ADVANCES XN CARBOHYDRATE CHEMISTRY VOLUME 10
This Page Intentionally Left Blank
t
Carbohydrate Chemistry Editor ME...
34 downloads
1699 Views
22MB Size
Report
This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form
ADVANCES XN CARBOHYDRATE CHEMISTRY VOLUME 10
This Page Intentionally Left Blank
t
Carbohydrate Chemistry Editor MELVILLE L. WOLFROM Arsistant Editor R. STUART TIPSON Board of Advisors C. B. PURVES J. C. SOWDEN ROYL. WHISTLER
HERMANN 0. L. FISCHER
It. C. HOCKETT
W. W. PIGMAN
Board of Advisors for the British Isles J4. L. HIRST
STANLEY PEAT
MAURICESTACEY
Volume 10
1955
ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N. Y.
Copyright, 1955, by ACADEMIC PRESS INC. 125 East 23rd Street New York 10, N. Y.
A12 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
PRINTKD I N T H E UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME 10 W. W. BINKLEY,Department of Chemistry, The Ohio State University, Columbus, Ohio*
G. P. ELLIS,Department of Chemistry, King's College, University of London, England A. B. FOSTER,Department of Chemistry, The University, Edgbaston, Birmingham, England
L. J. HAYNES,The University, Edinburgh, Scotland ,JOHN E. HODGE,Northern Utilization Research Branch, Agricultural Research Service, U . S . Department of Agriculture, Peoria, Illinois JOHN HONEYMAN, Department of Chemistry, King's College, University of London, England A. J. HUGGARD, Department Columbus, Ohio
0.f
Chemistry, The Ohio State University,
GEORGEG. MAHER,Research Laboratories, Clinton Foods Inc., Clinton, Iowa J. A. MILLS, Division of Biochemistry and General Nutrition, Commonwealth Scientific and Industrial Research Organization, Adelaide, Australia
F. H. NEWTH,The University, Cambridge, England W. J. POLGLASE, Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, Canada 'Present address: The New York Sugar Trade Lahoratorg Inc., 113 Pearl Street, N e w York
V
4 , N . 1'.
This Page Intentionally Left Blank
PREFACE With this Volume, “Advances in Carbohydrate Chemistry” comes of age through the completion of a series of ten issues. Herein, modern conformational analysis is applied to the carbohydrate field by J. A. Mills (Adelaide), The ever-recurrent and always puzzling subject of nitrogen chemistry is elaborated in a discussion of the glycosylamines and their rearrangement products by G. P. Ellis with J. Honeyman (London) and by J. E. Hodge (Peoria). The preparation and reactivity of the useful glycosyl halides is presented by L. J. Haynes (Edinburgh) and F. H. Newth (Cambridge). W. W. Binkley (Columbus) summarizes the present status of column chromatographic technique as applied to the sugar group. Our series of chapters on the methyl ethers is augmented by G. G. Maher (Clinton, Iowa). Polysaccharide chemistry is represented by a chapter on the non-cellulosic components of wood from the pen of W. J. Polglase (Vancouver) and by one on the biochemically significant subject of heparin from A. B. Foster and A. J. Huggard (Birmingham and Columbus). These, together with an obituary of the late, esteemed Dr. E. G. V. Percival of Edinburgh, complete this Volume and are offered as a contribution to the summarizing of progress in the ever-growing subject of carbohydrate chemistry.
M. L. WOLFROM Columbus, Ohio
vii
This Page Intentionally Left Blank
CONTENTS CONTRIBUTORS TO VOLUME 1 0 . .. . . . . . . , , . . , . . . . . . . . , , . . . , . . . . . . . . . . . . . . . . . . . . .
v
PREFACE.. . . . . . . , . _ _, . . . . , , . . . . , , . . . . . . . . . . . , . . , , . . . . . , . . , , . . . . . . . . . . . . . . . . . vi EDMUNI) GEORGE VINCENT
!?ERrIVAL. . . . . . . . . .
. , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . X ... lll
The Stereochemistry of Cyclic Derivatives of Carbohydrates BY J. A. MILLS,Department of Biochemistry and General Nutrition, Commonwealth Scientijic and Industrial Research Organization, Adelaide, Australia
I. Introduction . . . . . . . . . . . . . , . , . . , . _ _. ._ _ ,, . . . . . . . . . . . . . . . . . . . . . . _ _. . . . . . 2 11. Nomenclature and Methods of Illustration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 111. Steric Requirements of Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 IV. Fundamental Properties of Rings.. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 V. Cyclic Acetals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 VI. Anhydro Compounds.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Column Chromatography of Sugars and Their Derivatives BY W. W. BINKLEY, Department of Chemistry, T h e Ohio Stale University, Columbus, Ohio
I. Introduction . . . . _ _, . . . . , . . . . . . . . . . . , . . . . . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . 55 11. Adsorbents for the Column Chromatography of Sugars, Sugar Alcohols, 56 and Sugar Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Column Chromatography of Sugar Derivatives. . . . . . . . . . . . . . . . . , . . . . . . . 79 Glycosylamines BY G. P. ELLISAND JOHNHONEYMAN, Department of Chemisfry, King's College, University of London, England I. 11. 111. IV. V. VI. VII. VIII. IX.
Introduction ..... _ _. . . _ . . . . . . _ .. . . . . , , . . . . . . . . , . , . . . . . . . . . . . . . . . . . . . . Nomenclature . . . . . . . . . . . . . . . . . , . . . . . , . . . . _ .. _ _ _ . . . . _ .. _ . ._ _ _ . _ _. . .... Preparation . . . . , . . ,.. , . . . . . . . . . . . . , . . . . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . . Physical Properties.. . . . . . . . . . , . . . . . . . . . , . , , . . . . . . , , , . . . . . . . . . . . . . . . . . . Structure. . . . _ .. , . . _ .. _ ., . . _ _ . _. .. . . . . . . . . . . . , . . . _ .. . . . . . _ . . . . . . . . _. . . Diglycosylamines . . , . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diitmide Derivatives of Aldoses.. . . , . . . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses of Glycosylamines.. . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tables of Properties of Glycosylamines.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,
95 96 97 101 102 120 121 124 125
The Amadori Rearrangement BY JOHN E. HODGE,Northern Utilization Research Branch, Agricultural Research Service, 7J. S . Department of Agriculture, Peoria, Illinois
I. Introduction... ............... . . ... ... ... . .......... . . . . . . . . . . . . . . . . . . . 169 ix
CONTENTS
X
I1 . Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Scope of the Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Mechanism of the Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Physical Properties of Amadori Rearrangement Products . . . . . . . . . . . . . . . VII . Chemical Properties of Amadori Rearrangement Products . . . . . . . . . . . . . . . VIII . Proof of Structure of Amadori Rearrangement Products . . . . . . . . . . . . . . . . IX . Retrospect and the Future . . . . . . . ..................... .. X . Tables of Compounds . . . . . . . . . . . . ............................
172 173 175 178 185 187 199 201 203
The Glycosyl Halides and Their Derivatives BY 1, . J . HAYNES, l’he University, Edinburgh, Scotland A N D F . H . NEWTH, The l J n i v w s i t y , Cambridge, England I . Introduction., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Preparation of Glycosyl Halide Derivatives . . . . . . . . . . . . . . . . . . . . . . . . 111. Structure of Glycosyl Halides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 IV . General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 V . Reaction Mechanism and Effect of Structure on Reaction Rates . . . . . . . . 234 V I . Reactions of the Poly-0-acylglycosyl Halides . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 VII . Tables of Properties of Some Glycosyl Halide Derivatives . . . . . . . . . . . . . . 246 The Methyl Ethers of the Aldopentoses and of Rhamnose and Fucose BY GEORGEG . MAHER,Research Laboratories, Clinton Foods I n c . , Clinton, Iowa Table I . ,. . . . . . . . . . . . . . . . . . . . . . . . . . ...................................... 257 Table I1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 Table I11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 Table IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Table V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 Table V I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 Table V I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 Table VIII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 The Methyl Ethers of D-Galactose BY GEORQEG . MAHER,Research Laboratories, Clinton Foods I n c . , Clinton, Iowa Table Table Table Table
I......................................................................
I1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
273 274 277 279
Polysaccharides Associated with Wood Cellulose BY W . J . POLGLASE, Department of Biochemistry, University of British Columbia, Vancotcuer, British Columbia, Canada .................................... I . Introduction. . . . . . . . . . . . . . . . . I1 . Carbohydrate Constituents of Wood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11 . Carbohydrates in Wood Cellulose Preparations . . . . . . . . . . . . . . . . . . . . . . . . . IV . Preparation and Composition of Wood Cellulose . . . . . . . . . . . . . . . . . . . . . . . . V . Fine Structure of Wood Cellulose and Associated Polysaccharides . . . . . . .
283 285 287 316 328
CONTENTS
xi
The Chemistry of Heparin BYA . B . FOSTER. Chemistry Department. T h e University. Birniingh.am. England A N D A . J . HUOGARD. Chemistry Department. T h e Ohio State University. Columbus. Ohio I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 I1. The Discovery of Heparin., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 111. The IV . The V . The VI . The
Isolation and Purification of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 . . . . . . . . 348 Structure of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anticoagulant Activity of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 Biosynthesis of Mucopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
AUTHORINDEX .................................... SUBJECT
I N D E X .........................
CONTENTS O F
VOLUMES
1-9
........................................
. . . . . . . . 369
. . . . . . . . 391
........ 435
This Page Intentionally Left Blank
EDMUND GEORGEVINCENTPERCIVAL 1907-1 951 The death of E . G. V. Percival a t the early age of 43, when he was a t the height of his powers as a scientist, was a severe blow to carbohydrate chemistry. He was born on 10th November, 1907, in the Midlands of England a t Hinckley in the County of Leicester, being the younger son of Albert Henry and Elizabeth Percival. As a boy, he attended the King Edward VII Grammar School in the neighboring town of Coalville. His school record showed great promise, and in 1925 he entered the University of Birmingham where he studied for the honours degree in chemistry. His undergraduate career was one of unusual brilliance, and it was a foregone conclusion that he would obtain a First Class Honours in Chemistry a t the end of the course in 1928. A research career was clearly indicated for him, but, before choosing a field in which to specialize, Percival wisely decided to gain as wide an experience as possible. I n the fall of 1928, he joined William Wardlaw’s group of workers in the Birmingham University laboratories and took part in work on the chemistry of metallic co-ordination compounds. A year later, he gained a Research Fellowship awarded by the Canadian Pulp and Paper Association, tenable a t McGill University, Montreal, and there, in Harold Hibbert’s laboratory, he investigated various physicochemical problems concerned with addition compounds of cellulose. Before returning to England, he made an extensive tour of research centers in Canada and the United States, gaining much experience which was of value to him in his later work, and making, as was the case wherever he went, many lasting friendships. The experience in Montreal had given him a deep interest in carbohydrate chemistry, and this became his main field of work 011 his return to Birmingham University in October, 1930, as senior research assistant to W. N. Haworth (later Sir Norman Haworth). This period was one of rapid and successful progress, and a t this stage he decided to seek a post which would enable him to pursue a career in the academic world. The opportunity came three years later (1933), when he was appointed to a lectureship in organic chemistry in the University of Edinburgh. Here, in the Department presided over by James P. Kendall, F.R.S., Percival settled happily, and during the next 18 years he built up a research school in carbohydrate chemistry which had an international reputation. He found conditions so congenial in Edinburgh that he made no serious attempt to gain xiii
xiv
EDMUND GEORGE VINCENT PERCIVAL
promotion by moving elsewhere. In 1934, he married Ethel Elizabeth Kempson, herself a chemist, a graduate of the University of Birmingham where she had been a student in Sir Norman Haworth’s laboratories. After her arrival in Edinburgh, she took an active part in the development of Percival’s research activities, and assisted in the teaching of undergraduates. She and their two children survive him, and Mrs. Percival is COIItinuing, as a member of the University staff, some of the work initiated by her husband. Percival entered fully into the scientific and social life of both the University and the City of Edinburgh. He took a keen and sympathet,ic interest in student welfare and activities, and amongst his University duties was that of Director of Studies. He was elected t o membership in the Faculty of Science, and in 1948 was promoted to the status of Reader in the University. Shortly before his death he had been chosen to serve as a member of the Senatus Academicus. He gave his time and energy, with unselfish enthusiasm, to work for various scientific societies. For many years he had been interested in the work of the Pharmaceutical Societ,y and in British Chemical Abstracts. H e was concerned with work for the Institute of Brewing, he was one of the original pioneers in investigations on the chemistry of marine algal polysaccharides sponsored by the Scottish Seaweed Research Association, and he was an active member of this Association’s Chemical Advisory Committee. He served also on the local district committees of the Royal Institute of Chemistry and of the Society of Chemical Industry, and in addition acted for two years as Chairman of the Edinburgh and South East Scotland section of the Royal Institute. He was the local representative in Edinburgh of the Chemical Society (London), and at the time of his death was a member of the Society’s Council and of its Committee on Carbohydrate Nomenclature. His help was called for on all special occasions, and in 1948, when the Society of Chemical Industry held its annual general meeting in Edinburgh, and again in 1951, when he was Local Secretary for the Chemistry Section of the British Association for the Advancement of Science, his unselfish and unstinted efforts contributed in large part to the success of the meetings. It was characteristic of Percival that, to everything he undertook, he brought enthusiasm and intensity of purpose, whether it was in playing tennis or cricket, discussing painting (in which art he was deeply knowledgeable), walking on the Cheviot Hills, or in teaching and research. He had a brain which acted with lightning rapidity, and he had the power to carry through with accuracy (and with every attention to detail) a phenomenal amount of work in a very short time. He was a clear lecturer and expositor, and in consequence he was frequently invited to visit other universities and chemical societies. These visits, especially those which in-
EDMUND GEORGE VINCENT PERCIVAL
xv
volved traveling, he found highly congenial. He was a t his best as a teacher with advanced students of quick perception, who could follow without difficulty the logical but rapid development of his theme. Friendly, generous, sympathetic, and approachable, he was an inspiring leader for postgraduate research workers, who found him full of ideas and suggestions, and marveled a t his encyclopedic memory for details. All who had the privilege to work with him and enjoy his friendship will remember especially his kindliness, generosity, and unselfishness, his deep sense of loyalty, and his capacity to see and appreciate the humorous side of everyday happenings. Percival's outstanding ability was soon recognized, and awards came to him a t an early age-Fellowship of the Royal Institute of Chemistry in 1936, the D.Sc. degree of the University of Edinburgh in 1938, and, in 1941, election t o the Fellowship of the Royal Society of Edinburgh. Although Percival's main interest in chemistry lay in the carbohydrate field, his earliest research, carried out under William Wardlaw in Birmingham (1928-1929) was concerned with polynuclear co-ordination compounds of cobalt with amines.' During the following year, he held a Research Fellowship in Montreal, working with Hibbert on the constitution of sodacellulose, the hydrolysis of polysaccharides, and the absorption of aluminum ions on cellulose. It was demonstrated that a chemical compound is formed between the sodium hydroxide and the cellulose, containing about 15 % of alkali (or one molecule to each anhydro-D-glucose unit). Investigation of the rates of hydrolysis indicated that the lower rate for cellulose as compared wit,h that of starch is a reflection of the comparative rates of hydrolysis of cellobiose and maltose.2 I n the following three years (1930-33), working with Sir Norman Haworth, Percival began his studies in structural carbohydrate chemistry, and took part in the early work of the Birmingham School on the investigation of polysaccharides. Some of the most important results he obtained were concerned with the structures of starch and glycogen. Up to this time, strictly chemical evidence was lacking as to the presence of preformed maltose units in starch and glycogen, and concerning the nature of the ring present in the D-glucose residues. Percival developed a method of simultaneous deacetylation and methylation of the acetylated polysaccharides followed by acetolysis of the methyl derivatives. These yielded, amongst other products, a partially methylated maltose. The hexa-0methylbiose was oxidized to the bionic acid and subjected to further methylation. The resulting methyl octa-0-methylmaltobionate was then (1) E. G . V. Percival and W. Wardlaw, J . Chem. Soc., 1317, 1505, 2628 (1929). (2) A. C. Cuthbertson, H . Hibbert and E. G. V. Percival, J. Am. Chenz. SOC.,62, 3257, 3448 (1930); H. Hibbert and E. G . V. Percival, ibid., 62, 3995 (1930).
xvi
EDMUND GEORGE VINCENT PERCIVAL
hydrolyzed and the tetra-0-methyl-D-glucopyranose and tetra-0-methylD-glUCOniC 1,4-lactone were separated and identified, thus showing that the maltose structure must be present in starch and glycogen. Similar methods were applied to a di-0-methylxylan, in which the methoxyl groups were found to be attached to C2 and C3, showing that the linkage occurs a t C4 and that the D-xylose is present in the pyranose forrne3The methylation and hydrolysis technique was further applied to inulin, the methylated derivative of which gave rise to rather labile D-fructofuranose derivatives. In order to avoid unnecessary decomposition, the hydrolysis (after methylation) mas carried out using aqueous methanolic oxalic acid, and the yield of tetra-0-methyl-D-fructofuranose indicated the presence of one terminal nonreducing D-fructofuranose unit in 32 residues? One of Percival’s major contributions during his Birmingham period was the part he played, in collaboration with other members of the School, in determining the constitution of ascorbic acid (vitamin C). Oxidation studies were carried out which showed t,hat L-threonic acid is one of the products of oxidation, indicating ascorbic acid to be a derivative of L-gulose. The nature of the ring system was investigated by methylation studies, and vitamin C was proved to be the enolic form of 2-keto-~-gulonic1 , 4 - l a ~ t o n e . ~ Confirmation was obtained for this structure by the synthesis of vitamin C by the same group of workers. A t this point in his career, Percival moved to Edinburgh (1933), where he continued his work in the carbohydrate field. During the succeeding 18 years, his numerous publications covered a wide variety of subjects. From among the early work in Edinburgh may be ment,ioned the study of the compounds of alkali-metal hydroxides with sugars and polysaccharides, as a result of which he suggested that the alkali-suga,r complexes are definite compounds, and indicated how, by methylation, the points of attachment of the added hydroxides could be determined. Compounds of alkali-metal hydroxides with mono-, di-, and poly-saccharides were investigated.6 The methylation studies resulted in the production of partially methylated sugars whose identification required reference compounds, and Percival proceeded to study various mono-0-methylhexoses not previously charac(3) W. N. Haworth and E. G. V. Percival, J. Chem. SOC.,1342, 2850 (1931); 2277 (1932). (4) W. N. Hanorth, E. L. Hirst and E. G. V. Percival, J. Chew. SOC.,2384 (1932). ( 5 ) R. W. Herhert, E. L. Hirst, E. G. V . Percival, R. J. W. Reynolds and F. Smith, J. Chem. SOC.,1270 (1933) ; R . G. Ault,, D. Iionas reference compounds.$ At about the same time, work was commenced on other algal (7) Elizabeth E. Pereival and E. G. V. Percival, J . Chem. SOC.,1398 (1935); 1320 (1937); 750 (1941); F:. G. V. Percival, ibid., 1770 (1936); 1384 (1938); 783 (1945); J. R. Muir and E. ‘2. V. Percival, ibitl., 1479 (1940); W. J. Heddle arid E. G. V. Percival, ibid., 1511 (1940). (8) H. T . Macpherson and E. G . V. Percival, J . Chem. SOC.,1920 (1937). (9) €3. G. V. Percival and J. C. Somerville, J. Che,m. SOC., 1615 (1937); I. A. Forbes and E. G. V. Percival, ibid., 1844 (1939); T. L. Cottrell and E. G. V. Percival, ibid., 749 (1942).
xviii
EDMUND GEORGE VINCENT PERCIVAL
polysaccharides, notably those forming the mucilaginous extracts of Chondrus crispus and Gigartina slellata, collectively known as Irish moss. The polysaccharides from the two algae are essentially similar, in that both contain a considerable number of ester sulfate groups and consist of galactopyranose residues linked through C1 and C3, a portion of the galactose being in the L-form in the Chondrus polysaccharide. The complete elucidation of the structure proved difficult owing to the presence of the sulfate groups which interfere with methylation.1° In view of these difficulties and of the need for more knowledge of carbohydrate sulfuric esters, Percival then investigated the sulfates of D-glucose and D-galactose, their rates of hydrolysis with reference to the position of the sulfate group in the molecule, and the formation of 3,6- and 5,6-anhydro sugars on alkaline hydrolysis.]' With the collaboration of the newly-formed Scottish Seaweed Research Association, Percival commenced a further series of investigations in the marine algal field,lla the earlier work being concerned with the development of specific methods of analysis for the various constituents present in seaveed. Procedures were worked out for the estimation of D-mannitol, alginic acid, laminarin, and fucoidin.12 At the same time, structural studies were instituted on the last three polysaccharides.*3 Laminarinlla was shown to consist essent>iallyof (3 1) linked P-D-glucose units, and to occur in two forms, a soluble and an insoluble, depending on the species of seaweed used for extraction. I n fucoidin,lla the principal sugar is L-fucose, along with sulfate groups, and a branched structure was proposed in which 2 + 1a-L links predominate. Among other seaweed polysaccharides, the xylan from Rhodymenia palmata was found to be of unusual interest in that it was shown to contain both (3 --f 1) and (4--+ 1) linked D-XylOSe residues as part of the main structure of the polysaccharide. Percival had had previous experience with xylans while investigating the structures of the mucilages
-
(10) J. Buchanan, Elizabeth E. Percival and E. G. V. Percival, J . Chem. SOC., 51 (1943); E. T. Dewar and E. G. V. Percival, ibid., 1622 (1947); R . Johnston and E. G. V. Percival, ibid., 1994 (1950). (11) Elizabeth E. Percival and E. G. V . Percival, J . Chem. Soc., 1585 (1938); 874 (1945); E. G. V. Percival and T. H. Soutar, ibid., 1475 (1940); R. B. Duff and E. G. V. Percival, ibid., 830 (1941); 1675 (1947); E. G. V. Percival, ibid., 119 (1945); R. B. Duff, J . Chem. SOC.,1597 (1949). (Ila) See also, T. Mori, Advances in Carbohydrate Chem., 8, 315 (1953). (12) M. Christine Cameron, E. G. V. Percival and A. G. Ross, J . SOC.Chem. Znd. (London), 67, 161 (1948); E. G. V. Percival and A . G. Ross, ibid., 67, 420 (1948); W. A. P. Black, W. J . Cornhill, E. T. Dewar, E G. V. Percival and A. G . Ross, ibid., 69, 317 (1950). (13) E. G. V. Pereivsl a n d A. G . Ross, .I. Cl,eni. Sot-., 717 (1950); 720 (1951); J. Conchie and €2. G. V. Percival, itlid., 827 (1950) ; J . J. Connell, E. L. Hirst and E. G. V . Percival, ibid., 3494 (1950).
EDMUND GEORGE VINCENT PERCIVAL
xix
from t,he seeds of various meinhers of t'he plantain family (Plantago lanccolata, Plantago arenaria, and Plantago ov,uta). All t'hese sylans are complex in st,ructure and contain, in addit>ion t o D-xylose, ot,her pentoses, hexoses, and uronic acid residues; for example, the mucilage from Plantago arenaria contains D-xylose, L-arabinose, u-galactose, and wgalacturonic acid. The mode of' attachment of the uronic acid is not clear, and the struct,ure of the main D-xylopyranose chain includes various types of linkage and is highly branched. I n the mucilage from Plantago lanceolnta, for example, the following types of D-xylose residue were foundL4to occur as building unit,s: D-xylp 1, 3 D-xylp 1, 4 D-xylp 1, :D-xylp 1, iD-xylp 1, and :D-xylp ;. Further studies were carried out on the xylans from esparto and from pear cell-wall. I n the former, the xylan was shown to be free from L-arabinose and t o consist of a singly-branched molecule, the main chain containing about 75 D-xylopyranose units linked through the 4 position, the branching point occurring on C3. For the pear cell-wall polysaccharide, the general structure was found similar, but with the modification that t3he terminal residue consists of a D-glucuronic, acid unit and the main chain is rather longer (ca. 115 D-xylopyranose Percival also applied himself to the study of t.he polysaccharides from lichens, as, for example, tjhe complex product of alkaline extraction of Iceland moss (Cetraria islandica). As a result of this work, he concluded tJhat t,he polysaccharide consists of p-D-glucose residues united by various linkages: 1,2, 1,3, 1,4, and 1 , G , and includes also terminal D-galacto- and D-gluco-pyranose end groups. It was not possible to decide whether these linkages all occur in one polysaccharide.16 I n the later stages of his career, Percival published papers dealing with barley starch1? and wood starches,L8the former being found to be, in the main respects, similar to other cereal starches. Fructans also attracted his attention, and those from couch grass and perennial rye grass were particularly investigated; it was shown that the fructan from couch grass has both 2 , l and 2,G linkages in the molecule, whereas that from rye grass is essentJially a 2,G-linked, straight-chain p o l y s a ~ c h a r i d eFinally, .~~ mention may be made of the inulin from dahlia tubers, in which the presence of (14) J. Mullan and E. G. V . Percival, J . Chem. Soe., 1501 (1940); W . A. G. Nelson and E. G. V. Percival, i b i d . , 58 (1942); R. A. Laidlaw and E. G. V. Percival, ibid., 1600 (1949); 528 (1950); E. G. V. Percival and I. C. Willox, i b i J . , 1608 (1949). (15) S. K. Chanda, E. L. Hirst and E. G. V . Percival, J . Chem. SOC.,1240 (1951). (16) H. Granichstadten and E. G. V . Percival, J . Chem. SOC.,54 (1943). (17) I. C. MacWilliam and E. G. V . Percival, J . Chem. SOC.,2259 (1951). (18) W. G. Campbell, J. L. Frahn, E. L. Hirst, D. F . Pnckman and E. G . V. Perc i v d , J . Chem. SOC.,3489 (1951). (19) P. C . Arni and E. G. V . Percival, J . Chem. SOC.,1822 (1951).
xx
EDMUND GEORGE VINCENT PERCIVAL
u-glucose as an integral part of the molecule was demonstrated and possible structures were suggestedz0in which one n-glucose residue occurred as aii end group, linked to D-fructose by a sucrose type of link, whilst the second u-glucose residue was in the middle of the chain, linked through C1 and C3. Oiily a few days before his death, Percival took a prorninerit part in a conference 011 grass, sponsored by the Nutrition Society, a t which he read a p a p e P 011 the “Carbohydrate Coiistituerits of Herbage.” In this paper, he emphasized the importance of the fructaris as reserve carbohydrates in the metabolism of the plant, arid expressed the opinion that they would be found to play a considerable part, in the chemistry of the preservation of grass in the form of silage. E. L. HIRST A. G. Ross (20) E. I,. Hirst, D I. McGilvrag and E. G. V. Percival, J . Cheni. Soc., 1297 (1950). (21) E. G. V. Percivnl, Brit. J . Nutrztion, 6, 104 (1952).
THE STEREOCHEMISTRY OF CYCLIC DERIVATIVES OF CARBOHYDRATES* BY J . A . MILLS Division of Biochemistry and General Nutrition. Commonwealth Scientific and Industrial Research Organization. Adelaide. Australia
CONTENTS 1. 1ntroduct)ion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Nomenclature and Methods of Illustration . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Structural Formulas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 . Steric Requirements of Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Reversible and Irreversible Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Reactions with Specific Steric Requirements . . . . . . . . . . . . . . . . . . . . . I V . Fundamental Properties of Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Conformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Monocyclic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Five-membered Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Sis-membered Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . Other Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3. Structures Containing Fused Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Two Five-membered Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Two Sis-membered Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . One Five-membered and One Six-membered Ring . . . . . . . . . . . . . . . . . . . . d . Other Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Bridged Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . The Course of Irreversible Ring Closures . . . . . . . . . . . . . . . . . . . . . . . . . . V . Cyclic Acetals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Monocyclic Acetals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Five-membered Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Six-membered Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Acetals Containing Fused Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Fused Five-membered Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Fused Six-membered Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I . Anhydro Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The 1,4:3,6-Dianhydrohesitols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . 1 , 6-Anhydro Derivatives of Pyranose Sugars . . . . . . . . . . . . . . . . . . . . . 3 . The Scission of Epoxide Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~~~~~
~
~
~
*This review is based on literature available t o the author up t o July. 1954.
2 3 3 4 6 6 8 10 . 10 12 12 14 17 17 17 18 20 20 21 23 25 25 26 26 28 31 31
36 46 46 40 51
2
J. A . MILLS
I . INTRODUCTION The chief aim of this article is to make a comparison between carbohydrates and analogous alicyclic compounds, with emphasis on the stereochemistry of the ring systems involved. In alicyclic chemistry, notable additions have recently been made to our knowledge of the stereochemistry This new information of saturated rings, particularly six-membered already has had a considerable impact on carbohydrate chemistry, but has 6, The wider application attempted been restricted t o a few here will, it is hoped, assist in the coordination of considerable parts of the vohminous and diverse information available about carbohydrates, and may possibly lead to further development of stereochemical theory. I t is of interest to recall the development of the chemistry of terpenoids and steroids. Initially, these compounds were studied as problems in structural chemistry, but it was later realized14 that terpenoids of established structure provided first-class material for the study of the mechanisms of reactions, their availability in optically active forms being a notable advantage. Stereochemical theory has also greatly benefited in recent years from studies on steroids, largely because of the wider range of structural types available through the complexity of steroid molecules. Carbohydrates also possess the desirable features of optical activity, crystallinity, and availability, and display a diversity in structural types and reactions that cannot be matched in other fields. The writer, whose interests have lain mainly in the alicyclic field, has been greatly impressed by the scope that s t
(I) C. W. Beckett, K. S. Pitzer and R . Spitser, J . Am. Chem. Soc., 69,2488 (1947). (2) 0. Hassel and B. Ottar, dcta Chenr. Scand., 1, 929 (1947). (3) 0. Hassel, Research (London), 3, 504 (1950). (4) D. H. R. Barton, Experientia, 6, 316 (1950). (5) S. J. Angyal and J. A. Mills, Revs. Pure and A p p l . Chem. (Australia), 2, 185 (1952). (6) D. H. R. Barton, J. Cheni. Soc., 1027 (1953). (7) 0. Hassel, Quart. Revs. (London), 7, 221 (1953). (8) H. C. Brown, J. H. Brewster and H. Shechter, J . A m . Chern. Soc., 76, 467 (1954). (9) Further discussions are given by (a) R. B. Turner, in “Natural Products Related to Phenanthrene,” L. F. Fieser and Mary Fieser, eds., Reinhold Publishing Corp., New York, N. Y., 3rd Edition, 1949, p. 620; (b) V. Prelog, J . Chem. Soc., 420 (1950); (c) W. Klyne, in “Progress in Stereochemistry,” W. Klyne, ed., Academic Press Inc., New York, N. Y., Butterworths Publications Ltd., London, 1954, p. 36. (10) R . E. Reeves, J . A m . Chem. SOC.,72, 1499 (1950); and preceding papers in the series. (11) R. E. Reeves, Advances in Carbohydrate Chem., 6, 107 (1951). (12) R. J. Dimler, Advances in Carbohydrate Chem., 7, 37 (1952). (13) R. C. Cookson, Chemistry & Industry, 223 (1954). (14) See, for example, W. Huckel, Die Chemie, 66, 227 (1942).
CYCLIC DERIVATIVES OF CARBOHYDRATES
3
carbohydrate chemistry offers for elaborating and testing stereochemical hypotheses. Important contributions have, of cvursc, already come from this source, but usually as by-products of the development of carbohydrate chemistry, rather than through the deliberate choice of carbohydrate compounds as models for experimental study. To permit full advantage to be taken of the opportunities offered, a more detailed knowledge of the stereochemistry of cyclic carbohydrates is needed. The simple pyrariose and furanose sugars pose formidable problems of interactions between neighboring groups, but this difficulty largely disappears in their bicyclic and tricyclic derivatives. Accordingly, attentjion is confined mainly to compounds containing several rings, about which much information has been collected in recent reviews on cyclic acetalsI6 and anhydro I7 Research on these topics may seem to have lost some of its compounds,16~ initial impetus, but it will be seen that they still offer scope for further work of fundamental importance. Some of the conformational assignments are necessarily tentative a t present, and the opportunity is taken to point out directions in which more decisive evidence might be found. 11. NOMENCLATURE AND METHODS OF ILLUSTRATION 1 . Definitions
The convenient system of nomenclature for cyclic acetals introduced by Barker and Bourne15 will be used here. Rings formed by the engagement of hydroxyl groups attached to carbon atoms that are adjacent, separated by one atom, or separated by two atoms, are called a-,p-, or y-rings, respectively, and, if both hydroxyl groups are secondary, the designation C for two hydroxyl groups on t\he same side of the Fischer projection of the acyclic polyalcohol, or T for two groups on opposite sides, is added. Those parts of a polyol carbon chain not incorporated into an acetal ring, which appear as substituents in the ring, will be called residues. 2,4-O-Methylene-~-glucitolcontains a pC-ring, with the residues -CH20H and -CHOH-CH20H as substituents. The term fused rings wiH-bd used to denote the type of union found in decalin, where two rings have two atoms in common; the two atoms comprise a ring juncticn and are situated a t angular positions. Bridged rings are rings in which non-adjacent ring-atoms are joined by a bridge containing contains two one or more atoms. Methyl 3,6-anhydro-a-~-glucofuranoside (15) S.A. Barker and E. J. Bourne, Advances in Carbohydrate Chem., 7,137 (1952). (16) S.Peat, Advances in Carbohydrate Chem., 2, 37 (1946). (17) L.F.Wiggins, Advances in Carbohydrate Chem., 6, 191 (1950). (18) S. A. Barker and E. J. Bourne, J . Chem. SOC.,905 (1952).
4
J. A. MILLS
fused five-membered rings, whereas methyl 3 ,6-anhydro-a-n-glucopyranoside contains a bridged riug, and may be regarded as a six-membered ring bridged by two atoms or as a seven-membered ring bridged by one atom. 2. Structural Formulas
There is no settled convention for the representation of polyacetals and other complex cyclic derivatives of carbohydrate^.^^ To facilitate the comparison of polycyclic derivatives of carbohydrates with alicyclic compounds, the carbohydrate derivatives will be drawn in perspective, with single rings and fused rings placed in the plane of the paper, and with darkened or broken lines used to show the orientation of substituents and as is cusbridges above or below the plane of the rings, tomary for terpenes arid steroids. The convenience of the proposed usage is seen on comparing the Fischer projection (I) and the new representation (11) of 1,2:3,5-di-O-isopropylidene-a-D-glucofuranose; I1 shows clearly the nature of the ring-junctions and the relative positions of substituents, which are the factors controHing the stability and reactivity of the compound. The steroid type of formula is suitable for showing the configurations at new asymmetric centers in cyclic acetals, if these are known. Hydrogen atoms in the angular positions
I
CH,OH I
I1
111
a t ring-junctions must always be shown, but those a t points of substitution (as a t C5 in 11) may be omitted. The diagrams are more suitable for setting in type if drawn with bonds at angles of 45” and 90” only, but the true shape of the molecules is approached more closely by the representation 111. Fischer projections are still essential for showing genetic relations between carbohydrates, but are definitely unsuitable for precise stereochemical deductions; and reliance on them may have been responsible for some (19) W. W. Pigman and R. M. Goepp, J r . , “Chemistry of the Carbohydrates,” Academic Press Inc., New York, N. Y., 1948, p. 53. (20) J. A. Mills, Ph.D. Thesis, Cambridge, Engl., 1953. (21) J. A. Mills, Chemistry & Industry, 633 (1954).
CYCLIC DERIVATIVES OF CARBOHYDRATES
5
errors found in the literature. Formulas similar to I1 have already been used and for derivatives of D-glucofuranuronofor 1 4: 3 6-dianhydrohe~itols'~ r-lactone,22and the writer believes that they should be regularly used for depicting complex carbohydrate derivatives containing fused or bridged rings. Recourse to accurate models is necessary in studying intricate stereochemical problems, but with practice much information may be read from properly drawn diagrams of the type of 11. Because the plane of the paper is the plane of reference, the new formulas may be rotated in the plane of the paper without alteration of their significance, although this may not be true of all perspective formulas.23 Haworth perspective formulas24 are suitable for single pyranose and furanose rings and for some simple bridged structures, but when it is necessary to show conformations of six-membered rings, the non-planar "chair)' and "boat" forms will be drawn in perspective. A method has been devisedz5 for drawing chair rings with lines at angles of 45" and 90" only, suitable for setting in type; it is, however, not readily applicable to some bicyclic structures with cis ring-junctions. Two veiy useful rules for comparing Fischer projections with the new formulas may readily be deduced. (1) In a single ring, of any kind or size, resulting from closure a t two secondary hydroxyl groups on the same side. of a Fischer projection, the two residues will be in cis arrangement, whereas two hydroxyl groups on opposite sides of a Fischer projection will lead to a trans arrangement of residues. ( 2 ) When two adjacent carbon atoms of a Fischer projection become the ring-junction of a pair of fused rings, the ring-junction will be trans if the hydroxyl groups attached to the two carbon atoms are on the same side of the Fischer projection, but cis if the hydroxyl groups are on opposite sides. )
)
As an example, formula V will be transformed to a Fischer projection. Inspection
CH,OH
IV (22) (23) (24) (25)
1
V
K.-C. Tsou and A. M. Seligman, J. A m . Chem. Soc., 74, 5605 (1952). C . S . Hudson, Advances i n Carbohydrate Chent., 3, 1 (1948). H. D. K. Drew and W. N. Hnworth, .I. Chem. SOC.,2303 (1926). R . S. Cahn, J . Chem. Soc., 695, footnote (1952); 3520 (1951).
6
J. A . MILLS
of the steric arrangement a t C2 in V shows that i t is derived from a hydroxyl group on the left of a Fischer projection. Considering ring B only, it is seen t h a t the substituents C5 (in ring A) and C1 are trans; therefore the 2,4-ring is a OT-ring, and the hydroxyl group a t C4 is on the right of the Fischer projection. The ring-junction is cis, so the hydroxyl groups a t C3 and C4 are on opposite sides of the projection. By inspection, or by considering the substituents in ring A , the configuration a t C5 is (IV). Similar determined. Formula V represents 2,4:3,5-di-O-methylene-~-mannitol reasoning may be used t o derive steroid-type formulas from Fischer projections.
To assist comparisons between diagrams drawn according to different conventions, the ring-oxygen atom will, as far as is practicable, be placed at the top in furanose rings, and at the top right-hand corner in pyranose rings. The important class of bicyclic acetals represented by V will always be depicted with the orientation of oxygen atoms shown in V.
111. STERIC REQUIREMENTS OF REACTIONS I . Reversible and Irreversible Rbactions Carbohydrate chemistry provides many examples of reactions that could proceed by alternative pathways, to afford isomeric products that differ in configuration or structure. The effect of the configuration of the reactants on the course of such reactions may depend very largely on whether the reactions are reversible or irreversible. If all pathways are irreversible, the composition of the products depends on the relative speed along the pathways; it is kinetically controlled. These speeds are determined by entropy changes and by the stereochemistry of the traiisition states26concerned; the latter is often related to the stereochemistry of the reactants in a complex way, and has still to be elucidated for many reactions. On the other hand, if each pathway of reaction is reversible, and i f a true equilibrium is established, the composition of the products is quite independent of the mechanism of reaction, being determined solely by the relative thermodynamic stabilities of the constituents, even though the speed of the individual reartions may be very different. A study of such reversible reactions is therefore suitable for assessing the relative stabilities of isomeric compounds. Carbohydrate chemistry is especially rich in examples of ring structures formed by reversible reactions, such as g l y c ~ s i d e sand ~ ~ cyclic acetals,28 and provides a more convenient field for examining the factors governing the stability of rings than does alicyclic chemistry, in which ring closures are usually irreversible. Furthermore, the prominent reversible reactions (26)For reviews and references, see (a) E. D. Hughes, Quart. Revs. (London), 2, 107 (1948); (b) C. R. Ingold, “Structure and hfechanism in Organic Chemistry,” Cornell University Press, Ithsca, N . Y , 1953,pp. 40-49. (27)1’. A. Levene, A. I,. R:LJmond and It. T. Dillon, J . B i d . (‘hem ., 96, 699 (1932). (28) R. M. Hann and C.S. Hudson, J. Ant. C’hem. Soc., 66, 1909 (1944).
CYCLIC DERIVATIVES OF CARBOHYDRATES
7
of carbohydrates are nearly all catalyzed by acids, whereas the equilibria most studied in alicyclic chemistry, such as epimerizatioii of secondary hydroxyl groups4~ 2 9 , ao and of ring 29 are base-catalyzed. Epimerizations a t a positions in aldonic acids3' and 2 , 4 :3 ,&di-O-methylenehexaric acids5" are well established, but base-catalyzed epimerizatioii of secondary hydroxyl groups in the rings of g!ycosides and anhydro derivatives does not seem to have been investigated. Irreversible reactions of carbohydrates include the formation of glycosides and 1-deoxy-1-thioglycosidesfrom acylglycosyl halides33,34 and thioa c e t a l ~ , respectively, ~~-~~ esterifications by acyl and sulfonyl halides or anhydrides in ~ y r i d i n e ,40~displacements ~ of sulforiyloxy groups by various reagents,3Yand most examples of formation and scission of anhydro rings.l6V 17939 It is more difficult to define the influence of stereo effects on the course of these reactions; and some of the results obtained await explanation. I n using the results of reversible reactions in stereochemical analysis, a decision is often difficult as to whether a true equilibrium had in fact been established in experiments not specifically designed for quantitative studies. Recent syntheses of cyclic acetals are examples. When reducing sugars are condensed with acetone or acetaldehyde in the preseiice of strong acids, it is possible that decompositions caused by the catalyst will proceed more rapidly than the establishment of an equilibrium. This risk is less in reactions of alditols with benzaldehyde or formaldehyde, but again a true equilibrium may not result, because of the crystallization of insoluble acetals, or through interruption of the experiment before the rather difficultly hydrolyzable methylene acetals have come to equilibrium. The processing of reaction mixtures is often very incomplete. These uncertainties have been 1
(29) W. Huckel, Ahn., 633, 1 (1938). (30) W. von E. Doering and T. C. Aschner, J. Am. Chem. SOC.,71, 838 (1949), and references cited there. (31) E. Fischer, Ber., 23,799 (1890); W. N . Hnworth and C. W. Long, J . Chem. SOC., 345 (1929). (32) W. N. Haworth, W. G. M. Jones, M. Stncey and L. F. Wiggins, J . Chem. SOC., 61 (1944). (33) E. Pacsu, Advances in Carbohydrate Chem., 1, 77 (1945). (34) W. L. Evans, D. D. Reynolds and E. A. Talley, Advances in Carbohydrate Chem., 6, 27 (1951). (35) J. W. Green and E. Pacsu, J . Am. Cheni. SOC.,69, 1205, 2569 (1937). (36) E. Pacsu, J . Am. Chem. SOC.,61, 1671 (1939). (37) M. L. Wolfrom, D. I. Weisblat and A. R. Hanze, J . Am. Chem. Soc., 66, 2065 (1944). (38) A. L. Raymond, Advances in Carbohydrate Chem., 1, 129 (1945). (39) R. S. Tipson, Advances i n Carbohydrate Chem., 8, 107 (1953). (40) J. M. Sugihara, Advances in Carbohydrate Cheni.,8, 1 (1953).
8
J. A . MILLS
kept in mind when using data on cyclic acetals for the generalizations of Section V, and most weight has been assigned to the results of the experiments that seem to have more nearly approached the ideals of a true equilibrium and complete subsequent processing. 2. Reactions with Specific Steric Requirements
That the bimolecular displacement reaction (SN2) requires a collinear arrangement of three atomic centers is well known. Although few kinetic studies have been made, SN2reactions are probably fairly common in carbohydrate chemistry, especially intramolecular reactions leading to the establishment of anhydro rings. If the required steric arrangement is not attainable, the reaction may be entirely prohibited.16 The closure and scission of ethylene oxide rings is a special case of the intramolecular SN2reaction,16 and would seem to call for a tranx =-Cc,.- sition state with a specific arrangement (A) of four atomic ,. \o,.,, centers.l3. l 6 It is true that a trans arrangement of groups16 is necessary for the reaction, but this will not be sufficient A for ensuring reaction if molecular rigidity prevents the attainment of the transition state, as is shown by the following example. The reaction of 5,6-anhydro-l, 2-O-isopropylidene-cr-~-glucofuranose (VI) with bases is a fruitful source16r41 of D-glucose derivatives having a substituent a t C6. However, even when the opening of the anhydro ring of VI, by external attack of an anion, proceeds with difficulty, there is apparently little tendency for an intramolecular displacement by the free hydroxyl group a t C3; there is no mention4I of the formation of 3,6-a11hydro-1 ,2-O-isopropy~idene-cu-~-g~ucofuranose (VII), although this42 is a well-defined solid, and would have been readily recognized. On the other (VIII) reacts hand, 1,2-O-isopropylidene-5,6-di-O-tosyl-cr-D-glucofuranose
'
VI
VII
VIII
very readily with base, giving42the 5-0-tosyl derivative of VII. Scission of ethylene oxide rings through intramolecular attack by adjacent hydroxyl groups has often been observed,I6 and the failure of such a n attack to occur with VI must be ascribed to steric factors. Approach of C6 to the hydroxyl group a t C3 may occur with about equal ease for VI and VIII, but whereas (41) H. Ohle and K. Tessmar, Be?.., 71, 1843 (1938). (42) H. Ohle, L. von Vargha and H. Erlbach, Ber., 61, 1211 (1928).
CYCLIC DERIVATIVES O F CARBOHYDRATES
9
reaction of VIII involves only a simple displacement a t C6, reaction of VI will require a specific arrangement of C6, C5, and the attacking and displaced oxygen atoms, which apparently is unattainable. It is interesting to note that 1,4-anhydro-6-0-tosylsorbitol affords43I ,4:3,6-dianhydrosorbito1 on treatment with base; formation of the 5 ,6-anhydro compound may reasonably be postulated as an intermediate stage, and it seems that the greater flexibility of t,he single five-membered ring, compared with that of the bicyclic system of VI, permits the establishment of the transition state required for conversion of the 5,6-anhydro to the 3,6-anhydro compound. The bimolecular elimination reaction (E2) also requires a specific ar~ B, the groups X and Y are rangement (B) of four atomic ~ en ters.4In coplanar with the two carbon atoms and antiparallel (anti-trans, or “true
trans” relation). If the configuration of the reactant prevents the establishment of the transition state B, eliminat,ion by the E2 mechanism will not occur. It is doubtful whether this mode of elimination has been kinetically established for any carbohydrates, but for this, as for other stereospecific reactions, they provide a potentially useful testing ground. Some interesting results have recently been obtained45in studies on elimination reactions of esters of hydroxy acids. The mechanism is not fully established, but probably is of the bimolecular type. An especially interesting observation is that sodium iodide promotes the removal of two vicinal sulfonyloxy groups by a process of cis-elimination; a series of elimination reactions of this type is known in carbohydrate chemistry,39but apparently does not yet include an example from which the stereochemistry of the reaction could be deduced. The spontaneous decomposition of the diazonium ions obtained on treating non-aromatic primary amines wit,h nitrous acid in weakly acidic solutions (deamination reaction) is an irreversible reaction possessing great driving force under mild conditions. The steric consequences of the reaction in the aliphatic and alicyclic fields have proved to be quite diverse,4°-6n (43) (44) (45) (1953). (46)
V. G. Bashford and I,. F. Wiggins, J . Chem. Soe., 299 (1948). See ref. 26(b), p. 465. R. P. Linstead, L. N. Owen and R. F. Webb, J . Chem. SOC.,1211, 1218, 1225 Phyllis Brewster, F. Hiron, E. D. Hughes, C. K. Ingold and P. A. D. S. Rao,
10
J. A . MILLS
but amenable to classification. Its application to carbohydratesl6 altro, gulo > talo > a110 > galacto, manno > gluco.
The latest work, lgaa. 19*b however, appears to place allo before talo, and shows that the glum configuration permits some formation of anhydride. (198b) J. W. P r a t t and N. K. Richtmyer, Abstracts Papers Am . Chem. Soc., 126, 22D (1954); L. D. Ough and R. G. Rohwer, ibid., 126, 16D (1954).
CYCLIC DERIVATIVES OF CARBOHYDRATES
51
It is possible that, in t,he allo and gluco anhydrides, hydrogen bonding between a xkl hydroxyl groups may promote stability. A similar order should hold for ketoses, adthough the additional equatorial group (at the anomeric center) may increase the stability of individual isomers, and the diketose dianhydrides are actually always formed. 3. The Scission of Epoxide Rings Since t.his topic was last reviewed in this serieslL6much additional information about the scission of epoxy derivatives of pyranose sugars has become pvailable. Only this t)ype of epoxide will be discussed here. Several w o r k e r ~ ' ~ have.cornpiled 9-~~~ lists of t,he available evidence. There is general agreement that scission of the epoxide ring to provide two free substituenh always occurs with inversion of configuration16a t the point of attack by the reagent, and the chief problem is the predic~tioiiof t,he direct,ion of opening of the ring when two alternatives are possible, which is always the case for 2,3- and 3,4-anhydro derivatives of pyranose rings. For example, a 2,3-anhydride with the D-manno configuration may afford products with the D-gkKO configuration (attack a t C2), or with the D-altro configurat'ion (attack a t C3); products with the D-gluco and D-altro configurat'ions may also result from a 2,3-anhydride with the D-a,llo configuration, but in this case by attack a t C3 and C2, respectively. Some interesting regularities were noted. Derivatives with the D-altro configuration predominate in the products of scission, by bases, of methyl and methyl 2,3-an2,3-anhydro-4,G-0-benzylidene-a-D-allopyranoside hydro-4,6-O-benzylidene-a-~-mannopyranoside.~~~~ 204 Scission of methyl 2,3-anhydro-4,6-O-benzylidene-a(and P)-D-talopyranoside and methyl 2,3-anhydro-4,6-O-benzylidene-a(and @)-D-gulopyranosideaffords, almost ~ ~The ~ * action of ammoniazo6or sodium entirely, derivatives of ~ - i d o s e .*06 gives derivatives of methoxidez06 on 1 ,6: 2,3-dianhydro-p-~-talopyranose 1 ,6-anhydro-p-~-galactose,and t,he same reagentszo6with 1 ,6:3,4-dianhydro-P-D-talopyranose yield derivatives of I ,B-anhydro-P-~-manqo(199) F. H. Newth, W. C. Overend and L. F. Wiggins, J. C h e w . Soc., 10 (1917). (200) S. Mukherjee and H. C. Srivastilva, Proc. I n d i a n A c a d . Sci.,36A, 178 (1952). (201) A. I(.Bose, D. K . It. Chaudhuri and A. K. Bhattacharyya, Chemistry & Industry, 869 (1953). (202) F. H. Newtjh, Chenristry & I n d u s t r y , 1257 (1953). 1193 (1935); G. J. Robert(203) G . ,J. Robertson and C. F. Griffith, J . Chem. SOC., son and W. Whitehead, ibitl., 319 (1940); W. €I. Myers and G. J. Robertson, J . A m . ('hem. Soc., 66, 8 (1943). (204) N. K. Richtmyer and C . S. Hudson, J . A m . Chevr. Soc., 63, 1727 (1941); N . K. Richt,myer, ,~dva7iccsZ'TL ('arbohydrafe ('/ienr,, 1, 37 (1945). (205) L. F. Wiggins, J . f'hem. Soc., 522 (1944). (206) Syllil 1'. .James, F. Sinit!, M. Stacey and I,. F. Wiggins, J . collector.23n'umerous effluent fractionators have been proposed for column c h r o m a t ~ g r a p h y .24~ ~The . successful application of these devices was (22) R . I,. Whistler and D. F. Durso, J . Anz. Chem. SOC., 72, 677 (1950); 74, 5140 (1952). (23) D. F. Durso, E. D. Schall and R. L. Whistler, Anal. Chem., 23, 425 (1951); J . L. Hickson and R. I,. Whistler, ibid., 26, 1425 (1953). (24) I,. Hough, J. K. N . Jones a n d W. H. Wadinan, . J . Chwm. SOC.,2511 (1949); D. M. P. Phillips, Natctrc, 164, 545 (1949); S. 9. Itnndall arid A . J. 1'. Martin, Biochem. d . (I,ondon), 44, €'roc.. ii (1941)); A . R. Gilson, C h e n i i s f r g Le. I r ~ d z r s l l y29, , 155 (1951); R . A . Grcirit a n d S. R.. Stitch, ibitl., 230 (19.51); J . 0 , Harris, ibid., 255 (1951); I,. A. Boggs, I,. S. Caendct, M. Dulmis and F. Sniitli, Annl. Chem., 24, 1148 (1952); J.Etlelnian and It. V. Martin, Bioch,ern. J . (London), 60, Proc. xxi (1952); A . T.Carlander and S. Gardell, Arkiv Kemi, 4, 461 (1952); J . E. Varner and W. A . Bulen, J . Chem.
63
COLUMN CHROMATOGRAPHY O F SUGARS
exemplilied with a chromatogram of an acid hydrolyzate of an alpha Schardinger cleutrin, using gradient development with ethanol in water (the concentration of the ethanol in water was changed gradually and continuously from 0 to 20 % during the course of the development,; see Fig. 8).2b
0.4-
0
50
10
80
Fraction no.
FIG.8.-Chromatography of a Mixture of Mono- t o Hexa-saccharides from an Acid Hydrolyzate of a Schardinger Dextrin. [The Adsorbent was Carbon (Darco G-60) Pretreated with 1% Ethanolic Stearic Acid; the Developer was Gradually Changed from 0% to 15% Ethanol.]
The unusual utility of this adsorbent has been demonstrated by its role in the preliminary isolation of isomaltose,26(a)maltotriose,26(a)maltotetraose,26(b)and panose26(n) from the hydrolytic products of starch; in the preparation of D-xylo-biose, -triose, -tetraose, -pentaose, -hexaose, and -heptaose from a partially hydrolyzed xylanZ7;in the resolution of bimolecular dianhydrides of L-sorbose28(a) and D-fructose2s(b) ; in an improved procedure for the preparation of s t a c h y ~ s e and ~ ~ ; in the preparation of a series of maltodextrins (as high as maltoheptaose) from the partial, acid Educ., 29,625 (1952) ; H. J. Huisman and S. A. Krans, Chent. Weekblad, 48,1007 (1952) ; R. J . Dimler, J. W. Van Cleve, Edna M. Montgomery, L. R. Bair, F. J. Castle and J. A. Whithead, Anal. Chenz., 26, 1428 (1953); E. S. Sandcrson, ibid., 26, 944 (1954); D. Fraser, ibid., 26, 1858 (1954). (25) A. Tiselius, Endeavour, 11, No. 41, 13 (1952); R. S. Alm, R. J. P. Williams and A. Tiselius, Acta Chem. Scand., 6, 826 (1952); R . S. Alm, i b i d . , 6, 1186 (1952). (26) (a) M. L. Wolfrom, A. Thompson, A . N . O’Neill and T . T . Galltowski, J . Am. Chem. SOC.,74, 1062 (1952); A. Thompson and M. L. Wolfrorn, i b i d . , 74, 3612 (1952); 73, 5849 (1951); (b) R. L . Whistler and J. L . Hickson, zbid., 76, 1671 (1954); Edna M. Montgomery and F. B. Weakley, J . Assoc. O j i c . Agr. Chemists, 36, 1096 (1953). (27) R. L . Whistler and C.-C. T u , J . A m . Chem. SOC.,73, 1389 (1951); 74, 3609 (1952); 76,645 (1953); R. L. Whistler, J. Bachrach and C.-C. Tu, i b i d . , 74,3059 (1952). (28) (a) M. 1,. Wolfrom and H. W. Hilton, J . A m . Chem. Soc., 74, 5334 (1952); (b) B. Wickberg, A d a Chem. Scand., 6, 961 (1952). (29) M. L. Wolfrom, R C. Burrell, A. Thompson and S. S. Furst, J . A m . Chern. Soc., 74, 6299 (1952); R. A. Laidlaw and Clare B. Wylam, J . Chem. SOC., 567 (1953).
64
W. W. RINKLEY
hydrolysis of pot,ato amylase."" Carbon-column chromatograms of the sugars of Cmbilicaria pustulnta, when developed wit>h 1, 15, and 50% aqueous ethanol, yielded D-arabitol, D-marinitol, umbilicin [3-0-(@-~galactopyranosy1)-o-arabit,ol],a ,a-trehalose, and sucrose.31 The carboncolumn analysis of the acid hydrolyzates of the hemicelluloses of Scots pine and black spruce showed the presence of D-xylose, ~-arabinose,D-mannose, D-galactose, L-rhamnose, L-fucose, D-galacturonic acid, ~-xylose-(2-+ I ) 4-~-methyl-~-g~ucosiduronic acid, and 4-O-methyl-~-g~ucuronic acid.32 A crystalline nitrogen-containing tetrasaccharide was obtained from human milk by carbon c h r ~ m a t o g r a p h yUse . ~ ~ of this adsorbent made possible the isolation of certain oligosaccharides from enzymic transglycosidations using sucrose in the substrate; some examples are k e ~ t o s earid ~ ~n e o k e ~ t o s ewith ~~ yeast invertase, a new trisaccharide with T a k a d i a ~ t a s e and , ~ ~ a reducing O-(D-fructosyl)-D-glucoseamong the products from sucrose, D-ghcose, and yeast invertase.37 The salient chromatographic advantages of carbon are general availability, low cost, keen selectivity, and good capacity. Less attractive features of this adsorbent are that it is disagreeable to handle and that it usually requires some pretreatment before use. Furthermore, the rate of solvent flow through columns of finely divided carbon is too low unless up to an equal weight of an inert diluent38is added; a corresponding decrease in column capacity results from this addition. The last objection is met by employing columns composed only of carbon capable of passing through a 40- or 60-mesh screen but retained by ail 80-mesh screen.39 2. Fuller's Earth Clay
Zones formed in the chromatography of colorless substances on columns of colorless or light-colored adsorbents may be detected by streaking the (30) W. J. Whelan, J . M. Bailey and P. J. P. Roberts, J . Chem. SOC.,1293 (1953). (31) B. Lindberg and B. Wickberg, Acla Chem. Scand., 7 , 140 (1953). (32) A. R . N. Gorrod and J . K . N . Jones, J . Chem. Soc., 2522 (1954). The acidic disaccharide was also resolved chromatographically from an acid hydrolyzate of hemicellulose-B from corn cob; R. I,. Whistler and L. Hough, 6 .A m . Chem. Soc., 76, 4918 (1953); R. L . Whistler, H . E. Conrad and L. Hough, ibid., 76, 1668 (1954). (33) R . IZuhn, Adeline Gauhe and H . H. Baer, Chem. BeT., 86, 827 (1953). (34) N . Albon, D. J . Bell, P. H. Blanchard, D. Gross and J. T . Rundell, J . Chem. SOC.,24 (1953). (35) D. Gross, P. H. Blanchard and D. J . Bell, J . Chem. Soc., 1727 (1954). (36) J . S. D. Bacon and D. J . Bell, J . Chem. SOC.,2528 (1953). (37) D. J. Bell and J. Edelman, J . Chem. S O C .4652 , (1954). (38) Celite No. 535, a diatomaceous filter aid, is a product of the Johns-Manviile Co., New York, N. Y . , and is often used for this purpose. (39) R . L. Whistler, Science, 26, 899 (1954).
COLUMN CHROMATOGRAPHY OF SUGAHS
65
extruded columii with a suitable indicator (streak reagent).40The potent'ialities of this method are great. The rapid evaluation and selection of adsorbeiit,s and developers are made possible, using only milligram quantities of adsorbate. Aqueous, alkaline, potassium permanganate4' is often employed as a reagent for the detection of sugars and their colorless derivatives. The st,reaked adsorbent column is cut into sections as indicated by the reagent. The streak is scraped off the column section, and the zone is then eluted with a solvent mixture containing 10 to 20% more water than t.he developer employed in the chromatogram. The recovered adsorbate is oft,en obtained in crysballiiie form. The suitability of fuller's eart>h for the chromatography of carbohydrates was demonstrated by the establishment of an adsorpt,ioii series which included nearly 100 sugar subst,ances (see Table 11).43 The developers mere aqueous 2-propanol and aqueous et,hanol; the water content of these mixtures was gradually increased in order to develop more st'rongly adsorbed carbohydrates, particularly sugar Here, the developing effect of aqueous ethanol was the reverse of its role in the chromatography of sugars 011 carbon.45 In general, the adsorption of sugars on clay did not present a definite pattern. The adsorptive bond of the members of the D-glucose-sucrose-raffinose series did become stronger with an increase of molecular weight ; glycerol, erythritol, ribitol, and allit,ol behaved similarly. Glycosides are held more (30) 12. Zechmeister, L. de Cholnoky and (Mlle.) E. Ujhelyi, Bicll. soc. chim. bid., 18, 1885 (1936). (41) W. H. McNeely, W. W. Binkley and M. L. Wolfrom, J . A m . C'hem. Suc., 67, 527 (1945). (42) These clays are hydrated magnesium aluminum silicates containing some iron and calcium. (43) B. W. Lew, M. L. Wolfrom and R . M. Goepp, Jr., J . A m . Chem. SOC.,68,1449 (1946). A suitable fuller's earth clay was Flores XXX; it was produced by the Floridin Co., Warren, Pa. Other selective clays were Floridin XXX (Floridin Co., Warren, Pa.), Types A and AA Attapulgus Clays (Attapulgus Clay Co., Attapulgus, Ga.), Bleaching Clay 260 (Industrial Minerals and Chemical Co., Berkeley, Calif.), and J. Neutrol (Filtrol Corp., Los Angeles, Calif.). (44) Often, sugar acids can be detected with Congo Red indicator, and ascorbic acid with 2,6-dichlorophenolindophenol. (45) The detection of sugar alcohols in chromatograms developed with aqueous ethanol was difficult because the streak reagent, alkaline potassium permanganate, was rapidly decolorized. This difficulty was largely overcome in small exploratory chromatograms by the use of aqueous tert-butyl alcohol as the developer. The rate of flow of these solvents through fuller's earth clay was very slow and this developer was unsatisfactory for larger columns of this adsorbent. These columns were developed with aqueous ethanol, and extruded. They were wrapped with aluminum foil, leaving exposed t o the air an area, 1 cm. wide, from the top t o the bottom of the adsorbent column. Sufficient solvent had evaporated from the exposed surface in 18 hours to permit the use of alkaline permanganate.
66
W . W . BINKLEY
TABLE I1 Chroinatoyraphzc Adsoi ptzon Series,u on Fzrllci 's E a r t h Clay, oJ Curbohydrulcs and Some Related Substances43 Class I (10 cc. of 70% ethyl alcoholb) Potassium acid D-glwarate, D-glucosamine hydrochloride, 1)-galactosamine (chondrosaniine) hydrochloride (dertro)-Tartaric acid D-Gluconic acid, ammonium wgluconnte, sodium D-gluconate D-Arabonic acid, L-arabonic acid, potassium D-arabonate, citric acid D-Mannonic acid a-D-Galacturonic acid Class ZI {lo cc. of 90% ethyl alcohol) Lactitol, melibiitol L-Iditol, inyo-inositol (m. p. 225"),stitchyose, Schardinger a-dextrin Lactose D-Glucitol, dulcitol, L-perseulose, D-perseulose, Schardinger 8-dextrin, (Zeao)malic acid Raffinose Xylitol D-Mannitol, u-talitol, gentiobiose Class IZI (5 cc. of 90% ethyl alcohol) D-Gulose Melibiose, D-mannose, wribose, 3,6-anhydro-~-glucitol,u-psicose Cellobiose l-Deoxp-o-glucitol Ascorbic acid D-ArabitoI Class Ik' (4 cc of 95% ethyl alcohol) Maltose, u-galactose, D-fructose, melezitose, u-)r2unno-heptrIlose, trehalose, D glycero-D-yulo-heptose L-Fucitol, turanose, 1,5-anhydro-u-niannitol, 1,4-anhydro-~-mannito1, D-lyxose D-Rhamnitol, L-rhamnitol, 2-deoxy-~-"glucitol,"D-gluco-heptdose L-Arabinose, D-arabinose, ribitol, quercitol, diethylene glycol (2,2'-oxydiethanol), dipentaerythritol Sucrose, erythritol, L-altrose ~-G~ucurono-2,6-lactone L-Sorbose, succinic acid Anhydroenneahepti to1 n-Glucose, glycerol Class 1' (5 cc. of 90% isopropyl alcohol) L-Allose 1,4:3,6-Dianhydro-1)-mannitol, amygdalin ~-D-Ga~ncto-metasitccharinic acid 3-Hydroxypropyl 8-o-glucopyranoside rJ-Fucose
BT
COLUMN CHROMATOGRAPHS O F SUGARS
TABLEI1 (Contznued) - ______-____. ~-
_
_
~
Class VZ (6 cc of 97% isopropyl nlcoliol)
Methyl cu-i,-glucopyranosidc, methyl ~-u-glucopyranoside Methyl a-D-mannopyranoside, D-xylose, D-glucono-l,5-lactone 1,4-Anhydro-o-gluc~itol,dipropylene glycol, pentaerythritol, 1,5:3,6-dianhytIrou-mannitol, 1,4:3,6-dianhydro-~-glucitol D-Clucono-l , 4-lactone Class V I I (4 c c . of 97% isopropyl alcohol) L-Rliamnose, salicin Ethylene glycol (1,2-ethancdiol) Propylene glycol (I ,2-propnnediol) 1’11lori x i n 1 , 4 : 3 ,A-Dianhydro-~-idi to1 a Arranged in decreasing order of adsorptive strength. Adsorbent: 1.68 g. of Florex XXX43/Cclite38 (5/1). Dimensions of adsorbent column: 0.9 X 6 cm. of adsorbent. Adsorbate solution: I nig. in 0.5 cc. of developer shown. Developer: noted following the class heading. Prepared by adding 30 cc. of water t o 70 cc. of absolute ethanol. Other dilutions were made similarly, except, t h a t 95% ethyl alcohol was the axeotrope.
loosely than sugars, sugar acids more tightly. Columns of d a y are difficult, to extrude from the usual, straight, glass tubes; they are removed more readily from metal tubes or from glass tubes tapered uniformly and gradually outward from the bottom to the top.46The rate of developer flow through clay columns was slow; the rate was improved by the admixing of a filter aid.38The flow rate can be increased &fold when the clay is air-blown (to remove the finer particles) and the developer is subjected to a pressure of 120 Ibs. per sq. in.47The keen selectivity of clay was exemplified by the separation and isolation in gram quantities (by the extrusion method) of two new di-D-fructose dianhydrides, D-fructopyranose-Dfructofuranose 1,2’:2, l’-diaiihydride4* and a second of unestablished ~ t r u c t u r e The . ~ ~ interconversion, by heat, of D-fructose tlo D-glucose was discovered with the aid of extrusion, clay chromatography. bo 2,3-0-Isopropylideue-D-fructose was isolat,ed chromatographically from partially (46) Ref. 7(g), p. 7 . Tapered glass tubes are produced by the Scientific Class Co., Bloomfield, N . J. (47) D. F. Mowery, Jr., J . Am. C h ~ m SOC.,73, 5047 (1951). Heavy-walled Pyrex tubes were needed for these chromatograms. (48) M. L. Wolfrom and M. Grace Blair, J . Am. Chem. SOC.,70, 2046 (1948); M. I,. Wolfrom, W. W. Binkley, W. I,. Shilling and H . W. Hilton, ibid., 73, 3553 (1951). (49) M. L. Wolfrom, H . W. Hilton and W. W. Binkley, d Am. Chern. SOC.,74, 2867 (1952). (50) M. 1,. Wolfrom and W. L. Shilling, J . Am. Chem. SOC.,73, 3557 (1951).
-
68
W. W. BINKLEY
hydrolyzed “ij-diacetoiie-D-fru~tose,” showing the structure of thc latter to be 2,3:4,5-di-~-isopropylidene-~-fructopyraiiose.~~ Clay has been used effectively in the flowing or elutJioiichromatography of sugars. The residual sucrose was removed almost quantitatively from beet molasses, arid in 73 % yield from cane, blackstrap molasses.52Subscucose
0.19
0.16
u) u)
-
2
.+.
0
@h
2 L
g o.oe u
0.04
0.01
t
I
I
15
39
54
89
Fraction no.
FIG.9.-Chromatography on Fuller’s Earth Clay of a Fraction from Cuban, Blackstrap Molasses. [The Developer was Azeotropic Ethanol from X t o Y , and Ethanol/Water (90/10) from Y t o Z.]
quently, crystalline D-glucose and myo-inositol, and zones containing Dfructose and D-mannitol, were isolated from cane molasses (see Fig. 9); a similar chromatogram of its yeast-fermentation residue revealed the presence of D-glucose, sucrose, erythritol, D-fructose, D-arabitol, and (51) M. L. Wolfrom, W. L. Shilling and W. W. Binkley, J . Am. Chem. Soc., 74, 4544 (1950). (52) W. W Binkley and M. L. Wolfrom, J. Am. Chem. Soc., 69, 664 (1947); M. L. Wolfrom and W. W. Binkley, U. S. Pat. 2,504,169 (1950); Chem. Abstracts, 44, 6180 (1950).
69
COLUMN CHROMATOGRAPHY OF SUGSRS
D-mannitol (see Fig. 10).53 Crystalline D-fructose was prepared chromatographically from invert sugar (acid-hydrolyzed sucrose) .64 Fuller's earth clay is capable of some unusual and difficult separations of sugars, and because of its comparatively light color this adsorbent is adaptable to extrusion, streak-reagent chromatography; furthermore, it is cheap. Most organic solvents pass rapidly through columns of clay. The
D-Frucrose D-Arabitot
o . 0 6 ~, x Erythritol
5
10 13
D-Mannitol
19
29
35
49
Fraction no.
FIG.10.-Chromatography, on Fuller's Earth Clay, of a Fraction from the NonFermented (by Yeast) Residue of Cuban, Blackstrap Molasses. [The Developer was Aeeotropic Pzthanol from X t o Y, and EthanollWater (90/10) from Y to Z.]
addition of wat,er to these solvents markedly decreases tjhe rate of their percolation, and so the admixture of 15 t,o 20 % of filt,eraid t30t,he adsorbent is desirable. The capacity of fuller's eartjh clay is low, and some pretreat'ment is required when t>heclay is t,o be used for isolation work. Whereas the adsorptive propert,ies of clays from different areas are variable, this adsorbent from any particular geographical locatJion is except.ionally uniform. The poor extrusion propert.ies of the makrial are partially overcome by the (53) W. W .Binkley and hl. L. Wolfroni, J . A m . Chevr. Soc., 72, 4775 (1950). (54) D. F. Mowery, Jr., J . h ~ Cheni. . SOC.,73, 5049 (1951); D. F. Mowery, Jr., and G. R . Ferrante, i b i d . , 76, 4103 (1954), for the separation of methyl u-galactoside anomers.
70
W. W. BINKLEY
use of metal, or tapered-glass, chromatographic tubes. Certain inorganic substances are leached from the adsorbent during the elution process, particularly when water-rich solvents are employed, and these form troublesome suspensions. They are removed from an aqueous solution of the adsorbate by treatment with decolorizing carbon and passage through acid-washed asbestos under diminished pressure, followed by gravity filtrat,ion t,hrough hardened filter paper.49
3. Carbohydrates T s v ~ e t t himself, ,~~ recognized the potentialities of powdered sucrose as a chromatographic adsorbent, and employed t,his sugar in his investigations on chlorophyll. Soon after,56the branched form (smylopectin) and the h e a r form (amylose) of potato starch were separated on fibrous celliilose (washed cotton) ; corn starch has also been so f r a c t i ~ n a t e d Interestjingly, .~~ the linear form is the more st,roiigly adsorbed although its molecular weight is lower. Amylose may be able to form with t,he cellulose, a t best,, some t3ype of weak inclusion complex.58 Thus, the removal of amylose from amylopectin is not complete,59and some modifications of this adsorption procedure likewise were unsuccessful.60 The behavior of cellulose nitrate and of non-carbohydrate polymers, on carbon, is similar.6‘ The advent of modern, filt>er-paperchromatography,62and its extension to ~ u g a r s , 6have ~ produced a powerful, qualitative, analytical method. Often, only microamounts of adsorbat,es are handled by this procedure; but larger yuantities of resolved materials were needed for some quant,itative separations, isolations, arid proper identifications, and columns of tightly packed, powdered cellulose were found to be 64 A mixture containing 50 mg. each of L-rhamnose, D-ribose, L-arabinose, and D-galaCtOSe was adsorbed on a 12 by 1.25 in. (diam.) column of powdered cellulose7~ prewetted with t,he (55) RI. Tswett, Y i p o g r . Warshawskago iitsch,ebnago Okrwgn, Warsaw, 1910 (in Russian). (56) C. Tariret,, C o w p t . rend., 168, 1353 (1914). (57) F:.Pacsu :tnd J. W. Mullen, 11, J . A m . Cheni. Soc., 63, 1168 (1941). stsJlizat,ion,” i i i “Technique of Organic Chemistry,” A. (58) R. S. Tipson, ‘‘ Weissherger, ed., Interscience Publishers, I n c . , New York, N. Y., Vol. 111,2nd Edition, i n press. (59) T. J . Schoch, Arlaances in Corhohydrate Chenf,.,1, 247 (1945). (60) G . A. Gilbert, C. T. Greenwood and F. J. H y h r t , J. Chew. Soc., 4454 (1954). (61) 8. Claesson, A r h i v K e m i , Jlineral. Geol., 26A, No. 24 (1948). (62) R . Corisden, A . H. Gordon nnd A. J. P. Martin, Bioche,ni. .I. (London), 38, 224 (1048); A . J . P. Martin, P r i x N o b r l , 110 (1952). (63) S. M . Part,ritlge, .Vatitre, 168, 270 (1946). See G. 1;.Komkahany, Adzinnces in Cnrhoh!/drnta C h e n ~ .9, , 303 (19.54). (64) I,. Hough, J . K . N. Jones iind W. I€.Wildman, ;Vnfur.e,162,448 (1048). (65) A commodity of H. Reeve Angel and Co., Inc., 52 I3uane St., New Yorlr 7 , N.T.
COLUMN CHROMATOGRAPHY O F SUGARS
71
developer, 1-butaiiol saturated with water coiitaiiiiiig 1 % of ammoilia. The flowing chromatogram was employed, and the resolved, cystalline sugars were recovered in 95 % yield from the column effluent. Columns of wet, powdered cellulose are extruded readily i t ith compressed air, hut no suitable streaking reagent is available at present. Fortunately, the chromatographic behavior of powdered cellulose is identical with that of f lterpaper strips and sheets. The flowing chromatogram of this adsorbent, using an automatic, effluent-fraction collectorz4 coordinated with paper-strip analysis, has produced a strong tool. ITsually, the rate of' solveiit percolation through this adsorbent is sufficiently rapid that neither the application of pressure (or vacuum) nor the addition of an inert dilueut (filter aid) is required. The method is also very effective in the resolution of TABLE111 Flowing Chromatogram, on Powdered Cellulose, or 1.6 g. of the Sugars from the Incomplete Hydrolysis or the Gum of the Sterculia setigera Trees@(*) Cofiiponrnls
Yield, mg
I I1 111 IV V
15 413 145 373 640
a ketose L-rhamnose wtagatose and an aldose wtagatose, wgalactose, and an aldose 11-galactose
Total, 1,586 a
The developer was 1-butanol, saturated with water.
mixtures of methylated sugars (which is discussed in detail in a later Section). The broad applicability of this method is already apparent in the successful elucidation of the structure of certain naturally occurring, plant polysaccharides. Powdered-cellulose chromatography of the sugars from the incomplete, acid hydrolysis of t,he gum from the Sterculia setigera tree yielded pure, crystalline L-rhamnose hydrate and D-galactose, as well as D-tagatose (also crystalline, see Table III)66(a); other methods showed the presence of D-galacturonic acid residues. Pure crystals of' L-rhamnose and D-xylose were obtained by powdered-cellulose chromatography of a hydro(66) (a) E. L. Hirst, L. Hough and J. K. N. Jones, Nature, 163, 177 (1949); J . Chem. Soc., 3145 (1949); (b) P. Andrews, D. H. Ball and J . K. N . Jones, ibid., 4090 (1953),
for peach and cherry gums; (c) P. Andrews and J. K. N . Jones, ibid.,1724,4134 (1954), for lemon and golden apple gums; (d) E. L. Hirst and A . S. Perlin, ibid., 2622 (1954), fov the gum of Acacia pycn.antha.
72
W. W. BINKLEY
lyzed mucilage from flax seed; L-arabinose was also 67 The acidhydrolyzed mucilage of the slippery elm (Ulmus ,fuZva,) was found to contain ~-rhamnose-(2+ 1) D-galactosiduronic acid and a mixture of sugars, resolved by this chromatographic method int>o it,s components, D-galactose, L-rhamnose, and 3-O-methyl-~-galactose.~~ The application of the method, using a hydrocellulose, to the acid hydrolyzates of several galactomannans (from clover,'j9 and fenugreek seed70) gave crystalline D-mannose and D-galactose; n-glucose was obtained, in addition to these sugars, from certain Iris The residues of D-galactose, Larabinose, and 4-O-methyl-~-glucuronic acid (isolated by another procedure) were shown, using powdered-cellulose columns, to be present in gum myrrh72;and D-xylose and D-galactose residues were found to be components of the polysaccharide of tJhefresh water alga, N o s l o ~An . ~ ~L-arab. inopyranose disaccharide [3-O-(P-~-arabinopyranosyl)-~-arabinose] was separated from the hydrolytic products of larch g gal act an.^^ Chromatography, on powdered hydrocellulose, of the sugars from the hydrolyzed, animal polysaccharide of frog-spawn mucin led to the identification of L-fucose, D-mannose, D-galactose, D-glucosamine, and D-galactosamine among the components (see Table IV).75 Further evidence of the diversity of chromatographic applicability of hydrocellulose was shown in its ability t o separate a hexitol from a hexose; crystalline D-arabitol and D-galactose were isolated from acid-hydrolyzed umbilicin of Umbilicaria p ~ s f u l a t a . ~ ~ The powdered-cellulose column has performed valuable service in the study of sugar synthesis. DL-Xylose was resolved from the products of the action of heat and alkali on paraformaldehyde; DL-xylose and DL-arabinose were isolated after the action of alkali a t 20" on a mixture of glyceraldehyde and gly~olaldehyde~';subsequently, DL-ribose (identified as the p-tolylsulfonylhydrazone7*) was obtained79 using a column operating a t 60". (67) D. G. Easterby and J . K. N. Jones, Nature, 166, 614 (1950). (68) L. Hough, J. K. N . Jones and E . L. Hirst, Nature, 166, 34 (1950); E.L. Hirst, I,. Hough and J . K. W. Jones, J . Cheni. SOC.,323 (1951); for okra mucilage, R . L. Whistler and H. E. Conrad, J . A m . Chem. SOC.,76, 1673, 3544 (1954). (69) P. Andrews, L:Hough and J. K. N. Jones, J . A m . Chem. SOC.,74, 4029 (1952). (70) P. Andrews, L. Hough and J. K. N. Jones, J . Chem. SOC.,2744 (1952). (71) P. Andrews, L . Hough and J. K . N . Jones, J . Chem. SOC.,1186 (1953). (72) L. Hough, J . I(. N. Jones and W. H. Wadman, J . Chhem. SOC.,796 (1952). (73) L. Hough, J . Ihrose,B" 5-deosy-~-threo-pentulosefrom acetaldehyde and a triose phosphate,81 and D-ido-heptulosan from D-xylose.82 The powdered-c*ellulose, flowing chromatogram of an unfermentable residue from the mixture obtained by the action of heat, on D-fructose yielded a sirup containing a ketose having a mobility greater than that of D-fructose; i t may be ~-psicose.*~ D-Psicose (identified as the phenylosazone TABLEIV 17lowing Chrornntograrn on Powdered Cellulose of the Sugars from the Hydrolysis of 1.082 y. of Frog-spawn Mucin7$ Ej4uent fractiona
Yield, mg.
I I1
59 16
111 IV V VI
15 148 108 -
Component
L-fucose o-mannose (identified as the phenylhydrazone) amino sugar o-galactose D-glucosaminc o-galactosamine (chondrosamine)
1
Physicel form o//raclion
crystalline sirup sirup crystalline crystalline hydrochloride crystalline hydrochloride
~
Total, 346 0
The developer was 1-butanol, half saturated with water.
and as 1,2 : 3 ,4-di-O-isopropylide11e-~-psicofuranose)was found among the products of the action, a t 37") of ammonia on ~ - g l u c o s e . ~Chromato~(~) graphic assay of the products formed by the action of this base on maltose and lactose revealed their partial conversion to m a l t u l o ~ e ~and ~ ( ~lactu) 1 0 ~ e , ~respectively; ~(~) ammoniacal melibiose yielded melibiulose, ti-O-(c~-~galactopyranosy1)-p-D-mannose, D-tagatose, and ~ - g a l a c t o s e . ~ ~ ( ~ ) A fine example of the rapacity and selectivity of powdered cellulose is (80) L. Houyh and J . K. N Jones, J . Chenr. Sac., 4047 (1952); 4052 (1952); 342 (1953). (81) P. A ,J. Gorin, L. Ho~ighand J . K. N . Jones, d . Chem. Sac ,2140 (1953). (82) J. K. N. Jones, J . Chern. Sac., 3643 (1954). (83) L. Sattler, F. W. Zerhan, G . I,. Clark, C.-C. Chu, N . Albon, D. Gross and H. C. S. de Whalley, Ind. Eng. Chem., 44, 1127 (1952). (84) (a) L . Hough, J . K. K.Jones and E. L. Richards, J . Chem. Sac., 2005 (1953); (b) 295 (1954).
74
W. W. BINIiLEY
the quantitative determination of raffinose in raw-beet sugars.85h chromatogram of 20 g. of the raw sugar, on a 30 by 7.5 cm. (diam.) column of adsorbent, sharply separated 0.5 % of raffinose (as the pentahydrate) from the very large amount of sucrose and extremely small quantity of stachyose; the developer was 2-propanol/l-butanol/water(7/1 /2).a6 Starch columns were found suitable for the separation of certain monos a c c h a r i d e ~These . ~ ~ columns, pretreated with aqueous I -butanol and the developer 1-butanol/l-propanol/water (4/1/1), and developed under
2oot
a
c
c
3
L
150-
L L
f
-
E
e
l
100P aJ
: 5 Fn
x
50 -
f.n
01
I
I
I
20
50
100
Effluent volume, mi.
FIG.11.-Chromatogram, on a 20 x 0.9 cm. Potato-Starch Column, of (a) L-Rhamnose, (b) L-Fucose, (c) D-Ribose, (d) D-Xylose, (e) D-Mannose, ( f ) D-Glucose, and (g) o-Galactose. [The Developer was 1-Butanol/l-Propanol/Water(4/1/1).]
pressure, \wre capable of sharp separations (see Fig. 11). The capacity of starch was found to be lorn; the maximum weight-ratio of adsorbate to adsorbent was ca. 1:1000. The coordinated use of powdered-cellulose and starch chromatography has far-reaching potentialities. Cellulose powder has moderately good capacity, and the rate of flow of developer through its columns is good if slight pressure is applied. Filterpaper strips and the cellulose powder have identical adsorptive properties, (85) D. Gross and N . Albon, Arinlyst, 78, 191 (1953). (86) Unless otherwise noted, solvent ratios will be expressed on a volume basis. (87) S. Gardell, Acta Chem. Scand., 7, 201 (1953). Potato starch was conditioned by suspension in a mixture of 1-butanol and water; the total moisture in the system was in the weight ratio of waterlanhydrous starch:3/10.
COLUMN CHROMATOGRAPHY OF RTJGARS
75
and they form a useful chromatographic partnership. The progress of colored and of fluorescent (iii ultraviolet light) suhstaiices, moving on cellulose, is readily followed. Chromatographic cellulose is expensive, and it usually requires some treatment before use. Starch is capable of separating closely related sugars. I t is inexpensive, but t,he rate of flow of developer through starch columns under pressure is low, and the adsorbate capacity of starch is very low. This carbohydrate must be conditioned and prewashed before it is suitable for chromatography. 4. Ion-Pxchange Resins
Serious thought and a considerable amount of effort have been devoted to the utilization of ion-exchange resins in the sugar industry.88Some success has been achieved in the beet industry, but the situation is much less certain in cane-sugar production. The principal objectives are the retention by the resins of the ionic (organic and inorganic) impurities and the maximum recovery of sucrose. Quite different is their use in the separation of sugars. A column of an anion-exchange resin (Amberlite IR-400,s9 regenerated with sodium hydroxide) retained, in general, reducing sugars when developed with water. Sugar alcohols and methyl a-D-glucopyranoside were not adsorbed,g0 and sucrose was only partially retained. The sugars were completely eluted by 10 % sodium chloride. The chromatographic separation of sugar mixtures was not attempted. The polyhydroxy functions of sugars and related substances permit the formation of complexes with the borate ion of boric acid and borate salts.g1 These complexes are readily formed in low concentrations of borate and display different adsorptive strengths when added to a column of an anionexchange resin (Dowex-la2) regenerated to the borate form.93 The chromatogram was developed first with 0.018 M sodium borate; D-fructose and D-galactose, in succession, appeared in the effluent, followed by D-glucose, which required 0.03 M sodium borate. The adsorptive strength of nonreducing sugars is dependent upon their molecular weights, stachyose being more strongly bonded than raffinose, and raffinose more tightly held than (88) R . Kunin and R . J. Myers, “Ion Exchange Resins,” John Wiley and Sons, Inc., New York, N . Y., 1950; R . Kunin, Chem. Eng. News, 32, 3046 (1954). (89) A product of the Resinous Products Division of Rohm and Haas Co., Philadelphia, Pa. (90) S. Roseman, R . H. Abeles and A . Dorfman, Arch. Biochem. and Biophys., 36, 232 (1952). (91) J. Boeseken, Advances i n Carbohydrate Chem., 4, 189 (1949). (92) A product of the Dow Chemical Co., Midland, Mich. (93) J. X. Khym and L. P. Zill, J. A m . Chem. Soc., 73, 2399 (1951).
76
W. W. RINKLET
The effect of a functioiial group was even greater, the adsorptive strength of D-fructose being higher than that of the oligosaccharides. A mixture of the pentoses, D-ribose, D-arabiiiose, and D-xylose, was resolved .~~ by Dowex-1 when developed with 0.015 M potassium t e t r a b ~ r a t e The fine selectivity of this method was established by its successful application to a series of disaccharides (see Table V). The progress of these chromatograms was followed quantitatively (for total sugar) with anthrone963 y7 and orcinol r e a ge nt ~ .~The 8 individual sugars were identified by paper chromatography after the removal of potassium ions with a cation-exchange resin. The isolation of pure sugars was completed after the formation, and removal under diminished pressure, of the boric acid as its methyl ester.99 This method is also applicable to sugar alcohols, as exemplified by the TABLE V Chromatography; on an Anion-exchange Resin ( D o w e x - I ) , of Certain DisaccharidesQs Disaccharide (order of appearance in efluenl)
Volume required lo produce maximum concentralion in eBuent, 1nl.b
Sucrose Trehalose Cellobiose Maltose Lactose
175 370 660 750 X1@ ~
The developer was 0.005 M K2B,07 . 6 An amount of 10-25 mg. of each sugar waa added t o an 11 by 1 cm. (diam.) column of resin. a
separation of D-glucitol, galactitol and D-mannitol; to sugar acids in the resolution of D-galacturonic and D-glucuronic acids on Dowex-1,92 acetate or formate form, with aqueous acetic or formic acid as the developerloo; and to amino sugars in the chromatography of D-glucosamine and D-galactosSedoheptulosan is readily sepamine (as hydrochlorides) lol on D0wex-50.~~ arated from the more strongly adsorbed s e d o h e p t u l o ~ e . ~ ~ The chief disadvantage of the method is the unusually large volume of (94) G. R. Noggle and L. P. Zill, Arch. Biochem. and Biophys., 41, 21 (1952). (95) J. X. Khym and L. P. Zill, J . Am. Chem. Soc., 74, 2090 (1952). (96) R. Dreywood, Ind. Eng. Chem., Anal. Ed., 18, 499 (1946). (97) D. L. Morris, Science, 107, 254 (1948). (98) A. H. Brown, Arch. Biochem., 11, 269 (1946). (99) L. P. Zill, J. X. Khym and G. M. Cheniae, d . A m . Chenz. Soc., 76, 1339 (1953). (100) J. X. Khym and D . G. Doherty, J . A m . Chem. SOC.,74, 3199 (1952); J. X. Khym and W. E. Cohn, ibid., 76, 1818 (1954); J. X. Khym, D. G. Doherty and W. E. Cohn, ibid., 76, 5523 (1954) for the chromatography of o-ribose phosphates. (101) S. Gardell, Acta Chem. Scand., 7,207 (1953); S . Gardell and S. Rastgeldi, ibid., 8, 362 (1954); E. Drake and S. Gardell, Arkiw Kemi, 4 , 469 (1952).
COLUMN CHROMATOGRAPHY OF S U G A R S
77
developer required in order to achieve the desired separations. The durutiori of the experiments is long, and the recovery of small amounts of separated sugars from large volumes of solution is tedious. TABLEV I Chromatographic Adsorption Series,= on Calcium Acid Silicutc, of Some Sugars and Related S r ~ b s t u n c e s ~ ~ ~ CInss Z (10 cc. of 90% dioxane) a-D-Galacturonic acid Lactose monohydrate 1,actitol Drilcitol Melezitose, raffinose pentahydrate, gentiobiose, D-glyccro-D-gubheptose Sucrose, maltose monohydrate, cellobiose, D-glucitol (sorbitol) D-Galactose, D-mannitol D -Glucose, o -fructose, I) -mannose, L-sorbose L-Fucitol L-Arabinose Class IZ (5 cc. of 90% dioxane) L - Fu cose o-Xylose, L-rhamnose monohydrate Methyl a-D-ghcopyranoside Class I11 (10 cc. of 90% diosane) D-glycero-D-gulo-Heptonamide o-Galactonamide, D-glnconamide Class ZV (5 cc. of 90% dioxane) D-Lyxonamide D-Ribonamide L-Fuconamide a Arranged in decreasing order of adsorptive strength. Adsorbent: 1.8 g . of Silene EF10Z/Celite38 (5/1) (wt. ratio). Dimensions of adsorbent column: 0.9 X 11 cm. Adsorbate solution: 2 mg. in 0.2 cc. of 90% dioxane. Developer: noted following the class heading.
5 . Calcium Acid Silicate In addition to fuller's earth clays,43certain synthetic silicates are useful in the column chromatography of sugars. Calcium acid silicate (Silene EF102) was, in its original e v a l ~ a t i o n , found ' ~ ~ to be suitable for the separation of such groups of sugars as pentoses from hexoses, and mono- from oligo-saccharides, following the general pattern of increased adsorptive strength with molecular weight (see Table VI) . The influence of molecular (102) A product of the Columbia Chemical Division, Pittsburgh Plate Glass Co., Barberton, Ohio. (103) I,. W. Georges, R . S. Bower and M. L. Wolfrom, J . Am. Chem. Soc., 68, 2169 (1946).
78
W. W. BINKLEY
weight was as usual overshadowed by that of the carhosyl group of the sugar arid ; in the series studied, tlic 1,oud het~\veeua-D-galacturouic acid and this silicate adsorbent was the st,rongest. C'ertain properties of this adsorbent, preseiit, a st,roiig rase for its c-hromatographic~use. ITnlikcfuller's eart'h clays, t,his syiit,hetic: silicate is readily estjrricled from st,rsight, glass tubes. The adsorlieiit is white, arid if it is used with t,he proper dcvelopers (aqueous dioxilrie or aqueous lert-butyl alcohol), dctectioii of slowly reduring sugar substances is possible with the permangallate-streak reagent,. Separate lot,s of t,his silicate varied greatly in adsorbent properties when test'ed under st,aiidard conditions with il mixture of D-glucaose and maltose."'" The adsorbents were gradually deact,ivated by continuous, water washing; they were reactivated by sodium hydroxide and certain salts. The pH of an aqueous susperisioii of the alkali-reactivat,ed adsorbent is 10-1 2; and so TABLE VII T h e pH of th,e Supernatant Liquors froni the M i x t u r e of 1 9. of Various Silicate Adsorbents with 16 ml. of Water'"" Adsorbent*
- ~ _ _ ~ _ _ _ _
I
__-_~
Hgdrnt,etl c:ilciiim :wid silic:ite, lot. K O . 1" Hytlr:Lt,ed c:ilciuu x i d silicate, lot, N o . 2" Hydrat.erI calcirini acid silicate, lot, N o . 3" Hydrated magnesium acid silicate,, Fuller's earth clay"
PfJ
10.3 10.4 10.6 8.3 8.6
a The adsorbents were mixed with :t filter aid in the weight-rtit,ios of 5/1. EF.'a2 c Magnesol.'o5 d Florex XSX.43
* Silene
the adsorbent is then unsuitable for the resolution of alkali-sensitive sugars. The alka1iiiit.y of untreated calcium acid si1icat.e was greater t,hsn that, of some other si1icat.e adsorbents (see Table 1'11). Sufficient. colloidal and soluble iiiorganir substances are roiitributed to the eluat,e by the adsorbent, during zoiie elution, t,hat a tedious procedure must, be followecl hi order t o recover the resolved adsorbate. These difficulties are not. encountered in t,he chromat,ography of sugar derivatives which require much less polar developers. The principal advantages of t,his adsorbentJ are it,s low cost, its \vhite color, the fairly high rate of solveiit percolation through it, and t,he ease of its ext,rusioii. (104) D . 0 . IIotTrnaii (with h l . I,. Wolftom), P1i.D. Ilissertation, The Ohio St,:it,e Universit,y, 1048. A n mioiirit of 0.2 ml. of :L solut>ioriof r)-glucosc (100 mg.) anti malt,ose (100 ing.) in a Inixcture of I nil. of w:iter :mtl $1 ml. of dioxane was added a t the t q of a 120 X 0 mm. (tlinm.) colu~niiof Silenc E;F/filter aid (.5/1, \ v t . rat,ios). The clirornnt,ograin was developed wit,h 8 ml. of :L soliition prel)*.red by mixing 1 i d . of vi:tt,er wit,h 10 ml. of diossrie.
COLUMN CHROMATOGRAPHY O F SITGARS
79
6. Other Adsorbents Certain adsorbents have been selected for specific, sugar separations, without consideration of t>heirpossible general applicability t,o carbohydrates. Mixt'ures of C14-labeledD-glucose and D-fructose were resolved on a column of hydrated magnesium acid silicate,lo6employing 05 % ethanol as the developer."J6The acid-treated silicate (essentially silicic acid) was useful as an adsorbent in the investigation of the structure of sapote gum.107 Alumina,'O* one of the most popular chromatographic adsorbents, has rarely been used for uncombined sugars. A sharp separation of the branched (upper zone) from t,he linear (lower zone) constituent of starch was stated to be effected on a column in which the upper section of the alumina was adjusted to pII 4.5 and t,he lower section tJopH 8.2; t'he developer was 0.0025 N iodine in pot)assium iodide.logA multiplicity of zoiies resulted on chromatographing animal glycogens on freshly precipitated calcium carbonate; aqueous iodine was employed as the developer and zone indicator.l10 The adsorption on silica gel of the polysaccharides of Mycobacterium tuberculosis and successive development with hexane, chloroform, methanol, and water produced 21 distinct fractions.11*
111. COLUMN CHROMATOGHAPHY O F SUGAR DEarvATIVEs I . p-Phenylaxobenxoyl Esters
The epic of the column chromatography of sugars began a little more than a decade The sugars D-glucose and D-fructose, being colorless, were esterified with a colored acyl halide, to yield colored sugar derivat,ives. The acylating agent chosen was p-phenylazobenzoyl ("azoyl") chloride, and red-colored, crystalline pentaazoates were obtained. A mixture of these esters was adsorbed on a column of silica, and developed under pressure with a 1/1 mixture of benzene/petroleum ether (b. p. SO-SO"). The progress of the resolution was followed visually; a typical Tswett chromatogram was (105) hlagnesol, it hydrated magnesium acid silicate produced by the Food Machinery and Chemical Corporation, Westvaco Chemical Division, South Charleston, W. Virginia. This adsorbent has been useful in the chromatography of several types of acyl sugar derivative. (106) G. R. Noggle and R. A . Bolomey, Plant Physiol., 26, 174 (1951). (107) E.V. White, J . A m . C h m . SOC.,76,4906 (1954). (108) H. Brockmann and H. Schodder, Ber., 74, 73 (1941). (109) pvl. Ulmnnn, Makrorrrol. Chem., 9,76,97 (1952); 10, 147,221 (1953); Die Sturke, 4, 73 (1952); Pharmazie, 7, 787 (1952). (110) V . V. Koval'skil, Doklady A k a d . Nauk S. S . S. R., 68, 1083 (1947); Chem. Abstracts, 44, 7902 (1950). (111) B. Siege], G. A . Candela and R. M. Howard, J . Am. Ch.e,m. SOC.,76, 1311 (1954). (112) W. S. Reich, Compt. rend., 208, 589, 748 (1939); Biochem. J . (London), 33, 1000 (1939).
80
W . W. BINKLEY
produced. A red-colored zone containing the more strongly adsorbed D-fructose ester remained near the top of the column; below it was an orange-colored interzone, and lower still on the column was a second redcolored zone containing the D-glucose ester. This classical demonstration initiated the modern, column chromatography of sugars and led to the preparation of a new and interesting series of sugar esters (see Table VIII) In addition to alumina and silica, &lagneso1106 and silicic were found to be useful adsorbents for the separation of sugar azoates; the TABLE VIII Sugar Azoates Suitable f o r Clhromatograph?/‘13 Azoafe o/
a-D-Glucose 0-D-Glucose a-D-Galactose p-D-Galactose 0-D-Fructose a-D-XylOSe (3-D-Arabinose p - L - Arabinose Sucrose a ,a-Trehalose (3,fl-Trehalose a-Lactose @-Lactose a-Gentiobiose p-Maltose &Cellobiose p-Melibiose Meleeitose
Sinfers at, “C.
265
268
.Welling poinf, “C. (mi-.)
265-266: 252-253 275-276 255-255.5 124.5-125.5 15C-157 261.5-262 262-262.5 125-125.5 134-134.5 328-329 287-288 199-204 232-233 274275 272-273 279.5-280
127-130 143-145
223
-50
436 170 -440 244 -755 755 35 210 17 320 167 62 2 105 172 188 146
selectioii of the proper developers is critical in the evaluation of these adsorbeiits (see Table IX) . l I 3 “Azoyl chromatography” of sugars, employing Magnesol, separated mono- from di-saccharides, such as D-galactose from lactose, as well as the two disaccharides, sucrose from lactose.115Application of this method, with silicic acid as the adsorbent, resolved these binary mixtures of sugar azoates: D-galactose from D-fructose, D-glucose from melezitose, and a- from P-D-galactose; and was extended successfully to ternary and quaternary (113) G. H. Coleman end C. M. McCloskey, J . Am. Chern. Soc., 66, 1588 (1943). (114) Reagent grade, Merck and Go., Rahway, N. J. (115) G. H. Coleman, 8.G. Farnhani and A . Miller, J . Am. Chem. SOC., 64, 1501 (1942).
COLUMN CHROMATOGRAPHY OF SUGARS
81
Several factors have limited the general acceptability of this method. Completely azoylated sugars may contain up to 85% of the azoyl group. In addition to the tedious procedure for the preparation of these esters, coiisiderahle quantities of the comparatively rare axoyl chloride are (YJI~sumed. Reconversion of the axoates isolated t80 the free sugars is uiiTABLE IX InJ?iiPnce of the Dciirloprr on the Bvuluatzon of Adsodents for the Separation p-o-Gltirose Penfaazonfe and p-Cellohzosr Clctanzoatc'13 Adsnrbenl
I
/)eveloper
:
(ij
Re.solulioir
Magnesol and Dicalitee Silicic acid
I,igroine/benzene/chloroforni 1/1/1 (Developer A)
Magnesol and Dicalite Silicic acid Magnesol and Dicalite Silicic acid Silicic acid
A containing 0.4% of ethanol"
Good; two zones; cellobiose zone remained a t top of column Good; two zones
A Containing 0.4% of ethanol A containing ]-lo% of ethanol
Fair; two zones Poor t o none
A containing ]-lo% of cthanol Chloroform
Poor t o none Adequate; two zones; cellobiose zone remained a t top of column Adequate; two zones; cellobiose zone remained at top of column Adequate; two zones; cellobiose zone moved down somewhat from top of column Poor
A
Silicic acid
Chloroform with 0 1% of ethanol
Silicic acid
Chloroform with 0.2% of ethanol
Silicic acid
Chloroform with more than 0 2% of ethanol
None
A diatomaceous filter-aid produced by the Dicalite Co., New York, N . Y. cent,
* Volume per
satisfactory. However, the idea of combination of a colored group with sugars to be chromatographed was fuiidameritally sound, arid some of these objections have now been eliminated. Partially methylated D-glucoses were azoylated and then chromatographed 011 alumina, using chloroform as the developer,L16and 011 silica (prepared from sodium silicate) with chloroform/beneene/petroleum ether (b. p. 65-1 10"):l / l / l . I L 7 The color (116) K. Myrback and C. 0. Tsmm, Svensk Kern. l'idskr., 63,441 (1941). (117) J. K. Mertzweiller, D . M. Carney and F. F . Farley, J . Am. Chem. Soc., 66, 2367 (1943).
82
w.
U'. %INKLEY
iiitroduced into a hexose molecule I)y a single azoyl group is suficient to permit visual observat,ion of t.he progress of t,he adsorbat>edown the column. The adsorpt>ivebonds become weaker with increase in number of azoyl groups and with devrease in number of methoxyl groups. The yields in the formation of this type of compound were 95 ?4 or better, and the recovery of resolved substances was nearly quantitative. Hardly believable though it may seem, a mixture of methylated sugars had been separated, virtually quantitat,ively, at room temperature with very simple apparatus. A new and powerful tool for the elucidation of structure of polysaccharides had been forged; but, until recently, it was rarely used. This method was further refined"* before actual use in the structural studies of polysaccharides. Partially methylated D-glucoses were reduced t o their corresponding hexitols, which were converted to their azoyl esters, thus avoiding the complication of anomers. Markedly simplified chromatograms were obtained with alumina.118- The utility of this method was demonstrated by its successful application120to the hydrolyzate from fully methylated lichenin. Elution of the zones from the chromatogram on alumina led to the recovery of 68 f 4 % of 1,4,5-tri-0-azoyl-2,3,6-tri-O-methy~-D-glucitol (denoting 1 -+ 4 links) and of 32 f 4 % of 1,3,5-tri-O-azoyl-2,4,6-tri-O-rnethyl-~glucitol (denoting 1 -+ 3 links), which confirmed the results obt,ained by a different chromatographic method.121 Another interesting variation of the chromatography of sugar derivatives containing both colorless and colored substit,uent groups is the use of the acetates of p-phenylazophenyl glycosides.lz2These compounds are prepared from interaction of poly-0-acetylglycosyl chlorides (formed more easily than methylated sugars) with p-hydroxyazobenzene (more readily obtained than azoyl chloride). They were adsorbed on silica gel'23and then developed with chloroform or benzene, or with a mixture of these solvents with petroleum ether. Typical separations were D-xylose and D-glucose; (118) R. A. Boissonnas, Erperienlia, 3,238 (1947) ;Helv. Chim. Acta, 30, 1689 (1947). (119) The alumina must be exactly neutralized with acid prior t o use as an adsorbent for sugar esters since these are decomposed by alkali. R. W . Jeanloz, H . G. Fletcher, Jr., and C. S. Hudson, J . Ant. Chern. SOC.,70,4054 (1948). H . E. Stavely and J. Fried, ibid., 71, 137 (1949). (120) R. A. Boissonnas. Helv. Chim. Acta, 30, 1703 (1947). (121) K. H . Meyer and P. Gurtler, Helv. Chim. Acta, 30, 751 (1947). (122) C. D. Hurd and R. P. Zelinski, J . Am. Chem. Soc., 69, 243 (1947). (123) The silica gel was prepared by slowly adding 200 ml. of 3 N hydrochloric acid t o a mechanically agitated solution of 100 g. of water-glass in 200 ml. of water. The gel was broken up; it was heated on a steam bath in a current of air t o remove most of the excess water and acid. The friable residue was crushed, washed with water until neutral t o litmus, and dried a t 70" for 3-4 days. It was ground t o 40-60 mesh before use.
COLUMN CHROMATOGRAPHY O F SUGARS
83
D-galactose, D-glucose, and maltose; lactose and maltose; and D-xylose, D-glucose, and lactose, all as their acetylated p-phenylazophenyl glycosides. 2. Acetate Esters
The introduction of the “brush” or “streak” technique40for the locating of zones of colorless substances added vital impetus t’o the progress of modern, column chromatography. Colorless compounds are adsorbed on columns of white or light-colored adsorbents; the chromatogram is developed, and the moist column of adsorbent is extruded. The column is then brushed or streakedI24lengthwise with a reagent sufficiently sensitive to undergo a color change in the presence of the adsorbates. The resolved substances are located; the column is cut into appropriate sect’ions,and the portion of the adsorbent wetted by the streaking reagent is removed by gentle scraping. These sections are then eluted, and the individual materials are recovered. This method has been extended to sugars and to several of their colorless derivatives. Its first and most extensive app1icat)ion has been to sugar acetates. Several factors influenced the selection of this group of compounds. They are a simple type of sugar derivative, are easily prepared, and are soluble in many organic solvents. Often, sugar acetates crystallize readily, and they are most extensively described in the sugar literature. Further, the individuality of the sugars is masked far less in the acetates than in their benzoate and azoate esters, and so sharper chromatographic separations can be expected. Directive acetylation was employed in order to encourage the formation of one anomer of each sugar; thus, each resolved sugar would be represented by a single zone. Hydrated magnesium and calcium acid silicates (MagnesolIo5and Silene EF,lo2respectively) are often suitable for sugar-acetate chromatography. The original extension of the brush technique to sugar acetates employed Magnesol,4* and this adsorbent has since been generally preferred. A major factor in the subsequent success of the method was t,he discovery, during its adaptat,ion to sugar acetates, of an unusually versatile txush or streak reagent., namely, alkaline ~erma1iganate.l~~ The chromatographic selectivity of Magnesol is notably dependent upon its moisture content.Iz6 The octaacetates of p-gentiobiose and p-maltose are st,rongly adsorbed and (124) This may he accomplished with a medicine dropper the tip of which is drawn out fine. (125) The streak reagent was prepared by dissolving 50 mg. of potassium permanganate in 5 ml. of 2.5 N sodium hydroxide. It was most, sensitive immediately after preparation. (126) M . L. Wolfrom, A . Thompson, T . T . Galkowski and E:. J. Quinn, Annl. Chcm., 24, 1670 (1952). This interesting eti’ect is not uniisual with adsorbeIit,s.
84
\V. W. BINKLEY
are only slightly separated at low moisture contents; zones of these substances are diffuse and widely separated when this adsorbent possesses over 20 % of moisture. The best results (based on the consequent separation of these acetates) mere obtained in the moisture-content range of 12-18 %. A benzeneltert-butyl alcohol (125/1) developer was used in these evaluations, and i t has frequently been preferred to benzene-ethanol mixtures because of its greater stability to alkaline ~ e r m a n g a n a t e . ~ ~
~ZZZZ-
a-Cellohexaose eicosaacetate
a-Celiotriose hendecaacetate
a-D-Glucopyranose pentaacetate FIG.12.-Chromatograms of Cellulose Acetolyzate on (a) Hydrated Magnesium Acid Silicate arid (b) Hydrated Calcium Acid Silicate. [The Chromatograms were Developed with Benzene/Ethanol (100/1) .] See Addendum, p. 94.
Mono-, di-, and tri-saccharide acet,ates were easily separated, using benzene -ethanol developer, into groups whose composition reflected the influence of molecular weight (see Fig. 12) .Iz7 The influence of t.he position of (see Fig. l3),lz6.128 and t,he loss of polarit,y of, a funct,ional group (see Fig. 14)Iz9on the adsorbabilit,y of acet,ates was interesting. Transformat,ion of t,he carbosyl group of pentn-O-acet,yl-D-gluconic acid t,o cert,ain nitro(127) E . E. Dickey and M. I,. Wolfrom, J . A m , Chptn. Soc., 71, 825 (1949); M. I,. Wolfrom and J . C . Dacons, ibl'tE.,'74, 5331 (1952); J. G . Leech, T a p p i , 36, 249 (1952). (128) W. W. Binkley and hf. 1,. Wolfrom, ./. Ant. Chevt.SOC.,68, 1720 (1946). (129) Ref. 7 ( g ) , p. 28.
85
COLUMN CHROMATOGRAPHS OF SUGARS
p-Gentiobiose octaacetate
@-Maltose octaacetate
-a-D-Glucopyranose pentaacetate a-D-Glucopyranose pentaacetate
~
~
FIG.13 -Chromatograms, on Hydrated Magnesium Acid Silicate, of (a) 01- and p-i>-Glucopyr:tnose Pentaaretntcs and of (b) p-Gentiobiose Octaacctate and p-Maltose 0 c t aa ce t at e .
pentaacetate
+ ’L’//,’///,/////:
fi-D.Glucopyranose __f
2,3,4,64etraacetate
OAC
H @.D.Glucopyranose
,’, / ,/
pentaacetate
A
c--- c‘’
’
‘OR‘
I
FIG.14.-Relationship Between the Adsorptive Strength and the Polarity of C1 of Certniri D-Glucose Acetates Chromatographed on Hydrated Magnesium Acid Silicate.
8G
W. W. BINRLET
genous and other derivatives produred, 011 a silicic acid adsorbeiit’”Owith henzenc-acetic acid developers, the following series (in order of derreasing adsorptive strength) : amides, phenylhydrazides, acids, lactones, and nitri1es.la1 Kumerous publit-atious i n the last decade have testified to the soundness of sugar-acetate chromatography on Magnesol as a separative procedure. The potentialities of the method are demonstrated in the elucidationz6, of the structure of the trisaccharide, p a n 0 ~ e . lThe ~ ~ acetate of its sugar alcohol (panitol dodecaacetate) was partially hydrolyzed and then chromatographed. The five components of the hydrolyzate were separated, recovered as crystalline materials, and properly identified. They were acetates of D-glucitol, p-D-glUCOpyranOSe, 0-isomaltose, maltitol, and unreacted panitol; thus, the structure of the trisaccharide was unequivocally established as 4-0-(cr-isomaltopyranosyl)-~-glucose.The presence in amylopectin of glycosidic 1 -+ 6 links (indicating branched structures) was proved by the chromatographic isolation of the acetates of p-isomaltose [~-O-(LY-Dglucopyranosyl)-p-~-glucose]~~~ 135 and panose.26(a) Maltotriose [4-0-(0(maltopyranosyl)-~-glucose]~~6 was found1” among the hydrolytic products of starch, and the structure of this triose was confirmed26(a)by “acetate chromatography.” Application of the method to glycogen revealed the .~~~ existence of glycosidic 1 + 6 links in this animal p o l y ~ a c c h a r i d eCrystalline aldehydo-D-galactose heptaacetate and aldehydo-D-xylose hexaacetate were separated chromatographically from the acetolyzates of guaran acetate and xylan acetate, respe~tive1y.l~~ 1
(130) A product of the Mallinckrodt Chemical Works, St. Louis, Mo. The adsorbent mixture was silicic acid/Celite No. 535 (3/1). (131) J. F. Haskins and M. J. Hogsed, J. Org. Chem., 16, 1275 (1950). (132) M. L. Wolfrom, A . Thompson and T . T . Galkowski, J . A m . Chem. SOC.,73, 4093 (1951); A . Thompson, M. L. Wolfrom and E. J . Quinn, ibid., 76, 3003 (1953). (133) S. C. Pan, A . A. Andreasen and P. Kolachov, Science, 112, 115 (1950); S. C. Pan, L. W. Nicholson and P. Kolachov, J . Am. Chem. SOC.,73,2547 (1951). (134) M. L . Wolfrom, J. T . Tyree, T. T . Galkowski and A . N . O’Neill, J . Am. Chem. Soc.,73,4927 (1951). (135) The first isolation of crystalline p-isomaltose octaacetate was accomplished by means of Magnesol chromatography; I,. W. Georges, I . L. Miller and M. L. Wolfrom, J . Am. Chem. SOC.,69, 473 (1947); M. L. Wolfrom, L. W. Georges and I. L. Miller, ibid., 71, 125 11949). (136) J. M. Sugihara and M. L. Wolfrom, J . A m . Chem. Soc., 71,3357 (1949). (137) M . L. Wolfrom, L. W. Georges, A . Thompson and I. L. Miller, J . Am. Chem. Soc .,71,2873 (1949). (138) M. L. Wolfrom, E. N. Lassettre and A. N . O’Neill, J . Am. Chem. Soc., 74, 3162 (1952). (139) R . 1,. Whistler, Eileen Heyne and J. Bachrach, J . A m . Chem. Soc., 71, 1476 (1949).
COLUMN CHROMATOGRAPHY OF SUGARS
87
In addition to its use in carbohydrate structural studies, sugar-acetate chromatography has been employed as an analytical tool. The first isolation of D-glucose and D-fructose (as their crystalline acetates) from cane juice was achieved by its aid.128D-Glucose, n-fructose, D-mannitol, and myoinositol were found in cane, blackstrap molasses (see Fig. 9),53.140 and, in addition to these, D-arabitol and erythritol in its fermentation residue (see Fig. The interconversion, by heat, of D-fructose to n-glucose was column chroreadily predicted on the basis of enolizatiori matography supplied the proof.50hiialysis of the acid reversion products of D-glucose by a combination of carbon chromatography and magnesium acid silicate chromatography led to the isolat,iori of gent.iobiose, isomaltose, maltose, cellobiose, sophorose, P ,P-trehalose, and 1evoglClcosan (all as acetates), and of the acetate of an unknown disaccharide, temporarily designated y - a ~ e t a t e . ' ~ ~ Sugar-acetate chromatography was instrumental in the preparation of crystalline, key-intermediate compounds in some intricate syntheses of sugars, such as keto-D-glycero-D-gulo-octulose heptaacetate in the format,ion of ~-glycero-~-gulo-octulose.~~~ Sugar diazo compounds have often been purified by this technique; some derivatives responding were l-deoxy-ldiazo-keto-L-galacto-heptulose p e n t a a ~ e t a t e ,1-deoxy-1 '~~ -diazo-keto-D-glyceroD-galacto-octulose h e ~ a a c e t a t e , ' ~and ~ 1-deoxy-1-diazo-keto-L-manno-heptulose p e n t a a ~ e t a t e . The ' ~ ~ anomeric a- and @-D-glucosaminepentaacetates A chromatogram of an acet'ylated, residual were resolved on Magnes01.l~~ sirup from tjhe preparation of N-methyl-L-glucosaminic acid nitrile separated the acet,ate thereof from its epimer, penta-0-acetyl-N-methyl-Lmannosaminic a~id.1~8 Magnesol chromatography was one of the keystones in the actual isolation of sucrose (as its octaacetate) after its first chemical synthesis.149This (140) W. W. Binkley, NI. Grace Blair and M. L. Wolfrom, J . A,tn. Chem. SOC., 67, 1789 (1945). L:Wolfrom I. and W. L. Lewis, J . Am. Che,m. Suc., 60, 837 (l(328). (141) & (142) A. Thompson, Kiiniko Anno, M. 1,. Wolfrom and M. Inatome, J . Atti. Chma. SOC.,76, 1309 (1951) ; the y-acetate is now known t o be 3 - O - ( o l - ~ - g l u c o p y r a n o sy l ) - ~ - ~ glucopyranose octaacetate. (143) M. L. Wolfrom and A . Thompson, J . Am. Chem. SOC., 68, 1453 (1946). (144) M. L . Wolfrom, J. M. Berkebile and A . Thompson, J . Am. Chem. SOC., 71, 2360 (1949). (145) RI. I,. Wolfrom and 1'. W. Cooper, J . A m . Chein. SOC., 72, 1345 (1950). (146) M. I,. Wolfrom and H. B. Wood, J . A m . Chem. SOC.,73, 730 (1951). 71, 2870 (147) M. I,. Wolfrom, R . U. Lemieux and S. M. Olin, J . A m . Cheni. SOC., (1949). (148) M. L. Wolfrom and A. Thompson, J . h a . Chem. SOC.,69, 1847 (1947). 76, 4118 (1953). (149) R . U. Lemieux and G. Huber, J . Am. Chem. SOC.,
88
W. W. BINKLEY
adsorbent can distinguish the difference between the glycosidic bonds of sucrose octaacetate and isosucrose octaacet at.e.150 A chromatographic series showing t.he adsorptive strength between certaiii sugar acetates and a hydrated calcium acid silicate (Silene EF) has been prepared (see Table X) .lo3 151 Silene EF is a weaker acetate adsorbent than is Magnesol; thus, the former has greater utility in the separation of the acetates of t,he larger oligosaccharides, such as those cf a-cellotetraose, a-cellopentaose, a-cellohexaose, and a-cellohept)aose (see Fig. 12).lZ7 T h e 9
TABLE
Chroriialoqraphic Adsorption Series; on Calcium Acid Silicate, of Some Sugar Derivatives'~3 J
Class I[I5 cc. of benzenelethanol (250/l)b] Raffinose hendecaacctate 8-R.lelibiose octaacetate, sucrose octaacetate 8-Maltopyranose octaacetate Class ZZ 115 cc. of benzene/ethanol (500/1)] keto-wFructose pentnacetate D-Glucitol (sorbitol) hexaacetate, D-mannitol hexaacetate 6-D-Glueopyranose pentaacetate ~ - - ~ - A m b i r i o p y m n otetraacetate se CIoss I I Z [15 cc. of benzenc/ethanol (lOOO/l)] WL-FUCO t>et,raacetate S~ Class I T T (15 cc. of benzenelethanol ( l O O / l ) ] 2,3-Di-O-methyl-~-glucose 2 , 3 ,B-Tri-O-methyl-n-glucose Class V [12.5 cc. of benzene/ethanol (250/1)] 2,3,4,6-Tetra-O-methyl-~-glucose a Arranged in decreasing order of adsorptive strength. Adsorbent: 1.8 g. of Silene EF1°2/Celite" (5/1) ( a t . ratio). Dimensions of adsorbent column: 0.9 X 11 cm. Adsorbate solution: 2 mg. in 0.5 cc. of absolute benzene or chloroform. Developer: noted in class heading. b The benzene was thiophene-free, and the ethanol was absolute.
increased adsorptive strength of this series reflected the increase in their molecular weights. 3. Methyl Ethers Nearly all polysaccharides are st,ableto alkalis, aiid often they are met,hylat,ed almost completely by ail appropriate, single t>reatment with methyl su1fat)e aiid a strong hase.15' Vsually, succwsive t'reat,ments with this or (150) W . W . I h k l e y atid hI. I,. Wolfroni, J . A v i , C h n . Soc.., 68, 2171 (1946). (151) The variability in adsorptive strength of various lots of Silene EF for sugaracetate chromatography was small, and its relatively large content of alkali and other water-soluble material was not troublesome. (152) W. N. Haworth, J . Chenr. Soc., 107, 8 (1915).
COLUMN CHROMATOGRAPHY O F STJG.4 RR
89
other methylatiiig s g e ~ l t s produce l~~ fully methylated products which may he used a.s at.art,iiig materials for t.hc elucidation of the structure of the polysaccharides. Formerly, t,licse et,heritied subst,aiices wcrc hydrolyzed, and the rt:sulting partiadly rnethylated proclucts were subjected to frart>ional distillatiou under diminished pressure. Rarely is this rectification completely satisfactory because of the inaccuracies introduced by demethylation, pyrolysis, arid incomplet,e recovery of the methylated sugars. These dificult.ies have tjo a great extent been eliminat'ed by chromat,ography. The colored azoyl esters of partially methylat,cd sugars were prepared, and the conditions established for t.heir resolution cn and silica.123Separat.ion of the methyl glycosides of 2 , 3 ,6-tri-O-met.hyl- and tetra-0-methyl-D-glucopyranose was accomplished with a flowing rhromatogram on The completely methylated hesoside moved rapidly on development with an ether-petroleum ether mixture, and was recovered in 94% yield from a portion of the column effluent,. The more strongly adsorbed methyl tri-0-methyl-P-D-glucoside appeared next in the effluent; however, development n-ith methanol was necessary in order to release the a - anomer; ~ the total yield of the anomers was 88%. These recoveries were good, but not quantitative. The magnitude of t.he repeat,ing units in rice starch and banana starch was found by this method to be 33 and 26 D-glucose residues, respectively; these values confirmed the rcsult,s obtained by fractional-distillation 156 The ariomeric met,hyl glycosides of fully methylated D-glucose and D-galactose have been partially separated on alumina, with chloroform as the developer.157The formation of anomers during the preparation of these met,hyl glycosides is a complication; often, two zones represent a single component of the original mixture. Polymerization and demethylation may also occur during glycoside formation. These object,ioris have for the most part been met by using a combination of (a) extraction with part,ition solvents and (b) chromatography on silica-water columns, for the resolution of di-, t,ri-, and tetra-0-methyl-Dg l ~ c o s e s The .~~~ Molisch reagent was used for detecting the zones in the extruded chromatograms of these substances. EssentJially the same separa1
(153) T. Purdie and J. C. Irvine, 6.Chem. Soc., 83, 1021 (1903). (154) J. K. N. Jones, J . Chern. Soc., 333 (1944). (155) E. L. Hirst and G. T. Young, J . Chem. Soc., 1471 (1939). (156) E. G . E. Hawkins, J . Ihatacetylation of each of their two distinct crystalline N-phenyl-D-ribosylarniiies gave a different glass-like triacetate, Todd and coworkersb1present evidence for believing t,hat these amorphous triacetates arc, in fact,, one compound. Thus, hydrolysis of t,hc aniline residue, followed by ncetylatioii of the result,ing triacet,at,e,gives, from each, D-ribose (61) M. FrBrejacque, C o r q t . rend., 202, 1190 (1936). (62) K. Butler, F. Smith and M. Stacey, J . Chem. SOC.,3371 (1949).
112
G. P. ELLIS AND JOHN HONEYMAN
1,2,3,4-tetraacetate. Consequently, when such changes in structure may accompany acetylation, caut,ion is necessary in applying the results of acetylation in structural studies. If mutarotation precedes acetylation, the structure of the derived acetate has little relevance to the problem of determining the structure of the unest,erified glycosylaniine. Indirect methods for preparing such acetates include the condensation of 0-acetyl-aldosyl bromides or aldohexose 2 , 3 , 4 , G-tetraacetates with the appropriate amine, usually in chloroform or ether solution. Another method, introduced by Frkrejacque,61consists in heating an aldohexose 1,2 ,3 ,4 ,6 pentaacetate in aqueous, ethanolic, acet,ic acid and adding the amine to the cooled solution. These methods lead to the isolation of the compound with the 0-D-pyranose structure. Frhrejacque examined some of these acetates and showed that, by keeping the compound a t its melting point for an hour, a mixture of the original compound and its anomer is obtained. These were separated by fractional recryst,allization, so making available several anomers previously unobtained. From the specific rotation then det,ermined, F r k r e j a ~ q u ereported ~~ that8 such acet'ates do not conform to Hudson's rules of isorotation, but berth^^^ has shown that the acetates of primary glycosylamines do conform t,o these rules, the contribution of C1 being 16,000. Helferich and PortzZ5found trhat addition of benzylamine t o a- or 0-D-glucopyranose peutaacetate removes the acetyl group on C 1, giving an addition compound of D-glucose 2 ,3 ,4 ,G-tetraacetat,e with benzylamine, which, on heat>ing, yields fv-benzyl-D-ghcosylamine t,et)raacetate. Another method for preparing this compound is of interest. Helferich and Mitrowsky6$found that D-glucosylamine tetraacetate readily gives Schiff bases on reaction with aromatic aldehydes. They prepared in this way N-benzylidene-D-glucosylamine tetraacetate which was readily hydrogenated in the presence of Raney nickel to N-benzyl-D-glucosylamine t,etraacetate. When a- or P-D-glucopyranose pentaacetate is treated with piperidine (t,hreemolecular proport,ions are best), deacetylation occurs on C1 and C2, whose and the product is N-(3,4, G-tri-O-acet,yl-D-glucosyl)piperidine,66 structure was proved by conventional methods, including its conversion into 2-O-methyl-~-g~ucose. The same triacetate is obtained when piperichloride or with D-glucose dine reacts witah 3,4,G-tri-O-acet~yl-~-glucosyl 2 , 3 , 4 ,G-tet,raacet'ate. Loss of t,he acetyl group on C2 must accompany or
+
(63) M. Frhrejacque, Conipt. r e n d . , 204, 1480 (1937). (64) A. Bertho, A . m . , 662, 229 (1949). (65) B. Helferich and A . Mitrowsky, Che,nz. Ber., 86, 1 (1952). (66) J. E. Hodge and C. E. Rist, J. A m . Chem. SOC.,74, 1498 (1952).
GLYCOSTLAMINES
113
precede formation of the cart,on-to-iiitrogeii bond, since N-(%,3,4,&tetrao-acet,yl-D-glu(,osyl)piperiditieis unaffected by piperidiiie. Deacetylat'ioii of the esters, which proceeds normally, has hecn successfully carried out with ammonia, sodium methoxide, or harium methoxide in methanol. In this way, for example, N-p-tolyl-p-D-glucosylamiiie 2 , 3 , 4 , (i-tetraacetate is converted into N-p-tolyl-p-D-glucosylamine, and N-acetyl-p-D-galactosylaniiiie tetraacetate into N-acetyl-p-D-galactOSylamine.5z All tfhe known crystalline acetJat#esof the glycosylamines, whether prcpared directly or indirect,ly, have the pyranose ring st,ructure. One reaction sequence carried out by Kuhn and DansiZ7is, from the s h c t u r a l point of view, typical of the method used to determine structure. Condensation of p-toluidine with D-glucose 2 , 3 , 4 ,6-tetraacetate or with 2,3,4,6-tetra-0acetyl-a-D-glucosyl bromide leads to the same compound, which is, without, doubt, N-p-tolyl-/3-D-glucosylamine 2,3,4,G-tetraacetate. The same rompound is obtained by the acetylation of N-p-tolyl-p-D-glucosylamine. In addition, Ellis and H ~ n e y m a nhave ~ ~ successfully used acid hydrolysis of N-p-t801yl-p-D-ghicosylaminetetraacetate (prepared from N-p-tolyl-p-Dglucosylamine) as a method for preparing D-glucose 2 , 3 , 4 ,&tetraacetate. Incidentally, t,his makes an att,ractivepreparative method for such acetates, alternative to that involving the 0-acetylaldosyl bromide. The various stages that have been achieved are shown on p. 114. A somewhat similar reaction sequence was carried out by Butler, Smith and StaceyfiZfor N phenyl-D-galactosylamine. The method has been extended to the ketose series by Barry and H o ~ i e y m a nshowing ,~~ that the crystalline tetraacetates, readily obtainable from N-phenyl- and N-p-tolyl-D-fructosylamine by acetylation in pyridine, are also pyranose. These tetraacetates are hydrolyzed to D-fructose ll3,4,5-tetraacetate. This tetraacetate does not condense with aniline, but 1 , 3 , 4,5-tetra-O-acet~yl-~-fructosyl chloride does, to give the above N-phenyl-D-fructosylamine 1,3,4 ,&tetraacetate as the only recognized product, in 10 % yield. One interesting and, at first, surprising instance was described by Zemp1611, Csuros and B r u c k i ~ e r who , ~ ~ found that trimethylamine reacts with the heptaacetates of cellobiosyl bromide and of lactosyl bromide, respectively. Later work by Zempl6n and BruckneP showed that the product of such reactions is the same as that obtained when dimethylamine is used. Thus, the cellobiose reaction product is N ,N-dimethylcellobiosylamine (67) G. ZemplBu, Z . Csiirijs and Z.Bruckner, Ber., 61, 927 (1928). (68) G . Zempi6n and Z . Bruckner, B e r . , 61, 2481 (1928).
114
G . P. ELLIS AND JOHN HONEYMAN
H OH iV-p-Tolgl-P-D-glucopyranosyhmine
>
CHiOAc
c-
H
H < AcO
H ,OAc
H
OAc
A’-p-Tolyl-P-Dglucopyi anosylamlne tetraacetate
OAc
D-ClucoW 1,2,3,4,6pcnt aacetate
tl H OAc ~ ~ - G l u c o sbromide yl 2,3,4.6-tetraacetate
H OAc D-Glucose 2,3,4,6tetraacetate
FIG.1
heptaacetate. This reaction appears comparable with that between tertiary amines and alkyl halides,69as exemplified by the following. 2 CsH5N(CHJ)2f C6HllBr + CsH6N(CHR)J3r f C6H5K(CI-T3)C.5H~1
The reaction betweeu tertiary amines and “acetobromo” sugars was further studied by F. Micheel and Hertha M i ~ h e e 71I ~who ~ ~ found that trimethylamine forms quaternary ammonium salts with some acetylated glycosyl bromides (for example, those of D-glucose and D-galactose) but not with others (for example, those of D-mannose and L-rhamnose). This was attributed t o the C2 hydroxyl group’s being on the same side as the oxygen hridge to C l in D-glucose and D-galactose, but 011 the opposite side (69) A C l a w and P. Rnutenberg, Ber., 14, 620 (1881). (70) F Michcel and Hertha Micheel, Be? , 63, 386 (1!)30) (71) F hIichce1 arid Herthn Michcel, Ber , 63, 2862 (1030).
GLYCOSYLAMINES
115
in D-mannose and L-rhamnose. The formation or nonformation of a quaternary ammonium salt is thus useful for determining whether a sugar has the Q or ,B configuration. Most glycosylamine acetates are readily obtainable in crystalline condition. Their stability is excellent; they can be stored indefinitely without occurrence of any decomposition. Since their melting points are sharp and reproducible, and their chloroform solutions do not mutarotate, they are derivatives suitable for identifying sugars and glycosylamines.
5. Benzoylation
The preferred method for the preparation of crystalline benzoates of g l y c ~ s y l a m i n e sis~ ~to treat the glycosylamine in pyridine with benzoyl chloride a t 0" for periods of up to four hours; longer reaction times are detrimental. I n one instance, reaction with benzoyl chloride in aqueous sodium hydroxide solution was used successfully, but the yield was lower than by the pyridine method. By these methods, all of the hydroxyl groups were benzoylated and the resultant ester was obtained by pouring the reaction solution into water and, if necessary, extracting with ehloroform and purifying and evaporating the chloroform solution. In this way, the tetrabeiizoates of N-p-tolyl-D-glucosylamine and N-p-tolyl-D-mannosylamine (and N-p-tolyl-n-xylosylainine tribenzoate) were obtained in high yield. From certain N-phenylaldosylamines treated in this way, benzanilide was the only crystalline product isolated. Similar treatment of N-phenyl-Df r ~ c t o s y l a m i n egives ~ ~ the tetrabenzoate in high yield, but a comparatively low yield (30%) was obtained from the N-p-tolyl derivative. The benzoates are more resistant to acid hydrolysis than are the corresponding acaetates,but by boiling with hydrochloric acid in acetone the amine radical is removed, giving good yields of D-glucose 2,3,4,6-tetrabenzoate, ~ - i n a i i n o x2,3,4,&tetrabenzoate, D-xylose 2 , 3 , -k-tiit)enzoate, and D-fructose 1,3,4,thtetrabenzoate, compounds of known structure, previously prepared and c h a r a c t e r i ~ e d . ~ ? - ~ ~ Benzoylation thus resembles acetylation in that the glycosylamine rcacts in the pyranose form. However, only one anomer of each benzoate has heen isolated. Again, this is an attractive method, alternative to the use of 0-bcnzoylaldosyl bromides, for preparing aldose benzoates with C1 unsuhtituted. (72) 5:. Fisclin :mcI €I. N o t h , Bcr., 61, 332 (1918). (73) R.I
5
3m
+
e
In]",degrees
R~iere,rre>
-
~
-96 4 , s diniethyl-l,2 AT,N'-phen!.lenedi-, octaacetatt [ ~ ~ , N ' - d i - ( t e t r a - O - a c e t ~ l - u - g l u c -4,5os~l) dimeth?.l-o-pheri3.lenedisnii1ie] N-1 -dodecylS-(3-etho.tyethylideiieamino-4 niethylphenyl)-, 2,3,4,6-tetraacetate ,\J-2-(l-ethoxyeth~4idenea1nino)phenyl-, 2,3,4,6tetraacetate .V-o-ethosyphenylS-p-ethougphenyl-
tetraacetate A-ethylA-(Ar-ethylthiocarbamoy1)-, tetraacetate Xl-heptylN-l-hexadecylN-1 -hexyl-
AV-(2-hydroxy-3,5-dinitrophenyl)4,6-O-ethylidene-2,3-oxidoethylideneS-2-hydroxyethyl.V- (2-hydroxv-5-nitrobenzylidene)
IS1
--t -30 -19.4
47 34
105.5 138
-65.7
4G 116
189
-91.7
116
157 110-120 (clec.) 11s 115-116 92 132 107- 105 157-1 59 97 97-98 105105 79-80 93-95
-96.1
4
-35.3
c
-80.5 -22 -28.6
+
-27.4
-12.5 -12.3 +15.4
-13
+
4
+
-7
4-10.1
+
t29.5
-25.3
4
13.5
165
200 11S116 116 (dec.) 170
173 12 20 25 PO, l i l 90 90 92 117 21 21 117 46 65 175 163 45
65
N-o-hydroxyphenylN-p-hydroxyphenyltetraacetate pentaacetate N -3-hydroxy-4-propoxycarbon ylphenylN-(4-iodo-2-nitropheny1)tetraacetate N -0-methoxyphenylN-p-methoxyphenyl2,3,4,6-tetraacetate 2 , s ,4,6-tetramethyl ether
N - (4-methyl-2-nitrophenyl). tetraacetate, I tetraacetate, I1 N-methyl-N-phenyl-, 2,3,4,64etraacetate N-methylsulfonyl-, tetraacetate N-(N-methylthiocarbamoyl)-, tetraacetate N-[2-methyl-3- (0-tolylazo)phenyl]N-l-naphthylN-2-naphthyl-
-+
-+
- 126 +8.6 -69.8 -38
+297 f259 f257
---t -+ -+
+lo7 +I57 +145
AcOEt EtONa-EtsO EtONa-Et?O
78-80 207 215 (dec.) 120-128 1so 129 102 160-162 182-1 84 135 92 117 113-120 (dec.) 172.$1 73 70-75 184 172-186 178
-30.3 -31.8 -51.7 +107.7 49 $51 +10.3 $13.1
+
-86.5
-+
-111
-+
-58.0 -48.1 -133.5
-136.3 -114 +26.1 -75.2 -+
-171
CHCI, CHCl p AcOEt AcOEt-HCI CHCl3 CHCI, RleOH AleOH C&H,N (?)
C5HbN AcOPvIe
19 65 25 65 124 116 116 173 8 161 162 I62 162 117 176 165 113 34 34 34 161 161 65 92 177 57 12 178 178 17 17, 34 39 35
143
2,3,4,6-tetraacetate N-o-nitrophenyltetraacetate N-m-ni trophenyl-
+I2 -68 -12.4
-88 -89.4
CLY COSYLAMINES
N-methylN- (N-methylcarbamoyl) -
148-150 148-149 140 133 135-137 (dec.) 136 (dec.) 135 146 86 129 110
TABLEXI1 (Continued) Con+ound
Melting point, "C.
[a],, , degrees
n-Glucosylamine, A-m-nitrophenyl- (Continued) -169 tetraacetate N-p-nitrophenyldihydrate
2,3 ,4-triacetate 6-trityl ether tetraacetate N-acetyl3-methyl ether, monohgdrate
N-(N-1-octadecylcarbainoyl). N-l-octadecylN-l-octglN-l-pentylWphenyl-
175 136 175 184
219 80-140 180 155 161 182
181-189 (dec.) 103.5 102 9697 crc. 147 (dec.)
147 (dec.) 110-150 (dec.) 140
-1
-81.5 -212 -192 --t -202 -150 + -215 -125 --t -161
CjHJ-AcOH CjH5N CHClr CsH5N CjHj?; CjH 5SHrO C jHjS-AcOH
- 120 - 101 +lo0 -319 --t -192.5 -198.5 4 -196.7 -215 --t -198
CjHjX CHCI. CHClz C jH 5N C5HjX-HjO CjHjN-AcOH
--t
- 132
-100
Rolalion soloenl
Reievences
35 18 18 18 35 35 35 35 35 35 35 18 35 35 35 135 46 46
-22
--t
-44 -47
-8
4-10.2 --t -52.3 -15 + -52 f11.25 + -51.9 +14.8 + -14.3 +37.2 -+ -85.6 +36.9 +27.7
EtOH EtOH MeOH MeOH MeOH MeOH H20 NaOH-H?O HCl-HJO HCI-HrO
21, 117 6 6 11
23 32 46 46 46 46
l-
r. e
134-135
tetraacetate N-acetyl2,3,4,6-tetraacetate, a 2,3,4,6-tetraacetate, p-
2-methyl ether 3-methyl ether 4-methyl ether 6-methyl ether 2,4-dimethyl ether 3,4-dimethyl ether 2,3,4-trimethyl ether 2,3,6-trimethyl ether 2,4,6-trimethyl ether 2,3,1,6-tetramethyl ether
131-136 9696 118 100 143 149-1 50 98 97 97-98 161 154-155 182-1 83 130 196 2 19-220 177-178 145-116 140 sirup 160-162 163-165 135 135
137-139 13s 133-135 134-135 N-1-phenJ-lztzophenyleneN - (I\i-plienylcarbamoyl)2-deosy -h-phenyl-
199 223 (dec.) 193-194
+53
+10.5
+
-20.6
+76.1 +180 + +41.6 +180 -73.7 -54.8 + t 4 1 . 6 -57 -106.6 +38.3 -108.5 + -50.3 -84.0 4 -39
+30 f 3 - 166
-79 -81 +238.4 +224 4 +17 +285 + +lo6 +266.6 + +162.9 +290 + +160 +228.7 + +58.9 +266 + +58.3 +227.8 + f 5 7 . 5 240 +238.5 -114
.--)
+
--t
CHCl, CHCl,-ScOH CHCI, EtOH CHCl1-AcOH CHCl, AIeOH SIeOH MeOH
MeOH EtOH
MeOH MeOH AIe2CO% MeOH AcOH-AcOEt EtOSa-EtiO EtOSn-Et~O AIeOH-HC1 MeOH-HCI MeOH ;LIe2C0 CHClj
H,O
-55
-138
16 33
-52.2 -50.9 --*
-106
c 5H 6N
23 179 161 63 32 161 63 32 57 1so 1803 57 181 lROa 58 59 152 141 183 183 184 11 162 162 162 185 56 55 55 55 111 165 156
0
2
0
% 2
* 3P
F
5
Y
?P iil
[elD, degrees
Compound
o-Glucosylamine, A-phenyl-2-deosy- (Continued) 3,5,6-trimethyl ether N-2-phenylethyl-, monohydrate N - (N-phenylthiocarbamoyl)-, tetrascetate N-p-sulfacetamidophenyl2,3,4,6-tetraacetate :V-p-s:ilfamylphenyl-
18&189 135-136 92-93 110 (dec.) 159 197-198 (dec.) 195 204 210 204
tetraacetate hexaacetate N-p-sulfamylphenyl-or-,2,3,4,6-tetraacetate N-p-sulfamylphenyl-~-,2,3,4,6-tetraacetate N-p-sulfophenylN-thiocarbamoylN’-benzoyl-, tetrabenzoate N-o-tolpl-
N-m-tolyl-
189 115 204-205 (dec.) 204
>300 210-212 (dec.) 215-216 205 101 97-98 97 95-96 117
106-107
-153 -64
-102 -60 -19.2 -25 ---t +2 - 22 - 56 +12.8 4
---t
- 123 - 125 - 122 -119.6 +29.7 -86 77 203 +197 -81
+
+
- 36 -35.7 +45 -99 --t -51 -103 4 -22
-79 -102.9 -102 -97.5
+
+
+ +
-50 -50.3 -32 -49.5
Rolalioa solvenl
CsH5Y IIeOH EtOH CsHsN CHCl3 H,O
CsH,?j HLO aq. SstlCOa
H?O H?O aq. HC1 CjH5N C5HsS C sH s N CHCI, C5HsS
H2O H,O C .H sN XeOH EtOH
MeOH MeOH EtOH hIeOH
40 40 187 36 65 125 121 19 19 19 125 188 188 19 124 124 124 124 125 92 166 113 47 47 20 173 33 47 47 33
t, Y
t 2:
3
N-p-tolyl-
N -p-tolyl-cutetraacetate N-p-tolyl-Bhemihydrate monohydrate
135-136 125 117-119 (dec.)
-94.6 + -21.1 --t -97.6 + -92.5 ---t -101.2 +
-82 -85
3,4,6-triacetate 2,3,4,6-tetraacetate
,V-acetyl2,3,4,6-tetrabenzoate
133-134 147 143-146 148 143-1 44 144-145 145-146 141-1 42 147-1 48 146 142 209
2-methyl ether 2,3-dimet,hyl ether
15G151 151
-47.3 -45 -45.2 -35.5 -45.8
-47.6
MeOH EtOH MeOH MeOH CHC13-AcOH MeOH MeOH MeOH EtOH MeOH
-40 -44.5
HzO EtOH CHCl,
-57.5 + +34.2
AcOMe CHClrAcOH
-33.3 -34.2 -35.0
CHC1, CHC13 CHClz
---t
-26.6 +64.2 t14.1 +50.0 EtOH
12 12 12 27 32 20 9 9 189 161 27 63 20 32 32 55 179 189 61 55 55 21 135
147
+26.6 (equilibrium value)
6 6 12 16 63
GLYCOSYLAMINES
115-120 (dec.) 114-115 117-1 18 112-1 13 115
-43.9 -38.8 +l81.9 + -45 $208.9 + -44.6 4-119 + +34.2
TABLEXI1 (Continued) c Rololioia solvenl
n-Glucosylamine, N-p-tolyl-P- (Continued) 2,3,4,6-tetramethyl ether
I44 147-150 151
151-152 192 (dec.)
.v-p-tolylsulfonyl2,3,4-triacetate, 6-p-toluenesulfonate tetraacetate 6-p-toluenesulfonate N-u-Glucosylhenznmide, 2-thioet hyl ether N-n-Glucosylcarhnmic acid, ethyl ester meth?l ester pentyl ester N-~-Glucosyl-3-carbanioyl-l,2(or 1,6)-dihydropyridiiiium bronlide 6’-tetraacetate 2’, 3’ ,4’, i~-~-Glucosyl-3-carbamoylppridinium bromide
N-n-Glucosyl-3-carhethoxy-l, 2-dihydropyridine, 2’, 3’, 4’,6’-tetraacetate ~-n-Gliicosyl-3-cyano-l, 2-dihydropyridinium bromide, 2‘, 3’,4‘, 6’4etraacetate N - ~ - G l ~ c o ~ y2-dihydropyridine, l-l, 2‘,3‘,4’,6‘tetraacetate
I9i 174-176 163 132 186-189 (dec.) 66-72 75-8 1 88
203-205 157-158 192-200 151-152 (dec.) 146.5
156 154156
+I63 --+ ? f272 -+ +I10 +249 + f153 +220.5 -+ +157.5 $221.4 --+ +60.0 +207.0 -67.5 --+ +10 -67.5 -+ $10 + +42.5
MeOH AcOEt-AcOH
EtONa-EtZO EtONa-EtzO MeOH CHCl,
EtOH-H20 EtOH-HCl
+33.5 f29.6 -5.5
CsHsS
-24.5 -13.7 -6.6 +4 .9
HrO H.?0 Et,O H2 0
-11.1 -18.3
+28.6
CHCl, CHClx
Refeerniaces
13 27 162 162 162 55 55 27 27
hb
00
? ’j M F
$ 65 65 65 65 94 189a 159a
155 190 190 190 190Q 190
190 190
* 15 cl
Z
m
0
e:
5z
ethyl ester sodium salt N-n-Glucosylguanidine N-D-Ghcosylguan ylgl ycine N-D-Glucosylguanylglyc ylglycine N-~-Glucosylhydantoicacid ethyl ester, tetraacetate potassium salt N -n -Glucosylhydantoin n-Glucosyl isocyanate, tetraacetate D - G ~ U C isothiocyanate OS~~ tetraacetate
N-n-Glucosyllysine N-n-Glucosylpiperidine
hydrochloride 2,3,4,6-tetraacetate 3,4,6-triacetate 2-carbanilate 2-p-nitrobenzoate 2-p-toluenesulf onate 2-methyl ether
+ +18.2 +24.8 -5 -8 + +20 4-31.96 + -0.57 -115
-6.6
N-D-Glucosylglycine, barium salt 108 94 (dec.) 208 (dec.) 180 (dec.) 169.5170 149-149.5 134-137 (dec.) 27G271 117-1 18 112-114 111.&113 100 75 (dec.) 115 129-130 130 (dec.) 144 150-151 123; 136 125 (dec.) 164 158-159 (dec.) 154 (dec.) 113
-25.8 -3
+5 -7.5 -12.8 +6.02 -8.5 -16.9 -43 + -13 +8.5 +3.0 +4 4 +33.5 -1 + $33 +31.6 +37 f52.3 17 +23
+
46 46 190b 154a 132 154 154 191 191 191 191 166 189a 191 166 166 192 19 20 45 45 45 45 193 66 66 66 66 66
d
84 F
>
z
M
TA
TABLE XI1 (Continued) Compound
Herling point, "C
[ a l,degrees ~
Kolalion S d U e n l
____
N-o-Glucosylpiperidine (Continued) 2-carbanilate 2-p-toluenesulfonate 2-methyl ether 3-methyl ether tetramethyl ether N-o-Glucosylpyridinium bromide, tetraacetate N-D-Glucosylpyrrole N-D-Glucosyl-DL-serine N-D-Glucosylsuccinamide tetraacetate N-o-Clucosylsuccinimide dihydrate N-o-CIucosylsulfapS.ridine N-D-Glucosylthiocarbamicacid, ethyl ester, tetraacetate N-D-Glucosylthiohydantoicacid ethyl ester ethanolate tetraacetate potassium salt
N-~-Glucosyl-2-thiohydantoin N-D-Glucosyltrimethylammoniunibromide 6-trityl ether N-D-Glucosyltrimethylammonium chloride, tetraacetate N-D-Glucosyltrimethylammoniumchloroplatinate, tetraacetate N-D-Glucosyltrimethylammoniuniiodide N-D-Glucosyltrimethylammoniumpicrate tetraacetate N-D-Glucosyltrimethylarnmoniumperchlorate, tetraacetate 0
3
Ll
0
152 (dec.) 111-112 (dec.) 114 13Q-131 74 174 190
+63 3-4
-2.5
-15 203-204 192 88-90 103-104 159-160
4 -6.0 +8.8 +91.7 -6.43
+19 +12.8 -17.4 --t
+11.5
CjHsN CsHsS C,HsS C sH sS MeOH
HLO
H,O (CHC1,)i
HZO
(CHCI?)*
66 66 66 36 60 194 195 154a 166 166 166 152b
z
113
>
3
? 3 F F
2:
3
2 152-1 55 119-1 21 151-152.5 137-141 (dec.) 224-225 161-162 183-185 173
-31.3
+a +22.8
+5.0 +14.8 4-6.26
EtOH
191 191 191 191 191 196 153
HZO
196
HzO CHCI,
H?O HrO
209-210
196
162-163 144 133 190
196 196 196 196
The elementary analysis given by the authors does not accord with this formulation.
2
2,
z
2 3
5z
151
GLYCOSYLAMINES
TABLEXIIA D-Idosylamine Derivative Con,pound ~~
D-Idosylamine 6-deoxy-N-phenyl-, 3-methyl ether
1
1
62-63
I
~
196a
TABLEXI11 Lactosylaniines and T h e i r Derivatives Compound
f e l l i n g poinf, "C
degrees
Iefer:nces
(?I
-98.3
+
+24
CHCla-AcOH
192
-14.3
-+
+24
CHC13-AcOH
63
HL' H,O CHCli MeOH
HZO
165
-89
H,O
I04 167b
-21.4
C6H6
130
+2.1
30-240 (dec.
208-21 2 154 17-110 (dec. 139 (dec.) 197 152
N-p-sulfamylphenyl trihydrate
190 210-212
N-p-tolyl-a-, heptaacetate N-p-tolyl-@-,heptaacetate
189 202 208 169-170
Lnctosyl isothiocyanate, heptaacetate N-Lactosylthiocarbamic acid ethyl ester, heptaacetate
Rololion solvenl
44-148 (dec. 209
246-248 142-146
+39.5 +38.5 +1.5 f 0.5 4-2.7 +7.4
-26 +90.2 $21 +lo1 -31 -31 4 +21 --f
N -phen y1-p-, hep twncctate
-
12 128 1961) 196b 196b 46 63
Lactosylamine N-acetyl-, dihydrate heptaacetate, monohydrate N-4-biphenylN-p-bromophenyl-a-, heptaacetate N-p-bromophenyl-p-, heptaacetnt e N -carbarnoylN-(4-cnrboxy-3-hydroxyphenyl) sodium salt phenyl ester, tetrahydrate N , N-dimethyl-, heptaacetate N-l-dodecylN-p-ethoxyphenylN-phen yl -a- , heptnacet at e
,
'[a],
119
-69 -79 182.3 + +24
-29
-+
+24.
46 19 61 63 61 63 152b 124 C 6H 6N H?O 124 CHCls-AcOH 63 61 CHCla-AcOH 63 114
C,H,N CHCl2 CHCla-AcOH CHCl3 CIIC~:S-ACOII
114
-
152
G . P. ELLIS AND JOHN HONEYMAN
TABLE XIV 1)-Lyxosylamines and Their Derivatives ~
Melting
Compound
point, “C.
[a]” , degrees
Rotation soluenl
References
______
D-Lysosylamine iV-3,4-dimethyIphenylN- (1,5-dimethyl-2-nitrophenyl) 2‘,3 ‘ , 4’-triacetate N-p-nitrophenyl-
142-143 146-147 198-199 190-191 143-145
-109 f 3 -154 f 2
C5H5X CsHsN
197 197a 108 108 198
TABLEXV L-Lyxosylartziiie Derivatives Compound
Melting
poinf,v-,
L-Lyxosylamine N-(4,5-dimethyl-2-nitrophenyl)- 195196 2’, 3’, 4’-triacetate 184-185
[=ID, degrees
Rotatiotz sotuenf
Refer-
+112 f 2 +155 f 2
C5H5N C5H5N
108 108
ence
TABLEXVI Maltosylamines and Their Derivatives j f e l l i n g point, "C.
N , N'-Dimaltosylbenzidine Maltosylamine heptaacetate N - (4-carbomethoxy-3-hydroxypheny1)N - (4-carboxy-3-hydroxyphenyl)N -0-carboxyphenylN-l-dodecyl-
175 ( d e c . ) ca. 165 ( d e c . ) 191-191.5 235-240 13&145 153-155 4&54
Rotation solvent
Rejerences
+I3 +I18 +73.7 -35.7 -42.4 t48.9 -+ +68 +67 +73 +72 -+ +86 +68 +65 +97.4 +92 +93.4
HzO Hz0 CHC1, C 5H sN C sH sN MeOH Ha0 HCl-H*O AcOH-H~O AcOH Hz0 HCL-HzO
127
+76.5 +37.5 +92.5 +36.9 - 186
H2 0 CHC13-AcOH CHCla CjHsN
+39 +94.4 +39.3
CHCls-BcOH CHC13
[&,degrees
-+
N-l-hexylN-l-octadecylN-phenylheptaacetate N -p-sulf amylphenylN-p-tolyl-, heptaacetate
-+
8&115 90-100 205 212-214 236 182
-+
-+
112 64 104 104 13 46 46 46 46 46 46 46 57 63 61 124 199 63 61
0
3 g 4 t?
k
z
M
m
TABLE XVII D-Vannosylnmines Coiiipound
N,N’-Di-(D-mannosyl)urea D-Mannose-1-diacetamide D-Mannose-1 dibenzamide pentabenzoate D-Mannosylamine N-benzoyltetraacetate N-n-butylN-p-carbethoxyphenylN-o-carbomethoxyphenylN - (4-carboxy-3-hydroxyphenyl). sodium salt phenyl ester N-o-carboxyphenylN-m-carhoxyphenylN-p-carboxyphenylN-(3,4-dimethyl-2-nitrophenyl) tetraacetate N - (4,5-dimethyl-2-nitrophenyl)triacetate, 6-trityl ether tetraacetate N-3, C-dimethylphenylN-p-ethoxyphen yl-
(irirl
T h e i r Derivatives
Melting p o i n f , “C.
[aID, degrees
188 219 21&219 226 (dec.) 22.5226 140-142
-45.8
H,O
-13.8 +3.6 t2.8 +25.3
HZO CsH5N C 5H 5N
+6.4 -28.8
CjH5N CHCl,
+7.2 -54 - 191 -97.5
EtOH C 5H 5 s
ca. 254 253-254 135-136 71-72 179-180 177-1 78 140-240 (dec.) 191.5-193 126 136 182 215-216 (dec.) 154-155 213 (dec.) 130 218 18&185 157 (dec.)
-29.4
+ -21.1 - 10 t35.7 -28.5 258 -35.0 -41.1
-
--t
CHC1,
EtOH
H,O
hIeOH EtOH EtOH CHCl, CHCla C 5H 5N C sH 5N AcOMe
-93.8
- 174 -155
Rolafion solvent
-145
C 5H 5N C 5H 5N
Pejerences
165 94 iai 94 101 101 94 101 101 117 115 137 104 104 167h 13 13i 115 34 34 34 17 17 I7 17 15
N-l-hexylN-1- (2-hydrosgnaplithyl) -M-phenylethylN-(4-niethy-2-nitrophenyl)tetraacetate N-2-naphthglN-o-nit rophenyLj3tetraacetate N-rn-nitrophenyl-pN-p-nitrophenyLj3dihydrate
tetraacetate N-l-pentyl-
N-4-phen ylazophenylene N-phenyl-
2,3-dimethyl ether 2,3,6-t rimethyl ether
2,4,6-trimethyl ether 3,4,6-trimethyl ether 2,3,1,6-tetramethyl ether
75 207-208 (dec .) 205-206 (dec.) 144-145 195-196 196 214-21 5 126 127-128 199 209 219
184 70-71 247 181 (dec.) 180-1 81 181 sirup 127-128 131 234 134 140- 143 112-143
144
-97.8 -224 -22.5
- 107 - 103 - 187 -336 -406 -+ -325 -306 4 -282 -333 3 -322 - 150
-178.5
4
-81.5
-179.3 -101.4 -+ -45
-155
4
-39
-150 -+ +8 +154.5 +55.5 -87.9 3 - 8 . 3 -95.5 ---t -38.9 -8 (final value)
CHCli CJHJN CbH5N CHCL CHCl, C5H5N C5Hhh’ C )H sN CjHJ”H3O C~ H ~ N - A c O H CHCL,
CsHjS C 5H 5 s MeOH
MeOH MeOH MeOH MeOH Me&O MeOH-HCl
117 140 34 34 110 18 34 18 34 18 18 35 35 35 18 117 111 54 20 16 16 144 200 20 1 144 202 202 54 54
56 20 1
156
TABLE XVII (Continued) Compound
didring point, "C.
[LII],,,degrees
Rolalion solvent
Yeferences
A-p-sulf amylphenyl-
monohydrste N-p-sulfophenylN-p-tolyl-
tetrascetate 2,3,4,6-tetrabenzoste N-D-Mannos ylguanidine N-D-nIsnnosylpiperidine
14G-145 145-146.5 202 204 194 >300 184 183-184 175 183 165-167 133-134 80 (dec.) 11G-117 11.5116 (dec.)
-84.0
4
-7.5
MeOH
- 163 - 103 - 186
C jH S I X B,O C jH sN
- 181
CsHjN -12 -45.0
MeOH MeOH
-125.6 +10.03 4 -0.72 -17.5 4 +12 -21.6 4 +13.3 -27.7 + +24.2
CHClz HzO MeOH MeOH C SH sN
-44 -101.4
+ --*
202 203 19 125 124 125 204 20 126 16 126 16 132
60 45 45
G . P. ELLIS AND JOHN HONEYMAN
D-Alannosylamine, N-phenyl-,2,3,4,6, - tetramethyl ether (Cont i m e d )
TABLE XVIII L-Rhanmosulamines and Their Derivatives Coin pound
L-Rhamnosylamine 0.5 MeOH 0.5 EtOH N-n-butylN-p-carbethoxyphenylN-o-carboxyphenyl-
N-p-carboxyphenylN , N-dimethyl-, 0-isopropylidene acetal N - (4,5-dimethyl-2-nitrophenyl) triacetate, I triacetate, I1 N -ethyl N-l-heptylN-l -hexylN-l-(2-hydroxynaphthyl)-N-phenylmethylN-methylN- (4-methyl-2-nitrophenyl) triacetate N-o-nitrophenyltriacetate N-m-nitrophenylN-p-nitrophenyltriacetate N -1-pen tyl-
Melfing p o d ,
T.
116 (dec.) 80 136-137 194 167-1 68
165-166 169-170 b. p. 82-84 a t 1 mm. 169 106-107 141-142 138 132-133 192 (dec.) 126-127 215 (dec.) 161-162 225 (dec.) 185 150 208 209 139-140
Rolafion solven
+38 +28 f14 +66.4
$51.2 +42.9 +148.8 + f100.2 f 52 -28.6 -20.2 --t
Hz0 H20 EtOH MeOH EtOH CsH& EtOH EtOH Hz0
References
112 112 126 115 13 13 13 137 115 205
g 4 4
+100.8
CHC13
+104.9
CHCh
f117.6 +191 +320 123
CHCla CsHsN C5H;N CHCl,
+
;:
117 117 117 140 117 34 34 34 34 18 18 l8 117
+
P
B
+ u1 ~
TABLEXVIII (Continued) Comjound
N-phenyl-
2,3-dimethyl ether
2,4-dimethyl ether 2,3,4-trimethyl ether
N -4-phenylazophenyleneN-n-propylN-p-sulfamylphenylN-p-sulf ophenylN-p-tolyl-
'otalion soloenl
Melting point, OC.
118 121-127 (dec.) 144 138-139 135-136 141-142.5 141-142 111-1 13 111 227 145 208-210 (dec.) 181 >300 151-154
-50.4 +77.1 +136.9 --f
EtOH EtOH
206 118 54
207 208 +147.8 +128.5 +110 +138.5 +138.3
+42.8 +5.6 4 +7 4 +16.9 +46.9 +
+
--f
+lo1
+92
+
+80
EtOH EtOH EtOH EtOH Me&O
H20
MeOH
209
m 196a 54 54 210 111 117 211 125 125 126
TABLEXIX o-Ribosylamines and Their Derivatives Compound
n-Ribosylamine N- (2-amino-4,5-dimethylphenyl) -, 2,3-di-O-acetyl5-0-tritylN-p-carboxyphenyl-a-, (pyranose) N-o-chlorophenyl-a-, (pyranose) N- (4,5-diethyl-2-nitrophenyl)-, (pyranose) N- (4,5-dimethyl-2-nitrophenyl) -
Melling p o i n f , "C.
N - (4,5-dimethyl-2-thioformamidophenyl)-, 2', 3' ,4'triacetate N- (4-ethyl-2-nitropheny1)N-ethyl-N-2-nitrophenylN- (3-hydroxy-4-methylphenyl)-,(pyranose), 2 EtOH N-4-methoxyphenyl-a-, (pyranose) N-1-naphthyla-, (pyranose) N-2-naphthyl-, (pyranose) N-o-nitrophenylisomer A isomer B N-phenyl-, Ac
Rotalion solvenf
References
C sH jN
212 172
137-138 (dec.) f10.7 129-130 (dec.) 152-153 171-177 164
2,3-di-O-acetyl-5-0-trityltriacetate 5-trityl ether N-3,4-dimethylphenylisomer Am isomer B*
[a]., degrees
163 118 (dec.) 128-130 (dec.) 110-112 (dec.) 9&98 190.5194.5 130-134 (dec.) 133-135 (dec.) 109-110 (dec.) 146-147 (dec.) 119-120 183-185 (dec.) 167-168 193-194 138-140 (dec.) 126-127 123-124
+231 +136
---* ---*
+70.2 +125
-85 +go 68 160 24 +172 +171.7 4 +56.5 +94.5 4 +53.0
+ + +
-107.5 +116 4 +32.4 +I22 + +40.8 +I22 4 +29.2 +96.6 - 109 -122.5 -109.1 +176.5 -+ +156.6 +180 +180 4 +161 +I82 .+ +52.3
C sH sN C gH 5X C gH gN C5H5N C 5H jN AcOMe C 5H :N
CsH5N C 5H 5 3 C 5H 5N C gH jN
15 15 213 17 172 17 172 17 14 14 172 214 212 15 15 15 15 15 16 16 14 51 51 40
0
r
2
3s
k 3
;R
CL
cn a
TABLEXIX (Continued) Melting point. "C.
Compound
[4,, degrees +135 4-12 +176.4 +134.9 -+ +14.1 +63.4 f48.6 $60 60 +48.8 -+
133-134 (dec.)
Bd, hemihydrate
125-127 (dec.) 119 114-116
-+
+
-+
+62 --* +50 +23 +13 +60.2 +23.8 +8.3 32 ---f
112-114 (dec.)
-+
N-p-tolyl-
resinous 56.5 165-166 168 172-173 169-170 1712172 130 (dec.)
2 EtOH 0.5 HzO
102-103 (dec.) 123 (dec.)
triacetate 2,3,5-trimetJ1iylether 2-deoxy -N-phenyl-
+
+19.5 +17.4 f141 -+ +47 +164 -+ +64 +136.2 -+ +12.5 +178.2 +76.0 f53.2 +58.6 +22.1 -+ +11.7 -+
2~deoxy-N-p-tolylN-~-Ribosyl-3-carbamoylpyridinium bromide
167-168 141-142 (dec.) 147 (dec.) 170 142
N'-acetylN-~-Ribosyl-3-carbamoylpyridine 0 Claimed t o be furanose. some authors.
b
Claimed t o be pyranose.
c
Claimed t o be furanose by some authors.
Rolalion solocnl
References
MeOH C 5H sN MeOH C 5H sN C6Ht.N CSH sN-HZ0 C sH sN MeOH C 5H 5N MeOH CHCI,
40 16 16 14 51 51 40 40 16 16 51 215 216 217 218 219, 220 219 16 16 15 16 16 218 221 19Oa 19Oa 190a
EtOH EtOH C5HsN C sH sN MeOH C sH sN C sH 5N C 5H 5N MeOH HzO HzO H?O HzO d
Claimed t o bepyranose by
161
GLYCOSYLAMINES
TABLE XX L-Ribosylamine Derivative Compound
L-Ribouylamine 2-deoxy-N-phenyl-
Compound
L-Sorbosy laniine N -p-ethoxyphenyl-
217 40
169.b170.5 172-173
p$$:!t. 160
[u]., degrees
Rofalion soloenl
Reference
- 191
C 6H 6PvT
17
compound
N ,N’-Di-D-xylosylurea D-Xylosylamine triacetate N-o-aminophenyl-, triaeetat e N-n-butylN-p-carbethoxyphenylN-o-carbomethoxyphenylN-o-carboxyphenylN -p-carboxyphenylN- (4-chloro-2-nitrophenyl) triacetate, I triacetate, I1 N-(4,5-dimethyl-2-nitrophenyl) triacetate N-o-(1-ethoxyethy1idenearnino)phenyl-, triacetate N-l-hexylN-(4-methyl-2-nitrophenyl) triacetate, I triacetate, I1 N-o-nitrophenyltriacetate N-p-nitrophenyl-a-
N -p-nitrophenyl-p-
Mclfing poinf, ‘C.
230-235 (dec.) 130 101-102 179 81-82 116-117 170 167 172 180 162 117 212-213 (dec.) 168-169 139 87 ca. 170 183 13&132 172-176 149 192
101; 109
dcgrecs
-20 -18.1 -15.9 -22.4 -50
- 28 - 22
Rotation solucnt
H2 0
0) H20 CHCL CHCI,
-11 +61.6 -59.6 -90.1 +96.7,
EtOH EtOH EtOH EtOH C5HsN CHCl, CHCls
-62.1
CHCli
-87.3 f7.9
CHCla CHCla
-109.5 +292.5 +260 4 $280.4 f260 --t +285 -95.6
AcOMe C5H5N C,H,N-H,O C~H~S-ACOH CsHjN
References
113 112 222
6.4 116 117 115 137 137 115 116 116 116 34 34 116 117 34 32 34 34
17 35 35 35 3.5
N -phenyl-
triacetate 2-methyl ether
3-methyl ether 2,3-dimethyl ether
2,4-dimethyl ether
148 140-141 142 142-144 143-144 151 125-126 128 123-124 138 136 146 145
-94.3 -79.6
2,3,4-trimethyI ether
2-deoxy -N -phenyl-
-70.8 -24
-87
+214 +23.7 +77
AcOEt
-+
-48 -21.9
+185 190 +118 + +75
+
-40
-84
-97 -37.4
C~H~X-ACOH MeOH CsHsN MeOH MeOH CHCli AcOEt AcOEt
4
+25
120-122 122-123 123 128 174 155-157 170 121 126 98-100 100-101 102 104 97-98 137
+
-90 -84.1
-7 3,4-dimethyl ether
3
+87 -82 +
AcOEt .ScOEt AcOEt-AcOH
AcOEt AcOEt -AcOH Dioxane
-+
+47
EtOH
+
+32.8 -20
MeOH H2 0
-+
35 39 20 51 40 16 51 223, 224, 225 224a 226 223 227 228 229 229 230 226 227 231 148(a) 232 232 233 234 235 223 225 236 122 224a 237
L3
F
r!
n 0 7l 4
F
>
5
3
n
L
3
w
TABLEXXII (Continued) Compound
Melting point, "C.
[aID, degrees
Rotation solvent
I
References
D-Xylosylamine (Continued) N-p-sulfacetamidophenylN-p-sulf amylphenylN-p-sulfophenylN-p-tolyl-
trihenzoate N-D-Xylosylpiperidine, hydrochloride N-D-Xylosyltrimethylammoniumbromide, 2,3,4-triacetate
139 121 168-169 157 >300 124-125 122-124 124-125 180-181 125 181
-58
-62.3 -48 -41.5 -44-t -12 -76.6 + + 21.2 -59.0 +48.3 +9.6 -20.8
238 1 25 124 125 125 20
126 16 16 55 77 153
GLYCOSYLAMINES
165
(108) Dorothea Heyl, Edith C. Chase, C. H . Shunk, Marjorie U. Moore, Gladys A . Emerson and K. Folkers, J . Am. Chem. SOC.,76, 1355 (1954). (109) V. A. Kon’kova, Zhur. ObshcheZ K h i m . , 22, 1896 (1952); Chem. Abstracts, 47, 5361 (1953). (110) H. Lehr and H. Erlenmeyer, Helv.Chim. Acla, 29, 66 (1946). (111) J . Guilhot, Compt. rend., 223, 89 (1946). (112) C. A. Lobry de Bruyn and F. H. van Leent, Rec. trav. chim., 14, 134 (1895). (113) B. Helferich and W. Kosche, Ber., 69, 69 (1926). , 1916 (1938). (114) T. B. Johnson and W. Bergmann. J . A m . Chem. S O C . 60, (115) Y. Inoue, 0. Konoshin and S. Kitaoka, J . Agr. Chem. SOC.J a p a n , 26, 59 (1951-52). (116) H. Antaki and V. Petrow, J . Chem. S O C .2873 , (1951). (117) E. VotoEek and F. Valentin, Collection Czechoslov. Ghem. Comniuns., 6, 77 (1934). . S O C .37, , 119 (1905). (118) 1’. Hermann, J . R i ~ s s Phys.-Chem. (119) E. 1,. Hirst, J. K. N. Jones and Elin M. L . Williams, J . Chcm. Soc., 1062 (1947). 753 (1939). (120) F. Smith, J . Chem. SOC., (121) F . Smith, J . Chcm. SOC.,744 (1939). (122) P. Andrews, D . H. Ball and J. K . N . Jones, J . Chem. SOC.,4090 (1053). , (1948). (123) W. N. Haworth, P. W. Kent a n d M . Stacey, J . Chem. S O C .1211 (124) R. Bogn&r and P . NBnBsi, J . Chem. SOC.,1703 (1953). (125) Y. Inone, K. Onodera, S. Kitaoka and J. Shishiyama, J . Agr. Chem. SOC. J a p a n , 26, 48 (1952). (126) Y. Inoiie and K . Onodera, J . Agr. Chem. SOC.J a p a n , 22.70 (1948). (127) 0. Adler, Ber., 42, 1742 (1909). (128) F . Micheel, R . Frier, Elizabeth Plate and A. Hiller, Chem. Ber., 86, 1092 (1952). (129) L. Zechmeister, G. T6th and I. Pinczesi, Ann., 626, 14 (1936). (130) P. Karrer and J . C. Harloff, Helv.Chim. Acta, 16, 962 (1933). (131) Adrienne Wilhelms, Magyar Biol. KutatbinlCzet M u n k d i , 13, 525 (1941); Chem. Abstracts, 36, 41 1 (1942). (132) R. S. Morrell and A. E. Bellars, J . Chem. SOC.,91, 1010 (1907). (133) Sybil P. James and F. Smith, J . Chem. Soc., 746 (1945). (134) B. Helferich and H. Schirp, Chem. Ber., 86, 547 (1053). (135) R. hllerton and W. G. Overend, J . Chem. SOC.,35 (1952). (136%) A. Bertho and J . Maier, Ann. 498, 50 (1932). (136b) G . N. Vyas, Nitya Anand and M. L. Dhat, Currcnt Sci. (India), 21, 103 (1052); Cheai. Abstracts, 47, 4856 (1953). (137) Y. Inoue, K . Onodera and S. Kitaolia, J . Agr. Chetn. SOC.J a p a n , 26, 291 (1951-52). (135) J. G. Erickson and Joan E. Keps, J . A m . Chem. Soc., 76,4339 (1953). (130) W. H . Claus and A. n e e , German P a t . 97,736 (1897); Chem. Centr., 69, 11, 695 (1898). (140) M. Betti, Gazt. c h i m . ital., 42, I, 288 (1912); Chcrn. Abstracts, 6, 2417 (1912). (141) D . McCreath and F. Smith, J . Chem. S o c . , 387 (1939). , (1946). (142) E. L. Hirst and J. K . N . Jones, 1.Chert!. S O C .506 (143) G. J. Robertson and R . A. Lamb, J . C h e w . SOC.,1321 (1934). (144) I . Ehrenthal, M. C. Rafiqne and F. Smith, .I. Am. Chem. SOC.,74, 1341 (1952).
1GG
G . P. ELLIS AND JOHN HONEYMAN
E. T. Dewar and E . G. V. Percival, J . Chem. SOC., 1622 (1947). E. L. Hirst and J . K. N . Jones, J . Chem. SOC.,1482 (1939). E . V. White, J . A m , Chem. SOC.,64, 1507 (1942). (a) J . K. N . Jones, J. Chenz. SOC., 3141 (1949). (b) W. G. Campbell, E. L. Hirst and J . K. N. Jones, i b i d . , 774 (1948) (149) P. Andrews, I,. Hough and J . K. N . Jones, J. Chem. SOC., 2744 (1952). (150) G. J. Lawson and M. Stacey, J . Chenz. SOC.,1925 (1954). (151) W. G. Overend, F . Shafizadeh and M . Stacey, J . Chem. SOC.,671 (1950). (1528) H. Lehr, H . Bloch and H. Erlenmeyer, Helv. Chirn. Acta, 28, 1415 (1945). (f52b) G . Cavallini and A. Saccarello, Chimica e industria (Milan), 24, 425 (145) (146) (147) (148)
(1942). (152c) E. G. V. Percival and T . G. H . Thomas, J . C h e m . SOC.,750 (1942). (153) F. Micheel and Hertha Micheel, B e r . , 66, 253 (1932). (154) F. Micheel and W. Berlenbach, Cheni. Ber., 86, 189 (1952). (154a) F. Micheel and Almuth Klemer, Chem. Ber., 86, 1083 (1952). 64,3360 (1932). (155) T. B. Johnson and W. Bergmann, J. Am. Chem. SOC., (156) W. E. Stone, A m . Cheni. .J., 17, 192 (1895). *(157) C. A . Lobry de Bruyn, Rec. trnv. chim., 14, 98 (1895). { (158) A . R. Ling and D. R. Nanji, J . Chem: SOC.,12!, 1682 (1922). (159) K. Josephson, Ber., 60, 1822 (1927). (160) C. N. Cameron, J . Am. Chem. SOC.,49, 1759 (1927). (160a) B. Helferich and W. Porta, Chem. Ber., 86, 1034 (1953). (161) J . W. Baker, J . Chem. SOC.,1583 (1928). 1979 (1928). (162) J . W. Baker, J . Chem. SOC., 69, 969 (1947). (163) A . Mohammad and H. S. Olcott, J . Am. Chem. SOC., (164) C. Sanniit, C o a i p t . r e n d . , 226, 182 (1948). (165) N . Schoorl, R P C .trav. chim.,22, 31 (1903). (166) E. Fischer, Rer., 47, 1377 (1914). (167a) C. Sanni6 and H . Lapin, Bull. SOC. chim. F r a m e , [V] 17, 1234 (1950). (167b) W. 0. Godfredson, E. J. Nielsen, R. Reiter, E. Schgfnfeldt and I. Steengaard, Actn Chem. Scand.,7,781 (1953). (168) P. I h r r e r , C. Nkgeli and H. Weidmann, Helv. Chiin. Acto, 2, 242 (1919). (169) A. Dansi, Farm. sci. e tec. (Pavia), 2, 195 (1947); Chem. Abstracfs, 42, 639 (1948). (170) (a) 11. Grwshof and Ursul:L Tippold, drznei,~rLillcl-Fot,sch.,3, 42 (1953) ; C h w i . L4bs/racls,48, 2001 (1954). (LI) C , Saririik :rnd hl. Vincent, Conipt. rend. soc. b i d . , 142, 493 (1948). (c) G. Guillot-lirbain and C. Sanniit, J . ph,ysiol. (Paris), 42, 7 3 (1050). (d) J. S. Cannell, Phnrm. J . , 167, 231 (1951). (e) J . S.C:annell, J . Phat,nt. Phurnzacol., 3, 741 (1951). (171) K. Frendenberg and E. Braun, Ann., 460, 288 (1928). (172) F. W. Holly, C. H . Shunk, Elizabeth W. Peel, J . J. Cahill, J . B. Lavigne ant1 K. Folkrrs, .J. A m . C h c . ~Soc., . 74, 4521 (1052). (173) M. A m d o r i , A f l i reale acccrd. n a z l . Lincei, 161 13, I95 (1931). (171) S. Mostmowski,An:. A k n t l . Il.iss. KruXn~r,641 (1909); C h e n l . A b s f r o c l s , 4, . e t r f r . , 80, 11, 1267 (1909). 2321 (1910); C h ~ t r i C (17.5) €3. Helferich and A . Porck, A n n . , 686, 239 (1954). ~ i (IJondon), . 20, 205 (1926). (176) A . Hyrid, L l i ~ e h e ~.I. (177) Ett,rl and J . Hpt)liG, f ~ o / / r ~ l ; oc n , oslov. ( ' R c m . Concmuns., 16, G5 (1950). (178) V. Cuculescu, Bull. S O C . chim.France,[V] 6, 970 (1938).
v.
GLYCOSYLAMINES
107
(179) Y. Inoue and Iionof increasing amounts of hydrochloric acid to N-aryl-Dglucosylamines, in quantities up to one mole per mole of the glucosylamine, greatly increases the rate of color formation. When one equivalent of acid has been added, the browning is a t a maximum and no ketose derivative can be isolated. When more than one equivalent of acid is added, the reaction is apparently of a different type. There is no longer an induction period preceding the browning, and less color is Strong acid is not required for decomposition to occur; the browning discoloration also 4 6 , 4 7 , 48 takes place quite rapidly in the presence of glacial acetic As to the nature of the decomposition, both dehydration and fission of the 4 7 , 49 but exact knowledge of the rethe sugar are known to occur 2 2 , actions is lacking. Two a1ternat.e pathways for the production of tarry decomposition products from glycosylamines were considered by Bayne and H 01m s. ~~ According to their speculations, tar could be formed either directly from the Aniadori rearrangement product or from “unsaturated intermediates” which could be produced from the glycosylamine and would be in equilibrium with the ketose derivat,ive. GottschalkZ2has proposed t,hat it is the 1,2-enol, which may be formed from the glycosylamine and also from the ketose, that leads to the formation of humin,like substances -presumably t,hrough 5-hydroxymethyl-2-furaldehyde or a Schiff base thereof. Decomposition of the 1,2-enol (which should be considered to be the ammono analog of the 1,2-enediol form of unsubstituted sugars), would channel the course of the reaction through that form without allowing the production of much ketose derivative. The following scheme shows the 23v
38s
(45) L. Rosen, K. C. Johnson and W. W. Pigman, J. Am. Cheni. SOC.,76, 3460 (1953). (46) C. N. Cameron, J . Ant. Chent. SOC.,48, 2233, 2737 (1926). (47) C. N. Cameron, J . A m . Cheni. SOC.,49, 1759 (1927). (48) W. W. Pigman, €3. A . Cleveland, D. H. Couch and J. H. Cleveland, J . Am. Chem. Soc., 73, 1976 (1951). (49) A. Gottschalk and S.h1. Partridge, N u t w e , 166, 684 (1950). (50) S. Bnyne and W. H. Holins, J. Chela. SOC., 3247 (1952).
AMADORI REARRANGEMENT
181
chief stages of the rearrangement for aldohexoses and the known decomposition reactions (see Section VII) which are believed to arise from the 1,2eno1.22, 2 3 , 49, 60 Aldohesose+ arnine
11-
Ha0
A'-Substituted glycosylan~~ne (VI)
5-Hydroxymeth yl2-furaldehyde
amino-reductones
I Highly colored melanoidins 1 2. Proposed Mechanisms
From the experiment,al evidence presented in the preceding Section on the proton acceptor-proton donor relationship of glycosylamine and acid catalyst, the Amadori rearrangement must be classed as another of the many examples of acid-base ~ a t a l y s i sPresumably, .~~ the reaction involves merely the acceptance of a proton by the glycosylamine base, prototropic shifts, and the subsequent discharge of a proton from the cation originally formed. Thus, the rearrangement is pictured as the ammono analog of aldose enolization when the latter is catalyzed by acids. For example, Petuely62 gave the following representation of sugar enolieation by acid catalysis, for 6 3 * 64 which there is now definite experimental (51) (52) (53) (54)
W. F. Luder and S. Zuffanti, Chem. Revs., 34, 345 (1944). F. Petuely, Monatsh., 84, 298 (1953). M. L. Wolfrom and W. L. Shilling, J. A m . Chem. Soc., 73, 3557 (1951). E. Berner and S. Sandlie, Chemistry & Industry, 1221 (1952).
182
JOHN E. HODGE
HC=O
+ H+ ,
I
HC=O+H
HCOH
- H+
I
II
CHZOH
CHzOH
- I{+
I - 1 C=O+H I
C=O
I
The ammono analog of the above reaction mechanism would be as follows. HC-N-R HC=N+H-R
I
+ H+
-
HCOH
- H1 +
I
IIC-NF-R
II COH I
H+
-
+ H+
HCOH
P
I
CHZNH-R
- +I H+
P
- H+
- H+
C=O+H
' +H+
I
CHzNH-R
' I
c=o I
This is the mechanism that was proposed originally by Kuhn and Weygand.2*l6 The nitrogen of the N-substituted glycosylamine (VI, R' = H or a radical) accepts a proton from the acid catalyst, to form the ammonium ion (VII) which presumably is in equilibrium with the cation of the Schiff base (VIII). Isbe11b6then explains the shift of the double bond on the basis e R-NH-R'
fi!!l I
El
HC=NRR'
I
HCOH
HOCH
-
+ HIQ
HCOH
-
HCOH
HCOH
HFO
HFOH
I
HCO
I
I
R-N-R'
-
CH2 HOCH I
I HCOH I HCOH I
CHiOH X
~
I
HOYH HCOH
I I
HCOH
CHzOH VIII
~~~~
HCOH I HCO
CHzOH IX R-N-R'
I
CHI
I
-
- H@
I
R-N-R' I CH1
I
I c=o
-
I
CH2OH VII
CHzOH VI
7
I HOCH
L
HCNRR' II COH
-
I
CHiOH XI
(55) H. S. Isbell, Ann. Rev. Bioehem., 12, 206 (1943).
I I
HOC -
H°FH HCOH I I
HCOH HzCOXI1
183
AMADORI XEAHHANGEMENT
of flow of electrons toward the positive nitrogen atom, which makes C1 transiently positive. The secondary flow of electrons from C2 to C1 then weakens the C-H bond on C2, causing the expulsion of that proton to give the enol form of the ketose derivative (IX). The tautomeric shift to the keto form (X) would presumably be driven by the strong tendency of X to go over into one of the stable ring forms (XI or XII). The foregoing mechanism is also applicable to N-glycosyl derivatives of secondary amines (R’ = R or another radical, or R’ N . R’ = a heterocyclic, secondary amine). I n the mechanism proposed by Gottschalk,22 the catalyzing proton is presumed to add to the ring oxygen of VI to give XI11 (R’ = H or a radical). This mode of proton addition has also been suggested by Isbell and Frush6‘j and by Petuely67 to explain the mutarotation of sugars and glycosylamines. Opening of the ring of the cation XI11 would leave C1 with a transient positive charge (as shown by XIV) to which an electron pair from C2 could be attracted to form the enol (XV) directly, without formation of a Schiff-base intermediate.52 A similar mechanism was proposed by GottschalkZ2for conversion of the 1-amino-1-deoxy-2-ketose to the labile 1,2-en01 (XV).
I
CHrOH XI11
I
CHzOH XIV
&H~OH
xv
Whether or not enolization of a sugar or glycosylamine can occur without the formation of an intermediate carbonyl or azomethine group, respectively, is a moot question, discussion of which is beyond the domain of this it is difficult to imagine in the first Chapter (see P e t ~ e l y57). ~ ~However, . place how the weakly basic oxygen atom of the sugar ring could preferentially take on the catalyzing proton in face of the more strongly basic nitrogen atom which is present in the same molecule. Moreover, on present evidence, the Amadori rearrangement does not Occur unless the nitrogen atom is sufficiently basic to accept a proton; hence, any reaction mechanism which ignores t,he primary role apparently played by the amino-nitrogen atom is to be questioned ad this time. (56) H.S. Isbell and Harriet L. Frush, J. Research Natl. Bur. Standwds, 46, 132 (1951). (57) F. Petuely, Angew. Chem., 66, 268 (1953).
184
JOHN E. HODGE
3. Comparison with the Lobry de Bruyn-Alberda van Ekenstein Transformation The Aniadori rearrangement has some features of the Lobry de BruyiiAlberda van Ekenstein transformation,68as can be seen from the ammono analogy to sugar enolization formulat>edin Part 2 of this Section. 130th reactions occur in basic media, and each doubtless involves 1,2-enolization of the sugar. However, the Amadori rearrangement proceeds by acceptance of a proton from the acid catalyst, whereas the Lobry de Bruyii-Alberda van Ekeiistein transformation proceeds by delivery of a proton to the base catalyst. Aside from what may be argued as to the enolizatiori mechanism, there are other important differences. -As no demonstration of reversibility or of a quasi-equilibrium has been made for the Amadori rearrangement. Either epimer of an aldosylamine (e.g., either N-substitut,ed D-glucosylaniine or N-substituted D-mannosylamine) may be used as starting material, with production of the ketose derivative in good 3 7 , 38 But, despite their excellent crystallizing properties, N-substituted ~-mannosylamineshave not been found during the isomerization of N-substituted D-glucosylamines. Nor have both the N-substituted glycosylamine and the Amadori rearrangement product been isolated from the same reaction mixture. By the Lobry de RruynAlberda van Ekeiistein transformation, both D-glucose and D-mannose can be produced, starting with D-fructose. But neither epimer of a N-substituted aldosylamine has been obtained from a N-substituted l-amino-l-deoxy-2ketose. The reason for these differences probably lies in the greater lability of the intermediate eneamiriol (XV), supposedly formed in solution during the Amadori rearrangement. Decomposition accompanied by browning occurs in both the Lobry de Bruyn-Alberda van Ekensteiri t r a n s f ~ r m a t i o n and ~ ~ the Ainadori rearrangement. In aqueous solutions under conditions of equal p H , browning is more severe in the presence of amines than in the presence of strong alkali.38This can be explained by the different nature of the decomposition products in the two reactions. Decomposition in aqueous alkali arises mainly from sugar fission and saccharinic acid rearrarigemeiit~.~~ 5 9 Sugar acids, including lactic acid and fatty acids, are known to be relatively stable toward further decomposition with browning.23On the other hand, in the Amadori rearrangement, the main decomposition reactions are sugar fission and sugar dehydration, both of which produce very reactive intermediates (for example, a-dicarbonyl compounds, a ,b-unsaturated aldehydes, and reductones) which continue to react, in the absence of high (58) L. Sattler, Advances zn Carbohydrate Cheni., 3, 113 (1948). (59) J , B. Gottfried and D. G. Benjamin, Z 7 d . Eng. Chem., 44, 141 (1952).
AMADORI REARRANGEMENT
185
concentrations of hydroxyl ions, to form colored by product^.'^ Therefore, reversihi1it)y and estahlishnient of a quasi-equilibrium during the Amadori rearrangement cannot be expected, unless sugar decomposition is blocked.
VI. PHYSICAL PROPXRTIES OF AMADORI REARRANGEMENT PIEODUCTS 1. Color and Taste
Like the N-suhstitut,ed glycosylamines, the N-substituted 1-amino-1deoxy-2-ketoses are colorless when the parent amine is colorless. 1-DeoxyI -p-toluidiiio-D-fructose is t n ~ t ~ e l e sbut s , ~ derivatives of the more strongly basic. amiiies are bitter. 2. Crystallization The rearrangement products derived from aromatic and non-aromatic heterocyclic amines crystallize readily from the lower alcohols. Unlike those of many of the N-substituted glycosylamines, the crystals are not solvated. On the other hand, the ketose derivatives of aralkyl- and alkyl-amines, such as 2-phenylethylamine, ethanolamine, diethanolamine, glycine ethyl ester, and phenylalanine (see Table II), are hydrated or alcoholated, or both, and are difficult to isolate in pure crystalline form. The crystals which have been isolated were hygroscopic.Z2 38 Alcohols, aqueous alcohols, and water are the most commonly used solvents for crystallization. Acetone, ether, or benzene have been added to the alcoholic media in order to increase the yield of crystalline compound. The use of solvents that contain peroxides promotes decomposition of the crystals during storage.60 The crystalline form of the 1-amino-1-deoxy-2-ketoses varies. Most commonly, acicular needles or lamellar plates are formed, but columnar prisms are also known. Amadori3' reported that, a t different times, his stable isomers crystallized from the same solvent as either needles or flakes. 9
3. Melting Amadori rearrangement products, like the N-substituted glycosylamines, generally melt with decomposition. I n conformity with the greater stability of the rearrangement products as compared with that of the glycosylamines, the former usually melt and decompose a t higher temperatures, or with slower decomposition a t a given temperature. I n the aromatic series, virtually all of the currently known rearrangement products melt a t some 30 (or more) degrees above the melting points of the corresponding glycosylamines. With strongly basic piperidine derivatives, there is little difference in the decomposition temperatures. However, the ketose derivative does decompose more slowly than the aldose derivative a t temperatures below the melting point.37*38
186
JOHN E. HODGE
Kuhn and Birkofer13 reported that aromatic N-substituted glyrosylamines generally foam upon melting, whereas the Amadori rearrangement products melt and decompose without foaming. 4. Optical Rotation
All the Amadori rearrangement products so far isolated in crystalline form belong to the D-series. As all but one of them are D-fructose derivatives, they are rather strongly levorotatory. Mutarotation of the rearrangement products is commonly observed in absolute methyl alcohol or ethyl alcohol solut,ions, and also in dry pyridine (which is the best solvent)).The change in rotation is generally slow, and the rotation decreases from a large negative value to a smaller, nearly constant, negative value. On the other hand, solutions of these products in water or dilute acid display a constant rotation over a period of several hours. Unfortunately, most of the aminodeoxyketoses derived from aromatic amines are not appreciably soluble in water. They are, however, soluble in dilute acids; hence, it, is recommended that water containing at least one equivalent of hydrochloric acid be used in determining the optical rotations of Amadori rearrangement products. A small excess of acid does not alter the rotation. Aqueous solutions of the hydrochloride salts of aminodeoxyketoses, and also of the aminodeoxyalditols, generally exhibit an optical rotation of higher magnitude than that of the parent basic a m i n e ~ .3~7 *. 3 * , 6n Another advantage is gained by the use of dilute hydrochloric acid: because the isomeric N-substituted glycosylamines are easily hydrolyzed-to give, finally, the rotation of the unsubstituted sugar-measurement of the rotation in acid solution affords a check on the identity of the compound. The optical rotations of the known 1-amino-1-deoxy-2-ketoses are recorded in Table I (page 203). 5. Light Absorptian According to ultraviolet-absorption measurements made by Kuhn and Dansi,l the spectrum of 1-deoxy-1-p-toluidino-D-fructosein alcohol is insignificantly different from that of N-p-tolyl-D-glucosylamine (or of pure p-toluidine61*'j2).Because an unsaturated linkage in conjugation with those of the aromatic nucleus is not indicated by the spectra, neither N-p-tolylD-glucosylamine nor the rearrangement product shows the structure of a Schiff base in alcohol. L e g a ~ came ~ ~ to the same conclusion on the basis of infrared measurements on the same two compounds in the solid state. No absorption a t 6.05 p, corresponding to a -C=Nlinkage, was found. (60) (61) (62) (63)
K . Zeile and W. Kruckenberg, B e r . , 76, 1127 (1942). R . G . Cooke and A . K . Macbeth, J. Chem. Soc., 1595 (1937). F. Pruckner and B. Witkop, Ann., 664, 132 (1943). F. Legay, Conzpt. rend., 234, 1612 (1952).
AMADORI
REARRANGEMENT
187
However, he did find an absorption band a t 6.05 p for solid N-o-tolyl-Dglucosylamine, and also for N-2-naphthyl-~-glucosylamine,indicating for the first time the existence of a Schiff-base structure for N-substituted glucosylamines in the solid state. For the other N-substituted D-glucosylamines. tested (N-phenyl, N-p-nitrophenyl, and N-rn-nitro-p-tolyl), no Schiff-base structure was indicated. All compounds exhibited a band a t 2.88 p which could have been given by either an NH or an OH grouping. VII. CHEMICAL PROPERTIES OF AMADORI REARRANGEMENT PRODUCTS 1. Enolization and Oxidation
Ketoses are more easily dehydrogenated than aldoses, to yield the mutual osone. For example, aldoses are not attacked by hot selenious acid whereas, under the same conditions, D-fructose reduces the reagent to selenium.64The oxidation product, at least in part, is D-glucosone.66Hydrazine also dehydrogenates D-fructose, but not D-glucose, in weakly acid or weakly alkaline solution at 100’. That the chief product is D-glucosone (or an imino analog of D-glucosone) was shown by Weygandand Bergmann18 through the quick formation of the phenylosazone or tetrahydroxybutylquinoxaline derivatives a t low temperatures. Furthermore, D-fructose and * 68 or D-threo-pentulose are oxidized by cupric salts,@ o-dinitr~benzene,~~ 2 , 6-dichlorophenolindophenol,69in alkaline solution a t ordinary temperatures, much more quickly than are the corresponding aldoses. The ketotriose, 1,3-dihydroxy-2-propanone(dihydroxyacetone), is dehydrogenated or 2 , 6-dichlorophenolindopheno169in alkali, by either o-dinitr~benzene~o and the reaction is quantitative for one mole of enediol, using the latter reagent .71 If N-substituted I-amino-1-deoxy-D-fructoses form ammono analogs of enediols with the same ease that D-fructose is transformed to an enediol, then dehydrogenating agents would be expected to distinguish between N-substituted aldosylamines and the corresponding Amadori rearrangement products. Such has been found to be the case. The distinction between (64) H. L. Riley, J. F . Morley and N. A. C. Friend, J . Chem. Soc., 1875 (1932). (65) K. C. Dixon and K. Harrison, Biochem. J. (London), 26, 1954 (1932). (66) 0. T. Schmidt and R . Treiber, Ber., 66, 1765 (1933). (67) H. L. J. Chavassieu and A. Morel, Compt. rend., 143, 966 (1906). (68) R . Kuhn and F. Weygand, Ber., 69, 1969 (1936). (69) H. von Euler, H . Hasselquist and G. Hanshoff, Arkiu Kemi, 6, 471 (1954). (70) W. R. Fearon and E. Kawerau, Biochern. J . (London), 37,326 (1943). (71) H. von Euler and H. Hasselquist, “Reduktone,” Sammlung Chemischer und Chemisch-technischer Vortriige, No. 50, Ferdinand Enke, Stuttgart, Germany, 1950, p. 6.
188
JOHN E. HODGE
the two is quite sharp when use is made of such oxidation-reduction indicators as o-dinitrobenzene,z' 1 3 , 2 2 , 38 Methylene Blue,l3,2 2 ~ 2 8 38 , and 2,6-dichlorophenolindophen~l~~~ 3 7 , 38 in alkali a t 25". If the test with o-dinitrobenzene is carried out according to Fearon and Kawerau's direction^,^^ one may distinguish between true reductones (that is, stable enediol-acarbonyl compounds, such as ascorbic acid, triose-reductone, and reductic acid, which produce the purple color immediately), Amadori rearrangement products (which form the color after about 1 minute), and N-substituted glycosylamines (which give no color within 15 minutes or longer).38Other oxidizing agents, such as permanga~iate,~ Benedict,2" Fehling,l3. and toll en^'^ solutions, also show a faster rate of reaction with the aminodeoxyketose derivative, but the distinction is not as marked as with the use of enediol reagents. A strongly reducing enediol(s) is formed from an Amadori rearrangement product only in strongly alkaline solution. For example, in methanolpyridine-water, N-substituted 1-amino-1-deoxy-2-ketoses give a negative test with titanium trichloride by the method of Weygand and C s e n d e ~ , ' ~ indicating the absence of an a-enediol gr~uping.'~ The explanation for the lack of a definite end-point upon titration of Amadori rearrangement products in alkali with Methylene Blue or 2 , 6-dichlorophenolindopheno113 may lie in the finding of P e t ~ e l that y ~ ~D-glucosone reacts with alkali t o form one or more reductones (a-enediol-a-carbonyl compounds which are strongly reducing, even in acid solution). Because the phenylosazone and tetrahydroxybutylquinoxaliiie derivatives of D-glucosone have been isolated from 1-deoxy-1-p-toluidino-D-fructose in high yields,l8' l9 1,2-enolization of the aminodeoxyketose must predominate over 2,3-enolization. No proof for 2,3-enolization is known a t present,. The sequence of reactions (in alkali) which lead from tbe aminodeoxyketose (XVI) through the 1,2-enediol (XVIII) to the glycosone (XIX) are thought to be as follows: f
XVI
XVII
XVIII
Hydrolysis of the eneaminol (XVII) must precede the dehydrogenation in (72) F. Weygand and E. Csendes, Chem. Bw.,86, 45 (1952). (73) J. E. Hodge, unpublished observations. (74) F. Petuely, Monatsh., 83, 765 (1952).
189
AMADORI REARRANGEMENT
alkali. Although it is true that, if R‘ = H in XVII, dehydrogenation of the eneaminol could occur before hydrolysis, such would not be t,he case when -NRR’ is a secondary amino radical-yet secondary amino rearrangement products are as readily dehydrogenated in alkali as are those derived from primary a m i n e ~ .38~ It ~ , has been shown that the anilino derivative of a grouping such as XVII (R = phenyl, R,’ = H) will reduce o-dinitrobenzene in alkali, whereas the N-methylanilino derivative (R = phenyl, R’ = CH3) will 76 The initial reversibility of the enolization (XVI XVII) was demonstrated by Kuhn and D a n ~ i Dissolution .~ of 1-deoxy-1-p-toluidino-D-fructose in alcoholic sodium hydroxide caused a lowering of the specific rotation from -31.5 to -3.Go, and acidification immediately thereafter restored the initial rotation. Furthermore, addition of sodium hydroxide did not cause displacement of the wavelengths of maximum absorption in the ultraviolet region, showing that D-glucosone or a stable reductone is not formed immediately. Nevertheless, alkaline solutions of aminodeoxyketoses soon turn golden yellow 011 standing, arid later become reddish and brown in the known manner of osone preparations.4’ 5 . 6 , 7 , 38 Amadori4v 5 , showed by diazotization reactions that the amine actually is liberated from the rearrangement products on stJanding in aqueous or alcoholic alkali, and GottschalkZ2recovered nearly all of the amino acid after heating l-deoxy-lphenylalanino-D-fructose in dilute sodium carbonate solution. I n the latter reaction, none of the starting carbohydrate could be detected chromatographically after the alkali treatment. In contrast to the Amadori rearrangement products, the N-aryl-D-glucosylamines are relatively stable in alkali. They do not readily liberate the amine, for their solutions give a constant optical rotation while remaining 6 , 6 ’ However, the N-alkyl-Dcolorless in alkali over extended glucosylamines are much more labile than the N-aryl derivatives in alkali.34 35 36 37 Alkaline solutions of Amadori rearrangement products give an isonitrile odor when heated,7 indicating fission between CI and C2 of the sugar radical. Weygand and B e r g ~ n a n nproduced ~~ this fission oxidatively by shaking the aminoketose with oxygen and a platinum-on-charcoal catalyst in ammonium hydroxide. From the ammonium salt of tjhe enol form (XX, R = CH3, OCH3 , or OC&), the ammonium salt of D-arabonic acid (XXI)
+
l
I
(75) W. Cocker, R . A . Q. O’Meara, J. C. P. Schwarz and E. R . Stuart, J . C h m . SOC.,2052 (1950). (76) W. Cocker, I). S. .Jerikinson a n d P. Scliwarz, .I. Cheiu. SOC.,1628 (1953). (77) F. Weygand and Annemarie Bergmann, Chrm. B e i . , 80, 261 (1947).
190
JOHN E. HODGE
HC-NH-C6H4-R
HC-NH-CsH4-R II c.00 NHe@
I
HOCH I H~OH HCOH I
I
II
0
--c Ha0
R-C,H,-NH,
+ COz -I- H 8
0
9/1
XXII
XXIX > XXVII, and the explanation of this difference must lie in the spatial arrangement of the acetyl groups in relation to C1. Accurate models of the four compounds were constructed on the basis of Reeves' C1 conformation,156and the groups were positioned in such a way that their fractional charges would represent a structure with minimum potential energy. It was found that the steric hindrance or amount of "crowding" of groups a t C1 is inversely proportional to the rate of methanolysis. (The effect of the acetyl group a t C3 could not be evaluated, since the appropriate derivatives of D-allose were not available, but Howards6had suggested that, in a cis-2 ,3-diacetoxy system, the 3-acetoxyl group would restrict movement of the 2-acetoxyl group t o the neighborhood of Cl.) Further support for the view that variation in rate of unimolecular alcoholysis is a function of hindrance a t C1 was given by a study of hepta-0-acetyl-a-cellobiosyl bromide and hepta-0acetyl-a-gentiobiosyl bromide. Their models showed that there should be little difference in rate from that of tetra-0-acetyl-a-D-glucopyranosyl bromide, and this is borne out by their rate constants, given in Table I ; the higher value for the gentiobiose derivative is accounted for by the p-r)-biose linkage. The discussion so far has been concerned primarily with the mechanism of solvolysis of the cis halides. There is no reason to suppose that the trans halides react by a different mechanism, and indeed, tetra-0-acetyl-a-1,mannosyl bromide has been shown to undergo unimolecular solvolysis. On the basis of an SN1mechanism as promoted by the lactol ring oxygen, and of the steric influence of all the groups in the molecule, it should be possible to interpret the reactions of both the cis and the trans halides and to explain the nature of the products from each. The neighboring-group concept of Winstein has been used to explain the difference in reactivity exhibited by anomeric pairs of halides.a6 8K This suggestion is by no means improbable, and would explain the facts that tri-0-benzoyl-P-D-ribopyranosyl bromide is 19 times more reactive a t 20" in 1:9 dioxane-methanol than is the a anomer, and that the rate for the trans-0 chloride shows an 85-fold increase over that of the cis-a chloride. However, in view of the properties of the series XXVII to XXX, steric effects from the whole molecule may be equally important, although the disadvantage of this concept is that hindrance as assessed from models is difficult t o define precisely. It is of interest to compare the rates of methanolysis of tri-0-benzoyl-a-~-xylopyranosyl bromide36and of tri-0-acetyla-D-xylopyranosyl bromideL6'[105k200= 51 (sec-l) in 1:9 dioxane-methanol and 105k210= 139(sec-l) in 100 % methanol, respectively]. Although the value of k for the former compound would he higher a t 21" in 100 % meth-
238
L. J. HAYNES AND F. H . NEWTH
anol, a satisfactory explanation would, in the absence of directly coinparable figures, seem to be in terms of greater hindrance by the benzoyl groups. The extensive work of H. G. Fletcher, Jr., arid the late C. S. Hudson and their c o l l e a g ~ e *Os ~ 8~5 -~8x 162 on the reactions of the poly-0-benzoylglycosyl halides has shown that glycoside formation occurs with the expected inversion when the halogen atom is cis to the benzoyl group a t C2, but proceeds with retention of configuration when the groups are trans. Although the cationic, cyclic intermediate XXXI has been postulated in order to account for the latter reaction, the fact that no 1,2-orthobenzoate in the pyranose series has been isolated (although its presence was sought) may be significant. It was found, however, that the frans halides do give rise to a small amount of cis-glycoside (together with the main product). The manner in which these compounds and the corresponding acetates react now seems clear.
Q7
Q 0-c:?
0-c? \
XXXI
‘CHa
Ph
XXXII
The reactions of both the cis-acetohalogeno sugars and cis-benzoylhalogeno sugars involve inversion of configuration. The initial ionization of the C1-X bond followed by the departure of the halide ion on the same side as the neighboring acyl g o u p which can protect the C1 cation, leaves only the opposite side open for attack by solvent molecules, and so inversion is complete. When, however, the halogen atom is trans to an acetyl group, its departure, which may well be facilitated by this situation, is followed by a competitive, nucleophilic attack from the opposite side by solvent (ROH) molecules and by the oxygen of the polarizable carbonyl group. Thus, both the cyclic cation XXXII and the cis-glycoside can be formed. Compound XX X I I can then itself undergo further unimolecular solvolysis, to give the trans-glycoside or react per se to form the orthoacetate. This mechanism is essentially the same as that formulated by Frush and Isbe11lS6and by Pacsu,l except that the carbonyl oxygen of the neighboring acyl group is not required t o participate directly in an initial bimolecular transition state as pictured by these authors, and it thus explains the formation of some of the cis-glycoside. The failure of 3,4,6-tri-0-acetyl-2-0-trichloroacetyl-/3-D-glucosyl chloride to give a 1,2-0rthotrichloroacetate is impor(162) (a) R . W. Jeanlor, H. G. Fletcher, Jr., and C. S. Hudson, J . Am . Chem. SOC.,70,4055 (1948); (b) R. K. Ness, H. G. Fletcher, Jr., and C. S. Hudson, ibid., 73, 296,3698 (1951); (c) R. K. Ness and H. G. Fletcher, Jr., ibid., 74,5344 (1952).
(:LYCOSYL HALIDES .1ND THEIR DEIilVa\'171VES
239
tant, and may be explained on the grounds that the inductive effect of the trichloromethyl group so diminishes the polarization of the carbonyl group that the nucleophilic activity of the carbonyl oxygen is insufficient to permit formation of the cyclic, orthoester cation. In such a trans system the steric effect of the trichloroacetyl group will still be operative and will permit the formation of both the a- and the p-glycoside.6RAlthough acid strength is satisfactory in explaining the properties of the trichloroacetyl group, it does not account for the difference between acetyl and benzoyl derivatives in the pyranose series, where the latter do not form orthobenzoates. If acid strength were the only consideration, the orthobenzoate structure should be formed as readily as is the orthoacetate. The present picture is further complicated by the recent discovery that hydrolysis of tri-0-benzoyl-Dribofuranosyl bromide gives u-ribofuranose 1,2-(orthobenzoate) 3,5-dibenzoate as a major product of the reaction, and that this compound is converted t o 2 ,3 ,5-tri-O-benzoyl-p-~-ribose through t,he action of pyridine.163 The kinetics of the reactions of poly-0-acylglycosyl bromides with amines With such in acetone have been described in a recent comrnunicati~n.'~~ strongly nucleophilic, secondary amines as piperidine, di-n-butylamine, and diethylamine, biniolecular reactions are undergone by tetra-o-acetyland tri-0-acetyl-Da-D-glucopyranosyl, tetra-0-acetyl-D-galactopyranosyl, xylopyranosyl bromides, and these show second-order kinetics in pure acetone, whereas tetra-0-acetyl-a-D-mannosyl bromide exhibits the incursion of some unimolecular reaction. With the weakly nucleophilic reagents N-methylaniline, pyridine, and 3-picoline1 unimolecular reactions bromide; and, with were observed for tetra-0-acetyl-a-D-mannopyranosyl tetra-0-acetyl-a-D-glucopyranosyl bromide, the reactions pursued a mixed mechanism. The influence of neighboring acyl groups on the rate of reaction appears to be smaller than that observed in the unimolecular solvolytic reactions. 61 VI. REACTIONS OF THE POLY-O-ACYLGLYCOSYL HALIDES I n the preceding Section, the poly-0-acylglycosyl halides have been discussed with particular reference to the effect of their structure on the reactivity of the halogen atom and on the configuration of the product resulting from the unimolecular, nucleophilic substitution. I n the present Section is given a general account of the reactions of the halides which refers more to the type of product which may be obtained from these compounds. (163) R. K. Ness, H. W. Diehl arid H. G. Fletcher, Jr., J . A m . Chem. Soc., 76,763 (1954); R. K. Ness and H. G. Fletcher, Jr., ibid., 76,1663 (1954). (164) N. B. Chapman and W. E. Laird, Chemistry & Industry, 20 (1954).
240
L. J. HAYKSS AND F. H .
Nmvm
1. Formation of Glycosides Synthesis in this very large group of natural products165 has been achieved mainly through the reaction of poly-0-acylglycosyl halides with hydroxylic compounds. Since t’he early formation of the phenolic glycosides, arbutin and methylarbutin, by Michael,166most of the naturally occurring p-1)glycosides have been synthesized. The procedure is straightforward, and when, for example, tetra-0-acetyl-a-D-glucopyraliosyl bromide in an inert solvent is allowed t o react with a hydroxylic compound in the presence of a suitable base, the tetra-O-acetyl-P-D-ghcopyranoside is formed in adequate yield. It is unnecessary to enumerate all the glycosides made in this way, but two examples of interest may be given. I n 1924, amygdalin, one of the earliest-known natural glycosides, was synthesized independently by Haworth, by Zemplh, and by I h h 1 1 I ~and ~ their coworkers by condensing hepta-0-acetyl-a-gentiobiosyl bromide with ethyl m-mandelate in the presence of silver oxide. Resolution of the amide, followed by dehydration and deacetylation, provided marldelonitrile 0-gentiobioside identical with the natural product. Ailot,hcr important application was made in 1932, when Robinson and Todd’6s synthesized the anthocyanins peonin, pelargonin, malvin, and cyanin chlorides. The intermediate 2-(tetra-O-acetyl-P-~glucopyranosy1)phloroglucinaldehyde was obtained by condensing tetra-0acetyl-a-D-glucopyranosyl bromide with phloroClo OCH, glucinaldehyde in acetonitrile containing aqueous alkali, but in acetone the yield was higher HO OAc and less “diglucoside~’was formed. The other OG(OAc)4 intermediate, t,he substituted w-hydroxyacetoOG(0Ac)r phenone D-glucopyranoside, was prepared using XXXIII the Koenigs-Knorr conditions.16g When the two components were condensed in the presence of acid, the acet>ylated 3,5-bis(~-glucopyranoside) (XXXIII) of peonin was formed. The oligosaccharides represent a most important group of glycosides. Synthesis ill this field has been extensive and has been reviewed by Evans, Reynolds and T a l l e ~ . ~ ‘The “ ) formation of bhe p-biose linkage presents no difficulty when the 2-0-acetyl group of the poly-0-acylglycosyl halide is (165) E. F. Armstrong and K. F. Armstrong, “The Glycosides,” Longmans, Green and Company, New York, N . Y., 1931. J. Honeyman, “The Plant Glycosides,” Arnold, London, 1949. (166) A. Michael, Ber., 14, 2097 (1881). (167) R.Campbell and W. N. Haworth, ,I. Chei>t.SOC.,125,1337 (1924). G. Zempl6n and A. Kunx, B e T . , 67, 1357 (1924). R. Kuhn and €1. Sobotka, ibid., 57, 1767 (1924). (168) R. Robinson and A. R . Todd, J. Cherii. SOC.,2290, 2488 (1932). (169) W. Koenigs and 13. Knorr, Ber., 34, 957 (1901).
GLYCOSPL HALIDES AND THEIR DEKIVATIVES
241
cis to the halogen atom, as in tetra-0-acetyl-a-D-glucopyranosyl bromide. There is then no neighboring-group interference which could lead to an orthoester, and the p-glycosidic linkage is smoothly formed. Several refinements have been incorporated in the original Koenigs-Knorr condition^,^(^) among which is the introduction of iodine as a catalyst into the reaction mixture.I7OThis catalytic effect may well be due to an increase in the polarity of the solvent chloroform by the iodine, an effect which would enable the SN1reaction of the halide to proceed more smoothly. With all thenknown refinements, Reynolds and Evans171 described a practical method of obtaining octa-0-acetyl-P-gentiobiose in 74 % yield. A yield comparable to this has been recorded when tetra-0-acetyliu-D-glucopyranosylbromide was allowed to react with the melt obtained by dissolving sodium in molten 1,2,3,4-tetra-O-acetyl-P-~-glucose. Sodium bromide is eliminated, and an 80 % yield of octa-0-acetyl-0-gentiobiose is obtained.172The synthesis of octa-0-acetyl-P-cellobiose was also achieved in this way, using 1,2 , 3 , Gtetra-O-acetyl-@-D-glucopyranose, but the yield was only about 40 %. The direct formation of the a-D-glucosyl linkage has been a subject of study for a number of years. The problem is still of great interest, particularly in connection with the synthesis of maltose, isomaltose, and sucrose. A successful, chemical synthesis of sucrose has been described by Lemieux and H ~ b e r ,who ~ ~ ~heated 3,4,6-tri-O-acetyl-l , 2-anhydro-a-~-glucose and, after (Brigl’s anhydride) with 1,3 ,4,6-tetra-O-acety~-D-fructofuranose acetylating the product, obtained octa-0-acetylsucrose in small yield. Although this long-outstanding objective in sugar chemistry has been achieved, the method employed does not settle unequivocally the a , @ nature of the biose linkage, since the D-fructose derivative was not of a particular configuration and the reactions of Brigl’s anhydride usually lead to the formation of @-I,-glucosides.The problem as t o how a-D-oligosaccharides may be formed through the use of the 0-acylglycosyl halides remains. In view of the discussion in the previous Section, it would seem that the formation of an a-D-glycosyl linkage by employing the &halide must be difficult, since orthoester formation will be concomitant in the reaction. Indeed, in the reaction product of tetra-0-acetyl-a-u-mannopyranosyl bromide with 1 , 2 , 3,4-tetra-O-acetyl-@-~-glucose,Reynolds, Evans and Talley126found two isomeric disaccharide orthoesters. The normal biose, of undefined configuration, was also formed and its yield was greater in the presence of iodine. The compound likely t o be of greatest use in the forma(170) B. Helferich, E. Bohn and S. Winkler, Ber., 63, 989 (1030). (171) D. D. Reynolds and W. 12. Evans, J . A m . Chem. SOC.,60,2559 (1938). (172) Violet E. Gilbert, F. Smith and M. Stacey, J. Cliem. Soc., 622 (1946). (173) R. U. Lemieux and G. Huber, Abstracts Papers Am. CItcni. SOC.,134, 181) (1953); J. A w . Chem. Soc., 76,1118 (1953).
242
L . J. HAPNES AND F. H. NEWTH
tion of a-D-glucosyl disaccharides is Brigl's 3 ,4 ,6-tri-O-acetyl-@-~-glucosyl ~hloride.6~6* I t s reactivity with nucleophilic reagents is much higher than that of many other halidePo (see Table I), and the absence of a 2-0-acyl group obviates any danger of forming a 1 ,2-orthoester during the reaction of the @-halogenatom. Inversion of configuration would give primarily the a-u isomer, although a certain amount of @-D form would be produced because of the unimolecular character of the reaction. Further investigation may well show that condensation of a poly-0-acylglycosyl halide with a hydroxylic molecule proceeds more readily in a polar solvent (such as acetonitrile or dimethylformamide) than in the conventional chloroform. was The synthesis of melibiose [6-0-(a-~-galactopyranosyl)-~-glucose] ~ ~ quinoline as a condescribed in 1928 by Helferich and B r e d e r e ~ k . 'Using densing agent in the reaction between tetra-0-acetyl-a-D-galactopyranosyl bromide and 1,2 ,3 ,4-tetra-O-acetyl-@-~-glucose,they were able to isolate a small amount of octa-0-acetylmelibiose. I n the presence of silver oxide, only the 0-Dlinkage was formed.I76 The reaction of the halides with nitrogen compounds is discussed later in this Section, but a possible function of the quinoline may be mentioned here. Reaction of the a-D-galactosyl bromide with quinoline could give a quaternary ion having the 0-D configuration, and reaction of this with the 6-hydroxyl group of the tetra-0-acetyln-glucose would then give the a-D-biose linkage. I n view of the small yield, however, the full course of the reaction is likely to be more complicated. The difficulties encountered in the synthesis of a-Dlinked oligosaccharides are not so apparent in the formation of a-D-glycosides from simple alcohols. Zemplen and his coworkers176examined the reaction of hepta-0-acetyl-acellobiosyl bromide with methanol, and found that, in the presence of mercuric acetate, both methyl a- and P-cellobioside are formed. Solvolysis by alcohol in the presence of mercuric acetate gives mainly the @ form, whereas with a molar quantity of alcohol in benzene the a form is obtained. With silver salts, only the /3 form is obtained. Then, Helferich and Wedem e ~ e r "examined ~ the effect of several metallic oxides and salts on the methanolysis of tetra-0-acetyl-a-D-glucopyranosylbromide. They found that although the yield of methyl tetra-0-acetyl-P-D-glucopyranoside is lorn in the presence of zinc, cadmium, and mercuric oxides, much higher yields of glucoside result with zinc acetate or mercuric cyanide. A comparably high yield of benzyl tetra-0-acetyl-@-D-glucopyranoside was ob(174) B. Helferich and H. Bredereck, A m . , 466, 166 (1928). (175) B.Helferich and H . Rsuch, Ber., 69,2655 (1926). R . Hclferich and G . Sparniberg, ibid., 66, 806 (1933). (176) G.ZemplBn, Fortschr. Chem. org. Naturstoffe, 1, 1 (1938). This review covers work carried out between 1929 and 1932. (177) J3. Helfcrich and K. F. Wedemeyer, A n n . , 663, 139 (1949).
GLYCOSYL HALIDES AND T H E I R DERIVATIVES
243
tained in the presence of zinc oxide. The homogeneous reaction in the presence of aryl mercuric acetate gave only P - ~ - g h c o s i d e sand ~ ~ the ~ kinetic analysis is c~mplicated.’~~ The factors governing this reaction (and all those which are heterogeneous) are by no means yet understood, since surface effects and complex formation make interpretation difficult. 2. Formation of Glycosylamines ( “ N - G l y c o ~ i d e s ” ) ~ ~ ~ ~
Xucleophilic replacement of the halogen atom in the poly-0-acylglycosyl halides occurs with nitrogenous compounds and a “N-glycoside” is formed. In general, the stereochemistry follows the same course as in the formation of glycosides, and inversion occurs. Different types of product may, however, be obtained, depending on whether the amine is primary, secondary, or tertiary. Furthermore, as was pointed out in the preceding Section, the reactions are non-solvolytic. There is evidence that they are b i m o l e ~ u l a r , ~ ~ ~ arid the finer steric considerations will not necessarily be the same as those which have been evaluated for the solvolytic reactions. An example of the formation of a “N-glycoside” is the reaction of tetra-0acetyl-a-D-galactopyranosyl bromide with aniline, when tetra-0-acetyl-P1)-galactopyranosylanilineresults in good yield With secondary amines, dehydrohalogenation occurs, and a 1,2-glycoseen is obtained.181Quaternization is the first step in the reaction with tertiary amines, and treatment of the quaternary ammonium salt with barium hydroxide leads to the formation of a 1,6-anhydro ring. In this way, 1,6-anhydro-P-~-galactohave been prepared. The pyranosela2and 1,Ci-anhydro-P-~-glucopyranose~~~ formation of 3,4,6-tri-O-acetyl-l,2-anhydro-a-D-glucose by treating 3 ,4 , ti-tri-0-acetyl-P-D-glucosyl chloride with ammonia in benzene solution may also be mentioned.67 An important use of the poly-0-acylglycosyl halides in synthesis of “iV-glycosides” has been in their coupling with heterocyclic bases to form the naturally occurring nucleosides. Early synthetic experiments on the formation of pyrimidine riucleosides by treating a diethoxypyrimidine with a poly-0-acetylglycosyl halide, to give the analogs of cytidine and uridine, have been reviewed by LythgoelB4;by using tri-0-acetyl-a-u-ribofuranosyl hromide, this method has led to the successful synthesis of uridine and cytidine.15 The thymine nucleosides have been synthesized in a similar (178) B. Helferich and I $17 tl9.5 f 4 5 + +19
L53-155
f G -+
3-O-Methyl-N-phenyl-a-~-sylopy- I37 ranosylamine
3-O-Methyl-~-xylonolactone
14
4-O-Methyl-~-xylose phenylosazone
sirup 158-158.5
160-161 Methyl 4-O-methyl-p-~-sylopyran-35 oside sirup 5-0-Methyl-D-xylose 170-171 p-bromophenylosazone
1,2-0-isopropylidene acetal, 3 - p toluenesulfonate Methyl 5-0-methyl -a-D-xyloside, 3p-toluenesulfonate Methyl 5-O-methyl-p-~-xyloside,3p-toluenesulfonate 2,3-Di-O-methyl-a-~-xylose
[..ID,
Rotalion Aolzrent
H1 0 K O HzO
J, 11
12 10 109 13 B
-14
+80 +77
+76 + t-40 (820 hrs.) +9 f 2 +25 + 0
ChH5NEtOII EtOAc EtOAr HzO
HzO C5H5NEtOH
9 10, 11, 12 14 10
15 16
15 15
-69
HzO
+32.8 --+ +36 -50 + -30
9 9
81-82
-31.8
HzO CbH5NEtOH CHCl,
sirup
+44.5
CHC1,
8
89
-51.7
CHCl,
8
79-80
+70 + f 2 3 (1 H,O day)
Methyl 2,3-di-O-methyl-a-~-xylo-sirup side Methyl 2,3-di-O-methyl-p-o-xylo- sirup side 63 56-59 4-p-toluenesulfonate 2,3-Di-O-methyl-N-phenyl-~-xylo-145-146 pyranosylamine 121-123, 126 131-132
8
+61.8
CIIIOH
18, 19, 20 I 21 18
-5.8
CHC13
1
-47.3 -8.8 +182.5 f 2 . 5
H2O CHCla EtOAc
+180
EtOAc
12 1 17, 18, 22 3, 22, 23 24
250
METHYL ETHERS OF PENTOSES AND 6-DEOXYHEXOSES
TABLEI (Conlinued) ~~
Substance
Melting
[a]~ degrees ,
point, "C.
2,3-Di-O-methyl-~-xylono-~-~acsirup tone
132-134
2,3-Di-O-methyl-~-xylonic acid, phenylhydrazide p-bromophenylhydrazide 2,4-Di-O-methyl-p-~-xylose
107-108 114-115 150-151 108 108 111 116-118 2,4-Di-O-methyl-N -phenyl-~-xylo- 170 155-157 sylamine Methyl 2,4-di-O-methyl-p-~-xylo-77.5-78.5 side 60-61 3-p-toluenesulfonate 88 75-76 2,4-Di-O-methyl-~-xylono~actone sirup
Rolafion solvent
ReJerences
H?O
18 17 24
$46
HzO 95% EtOH H2 0
+30
EtOH
+97 +69 (400 hrs.) 87 +94.3 --f
+
HzO CHCla dioxane EtOAc CHClz CHCI, CHCI, CHC13 HzO
17, 23, 25 18 23 18 26 109 4 , 10 16 26 16 16 1 1 16 3, 26
H,O H9 0 EtOH
16 16 16
+36 +16.4 +33.7
H,O EtOE-I CHCl,
8 8 8
-49.9 $24.9 -+ +20.5 +5.3 f40, f 3 0 $13 +31 f 5 -82 -33 -71 -34.8 -56 -+ -27 (65 hrs.)
CHCI,
8 1, 13
-30
--f
$22
-13 4 +23 -26 - 82 -40 - 70 -82.4 -58.0 +28.8 -15 --t +30 (3
HI0
days)
2,4-Di-O-methyl-~-xylo~amide 2,4-Di-O-rnethyl-r,-xylonic acid, phenylhydrazide 2,5-Di-O-methyl-~-xylose
98-100 143.5144.5 sirup
Methyl 2,5-di-O-methyl-a-~-xylo-sirup side, 3-p-toluenesulfonate p anomer sirup 3,4-Di-@methy1-D-xyl ose sirup
Methyl 3,4-di-O-methyl-p-~-xylo- 89-90 side 2-p-toluenesulfonate 105 3,4-Di-O-met,h~~l-1~-x~Ionolacto1ie 68
-13 +51 +47
--f
+29.5
H 2 0
CHCI3 CHCla MeOH MeOH CHCla CHCI, HP O CHCI? r-I .o
1 28 15 27 1 5 15 1 13, 15, 21,
27, 20, 30
260
GEORGE G. MAHER
TABLEI (Continued) Substance
3,4-Di-O-methyl-~-xylonic acid, phenylhydrazide 3,5-Di-O-methyl-o-xylose
1,2-0-isopropylidene acetsl p-bromophenylosazone
Melting point, "C.
15
sirup sirup 107-108
+11 +25 -60 -46 + -30 +72 + +41 (48 days) +75 -+ +27 (33 days) ~ 0 1 1 6 7 8 0 +81.5 + +39 (49 days)
95-96
+6
91-92
+64
+
+18
Methyl 2,3,4-tri-O-methyl-a-~-xysirup loside
+86 +49.5
Methyl 2,3,4-tri-O-methyl-8-~-xy49-50 loside 2,3,4-Tri-O-methyl-N-phenyl-o-xy-102 losylamine 97-98 2,3,4-Tri-O-methyl-~-xylonolac- 54,55,56 tone 2,3,4-Tri-O-methyl-u-sylonic acid, 138 phenylhydrazide 2,3,5-Tri-O-methyl-~-sylose sirup
-73 -66 -84 + +47
Methyl 2,3,5-tri-O-rnethyl-u-xylo-sirup side 2,3,5-Tri-0-methyl-I-xylonolac t,one
sirup
2,3,5-Tri-O-meth~.l-o-sylonamide 84-85 2,3,5-Tri-O-methyl-u-sylonic acid, 89
phenylhydrazide
References
132
sirup
3,5-Di-O-methyl-~-xylonic acid, phenylhydrazide 2,3,4-Tri-0-methyl-o-xylose
-
[ a ] degrees ~,
CHC13
7
HzO
7
CHCl, 7 CsHsN- 9 EtOH HzO
7
H20
5
H2O
9,31
EtOH
9
Hz0
4,11, 25, 29, 32, 33I 34, 35, 50 32 32
MeOH HC1EtOH CHCl, MeOH EtOH
16 15,35 10,14 17,22 11,16, 50 36
-97 + +32.8 -4 + +21 (120 hrs.)
MeOH
4-24.7+ +29.5 f32 $114 +134 +74 -+ +61.4 (504 hrs.) [alsrtI1 +I00
H,O
37
MeOH MeOH HzO
37 5 5 31,38
Hz0
H20
H?O
18 39 18
METHYL ETHERS OF PENTOSES AND
G-DEOXYHEXOSES
2G I
TABLE I1 The M e t h y l Et rs of D-) ubin ose Subslance
2-O-Methyl-n-arabinose
&felling
poin6, "C.
sirup
143 (dec.) phenylhydrazone 113 Methyl 2-0-mcthyl-p-~-nrsbinopyiano- 48 side 62-63 Methyl 2-O-niethyl-~-arnbiriosicle 2-O-Methyl-~-ara\~onolactorie 87 p-tolylsulfonylhydraxone
2-0-Me thy1-D -arabonamide hydrate 2-O-Methyl-~-arabonic acid,phenylhydrazide Ammonium 2-O-methyl-~-arabonate 3-0-Methyl-D-arabinosz
131 9697 158-159 (dcc.) 146 sirup
3-O-Methyl-~-arabonolactone
81
3-0-Methyl-D-arabonamide 2,4-Di-O-methyl-~-arabinose
132 sirup
2,4-Di-O-methyl-N-phengl-~-ar~ _inosy1amine 2,5-Di-O-niethyl-~-arabinose 2,5-Di-O-methyl-~-arabonolactone
142-143
2,5-Di-0-methyl-D-arabonamide
sirup sirup 59-60 131-132 30.5-131 166-167
2,5-Di-O-methyl-u-arabonic acid, phenylhydraaide 3,5-Di-O-methyl-~-arabinose sirup 3,5-Di-O-methyl-N-phenyl-~-arabino-118 sylamine 3,5-Di-O-niethyl-~-arabonolactone 74-75
3,5-Di-O-methyl-~-arabonamide 144 3,5-Di-O-methyl-o-arabonic acid, 144-145 phenylhydrazide Methyl 2,3,4-tri-O-methyl-o-arabi110-sirup side
[ a ] degrecs ~.
Rolnlion solvcnl
- 102
HzO
- 17
HzO
-205 -15.4 +52.7 4 +47.4 (90 hrs.) -53.2
Re/-
E P e nc E s
40,41, 42 41
41 41 M e O H 42 40 HI0 1% 2 0
40
H20
40 40
HzO
40
-27.7 HzO -90 + -43 (3 H 2 0 days) f99 -+ +-75 Hso (22 days) (passes through a max.)
40 5
-23
5
5
43 MeOH 43 43
-30.8 -37.8
HzO
+23 -51 -+ -32 (20 days) +62.2
HsO HzO
HzO
103 4
103 4
-34
HzO
103 4
447
1120 HzO
5 44
H2
0
5
H2
0
44
-28.8
$85 -+ +57 (22 days) -11
5
-248
H2
0
45
-
GEORGE G . MAHER
TABLEI1 (Continued) Melting poinl, “C
Substance
2,3,4-Tri-O-methyl-~-araboni~mide 96 2,3,5-Tri-O-methyl-~-arabinose sirup Methyl 2,3,5-tri-o-methyl-~-arabino- sirup
[.ID,
degrees
-25 $40 80
+
side 2,3,5-Tri-O-methyl-~-arabonolactone 30 2,3,5-Tri-O-niethyl-~-arahonnmide 134-135 -25 137-138 - 19 135-136 - 14 137-139 -16.5 sirup +18.4 144 -35.2
Rololion solvent
MeOH 47 MeOH 46 46 HzO 103
EtOH 44 MeOH 47 H?O
HzO H?O EtOH
TABLEI11 The M e t h y l Ethers of L-Arabinose Subslance
Melting boint, “C.
[a]~ degrees ,
Rejerences
4,44, 46 103 46 47
Rototion solvent
References
~~
2-O-Methyl-~-arabinose
sirup
+100
pheriylhydrazone
114, 116 p-tolylsulfonylhydrazone 145-145 Methyl 2-O-methyl-p-~-arabinopyrano- 63-65 side hydrate 46-47 2-O-Methyl-~-arabonolactone sirup 2-O-Methyl-~-arabonamide 131 3-0-Me thyl-L-arabinose sirup phenylosazone 163 3-O-Methyl-N-phenyl-~-arabinopyrano-117 sylamine 3-O-Methyl-~-arabonolactone 78 3-O-Methyl-~-arabonamide 132 2,3-Di-O-methyl-~-arabinose sirup
+208 -44 + -40 +52 $110
-74
+
107 +101
2,3-Di-O-methyl-N-phenyl-~-arabinopy138,
ranosylamine
139 35 30
-38 + -25 (12 days) -38 + --30 (7 days) -33
41,48, 49 41,49 50 48 48 49 49 51 52 51
51 51 49,52 53 49,53, 547 52 52 53
95
263
METHYL ETHERS O F PENTOSES AND 6-DEOXYHEXOSES
TABLE I11 Continued) Substance
2,3-Di-O-me thyl-L-arabonamide
Melting point, "C
162 156, 160 sirup
2,4-Di-O-methyl-N-phenyl-~-arabino- 145sylamine 146 126 2,4-Di-O-methyl-~-arabonolactone sirup
158 sirup Methyl 2,5-di-O-rnethyl-~-arabinoside sirup 2,5-Di-O-methyl-~-arabonolactone 60 131, 132 2,5-Di-O-methyl-~-arabonic acid
phenylhydrazide 3,4-Di-O-methyl-~-arabinose
[a]D,degrees
+17 +30. 85 +37.8= +I18 +129 i 4
+55 + +27 (14.5hrs.) +99 + +39 (17 hrs.)
+60
-2 -60 -60 -+ -44.8 (320 hrs.) - 19 +38
+I16 +lo4 +125
phenylosazone 132 Methyl 3,4-di-O-methyl-p-~-arabinosidesirup 2-benzoate sirup 2-p-toluenesulfonate 111112 3,4-Di-O-methyl-~-arabonolactone sirup 3,4-Di-O-methyl-~-ara bonamide 133 3,5-Di-O-methyl-~-arabinose phenylosa- 170 zone
Re/eTences
49, 52 23,53, 65, 95 43 43 53 55 53, 55 53 43 55 43 57
56 56 43,56, 57 50 4, 50, 56 43, 57 56, 43 2
+25.8 + -16 (120hrs.)
162, 163 sirup
Rulalion solvent
50,56, 43 58 53 111 53
+210.6 +143.5
58
+44 + -1 (6 hrs.) +28.2
111
58 58
111 59
264
GEORGE G. MAIlER
TABLE 111 (('ontini~erl) Subslance
3,5-Di-O-methyl-~.nrabonic acid, phenylhydrazide 2 , 3 , 4-Tri-0-methyl-L-arabillosc
M elling Poinl, "C.
[ a ] d~p , grees
-84
HpO
60
73 78 145 144 114
-83 - 43
1120 CHCI, Hp0
51 50 59,60 51
sirup
+l20, +122, 127
H,O
+46.2b
1120 MeOH HzO MeOH HIO
sirup
+10
51
+
+24b
+250b +223b +I45 + +22.4 hrs.)
+24
+45
62 75, 63 75 43, 63
MeOH
43 43, 53,
63, 64 53, 55
159, 160 0
H20
-43
-45 4 -24 (50 hrs.) -47, -44
H.0
138138
+20, +21
I120
138
+I6
H2
137 130
+17.4 +21, +24
Hp0
28,30 20, 31
62
EtOH HpO
156, 157
2,3,5-Tri-O-mcthyl-1,-arabinose sirup Methyl 2,3,5-tri-O-mrth~l-~-arabirioside sirup 2,3,5-Tri-O-niethyl-~-araborlolactone 33
53,55, 61, 62 62 62
(24
+I36 107, 103 2,3,4-Tri-O-meth.vl-r, arabonic arid, phenyl hydrazide
erences
75
Methyl 2,3, 4-tri-O-methyl-~-r,-:trabino- 46-48 side B anomcr 44-46
-+ -69 (28 days)
Ref-
Rolnlion solvent
HpO
57 60 43 05, 43 40, 50, 65 14,23, 59 43 49,60, 65, 66 104 17, 66 9
0
EtOH
These rotations wcre deduced from the rotations of the isomeric D compounds. * T h e rotations of the anomers have been reversed from those in the original paper, in view of Hudson's rule. a
T A B L EIV*
-
'I'he Methyl Ethers of D-Ribose Subslance
Meltiag poinl, "C.
Methyl 2,3-3nhq'dro-~-O-nict2lyl-p- 75-77 D-rihoside sirup 5-0-Met hyl -D -ribose 161-162 p-bromophenylosazone 2,3-Di-O-methyl-D-rihose
3,5-Di-O-methyl-~-ribosc phrnylosazone 2,3,4-Tri -0-methyl-D-ribose
35-86 Jaioo
2,3,4-Tri-O-methyl-~-ribonic acid 2,3,4-Tri-O-methyl-~-rihono-6-lac- sirup tone
sirup
2,3,5-Tri-O-meth?.l-N-phenyl-~ri- 55.5, 56.5 bosj~lamine sirup Methyl 2,3,5-tri-O-methyl-~-riboside 2,3,5-Tri-O-rnethyl-o-ribonicacid
2,3,5-Tri-O-methyl-u-ribono-r-lac-18.5-19 tone
phenylhydrazide
-~
~-
-7
€I20
-48
CsHhN8:to13
108.5109.5
Re/erenm
15 67 67 68 60
sirup 161
sirup
2,3,5-Tri-O-methyl-~-ribonic acid,
[a]D,degrees
Rolalron solvenl
-51.7
4
-40
I3rO
70 105 72 70
-26.7 -35
MeOH HIO
-24.2 f34.0 +69.3
MeOH CHCI,
70 70 70
f85.4 $114.1 -4.4++17.1 (191 hrs.) +46.2 f41.4
C 611 6 EtzO Hz0
70 70 70
MeOH MeOH
+40.6 f51.6
HzO HzO
72 69 7 106 67 72, 107 69, 71
$59.1
MeOH
69
+40.9 -+ $11.9 (448 hrs .) -20.2 3 -10.6 (141 hrs.) -18.9 3 $7.6 (703 hrs.) $55.9 +124.0 f83.1
Hz0
72
HzO
67, 69
Hz0
72
CHCls EtzO C6H6
72 72 72 60
HIO
* For additional material see G. R. Barker, T . M Noone, D. C. C. Smith and J. W Spoors, J . Chem. SOC.,1327 (1055); and G. R. Barker and D. C. C . Smith, .I. Chem. Soc., 1323 (1955). 265
TABLEV The Methyl Ethers of o-Lyxose
Melting oint, "C
Subslance
-
[u]D.
Rolnlion solvenl
degrees
+60
Methyl 2,3-anhydro-5-0-methyl-cu-u-lyxo- 43 side 2,3,4-Tri-O-methyl-~-lyxose 79 Methyl 2,3,4-tri-O-methyl-o-lyxoside sirup
-10
5 73 73 73 73
-22 +lo
--f
[a16461
+37.3 +35.5--9.3 (66.5 hrs.)
2,3,4-Tri-O-methyl -~-lyxonolactone
sirup
2,3,4-Tri-O-methyl-o-lyxonic acid, phenylhydrazide 2,3,5-Tri-O-methyl-o-lyxosc 2,3,5-Tri-O-methy~-~-lyxonolactorle
180181 sirup +39 41 +82.5 4 +56.5 (1000 hrs.) sirup -20.8 --L +25.6 (500 hrs.) 140 142
2,3,5-Tri-O-methy~-~-lyxonic acid phenylhydrazide
References
36, 73 74 74
74 74 36
TABLEVI* The Methyl Ethers oj" L-Rhamrkose Substance
Mellinx point, "C.
sirup 113-114 2-0-Methyl-N-phenyl-L-rhamnosyl-152 amine Methyl 2-0-methyl-~-rhamnopy138-140 ranoside 2-O-Methyl-~-rhamnono-y-lactone 116-1 17
2-O-Methyl-~-rhamnonamide 3-O-Methyl-~-rhamnose phenylosazone hydrate 4-O-Methyl-~-rhamnose
phenylhydrazone phenylosazone
117-118 113 128-130 118 (dec. 125-126 122
176 (dec. 162-163
triacetate
[a]D,
Rotirlion solvent
+31, +24
H,O
+43
CsHGN
References
76,110 77 110 76
-62 4 -64 (117 hrs.) +57 (17 hrs.)
HzO?
CfiHsNEtjOH
110 110 28 78 78
+13
-12.9
+26 $25.8 -12.2
266
degrees
-+
f14.3
MeOH MeOHNH3 H20
79 80
CfiH6NEtO H MeOH
79
110 80
80
TABLE VI (Continued) Subslance
Melling
Methyl 4-O-methyl-a-~-rhamnosidesirup j3 anomer sirup 4-O-Methyl-~-rhamnono-6-lactone82 80- 81
5-0-Methyl-L-rhamnose
[a]~ degrees ,
)aid, "C.
pheny lhydrazone
102-103 162-163
phenylosazone
123-124
triacetate 2,3-methylene acetal
115-116 77-79
Methyl 5-0-methyl-a-~-rhamnoside 59-60 5-O-Methyl-~-rhamnono-~-lactone 164-166 2,3-Di-O-methyl-~-rhamnose sirup 1,5-di-benzyl ether
119 2,3-Di-O-methyl-N-phenyl-~-rham138 136-137 nopyranosylamine
-50.2 -13.9 -141+ -115 (14 hrs.) -140 + -112 (18 hrs.) -4.3 -18.4 + +8.1 (7 days) $65.3 -+ +44.4 (3 days) -76.3 $6.4 + +4.5 (46 hrs.) -89.2 -36 f 4 $47.6, +42.5 +71.7 +147.8+ +42.8 (70 hrs.) -6, -14
Methyl 2,3-di-O-methyl-a-~-rhamsirup noside Methyl 5-0-benzy1-2,3-di-O-methyl93 -72 L-rhamnoside 2,4-Di-O-methyl-~-rhamnose 01-93 +10.6 2,4-Di-O-methyl-N-phenyl-~-rham+128.5 + f 5 . 6 141nosylamine 142.5 +136 + +4 3,4-Di-O-methyl-~-rhamnose -10 + +18.6 91-92 +18.2 (equilib94-95 rium) $24 + +18.5 98-99 19 102-103 3,4-Di-O-methyl-~-rhamnose, 1,2- 67 +36 (methyl orthoacetate) +40.6 67-68 3,4-Di-O-methyl-~-rhamnonic acid -15.9+ -118.1 3,4-Di-O-methyl-~-rhamnonolac- 76-78 -158.5 + tone -116.6 -154 + -116 78-79 (48 hrs.) -148 -+ -117 (72 hrs.) -153 --t -119 66-68 (150 hrs.) 152155, 154150 sirup
+
267
Rotation solvent
HnO
References
H20 HzO
79 79 81
HzO?
110
H*O C6H5N5
79 79
CsHbNEtOH MeOH
79
HzO? H2 0 HzO?
79 110
EtOH
79 110 78, 53 78 82, 83 108
H?O
82, 83
Me&O
78
1320
lOX(1)) 108(a) 108(b) 14, 84 85
H20 MenCO
EtOH EtOH H?O
HzO
H2
H rO HZO H?O H2 0
86 4 87 84 87 81 85, 88
HtO
89
H*0
4
Hz0
84
0
H20
84, 88
H2O
%90, 91, 92 ___
TABLE VI (Continued)
-
~
Melting )oant, "C.
Subslance
[WID,
2,3,4-Tri-O-methyl-N-phenyl-~- 111,112
Rotation solvent
degrees
References
44, 56
+127
rhamnosylarnine 110 119-120 sirup
hfethyl 2,3,4-tri-o-methyl-a-~rhamrioside 53-54 ,3 anomer 40-41 2,3,4-Tri-O-rnethyl-~-rharnnonolactone 2,3,4-Tri-O-methyl-~-rhamrionic 177 acid, phenylhydrazide 2,3,5-Tri-O-methyl-~-rhamnonic 160 acid, phenylhydrazide
+130 (14 hrs.)
Me&O
-15.1
HzO
+106 -130
84 94
H20 +
-78
93 92 91
HzO
56, 95 56
a No ethanol was used in the rotation solvent, according t o the authors. *;For additional material see G. R. Barker, T. M. Noone, D. C. C. Smith and J. W. Spoors, J . Chent. Soc., 1327 (1955); and G. R. Barker and D. C . C. Smith, J . Chem. Soc., 1323 (1955).
TABLE VII The Methyl Ethers of o-Fucose Melting boinl, "C.
Subslance
[..ID,degrees
Rotation solvent
5 ) O
$5 -
2-0-Methyl-n-fucose
155161 Methyl 3,4-0-isopropylidene-2-O-mcthyl-98-100 n-fucoside sirup 3-O-Methyl-~-fucose 106, 119 178, phenylosazone 179 170180 Methyl 3-0-met.h~l-cu-~-fucopyrarioside sirup 97-99 0 anomer 3-0-Methyl-u-fuconolactorie 136137 137138 2,3,4-Tri-O-methyl-~ -fucose hydrate
sirup 65
1332,3,4-Tri-O-meth~-l-S-p1ieriyl-~-fucosyl135 amine hlcthyl 2,3,4-tri-0-methyl-~-~-fucosidc03-98 268
+73
+
+87
H20
76 76
+ +
[I2 0 HzO e10
103 +110 106
96 98 97 99
-> +18 (14 hrs.) +124.4
C,H&
-75.3 f 4
h20
01 98 98
-92.5 + -74.9 (16 (lays) 106 +183 +128.8 (cnlc'd. as nnhydrous) +76
H20
00
H?O H2 0
13 00
EtOII
13
+11.2
HzO
f0.5
+
---f
EtOH MezCO
00
13
-
METHYL ETHERS OF PENTOSES AND G-DEOXYHEXOSES
269
TABLEVIII The Methyl Ethers of L-Fucose
-
~
M.eZfing p o d , "C.
Substance
[.ID,
L:
degrees
p
-
Me thy1 3,4-0-isopropylidene-2-0-methylB-L-fucoside 3-0-Methyl -L-fucose p heny 1osaz one Methyl 3-O-methyl-a-r,-fucopyranoside 3-O-Methyl-~-fuconolactone
88-92
-10.9
100
sirup 172-176 130-132 sirup
-94
102 102 102 102
3-O-Methyl-~-fuconamide 176180 2,3-Di-O-methyl-~-fucose sirup Methyl 2,3-di-0-methyl-a-~-fucopyranoside49-51 2,3 -Di-0-methyl-r. fuconolactone sirup 2,3-Di-O-methyl-~-fuconamide 3,4-Di-O-methyl-~-fucose Methyl 3,4-di-O-methyl-a-~-fucoside 2,3,4-Tri-O-methyl-~-fucose
hydrate
78-79 76, 82 100
36-37
65
2,3,4-Tri-O-methyl-N-phenyl-~-fucosyl- 133-134 amine 97-98 Methyl 2,3,4-tri-O-methyl-01-L-fucoside 85-92 p anomer 101.5102.5 2,3,4-Tri-O-methyl-~-fuconolactone sirup 102
- 173 +25 -+ +75 (62 hrs.) +16.4 +4.6 - 190 f 9 4 +47 (22 hrs.) +30.2 - 118 -213 -184 -+ - 128 -111 -169 -+ -118 (24 hrs.) -77 -209 - 196 -21.1
-1384 -36 (48 hrs.) -35
102 102 102 102 102 82 82 100
13 100
13
100 13 100 13 13
-
References (1) G. J . Robertson arid T. H. Speedie, J. Cheni. Soc., 824 (1934). (2) R. J. McIlroy, J . Cheiiz. SOC.,100 (1946). ( 3 ) G. 0. Aspinall and R. S. Mahomed, J . Chem. Soc., 1731 (1954) (4) E. L. Hirst,, E. G. V. Percival and Clare B. Wylam, J . Chem. SOC.,189 (1954). (5) Elizabct,h E. Percival and R. Zohrist, J . Chem. SOC.,564 (1953). (6) E. G. V. Percival arid I. C. Willos, J . ('hem. Soc., 1608 (1949). (7) R. A . Laidlaw, J . ( ' h w i . SOC.,2941 (1952). (8) G. J. Robertsoii and D. Gall, J . Chein. Soc., 1600 (1937). (I)) 1'. A . 1,evene and A . I,. Raymond, .I. B i d . ('hem., 102, 331 (1933). (10) R. A . 1,aidl:tw and IS. G. V . Percival, .I. P h t . SOC.,528 (1950). (11) E. V. White, J . Am. Chem. Soc., 76, 257, 4692 (1953).
270
GEORGE G. MAHER
(12) G. 0. Aspinall, E. L. Hirst and R . S. Mahomed, J . Chem. SOC.,1734 (1954). (13) Sybil P. James and F. Smith, J . Chem. Soc., 739, 746 (1945). (14) R. A. Laidlaw and E. G. V. Percival, J . Chem. SOC.,1600 (1949). (15) L. Hough and J. K . N. Jones, J . Chem. SOC.,4349 (1952). (16) 0. Wintersteiner and Anna Klingsberg, J . Am. Chem. SOC., 71, 939 (1949). (17) I. Ehrenthal, R. Montgomery and F. Smith, J . Am. Chem. SOC.,76, 5509 (1954). (18) H. A. Hampton, W. N. Haworth and E. L. Hirst, J . Chem. SOC.,1739 (1929). (19) S. K . Chanda and E. G. V. Percival, Nature, 166, 787 (1950). (20) S. K . Chanda, Elizabeth E. Percival and E. G. V. Percival, J . Chem. SOC., 260 (1952). (21) R. L. Whistler antl L. Hough, J . Am. Chem. SOC., 76, 4918 (1953). (22) I. Ehrenthal, M. C. Rafique and F. Smith, J . A m . Chem. SOC.,74,1341 (1952). (23) G. 0. Aspinall, E . I,. Hirst, R. W. Moody and E. G. V. Percival, J . Chem. SOC.,1631 (1953). (24) J. Tachi and N. Yaniamori, J . A g r . Cheni. SOC.Japan, 26, 12 (1951-1952); Chem. Abstracts, 47, 10486 (1953). (25) R. A. S. Bywater, W. N. Haworth, E . I,. Hirst and S. Peat, J . Chem. SOC., 1983 (1937). (26) C. C. Barker, E. L. Hirst and d. K . N. Jones, J . Chenz. SOC.,783 (1946). (27) J . K . N. Jones and L. E. Wise, J . Chem. SOC., 3389 (1952). 2522 (1954). (28) A. R. N . Gorrod and J. K . N. Jones, J . Chem. SOC., (29) E. V. White and P. S. Rao, J . Am. Cheni. Soc., 76, 2617 (1953). (30) R. L. Whistler, H. E. Conrad and L. Hough, J . Am. Chem. SOC.,76, 1668 (1954). (31) W. N. Haworth and C . It. Porter, J . Chern. SOC.,611 (1928). (32) A. E. Carruthers and E. L. Hirst, J. Chem. SOC.,121, 2299 (1922). (33) S. K . Chanda, E. L. Hirst, J . K . N. Jones and E. G. V. Percival, J . Chem. Soc., 1289 (1950). (34) S. K . Chanda, E. L. Hirst and E. G. V. Percival, J . Chenz. SOC.,1240 (1951). (35) F. P. Phelps and C. B. Purves, J . A m . Chem. SOC.,61, 2443 (1929). (36) W. N. Haworth and C. W. Long, J . Chem.. SOC.,345 (1929). (37) W. N . Haworth and G. C. Westgarth, J . Chem. SOC.,880 (1926). (38) H. D. K . Drew, E. H . Goodyear and W. N . Haworth, J . Chem. SOC.,1237 (1927). (39) R. 1,. Whist,ler, J . Bachrltch and C.-C. T u , J . A m . Chem. SOC.,74,3059 (1952). (40) 0. T . Schmidt antl A . Simon, J . prakt. Cheni., 162, 190 (1939). (41) J . K . N . Jones, P. W. Kent and M. Stacey, J . Chem. SOC.,1341 (1947). (42) G. J. Halliburton and R. J. McIlroy, J . Chenz. SOC.,299 (1949). (43) F. Smith, J . Chern. SOC.,744 (1939). (44) W. N . Haworth, P.W. Kent and M. Stacey, J . Chenz. SOC.,1211, 1220 (1948). (45) J. W. P r a t t , N. K . Richtmyer and C. S. Hudson, J . Am. Chem. SOC.,74,2200 (1952). (46) W. N . Haworth, S. Peat and J . Whetstone, J . Chem. SOC.,1975 (1938). (47) P. W. Kent and M. W . Whitehouse, J . Cheni. SOC.,2501 (1953). (1s) Mary Ann Oltlham and J . Honeyman, J . ('hem. SOC.,986 (1946). (49) E. I,. Hirst and J . K.N . JOIRS, J. ('hem. Soc., 1221 (1947); 2311 (1948). (50) F. Brown, E. 1,. Ilirst. and J . K.N . Jones, J . Chern. Soc., 1757 (1949). (51) E. I,. Hirst, J. Iheestablished techniques of carb0hydrat.e chemistry. Ligiiin can be removed by a number of other procedures, but these, in general, result in incomplete delignification or in serious loss or degradation of polysaccharides. The classical procedure of Cross and Bevanli4 employs extraction with 1 % sodium hydroxide, followed by repeated chlorinations and extractions with hot 2 % sodium sulfite solution. The yields of cellulosic material obtained in this procedure are considerably lower t'han those obtained by the holocellulose procedure. Van Beckum and R i t t ~ showed ' ~ ~ that hydrolysis of holocellulose nit'h hot 1.3 % sulfuric acid for 2 hours yields residues which are equal in and similar in chemical properties,176to Cross-and-Revan cellulose obtained from the same wood. (171) H. F. Lewis, T a p p i , 33, 299 (1950). (172) R. I,. Mitchell and G. J. Rit,ter, J . A m . Chent. Soc., 62, 1958 (1940). (173) L. E. Wise, Ref. 9, p. 408. (174) E. J. Bevan and C . F. Cross, J . Chem. SOC.,38, 666 (1880); C. F. Cross, E. J. Bevan and C. Beadle, ''Cellulose," Longmans, Green and Co., London, 1895. (175) W. G. Van Beckum and G . J. Ritter, Paper Trade J., 108, No. 7, 27 (1939).
POLYSACCJIARIDES ASSOCIATED WIT11 WOOD CELLUT,OGE
32 I
h iium\m of other deligiiificatioii procedures have heen devised in which the primary objective has been to obtain, for various purposes, a measure of the total-carbohydrate fraction. These procedures have employed as the active delignifying agents such materials as nitric acid,li6 monoethanola* l i 8 atid other amines,17* neutral h y p o ~ h l o r i t eavid , ~ ~ ~hypochlorite,’sO etc. These methods have recently been discussed by RrowningLS1and by .Jahn.L82Although such procedures are of general interest, their application has not yielded much information that could be considered fundamental to an understanding of the chemistry of wood cellulose. Neither have these procedures herome important in the commerrial production of wood cellulose. 2 . Commercial Wood Cellulose
a. General.-Technical wood pulp is prepared by one of the following three pulping procedures: the sulfite, the sulfate, and the soda processes. In t’he first of these, the cooking liquor is acidic; in the other two, it is alkaline. The scope for variation of reaction conditions is enormous. Some of the more obvious variables are: type of raw material, dimensions of wood chips, concentration of active ingredients of pulping liquor, liquor-to-wood ratio, time-temperature relationship, washing and screening procedures, etc. Added to this are the possibilities for endless variations of methods for bleaching and purification, or for treatment of wood chips prior to pulping. The selection of specific conditions depends on the u1t)imate use for which the cellulosic material is intended. For a full understanding of pulping processes,18~a knowledge of the anatomy of ~ 0 0 d ~is~important, ~ - ~ 8 ~since marly of the problems of pulping are associated with such factors as ensuring penetration of wood chips by cooking liquors and avoiding mechanical damage to fibers. The nature of the reaction of pulping reagents with lignin is also of prime importance to (176) G. dayme arid P. Schorning, Papier-Fabr., 36, No. 21/25, 235; No. 37, 393 (1938). (177) L. E. Wise, F. C. Peterson and W. M. Harlow, Ind. Eng. Chem. A n d . E d . , 11, 18 (1939). (178) P . Bloom and E. C. Jahn, Tech. Assoc. Papers, 24, 127 (1941). (179) A. G. Norman and S. H. Jenkins, Biochem. J . (London), 27, 818 (1033). (180) J . H. Ross, A. L. Davidson and E. 0. Houghton, Pulp & Paper Mag. Can., 27, No. 25, 925 (1929). (181) B. L. Browning, Ref. 9, p. 1153. (182) E. C. Jahn, Ref. 9, p. 1021. (183) J. N. Stephenson, ed., “Pulp and Paper Manufacture,” McGraw-Hill Book Co., Inc., New York, N . Y., 1950. (184) H. P. Brown, Ref. 9, p. 7. (185) W. M. Harlow, Ref. 9, p. 99. (186) G. J . Ritter, Ref. 1, p. 286.
322
W. J. POLGLARE
an understanding of pulping processcs. Such aspects will receive oiily cursory att,ention in the following discussion. The chief object'ive will be to outline the apparent effect of certain chemical pulping processes on the carbohydrate composition of wood pulps. 0. Sulfcte Pulps.-The process for making white pulp from wood by treatment with sulfurous acid or calcium bisulfite under pressure was discovered by Tilghman.ls7,lS8 In spite of many difficulties, the process eventually permitted product,ion of pulp a t a profit, and, t,oday, it is still one of t'he most important processes for the preparation of wood cellulose. The chemistry of delignification in the sulfit,e process has been discussed recentJy by Hagglu1id~8~ and by E r d t m a i ~ 'Depolymerization ~~ of polysaccharides occurs to an extent dependent, on the acidit,y of t8hecooking liquors, the reaction time, and the temperature of reaction. This has been shown by reducing-sugar analysis on liquors during t3he sulfite cook.1g1Evidently, complex sugars are liberated during the early stages of cooking, since acid hydrolysis increases t,he reducing value of sampled cooking liquors. According t,o H a g g l ~ n dthe , ~ ~final, ~ wast,e, sulfite liquors contain, chiefly, monomeric sugars and aldonic acids (formed as the result of the oxidation of sugars by bisulfite ions). Both D-mannonic and n-xylonic acids have been isolated from sulfite, waste liquors.18g The order of appearance of different sugars in sulfite cooking liquors has been studied with filter-paper chromatographic techniques by Sundman.112 The liquors were sampled a t various stages during cooks of spruce, pine, birch, and aspen. Arabinose appeared in the cooking liquors before the temperature had reached loo", and increased rapidly ill amount, eventually decreasing toward the end of the cook. Xylose and galactose appeared a t the same time, and a little later than arabinose, but their proportion did not decrease on prolonged cooking. Mannose was detected when t,he ternperature of digest>ionreached I 30°, and glucose at 140". The quantitative data obtained by Isbell and Frush1g2on hydrolysis by dilute hydrochloric acid of met,hyl glycosides are of interest, in this connection. From these studies,lg2 it may be deduced that the rat,es of hydrolysis of the methyl 0-D-pyranosides of t,he wood sugars arc i n the following order: D-xylOSe = n-galactose > n-mannose > D-glucose. The ext,reme acid(187) B. C. Tilghman, U. s. P a t . 70,483 (1867). (188) For a history and an outline of the sulfite process, see G . C . McGregor, Ref. 183, Vol. 1, p. 252. (189) E. Ragglund, "Chemist,ry of Wood," Academic Press Inc., NPWYork, N . Y., 1951. (190) H. Erdtman, Ref. 9, p . 999. (191) E. Hiiggluntl, Ref. 189, p. 430. (102) H. S. Isbell and Harriet I,. Frush, J . Research N a t l . Bur. Slandards, 24, 125 (1940).
POLYSACCHARIDES ASSOCIATED W I T I I WOOD CELLULOSE
Analysis
I
Spruce Spruce Pine Aspen Aspen Birch Birch
3.75 6.50 6.50 2.50 5.25 2.75 5.50
I
0.f
323
TABLE XV Unbleached, Sulfite Pulps46
-_
58.3 52.1 54.0 55.6 51.5 54.6 48.9
87.1 88.1 85.6 83.3 86.3 77.1 81.6
_____
89.5 90.1 86.2 88.0 90.4 83.4 87.5
6.3 6.0 7.7 2.1 2.0 1.4 1.5
4.2 3.9 6.1 9 .9 7.6 15.2 11.0
Cooking liquor: total 802 , 71.1%; CaO, 1.3%. Time t o 60°, 0.5; time from 60' t o 105", 3; time from 105" to 130°, 0.5 hours. Maximum temperature, 130".
lability of L-arabinose units in polysaccharides is well known and is related to the furanose-ring form usually exhibited by this sugar in polysaccharidcs. Thus, the order in which these sugars appear in sulfite cooking liquors is consistent with the known stability, toward acids, of the glycosidic bonds of model substances, The final liquors from pine and spruce mere rich in mannose and galactose, whereas birch and aspen liquors were high i n xylose.l I 2 I n further studies on the sulfite pulping process, Sundman, Saarnio and G u s t a f s ~ o nanalyzed ~~ a number of sulfite pulps by hydrolysis, followed by paper chromatography. Some of their results are shown in Table XV. Although galactose and arabinose had been detected in complete hydrolyzates of wood, by chromatographic methods (see Table XV), neither of these sugars could be detected in the hydrolyzates of sulfite pulps. After chlorination and hypochlorite bleaching of these sulfite pulps, the mannan and xylan content did not change a p p r e ~ i a b l y . ~ ~ For the purpose of conversion to textile fibers, dispersions or solutions of cellulose or its derivatives are achieved by various means, in order to make possible the extrusion of the fiber-forming material through the small orifices of the spinning jets. Wood pulps prepared for these and similar uses (such as the manufacture of cellophane) are known as "dissolving pulps.'7 The manufacture of dissolving pulps is a highly developed art, with processes protected by or, more effectively, within company files. The purification of siilfite pulps to a degree suitable for manufacturc of textile rayon (YO to 9.1%) alpha-cellulose), tire cord (94 to 95% alpha-cellulose), ' alpha-rellulosc) requires some kind of and cellulose ncetnte (95 to 96 % (193) Sce G. rJ:Lyme, Paper Trade J . , 106, NO. 21, 37 (1938).
324
W. J. POLGLASE
alkaline treatment,Ig4in addition to treatments for removal of residual lignin. Such pulps are frequently termed "high-alpha pulps." According to Hisey,Ig4the increase in alpha-cellulose content results from the removal of hemicelluloses and short-chain cellulose by dispersal in alkali, which leaves the longer cellulose molecules intact. Sodium hydroxide appears to be the most important alkali employed in commercial operations, a.nd two general methods for its use are of importance. One method employs dilute, sodium hydroxide solutions a t elevated temperatures, and t,he other uses concentrated solut'ioiis a t low temperatures. In the hot, alkaline refining process, temperature, time, concentration of alkali, and consistency of pulp may be varied over wide limits. The literature contains only a limited amount of data showing the effect of alkaline treatment on sulfite-pulp composition. However, from the available data, certain principles are apparent. Norman116has studied the effect on xylan of hot, alkaline extraction of cellulose prepared from oak, wheat, barley, and jute by the hypochlorite method of Korman and ,Jenkins.179It was observed'I6 that the xylan content of t,hese cellulose preparations can be diminished by boiling with various concentrations of alkali, but, in agreement with previous work by Heuser and Haug,Ig5an appreciable percentage of the original xylan could not be removed. Furthermore, it was noted that continued boiling with alkali removes hexosan (of undetermined configuration) a t a greater rate than xylan. Examples of the diminution of th.e pentosan of wood pulps by alkaline purification may be taken from the work of Richter.Ig6In a series of papers dealing chiefly with the pulping of Richter showed that either hot, dilute, sodium hydroxide or more concentrated sodium hydroxide a t ordinary temperatures removes a substantial part of the pentosan from sulfite pulps. Some of Richter's data are shown in Table XVI. In attaining a high alpha-cellulose with simultaneous removal of pentosail, there is a considerable loss of other material, presumably hexosan in nature. Although higher purity can be attained by treat'ment with cold, concentrated sodium hydroxide, this results in mercerization of the cellulose. Mercerized cellulose is of no use in the acetylation process unless it is specially treated i n order t,o avoid inactivation by The effect of alkaline treatment, on the mannan content of wood pulps (194) W. 0. Hisey, R.ef. 9, p. 1055; M. Martin, Can. Pulp and Paper, 6. No. 6, 8 (1952); L. K. Bickell, ibid., 6, No. 3, i 2 (1953). (195) E. Heuser and A . Haug, 2 . angew. Cheni., 31, 166 (1918). (196) G . A. Richter, Znd. Eng. ( ' h e m . , 33, 1518 (1941). (197) G . A. Itichter, Znd. Eng. ('hem., 33, 75, 532 (1941). (198) F. Olsen, Znd. E,ng. C h w i . , 30, 524 (1938).
POLYSACCIIARIDES ASSOCIATED WIT11 WOOD CELLULOSE
325
TABLEXVI Reniooal of Pentosans from Unbleuched, Suljife P u l p s l g ~ Pulp
'".*
h'aOIl added
Yield, %
Alphacellulose,
%
%
Penlosan,
~~~
Softwood
Mixed hardwoods
White birch
0 10% (on pulp)" 15% (solution concentration)
125 25
82.2 86.5
87.7 94.6 06.9
4.5 2.5 0.93
0 10% (on pulp)"
125
84
87.8 94.0
7.2 3.1
83.6
86.8 96.3
8.4 1.9
0 18% (solution concentration)
30
a At a consistency of lo%, this is equivalent t o a 1% solution concentration of sodium hydroxide.
has not been reported. However, Sundman and coworkers45state that a pulp having low manna11 content ( % unspecified) was obtained when a spruce, sulfite pulp was digested by a mild, sulfate cook. Presumably, the mannan content of sulfite pulps also is reduced by refining in alkaline solutions. It has been noted (page 319) that mannan is not as readily removed from softwood holocellulose by cold, concentrated alkalis as is xylan. It would appear, then, that the effect of the sulfite process on wood polysaccharides is primarily a depolymerization which leads in part to soluble sugars (predominantly from the more readily hydrolyzed, non-glucose polymers). The sulfite pulp, as normally obtained, contains a certain amount of low molecular-weight xylan, mannan, and glucan. Subsequent alkaline treatments disperse the main part of this short-chain material, to yield high-alpha cellulose containing residual amounts of inaccessible or high D. P., non-glucose polyoses. It has been shown120 that pulps prepared by the sulfite process contain uronic acid anhydride residues (apparently, 4-O-methyl-~-glucuronic acid anhydridelZ0). The uronic acid content of such pulps can be diminished by hot alkaline refining.lZ0 c. Alkaline Pulps.-Two general processes for pulping wood with alkali are important. In the soda process,1ggthe active component of the cooking liquor is sodium hydroxide. The stability of the glycosidic bond, together with the insolubility of cellulose, permits the use of high temperatures and high concentrations of alkali in effecting delignification. According to the theory of Koon,200lignin is dissolved as sodium lignate, and part of the hemicelluloses are removed by a combination of dissolution, colloidal dis(199) c. Watt and H. Burgess, U. S. Pats. 1,448 (1854); 1,449 (1854). (200) C. M. Koon, Ref. 1, p. 502.
32G
W . S. POLGLASE
persion, and degradation. Koon200 discusses the work of I3rauris and Grimes,'O' who had concluded that about half of the total alkali ronsumcd is used up by unknown mechanisms, t)o dissolve the carbohydrates. HagglundZo2 found lactones and hydrosy-acids (saccharinic, lactic), 1o the extent of 18.2 % of the wood, in the waste liquor ohtained from the alkaline pulping of spruce."? These are undoubtedly the products of alkaline degradation of the hemicelluloses removed from the wood. The sulfate process or kraft process has the advantage of higher pulp yield than is afforded by the soda process. The so-called "sulfate liquor" is a mixture of two components, sodium hydroside arid sodium sulfide. I n industrial practice, sodium sulfide is formed by the reduction of added sodium sulfate when the organic matter from a previous pulping operation is burned for chemical recovery. I n a review of alkaline processes, HolzerZo4 cites experimental work which shows that the higher pulp yield in the sulfate process is ascribable to the rate of dissolution of lignin, which is higher than in the soda cook, whereas the rate of dissolution of carbohydrate is the same in the two procedures. This results in a shorter reaction time for delignification, which, according to H a g g l ~ n d is , ~the ~ ~ most important reason for the favorable effect of sulfide. In a recent study on the dissolution and destruction of carbohydrates during the sulfate cook, Saarnio and Gustafssonlll reported a higher rate of destruction of dissolved hexosans than of dissolved pentosans. These workers"' isolated polysaccharide fractions from samples (taken hourly) of sulfate cooking liquor, and analyzed the fractioiis (after hydrolysis to monosaccharides) by quantitative, paper c h r ~ m a t o g r a p h y .They ~~ observed"' an increase in the proportion of polysaccharides in the cooking liquor in the early stages of the cook, when dissolution predominates. Soon after reaching the maximum temperature, however, dissolution decreases and destruction of polysaccharides becomes predominant. On comparing data on the carbohydrate composition of wood3* 206-208 with those on sulfate pulps, Sundman and coworkers45concluded that the galactans and arabans of wood are almost completely dissolved during the sulfate cook. About 70 % of the maiinan is dissolved from pine wood, as is about 60% of the mannan from birch. From pine, about 50 to 70% of the (201) F. E. Brauns and W. S. Grimes, Paper Trade J . , 108, No 11, 40 (1939). (202) E. Hagglund, Cellulosechemie, 6, 81 (1924). (203) Ref. 189, p. 467. (204) W. F. Holzer, Ref. 9, p. 975. (205) E. Hagglund, Ref. 189, p. 476. (206) C. Gustafsson, P. I. Ollinman and J. Saarnio, Acta C'hem. Scand., 6, 1299 (1952). (207) J. Larinkari, Finnzsh Paper Tinrber J . , 27, 143 (1945). (208) W. Jensen, Finnish Paper Timbe, J . , 31, No. 7A, 20 (1949).
POLYSAC(:IIhHIDES
327
ASSOCIATED WITH WOOD CELLULOSE
TABLEXVII ('urbohydrate Composition, of Unbleached, Sulfate Pulps" lannan, 7" Xylan, ?4
Araban.
76
~~
Spruce Pine Pinee Pine * Birch Birch"
47.7 44.2 39.9 37.8 57.3 48.7
89.3 90.2 90.9 92.9 81.4 86.1 81.7
84.4 84.4 88.3 92.8 74.5 76.4 82.8
4.7 5.6 5.9 2.8 1.2 1.2 0.5
9.9 9.0 5.3 4.4 23.8 22.4 16.8
a Cooked a t higher chemical concentration than the previous sample. Pre-hydrolyzed (2 hours a t 140'C.). hydrolyzed (2 hours a t 150°C.).
1 1 0.5 0 0.5
+ 0
* Pre-
xylan is removed, and from birch about 50 to 6075, depending on the degree of cooking. About 15% of the glucan in pine wood, but practically none of the glucan of birch wood, is dissolved in the cooking liquor.111 This glucan was determined"' as a yield loss, not as glucan in the alkaline cooking liquors. Since definite evidence for presence of glucan in the cooking liquors could not be obtained, it was suggested that pine may contain an easily destroyed glucan, either of low molecular weight or of a constitution other than that of a poly-(4 -+ 1 p-D-ghcoside). The carbohydrate analyses reported by Sundman, Saarnio and Gustafsson for sulfate pulps are listed in Table XVII. These results were obtained by quantitative, paper c h r ~ m a t o g r a p h yon ~ ~completely hydrolyzed samples. I n another study,'*O the uronic acid anhydride content of some wood pulps was determined. It was notedLz0that sulfate pulps prepared from hardwoods retain a considerable proportion of uronic acid, whereas sulfate pulps from softwoods may be entirely free from polyuronides. Some comparisons between the composition of sulfate and sulfite pulps are of interest (see Tables XV and XVII). The alpha-cellulose content of sulfate pulps is generally higher than that of sulfite pulps from the same species, but the glucan content is higher for the sulfite pulps. In every case, escept for prehydrolysed samples, the alpha-cellulose content of sulfate pulps is higher than the total, true cellulose (glucan) content, and, for sulfite pulps, the alpha-cellulose content is always lower than the true cellulose content. In the sulfate pulps, some non-cellulose polysaccharide is retained in the alpha-cellulose, whereas in sulfite pulps, at least some of the glucan must enter the beta- or gamma-fractions or both. When yields are considered in terms of the anhydro-D-glucose content of the pulp, the sulfite process is superior. The pre-hydrolyzed pine and birch sulfate pulps (see Table XVII) war-
328
W. J . POLGLASE
rant special comment, since the preparation of commercial, wood pulps by processes involving steam, water, or acid pre-hydrolysis is becoming increasingly popular. I n the case of pine, pre-hydrolysis yields a sulfate pulp containing about half as much xylaii and mannan as does the nonprehydrolyzed pulp. Similarly, for birch, a reduction in non-cellulose polyoses may result from pre-hydrolysis. When certain softwoods are pulped by the sulfite process, the resulting pulp usually contains a considerable proportion of uronic acid anhydride which cannot be removed entirely in subsequent alkaline refining. When the sulfate process is used 011 softwoods, a pulp \vhich is free from uronic acid anhydride may be obtained. This important difference between the sulfite and sulfate pulps from softwoods does not apply to hardwoods, which, when pulped by either process, still retain a considerable proportion of uronic acid anhydride in the final product.120
V. FINESTRUCTURE OF WOODCELLULOSE A N D ASSOCIATED POLYSACCHARIDES 1. Alpha-, Beta-, and Gamma-cellulose
.4 number of methods of analysis are important in wood-cellulose chemistry. One of the most important is the determination of solubility in sodium hydroside solution a t room temperature. This distinguishes between alphaccllulose (not dissolved by 17.5 % sodium hydroxide), beta-cellulose (dissolved by 17.5 % sodium hydroxide, but precipitated when acidified with acetic acid), and gamma-cellulose (dissolved by 17.5 % sodium hydroxide, and not precipitated on acidification). The empirical nature of this fractionation may be appreciated by comparing the composition of a number of different preparations of alphacellulose. The alpha-cellulose from such coniferous woods as spruce, pine, and hemlock may contain appreciable amounts of mannan, although the xylan content is usually low. Similarly, the alpha-cellulose from hardwoods may contain appreciable quantities of non-cellulosic polyoses, although, in this case, the chief polyose is xylan, not mannan. On the other hand, certain types of wood pulp, prepared by sequences involving both acidic and alkaline treatments, show a linear relationship between gamma-cellulose and non-cellulosic polyoses. This may be illusstrated from the studies of White, Steinman and Workb0on acetylation pulps. Acetylation-grade pulps are usually prepared from softwoods by sulfite cooking followed by hot-caustic refining. When the total polyose content of a number of such pulps was compared with their gamma-cellulose content, a linear relationship was obtained (see Fig. 3 ) . In spite of this excellent correlation, it cannot be assumed that all of the non-cellulosic polyoses are contained in the gamma fraction of these pulps (see below).
POLYSACCHARIDES ASSOCIATED W I T H WOOD CELLULOSE
:329
u) W
0 J
5.0
J
W 4 0 0
a
$ 3.0 a
0
0 lL 2.0
:: 1.0 W
b-
z :0 ~
g
t o o
10
2 .O
3 0
PERCENTAGE O F M A N N A N
PLUS XYLAN
FIQ.3.-Relationship Between the Gamma-cellulose Content and the Mannan Plus Xylan Cont.ent of Wood Pulp.’13
The limitations of the alpha-cellulose determination have become more apparent as the need for pulps of greater purity has increased. For certain uses, particularly in the manufacture of cellulose-acetate rayon, it has been deemed desirable to prepare a “high-alpha” pulp, since this was believed to be synonymous with high purity. This is not surprising in view of the high-alpha content of purified, cotton linters (99 %). High-alpha, cellulose pulps can be most readily obtained by a modified, alkaline (kraft), pulpiiig process. Yet, despite their lower alpha-cellulose content, hot-caustic refined, sulfite pulps produce superior acetates. Current studies on woodcellulose composition point to the non-cellulosic polyoses as of importance to the properties of the product. In certain cases, the higher-alpha pulps may actually have a lower, true-cellulose content than have pulps with lower alpha-content. The physical characteristics of alpha-, beta-, and gamma-cellulose fractions have been studied with the electron microscope and x-ray camera, by Rhby.2°9 The investigations were made on commercial, softwood pulps prepared both by the sulfite and the sulfate processes. The electron-microscope investigation showed that alpha- and gamma-celluloses are two separate phases; the alpha consists of micellar strings, and the gamma is a dispersed phase without defined structural elements. The beta-cellulose could not be classified directly from the electron micrographs, but the x-ray diagrams were similar t o those of the alpha-cellulose, implying that the betacellulose from softwoods consists of disordered and broken micelle strings, containing only short-chain cellulose. This assumption receives support from the recognized fact that the beta fraction increases as a result of various different types of degradation of technical, wood pulps. (209) I3. G. Rbnby, Svensk Papperstidn
, 66,
115 (1952).
330
W. J. POLGLASE
These coiiclusions are in agreement with Mitchell’s studies210on the chain length of nitrated, cellulosic constituents of wood. In these studies,210 only two distinct fractioiis were obtained from nitrated, Western-hemlock wood, namely, hemicellulose nitrate which had a D. P. of about 70 and amounted to 30 % of the total, and alpha-cellulose nitrate with an average D. P. of about 2000 to 2500, representing the remaining 70%. Mitchell suggests that his resultsindicate that the beta portion of an industrial, wood cellulose is probably made in the pulping and bleaching operations, and that it consists of short-chain fragments resulting from cleavage of the long-chain, alpha-cellulose component. Mitchell speculates, further, “that wood cellulose may consist of only two portions-one that is water-insoluble, alpha, and one that is potentially water-soluble, gamma. The difference in solubility would be due not only to differences in chain length but, perhaps primarily, to differences in chain unit, end groups and side groups. Gammacellulose may be held in the fiber by cross links or secondary bonds only to become soluble when liberated-for example, by the swelling action of sodium hydroxide.” Simmons211has studied the carbohydrate composition of the alpha-, beta-, and gamma-fractions of bleached, coniferous, chemical wood-pulps. He concluded211that the gamma fraction consists largely of mannan and xylan. The alpha and beta fractions may contain appreciable proportions of mannan and xylan. However, the mannan and xylan in the beta fraction are occluded during precipitation of the beta-cellulose. These results211are in agreement with the ideas expressed above that alpha-cellulose and betacellulose are chemically similar, and differ only in D. P. 2 . Accessibility of Cellulose
An analytical method for the determination of accessibility of cellulose was developed by Nickerson212in 1941. Since that time, the procedure, or a modification 214 has been used extensively in the examination of cellulosic materials.216 The original method212was based on the observation that a boiling mixture of ferric chloride and hydrochloric acid causes relatively rapid evolution of carbon dioxide from D-glucose. When cellulose is heated with these reagents, D-glucose is formed by hydrolysis, and is then oxidized to yield (210) R. L. Mitchell, I n d . Eng. Chem., 38,843 (1946). (211) J. R. Simmons, B r i t . Paper and Board Makers’ Assoc., Proc. Tech. Sect., 33, 513 (1952). (212) R . F. Nickerson, I n d . Eng. Chem. A n a l . E d . , 13, 423 (1941). (213) C. C. Conrad and A. G . Scroggie, I n d . Eng. Chem., 37,592 (1945). (214) E. L . Love11 and 0 . Goldsrhmid, I n d . Eng. Chem., 38, 811 (1946). (215) R . F. Nickerson, Advances in Carbohydrate Chem., 6 , 103 (1950).
33 1
POLYSACCHARIDES ASSOCIATED W I T H WOOD CELLULOSE
carbon dioxide, which may be collected in absorption tubes. Since, under controlled conditions, the rate of evolution of carbon dioxide is proportional to the concentration of D-glucose, the course of the hydrolysis of cellulose may be followed by measuring the evolution of carbon dioxide. For similar determinations on wood pulps, Love11 and G ~ l d s c h m i d ,recognizing ~~~ that hemicelluloses in mood pulps might not give a satisfactory rate of evolut,ion of carbon dioxide, proposed that the sample be weighed after given times of hydrolysis. By either method, and for a large number of cellulose samples, it has been found that an initially rapid rate of hydrolysis is followed by a slower, relatively constant rate of hydrolysis. The iniTABLEXVIII A m o u n t of Easily Accessible Material, and Hydrolysis Rate Constant, k , for the Dificultly Accessible Fraction, f o r Diferent Pulps217 Ftber material
Easily accessible, 7/0
k
Cotton Hot-alkali refined linters Flax pulp Holocellulose Spruce sulfite, strong Spruce sulfite, rayon Spruce sulfite, hot-alkali refined Pine sulfate Pre-hydrolyzed sulfate Aspen sulfite Birch sulfate Viscose rayon
11.9 6.2 10.0 35.6 15.7 10.0 6.6 16.8 10.2 11.3 28.0 35.9
0.0062 0.0065 0.0066 0.014 0.012 0.013 0.013 0.012 0.011 0.012 0.017 0.039
tial, rapid reaction is presumed to denote hydrolysis of amorphous cellulose, whereas the later, slower reaction is believed to result from the hydrolysis of crystallites. During the first 30 minutes of the rapid reaction, thc ciiprammonium viscosity falls to a value which remains nearly constant for several hours,216 despite continuing hydrolysis. A possible explanatioiF is that disordered cellulose lying between crystallites is first attacked and that, thereafter, attack occurs mainly on the lateral surfaces of crystallites. If this is true, the length of the crystallites is indicated by the minimum viscosity attained. This crystallite length is usually expressed as a ch.ainlength “limiting 1). P.” 111 this andyticaal method, wood pulps in general differ from cotton linters in showing a greater acctessibility, as shown in Table XVIII (from the data (21G) R F Nickchrson ant1 .J. A Hatbile, I n d . Eny ( ’ / / e n / , 39, 1507 (1947). (217) W. €1 Algar, H W Giritz and A M. Custafsson, Ssensk Pappershdn., 64, 335 (1051).
332
W. J. POLGLASE
TABLE XIX Removal o j Carbohvdrates During Sul-fite Cookin Yield of bleached Pulp, %
Pulp -.
Wood Holocellulose Extra strong Greaseproof Medium Soft Rayon Acetylation pulp"
%
___
67.9 50.2 49.7 48.8 46.6 44.6 33.4
Cellulose, % '
Easily accessible
Beta-
Gamma-
77.3 86.3 86.6 87.2 88.9 90.1 95.0
traces tracef 1.4 1.6 2.9 2. 9
20.0 12.8 11.0 9.8 8.8 6.9 2.1
61.2
0. 4
34.2b
Alpha-
17
AlphaDificultl y ceiklose, accessible the wood) 96
IA
~
34.5 35.6 17.4 16.0 15.3 13.8 11.3 6.6
Holocellulose (alpha-, beta-, garnma.de. termination with hydrolytic pretreatment)
1
.o
43.4 43.4 41.5 41.6 41.4 40.3 40.1 31.1
52.5 43.3 43.0 42.6 41.4 40.2 31.7 42.6
Hot alkali refined, 10% NaOH, 120°C. b This value includes the amount of material dissolved during the short, hydrolytic pretreatment.
of Algar, Giertz and G u s t a f s s o ~ i ~These ~ ~ ) . workers217note a very good correlation between the gamma-cellulose fraction and the easily hydrolyzable fraction of wood-cellulose. As a result, the fiber is picturedZ17as consisting of a well-ordered, well-defined micelle-string structure embedded in amorphous hemicellulose. Giertz and coworkers217prepared a chlorite holocellulose, and a series of sulfite pulps representing a yield range of 68 t o 33%. On these cellulose preparations they performed accessibility estimations by the method of Nickerson,212as modified by Love11 and Gold~chmid,2~~ as well as conventional alpha-, beta-, and gamma-cellulose determinations. Their results are given in Table XIX. I n the range of sulfite pulps from the extra strong pulp to the rayon pulp, the variation in alpha-cellulose is relatively small, whereas both the gamma content and the accessibility decrease appreciably. The gamma content of holocellulose is low, compared to the percentage of accessible holocellulose. Giertz and associates217suggest that this is ascribable to the structural strength of the holocellulose fibers, which do notJswell sufficiently to allow outward diffusion of the interfibrillar substance. After a short,, vigorous hydrolysis of the holocellulose, the gamma-cellulose increased to a value consistent with the accessibility value, and the alpha-cellulose decreased proportionately. Since there exists a close connection hetween easily hydrolyzable material and gamma-cellulose, Giertz and c o ~ ~ o r l i ~point r s ~ ~ out 7 that therc must also be n relationship between difficultly hydrolyzable material and alpha- plus beta-cellulose. This means that the beta-cellulose is, from the hydrolysis viewpoint, of the
POLYSACCHARIDES ASSOCIATED WITH WOOD CELLULOSE
333
same character as the alpha-cellulose, and consequently is difficultly accessible. Just as Mitchell Giertz and associate^^'^ believe that the low beta-cellulose values of holocellulose and strong-sulfite pulp indicate that there is no beta-cellulose in wood, but that it is an artifact formed during the refining operation. Furthermore, it is that the betacellulose is formed, during pulping, from the difficultly accessible parts of the fiber, and is actually degraded alpha-cellulose. Thus, from nitrate studies, chemical analyses, electron-microscopic and x-ray diffraction work, and accessibility studies, come the same conclusions-namely, that wood cellulose consists of two phases: an ordered, cellulosic phase of high molecular weight, and an amorphous, accessible, hemicellulosic phase of low molecular weight. The cellulosic phase is alkaliinsoluble (alpha-cellulose) except for the short chains formed as a result of attack by chemical reagents during pulping (beta-cellulose). The hemicellulosic phase (gamma-cellulose) is water-soluble when once it has been liberated from the cellulose matrix by the swelling action of alkali. Accumulated evidence indicates that part of the non-cellulose polyoses are in the cellulosic phase (the “cdlulosans” of NormanI4) and part are in the hemicellulosic phase.
This Page Intentionally Left Blank
THE CHEMISTRY OF HEPARIN
BY A . B . FOSTER* A N D A . J . HIJGGARD f l r pmistry Departments, T h e Ilniaersity. Birmingham. lhgland. nnd The Ohio Sfate University. Columbus. Ohio
CONTENTS I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 I1. The Discovery of Heparin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 111. The Isolation and Purification of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 2 . Methods of Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 a . Early Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 b . Renzidine Salt Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 3 . The Homogeneity of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 4 . The Action of Enzymes, Alkali, and Acid on Heparin . . . . . . . . . . . . . . . . . 345 a . Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 b . Alkali . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 c . Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 348 5 . Other Methods of Extractlion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I V . The Structure of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 1 . Acidic Hydrolysis of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 a . Total, Acidic Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 b . Hexosamine and Hexuronic Acid Content . . . . . . . . . . . . . . . . . . . . . . . . 351 c . Sulfur and Nitrogen Content.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 ............................ 352 d . Partial, Acidic Hydrolysis . . . . 2 . Acetylative Desulfation Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 3 . Other Methods of Degradation of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 a . Action of Nitrous Acid on Derivatives of 2-Amino-2-deo.uy-1~-glucose and Heparin., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 b . Oxidation in the Presence of Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . 35'3 V . The Anticoagulant Activity of Hcparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 I . The Biological Activity of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 2 . The Structure and Anticoagulant Activit.y of Heparin . . . . . . . . . . . . . . 360 a . Degree of Sulfation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 b . The N-Sulfate Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 c . Distribution of the Sulfate Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 d . Molecular Weight of Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
* Fellow of the Rockefeller Foundation at The Ohio State University. 1953.54 . The authors wish t o thank Professor M . L . Wolfrom for access t o much unpublished data on heparin during the preparation of this Chapter. and for valuable criticism and advice . 335
336
A.
B. FOSTER A N D A . J . HUGGARD
3. The R.Iolccula,r Shape of Heparin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . Sulfat,ion of Heparin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Inactivation of Heparin by Dilut,e Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Ot,her Considerations... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I . The Biosynthcsis of Muco1)olgsaccharidcs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
364 364 365 366 367
I . INTRODUCTION Heparin,* the blood anticoagulant present in circulatory tissue is now recognized to be an important and chemically unique polysaccharide of considerable biological significance. Since its discovery almost forty years ago, heparin has been intensively investigated by numerous workers. Much of its chemistry has consequently been evaluated and it is rather surprising that no comprehensive chemical review has recently appeared. In his excellent book, Jorpesl has described in some detail the history and early chemistry of heparin and the development of its medical applications. This Chapter is intended to present an account of the more recent developments in the chemistry of heparin together with the significant features of the earlier work.
11. THE DISCOVERY OF HEPARIN Heparin was discovered in 1916 by McLean2 who was working under the direction of Howell a t Johns Hopkins University. I n order to ascertain the origin of its blood-coagulating properties, crude cephalin was submitted to a careful fractionation. Fractions were obtained which unexpectedly inhibited the coagulation of oxalated, dog serum. This was a significant discovery since no anticoagulant had previously been found in mammalian tissue. Because of its abundance in liver, the anticoagulant material was named h e ~ a r i n Howell .~ was quick to recognize the potential therapeutic value of heparin, and in his Harvey Lecture; in 1917, he expressed the hope that the substance would find “suitable application . . . in the therapeutic treatment of disorders of coagulation.” Initially, heparin was thought to be a phosphatide because of its closely similar behavior to cephalin in the extraction procedure, but it is now recognized to be a highly sulfated mucopolysaccharide.
* The term heparin has not been used consistently since its introduction; it usually implies the sodium salt of the sulfated polysaccharide, but many other salts are known. Perhaps a more logical name would be heparinic acid; the sodium salt would then be termed sodium heparinate, and the naturally occurring complex which contains the anticoagulant, protein heparinate. In this Chapter, theterm heparinsignifies the sodium salt, unless otherwise qualified. (1) (J.) E. Jorpes, “Heparin,”Oxford University Press, London, 2nd Edition, 1946. (2) J. McLean, Am. J . Physiol., 41,250 (1916). (3) W. H. Howell and E. Holt, Am. J. Physiol., 47, 328 (1918). (4) W. H. Howell, Harvey Lectures, Ser. 12 (1916-17).
THE CHEMISTRY O F HEPARIN
337
111. THEISOLATION AND PURIFICATION OF HEPARIN 1. Introduction
I n the chemical literature prior to 1940, the majority of papers published on heparin were devoted to the isolation and purification of the polysaccharide. The enrlicst workers were concerned with the isolation of highly active, non-toxic preparations. Later, as attention was focused on the problem of heparin structure, many further attempts were made to isolate the pure substance, and extensive data on the activity and analytical composition of the various preparations have consequently accumulated. During the early period, the different research laboratories each claimed to have isolated pure heparin, but it is only within the last decade, with the application of physicochemical methods, that relatively homogeneous preparations have become available. 2 . Methods of Isolation a. Early Procedures.-Some details of the original procedures for the extraction of heparin will be given, since they form the basis for subsequent methods. Heparin does not exist in the free state in the body under normal conditions, but is invariably linked with a protein which in one case (in mast cells, see page 360) has been shown to be a l i p ~ p r o t e i nI. n~ certain abnormal conditions, free heparin may appear in the body. Thus, heparin is released from the liver into the blood stream in dogs in anaphylactic shock.6 In the isolation of heparin from normal tissue, a stepwise purification procedure is required. The earliest method for the isolation of heparin, developed by H ~ w e l l , ~ involved the treatment of dried and minced dog liver with boiling methanol, and subsequent extraction with physiological saline solution from which the active principle was precipitated by acetone. Glycogen was then removed by digestion with Takadiastase and the proteins were split off with cadmium chloride. As a result of this work, heparin was made available commercially. Subsequently, Howell8 improved the separation of heparin from protein by introducing the use of Lloyd's reagent (aluminum silicate) in acetic acid. The final stage then involved the precipitation of the heparin nith excess barium hydroxide. Howell claimeds that his heparin preparation con(5) 0. Snellman, B. S y l v h and Christina JulBn, Biochim. et Biophys. Acta, 7, 98 (1951). (6) L. B. Jaques and E. T. Waters, A m . J . Physiol., 129, 389 (1940); J. Physiol. (London), 99, 454 (1941). (7) W. H. Howell, Am. J. Physiol., 63, 434 (1922-23); 71, 553 (1924-25). (8) W. H. Howell, Bull. Johns Hopkins Hosp., 42, 199 (1928).
338
A. B . POSTKR A N D ,4. J. HUGGARD
tained 110 nitrogen (undoubtedly because of the insensitive test used), phosphorus, or sulfur, in spite of the fact that inorganic sulfate had been identified amongst the products after hydrolysis with hydrochloric acid. He thought that heparin was consequently not a protein or phosphatide, and he showed it to be thermostable, insensitive to bacteria, and resistant to the common enzymes. Howell suggested that heparin was carbohydrate in nature and contained uronic acid. I n 1933, Fischer and Schmitz9 first claimed to have isolated a pure heparin preparation. Their lengthy extraction and isolation procedure culminated in the isolation of a “microcrystalline” hruciiie salt 32 times as active as the starting material. KO evidence other than that of appearance was quoted in support of the claimed crystallinity of the product. It was concludedg that heparin was a carbohydrate (Molisch test) and contained a uronic acid. Fischer and Schmitzlowere also interested in the interaction of heparin with proteins, such as casein and serum albumin. They reported that heparin appeared to shift the isoelectric point of the proteins to the acid side, probably by the formation of a molecular complex, and that combination with the protein occurred only near the isoelectric point and on the acid side.” The complex was reversibly dissociated by the addition of alkali. These results are of irnportance in selecting conditions for the purification of crude heparin preparations, and constitute the basis for the extraction procedure developed by Charles and Scott,12which has subsequently been used extensively. Charles and Scott employed the cheaper and more readily available beef liver’? and beef as the source material. Beef liver (or lung) a a s minced and then autolyzed for twenty-four hours before extraction with an alkaline solution saturated with ammonium sulfate. Protein was precipitated by warming the extract, and the heparin-protein complex was precipitated from the supernatant liquor on acidification. Extraction of the complex with ethanol removed fatty material, and tryptic digestion removed most of the protein. The heparin u a s precipitated with ethanol, redissolved in h a r m alkaline solution to destroy trqpsin, and reprecipitated \titti acetone. This material, “crude heparin,’’ a a s isolated in a lield of 15-50 g. per 100 Ib. of animal tissue. In a later paper,14 the purification of crude heparin by fractionation successively n i t h Lloyd’s reagent, cadmium chloride, and acetone, mas described. The “purified heparin” was 100 times as active as the crude material. Scott and Charles“ reported the presence of nitrogen (9) A . Fischer and A Gchmitz, Hoppe-Sqilet’s Z . physzol. (’hem., 216, 264 (1933). (10) A. Fischer, ~vnlirrwissenfichnSten,19, 965 (1931); A . Fischer and A. Schmit7 tbid, 20, 471 (1932); B?orhein. Z , 269, 61 (1933) (11) See I,. R . Jnques, Biochew. . J . (London), 37, 189 (1043). (12) A F. Charles and D. A . Scott, J. Biol. (‘hem., 102, 425 (1933) (13) A . F. Charles and D. A. Scott, Trans. Roy. Sac. Can., B, 28, 55 (1935). (14) D. A. Scott and A. F. Charles, J. B i d . Chem., 102, 437 (1933).
THE CHEMISTRY OF HEPARIN
339
and inorganic ash, but failed t o obtain a positive naphthoresorcinol test for uronic acid.
A few years later, Jorpes confirmed much of the work done by Charles and Scott. Heparin was purified by their procedure and, after several successive treatments with Lloyd’s reagent, the product ivas found to coiitairi 1.64 % of nitrogen. The presence of sulfur (as both bound and free sulfate), inorganic ash, uronic acid, hexosamine, and acetic acid was reported. Electrodialysis of the purified heparin removed free sulfate, and afforded a highly active preparation which could be precipitated as an amorphous brucine salt.16 Lipmann and Fischer’G later confirmed the presence of nitrogen and sulfur in heparin isolated by the procedure of Charles and Scott.l2-l4 There was now fairly good agreement between the various laboratories regarding the qualitative composition of heparin. Jorpes and Berg~trorn’~ offered an explanation for the failure by Charles and ScottI4 to obtain a positive naphthoresorcinol test for uronic acid when they showed that highly active heparin is resistant to depolymerization by acid. It was well known, a t this time, that heparin occurs widely distributed in mammalian tissues. Its presence in dog liver,3 lymph glands; and blood ve ~se l s ,in ~ uterine mucous membrane,18 and in extracts of milk, kidney, lung, heart, arid other had already been demonstrated. Charles and Scottz0 determined the quantitative distribution in various tissues, organs, and fluids, as recorded in Table I . Heparin is now thought to originate in the mast cells. These cells are generally located in the connective tissue, in the vicinity of the capillaries, and in the walls of the blood vessels. Mast cells are especially plentiful in liver and lung tissue, which are consequently the best sources of heparin; and they give a purple stain with Toluidiiie Blue, owing to the interaction of the dye with heparin. A detailed account of the histological demonstration of heparin has been published elsewhere.’ b. Benzidine Salt Procedures.-In 1936, Charles and Scottz1described a method for the purification of heparin (through the benzidirie salt) which was a significant development in heparin chemistry. When “purified” heparin was treated with benzidine hydrochloride, a salt was obtained which was (15) (a) (J.) E. Jorpes, Nalurwissenschuften, 23, 196 (1935) ; (b) Biochern. J . (London), 29, 1817 (1935); (c) Acta Med. S c a d . , 88, 427 (1936). (16) F. Lipmann and A. Fischer, Hoppe-Seyler’s 2. physiol. Chem., 237,273 (1935). (17) (J.) I%. Jorpes and S. Bergstrom, Hoppe-Seyler’s 2. physiol. Chem., 244. 253 (1936). (18) J. I,. King, Am. J. Physiol., 67, 444 (1921). Igakkni Zasshi, 3, 61 (1926). (19) 0. Kashiwamura and R. Katsuki, K?~mamoto (20) A. F. Charles and D. A. Scott, J . B i d . Chein., 102.431 (1933). (21) A. F. Charles and D. A. Scott, Biochem. J. (London), 30, 1927 (1936).
340
A . R . FOSTER AND A . J. HUGGARD
readily decomposed with dilute ammonium hydroxide to afford the aniinoniuni salt of a highly active heparin which contained only 0.7 % of ash. The benzidine salt was readily convertible into metallic salts; and, when treated with barium acetate in acetic acid, a barium acid salt of heparin was ohtained whose photomicrograph showed it to be crystalline. Charles arid Toddz2re-examined this material and stated that “the crystals N ere tvv small and heavily twinned for an x-ray diagram”; the crystals displayed Most workers from 1936 onward regarded the only low birefringen~e.~~ barium acid salt as a good starting point for chemical investigations on heparin. The analysis of the barium acid salt, originally made by Charles and Distribution
TABLE I Heparin i n the Bodyz0 .
0.f .
Heparin,
n,g./kg. of’ tissue
Location
Reef liver spleen heart blood thymus serum lung muscle Hog liver Dog liver
“Crude”
“Parijied”
780 1000 200 260 640 24 840 2000 1400 900
190 230 54 66 310 230 600 340 330
Scott,21was performed on material that was not anhydrous; likewise, Meyer and S m ~ t analyzed h ~ ~ an incompletely dried sample obtained from Charles (see Table 11, column 1). Astrup and J e n ~ e nconverted ~~ the crystalline barium acid salt to the sodium salt of heparin, which they subjected to elemental analysis. Results in good agreement with those of Charles and Scottz1were claimed when allowance was made for the “hydration.” Reinert and WintersteinZ6also prepared heparin by the benzidine-salt procedure and obtained 25-50 mg. (22) A. F. Charles and A. R. Todd, Biochem. J . (London), 34, 112 (1940). (23) M. L. Wolfrom, D. I. Weisblat, J. V. Karabinos, W. H. McNeely and J. McLean, J . Am. Chem. Soc., 66, 2077 (1943). (24) K. Meyer, Cold Spring Harbor Symposia Quant. Biol.,6, 97 (1938). (25) T. Astrup and H. B. Jensen, J . Biol. Chem., 124, 309 (1938). (26) M. Reinert and A. Winterstein, Arch. intern. pharmacodynamie, 62,47 (1939).
34 1
T H E CHEMISTRY OF H E P A R I N
of heparin per kg. of liver. The highest sulfur content (sodium salt basis) recorded by these workers was 12 %. Charles and Toddzzreported that the barium acid salt and the ammonium salt were found to be readily interconvertible, which indicated that both were definite compounds. Analytical data (see Table 11,column 2 ) reported for incompletely dried material were in agreement with those of earlier workers when calculated on a “hydrated” basis. Masamune, Suzuki and Kondohz7 also reported analytical data on an incompletely dried sample (degree of hydration not stated) of the barium (acid) salt (see Table 11, column 3). No significant chemical differences were found in samples of
Authors
l+D
I
degrees
C
H
Ba
Conimenls
_ _ _ - _ ~ _ _ - ~ - Meyer and Smyth24 Charles and Todd21
+53 +5W
17.8 3.1 1.9 20.2 3.5 2 . 2
Masamune and coworkers25 Wolfromandcoworkers23
9.8 2.2
2.36
+36
2.1
+47.5 20.2 2 . 9 1.9
0.0
9.6 1.32.9 11.0
16.3
20.1 33.3 33.4
Incompletely dried sample 19.7 Moistnre content, 12.1% 22.5 Recalculated
to anhydrous basis Degree of hydra0.0 8 . 7 0 . 0 26.4 28.7 27.7 tion got stated 0.3*11.3 0 . 0 0 . 0 1 7 f 1 18 40.4’ 23.8 Anliydrous sample
heparin (barium acid salt) isolated from dog, beef, swine, and sheep tissues.z8 29 I n 1943, Wolfrom and coworker^?^ published the results of a thorough investigation of the elemental composition of the barium acid salt of heparin, and special attention was given to the preparation of anhydrous material. The results are quoted in Table 11, column 4. Summation of the analytical results apparently accounted for 88.6 % of the material present. However, there was by no means unanirrious agreement that the barium acid salt of heparin was the most pure and active material prepared. In particular] J o r p e ~ who , ~ ~ had been very critical of the evidence for the crystallinity of the barium acid salt, continued t o purify his heparin prepa(27) H. Masamune, ill. Susuki and P.Kondoh, J. Bioehem. (Japan), 31,343 (1940). (28) 1,. B. Jaques, Scienre, 92, 488 (1940). (29) I,. B. Jaqutw, 13. T. Waters and A . F. Charles, .I. Riol. C h ~ m . 144, , 229 (1942) (30) (J.)E.Jolprs, Riochsni. .I. (T,oridon), 36,203 (19-12); Hoppr-Scylcr’s Z . physiol. Chew/., 278, 7 (1943).
342
A . B. FOSTER AND A. J. HUGGARD
rations by fractionation of the brucine salts. It was m a i n t a i i ~ e dthat ~ ~ further purification could be effected by this procedure, but this claim was specifically denied by Charles and Todd.22Jorpes’ procedureL5involved electrodialysis of the sodium salt (purified through the benzidine salt) against distilled water. The acidic, cathodic solution was treated with brucine sulfate, and the resultant salt was fractionated by repeated freezing and thawing of its aqueous solution. Jorpes30 associated activity with degree of sulfation (see page 361), and he reported that his amorphous preparations had a sulfur content of 13.5 % (sodium salt basis). The fractionation procedures (brucine salt) were performed on heparins obtained from various tissues:O and it was postulated that the heparin material was not a definite compound but was a mixture of “mucoitin polysulfuric acid esters.” Fractions corresponding in their sulfur content t o mono-, di-, and tri-sulfates (per disaccharide period) were obtained, together with sulfur-free fractions32(see also Marbet and Winterstein33).The most active material had a sulfur content of 13.8%, corresponding t o three sulfate groups per disaccharide unit. In 1943, Kuizenga and Spaulding3* advanced more evidence that a barium acid salt of heparin that they had prepared was not homogeneous. A fractionation mas achieved using aqueous acetone, to give two products xvhich, after conversion to the sodium salts, showed different activities and sulfur contents. This result would appear to support the contention that heparin is a mixture of closely related substance^.^^ It should be emphasized that the barium acid salt described by Kuizenga and S p a ~ l d i n gwas ~~ obtained by a procedure different from that by which the barium acid salt described by Charles and Scott2‘ and by Wolfrom and coworkers23was obtained. The latter salt was reported to be electrophoretically homogene~us.~~ More recently, it has been reported by Wolfrom and coworkers36that a relatively homogeneous polymer fraction with a high degree of sulfation may he obtained from commercial heparin, after fractionation as the (31) S. Bcrgstrom, (J.)E. Jorpes a i d 0. Wilander, Skand. Arch. PhysioE., 76. 175 (1937); (J.) 13. Jorpes and S. Bergstr6m, Biochern. J. (London), 33, 47 (1939). (32) (J.) E. Jorpes and S . Gardell, J . Biol. Chem.,176,267 (1948). (33) R.Marbet and A . Winterstein, Helv. Chim. Acta, 34, 2311 (1951). (31) M. H. Kuizenga and L. B. Spaulding, J . B i d . Chem., 148, 641 (1943). (35) AT. I,. Wolfrom, R. K. Madison and M. J. Cron, J . Am. Chem. SOC.,74, 1491 (1952). This paper reportrtl a correction t o the earlier work of M. L. Wolfrom and F.A 11. Rice, J . A n , . Chein. Soc., 69,2918 (1947). What was originally suspected as a second component in heparin was shown to be an abnormally large boundary anomaly. (36) Af. I,. Wolfrom, R. Montgomery, ,J. V. I<ar:it)inos and P. Rathget), .I. A M . (‘hem. SOC., 7 2 , 5796 (1950).
THE CHEMISTRY OF HEPARIN
343
neutral barium salt with subsequent purification using cold acetic acid.3GJ Charles and Scott'2*l4 recognized that heparin could be precipitated as the barium salt, and fractions of low sulfate content were also obtained by its use.39The neutral barium salt is also prepared through the henzidiiie salt. Other derivatives of heparin have been reported to have a crystalline appearance; for example, the salts formed with piperidine, n-pentylamine, and i~opentylamine.~~ D o d e ~ y l a m i n eand ~ ~ de~amethylenediamine~~ have also been used in the preparation of heparin. It is clear from the preceding account that much of the confusion which arose in the earlier work on heparin was due primarily to insufficient purification of the preparations. In actual fact, no satisfactory criterion of purity had been established. Most workers strove to obtain preparations of high anticoagulant activity, and the situation was complicated by the fact that, during this period, several different methods for the assay of anticoagulant activity were in use. Jorpes and his associates had associated activity with sulfur content, and the latter was used as a criterion of purity. This is currently thought to be an over-simplification (see page 360). Bell and IG-antz40 studied the ultraviolet absorption of a series of heparin preparations, and found no correlation between absorption and activity. Some of the highly active preparations showed no absorption in the range 230-300 mp, whilst others showed a maximum at 265-292 m p and a minimum a t 240-260 mp. These results would appear to indicate the presence of impurities, since, on re-investigation of the ultraviolet absorption of heparin, no absorption in this region was This inference is supported by the results of a chromatographic study of heparin.42The movement of heparin on paper, under the influence of a 1-propanol-water solvent system, showed two components (RF0.00, 0.57). The original heparin preparation showed absorption a t 270 mp and, after chromatography, the more rapidly moving component, which had all of the anticoagulant activity, showed no absorption a t 270 mp. (364 M. L. Wolfrom and P. Rathgeb, unpublished results. (37) D. A. Scott, A. F. Charles and A. M. Fischer, Trans. Roy. SOC.Pan., V , 36, 49 (1942). (38) A. E. O'Keeffe and J. A. Shannon, U. S. P a t . 2,552,507 (1951); Chem. Abstracts, 46, 6806 (1951). (39) J. Lee and L. Berger, U. S. Pat. 2,561,384 (1951); Chem. Abstracts, 46, 10515 (1951). (40) F. K. Bell and J. C. Krantz, J . Am. Pharm. Assoc., S c i . Ed., 39, 94 (1950). (41) A. B. Foster and E. F. Martlew, unpublished results. (42) D. Molho and L u c k Molho-Lacroix, Compt. rend., 236, 523 (1952).
344
A.
R. FOSTER AND A. J. IIUGGARD
3. The Homogeneity of Heparin
In the last decade, physicochemical methods have been applied in assessing the homogeneity of heparin. The first recorded electrophoretic study of heparin was made by Wilander,4"awho demonstrated the presence of two components, one of which was thought to be protein. Chargaff, Ziff and Moore43studied Roche heparin44and concluded that it was homogeneous, but this result was not confirmed by later workers. Jensen, Snellman and S y l ~ 6 nreported ~~ that both Roche and V i t r ~ mheparin ~~ contained two fractions. The faster-moving component contained 75 % of the biological activity and had the higher sulfur content (9.3 %) ; it was apparently homogeneous. The slower-moving fraction (S, 7.3 %) probably contained more than one substance. These findings were confirmed by Meyer and S c h ~ v a r t zin~ a~ later study of Roche heparin. The metachromatic activity of heparin has also been used for demonIt has been known for some time that heparin strating its i~ihornogeneity.~~ (and other sulfated polysaccharides) gives a staining reaction with certain basic dyes such as Toluidine Blue,@and this reaction has been used extensively for investigating the distribution of heparin in body tissue.' When commercial heparin preparations were submitted to fractionation using alcohol, dioxane, or acetone, the biological activity and metachromasia of the fractions showed a marked discrepancy, indicating the precipitation of more than one substance, although no separation could be effected. The use of adsorption-front analysis49also indicated the inhomogeneity of commercial heparin. Various unspecified samples of the sodium salt of heparin have been submitted to Craig counter-current distribution between an aqueous buffer solution and n-pentyl Three fractions were obtained, two of which showed anticoagulant activity. (42a) 0. Wilander, Skand. Arch. Phusiol., Suppi!. 16, 89 (1939). (43) E. Chargaff, M. Ziff and D. H. Moore, J. B i d . Chem., 139, 383 (1941). (44) Commercial samples of heparin referred t o in this Chapter are Roche (United States) and Vitrum (Sweden). (45) R. Jensen, 0. Snellman and B. SylvBn, J. Biol. Chem., 174, 265 (1948). (46) K. H. Meyer and D. E. Schwarta, Helv. Chim. Acta, 33, 1651 (1950). (47) L. B. Jaques, Margaret B. Mitford and Ann G. Ricker, Rev. can. biol., 6, 740 (1947). (48) A spectrophotometric study of the metachromatic reaction of Toluidine Blue with sulfated polysaccharides has recently been made; J . Ball and D. S. Jackson, Stain Technol., 28, 33 (1953). (49) 0. Snellman, R. Jensen and B. Sylven, Acta. Chem. Scand., 3, 589 (1949). (50) A. E. O'Keeffe, F. M. Russo-Alesi, M. A. Dolliver and C. J. Stiller, J . Am. Chem. Soc., 71, 1517 (1949).
THE CHEMISTRY OF HEPARIN
345
The behavior of heparin in filter-paper electrophoresis has also been studied.61 More recently,36Wolfrom and coworkers have obtained a heparin preparation (by fractionation of the neutral barium salt) which appeared to be homogeneous in both electrophoresis and counter-current distribution studies. Consequently, it would seem that this material is the best available for structural studies. There can be little doubt that the isolation of a homogeneous heparin preparation is difficult. In fact, Jorpes considered that heparin is not a definite compound but a mixture of inucoitin polysulfates. He suggested32 that the isolation of a range of heparins with increasing sulfur content could be explained in two mays; firstly, it is possible that heparin is elaborated and broken down in the body in such a way that heparins with different levels of activity exist side by side; secondly, the purest form of heparin may undergo chemical modification during the isolation procedure, t o yield less active materials. Jorpes favored the former alternative. 4. The Action of Enzymes, Alkali, and Acid on Heparin
At this point, it may be worthwhile to consider the various stages in the extraction and isolation of heparin in terms of present knowledge of the stability and activity of the molecule. During the process of isolation, heparin is treated with enzymes, alkali, and acid; these stages will be considered in turn. a. Enzymes.-Very little appears to be known about the enzymes, in animal tissue, which are capable of modifying heparin. Jacques and cow o r k e r ~53 ~have ~ ~ reported the inactivation of heparin by an enzyme “heparinase” isolated from rabbit liver. The optimum conditions for the autolysis of minced liver in the initial stages of heparin isolation have been is added to freshly investigated by Kuizenga and S p a ~ l d i n g If . ~ water ~ minced liver and incubation is allowed to proceed a t 35” for 30 minutes and a t room temperature for a day, heparin of twice the purity and in twice the yield (over earlier procedures) is isolated. Jorpes30reported that the degree of autolysis had little effect on the activity and sulfur content of the heparin subsequently extracted, but no experimental data were cited. From the available evidence it would appear that the autolysis procedure increases in efficiency when made slightly more drastic, and hence it is not probable that degradation of the heparin occurs. Neither is there any evidence that tryptic digestion of crude heparin causes any modification of the molecule. (51) K. G . Rienits, Biochern. J . (London), 63,79 (1953). (52) I,. B. Jnques, J . Biol. Cheni., 133, 445 (1940). (53) L. B. Jaques and Eve Kuri-Szantie, Can. J. M e d . Sci., 30, 353 (1952).
346
A . B. FOSTER AND A . J. HUGGARV
Jorpes30showed that a highly active sodium salt of heparin was not inactivated under the conditions of tryptic digestion. b. Alkali.-Much more is known of the stability of heparin toward alkali (and acid). Charles arid Scott12investigated the inactivation of impure heparin, and found that treatment with 0.25 N alkali a t 80" caused loss of half the activity in two hours. J ~ r p e reported s~~ that a highly active sodium salt of heparin was not inactivated in 10 N alkali and 50% amit was reported that heparin monium sulfate a t 80" for one hour. dissolved in N sodium hydroxide a t 100" lost 75% of its activity in five minutes. During Charles and Scott's alkaline extraction procedurez0 of minced tissues, the crude material was maintained in 0.5 N sodium hydroxide, saturated with ammonium sulfate, a t 50" for 30-60 minutes. It would seem highly probable that some modification of the heparin molecule occurs a t this stage. Certain recent results are of interest in this respect. It has been pointed outs5that the results of Gilbert and may have some bearing on the isolation of heparin. These workers demonstrated that starch isolated from potatoes by alkaline treatment under anaerobic conditions was much more highly polymerized than starch similarly isolated under normal conditions. Further, it was shown that highly polymeric starch was but little degraded by the action of 0.2 N sodium hydroxide at 100' in the absence of oxygen, but was rapidly depolymerized when oxygen was introduced. Cellulose is also degraded by alkali and oxygen.6saRelated results were reported by Blix and Snellmans7 who found that hyaluronic acid and chondroitinsulfuric acid, chemical relatives of heparin, were more highly polymerized when isolated under anaerobic conditions in the absence of alkali. The possibility that the action of alkali on heparin, during the isolation procedure, may lead to fragmentation of the molecule, must be borne in mind when considering the data on the homogeneity of heparin. It would be of interest to isolate heparin under anaerobic conditions. c. Acid.-The acid-hydrolytic characteristics of heparin have received close attention, especially in recent years. Schmitz and Fischer6*noted that heparin lost 70% of its activity in one minute when treated with 0.1 N hydrochloric acid at 70", arid Charles and Scott14reported that, a t pH 2.5, crude heparin lost half its potency in two hours a t 80". Recent studies36 (54) (J.) E. Jorpes, R. Bostrcm and V. Mutt, J . B i d . Cheni., 183,607 (1950). (55) .4. B. Foster, E. F. kfartlen arid M. Stacey, Chemistry & Zndiistry, 899 (1953). (56) R. J Bottle, C, A Gitlwrt, C. T. Greenwood arid N. K. Saad, Chemistry d Zndiistry, 541 (1953). (5th) E. Heuser, "Ctieniistry of Cellulos~,"John Wiley arid Sons, Inc., New York, N. Y., 1941. (57) G . Blix arid 0. Snellman, Nature, 163, 587 (1944). (58) A. Schinitz and 8 . Fischer, Hoppe-Seyler's 2. physiol. Cherra., 216, 264 (1933).
THE CHEMISTRY O F HEPARIN
347
have shown that heparin is inactivated rapidly in warm, dilute, acetic acid (see p. 365). During the extraction and purification procedures, heparin is treated in acidic conditions a number of times: (1) heparin is precipitated along with protein from an alkaline extract of liver by acidification, ( 2 ) the precipitate is washed n ith warm acid, (3) coagulation of inert material is effected by warming the solution of heparin a t p1-I 4.1 in the presence of ammonium sulfate to SO”, and (4) removal of protein-like impurities is carried out with precipitating and absorbing agents in acidic solution. Charles and Scott’2. 21 assayed the biological activity of their heparin samples a t different stages in the extraction and isolation procedure, and thus calculated the “total activity” at each point. However, it should be emphasized that, especially in the early stages of separation from protein, assay of “total activity” does not give any indication of chemical modification of the heparin molecule. Astrup and J e n ~ e nexamined ~~ the total activity of heparin samples at various stages during the isolation, and found that a t certain steps, such as deproteinization, a n increase in total activity occurred. These workers noted that binding with protein may actually mask the total potential activity of a crude heparin sample; this may be especially true of in vitro assays. Treatment of the crude heparin-protein complex may increase the total activity by liberation of protein, and decrease the potency by modification of the heparin molecule itself. Jacpes52 believes that if the affinity of heparin for proteins (not concerned with the coagulation system) is sufficiently great, linkage with such proteins might inhibit heparin activity in uiuo. To assay, accurately, the effect of a certain chemical procedure on crude heparin, the conditions must be simulated on a purified sample. That some inactivation of heparin will occur when it is treated with warm acid solutions is to be expected. However, many of the stages outlined above involve acidic treatment, either a t room temperature or when the heparin is precipitated from solution and a two-phase system exists (so that inactivation may not be extensive). When heparin is purified as the barium acid salt by repeated recrystallization from warm, dilute acetic acid, some inactivation is to be expected. Inactivation a t this stage has been cited as the cause of so-called species specificity of heparin. Jaques and Waters,Z9 in 1940, claimed that the use of Charles and Scott’s procedure12r21 for the isolation of heparin (as the barium acid salt) from tissues of different animals gave preparations of quite different activity. Thus heparin preparations from dog, beef, swine, and sheep lungs had relative potencies of 10, 5, 2, and 1. Wolfrom and coworkers60noted that Jaques had used slightly different conditions to crystallize the barium acid salt of (59) T. Astrup and H. B. Jensen, Skand. Arch. Physiol., 79, 290 (1938). (60) M. I,. Wolfrom, J. V . Karabinos, C. S. Smith, P. H. Ohliger, J . JPC m i l 0. Keller, J . A m . Chem. Soc., 67, 1624 (1945).
348
A . B. FOSTER AND A. J. HUGGARD
heparin from each species, alld suggested that this might, he respoiisible for the different levels of activity. When the procedure was standardized,6" heparin from dog and beef liver showed the same activity. Later, it, was confirmedG1that the barium acid salt of heparin isolated from pig liver and beef liver had the same potency. The isolation of heparin as thc rieutral barium salt36,36u. itivolves no warm-acid treatment. 5 . Other Methods of Extraction
It appears that chemical modification of heparin is possible during the most commonly used extraction and isolation procedures. In recent years, milder techniques have been applied to the isolation of heparin. Snellman, Jensen and SylvBnG2 circutnvented the use of alkali extraction by employing solutions of potassium thiocyanate; these rapidly and efficiently extract the mast-cell, granular substance from liver and skin. Subsequent removal of the thiocyanate by dialysis led to the precipitation of a heparin-protein cotnplex which was carried forward to the tryptic digestion stage as before. It is of interest that the calcium chloride extraction procedure, introduced and used extensively by Meyer and S n i ~ t in h ~the ~ extraction of tissue mucopolysaccharides, did not extract the heparin present in the granular substance of tissue mast cells. This is rather surprising in view of the fact that Howell' effected extraction by the use of sodium chloride solutions. For the separation of heparin from heparin-protein complexes, Homan and Lens64have developed a method which avoids the use of acid media. The heparin-protein complex is dissolved in aqueous solution a t pH 7.5 and is extracted with phenol (which removes most of the protein). The method also facilitates the removal of colored impurities which are normally difficult to eliminate. Extraction of the heparin-protein-octylamine complex with phenol has been studied.'jS I n conclusion, satisfactory methods are now available for the extraction and purification of heparin; these have been developed in the light of modern knowledge of heparin activity. The homogeneity of heparin preparations may also be ascertained before structural studies are begun. IV. THE STRUCTURE OF HEPARIN 1. Acidic Hydrolysis of Heparin Considerable information concerning the structure of heparin has been obtained from hydrolytic studies. Controlled, acid hydrolysis is a valuable W. C . Risser, J. Am. Chem. S o c . , 68, 341 (1946). 0. Snellman, R. Jensen and B . SylvBn, Nature, 161, 639 (1948). K. Meyer and Elizabeth M. Smyth, J. Biol. Chem., 119,507 (1937). J. D. H. Homan and J. Lens, Biochim. et Biophys. Acta, 2,333 (1948); J. Lens and J. D . H . Homan, Dutch Pat. 62,276 (1949); Chem. Abstracts, 43, 4817 (1949). (65) F. C. Monkhouse and L. B. Jaques, J . Lab. Clin. Med., 36.782 (1950). (61) (62) (63) (64)
THE CHEMISTRY OF HEPARIN
349
technique currently in extensive use in the study of polysaccharide structure. Complete hydrolysis of a polysaccharide to the nionosaccharide stage, and analysis of the hydrolyzate, serves to identify the sugar components present in the polymer. Graded, acid hydrolysis yields fragments, the identification of which may throw light on the structure of the parent polysaccharide with respect to (1) the sequence of sugar units aloiig the polysaccharide chain, ( 2 ) branching of chains, and (3) position and configuration of the glycosidic linkages. Thus, partial, acid hydrolysis of amylopectintic yields maltose and isonialtose, thereby confirming the presence of a-w(l+ 4) and a-r)-(l+ 6) linkages previously inferred from methylation studies. A general account, a t this point, of the acidic hydrolysis of heparin will provide a perspective for later, detailed discussion. The application of controlled, acid hydrolysis to heparin has been only partially successful owing to the unique, chemical structure of the mucopolysaccharide. If heparin is represented by the partial formula (I) (see page 357 for the detailed structure), on acidic hydrolysis the first stage will involve rapid cleavage of the labile, sulfamic acid groupsti7 [together with slower hydrolysis of the 0-sulfate groups, not shown in I] to yield “$-heparin” (11). Subsequent hydrolysis of I1 will be controlled to a large exteiit by the positive charges of free amino groups in acid solution which will provide an effective electrostatic shield around the adjacent hexosamiiiidic linkages b (and also, t o a much smaller extent, around the linkages a). Hydrions, the effective hydrolyzing agents, will be repelled from the molecular locality of the hexosaminidic linkages, and hydrolytic scission will tend t o occur predominantly a t the D-ghcosiduronic linkages a. The establishment of -NH,+ groups along the heparin molecules in the early stages of acidic hydrolysis accounts for the greater general stability of heparin over that of other mucopolysaccharides, such as hyaluronic acid and chondroitinsulfuric acid, which have acetylated amino groups.32 This type of electrostatic shielding has been studied in simple derivatives of 2-amino-2-deoxy-~-g~ucose(D-glucosamine) .68 Thus, methyl 2-amino-2-deoxy-a-~-glucopyranoside is much more resistant to acidic hydrolysis a t the glycosidic center than is the N acetyl derivative, which is hydrolyzed a t approximately the same rate as methyl a-D-glucopyranoside. A similar effect has been observed with the 1-phosphates of 2-amino-2-deoxy-~-glucose~~ and 2-amino-2-deoxy(66) M. L. Wolfrom, J . T. Tyree, T. T. Galkowski and A. N. O’Neill, J . A m . Chem. SOC.,72, 1427 (1950) ; 73. 4927 (1951); see A. Thompson and M. L. Wolfrom, ibid., 73, 5849 (1951). (67) A. B. Foster, E. F. Martlew and M. Stacey, Abstracts Papers Am. Chem. SOC.,126, 6D (1954). (68) (a) R. C. G. Moggridge and A. Neuberger, J . Chem. Soc., 745 (1938); (b) A. B. Foster and M. Stacey, Advances in Carbohydrate Chem., 7, 271 (1952). (69) D. H. Brown, J . B i d . Chem., 204, 877 (1953).
350
A. B . FOSTER AND A. J. HUGGARD
COOH
CHZOH
CHiOH
CHiOH
COOH
CHZOH
COOH
-0 NHSOIOH
NHSOIOH
i
warm dilute acid
a
a
CHiOH
n
U
COOH
I
TO I I
NH? ;
NH? I
b
b
NH?
I
I
L
I1 $-Heparin
+
other oligosaccharide fragments
I11
/
CHjOH
0 +
C01 and other uronic acid
decomposition products
NH?
IV
D-galactose (D-galactosamine, chondrosamine) which are more stable toward acidic hydrolysis than are the corresponding derivatives of D-glucose and D-galactose. Acidic hydrolysis of +-heparin (11) will result in predominant fragmentation of the chain a t the D-glucosiduronic linkages a to give a series of oligosaccharides, the smallest of which will be the disaccharide 111. Further hydrolysis leads to the destruction of the D-glucuronic acid moiety, leaving 2-amino-2-deoxy-~-glucose(IV) as the main final product of hydrolysis. (70) C . E. Cardini and Id.F. Leloir, A r c h . Biochem. and R i o p h y s . , 46, 55 (1953).
THE CHEMISTRY OF HEPARIN
35 I
a. Total, Acidic Hydrolysis.-Complete, acid hydrolysis was the first hydrolytic procedure applied to heparin, and 2-amino-2-deoxy-a-~-glucose hydrochloride was subsequently identified.'7 Isolation and identification of the D-glucuronic acid moiety of heparin presented some difficulty. Qualitative indications of the presence of uronic acid were early obtained8 and were followed by quantitative studie~.~5,*8 Wolfrom and Karabinos,?l in a study of the hydrolytic characteristics of heparin showed that the rates of liberation of 2-amino-2-deoxy-~-glucose and destruction of u-glucuronic acid were roughly parallel. It was suggested by these authors that decarboxylation of the uronic acid might occur whilst it was glycosidically bound in the hydrolytic fragments of the polysaccharide. The isolation of D-glucuronic acid following direct acid hydrolysis of heparin would appear to be precluded. The evidence for the presence of D-glucuronic acid was put on a definitive crystalline basis by Wolfrom and Degradation of heparin using a bromine-sulfuric acid mixture resulted in oxidation of the glycosidic carbon atom of the D-glucuronic acid moiety following hydrolysis of the D-glucosiduronic linkages. The D-glucaric (saccharic) acid so formed was stable under the hydrolytic conditions employed. It was isolated as the crystalline potassium acid salt which had a specific optical rotation identical with that of authentic material. Potassium acid D-glucarate also has a characteristic x-ray diffraction pattern.73 2-Amino-2-deoxy-~-gluconic acid was also isolated following oxidative hydrolysis. At this point, it is pertinent to review the information that may be gained from analytical data concerning the structure of heparin. Since 1935, many analytical data on heparin have been accumulated, but, as noted earlier, much of the work was performed on inhomogeneous or incompletely dried samples. The most thorough quantitative examination of the composition of heparin was made by Wolfrom and coworkers23on the barium acid salt. 0. Hexosamine and Hcxuronic Acid Content.-,Jorpe~~~ has estimated the presence in heparin of 17-19 % of hexuronic acid and the amount of hexosamine t o be that calculated for one molecule of hexosamine per molecule of hexuroriic acid. The hexuronic acid was estimated as carbon dioxide, foland the lowing acid hydrolysis by a modified Tolleiis-LefBvre hexosamine by the Elson-Morgan colorimetric method.75 (71) M. I,. Wolfrom and J. V. Karabinos, J . A m . Chetn. SOC.,67, 679 (1945). (72) M . L. Wolfrom and F. A . H. Rice, J . Am. C h e m . Soc., 68, 532 (1946). (73) M. I,. Wolfrom and W . B. Neeleg, J . A m . f'herr/. Soc., 76, 2778 (1953). (74) A D. Dickson, H. Otterson and K. P. Link, J. A m . C h e m . SOC., 62,775 (1930). (75) L. A. Elson and W. T. J. Morgan, Biochem. J . (London), 27, 1824 (1933); see Ref. 68(b), p. 257.
352
A. B. FOSTER AND A. J . HUGGARI)
MeyerZ4and Wolfrom and their detected the uronic acid in the barium acid salt of heparin both qualitatively and quantitatively, and considered it to be the source of the acidity in the acid salt. Amino sugar was also estimated. These results, which cannot be said to be as precise as the elemental data, indicate a ratio of 1: 1 for amino sugar and hexuronic acid. c. Sulfur and Nitrogen Contmt.-Jorpeslb recognized that the high ash content of heparin was due to the presence of ester sulfate, and in his early 3 0 ' 31 he reported that heparin contained 2.5 sulfate groups per disaccharide period. Later, by fractionation with brucine, fractions n ere obtained which, according to sulfur analysis, had 3 sulfate groups per disaccharide unit, and in one case30 an even higher sulfate content. The homogeneity of these samples has not been assessed. Electrometric titration of free heparin obtained by electrodialysis showed both weak acid (uronic acid) and strong acid (ester sulfate) There appeared to be three sulfate groups per uronic acid. Wolfrom and coworkers23have summated their analytical data on the barium acid salt of heparin, and have expressed the data in terms of a tetrasaccharide unit which comprises two molecular proportions each of 2-amino-2-deoxy-~-glucose residue and D-glucuronic acid residue, and 5 (rather than 6) ester sulfate groups, in an K :S ratio of 2 :5. The barium :sulfur ratio was 1:2, which indicated that the sulfur is essentially present as ester sulfate, and hence the acidity must be due t o the carboxyl group, in accordance with the shape of the titration curve.42a d. Partial, Acidic Hydrolysis.-As indicated previously, the first step in the hydrolysis of heparin is the cleavage of the N-sulfate groups to give +heparin (11).Although this step is now well established, its elucidation was a problem of considerable difficulty. Confusion was created by the fact that heparin, although containing an amino sugar, does not contain any N-acetyl groups. It had been suggested76that all mucopolysaccharides have the amino sugar N-acetylated, and, in this respect, heparin is unique within the class of mucopolysaccharides. (Mucopolysaccharides have been classified by M e y d 7 and S t a ~ e y . ~It * )had been known for some time that the nitrogen in heparin is not present as the free amino group,21-y 2 , 587 79 since only small amounts of nitrogen are formed from heparin on treatment with nitrous acid (Van Slyke). Charles and Scott" noted that the action of nitrous acid completely inactivates heparin, and Charles and Todd2*observed that the amino-nitrogen value increases slowly in the Van Slyke esti(76) (77) (78) (79)
0. Furth, H. Hwmnnn and R. Scholl, Biochem. Z . , 271, 395 (1934). K. Meyer, Advances in Protein C ' h e ? ~ .2, , 249 (1945). M. Stacey, Advances zn Carbohydrate C h e m . , 2, 161 (1946) (J.) E. Jorpes and S. Bergstrom, J . B i d . Cherri., 118, 447 (1937).
THE CHEMISTRY OF HEP.4RIN
353
illation uiitil, aftcr 1ti hours, all the iiitrogeii is accouiited for. 2-AcetJaniido2-deosy-D-glucose (N-ncetyl-D-glucosatnine) gives no nitrogen under these condit'ions. All the nitrogen was present, in the amino sugar,?:!and evidcnre tJhatJthe amino group is substituted had been advanced by Wilander,"S;L who could detect no sign of buffering between pH 7 and 10 in the electrometric titration of free heparin. Heparin was assayed for N-acetyl by various workers, a.nd a range of N-acetyl contents was reported, t,he value being invariably lcss than t,he theoretical amount for acetylation of all the amino groups.15 2z Complet,e absence of N-acet,yl groups was claimed by other workers.23'z 5 , 27 In 1950, Meyer and Schwartz46 demonstrated that commercial heparin which had a low acetyl content was split, on electrophoresis, into two components. The more rapidly moving component contained all of the anticoagulant activity but had zero acetyl content. The slower moving component had an acetyl content corresponding to that, of a polymer of 2-acetamido-2-deoxy-~-glucose. It has been suggestedz3 8o that the small quantities of acetic acid in heparin may originate in the purification procedure when acetic acid is used. Heparin appears to have a definite capacity for the absorptive retention of acetic acid. A l t e r n a t i ~ e l y , ~ ~ t,he acetyl content may arise from an impurity which accompanies heparin in the isolation procedure and which is difficult to remove. 56 it has been shown by interaction of heparin with nitrous acid and l-fluor0-2,4-dinitrobenzene that 6 to 10% of tthe amino groups are present as -NH2 . Various suggestions as to the mode of linkage of the remaining amino groups have been made by several workers. It was shownz3 t,hat the residue which blocks the amino group is easily cleaved by acid, and I
hence it could not be a
\
N-CH3
grouping. The amino group is not in-
/
volved in a glycosidic linkage since, on release of the amino groups under mild, acid hydrolytic conditions, there is not a corresponding increase in the reducing power.54.81 The presence of a linkage involving the carboxyl group of the uronic acid and the amino group was also pre~luded,4~ since the potentiometric tit,ration of heparin indicated the presence of one carboxyl group per nitrogen atom and showed that t,he only acidic groups in the polysaccharide are -C02H and -S020H. Masamune, Suzuki and Kondohz7 were the first to suggest a sulfuric group in heparin in the form of bridges, -C-0-S02-NH-C-, but they presented no experimental evidence therefor. This suggestion was criticized by Jorpes, Bostrom and who pointed out that W i l a ~ i d e r ~ ~ * had shown that all the sulfate groups in heparin (acid form) are free and (SO) M. L . Wolfrom, Ohio State Univ. Eng. Espt. Sta. News, 26, No. 5, 22 (1953). 67, 748 (1945). (81) M. L. Wolfrom and W. H. McNeely, 6.Am. Chem. SOC.,
354
A . B. FOSTER AND A. J. HTJGGARD
titratable. The presence of a sulfamic acid groupiiig, -NI-I-S02011, was again suggested by Wolfrom and McNeely8*on the basis of the results of inactivation studies (see p. 365). The properties of heparin with respect to the presence of a sulfamic acid group accord with the known general chemistry of substituted sulfamic acids.82The stability of substituted sulfamic acids is related to the basicity of the parent aminesSs3Thus, an amine of low basicity gives a substituted sulfamic acid (arylsulfamic acid) which is very unstable in acid solution. Alkylsulfamic acids are comparatively stable in cold aqueous solution. It would appear that the acidic hydrolysis of alkylsulfamates has a high, temperature coefficient. Cyclohexylsulfamic acid, which is describeds4 as a “fairly strong acid,” is hydrolyzed in hot water. Since the basicity of 2-amino-2-deoxy-~-glucoseis intermediate between that of methylamine (and cyclohexylamine) and aniline,s5the properties of “N-sulfate” might be expected to be intermediate between those of the two classes of substituted sulfamic acids. Recent on the hydrolysis of the “N-sulfate” group in heparin showed that, when a 1% solution of the sodium salt in 0.04 N hydrochloric acid is heated a t loo”, hydrolysis of the “N-sulfate” is complete in 90 minutes. Meyer and in a study of model compounds structurally related to heparin, showed that the hydrolytic release of sulfate under acid conditions from 2-amino-2-deoxy-~-ghcoseN-sulfate is more rapid than from D-glucose 6-0-sulfate. These workers also obtained a biologically inactive, nitroso derivative of heparin, and offered this as evidence for a sulfamic acid linkage in the molecule. Wolfrom, Shen and Summerss6prepared dibarium methyl 2-am~no-2-deoxy-N-sulfo-tr~-0-sulfo-~-~-g~ucopyranos~de salt, and found that, on heating a 3 x loT4M solution in 0.004 N hydrochloric acid, the “N-sulfate” is lost in 20 minutes and the 0-sulfate after 12 hours. Some difficulty was experienced in establishing a strict correlation between liberation of amino group and sulfate release. For example, Wolfrom and McNeely,81and Jorpes, Bostrom and did not obtain correspondence between these when heparin was treated with hot, dilute acid. It was suggestedb4that retention of the barium sulfate in the colloidal state (peptization) by the heparin might be responsible for inaccurate results in sulfate analysis. If, however, heparin was first treated with hot, dilute alkali, then, on subsequent acid treatment, correspondence between release
H,oH CHzOH
COOH
~OSOZO
H
NH,'
H
OH
V
2 . Acetylative Desulfation Procedures
The application of the classical niethylation t,echiiiyues to hepariii has not yet been s ~ c c e s s f u l .The ~ ~ presence of a high percentage of sulfate groups in the heparin molecule is undoubtedly one of the complicating factors. Thus, a satisfactory method of desulfation would be of value in furthering structural studies of heparin. Considerable progress toward this end was made with the introduction of a chemical method of desulfation The by Wolfrom and M o n t g ~ i n e r y .94~ ~ , t,echnique involves the use of absolute sulfuric acid and acetic anhydride. It is most probable that the act,ivc desulfating agent is the acetylium ion, CH3-CO+, which is known to be produced when strong acids are added to acetic anhydride.9b A possible mechanism of desulfatioii is as follows:
(CH,--CO)zO CHa-CO+ HO-SOz+
+ HzS04 + R-0-SOzOH + HSO,
--+
+ HSO4- + CH;r-COzH R-O-COCH3 + +SOz-OH
+
HzSz07
CH3-C0+
(92) h1. Stacey, private communication; hl. L. Wolfrom, private communication. (93) M. L. Wolfrom and R. hlontgomery, J . A m . Chevi. SOC.,72, 2859 (1950). (94) No use has yet been reported of enzymic desulfation in t,he study of heparin. Enzymes are known which will effect, the hydrolysis of mucopolysaccharitle sulfate groups; see K. S. Dodgson and B. Spencer, Biochem. J . (London), 63, iv (1953). (95) H. Burton and P. F. G. Praill, Quavt. Revs. (London), 6, 302 (1952).
357
T H E CHEMISTRY O F HEPARIN
The use of absolute sulfuric acid tends to create experimental difficulties, and may cause dehydration and degradation. It would be of interest to examine other sources of acetylium ions as potential desulfating agents with respect t o thc obviation of degradation. The effect of the acetic anhydridesulfuric acid reagent on a wide range of simple carbohydrates was studied,93 and permitted evaluation of the stability of glycosidic linkages under reaction conditions. Acetylative desulfation of D-glucose 3-0-sulfate proceeded without Walden inversion, thus suggesting the possible mechanism of desulfation shown below. R-O--SO~-OH
t CHSCO
- @1
R-0-SO,-OH --.---I
R-0-CO-CH~
co
I CHI
@
+
HO-SOz@
Application of the procedure to heparin gave a degraded product which was sulfur-free. I n work described in a later paper,36this material was de-0acetylated and then was found to consume 2 moles of periodate per tetrasaccharide unit, without the formation of formic acid or formaldehyde. The original heparin (see p. 355) consumed 1 mole of periodate, and contained 5 sulfate groups, per tetrasaccharide unit. From these data, together with those reported for heparosinsulfuric acid (see p. 356), Wolfrom and coworkers36have postulated VI as a probable structure for the barium acid CH,OH
p-ko0
COOH
-0 Q0QO
OH
Ba@3020 H
hHSO,OBa(
H
coon
CH,OH
OH
Ba~OSO~O H VI
H
OH
NHS020Ba,
H
0-
OSOIOBat
salt of heparin. Certain structural features of heparin are not yet known or have not been proved unequivocally. They are: (1) the distribution of the 0-sulfate groups, (2) the identity of the sugar unit a t the reducing and nonreducing ends of the chains, (3) the configuration of the D-glucosiduronic linkage, and (4) the presence or absence of branching in the chains. The determination of these points may be important, especially in connection with the relationship of the anticoagulant activity of heparin to its molecular architecture.
3. Other Methods of Degradation of Heparin a. Action of Nitrous Acid on Derivatives of 2-Amino-2-deoxy-~-glucoseand Heparin.-The effect of the action of nitrous acid on 2-amino-%deoxy-~glucose or chitosan (de-N-acetylated chitin) has been known for many
358
A. B . FOSTER AND A. J. HUGGARD
years; 2,5-anhydro-u-mannose (VII) is aff ~ r d e d A. ~monomolecular ~ substance, later recognized as VII,96& was obtained very readily by the action of nitrous acid upon chit0san.~6~ Substance VII is also formed when methyl 2-amino-2-deoxy-a- or 0-D-glucopyranoside (VIII) is treated with nitrous CHiOH
qH,oH
HO H(-+Ho
H
H VII
H
hHg VIII
acid.g7I n both cases, the glycosidic methyl group is split off, but the rate of reaction is much more rapid for the 0-D anomer. A study of treatment of $-heparin (11) and chitosan with nitrous acid showed that the rates of deamination of the polymers closely follows those of methyl 2-amino-2deoxy-a- and -0-D-glucopyranoside (VIII), respectively. The linkages in chitosan are known to be 0-D,and thus there is strong indication that the hexosaminidic linkages in heparin are a-Din configuration, in agreement with the heparin formula (VI) suggested by Wolfrom and The nitrous acid treatment of $-heparin, in addition to providing a correlation between the rate of deamination and the configuration a t the glycosidic center, constitutes a new method of degradation of heparin. Although little has yet been reported on the structure of the products of the deamination, the method is of interest since it leads to the selective cleavage of the hexosaminidic linkages (which are those most stable in acidic hydrolysis). A study of the infrared absorption spectra of D-glucopyranose and its derivativesg8has shown that definite absorption bands may be associated with a-D or fl-D configuration a t the glycosidic center. The absorption is characteristic in mono-, oligo- and poly-mglucose derivatives and in deUnfortunately, the infrared rivatives of 2-amino-2-deoxy-~-g~ucopyranose. spectrum of heparin appears to be considerably influenced by the high percentage of sulfate ester groups present and, a t the present time, information on the configuration of the glycosidic links in heparin cannot be gained thus.99 The infrared absorption spectra of certain mucopolysac(96) For a detailed account of the deamination of 2-amino-2-dcoxy-~-glucose, see S. Peat, Advances i n Carbohydrate Cheni., 2 , 37 (1946). (96a) Personal communication from the late Prof. K. H. Meyer. (96b) K. H. Meyer and H. Wehrli, Helv. Chim. Acta, 20, 361 (1937). (97) A. B. Foster, E. F . Martlew and M. Stacey, Chemistry J1- Industry, 825 (1953). (98) S. A. Barker, E. J. Bourne, M. Stacey and D. H. Whiffen, J. Chem. Soc., 171 (1954). (99) A. B. Foster and M. Stacey, unpublished data.
THE CHEMISTRY OF HEPARIN
359
charides have been studied with respect to -C02H, -NHCOCH, , and -S020H groups.'Oo b. Oxidation in the Presence of Ascorbic Acid.-Skanse and Sundblad'O' have reported an interesting degradation of polysaccharides. They have shown that heparin, hyaluronic acid, starch, and cellulose are degraded in the presence of ascorbic acid and oxygen, with Cu++or hydrogen peroxide also present. The products formed are nonreducing but are acidic and dialyzable. I n the case of hyaluronic acid, the product appeared to be a disaccharide in which the D-glucosiduronic linkage was intact. Few details mere given on the products of heparin degradation.
V. THE ANTICOAGULANT ACTIVITYOF HEPARIN 1. The Biological Activity of Heparin The most familiar property of heparin is its activity as a blood anticoagulant. The coagulation of blood is an efficient and complicated process, and, although the mechanism has not been completely elucidated, the broad outlines are known and may be represented in the scheme shown below.1o2* The effective step in the sequence is the conversion of fibrinogen, a soluble protein present in blood, into its insoluble form, fibrin. The fibrillar network of the fibrin is responsible for the conversion of the liquid phase of normal blood to the solid phase of the clot. The conversion of fibrinogen to fibrin is controlled by the protein thrombin, the formation of which from prothrombin is catalyzed by thrombokinase. Heparin, together with Caff and a plasma co-factor, controls the level of thrombokinase in the blood under normal conditions. In the event of mechanical or chemical injury to tissue or blood platelets, the thrombokinase contained therein is released a t the site of the injury, counteracts the action of heparin, and initiates the coagulation process. I n the condition of thrombosis, where blood clots develop in the veins and arteries, the injection of heparin may cause dispersion of the clots.' Thrombokinase
i -
_ _ _ _ _ - - _ --_- - --Heparin,
Prothrombin
Thrombin and a plasma co-factor
Fibrinogen (100) (101) (102) (103) (1955).
Ca++
I
___c
Fibrin
S. F. D. Orr, R. 6.C. Harris a n d B. Sylven, Nature, 169, 544 (1952). R . Skaiise and L. Sundblad, Acta Physiol. Scarid., 6. 37 (1943). K. H. Meyer, R . P. Piroue and M. E. Odier, Helv. Chirn. Acta, 36,574 (1952). T . Astrup, Advances i n Enzynrol., 10, 1 (1950); W. € 3 . Seegers, ibid., 16, 23
360
A. B. FOSTER AND A. J . HUGGARD
Snellnian, S y l v h and Ju16n5have isolated what appears to be the plasma co-factor from tissue, mast-cell cytoplasm. The co-factor was a lipoprotein containing lecithin, cholesterol, and neutral fats, together with a low molecular-weight polypeptide. Only the six amino acids cysteine, threonine, tyrosine, glycine, leueine, and tryptophan were present in the polypeptide. I n addition to its role in the blood-coagulation process, heparin shows other types of biological activity, such as its action in clearing fat globules from the blood stream (alimentary lipemia)In4and its use in the treatment of frost-bite.' For ail extensive account of the development and use of heparin in medical and surgical practice, the reader is referred to Jorpes' book.' Heparin also possesses activity toward certain strains of bacteria; for example, in a protein-free medium, heparin was found to be bacteriostatic toward Micrococcus pyogenes a t 100 p.p.rn.ln6 At 100,000 p.p.m., heparin was riot bactericidal but caused the development of a high percentage of mutants, smooth + rough, which did not revert. The mutagenic action of heparin is probably related to its ability to displace ribo- and deoxyribonucleic acid from cells.106Heparin can also function as an inhibitor for pancreatic r i b o n u c l e a ~ eA . ~series ~ ~ of derivatives of +heparin (11)in which the amino group was substituted by various organic residues, such as nicotinyl, isonicotinyl, and trifluoromethylphenyl, showed little activity against several species of b a ~ t e r i a . ~The ' bacteriostatic activity of heparin in certain cases appears to be due to the prevention of gelation of cytoplasmic material, which is an essential preliminary to cell division (mitosis).loSThe effect is reversible. 2 . The Structure and Anticoagulant Activity of Heparin
The mechanism whereby heparin functions as an anticoagulant, and the architectural features of the molecule which are associated with the activity, have not been fully elucidated. In the following Sections, an attempt has been made to integrate and evaluate the work done to date on the structure of heparin in relation to its activity. The suggestions subsequently made are not necessarily the oiily interpretations of the known data. It would appear that the high anticoagulant activity of heparin is due to the concerted effect of a series of molecular features which include (1) degree of sulfation (and distribution of the sulfate groups), ( 2 ) molecular size, and (3) molecular shape. Many attempts have been made to imitate (104) (105) (106) (107) (108)
E. A. Nikkila and E. Haahti, Acla Cheni. Scund., 8, 363 (1954). J. R. Warren and F. Graham, J . Bacteriol., 60, 171 (1950). N. G. Anderson and I(.M. Wilbur, Federation Proc , 9, 254 (1950). J . S. Roth, Arch. Biochem. and Biophys., 44, 265 (1953). L. V. Heilbrunnand W. L. Wilson, Proc. Soc. E x p t l . Biol. Med., 70,179 (1949).
THE CHEMISTRY O F HEPARIN
361
oiie or more of these features in the preparatioii of synthetic, sulfated polysaccharides. These studies derived their impetus from the need for a cheap, synthetic anticoagulant to replace the expeiisive natural product. I n view of the iiicompletcness of our knowledge of the structure of heparin in relation to its activity, the preparation of sulfated polysaccharides as synthetic anticoagulants has been undertaken in the past on a somewhat empirical basis. a. Degree of Sulfation.-The recognition of a high sulfate-ester content in heparin led to the examination of synthetic, highly sulfated polysaccharides as potential anticoagulants. Many polysaccharides have been sulfated and studied,1ogincluding chondroitinsulfuric acid, cellulose, inulin, xylan, chitin, chitosan, alginic acid, and dextran. All of the products were found to have anticoagulant activity, but of an order much lower than that of heparin. Further, the toxicity of many of the synthetic, sulfated polysaccharides was such as to contraindicate their therapeutic use. Meyer and coworkers102have studied in detail the sulfation of chondroitinsulfuric acid, since, a t the time, this appeared to be the most active of all the synthetic products. Various methods are available for the sulfation of polysaccharides.ln2Most commonly used is the mixture pyridine-chlorosulfonic acid, but chlorosulfonic acid and the dioxane-sulfur trioxide system have also been employed ; these reagents lead to extensive degradation of the polysaccharide. Meyer and coworkers102used sulfur dioxide as the liquid phase and sulfur trioxide or chlorosulfonic acid as the sulfating agent, and claimed that these systems caused very little degradation of the polysaccharide. Recently,llo formamide and N ,N-dimethylformamide have been used as the liquid phase, with sulfur trioxide as the sulfating reagent. This system, which facilitates a homogeneous reaction, also leads to some degradation of the polysaccharide. Using chondroitinsulfate (molecular weight 30,000) from hog nasal cartilage, and sulfating with the sulfur dioxide and sulfur trioxide system, Meyer and coworkers102obtained a high yield of product; this was little degraded and contained 12-14 % of sulfur (the sodium salt of heparin with a N :S ratio of 2 :5 has 12 % of sulfur). The activity of this product, measured by in vitro methods, was 10-25% of that of standard heparin,"' but in vivo methods (injection into rabbits) indicated 30-45% of the activity (109) See Reference 102 and the references given therein. (110) M. L. Wolfrom, (Mrs.) T. M. Shen Han and T. Y. Shen, private rommunication. (111) A preparation of heparin from beef liver was selected in 1942 by the Department of Biological Standards of the National Institute of Medical Research in London to serve as the provisional international standard. Quoted by (J.) E. Jorpes and S. Gardell, Ref. 32.
362
A. B. FOSTER AND A . J. HUGGARD
of standard heparin. Wolfroni, Shen and Summers86 also reported a low i n vitro activity for sulfated chondroitinsu1fat)e. The variety of methods that have been used for assaying anticoagulant activity makes difficult a comparison of the results of different workers."' It is claimed that the most satisfactory, although not the most convenient, method involves animal tests. By the use of three groups of animals, for control and for injection of standard heparin and of synthetic products, the effect of the latter group is thought to be most accurately assessed. Subsequent references to the activity of sulfated polysaccharides will be limited to comparisons with that of the heparin preparations used by individual authors and not with that of a common standard. Recently, a detailed study of dextran sulfate has been made.113Dextran ( polyglucose elaborated by Leuconostoc is essentially an a - ~ -1+6)-linked mesenteroides. The molecules as synthesized by the micro-organism are gigantic, but they may be degraded to molecules of specific molecular weights by various methods. Two variables were studied in dextran sulfate: (1) degree of sulfation, and (2) molecular weight. It was found that a degree of sulfation higher than 5.2 sulfate groups per tetrasaccharide period, compared to the 5 sulfate groups in heparin, is necessary in order to give appreciable activity, and this was ca. 10 % of the activity of heparin. Dextran sulfates with a high molecular weight (30,000 and 300,000) had a high toxicity. As the molecular weight was reduced, the toxicity fell, and this effect appeared to parallel a decreasing ability of the dextran sulfate to precipitate fibrinogen. It was suggested that, on injection, the high molecular-weight dextran sulfates cause precipitation of fibrinogen, with subsequent agglutination of the platelets around the precipitate and consequent toxic effects. A dextran sulfate of molecular weight of 7,000-8,000 was found to have a low toxicity and to be satisfactory for clinical use. The administration of synthetic, polysaccharide sulfates in large doses may lead to death from internal bleeding.lI4 The preceding data suggest that the activity of heparin is not simply a function of the degree of sulfation, since heparin preparations of high activity contain fewer sulfate groups than the more weakly acting, synthetic products. Karrer and associates116also point out that the relatively rapid disappearance of heparin activity in vivo, compared to the slow disappearance of activity of synthetic, sulfated polysaccharides, may be due to a different mode of action. (112) See B. Blombiick, Margarita Blomback, E. V. Corneliusson and (J.) E. Jorpes, J. Pharm. Pharmacol., 6, 1031 (1953). (113) C. R. Ricketts, Biochem. J. (London), 61,129 (1952); C. R. Ricketts and K . W. Walton, Chemistry & Industry, 869 (1952). (114) T. Astrup and J. Piper, Acta Physiol. Scand., 9, 28 (1945); 11, 1211 (1946). (115) P. Karrer, H. Koenig and E. Usteri, Helv. Chim. Acta, 26, 1296 (1943).
THE CHEMISTRY OF HEPARIN
363
b. The N-Sulfate Group.-The presence of the “N-sulfate” group in heparin and the physiological properties of the polysaccharide, which are unique within the class of mucopolysaccharides, suggest that there may be a relationship. This possibility has been examined recently, following the synthesis of sulfated polysaccharides which contain the “N-sulfate” grouping. I n a study of chitosan sulfate (obtained by the sulfation of de-Nacetylated chitin) , Doczi, Fischman and King116reported obtaining a product with a n activity ca. 50% of that of heparin. No details of the method of sulfation were given, but they indicated that the activity was assayed by an in vivo method. Similar results were simultaneously reported by Wolfrom, Shen and Summers,86who sulfated chitosan with the pyridinechlorosulfonic acid system and isolated a product with ca. 50% of the activity of heparin but with greater toxicity (because of high molecular weight). It was found that the product obtained by sulfation of de-Nacetylated chondroitinsulfate also had an activity ca. 50% of that of heparin. However, direct sulfation of chondroitinsulfate gave a product with < l o % of the activity of heparin. Rickettsll’ did not succeed in obtaining a chitosan sulfate of high activity. The fact that synthetic polysaccharide sulfates have been obtained which have “N-sulfate” groups present and which display activity considerably higher than when these groups are absent would seem to indicate that the “N-sulfate” group per se may be associated in an important way with the activity. Heparin preparations may, however, have a high percentage of “N-sulfate” groups but a low Perhaps significant is the observation that the sulfur content of the active chitosan sulfates (13-17 % for the sodium salts) is considerably higher than that of active, heparin preparations (12%). It would be of interest to compare the activities of preparations of heparin and chitosan sulfate which have similar sulfur content and molecular weight. c. Distribution of the Sulfate Groups.-The distribution of the 0-sulfate groups in heparin has not been fully established; more hydroxyl groups than sulfate ester groups are present in the molecule, and hence several distributions are possible. Enzymic sulfation of the natural heparin polysaccharide most probably leads to a regular distribution of the sulfate groups, whereas the laboratory synthesis of polysaccharide sulfates probably gives a random distribution. The heterogeneity of the sulfation procedures used most extensively to date may also tend to give an uneven distribution of sulfate groups in the final product. d. Molccidar I’C’eighi of Heparin.-Several measurements have been made on the molecular weight of heparin, and the values obtained approximate (116) J. Doczi, A . Fischman and J. A. King, J . Am. Chem. Soc., 76, 1512 (1953). (117) C . R . Ricketts, Research (London), 6, 17-S (1953).
364
A. B. FOSTER AND A.
J. HUGGARD
to 20,000. It is possible that the results may be influenced by the potent polyelectrolyte character of heparin. The values are as follows: 17,00018,000 by the reducing value46;17,000,4520,000,36and 15,00011s from diffusion measurements. 3. The Molecular Shape of Heparin Recent 1vorkS5indicates that heparin preparations may have a low activity and yet have a high percentage of “N-sulfate” groups. It was suggested5*that this and other observations might be explained by postulating the presence of a small number of intramolecular, sulfate bridges of the type IX. The presence of such bridges in sniall amount could hold the heparin in a shape, specific for activity, which would be irreversibly changed by cleavage with acid or alkali, as shown (IX). Detection of these bridges on the basis of analytical data would be difficult, acid ; since the consequent reduction in cationic content I I would be within the normal variation of experiC H - K H - ~ O ~4-0-CH 1 ; ; 1 mental error. The report by Wilander42athat all of the sulfate groups in heparin were free and i bise titratable is also subject t o the same limitation IX (S analysis). There is also some doubt concerning the purity of the heparin preparation used by Wilander, since only recently have methods been established which permit the isolation of (chemically) well defined and (physically) relatively honiogeneous heparin preparations. It is of interest to consider some of the known facts about heparin in the light of the above suggestion of the presence of sulfate bridges. a. Sulfation of Heparin.-The sulfation of a highly active heparin preparation (S, 12 % for the sodium salt) with the pyridirie-chlorosulfonic acid system, used in the preparation of chitosan sulfates,E6causess1’86 an increase in the sulfate content (S, 14.4%) but a reduction in activity of more than 50%. I n fact, the activity of the sulfated heparin was closely similar to that of chitosan sulfate (S, 15.3%) and of sulfated, de-N-acetylated chondroitinsulfate. Possibly, in the process of sulfation, sulfate bridges, if present, would be cleaved and the increase in sulfate content would then give a molecule structurally related to the sulfated, de-N-acetylated chondroitinsulfate. That, in the sulfation, the heparin is degraded below a critical, molecular size would appear unlikely, since the product has appreciable activity, and toxicity (rather than activity) varies with molecular weight.13 It appears that some degree of chemical modification of heparin may he #
I
(118) K. H. Meyer, “Xatural and Synthetic High Polymers,” Intersckncc Pub lishers, New York, N. Y., 2nd Edition, 1050, p. 456.
THE CHEMISTRY OF HEPARIN
365
achieved without decreasing the activity. Thus, Bell and J a q ~ e s reported I~~ that acetylation of active heparin with ketene introduced 4.02 % of acetyl groups without reducing the activity. Deacetylation, again without loss in activity, could be effected by heating in neutral solution. The ease with which the acetyl groups were removed makes difficult any suggestion as to the mode of t,heir attachment to the heparin molecule. That the acetyl content is due t o absorbed acetic acid is not precluded. b. Inactivation of Heparin by Dilute Acid.-Heparin rapidly loses its acThe inactivation has tivity in warm, dilute, acid solution.g*1 6 * 2 3 , 5 9 , been studied in detail by Wolfrom and McNeely,81who found that a 2 % solution of the barium acid salt in 11 % acetic acid, a t 68 f 2", lost most of its activity in 48 hours. Several significant observations were made. (1) The inactivation was solely a function of hydrions; (2) there was no significant change in the low reducing value; (3) there was no appreciable change in the optical rotation; (4)the inactivated product still gave the Toluidine Blue test characteristic of sulfated p o ly s a c ~ h a rid e s ~ lZ0; ~ '~and ) ( 5 ) the inactivated product could be precipitated from aqueous solution by means of excess acetic acid or ethanol. These observations would appear to indicate that there was no marked change in molecular size during the inactivation. Release of amino groups was also observed to occur during the inactivation, although most of the activity had disappeared when an appreciable portion of the amino groups were still combined. Only a small apparent loss of sulfate groups occurred during inactivation (S 12.9% + 11.8 %). Thus, during the inactivation process, the heparin molecule would appear to have undergone little change in elemental content. Wolfrom and McNeelylsl commenting on the result of Charles and Todd,22point out that the action of dilute, methanolic, hydrogen chloride on heparin leads to a much greater rate of inactivation than of sulfate release. It is interesting to note that Jaques and associate^^^ have reported the isolation of an enzyme preparation which inactivated heparin without causing loss of sulfate. The suggested presence of sulfamic bridges in heparin would not be a t variance with the preceding results. Charles and Scott21 reported that heparin was inactivated by dilute nitrous acid. Heparin is known to contain a small percentage (&lo%) of 81 The action of nitrous acid would be expected to free amino cleave the polysaccharide chains wherever a free amino group occurs (see p. 357) and, if these groups are randomly distributed, fragmentation and consequent inactivation would follow. 7 9 8
(119) H. J . Bell and L. B. Jaques, Can. J . Research, 26B,472 (1917). (120) I,. Lison, Conipt. rend. soc. biol., 118, 821 (1935); (J.) E. Jorpes, Actu Med. Scand., 88, 427 (1936).
366
A. B. FOSTER AND A. J. HUGGARD
c. Other Considerations.-Jensen, Snellman and S y l ~ B nhave ~ ~ studied the change in physical properties of heparin during the inactivation caused by recrystallization from warm, dilute acetic They found no decrease in the sulfate content during the inactivation (few sulfur analyses were reported in the paper). The sulfur content of the heparin studied was low and its absolute activity was not stated; the fall in activity was expressed as a percentage of the original activity. As the activity fell (100 + 44 %) a continuous increase in the sedimentation rate occurred (8202.07 -+ 2.7), and the ultracentrifugation diagrams indicated an increased degree of polydispersion. It was suggested that a “structural rearrangement” had occurred, such as a change in shape or hydration. That a large change in molecular weight had not occurred was indicated by the fact that the molecular weight, as determined from the diffusion constant, was 17,000 for the original heparin and 16,600 after inactivation. During inactivation, the frictional ratio fell from 2.5 to 1.81. Assuming that the original heparin molecule was an ellipsoid, these figures would indicate a change in axial ratio of 35:l .--f 16:l. The above data further substantiate the hypothesis of a change in shape rather than in size during inactivation. The shape of the heparin molecule may also be controlled to some extent by the sequence and configuration of the glycosidic linkages. In general, an Q-D-( 1 .--f 4)-linked, unbranched poly-D-glucopyranose tends to be helical in structure, as is evidenced by the classical example of amylose.121When the a linear molecule results; this has fibrous characteristics configuration is 0-D, if the molecular weight is sufficiently high (compare, cellulose). The mucopolysaccharides chondroitinsulfate and hyaluronic acid contain predominantly p-D linkages and, whilst the shape of the former is probably influenced by the D-galactose configuration of the amino sugar, the fibrous nature of the latter is well known. Heparin, however, appears to have predominantly a-D-glycosidic linkages, again rather unusual in the class of mucopolysaccharides. A helical structure somewhat distorted from that of -D amylose could result from the alternating sequence of (3 -+ ~ ) - ( Y and (4 l ) - a - linkages ~ in heparin. The presence of sulfate bridges which could endow the molecule with a certain degree of rigidity, and the possibility of a subtle change in shape on cleavage of these bridges, mould not be difficult to envisage in such a structure. An alternating sequence of (4 1 ) a - i ) - and (3 + I)-a-D-glucosidic links has also been shown to occur in a polysacncharide synthesized by Aspergillus niger,122but little appears to be known about the shape of this molecule. -+
.--)
(121) “Chemistry and Industry of Starch,” R . W. Kerr, ed., Academic Press Inc., New I7ork, N. Y.,2nd Edition, 1950,pp. 170, 185. (122) S. A. Barker, R. J. Bourne and M. Stacey, J . Vltem. Soc., 3084 (1953).
THE CHEMISTRY O F HEPARIN
307
An interesting sulfated polysaccharide which has anticoagulant activity has been described by Marbet and Winterstein.33 They reported the isolation of “@-heparin”from the byproducts obtained in the preparation of heparin from beef lung. It was found that &heparin had (1) a molecular weight of ca. 16,000 (from viscosity measurements) , (2) predominantly p-D-glucosidic linkages, (3) uronic acid and 2-acetamido-2-deoxy-~-galactose (N-acetylchondrosamine) in 1 : l ratio, and (4) 2 sulfate ester groups per tetrasaccharide unit. Chemically, 8-heparin is closely related to chondroitinsulfate, but it has a much higher activity (ca. 25 % of that of heparin). The facts that the &heparin molecule is of approximately the same size as that of heparin and that it has a low sulfate content suggest that its appreciable activity may well be a function of the shape of the molecule. Summarizing, it may be stated that the combination of size, shape, degree of sulfation, and distribution of sulfate groups endows heparin with a specific, anticoagulant activity of a type which may be different from that shown by synthetic, sulfated polysaccharides,l16 and that inactivation appears to take place with relatively little chemical change in the molecule. The sulfation of heparin may well result in its conversion to the class of synthetic, sulfated mucopolysaccharides.
VI. THEBIOSYNTHESIS OF MUCOPOLYSACCHARIDES I n conclusion, it may be of interest to summarize briefly our present knowledge of the biosynthesis of mucopolysaccharides, including heparin. This would appear a t first sight to be more complex than the biosynthesis of poly-~-glucoses~~3 (which is fairly well understood), since more than one sugar species is involved. Hyaluronic acid, one of the mucopolysaccharides, has been the most studied with respect to its biosynthesis, since it is elaborated by certain micro-organisms and is thus readily accessible. Probably, the biosynthesis of individual mucopolysaccharides follows the same broad outline, especially with respect to the elaboration of the amino sugar and uronic acid, so that observations on one mucopolysaccharide may have a general relevance. Some studies have been made on the biosynthesis of chondroitinsulfate in cartilage tissue slices, and it is probably only a matter of time before heparin will be studied in a similar manner. Both D-glucuronic acid and 2-amino-2-deoxy-~-glucoseappear to be incorporated into the mucopolysaccharides through utilization of intact D-glucose, with prior or subsequent oxidation or amination, respectively. This has followed from tracer studies, using ~-glucose-6-C’*and D-glucose(123) S. A. Barker and E. J. Bourne, Quart. Revs. (London), 7,56 (1953); M. Stacey, Advances i n Enzymol., 16,301 (1954).
368
A . B . FOSTER AND A. J. HUGGARD
l-C14, made by Roseman and coworkers,124and by Topper and L i p t ~ n . ~ ? ~ Topper and L i p t ~ n found ~ ? ~ that 2-aniino-2-deoxy-D-glucose and 2-acet%mido-2-deoxy-D-glucose,but not D-glucuronic acid, are incorporated into microbial hyaluronic acid. Thus, D-glucose may be oxidized after its incorporation into the mucopolysaccharide. D-Glucosone may be involved in the reaction sequence which leads to 2-amino-2-deoxy-~-glucose.~~~ Iz6 Thus, D-ghCOSe-l-C14and ~-glueosone-l-C'~ are converted to serum %amino-2-deoxy-~-glucosein rats, but ~ - g l u c o s o n e - l - Cis~ ~the more effective precursor. Lowther and Rogers127have showii that L-glutamine may be involved in the synthesis of 2-amino-2-deoxy-~-glucose in microbial hyaluronic acid. A partially purified, enzyme extract from the mold Neurospora crassa has been found to catalyze the conversion of hexose 6-phosphate to 2-amino2-deoxy-~-glucosephosphate in the presence of L-glutamine.128Phosphorylation of 2-amino-2-deoxy-~-glucoseto give the 6-phosphate has been effected with brain extractlZ9and with crystalline yeast h e ~ o k i n a s e . ~Phospho~" glucomutase from rabbit muscle converts 2-amino-2-deoxy-~-glucose 6-phosphate to the l - p h o ~ p h a t e , ~and 3 ~ 2-amino-2-deoxy-~-glucose 1 ,6-diphosphate may function as a coenzyme in this process, analogously to D-glucose 1,6-diphosphate in the D-glucose series. Similar results have been reported with 2-amino-2-deoxy-~-galactosc.~~ None of these phosphate derivatives have been synthesized by chemical methods. The exchange of acetyl and the uptake of S3504"in chondroitinin slices of cartilage tissue have been studied. The mechanism whereby the backbone chains of the mucopolysaccharides are elaborated is a t present unknown. At least two possible sequences in the biosynthesis could involve stepwise addition of either (1) (alternately) amino sugar and uronic acid or ( 2 ) initially formed disaccharide units which may contain a hexuronic or hexosaminic linkage. I t is, however, clear that, a t this point, a tremendous gap in our knowledge of heparin and other mucopolysaccharides exists. 7
(124) S. Roseman, J. Ludowieg, Frances E. Moses and A. Dorfman, Arch. Biochem. and Biophys., 42, 472 (1953); J. Biol. Chern., 206, 665 (1954); S. Roseman, Frances E. Moses, J. Ludowieg and A. Dorfman, ibid., 203, 213 (1953). (125) Y. J. Topper andM. M. Lipton, J . Biol.Chem., 203, 135 (1953); see also J. F. Douglas and C. G. King, ibid., 202, 865 (1953). (126) C. E. Becker and H. G. Day, J . B i d . Chem., 201, 795 (1953). (127) D. A. Lowther and H. J. Rogers, Biochem. J . (London), 63, xxxix (1953). (128) L. F . Leloir and C . E. Cardini, Biochim. et Biophys. A c t a , 12, 15 (1953). (129) R. P. Harper and J. H. Quastel, N a t u r e , 164, 693 (1949). (130) D. H. Brown, Biochim. et Biophys. Acta, 7, 487 (1951). (131) D . H. Brown, J . B i d . Chem., 204, 877 (1953). (132) H. Bostrom and B. Mansson, Acta Chem. Scand., 6, 1559 (1952). (133) H. Bostrijm and S. Aqvist, Acta C h e w Scand., 6, 1557 (1952) and references cited therein.
Author Index Numbers in parentheses are footnote numbem. They are inserted to indicate the reference when an author's work is cited but his name is not mentioned on the page.
A Abdel-Akher, M., 277(34) Abeles, R.H., 75 Abramovitch, R.A. A,, 217 Adams, G. A., 318 Adler, O., 128(127), 136(127), 153(127) Ahmed, Z . F., 280(36) Aichner, F. X., 214,251(43), 254(43) Alberda van Ekenstein, W., 30 Albon, N., 64,73,74 Alder, K., 16,21(78) Alesander, B.H., 278(42) Algar, W. H., 331,332(217), 333(217) Allerton, R.,131(135), 147(135) Allison, J. B.,221,354 Alm, R. S.,63 Amadori, M.,97, 142(7, l73), l43(8),
146(173), 147(9), 170,171,175,176(3), 178(3' 5)' lS5' 189(4' 5' 6)' 195(4)1 203(3, 4, 5) Anand, N., 131(136b), 132(136b), 134 (136b) Andersen, C. C., 36 Anderson, A. B.,275(22), 280(22), 317 Anderson, A. S.,265(71) Anderson, C. G., 31 Anderson, E.,286,287,302,312,313(76, 77,79) Anderson, N. G., 360 Anderson, R. H., 171, 174, 175(15, 29) 176(29), I88(28), 204(28) Andreasen, A. A,, 86 Andrews, P., 71, 72, 90(69, 70, 71), 128 (122), 133(149), 163(122), 262(53), 263(53), 264(53), 266(110), 267(110), 275(15), 278(15), 279(15, 60,61), 289
Annison, E. F., 355 Anno, K., 40, 50(198a), 87 Antalti, H., 127(116), 137(116), 140(116), I42 (116), I43(1 16), 162(116) Ant-Wuorinen, O., 315
% ;:! &',;.
v., 159(214)
Appel, H., 28 Appling, J. W . , 306 Aqvist, S.,368 Armstrong, E.F., 210, 211,213(13), 229, 240, 247(19), 252(19) Armstrong, K' F'*240 Arni, 1'. C., 91 Arth, G. E., 43 Asahina, Y., 35 Asrhner, T. C., 7 91, 163(226,2277 230)~ Aspina11,G . O',
257(3), 258(3, 23, 12), 259(23), 263 (23), 264(23), 275(16), 278(16), 279 (16),289,303 Astbury, W. T., 307,309(113), 329(113) Aston, J. G., 11, 12(57) Astrupy T'y 3403 3479353(25)9 3597 362* 365(59) Atchison, J. E., 317 AudriethJ L' F'l 354 Auerbach, I., 90 Avery, J., 268(94)
B Babers, F. H., 247(216,217) Bachrach, J.,63,86,260(39) Bacon, J. S.D., 64,276(27, 28) Bachli, P., 254(275) Baer, H.H., 64,252(258) Bailev, J. M., 64 Angel], S.,304 Angyal, S. J., 2, 12(5), 14(5), 15, 16(5), Bair, I,. R., 63,70(24), 71(24) 20(68), 28, 37(5), 47, 48(190), 228, Baker, J. L.,290 253(130) Baker, J. W., 138(161, 162), 141(161,
369
370
A4UTHOR INDEX
162), 143(161, 162), 145(161, 162), 147(161), 148(162), 149(193) Baker, S.B., 23, 47 Baker, W., 267(92), 268(92) B a l d e h , E. R., 194 Baldwin, E., 275(19, 75), 278(75), 280(19) Ball, D. H., 71, 128(122), 163(122), 262 (53), 263 (53), 264 (53) Ball, J., 344 Ballou, C. E., 229 Balston, J. N., 56, 70(7) Barclay, J. L., 106 B&rczai-Martos,M., 214 Barker, C . C., 163(233), 259(26) Barker, G. R., 160(215), 265(68, 69, 71) Barker, S. A , , 3, 12, 13(58), 24(58), 25 (15, 18), 26,27(58), 28(15, 18), 29(15), 30(15), 31(58), 36(58), 37(15), 38(15, 58), 39(15, 58), 40(15), 44(58), 46(15) 89,91, 110, 145(183), 150(60), 156(60), 358, 366, 367 Barrett, E. P., 56 Barrett, F. C . , 56, 70(7) Barrett, J. W., 17, 22(81) Barry, C. P., 99, 102(24), 113, 115(24), 130(24), 208, 217(8) Bartlett, J. K., 90 Barton, D. H. R., 2, 7(4), 12(4), 14, 15 (1,6, 65), 18(4), 21, 29, 53 (92) Bashford, V. G., 9, 10, 23(43, 51), 48, 49 (193) Bastiansen, O., 18, 19(83) Battenberg, E., 311 Bauerlein, K., 246(201) Baur, L., 251(244), 319 Bayly, R. T., 121 Bayne, S., 101, 102(33), 134(33), 139(33), 140(33), 141(33), 145(33), 146(38), 180, 181(50), 185(50), 203(50) Beadle, C., 320 Beaven, G. H., 262(95), 263(95), 264(95), 268 (95) Beck, F., 223, 227(97) Becker, C. E., 368 Beckett, C . W., 2, 12(1), 14(1) Beevers, C. A., 10, 13(54), 16(54), 231 Behrend, R., 229 Bell, D. J., 64, 89, 90, 110, 145(58, 181), 273(1, 4), 274(1, 77), 275(1, 19, 23, 75, 77), 276(1, 4, 23, 27, 28), 277(23), 278(4, 75), 280(19, 65)
Bell, F. K., 343 Bell, H. J., 365 Bellars, A. E., 130(132), 149(132), 156 (132) Bels, W., 306 Benjamin, D. G., 184 Bennett, E., 318 Bennett, E. C., 302,313(79) Berend, G., 32 BerezovskiI, V. M., 152(197a) Berg, S., 252(258) Berger, L., 98, 102(14, 15), 110, 111, 116, 124, 126(15), 159(14, 15), 160(14, 15), 343 Berglund, E., 355 Bergmann, A., 172, 187, 188(18), 189, 197 (18), 198, 200(1S) Bergmann, M., 219, 223, 227(97), 228, 247 (136), 251(239), 253(128, 266, 270) Bergmann, W., 127(114), 136(155), 140 (155), 1481155), 151(114) Bergstrom, S., 339, 342, 351(17), 352, 365 (79) Berkebile, J. M., 87 Berlenbach, W., 136(154), 149(154) Berner, E., 181 Bernoulli, A. L., 215,248(50) Bertho, A , , 112, 129(64), 131(136a), 137 (64, 136a), 153(64), 162(64) Betti, M., 132(140), 155(140), 157040) Bevan, E. J., 320 Beyler, R. E., 43 Bhattacharyya, A. K., 51, 53(201) Bickell, L. K., 324 Binkley, W. W., 33, 56, 65, 67, 68, 69, 70 (7, 49), 83(41), 84,87, 88, 295 Birkofer, L., 98, 101(19), 104, 128(19), 130(19, 132(19), 141(19), 143(19), 146(19), 149(19), 151(In), 156(19), 171, 176(13), 177(13), 186, 188(13), 191(13), 192, 200(13), 201, 203(13), 205(13) Bjorkqvist, C., 314 Blair, M. G., 67, 87, 229 Blanchard, P. H., 64 Blindenbacher, F., 93 Blix, G., 346 Bloch, H., 134(152a), 139(152a) Blomback, B., 362 Blombiick, M., 362 Bloom, P., 321
37 1
AUTHOR INDEX
Bodnrt, A , , 213 Boeseken, J., 21, 75, 231 Bottger, S., 225 Boggs, L. A., 62, 70(24), 71(24) B o g d r , R., 100, 128(124), 129(124), 131 (124), 134(124), 138(124), 139(124), 143(124), 146(124), 151(124), 153(124), 156(124), I64 (124), 249(233) Bohn, E., 241 Boissonnas, R. A , , 82 Bolliger, H. R., 92, 93 Bolomey, R. A,, 79 Bonhoeffer, K. F., 106 Bonner, W. A , , 215,245(56), 248(227), 253 (227) Bose, A. K., 16,51,53(201) Bostrom, H., 346, 353(54), 354, 355, 364 (54), 368 Bott, H. G., 266(74) Bottle, R. J., 346 Bourjau, W., 247(207), 248(207), 251(207) Bourne, E. J., 3, 12, 13(58), 24(58), 25(15, 18), 26, 27(58), 28, 29(15), 30(15), 31 (58), 36(58), 37(15), 38(15, 581, 39 (15,58), 40(15), 44,46(15), 89,91,110, 121, 145(183), 150(60), 156(60), 358, 366, 367 Bourquelot, E., 291 Bower, R. S., 77,88(103), 94 Brading, J. W. E., 268(93) Bratt, L. C., 285, 291, 292(38) Braun, C. E., 146(188) Braun, E., 21, 141(171), 248(226) Brauns, D. H., 210,212, 215,216,219,223, 231, 246(14, 21, 22, 49, 79, 100, 205), 247(49,64,219), 248(220,231), 249(21, 100, 205), 251(243), 253(14, 79, 220), 254(21, 100, 205), 256(21, 49, 79, 100, 205) Brauns, F. E., 326 Breddy, I,. J., 306 Bredereck, H., 225, 242, 246(203), 247 (203) Brewster, J. H., 2, 12(8), 13(8), 14(8), 17 (8)I 20 (8) Brewster, P., 9,49(46) Brice, C., 219 Brigl, P.,33,115,121,136(91,94),137(91), 148(94), 154(94), 217, 219(67), 222, 242 (67), 243 (67), 248 (67), 249 (69), 252 (246)
Brimley, R. C., 56,70(7) Bristow, N . W., 244, 251 (237) Brockmann, H., 79 Bromund, W. H., 148(189a), 149(189a) Brown, A. H., 76 Brown, D. H., 349, 368 Brown, F., 89, 158(208), 260(50), 262(50), 263(65), 264 (50, 65), 267 (83), 273(6), 275(6, 17), 278(17) Brown, G. B., 244 Brown, H. C., 2,12(8), 13(8), 14(8), 17(8), 20 (8) Brown, H. P., 321 Brown, R. L., 200 Browne, C. A., 303, 305(83), 306(83) Browning, B. I,., 286, 291, 292(39), 303, 306, 312,314,315, 318,321 Bruce, G. T., 44,89 Bruckner, Z., 113, 129(67, 68), 219 Bublitz, L. O., 318 Bublita, W. J., 318 Buchanan, J. G., 244 Biichi, J., 28,148(190), 245 Bulen, W. A., 62, 70(24), 71(24) Burgess, H., 325 Burke, W. J., 43 Burkhart, B., 319 Burrell, R. C., 63,91(29) Burton, H., 356 Butler, K., 102, 105(40), 111, 116(62), 132 (40), 133(40, 62), 134(40, 62), 146(40), 158(209), 159(40), 160(40), 161(40), 163(40), 243, 267(108) Butler, M. J., 354 Bywater, R. A. S., 259(25), 260(25) C
Cadenbach, G., 225 Cahill, J. J., 141(172), 159(172) Cahn, R. S., 5 Calderbank, K. E., 15 Cameron, A., 279(54) Cameron, C. N., 137(160), 180, 195(46, 47), 201 Campbell, I. G. M., 16,21(79) Campbell, R., 240 Campbell, W. G., 133(148) Candela, G. A., 79 Cannell, J. S., 140(170d,e) Cantor, S. M., 248(227), 253(227) Cardini, C. E., 350, 368 Carlander, A. T., 62, 70(24), 71(24)
372
AUTHOR INDEX
Carney, D . M., 81 Carrington, T. R., 91 Carruthers, A. E., 160(32) Carson, J. F., 175 Cassidy, H. G., 56,70(7) Castagne, A. E., 318 Castan, P., 247(219) Castle, F. J., 63, 70(24), 71 (24) Cavalieri, L. F., 149(19Oh), 176, 189(35, 36) Cnvallini, G., 134(1521)), 150(152b), 151 (152b) Challinor, S. W., 277(39) Chanda, S. K., 90, 92, 163(229), 258(19, 20), 260(33, 34), 299,300 Chandler, L. B., 118, 137(85) Chang, P., 244 Chapman, N. B., 239, 243(164) Charalambous, G., 151(196a), 158(196a) Chargaff, E., 344 Charles, A. F., 338,339,340,341,342,343, 346, 347, 352, 353(22), 364(13), 365 Charlton, W., 31, 279(73), 280(73) Chase, E. C., 126(108), 152(108) Chatterjee, A., 16 Chaudhuri, D . K. R., 51,53(201) Chavassieu, H. L. J., 187 Cheniae, G. M., 76 Christman, C . C., 219, 267(85) Chu, C.-C., 73 Claesson, P., 248(229) Claesson, S., 58, 61, 63(21), 70 Clark, E. P., 253(267) Clark, G. L., 73 Claus, A., 114 Claus, W. H., 132(139), 171, 176(9) Cleveland, E. A , , 105, 137(46), 138(46), 141(46), 142(46), 144(46), 149(46), 151 (46), 153(46), 180 Cleveland, J. H., 105, 137(46), 138(46), 141(46), 142(46), 144(46), 149(46), 151 (46), 153(46), 180 Cochran, W., 10, 13(54), 16(54) Cocker, W., 189 Cohn, W. E., 76 Cole, W., 190 Coleman, G. H., 80 Collatz, H., 252(249), 254(249), 256(249) Colley, A , , 215 Compton, J., 32, 251(238, 242), 265(105), 266(79), 267(79)
Conchie, J., 260(102) Connell, J. J., 145(183), 278(45) Conrad, C. C., 330 Conrad, H. E., 64, 72, 259(30), 267(89), 277(37), 279(37) Consden, R., 70, 285, 292(4) Conway, H. F., 56 Cook, A. H., 17 Cook, E. W., 115, 218(232), 249(232), 253 (232) Cooke, R. G., 186 Cookson, R. C . , 2,8(13), 16,21,52(13), 53 Cooper, P. W., 87 Cope, A. C., 179 Corneliusson, E. V., 362 Cortese, F., 251 (240), 252(240) Cottrall, L. G., 310 Couch, D. H., 105, 137(46), 138(46), 141 (46), 142(46), 144(46), 149(46), 151 (46), 153(46), 180 Councler, C., 305 Counsell, J. N., 72 Couvert, H., 231 Cox, E. G., 273(2), 277(2) Cramer, F. B., 222,227(95) Cremlyn, R. J. W., 10, 19(50) Criegee, R., 294 Cron, M. J., 342 Cross, C. F., 320 Csendes, E., 188 Csuros, Z., 113, 129(67), 228,247(131), 253 (130) Cuculescu, V., 143(178) Cuendet, L. S., 56, 62, 70(24), 71(24) Cunneen, J. I., 264(60)
D Dacons, J. C., 84, 88(127), 295 D’Addieco, A. A , , 309,317(119) Dale, J. K., 215,229,247(135) Danehy, J. P., 96 Dansi, A., 99, 110, 113, 140(169), 141(27), 147(27), 148(27), 170, 175, 185(7), 186, 188(7), 189, 191(7), 193, 195, 196, 199, 200, 203(7) Davidson, A. L., 321 Davies, G. F., 19 Davoll, J., 119,212,228,244,245,248(24), 251 (135) Day, H. G., 368 de Cholnoky, L., 65, 83(40)
373
AUTHOR INDEX
Dedonder, R., 89 Deferrari, J. O., 123, 124, 131(101), 136 (loo), 137(100), 154(101) Deriae, R. E . , 160(217), 161(217), 210, 214(12) Derx, H. G., 21 Deulofeu, V., 121, 123, 124, 126(99), 131 (101), 136(100), 137(100), 154(101) Dewar, E . T., 133(145), 274(24), 275(24), 276(24), 279(24) d e Whalley, H . C. S., 73 Dhat, &I. L., 131(136b), 132(136b), 134 (l36b) Dickey, E. E., 84, 88(127), 295 Dickson, A. D., 351 Diehl, H. W., 20,23, 44, 239 Dienes, M. I,., 294 Dillon, R. T., 6 Dimler, R. J., 2, 21(12), 56, 63, 70(7, 24), 71 (24), 278 (42) Distelmaier, A., 32, 36(134), 268(100), 269(100) Dittmer, K., 244 Dixon, K . C., 187 Doczi, J., 363 Dodgson, K. S., 356 Doering, W. von I-, 130 -, 2-O-methyl-~-,268 -, N-(p-hydroxypheny1)-D-, 99, 130 -, 3-O-methyi-o-, 268 -, N-(p-methoxypheny1)-D-, 99 phenylosazone, 268 -, N-phenyl-D-, 99, 124, 130 -, S-O-methyl-~-,269 preparation of, 97 phenylosaxone, 269 reaction with hydrogen cyanide, 102
402 -, tetra-0-acetyl-a-L-,88 -, 2,3,4-tri-O-methyl-~-,268
SUBJECT INDEX
G
Galactan, 284 hydrate, 268 of larch, 72 -, 2,3,4-tri-O-methyl-~-,269 of wood, 326 hydrate, 269 Galactaric acid, 313 Fucoside, methyl 3,4-di-O-rnethyl-a-~-, -, 2-O-methyl-, diamide, 273 -, 2,3,4-tri-O-methyl-, 277 269 - , methyl 3,4-0-isopropylidene-2-0- dimethyl ester, 277 Galactitol, 76, 174 methyl-I)-, 268 -, methyl 3,4-0-isopropylidene-2-0- -, I-amino-1-deoxy-u-, 205 -, 1,4-anhydro-~-,245 methyl-O-L-, 269 27 -, methyl 2,3,4-tri-O-methyl-8-~-, 268 -, 1,2:4,5-di-O-isopropylidene-~~-, -, methyl 2,3,4-tri-O-methyl-a-~-, 269 Galactofuranose, 1,2-O-isopropylideneD - , 33 p anomer, 269 Galactofuranoside, methyl 6-0-methylFucosylamine, A-n-butyl-L-, 130 @-I-, 274 -, N-n-heptyl-L-, 130 Galactomannans, 72, 90 -, A'-n-hexyl-L-, 130 Galactonic acid, D - , amide, 77 -, N-methyl-L-, 130 -, 2,4-di-O-methyl-~-,amide, 275 -, N-1-pentyl-L-, 130 lactone, 275 -, N-phenyl-D-, 2,3,4-trimethyl ether, phenylhydrazide, 275 130,268 -, 2,6-di-O-methyl-~-,276 -, N-phenyl-L-, 130 amide, 276 lactone, 276 2,3,4-trimethjd ether, 130, 269 phenylhydrazide, 276 -, N-n-propyl-I,-, 130 Fuller's earth clay, adsorption of carbo- -, 3,4-di-O-rnethy-o-,amide, 276 lactone, 276 hydrates on, 66 -, 4,6-di-O-niethyl-1-, amide, hydrate, use in column chromatography, 64 276 2-Furaldehyde, compounds resembling, lactone, 276 305 -, 2-O-methyl-u-, amide, 273 conversion tables, for phloroglucinol lactone, 273 compound, 305 -, B-O-methyl-~-,274 determination of, 305 lactone, 274 colorimetric methods, 306 phenylhydrazine salt, 274 gravimetric methods, 305 -, 2,3,4,6-tetra-O-methyl-~-, 280 reagents for, 305 amide, 280 volumetric methods, 305 lactone, 280 from oxycellulose, 304 phenylhydrazide, 280 from pentosans, 303 -, 2,3,5,6-tetra-o-methyl-~-, amide, 280 from nronic acids, 304 lactone, 280 -, 5-hydroxymethyl-, 180, 192, 193, 305, -, 2,3,4-tri-O-methyl-~-, 277 306 amide, 277 -, methyl-, 305,306 lactone, 277 Furan, 2-hydroxyacetyl-, 191 phenylhydruzide, 277 -, tetrahydro-, 13, 24 -, 2,3,5-tri-O-methyl-~-,amide, 278 Furfuraldehyde. See 2-Furaldehyde. lactone, 278 Puroic acid, 306 phenylhydrazide, 278
SUBJECT INDEX
403
-, 1,6-anhydr0-2,3,4-tri-O-meth3.l-p-i)-, amide, 278 277 Iactone, 278 -, I , 2 : 3 , 4 - d i - O - i s o ~ ~ r o ~ ~ y l i d e32, n e 33 -~-, phenylhydrazitle, 278 -, 2,3-di-O-met,hyl-p-i)-, 274 -, 2,4,6-t,ri-O-met,hyl-~-,amide, 278 -, 2,4-di-O-methyl-ou-u-,275 lactone, 278 nionohydrate, 275 -, 3,4,6-tri-O-methyl-u-, Iactone, 279 -, 2,6-di-O-methyl-p-~-,275 Galactopyrunose, 1,6-anhydro-&~-,243 monohydrate, 276 322 Galactopyranoside, methyl @-I-, -, 3,4-di-O-methyl-P-i~-,276 -, methyl 2,6-di-O-rnesyl-a-~-,25 -, 4,6-di -0-methyl-01- I) -,276 -, methyl 2,3-di-O-methyl-a-~-,274 hydrate, 276 4,6-O-benzylidene acetal, 275 phenylosazone, 276 p anomer, 275 -, 1,2:3,4-di-O-methylene-n-, 33 -, methyl 2,6-di-O-methyl-P-~-,276 -, hept,a-O-acetyl-alde~i~(lv-i~-, 86 3,4-O-isopropylidene acetal, 276 -, 2-O-methyl-p-~-,273 01 anomer, 276 -, 3-O-methyl-o-, 72 -, methyl 2-O-methyl-01-~-, 273 01 anomer, 274 4,6-0-benzylidene acetal, 273 phenylosaeone, 274 3-tosylate, 273 -, 4-O-methyl-P-o-, 274 3,4-0-isopropylidene acetal, 273 phenylosazone, 274 6-tosylate, 273 -, 6-0-methyl-cr-o-, 274 -, methyl 2-O-methyl-p-o-, 273 phenylhydrazone, 274 4,6-0-benzylidene acetal, 273 phenylosazone, 274 3-tosylate, 273 -, penta-0-acetyl-D-, a! and P anomers, 3,4-O-isopropylidene acetal, 273 230 -, methyl 3-O-methyl-p-r)-, 274 -, 1,2,3,4,6-penta-O-acetyl-u-, 21 1 4,6-dibenzyl ether, 274 -, penta-0-acetyl-aldehydo-v-, 124 a anomer, 274 -, 2,3,4,6-tetra-o-methyl-v-, 91 Galac topyranosylamine, 2,6-di-001 anomer, 279 methyl-N-phenyl-D-, 276 @ anomer, 279 -, 2-0-methyl-N-phenyl-o-, 273 -, 2,3,5,6-tetra-O-methyl-~-, 280 Galactopyranosyl chloride, W D - , 247 -, 2,3,4-tri-O-acetyl-l,6-anhydro-D-o-, -, 6-0-(~-z-rhamnopyranosyl)-cr-o-, 228 hexaacetate, 249 -, 1,2,4-tri-O-acety1-6-deoxy-3-0Galactosamine, I>-, See Galactose, 2methyl-D-, a! and p anomers, 92, 93 amino-2-deosy-~-. -, 2,3,4-tri-O-methyl-1-, 91 Galactose, v-, 64, 66, 77, 174, 318, 322 01 anomer, 277 I-diacetamide derivative, 124, 131 monohydrate, 277 pentaacetate, 131 -, 2,3,4-tri-O-methy1-6-O-trityl-r-, 277 1-dibenzamide derivative, 131 1-acetate, 277 methyl ethers of. See tables on pages -, 2,3,5-tri-O-methyl-~-,278 679 to 680. -, 2,3, g-tri-o-methyl-~-, 278 pentaazoate, 01 and @ anomers, 80 -, 2,4,6-tri-O-methyl-o-~-, 278 -, 2-acetarnido-2-deoxy-o-, 367 -, 3,4,6-tri-O-methyl-~-,279 1,2-0-isopropylidene acetal, 279 -, 2-amino-2-deoxy-o-, 72, 73, 350, 368 Galactoseen-l,2, 2,3,4,6-tetra-Ohydrochloride, 66, 76 acetyl-D-, 229 -, 1,6-anhydro-B-~-,51 -, 1,6-anhydr0-3,4-0-isopropylidene-2-Calactoseptanosyl chloride, 2,3,4,5tetra-0-acetyl-P-D-, 247 0-methyl-D-, 273 -, 2,3,6-tri-O-methyl-o-, 278
403-
SUBJECT INDEX
Galactoside, methyl 3,G-anhydro-2-0- -, N-o -carbomet hoxyphen yl -D -, 131 iiieyyl-a-u-, 25 -, N-(.l-carboxy-3-hydroxy~lie1i~~l)-u-, -, methyl 4,G ~ - b e l l Z ~ ~ i d e l l e - a -43, D -44 , 131 B anomer, 43, 44 -, N-o-cait~oxyphenyl-~-, 98, 131 -, methyl 2 , 4-di -0-methyl -01 -u -, 275 -, N-p-carhoxyphenyl-D-,131 anomer, 275 -, 2 deoxy-N-phenyl-u-, 132 -, methyl 3,4-di-O-iiiethyl-p-u-,276 -, 2-deoxy-N-p-tolyl-u-, l O G , 134 -, methyl 4,6-di-O-methyl-p-~-,276 -, N , N-diacetyI-2,3,4,5,6-penta-O2,3-dibenzyl ether, 276 acetyl-1,-, 131 2,3-ditosylate, 276 -, N - (3,4-dimethyl-2-nitrophenyl) -I)-, -, methyl tetra-0-methyl-D-, 89 tetraacetate, 131 -, methyl 2,3,4,G-tet rn-0-met hyl-a-r)-, -, N - (4,5-dimethyl-2-nitiophenyl) -w, 280 tetraacetate, 131 nnomer, 280 -, 2,3-di-O-methyl-N-l,henyl-a-, 274 -, met hy1 2,3,5,G-t etra-0-methyl-w, p anomer, 133 280 -, 2,4-di -0-methyl-N-yhenyl-u-, 275 -, methyl 2,3,4-tri-O-methyl-a-~-,277 p anomer, 133 -, methyl 2,3,5-tri-O-methyl-~-,278 -, 2,6-di -0m e t h y l -N-phenyl-p-u -, 133 -, methyl 2,4,6-tri-O-rnethyl-a-~-, 278 -, 4,G-di-O-methyl-N-phenyl-~-, 276 hydrate, 278 0 anomer, 133 3-tosylate, 278 -, N-p-(p-dodecylaminobiphenylsul-, methyl 2,4,6-tri-O-methyI-B-~-, 278 fone)-D-, 132 hemihydrate, 278 -, N-(N-n-dodecylcarbarnoy1)-o-,132 3-tosylate, 278 -, N-p-ethoxyphenyl-n-, 132 -, methyl 2,3,4-tri-O-methyl-6-0-trityl-, N-p-(p-ethylaminobiphenylsu1fone)D-, 277 D-, 132 Galactosiduronic acid, ~-rhamnose-(2+ -, N-n-heptyl-D-, 132 1) D-, 72 -, N - p - (p-hexylaminobiphenylsulfone) Galactosiduronic acids, di- and tri-, 91 D-, 132 Galactosylamine, D - , 108, 131 -, N-1-(2-hydroxynaphthyl)-N-phenyla - ~ -ammonia , complex, 131 methyl-D-, 132 B-D-, 131 -, N-p-(p-isobutylaminobiphenylsulacetylation of, 111 fone)-D-, 132 tetraacetate, 131 -, N-p-(p-isopentylaminobiphenylsul-, N-acetyl-a-o-, 131 fone)-D-, 132 periodate oxidation of, 118 -, N-(4-methyl-2-nitrophenyl-o-, 132 tetraacetate, 131 -, 2-O-methyl-N-phenyl-B-~-,133 -, N-acetyl-p-D-, 113, 131 -, 4-O-methyl-N-phenyl-~-,274 periodate oxidation of, 118 p anomer, 133 tetraacetate, 131 -, N J - n a p h t h y l - ~ - 132 , -, N-acetyl-2,3,4,6-tetra-O-acetyl-a D-, -, N-o-nitrophenyl-D-, tetraacet ate, 132 111 -, N-p-nitrophenyl-p-D-, 132 p anomer, 111 tetraacetate, 132 deacetylation of, 113 -, N-p-(p-octy1aminobiphenylsulfone)-, N-acetyl-tetra-O-acetyl-N-p-nitroD-, 132 phenyl-pa-, 132 -, N-1-pentyl-D-, 132 -, N-n-butyl-D-, 131 -, N-p-(p-penty1aminobiphenylsulfone)-, N-p-(p-1iutylaminobiphmylsulfone)D - , 132 D-, 131 -, N - p h e n y h - , 113, 124, 134 -, N-carbamoyl-D-, 131 acetylation of, 111
SUBJECT INDEX
405
-, 3 , 4 , 6 - t r i - O - acet y l - ~ - 248 , p anomer, 132, 133 2 trichloroacetate, 248 preparat,ion of, 97 -, 3,4,6-t,ri-O-acet yl-2-chloro-2-deoxyreaction wit.h hydrogen cyanidt., 102 LY-D-, 247 -, N-(4-phenylaeophenVlene)-1~-,132 -, 2,4,6-tri-O-iiieth\~I-3-0-tosyl-a-~-, -, N-n,-propyl-D-, 134 247 -, N-p-(p-propylaminohiphenylsul- Galactosyl phosphate, I > - , 350 fone)-o-, 134 -, 2-amino-2-deoxy-~-,350 -, N-p-sulfamylpheiiyl-D-, 134 Galacturonic acid, 313 monohydrate, 134 D - , 64, 76 -, N-p-sulfopheiiyl-u-, 134 LY anomer, 66, 77, 78 -, 2,3,4,6-tet ra-O-acet).l-N-pheiiyI-Wu-, of aspen-wood hcmicellulose, 286 D - , 13-1 in beech wood, 301 p anomcr, 133 -, 2,3,5-tri-O-acetyl-l -hromo-1-deoxy-, 2,3,4,6-tet,ra-O-mcthyl-~-,135 LY-D-, methyl ester, 251 -, tetra-O-methyl-N-phenyl-p-n-, 133 Gentiobiose, 66, 77, 87, 223 -, 2 , 3 , 4 , 6 - t etra-O-met hyl-N-phengl-n-, LY , azoate, 80 279, 280 -, octa-0-acety-p-, 83 p anomer, 133 synthesis of, 241 -, N-f-tolJTl-D-,134 Gentiobioside, maridelonitrile p-, 240 -, N-p-tolJrl-D-, 98, 134 Gentiobiosylamine, N-p-nitrophenyl-8-, hydrolysis of, 105 135 -, 2 , 3 ,4-t,ri-o-methyl-N-p~iciiyl-o-,277 heptaacetate, 135 p anomer, 133 Centiobiosyl bromide, hc.pta-0-acetylL Y - , 236, 237, 240, 254 6-trityl ether, 133 -, 2,4,6-tri~O-met,hyl-N-phenyl~r)-, 278 -, hexn-O-acetyl-6’-bromo 6’-deoxy-a-, 6 anomer’, 133 254 Galact osyl bromide, 2,3,4,6-t et r a - 0 - Gentiobiosyl chloride, hepln-0-acetylacetyl-ol-D-, 242, 243, 251 a-,249 methitnolysis of, 236 Gentiobiosyl fluoride, 246 reaction of, with quinolime, 242 heptaacetat e , 246 with secondary amines, 239 -, 2‘, 3’, 4‘, 6’-tet ra-O-ilcetyl-2,3,4-triwith t.rimet,hylamine, 111 0-benxoyl-. 246 -, 2,3,5,6-t.et.ra-O-acet.yI-~-~-~ 211, 251 (:entiol)iosyl halidcs, poly-0-acetyl-, -, 2,3,4-t~ri-O-acetyl-6-brorno-6-~lcoxy- 232 a - ~211, , 251 (>entiobiospl iodidc, ~iept:L-O-acctyl-c-, -, 2,3,5-t ri-0-ace1 yl-6-bl.oino-6-ileosy256 @-I-, 251 Glucal, tri-O-acetyl-i)-, 228 -, 3,4,6-t ri-0-ace1 yl-2-bl.oriio-2-tlt:osy- Glucamiiie. Scr Glucitol, 1 arnino-la - ~251 , dcoxy-. -, 2,3,4-t ri-O-:tcet )I-6-0-t osyl -LY-D-, 251 (>Incam, from bntleu, !)1 G:tlartosyl chloride, 2,3,4,6-tetrit-0hiosynthesis of, 367 : i c e t y l - ~ - ~247 -, of I)irch, 327 p iinomer, 247 of pine, 327 -, 2 . 3 , s ,n-tetr:L-O-:~CPtyl-D-,230 in sulfate pulps, 327 p itnoini’r, 234, 247 in sulfite pulps, 323, 327 retlnrtion with lithirim alumininn h y the t e r m , 327 tlridr, 245 of M nods, 2% -, 2 , 3 , 4 - t t ~ i - O - n c ~ t y I - ~22S, - i ~ - 2/17 , Glno:iric arid, D-, 351 6-tosplate, 247 potassium :wid salt, 66, 286, 314, 351 a anomer, 134
406
-,
SUBJECT INDEX
Glucofuranosylamine, N-acetyl-o-, sup2,4:3,5-di-O-met hylene-I)-, 39 epimerization of, 41 posed, 119 Glucoheptonic acid, hexa-0-acetyl-"a"formation of, 42 D- , nitrile, reaction with ammonia, Glucitol, D-, 66, 76, 77 118 acetate, 86 Glucomannan, of Amorphophallus konhexaacetate, 88 1,3:2,4:5,6-triacetals, 39 j a c , 289 Gluconic acid, D-, 66, 314 -, 1-amino-1-deoxy-D-, 104, 205 amide, 77 N-substituted, 104 -, 1,4-anhydro-~-,67 ammonium salt, 66 1,4-lactone, 67, 314 6-tosylate, 9 1,5-lactone, 67 -, 1,5-anhydro-~-,245 sodium salt, 66 -, 3,6-anhydro-~-,66 -, %amino-2-deoxy-~-,351 -, 1-deoxy-o-, 66 -, 2-deoxy-~-,66 -, 2,4 :3,5-di-O-methylene-u-, 39 -, penta-0-acetyl-D-, 84 -, 1-deoxy-1-piperidino-u-,192 nitrile, 123 -, 1-deoxy-1-p-tohidino-D-, 199 -, 2,5-diamino-l,4:3,6-dianhydro-2,5-Glucopyranose, a-D-,120 W D- , crystal structure of, 231 dideoxy-o-, 48 -, 1,4:3,6-dianhydro-~-,9, 46, 67 0-D-, acetate, 86 D-, and derivatives, infrared absorp2,5-ditosylate, 47, 48 tion spectra of, 358 -, 1,5:3,6-dianhydro-2,4-O-methyleneD-, 23 -, 2-amino-2-deoxy-~-, infrared rtbsorption spectra of derivatives of, -, 2,4: 3,5-di-O-benzylidene-l,6-di358 h o m o - l ,B-dideoxy-~-, 42 -, 1,6-dichloro-l,6-dideoxy-2,4:3,5- -, 1,6-anhydro-p-u-, 22, 49, 51, 87, 227 preparation of, 243 di-0-methylene-o-, 42 triacetate, 223 -, 2,4 :3,5-di-O-methyIene-o-, 39 tribenzoate, 228 acetolysis of, 40 trimethyl ether, 228 oxidation of, 42 -, 6-O-(or-D-galactopyranosyl)-p-~-. See 1,6-ditosylate, 42 Melibiose. -, 2,4-0-methylene-~-,3, 29 oc-, 1,3,5-tri-O-azoyl-2,4,6-tri -0-methyl- -, 6-O-(p-D-galactopyranosyl)-p-~-, taacetate, 218 D - , 82 OCta-, 1,4,5-tri -0-azogl-2,3,6-tri-O-methyl--, 3-0-(or-D-glUCopyraKloSy1)-p-D-, acetate, 87 I)-, 82 -, 1 , 3 :2,4:5,6-tri -0-benzylidenc-u-, 2s -, tctra-0-acetyl-a-r-, configuration and structure of, 231 -, 1,3:2,4:5,6-tri-O-ethylidene-~-, 28 -, 1,2,3,6-tetra-O-acety1-8-1,-, 241 Glucofuranose, a - u - , I,a-acetals of, 31 -, tetra-0-methyl-D-, 221 -, 1,6-anhydro-p-~-,22 -, 6-O-henzoyl-l , 2-0-isopropylidene-I-> Glucopyranoside, (substituted) o-hydroxyacetophenone I>-, 240 225 -, 3-hydroxypropyl WD-, 66 -, 1,2-O-henzylidene-a-~-,35 -, methyl cu-u-, 67, 77 -, 1,2-O-isoprop~lidene-3-O-tosyl-r,-, p anomer, 67, 322 225 hydrolysis of, 349 -, 1,2-0-isopropylidrn~~ 6 O-tOS).l-D-, -, methyl 2-amino-2-deoxy-a-~-, 349. 23,225 358 Clurofurnnositlr, methyl 3,G-anhytlrop anomrr, 349, 358 a-u-, 3 -, methyl 3,g-anhydro a-D-,4, 22
SUBJECT INDEX
-,
407
methyl 3,6-suliydio-2-0-tosyl-~-i)-, from u-fructose. 67, 87 25 latbelcd with CP, 79 -, methyl 2,3,6-1ri-O-methyl-u-, 89 monoacetamide derivative, cyclic a arid p anomers, 89 form, 123 p:trtiallq methylated, azoylation of, 81 -, peoni11-3,5 his-(D-, octaacctate, 240 Glucopyranosylaniine, N -phenyl -u-, 97, “penta-0-acctyl-monoacetone” deriv109 ative, 222 pentaazoate, 79 Glucopyranosyl bromide, 6-0-(p-cellobiosyl)-a-D-, decaacetate, 254 a anomer, 80 -, 2,6 -di -0-acet yl-3-0-mesyl-a- D -, 226, p anomer, 80,81 252 phenylosazone, 200 reaction of, with aliphatic amines, 100 -, 6-0- (8-gent iohiosy1)-a-a-, drcawith aniline, 97 acetate, 254 -, 2-O-(P-~-glucopyranosyl)-a-~-, heptawith p-anisidine, 97 with dibeneylamine, 200 acetate, 254 with morpholine, 200 -, 3-O-(p-~-glucopyranosyl)-o-, heptaacetate, 254 with p-phenetidine, 97 with piperidine, 200 -, 6-O-(a-D-glUCOpyranOSyl)-a-D-,heptaacetate, 254 with p-toluidine, 97, 200 separation di-, tetra-, and tri-methyl Glucopyranosyl chloride, 6-O-(p-cellobiosyI)-a-o-, decaacetate, 249 ethers of, 89 -, 6-O-(P-~-glucopyranosyl)-cx-~-, hep3-sulfate, simultaneous acetylation taacetate, 249 and desulfation of, 357 -, 2,3,6-tri-O-methyl-a-, 224 6-sulfate, 354 a anomer, 248 -, 2-acetamido-2-deoxy-n-, 122, 353, Glucopyranosyl fluoride, a - ~ -246 , 368 -, 6-chloro-6-deoxy-a-1,-, 246 diethyl thioacetal, 87 -, 6-O-trityl-a-~-,246 polymer of, 353 triacetate, 246 -, 2,4-di-O-acetyl-1,6-anhydro-3-0tribenzoate, 246 tOSyl-fi-D-, 228 Glucosamine, D-. See Glucose, 2-amino-2- -, 5,6-di-0-acetyl-l,2-O-(l-bromodeoxy-D-. et hylidene)-3-0-mesyl-~-,227 Glucosaminic acid, N-methyl-L-, nitrile, -, 5,6-di-O-acetyl-l,2-0-isopropyli87 dene-3-0-tosyl-~-,226 Glucosan, POIY-D-,225 -, 5,6-di-0-acetyl-3-0-mesyl-~-, 226 -, 2-amino-2-deoxy-~-,72, 73, 122, 173, Glucose, D - , 66, 77, 173, 314, 322 a,p , “a,” and (‘7’’ forms, 229 175, 349, 368 acid reversion products from, 87 action of nitrous acid on derivatives of, action of ammonia on, 73 357 anilide. See Glucosylamine, N-phenylbasicity of, 354 D-. 1,6-diphosphate, 368 condensation with aniline, 102 electrostatic shielding, in derivatives condensation with aromatic amines, of, 349 170,175 hydrochloride, 66, 76, 351 configuration of a-D-,231 incorporation into mucopolysaccha1-dibenzamide derivative, 121, 123, rides, 367 136 pentaacetate, WD-, 87 pentaacetate, 136 0 anomer, 87 1-di-(cyclohexylamine) derivative, 136 6-phosphate, 368 1,6-diphosphate, 368 N-sulfate, 354
408
SUBJECT INDEX
-, 5 , ~ - : ~ 1 i h y d r o - 1 , 2 - ~ - i s o ~ ~ r o ~ ~ y-,i i ~ peilta-O-be~izoyl-alrleh!/do-u-, c11~-~action 8 of met hanolic itinnionia on, 121, 124 3,6-anhydro-l,2-~-isopropylidene--, 1,2,3,4-tetra-O-acetyl-p-~-, 241 5-0-tosyl-m-~-,8, 48 -, 2,3,4,6-tet ra-0-acet y1-n-, compound -, 3,5-O-heiizylidene-l,2-O-isopropyl-- with benzylamine, 112 idene-a-D-, 36 prepamt.ion of, 113 -, 6-deoxy-l , 2:3,5-di-O-isopropylidenc- -, 1,3,4,6-tetra-O-acctyl-2-amino-2-~~6-nitro -a-D -, 36 OXY-D-, hydrochloride, 122 -, 5,B-di-O-benzoy-l , 2-0-isopropyl- -, 2,3,4,6-tetra-O-benzo,1~l-n-, 115 idene-3-O-tosyl-o-, 226 -, 2,3,4,6-tet ra-O-met~hyl-o-, 88, 92, -, 1,2 :3,5-di-O-isopropylidene-ff~n - , 36 109 graphic reprcsent,at,ion of, 4 -, 2,3,4-t,ri-O-acetyI-l,G-anhydro-p-~-, -, 1,2:5,6-di-O-isoprop~Iideiie-n-, 225 227,228 3-benzoate, 225 -, 3,4,6-t~ri-O-acetyl-l,2-nnhydro-~-n-, 3-mesylate, 226 241,243 3-tosylate, 225 , 1,2,4-tri-O-acetyl-6-deoxy-3-O-, 2,3-di-O-methyl-~-,88 m e t h y h - , OL and 6 anomers, 92 -, 1,2:3,5-di-O-methylene-a-~-, 36 -, 3,5,6-tri-O-acetyl-l, 2-0-isopropyli-, O - ( D - f r U ctO S J'l) - D -, 64 dene-o-, 226 -, 6-O-(a-D-galactopyranosyl)-~-. See -, tri-O-methyl-D-, isomers of, 92 Melibiose. -, 2,3,4-t,ri-O-met,hyl-i,-, 91 -, 6-O-(ay-o-glucopyranosyl)-p-~-.See -, 2 , 3 , 6-tri-0-methyl-D-,88 Isomaltose. Glucose-l-C14, I)-, 368 -, .l-O-(or-isornaltopyranosyl)-o-. See Glucose-6-Cl4, D-, 367 Panose. Glucoseen-l,2, 2,3,4,6-tetra-O-acelyl-, 1,2-O-isopropylidene-5, 6-d-O-t,osylD - , 229 ff-D-, 8 Glucoside, aniline D - . See Glucosyl-, 1,2-0-isopropylidene-3,5,6-tri-O- itmine, N-phenyi-o-. toSyl-D-, 226 -, aniline N-D-.See Glucosylamine, N -, 4-0 - (or-maltopyranosyl) -D-. See Malt ophenyl-D-. triose. -, benzyl 2,3,4,6-tjetra-0-acetyl-p-u-, -, 2-0-1nethy1-~-,112 242 -, penta-O-acet,yl-D-, 216 -, hesperidin, 94 01 and p anomers, 229 -, methyl W D - , 229 action of ammonia on, 121 anomer, 229 -, 1,2,3,4,6-penta-O-acetyl-a-~-, 21 1, con6gurat)ion of, 231 212,219,223 -, methyl 2-amino-2-deosy-N-sulfop anomer, 88, 211, 217 3,4,6-tri-O-sulfo-p-~-, diharium salt, conversion t o 01 anomer, 219 354 react.ion wit>h aluminiim chloride, -, methyl 4,6-0-benzylidene-p-o-, 37 221 -, methyl 4,6-0-benzylidene-2,3-di-Oanomers, reaction of, with benzyltoSyl-a-D-, 93 amine, 112 -, methyl 4,6-0-benzylidene-2-O-tosylwith piperidine, 112 a - D - , 93 with titanium tetrachloride, 219 -, methyl 4,6-0-benzylidene-3-0-tosyl-, penta-O-acetyl-aldeh?/do-D-, action a - n - , 93 of ammonia on, 118 -, methyl 3,4-di-O-acety-2,6-di-OtoSyl-p-D-, 25 -, 1,2,3,4,6-penta-O-berizoyl-o-, 01 and p anomers, action of ammonia on, -, methyl 2,3,4,6-tetrn-O - s c e t g l - p - ~ - , 123 222,242 D-,
,
-
SUBJECT I N D E X
-,
me thy1 2,3,4,6-tet rit -0-mct hyl -u - , 89 inethyl 2,3,4-t,ri-0-acetyl-6-O-benzoyl-b-n-, 225 -, methyl 2,3,4-t ri-O-acet.yl-6-O-tosy1@I>-, 225 -, naringin, 94 -, rutin, 94 Glucosidurouic acid, 1-l)romo-l-drosy01-u-galactopyr~nose-(6+ 1) 0-D-, hesaacetat,e, methyl ester, 254 -, 1-bromo-1-deoxy-ol-o-glucoDyranosc(4 + 1) b-u-, hesascetate, met.hgl ester, 254 -, u-xylose-(P + 1) 4-0-mct,hyl-u-, 64 01 auomer, 303 Glucosone, D-, 187, 188, 198, 368 imino analog, 187 phenylosazone, 187,188 tetrahydroxybutylquitioxali~iederivative, 187, 188 G l u c ~ s o n e - l - CD ~ -~, ,368 Glucosylamine, I>-, 96, 136, 137 acet.ylation of, 121 N-acet,ylation of, 117 hydrolysis of, 105 reaction of, with ethyl m:tlonat.e, 201 with 2,4-pent,anedione, 201 N-suhstituted derivatives of, 171, 184 Schiff-base structure of, 187 -, N-p-acetox?iphenyl-D-, tet,raacetate, 137 -, N-itcctyl-D-, 123, 137, 179 furanose form, supposed, 137 oxidation of, with lead tetraacetate, 118 with pcriodate, 117 prcparation of, 117 second isomer of, 118 t.etraacet.ate, 117, 137 -, N - acctyl - N - p - bromophenyl-i)-, 2 , 3 , 4 , 6 - t d r a a ~ e t a t ~111, e , 137 -, N - (2 -amino - 3,4 - dimethylpheny1)D - , t e t r u c e t a t e , 137 -, N - (2 -amino - 4,5 - dimet,hylphenyl)I ) . , tetraacetate, 137 -, N - (2 - amino - 4 - methylphenyl)-o-, t,etraacetate, 137 -, N - (o-nniinophenyl)-D-, 2,3,4, 6-tetra+cetat.e, 137
-,
40'3
-, N - (2 - I)euzisothiazoliii 3-0nc-1,l-dioxide) -D-,2,3,4,6-teti aacetat e, 137
-, N-henzyl-o-, 137 tetraacetate, 112,137 preparation of, 112 -, N-bcnzylidmc I)-, tetraacetate, 112, 137 -, D - (N-benzyl - N-met h yl) -1) -,t et raacr tntc, 137 DL-, 137 I.-, 137 -, N-4-biphenyl-1,-, 138 -, N-p-hromophen?ll-ol-u-, 2,3,4,6-te t raacetate, 138 p anomer, 2,3,4,6-tetraacetate, 138 2,3,4,6-tetramethyl ether, 138 -, N-n-bUtJrl-D-,138 hydrolysis of, 105 -, N-carbamoyl-u-, 138 N-acetyl-tetra-0-acetyl derivative, 138 N'- benzoyl - tetra - 0 - acetyl derivative, 138 tetraacetate, 138 tetrabenzoate, 138 -, N - (4 - carbethoxy - 3 - hydroxyphenyl)-D-, 138 -, Wo-carbet hosyphenyl-D-, 138 -, N-p-carbethoxyphenyl-u-, 138, 139 tetraacetate, 139 -, W ( 4 - carbomethosy - 3 - hydroxyphenyl)-o-, 139 -, N-o-carbomethoxyphenyl-o-,139 tetraacetate, 139 -, N-p-carbomethoxyphetiyl-o-, 139 tetraacetate, 139 -, N-(4-carboxy-3-hyd1oxyphenyl)-n-, 139 hemihydrate, 139 monohydiate, 139 pentaacetate, 139 phenyl ester, 139 sodium salt, 139 tuherculostatic activity of, 125 2,3,4,6-tet raaret ate, 139 -, N-m-carhoxypheny~-~-, 139, 140 tetraacetate,l40 -, N-o-carboxyplienyl-u-, 98, 139 N-acetyl derivative, sodium salt, 139 tetraacetate, 139 tetraacetate, 139
410
-, N-p-carboxyphenyl-u-, 1-40
SUBJECT INDEX
-, N - 2 - (1 - ethoxyethylideneamino)butyl ester, 138 phenyl-D-, 2,3,4 ,g-tetraacetate, 142 dihydrate, 138 -, N-o-ethoxyphenyl-D-, 142 tetraacetate, 138 -, N-p-ethoxyphenyl-o-, 142 2-diethylaminoethyl ester, monohytetraacetate, 142 drochloride, 140 -, N-ethyl-u-, 142 monohydrate, monohydrochloride, -, N - ( N - ethylthiocarbamoyl) - D - , 140 tetraacetate, 142 tetraacetate, 140 -, N-1-heptyl-D-, 142 -, N-o-carboxyphenylsulfonyl-u-, 140 -, N-1-hexadecyl-D-, 142 sodium salt, 140 -, N-1-hexyl-D-, 142 -, N-2-chloroethgloxycarbonyl-~-,tethydrolysis of, 105 raacetate, 140 -, N-(2-hydroxy-3,5-dinitrophenyl) -u-, -, N-(4-chloro-2-nitrophenyl) -D-, 140 142 tetraacetate, forms I and 11, 140 4,6 - 0 - ethylidene - 2 , 3 - oxidoethyli-, N - n f-chlorophenyl-o-, 140 dene metal, 142 -, N-o-chlorophenyl-u-, 140 -, N-2-hydroxgethyl-1-, 142, 201 -, N-p-chlorophenyl-o-, 141 -, N-(2-hydroxy-5-nitrobenzylidene) -D2,3,4 ,6-tetraacetate, 141 142 2,3,4,6-tet ramethyl ether, 141 -, N-o-hydroxyphenyl-o-, 143 -, N-n-decyl-D-, 141 -, AT-p-hydroxyphenyl-D-, 143 hydrolysis of, 105 pentaacetate, 143 -, 2-deosy-N-phenyl-o-, 145, 146 t etraacetate, 143 infrared absorption spectrum of, 102 -, N-(3 - hpdroxy - 4 - propoxycarbonyl3,5,6-trimethyl ether, 146 phenyl)-o-, 143 -, 2-deoxy-N-p-tolyl-o-, 148 -, N-(4-iodo-2-nitrophenyl)-~-, 143 -, N , N-dibenzyl-D-, supposed, 104 tetraacetate, 143 -, N , N-diethyl-D-, 141 -, N - o -methox ypyen yl -D -, 143 -, N , N-di-(2-hydroxyethyl)- D - , 141 -, N-p-methoxyphenyl-D-, 143 hexaacetate, 141 2,3,4, &tetraacetate, 143 -, N,N-dimethyl-D-, 141 2,3,4,6-tetrarnethyl ether, 143 2,3,6-trimethyl ether, 141 -, N-methyl-D-, 143 hydrochloride, 141 -, N-(N-methylcarbamoyl)-~-, 143 hydriodide, 141 -, N-(4-methyl-2-nitrophenyl)-~-, 143 -, N-(N,N-dimethylcarbamoyl)-o-,141 tetraacetate, forms I and 11, 143 -, N-(3,4-dimethyl-2-nitrophenyl) -D-, -, N-meth yl -N-phenyl -D -, t e t raacet ate, tetraacetate, 141 104 -, N-(4,5-dimethyl-2-nitroplie1iyl)-~-, 2,3,4, &tetraacetate, 143 99, 141 -, N-methylsulfonyl-o-, tetraacetate, tetraacetate, 141 124, 143 forms I and 11,141 -, N-(N-methylthiocarbamoy1)-D-,t e t -, N-2,3-dimethylphenyl-~-, 141 raacetate, 143 -, N-2,4-dimethylphenyl-o-,141 -, N-[2 - methyl-3-(0 - tolylazo)phenyl]-, N-2,5-dimethylphenyl-~-, 141 D - , 143 -, N-3,4-dimethylphenyl-o-,141 -, N-1-naphthyl-o-, 143 -, N-3,5-dimethylphenyl-~-, 141, 142 -, N-2-naphthyl-D-, 143 -, N-1-dodecyl-D-, 142 infrared absorption spectrum of, 103, -, N-(3-ethoxyethylideneamino-4-meth187 ylphenyl)-D-, 2,3,4,6-tetraacetate, 2,3,4 ,&tetraacetate, 143 142 -, N-rn-nitrophenyl-D-, 125, 143, 144
SUBJECT INDEX
periodate oxidation of, 119 tetraacetate, 144 -, N-o-nitroplieiiyl-u-, 1Y3 tetraacetate, 143 -, N - p nitrophenyl-o-, 125, 141, 187 dihydrate, 144 infrared absorption slmh-uni, 103 3-methyl ether, monohydrate, 144 periodate oxidation of, 119 tetraacetate, 144 N-acetyl derivative, 144 2,3,4-t riacetate, 144 6-trityl ether, 144 6-trityl ether, 116 -, N-(3-nitro-p-tolyl) - D - , 187 -, N-1-octadecyl-u-, 144 -, N - (N-1-octadecylcarbamoyl) -w, 144 -, N-l-OCtyl-D-, 144 hydrolysis of, 105 -, N-1-pentyl-o-, 144 -, N-p-phenetyl-D-, 171 -, N-phenyl-D-, 96, 97, 101, 144, 145, 187 N-acetyl - tetra - 0 - acetyl derivative, 111, 145 2,4-dimethyl ether, 145 3,4-dimethyl ether, 110, 145 hydrolysis of, 105 methylation of, 109 2-methyl ether, 110, 145 3-methyl ether, 145 4-methyl ether, 145 6-methyl ether, 110, 145 preparation of, 97 reaction with hydrogen cyanide, 102 relative stability of, 102 tetraacetate, 145 2,3,4,6-tetraacet ate, a anomer, 111, 145 B anomer, 111,145 complex with carbon tetrachloride, 111
2,3,4,6-tetramethylether, 109,110,145 2,3,4-trimethyl ether, 110,145 2,3,6-trimethyl ether, 145 2,4,6-trimethyl ether, 110, 145 -, N-4-phenylazophenylene-~-, 145 -, N-(N-pheny1carbamoy.I)-a-, 145 -, NS-phenylethyl-~-, monohydrate, 146
41 1
-, N-(Npheriylthiocarbamoyl)-i)-,tet raacetate, 146
-, N-p-sulfacetamidol,henyl-D-, 146 2,3,4,6-tet raacetat e, 146 -, N-p-sulfainylphenyl-D-, 146 hexaacetate, 146 tetraacetate, 146 2,3,4,64etrititcetate, CY anomer, 146 /3 anomer, 146 -, N-p-sulfophenyl-D-, 146 -, tetra-0-acetyl-u-, 121, 1.71 hydrochloride, 137 reaction with aromatic aldehydrs, 112 -, tetra-0-acetyl-N-mesyl-o-, 124, 143 -, N-thiocarbamoyl-D-, 146 N’-benzoyl -tet r:t-0-benzoyl dcrivat i ve, 146 -, N-m-tolyl-u-, 146 hemihydrate, 101 relative stability of, 102 -, N-0-tolyl-D-, 146 hemihydrate, 101 infrared absorption spectrum, 103, 187 relative stability of, 102 -, N-p-tOlyl-D-, 98, 99, 101, 147, 175, 179 N-acetyl-tetra-0-acetyl derivative, 111 LY anomer, 147 tetraacetate, 147 B anomer, 113 acetylation of, 113 N-acety1-2,3,4,6-tetra-O-acetyl derivative, 147 2,3-dimethyl ether, 147 hemihydrate, 147 2-methyl ether, 147 monohydrate, 147 2,3,4,6-tetraacetate, 111, 113, 147 deacetylation of, 113 2,3,4,6-tetrabenzoate, 147 2,3,4,64etramethyl ether, 148 3,4,6-triacetate, 147 hemihydrate, 101, 147 infrared absorption spectrum, 103, 186 methylation of, 110 monohydrate, 101, 147 relative stability of, 102 tetraacetate, hydrogenation of, 04 tetrabenzoate, 115 2,3,4,6-tetramethyl ether, 110 ultraviolet absorption spectrum 186
412
SUBJECT INDEX
-, ~ V - p - t o l ~ l s u l f o I ~ ~ ~148 l-I,-,
reaction o f , with active silver chloride, 21!) tet rxtcet at e , 145 with silver fluoride, 221 6-p-toluenesulfon:~tte,1-18 with secondary ainines, 239 2.3,4-t.riacetat e , 1-18 %-ith t~rin~cthylamine, 114 Glucosylamines, iV-alkyl-w, !IS, 189 rcduction with lithium aluniinuin hy1Lydtogen:ttion of, 104 dricle, 245 -, N-aryl-u-, 180, lS!), 1!J5 from rice starch, 223 hydrogenat,ion of, 104 solvolytic reactions of, 234 hydrolysis of, by sulfuric acid, 105 for synthesis of glycosides, 240 order of stability of, 102 -, 2,3,4,6-tet ra-O-ncet,yl-a-o~-,253 -, !V-benzyl-i)-, 195 Glricosyl bromide, 2-0-ac.etylB-O-t)rn- -, 2,3,4,6-t'etra-O-acetyl-cu-r,-, 253 -, 2,B,4,6-tetr:~-O-l,enzoyl-~u-l,-,253 my1 -3,5-di -0-tosyl- a - i ) - , 252 -, 2,3,4,6-tetra-0-propionyl-cu-u-, 253 -, 4-O-aeetyl-6-deoxg-6-iodo-2,3-di-0-, 2,3,4-tri-O-acetyl-cu-1)-, 228, 252 tosyl-a-u-, 224, 253 6-benzoatej 252 -, 2-0-acetyl - 5,6-di-O-benzoyl - 3 - 0 6-bromo-6-deoxy derivative, 211, 228, tospl-a-u-, 252 252 p anomer, supposed, 226 -, 6-0-acetyl-2,3,4-t,ri~O-l)enzoyl-a-~-, 6-p-bromophenyl ether, 252 6-chloro-6-deoxy derivat,ive, 252 228, 253 6-deoxy-6-fluoro derivative, 252 -, 6 -0-:tcet.yl-2,3,4- t ri -0-l)enzyl-cu - I)., 6-deoxy-6-iodo derivative, 252 253 6-deoxy-6-t,hiocyano derivative, 252 -, 0-acetyl-tri-0-tospl-u-, 226 6-di phen yl p h ~ s p l i a t ~252 e, -, 2-0-acet,yl-3,5,6-tri-0-t osyl-a -D-, 252 6-mesylate, 252 -, 4-0-acet y1-2,3,6-tri-O-t~osyl-a-r-,224, 6-methyl ether, 252 252 6-(2-naphthyl) ether, 252 bis-(2,3,4-tri-O-acet y l - a - ~ - 6,6-car, 6-phenyl ether, 252 bonate, 253 3,4-di-0-acetyl-6-deoxy-6-iodo-2-0- 6-tosylate, 252 -, 2,4,6-tri-O-acetyl-a-~-, 225 toSyl-a-D-, 252 3,4-di-O-acetyl-2,6-di-O-bensoyl-a-3-benzoat,e, 225, 252 3-mesylate, 252 D - , 252 3-tosylate, 252 3,4-di-O-acetyl-2,6-di-0-tosyl-a-~-, -, 3,4,6-tri-O-acetyl-2-amino-2-deosy252 CPD-, hydrobromide, 252 2,4-di-O-acety1-3,6-di-0-(tri-0-acehydrochloride, 252 tyigailoyl) -a-D-,252 tetra-0-acetyl-a-o-, quaternization -, 2,4,6-tri-0-acet,yl-3-0-henzoyl-a-~-, 252 mith pyridine, 245 reaction with ammonia, 121 -, 2,3,4,6-tetra-O-acetyl-~-, 94 naming of anomers of, 230 stable form of, 232 -, 2,3,4, 6-tetjra-O-acetyl-cY-D-, 208, 222, 223, 224,237, 240, 241, 244, 252 configuration of, 231 from levoglucosan, 228 methanolysis of, 236, 242 rate of, 235 preparation of, 214
-,
2,3,4-tri-O-acetyl-6-bromo-6-deoxy-
WD-,
252
-, 2,3,4-tri-o-acetyl-6-deoxy-a-o-, 251 -, 3,4,6-tri-o-acety1-2-deoxyu-, 228 anomer, 251 2 , 5 , 6 - tri - 0 - acet,yl-3-O-mesyl - a I>-, supposed, 227, 252 -, 2,3,6-tri - 0 - acetyl - 4-04osyl - LY D-, 252 -, 2,4,6-tri-O-acetyl-3-O-tosyl-ol-o-, 225, 228 -, 2,5,6-t,ri-O-acety1-3-O-tosyl-, 252 01
-,
SUBJECT INDEX
413
-, 3,4,6-tri-O-acety1-2-O-tosyl-a-u-, 252 Glucosyl isocyanate, tetra-0-acetyl-D-, -, 3,4,6-tri-O-benzop1-2-deo~g-a-i~-, 251 149 Glucosyl chloride, 4-0-acetyl-2,3,6-tri- Glucosyl isothiocyanute, u-, 14‘3 @met hyl-a-D-, 247 tetraacetate, 149 -, 4-0-acetyl-2,3,6-tri-O-tosyl-ol-u-,247 Glucosyl phosphate, u-, 350 -, 5 -0-henzoyl-2,3,6- t r i - f ~ - i n e t h y Dl -- , -, 2-aniino-2-dcoxy-~-,340, 368 G lucosylpyridi ni uni bromide tleriva247 -, 3,4-di-0-acetyl-6 -deoxy -a-D -,248 tives. See un.tler l’yridinium bromide. p anomer, 240 Glucosyl thiocyanute, t,etra-O-scetyl-a-, 3,4-di-0-acetyl-6-deosy-2-0-trichlo- I>-, 121 roacctyl-P-u-, 249 Glucosyltrimethglsmi~io~~iiim halide dc-, tctra-O-acetyl-I,-, 216 rivat>ives. See u n d e r respecfive h m -, 2,3,4,6-tetra-o-acetyl-o-, 215 nionirim halide. DI anomer, 209, 211, 219, 222, 247 Glucuronic acid, I>-, 76, 314 p anomer, 209,211,212,210,221,248 in beech wood, 301 rearrangement of, 234 incorporation of, into mucopolgsac-, 2,3,5,6-tetra-O-acetyl-u-, 247 charides, 367 supposed, 222 2,6-lactone, 66 -, 2,3,4,6-tetra-0-benzoyl-a-~-, 248 -, 2,5-di-O-acetyl-l-hromo-I -deoxy-a-, 2,3,4,6-tet,ra-O-mesyl-a-u-, 248 u-, 6,3-lact,onc, 252 -, 2,3,4,6-tetra-O-iiiethyl-a-u-, 221, 248 -, 2,5 - di- 0 - acetyl-1- chloro -1-deoxy-, 2,3,4,6~t,etra-O-propiorlyl-a-1,-,248 a - D - ,6,3-lactone, 247 -, 2,3,4,6-tetra-O-Sulfo-a-o-, 248 methyl ester, 247 -, 2,3,4,6-tct.rn-O-tosylol-~-,215, 243 -, 4-O-methyl-~-,64, 72, 91, 286, 303, -, 2,3,4-tri-O-acetyl-au-u-, 228, 247 313, 325 -, 3,4,6-t,ri-o-acet3-1-p-~-, 217, 242, 248 Glutamine, I,-, in synthesis of 2-nmino-2methanolysis of, 236 deoxy-D-glucose, 368 reaction with ammonia, 243 Glycals, 205 -, 2,3,4-tri -0-acetyl-6-chloro-6-deoxy-aaddition t o , of halogen acids and of D - , 247 halogens, 228 -, 3,4,6-tri-O-acetyl-2-chloro-2-deoxyGlyceraldehyde. See Glycerose. WD-, 235, 247 Glyceritol, 66 -, 2,3,4-tri-O-acetyl-6-deoxy-a-~-, 248 -, 1,3-O-benzylidenc-, isomers of, 30 -, 3,4,6-tri-0-acetyl-2-dcoxy-o-, 228 -, 2,3-S-benzylidene-2,3-dideoxy-2,3-, 3,4,6-tri-0-acetyl-2-0-tosgl-a-~-, 247 dithio-DL-, 28 -, 3,4,6-tri-O-acety1-2-O-t richloroace- Glyceronic acid, D - , 117 tyl-p-D-, 217, 238, 248 Glycerose, 72, 174,204 methanolgsis of, 235, 236 -, 3-phenyl-, 174 Glucosyl fluoride, 2,3,4,6-tctra-O-acedimeric, 204 t,J’l-a-D-, 246 Glycine, 360 ethyl ester, 185 p anomer, 221,246 -, 2,3,4,6-tetra-O-henzoyl-cu-1,-, 246 -, N-o-glucosyl-, barium salt,, 149 -, 2,3,4-tri-0-acet~.vl-6-chloro-6-deoxy- ethyl ester, 140 hydrolysis of, 105 a-~246 , Glucosyl halides, tetrn-0-acetyl-D-, 232 sodium salt, 149 Glucosyl iodide, 2 , 3 , 4 , 6 4 e tra-0-acetyl- -, N-D-glUCoSylg~laIlyl-,149 W D - , 256 -, N-D-glUCOSylgUaIlylglycyl-, 149 -, 2,3,4,6-tet,ia-O-benzoyl-a-D-, 256 Glycitols, 1-amino-1-deoxy-, 200 -, 2,3,4-tri-O-acet~yl-6-deoxy-6-iodo-ay-, anhydro-, formation of, 245 D - , 256 -, 1-arylamino-1-deoxy-, 191
414
SUBJECT INDEX
Glycofuranosides, from thioacetals, 23 methyl 3,6-anhydro-, formation of,
-,
22 Glycofuranosyl halides, poly-0-acetyl-,
preparation of, 97 structure of, 102 N-substituted, hydrogenation of, 103,
200
reaction with hydrocyanic acid, 201 the term, 97 Glycogens, animal, fractionation by tritylation of, 116 colunin chromatography, 79 uses of, 124 structure of, 86 -, N-aryl-, hydrolysis of, 105 Glycolaldehyde, 72,73 oxidation of, 119 Glycolic acid, 2,3,4,6-tetra-O-acetyl-p-, Karyldeoxy-, hydrolysis of, 105 D-glucoside, ethyl ester, 222 Glycols, cis-l,3-, cuprammonium rom- -, N-0-carboxyphenyl-, 98 -, N-phenyl-D-, separation of anomeric plexes of, 23 tetraacetates, 111 Glycopyranoses, 1,4-anhydro-, 21 Glycopyranosides, methyl 3,6-anhydro , -, N-p-sulfophenyl-D-, 99 -, Ni-p-tolyl-, 98 rearrangement of, 22 Glycosans, conversion t o poly-0-acyl- Glycosyl bromides, poly-0-acetyl-, staglycosyl halides, 227 bility of, 233 -, 1,2-cis-poly-O-acetyl-, 220 Glycoseen-l,2, 229 -, poly-0-acyl-, preparation of, 213 formation of, 243 Glycosides, alkyl and aryl, 229 reaction with amines, 239 -, poly-0-benzoyl-, stability of, 233 cardiac, 209 Clycosyl chlorides, poly-0-acetyl-, 82, conversion t o glycosyl halides, 222 233 flavonoid, 93 formation of, 240 1,Z-transisomers, 220 -, poly-0-acyl-, preparation of, 212 hydroxyanthraquinorie, 209 infrared absorption spectra of snomers, Glycosyl fluorides, 233.See fable of properties on page 246. 358 -, poly-0-acetyl-, stability of, 233 methyl, hydrolysis of, 322 naturally occurring P-D-, 240 -, poly-0-acyl-, 212,221 in seeds, 94 preparation of, 210 -, 1-deoxy-1-thio-, reductive desulfuri - Clycosyl halides, 01 and 6 anomers of, 209 aation of, 245 of the ‘‘0-series,” 210,219 -, glycolic ester p-D-, 2,3,4,6-tetraacepreparation of, 2@9 tate, 222 simultaneous formation and acetylation of, 214 -, p-phenylazophenyl poly-0-acetyl-, 82 “N-Glycosides.” See Glycosylamines. the term, 207 Glycosylamines, 95, 106, 243. See also -, 2-deoxy-, preparation of, 228,229 tables of properties on pages 126 to 164. -, poly-0-acetyl-, of the “p-series,” 234 acetylation of, 110 chemical properties of, 233 as antioxidants, 125 optical rotations of, 231 benzoylation of, 115 optical rotatory properties of, 230 decomposition w i t h benzaldehyde, 105 physical properties of, 233 formation of, 243 reduction with lithium aluminum hyhydrolysis of, 104 dride, 245 methylation of, 109 solvolytic reactivity of, 235 stable forms of, 232 mutarotation of, 104,183 nomenclature of, 96 -, poly-0-acyl-, configurations of, 231 physical properties of, 101 from glycosans, 227 in plastics, 125 from ortho esters, 227
234
415
SUBJECT INDEX
preparation of, from glycosides, 222 with hydrogen halide in acetic acid,
sapote, 7!) Slerculia setigera, 71
213 with hydrogen halide in ether, 212 with liquid hydrogen halide, 210 with phosphorus halides, 215 with titanium tetrahalide, 218 reaction of, with nucleophilic reagents,
234 with primary, secondary, and tertiary amines, 243 reactions of, 239 stable anomers of, 210 structure of, 229 unstable anomers of, 210 -, poly-0-benzoyl-, reactions of, 238 Glycosyl iodides, 209 -, poly-0-acetyl-, stability of, 233 -, poly-0-acyl-, 219 Glyoxylic acid, 312,314 Grapes, 93 Grass, couch, rhizomes of, 91,92 esparto, 90 leafy cocksfoot, fructan of, 91,92 rye, fructan of, 91,92 Grignard reagents, for scission of anhydro sugars, 53 Guanidine, amino-, 194 -, N-D-frUCtOSyl-, 130 -, N-D-glUCOSyl-, 149 -, N-D-mannosyl-, 156 Guanosine, synthesis of, 244 Guaran, acetate, 86 Gulitol, 1,5-anhydro-~-,246 Gulopyranose, 1,6-anhydro-b-~-,50 Gulose, D-, 66 Guloside, methyl 2,3-anhydro-4,6-0benzylidene-D-, 52 a anomer, 51,93 p anomer, 51 -, methyl 2,6- dideoxy - 6 - iodo - 3 - 0methyl-4-0-tosyl-or-~-,92 p anomer, 92 Gum, Acacia pycnanlha, 71 apple, golden, 71 cherry, 71,90 Karaya, 90 lemon, 71 myrrh, 72 peach, 71
H Halogens, radii of atoms of, 231 Hardwoods, alpha-cellulose of, 328 hemicelluloses of, 302,313 isolation of carbohydrates from, 317 mixed, sulfite pulp from, 325 pulping of, 324 sulfate pulps from, 327 Heartwood, of oak, 301,302 Hemicellulose-A, of beech wood, 91 Hemicellulose-A, -B,and -C, of black spruce, 296 of slash pine, 296 Hemicelluloses, of American white oak,
300 of aspen wood, 286 of beech wood, 300 of black spruce, 64,313 of conifers, 313 effects of, on pulps, 310,311 of hardwoods, 302,313 of jute fiber, 304 of mesquite wood, 286 nitrate, from Western hemlock wood,
330 of oak, 301 of oak wood, 313 preparation of, from holocelluloses, 318 of Scots pine, 64 of slash pine, 313 of softwoods, 302 of spruce, 290 of white pine, 313 wood, alkaline degradation of, 326 uronic acids of, 313 Hemlock (the t r e e ) , alpha-cellulose of,
328 Western, 292,319,320,330 Heparin, acetylation of, 365 N-acetyl content of, 353 acid hydrolysis of, 348 action on, of acid, 346 of alkali, 346 of enzymes, 345,365 of nitrous acid, 357 adsorption-front analysis of, 344 antibacterial action of, 36@
416
SUBJECT INDEX
anticoagulant activity of, 336,343,353, 359, 360 ash content of, 352 bacteriostatic activity of, 360 biological activity of, 359 commercial, electrophoresis of, 353 fractionation of, 344 complex of, with protein, 347 with Toluidine ]Hue, 339, 344 composition of, 339 countercurrent distribution of, 344 degradation of, 357 N- (2,4-dinitrophenyl) derivat ive, 355 discovery of, 336 distribution of, in hotly tissues, 344 distril)ution of sulfate groups in, 363 electrometric titration of, 352, 353 electrophoretic study of, 344 enzymic inactivation of, 365 enzymic sulfation of, 363 extraction of, 348 filter-paper electrophoresis of, 345 fractionation of, Nith brucine, 352 from beef liver, 335 from beef lung, 33s from dog liver, 337 n-glucuronic acid of, 351 helical structure of, 366 hexosaminc content of, 351 hexuronic acid content of, 351 histological demonstration of, 339 homogeneity of, 344 inactivation of, by acetic acid, 366 by dilute acid, 365 by enzymes, 365 by nitrous acid, 352, 365 infrared absorption spectrum of, 358 as inhibitor of pancreatic ribonuclease, 360 interaction of, p i t h proteins, 335 isolation of, 337 in mammalian tissues, 330 medical uses for, 360 metachromatic activity of, 344 molecular shape of, 364 molecular weight of, 363 mutagenic action of, 360 nitrogen content of, 352 nitroso derivative of, 354 occurrence of, 337,339, 340
octylamine-protein complex of, 348 oxidation of, in presence of ascorbic acid, 359 paper chromatography of, 343 partial acid hydrolysis of, 352, 355 partial formula of, 349, 350 polyelectrolyte character of, 364 potency of, from various sources, 347, 348 potentiometric titration of, 353 properties of, 337, 338 provisional international standard for, 361 purification of, 338, 339, 355 purity of, criteria of, 343 regeneration of, from heparin-octyl:tmiiie-protein complexes, 345 from heparin-protein complexes, 345 rctention of acetic acid by, 353 Roche, 314 simultaneous acetylation and desulfation of, 356 simultaneous hydrolysis and oxidation of, 351 structure of, 348, 360 sulfaniic acid groupings of, 354 sulfate bridges in, 364 “N-sulfate” group of, 354, 363 sulfation of, 364 sulfur content of, 352 supposed species-specificity of, 347 surgical uses for, 360 the term, 336 total, acid hydrolysis of, 351 ultraviolet absorption spectrum of, 343 Vittum, 344 p-Heparin, 367 $-Heparin, 349, 350,352 acid hydrolysis of, 350 action of nitrous arid on, 358 derivatives of, 360 -, N-isonicotinyl-, 360 -, N-nicotinvl-, 360 -, N-trifluoromethylphenyl-, 360 Heparinnse, 345 Heparinic acid, 336, 353 ammonium salt, 340,341 barium acid salt, 340, 341, 347, 352 analysis of, 31
RUBSECT INDEX
417
barium-sulfur ratio of, 352 CJf y C a S t , , 36s ~~CXOkill&SC, coni1)osit ion of, 351 Ilesonic u i d , di-O-iiict.Ii~leiic-, 42 iiitrogeri-sulfur ratio of, 352 Hesosans, 326 slruct~ii~.e of, 357 of cellulose prqi:ir:~ti~~iis, 321, :;25 bariiun salt, 343, 348, 3% Hcose-:i.niiiioni:i, I06 honwgcneous, 345 Ilesositle, methyl 2,6-tlideosy-3,4-cli-0benzitline salt,, 33‘3,355 1,osyI-01- I ) -.ryfo-, !U brucine s:tlt, 338, 339 p anomer, 92 tlecamethylenediamine salt, 343 Hexulose, 1 ,6-dideosy-l -(p-tnIui~litin)dodecylamine salt, 343 L-orrrbino-, 205 isoperitylamine salt, 343 -, r.ibo-. See T’sic,ose. n-pent>ylaminesalt, 343 Hexiironic acid, O-metli~.l-,313 piperidine salt, 343 of hardwoods, 302 prot,ein complex of, 336 of oak heniicelluloses, 301 sodium salt, 336, 346 of softwoods, 302 analysis of, 340 Ilist,idine, N , N ’ - d i - ( n - g l i ~ c o s ~ ~ l g i i a n ~ ~ l ) Heparosinsulfuric acid, 356 L-, 136 -, N-acetyl-, 356 Holocellulose, 288, 313, 318, 331 Heptonic acid, ~ - g l ~ c e r . o - ~ ) - g uamide, lo-, of birch wood, 303, 314 77 of black spruce, 296,319 Heptose, r~-glycero-o-qulo-,66, 77 carbohydrate content of, 319 Heptosyl hromide, 2,3,4, 6,7-pentma-0- chlorite, 332 acetJ.l-P-D-gl!/cer.o-I,-ynlacto-, 253 from aspen wood, 308, 309, 310 -, 2 , 3 , 4 , 6 , 7 - p e n t ~ ~ - O - n c e t g l - ~ - n - g Z ~ ~ r e ~ oprocedure for, 317, 318 w y u l o - , 253 of slash pine, 318 -, 2,3,4,6,7-pent,a-O-acet~yI-ol-~-glyce~o- delignified, 312 D-ido-, 253 of Douglas fir, 319 -, 2,3,4,6,7-penta-O-acet,yl-ol-~-gl~~cer.o- hydrolysis of, 320 ~ - t n l o -253 , isolation of, 317 _ _, 2,3,4,6-tetra-O-ncet~.l-i-~~eoxy-01-~~of loblolly pine, 319 glycero-L-gulncto-, 251 maple, analysis of, 316 Heptosyl chloride, 2,3,4,6,7-penta-Ofrom non-woody plant material, 318 Ltcetyl-ol-D-glycCrO-D-g~ZO-,248 of overcup oak, 319 -, 2,3,4,6,7-penta-O-acetyl-p-D-gl?/ce,.oof slash pine, 290, 295, 296, 319 D - g U b , 248 of softwoods, 325 -, 2,3,4,6-tetra-U-acet~~1-7-deosy-~-gly- of Southern red oak, 319 cero-L-galacto-,2-17 of spruce, 314 Heptulosan, ~ - i d o - 73 , of sugar maple, 320 Heptulose, D-gluco-, 66 the term, 316 D-~ZU.TWLO-,66 of Western hemlock, 319 -, I-deosy-I-diazo-keto-L-guluc~o-, penof Western red cedar, 319 taacetate, 87 from wood, 312 -, 1-deoxy-I-diazo-keto-L-nlanno-, pen- Horses, polysaccharide from intestines taacetate, 87 of, 90 Hexaric acids, 2,4:3,5-diacetaIs of, 40 Huckleberry, leaves, 93 -, 2,4:3,5-di-O-methylene-,7 Hudson’s rilles, of isorotation, 112 Hexitols, 1-amino-1-deosy-, deaminafor naming anomers, 230 tion of, 23 Humin, formation of, 193 -, 1,4-anhydro-,23 Hpaluronic acid, 346,349,366 -, 1-arylamino-1-deoxy-, 200 biosynthesis of, 367
418
SUBJECT INDEX
microbial, 368 oxidation of, in presence of ascorbic acid, 359 11) dantoic acid, N-o-glucosyl-, 119 ethyl ester, tetraacetate, 1.1-9 potassiiim salt, 149 -, N-(o-glucosy1)thio-, ethyl ester, 150 ethanolate, 150 tetraacetate, 150 potassium salt, 150 Hydant oin, N - o -gluc osyl-, 140 -, N-(1~-glucosyl)-2-thio-,150 Hydrasine, action on o-friictose, 187 -, (2,4-dinitrophenyl)-, 305 -, (p-nitropheny1)-, 195, 305 -, phenyl-, 305 Hydrindan. See Indan, hesahydro-. 1-Hydrindanone. See 1-Indanone, hexahydro-. Hydrobenzoin. See 1,2-Ethanediol, 1,2diphenyl-. Hydrogen bromide, liquid, 211, 228 Hydrogen chloride, liynid, 211, 222, 224 Hydrogen cyanide, 102 reaction with X-suhstituted glyrosylamines, 201 Hydrogen fluoride, anhydrous, 225 liquid, 212 Hydrogen iodide, 219 Hydrogen-ion concentration, effect on glycosylamines, 106 Hydrophilicity, of wood pulps, 315 Hydroxylamine, hydrochloride, 306
, 2,1:3,5-di-0-metIiy.Icnc
- 1,6-di-Otosyl-lr-, 12 -, 1 , 3 :2,1:5,6-tri-O-l,eiixyliclene-~-, 39 Idopj-ranow, 1,6 anhydro-p-r-, 50 Idose, 6-deouy-l , 2:3,5-di-O-isopropylidene-6-nitro-1-, 36 Idoside, methyl 4,6-O-benzylidene-a-o-, 43 4 anomer, 44 Idosylamine, 6 - d e o ~ g - h ' - p h e n y l - ~ -3-, methyl ether, 151 Indan, hexahydro-, 20 1-Indnnone, hexahpdro-, 20 Indophenol, 2,6-dirhlorophenol-, 187, 188, 201, 202 use in column chromatography, 65 Infrared absorption spectra, 100, 102, 103, 186 Inositol, rnyo-, 66, 87 Interferometer, use in column chromatography, 58 Iiiulin, 91, 92 D-glucose content of, 91 sulfated, 361 Invertase, of yeast, 64 Ion-exchange resins, for column chromatography, 75 Iris, galactomannan of seed, 72, 90 Iris ochroleuca, seeds, 289 Iris sibirica, seeds, 289 Isoglucosamine, 173 -, N-p-tOlyl-D-, 173, 199 Isoguanosine, synthesis of, 244 Isomaltose, 86, 87 I p - , acetate, 86 isolation of, b y column chromatogIdaric acid, 2,4:3,5-di-O-meth~lene-r,-, 40 raphy, 63 epimerieation of, 41 p-, octaacetate, 86 Iditol, L-, 66 Isoquercitrin, 94 2,4:3,5-diacetals, 39 Isorhamnosylamine. See Glucosylamine, -, 2,5-diamino-l,4:3,6-dianhydro-Z, 56-deoxy-. dideoxy-L-, 48 Isosucrose, octaacetate, 88 -, 1,4:3,6-dianhydro-~-,46, 49, 67 Ivory nut, mannan of, 289,290 -, 1,4:3,6-dianhydro -5 -chloro-5-deoxy 2-O-mesyl-~-,48 J -, 1,4:3,6-dianhydro-2,5-di-O-tosyl-~-, Juniperis commzinis, 286 47, 49 -, 1,4:3,6-dianhydro-2,5-O-methylene- Jute, 324 fiber, hemicelluloses of, 304 L - , supposed, 47 -
.
SUBJECT INDEX
K Karaya gum, 90 Kestose, 64 Ketohexopyranosyl halides, poly-0-acetyl-, stable forms of, 233 Ketoses, condensation with aliphatic amines, 100 dehydrogenation of, by hydrazines, 197 differentiation from aldoses, 197 reagents for detection of, 193, 194 Seliwanoff test for, 201 -, 1-amino-I-deoxy-, formation of, 169, 170 nomenclature of N-substituted, 173 optical rotations of, 186, 203 -, 1-arylamino-1-deoxy-, hydrogenation of, 191 for preparation of phenylosazones, 197 -, 1 - deoxy-1-(N-methylanilino)- 3 phenyl-, 174 semicarbazone, 175 Ketosylamines, N-substituted, 99 Knoevenagel reaction, 179 Koenigs-Knorr reac'tion, 209, 231, 240 improvements in conditions for, 241 Kojic acid, synthesis of, 229 Konjac mannan, 289 Kraft process, for pulping, 326,329
L Lactaldehyde, DL-, 73 Lactic acid, 184 in pulping waste liquors, 326 Lactitol, 66, 77 Lactobacillus gayonii, 306 Lactobacillus nrannitopoeus, 306 Lactose, 66, 76, 80, 83 action of ammonia on, 73 azoate, a-,80 p nnomer, 80 monohydrate, 77 octancetate, 215, 216 I,actosylnmine, N-acetyl-, dihydrate, 151 heptaacetate, monohydrate, 151 -, N-4-biphenyl-, 151 -, N-p-bromoplienyl-a-, heptaacetate, 1.51
0 anomer, 151 -, N-carbamoyl-, 151
, N
419
- (4-carboxy-3-hydroxy.phenyl)-, phenyl ester, tetrahydrate, 151 sodium salt, 151 -, N,N-dimethyl , heptaacetate, 151 -, N-I-dodecyl-, 151 -, N-p-ethoxyphenyl-, 151 -, N-phenyl-a-, heptaacetate, 151 @ anomer, 151 -, N-p-sulfamylphenyl-, 151 trihydrate, 151 -, N-p-tolyl-a-, heptaacetnte, 151 P anomer, 151 Lactosyl hromide, hepta-0-acetyl-a-, 254 I,actosyl chloride, hepta-O-acet~l-,215, 216 a anomer, 249 I~actosylfluoride, 246 heptaace t at e, 246 1,actosyl iodide, hepta-0-acetyl-a-, 256 Lactosyl isothiocyanate, hepta-0-acetyl-, 151 Lactulose, 73 Laminaribiose, a-. See Glucopyranose, 3-0-(p-o-glucop~ranosgl) -1)Larch, e-galactan of, 72 Lead tetraacetate, oxidation I)y. See Oxidation. I>ecithin, 360 Lemonflavin. See Oak, bark. Lemon gum, 71 Leucine, 360 Leuconostoc mesentei oitbs, 362 Levans, bacterial, 89 Levoglucosan. See Glucopyranose, 1,6anhydro-p-o-. Levulinic acid, 305 lichenin, fully methyhted, 82 triacetate, 223 J,ignin, 284, 316 association with xylan, 310 dissolution of, 326 i n hemirelluloscs, 287 of maple holort.llnlose, 316 of maple wood, 316 sodium salt, 325 -, chloro-, 316, 317 h p e m i a , alimentnrp, 360 Lithium aluminum hgdride. See Aluminum lithium hydride. -
420
SUBJECT INDEX
Lobry de Brngn and Alhcrda van Ekenstein transformation, 184, 195 LoIZ'IL~Z perenne, 91 Lucerne, galnctornannan of, 72 galactomannan of seeds, 90 Lysinc, N2,N6-di-(D-glucosy1) - DL- , 136 -, N , Nl-di- (D-glucosylguanyl)- D L - , 136 -, N-D-glUcosyl-, 149 Lyxitol, 1-amino-1-deoxy-n-, 173, 205 Lysonic acid, D-, amide, 77 -, 2,3, 4-tjri-O-methyl-~-,lactone, 266 phcnylhydrazidc, 266 -, 2,3,5-tri -0-methyl-o-, 266 lactone, 266 phcnylhydrazide, 266 Lyxosc, D-, 66 -, 2,3,4-tri-O-methy1-1-,266 -, 2,3,5-tri-O-mcthyl-~-, 266 Lyxosidc, methyl 2,3-anhgdro-5-0methyl-a-o-, 266 -, methyl 2,3,4-tri-O-nicthyl-~-,266 Lyxosglaminc, D - , 152 -, N-(4,5-dimcthyl-2-nitrophcnyl) -D-, 152 2,3,4-triacctate, 152 -, N-(4,5-dimethyl-2-1iitrophenyl)-~-, 152 2 , 3 ,4-triacctate, 152 -, N - (3,4-dimethylphcnyl) - D- , 152 -, N-p-nitrophcnyl-I)-,152 -, iv-phcnyl-D-, comples with sodium sulfate, 124 I ~ g ~ o s ybromide, l 2,3,4-tri-O-acctyl-aD - , 253 1,ysosyl halides, tri-O-:tcet,yl-i)-, 232
M nragllcsol, 7s, 70, SO, s 1 , m , SG, 8 7 , N for coliimn chroiiiat,ogr:tl,liy, 93 hlaillard reactiou, 172, 103 Malic acid, levo-, 66 Rfaloriic acid, 177 ethyl e s k r , 177, 201 hlultitol, acetate, 8G Maltotlextrins, prep~r;~!ion of, 63 Malt.ohept.aose,prep:irat.ion of, 03 Rlsltose, 66, 76, 83, 87 action of ammonia on, 7:; P - , aeoatc, 80 degradation of acetate, 224
heptaacetate, 213 monohydratc, 77 g-, octaacctatc, 83,88, 213 -, hexa-O-aoetyl-l,2-0-(1-chlorocth~lidene)-, 213, 227 Maltosylaminc, 153 heptaacctatc, 153 -, N-(l-carbomethoxy- 3 -hydrosyphcny1)-, 153 -, N - (4-cnrboxy-3-hydroxyphenyl)-, 153 -, N-(o-carbosyphcny1)-, 98, 153 -, N-(l-dodccyl)-, 153 hydrolysis of, 105 -, N-l-hcryl-, 153 -, N-1-octadecyl-, 153 -, N-phcnyl-, 153 hcptaacctate, 153 -, N-p-sulfamylphenyl-, 153 -, N-p-tolyl-, hcptaacctatc, 153 Maltosyl bromide, hepta-0-acetyl-, 214, 222 01 anomcr, 254 from potato starch, 223 Maltosgl chloride, hcpta-0-acctyl-a-, 213, 249 -, hesa-O-acet~l-2-O-tI.ichloro~~cctyl-p~, 217, 249 Ma1tosyl fluoride, hept:t-O-acetyl-, 216 Pvlaltosyl halides, poly-0-acctgl-, 232 Maltotetraosc, isolation of, by column chromatography, 63 Rf a1tot riosc, 86 isolat,ion of, by column chromatography, 63 hfaltulosc, 73 Malviri chloride, 2440 hlandelic acid, D L - , ethyl ester, 240 Mannans, 284, 319, 320 of alpha-ocllnlose, 328 of birch, 326 compound wit,h cellulose, 296 content of, in coniferous woods, 296 detcrrninatior~of, 290, 294 optical rotntion of'. See tuble on prc~lc 290.
oxidation wit,li lcail t.ptr:i:t of pine wood, 326 properties of, 205 st,ructurc of, 288 of sulfitc pulps, 323, 325
SUBJECT IXDEX
42 1
the term, in wood chemistry, 284 Mannopyranoside, methyl WD-, 67 of unbleached, sulfate pulps, 327 B anomer, 16, 322 of woods, 285,290 -, methyl di-0-benzylidrne-oI-u-, 35 -xylaii combination, of wood pulp, 329 -, methyl 6 - o - t o s y l - a - ~ -22, illarman A , 288,289,290 Maniiopyranosyl iodide, O - ( B - ~ - g l u c o of ivory nut, 290 pyranosyl) - o ~ - D - , heptaacetate, 256 Mannan B, 288, 289, 290 Mannosaminic w i d , penta-0-acetyl-NMannaric acid, 2,4:3,5-di O-methylenemethyl-L-, 87 D - , epimerization of, 41 Mannose, 318, 322 Mannitol, D-, 64, 66, 76, 77,87 D-, 64, 66, 77, 173 effect of hot hydrochloric acid on, 23 determination of, 293 hesaacetate, 88 I)-, 1-dincetamide derivative, 154 -, 1-amino-1-deoxy-I)-, 205 I ) - , 1-dibenzamide derivative, 124, 154 -, 1,4-anhydro-~-,23,66 pentabenzoate, 154 -, 1,5-anhydro-1)-,23, 66, 245 disaccharide of, 296 di-0-benzylidene acetal, 35 n-, monobenzamide derivative, 124 6-tosylate, 23 phenylhydrazone, 291, 292 -, 6-deoxy-l , 3:2,5-di-O-methylene-r-, D - , phenylhydrazone, 102 44 D - , reaction of, with dihenzylamine, -, 1-deoxy-1-p-toluidino-o-, 199 200 -, 1,4:3,6-dianhydro-1-, 46, 66 with morpholine, 200 -, 1,5:3,6-dianhydro-~-,67 with p-phenetidine, 98 -, 1,4:3,6-dianhydro-2,5-dideoxy-2,5- with piperidine, 200 imino-u-, 48 with p-toluidine, 200 -, 1,4 :3,6-dianhydroS,5-di-O-t,osyl-r)-, -, 2,5-anhydro-o-, 358 47 -, 2 , 3 :5,6-di-O-isopropylidene-u-,32, reaction with ammonia, 48 34, 221 -, 1 , 4 :3,6-dianhydro-2,5-O-methylene--, 6-O-(~~-~-galactopyranosyl)-p-1,-, 73 n-, 47 -, O-glucosyl-, 296 -, 1,B-di-O-benzoyl-o-,41 -, 1,2,3,4,6-penta-O-benzoyl-~-, action 2,5-0-methylene acetal, 35 of ammonia on CY and p anomers, 124 -, 1 ,6-di-O-benzoyl-3,4-O-benzylidene--, 2,3,4,6-tetra-O-benzoyl-~-, 115 D-,35 -, tetra-0-methyl-o-, 288 -, 2,3,4-tri-0-acetyl-I,6-anhydro-o-, 228 2,5-O-methylene acetal, 35 -, 1,6-dichloro-l,6-dideosy-~-, 2,3,4,5- Mannoside, methyl 2,3-anhydro-4,6-0benzylidene-a-D-, 51, 52 di-0-ethylidene acetal, 41 isomeric di-0-isopropylidene acetals of, -, methyl 2,3-anhydro-4,6-di-O-methyI6-D-, 52 41 Mannosylamine, D-, N-substituted, 184 -, 2,j:3,5-di-O-methylene-u-, 6, 40 -, N-benzoyl-D-, 154 1,6-ditosylate, 43 tetraacetate, 154 -, 2,5-o-methylene-o-,45 -, N-n-butyl-D) 154 -, mono-0-benzylidene-mono-0-meth- -, N-p-carbethoxyphenyl-D-, 154 ylene-D-, 41 -, N-o-carbomethoxyphenyl-o-,154 -, 1,3:2,5:4,6-tri-O-ethylidene-~-, 44, -, N - (4-carboxy -3-hydroxyphenyl)-D-, 45 154 -, 1 , 3 :2,5 :4,6-tri-O-methylene-, 44 phenyl ester, 154 Mannonic acid, D-, 66 sodium salt, 154 from sulfite waste liquors, 322 -, N-ni-carboxyphenyl-D-, 154 51 Mannopyranose, 1,6-anhydro-p-~,-, -, N-a-carboxyphenyl-n-, 98, 154
422
-, N-p-carboxyphenyl-D-, 154
SUBJECT INDEX
-, 4-0-(~-~-glucopyranosyl)-~-1t-, hepN- (3,4-dimethyl-2-nitrophenyl) -D-, taacetate, 24'3 154 -, 2,3,4,6-tetra-O-acetyl-c-~-, 220, 248 -, 2,3,4,6-tetra-O-benzoyl-a-~-, 248 tetraacetate, 154 -, N-(4,5-dimethyl-2-nitrophenyl)-u-, -, 3,4 ,6-tri-0-acetJy1-o-,249 154 2-trichloroacetate, 249 Mannosyl fluoride, 3,6-di-O-acety1-4-0tetraacetate, 154 (2,3,4,6 - tetra - 0 - acetyl - 0 - D 6-trityl ether, triacetate, 154 glUC0Syi)-a-0-, 212, 246 -, N- (3,4-dimethylphenyl)- D - , 154 -, N-p-ethoxyphenyl-1)-, 154 -, 4-O-(p-~-glucopyranosyl)-a-~-, heptaacetate, 246 -, N-1-hexyl-o-, 155 246 -, N-l-(2-hydroxynaphthyl)-N-phenyl- -, 2,3,4,6-tetra-O-acetyl-c~-~-, Mannosyl halides, poly -0-srety 1-1-0ethyl-D-, 155 (D-glUCOSyl)-D-,232 -, N-(4-methyl-2-nitrophenyl) -D-, 155 -, tetra-0-acetyl-D-,232 tetraacetate, 155 Mannosyl iodide, 2,3,4,6-tetra-O-ace-, N-2-naphthyl-~-,155 tyl-, 256 -, N-nL-nitrophenyl-0-D-, 155 -, 2,3,4,6-tetra-O-benzoyl-, 256 -, N-o-nitrophenyl-B-D-, 155 Maple, holocellulose, analysis of, 316 tetraacetate, 155 holocellulose of sugar-, 320 -, N -p-nitrophenyl-0-o-, 155 wood, 316 dihydrate, 155 analysis of, 316 tetraacetate, 155 Melanoidins, 193 -, N-1-pentyl-u-, 155 Melezitose, 66, 77,80 -, N-phenyl-o-, 102, 155 azoate, 80 complex with sodium sulfate, 124 Melibiitol, 66 2,3-dimethyl ether, 155 Melibiose, 66, 218, 242 2,3,4,6-te tmme thy1 ether, 155, 156 action of ammonia on, 73 2,3,6-trimethyl ether, 155 0-, azoate, 80 2,4,6-trimethyl ether, 155 octaacetate, 218 3,4,6-trimethyl ether, 155 8-anomer, 88 -, N-4-phenylazophenylene-~-, 155 synthetic, 242 -, N-p-sulfamylphenyl D-, 156 synthesis of, 242 monohydrate, 156 Melibiosyl bromide, hepta-0-acetyl-a-, -, N-p-sulfophenyl-o-, 156 254 -, N-P-tolyl-D-, 102, 156, 199 Melibiosyl chloride, hepta-0-acetyl-a-, tetraacetate, 156 249 tetrabenzoate, 115 Melibiosyl fluoride, hepta-0-acetyl-, 246 2,3,4,6-tetrabenzoate, 156 Mannosyl bromide, 4-o-(@-D-glUCOpy- Melibiosyl halides, poly-0-acetyl-, 232 Melibiosyl iodide, hepta-0-acetyl-a-, 256 ranosyl)-a-o-, heptaacetate, 254 -, 2,3,4,6-tetra-O-acetyl-a-~-, 220, 241, Melibiulose, 73 Metasaccharinic acid, a-o-galacto-, 66 245, 253 Methane, diphenyl-, 177 methanolysis of, 236 Methylamine, 354 reaction with secondary amines, 239 Methylation, of glycosylamines, 109 solvolysis of, 236, 237 Methylene Blue, 188,201, 202 -, 2,3,4,6-tetra-O-benzoyl-a-~-, 253 Micrococcus pyogenes, 360 Mannosyl chloride, 2,3 :5,6-di-O-isopro- Milk, nitrogenous tetrasaccharide from pyiidene-D-, 221 human, 64 a anomer, 248 Mill, Lamp&, 308 -,
423
SUBJECT INDEX
Mit,osis, 360 Molasses, beet, 68 cane, blackstrap, 68, 87 fermentation residue of, 87 cane, yeast-fermentation residue of, 68 Molisch reagent, 89 Monosaccharides, separation from disaccharides, 57 Morpholine, 177, 200 Mucilage, flasseed, 72 slippery elm, 72 MucopolysBccharides, biosynthesis of, 367 classification of, 352 enzymic desulfation of, 356 infrared absorption spectra of, 358 Mucoproteins, linkages in, 172 Mycobacterium tuberculosis, polysaccharides of, 79 Myrrh, gum, 72
N Napthalene, ris-decahydro-, 18 trans isomer, 18 Neighboring-group effect, 235, 237 Neokestose, 64 Neolactosyl chloride, hepta-0-acetyl-, 216 a anomer, 249 Neurospora crassa, 368 Nicotinamide, glycosyl , 245 -, o-ribofuranosyl-, 245 Nigeran, 91 Norcamphane, 21 Nortropane, 21 Noslac, polysaccharide of, 72 Novocaine. See Procaine. Kucleic acid, deoxyribo-, 360 ribo-, 360 Nucleosides, purine and pyrimidine, permanganate oxidation of, 119 synthesis of, 243
0 Oak, 324 Ameriran white, 300 bark of, 93 hemicellulose-A and -B, from heartwood of, 301 from sapwood of, 301
hemicelluloses of, 301 overcup, 319 Southern red, 319 wood, hemicelluloses of, 313 Obituary, of Edmund George Vincent Percival, xiii Octadecylamine, reaction with o-fructose, 100 Octulose, D-glycero-D-g?do-, 87 keto form, heptaacetate, 87 -, 1-deoxy-1-diazo-keto-D-glycero-D-galacto-, hexaacetate, 87 Oligosaccharides, 208 acetates of, 295 a-D-linked, synthesis of, 241, 242 synthesis of, 240 of D-xylose, 300 Optical rotations, of poly-0-acetylglycosy1 halides, 231 relation to structure, of sugars, 230 Optical superposition, Van’t Hoff’s principle of, 230 Orcinol reagent, 76 Osazones, phenyl-, formation of, 197 mechanism of formation of, 197 preparation of, 172 7-Oxabicyclo[4.1.O]heptane, 21 Osalic acid, 314 Oxidation, with alkaline hypoidite, 312 with bromine, 312 with bromine-sulfuric acid, 351 with chlorous acid, 312 with chromium trioxide in pyridine, 43 with cupric salts, 187 187 with 2,6-dichlorophenolindophenol, with 0-dinitrobenzene, 187 with hydrazine(l87 with lead tetraacetate, 118,294 with nitric acid, 286 with nitrogen dioxide, 312 waith nitrogen tetroxide, 314 with oxygen in the presence of ascorbic acid, 359 with periodate, 116, 190, 196, 200, 312, 356, 357 with selenious acid, 187
P Panitol, dodecaacetate, 86 Panose, 86
424
SUBJECT INDEX
acetate, 86 -, 1-deoxy-1-piperidino-D-lhreo-, 203 isolation of, by column chromatog5-trityl ether, 196,203 raphy, 63 hydrochloride, 203 Paper, beater additive for, 289 -, 1 - deoxy - 1 - (p-toluidino)-L-erythl.o-, determination of pentosan in, 304 205 Paper birch, wood, 318 -, l-deosy-l-(p-toluidino)-D-threo-, 205 xylan of, 299 Peonin chloride, 240 Paper chromatography, 76,90,106, 287, Percival, Edmund George Vincent, obit295,307,318,322,326,327 uary of, xiii formation of glycosylamines during, Periodate oxidation. See Oxidation, with 121 periodate. of heparin, 343 Perseitol, 1,3:5,7-di-O-benzylidene-o-, quantitative, 292 30 of wood hydrolyzates, 285 -, 1,3:5,7-di-O-henzylidene-~-, 30 Papermaking, 315 Perseulose, D-,66 Pager pulp. See Pulps. L-, 66 Paraformaldehyde, 72. See also s-Tri- pH. See Hydrogen-ion conceptration oxane. Phenethylamine, 185 Paraldehyde, 15 p-Phenetidine, reaction of, with D-frucPeach, gum, 71 tose, 98 Pears, xylan from, 300 with D-galactose, 171 Peas, enzyme of, 73 with D-glucose, 97,170, 171,175 Pectic acid, 91 with D-mannose, 98 from wood, 313 with L-sorbose, 98 Pelargonin chloride, 240 Phenol, relative affinity for carbon, 59 Pentaerythritol, 67 -, p-phenylazo-, 82 Pentalene, octahydro-, 22 o-Phenylenediamine, 96, 198 cis form, 17 -, N , AT’-di- (tetra-0-acetyl-D-glucosyl)trans form, 17 4,5-dimethyl-, 112 2,4-Pentanedione, 177,201 I’hlorizin, 67 Pentitols, 1-arylamino-1-deoxy-, 200 Phloroglucinaldehyde. See BenzaldePentosans, 326 hyde, 2,4,6-trihydroxy-. alkali-insoluble, of wood, 308 Phloroglucinol, 305 of maple holocellulose, 316 Phosphoglucomutase, 368 of maple wood, 316 Phosphorus pentabromide, 228 removal of, from unbleached sulfite Phosphorus pentachloride, 215,221 pulps, 325 Photometer, Zeiss Schnell-, 291 resistant, of wood, 308 Picea ezcelsa,286,314 of wood pulps, 324 3-Picoline, 239 Pentulose, D-threo-, 73 Pine, alpha-cellulose of, 328 oxidation of, with cupric salts, 187 hemicelluloses of, 302 with 2,6-dichlorophenolindophenol, loblolly, 319,320 187 pectic material from, 313 with o-dinitrobenzene, 187 mannan of, 290 -, 1-alkylamino-l-deoxy-o-th,eo-,203 pulp from, 327 -, 5-deoxy-~-fhreo-, 73 slash, 319,320 -, 1 - deouy-l-(3,4-dimethylanilino)-~chlorite holocellulose of, 318 erythro-,171,205 -, 1 - deoxy-l-(3,4-dimethylanilino)-~- hemicelluloses of, 313 sulfate pulp from, 331 erythro-, 205 hydrogenation of, 192 sulfite cooking of, 307,322
SUBJECT INDEX
srilfit,e p u l p from, 310, 328 whit,e, hemicellulose of, 313 wood, glucan and niannari of, 326, 327 s y l a n of, 327 Pinus caribaea, 318 Pinus silvestris, 286 Piperidine, 200, 239 acetat,e, 179, 195 action of, on penta-O-ncet,yl-o-glucopyranoses, 112 on 2,3,4,6-t,etra-O-:icet,yI-D-gliicosr, 112 on 3,4,6-tri - 0 - acet,yl - D - glucosgl chloride, 112 -, N-o-arabinosyl-, 126 -, N-~-arabinosyl-,128 -, N-o-galactosyl-, 134 mutarotation of, 105, 194 -, i\i-D,-glUCOSyl-,97, 140 2-carbanilate, 150 decomposition of, 101 hydrochloride, 149 hydrogenation of, 104 2-methyl ether, 150 3-methyl ether, 150 reaction with hydrogen cyanide, 102 supposed mutarotation of, 104 2,3,4,6-tetraacetate, 113, 149 tetramethyl ether, 150 2-p-toluenesulfonate, 150 3,4,6-triacetate, 112, 149 2-carbanilate, 149 2-methyl ether, 149 2-p-nitrobenzoate, 149 2-p-toluenesulfonate, 149 -, N-D-mannosyl-, 156 mutarotation of, 105 -, A’%-xylosgl-, hydrochloride, 164, 196 Plasma. See Blood plasma. Plastic materials, use of glycosylnmines in, 125 Polygalitol. See Glucitol, 1,5-anhydroD-.
Poly-D-glucoses. See Glucans. Polyose, the term, 284 of wood, 311 Polysaccharides, associated with wood cellulose, 283 from horse intestines, 90 noncellulosic, of wood, 328 from sheep rumen, 90
425
from sprnce wood, 318 sulfated, reaction with Tolrridine Blue, 344 synthetic, 361 test for, 365 sulfatiori of, 361 triacetates of, linked 3 --f ~ - c Y - D - 224 , linked 4 -+ LwD-,224 linked 6 --f I-WD-, 224 Phlyuronides, of pulp, 313 Porphyrn uinhi/iraZis, mannan o f , 289, 290 Primeverasyl chloride, hexa-0-acetyl-a-, 249 Procaine, N-o-ghicosyl-, monohydrochloride, 140 monohydrate, 140 1,2-Propanediol, 67 2-Propanone, 1,3-dihydroxy-, oxidation of, with 2,6-dichlorophenolindophenol, 187 with o-dinitrobenzene, 187 -, hydroxy-, 195 -, phenyl-, 177 Propionic acid, 3-mercapto-, 177 Propylene glycol. See 1,2-Propanediol. Proteins, complexes with heparin, 338 Prothrombin, 359 Psicofuranose, 1,2:3,4-di-O-isopropylidene-D-, 73 Psicose, D - , 66, 73 phenylosazone, 73 Pulping, alkaline, 307 of hardwoods, 324 procedures for, 321 soda process, 321 sulfate process, 321 sulfite process, 321 Pulps, acetylation grade of, 295, 328 alkaline, 325 from hardwoods, 308 alpha-cellulose content of, 298 from aspen wood, 308, 309 bleached kraft, 309 bleached, soda, 308, 309 bleached, sulfate, 308, 309 bleached, sulfite, 308, 309 chlorite holocellulose, 308, 310 kraft-cooked holocellulose, 309 overhleached sulfate, 309
426
SUBJECT INDEX
resistant xylnn of, 309 xylan of, 309 bleached coniferous, 330 carboxyl content of, 298 for chemical conversion, 311 determination of pentosan in, 304 difficultly and easily accessible material of, 331 dissolving, 323 folding resistance of, 311 hardwood, 292 high-alpha, 324, 329 hydration capacity of, 311 mannan content of, 298 of mixed hardwoods, 325 for papermaking, 310 pentosan content of, 298, 324 pine sulfite, 290 rag, free from hemicellulose, 311 softwood, 296, 325 sulfate, alpha-cellulose content of, 327 carbohydrate composition of, 326 comparison with sulfite pulps, 327 glucan content of, 327 from hardwoods, 310, 327 prehydrolyzed, 327 , from softwoods, 327,328 sulfite, 310 alpha-cellulose content of, 327 comparison with sulfate pulps, 327 glucan content of, 327 hydrolysis of, 323 pentosans of, 324 from softwoods, 308, 310, 328 sulfite cooking of, 332 tear resistance of, 311 technical, uronic acid of, 313 tensile strength of, 311 unbleached sulfate, 292 carbohydrate composition of, 327 unbleached sulfite, removal of pentosans from, 325 uronic acid anhydride of, 328 white birch, 325 wood, 284, 321 alkaline extraction of, 308 carboxpl content of, 315 carboxyl groups of, 315 carboxylic acids in, 312 effect of beating, 308
effect of drying, 308, 309 effect of grinding, 308 gamma-cellulose of, 329 hydrolyzates of, 292 hydrophilicity of, 315 mannan content of, 298 mannan plus xylan content of, 329 pentosan in, 303 rate of beating of, 315 technical, 321 for viscose rayon, 311 xylan content of, 298 Pyran, 2,3-dichlorotetrahydro-,235 -, tetrahydro-, 16 Pyranose ring, conformations of, 232 Pyridine, 239 -, N-~-ribosyl-3-carbamoyl-,160 -, N-(2,3,4,6-tetra-O-acetyl-~-glucosyl) -3-carbethoxy-l , 2-dihydro-, 148 -, N - (2,3,4,6-tetra-O-acetyl-o-glucosyl)-l,2-dihydro-, 148 Pyridinium bromide, N-~-glucosyl-3carbamoyl-, 148 -, N-~-glucosyl-3-carbnmopl-l ,2 (or 1,6)-dihydro-, 148 2’, 3’, 4’, 6’-tetraacetate, 148 -, N-~-ribosyl-3-carbamoyl-,160 N’-acetyl derivative, 160 - , N-(tetra-0-acetyl-D-glucosyl)-, 150 -, N-(2,3,4,6-tetra-O-acetyl-u-glucosyl)-3-cyano-l,2-dihydro-, 148 Pyrrole, N-D-glucosyl-, 150 Pyruvaldehyde, 174, 195 bis-p-nitrophenylhydrazone,195 m-nitrobenzoylosazone, 174 p-nitrophenylosazone, 198 -, 3-phengl-, m-ni t rohenzoylosazone, 174
Q Quercetin, 94 -, glycosyl-, 93 Quercetrin, 94 Quercitol, 66 Quercitrin, 93 Quercus robur ,286 Quinoxaline, 2-(u-arabino-tetrshydroxybutyl)-, 198,200 Quinoxalines, preparation of, 172
SUBJECT INDEX
R Rafinose, 66,75 azoate, 80 hendecaacetate, 88 pentahydrate, 77 in raw beet sugars, 74 separation of, by column chromatography, 57 Rayon, cellulose acetate, 329 textile, 323 viscose, 331 Redox indicators, 188 Reductic acid, 188 Reductones, 101, 184, 188 formation of, 194 six-carbon, 193 the term, 188 Resorcinol, 193 Rhamnitol, 174 D - , 66 L - , 66 -, 1-amino-1-deoxy-L-, 205 Rhamnoglucosides, 94 Rhamnonic acid, 3,4-di-O-metliyl-~-, 267 amide, 267 lactone, 267 -, 2-O-methyl-~-,amide, 266 l,.l-lactone, 266 -, 4-O-methyl-~-,lactone, 267 -, 5-O-methyl-~-,lactone, 267 -, 2,3,4-tri-O-methyl-~-,lactone, 268 phenyIhydrazide, 268 -, 2,3,5-tri-O-methyl-~-,phenylhydrazide, 268 Rhamnopyranoside, methyl 2-0-methylL-, 266 I~harnnopyranosyluniine, 2,3-di-0methyl-N-phenyl-~.,267 Hhamnose, L - , 64, 67, 174 of aspen Nood, 287 henzoylhydrazone, 287 methyl ethers of. See the tables on pages 266 lo 268.
monohydrate, 77 -, 2,3-di-O-methyl-r-, 267 1,5-dit)enzyl ether, 267 -, 2,4-di -@methyl -I,-, 267 - , 3,4-di-O-methyl-r , 267 1,2-(methyl orthoacetate), 267
427
-, 2-O-methyl-1~-,266 -, 3-O-methyl-~-,266 phenylosazone, 266 hydrate, 266 -, 4-O-methyl-~-,266 phenylhydrazone, 266 phenylosazone, 266 triacetate, 266 -, 5-O-methyl-~-,267 2,3-0-methylene acetal, 267 phenylhydrazone, 267 phenylosazone, 267 triacetate, 267 -, 1,2,3,4-tetra-O-acetyl-~-, 218 -, 2,3,4-tri-O-methyl-~-,267 Rhamnoside, methyl 5-0-benzoyl-2,3di-0-methyl-L-, 267 -, methyl 2,3-di-O-methyl-a-~-,267 -, methyl 4-O-methyl-a-~-,267 p anomer, 267 -, methyl 5-O-methyl-a-~-,267 -, methyl 2,3,4-tri-O-methyl-a-~-, 268 @ anomer, 268 Rhamnosylamine, L-, with ethanol of crystallization, 157 with methanol of crystallization, 157 -, N-n-butyl-L-, 157 -, N-p-carbethoxyphenyl-L-,157 -, N-o-carboxyphenyl-L-, 98, 157 --, N-p-carboxyphenyl-L-, 157 -, N,N-dimethyl-L-, isopropylidene acetal, 157 -, N-(4,5-dimethyl-2-nitrophenyl) -L-, triacetate, forms I and 11, 157 -, N-ethyl-L-, 157 -, N-1-heptyl-L-, 157 -, N-1-hexyl-L-, 157 -, N-l-(2-hydroxynaphtliyl)-N-phenylmethyl-L-, 157 -, N-methyl-L-, 157 -, N-(4-methyl-2-nitrophenyl)-~-, 157 triacetate, 157 -, N-m-nitrophenyl-L-, 157 -, N-o-nitrophenyl-I,-, 157 triilcet:rte, 157 -, N-p-nitrophenyl-L-, 157 triacetate, 157 -, N-I-pentyl-I-, 157 -, K-pheny-L-, 168 2,3-dimethyl ether, 158
428
SUBJECT INDEX
2,4-dimethyl ether, 158, 267 I) , phosphates, chromatography of, 76 2-methyl ether, 266 D-, separation from o-arabinose, 124 2,3,4-trimethyl ether, 158, 268 DL-, 72 -, N-4-phenylazophenylene-L-, 158 DL-, p-tolylsulfonylhydrazone,72 -, N-n-propyl-L-, 158 -, 3,5-di-O-benzoyl-~-,1,2-0rthobenzoate, 239 -, N - p sulfamylphenyl-L-, 158 -, N-p-sulfophenyl-L-, 158 -, 2,3-di-O-methyl-o-, 265 -, N-p-tolyl-L-, 158 -, 3,5-di-O-methyl-o-, phenylosazone, Rhamnosyl bromide, 2,3,4-tri-O-acetyl265 a - ~218, , 230, 253 -, 1,2-0-isopropylidene 3,5-di-O-tosyl-, 2,3,4-tri-O-benzoyl-a-~-, 253 I ) - , 35 Rhamnosyl chloride, 2,3,4-tri-O-acetyl- -, 5-O-methyl-o-, 265 W L - , 248 p-bromophenylosazone, 265 -, 2,3,4-tri-O-benzoj.l-a-~-, 248 -, 1,2,3,4-tetra-O-acetyl-o-, 112 Rhamnosyl halides, tri-0-acetyl-L-, 232 -, 1,2,3,5-tetra 0-acetyl-D-, 211, 212 Rhamnosyl iodide, 2,3,4-tri-O-henzoyl- -, 2,3,4,5-tetra-O-acetyI-aldehydo-D-, a-L-,256 100 Rhaninus jranguln, 94 -, 1 , 2 , 3 , 4 tetra-0-benzoyl-p-D-, 220 Ribitol, 66 --, 2,3,5-tri-O-benzoyl-13-~-, 239 -, 1-amino-1-deoxy-, 173 -, 2,3,4-tri-O-rnethyl-o-, 265 -, 1-amino-1-deoxy-D-, 206 -, 2,3,5-tri-O-methyl-~-,265 -, 1-amino-1-deoxy-L-, 205 Rihoside, methyl 2,3-anhpdro-4-0-, 1-deoxy-1- (3,4-dimethylanilino) -D-, methj l - p - ~ . , 265 171 -, methyl 2,3,4-tri-O-methj~l-o-, 265 -, l-deoxy-l-(3,4-dimethylanilino)-~-,-, methyl 2,3,5-tri 0-methyl-D-. 265 Rihosylamine, D-, 159 192 Riboflavine, preparation of, 124, 171 -, N - a r y h - , 98 Ribofuranose, 2,3-0-isopropylidene-~-,-, 2-deoxy-N-phenyl-1-, 160 34 - , 2-deoxy-N-phenyl-~-,161 derivatives of, 32 -, 2-deoxy-N-p-tolyl-~-,160 tosylation of, 34 -, 2,3-di-O-acetyl-N-(2-amino-4,5-diRibonic acid, D-, amide, 77 methylphenyl)-5-O-trityl-~-, 159 -, 2,3,4-tri-O-methyl-o-, 265 -, N-(4,5-dimethyl-2-nitrophenyl) -D , 159 lactone, 265 -, 2,3,5-tri-O-methyI-~-, 265 triacetate, 159 tritylation of, 116 lactone, 265 5-trityl ether, 159 phenylhydrazide, 265 2,3-diacetate, 159 Ribonuclease, pancreatic, 360 Ilihopyranosylamine, N-p-carl)oxy- -, N-(3,4-dimethylphenyl)-o-, 150 p heny 1-a-o-, 159 isomers A and B, 159 -, N-o-chlorophenyl-a-D-, 159 -, N-ethyl N-2-nitrophenyl-~-,159 -, N-(4,5-diethyl-2-nitrophenyl)-~-, 159 -, N - (4-ethyl-2-nitrophenyl)-I)-, 159 -, N-(3-hydroxy-4-methylphenyl)-~-, -, N-o-nitrophenyl-I)-, 159 isomers A arid B, 159 159 -, N -4-methoxyphenyl - a - ~ -159 , -, N-phenyl-D , 109 -, N-l-Ilaphthyl-ol-I>-,159 acetvlation of, 111 -, N-2-naphthyl-n-, 359 attempted methylation of, 110 comple\ with sodium sulfate, 124 -, N-phenyl-i)-, acet:itc, 101 infrared absorption spectrum, 102 Rihose, D - , 66 isomer A, 159, 160 D - , condensation with amines, 124
SUBJECT INDEX
isomer B, hemihydrate, 160 isomeric forms, 98 triacetate, 160 2,3,5-trimethyl ether, 160,265 tritylation of, 116 -, N-p-tOlyl-D-, 160 hemihydrate, 160 with 2 ethanol of crystallization, 160 -, 2,3,4-tri-O-acetyl-N- (4,5-dimethyl2-thioformamidophenyl)-~-,159 Ribosyl bromide, 2,3,4-tri-O-acetyI-pD-, 253 -, 2,3,4-tri-O-acetyl-~-,253 -, 2,3,5-tri-O-acety1-11-,210, 253 LY anomer, 243, 244, 245 -, 2,3,4-tri-O-benzoyl-a-~-, 237,253 p anomer, 220, 237, 253 -, 2,3,5-tri-O-benzoyl-~-,239 Ribosyl chloride, 3,4-di-O-acetyl-2-deOXY-D-, 245, 247 -, 2,3,4-tri-O-acetyl-p-o-, 248 -, 2,3,5-tri-O-acetyl-~-,212 a anomer, 248 -, 2,3,4-tri-O-bensoyl-a-~-, 220,237,248 p anomer, 220, 237, 248 Ribosyl halides, poly-0-acyl-, 208 Robinobiosyl chloride, hem-0-acetyla-,249 Rutin, 94 Itutinosyl chloride, hem-0-acetyl-a-, 249 S
Saccharic acid. See Glucaric acid, D - . Saccharin, tetra-O-acetyl-I)-glucosyl-,137 Saccharinic acid, 184 in pulping waste liquors, 326 Sacchnrorrryces ba!lanus, 294 Salep, mannan of, 288,290 Salicin, 67 Salicylic acid, 4-amino-, 125 Saponins, 209 of wood, 70 Sapote, gum, 79 Sapwood, of oak, 301, 302 Scots pine, hemicelluloses of, 64 Sedoheptulosan, 76 Sedohept.ulose,73,76 Selenions acid, act,ion on D-fructose, 187 Seliwanoff reagent, 193
429
Seliwanoff test, for ketose, 201 Serine, acyl derivatives, periodate oxidation of, 117 -, N-D-glucOSJ’l-DL-,150 Sheep, polysaccharide from riimen of, 90 Shock, anaphylactic, 337 Silene EF, 77, 78, 83, 88 Silica, f o r column chromatography, 89 Silicic acid, 80, 81, 86, 94 for column chromatography, 93 Silicon tetrachloride, 219 Silver chloride, activated, 210, 219, 220 Silver fluoride, 221 Slash pine, alpha-cellulose of, 295, 296 holocellulose of, 290 mannan of, 290, 296 Slippery elm, mucilage, 72 Soda process, for pulping wood, 321, 325 Sodium nitroprusside, 195 Sodium sulfate, complexes with glycosylamines, 124 Softwoods, hemicelluloses of, 302 holocellulose of, 325 sulfate pulps from, 327, 328 sulfite pulps from, 325, 328 Sophorose, 87 Sorbitol. See Glucitol, D Sorbose, L-, 66, 77, 175 bimolecular dianhydrides of, 63 reaction of, with aliphatic amines, 100 with p-phenetidine, 98 -, 6-deoxy-~-,73 Sorbosylamine, N-p-ethosyphenyl-I,-, 161 -, N-phcnyl-L-, 124 Sorbosyl chloride, 1,3,4,5 tetra-0)-acetyla - ~248 , Spruce, alkaline pulping of, 326 alpha-cellulose of, 328 black, 319, 320 ’ hemicelluloses o f , 64, 313 holocellulose of, 296 mannan in, 296 pectic materiel from, 313 hemicellulose of, 290 holocellulose of, 314 mannan of, 290 pulp from, 327 sulfite cooking of, 307,322
430
SUBJECT INDEX
sulfite pulp from, 310,323,325,331 wood, 318 Stachyose, 66,75 preparation of, 63 structure of, 91 Stannic chloride, 219 Starch, as adsorbent in column chromatography, 74,75 banana, 89 corn, fractionation of, 70 degradation of, 223 fractionation of, by column chromatography, 79 highly polymerized, 346 hydrolysis products from, 86 separation of, by column chromatography, 63 oxidation of, in the presence of ascorbic acid, 359 potato, 223 fractionation of, 70 rice, 89,223 structure of, 224 -, 6-deoxy-6-iodo-2,3-di-O-tosyl-, 224 -, tri-0-methyl-, 224 -, tri -0-tosyl-, 224 Sterculia setigera, gum, 71 Steric hindrance, 237 Straw, of wheat, 90 Styracitol. See Mannitol, 1,5-anhydroD-.
“Substance X,” from hemicelluloses, 287 Succinamide, N - (tetra-0-acetyl-D-gluco~ y l ) - ,150 Succinic acid, 66 Succinimide, N-o-glucosyl-, 150 dihydrate, 150 Sucrose, 64,66, 76, 77, 80 azoate, 80 octaacetate, 88 chemical synthesis of, 87,241 synthesis of, 241 x-ray study of, 13 Sugar acids, column chromatography of, 65 Sugar beets, aralmn of, 90 Sugars, alkyl orthoesters of, reaction of, with hydrogen halides, 227 with tit nriium tetrachloride, 227 aiihgdro derivatives of, 208
arylhydraxones of, 99 azoates. See table of sugar azoates suitable f o r chromatography, on page 80.
complexes with borate ion, 75 deoxyseleno derivatives, 208 deoxythio derivatives, 208 diazo derivatives, 87 effect of boric acid on electric conductivity of, 231 invert, 69 methyl ethers, characterization of, 124 mutarotation of, 106, 107 orthoesters of, 208 partially methylated, azoates of, 89 p-phenylazobenzoates, 79 raw beet, raffinose in, 74 reducing, reaction with ammonia, 106 relative affinity of, for carbon, 59 Sulfamic acid, 354 -, cyclohexyl-. See Cyclohexanesulfamic acid. Sulfapyridine, condensation with sugars,
125
-,
N-D-galactosyl-, 134 -, N-D-glUCOSyl-, 150 Sulfate cook, of sulfite pulp, 325 Sulfate process, for pulping wood, 321,
326 sulfate liquor for, 326 Sulfation, of polysaccharides, 361 Sulfite cooking, removal of carbohydrates during, 332 Sulfite process, for pulping wood, 321,
322 Sulfite pulp, mnnnan content of, 325
T Tagntose, D-, 71, 73 -, 1 -deoxy-1-(p-to1uidino)-u-, 205 Taka-diastase, 64 action on oak hemicelluloses, 301, 302 Talitol, u - , 66 -, 1 , B : 2,4:5,G-tri-O-l)eiizylidciir-i~-,38 -, 1 , 3 :2,4:5,6-tri-0-mrt hylene-I)-, 38 Talopyranose, 1,G:2,3-dianhydro-p-~-, 51
Talose, 1 ,G:2,3-cliatilri~dro-P-u-,52 Tnlose, 1,6:3,4-dianhydro-p-~-,51, 52
SUBJECT INDEX
43 1
Taloside, methyl 2,3-unhydro-4,6-0itcetylcellobiosyl bromide, 113 bcneylidene-D-, 52 on hepta-0-acetyllactosyl bromide, 01 anomer, 51, 93 113 p anomer, 51 quaternary ammonium salts from, 114 Talosyl bromide, 2,3,4,6-tetra-O-acetyl- Triose, phosphate, 73 D-, 253 Triose-reduct one, 188 Tartaric acid, 314 s-Trioxane, 15 dextro-, 66 Triphenylmethyl ethers. See under reTextiles, fibers for, 323 spective tri tyl ethers. Theophylline, silver salt, 228, 229, 245 s-Trithiane, 15 -, D-arahinofuranosyl-, 244 -, 2,4,6-trimcthyl-, 15, 31 -, (2-deoxy-o-glucopyranosy~)-,229 Y‘rilicum repens L., 91 -, (2-deoxy-~-ribopyranosyl)-,229 Triulose, 1 deoxy-1-(N-met hylnnilino) -, 7-(3,4-di-O-acetyl-2-deoxy-or,p-~3-phenyl-, 204 ribosy1)-, 245 hydrochloride, 204 -, D-xylofuranosyl-, 244 semicarbaxone, 204 Thiapyran, 2,3-dichloro-tetrahydro-, 235 Tryptophan, 360 Threitol, 1 , 3:2,4-di -0-methylene-w , 19 Turanose, 66 Threonine, 360 Turanosyl bromide, hepta-0-acetyl-p-, Thrombin, 359 254 Thrombokinase, 359 Turanosyl chloride, hepta-0-acetyl-p-, 249 Thrombosis, 359 Thymine, nucleosides of, 243 Turanosyl iodide, hepta-0-acetpl-, 256 Tires, cord for, 323 Tyrosine, 360 Titanium tetrabromide, 218, 228 Titanium tetrachloride, 218,222, 227,228 U Titanium trichloride, 188 0-Toluidine, reaction with o-glucose, 176 U h u s julva. See Slippery elm. Ultraviolet absorption spectra, 186 p-Toluidine, 99, 200 action of, on 2,3,4,6-tetra-o-acetyl-n-Unibilicaria pustulata, 72 sugars of, 64 glucose, 113 See Arabitol, 3-0-(p-~-galacUmbilicin. on 2,3,4,6-tetra-0-acetyl-01-~-glucotopyranosy1)-D-. syl bromide, 113 Urea, N,N’-di-(L-arabinosyl)-, 128 condensation with aldose methyl hexabenzoate, 128 ethers, 110 -, N , N’-di-(D-gIucosyl) -, 136 reaction with D-glucose, 97, 170, 175 octaacetate, 136 ultraviolet absorption spectrum of, octabcnzoate, 136 186 -, N , N’-di- (D-mannosyl) -,154 -, N-formyl-, 190 -, N,N’-di- (D-xylosyl) -, 162 -, N-methyl-, 196 Uridine, synthesis of, 243 Toluidine Blue, 339, 344, 365 Uronic acids, associated with cellulose, Tosylation-iodination, of starch, 224 284 Transglycosidation, 218, 219 column chromatography of, 94 Transglycosylation, of aldosylamines, 99 determination of, 286 Trehalose, 66, 76 in cellulose and wood, 314, 315 01,01-, 64 2-furaldehyde from, 304 azoate, 80 of holocellulose, 319 B,B-, 87 moieties of, in wood, 284, 286 azoate, 80 monomethyl ether, 306 Trimethylamine, action of, on hept,a-0-
432
SUBJECT INDEX
in wood hemicelluloses, 313 of wood pulps, 327 Uronic anhydrides, of alpha-cellulosc, 320 of holocelluloses, 319 in pulps, 328 of woods, 285
V Vaccznium n2 yrtzlliis, 93 Vrgetahle ivory, mannan of, 288 Viscose, process for, 311 rayon, 284 effect of mannan and sylan on tensile strength of, 297 laundering resistance of, 311 wood pulps for, 311 Viscosity, intrinsic, of cellulose acetate, 297 Viscosity ratio, the term, 297 Vitamin B,? , 244
W Walden inversion, 230, 234 of glycosyl halides, 210 Western hemlock, holocellulosc from, 312 pectic acid from, 313 Wheat, 324 Wohl degradation, 123 Wood, anatomy of, 321 ash, 286 aspen, 286, 303 hydrolysis of, 287 birch, 286 hlack spruce, 285 carbohydrate composition of, 285, 326 cedar, 286 cellulose from, 284, 328 carbohydrates in, 287 preparation of, 316 characterization of, 317 commercial cellulose from, 321 coniferous, 285, 327 isolation of Carbohydrates from, 317 mannan content of, 296 deciduous, 286 delignification of, 316 determination of mannan in, 290 Douglas fir, 285
extraction with a l l d i , 318 cvtractivcs of, 284 hemicelluloses of, 313 hydrolyzates of, 292 isolation of carbohydrates from, 317 loblolly pine, 285 maple, 316 analysis of, 316 mesquite, 286 nitrated cellulosic material from, 330 oak, 286 paper birch, 318 pectic material in, 312, 313 pine, 286 polysaccharides of, 283, 328 pulps. See Pulps, wood. saponins of, 90 Southern red oak, 285 spruce, 286, 318 sulfate cook of, 307 sulfite cook of, 307 summative analysis of, 304 Western hemlock, 285 nitration of, 330 Wrstcrn red cedar, 285
X Xanthic acid, glycosyl esters, reductive desulfurization of, 245 Xanthorhnmnin, 94 X-rays, for determining crystal strncturc of a-o-glucopyranose, 231 Xylan, 284, 296, 299, 319, 320, 324 acetate, 86 acetylation of, 311 acidic, 310, 313 from birch, 303 of alpha-cellulose, 328 association of, u i t h cellulose, in wood, 307,309 with lignin, 310 from barley straw, 299 of birch, 327 from corn cobs, 300 crystalline, properties of, 299 determination of, 303 diacetate, 311 effect of different alkalis on, 308 from esparto grass, 90, 299 hemicellulosic. 311
SUBJECT INDEX
433
neutral type of, 310 -, 3-O-methyl-~-,lactone, 258 -, 2,3,4-tri-O-rnethyl-o-, lactone, 260 from paper birch, 299 phenylhydrazide, 260 partial hydrolysis of, 63 -, 2,3,5-tri-O-methyl-o-,amide, 260 from pears, 300 lactone, 260 of pine, 327 phenylhydrazide, 260 properties of, in wood cellulose, 307 resistant, 311 Xylopentaose, D-, 63 of aspenwood pulp, 308, 309 Xylopyranoside, methyl B-D-, 322 isolation of, 309 -, methyl 2-0-methyl-p-o-, 257 cf wood, 308 3,4-diacetate, 257 structure of, 299, 300 3,4-ditosylate, 257 sulfated, 361 Xylopyranosylamine, 2,3-di-O-methyl of sulfite pulps, 323 N-phenyl-D-, 258 -, 2-O-methyl-N-phenyl-~-, 257 the term, in wood chemistry, 284 -, 3-O-methyl-N-phenyl-~-D-,258 of unbleached sulfate pulps, 32i of \+heat straw, 90 Xplose, 318, 322 D-, 64, 67, 77, 173, 314 of woods, 285,299,300 determination of, 293, 306 3,4-Xylidine, condensation with uarahinose, 171 D-, fermentation of, 306 Xylitol, 66 D-, 2-furaldehyde from, 305 -, 1,3-anhydro-2,4-0-methylene-~~-, 20 W D - , tetraazoate, 80 -, l-deoxy-2,4-0-methylene-~-, 30 D - , 5-trityl ether, 173 -, l-deoxy-2,4-0-methylene-3,5-di-O- DL-, 72 tOSyl-D-, 30 -, 3,5-anhydro-1,2-0-isopropylidene-, 2,4:3,5-di-0-benzylidene-l-0-tosylu-, 20 DL-, 43 -, di-0-benzylidene-D-, dimethyl acetal, -, 2,4: 3,5-di-O-methylene-l-O-tosyl306 D L - , 43 -, di-0-benzylidene-L-, dimethyl acetal, Xylobiose, D-, 63, 300 306 X ylof uranose , 3-O-met h yl -D -, 257 -, di-0-(p-isopropylbenzy1idene)-D-,di1,2-O-isopropylidene acetal, 257 methyl acetal, 306 5-tosylate, 257 -, di-0-(p-isopropylbenzy1idene)-L-,diXyloheptaose, D-, 63 methyl acetal, 306 Xylohexaose, D - , 63, 300 -, 1,2: 3,5-di-O-isopropylidene-ol-o-, 32, Xylonic acid, compound with cadmium 36 bromide, 306 -, 2,3-di-O-methyl-a-~-,91, 258 D - , from sulfite waste liquors, 322 -, 2,4-di-O-methyl-p-n-, 259 -, 2,3-di-O-methyl-~-,amide, 259 -, 2,5-di-O-methyl-~-,259 p-bromophenylhydrazide, 259 -, 3,4-di-O-methyl-o-, 259 lactone, 259 -, 3,5-di-O-methyl-~-,260 phenylhydrazide, 259 p-bromophenylosazone, 260 -, 2,4-di-O-methyl-o-, amide, 259 1,2-O-isopropylidene acetal, 260 lactone, 259 -, hexa-0-acet yl - d d e hydo -D -, 86 phenylhydrazide, 259 -, 1,2-0-isopropylidene-3-0-methyl-5-, 3,4-di-o-methyl-o-,lactone, 259 0-tosyl-D-, 257 phenylhydraxide, 260 -, 1,2-O-isopropylidene-5-0-methyl-3-, 3,5-di-O-methyl-1-, lactone, 260 0-tosyl-D-, 258 phenylhydrazide, 260 -, 2-0-methyl-p-~-,257 -, 2-O-methyl-n-, amide, 257 triacetate, 257 -, S - o - m e t h y l - ~ ~258 -, lactone, 257
434
SUBJECT INDEX
p-hromophcnylosazone, 258 -, N-o-nitrophenyl-u-, 162 phenylosazone, 258 triacetate, 162 -, 4-O-methyl-~-,258 -, N-p-nitrophenyl-a-u-, 162 phenylosaxone, 258 p anomer, 162 -, 5-O-methpl-~-,258 -, N-phenyl-D-, 124, 163 p-bromophenylosazone, 258 2,3-dimethyl ether, 163 -, 2,3,4-tri-O-benzoyl-~-,115 2,4-dimethyl ether, 163, 259 -, 2,3,4-tri-O-methyl-o-, 260 3,4-dimethyl ether, 163 -, 2,3,5-tri - O - m e t h y - ~ -260 , 2-methyl ether, 163 Xyloside, methyl 2,3-di-O-methyl-a-~-, 3-methyl ether, 163 258 triacetate, 163 p anomer, 258 2,3,4-trimethyl ether, 163,260 4-tosylate, 258 -, N-p-sulfacetamidophenyl-u-, 164 -, methyl 2,4-di-O-methyl-p-u-, 259 -, N-p-sulfamylphenyl-n-, 164 3-tosylate, 259 -, N-p-sulfophenyl-o-, 164 -, methyl 3,4-di-O-methyl-p-o-, 259 -, N-P-tolyl-D-, 164 2-tosylate, 259 tribenxoate, 115, 164 -, methyl 2,5-di-0-methyl-3-0-t osyl-a- Xylosyl bromide, 2,3,4-t ri-0-acctyl-aD-, 259 D- , 237, 253 0 anomer, 259 methanolysis of, 236 -, methyl 4-O-rnethyl-B-o-, 258 reaction with secondary amines, 239 -, methyl 5-0-methyl-3-0-tosyl-a-~-, -, 2,3,4-tri-O-acetyl-~-, 253 258 -, 2,3,4-tri-O-benxoyl-n-, 253 p anomer, 258 01 anomer, 237 -, methyl 2,3,4-tri-O-metliyl-a-~-, 260 -, 2,3,4-tri-U-l,enzoyI-~-, 253 p anomer, 260 Sylosyl chloride, 3,4-di-O-benxoyl-2-, methyl 2,3,5-tri-O-methgl-u-,260 ch~oro-2-deoxy-a-o-, 248 Xylosylamine, D-, 162 -, 2,3,4-t ri-0-acetyl-a-D-, 248 triacetate, 162 p anomer, 248 -, N-o-aminophenyl-o-, 162 Xylosyl fluoride, 2,3,4-tri-U-acetyl-atriacetate, 162 D-, 246 -, N-n-butyl-D-, 162 Xylosyl halides, tri-0-acety-D-, 232 -, N-p-carboethoxyphenyl-u-, 162 Xylosyltrimethylammonium bromide de-, N-o-carbomethoxyphenyl-D-, 162 rivative. See under Ammonium -, N-o-carboxyphenyl-D-, 162 bromide. -, N-p-carboxyphenyl-D-, 162 Xylotetraosc, D-, 63 -, N-(4-chloro-2-nitrophenyl)-~-,162 Sylotriose, D - , 63 triacetate, forms I and 11, 162 Xylulose, u-. See Pentulose, D-threo-. -, 2-deoxy-N-phenyl-~-,163, 164
, N-(4,5-dimethyl-2-nitrophenyl)-~-,
-
162 triacetate, 162 -, N - o - (1-ethoxyethylideneamin0)phenyl-D-, triacetate, 162 -, N-1-hesyl-D-, 162 -, N - (4-methyl-2-nitrophenyl) -D-, 162 triacetate, forms I and 11, 162
Y Yarns, rayon, 297 viscose, strengt,h of, 311 Teasts, 306 crystalline hexokinase of, 368 mannan of, 289, 290
ADVANCES IN CARBOHYDRATE CHEMISTRY Volume 1 C . S . HUDSON. The Fischer Cyanohydrin Synthesis and the Configurations of Higher-Carbon Sugars and Alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NELSONK . RICHTMYER, The A1t)rose Group of Substances . . . . . . . . . . . . . . . . . . . . EUGENE PAC.SU.Carbohydrat.e Orthoesters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ALBERTI, . RAYMOND. Thio- and Seleno-Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROBERTC . ELDERFIELD, The Carbohydrate Components of the Cardiac Glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . JELLEFF CARRand JOHN C . KRANTZ, JR., Metabolism of t.he Sugar Alcohols and Their Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . STUARTTIPSON, The Chemistry of the Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . THOMAS JOHN SCHOCH, The Fractionation of Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . ROYL . WHISTLER,Preparation and Properties of Starch Esters . . . . . . . . . . . . . . . CHARLESR . FORDYCE, Cellulose Esters of Organic Acids . . . . . . . . . . . . . . . . . . . . . ERNESTANDERSONA N D LILA SANDS,A Discussion of Methods of Value in Research on Plant Polyuronides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 37 77 129
147 175 193 247 279 309 329
Volume 2 C . S . HUDSON, Melezitose and Turanose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . STANLEY PEAT, The Chemistry of Anhydro Sugars . . . . . . . . . . . . . . . . . . . . . . . . F . SMITH,Analogs of Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . R . LESPIEAU,Synthesis of Hexitols and Pentitols from Unsaturated Polyhydric Alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HARRY J . DEUEL,JR. and MARGARET G . MOREHOUSE, The 1nterrelat)ionof Carbohydrate and F a t Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M . STACEY, The Chemistry of Mucopolysaccharides and Mucoproteins . . . . . . . . . TAYLOR H . EVANSand HAROLD HIBBERT,Bacterial Polysaccharides . . . . . . . . . . E . L . HIRSTand J . K . N . JONES, T h e Chemistry of Pectic Mat.erials. . . . . . . . . . EMMAJ . MCDONALD, The Polyfructosans and Difructose Anhydrides . . . . . . . . . . JOSEPH F . HASKINS,Cellulose Ethers of Industrial Significance . . . . . . . . . . . . . . .
1
37
107 119 161 203 235 253 279
Volume 3 C . S. HUDSON, Hist.orical Aspects of Emil Fischer’s Fundamental Conventions for Writing Stereo-Formulas in a Plane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . G . V . PERCIVAL, The Structure and Reactivity of the Hydrazone and Osazone Derivatives of the Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HEWITTG . FLETCHER, J R . , The Chemistry and Configuration of the Cyclitols., BURCKHARDT HELFERICH, Trityl Ethers of Carbohydrates. . . . . . . . . . . . . . . . . . . . . LOUIS SATTLER,Glutose and the Unfermentable Reducing Substances in Cane Molasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JOHNW . GREEN,The Halogen Oxidation of Simple Carbohydrates, Excluding the Action of Periodic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
1 23 45 79 113 129
43G
ADVANCES IN CARBOHYDRATE CHEMISTRY
JACK ConirToN. The Molecular Coiist.it.ut.ionof Cellulose .
. . . . . . . . . . . . . . .
SAMUEL OURIN. 1sot.opic Tracers in the Study of C:tabohydrate htet.abolism . . KARL~ Z Y R B A C K . Products of the Enzymic Degradation of Starch and Glycogen . S. rAcm and P . . KENT.The Polysaccharides of MycoDacteriirni tuberculosis. It . 17 . 1,E M I E U X :tnd M . L . WOLFROM. The Chemistry of Streptomycin . . . . . .
w
185 229 251 311 337
Volume 4
IRVING LEVI A N D CLIFFORD B . PURVSS. The Structure and Configuration of Sucrose (alpha-D-GlucoIJyrnnosyl Deta-D- Fructofuranoside) . . . . . . . . . . . . . . 1 H . G . BRAYand M . STACEY. Blood Group Polys.iccharides . . . . . . . . . . . . . . . . . 37 C . S. HUDSON. Apiose and the Glycosides of t.he Parsley Plant . . . . . . . . . . . 57 CARLNEUBERG, Diochemical Reductions a t t.he Expense of Sugars . . . . . . . . . . 75 VPNABCIODEIJLOKEU, Thc Acylated Nit.riles of Alclonic Acids and Their Degra119 dation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ELWINE . HARRIS.Wood Saccharificat.ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 J . BBESEKEN. The Use of Boric Acid for the Determination of the Configuration of Carbohydrat.es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 ROLLANDLOHMAR and R . M . GOEPP.JR.,The Hexit.ols and Some of Their Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 J . K . N . JONES and F . S%cI.rH.Plant. Gums and Mucilages . . . . . . . . . . . . . . . 243 I. . F . WIGGINS, The Utilization of Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Volume 6 HEWITTG . FLETCHER. J R . ,and NELSON K . RICHTMYER. Applications in t.he Carbohydrate Field of Reductive Desu1furizat)ion by Raney Nickel . . . . . . . . . 1 W . Z. HASSII)and M . DOUDOROFF. Enzymatic Synthesis of Sucrose and Other 29 Disaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ALFREDGOTTSCHALK. Principles Underlying Enzyme Specificity in the Domain of Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Z . I . KERTESZ and R . J . MCCULLOCH. Enzymes Acting on Pectic Subst.ances. . . . 79 R . F . NICKERSON. T h e Relative Crystallinity of Celluloses . . . . . . . . . . . . . . . . . . . 103 G . R . DEANand J . B . GOTTFmED. The Commercial 1’roduct)ion of Crystalline 127 Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . J . BOURNE and STANLEY PEAT,The Methyl Ethers of n-Glucose . . . . . . . . 145 L . F . WIGGINS,Anhydrides of the Pentitols and Hexitols . . . . . . . . . . . . . . . . . . 191 MARYL . CALDWELL and MILI)RED ADAMS, Action of Certain Alpha Amylases . . . 229 ROYI, . WHISTLER. Svlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Volume 6 E . L . HIRST, Obituary of Walter Norman Haworth . . . . . . . . . . . . . . . . . . . . . . . . . . D . J . BELL,The Met.hyl Ethers of D-(falactose . . . . . . . . . . . . . . . . . . . . . . . . . W . L . EVANS. D . D . REYNOLDS arid E . A . TALLEY, The Synthesis of Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . H . NEWTH.The Formation of Furan Compounds from Hexoses . . . . . . . . . . . . . RICHARD E . REEVES,Cuprammonium-Glycoside Complexes . . . . . . . . . . . . . . . . . ROGERW . JEANLOZ and HEWITTG . FLETCHER. J R . , The Chemistry of Ribose . . . NELSONK . RICHTMYER. The 2-(Aldo-polyli~~droxyalkyl)benzimidazoles ....... ELLIOTTP. BARRETT, Trends in the Development. of Granular Adsorbents for Sugar Refining., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
11
27 83 107 135 175 205
A DV.kNCES I N CAIEBOHYDIEATE CRISMISTRY
437
ROBERTELLSWORTH MILLERand SIDNEY M . CAN.roR. Aconit.ic Acid. a Byproduct. in the Manufacture of Sugar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 WrrmAM A . RONNER. FriedelLCrafts and Grignard Processes in the Carbohydrat.e Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 JOHN C . SOWDEN. T h e Xitromethane and 2-Nitroethanol Syntheses . . . . . . . . . . 291
Volume 7
R . -4. LAIDLAW and I