111. FRACTIONATION A N D ~’IJILIFIC*ATION OF TIII: SCHAHDINGEH DEXTRINS As l+eudeaberg has repeatedly pointed out,7? much of the confusion surrounding the Svhardinger dextrins has originated in work done with impure preparations, even though they may have heen crystalline or partially so.

212

DEXTER FRENCH

The original separation method of Schardiriger depended on the ease of crystallization of the P-dextrin from water and its low solubility (about 1.5% a t room temperature) ; the a-dextrin was obtained from the mother liquor by treatment with alcohollo (see Scheme 11). A substantial advance in terhnique mas provided by Freudenherg and Jacobi,Z0who relied not only on solubility differcmies of the dextrins themselves, but also 011 the differences of solubilities and rates of crystallization of the dextrin acetates. Although the scheme appears somewhat tedious, SCHEME I1 Fractionation Scheme of Sehardinger dextrin mixture ‘lot [on cooling water

a-dextrin fine hexagonal plates (“Schlamm”) and solution recrystallize from water

1 I i alcohol

gummy precipitate (discard)

m-destrin

(hexagonal plates or blade-shaped prisms)

1

from water recrystallized by adding alcohol pure a-dextrin

especially insofar as it requires several acetylations and saponifications, it has the distinct merit of producing an a-dextrin quite free from other dextrin impurities. The hitherto undescribed y-dextrin appeared in the fractionation, although it was probably contaminated with other dextrins. Fravtions 6 and E do riot represent homogeneous materials7’; most likely they are mixtures of the crystallirie dextriris with amorphous limit dextrins (see Scheme 111). ~~ In further studies, Freudenberg, Plaiikenhorn and I i i i a ~ b e rmodified the previous fractioiiatiori scheme, without decreasing its complexity. Thcse (77) K. Freudenberg, E. Plankenhorn arid H. Knauber, Chewistry & I n d u s t r y , 731 (1917); Ann. 668, 1 (1947).

213

T H E SCHARDINGER DEXTRINS

SCHEME 111 Fractionation Scheme of Freudenbery and Jarobi A. raw product (50 g.) from water

1

sedimen t (“Schlamm”)

13. p-

1

i

+ 8-dextriii t,o

solution 13

I

fraction J

1.1

‘ I+

C. 6-dextrin

sediment (stirred with methanol and combined with D)

filt.rate C

:tlcohol

L partially crystalline

I). dextrin mixture (35 g . ) I

acetyl:tt,e acetate mixture (70 9 . )

1

.1

R. mother liquor: coticelitrate

E. acetate (45 g . )

t

ethyl acetate

I

I

.~

1

F. mother liquor €-acetate

a-acetate (42 9.)

G . p-

(12 9.1

t,oliieiie -~

.1 H. mother liquor

p-dext riri

L

J. p-

+ &dextrin+--i (2-3 g.)

I p-

2

G . mother liquor petxoleum ether p,y, 6-acet,atje saporiify

1

H. @-acetate (6 9.)

6-dextrin

1

2-

+ 6-acet,ate

1

1 mother liquor methyl alcohol

I

1.

8-dextrin

1

more

1 : [ em

+ 6-dextrin

mother liquor

1

mother liquor concentrated

i

B-dextrin

1

p-dextriii :wet ate

7-deutrin acetate in solutiori saponify

1

y-dextrin

SCHEME IV Fractionation Scheme of Freudenberg, Plankenhorn, and Knauber77 Solution from 250 g. of starch

I

I

A. s-destrin

decanted portion trichloroet,hylene

I

I

1. precipitate (83 g.)

l

I

mother liquor Ca lactate, reducing and amorphous dextrins

2. precipitate (17 g.)

B. (100 g.) hot H20 I

I C. crude crystals 8-dextrin (5 9.) combined wit.h R

,

I

1

E. solution ofI

a-, B-, and 7-dextrin, concentrated and acetylated (145 g.)

D. mud r-, s-, and 8-dextrin ( 3 4 g.) water, 20"

hot toluene

I

-

F. residue: r- and s-dextrin EtOH, HzO

I

d

r-dextrin (0.5 9.)

s-dextrin (0.5 g.) added t.0 A

K. a-dextrin acetate (90 9.) with some r(?)-acetate, acetic ester I

M. a-dextrin acetate (80 g.) saponified

S . mother liquor, concentrated a-dextrin acetate (6 g.),

a-destrin (33 g.)

also mixture of j3- and e - ( ? ) dextrin acetate (1-2 9.)

I

G. mother liquor concentrated

I

J. insol. residue (3-4 g.) incompletely acetylated dextrins?

H. j3, r, s-dextrin combined with F

I L. mother liquor concentrated I

I

I

U M

1 0

3

m

b

filtrate cooled

3m

I

I

0. 8-dextrin acetate (40 g.) with traces of s-dextrin acetate

mother liquor pet. ether

saponified

(15-16 g.) sap. H20 bromobenzene

I

I

P. 8-, 7-dextrin

H,O

I

Q . residue t.r:tee s-dextrin

--

1

solution oornbined with C and W, crystallized from H20

I

R. 0-dextrin (27 g . ) I1 U. residue

_1

S. addition compoiind treated with acetone, then extracted with hot pyridine

T. mother liquor coutains gummy reducing components

I ,

8M u,

F

?-!

s

V. pyridine solution concentrated

I

3 G3

m 3

I U I m I

I

water

W.8-dextrin (1-2

g.)

united with R undissolved trace united with Q

II

I

dissolved y-dext,rin (4.59 . )

X. mother liquor pyridine HzO sep.

uE z

i

T.undissolved t,race united with Q

(s)

Z.mother liquor unit.ed with saponified product from P

216

DEXTER FRENCH

authors made use of the o b serv atio ~that i~ ~ bromobenzene does not precipitate a-dextrin , but the p- and y-dextrins are readily precipitated. p-Dextrin was separated from y-dextrin by differential dissolution of the y-dextrin in warm pyridine. The additional r and s-dextrins (similar to Schardinger’s “Schlamm”) were considered to be organic complexes of a- and p-dextrins (see Scheme IV). French and coworkers3 sought to decrease the complexities of previous fractionation schemes. Fractionation was achieved, without acetylation of the dextrins, by taking advantage of the low solubility of p-dextrin in water, the differential precipitating action of bromobenzene on 0- and y-dextrin (leaving a-dextrin in solution), and the low solubility of ydextrin in 60 % 1-propanol. These authors also made use of the fact that conditions during the original enzymolysis of the starch can be preadjusted, depending 011 which dextrin is de~ired.7~ Large yields of p-dextrin are obtained if the enzymolysis is conducted in the presence of a precipitant (for example, toluene or trichlorethylene). Maximal yields of a-dextrin are obtained, in the absence of a precipitant, with a relatively short conversion. For preparation of y-dextrin, the enzymolysis should be very extensive, again in the absence of a precipitant (see Scheme V). Purification of the isolated a-dextrin may be effected by selective precipitation from aqueous solution by cyclohexane, recrystallization from 60 % 1-propanol, and finally recrystallization from water. It is absolutely necessary t o avoid contamination by greasy materials or by mineral matter found in commercial filter pads and charcoal, as these contaminants cause turbidity or opalescence. Before the final crystallization, it is well to filter the aqueous solution through a fine fritted-glass filter. To refine purified p-dextrin further, it may be dissolved in water (about a 2 % solution) a t room temperature and allowed to stand a considerable time in order to promote aggregation of insoluble impurities and complexes. After filtration through a fine fritted-glass filter, the solution should be concentrated to about one tenth of the original volume and allowed to crystallize. Purified y-dextrin is handled in much the same fashion as is a-dextrin, except that, toluene may be used as the specific precipitant. Crystallization from 60% 1-propanol should be repeated until there is no evidence for pdextrin in the mother liquors (when allowed to crystallize 011 a microscope slide). The @-dextrincrystallizes with ease from the mixture, and it can be recognized by its characteristic crystal form (see Fig. 3). Table I11 lists various properties of purified Schardinger dextrins. (78) K. Freudenberg and H. Boppel, Ber., 73, 609 (1940). (79) W. S . McClenahan, Evelyn B. Tilderi and C . S. Hudson, J . A m . Chern. SOC., 64, 2139 (1942).

217

T H E SCHARDINGER DEXTRINS SCHEME

v

Fractionation Scheme of French, T,enine, Pasirr and Norberg3

precipitants centrifuge

I

discard effluent

/

/

or ppt. with trichlorethylene

remove t 01- concentrate uene or bro- t o 25% solmobeneene ids, cool hy boiling with water;

!

recryat,:tllize from 60% 1-propanol

.1

boil with water

8-dextrin

I dilute to 3% solids,

recrystallize from boiling water

dilute to ppt. with 2% solids bromobenzene

i

pure p-dextrin

ppt. wit,h bromobenzene

pnre a-dextrin

I I

boil t o remove bromobenzene, add 1.5 vol. of 1-propanol

dissolve in boiling water

solution (discard)

7-dextrin-1-propanol complex repeated crystallization from 60% I-propanol

-

-

1

boil with concentrate t o witter 4&50% solids, cool pure 7-tlextrin

remove 1-propanol and return t o P-7 crudes

(discard)

218

DEXTER FRENCH

TABLE111 Properties of Sehardinger dextrins Dexlrin :efer-

Properly

mces a

B

Y

f151.4" f 0 . 5 t161.9" f 0.5 +150.5" f 0 . 5 t162.5" f 0 . 5 f177.4" f 0 . 5 f158" f 2 +160, 168" f 2 f148" f 2 4-150.5" f 0.5 f162.5" f 0 . 5 23.2 1.85 14.5 Solubility" in HzO 10 1.5, 1.4 20, 21 0.4-1.2 0.27 0.54 Solubility in 60% 1-propanola 17.7, 8.13 10.2 14.2, 14.5 Water of crystallization, 9 0.9 0.06 0.04 Solubility" in Hz0 in presence of toluene 0.9 0.02 0.06 p-xylene 3.3 0.04 0.17 p-cymene 0.03 0.26 0.03 trichloroethylene 0.08 0.12 0.03 tetrachloroethane 0.004 tetrachloroethylene 0.7 0.01 0.03 2.4 0.01 bromobenzene yellow needles Iodine complex with 12-K blue hexagonal yellow-brown plates or dimonoclinic or brown recchroic prisms tangular needles or plates prisms monoclinic Crystal form from 60% Hexagonal square or recparallelplates or 1-propanol tangular blade-shaped ograms prisms needles (tetragonal) !(YID (of

pure), in water

27 3 0,77 79 3 0,77 3

7, 3 3 3 3 3 3 3 3

Dexlrin Acelales

f105.5" +107.5"

Solubility" in toluene methanol ethyl acetate butyl acetate Melting point

0.22 1.39 8.84 0.67 242-3"

0

+122.0" +121" f125.5" 0.24 2.54 very sol. 17.8 196-196.5" 200"

Solubilit,y measured in g. per 100 ml. of solution.

f 138.5" f137"

very sol. very sol. very sol. 11 .0

3 20 79 3 3 3 3 70 77

THE SCHARDINGER DEXTRINS

219

IV. Bacillus macerans AMYLASE 1. Source

In 1‘33‘3 Tilden and H ~ c I s o l iannounced ~~ the discovery of a (:ell-free enzyine preparat.iou [from cultures of B. maceruns (“Aerobacillus macerans”)] which had the ability to convert starch to the Svhardinger dextrins. Prior to this time the cry~t~alline dextrins had beer: produced by the action of bacterial cultures 01: starch pastes, and it was not clear whether they were to be thought of as starch degradation products, transformation products, or products of bacterial metabolism. The discovery of the unique amylase (“Schardinger dextrinogenase”)8nhas made it possible to study in greater detail the mechanism of formation of the Schardinger dextrins and hence the relationship between their constitution and that of starch. The Bacillus macerans enzyme is distinctive in that it degrades starch with the production of almost no reducing p0wer.7~Even though most enzyme preparations have a detectable hydrolytic activity (as measured by increase in copper reducing values) yet this is exceedingly small in comparison with other amylases at extents of conversion which are similar as judged by decrease in viscosity or iodine-staining ability. At the present it is still debatable whether B. macerans amylase preparations are mixtures of two (or more) amylases, of which one has hydrolytic activity and another produces crystalline dextrins, or whether the hydrolytic activity is an intrinsic property of the same enzyme that produces crystalline dextrins. Although the enzyme has been enriched many-fold, it has so far eluded attempts to 38, bring it into well-defined crystalline Considerable effort has been spent on working out methods for the production of B. macerans a m y l a ~ e , s and ~ - ~ ~although high yields have been obtained in some cases, there is need for improvement in this respect. The usual laboratory methodz6’8 5 * 86 for obtaining B. macerans amylase is t o culture the organism on an autoclaved potato or oatmeal medium in the presence of calcium ctarbonate a t 37-45” for 2-4 weeks. The culture fluid (filtrate or centrifugate) contains enzynie which can be stored as such, 8. Schwimmer and ,J. A . Garibaldi, Cereal Chert)., 29, 108 (1952). S. Schwimmer and A. K. Balls, Federation P r o c . , 10, 245 (1951). S. Schwimmer, Federation Proc., 11. 283 (1952). S. Schwimnier, Arch. Biorhem. und Riophys., 43, 108 (1953). (84) W. S. Hale arid Lydia C. Rawlins, Cereal Cheni., 28, 49 (I!J51). (85) Evelyn B. Tilderi and C. S. Hudson. J . Baderiol., 43, 527 (1942). (86) Evelyn B. Tildcn, hliltlred H. Atlams :ind C. 6.IIudson, J . Ant. Chem. So?., 64, 1432 (1942). (87) It. W. Liggett mid W. C. Miissulm:~n, U. S. l’:it,. 2,494,514 (1950); Cheni.A h struck, 44, 2083 (1950). (88) Hilda S. Daniels and G. L. Stahly, J . Bueteriol., 62, 351 (1946). (80) (81) (82) (83)

220

DEXTER FRENCH

lyophilized, or concentrated by acetone precipitation. Further procedural details80.84 are beyond the scope of this review. The ability of culture filtrates to produce Schardinger dextrins (identified as their crystalline iodine complexes) has been usedx5as a means for thc identification of B. macerans. So far no other source, microbial or otherwise, has been found for this unique enzyme. 2. Measurement of B. macerans Amylase Activity

Of the schemes which have been proposed for the assay of the unique amylase activity of B. macerans, the simplest and most direct is the Tilden and Hudson microscopic test.85 One ml. of 3% starch solution was incubated a t 40" with 0.5 ml. of clear supernate or filtrate from a culture grown for 2-4 weeks on a suitable medium. At intervals 3 drops of the digest were transferred to a spot plate and a drop of 0.1 N iodine solution added. The microscopic appearances shown in the illustrations are typical of those seen when a loopful of the mixture was transferred t o a microscope slide and examined after evaporation. The appearance of small blue dots (see Fig. 17), which the high power showed t o be hexagonal crystals, characterized the first stage, just as the blue color began t o change t o violet. Later, long needles gradually spread out from the center until they covered the area of the drop and were noticeable without microscopic examination (see Fig. 18). This stage, when the color was a brown-violet, although not indicative of completion of the reaction, served as a suitable end-point with which t o compare different cultures for enzyme content and t o determine the activity of purified enzyme preparations. One unit of enzyme is defined as t h a t quantity which will convert 1 ml. of 3% starch (30 mg.) t o this point in 30 minutes a t 40" at the optimal pH. The brown-violet, stage very soon gives way t o a brown stage (see Fig. 19) which lasts for many hours without further change. This test is based upon the fact t h a t the characteristic crystJal form of the or-dextrin-iodine complex in the presence of potassium iodide depends upon the ratio of a-dextrin to iodine-iodide. A t low a-dextrin levels, the complex forms on evaporation of the solution in hexagonal plates or prisms (see Fig. 17) ; at higher a-dextrin levels the very distinctive dichroic needles penetrate the bulk of the solution (see Figs. 18 and 19). The endpoint of the reaction represents that point where there is a transition from the hexagons t o the needles (see Fig. 18).

Although the Tilden-Hudson test is very useful it has certain drawbacks in precise work. (a) The exact time a t which needles appear is difficult to determine. (b) Enzyme activity is slowed but not stopped by the addition of iodine solution; in fact, it is possible t o add starch to a mixture of enzyme and iodine solution and observe the gradual formation of the crystalline dextrin-iodine complexes. (c) The apparent "activity" depends very much on the presence of such foreign materials as salts, D-glucose, maltose, etc., which may be present in the enzyme solution. In some enzyme preparations which seem to have fair activity, the needles are never observed. Hale and Rawliris have published a colorimetric method84 which is well

THE SCHARDINGER DEXTRINS

22 1

FIG.17.-Iodine test for Schardinger d e ~ t r i n s . 8 ~ Microscopic appearance typical of the early stage of the decomposition of starch by B. macerans amylase. Under higher power the crystals are seen t o be blue hexagons. FIG. 18.--Iodine test for Schardinger dextrins.s5 Microscopic appearance typical of the “brown-violet, stage” of the decomposition of starch. Notice that where the fine crystal needles cross each other at right angles the light is extinguished, due t o the dichroism of the needles. FIG.lg.-Iodine test for Schardinger dextrins.R6 Microscopic appearance typical of the “hrown stage” of t,he decomposition of starch.

222

DEXTER FHENCH

adapted to the quantitative measurement of B. macerans amylase activity provided the enzyme preparation does not contain any significant quantity of a-amylase. One ml. of M calcium acetate-aretic acid buffer (pH 5.2) aritl4.0 ml. of water containing the enzyme are added 1 0 10 ml. of 2% starch (Lintner soluble) solution a t 40". After 5, 10, or 20 minutes (depending on the activity of the enzyme), 0.5 ml. of the digestion mixture is removed and added t o a mixture of 5 ml. of a solution of iodine in potassium iodide (0.0035 1M In in 0.25 M KI) and 0.2 ml. of 2 N sulfuric w i d . This mixture is then diluted with 10 ml. of water, and the light transmission of the diluted mixture is measured at 660 mp in an Evelyn photoelectric colorimeter a t 25" three minutes after the addition of the iodine. The colorimeter scale is set so t h a t the blank containing iodine, but no digest, gives a reading of 100 scale divisions. One unit of enzyme has been taken as that amount which would produce a color showing 50% transmission in 10 minutes. This unit is approximately 26 times t h a t used by Tilden and Hudson. Fig. 20 shows curves from which the activity of a p r e p aration may be read directly.

I-

0

2

3

I

4

5

ENZYME UNITS FIG. 20.-Assay of B. maceruns amylase based upon 50% transmission of light a t 660 mp through the dextrin-iodine solution after 5, 10, and 20 minutes.84

A method adapted to quantitative measurement of B. macerans amylase activity depends on the decrease in viscosity of a standard starch substrate during enzymolysis. Since other amylases also cause viscosity drops this 7 9 , 85 method lacks specificity, but it may be useful in some An interesting method depending on having available rather large amounts of pure p-amylase is suggested from the results of Schwimmer and Garibaldi.8" A starch solution is treated under standardized conditions M ith 13. tuacefalzs amylase. A t intervals aliquots are inactivated by heating to boiling, and then treated with sufficient @-amylaset o give complete conversioii t o maltose in 10 minutes. The maltose produced is determined by reducing-value determinations. The B . nzaterans amylase activity is proportional t o the rate at which the substrate becomes unavazlable for conversion to maltose. By plotting the maltose yields against time, a curve is obtained with a negative slope Rhich can be converted into moles of Schardinger

223

THE SCH.4RDINGEH DEXTRINS

dext.rin formed per rlriit time (average moIec1llar weight, of Schardinger dextrins, 1000). I n this manner, the “tiirnover number” of Schwimmer’s highly purified preparat.ion was found t o be 43,000 (moles of dextrin per 100,000 g . of enzyme per min.). Plotting the reciprocal of the reducing value itgainst time gave an approximately straight line, the slope of which could also lie taken as a incasure of the enzyme activity.

3.5

4.0 3.5 3.0 0

500

1000

1500

2l.-O)ptical rotatory changes cliiring coupling reactions with B . rwacerans amyl,zse.*9 A, 8-dextriii with maltose; B, p-dext.riii with sucrose; C, 8-dextrin with D-gliicose; D, 8-dextriri alone (control) ; E, a-dextriri and methyl a-D-glucopyranoside; F,a-ctextrin and sucrose; (1, a-dextriii and 11-glucose; H , a-dext,rin alone (control). FIci.

The method would appear t,o be capable of good quantitative enzyme assay; its main practical objection is the requirement for large amounts of pure P-amylase. A method capable of adaptatioii to B. macerans amylase assay is based upon the optical rotational shifts observed during coupling reactions (see p. 2263 and Fig. 2l).X9 A mixture of 0.25 g . of a-dextrin and 0.05 g. of u-glucose is treated with B. rnacerans amylase in a t,otal volume of 25 ml. The optical rotation is measured from time t o

_-

(89) D. French, M. L. Levine, Ethelda Norberg, P. Kordin; J. H. Pazur and C. M. Wild, J . AWL.Chem. Soc., 76, 2387 (1954).

224

DEXTER FRENCH

time until the observed value has increased 0.24.4”(2 dm. tube). The slope of the curve at any given temperature is proportional t o the enzyme activity. With 20 Tilden and Hudson units, an increase of 0.2” requires about 1 hour a t 25”.

Here i t would be necessary to be sure that the rotational shifts arise through the coupling reactions and not through such secondary effects as mutarotation of the D-glucose or temperature changes. The rotational shifts are not large, but they could be obtained with good accuracy on a precision polarimeter. Similarly it would be possible to make use of changes in rotation during the cyclization reactions, provided it is safe to assume that hydrolytic reactions, or other side reactions, are negligible. 3. Substrates for Production of Schardinger Dextrins by B. macerans Amylase

Practically all workers in the field have noted that different starchy substrates differ in their behavior with B. macerans amylase, both as regards the total yields of Schardinger dextrins and also with respect to the relative proportions of aIpha and beta dextrins. S ~ h a r d i n g e himself r~~ started a trend in giving the early names “crystallized amylose” t o a-dextrin and “crystallized amylodextrin” to p-dextrin. Pringsheim47 reported relatively higher yields of p-dextrin from glycogen and crude preparations of amylopectin, and postulated that amylose is polymerized a-diamylose ; amylopectin and glycogen are polymerized 0triamylose. McClenahan, Tilden and Hudson79pointed out that the ratio of a to p dextrin changed during the enzymolysis of starch, and further that it was possible t o increase the yield of p-dextrin to over 50% of the weight of the starch used by adding a dextrin precipitant during the enzymolysis. Similar results were obtained by French and coworker^.^ In comparative studies of starch fractions, Wilson, Schoch and Hudson,go and also 92 showed that the amylose fraction of starch gave much higher yields of Schardinger dextrins (up to about 70 %) than did the amylopectin fraction. Starch modification or degradation products in generai gave reduced yields; especially with beta amylase limit dextrins or acid hydrolyzed starch sirups there was no detectable production of crystalline dextrins. Kneen and B e ~ k o r d and , ~ ~ also Myrback and GjOrlingteg observed that the reducing values and amounts of fermentable sugars gradually increased (90) (1943). (91) (92) (93)

E. J. Wilson, Jr., T. J. Schoch and C. S. Hudson, J. Am. Chem. Soc., 66, 1380 R. W. Kerr, J. Am. Chem. Soc., 64, 3044 (1942). R. W. Kerr, J . Am. Chenz. Soc., 66, 188 (1943). E. Kneen and L. D. Beckord, Arch. Biochem., 10, 41 (1946).

T HE SCHARDINGER DEXTRINS

225

during enzymolysis of starch with B. maceruns amylase preparations. Further, the total Schardinger dextrin yield increased to a maximum and then declined to practically zero as the reducing value increased. Cramer and Steinle38have extended these observations using a highly purified B. macerans amylase with amylose and other substrates. With very dilute amylose (0.05%) the initial action appeared to give almost entirely a-dextrin ; then as enzymolysis proceeded p-dextrin and y-dextrin were produced in increasing amounts, together with small amounts of reducing oligosaccharides. Eventual yields of Schardinger dextrins were over 90 %. Although Keng1found that acid modification of corn starch led to greatly reduced yields of Schardinger dextrin, N ~ r b e r g Sameclg4 ,~~ and Cramer38 have shown that even very short-chain amylose dextrins can be converted in part into Schardinger dextrins. On the other hand, in the presence of substantial amounts of low molecular-weight sugars (such as D-glucose and maltose) , starch gives easily detectable amounts of Schardinger dextrins; however, these are subsequently converted into linear oligosaccharides by coupling reactions (see the following Section). For practical large-scale production of the Schardinger dextrins, the best source is whole unmodified starch (potato, defatted corn, or waxy maize). The cereal starches must be defatted prior to use, because otherwise a considerable quantity of an insoluble coagulum forms during the dige~tion.~~

4. Action Pattern of B. macerans Amylase The action of B. macerans amylase was originally considered to be essentially that of starch degradation, giving the Schardinger dextrins in much the same way that beta amylase gives maltose. The general impression at the time was expressed by Samec as follows.94“On the basis of these observations that the Schardinger dextrins are formed from low molecularweight degradation products of amylose, whose molecules contain no anomalous linkages, we must ascribe to Bacillus macerans and its enzyme a ring-closing as well as a hydrolytic action.” FreudenbergZ5had earlier expressed the view that if starch has a helical arrangement, the first and sixth D-glucose units would be in such close proximity that the scission of the sixth linkage could occur with the simultaneous formation of a ring. This concept was first recognized by CoriZs and MyrbaekZ9as a special case of transglucosylation. The possibility that B. macerans amylase is capable of effecting ring closure with short amylose chains has been tested by L e v i ~ i eUsing . ~ ~ mal(94) M. Samec and F. Cernigoj, Ber., 76, 1758 (1942). (95) M. L. Levine, Ph.D. Thesis, Iowa State College, 1947.

226

DEXTER FRENCH

tohexaose and maltoheptaose, Levirie found tbat the total reducing value did not change appreriably during the partial conversion of these materials to Schardinger dextrins. Thus glucosidic bonds in the cyclic molecules were lxing formed only a t the expense of other glucosidic bonds in the openchain compounds. Levine also observed that B. mucerans amylase converted a-dextrin into /?-dextrin in the presence of certain co-substrates (D-glucose, maltose, sucrose, etc.). Observatiorls of the type indicated that

FIG.22.-lteversible

action of R . niarerans amylase showing schematically the

way in which linear and cyclic compounds are interconverted through internal trans-

glycosylation reaction^.^^ The sugar radicals.

R group can be any of a wide variety of sugar or non-

B. macerans amylase action is reversible according t o the following eyuations.32. 33

G,=

GnA

G n = Gn-7

+

+P

etc. cyclization+ +-coupling Fig. 22 illustrates schematically the transfer of a glucosidic bond by

B. macerans amylase during reversible Srhardinger dextrin formation.96 The K. group can be any of a variety of sugar or non-sugar groups (such as D-glUCOSY1, D-fructosyl, methyl, phenyl, etc.) .32 89 Occurrence of coupling reactions has been confirmed*gby isolation of the coupled products as well ((36) E. J. IIehre, Advances in Enzyttiol., 11, 297 (1951).

THE! S C H A 1LI)ING ER I)EXTHINS

227

as by nunierous chromatographic rxperiiiient,s. I'ig. 23 shows the formation of tshe entire series of radioactive amyIci-oligosa(~~~harides from D-glucoseCL4and inactive a-destrin with B. maccrans amylase. In attempting t o acvount for the cwnversion of a-tlextrin into 0-dextrin and vice versa, Leviiw suggestedgs that tioupling reactions lead to the for-

FIG.2~.~Rnclioautogrrtph of pu.per chrom:it.ogr:tm sep:tratJion of mistailreobtained H. ttrnrernns :tmylnse.*9 0, , G g , C s , et,c., represent, 1)-glucose,maltose, mnltotriose, eto.;a, p , y represent t h e positions to which the S o h d i t i g e r dext,riris inove. Note that t,he Schnrdiriger dextrins themselves do not hecome rttdioact,ive. After the c:hromatogrum wtts sectioned, inactive a-dextrin was isolat,ed from the 1)iiiid lietween G a:mtl Cband it1ent)ifiedby means of the iodine test. 1)y t,reat.ing radio:wt,ive o-glucose arid iri:ictive n-dext.riri with

228

DEXTER FRENCH

rnation of linear chains which are in turn converted to cyclic dextrins plus linear chains of different length. Thus by repeated coupling and cyclization reactions, I?. macerans amylase could eventually establish an equilibrium between the various linear and cyclic compounds. The following sequence from Levine (using Cramer’s notation) illustrates how P-dextrin (7-ose)e y L i o and maltose (2-ose) could give a-dextrin together with the whole range of linear oligosaccharides :

+

(7-0se),~~~ 2-ose ~ 5 9-ose

4-3-ose 9-ose e (6-0se),,~~,

+ (7-ose),,,i0 16-ose (7-ose),,,l, + 3-ose + 10-ose 16-ose F2 2 (6-ose),yrlo+ 4-ose (G-ose),,,l, + 2-ose F2 8-ose 9-ose

$

etc. Working a t rather high substrate concentrations (5-10 %), Norberg and French33 showed that maltoheptaose is disproportionated by B. macerans amylase t o give higher and lower oligosaccharides (tentatively identified by electrophoretic analysis as 10-ose and 4-ose). Pazurg7has repeated this type of experiment, following the reaction by paper chromatography. In Pazur’s work, all the amylose oligosaccharides appeared simultaneously, indicating that maltoheptaose is disproportionated by a more random process.

Gn

+ G m S G(n+q +

G(m-z)

homologizing or disproportioriation The range of values of x (the number of D-glucose residues being transferred from G, t o G,) depends on the nature of the substrates, as we11 as the specificity of the enzyme. In homologizing reactions with maltose, x can be as small as 1. In this case, the reaction becomes equivalent to the amylomaltase reaction. Fig. 23 gives graphic evidence that homologizing reactions have occurred. The initial coupled product (7-ose) through further reactions has led to all the oligosaccharides from 2-ose to 10-ose; higher dextrins were apparent but not resolved on the radioautograph. More recently, Cramer and Steinle38have examined the action of B. macerans on maltohexaose and maltoheptaose, a t a lower substrate roiicen(97) J. H. Pazur, Ph.D. Thesis, Iowa State College, 1950

T H E SCHARDINGER DEXTRINS

229

tration (1 %). These authors failed to find evidence for a rapid disproportionation reaction and concluded that the ryclic dextriris are essential reactants or products in all reactions of the disproportionation type. It appears that, further experiment,al work is needed t>oresolve this question, but it may be noted that since disproportionation reactions are second order with respect to substrate, whereas cyclization reactions are only jirst order, it may be expected that a t suitably low substrate levels the cyclization reactions will preponderate whereas a t high substrate levels the disproportionation reactions will become more important. In an attempt to account for the eventual production of low molecularweight reducing sugars from starch by B. macerans amylase action, Cramer and Steinle propose the following

+ 7-ose 13-ose D-glucose + a + 6-ose 13-ose 2-ose + a + 5-ose 13-ose --+ 3-ose + + 4-ose 13-ose + 4ose + a + 3-ose 13-ose --+ a +

+

(Y

etc. By this scheme, short-chain dextrins are converted in such a way that a central part of the molecule becomes a cyclic dextrin, the two ends becoming reducing sugars. Although the scheme is ingenious, the evidence for i t is extremely tenuous. It appears to the writer that the more likely source of reducing sugars is the presence of traces of hydrolyzing enzymes knownB0to be present in crude B. maceruns amylase preparations. In the production of Schardinger dextrins from starch, B. macerans amylase resembles P-amylase, in that action begins a t the norireducing end of the starch chain, producing Schardinger dextrins (or maltose, with pamylase). Product formation stops when the enzyme comes to the end of a straight chain substrate or a branching point. On the other hand, B . macerans amyIase preparations show activity with a pronounced resemblance to alpha-type amylase activity as judged by reduction in viscosity of the substrate or by rapid changes in the color given with iodine. Dr. Ethelda J . Norberg, in unpublished experiments with beta amylase limit dextrin, showed that B. macerans amylase effect,s a rapid decrease in viscosity, without the formation of appreciable amounts of reducing sugars. No detectable amounts of Schardinger dextrins were produced. Here again more work is needed to clarify this aspect of the action of B. macerans amylase.

230

DEXTER FRENCH

Measurement~~7~ Y* of the concentrations of the various componentsy7of equilibrated digest of B. macerans amylase with defined substrates have shown that the equilibrium constant for the honlologizing reaction is essentially 1.

[G,+zI [Gm-zl tGn1 [Gml This experimental result is in agreement with the expectation that increasing the length of an oligosaccharide chain by one su glucose unit should give a characteristic change in free energy, regardless of the length of the chain. It may he pointed out that the same equilibrium holds for the distribution of oligosaccharides obtained by other enzymes, such as phosphorylase or amylomaltase, and in the case of B. macerans amylase the equilibrium derivations do not depend on whether equilibrium is reached by direct homologizing reactions or by multiple cyclization and coupling reactions. Similarly, it was possible to determine equilibrium constants for the formation of a-,6-, and y -d ex trii~ s.~ ~ Khomozogizz,rg

=

1=

The constants K,, Kg, and K , thus govern reactions in which the position of equilibrium is shifted with changing total substrate concentration. These equilibria imply that a t suitable substrate concentrations it should be possible to produce the Schardinger dextrins even from the starch oligosaccharides containing six or fewer u-glucose units. By going to suficientJy low concentrations, l'azur demonstrated the formation of a-dextrin with maltotriose (c = 0.1 %), but with maltose the concentrations required are so low (c less than 0.01 %) that the cyclization reactions could not be ohserved. It is easily possible to carry out a two-step reactsion with maltose such that oligosaccharides iri the range of :HiD-glUcOSe units are first produced by homologizing reactions; then these higher oligosaccharides may be freed from D-glucose and maItosc by alcohol fractionation or yeast fermentation and subsequently used as subst rates for the cyclization reactions. which so far has notj A variation suggested by Barker arid been carried out, is the simultaneous action on maltose of R. .ntmcrans (98) J. H. Pezur, Abstracts Papers A m . Cheni. Soc., 128, 6D (1955). (99) S. A. Barker and E. J. Bourne, Quart. Revs. (Imidon), 'I, 56 (1953).

T H E SCHABDINGEX UEXTBINS

231

amylase and a D-glucose-destroying enzyme such as D-glUcOSe oxidase. If the digest does not contain an enzyme leading to side reactions it should be possible to convert essentially all of the nonreducing D-glUcOSe units in maltose into Schardiriger dextrins. Contrary to the suggestion of Barker and Bourne, it would not be expected that high molecular-weight linear starch chains would be produced unless t,he original substrate concentration was very high. This reaction pattern is closely similar to the synthesis of amylose from maltose by amylomaltase and D-gliwose oxidase.Ici”

V. OTHERBIOCHEMICAL PROPERTIES OF THE SCHARDINGER DEXTRINS 1. Degradation by Amylascs

One of the rather remarkable properties of the Schardinger dextrins, noted by Schardinger, is their resistance to hydrolysis by the common starch-splitting enzymes. It has been repeatedly reported that the Schardinger dextriiis are completely resistant to beta amylase action. This resistance is explicable on the basis of the known action mechanism of beta amylase, which proceeds from the nonreducing end group of a starch chain and removes D-glucose units in pairs as maltose, this action continuing until the chain end is reached or a branch is encountered. The Schardinger dextrins, being cyclic, have no nonreducing end group and must therefore be immune to beta amylase attack. The Schardinger dextriris have also been reported’”I to be stable to alphatype amylases. However, in a study of the action of salivary amylase, French and coworkers1n2found that while the a-dextrin is essentially completely resistant,, the b-dextrin is attacked very slowly indeed and the y-dextrin is attacked about 1 % as rapidly as is starch. Here it is clear that the ring size exerts an effect; possibly the smaller rings have greater rigidity and heiice cannot adapt their shape to that required by the enzyme. Fungal amylase systems such as Takadiastase have been reportedz2 to degrade the Schardiriger dextrins completely t o D-glucose, although with p-dextrin some difficulty occurred. Ren-GershomJn2” has reported the pressence in fungal enzyme preparations of distinct enzymes specific for Schardinger dextrins. With a-dextrin, cyclohexaglucanase gave products showing an upward mutarotation (resembling P-amylase) ; with P-dextrin, cyclo(100) J. Monocl :tiid Anne-Marie Torrimi, Conapt. , e r ~ d . ,227, 240 (1948). (101) Early reports of the partial hydrolysis of t>lieGchsrdinger dextriiis by pancreat,ic amylase13 indicated t,hat “a-tetraamylose” is more susceptible than “p-hexaamylose.” This is moat likely a reflection of the relative degree of purity of t.he 8-dextrine; t,he rrude a-dext.rin prepnrations probably contained starchy impurities which were attacked by the enzyme used. (102) D. French, G . M. Wild and P. Nordin, unpublished work. (102a) E. Ben-Gershoni, Nature, 176, 593 (1955).

232

DEXTER FRENCH

heptaglucanase gave products showing a downward mutarotation. These preliminary results are most interesting and should be extended. With bacterial amylases, there is practically no information in the literature, but unpublished experiments of Wilson, Tilden, and Hudson indicate that the Schardinger dextrins are cleaved by an amylase from Bacillus pofymyxa (closely related to B . maceruns). The final products of action on 0-dextrin are D-glucose arid maltose in the ratio of one to three. Bacillus macerans amylase has been reported t o give a change in optical rotation with a-dextrin; the production of reducing sugars seems negligible and it is unclear what the products may l1e.7~With p-dextrin the action is reported t o be negligible.

2. Inhibition of Phosphorylase

It was first noted by Green and Stumpfl03that the Schardinger dextrins inhibit the action of potato phosphorylase. By varying the ratio of Schardinger dextrin to starch “primer,” these workers concluded that the Schardinger dextrins and the starch were competing for the same active group or active center in the enzyme. It seems reasonable that the Schardinger dextrins are able to react a t the binding sites normally used by the “primer,” but since they lack the necessary nonreducing end group, they are unable to participate as substrates in the actual reaction. This inhibition shows up rather remarkably on paper chromatograms containing mixtures of priming and inhibiting saccharides when the papers are sprayed with a mixture of phosphorylase and a-D-glucopyranosyl phosphate, incubated to allow starch synthesis t o occur, then sprayed lightly with dilute iodine soluThe priming areas show up as blue-ringed spots, the centers being colorless, yellow, red or purple, depending on the amount and nature of the primer used. With inhibitors, the areas show up as white patches on a light-blue background; in the case of a-dextrin, if the concentration is sufficiently high a yellow t o blue area of the a-dextrin-iodine complex will be observed in the center of the white patch. 3 . Utilization of Schardinger Dezlrins by Organisms Pringsheim conducted several tests t o determine whether the Schardinger dextrins are physiologically available, either t o plants or animals. Using destarched Spirogyra suspended in various sugar solutions, Pringsheim and M u l l ~mere r ~ ~not ~ able t o detect any starch formation with the Schardinger (103) D. E. Green and P. K . Stumpf, J. Biol. C‘hew., 142, 355 (1942). (104) 11. French and G. M. Wild, J. A m . Chenz. SOC.,76, 4490 (1953). (105) H. Pringsheim and K. 0. Muller, Hoppe-Seyler’s 2. physiol. Cheni., 118, 236 (1922).

THE SCHARDINGER DEXTRINS

233

dextrins, although D-glucose, glycerol and especially maltose gave rise to starch. Similar results were obtained using MesotaeniumP From the start it had been generally known t,hat the Schardinger destrim were not fermentable and hence not ut,ilized by yeast. McCloskey arid Porter1ofireported that of 18 tiarterial species arid 4 yeasts, only Bacillpis macerans arid B. polymg.ca were able to utilize the Sehardinger dextrins. A common observation, which constitutes a practical nuisance in the laboratory, is the growth of molds on u ~ ip rote c te d Schardinger ’~~ dextrin solutions; hence certain molds a t least must have the enzymic machinery for converting Schardinger dextrins int,o more conventional energy sources. With animal experiments, von Hoessliii and Pringsheim108were unable to detect any synthesis of glycogen when Schardinger dextrins were administered to fasted rabbits or guinea pigs. With diabetic patients, 50 g. of a-dextrin did not give rise to any noticeable increase in urine sugar. Since tests for fecal a-dextrin were negative, these authors concluded that the a-dextrin was “directly utilized.” Pringsheim comments: “Since [the Schardinger dextrins] can be used to combat acetonuria, within certain limits, they would be a suitable source of energy for diabetics, did they not occasionally cause nausea even though possessing an agreeable sweet taste; this nauseating effect was probably due to the adherence of impurities difficult to eliminate.” In unpublished attempts to investigate the ability of animals to utilize Schardiiiger dextrins, B. H. Thomas and D. French fed rats a diet in which a part of the carbohydrate was supplied by highly purified p-dextrin. The animals refused to eat the test diet except in very small quantities and within a week all animals 011 the ration were dead. Postmortem examination did not reveal the cause of death. In unpublished work from General Foods, Inc., Dr. R. R. Baldwin reports that with rats stomach-fed a suspension of 0-dextrin in vegetable oil, similar though erratic results were found. Although these experiments are very inconclusive, it would appear that the Schardinger dextrins exhibit a toxic effect, possibly by virtue of their remarkable complexing ability. In any case, the suggestion of Pringsheim that they be used as an “energy source” by diabetics looks risky. (106) C. M. McCloskey :tiid J. It. Porter, P w c . SOC. E x p t l . B i d . Med., 60, 269 (1945). (107) The materials cust,omltrily employed too prevent mold growth (toluene, chloroform, etc.) also act, t o precipit.ate the Schardinger dextrins. T h e best way to avoid mold growth is t o keep the solutions sterile, or t o heat them t o boiling and then cover or stopper while hot. (108) H. von Hoessliii and H. Pringsheim, Hoppe-Seyler’s 2. physiol. Chern., 131, 168 (1023).

234

DEXTER FRENCH

VI. MOLECULAR SIZEOF

THE

SCHARDINGER DEXTRINS’O’

The development of ideas regarding the exact molecular constitution of the Schardinger dextrins has necessarily involved an interplay between evidenre relating to niolecwlar weight on the one hand and that relating to the mode of attachment of the individual D - ~ ~ U Cunits O S Con the other. In all, a period of 38 years (from 1912 to 1950) elapsed hetween the first published estimates of the molecular weights and the substantially complete agreement on the final size and structure of the a , p, and y-dextrins. 1. Molecular Size jrom Measurements o j Colligative Properties

Schardinger himself did not attempt molecular-weight determinations of the crystalline dextrins. The first molecular-weight determinations mere based on freezing-point depressions in waterL1or on osmotic-pressure measurements.iLO* Other measurements were based on dialysis rates*O or on cryoscopic measurements with the acetates,“’ 2o nitrates,56 and methyl ethers.I5 In some cases microisopiestic methods were used.112 In most of the early work the importance of low molecular-weight impurities (such as alcohol or salts of crystallization) appears to have been ignored. Further, the necessity of extrapolation to infinite dilution has not always been appreciated, or in some cases the extrapolations were along curved lines.”‘ Although early workers had arrived a t a hexasarcharide character for one modification of a-dextrin (“a-hexaamylose”) this result was of questionable significance because of the simultaneous findings that vompounds now regarded merely as different c*rystallinemodifications or complexes of a-dextrin were reported as “a-dianiylose,” “a-tetraamylose,” “a-octaamylose,” “a-amylosaii,” “a-i~oamylosan,”and so on. If there was an element of truth here, it, was well concealed hy the surrounding confusion. The first reliable molecular-weight determination based upoii colligative properties was that caarried out by Gruenhut, Cushing and Caesar.ll2 These workers converted the a- and 0-dextrins into their crystalline nitrates. Application of the Barger microisopiestic method, and extrapolation to infinite dilution, gave 5.9 D-glucose residues per molecule for a-dextrin, and 7.0 for p-dextrin. Freudenberg and Cranierj7 applied the Barger method to a- and y-dex(109) “The main iriterest is naturitlly centered on the mol~riilitrsize of these substances.” (l’ringsheim nrid Langh:~rit;~~) (110) W. Bilts and W. Truth?, Re,., 46, 1377 (1913) (111) M Ulmann, Bzotheni. 2 , 261, 458 (1932). (112) N S Grurnhut, M. 1, Cunhing itrid B. V. C:whar, J . &4rn Chenz Soc , 70, 424 (1948).

THE SCHARDINGER UEXTBINS

235

trim (as their nitrates). Extrapolation of their results to zero concentration gave (iand 8 D-glucose residues. In the extrapolation it appears that their data gave a negative slope, whereas that obtained by Gruenhut, Cushing arid Caesar was positive. This curious discrepancy has not been resolved; however both sets of workers arrived at the same conclusions as to the molecular weights. In trying to account for the previous erroneous molecular weights assigned t o the a- and p-dextrins, Freudenberg and Cramer3"J7 examined the freezing-point depressions in cyc*lohexanolof the methyl ethers of the aand @-dextrinsover the conrentration range up to 4 or 5 % . The plotted values of apparent niolecular weights against concentration show a negative slope, so that a t the cioncentrations previously used the apparent molecular weights mere too low by about one D-glucose unit. The extrapolations t o zero concentration are stated to agree exactly with 0 and 7 D-glucose units for the a- and p-dextrins. It seems to this writer that the main lesson to be learned from the above is that if any confidence is to be placed in molecular weights obtained by cryoscopic measurements, or other measurements of colligative properties, it is absolutely essential that the measurements be made over a range of conceiitrations, and then the results must be obtained by extrapolating to zero concentration. Even so, unless the data are highly accurate, it is difficult to distinguish between adjacent members of a homologous series, since the relatioe diff erenc'es between the molecular weights are small. 2. X-ray Molecdar Weights

Because of the difficulties inherent in any precise molecular-weight determination depending on colligative properties, French and RundleZ7in 1942 applied the x-ray method to determination of the molecular weights of the a- and p-dextrins. This method is capable, under certain circumstances, of giving precise molecular weights. However, it has sometimes been applied incorrectly to the Schardinger dextrin problem; hence a few words of clarification are in order. The basic principle of the method is to determine by x-ray diffraction the volume of the crystal unit-cell. This, together with the measured crystal density gives directly the mass of the contentJs of a unit cell, and hence the mass of an integral number of niolecules of the material. In most cases of complex organic molecules the number of molecules per cell is small (2-6) and fixed by the crystallographic symmetry and the symmetry of the molecule. The case of the @-dextrinas crystallized from water is a particularly good example of the simplest case. Unit-cell and crystal-density measurements, when corrected for the amount of water of crystallization, show that the

236

DEXTEll FRENCH

mass of t,he unit cell is equivalent to 14 D-glucose re~idues.2~ However, the crystallographic space group P2, requires that the unit cell contain two identical molecules (or some multiple of a), regardless of possible molecular symmetry. Hence the molecule of p-dextrin must contain 7 D-glucose residues or some submultiple of 7. (CeH,,06), ('an be eliminated on chemical grounds or by approximate molecular-weight determinations by other methods. Moreover, with complex organic molecules there are seldom more molecules per unit cell than the minimum required by the crystal and molecular symmetry (2 in this case). The case of the a-dextrin represents an intermediate situation. Several different crystalline modifications27~ 61, 6 2 , 1 1 3 * 1 1 4 crystallize in such a way that the unit cell contains 24 D-glucose residues in orthorhombic unit cells (space group P212121); the crplallographic symmetry demands 4 n identical molecules, regardless of molecular symmetry. In this case n can be 6,3, 2, or 1 giving (C6H1005)r,(CBH1006)2, (CeHl,O6),1,or (C6HloO~)6. Inasmuch as n is generally 1, (CeH,,O,)e is most likely. In this case also, chemical information and rough determinations of the molecular size exclude all but (C6H&)s . There exist additional crystalline modifications114 115 which it must have a 2-fold axis demand that if the alpha dextrin is (C6H1005)6 of symmetry. With a-dextrin the chemical structure allows the required 2-fold axis; but it is to be noted that this type of evidence in itself has not excluded C6HI0Os,(C6HIO06)2, or (C6H1006)3. The y-dextrin represents the most indecisive case yet observed.31 The measurements indicate that the unit cell contains 48 D-glucose residues. The tetragonal space group P42, demands 8 n molecules per unit cell, or 4 m molecules having a 2-fold axis of symmetry, or 2 1 molecules having a 4-fold axis of symmetry, or some combination of these. Some of the molecules in the unit cell may display symmetry and others not, so that the required number of molecules is really 8n 4m 21. From this evidence alone the y-dextrin could he (C,H,o06), where x = I , 2, 3, 4, 6, 8, 12, or 24. As it actually turns out in this case, chemical evidence requires that x = 8 so that the x-ray evidence demands the presence of a 4-fold axis of symmetry in the y-dextrin molecule. Borchert113has studied the Schardinger dextriiis by the x-ray method. With a-dextrin from water he obtained an orthorhombic unit cell with 24 D-glucose residues per cell. The space group is not given (most likely P2,2,2,) but he states that it requires 4 molecules per unit cell giving 6 D-glucose residues per molecule. The P-dextrin methyl ether also gives orthorhombic

+

+

(113) W . Borciiert, Z Naturforsth., 3b, 464 (1948). (114) L). French, Ph.11. Thesis, Iowa S t a t e College, 1942. (115) D. French and R. E. Rundle, Ahsfracts Papers Am. ('hem. Soc., 103, 713 (1942).

THE SCHARDINBER DEXTRINS

237

crystals with 28 substituted D-glUCOSf2 residues per unit cell (7 D-glucose residues per molecule of 0-dextrin). With y-dextrin, the crystal system was not identified, but a pseudo-cell was obtained which contained 16 D-glUcOSe residues, in harmony with an 8-nlenlbered ring as currently accepted. Examples of the misapplication of the x-ray method may be cited. In the work of Ott,lI6 the innermost observed ring of an x-ray diffraction powder pattern was taken as giving a rough idea as to the maximum unitcell dimension, which in turn could be related to the molecular weight of a substance provided that an assumption as to the number of molecules per unit cell was made. This type of procedure could give a reliable molecular weight only in the case of simple cubic crystals for which the innermost ring on the powder pattern corresponds to the (100) reflection and for which it is known that there is only 1 molecule per unit cell (or some other definite number), a most unlikely combination of circumstances! In the usual case, this method cannot fail to give an erroneous value of molecular weight. The following numbers of monosaccharide residues per molecule were reported by Ott: a-diamylose, 22; a-tetraamylose, 12; a-octaamylose, 63; P-triamylose or 0-hexaamylose, 6; cellulose, 3; lichenin, 7 ; starch, 2; and inulin, 6. The x-ray powder patterns published by Ott appear weak and poorly resolved, lending an additional item of doubt to an already tenuous argument. Herzog117examined several known substances by Ott’s method; out of seven cases, only one was reasonably close to the correct value while the others were off by 40 to 500 %. Ott’s value of 6 D-glucose units for P-dextrin was later used by Freudenberg7?as ammunition in defense of his early molecular weights. Although Freudenberg now agrees that p-dextrin contains 7 units, it seems most incongruous that he should have placed much faith in the exact value of a number whose companions were obviously incorrect. A second example of misapplication of the x-ray method is one in which the x-ray results are not consistent with the chemical requirements. Thus in an investigation of a-dextrin by Kratky and Schneidmesser,lI8the lattice constants and density reported required 10 D-glucose residues per unit cell. The orthorhombic space group (P2,2,2 or I’2,2,2,) required that there be 472 molecules per unit cell if the molecules were devoid of symmetry, or 2m molecules if each molecule had a 2-fold axis of symmetry, or some combination of 4n and 2m molecules. Inasmuch as the number 10 is not divisible by 4, the authors concluded that there must be only 2 molecules per unit cell, with 5 D-glucose units per molecule. (This number was in harmony with Freudenberg’s views on the a-dextrin arid was substquently cited7?as agree(116) E. Ott, Physak. Z . , 27, 174 (1926). (117) R . 0. Herzog, Physik. Z., 27. 378 (1926). (118) 0. Kratky and B. Srhneidmesser, Ber., 71, 1413 (1938).

238

DEXTER FRENCH

irig with his molecular-weight determinations.) However, it must be noted that if there are only 2 molecules per unit cell, then the crystal symmetry requires each molecule to have a %fold axis of symmetry, a condition which is clearly impossible for any conceivable combination of 5 D-glucose units. Thus this application of the x-ray crystal density method is of no validity because i t is inconsistent. Actually, in this case the source of error has never been fully clarified. Kratky stated that the crystals used in the study were twinned or otherwise malformed. The identity period along the needle axis as given by Kratky (9.39 A.) is similar to that found in various a-dextrin crystals from water or dilute alcohols. However, the combinationoof spacings perpendicular to the needle axis as reported by Kratky (9.65 A. and 21.9 has not been subsequently observed.

w.)

3. Sedimentation and Diflusion

Recent improvements in techniques of sedimentation and diffusion analysis have permitted their a p p l i c a t i ~ n ~ ~ tog materials -~~~ in the molecularweight range of the Schardinger dextrins. These measurements supplement other types of determinations in that (1) they are not particularly sensitive to the presence of low molecular-weight impurities, and (2) the sedimentation and diffusion constants can be extrapolated to infinite dilution to eliminate aggregation and interaction effects. The primary technical limitation on the accuracy of molecular weights so determined is the sedimentation constant. Since the molecular weight is directly proportional to the sedimentation constant, where S is low, small errors in X will be relatively important. For p-dextrin with X = 0.47, if S is in error by only 0.02 (an average error for measurements of this sort) this will give an error of =t50 in the molecular weight. A second essential factor which so far has not been determined with high precision is the partial specific volume of the carbohydrate. Nevertheless, it has been possible to obtain molecular weights which are within a few percent of the theoretical values (see Table IV). Besides their use in obtaining inolrcular weights, sedimentation and diffusion data may also be interpreted in terms of the over-all size and shape of large molecules. In the case of the Schardinger dextrins, the theory has not been adequately tested for this low molecular-weight range. However, the slightly lower value of Sz0for maltoheptaose (0.418) in comparison with cycloheptaamylose (0.4’7) is probably significant. If it is, it would indicate a more compact structure for the cyclic molecule. This finding is compatible (119) L. G . Longswwrth, J . Z’hys. Cheiii., 68, 770 (1954). (120) H. K . Schachman and W. F. Harrington, J . Polymer Sci.,12, 379 (1954). (121) 11. V. Wehber, J A m . Cheni. Sac., 78, 536 (1956)

239

THE SCHARDINGER DEXTHINS

with the known chemical structure as well as with x-ray and viscosity results. 4. Partial Hydrolysis by Acid or Enzymcs

Ring opening of the Srhardinger dextrins by partial acid hydrolysis gives initially the corresponding linear oligosaccharide. Further hydrolysis gives the lower homologs and D-glucose. By working out the kinetics of acid hydrolysis of p-dextrin, French, Levine and l’azurI2* were able to establish conditions for the preparation of maltoheptaose (“amyloheptaose”) in essentially pure form. The optical rotation and reducing values of the hydrolTABLEIV Hydrodynamic Properties of Su! rs and Dextrinsa Substance

Molecular weight

Parlial s p . vol.

s20.10

u-Glucose Sucrose Raffinose Stacliyose Maltoheptaose Cyclohexaamyloee Cycloheptaamylose Cyclooctaamylose

180 342 504 666 1152 972 1134 1296

0.621 0.618 0.608

0.134 0.228 0.277

0.620 0.623 0.624 0.621

0.448 0.480 0.47 0.492

026

X 106

M o l . wt. from S and D

6.728 5.209 4.339 3.839

146 320 454

3.443 3.224 3.000

990 1140 1204

5 Data obtained from Webber,lZ1 Schachman and Harrington,lzo and Longs~0rth.l’~

ysis product were in harmony with expected values for the heptasaccharide homologous with maltose. Moreover, the nitrogen content of the phenyl(122) D. French, M. 1,. Levine and J. H. Pazur, J . Am. Cheni. Soc., 71, 356 (1949). Kinetic analysis shows that the rate of hydrolysis of the glucosidic bonds in the p-dextrin ring is only 22% as great as in the open-chain compounds. Other kinetic approaches have been published by Swanson and Cori’32and Mvrback.’31 The kinetics worked out by Myrbiick are on the basis of 5 , 6 , and 7-membered rings for the a,p and 7-dextrins, but, even 30, the conclusion is reached t,hat, the cyclic dextrins are more stable than one would expect if the glucosidic linkages were hydrolyxed at, t,he “normal” (that is, the open-chitin) rate. Earlier work by Freudenberg,21 using 51% $ull‘uric acid as the hydrolytic agent, is more difficult t o interpret in that t)he iodometric method used t o follow the reaction gave erratic results, especially with p-destrin. From the polarimetric data i t appears that in strong acid t,he hydrolysis of or-dextrin is only slightly slower than that of starch, hut, with p-dextrin, hydrolysis is considerahly inhibited. It has been generally appreciated since the earliest work by Villiers that the Schardinger dextrins are anomalously resistmaritt o acid hydrolysis.

240

DEXTER FRENCH

hydrazone and the potassium content of the potassium aldonate agreed showed that under closely with the theoretic,al values. A further certain conditions maltose and maltotriose are the sole products of hydrolysis by soybean P-amylase, and that these are produced in the molar ratio 2 : 1. Similarly, action of &amylase on maltoheptaonic acid gave 2 moles of maltose and one mole of maltotrionic acid (identified by electrophoresis), Thus a variety of chemical arid biochemical experiments point to t>heheptasaccharide character of maltoheptaose, and in turn this indicates that p-dextrin is a heptasaccharide. As stated previously (see p. 232) B. polymyxa amylase converts p-dextrin into a mixture of D-glUcOSe and maltose in the molar proportion 1:3. The simplest stoichiometry which can give this result is as follows. (CsHiuOs)~-k 4HzO p-dextrin

+

3c1?&011 4- C6H1206

maltose

D-glucose

The 1:s ratio was considered by Hudson to he substantial evidence in favor of the heptasaccharide character of the p-dextrin at a time when there was considerable doubt whether it is a hexasaccharide or a heptasaccharide. The basic arguments involved would have more force if the specificity of the B. polymyxa amylase and its action on related compounds were better known. Fairly extensive acid hydrolysis of the Schardinger dextrins gives Dglucose and the linear series of maltosaccharides. By paper chromatography it can readily he shown3' that this series terminates abruptly with the particular maltosaccharide which contains the same number of D-glucose units as the parent cyclic dextrin. Since the malto-oligosaccharides fall in a very regular chromatographic seque1ice,~~3 the determination of the molecular size of a given pure Schardinger dextrin can be reduced to a counting process. Handled in this manner, the a-,p-, and y-dextrins gave series terminating abruptly with maltohexaose, maltoheptaose, and maltooctaose.31 This is perhaps the most, direct evidence for the octasaccharide character of y-dextrin. Using the (newly developed) gradient-elution method of charcoal chromatography, Alm,124, lZ5showed that hydrolysis of a-dextrin gives rise to seven regularly spaced peaks which are presumably D-ghCOSe, the linear maltooligosaccharides up to maltohexaose, and unchanged a-dextrin (see Fig. 24). (123) D. French and G . M. Wild, d . A m . Chen7. Soc., 76, 2612 (1853). (124) R. S. A h , R. J. P. Williams and A. Tiselins, Acta Chmi. Scand., 6, 826 (1952). (125) R. S. Alm, Acta Chenz. Sr:and., 6, 1186 (1952).

24 1

THE SCHARDINGER DEXTRINS

5. Chromatography, Optical Rotation and Miscellaneous Observations Rearing on thc Molecular Size of the Schardinger Dextrins

The Schardinger dextrins may be located on paper chromatograms by taking advantage of their iodine-complexing ability. When exposed to iodine vapor, or sprayed lightly with iodine solution, the a-dextrin gives a blue color, the P-dextrin gives a yellow color, and the y-dextrin gives an orange color. The regularity which characterizes the paper-chromatographic mobility of the linear amylose oligosa~charides~~3 does not apply to the lower members of the Schardinger dextrin family (see Fig. 25). Rather, they move considerably more rapidly than the linear compounds, possibly because in the presence of the chromatographic solvent they form I

.b

-

7

1

-

I

0.4 -

I

I

-

Saccharides

0

c

dextrin

0 al x.- 0.2 VI

U

. l -

o ' 00

I

10

I

I

I

I

50

I

I

I

80

Fraction no. FIQ.24.-Elution analysis of oligosaccharides formed by partial acid hydrolysis of a-dextrin.1*4The last peak at fractions 75-80 consists of unchanged a-dextrin.

organic complexes and migrate as such. Beyond the p-dextrin, it appears that the series has a certain amount of regularity, but it must be borne in mind that at present the only evidence for 6- and e-dextrins is their appearance on the radioautograph of a paper chromatogram (see Fig. 9). [Quantitative determinationg7' 98 of Schardinger dextrins on paper chromatograms can be effected by sectioning the chromatogram, eluting the dextrin with water and determining the dextrin in the eluate by the quantitative diphenylamine or anthrone method.] The Schardinger dextrins also fall into a series in which their optical rotations increase with increasing molecular size (see Fig. 26). This fact has been used by Freudenberga as evidence that y-dextrin is the next higher homolog above 0-dextrin and therefore contains 1 additional D-glucose residue. The stoichiometry of the reaction with iodine or other complexing agents may be used in some cases as evidence for the molecular size of the Schar-

242

DEXTER FRENCH

dl

h

0.01

L

I

I 2

I

I

I

3

4

5

I 6

I

I

I

I

I.*.

7

8

9

10

II

NUMBER OF D-GLUCOSE UNITS PER MOLECULE

FIG.25.-Chromatographic mobility of Schardinger dextrins as compared with linear oligosaccharides from starch. The values for the 8 - and e-dextrins were obtained from Fig. 9 and should be considered tentative. The ordinate (logarithmic scale) is the distribution function a' as used by French and Wild.1°2

v)

w

w

a

W

w

0

.

z

0

.

I-

a

i-

0 ct

u

* I50 O 0 I

k W u

n

rn

0

0.1

03

0.2

04

05

I n

FIQ. 26.-Specific optical rotations of linear and cyclic oligosaccharides, plotted against the reciprocal of the number of o-glucose units per molecule. Circles, linear compounds; squares, Schardinger dextrins. Series for which Freudenberg's rule holds should fall on a straight line which extrapolates at infinite molecular size (I/n= 0) to the specific rotation of the high molecular-weight parent polysaccharide, in this case, starch.

T H E SCHARDINGER DEXTRINS

243

dinger dextrins. In the case of a-dextrin, potentiometric titrations126with iodine solution in a potassium iodide medium have shown that one mole of dextrin reacts with one mole of iodine.lZ7From the ratio of the amount of iodine found to the weight of dextrin used an approximate value for the molecular weight may be calculated. Similarly, analysis of the ratio of iodine to carbohydrate in the crystalline stoichiometric a-dextrin complexes can be converted into molecular-weight data. In some cases, especially with p- and 7-dextrins, it appears that non-stoichiometric compounds are formed. I n principlelZ8it is possible to calculate molecular weights for cyclic compounds by measuring their concentrations in equilibrated mixtures of widely varying total carbohydrate concentrations. As yet this method has not been aplied t o the Schardinger dextrins.

VII. MOLECULAR CONSTITUTION 1. The Points of Glycosidic Attachment

The evidence of primary significance here has been obtained using the methylation method. The first result which would appear clean cut by present day standards was that of Irvine, Pringsheim and MacD0na1d.l~These workers were able to prepare a methylated P-dextrin (OCH3 = 43.6% as against 45.6 % required by theory) which crystallized from ether. Methanolysis of the crystalline material, followed by fractional distillation and hydrolysis, gave 2,3,6-tri-O-methyl-~-glucoseas the exclusive product. These authors cautiously suggested that “/3-hexaamylose is now shown t o be a symmetrical molecule in which each glucose residue belongs to the butylene-oxide [furanoside] type and is substituted in positions 1 and 5.” Further structural commitments were withheld; it appears to this writer that Irvine and Pringsheim were not in full agreement with each other a s regards the structural interpretation, which today seems so obvious. A corresponding methylation of a-dextrin by these workerslZ9failed to raise the methoxyl value above 37-40%. FreudenbergZ2 24 was able to prepare fully methylated a- and p-dextrins. The starting materials, particularly the a-dextrin, were probably of higher purity than those used by Irvine and Pringsheim. Besides, Freudenberg applied the Muskat liquid ammonia technique to the methylation, which 1

(126) H. A. Duke, Ph. D. Thesis, Iowa State College, 1947. (127) R. W. Liggett (private communication) and Levineo6 have shown t h a t potentiometric iodine titrationIZ6can be applied directly to the nrlalytical deterrnination of a-dextrin. (128) H. Jacobson, C. 0. Beckrnann and W. H. Stockmayer, J. Chem. Phys., 18, 1607 (1950). (129) J. C.Irvine, H. Pringsheim and A. F. Skinner, Ber., 62,2372 (1929).

244

DEXTER FRENCH

was found more effective than the Irvine procedure. With both methylated a- and P-dextrins, the sole product of hydrolysis was 2,3,6-tri-O-methyl-

D-glucose, isolated in over 90 % yield and rigidly identified. A control experiment starting with 2,3, B-tri-O-methyl-~-glucosegave a 94 % yield of the starting material, which indicated a small but significant decomposition

9.

FIG.27.-Rate curves for the periodate oxidation of the Schardinger dextrinsI3O; V, amylodextrin; 0 ,r ; A, 8; 0,a. The pseudo-time 0 has been placed on a logarithmic scale in order to facilitate kinetic analysis and t o compress a rather wide range of real times;

8

=

6'

[periodatel dt

The initial rate of oxidation of the linear amylodextrin is more than 40 times as rapid as with the a-dextrin.

during the methanolysis, distillation and hydrolysis. No trace of a more volatile compound (methyl tetra-0-methyl-D-glucoside) was found in the product from the Schardinger dextrins. This fact, coupled with an approximate knowledge of the molecular size, made it quite certain that the Schardinger dextrins could not be open-chain compounds. The yields of 2,3,6-tri-O-methyl-D-glucose were sufficiently close to 100 % (when allowance was made for the loss and decomposition noted above) that it would be impossible to have in the parent compound one or more of the D-glucose (130) D. French and R. L. McIntire, J. Am. Chem. SOC.,72, 5148 (1950).

245

THE SCEARDINGER DEXTRINS

units which would give rise to any product other than 2,3,6-tri-O-methylD-glucose. Finally, the optical rotations observed24 during hydrolysis of the fully methylated a- and p-dextrins in 51 % sulfuric acid approached the rotation of 2 , 3 , 6-tri-0-methyl-D-glucosein the same solvent. This careful work, based upon authentic purified materials and executed using refined techniques, has firmly established that the Schardinger aand @-dextrins are so constituted that methylation and hydrolysis give exclusively 2 , 3 ,6-tri-O-methyl-~-glucose. Further evidence relating to the location of the free hydroxyl groups in the Schardinger dextrins has been obtained by periodate oxidation. A study of a-, /3-, and y-dextrins in comparison with a straight-chain amylodextrin by French and McIntire130 gave as the principle result that the a-,0-, and y-dextrins fall in a regular series, with each dextrin consuming one mole of periodate per “anhydro-D-glucose” unit. No formic acid or formaldehyde is produced from any of the Schardinger dextrins; hence they cannot be open-chain compounds. Examination of the kinetics of oxidation showed that the initial oxidation is hindered; the initial rate increases in the order a, 0,7, amylodextrin (see Fig. 27). Other periodate oxidations have been reported by Myrback and J a r n e ~ t r o r n l(0-dextrin), ~~ by Freudenberg and Cramer3’ (a-, p-, and y-dextrin) and by Akiya and coworker^^^^^ (a- and pdextrin) . The methylation and periodate data appear to eliminate all structural possibilities except the following: (a) D-glucopyranose units linked 1 4 4; (b) D-glucofuranose units linked 1 -+ 5 ; (c) a combination of (a) and (b); and (d) open-chain D-g1UCOSe units linked by acetal oxygen bridges 1 + 4, 1 -+ 5 t o the adjacent D-glucose unit.

HCOHCO

I

CHzOH (a)

‘A

HCO

I HCOI

CHZOH (b)

HbOI

I

HCO-

I

CHsOH

(4

(131) K . Myrback and T. Jarnestrom, Arkiv Kemi, 1, 129 (1949); K. Myrback, ibid., 1, 161 (1949).

246

DEXTER FRENCH

I t is obvious that, essentially the same structural possibilities result from the methylation or periodate analysis of starch, particularly amylose. With starch, the evidence has been predominantly in favor of (a). At one time the late C. S. Hudson argued t h a t (d) had never been rigidly eliminated as a possibility. Periodate oxidation of (d) would result in chain cleavage with the production of small fragments rather than the formation of a high molecular-weight polymeric oxidized material. The retention of polymeric character, even of some granule structure, in periodate oxystarch is not compatible with (d).

By analogy with starch, (a) is the preferred structure for the Schardinger dextrins. Freudenberg’s studies on the kinetics of ring opening (hydrolysis, acetolysis, etc.) of the Schardinger dextrins have eliminated the possibility of any linkage more labile than a glucopyranoside type. Structure (a) is in best agreement with the attack on the Schardinger dextrins by certain starch-splitting enzymes, though unfortunately these have never been tested on model substrates having structures of the (b), (c), or (d) type. Of considerable significance is the i s o l a t i ~ n69~ of ~ -maltose ~ ~ ~ (as the crystalline heptaacetate) following partial acetolysis of the Schardinger dextrins. If we assume that there has been no structural rearrangement concurrent with acetolysis, then a t least some of the linkages must be of the maltose type. Numerous studies by K a r r e ~ showed -~~ that acetolysis of Schardinger dextrins gave essentially the same yield of maltose as starch or maltose itself gave, when treated similarly. This type of observation was considered by Karrer t o be strong evidence against the trisaccharide character of p-dextrin (“/3-triamylose”). 122 produced during hydrolysis of the ScharHigher oligosa~charides~~ dinger dextrins are definitely of the amylose type as judged by (1) chromatographic comparison with the amylose series31;(2) behavior with starchsplitting enzymes (/3-amylase,T3 salivary amylase,96 and B. macerans amylase33) and with phosphorylasel31-137; and (3) optical rotation.lzZ 8

2. Anomeric Configuration

As regards the anomeric configuration of each of the D-glucosidic units, Freudenberg originally postulated that there was one P-D-glucosidic structure with the remainder in the (Y-D configuration. The proposal of a single fl-D-glucosidic unit, which was designed to account for the initial rise in (132) Marjorie A. Swanson and C. F. Cori, J . BioZ. Chem., 172, 797 (1948). (133) Marjorie A. Swanson, J . BioE. Chem., 172, 805 (1948). (134) Marjorie A. Swanson and C. F. Cori, J. B i d . Chem., 172, 815 (1948). (135) Marjorie A . Swanson, J . B i d . Chem., 172, 825 (1948). (136) Gerty T. Cori, Marjorie A . Swanson and C. F. Cori, Federation Proc., 4, 234 (1945). (137) S. Hestrin, J . B i d . Chem., 179, 943 (1949).

THE SCHARDINGER DEXTRINS

247

rotation during acid hydrolysis, was later withdrawn and replaced by the currently held view that all the glucosidic bonds have the a - configura~ tion. Freudenberg’s arguments2’’24 are as follows. (1) If the initial increase in rotation during hydrolysis of the Schardinger dextrins is caused by a p-D linkage, i t must be an unusually labile p-Dlinkage, otherwise the increase in rotation due to hydrolysis of the p-D linkage would be more than compensated for by the simultaneous decrease in rotation caused by the splitting of the more numerous a - linkages. ~ (2) Determination of the rate of liberation of reducing groups shows that there is no unusually labile bond present. (3) It is possible to account for the initial rise in rotation, without having /3-D linkages, if it is assumed that the formation of a large ring has a lowering effect on the optical rotation; on opening the ring the rotation would then increase. (4) The optical rotatory curves during hydrolysis, after the initial increase, lie very close to that given by starch. Presence of a single (non-labile) @-D linkage in the molecule would tend t o lower the rotations below the starch curve by an easily discernible amount. The x-ray symmetry requires that if there are “abnormal” linkages in the Schardinger dextrins, the a-dextrin (2-fold molecular axis of symmetry) would have at least two such linkages and the y-dextrin four (4-fold molecular axis of symmetry). This lends even more force to Freudenberg’s view that all the linkages are of the a - ~ - (-+ l 4) type. AND INCLUSION COMPOUNDS VIII. COMPLEXFORMATION

One of the striking properties of the Schardinger dextrins is their ability to form complexes with a variety of organic and inorganic compounds. Many of the complexes, especially with iodine and organic solvents, are relatively insoluble crystalline materials. 1. Complexes with Organic Molecules

The insoluble complexes or inclusion compounds have considerable utility in that they are effectively used in the ~ e p a r a t i o n , p~rification,~. ’~~ ’I and identification3! lo of the individual dextrins. French and coworkers3 measured the solubilities of the a-,p-, and y-dextrins in the presence of excess organic liquids. A few representative precipitants are listed in Table 111. Two rather striking facts emerge from experiments of this sort. In the first place, it is surprising to find that any water-soluble carbohydrate would form such insoluble complexes with unreactive hydrocarbons, halogenated hydrocarbons, or the like. In some eases the solubility of the dextrin is reduced t o less than 1% of its water solubility. Secondly, it is rather that the different dextrins are precipitated with widely dif(138) F. Lange, German Pat. 442,963 (1927); Chem Zentr., 98, I, 2948 (1927).

248

DEXTER FRENCH

fering effectiveness by different organic precipitants. For a-dextrin, bromobenzene is only one third as effective a precipitant as benzene, whereas with p-dextrin bromobenzene is twice as effective as benzene. These observations undoubtedly have to do with the way in which the organic solvent is able t o fit into the Schardinger dextrin rings. A model of the benzene molecule neatly fits a model of the a-dextrin ring, but with bromobenzene there is difficulty in accommodating the bulky bromo group?* The differential precipitating actions of bromobenzene and p-xylene have been incorporated by Freudenberg and by French into schemes for the separation and purification of the crystalline dextrins. In a series of article~l~~-l46 on inclusion compounds, mainly dealing with the Schardinger dextrins, Cramer reports that the Schardinger dextrins form occlusion compounds with such compounds as nitrosobenzene, methylene blue, and a-hydroxy ketones, causing shifts in the absorption spectra and redox potentials. Hydrolysis of indican, either b y emulsin or by aqueous acid, is retarded by formation of the P-dextrin inclusion compound. In Cramer’s opinion, the hollow space of a Schardinger dextrin is a region of high electron density, which behaves like a Lewis base. At p H 8.4 the oxidation of 3-hydroxyoxindole (which readily forms an enediol) is accelerated three-fold by inclusion. In some respects, where the cyclic dextrins are acting as microheterogeneous catalysts, they resemble enzymes. Inclusion phenomena have been used by Schlenkl4’, 148 in two connections. The Schardinger dextrins form complexes with certain labile or readily oxidizable vitamins or drugs, for example, vitamin A. The rate of deterioration may be slowed by incorporating them into molecular ,“packages.” In another application, a-dextrin followed by iodine has been proposed as a spray reagent for locating complexible materials on paper chromatograms. Monoglycerides, hydrocarbons, higher alcohols, fatty acids, etc., show up as white or yellow spots on a purplish background. (139) F. Cramer, Naturwissenschaften, 38, 188 (1951). (140) F. Cramer, Chem. Ber., 84, 851 (1951). (141) F. Cramer, Chem. Ber., 84, 855 (1951). (142) F. Cramer and W . Herbst, Naturwissenschaften, 39, 256 (1952). (143) F. Cramer, Angew. Chem., 64, 437 (1952). (144) F. Cramer, Ann., 679, 17 (1953). (145) F. Cramer, Chem. Ber., 86, 1576 (1953). (146) F. Cramer, Chem. Ber., 86, 1582 (1953). (146a) H. von Dietrich and F. Cramer, Chem. Ber., 87, 806 (1954). (147) H. Schlenk, D. M. Sand and J. Ann Tillotson, J. A m . Chem. Soc., 77, 3587 (1955). (148) H. K. Mangold, Beverly G. Lamp and H. Schlenk, Abstracts Papers Ant. Chem. Soc., 127, 52N (1955).

249

THE SCHARDINGER DEXTRINS

2. Complexes with Iodine and Iodide

Although exact structural studies by x-ray diffraction have been disappointing in that they have failed so far to reveal fine details of structure, there is much evidence that in these complexes the Schardinger dextrin forms a ring enclosing the complexing agent. Pringsheim66 has reported analytical values for crystalline complexes of iodine or bromine with a- and p-dextrins (see Table V). It is not intended that the detailed formulas in Table V should be taken seriously, but rather as an illustration of the state of confusion that atTABLEV Halogen Addition Products oj the Schardinger Dextrins According lo Pringsheim66 Total halogen, per cent Dextrin

a-Hexa-amylose a-Tetra-amylose a-Diamylose p-Hexa-amylose 8-Triamylose a-Hexa-amylose a-Tetra-amylose a-Diamylose 8-Hexa-amylose 8-Triamylose

Compound

Bromine products (C6HioOs)s , 2 Br (CsHioOr), , 135 Br (CeHioOs)a, % Br ( C E H I O O 2~ )Br ~, (CeHioOb)a, 1 Br Iodine products (C 6 Hd s )6 , 234 1 (CeHioOs)r, $6 I (CeHio0.de , 3G 1 ( C E H I O O 3~ )I~ , (CsHioOs)s, 1% I

’computed

found

14.1 15.6 17.8 14.1 14.1

14.2 15.4 17.4 13.7 16.1

22.8 22.8 22.8 28.1 28.1

24.3 23.4 24.3 27.0 27.5

Atomic halogen per cent, f o w l

9.9 14.6 11.3 13.4

18.2 18.7

-

-

tended much of Pringsheim’s work. The proportions of halogen in the hypothetical compounds are bizarre. It may be noted, however, that some of the halogen is not present in the free (“atomic”) form, but as halide. French has shown114that the nature of the halogen complexes (particularly the iodine complexes) depends very much on the amount and nature of the halides added. In the absence of added iodide, a-dextrin forms an iodine complex (cr.Iz.14 HzO) which crystallizes as tiny, tan needles from water. In the presence of low concentrations of iodides, even the small amount produced by the hydrolysis of iodine in water, a-dextrin forms fine crystal needles containing both iodine and iodide. With dilute potasl 8 is produced. With more concentrated sium iodide, [ a * I ~ ] 2 . K I -H2O potassium iodide, blue-black hexagonal plates or prisms of a -I2.KI-8 H2O appear. With cations other than potassium, the crystal appearances vary. Thus with sodium or lithium, either blue or bronze hexagonal crystals

250

DEXTER FRENCH

form, depending on the conditions. Barium gives tiny transparent triangular prisms. Only a few of the numerous possibilities have been examined, but enough to show the variety of different complexes possible. A similar variety of crystalline complexes is produced with the P-dextrin; y-dextrin also forms iodine complexes but these remain virtually unexplored.

FIG.28.-Crystal structure of the a-dextrin-iodine complex formed in the absence of i0dide.1~9The outlines of the Schardinger dextrin molecules have been chosen on the basis of possible packing arrangements; the iodine atoms have been located by x-ray crystal structure analysis. In this tan-brown complex, there is little or no interaction between the iodine molecules such as occurs in the highly-colored blue or black “canal” compounds.

The tan-brown complex a m 1 2 (formed in the absence of iodide) has been studied by x-ray diffracti~n.’~~ In this case, only the iodine atoms can be located by x-ray diffraction means alone, but consideration of possible means of packing the a-dextrin molecules indicates that each iodine molecule is enclosed by a cyclic dextrin molecule (see Fig. 28). It may be noted that, in this case, the iodine molecules do not approach each other closely; there is nu opportunity for cooperative effects or interaction with iodide ion such as is observed in the highly colored iodine-iodide complexes. The (149) W . J. James and D. French, Proc. Iowa Acad. S c i . , 69, 197 (1952).

THE SCHARDINQER DEXTRINS

251

a-dextrin crystallizes in very nearly isomorphous crystals as the iodine complex from water and as complexes from dilute methanol, ethanol, and 1-propanol, which is added evidence that the complexing agent has little to do with the packing of the dextrin molecules; the inside of the torusshaped molecule provides an empty place which will accommodate the complexing agent without interfering with the over-all packing arrangement. Perhaps the most remarkable of the iodine complexes are the so-called “canal” compounds in which cylindrical rows of a-dextrin molecules enclose long rows of iodine atoms. In the compound a - 1 2 . K I (form I) sheets of horizontally packed cylinders are layered over each other, in such a way that each successive sheet is rotated 60” from the preceding one. The result is a hexagonal plate or prism which in the thinnest crystals shows a beautiful blue color. Viewed from the side, the hexagonal prisms show a remarkable dichroism in harmony with the proposed arrangement of the iodine atoms. The crystallographic symmetry requires that the a-dextrin molecules lie along 2-fold symmetry axes and that they be arranged front-tofront and back-to-back. The iodine molecules are also constrained to lie along 2-fold axes, and although there are several possible 2-fold axes in the structure the simplest arrangement is the one in which the dextrin molecules and the iodine molecules lie coaxially on the same axes with the dextrin. Packing requirements of the dextrin molecule are also in harmony with this arrangement. A second “canal” type of iodine complex (form 11) is formed from relatively dilute solutions and has the composition (cu.Iz)z.KI. Here the basic unit seems t o be a pair of a-dextrin molecules enclosing a n :I ion. The a2-1: units pack in a hexagonal or pseudohexagonal arrangement which gives an x-ray diffraction pattern with essentially the same spacings and intensities as the amylose-iodine complex. With this compound a remarkable dichroism is also observed which indicates that the iodine molecules are aligned parallel to the needle axis of the crystal. The location of the cation in these canal compounds is not clear, but the cation definitely influences the nature of the crystal which is formed. With sodium and lithium iodides, a form I1 type of complex crystallizes as hexagonal plates. I n the sodium iodide-iodine complex, the inclusion compound is not stoichiometric but rather the iodine atoms are packed into the canals in linear rows, with a spacing not related to the spacing of the dextrin molecules. With barium iodide, the form I type of complex crystallizes as triangular plates, in which the iodine chains lie parallel to the trigonal or rhombohedra1 axis. Unfortunately the crystal structure appears to be rather complex so that further structuraI details are unknown. It is a remarkable fact that

252

DEXTER FRENCH

none of the hexagonal crystals of a-dextrin complexes so far observed have indicated hexagonal symmetry for the a-dextrin molecule, though it would seem reasonable from the "chemical" symmetry. CramerlS9in his studies of the iodine inclusion compounds considers that in some cases the iodine chains can be represented as a one-dimensional electron gas. From the position of the light absorption maximum (6200 A.) together with one-dimensional Fourier analyses of the iodine "gas," Cramer concludes that there are perhaps 14 or more iodine atoms, spaced with an 1-1 distance of 3.06 A. in a polyiodide ion. DubelzBmeasured by electrometric methods the equilibria in solution between the Schardinger a- and P-dextrins, iodine, and iodide. Most pronounced with the a-dextrin, there is a strong affinity for iodine and particularly for triiodide ion. a

+ r2=

K

a . ~ 2 ;

=

2.0

x

103

+ I ' d a.1:; K = 1.35 X lo6 a + 1 ' s a * I e ;K = 13.5 a.Ie + Is= cu-1:; K = 2.0 X lo7 p + .Ie= 8.1"; K = 1.45 P.1" + I z d 8.1:; K = 1 X lo6

a-12

Formation of a crystalline complex a . K I was also observed. The formation of these charged complexes in solution has been used as the basis for an electrophoretic separation3s of the Schardinger dextrins. Beckmann and Forster"JOalso found that complex formation with a-dextrin enhances approximately 245-f old the ultraviolet absorption maxima in iodine-iodide solutions at 290 and 350 mp. It is probable that the colored complexes of iodine with methyl ethers and with the tosyl and mesyl esters of the Schardinger dextrins are also inclusion compounds of the same general type.

LX. RING CONFORMATION IN THE SCHARDINGER DEXTRINS If a satisfactory structural model for the Schardinger dextrins is to be arrived at, the question of the conformation ("Konstellation") of the individual D-glucose units must be considered. In his earliest efforts along this line, FreudenbergZ6v78 used rigid Kekul6 models which did not allow free rotation about the individual bonds. Using the rigid boat conformation (150) C. 0. Beckmapn and E, 0, Forster, private communication to the author.

THE SCHARDINQER DEXTRINS

253

(B1 in Reeves's designation1K1)(see Fig. 29), Freudenberg concluded that the smallest cyclic molecule of the Schardinger dextrin type would be the pentasaccharide. At the time it was not appreciated that the boat form is extremely flexible162 and that it is possible to pass continuously via unstrained rings through all the Sachse boat forms. By use of the flexible unstrained boat form of the D-glucose ring with space-filling Fischer-Hirschfelder models which allow free rotation about the individual bonds, a cyclic trisaccharide (see Fig. 16) and indeed any higher saccharide, can readily

GI

s

Ob

83

38

FIQ. 29.-The eight pyranose strainless ring conformations and the corresponding symbols (Reeves).l61 By convention the heavy lines represent the sides of the threedimensional figures nearer the observer. The dark circles represent ring oxygen atoms, the numbered circles carbon atoms 1 t o 5 . It should be noted tha t the boat forms (B) are interconvertihle through strainless flexible intermediates.16*

be made apparently with perfect axial symmetry and without involving bond strain. On the other hand, using the C1 rigid conformation for the D-glucopyranose ring, the smallest cyclic saccharide which can be made is the hexasaccharide (see Figs. 11 and 12). This involves a certain amount of strain. The heptasaccharide (see Fig. 13) can be easily made, but higher homologs seem to require kinking of the supercyclic molecule. From the evidence with models alone, one would expect to find Schardinger dextrins smaller (151) R. E. Reeves, J . A m . Chem. SOC.,71, 215 (1949); see also Advances i n CUTbohydrate Chem., 6 , 107 (1951). (152) P . Hasebroek and L. J . Oosterhoff, Discussions Faraday Soc., 10, 87 (1951).

254

DEXTER FRENCH

than the a-dextrin if the D-glucopyranose ring is in the flexible conformation; the failure of l?. macerans amylase to produce small rings may be either a reflection of the enzyme specificity or it may be a consequence of the rigidity of the D-glucopyranose unit in the C1 conformation. Recently Freudenberg and Cramer37 have proposed that flexible ring forms intermediate between B1 and 3B are involved in varying degree in the Schardinger dextrins and starch. Reeves has considered the significance of the cuprammonium rotational shifts with the Schardinger dextrinsl63 in comparison with those of starch and other carbohydrate materials. Although the rotational shifts are rather large, they are not as large as those observed with starch and cellulose. In the case of p-dextrin, the rotational shifts correspond to those expected if 4 of the seven D-glucose units react with cuprammonium. It is Reeves's opinion that the failure of some of the units to react does not necessarily imply that they must be in the "unreactive" ring conformations (such as 1C). Possibly some of the units are prevented from reacting with cuprammonium by steric or electrostatic factors. A more detailed study of the kinetics of periodate oxidation might have a further bearing on the question of ring conformation in the Schardinger dextrins. As noted before, periodate oxidation is considerably inhibited by the cyclic structures, as compared with the behavior of linear starch molecules. It is known that in such bicyclic compounds as 1,6-anhydro-~-glucofuranose the ring rigidity is such that periodate oxidation is completely inhibited. Possibly, incorporation of the D-glucopyranose ring into a supercyclic molecule also enhances the rigidity of the ring to the extent that the ability to react with periodate is greatly reduced.

X. DERIVATIVES OF

THE

SCHARDINGER DEXTRINS

The acetates, nitrates, and methyl ethers of the Schardinger dextrins have been reported in crystalline form. There are over 20 reports on the preparation and properties (for example, the optical rotation) of a-dextrin acetate from the work of Pringsheim alone. No attempt can be made to review the entire literature in this field, since most of it is only of historical interest. Rather the most recent or most reliable results will be summarized. (153) R. E. Reeves, private communications t o the author, July, 1949, and July, 1950. Early work on cuprammonium-Schardinger dextrin reactions is reported by E. Messmer, 2. physik. Chem., 126, 369 (1927). According t o Reeves, the insignificant rotatory shifts observed by Freudenberg and CrarneF are due t o the low concentration of cuprammonium used; with suitable cuprammonium solutions the molecular rotatory shifts with the a-,8-, and y-Schardinger dextrins amount t o - 1230°, - 1008", and -990", as compared with -1760" and -1560" for starch and glycogen.

THE SCHARDINGER DEXTRINS

255

1. Acetates

Judging from reports in the literature and the author's experience, one would think the preparation of the Schardinger dextrin acetates would be nearly fool-proof. Even so, widely varying optical rotations and other properties have been reported for these compounds. As has been seen, the variation in optical rotations, melting points, and freezing-point depression with different dextrin preparations has led to an abundance of confusion in this area. A preparative method which works was reported3 by French and coworkers. The crude dextrin, oven-dry, was added in four equal parts a t intervals t o five parts of boiling acetic anhydride containing half a p a r t of anhydrous sodium acetate. After the final addition the mixture was refluxed for 30 minutes, allowed t o cool t o room temperature, and poured with stirring onto cracked ice and water. As t h e dextrin acetate hardened, the water was replaced. When all the acetic anhydride had been destroyed, the dextrin acetate was broken into small pieces and collected by suction filtration. The crude a- and @-acetateswere crystallized from 10-15 parts of boiling toluene by cooling to room temperature. y-Dextrin acetate was crystallized from 2-3 parts of hot butyl acetate. The dextrin acetates appear t o crystallize with solvent of crystallization which is gradually lost on exposure to air, giving in some cases glasses which retain the exterior form of the original crystals. The [aInvalues of the pure acetates were $105.5, +122.0, and +138.5" (c 1 , in CHCla). [Freudenberg, Plankenhorn and Knauber'7 report corresponding values of +107.5, +121, and $137". The agreement is fairly typical of samples obtained in different procedures.]

2. Nitrates Lcibowitz and SilmannK6prepared crystalline a- and @-dextrin di- and tri-nitrates. In their procedure, 1 part of Schardinger dextrin was added t o 15 parts of nitric acid (freshly distilled from a mixture of 1 volume of concentrated nitric acid and two volumes of concentrated sulfuric acid into an ice-cooled receiver). With continuous cooling and stirring, two volumes of concentrated sulfuric acid were slowly added, whereupon the nitrate separated as a solid precipitate. After one hour the mixture was poured onto ice and worked up by rubbing in ice water and decanting several times. After filtering and liberal washing, the nitrate was dried on a clay plate or in a vacuum desiccator over calcium chloride. The yield amounted to about 170% of the starting material. Crude a-dextrin nitrate was dissolved in boiling glacial acetic acid. On cooling, the dinitrate crystallized in fine silky needles. If any substantial amount of a-trinitrate was present it was rapidly precipitated as platelets from the acetic acid. With @-dextrin,the crude nitrate was extracted with boiling alcohol, from which the dinitrate crystallized as clusters. The alcohol-insoluble 8-trinitrate was obtained as "crystalline" flakes after a week in glacial acetic acid. The [a],values reported were: a-dinitrate, +96" (nitrobenzene); a-trinitrate, +80" (ethyl acetate) ; p-dinitrate, +122' (nitrobenzene); and 8-trinitrate, +90.5" (nitrobenzene).

256

DEXTER FRENCH

Nitration with dinitrogen pentoxide in acetonitrile at -20" has been used by Freudenberg and Crame1-.~7The specific rotations found for a(14.2 % N), P- (13.2 % N), and y-dextrin nitrate (12.7 % N) in chloroform were +78", 94", 98", all reported with an uncertainty of 5. The theoretical value for a trinitrate is N, 14.4 %. Dinitrogen pentoxide in chloroform with sodium fluoride at -15" was used by Gruenhut, Cushing and Caesar.Il2 These authors were interested primarily in using the nitrates for determination of the molecular weight by the Barger method and did not report the optical rotatory values. The nitrogen analyses were too low for trinitrate derivatives, even though crystalline products were obtained. 3. Methyl Ethers

Crystalline methylated P-dextrin having 45.4 % OCHs (theor., 45.6 %) and melting at 102-5" was obtained after a lengthy procedure by Irvine, Pringsheim and MacDonald.'h Freudenberg's procedures are more Four grams of a-dextrin (dried under high vacuum a t 100") was dissolved in 200 ml. of liquid ammonia. To this mixture, kept at -50", was added slowly a solution of 4 g. of potassium in 100 ml. of ammonia. The colorless potassium compound was precipitated a t once. After removing the ammonia, absolute peroxide-free ether was added and the remainder of the ammonia was driven off by gentle warming. An excess of methyl iodide in ether was added t o the mixture and refluxed 4 hours. After removing the separated potassium iodide the ether solution was evaporated (yield, 84% of theoretical). Recrystallisation (twice) from 60-90" petroleum ether, with addition of a 1itt)le animal charcoal, gave 3.8 g. of beautiful clusters of elongated prisms, melting a t 20&10"; [aID+162" (c 1, CHC13); 44.6% OCHs. For remethylation, the crude product was dissolved in 300 ml. of ether, mixed with 100 ml. of ammonia and treated with 1 g. of sodium in 100 ml. of liquid ammonia. After processing as before, there was obtained a product with the same melting point and rotation but with 45.4% OCH3. The 6-dextrin, similarly treated, gave a 50% yield of crude product with low methoxyl analysis. After remethylation, the crude product was dissolved in cold water. On warming t o 80' the dextrin ether separated as an oil and then crystallized; m. p. 156-8", [a],+157" (CHCl,); OCHa, 45.5%. Both methylated dextrins dissolve easily in the cold i n alcohol, chloroform, or water. In warm water they are slightly soluble. Ether dissolves only the O-methylor-dextrin easily; in petroleum ether hoth substances are only slightly soluble, especially the &derivative.

4. Miscellaneous Derivatives Karrer reports data for barium,Ia and potassiumee complexes with the Schardinger dextrins, starch, and other carbohydrates, None of these materials were reported to be crystalline and it would seem that they would be of no value as characteristic derivatives. Schardinger dextrin tricarbanilates were prepared by Wolff 164 in a com(154) I. A . Wolff and C. E. Rist, J . Am. Chem. SOC.,70, 2779 (1948).

257

THE SCHARDINGER DEXTRINS

parative study with amylose and amylopectin. Though the starch tricarbanilates were limited in their solubility to such solvents as pyridine, morpholine, and dioxane, the Schardinger dextrin tricarbanilates were soluble in a variety of organic solvents including benzene and acetone, and could conveniently be purified by reprecipitation from isopropyl alcohol. These substances display striking differences in optical rotation depending upon the solvent, as shown in Table VI. TABLE VI Properties of Carbohydrate Tricarbanilates Specij#c rotation, degrees Tricarbanilate ia pyridine

Corn amylose Corn amylopectin 8-Dextrin

-82.5 -62.0 69.5

I

Melting point, degrees in morpholine

-7 -4 22

259-65 250-60 214-5

Partial benzoates and phosphates of a- and P-dextrin have been reported by Pringsheim and coworkers, but none of the materials were well-defined crystalline substances. In unpublished work, H. R. Bolliger prepared Schardinger dextrin tosylates with a view to converting the Schardinger dextrins into cyclic polymeric deoxy compounds. Unfortunately, reaction sequences which had proved t o be workable with monosaccharides failed to give defined compounds with the Schardinger dextrins. Lautsch, Wiechert, and Lehmann166have prepared tosyl and mesyl esters by treatment of the anydrous a- and P-Schardinger dextrins in pyridine with tosyl or mesyl chloride (1 mole per D-glucose residue). Derivatization occurred presumably a t the hydroxyl on C6 of each D-glucose unit. The a- and ptosyl derivatives crystallized from methanol; m. p. 174" and 170'; [a], 95" and 105' (c 1, CHCL). The corresponding mesyl derivatives, though not crystalline, could be converted to crystalline diacetates; m. p. 170"(dec.) and 165'(dec.); [aID 106' and 114' (c 1, CHC13). However, none of the substitution products of the Schardinger dextrins have matched the acetates with respect to ease of preparation, definite composition, stability, recrystallization, and regeneration of the original dextrins.

+

+

XI. SIGNIFICANCE OF THE SCHARDINGER DEXTRINS WITH RESPECT TO THE CONSTITUTION AND BEHAVIOR OF STARCH Since the discovery of the Schardinger dextrins, these cyclic compounds have been of special interest as they relate to starch. In the case of complex (155) W. Lautsch, R . Wiechert and H. Lehmann, Kolloid-Z., 136, 134 (1954).

258

DEXTER FRENCH

formation, we have learned a considerable amount which can be transferred directly to the starch situation. However, with respect to various chemical reactions and especially biochemical behavior, the Schardinger dextrins vary from starch in important aspects. We are left with the enigma as t o why ring closure exerts such a profound effect on the starch chain. In particular, interest has been focused on the question as to which part of the starch structure gives rise to the cyclic molecules. Pringsheim thought of amylose and amylopectin as “polymerized a-diamylose” and “polymerized p-triamylose.” The nature of the association-polymerization was something of a mystic concept which seems strange in the light of modern knowledge of polymer structure. However, a somewhat similar type of 2ssociation-polymerization is currently in vogue with such substances as insulin (fibril formation), collagen, and nucleoprotein. At one time Freudenberg1b6seriously proposed that starch is based upon a cyclic Schardinger dextrin nucleus, with side branches. In the bacterial breakdown, the branches would be broken off according to the following scheme (adapted from Freudenberg).

Bacillus macerans

Hypothetical starch structure

-

Linear and cyclic breakdown products

Although this ingenious suggestion accounts in a nice way for the poverty of reducing groups in starch, the presence of nonreducing end-groups, and the formation of limited amounts of Schardinger dextrins, it had to be abandoned for obvious reasons. Up t o 1939 the Schardinger dextrins were known only as products of the bacterial breakdown of starch. Commenting on their significance, Tilden and Hudsonz6state: (‘. . . work by other investigators has not demonstrated with certainty whether these crystalline dextrins represent comparatively simple components of starch itself, or whether they are formed as the result of synthetic activity of the living organism. In the latter case they would seem t o be of little importance t o the study of the constitution of starch. We now find, however, that when Aerobacillus macerans is grown for several weeks upon a potato medium, and the culture fluid is then filtered through a Berkfeld N filter to remove the microorganisms, the filtrate contains an enzyme which will produce the Schardinger dextrins from starch rapidly, and in greater yield than has been previously reported. We infer from this (156) K. Freudenberg, Ann. Rev. Biochem., 8, 81 (1939).

THE SCHARDINGER DEXTRINS

259

fact that the crystalline dextrins are components of the starch structure, or are closely related to such components.” With the advent of cell-free B. macerans amylase preparations, it has been possible t o study the action patterns of this enzyme (see Sect. IV, 4) and relate this information to starch breakdown. Some of the significant results are as follows. (a) B. macerans amylase is capable of transfer reactions involving only ( Y - D - ( ~-+ 4)-glucosidic bonds of linear or cyclic compounds. (b) Linear starch materials (for example, amylose, amylodextrin, and maltoheptaose) are extensively converted to Schardinger dextrins. I n the case of high molecular-weight amyloses, failure to achieve complete convorsion is not in agreement with theory, unless the poor yields result from the amylose’s being hydrolyzed or becoming insoluble during the enzymolysis. KerrS2obtained about 70 % conversion of amylose to Schardinger dextrins; Wilson, Schoch and Hudsongoreported similar results. By using extremely dilute amylose solutions (0.05 %) with high enzyme activities, Cramer and Steinle38were able to achieve over 90% conversion t o Schardinger dextrins.lK6”(c) With amylopectin or glycogen, only the outer chains (nonreducing) are converted into Schardinger dextrins. Substantial yields may be obtained from amylopectin or waxy corn starch (4050%).90-92 These yields are commensurate with the yields of maltose obtained with beta amylase. To the extent that starch (or amylopectin) is converted to Schardinger dextrins, it is not available for conversion t o maltose by beta amylase, and vice versa.8nAmylopectin beta amylase limit dextrin does not give Schardinger dextrins with B. macerans a m y l a ~ e . ~ ’ (d) Derivative formation of starch, such as low degree of carboxymethylation or oxidation, greatly inhibits the action of beta amylase and B. maceTans Alpha-type amylases are less sensitive to minor modification in structure. (e) Inhibition of phosphorylase by Schardinger dextrins is caused by competition with the natural “primer” for the primer binding site on the phosphorylase molecule.1n3Since the Schardinger dextrins do not have a free 4-position to accept a D-glucose unit from D-glucosyl phosphate, phosphorylase action is blocked. (f) For several years, the only source of individual linear starch oligosaccharides was the controlled acid hydrolysis of the Schardinger dextrins.Iz2With maltoheptaose, especially, it was possible to learn many details of the action of amylases, phosphorylase, and other starch-metabolizing enzymes. (g) Possibly the most val(156a) For an ideal amylose chain of length 200 or more, the yield of Schardinger dextrins would be greater than 95% at substrate concentrations of less than 3%. However, similar yields less than theoretical have been encountered with @-amylase, and on the whole there seem to be many points of similarity between actions of B . ni.uceruns amylase and @-amylase. (157) E. Husemann and E. Lindemann, Die Stiirke, 6, 141 (1954).

260

DEXTER FRENCH

uable property of the Schardinger dextrins is their ability to enter into B. macerans amylase coupling reactions32239 with a variety of co-substrates, in which D-glucose or short starch chains become joined to the co-substrate. These coupling reactions can be used to prepare model compounds, for example, malto-oligosaccharides in which the reducing D-glucose units are radioactive,168or “branched” oligosaccharides related to branching points in amylope~tin.’~~ Some “anomalous” properties of the Schardinger dextrins have been mentioned previously, but they will be summarized here. (a) Acid hydrolysis of the cyclic compounds is considerably slower than that of the corresponding open-chain oligosaccharides. (b) Periodate oxidation is slow with Schardinger dextrins in comparison to that of straight-chain amylodextrin. (c) The Schardinger dextrins are oxidizedz1by hypoiodous acid or hypoiodite, possibly by a glycol-cleavage reaction. (d) Reaction with cuprammonium gives optical rotatory shifts which are relatively small in comparison to those for starch or cellulose. (e) The cyclic compounds, particularly cyclohexaamylose, are extremely resistant to the alpha-type amylases (resistance to beta-amylase is not considered anomalous, in that beta-amylase requires an end group for its action). (f) The Schardinger dextrins appear to be toxic to small animals. (g) The optical rotations of the Schardinger dextrins are too low, in comparison with those of amylose or even the linear malto-oligosaccharides of comparable molecular size. The optical rotations do not obey Freudenberg’s rule correlating the rotations of a homologous series. (h) The chromatographic mobility is higher than that of the corresponding open-chain compounds; lower members, especially, do not fall in a regular series like that characterizing the linear oligosaccharides. These intriguing irregularities and departures from expectation must have their origins in the cyclic nature of the compounds. Steric hindrance, the rigidity of a ring as contrasted to a flexible chain, the conformation of the D-glucose ring, restriction of rotation about the glucosidic bond, the unusual microscopic environment in the center of the cyclic molecule, the tendency to form inclusion complexes-all these factors may be involved in the “anamolous” behavior mentioned above, but so far none of these has been adequately explained. It is the writer’s expectation that the Schardinger dextrins, and the enzyme from B. rnacerans which produces them, will continue t o serve, delight, teach, and intrigue the carbohydrate chemist for many years to come. (158) J. H. Pasur and Tania Budovich, J. Biol. Chem., 220,25 (1956). (159) R. Summer and D. French, J. Biol. Chem., 222, 469 (1956).

THE MOLECULAR STRUCTURE OF GLYCOGENS

Department of Chemistry. The University of Edinburgh. Scotland

. I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 1. Historical Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .262 2 General Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .263 . 3 . Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 4 . Isolation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 5. Estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .268 . 6. Basic Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .269 . I1. Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 . 1. Molecular Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 2 . Molecular Shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .276 3. Interaction with Iodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 a . Absorption Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 b . Iodine-binding Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .278 4 . Interaction with Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .279 111. Structural Analysis by Chemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 1. End-group Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .280 . a . Methylation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 b . Periodate Oxidation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 2. Characterization of Inter-chain Linkages . . . . . . . . . . . . . . . . . . . . . . . . . . . .281 . a . Methylation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .281 . b . Acid Hydrolysis Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .282 . c . Periodate Oxidation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .283 IV . Structural Analysis by Enzymic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 . 1 . End-group Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 2 . Determination of Exterior and Interior Chain Lengt.hs. . . . . . . . . . . . . . . . 286 . 3 . Evidence of Random Branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 4 . Determination of Multiple Branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 V . Molecular Structure of Glycogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 1. Glycogens of Vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 a . Mammalian Glycogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 b . Fish Glycogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 c . Glycogen-storage Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 2 . Glycogens of Invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 3 . Bacterial and Yeast Glycogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 4. Comparison of Glycogens with Amylopectins . . . . . . . . . . . . . . . . . . . . . . . . . . 294 . VI . Biological Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 . I . In vitro Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 2 . In vivo Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .297 VII . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

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I. INTRODUCTION 1. Historical Introduction

A review on the molecular structure of glycogens is particularly appropriate at the present time, as 100 years have now elapsed since Claude Bernard’ announced, in March 1857, the isolation of glycogen from dog liver. Following his discovery, in 1850, of the glycogenic function of the liver, Bernard observed that liver contains a substance which slowly changes into sugar. He isolated this substance, ‘‘matibre glycogbne animale,” and found it to be a white amorphous powder, soluble in water to give an opalescent solution. It was precipitated by both alcohol and acetic acid. Bernard observed that, although glycogen is resistant to hot alkali, it is readily broken down by hot acids and by diastatic enzymes to give fermentable sugars; furthermore, aqueous solutions give a characteristic red-brown coloration with iodine. The presence of glycogen was then reported in skeletal muscle; placental tissue,3 surface epithelial cells; and cells of the intestinal mucosa4; none could be detected in bone or in glandular or nervous t i ~ s u eIn . ~ 1861, glycogen (isolated from human liver) was found6 t o have the empirical formula C6HI006(recalculated on the basis of modern atomic weights). During investigations on yeast, Errera6 noted the presence of a substance which gave a brown coloration with iodine, and nine years later, Cremer? isolated glycogen from yeast as a white powder, soluble in water +198.9’) and having the properties of the animal glycogen described by Bernard. Improved methods for the preparation of yeast glycogen, free from yeast-gum (mannan), have been described by several workers, including Harden and Young.8 Cremer’s observations on yeast glycogen were confirmed by C l a ~ t r i a u who , ~ also found that the properties of glycogen from rabbit liver and from two species of fungi were identical with those of yeast glycogen. Although it had been assumed that glycogen was a polymer of glucose, detailed proof that glucose was produced by the complete acid hydrolysis of glycogen was not published until 1881. Kulz and Borntragerlo compared C. Bernard, Compl. rend., 44, 578 (1857). A. Sanson, Compt. rend., 44, 1159, 1323 (1857). C. Bernard, Journal de la physiologie de l’homme et des a n i m a u x , 2, 30 (1859). C. Bernard, Journal de la physiologie de l’homme et des animaux, 2, 326 1859). E. von Gorup-Besanez, Ann., 118, 229 (1861). L. Errera, Cornpt. rend., 101, 253 (1885). M. Cremer, Miinch. n7ed. Wochschr., 41, 525 (1894). A. Harden and W . J. Young, J . Chem. Soc., 81, 1224 (1902); 101. 1928 1912). G. Clautriau, cited in ref. 8. (10) E. Kulz and A. Borntrager, PfEtigers Arch. ges. Physiol., 24, 28 (1881)

(1) (2) (3) (4) (5) (6) (7) (8) (9)

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the analysis, optical activity, reducing power, microscopic appearance, and compound formation with sodium chloride, of glucose and the sugar isolated from an acid hydrolyzate of horse-liver glycogen; they were identical. The enzymic degradation of glycogen received considerable attention in this period. I n 1879, Seegen” showed that diastase, saliva, and pancreatic juice rapidly bring about a 60-70 % degradation of dog-liver glycogen. The products were a sugar, which differed from glucose in having a lower reducing power and a higher specific rotation, and a mixture of “dextrins”; a proportion of the ‘Ldextrins”was unfermentable. Two years later, Kulzl2 identified the sugar from the enzymic degradation of glycogen as maltose. The structural significance of this finding did not, of course, become apparent until the constitution of maltose had been determined13many years later. During the period 1880-1920, interest in glycogen was focused mainly upon improving methods of preparation and estimation, and upon its physiological role as a carbohydrate reserve; progress in purely chemical studies (see p. 269) was not possible until the ring structure of D-glucose had been established. In addition, the superficial properties (specific rotation, opalescence, iodine coloration, D-glucose content, and analysis for carbon, hydrogen, oxygen, nitrogen, and phosphorus) of “glycogens” from a variety of biological sources were compared. 2. General Properties

I n this article, the term “glycogen” is used t o describe a group of highly branched polysaccharides, isolated from animals or microorganisms, which conform t o the following criteria. (1) Empirical formula C6H1oO6 (inorganic material, nitrogen, phosphorus and sulfur being absent).-The majority of workers now agree that the empirical formula of glycogen is C6H1006, although in the period 1875-1900 a number of analyses14had suggested 6 C 6 H ~ 0 0 6 * & Harden 0. and Youngs found that oyster glycogen, when dried a t 100’ in air, had the analysis required for 6 CsHloOs.HtO, whereas a sample dried over phosphoric oxide a t 100”under diminished pressure, gave analytical results corresponding t o C6HloOs. Carefully purified preparations of glycogen are free from significant amounts of inorganic material, nitrogen, phosphorus, and sulfur, despite (11) J . Seegen, PfEuyers Arch. yes. Physiol., 19, 106 (1879). (12) E . Kulz, Pjlagers Arch. ges. Physiol., 24, 81 (1881). (13) W. N. Haworth and S . Peat, J . Chem. Soc., 3094 (1926); J. C . Irvine and I. M. A. Black, ibid., 862 (1926). (14) For example, E. Kule and A . Borntrager, PfEiigers Arch. ges. Physiol., 24, 19 (1881).

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many reports to the contrary in the literature. For example, Pantlitschko and MatulaI6 have claimed that glycogen contains four phosphate ester groups (and one uronic acid group) per 500 anhydro-D-glucose residues, whilst Wajzerlfibelieved that glycogen from the livers of a rabbit or guineapig contained 0.2 % of organically bound phosphorus (mainly as a-D-glucopyranose 6-phosphate) . (2) The aqueous solution has a high dextrorotation of about +196O.Figures given in the literature range from +179 to +233', the majority being between 191-199'. In a typical comparative study, Harden and Youngs found glycogen from oysters, rabbit muscle, and yeast to have [aID+191, +191, and +198", respectively. (3) The aqueous solution gives a red-brown coloration with iodine.-Although the staining power of glycogen with iodine is a characteristic property, the tint and intensity of coloration vary with the source of glycogen. For example, the iodine coloration given by rabbit-muscle glycogen is reddish-~iolet~'whereas liver glycogens, under similar conditions, stain reddish-brown ; in general, the iodine colorations of glycogens from invertebrates are much paler than those from tissues of vertebrates, although a number of exceptions to this generalization have been reported.s The staining power of glycogen with iodine must therefore be regarded as a superficial property, having uncertain structural significance. The iodine coloration of glycogen has been widely used for the detection of this polysaccharide in various tissues; in some instances, it is regrettable that no alternative method of identification has been applied, since other substances (for example, certain varieties of rice starch) are also stained red with iodine. (4) The aqueous solution has a negligible reducing power.-The apparent reducing power of a glycogen solution varies with the reagent used. The alkaline dinitrosalicylic acid, ferricyanide, or copper reagents give reducing powers of the order of 0.1 % of D-glucose, whilst with the (less specific) alkaline hypoiodite reagent, values of the order of 1 % may be obtained. The accurate determination of the apparent reducing power of glycogen has formed the basis of a method for the determination of the molecular weight (see pp. 274-5). (6) The aqueous solution is opalescent.-Aqueous solutions of glycogen show a variable but pronounced bluish-white opalescence; this opalescence may be so great that 1 % solutions are unsuitable for polarimetric observations. Quantitative measurements of the turbidity of glycogen solutions have been used for estimation of the glycogen content of a solution (see pp. 268-9), and for determination of the molecular weight (see Section 11). (15) M. Pantlitschko and J. Matula, Monatsh., 81, 179 (1950); Chem. Abstracts, 44, 8969 (1950). (16) J. Wajzer, Compt. rend., 144, 808 (1950). (17) F. G . Young, Biochem. J . (London), 31, 711 (1937).

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(6) Hydrolysis by dilute mineral acid at 100” gives an almost quantitative yield of D-glucose.-Complete hydrolysis of glycogen may be effected by dilute mineral acid (0.5-1.0 N hydrochloric acid or 1.0-2.0 N sulfuric acid) at 100”within 2-5 hours, about 97 % conversion to D-glucose being obtained. A small amount of D-glucose is destroyed by the acid, and a further amount is lost by acid-reversion yielding isomaltose and gentiobiose.’**l9 Significant amounts of monosaccharides other than D-glucose have not been detected in acid hydrolyzates of glycogen. (7) Chemical analysis indicates that the n-glucose residues are united by Q-(1 -+ 4)-linkages, and that the ratio of non-terminal to terminal residues i s normally 11 to 1 .-Structural investigations (p. 269) established the relationship between glycogen and starch, in that both polysaccharides contain a - ~ - (-+l 4)-glucosidic linkages. In amylopectin, the branched component of starch, the average chain length (C. L.) is20 18-27. (8) The molecular weight i s of the order of 106.-Molecular-weight determinations have shown glycogens to be amongst the largest of natural polymers. The majority of samples have mean molecular weights of (1-10) X los, and are polymolecularZ1;in addition, some samples are polydisperse and contain molecules with molecular weights averaging around two or three different values; for example, a human-liver glycogen (glycogenstorage disease) contains22 two “components” with molecular weights of about 9 X lo6 and 2 X lo6.Accordingly, molecular-weight determinations on glycogens must be regarded as giving the order of magnitude of the mean molecular size, rather than absolute values (see Section 11). (9) Hydrolysis by @-amylase normally results in 45 f 5 % conversim to maltose.-This criterionz3serves to differentiate between glycogens, amylopectins, and a-amylodextrin (the 0-limit dextrin of amylopectin) , since amylopectins __ have 0-amylolysis limits of 55 f 5%, whilst the latter, in which C. L. = 10-12, is not attacked by @-amylase.24 (10) Glycogen shows the characteristic infrared absorption spectrum of starch-type po1ysaccharides.-The infrared spectrum of glycogen, in the frequency range 730-960 cm.-‘, has three absorption peaks, at 928 f 3, 838 =t3, and 760 f 2 crn.?; the absorption peak at 838 cm.-’ is displayed by all carbohydrates containing a-D-glucopyranose units, whilst the peaks (18) E. Elizabeth Bacon and J. S. D. Bacon, Biochem. J. (London), 68,396 (1954). (19) A. Thompson, Kimiko Anno, M. L. Wolfrom and M. Inatome, J. Am. Chem. Sac., 76, 1309 (1954). (20) D . J . Bell, Ann. Repts. on Progr. Chem. (Chem. SOC.London), 44,223 (1947). (21) For reviews, see C. T . Greenwood, Advances i n Carbohydrate Chem., 7, 289 (1952); 11, 387 (1956). (22) D. J. Manners, J. Chem. Sac., 3527 (1954). (23) D. J. Bell and D. J. Manners, J. Chem. Sac., 3641 (1952). (24) For a review see K. Myrback, Advances in Carbohydrate Chem., 3,251 (1948).

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at 928 and 760 cm.-1 are shown only by (1 4 4)-linked glucans.26 Hence, glycogen may be readily distinguished by infrared spectrophotometry from all other polysaccharides, except related CY-D-(1 + 4)-glucans. Infrared spectrophotometry over the frequency range 900-1,700 cm.-’ has been used for the preliminary identification and estimation of glycogen in cultures of enteric (11) X - r a y analysis shows glycogen to be amorphous.-Glycogens give rise to a diffuse x-ray in contrast to starches, in which certain regions of the granules exist in crystalline form, thereby producing definite x-ray diffraction patterns?* 3. Occurrence

Glycogen has been isolated from livers, brains, and skeletal and cardiac muscles of many mammals, and has been detected in most animal cells, including those of adipose tissue. Human liver may contain 1-10% (by wet weight) of glycogen, which is also present in the tissues of invertebrates (for example, Ascaris lumbricoides, Helix pomatia, and Mytilus edulis), bacteria (for example, Aerobacter aerogenes, Bacillus megatherium, and Neisseria perflava) and protozoa (for example, Tetrahymena pyriformis and Trichomonas gallinae). In all these organisms, glycogen is important as the storage form of carbohydrate, and hence, as a source of energy. Polysaccharides which are stained red-brown with iodine and have chain lengths of about 12 have been isolated from certain plants (for example, Zea mays)29;these have been termed “phytoglycogens.” Dvonch and Whistler30 consider that such polysaccharides should be regarded as highly branched amylopectins. 4. Isolation and Purification

Glycogen may be isolated from tissues by extraction with concentrated alkali a t loo”, with chloral hydrate a t 80”, or with cold aqueous trichloroacetic acid. The most widely used method, developed by Bernard and Pfluger, involves digestion of the tissue with concentrated potassium hydroxide solution (20-GO %) at 100”. Cellular constituents other than glycogen (for (25) S. A. Barker, E. J. Bourne, M. Stacey and D. H. Whiffen, J. Chem. Soc., 171 (1954). (26) S. Levine, H. J. R. Stevenson, E. C. Tnhor, R. H. Bordner and L. A. Chambers, J. 13ncteriol., 66. 664 (1953). (27) R. S. Bear and C. F. Cori, J . Biol. Chem., 140, 111 (1941). (28) R. S. Bear and D. French, d . Am. Chem. SOC., 63,2298 (1941). (29) S. Peat, W. J. Whelan and J. R. Turvey, J. Chem. Soc., 2317 (1956); see also K. H. Meyer and Maria Fuld, Helv. Chim. Acta, 32, 757 (1949). (30) W. Dvonch and R. L. Whistler, J . Biol.Chem., 181, 889 (1949).

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example, proteins and nucleic acids) are destroyed, and glycogen is precipitated by the addition of alcohol. Further purification may be effected by several reprecipitations from aqueous solution with alcohol. For many tissues (for example, mammalian liver) , repeated extraction with boiling water has proved satisfactory; the combined extracts are then deproteinized with 4 % trichloroacetic acid, and glycogen is precipitated with Alternatively, the aqueous extracts may be deproteinized by using excess, concentrated, aqueous picric acid solution.32Highly purified rabbit-liver glycogen has been prepared33by thoroughly grinding the tissue under 3 % trichloroacetic acid solution. Addition of alcohol to the supernatant liquor from the centrifuged extract yielded crude glycogen which, after further purification, contained only 0.20 % of ash and 0.03 % of phosphorus. Extraction of glycogen, by the use of hot water or cold trichloroacetic acid solution should, however, be limited to liver tissue from animals in good nutritional condition, since these solvents are inadequate for the extraction of skeletal muscles, or livers of low glycogen content.’73 34 For the latter tissues, the Pfluger technique is used. Many glycogen samples prepared by the above methods, and particularly by the Pfluger technique, contain small amounts of inorganic material, including silica; this can be removed by electrodialysis of an aqueous solution of the glycogen. By this means, the ash and phosphorus content of MytiEus edulis glycogen were reduced from 0.25 and 0.12% to 0.08 and 0.03 7%) r e s p e c t i ~ e l y .Alternatively, ~~ repeated precipitation of glycogen with 80% acetic acid has been found to be a simple method of obtaining virtually ash-free preparation^.^' The methods of isolation of glycogen outlined above have been subjected to a number of criticisms. It has been suggested that degradation of glycogen occurs during digestion of the tissues by the Pfluger technique, and, as an akernative, the use of chloral hydrate for the extraction of glycogen has been r e ~omr ne n d ed Evidence .~ ~ on the alkali-stability of glycogen is conflicting. Bridgman37 reported that glycogen extracted from one half of a rabbit, liver by 3 % trichloroacetic acid had a molecular weight of 5.2 X lo6 (sedimentation-diff usion measurements), whilst glycogen isolated by the Pfliiger technique from the remaining half had a molecular weight of 4.6 X 106; the difference was stated to be not significant. In a similar study, (31) D. J. Bell and F. G . Young, Biochem. J. (London), 28, 882 (1934). (32) L. G. Petree and C. L. Alsberg, J. Biol. Chem., 82, 385 (1929). (33) M. Sahyum and C. L. Alsberg, J. Biol. Chem., 89, 33 (1930). (34) W . L. Bloom, G . T. Lewis, Mary Z. Schumpert and T. Shen, J . Biol. Chem., 188, 631 (1950). (35) Margaret McDowell, Proc. SOC.Exptl. Biol. Med., 26, 85 (1927). (36) K . H. Meyer and R . W. Jeanloz, Helv. Chim. Acta, 26, 1784 (1943). (37) W. B. Bridgman, J . Am. Chem. Sac., 64, 2349 (1942).

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Greenwood and Manners38found that glycogen isolated by the Pfluger method from half of the livers of two rabbits had sedimentation constants (Szo)of 83 and 86 S ; glycogen extracted by means of hot water from the remaining liver tissue had Szo= 76 and 85 S, respectively. Further evidence of the apparent stability of glycogen to alkali was obtained by Staudinger,39who showed, by a light-scattering technique, that the molecular weights of samples of guinea-pig liver glycogen and muscle glycogen were unaltered after treatment with 15-30 % potassium hydroxide at 100" for one hour. In contrast, digestion of rabbit-liver glycogen with 8 % sodium hydroxide at 100" for 1.5 hours reduced3sthe sedimentation constant from 86 to 57 S. Moreover, S c h l a m o w i t ~found ~ ~ that the molecular weight of rabbitliver glycogen decreased to about one third of the original value on treatment with 30% potassium hydroxide for 2 hours a t 100". In view of the susceptibility of amylose and starch to alkali in the presence of oxygen,4l digestion should preferably be carried out under anaerobic conditions. In the tissue cells, glycogen is closely associated with protein; indeed it has been suggesteda that two forms of glycogen exist-an insoluble form (desmoglycogen) which is bound to protein by "residual valencies," and a free, soluble form (lyoglycogen) which is readily extracted. Thus, only 27 % of the glycogen content of sclerotia of Phymatotm'chum omnivorum could be extracted with hot water; digestion of the residue with 35 % potassium hydroxide solution yielded the remainder of the glycogen.43More recently, Bloom and coworkers34found that only 55 and 85 % of the glycogen in rat muscle and liver tissues, respectively, was extracted by cold 10 % trichloroacetic acid solution. The experiments of Meyer and J e a n l o ~would ~ ~ suggest that the association between protein and glycogen is of a physical nature, involving entrapping of protein by the glycogen chains, and not a true chemical combination. 5. Estimation

A number of methods are now available4 for the estimation of glycogen in tissues, either involving acid hydrolysis of the glycogen and determina(38) C. T. GreenwoodandD. J. Manners, Proc. Chem. Soc., 26 (1957); sedimentation constants (SX,)are givenin Svedberg units, where S = 1 X 10Fain c.g.s. units. (39) H. Staudiqger, Makromol. Chem., 2, 88 (1948). (40) M. Schlamowitz, J. B i d . Chem., 190, 523 (1951). (41) For example, R. T. Bottle, G. A. Gilbert, C. T. Greenwood and K . N . Saad, Chemistry & Industry, 541 (1953); H. Baum and G. A. Gilbert, ibid., 489 (1954). (42) R. Willstatter and Margarete Rohdewald, Hoppe-Seyler's 2. physiol. Chem., 226,103 (1934); see also E. M. Mystkowski, Biochem. Z.,278,240 (1935). (43) D . R. Ergle, J. Am. Chem. Soc., 69, 2061 (1947). (44) For reviews, see J. van der Vies, Biochem. J. (London), 67, 410 (1954), and

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tion of the D-glucose produced, or measurement of the iodine coloration” or of tissue extracts. For certain biochemical and clinical investigations, special methods for the estimation of microgram quantities of glycogen have been developed.4s 6. Basic Structure Chemical studies reported by Karrer,47 in 1921, indicated that glycogen and starch have closely related structures. Acidic or enzymic hydrolysis gave similar products from both polysaccharides, and, on methylation with methyl sulfate and barium or sodium hydroxide, methyl ethers of similar composition were isolated. Furthermore, both glycogen and starch degraded by acetyl bromide gave “acetobromomaltose” (in about 60 % yield). Details of the chemical structure of glycogen remained unknown, however, until the polysaccharide attracted the attention of Haworth, Hirst, and Bell, and their respective collaborators. In their first investigation on this subject, Haworth, Hirst and Webb@ examined the acetylation and methylation of glycogen. Treatment with acetic anhydride in pyridine, or with sulfur dioxide and chlorine catalysts gave a tri-0-acetyl derivative in almost quantitative yield; on deacetylation with alcoholic potassium hydroxide, a polysaccharide with properties (specific rotation, staining power with iodine) similar to the original glycogen was obtained. Further proof that D-glucose is the sole component monosaccharide was afforded by methanolysis of glycogen triacetate, which gave a 98 % yield of methyl a- and 8-D-glucopyranosides. Earlier attempts by Karrer4’ and by Macbeth and Mackay40to prepare tri-0-methylglycogen (OMe, 45.6 %) by direct methylation of the polysaccharide had not been successful, but gave partially methylated products of OMe 32-37 %. Haworth and coworkers48found, however, that by simultaneous deacetylation and methylation of glycogen triacetate, followed by five or six further methylations, a trimethyl ether (OMe, 43.7) could be isolated in 90 % yield. The preliminary acetylation, during which degradaT. R. Niederland, J. Gvoedjhk and M. Trienov6, Chem. Zvesti, 10,242 (1956); see also A. Kemp and Adrienne J. M. Kits van Heijningen, Biochem. J . (London), 66, 646 (1954). (45) R. G. Hansen, W. J. Rutter and E . M. Craine, J . B i d . Chem., 196, 127 (1952) ; L. Gyermek and G. Fekete, Nature, 176, 386 (1955); Acta Physiol. Acad. Sci. Hung., 8, 259 (1955). (46) N. G. Heatley, Biochena. J . (London), 29, 2568 (1935); 0. Walaas and E v a Walaas, J . Biol. Chem., 187, 769 (1950); Jean Fong, F. L. Schaffer and P. L. Kirk, Arch. Hiochem. and Biophys., 46, 319 (1953). (47) P. Karrer and C. Niigeli, Helv. Chim. Acta, 4, 263 (1921); I?. Karrer, ibid., 4, 994 (1921). (48) W. N. Haworth, E. L. Hirst and J. I. Webb, J . Chem. SOC.,2479 (1929). (49) A. K. Macbeth and J. Mackay, J . Chem. SOC.,126, 1513 (1924).

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tion of the molecule did not occur (see p. 276), thus facilitated etherification. Tri-O-methylglycogen was then hydrolyzed, giving 2 , 3 , B-tri-O-methyl-~glucopyranose in 76 % yield, thereby providing evidence that the D-glucose residues are united by (1 ---f 4) linkages. Further evidence for the presence of continuous chains of ff-D-glucopyranose units in glycogen (and in starch) was obtained by Haworth and Percival,KOwho degraded tri-0-methylglycogen (or tri-0-methylstarch) with acetyl bromide, and isolated from the resulting mixture a disaccharide which, on oxidation and methylation, gave methyl octa-O-methylmaltobionate. Control experiments showed that under similar conditions 2,3,6tri-0-methyl-D-glucose does not undergo resynthesis to a disaccharide. The same authors51 then examined the methanolysis of tri-0-methylglycogen, and from the resulting mixture of methyl D-ghcosides isolated, by fractional distillation, 8.9 % of methyl tetra-0-methyl-D-glucopyranoside. From all the above evidence, it was suggested that glycogen consists of a linear chain of 12 a-(14 4)-linked D-glucose residues. The proposed structure did not, however, explain the observed lack of reducing power of glycogen ; Haworth and Percival6I suggested that, during extraction of glycogen from animal tissues with concentrated alkali, modification of the reducing group occurs. The results of Haworth and Percival were fully substantiated by Bell and associates. In 1935, the preparation and properties of O-aeetyl and O-benzoyl derivatives of rabbit- and fish-liver glycogens were reported.K2 Glycogen regenerated from these acyl derivatives had the same optical rotation, iodine coloration, reducing power, and low phosphorus content as the original polysaccharides. Methylation of the above glyc0gens,6~by simultaneous deacetylation and methylation of their triacetates, yielded trimethyl ethers (OMe, 45.5%); after acid hydrolysis, 9 % of tetra-0methyl-D-glucopyranose could be isolated, together with 2 ,3,B-tri-Omethyl-D-glucose and about 15 % of di-0-methyl-D-glucose. From these findings, it was deduced that both fish- and rabbit-liver glycogen are built up of unbranched chains of 12 D-glucose residues. In the following year, a methylation end-group assay of rabbit-liver glycogen (formed after the ingestion of D-galactose) was performedm; hydrolysis of the trimethyl ether gave 6 % of 2 , 3 , 4 ,6-tetra-O-methyl-D-glucose, corresponding to a chain length of 18 D-glucose residues. Glycogen prepared by the Pfluger technique from the whole tissues of Mytilus edulis was also shownKK to be (50) (51) (52) (53) (54) (55)

W. N. Haworth and E. G. V. Percival, J. Chem. Soc., 1342 (1931). W. N. Haworth and E. G. V. Percivnl, J. Chem. Soe., 2277 (1932). D. J. Bell and H. Kosterlite, Biochem. J. (London), 29, 2027 (1935). D. J. Bell, Biochem. J . (London), 29, 2031 (1935). D. J. Bell, Biochem. J . (London), 30, 1612 (1936). D. J. Bell, Biochem. J . (London), 30, 2144 (1936).

MOLECULAR STRUCTURE OF GLYCOGENS

27 1

composed of 18-unit chains. It was therefore believed that glycogen could exist in two forms, with either 12-unit or 18-unit unbranched chains of a-(1 -+ 4)-linked D-glucose residues. It will be noted that no structural significance was attributed to the presence of di-0-methyl-D-glucose in acid hydrolyeates of tri-0-methylglycogen. Furthermore, attempts to detect glycosidic methyl groups in methylated glycogen were unsuccessful.66 In 1937, two different molecular structures of glycogen were postulated by Haworth and H i r ~ t and , ~ ~by Staudinger,* respectively. The former structure arose from a consideration of methylation data and molecularweight determinations on glycogen. Measurements of the osmotic pressure exerted by certain glycogen derivatives led Carter and Record69to suggest that glycogens have a molecular weight of the order of lo6, equivalent to a degree of polymerization (D. P.) of 3,400-17,000. Glycogen is therefore a highly branched molecule containing several hundred chains of some 12 t o 18 D-glucose residues. The di-0-methyl-D-glucose previously mentioned may have arisen from the branch points; since a proportion of it was shown to be 2,3-di-O-methyl-~-glucose,~~ the inter-chain linkages were believed to involve C6 of the branching residue. Glycogen was accordingly formulated as a singly-branched molecule as shown in Fig. l(a). This “laminated” structure was the simplest molecular structure compatible with the data then available from chemical studies; methylation assay would thus permit determination of the mean length of the chains. An alternative structure, proposed by Staudingerlhsoriginated with the belief that the glycogen molecule is almost spherical. The experimental evidence for this hypothesis, namely that from viscosity determinations, is discussed on p. 276. It was suggested that glycogen is composed of a central chain of up to 100 D-glucose units linked through C l and C4, to which is attached (at C2, C3, and C6 of each unit) a side chain consisting of 12 or 18 a-(1 -+ 4)-linked D-glucose residues (Fig. l(b)). The application of methylation end-group assay to such a polysaccharide would, in effect, allow determination of the length of the side chains. The Staudinger formulation of the glycogen molecule is no longer accepted; more recent physicochemical studies indicate that the molecule is not spherical (see pp. 276-7). Furthermore, methylation and hydrolysis of such a polysaccharide would give D-glucose from the central chain, whilst the origin of the di-0-methyl-D-glucose in the hydrolyzate is not explained.66 A i,hird molecular structure for glycogen was postulated by MeyerG0in 1941; it was based on the methylation assay of glycogen and of the limit~

(56) (57) (58) (59) (60)

D. J. Bell, Biochem. J . (London), 31, 1683 (1937). W. N. Haworth, E. L. Hirst and F. A. Isherwood, J . Chem. Soc., 577 (1937). H. Staudinger and E. Husemann, Ann., 630, 1 (1937). S. R. Carter and B. R. Record, J. Soc. Chem. Ind. (London), 66, 218 (1936). K . H. Meyer and Maria Fuld, Helv. Chim. Acta, 24, 375 (1941).

272

D. J. MANNERS

dextrin remaining after @-amylolysis. @-Amylase catalyzes a stepwise hydrolysis of alternate linkages in a chain of a-(1 -+4)-linked D-glucose 61 Enzyme action commences a t the residues, thereby liberating malt0se.~4~ nonreducing end of the chain and ceases when glucosidic linkages other

R (a)

(C)

FIG.1.-Molecular Structures for Glycogen: (a) Haworth “laminated” form, (b) Staudinger “comb” form, and (c) Meyer “tree” form. Key:-Linear chain of ~ ( -+1 4)-linked D-glucose residues; Inter-chain linkage [1+6-glucosidic in structures (a) and (c); 1 + 2, 1 3, and 1 6-glucosidic in (b)]; A, B, and C are types of chain (see p. 285) and R = free reducing group. -+

--f

than ~ r - ~ -+ ( l 4) are encountered. The action of @-amylaseon glycogen is therefore incomplete and is confined t o the exterior portions of the chains, the products being maltose and a high molecular-weight limit-dextrin (p-dextrin) containing all the inter-chain linkages. A sample of mussel glycogen (C. L. = 11) was treated with wheat ~

(61) C. S. Hanes, New PhytoEogist, 36, 101, 189 (1937).

MOLECULAR STRUCTURE OF GLYCOGENS

273

@-amylase,yielding 47 % of maltose and a /3-dextrin. Methylation and hydrolysis of this dextrin gave 18 % of tetra-0-methyl-D-glucose, corresponding to a chain length of 5.5 D-glucose residues. Since the exterior “stubs” of 0-dextrins were believed to contain one or two D-glucose residues,62 it was concluded that the exterior chains contain 6-7 D-glucose residues, and hence, the interior chains consist of 3 D-glucose units. This finding was interpreted as indicating that glycogen has a compact, multiply-branched “tree” structure as shown in Fig. l(c). Although it is now generally accepted that glycogen has a multiplybranched “tree” structure, the above evidence does not, in fact, constitute proof of multiple branching, and the 0-amylolysis data are equally in accord with a “laminated” formulation. @-Amylolysiseventuates only in a shortening of exterior chains, the number of nonreducing terminal groups in the molecule remaining constant. Hence, if 50% of the molecule, whether of a laminated, tree, or comb-type structure, is removed by P-amylase, the relative proportion of end groups in the residual &limit dextrin must be doubled. In parallel studies on amylopectin, evidence for multiple branching was deduced from the results of a stepwise degradation.62Treatment of amylopectin with @-amylasegave 55 % of maltose and 45 % of limit dextrin (Dextrin I). On incubation of Dextrin I with yeast “maltase” preparation, slow degradation to D-glucose (17%) and Dextrin I1 occurred; the latter was now susceptible to further attack by &amylase. Dextrin I1 thus yielded maltose and a second 0-dextrin (Dextrin 111) which gave a red-brown coloration with iodine. These findings were stated to be explicable only by a multiply-branched structure. If amylopectin had a “comb”-type structure it was claimed that Dextrin I1 would consist of a linear chain of D-glucose residues which would give a blue coloration with iodine, and be completely saccharified by P-amylase. Two years later, it was stated63that a proportion of the inter-chain linkages of glycogen P-dextrin are degraded by a yeast-enzyme preparation. The close structural relationship between glycogen and amylopectin was thereby confirmed, although conclusive proof of multiple branching in glycogen had not been obtained. It must be noted that the yeast preparation was heterogeneous, and that “debranching” was believed to be due to a phosphorylase. This view is now known to be incorrect; yeast extracts contain an enzyme (isoamylase) which can hydrolyze WD-(1 + 6)-glucosidic linkages.” (62) K . H. Meyer and P. Bernfeld, Helv. Chim. Acta, 23, 875 (1940). (63) K. H . Meyer and P. Bernfeld, Helv. Chim. Acta, 26, 399 (1942). (64) D. J. Manners and Khin Maung, Chemistry & Industry, 950 (1955).

274

D. J. MANNERS

Recent developments in the structural chemistry of glycogens are described in later Sections of this article, which is intended to supplement those published in 1943 by Meyer,66 and. in 1948 by

11. PHYSICOCHEMICAL PROPERTIES 1. Molecular Weight

Although estimates of the molecular weight of several glycogens have been published, only a brief discussion will be presented here, since many of the results have already been reviewed.21 The physicochemical methods employed include measurements of osmotic pressure, giving number-average values, and of the viscosity, sedimentation, and diffusion of glycogen and derivatives in various solvents. The latter methods provide weight-average values, which, for a polymolecular system, are larger than number-average molecular weights.21 The homogeneity and particle size of glycogen have also been studied by electrophoresisfi7and ultramicroscopy,68respectively. More recently, two forms of molecular-weight light-scattering technique have been used ;in the values ( M ) are calculated from a reduced form of the Rayleigh equation in which

M

=

rSp/K

where rapis the specific turbidity (that is, the absolute turbidity of a solution containing 1 g. of glycogen per liter) and K is a constant, dependent on the wave-length of the incident light. The value of K is determined by using either glycogen39or amandinGg of known molecular weight. Alternatively, light-scattering may be considered as a problem in fluctuation theory, and results are then evaluated from the equation7O

HC/r

=

(1/M)

+ 2BC

where H and B are constants, and C is the concentration of glycogen. By this method, six glycogens had molecular weights” in the range (3-15) X 106. Chemical methods of molecular-weight determination are based upon (65) K. H. Meyer, Advances in Enzymol., 3, 109 (1943). (66) D. J. Bell, Biol. Revs. Cambridge Phil. SOC.,23, 256 (1948). (67) D. H. Northcote, Biochem. J . (London), 68, 353 (1954). (68) E. Husernann and H. Ruska, Naturwissenschaften, 28, 534 (1940); J. prakt. Chem., 166, 1 (1940). (69) P. Putzeys and L. Verhoeven, Proc. Intern. Colloq. Macromolecules, Amsterdam, 267 (1949). (70) P. Debye, J . Appl. Phys., 16, 338 (1944); J . Phys. & Colloid Chem., 61, 18 (1947). (71) B. S. Harrap and r). J. Manners, Nature, 170, 419 (1952).

MOLECULAR STRUCTURE OF GLYCOGENS

275

measurements of the reducing power of glycogens (which are assumed to contain one reducing group per molecule) with alkaline 3,5-dinitrosalicylic acid72or with f e r r i ~ y a n i d ereagent. ~~ The results obtained are lower than those from physicochemical methods; protozoal and yeast glycogen ( M = (2-3) X lo6 from sedimentation data) gave M = (1-2) X 106 with the dinitrosalicylic acid reagent.74 These discrepancies are due to alkalineoxidative degradation of the Evidence that glycogen has a high molecular weight was published in 1923. Samec and Isaievic found that a sample of dog-liver glycogen had a molecular weight of 1 .I x lo5, from osmotic-pressure measurements.76 TABLEI Molecular Weight Determinations on Glycogens Sample

Rabbit liver Fish liver Dog liver Rabbit liver Rabbit muscle Guinea-pig liver Cat liver Commercial

Derivalive

unsubstituted acetate acetate unsubstituted unsubstituted unsubstituted unsubstituted p-iodobenzoate

Methoda of meawrement

Mean molecular weights (x 10-6)

References

0.P. 0. P. 0. P. S. D. s. D. L. s. L. s. U. M.

1.2-2.3 I . 3-3.5 1.5 3.9-13.9 2.6 3.7-7.6 10.0 3-6

77 78 73 37 79 39 71 68

a 0. 1’. = osmotic pressure; S. D. = sedimentation and diffusion; L . S. = light scattering; U. M. = ultramicroscopy.

This observation appears t o have been overlooked by many workers. A few results reported since 1936 are summarized in Table I. Essentially similar results have been obtained38by ultracentrifuge measurements on 20 samples of glycogen, all of which were polymolecular; the molecular weights varied between (2-6) X lo6, six of the preparations being polydisperse. Polglase and coworkerssolikewise found samples of human glycogen to be (72) (73) (1951). (74) Liddle

:K. H. Meyer, G. Noelting and P. Bernfeld, Helv. Chim. Acta, 31, 103 (1948). R . W. Kerr, F. C. Cleveland and W. J. Katzbeck, J . A m . Chem. Soc., 73, 111

11. J. Manners, A. R . Archibald, I. D. Fleming, I. G. Jones, A. Margaret and Khin Maung, unpublished observations. (75) R. T. Bottle and G. A . Gilbert, Chemistry & Industry, 1201 (1954). (76) M. Samec and V. Isajevic, Compt. rend., 176, 1419 (1923). (77) H. B. Oakley and F. G. Young, Biochem. J . (London), 30, 868 (1936). (78) S.R . Carter and B. R. Record, J. Chem. Soc., 664 (1939). (79) D. J. Bell, H. Gutfreund, R. Cecil and A. G. Ogston, Biochem. J. (London), 42, 405 (1948). (80) W. J. Polglase, D. M. Brown and E. L. Smith, J . Biol. Chem., 199, 105 (1952).

276

D. J. MANNERS

polydisperse; both liver glycogen and muscle glycogen contained two components. Examination of sedimentation diagrams has 79 that glycogens are extremely polymolecular, and glycogen preparations can be fractionated. Guinea-pig liver glycogen (mean molecular weight, 3.7 X lo6), on fractional precipitation with methanol, gave39 fractions with molecular weights of 19.6, 6.8,2.2, 1.7, 1.1, and 0.9 X lo6,whilst Ascaris lumbricoides glycogen ( M = 8.8 x lo6) contains a fraction, 3 % by weight, with71 M = 22.5 X lo6. Glycogen can be acetylated (by means of acetic anhydride and pyridine) without appreciable degradation; this is important, since acetylation is usually a preliminary to methylation. Glycogen samples (D. P., 410, 1,750, and 5,090, by osmometry) gave acetates with D. P. 390, 1,680, and 5,300, respectively; on deacetylation, the regenerated glycogens hads8 D. P. 410, 1,730, and 5,350. To summarize, physicochemical measurements show that glycogens have molecular weights of -lo7, and are therefore amongst the largest of natural polymers. 2. Molecular Shape The majority of measurements of the molecular shape of glycogens indicate that the molecules are asymmetric, although Staudinger and Husemann68had suggested that glycogen molecules were spherical. They found that the specific viscosity of three glycogens (D. P. 410, 1,750, and 5,000) in various solvents was the same; the specific viscosity of a series of spherical polymers is independent of molecular weight. The glycogens used (commercial preparations) had relatively low molecular weights (-106) ; similar viscosity-molecular-weight relationships do not for glycogens with molecular weights of (2-6) X lo6. Later viscometric observations have shown that glycogen molecules deviate from spherical form, and also vary in shape. Ascaris lumbricoides glycogen ( M = 7 x lo6) has a higher specific viscosity than have mammalian glycogens of molecular weight7g(2-4) X lo6. Baker's-yeast glycogenal has a greater viscosity than rabbit-liver glycogen ; measurements on the yeast glycogen indicate an axial ratio of 1:8 or 1:13 for a prolate or oblate ellipsoid, respectively. The axial ratio of tubercle-bacillus glycogen is about 1 :10 (from viscosity and diffusion measurements), or 1 :11 (from sedimentation studiess2). Further evidence that glycogen molecules are elongated has been ob~

(81) D. H. Northcote, Biochem. J . (London), 63, 348 (1953). (82) E. Chargaff and D. H. Moore, J . Biol. Chem., 166, 493 (1944).

MOLECULAR STRUCTURE O F GLYCOGENS

277

tained from ultracentrifuge measurements. The frictional ratios of various samples of r a b b i t - l i ~ e rand ~ ~ methylated, fish-liverB3glycogens were 1.8-2.8 and 1.7-2.1, respectively. According to Bridgman,37a frictional ratio of 1.9 “corresponds to an ellipsoid of revolution having an axis ratio of 1 t o 18 for a prolate ellipsoid or a ratio of 1 to 25 for an oblate ellipsoid.” It must be noted, however, that the observed frictional ratios are due to the combined effects of molecular asymmetry and hydration.

mP

FIG. 2.-Absorption Spectra of Polysaccharide-Iodine Complexes. (I) Mytilus edulis glycogen, (11) rabbit liver glycogen, (111) waxy-maize starch (amylopectin). [Solutions contained 0.01% of polysaccharide and 0.02% of iodine in 0.2% of potassium iodide, and were read against a n iodine-potassium iodide reference solution.]

Thus, glycogens from different sources differ not only in molecular weight, but also in degree of molecular asymmetry.

3. Interaction with Iodine

a. Absorption Spectrum.-The absorption spectra of the iodine complexes of several samples of glycogen have been measured74;in Fig. 2, the spectra of iodine complexes of rabbit-liver glycogen, Mytilus edulis glycogen, and amylopectin are compared. (83) B. R. Record, J. Chew&.SOC.,1567 (1948).

278

D. J. MANNERS

The absorption maximum of the iodine complex of short, linear a-~-(l+ 4)-glucans appears to be related to the chain lengths4;for branched a - ~ - (4 l 4)-glucans, there are indications of a similar relationship, although

neither the average nor exterior chain length is the sole factor (see Table 11).Thus, rabbit-liver glycogen and muscle glycogen = 12-13) form iodine complexes having different absorption spectra. The optical density a t the wavelength of maximum absorption also increases with average chainlength. In contrast with the above spectra (measured in aqueous iodine-potassium iodide), Schlamowitz40 found that, in the presence of half-saturated

(m

TABLEI1 Wavelength of M a x i m u m Absorption of Glycogen-Iodine Complexes

c. L.

A pproximale exterior chain

13 6 7 13 13 14 13 18 10-11 21

8-9 3 4-5 8 8 8-9 8-9 12 7-8 15

~

Sample

References

lengthb

-\

Mytilus edulis Human liver0 Helix pomatia Tetrahymena pyriforniis Rabbit liver Human livere Rabbit muscle Rabbit liver Bacillus megatherium Human liver"

420 430 430 440 460 470 490 490 520 530

74 74 74 74 74 85 74 74 86 85

Glycogen-storage disease samples; d a t a on normal, human-liver glycogen is not available. Calculated from and the 8-amylolysis or phosphorolysis limit (see Section IV).

m.

ammonium sulfate, rabbit-liver glycogens of different chain-lengths and molecular weights had a similar absorption maximum a t 490-500 mp. At this maximum, the optical density increased with apparent chain-length. In general, little information on the absorption spectra of iodine complexes of glycogens of known molecular structure is available, and attempts to relate spectra with details of fine structure appear to be premature. b. Iodine-binding Power.-Measurements of the iodine-binding power of glycogen by potentiometric titration have shown that iodine has a much (84) Marjorie A. Swanson, J. Biol. Chem., 173, 825 (1948); W. J. Whelan and J. M. Bailey, Biochem. J. (London), 68, 560 (1954). (85) Barbara Illingworth and Gerty T. Cori, J . Biol. Chem., 199, 653 (1952). (86) C. Barry, R . Gavard, G. Milhaud and J. P. Aubert, Ann. inst. Pasteur, 84, 605 (1953).

MOLECULAR STRUCTURE O F GLYCOGENS

279

lower s h i t y for glycogen than for starch cornp~nents.~T For accurate measurements, a differential, potentiometric titration method must be used, and by this techniquess the uptake of iodine by glycogen has been studied. The slopes of potentiometric titration curves show that the iodine-binding power of glycogen (C. L. = 12-13) is one quarter that of %unit glycogen, and about one tenth that of amylopectins (C. L. = 20-23). The interaction of iodine and amylose involves formation of inclusion complexes in which iodine molecules are arranged, endwise and axially, inside a series of helices of a-(1 .+ 4)-linked D-glucose residues; each helix of 6 D-glucose residues contains one iodine molecule.89 With branched L Y - D - ( ~+ 4)-glucans, similar complex formation, limited t o the exterior chains, probably occurs. Glycogen (C. L. = 12) hasz3an average, exteriorchain length of 8 ; only a proportion of the exterior chains will contain a helix of six D-glucose residues and form an inclusion complex with iodine. Amylopectins (average, exterior chain-length, 13-18) have a relatively greater iodine-binding power since every exterior chain comprises 2-3 helices which will form complexes with iodine. Under the above conditions, with very low iodine concentrations, adsorption effects would be negligible.ss There appears to be an approximate relationship between the iodine binding power of branched ( Y - D - (+ ~ 4)-glucans and the exterior chain lengthR9";evidence in support of the suggestionss that it is related to the degree of multiple branching is not available.

4. Interaction with Proteins In solution, glycogens interact with certain proteins (for example, serum albumin and globulin, and myosin) to form complexes which may be examined by such physicochemical methods as ultracentrifugal analysis,g0 nej~helometry,~'ultraviolet sp ectro p h ~ to me tr y ,~ and ~ electroph~resis.~~ Since glycogens have a higher a f i i t y for myosin than 4-dextrin (muscle (87) F. L. Bates, I).French and R . E. Rundle, J . A m . Chem. SOC.,66, 142 (1943). (88) 11. M. W. Anderson and C. T. Greenwood, J . Chem. Soc., 3016 (1955). (89) R. E. Rundle and I).French, J . A m . Chem. SOC.,66, 1707 (1943); R . R. Baldwin, R . s. Bear and R. E. Rundle, ibid., 66, 111 (1944). (89a) However, certain Zea mays polysaccharides = 12-13, exterior chainlength, 8-9) bind about three times more iodine than animal glycogens of similar branching characteris tics .a8 (90) E. M. Mystkowski, Biochem. J . (London), 31, 716 (1937). (91) S. J. von Przylecki, H . Andrzejewski and E. M. Mystkowski, KoZZoid-Z., 71, 325 (1935). (92) E. L. Rozenfel'd and E. G. Plyshevskaya, Biokhimiya, 19, 161 (1954); Chem. Abstracts, 48, 9423 (1954). (93) T. T. Bolotina and E. L. Roaenfel'd, Doklady Akad. Nauk S . S.S . R., 87,643 (1952); Chem. Abstracts, 47, 6461 (1953).

(a

280

D. J. MANNERS

phosphorylase limit-dextrin) or P - d e ~ t r i ncomplex ,~~ formation mainly involves combination of protein with exterior chains of glycogen; the nature of the linkages has not been clearly established. An unusual glycogen-protein interaction has been investigated recently.94 Addition of concanavalin-A, a globulin from jack-bean meal, to a solution of glycogen results in the formation of an insoluble complex. This interaction is most marked with short-chain glycogens and glycogen @-limitdextrins; amylopectin gives no reaction. Accordingly, concanavalin-A has been used for the identification and estimation of “glycogen” from various biological sources.

111. STRUCTURAL ANALYSIS BY CHEMICAL METHODS 1. End-group Assay Chemical determinations of nonreducing end-groups are based on methylation or periodate oxidation studies.96 a. Methylation Studies.-In these, acid hydrolyzates of gram quantities of methylated glycogen are analyzed for tetra-0-methyl-D-glucopyranose, which originates only from nonreducing terminal groups. Analysis of mixed methyl ethers of D-glucose was formerly done through fractional distillation96 of the methyl glucosides61,67 or by chloroform-water extraction of tetra-0-methyl-D-glucopyranose from the remaining More recently, chromatographic methods have been used. b. Periodate Oxidation Studies.-To a large extent, methylation endgroup assay of amylosaccharides can be replaced by simpler decigramscale methods involving periodate oxidation. In these procedures, the formic acid which arises only from the nonreducing terminal groups of the amylosaccharide is determined, for example, by titration with sodium hydroxide or barium hydroxide. Originallylg7sparingly soluble potassium metaperiodate at 15” was used as oxidant (see also ref. 23); modifications using sodium metaperiodate at temperatures ranging from 2-20’ for 1-7 days have since been d e v i ~ e d . ~ ~ - ~ ~ ~ (94) J. A. Cifonelli, R. Montgomery and F. Smith, J . Am. Chem. Sac., 78,2485 (1956). which repre(95) Results are usually expressed as average chain lengths sent the number of D-glucose residues per end-group. Individual chains i n glycogen molecules vary considerably in length, probably from 620 D-glucose residues. (96) For experimental details see (a) W. N. Haworth and H. Machemer, J. Chem. Soc., 2270 (1932); (b) E. L. Hirst and G. T. Young, ibid., 1247 (1938); (c) I. Levi, W. L. Hawkins and H. Hibbert, J. Am. Chem. Sue., 64, 1957 (1942); (d) J. S. D. Bacon, E, Baldwin and D. J. Bell, Biochem. J. (London), 38, 198 (1944). (97) T. G. Halsall, E. L. Hirst and J. K. N . Jones, J . Chem. Sac., 1399 (1947). (98) Under the conditions siiggested by A . L. Potter and W. Z . Hassid, J . Am.. Chem. SOC.,70, 3488 (1948), oxidation is incomplete, giving high values values (see ref. 101); chain lengths of 18-23 thus assessed [M. Schlamowitz, J . Biol. Chem., 188, 145 (1951)l in fact, represent 15-17-unit glycogens.

(m),

28 1

MOLECULAR STRUCTURE OF GLYCOGENS

More than 80 different samples of glycogen have now been assayed by periodate oxidation,108and of these, 70 had C. L. values of 10-14.23f979 9 9 , l o o , lol Many results agree with those from methylation assays of the same samples. TABLE111 End-group Assay of Glycogens by Methylation Source of glycogen

Ascaris lumbricoides Dog liver Helix pomatia Horse liver Horse muscle Rabbit liver (D-fructose fed) Rabbit liver (sucrose fed) Rabbit liver Rabbit liver Rabbit muscle

Method of reparation of methylaled sugarP

S . G. P. P. D. S . G. 1).

D. A. Q. P. S. G.

C.L.

References

15 12 11-12 18 12 11-12 18-19 18-19 11-12 11

103 104 105 106 103 96(d) 96(d) 97 107 108

A. Light petroleum-water partition of methyl glucosides. D. Fractional diutillation of methyl glucosides. P. Chloroform-water partition of methylated sugars. Q. P. Quantitative paper chromatography of methylated sugars. S. G. Partition chromatography of methylated sugars on silica gel. 5

2. Characterization of Inter-chain Linkages a. Methylation Studies.-On hydrolysis, a fully methylated, branched a-D-(1 -+ 4)-glucan should give a mixture of 2,3,4,6-tetra-, 2,3,6-tri-, and one or more di-0-methyl-D-glucoses. Since the last fraction, theoretically equimolar with the tetra-0-methyl-D-glucopyranose, arises from the branch points, its characterization would identify the inter-chain linkage. ~~

(99) K. H. Meyer and P. Rathgeb, Helv. Chim. A d a , 31, 1540, 1545 (1948); M. Morrison, A. C. Kuyper and J. M. Orten, J. A m . Chem. Soc., 7 6 , 1502 (1953). (100) M. Abdel-Akher and F. Smith, J. A m . Chem. SOC.,73, 994 (1951). (101) D. J. Manners and A. R. Archibald, J . Chem. Soc., 2205 (1957). (102) J. M. Bobbitt, Advances i n Carbohydrate Chem., 11, 1 (1956). (102%)By an alternative, periodate oxidation assay [M. Abdel-Akher, J. K. Hamilton, R. Montgomery and F. Smith, J . Am. Chem. SOC.,74, 4970 (1952)], a glycogen ( T L . = 12, by methylation) had a chain length of 11. (103) D. J. Bell, J . Chem. Soc., 473 (1944). (104) W . 2. Hassid and I. L. Chaikoff, J. Biol. Chem., l!A3,755 (1938). (105) E.Baldwin and D. J. Bell, Biochem. J . (London), 34, 139 (1940). (106) F. A. Isherwood, Ph.D. Thesis, Birmingham, Engl., 1936. (107) E. L. Hirst, L. Hough and J. K. N. Jones, J . Chem. SOC.,928 (1949); see also J. K. Bartlett, L . Hough and J. K. N. Jones, Chemistry & Industry, 76 (1951). (108) D. J. Bell, J. Chem. Soc., 992 (1948).

282

D. J. MANNERS

The liydrolyzate of methylated, 18-unit1rabbit-liver glycogen contained a proportion of 2,3-di-O-methyl-~-gIucose~~; Haworth and coworkers therefore concluded that the inter-chain linkage was probably (1 + 6). Several years the di-0-methyl sugars from methylated rabbit-liver and muscle glycogens were analyzed by a periodate oxidation procedure, 2,6-Di-O-methyl-~-glucosewas the main component, providing tentative evidence for (1 + 3) inter-chain linkages; the 2,3- and 2,6-isomers were also present . Experimentally, it seems to be impossible t o methylate glycogen completely, despite repeated treatment with various methylating reagents.lo8 Consequently, hydrolysis of methylated, 12-unit glycogens yields tetra-, tri-, and di-0-methyl-D-glucose in the molecular ratio of approximately 1:9:2, instead of 1: 10: 1 as expected.s3*b6 In addition, during acid hydrolysis, appreciable demethylation of tri-0-methyl-D-glucose can occur, yielding di-0-methyl-D-glucoses.108Undermethylation and hydrolytic demethylation therefore give rise t o di-0-methyl-D-glucose which cannot be differentiated from that arising from the branch point. Thus, paper-chromatographic ana1ysislo7of hydrolyzed, methylated, rabbit-liver glycogen gave 8.7 % of tetra-, 69.0 % of tri-, 8.9 % of 2,3-di, 10.8 % of 3 ,6-di-, and 2.4 % of mono-0methyl-D-glucose. Despite these difficulties, methylation was, until 1948, the only method available for characterization of inter-chain linkages. b. Acid Hydrolysis Studies.-Partial hydrolysis of glycogen yields a mixture of sugars; apart from D-glucose, maltose and maltotriose will arise from linear portions of the chains. Sugars other than these maltosaccharides will contain unhydrolyzed branch points, and analysis of such sugars will identify the inter-chain linkages. After partial (75 %), acid hydrolysis of 5 g. of rabbit-liver glycogen, chromatography of the acetylated sugars yielded 92 mg. of P-isomaltose octaacetatelog;none was formed by “acid reversion” during a similar treatment of amylose. The isolation of a derivative of isomaltose provides evidence for C Z - D - ( ~3 6)-glucosidic linkages in the polysaccharide. Isomaltose (164 mg.) has been obtained18 by carbon-Celite chromatography of a (neutralized) partial, acid hydrolyzate of 4 g. of rabbit-liver glycogen. In control experiments, D-glucose and maltose gave much less isomaltose by “acid reversion.” It was concluded “that the isomaltose structure is an integral part of the glycogen molecule.” Peat and have similarly characterized the inter-chain linkages in baker’s-yeast glycogen. D-Glucose, maltose, isomaltose, and (109) M. L. Wolfrom, E. N. Lassettre and A. N. O’Neill, J. Am. Chem. SOC.,73, 595 (1951).

(110) S. Peat, W. J. Whelan and T. E. Edwards, J . Chem. Soc., 355 (1955).

MOLECULAR STRUCTURE OF GLYCOGENS

283

panose (4-0-cr-isomaltosyl-~-glucose)were isolated from a partial hydrolyzate, thus showing the presence of (Y-D-(~ -+ 6) inter-chain linkages. I n addition t o isomaltose, isomaltotriose (about 0.7 %) and nigerose (about 0.001 %) have been isolated from partial, acid hydrolyzates of beefliver glycogen.110aA small proportion of the branch points are therefore directly joined t o a n adjacent branch point by a n CY-D-(~ * 6) glucosidic linkage, and a very small proportion of ( Y - D --+ ( ~ 3) linkages may also occur. Partial, acid hydrolyses therefore show that glycogens contain both CY-D-(~--+ 4) and (Y-D-(~ + 6) as the principal glucosidic linkages; however, small numbers of other linkages may also be present. c. Periodate Oxidation Studies.-A method for the detection of (1 + 2 ) and (1 -+ 3)-linkages in amylosaccharides has been developed by Hirst and 112 In a chain of aldopyranose residues, every residue except those substituted at C2 or C3 will be oxidized by periodate. Assuming complete oxidation, the finding of D-glucose in an acid hydrolyzate of the oxidized polysaccharide indicates that it originally contained (1 + 2) or (1 + 3) linkages. I n an application of this method to a glycogen (source unspecified), 6.0 mg. of periodate-oxidized glycogen yielded 0.016 mg. of D-glucose, showing that, a t the most, only 2-3% of the inter-chain linkages could involve C2 or C3.113 This n-glucose might have arisen from incomplete oxidation of the glycogen. In similar studies, glycogens (of cat liver, Helix pomatia, Mytilus edulis, and Tetrahymena pyriformis) after prolonged periodate oxidation, were hydrolyzed with The hydrolyzate of Helix glycogen yielded a trace of D-glucose, equivalent to < 1% of (1 + 2 ) or (1 + 3) linkages; the other hydrolyzates did not contain D-glucose. Acid hydrolyzates of periodateoxidized glycogens (brewer's human liver22)and oxidized a-dextrins from fetal-sheep liver,114rabbit and Ascaris lumbricoides glycogens,l14 likewise gave no evidence of (1 -+ 2) or (1 + 3) inter-chain linkages. The acid hydrolyzate of a periodate-oxidized glycogen of unspecified origin, and of the derived polyalcohol, contained 1% of D-glucose.102aAlthough it was suggested that this arose from (I + 2 ) or (1 +. 3) linkages, the (110a) M. L. Wolfrom and A . Thompson, J . Am. Chem. SOC.,78, 4182 (1956); 79, 4214 (1957). (111) T. G. Halsall, E. L. Hirst, J. K. N. Jones and A. Roudier, Nature, 160, 899 (1947). (112) E. L. Hirst, J. K. N. Jones and A . Roudier, J. Chem. SOC.,1779 (1948). (113) G. C. Gibbons and R. A. Boissonnas, Helv. China. Acta, 33, 1477 (1950). (114) D. J. Bell and D. J. Manners, J. Chem. SOC.,1891 (1954). (115) I>. J. Manners and Khin Maung, J. Chem. SOL,867 (1955).

284

D. J. MANNERS

possibility of incomplete oxidation remains, and, without additional information, the significance of this fmding is uncertain. Periodate-oxidation studies have thus shown that, in several glycogens, over 99 % of the inter-cha.inlinkages are (1 -+ 6), and experiments employing partial hydrolysis by acid indicate that these linkages have an a-D-configuration.

IV. STRUCTURAL ANALYSISBY ENZYMIC METHODS Glycogens are attacked by three known groups of enzymes; amylases and phosphorylases degrade a - ~ 1- (+ 4)-linkages, whilst a - ~ - ( l-+ 6) inter-chain linkages are hydrolyzed by “debranching” enzymes?** 117 In contrast with P-amylases (see p. 272), a-amylases catalyze random l 4) linkages in both exterior and interior chains of hydrolysis of a - ~ - ( + glycogens, giving maltose as the main end-product. ___Action of salivary a-amylase also yields maltotriose and a-dextrins (D. P. usually 5-8, containing one or more a - ~ - (+ l 6 ) linkages) as end products. Other a-amylases, from malt and Aspergillus oryzaez hydrolyze maltotriose so that glycogen breakdown fmally gives maltose, D-glucose, and a-dextrins. The rate of a-amylolysis of glycogen is lower than that of amylose or amylopectin since the enzymes have a much lower affinity for glycogen than for the starch components.”* Pho~phorylases,~~7 in the presence of inorganic phosphate, remove D-glucose residues from the exterior chains of glycogen, according to the equation

[GI,

+ m H0.P

--+ [GI,-m

+ m (2.1-P

where [GI, or represents a chain of n or (n - m) residues, and HO-P and G l-P represent inorganic phosphate and a-D-glucosyl phosphate. Phosphorylases cannot bypass inter-chain linkages. The affinity of phosphorylase for glycogen depends upon the enzyme source. YeastlrSand muscle120phosphorylases readily attack glycogens (and amylopectins) yielding 30-50 % of D-glucosyl phosphate. By contrast, potato phosphorylase, under similar conditions, gives about 10 and 40 % of D-glucosyl phosphate from glycogen and amylopectin, respectively.121Muscle phosphorylase does not

.

(116) W. J. Whelan, Biochem. SOC.Symposia (Cambridge, Engl.), 11, 17 (1953). (117) D. J. Manners, Ann. Repts. on Progr. Chem. (Chem. Soo. London), 60. 288 (1954); Quart. Revs. (London), 9, 73 (1965). (118) S. Schwimmer, J. Biol. Chem., 186, 181 (1950); Virginia M. Hanrahan and Mary L. Caldwell, J . Am. Chem. SOC.,76, 2191 (1953). (119) Khin Maung, Ph.D. Thesis, Edinburgh, Scotland, 1956. (120) Gerty T. Cori and J. Larner, J . Biol.Chem., 188, 17 (1951); Barbara Illingworth, J. Larner and Gerty T. Cori, ibid., 199,631 (1952). (121) A. Margaret Liddle, Ph.D. Thesis, Edinburgh, Scotland, 1956; A. Margaret Liddle and D. J. Manners, J. Chem. Soc., in press (1957).

MOLECULAR STRUCTURE OF GLYCOGENS

285

degrade all exterior chains to the same extent.lz0In the singly-branched “laminated” and multiply-branched “tree” structures for glycogen (and amylopectin) (see Fig. l), three different types of chain may be distinguishedln: A-chain (side chain), attached by a single (1 + 6) linkage from the reducing group; B-chain (main chain) to which one or more A-chains are linked and itself attached by the reducing group to an adjacent chain; and C-chain, to which other chains are linked, and which is probably terminated by a free reducing group. [In a molecule consisting of n chains, the the ratio of A-:B-chains (A. B.) for a “laminated” structure is 1:(n - 2); a ‘%ree”structure contains equal numbers of A- and B-chains.] In a glycogen (or amylopectin) muscle phosphorylase limit-dextrin (+dextrin), the A- and B-chain stubs contain one and 6-7 D-glucose residues, respectively.lZ0 Several “debranching” enzymes have now been discovered. R-enzyme (from higher plants) hydrolyzes (1 -+ 6)-linkages in glycogen a-dextrins, although it has no action on g1ycogen.ll6Amylo-(1 4 6)-glucosidase (from rabbit muscle) also does not attack glycogen, but will remove the A-chain stubs of a +dextrin as D-glucose.120 In contrast, yeast isoamylase hydrolyzes (1 + 6) linkages in glycogen, a-dextrin, and ~ # ~ d e x t r i n . ~ ~ The action patterns of the above enzymes were determined by using the starch components as substrates; with certain reservations, these enzymes may he used for studying the fine structure of glycogen. ~

1. End-group Assay

Glycogen-type polymers contain, in effect, equal numbers of nonreducing end-groups and (1 4 6) inter-chain linkages’23; determination of the proportion of either will enable the average chain-length to be calculated. In contrast to chemical methods, the enzymic methods of end-group assay measure the proportion of (1 -+ 6) linkages; two enzymes are required, one specific for a - ~ - ( -+ l 4)-linkages, and a debranching enzyme to hydrolyze the small proportion (5-10 %) of a - ~ - (--+ l 6)-glucosidic linkages. In the method of Cori and Larner,lz0glycogen (or amylopectin) is completely digested by the concurrent action of muscle phosphorylase and amylo-(1 -+ 6)-glucosidase.D-Glucose (which arises only from residues attached to C6 of an adjacent residue) and D-glucosyl phosphate (which is obtained from all other residues) are determined, and the branch-point content is calculated from the proportion of ~ - g l u c o s e . Only l ~ ~ 10-15 mg. of glycogen is required for each assay; the method has been applied to more (122) S. Peat, W . J. Whelan and Gwen J. Thomas, J. Chem. Soc., 4546 (1952). (123) A molecule with n inter-chain linkages has (n 1) end-groups; for glycogen, n may be 2,000. (124) In a glycogen containing 8.0% of branch-points (that is, end-groups), the average chain length C (L ). is 100/8.0 = 12.5.

+

286

I).

3. MANNERS

than 50 glycogens, 36 of which had chain lengthss6,120.126 of 10-14. Many of the results are in good agreement with those from assays of the same Samples by methylation or by potassium periodate oxidation. Whelan and Roberts have devised an alternative method involving the successive action of salivary a-amylase and R-enzyme on glycogen.ll6SIz6 By determining the number of reducing groups produced by action of Renzyme on the a-dextrins, the __ proportion of (1 + 6) linkages can be calculated. Rabbit-liver glycogen (C. L., 13.6, by periodate oxidation) had a chain length of 12.5 by this method. 2. Determination of Exterior and Interior Chain Lengths

p-Amylolysis of glycogen produces maltose and a high-molecular-weight dextrin (p-dextrin) with exterior chains consisting of perhaps two or three D-glucose resid~es.2~3 24, 61, 117 The exterior-chain length can therefore be calculated from the average chain-length and the @-amylolysislimit. Using crystalline, sweet-potato @-amylase, glycogens ( C T . 12 f 1) hadz3 @-amylolysislimits of 45 f 4%. The average, exterior and interior chainlengths are therefore about 8 and 3 D-glucose residues, respectively, assuming that the exterior chain stubs average 2.5 residues.127 Several results are given in Table IV; in general, the length of the exterior chains is roughly twice that of interior chains. Exterior chain lengths cannot be measured by purely chemical methods. 3. Evidence of Random Branching

Enzymic studies have shown that the interior structure of glycogens is randomly branched. For example, on @-amylolysis,the exterior chains of rabbit-muscle glycogen are shortened by 5.4 residues, although maltose is the only sugar produced by enzyme aeti0n.2~This result must represent a statistical average of the loss of even numbers of D-glucose residues from individual chains of different, exterior lengths. Furthermore, although the mean, interior, chain length of a 12-unit glycogen is 3-4, there is evidence that a proportion of the interior chains contain 7-8 residues. &-Amylases hydrolyze interior chains in glycogen and its @-dextrin,glucosidic linkages in the middle of these chains being most readily hydrolyzed. Some of the a-dextrins so produced contain exterior chains of about 4 D-glucose residues, as they are partially degraded by @-amylasez3and show priming ac(125) Barbara Illingworth, Gerty T. Cori and C. F. Cori, J. B i d . Chem., 218, 123 (1956). (126) W. J. Whelan and P. J. P. Roberts, Nature, 170, 748 (1952). (127) In ref. 23, exterior-chain lengths were calculated on the assumption that the stubs contained 1.5 residues (see refs. 60, 62, and 65); more recent evidence suggests that the stubs contain two or three residues.

287

MOLECULAR STRUCTURE OF GLYCOGENS

tivity toward potato phosphorylase.'28 Hence, the original interior chains must contain about 8 residues. A study of the absorption spectrum of the iodine complex of the p-dextrin from glycogen also led SwansonB4to suggest that glycogen contains some interior chains of 8 D-glucose residues. From the mean, interior chain length of 3-4, and the above evidence, it follows that a number of interior chains must comprise only 1-2 residues. Evidence in favor of this hypothesis has been obtained, during studies of R-enzyme action on glycogen a-dextrins,lZ6which shows that a few a-dextrins contain two branch points which are separated by only one D-glucose residue. TABLEIV Determination of Exterior and Interior Chain Lengths of Glycogens Sample

Cat liver IV Cat liver VI Human muscle I1 Mytilus edulis I Mytiliis edulis V Mytilws edulis VI Rabbit, liver IV Rabbit, liver V Rabbit liver VI Rabbit, muscle I1 Yeast (baker's) Yeast (brewer's)

-

c. L.

I-dmylolysii limit, % '

Exlerior chain length"

13 12 11 12 9 13 13 14 18 11 12 13

54 52 40 43 40 46 45 51 53 39 50 44

9-10 8-9 7 7-8 6 8-9

8-9 9-10 12 6-7 8-9 8

a Number of D-glucose residues removed by P-amylase, length - exterior chain length - 1.

(m.)

Interior

chain lengih

References

2-3 2-3 3 3-4 2 3-4 3-4 3-4 5 3-4 2-3 4

121 121 121 23 121 121 121 121 129 121 81 115

+ 2.5. * Average chain

It is therefore very probable that the interior structure of glycogens is irregular. 4. Determination of Multiple Branching

A recent development in glycogen chemistry is the recognition that multiple branching is an essential structural feature. Enzymic experiments provide the only means of differentiating between singly- and multiplybranched structures, - and methods for the qualitative and quantitative assessment of A. B. (degree of multiple branching) have been devised. (128) Marjorie A. Swanson and C. F. Cori, J . Riol. Chena., 172, 815 (1948). The minimum substrate requirement for both these enzymes is a linear chain of 4 D-glucose residues. (129) T. G. Halsall, E. L. Hirst, L. Hough and J. K . N. Jones, J. Chem. SOC., 3200 (1949).

288

D. J. MANNERS

One method involves the successive action of muscle phosphorylase and amylo-(1 -+ 6)-glucosidase on glycogen.130 Treatment of a muscle-phosphorylase limit-dextrin (+limit dextrin; L.D. 1) with amylo-(1 4 6)-glucosidase yielded D-glucose (about 5 %) and a polysaccharide which, on incubation with muscle phosphorylase, gave a second +-limit dextrin (L.D. 2 ) . Repetition of the digestion with amylo-(1 + 6)-glucosidase and muscle phosphorylase gave L.D. 3. For rabbit-liver glycogen, L.D. 1, L.D. 2, and L.D. 3 represent 64, 38, and 23 % of the original polysaccharide; 30 and 20 % of the original branch-points are removed in the successive digestions with amylo-(1 46)-glucosidase. These findings support the “tree” type of TABLE V Calculation of A X in Glycogens“ Sample

Chain length of +-dextrin

Human liver (normal)d Human liver (glycogenstorage disease)d Human liver (glycogenstorage disease)d Rabbit liver6 Rabbit liver’ Rabbit musclef

8.7

0.071

8. 7 10.8

0’081} 0.071 0.057

9.4 8.5 8.9

0.065 0.072 0.070

B .A .

4.3

::;} 4.6

0.024 0.0291 0.0261 0.025

1:2.0 1:1.8 1:1.7 1:1.3

2.7 3.2 5.4

0.015 0.018 0.030

1:3.3 1:3.0 1:1.3

}

a The sole C-chain in the molecule has been neglected in these calculations. * Expressed per 100 g. of +-dextrin, and equal t o 100/(162 X chain length of +-dextrin). c Expressed per 100 g. of +-dextrin and equal t o (per cent degradation of 4-dextrin by amylo-(l 46)-glucosidase)/l80. Seeref. 85 for experimental figures. Seeref. 120 for experimental figures. f See ref. 130 for experimental figures.

structure for glycogen, since a “laminated” structure would lose only one branch point with each treatment. Since the yield of D-glucose released from a qhdextrin by amylo-(1 + 6)glucosidase is dependent onthe proportion of A-chains, the above method can be used to determine A. B.; recorded in Table V are typical results which have been calculated from the experimental data of Gerty T. Cori 130 on the assumption that debranching of the +limit and dextrin was complete. Similar calculations have been made by Be~kmann,’~’ using the data in reference 130. (130) J. Larner, Barbara Illingworth, Gerty T. Cori and C. F. Cori, J. Biol. Chem., 199, 641 (1952). (131) C. 0. Beckmann, Ann. N . Y . Acad. Sci., 67, 384 (1953).

289

MOLECULAR STRUCTURE O F GLYCOGENS

(a

In a similar s t ~ d y , ”an ~ abnormal-liver glycogen = S), which approximated in structure to a 4-dextrin,22 was treated with isoamylase.o4 Some 7.3 % of D-glucose was liberated; if A. B. is 1: 1, 8.3 % of g glucose would be released. Further evidence that glycogens may vary in degree of multiple branching has been obtained from examination of their 0- and +limit dextrins,lz*o which differ only in exterior-chain length. If all glycogens contained equal numbers of A- and B-chains, the chain-length difference between the 4- and p-dextrin should be constant. A-Chain stubs of pJZzand 4-dextrinslZ0contain, on the average, 2.5 and 1 residue, respectively, whilst the B-chain stubs of a 8-dextrin contain n D-glucose residues (the most probable value of n is 2.5) and those of a +dextrin comprise (n 4) residues.120 The average length of the exterior chains of a 4-dextrin is therefore [l (n 4)]/2 and of a p-dextrin is (2.5 n)/2. The difference in chain length should therefore be 1.25 D-glucose residues. Experimentally, the chain length difference for fifteen glycogens varied from 1.2 to 2.7, equivvalues of 1:l.O to 1:2.9. alent to There can be little doubt that glycogens are multiply-branched molecules, as originally suggested by K. H. Meyer, although variations in the degree of multiple branching exist. However, in view of unavoidable limitations in the experimental and analytical procedures employed during the enzymic degradation of glycogen (each molecule may contain some 2,000 exterior chains), the numerical results for A. B. quoted in thissection represent approximate rather than absolute values.

+

+ +

+

~

V. MOLECULAR STRUCTURE OF GLYCOGENS Glycogens are highly branched macromolecules composed of several thousand chains; on the average, each chain contains 10-14 a-(1 + 4)linked D-glucose residues and is joined to an adjacent chain by a (1 -+ 6)glucosidic linkage. Individual chains vary considerably in length. Glycogen of molecular weight 5 X lo6 contains about 31,000 D-glucose residues and about 2,500 L Y - D - ( ~+ 6) inter-chain linkages. The interior of such a molecule is very compact, some 10,000 of the D-glucose residues being arranged in chains so that the branch points are separated by an average distance of only 3 4 n-glucose residues. The over-all structure is multiply-branched, being tree- or bush-like in form; glycogens do not appear to consist of ordered arrangements of chains of similar lengths. Although average chain-lengths of 10 -14 residues are usual, values ranging from 6 -18 are occasionally found. Glycogen preparations from a single biological source are polymolecular, and, sometimes, polydisperse. The molecules range in molecular weight (132) A, Margaret Liddle and D. J. Manners, Biochem. J . (London), 61, xii (1955);

J, Chem. SOC.,in press (1957).

290

D. J. MANNERS

from 106-107,but are believed to have similar structures. However, there is tentative evidence of structural inhomogeneity in mussel glycogen, since two fractions, separated by electrodialysis, differed in solubility and viscosity, and had @-amylolysislimits of 30 and 43 %, r e ~ p e c t i v e l y . ~ ~ 1. Gigcogens of Vertebrates

a. Mammalian Glycogens.-The gens are recorded in Table VI.

properties of several mammalian glyco-

TABLEV I Properties of Some Mammalian Glycogens Sample

Cat liver Dog liver Fetal-pig liver Fetal-sheep liver Guinea-pig liver Horse liver Horse muscle Human liver Human muscle Ox liver Rabbit liver Rabbit liver Rabbit liver Rabbit liver Rabbit muscle R a t liver

DI.[

degree; WaO)

+

193 +193 +191 +196 190 190 198 f 195 195 192 198 188 198 +200 +196 $193

+ + + + + + + +

Molecular weighta

x

10-a

10.0(1) 2.0(s)" 14.8(1) -

2.9(s) -

2.4(s) l.l(o) 1.9(s)

6.8(1) -

c. L.6

-Amylo. lysis imit, %

References

48 49 49 42 41 51 52 43 45 -

23, 71, 74 100 38, 74 23, 71, 74 100 100 23, 56, 79 100 23, 74, 79 100 52, 53, 77, 121 57, 83, 121 23, 71 54 23, 71, 79, 108 100

Methods of measurement : (1) = light-scattering; ( 8 ) = sedimentation-diffusion; osmotic pressure of the methylated glycogen. Methods of assay: (m) = methylation; (p) = periodate oxidation. c This sample was polydisperse; a minor component had a molecular weight of 0.5 X loE. a

(0)

=

Apart from differences in molecular weight, 14 of the 16 samples show little variation in branching characteristics; the constituent chains contain 10 -14 D-glucose residues per end-group, and 41-51 % of these are removed, as maltose, by P-amylase. The average interior and exterior chain-lengths are, therefore, approximately 3 and 8 D-glucose residues, respectively. It is of interest that liver glycogens of the fetal pig and sheep, formed only during the later stages of embryonic d e ~ e l o p m e n t , have ' ~ ~ structures similar t o adult, mammalian-liver glycogen. (133) Compare A. M. Nemeth, W. Insull, and L, B, Flexner, J . Bio2. Chem., 208, 765 (1954).

291

MOLECULAR STRUCTURE OF GLYCOGENS

The assumption that muscle and liver glycogens are chemically identical is partly confirmed by the data in Table VI, which show them to have similar branching properties. However, they differ in iodine coloration,'7 and muscle glycogens have a greater affinity for myosin than have liver glycogens (see p. 279). The reasons for these differences are not yet known. b. Fish Glycogens.-The properties of several fish-liver glycogens are summarized in Table VII. In general, fish glycogens show little variation in degree of branching TABLE VII Properties of Some Fish-liver Glycogens C L 6.

Sample

Bass (Micropterus dolamieu) Bullhead ( A m e i u r u s melas) Carp (Cyprinus carpio) Crappie (Pomoxis annularis) Dogfishc Gadidad

Garfish (Lepisosteus osseus) Haddock (Gadus aeglefinus) Hake (Merluccius vulgaris) Northern pike (Esoz estor) Skate Walleyed pike (Stizostedion vitreum)

+191 +190 198 194 195 196 +195 180 190 194 196 +197

+ + + + + + + +

References

100 100 100 100 134 31, 53 100 134 134 100 121 100

Determined by sedimentation-diffusion of the methylated glycogen (ref. 83). Methods of assay: (m) = methylation; (p) = periodate oxidation. c For dogfishmuscle glycogen, the figures are $190-195; 2.5 and lZ(m), respectively (refs. 83 and 134). Pooled livers of cod (Gadus morrhua), haddock (Gadus aeglefinus), and whiting (Gadus merlangus) .

and resemble most mammalian glycogens. Apart from skate-I21 and Northern pike-livers4 gIycogens which have P-amylolysis limits of 45 and 34 %, respectively, the enzymic degradation of fish glycogens has not been studied. c. Glycogen-storage Disease.-This disease, of which there are several distinct types, is characterized by the accumulation of glycogen in the liver and in other tissues. In some cases, this glycogen has an abnormal structure. These conditions result, in part, from a deficiency of one of the enzymes concerned in the interconversion of D-glucose and glycogen; glycogen breakdown is usually affected. The liver-enzyme system is shown in Fig. 3. (134) W. N. Haworth, E. L. Hirst and F. Smith, J . Chem. Sac., 1914 (1939).

292

D. J. MANNERS

Present knowledge of the biochemistry of this disease is due largely to Gerty T. Cori and 126. 136 and, in Table VIII, the characteristics of four types of the disease and the properties of several glycogens are recorded. The glycogen has a normal structure in types 1 and 2; in the former, there is deposition of fat in the liver and kidney, and a decreased activity of glucose-6-phosphatase. The enzyme deficiencies in type 2 are unknown. In types 3 and 4, which are less common, the glycogens have abnormal GLYCOGEN phosphorylase

phosphor ylase

a-D-Glucopyranosyl phosphate

11

p hosphoglucomutase

phosphoglucomutase

D-Glucose 6-phosphate 1 1

hezokinase/

D-Glucose

/

/

7

iI 11

\

glucose \6-phosphatase

\

L D-Glucose

Glycolytic system intermediates FIG.3.-Eneymic Interconversion of D-Glucose and Liver Glycogen. In this reaction, 5-10% of D-glucose is produced, in addition t o a-D-glucopyranosyl phosphate .I20

structures. In type 3, the short, exterior chains indicate absence of amylo-( 1 + 6)-glu~osidase~~~ 86; this has been confirmed experimentally in two cases of the disease.lZ6Glycogen in type 4 resembles amylopectin in degree of branching, solubility, and x-ray diffracting properties.s6 2 . Glycogens of Invertebrates

Table IX summarizes the properties of several invertebrate glycogens, including protozoan glycogens. (135) For a review, see Gerty T. Cori, Harvey Lectures, 48, 145 (1953).

293

MOLECULAR STRUCTURE OF GLYCOGENS

The variation in degree of branching of these glycogens probably reflects differences in the relative activity of phosphorylase and branching enzyme during glycogen synthesis. In contrast to the above-mentioned protozoa which synthesize glycogentype polysaccharides, certain ciliates (for example, Cycloposthium) contain an amylopectin-type of polysaccharide, whilst the flagellate Polytomella coeca stores a typical two-component starch.13B TABLE VIII Types of Glycogen-storage Disease,126-186 and Properties of Glycogens ~

(von Gierke's disease) 2

3

4

~

-

~~

Affected organ

Enzyme dejiciency

liver and kidney

glucose-6phosphatase

Samfilc of glycogen

liver muscle liver kidney ? liver generalized liver heart liver generalized amylo-(I -+ 6)- liver muscle glucosidase liverb heart psoas diaphragm probably gen- branching en- liver eralized zyme

-

;. L

13 12 11 11 14 11 13 12 9 8 6 8 8 8 21

-

Phosphorolysis !;mil, %"

36 38 36 33 35 33 42 40 12 3 1 6 5 3 51

References

85 85 85 85 85 85 125 125 85 85 22 125 125 125 85

-

Percent conversion t o a-n-glucopyranosyl phosphate by muscle phosphorylase; this is a measure of the exterior chain length. * This glycogen was incorrectly described as coming from a case of von Gierke's disease.22 a

3. Bacterial and Yeast Glycogens Polysaccharides which resemble animal glycogens in chemical and physical properties have been isolated from bacterial cells, including aviana2and human137 strains of Mycobacterium tuberculosis and surface cultures of enteric bacteria26 (for example, Escherichia and Salmonella montevideo), and also from yeasts.0. 81. ilo, 116*13% (136) E. J. Bourne, M. Stacey and I. A. Wilkinson, J . Chem. Soc., 2694 (1950); the properties of several protozoal polysaccharides are compared in ref. 140. (137) P. W. Kent and M. Stacey, Biochim. et Biophys. Acta, 3. 641 (1949). (138) H. Palmstierna, Acta Chem. Scand., 10, 567 (1956). (139) R. W. Jeanloz, Helu. Chim. Acta, 27, 1501 (1944).

294

D. J. MANNERS

4. Comparison of Glycogens with Amylopectins Glycogens and amylopectins are structurally similar in that both contain chains of a-(1 + 4)-linked D-glucose residues which are mutually TABLEIX Properties of Some G1ycogens of Invertebrates S)ecicr

Phylum

a],

,

/e rees d20

Trichomonas foetus Trichomonas gallinae Tetrahymena p yriformis expansa Platyhel- Moniezi (sheep tapeworms) minthes Nematods Ascaris lumbricoides (pig roundworms) Annelida Arenicola sp. (lugworm) Molluscad Anodonta" Fraction I Fraction I1 Fraction I11 Mytilus edulis I Mytilus edulis I1 Mytilus edulis IV Mytilus edulis IX CaTdium sp. Helix pomatia I Helix pomatia I1 Protozoa

Molecular weight,'

x

10-6

-~ +199 2 . 9 ( ~ ) ~ $197 3.5(s) $195 9.8(1)

C7L.b

,-Amy1 olysis imif, %

References

51 44

140 140 141

60

+194

-

-

100

+196

0.7(s)

49

23, 79, 142

$200

-

43

121

$192

6.1(0) 2.1(0) 3.0(0) 3.8(s) 2.6(s)f

43 34 30 43 47 52 51 14

36 36 36 23, 74 23, 55 38, 121 121 121 105 23, 38, 74

-

+192 +195 +196 +196 $201 +192 +182

37

a Methods of measurement: (0) = osmotic pressure of the acetylated glycogen; ( s ) = sedimentation-diffusion, (1) = light-scattering. b Methods of assay: (m) = methylation; (p) = periodate oxidation. This specimen was polydisperse, a minor component having a molecular weight of 0.3 X lo8. These invertebrates are commonly known as fresh-water mussel, common mussel, cockle, and edible snail, respectively. = Fractionation by electrodislysis. f This specimen was polydisperse; minor components had molecular weights of 12.9 X lo8 and 0.3 X 108.

interlinked by a - ~ - ( l+ B)-glucosidic linkages; the chains are multiplybranched to a similar degree. Approximately equal numbers of A- and B-chains are present in waxy-maize starch121143 and in wheat and corn (140) (141) (142) (143)

D. J. Manners and J. F. Ryley, Biochem. J. (London), 69, 369 (1955). D. J. Manners and J. F. Ryley, Biochem. J. (London), 62, 480 (1952). E. Baldwin and H. K. King, Biochem. J . (London), 36,37 (1942). S. Peat, W. J. Whelan and Gwen J. Thomas, J . Chem. Soc., 3025 (1956).

295

MOLECULAR STRUCTURE OF GLYCOGENS

(maize) amylopectins (as shown by calculations similar t o those in Table

V), as well as in glycogens. Differences between glycogen and amylopectin include the proportion of (1 -+ 6) linkages (average chain length), a f b i t y for iodine, molecular shape, and interaction with concanavalin-A. The average chain-length in amylopectin is approximately twice that of glycogenz0;most amylopectins contain 18-27 D-glucose residues per end-group, although samples with C. L. values 30 11-13 or1@36 have been reported. The relative position of branching appears to be similar, and the length of the exterior chains is ~

TABLE X Properties o j Some Bacterial and Yeast Glycogens Sample

Aerobacter aerogenes Bacillus megatherium Neisseria perflava Neisseria perflava Neisseria perflava Yeast (baker’s)e Yeast (baker’s) Yeast (brewer’s)

b.C .

Mmylolysi limit, yo

-

+200 192

+

46 57-59 55-59 49 50 44

-e

2

+196 +187 $184-188 +198

Rcjerences

26 86 145 146 100 139 81 115

Methods of measurement: (s) = sedimentation-diffusion; (d) = reducing-power estimation, usinga dinitrosalicylic acid reagent. Methods of assay: (m) = methylation; (p) = periodate oxidation. c 175” in 0.5 N NaOH. [ale 178” in 0.5 N NaOH. 6 The glycogen was fractionated by electrodialysis giving a “soluble” fraction (27%) with t h e properties recorded; the “insoluble” fraction (73%) had a 8-amylolysis limit of 46%. Q

+

+

normally twice that of the interior chains, as calculated from the p-amylolysis limit (50-60%) and the average chain-length. It follows that the interior of amylopectin, although randomly branched, is less compact than that of glycogen; it is therefore more susceptible to enzymic attack. For example, a-amylases degrade interior chains (of 6-9 residues) in amylopectin more readily than those of glycogen ( 3 4 whilst R-enzyme, which has no appreciable action on glycogen, hydrolyzes many of the a - ~ - ( -+ l 6) linkages in a m y l o p e ~ t i n . ’It~ is ~ ~probable that the inter-chain (144) A. L. Potter, V. Silveira, R. M. McCready and H. S. Owens, J . A m . Chem. Soc., 76, 1335 (1953). (145) E. J. Hehre, J . B i d . Chem., 177, 267 (1949). (146) S. A. Barker, E. J. Bourne and M. Stacey, J . Chem. SOC.,2884 (1950). (146a) S. Peat, W. J. Whelan, P. N. Hobson and Gwen J. Thomas, J . Chem. SOC., 4440 (1954).

296

D. J. MANNERS

linkages in glycogen are so closely arranged as to be inaccessible to R-enzyme. Moreover, the interior chains of amylopectin can interact with iodine. The p-dextrin from amylopectin gives a purple coloration with iodine (the absorption spectrum has A, at 530 mp), in contrast to glycogen P-dextrin which gives little or no col0ration.7~ The increased affinity of amylopectins for iodine is partly accounted for by the increase in exterior and interior chain-lengths. -Other factors are probably involved as sweet corn polysaccharides (C. L. 12-13) bind more iodine than does glycogen,898and glutinous rice-starch 18; P-amylolysis limit, 47 %) is stained red rather than purple with Viscosity measurements indicate that amylopectins are more asymmetric than Iz9 The limiting viscosity numbers of rabbit-liver glycogen __ (C. L. = 12; D. P.= 30,000) and amylopectin (from rubber-seed endosperm; = 23; D. P. = 6,000) are 0.10 and 1.02, re~pectively,’~~ whilst dog-liver glycogen (D. P. 5,300) has a much lower viscosity73 than amylopectins of D. P. 1200-1800. Since glycogen molecules are not spherical (see p. 276), amylopectins must have an appreciably greater degree of molecular asymmetry. The steric arrangement of the multiply-branched chains therefore differs, despite the similarity of the degree of multiple branching. The nature of this difference remains open to speculation. In addition to these differences, amylopectins, unlike glycogens, do not reactg4with concanavalin-A. However, certain samples of glycogen and amylopectin have atypical properties. -Thus, liver glycogen from a case of type 4 glycogen-storage disease (C. L. 21) reacts physicochemically as an amylopectin.86* Conversely, sweet-corn polysaccharides resemble animal glycogens in chain length, P-amylolysis limit, molecular and reaction94 with concanavalin-A.

(a

a

__.

VI. BIOLOGICAL SYNTHESIS 1. In vitro Synthesis For the synthesis of glycogen-type polysaccharides from a-D-glucopyranosyl phosphate, two enzymes are required. PhosphoryIases, in presence of a suitable primer, synthesize linear chains of a-(1 4)-linked D-glucose residues; these are then converted into a branched polysaccharide by a branching enzyme.’49 A synthesis of glycogen in witro was reported by Stepanenko and coworkers,160 who incubated a-D-glucopyranosyl phosphate and a small --f

(147) K. H.Meyer and Maria Fuld, Helv. Chim. Acta, 24, 1404 (1941). (148) C.T.Greenwood and J. S. M. Robertson, J . Chem. Soc., 3769 (1954). (149) Muscle phosphorylase synthesizes an “amylose” from ru-D-glUCOpyranOsyl phosphate, whilst impure heart and liver preparations give polysaccharides with the physicoohemical properties of a glycogen (ref. 27). (150) B. N . Stepanenko, A. S. Kainova and A. N. Petrova, Proc. Third Intern. Congr. Biockem., Brussels, 50 (1955).

297

MOLECULAR STRUCTURE OF GLYCOGENS

amount of glycogen primer with rabbit-muscle phosphorylase and branching enzyme. The synthetic glycogens, which contained about 94 % of anhydro-Dglucose, 4-6% of moisture, and <0.1% of nitrogen and phosphorus, are similar t o ex vivo glycogens, except that the degree of branching appears to be slightly less. 2. I n vivo Synthesis

Liver glycogen is synthesized from D-glucose in vivo by the enzyme system hexokinase-phosphoglucomutase-phosphorylase-branching enzyme (see Fig. 3). Glycogen is also formed from n-galactose and D-mannose TABLE XI Properties of Synthetic Glycogens160 Molecular weight X 10-6

Synthetic No. Synthetic No. Synthetic No. Synthetic No. Primer No. I Primer No. 2

O-Amylolysir limit, yo

0.L.C

Sample

Id 2d 3d 4d

Ab

BC

1.8 2.3 4.6 1.9

2.1 1.1 3.0 2.2 5. I 1.5

XW.X

22 17 16 17 15 13

37 34 39 38 38 40

,ms

530 500 500 530 500 500

errlax

1.00 0.80 0.82 0.86 0.95 0.70

Sodium periodate oxidation was used, giving relative rather than absolute results (compare ref. 98). b Number-average value from reducing-power determination using a dinitrosalicylic acid reagent. Number-average value from reducing-power determination using a ferricyanide reagent. d Synthesized under different experimental conditions. (1

through D-glucose phosphate intermediates,151 and from D-fructose through the triose phosphate and hexose phosphates of the glycolytic pathway.162 In general, the nature of the carbohydrate source does not appear to affect the degree of branching of the liver g1yc0gen.l~~ Glycogenesis in most muscle tissues appears to follow a similar pathway; in both liver and muscle tissues, the addition of D-glucose residues to the exterior chains of existing glycogen molecules proceeds rapidly.'54 (151) On administration of o - g l ~ c o s e - 1 - Cor~ ~I>-mannose-l-CI4t o fasted rats, 80-90% of the ClPis located in C1 of t h e u-glucose residues in the liver glycogen [Margaret Cook and V. Lorber, J . B i d . Chem., 199, l (1952)l. (152) H. G . Hers, J . B i d . Chem., 214, 373 (1955); the glycolyt,ic pathway is also important in the synthesis of liver glycogen from non-carbohydrate sources, see 1'. A . Marks and R. L. Horecker, J . Riol. Chem., 218,327 (1956). (153) Ref. 100, but see also refs. 54, 96 (d), and Schlamowitz (ref. 98). (154) Marjorie R. Stetten and D. Stetten, Jr., J . B i d . Chem., 213, 723 (1955).

298

D. J. MANNERS

Variations in the structure of glycogen may be due, in part, to differences in relative activity of phosphorylase and branching enzyme. With a deficiency in branching enzyme, a glycogen with a low degree of branching would be expected. The amount of active enzyme in a tissue depends on many factors; phosphorylase activity is related to the rates of enzymic synthesis and breakdown of the phosphorylaseeenzyme protein and nucleotide (adenosine 5-phosphate) activator,*66the number of primer molecules available, and the inorganic phosphate content of the cells. In higher animals, the enzyme systems are under hormonal contr01.*5~ The rate and type of glycogen formation will therefore depend on the meta.bolic condition of the whole animal. VII. SUMMARY AND CONCLUSIONS Glycogens, from vertebrate, invertebrate, bacterial, and yeast cells, are multiply-branched molecules (molecular weight -107) consisting of chains of a-(1 4)-linked D-glucose residues. The chains, which are arranged in a tree- or bush-like form, normally contain an average of about 12 D-glucose residues; 40-50% of these may be removed by @-amylase.The exterior portions of the chains are therefore longer than those in the interior of the molecules, where adjacent branch points are separated by only 3-4 D-glucose residues. Small variations in molecular structure are shown by glycogens from different biological sources. I t seems probable that, with the improved chemical and enzymic semimicro-analytical methods now available, the structure of glycogen from hitherto-unexamined biological sources will be characterized and that structural studies will also be directed toward the quantitative determination of multiple branching and investigation of the compact interior of the molecules. A small proportion of glycogens may be expected to have abnormal structures. In this connection, the isolation of maltulose (some 5 %) from an a-amylolytic digest of glycogenlK7of pregnant-rabbit liver would suggest that D-fructose may be an extremely rare but minor component of certain glycogens. ---f

(155) The enzymic synthesis and inactivation of mammalian phosphorylases is discussed in “Enzymes and Metabolism,” Elsevier, Amsterdam, 1956,pp. 69, 150; Biochim. et Biophys. Aclu, 20, No. 1 (1956). (156) For example, the conversion of D-glucose into liver glycogen is stimulated by insulin, and inhibited by adrenaline and glucagon [J. Berthet, P. Jacques, H. G. Hers and C. de Duve, Biochim. et Biophys. Actu, 20, 190 (1956)l. (157) S.Peat, P. J. P. Roberts and W . J. Whelan, Bioehern. J . (London), 61, xvii (1952).

THE BIOSYNTHESIS OF HYALURONIC ACID BY ROYL. WHISTLER AND E. J. OLSON Department of Biochemistry, Piirdire UnizJersity, Lafayetle, Indiana I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 1 . Structure.. . . . . . . . . . . . . . . . . . 11. Metabolism of D-Glucuronic Ac 1 . The 8-D-GlucopyranosiduronatePrecursor. . . . . . . . . . . 304 2. Biosynthesis of Uridine 5-(~-Glucosyluronic phate) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 3. 8-D-Glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 111. Metabolism of D-Glucosamine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 1. Phosphorylation . . .... ........ .... . . . . 308 2. Reactions of Phospho-D-ghcosamine Mutase . . . . . . . . . . . . . . . . . . . . . . . . . . 309 3. Acetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 4. Uridine Derivatives of N-Acetyl-D-glucosamine. ....................... 312 5. Biosynthesis of D-Glucosamine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 IV. Biosynthesis of Hyaluronic Acid. .................... 1. Incorporation of Hexose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 2. L-Glutamine.. ..................... 3. Incorporation and Exchange of Acetyl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

I. INTRODUCTION Hyaluronic acid is the only heteroglycan whose biosynthesis has been examined in detail. Since aminodeoxy sugar units are present, the polysaccharide is an aminodeoxypolysaccharide or, as it is more commonly termed, a mucopolysaccharide. Hyaluronic acid is a copolymer of two sugar units which alternate in a linear chain, and hence it may also be classified as a diheteroglycan.’ Biosynthetic investigations show that the hyaluronate chain is assembled by the alternate incorporation of preformed N-acetyl-D-ghcosamine and D-glucuronate units. In descriptive scientific nomenclature, the units are 2-acetamido-2-deoxy-~-~-glucopyranosyl and 0-D-glucopyranosyluronate, respectively. The presence of carboxyl groups on alternate units has given rise to the suffix “-uronic acid.” However, in the native state and during most chemi(1) R. L. Whistler and C. L. Smart, “Polysaccharide Chemistry,” Academic Press Ine., New York, N. Y., 1953, p. IS. 299

300

ROY L. WHISTLER AND E. J. OLSON

cal treatments of the polysaccharide, it is in the form of its salts and hence the designation “hyaluronate” may be more consistently appropriate. Hyaluronate is produced both by animals and bacteria. It was first obtained by Meyer and Palmer from the vitreous humor of cattle eyes.2 Since then, it has been isolated from many sources, such as Wharton’s jell~,~-’O connective tissue,” skin,12 cock’s comb,I3 synovial fluid,I4 Rous sarcoma,16myxoedemal fluids,lB and encapsulated strains of hemolytic streptoc~cci.‘~ Aerobacter aerogenes may also produce hyaluronate.18Group A streptococci have been principally used for the investigation of the biosynthesis of hyaluronic acid. Aqueous solutions of hyaluronate are quite viscous. Except in very dilute solutions, the shearing strain is not proportional to the stress applied.Ig Much of the viscosity of hyaluronate solution is believed to be due to intramolecular associations,,since addition of salts to saline concentrations which are capable of breaking salt bridges and polar associations reduces the viscosity of hyaluronate solutions.** Physiological solutions which contain hyaluronate are much more viscous than equivalent aqueous solutions of hyaluronate. The viscosities of these physiological fluids are greatly reduced by the addition of hyaluronidase, an enzyme which hydrolyzes hyaluronate.21In addition to raising the viscosity of synovial fluid, hyaluronate may also impart to the fluid its lubricating and shockabsorption properties.lg (2) K. Meyer and J. W. Palmer, J . B i d . Chem., 107, 629 (1934). (3) K. Meyer and J. W. Palmer, J . B i d . Chem., 114, 689 (1936). (4) E. Haas, J . B i d . Chem., 163, 63 (1946). (5) D. McClean and C. W. Hale, Biochem. J . (London), 36, 159 (1941). (6) 13. McClean, Biochem. J . (London), 37, 169 (1943). (7) J. Madinaveitia and M. Stacey, Biochem. J . (London), 38, 413 (1944). (8) Z. Hadidian and N . W . Pirie, Biochem. J . (London), 42, 260 (1948). (9) A. E. Follett, J . B i d . Chem., 176, 177 (1948). (10) R . W. Jeanloz and E. Forchielli, J . B i d . Chem., 186, 495 (1950). (11) G. Asboe-Hansen, Acta Dermato-Venereol., 30, 221 (1950) ; Chem. Abstracts, 44, 10903 (1950). (12) K. Meyer and Eleanor Chaffee, J . Biol. Chem., 138, 491 (1941). (13) N. F. Boas, J. Biol. Chem., 181, 573 (1949). (14) K. Meyer, Elizabeth M. Smyth and M. H. Dawson, J . B i d . Chem., 128, 319 (1939). (15) G. H. Warren, E. C. Williams, H. E. Alburn and J. Seifter, Arch. Biochem., 20, 300 (1949). (16) E. M. Watson and R. H. Pearce, A m . J . Clin. Pathol., 17, 507 (1947). (17) F. E. Kendall, M. Heidelberger and M. H. Dawson, J . B i d . Chem., 118. 61 (1937). (18) G . H. Warren, Science, 111, 473 (1950). (19) A. G . Ogston and 0. E. Stanier, J . Physiol. (London), 119, 244 (1953). (20) G . Blix and 0. Snellman, Arkiv Kemi, Mineral. Geol., A19, No. 32. (21) K . Meyer, Physiol. Revs., 27, 335 (1947).

BIOSYNTHESIS OF HYALURONIC ACID

301

Occurrence of hyaluronate in interfibrillar regions has also led to the belief that it functions as a protective agent against bacterial attack. However, clinical22* 23 and embry~logical~~ work shows no general correlation between the ability of a microorganism to break down hyaluronate and its tissue-penetrating ability or virulence. Nonetheless, there is evidence that hyaluronate facilitates primary or secondary bacterial infection by serving as an energy source. Sallman, Birkeland and Grey26found that hyaluronate stimulates the respiration of hemolytic streptococci and that the stimulation is especially pronounced with strains which produce hyaluronidase. Further experimental work26 by these investigators has shown that N-acetyl-D-glucosamhe (2-acetamido-2-deoxy-~-glucose)produces a larger respiratory stimulation in hemolytic streptococci than does hyaluronate. Little stimulation is produced by D-glucuronic acid. From this, it is concluded that the N-acetyl-D-glucosamine residues of hyaluronate are the energy sources for the bacteria. Hemolytic streptococci also stimulate the respiration of other bacteria, when the organisms are grown in the presence of hyaluronate. This stimulation is due, in large part, to hyaluronidase supplied by the hemolytic streptococci, since the enzyme alone produces a comparable respiratory stimulation. In several experiments, intraperitoneal injections of leech extractszsor of another hyal~ronidase~~ were found to lessen the susceptibility to hemolytic streptococci intraperitoneally administered. Therefore, it was suggested that hyaluronate in the streptococcal capsules protects the organisms and is responsible for their greater virulence. These results were not confirmed in a similar experiment by McClean.28Subsequently, McClean injected a hyaluronidase into mice infected with streptococci and found that the bacterial capsules were initially removed but returned after about one and a half Similarly, Kass and Seastone found that hyaluronidase injections had no effect on the virulence of pneumococci in Some investigations have been made with the object of determining a possible role for hyaluronate in rheumatoid arthritis. This disease is characterized by fibrinoid degeneration, collagen decomposition, and lesions. It has been suggested that changes in either the concentration or the structure (22) Naula Crowley, J . PathoZ. Bacteriol., 66, 27 (1944). (23) J . H. Humphrey, J . Pathol. Bacteriol., 66, 273 (1944). (24) Barbara E. Russell and N. P. Sherwood, J . Infectious Diseases, 84.81 (1949). (25) B. Sallman, J. Birkeland and C. T. Grey, Proe. Soc. Expll. BioZ. Med., 76, 467 (1951). (26) G. K. Hirst, J . Expll. Med., 73, 493 (1941). (27) E. H. Kass and C. V. Seastone, J. Exptl.Med., 79, 319 (1944). (28) D. McClean, J . Pathol. Bacteriol., 63, 156 (1941). (29) D. McClean, J . Pathol. Bacteriol., 64, 284 (1942).

302

ROY L. WHISTLER AND E. J. OLSON

of hyaluronate in connective tissue are related to the disease.30Possibly, the increased water-holding capacity and the swelling of rheumatoid, connective tissue results from a high concentration of hyaluronate. Clinical investigations by Ragan and Meyer31 indicate that hyaluronate in the synovial fluid of arthritic patients has a degree of polymerization lower than normal. 1. Structure

Complete hydrolysis of hyaluronate by a crude testicular extract produces N-acetyl-D-glucosamine and D-glucuronic For partial hydrolysis of the polysaccharide, pneumococcaP3 and t e ~ t i c u l a rhyaluronidases ~~ are useful. When the hydrolyzate obtained by using testicular hyaluronidase is further subjected to brief, acid hydrolysis, hyalobiouronic acid (a ‘ldisaccharide”) can be obtained in good yield through simple crystallizati~n.~~ The reducing end of this disaccharide is a D-glucosamine unit, and the nonreducing end is a P-D-glucopyranosyluronicacid moiety. To examine this structure, Weissmann and MeyeP first esterified the carboxyl group of the D-glucuronic acid residue with cold, methanolic hydrogen chloride (see Fig. 1). After conversion of the reducing group to a carboxyl group by means of yellow mercuric oxide, the ester group was reduced with sodium borohydride. Next, the 2-amino-2-deoxy-~-gluconic acid part of the molecule was degraded by ninhydrin to a D-arabinose residue. The heptaacetate of this arabinose-containing disaccharide gave no melting-point depression when admixed with 3 ,4,5-tri-O-acetyl-2-0-(2,3 , 4 ,6-tetra-O-acetyl-p-~glucopyranosy1)-D-arabinose.The &D-(1 + 2) glycosidic linkage demonstrated here shows that, in hyalobiouronic acid, the linkage must be @-D-( 1-+ 3). Thus, hyalobiouronic acid is 2-amino-2-deoxy-~-ghcose-(3 + 1) p-D-ghcopyranosiduronic acid. Since the hyalobiouronic acid can be obtained both by combined enzymic and acid hydrolysis and by direct acid hydrolysis alone,37it is considered (30) E. Kulonen, Acta Physiol. S c a d , 24, Suppl. 88 (1951). (31) C . Ragan and K. Meyer, J. Ctin. Invest.,28, 56 (1949). (32) K . Meyer, A. Linker and M. M. Rapport, J. Biol. Chem., 192, 275 (1951). (33) M. M. Rapport, A. Linker and K . Meyer, J. Biol.Chem., 192, 283 (1951). (34) B. Weissmann, K . Meyer, Phyllis Sampson and A. Linker, J. Biol. Chem., 208, 417 (1954). (35) M. M. Rapport, B. Weissmann, A. Linker and K . Meyer, Nature, 168, 996 (1951). (36) B. Weissmann and K. Meyer, J. Am. Chem. Soc., 74, 4729 (1952). (37) B. Weissmann, M. M. Rapport, A. Linker and K. Meyer, J . Biol. Chem., 206. 205 (1953).

303

BIOSYNTHESIS OF HYALURONIC ACID

to be a fragment of the polysaccharide rather than an artifact created by an enzymic transfer reaction. CHzOH

+ CH,OH

OH HO

H H

H

HCI, cold

NH,

OH CHzOH H,OH

ti

HO H

HgO

,

-

N H ~

OH CH20H

H

OH CH20H

H

OH CHzOH

H H

OH

FIG.1.-Reaction Sequence in Determining the Structure of Hyalobiouronic Acid,

A series of higher oligosaccharides with even numbers of sugar units seems to be obtained when a digest of umbilical-cord hyaluronate by testicular hyaluronidase is eluted from a Dowex-1, ion-exchange column with formic acid solution34; the tetrasaccharide was obtained crystalline. The oligosaccharides apparently have a 2-acetamido-2-deoxy-3-0-(&~-

304

ROY L. WHISTLER AND E. J. OLSON

glucopyranosyluronic acid)-D-glucopyranose repeating unit. Consequently, it may be assumed that the N-acetyl derivative of the first “disaccharide” unit shown in Fig. 1 repeats more or less throughout the hyaluronic acid molecule. Since periodate does not oxidize the interior of the hyaluronic acid, each N-acetyl-D-glucosamine unit may be linked to C3 of each D-glucuronic acid unit,.38 In the past, the difficulty encountered in the methylation of hyaluronic acid caused some workers to believe that the molecule is a highly ramified structure.39 However, electron-microscopic photographs show that the molecule is linea~-.~O Light-scattering data indicate that hyaluronic acid and hyaluronate probably exist in solution as semirigid, random coils.4o-41 These coils are believed to be 5000-6000 A. long and 2200 A. in diameter.41 Other estimates of the len8th of sodium hyaluronate40 by light-scattering range from 2900 to 6700 A. Estimates of the len th of hyaluronate from range from 4,800 to 10,000 . The molecular size of viscosimetric hyaluronate depends on its source and method of isolation.42 Molecularweight estimates based on light-scattering 41 range from 1,270,000 to 4,300,000. Streaming birefringence and light-scattering studies indicate that sodium hyaluronate molecules shrink in solution.40 The infrared absorption spectra of hyaluronic acids from different sources are essentially similar.43Identical infrared absorption spectra are given by hyaluronate from Rous sarcomas and from umbilical cords. Slight quantitative differences between the spectra of hyaluronic acid from umbilical cord and of those from both myxoedemaP3 and streptococcal h ~ a l u r o n a t e ~ ~ may be due to impurities.

d

OF D-GLUCURONIC ACID 11. METABOLISM

1. The p-D-Glucopyranosiduronate Precursor

In establishing the mechanism of the biosynthesis of hyaluronate, consideration must be given to the manner of forming the p-D-gIucosiduronic acid linkage. Mammalian organisms excrete phenols,4sand primary, secondary, and (38) K. H . Meyer, J. Fellig and E. H. Fischer, Helv. Chim. Acta, 34, 939 (1951). (39) M. A. G . Kaye and M. Stacey, Biochem. J . (London), 46, xiii (1950). (40) J. W. Rowen, R . Brunish and F. W. Bishop, Biochim. et Biophys. Acta, 19, 480 (1956). (41) T. C. Laurent and J . Gergely, J. Biol. Chem., 212, 325 (1955). (42) G. Blix and 0. Snellman, Nature, 163, 587 (1944). (43) S. F. D. Orr, Biochim. et Biophys. Acta, 14, 173 (1954). (44) D. A . Lowther and H. J. Rogers, Biochem. J . (London), 62, 304 (1956). (45) H. G. Bray, Brenda E. Ryman and W. V. Thorpe, Biochenz. J . (London), 41, 212 (1947).

305

BIOSYNTHESIS OF HYALURONIC ACID

tertiary alcohols,46-in part, as 0-n-glucopyranosiduronates.D-Glucosiduronate conjugation takes place primarily in the liver4’ and can occur in liver slices or homogenates. Boiled, liver extract induces conjugation of o-aminophenol with n-glucuronic acid in a suspension of broken, mouse-liver cells.4* The proportion of D-glucosiduronate formed is proportional to the proportion of liver extract added. It is not occasioned by magnesium ions, fumarate, a-keto acids, diphosphopyridine nucleotide, cytochrome C, or adenosine 5-phosphate, since the addition of any of these compounds does not increase D-ghcosiduronate formation. Later work suggests that uridine 5-(~-glucosyluronic acid dihydrogen pyrophosphate) (“uridine diphosphate D-glucuronic acid”) is the conjugation agent.49This compound, obtained by elution of liver extracts adsorbed on a Dowex-1 column, can stimulate conjugation of o-aminophenol in fresh, rat-liver homogenates. Further analysis of the hydrolyzed eluate (by paper chromatography and ionophoresis) indicates the presence of uridine 5-phosphate, uridine 5-(trihydrogen pyrophosphate), and n-glucosyluronic acid phosphate. The presence of a uridine 5-phosphate link is substantiated by its hydrolysis with uridine-5-phosphatase from snake venom. Thus, the structure of “uridine diphosphate D-glucuronic acid” must be that shown in formula I.

COOH

OH

OH

I

The observed cleavage of uridine 5-(2-acetamido-2-deoxy-~-glucosyl dihydrogen pyrophosphate) by rat-liver muscle60 might suggest that a (46) I. A. Kamil, J. N . Smith and R. T. Williams, Biochem. J . (London), 63, 129 (1953). (47) W. L. Lipschits and E. Bueding, J . Biol. Chem., 129, 333 (1939). See also, H. G. Bray, Advances in Carbohydrate Chem., 8 , 251 (1953); R. S. Teague, ibid., 9, 185 (1954). (48) G . J. Dutton and I. D. E . Storey, Biochem. J . (London), 67, 275 (1954). (49) I. D. E. Storey and G. J. Dutton, Biochem. J. (London), 69, 279 (1955). (50) Evelyn E. B. Smith and G. T. Mills, Biochim. et Biophys. Acta, 13,386 (1954).

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ROY L. WHISTLER AND E. J. OLSON

similar cleavage of “uridine diphosphate D-glucuronate” occurs, liberating D-glucosyluronic acid phosphate which could then take part in conjugation. However, evidence that such a rcaction does not occur is based on the observation that neither a- nor p-D-glucosyluronate phosphate conjugates with o-aminophenol in the presence of rat-liver homogenates.61 2. Biosynthesis of Uridine 5-(~-GlucosyluronicAcid Dihydrogen Pyrophosphate)

Since the carbon skeleton of D-glucose appears to be incorporated as a unit both into 0-D-ghcosiduronate conjugatess2 and into hyaluronate,s3’ 64 it is probable that the D-glucosiduronate units share common precursors. A study by Dziewiatkowski and Lewiss5 suggests that glycogen is the major carbon source of D-glucosiduronate units. They noted that rats fed a D-ghcosiduronogenic substance such as menthol are depleted of liver glycogen. An oxidative enzyme in supernatants of mammalian livers2 can oxidize “uridine diphosphate D-glucose” to a substance which appears to be “uridine diphosphate D-glucuronate” because of its properties on Dowex-1, its positive carbazole reaction,56and its ability to conjugate o-aminophenol. The purified enzyme system from calf’s liver does not catalyze the oxidation of ethanol, a-n-glucopyranosyl phosphate, or “uridine diphosphate 2-acetamido-2-deoxy-~-glucose” in the presence of oxidized diphosphopyridine nucleotide. The enzyme is, therefore not considered t o be a general dehydrogenase. When the initial ratio of oxidized diphosphopyridine nucleotide to “uridine diphosphate D-glucose” is four, the oxidation goes t o 90% completion. No compound with an intermediate oxidation state was detected. The positive carbazole reaction indicates that one mole of “uridine diphosphate D-glucuronic acid” is formed for every two moles of diphosphopyridine nucleotide reduced. At present, it seems that uridine 5-(~-glucosyluronicacid pyrophosphate) is formed only through the oxidation of uridine diphosphate D-glucose. Attempts t o join either a- or p-D-glucosyluronate phosphate to uridine 5-(trihydrogen pyrophosphate) through the reversal of pyrophosphorolysis, (51) G.A. Levvy and C. A. Marsh, Biochem. J . (London), 62. 690 (1952). (52) J. L. Strominger, H. M. Kalckar, J. Axelrod and Elizabeth S. Maxwell, J . A m . Chem. Soc., 76, 6411 (1954). (53) S. Roseman, J. Ludowieg, Frances E. Moses and A. Dorfman, J . Biol. Chem., 206, 665 (1954). (54) S. Roseman, Frances E. Moses, J. Ludowieg and A. Dorfman, J . Biol.Chem., 203, 213 (1953). (55) D.D.Dziewiatkowski and H. B. Lewis, J . Biol. Chem., 163, 49 (1944). (56) Z. Dische, J . Biol. Chem., 183, 489 (1950).

BIOSYNTHESIS O F HYALURONIC ACID

307

+ “uridine diphosphate D-glucuronate” S uridine-5-triphosphoric acid + D-glucosyluronate phosphate,

pyrophosphate

have not been successful with the catalytic systems, yeast extracts, or guineapig-liver n u ~ l e i . ~ Both ” extracts are capable of reversibly pyrophosphorylating “uridine diphosphate D-glucose.” It should also be kept in mind that, since neither a- nor p-D-glucosyluronate phosphate stimulates D-glucosiduronate conjugation,s1 it is possible that neither a- nor p-D-glucosiduronate can replace the D-glucosyl unit of “uridine diphosphate D-glucose” through a transfer reaction. In v i m investigations with rner1,~7rats,68and guineapigsS9indicate that the formation of uridine 5-(~-glucosyluronicacid pyrophosphate) occurs solely through the oxidation of “uridine diphosphate D-glucose.” Neither ~ - g l u c u r o n o l a c t o n e ~nor ~ - ~ ~sodium ~ - g l u c u r o n a t e68~ ~is significantly iricorporated into urinary D-glucosiduronate conjugates which are formed on oral administration of a D-glucosiduronogenic material. D-Glucuronolactone labeled a t C6 is not incorporated significantly into urinary D-glucosiduronates when it is administered either parenterally57-5g or 0rally.~7D-Glucuronate, likewise, is not incorporated into p-D-glucosiduronate conjugates when administered either intravenously67 or intrap e r i t ~ n e a l l y .Both ~ ~ ~ -g lu cu ro n o lacto n e~ ~ D-glucuronic acids8 are 59 ~ and metabolized and excreted. 8

3. P-D-Glucuronidase

The repeated administration of a D-glucosiduronogenic material such as menthol increases the 0-D-glucuronidase activity in mice and dogs.60 I n dogs, especially large increases occur in the liver and kidney. This might imply that p-D-glucuronidase induces the formation of p-D-glucosiduronates. However, such a belief is not substantiated by other experiments. No correlation is found between P-D-glucuronidase activity and D-glucosiduronate-producing ability in normal, rat tissue.61Furthermore, partial hepatectomy, the intraperitoneal injection of menthol, and the subcutaneous injection of chloroform (which increases liver 0-D-glucuronidase activity) fail to increase the o-aminophenol-conjugating ability of surviving liver(57) F. Eisenberg, Jr., J. B. Field and D. Stetten, Jr., Arch. Biochem. and Biophys., 69, 297 (1955).

(58) G. C. Butler and Marian A. Packham, Arch. Biochem. and Biophys., 66, 551 (1955). (50) J . F . Douglas and C. G. King, J . B i d . Chem., 203, 889 (1953). (60) W. H. Fishman, J . B i d . Chern., 136, 229 (1940). (61) M. C. Karunairatnam, Lynda M. H. Kerr and G. A . Levvy, Biochem. J . (London), 46, 496 (1949).

308

ROY L. WHISTLER AND E. J. OLSON

slices obtained from treated rats. Then, too, the inhibition of P-D-glucuronidase activity of mouse-liver cells with saccharic acid (D-glucaric acid) does not inhibit their ability to produce p-D-glucosiduronates.62 Whatever the nature of the system which produces P-D-glucosiduronates, it is probably present in microsomes since these are required for formation of p-D-glucosiduronate in rat-liver homogenate."

111. METABOLISM OF D-GLUCOSAMINE 1. P h o ~ ~ h o r ~ Z a t ~ ~ D-Glucosamine is phosphorylated by a hexokinase preparation from yeast.63The resulting phosphorylated derivative consumes four moles of periodate per mole and is, therefore, considered to be D-glucosamine 6-phosphate. The rate of D-glucosamine phosphorylation is about 70% of that for D-glucose pho~phorylation.~~ 64 Free D-glucosamine and adenosine-5-triphosphoric acid disappear at similar rates in the presence of yeast hexok i n a ~ e .64~ ~ , D-Glucosamine phosphorylation appears to be catalyzed by the same enzyme that effects phosphorylation of D-glucose and D-fructose. Evidence for this belief is obtained by measuring the carbon dioxide evolved from phosphorylating reaction mixtures which contain sodium bicarbonate. Evolution of carbon dioxide results from the increase in acidity accompanying the phosphorylation reaction. The addition of D-glucosamine to a mixture of either D-glucose or D-fructose with ox-brain extracts lowers the evolution of carbon dioxide.6sThis diminished production of carbon dioxide is believed to be caused by competitive inhibition of phosphorylation of D-fructose and D-glucose by n-glucosamine. Since a mixture of D-glucose and D-glucosamine is phosphorylated at a rate intermediate between that for D-glucose and that for D-glucosamine, it is assumed that the same en~ y m catalyzes e ~ ~ the phosphorylation of both sugars. It is also observed that N-acetyl-D-glucosamine lowers the production of carbon dioxide from ox-brain extracts containing either D-glucosamine or D-fructoses6 and it may therefore be a hexokinase inhibitor. At high concentrations, N-acetylo-glucosamine also inhibits the production of carbon dioxide from phosphorylation of D-glucose by ox-brain extracts. Although N-acetyl-D-glucosamine has inhibitory properties, it is apparently not phosphorylated. When N-acetyl-D-ghcosamhe is added alone to ox-brain extracts, no carbon dioxide is produced.66Yeast hexokinase 9

(62) (63) (64) (65)

M. C. Karunairatnam and G. A . Levvy, Biochem. J . (London), 44,599 (1949). D. H. Brown, Biochim. et Biophys. Acta, 7 , 487 (1951). P. T. Grant and C. Long, Biochem. J. (London), 60,xx (1951-52). R. P. Harper and J. H. Quastel, Nature, 164, 693 (1949).

BIOSYNTHESIS O F HYALURONIC ACID

309

does not appear to catalyze the phosphorylation of N-acetyl-u-glucosamine.63 D-Glucosamine 6-phosphate is hydrolyzed by the D-glucose-6-phosphatase of rat-liver mitochondria.66The rate of this hydrolysis is about 8 % of that of D-glucose 6-phosphate hydrolysis. A phosphatase which preferentially catalyzes the hydrolysis of D-glucosamine 6-phosphate has been prepared from Neurospora crassa.67 This enzyme is not stimulated by magnesium ions and has an optimum activity between pH 6 and 7.5. It appears to be distinct from acid, alkaline, and other specific phosphatases. 2. Reactions of Phospho-D-glucosamineMutase

When D-glucosamine 6-phosphate and catalytic amounts of 6-O-phospho-D-glucosyl phosphate are added to a solution of purified phosphoglucomutase, acid-labile phosphate develops in proportion to the decrease in reducing power toward alkaline copper.68This indicates a transfer of phosphate from C6 to C1. D-Glucosamine 6-phosphate and the acid-labile phosphate ester can be separated from each other by passage through a Dowex-1 column. The equilibrium ratioe8 of D-glucosamine &phosphate to D-glucosamine 1-phosphate is approximately 4, in the pH range of 7.1 to 7.8. With the purified enzyme, the phosphoglucomutase reaction is several hundred times faster than the phospho-D-glucosamine mutase reaction.e8 The conversion of D-glucosamine 6-pliosphate to the l-phosphate can be catalyzed by phosphoglucomutase in vitro as shown above, and may be catalyzed by phosphoglucomutase in vivo.68 This belief may be based on the observation that the addition of 6-O-phospho-~-glucopyranosylphosphate, in all but small proportions, inhibits the D-glucosamine isomerization.686-O-Phospho-~-g~ucopyranosy~ phosphate, when initially present in an amount equal to 4 % of ~-glucosamine6-phosphate ona molar basis, lowers the isomerization rate of phosphoglucosamine mutase to about 70% of the maximum observed rate. From a consideration of the probable reaction,

+ phosphoglucomutase * phosphoglucomutase phosphate +

6-O-phospho-~-glucopyranosyl phosphate

D-glucopyranosyl phosphate (or D-glucopyranose 6-phosphate), it seems likely that inhibition is attributable to the production of D-gluco(66) F. Maley and H. A. Lardy, J. Am. Chem. Sac., 78, 1393 (1956). (67) H. J. Blumenthal, Aletha Hemerline and S. Roseman, Bacterial. Proe. (Sac. Am. Bacteriologists), 66, P7 (1956). (68) D. H. Brown, J . B i d . Chem., 204, 877 (1953).

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ROY L. WHISTLER AND E. J. OLSON

pyranosyl phosphate and 6-O-phospho-~-glucopyranose, which competitively inhibit the isomerization of D-ghCOSamine &phosphate. Therefore, it is possible that, in living cells, D-glucopyranose phosphates may extensively inhibit the isomerization of D-glucosamine phosphate by phosphoglucomutase. An analogous mutase reaction occurs with N-acetyl-D-glueosamine phosphates when N-acetyl-D-ghcosamine 1-phosphate is added to extracts of Neurospora c ~ a s s or a ~of~ pig kidney.70An enzyme system, from Neurospora crassa, which preferentially catalyzes this reaction, has been partially purified by fractionation with ammonium sulfate and treatment with calcium phosphate gel.71When 6-O-phospho-~-glucosylphosphate is added as a cofactor, this enzyme is capable of converting N-acetyl-D-glucosamine 1-phosphate to an acid-stable, phosphate ester of N-acetyl-~-glucosamine.~~ As the reaction proceeds, good quantitative agreement is obtained between the N-acetyl-D-glucosamine equivalents gained and the acid-labile, phosphate equivalents lost. Like N-acetyl-D-glucosamine 6-phosphate7the acidlabile phosphate ester of N-acetyl-D-glucosamine is destroyed by a purified, kidney-protein fraction (see Section 111, 5 ) . This catalytic system also converts N-acetyl-D-glucosamine 6-phosphate to N-acetyl-D-glucosamine 1-phosphate.71 Starting with either N-acetylD-glucosamine 1-phosphate or 6-phosphate7 the final ratio71 of the aeidstable ester to the acid-labile ester is 6:l. A diphosphate ester of N-acetyl-D-ghcosamine, obtained from the acid hydrolysis of a uridine 5-pyrophosphate derivative of N-acetyl-D-glucosamine phosphate (see Section 111, 4), can replace 6-O-phospho-~-glucosyl phosphate as a ofa actor.^' This diphosphate ester of N-acetyl-D-glucosamine has slightly greater catalytic activity than 6-O-phospho-~-glucopyranosyl phosphate. A N-acetyl-D-glucosamine diester can be isolated from the reaction mixture when 6-O-phospho-~-glucosyl phosphate is initially added .71 One molecule of the isolated N-acetyl-D-glucosamine diphosphate yields, on acid hydrolysis, one molecule of stable N-acetyl-D-glucosamine phosphate and one molecule of inorganic phosphate. The N-acetyl-Dglucosamine diphosphate ester has the same catalytic properties as the diester obtained from hydrolyzed uridine 5-(2-acetamido-2-deoxy-Ophospho-D-glucosyl dihydrogen pyrophosphate) . It is not definitely known whether the a or the @ anomer of N-acetylD-glucosamine 1-phosphate is involved in this isomerization of N-acetylD-glucosamine 1-phosphate." However, an extremely acid-labile N-acetyl-D-glucosamine phosphate did contaminate some of the N-acetyl(69) L. F. Leloir and C. E. Cardini, Biochim. et Biophys. Acta, 12, 15 (1953). (70) L. F. Leloir and C. E. Cardini, Biochim. et Biophys. Acta, 20, 33 (1956). (71) J. L. Reissig, J . B i d . Chem., 219, 753 (1956).

BIOSYNTHESIS OF HYALURONIC ACID

311

D-glucosamine preparation. This labile phosphate derivative is inactive as a substrate. Since the anomer of D-glucosyl phosphate is more acid-labile than is the a! a n ~ m e r ,the ? ~ a anomer of N-acetyl-D-glucosamine 1-phosphate is considered to be involved in this reaction.71 Pig-kidney extracts have some phospho-N-acetyl-D-glucosamine mutase activity. The ratio of phosphoglucomutase to phospho-N-acetyl-D-glucosamirie mutase activity is 60:l in these extracts.?l In rabbit muscle, this ratio?' is 1200: 1. Phosphoglucosamine mutase activity was not checked in any of the phosphomutase preparations investigated by this worker. 3. Acetytation D-Glucosamine 6-phosphate can be readily acetylated by a N-acetylase obtained from a preparation of yeast h e ~ o k i n a s eThe . ~ ~ resulting N-acetyl~-glucosamine6-phosphate is identified by its Morgan-Elson reacti0n.7~ The acetyl-coenzyme A which appears to be required for this reaction may be generated by acetate, adenosine-5-triphosphoric acid, and coenzyme A (in the presence of an acetate-activating enzyme). Extracts of Neurospora c r a ~ s aand ~ ~ pigeon-liver acetone powder,76in contrast to the yeast N - a ~ e t y l a s ecan , ~ ~readily acetylate free D-glucosamine. However, N-acetyl-D-glucosamine cannot be phosphorylated by the hexokinase preparations thus far examined.63 66 Partially purified preparations from human liver also appear to catalyze the N-acetylation of D-glucosamine.76 Preliminary studies suggest that the enzyme catalyzing the acetylation of p-nitroaniline also catalyzes the acetylation of D-glucosamine. Other mammalian tissues (such as rabbit liver, kidney, and muscle, and dog kidney) have ~-glucosamine-6-phosphate-N-acetylase activity but not D-glucosamine-N-acetylase a ~ t i v i t yA . ~partially ~ purified catalytic system having the former, but none of the latter, activity has been prepared from Neurospora c r a ~ s aThe . ~ ~authors believe that the D-glucosamine-N-acetylase activity of the original Neurospora crassa extracts'j9 was attributable to the formation of D-glucosamine 6-phosphate1 catalyzed by the hexokinase present. Phosphorylation probably precedes acetylation when N-acetyl-D-glucosamine 6-phosphate is formed from D-glucosamine. Evidence that D-glucosamine l-phosphate is not acetylated was obtained by the acetylation of an equilibrium mixture of D-glucosamine 1-phosphate and D-glucosamine 6-phosphate with a preparation of yeast N-a~etylase.?~ Analysis of the reaction products by the Morgan-Elson procedure,74either (72) M. I,. Wolfrom, C. S. Smith, 11. E. Pletchcr arid A. E. Brown, J . A m . Chem. SOC.,64, 23 (1942). (73) D. H. Brown, Riockivn. et Biophp. A d a , 16, 420 (1055). (74) W. T. J. Morgan and L. A. Elson, Bioche?ti. J . (London), 28, 988 (1934). (75) T. C. Chou and M. Soodak, J . B i d . Chew., 196, 105 (1952). (76) E. A. Davidson, H. J. Blumenthal and S. Roseman, in press.

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ROY L. WHISTLER AND E. J. OLSON

before or after acid hydrolysis, showed that no increase in N-acetyl-Dglucosamine occurs after acid hydrolysis.

4. Uridine Derivatives of N-Acetyl-D-glucosamine “Uridine diphosphate N-acetyl-D-ghcosamine” has been isolated and structurally ~haracterized.~? The compound can be obtained from a nucleotide fraction of yeast??by elution from a Dowex-1 anion-exchange column, and from guineapig livers by precipitation as the barium salt.60 The structure of “uridine diphosphate N-acetyl-D-ghcosamine,” namely, uridine 5-(2-acetamido-2-deoxy-~-glucopyranosyl dihydrogen pyrophosphate) (11),was derived from the chromatographic identification of the acid hydrolysis products, uridine 5-phosphate and uridine 5-pyrophosphate, and from the alkaline hydrolyzate, N-acetyl-D-glucosamine l-pho~phate.?~ The lability toward acid of the N-acetyl-D-glucosamine phosphate bond in “uridine diphosphate N-acetyl-D-glucosamine” suggests that it is a 1-phosphate bond. Liberation of inorganic phosphate from the uridine phosphate fragment by a specific 5-nucleotidase indicates that this fragment is uridine 5-phosphate.

OH

OH

I1

A uridine pyrophosphate derivative of N-acetyl-D-glucosamine which contains an extra phosphate group has been isolated from a hot-water extract of hen oviducts.78Uridine 5-phosphate and uridine 5-pyrophosphate have been recovered from acid hydrolyzates of this compound.78A diphosphate ester of N-acetyl-D-glucosamine can also be obtained from acid hydrolyzates of this uridine compound.71Because of the catalytic activity’l of phospho-N-acetyl-D-glucosamine mutase toward it, it is probably N-acetyl-D-glucosamine 1,6-diphosphate. “Uridine diphosphate N-acetyl-D-glucosamine” can undergo enzymic pyrophosphorolysis in the presence of pyrophosphate, to form uridined(77) E. Cabib, L. F. Leloir and C. E. Cardini, J. B i d . Chem., 203, 1055 (1953) (78) J. L. Strominger, Biochim. et Biophys. Acta, 17, 283 (1955).

BIOSYNTHESIS OF HYALURONIC ACID

313

triphosphoric acid and N-acetyl-D-glucosamine 1-phosphate. Rat-liver nuclei catalyze this reaction, but yeast pyrophosphorylase does not.60Both preparations catalyze60 the reversible pyrophosphorolysis of “uridine diphosphate D-glucose.” The uridine compounds resulting from the pyrophosphorolysis reaction can be identified through ultraviolet analysis of paper chromatograms.60Both “uridine diphosphate D-glucose” and “uridine diphosphate N-acetyl-D-glucosamine”60undergo pyrophosphate cleavage during pyrophosphorolysis. Rat-liver nuclei also catalyze the formation of a uridine pyrophosphate derivative of D-glucosamine from a-n-glucosamine 1-phosphate and uridine5-triphosphoric a ~ i d . 7 ~ It is not yet known whether “uridine diphosphate N-acetyh-glucosamine” can be formed by a transfer reaction between N-acetyl-D-glucosamine and L‘uridinediphosphate D-glucose.” 5. Biosynthesis of D-Glucosamine Leloir and Cardinias observed that the acetone powder of Neurospora

crassa is capable of synthesizing hexosamines on addition of either D-fructose 6-phosphate or D-glucose 6-phosphate, together with L-glutamine as a nitrogen source. Ineffective additives tested were D-xylose 5-phosphate, a-D-galactopyranosyl phosphate, D-glucose 2-phosphate, 6-O-phospho-~glucosyl phosphate, D-fructose 1,6-diphosphate, D-fructose 1-phosphate, D-mannose, D-glucose, D-fructose, maltose, 1,3-dihydroxy-2-propanone (dihydroxyacetone), and D-glycerose. D-Fructose 6-phosphate and ammonia interact to produce hexosamine when they are added to a fraction of pig-kidney protein together with catalytic amounts of N-acetyl-D-glucosamine 6 - p h o ~ p h a t e . Neither ~~ L-glutamine nor L-asparagine can replace ammonium sulfate as a nitrogen source in this system, and D-fructose 6-phosphate cannot be replaced by D-fructose, D-xylose, D-glucose, or D-ribose.68The presence of N-acetylD-glucosamine is also essential for this reaction. D-Fructose 6-phosphate is probably a more direct precursor of D-glucosamine 6-phosphate than is D-glucose 6-phosphate. Evidence for this belief was obtained on examining the apparent reversal of hexosamine synthesis. When D-glucosamine 6-phosphate was added to a hexoseisomerase-free, kidney-protein fraction together with N-acetyl-D-glucosamine &phosphate, it was quantitatively converted to ammonia and a substance having the chromatographic properties of D-fructose 6-pho~phate.’~ The reaction is not catalyzed by “uridine diphosphate N-acetyl-D-glucosamine,” N-acetylD-glucosamine 1-phosphate, N-acetyl-D-glucosamine, acetamide, N-acetyl(79) F. Maley, Gladys F. Maley and H. A. Lardy, J . Am. Chem. Soc., 78, 5303 (1956).

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ROY L. WHISTLER AND E. J. OLSON

glycine, N-acetyl-L-tryptophan, N-acetylcholine, ammonium acetate, hexose 6-phosphates, pyruvate, acetate, citrate, or a-ket0glutarate.7~ The D-fructose 6-phosphate to hexosamine 6-phosphate interconversion is observed not only with kidney preparations but also with brain, liver, lung, and intestinal-tissue extracts.?O N-Acetyl-n-glucosamhe 6-phosphate is metabolized to the D-fructose ester more slowly (by preparations of kidney enzyme) than is D-glucosamjne 6-ph0sphate.7~Acetate inhibits the disappearance of N-acetyl-D-ghcosamine 6-phosphate, but does not affect the disappearance of D-glucosamine 6-phosphate. A possible sequence of reactions for these transformations is as follows.70 N-Acetyl-D-glucosamine6-phosphate 2 X

+ acetate + NH3 X D-Glucosamine 6-phosphate D-fructose &phosphate + NH3 D-fructose 6-phosphate

Compound “X” is believed to be N-acetyl-D-fructosylamine 6-phosphate. An extract of Eschericha coli, free from hexoseisomerase, is capable of converting D-glucosamine 6-phosphate to D-fructose 6-phosphate plus ammonia. No added cofactors are required for this reaction.80 Other possible metabolic pathways for the synthesis of D-glucosamine units have been investigated. On feeding g l y ~ i n e - l C ~ ~to - Nhens, ’ ~ the N16: CI4 ratio of the recovered ovomucoid D-glucosamine is fifty times higher than in the original glycine.81The low CL4content of the D-glucosamine from the ovomucoid shows that but little incorporation of the carbonyl carbon atom of the glycine had occurred. Therefore, it is unlikely that either glycine or L-serine produces hexosamine precursors through condensation reactions. 2-Amino-2-deoxy-~-gluconic acid, which is a conceivable endproduct of such a condensation; is metabolically inert to mammalian tissue.82 Unlike fractions of pig-kidney protein, Neurospora crassa extracts can use L-glutamine, but not ammonium salts nor ammonium salts plus adenosined-triphosphoric acid. No synthesis of D-glucosamine is stimulated in Neurospora extracts by L-glutamic acid, L-aspartic acid, L-asparagine; L-alanine, glycine, L-valine, L-leucine, L-lysine, L-arginine, L-serine, L-cysteine, L-citrulline, L-ornithine, butyramide, putrescine, or urea.69 Recently, a protein fraction has been discovered, in rat liver, that converts D-glucose (80) D. G . Comb and S. Roseman, Biochim. et Biophys. Acta, 21, 193 (1956). (81) S. V. Rieder, Univ.Microfilms (Ann Arbor, Mich.), Pub. No. 5690, 119 pp.; Dissertation Abstr., 13, 634 (1953). (82) Cecilia Lutwak-Mann, Bioehem. J . (London), 36, 610 (1941).

BIOSYNTHESIS O F HYALURONIC ACID

315

6-phosphate to he~osamines.8~ L-Glutamine can donate nitrogen for this conversion, but ammonia cannot. No added cofactor is required for this hexosamine synthesis from D-glucose 6-phosphate.

IV. BIOSYNTHESIS OF HYALURONIC ACID 1. Incorporation of Hexose u-Glucose appears to be incorporated as a unit into both the D-glucosamine and D-glucuronate units of hyaluronate. Hemolytic streptococci A l l l , incubated for 30 hours with ~-ghcose-6CI4, produce radioactive hyaluronate. The D-glucuronate units of the hyaluronate are labeled predominantly on carboxyl carbon atoms.G3When incubated with ~-glucose-l-C’~, well over five sixths of the radioactivity in D-glucuronate is found in the C1 to C5 fragment.G4When hemolytic streptococci are incubated with either ~-glucose-l-C’~ or -6-C14,the D-glucosamine units of hyaluronate are labeled almost exclusively on the respective C1 or C6 64 Other experiments show that the carbon skeleton of D-glucose is also ‘O D-Glucose seems t o incorporated into endogenous hexosamine be converted t o a D-glucosamine derivative before its incorporation into hyaluronate. Thus, D-glucosamine and N-acetyl-D-glucosamine, added to a suspension of hemolytic streptococci containing ~ - g lu c o s e - l- C ~greatly ~, reduce the amount of CI4incorporated into D-glucosamine units of hyaluronate.g4D-Glucosamine remains intact during incorporation into hyaluronate. This was directly demonstrated by the incubation of D-glucosamine-l,6C142-N16 with hemolytic streptococci.86 The N16:C14 ratios in the added D-glucosamine and in the D-glucosamine units of the resulting hyaluronate were similar. D-Glucosone does not appear to be a metabolic intermediate for the D-glucosamine of streptococcal hyaluronate. After addition of uniformly labeled D-glucosone to hemolytic streptococci A1 11, the radioactivity in the carboxyl carbon atoms of n-glucuronate from hyaluronate is similar to the average radioactivity of the carbon atoms of D-glucosamine.86It is recognized8Gthat D-glucosone86ais chemically a highly labile substance. Radioactivity introduced into hyaluronate by “uridine diphosphate N-acetyl-D-glucosamine” labeled with acetyl-l-C14 in a homogenate of Rous chicken-wing sarcoma (free from nucleoside-triphosphoric acid) is (83) B. M. Pogell, Bioehim. et Biophys. A d a , 21, 205 (1956). (84) Y. J. Topper and M. M. Lipton, J . Biol. Chem., 203, 135 (1953). (85) A. Dorfman, S. Roseman, Frances E. Moses, J. Ludowieg and M. Mayeda, J . Bid.Chem., 212, 583 (1955). (86) A. Dorfman, S. Roseman, J. Ludowieg, M. Mayeda, Frances E. Moses and J. A. Cifonelli, J. Biol. Chem., 216, 549 (1955). (86a) S. Bayne and J. A. Fewster, Advances i n Carbohydrate Chem., 11, 43 (1956).

316

ROY L. WHISTLER AND E. J. OLSON

not lowered by excess acetate.87 Therefore, “uridine diphosphate N-acetylD-glucosamine” probably donates the complete N-acetyl-D-glucosamine unit for hyaluronate synthesis and it may be a direct hyaluronate precursor. This view is supported by other experimental data.87Increased radioactivity is found when uridine-5-triphosphoric acid is added to a homogenate of sarcoma (free from nucleoside-triphosphoric acid) that contains N-acetylD-glucosamine 6-phosphate labeled with acetate-l-C14. The Cl4-labeled uridine pyrophosphate derivative isolated from this solution is believed to be formed by the following reaction.87

+ N-acetyl-D-ghcosamine 1-phosphate “uridine diphosphate N-acetyl-D-ghcosamine” + pyrophosphate

Uridine-5-triphosphoric acid

Additions4of D-glucuronate to a culture of hemolytic streptococci does not significantly reduce the incorporation of ~-glucose-l-Cl~ into hyaluronate; hence, exogenous D-glucuronate is apparently not incorporated into hyaluronate. The lack of incorporation of free D-glucuronate units into hyaluronate and into 8-D-glucosiduronates (see Section 11, 2) makes possible the existence of a common precursor for these two products. The direct precursor for 0-D-glucosiduronate, namely, (‘uridine diphosphate ~-glucuronate,”~~ may also be the direct precursor for D-glucuronate units in hyaluronate. If this is true, “uridine diphosphate D-glucose” is probably a metabolic intermediate for the transformation of D-glucose into the D-glucuronate of hyaluronate.62 Hyaluronate molecules in an intermediate stage of synthesis have not been isolated. Loss of radioactivity on dialysis of sarcoma hyaluronate has been considered to be attributable to the diffusing away of hyaluronate oligosaccharide~.~~ However, the di-, tetra-, hexa-, and octa-oligosaccharides from hyaluronate are not precipitated along with hyaluronate when it is precipitated prior to dialysis. Any diffusible oligosaccharide which causes this loss of radioactivity is larger than an 0ctamer.~7The lower viscosities of diluted synovial fluid from patients with rheumatoid arthritis suggest that the hyaluronate is in a lower state of p~lymerization.~~ Possibly, rheumatoid arthritis interferes with hyaluronate polymerization. 2. L-Glutamine

L-Glutamine donates nitrogen for hexosamine synthesis in Neurospora crassa and for the D-glucosamine units of hyaluronate in ~treptococci.~9 Of twenty-three amino acids (and ammonium chloride) tested with group A streptococ~i,~~ L-glutamine was the only consistent nitrogen-donor for (87) L.Glaser and D. H. Brown, PTOC. Natl. Acad. Sci. U.S., 41, 253 (1955).

BIOSYNTHESIS O F HYALURONIC ACID

317

streptococcal hyaluronate. In these experiments, formation of hyaluronate is indicated by addition of acidified horse-serum to a medium freed from cells. A positive test is the observation of turbidity, produced by the interaction of hyaluronate with serum protein to form a mucin clot. Addition of hyaluronidase prevents the development of turbidity on addition of serum protein. Turbidity formed in the medium is not caused by hyaluronate from capsules, since the hemolytic streptococci used are not encapsulated. Hexosamine analysisss of the cell-free supernatants, before and after acid hydrolysis, indicates that bound D-glucosamine is synthesized. The D-glucosamine has been identified by x-ray analysis after isolation. Because there is no increase in bacterial nitrogen when these hemolytic streptococci are incubated under equivalent conditions with a standard bacterial broth, protein synthesis is probably not a factor in these experiments. L-Asparagine, L-serine, and ammonium chloride enhance hyaluronate synthesis in some fresh cultures of hemolytic streptoc~cci.~~ Both amino acids yield ammonia in these cultures, and, therefore, may stimulate hyaluronate synthesis in this way. Stimulation of hyaluronate formation by these two amino acids or by ammonium chloride does not occur in 4-day-old streptococcal suspensions unless L-glutamate is added.44 As L-glutamate by itself is inactive, this activation suggests that L-glutamate combines with the ammonia produced from L-asparagine, L-serine, or ammonium chloride, to produce L-glutamine which activates the hyaluronate synthesis. Hyaluronate present in the cell-free medium, after hemolytic streptococci have been incubated with ammonium L-glutamate, can be identified both by infrared and chemical analysis.44 Hyaluronate synthesis is sometimes decreased in the presence of L-glutamine when the suspensions age.44 The place of L-glutamine in hyaluronate synthesis is shown by experiments with methionine s ~ l f o x i d eSince . ~ ~ methionine sulfoxide (2-amino-4methylsulfinylbutyric acid) inhibits L-glutamine formation, the addition of the sulfoxide to a culture of hemolytic streptococci inhibits hexosamine synthesis and, consequently, hyaluronate formation.44This inhibition can take place at concentrations of methionine sulfoxide which are low enough not to inhibit hyaluronate synthesis with ~ - g l u t a m i n e . ~ ~ Adenosine-5-triphosphoric acid, magnesium sulfate, and cysteine bring about L-glutamine formation from ammonium L-glutamate. However, significant hexosamine synthesis is not observed when these three substances are incubated with ammonium L-glutamate in the presence of a residue-free supernatant from crushed, bacterial cells.44L-Glutamine stimu(88) L. A. Elson and W. T. J. Morgan, Biochern. J . (London), 27, 1824 (1933).

318

ROY L. WHISTLER AND E. J. OLSON

lates the synthesis of bound hexosamine when it is incubated under the same conditions. Adenosine-5-triphosphoric acid, magnesium ions, cysteine, and D-glucose are required for substantial synthesis of hexo~amine.~~ At a low concentration of ammonium L-glutamate or L-glutamine, synthesis of bound D-glucosamine appears to be directly proportional to the amount of amino acid When ammonium L-glutamate labeled with amm~nium-N'~ ions is incubated with streptococcal suspensions, a t least 80% of the N16 is recovered in the D-glucosamine of h y a l ~ r o n a t e . ~ ~ This high recovery of N16 in the D-glucosamine shows that its nitrogen is principally derived from the umide nitrogen of L-glutamine. 3. Incorporation and Exchange of Acetyl

Acetyl groups of hyaluronate are, most probably, introduced into Dglucosamine 6-phosphate (see Section III,3). This would be transformed to the N-acetyl-D-ghcosamine units, which are then incorporated into the developing hyaluronate molecule.85Although acetate exchange may take place with streptococcal hyaluronate, exchange probably does not occur in rabbit hyaluronate. A~etate-1-C'~~ incubated with hemolytic streptococci, is readily incorporated into hyaluronate.86Although a labeled acetyl group of N-acetylD-glucosamine is also incorporated into hyaluronate in a similar suspension, the amount introduced is decreased when free acetate is added simultane0usly.8~Furthermore, when the free acetate is so added, the radioactivity of the acetate in the medium is greater than the activity of the acetyl groups of the streptococcal hyaluronate. Extracts of Escherichiu coli are found to hydrolyze N-acetyl-~-glucosamine.~~ Since attempts to demonstrate phosphorylation of N-acetyl-D-glucosaminehave not been successful,63-66 N-acetyl-D-glucosamineis not a probable intermediate in hyaluronate formation. When D-glu~ose-1-C~~ is incubated with hemolytic streptococci, equal specific activities are found in derived acetate from the medium and derived acetate from h y a l ~ r o n a t eThis . ~ ~ indicates a ready exchange between free acetate and hyaluronate acetyl groups. On the other hand, acetyl groups of hyaluronate in the higher animals may not undergo exchange. This is indicated by the equivalent, biological half-lives of radioactive hyaluronate formed by subcutaneous injection into rabbits either ofg0acetate-l-CL4or of uniformly Iabeled ~ - g l u c o s e Thus, .~~ (89) S. Roseman, Federation Proc., 13, 283 (1954). (90) Sara Schiller, M. B. Mathews, L. Goldfaber, J. Ludowieg and A. Dorfman, J . B i d . Chem., 212, 531 (1955). (91) Sara Schiller, M. B. Mathews, J. A. Cifonelli and A. Dorfman, J . B i d . Chew,., 218, 139 (1956).

BIOSYNTHESIS OF HYALURONIC ACID

319

acetyl groups and D-glucosamine units are believed to “turn over” at equivalent rates. Furthermore, preliminary evidence indicates that acetyl groups are not exchanged in rabbit hyaluronate. This evidence is based on an analysis of radioactive, rabbit hyaluronate produced by injecting uniformly labeled ~ - g l u c o s eAnimals .~~ sacrificed at any time after receiving the injection have hyaluronate with the same specific activity as the Dglucosamine hydrolyzed from it.

This Page Intentionally Left Blank

Author Index for Volume 12 Numbers in parentheses are footnote numbere. They are ineerted to indicnte the reference when a n author's work is cited but his nnme is not mentioned on the page.

A Abdel-Akher, M., 113, 280 (loo), 281, 283 (102a), 290 (loo), 291 (loo), 294 (loo), 295 (loo), 297 (100). Abderhalden, E., 172 Adams, M. H., 139, 152 (20), 154 (20), 219 Adkins, H., 149 Akiya, S., 205, 206, 245 Alberda van Ekenstein, W., 75 Alburn, H. E., 300 Alexander, B. H., 177 Allerton, R., 126, 139, 151, 153 Alm, R. S., 240, 241 (124) Alsberg, C. L., 267 Amor&, L., 177 Anand, N., 142 Anbar, M., 133 Anderson, C. C., 141 Anderson, D. M. W., 279,296 (88 see 89a) Anderson, J. M., 141 (45), 142 Anderson, L., 104 Anderson, W., 141, 142 (37) Andrzejewski, H., 279 Angyal, S., 101, 103, 139, 143, 144 (14, 49), 147 (14), 151, 155 (14), 156 (14) Anno, K., 265 Ansell, E. G., 122, 123 (37), 126 (37), 128 (37), 132 (37) Appel, H., 180, 181 (124, 127) Wqvist, s.,110, 111 Araki, T., 38 Archibald, A. R., 275, 278 (74), 280 (101 see 98), 281, 283 (74), 290 (74), 294 (74), 296 (74) Armstrong, E. F., 181, 184 (130, 132) Asboe--Hamen,G., 300 Ashe, L. H., 197 Ashton, G. C., 106 Asp, L., 174, 175, 181, 184 (131)

Aspinall, G. O., 40,77 (13) Aubert, J. P., 278, 295 (86) Atherton, F. R., 142 Augestad, I., 171, 183 (69) Ault, R. G., 182 Axelrod, J., 306, 308 (52), 316 (52)

B Babers, F. H., 185, 186 (163) Bacon, E. E., 265, 282 (18) Bacon, J. S. D., 33, 121, 135 (24), 265, 282 (18), 297 (96d) Baddiley, J., 142 Badger, R. M., 21, 22 (%), 23 (24), 24, 25, 26 (24) Baer, E., 72, 138, 139 (12, 13), 141 Baer, H. H., 178 Bailey, J. M., 278, 287 (84) Bailey, R. J., 95, 96 (50) Baker, B. R., 163 Baker, J. W., 131 Baldwin, E., 280, 281, 294, 297 (96d) Baldwin, R. R., 279 Ballou, C. E., 139, 141, 148, 149, 152, 153 (27), 156 (27) Balls, A. K., 219 Barclay, J. L., 141 (45), 142 Bhrceai-Martos, M., 160 Barker, G. R., 182 Barker, S. A,, 20, 21, 23, 24, 25 (19), 26 (19, 20, 29), 27, 32, 33, 94, 95, 97, 107, 108 (80), 113, 115 (58), 230, 266, 295 Barnes, R. B., 18, 19, 21 (lo), 28 Barnwell, J. L., 141 (45), 142 Barr, E. S., 29 Barry, C., 278, 295 (86) Barry, C. P., 187 Barry, V. C., 60 Bartels, H. E., 183

321

322

AUTHOR INDEX, VOLUME

Barton, D. H. R., 102 Bartlett, J. K., 281, 282 (107) Bates, F. L., 279 Bath, J . , 30 Baum, H., 268 Baur, L., 176, 183 (105) Bay, I., 131 Bayne, S., 315 Bear, R. S.,266, 279, 296 (27) Bebbington, A., 113 Becker, E., 48 Beckett, C. W., 22 Beckmann, C. O., 243, 252, 288 Beckord, L. D., 224 Beensch, L., 182 Behrend, R., 119, 162 Beiser, A., 199 Bell, D. J., 33, 93, 94 (42), 101, 120, 121, 124, 125 (15), 126 (15, 19), 130, 135 (24), 139, 147 (23), 150 (23), 154 (23), 155 (23), 265, 267, 270, 271, 274, 275, 276 (79), 279 (23), 280, 281, 282 (53, 56, 108), 283, 286 (23), 287 (23), 290 (23, 52, 53, 54, 57, 79, 108), 291 (31, 53), 294 (23, 55, 79, 105), 295 (20), 297 (54, 96d) Bellamy, L., 21, 28 (23) Bembry, T. H., 169 Ben-Gershom, E., 231 Benjamin, D. G., 160, 166 (6), 169 (6), 176 (6), 177 (6), 180 (6), 181 (6), 184 03, 186 (6) Benoy, M. P., 66, 69, 71 (87a) Benson, A. A., 114 Bera, B. C., 101 Beran, K., 169 Berger, E., 141 Bergey, D. H., 197 Bergmann, M., 141, 166, 172, 176, 186 Bernard, C., 262 Berner, E., 171, 183 (69) Bernfeld, P., 273, 275, 286 (62 see 127) Berthet, J., 298 Biltz, W., 234 Binkley, W. W., 81, 172 Birkeland, J., 301 Bishop, F. W., 304 Black, I. M. A , , 185, 193 (16), 195, 201 (16), 263 Blair, M. G., 73, 172

12

Blanchard, P. H., 101 Blay, H., 177 Blinc, M., 192 Blix, G., 110, 300, 304 Block, R. J., 83, 84 (5a see 11), 91 (5a see 36) Blomquist, G., 193 (21), 195, 203 204 (21), 239 (21), 247 (21), 260 (21) Bloom, W. L., 267,268 Blumenthal, H. J., 309, 311 Boas, N. F., 300 Bobbitt, J. M., 280 (102), 281 Boeseken, J., 86, 92, 98 Bohn, E., 164 Boissonnas, R. A., 283 Bollenback, G. N., 160, 166 (6), 169 (6), 176 (6), 177 (6), 180 (6), 181 (6), 184 (6), 186 (6) Bolliger, H. R., 61, 141, 149 (41), 257 Bolotina, T. T., 279 Bonner, W. A., 169, 173, 175 (62) Boppel, H., 193 (23), 195, 203 (23), 216, 247 (78), 248 (78), 252 (78) Borchert, W., 236 Bordner, R. H., 266, 293 (26), 295 (26) Borntrager, A., 262, 263 Boschan, R., 117 Bosse, R., 169, 180 (591, 181 (59) Bottle, R. T., 268, 175 Bourne, E. J., 20, 21, 23, 24, 25 (19), 26 (19, 20, 29), 27 (291, 32, 33, 89, 90 (27), 94, 95, 96 (50), 97, 102 (27), 103, 107, 108 (80), 109 (27), 110, 113, 115 (58), 122, 230, 266, 293, 295 Bouveng, H., 93 Bower, R. S., 123, 124 (42), 125, 127 Brandt, S., 139, 147 (22), 155 (22) Brattain, R. R., 28 (41), 29 Braun, G., 162, 185 (23) Brauns, D. H., 172, 183, 185 (145), 186 Bray, H. G., 304, 305 Bredereck, H., 141,148 (361,166,180 (45), 184 (45) Breed, R. S., 197 Bretschneider, H., 169 Brewster, J. F., 86 Bridgman, W. B., 267, 275 (37), 277 (37) Briggs, D. R., 109 Brigl, P., 141, 187 Brink, N. G., 30

AUTHOR INDEX, VOLUME

Brissaud, L., 121, 123 Brough, G. W., 124 Brown, A. C., 120 Brown, A. E., 311 Brown, D. H., 308, 309, 311, 313 (68), 318 (63) Brown, D. M., 94, 142, 275 Brown, G. B., 186 Brown, J . R., 129 Brown, L., 30 Bruce, G. T., 32, 94 Brunish, R., 304 Buchler, C. C., 138, 142, 156 (1) Budovich, T., 260 Biicher, T., 96 Bueding, E., 305 Biirklin, E., 193 (14), 194 (14), 195, 201 (14), 246 (14), 256 (14) Burke, D. C., 101 Burket, S. C., 21, 22 (24), 23 (24), 24, 25, 26 (24), 32 Burton, H., 151 Busch, K. G. A., 44 Butler, G. C., 307 Butler, K., 94 C Cabib, E., 312 Cadotte, J. E., 171, 182 (74), 183 (74) Caesar, G. V., 121, 234, 256 Caldwell, M. L., 284, 295 (118) Calvin, M., 114 Cardini, C. E., 163, 310, 311 (69), 312, 313 (69, 70), 314 (69, 70), 315 (69, 70), 316 (69) Carpenter, E., 123, 124 (42) Carrington, T. R., 33 Carter, M. E., 40, 77 (13) Carter, N. M., 138, 141 Carter, S. R., 271, 275 Cecil, It., 275, 276 (79), 290 (79), 294 (79) Cernigoj, F., 225 Chaffee, E., 300 Chaikoff, I. L., 281 Chambers, L. A., 266, 293 (26), 295 (26) Chambers, M. A., 114 Chaney, A., 123, 124 (42) Chanlaroff, M. B,. 56 Chargaff, E., 276, 293 (82) Cheniae, G. M., 96, 114 (55)

12

323

Chittum, J. W., 37 Chou, T. C., 311 Chrisman, C. H., Jr., 29 Christie, S. M. H., 142 Christman, C. C., 187 Cifonelli, J. A., 280, 291 (94), 296 (94), 315, 318, 319 (91) Clark, J. W., 44 Clark, R. K., Jr., 30 CILtrk, V. M., 142 Clautriau, G., 262 Cleveland, F. C., 275, 296 (73) Cleveland, F. F., 17 Coblene, W. W., 29 Cohn, W. E., 114 Coleman, G. H., 86, 139, 141, 147 (22), 148 (26, 34), 151, 154 (26), 155 (22, 65), 164 Colley, A., 118, 160 Coldn, A. A,, 177 Comb, D . G., 314 Combs, E. E., 141, 148 (34) Compton, J., 122 Conchie, J., 162, 168 (20), 177 (20), 180 (20), 181 (20), 183 (20), 184 (20) Conrad, H. E., 94 Conrad, M., 46 Consden, R., 82, 86, 87, 89 (17), 91, 92, 104 Cook, M., 297 Cookson, R. C., 102 Coolidge, T. B., 82 Cooper, C. J . A., 193 (17), 195, 201 (17 see 71) Corbett, W. M., 40, 53, 60, 70, 71, 72, 75, 77 (14), 78 (68), 97 Corby, N. S., 142 Cori, C. F., 193, 195, 225, 239, 246, 266, 286,287, 288,292 (125) , 293 (125), 296 Cori, G. T., 246, 278, 284, 285 (120), 286, 288, 289 (120), 292, 293 (85, 125, 135), 296 (85, 135) Counsell, J. N., 90 Craine, E. M., 269 Cramer, F., 193 (30), 194, 195, 219 (38), 228, 229 (38), 234 (37), 235, 241 (37), 245,246 (38), 248, 252, 254, 256, 259 Cramer, F. B., 185 Creite, C., 33 Cremer, H. D, 85

324

AUTHOR INDEX, VOLUME

Cremer, M., 262 Cristol, S. J., 126 Crowley, N., 301 Cuisinier, L., 48, 70 (36), 78 (36) Cushing, M. L., 234, 256 Czuros, Z., 139, 143, 144 (14), 147 (14), 151, 155 (14), 156 (14)

D Dale, J. K., 162, 182, 183 (137), 185, 186 (15, 18, 153, 158) Daniels, H. S., 219 Danilov, S. N., 37 Davidson, E. A., 311 Davoll, J., 186 Dawson, M. H., 300 Debye, P., 274 de Duve, C., 298 Deferrari, J. O., 187 Demant, S., 169, 180 (59), 181 (59) Demmig, W., 180 Dennison, J. C., 139, 150 (25), 154 (25), 155 (25) Denzler, A., 33 de Pascual, J., 148 Derksen, J. C., 200 Dernikos, D., 199 Deuel, H., 33 Deulofeu, V., 187 Deutsch, A., 142 Dewar, J., 120, 121, 123 (32), 124 (49), 127 (32), 135 (32,49) Dickey, E. E., 123, 124 (42) Di Domenico, J., 126 Diehl, H. W., 32 Dillon, R. T., 170 Dimler, R. J., 95 Dische, Z., 306 Ditmar, R., 183, 185 (150) Dobriner, K., 31 Doherty, D. G., 114 Doppstadt, A., 166, 167 (47) Dorfman, A., 172, 180 (80),181 (80), 184 (80), 306, 315, 316 (a),318, 319 (91) Dornfeld, C., 151, 155 (65) Dostrovsky, I, 133 Douglas, J. F., 307 Drehfahl, G., 176, 182 (112) Dube, H. A , , 243, 249, 252 Ihbrunfaut, A. P., 48, 70 (35), 78 (35 see c ) , 79 (35)

12

Dtirr, W., 138, 145 (4), 147 (4), 149 (4), 150 (4), 154 (4) Dumpert, G., 193 (25), 195, 225 (25), 252 (25) Durrum, E. L., 83,84, 91 (5a see 36) Durso, D . F., 97, 107 (59) Dutton, G. J., 305, 316 (49) Dvonch, W., 266, 295 (30) Dziewiatkowski, D. D., 306

E Easty, D . M., 131 Edgar, R. H., 66 Edwards, T. E., 96, 97 (52), 282, 293 (110) Effenberger, J. A., 206, 207 (75), 208 (75) Ehrenberg, J., 141 Eisenberg, F., Jr., 307 Eisenlohr, F., 53, 78 (50) Eissler, F., 199 Ellis, J. W., 30 Elrick, D . E., 129 Elson, L. A., 311, 317 Elving, P. J., 123 Engels, H., 138, 145, 147 (3), 154 (3) Ennor, K. S., 126, 129 (60) Ergle, D. R., 268 Errera, L., 262, 293 (6) Erving, J. J., 109 Erwig, E., 162 Evans, W. L., 44, 66, 69, 71 (87a), 163, 164 Ewald, L., 193 (21), 195, 203 (21), 204 (21), 239 (21), 247 (21), 260 (21)

F Farrar, K. R., 141 Fasman, G. D., 94, 142 Fekete, G., 269 Fellig, J., 31, 304 Fernbdez-Garcia, R., 177 Ferno, O., 142 Ferrante, G. R., 170 Fewster, J. A., 315 Field, C. W., 82 Field, J. B., 307 Finkelstein, H., 127 Fiorini, W., 201, 246 (69) Fischer, E., 47, 64, 72, 77 (loo), 79, 166, 170, 172, 180 (43), 181, 182, 183 (138), 184 (130, 132), 186

AUTHOR INDEX, VOLUME

Fischer, E. H., 31, 304 Fischer, F. E., 153 Fischer, H. 0. L., 72, 138, 139, 141, 146, 147 (53), 148 (27, 53), 152, 154 (27), 153 (27) Fischer, R., 141, 149 (41) Fishman, W. H., 307 Fittig, It., 46, 57 Fleming, I. D., 275, 278 (74), 283 (74), 290 (74), 294 (74), 296 (74) Flexner, L. B., 290 Fletcher, H. G., Jr., 32, 139, 151, 153, 161, 162 (9), 164, 173 (8),183 (7, 8), 187 Fleury, G., 121, 123 Flodin, P., 82, 83 (3) Flynn, 13. H., 30 Folkers, K., 30 Follett, A. E., 300 Fong, J., 269 Forchielli, E., 300 Forster, E. O., 252 Fort, G . , 120, 121, 123 (32), 124, 125 (49), 127 (32), 135 (32, 49) Foster, A. B., 84, 85 (12b), 86, 88, 89, 90 (27), 91 (12b), 92, 93 (22, 25, 41), 94, 95 (25), 97 (25, 261, 98, 99 (621, 100 (63), 101, 102 (27), 103, 104, 105 (41), 106 (26), 107, 109 (27), 110, 112, 113, 141, 142 Foster, M. C., 106 Fox, J. J., 28 Frahn, J. L., 102, 103, 106, 107 (69), 108, 109 Frazer, J. H., 123, 124 (42) French, D., 190, 193, 194, 195, 203, 206, 207 (75), 208 (75), 216, 217, 218 (3, 27), 219 (33), 223, 224 (3), 226 (32, 33, 89), 231, 232, 235, 236, 239, 240, 241 (123), 242, 244, 245, 246 (31, 33, 73, 122), 247 (3), 250, 252 (331, 255, 259 (122), 260, 266, 279 Freudenberg, K., 120, 138, 139, 141, 145, 147 (3, 4, 21,24), 148 (21, 241, 149 (4), 150 (4), 151 (15), 152 (15), 154 (3, 4, 15, 24), 155 (21, 24), 193, 194, 195, 198, 203, 204 (21), 205 (241, 206 (22), 211 (72), 212, 213, 214, 216, 218 (20, 77), 225, 231 (22), 234 (20, 37), 235, 237, 239, 241, 243, 245, 247, 248 (78), 252, 254, 265, 256, 258, 260 (21)

325

12

Friedemann, T. E., 67, 72 (83) Friedlaender, P., 138 Frush, H. L., 86, 26,33, 163, 165 (30), 178 183 (120), 185 (120) Fuld, M., 266, 271, 286 (60 see 127), 295 (29), 296 Fuller, K. W., 110 G Gartner, E., 138, 145 (4), 147 (4), 149 (4) 150 (4), 154 (4) Gakhokidze, A. M., 37, 185 Gardell, S., 110, 111 Garibaldi, J. A , , 219, 220 (80), 222, 229 (80), 259 (80) Garner, E. F., 109 Garton, G. A., 181 Gauhe, A., 171, 173 (73), 182 (73), 185 (73) Gavard, R., 278, 295 (86) Genung, 1,. B., 44 Gerecs, A., 166, 172 Gibbons, G. C., 283 Gibbs, M., 76 Gibson, G. E., 120 Gilbert, G. A., 268, 275 Gjorling, L. G., 193 (29), 195, 224, 225 (29) Gladding, E. K., 120, 131, 132 (74) Glaser, E., 166 Glaser, L., 316 Glasstone, S., 17 Glattfeld, J. W. E., 37 Gnuchtel, A., 127 Goebel, W. F., 139, 152 (20), 154 (20), 153, 185, 186 (163) Goerdeler, J., 169, 180 (59), 181 (59) Gohlke, B., 141 Goldberg, L., 135 Goldfaber, L., 318 Goldfrank, M., 121 Goldstein, K., 199 Gomberg, M., 138, 142, 156 (1) Goodman, M., 114 Gootz, R., 180, 181 (127), 184 (126) Gordon, A. H., 82, 110, 111 Gore, R. C., 19, 28 Gorin, P. A. J., 141 (45), 142 Gottschlich, A., 166, 167 (47) Gottstein, L., 46 Grabilina, G. O., 47, 78 (32)

326

AUTHOR INDEX, VOLUME

Grant, P. M., 89, 90 (27), 102 (27), 103, 107, 108 (80), 109 (27), 110 Grant, P. T., 308, 318 (64) Grassmann, W., 84 Green, D. E., 232, 259 (103) Green, J. W., 69, 77 (85), 78 (85), 79 (85), 178 Greenway, R. M., 112 Greenwood, C. T., 265, 268, 274 (21), 275 (38), 276 (38), 279, 290 (38), 294 (38), 296 Greiner, W., 141, 148 (36) Grey, C. T., 301 Griebel, R., 181, 184 (134) Gross, D., 85, 90, 91, 101, 102, 103, 104, 105, 107 Gruen, D. M., 31 Gruenhut, N. S., 234, 256 Gunther, E., 180, 184 (126) Gulland, J. M., 141 Gutfreund, H., 275, 276 (79), 290 (79), 294 (79) Gutzheit, M., 46 GvozdjBk, J., 268 (44), 269 Gyermek, L., 269

H Haas, E., 300 Haacsy, I., 172 Hadidian, Z., 300 Hale, C. W., 300 Hale, W. S., 219, 220 (84), 222 (84) Hall, R. H., 142 Halsall, T. G., 280, 281 (97), 283, 287, 296 (129) Hamilton, J. K., 281, 283 (102a) Hamlin, K. E., 153 Henes, C. S., 272, 286 (61) Hann, R. M., 145, 147 (51, 52, 64), 150 (51), 151, 153 (511, 154 (51, 52), 156 (51, 52), 186 Hannig, K., 84 Hansen, R. G., 269 Hanrahan, V. M., 284, 295 (118) Hardegger, E., 148, 176 Harden, A., 262, 263, 264 Harding, T. S., 32 Harper, R. P., 308, 311 (65), 318 (65) Harrap, B. S., 274, 275 (71), 276 (71), 290 (71) Harrington, W. F., 238, 239

12

Harris, R. J. C., 31 Harrison, G. R., 19 Hartung, W.H., 148 Hartwell, H., 28 (42), 29 Harvey, W. E., 142 Hashimoto, Y., 86, 89 (18) Haskins, W. T., 145, 147 (51, 52, 64), 150 (51), 151,153 (51), 154 (51,52), 156 (51,521 Hassel, O., 22 Hassid, W. Z., 280, 281, 297 (98) Hawkins, W. L., 280 Haworth, W. N., 182, 193, 195, 201, 263, 269, 270, 271, 280, 282 (57), 290 (57), 291 Hawse, V. P., 118 Hayes, D. H., 141, 142 (37) Haynes, L. J., 142, 160, 163 (4), 178 (4), 179 (4) Hayward, L. D., 119, 129 Hazebroek, P., 253 Heatley, N. G., 269 Hehre, E. J., 226, 295 Heidelberger, M., 300 Heidt, L. J., 149 Heinaelmann, R. O., 153 Heiten, E., 77 (104 see b), 79 Helferich, B., 127, 161, 164, 165, 166, 167, 168, 169, 172, 173, 180, 181, 183 (88), 184 (10, 45, 125, 126, 128, 134), 185 (88)

Hemerline, A., 309 Herbst, W., 248 Herling, F., 31 Hermans, P. H., 102 Herrman, R. F., 135 Herold, F., 50 Hers, H. G., 297, 298 Herzberg, G., 13, 17 (see 8) Herzog, R. O., 237 Hess, K., 200, 236 (61, 62) Hestrin, S., 246 Heyns, K., 167, 176 (51), 177 (51) Hibbert, H., 280 Hintikka, S. V., 48, 70 (37) Hirst, E. L., 90, 93 (311, 182, 269, 271, 280, 281, 282 (57, 107), 283, 287, 290 (57), 291, 296 (129) Hirst, G. K., 301 Hitchens, A. P., 197 Hixon, R. M., 145

AUTHOR INDEX, VOLUME

Hjelt, E., 57 Hobbs, R. B., 109 Hobday, G. I., 141 Hobkirk, R., 112 Hobson, P. N., 295 Hocket,t, R. C., 69, 71 (87s) Hormann, O., 161 Hoff, G. P., 66 Hoffma,n, D. O., 123, 124 (42), 127 Hoffman, E. J., 118 Holliday, P., 30 Holt, N. B., 86 Holemzmn, H., 185 Honeyman, J., 121, 123 (30,37), 124 (30), 125 (30), 126, 128 (30, 37), 129 (60), 130 (30), 132 (30, 37), 133 (30), 134 (30), 152, 187 Hooghwinkel, G. J., 111 Hoppe-Seyler, F., 38 Horecker, B. L., 297 Hough, L., 90, 141 (45), 142, 281, 282 (107), 287,296 (129) House, L. R., 32 Howard, H . T., 142 Hudson. C. S., 32,43, 47 (18), 51 (17, 18), 52, 59 (17, 18), 61 (18), 145, 147 (51, 52, 64), 150 (51), 151,153 (51),154 (51, 52), 156 (51, 52), 161, 162, 163, 164, 169 ( 5 5 ) , 171, 172, 173 ( 8 ) , 175, 178, 180 (55), 181, 182, 183, 184 (55, 133), 185, 186, 187, 190, 193 (26), 195, 216, 218 (79), 219, 220 (85), 221 (85), 222 (26, 79, 85), 224, 232 (79), 258, 259 Huebner, C. F., 176 Hull, G., 139, 141 (21), 147 (21), 148 (21), 155 (21) Humphrey, J. H., 301 Huniphries, P., 31 Hurd, C. D., 169, 175 (62) Hurwitz, O., 193 (13), 194 (13), 195, 201 (13), 231 (13 see 101), 246 (13), 256 (13) Husemann, E., 159, 271, 274, 275 (68), 276 (58) Hutton, D., 37

I Illingworth, B., 278, 284, 285 (120), 286, 288, 289 (120), 292 (85, 120, 125), 293 (85, 125), 296 (85) noE, A., 165, 172

327

12

Inatome, M., 265 Insull, W., 290 Irvine, J. C., 122, 174, 185, 193, 194, 195, 201 (16), 243, 256, 263 Isajevic, V., 275, 296 (76) Isbell, H. S., 26, 33, 65, 67, 86, 163, 165 (30), 173, 178, 183 (120), 185 (120) Isherwood, F. A., 271, 280 (57), 281, 282 (57), 290 (57)

J Jackson, E. L., 182, 183 Jacobi, R., 193 (20), 194 (20), 195, 203, 211 (72), 212 (20), 213, 218 (20), 234 (20) Jacobi, W., 138, 145 (4), 147 (4), 149 (4), 150 (4),154 (4) Jacobson, H., 243 Jacques, P., 298 Jaenicke, L., 86, 89 (20) Jarnestrom, T., 239 (131), 245, 246 (131) James, A. E., 32 James, W. J., 250 Jayme, G., 180 Jeanes, A., 32, 95 Jeanloz, R. W., 139, 150 (31), 156 (31), 161, 164 (8), 173 (8), 183 (7, 8), 187 (7), 267, 268, 276 (36), 290 (36), 293, 294 (36), 295 (139), 300 Jermyn, M. A., 169 Jette, E. R., 82 Johnson, J. M., 162, 185, 186 (161, 162) Jones, A. S., 86 Jones, I. G., 275, 278 (74), 283 (74), 290 (74), 294 (74), 296 (74) Jones, J. K. N., 90, 93 (31), 141 (45), 142, 171, 280, 281, 282 (107), 283, 287, 296 (129) Jones, R. N., 31 Joseph, J. P., 163 Jung, K.-H., 167

K Kabler, P. W., 31 Kainova, A. S., 296 Kalckar, H . M., 306, 308 (52), 316 (52) Kamil, I . A., 176, 305 Karabinos, J. V., 92 Kariyone, T., 138, 141 (8) Karrer, P., 126, 153, 193, 194, 195, 201,

328

AUTHOR INDEX, VOLUME

231 (13 see 101), 246 (12-14, 69), 256, 269 Karunairatnam, M. C., 307, 308 Kass, E. H., 301 Kata, J. R., 200 Katsbeck, W. J., 275, 296 (73) Kaye, M. A. G., 304 Kelch, C., 167, 176 (51), 177 (51) Kemp, A., 268 (44), 269 Kendall, F. E., 300 Kendall, J., 82 Kenner, G. W., 139, 142, 145 (32), 147 (32), 149, 152, 153 (32), 154 (32) Kenner, J., 53, 60, 69, 70, 71, 72, 74, 75, 78 (68), 97, 139, 141 (18), 149 (18), 155 (18) Kent, P. W., 111, 112, 293 Kerr, L. M. H., 307 Kerr, R. W., 224, 225, 259 (91, 92), 275, 296 (73) Khin Maung, 273, 275, 278 (74), 283, 284, 285 (64), 287 (115), 289 (64, 119), 290(74),293 (115),294(74),295(115), 296 (74) Khorana, H. G., 142 Khym, J. X., 96, 114 Kiliani, H., 42, 43, 44,45, 48, 49, 50, 53, 54, 55, 56, 57, 58, 59 (54, 65), 62 (62 see 74), 77 (20 see b, 54, 101), 78 (50, 52, 54, 56, 101), 79 Kimura, M., 86, 89 (18) Kimura, Y., 138, 141 (8) King, C. G., 307 King, H. K., 294 Kirk, P. L., 269 Kirschenlohr, W., 167, 173 (50), 183 (50), 185 (50) Kits van Heijningen, A. J. M., 268 (44), 269 Kleemann, S., 57 Klota, I. M., 31 Knapp, D. W., 193 (31), 194 (31), 195, 236 (31), 240 (31), 246 (31) Knauber, H., 212, 214, 218 (77), 237 (77), 255 Kneen, E., 224 Knorr E., 119, 124 (8), 132 ( 8 ) , 160, 163, 164 Koch, R., 196 Kocourek, J., 166

12

Koehler, W. L., 173 Konig, J., 172 Koenigs, W., 119, 124 (8), 132 (8), 160, 162, 163, 164 Korosy, F., 160 Kondo, K., 138 Kosterlitz, H., 270, 290 (52) Kowkabany, G. N., 81 Kratky, O., 237 Krauss, W., 138 Kremann, R., 124 Kroon, D. B., 111 Kubler, F., 139, 143 (30), 147 (30), 148 (30), 156 (30) Kuehl, F. A., Jr., 30 Kulz, E., 262, 263 Kuenne, D. J., 47, 73, 77 (96), 146, 147 (54), 148 (M), 154 (54) Kuhn, L. P., 32, 33,123, 124 (42), 126 Kuhn, R., 167, 171, 173 (50, 73), 178, 182 (73), 183 (50), 185 (50, 73’) Kulonen, E., 302 Kunkel, H. G., 84, 86 Kuyper, A. C., 280 (99), 281 Kusin, A., 75

L Lamb, R. A., 119 Lamp, B. G., 248 Lampen, J. O., 114 Landel, A. M., 104 Lange, F., 247 Langhans, A., 193, 194 ( l l ) , 199 (ll), 234 (11) Lardy, H. A., 309, 313 Larner, J., 284, 285 (120), 286 (120), 288, 289 (120), 292 (120) Lassettre, E. N., 282 Laurent, T. C., 304 Lautsoh, W., 257 Leake, C. D., 135 Leavell, G., 37 Lecocq, J., 141 Lehmann, H., 257 Lederer, M., 83 Leibowits, J., 199, 234 (56), 255 Leibowita, Y., 118 Leitch, G. C., 201 Leloir, L. F., 163, 310, 311 (691, 312, 313

AUTHOR INDEX, VOLUME

12

329

(69, 70), 314 (69, 70), 315 (69, 70), Long, J. W., 160, 166 (6), 169 (6), 176 (6), 177 (S), 180 (S), 181 (S), 184 (6), 186 316 (69) Lemieux, R. U., 30, 119, 163, 169, 174, (6) Longsworth, L. G., 238, 239 178 (26) Loofborrow, J. R., 19 Lenze, F., 118, 123 (3), 125 (3) Levene, P. A., 43, 47 (18), 51 (18), 59, Lorber, J., 139, 147 (23), 150 (23), 154 (23), 155 (23) 61 (18), 122, 141, 170, 173, 185, 186 Lorber, V., 297 187 Lord, R. C., 19 Levi, I., 280 Levine, M. L., 190, 193 (32), 195, 203 Los, M., 40, 77 (13) 216 (3), 217, 218 (3), 223, 224 (3), Lowther, D. A., 304, 316 (44), 317 (44), 318 (44) 225, 226 (32, 89), 227, 232 (89), 239, 243, 246 (73, 95, 122), 247 (3), 255 Lowy, B. A., 186 Ludowieg, J., 306, 315, 316 (84), 318 (3), 259 (122), 260 (32, 89) Lutwak-Mann, C., 314 Levine, S., 31, 33, 266, 293 (26), 295 (26) Levvy, G. A., 162, 168 (20), 177 (20), M 180 (20), 181 (20), 183 (20), 184 (m), McArthur, C. S., 141 306, 307, 308 McArthur, N., 120 Lewis, G. T., 267, 268 (34) Macbeth, A. K., 269 Lewis, H. B., 306 McClean, D., 300, 301 Lhoste, P., 123 McClenahan, W. S., 216, 218 (79), 224, Lichtenstein, S., 199, 224 (47) 232 (79) Liddel, U., 18, 19, 21 (lo), 28 (10) Liddle, A. M., 275, 278 (74), 283 (74), McCloskey, C. M., 139, 141, 147 (19, 22), 148(26, 34), 150 (19), 151, 152 (19, 284, 287 (121), 289, 290 (74, 121), 291 26), 155 (19, 22,65), 164, 233 (121), 294 (74, 121), 296 (74) McCready, R. M., 186,295 Liebermann, C., 46, 161 McDonald, D. L., 141 Liggett, R. W., 219, 243 McDonald, H. J., 83 Lilienfeld, L., 138 Macdonald, J., 193 (15), 195, 243, 256 Limpach, L., 45 Lindberg, B., 93, 97, 167, 174, 175, 181 McDowell, M., 267 McElroy, W. R., 123 184 (131) McGilvray, D. I., 139, 150 (17, 25), 152 Lindemann, E., 259 (17), 154 (25), 155 (17, 25) Lindquist, J. A., 160, 166 (6), 169 (6), 176 (6), 177 (6), 180 (6), 181 (6), 184 Machemer, H., 280 McHugh, D. J., 101, 103 (6), 186 (6) Link, K. P., 148, 149, 176, 182, 183 (105), McIntire, R. L., 244, 245 Mackay, J., 269 185, 187 (164) McKeown, G. G., 119, 129 (7b) Linker, A., 31, 302, 303 (34) McWain P., 123, 124 (42) Lipschitz, W. L., 305 Madinaveitia, J., 300 Lipton, M. M., 315 Magrath, D. I., 94, 142 Lloyd, P. F., 94 Maher, G. G., 125 Lobry de Bruyn, C. A., 75 Makarowa-Semljanskaja, N. N., 52 Lock, M. V., 114 LoefRer, P., 54, 56, 57, 59 (541, 77 (54, Maley, F., 309, 313 Maley, G. F., 313 101), 78 (54, 101), 79 Mangold, H. K., 248 Loevenhart, A. S., 135 Mann, F. G., 161, 162 (13) Lowa, A., 173, 183 (88), 185 (88) Manners, D. J., 265, 268, 273, 274, 275 Lohmar, R., 32 276 (38, 71), 278 (74), 279 (23), 280 Long, C., 308, 318 (64)

330

AUTHOR INDEX, VOLUME

(23, 101 see 98), 281, 283, 284, 285 (64), 286 (23, 117), 287 (23, 115, 121), 289, 290 (23, 38, 71, 74, 121), 291 (121), 292 (22), 293 (22, 115, 140), 294, 295 (115), 296 (74) Mannich, C., 163 Marans, N. S., 129 Mark, H., 30 Markham, R., 84 Marks, P. A., 297 Marsh, C. A., 162, 168 (20), 177, 180 (20), 181 (20), 182 (118), 183 (20, 118), 184 (20), 306, 307 (51) Martin, A. E., 28 Martin, A. J. P., 82 Martlew, E. F., 98 Mason, H. S., 142 Mathews, M . B., 318, 319 (91) Matthes, O., 42, 50, 77 (101), 78 (101), 79 Matthews, A. S., 91 Matula, J., 264 Mataelt, D., 96 Maurer, K., 176, 182 (112) Maxwell, E. S., 306, 308 (52), 316 (52) Mayeda, M., 315, 316 (84), 318 (84) Maaaero, L. W., 174 Mehltretter, C. L., 177 Meister, A. G., 17 Mellies, R. L., 177 Melvin, E. H., 32 Merrow, R. T., 117, 126 Messmer, E., 254 Meyer, A. S., 139, 147 (28), 149, 154 (28), 156 (28) Meyer, G. M., 43, 47 (18), 51 (18), 59 (IS), 61 (18), 187 Meyer, H., 141 Meyer, K., 300, 302, 303 (34), 316 (31) Meyer, K. H., 31, 266,267, 268,271,273, 274, 275, 276 (36), 280 (99), 281, 286 (60, 62, 65 see 127), 290 (36), 294 (36), 295 (29), 296, 304 Meyer-Delius, M., 193 (23, 24), 195, 203 (23, 24), 205 (24), 243 (24), 245 (24), 247 (24), 256 (24) Meyersohn, P., 199 Michael, A., 158 Michalski, J . J., 142 Micheel, F., 141 Michelson, A. M., 141, 142 Michl, H., 84, 86, 89 (19)

12

Miekeley, A., 193, 195, 200, 201 Milhaud, G., 278, 295 (86) Miller, A., 86 Miller, F. A., 19, 21 (14 see 22), 28 (40, 45), 29 Miller, G. E., 37 Mills, E. J., 125 Mills, G. T., 305, 307 (50), 312 (501, 313 (50) Mills, J. A,, 102, 103, 106, 107 (69), 108, 109 Missimer, J. K., 109 Mitchell, W. A., 173 Moggridge, R. C. G., 171 Mondori, B., 106 Monod, J., 231 Montgomery, E. M., 163, 169 ( 5 5 ) , 171, 175 (55), 180 (55), 181, 182 (70), 184 (55, 133), 186 Montgomery, R., 38, 280,281, 283 (102a), 291 (94), 296 (94) Montgomery, T. N., 120, 126 (23), 135 (23) Moore, D. H., 276, 293 (82) Morell, S., 176, 182, 183 (105), 185, 187 (164) Morgan, J. W. W., 121, 123 (30), 124 (30), 125 (30), 128 (30), 130 (30)) 132 (30), 133 (30), 134 (30), 152 Morgan, W. T. J., 311, 317 Mori, I., 86, 89 (18) Mori, T., 59 Morrison, M., 280 (99), 281 Moses, F. E., 306, 315, 316 (84), 318 (54) Mowery, D. F., Jr., 170 Moyer, J. D., 26, 33 Moaingo, R., 148 Muehlberger, C. F., 135 Muller, H., 141 Muller, K. O., 232 Mulder, G. J., 36 Murray, E. G . D., 197 Murray, G. E., 124 Murray, M. J., 17 Murumow, J. J., 48 Mussulman, W. C., 219 Myrback, K., 193 (29), 195, 224, 225, 239, 245, 246 (131), 265, 272 (24), 284 (24), 286 (24) Mystkowski, E. M., 268, 279

AUTHOR INDEX, VOLUME

N Nageli, C., 193, 194 (12, 13), 201, 231 (13 see 101), 246 (12, 13), 256 (13), 269 Naegell, H., 54, 56 (55) Narazio, F., 106 Neely, W. B., 20, 26, 32, 94 Nef, J. U., 38, 39, 40, 46, 47 (12), 48, 52, 54 (la), 55, 58, 59, 61, 63, 65, 77 (12), 78 (12), 79 (12), 130 Neil, N. W., 105 Neish, W. J. P., 106 Nemeth, A. M., 290 Nencki, M., 62, 72 (75) Ness, R. K., 161, 162 (9), 164, 187 Neuberg, C., 64 Neuberger, A., 171 Newth, F. H., 160,163 (4), 178 (4), 179 (4) Newton-Hearn, P. A., 89, 112, 113 Nicholas, S. D., 162 Nicolet, B. H., 67 Niederland, T. R., 268 (44), 269 Niemann, C., 162 Niemann, C. G., 139, 147 (19), 150 (19), 152 (19), 155 (19) Nippe, W., 173, 183 (88), 185 (88) Noe, A , , 138, 145 (4), 147 (4), 149 (4), 150 (4), 154 (4) Noelting, G., 275 Noggle, G. R., 114 Norberg, E., 190, 193, 195, 203, 216 (3), 217, 218 (3), 219 (33), 223, 224 (3), 225, 226 (32, 33, 89), 232 (89), 246 (33, 73), 247 (3), 252 (33), 255 (3), 260 (32, 89) Nordin, P., 223, 226 (89), 231, 232 (89), 242 (102), 260 (89) Northcote, D. H., 93, 94 (42), 110, 114, 274, 276, 287 (81), 293 (81), 295 (81) Northrop, J. H., 197 0 Oakley, H . B., 275, 290 (77) O’Donnell, M. J., 19 Oetker, R., 186 Oettinger, W. F., 135 Ogston, A. G., 275, 276 (79), 290 (79), 294 (79), 300 Ohle, H., 148, 154 (55), 155 (55) Openshaw, H. T., 142

331

12

Ohlmeyer, P., 194 (36), 195,202 (36) Okui, S., 205, 245 (74c, d) Oldham, J. W. H., 119, 120, 121, 122 (12), 123 (11, 31), 124 (19), 125 (12), 126 (19), 127 (11, 12), 130 (11, 19), 132 (11), 174 O’Leary, M. J., 109 O h , S. M., 123, 124 (42) O’Loane, J. K., 14 O’Mant, M., 97 O’Meara, D., 124,125 (47) O’Neill, A. N., 282 Oosterhoff, L. J., 253 Orr, S. F. D., 31, 304 Orten, J. M., 280 (99), 281 Ott, E., 237 Ottar, B., 22 Overend, W. G., 62,91,94, 126, 141, 142 Overman, R. S., 176 Owens, H . S., 295

P Packham, M. A., 307 Pacsu, E., 172, 174, 175, 178, 185 PalmBn, J., 48 Palmer, J. W., 300 Palmstierna, H., 293 Pantlitschko, M., 264 Parker, H. O., 162, 185 (17), 186 (17) Parker, L. F. J., 83 Parrotta, E. W., 126 Partridge, S. M., 90, 91 Passmore, F., 77 (loo), 79 Pasternak, C. A., 111 Patterson, T. S., 118, 182, 183 (136) Pazur, J. H., 190, 193 (31, 32), 194 (31), 195, 203, 216 (3), 217, 218 (3), 223, 224 (3), 226 (32, 89), 228, 230, 232 (89), 236 (31), 239, 240 (311, 241 (97, 98), 246 (31, 73, 122), 247 (3), 255 (3), 259 (122), 260 Pearce, R. H., 300 Peat, S., 96, 97 (52), 113, 193 (17), 195, 201 (17 see 71), 263, 266, 282, 285, 289 (122), 293 (110), 294, 295, 296 (29), 298 Peligot, E., 36, 37, 43, 77 (19) Percival, E. E., 141 (45), 142 Percival, E. G. V., 270, 280 (51) Persch, W., 199 Peters,‘ 0.,’180, 184 (126)

332

AUTHOR INDEX, VOLUME

12

Redemann, C. E., 162 Rees, D. E., 139, 148 (26), 154 (26) Reeves, R. E., 25, 26, 139, 152 (20), 154 (20), 174, 253, 254 Reichstein, T., 139, 141, 147 (28), 149, 154 (28), 156 (28) Reinwein, H., 19 Reissig, J. L., 310, 311 (71), 312 (71), 314 (71) Reynolds, D. D., 163, 164 Reynolds, V. H., 165 Richards, G. N., 53, 61, 62 (71), 69, 70, 71, 72,74, 114, 139, 141 (18), 149 (18), 155 (18) Richards, R . E., 28 (42,44), 29 Richtmyer, N. K., 138, 148 (6), 168, 169 (55), 175 (55), 180 (55), 181, 184 (55, 133) Ricketts, C. R., 90 Riedel, H., 173, 183 (88),185 (88) Rieder, S. V., 314 Rienits, K. G., 111 Rimbach, E., 77 (104 see b), 79 Rist, C. E., 177, 256 Rievi, S. B. H., 86 Roberts, P. J. P., 286, 287 (126), 298 Robertson, G. J., 119, 130 (13) Robertson, J., 182, 183 (136) Robertson, J. S. M., 296 Robinson, D., 181 Rogers, H. J., 304,316 (44), 317 (44), 318 (44) Rogers, R. H., 29 Rohdewald, M., 268 Q Rosanoff, M. A., 41 Roseman, S., 148, 149, 172, 180 (80), 181 Quastel, J. H., 308, 311 (65), 318 (65) (80), 184 (80), 306, 309, 311, 314, 315, R 316 (84),318 Rosenmund, K. W., 138 Ragan, C., 302, 316 (31) Rosenthal, A., 121 Ramsay, D. A., 22 Rapp, W., 193 (22), 195, 203 (22), 206 Roth, P., 119, 162 Roudier, A., 283 (221,231 (22), 243 (22) Rowen, J. W., 30, 304 Rapport, M. M., 31,302 Rozenfel’d, E. L., 279, 280 (92) Raske, K., 166 Ruttner, O., 153 Rasmussen, R. S., 28 (41), 29 Ruff, O., 51, 52 Rathgeb, P., 280 (99), 281 Rundle, R. E., 193 (27), 194 (27), 195, Rawlins, L. C., 219, 220 (84), 222 (84) 218 (Z), 235, 236, 279 Raymond, A. L., 141, 170, 183 (65a) Record, B. R., 271, 275, 277, 290 (83), Ruska, H., 274, 275 (68) Russell, B. E., 301 291 (83)

Petersen, J. C., 99 Petersen, S. R., 180, 184 (128) Petree, L. G., 267 Petrova, A. N., 296 Pette, D., 96 Phillips, G. O., 177 Pippen, E. L., 186 Pirie, N. W., 300 Piteer, K. S., 22 Plankenhorn, E., 120, 139, 143 (30), 147 (24, 30), 148 (24, 30), 151 (15), 152 (15), 154 (15, 24), 156 (30), 212, 214, 218 (77), 237 (77), 255 Pletcher, D. E., 311 ’ Ploete, T., 193 (25), 195,225 (25), 252 (25) Plyler, E. K., 30 Plyshevskaya, E. G., 279, 280 (92) Pogell, B. M., 315 Pohlen, E. K., 76 Polglase, W. J., 275 Potter, A. L., 280, 295, 297 (98) Porter, J. R., 233 Powell, G., 141, 169 Praill, P. F. G., 151 Preckel, R. F., 129 Preece, I. A., 112 Pringsheim, H., 192, 193, 194, 195, 198, 199, 201, 202, 224, 232, 233, 234 (ll), 243, 249, 256 Prins, D. A,, 61 Purves, C. B., 120, 123, 124, 129, 131, 132 (74), 149, 171, 175, 182 (68), 183 (104) Puteeys, P., 274

AUTHOR INDEX, VOLUME 12

333

Schmitz, E., 73 Schmitz-Hillebrecht, E., 161, 168 (lo), 169 (lo), 180 (lo), 181 (lo), 184 (10) Schneider, J. M., 37 Schneidmesser, B., 237 Schoch, T. J., 183, 185 (148), 224, 259 S Schorigin, P., 52 Schroeder, E. F., 44, 170, 183 (65a) Saad, K. N., 268 Schriiter, G. A., 163, 166 (27), 174 (28), Sack, J., 48 182 (27, 28), 185 (27) S&da, J., 178, 182 (123) Schumacher, J. N., 73 Sahyum, M., 267 Schumpert, M. Z., 267, 268 (34) Sallentien, H., 199 Schwager, A., 118 Sallman, B., 301 Schwalbe, C. G., 48 Salmon, M. R., 141 Schwimmer, S., 219, 220 (80),222, 229 Samec, M., 192, 225, 275, 296 (76) (80), 259 (80),284, 295 (118) Sampson, P., 302, 303 (34) Seastone, C. V., 301 Samuel, D., 133 Seegen, J., 263 Sand, D. M., 248 Segall, G. H., 129 Sanda, TI., 54, 56 Seifter, J., 300 Sander, F. V., 37 Seligman, A. M., 173, 176, 177 (109), Sanson, A., 262 (89, 109, 110), 184 (89, 110), 186 Santiago, E., 177 Saunders, B. C., 161, 162 (13) (110) Senior, J. K., 197 Saunders, W. A , , 141 (45), 142 Shaffer, P. A., 67, 72 (83) Sautermeister, A., 57, 59 (65) Shafizadeh, F., 52, 91 Sayteeff, A., 46 Schaaf, E., 193 (25), 195, 225 (25), 252 Shen, T., 267, 268 (34) Shepherd, D. M., 124, 125 (47), 131 (44) (25) Sheppard, N., 28 (39), 29 Schachman, H. K., 238, 239 Sherman, L. P., 37 Schaffer, F. L., 269 Sherwood, N. P., 301 Schaffer, R., 75 Schardinger, F., 190, 193, 194, 195, 196, Shyluk, W. P., 169, 174 Sieber, N., 62, 72 (75) 197, 198, 212 (lo), 224, 247 (10) Silmann, 5. H., 199, 234 (561, 255 Schaub, R. E., 163 Scheibler, C., 37, 43, 44,46, 47, 77 (7), 79 Silveira, V., 295 Simonoff, R., 148 (7) Simpson, D. M., 28 (39). 29 Scheidt, U., 19 Singer, R., 173 Schiller, S., 318, 319 (91) Sintenis, F., 150 Schinle, R., 187 Schlamowitz, M., 268, 278, 280, 297 Sisido, K., 168 Schlenk, H., 248 Skinner, A. F., 174, 243 Schlubach, H . H., 122, 123 (34), 163, 166 Skraup, Z. H., 124, 172 (27), 174 (28), 182 (27, 28), 183, 185 Smart, C. L., 299 (27) Smirnoff, A. P., 201 Schmale, K., 199 Smith, C. S., 311 Schmid, H., 126 Smith, D. C. C., 182 Schmidt, O., 65 Schmidt, 0. T., 52, 139, 143 (30), 146, Smith, E. E. B., 305, 307 (501, 312 (501, ' 313 (50) 147 (29, 30), 148 (29,30), 151 ( a ) , Smith, E. L., 275 152, 153 (291,156 (29, 30)

Rutherford, J. K., 119, 122, 125 (12), 127 (12) Rutter, W. J., 269 Ryley, J. F., 293 (140), 294 Ryman, B. E., 304

334

AUTHOR INDEX, VOLUME

Smith, F., 109, 113, 162, 170, 171, 176, 182 (74), 183, 280, 281, 283 (102a), 290 (loo), 291, 294 (loo), 295 (loo), 296 (941, 297 (100) Smith, F. A., 26, 33 Smith, J. D., 84, 106 Smith, J. N., 176, 305 Smits, G., 111 Smyth, E. M., 300 Snellman, O., 300, 304 Soff, K., 193 (21), 195, 203 (21), 204 (21), 239 (21), 247 (21), 260 (21) Soffer, L. M., 126 Sokoloff, N., 118 Solms, J., 33 Soltero-Diaz, H., 177 Soodak, M., 311 Sorokin, B., 78 (102), 79 Sowden, J. C., 61, 62 (70), 73, 75, 77 (96), 146, 147 (53, 54), 148 (53, 54), 153 (54) Speedie, T. H., 119, 130 (13) Speeter, M. E., 153 Spieth G. E., 37 Spits, D., 176 Spitser, R., 22 Spriestersbach, D., 171, 182 (74), 183 (74) Stacey, M., 21, 23, 24 (19), 25 (191, 26 (19, 20), 30, 32, 62, 89, 91, 92, 93 (41), 94, 95, 98, 99 (62), 101, 103, 104, 105 (41), 107, 108 (80), 112, 113, 122, 141, 171, 266, 293, 295, 300, 304 Stadler, P., 122, 123 (34) Stafford, R. W., 28 Stahly, G. L., 219 StanBk, J., 166, 178, 182 (123) Stanier, 0. E., 300 Stanier, W. M., 86, 87, 89 (17), 91, 92, 104 Staub, M., 201, 256 (66) Staudinger, H., 268, 271, 274 (39), 275 (39), 276 (39, 58) Steingroever, A., 199, 249 (55) Steinle, D., 194 (38), 195, 219 (38), 228, 229 (38), 246 (38), 259 Stening, T. C., 126, 129 (60) Stepanenko, B. N., 296 Stephens, R., 24, 26 (29), 27 Stetten, D., Jr., 297, 307

12

Stetten, M. R., 297 Stevenson, H. J. R., 31,33, 266, 293 (26), 295 (26) Stewart, J. E., 26 Stimson, M. M., 19 Stockmayer, W. H., 243 Storey, I. D . E., 305, 316 (49) Stone, W. E., 186 Streeck, H., 165 Strominger, J. L., 306, 308 (52), 312, 316 (52) Stumpf, P. K., 232, 259 (103) Sugihara, J. M., 99 Summer, R., 260 Sundberg, R. L., 139, 141, 148 (26, 34), 154 (26) Susuki, S., 205, 245 (74d) Swanson, M. A., 239 (132), 246, 278, 287 S y l v h , B., 31 Synge, R. L. M., 120, 121 (15), 124, 125 (15), 126 (15), 130

T Tabor, E. C., 266, 293 (26), 295 (26) Tafel, J., 72 Talley, E. A., 163, 164 (25) Tanret, C., 161 Tatlow, J. C., 122 Tauber, H., 106 Tausz, J., 149 Taylor, C. W., 139, 145 (32), 147 (32), 149, 152, 153 (32), 154 (32) Teague, O., 82 Teague, R. S., 305 Tedder, J. M., 122 Tessmar, K., 148, 154 (55), 155 (55) Theander, O., 97, 107 (58), 115 (58) Thomas, G. J., 285, 289 (122), 294, 295 Thompson, A , , 96, 265, 283 Thompson, H. W., 15, 19, 21, 28 (21, 38, 43, 44), 29, 31 Thorpe, W. V., 304 Tilden, E. B., 193, 195, 216, 218 (79), 219, 220 (85), 221 (85), 222 (26, 79, 85), 224, 232 (79), 258 Tillotson, J. A., 248 Timell, T. E., 123 Tipson, R. S., 122, 126 (361, 173, 185, 186 Tiselius, A., 82, 83 (3), 84, 85, 86, 240, 241 (124)

AUTHOR INDEX, VOLUME

Todd, A. R., 94, 118, 139, 141, 142, 145 (32), 147 (32), 149, 152, 153 (32), 154 (32) Toepffer, H., 141 Tollens, B., 36, 48 Topper, Y. J., 315 Torkington, P., 19, 28 (43), 29, 31 Torriani, A.-M., 231 Touster, O., 165 Trauth, O., 139, 147 (16), 148 (16), 150 (16), 156 (16) Treiber, E., 30 TriznovB, M., 268 (44), 269 Trogus, C., 200, 236 (61, 62) Trotter, I. F., 28 (38), 29, 30 Truthe, W., 234 Tsou, K.-C., 173, 176, 177 (109), 181 (89, 109, 110), 184 (89, 110), 186 (110) Turvey, J . R., 266, 295 (29), 296 (29)

U Ulmann, M., 200,234,236 (61,62) Utkin, I,. M., 47, 72, 78 (32)

V Van Cleve, J. W., 170, 183 van der Vies, J., 268 Van Dolah, R. W., 117, 126 Van Duzee, E. M., 149 Vaughan, G., 94 Verhoeven, L., 274 Vignon, L., 131 Villiers, A., 193, 194, 195, 247 (7) Vollbrechthausen, P., 86 vom Hove, 138, 145 (4), 147 (4), 149 (4), 150 (4), 154 (4) von Dietrich, H., 248 von Faber, O., 48 von Gorup-Besanez, E., 262 von Grote, A. F., 36 von Hochstetter, H., 138, 145, 147 (3, 4), 149 (4), 150 (4), 154 (3, 4) von Hoesslin, H., 233 von Mechel, L., 166, 172 (43), 180 (43), 181 (43) von Przylecki, S. J., 279 von Putnoky, N., 149 Voto&ek, E., 46, 47

335

12 W

Wadman, W. H., 90 Wiilti, A., 193 (13), 194 (13), 195, 201, 231 (13 see 101), 246 (13), 256 (13), 256 (66) Wajzer, J., 264 Walaas, E., 269 Walaas, O., 269 Walker, D. G., 105 Ward, R. B., 107 Warren, G. H., 300 Watanabe, T., 205, 206 Watson, E. M., 300 Watson, R. W., 141 (45), 142 Webb, J. I., 269 Webb, R. F., 142 Webber, R. V., 238, 239 Wedemeyer, K.-F., 165, 167 (38) Weerman, R. A , , 51, 78 (44, 103), 79 Weidinger, A., 194 (35, 36), 195, 199, 202 (35, 36) Weigel, H., 20, 24 (18) Weigl, J. W., 30 Weigner, E., 171, 183 (69) Weis, K., 168 Weisblat, D. I., 153 Weissmann, B., 31, 302, 303 (34) Weltzien, W., 173 Wernicke, E., 139, 146, 147 (29), 148 (29), 151 (as),152, 153 (29), 156 (29): West, W., 82 Westhall, R. G., 90 Westphal, O., 185 Weygand, F., 139, 147 (16), 148 (16), 150 (16), 156 (16) Weymouth, F. J., 142 Whelan, W. J., 96, 97 (52), 266, 278, 282, 284, 285, 286, 287 (84, 126), 289 (122), 293 (110) 294, 295, 296 (29), 298 Whiffen, D. H., 14, 20, 21, 23, 24, 25 (19), 26 (19, 20, 29), 27 (29), 32, 266 Whistler, R. L., 32, 40, 77 (14), 94, 97, 107 (59), 266, 295 (30), 299 Whitehouse, M. W., 112 Wickberg, B., 97 Wieehert, R., 257 Wiener, A., 194 (35), 195, 202 (35) Wigner, J. H., 119, 129 (7a) Wild, G. M., 223, 226 (89), 231, 232, 240, 241 (123), 242, 260 (89)

336

AUTHOR INDEX, VOLUME

Wilham, C. A., 32 Wilkins, C. H., 28 (45), 29 Wilkinson, I. A., 113, 293 Will, W., 118, 123 (3), 125 (3) Williams, D., 29 Williams, E. C., 300 Williams, J. H., 163 Williams, R. J. P., 240, 241 (124) Williams, R. T., 176, 181, 183, 305 Williams, V. Z., 18, 19, 21 (lo), 28 Williamson, S., 120 Willstatter, R., 268 Wilson, E. J., Jr., 183, 185 (148), 224, 259 Windaus, A., 62 Winkler, S., 164, 180, 181 (125), 184 (125, 126) Wise, C. S., 95 Wislicenus, J., 45 Wohl, A., 64 Wolf, I., 122, 123 (34) Wolfes, O., 138 Wolff, I. A., 256 Wolff, W. W., 176 Wolfrom, M. L., 30, 73, 92, 96, 121, 123, 124 (42), 125, 127, 172, 187, 265, 282, 283, 311 Woodin, A. M., 92

12

Woodruff, S., 37 Wotia, J. H., 28 (401, 29 Wright, R. S., 142 Wulwek, W., 166

Y Yagoda, H., 124 Yoffe, A. D., 133 Young, F. G., 264, 267, 275, 290 (77), 291 (17, 31) Young, G. T., 280 Young, W. J., 262,263

z Zeile, K., 141 Zelinski, R. P., 169 Z e m p l h , G., 139, 143, 144, 147 (14), 151, 155 (14), 156 (14), 166, 169, 172 Zervas, L., 141, 142, 186 Zetzsche, F., 138 Zill, L. P., 96, 114 Zilliken, F., 171, 173 (73), 182 (73), 185 (73) Zinner, H., 185 Zittle, C. A , , 86 Zweig, G., 83, 84 (5a see l l ) , 91 (5a see 36)

Subject Index for Volume 12 A Acetamide, N , N-dimethyl-, 174 Acetic acid, methylpropyl-, DL-. See Valeric acid, methyl-^^-. -, trichloro-, 266 -, trifluoro-, anhydride, 122 Acetolysis, of polysaccharides, 201 Acetone, from starch, 197 -, dihydroxy-. See 2-Propanone, 1,3dihydroxy-. Acetonitrile, 122,128 N-Acetylase, from yeast, 311 Acetyl coenzyme A. See Coenzyme A, acetate. Adenosine, 139 2-phosphate, 101 3-phosphate, 101 5-phosphate, 101, 298 Adenosine-5-triphosphoricacid, 308,311, 314,317 Adipic acid, 56,58 -, 2,3,5-trihydroxy-, 56, 59, 61 Adrenaline, 298 “Aerobacillus macerans,” 219, 258 Aerobacter aerogenes, 266, 300 Albumin, of serum, 279 Alcohols, polyhydric, M o values of. See the table on page 108. reaction with borate ion, 86 lone electrophoresis of, 102 Aldohexoses, 3-C-methyl-, 47 Aldonic acids, 152 2-deoxy-, 37 w-deoxy-, 37 deoxy-, lactone, 37 Aldopentose, 3-deoxy-2-C-(hydroxymethyl)-(D-erythro or D-threo)-, 52 Aldoses, bisulfite complexes of, 108 -, 1,2-dideoxy-l,2-dihalogeno-,37 Aldotetrose, 2,3-dideoxy-2-dimethylamino-4-0-methyl-, 30 Alkyl sodium sulfate, infrared absorption spectrum of, 31

Alloside, methyl 2,3-anhydro-4,6-0benzylidene-a-D-, 132-134 Altrose, 3-deoxy-3-C-ethyl-~-, 94 1 ,g-anhydride, 94 Aluminum lithium hydride, 126, 153 Amandin, 274 Amino acids, separation of, from sugars, 91 zone electrophoresis of, 91 Amylases, 284 of Bacillus macerans, 190, 206, 219, 246 action pattern of, 225 cell free, 259 of Bacillus polymyxa, 240 bacterial, 232 fungal, 231 salivary, 231, 246 a-,231,259,284,286,295 from Aspergillus oryzae, 284 from malt, 284 salivary, 284, 286 8-, 207, 229,231,246,259, 265, 272, 273, 284 crystalline, from sweet potato, 286 of soya beans, 240 Amylodextrin, 245, 259 01-, 265 Amylo-(1 --t 6)-glucosidase, 285, 288, 292 Amyloheptaose. See Maltoheptaose. Amylomaltase, 231 Amylopectins, 224, 258, 265, 273 a-amylolysis of, 284 comparison of, with glycogens, 294 inclusion complex with iodine, 277-279 iodine-binding capacity, 279 tricarbanilates, 257 zone electrophoresis of, 112, 113 Amylosaccharides, 112, 113 Amyloses, 112, 113, 224, 258, 259 a-amylolysis of, 284 cyclo-, 208 cyclodeca-, 208 cyclohepta-, 238

337

338

SUBJECT INDEX, VOLUME

12

Bacillus macerans, 190, 246, 258 cyplohexa-, 208 cyclonona-, 208 amylase of, 206, 219 effect of alkali on, 268 cell-free, 259 inclusion complexes with iodine, 279 the name, 197 Bacillus megatherium, 266 tricarbanilates, 257 Angina pectoris, 134 Bacillus polymyxa, 232, 240 Aniline, p-nitro-, acetylation of, 311 Bacteria, enteric, 266,293 Anomers, infrared absorption spectra of, Benzene, bromo-, 216, 248 25 -, nitroso-, 248 Arabinitol, 1,5-anhydro-~-,98, 99 Benzidine, N , N‘-diphenyl-, 123 , 171 Benzimidazole, 73 Arabinofuranoside, methyl a - ~ -100, ,9 anomer, 100 C14-labeled, 73 -, methyl WL-, 178 2-Benzimidazolecarboxylic acid, 73, 74 Arabinonic acid, D-, 46 Benzyl nitrate, 130 Arabinopyranoside, methyl a - ~ -99, , 100 Betacoccus arabinosaceous, 32 ,9 anomer, 99, 100, 171 dextran of, 94 -, methyl p-L-, 171 Borax, use of, in zone electrophoresis, 86 Arabinose, L-, action of alkali on, 41 Brigl’s anhydride, 148 L-, diethyl dithioacetal, 178 1,4-Butanediol, 102 D-, and L-, infrared absorption spectra 2,3-Butanediol, erythro-, 102 of, 29 threo-, 102 M G value of, 114 Butyl nitrate, 126 L-, tetranitrate ester, 118 1,4-Butynediol, diacetate, 40 Butyric acid, 2-amino-4-(methylsulL-, 1,2,3,4-tetranitrate ester, 123 -, 1,2,3-tri-O-acetyl-~-, 5-nitrate ester, finy1)-, 317 122 -, 2,4-dihydroxy-, DL-, 38 -, 1,2,3-tri-O-acetyl-5-deoxy-5-iodo-~-, brucine salt, 52 122 1,4-1actone, 60 -, 3,4,5-tri-O-acety1-2-0-(2,3,4,6-tetra- structure of, 39 0-acetyl-,9-D-glucosyl)-D-, 302 -, 3,4-dihydroxy-, DL-, 39 -, 2-O-(~-xylopyranosyl)-~-, action of -, 2,4-dihydroxy-2- (hydroxymethy1)-, alkali on, 71 DL-, 40 Arabinoside, phenyl 2,3,4-tri-O-acetyllactone, 40 0-L-, 149 -, 2-ethyl-4-hydroxy-, DL-, 56, 58 ,9 anomer, 149 1,4-lactone, 56 Arabinosyl bromide, 2,3,4-tri-O-acetyl- -, 2-methyl-, 40 B-L-, 160 -, ~-oxo-,105 Arbutin, 138 C Arthritis, rheumatoid, 301,316 Ascaris lumbricoides, 266, 276, 283 Carbohydrates, acidic derivatives of, 104 Ascorbic acid, infrared absorption specbasic derivatives of, 104 trum of, 30 complexes with borate, 92 Asparagine, L-, 317 electrophoretic mobility of, i n borate Aspartic acid, peptides of, 91 solution, 87, 88 Aspergillus niger, 33, 95 infrared absorption spectra of, 13, 23, Aspergillus oryzae, 284 27, 33 Aucubin, 138 molecular size of, 107 nitrate esters of, 117-135 B alkaline hydrolysis of, 130 “Bacillus acetoethylicum,” 197 analysis of, 123 Bacillus amylobacter, 195 denitration of, 125

BUBJECT INDEX, VOLUME

preparation of, 118 reaction of, with acids, 125 with pyridine, 129 with sodium iodide, 127 with sodium nitrite, 128 reactions of, 124 reductive denitration of, 125 stability of, 122, 123 uses of, 134 separation of, by ion-exchange resins, 114 hy zone electrophoresis, 91 Cation-exchange resins. See Resins, cation-exchange. Cellobiose, action of lime-water on, 48, 69, 70 MQ value of, 95 -, octa-0-acetyl-a-, 162,169 Cellobioside, ethyl hepta-0-acetyliu-, 169 -, methyl (I-, heptaacetate, 167, 174 -, methyl 8-, heptaacetate, 167, 174 heptanitrate, 121 -, phenyl hepta-0-acetyl-8-, 166, 169 Cellcihiosyl bromide, hepta-0-acetyl-, 160 (I anomer, 166 Cellobiulose, action of lime-water on, 70 Cellotetraose, action of lime-water on, 70 Cellulose, fibers of, in plants, 29 infrared absorption spectrum of, 29 nitrate esters, 118, 123, 129, 134 oxime, 129 partially degraded, action of limewater on, 48 partially nitrated, 124 “Cellulosine,” 190 Chloral hydrate, 266, 267 Chondroitinsulfuric acid, 110, 111 infrared absorption spectrum of, ill Chromatography, on carbon, 240 column. See Column chomatography. electro-. See Zone electrophoresis. Clostridium butylicum, 195 Codehydrogenase I, 305, 306 Coenzyme A, 311 acetate, 311 Collagen, 258 s-Collidine, 167 Column chromatography, on alumina, 122 on carbon-Celite, 96, 97

12

339

plus borate, 107,115 plus molybdate, 107 Complexes of borate, with carbohydrates, 92 with methyl a-and ,C?-D-ghiCOpyranOsides, 113 with (I- and 8-Schardinger dextrins, 113 of ferric chloride with octa-0-acetyla-cellobiose, 169 of methyl 0-D-glucopyranoside with potassium acetate, 170 of nitric acid, 121 of sodium fluoride, 121 tridentate, 101, 103 Complexing agents, for zone electrophoresis, 106 Concanavalin-A, 280, 295, 296 Cycloamyloses. See Amyloses, cyclo-. Cyclodecaamylose. See Amylose, cyclodeca-. Cycloheptaamylose. See Amylose, cyclohepta-. Cycloheptaglucanase, 232 Cyclohexaamylose. See Amylose, cyclohexa-. Cyclohexaglucanase, 231 Cyclohexane, infrared absorption spect r u m of, 22, 25 1,2-Cyclohexanediol, trans-, 88 Cyclononaamylose. See Amylose, cyclonona-. Cycloposthium, 293 Cysteine, 317

D Devarda’s alloy, 124 Dextrans, bacterial, infrared absorption spectra of, 32 of Betacoccus arabinosaceous, 94 infrared absorption spectrum of, 24 sulfate, 90 Dextrins, I, 11, and 111, from amylopectin, 273 a-,284, 286 from glycogen, 285-287 p-, 272, 280 from amylopectin, 296 from glycogen, 286, 289, 296 inclusion complex with iodine, 287

340

SUBJECT INDEX, VOLUME

muscle phosphorylase limit. See Dextrins, +-. 9-, 279, 280, 285,288,289 from glycogen, 289 Schardinger. See Schardinger dextrins. Diastase, 263 Diethyl magnesium, 94 Digitalose, D-. See Galactose, 6-deoxy3-0-met hyl-D-. Dimethylamine, 174 for deacetylation, 130 Dioxindole. See Oxindole, 3-hydroxy-. Diphenylamine, 123 Diphosphopyridine nucleotide. See Codehydrogenase I. Disaccharides, Mo values of. See table on page 96. zone electrophoresis of derivatives, 109 Disease, glycogen-storage, 265, 291. See the table on page 293. von Gierke’s, 293 Dowex 1, 114

E Electrochromatography. See Zone electrophoresis. Electroendosmotic flow, 88 Electromigration. See Zone electrophoresis. Electrophoresis, boundary, 82, 114 Electrophoresis, zone. See Zone electrophoresis. Emulsin, 248 Enediols, 6446,68,70,75,76 Enzymes, branching, 296-298 debranching, 284 P- and Q-, 113 Erythritol, nitrate ester of, 131 Escherichia coli, 293, 314, 318 1,2-Ethanediol, l,a-diphenyl-. See Hydrobenzoin. Ethanethiol, 152 Ether, benzyl phenyl, 150, 153 Ethers, benzyl, of sugars. See the table on pages 164-166. Ethylene glycol, dimethyl ether, 146 nitrate ester, 131 Explosives, 134

F Fibrin, human, 92 Finkelstein’s reagent, 127

12

Formic acid, from hexoses, 36 Fructofuranoside, methyl WD-, 101, 171 p anomer, 101,171 Fructopyranoside, benzyl P-D-, 175 -, methyl WD-, 100,163,179 p anomer, 100,175 -, methyl tetra-0-acetyl-cu-D-, 166, 174 Fructose, D-, 103 D-, action of alkali on, 73 D-, action of calcium hydroxide on, 43, 71 1,6-diphoephate, 105 D-, electrophoretic mobility of, 87 D-, infrared absorption spectrum of, 29 D-, methyl ethers of, 135 M o value of, 114 D-, mobilities of methyl ethers of. See table on page 0.6. D-, 3-nitrate ester, 125 di-0-isopropylidene acetal, 125 D-, 6-phosphate, 313 D-, phosphorylation of, 308 -, anhydro-n-, isomeric trinitrate esters, 118 -, 1,2:4,5-di-O-isopropylidene-~-, 120 3-nitrate ester, 120 -, ~ - O - ( C Y - D - ~ ~ U Caction O S ~ ~of )-D lime-, water on, 70 -, 1-0-methyl-D-, action of lime-water on, 70 -, 3-O-methyl-~-,action of lime-water on, 70 -, 4-O-methyl-n-, 70 Fructoside, phenyl tetra-0-acetyl-P-D-, 169 Fructosylamine, N-acetyl-, 6-phosphate, 314 Fuconic acid, 2-0-benzyl-D-, 152 Fucose (6-deoxygalactose), 139 -, 2-0-benzyl-~-,152 -, 4,5-O-isopropylidene-~-,dimethyl acetal, 146 2-benzyl ether, 146 Fucoside, methyl 2-0-benzyl-o-, 151 3,4-0-isopropylidene acetal, 151 2-Furaldehyde, 5- (hydroxymethyl) -, 72 Furan, tetrahydro-, 148,153 G

Galactal, D-, 91 Galactaric acid, tetranitrate ester, 120

SUBJECT INDEX, VOLUME

Galactitol, 103, 107 hexanitrate ester, 119, 129 nitrate esters of, 131 pentanitrate ester, 119 1,2,3,5,6-pentanitrate ester, 129 -, lJ5-anhydro-, 98 -, 2,5-di-O-benzyl-, 150,151 1,3:4,6-di-O-beneylidene acetal, 150 1,3:4,6-di-O-methylene acetal, 151 Galactometasaccharinic acids, D-. See Saccharinic acids, D-galactometa-. Galactonic acid, D-, pentanitrate ester, 121 Galactopyranose, CY-Dand 8-n-, infrared absorption spectra of,25 -, 6-deoxy-~-,infrared absorption spectrum of, 27 Galactopyranoside, methyl a-D-, 100 monohydrate, oxidation of, 177 oxidation of, 176 -, methyl D-D-, 100 2,6-dinitrate ester, 125 3,4-0-isopropylidene acetal, 125 6-nitrate ester, 124, 130 2,3,4-triacetate, 130 -, methyl 2,3-di-O-methyl-cu-~-,119 4,6-dinitrate ester, 119 -, methyl 2,6-di-O-methyla-~-,120 3,4-dinitrate ester, 120 3,4-0-isopropylidene acetal, 120 Galactopyranosiduronic acid, methyl CY-D-, 176 dihydrate, 177 lead salt, 177 methyl ester, 171 -, methyl 8-D-,176 Galactose, D-, acid reversion products of, 109 action of alkali on, 48 action of lime-water on, 54,73 D-, infrared absorption spectrum of, 29 M ovalue of, 114 D-, methyl ethers of, 135 a - ~ -1,2,3,4,6-pentanitrate , ester, 118, 123 8 anomer, 118, 123 D-, separation of, from D-glucose, 91 -, 2-acetamido-2-deoxy-~-, 106 -, %deoxy-~-.See Hexose, P-deoxy-~lyxo-. -, 6-deoxy-3-O-methyl-~-,139

12

34 1

-, 1,2:3,4-di-0-isopropylidene-~-, 127 6-nitrate ester, 127, 129 Galactose-l-W, D-, 73 Galactoside, p-cresyl 2,3,4,6-tetra-Oacetyl-CY-D-, 167 -, ethyl 2,3,4,6-tetra-O-acetyl-c~-~-, 174 8 anomer, 174 -, methyl D-, 170 -, methyl & D - , 2,3,4,6-tetranitrate ester, 121 -, methyl 3,4-0-isopropylidene-p~-,121 2,6-dinitrate ester, 121 -, methyl 3,4-0-isopropylidene-2-0methyl-p-D-, 126 6-nitrate ester, 126 -, methyl 2,3,4-tri-O-acetyl-p-~-, 120 6-nitrate ester, 120 6-trityl ether, 120 -, phenyl CY-D-, 166 -, phenyl 2,3,4,6-tetra-O-acetyl-a-~-, 167 8 anomer, 169 Galactosiduronic acid, poly-D-, 33 Galactosyl bromide, 2,3,4,6-tetra-0acetyl-D-, 160, 167 Galactosyl phosphate, D-, 105 Galacturonic acid, D-, 104 1-phosphate, 105 -, 2,3,4-tri-O-acetyl-l-bromo-l-deoxyLZ-D-, 176 Gentiobiose, 265 Ma value of, 95 Glass, fiber, use in zone electrophoresis, 109 Globulin, from jack-bean meal, 280 of serum, 279 Glucagon, 298 Glucal, D-, 91 Glucan, poly-, of Aspergillus niger, 95 Glucaric acid, D-, 308 “Glucinic acid,” 36 Glucitol, D-, 103, 107 hexanitrate ester, 122 nitrate esters, 134 -, 1,&anhydro-~-,98 -, 1,4:3,6-dianhydro-~-, 2,5-dinitrate ester, 135 Glucofuranose, 1,6-anhydro-n-, 254 -, 1,2-0-isopropylidene-a-~-, 101, 146 6-acetate, 3,5-dinitrate ester, 130 6-benzyl ether, 148

342

SUBJECT INDEX, VOLUME

12

2,3,4,6-tetranitrate ester, 129 3-benayl ether, 5,6-diacetate, 146 2,3,6-trinitrate ester, 129 5-nitrate ester, 130 oxidation of, 177 -, methyl 2,3-di-O-acetyl-p-~-,122 Glucofuranosides, D-, 101 6-nitrate ester, 122 6-tosyl ester, 122 Glucofuranosiduronic acid, alkyl B-D-, 6,3-lactone, 176 -, methyl 2,3-di-O-methyl-@-o-,119 4,6-dinitrate ester, 119 -, methyl D-, 6,3-lactone, 171 Glucofuranuronic acid, 6,3-lactone, 176 -, methyl tetra-0-methyl-n-, 270 Glucometasaccharinic acids, D-. See Sac- -, phenyl WD-, monohydrate, 177 charinic acids, D-glucometa-. -, phenyl p-D-, 158 Gluconic acid, 105 Glucopyranosiduronic acid, alkyl D-D-, D-, pentanitrate ester, 121 305 -, 2-amino-2-deoxy-~-,314 -, 2-amino-2-deoxy-~-glucose-(3 + 1) -, 2-keto-. See Hexonic acid, z - k e t o - ~ P-D-, 302 arabino-. -, (1evo)-menthyl CY-D-, 176 Glucopyranose, D-, infrared absorption - methyl CY-D-, 176 spectra of derivatives of, 23-25. See p anomer, 176 also table on page 29. -, phenolphthalein mono-p-w, 178 CY-D-, 6-phosphate, 264, 309, 310 -, phenyl (Y-D-, 177, 178 -, 2-acetamido-2-deoxy-3-0-(~-~-gluco-sodium salt, 177 pyranosyluronic acid)-D-, 304 -, phenylp-D-, 176,177 --, 1,6-anhydro-p-~-,132, 203-205 -, pregnanediol-3 P-D-, 176 benaylation of, 144 Glucopyranosyl phosphate, (Y-D-, 232. 2,4-dibenzyl ether, 144, 145 259, 284, 285, 296, 306 3-acetate, 145 6-phosphate, 309 2,3,4-triacetate, 144 Glucosaccharinic acid, “a”-D-.See Sac2,3,4-tribenzyl ether, 144 charinic acid, “a”-D-gluco-. -, 1,6-anhydro-4-0-(a-~-glucopyrano-Glucosamine, D - . See Glucose, 2-aminoSyl)-D-, 205 a-deoxy-~-. -, 4,6-O-benaylidene-~-,infrared ab- -, N-acetyl-D-. See Glucose, 2-acetamsorption spectra of derivatives of, 26 ido-2-deoxy-~-. -, tetra-0-methyl-D-, 270, 28fk282 Gl~cosamine-l,6-C’~2-N16, D-, 315 -, 2,3,6-tri-O-methyl-~-,270, 281, 282 D-Glucosamine-N-acetylase,311 Glucopyranoside, alkyl (Y-D-, 178 D -Glucosamine-6-phosphate-N-acetyl8 anomer, 178 ase, 311 -, methyl a-D-,171, 269 Glucosaminide, alkyl N-acetyl-. See Glu3,6-anhydride, 132 coside, alkyl 2-acetamido-2-deborate complex of, 113 OXY-D-. mobility of, 99 -, phenyl tri-0-acetyl-8-n-. See Glucocondensation with metaboric acid, 99 side, phenyl tri-O-acetyl-2-amino-2nitrate esters, 134 deoxy-p-D-. -, methyl p-D-, 132, 163, 269 Glucose, D-, 103 borate complex, 113 D-, action of alkali on, 48, 62, 69 mobility of, 99 action of barium hydroxide on, 36 complex with potassium acetate, 170 D-, action of basic resin on, 73 2,3-dinitrate ester, 124 action of calcium hydroxide on, 36, 43 4,6-dimethyl ether, 124 D-, action of hot, concentrated sodium 3,6-dinitrate ester, 128, 129 hydroxide on, 75 6-nitrate ester, 130 D-, action of lime-water on, 75 2,3,4-triacetate, 130 D-, action of sodium hydroxide on, 60

SUBJECT INDEX, VOLUME

12

343

electrophoretic mobility of, 87 -, 6-deoxy-l , 2-O-isopropylidene-~-,149 infrared absorption spectrum of, 29 -, 1,2:5,6-di-O-isopropylidene-~-, 145, D-, methyl ethers of, 135 146 3-benzyl ether, 146 Mo value of, 114 the name, 36 -, di-0-methyl-n-, 270, 271 D-, nitrate esters, 123, 134 -, 2,3-di-O-methyl-~-,92,271, 282 LX-D-, lf2,3,4,6-pentanitrate ester, 121, action of lime-water on, 72 123 -, 2,4-di-O-methyl-~-,94, 139 /3 anomer, 121, 123 -, 2,6-di-O-methyl-~-,139, 282 D - , 6-phosphate, 313 -, 3,5-di-O-methyl-~-,139 D - , phosphorylation of, 308 -, 3,6-di-O-methyl-~-,282 D-, separation of, from D-galactose, 91 -, 4,6-di-O-methyl-~-,139 D-, uniformly labeled, 318 -, 5,6-di-O-methyl-~-,139, 141 -, 2-acetamido-2-deoxy-o-, 106,167,299, -, 6-0-(01-~-galactosyl)-~-, action of 301, 302, 308 lime-water on, 71 diphosphate ester, 310 -, 3-0-(/3-D-glUCOSYl)-D-,action of lime6-phosphate, 310, 313 water on, 70 labeled with acetate-l-C'*, 316 -, 4-0-01-isomaltosyl-~-,283 tetraacetate, 171 -, l-O-methyl-~-,101,139 -, 2-amino-2-deoxy-~-,167, 308 -, 3-O-methyl-~-,action of lime-water N-acetylation of, 311 on, 70 biosynthesis of, 313 -, 4-O-methyl-~-,92,113, 139 6-methyl ether, 139 action of lime-water on, 53, 70 6-phosphate, 309, 310, 313, 318 -, 6-O-methyl-~-,action of lime-water acetylation of, 311 on, 71 hydrolysis of, 309 -, mono-0-methyl-D-, 282 periodate oxidation of, 308 -, 1,2,3,4,6-penta-O-acetyl-01-~-, 119, phosphorylation of, 308 151, 162, 168, 170 -, 5,6-anhydro-1,2-0-isopropylidene-a- p anomer, 119, 124, 168, 169 D-, 148 -, 1,2,4,6-tetra-O-acetyl-@-~-, 139 3-benzyl ether, 149 3-benzyl ether, 120, 152 -, 1,6-anhydro-2,3,.l-tri-O-acetyl-@-~-, -, 1,2,3,6-tetra-0-acetyl-4-o-mesyl-~-, 119 127 -, I , 6-anhydr0-2,3,4-tri-O-methyl-B-~-, -, 2,3,4,6-tetra-O-methyl-~-, 88 119 -, 2,3,4-tri-O-acetyl-a-~-, 1,6-dinitrate -, :M-benzyl-D-, 138, 145 ester, 119 1,2:5,6-di-O-isopropylidene acetal, 145 -, 3,4,6-tri-O-acetyl-l,2-anhydro-01-~-, 2,4,6-trimethyI ether, 151 148, 178 -, 6-0-benzyl-3-O-methyl-~-, action of -, 2,3,4-tri-O-benzyl-~-,151 lime-water on, 70 l,B-anhydride, 0, 151 -, 4,6-0-benzylidene-~-, action of 1,6-diacetate, 151 lime-water on, 70 -, 3 5 , 6-tri-0-benzyl-D-, 150 3-methyl ether, action of lime-water 1,2-0-isopropylidene acetal, 150 on, 70 -, 3,5-O-benayIidene-I,2-0-isopropyli- -, tri-0-methyl-n-, demethylation of, 282 dene-a+-, 129, 145 , 2,3,4-tri-O-methyl-~-,151 6-benzyl ether, 145 6-benayl ether, 151 6-nitrate, 129 -, z-deoxy-~-.See Hexose, 2-deoxy-~- 1,6-dinitrate ester, 0 1 , 119 arabino-. -, 2,3,5-tri-O-methyl-~-,139 D-,

D-,

~

-

344

-,

SUBJECT INDEX, VOLUME

12

2,3,6-tri-O-methyl-~-,202, 203, 243, -, methyl 3-0-benzyl-2,4,6-tri-O244 methyl+, 151 -, 2,4,6-tri-O-methyl-~-,139 -, methyl 6-0-benzyl-2,3,4-tri-O-, 3,4, B-tri-O-methyI-~-,139 methyl-D-, 151 “Glucose,” 1,2,3,6-tetra-O-acetyl-4-de--, methyl 4,6-0-benzylidene-a-~-,121, oxy-4-iodo-~-,127 126, 132 Glucose-l-Cl4, D-, 315, 316, 318 2,3-dinitrate ester, 121, 124, 126, 128, D-, action of potassium hydroxide on, 130, 132 76 2,3-di-p-toluenesulfonate,127, 132 Glucose-6-C“, D-, 315 2-nitrate ester, 132, 134 D-Glucose oxidase, 231 3-tosylate, 134 ~-Glucose-6-phosphatase, 292,309 3-nitrate ester, 125, 128, 132, 133 Glucoside, a1kyl B-D-, 2,3,4,6-tetrani2-acetate, 125 trate ester, 131 2-tosylate, 134 -, alkyl 2-acetamido-2-deoxy-~-, 163 -, methyl 6-deoxy-6-iodo-2,3-di-O-, ally1 2,3,4,6-tetra-O-acetyl-a-~-, 175 methyl+D-, 125, 127 j3 anomer, 175 4-nitrate ester, 125, 127 -, benzyl 2,3,4,6-tetra-O-acetyl-a-n-, 4-tosylate, 127 175 -, methyl 3,4-di-O-acetyl-a-~-, 2,6j3 anomer, 175 dibenzoate, 100 -, benayl 2,3,4,6-tetra-O-benzoyl-p-~-, -, methyl 3,4-di-O-acetyl-p-~-,2,6-di168 nitrate ester, 128 -, benayl 3,4,6-tri-O-acetyl-p-~-, 148 -, methyl 4,6-di-O-acety1-2,3-di-O-, ethyl 2,3,4,6-tetra-O-acetyl-a-~-, 175 b e n z o y l e - ~ - ,1ocl j3 anomer, 175 -, isopropyl 2,3,4,6-tetra-O-acetyl-a--, methyl 2,3-di-O-methyl-j3-~-, 4,6dinitrate ester, 127 D-, 175 4,6-ditosylateJ 127 j3 anomer, 175 -, methyl 4,6-di-o-rnethyl-j3-~-,125 -, (2euo)-menthyl a-D-, 166 2J-dinitrate ester, 126 -, (2evo)-menthyl 2,3,4,6-tetra-O-benz-, methyl 4,6-0-ethylidene-a -D-,2,3OYl-p-D-, 168 dinitrate ester, 124, 128, 133 -, methyl WD-, 2,3,4,6-tetranitrate 3-nitrate ester, 128 ester, 118, 121, 123, 129 -, methyl B-D-,3,4-dinitrate ester, 130 -, methyl 4,6-O-ethylidene-j3-~-, 120, 121, 125 2,6-diacetate, 130 2,3-diacetate, 128 2,3.4.6-tetranitrate ester, 120. 121. , . 2,3-dinitrate ester, 120, 121, 125, 128, 123, 127, 129, 131 133 methyl 2-acetamido-2-deoxy-u-, 171 methyl 2-acetamido-3,4,6-tri-O-ace- 3-nitrate ester, 128 2-acetate, 128 tyl-2-deoxy-j3-~-,167 2-benzoate, 124 methyl 4-O-(l-acetoxyethyl)-6-02-p-toluenesulfonate, 124 acetyl-B-D-, 2,a-dinitrate ester, 120, -, methyl 4,6-O-(nitrobenaylidene)-j3125 D-, 124 methyl 6-O-acetyl-p-~-, 2,3,4-tridinitrate ester, 124 nitrate ester, 120 methyl 3-0-acetyl-2-0-methyl-j3-~-, mononitrate ester, 124 4,6-dinitrate ester, 127 -, methyl 2,3,4,6-tetra-O-acetyl-a-~-, methyl 4-0-acetyl-6-O-trityl-j3-~-, 175 2,3-dinitrate ester, 124 @ anomer, 132, 165,167, 169, 175

SUBJECT INDEX, VOLUME

12

345

-, methyl 2,3,4,6-tetra-O-benzoyl-a-~-,lactone, 171, 176, 307 174 labeled a t C6, 307 anomer, 174 metabolism, 304 -, methyl 2,3,4-tri-O-acetyl-a-~-, 6a-D-, 1-phosphate, 105, 305, 306 nitrate ester, 120, 132 I3 anomer, 306 6-trityl ether, 120 sodium salt, 307 -, methyl 3,4,6-tri-O-acetyl-a-~-, 2- -, 1,2,3,4-tetra-O-acetyl-j3-~-, methyl nitrate ester, 132 ester, 169 -, methyl 2,3,4-tri-O-methyl-a-~-, 132 -, 1,2,5-tri-O-acetyl-&~-,6,3-lactone, B anomer, 125 186 6-nitrate ester, 132 -, 2,3,4-tri-O-acetyl-l-bromo-l-deoxy8 anomer, 125 a - D - , 166, 176 -, o-nitropheny12,3,4,6-tetra-O-acetyl- methyl ester, 167 D-D-,166 b-D-Glucuronidase, 307 -, phenyl2,3-di-O-benzy1-8-~-, 150 Glutaconic acid, 50 4,6-O-benzylidene acetal, 150 -, I - m e t h y l - ~ ~46 -, -, phenyl 2,3,4,6-tetra-O-acetyl-a-~-, Glutamic acid, L-, 317 149, 168, 175 ammonium salt, 317 j3 anomer, 149, 168, 169, 174, 175 labeled with ammonium-N16,318 -, phenyl tri-O-acetyl-2-amino-2-deoxy- peptides of, 91 @-D-, 165 Glutamine, L-, 313, 314, 316, 317 Glucosiduronic acid, methyl 2,3,4-tri-0Glutaric acid, 2,3-dihydroxy-, 50 acetyl-a+-, methyl ester, 176 -, 2,4-dihydroxy-, 50, 59 j3 anomer, 176 meso-, 42 -, phenyl 2,3,4-tri-O-acety14-n-, 167 n-threo-, 42 methyl ester, 166, 168, 169 L-threo-, 42 Glucosone, D-, 315 -, 2-keto-, 105 Glucosyl bromide, 2-acetamido-3,4,6, a - m e t h y l - ~ ~45 -, tri-O-acetyl-2-deoxy-n-,167 -, 2,3,4,6-tetra-O-acetyl-a-~-, 119, 160, Glyceraldehyde. See Glycerose. Glyceritol, 233 163, 165-167 nitrate esters of, 131, 134 -, 2,3,4,6-tetra-O-benzoy1-m-~-, 168 3-phosphate, 105 Glucosyl chloride, 2,3,4,6-tetra-O-acetrinitrate ester, 135 tyl-a-D-, 118, 122, 160, 169 Glucosyl nitrate, 2,3,4,6-tetra-O-ace- -, 1-chloro-1-deoxy-, 39 Glycerol, a-chlorohydrin. See Glyceritol, tyl-a-D-, 118, 119, 123, 124, 132 1-chloro-1-deoxy-. 13 anomer, 122-124 - , 65 -, 2,4,6-tri-O-acetyl-3-0-(nitrobenzyl)- Glyceronic acid, 3 - d e o x y - ~ ~38, B-D-, 120, 152 Glycerose, 62, 65, 72, 138 -, 2,3,4-tri-o-methyl-a-~-, 6-nitrate action of alkali on, 38, 73 ester, 132 D-, 74 Glucosyl phosphate, 105 D-, 3-phosphate, 139 WD-, 105, 113 -, 2-O-benzyl-~-,139, 152 -, 2-acetamido-2-deoxy-a-~-, 310-313 diethyl dithioacetal, 139, 152 6-phosphate, 312 dimethyl acetal, 139 -, 2-amino-2-deoxy-a-~-,309, 313 -, 3-O-methyl-~-,139 Glucosylamine, N-benzyl-D-, 107 Glycerose-3-C14, D-, action of lime-water on, 76 Glucuronic acid, D-, 104, 105, 178, 301, action of sodium hydroxide on, 76 302,307,316 j3

346

SUBJECT INDEX, VOLUME

Glycerulose. See 2-Propanone, 1,3-dihydroxy-. Gly~ine-l-C'~-N15, 314 Glycofuranosides, M o values of. See table on page 101. zone electrophoresis of, 100 Glycogens, 96, 113,233,259, 306 acetates of, 276 acetylation of, 269, 276 acid hydrolysis of, 265, 282 action of pamylase on, 265 a-amylolysis of, 284 P-amylolysis of, 272, 286 apparent reducing power of, 264 association with protein, 268 bacterial, 293. See the table on page 295. biological synthesis of, 296 coloration with iodine, 264 comparison of, with amylopectins, 294 degree of multiple branching in. See the table on page 288. desmo-, 268 discovery of, 262 empirical formula of, 262 end-group assay of, 280, 285. See also table on page 281, ensymolysis of, 263 estimation of, 268 exterior chain lengths of, 286. See table on page 887. ex vivo, 297 from liver, 262 from various sources, 113 from yeast, 262 general properties of, 263 inclusion complexes of, with iodine, 277. See table on page 278. with proteins, 280 infrared absorption spectrum of, 265 interaction of, with iodine, 277 with proteins, 279 inter-chain linkages of, 281 interior chain lengths of, 286. See table on page 987. i n vitro synthesis o f , 296 in vivo synthesis of, 297 iodine-binding capacity, 278 isolation of, 266 lye-, 268 methylation of, 269, 280, 281 methyl ethers of, 269,277

12

molecular shape of, 276 molecular structure of, 261-298 molecular weight of, 265, 267, 271, 274. See the table on page 276. multiple branching in, 287 occurrence of, 262, 266 of fish, 291 of invertebrates, 292. See the table on page 294. o f mammals, 290 of vertebrates, 290 of yeast, 293. See the table on page 295. optical rotation of, 264 periodate oxidation of, 280, 283 physicochemical properties of, 274 phyto-, 266 properties of, 262 purification of, 266 radioactive, 206 random branching in, 286 sedimentation constant of, 268 specific viscosity of, 276 stability t o alkali, 267, 268 structural analysis of, by chemical methods, 280 by enzymic methods, 284 structure of, 265, 269 synthetic. See the table on page 297. the term, 263 triacetate, 269, 270 tribenzoate, 270 trimethyl ether, 269-271 ultraviolet absorption spectra of iodine complexes, 277 x-ray analysis of, 266 Glycogen-storage disease, 265, 291. See the table on page 295. Glycolic acid, 45 Glycopyranosides, alkyl, zone electrophoresis of, 98 phenyl, 149 Glycosides, acetates of, deacetylation of, 171 benzyl, 138, 148 flavonoid, 104, 105 infrared absorption spectra of, 32 methyl, 157-187 methyl a-. See the table on page 182. polyacetates of. See the table on page 186.

SUBJECT INDEX, VOLUME

methyl 8-. See the table on page 185. polyacetates of. See the table on page 186.

oxidation t o glycosiduronic acids, 175 phenyl, 157-187 phenyl Q - . See the table on page 180. polyacetates of. See the table on page 184.

phenyl B-. See the table on page 181. polyacetates of. See the table on page 184.

synthesis of, special methods for, 174 Glycosiduronic acids, preparation from glycosides, 175 Glycosylamines, N-benzyl-, 95, 107 Glycosyl bromides, poly-0-benzoyl-, 167 Glycosyl halides, poly-0-acyl, 158, 160 condensation of, with alcohols and phenols, 163 preparation of, 158 Gulopyranoside, methyl WD-, 99 -, methyl S-D-,100

H Hamameloside, methyl, 52 Heliz pomatia, 266, 283 Heparin, 111 acid hydrolysis of, 92 n-Hexanoic acid, 55 -, 4-hydroxy-, 57 1,4-lactone, 55 Hexitols, zone electrophoresis of, 103, 106, 107 Hexokinase, 297 from yeast, 308 possible inhibitor of, 308 Hexonic acids, 3-deoxy-, 37 -, 3-deoxy-~-arabino-,59 lactone, 61 phenylhydrazide, 61 preparation of, 60 -, 3-deoxy-~-lyxo-,53 preparation of, 55 structure of, 56 -, 3-deoxy-n-ribo-, 59 preparation of, 60 -, .%deoxy-~-zylo-,53, 73 calcium salt, 54 preparation of, 54 structure of, 55 -, 2-keto-~-arabino-,105

12

347

Hexopyranosides, M Q values of. See table on page 98. Hexosamines, zone electrophoresis of, 104 Hexose, phosphate, 105 -, 2-deoxy-~-arabino-,91 -, 2-deoxy-~-lyxo-,91 Hexoses, action of alkali on, 38 oxidation of, 176 4-0-substituted, action of alkali on, 70 Hexoside, methyl anhydro-a-D-, 132 -, methyl 4,6-0-benzylidene-2,3-dideoxy-2,3-diketo-a-~-erythro-, dioxime, 130 quinoxaline derivative, 133 -, methyl 3-deoxy-~-arabino-,61 Hyalobiouronic acid, 302 Hyaluronic acid, 110, 111 acetyl groups of, 318 biosynthesis of, 299-319 discovery of, 300 infrared absorption spectrum of, 31, 304 methylation of, 304 molecular shape of, 304 occurrence of, 300 oligosaccharides from, 316 periodate oxidation of, 304 properties of, 300 structure of, 302 Hyaluronidase, 300, 317 pneumococcal, 302 testicular, 302 Hydrazine, for reduction of nitrate esters, 126 Hydrobenzoin, erythro-, 102 threo-, 102 Hydrogen-ion concentration, effect on electrophoretic mobility, 87 Hydrogenolysis, catalytic, 148 chemical, 149 of benzyl ethers, 148 of sugars, 137, 138 of benzylidene acetals, 141 of benzyloxycarbonyl esters, 141 of beneyloxymethyl ethers, 141 of trityl ethers, 141 Hydroxylamine, 129 -, 0-methyl-. See Methoxyamine. Hyflo Supercel, 110 Hypertension, 134

348

SUBJECT INDEX, VOLUME

I

of of of of of of of of of of of

12

glycosides, 32 hyaluronic acid, 31 Idose, L-, 139 scyllo-inositol, 24 -, 5,6-anhydro-3-0-benryl-l, 2-0-isoproinositols, 24, 26 pylidene-L-, 149 isomaltose, 24 -, 6-deoxy-~-,139 D- and L-lyxose, 29 1,2-0-isopropylidene acetal, 149 a- and fi-D-mannopyranose, 25, 29 Inclusion complexes, 113 melibiose, 33 canal type, 251 mucopolysaccharides, 30 of amylopectins with iodine, 277, 279, nigeran, 24 296 oligosaccharides, 32 of amylose with iodine, 279 of pneumococcal polysaccharides, 31 of dextrins with iodine, 296 of poly-D-galactosiduronic acid derivaof 8-dextrin (from glycogen) with iotives, 33 dine, 287 of polysaccharides, 24 of glycogens, with iodine, 277, 296 of quercitols, 26 with proteins, 280 of starch, 24 of rice starch with iodine, 296 of sugar acetates, 33 of Schardinger dextrins, 191, 197-200, of sugars, 32 216, 220, 241, 247-252 of sulfates, 31 with iodide, 249 of the tetrahydropyran nucleus, 21, with iodine, 249 25, 26 Indican, 248 Inositols, borate complexes of, 103 Infrared absorption spectra, 15 cis-, 104 determination of, 18 ep i - , 104 interpretation of, 21 infrared absorption spectra of, 24,26 of acetates, 31 M G values of. Se e the table on page 10.9. of 3-acetoxysteroids, 31 monomethyl ethers, 104 of d k y l sodium sulfate, 31 myo-, 104 of anomers of a sugar, 25,32 scyllo-, infrared absorption spectrum of D- and L-arabinose, 29 of, 24 of ascorbic acid, 30 zone electrophoresis of, 103 of bacterial dextrans, 32 Insulin, 258, 298 of 4,6-0-benzylidene-ol-~-glucose de- Invertase, of yeast, 90, 101 rivatives, 26 Ion exchange, borate-anion exchangers, of carbohydrates, 13, 23, 27, 33 114 of cellulose, 29 resins, 96 of chondroitinsulfuric acid, 31 for separating carbohydrates, 114 of cyclohexane, 22, 25 Ionography. See Zone electrophoresis. of 6-deoxy-~-galactopyranose, 27 lonophoresis. See Zone electrophoresis. of 6-deoxy-~-mannopyranose, 27 Isoamylase, 289 of deoxy sugars, 26 of yeast, 273, 285 of dextran, 24 Isomaltose, 265, 282 of 2,3-dideoxyS-dimethylamin0-4-0- infrared absorption spectrum of, 24 me thyl-aldotet rose, 30 M Qvalue of, 95 of D-fructose, 29 separation of, from maltose, 91 of furanose sugar derivatives, 27 -, octa-0-acetyl-8-, 282 of a- and 8-D-galactopyranose, 26, 29 Isomaltotriose, 96 of D-glucopyranose derivatives, 23-25, Isosaccharinic acid. See Saccharinic acid, 29 iso-. of glycopyranoses, 27 Isosucrose, octa-0-acetyl-, 174

SUBJECT INDEX, VOLUME

K

12

349

M a value of, 95 nitrate ester, 118 octaacetate, 169 separation of, from isomaltose, 91 MaItoside, ethyl hepta-0-acetyl-a-, 169 L Maltosyl bromide, hepta-0-acetyl-, 160, 269 Lactic acid, 62, 65, 71 Maltotrionic acid, 240 CI4-labeled, 76 DL-. See Glyceronic acid, 3 - d e o x y - ~ ~ - .Maltotriose, 207,209, 230, 240, 282, 284 Ma1tulose, 298 from hexoses, 36 action of lime-water on, 70 -, 2,3-diphenyl-, 67 Lactose, action of lime-water on, 48, 53, Mannan, 178 Mannitol, D-, 103, 107, 125 54, 69, 70 D-, hexanitrate ester, 118,122, 125,129, nitrate ester, 118 134 Lactoside, ethyl a-,178 nitrate ester of, 131 Lactosyl bromide, hepta-0-acetyl-, 160 1,2,3,5,6-pentanitrate ester, 119, 129 Lactulose, action of lime-water on, 70 Laminaribiose, 70, 97. See also Glucose, -, I,S-anhydro-~-,98 -, 2,S-anhydro-~-,101 3-0-(@-D-glUCOSyl) -D-. -, 2,5-di-O-benzyl-~-,139 Laminarin, action of lime-water on, 60 1,4:3,6-dianhydride, 153 Leeches, extracts of, 301 1,3: 4,5-di-O-methylene acetal, 139 Levene, Phoebus Aaron Theodor, obituMannopyranose, a-D-and 8-D-,infrared ary of, 1 absorption spectra of, 25 Levoglucosan. See Glucopyranose, 1,6-, B-deoxy-~-,infrared absorption specanhydro-j3-Dtrum of, 27 Levulinic acid, from hexoses, 36 Lithium aluminum hydride. See Alumi- Mannopyranoside, methyl WD-, 171, 172, 178 num lithium hydride. borate complex of, 100 2,6-Lutidine, 167 -, methyl 8-D-,99, 178 Lyxopyranoside, methyl a-D-,99 borate complex of, 100 @ anomer, 99 Lyxose, D- and L-, infrared absorption Mannose, D-, 103 infrared absorption spectrum of, 29 spectra of, 29 nitrate esters of, 134 M -, 6-deoxy-2,3-di-O-rnethyl-~-, 139 -, 2,3 :5,6-di-O-isopropylidene -D-, ac Malic acid, D- and L-, 40 tion of lime-water on, 70, 72 DIP, 39 -, 1,2,3,4,6-penta-O-acetyl-P-~-, 162 Maltase, from yeast, 273 Maltobionic acid, octa-0-methyl-, -, 3,4,6-tri-O-acetyl-l ,2-0-(1-methoxymethyl ester, 270 ethylidene)-D-, 165 Maltoheptaonic acid, 240 Mannose-l-C14, D-, action of lime-water Ma1tohept aose, 226, 228, 238-240, 259 on, 73 Mannoside, methyl CPD-, 2,3,4,6-tetrapreparation of, 239 nitrate ester, 118 Maltohexaose, 89, 226, 228, 240 -, methyl 2,3-anhydro-4,6-0-benzylMaltooctaose, 240 idene-a-D-, 94 Maltose, 89, 97, 207, 230, 233, 240, 263, -, methyl 2,3,4,6-tetra-0-acetyl-a-~-, 265, 272, 282, 284, 286 165, 172, 174 acid hydrolysis of, 204 action of lime-water on, 48, 53, 69, 70 p anomer, 165, 174 Mannosyl bromide, 2,3,4,6-tetra-O-acheptaacetate, 201, 246 etyl-a-D-, 165 methylation of, 201

Kes t ose, 101 2-Keto acids, phenyl hydrazones, 106 Koenigs-Knorr reaction, 148, 163, 164

.

350

SUBJECT INDEX, VOLUME

Melibiose, 32,33,71,97. See also Glucose, 6-0- (a-~-galactosyl) -D-. Menthol, 306, 307 Mercaptan, benzyl. See a-Toluenethiol. ethyl. See Ethanethiol. Mesotaenium, 233 Metasaccharinic acids. See Saccharinic acids, meta-. Methane, diazo-, 178 Methionine, sulfoxide. See Butyric acid, 2-amino-4-(methylsulfinyl) -. Methoxyamine, 129 Methylene Blue, 248 Methyl nitrate, 130 M Q values. See table on page 86. of arabinose, 114 of fructose, 114 of galactose, 114 of glucose, 114 of glycofuranosides, 101 of hexopyranosides, 98 of inositols, 103 of pentopyranosides, 98 of polyhydric alcohols, 102 of ribose, 114 of xylose, 114 the term, 88 Molecular spectra, 14 Monosaccharides, M Q and Rp values of. See the table on page 90. Muck acid. See Galactaric acid. Mucilage, okra, 94 Muconic acid, 3-hydroxy-, lactone, 61 Mucopolysaccharides. See Polysaccharides, muco-. Mycobacterium tuberculosis, 293 Myosin, 279 Mytilus edulis, 266, 267, 270, 277, 283

N Neisseria perflava, 113, 266 Neokestose, 101 Neurospora crassa, 309-311, 313, 314, 316 Nickel, Raney, 148, 149 Nigeran, infrared absorption spectrum of, 24 Nigerose, 97 Nitric acid, alkyl esters of, 131 carbohydrate esters of, 117-135 complex of, 121 red fuming, 152

12

Nitrometer, Dupont, 123 Nitron, 124 Nucleic acids, zone electrophoresis of components of, 105 Nucleoprotein, 258 5-Nucleotidase, 312 Nucleotide, diphosphopyridine. See Codehydrogenase I.

0 Oats, water-soluble polysaccharides of, 112 Obituary, of P. A. Levene, 1 Okra, mucilage, 94 Oligosaccharides, infrared absorption spectra of, 32 zone electrophoresis of derivatives, 109 Oxidation, with hypobromite, 176 with lead tetraacetate, 153 with nitrogen tetroxide, 176 with periodate. See Periodate oxidation. with silver oxide, 44, 45 Oxindole, 3-hydroxy-, 248

P Panitol, dodecaacetate, 96 Panose, 96, 283 Paper chromatography, 90, 91, 94, 95 Paper electrophoretograms. See Pherograms. Parasaccharin. See Hexonic acid, 3deoxy-D-lyxo-. Parasaccharinic acid. See Saccharinic acid, para-. 1,3-Pentanediol, 102 1,5-Pentanediol, 102 2,4-Pentanediol, 102 Pentonic acid, 3-deoxy-, 69 configuration of, 42 lactones, 43 phenylhydrazides, 43 preparation of, 41 structure of, 41, 42 -, 3-deoxy-~-erythro-,41, 52 -, 3-deoxy-~-erythro-,41 -, 3-deoxy-~-threo-,41, 42 -, 3-deoxy-~-threo-,41, 42 -, 3-deoxy-2-C-(hydroxymethyl)-,37, 50, 51 -3 3-deoxy-2-C- (hydroxymethyl) -

SUBJECT INDEX, VOLUME

(D-erythro or D-three)-. See Saccharinic acid, “cr”-D-iso-. -, 2-C-methyl-, 37 -, 2-C-methyl-~-arabino-,46 -, 2-C-methyl-~-ribo(?)-,preparation of lactone, 43 Pentopyranosides, M a values of. See table on page 98. Pentoses, action of alkali on, 38, 41 metasaccharo-, 56,57. See also Pentose, 2-deoxy-~-threo-. parasaccharo-, 57 -, 2-deoxy-~-erythro-,61, 62 -, 2-deoxy-~-threo-,59, 73 Pentosylamine, 2-deoxy-N-phenylD-erythro-, 62 Pentdose, S-deoxy-~-qlycero-,52 P enzyme, from potatoes, 113 Periodate oxidation, 40, 47, 139, 153, 245 of D-glucosamine 6-phosphate, 308 of glycogens, 280, 283 of hyaluronic acid, 304 of Schardinger dextrins, 245 pH. See Hydrogen-ion concentration. Phenol, o-amino-, conjugation of, 305307 -, o-nitro-, 166 o-Phenylenediamine, 73, 74, 133 Pherograms, 96, 97, 107-109 Phospho-N-acetyl-D-glucosamine mutase, 311, 312 Phosphoglucomutase, 279, 309, 311 Phospho-D-glucosamine mutase, 309, 311 reactions of, 309 Phosphorochloridic acid, dibenzyl ester, 142 diphenyl ester, 139, 141 Phosphorylases, 246, 284, 296-298 inhibition of, 232 by Schardinger dextrins, 259 of muscle, 285, 288 of potato, 287 Phymatotrichum oninivorum, 268 Pig, extract of skin, 110, 111 Plants, cellulose fibers of, 29 Pneumococcus, Type 11, polysaccharide of, 94 Poly-D-galacturonic acid. See Galactosiduronic acid, POlY-D-. Polysaccharides, acidic, zone electrophoresis of, 110

12

351

infrared absorption spectra of, 24 methanolysis of, 178 methylated, analysis of, 141 mobilities of. See the table on page 11 1 . muoo-, 30, 110, 111 corneal, 92 infrared absorption spectra of, 30 from Neisseria perflava, 113 neutral, boundary electrophoresis of, 114 zone electrophoresis of, 112 pneumococcal, infrared absorption spectra of, 31 of Pneumococcus, Type 11, 94 proteinaceous, study of, by zone electrophoresis, 91, 92 water-soluble, of cereals, 112 Polytomella coeca, 293 1,2-Propanediol, 3-chloro-. See Glyceritol, 1-chloro-1-deoxy-. 2-Propanone, 1,3-dihydroxy-, 72, 74, 76 Propiophenone, 2-hydroxy-3-methoxy3-phenyl-, 67 Proteins, inclusion complexes with glycogens, 280 Pyran, tetrahydro-, infrared absorption spectrum of, 21, 25, 26 Pyrophosphorylase, of yeast, 313 Pyruvaldehyde, 76

Q Q enzyme, from potatoes, 113 Quercitol, cis-, 104 epi-, 104 infrared absorption spectra of, 26 Scyllo-, 103, 104

R Raffinose, 32 R enzyme, 285-287, 295 Resins, cation-exchange, 91, 170, 171 ion-exchange, 91, 172 R p values. See table on page 93. Rhamnitol, 1,5-anhydro-~-,98 Rhamnopyranoside, methyl a-L-,171 p anomer, 99 Rhamnose, 92. See also Mannose, 6deoxy-. L-, nitrate ester, 118 -, 2,4-di-O-methyl-~-,92, 94 -, 3,4-di-O-methyl-~-,92

352

SUBJECT INDEX, VOLUME

Ribitol, 1,5-anhydro-, 98 Ribopyranoside, methyl B-D-, 179 Ribose, 92 electrophoretic mobility of, 87 M G value of, 114 D-, mono- and di-methyl ethers of, 94 -, B-O-benzyl-~-,152 diethyl dithioacetal, 152 -, 2-deoxy-~-. See Pentose, 2-deoxyn-er ythro-. anilide. See Pentosylamine, 2-deoxyN-phenyl-D-erythro-. -, di-O-methyl-D-, 94 -, 2,5-di-O-methyl-~-,94 -, 3,5-di-O-rnethyl-n-, 94 Rosanoff’s convention, 41 “Rottebazillus I,” 197 Ruff degradation, 47, 51, 52, 56, 57, 61, 62, 73 Rye, water-soluble polysaccharides of, 112 S

Saccharic acid. See Glucaric acid, D-. Saccharin, the term (in sugar chemistry), 37 “j3”-D-galactometa-. See Hexonic acid, S-deoxy-~-l yxo-. (‘a 9 , -D-glUCO-, 37 Saccharinic acids, 35, 63, 64 derivatives of. See table of properties on pages 77 to 79. formation of, by various bases, 75 from hexoses , 36 D-galactometa-, 53, 62 configuration of, 59 preparation of, 54 1‘ a 1 , -D-galactometa-, 73. See also Hexonic acid, 3-deoxy-~-xylo-. isomerization of, 58 “8”-D-galactometa-. See Hexonic acid, 3-deoxy-~-lyxo-. D-glUCO-, 62 ‘‘a’’-D-glUCO-, 62, 69, 71, 74, 75 configuration of, 46 lactone, 46 2,3-0-isopropylidene acetal, 47 preparation of, 43 phenylhydrazide, 47 reactions of, 47 structure of, 44

12

D-glucometa-, 59, 62 configuration of, 61 degradation of, 62 preparation of, 60 structure of, 61 a 1 , -n-glucometa-. See Hexonic acid, 3-deoxy-~-ribo-. I ( J t-D-glucometa-. See Hexonic acid, 3-deoxy-~-arabino-. iso-, 37, 63, 64,70 five-carbon, 40 D-iso-, 62 I ( ,I a -D-iso, 42, 43, 48, 62, 75 amide, 51 anilide, 52 brucine salt, 52 calcium salt, 48, 54 configuration of, 51 lactone, 49, 51 preparation of, 48 lead salt, 52 phenylhydrazide, 51 reactions of, 52 structure of, 49 “,”’-D-iSO-, 48, 52 brucine salt, 52 calcium salt, 52 lactone, 52 phenylhydrazide, 52 quinine salt, 52 mechanism of formation of, 42, 43, 46, 62 meta-, 37, 62, 64, 67, 69, 70, 75 five-carbon, 41 formation of, 63 four-carbon, 38 mechanism of formation of, 59 para-, 54, 56, 57, 62, 63, 69. See also Hexonic acid, 3-deoxy-~-lyxo-. preparation of, 39 the term, 37 Sa~charinic-l-Cl~acid, (‘a”-D-galactometa-, 73 “Saccharon,” 45 reduction of, 45 “Saccharonic acid,” 45 Salicylic acid, 3,5-dinitro-, 275 Salmonella montevideo, 293 Schardinger dextrinogenase, 219 Schardinger dextrins, 189-260 a-,89,113,206,207 <(

SUBJECT INDEX, VOLUME

8-, 89, 113, 206, 207 7 - , 205, 207 6-, 205, 207 e-, 205, 208 acetates of, 234, 254, 255 acid hydrolysis of, 203 anomeric configurations of unions in, 246 barium complexes of, 256 a-,canal inclusion complexes of, 251 coupling reactions of, 226-228, 260 cuprammonium reactions of, 254 degradation of, by amylases, 231 derivatives of, 254 di-0-acetyl-mono-0-mesylesters, 257 electrophoretic separation of inclusion complexes of, 252 fractionation of, 211 glycosidic unions in, 243 history of, 192 hydrodynamic properties of. See the table on page 839. inclusion complexes of, 199, 200, 241, 247-252 with bromobensene, 216 with iodide, 249 with iodine, 192, 198, 220, 232, 249 with organic molecules. See the table on page 818. literature on, 192 mesyl esters of, 252, 257 methylation of, 201, 203 methyl ethers of, 234, 235, 243, 252, 254,256 a-,modifications of, 200 molecular constitution of, 243 molecular size of, 234 molecular weights of, by sedimentation and diffusion, 238 by x-ray diffraction, 235 nitrates of, 234, 254, 255 optical rotations of, 241 paper chromatography of, 241 partial acetolysis of, 246 partial bensoates of, 257 partial hydrolysis of, by acids, 239 by enzymes, 239 partial phosphates of, 257 periodate oxidation of, 245 polyacetates of, 212 possible toxicity of, 233

12

353

potassium complexes of, 256 properties of, 190 properties of purified. See the table on page $18. pure a-,203 pure 8-, 203 purification of, 211 relation t o starch, 257 resistance to &amylase, 203 “r” form, 216 ring conformations in, 252 “8” form, 216 sodium complexes of, 256 from starches, 198 substrates for production of, 224 tosyl esters of, 252, 257 tricarbanilates of, 256. See also the table on page 867. unusual properties of, 260 utilization of, by organisms, 232 Serine, L-, 317 Sodium benzoxide, 146 Sodium borohydride, 96 Sodium fluoride, complex of, 121 Sorbose, DL-, 72 L-, 103 Spirogyra, 232 Starches, 246, 265 acid hydrolysis of, 203 breakdown of, 190 complete nitrate-esterification of, 121 effect of alkali on, 268 enzymolysis of, 216 infrared absorption spectrum of, 24 nitrate esters, 134 of Polytomella coeca, 293 relation to Schardinger dextrins, 257 rice, coloration with iodine, 264 ring conformations in, 254 Schardinger dextrins from, 198 structure of, 269 trimethyl ether of, 270 x-ray analysis of, 266 -, 0-(carboxymethy1)-, 259 -, OXY-,246, 259 Steroids, 3-acetoxy-, infrared absorption spectra of, 31 Streptamine, 106 Streptidine, 106 Streptococci, group A, 316 hemolytic, 315

354

SUBJECT INDEX, VOLUME

A 111, 315 group A, 92, 300 Streptomycin, 106 -, mannosido-, 106 Streptothricin, 106 Sucrose, 90, 101 borate complex of, 100 nitrate esters of, 134 octanitrate ester, 118 Sugars, acetamido, M Q and R p values of. See table on page 93. acetates, condensation of, with phenols, 168 preparation of, 161 aldehydo form of, 93 aminodeoxy derivatives of, 106 benzyl ethers of, 137. See the table on page 147. chemical properties of, 150 hydrogenolysis of, 148 physical properties of, 153 preparation of, 142 beneylidene acetals of, 141 condensation of, with alcohols and phenols, 170 deoxy derivatives, infrared absorption spectra of, 26 M Q and R p values of. See the table on page 93. (diphenyl phosphate) esters of, 142 furanose, infrared absorption spectra of, 27 hydrodynamic properties of. See the table on page 239. invert, action of lime-water on, 43 methylated, M a and RF values of. See the table on page 93. nitrate esters of, 117-135 physical properties of, 122 purification of, 123 oxidation of, with silver oxide, 44, 45 phosphates of, 142 polyacetates, of a-pyranoses. See the table on page 186. of 8-pyranoses. See the table on page 186. polybenzoates of furanose and pyranose. See the table o n page 187. pyranose, infrared absorption specha of. See the table o n page 27.

12

separation of, from amino acids, 91 thioacetals of, 152 trityl ethers of, 141 detritylation of, 152

T Takadiastase, 33, 231 Tartaric acid, 120 D-, 59, 61 dinitrate ester, 120 leuo-, 58 Tetrahymena pyriformis, 266, 283 Tetronic acids, deoxy-, 37 -, 3 - d e o x y - ~ ~36, - , 38 -, 3-deoxy-2-C-(bydroxymethyl)-DL-,40 Thyroid, mucopolysaccharides from follicle, 111 a-Toluenethiol, 152 Toluidine Blue, 111 Trehalose, borate complex of, 100 nitrate ester of, 118 Trichomonas gallinae, 266 Triose phosphate, 105 Turanose. See Fructose, S-O-(a-~-gluCOSyl) -D-.

U Uridine, 5-(2-acetamido-2-deoxy-~-glucosy1 dihydrogen pyrophosphate) , 305, 306, 312, 315, 316 5-(2-acetamido - 2-deoxy-0- phospho-Dglucosyl dihydrogen pyrophosphate), 310 “diphosphate 2-acetamido-2-deoxy.~glucose.” See Uridine, 5-(2-acetamido - 2- deoxy-D - glucosyl dihydrogen pyrophosphate) . “diphosphate N - acetyl - D - glucosamine.” See Uridine, 5-(2-acetamido-2-deoxy-D-glucosyl dihydrogen pyrophosphate) . “diphosphate D-glucose.” See Uridine, b-(D-glUCOSyl dihydrogen pyrophosphate). “diphosphate o-glucuronic acid.” See Uridine, 5-(~-glucosyluronicacid dihydrogen pyrophosphate). 5-(~-glucosyl dihydrogen pyrophosphate), 306, 313, 316

SUBJECT INDEX, VOLUME

5-(~-glucosyluronic acid dihydrogen pyrophosphate), 305, 306, 316 biosynthesis of, 306 5-phosphate, 305, 312 5-(trihydrogen pyrophosphate), 305, 306, 312 Uridine-5-phosphatase, 305 Uridine-5-triphosphoric acid, 313, 316 Uridylic acid, “b,” methylated, 94 Uronic acids, zone electrophoresis of, 104

V Valeric acid, 4-hydroxy-, 1,4-1actone, 56 silver salt, 56 -, 4-hydroxy-2-methyl-n~-,1,4-lactone, 46, 49 -, 2 - r n e t h y l - ~ ~46, - , 49 -, trihydroxy-, 56 -, 1,3,4-trihydroxy-. See Pentonic acid, 3-deoxy-. Vitamin A, 248

W Walden inversion, 161, 163-167 Water, containing 01*,133

X Xylan, 40 p-Xylene, 248 2,4-Xylenol, 5-nitro-, 124 Xylitol, 1,5-anhydro-, 98 Xylobiose. See Xylose, 4-O-fi-D-XylOpyranosyl-D-. Xylofuranoside, methyl LY-D-,100 j3 anomer, 100 Xylopyranoside, alkyl D-, 98 -, methyl WD-, 89 j3 anomer, 130 3-nitrate, 130 2,4-diacetate, 130 Xylose, D-, action of alkali on, 41 I-. See Xylose, D-. M o value of, 114 D-, 1,2,3,4-tetranitrate ester, 123 -, 2-deoxy-~-. See Pentose, 2-deoxyn-threo-. -, 4-0-j3-~-xylopyranosyl-~-, 40 Xyloside, methyl D-, 2,3,4-trihitrate ester, 123

355

12

-, methyl 2,4-di-0-acetyl-fi-~-,119 3-nitrate ester, 119

-, phenyl 2,3,4-tri-O-acetyl-c-~-, 149 j3 anomer, 149, 169 Xylotriose, 40

Y Yeast, N-acetylase from, 311 baker’s, 96 hexokinase from, 308 invertase of, 90, 101 isoamylase of, 273, 285 “maltase” from, 273 pyrophosphorylase of, 313 Z

Zea mags, 266 polysaccharides of, 279 Zone electrophoresis, advantage of, 105 apparatus for, 84 using an enclosed strip, 84 using an immersed strip, 84 using a suspended strip, 84 complemented by paper chromatography, 90 detection of zones in, 109 for molecular-size determination, 107 for separating amino acids from sugars, 91 for separating carbohydrates, 91 in presence of arsenite buffer, 106 of barbiturate buffer. See the table on page 111. of basic lead acetate, 106, 107 of borate, of acidic carbohydrates, 104 of basic carbohydrates, 104 of disaccharides, 95 of flavonoid glycosides, 104 of glycofuranosides, 100 of glycopyranosides, 98 of methylated sugars, 92 of monosaccharides, 89 of neutral carbohydrates, 89 of oligosaccharides, 95 of polyhydric alcohols, 102 of borax, 106 of complexing agents, of carbohydrates. See the table on page 106. of formate buffer, 107

356

SUBJECT INDEX, VOLUME

of metavanadate, 107 of tungstate-molybdate, 107 location of zones in, 83 matrix for, 83, 110 mobility in, of carbohydrates, 87 effect of pH on, 87 of 2-acetamido-2-deoxy-~-galactose, 106 106 of 2-acetamido-2-deoxy-~-glucose, of acidic polysaccharides, 110 of acid reversion products of D-galactose, 109 of aldose-bisulfite complexes. See the table on page 108. of D-altrose, 91 of amino acids, 91 of aminodeoxy sugars, 106 of amylopectin, 112, 113 of amylosaccharides, 112, 113 of amylose, 112, 113 of aspartic acid peptides, 91 of N-benzylglycosylammonium ions. See the table on page 108. of carbohydrates, 81 in presence of borate, 83, 86 of cereal polysaccharides. See the table on page 112. of chondroitinsulfuric acid, 111 of 2,4-di-O-methyl-~-rhamnose, 92 of 3,a-di-O-methyl-L-rhamnose, 92 of di- and mono-0-methyl-D-riboses, 94 of flavonoid glycosides. See the table on page 106. of fructose 1,6-diphosphate, 105 of galactitol, 107 of D-galactose, 89, 91 of D-galactosyl phosphate, 105 of D-galacturonic acid 1-phosphate, 105 of D-glucitol, 107 of gluconic acid, 105 of D-glucose, 86, 89, 91 of a-D-glucosyl phosphate, 105 of D-glucuronio acid 1-phosphate, 105 of glutamic acid peptides, 91 of glyceritol 3-phosphate, 105 of glycogens, 113

of of of of of of of

12

D-gulose, 91 heparin, 111 hexitols, 103, 106, 107 hexose monophosphates, 105 hexose phosphate, 105 hyaluronic acid, 111 hydrolyzate, of corneal mucopolysaccharide, 92 of Group A hemolytic streptococci, 92 of heparin, 92 of human fibrin, 92 of inositols, 103 of isomaltose, 91 of 2-ketobutyric acid, 105 of 2-ketoglutaric acid, 105 of 2-keto-~-arabino-hexonic acid, 105 of maltose, 86, 91 of o-mannitol, 107 of D-mannose, 89, 91 of mannosidostreptomycin, 106 of mucopolysaccharides, 110 from thyroid follicle, 111 of Neisseria perjlava polysaccharide, 113 of neutral polysaccharides, 112 of nucleic acid components, 105 of nucleic acid hydrolyzate, 86 of phenylhydrazones of 2-keto acids, 106 of pig-skin extract, 110, 111 of proteinaceous polysaccharide hydrolyzate, 91, 92 of streptamine, 106 of streptidine, 106 of streptomycin, 106 of streptothricin, 106 of triose phosphate, 105 on cellulose sheets, 110 on glass paper, 109 paper strip for, 85 potential gradients in, 85, 91 potential gradients for, 110 speed of, 105 spray reagents for, 109 technique of, 83 the term, 82

Cumulative Author Index for Volumes 1-11 A Adams, Mildred. See Caldwell, Mary L. Anderson, Ernest, and Sands, Lila, A Discussion of Methods of Value in Research on Plant Polyuronides, I, 329-344 Aspinall, G. O., The Methyl Ethers of Hexuronic Acids, IX, 131-148 Aspinall, G. O., The Methyl Ethers of D-Mannose, VIII, 217-230

Bonner, William A,, Friedel-Crafts and Grignard Processes in the Carbohydrate Series, VI, 251-289 Bourne, E. J., and Peat, Stanley, The Methyl Ethers of D-Glucose, V, 145190 Bourne, E . J. See also, Barker, S . A. Bray, H. G., D-Glucuronic Acid in Metabolism, VIII, 251-275 Bray, H. G., and Stacey, M., Blood Group Polysaccharides, IV, 37-55

B Ballou. Clinton E., Alkali-sensitive Glscosides, IX, 59-95 Barker, G. R.3 Nucleic Acids, XI, 285-333 Barker, S. A., and Bourne, E. J., Acetals and Ketals of the Tetritols, Pentito18 and Hexitols, VII, 137-207 Barrett, Elliott P., Tmnds in the Development Of for Sugar Refining, VI, 205-230 Barry, C. P., and Honeyman, John, Fructose and its Derivatives, VII, 53-98 Bayne, S., and Fewster, J. A., The Oaones, XI, 43-96 BeBlik, Andrew, Kojic Acid, XI, 146183 Bell, D. J., The Methyl Ethers of D-Galactose, VI, 11-25 Binkley, W. W., Column Chromatography of Sugars and Their Derivatives, X, 55-94 Binkley, W. W., and Wolfrom, M. L., Composition of Cane Juice and Cane Final Molasses, VIII, 291-314 Blair, Mary Grace, The 2-Hydroxyglycals, IX, 97-129 Bobbitt, J. M., Periodate Oxidation of Carbohydrates, XI, 1-41 Boeseken, J., The Use of Boric Acid for the Determination of the Configuration of Carbohydrates, IV, 189-210

C

Caldwell, Mary L., and Adama, Mildred, Action of Certain Alpha ~ ~ v, 229-268 Cantor, Sidney, M. see ill^^, ~~b~~~ Ellsworth. Carr, c. je1leff, and Krantz, John c., Jr., Metabolism of the Sugar Alcohols and Their Derivatives, I, 175192 Compton, Jack, The Molecular Constitution of Cellulose, 111, 185-228 D Dean, G. R., and Gottfried, J. B., The Commercial Production of Crystalline Dextrose, V, 127-143 Deits, Victor R. See Liggett, R. W . Deuel, Harry J., Jr., and Morehouse, Margaret G., The Interrelation of Carbohydrate and Fat Metabolism, 11, 119-160 Deulofeu, Venancio, The Acylated Nitriles of Aldonic Acids and Their Degradation, IV, 119-151 Dimler, R. J., 1,6-Anhydrohexofuranoses, A New Class of Hexosans, VII, 37-52 Doudoroff, M. See Hassid, W. Z.

357

~

358

CUMULATIVE AUTHOR INDEX FOR VOLS.

E Elderfield, Robert C., The Carbohydrate Components of the Cardiac Glycosides, I, 147-173 Ellis, G. P., and Honeyman, John, Glycosylamines, X, 95-168 Evans, Taylor H., and Hibbert, Harold, Bacterial Polgsaccharides,. 11, . 203233 Evans, W. L., Reynolds, D. D., and Talley, E. A., The Synthesis of Oligosaccharides, VI, 27-81 l7

Fewster, J. A. See Bayne, S. Fletcher, Hewitt, G., Jr., The Chemistry and Configuration of the Cyclitols, 111, 45-77 Fletcher, Hewitt G., Jr., and Richtmyer, Nelson K., Applications in the Carbohydrate Field of Reductive Desulfurization by Raney Nickel, V, 1-28 Fletcher, Hewitt G., Jr. See also, Jeanloz, Roger W. Fordyce, Charles R., Cellulose Esters of Organic Acids, I, 309-327 Foster, A. B., and Huggard, A. J., The Chemistry of Heparin, X, 335-368 Foster, A. B., and Stacey, M., The Chemistry of the 2-Amino Sugars (2-Amino-2-deoxy-sugars), VII, 247288 Fench, Dexter, The Raffinose Family of Oligosaccharides, IX, 149-184

G Garcia GonzAlez, F., Reactions of Monosaccharides with beta-Ketonic Esters and Related Substances, XI, 97-143 Goepp, Rudolph Maximilian, Jr. See Lohmar, Rolland. Gottfried, J. B. See Dean, G. R. Gottschalk, Alfred, Principles Underlying Enzyme Specificity in the Domain of Carbohydrates, V, 49-78 Green, John W., The Halogen Oxidation of Simple Carbohydrates, Excluding the Action of Periodic Acid, 111,

129-184

1-11

Greenwood, C. T., Aspects of the Physical Chemistry of Starch, XI, 335-385 Greenwood, C. T., The Size and Shape of Some Polysaccharide Molecules, VII, 289332; XI, 385-393 Gurin, Samuel, Isotopic Tracers in the Study of Carbohydrate Metabolism, 111, 229-250 U *-

Harris, Elwin E., Wood Saccharification, IV, 153-188 Haskins, Joseph F., Cellulose Ethers of Industrial Significance, 11, 279-294 Hassid, W. Z., and Doudoroff; M., Enzymatic Synthesis of Sucrose and Other Disaccharides, V, 2 9 4 8 Haynes, L. J., and Newth, F. H., The Glycosyl Halides and Their Derivatives, X, 207-256 Hehre, Edward J., The Substituted-sucrose Structure of Melezitose, VIII, 277-290 Helferich, Burckhardt, The Glycals, VII, 209-245 Helferich, Burckhardt, Trityl Ethers of Carbohydrates, 111, 79-111 Hibbert, Harold. See Evans, Taylor H. Hindert, Marjorie. See Karabinos, J. V. Hirst, E. L., [Obituary of] James Colquhoun Irvine, VIII, xi-xvii Hirst, E. L., [Obituary of] Walter Norman Haworth, VI, 1-9 Hirst, E. L., and Jones, J. K. N., The Chemistry of Pectic Materials, 11, 235-251 Hirst, E. L., and Ross, A. G., [Obituary of] Edmund George Vincent PerciVal, X, xiii-xx Hodge, John E., The Amadori Rearrangement, x, 169-205 Honeyman, John. See Barry, C. P. Honeyman, John. See Ellis, G . P. Hough, L., and Jones, J. K . N., The Biosynthesis of the Monosaccharides, XI, 185-262 Huggard, A. J. See Foster, A. B. Hudson, C. S., Apiose and the Glycosides of the Parsley Plant, IV, 57-74 Hudson, C. S., The Fischer Cyanohydrin Synthesis and the Configurations of

CUMULATIVE AUTHOR INDEX FOR VOLS.

Higher-carbon Sugars and Alcohols, I, 1-36 Hudson, C. S., Historical Aspects of Emil Fischer’s Fundamental Conventions for Writing Stereo-formulas in a Plane, 111,1-22 Hudson, C. S., Melezitose and Turanose, 11, 1-36

J Jeanloz, Roger W., [Obituary of] Kurt Heinrich Meyer, XI, xiii-xviii Jeanloz, Roger W., and Fletcher, Hewitt G., Jr., The Chemistry of Ribose, VI, 135-174 Jones, J. K. N., and Smith, F., Plant Gums and Mucilages, IV, 243-291 Jones, J. K. N. See also, Hirst, E. L. Jones, J. K . N. See also, Hough, L.

6: Karabinos, J. V., Psicose, Sorbose and Tagatose, VII, 99-136 Karabinose, J. V., and Hindert, Marjorie, Carboxymethylcellulose, IX, 285-302 Kent, P. W. See Stacey, M. Kertesz, Z . I., and McColloch, R. J., Enzymes Acting on Pectic Substances, V, 79-102 Kowkabany, George N., Paper Chromatography of Carbohydrates and Related Compounds, IX, 303-353 Krantz, John C., Jr. See Carr, C. Jelleff.

L Laidlaw, R. A , , and Percival, E. G. V., The Methyl Ethers of the Aldopentoses and of Rhamnose and Fucose, VII, 1-36 Lemieux, R. U., Some Implications in Carbohydrate Chemistry of Theories Relating to the Mechanisms of Replacement Reactions, IX, 1-57 Lemieux, R. U., and Wolfrom, M. L., The Chemistry of Streptomycin, 111, 337-384 Lespieau, R., Synthesis of Hexitols and Pentitols from Unsaturated Polyhydric Alcohols, 11, 107-118

1-11

359

Levi, Irving, and Purves, Clifford B., The Structure and Configuration of Sucrose (alpha-o-Glucopyranosyl beta-D-Fructofuranoside), IV, 1-35 Liggett, R. W., and Deitz, Victor R., Color and Turbidity of Sugar Products, IX, 247-284 Lohmar, Rolland, and Goepp, Rudolph Maximilian, Jr., The Hexitols and Some of Their Derivatives, IV, 211241

M Maher, George G., The Methyl Ethers of the Aldopentoses and of Rhamnose and Fucose, X, 257-272 Maher, George G., The Methyl Ethers of o-Galactose, x, 273-282 McColloch, R . J. See Kertesz, Z. I. McDonald, Emma J., The Polyfructosans and Difructose Anhydrides, 11, 253-277 Mehltretter, C. L., The Chemical Synthesis of D-Glucuronic Acid, VIII, 231-249 Miller, Robert Ellsworth, and Cantor, Sidney M., Aconitic Acid, a Byproduct in the Manufacture of Sugar, VI, 231-249 Mills, J. A., The Stereochemistry of Cyclic Derivatives of Carbohydrates, X, 1-53 Morehouse, Margaret G. See Deuel, Harry J., Jr. Mori, T., Seaweed Polysaccharides, VIII, 315-350 Myrblck, Karl, Products of the Enzymic Degradation of Starch and Glycogen, 111, 251-310

N Neuberg, Carl, Biochemical Reductions at the Expense of Sugars, IV, 75117 Newth, F. H., The Formation of Furan Compounds from Hexoses, VI, 83106 Newth, F. H. See also, Haynes, L. J. Nickerson, R. F., The Relative Crystallinity of Celluloses, V, 103-126

360

CUMULATIVE AUTHOR INDEX FOR VOLS.

0

Overend, W. G., and Stacey, M., The Chemistry of the 2-Desoxysugars, VIII, 45-105

P Pacsu, Eugene, Carbohydrate Orthoesters, I, 77-127 Peat, Stanley, The Chemistry of Anhydro Sugars, 11, 37-77 Peat, Stanley. See also, Bourne, E. J. Percival, E. G. V., The Structure and Reactivity of the Hydrazone and Osazone Derivatives of the Sugars, 111,2344 Percival, E. G. V. See also, Laidlaw, R. A. Polglase, W. J., Pdysaccharides Associated with Wood Cellulose, X, 283333 Purves, Clifford B. See Levi, Irving.

R Raymond, Albert L., Thio- and Selenosugars, I, 129-145 Reeves, Richard E. , CuprammoniumGlycoside Complexes, VI, 107-134 Reynolds, D. D. See Evans, W. L. Richtmyer, Nelson K., The Altrose Group of Substances, I, 37-76 Richtmyer, Nelson K., The 2-(aZdoPolyhydroxyal kyl) benzimidazoles, VI, 175-203 Richtmyer, Nelson K. See also, Fletcher, Hewitt G., Jr. Ross, A. G. See Hirst, E. L.

S Sands, Lila. See Anderson, Ernest. Sattler, Louis, Glutose and the Unfermentable Reducing Substances in Cane Molasses, 111, 113-128 Sohoch, Thomas John, The Fractionation of Starch, I, 247-277

1-11

Shafizadeh, F., Branched-chain Sugars of Natural Occurrence, XI, 263-283 Smith, F., Analogs of Ascorbic Acid, 11, 79-106 Smith, F. See also, Jones, J. K. N. Sowden, John C., The Nitromethane and 2-Nitroethanol Syntheses, VI, 291318 Stacey, M., The Chemistry of Mucopolysaccharides and Mucoproteins, 11, 161-201 Stacey, M., and Kent, P. W., The Polysaccharides of Mycobacteriurn tuberculosis, 111,311436 Stacey, M. See also, Bray, H. G. Stacey, M. See also, Foster, A. B. Stacey, M. See also, Overend, W. G. Sugihara, James M., Relative Reactivities of Hydroxyl Groups of Carbohydrates, VIII, 1 4 4 T Talley, E. A. See Evans, W. L. Teague, Robert S., The Conjugates of D-Ghcuronic Acid of Animal Origin, IX, 185-246 Tipson, R. Stuart, The Chemistry of the Nucleic Acids, I, 193-245 Tipson, R. Stuart, Sulfonic Esters of Carbohydrates, VIII, 107-215

W Whistler, Roy L., Preparation and Properties of Starch Esters, I, 279-307 Whistler, Roy L., Xylan, V, 269-290 Wiggins, L. F., Anhydrides of the Pentitols and Hexitols, V, 191-228 Wiggins, L. F., The Utilization of Sucrose, IV, 293-336 Wolfrom, M. L., [Obituary of] Claude Silbert Hudson, IX, xiii-xviii Wolfrom, M. L., [Obituary of] Rudolph Maximilian Goepp, Jr., 111, xv-xxiii Wolfrom, M. L. See also, Binkley, W. W. Wolfrom, M. L. See also, Lemieux, R. U.

Cumulative Subject Index for Volumes 1-11 A Acetals, of hexitols, pentitols, and tetritols, VII, 137-207 Aconitic acid, VI, 231-249 Adsorbents, granular, for sugar refining, VI, 205230 Alcohols, higher-carbon sugar, configurations of, I, 1-36 unsaturated polyhydric, 11, 107-118 Aldonic acids, acylated nitriles of, IV, 119-151 Aldopentoses, methyl ethers of, VII, 1-36; X , 257272 Altrose, group of compounds related to, I, 3776 Amadori rearrangement, X , 169-205 Amino sugars. See Sugars, 2-amino-2deoxy-. Amylases, certain alpha, V, 229-268 Anhydrides, difructose, 11, 253-277 of hexitols, V, 191-228 of pentitols, V, 191-228 Anhydro sugars. See Sugars, anhydro-. Animals, conjugates of D-glucuronic acid originating in, IX, 185-246 Apiose, IV, 67-74 Ascorbic acid, analogs of, 11, 79-106

Biochemical reductions, a t the expense of sugars, IV, 75-117 Biosynthesis, of the monosaccharides, XI, 185262 Blood groups, polysaccharides of, IV, 37-55 Boric acid, for determining configuration of carbohydrates, IV, 189-210 Branched-chain sugars. See Sugars, branched-chain. C

Cane juice, composition of, VIII, 291-314 Cane molasses. See Molasses, cane. Carbohydrates, applications of reductive desulfurization by Raney nickel, in the field of,

v, 1-28

as components of cardiac glycosides, I, 147-173 determination of configuration of, with boric acid, IV, 189-210 enzyme specificity in the domain of, V, 49-78 Friedel-Crafts and Grignard processes applied to, VI, 251-289 halogen oxidation of simple, 111, 129184 mechanisms of replacement reactions in chemistry of, IX, 1-57 metabolism of, 11, 119-160; 111, 229250 orthoesters of, I, 77-127 periodate oxidation of, XI, 1 4 1 and related compounds, paper chromatography of, IX, 303-353 relative reactivities of hydroxyl groups of, VIII, 1 4 4 stereochemistry of cyclic derivatives of, X , 1-53

B Bacteria, polysaccharides from, 11, 203-233 Benaimidazoles, 2- (aldo-polyhydroxyalkyl) -, VI, 175203 361

362

CUMULATIVE SUBJECT INDEX FOR VOLS.

sulfonic esters of, VIII, 107-215 trityl ethers of, 111, 79-111 Carboxymethyl ether, of cellulose, IX, 285-302 Cellulose, carboxymethyl-, IX, 285-302 esters of, with organic acids, I, 309327 ethers of, 11, 279-294 molecular constitution of, 111, 185228 of wood, polysaccharides associated with, X, 283-333 Celluloses, relative crystallinity of, V, 103-126 Chromatography, column. See Column chromatography. paper. See Paper chromatography. Color, of sugar products, IX, 247-284 Column chromatography, of sugars and their derivatives, X, 55-94 Complexes, cuprammonium-glycoside, VI, 107-134 Configuration, of carbohydrates, determination of, IV, 189-210 of cyclitols, 111, 45-77 Conjugates, of D-glucuronic acid, IX, 185-246 Crystallinity, relative, of celluloses, V, 103-126 Cuprammonium-glycoside complexes, VI, 107-134 Cyanohydrin synthesis, Fischer, I, 1-36 Cyclic derivatives, of carbohydrates, stereochemistry of, 1-53 Cyclit 01s, chemistry and configuration of, 111, 45-77

x,

1-11

Dextrose, commercial production of crystalline, V, 127-143 Difructose, anhydrides, 11, 253-277 Disaccharides, enzymic synthesis of, V, 2 9 4 8

E Enzymes. See also Amylases. acting on pectic substances, V, 79-102 degradation by, of starch and glycogen, 111, 251-310 specificity of, in the domain of carbohydrates, V, 49-78 synthesis of sucrose and other disaccharides by, V, 2 9 4 8 Esters, of cellulose, with organic acids, I, 309-327 beta-ketonic (and related substances), reactions with monosaccharides, XI, 97-143 of starch, preparation and properties of, I, 279-307 sulfonic, of carbohydrates, VIII, 107215 Ethanol, 2-nitro-, syntheses with, VI, 291-318 Ethers, carboxymethyl, of cellulose, I X , 285302 of cellulose, 11, 279-294 methyl, of the aldopentoses, VII, 1-36; X , 257-272 of fucose, VII, 1-36; X , 257-272 of D-galactose, VI, 11-25; X, 273-282 of D-glucose, V, 145-190 of hexuronic acids, IX, 131-148 of D-mannose, VIII, 217-230 of rhamnose, VII, 1-36; X, 257-272 trityl, of carbohydrates, 111, 79-111

D

F

Degradation, of acylated nitriles of aldonic acids, IV, 119-151 Deoxy sugars. See Sugars, deoxy-. Desulfurization, reductive, by Raney nickel, V, 1-28

Fat, metabolism of, 11, 119-160 Formulas, stereo-, writing of, in a plane, 111, 1-22 Fractionation, of starch, I, 247-277

Friedel-Crafts process, in the carbohydrate series, VI, 251-289 Fructans, 11, 253-277 Fructofuranoside, a-D-glucopyranosyl p-D-, IV, 1-35 Fructosans, poly-. See Fructans. Fructose, and its derivatives, VII, 53-98 di-, anhydrides, 11, 153-277 Fucose, methyl ethers of, VII, 1-36; X, 257272 Furan compounds, formation from hexoses, VI, 83-106

G Galactose, methyl ethers of D-, VI, 11-25; X, 273282 Glucose. See also Dextrose. methyl ethers of D-, V, 145-190 Glucuronic acid, Dchemical synthesis of, VIII, 231-249 conjugates of, of animal origin, IX, 185-246 in metabolism, VIII, 251-275 Glutose, 111, 113-128 Glycals, VII, 209-245 -, 2-hydroxy-, IX, 97-129 Glycogen, enzymic degradation of, 111, 251-310 Glycoside-cuprammonium complexes, VI, 107-134 Glycosides, alkali-sensitive, IX, 59-95 cardiac, I, 147-173 of the parsley plant, IV, 57-74 Glycosylamines, X , 95-168 Glycosyl halides, and their derivatives, X , 207-256 Goepp, Rudolph Maximilian, Jr., obituary of, 111, xv-xxiii Grignard process, in the carbohydrate series, VI, 251-289 Gums, of plants, IV, 243-291

H Halogen oxidation. See Oxidation, halogen.

Haworth, Walter Norman, obituary of, VI, 1-9 Heparin, chemistry of, X , 335-368 Hexitols, acetals and ketals of, VII, 137-207 anhydrides of, V, 191-228 and some of their derivatives, IV, 211241 synthesis of, 11, 107-114 Hexofuranoses, l,g-anhydro-, VII, 37-52 Hexosans, VII, 37-52 Hexoses. See also Hexofuranoses. formation of furan compounds from, VI, 83-106 Hexuronic acids, methyl ethers of, IX, 131-148 Hudson, Claude Silbert, obituary of, IX, xiii-xviii Hydrazones, of sugars, 111, 2 3 4 4 Hydroxyl groups, relative reactivities of, VIII, 1-44

I Irvine, James Colquhoun, obituary of, VIII, xi-xvii Isotopic tracers. See Tracers, isotopic.

K Ketals, of hexitols, pentitols, and tetritols, VII, 137-207 Kojic acid, XI, 145-183

M Mannose, methyl ethers of D - , VIII, 217-230 Mechanisms, of replacement reactions in carbohydrate chemistry, IX, 1-57 Melezitose, 11, 1-36 structure of, VIII, 277-290 Metabolism, of carbohydrates, 11, 119-160 use of isotopic tracers in studying, 111, 229-250 of fat, 11, 119-160 of the sugar alcohols and their derivatives, I, 17b192 D-glucuronic acid in, VIII, 251-275

364

CUMULATIVE SUBJECT INDEX FOR VOLS.

Methane, nitro-, syntheses with, VI, 291-318 Methyl ethers. See Ethers, methyl. Meyer, Kurt Heinrich, obituary of, X I , xiii-xviii Molasses, cane, 111, 113-128 cane final, composition of, VIII, 291314 ,Monosaccharides, biosynthesis of, X I , 185-262 reactions of, with bete-ketonic esters and related substances, XI, 97-143 Mucilages, of plants, IV, 243-291 Mucopolysaccharides. See Polysaccharides, muco-. Mucoproteins. See Proteins, muco-. Mycobacterium tuberculosis, polysaccharides of, 111, 311-336

' N Nickel, Raney. See Raney nickel. Nitriles, acylated, of aldonic acids, IV, 119-151 Nucleic acids, I, 193-245; XI, 285-333 0

Obituary, of Rudolph Maximilian Goepp, Jr., 111, xv-xxiii of Walter Norman Haworth, VI, 1-9 of Claude Silbert Hudson, IX, xiiixviii of James Colquhoun Irvine, VIII, xixvii of Kurt Heinrich Meyer, X I , xiii-xviii of Edmund George Vincent Percival, X, xiii-xx Oligosaccharides, the raffinose family of, IX, 149-184 synthesis of, VI, 27-81 Orthoesters, of carbohydrates, I, 77-127 Osazones, of sugars, 111, 23-44 Osones, X I , 43-96

1-11

Oxidation, halogen, of simple carbohydrates, 111, 129-184 periodate, of carbohydrates, XI, 1 4 1

P Paper chromatography, of carbohydrates and related compounds, IX, 303-353 Parsley, glycosides of the plant, IV, 57-74 Pectic materials, chemistry of, 11, 235-251 enzymes acting on, V, 79-102 Pentitols, acetals and ketals of, VII, 137-207 anhydrides of, V, 191-228 synthesis of, 11, 107-118 Percival, Edmund George Vincent, obituary of, X, xiii-xx Periodate oxidation. See Oxidation, periodate. Physical chemistry, of starch, XI, 335-385 Plants, glycosides of parsley, IV, 57-74 gums of, IV, 243-291 mucilages of, IV, 243-291 polyuronides of, I, 329-344 Polyfructosans. See Fructans. Polysaccharides. See also Carbohydrates) Cellulose, Fructans, Glycogen, Starch, and Xylan. associated with wood cellulose, X, 283333 bacterial, 11, 203-233 blood group, IV, 37-55 muco-, chemistry of, 11, 161-201 of Mycobacterium tuberculosis, 111, 311-336 of seaweeds, VIII, 315-350 shape and size of molecules of, VII, 289-332; XI, 385-393 Polyuronides, of plants, I, 329-344 Proteins, muco-, chemistry of, 11, 161-201 Psicose VII, 99-136

CUMULATIVE SUBJECT INDEX FOR VOLS.

R Raffinose, family of oligosaccharides, IX, 149-184 Raney nickel, reductive desulfurization by, V, 1-28 Reactivities, relative, of hydroxyl groups of carbohydrates, VIII, 1-44 Rearrangement, the Amadori, X, 169-205 Reductions, biochemical, a t the expense of sugars, IV, 75-117 Replacement reactions, mechanisms of, in carbohydrate chemistry, IX, 1-57 Rhamnose , methyl ethers of, VII, 1-36; X, 257-272 Ribose chemistry of, VI, 135-174

S Saccharification, Of Wood, IV,153-188 Seaweeds, polysaccharides of, VIII, 315-350 Seleno sugars. See Sugars, deoxy-seleno-. Shape, of some polysaccharide molecules, VII, 289-332; XI, 385-393 Size, of some polysaccharide molecules, VII, 289-332; XI, 385-393 Sorbose, VII, 99-136 Specificity, of enzymes, in the domain of carbohydrates, V, 49-78 Starch , enzymic degradation of, 111, 251-310 fractionation of, I, 247-277 physical chemistry of, XI, 335-385 preparation and properties of esters of, I, 279-307 Stereochemistry , of cyclic derivatives of carbohydrates, X, 1-53 Streptomycin , chemistry of, 111, 337-384 Sucrose,

1-11

365

enzymic synthesis of, V, 2948 structure and configuration of, IV, 1-35 utilization of, IV, 293-336 Sugar, aconitic acid as by-product in manufacture of, VI, 231-249 Sugar alcohols, higher-carbon, configurations of, I, 1-36 and their derivatives, metabolism of, I, 175-192 Sugar products, color and turbidity of, IX, 247-284 Sugar refinining, granular adsorbents for, VI, 205-230 Sugars, biochemical reductions at the expense of, IV, 75-117 branched-chain, of natural occurrence, XI, 263-283 higher-carbon, configurations of, I, 1-36 hydrazones of, 111, 23-44 osazones of, 111, 2344 and their derivatives, column chromatography of, X, 55-94 related t o altrose, I, 37-76 -, 2-amino-. See Sugars, 2-amino-2deoxy-. -, 2-amino-2-deoxy-, VII, 247-288 -, anhydro-, chemistry of, II,37-77 -, 2-deoxy-, VIII, 45-105 -, deoxy-seleno-, I, 144-145 -, deoxy-thio-, I, 129-144 Synthesis, biochemical, of monosaccharides, XI, 185-262 chemical, of D-glucuronic acid, VIII, 231-249 enzymic, of sucrose and other disaccharides, V, 2948 Sulfonic esters, of carbohydrates, VIII, 107-215

T Tagatose, VII, 99-136 Tetritols, acetals and ketals of, VII, 137-207

366

CUMULATIVE SUBJECT INDEX FOR VOLS.

Thio sugars. See Sugars, deoxy-thio-. Tracers, isotopic, 111, 229-250 Trityl ethers, of carbohydrates, 111, 79-111 Turanose, 11, 1-36 Turbidity, of sugar products, IX, 247-284

1-11

W Wood, polysaccharides associated with cellulose of, X, 283-333 saccharification of, IV, 153-188

X Xylan, V, 269-290

ERRATA

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