Advances in Carbohydrate Chemistry, Volume 8

ADVANCES IN CARBOHYDRATE CHEMISTRY

VOLUME 8

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Advances in

Carbohydrate Chemistry Edited by MELVILLE L. WOLFROM CLAUDE S. HUDSON National Institutes of Health Bethesda, Maryland

Department of Chemistry The Ohio State Universily Columbus, Ohio

Associate Editors for the British Isles STANLEY PEAT MAURICE STACEY The Universily Birmingham, England

University College of North Wales Bangor, Caernarvonshire, Wales

E. L. HIRST The University Edinburgh, Scotland

Board of Advisors C. B. PURVES J. C. SOWDEN ROYL. WHISTLER

WILLIAML. EVANS HERMANN 0. L. FISCHER R. C. HOCKETT W. W. PIGMAN

Volume 8

1953

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

Copyright, 1953, by ACADEMIC PRESS INC. 125 EAST2 3 STREET ~ ~ NEWYORK10, 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 Congress Catalog Card Number (45-11351)

PRINTED I N THE UNITED STATE8 OF AMERICA

CONTRIBUTORS TO VOLUME 8 G. 0. ASPINALL, The University of Edinburgh, Scotland W. W. BINKLEY,Department of Chemistry, The Ohio State University, Columbus, Ohio

H. G. BRAY,Department of Physiology, Medical School of the University, Birmingham, England EDWARD J. HEHRE,Department of Bacteriology and Immunology, Cornell University Medical College, New York, New York C. L. MEHLTRETTER, Northern Regional Research Laboratory, Agricultural Research Administration, U. S. Department of Agriculture, Peoria, Illinois T. MORI,Tokyo University, Tokyo, Japan W. G. OVEREND, The Pennsylvania State College, U . S. A., and Chemistry Department, University of Birmingham, England M. STACEY, Chemistry Department, University of Birmingham, England JAMESM. SUGIHARA, Department of Chemistry, University of Utah, Salt Lake City, Utah R. STUART TIPSON,Department of Research in Organic Chemistry, Mellon Institute, Pittsburgh, Pennsylvania M. L. WOLFROM, Department of Chemistry, The Ohio State University, Columbus, Ohio

V

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PREFACE The sudden death of Claude S. Hudson on December 27, 1952, in his home at Washington, D. C., removes from carbohydrate chemistry one of its great and inspiring leaders. Thus, in the space of a few years, have passed away three great pioneers in this field, W. N. Haworth (1950), J. C. Irvine (1952), and C. S. Hudson (1952). The last had been a guiding spirit for ((Advances in Carbohydrate Chemistry” since its inception in 1944. For the past four years he had been an active editor and, indeed, since his retirement in January, 1951, the editorship of the “Advances” had occupied the greater portion of his time. Dr. Hudson set up exacting standards for his own writing and research and held to a high quality of scholarship in these endeavors. He laid down the policy that the attempt should be made to hold all of the chapters in “Advances in Carbohydrate Chemistry ” to the criteria established in his own writing, while maintaining the integrity of the authors concerned and changing nothing without their full consent. The enforcement of such a policy is not without attendant difficulties; its degree of success may be judged by the readers of these volumes. The manuscripts for the present edition were in the hands of Dr. Hudson at the time of his demise and all had received his editorial attention. Carbohydrate nomenclature has been an ever-present problem in this series. It has recently been the subject of rather extensive consultations between representatives of the American and British carbohydrate chemists, and the final results have appeared in printed form, Chem. Eng. News, 31, 1776 (1953) and J . Chem. SOC.,5108 (1952). Meanwhile, the present volume represents rather a transition stage in this development, particularly as regards the use of the 0-substitution indication, which has been employed only in part. More.uniform usage may be expected in the future. Dr. R. Stuart Tipson has rendered important service in the editing of this volume and has prepared the index. M. L. Wolfrom Columbus, Ohio

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CONTENTS CONTRIBUTORS TO VOLUME 8 . . . . . . . . . . . . . . . . . . . . . . .

v

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

JAMES COLQUHOUN IRVINE . . . . . . . . . . . . . . . . . . . . . . . .

xi

Relative Reactivities of Hydroxyl Groups of Carbohydrates

BY JAMES M . SUGIHARA, Department of Chemistry. University of Utah. Salt Lake City. Utah

. Introduction .

I I1. I11. IV . V VI

. .

1

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

Configurational Relationships and Neighboring-group Effects . . . . . . Selective Etherification . . . . . . . . . . . . . . . . . . . . Selective Esterification and Hydrolysis . . . . . . . . . . . . Selective Oxidation . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 16 24 38 44

The Chemistry of the 2-Desoxysugars BYW . G . OVEREND, The Pennsylvania State College, U . S . A . , and Chemistry Department, University of Birmingham, England, A N D M. STACEY,Chemistry Departmen.1, University of Biriiiinghain, England

I. I1. I11. IV . V. VI .

Introduction . . . . . . . . Nomenclature . . . . . . . Occurrence and Isolation . . Detection . . . . . . . . . Synthesis of 2-Desoxysugars . Transformation Products . .

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

45 46

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

53 66 91

49

Sulfonic Esters of Carbohydrates

BY R . STUARTTIPSON, Department of Research in Organic Chemistry, Mellon Institute, Pittsburgh, Pennsylvania

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Methods for Sulfonylation of Carbohydrates . . . . . . . . . . I11. Physical Properties and Chemical Stability . . . . . . . . . . IV . Reductive Desulfonylation and Desulfonyloxylation . . . . . . V. Action of Some Alkaline Reagents on Sulfonic Esters . . . . . . . VI . Action of Alkali-Metal Halides on Sulfonic Esters . . . . . . . . VII . Action of Other Salts on Sulfonic Esters . . . . . . . . . . . . .

. . . . . .

. . . . . .

108 . 111 . 140 . 161 . 165 . 180

. . . 212

The Methyl Ethers of D-Mannose

BY G . 0. ASPINALL,The University of Edinburgh, Scotland . . . . . . . . . . . . . . . . . . . . . .

I . Introduction . . . . . . I1. Monomethyl-D-mannoses I11. Dimethyl-D-mannoses . . IV. Trimethyl-D-mannoses . V . Tetramethyl-D-mannoses

217 . . . . . . . . . . . . . . . . . . . . . . 218 . . . . . . . . . . . . . . . . . . . . . . 220 . . . . . . . . . . . . . . . . . . . . . . 224 . . . . . . . . . . . . . . . . . . . . . . 228 ix

CONTENTS

X

The Chemical Synthesis of D-Glucuronic Acid BY C.L. MEHLTREFTER. Northern Regional Research Laboratmy. Agrakultural Research Administration. U S. Department of Agriculture. Peoria. Illinois I Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 I1 Reduction of 1.4. ~.Glucosaccharolactone . . . . . . . . . . . . . . . 233 111 Oxidation of D-Glucose Derivatives by Various Agents . . . . . . . . . 236

.

. .

.

D-Glucuronic Acid in Metabolism

. .

BY H G BRAY.Department of Physiology. Medical School of the University. Birmingham. England

. . . . . . .

I Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 I1 D-Glucuronide Formation in Vivo . . . . . . . . . . . . . . . . . . . 252 I11 Structure of Glucuronides . . . . . . . . . . . . . . . . . . . . . . 254 IV Origin of D-Glucuronic Acid and Mechanism of D-Glucuronide Synthesis . 257 V Site of D-Glucuronide Formation . . . . . . . . . . . . . . . . . . . 259 VI Kinetics of D-Glucuronide Formation . . . . . . . . . . . . . . . . . 260 VII Enzymes and PGlucuronide Formation . . . . . . . . . . . . . . . . 261 The Substituted-Sucrose Structure of Melezitose

.

BYEDWARD J HEHRE.Department of Bacteriology and Immunology. Cornell University Medical College. New Ymk.New York: 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 I1 The Concept of Structural Relationship of Meleeitose and Sucrose . . . . 278 I11 A Bacterial Degradation of Melezitose to Sucrose. . . . . . . . . . . . 282 IV Meleritose Degradation by Cell-free Proteus Enzymes . . . . . . . . . . 287 V Melezitose as a Sucrose-ended Sugar . . . . . . . . . . . . . . . . . 288

. . .

. .

Composition of Cane Juice and Cane Final Molasses

. .

.

BY W W BINKLEY AND M.L WOLFROM. Department of Chemistry. The Ohio Shte University. Columbus. Ohio I Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 I1 Composition of Cane Juice . . . . . . . . . . . . . . . . . . . . . 292 I11 Composition of Cane Final Molasses . . . . . . . . . . . . . . . . . 303

. . .

Seaweed Polysaccharides

BY T. MORI.Tokyo University. Tokyo. Japan I Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11 Mucilage of B k e a Edulis . . . . . . . . . . . . . . . . . . . . . . IV Carrageenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . V.Fucoidin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI Laminarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII Other Polysaccharidee . . . . . . . . . . . . . . . . . . . . . . . A~TEIORINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJEOTINDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ERBATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contents of Volumes 1-7 . . . . . . . . . . . . . . . . . . . . . .

. . . . .

.

316 317 328 330 340 344 347 351 370 405 . 406

JAMES COLQUHOUN IRVINE 1877-1952 The importance of Sir James Irvine’s contributions to carbohydrate chemistry can best be appreciated by recalling, in the first place, the position attained by this branch of science at the close of the nineteenth century. The significance of the carbohydrates had been recognized both by biologists and by chemists and spectacular advances in our knowledge of this group had been made by Emil Fischer. Despite his genius, however, the available methods of investigation had failed to provide a solution to many fundamental structural problems. Fischer began to turn to other fields of enquiry and there was a feeling that for the moment the limit had been reached in structural work on the sugars. It was Irvine’s great achievement to realize that Purdie’s recently discovered methylation process provided a technique which could be applied in structural work in all branches of sugar chemistry. But to understand more clearly how this came about and why Irvine, still a very young chemist, proceeded to undertake a systematic exploration of the methylated sugars, we must go back a little. James Colquhoun Irvine was born in Glasgow, in the west of Scotland, on 9th May, 1877. His early education was a t Allan Glen’s school in that city and this was followed by a period a t the Royal Technical College, Glasgow. Then in the autumn of 1895 he matriculated at the University of St. Andrews. Here he became a student of the oldest of the Scottish Universities and he quickly acquired an abiding love for the city and for the University in which he was to serve so brilliantly as student, lecturer, professor and principal. When Irvine commenced his chemical studies a t St. Andrews the head of the department was Professor Thomas Purdie, F.R.S., who was quick to appreciate the quality of his new pupil. Purdie was one of the great figures in chemistry and Irvine retained a lifelong regard and veneration for him as teacher and friend. After gaining the B.Sc. degree in 1898 Irvine went to study with Wislicenus a t Leipzig. It was during this period that the idea came to him of using Purdie’s methylation process as a means for the study of the molecular structure of the sugars. He realized from the start the full significance of the new procedure which has been found to be so powerful a weapon that its usefulness is by no means exhausted after fifty years of intensive application by chemists in all xi

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parts of the world. Irvine returned to St. Andrews to develop the new method and so successful was he during the next few years that when Purdie retired in 1909 he was appointed to the chair of chemistry in St. Andrews. I n 1905 he married Mabel Violet, younger daughter of John Williams of Dunmurry, County Antrim, and throughout his career he owed much to her neverfailing devotion and counsel. Sir James and Lady Irvine had three children: two daughters, and a son who died while on active service during the second world war. From 1909-1914, a period which included the celebration in 1911 of the 500th anniversary of the foundation of the University, the work on sugars was continued without interruption but at the outbreak of the first world war in 1914 academic work ceased and the laboratories were hurriedly re-organized for the preparation on a large scale of fine chemicals and medicinal substances which were urgently required by the British and Allied Governments. Few trained chemists were available and facilities for the manufacture of fine chemicals were woefully inadequate. With the assistance of W. N. Haworth (afterwards Sir Norman Haworth) Irvine organized the laboratories for the preparation of dulcitol and other carbohydrates, orthoform and novocaine. During the later stages of the war much difficult and dangerous work was carried out on the manufacture of mustard gas. It would take long to tell how the many steps of the various syntheses were itemized and reduced to a series of routine operations which could be performed successfully by voluntary assistants, many of whom had never previously handled a piece of scientific apparatus. Fundamental research was resumed in 1919 and in a very short time an enthusiastic group of workers had gathered together for a systematic study of the sugar group. Irvine was then a t the height of his powers as teacher and director of research. No one who had the good fortune to attend his lecture courses will ever forget them. The chemistry lecture was assuredly one of the events of the day, with the long lecture bench crowded with experiments by which all the important points were illustrated. The timing of these was so exact that there was no interruption of the argument, and speech and experiment became perfectly integrated. The lectures themselves were models of clear exposition inspiring the audience with a keen desire for further understanding. They were delivered in the grand manner and in the tradition of the old masters of chemical science. Irvine’s method was different, but equally stimulating and effective in the informal discussions with small groups of research workers. On these occasions concise summaries of recent publications were given with an ease and clarity which delighted and amazed his hearers. Equally memorable were his talks a t the bench with

JAMES COLQUHOUN IRVINE

...

Xlll

research students whose difficulties appeared much less formidable after the suggestions and kindly encouragement they received during Irvine’s rounds of the laboratories. I n 1921 Irvine was appointed Principal and Vice-Chancellor of the University of St. Andrews. A new stage in his life now began, and he had to face tasks which might well have daunted a man of lesser faith and spirit. I n his early days at St. Andrews he found the University, old in years and proud of its tradition of learning, suffering from lack of buildings and equipment for scientific research. The number of students was small and the financial position was difficult. But Irvine possessed an unconquerable faith in the destiny of St. Andrews and as the result of a rare combination of vision and driving power in practical affairs he succeeded during his thirty-one years of office as Principal in changing the face of the University. It stands indeed today a monument to the inestimable services of this distinguished Principal and Vice-Chancellor. Developments took place in all faculties. New buildings were constructed in St. Andrews and Dundee, where rapid advances were being made, particularly in the schools of medicine and engineering. In St. Andrews a graduation hall was built and the student life of the University was transformed by the construction of new halls of residence and the extension of existing buildings. This and the institution of a system of Regents responsible for guiding and advising small groups of students were projects to which Irvine gave special at,tention and he was greatly helped in reaching this goal by the generous Harkness benefactions to the University. Sir James Irvine’s services to his University were so great that no adequate account can be given in a brief notice. It is no secret that on various occasions he was asked to undertake positions involving wider spheres of responsibility and activity, yet he chose to remain in St. Andrews, feeling that his real life-work lay in the University and city he loved so well. Nevertheless he could not escape calls to act as adviser and administrator outside the walls of St. Andrews and as the years passed he found himself called upon to play a more and more prominent part in these extramural activities. He gave much time and thought to the preservation of the historical buildings in St. Andrews. He served on many boards and committees, often as Chairman. These included the Scottish Universities Entrance Board and the Forest Products Research Board of the Department of Scientific and Industrial Research (Chairman, 1927-1939). He was Chairman of the Advisory Council of the Scottish Education Department (1925-31), of the Inter-University Council for Higher Education in the Colonies (1946), of the Viceroy’s Committee on the Indian Institute of Science (1936), and of the Adult

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Education Committee for Scotland (1927-1929). He was a member of the Prime Minister’s Committee on the Training of Biologists (1931). He played a notable part in the foundation of the University College of the West Indies. This was work in which he was deeply interested and it was a great satisfaction to him to be present at the inauguration of the College and to realize that in many respects, including the adoption of the Red Gown by its students, it was founded on the traditions of his own University in Scotland. He had a warm regard and respect for the United States of America and took special pride in his work for the Pilgrim Trust and the Commonwealth Fellowship Fund. He travelled widely in the U. S. A. where he made many lasting friendships. Amongst these many activities may be mentioned his visits to Williamstown as Foreign Lecturer at the Institute of Politics (1926), t o Princeton as Vanuxem Lecturer (1929) and to Yale as Woodward Lecturer (1931). In his character and personality Irvine displayed a wide versatility. He was a great scientist yet he possessed in full measure a love and understanding of humanistic studies which were reflected in his writing and in the appeal of his oratory. He had unusual powers in reducing complicated problems to their essentials, yet his memory could retain the minutest detail. Persuasive in argument, he was forceful and determined in carrying through cherished projects once the decision had been made. He was genial and charming in human relationships and one to whom his students turned as a trusted friend and counsellor. He was deeply sensible of the traditions and aspirations of the University as a whole, but he could always find time, no matter how pressing the business of the moment, to write in his own hand letters of kindly encouragement and congratulation t o his youthful colleagues and acquaintances. Throughout his life he spent his energies freely, finding relaxation during such vacations as came his way, in swimming, fishing and reading. The war-time years had been a great strain but he faced the post-war problems with his usual zest. It was his great desire to pilot the University through the difficult period of re-adjustment and reconstruction but this wish remained unfulfilled. He had a very serious illness in the summer of 1951 and although he made a remarkable recovery he was never again able to undertake in full the multifarious duties of the Principalship. He insisted, nevertheless, in doing more than his strength could bear, and only two days before his death, which came suddenly on June 12th, 1952, he had presided over a long and important meeting of the University Court at St. Andrews. His contributions to learning as teacher and investigator, his devotion to College and University, his achievements in many-sided activities in the widest fields of scholarship and statesman-

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ship ensure for him a lasting place as one of the most illustrious in the long line of Principals of the University of St. Andrews. Irvine received many honors in recognition of his achievements. The work he carried out during the first world war brought him the C.B.E. in 1920. Five years later he was given a Knighthood and in 1948 he was accorded the high honor of K.B.E. His scientific work was recognized by his election to the Fellowship of the Royal Society in 1919. He received the Davy Medal of the Royal Society in 1925 and the Longstaff Medal of the Chemical Society in 1933. He became a Fellow of the. Royal Society of Edinburgh in 1917, being Vice President (1922-25) and Gunning Victoria Jubilee Prize winner, one of the Society’s highest honors, in 1940. He was an honorary member and Willard Gibbs Medallist of the American Chemical Society, Medallist and honorary member of the Franklin Institute and honorary member of the American Philosophical Society. He was President of the Chemistry Section of the British Association for the Advancement of Science at the Hull meeting in 1922. Academic honors included the degrees of Ph.D. (Leipzig), D.Sc. (St. Andrews), Hon. D. Sc. (Liverpool,Princeton and McGill), Hon. Sc. D. (Cambridge, Pennsylvania and Yale), Hon. D.C.L. (Oxford and Durham), Hon. LL.D. (Glasgow, Aberdeen, Edinburgh, Wales, Toronto, Columbia and New York). He was a Justice of the Peace and Freeman of the City of St. Andrews. Irvine’s scientific work was essentially that of a pioneer. He understood the full power of Purdie’s methylation process as a method for structural investigations in the sugar group and while he was still working for his doctorate at Leipzig he wrote to Purdie outlining the whole field. At that time little was known with certainty concerning the fine structure of the simple sugars and still less of the disaccharides and polysaccharides. Irvine realized that the transformation of the reactive hydroxyl groups of the sugars into stable methyl ethers by the use of Purdie’s reaction with silver oxide and methyl iodide provided a method by which the next stage in carbohydrate chemistry could be initiated. He indicated how the procedure could be elaborated to determine the position of the linkages in the disaccharides and in the higher sugars. As a necessary preliminary to such work, however, it was essential to have a series of reference compounds and on returning to St. Andrews he began a systematic study of the methylated sugars. Purdie and he reported on the alkylation of sugars at the meeting of the British Association for the Advancement of Science in 1902, and this was followed by a note on applications t o disaccharides, given at the following meeting of the Association in 1903. At St. Andrews work was continuing on the preparation and properties of the methyl derivatives of the simple sugars, amongst which mention may

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be made of tetramethylglucose and its a- and P-methyl glucosides, and the carresponding derivatives of galactose, mannose and other sugars. Derivatives of these methylated sugars, for example oximes, anilides and hydrazones were also prepared and their properties studied. It was shown also that a non-reducing octamethyldisaccharide of the trehalose type was formed by the condensation of two molecules of tetramethylglucose under the influence of an acid catalyst in a non-aqueous medium. Another stage in the development of the sugar work came with the preparation of the partly methylated derivatives. These were obtained in various ways and the investigations led to detailed studies of the isopropylidene derivatives of glucose, fructose, and mannose. Much was learned in this difficult field but in many instances exact structural formulas could not be assigned until the nature of the ring systems present in the stable and labile forms of the sugars became known in later years. Irvine's first publication (1899) dealt with the rotatory powers of the optically active methoxy- and ethoxy-propionic acids prepared from lactic acid. In those early days the Purdie reactJionafforded so rich a field for investigation that Irvine and his collaborators continued work on various types of hydroxy bodies in addition to the sugars. There appeared papers on the isopropylidene derivatives and methyl ethers of glycerol and mannitol, and on the chemistry of benzoin and benzoin-like materials. The constitution of the glucoside salicin was studied and its pentamethyl ether was synthesized. Another example from the monosaccharide group of the insight which led Irvine to discern which problems were of special importance is found in his work on glucosamine, in the course of which'he endeavored to decide whether this substance was related stereochemically to D-glucose or to D-mannose. He discovered that it could be transformed at will into derivatives of D-glucose or D-mannose by alternative procedures. Here again much important information was gleaned, but the final resolution of the problem came only many years afterwards when later workers, by using entirely novel methods, provided a proof of the presence of the D-glucose type of configuration in glucosamine. It had been realized from the beginning that the final goal was the application of the new methods to structural studies in the wider fields of oligosaccharides and polysaccharides. The important reference substance, 2,3,6-trimethyl-~-glucosewas isolated by Denham in the course of work at St. Andrews on the methylation of cellulose in which he made use of dimethyl sulfate as the methylating agent. At about the same time (1912-13) W. N. Haworth who was then a member of staff a t St. Andrews, became interested in carbohydrate

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chemistry and developed the dimethyl sulfate method, applying it to sucrose, cellobiose, lactose, maltose and raffinose. Irvine’s own special interests now leaned more towards the polysaccharides and he began (with Miss E. S. Steele) a series of studies on the methylation of inulin and on the partly methylated fructoses obtainable from the methylated polysaccharide. Investigations into the structure of cellulose were also undertaken and reference may be made to the quantitative transformation of cellulose into methyl D-glucoside, the preparation of trimethylcellulose and the proof, derived from examination of its hydrolysis products, that methylated cellulose is built up exclusively, or almost exclusively, of residues of 2,3,6-trimethyl-~-glucose. Pioneering work was carried out also on the structure of starch and certain degradation products of starch, using the methylation technique. These studies involved a reconsideration of the then-accepted structure of the disaccharide maltose and proof was given that octamethylmaltose yields on hydrolysis 2,3,6-trimethyl-~-glucose. Residues of this sugar were shown therefore to constitute some of the units of which the starchmoleculewascomposed. Irvine had always been specially interested in sucrose and he devoted much attention to attempted syntheses of this important sugar. He examined with meticulous thoroughness the condensation of D-glucose with derivatives of D-fructofuranose but, in no case could any trace of sucrose be detected, a conclusion which has been confirmed by many subsequent investigators. An isomeride of sucrose was obtained the properties of which were studied. In later years much important work was carried out in the St. Andrews laboratories on anhydro-sugars and their derivatives (G. J. Robertson) and on the nitrate esters of methylated sugars (J. W. H. Oldham). Irvine never diminished his keen interest in this work but it was inevitable that less time could be devoted to chemical research after he had assumed the onerous duties of Principal and Vice-Chancellor in 1921. It was characteristic of him, however, that only a few days before his death he was busy propounding long-term schemes of research on possible developments of sugar derivatives for use in chemotherapy. Irvine’s pioneering activities covered a wide range of problems in carbohydrate chemistry. His publications are characterized by the clear and precise thinking and by the elegance of style which are evident in all his writings. The ideas he put forward have been singularly fruitful and the exploits of the small band of research workers in St. Andrews half a century ago inspired an ever-increasing volume of important work which has been carried out since those days in many different laboratories in all parts of the world. E. L. HIRST

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RELATIVE REACTIVITIES OF HYDROXYL GROUPS OF CARBOHYDRATES

BY JAMESM. SUQIHARA Department of chemistry, University of Utah, Salt Lake City, Utah

CONTENTS I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . 11. Configurational Relationships and Neighbo 1. Acyl Migration.. . . . . . , . . . . . . . . . . . . . . 2. Anhydride Formation. . . . . . . . . . . . . . . . . 3. Osazone Formation... . . . . . . . 4. Glycol Complexes.. . . . . . . . . . . . . . . . . . 111. Selective Etherification. . . . . . . . . 1. Methyl Ethers.. . . . . . . . . . . . . . . . . . . . . . 2. Other Ethers.. . . . . . . . . . . . . . . IV. Selective Esterification and Hydr 1. Tosyl and Mesyl Estere.. . . . . . . . . . . . . .

..........

16

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

3. Other Esters.. . . . . . . . . . . . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . 33 V. Selective Oxidation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 VI. Conclusions. . . . . . . . . . . . . . . . . ................ . . . . . . . . . . . . . 44

I. INTRODUCTION Differences in reactivities of the hydroxyl groups of carbohydrates have been recognized for some time. Certain of these differences are understandable from the point of view of classical organic chemistry; in this respect the differencesin reactivity of primary and secondary alcohols may be cited. The advent of a much better knowledge of mechanisms of organic reactions, in many cases initiated and promoted in the area of carbohydrate chemistry, has clarified some of the complex properties of the polyfunctional carbohydrates. Further impetus and stimulus in this direction should be of material value in clarifying the reactions and possibilities of carbohydrate chemistry. A better understanding of the relative reactivities of hydroxyl groups in carbohydrates may be of considerable value in the field of syntheses. The usual approach t o the preparation of partially or heterogeneously substituted polyhydroxy compounds requires the use of blocking groups, which are subsequently removed. Any synthesis which permits the 1

2

JAMEB M. SUGIHARA

elimination of several steps in a multistage process, is obviously desirable provided that the yields and operations are comparable. Some direct synthetic reactions that have been found to be possible by the proper control of conditions are well established. Further progress in this direction has been aided, and should continue to thrive, by the proper application of refined separation methods, such as chromatography, either alone or in combination with ion-exchange purification. The resolution of complex reaction mixtures, containing substances of very similar properties, has been realized in many cases. Thus the execution of a reaction under optimum conditions to realize the maximum amount of the desired substance, and followed by chromatographic and ion-exchange separation of the products and excess reactants, should permit many new direct syntheses to be attainable. Much of the earlier work on selective reactivity was motivated by the preparation and study of polysaccharides of industrial importance. It appears that more effort may have been exerted in this field than in that of monomeric chemistry. Some of the topics that will be discussed in this chapter have been included in previous volumes of this publication and in other review articles. Such items will be briefly reconsidered from the point of view of selective reactivities of hydroxyl groups.

11. CONFIGURATIONAL RELATIONSHIPS AND NEIGHBORING-GROUP

EFFECTS Configurational relationships and neighboring-group effects are undoubtedly of primary importance in modifying the behavior of carbohydrates. In this connection certain types of reactions, which are reasonably well established, are described in this section because they are of importance for the explanation of the selective reactions of carbohydrate hydroxyl groups. 1. Acyl Migration The isomerization of a compound by intramolecular transesterification has been observed many times.' Fischer2 was the first to observe

H-C-OH

I

H-C-0

I

/ \

OH

H-C-0-

I

8

(1)

-R

(1) For a brief review see E. L. Hirst and S. Peat, Ann. Repts. on Progr. Chem. (Chem. SOC.London), 81,172 (1934). (2) E. Fischer, Ber., 63, 1621 (1920);see A. P.Doerschuk, J. Am. Chem. Soc., 74, 4202 (1952).

RELATIVE REACTIVITIES O F HYDROXYL GROUPS

3

this reaction and to postulate a mechanism based upon a neighboring group effect with an ortho acid ester as an intermediate (1). I n most instances where acyl migration has been observed, such as in the methylato form methyl tion of methyl 2,3,4-tri-0-acetyl-a-~-glucopyranoside 2-0-methyl-3,4,G-tri-O-acetyl-a-~-glucopyranos~de,~ the proximity in space (2) of the migrating group to the free hydroxyl group would be sufficiently close to permit an intermediate cyclic structure as proposed by Fischer.2 The methylation per se is not the determining factor; the

alkaline conditions imposed in using silver oxide doubtless must play the important role. When the analogous benzoyl ester, methyl 2,3,4-tri-Obenzoyl-a-D-glucopyranoside, was methylated, the expected 6-0-methyl derivative was ~ b t a i n e d . ~Acyl migration has been observed in other methylation reactions. The methylation of 1,2,3,6-t,etra-O-acetyl-fi-~glucopyranose (I) with methyl iodide and silver oxide gives methyl 2,3,4,6-tetra-0-acetyl-fi-~-glucopyranoside (11); 5 migration of acetyl from position 1 t o position 4 occurs. A study of molecular models appears t o indicate the probability of a shift sequence 1 + 6 + 4 through orthoester intermediates. 5-0-Acetyl- 1,2-0-isopropylidene-3-0-methyl-G-O-tritylD-glucofuranose (IV) is obtained in the methylation of 3-0-acetyl-1,2-0isdpropylidene-6-0-trityl-D-glucofuranose (III).6 Robertson' obtained AcO

b

-1

y

MeOCH

I

HCOAc

HkOAc

b

AGO H HkOH

L

H 0-

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

hiel

-

AoO

b

AcO H

I

HCOAc

h

H 0-

W. N. Haworth, E. L. Hirst and Ethel G. Tcece, J . Chem. Soc., 2858 (1931). B. Helferich and E. Giinther, Ber., 64, 1276 (1931). B. Helferich and W. Klein, Ann., 466, 173 (1927). 1,. v. Vargha, Ber., 67, 1223 (1934). G. J. Robertson, J . Chem. Soe., 737 (1933).

T

A

(4

HO-lA%,d

HO-

HhOH

H OAc

LH*OCPhs

A

AHnOCPha

I11

IV

a mixture of methyl 2,3,6-tri-O-methyl- and 2,3,4-tri-0-methyl-a-~-g1ucopyranosides from the methylation of methyl 4-0-acetyl-2,3-di-O-methyla-D-glucopyranoside, and further examples of acyl migration have been noted. In the majority of the cases the movement of acyl is toward the primary hydroxyl group. The comparative values of the equilibrium constants of acetates of primary and secondary alcohols*would favor the shift in this direction. Helferich and Kleine found that 1,2,3,4-tetra-Oacetyl-0-D-glucose is isomerized by the alkali present in soft glass to form Molecular models again show the lJ2,3,6-tetra-O-acety1-~-D-glucose. probability of an acetyl shift from position 4 to 6. 3-0-Acetyl-1,2-0isopropylidene-D-glucofuranose (V) forms the 6-0-acetyl derivative (VI) in good yield.'O The same rearrangement occurs with 3-0-benzoyl-1,20-isopropylidene-D-glucofuranose." These reactions are catalyzed by alkali. I n all the investigations cited so far, suitable cyclic ortho acid ester structures with little strain are sterically possible. \ HCO

HCO

HAOH

HAOH

A&OH V

AHaOAc VI

(8) G. B. Hatch and H. Adkins, J. Am. Chem. Soc., 69, 1694 (1937). (9) B. Helferich and W. Klein, Ann., 460, 219 (1926). (10) K. Josephson, Ann., 472, 217 (1929); H. Ohle, E. Euler and R. Lichenstein, Ber., 62, 2885 (1929). (11) H. Ohle, Ber., 67, 403 (1924).

RELATIVE REACTIVITIES OF HYDROXYL GROUPS

5

Acyl migration has been shown to occur in an acid solution. Brigl in boiling acetylene and Gruner l 2 placed 1,6-di-O-benzoyl-~-mannitol tetrachloride with a little p-toluenesulfonic acid as catalyst and obtained three compounds, which they designated as 2,4-monoanhydro-~-mannito1 1,6-dibenzoate, 2,4 :3,5-dianhydro-~-mannitol l,Bdibenzoate, and 2,5-monoanhydro-~-mannitol1,6-dibenzoate. However, Hockett and coworkers18 reinvestigated this reaction and they have established the correct structures as being 1,4-monoanhydro-n-mannitol2,6- (or 3,6)dibenzoate (VII), 1,4 :3,6-dianhydro-~-mannitol 2,5-dibenzoate (VIII), 1,6-dibenzoate (IX). These findings and 2,5~-monoanhydro-~-glucitol were based upon lead tetraacetate oxidation studies. I n VII and VIII HZC? B ? i f r i HOCH

HbOH

I

CH~OBZ VII

BeOk

1

1

H~COBZ

HO H HLOH

B e!:

0 H2 VIII

H 0AH*OBz IX

benz,oyl migration could have occurred through an intermediate cyclic ortho ester. Anhydro formation may have involved the initial formation of a tosyl ester with p-toluenesulfonic acid. Such an intermediate would permit the necessary inversion about carbon atom 2 in the formation of IX (see the following section on anhydride formation, page 7). I n the classical Wohl degradati~n,'~ a diacetamido derivative is formed as an intermediate. Isbell and Frush16 have proposed a mechanism for its formation, based upon two successive acetyl migrations. The migration of acyl from nitrogen to oxygen has also been observed to occur. In epimeric acetylinosamines McCasland16 noted that a cis relationship of acetylamino to hydroxyl is necessary for facile migration in dilute acid in this rearrangement reaction in which configuration is retained. Among (12) P. Brigl and H. Grliner, Ber., 60, 1945 (1933); 67, 1582 (1934). (13) (a) R. C. Hockett, H. G. Fletcher, Jr., Elizabeth L. Sheffield and R. M. Goepp, Jr., J . Am. Chem. Soc., 68, 927 (1946); (b) R. C. Hockett, H. G. Fletcher, Jr., ElizabethL. Sheffield, R. M. Goepp, Jr., and S. Solteberg, ibid., 930; (c) R. C. Hockett, M. Zief and R. M. Goepp, Jr., ibid., 935. (14) A. Wohl, Ber., 26, 730 (1893). (15) H. S. Isbell and Harriet L. Frush, J . Am. Chem. Soc., 71, 1579 (1949). (16) G. E. McCasland, J . Am. Chem. Soc., 73, 2295 (1951). Further references are contained in this article.

6

JAMES

M. SUGIHARA

the recent studies concerned with this migration, van Tamelen17 has obtained further evidence favoring a cyclic intermediate in the relative ease of acyl migration of Cis and trans N-(pnitrobenroyl)-2-aminocyclopentanols. The &-isomer (X) reacts readily to form the hydrochloride OH

-

.-T=O

NHS

-c-o-c-cH~

I

cH3-!T-

b

OH

I

H-C-FHt

I “ir I 8

-C-O-C-CH3

cH’-8-r

-

,*‘

iHa

I

CHS-C-0-COH

‘

I 1

-C-OH

I

CH3-C-C-

1

A I I

0

I

NI13

-4-OH

t--

A

1

CH3-C-C-

d I

I

1 8 I CH~-C--COH -c-OH I C-I GO-

I /C‘’ *OH

-C-O$

H-C-KH-C-CHI

I

H 1 NH-C-NHC-CHI

CHS

OH

CYH

Hescan be attributed to the fact that sugar chemists were so accustomed to thinking of sugar esters in terms of carboxylic esters (particularly, acetates and benzoates) that they overlooked an earlier (14) S. OdPn, Arkiv Kenti. Mzneral. Geol., 7, No. 15, 1 (1918);Chem. Abstracts, 14, 2170. (15) S. O d h . Arkiv Kenii. Mzneral. Geol., 7 , No. 15, 23 (1918); Chem. Abstracts, 14,2170. (16) K.Freudenberg and 0. Ivers, Ber., 66, 929 (1922). (17) K . Freudenberg and F. Brauns, Ber., 66,3233 (1922). (18) B. Helferich and M. Vock, Ber., 74, 1807 (1941). (19) I”c:

(Inversion)

H+-

t!

H2N H

HAOH

A1 +

t!I

H OH

I

H2N H

For example, treatment of 2,3-anhydro-l,4-dideoxy-erythritol(meso2,3-epoxybutane) with ammonia aff ~ r d s ~ D( ~ -), *L( +)-threo-3-amino2-butanol:

ra 8““ L! Hr

CHs

H

(Inversion)

HA>-

H2N H

H OH

AHS

AH a

D(

-1

+ HaN

H

AH*

L(+)

Of course, if the original molecule is not symmetrical, equimolecular proportions of the two aminodeoxy derivatives will not result. Such conversions of sugars311have been by Stacey. An example is the preparation of methyl 3-amino-3-deoxy-/3-~-altroside (methyl “epiglucosaminide”) in 40% yield from276methyl 3,4,6-tri-O-acetyl(322) W. 0. Cutler and S. Peat, J . Chem. SOC.,782 (1939). (323) F. H. Dickey, W. Fickett, and H. J. Lucas, J . Am. Chem. SOC.,74, 944 (1952). (324) M. Stacey, “The Chemistry of Mucopolysaccharides and Mucoproteins ” in Advances in Carbohydrate Chem., 2, 161-201 (1946).

177

SULFONIC ESTERS OF CARBOHYDRATES

2-O-tosyl-p-~-glucoside, presumably via methyl 2,3-anhydro-p-~-mannopyranoside :

HacoF 1: H OTs

AGO H

(Inversion)

HAOAc

--

(Inversion)

1:

jH NHz HAOH

(LI

H 0-

H 0-

CHzOH

AH.O*e

Such reactions have proved valuable in elucidating the structure of naturally occurring aminosugars, e.g., chitosamine and chondrosamine. Sulfonic esters of sugar alcohols often react similarly with ammonia; and -sorbitol give326the thus, 1,4 :3,6-dianhydro-2,5-di-O-tosyl-~-mannitol corresponding 2,5-diamino-2,5-dideoxy derivatives. However, 1,4:3,6dianhydro-L-iditol aff ords328a compound thought to be 1,4:3,6-dianhydro2,5-dideoxy-2,5-imino-~-mannitol, 6. Desulfonyloxylation with Amines.-As previously mentioned, Ullmann and N&dai27found, in 1908, that alkyl sulfonates, like alkyl halides, can alkylate primary and secondary amines. I n 1922,investigation17 of the behavior of sulfonic esters of sugars commenced with the p r e p a r a t i ~ n 'of ~ ~the ~ ~ 3-deoxy-3-hydrazino derivative (together with some of the 3-deoxy-~-glucoseen-3,4derivative) by action of anhydrous hydrazine a t 140' on 1,2:5,6-di-O-isopropyl~dene-3-0-tosyl-~-glucose. Attention should be drawn to a fascinating conversion said to be undergonez6by the product, on hydrolysis of the isopropylidene groups with hydrochloric acid: N%H

H~OH HA0

\

c1

€1 OH

hH20H

/!H22IP

Replacement can also be caused at a primary carbon atom; thus, 1,2:3,4di-0-isopropylidene-6-O-tosyl-~-galactose reacts l Z 2with anhydrous hydra(325) R. Montgomery and L. F. Wiggins, J. Chem. SOC.,393 (1946). (326) V. G. Bashford and L. F. Wiggins, J . Chem. SOC.,371 (1950).

178

R. STUART TIPSON

zine a t 60" t o give the corresponding 6-deoxy-6-hydrazino derivative plus some of the unsymmetrical bis-(6-deoxy-di-0-isopropylidene-galactose)hydrazine. Hydrazine reacts with methyl 2,3-anhydro-4,6-0-benzylidene-a-D-alloside to give2I4 methyl 2-deoxy-2-hydra~ino-4~6-O-benzylidene-a-D-altroside, and with methyl 2,3-anhydro-4,6-O-benzylidene-a-~mannoside t o give214 methyl 3-deoxy-3-hydra~ino-4~6-0-benzylidenea-D-altroside. Other nitrogenous bases, e.g., dimethylamine,84~1s8~327 behave similarly. Secondary tosyloxy derivatives may give risex6*to unsaturated compounds by dehydrogenation-desulfonyloxylation.

3. Reaction with Alkali-Metal Mercaptides and Sulfides a. Desulfonyloxylation with Allcali-Metal Mercaptides.-In 1925, Gilman and Beaber observed328that alkali-metal mercaptides (e.g., sodium n-butyl mercaptide) desulfonyloxylate simple, primary, alkyl sulfonic esters with formation of the corresponding thio-ether plus sodium p-toluenesulfonate, and Raymond329first applied the reaction to sulfonic esters of sugars in 1934. Thus, treatment of l12-O-isopropylidene5-O-tosyl-~-xylose with potassium methyl (or ethyl) mercaptide in dry acetone at 100" for 2 hours (in a sealed flask) gave 5-deoxy-l,2-0-isopropylidene-5-methylthio- (or -5-ethylthio-)~-xylose in 75 % yield; and 1,2-0-isopropylidene6-O-tosyl-~-glucofuranose with potassium methyl mercaptide in dioxane, during 75 minutes a t 100°, aff ~ r d e d 6-deoxy~~Q 1,2-0-isopropylidene-6-methylthio-~-glucofuranose.Similarly, methyl 2,3-O-isopropylidene-5-O-tosyl-n-ribofuranoside gives33aa 62% ' yield of the corresponding 5-deoxy-5-methylthio derivative; and 9'-(2,3-O-isopropylidene-5-0-tosyl-~-~-ribofuranosyl)-hypoxanthine~~~~~~~ yields, with sodium methyl mercaptide in acetone,332or with potassium methyl mercaptide in dry dimethylformamide, 9'-(5-deoxy-2,3-0-isopropylidene5-methylthio-~-~-ribofuranosyl)hypoxanthine, probably identical with the O-isopropylidene derivative of the nucleoside obtained by deaminating from yeast. Attempts to apply the deoxy-methylthiopentosyl-adenine334 the same reaction for direct synthesis of this adenine nucleoside (by action K. Freudenberg and K. Smeykal, Ber., 69, 100 (1926). H. Gilman and N. J. Beaber, J. Am. Chem. Soc., 47, 1449 (1925). A. L. Raymond, J . Biol. Chem., 107, 85 (1934). W. G. Overend and L. F. J. Parker, Nature, 167, 526 (1951). P. A. Levene and R. S. Tipson, J. Biol. Chem., 111, 313 (1935). K. Satoh and K. Makino, Nature, 167, 238 (1951). J. Baddiley, 0. Trauth, and F. Weygand, Nature, 167, 359 (1951); J. Baddiley, J. Chem. Sac., 1348 (1951); F. Weygand and 0. Trauth, Chem. Ber., 84, 633 (1951). (334) A. L. Raymond, "Thio- and Seleno-Sugars" in Advances in Carbohydrate C h m . , 1, 129-145 (1945). (327) (328) (329) (330) (331) (332) (333)

SULFONIC ESTERS O F CARBOHYDRATES

179

of sodiumaa2or potassiumaaamethyl mercaptide o n a supposed 2,3-0-isopropylidene-5-0-tosyl-adenosine, X ) gave a product which, though allegedly consisting of 5-deoxy-2,3-0-isopropylidene-5-methylthio-adenosine, can at present only be accepted with dubiety in view of the fact that tosylation of adenosine derivatives (to yield compounds of the type of X ) actually give@ N-tosyl-O-tosyl derivatives. Like sodium hydroxide and sodium methoxide, sodium methyl mercaptide merely detosylates an isolated secondary sulfonyloxy group, as in methyl 6-deoxy-3,4-0-isopropylidene-2-0-tosyl-~-~-galactos~de~~~ and methyl 4,6-0-benzylidene-3-O-methyl-2-O-tosyl-t-~-galactoside. ls2 b. Desulfonyloxylation with Alkali-Metal Sulfides.-In 1935, it was notedao6(') that the p-toluenesulfonate of ( -)P-octanol reacts with hydrogen sulfide to give ( --)P-octyl thiol, and that the p-toluenesulfonate of a-benzylethanol, with disodium sulfide in ethanol, yields bis-a-benzylethyl sulfide. Simple dialkyl sulfides and dialkyl disulfides were prepared336by stirring a boiling, saturated aqueous solution of disodium sulfide or disodium disulfide with the alkyl sulfonate, but such reactions have apparently not yet been applied to sulfonic esters of monosaccharides. Disulfide cross-links have been introduced into celluloseaa6 by treating tosylated cellulose acetate with hydrogen sulfide (in pyridine) , or with sodium thiosulfate or thiourea, and then gently oxidizing the product. (Polyvinyl p-toluenesulfonate was similarly treated with thiourea.) The action of disodium sulfide on tosyloxyethyl-cellulose has also*l been studied. 4. Reaction with Other Hydrolysts Soda-lime, finely powdered, intimately mixed with half its weight of 1,2:3,5-di-0-isopropylidene-6-0-tosyl-~-gluco~e, and the mixture heated a t 200" under high vacuum during 30 minutes, transforms the glucose ester intoaa76-deoxy-1,2:3,5-di-0-isopropylidene-~-glucoseen-5,6. Similarly, 1,2:5,6-di-O-isopropylidene-3-O-tosyl-~-glucose givess3* 3-deoxy1,2:5,6-di-O-isopropylidene-~-glucoseen-3,4 (in 68% ' yield) , identical with that obtained" by treating the 3-O-tosyl derivative with hydrazine. The 3-O-mesyl derivative gave only a 4.2 % yield on identical treatment with soda-lime. In the same way, methyl 4,6-0-benzylidene-3-deoxy2-O-tosyl-a-~-mannoside giveszE9methyl 4,6-0-benzylidene-2,3-dideoxya-~-"mannoside"-2,3-een : (335) (336) (337) (338)

F. Drahowsal and D. Klamann, Monatah., 82, 970 (1951). E. F. hard and P. W. Morgan, Znd. Ens. Chem., 41, 617 (1949). H. Ohle and R. Deplanque, Ber., 66, 12 (1933). F. Weygand and H. Wolz, Chem. Ber., 86, 256 (1952).

180

R. STUART TIPSON

CHzO'

Potassium carbonate in aqueous methanol transforms162methyl 4,6-0-benzylidene-3-0-tosyl-a-~-galactoside into methyl 2,3-anhydro4,6-0-benzylidene-a-~-guloside, on boiling under reflux for 15 hours.

VI. ACTIONOF ALKALI-METAL HALIDESON SULFONIC ESTERS In 1897,it was observed88@that potassium iodide (in hot alcohol or acetone) converts ethyl p-bromobenaenesulfonate to ethyl iodide. Many years later, methyl and ethyl i0dides8~0~~~1 and ethyl a-iodopropionate*05" were prepared in good yield by the action of the same iodide (in boiling water or alcohol) on the corresponding p-toluenesulfonates. In these examples, the reaction obviously proceeded as an alkyl-oxygen fission,**in accordance widh the following equation:

Not until 1927 was the reaction applied841to a sulfonic ester o j a sugar derivative. The idea of trying it in this connection presumably arose from (a) the earlier observations*6ea' that, toward nucleophilic reagents, sulfonic esters usually act similarly to alkyl halides and eaters of nitric acid, and (b) the then-recent discovery that Finkelstein's reagent848 (uit., a solution of anhydrous sodium iodide in anhydrous acetone, which had been used for converting alkyl bromides and chlorides to the corresponding iodides) transforms both methyl 6-bromo-6-deoxy-2,3,4-tri-~-methyl-~-glucoside~~~ and methyl 2,3,4-tri-O-methyl-~-glucoside 6-nitrate24a to methyl Bdeoxy-6-iodo2,3,4-tri-O-methyl-~-glucoside, in good yield; treatment with 2 molecular proportions

AHp:-ONO*

I

! Na

of sodium iodide in acetone, during 6 hours at 100" (in a sealed tube) was employed.

(339) J. H. Kastle, P. Murrill, and J. C. Fraeer, Am. Chern. J . , 18, 894 (1897). (340) D.H. Peacock and B. K. Menon, Quart. J . Indian Chem. Soc., 2,240 (1925). (341) W.Rodionow, Bull. 8oc. chim. France, [4],3@,305 (1926). (342)K.Freudenberg and K. Rsschig, Bw., 60, 1633 (1927). (343) H.Finkelstein, Ber., 43, 1528 (1910). (344) J. C. Irvine and J. W. H. Oldham, J . Chem. Soc., 187, 2729 (1925).

SULFONIC ESTERS OF CARBOHYDRATES

181

1. Action of S o d i u m Iodide o n Primary Sulfonyloxy Groups

For work in the sugar series, the advantage of acetone as a solvent (compared with w a t e F or ethanol, employed in the earlier studies mentioned) is that it dissolves the sodium iodide, the sulfonic ester, and the deoxyiodosugar derivative, but dissolves practically none of the sodium sulfonate formed. Use of other solvents is discussed on p. 197. a. Primary Sulfonyloxyl Group of a Mono-0-sulfonylated A1dose.-On treating 1,2:3,4-di-O-isopropylidene-6-0-tosyl-~-galactose with a 10% solution of sodium iodide in acetone during 36 hours a t 125' (sealed tube), desulfonyloxylation occurred, giving342,34b the corresponding 6-deoxy-6odo derivative:

-+

CHJ

Prior to 1932, a year of some significance in this field (see p. 191), the reaction was, for example, applied to the preparation of methyl 2,3,4tri-0-acetyl-6-deoxy-6-iodo-a-~-g~ucoside~*~ (25 hours at 130"), 2,3,4,2',(24 hours a t 130"; 3',4'- hexa-0-acetyl-6,6'-ddeoxy-6,6'-diiodo-trehalo~e~~~ 89 % yield) , and methyl 2,3,4,2',3'-penta-0-acetyl-6,6'-dideoxy-6,6'diiodo~-cellobioside"4s (60 hours at 100"; 94% yield of product; 100% yield of sodium p-toluenesulfonate) . Such products are valuable in the preparation of other w-derivatives, e.g., w-deoxy sugars (see p. 157). Some other mono-0-sulfonylated-aldose derivatives to which the iodination reaction has since been successfully applied (either preparatively or diagnostically, or both) are listed in Table 111. The yields given therein are those recorded by the authors, and have not been recalculated. Where the yield approaches the theoretical, there is seldom any indication that the same yield might not have resulted a t a lower temperature or after a shorter reaction-time, or both. Low yields might, in some instances, be attributable to poor experimental technique. However, despite its deficiencies, Table I11 contains sufficient information to warrant a few conclusions, some of which are to be found scattered through the literature. (345) (346) (347) (348)

K. Freudenberg and K. Raschig, Ber., 62, 373 (1929). B. Helferich and E. Himmen, Ber., 61, 1825 (1928). H. Bredereck, Ber., 8S, 959 (1930). B. Helferich, E. Bohn, and S. Winkler, Ber., 63, 989 (1930).

182

R. STUART TIPSON

TABLEI11 Replacement,' b y Iodine, of Primary Sulfonyloxy G o u p of Mono-0-sulfonyl-aldose Derivatives

NO.

__

1 2

3 4 5 6 7 8

9 10

11

~

~~

Temp., Time, Compound "C. hrs. ~1,2,3-Tri-O-acetyl-5-O-tosyl-~-arabinose 100 1 lJ2-O-Isopropylidene-5-O-tosyl-~ 100 6 arabinose Methyl 2,3-di-O-acetyl-5-O-tosyl-cu-~arabinoside Methyl 2,3-0-isopropylidene-5-0-tosyln-ribofuranoside 100 2 9'- (2,3-Di-O-acetyl-5-O-tosyl100 2 99c j3-D-ribofuranosyl)-N-tosyl-adenine 9'-( 2,3-O-Isopropylidene-5-0-tosyl-~-~99c 100 2 ribofuranosy1)-hypoxanthine 3'-(2,3-Di-0-methyl-5-0-tosyl-p-~100 2 ribofuranosy1)-uracil 3'-(2,3-0-Isopropylidene-5-0-tosy~-~-~100 2 ribofuranosy1)-uracil 3-O-Acetyl-l,2-0-isopropylidene-5-0100 tosyl-D-xylose 59 32d 3-0-Benzoyl-l ,2-O-isopropylidene-5-0100 38d 62 tosyl-D-x ylose

{f { 100 {f

References

134 134 134 207 111 331 107

I I I

1,2-0-Isopropylidene-5-0-tosyl-~-xylose 88 67d 11 1,2-0-Isopropy~idene-5-0-tosyl-~-xylose 100 6 98b 12 1,2-O-Isopropylidene-5-O-tosyl-~-xylose 100 2 4 ~ 13 Methyl 3,4di-O-acetyl-2-deoxy-6-080 2 tosy1-a-D-alloside 54c 14 Methyl 2-deoxy-3-0-methyl-6-O-tosyl80 CY-D-"allopyranoside '' 3 15 Methyl 4-0-acetyl-2,3-anhydro-6-080 4 tosyl-cu-D-alloside 70c 16 Methyl 2-0-methyl-6-0-tosyl-cu-~100 6 altropyranoside 17 Methyl 3,4-di-O-acetyl-2-0-methyl-6-0100 6 bsyl-cu-D-altroside 77b 18 Methyl 2,3,4tri-0-acetyl-6-O-tosyl-a-~100 4 altroside 19 Methyl 2,3,4-tri-O-benzoyl-6-0-tosyla-D-altroside 70' 18 93" 20 3,4-O-Isopropylidene-6-o-mesyl-~115-25 galactal 5.: 11 .2c.g 21 3,4-0-Isopropylidene-6-O-tosyl-n115-25 galactal 5.1 13.8evu 22 1,2:3,4-Di-O-isopropylidene-6-O-mesyl- (115-25 5.t 1130-35 40 93°C D-galactose

349 133 133 133 263 350 181 269 181 184 184 351 352 169 169 169

183

SULFONIC ESTERS O F CARBOHYDRATES

TABLEI11 (Continued)

No.

Compound

Temp.,

"C.

V'

an hri

Yield,b*c* References percent

-

22 1,2:3,4Di-O-isopropylidene-6-0-mesylD-galactose 100 96 23 1,2:3,4-Di-O-isopropylidene-6-0-tosylD-galactose 105-10 36 23 1,2:3,4-Di-O-isopropylidene-6-O-tosyl- f 115-25 5 D-galactose I 115 20 24 6-O-Tosyl-D-galactose 115-25 5 25 Methyl 2-0-acetyl-3,40-isopropylidene6-O-tosyl-a-~-galactoside 120 6 26 Methyl 2-deoxy-3,4-0-isopropylidene6-O-tosyl-a-~-"galactoside " 140 5 27 Methyl 2-0-methyl-3,40-isopropylidene-6-0-tosyl-a-~-galactoside 140 5 28 Methyl 3,4-0-isopropylidene-6-0-tosyl112 15 a-D-galactoside 29 Methyl 2,4di-O-acetyl-3-0-methyl-6-0tosyl-a-D-galactoside 124 36 30 Methyl 2,4di-O-acetyl-3-0-methyl-6-0125 6 tosyl-p-D-galactoside 31 Methyl 2-deoxy-3-0-methyl-6-O-tosyla-D-"galactopyranoside " 80 B 32 1,2,3,4-Tetra-O-acetyl-6-O-mesyla-nglucose 100 2 33 1,2,3,PTetra-O-acetyl-6-0-mesyl-@-~glucose 100 2 33 1,2,3,4Tetra-O-acety1-6-~-mesy~-@-~-I 20 36 Ireflux 3 glucose 34 1,2,3,4-Tetra-0-acetyl-6-0-tosyl-a-~refluxh 1 glucose 35 1,2,3,4Tetra-0-acetyl-6-0-tosyl-~-~refluxh glucose 1 36 Ethyl 2,3,4-trideoxy-6-0-mesyl-a-~"glucoside" 115-20 5 37 Methyl 2,3,4-tri-0-acetyl-6-O-to~yl~-~100 1 glucoside 37 Methyl 2,3,4-tri-0-acety1-6-0-tosyl-a-~refluxi glucoside 3 38 Methyl 2,3,4-tri-O-acetyl-6-0-tosyl-fi-~glucoside 100 1 39 Methyl 2,3,4-tri-O-benzoyl-6-O-tosyla-D-glucoside 40 Methyl 6-O-tosyl-fi-~-glucopyranoside 100 2 41 Methyl 2,4-di-O-acetyl-3-0-methyl-6-0tosyl-@-D-glucoside 100 20

-

99b 85b

1 ooc 1 23 .9c*o 1 9ib 1 76.8c90 67b 1 8W I 36c ca.

50b

190 72, 353 169 169 226 354 232 355

81b

309

60"

268

75 c

270

65b

190

96b

190 24

396

97

95b

97

53c

356 171 357 171 260 72, 358

72b

359

184

R, STUART TIPSON

-

TABLEI11 (Continued)

No.

Temp.,

Compound

“C.

zm Yield,b+*’ References hrs percent

1.

-

42 Methyl 2,3,4tri-O-methy1-6-O-toayl100

cu-D-ghcoside

43 3,5-Di-O-acetyl-l,2-0-isopropylidene6-O-tosyl-~-gluoose

44 3,5-O-Benzylidene-l,2-0-isopropylidene45 46 47

48 49 50 51

-

100 -74

64b SOb

4 reflux 6-O-mesyl-~-ghcose 3,5-O-Benzylidene-l,2-0-isopropylidene90 3 6-O-tOt3yl-D-ghCOSe 1,2-O-Isopropylidene-6-O-tosyl-~refluxh 2. glucofuranose 1,2:3,5-Di-O-isopropylidene-6-0-tosylD-glucose 80 3 Methyl 2,3,4,2’,3’-penta-O-acetyl-6,6’di-0-mesyl-p-oellobioside Methyl 2,3,4,2’,3’-penta-O-acetyl-6,6’di-0-tosyl-8-cellobioside 100 60 Methyl 2,3-di-O-acetyl-4-0-methyl6-O-tosyl-a-~-mannoside 100 2 Methyl 2,3,4-tri-0-bensoyl-6-O-tosyla-D-mannoside 100 2

*

360

5

-

LOO“

1 I

113,359 321 146 (34,361)

86b

362 246 946 LOOC

1 I

284 172,348

LOOb*C

251

l0OC

173

a Unless otherwise noted, acetone was the solvent. Yield of deoxyiodo-sugar derivative. 0 Yield of sodium sulfonete. d Yield by determination of sodium iodide consumed. Free iodine liberated. I Acetonylacetone. 0 Corrected for solubility of sodium sulfonate in acetone. Acetic anhydride. Isobutyl methyl ketone.

(349) P. A. Levene and R. 8. Tipson, J . Biol. Chem., 106, 113 (1934). (350) H. Miiller and T. Reichstein, Helv. Chim. Acta, 21, 251 (1938). (351) M. Gut and D. A. Prim, Helv. Chim. Acta, 29, 1555 (1946). (352) D.A. Rosenfeld, N. K. Richtmyer, and C. S. Hudson, J . Am. Chem. SOC., 70, 2201 (1948). (353) A. L.Raymond and E. F. Schroeder (to G. D. Searle & Co.), U. S. Pat. 2,365,777(Deo. 26,1944); Chem. Abstracts, 39, 4434. (354) A. B.Foster, W. G. Overend, and M. Stacey, J . Chem. Soc., 974 (1951). (355) 0. T.Schmidt and E. Wernicke, Ann., 668, 70 (1947). (356) S.Laland, W. G. Overend, and M. Stacey, J . Chem. Soc., 738 (1950). (357) M.Zief and R. C . Hockett, J . Am. Chem. SOC.,67, 1267 (1945). (358) A. L. Raymond and E. F. Schroeder (to G. D. Searle & Co.), U. S. Pat. 2,365,776(Dec. 26,1944); Chem. Abstracts, 89, 4434. (359) B. Helferich and 0. Lang, J . prakt. Chem., 132,321 (1932). (360) C. C.Barker, E. L. Hirst, and J, K. N. Jones, J . Chem. Soc., 1695 (1938). (361) P. A. Levene and A. L. Raymond, Ber., 66, 384 (1933). (362) G. R. Barker and R. W. Goodrich, J . Chem. SOC.,S 233 (1949).

SULFONIC ESTPRS O F CARBOHYDRATES

185

For D-galactose, a 64osyloxy group (compounds 21, 23) i s somewhat more reactive than the (smaller) 6-mesyloxy group (compounds 20, 22), and the same is probably true of D-glucose derivatives (compounds 34 and 32; 35 and 33). Secondly, a conjigurational eflect (e.g., as between one sugar and an isomer) is noticeable. Allose, altrose, mannose, and ribose derivatives react almost quantitatively under relatively mild conditions, and so do many glucose derivatives. However, there seems to be no doubt that D-galactose derivatives (compounds 20 to 31), and, probably, xylose derivatives (compounds 9 to 12) require much more drastic conditions for accomplishing desulfonyloxylation than do derivatives of D-glucose (compounds 32 to 49) and, possibly, of L-arabinose (compounds 1 to 3). For example, a 6-O-mesyl-~-glucose (compound 33) is much more reactive than a 6-O-mesyl-~-galactose (compound 22) ; the same appears to be true of the corresponding 6-O-tosyl derivatives (cf., compounds 47 and 23). This observation suggests a steric effect arising from spatial proximity of the 6-sulfonyloxy group t o differing groups in t8he respective sugar molecules. A somewhat related influence appears to be exhibited by anomers. Thus, derivatives of P-D-galactose (e.g., compound 30) are more reactive than derivatives of a-D-galactose (compound 29) ; similarly 8-D-glucose derivatives (e.g., compounds 33 and 35) are more reactive than a-D-glucose derivatives (compounds 32 and 34). Presence of a methylene group in the sugar chain (e.g., the 2-deoxy group of compound 31) greatly increases the reactivity; this effect will be discussed subsequently (see p. 195). An acetal ring reduces the reactivity of the 6-sulfonyloxy group, and two acetal rings may reduce it even more; compare, compounds 20 and 21; 26; 23 and 24; 32 and 43. The effect of an acetal ring (on reactivity) is greater with a mesyloxy group than with a tosyloxy group (cf., compounds 20 and 21). Finally, the nature of the group on the carbon atom contiguous to that bearing the sulfonyloxy group has an effect on the behavior of the latter group. An influence is noticeable with the following groupings attached at the penultimate carbon atom : hydroxyl, carboxylic ester, acetal ring, (anhydro ring, see p. 189), and sugar ring. In 6 of the 7 cases in which liberation of free iodine has been definitely reported (compounds 12, 23, 25, 29, 30, 31, 43) the primary sulfonyloxy group is contiguous to the carbon atom engaged in ring formation; presumably the hydrogen of this LObH bHeI --t

AH,

carbon atom is detached, with formation of a deoxy-glycoseen by desul-

186

R. STUART TIPSON

fonyloxylation-dehydrogenation. (In compound 43, the secondary hydroxyl group contiguous t o the sulfonyloxy group is acetylated, and the nature of the by-product, possibly an anhydro-sugar, was not estab-

'J

HCO I

H0-J I

lished.) In none of the examples cited did this side-reaction (involving iodine liberation) occur to a pronounced extent, although Raymond and Schr~eder'~ point out that, under their conditions, compound 23 gave an 85% yield of desired 6-deoxy-6-iodo derivative (with liberation of only a trace of iodine) , whereas, by use of Freudenberg and Raschig's original conditions, 5 4 2 a considerable amount of decomposition occurred. However, one compound is known with which formation of the 5-deoxyglycoseen-5,6 proceeds34to completion (in acetone) in 2 hours a t 100'; this compound is 1,2-0-isopropylidene-6-O-tosyl-~-glucofuranose.However, if the reaction is conducted in boiling acetic anhydride under reflux (Table 111, compound 46), acetylation362 takes place, and 3,5-di-O-acetyl6-deoxy-6-iodo-1,2-O-isopropylidene-~-glucose may be isolated (as from compound 43). As regards compound 5 (Table 111),it may be noted that the O-tosyloxy group reacts, but the N-tosyl group does not. A so-called 2,3-0isopropylidene-5-0-tosyl-adenosineis saida6ato undergo the iodination reaction with simultaneous cyclization, to yield 2,3-0-isopropylidene-5,3'cyclo-adenosine iodide. b. Primary Sulfonyloxy Group(s) of a-, W-, or a,w-0-Sulfonylaled A1ditols.-Much the same behavior characterizes the primary sulfonyloxy groups of sulfonylated sugar alcohols. Some compounds to which the reaction has been applied (either preparatively, or diagnostically, or both) are listed in Table IV. Here again, iodine is liberated with a compound having a primary sulfonyloxy group next to a secondary carbon atom whose oxygen atom is engaged in ring formalion (as in the isopropylidene acetal, compound 13) or is present as a free hydroxyl group (cf., compounds 20 and 21). In the latter category is 1,4-anhydro-6-0-tosylsorbitol which, on treatment12 with the hot reagent, "decomposes with liberation of iodine." Furthermore, when 3,6-anhydro-l-deoxy-l-iodo4,5-O-isopropylidene-~-mannitol is with this reagent during 12 hours a t 210-1501 3,6-anhydro-4,5-0-isopropylidene-~-mannitoleen-l,2 (363) V. M. Clark, A. R. Todd, and J. Zussmm, J . Chem. SOL, 2952 (1951).

SULFONIC ESTERS OF CARBOHYDRATES

187

TABLEI V Replacement,a b y Iodine, oj Primary Sulfonyloxy Group(s) of a-, W - , or a,@-0-Sulfonylated Alditols

--

No.

Compound

1 [Di-0-tosyl ethylene glycol]

Temp.

"C. 25

I

28

I24e

16d' 7sdJ 364 48d 179 1OOd 365

2 2-O-Benzoyl-3-deoxy-l-0-tosyl-~, Lreflux 2.5 glyceritol 2 95-10 3 2,3-O-Isopropylidene-1,4-di-O-tosylD-threitol 100 2.7 4 1,3:2,4-Di-O-methylene-5-O-tosyl-~,~-ribitol801 4 1OOb 5 1,3-Anhydro-2,4-0-methylene-5-0-tosyl88b1 D,GXylitOl 100' 46 9 PJ 6 3,5-O-Benzylidene-2,4O-methylene-l-Otosyl-D,L-xylitol 1201 48-72 96b 7 2,4:3,5-Di-O-methylene-l-0-tosyl-~, L-xylitol 100 4 50d] 1201 4 1OOd reflux' 1 iwdj 8 2,3,4.5-Di-O-isopropylidene-l-0-tosyl100 2 94d1 D,Gxylitol reflux 2 i8dJ 8 2,3,4,5-Di-O-isopropylidene-l-0-tosyln,Lxylitol 601 19 88b 9 2,4:3,5-Di-O-methylene-l,6-di-O-tosyl-allitol 100 7.5 85d 10 2,3,4,5-Di-O-benrylidene-l, 6-di-0-tosyldulcitol (I and 11) refluxh 1 100" 11 2,3,4,5-Di-O-isopropylidene-1,6-di-0-tosyl- 100 2 81d dulcitol refluxh 1 1OOd 12 I,5-Anhydro-2,3,4tri-0-benzoyl-6-O-tosylD-galactitol 100 2 21-23d 13 2,3,4,5-Di-O-isopropylidene-l-O-tosylL-fucitol 100 1" 2;. lOOd :2,4-di-O-ethylidene-614 5-0-Acetyl-l,3 0-tosyl-sorbitol 00-10 5 80d 15 2,3,4,5-Tetra-O-benzoyl-l,6-di-O-tosylsorbitol 100 2.5 16 l,PAnhydro-2,3,5-tri-0-benzoyl-6-0-tosylsorbitol 100 1 85b (or 2),5-0-benzylidene17 1,4-Anhydro-3 6-0-tosyl-sorbitol 100 2 80b 18 1,4,5-Tri-O-acetyl-3,6-anhydro-l-0-tosylsorbitol 110 80d PO-methylene-1,619 1,5-Di-O-acetyl-2, 100 2 di-0-tosyl-sorbito1 801 5 20 i-Deoxy-6-iodo-2,4-O-methylene-l-O-tosylsorbitol 100 3 1;" 50d 5' :a,&di-O-methylene21 j-Deoxy-6-iodo-2,4 1-0-tosyl-sorbitol .efluxh 2 92c

-

-

Time, Yield,b-c*c Rejerhrs. Percent encea

366 266 180

180 367 368 369 370 371 273 87 314 372 373 374 72 72 139 170 170 170 -

188

R. STUART TIPSON

-

TABLE IV (Continued)

No.

--

Compound

Pemp., rime, Yield,b+Id Zeferhrs. Percent mces "C.

-

22 2,4-0-Methylene-l,6-di-O-tosyl-sorbitol 22 2,4O-Methylene-l,6-di-O-tosyl-sorbito1 23 2,4:3,5-Di-O-methylenel,&di-O-tosylsorbitol 24 2,5-Anhydro-l,6-di-0-tosyl-~-iditol 25 2,4:3,5-Di-O-methylene-l,6-di-O-tosyl-~iditol 26 2,4,5-Tri-O-acetyl-1,3-0-ethylidene-6-0tosyl-D-mannitol 27 1,3,4-Tri-0-acetyl-2,5-0-methylene-6-0tosyl-D-mannitol :3,4-di-O-isopropylidene-628 5-0-Acetyl-1,2 0-tosyl-D-mannitol 29 1,5-Anhydro-2,3,4-tri-O-benaoyl-6-0tosyl-D-mannitol 30 3,6-Anhydro-2,4,5-tri-O-acetyl-l-O-tosylD-mannitol 31 3,6-Anhydro-2-O-acetyl-4,5-O-isopropylidene-l-O-tosyl-D-mannitol 32 3,6-Anhydr0-4,5-O-isopropylidene-l-Otosyl-D-mannitol 33 2,3,4,5-Tetra-O-bensoyl-1,6-di-O-tosylD-mannitol 34 2,3,4,5-Di-O-benaylidene-l,6-di-O-tosylD-mannitol 35 2,3,4,5-Di-0-methylene1,6-di-O-tosylD-mannitol 6-di-0-tosyl36 2,4:3,5-Di-0-methylene-I, D-talitol 37 2,5-Anhydr0-3,4-deoxy-1,6-di-O-tosyler ythro-hexitol

-

100 100 110 *efluxh 100 100 100 110' Sefluxh

5 2 80d 8 91" 2 2 100b 70c 5 2 95;"97d 24 1.5

100

3

100

2

05-10

2.5

96d

170 375 376 170 204 377 378

100"d

251

90d

373

100

3

100

12

78d

380

15-20

4

88d

381

80-90

4

91d

382

379

76

100

2.5

100

2

9gC

235

100

2

99

383

reflux*

1

low

384

05-10

5

1OOd

385

-

0 Unless otherwise noted, acetone was the solvent. b Yield of monodeoxy-monoiodo-alditol derivative. 0 Yield of dideoxy-diiodo-alditol derivative. d Yield of sodium sulfonate. Free iodine liberated. Aaetonylacetone. 0 Plus sodium bicarbonate to prevent cleavage of the benaylidene group. h Acetic anhydride.

(364) R.S.Tipson,Mary A. Clapp, and L.H. Cretcher, J. Org. Chem.,12, 133 (1947). (365) W. C . J. Ross, J.Chem.Soc.,2257.(1950). (366) L. J. Rubin,H.A.Lardy,and H.0.L.Fischer, J.Am. Chem.Soc., 74, 425 (1952). (367) R. M. Hann, A. T.Ness, and C.S. Hudson,J. Am. Chem. Soc., 66, 670 (1944).

SULFONIC ESTERS O F CARBOHYDRATES

189

is formed. After the discovery that a l-0-tosyloxy group of a tosylated ketose is resistant to the reagent (see p. 190), the same kind of behavior was found with the 1-0-tosyloxy group of tosylated hexitols (e.g., compounds 22 and 23) ; it is therefore possible to prepare both a 6-deoxy-6iodo-l-O-tosyl- and a 1,6-dideoxy-l,6-diiodo-hexitolderivative from a 1,6-di-0-tosyl-hexitol. With acetone as solvent, the yield of the dideoxydiiodo derivative is improved170if all free hydroxyl groups are first acetylated; hence acetic anhydride is preferable to acetone as the reaction medium in such iodinations. These a- and w-deoxyiodo and a,w-dideoxydiiodo derivatives are useful for the preparation of other a- and w-derivatives, including a- and w-deoxy- and a,w-dideoxy-alditols (see p. 157). Probably most significant is the observation that presence of an anhydro ring may, like presence of an acetal ring, confer stability on a primary sulfonyloxy group (cf., compounds 5 and 8; 12 and 13; 27 and 30, for example). c. Primary Sulfonyloxy Group of a Mono-O-sulfonylated Aldonic Acid. Very little information is as yet available regarding the behavior of compounds of this class towards Finkelstein's reagent. TreatmentaBo of methyl 2,4 :3,5-di-O-bensylidene6-O-tosyl-~-idonate with the reagent (368) R. S. Tipson and L. H. Cretoher, J. Org. Chem., 8, 95 (1943). (369) R. M. Hann, A. T. Ness, and C. S. Hudson, J. Am. Chem. Soc., 66, 73 (1944). (370) M.L. Wolfrom, B. W. Lew, and R. M. Goepp, Jr., J. An. Chem. Soc., 68, 1443 (1946). (371) W. T.Haskins, R. M. Ham, and C.S. Hudson, J. Am. Chem. Soc., 64, 137 (1942). (372) A. T. Ness, R. M. Ham, and C. S. Hudson, J. Am. Chem. Soc., 64, 982 (1942). (373) L. F. Wiggins, J. Chem. Soc., 388 (1946). (374) Y. Hamamura, J . Agr. Chem. Soc. Japan, 18, 581 (1942); Chem. Abstracts, 46, 4652. (375) E.J. Bourne and L. F. Wiggins, J . Chem. Soc., 517 (1944). (376) R. M. Hain, J. K. Wolfe, and C. 8. Hudson, J. Am. Chem. Soc., 66, 1898 (1944). (377) R. M. Hann and C. S. Hudson, J. Am. Chem. Soc., 67, 602 (1945). (378) E.J. Bourne, a. T. Bruce, and L. F. Wiggins, J . Chm. SOC.,2708 (1951). (379) L. Zervas and Irene Papadimitriou, Ber., 73, 174 (1940). (380) L. F. Wiggins, J. Chem. Soc., 4 (1945). (381) A. B. Foster and W. G. Overend, J. Chem. Soc., 1132 (1951). (382) A. B. Foster and W. G. Overend, J. Chem. Soc., 3452 (1951). (383) W.T.Haskins, R. M. Ham, and C. S. Hudson, J. Am. Chem. Soc., 6.5, 67 (1943). (384) R. M.Hann, W. T.Haskim, and C.S. Hudson, J. Am. Chem. Soc., 69,624 (1947). (385)L. F. Wiggins and D. J. C. Wood, J. Chem. Soc., 1566 (1950). (386) E. Seebeck, E.Sorkin, and T. Reichstein, Helu. Chim. Ada, 28, 934 (1945).

190

R. STUART TIPSON

(during 5 hours a t 100") gave a sulfur-free product, said to be methyl

2,4:3,5-di-O-benzylidene-~-idonate (on the dubious basis aff orded by a mixed melting-point determination). On the other hand, the corresponding 6-0-mesyl derivative remained completely unchanged on treatment as above (or even during 5 hours at 120"); this is a further example of the lower reactivity of a 6-0-mesyl group as compared with a 6-O-tosyl group. In contrast to these results, methyl 2,4:3,5-di-Omethy~ene-6-0-tosyl-~-gluconate givesas7methyl 6-deoxy-6-iodo-2,4 :3,5di-0-methylene-D-gluconate,in practically quantitative yield, on treatment with the reagent during 2.5 hours a t looo. d. Primary Sulfonyloxy Group of a-,W - , or a,w-O-Sulfonylated Ketoses. Because'the reactivity of the primary hydroxyl group at carbon atom 1 is profoundly affected by the presence of the adjacent ketonic function at carbon atom 2 of k e t w s , Levene and Tipson"8 became curious as t o the behavior of a lone 1-0-tosyl group (towards Finkelstein's reagent). They therefore tested 2,3 :4,5-di-O-isopropy~dene-l-O-tosy~-~-fructose, CH~OTS I

and discovered388that it is completely unagected at lOO", during either 2 or 8 hours; the solution remained colorless, and no sodium p-toluenesulfonate was formed. The experiment was repeated and, even after 5 days at 130-135", only a trace of sodium p-toluenesulfonate was isolated; a trace of free iodine was liberated, but a large proportion of the starting material was recovered unchanged. Similarly, the reagent is without effect208 on phenyl 3,4,5-tri-O-acetyl-l-O-mesyl-~-~-fructoside (during 40 hours a t 125-130'). However, on treating2666-deoxy-2,3-0isopropylidene-1-0-tosy1-D-fructose during 48 hours at 125", sodium p-toluenesulfonate and some free iodine are formed, and some of the 1-deoxy-1-iodo derivative (yield, not stated) may be isolated. 6-Deoxy2,3-0-isopropylidene-l-O-tosyl-~-sorbose b e h a v e P similarly (36 hours at 125'). Under even more conditions, uiz., 100 hours at 100" under nitrogen pressure of 1,000 lbs. per sq. in., the exchange reaction (387) C.L. Mehltretter, R. L. Mellies, C. E. Rist, and G. E. Hilbert, J . Ant. Chern. 69,2130 (1947). (388) P.DA. Levene and R. S. Tipson, J . Biol. Chm., 120, 607 (1937).

Soc.,

SULFONIC ESTERS O F CARBOHYDRATES

191

proceeded to the extent of some 50% with 2,3 :4,6-di-O-isopropylidene1-0-tosyl-L-sorbose ; re-treatment under the same conditions afforded some of the 1-deoxy-1-iodo derivative (yield, not stated). I n contrast, the 6-tosyloxy group of 1,6-di-O-tosylketohexofuranose derivatives is more readily re'placed by iodine. Thus, by reaction during 24 hours a t 90-lOO", 2,3-0-isopropylidene-1,6-di-0-tosyl-~-sorbose gives284 the corresponding 6-deoxy-6-iodo-1-0-tosylderivative (possibly accompanied by some unisolated 1,6-dideoxy-l,6-diiodo derivative) ; and 2,3-0isopropylidene-1,6-di-0-tosyl-~-fructose behaves2s6 similarly (16 hours at 100"). 2. Action of S o d i u m Iodide o n Secondary Sulfonyloxy Groups

I n all the examples so far considered, the reaction of sodium iodide with one or more primary sulfonyloxy groups, only, was involved. However, in 1932, Oldham and RutherfordZ0published the first study of the behavior (towards this reagent) of some D-glucose derivatives containing secondary sulfonyloxy groups, thereby opening up a field of research whose possibilities have still not been fully explored. a. Secondary Sulfonyloxy Groups Non-contiguous to, or N o t Accompanied by, a Primary Sulfonyloxy Group, in Cyclic-Sugar Derivatives.-On treating methyl 2,3-di-O-methyl-4,6-di-O-phenylsulfonyl-~-~-glucoside with an equal weight of sodium iodide in acetone (volume, not stated) during 2 hours a t lOO", these authors20 found that, in conformity with the experience of the earlier workers (see p. 181), the 6-phenylsulfonyloxy group reacts readily and quantitatively, but they also found that the secondary sulfonyloxy group does not react appreciably under these conditions, and the product was recognized to be methyl 6-deoxy-6-iodo-2,3di-0-methyl-4-0-phenylsulfonyl-/3-~-glucoside. They stated20 that "it

is important to limit the time of heating t o two hours, since prolonged treatment seems t o lead also to the replacement of the 4-group and t o other complications." By using these very clearly stipulated conditions, they found that non-contiguous primary and secondary tosyloxy groups

192

R. STUART TIPSON

of tosylated D-glucose derivatives similarly display differential reactivity, and that, in the absence of a primary tosyloxy group, secondary tosyloxy groups are practically unreactive. Thus, methyl 2,3-di-O-acetyl-4,6-diO-tosyl-P-~-glucosidegavez0a 92 % yield of the corresponding 6-deoxy-6iodo-4-0-tosyl derivative, and methyl tetra-0-tosyl-P-D-glucopyranoside gave a 93% ’ yield of the 6-deoxy-6-iodo-2,3,4-tri-O-tosylderivative. Furthermore, under their conditions, the following compounds (having no primary sulfonyloxy group) did not react20 with sodium iodide in acetone: 2,3,4-tri-O-phenylsulfonyl-glucosan, methyl 2,3,6-tri-O-methyl-5-O-tosylD-glucoside, methyl 4,6-di-O-rnethyl-2,3-di-O-tosyla-~-glucoside, and 1,2:5,6-di-0-isopropylidene-3-0-tosyl-~-glucose. Hence, they clearly demonstrated20that, in such compounds of D-glucose, under the prescribed conditions of treatment, (a) a phenylsulfonyloxy or tosyloxy group at position 6 reacts almost quantitatively ; (b) these substituents a t positions 2,3,4, or 5 are unreactive; and (c) these properties are independent of the ring structure (furanose or pyranose) of the D-glucose derivative. These principles have often been referred to as “Oldham and Rutherford’s Rule.” Oldham and RutherfordZ0therefore proposed use of the following method for ascertaining the presence of a free primary hydroxyl group in substituted D-glucopyranose derivatives: (1) tosylate the compound ; ( 2 ) permit the tosylation product to react with sodium iodide (used in excess) in acetone during 2 hours a t 100”; and (3) treat the iodinat,ion product with silver nitrate in acetonitrile (see p. 155), and weigh t,he resulting silver iodide. (They did not imply that this method would be applicable either to D-glucofuranose derivatives having a secondary sulfonyloxy group contiguous to the primary sulfonyloxy group, or to O-sulfonyl derivatives of other sugars, sugar alcohols, etc.) It may be noted that Oldham and Rutherford20 recommended use of a standard temperature and reaction time, but only once mentioned the proportion of sodium iodide they employed (“an equal weight,” as already noted); in no case did they indicate how much acetone they employed. However, experience has shown that neither too great nor too small an excess of sodium iodide should be used, and that the acetone solution should not be too dilute. A series of comparative experiments has been performed389 under the followinga64standard conditions: a weighed amount of the sulfonic ester is treated with one hundred per cent excess (2 molecular equivalents per sulfonyloxy group) of a 10%solution of anhydrous sodium iodide in anhydrous acetone, in a sealed tube at 100” during 2 hours. Nowadays, the third step of Oldham and Rutherford’s diagnostic procedure is usually omitted, since a highly satisfactory indicationSB4 of the (389) R. S. Tipson and P. Block, Jr., J . Am. Chem. SOC.,66, 1880 (1944).

SULFONIC ESTERS OF CARBOHYDRATES

193

extent of reaction is afforded by weighing the sodium p-toluenesulfonate which crystallizes out in the second step, because the solubility of the sodium sulfonates in the reaction solution is slight. However, a correction (not physico-chemically proper) has been introduced169by assuming that the solubility in the reaction solution is the same as in pure acetone, viz., for sodium methanesulfonate, 0.04 g.; and for sodium p-toluenesulfonate, 0.12 g. per 100 cc. of acetone a t 18". No case has been reported in which the solubility of the sodium sulfonate is appreciably modified, or its crystallization inhibited, by a sulfonic ester or its iodination product. However, a method which is unaffected by these possibilities and is adaptable t o use on a micro scale, consists133in determination of the sodium iodide consumed during the reaction, by titration of aliquots of the reaction solution, for in-organic iodide, with staadardized silver nitrate-ammonium thiocyanate (and ferric alum as indicator). Should free iodine be liberated, the sealed tube should be cooled in Dry Icechloroform immediately after elapse of the heating period, opened, and the iodine estimated3" by titration with standardized sodium thiosulfate solution (with starch as indicator); if the iodination reaction is prolonged, the results may be deceptive, because of some iodination of the acetone. I n the iodination of some tosyl esters of primary monohydric alcohols, three molecular equivalents of dry sodium iodide in pure acetonylacetone were employed, and the reaction was followed182by observing the change in refractive index of the reaction mixture; as for the action of sodium ethoxide on ethyl sulfonates, 390 the iodination reaction followed secondorder kinetics. Confirmation of Oldham and Rutherford's Rule, as applied t o D-glucose derivatives, was not long in forthcoming; and little time elapsed before attempts were made to find out whether the Rule could be extended to other sugars. Table V lists the behavior of some cyclic-sugar derivatives bearing one or more secondary 0-sulfonyl groups non-conliguous to the primary 0-sulfonyl group. I n 1933, confirmation of the fact that, under drastic conditions, a secondary as well as a primary tosyloxy group can react, was afforded24O by the isolation of some methyl 4-0-acetyldideoxy-diiodo-mono-0-tosyl-P-D-" glucoside" on treating compound 19 (Table V) with 4.6 molecular equivalents of sodium iodide in acetone during 25 hours at 130"; similar behavior, to a lesser extent, was observed in 1937 with compound 11, treated a t 90" and then 115". I n these, and all other, instances of replacement of a secondary sulfonyloxy group, Walden inversion may have occurred, but this point is, as yet, not settled for the iodination reaction. A 6-0-mesyl group (as in compound 23) may be so reactive that, by correct choice of conditions, only the (390) M. S.Morgan and L. H.Cretcher, J. A m . Chern. Soc., 70, 375 (1948).

194

R. STUART TIPSON

TABLEV Replacementla by Iodine, of Non-contiguous Primary and Secondary Suljonyloxy Groups in Derivatives of Cyclic Sugars

-

-

No.

Compound

1

2 3 4 5 (i

7

7 8 9 10

11

feferences

Temp., Time, hrs. “C.

if-(2-Deoxy-3,5-di-O-tosyl-p-~-“ribosyl”)-5’00 methyluracil Methyl 2-deoxy-3,5-di-O-tor~yl-a,B-~.05-10 “riboside” Methyl 3-0-methyl-2,4,6-tri-0-tosyl-a-~10 altroside Methyl 2,3-di-O-acety1-4,6-di-O-tosyl-j3-~40 galactoside Methyl 2-deoxy-3-0-methyl-4,6-di-O-tosyl10-90 a-n-“galactoside ” Methyl 3,4-0-isopropylidene-2,6-di-O-mesylL 15-25 a-D-galactoside Methyl 3,4-O-isopropylidene-2,6-di-O-tosyl115-25 a-D-galactoside Methyl 3,4-0-isopropylidene-2,6-di-O-tosyl120 a-D-galactoside 115-25 Methyl 2,6-di-O-mesyl-a-~galactopyranoside 115 Methyl 2,G-di-0-tosyl-a-~-galactopyranosideL 15-25 Methyl 3-0-methyl-2,4,6-tri-O-tosyl-P-~I20 galactoside $0then 1,4Di-O-acetyl-2,3,6-tri-O-tosyl-P-~-glucose

115

2,

1OO;d 430

110

3

LOOd

274

16

>Od

267

16f

30d

154

8f

35d

270

5.5 3.9d

169

5.5 17.gd

169

G 5.5 30 5.5

3 6 ; b 91d 37.5d 36.4d 31.2d

9, 77d 15the1 lOld 2f 55b

refluxu 0.083 12 1,3,4Tri-0-acetyl-2,6-di-O-tosyl-~-~-glucose 4Tri-0-acety l-2,6-di-O-tosyl-~-n-glucose refluxu 0.083 13 1,3, 14 Benzyl 2,3-di-O-acetyl-4,6-di-O-tosyl-j3-~2 99b 100 glucoside 15 Ethyl 2,3-dideoxy-4,6-di-0-mesyl-a-~105-10 3 “glucoside 16 Ethyl 2,3-dideoxy-4,6-di-O-tosyl-u-~-

110 “glucoside ” 17 Ethyl 2,3-didehydroxy-4,6-di-O-mesyl-a-~1105-10 “glucoside ” 18 Ethyl 2,3-didehydroxy-4,6-di-O-tosyl-a-~110-15 “glucoside” 18 3thyl 2,3-didehydroxy-4,6-di-O-tosyl-a-~- 18 “ glucoside ” 25 30

169 169 268 124 237 237 391 356

6

lOOd

356

3

93d*

356

3

934. 75’ 83. 100’ 130d

356

216, 216 216 25

19 Methyl 40-acetyl-2,3,6-tri-O-tosyl-p-~-

130

glucoside 20 Methyl 3,4di-~-acetyl-2,6-di-O-tosyl-,3-~glucoside

reflux0 0.083 lOOd

-

226

356 240 (109) 142

SULFONIC ESTERS OF CARBOHYDRATES

TABLEV (Continued) NO.

D-glucose Sucrose

I This formulation received support from studies showing that a similar type of structure is possessed by other sugars. In the case of raffinose, for example, Neuberg’ was able to obtain D-galactose and crystalline sucrose following the action of the a-D-galactosidase of almond emulsin. Similarly, Bourquelot and Bridela obtained D-glucose and crystalline sucrose from gentianose hydrolyzed by the p-D-glucosidase component of almond emulsin. The existence of these trisaccharide analogues of sucrose made it seem the more credible that melezitose was of the same class. The insusceptibility of melezitose to yeast invertase,6 which readily hydrolyzes raffinose and gentianose as well as sucrose, was, however, not easy to reconcile with that view. I n 1926, Kuhn and von Grundherrg seriously argued for the concept of melezitose as a derivative of sucrose after excluding a cyclic arrangement for the three hexose units on the basis that Alekhine6 had obtained a crystalline hendecaacetate from melezitose. (Through elimination of a n extra molecule of water, a cyclic structure would present only nine hydroxyl groups.) These investigatorsg reported no enzymic or chemical cleavage of melezitose yielding sucrose or other non-reducing disaccharide, but they offered evidence suggestive for their hypothesis by analogies drawn between various hydrolyses of melezitose and sucrose. Melezitose was found, for example, to be split to D-glucose by the so-called “glucosaccharase l 1 enzyme preparations from Aspergillus oryzae and Lowenbrau yeast which hydrolyzed sucrose. The long-recognized inability of ordinary yeast invertase to attack melezitose6*10was explained by the assumption that this enzyme is a type of “fructosaccharase” that requires an unsubstituted (terminal) D-fructose molecule. Actually, the experiments of Kuhn and von Grundherr with the aspergillus and Lowenbrau yeast enzymes cannot be taken as evidence that melezitose contains the (7) C. Neuberg, Biochem. Z., 3, 519 (1907). (8) E. Bourquelot and M. Bridel, Compt. rend., 171, 11 (1920). (9) R. Kuhn and G. E. von Grundherr, Ber., 69, 1655 (1926). (10) C. S. Hudson and S. F. Sherwood, J . Ant. Chem. Soc., 42, 116 (1920).

280

EDWARD J. HEHRE

sucrose moiety because, as Bridel and Aagaard" and Weidenhagen12 showed subsequently, these enzymes act on various a-D-glucosidic bonds and not exclusively and specifically on that of sucrose; that is, according to the latter authors these enzymes should be classed as a-wglucosidases rather than as D-glucosaccharases. Likewise, the explanation assumed by Kuhn and von Grundherr for the inactivity of yeast invertase on melezitose does not indicate that the sucrose structure is necessarily present, although it does remove an important objection to the idea. One of the major points advanced by Kuhn and von Grundherrg for the substituted-sucrose structure of melezitose was the extreme ease of acid hydrolysis of one of the linkages, which had earlier led Alekhine6 to the discovery of turanose and the trisaccharide nature of melezitose. For years this feature was accepted as virtual proof of the presence of sucrose in the melezitose molecule. However, the opinion that the ease of hydrolysis was evidence for the furanoid form of the D-fructose ring lost all validity with the discovery by Purves and Hudson1*that methyl D-fructopyranoside is hydrolyzed by acids with the same ease as methyl D-fructofuranoside; moreover, the ease of hydrolysis never was evidence concerning the a- or j3-configuration of the D-fructose unit in melezitose.a Nevertheless, over the years, the concept of melezitose as a D-glucosylsucrose was widely held, especially since no D-glucose < > D-fructose moiety had ever been found in Nature that was not a-D-glucopyranose < > j3-D-fructofuranose (i.e., sucrose). Indeed, this more or less intuitive formulation came to be regarded as a proven structure by many chemists, and was assigned without reservation in many works of reference before the clarifying account given by Hudson.2

2. Incomplete Status of Earlier Evidence on Individual Ring Structures and Configurations Experimental support for the hypothesis that melezitose may be a substituted sucrose came from determinations of the ring structures and configurations of the individual sugar components involved. Actually, final proof was not achieved for the lack of a single point of information. Conclusive evidence for the pyranoid-ring structures of the D-glucose units in melezitose was obtained through the methylation experiments of Zemplh and BraunI4 and especially through those of Leitch16 who isolated two molecular equivalents of crystalline tetramethyl-D-gluco(11) M. Bridel and T. Aagaard, Bull. soc. chim. bid., 9, 884 (1927);T.Aagaard, Tidsskr. Kjemi og Bergvesen, 8, 5, 16, 35 (1928);Chem. Abstractrr, 24, 1089 (1930). (12) R. Weidenhagen, Z. Ver. deut. Zucker-Ind., 78, 406, 781 (1928); 79, 115 (1929);80,383 (1930). (13) C.B. Purves and C. S. Hudson, J . Am. Chem. Soc., 60, 1170 (1937). (14) G. Zemplh and G. Braun, Ber., 69, 2230 (1926). (15) Grace C.Leitch, J . Chem. Soc., 588 (1927).

STRUCTURE O F MELEZITOSE

281

pyranose from the hydrolyzate of hendecamethylmelezitose. The methylation experiments of the latter author also showed that the D-fructose fragment of melezitose in all probability has the same (furanoid) ring structure as the D-fructose component of sucrose. That is, when the trimethyl-D-fructose portion of the hydrolyzate was further methylated and the resulting methyl tetramethyl-D-fructoside hydrolyzed, a reducing sugar (presumably 1,3,4,6-tetramethyl-~-fructofuranose) was obtained that agreed in physical properties with the tetramethyl-Dfructose obtained from the hydrolysis of octa- or heptamethyl-sucrose.'6 By the use of the periodate oxidation technique, Richtmyer and Hudson16 confirmed the pyranoid form of the two D-glucose units in melezitose, and in addition conclusively proved the presence of a furanoid D-fructose unit. Their data show that four moles of periodic acid are consumed, with two moles of formic acid (but no formaldehyde) liberated per mole of melezitose. Some formaldehyde would have been produced if either of the D-glucose rings were of furanoid form, while more than four moles of periodate would have been reduced if either of the D-glucose rings were larger than the six-membered form. Furthermore, since the pyranoid structures thus indicated for both D-glucose units account exactly for the four moles of reagent used and for the two moles of formic acid released, it is evident that the D-fructose unit was not attacked by the periodic acid and therefore must be of the furanoid ring type with no pair of hydroxyl groups on adjacent carbon atoms. Support for this conclusion pertaining to the fructose ring form was obtained by Richtmyer and Hudson16 in additional experiments in which the presence of intact D-fructose in the oxidized melezitose molecule was demonstrated. All of the foregoing evidence is consistent with a D-glucosyl-sucrose structure for melezitose (I). Indeed, when supplemented by experimental data on the structure of turanose, which is now known with assurance to be 3-c~-~-glucopyranosyl-~-jructose, l7 the evidence permits D-glucomelezitose to be written as 3-a-~-glucopyranosyl-~-fructofuranosyl pyranoside. This expression lacks assignment only of the a- or p-form for the D-fructose unit and for the right-hand D-glucose unit, which features however are essential for a decision on the identity or nonidentity of melezitose with a D-glucosyl-sucrose. Reasonably good evidence that the D-glucose unit in question possesses the a-configuration was obtained from enzymic studies which showed that the 8-D-glucosidase of almond emulsin has no appreciable effect upon r n e l e ~ i t o s ewhile ~~~~ preparations containing a-D-ghcosidase from yeast, malt, Aspergillus oryzae, and other organisms hydrolyze melezitose into (16) N. K.Richtmyer and C. S. Hudson, J . Org. Chem., 11, 610 (1946). (17)The reader is referred to the review article2 of Hudson for a thorough treatment of the development of knowledge of the turanose structure.

282

EDWARD J. HEHRE

its three hexose components.1lP12 However, as mentioned a t the beginning, no experimental data were obtained bearing on the question of the a- or P-configuration of the D-fructose unit. The well established observation that yeast invertase is without action on melezitose is of no help in choosing the proper allocation, Insusceptibility to invertase cannot be taken as evidence that melezitose contains an a- rather than a P-type D-fructose linkage because the presence of a glucosyl substituent on the D-fructose unit might suffice to prevent the enzyme from acting.Qn12018On the other hand, neither of course does the failure of invertase, or P-D-fructofuranosidase, indicate the presence of a P-D-fructose linkage and consequently sucrose in the molecule. Isolation of the non-reducing disaccharide portion of melezitose would permit the last point of structure to be settled, but this direct type of solution has long been regarded as not likely to be accomplished. Treatment of melezitose with acid causes the preferential rupture of the unknown glycosidic bond, which precludes isolation of the D-glucose < > D-fructose fragment. Moreover, treatments with a-D-glucosidase preparations cause the release of all three hexose components rather than yield any non-reducing disaccharide, presumably, according to Weidenhagen,l2.l9because of the presence and equal susceptibility of the two a-D-glucosidic bonds in melezitose. Weidenhagenlo (p. 187) in fact expressed the futility of seeking the non-reducing moiety through selective enzymic splitting in strong and unequivocal terms, viz., “Die Annahme, dass auf enzymatischem Wege eine Spaltung der Melezitose in Glucose und Rohrzucker oder Glucose und Turanose moglich sei, ist ad absurdurn gefuhrt.” Thus, the final point required to complete the knowledge of the structure of melezitose and show its relationship to sucrose (i.e., a decision on the configuration of the D-fructose unit) not only remained undetermined, but its solution also seemed remote because of the peculiar circumstances noted above. Solution was achieved, however, with the finding by Hehre and Carlson3 of a biological degradation yielding the non-reducing disaccharide entity which, with the kind help of Professor C. S. Hudson, was conclusively proved to be sucrose.

111. A BACTERIAL DEGRADATION OF MELEZITOSE TO SUCROSE 1. Recognition of the Selective Action of Proteus Bacteria

On the premise that previous observations with a few a-D-glucosidase preparations were too limited to establish the biological equivalence (18) Mildred Adams, N. K. Richtmyer, and C . S. Hudson, J . Am. Chem. Soc., 66, 1369 (1943). (19) R.Weidenhagen, Ergeb. Enzymforsch., 1, 168 (1932).

STRUCTURE O F MELEZITOSE

283

claimed by Weidenhagen l9 for the two a-D-glucosidic linkages in melezitose, a survey was undertaken in which diverse bacteria were examined for the capacity to split the acid-stable linkage of the trisaccharide a t a faster rate than the acid-labile linkage. The survey was made with the aid of an enzymic-serological test for sucrose, capable of detecting that sugar in exceedingly minute quantities in the presence of large quantities of other sugars, including meleaitose. This test, which seemed ideally suited for recognition of the slightest degradation of melezitose to sucrose, is based on the specific conversion of sucrose to the polysaccharide dextran by the enzyme dextransucrase of Leuconostoc mesenteroides20s21with recognition of the dextran so formed, by serological means.20,22 Suspensions of various barteria wcre incubated with melezitose in the presence of dextransucrase, and the supernatant fluids of such mixtures examined for their capacity to give serological precipitation with dextran-reactive antiserums. Negative results were obtained with bacteria of many genera and species. However, fluids from incubated mixtures of melezitose with variants of Proteus bacteria commonly used in the laboratory diagnosis of rickettsia1 diseases (i.e., Proteus 0x2, X2, 0x19, X19, OXK, XK) gave precipitin reactions for dextran when diluted several hundredfold. This was taken as presumptive evidence that sucrose had been released from the melezitose by the Proteus bacteria; the amount produced in the initial experiment, however, could not have exceeded one per cent of that theoretically obtainable from a D-glucosyl-sucrose, since fluids with degrees of serological reactivity similar to those of the mixtures of Proteus cells plus 10% melezitose could be produced by incubating solutions of 0.02 to 0.1% sucrose with the same dextransucrase preparation under the same conditions.

2. Production of Invertase-sensitive Material from Melezitose by Proteus Vulgaris O X 2 By altering the culture medium used for growing the Proteus bacteria, and by using cell suspensions of greater density, the production from melezitose of abundant amounts of material susceptible to invertase action, as well as of reducing sugars, was readily shown.3 Figure 1 illustrates the degradation of a 5 % solution of melezitose monohydrateZ3 (20) E. J. Hehre, Science, 93, 237 (1941); E. J. Hehre and J. Y. Sugg, J . Exptl. Med., 76, 339 (1942). (21) E. J. Hehre, J. Bid. Chem., 163, 221 (1946). (22) J. Y. Sugg and E. J. Hehre, J. Zmntunol., 43, 119 (1942). (23) The melezitose (Pfanstiehl) used in our experiments was isolated from “honeydew honey,” one of the important sources discovered by Hudson and Sherwood.lo

284

EDWARD J. HEHRE

by young, washed cells of Proteus vulgaris, OX2 variety. Three distinct phases are seen: an initial or lag period of two or three days during which little change is found; a phase of rapid production both of invertasesensitive material and of free reducing sugars; and a final phase in which the free reducing sugar content further increases while the “sucrose ”

DAYS

FIQ. 1.LAction of Proteus OX2 bacteria on melezitose monohydrate (initial concentration, 5.0%) in system held at pH 5.6 and 23°C. Open circles represent free reducing sugars, calculated as glucose; solid circles represent invertase-sensitive material, calculated as sucrose.

content declines. The formation and subsequent partial disappearance of invertase-sensitive material, together with the production of reducing sugars in progressively increasing quantity suggests that the Proteus bacteria, possibly after a period of autolysis, had released an enzyme or enzymes catalyzing the reactions: Melezitose Sucrose

+ HzO+ Sucrose + D-Glucose + HzO-+ D-Fructose D-Glucose

Moreover, the disproportionately large amount of reducing sugar liberated in the relatively early period suggests that other degradative reactions also had occurred. The maximal concentration of the invertase-sensitive product, found

STRUCTURE O F MELEZITOSE

285

on the sixth and seventh days of the experiment, was 43% of that theoretically expected from a 5 % solution of a D-glucosyl-sucrose (monohydrate). In other experiments, maximal “sucrose” yields from 38 to 56% were found a t corresponding periods. In view of these favorable results, isolation of the invertase-sensitive material was undertaken. Part of the Proteus-melezitose mixture of Fig. 1, for example, was taken on the seventh day of incubation and, after removal of bacteria and heatcoagulable protein, was placed on a carbon-Celite columnz4for fractionation. After adsorbed reducing sugars (D-glucose and D-fructose) had been eluted with water, a fraction was obtained by elution with 0.5% aqueous phenol that contained about half of the invertase-sensitive material present in the original digest, with little accompanying free reducing sugar or undegraded melezitose. On vacuum evaporation of this fraction and treatment of the resulting sirup with ethanol, a crystalline product was readily obtained.3

3. IdentiJication of the Invertase-sensitive Product as Sucrose The crystalline sugar was non-reducing toward alkaline copper and ferricyanide reagents and, on paper chromatograms, migrated as a single component with the velocity of natural sucrose. Its specific optical +65.8”, in rotation, measured in Dr. Hudson’s laboratory,2bwas good agreement with the rotation of pure sucrose. Moreover, when hydrolyzed with acid or with yeast invertase and analyzed for reducing sugars, the melezitose-derived product yielded an amount of invert sugar corresponding t o sucrose. Chromatograms26 of the acid-hydrolyzed material confirmed the presence of two components that migrated with velocities corresponding to D-glucose and D-fructose. The rate of hydrolysis of the melezitose-derived material by acid was, furthermore, identical with that of natural sucrose (Fig. 2). Additional evidence that the product obtained from melezitose is sucrose was provided by X-ray diffraction patterns made in Dr. Hudson’s laboratory26(Fig. 3). I n common with natural sucrose, the melezitose-derived sugar was found to give an olive-green color reaction with the diazouracil reagent of RaybinZ7whereas melezitose and turanose give negative reactions. By acetylation, Drs. Hudson and Fletcher2b prepared a crystalline (24) Edna M. Montgomery, F. B. Weakley and G. E. Hilbert, J. Am. Chem. SOC., 71, 1686 (1949). (25) The data on the specific optical rotation, X-ray diffraction, and on the octaacetate of the meleeitose product were obtained by Professor C. S. Hudson, Dr.

Hewitt G. Fletcher, Jr., and Dr. Nelson K. Richtmyer of the Laboratory of Chemistry and Chemotherapy, National Institutes of Health. (26) S. M. Partridge, Biochem. J., 42, 238 (1948). (27) H. W. Raybin, J. Am. Chem. SOC.,66, 2603 (1933); 69, 1402 (1937).

286

EDWARD J. H E H R E

FIG. 2.cChanges in optical rotation, (Y X 50, observed during hydrolysis of 1.0% “sucrose” from melezitose (solid circles) and of 1.0% Bureau of Standards sucrose (open circles) by 1.0 N HCl a t 22°C.; 1, 2 dm.

FIG.3.8,*6-X-Ray diffraction patterns of melezitose, “sucrose ” from melezitose, natural sucrose, and turanose. The diagrams were made by Mr. William C. White of the National Institutes of Health.

STRUCTURE O F MELEZITOSE

287

derivative with [,IDz0 +60.5” (c 1.4, chloroform) and a melting point of 87-89’ which was not depressed on admixture of the test material with authentic sucrose octaacetate. Rotation of the latter substancez8 is +59.6’; the melting point of the stable allotropeJZg89’. The [,ID melezitose-derived sugar, finally, was found3 to serve as a substrate for three different polysaccharide-synthesizing enzymes (dextransucrase from Leuconostoc mesenteroides,20~21 amylosucrase from Neisseria perJ ~ U V U and , ~ ~ levansucrase from Streptococcus s ~ l i v u r i u s ~ that ~ ) are operative upon sucrose but not upon melezitose, turanose, or the common natural di- or mono-saccharides.

IV. MELEZITOSE DEGRADATION BY CELL-FREE Proteus ENZYMES On the basis of the above evidence, there can be no doubt that the product obtained from melezitose by the action of Proteus bacteria is sucrose. The rather complicated course of the action of the Proteus bacteria on melezitose (Fig. l), nevertheless, might make somewhat uncertain the assumption that the sucrose had been formed by direct hydrolytic degradation rather than through a more complex series of reactions perhaps dependent upon the living bacteria. All uncertainty was dispelled, however, when it was found3 that suspensions of Proteus bacteria in acetate buffer kept a t room temperature for several days attack melezitose without delay, and that active, soluble enzyme preparations could be obtained from such “aged” suspensions. Figure 4 illustrates the action of a cell-free, Proteus enzyme preparation on melezitose. It is evident that a lag period was not present and that essentially equimolecular amounts of free reducing sugar (as glucose) and of invertase-sensitive material (as sucrose) were released during the first four or five days of incubation. On the fifth day, when approximately twothirds of the melezitose had been degraded, paper chromatograma showed the presence of three components, which corresponded to melezitose, sucrose and D-glucose; other sugars, including turanose and D-fructose, were not detected. The maximal quantity of sucrose released corresponded to about 75% of that theoretically expected from a D-glucosyl-sucrose. The disproportionately large amount of reducing sugars present in the mixture after the fifth day of incubation can be accounted for on the basis of degradation of some of the newly formed sucrose. I n separate tests, the (28) C. S. Hudson and J. M. Johnson, J . Am. Chem. SOC.,37, 2748 (1915). (29) R. P.Linstead, A. Rutenberg, W. G . Dauben and W. L. Evans, J . Am. Chem. SOC.,62, 3260 (1940).

(30) E.J. Hehre and Doris M. Hamilton, J . Biol. Chem., 166, 777 (1946);J . Bact., 66, 197 (1948);E.J. Hehre, Advances i n Enzymol., 11, 297 (1951). (31) E. J. Hehre, Proc. SOC.Exptl. Biol. Med., 68, 219 (1945).

288

EDWARD J. HEHRE

same enzyme preparation was found capable of attacking sucrose; whether one and the same enzyme is involved in the action on sucrose and on the turanose linkage of melezitose is not known. However, the important point established by the experiment of Fig. 4 is that the pro30

t

10

D AVS

FIG.4.8-Action of cell-free, Proteus enzyme solution upon melezitose, initial concentration 20 m M . Solid circles represent sucrose; open circles represent reducing sugars, calculated as glucose.

duction of sucrose from melezitose by Proteus action is the result of direct enzymic hydrolysis.

V. MELEZITOSE AS

SUCROSE-ENDED SUGAR Conclusive proof is thus at hand that, as long surmised, the structure of melezitose includes the sucrose moiety; and, since the constitution of sucroses2is known to be fi-D-fructofuranosyl a-D-glucopyranoside, that the full structure of melezitose is 3-ff-D-glucOpyranOSyl-fi-D-frUCtOfUrUnOSyl a-D-glucopyranoside (11). The identification of sucrose as the nonreducing disaccharide portion of melezitose actually establishes only one new point, namely, that the D-fructose unit of the trisaccharide has the fi- and not the a-configuration; but this was the final point required to complete the knowledge of the structure of melezitose. Moreover, the degradation to sucrose is significant beyond resolving the question of the (32) I. Levi and C. B. Purves, Advances in Carbohydrate Chem., 4, 1 (1949). A

STRUCTURE OF MELEZITOSE

289

configuration of the D-fructose unit because it confirms completely and in an easily understandable way all the other details of structure of the D-fructose < > D-glucose entity of melesitose that had been established earlier. The presence of sucrose, for example, proves the pyranoid ring form of the D-glucose unit of the non-reducing entity, in confirmation of the methylation experiments of Zempl6n and Braun14 and Leitchlb and of the periodate oxidation experiments of Richtmyer and Hudson;lB it likewise confirms the a-configuration of the same D-glucose unit, in HOCHI

H

OH

H

OH

H

I1

keeping with the earlier enzymic studies of Weidenhagen.12 The isolation of sucrose also furnishes an independent proof of the furanoid ring form of the D-fructose unit in melesitose, which originally was indicated by the methylation studies of Leitch16 and established through the periodate studies of Richtmyer and Hudson. l6 The complete concurrence with these diverse earlier findings adds strong support to the conclusion that melesitose can now definitely be classed as one of the “sucrose-ended ” sugars of Nature. Melezitose, however, differs in certain respects from most of the currently known members of this rapidly growing (and evidently large) class of saccharide. For example, sucrose, raffinose, gentianose, s t a c h y o ~ e and , ~ ~ v~e~r ~b a s c ~ s e ,as ~ ~well ~ as the recently described “glucofructosane B ” or “inulobiosyl glucose” (l-g-~fructofuranosyl-P-D-fructofuranosyl a-~-glucopyranoside~~~), “kestose” (64D-fructofuranosyl-P-D-fructofuranosyla-D-ghcopyranosideJh),and “erlose ” (33) C. Tanret, Bull. soc. chim., 131, 27, 947 (1902); M. Onuki, Sci. Papers Inst. Phys. Chem. Research (Tokyo), 20, 201 (1933); R. A. Laidlaw and Claire B. Wylam, J . Chem. SOC.,567 (1953). (34a) S. Murakami, Proc. Imp. Acad. (Tokyo), 16, 12 (1940). (34b) R. Dedonder, Bull. SOC.chim. biol., 34, 144, 157, 171 (1952); J. H. Pazur, J . Biol. Chem., 199, 217 (1952). (340) N. Albon, D. J. Bell, P. H. Blanchard, D. Gross, and J. J. Rundell, J. Chem. Boc., 24 (1953).

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EDWARD J. HEHRE

(4-a-~-glucopyranosyl-a-~-glucopyranosyl P-D-fructofuranosideaM),all possess a terminal (unsubstituted) /%linked D-fructofuranose unit; all are susceptible to the action of yeast invertase; and all (except possibly verbascose, for which no report of a test was found) give a positive color reaction with d i a z o u ra ~ i l . ~Only ~ planteose, recently to be 6-cu-D-ga~actopyranosy~~-~-fructofuranosy~ a-D-glucopyranoside, resembles melezitose in lacking a terminal 8-linked D-fructofuranose unit, in being insusceptible to yeast invertase, and in giving a negative reaction with diazouracil. The effects of substitution in the &fructose moiety of sucrose, at least by an a-linked D-glucopyranosyl unit at the third carbon atom (melezitose) or by an a-linked D-galactopyranosyl unit at the sixth carbon atom (planteose), show that neither yeast invertase nor diazouracil is a completely reliable reagent for the recognition of the chemicallycombined sucrose moiety (see Levi and Purvesa2). The presence of a substituent on the &fructose unit of melezitose is associated with still another effect. That is, the a-D-glucopyranosyl residue at the sucrose end of the melezitose molecule, unlike that of sucrose, does not undergo polymerization to dextran or glycogen under the influence respectively of Leuconostoc mesenteroidessJ6or Neisseria per$avaaO systems. By contrast, it has long been known that the /3-D-fructofuranose residue of raffinose, like that of sucrose, does undergo biological polymerization to levan.a1*s6 Molecular models as well as the perspective formula of melezitose (11) suggest the possibility that a sort of structural “ overlapping ” may underlie the failure of certain reactions expected of the sucrose union. The preferential splitting of the turanose(over the sucrose-) linkage, by Proleus enzymes, may also perhaps be based on such a hindrance, though ample evidence has now been obtained87 t o show that the substrate specificity of a-D-ghcosidases is not invariably as broad as originally postulated by Weidenhagen. l8 (34d) J. W. White, Jr., and Jeanne Maher, J. Am. Chem. Soc., 76, 1259 (1953). A ample of this sugar, furnished by Dr. White, was found to give a strongly positive

Raybin” test. (34e) N. Wattiez and M. Hans, BUZZ. acad. roy. med. Belg., 8, 386 (1943); D. French, G. M. Wild, B. Young, and W. J. James, J. Am. Chem. Soc., 76, 709 (1953). (35) H. L. A. Tarr and H. Hibbert, Can. J . Research, B, 6, 414 (1931). (36) M. W. Beijerinck, Folia Microbiol., 1, 377 (1912); F. C. Harrison, H. L. A. Tarr and H. Hibbert, Can. J . Research, B, S, 449 (1930); E. J. Hehre, Dorothy S. Genghof, and J. M. Neill, J . Immunol., 61, 5 (1945). (37) B. Helferich, in J. M. Sumner and K. Myrbitck, “The Enzymes,” Academic Press, New York, 1950, Vol. 1, pp. 95-96; C. Neuberg and Ines Mandl, ibid., pp. 542-544; A. Gottschalk, ibid,, pp. 577-580.

COMPOSITION OF CANE JUICE AND CANE FINAL MOLASSES

BY W. W. BINKLEYAND M. L. WOLFROM Department of Chemistry, The Ohio Slate University, Columbus, Ohio

CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 11. Composition of Cane Juice.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 1. Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 a. Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 b. Bacterial.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 2. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 3. Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 4. Nitrogen Compounds.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 5. Non-nitrogenous Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 6. Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 7. Waxes, Sterols and Lipids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 8. Inorganic Components.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 .......................................... 301 9. Summary. . . . . . 111. Composition of Ca . . . . . . . . . . . . . 303 1. Carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 . . . . . . . . 308 2. Vitamins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 3. Nitrogen Compounds. . . . . . . . . . . . 4. Non-nitrogenous Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 5. Pigmented Materials. . . . . . . . . . . . . . . . . . . . . . . . . 311 . . , 311 6. Waxes, Sterols and Lipids. . . . . . . . . . . . . . . . 7. Odorants.. . . . . . . . . . . . . . . . . . . . . . . . . . . 312 8. Inorganic Components.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 9. Summary . . . . . . . . . . . . ........................................ 312 Addendiim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

I. INTRODUCTION The expressible juice from sugar cane is the precursor of blackstrap or final molasses. Components of the juice constitute the major part of this by-product. However, a significant portion of the final molasses consists of the altered reaction products formed under factory conditions from the juice constituents. A summary of t,he composition of cane juice is presented as a necessary background for any discussion of cane final molasses. Cane juice is an aqueous solution circulating in the living plant and carrying materials required for growth and metabolism. It is therefore extremely complex. It distinguishes itself from other plant juices or saps by its characteristically high content of sucrose. The 291

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W. W. BINKLEY AND M. L. WOLFROM

cane juices referred t o in this writing are “screenedyycrusher juicea; they closely approximate the normal plant juice. The range in variability of these juices is demonstrated by their empirical analyses’ (basis whole juice) : water, 78-86 % ; sucrose, 10-20 %; reducing sugars, 0.5-2.5 % ; other organic compounds, 0.5-1 .O%; ash, 0 . 3 4 7 % ; p H 5.2-6.2. In the early part of the 1951 season a typical Florida crusher juice showed a pH of 5.8 and contained (basis whole juice) : 18.55’ Brix solids at 2OOC. ; sucrose, 16.22%; reducing sugars, 0.5%.2 The pressure on the crusher rolIs which express this juice from the shredded cane may be as much as 75 tons per foot of roll width.’ Leaving the crushers the cane (bagasse) is extracted during passage through a series of 3-roll mills; considerable water is added and these liquids are then much less representative of the actual juice in the cane.

11. COMPOSITION OF CANEJUICE 1. Carbohydrates a. Normal.-The production of sucrose from sugar cane is revealed in the earliest records of modern ~ i v i l i z a t i o n . ~The ~ ~ application of the scientific method to this process began slightly more than a century ago.6 Three species of the genus Saccharum are grown for the industrial production of sugar; they are spontaneum, robustum and o$cinarum.a The sugar cane is a reed or grass and is propagated by joint cuttings (“plant cane”). Several cuttings (“stubble cane”) are made from each planting.6 Seedlings are required for new varieties and hybrids. As the seeds are very difficult to germinate, this work has been carried out in the experiment stations and has led to such variety designations as POJ 234 (variety 234 of Proefstation Oost Java), BH (Barbados Hybrid) or D (Demarara, British Guiana).8 The cane seldom matures before harvest in Louisiana, the danger of frost there confining the growing period to about nine months. This period is about fifteen months in Cuba and is eighteen to twenty-four months in Hawaii. (1) E. R. Riegel, “Industrid Chemistry,” 5th ed., Reiiold Publishing Corp., New York, 1949. (2) Private communication from Dr. B. A. Bourne of the United States Sugar Corp., Clewiston, Florida. (3) Noel Deerr, “History of Sugar,” Chapman and Hall, London, 1949, Vol. I, p. 12. (4) E. 0. von Lippmann, “Geschichte dea Zuckers,” 2nd ed., J. Springer, Berlin, 1929. (5) J. B. Avenquin, J . chim. ma,pharmacie tosicologie, [2] 1, 132 (1836). (6) A. Van Hook, “Sugar,” The Ronald Press Co., New York, 1949, p. 23.

COMPOSITION

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293

Reducing sugars, 4-8%,’ are known to be present in cane juice; they are n-glucose and D-fructose. The acetylation of lyophiliaed (freeaedried) normal cane juice solids followed by chromatography on a magnesium acid silicate of these acetates led to the iso1at)ion and proper identification of the sugars as crystalline derivatives.8 Methods of approximate analysis in solution for sucrose, D-glucose and D-fructose have been extensively developed and refined.g They depend upon the fact that sucrose is a non-reducing disaccharide of rather high optical rotation, [a],, +66”, and that on acid hydrolysis to its reducing sugar components, the mixture is levorotatory because of the high equilibrium levorotation, [aID -92”, displayed by D-fructose over that of D-glucose, [.ID +52.5”. The fact that n-fructose is highly tautomeric and changes its equilibrium rotation markedly with temperature is also employed in analysis. The other carbohydrates in cane juice are the soluble polysaccharides vaguely classified under the terms “hemi-celluloses, soluble gums and pectins.” It is possible that some of these polysaccharides may enter the juice during the milling of the cane as the plant cell structure is destroyed. A gummy product has been isolated from cane fiber by alkali extraction followed by alcohol precipitation, Acid hydrolysis of this substance yielded crystalline D-xylose and L-arabinose.l o Such gums in Trinidad cane juices were isolated by alcohol precipitation at suitable hydrogen ion concentration and assayed for pentose content by the Tollens 2-furaldehyde assay; the results showed an “apparent pentosan” content of 0.04-0.07%” of the Brix solids. Pectins are present in plant juices. These are now considered to be polymers containing a basic chain, designated pectic acid, of 4-linked Pgalactopyranuronic acids, the. carboxyl groups of which exist in part as methyl esters and in part as salts. Variable amounts of an araban and a galactan are associated with this polysaccharide.12 A method of approximate assay for pectic substances13 depends upon saponification (7) Unless otherwise noted, constituent concentrations of cane juice and of cane final molasses are expressed on the basis of the solids contents of the source materials. When the solids contents are unavailable, the normal values of 17% and 80% for cane juice solids and cane final molasses solids, respectively, have been employed to closely estimate these constituent concentrations. (8) W. W. Binkley and M. L. Wolfrom, J . Am. Chem. Soc., 68, 1720 (1946). (9) C. A. Browne and F. W. Zerban, “Physical and Chemical Methods of Sugar Analysis,” John Wiley and Sons, Inc., New York, 3rd ed., 1941. (10) C. A. Browne, Jr., and R. E. Blouin, Louisiana Expt. Sta. Bull. 91 (1907). (11) R. G. W. Farnell, Intern. Sugar J., 26, 480 (1924). (12) E. L. Hirst and J. K. N. Jones, Advunces in Carbohydrate Chem., 2,235 (1946). (13) M. H. Carre and D. Haynes, Biochem. J . , 16, 60 (1922).

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followed by the precipitation and weighing of the water-insoluble (but impure) calcium pectate. Application of this procedure to Trinidad cane juicesll showed the presence of 0 . 0 4 1 % of pectins (as calcium pectate) in the Brix solids. Uronic acids are readily decarboxylated on heating with hot mineral acids. An assay for their presence depends upon the determination of the evolved carbon dioxide14and when applied to Louisiana cane juice indicated a uronic acid content of 0.44% of the ash-free solids.16 The uronic acids are derived from the cane fiber and pectins. The methoxyl content of the juice was found by a modified hydriodic acid digestion method16 to be 0.08% of the ash-free solids.Is It is presumably derived from the pectins present. Starch is readily detected by iodine coloration and has been reported in the Uba cane juice of South Africa.” The starch content of Louisiana cane juice has been determined quantitatively's by extraction of the nondialyzable juice fraction with perchloric acid, precipitation of the starch with iodine, decomposition of the starch-iodine complex with alcoholic sodium hydroxide, hydrolysis of the starch with aqueous hydrochloric acid and estimation of the starch content from the copper reduction value of the hydr01yzate.l~ The average starch content of this juice was found to be 0.012-0.018%;the maximum value was 0.035%.18 The starch is present in the cane just above the nodes, where it apparently serves as a reserve carbohydrate for growth. It is probably not a normal constituent of the plant juice but enters the crusher juice through the mechanical disintegration of the nodes. Cane juice is a rich source of the cyclic alcohol, myo-inositol (the definitive prefix my0 has been suggested to replace meso for the common inositol, m. p. 225OZ0). It was isolated from cane juice by chromatographyzl; bioassay (Beadle methodzz) of the juice showed 0.041% myoinositol.zl A logical companion of this constituent is phytin (a calcium(14) K. U. Lefhvre and B. Tollens, Ber., 40,4513 (1907); A. D. Dickson, H. Otterson and K. P. Link, J . Am. Chem. SOC.,62, 1775 (1930). (15) C. A. Browne and M. Phillips, Intern. Sugar J., 41, 430 (1939). (16) M. Phillips, J . ASSOC. 03.Agr. Chemists, 16, 118 (1932). (17) L. Fevilherade, Proc. S. African Sugar Tech. Assoc., 71 (1929); Chem. Abstracts, 24, 3127 (1930). (18) R. T. Balch, B. A. Smith and L. F. Martin, Sugar J., 16, No. 6, 39 (1952); R. T. Balch, ibid., No. 8, 11 (1953). (19) G. S. Pucher, C. S. Leavenworth and H. B. Vickery, Anal. Chem., 20, 850 (1948). (20) H. G. Fletcher, Jr., L. Anderson and H. A. Lardy, J . Org. Chem., 16, 1238 (1951). (21) W. W. Binkley, M. Grace Blair and M. L. Wolfrorn, J . Am. Chem. Soe., 67, 1789 (1945). (22) G. W. Beadle, J . Biol. Chem., 166, 683 (1944).

COMPOSITION

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AND CANE FINAL MOLASSES

295

magnesium salt of myo-inositol hexaphosphate) ; it was not found in cane juice.21 This juice contained iron which forms a very insoluble salt with myo-inositol hexaphosphate and the modifiedz3phytin assay,z4depending upon the precipitation of ferric phytate, was thus not applicable. Phytin must certainly be a juice constituent since it is found in blackstrap molasses.21 The amount of organic phosphate in cane juice (average ca. 18.7 mg. of phosphoric anhydride per 100 g. of Brix solids) has been estimated from the difference between the total and inorganic phosphorus contents; the presence of hexose phosphates is suggested.26*26a b. Bacterial.-Slime-producing bacteria are associated with the cane plant and these attack cane damaged by frost or by other agents. Some of these bacteria belong t o the Leuconostoc mesenteroides and Leuconostoc dextranicum classifications and form many strains that are difficultly distinguishable. They contain a transglycosidase enzyme system which acts upon sucrose to transfer the D-glucose glycosidic linkage from D-fructose to the D-glucose of another sucrose molecule with the concomitant liberation of D-fructose. This process continues and a polysaccharide molecule is built up containing a-D-glucopyranose units linked 1 -+ 6 and 1 --+ 4 with the former predominating. I n some products other linkages may be present.25 These substances are termed dext r a n ~ ~ ’and - ~ ~have been the subject of considerable investigation since the recently indicated use of acid-modified dextrans as blood extenders. Other bacteria associated with cane and cane products, such as Bacillus mesentericus, contain a transglycosidase enzyme system that transfers the D-fructose glycosidic linkage in sucrose to the D-fructose unit of another sucrose molecule with the liberation of D-glucose. The polysaccharides formed are termed lev an^^*-^^ and consist of D-fructofuranose units joined 2 -+ 6. The dextran-producing bacteria are thermolabile and are destroyed in the mill processing whereas the levan-producing bacteria persist. D-Mannitol is not a normal constituent of cane juicez1but is always (23) E. B. Earley, Ind. Eng. Chem., Anal. Ed., 16, 389 (1944). (24) W. Heubner and H. Stadler, Biochem. Z., 64, 422 (1941). (25) P. Honig, “Phosphates in Clarified Cane Juice,” West Indies Sugar Corp. Rept. 1 (1952); Abstracts Papers Am. Chem. Sac., 121, 15P (1952). (25a) L. F. Wiggins, Intern. Sugar J., 64, 324 (1952). (26) R. L. Lohmar, J . Am. Chem. SOC.,74, 4974 (1952). (27) E. Durin, Compt. rend., 83, 128 (1876). (28) T. H. Evans and H. Hibbert, Advances in Carbohydrate Chem., 2, 203 (1946); Sugar Research Foundation, Sci. Rept. Series, 6 (1947). (29) E. J. Hehre, Transactions N . Y . Acad. Sn’., [11] 10, No. 6, 188 (1948). (30) R. G. Smith, Intern. Sugar J., 4, 430 (1902); R. G. Smith and T. Steel, J . SOC.Chem. Ind., 21, 1381 (1902).

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present in final molasses.21 It is introduced by bacterial action. There are many types of bacteria that produce Dmannitol from carbohydrates, especially from D-fructose.al The occurrence of gross amounts of D-mannitol in damaged and bacterially infected cane has been described.a2 2 . Enzymes Hydrolyzing and color-producing enzymes or enzyme systems are active in raw cane juice and both contribute to the formation of molasses. The cane plant contains the sucrose-hydrolyzing enzyme invertasea2and thus differs from the sugar beet, where it is absent. This enzyme is present in the juice and produces a simultaneous decrease of sucrose and an increase in reducing sugars. The color-producing enzyme systems are represented by an oxidase (laccase), a peroxidase and t y r o s i n a ~ e . ~ ~ - ~ ~ These are oxidizing enzymes that produce phenols and quinones from aromatic compounds present in the plant juice which then react with ferric ion to produce dark colored complexes.

3 . Vitamins Raw cane juice is not a rich source of vitamins; it does contain a little vitamin A, some of the B vitamins and probably a trace of vitamin D.aa A more complete assay of the B-group vitamins revealed the presence of thiamine, riboflavin, pantothenic acid, niacin, and biotinSa7 Cane juice is a good source for the reported fat-soluble “antistiffness factor,” a yield of 0.1 g. (50,000,000 units) being obtained from 55 gal. of raw juice.s8 Application of counter-current distribution with a methanolisooctane system to a cane juice concentrate containing this “antistiffness factor ’) and sublimation of one of the three resulting fractions produced a sublimate with an infrared spectrum like ~ t i g m a s t e r o l . ~This ~ sterol has the same chemical and biological properties as the “antistiffness (31) H. R. Stiles, W. H. Peterson and E. B. Fred, J . Biol. Chem., 64, 643 (1925). (32) C. A. Browne, Jr., J . Am. Chem. SOC.,28,453 (1906); C. F. Walton, Jr., and C. A. Fort, Ind. Ens. Chem., 23, 1295 (1931). (33) M. Raciborski, Jaarverslag 1898 van het proefsta. voor Suikerriet West Java, 15 (1899). (34) F. W. Zerban and E. C . Freeland, Louisiana Agr. Expt. Sta. Bull. 106 (1919). (35) F. W. Zerban, Ind. Eng. Chem., 12, 744 (1920). (36) E. M. Nelson and D. B. Jones, J . Agr. Research, 41, 749 (1930). (37) W. R. Jackson and T. J. Macek, Znd. Eng. Chem., 36, 261 (1944). (38) J. Van Wagtendonk and Rosalind Wulzen, J . Biol. Chem., 164, 597 (1946). (39) H. Rosenkrantz, A. T. Milhorat, M. Farber and A. E. Milman, Proc. SOC. Exptl. Biol. Med., 70, 408 (1951).

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297

myo-Inositol, a vitamin complement and growth factor, is present in relatively large quantities. It is generally included as a member of the B-group of vitamins although its position in human nutrition is still not adequately defined.41

4. Nitrogen Compounds Both simple and complex organic nitrogen compounds occur in cane juice. Loosely defined albumins and simpler proteins are the complex substances and they may represent as much as 25% of the organic nitrogen compounds of the juice.42 None of these proteins has been isolated or characterized. Analysis of a high-nitrogen juice (Florida) with ion-exchange resins showed that at least 75 % of the nitrogen substance behaved like amino a ~ i d s . 4 ~L(1evo)-Asparagine is the most abundant amino acid of cane juice, from which it has been isolated and ctdequately identified.44 L(deztro)-Glutamine and tyrosine were likewise isolated and identified44; the cane juice employed was Puerto Rican. Application of amino acid paper chromatography to the cations removed from cane juice by ion-exchange resins indicated the presence of leucine (or isoleucine), valine, y-aminobutyric acid, alanine, glycine, serine, asparagine, glutamic and aspartic acids, lysine and gl~tamine.4~~46 A preliminary chromatographic fractionation on clay enhanced the clarity of the spots, which were further identified by spot enhancement with authentic specimens of these substance^.^^ Tyrosine was detected only after a preliminary concentration on a column of powdered celluMicrobiological amino acid assays46 of the organic cationic fraction of a Florida cane juice showed l leu cine,^'-^^ 0.0025%; L-iso,~~ l e ~ c i n e , *0.0010%; ~ * ~ ~ ~ - v a l i n e ,0.0018%; ~ ~ * ~ ~ ~ - t r y p t o p h a n 0.0034%; (40) E. Kaiser and Rosalind Wulzen, Arch. Biochem. Biophys., 31, 327 (1951). (41) D. W. Woolley, J. Nutrition, 28, 305 (1944). (42) F. A. F. C. Went, Jahrb. wiss. Bolan., 31, 289 (1898); Chem. Zentr., 11, 367 (1898). (43) G. N. Kowkabany, W. W. Binkley and M. L. Wolfrom, Abstracts Papers 12th Intern. Congr. Pure Applied Chem., 166 (1951); Agr. Food Chem., 1, 84 (1953). (44) F. W. Zerban, 8th Intern. Congr. Pure Applied Chem., 103 (1912); Chem. Abstracts, 6, 3337 (1912). (45) 0. E. Pratt and L. F. Wiggins, Proc. Brit. West Indies Sugar Technol., 29 (1949); L. F. Wiggins and J. H. Williams, ibid., 40 (1951). (46) Performed by the Food Research Laboratories, Inc., Long Island City, N. Y., for the Sugar Research Foundation, Inc., New York, N. Y. (47) Method of S. Shankman, J . Biol. Chem., 160, 305 (1943). (48) Method of J. R. McMahan and E. E. Snell, J . Biol. Chem., 162, 83 (1944). (49) Method of R. D. Greene and A. Black, J . Biol. Chem., 166, 1 (1944).

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W. W. BINKLEY AND M. L. WOLFROM

L-methionine,600.0009 %. L-Lysine, L-arginine, L-threonine, L-histidine and L-phenylalanine were sought but not d e t e ~ t e d . Although ~~~~~ qualitatively the general amino acid picture was always the same, the total amounts of amino acids in the top portions of Barbados cane varied with the variety from 1 to 12.5 millimoles per liter of cane juice; it was highest in the top and lower portions of the cane and was at a minimum near the middle. It was at a maximum in the early stages of

5 . Non-nitrogenous Acids Juice expressed from normal cane does not contain volatile acids; soured juice or juice from damaged cane possesses volatile acids, largely acetic.61 The principal organic acid of cane juice is aconitic. Its presence was based, as early as 1877,62on the isolation of a nearly pure specimen of the acid but proper identification was not recorded until 1919.63 The concentration of this acid in Louisiana juice solids varies from 0.3 to 1.6%and it may represent as much as 90% of the non-volatile acids.64 The occurrence in cane juice of such acids as malic,s6-6*suc~ i n iglycolic,6o ~ , ~formic,61 ~ ~ and ~ ~ ~ ~ ~ has been based only on qualitative tests and analytical methods. The recent application of a chromatographic p r o ~ e d u r eemploying ,~~ silicic acid, to an anionic fraction of cane juice has led to the isolation and identification of fumaric and succinic acids in addition to aconitic acid.'l6 Aconitic acid exhibits geometric isomerism and exists in cis and trans forms.66 These are interconvertible in solution, temperature and pH being factor^.^' It is (50) Method of J. L. Stokes, M. Gunness, Irla M. Dwyer and Muriel C. Caswell, J . Biol. Chem., 160, 35 (1945). (51) C. A. Fort and J. I. Lauritzen, Ind. Eng. Chem., Anal. Ed., 10, 251 (1938). (52) A. Behr, Ber., 10, 351 (1877). (53) C. S. Taylor, J. Chem. SOC.,110, 886 (1919). (54) R. T. Balch, C. B. Broeg and J. A. Ambler, Intern. Sugar J., 48, 186 (1946). (55) A. Payen, Compt. rend., 28, 613 (1849). (56) H. Winter, Z. Ver. deut. Zuckerind., 38, 780 (1888). (57) J. van Breda de Haan, Jaaruerslag 1891 van het proejsta. voor Suikerriet West Jaua, 9 (1892). (58) P. A. Yoder, J . Ind. Eng. Chem., 3, 640 (1911). (59) T. Tanabe, Repts. Tianan Formosa Ezpt. Sta., 4, 33 (1937). (60) E. C. Shorey, J . Am. Chem. Soc., 21, 45 (1899). (61) F. W. Zerban, J . Assoc. Of. Agr. Chemists, 16, 355 (1932). (62) J. E. Quintus Bosz, Arch. Suikerind., 28, 969 (1920). (63) H. C. Prinsen Geerligs, Arch. Suikerind. Nederland en Ned.-Indie, 1, 230 (1940). (64) C. S. Marvel and R. D. Rands, J . Am. Chem. doc., 72, 2642 (1950). (65) See Addendum, p. 314. (66) R. E. Miller and S. M. Cantor, Advances in Carbohydrate Chem., 6,231 (1951). (67) J. A. Ambler and E. J. Roberts, J . Org. Chem., 13, 399 (1948).

COMPOSITION OF CANE JUICE AND CANE FINAL MOLASSES

299

generally assumed that the natural form of aconitic acid is mainly cis because the enzyme system aconitase, widely distributed in plants and animals, converts citric acid to isocitric acid through the intermediate cis-aconitic acid. The pH of cane juice lies in the range 5.2-6.2 where the equilibrium would favor the ~is-forrn.~’

6. Pigments Normal cane juice, as it circulates in the intact plant, is colorless34; however, the tissue breakdown produced in grinding the cane for sucrose extraction permits the colloidal suspension and solution of pigmented substances not normally present. The tanninsa8 and water-soluble an tho cyan in^^^ are the major color contributors together with the colorproducing enzyme systems described previously (p. 296). One of the anthocyanins has been isolated from the rind of Purple Mauritius cane and converted with methanolic hydrogen chloride to a red-brown crystalit is a diglucoside and it possesses one line solid, CzsH33O17C1.4HzOB9; methoxyl group. Qualitative tests show that the aglycon is probably a mono-0-methyl-delphinidine. Chlorophyll is largely insoluble in the cane juice and is removed in the mill scums.34 That a small amount is soluble, however, is established by the chromatographic isolation of a green-colored fraction from final rn0lasses7~giving the ultraviolet absorption spectrum of chlorophyll a. An amorphous, incrustating solid, resembling lignin and designated “saccharetin ” is mechanically dispersed in the juice during the grinding of the cane. It is a water-insoluble, weakly acidic substance with the reported formula (C5H702),.7’ It dissolved readily in alkali to yield an intense yellow solution, gave a red color with phloroglucinol and hydrochloric acid, and an orange-yellow color with aniline sulfate and sulfuric acid. Dry distillation of “ saccharetin ” yielded pyrogallol; alkali fusion gave protocatechuic acid and pyrocatechol ; acid hydrolysis produced vanillic acid and vanillin. The lignin of bagasse has recently been isolated by an enzymic method and c h a r a c t e r i ~ e d . ~ ~ Anthocyanins, tannins and “saccharetin ” contain phenolic groups; their c,olor contribution is increased by the iron dissolved from the (68) F. W. Zerban, J . Znd. Eng. Chem., 10, 814 (1918). (69) C. J. Dasa Rao, D. G. Walawalkar and B. S. Srikantan, J . Indian Chem. Soc., 16, 27 (1938). (70) W. W. Binkley and M. L. Wolfrom, J . Am. Chem. Soc., 70, 290 (1948). (71) L. G . Langguth Steuerwald, Arch. Suikerind., 19,1543 (1911); Chem. Abstracts, 6, 691 (1912). (72) G . de Stevens and F. F. Nord, J . Am. Chem. Soc., 73, 4622 (1951); 74, 3326 (1952); 76, 305 (1953); Proc. Nail. Acad. Sn’. U.S., 39, 80 (1953).

300

W.

W. BINKLEY AND M.

L. WOLFROM

sugar mill and by the contact of air with the warm alkaline defecation solutions.T8

7. Waxes, Sterols and Lipids A small amount of the wax coating of sugar cane is dispersed in the juice during grinding. Extraction with petroleum ether will remove it from the raw j ~ i c e . 7 ~The wax and other similar substances are almost completely removed by the normal juice defecation and are found in the settlings (muds, filter-press cake) ; a minute quantity is carried through the entire process and is found in the final molasses (p. 311). The crude wax content of Louisiana whole cane is 0.2% and that of press cake may run to 22% (dry basis).16 Dried filter-press muds from the northern British West Indies islands and from Jamaica, Trinidad and British Guiana have an average wax content of 14.7, 11.3, 8.8 and 7.0%, respecti~ely.7~ Chromatography, on alumina, fractionated Louisiana cuticle cane wax (scraped from the stalk surface) into three groups: free acids, free alcohols and other substances (esters, ketones, hydrocarbons).77 Slight to almost complete hydrolysis occurred during the formation of these chromatograms. Identification of the individual components of the fractions was not completed. An empirical analysis of Louisiana cuticle cane wax is presented in Table I. TABLEI Chemical Data on Louisiana Sugarcane Cuticle Waxes7b

0

source

Saponification Value

Acid Value

Iodine Value

Acetyl Value

Corn290 Co5 281

40.5 56.7

18.0 23.8

8.0 15.6

91.8

-

Seedling variety from Coimbatore Experiment Stetion (India).

The nature of the non-acid fraction of the wax ia of interest.76 Some glycerol is present but the main alcohols are the higher-carbon monohydric alcohols, ceryl (n-C26Hsl-CH20H, isolated from Louisiana wax) and melissyl (isolated from Cuban molasses70). The latter is probably a (73) F. W. Zerban, Sugar Research Foundation, Tech. Rept. Series, 2 (1947). (74) N. G.Chatterjee, J . Indian Chem. SOC.,Ind. and News Ed., 8, 183 (1940). (75) R. T. Balch, Sugar Research Foundation, Tech. Rept. Series., 8 (1947). (76) L. F. Wiggins, Proc. E d . Wed Indiee Sugar Technol., 16 (1950). (77) T. W. Findley (with J. B. Brown), Ph.D. Dissertation, The Ohio State University, 1950.

COMPOSITION O F CANE JUICE AND CANE FINAL MOLASSES

301

mixture of n-CZSHss-CHtOH and n-Ca1Hss-CHz0H. The sterol content (free and combined) of Louisiana wax is in the range of 5-10%.76 The sterol fraction consists76 of 3 parts of stigmasterol (m. p. 171", [a], -51" in chloroform; acetate: m. p. 144",DI.[ -55" in chloroform) and 7 parts of a mixture of p-sitosterol and 7-sitostierol (m. p. 137-143", [.ID -38" in chloroform).

8. Inorganic Components Crusher juice and whole raw cane juice contain representative i riorganic constituents; clarified juice is limed. The inorganic substances TABLEI1 Inorganic Components of Juice from Louisiana-grown Cane Varieties Coimbatore 281 and %lo78

Co. 981a

Co. 29O0

KaO NanO CaO MI30 Alz08 FeaOs MnO

1.603% 0.060 0.216 0.248 0.088 0.0104 0.0034

1.792% 0.056 0.189 0.263 0.067 0.01 0.0031

SO8

0.543 0.415 0.191 0.094

0.455 0.400 0.246 0.096

Component

PaOs

c1

SiO2 Expraasad ran per cent on aolid~.

or ash contents vary greatly among the different varieties of cane within a confined area and in a single variety grown in the different cane producing areas. The principal cations of cane juice are potassium, magnesium, calcium, aluminum, sodium, iron and manganese; anions are sulfate, phosphate, chloride and silicate (Table 11). Raw juice contains more inorganic cations than inorganic anions even though it is acid, pH 5.2-6.2; this acidity is due to the presence of organic acids. 9. Summary

Table 111 summarises the data on cane juice constituents. (78) C. A. Fort and N. McKaig, U . S. Dept. Agr., Tech. Bull. 688 (1939).

302

W. W. BINKLEY AND M. L. WOLFROM

TASLE~ I11 Cane Juice Constituents Component Carbohydrates Sucroie D-Glucose D-Fructose myo-Inositol Phytin Pentosans Methoxyl Pectins Uronic acids Starch Organic phosphate Enzymes Invertase Oxidase Peroxidase Tyrosinase Vitamins Vitamin A Biotin Vitamin D (?) Niacin Pantothenic acid Riboflavin Thiamine Nitrogen compounds Amino acids Alanine 7-Aminobutyric acid L(leoo)-Asparagine Aspartic acid Glutamic acid L(deztro)-Glutamine Glycine dsoleucine *Leucine Lysine L-Methionine Serine *Tryptophan Tyrosine L-Valine

Juice origin" La.

}

La. La. Cuba Trin. La. Trin. La. La. Cuba

Concentration, % 78-84 4.3-7.8

78 78

0.041

21 21 11 15 11 15 18 25

-

0.04-0.07 0.08 0.0-0.1 0.44 0.012-0.018 0.0187

La. La. La. *La. La. La. Cuba La. La. Cuba La. Cuba La. Cuba La. Cuba

Reference

32, 34 34 34 34 2.2 x 10-6 17.6 x 10-6

36 37 37

4.9 x 10-6 4..5 x 10-6 218 x 10-6 99.4 x 10-6 3.1 X 4.9 x 10-6 5.3 x 10-6 10.5 X 10-6

36 37 37 37 37 37 37 37 37

-

Barb. 1 - 12.5 X lO-'JM 45 Fla., Jam. 43,45 Fla., Jam. 43,45 Fla., P. R., Jam. 4345 Fla., Jam. 43,45 Fla ., Jam. 43,45 Fla., P. R., Jam. 4345 Fla., Jam. 43, 45 Fla. 0.0010 46 Fla. 0.0025 46 Jam. 45 Fla. 0.009 46 Fla., Jam. 43, 45 Fla. 0.0034 46 Fla., P. R. 43, 44 Fla. 0.0018 48

COMPOSITION O F CANE JUICE A N D CANE FINAL MOLASSES

303

TABLEI11 (Continued) Component Proteins Non-nitrogenous acidsa6 Aconitic Fumaric Glycolic Malic Oxalic Succinic Pigments Anthocyanins Chlorophyll “Saccharetin ” Tannins Waxes, sterols and lipids Nonsaponifiable fraction Palmitic acid Oleic acid Linoleic acid Linolenic acid Inorganic components

Java La. La. Trin. La. La. Hawaii Java

b

0.33-0.49 1.79-3.48 0.6-1.2 0.3-2.1

-

La. La. Trin. La. La., Cuba La. La. La., Trin. La. La. La. La. La. La. Trin.

Barb. = Barbados; Fla. = Florida; Jam. = Jamaica; La.

Trin. = Trinidad.

Concentration, %’

Juice Origin”

-

0.73-1.58

-

0.73-1.58 38. Qb 16.96 9.16 31 .4b 1 .Ob 2.6-3.6 1.2-3.0

-

Reference 42 78 78 79 54, 65 65 61 55-58 55,62,63 65 79 34 34, 70 34 68 78, 79 75 75 75 75 75 78 79

Louisiana; P. R. = Puerto Rico;

Percentage of crude cane wax from press oake.

111. COMPOSITION OF CANE FINALMOLASSES During the production of sucrose from cane juice, crystallization inhibitors collect in the residual sirups or mother liquors; these sirups are called molasses. When the accumulation of these inhibitors is so great that the recovery of sucrose is no longer economically feasible, this molasses is known as the final or blackstrap molasses. The term “blackstrap” originated in the Dutch sugar industry from black “stroop” meaning black sirup.80 The molasses obtained in the early stages of sucrose production has a pleasant, palatable flavor and is used in the preparation of edible molasses. While some molasses intermediate between edible and blackstrap are best suited for the production of calcium magnesium aconitate,81far more aconitate is recovered from blackstrap (diluted t o 55’ Brix) which is available all year. In the recrystalli(79) F. Hardy, PZanter Sugar Mfr., 70, 445 (1927). (80) Noel Deerr, Intern. Sugar J . , 47, 123 (1945). (81) L. Godchaux, 11, Sugar J., 10, 2 (1947).

304

W. W . BINKLEY AND M. L. WOLFROM

zation or refining of the crude sucrose, the final mother liquor concentrate is known as refinery blackstrap molasses. I t can be considered as consisting of the accumulated blackstrap molasses originally adherent to the crude sucrose crystals. I n the crystallization of sucrose from cane juice, the pH of the normal juice entering the process lies in the range 5-6. This juice contains a little water added during the crushing process. Calcium hydroxide is added to bring the pH to 8 f 0.5 and the mixture is heated to 220°F. and maintained around 200°F. for several hours. This is the defecation process and resdts in a clarification of the liquid with the precipitation of suspended materials, proteins, waxes and fats.81a It is closely controlled and varies slightly with the processor. After passing through the settlers the pH is now approximately 7. The muds from the settlers are removed with continuous rotary filters, resulting in filter cake. The clarified juice is heated to 225°F. in the first of a bank (usually four) of multiple-effect vacuum evaporators for a period of ten minutes or less and for longer periods at lower temperatures in subsequent evaporators as the juice is concentrated to a sirup. The crystallization of sucrose from this sirup is a batch operation and is accomplished with single vacuum evaporators (“pans ”). During these processes the mother liquors are recirculated and fresh defecated juice is added. The final residual sirup is blackstrap molasses; its pH is 5.8. 1. Carbohydrates

As in cane juice, sucrose is the principal sugar of cane blackstrap molasses. However, the ratio of sucrose to apparent reducing sugars has dropped from 10-15: 1in the juice to 1.5-2.5: 1 in the molasses. Patents have been issued for the recovery of sucrose from molasses but general acceptance of these processes by the industry is still lacking. Some of the methods depend on the removal from molasses of the reducing sugars and other impurities by lime,82invertase-free yeast,83 barium hydroxide,8* or fuller’s earth clay.86 Other methods are based upon the use of solventsseand of ion-exchange resins. ST The significant simple sugars are (81a) P. Honig, Sugar, 47, No. 6,31 (1952). (82)E.E.Battelle, U. 5. Pats. 1,044,003(1913),1,044,004(1913). (83)H.De F.Olivarius, U. S. Pats. 1,730,473 (1929),1,788,628(1931). (84)A. L. Holven, U. S. Pats. 1,878,144(1933),1,878,145(1933). (85) M. L. Wolfrom and W. W. Binkley, U. S. Pat. 2,504,169 (1950);J . Am. Chem. SOC.,69, 664 (1947). (88) J. H. Payne, U. S. Pat. 2,501,914(1950). (87) N. V. Octrooien Maatschappij, Dutch Pat. 68,496 (1946); Chem. Abdracts. 41,4667 (1947).

COMPOSITION

OF CANE JUICE

AND CANE FINAL

MOLASSES

305

D-glucose and D-fructose. The actual isolation of crystalline D-glucose was accomplished by the chromatography of Cuban blackstrap molassesss on fuller’s earth claysg;D-fructose was obtained as a sirup which yielded crystalline acetates of the B-D-pyranose and keto forms of this sugar after acetylation and chromatographyg0on magnesium acid silicate. The precipitation from molasses of D-mannose as its phenylhydraeone was reported over 50 years Recent investigation of the phenylhydrazone from Cuban molasses did not reveal any D-mannose phenylhydrazoneg2;the presence of this sugar in cane molasses cannot be considered as demonstrated. Early investigatorsgs considered an unestablished sugar designated “glutose” to be an unfermentable, molasses component and to be isolable from this unfermentable residue as its phenylosaeone. Later workg4showed that “ glutose phenylosaeone ” was an impure D-glucose phenylosazone. The presence of D-psicose (D-rib-hexdose, D-allulose) in distillery slop (from the fermentation of cane molasses) is claimed,g5but sharp experimental support is lacking. Diheterolevulosans (difructose dianhydrides) are obtained by refluxing concentrated aqueous solutions of ~ - f r u c t o s e . ~ Chromatography ~~~~ of Cuban blackstrap molasses in a pilot-plant-scale chromatogram on fuller’s earth clay did not reveal the presence of these s u b s t a n ~ e s . ~ ~ Traces of the complex carbohydrates contained in the juice survive the processing and appear in the molasses; analytical data indicate that they probably consist largely of pectins and pentosans. lo Quantitative estimations based on the ash-free solids of Louisiana molasses revealed the presence of 2% uronic acids and 0.5% methoxyl.16 Heat-modified (88) W. W. Binkley and M. L. Wolfrom, J . Am. Chem. SOC.,73, 4778 (1950). (89) B. W. Lew, M. L. Wolfrom and R. M. Goepp, Jr., J. Am. Chem. SOC.,68, 1449 (1946). (90) W. H. McNeely, W. W. Binkley and M. L. Wolfrom, J. Am. Chem. SOC., 67, 527 (1945). (91) C . A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trav. chim., 16, 260, 280 (1897). (92) M. Grace Blair (with M. L. Wolfrom), Ph. D. Dissertation, The Ohio State University, 1947. (93) H. C. Prinsen Geerligs, Intern. Sugar J., 40, 345 (1938). Review article. (94) L. Sattler, Advances in Carbohgdrate Chem., 3, 113 (1948). (95) F. W. Zerban and L. Sattler, Znd. Eng. Chem., 34, 1180 (1942). (96) L. Sattler and F. W. Zerban, Znd. Eng. Chem., 37, 1133 (1945). (97) M. L. Wolfrom and M. Grace Blair, J. Am. Chem. SOC.,70, 2406 (1948). (98) W. W. Binkley and M. L. Wolfrom, Sugar Research Foundation, Member Rept. 26, 21 (1950).

306

W. W. BINKLEY AND M. L. WOLFROM

starches are probably contained in the molasses from the starch-bearing cane juice^.^^^^^ Crystalline myo-inositol (see p. 294) was isolated from Cuban final molasses by chromatography on fuller's earth clay.ss The myo-inositol content (0.261%) of this molasses was determined by fermentation with yeast, acetylation of the unfermented residue, chromatography on magnesium acid silicate of the acetylated residue and the isolation of crystalline myo-inositol hexaacetate. The value obtained by bioassay was 0.238%.21 The phytin content of Cuban molasses was estimated to be 0.22-0.23% by bioassay.21 A small amount of D-mannitol as the crystalline hexaacetate was obtained from this molasses by these chromatographic procedures.88 I n addition to the D-mannitol produced by the bacteria in the sugar mill, this hexitol may be introduced bacterially in the cane juice, thus leading to its accumulation in the final molasses. Extension of these chromatographic techniques to the unfermented residue of Cuban molasses led to the isolation of erythritol and D-arabitol (as their respective acetates) as trace constituentsss; the yeast may be the source of these substances. Another important carbohydrate group of cane final molasses, and one which has been little studied, consists of the products formed by the action of heat, alkali and amino acids upon the reducing sugars. This is sometimes termed the non-fermentable fraction as it remains after yeast fermentation although it is thereby badly contaminated with metabolic products from the yeast. The reducing sugars D-glucose and D-fructose are first formed by the action of invertase on sucrose. These are then subjected to the action of hot alkali (pH ca. 8) during the defecation and to considerable heat treatment a t pH 6-7 thereafter (see p. 304). Monosaccharide decomposition involving dehydration, disproportionation, and fragmentation, is involved. Dehydration leads necessarily to dicarbonyl compounds99~100 which are probably significant reaction intermediates and may form furan derivatives (Fig. 1 and Sections 111-4 and 111-5). Chain fragmentation by reverse aldolizationlol may play a part (Fig. 1 and Section 111-4). In addition, the amino acids present undoubtedly react with the reducing sugars, or their dehydration products, to yield dark colored polymeric substances containing nitrogen. This is known as the Mail(99) M. L. Wolfrom, E. G. Wallace and E. A. Metcalf, J. Am. Chem. SOC.,64,265 (1942). (100) M. L. Wolfrom, R. D. Schuetz and L. F. Cavalieri, J. Am. Chem. SOC.,71, 3518 (1949). (101) J. F. Haskins and M. J. Hogsed, J . Org. Chem., 15, 1275 (1950).

COMPOSITION OF CANE JUICE AND CANE FINAL MOLASSES

307

lard102 or “browning” rea~tion.1~3-1~6It leads to the production of color and finally to the separation of brown to black solids known as “ melanoidins.” O=? HO'H3

HO'

+

HO'H3

*?

7

3*

0=3H

H?oH

HofH

H03H

Hof

=

?

Ho?H H OH=

Ho?H

HozHf

HO~H

Ho'H?

HO'HHQ

Ho?H 0=3H

f

H OH= HO~H

HO'HHQ

I H03H

HofH H?oH

Ho?H -0OH HO'H?

y' HO? 0=3H

0=3H

0=3H

HP

0=3

zHf

0=3

I

I

OEH-

H3-H3 O=g-f

HY-'H30H

/ O\

+-

p

HozHP

OKH-

Ho?H H0;7H

1

HO'H3

Hay

HozH7

These various products of sugar decomposition are reducing88*10s so that the consideration of this analytically measurable property as being due solely to D-glucose and D-fructose is in error. Their total content has been estimatedlo7to be 10.1 and 10.7% of the solids in final molasses samples from Louisiana and Cuba, respectively. This fraction was separated by clay chromatographyss and its more complex components were segregated by dialysis and freeze-drying. The product so obtained was a brown, bitter, non-hygroscopic solid that contained nitrogen (1.7 %) and exhibited a slight Fehling reduction. The major fraction of the (102) L.-C. Maillard, Compt. rend., 164, 66 (1912); Ann. chim., [9] 6, 258 (1916). (103) J. P. Danehy and W. W. Pigman, Advances in Food Research, 3, 241 (1951). (104) M. L. Wolfrom, R. C. Schlicht, A. W. Langer, Jr., and C. S. Rooney, J . Am. Chem. Soc., 76, 1013 (1953). (105) L. Sattler and F. W. Zerban, Znd. Eng. Chem., 41, 1401 (1949). (106) C. Erb and F. W. Zerban, I d . Eng. Chem., 39, 1597 (1947). (107) C. A. Fort, Sugar, 41, No. 11, 36 (1946).

308

W. W. BINKLEY AND M. L. WOLFROM

material when subjected to dialysis passed through the membrane in the manner of relatively low molecular weight substances.108 2. Vitamins The desugaring of cane juice concentrates the heat- and alkali-stable vitamins in the final molasses. Even after this accumulation, only mgo-inositol may have reached the level of minimum dietary requirem e n t ~ . ’ Niacin, ~~ pantothenic acid and riboflavin are also present in significant quantities109;the thiamine, pyridoxin, pantothenic acid, biotin and folic acid contents of molasses have been estimated by bioassay.llOslll The biotin content of Hawaiian and Cuban molasses was 2.1 and 1.7 gammas per gram, respectively.112 The “antistiffness factor” (closely related to stigmasterol) has been found in cane m o l a s ~ e s . The ~~~~~ distillery slop from the yeast fermentation of molasses is marketed as a vitamin concentrate; this product also contains vitamins originating in the yeast.

3 . Nitrogen Compounds The nitrogen content of the cane blackstraps of North America varies between 0.4 and 1.4%. A surprisingly large fraction, as much as 60-70%, of this nitrogen is present in a relatively simple form.43*113 Isolations based on identified crystalline products, obtained from Hawaiian molasses with the aid of ion-exchange resins, are recorded113 for aspartic acid (as the free acid), glutamic acid (as the hydrochloride), lysine (as the picrate) and the purines guanine (as the hydrochloride) and xanthine (as the free base); qualitative tests were obtained for hypoxanthine and the pyrimidine 5-methyl-cytosine. Aspartic and glutamic acids occur in the plant mainly as their amides asparagine and glutamine but in the mill processing these are hydroly~edwith the evolution of ammonia. The amino acids from Florida molasses were concentrated on fuller’s earth clay and were resolved by paper chromatography employing spot enhancement with known specimens43;the presence of asparagine, aspartic acid, glutamic acid, y-aminobutyric acid, alanine, glycine, leucine or isoleucine, and valine were so established. Albumins and other protein-like substances of cane juice are precipi(108)W. W.Binkley and M. L. Wolfrom, unpublished results. (109)R: C. Hockett, J . California State Dental Assn., 26, No. 3 Suppl., 72 (1950). (110) D.Rogers and M. N. Mickelson, Znd. Ens. Chem., 40, 527 (1948). (111) W.A. Krehl and G. R. Cowgill, unpublished results. (112) E. E. Snell, R. E. Eaken and R. J. Williams, J . Am. Chem. Soc., 62, 175 (1940). (113)J. H. Payne with R. F. Gill, Jr., Hawaiian Planters’ Record, 60, No. 2, 69 (1946).

COMPOSITION OF CANE JUICE AND CANE FINAL MOLASSES

309

tated during defecation and only traces of them reach the final molasses. The principal bearers of complex nitrogen are the molasses “browning” products mentioned in the preceding section.

4. Non-nitrogenous Acids Aconitic acid was the first organic acid properly established as a component of cane molasses.63 Louisiana molasses is a rich source and contains more than 6% of this acid in some ca~es.1~4With a potential annual harvest of over 5,000,000 pounds of aconitic acid from Louisiana alone, it is nevertheless only recently that the commercial production of this acid from molasses has been initiated in Louisiana.l16 Its isolation depends upon the insolubility and ready crystallizability of dicalcium Final, or an intermagnesium trans-aconitate hexahydrate. 114,116-118 mediate or “ B ” , molasses is diluted to 55% solids (55” Brix) with the appropriate wash water from the process (see below) and the pH is adjusted to 7 with lime. The solution is heated to 200°F. and calcium chloride is added. Heating is continued for forty-five minutes whereupon the precipitate is collected. It is resuspended a t 195°F. in a volume of water sufficient to adjust the next lot of molasses to 55” Brix. The precipitate is again centrifuged; it contains about 56% aconitic acid.l16 Aconitic acid, either as such or after conversion to itaconic acid, is of present-day commercial interest. llsa Malic and citric acids have been adequately identified from molasses as their crystalline h y d r a ~ i d e s . ~It~is~ probable that at least the former is a normal juice constituent. Lactic acid was identified as its zinc salt in molasses11g;it arises from bacterial action. Formic acid is presentllg; it probably has an origin, at least in part, in sugar decomposition. Acetic and propionic acids are components and their amounts serve as a rough index of the activity of the microorganisms introduced into the molasses. The microbial count of cane juice, molasses and related products has been determined (Table IV). Application of modern ion-exchange and silica-gel chromatographic techniques to cane distillery slop (cane molasses yeast fermentation (114) R. T. Balch, C. B. Broeg and J. A. Ambler, Sugar, 40, No. 10, 32 (1945); 41, No. 1, 46 (1946); see Ref. 133. (115) L. Godchaux, 11, Sugar J., No. 4, 3 (1949). (116) E. K. Ventre, J. A. Ambler and S. Byall, U. S. Pat. 2,359,537 (1944). (117) J. A. Ambler, J. Turer and G. L. Keenan, J . A m . Chem. Soc., 87, 1 (1945). (118) E. K. Ventre, U. S. Pat. 2,469,090 (1949). (11%) R. N. Evans, Abstracts Papers Am. Chem. Soc., 119, 3Q (1951). (119) E. K. Nelson, J . Am. Chern. SOC.,61, 2808 (1929). (120) C. H. Millstein, L. C. Tobin and C. S. McCleskey, Sugar J., 3, No. 9, 13 (1941).

310

W. W. BINKLEY AND M. L. WOLFROM

residue) led t o the isolation of crystalline aconitio acid as the major organic acid component.B6 The steam distillation of acidified cane distillery slop yielded formic and acetic acids ; they were identified by qualitative tests. 121 Esterification and ester distillation followed by the formation of crystalline hydrazides led t o the adequate identification of succinic, tricarballylic and perhaps citric as component acids.lal Ether extraction of diluted acidified slop and fractional precipitation in aqueous ethanol of the barium salts of the extracted acids placed succinic and TABLEIV

Microbial CounP of Sugar Mill Products‘s0

Raw juice Clarifier effluent Press juice Evaporator sirup Storage tank sirup Crystallizer contents Masseouite Raw sugar Molaaaes a

Number of bacteris per ml.

Thermophiticc Bacteria

Mesophili@ Bacteria

Product

Low 8,000,000 0 0 200

1,300 2,000 1,200 340 300 a

High 750,000,000 11 51,000 3,300 7,100 44,000 10,600 5,100 310.OOO

Optimal growth at 15-4CPC.

Low 14 0 3,700 300 16,100 350 1,700 100 1.200

High 170 8 250,000 15,500 38,500 15,000 17,100 2,200 16.500

O p t i d growth at 40-600C.

tricarballylic with a trace of aconitic acids in the precipitate and lactic acid (isolated as the crystalline zinc salt) in the supernatant liquor.121 Other acids (often as esters) have been found in fermented molasses. Usually these substances are products of bacteriological action and they are not normal constituents of unfermented molasses. “Bauer ” oil from the yeast fermentation of Cuban blackstrap consists chiefly of the ethyl esters of capric, lauric, myristic and palmitic acids.lZ2 The fat from the scums of hot-room Louisiana molasses contained hexanoic (caproic) and octanoic (caprylic) acids.10 The occurrence of such volatile acids as propionic, lZ3b ~ t y r i c ~ and * ~ Jvaleric ~ ~ acids124 requires more adequate establishment. Qualitative tests showed the probable presence of 5(hydroxymethyl)2-furaldehyde, acetoin, levulinic and formic acids, and methylglyoxal (121) E.K. Nelson and C. A. Greenleaf, Znd. Eng. Chem., 21,857 (1929). (122) C. S. Marvel and F. D. Hager, J . Am. Chem. SOC.,46,726 (1924). (123) N. Srinivasan, Intern. Sugar J., 41, 68 (1939). (124) E.Humboldt, Facts About Sugar, 96, 18 (1930).

COMPOSITION OF CANE JUICE AND CANE FINAL MOLASSES

311

(or acetol) in the volatile decomposition products from the unfermentable residue of a heated D-fructose solution'os; these are sugar decomposition products. The volatile products from a fermented sucrose mixture contained small quantities of acetylmethyl carbinol. lo 5. Pigmented Materials

The principal pigmented substances of final molasses are probably the complex products of the reaction between the reducing sugars and the amino-containing components of the cane juice (see Section 111-1). The reducing-sugar self-decomposition in the presence of organic anions, especially aconitate, is probably also a factor and here the anion may serve principally as a buffer and probably does not enter into reaction with the reducing sugar or with its decomposition products. Model experiments126at pH 8 indicate that the main chemical color-producing system in molasses is that of D-fructose and D-glucose with asparagine followed by that of D-fructose and D-glucose in the presence of aconitate ion. Concentrated aqueous solutions of D-fructose heated at sugar mill temperature will produce a dark colored solution in the absence of amino a ~ i d s . ~ ~ , ~ ~ The polyphenolic colored substances of cane juice, largely tannins and an tho cyan in^,^^ form even more intensely colored iron complexes. Some of these compounds survive defecation and darken further with prolonged exposure to air and ferric ion a t elevated temperatures. Virtually pure chlorophyll a was isolated from Cuban molasses by chromatographic and extraction proceduresT0;it was identified by its absorption spectrum; its estimated concentration was 0.00005%. 6. Waxes, Sterols and Lipids

The concentration of fats and related substances in molasses is low; analytical values depend on the extracting solvent.126 These tenaciously retained materials can be removed by fractionation of blackstrap on fuller's earth clay.?O Chromatography on a calcium silicate of the fat fraction of Cuban molasses led to the isolation of melissyl alcohol, a phytosterol fraction, chlorophyll a and a fat fraction containing a glyceride of linoleic acid.'O Stigmasterol and syringic acid are reported as ether-extractable constituents of molasses.127 (125) J. N. Schumacher (with M. L. Wolfrom), M. Sc. Thesis, The Ohio State University, 1952. (126) C. F. Bardorf, Can. Chem. Met., 11, 231 (1927). (127) S, Takei and T. Imaki, Bull. Znst. Phys. Chem. Research (Tokyo), 16, 1055 (1936).

312

W..W. BINKLEY AND M. L. WOLFROM

7 . Odorants The ether extract of cane molasses yields an acidic substance with the characteristic odor of raw sugar.128 The steam distillation of molasses is stated to yield a “rum Fractionation of cane final molasses on fuller’s earth clay produces a concentrate with a strong molasses 0dor.7~ The infrared spectra of the volatile portion of this concentrate indicated the absence of hydroxyl and carbonyl and the presence of a substituted benzene structure, of paraffinic methylene and methyl groups, of an acetate group, and of the > C=C < and -C=Clinkages. The presence of a sulfur function is probable. Further chromatography indicated complexity in this volatile c ~ n c e n t ra t e.’~~ The deionized unfermentable (by yeast) residue from Cuban final molasses has a raisin-like odor.21 8 . Inorganic Components

The mineral constituents of the raw cane juice persist in the final molasses. The principal difference in relative amounts of these substances in molasses arises from the use of lime in defecation which causes an increase in calcium. Egyptian cane molasses solids contained 0.66% of titanium.131 The cations are believed to complex with the sugars and to thus inhibit the crystallization of sucrose, which latter is known to form compounds with inorganic salts, such as its well known compound with sodium chloride. Decationization of cane juice with ion exchange resins greatly reduces molasses formation but sucrose inversion is a concomitant problem. 192 9. Summary

Table V summarizes the data on the constituents of cane final molasses. (128) S. Takei and T. Imaki, Bull. Znst. Phys. Chem. Research (Tokyo), 16, 124 (1936). (129) E. Arroyo, Univ. Puerto Rico Agr. Expt. Sta. Research Bull. 6 (1945); D. KervBgant, “Rhums et eaux-de-vie de came,” Lea Editions du Golfe, Vannes, France, 1946. (130) M. L. Wolfrom, W. W. Binkley an&IFlorinda 0. Bobbio, El Crisol, i n press (1953). (131) E. 0. von Lippmann, Ber., 68, 426 (1925). (132) J. H. Payne, H. P. Kortachak and R. F. Gill, Jr., Ind. Eng. Chem., 44, 1411 (1952).

313

COMPOSITION OF CANE JUICE AND CANE FINAL MOLASSES

TABLEV Cane Final Molasses Constituents Component

Carbohydrates Sucrose Reducing sugars " D-Glucose D-Fructose myo-Inositol Phytin D-Mannitol Uronic acids Methoxyl Sugar "reaction products" Vitamins Biotin Folic acid Nicotinic acid Pantothenic acid Pyridoxine Riboflavin Thiamine Nitrogen Compounds Total nitrogen

Amino Acids Alanine 7-Aminobutyric acid Asparagine Aspartic acid Glutamic acid Glycine Leucine (or isoleucine) Lysine Valine Nucleic acid bases Guanine Hypoxanthine 5-Methylcytosine Xanthine

Molasses Origin"

Concentration, Reference

%'

La. Cuba La. Cuba La. Cuba Cuba Cuba Cuba Cuba Cuba La. La. La. Cuba

80.52b 78.90b 37.39 47.28 32.72 20.98 6.9 1.6 0.261 0.225 0.6 2.0 0.8 10.1 10.7

107 107 88 88 21 21 21 15 15 107 107

Cuba Cuba Cuba Cuba Cuba Cuba Cuba

17.0 X 0.43 X 222 x 635 X 19.1 x 24.4 X 8.5 X

111 111 111 111 111 111 111

Cuba Fla. Hawaii La. P. R. Fla. Fla. Fla. Fla., Hawaii Fla., Hawaii Fla. Fla. Hawaii Fla. Hawaii Hawaii Hawaii Hawaii

0.89 1.40 0.71 0.38 1.16

-

-

-

-

107 107 107

107

10-6 lo-& 10-6

lo-'

108 108 108 108 108 43 43 43 43, 113 43, 113 43 43 113 43 113 113 113 113

314

W. W. BINICLEY AND M. L. WOLF’ROM

TABLBV (Continued) Molasses Origin“ Cuba La. La. P. R. P. R. P. R. P. R. P. R. P. R.

Component Non-nitrogenous acids

Aconitic Malio Citric Formic Lactic Acetic Bacteria Mesophilic Thermophilic Pigments Chlorophyll a “Browning products” Tannins Anthocyanins Waxes, sterols and lipids Melissyl alcohol Phytosterol Stigmasterol Syringic acid Odorants Molasses odor fraction Inorganic components

-

Concentration, %7

7.59 7.39 4.95 0.95

-

0.12 0.60 0.24

Reference 107 107 133 119 119 119 119 119 119

La. La.

300-310,0OOc 1,200-16,5OOc

120 120

Cuba La., Cuba La. La. Cuba Cuba Cuba Formosa ( 1 ) Formosa (?)

5 x 10-6 10.1-10.7

70 107 34 34 70 70 70 127 127

-

0.50 -

-

Cuba, Hawaii La. 13.46d Cuba 13. 76d

6 Fla. = Florida; La, = Louisiana; P. R. Puerto Rioo. ber of bacteria per ml. of molasses. d Carbonate ash.

b

70, 130 107 107

Total aotids in the molasses.

Num-

ADDENDUM

Non-nitrogenous Acids in Cane Juice Application134to Louisiana cane juice of the procedure of Ramsey and Patterson,136as modified by Marvel and Itands,64 leads to the following acid assay: aconitic, 2.07 % ; malic, 0.28% ; citric, 0.22%; mesaconic, 0.058%; succinic, 0.04%; fumaric, 0.023 %; glycolic, oxalic and an unknown acid, present. WigginszbShas tentatively identified, by paper chromatography, succinic, malic, aconitic, citric, glycolic, and possibly glyoxalic acids. (133) C. A. Fort, B. A. Smith, C. L. Black and L. F. Martin, Sugar, 47, No. 10, 33 (1952). (134) E. J. Roberts, C. A. Fort and L. F. Martin, Abstracts Papers Am. Chem. SOC.,124, 1OD (1953). (135) L. L. Ramsey and W. I. Patterson, J . Assoc. Ofic. Agr. Chemists, 28, 644 (1945).

SEAWEED POLYSACCHARIDES

BY T . MORI* Tokyo University. Tokyo. Japan

CONTENTS I . Introduction . . . . . . . . . . . . . . . . . . . . . . . I1. Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . 316

317 318 b cGalactose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 c . 3,6-Anhydro-~-galactose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 d . Ester Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 e . Other Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 2. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 I11 . Mucilage of Dilsea Edulis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 1. Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 2. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 IV . Carrageenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 1. Cold-water Extract and Hot-water Extract . . . . . . . . . . . . . . . . . . . . . . . . . 330 a Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 b . Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 2. Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 a . D-Galactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 b . IcGalactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 c. “Ketose” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 d . D-GIucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 e . 2-Keto-~-gluconicAcid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 f . Pentose and Methylpentose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 g . Summary of Carbohydrate Constituents . . . . . . . . . . . . . . . . . . . . . . . 335 h . Ester Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 3. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Hydrolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 V. Fucoidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 1. Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 2. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 VI . Laminarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 . . . . . . . . . . . . . . . . . . . . . . . 344 1. Composition . . . . . . . . . . . . . . . . . . 2. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 3. Hydrolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 ............................................ ............................................

.

.

* Contribution from the Laboratory of Fisheries Chemistry of the Faculty of Agriculture of the University The writer wishes to express his sincere thanks to Professor M . L Wolfrom for his kind revision of the manuscript; to D r Kimiko Anno for checking the references; and to D r E Kylin and to Professors V . C . Barry, T. Dillon and C . Araki for sending reprints of their publications . 316

.

.

. .

.

316

1. MORI

VII. Other Polysaccharides. . . . . . . . . . . . 1. Mucilage of Dumontia Incrassatas . . . . . .. . . . . . . . . . . . ... 347 2. Algal Cellulose ...................... 348 3. Algal Xylan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Alginic Acid.. . . . . . ........................ 349 5. Floridean Starch.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Cyanophycean Starch.. .......................... 350

I. INTRODUCTION Seaweeds are classified into four groups: the Chlorophyceae or green algae; Phaeophyceae or brown algae; Rhodophyceae or red algae; and Cyanophyceae or blue-green algae. Of these, Phaeophyceae and Rhodophyceae are the more significant from the viewpoint of output and utilization. The major components of seaweed are carbohydrate in nature and thus the use of seaweed in food and industry is due t o its carbohydrate content and especially t o such polysaccharides as agar, algin, carrageenin and funorin’ (the polysaccharide of Gloiopeltis). In some cases seaweed is utilized for the manufacture of iodine and potassium chloride. Chemical studies on seaweed have centered mainly on their carbohydrates, especially those of Phaeophyceae and Rhodophyceae where material is in good abundance. Seaweed polysaccharides may be differentiated into reserve polysaccharides, such as laminarin and Floridean starch, and structural polysaccharides. Except for cellulose, the latter are mainly mucilages serving as membrane-thickening materials. Examples of these are algin, carrageenin, fucoidin and funorin. With the exception of algin, these mucilage polysaccharides are characterized by their combined sulfate content, first reported by Ham2 in carrageenin. Little progress in knowledge of the structure of these polysaccharides was made until Percival and Somerville3 succeeded in methylating agar and isolating 2,4,6-trimethyl-~-galactose, proving that D-galactopyranose units are joined C1-+ C3. This was also the first demonstration that D-galactopyranose units could form such a polysaccharide linkage. Thereafter many studies have been carried out on seaweed polysaccharides by Percival and by other investigators. Nevertheless, the structure of none of the polysaccharides is fully clarified. This is due t o their complexity, caused by the presence of several sugars and, save for algin and laminarin, of combined or ester sulfate. A carbohydrate sulfate ester is difficult to manipulate experimentally because of the extreme ease with which it (1) C. K. Tseng in “Colloid Chemistry” (J. Alexander, editor), Rheinhold Publishing Corp., New York, 1946, Vol. VI, p. 629. (2) P. Haas, Biochem. J . , 16, 469 (1921). (3) E. G. V. Percival and J. C. Somerville, J . Chem. Soc., 1015 (1937).

SEAWEED POLYSACCHARIDES

317

undergoes desulfation and carbonization in acid media. Although the difficulties are formidable, the elucidation of the structure of these polysaccharides is a desirable objective. We may include among these polysaccharides the substance fucoidin, recently discovered by Vasseur' in the jelly coat of the sea-urchin.

11. AGAR In Florideae, one of the subclasses of Rhodophyceae, the cell wall is composed of an inner layer consisting mainly of cellulose and an outer layer of pectic substances, probably of a complex nature. Commercial agar is derived from the pectic layers of some species of Florideae such as Gelidium Amansii, G . cartilagineum (U. S. A.), G . japonicum, G . pacijicum, G . subcostatum, Acanthopeltis japonica, Ceramium Hypnaeoides, and Graeilaria conferuoides. Since Japanese agar has a world distribution, is often used as research material, and since chemical changes in the agar molecule may be introduced in its manufacture, this process as used in Japan will be briefly described. Japanese Method of Agar Manufacture.-After soaking and washing, the agar-bearing seaweeds or " agarophytes," mentioned above, are boiled with 25 times their weight of water for 2 hours, followed by the addition of 1 part of concentrated suIfuric acid per 500 parts of raw material and additional heating at about 80"for 14 hours. Approximately 3 hours before the solution is removed from the heating tank, 1 part of sodium hydrosulfite per 50 parts of agarophytes is introduced for bleaching. The pH of the solution is 5-6 just after the sulfuric acid is added and is 6-7 at the end of the heating. The liquid is filtered and the agar gel obtained on cooling is cut into suitable shape, frozen during the night and then thawed during the day, whereupon the water in the agar gel flows away, carrying with it water-soluble impurities. After sun drying, commercial agar results. 1. Composition Payen6 was the first to study the carbohydrate of agarophyte. He termed it gelose and assigned to it the formula CaHloOs. In 1884 Bauers found it to be a galactan. Neuberg and Ohle7discovered the presence of ester (ethereal) sulfate in commercial agar although prior to this Haas2 had proved its presence in carrageen mucilage or carrageenin. Recently, (4) E. Vasseur, Acta Chem. Scand., 2 , 900 (1948). (5) A. Payen, Compt. rend., 49, 521 (1859). (6) R. W. Bauer, J . prakt. Chem., [2] 30, 367 (1884). (7) C. Neuberg and H. Ohle, Biochem. Z.,126, 311 (1921).

318

T. MORI

Hands and Peat,* and Percival, Somerville and Forbesg isolated from methylated agar a compound which was found to be a derivative of 3,6-anhydro-~-galactose.The components of agar and their relative amounts will be described in some detail in the following sections. a. D-Galactose.--Yanagawa'O prepared crude agar from six species of agarophyte by extracting the raw materials with hot water, precipitating the agar with alcohol, and repeating the dissolution and precipitation twice. He found that this crude agar contained 65.2% of sugar as hexose. Galactose assays by the mucic acid method ranged from 37% in the agar from Gelidium subcostatum to 48% in that from Gracilaria conferuoides, while commercial agar gave a value of 41%. Araki'l prepared crude agar from Gelidium Amansii, the most important agarophyte, by the procedure of Yanagawa, except that he precipitated his product with acetone rather than with ethanol. The crude agar thus obtained, though contaminated with some nitrogenous compounds (N, 0.89 %), contained 67% of hexose assaying 32% galactose. Hayashi12found 30% of galactose in a more impure preparation from G. Arnansii. Percival and Somerville3isolated 65 % of a trimethyl-D-galactose (as its crystalline methyl glycoside anomeric mixture) from methylated commercial agar. From these results it may be concluded that D-galactose composes about two-thirds of the sugar content of agar. b. L-Galactose.-The presence of D,L-galactose in Porphyra laciniata, one of the most important seaweed foods of Japan, was reported by Oshima and Tollens13 as early as 1901. Piriel* isolated a crystalline derivative of D,L-galactose from commercial agar. L-Galactose was obtained by an enzymic procedure by Araki16from the agar of G. Amansii, G. subcostatum and Acanthopeltis japonica. According to Pirie,I4 agar contains 0.8% of L-galactose. (8) S. Hands and S. Peat, Nature, 142, 797 (1938); Chemistrg & Industry, 16, 937 (1938). (9) E. G . V. Percival, J. C . Sornerville and I. A. Forbes, Nature, 142, 797 (1938); E. G. V. Percival and I. A. Forbes, ibid., 1076 (1938); I. A. Forbes and E. G. V. Percival, J . Chem. SOC.,1844 (1939). (10) T. Yanagawa, Repts. Imp. Ind. Research Inst., Osaka, Japan, 14, No. 5, I (1933); Chem. Abstracts, 30, 3541 (1936). (11) C . Araki, J . Chem. SOC.Japan, 68, 1214 (1937); Chem. Abstracts, 32, 4172 (1938). (12) K. Hayashi, J . SOC.Trop. Agr. Taihoku Imp. Univ., 14, 5 (1942); Chem. Abstracts, 42, 5425 (1948). (13) K. Oshima and B. Tollens, Ber., 34, 1422 (1901). (14) N. W. Pirie, Biochem. J . , SO, 369 (1936). (15) C . Araki, J . Chem. SOC.Japan, 69, 424 (1938); Chem. Abstracts, 36, 7946 (1941).

SEAWEED POLYSACCHARIDES

319

c. 5,6-Anhgdro-~-galactose.-Takao~~ concluded that agarophyte contained fructose because it gave the Seliwanoff ketose reaction. On the other hand, Hands and Peatla and Percival, Somerville and for be^,^ obtained methyl 3,6-anhydro-2,4-dimethyl-P-~-galactoside (I) in crystalline form on further methylation of hydrolyzed methylated commercial

H

H I

agar. The melting point, single crystal x-ray diffraction photograph, and specific rotation demonstrated that I was enantiomorphous with the corresponding member of the D-series synthesized by Haworth, Jackson and Smith.'? This 3,g-aahydro derivative of galactose exhibits the Seliwanoff reaction with resorcinol and the Bredereck reaction with ammonium m ~ l y b d a t e . ~ The substances considered to be fructose by Takao and others may then well be 3,6-anhydro-~-galactose. Araki and from the Arai'* also isolated 3,6-anhydro-2,4-dimethyl-~-galactose methylated agar of Gelidium Amansii. Now the question arises whether 3,6-anhydro-~-galactosepre-exists in agar or is formed from an L-galactose sulfate component during methylation in the alkaline medium. Anhydro ring formation is known t o be a possibility in the alkaline hydrolysis of some sugar s u l f a t e ~ . 1 ~ -Jones ~~ and Peat23 consider it logical t o assume that during the methylation process the 3,G-anhydro ring appears in the L-galactose component on sulfate removal. This view is supported by the fact that Percival and c o w ~ r k e r s obtained * ~ ~ ~ ~ 3,6-anhydro-~-galactosefrom barium D-galactose sulfate in a manner analogous to its formation from the corresponding 6-p-toluenesulfonate. However, the washed agar employed in these experiments contained only a trace (0.1%) of sulfurgso that the amount (10-13 %) of 3,6-anhydro-2,4-dimethyl-~-galactose produced from meth(16) Y. Takao, Repts. Central Inst., Government of Formosa, 6, 13 (1918). (17) W. N. Haworth, J. Jackson and F. Smith, Nature, 142, 1075 (1938); J . Chem. Sac., 620 (1940). (18) C . Araki and B.Arai, J . Chem. Sac. Japan, 61, 503 (1940); Chem. Abstracts, 87, 89 (1943). (19) E . G . V. Percival and T. H. Soutar, J . Chem. Sac., 1475 (1940). (20) R. B. Duff and E. G. V. Percival, J . Chem. Sac., 830 (1941). (21) E. G. V. Percival, J . Chem. Sac., 119 (1945). (22) E. G. V. Percival, J. Chem. Sac., 1675 (1947). (23) W. G. M. Jones and S. Peat, J . Chem. Soc., 225 (1942).

320

T. MORI

ylated agar is not accounted for by such a desulfation process. Barry and DillonZ4prepared agar from bleached Gelidium latifolium by the same procedure as described previously for commercial agar except that the sodium hydrosulfite addition was omitted; the product contained 0.364 % S. PercivalZ6likewise prepared agar from Gracilaria confervoides and Gelidium crinale by a similar mild treatment and found 0.43% S and 0.47% S, respectively, in the products. Thus, none of these preparations would have contained sufficient sulfate residue, if it were all L-galactose 6-sulfate, to account for the amount of methy1 3,6-anhydro-2,4-dimethyl19-L-galactoside obtained. Barry, Dillon and McGettrick2Enoted that agar was essentially not attacked by periodic acid and that therefore no a-glycol groups were present in its structure. This demonstrates that 3,6-anhydro-~-galactose is a constituent of agar, since, if this unit were produced from L-galactose components in which C3 did not take part in a linkage, such L-galactose residues would present a-glycol units for periodate attack. ArakiZ7isolated a new disaccharide, agarobiose, from the agar of Gelidium Amansii. The agar was partially hydrolyzed with N sulfuric acid for one hour a t loo", neutralized, concentrated and precipitated by ethanol. The ethanol-soluble fraction was fermented with Saccharomyces sake and the non-fermented residue was isolated and crystallized from ethanol. The disaccharide, [(Y]D -5.8" in water, showed a strong Seliwanoff reaction and was sulfate-free. It was further characterized - 135.5' + - 111" in pyriby its phenylosazone (m. p. 218-219", dine-ethan~l~'~) and hexabenzoate (m. p. 142", [ a ] D -80.3' in chloroform). From methylated agarobiose Araki28isolated 3,6-anhydro-2,5-dimethylL-galactose dimethyl acetal (11). Since the disaccharide contained no

I1

sulfate group, this anhydro derivative could not have been formed by a (24) V. C . Barry and T. Dillon, Chemistry & Industry, 22, 167 (1944). (25) E. G. V. Percival, Nature, 164, 673 (1944). (26) V. C. Barry, T. Dillon and W. McGettrick, J . Chem. Soc., 183 (1942). (27) C. Araki, J . Chem. SOC.Japan, 66, 533 (1944); Chem. Abstracts, 42, 1210 (1948). (27a) C. Araki, private communication. (28) C. Araki, J . Chem. Soe. Japan, 66, 627 (1944); Chem. Abstracts, 42, 1210 (1948).

321

SEAWEED POLYSACCHARIDES

desulfation reaction. According t o E. E. P e r c i ~ a la, ~galactose ~ sulfate, pre-existent in agar, could not have produced a 3,6-anhydro ring on being subjected to the acid treatment employed by Araki in his isolation of agarobiose. In addition, Arakiao isolated 3,6-anhydro-~-galactose H

O

f

F

ActO

A c O C HP M e

E

H

H

H

I11

= H

Ha504 Ao,O

A

IV

OAc

c

O

C

P

H

H

H

OH

-

H

/

CHO

U

OAc

1

;c:$3-] OAc VII 1 . 0

CH~OAC AH co