Advances in Carbohydrate Chemistry, Volume 16

ADVANCES IN CARBOHYDRATE CHEMISTRY VOLUME 16

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Advances in Carbohydrate Chemistry Editor MELVILLE L. WOLFROM Associate Editor R. STUART TIPSON Board of Advisors J. C. SOWDEN ROYL. WHISTLER

R. C. HOCKETT W. W. PIQMAN C. B. PIJRVEB

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

STANLEY PEAT

MAURICESTACEY

Volume 16

1961 ACADEMIC PRESS

NEW YORK and LONDON

Copyright 0, 1961, by Actldemic Press Lac, ALL RIGHTB REBEBVED NO PART O F T H I S BOOK MAY B E BEPRODUCED I N ANY FORM, BY PHOTOBTAT, MICROFILM, OR ANY OTHER MEANB, WITHOUT WRITTEN PERMIBBION FROM THE PUBLIBHERB.

ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORE3, N. Y. United Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON) LTD. 17 OLDQUEENSTREET,LONDON, S.W.1

Libraw of Congreee Culolog Card Number: 46-11361

PBINTED I N T H E UNITED STATE8 O F AMERICA

'

LIST OF CONTRIBUTORS

T. G. BONNER, Department of Chemistry, Royal Holloway College, University of London, Englejield Green, Surrey, England

JOHNR. CLAMP, Department of Chemistry, The University, Bristol, England H. DEUEL, Laboratory of Agricultural Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland P. DUBACH, Laboratory of Agricultural Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland

R. D. GUTHRIE,Shirley Institute, Manchester, England* JOHNL. HICKSON, Sugar Research Foundation, Inc., New York, New York L. HOUGH,Department of Chemistry, The University, Bristol, England ALMUTHKLEMER, Organisch-Chemisches Institut der Universitdt, Munster, Westfalen, Germany EDGARLEDERER,Labordoire de Chimie biologique, Facult6 des Sciences, Paris, and Institut de Chimie des Substances Naturelles, Gif sur Yvette, Seine et Oise, France OMPRAKASH MALHOTRA ,Chemisches Laboratorium deer Universitlit, Freiburg im Breisgau, Germany N . C . MEHTA,Laboratory of Agricultural Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland FRITZMICHEEL,Organisch-Chemisches Institut der Universitat, Munster , Westfalen, Gemnany J. MUETGEERT, Plastics Research Institute T.N.O., Delft, Holland GLYN0. PHILLIPS,Department of chemistry, University College, Cardig, Wales

R. STUART TIPSON, Washington, D. C. KURTWALLENFELS, Chemisches Laboratorium der Universitdt, Freiburg im Breisgau, Germany

ROY L. WHISTLER,Department of Biochemistry, Purdue University, Lafayette,Indiana

* Present address: Chemistry Department, The University, Leioester, England. V

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PREFACE This sixteenth Volume of the Advances in Carbohydrate Chemistry continues with its task of providing comprehensive reviews on matters of interest in the general chemistry of the carbohydrates. A significant problem is treated by Phillips (Cardiff)-the effects produced in carbohydrates by ionizing radiation-a topic which is in its infancy and which can be expected to undergo extensive development. Fluorine chemistry is likewise a modern subject undergoing intensive study, and some phases of its application to carbohydrates are detailed by Bonner (London) and by Micheel and Klemer (Munster). The effects of various glycol-splitting reagents on the carbohydrates have been summarized in previous issues of this Series, and recent results now allow an elaboration of the ring structures of the dialdehydes produced from the pyranoid sugar rings by periodate ion (Guthrie, Manchester). In the early issues of the Advances, the late Claude S. Hudson initiated a set of articles on single sugars (or simple groups of sugars), and lactose was one of those selected for discussion. This account, started in 1954 by Whistler (Purdue) but never completed to his satisfaction, has at last been finished by Hough and Clamp (Bristol). Biochemical aspects have been treated authoritatively by Lederer (Paris), who reports on the interesting new sugars found in the glycolipids of the acid-fast bacteria; Wallenfels and Prakash Malhotra (Freiburg i. B.) detail the fascinating subject of the first isolation and crystallization of a simple glycosidase; and Deuel and associates (Zurich) discuss the carbohydrate residues isolable from the soil. In the first Volume of this Series, T. J. Schoch described a fractionation of starch by which he firmly established the existence of the amylose and amylopectin fractions. His process remained a laboratory procedure only, but, recently, Dutch chemists have developed a large-scale fractionation of potato starch, and pure amylose is now obtainable in commercial quantities; the new process is herein described by Muetgeert (Delft). Finally, an obituary of the late Harold Hibbert is offered by one of his former associates. The Subject Index has been prepared by Dr. R. David Nelson.

M. L. WOLFROM R. STUARTTIPSON

Columbus, Ohio Washington, D. C.

vii

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CONTRIBUTORS TO VOLUME 16 ................................................

v

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

v i

HAROLD HIBBERT ............................................................

1

Radiation Chemistry of Carbohydrates GLYN0. PHILLIPS

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Primary and Secondary Effects of Radiation .. . . . . . . . . . . . . . . . . . . . . . . . . . . I11. The Effect of Radiation on Compounds Related to Carbohydrates . . . . . . IV . The Effect of Radiation on Carbohydrates..............................

13 14 22 32

Applications of Trifluoroacetic Anhydride in Carbohydrate Chemistry

.

T. G BONNER I . Introduction ........................................................... I1. Trifluoroacetylation with Trifluoroacetic Anhydride .................... I11 The Trifluoroacetyl Group as a Blocking Group ........................ IV . Acylation with Carboxylic Acid-Trifluoroacetic Anhydride Mixtures ..... V . Selective Ring-opening of Cyclic Acetals with Carboxylic Acid-Trifluoroacetic Anhydride Mixtures ............................................. V I . The Synthesis of Linear Polymeric Esters from Cyclic Trimethylene Acetals and Dibasic Carboxylic Acids ...................................... VII . The Mechanism of Acylation by Acyl Trifluoroacetates . . . . . . . . . . . . . . . . .

.

59

60 63 67 69 77 79

Glycosyl Fluorides and Azides

FRITZMICREELAND ALMUTHKLEMER I . Introduction ........................................................... I1 Preparation of the Glycosyl Fluorides .................................. I11. Reactions of the Glycosyl Fluorides .................................... IV . The o-Fluoro Carbohydrates ........................................... V . The Aldosyl Azides .................................................... VI . Tables of Properties of Glycosyl Fluoride Derivatives ..................

.

is

85 86

88 95 95 97

X

CONTENTS

The “Dialdehydes” From the Periodate Oxidation of Carbohydrates

. .

R D GUTHRIE

I . Introduction ........................................................... 106 I1. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 I11. General Properties of the Oxidation Products ........................... 108 IV . Oxidation Products from Monosaccharide Derivatives and Related Compounds ................................................................ 108 V. Oxidation Products from Di-, Tri-, and Oligo-saccharides . . . . . . . . . . . . . . . 134 V I Oxidation Products from Polysaccharides ............................... 137 V I I . Alkaline Degradation of Periodate-oxidized Carbohydrates.............. 153 VIII . Uses of Periodate-oxidized Carbohydrates .............................. 157

.

Lactose

.

JOHN R . CLAMP.L . HOUOH.JOHN L HICHSON.A N D ROY L . WHISTLER

I . Introduction ........................................................... I1 The Structure of Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Occurrence and Biochemical Properties of Lactose ...................... I V . Chemical Properties of Lactose ......................................... V. Some Physical Properties of Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

159 180 165 181 201

Glycolipids of Acid-Fast Bacteria

EDGAR LEDERER I . Introduction ........................................................... 207 I1. Chemistry of Glycolipids of Acid-fast Bacteria ......................... 209 I11. Biological Activities of Glycolipids of Acid-fast Bacteria . . . . . . . . . . . . . . 230 Galactosidases

KURTWALLENFELS AND OM PRAKAEIH MALHOTRA I. Introduction ........................................................... I1. 8-Galactosidases . ...................................................... I11. a.Galactosidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239 240

290

The Fractionation of Starch J . MUETQEERT

I . Introduction ........................................................... I1. Fractionation by Complexing Agents ................................... I11. Fractionation by Leaching Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

299 300 306

CONTENTS IV. Fractionation by Fractional Precipitation. ............................. V. Industrial Methods of Fractionation.. .................................. VI. General Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi 309 325 332

Carbohydrates in the Soil N. C. MEHTA, P. DUBACH AND H. DEUEL

I. Introduction.. .................................................... 11. Isolation and Characterization.. . . 111. Quantitative Determination.. . . . . . IV. Source and Transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 V. State and Function.. ........ .... 352 VI. Summary .............................................................. 354 AUTHORINDEX FOR VOLUME16.... . . 35; SUBJECT INDEX FOR VOLUME16... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 CUMULATIVE AUTHORINDEX FOR VOLUMES 1-16.. . . . . . . . . . . . . . . . . 396 CUMULATIVE SUBJECT INDEX FOR VOLUMES1-16 .............................. 402 ERRATA ......................................................................

410

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HAROLD HIBBERT 1877-1945

To few is it given to spend such a varied life, and one so rich in achievement, as that of Harold Hibbert. He was born in Manchester, England, on August 27th, 1877, the second of the four sons of Isaac and Martha (Scholes) Hibbert. All four boys were to make their marks in life. Frank, the eldest, eventually engaged in manufacture in London; Ernest, a British-trained mining engineer was involved in the technical development of the great Noranda strike in the wilds of northwestern Quebec; and Arthur, the youngest, who also was a well-known mining engineer (in Cyprus, India, Peru, and Spain), distinguished himself as a Major in the British Corps of Engineers in the First World War, being in charge of the tunnelling of Hill 60, and receiving the D.S.O. and M.C. Undoubtedly, their accomplishments were partly attributable to their upbringing-their father was a Wesleyan Methodist, a staunch Liberal, and a teetotaler and nonsmoker. Harold attended the Central Board School in Manchester and, at the age of seventeen, was awarded a Manchester Corporation Scholarship. In 1894, he entered Owens’ College of the federal Victoria University, ManChester, and, three years later, graduated with a B. Sc. with First Class Honours in Chemistry. He was awarded the Levinstein Exhibition fellowship, proceeded to conduct his first researches in organic chemistry under Professor William H. Perkin, Jnr., and received his M. Sc. degree from the Victoria University in 1900, the year of his first publication. I n 1899, Hibbert accepted an appointment as Senior Demonstrator and Assistant Lecturer in Chemistry at the University College of Wales, in Aberystwyth. Two years later, Dr. J. J. Sudborough was appointed Professor of Chemistry, and Hibbert published four papers with him, during 1903-04, on addition compounds, on the differentiation and estimation of primary, secondary, and tertiary amines, and on the estimation of hydroxyl groups in organic compounds. Hibbert now decided to study abroad for his doctorate, and he arrived a t the University of Leipaig in October, 1904, to work under Professor Arthur Hantasch on addition products of trialkyl derivatives of arsines, phosphines, and stibines. Hibbert published an article on the preparation of the trialkyl derivatives (by means of the Grignard reaction) in Berichte for 1906, and, in the following year, he and Hantasch described the addition products in the mme Journal. In 1906, Hibbert was awarded the Ph. D. degree summa cum l a d e by the University of Leipaig. During his two years 1

2

OBITUARY-HAROLD

HIBBERT

in Germany, Hibbert learned to speak German fluently and idiomatically, and he acquired a broader knowledge of philosophy, music, and art than he had previously had. The young Englishman also became friendly with several American students who were later to become well-known chemists: W. C. Bray, Colin G. Fink, Arthur B. Lamb, and S. C. Lind. In 1906, at the age of twenty-nine, Hibbert came to the United States on a two-year appointment at Tufts College, in Boston, Massachusetts. There he worked under Professor Arthur Michael on keto-enol tautomerism and the effect of solvent on the equilibrium. Hibbert’s association with Michael, with whom he published some half-dozen papers, was to have a profound and lasting influence on his subsequent career. Hibbert now returned to England and obtained a Chemical Society grant which enabled him to conduct independent research with Sir William Tilden at the Imperial College of Science in London. He completed earlier work, begun a t Aberystwyth, on some quantitative applications of the Grignard reaction (in what is now known as the Tschugaeff-Zerewitinoff method). In 1904, Sudborough and Hibbert had shown the value of a high-boiling ether in this reaction, and they were the first to devise a quantitative procedure. For two years, Hibbert fruitlessly sought an academic position in Great Britain; he therefore came back to the United States and, in 1910, succeeeded in obtaining a post as a research chemist with the E. I. du Pont de Nemours Powder Co. at their Experimental Station in Wilmington, Delaware. Here he did important work on the stability of frozen and liquid glycerol trinitrate (nitroglycerin) which won him international recognition. In 1912, he was awarded the highly coveted D. Sc. degree of the (Victoria) University of Manchester. With the outbreak of the First World War in 1914, Hibbert accepted a position as Research Fellow at the Mellon Institute of Industrial Research in Pittsburgh, Pennsylvania, on a Gulf Refining Co. Fellowship (1914-15); he was then a Senior Fellow on the Union Carbide Acetylene Fellowship (1915-16) and studied new methods for synthesizing (and manufacturing) acetone, acetaldehyde, and acetic acid from acetylene. As a result of these investigations. Hibbert was called into consultation by the Shawinigan Water and Power Company of Montreal, and one of his former associates, Mr. Howard Matheson, was put in charge of erecting, at Shawinigan Falls, Quebec, a large plant for the manufacture of acetic acid. Hibbert also devoted much attention to the syntheais and properties of ethylene glycol and its derivatives-compounds which have found numerous industrial applications-and his patents on glycols were purchased by the Union Carbide Company. The best known of his patents, namely, U. s. Patent 1,213,368 (1917), relates to the use of ethylene glycol as an antifreeze for car and aeroplane radiators. This product was marketed as Prestone, named after the Prestolite Company which sold calcium carbide for acetylene lamps

3

R. STUART TIPSON

and which was bought out by Union Carbide. In addition, he was the first to apply for a patent on the use of ethylene gas for welding and cutting. He published his well-known method for dehydrating alcohols with a trace of iodine, and (with B. T. Brooks) a procedure for synthesizing the higher aliphatic alcohols by high-pressure reactions involving chlorinated petroleum hydrocarbons. He also showed the usefulness of ethylene glycol dinitrate as a liquid explosive; this was tested, with very favorable results, by the U. S. Navy Department, and Dr. Hibbert offered the United States Government the use of his patents, free of all royalties. Hibbert left the Mellon Institute in 1916 to become a private consultant, first in Toronto and then in New York City. During 1917 and 1918,he was Chemical Adviser (on gas warfare) to the British War Mission in Washington, D. C. He also became the Director of the Research and Technical Divisionof RalphL. Fuller and Co., New York, N. Y. (a company organized in order to manufacture certain pharmaceuticals hitherto obtained from Germany), and, in co-operation with the British-American Chemical Co., devoted himself to the erection and operation of chemical plants in Canada and the U. S. A. He married Beulah Virginia Cole on May 14,1917;at this time, she was a teacher of physiography a t the Julia Richman High School in New York City. Much of his subsequent success is attributable to her inspiration and guidance. In 1919, a t the age of forty-two, he realized that the academic life meant more to him than the less challenging, although more lucrative, career of industrial chemist, and he accepted an appointment as Assistant Professor of Chemistry at Yale University in New Haven, Connecticut. In 1921,he was promoted to an Associate Professorship and, two years later, he became an American citizen. He soon embarked on extensive investigation of the chemistry of cellulose, which eventually led to some sixty-nine papers in a series entitled “Studies on Reactions Relating to Carbohydrates and Polysaccharides.” Denham and Woodhouse had previously methylated cellulose and had hydrolyzed the product to 2,3,6-tri-O-methyl-~-glucose. For the supposed “cellulose monomer” (which, according to the ideas then held, could associate through “secondary valence forces” to give the “cellulose polymer”), Hibbert proposed the following formula. CHor

ii”’”” H-0

4

OBITUARY-HAROLD

HIBBERT

However, he did not rule out the then-unpopular possibility that the polymer might consist of units joined by main valences available on opening of the 1 ,Boxygen bridges (at that time, the sugar ring was assumed to be 1 ,4), as follows.

-?“I HH: A! 0Y

or

c:

H 0-

[q:LoCHiOH I

HOH-CHOH-CH-

(!XIOH

He and his students then tried to synthesize simple analogs of the above “monomeric unit,” an endeavor which proved fruitless, but, in the course of their studies, they accumulated a large amount of knowledge regarding the preparation and properties of cyclic acetals. Thus, they found that a trace of acid catalyzes the formation of a polymer from 5,6-dihydroxy-2hexanone, and that aliphatic aldehydes (RCHO) readily combine with ClaCCHO), . chloral to give polymers of the general formula (2 RCHO The condensation of glycerol with aldehydes and ketones was studied; with benzaldehyde, a mixture of the cyclic acetals having the five- and the six-membered ring resulted, showing that presence of hydroxyl groups on vicinal carbon atoms is not essential for occurrence of the reaction. On the other hand, on condensation of acetone with various other polyhydric alcohols, the cyclic acetal having the five-membered ring was alway formed exclusively. The migration of acyl groups was another field of interest. Emil Fischer had suggested that a compound having the dioxolane ring (“orthoester”), m in (l), was the intermediate in the migration, and Hibbert placed the theory on a firm footing.

+

0

I

I

I (1)

For glycerol esters, the migration was found to be toward the primary hydroxyl group. He stated that “The tendency and ease of ring formation will be dependent on: (a) the relatively labile character of the hydrogen attached to the hydroxyl group, (b) the negative polarity of the carbonyl

R. STUART TIPSON

5

group in the acyl radical, and (c) the spatial relationship of the migratory hydrogen atom with reference to the carbonyl group.” Consequently, he predicted that the orthoester structure would be stabilized by the trichloroacetyl radical; confirmation came from the discovery that the trichloroacetate of ethylene glycol can only exist in the cyclic form. On heating, this orthoester decomposes into the carbonate plus chloroform.

These studies involved the concept of “neighboring-group effects” and laid the basis for interpretations of reaction mechanisms that were later to be developed by other carbohydrate chemists and then, eventually, be adopted by organic chemists in general. In contrast to the behavior of esters, no tendency to migrate was found with methyl ethers, an observation of importance at that time, when methylation procedures were being extensively employed in the determination of ring structures of sugar derivatives. Hibbert waa intimately associated with the founding of the Division of Cellulose Chemistry of the American Chemical Society and served as the first Chairman of the Division (1920 to 1922). Formulation of procedures for defining a Standard Cellulose preparation was an early project. Hibbert was a stimulating and vigorous leader of the discussions of the Division, some of which became so heated that they will not be forgotten by those who were in attendance. Outstanding in this regard was one session of the A. C. S. Organic Symposium at Princeton University in 1929, after Hibbert had risen to comment on a lecture on polymerization by Wallace H. Carothers (the inventor of Nylon). After six years at Yale University, Hibbert was honored by appointment, at the invitation of Sir Arthur Currie, to the chair of the E. B. Eddy Professorship of Industrial and Cellulose Chemistry at McGill University, Montreal, Canada, a position he was to hold for eighteen years, while still retaining his American citizenship. In 1925, the Pulp and Paper Research Institute of Canada, erected on the McGill campus by the Canadian Pulp and Paper Association, had just been completed, and Hibbert’s Department moved into part of the magnificent new building. The modern facilities that were placed a t his disposal, together with the unflagging energies of a group of enthusiastic graduate students, needed only Hibbert’s stimulus and inspiration to make this a new center for productive research. His almost 100 predoctoral and postdoctoral students (to whom he was affectionately known, although not to his face, as “Pa Hibbert”) came from abroad and

6

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HIBBERT

from all parts of the Dominion; they were fired by his enthusiasm, respected his ability and his vision regarding research problems, and soon learned to emulate his enormous capacity for work. Nevertheless, he did not believe in “all work and no play.” He would make the rounds of the laboratories at unexpected hours; and the author well remembers being caught running an experiment one glorious, sunny Saturday afternoon in the Fall and being told, in Hibbert’s north-country accent, to “get out of here, and go and play a game of tennis!” Such visits were sometimes embarrassing, as when, one night, Hibbert brought in a visitor to see the library, shortly after midnight, and found one of the research chemists stretched out on one of the library tables, fast asleep after some exhausting experiments; the two tiptoed quietly away and left the student to his slumbers. Hibbert’s valuable library was always available to his students, to whom it was known as “Hibbert’s Cadillac.” The reason for this name throws some light on Hibbert’s interests; his brother Ernest, when visiting him, had been annoyed a t the old car that Harold then drove, and so he gave Harold the money to buy a new one and recommended a Cadillac. (This was the brother who had made a good stake in the Noranda gold-silver-copper strike.) Harold thought the matter over and decided that he would, instead, use the money to develop his personal library, which he kept at home. Hibbert was a taskmaster, but he was also a father to “his boys’’ (and several of “his girls”); besides making sure that each developed himself to the extent of his capabilities, he helped in planning the future of his students and finding a place for them in industry or teaching after they had received their Ph. D. degrees. He never rested until he had found a suitable opening for each of them. Sometimes, a grant would be obtained (often, surreptitiously, out of his own pocket) for those in need of financial assistance. This deep concern for the welfare of his students was fully shared by Mrs. Hibbert, and, together, they established the Hibbert-Cole Scholarship for students at McGill. They especially delighted in entertaining students who were far from home; his students were often invited on a picnic in the summer, a car-ride in the spring or autumn, or to the Hibberts’ home. On Christmas Day, 1929, E. G. V. Percival and the author had the pleasure of a delightful Christmas dinner at their home (and were amazed to receive totally unexpected Christmas presents). In 1929, the first organic microanalytical laboratory in Canada (and one of the first in North America) was started in his Department, under the direction of the author, and graduate students and professors came from all parts of the Dominion to learn the specialized techniques of Fritz Pregl’s procedures (which had been passed from Pregl to H. D. K. Drew and, from him, to the author). It was then that we discovered that all chemists can be divided into two groups-those who, unable to acquire the necessary

7

R. STUART TIPSON

manipulative skills, can never be taught to perform a quantitative microanalysis, and those who learn the essentials with ease, often in a fortnight of concentrated effort. By this time, Hermann Staudinger and Wallace H. Carothers had firmly established that primary (not secondary) valence forces are involved in polymerization. Hibbert and his coworkers then synthesized a series of individual, linear polymers, each of known chain-length, and studied their properties as a function of chain length. The kind of reaction used in these syntheses, for polymers containing 4, 6, 8, 12, 18, 42, 90, and 186 units, was as follows. H 2

H2CONa HOHs

b

\c/

ClCH2 +H2hCI

-+

HOHzC

H H

/ \o/

\c/

H

0

LC/ \c/

d

\H

CHiOH

€f\H

Another example of polymerization that intrigued Hibbert was that brought about by the slime-producingbacteria, that can take sugar residues from certain di- and tri-saccharides and combine them to give polysaccharides. The first such polymer we studied was a fructan, which he renamed levan, produced by the action of Bacillus mesentericus on sucrose; it was the cause of considerable trouble in the sugar industry. Hibbert and coworkers found that the bacillus utilizes the D-glucose moiety and that the nascent D-fructofuranose moieties combine to give levan, a polymer of D-fructofuranose, which differs from inulin [whose structure had already been shown by Haworth and coworkers to be (2+l)-~-fructofuranoid] in having (2+6)-linkages. Methylation of levan, followed by hydrolysis, afforded crystalline 1,3,4-tri-0-methy~-~-frctose. Hibbert and his school were also the first to conduct extensive studies on dextran, a polymer (of D-glucose) produced from sucrose by various strains of Leucmostoc mesenteroides; the principal linkage present was shown to be a-~-(l-+6).Dextran has since found use as a blood extender. Another pioneer study was on the polysaccharide produced, as a membrane, by Acetobacter xylinum; this carbohydrate was shown to be cellulosic and has been called “bacterial cellulose.” I t could be acetylated and spun into a cellulose acetate fiber. In these studies, the general chemical identity of wood cellulose with cotton cellulose was established. He also became interested in the polymeric “humic acid” that is formed by the action of mineral acids on hexoses; a use was developed for it as an extender in lead accumulators (“storage batteries”). All of these investigations bore a relationship to polymerization and to carbohydrate chemistry, but, since the Pulp and Paper Research Institute

8

OBITUARY-HAROLD

HIBBERT

was primarily interested in the chemistry of wood, Professor Hibbert and his associates engaged in an intensive study of cellulose, its behavior with alkali, and the complicated changes occurring during its oxidation. However, Hibbert’s main interest gradually became the other main component of wood, namely, the lignin, which constitutes 30 % of all woods and which was being run into the streams as a total loss in the manufacture of sulfite pulp. Although lignin had been the subject of numerous investigations during the preceding seventy years, but little progress had been made, largely because of the difficulty in isolating it in unchanged form. Hibbert was to establish an international reputation as one of the foremost workers in this field. He developed techniques for isolating lignin in as unchanged a condition as possible, free from the other constituents of wood. The pulp-bleaching process was studied and improved, and the preparation of vanillin from sulfite-pulping waste-liquors was developed and made commercial. Eighty-seven papers were eventually published in a series, starting in 1930, entitled “Studies on Lignin and Related Compounds.” The alkaline degradation products of ligninsulfonic acids from softwoods were found to be guaiacol, vanillin, and acetovanillin, whereas those from hardwoods were the analogous compounds 1 ,3-di-O-niethylpyrogallol, syringaldehyde, and acetosyringone. In addition, wood meal (pre-extracted to remove fats, resins, tannins, and waxes) was extracted with acidified alcohols (for example, ethanol), and the water-soluble fraction of the lignin was found to contain 1-(4hydroxy-3-methoxypheny1)-1,2-propanedione (“methyl vanilloyl ketone”), its 5-methoxy derivative (“methyl syringoyl ketone”), a-ethoxypropiovanillone [2-ethoxy-1- (4 - hydroxy-3 -methoxypheny1)-l-propanone], and a-ethoxypropiosyringone [2-ethoxy-l-(4-hydroxy-3,5-dimethoxyphenyl)-l-propanone] ; the two ethyl ethers were thought to have been formed from the corresponding hydroxy compounds during the ethanolysis. These products are, or are derived from, the building units of lignin. Finally, Hibbert’s work on phenol lignin and related products has been used in the lignin-Bakelite industry. In recognition of his outstanding contributions to both pure and applied chemistry, Harold Hibbert was, in 1936, honored with the LL. D. degree honoris mu8u by the University of British Columbia. He was made an Honorary Member of the Society of Chemical Industry (London) in 1943; on this occasion, the President of the Society stated: “The Council, in deciding to bestow this honour, selected with great care one they considered worthy, for his career illustrates to a remarkable degree the influence which a man of high scientific attainments can exert on industry and the wellbeing of the community.” Two years later (only a few weeks before his death), Hibbert was accorded the highest honor bestowable by the scientists of his adopted country by his election to membership in the National Academy of Sciences (U.S.A.).

R. STUART TIPSON

9

On his retirement from McGill in 1943, his past and then-present students joined in attesting to their high regard for him by presenting him with a bronze plaque engraved with the signatures of the students who had received their advanced training under him,and Mrs. Hibbert was given an Audubon print. Hibbert was honored by election as a Fellow of the Royal Society of Canada, and he was a member (or Fellow) of the American Association for the Advancement of Science; American Chemical Society (Chairman, New Haven Section, 1920, and Chairman, Division of Cellulose Chemistry, 19201922); American Pulp and Paper Association; Canadian Chemical Association; Canadian Pulp and Paper Association; Deutsche chemische Gesellschaft ; Royal Institute of Chemistry (London); The Smithsonian Institution (Washington, D. C.) ; Society of Chemical Industry (Chairman, Montreal Section, 1930); Society of the Sigma Xi; Technical Association of the Pulp and Paper Industry; and the Textile Institute (England). In addition, he served on the editorial board of Cellulosechemie for several years. He was author or co-author of 253 scientific papers and 50 patents (British, Canadian, and U. S.). Hibbert was tall and well-built, and had a bush of hair which, in his later years, was snow-white; he was strikingly handsome and an immaculate dresser. He had a vivid, colorful personality, was always optimistic and high-spirited, and was an indefatigable worker. He had a fine baritone voice, and delighted in singing airs from The Messiah and the rollicking songs of Old England and of the Germany of his student days. He and Mrs. Hibbert were lovers of the great outdoors and were enthusiastic birdwatchers. His country ramblings and canoe trips renewed his strength and helped maintain his buoyancy of spirit. He was a180 keen on golf and tennis, and was a practitioner of daily setting-up exercises. Dr. Hibbert read widely, not only in Chemistry but in other fields, for extension of his knowledge and for pleasure and relaxation. He had an excellent background in English literature and loved to recite the verses of the great English poets, from memory. He was known for his ability to write and speak with forcefulness and clarity, and his skill in debate was particularly in evidence at the meetings of the American Chemical Society. Indeed, his charm and vitality gave color to any gathering he attended. He was keenly interested in baseball, and played the game during his early days in the U. S. He belonged to the Unitarian Church, was a thirty-second degree Mason (Scottish Rite), and was a member of the University Club of Montreal, the Chemists’ Club of New York, the Faculty Club of McGill University, and Golf, Tennis, and Canoe Clubs. Two years after his retirement, Harold Hibbert died of cancer of the pancreas, on May 13, 1945, the day before his twenty-eighth wedding anniversary. His memory is kept alive a t McGill University by the Harold

10

OBITUARY-HAROLD

HIBBERT

Hibbert Memorial Fellowship, established by his millionaire brother Colonel Ernest Hibbert (1879-1948) with an endowment of $1 00,000, which supports a post-Ph. D. Fellowship in the Department of Chemistry; holders to date have been Drs. Conrad Schuerch, Alan H. Vroom, Tore E. Timell, Necmi Sanyer, John Honeyman, Bengt 0. Lindgren, Terrence J. Painter, Ingemar Croon, and Iqbal R. Siddiqui. In 1954, Mrs. Hibbert presented almost $l,OOO to the University of Manchester in his memory; this has been used for endowing two Harold Hibbert Memorial Prizes, awarded annually by the Department of Chemistry for the two best Ph. D. theses submitted during the previous year. His personal chemical library, consisting of some 1,500 volumes (many of them irreplaceable), was kept intact and became the Hibbert Library in the research laboratory of the Crown Zellerbach Corporation in Camas, Washington. His scientific achievements live on in his publications and in the accomplishments of the many students he inspired. As Dr. Emil Heuser remarked, some two weeks after Hibbert’s death, in an address (on Hibbert’s work) before the North-East Wisconsin Section of the American Chemical Society: “Cellulose and lignin chemists, the world over, have lost a great deal through Professor Hibbert’s death. He will long be remembered, not only by his personal friends but also by those who have benefitted from his work and those who will do so for many years to come.”

R. STUARTTIPSON APPENDIX The following is a list of the 118 scientists who published articles in collaboration with Dr. Harold Hibbert. J. S. Allen; C. G. Anderson; W. R. Ashford; S. B. Baker; R. H. Ball; J. Barsha; S. H. Beard; A. Bell; E. M. Bilger; J. R. Bower, Jr.; F. Brauns; C. P. Brewer; L. Brickman; B. T. Brooks; Irene K. Buckland; C. Pauline Burt; Laura T. Cannon; N. M. Carter; J. Compton; L. M. Cooke; A. B. Cramer; R. H. J. Creighton; A. C. Cuthbertson; R. M. Dorland; A. M. Eastham; H. Essex; T. H. Evans; E. C. Fairhead; H. E. Fisher; J. H. Fisher; R. Fordyce; Frances L. Fowler; G. P. Fuller; A. F. Gallaugher; J. A. F. Gardner; R. D. Gibbs; W. F. Gillespie; H. P. Godard; K. R. Gray; Margaret E. Greig; E. G. Hallonquist; A. Hantzsch; F. C. Harrison; S. M. Hassan; W. L. Hawkins; W. F. Henderson; W. B. Hewson; Bertha Hibbert; A. C. Hill; H. s. Hill; E. 0. Houghton; F. Howett; M. J. Hunter; E. C. Jahn; B. Johnsen; E. G. King; M. Kulka; F. Leger; I. Levi; M. Lieff; E. L. Lovell; 0. Maas; J. L. McCarthy; W. S. MacGregor; A. S. MacInnes; H. W. MacKinney; L. Marion; H. B. Marshall; A. Michael; M. Michaelis; J. P. Millington; L. Mitchell; W. Mitchell; W. 0. Mitscherling; R. E. Montonna; L. P. Moore; R. G. D.

R. STUART TIPSON

11

Moore; J. G. Morazain; H. A. Morton; A. C. Neish; A. Paquet; J. L. Parsons; R. F. Patterson; Q . P. Peniston; J. M. Pepper; E. G . V. Percival; S. Perry; S. Z. Perry; J. B. Phillips; Muriel E. Platt; M. Plunguian; J. C. Pullman; J. J. Pyle; R. R. Read; W. L. Reinhardt; R. P. Roberts; H. J. Rowley; C. A. Sankey; H. Schwartz; W. H. Steeves; M. G. Sturrock; J. J. Sudborough; R. F. Suit; J. N. Swartz; H. L. A. Tarr; K. A. Taylor; J. A. Timm; R. S. Tipson; G. H. Tomlinson, Jr.; G. H. Tomlinson, 2nd.; S. M. Trister; E. West; K. A. West; M. S. Whelen; E. V. White; A. Wise; L. E. Wise; and G. F. Wright.

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RADIATION CHEMISTRY OF CARBOHYDRATES

BY GLYN0. PHILLIPS Department of Chemistry, University College, Cardiff, Wales I. Introduction.. .............................. . . . . . . . . . . . 14 11. Primary and Secondary Effects of Radiation.. . 1. Interaction of High-energy Radiations with 2. Radiolysis of Water and Aqueous Solutions 3. Chemical Dosimetry.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 111. The Effect of Radiation on Compounds Related to Carbohydrates. . . . . . . . . . . 22 1. Alcohols.. ................................. ............... 2. Nucleic Acids.. ............................ .................... 26 3. Hydroxy Acids. .......................................... IV. The Effect of R 1. Polysaccharides 2. Aldohexoses, 3. Glycosides, Disaccharides, Trisaccharides, and Lactones. 4. Absorption Spectra and Post-irradiation Processes.. . . . . . 6. Self-decomposition of C14-labeled Carbohydrates.. . . . . .

I. INTRODUCTION Although about 50 years have elapsed since ionizing radiations were first shown to produce chemical systematic progress in the field has taken place only within the last decade. This advance may be attributed to the large range of comparatively cheap radiation-sources which have become available in this period as a result of developments in the nuclearpower industry. The general field of radiation chemistry has been well reviewed? Interest in the biological action of radiations, chemical utilization of fission-product radiations, and the use of radiation in the sterilization of food has stimulated an intensive study into the effects of ionizing radiations on organic compounds?*OIn contrast, chemical studies on the behavior of (1) F. Geisel, Bet., 36, 3608 (1902); 36. 342 (1903). (2) A. T. Cameron and W. Ramsey, J . Chem. Soc., 91, 931 (1907); 93, 966 (1908). (3) A. Debierne, Compt. rend., 148, 703 (1909). (4) M. Kembaum, Compt. rend., 148, 706 (1909); 149. 116 (1909). (6) F. L. Usher, Jahrb. Radioakt. u. Elektronik, 8, 323 (1911); Chem. Abstracts, 6, 322 (1912). (6) W. Duane and 0. Scheuer, Radium, 10, 33 (1913). (7) See Ann. Rev. Phys. Chem., 1 (1960)-10 (1969). (8) E. Collinson and A. J. Swallow, Quart. Revs. (London), 9, 311 (1966). (9) E. Collinson and A. J. Swallow, Chem. Revs., 66, 471 (1966). 13

14

G. 0. PHILLIPS

carbohydrates are meager, although a great deal of attention has been directed toward the physical changes observable during irradiation. However, in reccnt years, in view of the important physiological role of carbohydrates and their wide occurrence in foods, a more fundamental approach has been adopted toward the study of the radiation chemistry of carbohydrates. Consequently, at the present stage in the development of the subject, it is considered that a review will be of greatest value to investigators in this and allied fields if it is broadly based. Therefore in this review, the basic principles of radiation chemistry will be discussed, particularly with regard to the behavior of aqueous solutions. Investigations into compounds which are structurally related to the carbohydrates and which have received more systematic study will be considered, and the present position with regard to carbohydrates will be reviewed. In the broad sense, radiation chemistry embraces photochemistry-the chemistry of rcartions which occur in electrical discharges, and reactions in the atomic nucleus by the agency of neutrons and high-energy radiat]ions. In this review, however, attention will be confined mainly to the chemical changes induced by a-rays, @-rays,x-rays, and y-rays.

11. PI~IMARY AND SECONDARY EFFECTS OF RADIATION 1. Interaction of High-energy Radiations with Matter The processes by which high-energy radiations and particles interact with matter have been described in Whether the radiation be electromagnetic (x-rays or y-rays) or corpuscular (a-rays and @-rays), thc final transfer of rriergy occurs by way of charged particles. With electromagnetic radiation, interacfion of high-energy quanta with atoms of thc medium through which they pass leads to ionization, since the energy of the quanta is substantially greater than the binding energy of an electron. Although there are several mechanisms by which this process may bo brought about,14 it is clear that high-speed electrons are ejected and these give rise to chemical changes. The single atom initially affected by the radiation makes a negligible contribution to the total chemical change, which arises mainly from the ionization and excitation processes initiated by the secondary, high-speed electrons. For corpuscular radiations, the high-speed particles directly applied give rise to effects similar to those of the secondary electrons. (10) F. 8. Dniritori, J . Phys. & Colloid Chem., 62, 490 (1948). (11) J. L. Mugee, A n n . Rev. Nuclear Sci., 9, 171 (1954). (12) D. E. h a , “Actions of Radiations on Living CellB,” Cumbridge Univcrsity Press, London, 1955. (13) J. L. Magee and M. Burton, J . Am. Chem. Soc., 79, 523 (1951). (14) M. Burton, J . Chem. Educ., 28, 404 (1951).

RADIATION CHEMISTRY OF CARBOHYDRATES

15

Charged particles, in the main, interact with the electronic cloud around the molecule. This interaction is directly related to the charge of the particle and inversely related to its velocity. Thus, the energy of the particle may be transferred to the electron cloud and it gives rise to a displacement of the electrons. If the displacement is sufficient and the electron is no longer associated with the parent molecule, ionization has occurred. If, however, the displacement is not so pronounced, excitation has occurred. Such an excitation may be related to the excitation processes encountered in the photochemical, primary act. Other excitation states may arise as a result of slow, secondary electrons (20-100 e.v. of energy). Eyring, Hirshfelder, and Taylor16 first took into account the chemical contribution of such excited molecules, and the present position with regard to their contribution has been summarized by Magee and Burton.1s The density of excitation and ionization is not necessarily the same for all radiation qualities. For example, it is greater along the track of an a-particle than for an electron track. For a primary-recoil electron produced by C060 y-rays in water, the distance between successive ionizations is about 1000 d. The ionized track is, therefore, sparse. At each point of ionization, secondary electrons give rise to further ionizations, forming a group of ionpairs. In contrast, a-particles form a continuous track as a result of overlapping between the spheres of ionization. Experimentally, it has been shown for gases that approximately 25-32 e.v. of energy are required for forming an ion-pair, whereas ionization potentials for gases are in the range of 9-15 e.v. For this reason, it has been suggested that, if the excess energy is dissipated in electronic and excitation processes, half of the energy of the radiation goes into ionization and half into excitation. When ions and excited molecules have been formed in this manner, a variety of secondary processes may occur before the final chemical change takes place. To illustrate the nature of such primary and secondary processes, the behavior of water on exposure to ionizing radiations will be considered; this is a matter of fundamental importance in the present review, because the majority of carbohydrate investigations in this field have been undertaken in aqueous solution. 2. Radiolysis of Water and Aqueous Solutions Probably, no other system in radiation chemistry has been studied in so much detail as the action of ionizing radiations on water and aqueous systems. Nevertheless, knowledge about the detailed processes occurring is still incomplete. The over-all effects are, however, well established. It is generally acknowledged that absorption of energy by water results in the (15)

H.Eyring, J. 0. Hirshfelder and H. S. Taylor, J . Chem. Phys., 4,479 (1936).

16

0. PHILLIPS

Q.

formation of hydroxyl radicals and solvated electrons or hydrogen atoms. Molecular hydrogen and hydrogen peroxide are also produced. The present position of our knowledge of the radiation chemistry of aqueous systems has been demxibed.*6-a1 a. Formation of Hydrogen Atmns and Hydroxyl Radicals,--When electromagnetic radiation or charged particles interact with water, ionization occurs along the track of the particle or primary-recoil electron. At each point of ionization, the secondary electrons possess sufficient energy to induce further ionizations within about 20 A. of the track. These clusters of ionizations are known as b-raya or spurs. At a greater distance, the water molecules will only become electronically excited. The situation22 up to lo-'*10-" sec. after passage of the charged particle may be summarized as: HsO

+ el

-+

H20'

+ es + es

(1 1

where el and e2 are recoil electrons, and erris the secondary electron which can initiate ionization processes of the type: HIO

+

eB

+

Ha"

+ eal + es .

(2)

Reaction ( 1 ) is the primary ionization-process initiated by a recoil electron. It is thought that, subsequently, H20@is converted to a hydroxyl radical within lW1lseconds: HnO'

+ HnO + HsO' + .OH.

(8)

There are two views about the fate of the secondary electron. Samuel and Magee2882' assume that the electron does not leave the field of the parent ion, and that it eventually forms a hydrogen atom by charge-neutraliaaand Baxendale and Hughes:' tion with [email protected] and Frohlich*6*26 on the other hand, favor the idea that the hydrogen atom is created at a considerable distance from the parent ion, mainly by subexcitation electrons. These electrons come principally from the primary ionization of wa(16) F. 5. Dainton, Radiation Research, Suppl. 1, 1 (1969). (17) E. J. Hart, Proc. Intern. conf. Peaceful Uses Atomic Energy, Geneva, OB, 6 (1968). (18) E.J. Hart, J . Chem. Educ., 84, 686 (1967). (19) N.Miller, Revs. Pure and Appl. Chem. (Australia), 7 , 123 (1967). (20) M. Haissinsky, Acta. Chim. Acad. Sci. Hung., 12, 241 (1967). (21) J. Weies, Intern. J . Appl. Radiation and Isotopes, 6, 62 (1969). (22) H. A. Dewhurst, A. H. Samuel and J. L. Magee, Radiation Research, 1, 62 (1964). (23) J. L.Magee, J . A m . C h m . Soc., 78,3270 (1961). (24) A. H.Samuel and J. L. Magee, J . Chem. Phys., 21, lOs0 (1963). (26) R.L.Platemann, Radiation Research, 2, 1 (1966). (26)H. Frohlich and R. L. Platemann, Phys. Rev., 02, 1162 (1963). (27) J. H.Baxendale and C. Hughes, 2.phyeik. Chem. (Frankfurt), 14,306 (1968).

RADIATION CHEMISTRY OF CARBOHYDRATES

17

ter and they lose energy by inelastic collision. When the energy falls below the excitation energy of water, solvation of the electrons may occur and these solvated electrons subsequently form hydrogen atoms. From a general point of view, however, there does not appear to be any serious objection to the view proposed by Leal2 and Gray,28 namely, that the secondary electron is captured by the water molecule, to give H200, which leads to hydrogen atoms according to the equation :

+ OHe.

HzOe + H

It is also probable that free radicals are formed from excited water-molecules outside the ionization spurs. These would contribute to the net formation of hydrogen and hydroxyl free radicals. Therefore, although important refinements have been added, the fundamental description of the action of ionizing radiations on water remains as summarized by A l l e ~ 2 ~ H*O

*

+ ee + HzO OHe + H:q.+

HZ0@ aq. ---t ---t

Net result, HZ0

-+ 11

+ €€&.+ OH OHe + H

H ~ O ee~

Ha0

OH

According to this theory, hydrogen and hydroxyl free radicals would be distributed along the track of the original particle or primary-recoil electron, with the hydroxyl radicals situated near the track. The location of the hydrogen atoms is less certain, and they may be situated several A. units away from the site of electron formation. b. Formation of Hydrogen and Hydrogen Peroxide.-The view most generally held is that the formation of hydrogen and hydrogen peroxide occurs by pair-wise combination of hydrogen arid hydroxyl radicals : (4)

H+H+Hz OH

+ OH

+

HzOz

.

(6)

On this basis, about half of the free radicals would recombine to form water: H

+ OH

---t

HzO.

(6)

Thus, competition is set up between combination and diffusion of the radicals. On the basis of this diffusion-combination model, it is possible to account satisfactorily for the production of hydrogen peroxide and hydrogen. The mathematical treatment permits calculation of the theoretical fraction of the radicals reacting with a solute, and the resulting value is in good

L.H.Gray, J . chim. p h y s . , 48, 172 (1951). (29) A. 0. Allen, J . Phys. & Colloid. Chem., 62, 479 (1948). (28)

a.

18

0. PHILLIPS

agreement with experimental 0bservations.2~ * 2 6 ~ 8 0 * 3 1The combination reactions 4-6 occur within lO-'sec. after passage of the charged particle. When the solute is present in concentrations greater than lo-' M , the hydrogen atoms and hydroxyl radicals which escape by diffusion may react with the solute in an area well removed from the initial ionization. In the absence of a solute, water stabilization results from the following reactions: OH

+ Hz

-+

H + HzOz 3

+H Hz0 + OH, Hz0

These reactions are probably responsible for the low G values of hydrogen and hydrogen peroxide in liquid water. The G value in radiation chemistry refers to the chemical yield in units of molecules formed or disappeared per 100 electron volts of energy input. An alternative method for producing molecular hydrogen and hydrogen peroxide, proposed by Johnson and Weiss," is based on the direct interaction of excited water-molecules. 2 HzO* --t HzOz 2 H a * +Hn

+ HZ

+ 2 OH

More recently, WeissZ1put forward another theory, based on the interaction of ions according to the equations 2 HzO" 4HzOz + 2 He and 2 HsOe -+ Hz

+ 2 OH'.

c. Free-radical and Molecular-product Yields.-Although doubt still remains concerning the mode of formation of free radicals and molecular products, the net process following the interaction of electromagnetic radiation or of charged particles with water may be represented:

Ha0

+a

HZ+ b HZOZ+ c H

+ d (OH).

Accurate measurement of free-radical and molecular-product yields is important in radiation-chemistry studies on aqueous solutions, for these measurements enable quantitative predictions to be made regarding the extent of chemical changes during irradiations, and lead to an understanding of reaction mechanisms. Therefore, recent research has been directed toward the measurement of these yields, which are generally expressed as G values. An excellent account of the chemical methods used for determining G values (30) H. Fricke, Ann. N. Y . dcad. Sci., 69, 667 (1955). 77,4960 (1966). (31) H. A. Schwarr, J . Am. Chem. SOC., (32) G. R. A. Johnson and J. Weiss, Proc. Roy. SOC.(London), A040, 189 (1957).

RADIATION CHEMISTRY OF CARBOHYDRATES

19

of primary products formed in the radiolysis of water is given by Dainton.16 Allenas has surveyed in detail the yields which have been reported. The yields respectively designated G(OH), G(H), G(H2), and G(H202)depend on the reactivity and concentration of the solute, and on the density of energy release along the particle track. Such light-particle radiations as x-rays, y-rays, and electrons generate G(0H) varies between 6 and 7, free radicals mainly. The sum G(H) gradually increasings4as the concentration of solute increases from 10-4 M to M. Yields of molecular hydrogen and hydrogen peroxide are 0.4-0.5. Heavy-particle radiations produce mainly molecular hydrogen and hydrogen peroxide. The highest value yet obtained is with fission recoils, where G(H2) is 1.83, with the G(0H) and G(H) almostss zero. More hydrogen atoms than hydroxyl radicals are formed in y r a y irradiated acid solutions. In 0.8 N sulfuric acid, G(H) is 3.65 and G(0H) is36 2.95. As the pH increases above 3, the difference in G(H) - G(0H) decreases. Recent determinations of radical and molecular yields are in reasonable agreement with these values.2g~37-ag The relative yields of hydrogen atoms and hydroxyl radicals may be substantially affected by addition of either hydrogen or hydrogen peroxide to the system which is irradiated. Hydrogen provides hydrogen atoms and hydrogen peroxide increases the concentration of hydroxyl radical.

+

OH

H

+ Hz + H + HzO

+ Hz0z + OH + Ha0

Consequently, if suitable concentrations of hydrogen and hydrogen peroxide are chosen, it is possible to study the actions of hydrogen and hydroxyl radicals separately. The yields G(H2) and G(H202) decrease, also, with increasing concentration of solute. If a solute reacts with hydrogen atoms, the yield of hydrogen decreases as a result of competition between the reactions: H+H-+Ht H

+ solute + product.

(33) A. 0. Allen, Radiation Research, 1, 85 (1954). (34) E. J. Hart, Radiation Research, 2, 33 (1955). (35) J. W. Boyle, W. F. Kieffer, C. J. Hochanadel, T. J.

Sworski and J. A. Ghormley, Proc. Intern. Conf. Peaceful Uses Atomic Energy, Geneva, 7 , 576 (1956). (36) N. F. Barr and R. H. Schuler, Radiation Research, 7, 302 (1957). (37) M. Lefort and X. Tarrago, Compt. rend., 247,454 (1958). (38) M. Daniels and J. Weiss, J . Chem. Soc., 2467 (1958). (39) W. M. Garrison, B. M. Weeks, J. 0. Ward and W. Bennett, J . Chem. Phys., 27, 1214 (1957).

20

Q. 0.

PHILLIPS

Similarly, an oxidizable solute decreases G(H209): OH

OH

+ OH

+ solute

-+

HnOs

4

product.

When solute concentrations are below 0.1 M ,the chemical change occurs by indirect action. Under these conditions, the energy is absorbed by the water, and chemical changes result from the effect of the species produced in the primary radiolysis of water. At higher concentrations, direct-action effects may become important. Thus, when solid carbohydrates are irradiated, direct-action effectsare responsible for the chemical change, whereas, in solution, the concentration of solute may influence the contribution of indirect- and direct-action processes. d. New Radical Species.-Evidence for new radical species, which has been accumulating recently, has been summarized by Hart." Suggested species which may be present in irradiated water under particular conditions are the hydroperoxy radical, HOz , hydrogen-molecule ion, Ha@,oxygen atom ion, Oe, and subexcitation electrons. There is every reason to believe that the hydroperoxy radical is present in the track of the charged particle. When the concentration of solute is high, this radical is not found, and it may be formed, therefore, within the regions of intense ionization by the reaction: OH

+ HnOa

+ HnO

+ HOI .

(7)

Therefore, if the hydroxyl radicals are effectively scavenged by the solute, reaction (7)cannot take place, and G(H0a) is low. For 7-rays, the value is 0.026, but the value increases to 0.15 for low-energy a-rays.84,'O Evidence for the hydrogen-molecule ion is not definite. This species is proposed in order to explain yields of ferric ion in de-aerated, acid solution.41 Few

+ HI@+ Few + HI

Other explanations have, however, been put forward in order to account for this behavior.@ Hart, Gordon, and Hutchinson," on the basis of pH studies, propose ionization of the hydroxyl radical at pH's above 9: OH Oe + HS There are indications that Oe is more stable than the hydroxyl radical. (40)T. J. Sworski, Radiation Research, 6, 247 (1956). (41) W. G. Rothschild and A. 0. Allen, Radiation Research, 8, 101 (1953). (42) S. Gordon and E. J. Hart, J . Am. Chem. Soc., 77, 3981 (1955). (43) A. Charlesby and A. J. Swallow, Ann. Rev. Phyu. Chem., 10, 295 (1959). (44) E. J. Hart, S. Gordon and D. A. Hutchineon, J . A m . Chem. Soc., 76, 0105 (1963).

RADIATION CHEMISTRY OF CARBOHYDRATES

21

The possibility that subexcitation electrons may be responsible for the formation of hydrogen atoms was mentioned previo~sly.2~~a7 Attempts to confirm reports of solvated electrons have not been successful. No visible color is observable when metallic potassium, distilled onto the walls of a silica absorption-cell, reacts with water a t 0". 3. Chemical Dosimetry For quantitative studies in radiation chemistry, it is essential that the energy input into the irradiated volume should be accurately determined. For this purpose, the most versatile and reliable method is the ferrous sulfate dosimeter, proposed by Fricke and M0rse.~6The method involves the use of an air-saturated solution of 10-' M ferrous sulfate and 10-8 M sodium chloride in 0.8 N sulfuric acid. On exposure of the solution to ionizing radiations, the ferrous ion is oxidized to ferric ion, which may conveniently be determined accurately by spectrophotometry. The amount of chemical change is proportional to the total energy-input, independent of dose rate, and (within wide limits) independent of the concentration of ferrous ion, ferric ion, and oxygen. The main reactions involved are as follows.

+ Ha02 + F e w + OH + OHe Fern + OH + F e w + OHe H + 02 + HOz Few + HOS + Fe- + HOae H O P + H' + H a ,

Few

Thus, according to this mechanism, each peroxide molecule oxidizes two ferrous ions, each hydroxyl radical oxidizes one ferrous ion, and each hydrogen atom oxidizes three ferrous ions. Owing to the high preferential reactivity of organic substances toward hydroxyl radicals, care must be taken to use highly pursed water, free from organic impurities, in preparing the dosimeter. For this reason, also, sodium chloride is generally added. In the presence of chloride ions, the hydroxyl radicals readily react according to the equation : OH

+ Cle + OHe + C1.

The chlorine atom formed is still able to oxidize one ferrous ion, but it is much less reactive toward organic molecules. A constant G value, in the absence and presence of chloride ions, is, therefore, a convenient test for organic impurities in water. Calorimetric methods and ioniaation chambers were used for calibration (46) H.Fricke and 8.Morse, Phil. Mag., 7, 129 (1929).

22

a.

0. PHILLIPS

in this method, but, for a considerable period, there was a lack of correlation between the two methods. The conflict was eventually resolved, and Hochenadel and G h ~ r m l e y using , ~ ~ the (more reliable) calorimetric technique, first established the accepted value G(Feae) as 15.6 f 0.3 for Coeo y-rays in 0.8 N sulfuric acid. Energy input can, therefore, be readily calculated from G(Fe3@)for the radiation quality used, and thc amount of ferric ion formed is given by the expression: E(e.v./l.) = [lo0X 6.02 X 10W]/G(FeSe),

where C is the increase in ferric concentration (in moles/liter) produced by the particular radiation. The value of G(Fe3@)varies from 15.6 for such light particles as electrons, y-rays, and cr-rays to about 3 for fission recoils. Radiations having intermediate, linear-energy transfer have values between these two limit^.^^-^^

111. THEEFFECT OF RADIATION ON COMPOUNDS RELATED TO CARBOHYDRATES 1. Alcohols

Alcohols have been irradiated in the pure state and in aqueous solution, and it is therefore necessary to consider the two cases independently. Cyclotron a-rays were used by McDonnell and Newton63 to irradiate pure alcohols ranging from methanol to decyl alcohol, including the two isomers of propanol and the four isomers of butanol. The chemical changes induced were more specific than would have been anticipated from purely theoretical considerations. Analysis of the gaseous products by mass spectrometry, and of the liquid-phase products for water, carbonyl compounds, aldehydes, total glycols, and vicinal glycols, showed that, although a large number of products were formed, their nature was considerably restricted. Apart from carbon monoxide, no product is formed that is removed from the original alcohol by more than one oxidation state. The only glycols formed are vicinal glycols. Therefore, bonds are not broken indiscriminately by radiation, and the major reaction involves groups di(46) C. J. Hochanadel and J. A. Ghormley, J . Chem. Phys., 21, 880 (1953). (47)E . J. Hart, W. J. Ramler and R. 5.Rocklin, Radiation Research, 4,378 (1956). (48)R.H. Sohuler and A. 0. Allen, J . Am. Chem. SOC.,79, 1565 (1957). (49) W.R. McDonnell and E. J. Hart, J . Am. Chem. SOC.,76,2121 (1954). (50) R.H.Schuler and N. F. Barr, J . Am. C h m . SOC.,78, 6756 (1956). (51)L.Ehrenberg and E . Sneland, JENER Puhls., No. 8, 25 (1954). (52) N . Miller, in “Introduction A la Dosimetrie des Radiations, Actions chimique et biologiques des Radiations,” M. Haissinsky, ed., Masson et Cie, Paris, France, 1966. (53)W.R. McDonnell and A. S. Newton, J . Am. Chem. SOC.,76, 4G51 (1954).

RADIATION CHEMIBTRY OF CARBOHYDRATES

23

rectly attached to the C-OH group. It appears that fission of one of the =C-H bonds occurs, to give a hydrogen atom and a radical. The radical may dimerize to form a glycol, or it may be further oxidized to a carbonyl compound. Combination of the hydrogen atoms gives hydrogen. For ethanol, this may be represented as follows.

CY

CHjCHzOH 2 CHjCHOH

+ CHaCHOH -+

+H

CHaCHOH

I

CHjCHOH H+H+Hz

The l-hydroxyethyl radical may be further oxidized, to form aldehydes from primary alcohols, and ketones from secondary alcohols. The only tertiary alcohol to be studied in detail gave the corresponding ketone on irradiation. Support for the intermediate formation of the l-hydroxyethyl radical and a hydrogen atom was given by Burr.64 During radiolysis of CH3CH20H, CD3CH20H1 CH3CD20H1 CH3CH20D, and CD3CD20H, the proportion of deuterium in the evolved hydrogen was measured. Mass spectrometry showed that the loss of hydrogen was large for CH3CH20Hand CD3CH2OH, and small for CH3CD20H,although the isotope effect should lead to the opposite behavior if there is no preferential attack at the CH2 position. The total yield of hydrogen decreaseafrom G 3.7 to G 3.0 when the hydrogen atoms of the CH2 group are replaced by deuterium.66The main process is, therefore: CHaCHzOH -+ CHsCHOH

+ Hz .

However, deuterium also appears in the radiolytic gas when CH3CH20D is irradiated. This suggests, as a secondary process, the following. H

+ CHaCHzOH

-+

HZ

+ CHjCH,O*

The CH3CH2O* radicals disproportionate to give acetaldehyde and ethanol. On irradiation of liquid ethanols6with helium ions, the radiation yields for hydrogen, total carbonyl compounds, and vicinal glycols decrease markedly over the range 0.029 to 2.7 X 102* e.v./ml. When acetaldehyde or l-hexene were added, even in concentrations of 1%, they were sufficient to decrease the hydrogen yield, indicating a pronounced protective action by the products. It has been suggested that thermalized hydrogen-atoms may be responsible for at least part of the hydrogen formed during the radiolysis (54) J. G. Burr, J . Am. Chem. Soc., 79, 761 (1957). (55) J. G. Burr, J . Phys. Chem., 61, 1477 (1957). (56) W. R. McDonnell and A. S. Newton, J . Am. Chem. Soc., 78, 4554 (1956).

24

0. 0. PHILLIPS

of liquid ethanol, and McDonnell and GordonB7put forward a similar postulate to permit interpretation of the irradiation of methanol. By use of Co60 y-rays and 28 MeV a-rays, it was found that the amount of hydrogen released is similar in both cases, but the formation of formaldehyde is favored by the heavy-particle radiat,ion. Alcohols having very long chains appear to give less aldehyde and less glycol than alcohols having shorter chains. On prolonged irradiation, the primary products are affected, and initial aldehydes give rise to polymers. Finally, there is an increase in the amount of gaseous products formed.68 The effect of oxygen appears to be an increase in the amount of acid formed.6g An interesting aspect of the effects of radiation on alcohols is the extensive changes which may occur in C"-labeled alcohols under the agency of their own radiation (0.155MeV &rays). Methanol-@*undergoes considerable self-decomposition, with consequent formation of compounds of higher molecular weight. Methane is formed, and, in addition, ethylene glycol, glyceritol, and erythritoPO in the ratio 1360:14.9:1. Further consideration of self-decomposition is given for Clclabeled carbohydrates. The indirect action of radiation on aqueous alcohol leads to changes which are broadly similar in pattern to the direct-action effects described for pure alcohols. Hydrogen, aldehydes, glycols, and acids are formed in general, although it has not been established that acids are primary products."'-66 formaldehyde is formed During irradiation of methanol-water in yields proportional to the number of alcohol molecules exposed to radiation. Excess of the "dimer" (ethylene glycol) was formed-no doubt as a result of attack by free radicals formed during the primary radiolysis of water. (57) W. R. McDonnell and 5.Gordon, J . Chem. Phys., Is, 208 (1955). (58) J. C. McLennan, M. W. Perrin and H. J. C. Treton, Proc. Roy. SOC.(London), A126, 246 (1929). (5s) A. Kailan, Silzber Akad. Wiss. Wien, Mulh. nolurw. K l . , Abt. 1111, 140, 419 (1932). (60) W. J. Skraba, J. G. Burr and D. N . Hess, J . Chem. Phys., 21, 1296 (1953). (61) A. Kailan, Sitzber. Akad. Wiss. Wien, Math. nalurw. K l . , Abt. IIa, 143, 163 (1934). (62) H. Fricke, E. J. Hart and H. P. Smith, J . Chem. Phys., 6 , 229 (1938). (63) J. Loiseleur, R. Latarjet and C. Crovisier, Compt. rend. soc. bioZ., 136, 57 (1942). (64) W. R. McDonnell, J . Chem. Phys., 99, 208 (1955). (65) G. Scholes, J . chim. phys., 62, 640 (1966). (66) A. J. Swallow, Biochem. J . , 64, 253 (1953).

RADIATION CHEMISTRY OF CARBOHYDRATES

25

HzO + H + OH + CHsOH + HZ+ *CHsOH OH + CHaOH + Hz0 + *CH*OH H

2 *CHIOH 4CHiOH

I

CHIOH

The mechanism of radiolysis of aqueous solutions has been studied by Jayson, Scholes, and Weiw.67 The amounts of acetaldehyde, hydrogen peroxide, hydrogen, and 2,3-butanediol were measured at various pH’s and ethanol concentrations in oxygenated and in evacuated solutions. The yield of acetaldehyde is not independent of the concentration of ethanol. I n oxygen, the curve for yield of acetaldehyde against concentration of ethanol flattens out at a concentration of lo-* M , but the yield of acetaldehyde increases a t higher concentrations. The range of concentration studied was 1W6to 1 M ethanol. For the initial portion of the curve, the following reactions have been postulated.

+ OH + CHsCHOH + H20 H+ HO2 CHjCHOH + Oz + CHaC(0z)OH HOz + OH + Hz0 + On CH&(O2)OH + H o t + CHsCHO + CHaCHzOH

0 2

0 2

The yield, therefore, should be the over-all result of the competition between the last two reactions, and a G value of about 2 is attained, which corresponds to the available amounts of hydroxyl radicals. For higher concentrations of ethanol, the yield of hydrogen peroxide stays almost constant until the concentration of ethanol approaches M . The increase in aldehyde is, however, attributed either to competition between the reactions: H

+

01 4

HOI

and CHsCHIOH

+ H + CHaCHOH + Hz

or to electronic excitation of the ethanol molecules by subexcitation electrons produced by the (67) G . G. Jayson, G. Scholes and J. Weiss, J . Chem. SOC.,1368 (1957). J. T. Allen, E. M. Hayon and J. Weiss, ibid., 3913 (1969). (68) J. Weiss, J . chim. phys., 62, 40 (1966).

26

0. 0. PHILLIPS

Since, on this view, hydrogen atoms and hydroxyl radicals react to form the products, the sum of theyield of aldehyde (G 1.9) and of 2,3-butanediol (G 1.6) at pH 1.2 under vacuum should approximate to G(H) G(0H). The value 3.55 is in reasonable agreement with values from other Bystems at this pH. It would not appear necessary, therefore, to invoke the concept of sub-excitation electrons. The concentration dependence may be an indication that a solute concentration of 3 or 4 X 1CSM is necessary for scavenging all of the hydroxyl radicals and hydrogen atoms available. More recently, the effect of increasing the ethanol concentration beyond 1 M was examined. The yields of products further increased. Up to 5 M ethanol, the increase could be accounted for on the basis of a decrease in the back reaction H OH 4 HaO, and therefore provide an increase in the number of reactive species. However, above 5 M ethanol, the yields are so great that they cannot be the result of simple radical-solute reactions.

+

+

2. NucleicAd8

Although extensive investigations have been undertaken into the effects of ionizing radiation on nucleic acids, the precise chemical changes which are induced remain uncertain. Attention has been mainly concentrated on the changes in viscosity observed during and after irradiation. On irradiation in aqueous solution, the viscosity decreasesJ69J0and it continues to decrease for many hours after irradiation is terminated?0-73The evidence regarding the effect of oxygen on this process is rather Measurements of streaming birefringence7O and of sedimentation and diff usion constants support the view that degradation is the main effect of irradiation, fragments having molecular weights above 10,000 being produced?' This interpretation is also in keeping with the observed viscosity changes. Numerous investigations have centered on the physical changes accompanying irradiation , particularly changes in molecular weight and hydrogen bonding. A review of this aspect is given by Butler.lo There is ample evidence, from comparisons of the action of radiation A. H. Sparrow and F. M. Rosenfeld, Science, 104, 246 (1946). J. A. V. Butler, Radiation Research, Suppl. 1, 403 (1969). B. Taylor, J. P. Greenstein and A. Hollaender, Science, 106, 263 (1947). B. Taylor, J. P. Greenstein and A. Hollaender, Cold Spring Harbor Symposia Quant. Biol., la, 237 (1947). (73) B. Taylor, J. P. Greenstein and A. Hollaender, Arch. Biochem., 16, 19 (1948) (74) B. E. Conway, Brit. J . Radiol., N, 49 (1964). (76) B. E. Conway, Nature, 175, 679 (19S4). (76) J. A. Crowther and H. Liebmann, Nature, 115, 698 (1939). (77) M. Daniels, G. Scholes and J. Weiss, Nature, 171, 1163 (1966). (78) M. Daniels, G. Scholes and J. Weiss, Ezpen'entia, 11, 219 (1965). (69) (70) (71) (72)

RADIATION CHEMISTRY OF CARBOHYDRATES

27

with the behavior of Fenton’s reagent7g-81 and the effect of hydrogen peroxide photolyeed with ultraviolet light, that hydroxyl radicals79~*2*~~ are, in part or entirely, responsible for the observed changes. Hydrogen-platinum black has no effect on the nucleic acids, indicating that hydrogen atoms do not play a prominent although this observation is by no means proof positive. Attack by free radicals formed during radiolysis of aqueous solutions of nucleic acid does not appear to be specific at particular sites in the molecule. Deamination, liberation of inorganic phosphate, decrease in optical density at 265 mp, increase in Van Slyke amino nitrogen, decrease in purine nitrogen, and an increase in the number of titratable acid groups have all been 0bserved.7~ Nucleosides and nucleotides appear to behave similarly on irradiation. On the information available at present, it is not possible to identify the primary degradation processes following the irradiation of nucleic acid solutions. The following over-all changes have, however, been observed: (a) fission of glycosidic links and liberation of the purine base, (b) deammoniation and ring opening in the bases, (c) breaking of ester links to give inorganic phosphate, and (d) splitting of internucleotide links. It is probable, therefore, that the radiation-induced loss in viscosity is due to a reduction in hydrogen bonding between the molecules as a result of the loss of vital groups, as well as to direct degradation of the polynucleotide chain. The slow, post-irradiation decrease in viscosity (“after-effect”) was investigated by Daniels, Scholes, Weiss, and Wheeler,gOwho relate this phenomenon to the labilization of phosphate bonds by the intermediate formation of labile phosphate esters. Leading to this conclusion is the observation that about fifteen times as much inorganic phosphate can be obtained by acid hydrolysis of irradiated, aqueous solutions of nucleic acid as is formed directly by the radiation. It is, therefore, thought that, after the labile phosphate esters have been formed, they undergo slow acid hydrolysis, and that this mild hydrolysis occurring at the diester phosphate groups, even (79) J. A. V. Butler and B. E. Conway, Proc. Roy. Soc. (London), B141,562 (1953). (80)E. L. Grinnan and W. A. Mosher, J . Biol. Chem., 101, 719 (1951). (81) G. Limperos and W. A. Mosher, Am. J . Roentgenol. Radium Therapy, 63, 681 (1950). (82) J. A. V. Butler and K. A. Smith, Nature, 166,847 (1950). (83) B. E. Conway, Brit. J . Radiol., 27. 42 (1954). 77, 258 (1951). (84) D. B. Smith and G. C. Butler, J . Am. Chem. SOC., (85) E. S. G. Baron, P. Johnson and A. Corbure, Radiation Research, 1, 410 (1951). (86) G. Scholes, G. Stein and J. Weiss, Nature, 164, 709 (1949). (87) G. Scholes and J. Weiss, Ezpll. Cell Research, Suppl. 2, 219 (1952). (88) G. Scholes and J. Weiss, Nature, 166, 640 (1960). (89) G. Scholes and J. Weiss, Biochem. J . , 63, 667 (1963). (90)M. Daniels, G . Scholes, J. Weiss and C. M. Wheeler, J . Chem. SOC.,226 (1957).

28

G. 0. PHILLIPS

though it might not lead to the formation of inorganic phosphate, is sufficient to lead to a decrease in viscosity. Model experiments using purine and pyrimidine nucleotides support this view.e1 When these are irradiated in aqueous solution with x-rays, there is a post-irradiation release of inorganic phosphate in the presence and absence of oxygen. The post-irradiation process is first-order, and it is suggested that this behavior is due to the introduction of activating carbonyl groups in the sugar component. This interpretation of the after-effect appears more convincing than a previous explanation in which it was attributed to the formation of an unstable peroxide which decomposes slowly when irradiation is terminated.81 Hydroperoxides have been detected in nucleic acid solutions irradiated in oxygen, but these appear to be associated with the pyrimidine residue rather than with the sugar moiety.go Related to the interpretation of the effects of radiation on nucleic acids are the studies on the formation of labile phosphate esters in solutions of simple phosphates by irradiation. When glyceritol 1- and 2-phosphates are irradiated with 200 KV x-rays, inorganic phosphate is liberated. The former gives 1,3-dihydroxy-2-propanonephosphate, and the latter, an acid-labile phosphate e ~ t e r . Detailed ~ ~ - ~ ~studies on methyl, ethyl, propyl, butyl, and amyl phosphates have indicated the mode of formation of such labile phosphate esters.96 Two reactions have been recognized: 0

II + 2 (H + OH) + 0% RCH + HsPO, + HaOa + H i 0 RCHaOPOaH3 + (H + OH) + 1.6 Oa RCOPOaHt + H i 0 1 + Hz0.

RCHIOPOaHz

+

4

II

The latter reaction (producing the labile acyl phosphate) is less favored at increasing chain-lengths, and attack occurs along the hydrocarbon chain, with formation of an organic peroxide. In the absence of oxygen, no acyl phosphate or peroxide is formed, but inorganic phosphate is liberated. 3. Hydroxy Acids

When irradiated in aqueous solution, hydroxy acids are converted into the corresponding keto acids, particularly in the presence of oxygen. Lactic acid, for example, gives pyruvic acid,W and malic, citric, and 3-hydroxy(91) (92) (93) (94) (96) (96) (97)

M. Daniels, G. Scholes and J. Weiss, J . Chem. Soc., 3771 (1966). J. A. V. Butler and B. E. Conway, J . Chem. Soc., 3418 (1948). G. Scholes, W. Taylor and J. Weiss, J . Chem. Soc., 236 (1967). G. Scholes and J. Weiss, Nature, 171, 920 (1966). G. Scholes and J. Weiss, Brit. J . Radiol., 27, 47 (1964). R. W. Wilkinson and T. F. Williams, J . chim. phye., 61,600 (1966). G. R. A. Johnson, G . Scholes and J. Weiss, J . Chem. Soc., 3091 (1963).

RADIATION CHEMISTRY OF CARBOHYDRATES

29

butyric acids each give the related keto acid.98 In all the examples studied, the effect of oxygen is similar and leads to an enhanced yield of the product. Many of the reactions induced in organic molecules on irradiation in oxygenated aqueous solution follow a common mechanistic pattern. In this, the hydroxy acids conform strictly, and, because of the obvious relevance of such mechanisms to carbohydrate irradiations, the general pattern will be considered here.90 For a wide group of organic solutes in water, it is generally assumed that hydrogen atoms and hydroxyl radicals formed during primary radiolysis of water are removed by the reactions: H OH

+ Oz + HOz

+ HzM + HMO+ HsO,

where H2M is the solute molecule. Studies based on the measurement of the over-all stoichiometry of such reactions lead to the conclusions that HO2 does not react with organic solutes in the initial step and that the subsequent reactions involve: HMO+ O t + M + H O z 2 H0z

+

HzOz

+ 02.

The following yield-relation may therefore be expected: G(-H&f) = Gp(0H) G(Hz0z) = Gp(Hz0z)

+ 0.5 GP(OH) + 0.5 GP(H),

where Gp(OH), Gp(H), and Gp(H202) represent the primary yields of hydroxyl radicals, hydrogen atoms, and hydrogen peroxide, respectively. The value G(-H2M) is the over-all G for the disappearance of solute, and G(H202)is the observed yield of hydrogen peroxide. Several reactions have been interpreted on this basis.WJo0Jo1Termination occurs by the reaction :

+

2 HOa + H ~ 0 2 0 2 .

Modifications of this general pattern have been encountered, indicating clearly that no mechanism should be proposed unless accurate, initial-yield measurements have been undertaken. Malic acid solutions, for example, on irradiation with x-rays in oxygen, give oxalacetic acid in accordance with the mechanism outlined, with G( -malic acid) initially equal to 2.6. How(98) A. W. Pratt and F. K . Putney, Radiation Research, l, 234 (1964). (99) For a general summary of such mechanisms, see W. M. Garrison, Ann. Rev. Phys. Chem., 8 , 129 (1967). (100) E.J . Hart, J . Am. Chem. Soc., 76,4198 (1964). (101) W. M. Garrison and B. M. Weeks, J . Chem. Phys., 26, 585 (1956).

30

0. 0. PHILLIPS

ever, a parallel oxidation-path yields hydroxyoxalacetic acid with the overall stoichiometry : H N

+ 02

+

MO

+ Hz0.

Therefore, it would seem that this product is formed through an organicperoxy radical as intermediate, rather than through the reaction of a malic acid radical as required by the basic mechanism. An even more marked deviation occurs when L-ascorbic acid is irradiated with Coeo y-rays in oxygenated 0.8 N sulfuric acid.'" Here, the over-all stoichiometry is such that quantitative interpretation is impossible unless hydroxyl radicals and HOz radicals are involved in an initial, abstraction step:

+ H N HMO+ Hz0 HzM + H0z + HMO+ HzOz , OH

-+

followed by reaction of the one-electron oxidation-product HM with oxygen to form a peroxy radical: HMO

+ 02

-+

HM*(Oz).

Finally, HMO(02) is removed through the termination reaction: HM*(Oz)

+ HMO

+2

M

+ HzOz

or 2 HM*(Oz) --* 2 M

+ HzOz + 02 .

For carbohydrate irradiations in solution, also, a general difficulty exists if the mechanism is interpreted on the basis of initial abstraction by hydroxyl radicals only. The value G(-sugar), as will be seen from the subsequent discussion, is greater than the primary yield of hydroxyl radicals. In this respect, therefore, the behavior of carbohydrates on irradiation in solution resembles that of alcohol more closely than that of hydroxy acids. In the absence of oxygen, the mechanism of radiolysis of aqueous hydroxy acids is modified somewhat. During the y-irradiation of glycolic acid solutions under vacuum, the products are glyoxylic, oxalic, and tartaric acids.loSThese acids may be formed as follows. Hz0

--HCI

H

+ OH

HOCH~COZH2*CH(OH)COzH (102) N. F. Barr and C. G. King, J . Am. Chem. Soc., 78, 303 (195G). (103) P. M. Grant and R. B. Ward, J . Chem. Soc., 2654 (1959).

RADIATION CHEMISTRY OF CARBOHYDRATES

31

2 *CH(0H)CO ZH+ HOzCCH(0H)CH (0H)COzH

*CH(OH)COzH

(H0)zCHCOzH [-t CHOCOzH

-

(H0)zCHCOzH *C(OH)zCOzH

OH

+ HzO]

3 *C(OH)&OzH

(H0)sCCOzH [+ HOzCCOzH

+ HzO]

Dehydrogenation of glycolic acid by radicals from water may be anticipated, because of the activation of the a-hydrogen atom by the carboxyl groups. Further strong evidence for the facility of the first step comes from the observation that the direct action of y-rays on polycrystalline glycolic acid results in almost exclusive formation of the (carboxyhydroxymethyl) Quantitative studiesLosindicate that glyoxylic and tartaric acids are primary products and oxalic acid is a secondary product. By including the amounts of formic acid, formaldehyde, and carbon dioxide, a mass balance for carbon may be obtained. Therefore, the over-all degradation pattern under vacuum may be represented as follows. 0 Dimer i so% HOCHZCOZH (Tartaric acid) glycolic acid

glyoxylic acid

I

% ’

I

&HZ formaldehyde

formic acid

HzCzO4 oxalic acid

In oxygen, only a trace of tartaric acid is formed by dimerization of the (carboxyhydroxymethyl) radicals, but the yield of glyoxylic acid is increased to 70%. This is in accordance with the general mechanism for oxygenated solutions previously described. Dimeriaation is diminished following the removal of carboxyhydroxymethyl radicals by the following reactions. CH (OH)CO ZHA *OOCH(OH) COzH

0 *OOCH(OH)COzH

+ HOz

II

+ HC-COzH

+ HzOz + 0 2

Prolonged irradiation of hydroxy acids in the absence of oxygen leads to (104) P. M. Grant, R. B. Ward and D. H. Whiffen, J . Chem. Soc., 4635 (1958). (105) P. M. Grant and R. B. Ward, J . Chem. SOC.,2659 (1959).

32

0. 0. PHILLIPS

the formation of an acidic polymer,106presumably by an extension of the dimeriaation process, with combination of radicals formed by primary and secondary abstractions. This reaction leading to polymer formation is a general feature of irradiations of sugars, hydroxy acids, and amino acids in the absence of oxygen. The products from the irradiation of pure hydroxy acids have not been studied, but calcium glycolate-CI4is degraded, under the action of its own &rays, to formic acid and oxalic acid.1°7J0* Electron-resonance spectroscopy was used for identifying the radical obtained on x-irradiation'OD and 7-irradiation'w of crystalline glycolic acid. The evidence supports the structure HOCHC02H for the trapped radical, with only very slight indications of the presence of another radical. One possihlc step in the formation of the (carboxyhydroxymethyl) radical is as follows.

+ H O C H ~ C O ~+H HOCHCO~H + H This process may, however, occur in two stages - loss of an electron, followed by loss of a proton.

IV. THEEFFECT OF RADIATION ON CARBOHYDRATE^ One of the earliest workers to study the effect of ionizing radiations on carbohydrates was Kailan,lloJ1lwho observed that the radiations emitted from radium salts can induce hydrolysis in sucrose and D-glucose. Later interest centered on the physical changes which accompany irradiation, including changes in pH, optical rotation, reducing power, viscosity, and ultraviolet absorption ~ p e c t r a . ~ l 2Recently, -l~~ however, more attention has been given to the nature of the chemical changes which accompany irradiation, and, in this Section, the emphasis is placed on this aspect, wherever information is available. The subject is, however, far from complete, and, for many of the compounds studied, indications only can be given regarding the chemical changes involved. (106) S. A. Uttrker, P. M. Grant, M. Stacey and R. 13. Ward, J . Chem. Soc., 2648 (1959). (107) R . M . Lemmon, Nucleonics, 11. 44 (1963). (108) B. M. Tolbert, P. T. hdams, E. C. Bennett, A. M. Hughecl, M. R. Kirk, R.M. Lemmon, R . M. Noller, R. Ostwald and M. Calvin, J . Am. Chem. SOC.,76, 1867 (1953). (109) W. Gordy, W. B. Ard, Jr., and H . Shields, Proc. Natl. Acad. Sci. U . S . , 41, 996 (1965). (110) A. Kailan, Monalah., S!l, 1361 (1912). (111) A. Kailan, Monalah., S4, 1269 (1913). (112) P. Holtz and J. P. Becker, Arch. ezptl. Pathol. u.Pharniakol., 182, 160 (1936). (113) P. Holtz, Arch. ezptl. Pathol. u . Pharmakol., 182, 141 (1936). (114) A. Nome, Compt. rend. soc. b i d , 89, 96 (1923).

RADIATION CHEMISTRY OF CARBOHYDRATES

33

1. Polysaccharides

When polysaccharides are irradiated in the solid state or in solution, degradation is the most predominant feature observed. This statement is true for such naturally occurring polysaccharides as cellulose,l16 dextran,l17 starch,119,120 agar,’2l alginic acid,122various gums,119pectin~,l2~-’2~ and hyaluronic acid.126-128 That degradation occurs when aqueous solutions are irradiated is generally inferred from decreases in viscosity observed120,122,127,128 and the formation of reducing substances.l20 It has been claimed that larger doses are needed for degrading polysaccharides in the solid state than in aqueous solutions, and this claim is in keeping with general experience in radiation chemistry. For carbohydrates, however, the claim is based on very scanty evidence, and a quantitative comparison between irradiations of the pure solids and solutions of polysaccharides is urgently required. l ~ ~irWhen pectin powder (9.4% of moisture)lz6 or dry a p p l e - p e ~ t i nare radiated with x-rays or fast electrons, the viscosity of the solution after irradiation is lower than that of the unirradiated controls, and the decrease in viscosity is more pronounced at higher doses. Only slight changes in reducing power were, however, observed. Similar changes occur when aqueous solutions are irradiated. Sucrose, D-glucose, and D-fructose added to a pectin solution exert a protective action, presumably as a result of their scavenging of the hydrogen atoms and hydroxyl radicals formed during the primary radiolysis of water. D-Fructose is reported to be “by far” the most effective protective agent, although, from work discussed later in this article, D-fructose does not appear to be more susceptible to the action of s118

(115) E. J. Lawton, W. D. Bellamy, R. E. Hungate, M. P. Bryant and E. Hall, Tappi, 34, 113A (1951). (116) J. F. Saeman, M. A. Millett and E. J. Lawton, Ind. Eng. Chem., 44, 2848 (1952). (117) F. P. Price, W. D. Bellamy and E. J. Lawton, J . Phys. Chem., 68, 821 (1954); P. 0. Kinell, K. A. Granath and T. Vanngard, Arkiu Fysik, 13, 272 (1958). (118) C. R. Ricketts and C. E. Rowe, Chem. & Ind. (London), 189 (1954). (119) A. Branch, W. Huber and A. Waly, Arch. Biochem. Biophys., 39, 245 (1952). (120) M. A. Khenokh, Zhur. Obshchei Khim., 20, 1560 (1950). (121) H. Kersten and C. H. Dwight, J . Phys. Chem., 41, 687 (1937). (122) R. N. Fenstein and L. L. Nejelski, Radiation Research, 2, 8 (1955). (123) C. H. Dwight and H. Kersten, J . Phys. Chem., 42, 1167 (1938). (124) E. A. Roberts and B. E. Procter, Food Research, 20, 254 (1955). (125) Z. I. Kertesr, B. H. Morgan, L. W. Tuttle and M. Lavin, Radiation Research, 6, 372 (1956). (126) A. Caputo, Nature, 179, 1133 (1957). (127) C. Ragan, C. P. Donlan, J. A. Coss and A. F. Grubin, Proc. Soc. Exptl. Biol. Med., 88, 170 (1947). (128) M. D. Schoenberg, R. E. Brookes, J. J. Hall and M. Schneiderman, Arch. Biochem., SO, 333 (1951).

34

Q. 0 . PHILLIPS

hydrogen atoms and hydroxyl radicals when irradiated in dilute, aqueous solution. Depolymerization of cellulose fibers during irradiation is accompanied by a reduction in crystallinity, and, at high doses, extensive decomposition occurs. A dose of 5 X lo1*equivalent roentgens brings about marked degradation and is sufficient to convert cotton linters into water-soluble materials.116J2e-131 After irradiation, cellulose is more susceptible to acid hydrolysislle and exhibits an after-effect.lgl When irradiation is terminated, the intrinsic viscosity of cupriethylenediamine solutions of the irradiated cellulose continues to decrease. This behavior is initiated by oxygen and terminated by water. A similar effect is encountered with pectins after irradiation. Purified cotton subjected to y-irradiationls0 shows base-exchange properties, and the number of groups exhibiting these properties increases with increasing radiation-dose. Formation of carbonyl and carboxyl groups, decrease in tensile strength of the fibers, and increased solubility in water and alkali accompany irradiation in oxygen and nitrogen. Consideration has been given to the mechanism of the process, and it appears that (a) a fraction of the absorbed energy leads directly to ionization and (b) the remainder is transmitted as r-radiation of lower energy and as projected electrons. When aqueous solutions of amylose or starch are irradiated, degradation occurs, to give lower saccharides and dextrins.1aJ33The process may, however, not be related entirely to simple hydrolysis, for acid is formed and an absorption maximum appears at 265 mp after irradiati~n.'~~ Absorption in this region appears to be a feature characteristic of irradiated-carbohydrate solutions. The degradation of a m y l ~ s was e ~ ~followed ~ by measuring the increase in reducing power and by the reduction in intensity of the color formed on adding iodine to the solution. According to the results obtained by both methods, oxygen inhibits the degradation. Although the products formed have not been identified with certainty, there are indications from paper chromatography that (in addition to D-glucose) maltose, maltotriose, glyoxal, and a tetrose are formed. Production of acid is independent of the (129) A. Charlesby, U.K . Atomic Energy Reeearch Establishment Rept., A.E.R.E. M/R 1342 (1954); U.S . Atomic Energy Comm. Nuclear Sci. Abstr., 8, No. 3288 (1954); A. Charlesby, J . Polymer Sci., 16, 263 (1966). (130) J. C. Arthur and F. A. Blouin, Teztile Research J.,28,198 (1958); J. C. Arthur, ibid., 28, 204 (1968); J. C. Arthur and R.J. Demint, ibid., 2Q, 276 (1959). (131) R.E. Glegg and Z. I. Kertesz, J . Polymer Sn'., 28,289 (1967). See rtlso, Idem, Radiation Reeearch, 8, 469 (1957) and Science, 124, 893 (1966). (132) E. J. Bourne, M. Stacey and G . Vaughan, Chem. & Znd. (London), 189 (1954). (133) H. A. Colwell and 5. Russ, Radium, Q, 230 (1912). (134) M. A. Khenokh, Doklady Akad. Nauk 8.S . S . R . , 104,746 (1966).

RADIATION CHEMISTRY OF CARBOHYDRATES

35

extent of degradation of the amylose, and depends only on the dose. Initial G(acid) in oxygen is 1.5 and, under vacuum, is 1.4. Thus, a t least two primary processes take place simultaneously. One process leads to formation of acid and appears to be independent of oxygen, and the second process leads to degradation and is inhibited by the presence of oxygen. It must also be appreciated that, at any particular irradiation dose, some of the products have arisen by secondary processes. For example, D-glucose, present in Possufficient concentration, gives rise to acids, a tetrose, and gly0xa1.l~~ sibly, acids formed by secondary attack on D-glucose may lead to simple ionic hydrolysis to give lower saccharides. Therefore, unless the products and the primary and secondary degradation-processes are clearly identified, the conclusion is not justifiable that the breakdown of starch, amylose, and amylopectin with 200 KV x-rays and y-rays is similar in pattern to that obtained by biological agencies.136 Irradiation of solid, potato-starch granules (moisture content, 18.5 %) leads to changes similar to those observed in solution.ln After irradiation, the intrinsic viscosity and iodine-staining power of the starch specimens decreased, whereas the reducing power, lability to alkali, carboxyl value, and carbonyl number of the specimens increased with radiation dose. X-ray diffraction patterns showed that the crystalline part of the potato starch is damaged after doses of up to 1.5 X 107 roentgens. By paper chromatography, indications were obtained that D-glucose, maltose, D-arabinose, Dgluconic acid, D-glucuronic acid, and a series of dextrins of low molecular weight are formed by irradiation, with n-glucose and maltose predominating. The gases produced during y-irradiation are hydrogen, carbon monoxide, and carbon dioxide. When aqueous solutions of D-glucose are irradiated, hydrogen is the main gas produced, with carbon dioxide and carbon monoxide being formed in smaller amounts. D-kabinose, D-gluconic acid, and D-glucuronic acids are also among the products.136Therefore, it appears probable that, as for starch solutions, irradiation of starch granules leads, by oxidative and hydrolytic processes, to lower saccharides which subsequently undergo secondary degradation. The irradiation of dextran in the solid state"? and in solution138has been studied in detail. When dry, native dextran of high molecular weight (from Leuconostoc mesenteroides) is irradiated with 800 KV peak electrons,117the initial, molecular weight amounting to several million is diminished to about (135) G. 0. Phillips, G. J. Moody and G. L. Mattok, J . Chem. SOC.,3522 (1958). (136) L. Ehrenberg, M. Jaarma and K. G. Zimmer, Acta. Chem. Scand., 11, 950 (1957). (137) A. Mishina and Z. Nikuni, Mem. Inst. Sci. and Ind. Research Osaka Univ., 17, 215 (1960). (138) G . 0 . Phillips and G. J. Moody, J . Chem. SOC.,3634 (1958).

36

G. 0. PHILLIPS

fifty thousand by doses of about 1oB roentgen equivalents. Irradiation in nitrogen slightly reduces the extent of degradation, indicating that oxygen is involved in secondary processes only. The actual decrease in molecular weight is much less than the apparent degradation indicated by end-group methods of assay. Comparison of the number of end groups with the calculated number of ion pairs produced at each irradiation dose shows that, up to 6 X lo7 roentgen equivalents, there are, on the average, 2.1 f 0.2 end groups formed in the material per ion pair. This implies that the end result of each ionizing act, wherever it occurs in the molecule, is the production of two reducing end-groups. As degradation continues during irradiation, an increase in branching occurs, since, for a twenty-fold decrease in molecular weight, the branching per molecule drops by only a factor of two. All the branch points are probably tetrafunctional. Out of five ionpairs produced by the radiation, 1.0 produces a tetrafunctional branch, 1.1 produce a break in the chain, and the rest lead to a rupture of the D-glucose ring without occurrence of either degradation or eventual branching. The main degradation products have been identified in solution and estimated quantitatively.188 From their nature, it appears that one of the main processes is hydrolysis to D-glucose, isomaltose, and isomaltotriose. Moreover, the yield-dose curve (increase in the concentration of product with the dose) for reducing substances indicates that formation of D-glucose and other lower saccharides is a primary process. Two other major products arc D-gluconic acid and D-glucuronic acid, and the over-all yield-dose curve for acid formation indicates that these acids are also primary products, since they comprise the major acid constituents. It is evident, therefore, that, on irradiation, dextran in solution undergoes degradation by a t least two independent processes involving scission at the (1 + 6)-linkage in the molecule. One leads to D-glucose and lower saccharides, and the other, to Dgluconic acid and D-glucuronic acid. Glyoxal and crythrose, which are prrseiit in smaller proportions, are secondary products from D-glucose. Thus, random attack along the chain, us indicated on p. 37, would lead to all of the main products. 2. AIdohexoses, Ketohexoses, and Hexitols

Unbranched, six-carbon sugars and their derivatives are considerably more stable toward ionizing radiations in the solid state than in solution.139140 This behavior could be anticipated , since recombination of radicals initially formed by irradiation, aided by the “cage effect” of the lattice, would reduce the extent of the reaction with the solids, compared with similar (139) M. L. Wolfrom, W. W. Binkley, L. J. McCabe, T. M. Shen Han, and A, M. Michelakis, Radiation Research, 10, 37 (1969). (140) G. 0. Phillips and G . J. Moody, Chem. d Ind. (London), 1247 (1969).

RADIATION CHIMISTRY OF CARBOHYDRATES

37

changes in solution (which result from the action of the free radicals formed by primary radiolysis of water). It is clear, however, that free radicals are formed within the solid lattice during irradiation ; this was demonstrated

-?

-0

-?

I

7 I i

{>?{

Q

(Lo;o2H+

L 4

by the results of a paramagnetic-resonance study of each of sixteen carbohydrates after irradiation in the powder form.141On exposure of solid carbohydrates to strongly ionizing radiation, electrons can be removed from ground-state, molecular orbitals possessing sufficient energy to free them (141) D. William, J. E. Geusic, M. L. Wolfrom and L. J. McCabe, Proc. Nall. Acad. Sci. U.S., 44, 1128 (1958);G. Abstrom and C. Ehrenstein, Acta Chsm. Scand., 13, 856 (1959).

38

0. 0. PHILLIPS

from the molecule. If, on losing an electron in this way, the ionized molecule does not break up, it will have an unpaired electron in one of its orbitals. The electron removed from one molecule may attach itself to a neighboring molecule and form an excited orbital of this molecule, or it may be trapped at imperfections in the crystal lattice. Ionizing radiation may give rise to positively or negatively charged ions. These ions will be short-lived and will probably become stabilized as uncharged free radicals having unpaired electrons. In most cases, the formation of the final radical will be a complicated process, in which unstable entities are initially produced by irradiation and these, in turn, decay to others, until a stable radical is formed. If the barrier to the return passage of the electrons between the molecules is large, concentrations of free radicals can be built up that are sufficiently high to give a detectable electron-spin resonance.141For a-Dglucopyranose monohydrate, D-glucitol (sorbitol), a-D-galactopyranose, and myo-inositol, the paramagnetic-resonance spectra of samples irradiated either with fast electrons or with x-rays were identical, indicating that the final, radical products produced by x-irradiation are the same as those produccd by fast-electron irr~idiati0n.I~~ Radicals produced in this way may possess a half-life of up to 12.5 weeks at 20"and approximately 8.5 hrs. a t !i0°.117 The formation of colors and fluorescence in the irradiated Samp l e ~ ' ~ is~ a further indication of the excitation of the molecule. Definite physical and chemical changes have also been observed. Changes in optical rotation, mclting point, and acidity show that degradation occurs when u-glucitol, D-glucose, and wfructose are irradiated. Indications were obtained from ultraviolet absorption spectra that keto groups are introduced into the rn0lccule.~40The reducing values of D-glucose and D-fructose decreased on irradiation,I39 and, on the basis of paper-chromatographic evidence, it would appear that D-fructose is more susceptible to radiation damage in the solid state tban are D-glucose and ~ - g l u c i t o l . ~ ~ ~ J ~ ~ The precise nature of the chemical changes have been examined in detail for irradiations of aqueous solutions only.136J3eJ42-148 Oxygen exerts an important influence on the nature of the products, and, in radiation-chemical studies on carbohydrates, it is therefore essential to maintain either evacuated or oxygenated conditions throughout a particular irradiation. If initially air-equilibrated solutions are used, and no provision is made for reC. T. Bothner-By and E. A. Balazs, Radiation Research, 6, 302 (1957). G. 0. Phillips and G. J. Moody, J . Chem. SOC.,754 (1960). G. 0. Phillips and W. J. Criddle, J . Chem. Soc., 3404 (1960). G. 0.Phillips, Nature, 175, 1044 (1964). P. M. Grant and R. R. Ward, J . Chem. Soc., 2871 (1959). G. 0. Phillips, G . L. Mattok and G. J. Moody, Proc. Intern. Conf. Peaceful Uses Atomic Energy, Geneva, 29, 92 (1958). (148) G. 0. Phillips and G. J. Moody, Intern. J . Appl. Radialion and Isotopes, 6 , 78 (1959). (142) (143) (144) (145) (146) (147)

RADIATION CHEMISTRY OF CARBOHYDRATES

39

placing the oxygen consumed during irradiation,142the observations cannot be related either to fully oxygenated or to evacuated conditions, and quantitative measurements undertaken would be difficult to reproduce accurately. Moreover, it should be emphasized that the method of de-aerating the solution by passing nitrogen through is not so satisfactory as the complete-evacuation procedure, and Bourne, Stacey, and Vaughanla2have shown that there are appreciable differences between sugar solutions irradiated under vacuum and under nitrogen. The action of ionizing radiations on ~ - g l u c o s e and * ~ ~ D-mannose solution~~ in~oxygen ' has been studied by use of paper-chromatographic and radioactive-tracer methods. These methods for identification and quantitative estimation of the constituents present in the irradiated solutions have been de~cribed.1~9 Initially, ~-glucose-C~~ or ~ - m a n n o s e - Cis~added ~ to the solution to be irradiated. After a preliminary survey, uing paper chromatography and autoradiography to reveal the nature of the products, carrierdilution analysis is used for confirming or rejecting the indications given by paper chromatography. Crystalline derivatives are necessary for use in carrier-dilution analysis. Thereafter, the yield-dose curves are obtained for the main products by application of individual, carrier-dilution estimations a t each dose level and by direct scanning of spots separating discretely on paper chromatograms at various energy-inputs, by use of an endwindow, Geiger-Muller counter. Correlation between the two methods is therefore possible, and primary and secondary products may be distinguished by reference to the form of the yield-dose curves. The over-all pattern of degradation for D-glucose and D-mannose is similar. Therefore, detailed consideration is given for D-mannose solutions only.144 Table I shows the nature and amounts of the main products after total energy inputs of 3.7 X lon and 2.25 X loz3e.v. In these analyses, the unchanged D-mannose and the sum of the products account for 72 % and 85 % (by weight), respectively, of the original D-mannose present. It is evident that, a t high energy-inputs, part of the complexity of the system is attributable to secondary degradation. Therefore, to elucidate the nature of the primary degradation, particular attention was given to the yield-dose curves for the main products at low energy-inputs. The formation of acid is a primary process and the yield of it is independent of the concentration of D-mannose over a ten-fold range, indicating that the radiation energy is absorbed by the water and that chemical reactions are initiated by the reactive species formed. Since the rate of formation of acid increases with increasing input of energy, it appears that acids are also formed by secondary processes. The initial G for total acid is 1.6 and, for n-mannonic acid, is 0.6-0.7. It is probable that D-mannuronic acid represents the major portion (149) G . 0. Phillips and W. J. Criddle, Proc. Intern. Conf. Radioactive Isotopes (Copenhagen), 1960 (in the press).

TABLE I Constituents Present in Aqueous D-hfannose Solutions After Irradiation with CoaO -prays Yickl Conditwns4 (ma,imolc)

Prtdud

D-Mannoae

A B

1.20 0.16 0.50

A B

0.44 0.26 0.03

A B

0.06

A

1.40 0.40 0.49

A

0.06 0.31 0.21

C

D-Arabinose

C

D-LyXOSe

C

Two-carbon aldehydic fragments

Three-carbon aldehydic fragments

B C €3

C Oxalic acid

A B

0.04 0.74 0.19

A B

0.18 0.18 0.08

A

0.46 0.67

C

Formaldehyde

C

Sugar acids

B

C

D-Erythrose

0.17 0.001

0.38b

A B

0.12

C

0.69 0.10

D-Glucosone

C

0.20

Formic acid

A B

0.22 6.34

Carbon dioxide

A B

0.03 2.33

Key: A. Initial ~-Manno@e, 5.6 millimoles; ener input, 3.7 X lo**e.v. (vol., 40 d.) in oxygen, B. Initial D-Mannose, 5.48 millirn%s; energy input, 2.26 X lopa e.v. (vol., 100 d.) in oxy en. c. Initial D-Mannose, 2.42 millimoles; energy lnput 3.9 X 10" e.v. (vol., 100 m f ) under vacuum. Sum of D-gluconic acid and D-arabznohexuloaonic acid.

40

RADIATION CHEMISTRY OF CARBOHYDRATES

41

of the remaining acid formed initially. D-Arabinose is a primary product and is formed with an initial G of 0.54.6.The rate of formation increases at high energy-inputs, indicating that D-arabinose is formed by a secondary process also. The primary formation arises as a result of scission of the C - 1 4 - 2 bond in D-mannose, and decarboxylation of D-mannonic acid may account for the secondary formation. Experiments with ~-rnannose-I-C'~ show that primary scission of the C-1-C-2 bond leads also to formaldehyde. Initially, the yield-dose curves

m r (lOlev/rnl.).

Fro. 1.-Rate of Formation of Products During Irradiation of Oxygenated Solutions of D-Mannose (A 0 ,D-Arabinose;B 0 , formaldehyde;C A , formaldehyde from n-mannose-1-C9.

for formaldehyde from ~-mannose-I-C1~ and generally-C14-labeled u-mannose are identical, but, at increased energy-inputs, the apparent yield of formaldehyde from ~-mannose-I-C~~ decreases (see Fig. 1). Since, in the latter estimation, only formaldehyde-I-CI4is measured, the fall in concentration probably results from dilution by the formaldehyde formed from the remaining (inactive) portion of the D-mannose molecule. As required by this mechanism, the D-arabinose formed in experiments with D-mann0se-1-C~~ is inactive. Primary scissioii of the hexose molecule into four- and two-carbon, aldehydic fragments occurs, probably to give erythrose and glyoxal. The initial G value for total, two-carbon, fragment formation is 0.64,Using Dmannose-I-P, it is possible to measure the primary formation of those

42

G. 0. PHILLIPS

two-carbon fragments which contain carbon-14 and which are thus formed by scission of the C-2-C-3 bond; this process should also lead to simultaneous formation of a four-carbon fragment. The initial G value for two-carbon fragments derived from measurements of carbon-14 is 0.2, a value in reasonable agreement with the initial G(erythrose) of 0.18. The appreciable difference between these values and the over-all formation of two-carbon fragments (G 0.64) leads to the conclusion that such fragments must be formed by two primary processes, which may be represented as follows. CH,OH

0

+

F"

$--OH

Ho

0

II

/""

6"

$qlH CH,OH

1

HO

/

/

Further oxidation of glyoxal (to oxalic acid) occurs, but this is a secondary process. Symmetrical scission of the hexose molecule into three-carbon fragments takes place, but to tin extent smaller than by the process described. Irradiated solutions of D-mannose show maximum absorption at 275 mp, and it is probable that more than one constituent may account for the absorption spectrum. Enediols absorb strongly in this region and, in particular, reductone, a possible constituent present in irradiated solutions of D-mannose, absorbs a t 290 mp in alkali, The over-all consumption of Dmannose during irradiation shows an initial G 3.5, in excellent agreement with irradiations of D-glucose (G 3.5) .la The main processes may therefore be represented as follows. D-Mannose

n-Erythrose and glyoxal

L

glyoxal I(

Oxalic acid

/

D-mannuronic acid

l.

D-lyxose

D-mannonic acid

1

D-arabinose

D-arabinose

+

formaldehydc

43

RADIATION CHEMISTRY OF CARBOHYDRATES

These primary processes proceed with an initial G 2.84, and therefore account for the main processes of degradation, although a further degradative path is not precluded. Under vacuum, the complications of secondary reactions involving oxygen are excluded, and the degradative pattern follows a rather different path.160The constituents present in evacuated solutions of D-mannose after e.v. are shown in Table I, irradiation to a total energy input of 3.9 X and these account for about 97 % (by weight) of the initial D-mannose present. The initial formation of acid (G 0.5) agrees well with the initial G (mannonic acid), indicating that the acid first produced is D-mannonic acid. Other acids are formed at higher doses by secondary processes, for example, Darabino-hexulosonic acid. D-Mannuronic acid is not present in detectable amounts in irradiations under vacuum, although it is a major product on irradiation in oxygen. D-Glucosone (D-arabino-hexosulose)is a primary product having initial G 0.5. The primary scission between C-1 and C-2 (to give formaldehyde and D-arabinose), encountered in oxygenated solution, does not occur under vacuum, and D-arabinose arises by a secondary decarboxylation of D-mannonic acid. Ring-scission processes similar to those observed in oxygen are indicated by the yielddose curves for two- and three-carbon, aldehydic fragments, although the latter fragments are formed in much higher yields than in the oxygenated system. The primary scission between (2-24-3 and (3-4-C-5, to give three two-carbon fragments, occurs with initial G 0.95, and comparison with the initial G 0.25 for D-erythrose formation indicates that scission to give two- and four-carbon fragments takes place simultaneously with the formation of three two-carbon fragments. Another primary process which is not so pronounced in oxygen is the symmetrical scission giving two three-carbon aldehydic fragments (G 0.5). The degradation under vacuum may, therefore, be represented as follows. D-Mannose

I

1

I

D-Mannonic acid D-glucosone

1

D-Arabinose

I three-carbon aldehydic fragments

1

two-carbon aldehydic fragments

1

four-carbon two-carbon aldehydic fragments

+

D-arabinohexulosonic acid

A distinctive feature (not encountered in oxygen) of irradiations under vacuum is the formation of a polymer at high doses. This behavior was first reported by Stacey and coworkers.10gFrom the yield-dose curve for polymer, (160) G. 0. Phillips and W. J. Criddle, (in the press).

44

Q. 0.

PHILLIPS

Phillips and Criddlelso conclude that polymer is formed by secondary processes, with the rate of formation increasing markedly at high doses. The primary processes described for irradiations under vacuum account for an initial G for the degradation of D-mannose of 2.4, in contrast to the observed value of G 3.5 for disappearance of D-mannose. The primary processes that are unaccounted for probably arise by dimerisation of radicals formed aa the preliminary step in the production of the polymer. The nature of the identified products demonstrates that attack by free radicals formed by the primary radiolysis of water occurs throughout the molecule. Attack at C-1 leads to D-mannonic acid; at the C-34-4 bond, to threecarbon fragments; and at the C - 2 4 - 3 and C-4-C-5 bonds, to give three two-carbon fragments. Thus, the various sugar radicals formed initially (which lead to these products) may dimerize and eventually build up into a branched polymer of complex composition. Further radicals arising from primary and successive degradation products may well account for the acidic character of the polymer. Polymer formation is, therefore, an inefficient coupling-processrather than a chain reaction. Because of the general similarity between the radicals formed in water during irradiations under vacuum and in oxygen, some correlation should be possible between the two systems, and appears to have been made. A striking feature of the processes is the identical rate of disappearance of Dmannose under both conditions (G 3.6), which points to comparable initialabstraction processes. Subsequently, the secondary effect of oxygen and HOBradicals leads to somewhat differing products. Under both conditions, D-mannonic acid is formed, and the products arising from ring scission are also similar. Symmetrical scission of D-mannose in oxygen does not occur easily, although this circumstance is more significant for oxygenated solutions of D-glucose,l**However, in oxygen, attack at C-2 causes ring scission leading to D-arabinose and formaldehyde, but, under vacuum, such attack affords D-glucosone (D-arabino-hexosulose). These reactions may be represented as follows. Under vacuum

RADIATION CHEMISTRY OF CARBOHYDRATES

45

In oxygen H2C=O formaldehyde

HC=O

+

I

HC-0 HObH

I

&Mannose

D-arabinose

This behavior is analogous to that of queous glycolic cid. The carboncarbon scission which occurs in oxygen is diminished under vacuum.1MJO6 Similarly, dimerization is only observed in the absence of oxygen. D-Mannuronk acid is formed in oxygen only, and it is probable that, under vacuum, the eame initial step leads to dimerization, as is observed for irradiations of D-glucitol in the absence of oxygen.lal RCHnOH

5

+ OH + RCHOH + HzO in o x y g e n l A

RCOzH

under vaouum

RCHOH RbHoH

D-G~UCOW solutions behave similarly on irradiation. Grant and Ward146 detected D-gluconic acid and D-glucosone in solutions of D-glucose irradiated under vacuum. They postulated a degradative mechanism analogous to the radiation-induced degradation of glycolic acid: HC-O HbOH (bHOHir

-b

c=o

HbOH

5

( HOH)a

bH,OH

HC=O

+

bOH kHOHil

bHzOH OHIA

O H ~ ~ ' ~ , m d i caddition a l

D-Gluconic polymer D-gluco- polymer acid sone

Summarizing therefore, it would appear that, when aldohexoses are irradiated in dilute solution, attack is not confined to any particular part of the molecule. The products formed in oxygen and under vacuum, respec(161) W. J. Criddle, Ph.D. Thesis, University of Wales, 1960.

46

GI. 0 . PHILLIPS

tively, demonstrate that all bonds are affected. Oxidation occurs at the extremities of the molecule and, simultaneously, ring scission leads to lower fragments. Similar initial processes probably take place in oxygen and under vacuum, although secondary reactions involving oxygen may considerably modify the nature of the end product. Support for this view comes, not only from the nature of the products, but also from the identical G values (3.5) for the disappearance of aldohexose in oxygen and under vacuum. The G value is significant and demonstrates that hydroxyl radicals are not the only species which may initiate reaction, following abstractions of the type: RH

+ OH - + R e + H@.

If all hydroxyl radicals were scavenged by this process, G for the disappearance of the aldohexose could not rise above G,(OH), the primary yield of hydroxyl radicals. The situation is similar to that encountered with alcohols irradiated at high concentration^,^^ and it is probable that hydrogen atoms may also participate in the initiation process. The alternative possibility is that HOs radicals may initiate reaction, as proposed for irradiations of L-ascorbic acid.1M Clearly, therefore, on irradiation in solution, aldohexoses exhibit changes similar in character to those observed under comparable conditions in related compounds, particularly hydroxy acids and alcohols. Further kinetic work is, however, necessary, before intelligent, detailed mechanisms can be advanced in order to explain the observed changes. When D-fructose is irradiated in aqueous solution in the presence of oxygen, the following inter-related degradation processes have been distinguished.14* CHiOH L

HC-0 L

O

COaH O

L

I HOCH

HOkH d

O

HOAH __f

HAOH

H h H

HLOH

HAOH

HLOH

HAOH

bH2OH LHZOH AH20H D-arabino-Hexulose D-arabh-hexosuloee D-arabino(D-Fructose) (D-glucosone) hexulosonic acid

I I - I -

CHzOH

0-AH Glycolaldehyde

HC-0

H =O glyoxal

COiH

A02H oxalic acid

RADIATION CHEMISTRY OF CARBOHYDRATE]S

47

Evidence has also been educed for the formation of two constituents, reductone and 1,3-dihydroxy-2-propanone,by symmetrical scission of the D-fructose molecule; these constituents are mainly responsible for the peak at 285-290 mp in the ultraviolet absorption spectrum of the D-fructose solutions. CHzOH

CHpOH

b=O

b=O

ROAH

b

H =O

H OH

+ +

HAOH

HboH

AHtOH

HC=O

LH2OH

HCOH

e

&OH

A

H =O CHzOH

e b=o bH@H

For the decomposition of D-fructose, G is 4.0, a value of the same order as for the rate of consumption of D-glucose and D-mannose (G 3.5) under comparable conditions. Another primary process may involve oxidation of the primary alcohol group a t C-6 to form ~-2gzo-5-hexulosonicacid, since there is accumulating evidence from the behavior of ~ - g l u c o s e D-glu,~~~ ci t0 1 , ’ ~ ~and J ~ ~~ - m a n n i t o P that ~ J ~ the ~ primary alcohol groups are more reactive than secondary alcohol groups toward free radicals formed by the action of radiation on water. The degradation induced in hexitols on irradiation in aqueous solution is more specific than for hexoses under comparable conditions. In 1954, it was reported that, when D-mannitol solutions (1 %) are irradiated with fast electrons in oxygen, D-mannose is the main product, and that, after prolonged irradiation, D-mannuronic acid is forrned.l45By use of more concentrated solutions (50 %), similar results were subsequently obtained by Wolfrom and coworker^,^^^ and penta-0-acetyl-p-D-mannopyranose was isolated. D-habinose was formed as a secondary product, in addition to D-mannuronic acid (see p. 48). Oxidation of either of the primary hydroxyl groups in D-mannitol would give D-mannose, and hence the characterization of Dmannose is easier than that of the products from irradiations of D-glucitol. Paper-chromatographic evidence reveals the presence of four main products from D-glucitol, namely, glucose, gulose, xylose, and arabin0se.’39J~7J~ Carrier-dilution analyes demonstrate that the stereochemical forms present are D-arabinose, L-xylose, and ~ - g l u c o s e On . ~ ~configurational ~ grounds, therefore, it is deduced that gulose is present as the L isomer. Oxidation of the primary alcohol group at one extremity of the D-glucitol molecule leads to D-glucose, and oxidation of that a t the other end, to ~-gulose.Sim-

a.

48

0. PHILLIPS

ilar considerations apply to formation of pentoses. Simultaneously, the presence of formaldehyde was detected by isotope-dilution analysis. Thus, the degradation may be represented as follows. HC-0 HbOH

HC-0

b

HO H HbOH HboH

b + HbOH Hb ,OH

HO H

+

H HC=O formaldehyde

I

bI

HO H

~ H ~ O H ~ H ~ O H D-glucose D-srabinose CHiOH I

HC-0 I

RADIATION CHPJMISTRY OF CARBOHYDRATES

49

The yield-dose curvesI47for the hexoses and pentoses demonstrate that D-glucose and ~-guloseare the primary products, formed at identical rates (G 1.2), with the formation of D-arabinose and ~-xyloseoccurring subsequently. Reactions proceeding with ring scission are also of a secondary character.14Q As for hexose degradations, G(-D-glucitol) is 3.5 for 1% solutions. To establish whether products formed initially at low rates are formed by primary or secondary processes is a common difficulty. This applies particularly to D-gluconic acid and L-gulonic acid produced during the irradiation of D-glucitol solutions. Potentiometric measurements indicate only slow initial formation of acid (G 0.3),but, from accompanying carrier-dilution estimations, it is probable that the direct conversion to acid, RCHZOH--.) RCO2H, is a primary process with an initial G of about 0.15. Wolfrom and cow~rkers'~Q commented on the similarity of the products from the irradiation of alditol solutions and from the action of Fenton's reagent (ferrous ions and hydrogen peroxide) thereon. Few

+ Ha02 + Fern + .OH + OH-

D-Mannose was synthesized from D-mannitol, in 40% yield, by the latter method'" through the agency of hydroxyl radicals, and it seems probable that a comparable explanation applies to the irradiation process. When conditions of strict evacuation are maintained during irradiations of D-glucitol,l6' the yields of hexoses are lower than in oxygen (G 0.7), although the rate of disappearance of D-glucitol is identical with the rate in oxygen (G 3.5). Under vacuum, ring scission gains in significance, and glycolaldehyde and tetrose are formed. The yieldaose curves for these fragments demonstrate that they are formed by primary processes, with an initial G of about 1.0. CHaOH H LI o H H O ~ H

1"'+""

H =O

CHaOH HboH I

H O ~ H

A

H=O ......I...... H ~ O H

LH,OH

I

H~OH

AHSOH

+

HC=O bHaOH

Gas analysis indicates that primary abstraction processes under vacuum involve hydrogen atoms in addition to hydroxyl radicals.

+ OH + RCHOH + HIO RCHIOH + H -+ RCHOH + Hn

RCHIOH

(162) F. Haber and W. Weiss, Proc. Roy. SOC.,A147.332 (1934).

50

0. 0. PHILLIPS

Under these conditions, initial dimerisation occurs as the primary step in the formation of the polymer-which may easily be isolated from irradiations of D-glucitol in the absence of oxygen (as from hexose irradiations under vacuum). 2 RCHOH + RCHOH

RtrHoH

Three primary processes have, therefore, been identified as occurring when D-glucitol solutions are irradiated under vacuum. D-Glucitol

8

Hexoses

dimer

glycolaldehyde

+ tetroses

With the information at present available, it is not possible to advance detailed mechanisms for the proceases described, and further kinetic investigations are necessary. The behavior of alditols and D-fructose on irradiation in solution support the view that primary alcohol groups are more susceptible to attack than normal secondary alcohol groups, the group at the lactol carbon atom providing an understandable exception. 3. GlycoSides, Disaccharides, Trismharides, and Lactimes

When maltose and cellobiose are irradiated with fast electrons in airequilibrated, 50 % solutions, the predominating reaction is hydrolysis, and the results suggest that the W D linkage is the more labile, a conclusion in accord with the relative ease of hydrolysis of these disaccharides by acids. A number of samples of aqueous maltose solutions (20%) were irradiated with electrons to doses of 20 to 100 M rep. The apparent hydrolysis, as determined by the reducing power, increased linearly with dose, and this result has been interpreted in terms of the hydrolysis of only one bond.139 Hydrolysis at the glycosidic bond occurs also during irradiation of sucrose in aqueous solution, and acid is produced.16a-*aa Changes in the ultraviolet absorption spectrum have also been noted,’u and the over-all change in optical rotation accompanying inversion was proposed by WrightlsBfor use as a pile-radiation dosimeter. The chemical effects of radiation on sucrose solutions have been investigated by Wolfrom, Binkley, and McCabe16’ (163) M. C. Reinhard and I(.L. Tucker, Radiology, 12, 161 (1929). (164) G. L. Clark and K. R. Etch, J . Am. Chem. rSoc., 62,466 (1930). (165) G. L. Clark, L. W. Pickett and E. D. Johnson, Radiology, 18, 245 (1930). , 60 (1962). (156) J. Wright, Discusdons Faraday ~ o c . 1P, (157) M. L. Wolfrom, W. W. Binkley and L. J. McCabe, J . Am. Chem. Soc., 81, 1442 (1969).

RADIATION CHEMISTRY OF CARBOHYDRATE8

51

and by Phillips and Moody.'@ Different experimental conditions were used in the two investigations. Wolfrom and cow0rkers~~7 irradiated 50 % aqueous solutions of sucrose with fast electrons without oxygenation in open, aluminum containers cooled in ethanol-solid carbon dioxide, ice and water, and ambient air, whereas Phillips and Moody1@irradiated dilute solutions (2.9 X 10-2 M ) with CosOy-radiation in oxygen at room temperature. In the former investigation,lS7attention was focused on comparing the effects of fast electrons on sucrose and on methyl a-D-glucopyranoside, since preliminary had suggested that the glycosidic bond might be especially sensitive to ionizing radiations. The only products detected in irradiated sucrose solutions were D-fructose and D-glucose, the former by paper chromatography and the latter as the pentaacetate. Paper-chromatographic evidence indicated the formation of substantial proportions of D-glucose in irradiated solutions of methyl a-D-glucopyranoside. In comparison with sucrose, methyl a-D-glucopyranoside is more resistant to hydrolysis under the same experimental conditions. After an energy input of 104 megareps, the extent of hydrolysis of aqueous sucrose solutions was 22.2, 27.0, and 37.8 % when cooled with ethanol-solid carbon dioxide, ice and water, and ambient air, respectively, whereas, a t the same energy input a t ice-water temperature, methyl a-D-glucopyranoside was hydrolyzed to the extent of 6.3% (based on conversion to D-glucose). Evidence was obtained that about 10 % of the sucrose is transformed into nonreducing substances a t all three temperatures. It should be borne in mind that, in comparison with the energy inputs used in this investigation, a dose of about 3 megareps is required for the sterilization of food. From measurements of the increase in reducing power with dose, reported by Wolfrom and coworkers,'67 the extent of apparent hydrolysis at 3 megareps is about 4, 2, and 1 % when the solution is cooled in ethanol-solid carbon dioxide, icewater, and ambient air (at about 27"), respectively. Yield-dose curves obtained by carrier-dilution analysis and by paper chromatography reveal that D-glucose and D-fructose are primary products of the y-irradiation of dilute, aqueous, sucrose solutions in oxygen, together with smaller amounts of D-glucosone and D-gluconic acid.'& D-G~ucuronic acid, D-arabino-hexulonic acid, D-arabinose, and two- and threecarbon, aldehydic fragments arise by secondary processes. In the final stages, carbon dioxide and formic acid are formed. Hydrogen peroxide is produced continuously. The initial G values for D-glucose and D-fructose are 1.5-1.6; D-gluconic acid and D-glucosone are formed with G 0.4 and 0.6, respectively. (158) G.0.Phillips and G. J. Moody, J . Chem. Soc., 166 (1960). (169)M. L.Wolfrom, W.W.Binkley and L. J. McCabe, Abstracts Papers Am. Chem. Soc., 190. 16A (1956).

52

0. 0. PHILLIP8

The primary formation of D-gluconic acid and D-glucosone simultaneously with D-glucose and n-fructose may be accounted for by two types of oxidative scission of the disaccharide linkage, a t a and b. The former leads to D-fructose and

D-gluconic acid, and the latter, to D-glucose and D-glucosone. A similar type of process was envisaged for the degradation of aqueous dextran with y-radiation.'" If the two types of scission occurred to a comparable extent, the amounts of the four main products would be of the same order. The results show, however, that, although comparable amounts of D-gluconic acid and D-glucosone are formed, the proportions of D-glucose and D-fructose are higher. It appears, therefore, that hydrolysis is the dominant process, but that it is accompanied, to a smaller extent, by the oxidative scission described. The over-all degradation pattern for sucrose has been formulated as follows. Sucrose

3 -

D-Glucose __t D-gluoonio acid D-glucosone

I

I(\

alyoxal

D-glucuronic acid

n-arabinose

I

D-fructose

D-arabino-hexuloeonic acid

Hydrolysis is the predominating process when the trisaccharide raffinose is exposed to y-rays in 2% aqueous solution.luJ*O The extent of hydrolysis increases with dose and, since two bonds are hydrolyzed, the reducing power-dose curve is non-linear. It has been reported by Coleby'eo that, when solutions of D-glucono-1,4lactone, D-gulono-1 ,.Q-lactone,L-galactono-1,4-lactone and L-gulono-1 ,4lactone are irradiated with x-rays (210kV,, lOmA), y-rays (Coao), and fast electrons (4 MeV) under vacuum, the lactones are converted into the corresponding ascorbic acids. (180) B. Coleby, Chem. & Ind. (London), 111 (1967).

RADIATION CHEMISTRY OF CARBOHYDRATES

53

CHOH AH20H

bH2OH

Evidence for the production of ascorbic acids is that, after irradiation, the solution was able to decolorize solutions of 2,G-dichlorophenolindophenol and to produce a red color with an alkaline solution of triphenyltetrazolium chloride. The solution also displayed an absorption maximum at 245 mp similar to that of L-ascorbic acid, and paper chromatography indicated the presence of a product running identically with L-ascorbic acid. The yield of the ascorbic acid was a function of the concentration, rising from G 0.16 at 5 X M to G 0.95 at 4 X 1W2M for a dose of 2.8 X lo1*e.v. ml.-1 of x-rays. The route to the ascorbic acid from the lactone may involve abstraction of a hydrogen atom at C-2 or C-3,followed by enolisation.

4. Absorption Spectra and Post-irradiation Processes Irradiated solutions of carbohydrates have similar, characteristic ultraviolet absorption spectra.'34~'8"18e~142,148.146 A broad absorption occurs in the region of 240-300 mp, and the maxima, which may vary for individual carbohydrates, fall in the region of 260-290 mp; the intensity of the peak increases very markedly on addition of alkali.136For the absorption maximum, a shift to higher wave-lengths and an increase in intensity may accompany the addition of alkali.'" Several of the identified products absorb in this region, either in acid or alkaline solution, and it is probable that more than one absorbing constituent is responsible for the resulting absorption spectrum. One compound which may contribute to the composite spectrum may be 1,3-dihydroxy-2-propanone(A, 265 mp, in neutral solution), formed by isomerization of glycerose, a change which occurs readily (particularly in alka1i).lS6Reductone (Ama in alkali, 287 mp, and, in acid, 268 rnplB1),D-glucosone (Amax in alkali, 265 m ~ ) , and ' ~ ~the dienol (161)

T.C. Laurent, J . Am. Chem. Soc., 78, 1876 (1966).

54

0. 0. PHILLIPS

form of D-urabino-hexulosonic acid (A, in alkali, 275 mp, and, in acid, 230 inp)'61 are other possible absorbing species. However, all enediol structurcs [such as L-ascorbic acid (A, in neutral or acid solution, 265 mp14E)] would be expected to absorb strongly in this region, and, since "D-ghco-ascorbic" acid has been claimed to be a product from the irradiation of D-gluconolactone in aqueous solution,lEOsecondary products may also contribute to the over-all spectrum. There is evidence for the presence of slow post-irradiation processes when sugar solutions are irradiated in oxygen and under vacuum. When D-glucose solutions are irradiated under vacuum, the absorption at 265 mp increases steadily and attains a maximum at 20-30 hr. after irradiation has ceased.186 Similarly, for D-fructose solutions irradiated in oxygen and under vacuum, the absorption maxima continue to increase for several days after irradiation has ceased. This process may be associated with the postirradiation decrease in concentration of hydrogen peroxide at a rate of 3 4 X lo'* molecules min.-l ml.-l in irradiated ~-fructosel~~ and D-glucose13b solutions. The occurrence of post-irradiation reactions is further demonstrated in these systems by a liberation of gas for 2 6 3 0 hr. after irradiation is terminated.186 Further detailed examination of these interesting reactions is necessary, before it will be possible to speculate about their association with several important post-irradiation processes encountered when biological systems are irradiated.'* 5. Self-decomposition of Cl4-hbeledCarbohydrates

When carbon-14 tracer techniques are applied to chemical problems, it is important that the C14-labeled compounds used should be chemically pure. However, it has frequently been assumed that such compounds are virtually stable when stored prior to use. Evidence is now accumulating that this assumption is unwarranted, and that a considerable degree of degradation occurs in CWabeled amino acids, amino alcohols, purine derivatives, calcium glycolate, cholesterol, thyroxine, and succinic acid during st0rage.1~7J~ As noted previously, rnethan01-C~~ undergoes decomposition under the action of its own radiation.60 Wagner and GuinnlE2studied the self-decomposition of methyl-C14 iodide and, from the limited literature on this s u b j e ~ t , l it ~ ~would J ~ ~ appear that various groups of C14-labeled compounds have widely differing susceptibilities to radiation self-decomposition. A reference to the self-decomposition of carbohydrates appeared in 1956,1h when it was discovered that ~-glucose-C~~ solution requires (162) C. D. Wagner and V. P. Guinn, J . Am. Chem. SOC.,76, 4861 (1963). (183) See, in addition to the cited references, B. M. Tolbert and R. M. Lemmon, Radiation Research, 3, 62 (1956). (164) W. G . Dauben and P. H. Paycot, J . Am. Chem. SOC.,78,6667 (1966). (l64a) P. J. Allen and J. S. D. Bacon, Biochem. J . , 65,200 (1966).

RADIATION CHEMISTRY OF CARBOHYDRATES

55

purification, as 2 % of its activity migrates (during paper chromatography) in the disaccharide region. Although this effect was not attributed to selfdecomposition, this explanation now appears likely. Clearly, it is of supreme importance that the user of C14-labeled carbohydrates for tracer studies be aware of the phenomenon of self-decomposition, since the products resulting can otherwise lead to erroneous interpretation of the results of experiments. It is also important that the method of storage that will cause minimal degradation should be ascertained and the products be recognized, so that purification methods may be devised. Therefore, although but little published material is available166J66 a t the time of writing, the author has compiled this Section (consisting of preliminary information) in order that this important aspect of the radiation chemistry of carbohydrates may be included. The author is indebted to Professor E. J. Bourne, Dr. H. Weigel, and Dr. R. Bayly for making their complete results available prior to publication. The decomposition of a compound labeled with a radioactive isotope can be due to one or more of four effects,lg as follows. (1) A primary (internal) radiation effect, wherein the decomposition of the molecules arises as a result of the disintegration of their unstable atomic nuclei. (2) A primary (external) radiation effect, in which decomposition occurs by interaction of the molecule with a nuclear particle. (3) A secondary radiation effect, where decomposition arises from reaction with a reactive species produced by the radiation. An example would be that of free radicals produced by the radiolysis of residual water in freeze-dried carbohydrate samples. (4)A chemical effect, whereby decomposition arises from chemical reactions which are not connected with radiation. For C14-labeled carbohydrates stored as freeze-dried samples under vacuum at room temperature, self-decomposition arises mainly by the primary (external) radiation effect and the secondary radiation effect. However, it has been observed that the alkalinity of normally washed, Pyrex glass is detrimental to the stability of C14-labeled carbohydrate sirups, and it is desirable to store the samples at as low a temperature as possible in order to reduce the rate of such unavoidable chemical reactions. Table I1 shows the extent of self-decomposition of initially pure samples of sucrose-C14 and ~-glucose-C14which had been stored as uniform films on Whatman No. 3 paper or as freeze-dried samples in Pyrex tubes (which had been filled with water and autoclaved for 2 hr. at 151 lb./sq. in. to remove surface alkalinity). The tubes of samples which were stored under vacuum were evacuated to a pressure of 0.01 mm. Hg for several hours before being sealed (to reduce the moisture content to a minimum). (165) N. Baker, A. P. Gibbons and R. A. Shipley, Biochim. et Biophys. Acta, 28, 68 (1958). (166) A. Walton and H. Weigel, Nature, 189, 981 (1959).

a.

56

0 . PHILLIPS

It would appear that, for the freeze-dried samples of D-glucose (tubes 749, secondary-radiation effects play a major role in the decomposition, due probably to retention of non-bonded water by the D-glucose. Sucroae, TABLE I1 Sel decompositim1~of Swro8e-C14 and ~-Orlucoee-C~~

-

-

Slorags conditions

compoumi

Pub8 no.

-

TyP., Prar-

Form

C.

sure

freaadried room froow-dried --Boo on paper room --Boo on PSW room on psper

Mo.

1

a a 4 6 0

-

Im-

,i:s, %

-

on paper

--Boo

atm. atm.

16.4 16.1 16.7 4.9

WO.

a.4

VIM.

1.8

VBO.

7 8

room

atm.

--Boo

atm.

9

room -80'

-0.

room -80'

-0.

10 11

ia

-

vao.

18.9 0.0 13.1 3.0 1.a 0.7

- -VIM.

I-

I-I-

0.071

0.36

0.086

0.96

0.0dB

o.oao

0.010

-

-

0.007 -0.77 0.13 0.61 o.1~ 0.044 0.024

o.a o.a o.a 0.1

-a1 a1 ai

ai

-

-

16 16 0.3 0.3 0.3

4.0 4.a a34 68 33

0.8

16

10 10 10 10

79 a3 63 14 900

- 47 0.m - 47 0.05 6!26 * To calculate G(-M),

The weight of the Whatman No.3. pa er waa 18 m om-'. it is necessary to estimate the fraotion of the tot8energy liberated (during the decomposing period) that is absorbed by the sugar or su ar sirup. For a pure 0emitter distributed over a relatively large area in a layer 1 o f even thickness (in om.), this fraction F, is given by:

(8)

0

F

-

+

1 ~ 1 0 . 5 log, r/pO1/2r where p is the density (me. om.-#) and r is the mean range of the particles expressed in m om.* units. Alternatively, the fraction of the energy absorbed by the sugar may t e calculated by Besumin that the compound and the paper absorb all the radiation in the ratio of their weigtts. 0 Defined aa the number of molecules permanently altered or decomposed per 100 0.v. absorbed by the sugar or sugar sirup. * Each sucrose tube contained 600 pc in 1.16 mg. of sucrose (149 mc. per m. mole; abundance of CI', 19.5p) and was stored for 88 weeks. Calculation shows that primary (internal) radiation ecompoaition contributed 0.06% to the observed impurity. Other experiments with s a m lea of freese-dried sucrose have shown that, a t -Ma, decomposition is considera%l increaaed if the sugar is stored under atmospheric pressure instead of being sealedrunder vacuum. The difference is even greater with samples held at room temperature. Each tube of D- lucose contained 100 pc in 0.43 mg. of D-gluoose (42 mc. perm. mole; abundance of &4,11.0%) and waa stored for 34 weeks. Calculation shows that primary (internal) radiation decomposition contributed 0.006% to the observed impurity.

on the other hand, can, with efficient freeze-drying, be obtained anhydrous, and the extents of decomposition for tubes 1 and 2 suggest that this small, constant amount of degradation is attributable to primary-radiation effects. This may be inferred from the very small difference in the extent of decomposition of freeze-dried aucroee at room temperature and at -80'. For D-glucose, a much greater temperature-dependence is shown, due, pre-

RADIATION CHEMISTRY OF CARBOHMRATES

57

sumably, to a reduction in the mobility of radicals formed by secondary effects. Radicals would, therefore, appear not to play an important part in the decomposition of freeze-dried sucrose samples. When sugars are distributed on paper (tubes 3-6, 11, and 12), the decomposition is more marked, presumably because of increased secondary effects arising from the moisture in the paper. The importance of secondary-radiation effects may be seen from the decomposition of dextran-CI4sulfate containing about 20 D-glucose residues per molecule, observed by Bayly and Weigel.1g7JB8 It had the relatively low specific radioactivity of 22.4 mC. per g.-atom of carbon (about 3 mC/ millimole of dextran sulfate). After three weeks in the freeze-dried form, it had charred and become a total loss. The decomposition was presumably attributable to a secondary-radiation effect arising from a prior liberation of sulfuric acid, which then released more sulfuric acid to destroy the material. Arising from the decomposition of C14-labeled carbohydrates are an enormous variety of products. For example, when a sample of ~-glucose-C'~ (about 6 mg., having a specific activity of about 14.44 mC. per millimole) was stored as a freeze-dried sample in the dark for 26 months, a 14.5% decomposition of the D-glucose occurred and, by use of two-dimensional paper chromatography-paper electrophoresis, the presence of 37 constituents was revealed.la The greater complexity of this system in comparison with that of +radiated solutions of ~-glucose185supports the view that direct-action effects supplement the decomposition caused by secondaryradiation effects, which are entirely responsible for the decomposition when dilute solutions are irradiated. Two methods for reducing the magnitude of decomposition from primary (external) radiation are (a) dispersion over a large area and (b) dilution. These methods also reduce decomposition caused by secondaryradiation effects'"; this is borne out by experimental results obtained168 with ~-mannose-C'~.Aliquots (5 ml.) of pure ~-mannose-C'~ (about 100 pC.) in water (100 ml.) were stored under vacuum. By isotope-dilution analysis, it was estimated that the rate of decomposition in the freeze-dried state was 7 % a year and, in the frozen state, 1 % a, year. The frozen state would, therefore, appear to be the most satisfactory method of storage over long periods of time.

Note Added in Proof Since this review was prepared, an important development has been reported by Dr. F. C. Leavitt at the 3rd. Cellulose Conference, Syracuse, (167) R. J. Bayly and H. Weigel, Nature, 188, 384 (1960). (108) E. J. Bourne, D. H. Hudson and H . Weigel, J . Chem. Soc., 5153 (1960). (169) G. 0. Phillips and W. J. Criddle (unpublished reaults).

58

Q. 0. PHILLIPS

N. Y. (October, 1960). High-energy radiation unexpectedly produces crosslinking in some cellulose compounds. An important factor which determines whether cross-linking or degradation predominates is the viscosity of the system. Highly viscous solutions give limited freedom of motion to polymer chains, and high-energy radiation degrades these systems until the fall in viscosity permits free, bimolecular coupliig-reactions, At this point, the system immediately gels. There is evidence that the cross-linking process involves an indirect effect of radiation, occurring through the agency of free radicals.

APPLICATIONS OF TFUFLUOROACETIC ANHYDRIDE IN CARBOHYDRATE CHEMISTRY

BY T. G. BONNER Department of Chemistry, Royal Holloway College, University of London, Engle$eld Green, Surrey, England

.......................... n with Trifluoroacetic Anh 1. 0-Trifluoroacetylation.

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

59

60

2 . N-Trifluoroacetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 I11 The Trifluoroacetyl Group &B a Blocking Group .......................... 63 1 The Synthesis of 2- and 3-0-Substituted D-GIucoses .................... 63 2 . The Synthesis of 2,4.Di.O.methyl.~.rhamnose ......................... 64 3 . Other Synthetic Uses of Trifluoroacetyl Derivatives . . . . . . . . . . . . . . . . . . . 65 IV. 1. Acylation with Carboxylic Acid-Trifluoroacetic Anhydride Mixtures 67 . . . . . . ............ 67 Acylation of Hydroxy Compounds.. . . . . . . . . . . . . . . . . .

.

.

V. Selective Ring-opening of Cyclic Acetals with Carboxylic Acid-Trifluoroacetic Anhydride Mixtures, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Methylene Acetals of D-Glucitol.. . . . . . . . . . ............ 2. Methylene Acetals of D-Mannitol.. . . . . . . . . . . ........... 3. Other Acetals.. ............................. ............ VI. The Synthesis of Linear Polymeric Esters from C and DibMic Carboxylic Acids. ....................... .............. VII. The Mechanism of Acylation by Acyl Trifluor 1. The Formation of Acyl Trifluoroacetates.. . ................

69 69 75 76 77

79 2. The Reaction of Acyl Trifluoroacetates with Hydroxy Compounds.. .... 81 3. Other Acylation Reactions of Acyl Trifluoroacetates.. . . . . . . . . . . . . . . . . . 83

I. INTRODUCTION Trifluoroacetic anhydride was first obtained by Swarts by dehydration of the acid, and his initial studies of organic compounds containing the trifluoroacetyl group began with an examination of ethyl trifluoroacetate, synthesized by esterification of ethyl alcohol with trifluoroacetic acid in the presence of concentrated sulfuric acid.1 A few other organic trifluoroacetates were later prepared in the same way, but the potential use of the anhydride of tritluoroacetic acid as a preparative agent in general organic chemistry, and , in particular, in carbohydrate chemistry was not realized until recently.2J The importance of this reagent became apparent with (1) F. Swarts, Bull. clasae sci., Acad. roy. Belg., 8, 343 (1922). (2) E. J. Bourne, M. Stacey, J. C. Tatlow and J. M. Tedder, Nature, 164,705 (1949). (3) J. M. Tedder, Chem. Revs., 66, 787 (1956).

59

60

T. 0. BONNER

the discovery that, apart from its use as a direct trifluoroacetylating agent, its addition to a carboxylic acid in slight excess of the equimolecular proportion provided a powerful acylating solution for the conversion of a suitable substrate to the acyl derivative of the carboxylic acid.4 The original investigation which revealed this latter property of trifluoroacetic anhydride was concerned with an attempt to prepare cellulose trifluoroacetates by direct treatment of cellulose with the anhydride. No reaction was apparent, but, when the cellulose was pretreated with acetic acid, it subsequently dissolved slowly in trifluoroacetic anhydride, with the formation of a chloroform-soluble product containing no fluorine and having the properties of cellulose acetate. This novel use of trifluoroacetic anhydride has since been widely extended. At the same time, the more conventional function of introducing the trifiuoroacetate group into hydroxy compounds is of considerable interest and will be briefly dealt with first. Trifluoroacetic anhydride is conveniently prepared in high yield by distillation of trifluoroacetic acid over phosphorus pentaoxide.le4 An alternative procedure, for which a high yield is also claimed, utilizes sulfur trioxide to convert trifluoroacetic acid into trifluoroacetylsulfuric acid which, on further treatment with trifluoroacetic acid, affords trifluoroacetic anhydride.' Although highly volatile (b.p. 34-40'), trifluoroacetic anhydride is much more convenient to handles than the acyl chloride (b.p. -27") or the acyl bromide (b.p. -5").

11. TRIFLUOROACETYLATION WITH TRIFLUOROACETIC ANHYDRIDE 1. 0-Trifluoroacetylatirm

The replacement of the hydrogen atom in a hydroxyl group by the trifluoroacetyl group is effected by the usual acylation procedure of warming the hydroxy compound with trifluoroacetic anhydride in the presence of dry sodium trifluoroacetate under anhydrous conditions.? The recovery of the ester requires, however, a modified procedure, since the usual method of destroying the excess acid anhydride by means of aqueous sodium bicarbonate simultaneously brings about hydrolysis of the alkali-labile trifluoroacetate group. In order to remove excess trifluoroacetic anhydride and the trifluoroacetic acid present under anhydrous conditions, the reaction mixture is distilled several times with dry carbon tetrachloride and (4) E. J. Bourne, M. Stacey, J. C. Tatlow and J. M. Tedder, J . Chem. Soc., 2976 (1949). (6) J. F. Dowdall, U. S. Pat. 2,628,263 (1963); Chem. Abstracts, 48, 1426 (1964). 66,389 (1943). (6) J. H. Simons and E. 0. Ramler, J . Am. Chem. SOC., (7) E. J. Bourne, C. E. M. Tatlow and J. C. Tatlow, J . Chenc. Soc., 13G7 (1960).

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

61

the ester is finally removed from the residual sodium trifluoroacetate by extraction with the same solvent or dry hexane. Original experiments resulted in good yields, from the appropriate sugar derivatives, of D-mannitol hexakis(trifluoroacetate)and methyl 4,6-O-benzylidene-2,3-di-O-(trifluoroacetyl)-cy-D-glucoside,both of which are crystalline, and methyl 2,3,4,6tetra-0-(trifiuoroacetyl)-a-D-glucosideand 1 ,2-0-isopropylidene-3,5,6-tri0-(trifiuoroacety1)-D-glucose, isolated as pure liquids. The method was later applied successfully to a wide variety of hydroxy compounds, both aliphatic and phenolic, and additional procedures were adopted for the isolation of the product? The trifluoroacetates are reasonably stable when pure and dry, but, in the presence of water, they are hydrolyzed readily (apparently autocatalytically, through the trifluoroacetic acid liberated). The hydrolysis is very rapid with esters of polyhydroxy compounds, indicating a unique lability of trifluoroacetyl groups when they are adjacent to each other in the same molecule. An alternative method of removing the trifluoroacetyl groups is provided by the use of dry methanol; in this solvent at 17",the observed rotation of a dry methanolic solution of the product fell to 70% of its original value in 1 hour and to zero overnight. Both detrifluoroacetylation procedures produce the parent hydroxy compound without occurrence of an accompanying Walden inversion or formation of an anhydro ring. The above method of trifluoroacetylation was utilized to convert benzyl @-D-glucosideinto its 2,3,4,6-tetrakis(trifluoroacetate), with the intention of using this product in the synthesis of D-glucosyl ester^.^ It was hoped that the benzyl group could be removed by catalytic hydrogenation, and then position C-1 could be esterified with a carboxylic acid in the presence of trifluoroacetic anhydride, and the product detrifluoroacetylated with dry methanol; conditions could not, however, be found for the effective removal of the benzyl group from the trifluoroacetyl derivative. Swarts used a mixture of trifiuoroacetic acid and concentrated sulfuric acid for esterifying monohydroxy compounds and, although the method has subsequently been used successfully, other workers have found that either trifiuoroacetic acidlo or its anhydride aloneu J* are often effective esterifying media. Another method employs silver trifluoroacetate for (8) E. J. Bourne, M. Stacey, J. C. Tatlow and R. Worrall, J . Chem. SOC.,3268 (19%). (9) F. Weygand and E. Rauch, Chem. Ber., 87, 211 (1954). (10) A. Kalusayner, S. Reuter and E. D. Bergmann, J . Am. Chem. SOC.,77, 4164 (1955). (11) H. W. Coover and J. B. Dickey, U.S. Pat. 2,759,912 (1956); Chem. Abslracls, 61, 2327 (1957). (12) R. F. Clark and J. H. Simons, J . A m . Chem. SOC.,76, 6305 (1953). (13) V. T. Oliverio and E. Sawioki, J . Org. Chem., 20,363 (1956).

62

T. 0. BONNER

converting sn alkyl iodide into the corresponding alkyl trifluoroacetate in high yield.I4 A detailed study has been made of the action of pure trifluoroacetic acid on cellulose and cellobiose (and their acetates) .l6 Dissolution of cellulose occurs, and swelling takes place with rupture of hydrogen bonds and with micellar dispersion; esterscation takes place without occurrence of degradation, the cellulose being fully recovered on hydrolysis of the trifluoroacetylated product. It appears that there is a more rapid rate of trifluoroacetylation of primary than of secondary alcohol groups. Although pyridine has been reported to be an unsuitable medium for trifluoroacetylation (as it reacts with trifluoroacetic anhydride),? this solvent has been successfully employed in the trifluoroacetylation of 1l-epicorticosteronel6; in aqueous pyridine a t 20°, the trifluoroacetyl group introduced was found to undergo slow hydrolysis without further aid. 2. N - T r i J u o r ~ t y l a t i o n

An interesting contrast is provided by the methods of synthesizing 0trifluoroacetyl and N-trifluoroacetyl derivatives. The latter are very readily formed by the direct action of trifluoroacetic anhydride on primary or secondary amines, and are stable to prolonged boiling with dry methanol.” Since O-trifluoroacetylation usually requires the presence of sodium trifluoroacetate, selective trifluoroacetylation of amino groups is possible ; any adventitious introduction of O-trifluoroacetyl groups can be dealt with by subsequent treatment with methanol (to remove these groups selectively). Investigations of N-trifluoroacetylation have been mainly confined to amino acids, for which, excellent yields (70-95%) are obtained by the use of tritluoroacetic anhydride in anhydrous trifluoroacetic acid .u The presence of the strong acid prevents ionization of the carboxylic acid group of the amino acid and ensures nonformation of a mixed anhydride. This medium has also been found to trifluoroacetylate the amino group of a peptide linkage.lg Excellent yields of the N-trifluoroacetyl derivatives of amino acids and peptides, usually without occurrence of racemiaation, have been obtained20 by heating with phenyl trifluoroacetate in phenol at 1 2 0 - 1 5 0 O . A novel N-trifluoroacetylating reagent which has been used for (14) R. Filler, J. F. O’Brien, I. V. Fenner and M. Hauptechein, J . Am. Chern. SOC., 76, 966 (1963). (16) A. L. Geddee, J . Polymsr Sci., II,31 (1966). (16) A. Lardon and T. Reichatein, Helu. Chim. Acta, 87,388 (1964). (17) E. J. Bourne, 8.H. Henry, C. E. M. Tatlow and J. C. Tatlow, J . Chem. Soc., 4014 (1962). (18) F. Weygand and R. Geiger, Chem. Ber., 89,047 (1966). (19) F. Weygand, R. Geiger and V. Gloakler, Chem. Ber., 89,1643 (1966). (20)F. Weygand and A. Rapech, Chem. Ber., 81,2095 (1959).

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

63

amino acid anions in aqueous solutionz1is S-ethyl trifluorothioacetate CF3COSEt; the product was found to be stable in aqueous acid, but, in solutions of pH > 10, the trifluoroacylamide bond underwent rapid hydrolysis.

111. THETRIFLUOROACETYL GROUPAS

A

BLOCKING GROUP

1. The Synthesis of 2- and 3-0-SubstitutedD-Glucoses

The ease of hydrolysis of the trifluoroacetyl group, without occurrence of complicating side-effects, has obvious synthetic possibilities through the provision of a readily removable blocking group. Removal of the group has been achieved under extremely mild conditions at room temperature with (a) anhydrous methanol,7*22(b) aqueous acetone, when an autocatalytic acid hydrolysis appears to accompany the release of trifluoroacetic and (c) aqueous pyridine.I6 The first examination of this use of the trifluoroacetyl group in carbohydrate studies was concerned with the conversion of methyl 4,6-0-benzylidene-2,3-di-0-(trifhoroacety1)-cr-~-glucoside (1) into 2- and 3-substituted D-glucoses.22The compound (1) , although losing both trifluoroacetyl groups readily in methanolic solution on prolonged standing (18 hr.), can undergo partial de-esterification to the monotrifluoroacetyl derivative (2), either by suitable treatment with dry methanol-carbon tetrachloride or by use of a concentrated solution of the bis(trifluor0acetate) in methanol, when the mono ester gradually separates. In establishing the location of the surviving trifluoroacetyl group, it was found that, apart from its lability and ease of replacement, this group may migrate under alkaline conditions. Methylation of the mono ester (2) with methyl iodide and silver oxide was inconclusive, the sole product being the 2,3-dimethyl ether; this replacement of trifluoroacetyl groups by methyl groups during methylation, which had been noted previously, is variable and appears to depend on such factors as the purity of the reagents.' The reactions of the mono ester which indicated the position of the trifluoroacetyl group and its propensity to migrate were (a) acetylation with an acetic acid-trifluoroacetic anhydride mixture, (b) acetylation with acetic

L!2)oM.

''L

(1); R = R' CFsCO (2); R = CFsCO, R' = H (3); R = R' = H

OR

R'

(4); R CFsCO, R' = BZ (5); R = Bz,R' = H

anhydride in pyridine, and (c) tosylation in pyridine. I n each case, the (21) E. E. Schallenberg and M. Calvin, J . Am. Chem. Soc., 77, 2779 (1955). (22) E. J. Bourne, M. Stacey, C. E. M. Tatlow and J. C. Tatlow, J . Chem. Sac., 826 (1951). (23) E. J. Bourne, A. J. Huggard and J. C. Tatlow, J . Chem. Soc., 735 (1953).

64

T. 0. BONNBR

free hydroxyl group was substituted, the trifluoroacetyl group being retained. On removal of the latter group, the products were found to be the following derivatives of methyl 4,6-O-benzylidene-cu-~-glucoside (3): from reaction (a), the 2-acetate; from (b), the 3-acetate; and from (c), the 2-0tosyl derivative. Since migration of acyl groups is favored by alkaline conditions, it was concluded that migration of the trifluoroacetyl group occurs during the aeetylation in pyridine solution, but not in the aeetylation under acid conditions nor in the p-tolylsulfonation. The displacement can be regarded as a process similar to the removal of the trifluoroacetyl group in methanolysis, since both processes involve transfer of the group from one alkoxy oxygen atom to another. It is known that, whereas p-tolylsulfonylation of (3) proceeds preferentially a t the C-2 hydroxyl group, the 3-acetate of (3) is more stable than the 2-acetate. The trifluoroacetyl group appears, therefore, to be readily displaced when, by this occurrence, the incoming group is enabled to take up its most favorable position. The mono(trifluoroacetate) (2) is, therefore, the 3-ester. This conclusion has been confirmed by a study of the benzoylation of this isomer.a* A monobenzoate (4) was obtained which gave, either on acid hydrolysis or on alcoholysis, a product which, on p-tolylsulfonylation, formed the 2-0benzoyl-3-0-tosyl derivative of (3). Attempted removal, by dilute alkaline hydrolysis, of the trifluoroacetyl group from the monobenzoyl derivative (4) led to simultaneous migration of the benzoyl group from the C-2- to the C-3-hydroxyl group, to give methyl 3-0-benzoyl-4,6-O-benzylidenea-D-glucopyranoside (5) ; this migration also occurs when the 2-benzoate (4) is treated with dilute alkali. 2. The Synthesis of d ,4-Di-O-methyl-~-rhamnose Although the 2 ,&dimethyl and 3 ,Cdimethyl ethers of L-rhamnose were known, the 2,4-dimethyl ether had not been synthesized prior to its preparation through a trifluoroacetyl intermediate.2412sThe synthesis started from methyl 2,3-O-isopropylideneiu-~-rhamnopyranoside; this was methylated and the acetal group removed, to give methyl 4-O-methyl-cu-~rhamnopyranoside (6). Conversion to the 2,3-bis(trifluoroacetate) (7) was readily achieved with trifluoroacetic anhydride in the presence of sodium trifluoroacetate. As expected, the trifluoroacetate (7) was completely deacylated by treatment with alcohol, regenerating (6) ; this process was complete after 25 min. a t room temperature. The procedure for selective de-esterification was based on the observation that, if excess carbon tetrachloride (6 vol.) is present, very little methanolysis occurs. By use of a mixed methanol-carbon tetrachloride solvent (65: 35 vol./vol.), the meth(24) K. Butler, P. F. Lloyd and M. Stacey, Chem. & Ind. (London), 107 (1954). (26) K. Butler, P. F. Lloyd and M. Stacey, J . Chem. Soc., 1631 (1966).

65

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

anolysis was allowed to proceed to the point of maximum optical rotation, and the de-esterification was then effectively stopped by pouring the solution into a large volume of carbon tetrachloride. On evaporation of the solvent, a sirup was obtained having the methoxyl content of a mono(trifluoroacetate). On acetylation with pyridine and acetic anhydride, followed by methanolysis to remove the remaining trifluoroacetyl group, methyl 3-0-acetyl-4-0-methyl-cu-~-rhamnopyranoside (8) was obtained. This was converted to its 2-methyl ether (9), with silver oxide and methyl iodide; and then subjected to deacetylation and acid hydrolysis to give, predominantly, 2,4-di-O-methyl-~-rhamnose, together with a small pro(6); R = R' = H MeO,I.&po,eyMe

W I

OR

I

OR'

(7): R R' = CFSCO (8); R = Ac, R' = H (9); R = Ac, R' = Me (10); R = CFsCO, R' = H

portion of the 3,4-dimethyl ether. The reaction sequence employed is similar to that used with the D-glucose analogs above, and, although not established so conclusively, it was assumed that methanolysis of the bis(trifluoroacetate) proceeds more rapidly at the C-2- than at the C-3-hydroxyl group. Methanolysis of the di-ester was found to be much faster than that of the residual mono(trifluoroacetate), confirming the unique lability noted previously in trifluoroacetyl groups adjacent to each other in the =me molecule. The mono(trifluoroacetate) obtained by selective methanolysis of the diester is, therefore, regarded as having the structure (10) and the formation of the monoacetate (8) must involve migration of an acyl group at some stage. As in the analogous reaction in the previous Section, it is considered most likely that transference of the trifluoroacetyl group from the C-3- to the C-2-hydroxyl group occurs during the acetylation of (10) in pyridine. 3. Other Synthetic Uses of Trijiuoroacetyl Derivatives

Use has been made of the trifluoroacetyl derivatives of 1,3: 2,4-di-0ethylidene-D-glucitol (11) in the synthesis of some 5- and 6-substituted R = R' = H Ac, R' CFsCO (12); R (13); R = Ac, R' = H (14); R = Me, R' = Ac (16); R = Me, R' = H

(11);

p

66

T. Q. BONNER

D-glucitols.26 The 5,6-bis(trifluoroacetate) of (11) was obtained by the usual procedure, and controlled alcoholysis with isopentyl alcohol removed one trifluoroacetyl residue. In contrast to the results obtained with methyl 4,6-0-benzylidene-cu-~-glucoside, acetylation of the free hydroxyl group with either acetic anhydride and pyridine or acetic acid-trifluoroacetic anhydride gave the same 5-0-acetyl-6-0-trifluoroacetyl derivative (12), both acylations proceeding without accompanying migration of the trifluoroacetyl group. Alcoholysis of (12) with methanol gave the 5-acetate (13), and treatment of this product (13) or of its precursor (12) with methyl iodide-silver oxide gave the same 6-0-acetyl-5-0-methyl derivative (14). The trifluoroacetyl group in (12) is removed in the methylation reaction, and there is an accompanying migration of the acetyl group from C-5 to C-6. The migration also occurs in the 5-acetate (13) and is known to be common during methylations with Purdie's reagents?' Deacetylation of (14) yields the 5-methyl ether (15). An interesting application of the use of trifluoroacetic anhydride to provide blocking groups has been reported in the synthesis of D-glucosides and D-glucosiduronic acids of phenolic amino acids.28 Tetra-0-acetyl-a+glucopyranosyl bromide (or methyl tri-0-acetyl-1-bromo-1-deoxy-D-glucuronate) is coupled with the ethyl ester of the N-(trifluoroacety1)amino acid, and the N-(trifluoroacetyl) group is readily removed by treatment with 0.2 N sodium hydroxide or 0.2 N barium hydroxide; this procedure does not affect the D-glucosidic (or D-ghcosiduronic) linkage. For diiodotyrosine, this is a much more useful method of blocking the amino group than the more usual benayloxycarbonyl substitution, since the catalytic hydrogenation procedure employed for removing the latter group can also cause de-iodination. Trifluoroacetylation of a hydroxyl group (in order to prevent reaction at this group) has also found application in steroid synthesis; for example, methyl 3/3-hydroxy-5-etiocholenate has been converted to 1l-epi-corticosterone by way of its trifluoroacetyl ester.'* A possible application of the blocking effect of trifluoroacetyl groups, making use of the difference in reactivity of the N-(trifluoroacetyl) and 0-(trifluoroacetyl) groups toward hydrolytic attack, would be for conversion of a hexosamine into its tetra-0-acetyl derivative containing the free amino group. The procedure for this synthesis would require the conversion of the hexosamine to its pentakis(trifluoroacetate), the selective removal of the 0-(trifluoroacetyl) groups with dry methanol, followed by (26) E. J. Bourne, C. E. M. Tatlow, J. C. Tatlow and R. Worrall, J . Chem. SOC., 3946 (1968). (27) J. M. Sugihara, Advances in Carbohydrate Chem., 8,l (1963). (28) A. Taurog, 8.Abraham and I. L. Chaikoff, J . Am. Chem. Soc., 76,3476 (1953).

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

67

acetylation of the free hydroxyl groups and hydrolysis of the N-(trifluoroacetyl) group by mild treatment with alkali.

IV. ACYLATION WITH CARBOXYLIC ACID-TRIFLUOROACETIC ANHYDRIDE MIXTURES 1. Acyhtion of Hydroxy Compounds

The selective acylating action of a mixed anhydride of two carboxylic acids was first correctly diagnosed by B6ha1,2e who showed that, in the acylation of an alcohol by a mixed anhydride, there preponderates (in the product) the ester formed from the acid having the smaller number of carbon atoms. The formation, from a mixture of acetic anhydride and either mono-, di-, or tri-chloroacetic acid, of an acetylating agent sufficiently powerful to effect p-acetylation of anisole was later demonstrated by Unger?e* Trifluoroacetic acid was first used in this connection by Newman,aowho found that a mixture of this acid with acetic anhydride converted anisole into 4'-methoxyacetophenone in 63 % yield (with recovery of 31 % of the anisole) at a much lower temperature than that previously employed with chloroacetic acids. The modified technique adopted by Bourne and co. workers, which has now been extensively applied to acylation reactions,'~~~ consists in treating the hydroxy compound with a slight excesa of an equimolar mixture of the requisite carboxylic acid in the presence of trifluoroacetic anhydride; the reaction mixture is poured into aqueous sodium bicarbonate, and the ester is isolated. Esterification by the lower fatty acids usually proceeds spontaneously and exothermally, and is sometimes complete by the time the temperature has returned to room temperature. For benzoylation, warming of the reaction mixture is necessary and it is recommended that, in general, any carboxylic acid should first be heated gently with trifluoroacetic anhydride before adding the hydroxy compound. The mild conditions of this acylating technique enable acyl derivatives of acidlabile glycosides to be prepared in good yield, as illustrated by the resulting yields of the tetraacetate (55 %) and the tetrapropionate (77 %) of methyl a-D-glucoside and the octaacetate of ap-trehalose (68 %). Sucrose, which is extremely sensitive to acid, affords its octaacetate in 67 %yield. Both cellulose and amylose gradually dissolve at 50-60" in a mixture of acetic acid and trifluoroacetic anhydride, with the formation of chloroform-soluble, fibrous (29) A. BBhal, Compt. rend., 128, 1460 (1899); Ann. chim.phys., [7] 20, 417 (1900). (2Qa) F. Unger, Ann., 604,267 (1933). (30) M. S . Newman, J . Am. Chem. sbc., 67,345 (1945). (31) E. J. Bourne, M. Stacey and J. C. Tatlow, British Pat. 684,754; Chem. Ab8 h C l 8 , 48, 2095 (1954).

68

T. 0. BONNER

acetates having acetyl contents greater than 40 %, with no evidence of extensive degradation of the polysaccharide chains. Conversion of cellulose to its benzoate was similarly achieved. 2. Composition of the Acylating Medium The role of trifluoroacetic anhydride in these acylations was indicated by the diminished yield of acylation product obtained when the ratio of trifluoroacetic anhydride to hydroxy compound lay below unity, optimum conditions requiring a slight excess of this reagent. A catalytic function was, therefore, excluded, and the view was advanced that trifluoroacetic anhydride serves the purpose of converting the added carboxylic acid into the corresponding acyl trifluoroacetate. Later work on the nature of the equilibria between acyl anhydrides and acids in the presence of trifluoroacetic anhydride showed that the acylating capacity of a mixture of a carboxylic acid and trifluoroacetic anhydride is enhanced by the trifluoroacetic acid liberated when the unsymmetrical anhydride is formed.= Further, cryoscopic studies on solutions in acetic acid of the pure, unsymmetrical anhydride, acetyl trifluoroacetate, have shown that, contrary to an earlier conclusion that acetic anhydride is not formed to any appreciable extent,as acetyl trifluoroacetate is, in fact, almost completely converted into acetic anhydride in excess acetic acid.84In carrying out an acylation with trifluoroacetic anhydride and a carboxylic acid, it is, therefore, important to avoid an excess of the acid, so that the maximum concentration of the unsymmetrical anhydride is present in the equilibrium system. Extensive studies made on the action of acyl trifluoroacetates on hydroxy compounds under different conditions, with the simultaneous formation of the acyl and trifluoroacetyl derivatives, will be discussed later. The rate of acetylation of 0-(hydroxymethy1)cellulose (and other hydroxy compounds) by mixtures of carboxylic acids and their anhydrides has been found to increase greatly in the presence of trifluoroacetic acid. The acceleration is very much less with mono- and tri-chloroacetic acids, presumably because they form unsymmetrical anhydrides which are less effective acylating agents than acyl trifluoroacetates.*6The exceptional acylating power of the latter anhydrides is shown by their use in the synthesis of alkyl aryl ketones from polyalkylbenzenes, phenyl ethers, furan, and thiophene under mild The principle has been extended to include acids (32) (1964). (33) (34) (36) (3G) (1951).

E. J. Bourne, M. Stacey, J. C. Tatlow and R. Worrall, J . Chem. Soe., 2006 P. W. Morgan, J . A m . Chem. SOL, 75,860 (1961). E. J. Bourne, J. C. Tatlow and R. Worral1;J. Chem. SOC.,316 (1967). P. W. Morgan, U. s. Pat. 2,629,716 (1963); Chem. Abstracts, 48, 716 (1954). E. J. Bourne, M. Stacey, J. C. Tatlow and J. M. Tedder, J . Chem. SOC.,718

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

69

other than those of the carboxylic type; for example, a mixture of p-toluenesulfonic acid and trifhoroacetic anhydride forms sulfones by reaction with suitably activated aromatic compounds. By the same technique, the hexanitrate esters of D-mannitol and D-glucitol were obtained" by use of solutions of fuming nitric acid in trifluoroacetic anhydride at 0". The probable reaction mechanisms of these and other examples of the conversion of oxy acids into reactive entities have been briefly considered." Similar use has been made of trifluoroacetic anhydride in the preparation of the cyclic 2,3-phosphate of adenosine from adenosine 2-phosphate ; the latter appears to be converted into the unsymmetrical anhydride, which then acts as an internal phosphorylating agent toward the C-3-hydroxyl group.89Treatment of the product with ethanolic ammonia removed the

HO-

B

-0-COCFS

trifluoroacetyl groups present. In the field of nucleotides, many similar preparations have been effected, all of which appear to proceed through the unsymmetrical phosphoryl trifluoroacetic anhydride~.~O-e

V. SELECTIVE RING-OPENING OF CYCLIC ACETALS WITH CARBOXYLIC ACID-TRIFLUOROACETIC ANHYDRIDE MIXTURES 1. Methylene Acetals of D-Gluca*tol

The use of an acylating medium for effecting scission of the acetal ring of a cyclic acetal of a sugar was first demonstrated in the conversion of methyl 4,6-O-ethylidene-&~-glucosideinto methyl 4 - 0 4 1-acetoxyethy1)6-O-acetyl-~-~-glucoside.~ The reagent employed was a 0.1 % (vol./vol.) solution of concentrated sulfuric acid in acetic anhydride at room tempera(37) E. J. Bourne, M. Stacey, J. C. Tatlow and J. M. Tedder, J . Chem. SOC.,1695 (1952). (38) E. J. Bourne, J. E. B. Randles, M. Stacey, J. C. Tatlow and J. M. Tedder, J . Am. Chem. SOC., 76, 3206 (1954). (39) D. M. Brown, D. I. Magrath and A. R. Todd, J . Chem. SOC.,2708 (1952). (40) S. M. H. Christie, D. T. Elmore, G . W. Kenner, A. R. Todd and F. J. Weymouth, J . Chem. Soc., 2947 (1963). (41) L. Schuster, N. 0. Kaplan and F. E. Stoleenbach, J . Biol. Chem., 216, 195 (1955). (42) C. Deluca and N. 0. Kaplan, J . Biol. Chem., 228,569 (1956). (43) H. H. Schlubach, W. Rauchenberger and A. Schultse, Ber., 66, 1248 (1933).

70

T. a. BONNER

ture. In a slightly modified form,"-'e this procedure has been extensively employed for the selective ring-scission of cyclic acetals of polyhydric alcohols at 0". When the ring-opening reaction occurs with a cyclic methylene acetal, the product contains an 0-acetyl group attached to one and an 0-(acetoxymethyl) group to the other of the two oxygen atoms originally forming the methylenedioxy ring. The acetylating entity attacking the ring is presumed to be the acetylium ion, CHaCO', or the conjugate acid of acetic anhydride, (CHaCO)20H' ; the formation of either species requires the presence of a strong acid. Since a mixture of a carboxylic acid and trifluoroacetic anhydride also gives rise to a strongly acylating entity, it was evident that this reagent could react similarly with a cyclic methylene acetal. Assuming that, in this case, the acylating species originates in the unsymmetrical anhydride, that is, the acyl trifluoroacetate, the product of the ring-opening reaction might be expected to contain an 0-(trifluoroacetoxymethyl) group, in addition to the 0-acyl group. The procedure was tested4' by treating 1,6di-O-benzoyl-2,4:3,5-di-O-methylene-~-glucitol(16) with a nine-fold excess (necessary for effecting complete dissolution) of an equimolar mixture of acetic acid and trifluoroacetic anhydride at 25". After 3 hours, the optical rotation had become constant, and the product was a fluorine-containing sirup which decomposed, on exposure to air, with evolution of formaldehyde and trifluoroacetic acid. Treatment of the product with dry methanol (to remove any 0-trifluoroacetyl groups) gave an 0-acetyl-1 ,6-di-O-benzoyl2,4-O-methylene-~-glucitol. Assignment of the acetoxyl group to C-5 (17) was indicated by the formation of a product identical with compound (17) 6-di-0by controlled, acid hydrolysis of 3-0-(acetoxymethyl)-5-0-acetyl-l, benzoyl-2,4-O-methy~ene-~-glucitol to remove the acetoxymethyl group. Confirmation was provided" by the further employment of trifluoroacetic anhydride in conjunction with benzoic acid to open the 1,3-acetal ring of 5-0-acetyl-6-O-benzoyl-l ,3 :2,4-di-O-methylene-~-glucitol (18) and produce, after removal of the trifluoroacetoxymethyl group by methanolysis, compound (17); attack by the benzoylating agent had clearly taken place a t C-1, and the subsequent ring-scission was followed by the appearance of a trifluoroacetoxymethyl group at C-3. The attempted benzoylation of the free hydroxyl group in (17) revealed some interesting stereochemical features (of this and similar molecules) which determine the reactivity to further attack by different reagents. Although acetylation of the C-3-hydroxyl group with acetic anhydride in A. T. Ness, R. M. Hann and C. 8. Hudson, J . A m . Chem. Soc., 66,2216 (1943). R. M. Hann and C. S. Hudson, J . Am. Chem. Soc., 66, 1906 (1944). A. T. Ness, R. M. Hann and C. S. Hudson, J . A m . Chem. SOC.,70,766 (1948). E. J. Bourne, J. Burdon and J. C. Tatlow, J . Chem. SOC.,1274 (19%). (48)E. J. Bourne, J. Burdoa and J. C. Tatlow, J . Chem. rSoc., 1864 (1969). (44) (46) (46) (47)

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

CHzOR

71

CHZOBZ

I

I

I

~H~OR' (16); R = R' = Bz (23); R = R' = Ac (29); R = R' = Pr

(17); R (19);R (20); R (21);R

= H, R' = Ac

= Bz, R' = kc = H, R' = Bz = Ac, R' = Bz

CHzOR

HbOA

HAOJ HAOR!' AHzOR"'

(24) (18); R = Ac, R' = Bz (22); R = R' = Ac

(25); R = R"' = Ac, R' = R" = H (26);R = R' = R" = R'" = Ac (27); R = R"' = Pr, R' = R" = H (28); R = R'" = Pr, R' = R" = Ac

pyridine a t room temperature is quite successful, benzoyl chloride in the same medium is much less effective. Benzoic acid and trifluoroacetic anhydride at 60°, on the other hand, achieve a good yield of the 3-benzoate (19). It might appear that, since the C-3-hydroxyl group under attack is axial with respect to the acetal ring, an effect operates that is similar to that recognized with axial hydroxyl groups in cyclohexane derivatives. These hydroxyl groups are difficult to esterify because of the steric hindrance offered by the two &hydrogen atoms; the corresponding @-positionsin the cyclic acetal are, however, occupied by the ring-oxygen atoms. Models of (16) show that the three large groups attached to C-2 and C-5 hinder access of reagents to the C-3-hydroxyl group (see Fig. 1). This observation suggests that the successful acetylation in pyridine involves a less bulky reagent than benzoyl chloride in the same medium, and that the benzoylating agent in the benzoic acid-trifluoroacetic anhydride mixture is either of a size that makes it less easily obstructed or is a very powerful benzoylating species.

72

T.

a. BONNER

The relative inacceeaibility of the C-3-hydroxyl group is again indicated in which the Schotten-Baumann benzoylation of 2,4-O-methylene-~-glucitol, as that obtained gives the same tri-O-benzoyl-2,4-O-methy~ene-~-gluc~tol by aqueous hydrolysis of the product obtained from the reaction of 1,6di-O-benzoyl-2,4: 3,5-di-0 methylene-D-glucitol (16) with a nine-fold excess of an equimolar mixture of benzoic acid and trifiuoroacetic anhydride at 25" for 12 hours. The expected product in the latter reaction is the 1,5,6tribenzoate (20), by analogy with the similar reaction of acetic acid-trifluoroacetic anhydride with (16) to give (17), and this expectation was confirmed by acetylation to give a product different from the isomeric 5-0acetyl-1 ,3,6-tri-O-benzoyl derivative (19). The only possible alternative is the 3-O-acetyl-l , 5,6-tri-O-benzoyl derivative (21).

$

FIG. 1.4onformation of (16). (+ = Favorable route for RCO'; .-+ able route for RCO".)

unfsvor-

The stability of the 2,4-ring in the above acylating media was further demonstrated by the failure of prolonged action (24 hr. at 25') of acetic acid and trifluoroacetic anhydride on the parent compound (2 ,CO-methylene-D-glucitol) to produce any appreciable ring-scission. This acetal ring is a BC-ring, which is known to possess much greater stability than other types of ring formed in these cyclic acetals of hexitol~~~~50; in accordance with the views of Mills,K1the stability is attributed to the fact that the large benzoyloxymethyl group occupies an equatorial position in the 2,4-ring, whereas, in contrast, the axial position of this group with respect to the 3,B-ring @T) makes the latter relatively unstable and labile to attack. Although it is not unexpected, therefore, that the dibengoate (16) undergoes ring scission a t the 3,5414 only, it is significant that this scission occurs solely in one direction, that is, the acylating species attacks uniquely at the C-5 position. The explanation offered is that the most "favored" conforma(49) 8.A. Barker and E. J. Bourne, Aduancee in Carbohydrate Chem., 7,137 (1962). (60)S.A. Barker, E. J. Bourne and D. H. Whiffen, J . Chem. Soc., 3866 (1962). (61) J. A. Mills, Aduancee in Carbohydrate Chem., 10.2 (1966).

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

73

tion of (16), which is analogous to cis-decalin, allows attack of the acylating agent in an equatorial direction a t the Cd-oxygen atom to be the least hindered (see Fig. 1).Approach of the reagent in an equatorial direction at the C-3-oxygen atom is hindered by non-bonded interaction with the benzoyloxymethyl group attached to (3-2, and attack in axial directions either at position 3 or 5 is ruled out for the usual conformational reasons. Molecular models confirm that an oxonium complex is possible at the C-5-oxygen atom, but not at that a t C-3. It was found" that, when a 1O:l molar ratio of acetic acid to trifluoroacetic anhydride was caused to react with (16), the product (after 12 hr. at 25") was 3-0-(acetoxymet hyl)-5-0-acet yl- 1,6-di-O-benzoyl-2,4-0-met hylene-D-glucitol, formed in 90% yield. Since this is the product which would be obtained in the Hudson acetolysis reaction, its formation suggests that, in the presence of excess acetic acid, trifluoroacetic anhydride does not afford acetyl trifluoroacetate (as in the equimolecular mixture), but is converted into trifluoroacetic acid, with acetic anhydride as the accompanying product; the mixture would then be similar to that used in acetolysis, that is, acetic anhydride in acetic acid in the presence of a strong acid. This result emphasizes the advantage of using the 1:1molar ratio for selective scissions of cyclic acetals, since the trifluoroacetoxy groups which result from the ring opening with this mixture are readily removed by methanol, without effect on other groups, whereas the removal of acetoxy groups requires a more vigorous, hydrolytic procedure, with a concomitant low yield of product. Further examples of selective ring-opening in cyclic acetals by an equimolecular mixture of a carboxylic acid and trifluoroacetic anhydride have been provided by 8- and a-methylene derivatives of D-glucitol where the initial attack appears to occur a t the primary carbon atom of these two types of ring. With acetic, benzoic, and propionic acids, the product obtained, after ring opening and removal of the trifluoroacetoxymethyl groups, contains the 0-acyl group a t the primary carbon atom, with free hydroxyl groups at the secondary position; for example, treatment of 5,6-di-O-acetyl1,3:2,4-di-O-methylene-~-glucitol (22) with a mixture of acetic acid (3 moles) and trifluoroacetic anhydride (3 moles) for 7 hr. at 25" yielded, after mild hydrolysis with sodium bicarbonate solution, a crystalline tri-0-acetyl2,4-0-methylene-~-glucitol identical with that obtained by the similar treatment of 1,6-di-O-acety1-2,4:3,5-di-0-methylene-~-glucitol (23). As it had been established that the 3,5-ring in the latter is selectively opened and the center of attack is at the C-5-oxygen atom, it is clear that the product is 1,5,6-tri-0-acetyl-2,4-O-methylene-~-glucitol. Hence, in the diacetal (22), the 1,3-ring @) is broken by attack of the acylating reagent at the C-1-oxygen atom, leaving the 2,4-ring intact. Studies of acid hydrolysis, acetolysis, and ease of formation of cyclic

74

T. 0. BONNER

acetals of hexit0ls4~*4~-6~ show that an a-ring is likely to be more labile than a &ring. It follows that interaction of 1,3:2,4:5,6-tri-O-methylene-~glucitol (24) with the acetic acid-trifluoroacetic anhydride mixture should result in scission of the 1,3-ring and the 5,6-ring (a), with the formation, after mild hydrolysis of the product, of l ,6-di-O-acetyl-2,CO-methylene-D-glucitol (25). The reaction yielded a sirup which had the correct analytical values and which could be acetylated to the known 1,3,5,6tetra-O-acetyl-2,4-0-methylene-~-glucitol (26), but attempts to prepare other derivatives by substitution at the two free hydroxyl groups failed. However, similar treatment of the tri-O-methylene-D-glucitol with an equimolecular mixture of propionic acid and trifluoroacetic anhydride gave the

(a)

H

' H

FIG.2.-Conformation of a 1,3:2,4-Diacetal of D-Glucitol. (4= Favorable route for RCO';

--+

= unfavorable route for RCO'.)

expected 2 ,4-O-methylene-.L,6-di-O-propionyl-~-glucitol(27), whose structure was shown by its hydrolysis to 2,4-0-methylene-~-glucito1 and by its acetylation to the 3,8diacetate (28). The structure of the diacetate was proved by its synthesis by an alternative route, which involved treatment of 2,4:3,5-di-0-methylene-l,6-di-O-propionyl-~-glucitol (29) with acetic acid-trifluoroacetic anhydride, followed by mild hydrolysis and acylation of the free hydroxyl group at C-3. Examination of the probably most stable conformation of a 1,3:2,4-diacetal of D-glucitol (see Fig. 2) shows that an axial approach to either oxygen atom of the 1,3-ring is improbable, because of non-bonded interactions analogous to the 1,&diaxial interactions of cyclohexane.@The same type of interaction would impede an equatorial approach to the C-3-oxygen atom, and the most probable attack of the acylating agent is, therefore, through an equatorial approach to the C-l-oxygen atom, to give the l-ester

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

75

of the 2 ,4-acetal. In the 5 ,6-ring of the tri-o-methylene-D-glucitol (24), the more accessible oxygen atom is a t C-6, with the result that, after reaction, the acyl group is found attached a t this position in the product. 2. Methylene Acetals of D - M a n n i t o l Some significant experiments have been carried out with 1 ,3 :2,5 :4 ,6tri-0-methylene-D-mannitol(1 mole) with acetic acid (4.5 moles) and trifluoroacetic anhydride (4.5 moles) at room t e m p e r a t ~ r e An . ~ ~initial, rapid increase in optical rotation was observed, culminating in a sharp maximum after 2 hr., with a subsequent slow fall in rotation spread over several days. This suggested that fission of the 1,3(@)-and 4,6@)-rings had occurred rapidly (since these rings are opened preferentially in the Hudson acetolysis procedureu), and that this was followed by a slower attack on the 2,5(yT)ring. By use of a solvent (to moderate the reaction), it was found that dilution of the reaction mixture with chloroform completely prevented ring scission, tri-0-methylene-D-mannitolbeing recovered in 93 % yield. In pure nitromethane, however, and in a mixture of this solvent with chloroform ( 1 : l by vol.), the initial, rapid change in optical rotation was again observed, but without the appearance of a sharp maximum rotation; samples of the reaction mixture taken within one hour of the start of the reaction were found to contain appreciable proportions (about 50%) of a mixture of an O-(acetoxymethy1)-O-acetyl-1, 3 :2,5-di-O-methylene-~-mannitol (30) and a di-O-(acetoxymethyl)-di-O-acetyl-2,5-O-methylene-~-mannitol (31). This result is quite different from that obtained with tri-o-methyleneD-glucitol, since the product of partial ring-scission of the latter gave no evidence of the presence of an O-(acetoxymethyl) group. It is probable that the 1 ,3- and 4,6-rings in tri-0-methylene-D-mannitol are opened in the expected way, to give O-acetyl-O-(trifluoroacetoxymethyl)derivatives, but that the latter group undergoes replacement by the acetoxymethyl group as a result of further attack by the acylating agent. Why this reaction occurs only for tri-0-methylene-D-mannitol and not for tri-0-methylene-D-glucitol is not clear, but the behavior is probably related to the different conformations of the two systems. Further experiments have established that, when tri-0-methylene-D-mannitol is treated with a larger proportion of the reagent, the 2 ,Bring does not remain intact, although it is evidently the most resistant to attack. Tri-O-methylene-D-mannitol(1 mole), on treatment with the 10: 1 mixture, that is, acetic acid (45 moles) and trifluoroacetic anhydride (4.5 moles), showed a similar differentiation in reactivity of the acetal rings. The only product recoverable in the early stages of the reaction a t 50" was (30), with the further product (31) appearing much later. Higher temperatures were (62)

T.G . Bonner, E.J. Bourne and D. Lewis, unpublished work.

7G

T. 0. BONNER

necessary in order to bring about ring opening of the 2,5-ring in (31), although, even near 100°, prolonged treatment appears necessary in order to achieve substantial fission of this ring. 3. Other Acetals Acetolysis by the Hudson procedure is known to remove a benzylidene An equimolar mixture acetal ring, to give the corresponding diacetate.s8164 of acetic acid and trifluoroacetic anhydride reacts in the same way with both benzylidene and isopropylidene aceta1s.a Treatment of 3 ,4-di-0acetyl-l,2:5,6-di-0-isopropylidene-~-mannitol with this reagent for 2 hr. at 25' gave a small yield (21%) of D-mannitol hexaacetate. 1,3:2,5:4,6Tri-0-bensylidene-D-mannitol,treated similarly for 24 hr., gave the same product in 39 % yield, together with a 73 % yield of benzaldehyde; the expected bis(trifluor0acetate) of the benzylidene acetal could not be isolated. 1,3,5 ,6-Tetra-O-acetyl-2,4-0-benzylidene-~-glucitol gave a high yield (85%) of D-glucitol hexaacetate under the same conditions, so that, with a phenyl substituent a t the methylenic carbon atom, the 2,4(BC)-ring is easily opened. In these three experiments, considerable darkening of the reaction mixtures occurred, although the single substances, acetone, benzaldehyde, D-glucitol, and D-mannitol do not themselves undergo this color change under these conditions. The different mechanisms of reaction of benzylidene and isopropylidene acetals, compared with that of methylene acetals, have not yet been elucidated, but, assuming, in both cases, that the acetal ring is ruptured to give an acetyl substituent together with a trifluoroacetoxymethyl substituent, in place of the alkylidene or arylidene group, it is evident that, when either

4 I

-bOAc

-bOAo

-bO-&OCOCF, I 1

-bOAc

I

R or R' is a phenyl group or when both are methyl groups, the oxygen atom attached to the carbon chain of the hexitol is likely to be more nucleophilic than if an unsubstituted methylene group is present. Further attack by the electrophilic, acylating entity is, therefore, facilitated, and this could result in the replacement of the trifluoroacetoxymethyl substitutuent by an acetyl group. (53) W. T. Hsskins, R. M. H a m and C. S. Hudson, J . A m . Chem. Soc., 64, 132, 136, 1614 (1042). (64) J. K.Wolfe, R. M. Hann and C. 8. Hudson, J . A m . Chem. floe., 64,1493 (1942).

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

77

In spite of the ring-opening reactions which can occur, free hydroxyl groups in cyclic acetals can be acetylated in reasonable yield without accompanying ring-scission, provided that only a slight excess (1.2 mole per hydroxyl group) of the equimolecular mixture of acetic acid and trifluoroacetic anhydride is employed. By this means, 1,3:2,4-di-O-methylene-~glucitol, its ethylidene analog, and 2,4-0-benzylidene-~-glucitol, respectively, were converted into their fully acetylated derivatives in excellent yield. Isopropylidene acetals of D-glucitol and D-mannitol under the same conditions, however, gave only negligible amounts of the acetal acetates.@ VI. THESYNTHESIS OF LINEARPOLYMERIC ESTEFSFROM CYCLIC TRIMETHYLENE ACETALSAND DIBASICCARBOXYLIC ACIDS In the original investigation of the use of trifluoroacetic anhydride in promoting acylation of hydroxy compounds by carboxylic acids,' it was noted that long-chain polyesters might result either from the combination of a dihydric alcohol and a dibasic acid or from a hydroxy carboxylic acid. Treatment of p-hydroxybenzoic acid with trifluoroacetic anhydride for 15 min. at 75" did, in fact, produce a polyester having m.p. 360". With pure reactants, this poly-ester should take the form of a linear polymer, but the product obtained did not appear to have this characteristic property. The is selectively later discovery that 1 ,3:2,4:5,6-tri-0-methylene-~-glucitol attacked at only two of its three acetal rings by an equimolecular mixture of a carboxylic acid and trifluoroacetic anhydride a t room temperature pointed to an alternative route for the synthesis of linear polyesters by the substitution of a dibasic acid for acetic acid. Since the product obtained in the acetic acid reaction, after removal of excess reagent and mild hydrolysis with methanol, is 1,6-di-O-acetyl-2,4-O-methylene-~-glucitol, the polyester resulting from the use of a dicarboxylic acid would be expected to possess two free hydroxyl groups per D-glucitol unit in the polymer chain. The reaction sequence for a cyclic, methylene acetal containing two labile rings is shown in Fig. 3. The use of a cyclic acetal having more than two reactive centers would lead to branching in the poly-ester chain and, probably, to a cross-linked product. There is the possibility that an intramolecular reaction could occur, but, as the product would possess a large, unstable ring, this is unlikely. Benzylidene or isopropylidene acetal rings are destroyed by acetic acid and trifluoroacetic anhydride, with the appearance of two acetate residues per acetal ring; a dibasic acid in place of acetic acid could, therefore, give rise to a linear poly-ester, but the product would not provide free hydroxyl groups. The presence of free hydroxyl groups in a linear polymer is valuable for certain applications; for example, if the polymer has use as an artificial fiber, the free hydroxyl groups enable the fiber to absorb water and they facilitate uptake of dye.

T.

78

0. BONNER

The investigation of polyester formation has been carried out using equimolecular proportions of 1,3 :2,4 :5,6-tri-O-methylene-~-glucitol and adipic acid in excess trifluoroacetic anhydride, the latter serving as the solvent.b6 After 3 hr. at room temperature, the volatile constituents were removed, and the reaction mixture was treated with an aqueous sodium bicarbonate (CHJ4(COZH),

+ 2

(CF,CO),O

=(CHJ~(CO-OCOCFJa

+ 2 CF&OaH

where T = CF,CO,CH,.

Fro. 3.-Synthesis of a Polyester.

solution and kept for a few days. The insoluble product was a colorless, brittle solid, melting a t 130-150" to a viscous liquid which could be drawn into brittle threads. The solid became swollen in some solvents and dissolved completely in pyridine. Alkaline hydrolysis yielded 2 ,bO-methylene-~glucitol and adipic acid; the only other product detected by paper chromatography was IL trace of D-glucitol. The infrared absorption spectrum of the (56) T. G. Bonner, E. J. Bourne and N. M. Saville, J . Chem. Soc., 2914 (1980).

APPLIC ITIONB OF TRIFLUOROACETIC ANHYDRIDE

79

product showed the presence of aliphatic carboxylate ester (but not of trifluoroacetate) and cyclic ether groups. The carboxylate ion was also found, presumably as the end group in some of the molecules, and the carboxylic acid group appeared on treatment with acid. If the assumption is made that the absorption coefficients of the -COOR and -COOe groups have closely similar values, the ratio of the corresponding absorptions indicated the presence of about ten of the former groups to one of the latter. As the molecules may contain two, one, or no carboxylate end-groups, the average chain-length could not be calculated from these data. Viscosity measurements in pyridine indicated an average, molecular weight of about 5,000; if the product is a linear polymer having, as the repeating unit, a monomethylene-D-glucitoladipate residue, the polymer chains would contain an average of 16 units. The expected repeating-unit is shown in Fig. 4. The molecular weight of the poly-ester is too low to provide the basis for a useful fiber, but a modification of the experimental conditions and, possibly,

FIG.4.-Repeating Unit of the Polyester Obtained from Tri-0-methylene-nglucitol and Adipic Acid.

of the dibasic acid constituent could lead to the synthesis of a more suitable product by this method of polymer formation.

VII. THE MECHANISM OF ACYLATION BY A c n TRIFLUOROACETATES 1. The Formation of Acyl Trijeuoroacetates

When a carboxylic acid and trifluoroacetic anhydride are mixed, the following equilibria are assumed to be established.as-ba XOH

+ (CF,CO)nO * XOCOCFa + CF&OaH

XOCOCFS XlO

+ XOH S XeO + CFaCOzH

+ (CFsCO)*O+ 2 XOCOCFa

(1 1 (2)

(3)

whereX = RCO. It is frequently postulated that the unsymmetrical anhydride undergoes partial ionization to X' and CFFCO,~, but direct evidence for this further step is meager. The evidence for the formation of the unsymmetrical anhydride in similar systems is, however, well established from previous (66)E.J. Bourne, J. E. B. Randles, J. C. Tatlow and J. M. Tedder, Nature, 168, 942 (1961).

80

T.

0. BONNER

investigations on mixtures of carboxylic acids and other anhydrides, particularly as a result of infrared-absorption studies." The latter technique, applied to a mixture of acetic anhydride and trifluoroacetic anhydride in carbon tetrachloride, revealed the gradual development of a new absorption band at 1072 cm.-', with an accompanying diminution of the original bands due to the primary components; the new band almost certainly indicated the formation of acetyl trifluoroacetate. In cryoscopic studies initiated by Morganw on solutions (in acetic acid) of trifluoroacetic acid and of trifluoroacetic anhydride, van't Hoff factors of 1 and 2, respectively, were reported. This result indicated that trifluoroacetic anhydride does not react in accordance with equation 4 (which would result in an i factor of 3), but probably forms the unsymmetrical anhydride as shown in equation 6.

+ 2 CHaCOzH (CHaC0)rO + 2 CFsCOaH (CF&O)P + CHaCOsH CHaCO4COCFS + CFaCO9H (CF&O)*O

+

(4) (6)

However, further infrared investigations,@based on comparisons of these systems with pure acetyl trifluoroacetate, although confirming the formation of the latter in 95 % yield from an equimolecular mixture of trifluoroacetic anhydride and acetic acid as represented in equation 6, showed that, in an equimolecular mixture of acetyl trifluoroacetate and acetic acid, acetic anhydride is formed to the extent of 60%. This result suggested that, in a large excess of acetic acid (as used in the cryoscopic studies), acetyl trifluoroacetate (and, hence, trifluoroacetic anhydride) should be largely converted into acetic anhydride, as represented by equation 4. New cryoscopic studies in acetic acida4showed an initial depression of freezing point corresponding to the formation of 2.5 particles per molecule of trifluoroacetic anhydride, but the freezing point was found to increase with time, the final result being an i factor of 2. The difficulties were attributed to ingress of atmospheric moisture, and, by use of (a) a special apparatus designed to avoid this ingress and (b) a more reliable cryoscopic constant for acetic acid, the cryoscopic behavior of trifluoroacetic anhydride was found to correspond to equation 4, and of acetyl trifluoroacetate, to equation 6. Probably, the original investigation,= although apparently providing evidence for the formation of acetyl trifluoroacetate, gave an erroneous result through interference by atmospheric moisture. CHaCO4COCFa

+ CHaCOnH

(CH&O)(O

+ CFsCOzH

(6)

In a detailed study of the electrical conductivities of the ternary system (CFaCO)~O-H&(CHaGO)nO, it was found that a dilute solution of trifluoroacetic anhydride in acetia acid has a small but definite conductivity which is slightly greater than twice that of a solution of trifluoroacetic acid (57) L.Brown and I. F.Trotter, J . Chem. floc., 87 (1961).

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

81

of the same molar concentration in the same solvent.@Also, in anhydrous mixtures of acetic anhydride and trifluoroacetic anhydride, a maximum conductivity appears near the equimolecular composition. These facts are interpreted as evidence for the formation of acetyl trifluoroacetate, and its partial ionization according to equation 7. CHICO~-COCFI

CHsCOe

+ CFpCOie

(7)

The production of these ions is assumed to occur more readily from the unsymmetrical anhydride than from the symmetrical anhydrides, resulting in the higher conductivities observed. The preparation of pure acetyl trifluoroacetate for the infrared studies referred to above was achieved by (a) fractional distillation of an equimolecular mixture of acetic anhydride and trifluoroacetic anhydride, or (b) the addition of pyridine to a mixture of acetic acid and trifluoroacetic anhydride, and fractional distillation of the fi1trate.a Acetyl trifluoroacetate is a colorless liquid, b.p. 95", which gradually becomes colored on ~tanding.6~ In solution in carbon tetrachloride or other inert solvent, no coloration occurs and the solutions are stable. Similar methods of preparation have been used by other workers, and several different acyl trifluoroacetates have been reported.60A different procedure for the preparation of acyl trifluoroacetates is the addition of the appropriate acyl chloride to a solution of silver trifluoroacetate in ether.61 The unsymmetrical anhydrides were found to be stable during the subsequent distillation, but disproportionation occurs in the presence of silver trifluoroacetate. 2. The Reactions of Acgl Tm'jeuoroacetateswith Hydroxy Compounds

The f i s t quantitative analyses of the products obtained by treating alcohols and phenols with an acyl trifluoroacetate showed that the anhydride can act simultaneously as an acylating and a trifluoroacetylating agent."*l-Butanol and acetyl trifluoroacetate in ether solution at 20" give a much greater yield of butyl trifluoroacetate than of butyl acetate; in the presence of trifluoroacetic acid, the proportions are reversed. In the absence of trifluoroacetic acid, sec-butyl alcohol gives about twice as much of the acetate as of the trifluoroacetate, and tert-butyl alcohol gives the acetate almost exclusively. It was confirmed with other hydroxy compounds that, when the acetylation predominates, the yield of product is much the same if the acetyl trifluoroacetate is replaced by an equimolecular mixture of (68) J. E. B. Randles, J. C. Tatlow and J. M. Tedder, J . Chem. Soc., 436 (1954). (69) J. M. Tedder, J . Chem. Soc., 2646 (1954). (60) W. D. Emmons, K. S. McCallum and A. F. Ferris, J . Am. Chem. Soc., 76, 6047 (1953). (61) A. F. Ferris and W. D. Emmons, J . Am. Chem. Soc., 76,232 (1953).

82

T.

a.

BONNER

acetic anhydride and trifluoroacetic anhydride. It was tentatively suggested that an acyl trifluoroacetate operates as a trifluoroacetylating agent in its molecular form, whereas the alternative acylation proceeds through the acylium ion (RCO@),the formation of which is enhanced by the addition of trifluoroacetic acid. The latter is always present in the acylating medium when a mixture of a carboxylic acid and trifluoroacetic anhydride is used in accordance with equation 1. More extensive studies* on a variety of hydroxy compounds, together with some rate measurements based on infrared analysis, appear to provide general support for the original interpretation of the mechanism of these reactions, although indicating many features which need further investigation. In these studies, the hydroxy compound was treated at 20" with a 30 per cent molar excess of acetyl trifluoroacetate in different environments which included the pure reactants only and the pure reactants with addition of trifluoroacetic acid, carbon tetrachloride, and sodium trifluoroacetate. The persistent formation of a high proportion of acetate in certain cases, under non-polar conditions in the presence of sodium trifluoroacetate (which would be expected to suppress formation of acetylium ions according to equation 7), clearly indicated that acetylation may also occur through the molecular form of acetyl trifluoroacetate. Possibly, this function only appears when a relatively less accessible hydroxyl group is present, to which the approach of the (larger) trifluoroacetyl group is more hindered than that of the acetyl group, as previously postulated62in acylation reactions of anhydrides of the chloroacetic acids. This steric factor is probably an important factor in the predominance of acylated derivatives when carbohydrates are treated with acetyl trifluoroacetate. Acylation of hydroxyl groups through acylium-ion attack, when a mixture of the acid anhydride and trifluoroacetic anhydride is employed, is probably preceded by formation of such acylium-ion derivatives as RC02H2@,(RCO)aOH@,and RCOOCOCFa:H@,which may, in themselves, act as acylating species. The function of trifluoroacetic acid in catalyzed acylations is not fully understood. The dielectric constant of trifluoroacetic acid (€20" = 8.4) does not suggest a strongly ionizing medium, but the capacity of the acid to solvate its own negative ions may be important, since such solvation could facilitate heterolysis of the unsymmetrical anhydride to acylium and trifluoroacetate ions.68Another mechanism was observed to operate with tert-butyl alcohol treated with acetyl trifluoroacetate, both in the absence of a solvent or in the presence of trifluoroacetic acid? The major product was tert-butyl trifluoroacetate, a 90 % yield being (62) A. R. Emery and V. Gold, J . Chem. Soc., 1443,1447,1466 (1960).

(63) J. J. Throssell, S. P. Sood, M. Szwarc and V. Stannett, J . Am. Chem. Soc., 78, lln (1966).

APPLICATIONS OF TRIFLUOROACETIC ANHYDRIDE

83

obtained with trifluoroacetic acid present. Since both the acetate and the alcohol were converted by trifluoroacetic acid alone into the trifluoroacetate, it is likely that alcohols of this type are first protonated by the acid, to give a conjugate acid, one molecule of which loses a molecule of water and then reacts with a trifluoroacetate anion. This mechanism of alkyl-oxygen fission is only likely to operate when there are structural factors tending to stabilize the carbonium ion formed. Measurements of rate of reaction of butano1 and sec-butyl alcohol showed that trifluoroacetate esters are formed much more rapidly with acetyl trifluoroacetate than with trifluoroacetic acid, so, in these cases, direct trifluoroacetylation by the acid is of minor importance. 3. Other Acylation Reactions of Acyl Tri$uoroacetates Other acylation reactions brought about by acyl trifluoroacetates and suggesting the operation of an acylium-ion mechanism are the acylation of aromatic compoundsPo~6 and the ring-opening reaction of cyclic acetals (see Sections V and VI). Successful acylations have been carried out on anisole, phenetole, mesitylene, thiophene, and furan, by use of trifluoroacetic anhydride in conjunction with acetic acid, benzoic acid, and cinnamic acid. Acetyl trifluoroacetate has been used directly for the acetylation of phenetole and thiophene, and benzoyl trifluoroacetate for the conversion of anisole to 4-methoxybenzophenone82;with both reagents, yields appear to be higher than with the carboxylic acid-trifluoroacetic anhydride mixtures. Although there is every likelihood that acylation of the aromatic ring proceeds through electrophilic attack by an acylium ion, the alternative mechanism of nucleophilic attack of aromatic compound on unsymmetrical anhydride (whether in the form of an ion-pair or of a highly polar molecule) is equally acceptable. In the reactions of acyl trifluoroacetates with cyclic acetals, selective attack of the reagent at certain sites of the acetal is interpreted by conformational analysis as discussed above. This interpretation represents the reaction as proceeding through attack of the acylium ion, with the implication that only this entity is capable of penetrating to the appropriate oxygen center. Conformational analysis, however, establishes which sites are most accessible to attack, without regard to the nature of the reagent. It is quite possible that a highly polar molecule could be the reacting agent and that this only undergoes heterolysis when within bond-forming distance of the reaction site. Such a species could not be acetyl trifluoroacetate alone, since this reagent has no capacity for opening any cyclic acetal rings under the usual conditions in the absence of trifluoroacetic acid.& There is thus a (64)P. H. Gore, Chem. Revs., 66, 229 (1955).

T.

84

Q. BONNER

strong likelihood that the conjugate acid of acetyl trifluoroacetate (or a species derived from it) is the acetylating agent when the acid is present. The difficulty of deciding between alternative reaction-intermediates with this type of reagent is evident from the analogous study of the cryoscopic behavior of acetic anhydride in anhydrous sulfuric acid. The discovery66 that the depression of freezing point corresponds to the formation of nearly four particles per mole of acetic anhydride led to an acceptable representation of the ionization as shown in equation 8. (CH&O)rO

+ 2 His04 + CHICO" + CHaCOOHa" + 2 HSO"

(8)

However, the results of other studies on this system suggested that quite different species are formed, and it has now been confirmed, by further that equation 8 is erroneous cryoscopic and conductivity and that the interaction taking place between acetic anhydride and sulfuric acid is that shown in equation 9. (CH&0)20

+ 3 HsSOI + 2 CHICOOH~"+ HSnOie + HSOP

(9)

Although this result does not constitute evidence against the occurrence of the acetyl ion as a reaction intermediate (since thie could be present in trace amounts in this and other acylating systems), it does transfer the onus of demonstrating the existence of this species in acetylation reactions to other, more sensitive, techniques. The method most likely to provide information concerning the nature of the reaction intermediates is that of detailed, kinetic analysis under a variety of carefully controlled conditions. It is evident that, in the application of this method, there is an extensive field of investigation to be surveyed, not only of the action of acyl trifluoroacetates on hydroxylic compounds under the infiuence of different media and catalysts, but also of the peculiar differences between the ring-opening reactions of cyclic acetals with this type of reagent and with that which is employed in the Hudson acetolysis procedure. (66) R. J. GiIlespie, J . Chem. 8m., 2997 (1960). (66) R. J. Gillespie and J. A. Leisten, Quart. Revs. (London), 8,40 (1964). (67) J. A. Leisten, J . Chem. ~ o c . 298 , (19%). (68) R. Flowers, R. J. Gilleepie and 9. Wasif, J . Chem. Soc., 607 (1966).

GLYCOSYL FLUORIDES AND AZIDES

BY FRITZMICHEELAND ALMUTHKLEMER* Organisch-Chemisches Institut der Universitdt, Milnater ,WestfaZen, Gemzany I. Introduction ............................................................ 11. Preparation of the Glycosyl Fluorides.. ................................. 111. Reactions of the Glycosyl Fluorides.. ................................... 1. Reactions Involving Participation. ................................... 2. Reactions Not Involving Participation. ............................... 3. Structure of the D-Fructose Moiety in Sucrose.. ...................... 4. Other Reactions.. .................................................... IV. The w-Fluoro Carbohydrates. ........................................... V. The Aldosyl &idea. .................................................... VI. Tables of Properties of Glycosyl Fluoride Derivatives ...................

85 88 88 89 92 93 93 95 95 97

I. INTRODUCTION In an earlier review’ of the glycosyl halides and their derivatives, the discussion of the fluoro compounds was necessarily limited by the restricted knowledge of these interesting compounds. Recent investigations have provided sufficient information to permit correlation of this rapidly expanding area of carbohydrate chemistry. The glycosyl fluorides and their derivatives occupy an exceptional position a~the result of the very high bond-energy of the carbon-fluorine linkage. Although the energy of the aliphatic carbon-fluorine bond is more than 50 % greater than that of any of the other aliphatic carbon-halogen bonds? the magnitudes of the bond energies for the various glycosyl halides are unknown. Certainly, the same relative order would be expected, but all the values should be lower. The experimental evidence so far obtained supports these expectations. Although the major portion of this review is concerned with the glycosyl fluorides and their derivatives, short Sections on the closely related glycosyl azides and on carbohydrates containing fluorine at non-glycosidic carbon atoms have been included. *The original German manuscript waa translated by Dr. Walter von Bebenburg and then revised by Mr. Alan Chaney of the Department of Chemistry of The Ohio State University, Columbus, Ohio. (1) L. J. Haynes and F. H. Newth, Advances i n Carbohydrate Chem.,10,207 (1965). (2) L. Pauling, “The Nature of the Chemical Bond,” Cornell University Press, Ithaca, N. Y., 2nd Edition, 1940, p. 53. 85

86

F. MICHEEL AND A . KLEMER

11. PREPARATION OF THE GLYCOSYL FLUORIDES The first acetylated glycosyl fluoride derivative was prepared by BraunsS in 1923 and, in subsequent papers, he explored the synthesis of a number of poly-0-acetylglycosyl fluoride^.^-^ I n addition, Brauns prepared the other poly-0-acetylglycosyl halides of the same carbohydrates and investigated the proportionality relations which exist between their optical rotations and the diameters of the respective halogen atoms.lO The method of synthesis employed in this early work involved the action of anhydrous, liquid hydrogen fluoride on the fully acetylated carbohydrate. In recent years, the process has been simplified by the usell of the anhydrous hydrogen fluoride commercially available, and by substitution1* of polyethylene equipment for the platinum vessels used earlier.8 This process should be capable of extension to carbohydrates with functions other than acetyl blocking the hydroxyl groups. However, only various partially methylated substances and the nitrogen-substituted (methyl, acctyl, and p-tolylsulfonyl) derivatives of 3,4,G-tri-O-acetyl-2-amino-2deoxy-D-glucose have been subjected to the reaction. Although reaction of the acetylated carbohydrates with hydrogen fluoride normally effects the desired replacement without complications, prolonged treatment sometimes causes deep-seated structural changes. Thus, octa-0-acetylcellobiose, after a reaction time of thirty minutes, gives a moderate yield of hepta-O-acet,ylcbellobiouyl fluoride,*,' but, after five hours, the major product is 3,6-di-0acetyl-4-0-(2,3,4,6-tetra-O-acetyl-P-D-glucosyl) -a-D-mannosyl fluoride.6 Prolonged treatment of penta-0-acetyl-P-D-fructopyranose with liquid hyfluoride.# drogen fluoride afforded 3,4,5-tri-0-acetyl-~-~-fructopyranosyl In the first example, both inversion and acetyl removal occurred a t the carbon atom adjacent to the potential reducing center, whereas, in the latter, only acetyl removal at the primary hydroxyl group was effected. The compounds prepared by this method are listed in the Tables. Examples of the newer methodsl'JY of synthesis will now be described. Hydrofluoric arid (50 ml., from a tank) is added to 20 g. of 1,2,3,4,6-penta-Oacetyl-a(or P-)-D-glucose cooled to -15" in a polyethylene flask. Thc acetatc dis(3) D. H . Brauns, J . A m . Chem. SOC.,46,833 (1923). (4) D. H. Brauns, J . A m . Chem. Soc., 46, 2381 (1923). (5) D. H. Brauns, J . A m . Chem. SOC.,46. 1484 (1924). (6) D. H. Brauns, J . A m . Chem. SOC.,48.2776 (1926). (7) D. H. Brauns, J . Am. Chem. Soc., 49, 3170 (1927). (8) D. H. Brauns, J . A m . Chem. Soc., 61, 1820 (1929). (9) D. H. Brauns and H. L. Frush, Bur. Standards J . Research, 6,449 (1931). (10) D. H. Brauns, Rec. Irau. chim., 69, 1175 (1950). (11) F. Micheel, A. Klemer, M. Nolte, H. Nordiek, L. Tork and H. Westermann, Chem. Ber., 90, 1612 (1957). (12) F. Micheel and H . Wulff, Chem. Ber., 89, 1521 (1956).

87

GLYCOSYL FLUORIDES AND AZIDES

solves in 2 to 3 min., and, after 20 min. at this temperature, the solution is kept a t room temperature for 10 min. The reaction mixture is then poured into a mixture of ice, water, and chloroform, and the organic layer is extracted with water several times. After drying the solution and evaporating the solvent, the product usually crystallizes. Recrystallization from hot ethanol gives pure 2,3,4,6-tetra-O-acetyla-D-glUCOSY1 fluoride (62%).

Most of the acetylated monosaccharides afford the more stable anomeric fluoride in this process.s However, the action" of a solution of hydrogen triacetate fluoride in acetic anhydride on 1,6-anhydro-p-~-glucopyranose (1) gave tetra-0-acetyl-P-D-glucopyranosyl fluoride (3). This result is prob-

ii

F

CHI-0-C-CH:

I

I

OAC

6AC

OAC

ably dependent on attack of the active species, acetyl fluoride, a t the anhydro-ring oxygen atom, as indicated. The intermediate structure [(2)] is hypothetical. The normal method of preparation of the less stable anomers of the acetylated glycosyl fluorides involves the action of silver fluoride on the acetylated glycosyl halide (bromide or chloride) of the opposite configuration a t the glycosidic carbon atom.13 Thus,'3 ti 50-g. sample of tetra-0-acetyl-a-D-glucosyl bromide in 150 ml. of dry acetonitrile containing 50 g. of anhydrous silver fluoride is shaken for 1 hr. The mixture is filtered and the filtrate is evaporated under diminished pressure a t a bath temperature of 3 5 O , with precautions to exclude moisture. The residue is dissolved in ether and cooled. Subsequently, crystallization is completed by the addition of petroleum ether. A 50 % yield of tetra-0-acetyl-P-D-glucopyranosyl fluoride is obtained.

Similar methods have been employed for the synthesis of the fluoride anomers of a number of variously substituted carbohydrate derivatives (see Tables), However, treatment of tri-0-acetyl-a-D-xylopyranosyl bro(13) B. Helferich and R. Gootz, Ber., 61, 2505 (1929). (14)F. Micheel, A. Klerner and R. Flitsch, Chem. Ber., 91, 663 (1958).

88

F. MICHEEL AND A. KLEMER

mide" and tri-0-acetyl-8-u-arabinopyranosyl bromide16 gave the acetylated glycosyl fluorides of the same configuration at the glycosidic carbon atom as that of the original bromides. Apparently, the unstable anomers, which are presumably formed a t first, readily isomerize to the more stable anomers. Unlike any of the other atetylated glycosyl halides, the fluorides may be deacetylated without loss or isomerization of the halide function. This unique reaction can be effected either with alcoholic ammonia17J8 or with a catalytic amount of sodium methoxide in alcoh01.~7J~ In some instances, however, depending on the concentration of base and on the configuration and type of substitution at the carbon atom next to the glycosidic center, side-reactions occur that lead to glycoside or anhydride structures.12.'4'1*'20

111. REACTIONS OF THE GLYCOSYL ~~'LUORIDES It R i of interest to note that, historically, the first chemical synthesis17 of a disaccharide, gentiobiose, involved the condensation of tetra-o-acetyla-D-glucopyranosyl bromide with 2,3,4-tri-O-benzoyl-a-~-glucopyranosyl fluoride. The resulting 8-0-(tetra-O-acetyl-8-D-glucopyraiiosyl)-tri-O-benzoyl-a-D-glucopyranosyl fluoride was saponified with methanolic ammonia, and the fluorine was removed by boiling with an aqueous suspension of calcium carbonate, to yield the free disaccharide (characterized as the 8-octaacetate). The procesees used in this synthesis are further discussed under the appropriate headings. When the halogen is fluorine, the usual methods' for the preparation of glycosides and oligosaccharides from the acetylated glycosyl halides (by reaction with hydroxylic compounds) are only successful in certain cases. The formation of a,/3-trehalosez1 may be cited as one of the few successful syntheses. However, with metal alkoxides, glycosides may be obtained having either the same or the opposite configuration at the glycosidic carhon atom. Frequently, internal glycosidation or anhydro-ring formatioii occur8 as a competing reaction; high concentrations of alkali favor this competition. In any event, the type of substitution Ltt the carbon atom adjacent to the carbon atom bearing the fluorine atom, and the Rputial relations of the groups involved, play dominant roles in the reactions of the glycosyl fluorides,12J4J*~20 The reactions may be divided into two types. (15) (16) (17) (18) (1960). (19) (20) (21)

H . Nordiek, Diplomarbeit, Miinster, 1964. A. Klemer and J. Ridder, Diplomarbeit, Muneter, 1958. B. Helferich, K . Bauerlein and F. Wiegand, Ann., 447, 27 (1926). F. Micheel and L. Tork, Diplomarbeit, Munster, 19M; Chem. Ber., 93. 1013 F. Micheel and A. Klemer, Chem. Ber., 86, 187 (195"). F. Micheel and E. Michaelis, C k m . Ber., 91, 188 (1958). V. E. Sharp and M. Stacey, J . Chem. Soc., 285 (1951).

89

QLYCOSYL FLUORIDES AND AZIDES

The first kind of reaction occurs when the substituent adjacent to the fluorine atom exists in a configurationally cis relation to it or contains no acidic hydrogen atom. The second and much faster type of reaction requires that the neighboring substituent exist in a configurationally trans relation to the fluorine atom and that it bear a removable hydrogen atom. In this latter circumstance, the reactions are believed to proceed through intermediates very similar to Brigl's anhydride,22 tri-O-acetyl-l , 2-anhydroa-D-glucopyranose, although such compounds have never actually been isolated from the reactions. Thus, the two types of reaction are those in which neighboring-group participation plays a role and those in which no participation can occur. 1. Reactions Involving Participation

If a hydroxyl group is situated trans to the glycosidic fluorine atom, a rapid reaction with sodium methoxide occurs, with elimination of hydrogen fluoride, as shown for the reaction14of 8-D-glucopyranosyl fluoride (4).The , was not isolated, depends on the confate of the intermediate [ ( 5 ) ] which centration of alkoxide. In dilute solution, methyl P-D-glucopyranoside (6) is formed, whereas, in concentrated solution, the product is 1,6-anhydroP-D-glucopyranose (7). If the C-6-hydroxyl group has been blocked with the triphenylmethyl group, methyl P-D-glucopyranoside is the sole product. NaOM in MeOH

bH (4)

a Coned. NaOMe

no

Ho

6H (6) (22) P. Brigl, 2.phy8iOl. Chem., 122,245 (1922).

90

F. MICHEEL AND A. KLEMER

Similar reactions have been observed with 8-D-galactopyranosyl fluoride2* and a-D-mannopyranosyl flu0ride.2~In this latter example, the epoxide ring in the supposed intermediate [(S)] would not be favorably located for internal-glycoside formation with the C-6-hydroxyl group ; the oiily observed product is methyl a-D-mannopyranoside (9).

0 CHzOH

HO OH

CC 8 11

(9) Depending on the alkoxide concentration, either the methyl 8-D-glucoside (12) or the anhydride (13) arises from 2-deoxy-2-p-toluenesulfonamidop-D-glucopyranosyl fluoride ( 10).l2,*O The presumed intermediate imine [(Il)] has not been isolated.

0

HO

HNT8

( 10)

Dilutr

HNT1

NoOMe MeOH in

-

FAT8

HO

“II)]

Concd.

HIITS

(13) (12) When the methanolic alkoxide is replaced by an aqueous solution of an inorganic base (preferably barium hydroxide), completely analogous reac(23) F. Micheel, A . Klemer, G . Baum, P. RistiE and F. Zumbulte, Chem. Ber., 88, 475 (1955). (24) F. Micheel and D. Borrmann, Dissertation, Miinster, 1960; Chem. Ber., 98, 1143 (1960).

91

GLYCOSYL FLUORIDES AND AZIDES

tions 0ccur.l9,*~ In dilute base, the free aldoses are formed preponderantly and, at higher concentrations, the anhydro glycosides result unless the C-6-hydroxyl group is blocked or is situated cis to the potential epoxide. Where internal-glycoside formation is possible, high yields of these compounds can be obtained by employing strongly basic ion-exchange resins.26 The action of aqueous base on a-D-mannopyranosyl fluoride (14) does since the epoxide internot afford any 1,6-anhydr0-/3-~-mannopyranoside, mediate (15) would be unfavorably situated with respect to the C-6-hydroxyl group. two disaccharides of the trehalose type are formed

1

Alkdi

CH2OH

obviously by reaction of the epoxide (15) with the anomeric I)-maniioses, (16) and (17), produced by hydrolysis. The disaccharides (18) and (19) are accompanied by polymeric D-mannans. The main products obtained, however, are nonreducing oligo- and poly-saccharides which, on the basis of their degradation with periodic acid, contain essentially linear (1 -+ 6)linkages. These linkages obviously arise by reaction of (15) with (18) and

(25) F. Micheel and

G. Baum, Chem. Ber., 88, 479 (1955).

92

F. MICHEEL AND A. KLEMElt

(191,

[a]+124"

(19). In a similar way, raffinose is transformed by (15) to oligo- and poly-

saccharide^.^^ 2. Reactions Not Involving Participation For the glycosyl fluorides, those reactions which do not involve participation are normally much slower than those which may proceed through cyclic intermediates (5, 8, 11, and 15). Two situations may force the glycosyl fluorides t,o react with bases without participation of the neighboring group. The C-2 function may be cis to the fluorine atom at C-1 or, if the relation is trans, may bear no removable proton. In the first case, typical reactions are the formation of the methyl 8-Dglucoside from a-D-glucopyranosyl fluorideI4 and of its 2-amino-2-deoxy on treatderivative from 2-amino-2-deoxy-cr-~-glucopyranosyl ment with sodium methoxide in methanol. The products from the reaction of aqueous bases with the glycosyl fluorides depend on the concentration of alkali. At low concentrations, the normal hydrolysis products are f ~ r m e d At . ~higher ~ ~ ~ concentrations ~ of base, if the proper (trans) steric relation exists between C-6 and the fluorine atom at C-1, anhydro compounds are formed,2a,za-za as in (21) from (20).

6)+ q CHg-0

HO

H

(20)

(21)

R = OH, NHTs or OMe and R ' = OH or R = R ' = OMe

In the second case, as with the 8-fluorides of 2-O-methyl-~-glucopyranose~4 and 2-deoxy-N-methyl-2-p-toluenesulfonamido-~-glucopyranose,26 the less readily obtainable methyl a-D-glycosides are produced, as in (23) from (22). (26) E. Michaelie, Diesertution, Miinster, 1959. (27) F. Micheel, A. Klemer and R. Flitsch, Chem. Ber., 91, 194 (1958). (28) C. Holthrtus, Diplomurbeit, Miinster, 1957.

QLYCOSYL FLUORIDES AND AZIDES

R = OMe

OT

93

CH~NTS

3. Structure of the D-Fructose Moiety in Sucrosc. Although the structure of the D-glucose moiety of sucrose had been established as a-~-gl~copyranosy1,2~ the only evidence for the structure of " the 1)-fructose moiety had been of a hiochemical n a t ~ r e . ~However, direct chemical proof31for the 0-wfructofuranosyl structure was obtained in 1958. The behaviorla of the anomeric 1-0-methyl-D-fructopyranosyl fluorides toward methoxide ion provides an independent proof of this structure. Thus, 1-0-methyl-p-D-fructopyranosyl fluoride (24), treated with methoxide ion (slow reaction) and then acetylated, affords methyl 3,4,5-tri-O-acetyl-l-O-methyl-a-u-fructopyranoside(25). Deacetylation with sodium methoxide results in the formation of methyl 1-0-methyl-a-ufructopyranoside (26).This slow replacement of fluorine by the methoxyl group requires that the fluorine be cis to the C-3-hydroxyl. In accord with this, 1-0-methyl-a-u-fructopyranosyl fluoride (27),having a trans situation, has been found to undergo a fast reaction with methoxide, affording the same methyl glycoside (26)on isolation through its acetate (25).These reactionsl8 establish the anomer (0-D) of negative rotation as (24),having the cis relation of the C-3-hydroxyl group to the fluorine atom; the other anomer (27)must be CY-Dand possess the trans relationship. Since the optical rotations of the D-fructopyranoses differ from those of the D-fructofuranoses in magnitude only (and never in sign), the anomeric configurations of the D-fructofuranoses are established. The /3 configuration derived in this way for the D-fructose moiety in sucrose coincides with the results of previous investigat~rs.~'JJ'

4. Other Reactions The aldosyl fluorides can react with pyridine to give pyridinium compounds.16The degree of reactivity depends on the steric arrangement of the glycosidic fluorine atom and on the nature of the substituent a t the ad(29) M. L. Wolfrom and F. Shafizadeh, J . O T ~Chem., . 21.88 (1956). (30) C. S. Hudson and C. B. Purves, J . Am. Chem. SOC.,69, 49 (1937). (31) R. U. Lemieux and J. P. Barrette, J . Am. Chem. SOC.,80,2243 (1958).

94

F. MICHEEL AND A. KLEMER

(-47

Ho I OH

(241,

OH

[&I -llOo

[a]+12.6"

(27X

r(G$Y] OH

dH

(261,

t34.1"

jacent carbon atom. Insufficient data have been accumulated to permit a general theoretical treatment. Pyridine containing pyridinium chloride reacts in the cold with @-D-glucopyranosylfluoride to give D-glucopyranosylpyridinium chloride (29, R = H, [CY]D+49.5"), but the same product results from the CY-Danomer on heating only. Both of the snomers of 2-0methyl-D-glucopyranosyl fluoride require heating with this reagent i n order to afford (2-0-methyl-~-glucopyranosyl)pyridinium chloride (29, R = Me, [CY]D +39.7").

(291, R =

H

or Me

GLYCOSYL FLUORIDES AND AZIDES

95

The lower reactivity of a-D-glucopyranosyl fluoride permits its etherification with triphenylmethyl chloride in pyridine without formation of the quaternary compound; the product is 6-O-trityl-cr-~-glucopyranosylfluoride." A similar process, leading, however, to the @-D anomer, can be carried out if a sterically hindered base, 2, 6-dimethylpyridine1 is used instead of the pyridine." Benzylidene acetals of the aldosyl fluorides can be prepared" by treatment with benzaldehyde-zinc chloride, if two suitably situated hydroxyl groups are present in the sugar derivative. This method has afforded 4,6-0benzylidene-P-D-glucopyranosyl fluoride.

IV. THEW-FLUORO CARBOHYDRATES and of 5-deoxy-5Several derivatives of 6-deoxy-6-fluoro-~-glucose~~ f l u o r o - ~ - r i b o s ehave ~ ~ * ~been ~ prepared (see Table 111). Because of the high strength of the carbon-fluorine bond12no displacement reactions of the fluorine atoms in these compounds are known. However, it has been reis strongly reported32that methyl 6-deoxy-6-fluoro-@-~-glucopyranoside ducing toward Fehling solution, in contrast with the behavior of the respective 6-chloro and 6-bromo compounds. The reaction of almond emulsin with a series of glycosides of 6-deoxy-6-fluoro-~-glucosehas been thoroughly inve~tigated.~~ The usual method for the synthesis of the u-fluoro aldoses is based on the displacement of a methylsulfonyloxy group by fluorine (supplied by either potassium fluoride or calcium fluoride). The reaction may be accomplished in aqueous methanol,32 ethylene or N , N-dimethylf ~ r m a m i d e employing ,~~ carbohydrate derivatives that have all the hydroxyl groups, except that to be replaced, blocked by groups stable to bases. The following example illustrates the synthetic meth0d.~2 Esterification of 3 ,5-O-benzylidene-l , 2-O-isopropylidene-c~-~-glucofuranose with pyridine and methanesulfonyl chloride in pyridine affords the 6-0-methylsulfonyl ester, which is treated with potassium fluoride (dihydrate) in methanol a t 100". Cleavage of the two acetal groups, with sulfuric acid in aqueous methanol, yields 6-deoxy-6-fluoro-~-glucose,which can be purified as the acetate. V. THEALDOSYL AZIDES The aldosyl azides resemble the aldosyl halides, especially the fluorides; this is to be expected from the similarities of hydrazoic acid and the halogen (32) B. Helferich and A. Gniichtel, Ber., 74, 1035 (1941). (33) N . F. Taylor and P. W. Kent, J . Chem. SOC.,872 (1958); P. W. Kent, A. Morris and N. F. Taylor, ibid., 298 (1960). (34) H. M. Kissmann and M. J. Weiss, J . A m . Chem. SOC.,80, 5559 (1959). (35) B. Helferich. S. Grunler and A. Gnuchtel, 2.physiol. Chem., 248,85 (1937).

96

F. MICHEEL AND A. KLEMER

acids. The aldosyl azides can be prepared easily by treating the readily available poly-0-acetylaldosyl halides with sodium azide or silver azide, the resulting displacement react ion usually produring an inverted configuration a t the glycosidic carbon atom. The first poly-0-acetylaldosyl azide was synthesized by I 3 ~ r t h ohy ~~ treating tetra-0-acetyl-a-u-glucopyranosylbromide with a metal azide. I n addition to the aldosyl azide derivatives that are k n o w (see Table IV), derivatives of 2-aniin0-2-deoxy-~-glucosylazide have been prepared" ,x7 (see Table V ) . If the trans hydroxyl group a t C-2 is not ncetylated, an iutermediate epoxide of the type suggested for the aldosyl fluorides call form, and the azide obtained has the same anonieric coilfiguration as thc original halide, as shown by the formulas. The acetylated azides, like the

cT - cko-T r CH,OAc

AgN,

ACO

AcO

WOH

H

acetylated fluorides, can be saponified withou 1 loss of the azide function.12* 2 3 , 3 8 3 8 The aldosyl azides obtained are much more stable thari the aldosyl fluorides and have not as yet been converted to glycosides. With conceritrated alkali, aldosyl azides having the proper steric arrangement (the C-2-hydroxyl group trans to both the azide group and the C-G group) react ' (like the corresponding fluorides) to produce the stable anhydro dcmvat i ~ e s . 2The ~ conversion of 8-D-glucopyranosylazide (30) to 1,6-anhydro-P-uglucopyranose (7) is illustrated.

..%y' - HgAq .I;;;

__c

H

(30)

CC511

(71

Reduction of the acetylated aldosyl azides afford~"~-~O the corresponding acetylated aldosylamines, as exemplified by the preparation*' of t ri-0acetyl-2-amino-2-deoxy-~-~-g~ucopyranosy~am~ne (31 -+ 33). A. Bertho (with H. Nussel), Ber., 88, 836 (1930). A. Bertho and A. RBvBss, Ann., 681, 161 (1953). A. Bertho (with M. Bentler), Ann., 661, 229 (1949). A. Bertho and J. Maier, Ann., 498, 50 (1932). (40) A. Bertho and D. Aures, Ann., 692, 54 (1955). (36) (37) (38) (39)

GLYCOSYL FLUORIDES AND AZIDES

97

Analogous to the reaction of other azides with acetylene and its derivatives to produce trinzoles, tetra-O-acet,yl-p-u-glucopyranosyl azide (34) reacts with phenylacetyle~ie.~~ Although the linear azide group might, permit addition in two ways to give either the 4-or the 5-phenyltriazole ring, only a single product, results. On the basis of theoretical con~iderations,~~ the preferred structure for this substance is 4-phenyl-1- (tetra-O-acetyl-p-r,glucopyranosyl) triazole (35).

OAC

(34)

Ac

(35)

VI. TABLES OF PROPERTIES OF GLYCOSYL FLUORIDE DERIVATIVES The five tables record the properties of the glycosyl fluoride derivatives. The refererice~~?-6~ not given in the text,, but cited in the Tables, are collected here. (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51)

F. Micheel and G. Baum, Chem. Ber., 90, 1595 (1957). F. Micheel and J. Reinbold, Dissertation, Munster, 1960. F. Micheel and H. Westermann, Dissertation, Munster, 1958. B. Helferich and H. Bredereck, Ber., 60, 1995 (1927). F. Micheel and H. Kochling, Dissertation, Munster, 1960. F. Micheel and El Baya, unpublished work. F. Micheel and D. Bartling, Diplomarbeit, Munster, 1953. F. Micheel and G. Baum, Dissertation, Munster, 1956. B. Helferich and M. Vock, Ber., 74, 1807 (1941). B. Helferich and 0. Peters, Ann., 494, 101 (1932). D. H. Brauns, Bur. Standards J . Research, 7, 573 (1931).

TABLEI Properties of Some Gl ycos yl Fluoride Derivatives

W

00

Fluoride 8-D-Arabinopyranosyl t ri -0-acet yl 8-L-Arabinopyranosyl, tri4-acetyla-Cellobiosyl, hepta4-acetyl8-Cellobiosyl hepta-0 -acetyla-D-Fructopyranosyl 1-0-methyltri -0-acet yl 8-~-Fructopyranosyl 14-methyltri-0-acetyltetra-0-acetyl3,4,5-tri-O-acetyltri-0-acetyl-34-methylB-D-Galactopyranosyl tetra-0-acetyla-Gentiobiosyl hepta-0-acetyl2',3', 4',6'-tetra-O-acetyl2,3,4-tri-O-benzoyla-D-Glucopyranosyl 6-bromo-6-deoxytri -0-acet yl 6-chloro-6-deoxytri-0-aeetyl2,3-di-O-methyldi-0-acetyl-

ROrorion sdneni

Melting point, "C.

EtOH EtOH HzO EtOH (amorph.) CHClr/Et 00 amorph . EtOH EtOH EtOH Et &/Pe t .* EtOH-EtzO EttO ?

EtOH EtzO MeOH MeOH EtOH MeOH/Et20 EtOH MeZCHOH MeOH/Et 20 CHCla/Pet EtOH/Et 2 0 .O

95-96 (dee.) 115 117-118 187

-182 -140 +138.2 +30.6

+7 173

-4

20 20

m

20

H2 0 CHCli CHCla CHCl: MeOH CHClg

References

16 16 5

3,6

23

23

ctl

?:

54-56

110-119 (dec.) 102-109 (dec.) 94 112 134-135 113-114 110-118 (dec.) 98-99 215-220 (dec.) 168-169 195-196 118-125 (dec.) 131 (dec.) 149 138 (dec.) 151-152 105-108 b.p. 114-116 (0.oOPmm.)

-12.6 +52.9 -119 -110 -116 -90.4

-128.8 -88.7

+a +n

+33.5

+43.8 +I5 +96.7 +82 +I04 +88.8

+107 +94

+60

20 20 20 20 20 19 18 20 20 23 18

20 20 20 20

HzO CHCla HIO HtO CHCla

ma: CHCla CHClr H20 MeOH HIO CHCl,

18 18 11 18 9 4, 9 9 9 23 23 17 7

CHClr H to HtO CHClr HR CHC13 0 H2

17 17, 50

EtOH

27

11 11

44 44 27

i;

B M

P 9 1:

W

9

PM k2 P

~

2-0-methyltri-0-acetyltetra-0-acetyltetra-0-benzoyl2,3,6-tri-C-methyl4-0-acetyl6-0-trityltri-0-acetyltri-0-bensoyl6-D-Glucopyranosyl 4,6-0-benzylidene6-bromo-6-deoxytri-0-acetyl2,3-di-0-benzyldi-0-acetyl2-0-methyltri-0-acetyltetra-o-acetyl6-0-trityltri-0-acetyla-D-Mannopyranosyl tetra-o-acetyla-D-Xylopyranosyl tri-0-acetyl4-0- (6-D-Glucopyranosyl)a-D-mannosyl hepta-o-acetyl3,6,2’,3’,4‘,6’-hexa-O-acetyl a-Lactosyl a-Ma1tosyl, hepta-o-acetyla -Melibiosyl , hepta -0-acet ylPet. = petroleum ether.

EtOH EtOH EtOH E t zO/Pet.a Etz0 Pet MetCO/Pet .a EtOH (amorph.) EtOH/Et 2 0 EtzO EtOH MeZCHOH EtzO/Pet.‘ (sirup) EtOH/Et SO EtrO EttO CHC1a/Pet.a MesCHOH EtOH

115 (dec.) 77-79 108 110-1 12 56- 57 42 135-140 (dec.) 147-148 99-102 (dec.) 156 (dec.) 11CL112 (dec.) 99 112-114

EtzO MezCHOH EtOH

108-112 (dec.) 73-75 98 70-80 123 96-97 114 (dec.) 68-69 105 (dec.) 87

MeOH MeOH HzO/MeOH EtOH MeOH/H20

155-156 145 180-195 (dec.) 174-175 135

+99.7 +I16 +W.l +110 +70.5 +77.2 +58.4 +119.6 75 +25 -74.5 -36 +35 +36 +8.6 +I1

I

+

20 20

20

+25

22 21 18

+58 +21.9 +17.9 +17.9 +16.1 +21.5 +76 +67.2

22 14 20 18 22 20

20

Hz 0 CHCla CHCla CsHsN CHCla CHCla CIH~N C sH sN CsHsN H ZO CsHsN dioxane H9 0 CHC13 CHCla CHCla H SO CHCla CHC13 CsHsN CsHsN

28 28 3 17 46 46 17 17 17 13, 19 11 11 11

11 42 42 14 14 13, 21 11 43

cc

d

4 l? crl

F

2

Ee3

P

z

tr P

I

H SO CHClr EtOH CHCla

24 51 11 3

CHCla CHCla CHCl a CHCI, CHCla

6 6 13 8 8

E c

e3

I

+13.6

20

+20.8

20 16 20 20

+83.2 +111.1 +149.7

co co

TABLE I1 Properties of Some i-Amino-d-deozr-D-alucosul Flwn'des Melling point, "C.

2-Amino-~-deoxy-a-~-~ucopyrsnosyl N-acetyl-

t ri -0-acet yl N-tosylM -methylt ri -0-acetyltri-0-acetyltri-0-acetyl-N-benzoyl2-~ino-2-deoxy-~-~-glucopyranosyl N-methyl-N-tosyltri-0-acetyltri-0-acetyl-N-tosyl-

' a ] D , degrees

Rolatimr SOlVd

Refcrnrcw

?

z

i;

z

Me&HOH/EttO EtOAc HtO Me&O/C6H MeOH MeOH MeOH MeOH MeOH EtOAc/Pet.

6

161.5-162 (dec.)

+96

186-187 (dec.) 136 (dec.) 83 117-118 146-147 lG4

+54.4 +55.5 +56 +62.8 +66 +lo7

148-150 147-118 147-148

-6.2 +6.9 +2

EtOH/dioxane CHClr MeOH MeOH CHClr CHCl 8 CHCla MeOH CHCls CHCla

12,47

12,47 20 20 20

m

45

20 20 12

m

m

20 ?

Pm z

m

::

TABLE I11 Properties of Some w-Fluoro Alddse Derivatives Compound

Crystallization SOlFent

Potation temp., "C.

Mdting point, "C.

Rotation solvent

Referentes

-

6-Deoxy-6-fluoroa-D-galactopyranose 1,2:3,4-di-O-isopropylidene-

MeOH/Et 2O

methyl pyranoside 6-Deoxy-6-fluoroa-D-glucofuranose, 1,20-isopropylidene acetal 3,5-O-benzylidene 3,5-di-O-acetyl3,5-di-O-mesyl6-Deoxy-6-fluoroa-D-glucopyranose methyl glycoside 2,3,4-tri-O-mesylG-Deoxy-G-fluoro-8-n-glucopyranoside phenyl triacetate vanillyl triacetate Tetra-0-acetyl-6-deoxy-6-fluoro-D-glucopyranose Tri-0-acetyl-6-deoxy-6-fluoroa-D-glucopyranosyl bromide 5-Deoxy-5-fluoro-p-~-ribofuranose methyl glycoside, 2,3-O-isopropylidene acetal tri-0-acetyl-

hfe2CO/Et20

-135 +7i lG0 b.p. 7&72O (0.015 -51.4 mm.) 139 +I94 -+

20 20

H2 0 CHCl3

33 33

20

H2 0

33

MeOH MeOH/H20 EtOH EtOH CHCla/Pet. MeOH

104- 105 112 109 155 109-110 133-131

+I4 +23 -24.5 -86 -+ +4i +43 +93

21 20 20 19 21 22

C6H6 CHClr CHCla 0 H2 H2 0 C 5H 5N

32,33 49 49 32, 49 33 32

H2 0 EtOH H2 0 EtOH EtOH

148-119 167-168 181-182 1%-167 125-126

-79 -82

21 19 19 20 19

H $0 CHClr CsHSN CHCla CsHjN

32 32 32 32 32

CHCIa/Pet.

127-1 28

+234

21

CHCls

32

b.p. 32O (0.025 mm.)

-92

20

CHCl3

33, 34

100-101 (subl.)

-26.8

25

CHCli

34

EtzO

-48.6 -35.7 +20

TABLEIV Properties of Some Glycosyl Azides Melting point, "C.

a-L-Arabinopyranosyl, tri-0-acetyl&Cellobiosyl, hepta-o-acetyl6-D-Galactopyranosyl tet ro-0-acetyla-D-Glucopyranosyl tetra-0-acetyl3,4,6-tri-O-acetyl2-0- (trichloroacetyl) @-D-Glucopyranosyl tetra-0-acetyl3,4,6-tri-O-acetyltri-O-acetyl-6-bromo-6-deoxy8-Maltosyl, hepta-o-acetyl8-D-Xylopyranosyl, tri-0-acetyl-

MeOH/H *O MeOH MeOH EttO/Pet. Et,O/Pet. Me&O/EtOH n-C sH 1i0H EtOH/MeOH EteO/Pet. MeOH MeOH/HsO MeOH/HzO

[CI~D,

degrees

"C.

88-89 182-182.5 152 96

-11.0 -30.9

106.5 66 139.5 89 129 (dec.)

+I80 +223.1 +145.7 -29.6 -33.0 -13.7 -15.2

155 137-138 (dec.) 91 87.5

'2o.ktion temp.,

20 16

+8.5

-16.2

+53

-79.3

20 18 19.5 19 20 19 18 23

18 16

Rotation solvent

References

'tl

5

CHCla CHCla HZ0 CHCla

38 38

c1

48

F

CHClt CHClz CHCla HeO CHCla CHCla CHC13 CHCli CHCI,

40 40 40 23,36 36 40 36 38 38

39

z

M

m

*2: tr

?

P

E

3

TABLE V Properties of Some 2-Amino-2-deozy-8-D-glucopyranosyl Azides 2-A mino-2-deoxy-B-D-glzopyranosyl az&

Unsubstituted N -acetylN 4sopropylidene3,4,6-tri-O-acetylN-acetylN-anisylideneN -p-nitrobenzylideneN -salicylidene-

crystallizalion

solvent

amorph. EtOH/Et 2 0 Me 2CO/H 2O EtOH EtOAc/Pet. MeOH MeOH MeOH

Mdting point, "c.

142 166-167 123 160-161 (dec.) 134 102 95

1

[a]D,degrees

-58.6 -30 -53 -11.5

-43

1 1 ygLy Rotation te;?,

20 20 20 20 20

-

Hz0 HzO H20 CHClr CHCla

-

References

12 12 12 37 12 37 37 37

k.

This Page Intentionally Left Blank

THE “DIALDEHYDES” FROM THE PERIODATE OXIDATION OF CARBOHYDRATES

BY R. D. GUTHRIE Shirley Instilute, Manchester, England* . . . . . . .................... ............ I. Introduction . . . . . . . . . . . . . . . . 11. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. General Properties of the Oxidation Products, . . . . . . . . . . . . . . . . . . . . , . . . . IV. Oxidation Products from Monosaccharide Derivatives and Related Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction.. . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Oxidation Products Forming Hemialdals , , . . . . . . . . . . . . . . , . . . . . , , . . . . 3. Oxidation Products Forming Internal Hemiacetals. . . . . . . . . . . . . . . . . . . 4. Reactions of Non-carbohydrate Analogs., . , . . . . . . . . . . . . . . . . . . . . . . . . . V. Oxidation Productasfrom Di-, Tri-, and Oligo-saccharides . . . . . . . . , , . . . . 1. Introduction.. . . , . , , , . . . . . . . . . . . . . , , . . . . . . , . . . . . . . . . . . . . . . . . . . . , . . . . 2. From Sucrose.. . . . . . 3. From Other Disacch heir Derivatives.. . . . . . . . . . . . , . , . . . . ......................... 4. From Tri- and OligoVI. Oxidation Products ......................... 1. Introduction.. . . . . . . . . . . . 2. From Starch . . . . . . _ _ _. ... . . . . . . . _, _ ., . , . . _ ., . . . . . . . . . . . . . . . . . . . . 3. From Cellulose. . 4. From Xylan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. From Other Polysaccharides . . . . . . . . . . . . . . . VII. Alkaline Degradation of Periodate-oxidized Carbohydrates VIII. Uses of Periodate-oxidized Carbohydrates.. , . , . . . . . . . . . . . . . . . . . . . . . . . . .

105 106 108 108 108 109 123 132 134 134 134 135 137 137 137 140 146 152 153 153 157

I. INTRODUCTION The splitting of a-glycol groups by periodate was discovered by Malapradcl-*in 1928, and the reaction has since found wide application. Icrom a n acyclic glycol, the products of the reaction are two carbonyl compounds; an a ,w-dicarhonyl compound results from a cyclic glycol. The Rl RLoH

I

It’

R1

(5

R =O RC=O

I

R*

* Present address: Cheniistry Department, The University, Leicesler, England. (1) L. Malaprade, Compt. rend., 186,382 (1928). (2) L. Malaprade, Bull. soc. china. (France), 43, 683 (1928).

105

106

R. D. GUTHRIE

carbonyl compounds are usually aldehydes, because carbohydrate oxidations generally involve secondary hydroxyl groups (R = H). Periodate will also cleave an a-amino alcohol group, to give the same product as thc corresponding a-diol; as the former group does not occur widely in carhohydrate chemistry, the products discussed are nearly all derived from a-diols. Periodate oxidation has found its main use as an analytical tool for the detection of a-glycol groups, especially in polysaccharide chemistry. Only in the last decade has any real study been made of the reaction products. This situation is probably attributable to several factors: (a) the alleged instability of the products, (b) the use by Jackson and Hudson in the late 1930’s of the acids prepared by further oxidation (see later for detailed references), and (c) the lack of knowledge of aliphatic and other dialdehydes, of which no systematic study has yet been made. Other aspects of the reaction, such as experimental conditions, stereochemical effects, mechanism, and analytical applications, will not be discussed, as these have been reviewed elsewhere.*-6The structure and reactions of the oxidation products have been reviewed previously, but only The application of the formazan reaction to periodate-oxidized carbohydrate derivatives has been reviewed.’ Throughout this review, “oxidation product(s) ” refers to that from periodate oxidation, unless otherwise stated. This review will be limited to a discussion of the structures and reactions of the aldehydes obtained from oxidation of cyclic carbohydrate derivatives. Such products as that from the oxidation of 1,2-O-isopropylideneu-glucofuranose, which is a monoaldehyde, will not be dealt with. A monosaccharide derivative will normally give rise to a dialdchyde, a disaccharide derivative to two dialdehyde units linked together (or a tetra-aldehyde) , and a polysaccharide will give a dialdehyde polymer (a polyaldehyde). All of these classes of compounds will be discussed. The same aldehydic products may be obtained, in some cases, by oxidation with lead tetraacetak? although this procedure has not been so widcly used as periodate oxidation in carbohydrate chemistry. 11. NOMENCLATURE

A hemialdal group9 is formed by the addition of the elements of a molecule of water across two aldehyde groups; these groups are usually, but (3) E. L. Jackson, Org. Reactions, 2, 341 (1944). (4) J. R. Dyer, Methods of Biochem. Anal., 3, 111 (1956). (5) J. M. Bobbitt, Advances in Carbohydrate Chem., 11, 1 (1956). (6) I(. Takiura and K. Koizumi, Yakugaku Kenkyu, 30, 809 (1958). (7) L. Meeter, Advances i n Carbohydrate Chem., 13, 105 (1958). (8) A. S. Perlin, Advances in Carbohydrate Chem., 14, 1 (1959). (9) V. C. Barry and P. W. D. Mitchell, J . Chem. Soc., 3631 (1953).

PERIODATE-OXIDIZED CARBOHYDRATES

107

H

rc=o

+Ha0

Lc=0 H

-n,o

rCHOH

\

I

0 I LCifoH

Hemialdal

not necessarily, in the same molecule. The hemialdal and the dialdehyde will be in equilibrium in water. In this review, the term dialdehyde methanolale1° will define the group resulting from the addition of a molecule of methanol across two aldehyde groups in the same molecule. These forms will be in equilibrium as above. The name dialdehyde methanolate does H p = 0 I I L C = 0 H

+ MeOH

A

or

P

- MeOH

Dialdehyde methanolate

not imply methanol of crystallizat,ion. Similar dialdehyde alcoholates could be made with other alcohols. The oxidation products and their derivatives, which generally exist in cyclic forms, cannot be named by the usual rules of carbohydrate nomenclature. For example, the cyclic form of the oxidation product (I) from

CH,OH--

(2)

( 1)

methyl 4 ,6-O-benzylidene-a-~-glucoside is namedl0 7,O-dihydroxy-kmethoxy-2-phenyl-trans-m-dioxano-[5,4-e][ 1,4]-dioxepan; the oxidation product (2)from methyl a-L-rhamnoside is named” 3,5-dihydroxy-2-~”methoxy-6-~”-methyldioxane. I n view of the difficulty for the reader to become familiar with new nomenclatures, the oxidation products will be referred to as “the oxidation product from. .,” or “the polyaldehyde from . . .,” and so on. The original paper may give the systematic name for any particular oxidation product. However, where the unoxidized compound is described by one trivial name (for example, sucrose; or cellulose), the

.

(10)R. D.Guthrie and J. Honeyman, J . Chem. Soc., 2441 (1959). (11) I. J. Goldstein, B. A. Lewis and F. Smith, J . A m . Chem. Soc., 80,939 (1958).

108

R. D. QUTIIRIE

periodate-oxidation product will bc denoted by using the prefix (‘oxy,’’ as in oxysucrose and oxycellulose. Should the compound be oxidized by some oxidant other than periodatc, that oxidant will precede thc namc, as in sodium dichromate oxycellulosc.

111. GENERALPROPERTIES OF THE OXIDATION 1’RODUCTS No product from the oxidation of a cyclic carbohydrate derivative has yet been isolated directly as the free dialdehyde, all being found as hemialdals or as internal hemiacetals, although the dialdehydes or their derivatives may be obtained from the oxidation products. Some of the oxidation products are crystalline, although many are sirups. The infrarcd absorption spectra of niost oxidation products show little or no carbonyl absorptiori, even those in which (from chemical react,ions) there is apparently a frw aldehyde group. Other physical methods yield similar results. Because the oxidation products exist in complex, cyclic forms, they display mutarotation in such solvents as water, pyridine, and alcohols1*;these mutarotation8 suggest that, in solution, equilibria occur between various forms. Early workers often sought one definite structure for a particular product, and results from different reactions gave different answers. The formation of cyclic forms of the oxidation products is interesting conformatZionally, although few authors have considered the products from this point of view. The main reactions studied have been oxidation to the corresponding acids, reduction to the corresponding polyhydric alcohol, or partial reduction to an intermediate “aldehyde-alcohol” ; reaction with nitrogenous bases has been studied, and also the reaction of the products wit,h alkylating and acylating reagents. Many of the oxidation products are extremcly labile to alkali, and this has been a wide field of study, particularly with the oxidation products from polysaccharides. The oxidation products will be divided into classes, and dealt with compound by compound, except for the reaction with alkali, which will be discussed separately. IV. OXIDATIONPRODUCT^ FROM MONOSACCHARIDE DERIVATIVES AND RELATED COMPOUNDS 1. Introduction

T o yield a “dialdehyde” on periodate oxidation, a monosaccharide derivative must be held in a ring form by suitable substituents. The oxidation products can be divided into two classes: those which generally do not contain hydroxyl groups elsewhere in the molecule, and form hemialdals, for example (3), and those which can form internal hemiacetals with a hydroxyl group elsewhere in the molecule, for example (4);the latter class (12) I. J. Coldstein, B. A. Lewis and F. Smith, Chem. & Ind. (London), 596 (1958).

PERIODATE-OXIDIZED CARBOHYDRATES

109

(3)

F)oMe k,))

O=CH

O=CH

e

O=CH

CH,OH Me

may, of course, form hemialdals as well. These two classes of products will be dealt with separately. Oxidation destroys the asymmetry of the sugar molecule and, consequently, the same oxidation products may be obtained from the same derivative of several diflerent sugars. For example, oxidation of any methyl a-D-pentopyranoside would yield (3) as the product.

2. Oxidation Products Forming Hemialdals a. From N ,N’-Dibenzoylstreptamine ( 5 )P-In structural work on streptomycin, the streptamine derivative ( 5 ) was oxidized with periodate and yielded a crystalline “dialdehyde monohydrate,” which gave a triacetate. The cyclic hemialdal structure (6b) was, hence, proposed for this oxidation product which, on oxidation with bromine water, gave the di(benzamid0)hydroxyglutaric acid (7), derived from the dialdehyde (6a). This was the first instance of a hemialdal structure’s being proposed for an oxidattion product of this type. b. From Methyl 6-Deoxyaldohexopyranosides.-Jackson and HudsonI4 noted that the crystalline oxidation product from methyl a-L-rhamnopyranoside has an analysis corresponding to that calculated for a dialdehyde monohydrate. Later work16 showed that several methyl 6-deoxyaldohexopyranosides give crystalline dialdehyde monohydrates. It was noted that the products could be sublimed a t 65” in vacuo over Anhydrone, without (13) H. E. Carter, R. K. Clark, Jr., S. R. Dickman, Y. H . Loo, P. S. Skell and W. A. Strong, Science, 103, 540 (1946). (14) E. L. Jackson and C. S. Hudson, J . A m . Chem. SOC.,69, 994 (1937). (15) W. D. Maclay, R. M. Hann and C. S. Hudson, J . Am. Chem. SOC.,61, 1660 (1939).

R. D. CiUTHRIE

110

=:OT HO

OH NHBZ

]H,O

O=

H

NHBz

OH

*

triacetate

COJi

loss of water; the water was, therefore, assigned as water of constitution although no structure was proposed for the products. The dialdehyde monohydrate from methyl cu-L-rhamnopyranoside has been shownll to possess the hemialdal structure (8) since it forms a di-pnitrobenzoate, and methylation with methyl iodide and silver oxide introduces two more methoxyl groups. Also, the dialdehyde monohydrate, and its enantiornorph,l6 show no infrared carbonyl absorption, but show hyMe

Me0 (8 )

(9)

droxyl absorption. The conformation shown in (8) has been suggested for this oxidation product,11which is not reduced by hydrogen in the presence of a palladium-charcoal catalyst, behavior characteristic of the hemialdal group.17J8It is, however, reduced, either with hydrogen and a Raney nickel catalyst or with sodium borohydride, to the corresponding diprimary al(16) D. Walters, J. D. Dutcher and 0. Wintersteiner, J . A m . Chem. Soc., 79, 5076 (1957). (17) J. E. Cadotte, G . G . S. Dutton, I. J . Goldstein, B. A. Lewis, J. W. Van Cleve and F. Smith, Abslracts Papers Am. Chem. sbc., 119, 5D (1956). (18) J. E. Cadotte, G . G . S. Dutton, I. J. Goldstein, B. A. Lewis, F. Smith and J. W. Van Cleve, J . A m . Chem. SOC.,79, 691 (1957).

111

PERIODATE-OXIDIZED CARBOHYDRATES

coho1 (9).I8 Oxidation of the periodate-oxidized methyl 6-deoxyaldohexopyranosides gave the corresponding dicarboxylic acids, isolated as their strontium or barium ~a1ts.I~ c. From Methyl 4 ,6-O-Alkylidene- and -Arylidene-D-g1ycosides.-The most completely studied compound in the group of, compounds forming hemialdals is the crystalline oxidation product from methyl 4,6-O-benzylidene-a-D-glucoside. This has been shown to be a dialdehyde dihydrate,10J2. 20-B for which the hydrated dialdehyde structure (10) was proposed,20 as there is hydroxyl absorption but no carbonyl absorption in its infrared spectrum. Recrystallization of the dialdehyde dihydrate from nitromethane10*22 or from dimethyl sulfoxide12 showed that one molecule of water was water of constitution, and the other, of hydration. One molecule of Reaction of water per molecule was also removed by drying in u(1cu0.~~ either the dialdehyde mono- or di-hydrate with acetic anhydride in pyridinelo gave the Same diacetate, showing that the original compounds sz2

PrCH2 \

PhCH0(-')OMe CH ,C,H Hd 'OH HO OH

(10)

0-CH,

ph(

(t)

0

/

CH I OH

\:Me

I

OH

(11)

were a hemialdal (11) and its hydrate; similar results were obtained by use of other acylating or alkylating reagents.10J2*22s28Attempts to prepare sulfonates of (11) gave only unchanged compound.1° Sublimation of the hemialdal hydrate in vucuo gave the crystalline, free dialdehyde, which had intense carbonyl absorptionI2; exposure to a moist atmosphere regenerated the hemialdal hydrate. The latter compound reduces Fehling solution, but does not restore the color to Schiff reagent,"Je22showing that a hemialdal group is chemically similar to a hemiacetal. The formation of the hemialdal (11) has been presumed to occur by hydration of one aldehyde group followed by ring closure,"Js22 as follows. Such a system allows for the possible formation of four isomers, although (19) M. Abdel-Akher, J. E. Cadotte, R. Montgomery, F. Smith, J. W. Van Cleve and B . A. Lewis, Nature, 171, 474 (1953). (20) J. W. Rowen, F . H. Forziatti and R . E. Reeves, J . A m . Chem. SOC.,73,4484 (1951). (21) J. Baddiley, J. G. Buchanan and L. Szabo, J . Chem. Soc., 3826 (1954). (22) R. D. Guthrie and J. Honeyman, Chem. & Znd. (London), 388 (1958). (23) I. J. Goldstein, B . A. Lewis and F. Smith, Abstracts Papers A m . Chem. Soc., 131, 17D (1957).

112

--

\

&CH

--

R. D. QUTHRIE ,

/ HC=O

\/OH/

&O

HC

HC=O

\OH

\ A ,’ CH

b

I

--‘a

\Ho\

/’

CH Le-O=CH ,,CH bH HO’

(11)

the crystalline product will presumably have that conformation having the two hydroxyl groups in the plane of the ring; also, the above scheme shows the free dialdehyde in equilibrium with the hemialdal in solution. The hemialdal and its hydrate showed complex mutarotation in pyridine.1° ,2z Dissolution of the hemialdal (11) or its hydrate in hot methanol, concentration, and cooling gtive a different derivative, whose analysis correRponded to that calculated for a dialdehyde methanolate.10~~ This derivative gave the @me methylation product as that from the hemialdal, but acetylation introduced only one ester group. Recrystallization of the methanolate from water gave the hemialdal hydrate, in contrast to the behavior of the fully methylated derivative, which was quite stable to boiling water. O-CHz

pl,cQ\) 0

c<

\CHOMe

OH I

OMe I

” v X ) ! ) M e CH CH dMe

(12)

AH

(13)

Such reactions are consist,ent with the formulation of the methanolate as (12) or (13), although it is not known which. The formation of the methanolate is believed t,o be analogous to hemialdal formation, as shown.22

\

@CH

/ HC=O

\

---

\H9/=O=CH CH _ M e 0/

‘ I

\ 10,

/’

HC HA AMe CH

(four possible isomers)

Similar “alcoholates” were obtained using ethyl, n-propyl, and benzyl alcohols1O~~; with the last alcohol, two different alcoholates were isolated. That the alcoholates are formed from the dialdehyde present in solution

PERIODATE-OXIDIZED CARBOHYDRATES

2k

113

\

1. H a 0

2. MeOH 3. PhCH,OH

was shown by carrying out the above series of reactions.'O Ethylene glycol has been shown to give the derivative (14), instead of (15).2'

(14)

(15)

Reaction of the hemialdal (11),or its hydrate, with amino and hydrazino compounds in water or methanol has been studied. Aromatic amines and benzylamine gave derivatives (16) of the hemialdal26; piperidine also gave a hemialdal deri~ative.2~ Reaction with ethylenediamine gave the tricyclic compound (17).24 Cy~lohexylamine,2~ hydroxylamine,26~emicarbazide;~(p-nitrophenyl) hyand isoni~otinoylhydrazine2~ drazine and (2 ,4-dinitrophenyl)hydra~ine?~ gave derivatives (18) of the free dialdehyde. Reduction of the cyclohexylamine derivative occurred with elimination of a molecular proportion of amine, to give the het,erocyclic compound (19).26 The above reactions proved t,he existence of an equilibrium between the dialdehyde and the hemialdal forms in solution. Reaction of the hemialdal with phenylhydrazine gave a yellow compound which resulted from the condensation of one molecule of phenylhydrazine with each dialdehyde molecule.10Structure (20) or (21) was tentatively'o proposed for this product, since it contained one hydroxyl group and did not afford a formazan.lO-nFurther studies27*have, however, shown that (24) R. L. Colbran, R. D. Guthrie and M. A. Parsons, J . Chem. SOC.,3532 (1960). (25) R. D. Guthrie, J. Honeyman and M. A. Parsons, J . Cheni. Soc., 2449 (1959). (26) R. D. Guthrie, Ph.D. Thesis, London (1958). (27) L. Mester and E. Moczar, Chem. & Znd. (London), 761 (1957). (27a) R. D. Guthrie, Proc. Chem. SOC.(London), 387 (1960).

114

R. D. GUTHRIE

HC

CH

I

I

NH

I CHz-CHz

NH

(21)

(20)

carbon-carbon bond formation occurred in the reaction, with formation of methyl 4,6-0-benzylidcn~-3-deoxy-3-(phenyla~o)-cr-~-glucoside (or -D-alloside) (21a) ; this compound was reduced to methyl 3-amino-4,6-O-benzylidene-cr-D-glucoside,and, hence, it provides a new route to the synthesis of derivatives of 3-amino-3-deoxy-~-glucose.

H,N=NPh

PERIODATE-OXIDIZED CARBOHYDRATES

115

Fkduction of the hemialdal (11) or its hydrate with sodium borohydride23828 or potassium borohydrideZ4gave the corresponding diprimary and alcohol (22), characterized as it,s d i a ~ e t a t e ?di-p-nitroben~oate,2~~~* ~

HO CbOH (22)

CHIOH

Cii,OMe H C = 0 (23)

dimethyl etherzs; the last compound was hydrolyzed to give 1-0-methylerythritol. Reaction of the hemialdal hydrate (11, hydrate) with methanolic hydrogen ~hloridez~ gave compound (23) which, after reduction with sodium borohydride, was hydrolyzed to D-erythrose and glycolaldehydeZ3;methylation of the borohydride-reduced compound, followed by hydrolysis, gave 2-O-methyl-~-erythroseand niethoxyacetaldehyde,25 confirming the correctness of structure (23). The same hemialdal hydrate (11, hydrate) was formed by oxidation of and of methyl 3-amino-4,6-0methyl 4,6-0-benzylidene-a-~-mannoside benzylidene-3-deoxy-a-~-altroside.~~ Similar hemialdal hydrates have also been prepared from 4,G-0-benzylidene derivatives of methyl p-D-glucoside, -D-galactoside, and - ~ - g u l o s i d e ,as ~ ~well as from methyl 4,6-0-0- and p-chlorobenzylidene-and methyl 4 ,6-O-o-bromobenzylidene-a-~-glucosides.~~ Oxidation of methyl 4,6-0-ethylidene-cu-~-glucosidegave a dialdehyde monohydrate to which a hemialdal structure was assigned.1° d. From Methyl A1dopentopyranosides.-A number of the methyl pentopyranosides were oxidized by Hudson and his c o w ~ r k e r s they ~ ~ ~did ~~~~~; not study the products, but oxidized them to the corresponding di-acids. The same product resulted from the same anomer in the same enantiomorphous series, because of the loss in asymmetry; for example, all methyl p-D-pentopyranosides gave the same product, and hence, on further oxidation, the same di-acid. This relationship was used by Jackson and Hudson Ionophoto correlate the structures of the methyl pentopyrano~ides.14~~~*~2 resis of the product from methyl 8-D-xyloside in sodium bisulfite buffer showed one spot, corresponding to a molecule with two aldehyde groups.33 However, from chemical studies, the sirupy products exist as hemialdals; (28) (29) (30) (31) (32) (33)

B. A. Lewis, Ph.D. Thesis, Minnesota (1957). I. J. Goldstein and F. Smith, Chem. & Znd. (London), 40 (1958). J. Honeyman and C. J. G . Shaw, J . Chem. SOC.,2454 (1959). W. D. Maclay and C. S. Hudson, J . A m . Chem. Soc., 60,2059 (1938). E. L. Jackson and C. S. Hudson, J . A m . Chem. SOC.,63, 1229 (1941). 0. Theander, Acta Chem. Scand., 11, 717 (1957).

110

R. D. QUTHRIE

this shows that the products will react in either form under different conditions. Methylation of the oxidation product (24) with methyl iodidesilver oxide gave a trimethoxy-dioxane derivative (25) .94

BCH

34)

(25)

It was observed by Jackson and Hudson14that the products from an anomeric pair of methyl pentopyranosides have equal, but opposite rotations. Since they believed that the products were dialdehydes, this was reasonable, as the anomeric carbon atom was the only asymmetric center. However, it is now known that these products exist as hemialdals, and, since the optical rotationu are high, it is certain that they are in the same form in solution. The hemialdal form has three asymmetric centers and, therefore, the two anomeric forms would be expected to have different rotations. It is the author's opinion that a conformational change occurs in the a-Danomer (assuming a CA86,36 conformation), such that the final hemialdals (26) and (27) are mirror images, as shown; this would then account for their displaying rotations numerically the same, but opposite in sign.

(26)

(34) I. J. Goldetein and F. Smith, J . A m . Chem. SOC.,82, 3421 (1960). (36) H. S. Iebell and R. 9. Tipson, Science, 180, 793 (1969). (36) H . S. Iebell and R. S. Tipson, J . Research Natl. Bur. Standards, 64A, 171 11960).

PERIODATE-OXIDIZED CARBOHYDRATES

I17

0-D-CA

(27)

The above theory assumes that the hemialdal hydroxyl groups are equatorial, but it would apply whatever conformation they assumed, provided that it was the same for both anomers. No crystalline derivatives of a pair of anomers are reported in the literature to prove this point. Reduction of the oxidation products with sodium borohydride or in the for example presence of Raney nickel gave the corresponding di01s,~~J7J7* (28). These diols have only one center of asymmetry and have been used as a method alternative to that of oxito correlate pentoside ~truct,ures,~'

(W dation mentioned ab0ve.~~JlJ2 The diols have been characterized as their crystalline di-p-nitrobenzoates. The diol (28) from the reduction of oxidized methyl a-D-xyloside was methylated% with methyl iodide (after dissolution (37) F. Smith and J. W. Van Cleve, J . A m . C h m . Soc., 77.3091 (1955). (37a) B. A. Lewis, R. Montgomery, F. Smith and J. W. Van Cleve, Abstracts Papers A m . Chem. Soc., 121, 4P (1952). (38) J . K. Hamilton, G. W. Huffman and F. Smith, J . Am. Chem. SOC.,81, 2173 (1969).

118

R. D. QUTHRIE

in liquid ammoiiia and treatment with sodium) ; methylation with Purdic's reagents was iiiromplete. The mcthylated diol was hydrolyzed to yield 2-methoxyethanol and methoxyacetaldehyde. Reduction of the oxidation product from methyl P-D- and @-L-arabinosideswith hydrogen and a palladium-charcoal catalyst gave only unchanged compound, consistent with the presence of a hemialdal gr0up.~7J* The sirupy oxidation product from methyl 8-L-arabinoside formed a crystalline methanolate,12 which was recrystallized unchanged from mcthanol, was distilled without loss of methanol, and formed a mono-p-nitrobenzoate. Treatment with water gave the original sirupy hemialdal. Reaction of the oxidation product from methyl a-D-xylopyranosidc with methanolic hydrogen chloride caused complex optical-rotational changc~2~; these were interpreted as formation of, and equilibria between, the trimethoxy-dioxane (25) isomers derived from the hemialdal form of the oxidation product. A very interesting synthetic use for periodate-oxidized methyl pcntopyranosides has been developed by Baer and F i ~ c h e r . ~Treatment ~*~" of the "dialdehyde" from methyl 8-u-pentopyranosides with nitromethanc-sodium methoxide gave a n aci-nitro compound which, after acidification and a small proporhydrogenation, gave methyl 3-amino-3-deoxy-@-~-riboside; was isolated as a by-product. tion of methyl 3-amino-3-deoxy-8-~-xyloside A similar sequence in the L series gave methyl 3-amino-3-deoxy-8-~-riboside. e. From 1,6-Anhydromonosaccharides.-levoglucosan (1 ,6-anhydro-O-~glucopyranose) (29)41has been oxidized to give the sirupy product (30), which was not studied but was oxidized to the diacid (31); this acid is remarkably resistant to acid hydrolysis, hot 2.5 N hydrochloric acid having no action on it. It would seem probable that the "dialdehyde" (30) exists as the hemialdal form (32). The same oxidation product, identified by comparison of [aID, has been prepared from altrosan,42 l e v o m a n n ~ s a n , ~ ~ galacto~an,~' g ~ l o s a n , 4and ~ allosan.46Reaction of periodate-oxidized levogliicosan with nitromethane and sodium methoxide in the same way as for the oxidized methyl pentopyranosides (see above) has led to the synthesis of 3-amino-l , 6-anhydro-3-deoxy derivatives of D-gulose, D-altrose, mid D-idose in yields of 13-15, 16, and 5 %, respe~tively.~~' (39) H. H. Baer and H. 0. L. Fischer, PTOC. Natl. Acad. Sci. U .S.,44, 991 (1958). (40) H. H. Baer and H . 0. L. Fischer, J . Am. Chem. SOC.,81,5184 (1959). (41) E.L. Jackson and C. S.Hudson, J . Am. Chem. SOC.,62,958 (1940). (42) N.K.Richtrnyer and C. S. Hudson, J . Am. Chem. SOC.,62,961 (1940). (43) A.E.Knauf, R. M. H a m and C. 5.Hudson, J . Am. Chem. Soc., 63,1447 (1941). (44) R. M.Hann and C. S. Hudson, J . A m . Chem. Soc., 64,2435 (1942). (45) L. C. Stewart and N . K. Richtrnyer, J . A m . Chem. SOC., 77, 1021 (1955). (46) J. W.Pratt and N. K. Richtrnyer, J . Am. Chem. SOC.,77, 1906 (1955). Chem. SOC.(London), 341 (46rt) A. C. Richardson and H. 0. L. Fischer, PTOC. (1960).

PERIODATE-OXIDIZED CARBOHYDRATES

119

(32)

(31)

f. From 3,6-Anhydromonosaccharide Osotria~oles.~7--Theosotriazoles of the 3 ,&anhydro derivatives of D-psicose and L-tagatose gave the same dialdehyde monohydrate, to which the hemialdal structure (33) was assigned. The mirror-image product (34) was obtained from oxidation of the HC=N

LEN/

'N-Ph

I

(33)

HC=N

i=N>-ph I

(34)

corresponding derivatives of either u-fructose or D-tagatose. Both hemialdals showed no infrared carbonyl absorption, but reduced Fehling solution. High-vacuum distillation of the hemialdal (33) at 140-145" gave a very viscous distillate which had an analysis corresponding to either the free dialdehyde (35) or its isomer (36). Acetylation of the distillate gave a crystalline monoacetate with an analysis corresponding to that for a derivative of (36); the same monoacetate could be prepared from the hemialdal (33) by acetylation for a longer time. A similar sequence of reactions was carried out on t,he hemialdal (34). This appears to be the only case of a (47) E. von Schreier, G. Stohl and E. Hardegger, Helu. China. A d a , 97,574 (1954).

R. D. GUTHRIE

120 HC=N

HC=N

A/

EH

0 HC=O (35)

'CH

HO'&&O(36)

dialdehyde hemialdal ;= enol-hemiacetal equilibrium recorded in the literature for carbohydrat,e dialdehydes. Reaction of the hemialdal (34) with (pnitrophenyl) hydradne gave an amorphous bishydrazone of the dialdehyde form. Treatment of (33) with the same base in the presence of acid gave the dialdehyde hishydrazone together with the hydrolysis product (37). Reaction of the hemialdal (33) with a-toluenethiol gave the glycolaldehyde derivative (38). ~

CH=N

PhCHzS

\

/

CH-C Ha-S-C

Hz-Ph

PhCHnS (37)

(38)

I t has been claimed that the phenylhydrazine derivatives of the above hemialdals, for which no structures were suggested, will not form forma2ans.n g. From Damhonitol.@-During structural work on thc myo-iiiositol derivative, dambonitol (39), a sirup was isolated, after periodate oxidation, which only crystallized aftcr treatment with ethanol; this product had an

~~

(48) A. K. Kiang and K. H . Loke, J . Chem. Soc., 480 (1956).

PERIODATE-OXIDIZED CARBOHYDRATES

121

analysis corresponding to that for a dialdehyde ethanolate, which was given formulation (41) ;it could be recrystallized from ether without change. The ethanolate reduced Fehling solution, but did not restore the color to Schiff reagent; the ethanol could not be removed by drying i n vacuo over phosphorus pentaoxide at 80". Reaction of the ethanolate with phenylhydrazine or its p-nitro- or 2,4-dinitro- derivatives gave crystalline bishydrazones of the dialdehyde (40). Reaction of the ethanolate with p-nitroaniline gave a compound having an analysis corresponding to that for a di-p-nitroanil monohydrate; the water in this compound could not be removed in vacuo over phosphorus pentaoxide at 100" during 36 hr., and so was regarded as water of constitution. No structure was proposed for this compound, but by comparison with the work on periodate-oxidized methyl 4,6-0-benzylidene-a-~-glucoside,2~ it was probably the hemialdal derivative (42).

3h--y

O,N('

'NH

q Me0

O . No N H

h. From the Anhydrides of the Condensation Products of Monosaccharides with &Dicarbonyl Compounds.-One mole of the furan derivative (43) (the anhydride of the ethyl acetoacetate-D-glucose condensation product4Q) reduced one mole of periodate to give a dialdehyde monohydrate60 to which structure (44) was assigned, since the molecule of water could not be removed over phosphorus pentaoxide a t 100" and since the compound showed no carbonyl absorption in the infrared.61Methylation studiesz' have now shown that structure (44) is correct, and have assisted in proving that the pyran structure originally proposed6z for compound (44) was incorrect. Similar hemialdals have been prepared from related condensation products from 0-dicarbonyl compounds, both by periodate and lead tetraacetate oxidation^.^^ w6l (49) F. Garcia GonzBlez, Advances i n Carbohydrate Chem., 11,97 (1956), and references therein. (50) F. Garcia GonrBlez, F. J. L6pez Aparicio and A. V&zquez Roncero, Anales real SOC. espafi. fis. y quim. (Madrid), 44B,243 (1948). (61) F. Garcia GonsBlea, F. J. L6pez Aparicio and M. Ortiz Rizo, Anales real soc. espafi.f l s . y quina. (Madrid), 62B,717 (1956). (52) J. K. N. Jones, J . Chem. Soc., 116 (1945).

122

R. D. QUTHRIE

(43)

(44)

The polarography of aqueous solutions of such hemialdals as (44) has shown that a reducing and a nonreducing form are both present,63t)hat.is, the dialdehyde and the hemialdal forms. i. From other Compounds.-Methylation of the sirupy oxidation product with methanolic hydrogen from methyl 6-0-methyla-D-galactopyranoside chloride2eor with Purdie's reagents34introduced two more methoxyl groups. No reduction of the oxidation product occurred with hydrogen and a palladium-charcoal catalyst18; the compound has, therefore, been assigned a hemialdal structure. This oxidation product has been used in the preparation of l-O-methyl-D-glyceritol.M The sirupy oxidation product from methyl 4 ,G-di-O-methyl-a-D-glucoside formed a crystalline ethanolate,12which could be recrystallized from metfhanol, and which formed a mono-p-nitrobenzoate; treatment of the ethanolate with water gave the original, sirupy hemialdal. The oxidation product from methyl 6-0-trityla-~-glucopyranosidehas been used in the preparation of l-0-methyl-~-glyceritol.~~ The oxidation product from methyl 5 ,G-di-0-methyl-a-D-glucoside has been used in the preparation of 1,2-di-O-methylerythrit01.~~ Lead tetraacetate oxidation of 2'5-anhydro-1 ,G-di-O-benzoyl-D-glucitol gave a crystalline dialdehyde dihydrate.K6 No dehydration or infrared CHZOBZ

(53) F. J. L6pez Aparicio and C. Piazza Molinf, Anales real soc. espail. fk.y qufm. (Madrid), 62B, 723 (1956). (54) I. J. Goldstein, J. K . Hamilton and F. Smith, J . Am. Chem. Soc., 79, 1190 (1957). (55) I. J. Goldstein and F. Smith, J . Am. Chem. SOC.,70, 1188 (1957). (56) R. C. Hockett, M. Zief and It. M. Goepp, Jr., J . A m . Chem. SOC.,68, 935 (1946).

PERIODATE-OXIDIZED CARBOHYDRATES

123

studies were carried out on this molecule, which was probably the hemialdal hydrate (45). Rather surprisingly, it is reported66 that “no derivatives could be obtained with methone reagent, phenylhydrazine, or 2,4-dinitrophenylhydrazine” ; it would be interesting to re-investigate this compound. 3. Oxidation Prodwts Forming Internal Hemiacetals

a. From Methyl Aldohexopyranosides and Methyl A1dopentofuranosides.Periodate oxidation of all methyl aldohexopyranosides [(46), n = 31 yield the same product (47), provided that the compounds are of the same anomeric form, and are all D series or all L series. For example, the methyl a-glycosides of D-mannose, D-glucose, D-galactose, and D-gulose all gave

AH20H &HIOH (46)

(47)

the same sirupy oxidation product, as shown by optical-rotation measurements.14*67Oxidation of the products with bromine water in the presence of strontium carbonate gave the same dicarboxylic acid salts from all four compound^.^^ This is the basis of Jackson and Hudson’s method for correlating glycoside structures. Similarly, it has been shown that the methyl P-glycosides of D-glucose, D-galactose, and D-mannose give the same oxidation product."^^ Also methyl aldopentofuranosides [(46), n = 21 give the same oxidation product (47) as the methyl aldohexopyranosides. Thus, methyl a-D-arabinofuranoside gave the same oxidation product as methyl a-~-glucopyranoside.’~ This relationship is also of great use in determining configurations. The most studied compound in this group is the oxidation product from methyl a-D-glucopyranoside, which, of course, can be prepared from other glycosides. At least six structures are theoretically possible for this “dialdehyde”: the free dialdehyde (48) or its hydrated form, a dioxane derivative formed by hemiacetal formation (49) or its hydrate, a fused tricyclic form (50) by further hemiacetal formation in (49), and a hemialdal form (51). Equilibria between the various forms should be possible, and, in different reactions detailed below, derivatives of the different forms arc produced. Some workers have sought one structure for the “dialdehyde,” (57) E. L. Jackson and C. S. Hudson, J . AWL.Chem. Soc., MI, 378 (1936). (58) E. L. Jackson and C. S. Hudson, J . Am. Chem. Soc., 61,959 (1939).

R. D. QUTHRTE

124

O=CH

HC=O

CHOH

although this is a false trail. The equilibria will be discussed after the chcmical reactions have been described. Ultraviolet spectra in water and dioxaneSe showed no carbonyl absorp~ the infrared spectrum of the product was assigned tion. A peak at 6 . 2 in to an aldehydic carbonyl group,EObut this assignment has been criticizedSe as being too high for such a group (aliphatic aldehyde carbonyl groups ~ ) 6.2-p ; band is probably attributable absorb between 5.71 and 5 . 8 1 ~ ~the to water.6ePolarography69 also showed the absence of carbonyl groups, but ionophoresis in a sodium bisulfite buffer” gave two spots corresponding to molecules with one and two carbonyl groups, respectively. Reduction of the oxidation product with hydrogen and a Raney nickel catalyst or wit#hsodium borohydride gave the corresponding tri-primary T h k is a, further method of correlating glycoside st,rucalcohol (52).1e*37#37a tures, as the only asymmetric center in the reduced compounds is the anomeric carbon atom. The trio1 (52) was characterized as a tri-p-nitrobenzoate and a trimethyl ether.88 Treatment with hydrogen in the presence of a palladium-carbon catalyst reduced only one aldehyde group to give the dioxane derivative (53) or (54). The half-reduced compound, methylated with methyl iodide-silver oxidem gave a sirupy mixture which, on (59) C. D. Hurd, P. J. Baker, Jr., R. P. Holysz and W. H. Saundars, Jr., J . Org. Chem., 18, 186 (1963). (60)L. P. Kuhn, Anal. Chem., 22, 276 (1960). (61) L. J. Bellamy, “Infrared Spectra of Complex M ~ l e c u l e s ,Methuen, ~’ London, 2nd Edition, 1958. (62) I. J. Goldstein and F. Smith, J . Am. Chem. Soc., 80,4681 (1958).

125

PERIODATE-OXIDIZED CARBOHYDRATES

(52)

methanolysis, yielded a racemic mixture of 1-0-methyl+- and L-glyceritol, showing that there was an equilibrium between (53) and (54).

(53)

i

(54)

C H(OMe),

Reaction of the “dialdehyde” with phenylhydrazine gave a derivative which formed an amorphous diphenylformazang (which, further, gave a monoacetate). Structure (55) was proposed for the formazan. Reaction of the “dialdehyde” with (p-nitropheny1)hydrazine gave an amorphous monoh y d r a z ~ n ewhich , ~ ~ was hydrolyzed by acid to glyoxal bis[(p-nitropheny1)hydrazone] and glycerose (as pyruvaldehyde) . Structure (.56) was assigned

(55)

(56)

to the monohydrazone, although it seems most unlikely, as it contains a free aldehyde group. The claim was made that, in the preparation of the monohydrazone, a bis[(p-nitrophenyl)hydrazone] was formed, although no practical details or analysis were given. (63) S. Akiya, S. Okui and S. Suzuki, Yakugaku Zasshi, 72, 785 (1952).

126

R. D. QUTHRIE

Acetylation of oxidized methyl a-D-glucopyranoside with ket,ene gave a product with 20.6 % acetyl content, in agreement with a monoacetate of structures (49) or Reaction of t,he oxidation product, with methanolic hydrogen chloride gave a product, containing a total of three methoxyl groups and one hyThis has been critidroxyl group, to which structure (57) was as~igned.2~ cieed22on the grounds that the hemiacetal group present in formulation (57) would be converted to a full acetal under the conditions used. The structure (39,derived from the hemialdal form (51) was suggested as a more plausible structure, Methylation of oxidized methyl a-D-glucoside

CH,OMe F \0-o j M e

H

CH,OMe (57)

(58)

with methyl iodide and silver oxide gave two crystalline compounds.34One, given structure (59), had ester absorption in the infrared and was thought to arise from partial oxidation of the hemiacetal form (49). The other crystalline product, which showed no hydroxyl or carbonyl absorption in the infrared and which was not reduced by sodium borohydride, was assigned structure (60). A further liquid fraction from the methylation had properties consistent with those of a mixture of (59) and (60) together with a small proportion of compound (61).

Me

0

(59)

(60)

(61)

Reaction of periodate-oxidized methyl a-D-glucopyranoside with nitromethane and sodium methoxide gave an aci-nitro compound which, after acidification and reduction, gave methyl 3-amino-3-deoxy-a-~-mannopyranoside hydro~hloride.~~ The above reactions suggest that equilibria are possible between the various forms of oxidized methyl a-D-glucoside. These equilibria have been discussed conformationally26;formation of the hemiacetal probably involves a conformational change, namely, (62) to (63) (not apparent from the (64)

H. H . Baer and H. 0.L. Fischer, J . Am. Chem. Soc., 81,3709 (1960).

127

PERIODATE-OXIDIZED CARBOHYDRATES

CH,OH

OMe (62)

(64)

(651

Haworth formulas) to form the dioxane derivative (65), rather than (64), which would have a 1,3-diaxial interaction between the aldehyde and methoxyl groups. The conformational changes involved in typical reactions of periodate-oxidized methyl a-D-glucoside are detailed in Fig. 1. Oxidized methyl a-D-glucopyranoside has been used in the preparation of l-O-methyl-D-glyceritol.Ma- or 8-Glucosidases did not attack oxidized methyl a- or P-D-glucopyranosides.66 b. From Nuc1eosides.-Periodate oxidation of adcnosirie (66) gave the

CH,OH

(65) J. E. Courtois and A. Valentino, Bull. 8oc. chim. biol., 26, 93 (1944).

128

OHr

FIG.1.-Conformational

Changes in Periodate-oxidized Methyl a - ~ - G l u c ~ p y -

ranoside.

same crystalline dialdehyde dihydrate as that from the oxidation of Q-P-Dglucopyranosyladenine.66Attempts to remove the water resulted in decomposition; treatment of the oxidation product with acid gave adenine. Oxidation of adenosine picrate gave a "dialdehyde monohydrate picrate," also obtained from the corresponding D-glucopyranosyl compound. Unfortunately, the infrared spectra of the above compounds were not taken. Similarly, oxidation of cytidine picrate gave the same dialdehyde picrate as Since these oxidation did the picrate of 3-j3-u-glucopyranosylcytosine.E0 products gave diphenylformazans, internal hemiacetal structures (67) havr been assigned to I t has been shown that periodate-oxidation products from adenosine, guanosine, uridine, and cytidine form bis(pheny1hydrazones) under neutral conditi0ns.M Degradation of the above compounds, or the original oxidation products, by Barry's method (see page 139) gave glyoxal bis(pheny1hydrazone). Distillation of the oxidation products from acid gave pyruvaldehyde (from the D-glycerose). Reduction of the oxidation products with sodium borohydride under alkaline conditions gave the corresponding triol, but, under slightly acid conditions, only that aldehyde group farther from (66) J. Davoll, B. Lythgoe and A. R. Todd, J . Chew. Soc., 833 (1946). (67) See Reference (7), p. 152. (68) J. X. Khym and W. E. Cohn, J . A m . Chem. Soc., 82, 6380 (1960).

PERIODATE-OXIDIZED CARBOHYDRATES

129

the glycosidic center was reduced. It was assumed that this partial reduction occurs because of the preferential formation of structures of type (67) under these reaction conditions. c. From Anhydro Compounds.-2,7-Anhydro-~-~-altro-heptulopyranose (sedoheptulosan) (68) has been oxidized to the sirupy “dialdehyde” (69), which was further oxidized to the diacid (70)69;this diacid, similar to that

(70)

prepared from levogl~cosan,~~ was remarkably resistant to acid hydrolysis; a bis(benzimidazo1e) could even be prepared under the drastic conditions of Moore and Link.”J The oxidation product (69), which was not isolated, was reduced (in the presence of Raney nickel) to the corresponding triol, which was hydrolyzed with acid to glycerit01.6~It would be of interest to see if the oxidation product (69) exists as an internal hemiacetal or as a hemialdal; the lattter would seem the more probable. The same oxidation product has been obtained from 2,7-anhydro-~-~-ido-heptulopyranose (D-idoheptulosan) ,?l and its enantiomorph from 2,7-anhydro-P-~-gulo.heptulopyranose (L-guloheptulosan) .’2 1,7-Anhydro-~-glycero-~-~-gulo-heptopyranose has been oxidized to a sirupy product, which was reduced with hydrogen and Raney nickel to the corresponding diprimary alcohol.ls (69) J . W. Pratt, N. K. Richtmyer and C. S. Hudson, J . Am. Chem. Soc., 74,2200 (1952). (70) S. Moore and K. P. Link, J . Biol. Chem., 133, 293 (1940). (71) J. W. Pratt, N. K. Richtmyer and C. 9. Hudson, J. A m . Chem. Soc., 74,2210 (1952). (72) L. C. Stewart, N . K. Richtmyer and C. S. Hudson, J . A m . Chem. SOC.,74, 2206 (1952). (73) L. C. Stewart and N. K. Richtmyer, J. A m . Chem. SOC.,77,424 (1955).

130

R. D. GUTHRIE

1,4-Anhydroxylitol has been oxidized to give a sirup, characterized by oxidation to the strontium salt of the corresponding dicarboxylic acid.74 1,5-Anhydro-D-glucitol (polygalitol) and 1,A-anhydro-D-mannitol (styracitol) have been oxidized with periodate, and then, further, to the corresponding strontium ~ a l t . 7 ~ The oxidation products from all of the above compounds have received no study. d. From Other Monosaccharide Derivatives.-Oxidation of phenyl p-Dglucopyranosylsulfonegave a sirupy dialdehyde, which presumably existed as the hemiacetal; reaction with phenylhydrazine gave glyoxal bis(pheny1hydrazone) and benzenesulfonic a ~ i d . 7Oxidation ~ by phenylhydrazine was postdated, in order to account for the observed fragmentation. This idea has been criticized,” since benzenesulfinic acid was isolated under conditions in which the disproportionation of the latter compound into benzenesulfonic acid and 8-phenyl thiobenzenesulfonate did not occur. The point was further proved77 when it was shown that hydroxylamine, which could not have any oxidizing action, gave glyoxime and benzenesulfinic acid. Oxidation of 1-(2-deoxy-~-“galactosyl”)benzirnidazole gave a crystalline product for which the authors suggested a hemialdal structure, although its analysis corresponded to that calculated for an anhydrous dialdeh~de’~; the probable structure is the hemiacetal(71). The oxidation product, which restored the color to Schiff reagent and reduced Tollens reagent, suggesting the presence of a free aldehyde group, also formed a crystalline “dialdehyde picrate.” The oxidation product was found to react with iodine (liberated in the reaction mixture used for titration) to give an iodinecontaining dialdehyde (72), probably as the hemiacetal; and it was shown

(74) J. F. Carson and W. D. Maclay, J . Am. Chem. SOC.,67, 1808 (1946). (76) N. K. Richtmyer and C. S. Hudson, J . Am. Chem. SOC.,66,64 (1943). (76) W. A. Bonner and R. W. Drisko, J . A m . Chem. SOC.,73, 3701 (1961). (77) E. Blanchfield and T. Dillon, J . Am. Chem. SOC.,76, 647 (1963). (78) A. J. Cleaver, A. B. Foster, E. J. Hedgley and W. G. Overend, J . Chem. SOC., 2578 (1969).

131

PERIODATE-OXIDIZED CARBOHYDRATES

that more iodine would react, presumably to form a di-iodo compound. This reaction has not been studied further, except for the observation that, for deoxy sugars and deoxyhexitols, the periodate uptake is always greater than the theoretical?s Oxidation of the D-fructose derivative (73) with lead tetraacetate gave a crystalline “dialdehyde” which showed no carbonyl absorption in the inReaction with frared spectrum, and to which structure (74) was as~igned.?~

r

1

C

i-7-y

-

_-C-Me

-0

CHOH

Me

-Me HC=O

Me

lithium aluminum hydride, however, gave the trio1 expected from reduction of the dialdehyde form. The oxidation product, treated with ethylmagnesium bromide or iodide in ether, gave, after hydrolysis, the compound (75), which, on further hydrolysis, gave 5,6-dideoxy-~-threo-hexulose, indicating a stereospecific, Grignard attack. No Grignard reaction on a dialdehyde from periodate oxidation appears to be recorded in the literature. Et-CH-CH2-0 bH

kH2-((

Et-CH-

(79) P. A. J. Gorin, L. Hough and J.

K. N. Jones, J . Chem. SOC.,2699 (1955).

132

R. D. OUTHRIE

4. Reactions of Non-carbohydrate Analogs As stated in the introduction, little is known about the reactions of dialdehydes, especially aliphatic dialdehydes, where, generally, only reactions with such reagents as (2 ,4-dinitrophenyl)hydrazine have been used for characterization purposes. Little is to be found in the literature regarding dialdehydes containing a hydroxyl group capable of forming a n internal hemiacetal. The compounds available for comparison are those potentially capable of forming a hemialdal group. o-Phthalaldehyde (76) is the most studied compound in this class, hut it is not a good model for sugar derivatives because of the rigidly-held aldehyde groups. In the solid state (as a Nujol mull), o-phthalaldehyde shows H

OH

OEt

OH

bEt

,c=o

o,,_,, H

(76)

(77)

(78)

carhonyl infrared absorption?O and, therefore, exists in the dialdehyde form (76). It is not very soluble in water, but, once dissolved, is very difficult to extract wit,h organic solvents. Study of the o-phthalaldehyde-water system showedR’t,he presence of a “monohydrate,” presumably (77) ;polarography showed the presence of reducing and nonreducing forms in solution.82 The ultraviolet spectrum of o-phthalaldehyde in 0.1 N sodium hydroxide solution is very similar to that of o-xylene, and its existence as the hemialdal (77) in this solvent was suggested?O o-Phthalaldehyde shows many typical aldehyde reactions, but there are several reactions in which derivatives of the hemialdal form (77) are obtained. Examples of these are: the reaction with ethanol in the prescnce of ammonium chloride to give thc diethyl derivative (78),Baand with dimethyl sulfate and sodium hydroxide to give the corresponding dimethyl comp0und.8~Similar chemical and physical properties have been demonstrated for gladiolic acid (79)*Oand flavipin (80).86The product from the lead tetraacetate oxidation of acenaphthenediol(81) was isolated as a monohydrates6; it would be interesting to examine the infrared spectrum of this product. (80)J. F. Grove, J . Chem. Soc., 3345 (1952). (81) L. Seekles, Rec. trau. chim., 4!4, 706 (1923).

(82)N. H. Furman and D. R. Norton, Anal. Chem., 26, 1111 (1964). (83) M. R. Powell and D. It. Rexford, J . Org. Chem., 18,812 (1953). (84) E. Schmitz, Chem. Ber., 81,410 (1958). (85) H . Raietrick and P. Rudman, Biochem. J . , 63, 395 (1956). (86) R. Criegee, L. Kraft and B. Rank, Ann., 607,159 (1933).

133

PERIODATE-OXIDIZED CARBOHYDRATES

Me,!$O2H

\

Ho,#cHo

/

H =O (79)

c=o

HO

H

OH

' '

/

OH

CHO

(80)

(81)

The reactions of such aliphatic a! ,w-dialdehydes as glutaraldehyde (82) would be expected to yield derivatives of the dialdehyde or of the hemialdal form (83), tetrahydropyran-2,6-diol.Such derivatives as the bis[(2,4-dinitrophenyl) hydrazone] are known, although few hemialdal derivatives are mentioned in the literature. Reaction of glutaraldehyde with ethanolic hydrogen chloride yields, amongst other products, 2,6-diethoxytetrahydropyran?7 Glutaraldehyde and benzenethiol gave the dialdehyde bis(diphenyl dithioacetal) and the tetrahydropyran derivative (84)

(83)

(82)\

(84)

Examples of dialdehydes which form the enol-hemiacetal group, as postulated by von Schreier and his coworkers for their oxidation products,'7 are homophthalaldehyde (85) ,88 iridodial (86),88 and periodate-oxidized codeine glycols.Qo

ac=; __ pJc;zH PH

H

,c=o

qHqjlH CH,

Cfi

(85)

T_ __L

c=o

Me

Me

(87) R. H. Hall and B. K . Howe, J . Chem. SOC., 2480 (1951). (87a) C. W. Smith, U. S. Pat. 2,619,491 (1952); Chem. Abstracts, 48, 8269 (1951). (88) K . T. Potts and R. Robinson, J . Chem. SOC.,2675 (1955).

134

R. D. GUTHRIE

V. OXIDATION PRODUCTS FROM DI-, TRI-, AND OLIGO-SACCHARIDES 1. Introduction

No broad division into two classes of oxidation products, similar to that made for the monosaccharide derivatives can be made for these oxidation products. There are nearly always several hydroxyl groups in the oxidation products and, therefore, there are generally several possibilities for hemiacctal formation. Many of the oxidation products were prepared in the course of structural work and, with the exception of oxysucrose, have received little study. 2. From Sucrose

Sucrose W ~ oxidized B by Fleury and C o ~ r t o i s ,who ~ ~ -obtained ~~ a tetraaldehyde (87) which was oxidized by bromine, in the presence of strontium or barium carbonate, to the corresponding tetracarboxylic acid salt; the latter compound was hydrolyzed with acid to yield D-glyceronic acid, glyoxylic acid, and hydroxypyruvic acid, which spontaneously decomposed into carbon dioxide and glycolaldehyde. It was founde1 that oxysucrose shows four aldehyde groups from hypoiodite estimation, but only two from mercurimetry ; the latter result was attributed to steric hindrance, but it would seem more likely that, under the conditions of the determination, CH,OH

O=CH HC=O

HC=O

HC=O

(89) G . W. K. Cavil1 and D. L. Ford, Australian J . Chem., 13, 296 (1960), and references therein. (90) H . Rapaport, M. 5. Chadka and C. H. Lovell, J . Am. Chem. SOC.,79. 4694 (1957). (91) P. Fleury and J. E. Courtois, Compl. rend., 214, 366 (1942). (92) P. Fleury and J . E. Courtois, Bull. soc. chim. (France), 10, 245 (1943). (93) P. Fleury and J . E;. Courtois, Bull. soc. chim. (France), 12, 648 (1945).

PERIODATE-OXIDIZED CARBOHYDRATES

135

oxysucrose exists in the bis(hemiaceta1) form (88). Hydrogenation of oxysucrose in the presence of Raney nickel gave the fully reduced polyhydric alcohol, which was hydrolyzed, the hydrolyzate reduced, and, from it, ethylene glycol and glyceritol obtained.g4 The reaction of phenylhydrazine with oxysucrose was found to be similar to that described for oxidized monosaccharide derivatives above, as only two molecules of the base reacted with the four potential aldehyde groupse6; the product was assigned structure (89). Mestere”found that this derivative forms an amorphous bis(diphenylf0rmazan) with benzenediazonium chloride, and so he assigned structure (90) to it. Acetylation of the formazan gave an amorphous product, identical with the formazan prepared from CH,OH

I NHPh

NNHPh (90)

acetylated (90). Reaction of oxysucrose with phenylhydrazine-acetic acid gave glyoxal bis(pheny1hydrazone) and glycerosazone, separated on an alumina c0lumn.~6Oxysucrose has also been obtained by oxidation of sucrose with lead tetraacet,ate in ~ y r i d i n e Oxysucrose .~~ was not attacked by sucram. 66 3. From Other Disaccharides and Their Derivatives Sirupy oxytrehalose (91) has been prepared97sMand oxidized to the corresponding tetracarboxylic acid,w which was hydrolyzed, and t,he hy(94)R. C. Hockett and M. Zief, J . A m . Chem. SOC.,72,2130 (1950). V. C. Barry and P. W. D. Mitchell, J . Chem. Soc., 4020 (1954). L. Mester and E. Mocaar, Chem. & Ind. (London), 764 (1957). E. L. Jackson and C. 8. Hudson, J . Am. Chem. SOC.,61, 1530 (1939). S. Akiya, S. Okui and S. Suauki, Yakugaku Zasshi, 72, 891 (1952); Chem. Abstracts, 47, 7447 (1953). (95) (96) (97) (98)

136

€2. D. QUTHRIE

drolyrate oxidized to give oxalic and glyceronic acids. The reaction of oxytrehalose (91) with (p-nitropheny1)hydrazinegave a product containing only two hydrazone groups, to which the improbable structure (92) was

(93)

assignedw; a more probable structure would be the bis(hemiaceta1) structure (93). Oxytrehalose wm not attacked by treha1ase.E6 Oxyamygdalin has been prepared and further oxidized to the tetracarboxylic acid, which gave mandelic, glyoxylic, and glyceronic acids on hydrolysi~.~~ The structures of methyl 8-lactoside and methyl /3 cellobioside have been correlated,'OO in the same way as for monosaccharide glycosides," by periodate oxidation followed by reduction. Both compounds gave the same (99) J. E. Courtois and A. Valentino, Bull. 8oc. chim. biol., 96,489 (1844). (100) J. K. Hamilton, G. W. Huffman and F. Smith, J . Am. Chem. Soc., 81,2176 (1969).

PERIODATE-OXIDIZED CARBOHYDRATES

137

tetra-aldehyde, which was reduced to the corresponding polyhydric alcohol. This was methylated in liquid ammonia and then hydrolyzed, to yield fragments useful in polysaccharide-degradationwork. Periodate oxidation has been used for correlating the structures of (1 + 6)-, (1 + 4)-, and (1 -+ 2)-linked disaccharides."J' 4. From Tri- and Oligo-saccharides

Raffinose has been oxidized to yield a hexa-aldehyde,wJOa characterized by oxidation to the corresponding hexa-carboxylic acid.IO2 OxyrafEnose reacted with three molecules of (p-nitrophenyl)hydrazine per mole, to give a compound to which a structure with three free aldehyde groups was assigned,* similar to that proposed for the corresponding oxytrehalose derivative. A more probable structure would be one containing three hemiacetal groups. Periodate oxidation has been used in the proof of structure of melezitose,'O' g e n t i ~ n o s eand , ~ ~ solanine1O6; ~ in all cases, the hexa-aldehydes were oxidized to the acids and hydrolyzed to identifiable fragments. Similar reactions were applied to the octa-aldehyde from the oxidation of stachyOSt?.'O6

VI. OXIDATION PRODUCTS FROM POLYSACCHARIDES 1 . Introduction

The products from the oxidation of polysaccharides are, generally, easily isolable. As a polysaccharide containing a-glycol groups is composed of a large number of potentially oxidizable units, a variable degree of oxidation (D.O.) from 0-100% is obtainable. (Where the D.O. is not specified, it is assumed to be 100%.) The possibilities for hemiacetal and hemialdal formation are much increased, because these links may be intermolecular as well as intramolecular. For (1 + 4)-linked polysaccharides, intramolecular-hemiacetal formation may occur, to give five- or six-membered rings. Authors generally draw all their formulas of this type with one or other ring, although there is as yet no definite evidence as to which is correct, except possibly for oxycelluloses. Some authors'07 favor the five-membered,hemiacetal ring, because of (101) A. J. Charlson and A. S. Perlin, Can. J . Chem., 34, 1804 (1956). (102) J. E. Courtois and A. Wickstrgm, Bull. S O C . chim.biol., 91, 759 (1950). (103) N. K. Richtmyer and C. S. Hudson, J . Org.Chem., 11, 610 (1940). (104) 11. HBrissey, A. Wickstrflm and J. E. Courtois, Bull. SOC. chim. biol., 99, 1768 (1951). (105) L. H. Briggs and L. C. Vining, J . Chem. SOC.,2809 (1953). (106) H. HBrissey, A. Wickstrgm and J. E. Courtois, Bull. SOC.chim. biol., 34,856 (1952.) (107) J. H. Michell and C. B. Purves, J . Am. Chem. SOC.,64,689 (1942).

138

R. D. GUTHRIE

“the known tendency of erythrose to form a furanoid ring”; this statenient is true for isolated erythrose molecules, but is not necessarily true for a molecule having other aldehyde groups available for cyclization. Formulation (94) is a hypothetical portion of an oxidized, (1 --$ 4)-linked polysaccharide; it illustrates some of the possible structures.

0-

I

CH,OH HC=O

/ \

CH

CH

OH

OH

I

O-

I

(94)

Formation of hemiacetal rings has oftentimes been postulated, without any consideration of the eonformational changes that might occur on for--+ 4)-linked mation of such a linkage. Consider a single unit in a /3-~-(1 polysaccharide such as cellulose, after oxidation with periodate to give (95). Ring closure between C-2 and the primary alcohol group is not possible unless rotation occurs about the C5-0 bond, when the rest of the polymer becomes axial; this is not apparent from the Haworth formula. If this situa-

(95)

(96)

tion is avoided by changing to the alternative “open-chair” conformation (96), ring closure could occur, but the polymer chain on C-1 would be axial. It is probable, therefore, that the ring-closed unit would take up some nonchair conformation. If these conformational changes were to occur in nearly every unit in a chain, it can be seen that cellulose would become non-linear on oxidation. Similar buckling of the chain would occur in the formation of

PERIODATE-OXIDIZED

CARBOHYDRATES

139

a five-membered, hemiacetal ring between the primary alcohol group and C-3. This theory is supported by the observation that filter paper10s~109 and cotton yarn lo9 shrink on oxidation. Oxystarch would not he so dcformed since, in the “open-chair” (96), both portions of the polymer are equatorial. Much of the work on the oxidation of polysaccharides has consisted of the determination of the number of a-glycol groups; in many cases, the polyaldehydes received no attention. The main reactions (of the oxypolysaccharides) that have been studied are their alkaline degradation (see p. 153) and their reaction with nitrogenous bases. The latter has given a degradation of great use in the structural chemistry of polysaccharides. The Barry degradation”0 involves the treatment of a periodate-oxidized polysaccharide with phenylhydrazine-acetic acid. Unbranched polysaccharides containing a-glycol groups give simple osazones which can be identified.“‘ A branched polysaccharide which is not oxidized at the branch-points will be degraded to the basic polysaccharide skeleton, which may then be re-oxidized and degraded. The degradation in the simple case of a (1 + 3)-linked polysaccharide is illustrated in Fig. 2. In the Barry degradation of beet arabinan112 and yeast mannan,Il3it was observed that fragments unexpectedly resistant to degradation can be formed, for example, 3-O-~-arabinofuranosylglycerosazone from beet arabinan.ll2 Another general use of the reaction with nitrogenous bases is the estimation of the number of a-glycol groups in a polysaccharide. Barry and his coworkers114found that each “dialdehyde unit” in an oxypolysaccharide reacts with only one molecular proportion of isonicotinoylhydrazine or thiosemicarbaside. The nitrogen content is, therefore, proportional to the number of oxidized glycol groups. Structures of the general type (97) were

P‘

(97)

(108) (109) (110) (111) (1955). (112) (113) (114) (1954).

E. L. Jackson and C. S. Hudson, J . Am. Chem. SOC.,69,2049 (1937). G . F. Davidson, J . Teztile Znst., S, T109 (1941). V. C. Barry, Nature, 162, 637 (1943). V. C. Barry, J. E. McCormick and P. W. D. Mitchell, J . Chem. SOC., 222 P. A. Finan and P. S. O’Colla, Chem. & Znd. (London), 493 (1958) P. A. Finan, A. Nolan and P. S. O’Colla, Chem. & Z71d. (London), 1404 (1958). V. C. Barry, J. E. McCormick and P. W. D. Mitchell, J . Chem. SOC., 3692

140

R. D. GUTHRIE

OH

~H,OH

OH

2 10,-

-0-

CH,OH

1 CH,OH

I

C=NNHPh

I HC=NNHPh +

+

P~NHNH,/A~OH

H{L-io CH,OH

-

(HC="HPh)?

FIG.2.-Barry Degradation of a (1 + 3)-Linked Polysaccharide. proposed for these products, whereD R' = CH20H or H; cornpare, the oxysucrose-phenylhydrazine d e r i v a t i ~ e Such . ~ ~ a structure, for which no evidence was given, would probably be very unstable, as it is quite similar to an aldehyde-amine compound. A reaction finding recent application in structural polysaccharide studirs is the reduction of an oxypolymmharidc to the corresponding polyhydric alcohol, which is readily hydrolyzed to identifiable fragments."b 2. From Starch Much of the chemistry of oxycelluloses is similar to that of oxystarshes, because of the common features in their structures. Their atudy has, howevery, been complementary. For example, much work has been done on the (116) M. Abdel-Akher, J. K . Hamilton, R. Montgomery and F. Smith, J . A m . Chem. SOC.,74, 4970 (1952).

PERIODATE-OXIDIZED CARBOHYDRATES

141

infrared spectrum of oxycelluloses, and none on oxystarches. Amine and substituted-hydrasine derivatives of oxystarches have been well studied; these derivatives of oxycelluloses have received little attention. Starch was oxidized by Jackson and Hudson,lMIwho used periodic acid at room temperature. Considerable study has recently been made of the oxidation of starch on a commercial basis, using electrolytically regenerated periodic acid.116-121 The reaction is carried out under what are normally drastic conditions for a periodate oxidation, namely pH 1.2-1.4and 100”F, apparently without occurrence of any over-oxidation. Oxystarch is known commercially as “dialdehyde starch.” The physical properties of oxidized corn-starch have been well studied.122 Contrary to an earlier report,lW oxystarches showed122increasing loss of birefringence with increasing D.O. ;oxystarches (D.O. 80-100 %) were nonbirefringent. X-ray studies showed little change up to a D.O. of 20%, but above this the amount of amorphous material increased so that, above 80 %, it was completely amorphous. Most granules were stained with iodine at 20% D.O., a t 40% about three-quarters only, and at 80-100% there was very little or no color. The only solvents for the whole 0-100 % D.O. range were glycerol and aqueous chloral hydrate; both required autoclaving for solution to occur. The usual solvents for starch dissolved only the low-D.O. oxystarches; N, N-dimethylformamide dissolved only the high-D.0. ones. The optical rotation in aqueous chloral hydrate was related to the D.O.; oxystarches (D.O. 100%) from several different sources all showed the same optical rotation. The properties of aqueous dispersions of a range of oxystarches have been studied.12aJz4 Various methods have been developed for determining the aldehyde content of oxystarches; these have been based on reaction with alkali,126re(116) W. Dvonch and C. L. Mehltretter, J . Am. Chem.Soc., 74, 5522 (1952). (117) C. L. Mehltretter, J. C. Rankin and P. R. Watson, Znd. Eng. Chem., 49, 350 (1957). (118) H. F. Conway and V. E. Sohns, Ind. Eng. Chem., 61, 637 (1959). (119) V. F. Pfiefer, V. E. Sohns, H. F. Conway, E. B. Lancaster, S. Dabic and E. L. Griffin, Jr., Ind. Eng. Chem., 62,201 (1960). (120) W. Dvonch and C. L. Mehltretter, U. S. Pat. 2,648,629 (1953); Chem. AbS t 7 U C t 8 , 47, 10884 (1953). (121) L. Mehltretter, 5. Pat. 2,713,663 (1955); chem. Abstracts, 49, 13806 (1956). (122) J. W. Sloan, B. T. Hofreiter, R. L. Mellies and I. A. Wolff, Znd. Eng. Chem., 48, 1165 (1966). (123) R. L. Mellies, C. L. Mehltretter and I. A. Wolff, Znd. Eng. Chem., 60, 1311 (1958). (124) S. Levine, H. L. Griffin and F. R. Senti, J. Polymer Sci., 86, 31 (1959). (125) B. T. Hofreiter, B. H. Alexander and I. A. Wolff, Anal. Chem., 27, 1930 (1966).

c.

u.

142

R. D. GUTHRIE

duction with sodium borohydride,126and reaction with (p-nitropheny1)hydrasine1*7or with hydroxylamine.* The last method appeared to show aldehyde groups of different reactivities; this could be accounted for by hemiacetal formation. When considered solely as the dialdehyde form, oxystarch is a polyacetal of D-erythroae and glyoxal (98), although it will actually contain hemiacetal and other linkages, mentioned in the introduction. After hydrolysis of oxystarch, the optical rotation was near that for the equilibrium value of D$!H,OH

(98)

erythrose.lM Oxyetarch reduced Fehling solution.108Acid hydrolysis gave glyoxal and D-erythrose in 25-33 % yield.1z9J30 Degradation of oxystarch with phenylhydrazine-acetic acid gave glyoxal bis(pheny1hydrazone) and ~-erythrosazone,~~ The hydrolysis of oxystarch with concentrated sulfurous acid gave D-erythrose and glyoxal in yields of 80 and 90 %, re~pectively.'~~ The success of this latter method was attributed to the prevention of hemiacetal formation by the blocking of the aldehyde groups in both the oxystarch and the hydrolyzate. Methanolysis of oxystarch with 10% methanolic hydrogen chloride, followed by steam distillation, gave glyoxal tetramethyl acetal (47 %) .Isa The remaining glyoxal formed a cyclic acetal with ~-erythrose~~7J~~JM (see below). When analysis was extended to cover both acetals, 90% of the glyoxal was accounted for. Reaction of oxystarches from several sources with 10 % methanolic hydrogen chloride yielded1*4 a crystalline solid, CloHla06(OH) (OCH3)3(earlier (128) J. C. Rankin and C. L. Mehltretter, Anal. Chem., 28,1012 (1956). (127) C. 5. Wise and C. L. Mehltretter, Anal. Chem., SO. 174 (1958). (128) E. K. Cladding and C. B. Purvee, Tappi, 116, 160 (1943). (129) E. L. Jackson and C. S. Hudson, J . A m . Chem. SOC.,80,989 (1938). (130) C. G. Caldwell and R. M. Hixon, J. Biol. Chem., 128,696 (1938). (131) J. W. Van Cleve and C. L. Mehltretter, Abstrael.3 Papers A m . Chem. SOC., 134, 2bD (1958). (132) D. H. Grangaard, E. K. Cladding and C. B. Purves, Paper Trade J., 116, 75 (1942). (133) D. H. Grangaard, J. H. Michell and C. B. Purves, J . A m . Chem. Soc., 61, 1290 (1939). (134) J. H . Michell and C. B. Purves, J. Am. Chem. SOC.,64, 585 (1942).

143

PERIODATE-OXIDIZED CARBOHYDRATES

given inc~rrectlyl~~), m.p. 150", in 1-2% yield. Structure (99) or (100) was suggested. Tosylation gave a monoester that did not react with sodium iodide in acetone at 100". The attempted isolation of erythrose monotosylate was unsuccessful. Methanolysis gave glyoxal tetramethyl acetal. A further crystalline product CsH?O3(0CH3)s,m.p. 97-98', was also formed in the methanolysis of oxystarch,1°7 and was also obtained in 36 % yield from the higher-melting product. Structure (101) or (102) was proposed for the I

I/O\

HC

A-0-

HA

A

H

HObH

-0CHn

AHz(loo)

MeO-

A

H-0-

A

H

(102)

low-melting compound, which also gave glyoxal tetramethyl acetal on methanolysis. The reaction of oxyamylopectin with methanol containing 0.4% of hydrogen chloridez9gave a polyaldehyde methyl acetal containing about two methoxyl groups per dialdehyde unit; a similar product was obtained from oxyamylose. The products were insoluble in N,N-dimethylformamide, mcresol, and alcohols; the formation of cross-links was suggested. Oxystarch, when left in liquid ammonia for 24 hr. and then precipitated with ethanol, gave a product containing two nitrogen atoms per dialdehyde unit1=; the product, for which no structure was suggested, decomposed on standing. Reductive ammonolysis of oxystarch (D.O. 90 %) in concentrated ammonium hydroxide under high pressure in the presence of Raney nickel at 100" caused the uptake of two moles of hydrogen per dialdehyde unit. A further slow reaction occurred at 190-245", with the uptake of three more moles of hydrogen. It was believed that two fleeting intermediates, aminoacetaldehyde and 3,4-pyrrolidinediol, were formed and underwent many

144

R. D. QUTHRIE

reactions to give a complex mixture of products, including diamines and heterocyclic compounds.lS6 Oxystarch (D.O. 95-99%) did not react with urea at room temperature in neutral or slightly acid solution; at alkaline pH, about one molecule of urea reacted with each dialdehyde unit.lla At higher temperatures, with excess urea a t neutral, acid, or alkaline pH, only one molecule of urea, again, reacted. These products showed the presence of one aldehyde group per dialdehyde unit, and the oxystarch was believed therefore to have reacted in a hemiacetal form. The reaction of oxystarch with thiourea gave a product with two thiourea groups per dialdehyde unit.Ia6 Reaction of this derivative with ethyl bromide, followed by an amino acid, gave the polymeric guanylamino acid (103). Hydrolysis of these compounds gave the original amino acid, the corresponding guanylamino acid, or its lactam.

4yy r/';

H--NH--C-NH

OH

Yii"

R

NH-C-NH-CH-CO,H

I

( 103)

Reaction of oxystarch with hydroxylamine under very slightly alkaline conditions gavelw glyoxime in yields of up to 30%. This reaction was suggested as an alternative to the Barry degradation, being somewhat cleaner, but there appears to be no such use reported in the literature. Cyclohexylamine reacted in a similar way1@to give N ,N'-ethanediylidenebiscyclohexylamine. Oxystarch reacted with phenylhydrazine to give a yellow compounde6Jm~ l30 which contained one basic group per dialdehyde Similar reactions were found with isonicotinoylhydrazine:s thiosemicarbazide,ls and paminobenzaldehyde thiosernicarba~one.~~~ Structures of the general type (97) were assigned to these po1ymers.QThat this structure is incorrect for the phenylhydrazine derivative was shown by its formation of a poly(diphenylformazan).l40 Mester therefore suggested structure (104) for the (136) L. A. Gugliemelli, R. V. Fitzsimmons, C. R. Russell and F. R. Senti, Abstracte Papers A m . Chem. Soc., 186, 6D (1969). (136) K. Maekawa and K. Ishimoto, Nippon Kaguku Zasshi, 76,601 (1967). (137) T.Dillon, Nature, 165, 646 (1946). (138) V. C. Barry and P. W. D. Mitchell, J . Chem. rSoc., 3610 (1963). (139) V. C. Barry, J. E. McCormick and P. W. D. Mitchell, Proc. Roy. Irish Acad.,

57B, 47 (1964). (140) L. Mester, J . Am. Chem. Hoe., 77,6462 (1866).

PERIODATE-OXIDIZED CARBOHYDRATES

145

phenylhydrasine derivative, although structure (105) is also feasible. The isonicotinoylhydrazine derivative was hydrolyzed with 50 % acetic acid alone or in the presence of cyclohexylamine, to give glyoxal bis(isonicotinoy1hydrazone), but, in the presence of phenylhydrasine, the glyoxal derivative of the added base was formed.9 This was interpreted as a breaking of either C-N bond in (97), depending on the reactants. Since this formulation is

probably wrong, some alternative explanation must be sought. The oxyalgl inic acid-isonicotinoylhydrasine polymer (see p. 153) gave the glyoxabis(azomethine) when hydrolyzed in the presence of cyclohexylamine,g so the results cannot be explained by an equilibrium between glyoxal derivatives with preferential formation of the more insoluble one. Oxystarch formed a tough, colorless gel with hydrasine in slightly acid solution.Is Oxidation of oxystarch to the corresponding polyacid has been studied with several r e a g e n t ~ . ' ~ Hydrolysis ~-'~~ of the polyacid gave glyoxylic acid and D-erythronic acid.141 Hydrogenation of oxystarch with Raney nickel catalyst, followed by hydrolysis, gave erythrito1.I" Simultaneous reduction and hydrolysis gave erythritol in yields of up to 71 %.rMJ4a Oxyamylopectin has been reduced with sodium borohydride to the corresponding polyhydric which has been methylatedrq; both derivatives have been hydrolyzed, to give fragments enabling the structure of amylopectin to he deduced. (141)A. R.Jeanes and C. S. Hudson, J . Org. Chem., 20, 1565 (1955). (142)R. L. Mellies and 0.L. Mehltretter, Abstracts Papers Am. Chem. Soc., 131, 5D (1957). (143)B. T.Hofreiter, I. A. Wolff and C. L. Mehltretter, J . A m . Chem. Sac., 79, 6457 (1957). (144)J. W. Sloan, B. T. Hofreiter, C. L. Mehltretter and I. A. Wolff, U. S. Pat. 2,783,283 (1957);Chem. Abstracts, 61, 10568 (1957). (145)J. W. Sloan and I. A. wolff, u. s. Pat. 2,796,447(1957);Chem. Abstracts, 61, 13434 (1957). (146) J. K.Hamilton and F. Smith, J . Am. Chem. SOC.,78,5907 (1956). (147) I. J. Goldstein, J. K. Hamilton and F. Smith, J . A m . Chem. SOC.,81, 6252 (1959).

146

R. D. QUTHRIB

3. From Cellulose

Jackson and HudsonlWJ** oxidized cellulose with periodic acid; hydrolysis of the resulting oxycellulose (106) gave glyoxal and D-erythrose (as Derythronic acid), but only in yields of 20 %. Jayme and his coworkers'" used a buffered, oxidizing solution, and obtained a better yield of oxycellulose and a better yield of hydrolysis products. The same product was obtained on oxidation of cellulose with either periodic acid or sodium peri0date.1~~ It was whilst studying the preparation of oxycellulose that HeadlSodiscovered that periodate oxidations should be carried out in the dark to minimize over-oxidation. Oxycellulose is characterized by great reducing CH,OH

CH,OH

HC=O

(106)

power arid by high fluidity in cuprammonium hydroxide solution (attributable to its alkali lability). The strength of yarn is much diminished by oxi~ * J ~ ~ has been oxidized dation, even at low degrees of ~ x i d a t i o n . ~ Cotton under conditions that opposed swelling.1s1Hydrolysis of oxycellulose gave a solution having an optical rotation near to that for the equilibrium value of D-erythrose.lm The infrared absorption spectra of oxycelluloses have been examined in some detail. The first study20 revealed that the spectra of oxycelluloses (D.O. 50,66, and 79 %) in Nujol mulls showed weak carbonyl bands at 5 . 8 ~ and stronger, adsorbed-water bands a t 6 . 1 ~ Structures . (107) and (108) were therefore proposed for oxycelluloses. The low intensity of the carbonyl absorption in the spectra has been confirmed.16a-166 Evidence for oxidation

dLy0, \It>\ CH,OH

CH

H d \OH

H/f&

HPoH

(148)G.Jayme, M.Siitre and 9. Maria, Nalum'ssenschaften, 29, 768 (1941). (149)G.F. Davidson, J . Tcztile Inst., 31, T81 (1940). (150)F.S. H.Head, Nature, 166,236 (1960);J . Textile Inst., 44, T209 (1953). (151)T.P.Nevell, J . Teztile Inet., 47, T287 (1956). (152) I. N . Ermolenko, R. C. Zhbandkov, V. I. Ivanov, I. Y. Lenshina and V. 8. Ivanova, Zzvest. Akad. Nauk S.S.S.R.Oldel. Khim. Nauk, 249 (1958);Chem. Abstracls, 62, 11408 (1958).

PERIODATE-OXIDIZED CARBOHYDRATES

147

at the primary alcohol group was cited,l= although no mention of it was made in later work.166It was claimed166that a band at 10.99~was connected with a hemiacetal group, and that its intensity was proportional to the D.O. The most thorough studies have been those of Higgins and McKenzielMand Spedding.lS6Both groups of workers showed that the intensity of the 5 . 7 8 ~(carbonyl) band was dependent on the D.O. and on the moisture content of the sample; this band increased from a weak shoulder at low D.O. to a distinct peak a t high D.O. Drying of oxycellulose filmsls6 caused a large increase in carbonyl intensity, first, with a decrease in the adsorbed water band, and, later, with this band at a minimum. This behavior was largely reversible and suggested the presence of two different sorts of dehydratable groups. The hydroxyl-group absorption had an intensity in the oxycellulose spectra lower than that of the cellulose spectra. This evidence led to the that oxycellulose contained about 70 % of its aldehyde groups as hemialdals and the remainder as hemiacetals or hydrated aldehydes. Higgins and M ~ K e n z i eshowed l ~ ~ that a band assigned to the primary alcohol group (at 9 . 5 2 ~ )disappears at very high D.O.; this was attributed to cycliation or oxidation. No clear evidence was found for the presence of hemiacetal groups. It was also shown’” that no band appeared at 5 . 8 ~until a D.O. of 20% was reached; since it was thought that all the aldehyde groups were masked, this absorption was assigned to carboxyl groups. I n view of the work described above, this conclusion is most unlikely. It has been shown, from x-ray ~tudies,~O~ that periodate attacks both the amorphous and the crystalline regions. Oxycelluloses are more hygroscopic than c e l l u l ~ s e . ~ ~ ~ J ~ ~ The reaction of oxycelluloses with hydroxylamine hydrochloride and determination of the freed acid, or of the nitrogen content of the product, has been used as a measure of the aldehyde content.’28Other methods for this determination are reduction with sodium borohydridelMor oxidation with chlorous Strole160has compared the hydroxylamine and borohydride methods and has shown that they give similar results. Oxidation with alkaline hypoiodite is not reliable for aldehyde-content determinations be(153) R. T. O’Connor, E. F. DuPr6 and D. Mitcham, Textile Research J . , 28, 382 (1958). (154) H. G . Higgins and A. W. McKensie, Australian J . Appl. Sci.,9, 167 (1958). (155) H. Spedding, J . Chem. Soc., 3147 (1900). (156) R. G. Zhbandkov, Optika i Spektroskopiya, 4, 318 (1968); Chem. Abstracls, 62, 11570 (1958). (157) A. Meller, Tappi, 36, 72 (1952). (158) B. Lindberg and 0. Theander, Svensk Papperstidn., 67,83 (1954). (159) G. M. Nabar and C. V. Padmanabhan, J . SOC.Dyers Colourisls, 69, 295 (1953). (160) U. Strole, Makromol. Chem., 20, 19 (1956).

148

R. D. GUTHRIE

cause of the instability of oxycelluloses toward alkali.1E1a2The “copper number”168-168b of oxycelluloses gives an empirical measure of reducing power. which Oxycellulose and phenylhydrazine gave a yellow formed a diphenylformazan,lm showing that the oxycellulose had reacted in one of the two possible hemiacetal forms. Aminophenols and oxycellulose gave derivatives which coupled with diazonium compounds, enabling chemically colored fibers to be prepared.lE4Reduction of oxycellulose oxime with lithium aluminum hydride, sodium borohydride, or sodium amalgam gave an “amino-oxycellulose” (109) in which up to 25 % of the oxime groups had been reduced.lE6 CH,OH

(109)

The reaction of diazomethane with oxycelluloses has been studied166-16R as a possible means of reducing their alkali lability. Head’” has shown that diazomethane in ether does not react with oxycelluloses unless water is also present, presumably to increase accessibility. Oxycellulose (D.O. 93 %) reacted to give a product with only one methoxy group per dialdehyde unit, and yet the copper number was almost zero (showing that all the aldehyde groups had been blocked). Structures containing epoxide rings (diazomethane can react with aldehyde groups to give epoxides16D),for example (1lo), or methylated double hemiacetals derived from two adjacent dialdehyde units, such as (lll),were proposed in order to account for the above prop(161) E. Pacsu, Teztile Research J., 16, 106 (1946). (162) R. L. Colbran and T. P. Nevell, J. Teztile Znst., 49, T333 (1968). (163) C. G. Schwalbe, Bet., 40, 1347 (1907). (163a) T. F. Heyes, J. SOC.Chem. Znd. (London), 47, 90T (1928). (163b) Tappi Standard Methods, T 216 m-60 (1960). (184) 2. A. Rogovin, A. C.Yaehunskaya and B. M. Bogoslovski, J . Appl. Chem. U.S.S.R. (English Translation), 33, 666 (1960). (166) Y. S. Koalova and Z. A. Rogovin, Vysokomolekulyarnye Soedineniya Vsesoyuz. Khim. Obshchestvo im. D . Z. Mendeleeva, 3 , 614 (1960), Chem. Abstracts, 56, 6392 (1961). (166) R.E. Reeves, Ind. Eng. Chem., 36, 1281 (1943). (167) R. E. Reeves and F. Darby, Jr., Teztile Research J . , 20. 172 (1960). (168) F. S. H. Head, J . Teztile Zmt., 43, T1 (1962). (169) B. Eistert, “Newer Methods of Preparative Organic Chemistry,” Interscience Publishers, Inc., New York, N. Y., 1948, p. 613.

PERIODATE-OXIDIZEDCARBOHYDRATE8

(111)

149

(112)

erties; the presence of epoxide rings in the products was not tested for. Double hemiacetals within the same dialdehyde unit, for example (112), were not considered. The reaction of diazomethane with periodate-oxidized monosaccharide derivatives would make an interesting study. Sodium borohydride reduces oxycelluloses to the corresponding polyhydric alcohols,11bJ70-17* which are almost completely stable toward alka1i.l7OJr1 The influence of pH, temperature, and concentration of the reactants have been investigated.171 Hydrolysis of the reduced oxycellulose gave glycolaldehyde and erythrito1.l" Attempts were made to reduce oxycelluloses with aluminum isopropoxide, with zinc and acetic acid, and with hydrogen over a platinum oxide catalyst; none of the products showed any decrease in alkali sensitivity and so, presumably, no reaction had occurred.16' Jayme and Maris"' hydrogenated oxycellulose in the presence of h n e y nickel to give a product which was hydrolyzed to erythritol and glyoxal, suggesting that partial reduction of the hemiacetal form (103)had occurred. The hydrogenation mixture was made alkaline with barium hydroxide, so it is possible that some degradation of the oxycellulose occurred. (170) A. Meller, Chem. & Ind. (London), 1204 (1963). (171) F. 8.H. Head, J . Teztile Inst., 46, T400 (1966). (172) I. J. Goldatein, J. K. Hamilton, R. Montgomery snd F. Smith, J . Am. Chem. Sac., 79, 8469 (1967). (173) N. Virkola, Papen'ja Puu, 40,367 (1968). (174) G. Jayme and 8.Maria, Ber., TI, 383 (1944).

R. D. GUTHRIE

150

CHPOH

AH,OH

CH,OH

(113)

p

B

r

AJ

CO,H

~

CH,OH

~

H

HCOH HTOH CH,OH I

o

+

Hr H =O

,

~

The same workers17*oxidized oxycellulose with bromine water, to give a product which was hydrolyzed to D-erythronic acid and glyoxal, again suggesting that oxycellulose reacted preferentially in the hemiacetal form (113). This behavior is in contrast to Pacsu's findingsl7'; he isolated glyoxylic acid after hydrolysis of an oxycellulose which had been further oxidized with bromine in bicarbonate solution. Oxidation of oxycelluloses with chlorous acid showed that one aldehyde group was oxidized more rapidly than the 0ther.l7~The polydibasic acids from complete oxidation of oxycelluloses with the latter reagent have been prepared by several groups of workers.l761808 It was noted that oxidation greatly dimiihea the alkali instability of 0xycelluloses.~7~--'7~ Nitrogen dioxide oxidation of oxycellulose gave181 the polytribasic acid (114). Oxycelluloee was degraded by 10 % methanolic hydrogen chloride to give the same products as oxystarch.l* Reaction with 0.4% methanolic hydrogen chloride introduced up to 25% of methoxyl, but, with ethanolic hydrogen chloride, only 3 % of ethoxyl was formedz8;this behavior is probably attributable to the different sizes of the reagents. Oxycelluloses conlB0

(176)E.Pacsu, Testile Research J . , 16, 364 (1946). (176)G.F. Davidson and T.P. Nevell, J . Testile Inst., 46, T407 (1966). (177) H.A. Rutherford, F. W. Minor, A. R. Martin and M. Harris, J . Research Natl. Bur. Standards, 89, 131 (1942). (178) A. G. Yashunskaya, N. N. Shorygina and Z. A. Rogovin, Zhur. Pm'klad. Khim., 28, 1037 (1849); Chem. Abstracts, 46, 3692 (1961). (179) W. K.Wilson and A. A. Padgett, Tappi, 98,292 (1966). (180) G. M. Nabar and C. V. Padmanabhan, Proc. Indian Acad. Sci., S A , 212 (1960). (180a) B. T. Hofreiter, I. A. Wolff and C. L. Mehltretter, U. 5. Pat. 2,894,946 (1969);chem. Abstracts, M, 2794 (1960). (181)V. I. Ivanov, N. Y. Lenshina and V. S. Ivanova, Doklady Akad. Nauk 8.S. 8.R., 119,326 (1969); Chem. Abstracts, M, 8664 (1960).

PERIODATE-OXIDIZED CARBOHYDRATES

151

COZH

(114)

taining 2.1-2.6 % of methoxyl have been prepared by use of 1% methanolic hydrogen chlorideB2;the products showed a diminished solubility in acetone, suggesting that cross-linking had occurred. A small proportion of methanol was retained after oxycellulose was washed with this solvent and exhaustively dried.18*This retention was attributed to the formation of a “polydialdehyde methanolate,” similar to those obtained with simple monosaccharide derivatives. The proportion of methanol retained is shown TABLEI Reaction of Ozycelluloses with Methanol OaMn1

% ’

Moles of MeOH pn

Chain m i l s r&ing wiCh MeOH, %

5.1 10.7 22.2

23

100 chain-unils

18.2 48.6 92.7

22 24

in Table I; the constant percentage of reacted chain units suggests an equilibrium, under these conditions, between about 25% of hemialdal groups and 75 % of other groups, probably hemiacetals. Nitrogen dioxide oxycellulose of a high D.O. also showed this phenomen0n.~~J8* Davidson”JgJ~~prepared nitrate esters from oxycellulose and noticed that, above about 2 % D.O., the products were insoluble in acetone; this observation has been confirrned.l8* The explanation put forwardlE2to account for the diminished solubility was that formation of hemiacetal cross-links occurred in the presence of anhydrous, strong acids. An alternative explanation is that the cross-links were already present in the oxycellulose and that J

~

~

-

~

~

~

(la) Z. A. Rogovin, A. G. Yashunskaya and N. N. Shorygina, Zhur. Priklad. Khim., 22, 866 (1949);Chem. Abstracts, 44, 835 (1960). (183)T.P.Nevell, Chem. & Ind. (London), 389 (1958). (184)T.P.Nevell, J . Feztile Inat., 42, T91 (1951). (185)H.Haas, E.Battenberg and D. Teves, Tappi, 36, 116 (1952). (186)Z. A. Rogovin, A. G. Yashunskaya and N. N. Shorygina, Zhur. Pn’klad. Khim., 22, 857 (1949);Chem. Abstracts, 44, 835 (1950). (187)B. Anthoni, Paperi j a Puu, 38, 504 (1956); Dissertation, Meden, Helsinki (1958).

152

R. D. QUTHRIEl

nitration stabilized them.l@ The physical properties of nitrated oxycelluloses have been studied in some Acetylation of oxycellulose gave a triacetate that, again, appeared to be cross-linked, and for which structure (115) was suggestedU* structures (116) and (117) would also be possible for the triacetate.

45-b $yP\Q\ EC CIi,OAc

CHOAc Acd%AC (115)

CHOAC

dAc

A C A C (116)

(117)

4. From Xylun Xyhn was oxidbed by Jayme and his coworkers1@,'88J8@ in a buffered solution; hydrolysis1"*W-1@0 of the product gave glyceroae (67 %) (as pyruThe devaldehydem) and glyoxal(62 %), as the bis(phenylhydraz0ne).l@J8@ gree of oxidation of the oxyxylan was calculated from the nitrogen content of the orange-yellow phenylhydrazine derivative, which was assumed to be the poly[bis(phenylhydrazone)l.'sg It has been recorded,@6however, that oxyxylan reacted with less than one molecule of phenylhydrazine per dialdehyde unit. Degradation of oxyxylan with phenylhydrazine-acetic acid gave glyoxal bis(phenylhydrazone),glycerosazone,and D-xylosazone.@6 I t has been averred that the oxyxylan-phenylhydrazine derivative formed a poly(diphenylforma~an)~*o; this olaim has been criticized'@'on the grounds that, unlike oxycellulose or oxystarch, oxyxylan could not form a hemiacetal, and, hence, there should be no free aldehyde groups to form true phenylhydrazones that would yield formazans. Hydrogenation of oxyxylan in the presence of Raney nickel, followed by hydrolysis, gave glycerol and glycolaldehydel@,lE2 Jg8; alternate reduction and hydrolysis gave glycerol and ethylene g1yco1.1"J@8Oxidation of oxyxylan with bromine water, followed by hydrolysis, gave D-glyceronic acid (isolated as the barium or brucine salts).'" #lea (188) G. Jayme and M. Btre, Ber., 76, 1840 (1942). (189) G. Jayme and M. &itre, Ber., 77,242 (1944). (190) M. Hamada and K. Maekawa, J . Fac. Agr. Kyuehu Uniu., 9, 311 (1950); Chem. Abstracte, 48,2602 (1964). (191) V. C. Barry and P. W. D. Mitchell, Chem. & Ind. (London), 1046 (1967). (192) I. Ehrenthal, R. Montgomery and F. Smith, J . Am. Chem. Soc., 76, 5609 (1964). (193) G. Jayme and M. Btre, Ber., 77,248 (1944).

PERIODATE-OXIDIZED CARBOHYDRATES

153

5. From Other Polysaccha&es

Oxyinulin has been hydrolyzed to yield glycerose. Oxidation of oxyinulin followed by hydrolysis gave glyceronic acid; reduction and hydrolysis gave g l y c e r i t ~ l Oxyinulin .~~~ and phenylhydrazine gave a yellow product which contained one phenylhydrazine group per dialdehyde unit,g6and which formed a formazan.l’O Isonicotinoylhydrazineg and paminobenzaldehyde thiosemicarbazonelsgalso reacted, to give products with one basic group per dialdehyde unit. Alginic acid was oxidized in the course of proof of its structure.l96 Hydrolysis of oxyalginic acid gave glyoxal, and, after further oxidation, it gave erythraric acid.l96 Oxyalginic acid reacted with isonicotinoylhydrazineg and p-aminobenzaldehyde thiosemicarbazonelsgto give products with one basic group per dialdehyde unit. The former compound was hydrolyzed with 50 % acetic acid to the corresponding glyoxal bis(hydrazone), but hydrolysis in the presence of phenylhydrazine or cyclohexylamine gave the glyoxal derivative of the added baseg; (compare oxystarch, p. 145). Oxydextran showed no carbonyl absorption in the infrared or ultraviolet spectra; hydration of the aldehyde groups was assumed.lg6Many other polysaccharides have been oxidized with periodate,6 but only in purely structural work; and no reactions have been studied (other than hydrolysis of the oxypolysaccharide or its oxidation or reduction products). OF PERIODATE-OXIDIZED CARBOHYDRATES VII. ALKALINE DEGRADATION The degradation by alkali of oxycelluloses containing carbonyl groups is of great importance in industries based on cellulose (for example, textiles and paper). A great deal of work has been carried out on periodate-oxidized cellulose as a typical example of these carbonyl oxycelluloses.196aThe majority of the work on periodate-oxidized monosaccharide derivatives has been carried out as a model for the oxycellulose system. The alkali lability of oxycelluloses was first observed by D a ~ i ds o n ,l O~J 4who ~J ~noted that, with as low as 2 % D.O., there was great alkali sensitivity. The characteristic feature of the degradation is the production of acidic fragments.lW Oxidized methyl a-L-rhamnopyranoside (118) undergoes a Cannizzaro

(194) K. Maekawa and T.Nakajima, Nippon Ndgei-kagaku Kaishi, 28,357 (1954); Chem. Abstracts, 48, 10078 (1954). (196) H. J. Lucas and W. T.Stewart, J . A m . Chem. SOC.,62, 1792 (1940). (196) J. W.Sloan, B. H. Alexander, R. L. Lohmar, I. A. Wolff and C. E. Rist, J . Am. Chem. SOC.,76, 4429 (1954). (196a) W. M. Corbett in ‘‘Recent Advances in the Chemistry of Cellulose and Starch,” J. Honeyman, ed., Heywood & Co., Ltd., London, 1969, p. 106. (197) G . F. Davidson, J . Testile Inst., I D , T196 (1938). (198) G . F. Davidson and T.P. Nevell, J . Testile Inst., 99, T102 (1948).

154

R. D. GUTHRIE

Me

(118)

Me

(119)

reaction in alkali, with the formation of a mixture of monobasic acids (119),’9nso it is possible that such a reaction might occur with oxycelluloses. Several theories have been proposed for the alkaline degradation of oxycelluloses; of these theories, two have predominated. Pacsu176 considered oxycellulose to be a hydroxyketene acetal(l20) ;degradation of such a compound would give glycolic acid and D-erythrose as the primary products. CHpOH

( 120)

However, Head200showed that glyoxal is produced by the treatment of both oxycellulose and periodate-oxidized methyl B-cellobioside (121) with alkali. The other theory was that of Haskins and Hogshead,2O1 based on the j3alkoxycarbonyl elimination mechanism of Isbell2O2for the formation of saccharinic acids on treatment of sugars with alkali. They suggested that fission of the C5-0 bond would yield glyoxal and D-erythrose. Recent work has indicated that the degradation is, indeed, based on a 8-alkoxycarbonyl elimination, but the products are different from those postulated above. Headloo showed that, whereas periodate-oxidized methyl j3-cellobioside (121) is quite labile to alkali and gives glyoxal, periodate-oxidized methyl j3-D-glucoside (122) reacts only slowly with alkali to produce acidic products,

j=oy

o

=

~

~

a CcH

HC=O ~

(199) E.M. Fry, E. J. Wileon and C. S. Hudson, J . Am. Chem. Soc., 64,872 (1942). (Zoo) F. S. H. Head, J . Teztile Znst., 88, T389 (1947). (201) J. F. Haskins and M. J. Hogsheed, J . Org.Chem., 16, 1204 (1950). (202) H. S. Isbell, J . Research Natl. Bur. Standards, 81, 46 (1944).

c

155

PERIODATE-OXIDIZED CARBOHYDRATES

presumably by a Cannizzaro reaction. Inspection of the formulas (121)and (122) reveals that the former contains both a- and /3-alkoxycarbonyl systems, whereas the latter has only an a-system. Alkaline degradation of periodate-oxidized methyl 4,6-O-benzylidene-aD-glucoside (123) with lime-water gave equal amounts of glycolic acid and 4-formyl-2-phenyl-2H,6H-1,3-dioxin (124), as well as a mixture of acids

7e~o~----(~hp

ph<(-),

HCOe

HC=OMe

O=CH

OH"

HC=O

0-

O=CH

HC=O

~H,OH

I

CH,OH

(125)

Hk=O

CH,OH

I

C0,H

(in small yield) resulting from an internal Cannizzaro reaction; no evidence for intermolecular reaction was found.*03The dioxin compound (124) was identified by acid hydrolysis, followed by treatment with lime-water to (203) D. O'Meara and G . N. Richards, J . Chem. SOC.,1204 (1958).

156

R. D. QUTHRIE

give a 2 ,4-dihydroxybutyric acid (a 3-deoxy-g2ycero-tetronicacid) (125). The debenzylidenated compound (126) was also treated with lime-water and gave equal amounts of glycolic acid and 2,4-dihydroxybutyric acid. The glycolic acid in both the above cases could have been formed by Pacsu's mechanism1176 instead of from the primary product, glyoxal. The dioxin compound and the 2,4-dihydroxybutyric acid could have been formed only by a p-alkoxycarbonyl mechanism as shown above. Degradation of oxycellulose gave glycolic acid and 2 ,4-dihydroxybutyric acid as the main products,lo' suggesting that it was broken down in the same way as the model compounds. Fragment (127), which contains an a-hydroxycarbonyl group, would not be expected to undergo further rapid alkaline degradation. The greater the degree of oxidation, the greater would be the number of units of the type (128) formed, giving more 2,4-dihydroxybutyric acid; this behavior was found in practice. The above mechanism accounts for much less than the theoretical quantity of products; other mechanisms are probably at work simultaneously, but that of p-alkoxycarbony1 elimination appears to be the main pathway. Similar studies have been carried out on the alkaline degradation of oxystarch,2°6oxyxylan, and oxydextran.*06* An interesting observation was made by Davidson and NevelP when studying the cuprammonium fluidity of oxycelluloses partially oxidized with chlorous acid, namely, that the more-easily oxidized aldehyde group contributed more to the alkali lability. This is, in fact, further evidence for the existence of the hemiacetal with a six-membered ring (113); this has a 8-alkoxycarbonylgroup and, from the work of Jayme and Maris,17' contains the more-easily oxidiaed aldehyde group. Much work has been performed on oxycelluloses in an effort to lessen their alkali-lability, Reduction,170,171 oxidation with chlorous acid,loe 3 7 ~ 7 6 * ~ 180,206 and reaction with diazomethane1e6*1w have been found t o be effective. Mild treatment of periodate-oxidized adenosine 5-phosphate or adenosine 5-(benzyl hydrogen phosphate) with alkali gave adenine and inorganic phosphate or benzyl pho~phate.2~7-2~ Periodate oxidation followed by alkali treatment would, therefore, be a means of degrading polynucleotides stepwise.2°7*208 A dinucleotide has been degraded in this way.200 (204) D. O'Meara and G. N. Riohards, J . Chem. Soc., 4604 (1968). (206) R. L. Whistler, P. K. Chang and G. N. Richards, J . Am. Chem. Soc., 81. 3133 (1969). (2088) R. L. Whistler, P. K. Chang and G. N. Richarde, J . Am. Chem. Soc., 81, 4068 (1969). (Zoe) A. Meller, Tappi, #, 171 (1961). (207)D. M. Brown,M. Fried and A. R. Todd, Chem. & Znd. (London), 362 (1963). (ZOS) D. M. Brown, M. Fried and A. R. Todd, J . Chem. Soc., 2208 (1966). ('209) P. R. Whitefield and R. Markham, Nature, 171,1161 (1963).

~ 7 ~

167

PERIODATE-OXIDIZED CARBOHYDRATES

CkCH

OH

HC=O

O=CH

HC=O

OH

I

t FH,OH

CH,OH

I

YHOH I

CO,H

OH0

CHIOH

g% I

H =O

1

HC=O

+ Hd=O HL=O

OH"_

HO

I

bH

OH0

p z

VIII. USESOF PERIODATE-OXIDIZED CARBOHYDRATES The uses of periodate-oxidized carbohydrates are thus far confined to uses for oxycelluloses and, more particularly, oxystarches ("dialdehyde starches"). Following the development of the electrolytic regenerative process for periodic oxystarch has been produced commercially in the United States by the Miles Chemical Company of Elkhart, Indiana. The earliest claim, before the development of the electrolytic process, was for the use of oxystarch as a textile sizing-agent; because of its alkali-lability, it could easily be removed from the yarn.21° Another use in textiles is for a highly alkali-soluble lace-backing from periodate-oxidized, cyanoethylated-cellulose textiles.211Because of its aldehydic character, oxystarch has been claimed as a useful tanning agent."2-214 Oxystarches (D.O. 40(210) W. C. E. Yelland, U. S. Pat. 2,606,188 (1962); Chem. Abstracts, 47,338 (1953). (211) H. Weisberg, U. S. Pat. 2,724,632 (1966); Chem. Abstracts, 60,8062 (1956). (212) M. L. Fein and E. M. Filachione, J . Am. Leather Chemists' Assoc., 62, 17 (1967).

158

R. D. GUTHRIE

100 %) can be molded to give translucent to semi-transparent articles.216 Other uses are in the preparation of tobacco sheets having great wet strength,l16and as a tub-sizing agent in paper man~facture.2~7 Methods for the preparation of water-soluble oxystarches by reaction with bisulfites have been Derivatives of oxystarch with isonicotinoylhydrazine (isoniazid), p aminobenzaldehyde thiosemicarbazone, and thiosemicarbazide have been claimed as agents for combatting t u b e r ~ u l o s i s . 2 The ~ ~ - ~bases ~ have been used singly or in combination. The thiosemicarbazide-oxystarch polymer has been found to be highly effective against isoniazid-resistant bacteria. Similar derivatives of oxyinulin and oxyalginic acid have been t e ~ t e d . 2 ~ ~ Reaction of oxidized cotton-duck of very low D.O. with phenylhydrazine was found to give a rot-proof material which was, unfortunately, yellow.n2 Oxidized paper of very low D.O. has been treated with Girard P reagent to yield paper of special usefulness in electrophoresis?*' Periodate-oxidized paper (no D.O. stated) has been claimed to have a greater wet-strength than untreated ~aper.2~' (213)E.M. Filachione, E. H. Harris, M. L. Fein, A. H. Korn, J. Naghski and P. A. Wells, J . A m . Leather Chemists' Aasoc., MI, 77 (1958). (214)P. A. Wells, E. M. Filachione and M. L. Fein, U. S. Pat. 2,886,401 (1959); Chem. Abatracts, MI, 16571 (1959). (216) R. L.Mellies and I. A. Wolff, U. S. Pat. 2,788,546(1957);Chem. Abstracts, 61, 10104 (1967). (216) S . Rosenberg and D. Bandel, U. 8. Pat. 2,887,414(1959);Chem. Abstracts, MI, 13620 (1959). (217) E.J. Jones, B. Wabers, J. W. Swanson, C. L. Mehltretter and F. It. Senti, Tappi, 43,862 (1969). 218) C. L. Mehltretter, J. W. Van Cleve and P. R. Watson, U. S. Pat. 2,880,238 (1969);Chem. Abstracts, MI, 127'20 (1969). (219) V. C. Barry, M. L. Conalty and E. E. Gaffney, Brit. J . Urol., 27, 35 (1956). (220) V. C. Barry, U.S. Pat. 2,837,W (1968);Chem. Abstracts, 61, 16239 (1958). (221) V. C. Barry and P. W. D. Mitchell, U. 8. Pat. 2,886,394(1969);Chem. Abstracts, 6S, 13638 (1959). (222) R. Thomaa, Teztile Research J . , 16, 669 (1966). (223) M. A. Jermyn and R. Thomaa, Nature, 171,728 (1053). (224) I. Yoshino, Sen4 Gakkaishi, 10,97 (1954);Chem. Abstracts, 61, 15124 (1957).

LACTOSE BY JOHNR. CLAMPAND L. HOUOH, Department of Chemistry, The University, Bristol, England

JOHNL. HICKSON, Sugar Research Foundation, Znc., 68 Wall Street, New York 6, N . AND

Y

ROY L. WHISTLER

Department of Biochemistry, Purdue University, Lafayette, Indiana

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

. . . . . . . . . . . . . . . . 160

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

..........

. . . . . . . . . . . . 167

IV. Chemical Proper

7. Thioacetals. ..............................................

.................... 198 11. Unsaturated Derivatives. ..................................... V. Some Physical Properties of Lactose. . . . . . . . . . . . . . . 201

I. INTRODUCTION The isolation of lactose was first described in the seventeenth century, when it was termed “milk sugar” after its source. Although other claims1*2 have been put forward, Whittiera attributed the discovery of lactose to Bartolettus,4 who, in 1633, isolated an “essential salt without nitrogen” (1) M. Nierenstein, Isis, 24, 367 (1936). (2) E. 0. von Lippmann, “Geschichte des Zuckers,” M. Hesse, Leipzig, 1929, p. 688. (3) E. 0. Whittier, Chem. Revs., 2,85 (1926); J . Dairy Sci., 27,505 (1944). (4) F. Bartolettus, “Methodus in Dyspnoeam seu de Respirationibus,” Libri V, Bononiae, 1633, p. 4-00. 159

160

CLAMP, HOUGH, HICRSON AND WHISTLER

from whey, the liquid remaining after the coagulation of milk. A more scientific preparation was described in 1688 by Ettmiiller,6 who isolated crude lactose from evaporated whey and purified it by recrystallization. Whilst there have been some reports of its presence in plants, lactose is largely a product of mammalian metabolism, being synthesized by the female and secreted in her milk. Since it can be so readily isolated in pure form from this source, a great deal of the fundamental knowledge about carbohydrates has been obtained from its study. Thus, the first research problem of C. S. Hudson was, at his own request, a physicochemical study of the mutarotation of milk sugar, a phenomenon that was not understood at that time. His first scientific paper, published in 1902, at the age of 21, was on the five forms of milk sugar. It is appropriate that, forty years later, the first chemical synthesis of lactose should also have been achieved by him. Lactose had been a recurring theme in his lie, and we should like to dedicate this review to his memory. Many of the methods that are now employed for the determination of hemi-acetal ring structures, and of the linkages present in oligo- and polysaccharides, were developed from their original application to lactose. Recently, interest in the properties of lactose has been stimulated by its importance in certain biological problems. Investigations into the metabolism of D-galactose have resulted in the elucidation of new enzymic pathways involving uridine coenzymes, and this has led to a knowledge of the route by which lactose arises. The realization that lactose can also occur as a component of certain oligosaccharides of milk has presented fresh problems concerning their structure and chemical synthesis. The reader is referred to a number of reviewss+9 for further details of the extensive literature on this carbohydrate, to supplement those aspects covered in this review.

11. THESTRUCTURE OF LACTOSE In 1812, Vogello identified glucose as a product of acid hydrolysis of lactose and, in 1855, Erdmann’l detected another component, which was shown by Pasteur12to be different from glucose. After its crystallization1*in 1856, this component was named galactose.lSFischer14established the con(5) M. Ettrnuller, “Opera Omnia,” Bd. 11, Frankfurt, 1688, p. 163. (6) P. Wagnet, Rev. prods. chim., 18, 5, 62, 116 (1920). (7) P. F. Sharp, Milk Plant Monthly, 19, No. 8 , 46 (1930). (8) E. Gaaaer, Monthly Bull. Agr. Sci. Pract., 11, No. 4, 135 (1931). (9) G. Cornalba, Boll. chim. furm.,71, 916 (1933). (10) H.A. Vogel, Ann. Physik, 4% 129 (1812). (11) E. 0. Erdmann, Juhrssber. Chem., 671 (1856). (12) L. Paateur, Compt. rend., 41, 347 (1868). (13) H. Fudakowski, Ber., 9. 43, 278, 1602 (1876). (14) E. Fiacher, Ber., 14, 1836, 2683 (1891).

161

LACTOSE

figuration of D-galactose and of D-glucose in 1891 and, on this basis, 0st16 confirmed the characterization of these two monosaccharides as components of lactose. By heating lactose with aqueous phenylhydrazine, Fischerl6 prepared a crystalline phenylosazone derivative which was converted into lactosone by means of fuming hydrochloric acid. Hydrolysis of this derivative with hot, dilute acid gave D-glucosone (D-arabino-hexosulose) and D-galactose. This evidence showed that, in lactose, D-galactose is linked by its reducing group to D-glucose and that the hydroxyl groups a t C-1 and C-2 of the latter are unsubstituted. Verification of this O-D-galactosyl-D-glucose structure was obtained by bromine oxidation of the reducing group of lactose to give lactobionic acid, followed by the isolation of D-galactose and D-gluconic acid from a hydrolyzate of this acid.” The configuration of the glycosidic bond at C-1 of the D-galactosyl residue was investigatedls by the use of a specific enzyme, now known as 8-D-galactosidase but originally termed lactase, which catalyzes the hydrolysis of methyl 8-D-galactopyranoside (1; R = Me), at that time thought to be a furanoside, to give methanol and D-galactose (2) ; the a anomer is not hydrolyzed. Since this enzyme hydrolyzes lactose (1; R = D-glucose), FischerIBconcluded that the inter-unit linkage is of the B-D configuration. CH,OH

CH,OH

OH

bH (1)

(2)

The attachment to the D-glucose residue was assigned to the hydroxyl group at either C-4 or C-5 by the identification of a tetra-0-methyl-Dgalactose, later recognized as the 2,3,4,64somer (3), and 2,3,6-tri-0methyl-D-glucose (4) in the hydrolysis products of a crystalline, fully methylated lactose.2O These results were verified by Irvine and Hirst21 and by Schlubach and H. Oat, Ber., Is, 3003 (1890). E. Fischer, Ber., 20, 821 (1887); 21, 2631 (1888). E. Fischer and J. Meyer, Ber., 22, 361 (1889). E. Fischer, Ber., 27, 2985, 3479 (1894). E. Fischer, Ber., 28, 1429 (1896). (20) W. N. Haworth and G . C. Leitch, J . Chem. Soc., 119,188 (1918); W. Charlton, W. N. Haworth and S. Peat, ibid., 89 (1926); W. N. Haworth, J. V. Loach and C. W. Long, ibid., 3146 (1927); W. N. Haworth and E. L. Hirst, ibid., 2616 (1930). (21) J. C. Irvine and E. L. Hirst, J . Chem. Soc., 121, 1213 (1922). (Z) H. H. Schlubach and K. Moog, Ber., 66, 1967 (1923). (16) (16) (17) (18) (19)

162

CLAMP, HOUQH, HICKSON AND WHIBTLER CH,OMe

bMe (3)

(4)

Prior to the late 1920's, it was thought that lactose, in common with other sugars, contained furanoid ring-structures, and this belief was erroneously supported by evidence derived largely from the influence of lactose on the conductance of boric acid solutions28and from molecular r0tations.2~More reliable evidence, however, led to the conclusion that both monosaccharide units in lactose have pyranoid structures. Several investigators solved this problem independently. Zemp16n2bdegraded lactose to O-p-D-galactopyranof3yl-D-arabinose by the simultaneous acetylation and dehydration of lactose oxime to an acetylated nitrile, followed by treatment with sodium methoxide in chloroform. R-CH(0H)-CH=NOH

R-CH(0Ac)-CzN 0 R-AH

NaOEt

+ NaOAc + NaCN

Repetition of this procedure on the galactosyl-arabinosegave O-j3-D-galactopyranosyl-D-erythrose (6) which did not give a phenylosazone. Hence, it was suggested that a B-D-(~ 4)-linkage is present in lactose (5). Conclusive evidence for this structure was also obtained by the following. 1. The isolation of tetra-0-methyl-D-glucono-l,4-lactone (8) from a hydrolyzate of the octa-0-methyllactobionic acid (7; R = Me).26 2. The formation of lactobiono-l,5-lactone (9) from lactobionic acid (7;R = H), the glycosidic linkage preventing its conversion to the more stable 1 , 4 - la ~ t o n e . ~ 3. By degradation of the D-glucose residue of lactose, with the removal of C-1 and C-2 to give 2-O-~-~-galactopyranosyl-~-erythronic acid, which could not be degraded any further without the liberation of D-galactose.% (23) (1942). (24) (26) (26) (27)

R. Vershuur, Thesis, Delft, 1926, p. 85;J. Baeseken, Rec. trau. chim., 81, 85

C. 8. Hudson, J . A m . Chem. SOC.,62, 1707 (1930). G. ZemplBn, Ber., 69, 2402 (1926); 60, 1308 (1927). W. N . Haworth and C. W. Long, J . Chem. Soc., 644 (1927). P. A. Levene and H. Sobotka, J . Biol. Chem., 71, 471 (1927); P. A. Levene and 0. Wintersteiner, ibid., 76, 316 (1927). (28)A. M. Gakhokidze, Zhur. Obehchet Khim., 20,120 (1960); Chem. Abslracts, 44, 6819 (1960).

LACTOSE

163

CH,OH

CH,OH

( 9)

The structure of lactose was, therefore, firmly established as 4-0-/3-~galactopyranosyl-D-glucopyranose ( 5 ) . It is of interest that, in 1882, lime-water treatment of lactose was found to yield29 the insoluble calcium “a”-D-isosaccharinak, a rearrangement product of the D-glucose component, and, in 1896 and 1899, Lobry de Bruyn and Alberda van Ekensteinm observed that treatment of lactose with either lead hydroxide or potassium hydroxide liberated D-galactose. KilianP established the structure of the saccharinate as that of a 3-deoxy2-C-(hydroxymethyl)pentonic acid and, had the mechanism of its formation been understood (see p. 188), this would have been sufficient evidence (29) L. Cuisinier, Monit. sci. Docteur Quesneville, [3] 19, 52 (1882); Bull. soc. chim. (France), [2] 98, 512 (1882). (30) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trav. china., 16, 92 (1896); 18, 147 (1899). (31) H. Kiliani, Ber., 18,631,2514 (1885); H. Kiliani and F. Herold, ibid., 38, 2671 (1905); H. Kiliani and 0. Matthes, ibid., 40, 1238 (1907).

164

CLAMP, HOUQH, HICKSON AND WHISTLER

to identify the (1 + 4)-linkage, prior to the application of the methylation method.sa A simple proof of the (1 4 4)-linkage was obtained by periodate oxidation of 1 mole of lactose phenylosazone, which gave 1mole of formaldehyde but no insoluble mesoxaldehyde 1,2-bis(phenylhydrazone) (11), a result that is typical of 4-O-substituted hexose phenylosazones (10) and that can be used for their diagnosis on a semi-micro HC-N-NHPh

HC-N-NHPh

h=N-NHPh

h=N-NHPh

HC-N-NHPh

HobH

I

-=+A

H OR

(11)

I

Hd=O

+

HaC=O

Additional proof for the j3-D-linkage in lactose was obtained by degradation to 2-O-j3-~-galactopyranosyl-glyceritol, to which Hudson's rules were then a~plied.~' This selective oxidation was achieved by two successive treatments with lead tetraacetate and sodium borohydride (see Fig. 1). CHIOH I

b=O

H

H D-Galp-0 I HH* O H

4

NaBHa

AHSOH

FIO.1.-Degradation of the D-Glucose Residue of Lactose to a Glyceritol Residue.

The ultimate proof of the lactose structure was provided in 1942 by Haskins, Hann, and Hudson,BK who synthesized lactose and its epimer, (32) (33) (34) (36) (1942).

J. Kenner, Chem. & Ind. (London), 727 (1966). L. Hough, D. B. Powell and B. M. Woods, J . Chem. Soc., 4799 (1966). A. J. Charlson and A. 8. Perlin. Can. J . Chem., 94,1804,1811 (1968). W. T. Haskins, R. M. Hann and C. 8. Hudson, J . A m . Chem.Soc., 64, 1852

165

LACTOSE

4-0-~-~-galactopyranosyl-~-mannose (epilactose) (15). This was the culmination of a lifetime’s research on lactose by C. S. Hudson. The essential step in their synthesis (see Fig. 2) was the Koenigs-Knorr reaction of

P

H&OH

Phenyl j3-Dmannopyranoside

CH,OAc

1,6- Anhydro-p -D

-

rnannopyranose

CqOAc

OAc

OAc

fi -D -Galactopyranoee pentaacetate

FIQ.2.-Haskins, Hann, and Hudson’s Synthesis” of Lactose. (Cont. on p . 166.)

tetra-0-acetyl-cw-D-galactopyranosyl bromide (13) with 1,6-anhydr0-2,30-isopropylidene-D-mannopyranose(12), under rigorously anhydrous conditions, in alcohol-free chloroform with silver oxide and iodine catalyst. Removal of the isopropylidene ring by acid hydrolysis, and subsequent opening of the 1,g-anhydro ring by acetolysis, gave “a-epilactose octaacetate” (14), which was converted into lactose by inversion of the configuration at C-2 through lactal hexaacetate (16). The over-all yield of bromide, was 8 %. lactose, based on the tetra-0-acetyl-a-D-galactopyranosyl Curtis and Jonesas simplified the synthesis of lactose by using 2,3:5,6di-0-isopropylidene-D-glucose diethyl acetal (17) in place of the anhydro compound (12) in the reaction with tetra-0-acetyl-cw-D-galactopyranosyl bromide (13). Subsequent removal of the acetyl groups by alkaline hy(36) E. J. C. Curtis and J. K. N . Jones, Cun.J . Chem., 37,368 (1959).

166

CLAMP, HOUOH, HICKSON AND WHISTLER

(16)

I

(1)

PhcoJi

(2) Ac,O/NaOAc

CHaOAc

OAc where (13) = RBr,

Pro. 2.-Haskins,

8-Lactose octaacetate Ham, uud Huduon’a Synthesisa6 of Lactose. (Con.from p. 166.)

drolysis, and of the isopropylidene residue by acid hydrolysis, gave lactose in 35 % yield.

LACTOSE

167

111. OCCURRENCE AND BIOCHEMICAL PROPERTIES OF LACTOSE 1. Occurrence

Lactose occurs in milk, either free or in the form of lactose-containing oligosaccharides. The concentration of free lactose may vary from 2 to 8.5% (wt./vol.) depending on the mammal,s cows’ milk containing approximately 4.5 % and human milk” 6 to 7 %. Variations also occur within a particular species, as a result of age, season, stage of lactation, and heredity .a The lactose-containing oligosaccharides of human milk have been investigated recently as a result of their association with the “bifidus factor,” a growth factor for Lactobacillus bimus, and reports on their concentration have varied from3B0.3 % to400.6 %. A number of reviews41have now appeared on the subject. It has been known42since 1900 that differences exist between the intestinal flora of breast-fed babies and those fed on cows’ milk, the flora of the former being composed almost exclusively of L. bifidus. In 1926, it was shown43that a growth factor for L. bimus occurs, associated with the lactose fraction in whey, in human milk, but it was not found in cream, in the proteins, or in the inorganic material. Later the nutritional requirements o f L. bifidus were rein~estigated~~ and, in the course of this work, there was isolated, from the feces of infants, a variant of L. bi$dus which gave only a scanty growth on the normal media, but which responded to the addition of human milk.46,46 This variant was named Lactobacillus bifidus var. pennsyl(37) M. Bell, J . Biol. Chem., 80, 239 (1928). (38) R. Waite, J. C. D., White and A. Robertson, J . Dairy Research, 23, 65, 82 (1956). (39) R. Kuhn, H. H. Baer and A. Gauhe, Chem. Ber., 88, 1135 (1955). (40) F. H. Malpress and F. E. Hytten, Biochem. J . , 68,708 (1958). (41) (a) D. J. Bell, Ann. Repts. on Progr. Chem. (Chem. Soc. London), 62,333 (1955). (b) R. Kuhn, Bull. S O C . chim. biol., 40,297 (1958). (c) P. Gyorgy, Ciba Foundation Symposium, Chemistry and Biology of Mucopolysaccharides, 140 (1958); J. Montreuil, Bull. SOC. chim. biol., 43, 1399 (1960). (42) H. Tissier, “Recherches sur la flore intestinale des nourrissons. (fitat normal et pathologique),” Thesis, G. Carre et C. Naud, Paris, 1900, Ann. M6d. Chir. infant, 4, 675 (1900); E. Moro, Wien. klin. Wochschr., 13, 114 (1900); Jahrb. Kinderheilk., 2, 38 (19ao). (43) H. Schonfeld, Jahrb. Kinderheilk., 63, 19 (1926). (44) R. F. Norris, T. Flanders, R. M. Tomarelli and P. Gyorgy, J . Bacleriol., 60, 881 (1950).

(46) P. Gyorgy, R. Kuhn, R. F. Norris, C. S. Rose and F. Zilliken, A m . J . Diseases Children, 84, 482 (1952). (46) P. Gyorgy, R. F. Norris and C. 5. Rose, Arch. Biochem. Biophys., 48, 193 (1954); P. Gyorgy and C. S. Rose, J . Bacteriol., 69,483 (1955).

168

CLAMP, HOUQH, HICKBON AND WHISTLER

vanim (L. bi@ue var. penn.). Attempts to isolate this L. ba@us growthfactor led to the recognition of the oligosaccharides of human milk, the bifidus factor being associated with those containing nitrogen." Polonovski and LespagnoP had previously found that human milk, unlike cows' milk, contains two carbohydrates other than lactose, to which they gave the names allolactose and gynolactose. Allolactose (6-O-~-~-galactopyranosylD-glucose) has since been synthesized chemi~ally~~ and found to have the same properties as the disaccharide originally isolated. However, its presence in milk has never been confirmed,"' and, since it can be ensymically synthesized by a 8-D-galactoaidase from E. coli, its presence might have been attributable to contamination by coliform organisms."(b)*W Gynolactose probably corresponds to a mixture of nitrogen-containing oligosaccharidea." Three types of lactose-containing oligosaccharides are present in human milk,"(b) namely, (a) those containing no nitrogen (fucosyl-lactose, lactodifucotetraose), (b) those containing 2-acetamido-2-deoxy-~-glucose (lacto-N-tetraose, lacto-N-fucopentaose I and 11, lacto-N-difucohexaose, and a series of higher saccharides), and (c) the sialic acid-containing oligosaccharides. The fucose referred to is 6-deoxy-~-galactose, Fuco~llactosecomprises about 10% of the total oligosaccharides and its m 0-a-L-fucopyranosyl-(1 4 2)-0-/3-~structure has been established ma@ galactopyranosyL(1 .--) 4)-~-glucopyranose(18). There is evidence, also, that another fucose derivative of lactose exists."(')

(18)

Fucosyllactose

Lacto-difucotetraose(less than 1% of the total oligosaccharides) is a (47) A. Gauhe, P. Gyorgy, J. R.E. Hoover, R. Kuhn, C. S. Rose, H. W. Ruelius and F. Zilliken, Arch. Biochem. Biophys., 46,214 (1964). (48) M. Polonovski and A. Leepagnol, Compt. rend. soc. bioZ., 104, 663 (1930); Compt. rend., 109, 1319 (1931); 1@6,466 (1932); Bull. soc. chim. biol., 16, 320 (1933). (49) B. Helferich and G. Sparmberg, Ber., 66,808 (1933). (4911) A. Leepagnol, J. Montreuil and E. Segard, Compt. rend. aoc. biol., 154. 130 (1880). (SO)

R.Kuhn, H. H. Baer and A. Gauhe, Chem. Ber., 88, 1713 (1966). (6l)(a) J. Montreuil, Compt. rend., 849, 192 (1956). (b) R.Kuhn, H. H. Baer and A. Gauhe, Chem. Ber., 80,2613 (1966).

LACTOSE

169

tetrasaccharide composed of lactose substituted on each of its component hexoses by a fucosyl residue,M,68 and, since these were later established as being a t C-2 of the D-galactose residue and at C-3 of the D-glucose,Mit is 0-a-L-fucopyranosyl-(1 -, 2)-0-~-~-galactopyranosyl-( 1 ---t ~)-[O-~-L-~UCOpyranosyl-(1 --+ 3)]-~-glucopyranose(19). CH,OH

0

HO I

HO (19) Lacto-difucotetraose

Lacto-N-ktraose, being the commonest oligosaccharide of human milk (15 % of the total oligosaccharides), was the first to be isolated,66and iso-

lation was soon followed by the determination of its structure,- which is 0-P-D-galactopyranosyl-( 1 + 3)-0-(2-acetamido-2-deoxy-~-~-glucopyranosy1)-(1---t 3)-0-/3-~-galactopyranosyl-( 1 + 4)-~-glucopyranose(20).

,OH

(20) Lacto-i+tetraose

Lacto-N-fucopentaose I (8% of the total oligosaccharides) is a derivative of lacto-N-tetraose in which the terminal D-galactose residue is substituted at C-2 by an a-L-fucosyl residues8and it has, therefore, been alternatively named monofuco-lacto-N-tetraoseI. The systematic name is O-a-L-fuco(52) M. Polonovski and J. Montreuil, Compt. rend., BE, 2203 (1964). (53) J. Montreuil, Compt. rend., 242,828 (1956). (64) R. Kuhn and A. Gauhe, Ann., 611.249 (1968). (55) R. Kuhn, A. Gauhe and H. H. Baer, Chem. Ber., 88, 827 (1963). (56) R. Kuhn, A. Gauhe and H. H. Bmr, Chem. Ber., 87,289 (1954). (57) R. Kuhn, Angew. Chem., 67, 184 (1955). (68) R. Kuhn and H. H. Baer, Chem. Ber., 89, 504 (1956). (59) R. Kuhn, H. H. Baer and A. Gauhe, Chem. Ber., 89, 2514 (1956).

170

CLAMP, HOUGH, HICKSON AND WHISTLER

pyranosyl - (1 + 2) - 0 - /3 - D - galactopyranosyl - (1 + 3) - 0- (2- acetamido -2deoxy-@-D-glucopyranosyl)- (1 +3) -0-/3-D-galactopyranosyl- (1 44) -Dglucopyranose (21).

f-;>I(>T~=p(=Q

HO

HO

no

,OH

OH

NHAc (21) Lacto-N-fucopentaose

OH

I

Lacto-N-fucopentaose 11 or monofuco-lacto-N-tetraose I1 (4 % of the total oligosaccharides) is also an a-L-fucosyl derivative of lacto-N-tetraose, but, in this case, the substitution is on C-4 of the 2-acetamido-2-deoxy-~glucose residue,E0that is, it is 0-8-D-galactopyranosyl-( 1 + 3)-0-[a-~fucopyranosyl-(1 --+ 4)]-0-(2-acetamido-2-deoxy-~-~-glucopyranosyl)(1 + 3)-0-8-~-galactopyranosyl-( 1 + 4)-~-glucopyranose(22).

H

(22)

Lacto-N-fucopentaoee 11

Lacto-N-difucohexaose (7 % of the total oligosaccharidcs) has not yet been fully characterized, but it appears to be a di-0-fucosyl derivative of lacto-N-tetraose in which the a-L-fucosyl residues are linked to C-2 of t,he terminal D-galactose residue (as in lacto-N-fucopentaose I) and to C-4 of the 2-acetamido-2-deoxy-~-glucose residue (as in lacto-N-fucopentaose 11).61

Many other oligosaccharides of greater complexity have been reported (60)R. Kuhn, H. H. Baer and A. Gauhe, Chem. Ber., 61, 364 (1968). (61) R. Kuhn, H. H. Baer and A. Gauhe, Ann., 611, 242 (1968). See R. Kuhn and A. Gauhe, Chem. Ber., 83, 647 (1960) for the structure of lacto-N-difucohexaose 11.

171

LACTOSE

to occur in human milks2-"; five of them have tentatively been formulated40 as di-(lacto-N-tetraose), monofuco-di-(lacto-N-tetraose), difuco-di-(lactoN-te traose) , monofuco-tri- (lacto-N-tetraose) , and dif uco-tri- (lacto-N-te traose). These authors4O could find only one lacto-N-pentaose, and they did not observe the corresponding, fucose-free tri-(lacto-N-tetraose). Human milk also contains acidic saccharides660'36as does the milk of a number of other animal species. It was, in fact, in the rat mammary gland ? similar ones that two compounds of this type were first d e t e ~ t e d , ~and were also found in the lactating cow's udder, although not in the nonlactating gland. The idea that these compounds might be derivatives of sialic acid67sreceived experimental later, when it was found that one of them yielded only sialic acid and lactose, whether hydrolyzed by acid70 or degraded by bacteria.?' It was, therefore, called neuraminolactose. Similar oligosaccharides have since been found, by paper chromatography, in guinea-pig mammary gland.72 Cows' milk contains at least seven oligosa~charides,?~ five of which release lactose on acid hydrolysis. Two of these oligosaccharides yield neuraminic as the two oligosacacid, in addition to lactose, and have the same RLaotose charides found in rat mammary gland by Trucco and Caputto.@ Colostrum is a richer source of these sialic acid derivatives than milk, and it was from cow colostrum that N-acetyl-0-acetylneuraminolactosewas isolated76 in about 0.025 % yield; however, owing to the extreme lability of the 0-acetyl group, most of the subsequent work has been performed on the N-acetylneuraminolactose. This compound has been split by influenza virus and Vibrio cholerae neuraminidase into lactose and N-acetylneurau73

(62) P.Gyorgy, J. R. E. Hoover, R. Kuhn and C. S. Rose, Arch. Biochem. Biophys., 48,m (1954). (63) J. Montreuil, Bull. SOC. chim. biol., 59, 395 (1957). (64) F.H.Malpress and F. E. Hytten, Nature, 180,1201 (1957). (65) J. R. E. Hoover, G. A. Braun and P. Gyorgy, Arch. Biochem. Biophys., 47, 216 (1953). (66) F. Zilliken, G . A. Braun and P. Gyorgy, Arch. Biochern. Biophys., 64, 564 (1955); 65, 394 (1956). (67) R.Caputto and R. E. Trucco, Nature, 169, 1061 (1952). (67a) F. Zilliken and M. W. Whitehouse, Advances i n Carbohydrate Chem., 13,237 (1958). (68) R. E. Trucco and R. Caputto, J . Bzol. Chem., 206, 901 (1954). (69) R. Heyworth and J. S. D. Bacon, Biochem. J . , 68, xxiv (1954). (70) R. Heyworth and J. S. D. Bacon, Biochem. J . , 68,41 (1957). (71) M. Shilo, Biochem. J . , 66, 48 (1957). (72) F. H. Malpress and A. B. Morrison, Nature, 169, 1103 (1952). (73) F.J. Reithel, M. G. Horowitr, H. M. Davidson and G . W. Kittinger, J . Biol. Chem., lB4, 839 (1952). (74) R. E.Trucco, P. Verdier and A. Rega, Biochim. el Biophys. Acta, 16, 582 (1954).

172

CLAMP, HOUQH, HICKSON AND WHISTLER

minic acid (lactaminic acid) .76,76 The N-acetylneuraminic acid is glycosidically linkedn to C-3 of the D-galactose moiety of lactose (23).

x%k+;mHs H OH

(23) N-Acetylneur aminolactose

It is probable that the “neuraminolactose” of rat mammary glandeBand the “sialido-lactose” of cow colostrumTsare identical and correspond to N-acetylneuraminolactose, the additional oligosaccharide found by Caputto and Trucco” (and others) being the diacetate isolated by Kuhn and Brossmer.7) In addition to its presence in milk, lactose occurs in small amounts in the urine of about one in ten healthy humans of both se~es,1~~*0 the condition being known as lactosuria. The percentage incidence and the concentration of lactose in the urine both increase markedly during the later stages of pregnancy and in the puerperium (the period immediately following deli~ery),1~**1-~~ and it waa from urine passed during the puerperium that the sugar was first isolated and identified in lact0suria.8~Unfortunately, there has been a recent tendency to rely exclusively on paper chroma(76) R. Kuhn and R. Brossmer, Chem. Bet., 89, 2013 (1966); Angezu. Chem., 88, 211 (1968). (76) A. Gottachalk, Biochim. el Biophys. Acta, 18, 045 (1967). (77) R. Kuhn and R. Brossmer,Angew. Chem., 70,26 (1968); Chem. Ber., 02, 1667 (1959) * (78) R.Heimer and I(.Meyer, Biochim. et Biophys. Acta, 27,490 (1968). (79) F. V. Flynn and C. Harper, Lumet, 466,698 (1953). (80) A. A. White and W. C. Heas, Arch. Biochsm. Biophys., 64, 57 (1956). (81) 0. Watkina, J . Biol. Chem., 80, 33 (1928). (82) L. B. Winter, J . Physiol. (London), 71, 341 (1931). (83) H. J. Brock and R. 8.Hubbard, Am. J . Digest. Diseases, 2,27 (1935). (84) F. Hofmeieter, 2.physiol. Chem., 1,101 (1877-78).

LACTOSE

173

tography for the identification of lactose in urine, although this method is by no means conclusive. For the isolation of lactose from the urine of a woman in the puerperium, there has been used a simple procedure involving the adsorption of the disaccharide onto activated carbon, followed by its desorption and characterization.s6 Lactose may also occur in the urine of normal babiesss in proportions that can be as great as 0.25 %, although the incidence is greater in those that are premature?' There appears to be no renal threshold for lactose,88 that is, there is no specific mechanism for its reabsorption in the kidneys, and so the appearance of lactose in the urine merely indicates its presence in the blood. Most authors agree that there are two main routes by which lactose may enter the blood stream and thereby give rise to lactosuria. It may originate from ingested disaccharide that has been absorbed directly from the alimentary canal. The increased amounts in late pregnancy and in the puerperium are due to its active synthesis in the mammary gland. A more unusual route is by skin absorption as, for example, in burns that have been treated with lactose-containing powders?g Although lactose is predominantly an animal sugar, there have been a few reports of its occurrence in plants. In 1871, its presence in the fruit of Achras sapota was claimede0and this claim has since been conikmed,91 the modern authors also finding it in the fruit of the related species Mimusops roxburghiana and Ponleria mmpechiana in which it occurs to the extent of between 0.023 and 0.043% of the total sugar. In addition, lactose has been found to constitute 25 % of the dry weight of the long-styled pollen of the Forsythia plant." 2. Biosynthesis

There is abundant evidence that blood D-glucose is the main source for the synthesis of lactose in the active mammary gland. This precursor was indicated, quite early in the study of this problem, by the accumulated evidence from a variety of physiological techniques used on the intact animal, although none of them would have been conclusive on its (85) P. Andrewe, L. Hough and D. B. Powell, Chem. & Znd. (London), 658 (1956). (86) H. Bickel and E. M. Hickmane, Arch. Diseaee Childhood, 27, 348 (1952); J. C. Haworth and D. McCredie, ibid., 31, 189 (1956). (87) J. C. Haworth and M. S. MacDonald, Arch. Disease Childhood, 32,417 (1957). (88) L. B. Winter, J . Physiol. (London), 77,100 (1933). (89) 9.Baar and J. P. Bull, Lancet, 262,978 (1952). (90)G. Bouchardat, Bull. soc. chim. (France), [2] 10,36 (1871). (91) F. J. Reithel and R. Venkataraman, Science, 123, 1083 (1956). (92) R. Kuhn and I. Liiw, Chem. Ber., 82,479 (1949). (93) For early literature, see the following reviews: S. J. Folley, Biol. Revs. Cambridge Phil. SOC.,15,421 (1940); 24, 316 (1949); W. E. Peteraen, J . Dairy Sci., 26, 71

174

CLAMP, IIOUOH, HICKBON AND WHISTLER

One of the first satisfactory demonstrations of lactose synthesis in vitro was that of Grant,Mwho used mammary-gland slices from lactating guineapigs. Of the hexoses tested, only D-glucose was readily converted into lactose, aa waa confirmed latere6 for guinea-pig, mammary-gland slices, but several other claims in this field could not be verified. Homogenates from this gland have also been successfully to synthesize lactose, and, eventually, a soluble protein from this source was claimed96 to achieve the m e result. The advent of compounds labeled with carbon-14 enabled a more vigorous study to be made of this problem, and their use confirmed the view that D-glucose is the most important precursor of l a c t o ~ e . ~ ~ ~ ~ * In the work using D-glucose uniformly labeled with carbon-14, it was found that the specific activities of the D-glucose and D-galactose moieties of lactose were the ~ a m ewhereas, , ~ with ~ ~ ~-glucose-l-C~~ ~ ~ ~ ~ as~the~precursor, the specific activity of the D-galactose moiety was slightly less than that of the D-glucose moiety101-104 In addition, the distribution of the label was different in the two hexoses, the D-glucose being labeled almost exclusively at C-1, whereas the D-galactose showed more randomization of the label.1OSThis tendency to randomization of the label in the D-galactose moiety occurred also when the D-glucose precursor was labeled at (2-2, but notlo6when it was labeled a t C-6. Similar tracer studiesl have been carried out using smaller compounds, such as acetate, although the interpretation of the results obtained from these biosynthetically more remote precursors is difficult, especially when they are used in the intact animal. Thus C14-labeledcarbonate, acetate-l-CI*, and acetate, butyrate, and caproate labeled a t C-2, injected intravenously into lactating cows, produced lactose having similar specific activities in the D-glucose and D-galactose residues.98This observation has been con(1942);Physiol. Revs., 24,340 (1944);J. A. B . Smith, J . Dairy Research, 17,349 (1950). For the present position, see the review by F. H. Malpress, Proc. Roy. SOC.(London), B149, 362 (1958). (94) G. A. Grant, Biochem. J . , 29, 1905 (1935). (95) F. H. Malpress and A. B. Morrison, Biochem. J . , 46,307 (1950). (96) G. W. Kittinger and F. J. Reithel, J . Biol. Chem., 106,527 (1953). (97) M. Kleiber, A. L. Black, M. A. Brown, C. F. Baxter, J. R. Luick and F. H. Stadtman, Biochim. el Biophys. A d a , 17,262 (1955). (98) C. F. Baxter, M. Kleiber and A. Black, Biochim. el Biophye. Acta, 21, 277 (1956). (99) T. H.French, G. Popj&kand F. H. Malpress, Nature, 169,71 (1952). (100) 0.K. Reiss and J. M. Barry, Biochem. J . , 06, 783 (1953). (101) J. M. Barry, Nature, 169, 878 (1962). (102) E.Dimant, V. R. Smith and H. A. Lardy, J . Biol. Chem., 101, 85 (1963). (103) P. Scharnbye, H.G. Wood and M. Kleiber, J . Biol. Chem., 226,1011 (1957). (104) J. H. Parur and C. L. Tipton, J . Biol. Chem., 124, 381 (1957). (106) H. G.Wood, R. Gillespie, S. Joffe, R. G. Hansen and H. Hardenbrook, J . Biol. Chem., W , 1271 (1968).

LACTOSE

175

firmed,"Jgalthough the labeling pattern was found to be different in the two hexoses. With a~etate-bC'~, both moieties were similarly and quite uniformly labeled, whereas, with NaHC1403and acetate-1 -P4,although C-4 of both hexoses had the highest activity, the distribution of the label was much more symmetrical in the D-glucose. The use of sodium formateC14 resulted in D-glucose and D-galactose residues having similar specific activities and labeling patterns.Ioe The biosynthesis of lactose by use of these simpler substances has been more satisfactorily studied'w with the perfused, isolated, cow's udder. It was found that, with acetate-1-Cl4 or pr~pionate-l-C'~, more than 90% of the total activity of the lactose was in the D-galactose portion, where it was concentrated mainly at C-4. A similar result was obtained'm with acetate-l-CI4, by use of a different technique. In this method, the labeled precursor was injected into the arterial supply to the udder on one side only, and then the milk constituents from the injected and non-injected sides were studied separately. It is interesting to note that the lactose obtained from the non-injected side had similar activities in its D-glucose and D-galactose residues, a result which parallels the result from work performed on the intact animal. Glyceritol-1 ,S-C142, administered by this method, produced similar 90.% of the total activity of the lactose from the injected side appearing in C-4 and C-6 of the D-galactose residue. The labeling pattern obtained in these experiments suggests107 that these smaller molecules are incorporated into the hexose by means of the tricarboxylic acid cycle and the anaerobic, glycolytic pathway. The results obtained with CI4-labe1ed compounds depend on a number of factors, for example, the type of compound used and the position of its label, whether the experiment is in vitro or in vim, and the duration of metabolism. Even differences in the experimental animal employed may be important.110 All these factors make the interpretation of this experimental work difficult, although certain general conclusions may be drawn. Any biosynthetic scheme proposed, therefore, must explain the origin of both hexose moieties from D-glucose. It must also explain a subsidiary source, from smaller metab~lites,~s contributing mainly to the biosynthesis of the u-galactose residue,1o3~ 0 6 ~ together 0 7 with a dilution and randomization of the label in this residue as compared with the D-glucose residue when (106) F. J. Carleton, H. R. Roberts and L. A. Nutting, Nature, 180, 915 (1957). (107) H. G. Wood, P. Schambye and G. J. Peeters, J . B i d . Chem., 226,1023 (1957). (108) H. G . Wood, P. Siu and P. Schambye, Arch. Biochem. Biophys., 69, 390 (1957). (109) H. G. Wood, S. Joffe, R. Gillespie, R. G . Hansen and H. Hardenbrook, J . B i d . Chem., 239, 1264 (1958). (110) S. J. Folley, "The Physiology and Biochemistry of Lactation,'' Oliver and Boyd Ltd., London, 1st Edition, 1956.

176

CLAMP, HOUOH, HICKSON AND WHIBTLER

~-glucose-l-C~~ is fed. D-Galactose would, therefore, seem to arise by way of intermediates that are in equilibrium to some extent with other enzyme systemslllJ12; these additional enzyme-systems may include the EmbdenMeyerhof glycolytic pathway, providing a link with the tricarboxylic acid cycle, the pentose phosphate cycle (including the D-glucose 6-phosphate dehydrogenase and transketolase-transaldolase pathways), and, possibly, the D-glucuronic acid cycle.11* The D-glucose residue, on the other hand, appears to be incorporated into lactose either (a) directly, by a route that contains few rate-limiting, equilibrating steps, or (b) in some cell organelle that limits admixture with other metabolic pathways. The biosynthesis of lactose, which is an aerobic p r o c e ~ s , ~must ~ ~ Jat~ ~ some stage require the direct conversion of D-glucose into D-galactose, since the tracer experiments show that, in the main pathway, at least, this conversion occura without rupture of the carbon chain. Such a process requires inversion at C-4 of the hexoses, and this process was postulated1lBquite early. A further step was elucidatedll’ later, when it was found that the conversion proceeds through the corresponding hexosyl phosphate derivatives. Not until 1949, however, did the whole subject cease to be a matter of conjecture and receivells a firm experimental basis. In yeast and mammalian liver waa discovered a thermostable factor which was necessary for the conversion of D-galactosyl phosphate into D-glucosyl phosphate. This coenzymic factor was later isolated from yeast, and was identifiedllg as uridine 5-(~-glucosylpyrophosphate) , the corresponding enzyme-complex effecting the change being called “galactowaldenase.” Eventually, both ,lZ1 uridine D-glucosyl pyrophosphate and galactowaldenase were found67JZ0 in mammary tissue. The over-all scheme of lactose biosynthesis is probably as follows, the enzyme catalyzing each step appearing in parentheses. (111) H. G. Wood and J. Kata, J . B i d . Chem., 188,1279 (1958). (112) G.J. Peeters and M. Debackere, Arch. intern. phy8iOl. el biochim., 84, 627 (1966). (113) G.E.Glock and P. McLean, Proc. Roy. Soc. (London), B l U , 354 (1954). (114)R. Heyworth and J. 9. D. Bacon, Biochem. J . , 61,224 (1956). (1lK) R. Venkataraman and F. J. Reithel, Arch. Biochem. Biophys., 70.205 (1957). (116)F.Rtihmann, Biochem. Z . , B8,237 (1919);R. Robinson, Nature, 190,44 (1927); H. K. Barrensoheen and N. Alders, Biochem. Z . , 9 1 , 87 (1932). (117) H.W.Kosterlita, Biochem. J., 87, 322 (1943). (118) R. Caputto, L.F. Leloir, R. E. Trucco, C. E. Cardini and A. C. Paladini, J . Biol. Chem., 179, 497 (1949). (119) R. Caputto, L.F. Leloir, C. E. Cardini and A. C. Paladini, J . B i d . Chem., 184, 333 (1960). (120)W. J. Rutter and R. G . Hansen, J . Biol. Cham., 909,323 (1963). (121) E.E.B. Smith and G . T. Mills, Biochim. el Biophy8. Ada, 18, 687 (1954).

177

LACTOSE

(Abbreviations used: A for adenosine moiety; U for uridine moiety; 0 Pfor-JO-; AH

A(5)-OPaH for adenosine 5-triphosphate; A(5)-OPzH for adenosine 5pyrophosphate; U(5)-OPaH for uridine 5-triphosphate; U(5)-OP2H for uridine 5-pyrophosphate ; and HPzH for pyrophosphoric acid). D-G

+ A(5)-OPsH D-G(S)-0PH

+

+

D-G(~)-OPH A(5)-OPtH (Hexokinase)

a - ~ - G ( l-0PH ) (Phosphoglucomutase)

+

a-D-G(l)-OPH 4- U(5)-OPaH U(~)-OP~-O(I)-D-GHOPtH (Pyrophosphate-uridyl transferme; uridine D-glucoeyl pyrophosphorylase) 1**-***

*

(1 1

(9) (3 1

U (6)-OPz-O(I)-D-G U (5)-OPz-O(l)-D-GallU (4) (Uridine 5- (D-galactosyl pyrophosphate) 4-epimerase; “galactowaldenase”~S~)~*”~*7 sU6

+

U ( ~ ) - O P I - O ( ~ ) - D - G~~-~D - G ( ~ ) - O + PH lactosyl-OPH (Galactosyl transferase)I**J W Lactosyl-OPH

--t

lactose

+ HOPH

(Phosphatase).1*0

J**

(6) (6 1

Reactions ( I ) and (9)are too well known in connection with the usual glycolytic pathways to need further elaboration. Both hexokinasela2 and phosphoglucomutaseee~l~ are present in mammary tissue, as are D-glucosyl (122) A. Munch-Petersen, H. M. Kalckar, E. Cutolo and E. E. B. Smith, Nature, 111, 1063 (1953) ; H. M. Kalckar, B. Braganca and A. Munch-Petersen, ibid., 172. 1038 (1953); E. E. B. Smith and A. Munch-Petersen, ibid., 173, 1038 (1953). (123) E. S. Maxwell, H. M. Kalckar and R. M. Burton, Biochim. et Biophys. Acta, 18,444 (19%). (124) Nicotine adenine dinucleotide (“diphosphopyridine nucleotide”) appears to be necessary in this transformation, although none of the reduced form accumulates; E. 5.Maxwell, J. A m . Chem. SOC.,78, 1074 (1956); see also, Ref. 125. (125) E. S. Maxwell, J. Biol. Chem., 229, 139 (1957). (126) The term galactowaldenase refers to the complex that catalyzes the over-all reaction a-D-glucosyl phosphate a-D-galactosyl phosphate; H. M. Kalckar and E. S. Maxwell, Biochim. et Biophys. Acta, 22,588 (1956). (127) L. F. Leloir, Arch. Biochem. Biophys., 38.186 (1951). (128) J. E. Gander, W. E. Petersen and P. D. Boyer, Arch. Biochem. Biophys., @I, 258 (1956). (129) J. E. Gander, W. E. Petersen and P. D. Boyer, Arch. Biochem. Biophys., 69, 85 (1957). (130) S . J. Folley and H. D. Kay, Biochem. J., 29, 1837 (1935). (131) S. J. Folley and A. L. Greenbaum, Biochem. J., 41,261 (1947). (132) C . Terner, Biochem. J.,62, 229 (1952).

178

CLAMP, HOUOH, HICESON AND WHISTLER

phosphate and lactosyl phosphate.lS3This biosynthetic route, each step of which is well authenticated, could occur in the mammary gland, since all the component enzymes have now been found there.la8* 3. Metabolism

The initial step in the metabolism of lactose is an enzymic hydrolysis to its constituent hexoses by (3-~-ga~actosidase.~*~ Since /3-~-galactosidases~~~ and p-D-glucosidases are difficult to separate from each other when they occur together, it had been postulated186 that a single enzyme catalyzes both actions; however, each enzyme can exist independently of the other.lS6191 p-D-Galactosidase activity is very widely distributed in Nature, occurring in micro-organisms, plants, and animals, including the snail,lascockand fly larva.140 In birds, a /3-D-galactosidase appears in their alimentary tract when they are fed 1act0se.l~~ In higher animals, the enzyme is found, not only in their pancreatic and small-intestinal secretions,141J42 but also in the kidney, liver, testis, epididymis, and vas deferens.14*JM However, the principal site of hydrolysis must be the intestinal tract, because, although this enzyme is present in many of the internal organs (for example, of the rat), intravenously administered lactose is largely excreted unchanged in the urine and only a small percentage is metab01ized.l~”/3-D-Galactosidase is also present in plants187,146 and certain marine algae,146but it has received the most attention in connection with (133) M. G. McGeown and F. H. Malpress, Biochem. J., 62, 606 (1952). (133a) See F.H. Malpress, Biochem. J.,78, 527 (1961) for one of the few quantita-

tive studies on lactose biosynthetic enzymes of mammary gland. (134) E. Fischer and E. F. Armstrong, Ber., 36, 3153 (1902). (134s) See K. Wallenfels and 0. P. Malhotra, This Volume, p. 239. (135) B. Helferich and H. Scheiber, Z . physiol. Chem., 226,272 (1934). (136) E. Hofmann, Naturu~issenschaSen,72,406 (1934) ;Biochem. Z., 273,198 (1934) ; 286, 429 (1936). (137) S. Veibel, in “The Enzymes,” J. B. Sumner and K. Myrbiick, eds., Academic Press Inc., New York, N. Y., 1960, Vol. 1, pt. 1, p. 623. (138) H. Bierry and J. Giaja, Compt. rend., 147, 268 (1908). (139) V. B. Wigglesworth, Biochem. J . , 21, 797 (1927). (140) M. Rockstein and A. S. Kamal, Phyeiol. Zool., 27, 65 (1954). (141) E. Fischer and W. Niebel, Silzber. prsuss. Akad. Wiss. Physik.-math. K l . , 6 , 73 (1896); T. S. Hamilton and H. H. Mitchell, J. Agr. Research, 27, 597, 605 (1924). (142) J. H. Landor, P. H. Brasher and L. R. Dragstedt, A . M . A . Arch. Surg., 71, 727 (1955); F. Alexander and A. K. Choudhury, Nature, 181,190 (19%). (143) A. M. Rutenburg, S. H. Rutenburg, B. Monis, R. 8. Teague and A. M. Seligman, J. Histochem. and Cytochem., 6, 122 (1958). (144) J. Conchie and A. J. Hay, Biochem. J., 73, 327 (1959). ( M a ) F. J. Carleton, 8.Misler and H. R. Roberts, J. Biol. Chem., 814,427 (1955). (145) E. Hofmann, Biochem. Z . , 267, 309 (1933); B. Helferich and F. Vorsatz, 2. physiol. Chem., 237, 254 (1935). (146) W. A. M. Duncan, D. J. Manners and A. G. Ross, Biochem. J . , 63.44 (1956).

LACTOSE

179

micro-organisms. The interesting phenomenon of induced (or adaptive) enzyme-formation can be studied by following the synthesis of P-D-galactosidase in response to certain inductors, and this matter has been investi, ~ ~ ~ and Staphylococcus a u ~ e u s8-D.~~~ gated in Escherichia ~ o l iNeurospora,l@ Galactosidases from different sources also hydrolyze various derivatives of lact0se.'~J~0 This lack of specificity may not, however, be characteristic of all /3-D-galactosidases, since there is some evidence that they may vary according to their source. Thus, the lactotriose produced by a P-D-galactosidase from Aspergillus is not hydrolyzed by the enzyme from Escherichia coZi.161The production of oligosaccharides by a transglycosylation reaction is characteristic of many so-called hydrolytic enzymes, including p-D-galactosidase. It is, in fact, similar to the acid-reversion phenomenon (see p. 181). It has been suggestedlK1 that lactose and P-D-galactosidasereact reversibly to form D-glucose and a D-galactosyl-enzyme complex which then transfers the D-galactosyl residue to a suitable acceptor, such as water, D-galactose, D-glucose, lactose, or some other hydroxylic compound. Thus, a wide variety of oligosaccharides can be formed initially,151J62 all of which must contain at least one D-galactosyl residue. After hydrolysis by p-D-galactosidase, the further metabolism of lactose will be that of its constituent monosaccharides. In all organisms, a variety of metabolic pathways, open to D-glucose, remain closed to D-galactose until inversion has occurred at C-4. However, there is evidence that, in micro-organisms,lKS other routes exist for the metabolism of D-galactose. The problem of the D-galactose-D-glucose interconversion was discussed on p. 176 in connection with the biosynthesis of lactose, although, in fact, the mechanism was largely elucidated by studying the metabolism of D-galactose in The initial step in this process is the formation of a-n-galactosyl phos(147) J. L. Koppel, C. J. Porter and B. F. Crocker, J . Gen. Physiol., 56,703 (1953); A. C. R. Dean and C. Hinshelwood, Proc. Roy. SOC.(London), B142, 225 (1954); S. Mvtrup, Biochim. el Biophys. Acta, 19,247,433 (1956). (148)0. E.Landman, Arch. Biochem. Biophys., 62, 93 (1954). (149) E. H.Creaser, J . Gen. Microbiol., 12, 288 (1955). (150) H.Bierry and A. Ranc, Compt. rend., 160, 1366 (1910);C..Neuberg and E. Hofmann, Biochem. Z., 266, 450 (1932); B. Helferich and W. (W.) Pigman, Ber., 72, 212 (1939). (151) K. Wallenfels, E.Bernt and G . Limberg, Ann., 664.63 (1953). (152) M. Aronson, Arch. Biochem. Biophys., 39,370 (1952);K.Wallenfels, E. Bernt and G . Limberg, Ann., 679, 113 (1953);H.R.Roberts and E. F. McFarren, J . Dairy Sci., 36, 620 (1953);Arch. Biochem. Biophys., 43, 233 (1953);J. H.Pazur, Science, 117, 355 (1953);J . Biol. Chsm., 208,439 (1954);J. H.Pazur, C. L. Tipton, T. Budovich and J. M. Marsh, J . Am. Chem. SOC.,80,119 (1958). (153) J. De Ley and M. Doudoroff, J . Biol. Chem., 337, 745 (1957).

180

CLAMP, IIOUQH, HICKSON AND WHISTLER

phate by the action of galactokinase, according to equation (7).a-D-GalacD-Gal

+ A(6)-OP,H

+ a-D-Gal(l)-OPH

+ A(6)-OPoIl

(7 1

tosyl phosphate may be converted into uridine B-(~-galactosyl pyrophosphate) by two different routes (Sa and Sb). (a-D-Galactosyl phosphate uridyl transferaee; uridyl transferase)1mJ a-D-ad

(1)-OPH

~ J M

+ U(6)-OPaH + U(~)-OPI-O(~)-D-G~~ + HOPsH

(8b)

(Uridine n-galactosyl pyrophosphorylase) .lmJ6'

The final step is the reversible inversion at C-4 (see page 177). This over-all scheme has so far been detected in animal liver and in micro-organisms,120J66 Certain yeasts can adapt themselves to utilizing D-galactose, and this adaptation has been variously attributed to the deJ~ of a-D-galactosyl uridyl transferase ,IKB velopment of galact~kinase,~~~ and of both of these enzymes and uridine €i-(D-galactosyl pyrophosphate) 4epimerase. 6o Certain biological systems possess the ability to oxidize lactose to lactobionic acid. This process has been found in P a e ~ d r n o n a aBacterium ~~~ anitrutum,16athe red alga Iridophycua Jlaccidum,'B"and Penicillium chrya0genum1~~ (which can also metabolize lactobionic acid further). The cellfree, ensyme preparation was stated to be specific only for the configuration at C-2 of the reducing hexose, and to consume oxygen; hence, it was called an oxidaee. Later, however, the purified-enzyme preparation was shown to be more specific for lactose and to be unable to utilize oxygen directly; it was, therefore, designated lactose dehydrogenase.166 (164) K. Kurahashi and E. P. Anderson, Biochim. el Biophys. Act4, 38,498 (1968). (166) K. J. Iseelbacher, Science, 136, 662 (1957);J . Biol. Cham.,882,429 (1968). (16f3)W.J. Rutter and R. G . Hansen, J. Biol. Chem.,909,311 (1963);R. G.Hansen and E. M. Craine, ibid., 808,293 (1964);B. Bloom, ibid., 889,166 (1967);H. M. Kalckar and H. de Robichon-Seulmajeter, Bull. soc. chim. b i d , U , 1308 (1969). (167) I.F. Wilkinaon, Biochem. J., 44,480 (1949). (168) R.E.Trucco, R. Caputto, L. F. Leloir and N. Mittelman, Arch. Biochem. Biophys., 18, 137 (1948). (169) G.T. Mills, E. E. B. Smith and A. C . Lochhead, Biochim. et Biophye. Ada, 86, 621 (1967). (180) H.de Robichon-Srulmajster, Science, 187, 28 (1968); Biochim. el Biophys. Acta, 49, 270 (1968). (161) F.H. Stodola and L. B. Lockwood, J . Bio2. Chem., 171,213 (1947);E.Masuo, F.Elhima and K. Yoshida, Shionogd Kenkylleho Nempd, 8,136 (1962). (162) P. Villecourt and H. Blachhre, Ann. imt.Paeteur, 88,623 (1966). (183) R. C. Bean and W.Z.Haesid, J. Biol. Chem., 818,426 (1966). (164) W.M. Cort, W. M. Connors, H. R. Roberts and W. Bucek, Arch. Biochem. BiOphys., 681477 (1966). (166) Y. Nishieuka, 8.Kuno and 0. Hayaishi, J . Biol. Chem.,885, PC13 (1980).

LACTOSE

181

IV. CHEMICAL PROPERTIES OF LACTOSE In common with other reducing disaccharides, lactose undergoes the usual reactions of aldoses, although precautions must be taken to prevent hydrolysis of the glycosidic bond. Furanoid structures cannot be formed, however, because of the presence of the (1 44)-linkage. 1. The Action of Acids Hydrolysis of lactose (3 % wt./vol.) in 0.05 N hydrochloric acid at 98” is found to be 50% complete in 125 rninutes,lBBthe rate of hydrolysis being more rapid than that of maltose. In 5-20 % hydrochloric acid, the hydrolysis is accompanied by degradation to humic substances.167A cation-exchange resin, such as Duolite C3 in the H’ form, is a convenient catalyst for the hydrolysis of lactose.lBB Oligosaccharides can be formed during acid hydrolysis by a process known as “reversion,” in which residues of lactose or its hydrolysis products are combined by condensation reactions.lB8The extent of reversion is dependent upon various factors, including the temperature, the concentrations of lactose and of acid, and the length of reaction time. The reversion tends to increase with increasing concentrations of carbohydrate and can be conveniently followed by paper chr0matography.l7~~7~ When crystalline lactose containing 0.3% of water was placed in an atmosphere of hydrochloric acid fumes for 7 days a t room temperature, a polymer was formed in 21 % yield.l’a 2. Oxidation

Nitric acid (25-35 % wt./vol.) causes hydrolysis of lactose and oxidation to give D-glucaric acid (“saccharic acid”) and galactaric acid (“mucic acid”),’7*J7‘ with further oxidation of these products to tartaric acid, oxalic acid, and carbon d i ~ x i d e . l ~ ~Oxidation -l ~ ’ to galactaric acid was suggestedl’s (166) F. P. Phelps and C. S. Hudson, J . A m . Chem. Soc., 48,503 (1926). (167) T. Takahasi, Nippon NBgei-kagaku Kaishi, 80, 553 (1944); Chem.Abstracts, 4 1 , 8 i t ~(1948). (168) R.J. Block, U. S. Pat. 2,592,509 (1952); Chem. Abstracts, 48, 5744 (1952). (169) G . Malyoth and H. W. Stein, Angew. Chem., 64,399 (1952). (170) K. Tiiufel, H. Iwainsky and H. Ruttloff, Biochem. Z . , 327,531 (1956). (171) L. Hough and J. B. Pridham, Chem. & Znd. (London), 1178 (1957). (172) C. R. Ricketts and C. E. Rowe, J . Chem. Sbc., 3809 (1955). (173) C. G. Scheele, in “Opuscula chemicrt et physica,” E. B. G. Hebenstreit, ed., Leipzig, 1789, Vol. 2. (174) J. von Liebig, Ann., 113, 1 (18eO). (175) A. P. Dubrunfaut, Compt. rend., 42,228 (1856); Jahresber. Chem., 643 (1856). (176) H. Hornemann, J . prakl. Chem., [l] 89,283 (1863). (177) W. H. Kent and B. Tollens, Ann., 117, 221 (1885). (178) B. Tollens and P. Rischbieth, Ber., 18,2616 (1885).

182

CLAMP, HOUGH, HICKSON AND WHISTLER

as a quantitative method for the estimation of lactose, but it has since been replaced by more accurate procedures. Lactose is oxidized by hypoiodite, prepared from iodine and alkali, to give lactobionic a~id.'7~-l~~ Under carefully controlled conditions, the reaction is stoichiometric and can be uMd for the quantitative determination of lactose on a micro scale by measurement of the iodine consumed.182-18K At pH 10.6, lactose is oxidized at a rate 0.81 times that observed for D-gluCOse.18S In acid solutions, the active oxidant can be either the free halogen (X) or hypohalous acid, the proportions of these two forms varying with the pH and the halogen used. Unless a buffer or neutralizing agent is present, such as barium carbonate ,IEe barium benzoate,'" or sodium hydrogen carbonate,lM the solution will become strongly acidic owing to the formation of halogen acid, with resultant hydrolysis of the glycosidic linkage. Thus, in the absence of a neutralizing agent, D-galactonic acid and D-gluconic

+

\c/

-0

HOX -0

XI HzO OH

-0

' '

\C/

\H

\

+x*+

\H OH

-0

+ HOX

+ HX /

\

--$

/

C=O

C=O

+ 2HX

+ HX + Ha0

acid are obtained in addition to lactobionic acid.l7Jsg-*92 Furthermore, the accumulation of halogen acid during the reaction inhibits the rate of oxidation, and, consequently, the presence of buffer results in high yields of lactobionic a ~ i d . ~ ~In7the J~~ presence of barium carbonate, bromine, not hypobromous acid, is the active oxidant, and lactose is directly oxidized as the hemiacetal, with the abstraction of two hydrogen atoms from the (179) E. Millon, Compl. rend., 21, 828 (1846). (180) G. Romijn, 2.anal. Chem., 56, 349 (1897). (181) W. F. Goebel, J . Biol. Chem., 71, 809 (1827). (182) K. Myrbhk, Suensk Kem. Tidekr., 61, 179, 206 (1939). (183) 0. G. Ingles and G. C. Israel, J . Chem. SOC.,810 (1948); 1213 (1949). (184) J. R. Hawthorne, Nature, 160, 714 (1947). (186) E. L. Hiret, L. Hough and J. K. N. Jones, J . Chem. Soc., 928 (1949). (186) H. A. Clowes and B. Tollens, Ann., 810,164 (1899). (187) C. S. Hudson and H. 8.Isbell, J . Am. Chem.SOC.,61.2225 (1929). (188) Y. Sahashi and T. Kakuda, Nippon Ndgei-kagaku Kaiahi, 24,176 (1960). (189) L. Berth and J. H. Hlaeiwetz, Ann., 110.96 (1862). (1W) H. Kiliani, Ber., 18, 1661 (1886); 69, 1486 (1926). (191) H. Hlasiwetz, Ann., 119.281 (1861). (192) H. Hlasiwetz and J. Herberman, Ann., 166, 120 (1870).

LACTOSE

183

anomeric center to give lactobiono-l,8lactone, from which the aldonic acid is generated by h y d r o l y s i ~ . ~ ~ ~ - ~ ~ ~ = 32.8),le6in 8-Lactose is more rapidly oxidized than a-lactose agreement with a C1 conformation for the D-glucose residue, a conformation in which, for the D series, the a-hydroxyl group is axial and less accessible to attack than the equatorial 8-hydroxyl group (24). W

H (24)

Electrolysis of a solution containing calcium bromide and lactose causes smooth oxidation of the disaccharide to lactobiono-1,5-lact0ne,’~7-’99presumably by the formation of free bromine at the anode. After hydrolysis of the lactone, lactobionic acid is usually isolated200,2O1as its insoluble calcium salt [ ( C I Z H Z I O Y Z )H2Ol. ~C~.~ 4-O-/.3-~-Galactopyranosyl-~-u~u~no-hexulosonic acid (“2-ketolactobionic acid”) was obtained by oxidizing lactosone with bromine, and the product was isolated as the mixed barium salt-barium bromide complex [(C12HIgOl2) 2Ba. BaBr2 - 4 H ~ 0 1 . 2 ~ 3-O-P-~-Galactopyranosyl-D-arabinosewas prepared from lactose o x h e by dehydration with acetic anhydride and degradation of the resultant nitrile with sodium methoxide in chloroform.26~20s This galactosylarabinose has also been prepared by the oxidation of calcium lactobionate with hydrogen peroxide and a ferric salt (the “Ruff degradation”) .25-204 Under H. S. Isbell, Bur. Standards J . Research, 8, 615 (1932). H. S. Isbell and C. S. Hudson, Bur. standards J . Research, 8,327 (1932). H. S. Isbell and W. W. Pigman, Bur. Standards J . Research, 10,337 (1933). R. Bentley, J . A m . Chem. SOC.,79, 1720 (1957). Rohm and Hass Co., German Pat. 558,379 (1931); French Pat. 715,176 (1931); Chem. Abstracts, 28, 1525 (1932). (198) E. L. Helwig, U. S. Pat. 1,895,414 (1933); Chem. Abstracls, 27,2389 (1933). (199) H. S. Isbell and H. L. Frush, Bur. Standards J . Research, 8 , 1145 (1931). (200) H. 9. Isbell, U. S. Pat. 1,980,996 (1935); Chem. Abstracts, !B, 478 (1935); Bur. Standarh J . Research, 11, 713 (1933). (201) C. S. Hudson and E. Yanovsky, J . A m . Chem. SOC.,SQ, 1013 (1917). (202) W. W. Walton and H. S. Isbell, J . Research Natl. Bur. Standards, 41, 119 (193) (194) (195) (196) (197)

(1948). (203) R. Kuhn, W. Kirschenlohr and W. Bister, Ann., 800, 135 (1956). (204)0. Ruff and G . Ollendorf, Ber., 32, 552 (1899); 33, 1798 (1900).

184

CLAMP, HOUQH, HICKSON AND WHISTLER

carefully controlled conditions, lactose in alkaline solution is oxidized by acid, atmospheric oxygen to yield 3-O-~-~-galactopyranosyl-~-arabinonic isolated as its brucine salt.206 The over-all reaction of 1 mole of lactose with an excess of sodium CHSOH

CH,OH

I

I OH

OH

1

\

1o.Q

1 10.0

metaperiodate, at room temperature and in the dark, gives 2 moles of formaldehyde, 9 moles of formic acid, and one mole of carbon dioxide, in common with the behavior of such other (1 3 4)-dihexose disaccharides as maltose and cellobiose.1°B-210 (205) E. Hardegger, K. Kreis and H. E. Khadern, HeZu. Chim. Acto, 86,618 (1962). ('206) J. E. Courtois and M. Ramet, Bull. aoc. chim. biol. ,99,240 (1947). (207) J. E. Courtois, A. WickstriSm and P. L. Diaet, Bull. uoc. chim. bioZ., 94. 1121 (1962).

LACTOBE

185

+ 11 1 0 d e + 2 H s C 4 + 9 HCOiH + COI + 11 IOae

ClrH~lOll

The initial step involves the oxidation of 1 mole of lactose (as the hemiacetal) with four moles of periodate, giving two moles of formic acid and

(27)

+

HCO,H

hydrolysis

I

(28)

I

hydrolysis 0

$=O

+

C=o

L

O

H

t

(30)

CO,

1

+

2 lore

II

+ HO,C -CH

&C=O

+

C q

+

I

1 10.e

HCOJ

4 HCO*H

2 HC0,H

FIG. 3.-The Periodate Oxidation of Lactose.

the formyl ester (25) which is relatively stable to hydrolysis209between pH 3 and 5. Consequently, further oxidation by periodate is impeded*11-212 ('208) P. F. Fleury, J. E. Courtois and A. Bieder, Bull. 8oc. chim. France, 118 (1962). (209) G. Neumiiller and E. Vaeseur, ArWu.Kemi, 6,236 (1963). (210) F. S. H. Head and G. Hughes, J . C h m . SOC.,603 (1864). (211) K. Meyer and P. Rathgeb, Helu. Chim. Acto, 81,1640 (1948).

186

CLAMP, HOUQH, HICKSON AND WHISTLER

within this range of pH. The rate of hydrolysis of esters of this type has been related to the inductive effect of electrophilic groups in the alcoholic component.21sAkaline or strongly acid conditions cause rapid hydrolysis of the formyl ester (25), and "over-oxidation" results from the formation of an intermediary malonaldehyde derivative (26) which contains an oxidizable hydrogen atom because of activation by the flanking carbonyl The resultant 1 mole of acetal of hydroxymalonaldehyde (27) is either oxidized through the glyoxylate (28) to give one mole of carbon dioxide, one mole of formaldehyde, and six moles of formic acid, or is hydrolyzed to give mesoxalaldehyde (29) and a dialdehyde (30) which are oxidized to the same products.2lSThus, two moles of formaldehyde were formed in four hours when 1 mole of lactose was oxidized2l6 with 0.05 M sodium metaperiodate at 18" and pH 8 (see Fig. 3). The theoretical yield of carbon dioxide was obtained in about 10 hours from lactose by oxidation2" with 0.06 M sodium metaperiodate in acetate buffer at pH 5.0 and 50". On the other hand, the use of phosphate buffer at pH 5.0 led to extensive over-oxidation of the products, giving carbon dioxide far in excess of one mole per m 0 l e . 2 ~ 7The ~ ~ concentration ~~ of periodate in the reaction mixture has a profound effect on the rate of oxidation of malonaldehyde derivatives and, in <0.01 M periodate, the reactions are very ~ 1 0 ~ . 2 ~ 9 , % ~ Lactose appears also to be oxidized as its hemiacetal by lead tetraacetate in acetic acid, to give a formyl ester (with the consumption of three to four moles of oxidant), without the liberation of any formaldehyde.221In addition, with various (1 3 4)-dihexose disaccharides at 27" in 90 % acetic acid containing potassium acetate as catalyst, the nonreducing unit gives rise to one mole of free formic acid per mole, and the reducing unit is oxidized to a tetrose diformate (31),m*na thus suggesting the following pathway. (212) M. Morrison, A. C. Kuyper and J. M. Orten, J . Am. Chem. SOC.,76, 1602 (lQ63). (213) L. Hough, T. J. Taylor, G. H. S. Thomas and B. M. Woods, J . Chem. Soc., 1212 (1968). (214) C. F. Huebner, S. R. Ames and E. C. Bubl, J . Am. Chem. Soc., 88, 1621 (1946)* (216) M. Cantley, L. Hough and A. 0. Pittet, Chem. & Znd. (London), 1126, 1263 (1969). (216) L.Hough and M. B. Perry, Chem. & Znd. (London), 768 (1966). (217) L. Hough and B. M. Woods, Chem. & Znd. (London), 1421 (1967). (218) G. Lindstedt, Nature, 166, 448 (1946). (219) J. R. Dyer, Methods of Biochem. Anal., 8 , 144 (1966). (2'20) T. G.Halsall, E. L. Hirat and J. K. N. Jones, J . Chem. Soc., 1427 (1947). (221) R. Criegee, Ann., 496.211 (1932). (222) A. S. Perlin, Anal. Chem., 27, 396 (1966). (223) A. J. Charlson and A. S. Perlin, Can. J . Chem., S4,1200 (1966).

187

LACTOSE

bH

HCOJi

+

I

3 PMOAc),

O=CH

HC=O

I

8

3. Reduction Early attempts to reduce lactose with sodium a m a l g a ~ ~ or2by ~ catalytic h y d r o g e n a t i ~ n ~under ~ ~ - ~pressure *~ with a nickel catalyst at 130°, caused degradation to galactitol, D-glucitol, and other alcohols. Lactitol (4-0-/3-~galactopyranosyl-D-glucitol)was obtained by catalytic hydrogenation under milder conditions,22a-228 by treatment with calcium amalgam,22gand, (224) G. Bouchardat, Ann. chim. et phys., (41 27,68 (1872). (225) V. Ipatieff, Ber., 46, 3218 (1912). (226) J. B. Senderens, Compt. rend., 170,47 (1920). (227) P. Karrer and J. Buchi, Helv. Chim. A d a , 20, 86 (1937). (22.8) T. Tanno, Nippon Kagaku Zasshi, 69,709 (1938). (229) C. Neuberg and F. Marx, Biochem. Z.,3,539 (1907).

188

CLAMP, HOUQH, HICKSON AND WHISTLER

more simply, by heating under reflux with freshly prepared Raney nickel in 70 % aqueous ethanol.2a0s2a1Reduction of lactose solutions with sodium borohydride is quantit8ative,2*2-ag4 but great care must be taken in the preparation of lactitol by this method, since the removal of sodium ions by a cation-exchange resin gives a strongly acidic solution which, on distillation with methanol to remove boric acid as methyl borate, can cause methanolysis of the lactitol, with the formation of D-glucitol and methyl D-galactosides.*la Electrolytic reduction of a lactose solution containing inorganic sulfite, between an amalgamated lead cathode and a graphite anode, is reported to give a 90 % yield of lactitol with a 90 % current-efficiency.286n286 Hydrogenolysis of lactose at a pressure of about 300 atmospheres with a copper-chromium oxide catalyst gave, amongst other products, methanol, ethanol, 1,2-propanediol, and three other compounds (that were tentatively identified as tetrahydro-4-hydroxyfurfurylalcohol, 1 , 2 ,5-hexanetriol, and 1, 2,5 ,6-hexanetetr01.2~7

4. Degradation with Base Eurlicr investigatorP ~ ~ 7 of- the 2 ~ alkaline-degradation ~ products of lactose found succinic acid, formic acid, and a crystalline lactone, later identified by Kilianial.241 as “a”-D-isosacch&riniclactone. This lactone was obtained in about 20% yield from the action of lime-water on lactose; also ~~~~ acid, ~~ detected as products of this reaction were ~ - g a l a c t o s e , 2lactic pyruvaldehyde,24 f0rmaldehyde,2~~ and other saccharinic acids.241 *248 The (230) M. L. Wolfrom, W. J. Burke, K. R. Brown and R. 5. Rose, J.Am. Chem. SOC., 60,571 (1938); M. L. Wolfrom, R. M. Hann and C. 8. Hudson, ibid., 74, 1106 (1952). (231) J. V. Karabinoe and A. T. Ballun, J . Am. Chem. SOC.,76,4501 (1953). (232) S. Peat, W. J. Whelan and J. G . Roberts, J . Chem. SOC., 2268 (1950). (233) P. D. Bragg and L. Hough, J . Chem. SOC.,870 (1957). (234) L. Hough, B. M. Woods and M. B. Perry, Chem. & Znd. (London), 1100 (1957). (235) R. A. Hales, U. S.Pat. 2,300,218 (1943); Chem. Abalracls, 37, 1680 (1943). (230) H. R. Hefti and W. Kolb, U. S. Pat. 2,507,973 (1960); Chem. Abslracls, 46, 2340 (1051). (237) W. H. Zartman and H. Adkins, J . A m . Chem. SOC.,66,4569 (1933). (238) H. Hlasiwete and L. Barth, Ann., 138,78 (1806). (239) F. Hoppe-Seyler, Ber., 4] 346 (1871). (240) M. Nencki and N. Sieber, J . prakt. Chem., [2] 24,498 (1881). (241) H. Kiliani, Ber., 41, 3903 (1909). (242) T. M. Lowry and G.L. Wilson, Trans. Faroday soc., 24,883 (1928). (243) J. U. Nef, Ann., 367, 301 (1907); 403, 382 (1914). (244) F. Fisohler, 2.phyaiol. Chem., 167, 1 (1920). (245) G. Klein, Biochem. Z . , 169, 132 (1920). (240) For further details, see J. C. Sowden, Advances i n Carbohydrate Chem., la, 36 (1957).

189

LACTOSE

presence of a small proportion of “p”-D-isosacch&rinicacid, the epimer of (6 a 9 , -D-iuosaccharinic acid, was also re~ealed.2*7-~~~ 3-Deoxy-~-xylo-hcxonic acid (36) (“a”-D-gelartometasaccharinic acid) was first rcrognizcd by Kiliani,260 in 1883, as a product of the prolonged action of lime-water on lactose a t room temperature, and it was later shown to arise from the Dgalactose (34) liberated during the initial ~ t a g e s . 2Montgomery ~~ and HudsonzK1isolated crystalline lac tulose (32) (4-O-@-~-galactopyranosyl-~arabino-hexulose) from the lime-water reaction. Subsequent studies by I s b e I P and Corbett and Kenner32s248 have shown that the major pathway in this degradation of lactose proceeds initially by a relatively slow conversion into lactulose (32) by the Lobry de BruyiiH

C

$

F

T

CH,OH i HOCH H

ye

H-COH OH

CH,OH I HOCH

H$’H-F@{&

+ -

OH

C&?oH

(32)

CHzoH

(33)

‘iOJ

H°FH CH2

HOiH+

other products

-

HFOH CH,OH

(36)

CH,OH

(34)

YHZOH HOCH

(247) (248) (249) (250) (251) (252)

J. U. Nef, Ann., 376,l (1910). W. M. Corbett and J. Kenner, J . Chem. SOC.,2245 (1953); 1789 (1954). J. Kenner and G. N. Richards, J . Chem. SOC.,1810 (1935). H. Kiliani, Ber., 16, 2625 (1883). E. M. Montgomery and C. S. Hudson, J . A m . Chem. Soc., 62,2101 (1930). H. S. Isbell, J . Research NaM. Bur. Standards, 26.35 (1941).

190

CLAMP, HOUGH, HICKSON AND WHISTLER

Alberda van Ekenstein followed by the rapid breakdown of this 8-alkoxy carbonyl derivative, probably through an enediol anioii (33) into D-galactose (34) and “a”-and “/iIJ’-isosacchariiiicacids (35). Ammonium hydroxide a t 37” isomcrizes lactose to lactulosc (32) and, in common with alkalis, causes fragmentation to give D-galactose (34) and its isomerization products (36), together with other substances in which D-lyxose and imidazole derivatives were tentatively identified.263 Lactulose and D-tagatose (D-lpo-hexulose) have been detected in heated milk.264 5. Reuctions with Hydrazines

Fischer first prepared lactose phenylosazone by heating the sugar with aqueous phenylhydrazine.266 He represented the structure of this osazone in acyclic form, but since, like other osazones, it shows mutarotation it was postulated that some form of tautomeric equilibrium or ring formation Ultraviolet-absorption data suggested an acyclic structure for the phenylo~azone.2~7 The extinction curve is characterized by three regions of maximum absorption (at 256 mp, 309-313 mp, and 396-399 mp), the constant value of B at the absorption maximum of the longest wavelength (emor = 20,360, on average) enabling its molecular weight to be determined to within f 2 % on micro-quantities, The spectra of the osazories differ appreciably from those of the bis(pheny1hydrazones) of glyoxal and pyruvaldehyde, owing to presence of the C-3 oxygen function.2u The results of application of the formazan reaction26eto D-arabino-hexose phenylosazone supported the acyclic structure. Periodate oxidation of lactose phenylin agreement with the osazone gave one mole of formaldehyde rapidly,ag*2“0 acyclic formulation. The mutarotation of osazones has been interpreted 261,282 in terms of an electron displacement during solvation of chelate-ring structures (37). In 1887,FischeP noted that lactose phenylosazone is readily converted (253) (254) (1952). (265) (256)

L. Hough, J. K. N . Jones and E. L. Richards, J . Chem. Sac., 2005 (1953). 5. Adachi, Nature, 181, 840 (1958); Nippon NByei-kagaku Kaishi, 32, 802

E. Fischer, Ber., 17, 579 (1884). W. N. Haworth, “The Constitution of the Sugars,” Edward Arnold and Co., London, 1929, p. 7. (257) V. C. Barry, J. E. McCormick and P. W. D. Mitchell, J . Chem. Sac., 223 (1965). (258) J. C. P. Schwarz and M. Finnegan, J . Chem. Sac., 3979 (1956). (259) L. Mester, J . Am. Chem. Soc., 77,4301 (1955). (280) J. E. Courtois, A. Wickstrom and P. L. Dizet, Bull. soc. chzm. France, [5] 19, 1006 (1952). (261) L. F. Fieser and M. Fieser, “Organic Chemistry,” D. C. Heath and Co., Boston, Mass., 1944, p. 351. (2G2) L. Mester and A. Major, J . Am. Chem. SOC.,79, 3232 (1957).

191

LACTOSE

HNPh

HNPh

into a mono-anhydro derivative on treatment with dilute sulfuric acid. Diels and MeyerZ63 showed that many sugar osazones are susceptible to dehydration when boiled in methanol containing a trace of sulfuric acid. The close similarity of the anhydro derivative prepared from D-glucosazone phenylosazone (38) led to its formulation to 3,B-anhydro-D-arabino-hexose as the 3,6-anhydro derivative, but their diacetate esters were later shown to have widely different properties?64

(38)

The “anhydrolactose phenylosazone” was also obtained by the deacetylation of lactose phenylosazone heptaacetate.266 The anhydro compound gave a pentaacetate, thus proving that an oxide ring is present. *Z67 later realized that inversion of configuraHardegger and S~hreier26~ tion occurs a t C-3 during anhydro-ring formation and, consequently, D-arabino-hexose phenylosazone gives rise to 3 ,6-anhydro-~-ribo-hexose phenylosazone (39; R = H). This structure was supported by the results HC;N- NH-Ph

(263) (264) (265) (266) (267)

0. Diels and R. Meyer, Ann., 619, 157 (1935). 0. Diels, R. Meyer and 0. Onnen, Ann., 626, 94 (1936). E. E. Percival and E. G. V. Percival, J . Chem. Soc., 1320 (1937). E. Hardegger and E. Schreier, Helv. Chim.A d a , 56,232,993 (1952). E. Schreier, G. Ytohr and E. Hardegger, Helu. Chim. Acla, 37, 35,574 (1954).

192

CLAMP, HOUQH, HICKSON AND WHISTLER

of the formazan reaction, and evidence was also obtained for chelate-ring formation BayneZBe observed that lactosazone is readily converted into “anhydrolactosazone” in hot alcohol, and he recommended extraction with hot water for purification of the osazone. The water-insoluble “anhydrolactosazone” was identified2B9as 3 ,6-anhydro-4-0-fl-~-galac topyranosyl-D-ribo-hexose phenylosazone (39; R = D-galactopyranosyl) by conversion with aqueous copper sulfate to its phenylosotriazole, which was then hydrolyzed to give D-galactose and the phenylosotriazole of 3,6-anhydro-~-ribo-hexose. Lactosazone was first converted into lactosone by treatment with hydrochloric a ~ i d , ~ ~ but n ~a7better ~ procedure consists in heating under reflux with b e n ~ a l d e h y d e . ~ ~ ~ Hydrogenation of lactose phenylosazone with hydrogen in the presence of a palladium-carbon catalyst affords a little 2-amino-2-deoxylactose (“lactosamine”), but mainly gives 1-amino-1-deoxylactulose.2~~ The mechanism of osazone formation has been studied by the isotopictracer technique, by using an W-labeled arylhydrazone, treating it with an unlabeled arylhydrazone, and making an isotopic assay of the reaction pr0ducts?7~The results were characteristic of Weygand’s mechanism involving the oxidation of the hydrazone to a l-imino-N16-2-ketoderivative (40) and subsequent osazone formation with the elimination of ammoniaNl6. HC=N16-NH-R HboH

I

HC=N”H +

L o I

HC=N-NHR 2

R-NH-NHa

L-N-NHR

+ “‘Ha

I

(40)

Although lactose phenylhydrazone has not yet been crystallized, several substituted phenylhydrazones, including N2-alkyl-N2-phenylhydrazones273 and N2-acylhydraaoncs,n4are suitable for identification purposes. The tolylhydrazones are particularly u~eful,27~ and lactose can he regenerated in high yield by heating the tolylhydrazone with aqueous sul(268) L. Mester and A. Major, J . Am. Chem. Soc., 77,4305 (1955); L. Mester, A d vances in Carbohydrale Chem., 13, 106 (1958). (269) S. Bayne, J . Chew. SOC.,4993 (1952). (270) E. Fischer, Ber.., 22.87 (1889); 44, 1903 (1902). (271) R. Kuhn and W. Kirschenlohr, Chem. Ber., 87, 1647 (1954). (272) E. M. Bamdas, K . M. Ermolaev, V. J. Maimind and M. M. Shemyakin, Chem. & Znd. (London), 1195 (1959). (273) W. Alberda van Ekenstein and C. A. Lobry de Bruyn, Rec. Irau. chtm., 16, 226 (1896). (274) B. Helferich and H . Schirp, Chem. Ber., 86,547 (1953). (275) P. P. T. Sah and C.-Z. Tseu, Sci. Repts. Natl. Tsing Hua Univ., Ser. A , 3 , 403, 409 (1936); Chem. Abstracts, SO. 7105 (1936).

193

LACTOSE

furic acid and ben~aldehyde.~’~ With N-[(methylthio)thiocarbonyl]hydrazine (methyl dithiocarbazinate) (NH2-NH-CS-SMe), lactose forms a highly crystalline h y d r a ~ o n e . ~ ~ ~ 6 . Other Nitrogenous Derivatives

I n alcoholic solution, lactose reacts with amrn0nia,2~~*8~ aniline,280dodecylamine,281urea,%2guanidine,28sand hydroxylamine,26 respectively, to give lactosylamine derivatives. N-Octadecyllactosylamine was formed by condensing octadecylamine with lactose in 2-propanol-water (5 :3 vol./ vol.) at room temperature.284 Occasionally, presence of a trace of acidic zinc chloride, or hydrogen chlocatalyst, such as ammonium chl0ride,2~~ rideza6may be necessary, especially if the amine is a weak base. p-Aminophen012~7and p-phenetidine286 condense with lactose under such conditions. Condensation of lactose with o-phenylenediamine gives a quinoxaline (41), which is transformed into a flavazole derivative (42) on reaction with phenylhydrazine.288The existence of a molecular compound of lactose with

k

I

R

(276) B. Helferich and D. Nachtsheim, Ann., 604, 26 (1957). (277) R. Hull, J . Chem. SOC.,2959 (1952). (278) C. A. Lobry de Bruyn and A. P. N . Franchimont, Rec. trau. chim., 12, 286 (1893). (279) C. A. Lobry de Bruyn and F. H. van Leent, Rec. trau. chim., 14, 134 (1895). (280) F. Micheel, R . Frier, E. Plate and A. Hiller, Chem. Ber., 86,1092 (1952). (281) W. W. Pigman, E. A. Cleveland, D. H. Crouch and J. H. Cleveland, J. Am. Chem. SOC.,73, 1976 (1951). (282)E. Hofmann, Biochem. Z . , 263,462 (1932). (283) H. Wolff, Ber., 88, 2613 (1896). (284)J. G . Erickson, J.Am. Chem. Soc., 77,2839 (1955). (285) R. Kuhn and R. Strobele, Ber., 70, 773 (1937). (286) R. Kuhn and L. Birkofer, Ber., 71, 621 (1938). (287) B. Helferich and A. Mitrowsky, Chem. Ber., 86, 1 (1952). (288) G . Neumuller, Arkiu Kemi, Mineral. Cfeol.,A21, No. 19, 13 (1946).

194

CLAMP, HOUGH, HICKSON A N D WHISTLER

pyridine was sugge~ted2~9 by thermal data, the solidification temperature, of and solubilities. Silver sulfate promotes a rather complex reacti0n2~~ hepta-0-acetyl-a-lactosyl bromide in pyridine to form l-(hepta-O-acetyl-P1actosyl)pyridinium hepta-0-acetyl-/3-lactosyl sulfate (43). ‘OsSOR

(43)

where R = hepta-O-acety1-/3-lactosyl. Interest in N-lactosylamine derivatives of various sulfa drugs has arisen from their ease of dissolution in water; such compounds include derivativw of s~lfanilamide,2~~ sulfapyridine, and sulfag~anidine.~~2 Lactosides of several alkaloids, including theophylline, theobroniine, and morphine, were prepared for the same purpose by their interaction2Yg2B4 with hepta-0-acetyl-a-lactosyl bromide. The development of a brown ~010r294aduring the processing of condensed milk has been attributed to the reaction of the amino groups of protein with la~tose.2~~-2~*The binding of lactose-l-P4with protein was found to occur prior to browning.2geTraces of copper and iron catalyze the reaction, but it is retarded by tin.800N-Lactosylglycine was prepared301 by condensing lactose with glycine in concentrated solution at 95”. Acidic conditions cause the rearrangement of glycosylamines to l-amino-l-deoxy2-ketoses (“the Amadori rearrangement”), as in the case of N-p-tolyllactosylamine.am When lactose was autoclaved with glycine, some maltol (3-hydroxy-2-methyl-4-pyrone)was 5-(Hydroxymethyl-)2(289) G.Pulcher and W. H. Dehn, J . A m . Chem. SOC.,43, 1753 (1921). (290) H.Ohle, W.Marecek and W. Bourjau, Ber., 62,833 (1929). (291) Laboratories Albert Rolland, French Pat. 937,719 (1948); Chem. Abslracls, 44, 2024 (1950). (292) E. W Tillitson, U.5. Pat. 2,374,791(1945);Chem. Abslracls, 40, lG35 (1BlG). (293) N.Froschl, J. Zellner and H. Zak, Monatsh., 66, 25 (1930). (294) E.Leinzinger, Pharm. Acta Helv., a,116 (1947). (294a) See G. P. Ellis, Advances i n Carbohydrate Chern., 14.63 (1969). (295) C. H.Lea, J . Dairy Research, 16, 369 (1948). (296) 8.Patton, J . Dazry Sci., 36, 1053 (1952). (297) J. B. Moeter and R. A. Chapman, Can. J . Research, F27.429 (1949). (298)B. B. Cook, J. Fraenkel-Conrat, B. Singer, A. F. Morgan, R. Buell and J. G . Moiees, J . Nutrition, 44, 217 (1951). (299) 5. Patton and R. J. Flipse, J . Dairy Sci., 36, 786 (1953). (300) B. H.Webb, J . Dairy Sci., 18, 81 (1935). (301) J. Dubourg and P . Devillers, Bull. soc. chim. France, [5]24.333 (1957). (302) S. Adachi, Chem & Znd. (London), 956 (1956). (303) 5. Patton, J . Biol. Chem., 184, 131 (1950).

LACTOSE

195

furaldehyde, furfuryl alcohol, and maltol were identified in autoclaved milk.304 Cyanohydrin formation from lactose was first described by Fischerao5 who used aqueous sodium cyanide in 1890 and later by Hann and in the presence of calcium chloride a t 0". After alkaline hydrolysis, 4-0-8D-galactopyranosy~-D-g~ycero-D-gu~o-heptonic acid was crystallized in 26 % yield.

7. Thioacetals Treatment of lactose with ethanethiol and concentrated hydrochloric acid produces the diethyl dithioacetal of lactose, together with those of ,~~~ to the accompanying hydrolysis. D-galactose and D - ~ ~ u c o s ~owing Lactose diethyl dithioacetal was isolated as a sirup by chromatography on a cellulose column, with 1-butanol half-saturated with water as the mobile phase. Oxidation of the dithioacetal with aqueous peroxypropionic acid gave a crystalline disulfone which was disproportionatedaOgin ammonium hydroxide (pH 10) t,o give bis(ethylsulfony1)methane and 3-0-8-~-galactopyranosyl-D-arabinose. The crystalline dibutyl,3lO d i h e ~ y l , ~and l l dibenzyl d i t h i o a c e t a l ~ of ~ ~lactose ~ - ~ ~ ~have been prepared, the latter in 92 % yield. From the dibenzyl dithioacetal, Stanek and SBda3lZprepared (by the usual procedures) crystalline ethyl a-lactopyranoside and its O-benzylidene, hepta-0-methyl, and hepta-0-acetyl derivatives. A l-thiolactose was prepared by treating 8-lactose with hydrogen sulfide in pyridine for three s308

8. Esters

The acetylation of lactose was examined in detail by Hudson and Johnwith the conclusion that earlier products were mixtures of isomers. Using the acetic anhydride-sodium acetate method,31Bthey were able to obtain a 55 % yield of octa-0-acetyl-8-lactose which, on treatment with zinc chloride in acetic anhydride, was transformed into the a anomer. This (304) (305) (306) (307) (308) (309) (310) (311) (312) (313) (314) (315) (316)

S. Patton, J . Dairy Sci., 93, 324 (1950). E. Fischer, Ber., 29, 930 (1890). R. M. Hann and C. S. Hudson, J . A m . Chem. Soc., 66, 1390 (1934). E . Fischer, Ber., 27, 673 (1894). T. J. Taylor, Ph. D. Thesis, Bristol University, Engl., 1956, p. 123. L. Hough and T. J. Taylor, J . Chem. Soc., 970 (1956). Y. Uyeda and .J. Kamon, Bull. Chem. SOC.Japan, 1.179 (1926). Z. E. Heweili, Chem. Ber., 86,962 (1953). J. Stan6k and J. S d a , Collection Czechoslou. Chem. Communs., 14,540 (1949). B. Gauthier and J. Maillard, Ann. pharm. franc., 11,509 (1953). W. Schneider and 0. Steihler, Ber., 62, 213 (1919). C. S. Hudson and J. M. Johnson, J . A m . Chem. SOC.,37, 1270 (1915). C. Liebermann and 0. Hormann, Ber., 11, 1618 (1878).

196

CLAMP, HOUQH, HICKSON AND WHIBTLER

inversion of configuration a t C-1 has also been achieved in ether-dioxanc containing sodium and sodium hydroxide, although some de-0-acetylation 0ccurred.~*7A crystalline lactose heptaacetate was obtained from the reaction of water, in the presence of silver carbonate, with hepta-0-acetyl-a,319 lactosyl bromide.318 Chromatography of mixed a ,&lactose octaacetates on columns of silica or calcium carbonate is preferable to that on alumina, which causes preferential hydrolysis3%of the acetyl group on the oxygen atom at C-1. Many lactose esters, including those of long-chain fatty havc becn prepared by using the respective acid anhydridesz2in either alkali or pyridine, and the respective acid chloride in p ~ r i d i n e . ~A~ 'mixture of lactose and phenyl isocyanate in pyridine gives an octaphenylurethan derivative of lactose.323The yellow a and p anomers of lactose octakis-[p(p-nitrophenylazo) benzoate] were prepared for chromatographic studies.a24 Lactose octanitrate and partially nitrated products were isolated from the reaction of lactose with a cold mixture of nitric acid and sulfuric acid.3a6m 32 6

There is evidence for the formation of monoalkyl carbonates of lactose when carbon dioxide is added to a solution of the disaccharide in aqueous sodium hydroxide.@? The l-phosphates of a- and &lactose have been synthesized by intcracting silver diphenyl phosphate with hepta-0-acetyl-a-lactosyl bromide, with the elimination of silver b r ~ m i d e , ~followed ~ * * ~ ~by hydrogenolysis of the phenyl group8 and fractional recrystallization of thc barium salts. P-Lactosyl phosphate is more readily hydrolyzed than the a anomer in N hydrochloric acid at 37" (04% and 26 % hydrolysis, respectively, after 80 minutes). The hepta-0-acetyllactosyl halides find important application in thc synthesis of lactosides. Hepta-O-acetyl-cr-lactosylchloride ("a-acetochlorolactose") was first prcparcd by trcatmcnt of octa-0-acetyllactose with hydrogen chloride in acetic anhydride.1Bss330 Later preparations utilizcd, as (317) M. L.Wolfrorn and D. R. Husted, J . Am. Chem. SOC.,SB, 364 (1937). (318)E. Fischer and H. Fischer, Ber., 48,2521 (1910). (319) C.S.Hudson and R. Sayre, J . A m . Chem. SOC.,S8, 1867 (1916). (320) H.Bredereck, H.Durr and K. Ruck, Chem. Ber., 87,526 (1954). (321) J. H.Schwartz and E. A. Talley, J . Am. Chem. SOC.,73,4490 (1951). (322) C. D. Hurd and K. M Gordon, J . Am. Chem. SOC.,65,2667 (1941). (323) L.Maquenne and W. Goodwin, Bull. soc. chim. (France), [3]31,430 (1904). (324) C.H.Coleman, J . Am. Chem. SOC.,67, 381 (1945). (325)w. de c. Crater, u. s Pat. 1,759,565(1930);Chem. Abstracts, 24,3649 (1930). (326) G. GB, Zhur. Russ. Piz.-Khim. Obshchestua, 14, 253 (1882); Ber., 16, 2238 (1W2). (327) B. Srnidt and J. Thomsen, Acta Chem. Scand., 10,1172 (1956). (328) F. J. Reithel and R . G. Young, J . Am. Chem. Soc., 74, 4210 (1952). (329) R. SastLki and K . Taniguchi, Nippon Ndgei-kagaku Kaishi, 39, 183 (1959). (330) Z.H. Skraup and It. Kremann, Monatsh., 22, 375 (1901).

LACTOSE

197

the halogenating agent, liquid hydrogen chloride,=’ phosphorus pentachloride-aluminum chloride in chloroform,w2and titanium t e t r a c h l ~ r i d e . ~ ~ ~ An isomeric chloro derivative termed “a-chloroacetylneolactose” was encountered in low yielda34when the phosphorus pentachloride-aluminum chloride reagent was used; the yield was increased to 20 % by using a large excess of a highly reactive aluminum chloride in chloroform. Octa-Oacetyl-a-neolactose was obtained by treating the chloride with sodium acetate in acetic anhydride, and subsequent deacetylation with barium methoxide yielded crystalline neolactose. Acid hydrolysis of t,his disaccharide gave D-galactose and D-altrose, and neolactose was subsequently (44), produced by showna34~33~ to be 4-O-~-~-galactopyranosyl-~-altrose CHaOH

nb

on (44)

inversion of the configurations of C-2 and C-3 of hepta-0-acetyl-a-lactosyl chloride by the agency of aluminum chloride. Hepta-O-ace tyl-cr-lactosyl bromide and iodide are conveniently prepared by treating octa-0-acetyl-P-lactose with hydrogen bromideal8332 and hydrogen i0dide,~~2 respectively, in glacial acetic acid. The fluoride, prepared by treating the octaacetate with hydrogen fluoride, was found to be appreciably less susceptible to dehalogenation than the other halides336; saponification with sodium methoxide in methanol produces a-lactosyl fluoride. 9. Lactosides Methyl P-lactoside monohydrate is prepared by treating hepta-O-acetyla-lactosyl chloride (or, preferably, the bromide) with methanol in the presence of silver carbonate (the Koenigs-Knorr reaction), followed by saponi7~~ unacetylfication with a trace of sodium methoxide in m e t h a n 0 1 . ~ ~The ated glycoside can be obtained directly from the bromide derivative by the use of magnesium ethoxide in methan01.~~9 (331) E. Fischer and E. F. Armstrong, Ber., 36, 833 (1902). (332) C. S.Hudson and A. Kunr, J . A m . Chem. Soc., 47,2052 (1925). (333) E.Pacsu, Ber., 61, 1508 (1928). (334) A. Kunz and C. 8.Hudson, J . A m . Chem. Soc., 48, 1978,2435 (1926). (335) N.K. Etichtmyer and C. S. Hudson, J . Am. Chem. Soc., 67, 1716 (1935). (336) B.Helferich and R. Coots, Ber., 62,2505 (1929). (337) R.Ditmar, Ber., 36, 1951 (1902);Monatsh., 23,870 (1902). (338) F. Smith and J. W. Van Cleve, J . Am. Chem. Soc., 74, 1912 (1952). (339) F. Smith and J. W. Van Cleve, J . A m . Chem. Soc., 77,3159 (1955).

198

CLAMP, HOIJGH, HICKSON A N D WHISTLER

The Koenigs-Knorr reaction has been used for preparing benzyl heptas 3 l 8 cholesteryl O-acetyl-p-lactoside, Z-menthyl hepta-O-acetyl-~-lactoside,283 hept~a-0-acet~yl-~-lactoside,340 2-hydroxyethyl hepta-0-acetyl-~-lactoside,293 dcoxycort,icosterone hepta-C)-acetyl-~-lactoside,3*1 3-chloropropyl hepta-0Siacetyl-p-lactoside, and 2-chloroethyl hepta-O-acetyl-@-la~toside.~~*-~~~ multaneous saponification and dechlorination of the latter gave3441,2-0cthylene-(4-O-~-~-ga~aetopyranosyl)-~-g~ucose (45). CH,OH

FH,OH

I

OH (45)

Higher oligosaccharides have been prepared by the Koenigs-Knorr reaction of heptJa-O-acetyl-cY-lactosylbromide with suitably protected sugars, followed by removal of the protecting groups. Thus, 6-0-@-lactosyl-nglucose was prepared by t,he use of 1,2,3 ,Ctetra-O-acetyl-~-~-glucopyra n o s ~and , ~ ~6-O-P-lactosyl-D-galactose ~ from 1,2 :3 ,4-di-O-isopropylideneD-galact o p y r a n o ~ e . ~ ~ ~ Hepta-O-ace tyl-a-lact,osyl bromide was reported to give an unknown oct,asaccharide (“tetralactose”) in the presence of silver carbonate.318Potassium benzenethiolate and ht.pta-O-acetyl-cY-lactjosylbromide in alcohol, with subsequent de-O-acetylation, gave phenyl 1-thio-P-lactoside, in which the t,hioglycosidic bond is remarkably stable to acid hydr0lysis.~~7 a general method for the prcparat#ionof Helferich and C r r i e b ~ Pdevised ~~ phcnyl lactoeides by t,reating the potassium phenoxide with hepta-0acetyl-a-lac tosyl bromide i n acetone. 10. Anhydro Derivatives On heating lactosc a t 185’ under 4-6 mm. pressure for 10-12 hours, one mole of wakr per mole is lost,, giving an a n h y d r ~ l a e t o s ewhich, ~ ~ ~ from its (340) (341) (342) 36, 7417 (343) (1938). (344) (345) (346) (347) (348) (349)

H . Lettrt! and A. Hagedorn, 2.physiol. Chem., 242,210 (1936). K . Miescher and C. Meystre, Helv. Chim. Acta, 26, 224 (1943). H. W. Coles and M. L. Dodds, U. S. Pat. 2,252,706 (1941); Chem. Abstracts, (1941). TI. W. Coles, M. L. Dodds and F. H. Bergeim, J . A m . Chem. Soc., 80, 1020 B. Helferich and J. Werner, Ber., 76, 595 (1943). B. Helferich and W. Schafer, Ann., 460,229 (1926). K . Freudenberg, A. Wolf, E. Knofp and S. H. Zaheer, Rer., 61, 1743 (1928). C . B. Purves, J . Am.. Chem. Soc., 61,3619 (1929). R. Helferich and R. Griebel, Ann., 644, 191 (1940). A. Pictet and M. M. Egan, Helv. Chim. A d a , 7,295 (1924).

199

LACTOSE

properties, is probably 1,2-anhydro-4-O-(~-~-galactopyranosyl)-~-glucose. The pyrolysis of a-lactose monohydrate under diminished pressure affords a sirup from which, after condensation with acetone, a 13% yicld of 1,6anhydro-p-D-glucopyranose(46) and a 17.5 % yield of 1,6-anhydro-3,4-0isopropylidene-p-D-galactose (47) were isolated.350 H2C

0

OH

Me

I

OH

1,5-Anhydro-CO-(p-D-galactopyranosyl)-D-glucitol (48) was prepared by the catalytic, reductive desulfurization of 2-naphthyl l-thio-P-lacto~idr.~~

1,6-Anhydro-4-O-(p-~-galactopyranosyl)-~-~-glucose (49) has been made by two methods, both of which involve the base-catalyzed elimination of an electrophilic group from C-1, namely, by the action of barium hydroxide on N-(hepta-O-acetyl-~-lactosyl)-tri-N-methylammonium or of 2.6 N potassium hydroxide on pheiiyl p - l a r t ~ s i d eatJ ~~ 100". ~

11. Unsaturated Derivatives The reduction of hepta-0-acetyl-a-lactosyl bromide with zinc and acetic acid produces hexa-0-acetyllacta1354.355 which, on deacetylation with am(350) A. E. Knauf, R . M. Hann and C. S. Hudson, J . A m . Chen. SOC., 63, 1484 (1941);64, 2435 (1942). (351) H. G.Fletcher, Jr., L. H. Koehler and C. S. Hudson, J . A m . Chern. SOC., 71, 3679 (1949). (352) P.Karrer and J. C. Harloff, Helv. Chim.Acla, 16,962 (1933). (353) E.M. Montgomery, N. K. Richtmyer and C. S. Hudson, J . Ana. Chem. SOC., 66, 1848 (1943). (354) E.Fischer and H. Thierfelder, Ber., 27, 2031 (1894). (355) E.Fischer and G. 0. Curme, Jr., Ber., 47, 2047 (1914).

200

CLAMP, HOUQH, HICKSON AND WHISTLER

monia in methanol y i e l d ~ ~ 6 lactal ~ J ~ 7 (50). Lactal does not reduce Fehling solution, and it behaves as an ethylene derivative, giving dibromo and CH,OH

VH,OH

ii

OH

CH,OH

bH

(51)

(50)

dihydro derivatives by addition reactions with bromine and hydrogen, respectively. Hexa-0-acetyllactal undergoes a facile intramolecular rearrangement on heating in water, to give penta-0-acetylpseudolactal by migration of the double bond to the 2 ,&positions, with the loss of an acetyl groupS66from C-3. Treatment of this acetate with methanolic ammonia generates pseudolactal (51). Attempted saponification of either hexa-0-acetyllactal or penta-0acetylpseudolactal with barium hydroxide gave another rearrangement p r o d ~ c ta, ketose ~ ~ ~ termed ~ ~ ~ isolactal (52). This product was hydrolyzed by &r)-galactotida@ to give u-galactose and D-isoglucal, and it gave B $!H,OH

(52)

pentaacetate with pyridine and acetic anhydride; the acetyl group on the oxygen atom a t C-2 of this pentaacetate can be hydrolyzed by boiling water. Ozonolysis of hexa-0-acetyllactal gives 3-0-/3-~-galactopyranosyl-~arabinose hexaacetate.86* Hydroxylation of lactal with aqueous peroxynamely 4-0-/3-~benzoic acid led to the isolation of only one is0mer,~~~J67 galactopyranosyl-D-mannose (53), although Watters and HudsonSeolater (366) M. Bergmann, M. Kobel, H. Schotte and E. Rennert, Ann., 434,79,86 (1923). (367) W. N. Haworth, E. L. Hirst, M. M. T. Plant and R. J. W. Reynolds, J . Chem. SOC.,2644 (1930). (368) M. Bergmann, L. Zervas and J. Engler, Ann., 608.26 (1933). (369) A . M . Gakhokidze, J . Qen. Chem. U.5.S. R . (EngZ. Transl.), 16,1907 (1946); Chem. Abslraete, 41, 6208 (1947). (360) A. J. Watters and C. 9. Hudson, J . A m . Chem. Soc., 611,3472 (1930).

201

LACTOSE

suggested the presence of an unknown sugar. I n methanol, this reaction gives methyl 4-0-p-~-ga~actopyranosyl-a-~-rnannoside.~~~ ,361 CH,OH

CH,OH

I

OH (53)

Elimination of the elements of hydrogen bromidc from hepta-o-acetyla-lactosyl bromide by means of dimethylamine gives hepta-o-acetyllactoseen-1 ,2 [hepta-O-acetyl-(2-hydroxylactal)](-54) which decolorixes bromine in chloroform and is sensitive to acidic and alkaline hydrolysis.362 CH,OAc

qH,OAc

OAc

OAc (54)

Deacetylation with sodium methoxide, followed by reaction with phenylhydrazine, gave a mixture of three osaxones, presumably D-lyxo-hexose phenylosaxone, 1 ,5-anhydro-D-erythro-hexulose2 ,3-phenylosazone1 and 1,5-anhydro - 4-0(p D - galactopyranosyl) - D - erythro - hexulose 2 ,3 - phenylfrom the intermediary 1,5-anosazone ( 5 5 ) , the latter being formedae3~as4 hydro-4-0-(~-~-galactopyranosy~)-~-arab~no-hexulose (56).

-

V. SOMEPHYSICAL PROPERTIES OF LACTOSE In 1855, Berthelot noticeda66that freshly prepared lactose solutions show a progressive fall in optical rotation with time. This phenomenon, which was soon confirmed,175had previously been reported for D-glUCOSe,s66and was later given the name r n u t ~ r o t a t i o nSeveral . ~ ~ ~ crystalline forms of lac(361) W. N. Haworth, J. Am. Chem. Soc., 62,4168 (1930). (362) K. Maurer, Ber., 69, 25 (1930). (363) M. Bergmann and L. Zervas, Ber., 64, 1432, 2032 (1931). (364) M. Bergmann and K. Grafe, J . Biol. Chem., 110, 173 (1935). (3ti5) M. Berthelot, Forlschr. Physik, 11, 316 (1855). (366) A. P. Dubrunfaut. Compt. rend., 23, 38 (1846). (367) T. M. Lowry, J . Chem. Soc., 76, 211 (1899).

202

CLAMP, HOUGH, HICKSON AND WHISTLER

I

NH

h

I

NH

I

Ph

(56)

tose were described368J6e which differed in their initial optical rotations. Tanret3T0recognized “ordinary” or “a”-lactose with [aID +SSO (initial, in + 5 5 O to +55.5’ (initial, in water)-the water), “@”-lactosehavingDI.[ precise value varying slightly with its rate of precipitation, and “y”-lactose withDI.[ +34.5’ (initial, in water). These substances were considered to be three distinct forms of lactose, the changes in optical rotation of aqueous solutions of “a”-and of “y”-lactose being ascribed to their conversion to the “p” form. In the rase of D-glucose, however, it was shown37’ that the “p” form is an equilibrium mixture of the “a”-and “y”-D-glucoscs. In addition, it was suggested that these two forms are stereoisomers (mated by the formation of a ring, supposedly a furanoid or, as it was then called, a y-oxide ring. This conclusion was extcnded to lactos~,~72 and the “a”-and y $ 9-1at:toscs w r c rcde~ignated~7~ ~7~ a-lactose hydratc and @-lac tow, rc((

spectively (see Tablc I). In addition, two other forms are known, anhydrous a-lactose which can be preparedsY6from a-lactose hydrate, and lactose (368) E. 0.Erdniann, Ber., 13, 2180 (1880). (369) M.Schmoeger, Ber., 13, 1915, 1922,2130(1880). (370) C.Tanret, Bull. soc. chim. (France), 18,349 (1896). (371) E.F.Armstrong, J . Chem. Soc., 83, 1305 (1903);T . M.Lowry, ibid., 83, 1314 (1903). (372) C.Tanret, Bull. soc. chim. (France), 33, 337 (1905). (373) C. S. Hudson and F. C!. Brown, J . A m . Chem. Soc., SO, 960 (1908). (374) C. S. HudRon, J . A m . Chem. Soc., 31, 66 (19011). (375) C. S. Hudson, Princeton Uniu. Bull., 13, 62 (1902).

203

LACTOSE

gla~s.~76 Other crystalline forms of lactose have been de~cribed,~77 but these appear to be molecular complexes of the a and p anomers. a-Lactose hydrate and p-lactose arc stahle a t room t e m p ~ r a t u r e , ~al7~ though their crystalliiie forms may vary to some a-Lactose hydrate, which is less hygroscopic than maltose or teiids to 3s2 its water of crystallization a t temperatures as low as 80", and the kinetics of this dehydration process have been Anhydrous a-lactose is meta~table,~8~-~86 being converted into p-lactose above 93.5'; below this temperature, in the presence of water, it reverts to the a-hydrate. a- and p-Lactose, whose anomeric configurations had been s u g g ~ s t c d ~ * ~ by their relative conductivities in boric arid solutions, arc interconverted by the mutarotation reaction. Calculations3*8-3Q0 have indicated that the transition temperature of this process is 93.5" and that the equilibrium constant is 1.54 a t room temperature, 1.65 a t 0", arid 1.41 (1.33) a t 100". TABLEI Melting Point and Optical Rotation of the Lactose Anomers Molecular weight a-Lactose hy- 360.3 drate (Y -Lac tose 342.3 &Lactose 342.3

Melting point, "C.

[a],, degrees

202

$85

223 252

$90 +34.9

+ 4

-+

Solvent

References

$52.6

HzO

+55.3 +55.3

He0 H20

384, 385, 389, 397, 398, 44G 201, 384 201, 385,389,397

The rate constants for the mutarotation reaction have also been determined391-594 under a variety of conditions, catalysis being more marked (376) B. L. Herrington, J . Dairy Sci., 17, 501 (1934). (377) R. C. Hockett and C. S. Hudson, J . A m . Chem. SOC.,63,4455 (1931). (378) M. G. ter Horst, Rec. trav. chim., 72, 878 (1953). (379) B. L. Herrington, J . Dairy Sci., 17, 533 (1934). (380)E. T. Wherry, J . Wash. Acad. Sci., 18, 302 (1928). (381) A. Sokolovsky, Znd. Eng. Chem., 29, 1422 (1937). (382) B. L. Herrington, J . Dairy Sci., 17, 595 (1934). (383) R. P. Choi, C. M. O'Malley and B. W. Fairbanks, J . Dairy Sci., 20,639 (194G). (384)A. Smits and J. Gillis, Proc. Acad. Sci. Amsterdam, W ,520, 573 (1918). (385) J. Gillis, Rec. trav. chim., 39, 88 (1920). (386) W. Mohr and J. Wellm, Milchwirtsch. Forsch., 17, 109 (1935). (387) J. Boeseken, Advances i n Carbohydrate Chem., 4,205 (1949). (388) C. S. Hudson, J . Am. Chem. SOC.,30, 1767 (1908). (389) J. Gillis, Rec. trav. chim., 39, 677 (1920). (390) R. Niguet, Znds. agr. et aliment. (Paris), 71,327 (1954). (391) C. S. Hudson, Sci. Papers Bur. Standards, 21,267 (1926). (392) H. C. Troy and P. F. Sharp, J. Dairy Sci., 13,140 (1930). (393) B. L. Herrington, J . Dairy Sci., 17,659 (1934). (394) J. C. Kendrew and E. A. Moelwyn-Hughes, Proc. Roy. Soc. (London), A176, 352 (1940).

204

CLAMP, IIOUGH, HICKSON A N D WHISTLER

in alkali2'2Jg6 than in acid. Salts of weak acids or bases also accelerate the pr0cess,~~3 but it is retarded by neutral salts and by deuterium oxide.8ge The proportions of a- and 8-lactose in an equilibrated solution have been calculateds97 to be 36.8 % and 63.2 %, respectively, from optical-rotation studies, and 37.5 % and 62.5 % by bromine oxidation; the D-glucose moiety cannot adopt the furanoid ring and the percentage of open-chain form was assumed to be negligible. The specific rotation of such a solution depends, amongst other things, on the concentration of the sugar, the temperature, and the presence of s<s.8g8-400 The temperature ( t ) coefficients have been determined401for the equations:

+ (1 - 20) 0.072" 61.94" + ( 1 - 20) 0.085".

[a]:= 52.40" [a]:460

Expressions for the dependence on temperature and concentration (where c is the concentration in g./lOO ml. of solution) are as f o l l 0 ~ ~ : ~ 7 ~

(a) for solutions of constant volume: [a]; = 52.00"

- 0.006c" -

(1

- 20) 0.037'

61.77"

- 0.007~'-

(1

- 20) 0.043'.

[a];4ao

5

(b) for solutions of constant weight: [a]: [aha0

= 52.60'

- 0.OoBc' -

(1

- 20) 0.065".

61.77'

- 0.007~"-

(1

- 20) 0.076".

0

The solubility of lactose in water is known over a wide temperature range,988the final solubility at825" being 17.8 g./100 ml. of solution, and its solubility in salt solutions403and in various organic solvents has also been reported,201,891,404,406 Adsorptive properties are exhibited by lactose, not only for certain 390,40*

(395) (396) (397) (1937). (398) (399) (400) (401)

F. Urech, Ber., 16, 2130 (1882); 16, 2270 (1883). J. Nicolle and F. Weisbuch, Compt. rend., 140,84,1340 (1955). H. 8. Isbell and W. W. Pigman, J . Research Natl. Bur. Slandarde, 18, 141

H.Trey, 2. phyeik. Chem. (Leipzig), 48, 620 (1903). W. H. Glover, J . Cheni. SOC.,98, 379 (1911). B. L. Herrington, J . Dairy Sci., 17, 701 (1934).

A. L. Bacharach, Analyst, 48, 521 (1923). (402)E. Saillard, Chim. & i d . (Paris), 2, 1035 (1919). (403) B. L. Herrington, J . Dairy Sci., 17, 805 (1934). (404) C. A. Lobry de Bruyn, 2. physik. Chem. (Leipzig), 10, 784 (1892). (405) J. G. Holty, J . Phys. Chem., 8, 764 (1905).

LACTOSE

205

vitamins as it crystallizes from sol~tion,~06-~~* but, with a wider application, as a column for chromatographic ~ e p a r a t i o n s . ~ ~ ~ - ~ ~ ~ The claim41s~414 that lactose shows the ultraviolet absorption that is characteristic of carbonyl groups has been di~puted.~l6 Further bands develop when lactose is warmed in alkaline s o l ~ t i o n . ~The 1 ~ infrared spectra of t42O have also been published. lactose417-419 and of various derivatives41@ Many thermodynamic properties of lactose are known, including its heat of c o m b u s t i ~ n , of ~ ~solution,s~s~a~b~424 ~-~~~ and of diluti0n~2~; its heat c a p a ~ i t y , ’ ~entropy,420 ~ , ~ ~ ~ ,and ~ free its mean energy and entropy of activation through a collodion membrane42*;and the specific heats of lactow Other physical properties of lactose solutions that have been investigated *432-4s3 v i s c o ~ i t y , 4rc~~,~~~ include the rate of crystalliaation,4s0~4a1 density,808 9429

(406) A Leviton, Znd. Eng. Chem., 36. 589 (1943) ; 36, 744 (1944). (407) W. Werner, hfilChWi88en8Chaft,1, 11 (1946). (408) W. L. Owen, U. S. Pat. 2,640,778 (1953); Chem. Abstracle, 47, 8397 (1953). (409) G. M. Henderson and H. G. Rule, J . Chem. SOC.,1568 (1939). (410) R. Fischer and W. Iwanoff, Arch. Pharm., 281,361 (1943). (411) V. Prelog and P. Wieland, Helu. Chim. A d a , 27,1127 (1944). (412) H. J. Lennartz, Z . anal. Chem., 128, 271 (1948). (413) J. E. Purvis, J. Chem. SOC.,123, 2515 (1923). (414) P. Niederhoff, Z . physiol. Chem., 166, 130 (1927). (415) L. Kwiecinski and L. Marchlewski, Bull. intern. acad. polun. sci., 379 (1927) ; Chem Abstracts, 22, 2108 (1928). (416) W. Gabryelski and L. Marchlewski, Biochem. Z . , 266,50 (1933). (417) L. P. Kuhn, Anal. Chem., 22, 276 (1950). (418) J. D. S. Goulden, J. Sci. Food Agr., 7,609 (1956). (419) Y. Tsuzuki and N. Mori, Nippon Kagaku Zasshi, 77, 993 (1956) ; ChenL. A b tJ~rWl8,62, 271 (1958). (420) H. S. Isbell, F. A. Smith, E. C. Creitz, H. L. Frush, J. D. Moyer and J. E. Stewart, J. Research Natl. Bur. Standards, 69,41 (1957). (421) A. G. Emery and F. G. Benedict, A m . J. Physiol., 28,301 (1911). (422) P. Karrer and W. Fioroni, Ber., 66, 2854 (1922); Helu. Chim. A d a , 6, 396 (1923). (423) T. H. Clarke arid G. Stegeman, J. A m . Chem. SOC.,61, 17’26 (1939). (424) W. F. Magie, Phys. Rev., 16, 381 (1903). (425) E. Lange and H. G. Markgraf, Z . Elektrochem., 64,73 (1950). (426) E. F. Furtsch and G. Stegeman, J. A m . Chem. SOC.,68,881 (1936). (427) A. G. Anderson and G. Stegeman, J . A m . Chem. Soc., 63, 2119 (1941). (428) K. E. Shuler, C. A. Dames and K. J. Laidler, J. Chem. Phya., 17,860 (1949). (429) K. Bennewitz and L. Kratz, Physik. Z . , 37,496 (1936). (430) J. D. Jenkins, J . A m . Chem. SOC.,47, 903 (1925). (431) E. 0. Whittier and S. P. Gould, Znd. Eng. Chem., 23,670 (1931). (432) W. Fleischmann and G. Wiegner, J. Landu*irtsch., 68,45 (1910). (433) E. J. McDonald and A. L. Turcotte, J. Assoc. O@c. Agr. c‘henaists, 31, 687 (1948); J . Research Natl. Bur. Standards, 41, 63 (1948). (434) 0. Pulvermacher, Z . anorg. allgem. Chem., 113,141 (1920). (435) H. S. Owens, J. A m . Chem. SOC.,62,930 (1940).

206

CLAMP, HOUGH, HICKSON AND WHISTLER

fractive ,436 8437 osmotic pressure:% vapor pres~ure,~~Q freezing-point d e p r e s s i ~ n,440 , ~,441 ~ velocity of dialysis,"z and interfacial tension with organic l i q ~ i d s . ~ ' ~ - ~ ~ ~ F. W. Zerban and J. Martin, J . Assoc. O,&. Agr. Chemists, 32, 709 (1949). H. P. Rieder, Ber. schweiz. botan. Gee., 61, 539 (1951). E. 0. Whittier, J . Phys. Chem., 37, 847 (1933). E. 0. Whittier and S. P. Gould, Znd. Eng. Chem., 92,77 (1930). E. H. Loomis, 2.physik. Chem. (Leipaig), 37, 407 (1901). W. J. Husa and 0. A. Roaai, J . Am. Pharm. Assoc., Sci. Ed., 31,270 (1942). F. Klages, Ann., 620, 71 (1935). N. S. Stroganov, Proloplasma, 24,431 (1936). (444)J. B. Mathews, Trans. Faraday Soc., 36. 1113 (1939). (446) H. W. Douglas, Trans. Faraday Soc., 46. 1082, 1090 (1950). (446) C. S. Hudson, 2.phyeik. Chem. (Leipzig), 44,417 (1903); J . A m . Chem. Soc.,

(436) (437) (438) (439) (440) (441) (442) (443)

32, 889 (1910).

GLYCOLIPIDS OF ACID-FAST BACTERIA

BY EDGARLEDERER Laboratoire de Chimie biologique, Facult6 des Sciences, Paris, and Institut de Chimie des Substances Naturelles, Gif sur Yvette, Seine et Oise, France I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 . . . . . . . . . . . . . . 209 11. Chemistry of Glycolipids of Acid-fast Bacte . . . . . . . . . . . . . . . 209 1 . Esters of Carbohydrates.. . . . . . . . . . . . . . . . . . . . 2. Glycosidic Glycolipids.. . . 111. Biological Activities of Glycolipids of Acid-fast Bacteria 1. Tissue Reactions. . . . . . . . . . . . . . . . . . . 231 2. Inhibition of Enzymes. . . . . . . . . . . . . . . . . . . . ............................................ 231 . . . . . . . . . . . . . . 232 G . Establishment of a Delay 7. Adjuvant Action.. . . . . . . . 8. Immunization.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237

I. INTRODUCTION Our knowledge of the chemistry and biological activities of glycolipids of acid-fast bacteria is fairly recent; it is only in the last few years that several of these compounds have been isolated in a pure state and that their chemical structure has been more or less fully elucidated. Separation in a pure state has only been possible through the application of chromatographic methods, and structural determinations have been greatly facilit,ated by infrared spectrometry. The biological activities of several glyco- and peptidoglyco-lipids of acidfast bacteria have been studied, and we shall see (in the corresponding Sections) that some most interesting properties have been discovered, such as a peculiar form of toxicity, adjuvant action, and immunization. Some of the glycolipids under consideration are specific for certain types of bacteria, and their presence in or absence from a particular strain can be used for taxonomical purposes. As concerns the chemistry of these glycolipids, they contain either an ester linkage between the sugar moiety and the lipid moiety, as in “cord factor” and in wax D of Mycobacteria, or they are glycosides of phenolic alcohols (as in mycoside B) or of inositol (as in the phosphoglycolipids), or they can belinked to the carboxyl group of D-alanine (as in mycoside C). 207

208

EDGAR LEDERER

TABLEI Carbohydrates Found i n cfiycolipids of Acid-fast Bacteria

GIycolipid

Carbohydralc

I. Pentose D- Arabinose

wax D

11. Hexoses D-Glucose

cord factor

D-Glucose D-Mannose

phospholipid phospholipid

D-Mannose D-Galactose

wax D

Type oj linkage of the carbohydrate in a polysaccharide, with D-galactose and D-mannose, esterified with mycolic acid as a,ru-trehalose, eeterified with mycolic acid linked to inoeitol as a - ~(1-1 - 6) -D-mannobiose linked t o inositol in a polysaccharide 8sterified with mycolic acid

111. 6 - D e o ~ y h e ~ a e s ~ 2-0-Methylfucose 2,4-Di-O-methylrham- mycoside A nose mycoside A and B 2-0-Methylrhamnose mycoside C 6-Deoxytalose (1 6-Deoxy-3-0-methyltalose ti 3,I-Di-0-methylrhamnose

1

-2 3-6 7-10

1

--

~

~~~

1, 12

~

IV. Amlno sugars D-Ghcosamine D -Galactosamine 0

I

The configuration (D or

wax D L)

as part of a polysaccharide

of these sugars is not yet known.

(1) J. Asselineau, H. Buc, P. Joll&sand E. Lederer, Bull. (1959) *

SOC.

chim. biol., 40, 1963

(2) H. Noll, H. Bloch, J. Awelineau and E. Lederer, Biochim. et Biophyu. Acta, 10, 299 (1966).

R.J. Anderson and E. Cf. Roberts, J . Biol. Chem., 89,611 (1930). (4) G. Michel and E. Lederer, Compt. rend., 240, 2464 (1966). (6) G. Michel, Bull. soc. ch,im. biol., 41, 1649 (1969). (6) E. Vilkas, Compt. rend., 248,604 (1969); Bull. soc. chim. biol., 42, 1006 (1960). (7) R. J. Anderson and A. G.Renfrew, J . Am. Chem. Sbc., 62, 1262 (1930). (8) R. J. Anderson, W.C. Lothrop and M. M. Creighton, J . Biol. Chem., 126, 299 (1938). (3)

OLYCOLIPIDS OF ACID-FAST BACTERIA

209

The carbohydrates found in the glycolipids of acid-fast bacteria are either very common sugars, such as D-arabinose, D-glucose, D-mannose, or a ,atrehalose, or are rare O-methylated deoxyhexoses. Table I gives a survey of the carbohydrates found in the glycolipids considered in the present article. The polysaccharides of Mycobacterium tuberculosis were reviewed some years ago by Stacey and Kent.la The lipid moiety of the glycolipids under consideration is either composed of branched-chain fatty acids (for example, mycolic acids in cord factor and in wax D) or can be a wax formed by the union of a phenolic alcohol with branched-chain fatty acids of the mycocerosic acid type, as in mycosides A and B. In the phosphoglycolipids, simple normal fatty acids have been found, accompanied by some methyl branched acids. Four principal types of glycolipid will be considered in the present review: (1) cord factor; (2) wax D; (3) type-specific mycosides; and (4) phosphoglycolipids. All recent work on these compounds is a development of the excellent work of Anderson and his published between 1926 and 1946. Of all the acid-fast bacteria, Mycobacteria have been studied most extensively: thus, most of our knowledge of glycolipids concerns Mycobacteria, and only occasional mention of Corynebacteria or other acid-fast micro-organisms can be found.

11. CHEMISTRY OF GLYCOLIPIDS OF ACID-FAST BACTERIA 1. Esters of Carbohydrates a. Esters of Treha1ose.-Anderson and Newmads reported that the “fat” of Mycobacteria contains trehalose (a-D-glucopyranosyl a-D-glucopyranoside), which they isolated in crystalline form from the acetone-soluble “fat” of three human strains; trehalose ha8 been isolated after saponification of the acetone-soluble fat of the human strain L‘Br&annes’’16and of M . phlei’7; until now, however, no fatty ester of trehalose has been isolated (9) E. Vilkas and E. Lederer, Bull. soc. chim. biol., 38, 111 (1956). (10) E. Vilkas and E. Lederer, Bull. soc. chim. biol., 41, 1013 (1960). (11) A. P. MacLennan, D. W. Smith and H. M. Randall, Biochem. J., 80,309 (1961). (12) D. W. Smith, H. M. Randall, A. P. MacLennan and E. Lederer, Nature, 186, 887 (1960). (13) M. Stacey and P. W. Kent, Advances in Carbohydrate Chem., 3, 311 (1949). (14) R. J. Anderson, Harvey Lectures, Ser. 35, 271 (1939); Fortschr. Chem. org. Naturstofle, 3, 145 (1939); Chem. Revs., 19, 225 (1941). (15) R. J. Anderson and M. S. Newman, J . Biol. Chem., 101,499 (1933). (16) A. Aebi, J. Asselineau and E. Lederer, Bull. aoc. chim. biol., 36, 661 (1953). (17) M. Barbier, Doctor’s Thesis, Univ. of Paris (1953).

210

EDGAR LEDERER

in a pure state from the acetone-soluble fat of Mycobacteria, and recent papers report that large proportions of glycerides are present in these fractions.18-20 Esters of trehalose with higher fatty acids have also been found in C . diphtheriae.21 ( 1 ) Cord Factor.-Robert KochZ2had long ago noticed that some strains of tubercle bacilli grow in long filaments. Dubos and c o ~ o r k e r sshowed ~~ that only virulent strains show “serpentine cords” when grown in vitro, and suggested that the cord-like growth-pattern is due to a chemical substance on the bacterial surface. BlochZ4confirmed this hypothesis, showing that petroleum ether disrupts the bacterial cords without destroying their viability, and he extracted a waxy material having a high toxicity for mice. The active material in this extract was called “cord factor.” The purification and isolation of “cord factor” was greatly facilitated by its particular delayed toxicity for mice (upon repeated injections of microgram quantities). For detailed reviews on cord factor, see Refs. 25 and 26. (2) Isolation of Cord Factor.-In Bloch’s first it had been shown that a petroleum-ether treatment of living bacteria extracts a crude lipid having the particular toxicity of cord factor. As the yield in these extractions was rather low, other lipid fractions extracted from whole bacilli were e ~ a m i n e d ~and 7 . ~it~was found that cord factor is present in preparations of wax C and wax D2e-principally, however, in the former. Pure cord-factor was finally isolated by repeated chromatography on magnesium silicate, silicic acid, and silica gel. From the H37 Rv strain and from BCG, cord factor was obtainedaoJ1as a colorless wax, melting at about 40°, [a]D +30°. (3) Chemical Structure of Cord Factor.-A first important clue to the (18) H. Bloch, J. Defaye, E. Lederer and H. Noll, Biochim. et Biophys. Acta, 23, 312 (1957). (19) H.No11 and E. Jackini, J . Rial. Chem., 2X2.903 (1958). (20) J. Asselineau and J. Moron, Rull. sac. chim. biol., 40,899 (1958). (21) E.L. Alimova, Biokhimiya, 20, 516 (1965);24, 785 (1959). (22) It. Koch, Mitt. kgl. Gesundh., 2. 1 (1884). (23)G.Middlebrook, R. J. Duhos and C. Pierce, J . E z p l l . Med., 76, 175 (1947). (24)H.Bloch, J . Expll. Med., 91, 197 (1950). (25) H.Noll, Bibliotheca Tuberc., Suppl. Schweie. 2. Tuberk. u. Pneumonol., 7 , 149 (1956). (26)E. Lederer, Festschr. Arthur Stall, 384 (1957). (27) H. No11 and H. Bloch, Am. Rev. Tuberc., 67, 828 (1953). (28)J. Asselineau, H. Bloch and E. Lederer, Am. Rev. Tuberc., 67, 853 (1953). (29)Mycobacteria were extracted according to the method of Anderson,*4first with alcohol-ether (l:l), and then with chloroform; the chloroform extract contains a fraction insoluble in boiling acetone (wax D) and one which is soluble in hoiling acetone and is precipitated on cooling (wax C). (30) H.No11 and H. Bloch, J . Biol. Chem., 214, 251 (1955). (31) J. Assclineau and E. Lederer, Biochim. et Biophys. Acla, 17, 161 (1955).

GLYCOLIPIDS OF ACID-FAST BACTERIA

21 1

chemical structure of cord factor was obtained by N011,~~ who found that saponification of more-or-less pure, cord-factor preparations gave 85 % of mycolic acid C S S H I ~f ~ O5 ~CH2 and a nonreducing glycoside which, on acid hydrolysis, gave D-glucose as the only sugar. Mycolic acids are characteristic constituents of Mycobacteria, discovered in 1938 by Lesuk and Anderson.38 For recent reviews on the structure of these “high molecular weight 8-hydroxy acids with a long a-side chain,” see Refs. 34-36. From degradative evidence and biosynthetic considerations, the two formulas (1) and (2), both corresponding to the molecular formula CBBH11804,can actually be considered to be the most probable for mycolic acids of human strains of Mycobacteria. The -OH group at C-5 or C-7 is, in human strains of M . tuberculosis, frequently methylated. c25H51-

8”

OH

H-CII--CH2-CH-C:H--CH--COIH AlEHl3

Cz5Hbi-CHz-CH-

(1)

AlEH33

Az,H~~

XH 8” H-CH-

H-CH-COZH

The following nomenclature has been proposed for the mycolic acids of Mycobacteria.3KThe term “mycolanoic acid” is used to designate the parent acid without its hydroxy, methoxy, or 0x0 groupings (c.&17&). The functional groups are designated in the usual manner (for example, 3-hydroxymycolanoic acid for CaHl740J. It is necessary to add the order of elution and the name of the strain from which the mycolic acid has been isolated. This is done in square brackets; for example, a-mycolic acid TestS6is designated as: 3-hydroxy-x-methoxy-mycolanoic acid [1-Test]; the y-mycolic acid of BCG (a mycolonic acid)3?is 3-hydroxy-x-0x0mycolanoic acid [3-BCG]. Anderson]‘ used the formula CBBHL7604 for a methoxylated mycolic acid; that is why a formula with 87 carbon atoms is used for the methoxylfree mycolanoic acid. All these formulas are considered to have a margin of error of * 5 CHI.

From the proportions of mycolic acid obtained by alkaline saponification (32) H. Noll, Intern. Congr. Microbiol. 6th Congr. Rome, 1, 191 (1953). (33) A. Lesuk and R. J. Anderson, J . Biol. Chem., 126,505 (1938). (34) J. Asselineau and E. Lederer, Fortsch. Chem. org. Naturstofle, 10, 170 (1953). (35) J. Asselineau and E. Lederer, Ciba Foundation Symposium Exptl. Tuberc. B a c i h s and Host, 14 (1955). (36) J. Asselineau and E. Lederer, in “Lipide Metabolism,” K. Bloch, ed., John Wiley and Sons, Inc., New York, N. Y.,1960,p. 337. (37) A Ginsburg and E. Lederer, Biochim. et Biophys. A d a , 9,328 (1952).

212

EDGAR LEDERER

of cord factor, it could be calculated that there was one molecule of mycolic acid present for each molecule of D-glucose. Then, the nonreducing, sugar component was identified its a,a-trehalose through the isolation of its crystalline octaacetate, m.p. 83") which was identified by mixed melting point and comparison of its infrared spectrum with that of an authentic sample. Thus, cord factor is a dimycolate of trehalose, and has the molecular formula ClssHaaaOlr f 10 CH2. In order to determine which of the hydroxyl groups of trehalose are esterified with mycolic acid, cord factor was methylated and the methylated cord-factor was then saponified; the hexa-0-methyltrehalose thus obtained was hydrolyzed by dilute acid, and from this, a tri-0-methyl-D-glucose resulted which gave only one spot on paper chromatograms; this proved that cord factor is symmetrical, that is, that, on each D-glucose moiety, the mycolic acid is esterified with the same-numbered hydroxyl group. A painstaking chromatographic and infrared study of the tri-0-methylD-glucose obtained from methylated cord-factor then showed that it was

T

( 3)

H1O- CO-

c

H-

r

H- CaoIi,p&OH)

PH

(4)

identical with 2,3,4-tri-0-methyl-~-glucose (3) ; thus, cord factor must be2 6 ,6'-di-O-mycoloyl-a ,a-trehalose (4). Table I1 illustrates the degradation of cord factor and of its octamethyl ether. (4) Syntheses of Cord Factor.-At an early stage in the work on cord factor, when it seemed that a mycolate of an amino sugar might be involved (the first preparations contained about 1 % of nitrogen, corresponding to one N atom per hexose mycolate), a series of mycolates of D-glucose, Dgalactose, and D-glucosamine were prepared, wherein mycolic acids isolated from Mycobacteria were esterified with the C-1-, C-2-, or C-6-hydroxyl group of these sugars [see, for example, formulas ( 5 ) ) (6)) (7a), (7b), and (8)l."" Some of these compound8 had a certain degree of cord-factor toxicity; (38) J. Asselineau and E. Lederer, Bull. 8oc. chim. France, 1232 (1966).

213

GLYCOLIPIDS OF ACID-FAST BACTERIA

in particular, those in which the C-6-hydroxyl group of the hexose was esterified with mycolic acid. As soon as structure (4) was certain for cord factor, efforts were made to prepare 6 ,6'-diesters of trehalose with mycolic acids, so as to confirm strucTABLE11 Degradation of Cord Factor26 Cord factor CISS&WOI~ (methylation) (reductive cleavage with LiAIH,)

(alkaline hydrolysis)

I

1

(alkaline hydrolysis)

.L

Mycolic acid CesH1~04

mycolic alcohol C.ssH17aOa

+ 2,3,4,2',3',4'hexa-0-methyltrehalose

I

Trehalose CinHni0I I

I

(acetylation) Trehalose octaecetate m.p. 81-82"

1

methylated mycolic acid

(acid hydrolysis)

2,3,4-tri0-methyl-D-glucose

acid hydrolysis)

I

D-glUCOSe (oxidation)

potassium D-gluconate m.p. 180"

ture (4) by synthesis ;also, a series of simpler 6 ,6'-diesters of trehalose were prepared, in order to permit study of the relationship of chemical structure and biological activity in this field. Four methods of synthesis of cord factor and its analogs have been developed. Method (i).-The esterification of one mole of trehalose with two moles of the acid chloride of an acetylated mycolic acid (in pyridine) gives a mix-

214

EDGAR LEDERER

CKOH

'

H4-?4H40 HO H

R OCH,

-HO q fl)

n

H

R-OH

1)) R

q

P

PH

NH-CO- (iH- CH- C e , H ~ 2 , ( O C H ~ C?4H,,

NH-COCH, ( 81

( 7)

ture of 6-mono-, G , G'-di-, and 2 , 6 ,6'-trimycolates which can be separated by chromatography on silicic acid.3e A di-0-acetyl-G,6'-di-0-mycoloyl derivative of trehalose (CIg2H374Olef 10 CHp , m.p. 35-37', [ a ]+25') ~ (9a), obtained by this method, had apHZO-CO-CH-

I

OR CH- C,,H,ZO(OCH,)

CZ4H4,

I! HO

-0

H

OH

OH

-C

OH

YR

H 0 -~H-CH-C,,Hl2,,(OCH~) CZ4H4Q

(39) J. Polonsky, G . FerrBol, It. Toubinna and E. Lederer, Bull. 1471 (1956).

BOC. chim. France,

215

GLYCOLIPIDS OF ACID-FAST BACTERIA

proximately the same biological activity as natural cord-factor, and its infrared spectrum was nearly identical with that of the natural product, except for a band a t 8.1 1.1 corresponding to the presence of acetyl groups. The deacetylation of this product with sodium methoxide or hydroxylamine (mycolic acid esters react very slowly with hydroxylamine) gives, in trehalose, rather low yield, a 6,6'-di-0-(3-hydroxy-x-methoxymycolanoyl) m.p. 4042O,[ a ] ~132O,C1&?H37001?f 10 CHZ(9b), with an infrared spectrum identical with that of the natural cord-factor; the biological activity of this compound was indistinguishable from that of natural cord-fa~tor.~" This method has the disadvantage of low yield, because of (a) the simultaneous formation of mono- and tri-esters and (b) the necessity for a preliminary protection of the free hydroxyl group of the mycolic acid by acetylation. Method (ii).-A method which seemed more promising for the specific synthesis of 6,6'-diesters of trehalose was then first studied with synthetic mycolic acids of lower molecular weight.39This method consists in heating the potassium salt of an acid with 2,3,4,2',3',4'-hexa-O-acetyl-6,6'-di-Op-tolylsulfonyltrehalose (lo), a compound already described by B r e d e r e ~ k . ~ ~

+

Y

FH,OTs

I

H

I

OAc

I H

OAc

(10)

It is known41athat the p-tolylsulfonyloxy group has the properties of a "pseudo-halogen" and that treatment of a p-toluenesulfonate by the salt of an acid can produce the replacement of the p-tolylsulfonyl group by the acyl group, through a SN2 reaction (for references, see Ref. 39). By using a natural mycolic acid (from the strain Test) and Bredereck's compound (lo), a 2,3,4,2', 3' 4'-hexa-0-acetyl-6,6'-di-O-mycoloyltrehalose (ll),C198H378023 f 10 CHZ,m.p. 39",[ a ]+44", ~ was obtained in 90 % yield. Deacetylation with hydroxylamine gave a 6,6'-di-O-mycoloyltre~ having all the physical and biological properhalose, m.p. 39-40', [ a ]+33", ties of natural c o r d - f a ~ t o r . ~ ~ Method (iii.)-As the deacetylation step in the previous synthesis is not easy to operate in good yield, a still simpler synthesis was devised.426 6'-Di)

)

(40) T. Gendre and E. Lederer, Bull. SOC.chim. France, 1478 (1956). (41) H. Bredereck, Ber., 63, 959 (1930). (41a) See R. S. Tipson, Advances i n Carbohydrate Chem., 8 , 107 (1953). (42) G. BrocherB-FerrBol and J. Polonsky, Bull. P O C . chim.France, 714 (1958).

216

EDGAR LEDERER

OH

H

OAc

(11)

0-p-tolylsulfonyltrehalose (12)was prepared by heating 1 mole of trehalose with 1.5 moles of p-toluenesulfonyl chloride in pyridine. After chromatography on silicic acid, 6,6’-di-O-p-tolylsulfonyltrehalose (12)was obtained as crystals melting at 118”,[ a ]+110 ~ f 4’ (CHCI,), in a yield of 28%. By heating two moles of the potassium salt of a mycolic acid with the above di-p-toluenesulfonate in N,N-dimethylformamide, the 6,6’-dimycolate can be obtained in a yield of about 45 %.

H

H (12) (13)

R = OTs R=I

Method (iv).-Another possible way of synthesizing cord factor is to react the 2,3,4,2’ ,3’,4’-hexa-O-acetyl-6,6’-dideoxy-6,6’-diiodotrehalose (13), already described by Bredereck,” with the silver salt of a mycolic acid; deacetylation of the hexa-0-acetyl-6,6’-di-O-mycoloyltrehalose thus obtained gives cord factor.lZe ( 5 ) Synthesis of Lower Homologs of “Cord Factor.” -The first threc methods mentioned above have also been used for the synthesis of trehalose esters of synthetic mycolic acids; mycolic acids of the general formula R-CH2-CH (OH)CH(R)C02H can be prepared by condensing two moleand then reducing the 8-keto ester cules of an ester R-CHa-C0&H3 RCHlCOCH (R)C0&H3 thus obtained.M v4

(42e) H . Noll, unpublished experiments. (43) E. Lederer, V. Portelance and K. Serck-Hanssen, Bull. 8oc. chim. France, 413 (1952) . (44) J. Polonsky and E. Lederer, Bull. soc. chim. France, 604 (1954).

217

OLYCOLIPIDS OB ACID-BAST BACTERIA

Esterification of trehalose with the synthetic 2-eicosyl-3-hydroxytetracosanoic acid Cn4Hss03(14) has given &mono-, 6 ,6’-di-, and 2,6,6’-triesters [see formulas (15a), (15b), (lsa), (16b), (17a), and (17b)].aQ OH

t:H-CH-COaH

CHa-(CHz)zo-

A’ZOH41 (14) CH,OR

(15) a R =

b

. .

U

b (17)

r \

- C - H-

H-(CI€JzO-CH3

OH

R’ = R” = H

R”’ = CH,CO

I*oH.l

,

.........,. O

(16)

H

R = R’ =

PR’”

R” = H

-!!-~H-CH-(CHZ),,,-CH3 lOH,,

- - - - .

e

R

. .

.

, ,

R’=R’’=R”=H

..

R‘” = CH&O

R”=R’’=H

PR”’

R=R’=R”=-C-CH-CH-(CH2)20-CH,

I

R”’ = CH,CO

C?OHII

With a view to studying the influence of the C-3-hydroxyl group of the mycolic acids on the biological activity of trehalose esters, trehalose was also esterified with the unsaturated acid (18) obtained by dehydration of the Ctr-mycolic acid (14). CHI-(CHn),o-CH=C-CO,H h20H41 (18)

Here, too, 6-mono-, 6,6‘-di-, and 2,6,6’-tri-esters of trehalose have been obtained.*@More recently, Diara and P ~ d l e have s ~ ~ prepared the 6,6’dicorynomycolate of trehalose, using natural corynomycolic acid (19) isolated from Corynebacterium diphthe~iae.~’ (45) A. Dims and J. Pudles, Bull. SOC. chim. biol., 41, 481 (1959). (46)E. Lederer and J. Pudles, Bull. SOC. chim. biol., 93, 1003 (1951).

218

EDGAR LEDERER

OH

I:

CIla(CHz)ir HCHCOzH I:14HZP (19)

(6) Variations of the Structure of Natural Cord-factor in Digerent Strains. is known that different strains frequently contain a different assortment of mycolic acids.34-86 Thus, it is not astonishing that cord factor of different strains sometimes contains different mycolic acids. It was found2 that cord factor of a streptomycin-resistant strain of H37-Rv and of the BCG strain contains a methoxyl-free 3, x-dihydroxymycolanoic acid C87H17404 f 5 CHa, whereas the virulent, human strain “Br6vannes” contains a cord factor wherein the mycolic acid isa0a 3-hydroxy-x-methoxymycolanoic acid C U H ~ T=t ~O 5~ CH2. The work of Demarteau-Ginsburg47and of Miche14*has shown that some bovine strains contain mixtures of mono- and di-mycolates of trehalose and that “atypical” or saprophytic Mycobacteria contain trehalose mycolates,

-It

OH

I:

H O - C ~ ~ H I I ~ H-CH-CO*H (!hH41 (20)

wherein the mycolic acid is of the type C84H16a04 f 5 CH2 (20), which is also found in the waxes of M . phlei and M. smegmati~.~~ The active, cordfactor fractions of the bovine strain Marmorek, as well as those of t,he “atypical” strain M . marianum, seem to be very complex; the water-soluble portion obtained after saponification contains, not only trrhalose, but also glucose, glycerol, and ethylene glycol. A “toxic lipid” isolatcd by Spitznagel and DubosKOby extraction of Mycobacteria with monochlorobenzene has been shown to contain cord factor as the only active compound.’* b. Esters of Po1ysaccharides.-Wax D of Mycobacteria.-All strains of Mycobacteria seem to contain a wax fraction which can be extracted with chloroform arid separated from other waxes by its insolubility in acetone. This fraction has been calleda4wax D. Two distinct groups of wax D have been described: (a) wax D fractions of bovine, avian, and saprophytic strains, which are nitrogen-free glycolipids, and (b) wax D fractions of human strains of M. tuberculosis, which are peptido-glycolipids. (47) H. Demarteau-Ginsburg,Doctor’s Thesis, Univ. of Paris (1958). (48) G. Michel, Doctor’s Thesis, Univ. of Paris (1958). (49) M.Barbier and E. Letlerer, Biochirn. et Biophys. A d a , 14, 246 (1954). (50) J. K.Spitznagel and 11. J. Dubos, J . Exptl. Med., 101.291 (1966).

219

GLYCOLIPIDS OF ACID-FAST BACTERIA

Wax D of Bovine Strains of M. tuberculosis.-These wax fractions usually represent 2% or less of the dry weight of the bacilli. No detailed study of the structure of these fractions is yet available. Demarteau-Ginsburg" has found that wax D fractions of the bovine strains Marmorek and Vall6e can be separated, by chromatography on silicic acid, into two fractions which differ in m. p. and percentage of mycolic acid (1st fraction; m. p. 200", 51 % of mycolic acid; 2nd fraction, m. p. 45", 80% of mycolic acid). The bovine strains Dupr6 and BCG also give two chromatographically-distinct, wax D fractions, both having a low melting point and containing about 70% of mycolic acid. The carbohydrate moiety of these fractions contains arabinose, mannose, and galactose. TABLEI11 Properties of Purified Wax D Isolated jrom Various Strains of Mycobacteria36 Strain

Human

Test H37Rv H37Rv, S.r. H37Ra

Bovine

Vallt5e B.C.G.

M . phlei

proportion in M. p., [WID, N, dry ba- degrees degrees % Cali, % ~

6.2 7.3 8.3 2.0

195-210 +22 195-210 190-205 +22 195-203 +20

0.8

4449 160-170 3841 140-170

1.6

--1.4 45-48 -

Amino acids

% -

+

glu1.59 0.15 alanine tamic acid cr,s-diamino1.4 0 . 2 pirnelic acid 1.65 0.28

---

-

p,

0.0 0.24 0.0 0.3

0

-

+

no amino acids

0.1

-

I1

I(

II

I<

Id

LI

0.2

-O

I

No amino acid has been found in these wax D preparations, and they are inactive as immunological adjuvants (see p . 235). Wax D of Avian and Saprophytic Strains.-These fractions arc also mycolic esters of polysaccharides; they have not yet been examined in detail. They are devoid of amino acids and are also inactive as immunological adjuvants (see p. 235). Wax D of Human Strains of M . tuberculosis.-The wax D fraction represents about 6 to 8 % of the dry weight of human, virulent strains of M . tuberculosis and only 2 % of the avirulent strain H 37-Ra (see Table 111). Most of the crude preparations of wax D are contaminated with phospholipids, from which they can be separated by chromatography on silicic acid' (see also Nojirna6').Even after this purification, 0.2 % of phosphorus is still found in wax D preparations.' (51) S.Nojima, J . Biochem. (Tokyo), 46,499,607 (1959).

220

EDGAR LEDERER

Wax D fractions of human strains of M.tuberculosis are nearly colorless, +28O; they are insoluble in amorphous powders melting above 2 0 ” , [ a ] ~ water, boiling acetone, or methanol, sparingly soluble in petroleum ether, and soluble in ether, chloroform, or benzene. The molecular weight and chemical composition of wax D varies from strain to strain. Wax D fractions from the strains Test and H37-RSahave a molecular weight of about 30,000, whereas wax D fractions of the strains “Canetti” and “BrBvannes” have a molecular weight of approximately 54,000. Saponification splits all these wax D fractions int,o equal weights of mycolic acid and a water-soluble peptido-polysaccharide. The sugars in the polysaccharide are: D-arabinose, D-mannose, D-galactose, D-glucosamine, and D-galactosamine; muramic acid is either absent or present in only small proportion.’ The peptide moiety of the polysaccharide of all of the wax D fractions thus far examined contains only three amino acids : alanine, glutamic acid, and mes0-2,6-diaminoheptanedioic acid (meso-a,€-diaminopimelic acid).6* In a recent study of the wax D of the strain “BrBvannes,” it was found that 1 molecule contains 2 molecules of glutamic acid, 2 molecules of meso-a,t-diaminopimelic acid, and 3 molecules of alanine. All of the glutamic acid isin the “unnatural” D form, whereas one molecule of alanine is D and the other two are L. Figure 1 gives an approximate picture of the structure of a wax D fraction.’ This structure has quite recently been expanded: the seven molecules of amino acids are linked in a heptapeptide having the structure: meso-a ,e-diaminopimelic acid-D-Ala-D-Glu-D-Glu-I,-Ala7neso-a, e-diaminopimelic acid-L-Ala. One of the two meso-a ,e-diaminopimelic acid molecules is linked to D-galactosamine, which is itself linked glycosidically to arabinose.62a The peptide moiety of wax D fractions of human strains seems to be necessary for the activity of these fractions as immunological adjuvants (see p. 235); this may be due to the close chemical similarity between the structure of the water-soluble portion of wax D and the cell wall of Mycobacteria, since the latter contains the same three amino acids (alanine, glutamic acid, and a,c-diaminopimelicacid) and the same sugars (arahinose, mannose, galactose, and aminohexoses),’*Wax D of human strains might be considered to be a “monomer” of the cell wall, heavily esterified with mycolic acid. (62) J. Asselineau, N. Cboucroun and E. Lederer, Biochim. et Biophys. Acta, 6 , 197 (1960). (62a) P.JollBs, H. Nguyen-Trung-Luong-Crosand E. Lederer, Biochim. et Biophys. A d a , 48, 669 (1960). (63) M. R. J. Salton, Bacterial Anat., Symposium SOC.Gen. Microbiol., 6th, London, 81 (1968); C. S. Cummins and H. Harris, J . Gen. Microbiol., 18, 173 (1968).

Polysaccharide moiety Heptapeptide moiety

3 Ma mesoGlu DAP D-

(2 L, 1 D)

amide

--------linkage

- [q glycosidic - ----linkage

(Molecular Weight

-

molecules of: arabinose, galactose, mannose, glucosamine, and galactosamine

Lipid moiety - - - - - - - - - 22 molecules of

(M. W. 26,000)

(M. W. 26,000) 54,OOO)

FIG.1.-Hypothetical Structure of Wax D of the Human, Virulent Strain BrBvannes.1 (Ala = alanine, Glu = glutamic acid, and DAP = a,c-diaminopimelic acid.)

222

EiDGAR LEDERER

Haworth, Kent, and Staceyh4examined the structure of a "lipid-bound" ~ and found that it contains polysaccharide of M . tuberculosis ( [ a ]+25O) u-arabinofuranose, D-galactopyranose, u-mannopyranosc, and u-glucosamine. From methylation and degradation studies, structure (21) has bccii proposed.

-

L-Rhap 1

1 2 n-Araj 1

1 IA-Rhap-(l 2)-~-Araf-(l--+ 2)-~-Manp-(l --f

6 2)-~-Manp-(l --f 2)-~-Manp-(1)

-I

-+

(21) where N = an aminodeoxyhexose.

This polysaccharide was free from amino acids; quite probably, it is either identical with or closely related to the polysaccharide of wax D mentioned above. It should also be mentioned that glycopeptides closely related to the peptido-polysaccharide of human-strain, wax D fractions are found in the culture filtrate of tubercle bacilli and in aqueous extracts; thus, K&ra and KeiP have described a giycopeptide, isolated from culture filtrates of virulent, human strains of M . luberculosis, which could be purified by preparative electrophoresis and which contains alanine, a,e-diaminopimelic acid, and glutamic acid in equimolecular proportions; structure (22) was proposed for the peptide portion of this glycopeptide. CH2-CH2-CH-NH-0 LO

I

A0

I

C-C!H--CH*--C

I NH I

HI-C H p-

8"'

H-C 0-NH-C

H -C 0 ,H AH3

(22)

The sugar moiety contains arabinose, galactose, mannose, and glucosamine. No indications were given as to the configurations of the amino acids or the sugars. K&ra and KeiP assumed that this glycopeptide is present in the cell membrane. Culture filtrates of avirulent strains of BCG, or of a murine strain, did not contain this glycopeptide. Foldes" has described similar compounds which he considers to be cell(64) (W.) N. Haworth, P. W. Kent and M. Stacey, J . Chem. Soc., 1211 (1948). (55) J. K&raand B. Keil, Collection Czechoslou. Chem. Communs., 23, 1392 (1958). (56) J. Fiildes, ~oturwi8senschuflen,48, 331, 432 (1959).

GLYCOLIPIDS OF ACID-FAST BACTERIA

223

wall components. A fraction PI contains glucose and arabinose, and a fraction Pz contains galactose and arabinose. Both polysaccharides have a peptide moiety containing the amino acids: a!,c-diaminopimelic acid, glutamic acid, aspartic acid, alanine, and muramic acid. 2. Glycosidic Glycolipids a. Type-specific Mycosides.-Studies initiated by Smith, Harrell, and Randall,67using the combined techniques of chromatography and infrared spectroscopy, of the lipids of 85 strains of Mycobacteria (including human, bovine, and avian species, as well as representatives of the atypical, acidfast groups) have led to the discovery that the distribution of certain lipids is limited to a single species of organism; the characteristic infrared spectrum of these compounds may thus serve for distinguishing between the various species of Mycobacteria. The distribution of these substances in the various species of Mycobacteria is as f o l l ~ (a) ~ ~Phthiocerol . ~ ~ dimycocerosateso is present in the lipids of 28 out of 30 human strains studied. (b) A compound first called GB is present in the lipids of 7 out of 7 bovine strains studied (G, is accompanied by phthiocerol dimycocerosate in virulent, bovine strains). (c) A compound first called G, is present in the lipids of 17 out of 17 photochromogenic strains. (d) A compound first called Jav is present in the lipids of 11 out of 13 avian strains, and also in the lipids of 3 out of 6 non-photochromogenic strains. A study of the chemical structure of the latter three t,ype-specific compounds having revealed that they are all glycolipids containing characteristic 0-methylated 6-deoxyhexosesel in glycosidic linkage,” a more rational nomenclature was proposed. The general name mycoside was coined for these compounds,12and a mycoside was defined as ‘(a type-specific glycolipid of mycobacterial origin.” The compound first called G, is now known as mycoside A , the compound GB as mycoside B , and the compound first named Jav as mycoside 6. The following data are available concerning the chemical structure of these compounds. (57) D. W. Smith, W. K. Harrell and H. M. Randall, Am. Rev. Tuberc., 69, 505 (1954). (58) E. H. Runyon, illed. Clin. N . Am., 43, 273 (1959). (59) D. W. Smith, H . M. Randall, A. P. MacLennan, R. K . Putney, and S. V. Rao, J . Bacteriol., 79, 217 (1960). (60) H. Noll, J . Biol. Chem., 224, 149 (1957). (61) For recent papers on the detection and identification of 6-deoxyhexoses on paper, see: A. P. MacLennan, H . M. Randall and D. W. Smith, Anal. Chem., 31,2020 (1959); M. T. Krauss, H. Jager, 0. Schindler and T. Reichstein, J . Chromalog., 3, 63 (1960).

224

EDGAR LEDERER

( 1 ) Mycoside A.-This compound has been obtained as a nearly colorless solid, melting a t 105", [a]:' -37" (CHCl,); C, 72.2; H, 11.3; OCH3, 8.6; N, 0; P, 0%.The ulwaviolet absorption spectrum shows maxims a t 222, 274, and 278 mp (in hexane). Mycoside A contains three different 0-methylated 6-deoxyhexoses, which have been identified as 2-0-methylThe lipid fucose, 2-O-methylrhamnose, and 2,4-di-O-methylrharnno~e.~~ moiety of mycoside A is a di- or tri-mycocerosate of an aromatic alcohol. ( 2 ) Mycoside B.-This substance is a colorless wax, melting at 25", [a]:' -22" (CHCla); it has about the same ultraviolet absorption maxima as mycoside A (222,274, and 281 mp) , The approximate molecular formula is CsaHlaoOlo; mycoside B containsonly one sugar, identified as 2-0-methylrhamnom. One molecular proportion of the lipid moiety, C7&48(1)6, of myroside B is a diester of a branched-chain acid fraction of the mean molecular weight of C:!4H480: with a hydroxy-phenyl-methoxy-diol C3lH& . In mycoside B, the 2-0-methylrhamnose is linked glycosidically to the one phenolic hydroxyl group of the lipid moiety.s1*In previous experiments, mycocerosic acid was found in mycoside BS9; possibly, the nature of the acid varies according to the strain and culture medium. ( 3 ) Mycoside C.-This material is a mixture of peptido-glycolipids and it can be separated by chromatography on silicic acid into several closely related compounds; the structure of one of these (fraction III/7 8) has been studied in detail.l2 This compound melts a t about 200", has [a111 -84" (CHCL) and an approximate molecular formula of C ~ ~ I I I N ~ O Z ~ . This particular mycoside C1 preparation contains three different deoxyhexoses : 6-deoxytalose, 6-deoxy-3-0-methyltalose, and 3,4-di-O-methylrhamnose.@The peptide portion of this mycoside C contains three diff erent amino acids linked in a pentapeptide. One molecular proportion contains one molecule of D-phenylalanine, two molecules of D-allo-threonine, arid two molecules of D-alanine; the pentapeptide has the structure D-PhrD-do-Thr-D-Ala-D-allo-Thr-D-Ala.The "unnatural" configuration of all of the constituent amino acids and the presence of D-allo-threonine arc rcmarkable f e a t u r c ~I. t~may ~ be recalled that D-amino acids have also been found in the peptide portion of wax D (see page 220) and arc usually fourid in the cell walls of bacteria. The lipid moiety of this mycoside C has not yet been obtained in a puro state, but it seems to be a mixture of hydroxy acids of approximate molecular formula CzoHgOa.Two 0-acetyl groups are also present in mycoside C, and can be readily recognized in the infrared spectrum by a band a t 8.1 p .

+

@la) H . Demarteau-Ginsburg and E. Lederer, unpublished results. (62) A. P. MacLennan, Biochem. J . , (1961) in press. (63) M. Ikawa, E. E. Snell arid E. Lederer, Nature, 188, 558 (1960).

225

GLYCOLIPIDS O F ACID-FAST BACTERIA

The t,entative structure (23) has been proposede4for this mycoside CI. 0(6-deoxy-3-O-methyltalose)

/ CI~HM 2-0-acetyl \ C-D-Phe-D-allo-Thr-D-Ala-D-allo-Thr-D-Ala-O-sugar-O-sugar I1 0

I

(23) (The sugar is 6-deoxytalose or 3,4-di-O-methylrhamnose.) .

I

Some uncertainties exist concerning the attachment of the sugar molecules and the exact formula of the hydroxy acid. The C-terminal carboxyl group of D-alanine is linked as an ester to a hydroxyl group of one of the sugar molecules. Two other mycoside C preparations, obtained from other fractions of the same chromatographic purification, seem to have closely related structures. Apparently, they mainly differ from fraction III/7 8 in the nature of the fatty acid and the number of deoxyhexose molecules. The peptide moiety, containing 5 molecules of D-amino acids, seems to be the same in t’hese three fractions (see Table IV). b. Phospho-g1ycolipids.-Andersone6 was the first to discover the presence of myo-inositol in a phospholipid, extracted from M . tuberculosis; the chemistry of mycobacterial phospholipids was then extensively studied by Anderson and ~ o w o r k e r s who , ~ ~ identified, ~ ~ ~ ~ ~ in hydrolysis products of these lipids, an inositol-phosphoric acid, an inositol-glycerol-diphosphoric acid, and “manninositose,” [a],+74”. One molecule of the last gave, on hydrolysis, two molecules of D-mannose (characterized by its phenylhydraxone, m.p. 194-195’) [aID+28” and one molecule of crystalline myo-inositol, m.p. 225”. (1) Nitrogen-free Phospholipids.-Mycobacterial phospholipids differ from those of other organisms by the absence of choline and colamine (for a review on bacterial phospholipids in general, see Ref. 36); the latter has only once been found-in a small fraction, after careful chromatography of a phospholipid of M . r n ~ r i a n u m . ~ * ~ K. Blochoewas the first to prepare nitrogen-free phosphatides from ICI. tuberculosis, followed later by PangbornsB(see also Macheboeuf and F a ~ r e ~ ~ ) .

+

(64) P. JollBs, F. Bigler, T. Gendre and E. Lederer, Bull. 8oc. chim. biol., 43, 177 (1960). (65) R. J. Anderson, J . Am. Chem. Soc., 61, 1607 (1930). (66) R. J. Anderson, R . L. Peck and M. M. Creighton, J . B i d . Chern., 136, 211 (1940). (67) G. I. de Suto-Nagy and R . J. Anderson, J . B i d . Chem., 171, 749, 761 (1947). (68) K. Bloch, Biochem. Z.,286. 372 (1936); 2.physiol. Chem., 244, 1 (1936). (69) M. C. Psngborn, Discussions Faraday Soc., 6, 110 (1949). (70) M. Macheboeuf and M. Faure, Compt. rend., aOB, 700 (1939).

TABLE IV Composition of Three Different Fractions of Mycoside C Y Products o j hydrolysis Mycosidc

c

Probable moletular jormula

M.P.,

dcgrees

C /aiI?l

COCHt

OCHa

Acdu acid,

Hydroxy acid

mdGF

DeoxyhGZOSf3,~

Amino acids, moles

moles

~~

1/17

-200

-44 58.718.613.81 6 . 6

6.3

CnHiaiN60zs II/7

III/7 (Cd

+8

-200

-34

95-200

-34

1 D-Phe, 2 D-alloThr, 2 D-Ala

5.6 (for 2)

Q

9

b ?t

H U

found

found 60.118.7i5.21 6.0 1 calculated C6~lllN6O23 59.818.315.11 6.8

1

6.6

1 D-Phe, 2 D-aEloThr, 2 D-Ala

6.3

The deoxyhexosesof these fractions have been identified by Dr. A. P. MacLennan;they are: 6-deoxytalose, 6-deoxy-3-0-methylcalose, and 3,4-di-O-methylrhamnose.

GLYCOLIPIDS OF ACID-FAST BACTERIA

227

(i) Phosphatidylinositol D-mannobioside.-More recent work has shown that a nitrogen-free phosphatidylinositol “dimannoside” can be isolated in fairly good yield, and in an apparently homogeneous state, from several J ~ compound can be considered to be the strains of M y c ~ b a c t e r i a . ~This typical phospholipid of Mycobacteria (m.p. 215’, [a], 32’). The structure of this compound has been investigated in some detail. (In some experiments, the phosphatidylinositol “dimannoside” was found to be heavily contaminated by an inositol-free phosphatidic acid which had probably been formed by autolysis during extraction of the bacteria.) By the method of H a n a h ~ i n(hydrolysis ~~ with 2 N hydrochloric acid for 20 min. a t looo), a diglyceride was isolated; this proves that two fatty acids of the phospholipid are both linked to glycerol. The fatty acids found in these preparations of mycobacterial phospholipids are generally a mixture of palmitic, hexadecenoic, st,earic, octadecenoic, and tuberculostearic acids. The linkage of the myo-inositol to the phosphatidic acid has not yet been determined with certainty; the inositolphosphoric acid, isolated after hydrolysis of the intact phosphatide (or of the water-soluble portion, obtained by alkaline hydrolysis), gives, on paper chromatography, a mixture of inositol 1(3)-phosphate and a small proportion of inositol 2-phosphate (separated on paper by the method of Pizer and BallouT3). This indicates that, in the phospholipid, the myo-inositol is probably linked by its C-1- or C-3-hydroxyl group to phosphoric acid. The result,s thus far described permit the conclusion that the mycobacterial inositol-phospholipids are mannosides of a typical phosphatidylfrom inositide, as isolated in recent years by Faure and Morelec-C~ulon~~ wheat germ and ox heart, by Hawthorne and ChargafF8) and by Okuhara and N a k a ~ a m a ? from ~ ( ~ )soy bean, and by Carter and coworker^'^ from sweet peas. The phosphoric acid is linked to the L-1-hydroxyl group of the myo-in~sitol.~~ We may now turn to the linkage of the sugar moiety in the mycobacterial phosphatidylinositol “dimannoside.” Hydrolysis of the permethylated, water-soluble portion gave an equimolecular mixture of a tri- and a tetra-0-methylmannose. This shows that the two D-mannose moieties

+

(71) E. Vilkas, Compt. rend., 246, 588 (1957). (72) D. J. Hanahan, Federation Proc., 16,826 (1957). (73) F. L. Pizer and C. E. Ballou, J . A m . Chem. Sac., 81,915 (1959). (74) M. Faure and M. J. Morelec-Coulon, Compt. rend., 238, 411 (1954); Bull. sac. chim. biol., 40, 1315 (1958). (75) (a) J. N. Hawthorne and E. Chargaff, J . Biol. Chem., 206, 276 (1954); (I)) E. Okuhara and T. Nakayama, ibid., 216, 295 (1955). (76) H. E. Carter, D. B. Smith and D. N. Jones, J . Biol. Chem., 232, 681 (1958). (77) C. E. Ballou and L. I. Pizer, J . A m . Chem. Sac., 81, 4745 (1959); 82, 3333 (1960).

228

EDGAR LEDERER

form a “dimannoside” which is linked to one of the hydroxyl groups of myo-inositol. The (1 --t 6)-linkage between the two mannose moieties was established by the following experiments.l0 (a) The tri-0-methylmannose obtained by hydrolysis of the permethylated phospholipid can be detected on paper with Partridge’s reagent, but not with periodate-benzidine; this shows that it has a methoxyl group a t C-2, and this excludes a (1 + 2)-linkage in the “dimannoside.” (b) Periodic acid oxidation of the inositol “dimannoside” (obtained aftcr alkaline hydrolysis of the phospholipid) destroys both of the mannose molecules; this excludes a (1 --j 3)-linkage between tthe two mannose molecules. (c) A (1 -+ 4)-linkage seems to be excluded by the negative result of the reaction of the tri-0-methylmannose with N , N dimethylaniline trichloroacetate; Hough, Jones, and W a d m a ~ have ~ ’ ~ shown that, methylated aldohexoses having a free 4-hydroxyl group give a violet color with this reagent. (d) A quantitative periodate oxidation of the inositol ‘Ldimannoside”showed the consumption of 8 moles of the oxidant per mole, in agreement with a (1 --+ 6)-linkage [an inositol “dimannoside” with a (1 --+ 2)- or a (1 t 4)-linkage would reduce 7 moles of oxidant per mole]. (e) Hough and PerryTg have shown that disaccharides having a (1 t 2)-, (1 -+ 3)-,or (1 + 4)-glycosidic linkage are susceptible to overoxidation with sodium metaperiodate in the dark, resulting in the liberation of formaldehyde, whereas (1 t 6)-linked disaccharides do not afford formaldehyde under the same conditions. Oxidation of the inositol “dimannoside” under Hough and Perry’s conditions did not liberate any formalde6)-linkage between the two hyde, which is again in agreement with a (1 mannose molecules. (f) Finally, the results of periodate oxidation as described by Greville and Northcotenowere also in agreement with a (1 + 6)linkage. The 2,3,4,6-tetra-O-methylmannose obtained after hydrolysis of the permethylated inositol “dimannoside” was characterized as its crystalline anilide, m.p. 141-143”. This proves that the corresponding mannose residue is present as the pyranoside. From molecular-rotation data, it follows that both of the D-mannose molecules are linked a-glyaosidically. The mannobiose of the phospholipid As a restudied is, thus, a 6-O-a-D-mannOpyranOSyl-a-D-mannOpyranOSe. sult of this work, structure (24)was proposed for the typical phosphatidylinositol D-mannobioside of Mycobacteria, wherein inositol is linked at the C-1 (or (3-3) hydroxyl group to phosphoric acid, but the location of the 6-0-a-D-mannopyranosyl-a-D-mannopyranose has not yet been determined. (ii) Other Phosphatidylinositol Glycoside8.-The complexity of the phos--f

(78) L. Hough, J. K. N . Jones and W. H. Wadman, J . Chem. Soc., 1702 (1950). (79) L. Hough and M. B. Perry, Chem. & Znd. (London), 768 (1956). (80) G. D. Greville and D. H. Northcote, J . Chem. Soc., 1945 (1952).

229

QLYCOLIPIDS OF ACID-FAST BACTERIA FHaOCOR1

FHOCOR,

d l

H,O- P - OH

~~ov(LFo-& HO HO

OH

HO

HO

HO

OH

OH (24 )

pholipid fraction of some strains has been demonstrated by Vilkass with a phospholipid isolated from a batch of BCG grown on a peptone-containing Sauton medium. Paper chromatography of the hydrolysis products showed the presence of some glucose, besides mannose; after dephosphorylation of the watersoluble portion by heating with ammonia, the phosphorus-free sirup was acetylated with acetic anhydride, and the acetylated product was chromatographed on magnesium silicate-Celite (2: 1). The following products were obtained (in order of elution) : tri-0-acetylglyceritol; inositol hexaacetate, m.p. 217'; an acetate of m.p. 78-80' and [a],+125", containing one mole of inositol and five moles of D-glucose per mole; an acetate, m.p. 178-180", [a],, +20.5', giving on hydrolysis one mole of inositol and one of mannose per mole; and an acetate of m.p. 135-6', [a], 54", which is the dodecaacetate of inositol "dimannoside." This experiment shows that the phospholipid of this batch was composed of (at least) five different compounds, namely, a sugar-free phosphatidylinositol (39 %) ; a phosphatidylinositol pentaglucoside (8 %) ; a phosphatidylinositol monomannoside (5 %) ; and a phosphatidylinositol "dimannoside" (48 %). Michel and Lederer4mbhave also described the isolation of a glucosecontaining phospholipid of M . marianum. Moreover, Pangbornel and Noj imaK1have isolated phospholipids having a composition which agrees very closely with that of a phosphatidylinositol pentamannoside. ( 2 ) Nitrogen-containing Phospholipids.-The absence of choline from mycobacterial phospholipids has already been mentioned; in one case, only, has colamine been ~haracterized.~.~ From several strains of Mycobacteria, phospholipid fractions containing amino acids have been obtained, the most frequent amino acid being L-ornithine.82

+

(81) M. C . Pangborn, Federation Proc., 17, 1133 (1958). (82) T.Gendre and E. Lederer, Ann. Acad. Sci. Fennicae, Ser. A ZZ, 60,313 (1955).

230

EDGAR LEDERER

In one case, hydroxylysine has been found,83 but,, on re-investigation of the phospholipid of a new batch of the same strain of M . phlei, only ornithine could bc isolated. Two explanations may be offered; either an error of identification was made in the first experiment, or a mutation of the strain had occurrrd. For other phospholipid preparations, papcr chromatography shows the presence of several amino acids. In view of the existence of peptido-lipids having solubility properties very similar to those of the phospholipids, it seems quite possible that t>hese amino acids arc, in reality, part of phosphorus-free peptido-lipids.

111. BIOLOGICAL ACTIVITIESOF GLYCOLIPIDS OF ACID-FAST BACTERIA

It is not intended to give here a very detailed discussion of the various biological activities concerned ; a recent review3"may be consulted for more information. 1. Tissue Reactions

Transformation of monocytcs into epithelioid cells and formation of giant cells of the Langhans type have been obtained by Sabins4with various lipid fractions of Mycobacteria prepared by Anderson; it is now known that the branched-chain fatty acids of the phthienoic, mycocerosic, and mycolic acid types are all ttctivc in this respect.8s-** Delaunay and co~orker,s*~ have shown that the peptido-glycolipid wax D (see p. 219) (which rcprescnts the main form in which mycolic acids arc present in human strains of tubrrcle bacillus) produces cellular modifications similar to those induced by mycolic acids; the nitrogen-free wax D from bovine strains is also active. Whitegohas observed large proliferation of epithelioid cells after the illjrction in tho foot,pad of guinea-pigs of 40 wg of wax D, using water-oil emulsions. The same proliferation is also found in popliteal lymph glands. (83) M.Bnrbier and E. Lederer, Biochint. el Biophys. Acta, 8, 590 (1952). (84)F.R.Sabin, Physiol. Revs., 12, 141 (1932);A m . Rev. Tuberc., 44,415 (1941). (85) H.Husseini and S. Elberg, A m . Rev. Tuberc., 66.655 (1952). (86)J. D.Chanley and N. Polgar, J . Chem. Soc., 1003 (1954). (87) J. Ungar, C. E. Coulthard and L. Dickinson, Brit. J . Ezpll. Pathol., 29, 322 (1948). n . dTennant, Yale J . Hiol. and Med., 16, 347 (1943);16, 1 (1943). (88) B.Cerstl ~ ~ R (89) A. Delaunay, J. Asselinenu and E. Lederer, Compl. rend. sac. biol., 146, 650 (1951). ('30)R . G . White, CaOa Foundation Symposiurri Erpll. Tuberc. Bacillus and Host, 83 (1955).

GLYCOLIPIDS OF ACID-FAST BACTERIA

23 1

2. Inhibition of Enzymes Kato and coworkersg1have made an extensive study of the mechanism of action of cord factor (see p. 210) and have found a significant decrease of the activity of the succinic and malic dehydrogenase systems of the liver of mice, about twenty-four hours after intraperitoneal injection of 0.1 mg. This finding seems to be related to the work of Martin and coworkers,82 who reported a decrease of succiriic dehydrogenase activity in the kidneys of tuberculous guinea-pigs. Later, Kato and coworkers93found that the injection of cord factor also decreased the activity of some other “diphosphopyridine nuc1eotide”-linked dehydrogenases in mouse liver (such as lactic and a-glycerophosphoric dehydrogenase). The decreased enzymic activity was restored by homogenizing the liver of mice (treated with cord factor) with the sucrose extract of normal mouse-liver. The metabolic lesion caused by the injection of cord factor was assumed to be a decrease of a soluble factor affecting the activity of the succinic dehydrogenase system of mouse liver. 3. Action on Leucocytes BlochZ4has found that crude preparations of cord factor show inhibitory effects on the migration of leucocytes. Inhibition of the chemotaxis of leucocytes toward starch granules by fiiie suspensions of Pmko (a peptido-glycolipid, chemically very similar to wax D of human strains) has been observed by Choucroun and c o ~ o r k e r s , ~ ~ using different experimental conditions. Meier and Scharg5have fourid that the wax D of tubercle bacillus has almost no action on the migration of leucocytes. Tubercle bacilli are known to multiply within monocytes of normal animals. According to Suter and White,gEmonocytes obtained from guincapigs which had been injected with wax D show inhibitory properties toward multiplication of phagocytized tubercle bacilli. (91) M. Kato, K . Miki, K . Matsunaga and Y. Yamamura, Am. Rev. Tuberc., 77, 482 (1958). (92) S. P. Martin, S. N . Chaudhuri, C. D. Cooper and R. Green, Ciba Foundotion Symposium Exptl. Tuberc. Bacillus and Host, 102 (1955). (93) M. Kato, M. Kusunose, K . Miki, K . Matsunaga and Y. Yamamura, Am. Rev. Respirat. Diseases, 80, 240 (1955). (94) N . Choucroun, A. Delaunay, S. Bazin and R. Robineaux, Ann. inst. Pasteur, 80, 619 (1951). (95) R . Meier and B. Schlir, Ezperientia, 10, 376 (1954). (96)E. Suter and R . A. White, Am. Rev. Tuberc., 70, 793 (1954); E. Suter, Ciba Foundatzon Symposium Exptl. Tuberc. Bacillus and Host, 198 (1955).

232

EDGAR LEDERER

4. Toxicity

The chemistry of “cord factor,” a toxic lipid of virulent or attenuated Mycobacteria has already been described in detail (see p. 210). It has also been mentioned that at least part of this biological activity can be explained by the action of cord factor on dehydrogenases dependent on “diphospho~ ~p.~ 231). ~~ pyridine nucleotide” as described by Kato and c o w ~ r k e r s(see The toxicity of cord factor is of a delayed type; five to ten micrograms kill adult mice within 5 to 8 days after injection. Repeated small doses are more toxic than a single large one. The cause of death is unknown; extensive pulmonary hemorrhages are the most conspicuous symptom.g7 A single injection of cord factor, which by itself has no lasting deleterious effects, causes tuberculous infections to progress more rapidly than they otherwise would; thus, mice receiving an injection of cord factor prior to infection die sooner than controls; their lungs, livers, and spleens contain a greater number of viable tubercle bacilli than do the organs of control animals. The enhancing effect of cord factor on murine tuberculosis seems to be specific: injections of mycolic acid or of bacterial-wax fractions other than cord factor do not influence the course of experimental tuberculosis. Infcctions of mice by gram-negative bacteria are not affected by injections of cord factor.w As concerns the relationship between chemical structure and cord-factor activity, the following preliminary statements have been made2’ on the basis of unpublished experiments by H. Bloch with compounds prepared in the author’s laboratory. (a) 6-Mycolates of monoses (D-glucose,D-galactose, D-glucosamine; for example, 7a and b) are toxic, but in a lesser degree than trehalose esters. The corrc:sponding 2-mycolates (6) and mycolamides (8) are not toxic. (b) As concerns esters of trehalose, the 6,6‘-dimycolates have the characteristic activity of cord factor, whereas the 2 ,2’-dimycolates are inactive. (c) 6 ,6’-Dimycolates of trehalose seem more active than thc 6-mono- or 2,6,6’-tri-mycolates. (d) Acetylation of the 8-hydroxyl group of mycolic acid diminishes the activity of cord factor only slightly. Fully acetylated cord-factor is inactive. (e) The influence of the structure of the acid that esterifies trehalose can be characterized as follows : even behenic (docosanoic) esters of trehalose are active, but doses of larger than 0.1 mg. are necessary. The 6 ,6‘-diester of trehalose with the synthetic mycolic acid ClrHeeOI(14) has about 50 % of the activityof natural cord-factor. Dehydration of the latter acid gives the unsaturated acid (18), whose 6,6’-diester of trehalose is inactive at dose levels of 0.1 mg. (97) H.Bloch and H. Noll, Brit. J . Ezptl. Pathol., 86, 8 (1955); H.Bloch, Ann N. Y . Acad. Sci., 88, 1076 (1QfIO)

GLYCOLIPIDS OF ACID-FAST BACTERIA

233

Carne and coworkersg8have described a toxic lipid extracted from Corynebacterium ouis, a pathogen which, in sheep, causes a wide-spread disease known as caseous lymphadenitis. The lipid extracted from living C . ovis with petroleum ether is toxic for leucocytes in vitro. A preliminary chemical investigation of this lipid fraction, kindly prepared by Dr. Carne, has not yet yielded definite information about the chemical nature of the active fraction.08* A synthetic 6,6’-dicorynomycolate of trehalose, prepared by Diara and P ~ d l e shas , ~ ~been found devoid of leucotoxic action. A mixture of trehalose esters extracted from the surface layer of C . diphtheriae has been found2I to have a toxic action on the skin of animals after subcutaneous injection, causing swelling, necrosis, and, finally, formation of a scab. It might well be worth while to study systematically the biological properties of various glycolipids; all the more so, as the toxic moieties of the endotoxins of gram-negative bacteria are also glycolipid~.~~ The simultaneous presence of several lipophilic and hydrophilic groups in the same molecule confers on glycolipids peculiar physicochemical properties (emulsifying, detergent, and so on) which might explain most of the toxic actions observed.

5 . Antigenicity Phospholipids arc amongst the most active antigenic lipid fractions; they fix complement in sera containing antibodies against the tubercle bacillus.1w The antigenic properties of the “antighe m6thylique” of Boquet and NBgre’O’ can be ascribed to phospholipid components. Purification of phos; two pholipid antigens has been particularly studied by active “phospholipopolysaccharides” have been isolated, one of them containing 1.7% of phosphorus, 10.5% of inositol, and 5 0 % of mannose. On the other hand, wax D of the tubercle bacillus appears to possess antigenic p r o p e r t i e ~ ~2~pg ~ Jof~ ~wax ; D induces the formation of hemag(98) H.It. Carne, N . Wickham and J. C. Kater, Nature, 178, 701 (1956). (98a) J. Asselineau and A. Diara, unpublished results. (99) 0. Westphal, A. Nowotny, 0 . Luderitz, H. Hurini, E. Eichenberger and G Schonholzer, Pharm. Acta Helv., 33. 401 (1958). (100) L. NBgre, “Les lipoides dans le Bacille tuberculeux et la tuberculose,” Maseon et Cie, Paris, France (1950).Y. Takahashi and K. Onos, Science, 127, 1053 (1958). (101) A. Boquet and L. NBgre, Compt. rend. SOC. biol., 86, 581 (1922). (102) M. C. Panghorn, N . Y . State Dept. Health Ann. Rept. Div. Labs. and Research, 18 (1955);11 (1956). (103) Y.Takeda, T . Ohta and Y. Sen, Zgaku to Seibutsugaku, 4 , 8 8 (1943). (104)Y.Takeda, N. Wakita, N . Watanabe and H. Suzuki, Japan. J . Tuberc., 2 , 3G1 (1954).

234

EDGAR LEDERER

glutinating antibodies in rabbits. Antigenic properties have also t m n ascribed to Pmko (a fraction very similar to wax D)loa;the circulating antibodies are detected by precipitation of the immune serum by thc polysaccharide moiety of Pmko.lo6 Several other components of the tubercle bacillus secm to be ablc to act as haptens, for example, phthienoic arid mycolic acidslo7or cord factor.lo6 Boyden and Sorkin,loQin an excellent review 011 antigens of Mycobacterium tuberculosis, think that “it is still not completely clear whether antibodies are formed against any of t,he lipid molecules, as distinct from polysaccharide or protein components of lipidic complexes.” 6 . Establishment of a Delayed Type of Hypersensitivity Tubercle bacilli induce in animals a peculiar type of hypersensitivity which is usually callcd the *‘tuberculin type” or “delayed type” of hypersensitivity (for reviews, see RaffellloJ1l). By injection of a crude preparation of Pmko [a glycolipid, isolated from tubercle bacillus (see p. 231) , containing protein components], Choucr01111~~~J~~ was able to induce a delayed type of hypersensitivity in guincapigs. The same author observed laterlo6that purified Pmko was inactive, and that the delayed type of hypersensitivity was only induced by the simultancous action of Pmko and a protein component. Meanwhile, Raffe1114,116 obtained similar results by using the “purificd wax” of Anderson (a mixture of wax C and wax D; see p. 210) and, as antigens, compounds as different as ovalbumin or picryl chloride116(sec also, Myrvik and Weiser”’). From further studies by Raffel and coworkers,l18it was concluded that the most-active lipid component isolated N. Choucroun, Am. Rev. Tuberc., 66, 203 (1947). N. Choucroun, Compt. rend., 229, 145 (1949). W. Catel and 8. Weidmann, Monatsschr. Kinderheilk., 101, 217 (1953). T. Ohara, Y. Shimmyo. I. Sekikawa, K . Morikawa and E. Sumikawa, Japan. J . Tuberc., 6 , 128 (1957). (109) S. V. Boyden and E. Sorkin, Bibliotheca Tuberc., Suppl. Rchweiz. 2. l’uberk. u . Pneumonol., 7, 17 (1956). (110) S. Raffel, Experientia, 6, 410 (1950). (111) S. Raffel, “Immunity, Hypersensitivity, Serology,” AppleLon-CenturyCrofts, Inc., New York, N . Y., 1953. (112) N. Choucroun, Compt. rend., 210, 749 (1940). (113) N . Choucroun, Con@. rend., 208, 1757 (1939). (114) S. Raffel, A m . Rev. Tuberc., 64, 564 (1946). (115) 8. Raffel, J . Infectious Diseases, 82, 267 (1948). (116) S. Raffel and J. E. Forney, J . Ezptl. Med., 88, 485 (1918); S. Raffel, L . E. Arnaud, C. D. Dukes and J. S. Huang, ibid., 90. 53 (1949). (117) 0. Myrvik and R. S. Weiser, J . Zmmunol., 88,413 (1952). (118) S. Raffel, J. Asselineau and E. Lederer, Ciba Foundatzon Symposium Exptl. Tuberc. Bacillus and Host, 174 (1955). (105) (106) (107) (108)

GLYCOLIPIDS OF ACID-FAST BACTERIA

235

from tubercle bacilli is wax D, and that simpler compounds, such as esters of mycolic acids with hexoses, are also active. Among many lipid substances isolated from various bacteria, only mycolic acid esters have becn found active. ForneylLghas produced a sensitization of guinea-pigs to Micrococcus pyoyencs by injection of killed bacillary bodies of this species together with crude wax of tubercle bacillus or M . smegmatis. Freund and StonelZohave found that the minimum amount of tubercle bacilli for sensitization of guinea-pigs was 0.0004 mg., whereas 0.2 mg. of wax D in a water-oil emulsion was required; the greater activity of the whole cells might be due, in part, to their greater surface, whereas it cannot be excluded that the activity of the wax D preparations might be due to an impurity.

7. Adjuvant Action Freund’s adjuvant, consisting of a water-oil emulsion containing killed Mycobacteria in the oil phase and the antigen in the aqueous phase, has been widely used by immunologists; Freund has reviewed our knowledgc of the mode of action of this type of adjuvant.121 After preliminary experiments by White and coworkers122with a “purified wax fraction,” it, was that wax D of human strains can effectively replace the whole bacilli in Freund’s adjuvant mixture. Other lipid fractions of human strains, as well as wax D of bovine, avian, and saprophytic strains, were inactive. The inactivity of the latter fractions of wax D suggests that the peptide portion of wax D of human strains (see p. 220) is essential for the adjuvant activity. Delipidated bacterial residues of human, bovine, avian, and saprophyte strains were active; this can be explained by the fact that these residues contain, essentially, cell walls. It is known that mycobacterial cell-walls are mainly composed of three amino acids (alanine, glutamic acid, and atediaminopimelic acid) linked to a polysaccharide containing arabinose, mannose, galactose, and muramic a ~ i d , and ~ ~ Jthus ~ ~ have an over-all composition very similar to that of the water-soluble part of the wax D of human strains. The adjuvant activity of the wax D of human strains of M . tuberculosis (119) J. E . Forney, A m . Rev. Tuberc., 69, 241 (1954). (120) J. Freund and S. H . Stone, J . Zmmunol., 82,560 (1959). (121) J. Freund, Bibliotheca Tuberc., Suppl. Schweiz. 2.Tuberk. u. Pneunaonol., 7. 130 (1956). (122) R. G . White, A. H. Coons and J. M. Connolly, J . Exptl. Med., 102,83 (1955). (123) R. G . White, L. Bernstock, R . G. S. Johns and E. Lederer, zrnmunobyg, 1, 54 (1958). (124) C. S . Cummins, Intern. Rev. Cytol., 6, 25 (1956).

236

EDGAR LEDERER

is probably due to its general chemical analogy with the cell wall, and the cellular reactions which result in the increase of antibody production might be considered a general reaction of the tissues of higher organisms to the contact of Mycobacterial cell-walls. Freund and Lipton126have shown that an Actinomycete, Nocardia asteroides, has the same adjuvant action as Mycobacteria. The cell wall of this organism has a structure very similar to that of Mycobacteria-alanine, glutamic acid, a ,c-diaminopimelic acid, arabinose, mannose, and galactose being the principal components. Whitel2b8has shown that the wax D of Nocurdiu asleroides is inactive as an adjuvant, whereas the delipidated cells are active. In guinea-pigs, the morphological changes which follow the injection of active fractions have been described by White and coworkers.12*The changes may be considered to be u result of a general stimulation of the reticuloendothelial system. The adjuvant action of wax D (of human strains of Mycobacteria) for the production of experimental, allergic encephalomyelitis has been reported by Colover**eand White and MarshalP7 (compare, previous work of Waksman and Adams128). PoundlZghas reported an adjuvant effect on antibody production against ovalbumin, using a lipid corresponding to the “hard wax of Anderson” (that is, wax C wax D). Freund and StonelZ0have studied the production of allergic encephalomyelitis and aspermatogenesis, and have found that 7 to 10 times more wax D than dried, whole, tubercle bacilli was required for inducing these allergic states. They think that these observations are not in favor of the role of wax D as an adjuvant; the results of Freund and Stone are, however, easily explained by the above-mentioned hypothesis that both the bacterial cell-walls and the wax D of human strains are active, because of the common general chemical structure; the cell walls have, of course, a much greater surface than the wax D, in paraffin oil, and can thus be more active. It should be mentioned that injection of the endotoxins (lipopolysaccharides) of gram-negative bacteria also produces an increase of antibody titer,Ia0but this action is linked with the properdin system and is of a very

+

(1%) J. Freund and M. M. Lipton, Proc. SOC.Exptl. Biol. Med., 68, 373 (1948). (12I5a) R.G.White, unpublished work. (126) J. Colover, Nature, 182, 105 (1968). (18) R. G.White and A. J. E. Marshall, Immunology, 1, 11 (1968). (128) B. Wabman and R. Adams, J . Infectious Dieeaaes, 83, 21 (1968). (129) A. W. Pound, J . Pathol. Bacteriol., 76, 66 (1958). (130) A. G.Johnson, 8.Gaines and M. Landy, J . Ezptl. Med., 103, 226 (1966).

GLYCOLIPIDS O F ACID-FAST BACTERIA

237

short duration (whereas, the action of Freund’s adjuvant canlast for several months). 8. Immunization

Killed tubercle bacilli are capable of conferring on experimental animals a certain amount of protection against infection with M . tuberculosis, although it seems that this protection is less effective than that conferred by living vaccines of attenuated or avirulent strains of bacilli.10QJs1-1a3 Many authors have reported attempts to immunize animals against tuberculous infection with various extracts of tubercle bacilli. Polysaccharides and proteins have generally been found ineffective, whereas certain lipid fractions have been found active. For a detailed discussion, see the recent review of C r ~ w l e . ’ ~ ~ Choucrounlo6has reported that her “Pmko” (which is chemically very similar to wax D, see p. 219) has immunizing activity when injected as a very fine and stable suspension in water. It seems that no other author has as yet confirmed this activity. Hoyt and coworkers1ahhave stated that “purified wax” (C D) loses its immunizing power when most of the bacillary debris it contains has been removed by centrifugation. Boquet and NBgre’O’ reported, in 1922, that an injection of a methanol extract of acetone-defatted, tubercle bacilli (the “antighe m6thylique”) confers a certain degree of protection against subsequent virulent infection. (For more-recent reports, see NBgre.’OO) Weiss and Dubos1a6have confirmed the protective effect obtained with methanol extracts of phenol-killed and acetone-defatted BCG or H37 Ra. NBgre’OO has stated that the active component of these extracts is a phosphatide, but no pure compound has yet been isolated from such extracts, nor is the phosphatidylinositol D-mannobioside described by Vilkas and LedererQ active.137 In the course of recent immunization experiments conducted in collaboration with H. Bloch (of Pittsburgh, Pa.), fractions of phospholipids were obtained which produce an increase in survival time of mice infected with virulent tubercle bacilli; the main constituent of these high-melting,

+

(131) R.J. Dubos, W. B. Schaefer and C. H. Pierce, J . Exptl. Med., 97, 221 (1953). (132) C. E. Palmer, H. Ferebee, S. N . Meyer and H. Bloch, Bull. World Health Organization, 12. 47 (1955). (133) H. Bloch and W. Segal, Am. Rev. Tuberc. Pulmonary Diseases, 71, 228 (1955). (134) A.J. Crowle, Bacterial. Rev., I 183 , (1958). (135) A. Hoyt, R.L. Dennerline, F. J. Moore and C. R. Smith, A m . Rev. Tuberc. Pulmonary Diseases, 76, 752 (1957). (136) D.W. Weiss and R. J. Dubos, J . Exptl. Med., 101, 313 (1955). (137) H. Bloch, unpublished experiments.

238

EDGAR LEDERER

strongly dextrorotrttory fractions seems to be a phosphatidylinositol o-mannotrioside. Acknowledgments. The author wishes to thank Dr. J. Asselineau for his help in preparing this review. The work of the author’s laboratory has been greatly facilitated by grants from the Fondation Waksman pour le DBveloppement des Recherches microbiologiques en France, and, more recently, by grant E 28-38 of the National Institute for Allergy and Infectious Diseases, National Institutes of Health, Bethesda 14, Md., U.S. A. (138) H. Bloch, E. Lederer and E. Vilkas, unpublished experiments.

GALACTOSIDASES

BY KURTWALLENFELS AND OM PRAKASH MALHOTRA Cheniisches Laboratorium der liniversitiil, Freibury irn Breisyau, Germany I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. p-Galactosidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Occurrence. . . . . . . . . . . . . . . . . . . . . . . . . . ............. 3. Purification . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . 242

9. Kinetics of Calf-intestine Enzyme. ..................... 111. a-Galactosidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Occurrence... . . . . . . . . . . . . . . . . . . . . . . . . . .........................

290 291

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

292

3. Purification. . . . . . . . . . . . . . . . .

I. INTRODUCTION It now seems fairly well settled that individual enzymes (glycosidases) are required for splitting various glycosidic bonds, except in some cases where the glycon ring roilformation is the same for more than one sugar. The enzymes bringing about the hydrolysis of the galactosidic linkage are termed galactosidases. There are two types of them, called a-galactosidases and 8-galactosidasrs depending on the configuration of the anomeric carbon atom of the substrate molecule. Specificity studies have shown that some of t,hese enzymes can also hydrolyze the configurationally analogous fucosides (6-deoxygalactosides) and arabinosides. To the best of our knowledge, no data exist to show whether special arabinosidases and fucosidases can also hydrolyze galactosides. These enzymes will not, therefore, be considered here. The p-galactosidase of Escherichiu coli, ML 309, was the first of the class of oligosaccharide-splitting enzymes to be obtained in pure and crystalline form. It is, therefore, well suited for studies on the mechanism of its action. 239

240

WALLENFELS AND MALHOTRA

Such studies may be helpful in understanding glycosidase activity, as well as glycosylt,ransferase actions in general. Moreover, this enzyme is the most extensively studied example of induced enzyme-formation, and it has been employed for investigating the mechanism of specific prot,ein synthesis and the genetic control thereof. An understanding of the constitution of the active center of this enzyme might help in identifying, in the bacterial cell, the apparatus which controls its production. a-Galactosidase has not as yet been obtained in pure form and has not been studied so extensively as 0-galactosidase. The results so far reported are discussed in Section 111. The scope of the present review will be limited to the enzyme and protein characteristics of galactosidases, and their occurrence, isolation, characterization, and properties as catalytic agents. The topic of induced enzyme-synthesis will only be touched on, and reference will be made to original and review articles on the subject. The biological role played by these enzymes will not be discussed. Throughout the article, all glycosides discussed will be understood t o be pyranoid, unless otherwise noted.

11. p-GALACTOSIDASEB 1. Occurrence

8-Galactosidase occurs in the emulsins of some Rosaceae (almond, peach, apricot, and apple trees),' kefir grains,' the hips of wild roses,2 and the seeds of alfalfa3 and coffee.' It is also present in Aspergillus oryzae (Takadiastase),S Aspergillus niger, Aspergillus jeavus, Escherichia coli,6 Saccharomyces fragilis? N e ~ r o s p ~ rand u , ~several other micro-organisms.1° Among the animals, it is found in snails (Helix pomatia), and in the intestines of dogs, rabbits, calves, sheep, and goats.l2JS I n the calf, a J

(1) E.Bourquelot and H. Hbrissey, Compl. rend., 187.66 (1903). (2) E.Hofmann, Biochem. Z . , 287, 309 (1933). (3) K.Hill, Ber. Verhandl. adlche. Akad. Wiaa. Leipzig, Math.-phya. K l . , 86, 115 (1934); Chem. Abstracts, 18, 6843 (1934). (4) B. Helferich and F. Vorsate, 2.phyaiol. Chem., 187,254 (1935). ( 6 ) C.Neuberg and 0. Rosenthal, Biochem. Z., 146,186 (1924). (6)E.Hofmann, Biochem. Z . , 272, 133 (1934). (7) C.J. Deere, A. D. Dulaney and I. D. Michaeleon, J . Bacteriol., 97,366 (1939). (8)B.van Dam, J. G. Revallier-Warfenius and L. C. van Dam-Sohermhorn, Neth. Milk Dairy J . , 4, 196 (1960);Chem Abalracts, 44, loo00 (1960). (9) 0.E.Landman, Arch. Biochem. Biophya., 62,93 (1954). (10)K.Nieizawa, J . Fm.Textile Sericult. Shinahu Univ., 1,213 (1961). (11) M.Cohn, Bacteriol. Revs., I1 140 (1967). (12) C.Porcher, Compt. rend., 140, 1406 (1906). (13) F. A. Cajori, Am. J . Md:d. Sci., 187. 296 (1934).

QALACTOSIDASES

241

high activity is observed in the mucous membrane of the small intestine, but the activity decreases with advancing age of the animal.**J6No activity is, however, observed in the mammary glands of nursing goats, cats, and cows.18 Human saliva” and human intestinal secretions*8have also been reported to contain this enzyme. 2. Standardization Lactose and o-nitrophenyl 8-D-galactopyranoside have mainly been employed as substrates for estimating the activity of p-galactosidase. With lactose, the liberated D-glucose can be estimated manometrically19~20or colorimetrically with notatin21p22or with hexokinase coupled either with or with pyruvate kinase and lactic dehydrogenase.24 Z~ischenferment2~ With o-nitrophenyl p-D-galactoside, the o-nitrophenol liberated is estimated spectrophotometrically. LederbergZ6and, later, Kuby and Lardy26 employed this substrate for a discontinuous method of estimation. Cohn and Monod20 and, later, Wallenfels and coworkersz7described continuous procedures which were especially suited to kinetic measurements. If it is desired to estimate high concentrations of the enzyme without dilution, phenyl or ethyl 8-D-galactosides can serve as convenient substrates. For the former, the rate of increase in optical density at 280 mp is noted. With ethyl p-D-galactoside as the substrate, the amount of ethanoI liberated in a definite time can readily be estimated2*with alcohol dehydrogenase and Codehydrogenase I. a. Continuous PToceduTe.27-Reagents: Buffer (0.05 M ) of Z-amino-Z-(hydroxymethyl)-1 ,3-propanediol hydrochloride, pH 7.6; M sodium chloride solution in buffer; 50 mg. of o-nitrophenyl @ - ~ - g a l a c t o s i d dissolved e~~ in 10 ml. of buffer; (14) N . S. C. Heilskov, Acla Physiol. Scand., 24,84 (1951). (15) K . Wallenfels and J. Fischer, 2.physiol. Chem., 321, 223 (1960). (16) H. C. Bradley, J . B i d . Chem., 13,431 (1913). (17) H. Chaucery, F. Lionetti, R. A. Winer and V. F. Lisanti, J . Dental Research, 55, 321 (1954); Chem. Abelracls, 48, 10076 (1954). (18) W. P. U. Jackson, Clin. Sci., 11,209 (1952); Chem. Abstracls, 49,11831 (1955). (19) D. Keilin and E. F. Hartree, Biochem. J . , 41,230 (1948). (20) M. Cohn and J. Monod, Biochim. el Biophys. Acta, 7 , 153 (1951). (21) J. D. Teller, Abstracls Papers Am. Chem. Soc., 190, 69c (1956). (22) A. S. G. Huggert and D. A. Nixon, Biochem. J . , 66, 1 2 (1957). ~ (23) J. Larner and C. M. McNickle, J . Biol. Chem., 216, 723 (1955). (24) G. Pfleiderer and L. Grein, Biochem. Z., 318,499 (1957). (25) J. Lederberg, J . Bacleriol., 80, 381 (1950). (26) S. A. Kuby and H. A. Lardy, J . Am. Chem. Soc., 76,890 (1953). (27) K . Wallenfels, M. L. Zarnitz, G. Laule, H . Bender and M. Keser, Biochem. Z., 331, 459 (1959). (28) K. Wallenfels, J. Lehmann and 0. P. Malhotra, Biochem. Z., 333, 209 (1960). (29) M. Seidman and K. P. Link, J . Am. Chem. SOC.,72, 4324 (1950).

242

WALLENFELS AND MALHOTRA

enzyme solution, properly diluted to give about 200 activity units (or about 0.4 y of pure crystalline enzyme) in 2.5 ml. of the test solution. Procedure: Take 0.5 ml. of o-nitrophenyl 6-D-galactoside solution, 0.125 ml. of the salt solution, 0.1-0.5 ml. of the enzyme solution, and make to 2.5 nil. with buffer. The buffer, sodium chloride, and substrate solutions are measured into a photometer cell (d = 1 cm.), and brought to the required temperature (20"). The reaction is started with enzyme and is followed by noting the optical density at 405 mp (against water) every 30 seconds. The increase of optical density (AE) in 2 minutesgo is taken for calculation of the "turnover number." The molecular extinction coefficient of o-nitrophenol a t 405 mp and pH 7.6 is 3.1 x lo3~ m ~ / m m o l e . ~ * b. Estimation of Activity an Bacterial Suspension.%-Reagents: Phosphate buffer (M/30), pH 6.8; toluene; 50 mg. of o-nitrophenyl @-D-galactosidedissolved in 100 ml. of buffer. Procedure: A diluted bacterial suspension (5 ml., containing 0.2 mg. of dry bacteria per ml.) is shaken with 0.1 ml. of toluene for 15 minutes in a ,I,-shaped tube fixed on a rotator inclined at, an angle of 5". This suspension (0.1-0.5 ml.) is then added to 4.0 ml. of the solution of o-nitrophenyl fi-D-galactoside and phosphate buffer is added to make 5.0 ml. The reaction mixture is incubated a t 40", and the optical density (at 405 mp) is noted (against water) after 15 minutes. The optical density of a reagent blank, run side-by-side is subtracted from the test valuc. c. Estimation of Activity during Purificati0n.n-Manganese acetate is added during purification of the enzyme (see below). As Mn"" ions activate P-galactosidase, all of the tests performed in order to follow the activity during purification are carried out in the presence of the optimum concentration of Mn@@ 11.1).The rest of the procedure is the same as is described under 11,2b. d. Units of Enzyme Activity.-Wallenfels and coworkersa7defined the unit of enzyme activity as that amount of enzyme which hydrolyzes 1 microgram of 0nitrophenyl P-D-galactoside in 15 minutes under the conditions described under 11, 2c. Hu, Wolfe, and Reithel,3a who crystallized P-galactosidasc from E. coli, ML 308, defined the unit of enzyme activity as the amount bringing about the hydrolysis of 10-~mole of o-nitrophenyl /3-n-galactoside per minute at Ad substrate concentration in 0.05 M sodium phosphate buffer, pH 7.0, a t 25".

3. I'urzjication a. Crystallization of /3-Galactosidase from E. coli, d1L 309.27-33---l-'reparationof cellfree eztract. The dried bacterial mass (50 g.) is finely powdered, and shaken overnight with 1,250 ml. of 111/1000 phosphate buffer (pH 7.3), 600 g. of acid-washed quartz sand, and 1.0 ml. of octanol. The temperature is maintained at 0". The mix-

(30) The authors took the interval between GO and 180 sec. after the addition of en-

zyme. (31) K. Wallenfels, 0. P. Malhotra and D. Dabich, Biochem. Z., 333, 377 (1960). (32) A. S. I,. Hu, R. C . Wolfe and F. J . Reithel, Arch. Biochem. Biophys., 81, 500 (1959). (33) Exact conditions for cultivation of the bacteria and for preparation of the dried powder are given in Ref. 27.

GALACTOSIDASES

243

ture is then centrifuged for 70 minutes at 16,000 r.p.m.3'; a clear, yellowish solution (990 ml.) is obtainrd (solution A), of specific activity, 42,000 units per mg. of protein. Removal of nucleic acids. Manganese acetate solution (250 ml. of M / l O ) is added dropwise, with constant stirring, to solution A (990 ml.). After 24 hours of standing a t O', the suspension is centrifuged for 20 minutes a t 16,000 r.p.ni. To the supernatant liquor (1160 ml.) is then added 116 nil. of 0.2%clupein sulfatc solution (pH 7.0),with constant stirring, and the solution is allowed to stand at 0" for 24 hours. The mixture is centrifuged for 20 minutes a t 10,000 r.p.m., and the supernatant liquor (1260 ml. of solution I3; specific activity, 83,000; yield, 72%) is treated further. Precipitation with alcohol. To solution R (1260 ml.) are added 125 ml. of 0.2 ill (ethylenedinitrilo) tetraacetic acid solution (pH adjusted with sodium hydroxide to pH 6.8). A total of 1385 ml. of purified, precooled ethanol (-15") is then added slowly, with constant stirring, while the solution is cooled in a freezing misture. The temperature of the bath is lowered gradually, so as to maintain the solution just above the freezing point. The solution is allowed to stand for 48 hours a t - 15" and is then centrifuged a t 2,500g a t the same temperature. The supernatant liquor is rejected, and the precipitate is freed from alcohol as completely as possible by sucking air around i t through a bent, plastic tube. The precipitate is suspended in 350 ml. of M/1000 phosphate buffer (pH 7.3) and allowed to stand at 0" for 3 hours, after which, it is centrifuged and a clear solution is obtained (solution C, 350 ml.; specific activity, 175,000; yield, 75%). Precipitation with ammonium sulfate. An equal volume (350 ml.) of saturated ammonium sulfate solution (pH 7.0) is added slowly, with stirring, to solution C, and the solution is allowed to stand for 2 hours a t 0". The suspension is centrifuged (20 minutes a t 20,000 r.p.m. a t 0"), and the supernatant liquor is removed as completely as possible by allowing it to drain away and then wiping the inside walls of the centrifuge tubes with filter paper. The precipitate is dissolved in a small amount of M/1000 phosphate buffer, pH 7.3, and the volume brought to about 15 ml. The solution is then dialyzed against the same buffer for 2 days, using a 20-mm. diameter, cellophane tube (Visking Corporation). The buffer outside the dialysis tube is changed a t intervals of 5 hours. Both solutions (inside, as well as outside, the dialysis tube) are stirred continuously. The volume of protein solution rises to about 20 ml. during the dialysis (solution D, specific activity, 360,000; yield, 90%). Precipitation with alcohol-ether mizture. To 20 ml. of solution D, a cooled mixture of 84 ml. of ether (purified, and distilled under nitrogen before use) and 36 ml. of ethanol is added dropwise with constant stirring. The temperature should be maintained only a little above the freezing point of the solution by cooling in a methanolDry Ice bath. The suspension is centrifuged immediately (5 minutes a t 20,000 r.p.m. at -35') in precooled centrifuge tubes, and the precipitate is freed from the organic solvents as completely as possible by sucking air through it, as described above. The temperature must be kept low ( -30°), because, otherwise, extensive denaturation takes place, with consequent loss of enzymic activity. A brownish, (34) The authors used a "Batch-bowl rotor" (capacity, 1.6 1.) and a Spinco Model L ultracentrifuge.

244

WAlrLENFELS AND MALHOTRA

granular mass is obtained which flows like an oil if the temperature is allowed to rise. This mass is dissolved in M/30 phosphate buffer, pH 6.0, containing 5% of sodium chloride. The volume of the solution is adjusted to a concentration of about 30 mg. of protein/ml. If the solution is turbid, it may be centrifuged and the insoluble residue rejected (Solution E; specific activity, 450,000; yield, 90%). Crystallization. A saturated solution of ammonium sulfate (pH 6.0) is added dropwise to solution E at O", with constant (but slow) stirring (60 r.p.m.). The rate of addition should be so adjusted that the precipitate formed by one drop is redissolved before the next drop falls. The addition of the ammonium sulfate solution is stopped at 23% of saturation by the sulfate (that is, s 0.23), and the solution is stirred overnight at 0". The turbidity produced is removed by centrifuging (20 minutes at 20,000 r.p.m. a t 0"). The ammonium sulfate concentration is raised to s = 0.235. If any amorphous product is formed at this stage, it is removed by centrifugation. The clear solution is nucleated with crystals from an earlier batch, and is stirred further at 0". Shining crystals begin to separate in 36-1 hour, and a thick suspension of crystals is obtained in 10-12 hours. Without nucleation, crystallization takes longer (1-2 days). The yield of crystalline 8-galactosidase (specific activity, 500,000;yield, 55%) from 50 g. of dried bacteria varies between 500 and 600 mg., depending mainly upon the activity of the bacterial powder. Recrystallization. The suspension of crystals obtained above is centrifuged (20,000 r.p.m. at 0") and the crystals are dissolved in M/30 phosphate buffer, pH 6.0, containing 5 % of sodium chloride, to a concentration of about 30 mg. of protein/ ml. The protein dissolves slowly, but completely, in 1-2 hours. The concentration of ammonium sulfate is estimated with Nessler reagent." By adding a saturated solution of ammonium sulfato as above, the concentration is raised to s = 0.23. The solution is then nucleated with P-galactosidase crystals, and is further stirred. Crystals begin to appear after 35 hour, and the process is complete in 24-36 hours. The product has a specific activity of 575,000; yield, 65%. If the above conditions are strictly adhered to, the final product is free from amorphous substances. Further recrystallization does not lead to any increase in the specific activity. The firpt crystals are thin, hexagonal plates (see Fig. 1A). On recrystallization, hexagon$ needles (see Fig. 1B) are obtained. b. Isolation of~-Gdactosidueefrom E. coli, M L 308.*-Bacterial cells are broken up by grinding them with alumina, added a t intervals until a total of about 2.5 times the wet weight of the cells is used. At this point, the mixture has the consistency of crumbly, moist sand. The paste is extracted four times with a total of 500 ml. of buffer A (0.05 M in acetic acid, 0.028 M in mercaptoacetic acid, and 0.01 M in magnesium chloride; pH adjusted to 7.0 with solid 2-amino-2-(hydroxymethyl)-l,3-propanediol) a t 0-5". The product has a specific activity of 5,000 unitsa6per mg. The extract is centrifuged for 1 hour at 25,0008 at 0", and the sediment is discarded. The clear, supernatant liquor is made 2.5% in streptomycin sulfate and,

-

(35) G . Beisenherz, H. J. Boltze, T. Bucher, R. Czok, K. H. Garbade, E. MeyerArendt and G. Pfleiderer, 2. Nalurforsch., 8b, 555 (1953). (38) For definition of the unit of activity in this procedure, see Section 11, 2.

GALACTOSIDASES

'145

B FIG.1.-Crystals of 8-G:iluctosidase of E . c o l i , R4L 309. (A: first crystallization;

B: third recrystitllization.)

246

WALLEXFELS AND MALHOTRA

after standing for 1.5 hours a t 0", is centrifuged for 1 hour a t 32,OOOg. The rc&luc* discardcd, and the supernatant liquor (specific activity, 5,000) ih trcatcd furthcr. The solution is made 64% saturated with respect to ammonium sulfatc and is :dlowcd to stand overnight a t @5". The precipitate is collected by centrifugation :iiid is dissolved in buffcr B l0.01 111 in acetic acid, 0.014 11.1in mercaptoacctic a d , :ind 0.01 M in magncsiurn chloride; pH adjusted to 7.6 with solid 2-amino-2(hydroxymethyl)-l,3-propanediol]to a conrentration of about 100 mg. of 1rotcbin/ml. (The product has a spccific artivitg of 9,600.) The solution is brought to 2S% ammonium sulfate saturation, is centrifuged after one hour, and thr Ireripitatc is discardd l'hc concentration of ammonium sulfate in the solution (spe(itic. activity, 9,300) is raised to 37 o/u saturation. The precipitate formed is separated by centrifugation and is dissolved in buffer C 10.01 J/ merraptoacetic acid solution cwntaining 0.01 A/ of magnesium chloride, adjustrd to pH 7.7 with solid 2-amino2-(hydrosymethyl)-l , 3-propanediol] to a concentration of about 80 mg./nil. (specific activity, 30,000). The solution is dialyzcd against three rhangcs of the s:tme buffer, to remove ammonium sulfate, and is then allowcd to percolate through a 3.7 x 24 mi. column of O-(i~iethylaminoethy1)rellulosea t 14 ml./hour. l'rotrin is then clutcd, using a concentration gradient of sodium chloride rising from 0 to 2$, and 5-ml. frartions are collected. .kctive fractions are poolcd (specific activity, Mi,OOo). Further purification is achieved by electrophoresis. The final product is an elrctro~~horctic.al1g homogenrous protein (sperifir activity, 146,000 units/rng. of protcin). .Utcrnativcly, the cnzynic Fractions obtained froni the O-(dicthylaminorthy1)rcllulosc column can be purified by crystallization in thr following way. The enzyme eluatc is dialyzrd against 100 ml. of buffer (0.05 119 2-amino-2-(hydrosynirthy1)-1 ,3-prol)anediol-acetic arid, pH 7.0, containing 0.01 M of magnesium chloride) to which 30 ml. of saturatrd ammonium sulfate solution, pH 7.0, has been :iddrd. After 12 hours, the small amount of precipitatc formed is removrd by rcntrifuging and is discarded. Diiilysis is continued, and saturatrd ammonium sulfate solution is uddcd a t thv rate of 1 nil. in 12 hours. After 24 hours, a copious, whitr precipitatr is obtained. The miAturc is transferred to a rcntrifuge tubc and is stirrcd vigorously. Strong birefringcncc is noted. After two recrystallizations, thr protein is elrrtrol)horcitically liomogcncous and possesses thc same specific artivity :is that obtained by electrophorek. c . Purtjkation of P-Galmtosidase from Calf Intestine.ls-Frcsh mucous Inembrane of thc small intrntinc of a calf H i homogenized with 3 times its weight of acrtone in a Waring 13lrndor a t -15". After allowing the misturc to stand a t -15" for 15 minutes, it is centrifuged a t 3,000 r.p.ni. The entire process is repeated with thr residue. The final residue is dried undcr vacuum over conccntrated sulfuric acid, finely ground, and preserved a t - 15". This powdcr (50 g.) is shaken for 5 hours at room temperature with 200 g. of quartz sand (A.R.) in 500 ml. of 61/30 phosphate buffer (pH 6.8), 500 ml. of water, and 1 ml. of octanol. On removal of the sand by centrifugation (60 min. a t 16,000 r.p.m. at 0°),34 850 nil. of a yellow, slightly turbid solution of specific activity, 8 units per mg. of protcin (unit of activity as defined by Wallenfels and coworker@ is obtained.

ik

G.4 LACTOSI D.4 SES

24i

To the ahovc solution, 45 ml. of M/lO manganese chlorid(h solution is atltlctl dropwise, with stirring, a t 0". The suspension is stirred for an extra 30 minutes niitl then 90 ml. of neutral protarnine sulfate solution (0.2%)is added slowly. The mi\ture is allowed to stand overnight a t 0' and is then centrifuged for 1 hour a t O", a t 16,000 r.p.m., affording 950 ml. of a clear, yellow solution of spec4fiv :tctivit\., 10.5; yield, 96%. .1r/io (EtIiy1enedinitrilo)tetraacetic acid solution (45 ml.) is addctl to the above supernatant liquor and then 1660 nil. of pre-cooled alcohol ( - 15') is adtl(~1 slo\vly, as the temperature is gradually lowered from 0 to -15' (final alcohol conrentrntion, 60%). The suspension is kept overnight a t -15" and is then centrifugeti :it 3,000 r.p.ni. for 90 minutes, a t the same temperature. The precipitate is suspenclcd in M/30 phosphate buffrr, pH 6.8, and is ccntrifuged a t 20,000 r.p.in., to obt:iiii 280 ml. of a colorless supernatant liquor of specific activity, 17.3; yield, 80%. The above solution is brought to pH 4.45 by adding 1 .If acetic acid solution (about 12 ml. are required) a t room temperature. After the mixture has been kcpt for 15 minutes a t room temperature, the precipitate is removed by centrifugation (30 minutes a t 20,000 r.p.m.) and discarded. T o the supernatant liquor (271 ml.; specific activity, 126; yield, 81 %), 1.94 g. of sodium acetate is added (pH 4.9). ilnother alcohol precipitation (at 50 % alcohol concentration) is carrirtl out a s above, and the precipitate is dissolved in a small volume of M/30 phosphate buffrr, pH 6.8, to obtain a conccntrated solution ( > I mg./ml.) before carrying out the nest step. The solutions obtained from several batches (the authors collected froin 3 bat(-lies;total vol., 39.3 ml.; specific activity, 200; yield, 81%) are collected, and ammonium sulfate solution (saturated a t 0') is added to a saturation of s = 0.15 (6.96 rnl. were added). The tenipcrature is then lowered to -15 to -20°, and prccooled alcohol ( - 15') is dropped in, to give a concentration of 50 %. The suspension is allowed to stand for 4 hours a t -15' and is then centrifuged. The 1Jrccipit:ite is suspended in 15 ml. of M/30 phosphate buffer, pH 6.8, and centrifuged, affording :i clear solution (specific activity, 1520; yield, 98%). The last step is repeated once (yield, 64%). The final solution has a specific activity of 17,000 units 1)cr mg. of protein. Starting from 50 g. of dry powder (obtained from 8-10 meters of intestine) oiily about 1 mg. of protein, at a final concentration of 0.22 mg./ml., is obtained. d. Separation of Carbohydrates and Enzyme in a Preparation from Calf Intestine.'; -Wallcnfels and Fischcr found that a highly purified 0-galactosidase preparation from calf intestine contained a large proportion of carbohydratesI5 (sre dso, Scction II,5e). They have attempted to separate the material having the enzyme activity froin the carbohydrates by chromatography on 0-(diethylaminoethyl)cellulose. A 12 x 1 cm. column of 0-(diethylaminoethy1)ccllulose (washed with 0.002 .If Na-K phosphate buffer, pH 6.8) was prepared in a glass tube (having a sintcwtlglass bottom) which could be cooled to the required temperature. -1 volumc of 6-8 ml. of enzyme solution (dialyzed earlier against 0.002 M phosphate buffer, pH 6.8, to remove ammonium sulfate) containing 10-13 mg. of protein (spc(4k activity, about 5,000) was pipetted onto the top of the column. Elution was carrim1 out with the same buffer (7-10 ml./hr.), and fractions were collected every 30

niinutcs and unrilyertl for carbohydrate (by the anthrone reaction) anc I for pga1:ictosidase activity. a f t e r h fractions had been collrctcd, :t 0.01 JI pliosphttte buffer, pH 6.8, was employed for elution. After collection of fraction 26, the buffer eniploycd had 0.01 ill concentration of ammonium sulfate. Until then, only c:irbohydrates (and no activity) could be cluted. Ak-tivcniaterial came down thc column when the conccntrittion of ammonium sulfate was raisctl to 0.05 111. Apart from a

1.5

1.0

2 E

-P 111 Y

t

*

U c

n

L

1.5

0

FIG.2.-Eepttration of 8-Galactosidase from Carbohydrates (from a Preparation of Calf Intestine), on O-(2-Diethylaminoethyl)celh1lose.~~ (0--0, enzyme nctivity; 0-- -0, carhohydratcs.)

slight activity observed in fractions 31-33, /3-galactosidase was mostly eluted in fractions 35-37. The activity was also paralleled b y a carbohydrate fraction (see Fig. 2). The active fractions showed a specific activity of 16,50&17.300 units per nig. of protein.

4 . Distinction betwecn P-Galactosidases from Diferent Sourccs

NisizawaL"compared t hc inhibition, specificity, and substrate affinity of p-galactosidasc preparations from different sources, but found no rcla-

GALACTOSIDASES

249

tionship common to all preparations. Cohii and Monod'l s37 ,38 have shown that the enzyme preparations from E . coli K 12, Aerobacter aerogenes, S'higella sonnei, and different mutants of E. coli ML are not distinguishable through their specificity and immunological behavior. The enzymes of Saccharomyces and Lactobacillus are, on the other hand, different from the above, as well as from each other. Widely different "turnover numbers" and ratios of the extinction a t 280 mp to that a t 260 mp are, however, observed for the crystalline 0-galactosidases of E. coli ML 308 and E. coli ML 309 although their sedimentation coefficients agree well with each other.flJ2 Different patterns for the influence of alkali ions on the enzyme activity are observed with preparations from E . coli strains K 12 and ML.2Oo 2 6 3 1 It has been shown that, in the D-galactose transfer reaction, the enzymes of E. coli and of the snail behaved similarly to each other, but those of calf intestine and of Aspergillusjlavus were different from them and from each other.39Crystalline 0-galactosidase of E. coli ML 309 and highly purified, calf-intestine enzyme exhibit very different specificities'5 828 (see also, Tables IV and V) and can be further distinguished by their different chemical compositions. 5 . Physical and Chemical Properties

a. Ultracentrifugation and Electrophoresis.-An enzyme fraction from E. coli ML was purified by Cohn'l and was heterogeneous in the ultracentrifuge, showing two peaks: a major, monodisperse component (80% of the , and a minor, polydisperse component 18-26). total; L S , , ~ O13.9) Both components were enzymically active. He concluded that the minor fraction consists of polymerized products-artefacts resulting from the handling. The molecular weight of the major component was estimated to be 700,000. As the details of calculation have not been published, it is difficult to know how Cohn arrived a t this high value of molecular weight with a relatively lower sedimentation constant (see also, the values reported by other workers). Cohn and Monod20 were able to separate, electrophoretically, two fractions which were identical in every respect except enzyme activity; one fraction was three times as active as the other. Reithel and coworkersg2have obtained, from E. coli ML 308, a preparat ion of p-galactosidase which was homogeneous on electrophoresis and in the ultracentrifuge (see Fig. 3) and had a diffusion constant of 2.12 X mi.? see.-' (referred to water at 20") and a sedimentation constant of 16.24 (referred to water at 20"). Assuming a partial specific volume of 0.750, they have estimated the iiolecular weight to be 7.47 X 105. On crystalliza(37) M. Cohn and A. hI. Torriani, Conapt. rend., 232, 115 (1951). (38)J. Monod and M. Cohn, Advances i n Enrymol., 13. 67 (1952). (39) K. Wallenfels, E. Bernt and G . Limberg, Ann., 684,63 (1953).

250

\VXLLENPELS A S I ) MrILHOTHA

tion, the preparation obtained ivas heterogelleous in the ultracentrifuge but still homogeneous by electrophoresis. Wallenfels and coworkersz7 found one major component (Sw, 2 u ., , 16.90) and several minor components of higher sedinientation constants. The relativc proportions of thc different fractions varied with the esperimental conditions (such as pH and ionic concentration), indicating that the enzyme molecule undergoes assoriation and dissociation. On incuhatioii

FIQ.3.-Ultrscentrifugation Pattern of 8-Galactosidase of E . coli, ML 308 (Purified by Prepnriitive Electrophoresis).8’

with p-(chloromercuri)berizoate, some slowly sedimenting fractions (S,,, .2u, 8, 12, and 14) were produced. For shorter incubation periods, this effcc-t could be reversed by the addition of reduced glutathione. The proportioir of the major component increased on repeated recrystallization or on the addition of zinc ions or of o-nitrophenyl l-thio-p-D-galact,opyraiiosidr. After six recrystallizations, the enzyme consisted of up to 98% of oirc component (s. 16.90)40(see Fig. 4). Ultracentrifugation of purified, calf-intestine enzyme showed two peaks; only the rapidly sedimenl ing component seemed to be erizymically active. b. Ultraviolet Absorption Spectrum.-The ultraviolet absorption spcc(40) K. Wallerifels and H. Sund, unpublished results.

GALACTOSIDASES

25 1

trum (see Fig. 5) of crystalline p-galactosidase from E. coli M L 309 showed a maximum a t 280 mp, with an inflection a t 290 mp. The value of the estiiiction coefficient a t 280 mp was 11.81 cm.*/mg. of nitrogen or 1.91

Flu. 4.-Ultracentrifugation tirncs recrystallized).

Pattern of 8-Gulactosidase of E . coli, RIL 309 (six

2.0

L

Wavelength (mu)

FIG.5.-Ultraviolet Absorption Spectrum of Crystalline j3-Galactosidase of E . coli, ML 309.*7( M / 3 0 Phosphate buffer, pH G.8.)

cm.Z/mg. of protein. The ratio of the extinctions a t 280 and 260 mp was to be 1.92. Cohn" gave the value 1.73 for the purified enzyme from E. coli ML, with an extinction coefficient (at 278 mp) of 1.56 cm.2/mg. of protein. Reithel and coworkers3*reported 1.52 as the value of the ratio E280n,,,/E26~mp for an electrophoretically homogeneous preparation from E.

252

WA4LLENFELSA N D MALHOTRA

coli lLlL 308. The extinction coefficient a t 280 mp (the maximum of absorption) was found to be 1.85 cm.*/mg. of protein. The ultraviolet absorption spectrum of the most artive fraction of culfiutestirie erizynie (purified 2,000 times) shows a maximum a t about 280 nip with the ratio E280mp/E280mp equall6 to 1.47. c. Elementary Composition of Crystalline P-Galactosidasc of E. cdi, AfL 309.-Crystalline P-galactosidase of E. coli, ML 309, is free from lionprotein matter. The enzyme contains2' 51.6 % of carbon, 6.33 % of hydrogen, 16.10% of nitrogen, and 0.93% of sulfur. Analysis for metals, in samples recrystallized a number of times in the authors' laboratories, wcre carried out by B. 1,. Vallee41in Boston. The results are shown in Table I. TABLEI Analysis for Afelais in the Crystalline 8-Galactosidase of E. coli, dlL S0941 Number of recrysldlizalionsa .Meld

a Nuinbers represent tested.

1

3

5

7

Ca Mg

358

195 -

Ba Na I< A1

X X

275 3 505 110 33

269 x X

gg. of mrtitl

-

2

x

1340 150

X

2G

x X

per g. of protein; - = not found; X = riot

Calc4um setms to be consistently present in all of the samples. Magnesium could not be detected at all. d. Amino Acid Analysls and Determination of End Groups in Crystalline 0-Calactosidase of E. coli, ML 309.-The results42 of amino acid analpis carried out by the diiiitropheiiylatioii technique are shown in Tahle 11. Six amino end-groups (four of threonine and two of glutaniic a d ) wrw found43for a molecular weight of 750,000. e. Chemical Composition of Calf-intestine Enzyme.-An wzyine prepnixtion, obtained by rhroniatography on ion exchangers and huviiig a spwific activity of 10,000 units/mg. of protein, was analyzed by Walleiifrls and Fischer for its chemical wmposition.16 The rrsults are showii in Table 111. As only small amounts of the more active frartions were available, diemical analyses could not lie performed on them, but chromatographic aiialysis indicated that their chemical compositions werc similar. (41) 13. 1,. Vullee, persond commrinicstion. (42) K . Wallenfels and .4. Arms, Biorhenb. Z., 333,247 (1960). (-13) K. Wirllriifelu uiitl A. A r m s , Riorhem. Z . , 333, 395 (19GO).

253

(JALACTOSIDASES

TABLEI1 Estimation o j Amino Acids in the 8-Galactosidase of E. coli, ML SOW Amino Acid

Alanine Arginine Aspartic acid Cystine/2 Glutamic acid Glycine Histidine isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophanb Tyrosine Valine Amide NHr

+

Total

Mole of amino aid/ 100,ooO g. of protein

g. of N/lOO g.

of protezn

g. of amino acid r e d u e per 100 g. of protein

60.51 f 1.17 46.04 f 0.98 110.98 f 2.08 16.39" 82.02 f 1.47 58.27 f 1.60 21.28 f 0.12 121.01 f 5.29 19.68 f 0.60 19.86 f 0.37 32.10 f 1.08 47.77 f 1.28 36.69 f 0.51 46.22 f 2.39 32.25 17.20 i 1.16 51.82 f 1.65 104.o o c

0.848 2.580 1.555 0.230 1.149 0.816 0.894 1.695 0.551 0.278 0.450 0.669 0.514 0.647 0.903 0.241 0.726 1.457

4.30 7.19 12.77 1.68 10.59 3.33 2.92 13.70 2.52 2.61 4.73 4.64 3.20 4.67 6.00 2.81 5.41 1.66

820.09

16.203

94.46

a Estimated as N-(dinitrophen 1)cysteic acid. As a maximum of 12.5 free sulfhydry1 groups are found with p-(c%loromercuri)benzoate, this value represents 12.5 cysteine and 2 cystine residues. b Tryptophan was estimated spectrophotometrically. c Not included in total.

TABLE I11 Chemical Composition of the p-Galactoaidasea of Calf

% Protein (after Folin) 7.0 Galactose 17.0 N-Acetylglucosamine (estimated 71.O after Boas") Moisture 9.0 Cb 46.0 H* 6.9 Nb 6.6 Ash 1.04 Acetylb 12.6 0 Specific activity, 10,OOO; purification, 1,250-fold. Calculated from above composition: C, 44.8; H, 6.05; N, 6.03, and acetyl 13.8%.

(44)N. F. Boas, J . Biol. Chem., 204,553 (1953).

254

WALLENFELS AND MALHOTRA

Nothing can be said at present regarding the biological implications of an extraordinarily high carbohydrate content, but it is of interest to recall that a 200-times eiirirhcd, ycast sucrasc was earlirr found to consist of up that rrystalline hcxokinasc rontains about to 70 % of polysa~rharidrs,~~ 50% of m a i i ~ i a n ,and ~ ~ that the cmulsins of sweet almo1ids~7-~9 and of snail60 contain carbohydrates. It has also been suggested6’that a glycosidase protein might, in some way, incorporate a sugar residue which would correspond to the area of specific adsorption of the glycon part of the substrate molecule; this would explain the high specificity shown for this part. Although it does not seem to be true of all glycosidases (for examplr, the crystalliiic P-galactosidase of I<. coli does not possess carbohydratrs), further results must be obtained before this question can be settled drfinitcly. Possibly, the high proportions of polysaccharides present in “purc” glycosidases might be related to their localization in the cell. Yeast invertase might be localized in the cell wall, which consists largely of mannan. Similarly, the association of calf-intestine 0-galactosidase with mucopolysaccharides niight be correlated with the fact that the enzyme occurs in the mucouR membrane of the small intestine. f. Stability.-(i) Influence of temperature. Cohri and MonodZ0have shown that the thrrmal inactivation of P-galactosidase is a first-order reaction. At 55”, the activity is completely lost in less than 1 minute, whereas, a t 47.3”, there is less than 5 % inactivation in 10 minutes. Similar results were reported by Z a r n i t P with crystalline 6-galactosidase of h?.coli, M L 309; a t lower temperatures, the enzyme is exceptionally stable. No loss in activity is observed on keeping the calf-intestine enzyme for 10 minutes a t 40” (pH 5.0). A steep fall in activity occurs as the temperature is raised. At temperatures below 30”, the activity showed no decrease in 20 minutes.I6 (ii) Influence of pH. There is almost no loss in enzyme activity when a solution of P-galactosidase of E. coli, ML 309 (250 pg./ml.; veronal buffers) was maintained for 30 minutes a t 40” and p H 6 to 8. The stability decreases sharply below p H 6, and slowly as the p H is raisedz7above 8. The calf(45) E. H. Fischer, L. Kohtes and J. Fellig, Heio. Chim. Acta, 34. 1132 (1951). (48) H. Boser, 2. physiol. Chem., 900, 1 (1955). (47) B. Helferich, W. Richter and 8. Grunler, Ber. Verhandl. sUchs. Akad. R’iss. Leipzig, Math.-phys. KZ.,89, 385 (1937); Chem. Abstracts, 32, 5859 (1938). (48) B. Helferich and W. W. Pigman, 2.physiol. Chem., 269,253 (1939). (49) B. Helferich, R. Hiltmann and W. W. Pigman, 2. physiol. Chem., 269, 150 (1939). (50) B. Helferich and J. Goerdeler, Ber. VerhundZ. siichs. Akad. W i s s . Leipzig, Math.-phys. KZ.,92, 75 (1940); Chem. Abstracts, 36, 5919 (1941).

(51) H. Bauman and W. W Pigman, in “The Carbohydrates,” W. W. Pigman, ed., Academic Press Inc., New York,N. Y., 1957, p. 636. (52) M. L. Zarnitz, Doctoral Dissertation, Freiburg, Gar., 1958.

255

GALACTOSIDASES

intcstine enzyme is most stable a t p H 5 (20", 30 minutcs). A steep fall in activity is observed below pH 4,but only a gradual fall takes ~ l a c e ' ~ , ~ ~ a t pH > 6. 6 . Hydrolytic, Synthetic, and Transfer Reactions

The glycosidases are known to catalyze hydrolytic as well as transfer reactions, that is, the sugar residue forming the glycon part of the substrate molecule may be transferred to water or to some other hydroxylic acceptor (such as another sugar or an alcohol). With P-galactosidases from various sources, this phenomenon was studied by Aronson, Ballio, Pazur, ,54-60 The synthesis of glycosides from Wallenfels and their co~orkers.~g free sugars and alcohols has also been shown to be catalyzed by these enzymes. Whether all three reactions are catalyzed by the same or by different enzyme proteins has long been a topic of discussion. With a crystalline p-galactosidase, Wallenfels and coworkers2' have shown that one and the same enzyme is responsible for these three reactions insofar as galactosyl transfer is concerned. To date, this is the only example in which it has been definitely established that these reactions are catalyzed by the same enzyme. In fact, an interesting relationship has been observed among these reactions. Of the various isomeric galactosylglucoses, the isomer most readily hydrolyzed is also that produced with the greatest speed by transfer and synthetic reactions (compare Fig. 5A, for L-arabinose transfer). The order of decreasing velocity of hydrolysis is (1 6 ) > (1 + 4) > (1 --+ 3) (see Table V), whereas the order for synthesisR1is (1 + 6) > (1 + 4) 2 (1 -+ 3). With purified, calf-intestine enzyme, this order is reversed, both for the hydrolytic and the transfer reactions.'6 On longer incubation, all of the products are hydrolyzed. These observations are readily explained if it is assumed that the aglycon and the acceptor occupy the same position on the enzyme molecule. The products of transfer can be detected in even lcss than 1 minute after the start of the reaction. Thc galactosyl transfer cannot be due to hydrolysis followed by synthesis, becausc this would involve synthesis a t exceedingly low D-galactosc concentrations, a phenorncnon which could not be ---f

(53) R. G . Young and F. J. Reithel, Biochim. el Biophys. Acla, 9, 283 (1952). (54) I<. Wallenfels, Naturwisaenschaften, 33, 306 (1951). (55) M.Aronson, Arch. Biochem. Biophys., 39, 370 (1952). (56) K.Wallenfels, E.Bernt iind G. Limberg, Ann., 679, 113 (1953). (57) J. H.Pazur, Science, 117. 355 (1953). (58) J. H.Pazur, J. M. Marsh and C. L. Tipton, J . Biol. Chem., 233,277 (1958). (59) J. H.Pazur, J. M. Marsh and C. L. Tipton, J . Am. Chem. SOC.,80,1433 (1958). (60) A. Ballio and S. Russi, Tetrahedron, 9, 125 (190). (61) K. Wallenfels, D. Beck and J. Lehmann, unpublished results. (GZ) K. Takano and T. Miwa, J . Biochem. (Tokyo), 40,471 (1953).

256

WALLENFELS AND MALHOTRA

a-L-Arabinose and /3-D-galactose differ only in the substituent on C-5 of the pyranose ring. The a-L-arabinosides are also hydrolyzed by 0-Dgala~tosidases,~~ ,28,0 and the transfer of an L-arabinose residue to D-glucose with enzyme of E. coli, ML 309, has now been The hydrolytic and transfer reactions of a-L-arabinosides, however, differ from those of 8-D-galactosides in two respects. (1) The hydrolysis of different galactosylglucoses shows diflerent orders of decreasing rate with enzyme preparations 1.0

0.8

1

0.6

E 0.4

0.2

0 Time ( M i n . )

-

Fro. 6A.-Formation and Disappearance of 0-a-L-Arabinosyl-n-glucose8with 8-Galactoeidaee of E . coli, ML 309.64 (Donor: o-nitrophenyl a-L-arabinopyranoside. Acceptor: D-glucose. 0.06 M 2-amino-2-(hydroxymethyl)-l,3-propanediolhydrochloride buffer, pH 7.6; 40".I, Oa-L-Arabinoeyl-(l --+ 3)-~-glucose;11, 0-a-L-arab;I I I , O ~ - ~ - a r a b i n o ~ y l -6)-~-glucose. (l+ The disaccharide8 inosyl-(l-+4)-~-glucose were estimated with triphenyltetrazolium chloride. The ordinate shows the extinction at 546 mp.)

from E. coli and from calf intestine, namely, (1 -+ 6) > (1 -+ 4) > (1 -+ 3) and (1 ---t 3) > (1 -+ 4) > (1 -+ 6), respectively, whereas the hydrolysis of arabinosylglucosesshows the same order, namely, (1 -+ 3) > (1 -+ 4) > (1 --t (i), with both the enzymes. (2) The a-L-arabinoside transfer reaction catalyzed by the E. coli enzyme shows another order of decreasing rate, (1 -+ 3) > (1 -+ 6) > (1 -+ 4), than is observed for hydrolysis. Kinetics of the formation and subsequent disappearance (by hydrolysis) of these compounds is shown in Fig. 5A. A special feature of the arabinose (63)J. Monod, G . Cohen-Raaire and M. Cohn, Biochim. et Biophys. A d a , 7, 585 (1951). (64) K. Wallenfele and D. Beck, Ann., W , 4 6 (1980).

GALACTOSIDASES

257

moiety with respect to the formation of enzyme-substrate complex will be discussed later. Several examples of enzymic synthesis of glycosides from monosaccharides and alcohols or other sugars have been described by Bourquelot and his coworkers, for example, the synthesis of ethyl and propyl P-D-galactop y r a n o s i d e ~ ~and ~ - " ~of ethyl a-L-arabinosidees with enzymes from almond emulsin and kefir. The conditions employed were not, however, physiological-very high concentrations had to be taken. The reaction was very slow and, therefore, long periods of incubation were necessary. Wallenfels and coworkerseQhave now demonstrated that synthesis is also possible under physiological conditions. They placed a layer of water-insoluble, but still fully active, p-galactosidase70on a carbon-kieselguhr column and circulated a 2-5% sugar solution containing equal amounts of D-glucose and D-galactose. The carbon column was saturated with oligosaccharides within a few days. Elution, followed by chromatography, showed that, (1 -+ 6), (1 4 4), and (I -+3) 8-D-galactosyl-D-glucoses had been formed.

7 . SpeciJicity The biological substrate for P-galactosidases from all sources is probably lactose. This, however, is not always the best substrate. The values of the Michaelis constants (K,) and maximal velocity (V,,,) of hydrolysis of several substrates under different conditions with 0-galactosidases of E. coli ML 309,28and calf intestinel6 are given in Table IV. I n other cases, M the hydrolysis has been studied a t a substrate concentration of only. The relative hydrolyzabilities of various substrates under these conditions can be read off from Table V.16,28Tables IV and V also give a comparison of the action of the enzymes of E . coli and calf intestine. In still other cases, semi-quantitative results with paper-chromatographic techniques have been reported.28These results are not reproduced in this article, but will be included in the discussion of specificity which follows. Results with less-pure enzyme preparations will also be discussed. The data in Tables IV and V suggest that specificity is to be attributed to a specific configuration of the enzyme rather than to the relative stabilities of various glycosides, for example, the ratio of V,,, for o-nitrophenyl 8-D-galactoside to that for a-lactose is 14.9 for E . coli enzyme and 0.3 for calf-intestine enzyme. The corresponding values for the ratio (66) E. Bourquelot and M. Bridel, Compl. rend., 166, 731 (1912). E. Bourquelot and H. HBrissey, Compt. rend., 166, 1552 (1912). M. Bridel, Compl. rend., 172, 1288 (1921). M. Bridel and C. Bgguin, Compt. rend., 182,812 (1926). K . Wallenfels, D. Beck and J. Lehmann, unpublished results. Water-insoluble, but still fully active, enzyme is obtained by carrying out the alcohol precipitation without pre-addition of (ethylenedinitri1o)tetraaceticacid. (66) (67) (68) (69) (70)

E5

01 00

TABLEIV Michaelis Constants (K,) and Maximal Velocity (V-=) of Hydrolysis of Various Glycosides zvith j3-Galactosidases of E. coli, ML 309** and of Calf Intestine*& E. coli, M L 309"; 0.05 M Z-amino-Z-(hy&oxymelhyl)-l,3-propanedwl- Calf intestine1s;Citrate-phosphate buyer, HC1 buffer, p H 7.6; 20" pH 5.3

I

I &D-Galactoside o -nitrophenyl p-nitrophenyl methyl salicylate phenyl 8-D-Galactosyl-(l 3)D-glUCOSe &D-Galactosyl-(l 4)D-glucose (lactose) -+

/

I

1

Withollt salt With 0.05 M With 0.05M NaCl KCl

0.95 4.45 1.0 3.23

With

Vitllotll o.o.5 salt

NaCl

20"

KCl

I

40"

~~

X lW3 1.61 X lW' 1.56 X lWa 134

X lo-' 5.13 X lW6 1.8 X 1WS X 1W' 2.5 X 1 P X lWa 1.47 X lWa -

18.5 6.31 6.55 -

178 22.4 4.2 10.4

-

83.5 4.67 X lWa 4.51 X lW3 29.5 139 54.5 1.0 X lo-' 1.14 X lW' 27.0 115

-

-

4.72

x lo-J 4.39

x 1Wa

-+

5.65 X 1.04 x 8equilibrium mixt. 5.55 x ,T-D-Galactosyl-(l -+ 6)- 8.32 X D-glucose a - L - Arabinoside o-nitrophenyl 7.14 X 1.05 X p-nitrophenyl a-

a

lWp 2.02 X 1Wa 1.8 X 10-* 9.0 lo-' 4.23 10-8 6.52 12.85

-

lWa ,3.92 X 1W' 1.82 X 1W' lW2 11.87 X 3.24 X 1W*

K , is expressed as mole per liter and V,

3

13.7 16.96

6.6

-

89.1 28.2

32.75 7.55 - 1.70 - 1.16 - 1.82 47.3 35.3

X X X X

lWa 7.63 X l(r* 1.38 X 1W* 1.0 X lWa 2.0 X

1W5 lW* 10-* l(r* 1.7

1.17 X lea3.63 X lW* -

as micromoles of substrate hydrolyzed per minute per mg. of protein.

2.76

_

3.8 8.05

-

~

259

QALACTOSIDASES

TABLBV Hydrolysis of Various Substrates" by the 8-Galactosidases of E. coli, ML and Calf Intestine"

Substrate

8-D-Galactoside d-ni trophenyl p-nitrophenyl phenyl methyl salicylate ethyl 6-deoxy- (ethyl 8-D-fucoside) "3-deoxy"- (ethyl 3-deoxy-8D-xylo-hexoside) phenyl 6-0-methylethyl 4-0-methylethyl 3-0-methyl ethyl 2-0-methylP-D-Galactosyl &D-glucoside 8-D-Gdectosyl-(l + 3)-~-glucose p-D-Galactosyl-(l + 4)-~-glucose(lactose) a-

Bequilibrium mixture fl-D-Galactosyl-(l --* 6)-~-glucose(allolactose) 6-Deoxy-~f-~-galactosyl(1 + 6) -D-gluCOSe (8-D-fUCOSyl-D-glUCOSe) a-L-Arabinoside o-nitrophenyl p-nitrophenyl phenyl a-L-Arabinosyl-(l -+ 3)-~-glucose a-L-Arabinosyl-(l 4)-~-glucose a-L-Arabinosyl-(1 + 6)-~-glucoee

309,28

E. coli, M L 309; 0.05 Calf Intestine; M 2-amino-2-(hydroxyIzethyl)-l,3-propanediol- citrate-phosphate bujer, pH 5.3 HC1 buffer, pH 7.6; 20" Rate of hydrolysisb

Relative rat@

20"

127.5 18.0 4.7 3.07 0.06 0.003 0.0

2125 300 78.2 50.1 1 0.05 0.0

20.0 13.8

0.0 0.0 0.0 0.0 2.0 2.11

5.56 1.99 4.2 7.0

90.3 33.2 70.0 116.6

11.80 8.16 0.27 3.36 0.55 0.24

-

-

0.0

0.0 0.0 0.0 33.3 35.8

0.288

-

40"

54.2 1.56

-

-

-

-

67.4

149.0

54.3

120 89.6 107 3.1

35.2

44.3 1.4

4.8

0.38

0.98

196 136 4.5 56.0 9.2 4.0

1.96

5.93

-

-

0.98 0.21 0.01

3.18 1.64 0.07

-

~

Substrate concentration, 10-2 M (except for eth 8-D-galactoside, 5 X 10W M ) . b Rates of hydrolysis are given in pmole of substrati iydrolyzed per minute per mg. of protein. The relative rate has been calculated b: :aking that for ethyl 8-D-galactoside as unity. 0

260

WALLENFELS AND MALHOTRA

Vmaxof a-lactose to that for allolactose (6-0-P-~-galactopyranosyl-D-glucose) are 0.7 and 56.5, respectively. Such values might be taken to be characteristic of various types of galactosidases. From the results reported so far, the following conclusions can be drawn regarding the glycon and aglycon specificities.

i

OH

(1)

I

a. Glycon SpeciJicity.-Like other glycosidases, 0-galactosidases exhibit strict specificity requirements for the structure of the glycon part of the substrate molecule. Only the changes in substituents on C-5 of the Dgalactose residue seem to be compatible with hydrolyzability. 1. The hydroxymethyl group on C-5 can be replaced by a methyl group, as in 0-D-fucosides (6-deoxy-~-galactosides) or by a hydrogen atom, as in a-L-arabinosides, without rendering the resulting compound completely immune to the action of 8-galactosidases. However, 0-D-fucosides are, in general, very poor substrates. 2. Methylation of one or more of the hydroxyl groups on C-2, C-3, C-4, and C-6 leads to complete loss of hydrolyzability with the E. coli enzyme,28~6a as well as with the calf-intestine enzyme.16 3. The D-galactopyranosidt? ring seems to bc essential. Thus, o-nitrophenyl 8-D-galactofuranoside could not be hydrolyzed by the E . coli enzyme.62Ethyl 0-D-galactofuranoside was not hydrolyzed by the calfintestine enzymc.16 4. a-D-Galactosides are not hydrolyzed.6a 5. 3-Deoxy-~-xylo-hexoside~ (3-deoxy-"~-galactosides") are not hydrolyzed by the E. coli enzyme OF by the calf-intestine enzyme. 6. Replacement of the D-galactosidic, anomeric oxygen atom by a sulfur atom was reported to lead to complete loss of activity, although the affinity ~ , ~ ~ showed that for the enzyme remains una1tered.I' J ~ , A~ re-investigation 0- and p-nitr6phenyl 1-thio-8-D-galactopyranosideswere split at a high concentration of enzyme. At 20" and pH 7.6, in 2-amino-2-(hydroxymethyl)-1 3-propanediol hydrochloride buffer, o-nitrophenyl P-D-galactopyranoside is split 7 X 106 times faster than its sulfur analog.78

QALACTOSIDASES

261

7. Epimerization a t any asymmetric carbon atom renders the compound unhydrolyzable by 8-galac tosidase. b. Aglycon Specifcity.-For both enzymes, wide tolerance is shown for changes in the aglycon, which may be another sugar residue, an alkyl group, or an aryl group. The rate of hydrolysis is, however, strongly influenced (see Tables IV and V). With the E . coli enzyme, aryl 8-D-galactosides are, in general, better hydrolyzed than D-galactose-containing disaccharides or alkyl 8-D-galactosides. With the enzyme of calf intestine, however, (1 --+ 3) and (1 + 4) 8-D-galactosyl-D-glucoses are the best substrates. With the bacterial enzyme, a-lactose shows a lower K , and a higher V,,, than 8-lactose. With calf-intestine enzyme, on the other hand, K , for a-lactose is lower than that for the 8-D anomer, whereas equal values of Vmaxare observed. Thus, at finite concentrations, a-lactose is better hydrolyzed by the calf-intestine enzyme, as well as by bacterial enzymes. It is interesting, in this connection, to recall the finding of Malyoth71 that 8-lactose is a better baby-food than the a-D anomer, and, also, the later discovery that &lactose favors the growth of Bifidus flora in the lower intestines of babies.72 o-j3-D-Galactosyl-( 1 -+ 3)-~-fructose,O-P-D-galactosyl-(1 + 3)-, (1 +4)-, and (1 --+ 6)-~-acety~-~-glucosamines, and lactotriose are also hydrolyzed. A slight hydrolysis is observed with O-/%D-galactosyl-(1-+ 6)-~-glucosazone, but not with O-p-D:galactosyl-( 1 44)-~-glucosazone.28 The aglycon determines not only the relative rates of hydrolysis but also the Michaelis constant (see Table IV). No correlation is, however, observed between K , and V,, . Although the exact nature of K , is not known in all cases, the data in Table IV do suggest that the aglycon exerts a strong influence on the enzyme-substrate affinity. This part of the substrate molecule must, therefore, also be bound in some way to the active site, but probably less specifically than the glycon. c. Acceptor Speczficity.-Takano and Miwa02n78 studied the acceptor specificities of purified-enzyme preparations from apricot emulsin and green leaves of the elder tree (Sambucus sieboldiana) and found that most of the aliphatic alcohols are able to act as acceptors, whereas lactate and malate are inactive. The efficiencies of various alcohols as acceptors in the transgalactosylation reaction are not the same for the two enzymes, indicating that the acceptor specificity of an enzyme may vary with the source. These authors believe that glycosidases might exist which perform hydrolysis exclusively, even in the presence of alcohols, and others which only catalyze the transfer reaction, representing two extreme cases of G.Malyoth, 2.Kinderheilk., 61, 3 (1939). (72) A. Adam, Monatsschr. Kinderheilk., 97, 500 (1949). (73) K. Takano, J . Biochem. (Tokyo), 43, 205 (1956).

(71)

262

WALLENFELS AND MALHOTRA

acceptor specificity. Similar views were expressed by Wallenfels as a result of studies with sugars as acceptor^.'^ Acceptor specificity of a different type was observed by Wallenfels ~ ~ , They 8 ~ found , ~ ~ that different, isomeric, transfer and coworkers.15~s9 products were formed at different rates with enzyme preparations from E . coli, calf intestine, Helix pomatia, and Aspergillus oryzae. These results have already been discussed. Studies are in progress in the authors' laboratories on the transfer of 8-D-galactosyl, P-D-fucosyl, and a-L-arabinosyl residues from aryl glycosides onto mono- and oligo-saccharides, in order to define the limits of acceptor specificity and to explore preparative possibilities for the synthesis of p-D-galactosyl-, 8-D-fucosyl-, and a-L-arabinosyl-oligosaccharides. The results obtained so far6' are shown in Table VI. Although not shown in Table VI, it has been found that the limits of acceptor specificity are much narrower with di- than with mono-saccharides, for example, sucrose is a much better acceptor than maltose or cellobiose. This suggests that a more or less specific binding takes place throughout the entire acceptor molecule. It is interesting that 2-, ;3-, or 6-deoxy-~-"galactoses" are not such good acceptors as D-galactose and that 3 ,6-dideoxy-~-"galactose" (colitose) completely inhibits the action of @-galactosidaseunder the conditions of the experiment. Further examination of this phenomenon might possibly lead to an explanation of the presence of deoxy sugars as end groups in many physiologically occurring polysaccharides. There is one common feature of all transfer studies. If a substance can function as an acceptor, it must evidently have a higher affinity for the enzyme than has water, because, in all these reactions, water (used as the solvent) is present in large excess. This holds for other glycosyl t,ransfer reactions as well. 8. Kinetics of E. coli 8-Galactosidase a. K , and V,,, .-The values of K , and V,,,, for various substrate?, and the influence of sodium chloride and potassium chloride thereon,** are shown in Table IV. b. E$ect of Temperature on K , and V,,, .-The influence of temperature on K , and VmaXhas been studied with o-nitrophenyl p-D-galactoside, which is the best substrate for this enzyme. The results are shown in Table VII. The Michaelin constant and V,,,,, (in the presence of sodium chloride) rise with increase in temperature; V,,, in the absence of sodium chloride shows a decrease as the temperature is raised above 30". The values in Table VII show that K, and V,, vary independently of each other, which might suggest that the Michaelis constant approaches the dissocia(74) K. Wallenfels, Colloq. Ges. physiol. Chem., 4, 180 (1953).

263

GALACTOSIDASES

TABLE VI Chromatographic Analysisa of Transfer Products with 'Various Acceptors and B-Galaclosidase of E. coli. M L SOgsr

-

No.

Subilrale

Acceptor

Transfer product

-

'mudensity of spol Nation lime min.) __

1

A

D-galactose

2

A

D-glUCOSe

-&D-Gal-(l -+ 6 ) - ~ - G s -&D-Gal-(l -+ 3 ) - ~ - G s 4)-D-G8 -j3-D-GaI-(l 6)-n-G -j3-D-Gal-(l ~)-D-G -fl-D-Gal-(l -p-D-Gal-(l -+ ~ ) - D - G D E c1 c2 El -+

-+ -+

3

A

L-arabinose

4

A

D-arabinose

E2

c1 c2

El E2 c1

atrong weak very weak very strong medium medium weak very weak strong medium weak very weak atrong medium weak very weak weak weak very weak strong very weak weak medium very weak

5

A

D-fructose

6

A

7

A

8

A

9

A

10

A

11 12

A B

13

B

14

B

c1 2-deoxy-"n-ga~acc2 tose" (2-deoxy-nE I yso-hexose) C 3-deoxy-"~-galacE tose" (3-deoxy-~zylo-hexose) C medium 6-deoxy-~-galactose E very weak (D-fucose) 3,6-dideoxy-~-xylo- inhibition hexose (colitose) strong c1 6-O-methyl-~-galacc2 very weak tose E weak strong F sucroae Oa-L-Ara-(l -.t ~ ) - D - G very atrong D-glucose 0-a-L-Ara-(l + 6 ) - ~ - c strong Oa-L-Ara-(l ~ ) - D - G medium 3,6-dideoxy-~-zylo- inhibition hexose D strong sucrose

c2 E

30 30

30

30

30

60 30 15 -

60 30 30

--$

-

30

y: 4, I. tri-

a 2-An~ino-2-(hydroxymethyl)-l,3-propanediol-HCI buffer, H 7.6;40". E phenyl B-D- alactoside; B, o-nitrophenyl a-L-ttrabinoside; C, lisaccharide : saccharide; by-product (o-galactosyl-D-galactose) ; F, O-@-D-galactosyl-(l A 6)O-a-D-glUCOSyl j3-D-fructoside.

%,

264

WALLENFELS AND MALHOTRA

TABLE VII Injluence of Temperature on K , and V,. of Hydr0ly8iSa of o-Nitrophenyl of E.coli, M L 50Oa8 @-~-Galactoside with the @-Ga~actosidase Michaelis conslad (K,)

Maximd velocitp ( V m A

Temperature, degrees

Without NaCl

Wilh NaCl

y!$

With NaCl

5 20 30 40

0.23 X 1 0 - 8 0.95 X lo-* 1.54 x 1 0 - 8 2.28 x 10-8

1.27 X 1 0 - 4 1.61 X 1 0 - 4 2.0 x 10-4 2.28 x 10-4

56.0 134.0 210.0 155.0

57.5 178.0 361.0 546.0

B

a 0.05 M 2-Amino-2-(hydrox methyl)-1,3- ropanediol-HCI buffer, pH 7.6; concentration of NaCl, when added: was 0.05 M. K , is expressed as moles per liter, and Vma. as micromoles of substrate hydrolyzed per minute per mg. of enzyme.

TABLE VIII Optimum pH for HydrOlySi8 of o-Nitrophenyl @-~-Galacloside b?j @-Galactoaidase of E. coli Strain

K 12 ML ML 309 ML 309 ML 309

ML 309 ML 309 ML 309

ML 308

Buffer phosphate phosphate veronal 2-amino-2- (hydroxymethyl)-lJ3-propanediol acetate 2-amino-2-(hydroxymethyl)-1 ,%propanediol acetate 2-amino-2- (hydroxymethyl) -1,%propanediol acetate 2-amino-2- (hydroxymethyl)-l,3-propanediol acetate 2-amino-2-(hydroxymethyl)-1 ,%propanediol acetate 2-aminoS-ihydroxymethyl) -1,3-propanedioLmercaptoacetic acid

-

Optimum PH

Rejerences

20

7.2-7.3 7 7.3 7.5

26 20 27 31

Nae

20

6.6

31

K@

20

6.6

31

none

5

7.5

31

Nae

5

7.1

31

Nae, Mgee

25

6.8

75

presenl N ae N a8 N a@ none

30 28 40

(75) F. J. Reithel and J. C. Kim, Arch. Biochem. Biophys., 90,271 (1960).

-

OALACTOSIDASES

265

tion const,ant of the enzyme-substrate complex. The energy of activation might be calculated from the variation of V,,, with temperature. A lower value for the higher temperature range is found in the presence of sodium chloride (12.5 kcal./mole for 5-30" and 7.77 kcal./mole for 3040"). Very similar values (13.1 kcal./mole for 0-30" and 7.3 kcal./mole for 30-37" a t p H 7.25) have been reported by Kuby and LardyZ6for the E. coli-K 12

PH

-

FIG.6.-Dependence on pH of Maximal Velocity (Vmx) of the Hydrolysis of o-Nitrophenyl j3-D-Galactosidebys*fl-Galactoeidase at 20". (Buffer: 0.05 M 2-amino2-(hydroxymethyl)-l,3-propanediol-aceticacid. X-X, in the absence of alkali; 0-0, in the presence of 0.05 M NaCl; A-A, in the presence of 0.05 M KCl. VrrmIis expressed in micromoles of o-nitrophenyl 8-D-galactoside hydrolyzed per minute per mg. of enzyme.)

enzyme. I n the absence of sodium chloride, a lower value (8.0-9.4 kcal./ mole) is observed which does not vary much with temperature. c. Influence of p H on Hydrolysis of o-Nitrophenyl /3-mGalacto,vide.Several values have been reported for the p H optimum of hydrolysis of o-nitrophenyl P-D-galactoside by enzyme preparations from various strains of E . coli. These are shown in Table VIII. From the behavior of K , and as the p H is varied (see Figs. 6 and 7)81with the crystalline P-galactosidase of E. coli ML 309, it can be deduced that two dissociable groups (pK 6.7 and 9.0 a t 20") of the enzyme molecule participate in the enzymic hydrolysis of o-nitrophenyl P-D-galactoside. From a comparison of these p K values with those given by EdsalP for various groups in proteins, the (76) J. T. Edsall, in "Proteins, Amino Acids, and Peptides," E. J. Cohn and J. T. Edaall, eds., Reinhold Publishing Corp., New York, N . Y., 1943, p. 445.

266

WALLENFELS AND MALHOTRA

group dissociating 011 the alkaline side of the pH optimum has been identified as a sulfhydryl group. This is supported by the inhibition of P-galactosidase by heavy-metal ions and p-(ch1oromercuri)benzoate discussed below. For the group dissociating on the acidic sidc, two possibilities arise, namely, an imidazolium group of a histidine residue or an a-ammonium group of a cystine residue. The latter possibility is excluded from the determination of amino cnd-groups; only threonine and glutamic acid were found.43 Moreover, the heat of dissociation (5.75 kcal./mole) ,31 as determined by parallel studies a t 5", agrees well with that reported for the imidazolium

T

PKm

11

I

I

5

6

I 8

1

7

I 9

PH __t

FIG.7.-Dependence on pH of the Michaelis Constant ( K , )of o-Nitropheny @-D-Galactosideat81 20". (Buffers: 0.05 M 2-amino-2-(hydroxymethyl)-l,3-propanediol-acetic acid. 0-0, in the absence of alkali salts; X-X, in the presence of 0.05 M NaCl; A-A, in the presence of 0.05 M KCI. The Michaelis constant is expressed in mole/liter.)

group (6.9-7.5 kcal./m0le)7~and not with that given for the a-ammonium group of cystine (10-13 kcal./rnole).76 This group is thus most probably an imidazolium group of a histidine residue. The effect of pH on the enzymic hydrolysis of o-nitrophenyl /3-~-galactosidecan thus be represented as shown in Scheme 1. The pK values of these groups, a8 well as the pH P

I?:

HN

HN H

I 3

SH '+H.

I

3

SH '+H' I

(pK = 6.7; 20') Inactive

13.:

HN

(pK Active Scheme 1

5

1

I

9.0; 20')

Inactive

267

GALACTOSIDASES

optimum, are shifted toward the acidic side in the presence of sodium chloride or potassium chloride. Shifting of the pK value of the imidazolium group toward the acidic side will lead to a higher activation a t lower pH than a t higher pH, as is actually the case. The shift in the pK value of the sulfhydryl group will, however, cause an inhibition a t higher pH, as is actually observed with potassium chloride. Apart from this parallel shift, sodium chloride also exhibits a net increase in the Vmax (when com-

FIG.8.-Activation of Hydrolysis of o-Nitrophenyl f?-D-Galactoside by Sodium Ch1oride.J' (0.05 M 2-amino-2-(hydroxymethyl)-l,3-propanediol-HC1buffer, pH 7.6; 20"; 3.33 X M o-nitrophenyl f?-D-galactoside. M = concentration of NaCl in test; M, = optimum NaCl concentration (3.2 X 10W M ) ; A E = increase in optical density (405 mp) in 2 minutes.)

pared a t the corresponding optimum pH). This might compensate for the possible inhibition a t higher pH. Friedenwald and Maengwyn-Davis77 have shown that if a substance is attached a t two points in an enzyme molecule, act,ivating at one and inhibiting at the other, the velocity of reaction is related to the activator concentration by the following equation:

v,/v

=

1

+ lc(Mo/M + M / M o )

where V = the observed velocity a t an activator concentration M; V, = the maximum velocity, M o = the optimum concentration of activator, and k = a constant. A plot of V-' against ( M o / M M/M,,) should then be a straight line. Such a plot for sodium chloride is shown in Fig. 8.

+

(77) J. S. Friedenwald and G . D. Maengwyn-Davies, Johns Hopkins Uniu. M c Collum-Pratt Inst. Contrib. N o . 70, 180 (1954).

WALLENFELS AND MALHOTRA

268

Similar straight lines are also obtained31for potassium chloride (M, = 2 X M) and ammonium chloride (M,= 5 X M). d . InjZuence of p H on V,, of Other Substrates.--Studies have also been reported on the influence of p H on Vmaxfor the hydrolysis of lactose, 50

10

T

30.

"rnax 20,

t PH

Fra. 9.-Dependence

-

pH of Maximal Velocity (Vmx) of the Hydrolysis of a-Lactose by @-Galactosidasea*at 20'. (Buffers: 0.06 M 2-amino-2-(hydroxymethyl)1,3-propanediol-acetic acid. VmaIis expressed in micromoles of a-lactose hydrolyzed per min. per mg. of enzyme. X-X, in the absence of alkali salts.) 011

o-nitrophenyl a-L-arabinoside, and p-nitrophenyl 8-D-galactoside in the absence, as well as in the presence, of alkali salts.*' The results are shown in Figs. 9, 10, and 11. It is noteworthy that, with lactose and o-nitrophenyl curves in the a-L-arabinoside, two maxima are observed in the V,.,-pH absence of alkali salts, as well as in the presence of potassium chloride (compare Ref. 20). In the presence of sodium chloride, which enhances the enzyme-substrate affinity in all cases, all the substrates examined

269

GALACTOSIDASES

show only one maximum in the Vmm--pHcurves. The pK values of groups on the active sites can then be readily read off. These values are given in Table IX, together with those found with o-nitrophenyl @-D-galactoside (in the absence of alkali salts, as well as in the presence of potassium chloride),

160

1

120

Vmax 80

40

1

I

I

6

I

1 PH

I

8

9

I

FIG.10.-Dependence on pH of Maximal Velocity (Vmax)of the Hydrolysis of o-Nitrophenyl a-L-Arabinopyranoside by @-Calactosidase**at 20". (Buffers : 0.05 M 2-amino-2-(hydroxymethyl)-l,3-propanediol-acetic acid. 0 -0, in the absence of alkali salts; X---X, in the presence of 0.05 M NaCl; A-A, in the presence of 0.05 M KCl. V,. is expressed as micromoles of o-nitrophenyl or-L-arabinoside hydrolyzed per min. per mg. of enzyme.)

Sodium ions are better activators for the hydrolysis of o-nitrophenyl 8-D-galactoside and o-nitrophenyl a-L-arabinoside, and K @ ions for that of lactose and p-nitrophenyl P-D-galactoside (compare Ref. 20). More substrates and alkali ions will have to be studied before any correlation or generalization can be made. It has been reported that whereas in the presence of o-nitrophenyl 8-D-galactoside, it is possible to measure the enzyme activity even a t pH 5 , with o-nitrophenyl a-L-arabinoside, exact measurements were not possible31 below p H 6. This shows that the generally known stabilizing

270

WALLENFELS AND MALHOTRA

effect of substrates on the enzyme activity can be difFerent for different substrates. e. Influence of Cations on the Hydrolysis of Diferent Substrates.-Figs. 6, I

X --

I

I

I

I

\ 0

6

I

7

I

I

8

I

I

9

PH FIG.11.-Dependence on pH of Maximal Velocity (Vmax) of the Hydrolysis of p-Nitrophenyl ,3-o-Galactopyranoside by 8-Galactosidase ats1 20'. (Buffers: 0.05 M 2-amino-2-(hydroxymethyl)-l,3-propanediol-aceticacid. 0-0, in the absence of alkali salts; X - - X , in the presence of 0.05 M NaCl; A-A, in the presence of 0.06 M KCI. V,, is expressed as micromoles of p-nitrophenyl 8-D-galactoside hydrolyzed per min. per mg. of enzyme.)

9, 10, and 11 show that, in the system enzyme-substrate-hydrogen ionsalkali ions, thc effect, of any one of the last three partners is determined by t,he concentration of the other two. Thus, the action of hydrogen ions arid that of alkali ions are interdependent, and both of them are further determined by tho nature and concentration of the substrate. No definite explanation can as yet be given of the different types of V,,,..-pH curves ob-

27 1

GALACTOSIDASES

tained with different substrates, especially the appearance of two maxima in some cases and the specific, activating action of Na@for the hydrolysis of o-nitrophenyl p-u-galactoside and o-nitrophenyl a-L-arabinoside, and of K @ for that of lactose and p-nitrophenyl 8-D-galactoside. To some extent, these differences might possibly be attributed to different structures of the enzyme-substrate complexes which, in turn, might be correlated with the enzyme-substrate affinity. It seems that, given similar conditions of enzyme-substrate affinity, these anomalies disappear to a great extent. Thus, the addition of sodium chloride, which always enhances the enzyme-substrate affinity, results in the disappearance of TABLEIX pK Valuesa1of Groupso on the Active Sites of the 8-Galactosidase of E. coli ML 309, at 20' (0.06 M 2-amino-d-(hydroxyrnethyl)-l ,d-propanediol in acetic acid buflers) Substrat&

Alkali ion

C C C

none K@ Nam

1)

Nam Na" Nam

E F

w A 6.7 5.9 5.8 (6.210 6.4 5.8 5.6

B 9.0 8.0 7.8 (8.6)~ 8.1 7.4 8.6

a A and B stand for the groups dissociating on the acidic and alkaline side of the pH optimum, respectively. C, o-nitrophenyl B-D-galactoside; D, lactose; E, o-nitrophenyl a-L-arabinosi:p%j p-nitrophenyl 8-o-galactoside. c The values in brackets have been taken from the pK,-pH curve.

second maxima. Moreover, the pK values read off under these conditions agree fairly well (see Table IX). A question which frequently comes to the foreground today is whether differences in structure exist for the different active sites of an enzyme. Until extensive structural investigations can be made on the enzyme, this question will remain debatable. However, even if such studies are not yet feasible, it is possible at least to establish the number of such sites per molecule. Cohn" had found 5.6 binding sites with phenethyl 1-thio-p-ugalactoside. The results of Cohn are supported by those of Wallenfels and coworker^.'^ Differences in the action of cations on the hydrolysis of various substrates have also been reported by Reithel and coworkers with @-galactosidaseof (78) K. Wallenfels, 0. P. Malhotra, B. Muller-Hill, D . Dabich and J. Fischer, unpublished results.

272

WALLENFELS AND MALHOTRA

E. coli, ML 308; for example, Mg@@ions activate the hydrolysis of onitrophenyl8-D-galactoside in the presence of Na@,but not that of lactose or of methyl P-D-galactoside.?6 The activating action of Na@ on the hydrolysis of o-nitrophenyl 8-Dgalactoside has been studied in greater detail. It has been found that activation is stronger at lower pH (compare Fig. S ) , higher temperature, and lower substrate concentration. Sodium chloride depresses K,, and increases V,,, . If the enzymic reaction is written according to the Michaelis-Menten scheme, E+

2 s, k-i (E 8)- % E + P Scheme 2

(where E = enzyme, S = substrate, and P = products), V m , x is prok2)/k1. As V , , x (or k2) is increased and portional to k2 and K, = (k-1 K , is simultaneously depressed by sodium chloride, the dissociation conmust be diminished or the enzyme-substrate affinity must, stant (k-&) be enhanced by the addition of sodium f. Determination of the Rate-determining Step.-Hydrolysis of 0- and pnitrophenyl 8-D-galactoside and of o-nitrophenyl a-L-arabinoside with the 8-galactosidase of E. coli, ML 309, has been studied in a “stopped-flow” apparatus at high enzyme concentrations, in order to determine the ratedetermining st,ep in the following scheme of reaction (see also, Section 11, 10).

+

EH

+ ROG

(EH ROG)

-%

k_l

EG -&p EH

+ ROH

+ GOH

Scheme 3

where EH = the enzyme, ROG = the substrate, ROH = the free aglycon, and GOH = D-galactose. It has been shown that ka is the ratedetermining step. It should be equal to Vmsx/rper sec., where V,,, is expressed in moles of substrate hydrolyzed per sec. per mole of enzyme, and T = the number of active sites per mole of enzyme on which the subk2, strate is hydrolyzed. The value ks is greater than 10k2 . Moreover LI>> so that the Michaelis constant ( K , = (kdl k 2 ) / k l ) approaches the disof the enzyme-substrate complex. The velocity sociation constant k-& constant kl is very high and seems to be limited only by the diffusion of substrate to the active site of the enzyme.?QI n an extreme case, where k3 is infinitely greater than k 2 , it is not possible to distinguish between Schemes 2 and 3.

+

(79) H. Gutfreund, 0. P. Malhotra and K. Wallenfels, unpublished results.

QALACTOSIDASES

273

g. Inhibition with Sulfhydryl Reagents.-The p-galactosidase of E . coli, ML 309, is inhibited by heavy-metal ions and by p-(ch1oromercuri)benzoate. An inhibitor concentration about ten times as great is necessary to bring about the same degree of inhibition if 0.05 M sodium chloride is

present in the incubation mixt~re.7~ Similar protection by the activating ions against inhibition was earlier observed with aldehyde dehydrogenase.EO The inhibitory action, as judged from the concentration of metal ion required for 50 % inhibition, decreases in the order Hg" 2 Age > Cd*@ > Zn*@ > Pb** Cue'. This agrees fairly well with the order reported by KlotzE1for the reaction of heavy metals with the sulfhydryl groups of serum albumin, namely, Hg'@ > Age > Pb** > Cd@* > Zn'*. The inhibition by these reagents supports the earlier conclusion (from pH studies) that free sulfhydryl groups are involved in the action of the pgalactosidase of E. coli, ML 309. According to Boyer 's method,@approximately 95 sulfhydryl groups are present in crystalline p-galactosidaseE**in 8 M urea. The slowness of the reaction of p-(chloromercuri)benzoate with the sulfhydryl groups of pgalactosidase permits correlation between the number of sulfhydryl groups reacted and t.he decrease in At least three types of sulfhydryl group are found in the enzyme. The first type reacts immediately with the reagent, without simultaneous loss of enzymic activity. The exact number of such sulfhydryl groups is difficult to determine, because comparatively small changes in optical density must be evaluated in the presence of an enzyme-p-(ch1oromercuri)benzoate mixture which has a high extinction initially. The best experimental evidence led to a value of 20 f 5 sulfhydryl groups per mole of enzyme. The second type of sulfhydryl group reacts more slowly than that described above and is involved with enzymic activity. The number of sulfhydryl groups which fall into this second category is temperature dependent. At 5", the number of groups found was 9 f 1.5per mole of enzyme, whereas 12 f 1 were found at 20". The third type of sulfhydryl group reacts after almost all activity is lost. I t is difficult to differentiate clearly between the three types of sulfhydryl groups when the reaction is carried out at 40".At this temperature, the

-

(SO) A. 0. M. Stoppani and C. Milstein, Biochem. J . , 67,406 (1957). (81) I. M. Klotr, Johns Hopkins Univ. McCollum-Pratt Inst. Contrib. N o . 70, 257 (1954). (82) P. D. Boyer, J . A m . Chem. Soc., 76,4331 (1954). (82a) All calculations are based on a molecular weight of 750,000, unless stated otherwise. (82b) K . Wallenfels and 0. P. Malhotra, in "The Enzymes,'' P. D. Boyer, H. Lardy, and K. Myrback, eds., Academic Press Inc., New York, N . Y., 2nd Edition, 1960, VOl. 4, p. 409.

274

WALLENFELS A N D MALHOTRA

reaction ends whcn approximately 95 f 5 sulfhydryl groups per mole of enzyme are found. Fig. 12 illustrates the results of an actual, single experiment in which the

Moles p- chloramercuribenzoote bound / mole enzyme (mol. w t 7 5 105 1

-

FIG.12.-Interaction of 8-Galactosidase of E . coli, ML 309, with p-(Chloromercuri)benzoate at 5 ( X X X ) , 20 (OOO), and 40" (AAA)J8 [Sulfhydryl groups were estimated by Boyer's methodas (p-(ch1oromercuri)benzoate 5 X 10-6 M , enzyme 250 pg./ml.).The activity was measured with phenyl 8-u-galaetoside (1 X 10-2 M in 2amino-2-(hydroxymethyl)-l,3-propanediol-HCI buffer, pH 7.6). A = Enzyme ucLivity in the presence of p-(ch1oromercuri)benzoate; A,, = enzyme activity in the absence of p - (ch1oromercuri)benzotite.]

percent activity remaining is correlated with the number of sulfhydryl groups reacted. The values stated in the text for the various types of sulfhydryl groups are median values derived from a number of similar experiments. One should keep in mind a t all times that the determiiiations of sulfhydry1 groups referred to above are based wholly on evidence gathered by use of Boycr's method. Cleavage of disulfidc bridges, or reaction of groups

GALACTOSTDASES

275

other than sulfhydryl with the reagent, remain as possibilities which could influence the values obtained. Because of the results found with p-(ch1oromercuri)benzoate and brcause P-galactosidase is believed to be a “sulfhydryl enzyme ,” it, seemed advisable to investigate the effects of other sulfhydryl reage1its.7~Virtually no inhibition of 0-galactosidase was found in the presence of iodoacetamM ) over a period of 7 hours. N-Phenylmaleimide, a t ide (9 X M , produced less than 10 % inhibition concentrations as high as 8.7 X over a 20-hour period. However, when the roncentration of N-phenylmaleimide was raised to 9.1 X lop3M , assay of the inrubation mixture revealed that 50 % inhibition of 0-galactosidase occurred within a 2-hour period when the experiment was performed in 2-amino-2-(hydroxymcthyl)1,3-propanediol-acetic acid buffer, pH 8.0. Virtually no activity was lost when the latter experiment was repeated in phosphate buffer (Na salts) a t the same pH. The N-ethyl derivative of maleimide, at a concentration of 1 X M, in 2-amino-2-(hydroxymethyl)-l, 3-propanediol acetate buffer of pH 7.5, did not inhibit the enzyme. When measurements were made in the same buffer a t pH 8.6 and at a N-ethylmaleimide concentration of 3 X M , 25% inhibition occurred within 3.5 hours. The pH-dependency of the maleimide derivative reactions has been explained in terms of reartion mechanisms (the RSe form reacting instead of RSH).820The effects of various buffers, that is, of salt composition, on the reaction, as well as the specificity of these reagents, remain to be investigated. h. Inhibition with BeTyllium.-A non-competitive inhibition is observed with beryllium chloride solutions. The inhibitory action cannot be reversed by adding Mg@@or tyrosine. The inhibition can be partly reversed by M o-nitrophenyl P-D-galactoincubating with the substrate (3.33 X side) or sodium chloride. In the case of sodium chloride, the reversing action increases with c ~ n c e n t r a t i o n If . ~ ~it is assumed that beryllium reacts with a specific group of the enzyme, which participates in catalysis, the pK value of this group can be determined by studying the effect of p H on the inhibitor constant ( K ) .A value of 7.15 is found for thepK of the group involved (see Fig. 13).83On comparing this value with those obtained from pH-activity data, it may be concluded that the imidazole group of a histidine residue is involved in the enzyme-Be” binding. Studies with model substances, however, have not revealed any such specific binding of Be@@to imidazole derivatives. It is, therefore, conceivable that beryllium inhibition might be attributed to the binding of polymeric beryllium hydroxide on a large area of enzyme protein.838 The different character of (82c) R. Cecil and J. R. McPhee, Advances i n Protein Chem., 14,256 (1959).

(83) K. Wallenfels and 0. P. Malhotra, unpublished results. (Ma) S. Bildstein, Doctoral Dissertation, Freiburg, Ger., 1960.

276

WALLENFELS AND MALHOTRA

t PKi

I

I

I

I

I

I

7

6

PH

8 _j

FIQ.13.-Inhibitionaa of 8-Galactosidase of E . coli, ML 309, with Beryllium Ions at Various pH's. (0.06 M 2-rtmino-2-(hydroxymethyl)-l,3-propanediol-aceticacid buffer at 20". Ki = [Be@@][Enzyrne]/[Enzyme-Be@@].) 100

80

T A

A.

60

XlOO

LO

20

0

I

0

I 20

I

I

CO

Time ( M i n )

I

I 60

FIQ. 14.-Inhibition of @-Galactosidaeeof E . coli, ML 309, with Beryllium Ions in Ordinary and Heavy Water. (0.06 M 2-amino-2-(hydroxymethyl)-l,3-propanediolHCl buffer, pH 7.6; 20".The pE (or pD) of heavy-water buffer was adjusted against the glass electrode and no correction was applied. A enzyme activity in the presence of beryllium; A, enzyme activity in the absence of beryllium.)

-

-

277

GALACTOSIDASES

beryllium inhibition as compared with the inhibition by heavy-metal ions and p(ch1oromercuri)benzoate (which probably react with sulfhydryl groups) is evident from the unusually large influence of heavy water on the rate of onset of inhibition (see Fig. 14) In sharp contrast, Age ion inhibition shows an isotope efTect18 of only 2.33. i. Inhibition with Agenk forming Complexes.-Reithel and Kim found that (ethylenedinitrilo)tetraacetic acid inhibits the activity of /3-galactoTABLBX Inhibition" of @-Galactoaidase of E. coli, M L 309, with Chelating Agentsn4 Chelaling agent Trade-name

Chel CD Chel DE EDTA Chel330 aci Chel ME Chel OC Chel DM acid Chel NTA

Systematic name

(1,2-cyclohexylenedinitrilo)tetraacetic acid [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (ethylenedinitri1o)tetraaceticacid [ (carboxymethy1imino)bis(ethylenenitri1o)ltetraacetic acid [ (oxybis (ethylenenitri1o)Jtetraacetic acid N- (carboxymethyl) -N'- (2-hydroxycyclohexyl) - N , N'-ethylenediglycine N- (carboxymethy1)-N'- (2-hydroxyethyl) -N-N'-ethylenediglycine Nitrilotriacetic acid

Concentralion (M) required to redwe the activity to

log K I of ca complex

55%

50-60%

12.06

2 X lo-'

11.0

2

10.7 10.03

2 X lea 10-6 2

10.05

lu-6

9.3

lo-'

-lo-*

8.0

lo-6

-10-8

6.4

6 X

x lo-'

2X

lee

lo-'

5

x

10-4

10-4

x lo-'

lo-'

In 0.05 M 2-amino-2-(hydroxymethyl)-l,3-propanediol-HC1 buffer, pH 7.6,20"; 3.33 X 1 0 - 8 M o-nitrophenyl 8-D-galactoside. 0

sidase of E. coli, ML 308, with o-nitrophenyl /3-D-galactoside as substrate?6 The inhibition could be reversed by Mg*@. As their enzyme was activated by Mg@@and these ions reversed the inhibition, they attributed the inhibition by (ethylenedinitrilo)tetraacetic acid to the removal of Mg@@ions. The hydrolysis of lactose was not activated by Mg@@ions and was also not inhibited by (ethylenedinitrilo)tetraacetic acid.I5 Crystalline p-galactosidase of E. coli, ML 309, does not contain magnesium, but considerable amounts of calcium are found. The calcium content remains practically constant through seven recryst,allizat,ions (see Table I). The effect of several chelating agents (see Table X), which differed from each other in the stabilities of their calcium complexes, on the activity

278

WALLENFELS AND MALHOTRA

of the P-galactosidase of E . coli, ML 309, has been ~tudied.8~ Typical curves tetraacetic acid (Chel CD) and obtained with (1,2-cyclohexylenedinitrilo) nitrilotriacetic acid (Chel NTA) are shown in Fig. 15. Similar curves were obtained with other chelating agents. An interesting feature of their action is that the inhibition takes place in two steps. In the first step, which, for the compounds studied, was independent of the value of log K for calcium-complex formation (with

-

0

25 0

-

Chel CD

1

I

I

I

I

I

6

5

3

2

7

6

5 L - l o g [Inhibitor]

-

2

100

15

25 0

3

Fro. 15.-Inhibition of 8-Galactosidase of E . coli, ML 309, with Chelating Agents, (1,2-Cyclohexylenedinitrilo)tetraacetic acid (Chel CD) and Nitrilotriacetic acid (Chel NTA).84 (See also Table X . 0.05 M 2-Amino-2-(hydroxymethyl)-l ,%propanediol-HC1 buffer, pH 7.6; 20";3.33 X 10P M o-nitrophenyl 8-D-galactoside. A = enzyme activity in the presence of inhibitor; A, = enzyme activity in the absence of inhibitor.)

one exception), t,he activity was reduced to about 5 5 % . The activity rcmained unaffected by increase in the inhibitor concentration and then showed a sudden fall. Apart from the one exception, the inhibitor concentration required for the second part followed the order that would be expected from the log K values for calcium-complex formation. Considering the large steric effects which might influence the interaction with proteins, the relationship is quite satisfactory. For weaker complexing agents, the fall of activity in the second step was less steep. In the second phase of inhibition, the activity can be partly rccovered by the addition of Ca@@, (84) K. Wallenfels and 0. P. Malhotra, unpublished results.

279

GALACTOSIDASES

while, during the first phase, the enzyme is slightly more sensitive to inhibition by Ca@@than is the untreated enzyme. Inhibition by (1,2cyclohexyleiiedinitrilo)tetraacetic acid proceeds very rapidly and is independent of the presence of Na@ion. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (Chel DE) does not fit in the series. Although the type of curve obtained is similar, higher concentrations of this reagent are required than would be expected from its log K value for formation of calcium-complex (see Table X). Another

I

01 5

I

I

4

2

3 - l o g [co++]

+

FIG.16.--Inhibition8' of 8-Galactosidase of E . coli, ML 309, with Ca@@Ions. (0.05 M 2-Amino-2-(hydroxymethyl)-l,3-propanediol-HC1 buffer, pH 7.6; 20";3.33 X 10-3 M o-nitrophenyl 8-D-galactoside. A = enzyme activity in the presence of Ca@@; A, = enzyme activity in the absence of Ca@@.)

difference between this and (1,2-cyclohexylenedinitrilo)tetraacetic acid is that, whereas in the latter case, o-nitrophenyl 8-D-galactoside does not exert a reversing action (it only increases the time required for attainment of equilibrium), with [ethylenebis(oxyethylenenitrilo)]tetraaceticacid, the inhibition can be partly reversed by o-nitrophenyl 8-D-galactoside. A definite explanation of these observations and their relationship with calcium present in the protein must await further results. j. Inhibition by Calcium.-Although there are indications that calcium forms a part of the enzyme molecule and that its presence might be of importance for the enzymic activity, inhibition is observed when Ca@@ ions are added to the enzyme solution (see Fig. 16).84It might be compared

280

WALLENFELS AND MALHOTRA

with the inhibition of the alcohol dehydrogenase of yeast with zinc, although the enzyme itself contains zinc (which is important for its activity).R6 Probably, the intrinsic calcium is important for maintaining the protein in an active conformation (about 5 calcium atoms are present per molecule; molecular weight, 750,000; calculated for 275 y of calcium/g. of enzyme), and extraneously added calcium is bound near the active site, thus blocking it, or reacts with one of the groups on the active site. The inhibition is instantaneous and of non-competitive character, with an inhibitor constant ( K J of 5.3 X lo-' M at 20" and pH 7.6 (compare the non-competitive inhibition by beryllium with Ki = 2.9 X 10-6 M under the same conditions). k. Inhibition by Sugars and Their Derivatives.-High concentrations of D-glucose and sucrose inhibit the 0-galactosidase of E . coli, K 12, noncompetitively.2BA competitive inhibition of the hydrolysis of o-nitrophenyl 8-D-galactoside with the enzyme of E . coli, K 12, has been observed in the presence of D-galactose, lactose, and some other p-D-galactosides.26 Monod and coworkers showed that phenyl l-thio-P-D-galactoside has almost the same affinity for the enzyme of E . coli, ML, as phenyl p-Dgalactoside.6aSimilar results were obtained by Wallenfels and coworkers with o-nitrophenyl l-thio-p-D-galactoside,which is a competitive inhibitor having an inhibitor constant of 1.2 X M at 20", whereas o-nitrophenyl p-D-galactoside has a Michaelis constant of 0.95 X lW3M under the same ,78 Apparently, the replacement of the D-galactosidic anomeric oxygen atom by a sulfur atom does not affect the enzyme-substrate affinity, suggesting, thereby, that the enzyme-substrate binding does not involve the D-galactosidic anomeric oxygen atom. 1. Inhibition with Cysttine and Reduced Glutathione.-In contrast with other sulfhydryl enzymes, the 0-galactosidase of E. coli, ML 309, is inhibited by cysteine and reduced glutathione (see Fig. 17).78The inhibition curves of both inhibitors are similar in shape. At 20", cysteine inhibition begins at 1 X M of cysteine, 35% of the activity is M . At 3 X left. The activity then remains constant until the inhibitor concentration has reached 3 X 10-*M . As the concentration of cysteine is increased beyond 3 X leaM , progressive inactivation of the enzyme is observed. The shape of the inhibition curves is similar to those obtained with metalchelating agents (see Fig. 15). To date, no analyses for possible, enzymebound, heavy-metal activators (for example, manganese and iron) have been carried out. The mechanism of inhibition must, therefore, remain an open question until either (a) participation of a heavy metal or (b) inactivation due purely to breaking of S-S bonds can be demonstrated. m. Number of Binding Sites.-Cohn determined the number of binding pea

(86) K. Wallenfels and B.. Sund, Biochem. Z . , 819, 17, 69 (1967).

28 1

GAWCTOSIDASES

sites of the 6-galactosidase of E. coli, strain ML, using the equilibriumdialysis methodsa with phenethyl l-thio-/3-D-galactosideas the competitive inhibitor." At 4", he found 5.6 sites, calculated on the basis of a molecular weight of 750,000. Wallenfels and MalhotrasZb~ 8 7 carried out similar experiments with crystalline /3-gslactosidaseof E . coli, ML 309 and o-nitrophenyl l-thio-8-Dgalactoside, employing the ultracentrifugation methodm 3 9 a t 4-6" and a t

6

5

L -Log

3

2

[ cysteine J

FIG.17.-Inhibition of Galactosidase of E. coli, ML 309, with Cysteine.78 (0-0-0, without NaCl at 20"; A-A-A, with 0.05 M NaCl at 20"; X-X-X, with 0.05 M NaCl at 40" and 0.05 M 2-amino-2-(hydroxymethyI)-l,3-propanediol-HC1 buffer, p H 7.0; o-nitrophenyl 8-D-galactoside, 3.33 X lo-' M. A = enzyme activity in the presence of cysteine; A. = enzyme activity in the absence of cysteine.)

20-22". On plotting the data by the method of S c a t ~ h a r d they , ~ ~ found 14.2 binding sites at 4-6Oand 35.2 at 20°, for a molecular weight of 750,000. The great discrepancy between the value of Cohn and those of Wallenfels and Malhotra led to a re-investigation of the problem.78 It was found that the competitive inhibitor o-nitrophenyl l-thio-j3-D-galactoside is not suited for binding-site studies, because it is slowly hydrolyzed at the enzyme (80) I. M. Klotz, in "The Proteins," H. Neurath and K . Bailey, eds., Academic Press, Inc., New York, N. Y., 1953, Vol. I, Part B, p. 727. (87) 0. P. Malhotra, Doctoral Dissertation, Freiburg, Ger., 1959. (88) J. E. Hayes, Jr., and S. F. Velick, J . Biol. Chem., 207,225 (1954). (89) K . Wallenfels and H. Sund, Biochem. Z., 319, 59 (1957). (90)G. Scatchard, Ann. N. Y . Acad. Sci., 61,660 (1949).

1

282

WALLENFELS AND MALHOTRA

concentrations used in the ultracentrifugation experiments (see also, p. 260). The product of hydrolysis, o-nitrothiophenol, has a lower extinction coefficient than o-nitrophenyl l-thio-8-D-galactoside a t 366 mp, the wavelength a t which the measurements of inhibitor concentration were made. Thus, as increasing amounts of inhibit,or were hydrolyzed, the more inhibitor was, seemingly, sedimented with the enzyme. The temperaturc dependence of the reaction, corresponding to an activation energy of some

4

5

6

7

PH

FIG. 18.-Dependence on pH of Maximal Velocity (Vmx) of the Hydrolysis of a-Lactose and o-Nitrophenyl 8-D-Galactoside with Calf-intestine 8-Calactosidsse at16 40". (V,,, is expressed aa micromoles of substrate hydrolyzed per min. per mg. of protein .)

25 Kcal., resulted in an increased rate of hydrolysis at 20°, in comparison to that at 5", and led to a misleading concept regarding the temperaturcdependence of the number of binding sites. Additional studies of binding wcre made with p-nitrophenyl l-thio-p-ugalactoside, using the ultracentrifugation technique. At O", under thc conditions used, hydrolysis was barely detectable. The measurements gave results in the range indicated by Cohn.

9. Kinetics of Calf-intestine Enzymelb

The K , values and rates of hydrolysis of various substrates have hceri given in Tables IV and V. In contrast to the E. coli enzyme, calf-intestine 0-galactmidastl is not affected by alkali ions. The energy of activation of

283

GALACTOSIDASES

hydrolysis of o-nitrophenyl 8-D-galactoside with this enzyme has been foundI5 to be 14.2 kcal./mole. Results reported so far on the variation of V,,,,, with pH, inhibition, and inactivation of urea are discussed below. a. Efect of pH.15-The effect of p H on the maximal velocity (V,,,) of the hydrolysis of lactose and o-nitrophenyl 0-D-galactoside is shown in Fig. 18. I n contrast to the E. coli enzyme, more or less perfectly symmetrical curves are found with both substrates. The optimum pH is, in both cases, 5.3. The same pK values for the dissociable groups are obtained with lactose (4.04and 6.5) and o-nitrophenyl 8-D-galactoside (4.04 and 6.6) as substrates. The effect of temperature on the pK values was studied with o-nitrophenyl 8-D-galactoside as substrate. The heats of dissociation of the two groups were found to be 0 and 5.65 kcal./mole, respectively. These groups have been identified as carboxyl and imidazolium groups. The effect of pH on enzyme activity can be represented as shown in Scheme 4. COtH

I

ay-r i

- H'

L

' +H'

(pK = 4.04;4 0', 20')

Inactive

:*CI NH

cooe

H

I

q

cooe

Y

i

-H'

L

' +H'

Active

(pK

=

1

6.6; 40" 6.87; 20"

)

i==

Inactive

Scheme 4

b. Inhibition.-(i) Inhibition with metal ions. I n contrast to E. coli 8-galactosidase, calf-intestine enzyme is not much inhibited by metal ions (see Table XI). Only high concentrations of Age and Hge@ show appreciable inhibition. Wallenfels and Fischer'6 have attributed this inhibition to a nonspecific binding of metal ions, which results in changes in the tertiary structure of the protein. (ii)Inhibition by amines. Histidine and 2-amino-2-(hydroxymethyl)-l, 3propanediol are inhibitory. The effect of various concentrations of these compounds is shown in Table XII. The inhibition by 2-amino-2-(hydroxymethyl)-1 ,3-propanediol is competitive, with an inhibitor constant ( K C ) of 1.55 x 10-3 M. (iii) Inhibition by monosaccharides and their derivatives. Like E. coli enzyme, the P-galactosidase of calf intestine is inhibited by monosac-

284

WALLENFELS AND MALROTRA

charides, but, in this case, D-galactose inhibits more strongly than D-glucose. o-Nitrophenyl l-thio-8-D-galactoside is a competitive inhibitor in this case, also. The value of the inhibitor constant ( K i ) at 40" (pH 5.3) is 6.9 X M , which is of the aame order as K,,, for o-nitrophenyl 8-u-galactoside. TABLEX I Inhibition. of Calf-Znieetine fl-C7alactosidascwith Metal Ions16 Concentration I n ~ ~ i o t t

Meld i o n Al3@ Hgee

lo-'

CU" Zn@@ Be@@ CO"'

10-8

39 35 10 6

10-4

10-8 10-3 10-8

Bae@

lo-' lo-'

p-(ohloromercuri)benzoate

The enzyme and inhibitor were incubated for 10 minutes a t 20', and the reaction was started with o-nitrophenyl 8-n-galactoside (10-W); citrate-phosphate buffer; p H 5.3.

TABLE XI1 Inhibitiona of Calf-iwleetine fl-Ohtacloeidase with Histidine and 8-Amino-8- ihydrozymelhyl)- 1 , S-propanediol''

% Inhibition at inhibitor concentration o j

Inhibitor

Histidine Z-Amino-2-(hydroxyrnethyl)1,3-propanediol

5XlP.M

1P'M

16 36

22

28

50

62

ZX10-8M

In 0.033 M Na-K phosphate buffer, pH 7.4; 40'; 10-*Mo-nitrophenylp-n-galactoside. 0

c. Inactivation with Urea. -The calf-intestine enzyme is rather sensitive to denaturation by urea, more so at 5" than at 20" (see Fig. 19). I n this respect, this enzyme is similar to the glutamic decarboxylase of E . coli, which has been showng1to be inactivated more rapidly at 0" than a t 25". The inactivation by urea is not reversed on 100-fold dilution. The high sensitivity to urea might possibly be due to the ready formation of ureacarbohydrate compounds (which were earlier described by SchoorlQ2).

(91) R. Shukuya and G. Schwert, J . Biol. Chem., 136,1658 (1980). (92) N. Schoorl, Rec. trau. chim., 29, 31 (1903).

QALACTOSIDASES

285

10. Postulated Mechanism of Action The inhibition experiments and pH-activity studies have demonstrated the presence of two groups, a sulfhydryl group and an imidazole group, on the active site of the P-galactosidase of E . coli, ML 309. Taking an analogy from esterases in which imidazole plays the part of a nucleophilic center, Wallenfels and MalhotragShave arrived a t the following mechanism for the transfer of a D-galactose residue from galactoside to an acceptor (water or another sugar or alcohol) (see Scheme 5 ) . This mechanism is in conformity

FIG.19.-Inactivation of Calf-intestine fl-Galactosidase with Urea.I6 (Citratephosphate buffer, pH 5.3; 10-* M o-nitrophenyl fl-D-galactoside. A = enzyme activity in the presence of urea; A, = enzyme activity in the absence of urea.)

with the conclusion that the aglycon has been removed from the enzyme before the acceptor enters the common binding-site and reaction. The extremely slow hydrolysis of l-thio-8-D-galactosides could be the consequence of the great weakness of the S-H-S bonds. In Scheme 5, the formation of products from the enzyme-substrate complex is a two-step process. It has been pointed out (see Section 11, 8f) that, so far, we cannot distinguish between this and a one-step process. A mechanism of the latter type, which may be called a “switch-over” mechanism, is shown in Scheme 6. I n this case, it is necessary to postulate the existence of two similar sites (for aglycon and acceptor, respectively) near (93) K . Wallenfels and 0. P. Malhotra, see Ref. 82b.

286

WALLENFELS AND MALHOTRA

-

Scheme 5

each other, because similar specificities are observed for aglycori and acceptor. The above considerations apply, also, to the calf-intestine enzyme, where the carboxylate ion (-COOe) will act as a nucleophilic center and the imidazolium group as an electrophile. According to Scheme 5, the intermediate enzyme-B-D-galtictoside compound will possess the nature of an ester. Evidently, Scheme 6 can apply equally well. It is not known, as yet, whether the large carbohydrate content of the most highly purified, calfintestine 8-galactosidase is a part of the enzyme molecule or not. Not,hing

GALACTOSIDASES

287

can be said a t present as to whether or not the carbohydrate part participates in some way in the bond-splitting or in the substrate-binding. Prom an examination of molecular models, it is found that the nucleophilic group can approach only from that side of the pyranoid ring on which the anomeric oxygen atom is linked (the front-side displacement of Koshlandg4). Schemes 5 and 6 involve the splitting of the bond from the D-galactosyl residue to the oxygen atom. In agreement with the action of certain other glycosidases, it has recently been shown (by carrying out the hydrolysis in H2018)that this bond is actually ruptured in the hydrolysis of o-nitrophenyl 0-D-galactoside with the E. coli enzyme.e6 That the apparent affinity for the enzyme is not affected on replacing the galactosidic, anomeric oxygen atom by a sulfur atom suggests that neither oxygen nor sulfur is involved in the formation of the enzyme-substrate complex. The results in Tables IV and V show that changes on hydroxyl groups of the n-galactose residue lead either to a complete loss of hydrolyzability (for the 2, 3, or 4 positions) or to a partial loss (at position 6). This suggests that these hydroxyl groups are involved in the specific binding of substrate to the enzyme. Apart from the unspecific van der Waals forces, which might operate with the glycon as well as with the aglycon, the former can also be attached to the enzyme by means of hydrogen bonds. If the sugar conformation ( C l according to Reevesg6)does not change during complex-formation, the electron pairs of the protein molecule, with which the hydrogen bonds are formed, must be located in a corresponding mirrorimage conformation (see Fig. 20). It has been concluded from an examination of molecular models that only alternate peptide groups would be in a position to form such bonds, so that about seven amino acid residues need to be present between cysteine and histidine residues in the E. coli enzyme or between carboxyl and imidazolium groups in the calf-intestine enzyme. The molecular models further show that the line joining the hydrogen atoms of various hydroxyl groups of D-galactose describes a helix having a height of 5-5.5 A. pcr turn. The inner diameter is bigger than that reported for protein helices (from x-ray measurements). It is, however, possible that some as-yet-unknown ways of winding of protein chains might exist, a t least in certain sections, which can form an envelope round the specificitydetermining glyconic part, as in Fig. 21. The fact that much wider specificity limits exist for the aglycon (but that it still can be presumed to be bound on several points) has led Wallenfels and coworkers to postulate attach(94) D.E.Koshland, Johns Hopkins Univ. McCollum-Pralt Znst. Contrib. No. 70, 608 (1954). (95) K.Wallenfels, 0.P. Malhotra, H. Dahn and H. Moll, unpublished results. (96) R. E. Reeves, J . Am. Chem. Soc., 73,1499 (1950).

288

WALLENFELS AND MALHOTRA

ment on the outer side of a helix, rather than an enclosure inside a helix. The accept,or will also be similarly bound in the case of a transfer reaction.28 a-L-Arabinosides have one hydroxyl group less than the 8-D-galactosides;

FIG. 20.-A Possible Structure of the 6-Galactosidase-substrate Complex for the E . coli enzyme.@*(For the calf-intestine enzyme, COOe and imidazolium' should probably be written for the iniidazole and SH groups, respectively.)

P

E'Io. 21.-Fixation

of Lactose on the B-Galactosidase Helix.28

they should, therefore, exhibit a lower affinity for the enzyme. Moreover, if the L-arabinoside molecule is rotated (within certain limits) about an axis passiiig t,hrough the center of the pyraiioid ring and perpendicular to the average plane of the ring, it will still be in a position to attach itself to the enzyme molecule through the same number of hydrogen bonds as before rotation. This rotation may possibly explain the peculiar behavior

289

GALACTOSIDASES

of a-L-arabiiiosides in the transfer and hydrolyt,ic reactions, as compared with 8-D-galactosides. It is well known that the pK value of a group is influenced by the surroundings of the g r o ~ p . ~The 7 * ~pK ~ value of the imidazolium group in the /3-galactosidase of E . coli lies near the alkaline end of the range given by It might be possible that certain negatively-charged groups, for Ed~all.7~ example, carboxyl groups, are in its neighborhood. Such groups will suppress the dissociation of the imidazolium group and thus raise its pK value. The influence of alkali ions can then be readily understood, as these ions will surround the negatively charged groups and thus shield the imidazolium group from their influence. The dissociation of the imidazolium group will be facilitated, and the pK value will shift toward the acidic side (as is actually observed). This effect will evidently enhance the enzyme activity, especially a t lower pH. Although several negatively-charged groups must be involved in such an effect, we have tried to represent it figuratively by showing only one carboxyl group near the imidazole ring in Scheme 7

Inarllvc

Artlvr

Inactive

Scheme i

(which is an extension of Scheme 1). The pK value of the sulfhydryl group is also displaced similarly, facilitating its dissociation. This, however, will result in inhibition of the enzyme activity (at higher pH). The mechanism of this pK-displacement might be similar to that for the imidazolium group, but no conjectures can be made about it, as yet. (97) H. Burchfield, Nature, 170, 630 (1957). (98) E. A. Barnard and W. D. Stein, Advances i n EnzymoZ., !dO, 51 (1958).

290

WALLENFELS AND MALHOTRA

11. Induced Synthesis of ,f?-Galactosidase

The subject of induced enzyme formation in general has been reviewed c ~ . ~ may ~ also be made to a review article hy in detail by P o ~ ~ oReference Cohn" and several outstanding original papers on this subject by Monod, Cohn, and others.loO-lM

111. a-GALACTOSIDASES Oligosaccharides with a-D-galactosidic bonds are fairly widely distributed.lO*It has been suggested that they might have been produced by the a-D-galactosyl-transferring action of a - g a l a c t o s i d a ~ e ~ ~(earlier ~ named melibiase). The possibility of this reaction with uridine 5-(D-galactosyl dihydrogen pyrophosphate) as the D-galactosyl donor has not been studied. The scope of such a mechanism for the synthesis of a-wgalactosyl oligosaccharides is, however, limited, as only low activities of a-galactosidases are encountered. That a-galactosidases are different from 8-galactosidases is shown by the difference in their behavior on precipitation with tannin (or alcohol), ~ ~ 1 Among ~ 1 2 themselves, the various aadsorption, and inactivati~n.~ galactosidases are similar in the effect of pH, but they can be differentiakd by virtue of their hydrolytic action on various substrates, for example, through the ratio of the rate of hydrolysis of melibiose to the rate of hydrolysis of phenyl b-D-galactoside (see Table XIII, specificity) .IoLi (99) M. R. Pollock, in Ref. 82b, Vol. 1,1959, p. 619. (100) F. Jacob and J. Monod, Compl. rend., 249,1282 (1959). (101) A. B. Pardee, F. Jacob and J. Monod, J . Mol. Biol., 1, 165 (1959). (102) M. Cohn and K. Horibata, J . Bacteriol., 78,601,613, 624 (1959). (103) J. Monod, Collop. Ues. physiol. Chem., 10, 120 (1960). (104) M. Cohn, E. Lennox and S. Spiegelman, Biochim. et Biophys. Acta, 39, 255 (1960). (105) F . Jacob, D. Perrin, C. Sanchez and J. Monod, Compt. rend., 260,1727 (1960). (106) T. Kameyama and G. D. Novelli, Racleriol. Proc. (SOC.Am. Bacteriologists), 148 (1960). (107) T. Kameyama and G. D. Novelli, Riochem. Riophys. Research Communs., 2 , 393 (1960). (108) D. French, Advance8 in Carbohydrale Chem., 9,149 (1954). (109) D. French, G. M. Wild, B. Young and W. J. James, J . Am. Chem. Soc., 76, 709 (1963). (110) J. E. Courtois, V.Jornadas Bioquim. Lalinas (Barcelona), 1 (1959). (111) B. Helferich, 5. Winkler, R. Gootz, 0. Peters and E. Giinther, 2.physiol. Chem., 108, 91 (1932). (112) T.Nagaoka, Tdhoku J . Ezpll. Med., 61, 137 (1949); Chem. AbShCl8, 46, 1600 (1952).

GALACTOSIDASES

291

1. Occurrence

a. Among Animals. -a-Galactosidase is found in Helix p ~ r n a t i aand ~~~ ,114 Patella vulgata,116the Australian blow-fly of sheep (Lucilia other snails,112 cuprina),116 and certain insects.117 Low activities are observed in the seminal fluid of rabbit and the seminal plasma of horse, but none is found in the genital secretions of bull, dog, sea urchin, monkey, or man.l18 The liver, kidney, and intestines of cow, dog, and pig are devoid of a-galactosidase activity.119 b. Among Plants. -a-Galactosidase occurs in almond emulsin,lZ0 the seeds of coffee: l ~ c e r n eand , ~ Pinus thunbergii,lZ1and in barley malt.122 c. Among Micro-organisms. -a-Galactosidase activity was first detected in brewers’ yeast.’Z3,lZ4Bakers’ yeast is devoid of this enzyrne.lz6a-Galactokefir,lZ7Bacillus sidase is found in Aspergillus niger,lZ6 Aspergillus 0ryzae,1~~ delbrfickii, sulfatase bacteria,6 Escherichia ~ o l iand , ~ Aerobacter ~ ~ aerogenes.lZ9 In the last two, it is not a constitutive but an inducible enzyme.1zQ ,l30 The J ~ ~not ; as a-galactosidase of E. coli is probably a surface e n ~ y r n e ~ ~itl has yet been obtained in cell-free form. (113) H. Bierry, Biochem. Z., 44,446 (1912); Compt. rend., 166,265 (1913). (114) M. Utsushi, K. Huji, S. Matsumoto and T. Nagaoka, TGhoku J . Esptl. Med., 60,175 (1949); Chem. Abstracts, 44,4942 (1950). (115) J. Conchie and G. A. Levvy, Biochem. J . , 66,389 (1957). (116) L. G. Weber, Australian J . Zool., 6, 164 (1957); Chem. Abstracts, 51, 13242 (1957). (117) H. Koike, Dokutsugaku Zasshi, 63, 228 (1954); Chem. Abstracts, 49. 7759 (1955). (118) J. Conchie and T. Mann, Nature, 179, 1190 (1957). (119) J. E. Courtois, C. Anagnostopoulos and F. Petek, Bull. SOC.chim. biol., 36, 731 (1953). (120) C. Neuberg, Biochem. Z . , 3, 519 (1907). (121) S. Hatori and T. Shiroya, Arch. Biochem. Biophys., 34, 121 (1951). (122) R. Weidenhagen and A. Renner, 2. Wirtschajtsgruppe Zucker-ind., 86, 22 (1936); Chem. Abstracts, 30. 3939 (1936). (123) A. Bau, Chem. Ztg., 19, 1873 (1895). (124) E. Fischer and P. Lindner, Ber., a8.3034 (1895). (125) M. Adams, N. K. Richtmyer and C. S. Hudson, J . A m . Chena. Soc., 66, 13139 (1943). 273, 198 (1934). (1213) E. Hofmann, Biochem. Z., (127) B. Helferich, Ber. Verhandl. sdchs. Akad. W i s s . Leipzig, Math.-phys. K l . , 96, 135 (1943); Chem. Abstracts, 40, 7253 (1946). (128) E. Hoeckner, 2. Hyg. Znfektionskrankh., 129.519 (1949); Chem. Abstracts, 44, 2070 (1950). (129) D. S. Hognem and E. H Battley, Federalion PTOC.,16, 197 (1957). (130) J. L. Koppel, C. J . Porter and B. F. Crocker, J . Gen. Physiol., 36,703 (1953). (131) C. J. Porter, B. F . Crocker and R. Holmes, J . Gen. Physiol., 37, 271 (1953). (132) R. Sheinin and B. F. Crocker, Congr. intern. biochim., 3rd. Congr. Brussels, Res. Comm., 1966, 91.

292

WALLENFELS AND MALHOTRA

2. Slandardizution

Melibiose is the test substrate most commonly employed. Liberated sugars are estimated with aldohexose-specific reagents (for details, see Ref. 133). Phenyl a-D-galactopyranoside is a more convenient substrate, and its hydrolysis can be readily followed photometrically at, 280-300 m ~ 4 . 31 6~ ~ Buffer and substrate are pipeted together, and the reaction is started with enzyme. (The final substrate concentration is 0.02 M , in 0.01 M acetate buffer, p H Ei.0;37”). After a definite time, the reaction is stopped by chilling the mixture in a freezing bath. An aliquot is diluted with sodium hydroxide solution (final concentration, 0.1 M ) and the extinction a t 285 mp is measured against that of a reagent blank. The amount of phenol liberated can then be calculated.la6 It has been shown that o-nitrophenyl a-D-galactopyranoside is hydrolyzed by the a-galactosidase of Aerobacter a~ogenes.12~ Analogously to the standardization of 8-galactosidase, o-nitrophenyl a-Dgalac topyranoside or p-nitrophenyl a-D-galactopyranoside can readily be employed as test substrates for estimating a-galactosidase activity.

3. PuriJication a-Galactosidases have not as yet been obtained in pure form. For isolation and purification from coffee, see Ref. 4; and, for separation of the a-galactosidase of almond emulsin from accompanying P-glucosidase and chitinase, see References 133 and 136. 4. Properties

a. Specifiity. -According to definition,I37 a-galactosidases should hydrolyze not only a-D-galactopyranosides but also 8-L-arabinopyranosides, a-D-fucopyranosides, and D-glycero-a-D-galacto-heptopyranosides.It has been shown that 8-L-arabinopyranosides are hydrolyzed, but only at diminished rates, by almond emulsinlll ,1a8 as well as by yeast No studies have been reported on the hydrolysis of a-D-fucopyranosides is not with a-galactosidases. Phenyl D-glycero-a-D-galato-heptopyranosidc hydrolyzed by e m u l ~ i or n ~by ~ ~yeast enzyrne,lz6even at high concentrations and after long periods of incubation. (133) S. Hestrin, D. S. Feingold and M. Schramm, in “Methods in Enzymology,’’ 8.P. Colowick and N. 0. Kaplan, eds., Academic Press Inc., New York, N. Y . , 1956, Vol. 1, p. 231. Intern. Congr. Biocheni., 4th Congr., Vienna, 1,140 (1969). (134) J. E. Courtois, PTOC. (136) M. Arnaud, Doctoral Dissertation, Paris, 1968. (136) L. Zechmeister, G. Tdth and M. B a h t , Enzynaologia, 6, 302 (1938). (137) W. W. Pigman, J . Ant. Chem. Soc., 62, 1371 (1940). (138) B. Helferich and H. Appel, 2.physiol. Chem., 106,231 (1932). (139) W.W.Pigman, J . Reeearch NalZ. Bur. Standards, 26, 197 (1941).

293

OAWCTOBIDASES

As with other glycosidases, wide limits of specificity exist for the aglycon. Thus, the a-galactosidase of Aerobucter aerogenes is capable of hydrolyzing melibiose, and methyl, ethyl, phenyl, or o-nitrophenyl a-D-galactopyranoAryl a-D-galactopyranosides are better substrates than alkyl a - ~ galactopyranosides or disac~harides.’3~J~~ In contrast to the action of pglucosidase, phenyl and o-cresyl a-D-galactopyranosides are hydrolyzed at almost equal rates.140 As has already been mentioned, the various agalactosidases can be distinguished by virtue of their specificity behavior. rate of hydrolysis of melibiose Thus, values of the ratio rate of hydrolysis of phenyl a-D-galnrtopyrarioside for enzyme preparations from yeast and almonds are different from those observed with barley and Takadiastase enzymes. The values in each group are similar among themselves (see Table XIII).108 TABLE XI11 Comparison of Hydrolysis of Melibiose and Phenyl a-D-Galactoside with a-Galactosidases from Various SourcesloB Soiirce

Ra of mdibiose R o j phenyl a-D-gdactoside

Bottom yeast Sweet almond Bitter almond Barley malt Aspergillus oryzae

0.67 1.1 0.8 0.15 0.1

R = rate of hydrolysis.

All of the naturally occurring a-D-galactopyranosides are hydrolyzed by a-galactosidases (see Table XIV).134The velocity of hydrolysis seems to be reduced by increase in the “D-galactosidic” ~hain-1ength.l~~ A free reducing group in the sugar molecule reduces the rate of hydrolysis; for example, melibiose is hydrolyzed less rapidly than raffin0~e.I~~ Oxidation of the reducing group (as in melibionic acid) is without effect on the rate of hydrolysis by enzyme preparations from almond emulsin or yeast.146 (140) S. Veibel, in “The Enzymes,” J. B. Sumner and K. Myrbiick, eds., Academic Press Inc., New York, 1st Edition, 1950, Vol. I, Part I, p. 621. (141) J. E. Courtois, C. Anagnostopoulos and F. Petek, Enzymologia, 17,69 (1954). (142) A. Wickstrom and A. B. Svendsen, Acla Chem. Scand., 10, 1199 (1956). (143) A. Archambault, J. E. Courtois, A. Wickstrom and P. L. Diset, Bull. SOC. chim. biol., 58, 1133 (1956). (144) J. E. Courtois, A. Wickstrom and P. L. Dizet, Bull. SOC. chim. biol., S8,851 (1956). (145) J. E. Courtois, C. Anagnostopoulos and F. Petek, Bull. 1059 (1958). (146) C . Cattaneo, Arch. sci. biol. (Bologna), 23,472 (1937).

SOC.

chim. biol., 40,

294

WALLENFELS AND MALHOTRA

Specificity of the acceptor will be discussed below, together with the D-galactose transfer reaction. b. Transgalactosylation. -Blanchard and Albon reported the formation of an unknown product during the hydrolysis of melibiose with yeast aga1a~tosidase.l~~ This unknown compound moved more slowly t,han melibiose on the chromatogram and was later identified by French as mannino(1 -+ 6)-~-glucose],evitriose [O-a-D-galactosyl-(1 -+ 6)-O-cu-~-galactosyldently formed by transfer of a D-galactopyranosyl residue to the D-galactose moiety of melibiose.108D-Galactosyl transferase action is the aspect of T A B L E XIV ~-GalaclosidesHydrolyzed by Coffee a - G a l a ~ l o s i d a s e ~ ~ ~

I

Oie'gosaccharides

Glycosides and Disacclaarides Sitbslrale

a-D-Galactosidc methyl phenyl p-nitrophenyl S)-DO-n-D-Galactosyl-(l glucose (melibiose) -+

O-a-D-Galactosyl-(l + 6)-Dmannose (epimelibiose) O-a-D-Gahctosyl-(l -+ 6)-Dfructose (planteobiose) Melibionic acid Manninotrionic acid

raffinose umbelliferose st achyose lychnose verbascose tetra-0-D-galactosylsucrose manninotriose galactomannans of lucerne

Product

Keferences

sucrose sucrose raffinose rafinose and a n un known trisaccharide stachyose

141

verbascose

144

melibiose 60% of the D-galactose liberated

144 145

142

141 143 144

a-galactosidases most extensively studied, especially by Courtois and his associates. The acceptor action of some compounds with coffee enzyme is shown in Table XV.14*On the other hand, no transfer to the following compounds could be observed with coffee e n z y m ~ ' ~all~ :of the pentoscs studied (D-ribose, L-arabinose, D-xylose, L-xylose), a 6-deoxyhexose (Lrhamnose), ke tohexoses (D-fructose and L-sorbose), D-glucosamine, and several glycosides (methyl p-D-ghcoside, methyl a-D-mannoside, and amygdalin) . On longer periods of incubation, hydrolysis products, only, are found (147) P. H. Blanchard and N. Albon, Arch. Biochem., 29,220 (1950). (148) C. Anagnostopoulos, J. E. Courtois and F. Petek, Arch. sci. biol. (Bologna), 39, 631 (1956).

295

GALACTOSIDASES

(compare the results with 0-galactosidases). Methanol has been termed a suitable acceptor, because the transfer product, methyl a-mgalactopyranoside, is only hydrolyzed a t a low It might be suggested that the most suitable conditions for transfer will be those under which the donor is split a t a certain optimal rate, and the transfer product is hydrolyzed at only a very low rate, if a t all. I n some cases (for example, with the P-galactosidase of E. coli), such conditions might be attained by regulating the p H and the ionic environment. No data as yet exist to confirm or contraindicate this hypothesis. Acceptor specificity and the kinetics of the transfer reaction have been studied in detail by Arnaud with phenyl a-D-galactopyranoside as the TABLE XV Acceptora Specificity of Cogee a - G a l a c t o ~ i d a s e ~ ~ ~ Transfer after Acceptor 6 hours

Methanol D-Galactose D-Glucose D-Mannose Lactose Maltose Cellobiose Gentiobiose Sucrose Gentianose 0

12 hours

24 hours

48 hours

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

Donor: 0.04 M phenyl a-D-galactoside; pH 5; 37"; 2 M acceptor.

donor and with enzyme preparations from coffee, lucerne, and Aspergillus omjzae. She estimated the liberated phenol (at 285 mp) and the free 1)galactose. A comparison of the two quantities gave the fraction of Dgalactose which had been transferred to an acceptor. If an acceptor interfered with any of these estimations, the transfer was estimated chromatographi~a1ly.l~~ Certain compounds (glycerol, D-mannitol, myo-inositol, trehalose, and methyl a-D-glucopyranoside) earlier reported to be nonacceptors14Rwere found by her to act as acceptors. The same proportion of transfer (that is, transfer/hydrolysis = ( P - G ) / G ,where P and G are the amounts of phenol and frcc D-galactose found) was observed with all three it was found enzymes. Consistent with the results of earlier that the D-galactose moiety is preferentially transferred to a primary J~~

(149) J. E. Courtois and F. Petek, Bull. SOC. chim. biol., 39,715 (1957).

29G

WALLENFELS AND MALHOTRA

alcoholic group in the acceptor molecule. In contrast with the action of P-galac tosidases, only one transfer product was observed in all the cases examined. The ratio of transfer to hydrolysis was independent of the concentrations of enzyme, donor, or liberated D-galactose, the source of the enzyme, the pH, or thermal inactivation. All of these factors affect transfer and hydrolysis similarly, so that the ratio of transfer to hydrolysis remains constant. These observations show that transfer and hydrolysis are carried out by the same e n ~ y m e . ' ~ * J ~ ~ The effect of the acceptor concentration on the transfer/hydrolysis ratio '~~ is Yhown in Fig. 22, which has been drawn from the data of A r n a ~ d . KO

FIQ.22.-Influence of Acceptor Concentration on the Transferring Action of a-Galactosidases. (Citrate-phosphate buffer, p H 6.5; 20"; 0.02 M phenyl a-wgalactoside; incubation time, 1 hour. A, Methanol; 0-0, coffee enzyme; .--a, lucerne enzyme; B, trehalose, coffee enzyme; C, sucrose, coffee enzyme.)

correlation could be observed between the acceptor efficiency and thc number of primary alcoholic groups or the molecular size. The differericch observed arc to be attributed to an acceptor specificity of the enzyme. It may bc asked whether water and organic acceptor are bound on the same site or on different sites. In the latter case, the ratio of transfer to hydrolysis should increase with the acceptor concentration arid then tend to be constant a t high concentrations of acceptor when the site for the organic acceptor is saturated. If, however, the two are bound on the same site, a linear increase in the transferlhydrolysis ratio should be observed. The data obtained so far (see Fig. 22) show that water and organic acceptors are bound on the same site. In other words, not only are the hydrolysis and transfer reactions catalyzed by the same enzyme, but they also take place on the same site of the enzyme molecule.

297

GALACTOSIDASES

Thc (+oncentrationof acceptor was varied between 0.1 and 5.0 M . The c*oiwentration of water (-55 M ) was always very high in comparison to that of the acwptor. Occurrence of a transfer reaction under thcse conditions shows that the organic acceptors have a higher affinity for the enzyme than for ~ a t e r . ’ ~ (Compare *J~~ the results with P-galactosidases.) c. Enzymic Synthesis of a-D-Galactosides. -Galactobiose and galactotriose were isolated as products when D-galactose was incubated with melibiase108; details of the conditions have not been given. d . lnjluence of pH.-The pH optima for the hydrolysis of various substrates with a-galactosidases from different sources are shown in Table XVI. A common featuro of all a-galactosidascs is a more-or-less flat pHTABLEXVI Optimal pH for the Hydrolysia of Diferent Substrates with Various a-Galaclosidases

-

Enzyme source

Yeast Lucerne Coffee Aspergillus oryzae Germinating barley Snail

Substrate

melibiose

Opiimurn p t l 3.5-5.5

A no change from pH 2.8 to 5.7 methyl a-D-galactoside 3.6-4.8 melibiose 3.5-5 A 3.5-5.5 A 3-6 3 and 6 (two maxima) A 3.5-6 A 5 A 3.2-3.8

RejerenceS 126,150 126 126 3 135 4 135 135 151 112

A = phenyl a-D-galactoside.

optimum in the region of pH 3.5-5.0. With coffee enzyme and phenyl a-wgalactopyranoside, two maxima (pH 3 and pH 6) are 0 b s e r v ~ d . lIt~ ~ is not yet clear whether this effect is due to the presence of more than one a-galactosidase or to some other factors. As already pointed out, the transfer reaction undergoes variation with pH similar to that of the hydrolytic reaction, so that the transfer/hydrolysis ratio is independent of pH with coffee, lucerne, and Aspergillus oryzae enzymes. e. Inhibition and Inactiziation. -Very little work has been reported on the inhibition or inactivation of a-galactosidases, so that no conjectures can be made a t present about the nature of the groups involved. (150) R. Weidenhagen, in “Die Methoden der Fermentforschung,” E. Bamann and K. Myrbiick, eds., G. Thieme Verlag, Leipzig, Ger., 1941, Vol. 3, p. 3046. (151) B. Helferich, S. Demant, J . Goerdeler and R. Bosse, 2. physiol. Chem., 283,

179 (1948).

298

WALLENFELS AND MALHOTRA

Lucerne enzyme lost 80 % of its activity3 in 3 hours a t 45". Snail enzyme was completely inactivated112in 30 minutes a t 70". LevvyI6*and C ~ n c h i ehave l ~ ~ suggested, from their work on @-glucuroiiidase and 8-glucosidase, that a glycosidase should be powerfully inhibited by the aldonolactone having identical configuration of the secondary alcoholic groups, irrespective of the size of lactone ring. Lucerne a-galactosidase is, however, an exception, in that it is not inhibited by D-galactono-1,4lactone The enzyme preparation from Aerobacter aerogenes was found by Hogriess and Battley t o be inactivated by oxygen, iodoacetamide, p-(ch1oromercuri)benzoate, and N-ethylmaleimide. After inactivation with oxygen, activity could be recovered on treatment with mercaptoacetate, cysteine, sodium sulfide, sodium cyanide, or 2-mcrcaptoethanol.~*BThe authors, however, have not drawn any conclusion as to whether these observations should 1)c taken to indicate that sulfhydryl groups are involved in the enzyme activity. D-Galactose is a competitive inhibitor for the hydrolysis of phenyl a - ~ galactopyranoside with lucerne enzyme, withISban inhibitor constant (Ki) of 2.4 x 10-3 M . 5 . Induced Sgnthesis of a-Galactosidase

Thc a-galactosidases of E . coli and Aerobacter aerogenes are not const'itutive, but can be induced by the presence of D-galactose or D-galactosidcs in the culture medium. Whereas D-galactose and a-D-galactosides induce both a-and @-galactosidases,@-D-galactosidesinduce only the latter enzyme . ~ ~further ~ information, see Refs. 129, 130, 131, 132, and in A . a e r o g e n e ~For 154. (152) 0. A. Lcvvy, Biochem. J . , 62,464 (1952). (153) J. Conchic, Biochem. J . , 68, 552 (1954). (164) D. S. Hogneee, Revs. Modern f h y s . , 31, 256 (1959).

THE FRACTIONATION OF STARCH BY J. MUETGEERT Plastics Research Institute, T . N . O., Deljt, Holland

. . . . . . . . . . 299 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

I. Introduction.. 11. Fractionation

300 302 304 111. Fractionation by Leaching Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 1. Alkaline Leaching.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 2. Aqueous Leaching.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 3. The Chloral Hydrate Technique.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 4. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 IV. Fractionation by Fractional Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 1. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 2. Aqueous Salt Solutions as Fractionating Media. . . . . . . . . . . . . . . . . . . . . . . 310 3. Aqueous Alcohols as Fractio . . . . . . . . . . . . . . . 319 4. Theoretical Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 5. Concluding Remark .... V. Industrial Methods of .................................... 325 1. Processing Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Resulting Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 3. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 VI. General Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

ns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION The fractionation of starch has been the subject of many publications in the past as well as in the present. The literature of the last twenty years, especially, shows a rapid accumulation of articles on starch research; this can he accounted for by a t least three major influences. These are, first, K. H. Meyer’s’ fundamental discovery that most native starches consist, to the extent of about 20%, of an essentially linear polysaccharide, which he called amylose. Second, T. J. Schoch’s2equally important demonstration of the ability of amylose to form water-insoluble, complex compounds with minor proportions of higher alcohols. Third, the fast-growing interest which Industry takes in useful polymers. In view of the great successes of cellulose chemistry, amylose chemistry could a t least be very promising. (1) K. H . Meyer, M. Wertheim and P. Bernfeld, Helu. Chim. Acto, 23, 865 (1940). (2) T. J. Schoch, Cereal Chem., 18, 121 (1941). 299

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As starch is a cheap material and as, moreover, Schoch's method was suggestive of the possibility of establishing relatively simple and cheap processes for the production of amylose from starch, all of the conditions necesary for intensifying starch research were fulfilled. Although many articles on starch fractionation have appeared since 1940, few of them, for good reasons, deal with industrial processes. In the first place, most industrial processes were patented; secondly, all of them were based on the principle of complex-formation and, hence, did not provide essentially new knowledge. However, the only fractionation method that has so far achieved the status of an industrial process actually applied is of an entirely different kind. Although this process had already been developed by 1952, its fundamentals have so far, for reasons of patent security, not been published in detail. As the process possesses several unique features and is, besides, of a rather universal character, the present survey will be largely devoted to it. Reviews covering the lit,erature on starch fractionation up to 1955 have already been the subject of two contribution^^^' appearing in this Series. Much of their contents might well have been repeated here, in order to round out the discussion of several of the new facts disclosed in this Chapter. For background information, the reader is referred to those articles. The greater part of the prevent survey concerns some of the results originally obtained at the Plastics Research Institute T. N. O., Holland.

11. FRACTIONATION BY COMPLEXINCI AGENTS 1. Qwlntilatzve Aspects

In 1041, Schoch demonstrated that slow cooling of a hot aqueous starch solution saturated with l-butanol gives a micro-crystalline precipitate of the linear starch component, which he called the A-fraction of starch? Since then, it has become generally recognized that practically any polar organic reagent-provided that its solubility in water be high enough-can form crystalline precipitates with amylose. Although there was (and still is) coilsiderable difference of opinion among the different investigators as to the relative merits of one or other type of complexing agent for starch fractionation, all of these workers have indiscriminately used these different substances at their respective saturation values of concentration in aqueous solution. With perhaps one exception,' no attempts seem to have been made to investigate the influence of the concentration of the complexing agent upon the course of its formation of complexes with starch. This situation is (3) T. J . Schoch, Advances i n Carbohydrate Chem., 1, 247 (1945). (4) C. T. Greenwood, Advances i n Carbohydrate Chem., 11,335 (1950). (5) R. L. Whistler and G. F:. Hilbert, J . A m . Chem. SOC.,67, 1161 (1945). (0) E. J. Bourne, G . H . Donnison, Sir Norman Haworth and S. Peat, J . Chem.

Soc., 1087 (1948).

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301

somewhat surprising because, as early as 1943,the problem of the distribution of an adsorband between the two starch fractions, a t least for iodine as the adsorbed substance, was quantitatively solved through use of the potentiometric, iodine-titration method? Whereas amylose adsorbs at a relatively low concentration of iodine, a level which remains practically constant during the better part of the adsorption process, amylopectin needs increasingly higher concentrations of iodine in order to reach a comparable degree of saturation. Evidently, these results point to a possible influence of the concentration of the organic complexing agents upon the course of complex-formation. Although Haworth and coworkersastudied cyclohexanol as a fractionating agent for starch a t various concentrations, their results do not seem to indicate any considerable influence of concentration upon formation of precipitate. Work done in the author’s laboratory, however, soon demonstrated that each of the complexing agents then known possesses its own spec@ region of concentration in which it shows optimum fractionating properties.* At these concentrations, the rate of precipitate formation and the specific, iodine-binding capacity of the precipitate are at their maximum; separation of the precipitated phase is easily effected, being quantitative within 5 minutes on centrifuging under a gravitational field of 1,000g. Even spontaneous settling during 24 hours (under the action of 1 g) often gives good results. Microscopic examination shows that the systems containing the complexing agent at its optimum concentration contain a greater number of crystalline particles, which are much better shaped and are of more uniform and relatively larger size than those developed at all other Concentrations of the same complexing agent. In accordance with these facts, separation of the amylose complexes from systems containing the complexing agent at its saturation concentration usually requires use of super-centrifuges operated at gravitational fields of 10,OOO g or higher. It is to be noted that these phenomena appear only if the complexing agents are applied at room temperature and the systems are not heated before or after their addition (see Section 11,2). The influence of Concentration is most strikingly observed in those cases where the complexing agent is very slightly water-soluble. Table I illustrates this effect for 2-octanol; only a t the optimum concentration (0.04 % by vol.) of this agent are crystals formed. At higher concentrations of 2-octanol, increasing amounts of branched material are precipitated, as may be seen from the decrease in the iodine-absorption values. A1t)hougheven the agents which are very slightly soluble show an optimal (7) F. L. Bates, L). French and R. E. Rundle, J . A m . Chem. Soc., 66, 142 (1943). (8) c. Bus and J. Muetgeert, U. S. Pat. 2,803,568 (1957); Chem. Abstracts, 61, 768 (1958).

w.

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J. MVETGEERT

region of concentration which is lower than their saturation-concentration values in water, the possible existence of compounds showing optimum fractionation a t values equal to or even higher than their saturation concentration cannot be overlooked. An example of a compound of this type, namely, isobutyl methyl ketone, has been r e p ~ r t e d . ~ Because the majority of complexing agents show a rather narrow region of concentration for the optimum effect, it seems desirable to designate this region by the term “critical.” I n the following account, therefore, the optimum concentration of a complexing agent will be called its “critical concentration.” I n Table 11, the critical concentrations of a number of comTABLEI Znjluence of Concentration of 8-Octanol on the Formation of Precipitate in an Aqueoua Solution of Potato Starch at 80” Centrifugal separations

0.02 0.04 0.06

0.08 0.10 0.12 0.14O

0.0 14.2 12.8 0.0 0.0 0.0 0.0

16.0 12.1

-

Centrifugal separationb

5.5 15.4 31 .O 26.7 27.2 30.8 32.4

11.8 14.5 13.9 12.0 11.9 12.0 10.6

0 At a gravitational field of 1,OOO g, during 5 minutes. At a gravitational field of 15,000 g, during 15 minutes. Saturation concentration.

plexing agents, together with thcir saturation concentrations in water, are given. 2 . Theoretical Considerations

I t is the writer’s opinion that the phenomena met with in the foregoing description can readily be accounted for, without resort to one or other of the more speculative concepts regarding the structure of the adsorption complexes. The potentiometrir, iodine titration clearly shows the adsorptive powers of both starch fractions, namely, their relative affinities for iodine in a quantitative way. As is well known, the iodine affinity of amylose depends on its chain length, the shorter chains having the lower affinity.’O Evidently, (9) Private communication from Dr. P. Hiemstra, AVEBE, Veendam, Holland. (10) I).French, in “Chemistry and Industry of Starch,” R. W. Kerr, ed., Academic Press Inc., New York, N . Y . , 2nd Edition, 1950, p. 171.

303

FRACTIONATION OF STARCH

amylopectin can, for two reasons, adsorb iodine at rather high iodine concentrations only: firstly, its branched structure causes strong steric limitations; secondly, the average chain-length of its outer branches, - of the order of 20-30 D-glucose units (compared to amylose, with a D.P. of 500-2,000), is very small. It is, however, clear that a t least the outer branches of the amylopectin molecule will adsorb iodine, and, as they have essentially the same structure as the amylose molecule, their complexes will possess precisely the same configuration as those of amylose, whatever this may be. As x-ray data" point to a general similarity in the structures of iodineamylose and butanol-amylose, it is highly probable that the complexformation of starch with organic complexing agents takes the same route as TABLE I1 Critical Concentrations of Some Complexing Agents and their Corresponding Saturation Concentrations i n Water Cornplem'ng agent

1-Butanol Amy1 alcoholc 1-Hexanol 2-Oc tan01 Isopropyl ketone Chloral hydrate Butyric acid Cyclohexanol Phenol Quinoline

Critical concentrationa Solubilityb 4.2 1.8 0.3 0.04 0.6 5-8 I1 . 0 0.5 2.5 0.6

7.9 3.1 0.59 0.13 1.o

470 m

5.7 6.7 6.0

In g. per 100 ml. of water. * In g. per 100 ml. of water at 20". c Fermentation amyl alcohol. (I

the adsorption of iodine by starch. Consequently, a discrete concentration of the adsorband is required in order to saturate the linear starch fraction, whereas higher concentrations are needed for making adsorption by the branched structures possible. Very probably, the latter phenomenon occurs a t those concentrations higher than the critical concentration. From their property of crystallizing spontaneously, it may be concluded that the amylose complexes possess a highly ordered structure. The outer branches of the complexed form of amylopectin molecules are, however, bound to have the same ordered structure; therefore, these structures are isomorphous with the amylose complex, and hence will take part in the crystallization reactions of the latter. As, however, only the complexed outer branches of the amylopectin molecules will fit the requirements of the lattice of the amylose complex, they evidently will act as so many dislocations (11) R. E. Rundle, J . Am. Chem. Soc., 69, 1772 (1947).

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J. MUETOEERT

and prevent the normal growth of the crystal. As a result, no regularly built, pure amylose-complex crystals are formed, and the precipitated particles contain substantial proportions of amylopectin. From the foregoing reasoning, it is clear that the critical concentration is a relative measure, as its value depends not only on the amount of amylopectin present but also on the D.P. of the amylose, in just the same way as this last factor influences the uptake of iodine. Most probably, the results of Whistler and coworkers,12who found that nitrobenzene, pentyl -acetate, and 2-heptanone will no longer give complexes with amylose of D.P. 20-40, whereas 1-butanol does give the normal complex, find their explanation here. With the exception of 1-butanol, the other compounds investigated are very slightly watersoluble. It is to be expected that their critical concentrations, relative to this particular amylose of low molecular weight, increased beyond the values of their saturation concentrations, thus making complex-formation impossible. 3. Practical Consequences a. Subfractionation.-As could be anticipated, the influence of the concentration of the complexing agent diminishes rapidly with increasing amylose content of the starch sample. Only minor influences can be found in recrystallization of amylose showing an iodine value of 15 % (that is, 150 mg. per g.) or higher. The rate of formation of precipitate in these systems is so high at room temperature that, even at the critical concentration, no well-shaped crystals are formed. However, it stands to reason that addition of increasing amounts of a complexing agent to solutions of amylose will precipitate different molecular species, as the amylose molecules of lower molecular weight will need higher concentrations of the complex-forming substance. In accordance with this hypothesis, Schoch and associates13could obtain any desired number of graded subfractions of amylose, after adding increasing amounts of 1-oct.ano1to a hot, aqueous solution of amylose. After cooling to 65", crystalline precipitates of amylose complexes were obtained, showing a range of intrinsic viscosities, in the order predicted above. Conversely, if it is desired to obtain amylopectin fractions showing a minimum iodine value, use of the highest concentration of a complexing agent is indicated. However, the amylopectin fractions resulting from such experiments must certainly be considered to be subfractions of the total amylopectin present in the native starch. b. Examples of Historical Interest.-Schoch2 reported that, in order to get well-shaped crystals of amylose complex, slow cooling of a hot solution (12) W. Dvonch, H. J. Yearian and R.L. Whistler, J . Am. Chem. Soc., 79, 1748 (1960).

(13) S. Lansky, M. Kooi and T. J. Schoch, J . Am. Chem. Soc., 71, 4086 (1949).

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FRACTIONATION OF STARCH

of starch saturated with 1-butanol (or mixtures of pentanols) was needed; at a temperature of 50-60", precipitation of crystals occurred. This result seems quite contrary to the foregoing results, but it apparently is not, as may be seen from Fig. 1, which shows the temperature-solubility relationships of the systems l-butanol-waterl* and pentanol-water.'S Just at the crystallization temperature of the pentanol-amylose complex (namely, 40-(jg0), the solubility of the complexing agent is a t its minimum value, which in this case is, apparently, sufficiently close to its critical concentration as a starch fractionating-agent (see Table 11).

0

10

Lo 60 -Tempemtufa,

eo

loo

OC.

FIQ. 1.-Influence of Temperature on the Solubility of l-Butanol14(Upper Curve) and Fermentation PentanolL6 (Lower Curve), Respectively, in Water.

Without any heating, the same or even better results can be obtained by adding the requisite amounts (to afford the critical concentrations) of these alcohols to a starch solution at room temperature. As the aqueous solutions of numerous other organic compounds show phase diagrams of the same type as that in Fig. 1, an interpretation of the results of experiments on starch fractionation with complexing agents at different temperatures should be undertaken with great care. Bauer and Pacsu,16in their study of alkaline dispersion methods as an aid to starch fractionation, describe some experimental results which can be fully explained by tjhe same arguments as are given in the foregoing example. Being (14) A. E. Hill arid W. M. MalisotT, in "Physikalisch-Chemische Tabellen," H. Landolt and R . Biirnutein, eds., Julius Springer Verlag, Berlin, 5th Edition, 1923, First Supplementary Vol. 1 , p. 300. (15) F. Fonteyn, in Ref. 14, Vol. 1, p. 752. (16) A. W. Bauer and E. Pacsu, Testile Research J . , 19.880, 871 (1953).

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J. MUETGEERT

unaware of the influences of temperature on the concentration of the complexing agent, these authors erroneously interpreted their results in terms of the unitary starch concept. Addition of 1-butanol or of 2-methyl-1butanol to dispersions of potato starch in 0.5-1.0 N alkali at room temperature, followed by neutralization with an acid (for example, hydrochloric acid), gave perfect, crystalline, amylose-complex precipitates, as has been amply verified in the present author’s laboratory. Several other examples of this sort could be gathered from the literature, but it is supposed that those given above will suffice to show the importance of the concept of the critical concentration, both for practical and theoretical purposes.

111. FRACTIONATION BY LEACHING TECHNIQUES Although most publications on starch in the past fifteen years have treated the properties of starch fractions, and not the fractionation of starch proper, a few deal with refinements of older fractionating techniques which are worth mentioning here. 1. Alkaline Leaching

In accordance with earlier investigation^,'^ Baum and Gilbert1*made use of the insolubility, in cold, dilute alkali, of the amylopectin fraction from undamaged starch-granules. They prepared their own starch from new potat,oes, and took extreme precautions to exclude oxygen during all operations; and they reported that 21% of the starch can be solubilized by treating it with 0.5 N sodium hydroxide solution at 15”during half an hour. The solubilized material possessed a “blue value” of 1.21 and showed an intrinsic viscosity of [q] 5.5 (measured in 0.5 N sodium hydroxide solution). 2. Aqueous Leaching Using the samc freshly prepared starch and working under anaerobic conditions, the same authorslo found that heating of starch suspensions in distilled water, during 5 minutes at loo”, likewise yields an amylose fraction with as high an intrinsic viscosity, [q] 4-5. The same technique, using (instead of distilled water) a 1 % sodium chloride solution, however, gave material of intrinsic viscosity values of about 3.0; this was somewhat vaguely ascribed by the authors to physical changes in the amylose solution. This last method was repeated in the author’s laboratory, using 1% sodium chloride solution (as the extractant) and potato starch from new Malta potatoes. Instead of hydrogen, use was made of oxygen-free nitrogen (17) Z. Gatin-Grusewska, Compt. rend., 146, 540 (1908). (18) H. Baum and G. A. Gilbert, J . Colloid Sci., 11, 428 (1956). (19) H. Baum and G . A. Gilbert, Chem. & Znd. (London), 489 (1954).

FRACTIONATION OF STARCH

307

as a n inert atmosphere. The amylose preparations showed iodine-capacity values up t o 15.7 O/o (by the potentiometric-titration method) and intrinsic viscosities, as measured in 1.0 N potassium hydroxide solution a t 25.00”, as high as [v] 4.10.

3. The Chloral Hydrate Technique Chloral hydrate displays several properties which make it an extremely useful tool in starch research. As these features were recognized as long ago as 1902 by MauchZ0and, seemingly, have been lost sight of in the intervening years, it may be of interest to mention some of the more important results of his work. In his study of the properties of aqueous solutions of chloral hydrate as solvents for starch, Mauch describes the following results. (i)Starch dissolves in aqueous solutions of chloral hydrate containing a t least 50% by wt. of chloral hydrate. (ii)No dextrins can be found in these solutions and, hence, no degradation of the starch occurs. (iii)Starch consists of a t least two components (which he called “amylogeen” and “amylodextrine”) and these components are present as separate entities in chloral hydrate solutions of starch. (iv) After dilution of the solutions with water, a precipitate is formed consisting of “flat spherocrystals” of the “amylodextrine” fraction. Unaware of these old findings, the present writer rediscovered the formation of complexes of chloral hydrate with amylose, and established its optimal conditions (Table I1 gives the critical region of concentration of chloral hydrate). Then, knowing the properties of chloral hydrate as a good solvent for starch, the development of a laboratory fractionation process became obvious. As, to date, no information regarding this work has been published, the details will be given here. Dissolve 60 g. of 100% chloral hydrate in 30 g. of distilled water. Buffer this solution by dissolving 4.0 g. of sodium acetate dihydrate in it. At room temperature, under constant stirring, add to this solution a suspension of 2-5 g. of starch in 10 ml. of distilled water. Keep the solution in the dark at 20’ for 72 hours. Supercentrifuge the solution (after diluting it with an equal volume of distilled water, if necessary). Wash the sediment several times with aqueous, 30% chloral hydrate solution, and keep the precipitate (see below). Combine the washings with the clear, supernatant liquor, dilute with distilled water until the chloral hydrate content is 5-8%, and keep for 24 hours. The amylose has then precipitated and can be separated in an ordinary, laboratory centrifuge at a gravitational field of 1,000 9. After being washed with a 5% solution of chloral hydrate in water (twice), followed by alcohol (twice) and ether (twice), the amylose can be dried at 60” and brought to constant weight in a vacuum desiccator over phosphorus pentaoside. (20) R. Mauch, Arch. Pharm., 240,166 (1902).

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From the mother liquor, the dissolved amylopectin is recovered by precipitation with ethanol. Combine the precipitate with the sediment obtained by the first centrifugation, to obtain the total amylopectin fraction. This method yields amylose fractions having high intrinsic viscosities, but-just as with both leaching methods described in the foregoing-of rather low iodine value. In Table 111, some of the results obtained by this method are given. The commercial starch-sample shows a significantly lower intrinsic viscosity than the one prepared in the laboratory. Compared with the results of hot, aqueous leaching (see Section III,2), the chloral hydrate method yields material having decidedly lower intrinsic viscosities. TABLE111 Fractionation of Potato Starch b y Chloral Hydrate a1 doo, i n the Presence and Absence of Oxygen Characleristics

Commercial Commercial

Laboratory prepared Laboratory prepared 0

A B A B

25.0 23.3 24.4 28.8

14.0 13.9 14.3 13.2

3.14 3.16 3.40 3.40

Key: A, in the presence of air; B, in the absence of air, and under nitrogen.

* Percentage of total starch.

Apparently, there is no influence of oxygen during the preparation of the fractions in the chloral hydrate procedure, as judged from the values of intrinsic viscosity of the experiments given in Table 111. Furthermore, it may be noted that the amylopectin fractions obtained by this method are insoluble in hot water (as well as in cold 1.0 N potassium hydroxide solution). 4. Concluding Remarks

There can be little doubt that, during most preparative, fractionation methods, a certain amount of degradation is inadvertently introduced. As a result, the intrinsic viscosities of both fractions of starch will be found to be lower than the corresponding values for their native state. In order to make a comparison possible, some method of fractionation has to be developed which gives no degradation whatsoever. In this respect, the techniques outlined in this Section might all have a fair chance of success. Restricting attention to potato starch as a substrate, and furthermore to the intrinsic viscosity of its amylose fraction as measured in 1.0 N potassium

FRACTIONATION OF STARCH

309

hydroxide solution a t 25.00”, the highest value which could be obtained by the “classical” fractionation experiments reported in the literature is 2.19 for a sub-fraction of potato amylose.la The industrial fractionation described in Section V yields amylose showing a value of [q] 2.15 for the total fraction. It is, however, very doubtful whether the extremely high values resulting from the methods described in Section I11 really have the same significance as the last-mentioned figures, for the following reasons. The industrial amylose of [q] 2.15 possesses this value at its maximum iodine-absorption capacity (20.0% by wt.). Recrystallization of this amylose by way of its dissolution in 1.0 N potassium hydroxide solution at 20” (under nitrogen) can be repeated indefinitely without changing its characteristic, intrinsic viscosity. The amylose fractions obtained according to (1) the leaching techniques with cold alkali and hot water, respectively, and (2) treatment with hot, 1% sodium chloride solution, as well as (3) the products from the chloral hydrate method, possess iodine values which are appreciably lower than 20 %. In order that valid comparisons of the intrinsic viscosities of different samples of amylose may be made, each of the latter should possess its highest iodine-absorption value, and, as it has been proved that appropriate recrystallization does not cause degradation, all samples should show constant properties with regard to their recrystallization behavior. This means that the products having high intrinsic viscosities-that is, [q] > 1.7-should all show the same, constant iodine-absorption value of 20.0 %. However, as has been amply verified in the author’s laboratory, recrystallization (even under extreme precautions, such as dissolving in air-free alkali at 0” under oxygen-free nitrogen) of the amylose preparations obtained by methods 2 and 3 of Section I11 gave successive products which showed a rapid decrease of their (originally high) intrinsic-viscosity values. Starting values of [v] 3.14.1 after the first recrystallization diminished to [q] 2.52.8, the latter values remaining sensibly constant during further treatments. Consequently, it appears that the methods discussed above can yield amylose fractions showing, after recrystallization, iodine values of 20.0% and intrinsic viscosities of, at most, [v] 2.8. Therefore, the original materials having the high values of [q] 3.4-5.5 might be artifacts, caused by the presence of molecular aggregates of amylose and amylopectin which survived during the fractionation processes, possibly because these processes are incapable of dispersing certain regions possessing a high degree of crystallinity or extensive mechanical entanglement, or both.

IV. FRACTIONATION BY FRACTIONAL PRECIPITATION 1. Introduction Gradual addition of a nonsolvent to a polymer solution causes the polymer to be precipitated in fractions showing decreasing molecular

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J. MUETGEERT

weights. This is the method of fractional precipitation, commonly used for the fractionation of homogeneous, polymolecular polymers. Because, apart from differences in their molecular weight, all species of a certain polymer have the same chemical structure, they show only minor differences with respect to their solubility in a binary solvent of given composition. It is obvious that, in general, much greater differences in solubility will be found to exist between polymers digem’ng in their chemical structure. Consequently, the fractionation of heterogeneous polymers by the method of fractional precipitation mostly effects sharp, quantitative separations. Although, as a rule, chemically dissimilar polymers cannot be combined in one solvent since they will mutually precipitate each other, starch affords an example of the few exceptions to this rule, as its aqueous solutions actually contain two chemically dissimilar polymers, namely, amylose and amylopectin. Nevertheless, until 1950, there were no reports of any systematic studies on the effects caused by the gradual addition, to aqueous, starch dispersions, of water-soluble substances possessing non-solvent character for whole starch. Likewise, no work seems to have been done on establishing the relative difference in solubility between amylosc and amylopectin in binary solvent mixtures.2l I n 1950,during the course of their studies on the possibilities of the fractionation of starch on an industrial scale, Busz2and Muetgeert,22 starting from the considerations just discussed, disclosed a new line of approach by using aqueous salt solutions as fractionating media for potato starch. In ~ general idea was developed into close collaboration with P. H i e m ~ t r a ?the a practical, working method which gave rise to a number of patents. For the following reasons, it is deemed useful to describe this method in some detail here. (i)The method has not hitherto been used as a means of starch fractionation. (ii)To date, the only practical, industrial fractionation has been based on the method. (iii)The principle is of universal applicability, and the results obtained may give rise to a better understanding of the phasc relationships in polymer systems. (iu) Except for a short siir~ e y , 2the ~ only publications concerning this method have issued in the patent literature. 2. Aqueous Salt Solutions as Fractionating Media

a. General Background.--As long ago as 1897,YoungzKreported the individual use of the sulfates of ammonium, magnesium, and sodium for (21) Those binary solvent systems causing the arnylose component to precipitate as an adsorption complex with one of the constituents of the Bolvent system have,

obviously, to be excluded here. (22) Of the Plmtics Research Iristitute T.N.O., Delft, Holland. (23) Of the Cooperatieve Verkoop- en Productievereniging van Aardappelnieel en Derivaten, AVEBE, G . A., Veendarn, Holland. (24) P. Hiernstra, W. C. Bus and J. Muetgeert, StUrke, 8, 235 (1056).

FRACTIONATION OF STARCH

311

the fractional precipitation, from their aqueous solutions, of the products of starch hydrolysis. As is obvious now, the fractions obtained differed only with respect, to their molecular weight, and, as Young used the different salts a t their respective saturation concentrations, he could only produce a very crude fractionation. In a note on the work of Gatin-Gru~ewska,'~ Maquenne2s claimed that 60% of starch is dissolved on treating it with aqueous solutions of sodium sulfate or sodium citrate a t their boiling temperatures. No indications were given as to the salt concentrations and the length of time of extraction used, and the dissolved material is considered to be amylose. Many other experimental studies on the interactions of starch with electrolytes have been published since then; however, as the majority of them are not concerned with the fractionation of starch proper, they will not be treated here. As a result of these studies, it was generally recognized that starch shows normal behavior with respect to the so-called lyotropic or Hofmeister series of ions. Among the more recent publications concerning the use of salts for the fractional precipitation of polymeric carbohydrates, the work of Preece and associatesz7 on water-soluble hemicelluloses of cereals is of interest. They demonstrated that mixtures of a- and 0-glucan can be effectively fractionated by ammonium sulfate, because 8-glucan is precipitated a t a much lower concentration of salt than a-glucan. b. Experimental Results.-Table IV shows the effect of adding various amounts of a saturated, aqueous, magnesium sulfate solution to a molecularly disperse, starch solution29a t 20". After the addition of the salt, the systems were kept for 10 minutes and were then centrifuged during 10 minutes a t a gravitational field of 10,000 g. The supernatant liquors were discarded, and the sediments (after being washed twice with 10 %, aqueous, magnesium sulfate solution, followed by washing with water until free of salts, and dehydrating them by washing twice with alcohol) were dried over phosphorus pentaoxide in a vacuum desiccator. Although the results point to a n unmistakable fractionating effect, the efficiency of the fractionation is very poor. The results of the same type of experiments, in which, however, the salt solution is added a t a temperature of loo", are recorded in Table V. After sz8

(25) R. A. Young, J . Physiol. (London), 22, 401 (1897-98). (26) L. Maquenne, Cornpt. rend., 146, 544 (1908). (27) I. A. Preece and K. G. Mackenzie, J . Znst. Brewing, 68, 353 (1952). (28) I. A. Preece and R. Hobkirk, J . Inst. Brewing, 69,385 (1953). (29) A starch solut,ion is considered to be molecularly disperse if its different molecular species are present as pure, spatially separated, chemical entities. For the present work, this state is arbitrarily defined as having been realized if, 24 hours after the addition of 2.2% (by vol.) of 2-methyl-1-butanol at a temperature of 20" to the starch solution, a yield of precipitate amounting to 23.5% by wt. (of the total starch dissolved) andshowing an iodine absorption value of 16.0% (by wt.) could be obtained.

312

J. MUETQEERT

the addition of the salt, the systems were kept boiling under reflux for 30 minutes, and then cooled to 60"and centrifuged a t this temperature. Washing and drying were performed in exactly the same way as described above for the amylose. Here, too, the fractionation efficiency is poor; however, the purity of the precipitated amylose, as compared with that obtained by TABLEIV Selective Salting-out of Potato Starch from Aqueous Solution"

0 18 20 22 24

26.2' 0.0 3.6 15.0 26.3

14.3 9.5 6.0 6.0

1.53

-

1.78 1.79 1.76

100 9 24 42

, At 20", at a constant concentration, of starch, of 2.5 g. er 100 ml. b Percentage of total starch; A. Measured by potentiometric titration'; * AB/3.75. 8 L'Blank"

5.

determination of total available amylose, by complex-fractionation with amyl alcohol a t its critical concentration.%

TABLE V Selective Salting-out of Potato Starch from Aqueous Solutiona Concentration Yield of of magnesium sul ate heptahydrate PLecipitdch per loo ml,) (AIby wt.)

4.

Iodine absorption

'$$!?

Intrinsic viscosity,

Fraclionalion efiI71 (WE.) ciencxd 70 ~

0 20 22 25

26.2' 6.9 10.0 9.1

14.3 17.5 17.7 14.4

1.56 1.64 1.78 1.66

100 32 47 35

b.

At lOO", a t a constant concentration, of starch, of 2.5 g. er 100 ml. b Percentage of total starch; A. Measured by potentiometric titration'; * AB(3.76. "Blank" determination of total available amylose, by complex fractionation with uniyl alcohol a t its critical concentration.*

fractionation with complexes, is very high. Moreover, the product is insoluble in water at room temperature and is only partially soluble in boiling water. I n order to study the influence of magnesium sulfate a t temperatures well above loo", experiments were done in another way. Potato starch was suspended in an aqueous, magnesium sulfate solution of the desired concentration. Sufficient magnesium oxide was added to ensure that the pH of the system a t the end of the fractionation process would be within the

FRACTSONATION OF STARCH

313

limits pH 7.5-6.5. The suspension was so heated in an autoclave that, within 20 minutes, a temperature of 160" was reached. The system was kept at this temperature for 15 minutes, and then so cooled (at a constant rate) that, after 25 minutes of cooling, its temperature had fallen to 70"; it was then kept a t 70" for 60 minutes. If necessary, enough distilled water was now added to reduce the magnesium sulfate concentration to 9.0 % by wt., and cooling was continued until a temperature of 20' was reached. The precipitated amylose was centrifuged in a supercentrifuge during 10 minutes at 2O', and the combined sediments were directly washed with water until free of salt. After dehydration by alcohol washing (twice) followed by ether washing (twice), and evaporation of the ether at 60', the precipitate was dried to constant weight in a vacuum desiccator over phosphorus pentaoxide. From the supernatant liquor, the amylopectin fraction was precipitated by adding sufficient magnesium sulfate to afford a total concentration of salt of 13.0% by wt. Allowing the precipitated amylopectin to remain in contact with the salt solution for 14 hours proved to be sufficient to make it perfectly insoluble in cold water. The amylopectin was then obtained in the dry state by the same technique used for the amylose precipitate. Potentiometric titration with iodine at 25.0" and measurement of the intrinsic viscosity at 25.00' were applied in order to characterize both fractions. Table VI shows the results of a number of these experiments, using different magnesium sulfate concentrations. The increase of fractionation efficiency with increase of the salt concentration is evident. Independent of the salt concentration, all experiments yield amylose fractions of maximum iodine-absorption capacity, as recrystallization with l-butanol did not show any increase in the original values. The foregoing description of some of the basic experimental work should provide a sufficiently clear idea of the type of experiments involved. In order to prevent this survey from becoming too long, the end results of these studies will be summarized in a more condensed form. The fractionation process will be divided into a number of unit operations, and the experimental results observed will be briefly stated. (i) Dispersion technique.-The gelatinization temperature of potato starch increases with increasing concentration of magnesium sulfate (see Fig. 2). In order to dissolve, a t a fixed temperature, a given amount of starch until a molecularly dispersez9solution is attained, a discrete heating time is required. Increase of salt concentration demands increasing the heating tinies. Increase of the solubilization temperature at fixed magnesium sulfate and starch concentrations causes an exponential decrease of the heating time. The logarithm of the reciprocal heating-time is, roughly, a linear function of the solubilization temperature. Hydrolytic and oxidative

3 14

J. MUETGEERT

degradation reactions of the starch are effectively diminished by addition of appropriate amounts of magnesium sulfite. Likewise, discoloration by thermal-degradation reactions normally occurring at temperatures of 140-160’ is strongly suppressed by the presence of SO$e ions.”” Less molecular degradation results a t high temperatures and, accordingly, shorter heating periods than at lower temperatures and their correspondingly longer heating times.30Consequently, it is important to use the highest TABLEVI Fractionation of Potato Starch froin its Homogeneous Dispersions i n Aqueous Solutions of Magnesium Sulfatea Amylose fraction Concentra lion o j Iodine magneslun Yield of sulfete, % Qrecipitateb, absorption % by wl. % by wl. valzle,c by wt.

0.0 2.3 4.6 6.4 8.5 9.7 10.0 10.3 10.7 11.0 12.1 13.2

0.0 23.46 8.0 11.4 13.O 12.7J 13.9J 15.4, 15.01 16.4J 17.2’ 16.8J

6.6 18.6 18.9 18.9 18.7 20.0 19.4 19.8 20.2 19.9 20.1

Amylopeclin fraction Iodine Yield of Intrinsic brecifdlate, bsorption value, yo viscosity % by wt. by wt. hl, d u g . 1.17 1.59 1.53 1.45 1.62 1.40 1.52 1.62 1.59 1.64 1.78

0.0 0.0

0.0 0.0 0.0 0.0 6.4 12.1 14.5 41.0 79.4 78.7

1.6 1.9 0.9 0.6 0.5 0.5

-

1.29 1.10 1.23 1.18 1.21 1.27

Fracionalion efi:iency,d

%

41 40 57 66 63 74 80 79 88 91 90

a At various concentrations o magnesium sulfate and a constant concentration, of starch, of 4y0 by wt. Indica d as A. Indicated as B. d AB/3.75. a Precipitate obtained after 24 hours. J Before cooling from 70” to 20°, water was added until the salt conccntration reached the value of 9.0%, in order to prevent precipitation of amylopectin.

possible rate of heat input, to reach the desired solubilization temperature. As gelatinization of the starch a t high rates of heating is simultaneous for all of its individual granules, infinitely high (structural) viscosity, lasting for restricted periods of time, is the result. Within 5-10 minutes a t 160°, the extreme viscosity has decreased to a value of 5-10 centipoises, for systems coutaining 5-10 % of starch. (ii) Cooling technique.-If the cooling of the molecularly disperse system has been effected “instantaneously” (for example, within 5 minutes), the (30) W. C. Bus, J. Muetgeert and P. Hiemstra, U. S. Pat. 2,829,987 (1958) ; Chern. Abstracts, 62, 13295 (1958).

315

FRACTIONATION OF STARCH

precipitated amylose consists of uniform particles of very small size, for example, about 0.2 micron. The lower rates of cooling, however, favor the formation of bigger particles. I n those cases, a gradient of particle sizes always occurs. Furthermore, microscopic examination reveals that the particles are perfect spheres and that most of them possess a definite, vacuolized structure. The size of the particles is a function of the molecular weight of the amylose material constituting them-the bigger particles consist of amylose of higher molecular weight than that in the smaller ones.

Temp.

'

OC

1LO-

100 120

-

80-

I' 60

LO

a

,/-

,'

I

I

I

I

1

FIQ.2.-Gelatinization of Potato Starch in Aqueous Solutions of Magnesium Sulfate. (Rate of heating: 2' per minute.) Table VII shows some results of the subfractionation (by particle size) of the amylose obtained from a single fractionation cycle. Increase of the starch concentration increases the average particle-size of the precipitated amylose fraction. Systems containing degraded starch (for example, soluble starch), irrespective of the rate of cooling, always yield amylose precipitates of very fine particle-size (about 0.2 micron). (iii) Recovery of the precipihted amylose fraction.-The precipitated amylose particles can be separated from the system by centrifuging with an ordinary, laboratory centrifuge a t 1,000 g. The sediments obtained contain as much as 25 % of dry amylose. As this amylose is insoluble in water a t

31G

J. MUETQEERT

20”, it is readily washed free of salts by repeatedly suspending the sediment in fresh water and centrifuging. No hysteresis phenomena accompany the decrease of the salt concentration in the wet amylose cake, as washing proceeds in perfect accordance with the mixing rule. (iu) Precipitation of amylopectzn and its recovery.-In order to precipitate the amylopectin, the salt concentration of the mother liquor has to be increased to 13% by wt. Rapid, quantitative flocculation of the amylopectin occurs. The freshly prepared precipitate is readily soluble in cold water. Increasing the time of contact of the precipitate with the salt solution decreases its cold-water solubility, until, ultimately, the material becomes insoluble in cold water. In contrast to the separation of amylose, amylopectin in the form obtained is readily recovered from its suspensions by filtering (or, of course, centrifuging). De-salting by washing with cold water proceeds in exactly the same way as for amylose. It should be noted that, after TABLEVII Mechanical Subfractionalion of Amplose Particles Obtained b y Fractional Precipitation of Potato Starch from an Aqueous Solution of Magnesium Sulfate Average ?article- Iodine absorplion szec, mwom 30 12 0.6

Intrinsic

vdw, % by wt. viscosity [9],dl./g.

19.4 19.8 18.4

2.04 1.67 0.66

precipitation of the starch, the salt concentration shows an increase of about 0.5 % as compared to its initial value. (v) Variants of the process.-So far, the use of magnesium sulfate has been described, but quite similar results are obtainable with other saltingout salts, provided that their thermal stability is high enough to withstand the solubilisation temperatures of up to 160”. For example, both sodium tartrate and sodium citrate exert strong salting-out action; however, because they decompose at temperatures above 100”in aqueous systems, they are of no use for starch fractionations of the type discussed. Of course, only “neutral” salts are of interest here, as these will not cause excessive hydrolytic degradation of the starch molecules; thus, all of the sulfates of the heavy metals are useless for the process under consideration. Both sodium sulfate and ammonium sulfate, although not quite so good as magnesium sulfate, give reasonably satisfactory results. Although the precipitation of amylose by salts does not depend on the complex-forming properties of amylose, it is to be noted that the addition of complexing agents to the salt system changes the process totally. Addition of 1.0 % of 2-methyl-1-butanol to the system (containing, for example,

FRACTIONATION OF STARCH

317

5 % of starch and 10 % of magnesium sulfate) yields, after cooling the system from 160' to go', the normal, sphero-crystalline, amylose complex. If, however, 13 % of magnesium sulfate is used instead of 10 %, no crystalline

precipitate is formed, but the normal, spherical, amylose particles are obtained. At salt concentrations between 10 and 13%, precipitates consisting of mixtures of the crystals of complex with non-crystalline, spherical particles can be obtained. From the salt system without complexing agent, a yield of 16.5%of amylose showing an iodine value of 20.0% (that is, 200 mg. per g.) and a yield of 83.0 % of amylopectin with iodine value of 0.6 % are obtained. The combination with complexing agents yields, on the average, 24.0% of amylose showing an iodine absorption of 15.6% and 75.0 % of amylopectin with an iodine absorption of -0.2 %. If, conversely, a dry amylose preparation, obtained by way of complex-formation with 2-methyl-l-butanol and showing an iodine value of 16.0 %, is dissolved a t 150" in an aqueous, magnesium sulfate solution (containing 10-12% by wt. of this salt), this solution gives, on cooling, about 75% of the total amylose as a non-crystalline precipitate with an iodine absorption of 19-20 %. c. Phase Relationshaps in the System Starch-Magnesium Sulfate-Water.The foregoing results are all indicative of the need for a study of the general, phase behavior of the systems involved. Although this could be said of virtually any type of fractionation technique, there is a general lack of this type of information with regard to polymer fractionation. In the case of starch, this is not so surprising, as its complex nature, the manifold phenomena met with in its physicochemical behavior, and, more specifically, the general dependence on time which manifests itself in the latter, are all arguments reasonable enough to discourage even an optimistic investigator from tackling this job. Although we are well aware of the limitations of the description, the present system can be described with the help of two conventional, triangular, phase diagrams, one for each of the starch fractions. Because of the general metastability of both starch fractions in the system involved, the experimental method employed for deriving these diagrams necessitated the use of a rather unusual technique which includes the addition of a fourth component. This work will not be treated in detail here, but some of its inherent limitations will be noted In contrast to their general use, the isotherms describing phase separation are not indicative of the existence of a real equilibrium between the phases; they only indicate incipient phase-separation at zero time. Although neither of the starch fractions separately studied was subfractionated (and, therefore, each represents in itself a polycomponent system), they will be treated as if they were pure, single components. Likewise, magnesium sulfate will be considered to be a pure single component. The system is, then, one con-

318

J . MUETGEERT

sisting of three components and, in the event of incipient phase-separation (at zero time!), of two phases. The phase rule requires that this system possess three degrees of freedom, one of which (pressure) can be disregarded.31 Hence, fixing the temperature and one of the concentration parameters (for example, the salt concentration) suffices to define the system. The phase diagrams were experimentally derived by using fractions of potato starch Solvent

Polymer IPolysoccharide I

FIQ.3.-Phase Diagrams of Two Ternary Systems : Amylose-Magnesium SulfateWater (Solid Curves) and Amylopectin-Magnesium Sulfate-Water (Dotted Curves).

obtained by the industrial fractionation process (see Section V). The amylose had an intrinsic viscosity of [q] 1.87, the amylopectin of [q] 1.20; the iodine values were 19.8 % for amylose and 0.60 % for amylopectin. For comparison, the results obtained from the two diagrams have been combined in one figure, which is, therefore, alternatively, a phase diagram for the systems amylose-magnesium sulfate-water or amylopectin-magnesium sulfate-water. Figure 3 presents that part of the phase diagram which is of interest for the starch fractionation. Although the Figure is (31) The influence of pressure on condensed systems is negligibly small; see, for example, A. Findlay, “The Phase Rule,” Dover Publications, Inc., New York, N. Y., 9th Edition, 1951, p. 340.

FRACTIONATION OF STARCH

319

essentially self-explanatory, the following particulars may be noted. Within the limits of the temperatures and concentrations stated, no formation of solid, salt phases is possible. As the phases could not be analyzed without changing the (pseudo-) equilibria, it was obviously impossible to estimate the position of the t,ie-lines. Since the isotherms for each fraction are parallel and at equidistant points, the critical temperature of phase separation is a linear function of the salt concentration. Moreover, for an equal change in salt concentration, the increase of the critical temperature of amylopectin is twice that for amylose; hence, increase of salt concentration or increase of polymer concentration, or both, will eventually lead to a reversal of the order in which the two fractions precipitate. The most noteworthy feature of these diagrams is that the shape of the binodials is in general agreement with the requirements of the theory on the phase behavior of polymer nonsolvent-solvent systems.32 The diagrams are in satisfactory agreement with the experimental facts observed in the actual fractionation experiments; moreover, they explain the phenomena described in the foregoing discussion with regard to combinations of complexing agents with the salt system (see Section IV, 2b, point v). Occurrence or non-occurrence of complex-formation evidently depends on the position of the isotherm for normal (non-complex) phase-separation of amylose. If the isotherm is located well above the temperature where amylose complex is precipitated (in the experiments described, this temperature was go"), crystalline complexes will not be formed, and the amylose precipitated will be in the normal, amorphous state. Conversely, if the isotherm of normal phase-separation is well below the temperature where amylose complex becomes insoluble, crystalline, amylose complex will be precipitated. It should be borne in mind that there are, in fact, as many isotherms as there are different molecular-weight species present in the polymer; also, that each of them occupies its own position. Similarly, it may be assumed that the crystalline complex of each molecular-weight species has its own isotherm, whose position (of course) differs from the one describing amorphous (liquid-liquid) precipitation. If the salt concentration is appropriately chosen, overlapping of both groups of isotherms occurs and evidently leads to precipitated phases containing both forms of amylose. 3. Aqueous Alcohols as Fractionating Media

The somewhat unusual idea of interpreting the influence of the salt in terms of a classical, non-solvent action seems to be supported by the characteristics of the phase diagrams obtained. Now, addition of non-solvents (32) A theoretical approach to the derivation of phase diagrams for ternary systems is given by H. Tompa, Trans. Furuday SOC.,45, 1142 (1949); C. H. Bamford and H. Tompa, ibid., 46, 310 (1950).

320

J. MUETGEERT

to polymer solutions invariably results in phase separation of the liyuidliquid type. Although the precipitated amylose proved, in all instances, to be in the solid state, its morphology is, nevertheless, indicative of its initial formation as a liquid phase, the latter occurring in the form of tiny droplets dispersed throughout the bulk of the solution. If these considerations are assumed to be justifiable, this fractionation principle must have a universal character, It is of interest, then, to ascertain whether, relative to starch, classical non-solvents can show the same phenomena as are met with in the salt solutions. As is well known, such TABLEVIII Fractional Precipdation of Potato Starch from its Homogeneous Dispersions i n Alcohol-Water Mixtures

Poor sohen6

Methanol

Ethanol

2-Propanol

0

Charatlnistics of the precipitate

by uol.

yield of precifdale, % by wl.

5 15 25

0.0 16.0 27.4

amorphous crystalline"

18.9 13.1

1.63 1.48

5 20 25 30

0.0 14.0 24.6 30.1

amorphous crystalline" crystalline"

19.4 15.2 11.8

1.58 1.49 1.52

15 35 45

14.7

crystalline"

16.2 12.9 8.4

1.65 1.41 1.29

Concmtro-

'$v~~$~

Iodine

Slate

ualzle,

70

by wt.

Inlrinsic viscosity

[TI, W

g .

A complex.

alcohols us methanol, ethanol, and 2-propanol are nonsolventrs for starch; therefore, experiments were performed in which various mixtures of each of these alcohols with water were used. Cooling to room temperature of homogeneous solutions of potato starch (obtained by autoclaving 5 % starch suspensions in water-alcohol mixtures during 15 minutes a t 160") gave the results shown in Table VIII. The same types of amylose precipitate as are normally found to be the results of fractionation in aqueous salt systems do, indeed, occur here, namely, spherical particles of 0.2-10 microns, insoluble in water a t 20°, and only partially soluble after prolonged boiling in water. As, however, superimposed on the normal phase-separation, complexformation between the alcohol and amylose is apt to occur, somewhat the

FRACTIONATION OF STARCH

32 1

same phenomena are met here as have already been reported as occurring if a complexing agent is added to the salt system (see point v, in Section IV,2b). It should be noted that the amorphous type of amylose precipitate is not formed with 2-propanol, as, apparently irrespective of its concentration, the formation of normal, crystalline, amylose complex is favored.

4. Theoretical Considerations I n the opinion of the writer, all of the phenomena discussed in this Section can, a t least qualitatively, be readily accounted for by existing theories on polymer fractionation. Starting from a few considerations regarding the nature of the two starch fractions, it will be seen that there is no need to resort to the more hypothetical effects often employed in explaining the interactions of electrolytes and hydrophilic colloids. Consequently, effects of electrical charge, ionic radii, or specific structural changes of the solvent (water) do not have to be introduced in order that the fractionating effect of salt solutions may be understood. It should first be noted that, in spite of all the differences between the two starch fractions, they have many points in common. Amylose is a linear, chain molecule composed of a-D-glucopyranose residues linked by a - ~ - ( l-+ 4)-glucosidic bonds, and each of the branches of the amylopectin molecule has the same structure as the amylose but contains, besides, a-~-(l---t 6)-glucosidic bonds at the points where branching occurs. Hence, both polymers contain the same chemical unit of structure in extensive parts of their molecules, and, moreover, all of these units have the identical stereochemical configuration. Therefore, amylose and amylopectin will show many of the properties of isotactic polymers; and, since the most outstanding property of the latter is their tendency to crystallize, both fractions of starch belong to the class of crystalline polymers. The difference between the two polymers, as regards their crystallization behavior, is thus seen to be one of degree rather than of kind. However, as is evident, amylose shows a far stronger tendency to crystallize than does amylopectin. Whereas aqueous solutions of the latter, containing as much as 20 % of polymer, will remain fluid for several hours a t room temperature, amylose solutions of the same concentration rapidly solidify at 100". Figure 4 shows a part of the solubility curve of amylose in water. Now, the precipitation of crystalline polymers from solution is very different from that of amorphous polymers. Whereas liquid-liquid phaseseparation is invariably the result if the latter type of polymer is caused to precipitate, the former type are precipitated mostly in the form of very small, solid particles. Nevertheless, it is possible to force a crystalline polymer to be precipitated also in the liquid state. Evidently, this will occur if the temperature a t which phase separation takes place is above the crys-

322

J. MUETGEERT

tallization temperature of the precipitate; this conclusion is borne out by experimental studies on the phase relationships of solutions of p~lyethylene?~ and 011 several other systenis of crystalline polymers. Now, all of the evidence points to the fact that amylose phase-separation caused by magnesium sulfate is just one more example of this type of precipitation of a crystalline polymer. As the critical temperature of phase separation increases with increase in the salt concentration, and as increase of the former (in the case of polymer-nonsolvent-solvent systems) is nor-

0

5

--

10

15 Arnylosc

20

2s

concentration

30

, % by w t

FIG.4.-Solubility of Amylose in Water, Derived from Measurements of the Temperature of Gelation at Different Concentrations of Amylose. (Intrinsic viscosity of amylose, [ q] 1.6. Rate of cooling: 2” per minute.)

mally found to be caused by increase of the nonsolvent-solvent ratio, it is clear that magnesium sulfate plays the role of the nonsolvent in the system. As is well e ~ t a b l i s h e dfrrtctionation ,~~ of crystalline polymers can only be effective if the phase separations are of the liquid-liquid type. Since the amylose concentration in the precipitates obtained from the salt systems is a t least 25 %, Figure 4 indicates that liquid-liquid phase-separation has a chance to occur only if the critical temperature of precipitation is higher than 100”. In order to reach precipitation temperatures above loo”, the salt concentration of the system has to be higher than 12 % (see Figure 3). This conclusion is confirmed by the experimental results, as practically (33) R . B. Richards, Trans. Faraday soc., 4, 10 (1946). (34) P. J. Flory, “Principles of Polymer Chemistry,” Cornell University Press, Ithaca, N . Y., 1953, p. 344.

FRACTIONATION OF STARCH

323

quantitative yields of amylose (at its maximum iodine value) are obtained from systems containing 13 % of magnesium sulfate. Because liquid-liquid separation can not occur a t temperatures below loo", very poor fractionation efficiency and fractions of amylose having sensibly lower iodine values are the result of salting-out experiments a t temperatures of 20-100" (see Tables I V and V). It is readily seen that, by fixing the nonsolvent-solvent ratio, the upper limit of molecular weight of the polymer such that it will just be soluble depends only on the temperature of the system. I n order to dissolve amylose having the maximum molecular weight, use of the highest permissible temperature is indicated. Now, the thermal instability of starch does not allow a temperature of 160" to be exceeded without occurrence of alteration. At this temperature, however, an aqueous, 13 % magnesium sulfate solution is still so poor a solvent that it will only dissolve starch polymers having sensibly lower molecular weights than those constituting the native material. Therefore, a certain amount of degradation has to take place before solubilization is possible. It is obvious that at least a part of the heating time needed for dissolving starch in magnesium sulfate solutions of the strength indicated has the character of a reaction time, namely, the time of hydrolysis needed for degrading both starch components until they will fit the requirements of the arbitrarily defined solvent a t its (equally arbitrarily) chosen temperature. I n accordance with this conclusion, it is observed that incipient precipitation of amylose occurs at temperatures which are only slightly lower than the solubilization temperatures; for example, after dissolving a t 160", the first visible precipitation of amylose occurs a t 145". Consequently, the material which is precipitated a t the higher temperatures constitutes the higher molecular-weight species, whilst, a t successively lower temperatures, amylose fractions of decreasing molecular weight are precipitated. At the higher temperatures, coalescence of submicroscopic particles into relatively big, liquid droplets is possible by reason of the fact that the viscosity of the precipitated, polymer phase is relatively low. At lower temperatures, however, the increase in viscosity and the incipient gelation3bof the concentrated polymer-phase increasingly impede coalescence. As a result, the particles remain discrete, that is, they do not merge into a coherent, liquid layer (sometimes called the coacervate layer), and the particle diameter is a function of the molecular weight (see Table VII). It is to be understood that the amylose from this type of precipitation will be insoluhle in wsltJer below 100" a s it really represents a retrograded form of nmylose. The (35) Superimposed on the temperature influence is an influence of the molecular weight of the amylose, ns the rate of gelation (= retrogradation) increases with dccreasing chain-length.36 (36) R. L. Whistler and C. Johnson, Cereal Chem., 26,418 (1948).

324

J. MUETGEERT

retrogradation, which is, in fact, crystallization is, however, a relatively slow process, whereas the phase separation is very fast; and hence the former is not essential for the mechanism of the precipitation of amylose. This fact has been confirmed by recent experiment^.^' From Fig. 3, it may be seen that lowering of the temperature to 60-70” will cause separation of amylopectin. I n general, this phase separation takes the same route as that for the amylose (except for the peculiar, morphological phenomena of the latter). As crystallization is much slower for the branched fraction of starch, the critical temperature of phase separation is sufficiently high to permit the existence of a coherent, liquid phase for short periods of time. The fact that freshly obtained amylopectin precipitate is soluble in cold water, whereas, after several hours, it is completely insoluble in cold water can only be interpreted as being the result of crystallization. In accordance with this conclusion, it is to be noted that this phenomenon is perfectly reversible. The increase of the salt concentration of the mother liquor, after the separation of both polymers, is the normal result of preferential solvation of the polymer by the solvent (water). 5 . Concluding Remarks

In the foregoing reasoning, only the main features of the method of fractionation with salts have been considered. With the help of prevailing theories on polymer fractionationas and of existing experimental facts on the physicochemical behavior of polymer systems,3g it is readily possible to account for the phenomena (which, in order to keep this survey within reasonable limits, have not been treated at length). Although separation of amylose from amylopectin is the main purpose of the process described, it has been observed that subfractionation of at least one of the two components of starch (namely, amylose) likewise occurs. In this connection, it is to be noted here that the experimental results strongly indicate that fractionation of amylose into its different molecular-weight species is remarkably efficient in salt solutions. In view of the great disparity in solvent power between the precipitant and the solvent in the case of aqueous salt solutions, this conclusion seems to confirm some predictions of Fl~ry’s.~O Several facts pertaining to the fundamental science involved have still (37) J. Ho116, J. Szejtli and C:. S.Gantner, Stdrke, 12,73 (1960). (38) Phase equilibria in polymer systems are excellently treated in P. J. Flory’s bo0k.~4 (39) More specifically, the work of H. G. Bungenberg de Jong on coacervrttes is recommended; see, for example, H. G. Bungenberg de Jong in “Colloid Science,” H. R. Kruyt, ed., Elsevier Publishing Company, Inc., New York, N. Y.,1949, Vol. 2. (40) P. J. Flory, Ref. 34, p. 663.

FRACTIONATION OF STARCH

325

to be evaluated. Thus, the values of the several interaction-parameters and their dependence on salt concentration and temperature are of prime importance. Furthermore, it would be of interest to determine the extent to which the solubiliaation temperatures are identifiable with the “theta temperature^"^^ of the systems involved. Obviously, such knowledge could only result from a more quantitative, thermodynamic treatment. In view of the linear dependency of the critical temperature of phase separation of both starch fractions on the solvent composition, however, a study of this kind would seem promising. V. INDUSTRIAL METHODSOF FRACTIONATION A number of patents on the fractionation of starch concern the industrial production of starch fractions. Although most of them have not been applied in actual practice (and, therefore, no adequate evaluation of their results is possible), it is of some interest to give a short description of their details. This situation does not apply to the method of fractionation based on the use of aqueous salt solutions, as this process has been in actual operat i ~ n for ‘ ~ several years and the resulting products are commercially available43;consequently, a more detailed description of this process is possible. The experimental results and the fundamentals of the mechanisms involved in the salt method have been treated a t length in Section IV; its technological features will now be mentioned briefly. 1. Processing Techniques

As early as 1947, Schoch devised an industrial method for starch fractionation4 based on the ability of amylose to form insoluble complexes with certain alcohols. In order also to recover the amylopectin fraction by precipitation, the total concentration of alcohol had to be sufficiently high; this, in turn, necessitated autoclaving, in order to solubilize the starch. Although the use of aliphatic alcohols having 1 to 5 carbon atoms was claimed, the examples given in the patent description dealt only with alcohols having at least 3 carbon atoms. The optimum concentrations recommended for alcohols having 3 to 5 carbon atoms ranged from 10 to 30 volume-percent. Starch concentrations of up to 7 % can be used; however, the best results are obtained with 3 to 4 % of starch. (41) At their “theta temperatures,” polymer solutions show ideal behavior; see Ref. 34. (42) In the starch-fractionation plant of the Coiiperatieve Productievereniging van Aardappelrneel en Derivaten, AVEBE G. A., Veendarn, Holland. (43) Amylose and amylopectin are sold in the U. S. A. by Stein, Hall & Co., Madison Avenue, New York, N . Y., under the respective trade names of Superlose and Ramalin. (44) T. J. Schoch, U. S. Pat. 2,515,095 (1950); Chem. Abstracts, 44, 11141 (1950).

326

J. MUETOEERT

According to the examples given in the patent, the process can be operated in several ways; one of them is described in the following. Corn starch (100 g.) is gelatinized in a boiling mixture of 2 liters of water and 400 ml. of 2-methyl-2-butanol. After adjustment of its pI-1 to 6.45 with a potassiuni phosphate buffer, the resulting paste is heated for 30 minutes in an autoclave a t 155"; cooling to 90" suffices to cause precipitation of the amylose complex (A-fraction), which is recovered as a dense precipitate by supercentrifuging. The amylopectin (B-fraction) is obtained from the supernatant liquor after keeping it at 3" for 1G24 hours. The yield of A-fraction is 30 % and its iodine value 12.5% ; the B-fraction comprises the remainder of the starch and shows an iodine value of 1.0 %, the fractionation efficiency in this case being 71 %. Besides corn starch, fractionatioii of several other kinds of starches by this method was claimed. Although 7 % is the upper limit of starch concentration which can be used for undegraded corn-starch, as high it is to be noted that thin-boiling starches can be f r a ~ t i o n a t e dat~ ~ a concentration as 17 %. The presence of relatively high concentrations of alcohol in the fractionating media has the additional advantage that defatting of the corn starch, prior to its fractionation, is unnecessary. It is clear that this process lends itself to continuous operation in a closed cycle, as the aqueous alcohol can, after removal of the amylopectin fraction, be used for dissolving and processing fresh portions of starch. In order to effect complete dissolution of starch at temperatures below loo", Bauer and P a c d rwommended the use either of dilute alkali or of dilute acid solutions. According to their so-called "alkali process,)' defatted corn starch is4sdissolved in 0.5 to 1.0 N alkali a t room temperature. Sufficient mineral acid is added to the resulting solution to bring its pH within the limits of 1 0 4 . After saturation with (for example) Pentasol (a mixture of primary amyl alcohols), the system is heated for several minutes a t a temperature between 60" and 100"; on cooling, an amylose precipitate is obtained which can readily be separated in an ordinary, industrial centrifuge. Addition of excess methanol to the supernatant liquor causes precipitation of the amylopectin. Different kinds of starches can be fractioriatcd by this method. Starch concentrations of up to 5 % are claimed to give about a 24% yield of amylose (showing an iodine value of 16.0%) and a 76 % yield of amylopectin (with a 0.9 % iodine absorption). If dilute acids are employed as fractionating media, far higher starch concentrations can be handled.16*47 Starch concentrations of up to 20 % (45) T. J. Schoch, U. S. Pat. 2,515,096 (1950); Chem. Abstracts, 44, 11141 (1950). and A. W. Bauer, U. S. Pat. 2,779,693 (1957); Chem. Abstracts, 61, (46) E. PRCSU 8459 (1957). (47) E. Pacsu and A. W. Elsuer, U. S. Pat. 2,779,694 (1957); Chem. Abstracts, 61, 8459 (1957).

FRACTIONATION OF STARCH

327

(and even higher) are claimed to be useful. The principle of this method is apparent from one of the examples given in the patent description. About 12.5 g. of dry, defatted corn-starch is mixed with 100 ml. of 0.1 N hydrochloric acid and 15 ml. of butanol, the mixture is heated during 35 minutes a t 85", and cooled to room temperature. After 24 hours, the amylose fraction has precipitated and it is separated in a centrifuge a t 2,000 r.p.m. The amylopectin fraction is precipitated by adding 200 ml. of methanol to the supernatant liquor. Each fraction is washed with methanol (twice) and ether (twice), and is dried in a vacuum oven a t 90".This method should give a 25.3'2, yield of amylose (with an iodine number of 16.7%) and a 74.7% yield of amylopectin (with an iodine value of 0.0). I t is clear that neither the alkali nor the acid method can be developed into continuous, recycling processes, since, in both methods, the recovery of the B-fraction necessitates addition of larger quantities of methanol. Moreover, in the alkali process, neutralization of the alkaline substance is necessary prior to precipitation of the A-fraction, and so, recycling is impossible. Whereas all of the methods proposed for large-scale fractionation of starch that have been discussed depend directly on the ability of amylose to form insoluble complexes with polar organic compounds, Cantor and Wimmer's process4*is based on a totally different principle. If a molecularly disperse solution of starchzYcontains a sufficient amount of calcium chloride and caustic alkali is added, a rapid and quantitative precipitation of the starch occurs, because of the formation of complexes (of calcium hydroxide with the starch polysaccharides) which are insoluble in an aqueous, saturated solution of calcium hydroxide. The same phenomenon is observed with the hydroxides of barium and strontium. It will be noted that this type of complex-formation is entirely different from that in which complexes are formed between amylose and certain polar, organic compounds. I n contrast to the precipitates of the latter complexes (which arc of a distinct, crystalline appearance), the starchalkaline-earth hydroxide complexes are amorphous, curdlike flocculates. These complexes dissociate on diluting them with water, and the starch redissolves. According to the patent description, the amylose complexes dissolve much more easily thaii the amylopectin complexes; hence, fractionation must occur if water is added stepwise. Likewise, fractionation will take place if the starch complexes are partially neutralized, by the gradual addition of an acid. For obvious reasons, such acids as carbonic acid arid sulfuric acid (which give insoluble calcium salts) are preferred. Furthermore, it is claimed that gradual addition of caustic alkali to a starch solu(48) S. M. Cantor and 13. L. Wimmer, U. 8. Pat. 2,779,692 (1957); Chent. Abstracts, 61. 8460 (1057).

328

J. MUETQEERT

tion in the presence of calcium chloride causes the amylose complex to be precipitated first, leaving amylopectin in solution. Likewise, gradual addition of calcium chloride solution to a sufficiently alkaline dispersion of starch causes preferential precipitation of the amylose complex. The recovery of the starch fractions from their complexes with calcium hydroxide is attained by different means, all of which are obvious and therefore need not be described here. As the method is, in general, very involved, and, in addition, comprises several chemically irreversible steps, it could only be applied as a batchwise process. The commercial production of starch fractions, based on the principle of fractional precipitation treated in Section IV, started in Holland around 1955. In the process employed,30 potato starch is dissolved under pressure, at an elevated temperature, in an aqueous solution of magnesium, ammonium, or sodium sulfate, or a mixture of two or of all three of them. For simplicity, attention will here be restricted to magnesium sulfate. In order to keep degradation of the starch within reasonable limits, the (high) solubilization temperature has to be reached within the shortest possible time. For this reason, batchwise processing (for example, by heating in autoclaves) is not adequate, and hence a continuous process is indicated. Technologically, the process is operated as follows. Potato starch is suspended a t room temperature in an aqueous solution of magnesium sulfate, in such a way that the suspension contains 10% by wt. of dry starch and 13% by wt. of magnesium sulfate. I n order to control the pH and rH of the system, minor amounts of magnesium sulfite are added.30This slurry is continuously fed into :t series of steam-operated, multipass, heat exchangers of the tube and shell type by means of a high-pressure, positivedisplacement pump. Within three minutes, the temperature of the system has reached 160°, and this is maintained during 15 minutes by passing the processing fluid through an appropriately designed residence-vessel. Connected with this vessel in :% closed system, a series of properly dimensioned heat-exchangers effect cooling of the solution to the desired temperature (80") within the desired time (25 minutes). The precipitated amylose fraction is then continuously separated in a series of closed, centrifugal separators, operating a t a gravitational field of about 2,000 g and at a temperature of 80'. The supernatiint liquor is continuously cooled to 20", and the precipitated amylopectin is kept for several hours in the salt solution a t this temperature, after which it is filtered off by means of a continuous, rotary filter. The resulting mother liquor can be re-used after adjustment of its salt concentration, and it is recycled. Both uf the resulting fractions are washed with water until free of salt, and the wet cakes obtained are dried on roll driers, or, alternatively, by spray-drying.

FRACTIONATION OF STARCH

329

As the separation of amylose a t 80" is technologically difficult to achieve, the following alternative procedure may be employed. Starch is dissolved in exactly the same way as described above; however, cooling is only carried to 90°, at which temperature, the salt concentration is decreased by injecting water into the system. If the salt concentration is adjusted to 10 %, the amylose may be separated a t 20" without any chance of precipitating amylopectin (see Fig. 3).4QI n order to precipitate the amylopectin, the salt concentration must now be increased to about 13 %. Several kinds of starches can be fractionated by the processes described, and starch concentrations of up to 20 % can be handled. Even starch-containing raw materials (for example, potatoes, corn, wheat, and other cereals), after being ground in order to destroy their cell structure, give reasonable results.60Use of these raw materials obviously makes the process more involved, and recycling becomes increasingly difficult. Degraded starches can, likewise, be fractionated; if, however, the molecular weight of their amylose fraction is less than 2 X 104, the process does not give satisfactory results. s1 In general, addition of a complexing agent to the salt solution is advantageous if starches of low molecular weights have to be fractionated. Washing of the amylose fractions has then to be performed with water containing a sufficient proportion of the complexing agent. 2. Resulting Products a. Properties Before Drying.-All of the industrial, fractionation processes discussed in this Section yield, initially, both of the starch fractions in the form of wet cakes, whose composition and structure differ considerably according to the processes employed for their production. If the amylose has been obtained by way of complex-formation with one or other organic, complexing agent, the material constituting the cake has a microcrystalline structure. As a rule, the proportion of (dry) amylose present in cakes having this structure never exceeds 12 % by wt. Even supercentrifugal separation does not increase this figure. Moreover, the proportion is independent of the fractionation process used; the Schoch method, the Bauer and Pacsu process, and the method of salt precipitation [if operated in combination with a complexing agent (see IV,2b, point v)] all yield amylose precipitates which, a t most, contain 12% of (dry) amylose. Inasmuch as the greater (49) W. C . Bus, J. Muetgeert and P. Hiemstra, U. 8. Pat. 2,829,988 (1958); Chem. Abetracts, 62, 14204 (1958). (50) W. C. Bus, J. Muetgeert and P. Hiemstra, U. S. Pat. 2,829,989 (1958); Cheni. Abstracts, 62, 13296 (1958). (51) W. C. Bus, J. Muetgeert and P. Hiemstra, U. S. Pat. 2,829,990 (1958); Chem. Abstracts, 62, 17768 (1958).

330

J. MUETQEERT

part of the occluded liquor has the composition of the mother liquor, washing (in order to remove the amylopectin dissolved in the latter) is unavoidable. As, however, amylose complex is readily soluble in water, washing has to be done with aqueous solutions of a complexing agent.62 The amylose cakes obtained from the salt-fractionation process as operated in the absence of complexing agents are of an entirely different nature. Owing, apparently, to the spherical shape of the amylose particles (which permits very dense packing), they contain as much as 30% by wt. of (dry) amylose. As, moreover, the amylose is in a retrograded state, washing can be performed with water, alone, without any loss of polymer. In accordanre with the fact that all fractionation methods yield amylopectin precipitates of an amorphous nature, no appreciable differences in the proportion of dry material present in the wet cakes is observed. Adequate separation, either by centrifuging or filtering, yields cakes containing as much as 2530 % (by weight) of dry amylopectin. However, whereas the amylopectin obtained by precipitation with alcohols, or other water-miscible organic solvents, is soluble in cold water, the inaterial resulting from salt precipitation is insoluble. Although, in the Schoch process, washing is not necessary, it, is essential in the acid as well as the alkali processcs of Bauer and I’arsu. In the latter proccsses, the acid and the (neutralized) alkali, respectively, are removed by washing with alcohol-water mixtures, or the like, roiitaining rather high proportions of the organic component. b. Properties After Drying.-Several changes in the solubility propertics of the starch fractions take place during the drying of the wet cakes. Itolldrying is applied, and temperatures of the rolls as high as 120’ are normal practice. As the type of amylose soluble in cold water, for example, the amylose complex, dissolves during the early stages of the drying operation, partial retrogradation occurs during the evaporation of thc water. As a result, the dry amylose is insoluble in cold water and dissolves only partially in boiling water. In addition, it shows a B-type of x-ray diagram, whereas, prior to drying, its x-ray diagram is of the V-type. Roll-drying of the amorphous, retrograded amylose resulting from frnctional precipitation, for example, with salting-out salts, does not introduce any considerable changes in its solubility. Only a small proportion of this type of amylose dissolves during the drying; the bulk of the dry material shows the same morphological characteristics as before drying, namely, spherical particles occurring in a gradient of sizes. Moreover both states, dry and wet, show identical x-ray diagrams (of the B-type) Of course, this

.

(62) It is not necessary that the complexing agent to he used in the washing liquid should be identical with the one employed in the friictionation process. Any type of complexing agent may be used, with the proviso that its concentration be not lower than its critical value (see Section 11).

33 1

FRACTIONATION OF STARCH

material, also, is insoluble in cold water and, compared with the dry product obtained by way of complex-formation, even is less soluble in water at the boiling temperature. The same phenomenon of intermediate solubilization in the early stages of the roll-drying is responsible for the fact that the dry amylopectin fractions, irrespective of the methods employed for their isolation, are all soluble in cold water. Whereas the wet, amylopectin cakes resulting from precipitaTABLE IX Properties of Starch Fractions Obtained by Fractionation of Potato Starch -4 ccordinq to Several Industrial Processes Properties of products obtained Fractionation process of

A-Fraction

Varianl nlsed

Iodine value, by wt.

cz IJ. 8. Pat. 2,515,095 T. J. Schoch

B-Fraction

Intrinsic Iodine Intrinsic viscosity, Valzle, viscosity, dl./g. % by wt. dl./g.

2-rnothyl-2-butanol

15.0

1.5

0.5

1.1

U. S. Patent 2,779,693 alkaline process E. Pacsu and A. W.

14.0

0.8

1 .o

0.7

acid process

12.0

0.3

0.2

0.3

2-methyl-1-butanol in niagnesium sulfate solu tion magnesium fiulfitte

16.0

1.8

0.3

1.1

20.0

2.0

0.8

1.2

Bauer

U. 8. Pat. 2,779,691 E. Pacsu and A . W Bauer

U. S. Pat. 2,829,987 W. C. Bus, J. Muctgeert,, and 1'. Hiemstra

tion by organic solvents arc, as a rule, soluble in cold water, those obtained from fractional precipitation by salts are not. Apparently, the intermediate solubilization on the hot rolls is sufficiently complete to destroy the crystal linity (which caused the initial insolubility, see Section IV, 4). The dry amylopcctin thus obtained is in an essentially amorphous state. However, it will be clear that, if the amylopectin insoluble in cold water has been dried at, temperatures below its solubilization temperature, in this case, GO", the dry product wilfbe insoluble in cold water. Products of this type may be obtained by vacuum, drum-drying or spray-drying of salted-out amylopectin a t any appropriate temperature below 60".

332

J . MUETGEERT

Apart from differences in their solubility, which, in a sense, might be regarded as secondary differences, the fractions obtained by the several industrial methods also show primary differences with regard to their iodine values and intrinsic viscosities. In Table IX, the (smoothed) averaged values of these fundamental properties have been listed for fractionation of potato starch according to the several industrial methods discussed. 3. Concluding Remarlcs

Although, for obvious remons, fractional precipitation from salt solutions is by far the most economical of the methods discussed for the industrial fractionation of starch, such processes as that of Cantor and Wimmer48 might possibly become of interest for the production of reactive intermediates of each starch fraction. Several types of chemical derivatives of both starch components can be synthesized by way of the calcium hydroxide complexes. It must be understood that the examples given in describing the several processes proposed for operation on an industrial scale by no means exhaust the possible applications of' these processes; in general, each of them has its own variants. This is obviously true for the fractional-precipitation technique, as the number of different nonsolvent-solvent systems which can be realized is practically infinite. Moreover, the large differences in the solubility of the starch fractions obtained by the last-mentioned technique offer additional possibilities for attaining a ~ e p a r a t i o n . ~ ~

VI. GENERAL CONCLUSIONS From the practical standpoint, the fractionation of starch may be considered to have finally reached a satisfactory status. In the opinion of the present writer, there is no need for the creation of fundamentally new methods. On the contrary, attention should be focused on several of the existing procedures, all of which could be greatly improved were their basic principles better understood; this conclusion seems true both for the industrial processes of fractionation and the laboratory methods. More specifically, the analytical type of laboratory procedure might well be refined. It would therefore be of great interest to investigate the mechanism of sorption in a far more quantitative way than has been done hitherto. Precise knowledge of the quantitative relationships existing between the chain length of amylose and the amount of foreign, molecular species adsorbed and desorbed, as functions of temperature and concentration parameters, would clarify many of the detaih of complex-formation. Prerequisite to studies of this kind is the availability of an adequate method of subfractionation for (53) W. C. BUR,J. Muetgeert and P. HiernRtra, U. S. Pat. 2,822,305 (1958) ; Chem. 62, 9635 (1958).

AbShCtS,

FRACTIONATION OF STARCH

333

amylose, a method still lacking. Likewise, more-quantitative knowledge concerning the fractional precipitation has to be gathered; hence, additional investigation of solvent-nonsolvent systems that show graded disparity as solvents for starch polymers seems indicated. Since the starch fractions are, as pointed out by Schoch,arather unusual in that they are composed of the mme structural unit, both starch fractionation proper and the fundamental science of the phase behavior of polymer systems would be promoted by such studies. The use of the Phase Rule and its enlightening descriptive representations cannot be overestimated as a tool for starch chemistry as a whole, and certainly it is indispensable for research into the fractionation of starch.

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CARBOHYDRATES IN THE SOIL BY N. C. MEHTA,P. DUBACH AND H. DEUEL Laboratory of Agricultural Chemistry, Swiss Feileral Znslitute o/ Technology, Zurich, Switzerland

I . Introduction. . . . . . . . . . . . . . . . . . . , . , , , . . . . . . . _ ._ . . . , . . . . . , . . . . . . . . , . . 335 11. Isolation and Characterization , , . . , . .. . , . , . , . , . , . , , . . , . . . . . . , . . _ ., . 337 1. Monosaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 2. Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 3. Other Carbohydrates.. . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ .343 111. Quantitative Determination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 1. Hexoses.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 2. Pentoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 3. Uronic Acids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 4 . Amino Sugars.. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . 346 . . . , . . . . . , . . . . . . . . . . . , . . . . . . . . . . . . . . . , . . . . . . . , . . . . . 347 6. Total Carbohydrates.. . . . . . , . . . . . , . . . . . . . . . . . , . . . . . . . . . , . . , . . . . , . . . . . 317 IV. Source arid Transformation. . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 1. Source.. . . . . , . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . , . . . , . . . . . . . . , . . . . . . . . . 348 2. Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 V. State and Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 1 . Interaction with Other Soil-constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 2. Function in the Soil.. . . . . . . . . . . . . . ..... 353 VI. Surumitry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 ,

I. INTRODUCTION The soil is one station in the geochemistry of carbohydrates. Originating in higher plants, animals, and micro-organisms, carbohydrates occur in soils and peats, in lakes, rivers, and oceans, and in lignites, brown coals, fossils, and sediments in general.’ Carbohydrates have even been isolakd from sedimentary rocks 180 to 300 million years The soil is a complex mixture of numerous inorganic and organic constit,uent>swhich vary in size, shape, chemical constitution, and reactivit,y, and it contains numerous organisms. The various constituents interact to form systems of higher order, thus contributing to the characteristic architecture of various soils.4 The soil structure (that is, the arrangement of the l2

(1) P. H. Abelson, Fortschr. Chem. org. Naturstoge, 17, 379 (1959). (2) J . R. Vallentyne, in “Organic Geochemistry,” I. A. Breger, ed., Pergamon

Press, London, in press. (3) J. G . Palacas, F. M. Swain and F. Smith, Nature, 186, 234 (1960). (4) H . Deuel, Trans. Intern. Congr. Soil Sci., 7th Congr., Madison, Wisc., in press. 335

336

MEHTA, DUBACH AND DEUEL

soil constituents in aggregates) determines to a large extent such properties as the air-water relationship, tillability, and stability toward erosion. The kind of soil which is produced in a given location is controlled by the following factors: climate, parent material, organisms, topography, and age.6 The formation of soil is brought about by three groups of processes: the weathering of the inorganic parent material, the incorporation and transformation of organic material (humification), and the interaction and translocation of the inorganic and organic constituents. A soil “profile” with characteristic “horizons” is formed, the top soil being, in general, richest in organic matter. Various combinations of these processes lead to different, well recognized soil-groups, such as podzols, brown forest soils, chernozems, and laterites.e Normal soils are well drained and well aerated. They generally contain less than 10 % of organic matter. Under conditions of poor drainage and poor aeration, an abnormal accumulation of salts (as in saline soils) or of organic matter (as in peats) occurs. This review deals mainly with normal, well drained soils; only brief designations of the soils are given. The organic constituents of the soil are collectively termed humus.’ The organic material originating from plants and animals (which is continually added to the soil) is rapidly decomposed. Under normal conditions, no continuous accumulation of any one substance is possible. While decomposition is taking place, new substances are synthesized which, in turn, are decomposed. The decomposing and synthesizing processes usually reavh an approximate dynamic equilibrium. Morphologically, two extreme humus forms can be distinguishede(”): (a) mor, which is incompletely decomposed plant material, not incorporated into the lower, inorganic part of the soil, and (b) mull, namely, well deromposed material, thoroughly mixed with the inorganic part of the soil. Soil organic matter is a mixture of a great number of compounds of low to high molecular weight>#sMany compounds, most of which are known to occur in plant and animal tissues and in micro-organisms, have been iso(5) H. Jenny, “Factors of Soil Formation,” McGraw-Hill Book Co., Inc., New York, N . Y . , 1941. (6) (a) J. S. Joffe, “Pedology,” Pedology Publications, New Brunswick, N . J., 2nd Edition, 1949. (b) W. L. Kubiena, “Bestimmungsbuch und Systematik der Boden Europas,” F. Enke, Stuttgart, 1963. (7) Monographs on soil organic matter: (a) S. A. Waksman, “Humus,” Williams and Wilkins Co., Baltimore, Md., 1936. (b) M. M. Kononowa, “Die Humusstoffe des Bodens,” Deutscher Verlag der Wissenachaften, Berlin, 1958. (c) J. Pochon and H. de Barjac, “Trait6 de Microbiologie des Sols,” Dunod, Paris, 1958. (d) F . Scheffer and B. Ulrich, “Humus und Humusdhgung,” F. Enke, Stuttgart, 1980. (8) F . E. Broadbent, Advance8 i n Agron., 6 , 153 (1953). (9) J. M. Bremner, J . Soil Scsci., 2, 67 (1961); 6, 214 (1964).

CARBOHYDRATES I N THE SOIL

337

lated from soils.'O but these compounds constitute only a n insignificant part of the total organic matter of soil. The major components of soil organic matter, usually over 50%, are the so called humic substances; these are colored, organic acids of unknown (probably aromatic) constitution and of low (fulvic acids) to high (humic acids) molecular weight. Nitrogen accounts for 3 to 5 % of soil organic matter. About 4.5 to 55 of the total organic nitrogen is a-amino nitrogen, corresponding to a protein content of 5 to 15 . % of soil organic matter. Total carbohydrates have been estimated to constitute between 5 and 20% (average 10%) of soil organic matter. Carbohydrates occur in the soil in living and in decaying organisms, as well as in extracellular form. As it is practically impossible to separate the living micro-organisms and tiny plant and animal tissues from the dead organic matter of soil, the whole system is usually treated as an entity. Carbohydrates have been studied in the most diverse (and not always clearly defined) soils. I n most cases, the top horizon of the soil profile has been examined, Although humus constitutes, generally, only a small part of the soil (below 10 %), it exerts a profound influence on the physical, chemical, and biological properties of the soil. Attempts have been made to attribute particular influences to specific compounds in humus. The observation of the ameliorative effect12of polysaccharides on soil structure has greatly stimulated research on the nature of soil carbohydrates. The following questions may be asked. Which monomeric sugars are present in soil carbohydrates and in what quantities? What kinds of carbohydrates occur in soil? What proportion of the organic matter of soil is carbohydrate? Are carbohydrates linked to other soil constituents? What is the source and what is the role of carbohydrates in soils? What are the differences between the carbohydrates of different soils?

11.

ISOLATION AND CHARACTERIZATION

1 . Monosaccharides

Only trace amounts of monosaccharides have so far been detected in soils. The reducing sugars in cold-water extracts of Norwegian amounted to less than 1 % of the total soil organic matter (2 % for peats). Glucose, galactose, xylose, and rhamnose have been identified by paper chromatography in sodium hydroxide extracts of various Scottish soils.14 (10) E. C. Shorey, U . S D e p t . Agr., Bur. Soils, Bull., 88, 1 (1913). (11) See Ref. 7(d), pp. 132-139. (12) J. P. Martin, W. P. Martin, J. B. Page, W. A. Raney and J. D. De hlent, Advances i n Agron., 7 , 1 (1955). (13) E. Alvsaker, Unio. Bergen Skrifter, 23, 1 (1948). (14) W. G. C. Forsyth, Chem. & Znd. (London), 515 (1948).

338

MEH‘PA, DUBACH AND DEUEL

For these ext,racts, t,he Elson-Morgan test for amino sugars was also positive. I n a cold-water extract of a Norwegian forest soil, glucose (0.2270 of the soil organic matter), galactose (0.02), fructose (0.035), xylose (0.03), arahinose (0.04), and ribose (<0.001%) have been det,ermined by quantitatdivepaper-chromatography.l6 The fructose was identified by x-ray analysis of it,s (2,4dinitrophenyl)hydrazone. This is, to date, the oirly reference in the likrature to a definite identification of fructose in the soil. Sugars and uronic acids were detected in solutions pressed out of peat^.'^^ nlonosaccharides are strongly adsorbed by clay mineralslG;the proportioii of such carbohydrates could, therefore, be considerably greater than is indicat,ed by the amount, that has been extracted under mild conditions.

2 . Polusaccharides Many methods have been used for the isolat,ion of polysaccharides from soil. Except for one ~ a s e , l 7 (the ~ ) maximum yield obtained awount,s for only about, 2 % of the soil organic matter. The average content of carbohydrat,e, however, is estimated to lie between 5 and 20% of soil organic matter.’ The nature arid state of the remaining carbohydrate material is not, known. So far, the primary aim of the various workers has actually been the isolation of an undegraded polysaccharide material free from inorganic and organic impurities, and the completeness of the extraction has only been of secondary interest. a. Extraction.-Polysaccharides have been extracted from the soil (see and acid;’ respectively. Table I) wit#hbuffers,l*Jghot water,2°-2ssalkali,24.26 (15) E. Alvsaker and K . Michelson, Acta Chem. Scand., 11, 1794,1795 (1957). (15a) I. V. Aleksandrova, Pochuouedenie, 11, 85 (1960);Soils and Fertilizers, C o v monwealth Bur. Soil. S c i . , 24, 99 (1961). (16) D.J. Greenland, J . Soil Sci., 7,319,329 (1956);R. A. Kohl and Y. A. Taylor, Soil Sci., 91. 223 (1961). (17)(a)J. W.Parson, “A Chemical Study of Polysaccharide Material Isolated from Soil,” Ph.T). Thesis, University of Reading, Engl., 1958. (b) J . W. Purson and J . Tinsley, Soil Sci. Sac. A m . Proc., 24, 198 (1960). (18) W.N. Huworth, F. W. Pinkard and M. Stucey, Nature, 168, 836 (1943). (19) B. Bernier, Riochem. J.,70, 590 (1958);A. G . Oyston, ibid., 70, 598 (1958). (20) R. B . Duff, J. Sci. Food Agr., 3 , 140 (1952);Chem. & Znd. (London), 1104 (1952);ibid., 1513 (1954). (21) 0.Theander, Suensk Kem. Tidskr., 64, 197 (1952);Acta Chem. Scand., 8 , 989 (1954). (22) R . L. Whistler and K . W. Kirby, J . A m . Chem. Sac., 78, 1755 (1958). (23) C. E . Clapp, “High Molecular Weight Water-soluble Muck: Isolation and Determinution of Constituent Sugars of a Borate Complex-forming Polysaccharide, Employing Electrophoretic Techniques,” Ph.D. Thesis, Cornell University, N . Y., 1957. (23a) J. 1,. Mortensen, Trans. Intern. Congr. Soil Sci., 7th Congr., Madison, W i s c . , in press.

339

CARBOHYDRATES IN THE SOIL

Polysaccharides were extracted from various British soils with buffers, in yields of 0.05 to 0.15% of the soils.'* No further details of the isolation procedure were given. More recently, soils were extractcd with a phosphate buffer of pH 7, and the polysaccharides were recovered from the dialyzed and concentrated extract by precipitation with ethan01.l~This procedure extracted less non-dialyzable, non-carbohydrate material than those employing dilute alkali or sodium pyrophosphate. Yields were about the same as with hot-water extraction, but the polysaccharides isolated by means of phosphate buffer had a higher viscosity. TABLE I Extraction and Yield o j Polysaccharide Preparations jrom various soils Yield of polysaccliaride preparalions Soils (Soil organic matter,

yo)

Extraction inetlzod

Refcir, s+ erences % of soil organac malter

--British soils British forest soils British and tropical soils Swiss soils (3.0-32.5%) Wisconsin soils (0.82.6%) Scottish soil (5.6%) Indiana soil (3.38%) New York muck soil British mineral soils, peats, and composts

buffers (room temperature) phosphate buffer (room temperature) alkali (0.5 N NaOH, room temperature) do. do. water (4 hrs., 85") water (6 hrs., 85", N2) water (24 hrs., Soxhlet) acid (98% HCOzH, 30 min. refluxing)

0.05-0.15 0.014.04

-

18 19

-

1.32-1.94

24

0.02-0.13 0.10-1.00 25 0.024.25 20, 27 0.3 0.05

-

20 1.45 22 1.8 23 3.5-11.5 17(a)

In a major investigation of their polysac~harides,~~ soils were extracted with 0.5 N sodium hydroxide. The humic substances of high molecular weight were precipitated by acidification of the extract to pH 2.5 to 3.0, and the centrifuged, light-colored solution was fractionated by chromatography on a column of carbon. Four fractions were collected. Fraction A was eluted with 0.1 N hydrochloric acid, and contained amino acids, purine bases, and sugars; fraction B was eluted with 90 % aqueous acetone, and, on drying, gave a red powder with indications of the presence of phenolic glycosides; fraction C was eluted with water, and contained the polysac(24) W. G . C. Forsyth, Biochem. J . , 41, 176 (1947); 46, 1401 (1950). (25) P. Dubach, G. Zweifel, R . Bach and H. Deuel, 2. Pflanzenernahr. Dung. 'u.. Bodenk., 69, 97 (1955).

340

MEHTA, DUBACH AND DEUEL

charides; fraction D was eluted with 0.5 N sodium hydroxide, and contained colored humic material. The polysaccharide material was precipitated from fraction C by the addition of acetone; the yields were 1.32 to 1.94% of the soil organic matter. Polysaccharides have also been isolated from alkaline extracts by a slightly modified p r o c e d ~ r e ~ ~the * ~ 7alkaline : extract was acidified and centrifuged, and the polysaccharides were precipitated by pouring the neutralized, concentrated, supernatant solution into acetone. Extraction of the polysaccharides with hot water20dissolved only a small amount of humic substances, mainly of low molecular weight.22 The method has the disadvantage that polysaccharides may be degraded a t elevated temperatures. The soil was twice extracted a t 85" for 4 hours, and the polysaccharides were precipitated by pouring the dialyzed, concentrated extract into acetone.20The original procedure has been modified to a 3-hour treatment21 a t 65" and to a 24-hour extraction in a Soxhlet apparatuv.23 A more complete e x t r a ~ t i o n ~of~ (polysaccharides ~) was attempted by refluxing the soil for two 30-minute periods with 98 % formic acid containing lithium bromide. The organic matter extracted was precipitated by the addition of ivopropyl ether and was redispersed in lithium chloride solution. The colored humic substances were then precipitated with hexadecyltrimethylammonium bromide, while the acidic and neutral polysaccharides were kept in solution by the lithium chloride. The possible degradative effects of hot formic acid on soil polymccharides have not yet been investigated. Except for the formic acid e x t r a c t i ~ n , l ~all ( ~the ) other methods yielded approximately the same amoun t of polysaccharide. However, even after more than 20 successive extractions of a Swiss brown-earth (Braunerde) with acid, water, and alkali, further extracts gave a positive anthrone reaction for sugars.28 The extraction of polysaccharides is probably made difficult by their interaction with inorganic surfaces and humic substanrcs. b. Purification.-The raw polysaccharides have been purified by re-preripitation, dialysis, formation of copper complexes, decolorization with carbon, and deproteinization with cadmium sulfate, and, also, by the Sevag method. These purification procedures always result, in appreciable loss of polysaccharide. None of the purified polysaccharide preparations were free from non-carbohydrates. Even after careful purification of the material, the carbohydrate ~ o n t e n t ~ 7was ( ~ )as ' ~low ~ as 50 %. The non-rarbohydrate part contained humic substances and proteins. (26) D. A. Rennie, E. Truog and 0. N . Allen, Soil Sci. SOC.Am. Proc.,18, 399 (1954). (27) G. Cheaters, 0. J. Attoe and 0. N. Allen, Soil Sci. SOC.A m . Proc.,21, 272 (1957).

(28) Unpubliehed work of this laboratory.

CARBOHYDRATES IN THE SOIL

34 1

c. Fractionation.-Hydrolyzates of the polysaccharide preparations usually contain more than ten sugars. Polysaccharides containing even 5 to 6 different kinds of sugar residues are rare, and none are yet known which have more than G different kinds of sugar residues.2ge30 The soil-polysaccharide preparations are, therefore, probably mixtures of different polysaccharides.17Jg*22, z3, 31 Consequently, many attempts have been made to fractionate the material, using the well established methods of polysaccharide chemistry. The shape of the precipitation curve obtained on gradual addition of ethanol to an aqueous solution of the polysaccharide material indicated heterogeneity.22 However, the five fractions collected showed no significant quantitative differences in component sugars. Another attempt to obtain fractionation of the polysaccharides by precipitation with ethanol from a solution in water or fonnamide was unsuccessful.1g Precipitation with hexadecyltrimethylammonium bromide likewise produced no clear separa- 2 Z , 2 3 , 2 a a ~some f preparations showed them to be tion.”(”)* l 9*32Electrophoresis1Q heterogeneous without, however, giving an unequivocal separation. Acetylation, followed by fractional precipitation in a series of solvents and by ultracentrifugation, produced no fractionation of the polysa~charides.’~ Some degree of fractionation was obtained by precipitation with Benedict copper s01ution.l~ Polysaccharides isolated from a Swiss river-soil by the carbon-adsorpt ion techniquez4 were fractionated by anion-exchange chromatography on a (2-diethylaminoethyl)cellulose column.31 Five fractions, having increasing uronic acid and decreasing (non-uronic acid) sugar content, were eluted with phosphate buffer and sodium hydroxide solutions of increasing concentrations. The major portion of the polysaccharides was very low in uronic acid, and a small fraction was very high in uronic acids. After hydrolysis of the fractions, no differences in the sugar components could be detected by qualitative paper-chromatography. The approach is promising; it, may eventually lead to the isolation of individual polysaccharides whose constitution and origin can be studied. d. Characterization.-The composition of polysaccharide preparations isolated from various soils is given in Table 11. Considering the differences in the soils and the methods used, the discrepancies in the findings are less (29) R. L. Whistler and C. L. Smart, “Polysaccharide Chemistry,” Academic Press Inc., New York, N . Y., 1953, p. 18. (30) H. Deuel and H . Neukom, Kolloidchem. makromol. Naturslofe, 18, 91 (1958). (31) M. Miiller, N . C. Mehta and H . Deuel, 2.PJanzenerndhr. Dung.u . Rodenk., 90, 139 (1960). (32) H . Streuli, N . C. Mehta, M. Muller and H . Deuel, Mill. Gebiele Lebensm. u . H y g . , 49, 396 (1958).

342

MEHTA, DUBACH AND DEUEL

surprising than the agreement in the main results. In the hydrolysates of extracted soil-polysaccharides, the following constituents have usually been found by paper chromatography and isolation of the pure sugars or their derivatives : glucose, galactose, mannose, xylose, arabinose, ribose, rhamnose, fucose, uroriic acids, amino sugars, and some unknown sugars. TABLEI1 Characterization of Polysaccharide P iarations Isolaled frotn Various Soils Soils -

Scar lid Soil'

I

I

British

mineral ndiana New E'ork soils,

7s:

soil22

peats, composts'7'a'

Equivalent weight N, % OCHa, % Reducing sugar, o/o Uronic anhydride, Yo Amino sugars, yo Component sugars, % of total sugars in preparation Glucose Galactose Mannose Arabinose Xylose Ribose Rhamnorre Fucose Unknown nugars

OOO 1 2

80

-

945 0.34 2.4

15.8 0

20 0

,185 0.3 0

20.8 20.0 21.9 11.7 23.6 1.5 0 0 0

-

36 29 10 4 11 0 7

-

-

-

-

9.1 trace

-

37.8-15.5 17.0-6.7 about 5

21.2 16.6 18.5 10.4 12.6 trace 14.2 0 6.5

-

127 .8-36.4 22.2-22.6 28.6-29.8 -

26.6-38.0 17.8-23.3 16.3-21 .0 6.8-8.2 7.5-9.0 7.7-13.6 8.2-19 .O 0 3.5-6.6 0

Three unknown sugars of high Rl value have been found in traces.?" They have tentatively been identified as 0-methyl-hexoses and O-niethylheptoses. These 0-methyl sugars may be the same as the unknown sugars The presence of 0of high Rl values detected by other workers.22023,26J1 methyl sugars has been demonstrated in nine out of ten soils and they seem to be of general occurrelice. One of them was found to be a constituent of a polysaccharide produced by Bacillus m~gatherium.~~ )34 The (33) Macaulay Inst. Soil Research, Ann. Rept., 1964/66, 32. (34) W. G. C . Forsyth, Trans. Intern. Congr. Soil Sci., 6th Congr., Lhopoldville, 3, 119 (1954).

CARBOHYDRATES IN THE SOIL

343

reported presence of fructose in soil polysaccharides'8 has not been confirmed, and no other ketoses have yet been found. Up to I 1 % of amino sugars has been determined (by the Elson-Morgan m e t h ~ d ' ~ ( ~in) Jthe ~) polysaccharide preparations. This extractable part of the amino sugars of soil is probably not chitin. Amino acids, also, have been found in the hydrolyzates of the p r e p a r a t i o n ~ . ~ ~ ~ ~ ~ J ~ No structural work has been done on the polysaccharide preparations. In the hydrolyzates, the sugar components have been estimated quantitatively by paper chromatography or other methods (see Table 11).It would be premature to try to interpret the ratios found between individual sugars or classes of sugars, or to discern a pattern in the variations between differen t soils, because the preparations were heterogeneous and represented only a small part of the total polysaccharides of the soil. Ultracentrif ugal studieslghave shown that the material isolated was polydisperse and that the macromolecules were highly anisometric. Certain polysaccharide preparations contained from 60 to 90 % of dialyzable component~.~~

3 . Other Carbohydrates The isolated monosaccharides and polysaccharides represent oiily a small part of the total carbohydrates of soil. The soil residue after extraction, and isolated fractions of soil organic matter (for example, humic substances), might contain sugars other than those which have been detected in the polysaccharidex isolated. However, hydrolysis of soils and of humic substances isolated, followed by chromatography of the freed sugars, showed that this was not the case.22*36,36 Amino sugars have almost exclusively been investigated in hydrolyzates of the total soil. The presence of glucosamine and galactosamine has been definitely established by paper chromatography and ion-exchange chroniatography37-42arid by the isolation of both sugars in crystalline form.40I t appears that 2-acetamido-2-deoxy-~-glucose (N-acetyl-~-glucosamiiie) is also present in the h y d r ~ l y z a t e sNO . ~ ~other amino sugars have as yet heeii detected .*2 Some other substances related to carbohydrates have been isolated from soils : glucaric acid,10mannitol,1° inositol (present partly as esters of phosD. L. Lynch, L. M. Wright and H . 0. Olney, Soil Sci., 84,405 (1957). D. L. Lynch, H . 0. Olney and L. M. Wright, J . Sci. Food Agr., 9,56 (1958). J. M. Bremner, J . Agr. Sci., 39, 183 (1949). J. M. Bremner, Biochem. J . , 47, 538 (1950). J. M. Bremner, J . Agr. Sci., 46, 247 (1955). J. M. Bremner, J . Sci. Food Agr., 9, 528 (1958). F. J. Stevenson, Soil Sci. SOC.Am. Proc., 18,373 (1954); 20,201 (1956). (42) F. J. Sowden, Soil Sci., 88, 138 (1959).

(35) (36) (37) (38) (39) (40) (41)

344

MEHTA, DUBACH AND DEUEL

phoric acid4a),ribonucleic acids, and deoxyribonucleic acids.44+46 All of these compounds are present in very small proportion. A rhamnoside has been isolated from an American soil.*O In the fractionat,ion of soil extracts on carbon,24the presence of phenolic glycosides in fraction B (see Section II,2a) has been claimed. Four g. of fraction B per 100 g. of soil organic matter was obtained. The material was very unstable, darkening on standing in the presence of air, until black tars were finally obtained. Ot,her worker^'^-^^ have obtained similar products. However, the evidence available doe8 not unequivocally show the presence of phenolic glycosides.47

111. QUANTITATIVE DETERMINATION Investigations on the influence of the carbohydrates on soil properties, and attempts to determine the composition of soil organic matter, have prompted many workers to determine, quantitatively, the proportion of carbohydrates in soils. As pointed out in the preceding Section, complete extraction of carbohydrates from the soil has not yet been accomplished. Moreover, the isolation of pure polysaccharides from the extracts is tedious and by no means quant,itative. It is, therefore, necessary to have quantitative methods for the determination of total carbohydrates or their individual components. The sugars have mostly been determined in hydrolyzates separated from the soil. It has not yet been found possible to determine either the completeness of hydrolysis or the losses occurring during hydrolysis. The diff ererit stabilities of the glycosidic linkages between the various sugars are not known. In addition, some of the sugars, particularly pentoses and uronic acids, may be partially destroyed during hydrolysis. It is difficult to determine accurately the substances or groups of substances present in such an extremely heterogeneous system as a soil. In no instance has a check, by isolation methods, of the results of the determinations yet been possible. 1. Hexoses

Attempts have been made to use colorimetric methods for t,he determination of the hexoses in soils. The reaction with anthrone has been tried directly on the soil, but was found to give irreproducible results.4* However, this method works with soil hydroly~ates.4~ In addition to what has been (43) R.K.Yoehida, Soil Sci., 80.81 (1940). (44)A. P.Adams, W. V. Bartholomew and F. E. Clark, Soil Sci. SOC.Am. PTOC., 18.40 (1964). (46) G . Anderson, Nature, 180, 287 (1967);Soil Sci., 86, 169 (1968). (40) T.V. Droadova, Pochuouedenie, 1, 83 (1966); Soils and Fertilizers, Commonwealth Bur. Soil Sci., 18, 27 (1966). (47)E.Schlichting, 2.Pflanzenerndhr. DzZng. u. B o d e d . , 61, 97 (1953). (48)Macaulay Znst. Soil Research, Ann. Rept., 186S/M,26. (49) R. H. Brink, P. Dubach and D. L. Lynch, Soil Sci., 88,167 (1960).

CARBOHYDRATES IN THE SOIL

345

said before concerning losses during hydrolysis, mention must be made of the fact that the various hexoses give different extinction coefficients in the anthrone method. The exact composition of the hexose mixture must, therefore, be known in order to permit of a calculation of the hexose content. The hexose content of various American soils, determined by the anthrone method and expressed as glucose, was found to be between 4 and 13 % of the soil organic matter.@-498 Hexoees of Delaware soils have also been determined, by quantitative paper-chromatography of soil hydrolyzates, as amounting to 1 to 2 % of the soil organic matte1-.~6 2. Pentoses

Pentoses have frequently been determined in soils by the furfural-phloroglucinol method.60But phloroglucinol also gives precipitates with a variety of other aldehydes, such as 5-methyl-2-furaldehyde, 5-(hydroxymethyl)-2furaldehyde, and formaldehyde. Orcino161 and aniline acetate62are much more specific reagents, and no aldehyde present in the hydrochloric acid distillate from soils has been found to interfere in the furfural determination by the orcinol method.63 The orcinol and aniline acetate methods give, for various Swiss and Norwegian soils, a “pentose anhydride” content of 0.5 to 8.5 % of the soil organic matter (see Table 111);no corrections were made for the furfural derived from uronic acids. Pentoses have also been determined in the hydrolyzates of soils (1 N sulfuric acid, 1 hour, 120°), after the removal of the uronic acids with anion exchanger, by Bial’s orcinol meth0d.6~8They constituted 3 to 5 % of soil organic matter. 3. Urmic Acids

The Lefbvre-Tollens decarboxylation method for the determination of uronic acids has been applied to s o i l ~ . This ~ ~ J method ~ gives good results with plant material adequately prepared. With soils, however, unbelievably high values for uroiiir acid, up to 40% of the soil organic matter, are ohtained. The decarboxylation method has been shown to be unsuited for the (49a) D. N. Graveland and D. L. Lynch, Soil Sci., 91, 162 (1961). (50)See Ref. 7(a), pp. 138-141, 160-163. (51) A. Johansson, Svensk Papperstidn., 66,820 (1952). (52) G. A. Adams and A. E. Castagne, Can. J . Research, BI. 314 (1948). (53) N. C. Mehta and H. Deuel, 2.PJlaneenernUhr. Dung. u . Bodenk., 90,209 (1960). (53a) R. L. Thomas and D. L. Lynch, Soil Sci., 91,312 (1961). (54) E. C. Shorey and J. B. Martin, J . A m . Chem. SOC.,63,4907 (1930). (55) For a review see: (a) H. Deuel, P. Dubach and R. Bach, 2. PJlanzenerndlhr. Dung. u. Bodenk., 81, 189 (1958). (b) H. Deuel and P. Dubach, i b i d . , 82, 97 (1958). (c) H . Deuel and P. Dubach, Helu. Chim. A d a , 41, 1310 (1958). (d) H. Deuel, P. Dubach and N. C. Mehta, Sci. Proc. Roy. Dublin Soc., 1, 115 (1960).

346

MEHTA, DUBACH AND DEUEL

determination of uroriic acids in soils (as well as in decomposed arid ccrtain fresh plant materials). Only a small part of the evolved carbon dioxide originates from uronic acids. Ready decarboxylation has been found to be a general property of the colored humic substances.66Preparations of humic substances that are free from uronic acids and sugars evolve carbon dioxide, even in a neutral medium a t 70". TJroiiic acids in soil extracts have been determined by the curt)azole method in proportions of 0.07 t o 0.16 % of the total soil.6fiIn another study, the uronic acids constituted 1 to 4 % of the organic matter of the The glycosiduroriic acids arc not completely extracted from the soil, and TABLE I11 Fudural-yieldinu Substances and Ked?tcing Sugars of Various Soils

Organic carbon of soils, of

Furfural-yielding substances as penlose anhydride, % of soil organic mallera

Soils

sod

range

zverage

Reducing sugars as glucose anhydride, Yoof soil organic mallera range

average ~

a

0.5-3.6 0.74.9 2.4-8.5

1.8 2.6 5.4

39 American soih

-

-

6.2-18.0 10

4 Swiss soils 2 Canadian soil8

1.5-3.5 6.6and 10.

2.6

15 Norwegian soils 'I I1 13

<5 <6

> 10

~

G.Ck18.1 12 5.2-21.6 12 1.8-45.2 24

<5 5-10 > 10

38

' I

I1

-

References

-

-

-

-

13 7(~),84, 68 53 52

Soil organic matter = organic carbon of soil X 1.72.

they are partially coprecipit,utJedwith the humic acids3Swhich have to t)c removed heforr the determinat#ion.The carbazolc method, t hercforc, givcs only a minimum value when applied t o extracts. The prohlem of the cstimation of the total uroiiic acids of soils remains to bc solved. 4. Amino Sugars

Although amino sugars in the soil were not detected unambiguously until a decade ag0,~7they are now the best investigated of the carbohydrates of the soil. Various independent, methods have been used for the determination of amino sugars in soil hydrolyzates. The alkaline deamination of amino sugars followed by the determinat,ion of the ammonia liberated,37J!J (56) D.L. Lynch, E. E. Hearns and L. J. Cotnoir, Soil Sci. SOC.Am. Proc., 21, 160 (1957);P.Dubach and D. L. Lynch, Soil Sci., 87,273 (1959).

CARBOHYDRATES IN THE SOIL

347

42,67 the Elson-Morgan colorimetric rnethodl40~67~~ and adaptations of the Moorestein method69 for the fractionation and determination of amiiio compouiids on ion-exchange resins41t41 have heen applied and found to give concordant results. There is also agreement on t,he optimal hydrolysis roilditions, namely, 6 N hydrocshloric acid at 100" for Ci to 9 hours. The values are multiplied by a factor (for example, 1.25) to compensate for losses occurring during hydrolysis. The results reported in the above-mentioned papers show consistently that between 5 and 10% of the organic nitrogen in surface soils consists of amino sugars. In many soils, the percentage increases with the depth of the soil sample removed, approaching 25 % in mature, clay-rich subsoils. I n somc tropical soils, there is no accumulation with d e ~ t h . 6 ~The " ratio of glucosamine to galactosamine varies with the kind of soi140~42*60 from 1.2 to 4.6, the highest ratio having been found in forest soils with acidic litter. If it is assumed that, in surface soils, the ratio of organic matter to nitrogen is 20:1, the content of amino sugar reported above corresponds to roughly 5 % of the organic matter. As the total carbohydrates account for about 5 to 20% of the organic matter, the amino sugars constitute a subs tantial proportion of the carbohydrates.

5. Other Sugars Among the other sugars detected in the soil, only the 6-deoxyhexoses have been quantitatively determined. In Delaware soils, rhamnose and fucose, determined by quantitative paper-chromatography, amounted to 20 % of the Under the conditions of furfural formation from pentoses, t,he 6-deoxyhexoses yield 5-met,hyl-2-furaldehyde;this has been determined by the differential solubilities of the phloroglucides in alcohol.61*6* The proportion of 6-deoxyhexoses in some cases exceeded that of pen toses.

6 . Total Carbohydrates The total carbohydrates of various soils13,63*64 have been determined, after hydrolysis, by measuring the reducing substances by the Bertrand and Hagedorn-Jensen methods. Soil hydrolysates always contain humic substances which reduce Fehling solutions6("),66; they do not,, however, (57) J. M . Brernner and K. Shaw, J . Agr. Sci., 44, 152 (1954). (58) F . J. Stevenson, Soil Sci., 89, 113 (1957); 84,99 (1957). (59) S. Moore and W. H. Stein, J. B i d . Chem., 192, 663 (1951). (59a) 8. Singh and P. K. Singh, J . Indian SOC.Soil Sci., 8 , 125 (1960). (60)F. J. Sowden and K . C. Ivarson, Plant and Soil, 11,249 (1959). (61) E. Michelet and J. Sebelien, Chemiker-Ztg., 30, 356 (1906). (62) R. Balks, Landwirtsch. Vers.-Sta., 103, 221 (1925). (63) For a review, see Ref. 7(a). (64) S. A. Wakeman and I. J. Hutchings, Soil Sci., 40,347 (1935). (65) T. B6res and I. Kiraly, AgrokCmia I s Talajtan, 6,245 (1956).

348

MEHTA, DUBACH AND DEUEL

interfere in the Somogyi method for the determination of reducing sugars.6 Soils with up to 10 % of organic carbon have, according to these methods, a carbohydrate content of about 12% of the soil organic matter (see Table 111).The proportion of carbohydrates seems to increase with the content of organic matter; soils high in organic matter usually contain a high proportion of poorly decomposed plant-material. By analogy with plant analysis, the reducing sugars in soil hydrolyzates obtained with dilute acid were presumed to originate from “hemicelluloses,” and those liberated from the residue (by digestion with 72% sulfuric acid and subsequent hydrolysis with dilute sulfuric acid) were believed to originate67from “cellulose.” Since most of the carbohydrates in aerated soils are probably not plant polysaccharides, these terms are inappropriate. Moreover, part of the so-called “cellulose” might actually be polymers of amino sugarsBB This terminology is, therefore, more applicable to soils containing humus of the more@type (poorly decomposed plant material) than of the mull type (well decomposed organic matter). The carbohydrates in soil hydrolyzates have been measured by quantitative paper-chromatography.96The summation of individual sugars gives, for two Delaware soils examined, a carbohydrate content of 2.3 and 5.6%, respectively, of the soil organic matter. Considering the complexity of the material, accurate determination of the individual sugars is probably the only way in which to estimate the total carbohydrate content of soils. Quantitative column-chromatography has been successfully used for the determination of individual sugars in the hydrolyzate of decomposing fore~t-litter.~~8

IV. SOURCE AND TRANSFORMATION In the soil, a continuous addition, degradation, and synthesis of carbohydrates takes place. A particular sample of soil gives a momentary glimpse into a dynamic (partly cyclic) system which might, except for seasonal variations, be in equilibrium. The relatively constant level of soil organic matter and of carbohydrates therein over a long period of time does not, therefore, reflect a long “life” of the individual carbohydrate molecules. 1. Source a. Plants.-The main primary source of carbohydrates is the added plant material; of this, carbohydrates comprise more than 50% of the dry matter. There is a wide variety of carbohydrates and carbohydrate-containing

(66) Unpublished results of this laboratory. (67) See Ref. 7(a), p. 408. (68) S. A. Waksman and K. R. Stevens, Soil Sci., 26, 113 (1928); SO, 97 (1930). (69) See Ref. 6(b), pp. 40-60. (69a) F. J. Sowden, personal communication.

CARBOHYDRATES IN THE SOIL

340

compounds in plants : mono-, oligo-, and poly-saccharides, glycosides, gallotannins, iiucleic acids, phytin, and so on; among these, polysaccharides predominate. Cellulose makes up the main part of the plant carbohydrates; other polysaccharides are starch, pectic substances, fructans, mannans, and xylans.70 The carbohydrates are incorporated into soils either as dead tissues or as exudates of living roots.71 b. Animals.-Animals are a minor source of carbohydrates for the soil. They may contribute glycogen, mucoids, chitin, nucleic acids, and so on. c. Microorganisms.-It is believed that micro-organisms (bacteria, actinomycetes, fungi, and algae) which decompose the primary plant, and animal material synthesize the major part of soil c a r h o h y d r a t e ~in~ ~ ~ ~ ~ ~ ~ aerated soils (see Section IV, 2b).

2. Transformation The carbohydrates in the soil are transformed mainly by endo- and exoenzymes.72 Most of the enzymes found in the soil are believed to be of microbial origin, the contribution of plant enzymes being small. Considering the presence of a multitude of micro-organisms, it is not surprising that numerous enzymes have been identified in soils-for example, amylase, cellulase, hemicellulase, polygalacturonase, and invertase. In addition to the above-mentioned carbohydrases, the soil must contain other enzymes involved in the transformation and synthesis of carbohydrates. Only phyt a ~ e and 7 ~ glucose oxidaseT4have been detected up to now. During the determination of soil-enzyme activity, the formation of additional enzymes has to be avoided. The effectiveness of toluene is a subject of contr0versy.~6 The enzymes in the soil may be adsorbed on other soil constituents; their activity is, thereby, decreased or increased, as shown by the results of model reactions.76 a. Decomposition of Carbohydrates.-Many studies have been made on the decomposition of total plant-materials and individual carbohydrates in (70) See Ref. 29, p. 1. (71) H. Borner, Botan. Rev., 26, 383 (1980).

(72) F. Richard, Mitt. Schweiz. Anstalt Forstl. Versuchswesen, 24, 297 (1945); H. Sqjrensen, Nature, 176, 74 (1955) ; G. 5. Davtyan, Pochvovedenie, 5.83 (1958) ; I. Kiss, Nature, 182.203 (1968); for citations of papers by E. Hofmann and coworkers, see G . Hoffmann, 2.Pjlanzenerndhr. Dung. u. Bodenk., 86.97 (1959) ; J. Drobnlk, Plant and Soil, 12, 199 (1960); J. Augier and R. Moreau, Ann. inst. Pasteur, 99. 130 (1960); V. Turkovh and M. bogl, Rdstlinnct Vyroba, 6, 1431 (1960). (73) R . H. Jackman and C. A. Black, Soil Sci., 73, 117 (1952). (74) A. S. Galstyan, Izvest. Akad. Nauk Armyan. S . S . R . , Biol. i Sels’skokhoz. Nauki, 12 (No.4), 75 (1959); Chem. Abstracts, 64, 11354 (1980). (75) D. Claus and K. Mechsner, Plant and Soil, 12, 195 (1960); J. Drobnlk, ibid., 14.94 (1961); E. Hofmann and G. Hoffmann, ibid., 14.96 (1961). (76) A. D. McLaren, Soil Sci. SOC.Am. PTOC., 18, 170 (1954).

350

MEHTA, DUBACH AND DEUEL

soils and in compost^.^ Many of the results have been obtained by the acidic hydrolysis (sce Section 111,6) and decarboxylation methods,77 and are thus subject to reservations. The results may be summarized as follows. All plant carbohydrates are more or less rapidly decomposed in the soil, a rough order of increasing stability of some of them being: monosaccharides, oligosaccharides, starch, pectin, mannan, xylan, and cellulose.7(c)-7(d),77s Plant carbohydrates persist in different soils for various periods of time. Relatively less cellulose is found in the lower than in the upper soil horizons. In highmoors, there is an accumulation of cellulose and hemicelluloses. Chitin of fuiigal or animal origin seems to be rather stable. The microbial polysaccharides are not resistant,, although the rate of decomposision is lower than that of some plant polysaccharides.22.78The carbohydrates are decomposed primarily by microbes. Purely chemical degradation is probably only important under special soil conditions, as in acid peats. The carbohydrat)es may be protected against decomposition by lignin,7(*)protein-phenol complexes,79 humic substances, and clays.80 The soil aggregates contain many micro-pores (of diameter less than 1 p ) which are inaccessible to microorganisme8*; polysaccharides situated in such pores would be comparatively immune to degradhon. Extracellular polysaccharides are more likely to be protected by the above mechanism than those polysaccharides which are part of plant and animal tissues. It has been shown that drying of the soil increases the amount of water-soluble mono- and oligo-saccharides.sl* The degradat,ion of carbohydrates leads, directly or indirectly, to various products, including carbon dioxide, organic acids, microbial polysaccharides, and humic substances. It! has often been maintained that carbohydrates are transformed into the dark-colored humic substances by chemical and microbial processes.’ The chemical degradation of carbohydrates, particularly under acidic conditions, produces reductones, furan derivatives, pyruvaldehyde, and so on, which can condense, either among themselves or with amino compounds (Maillard reactions), to produce dark-colored, amorphous products, similar t o humic subst,ances.s2Pyruvaldehyde, which has been held to be an intermediate in Maillard reactions, has been identified in many soils.8sSuch con(77) A. G. Norman and W. V. Bartholomew, Soil Sci. Soc. Am. Proc., 6, 848 (1940). (77a) H . K. Juin and A. K. Bhutt,acharya, 2.Pflanzenerndhr. Dung. u . Bodenk., 91, 233 (1980). (78) J . P. Martin, J . Bacteriol., 60,349 (1945); Soil Sci., 61, 157 (1946). (79) W. R . C. Handley, “Mull und Mor Formation in Relation to Forest Soils,” Her Majesty’s Stationery Ofice, London, 1954. (80) D. L. Lynch and L. J . Cotnoir, Soil Sci. Soc. Am. Proc., 20, 367 (1956). (81) A. D. Itovira and E. L. Graecen, Australian J . Agr. Besearch, 8,659 (1958). (81a) B. Bernier, Lava1 Uniu., Foresl Research Foundation, Contrib. NO.6, (1960). (82) See Ref. 7(d), pp. 120-125. (83) C. Enders and S. Sipirdsson, Biochem. Z., 313,174 (1942).

CARBOHYDRATES IN THE SOIL

35 1

densation reactions may occur in very acid soils, but are certainly of minor importance in the formation of soil humic substances in general. The suggestion that phenolic glycosides are intermediates in humic-substance formationZ4has not been substantiated. According to one theory, the synthesis of humic substances is supposed to be brought about, primarily, by the condensation of the autolysis products from micro-organisms growing on carbohydrates (mostly cellulose) .7 The biosyrithesis of aromatic compounds from ~arbohydrstes8~” may be of importance in the soil. It has been shown by tracer techniques that, when labeled glucose, hemicellulose, or cellulose is allowed to decompose in the soil, the activity is rapidly distributed in all soil organic fractions examined.84If the experiments were to be repeated on better defined fractions, the role of carbohydrates iii humic-substance formation could be considerably clarified. b. Synthesis of Carbohydrates.-Micro-organisms are capable of synthesizing polysaccharides and other carbohydrates, frequently as LZ major metabolic product.86 The polysaccharide formation may be endo- or exo-cellular. Polysaccharides produced by a few soil-bacterial species, in pure cultures, have been intensively investigated.8RNot much information is available about polysaccharides produced by the bulk of soil bacteria,34J8,86a*87 and even less is known about the fungal polysaccharides of soil.88Furthermore, the behavior of micro-organisms in pure cultures under optimum conditions does not indirate how they might behave under natural competitive conditions. It has been found that 5 to 16% of the bacterial species isolated from various British and tropical soils are capable of producing exocellular polysaccharides on synthetic rnedia.T8 A large majority of these produce either levans or glucose-uronic acid p o l y m e r ~ . ~Since ~ ~ ~t8he 7 levan-producing bacteria require a sucrose or raffinose substrate, and since these sugars have not been found in the soil, the contribution of levans to soil polysaccharides is probably negligible.I4 The levan-producing bacteria produce non-levan polysaccharides when grown on monomeric sugars. Two other groups of soil bacteria, present in lower numbers, produce polymers of the glucose-man(83a) See D. B. Sprinson Advances i n Carbohydrate Chem., 16,235 (1960). (84) See J. Mayaudon and P . Simonart, Plant and Soil, 11. 181 (1959) and earlier papers by these authors. (85) (a) T. H. Evans and H . Hibbert, Advances i n Carbohydrate Chem., 2,203 (1946). (b) M. Stacey and S . A. Barker, “Polysaccharides of Microorganisms,” Clarendon .Press, Oxford, 1900. (86) E. A. Cooper, W. D. Daker and M. Stacey, Biochem. J . , S2, 1752 (1938). (86a) D. L. Lynch, Can. J . Microbiol., 6 , 673 (1960). (87) (a) W. G . C. Forsyth and D. M. Webley, J . Gen. Microbiol., 3, 395 (1949). ( b ) Biochem. J . , 44,455 (1949). (88) B . Bernier, Can. J . Microbiol., 4, 195 (1958).

352

MEHTA, DUBACH AND DEUEL

nose-uronic acid and glucose-mannose-rhamnose-uronic acid type, respectively. Some strains of Bacillus megathetiurn produce polysaccharides containing glucose, fructose, mannose, rhamnose, xylose, uronic acids, and, probably, a methylated sugar. Paper chromatography of hydrolyzed, bacterial cultures invariably showed spots corresponding to ribose, probably from ribonucleic acid, and fucose.“ Nucleic acid might also be the source of ribose found in isolated soil polysaccharide preparations. Fucose, which is present in considerable amounts in the soil, is not known to be a common constituent of the higher plants, but it does occur in algal and bacterial polysaccharides in appreciable proportions. Soil bacteria are capable of producing polysaccharides containing all the sugars found in soils, except arabinose and galactose. An intensive searchs4 to isolate, from soil, micro-organisms capable of incorporating these two sugars into polysaccharides failed. Arabinose and galactose do occur, however, in polysaccharides produced by pathogenic bacteria.86(*) The glucosamine found in soil could originate partially from chitin, which is a constitutent of the cell wall of fungi and of the exoskeleton of various invertebrate spe~ies.8~ Part of the glucosamine may be a component of microbial polysaccharides. The origin of galactosamine, which occurs in large amounts in some forest soils, is not clear; however, t,here is an indication that it has a mainly bacterial rigi in.'^-^^ The proportion of glucose and xylose is lower in soils than in and other sugars are proportionally higher. It may be concluded that the major part of the polysaccharides in most aerated soils is, in fact, of microbial origin.20,22 This explains also their extreme heterogeneity. eP4

V. STATEAND FUNCTION The previous Sections have dealt with the chemistry, abundance, and transformation of soil carbohydrates, with little reference to the larger system, soil, in which they occur. The carbohydrates are in intimate contact with other organic and inorganic soil constituents and enter into interactions with them. Such interactions have an influence on the behavior of carbohydrates on the one hand and on soil properties and plant nutrition on the other. 1. Interactions with Other Soil-constitutents

a. Organic Constituents.-A part of the carbohydrates is associated with, and difficult to separate from, other organic substances, such as proteins and humic substances. This circumstance has led to the assumption that there is a covalent bond between the carbohydrate and the other material. Polysaccharide preparations isolated from soils always contain appreciable (89) P. W. Kent and M. W. Whitehouse, “Biochemistry of the Aminosugare,” Butterworths Scientific Publications, London, 1968,p. 92.

CARBOHYDRATES IN THE SOIL

353

amounts of non-carbohydrates. Carbohydrates have also been determined in humic acid preparation^,^^,^^'^^,^^ of which they were found to constitute 2 to 20%. However, by careful purification, sugar-free, humic fractions It seems that carbohave been obtained from some podeol soils.48, 5 6 ( a ) hydrates are linked to other organic constituents by van der Waals, hydrogen, or ionic bonds. b. Inorganic Constituents.-Sugars, nucleic acids, and polysaccharides are adsorbed on mineral s ~ r f a c e s ~ ~ ~ ~and ~ J 'are 2 J 'partially ~ protected from microbial degradation by this a d s ~ r p t i o n , ~Sugars ~ have been detected upon hydrolysis of a naturally occurring, clay-organic complex extracted from an Ohio The adsorption of neutral sugars and polysaccharides is, supposedly, mainly due to hydrogen bonding.le Acidic polysaccharides can form ionic and coordinate bonds with metal cations, either free or in mineral surfaces. Neutral soil-polysaccharides may form complexes with borate.23 Microscopic, electron-microscopic, and histochemical techniques would help in studying the actual state of carbohydrates in soils. sea

2. Function in the Soil

a. ZnfEuence on Physical Properties.-Perhaps the most important role attributed to polysaccharides is their influence on soil structure. Polysaccharides may flocculate2s or deflocculate clay minerals,96 affecting their mobility and distribution in the soil profile. It has repeatedly been shown that long-chain polysaccharides are capable of binding inorganic soil-particles into stable aggregates.12A statistical correlation has also been established between t,he amount of polysaccharides extracted26 from, or determinedge in, soils and their degree of aggregation; such a correlation does not, however, necessarily prove that polysaccharides are the main aggregating agents.g7 The specific destruction of carbohydrates of natural soil-ag(89a) D. E. Coffin, W. A. Delong and B. P. Warkentin, Trans. Intern. Cvnyr. Soil

Sci.,7th Congr., Madison, Wise., in press.

(90) F. Jacquin, Compl. rend., 960, 1892 (1960). (91) F. J. Sowden and H . Deuel, Soil Sci., 91, 44 (1961). (92) D. L. Lynch, L. M. Wright and L. J. Cotnoir, Soil Sci. Soc. A m . Proc., 20, 6 (1966); D. L. Lynch, L. M. Wright, E. E. Hearns and L. J. Cotnoir, Soil Sci., 84, 113 (1967). (93) C. A. I. Goring and W. V. Bartholomew, Soil Sci., 74, 149 (1952). (94) F. J. Stevenson, J. D. Marks, J. E. Varner and W. P. Martin, Soil Sci. Soc. Am. PTOC.,16, 69 (1962). (95) C . Bloomfield, Tram. Intern. Congr. Soil Sci., 6th Congr., Paris, B, 27 (1956). (96) J. A. Toogood and D. L. Lynch, Can. J . Soil Sci., 39,151 (1959); A . Kullmann and K. Koepke, Z. Pjlanzenerndhr. Dilng. u. Bodenk., 93.97 (1961). (97) N . C. Mehta, H. Streuli, M. Milller and H. Deuel, J . Sci. Food Agr., 11, 40 (1960).

354

MEHTA, DUBACH AND DEUEL

gregates by treatment with periodate, hot acid, and so on, failed to affect the stability of aggregates, whereas artificial aggregates, prepared with various polysaccharides, were destroyed by these treatment^.^' It was concluded that polysaccharides do not contribute essentially to the aggregation of the Swiss soils studied. b. Influence on Chemical Processes.-Carbohydrates may inhibit the precipitation of iron and aluminum by p h o ~ p h a t e and , ~ ~ favor the leaching of sesquioxides from the upper to lower horizons in some soils.QQ Bacteria isolated from soils can produce ~-arabino-2-hexulosonicacid (“2-ketogluconir arid”) , a chelatirig agent.loOChelating carbohydrates can accelerate the weathering of minerals. c. Influence on Microbial Activity.-Carbohydrates, particularly those from fresh plant-material, are a readily available source of carbon and energy for micro-organisms, and, consequently, they control to a great extent the microbial activity in soils. The decomposing carbohydrates are helieved by many workers to be the ultimate source of humic ~ubstances.~ On the other hand, the microbial decomposition of humic substances is accelerated in the presence of carbohydrates.101 d. Influence on Plant Nutrition.-Monomeric sugars can be absorbed and utilized by plants. Sugars niay stimulate seed germination and root elongation.1n2Soil rarbohydrates may have many different, indirect effects on plant nutrition. For instance, mineralization of phytin and nucleic acids supplies phosphorus to the plant. The carbohydrates may keep phosphate in u readily convertible form and prevent it from forming irisoluhle precipitates with calrium, iron, or aluminum. VI. SUMMARY A part of the carbohydrates of various soils has been isolated, purified, and shown to consist of polysaccharides composed of many sugars. Fructionation and characterization of these preparations showed the extreme heterogeneity of the polysuccharides, confirming their predomiiiant ly niicrobial origin. The quantitative determinations, although not always satisfactory, have shown the carbohydrates to constitute about 10% of soil organic matter. The major immediate problem is development of better methods for the determination of t,he total carbohydrates and their individual monosac(98)D. B. Bradley and D. H. Sieling, Soil Sci., 76, 175 (1953). (99) M. Schnitzer and W. A. DeLong, Soil Sci. SOC.A m . Proc., 19, 363 (1955). (100) R. B . Duff and D . M. Webley, C‘hem. & Ind. (London), 1376 (1959). (101) H. Thiele and G . Andersen, Zentr. Bakleriol. Parasilenk., Abt. 11, 107, 247 (1963). (102) R. Brown, A . W. Johnson, E. Robinson and A . R. Todd, Proc. Roy. SOC. (London), B136, 1 (1949); R . Brown and E. Robinson, ibid., B136, 577 (1950).

CARBOHYDRATES IN T H E SOIL

355

charide components. This is a prerequisite to (a) a more intensive study of the role and transformation of carbohydrates in the soil and (b) the elucidation of the nature and abundance of the remaining, possibly non-polysaccharide, part of soil carbohydrates. The study of the amount and kind of carbohydrates in different soils is of pedological interest.

Acknowledgment We wish to thank the Schweizerischer Nationalfonds zur Forderung wissenschajtlicher Forschung for financial support.

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Author Index for Volume 16 Footnote and reference numbers are given in parentheses for pages on which an author’s work is cited by this number only, without mention of his name.

A Abdel-Akher, M., 111(19), 117(19) 124, (19), 140(115), 149(115) Abelson, P. H., 335(1) Abraham, S., 66(28) Abstrom, G., 37(141), 38(141) Adachi, S., 190(254), 194(302) Adam, A., 261(72) Adams, A. P., 344(44) Adams, G. A., 345(52), 346(52) Adams, M., 291(125), 292(125) Adams, P. T., 32(108), 54(108) Adams, R., 236 Adkins, H., 188(237) Aebi, A., 209(16) Akiya, S., 125(63), 135(98), 136(98), 137 (98) Alberda van Ekenstein, W., 163, 192 (273) Albon, N., 294(147) Alders, N., 176(116) Aleksandrova, I. V., 338(15a) Alexander, B. H., 141(125), 153(196) Alexander, F., 178(142) Alimova, E. L., 210(21), 233(21) Allen, A. O., 17, 19(29), 20(42), 20(41), 22 (48) Allen, J. T., 25(67) Allen, 0. N., 339(26, 27), 340(26, 27), 353 (26) Allen, P. J., 54(164a) Alvsaker, E., 337(13), 338(15), 346(13), 347(13) Ames, 8. R., 186(214) Anagnostopoulos, C., 291(119), 294(141, 145, 148), 295(148) Andersen, G., 351(101) Anderson, A. G., 205(427) Anderson, E. P., 180(154) Anderson, G., 344(45)

Anderson, R. J., 208(3, 7, 8), 209, 211, 220(7), 225(8) Andrews, P., 173(85) Anthoni, B., 151(187), 152(187) Appel, H., 292(138) Archambault , A., 294 (143) Ard, W. B., Jr., 32(109) Arens, A,, 252(42, 43), 253(42), 266(43) Armstrong, E. F., 178(134), 197(331), 202 (371) Arnaud, L. E., 234(116) Arnaud, M., 292(135), 295(135), 296(135), 297 (135), 298(135) Aronson, M., 179(152), 255(55) Arthur, J. C., 34(130) Asselineau, J., 208(1, 2), 209(16), 210(20. 28,31), 211(34, 35,36), 212(2, 38), 218 (2, 34, 35, 36), 219(1), 220(1, 52), 230 (36, 89), 234(118) Attoe, 0. J., 339(27), 340(27) Augier, J., 349(72) Aures, D., 96(40), 102(40)

B Baar, S., 173(89) Bach, R., 338(25), 339(25), 342(25), 345 (55), 346(55), 353(25, 55a) Bacharach, A. L., 204(401) Bacon, J. S. D., = ( M a ) , 171(69, 70), 176 (114) Baddiley, J., lll(21) Baer, H. H., 118, 126(64), 167(39), 168(39, 50, 51b), 169(55, 56, 58, 59), 170(60, 61) Bauerlein, K., 88(17), 95(17), 98(17), 99 (17) Baker, N., 55(165) Baker, P. J., Jr., 124(59), 126(59) Balaas, E. A., 38(142), 39(142), 53(142) B a h t , M., 292(136)

357

358

AUTHOR INDEX, VOLUME

Balks, R., 347(62) Ballio, A., 255(60) Ballou, C. E., 227(77) Ballun, A. T., 188(231) Bamdas, E. M., 192(272) Bamford, C. H., 319(32) Bandel, D., 158(216) Barbier, M., 209(17), 218(49), 230(83) Barker, S. A., 32(106), 43(106), 72(49, 50), 74(49, M)), 351(85) Barnard, E. A., 289(98) Baron, E. S. G., 27(85) Rarr, N. F., 19(36), 22(50), 30(102), 46 (102) Barrenscheen, H. K., 176(116) Barrette, J. I)., 93(31) Barry, J. M., 174(100, 101) Barry, V. C., 106(9), 135(95), 139(110,111, 114), 140(9, 95), 142(95), 144(9, 95, 138, 139), 145(9, 138), 152(95, 191), 153(9, 95, 139), 158(219, 220, 221), 190 (257) Barth, I,., 182(189), 188(238) Burtholomew, W. V., 344(44), 350(77), 353(93) Bartling, I)., 97(47), lOO(47) Bartolettus, P., 159 Bates, F. L., 301(7), 312(7) Battenberg, E., 151(185) Battley, E . H., 291(129), 292(129), 293 (129), 298(129) Bau, A., 291(123) Bauer, A. W., 305, 326(16, 46, 47) B u m , G., 90(23), 91(25), 92(23), 96(23), 97(41, 48), 98(23), 102(23, 48) 13aum, H., 306(19) Bauman, H., 254(51) Baxendale, J. H., 16, 21(27) Baxter, C. F., 174(97, 98), 175(98) Bayly, R. J., 55(167), 57(167) Bnyne, S., 192(269) Bazin, S., 231(94) Bean, It. C., 180(163) Beck, D., 256(64), 262(64) Becker, J. P., 32(112) Bbguin, C., 257(68) Bbhal, A., 67 Beisenherz, G., 244(35) Bell, D. J., 167(41) Bell, M., 167(37) Bellamy, L. J., 124(61)

16

Bellamy, W. D., 33(115, 117), 35(117), 38 (117) Bender, H., 241(27), 242(27), 246(27), 249 (n), 250(27), 251(27), 252(27), 254 (27), 255(27), 264(27) Benedict, F. G., 205(421) Bennett, E. C., 32(108), 54(108) Bennett, W.,19(39) Bennewitz, K., 205(429) Bentler, M., 96(38), 102(38) Bentley, R., 183(196) Beres, T., 347(65) Bergeim, F. H., 198(343) Bergmann, E. I)., 61(10) Bergmann, M., 200(356, 358), 201(363, 364) Bernfeld, P., 299(1) Bernier, B., 338(19), 339(19), 341(19), 343 (19), 350(81a), 351(88) Bernstock, L., 235(123) Bernt, E., 179(151, 152), 249(39), 255(39, 56), 262(39, 56) Berthelot, M., 201 Bertho, A., 96(36, 37, 38, 39, 40), 102(36, 38, 39, 40), 103(37) Bhattacharya, A. K., 350(77a) Bickel, H., 173(86) Bieder, A., 184(208) Bierry, H., 178(138), 179(138, la),291 (113) Bigler, F., 225(64) Bildstein, S., 275(83a) Knkley, W.W., 36(139), 38(139), 47(139), 49(139), 50, 51(157, 159), 52(139), 53(139) Birkofer, I,., 193(286) Bister, W., 183(203) BlachBre, H., 180(162) Black, A. L., 174(97, 98), 175(98) Black, C. A., 349(73) Blanchard, P. H., 294(147) Blanchfield, E., 130(77) Bloch, H., 208(2), 210(18, 27, 28, 30), 212 (2), 218(2, 18, 30), 231, 232(97), 237 (132, 133) Bloch, K., 225 Block, R. J., 181(168), 196(168) Bloom, B., 180(156) Bloomfield, C., 363(95) Blouin, F. A., 34(130) Boas, N. F., 253

AUTHOR INDEX, VOLUME

Bobbitt, J. M., 106(5), 153(5) Boeseken, J., 162(23), 203(387) Borner, H., 349(71) Bogoslovski, B. M., 148(164) Boltze, H. J., 244(35) Bonner, T. G., 78(55) Bonner, W. A., 130(76) Boquet, A., 233, 237 Borrmann, D., 90(24), 91(24), 92(24), 99 (24) Boser, H., 254(46) Bosse, R., 297(151) Bothner-By, C. T., 38(142), 39(142), 53 (142) Bouchardat, G., 173(90), 187(224) Bourjau, W., 194(290) Bourne, E. J., 34(132), 39, 57(168), 59(2), 60(4,7), 61(8), 62(7, 17), 63(7, 22,23), 64(23), 66(26), 67(4, 31), 68(32, 34, 36), 69(37, 38), 70(47, 48), 72(49, 50), 73(47), 74(48,49,50), 76(48), 77(4, 48), 78(55), 79(38, 56), 80(32, 34), 81(32), 82(8), 83(32, 36, 48), 300(6), 301(6) Bourquelot, E., 240(1), 257(65, 66) Boyden, S. V., 234, 237 Boyer, P. D., 177(128, 1%), 273(82), 274 Boyle, J. W., 19(35) Bradley, D. B., 354(98) Bradley, H. C., 241(16) Braganca, B., 177(122), 180(122) Bragg, P. D., 188(233) Brasch, A., 33(119) Brasher, P. H., 178(142) Braun, G. A., 171(65, 66) Brauns, I). H., 86(3, 4, 5, 6, 7, 8, 9, lo), 87(3), 97(51), 98(3, 4, 5, 6, 7, 9), 99 (3J 6, 8, 51) Bredereck, H., 97(44), 98(44), 196(320), 215, 216 Bremner, J. M., 336(9), 343(37, 38, 39, 40), 346(37, 39), 347(40, 57) Bridel, M., 257(65, 67, 68) Briggs, L. H., 137(105) Brigl, P., 89 Brink, R. H., 344(49), 345(49), 353(49) Broadbent, F. E., 336(8) BrocherB-FerrBol, G., 215(42)* Brock, H. J., 172(83) Brookes, N. E., 33(128) Brossmer, R., 171(75), 172(75, 77)

* See also Ferdol.

G.

359

16

Brown, D. M., 69(39), 156(207, 208) Brown, F. C., 202(373), 205(373) Brown, K. R., 188(230) Brown, L., 80(57) Brown, M. A., 174(97) Brown, R., 354(102) Bryant, M. P., 33(115) Bubl, E. C., 186(214) Buc, H., 208(1), 219(1), 220(1) Bucek, W., 180(164) Buchanan, J. C., 111(21) Budovich, T., 179(152) Bucher, T., 244(35) Buchi, J., 187(227) Buell, R., 194(298) Bull, J . P., 173(89) Bungenberg de Jong, H. G., 324(39) Burchfield, H., 289(97) Burdon, J., 70(47, 48), 73(47), 74(48), 76 (48), 77(48), 83(48) Burke, W. J., 188(230) Burr, J. G., 23(55), 24(60), 54(60) Burton, M., 14(13, 14), 15 Burton, R. M., 177(123), 180(123) BUR,W. C., 301(8), 310(24), 314(30), 328 (30), 329(49, 50, 51), 332(53) Butler, G. C., 27(84) Butler, J. A. V., 26(70), 27(79,82), 28(92) But,ler, K., 64(24, 25) C

Cadotte, J. E., llO(17, 18), 111(19), 117 (19), 118(17, 18), 122(18), 124(19) Cajori, F. A., 240(13) Caldwell, C. C., 142(130), 144(130) Calvin, M., 32(108), 54(108), 63(21) Cameron, A. T., 13(2), Cantley, M., 186(215) Cantor, S. M., 327, 332 Caputo, A., 33(126) Caputto, R., 171(67), 172(68), 174(67), 176(67, 118, 119), 179(118, 119), 180 (158) Cardini, C. E., 176(118, 119), 179(118, 119) Carleton, F. J., 175(106), 178(144a) Carne, H. R., 233 Carson, J. F., 130(74) Carter, H. E., 109(13), 227 Castagne, A. E., 345(52), 346(52) Catel, W., 234(107)

360

AUTHOR INDEX, VOLUME

Cattaneo, C., 293(146) Cavill, G. W. K., 133(89) Cecil, R.,275(82c) Chadka, M. S., 133(90) Chaikoff, I. L., 66(28) Chang, R. P., 156(205,205a) Chanley, J. D., 230(86) Chapman, R.A., 194(297) Chargaff, E., 227 Charlesby, A., 20(43), 34(129) Charlson,A. J., 137(101),164(34),186(223) Charlton, W., 161(20) Chaucery, H., 241(17) Chaudhuri, S. N., 231(92) Chesters, G., 339(27), 340(27) Choi, R. P., 203(383) Choucroun, N., 220(52), 231, 234(105, l06), 237 Choudhury, A. K., 178(142) Christie, S. M. H., 69(40) Clapp, C. E., 338(23), 339(23), 340(23), 341(23), 342(23), 343(23), 3fi3(23) Clark, F. E., 344(44) Clark, G. L., 50(154, 155) Clark, R. F., el(l2) Clark, R. K., Jr., 109(13) Clarke, T. H., 205(423) Claus, D., 349(75) Cleaver, A. J., 130(78), 131(78) Cleveland, E. A., 193(281) Cleveland, J. H., 193(281) Clowes, H. A., 182(186) Coffin, D. E., 353(89a) Cohen-Bazire, G., 256(63), 260(63), 261 ( W , 280(63) Cohn, M., 240(11), 241(20), 249(20), 264, 256(63), 260(11, 63), 261(63), 264 (20), 269(20), 280(63), 281(11), 280 (102, 104) Cohn, W. E., 128(6X) Colbran, R. L., 113(24), 115(24), 121(24), 148(162) Coleby, B., 52, 54(160) Coleman, G. H., 196(324) Coles, H. W., 198(342, 343) Collinson, E., 13(8, 9) Colover, J., 236 Colwell, H. A,, 34(133) Conalty, M. L., 158(219) Conchie, J., 178(144), 291(116, 118), 298 (115)

16

Connolly, J. M., 235(122) Connors, W. M., 180(164) Conway, B. E., 26(74, 75), 27(79, 83), 28 (92) Conway, H. F., 141(118, 119), 157(118, 119) Cook, B. B., 194(298) Coons, A. H., 235(122) Cooper, C. D., 231(92) Cooper, E. A., 351(80) Coover, H. W., 61(11) Corbett, W.M., 153(196a), 189(248) Corbure, A,, 27(85) Cornalba, G., 160(9) Cort, W. M., lSO(164) Coss, J. A,, 33(127) Cotnoir, L. J., 346(56), 350(80), 353(80, 92) Coulthard, C. E., 230(87) Courtois, J. E., 127(65), 134(91), 135(65), 136(65, 99), 137(102, 104, l06), 184 (206, 207, 208), 190(280), 280(110), 291(119), 292(134), 293(134, 144), 294 (134, 141, 143, 144, 145, 148), 295(134, 148, 149), 296(134), %7(134) Craine, E. M., 180(156) Crater, W. de C., 196(325) Creaser, E. H., 178(149) Creighton, M. M., 208(8), 225(8, 66) Creitz, E. C., 206(420) Criddle, W. J., 38(144), 39(144, 149), 43 (150), 44, 45(151), 49(149, 151) Criegee, R.,132(86), 186(221) Crocker, B. F., 179(147), 291(130, 131, 132), 298(130, 131, 132) Crouch, D. H., 193(281) Crovisier, C., 24(63) Crowle, A. J., 237 Crowther, J. A., 26(76) Cuisinier, L., 163(29), 188(29) Cummine, C. S., 220(53), 235(53, 124) Curme, G. O., Jr., 189(355) Curtis, E. J. C., 165 Cutolo, E., 177(122), 180(122) Czok, R., 244(35)

D Dabic, S.,141(119), 157(119) Dabioh, D., 242(31), 249(31), 260(78), 264(31),

AUTHOR INDEX, VOLUME

265(31), 266(31), 267(31), 268(31), 269(31), 270(31), 271(31), 272(31), 274 (78), 281(78) Dainton, F. S., 14(10), 16(16), 19 Daker, W. D., 351(86) Dames, C. A., 205(428) Daniels, M., 19(38), 26(77, 78), 27, 28(90, 91) Darby, F., Jr., 148(167), 156(167) Dauben, W. G., 54(164) Davidson, G. F., 139(109), 146(109, 149), 147(109), 150(176), 151, 153(109, 149, 197, 198), 156 Davidson, H. M., 171(73), 174(73) Davoll, J., 128(66) Davtyan, G. S., 349(72) Dean, A. C. R., 179(147) Debackere, M., 176(112) de Barjac, H., 336(7c), 338(7c), 350(7c), 351(7) Debierne, A., 13(3) Deere, C. J., 240(7) Defaye, J., 210(18), 218(18) Dehn, W. H., 194(289) Delaunay, A., 230, 231(94) De Ley, J., 179(1&3) Delong, W. A., 353(89a), 354(99) Deluca, C., 69(42) Demant, S., 297(151) Dcmarteau-Ginsburg, H., 218, 219 De Ment, J. D., 337(12), 353(12) Demint, R. J., 34(130) Dennerline, R. L., 237(135) de Robichon-Szulmajster, H., 180(156, 160) de Suto-Nagy, G. I., 225(67) Deuel, H., 335(4), 338(25), 339(25), 341 30,31,32), 342(25,31), 343(31), 345(53, 55), 346(53, 55), 347(55c), 353(25, 55a, 91, 97), 354(97) Devillers, P., 194(301) Dewhurst, H. A., 16(22) Diarn, A., 217,233 Dickey, J. B., 61(11) Dickinson, L., 230(87) Dickmnn, S. R., 109(13) Diels, O., 191(264) Dillon, T., 130(77), 144(137) Dimant, E., 174(102) Ditmar, R., 197(337)

16

36 1

Dizet, P. L., 184(207), 190(260), 293(144), 294(143, 144) Dodds, M. L., 198(342,343) Donlan, C. P., 33(127) Donnison, G. H., 300(6), 301(6) Doudoroff, M., 179(153) Douglas, H. W., 206(445) Dowdall, J. F., gO(5) Dragstedt, L. R., 178(142) Drisko, R. W., 130(76) Drobnlk, J., 349(72,75) Drozdova, T. V., 344(46) Duane, W., 13(6) Dubach, P., 338(25), 339(25), 342(25), 344(49), 345(49, 55), 346(55, 56), 347 (55c), 353(25, 49, 55a) Dubos, R . J., 210, 218, 237(131) Dubourg, J., 194(301) Dubrunfaut, A . P., 181(175),201(175,366) Durr, H., 196(320) Duff, R. B., 338(20), 339(20), 340(20), 342(20), 349(20), 352(20), 354(100) Dukes, C. D., 234(116) Dulaney, A. D., 240(7) Duncan, W. A. M., 178(146) Du Pr6, E. F., 146(153), 147(153) Dutcher, J. D., llO(16) Dutton, G. G. S., llO(17, 18), 118(17, 18), 122(18) Dvonch, W., 141(116, 120), 157(116, 120), 304(12) Dwight, C. H., 33(121, 123) Dyer, J. R., 106(4), 186(219)

E Edsall, J. T., 265,266(76), 289 Egan, M. M., 198(349) Ehrenthal, I., 152(192) Ehrenberg, L., 22(51), 35(136) Ehrenstein, G., 36(141), 38(141) Eiehenberger, E., 233(99) Eistert, B., 148(169) Elberg, S., 230(85) Ellis, G. P., 194(294a) Elmore, D. T., 69(40) Emery, A. G., 205(421) Emery, A. R., 82(62) Emmons, W. D., 81(60, 61) Enders, C., 350(83) Engler, J., 200(358)

362

AUTHOR INDEX, VOLUME 16

Erdmann, E. O., 160,202(368) Erickson, J. G., 193(284) Ermolaev, K. M.,192(272) Ermolenko, I. N., 146(152), 147(152) Ettmiiller, M.,160 Evans, T.H.,351(85), 352(85a) Eyring, H.,15

Flory, P. J., 322(34),324 Flowers, R.,84(68) Flynn, F. V., 172(79) Foldes, J., 222 Folley, S. J., 173(93), 175(110), 177(130, 131) Fonteyn, F., 305(15) Ford, D. L.,133(89) F Forney, J. E., 234(116), 235 Fairbanks, €3. W., 203(383) Forsyth, W.G. C., 337(14), 338(24), 339 Faure, M.,225, 227 (24), 341(24), 342(34), 344(24), 349 Fein, M.L.,157(212,213,214) (24), 351(14, 24, 34, 87), 352(24, 34, Feingold, D.S., 292(133) 87),353(14) Fellig, J., 254(45) Forziatti, F. H., 111(20), 146(20) Fenner, I. V., 62(14) Foster, A. B.,130(78), 131(78) Fenetein, R.N.,33(122) Fraenkel-Conrat, J.,194(298) Ferebee, H., 237(132) Franchimont, A. P.N., 193(278) FerrBol, G., 214(39), 215(39), 217(39)* French, D.,290(108, log), 293(108), 297 Ferris, A. F., 81(60,61) (108), 301(7), 302(10), 312(7) Fieser, L. F., 190(261) French, T. H., 174(99) Fieser, M., 190(26l) Freudenberg, K.,198(346) Filachione, E.M.,157(212, 213, 214) Freund, J., 235, 236 Filler, R.,62(14) Fricke, H.,18(30), 21, 24(62) Finan, P. A., 139(112, 113) Fried, M.,156(207,208) Findlay, A.,318(31) Friedenwald, J. S.,267 Finnegan, M.,190(258) Frier, R., 193(280) Fioroni, W.,205(422) Froschl, N.,194(293), 198(293) Fischer, E., 160, 161(17, 18), 178(134, Frohlich, H.,16, 21(26), 25(26) 141), 182(17), 190(255), 190, 192(16, Frush, H.L.,86(9), 98(9), 183(199), 205 270), 195(307), 196(318), 197(318, 331), 198(318),199(354,355),291(124) Fry,(420) E. M., 154(199) Fischer, E. H., 254(45) Fudakowski, H., 160(13) Fischer, H.,196(318), 197(318), 198(318) Furman, N.H., 132(82) Fischer, H.0.L., 118(48a), 126(64) Furtsch, E.F., 205(426) Fischer, J., 241(15), 246(15), 247(15), 248 (15), 249(15), 252(15), 253(15), 154 G (15), 255(15), 258(15), 257(15), 258 (15), 259(15), 260(15, 78), 262(15), Gabryelski, W., 205(416) 274(78),281(78),282(15), 283(15), 284 Gaffney, E.E., 158(219) Caines, S.,236(130) (15),285(15) Gakhokidze, A. M.,162(28), 200(359) Fischer, R., 205(410) Galstyan, A. S.,349(74) Fischler, F., 188(244) Gander, J. E.,177(128, 129) Fitch, K. R.,50(154) Gantner, G. S.,324(37) Fitzsimmons, R.V.,144(135) Garbade, K.H., 244(35) Flanders, T.,l67(44) Garcfa, Gonetilez, F., 121(49,50,51) Fleiachmann, W.,205(432) Garrison, W. M.,19(39), 29(99, 101) Fletcher, H.G., Jr., 199(351) Gasser, E., 160(8) Fleury, P.F., 134(91), 184(208) Gatin-Gruzewska, Z., 306(17), 311 Flipse, R. J., 194(289) Flitsch, R.,87(14), 88(14), 89(14), 92(14, Gauhe, A., 167(39), 168(39, 47, 50, 51b), 169(54, 55, 56, 59), 170(60, 61) 27), 98(27), 99(14) Gauthier, B., 195(313) * See also Brooherd-FerrBol. G.

AUTHOR INDEX, VOLUME

GB, G., 196(326) Geddes, A. L., 62(15) Geiger, R., 62(18, 19) Geisel, F., 13(1) Gendre, T., 215(40), 225(64), 229(82) Gerstl, B., 230(88) Geusic, J. E., 37(141), 38(141) Ghormley, J. A , , 19(35), 22 Giaja, J., 178(138), 179(138) Gibbons, A. P., 55(165) Gilbert, G. A., 306(19) Gillespie, R. J., 84(65, 66, 68), 174(105), 175(109) Gillis, J., 203(384, 385, 389), 204(389) Ginsburg, A . , 211 (37) Cladding, E. K., 142(128, 132), 147(128) Glegg, R. E., 34(131) Clock, G. E., 176(113) Glockler, V., 62(19) Glover, W. H., 204(399) Gnuchtel, A., 95(32, 35), lOl(32) Goebel, W. F., 182(181) Goepp, R. M., Jr., 122(56), 123(56) Goerdeler, J., 254(50), 297(151) Gold, V., 82(62) Goldstein, I. J., 107(11), 108(12), llO(11, 17, 18), lll(12, 23), 112(12), 115(23, 29), 116(34), 118(12, 17, 18, 29), 122 (12, 18, 29, 34, 54, 55), 124(62), 126 (29, 34), 127(54), 143(29), 145(147), 149(172), 150(29) Goodwin, W., 196(323) Gootz, R., 87(13), 99(13), 197(336), 290 ( l l l ) , 292(111) Gordon, K. M., 196(322) Gordon, S., 20(42), 24 Gordy, W . . 32(109) Gore, P. H., 83(64) Gorin, P. A. J., 131(79) Goring, C. A . I., 353(93) Gottscbalk, A., 172(76) Could, S. P., 205(431), 206(439) Goulden, J. D. S., 205(418) Graecen, E. I,., 350(81) Grafe, K., 201(364) Granath, K. A., 33(117), 35(117), 38(117) Grangaard, D. H., 142(132, 133), 143(133) Grant, G . A., 174 Grant, 1’. M., 30(103), 31(104, 105), 32 (104, 106), 38(146), 43(106), 45(104, 105), 53(146), 54(146)

16

363

Graveland, D. N., 345(49a), 346(49a) Gray, L. H., 17 Green, R., 231(92) Greenbaum, A. L., 177(131) Greenland, D. J., 338(16), 353(16) Greenstein, J. P., 26(71, 72, 73) Greenwood, C. T., 300(4) Grein, L., 241(24) Greville, G. D., 228 Griebel, R., 198 Griffin, E. L., Jr., 141(119), 157(119) Griffin, H. L., 141(124) Grinnan, E. I,., 27(80) Grove, J. F., 132(80) Grubin, A. F., 33(127) Griinler, S., 95(35), 254(47) Gunther, E., 290(111), 292(111) Gugliemelli, L. A , , 144(135) Guinn, V. P., 54 Guthrie, R. D., 107(10), lll(10, 22), 112 (10, 22), 113(10, 25, 27a), 115(10, 24), 121(24, 25), 126(22) Gyorgy, P., 167(41, 44, 45, 46), 168(47), 171(62,65,66) €I Haas, H., 151(185) Haber, F., 49(152) Hagedorn, A., 198(340) Haissinsky, M., 16(20) Hales, R. A., 188(235) Hall, E., 33(115) Hall, J. J., 33(128) Hall, R. H., 133(87) Halsall, T. G., 186(220) Hamada, M., 152(190) Hamilton, J. K., 117(3X), 122(54), 134 (38), 127(54), 136(100), 140(115), 145 (146, 147), 149(115, 172) Hamilton, T. S., 178(141) Hanahan, D. J., 227 Handley, W. R. C., 350(79) Hann, R. M., 70(44, 45, 46), 74(46), 75 (44), 76(53, 5 4 ) , 109(15), 111(15), 118 (43, 44), 164, 165(35), 166(35) 167, 188(230), 195, 199(350) Hansen, It. G . , 174(105), 175(109), 176 (120), 180(120, 156) Hardegger, E., 119(47), 133(47), 184(205) 191 Hardenbrook, H., 174(105), 175(109)

364

AUTHOR INDEX, VOLUME 16

Hess, W. C., 172(8)0 Harloff, J . C ., 199(352) Hestrin, S.,292(133) Harnarda, M., 152(190) Heweili, 2.E.,195(311) Harper, C.,172(79) Harrell, W.K.,223 Heyes, T.F.,148(163a) Heyworth, R.,171(69,70),176(114) Harris, E. H.,157(213) Hibbert, H.,351(85), 352(85a) Harris, H., 220(53), 235(53) Hickmans, E.M.,173(86) Harris, M.,150(177), 156(177) Hart, E.J., 16(17,18), 19(34), 20(34, 42), Hiemstra, P.,310(24), 314(30), 328(30), 329(49, 50, 51), 332(53) 22(47,49),24(62), 29(100) HigginB, H. G., 146(154), 147 Hartree, E.F.,241(19) Hilbert, G.E.,300(5) Haskins, J. F.,154 Haskins, W.T.,76(53), 164, 165(35), 166 Hill, A. E.,305(14) Hill, K.,240(3), 290(3), 291(3), 297(3), (351,167 298(3) Hassid, W. Z., 180(163) Hiller, A., 193(280) Hatori, S., 291(121) Hiltmann, R.,254(49) Hauptschein, M.,62(14) Hinshelwood, C.,179(147) Haworth, J. C.,173(86, 87) Haworth, W. N., 161(20), 162(,26),190 Hirshfelder, J. O., 15 (256), 200(357), ZOl(357, 361), 222, Hirst, E.L.,161(20), 182(185), 186(220), u)o(367), 201(357) 300(6), 301, 338(18), 339(18) Hixon, R. M., 142(130), 144(130) Hawthorne, J. N., 227 Hlasiwetz, H., 182(191, 192), 188(238) Hawthorne, J. R.,182(184) Hlasiweta, J. H., 182(189) Hay, A. J., 178(144) Hobkirk, R., 311(28) Hayaishi, O.,180(165) Hochanadel, C.J., 19(35), 22 Hayes, J. E.,Jr., 281(88) Hockett, R.C., 122(56), 123(56), 135(94), Haynes, L. J., 85(1), 88(1) 203(377) Hayon, E.M., 25(67) Head, F. S. H., 146, 148(168), 149(171), Hoeckner, E., 291(128) 152(168), 154, 166(168, 171), 184(210) Hoffmann, G.,349(72,75) Hofmann, E., 178(136, 145), 179(150), Hearns, E.E.,346(56), 353(92) 193(282), 240(2, 6), 291(6, 126), 297 Hedgley, E.J., 130(78), 131(78) (126),349(75) Hefti, H. R.,188(236) Hofmeister, F.,172(84) Heilskov, N. S.C., 241(14) Hofreiter, B. T.,141(122, 125), 143(122), Heimer, R.,172(78) 144(122), 145(143, 144), 150(180a) Helferich, B.,87(13), 88(17), 95(17, 32, 35), 97(44, 49, 5 0 ) , 98(17, 44, SO), 99 Hogness, D. S., 291(129), 292(129), 293 (129),298(129, 154) (13,17), 101(32,49),168(49), 178(135, 145), 179(150),192(274), 193(276,287), Hogshead, M. J., 154 197(336), 198(344, 345), 240(4), 254 Hollaender, A., 26(71, 72, 73) (47,48,49,50), 290(111), 291(4, 127), Hollo, J., 324(37) Holmes, R.,291(131), 298(131) 292(111,138),297(4,151) Holthaus, G., 92(28),99(28) Helwig, E. L., 183(198) Holty, J. G.,204(405) Henderson, G.M., 205(409) Holta, P.,32(112, 113) Henry, 8.H., 62(17) Holysz, R. P.,124(59), 126(59) Herberman, J., 182(192) HBrissey, H.,137(104, l06), 240(1), 257 Honeyman, J., 107(10), 111(10,22), 112 (10,22), 113(10, 25), 115(10,30), 121 (66) (25), 126(22) Herold, F., 163(31), 188(31) Herrington, B.I,., 203(376,379,382,393), Hoover, J. R. E., 168(47), 171(62,65) Hoppe-Seyler, F.,188(239) 204(393,400,403) Horibata, K.,290(102) Hess, D.N.,24(60), 54(60)

AUTHOR INDEX, VOLUME

Hormann, O., 195(316) Hornemann, H., "176) Horowitz, M. G.,171(73),174(73) Hough, L.,131(79), 164(33), 173(85), 181 (171), 182(185), 186(213, 215, 216, 217), 188(213, 233, 234), 190(253), 190 (33),195(309), 228 Howe, B. K., 133(87) Hoyt, A , , 237 Hu, A. R. I,., 242(32), 244(32), 249(32), 250(32), 251 (32) Huang, J. S.,234(116) Hubbard, R.S.,172(83) Huber, W.,33(119) Hudson, C.S.,70(44, 45, 46), 74(46), 75 (44), 76(53, 54), 93(30), 109(15), 111 (15),115,116, 117(14, 31, 32), 118(41, 42, 43, 44), 123(14, 57, 58), 129(41, 69,71,72),130(75),135(97), 137(103), 139(108), 141(108), 142(108,129), 144 (108), 145(141), 146(108), 148(108), 154(199),162(24),164,165(35),166(35), 167, 181(166), 182(187), 183(194, Zal), 188(230), 190(251), 195, 196(319), 197(332, 334, 335), 199(350,351,353), 200, 202(373, 374, 375), 203(201, 377, 388,391,446), 204(201, 388,391), 205 (373,375), 291(125), 292(125) Hudson, D. H., 57(168) Huebner, C. F.,186(214) Huffman, G. W.,117(38), 124(38), 136 (100) Huggard, A. J., 63(23),64(23) Huggert, A. S.G., 241(22) Hughes, A. M.,32(108), 54.(108) Hughes, C.,16,21(27) Hughes, G.,184(210) Huji, K., 291(114) Hull, R.,193(277) Hungate, R.E.,33(115) Hurd, C.D.,124(59), 126(59), 196(322) Hurini, H., 233(99) Husn, W. J., 206(441) Husseini, H., 230(85) Husted, D. R.,196(317) Hutchings, I. J., 346(64), 347(64) Hutchinson, D.A.,20 Hytten, F. E., 167(40), 171(40, 64)

365

16 I

Ikawa, M., 224(63) Ingles, 0.G.,182(183) Ipatieff, V., 187(225) Irvine, J. C.,161 Isbell, H.S.,116(35, 36), 154, 182(187), 183(193, 194, 195, 199, 200,202), 189, 203(397), 204(397),205(420) Ishimoto, K., 144(136) Israel, G.C.,182(183) Isselbacher, K.J., 180(155) Ivanov, V. I., 146(152), 147(152), 150 (181) Ivanova, V. S., 146(152), 147(152), 150 (181) Ivarson, K. C., 347(60), 352(60) Iwainsky, H., 181(170) Iwanoff, W., 205(410)

J Jaarma, M., 35(136) Jackim, E.,210(19) Jackman, R.H., 349(73) Jackson, E. L.,106(3), 109, 115(14, 32), 116, 117(14, 32), 118(41), 123(14, 57, 58), 129(41), 135(97), 139(108), 141 (108), 142(108, 129), 144(108), 146 (108), 148(108) Jackson, W. P. U., 241(18), 287(18) Jacob, F.,290(100,101,105) Jacquin, F.,353(90) Jager, H., 223 Jain, H. K.,350(77a) James, W.J., 290(109) Jayme, G., 146, 149, 150(174), 152(148, 188, 189, 193), 156 Jayson, G.G.,25,46(67) Jeanes, A. R.,145(141) Jenkins, J. D., 205(430) Jenny, H., 336(5) Jermyn, M. A.,158(223) Joffe, J. S.,336(6a) Joffe, S.,174(105), 175(109) Johansson, A.,345(51a) Johns, R. G.S., 235(123) Johnson, A. G.,236(130) Johnson, A. W.,354(102) Johnson, C.,323(36) Johnson, E.D., 50(155) Johnson, G.R.A., 18,28(97), 29(97)

366

AUTHOR INDEX, VOLUME 16

Johnson, J . M . , 195 JohnRon, P.,27(85) JollBs, P.,208(1), 219(1), 220(1,52a), 225 (64) Jones, D . N., 227(76) Jones, E.J., 158(217) Jones, J. K . N., 121(52), 131(79), 165, 182(185), 186(220),190(253), 228

Kiraly, I., 347(65) Kirby, K. W., 338(22),339(22), 340(22), 341(22),342(22), 343(22), 349(22),350 (22),352(22),353(22) Kirk, M. R., 32(108), Sr(l08) Kirschenlohr, W.,183(203), 192(271) Kiss, I.,349(72) Kissmann, H.M.,95(34), lOl(34) Kittinger, G. W.,171(73), 174(73, 96), K 177(96) Kailan, A., 24(59,61),32 Klages, F., 206(442) Kakrtda, T.,182(188) Kleiber, M.,174(97, 98, 103),175(98, 103) Kalckar, H. M.,177(122, 123, 126), 180 Klein, G.,188(245) (122, 123, 156) Klemer, A., 86(11), 87(11, 14), 88(14, 16, Kaluszyner, A., 61(10) 19), 89(14), 90(23), 91(19), 92(14, 19, Kamal, A. S.,178(140) 23, 27), 93(16),95(11), 96(23), 98(11, 16,23,27),99(11,14, 19), 102(23) Kameyama, T.,290(106, 107) Kamon, J., 196(310) Klotz, I. M., 273,281(86) Kaplan, N.O.,69(41, 42) Knauf, A. E.,118(43),199(350) KAra, J., 222 Knopf, E.,198(346) Karabinos, J. V.,188(231) Kobel, M., 200(356) Karrer, P., 187(227),199(352), 205(422) Koch, R.,210 Kater, J. C.,233(98) Kochling, H.,97(45), lOO(45) Kato, M.,231, 232 Koehler, L.H.,199(351) Katz, J., 176(111) Koepke, K., 353(96) Kay, H.D., 177(130) Kohl, R. A.,338(16), 353(16) Keil, B.,222 Kohtes, I,., 254(45) Keilin, D.,241(19) Koike, H., 291(117) Kembaum, M.,13(4) Koizumi, K., l06(6) Kendrew, J. C.,203(394) Kolb, W., 188(236) Kenner, G.W.,69(40) Kononowa, M.M., 336(7), 338(7),350(7), Kenner, J., 164(32), 189(248, 249), 192 351 (7) Kooi, M., 304(13), 309(13) (249) Kent, P. W., 95(33), 101(33), 209,222,352 Koppel, J. I,., 179(147),291(130),21)8(130) Korn, A. H., 157(213) (89) Kent, W. H., 181(177) Koshland, D.E.,287 Kersten, H., 33(121, 123) Kosterlitz, H.W.,176(117), 179(117) Kcrtcsz, Z. I., 33(125), 34(131) Kozlova, Y. S., 148(165) Keuer, H.,241(27), 242(27), 246(27), 249 Kraft, I,., 132(86) (27), 250(27), 251 (27), 252(27), 254 Kratz, L.,205(429) (27),255(27),264(27) Krauss, M.T.,223(61) Khadem, H.E . , 184(205) Kreis, K., lM(205) Khenokh, M. A., 33(120), 34(134), 42 Kremann, R., 196(330) (134), 50(134),52(134), 53(134) Kubienu, W.L.,336(6b) Khym, J. X., 128(68) Kuby, S. A., 241, 249(26), 264(26), 265, Kiang, A. K., 120(48) 280(26) Kieffer, W. F.,19(35) Kiliani, H., 163,182(190),188(31,241),189 Kuhn, L. P., 124(60), 205(417) Kuhn, R., 167(39, 41), 167(45), 168(39, Kim, J. C.,216(75),272(75),277(75) 41b, 47, 50, 51b), 169(54, 55, 56, 57, Kinell, P.O.,33(117), 35(117), 38(117) 58, 59), 170(60, 6l), 171(62, 75), 172 King, C. G.,30(102),46(102)

AUTHOR INDEX, VOLUME

(75, 77), 173(92), 183(203), 192(271), 193(285, 286) Kullmann, A., 353(96) Kuno, S., 180(165) Kunz, A., 197(332,334) Kurahashi, K., 180(154) Kusunose, M., 231(93), 232(93) Kuyper, A. C., 185(212) Kwiecinski, L., 205(415)

L Laidler, K. J., 205(428) Lancaster, E. B., 141(119), 157(119) Landman, 0. E., 179(148), 240(9) Landor, J. H., 178(142) Landy, M., 236(130) Lange, E., 205(425) Lansky, S., 304(13), 309(13) Lardon, A., 62(16), 63(16), 66(16) Lardy, H. A., 174(102), 241, 249(26), 264 (26), 265, 280(26) Larner, J., 241(23) Latarjet, R., 24(63) Laule, G., 241(27), 242(27), 246(27), 249 (27), 250(27), 251 (27), 252(27), 254 (27), 255(27), 264(27) Laurent, T. C., 53(161), M(161) Lavin, M., 33(125) Lawton, E. J., 33(115, 116, 117), 34(116), 35(117), 38(117) Lea, C. H., 194(295) Lea, D. E., 14(12), 17, M(12) Lederberg, J., 241, 280(25) Lederer, E., 208(1,2,4,9,10,12), 209(16), 210(18, 26, 28,31), 211(34, 35, 36,37), 212(2, 38) 214(39), 215(39, 40), 216 (33, 44), 217(39, 46), 218(2, 18, 34, 35, 36, 49), 219(1), 220(1, 52, 52a), 223 (12), 224(12, 63), 225(4, 64),227(9), 228(10), 229(4, 82), 230(36, 83, 89), 232(26), 234(118), 235(123), 237 Lefort, M., 19(37) Lehmann, J., 241(28), 249(28), 256(28), 257(28), 258(28), 259(28), 260(28), 261 (28), 262(28), 264(28), 280(28), 288(28) Leinzinger, E., 194(294) Leisten, J. A., 84(66, 67) Leitch, G. C., 161(20) Leloir, L. F., 176(118, 119), 177(127), 179 (118, 119), 180(158) Lemieux, R. U., 93(31)

16

367

Lemmon, R. M., 33(107, 108), M(107, 108), 54(163) Lennartz, H. J., 205(412) Lennox, E., 290(104) Lenshina, I. Y., 146(152), 147(152), 150 (181) Lespagnol, A., 168(49a) Lesuk, A., 211 Lettrd, H., 198(340) Levene, P. A., 162(27) Levine, S., 141(124) Leviton, A., 205(406) Levvy, G. A.,291(115), 298(115) Lewis, B. A., 107(11), 108(12), llO(11, 17, 18), lll(12, 19, 23), 112(12), 115(23), 117(19, 37a), 118(12, 17, 18), 122(12, 18), 124(19, 37a) Liebermann, C., 195(316) Liebmann, H., 26(76) Limberg, G., 179(151, 152), 249(39), 255 (39, 56), 262(39, 56) Limperos, G., 27(81) Lindberg, B., 147(158) Lindner, P., 291(124) Lindstedt, G., 186(218) Link, K. P., 129, 241(29) Lionetti, F., 241(17) Lipton, M. M., 236 Lisanti, V. F., 241(17) Lloyd, P. F., 64(24, 25) Loach, J. V., 161(20) Lobry de Bruyn, C. A., 163, 192(273), 193(278, 279), 204(404) Lochhead, A. C., 180(159) Lockwood, L. B., 180(161) LWtrup, S., 179(147) Liiw, I., 173(92) Loiseleur, J., 24(63) Loke, K. H., 120(48) Lohmar, R. L., 153(196) Long, C. W., 161(20), 162(26) LOO,Y. H., 109(13) Loomis, E. H., 206(440) Lopez Aparicio, F. J., 121(50,51), 122(53) Lothrop, W. C., 208(8), 225(8) Lovell, C. H., 133(90) Lowry, T. M., 188(242), 201(367), 202 (371), 204(242) Lucas, H. J., 153(195) Liideritz, O., 233(99) Luick, J. R., 174(97)

368

AUTHOR INDEX, VOLUME

Lynch, L). L., 343(35, 36), 344(49), 345 (36, 49, 49a, 53a), 346(35, 49a, 56), 347(36), 348(36), 350(80), 351(86a), 353(35, 49,80,92,96) Lythgoe, B., 128(66)

M McCabe, L. J., 36(139), 37(141), 38(139, 141), 47(139), 49(139), 50, 51(157, 159),52(139), 53(139) McCallum, K. S., Sl(60) McCormick, J.E.,139(111,114), 144(139), 153(139), 190(257) McCredie, D., 173(86) McDonald, E. J., 205(433), 206(433) MacDonald, M. S.,173(87) McDonnell, W.R.,22(49), 23(%), 24(64) McFarren, E.F.,179(152) McGeown, M.G., 178(133) Macheboeuf, M., 225 McKenzie, A. W.,146(154), 147 Mackeneie, K.G., 311(27) McLaren, A. D., 349(76) Maclay, W.I)., 109(15), 111(15), 115(31), 117(31), 130(74) McLean, P.,176(113) MacLennan, A. P.,208(11,12),223(11,12, 59,el),224(11, 12,59,62) McLennan, J. C., 24(58) McNickle, C. M., 241(23) McPhee, J. R.,275(82c) Maekawa, K., 144(136), 152(190), 153 (194) Maengwyn-Davies, G . D., 267 Magee, J. L.,14(11, 13), 15, 16(22), 18 (24 Magie, W. F.,205(424) Magrath, I). I., 69(39) Maier, J., 96(39), 102(39) Maillard, J., 195(313) Maimind, V. J., 192(272) Major, A., 190(262), 192(268) Malaprade, L.,105 Malhotra, 0.P., 178(134a), 241(28), 242 (31), 249(28, 31), 256(28), 257(28), 258(28), 259(28), 260(28,78),261(28), 262(28), 264(28,31),265(31), 266(31), 267(31), 268(31), 269(31), 270(31), 271(31), 272(31), 273(82b), 274(78), 280(28), 281(78),285,288(28,93) Malisoff, W. M., 305(14)

16

Malpress, F. H., 167(40), 171(40, 64,72), 173(93), 174(95, 99), 178(133, 133a), Malyoth, G., 181(169), 261 Mann, T.,291(118) Manners, D. J., 178(146) Maquenne, I,., 196(323), 311 Maracek, W.,194(290) Marchlewski, L., 205(415,416) Maris, S., 146(148), 149, 150(174), 152 (148), 156 Markgraf, H. C.,205(425) Markham, R.,156(209) Marks, J. D.,353(94) Marsh, J. M., 179(152), 255(58, 59) Marshall, A. J. E., 236 Martin, A. R.,150(177), 156(177) Martin, J.,206(436) Martin, J. B.,345(54) Martin, J. P.,337(12), 350(78), 351(78), 353(12) Martin, S. P., 231 Martin, W.P.,337(12), 353(12, 94) Marx, F.,187(229) Masuo, E.,180(161) Mathews, J. B.,206(444) Matsunaga, K., 231(91,93),232(91,93) Matsumoto, S.,291(114) Matthes, O.,163(31), 188(31) Mattok, G. L.,35(135), 38(135, 147), 39 (135), 44(135), 47(135, 147), 49(147), 53(135), 54(135), 57(135) Mauch, R., 307 Maurer, K.,201(362) Maxwell, E. S.,177(123, 124, 125, 126), 180(123) Mayaudon, J., 351(84) Mechsner, K., 349(75) Mehta, N. C., 341(31, 32), 342(31), 343 (31),345(53, 55), 346(53, 55),353(97), 354(97) Mehltretter, C. L., 141(116,117,120,121, 123), 142(126, 127, 131), 145(142, 143, 144), 150(180a), 157(116, 117, 120, 121), 158(217,218) Meier, R., 231 Meller, A., 147(157), 149(170), 156(170, 206) Mellies, R. L.,141(122, 123), 143(122), 144(122), 145(142), 158(215) Mester, L., 106(7), 113(27), 120(27), 125 (27), 128(67), 135, 144(140), 148(140),

AUTHOR INDEX, VOLUME

152(140), 153(140), 190(259, 262), 192 (268) Meyer, J., 161(17), 182(17) Meyer, K. H., 172(78), 185(211), 299 Meyer, R., 191(264) Meyer, S. N., 237(132) Meyer-Arendt, E., 244(35) Meystre, C., 198(341) Michaelis, E., 88(20), 90(20), 92(26), 100 (20)3 Michaelson, I. D., 240(7) Micheel, F., 86(ll, 12), 87(11, 14), 88(12, 14, 18, 19, 20), 89(14), 90(12, 20, 23, 24), 91(19, 24, 25), 92(14, 19, 23, 24, 27), 93(18), 95(11), 96(12, 23), 97(41, 42, 43, 45, 47, 48), 98(11, 18, 23, 27), 99(11, 14, 19, 24, 42, 43, 46), lOO(12, 20, 45, 47), 102(23, 48), 103(12), 193 (280) Michel, G., 208(4, 5), 218, 225(4, 5), 229 (4, 5) Michelakis, A. M., 36(139), 38(139), 47 (139), 49(139), 52(139), 53(139) Michelet, E., 347(61) Michell, J. H., 137(107), 142(107, 133, 134), 143(107, 133), 150(107) Michelson, K., 338(15) Middlebrook, G., 210(23) Miescher, K., 198(341) Miki, K., 231(91, 93), 232(91, 93) Miller, N., 16(19), 22(52) Millet, M. A., 33(116), 34(167) Millon, E., 182(179) Mills, G. T., 176(121), 180(159) Mills, J. A., 72, 74(51) Milstein, C., 273(80) Minor, F. W., 150(177),156(177) Mishina, A., 35(137) Misler, S., 178(144a) Mitcham, D., 146(163), 147(153) Mitchell, H. H., 178(141) Mitchell, P. W. D., 106(9), 135(95), 139 (111, 114), 140(9, 95), 142(96), 144(9, 95,138,139), 145(9,138), 152(95,191), 153(9,95,139), 158(221), 190(257) Mitrowsky, A., 193(287) Mittelman, N., 180(158) Miwa, T., 255(62), 261 Moczar, E., 113(27), 120(27), 125(27), 135 (96) Moelwyn-Hughes, E. A., 203(394)

369

16

Mohr, W., 203(386) Moises, J. G., 194(298) Monis, B., 178(143) Monod, J., 241(20), 249(20), 254, 256(63), 260(63), 261(63), 264(20), 269(20), 280(63), 290(100, 101, 103, 105) Montgomery, E. M., 189, 190(251), 199 (353) Montgomery, R., 111(19), 117(19, 37a), 124(19, 37a), 140(115), 149(115, 172), 152(192) Montreuil, J., 167(41), 168(49a, 51a), 169 (52, 53), 171(63) Moody, G. J., 35(135, 138), 36(138, 140), 38(135, 140, 143, 147, 148), 39(135), 44(135), 46(143), 47(135, 147, 148), 49 (147), 51(158), 52(138), 53(135, 143), 54(135, 143), 57(135) Moog, K., 161 Moore, F. J., 237(135) Moore, S., 129, 347(59) Moreau, R., 349(72) Morelec-Coulon, M. J., 227 Morgan, A. F., 194(298) Morgan, B. H., 33(125) Morgan, P. W., 68(33, 35), RO(33) Mori, N., 205(419) Morikawa, K., 234(108) Moro, E., 167(42) Moron, J., 210(20) Morris, A., 95(33), lOl(33) Morrison, A. B., 171(72), 174(95) Morrison, M., 185(212) Morse, S., 21 Mortensen, J. L., 338(23a), 341(23a) Mosher, W. A., 27(80,81) Moster, J. B., 194(297) Moyer, J. D., 205(420) Miiller, M., 341(31, 32), 342(31), 343(31), 353 (97), 354 (97) Muller-Hill, B., 260(78), 274(78), 281(78) Muetgeert, J., 301(8), 310(24), 314(30), 328(30), 329(49, 50,51), 332(53) Munch-Petersen, A., 177(122), 180(122) Myrback, K., 182(182) Myrvik, O., 234

N Nabar, G.M., 147(159),150(180),156(180) Nachtsheim, D., 193(276)

370

AUTHOR INDEX, VOLUME

Nagaoka, T., 290(112), 291(112, 114), 297 (112), 298(112) Naghs ki , J ., 157(213) Nakajima, T.,153(194) Nakayama, T., 227 Nef, J. U . , 188(243), 189(247) NBgre, L., 233(100),237 Nejelski, L. L., 33(122) Nencki, M., 188(240) Ness, A . T., 70(44, 46), 74(46), 75(44) Neuberg, C., 179(150), 187(229;1,240(5), 291 (120) Neukom, H., 341 (30) Neumiiller, G., 184(209), 185(309), 193 (288) Nevell, T. P., 146(151), 148(162), 150 (176), lSl(183, 184), 153(198), 156 Newman, M. S., 67, 83(30), 209 Newth, F. H., 85(1), 88(1) Newton, A. S., 22, 23(56) Nguyen-Trung-Luong-Cros, H . , 220(52a) Nicolle, J., 204(396) Niebel, W., 178(141) Niederhoff, P., 205(414) Nierenstein, M., 159(1) Niguet, R., 203(390), 204(390) Nikuni, Z., 35(137) Nishizuka, Y., 180(165) Nisizawa, K., 240(10), 248 Nixon, D. A., 241(22) Nojima, S., 219,229 Nolan, A., 139(113) Noll, H., 208(2), 210(18, 19, 25, W ,30), 211, 212(2), 218(2, 18, 3011, 223(60), 232(97) Noller, R. M., 32(108), 54(108) Nolte, M., 86(11), 87(11), 95(11), 98(11), 99(11) Nordiek, H., 86(11), 87(11), 88(15), 95 ( l l ) , 98(11), 99(11) Norman, A. G., 350(77) Norrie, R. F., 167(44, 45, 46) Northcote, D. H., 228 Norton, D. R., 132(82) Novelli, G. D., 290(106, 107) Nowotny, A., 233(99) Niissel, H., 96(36), 102(36) Nutting, L. A., 175(106)

16 0

O’Brien, J. F., 62(14) O’Colla, P. S., 139(112, 113) O’Connor, R. T.,146(153), 147(153) Ogston, A. G., 338(19) Ohara, T.,234(108) Ohle, H., 194(290) Ohta, T., 233(103) Okuhara, E., 227 Okui, S., 125(63), 135(98), 136(98), 137 (98) Oliverio, V. T., 61(13) Ollendorf, G., 183(204) Olney, H. O., 343(35, 36), 345(36), 346 (35), 347 (36), 348(36), 353(35) O’Malley, C. M., 203(383) O’Meara, D., 155(203), 156(204) Onnen, O., 191(264) Onos, K., 233(100) Orten, J . M., 185(212) Ortiz Riro, M., 121(51) Ost, H., 161 Ostwald, R., 32(108), 54(108) Overend, W. G., 130(78), 131(78) Owen, W. L., 205(408) Owens, H. S., 206(435)

P Pacsu, E., 148(161), 150, 154, 15G(175), 197(333), 305, 326(16, 46, 47) Padgett, A. A., 150(179) Padmanabhan, C. V., 147(159), 150(180), 156(180) Page, J. B., 337(12), 353(12) Palacas, J. G., 335(3) Paladini, A. C., 176(118, 119), 179(118, 119) Palmer, C. E., 237(132) Pangborn, M. C., 225,229,233 Pardee, A. B., 290(101) Parson, J. W., 338(17a, 17b), 339(17a), 340(17a), 341(17a, 17b), 343(17a) Parsons, M. A., 113(24, 25), 115(24), 121 (24, 25) Pasteur, L., 160 Patton, S., 194(296, 299, 303), 195(304) Pauling, L., 85(2), 95(2) Paycot, P. H., 54(163) Pazur, J. H., 174(104), 179(152), 255(57, 58, 59)

AUTHOR INDEX, VOLUME

Peat, S., 161(20), 188(232), 300(6), 301(6) Peck, R. I,., 225(66) Peeterx, G. J., 175(107), 176(112) Percival, E. E., 191(265) Percival, E. G. U., 191(265) Perlin, A. S., 106(8), 137(101), 164(34), 186(222, 223) Perrin, D., 290(105) Perrin, M. W., 24(58) Perry, M. B., 186(216), 188(234), 228 Petek, F., 291(119), 294(141, 145, 148), 295(148, 149) Peters, O., 97(50), 98(50), 290(111), 292 (111) Petersen, W. E., 173(93), 177(128, 129) Pfiefer, V. F., 141(119), 157(119) Pfleiderer, G., 241(24), 244(35) Phelps, F. P., 181(166) Phillips, G. O., 35(135, 138), 36(138, 140), 38(135, 140, 143, 144, 145, 147, 148), 39(135, 144, 149), 43(150), 44(135), 46 (143), 47(135, 145, 147, 148), 49(147, 149), 51(158), 52(138), 53(135, 143), 54 (135, 143), 57(135) Piazza Molini, C., 122(53) Pickett,, L. W., 50(155) Pictet, A., 198(349) Pierce, C. H., 210(23), 237(131) Pigman, W. W.,179(150), 183(195), 193 (281), 203(397), 204(397), 254(48, 49, 51), 292(137, 139) Pinkard, F. W., 338(18), 339(18) Pittet,, A. O., 186(215) Pizer, F. L., 227(77) Plant, M. M. T., 200(357), 201(357) Plate, E., 193(280) Platzmann, R. I,., 16, 18(25), 21(26), 25 (26) Pochon, J., 336(7c), 338(7c), 350(7c), 351 (7) Polgar, N., 230(86) Pollock, M. R., 290 Polonovski, M., 168, 169(52) Polonsky, J., 214(39), 215(39, 42), 216 (a), 217(39) Popj&k,G., 174(99) Poreher, C., 240(12) Portelance, V., 216(43) Porter, C. J., 179(147), 291(130, 131), 298(130, 131) Potts, K. T., 133(88)

371

16

Pound, A . W., 236 Powell, D. B., 164(33), 173(85), 190(33) Powell, M. R., 132(83) l’ratt, A. W., 29(98) Pratt, J. W., 118(46), 129(69, 71) Preece, I. A., 311 Prelog, V., 205(411) Price, F. P., 33(117), 35(117), 38(117) Pridham, J. B., 181(171) Procter, B. E., 33(124) Pridles, J., 217(46), 233 Pulcher, G., 191(289) Pulvermacher, O . , 205(434) Purves, C. B., 93(30), 137(107), 142(107, 128, 132, 133, 134), 143(107, 133), 147 (128), 150(107), 198(347) Purvis, J . E., 205(413) Putney, F. K., 29(98) Pritney, R . K., 223(59), 224(59)

R Raffel, S., 234(116) Ragan, C., 33(127) Raiutrick, H., 132(85) Ramet, M., 184(206) Ramler, E. O., 60(6) Ramler, W. J., 22(47) Ramsey, W., 13(2) Ranc, A., 179(150) Randall, H. M., 208(11, 12), 223(11, 12, 59,61), 224(11,12,59) Randles, J. E. B., 69(38), 79(38, 56), 81 (58) Raney, W. A., 337(12), 353(12) Rank, B., 132(86) Rankin, J. C., 141(117), 112(126), 157 (117) Rao, S. V., 223(59), 224(59) Rapaport, H., 133(90) Rathgeb, P., 185(211) Rauch, E., 61(9) Rauchenberger, W., 69(43) Reeves, R . E., 111(20), 146(20), 148(166, 167), 149(166), 156(166, 167), 287 Rega, A., 171(74) Reichstein, T., 62(16), 63(16), 66(16), 223 (61) Reinbold, J., 97(42), 99(42) Reinhard, M. C., 50(153) Reiss, 0. K . , 174(100)

372

AUTHOR INDEX, VOLUME 16

Reithel, F. J., 171(73), 173(91), 174(73, 96), 176(115), 177(96), 196(328), 242 (32), 244(32), 249(32), 250(32), 251, 255(53), 264(75), 272(75), 277(75) Renfrew, A. G., 208(7), 220(7) Renner, A., 291(122) Rennert, E., 200(356) Rennie, D. A., 339(26), 340(26), 363(26) Reuter, S., 61(10) Revallier-Warfenius, J. G., 240 (8) R6v6sz, A., 96(37), 103(37) Rexford, D. R., 132(83) Reynolds, R. J. W., 200(357), 201(367) Richard, F., 349(72) Richards, E. L., 190(263) Richards, G. N . , 165(Iw3), lM(204, 205, 205a), 189(249),192(249) Richards, R. B., 322(33) Richardson, A. C., 118(46a) Richter, W., 254(47) Richtmyer, N. K., 118(42,45,46), 129(69, 71,72,73), 130(75),137(103j,197(335), 199(353), 291(125), 292(126) Ricketts, C. R., 33(118), 181(172) Ridder, J., 88(16), 93(16), 98(16) Rieder, H. P., 206(437) Rischbieth, P., 181(178) Rist, C. E., 153(196) Ristih, P., 90(23), 92(23), 96(!3), 98(23), 102(23) Rizo, M. Ortiz, 121(51) Roberts, E. A., 33(124) Roberts, E. G., 208(3) Roberts, H. R., 175(106), 178(144a), 179 (152), 180(164) Roberts, J. G., 188(232) Robertson, A., 167(38) Robineaux, R., 231(94) Robinson, E., 364(102) Robinson, R., 133(88), 176(116) Rocklin, R . S., 22(47) Rockstein, M., 178(140) Rahmann, F., 176(116) Rtipsch, A., 62(20) Rogovin, Z. A., 148(184, 165), 150(178), 161(182, 186), 162(182, lM), 156(178) Romijn, G., 182(180) Roncero, A. Vhzquez, 121(50) Rose, C. S., 167(46, 46), 168(47), 171(62) Rose, R. S., 188(230) Rosenberg, S., 158(216)

Rosenfeld, F. M., 26(69) Rosenthal, O., 240(5) Ross, A. G., 178(146) Rossi, 0. A., 206(441) Rothschild, W. G., 20(41) Rovira, A. D., 3W(81) Rowe, C. E., 33(118), 181(172) Rowen, J. W., 111(20), 146(20) Ruck, K., 196(320) Rudman, P., 132(86) Ruelius, H. W., 168(47) Ruff, O., 183(2U4) Rule, H. G., 206(409) Rundle, R. E., 301(7), 303(11), 312(7) Runyon, E. H., 223(58) Russ, S., 34(133) Russell, C . R., 144(135) Russi, S., 255(80) Rutenburg, A. M.,178(143) Rutenburg, 8. H., 178(143) Rutherford, H. A., 150(177), 156(177) Ruttloff, H., 181(170) Rutter, W. J., 176(120), 180(120, 166)

S Sabin, F. R., 230 Shda, J., 195(312) Saeman, J. F., 33(116), 34(116) SBtre, M., 146(148), 152(148, 188, 180, 193) Sah, P. P. T., 192(275) Sahashi, Y., 182(188) Saillard, E., 204(402) Salton, M. R. J., 220(53), 236(63) Samuel, A. H., 16(22), 18(24) Sanchez, C., 290(105) Sasaki, R., 196(329) Saunders, W. H., Jr., 124(59), 126(59) Saville, N . M., 78(55) Sawicki, E., 61(13) Sayre, R., 196(319) Scatchard, G., 281 Schaefer, W. B., 237(131) Schiir, B., 231 Schafer, W., 198(346) Schallenberg, E. E., 63(21) Sohambye, P., 174(103),175(103,107,108) Scheele, C. G., 181(173) Scheffer, F., 336(7d), 338(7d), 350(7d), 361(7) Scheiber, H., 178(136)

AUTHOR INDEX, VOLUME 16

Scheuer, O., 13(6) Schindler, O., 223(61) Schirp, H., 192(274) Schlichting, E., 344(47) Schlubach, H. H., 69(43), 161 Schmitz, E., 132(84) Schmoeger, M., 202(369), 205(369) Schneider, W., 195(314) Schneiderman, M., 33(128) Schnitzer, M., 354(99) Schoch, T. J., 299, 300(2,3), 304, 309(13), 325(44), 326(45), 333 Schoenberg, M. D., 33(128) Schonfeld, H., 167(43) Schonholzer, G., 233(99) Scholes, G., 24(65), 25, 26(77, 78), 27(86, 87, 88, 89), 28(90, 91, 93, 94, 95, 97), 29(97), 46(67) Schoorl, N., 284 Schotte, H., 200(356) Schramm, M., 292(133) Schrier, E., 191 Schuler, R. H., 19(36), 22(48, 50) Schultze, A., 69(43) Schuster, L., 69(41) Schwalbe, C. G., 148(163) Schwartz, J. H., 196(321) Schwarz, H. A., 18(31) Schwarz, J. C. P., 190(258) Schwert, G., 284(91) Sebelien, J., 347(61) Seekles, I,., 132(81) Segal, W., 237(133) Segard, E., 168(49a) Seidman, M., 241(29) Sekikawa, I., 234(108) Seligman, A.M., 178(143) Sen, Y., 233(103) Senderens, J. B., 187(226) Senti, F. R., 141(124), 144(135),158(217) Serck-Hanssen, K., 216(43) Shafizadeh, F., 93(29) Sharp, P. F., 160(7), 203(392) Sharp, V. E., 88(21), 99(21) Shaw, C. J. G., 115(30) Shaw, K., 347(57) Sheinin, R., 291(132), 298(132) Shemyakin, M. M., 192(272) Shen Han, T. M., 36(139), 38(139), 47 (139), 49(139), 52(139), 53(139)

373

Shields, H., 32(109) Shilo, M., 171(71) Shima, F., 180(161) Shimmyo, Y., 234(108) Shipley, R. A., 55(165) Shiroya, T., 291(121) Shorey, E. C., 337(10), 343(10), 344(10), 345(54) Shorygina, N. N., 150(178), 151(182,186), 152(182, 186), 156(178) Shukuya, R., 284(91) Shuler, K. E., 205(428) Sieber, N., 188(240) Sieling, D. H., 364(98) Sigurdsson, S., 350(83) Simonart, P., 351(84) Simons, J. H., 60(6), 61(12) Singer, B., 194(298) Singh, P. K., 347(59a) Singh, S., 347(59a) Siu, P., 175(108) Skell, P. S., 109(13) Skraba, W. J., 24(60),54(60) Skraup, 2. H., 196(330) Sloan, J. W., 141(122), 143(122), 144(122), 145(144, 145), 153(196) Slosse, A., 32(114) Smart, C. L., 341(29) Smidt, B., 196(327) Smith, C. R., 237(135) Smith, C. W., 133(87a) Smith, D. B., 27(84), 227(76) Smith, D. W., 208(11, 12), 223(11, 12, 59, 61), 224(11, 12, 59) Smith, E. E. R . , 176(121), 177(122), 180 (122, 159) Smith, F., 107(11), 108(12), llO(11, 17, 18), 111(12, 19, 23), 112(12), 115(23, 29), 116(34), 117(19, 37, 37a, 38), 118 (12, 17, 18, 29), 122(12, 18, 29, 34, 54, 55), 124(19, 37, 37a, 38, 62), 126(29, 34), 127(54), 136(37, loo), 140(115), 143(29), 145(146, 147), 149(115, 172), 150(29), 152(192), 197(338, 339), 205 (4201, 335(3) Smith, H. P., 24(62) Smith, J. A. B., 173(93) Smith, K. A., 27(82) Smith, V. R., 174(102) Smits, A., 203(384) Sneland, E., 22(51)

374

AU'J'HOR INDEX, VOLUME

16

Sund, H., 280(85), 281(89) Snell, E. E., 224(63) Suter, E., 231 Sohotka, H., 162(27) Suzuki, H., 233(104) S~rensen,H., 349(72) Sohns, V. E., 141(118, l l g ) , 157(118, 119) Suzuki, S., 125(63), 135(98), 136(98), 137 Sokolovsky, A., 203(381) (98) Svendsen, A. B., 294(142) Sood, S. P., 82(63) Swain, F. M., 335(3) Sorkin, E., 234,237 Sowden, F. J . , 343(42), 347(42, 60),352 Swallow, A. J., 13(8, 9), 20(43), 24(66) Swanson, J. W., 158(217) (42, 6'3,353(91) Swarts, F., 59(1), 60(1) Sowden, J. C., 188(246), 189c246) Sworski, T. J., 19(35), 20(40) Sparmberg, G., 168(49) Szabo, L., 111(21) Sparrow, A. H . , 26(69) Szejtli, J., 324(37) Spedding, H., 146(155), 147(153) Szwarc, M., 82(63) Spiegelman, S., 290(104) Spitznagel, J. K., 218 T Sprinson, D. B., 351(83a) Tiiufel, K., 181(170) Srogl, M., 349(72) St,acey, M., 32(106), 34(132), 39,43,59(2), Takahasi, T., lSl(167) 60(4), 61(8), 63(22), 64(24, 25), 67(4, Takahashi, Y., 233(100) 31), 68(32, 36), 69(37, 38), 77(4), 79 Takano, K., 256(62), 261 (38), 80(32), 81(32), 82(8), 83(32, 36), Takeda, Y., 233(103, 104) 88(21), 99(21), 209, 222, 338(18), 339 Takiura, K., l06(6) Talley, E. A., 196(321) (18), 351 (85, 86) Taniguchi, K., 196(329) Stadtman, F. H., 174(97) Tanno, T., 187(228) StanEk, J . , 195(312) Tanret, C., 202(372) Stannett,, V., 82(63) Tarrago, X., 19(37) Stcgeman, O . , 205(423,426,427) Tatlow, C. E. M., 60(7), 62(7, 17), 63(7, Steihler, O., 195(314) 22), 66(26) Stein, G., 27(86) Tatlow, J. C., 59(2), 60(4,7), 61(8), 62(7, Stein, H. W., 181 (169) 17), 63(7, 22, 23), 64(23), 66(26), 67 Stein, W. D., 289(98) (4, 31), 68(32, 34, 36), 69(37, 38), 70 Stein, W. H., 347(59) (47, 48), 73(47), 74(48), 76(48), 77(4, Stevens, K. R., 346(68), 348(68) 48), 79(38, 56), sO(32, 34), 81(32, 581, Stevenson, F. J . , 343(41), 347(41, 58), 82(8), 83(32,36,48) 353(94 ) Ttirrrog, A., 66(28) Siewtrrt., J. Is., 205(420) Taylor, n., 26(71, 72, 73) Stcwart, I,. C., 118(45), 129172,73) Taylor, H. S., 15 Stewart, W. T., 153(195) Taylor, N. F., 95(33), lOl(33) Stodola, F. H . , 180(161) Taylor, S. A., 338(16), 353(16) Stohr, G., 191(267) Taylor, T. J., 186(213), 188(213), 195(308, Stohl, G., 119(47), 133(47) Stolzenhuch, F. E., 69(41) 309) Taylor, W., 28(93) Stone, S. H., 235, 236 Teague, R. S., 178(143) Stoppani, A . 0. M., 273(80) Tedder, J. M., 59(2, 3), 60(4), 67(4), 68 Streuli, H., 341(32), 353(97), 3 54(97) (36), 69(37, 38), 77(4), 79(38, 56),81 Strohele, It., 193(285) Xtrble, U., 147 (58, 59), 83(36) Teller, J. D., 241(21) Stroganov, N. S., 206(443) Tennant, R., 230(88) Strong, W. A , , 109(13) ter Horst, M. G., 203(378), 204(378) Sugihara, .J. M., 66(27) Terner, C., 177(132) Sumikana, E., 234(108)

AUTHOR INDEX, VOLUME

Teves. D... 151(185) . Theander, O., 115(33), 124(33), 147(158), 338(21), 340(21) Thiele, H., 354(101) Thierfelder, H., 199(354) Thomas, G. H. S., 186(213), 188(213) Thomas, R. L., 158(222, 223), 345(53a) Thomsen, J., 196(327) Throssell, J. J., 82(63) Tillitson, E. W., 194(292) Tinsley, J., 338(17b), 341(17b) Tipson, R. S., 116(35, 36), 215(41a) Tipton, C. L., 174(104), 179(152), 255(58, 59) Tissier, H., 167(42) Todd, A. R., 69(39, a), 128(66), 156(207, 208), 354(102) Tolbert, B. M., 32(108), 54(108), 54(163) Tollens, B., 181(177, 178), 182(186) Tomarelli, R. M., 167(44) Tompa, H., 319(32) Toogood, J. A., 353(96) Tork, L., 86(11), 87(11), 88(18), 93(18), 95(11), 98(11, 18), 99(11) Torriani, A. M., 249(37) T&h, G., 292(136) Toubiana, R., 214(39), 215(39), 217(39) Treton, H. J. C., 24(58) Trey, H., 203(398),204(398) Trotter, I. F., 80(57) Troy, H. C., 203(392) Trucco, R. E., 171(67, 74), 172(68), 174 (67), 176(67, 118), 179(118), 180(158) Truog, E., 339(26), 340(26), 353(26) Tseu, c . - z . , 102(275) Tsuzuki, Y., 205(419) Tucker, K. L.,50(153) Turcotte, A . L.,205(433), 206(433) Tiirkova, V., 349(72) Tuttle, L. W., 33(125)

U Ulrich, B., 336(7d), 338(7d), 350(7d), 351 (71 Ungar, J., 230(87) Unger, F., 67 Urech, F., 204(395) Usher, F. L., 13(5) Utsushi, M., 291(114) Uyeda, Y., 195(310)

375

16

V Valentino, A., 127(65), 135(65), 136(65, 99) 9 Vallentyne, J. R., 335(2) Van Cleve, J. W., llO(17, 18), 111(19), 117(19, 37, 37a), 118(17, 18), 122(18), 124(19, 37, 37a), 136(37), 142(131), 158(218), 197(338, 339) van Dam, B., 240(8) van Dam-Schermhorn, 1,. C., 240(8) van Leent, F. H., 193(279) Vanngard, T., 33(117), 35(117), 38(117) Varner, J. E., 353(94) Vasseur, E., 184(209), 185(209) Vaughan, G., 34(132), 39 Veibel, S., 178(137), 293(140) Velick, S. F., 281(88) Venkataraman, R., 173(91), 176(115) Verdier, P., 171(74) Vilkas, E., 208(6, 9, lo), 227(9, 71), 228 (lo), 237 Villecourt, P., 180(162) Vining, L. C., 137(105) Virkola, N., 149(173) Vock, M., 97(49), lOl(49) Vogel, H. A., 160 von Liebig, J., 181(174) von Lippmann, E. O., 159(2) von Schreier, E., 119(47), 133(47) Vorsatz, F., 178(145), 240(4), 291(4), 297 (4)

W Wabers, B., 158(217) Wadman, W. H., 228 Wagner, C. D., 54 Wagnet, P., 160(6) Waite, R., 167(38) Wakita, N., 233(104) Waksman, B., 236 Waksman, S. A., 336(7a), 338(7a), 346(7a, 64,68), 347(64), 348(68), 350(7a), 351 (7) Wallenfels, K., 178(134a), 179(151, 152), 241(15, 27, 28), 242(27, 31), 246(15), 247(15), 248(15), 249(15, 27, 28, 31, 39), 250, 251(27), 252(15, 27, 42, 43), 253(15, 42), 254(15, 27), 255(15), 256 (15, 28, 64),257(15, 28), 258(15, 28), 259(15, 28), 260(15, 28, 78), 261(28),

376

AUTHOR INDEX, VOLUME

262(16, 28,39,56,64), 264(27>28,31), 265(31), 266(31, 43), 267(31), 268(31), 269(31), 270(31), 271(31), 272(31), 273(82b), 274(78), 280(28,85), 281(89), 282(16), 283(16), 284(15), 286(15), 288(28, 93) Walters, D., llO(16) Walton, A., 65(166) Walton, W. W., 183(202) Waly, A., 33(119) Ward, J. O., 19(39) Ward, R. B., 30(103), 31(104,106), 32(104, l06), 38(146), 43(106), 45(104, 105), 53(146), 54(146) Warkentin, B. P., 363(89a) Wasif, S., 84(68) Watanabe, N., 233(104) Watkins, O., 172(81) Watson, P. R., 141(117), 157(117), 158 (218) Watters, A. J., 200 Webb, B. H., 194(300) Weber, L. G., 291(116) Webley, D. M., 351(87), 362(87), 354(100) Weeks, B. M., 19(39), 29(101) Weidenhagen, R., 291(122), 297(150) Weidmann, S., 234(107) Weigel, H., 55(166, 167), 57(167, 168) Weisberg, H., 157(211) Weisbuch, F., 204(396) Weiser, R. S., 234 Weiss, D. W., 237 Weiss, J., 16(21), 18, 19(38), 25(68), 26(77, 78), 27(86, 87, 88, 89), 28(90, 91, 93, 94, 95, 97), 29(97), 46(67) Weim, M. J., 95(34), lOl(34) Weiss, W., 49(152) Wellm, J., 203(386) Wells, 1’. A., 157(213, 14) Werner, J., 198(344) Werner, W., 206(407) Wertheim, M., 299(1) Westermann, H., 86(11), 87(11), 95(11), 97(43), 98(ll), QQ(l1,43) Westphal, o., 233(99) Weygand, F., 61(9), 62(18, 19, 20) Weymouth, F. J., 69(40) Wheeler, C. M., 27, 28(90) Whelan, W. J., 188(232) Wherry, E. T., 203(380)

16

Whiffen, D. H., 31(104), 32(104), 45(104), 72(50), 74(50) Whistler, R . L., 156(205, 2054, 300(5), 304, 323(36), 338(22), 339(22), 340 (22) ,341 (22,29), 342(22), 343(22), 349 (22), 350(22), 352(22), 353(22) White, A. A., 172(80) White, J. C. D., 167(38) White, R. A., 231 White, R. G., 230,235(123), 236 Whitefield, P. R., 156(209) Whitehouse, M. W., 171(67a), 362(89) Whittier, E. O., 169, 160(3), 167(3), 205 (431), 206(438, 439) Wickham, N., 233(98) Wickstrgim, A., 137(102, 104, 106), 184 (207), 190(260), 293(144), 294(142, 143, 144) Wiegand, F., 88(17), 95(17), 98(17), 99 (17) Wiegner, G., 205(432) Wieland, P., 206(411) Wigglesworth, V. B., 178(139) Wild, G. O., 290(109) Wilkinson, J. F., 180(167) Wilkinson, R. W., 28(96) Williams, D., 37(141), 38(141) Williams, T. F., 28(96) Wilson, E. J., 154(199) Wilson, G. L., 188(242), 204(242) Wilson, W. K., 150(179) Wimmer, E. L., 327,332 Winer, R. A., 241(17) Winkler, S., 290(111), 292(111) Winter, L. B., 172(82), 173(88) Wintersteiner, O . , 110(16), 162(27) Wise, C. S., 142(127) Wolf, A , , 198(346) Wolfe, J. K., 76(54) Wolfe, R. G., 242(32), 244(32), 240(32), 250(32), 251 (32) Wolff, H., 193(283) Wolff, I. A,, 141(122, 123, 126), 143(122), 144(122), 145(143, 144, 145), 150 ( M a ) , 153(196), 158(215) Wolfrom, M. L., 36(139), 37(141), 38(139, 141), 47(139), 49, 50, 51(157, 159), 52 (139), 63(139), 93(29), 188(230), 196 (317) Wood, H. G., 174(103, 105), 175(103, 107. 108,109), 176(111)

AUTHOR INDEX, VOLUME 16 Woods, B. M., 164(33), 186(213, 217), 188 (213, 234), 190(33) Worrall, R., 61(8), 66(26), 68(32, 34), 80 (32, 34), 81(32), 82(8), S(32)

Wright, J . , 50 Wright, L. M., 343(35, 36), 345(36), 346 (35), 347(36), 348(36), 353(35, 92)

Wulff, H., 86(12), 88(12), 90(12), 96(12), 100(12), 103(12)

Y Yamamura, Y., 231(91, 93), 232(91, 93) Yanovsky, E., 183(201), 203(201), 204 (201)

Yashunskaya, A. G., 148(164), 150(178), 151(182, 186), 152(182, 186), 156(178)

Yearian, H. J., 304(12) Yelland, W. C. E., 157(210) Yoshida, K., 180(161) Yoshida, R. K., 344(43) Yoshino, I., 168(224) Young, B., 290(10I)) Young, R. A., 310

377

Young, R. G., 196(328), 255(63) Z

Zaheer, S. H., 198(346) Zak, H., 194(293), 198(293) Zarnits, M. L., 241(27), 242(27), 246(27), 249(27), 250(27), 251(27), 252(27), 254(27), 255(27), 260(52), 264(27) Zartman, W. H., 188(237) Zechmeister, L., 292(136) Zellner, J., 194(293), 198(293) Zemplh, G., 162, 183(25), 193(25) Zerban, F. W., 206(436) Zervas, L., 200(358), 201(363) Zhbandkov, R. G., 146(152), 147(152, 156 Zief, M., 122(56), 123(56), 135(94) Zilliken, F., 167,168(47), 171(66,67a) Zimmer, K. G., 35(136) Zumbute, F., 90(23), 92(23), 96(23), 98 (23), 102(23) Zweifel, G., 338(25), 339(25), 342(25), 353(25)

Subject Index For Volume 16 A Acetaldehyde, effect on EtOH irradiation, 23 from ethanol irradiation, 25 Acetals, cyclic, reaction with acyl trifluoroacetates, 83 cyclic, ring opening of, 69 Acetic acid, anhydride with trifluoroacetic acid, 80,81 reaction with cellulose, 60 with trifluoroacetic anhydride, 60 -, trifluoro-, anhydride, 79, 80 anhydride, acylation by mixtures of carboxylic acids and anhydrides and, 67 carhohydrat,e eRterification by, 61 prepn. of, 8 reuction of, with acet,ic acid, cellulose and, Go with amines, 02 with amino acids, 62 with peptides, 62 anhydrides with carboxylic acids, 79, 81

acylation by, 83 reaction of, with cyclic acetitls, 83 with hydroxy compds., 81 anhydride with acetic acid, 80. 81 esters, hydrolysis of, 61, 63 prepn. of, 61 reaction with methanol, A1 ethyl ester, 59 phenyl ester, reaction with amino acids and peptides, 62 reaction with cellohiose and cellulose, 62 -, trifluorothio-, S-ethyl ester, reaction with amino acids, 63 Acetic anhydride, 68 Acetone, dihydroxy-. See a-Propanone, 1,I-dihydroxy-. Acetophenone, 4’-methoxy-, 67 Acids, 7-ray effect on solutions o f , 19 hydroxy, keto acids from, hy irradiation, 28-32

in starch fractionation, 326 of soil, organic, 337 Adenine, 9-B-D-glUCOpyranOSyl-, oxidn. product, and picrate, 128 Adenosine, 5-(benzyl H phosphate), alkali effect on oxidized, 151; cyclic 2,3-phosphate, 69 oxidn. product, 127, 128 2-phosphate, B9 5-phosphate, alkali effect on oxidized, 156 picrate, oxidn. product, 128 5-pyrophosphate, 177 5-triphosphate, 177 Adipic acid, polyester from 1,3:2,4:5,Dtri-0-methylene-~-glucitol and, 78 Aglycons, specificity of, in enzymic reactions, 261 Alanine, esters with sugars, 207 Alcohols, complexes of higher, wit,h amylose, 299 complexes with amylose, 325 irradiation of, 22-26 reaction with acyl trifluoroacetates, 81 in starch fractionation, 320, 325 Aldehydes, di-, (non-carbohydrate), 132133 Aldohexopyranosides, methyl, oxidn. products, 123 -, methyl 6-deoxy-, oxidn. products, 109,111 Aldopentofuranosides, methyl, oxidn. products, 123 Aldopentopyranosides, methyl, oxidn. products, 116118 Aldoses, w-fluoro derivs., 101 prepn. of, 95 Alkalis, in starch fractionation, 326 starch precipn. by, 327 Allolactose. See Glucose, G-O-fl-~-gdactopyranosyl-D-. Allosan, oxidn. product, 118 Alloside, methyl 4,6-0-benzylidene-3deoxy-3-phenylazo-~-,114 Alpha rays, effect on alcs., 22

378

SUBJECT INDEX, VOLUME

Altrosan, oxidn. product, 118 Altrose, 3-amino-l ,6-anhydro-3-deoxyD-, 118 -, 4-O-fl-~-galactopyranosyl-~-, 197 octaacetate, 197 Altroside, methyl 3-amino-4,6-O-benzylidene-3-deoxy-~-,oxidn. product, 115 Amines, reaction with trifluoroacetic anhydride, 62 Amino acids, from B-galactositlase, 252, 253 of mycoside C, 224 reaction with 8-ethyl trifluorothioacetate, 63 with phenyl trifluoroacetate, G3 with trifluoroacetic anhydride, 62 of soil, 343 of wax D, 220 Ammonium iodide, (hepta-O-acetyl-8lactosyl ) trimet hyl -, 199 Ammonium sulfate, st,arch fractionation by, 328 Amygdalin, 294 Amy1 alcohols, complexes of isomeric, with amylose, 305 in starch fractionation, primary, 326 Amylase, in soil, 349 “Amylodextrine,” 307 “Amylogeen,” 307 Amylopectin, 309, 318, 325 alkaline leaching of, 306 complexes, 303, 304 with alkaline-earth hydroxides, 327 fractionation of, 304 insolubilization of, 313 iodine adsorption by, 301, 303 from org.-solvent precipitation, 330 precipn. and recovery of, 316 precipn. of, 321, 324, 328, 329 by MeOH, 326, 327 temp. effect on, 319, 324 radiation, effect on, 35 from salt-fractionation process, 330, 331 sepn. from amylose, 308, 310, 313, 317, 326 structure of, 321 system: magnesium sulfate-water-, 318

16

379

water (cold) insoluble, 313, 316, 330, 331 soluble, 330, 331 Amylose, 299, 300,307, 308, 309, 310, 318, 319, 320, 325 acetates, 67 by aqueous leaching, 30B complexes, 303, 304, 319 with alcs., 325 with alkaline-earth hydroxides, 327 with 1-butanol, 304, 306 with butanol, structure of, 303 with culcium hydroxide, 328 with chloral hydrate, 307 with iodine, struct,ure of, 303 with 2-methyl-l-butanol, 300, 317 with 2-1nethyl-2-butano1, 326 with pentanols, 304, 305 with 2-propanol, 321 crystalline, 300 fractionation of, 304, 315, 324 from fractionation with org. complesing agents, 329, 330 iodine adsorption by, 301, 302 particle size of precipd., 315 precipn. of, 312, 313, 310, 321-324, 320, 327, 328, 329 temp. effect on, 319 radiation effect on, 34, 35 recovery of precipd., 315 recrystallized, 309 retrograded, 323 from salt-fractionation process, 330 solubilization of, 323 solubility in water, 321 structure of, 321 system: magnesium sulfate-wster-, 318 water (cold) insoluble, 312, 330 soluble, 330 Arabinan, 328 Arabinofuranoside, methyl Q-D-, oxidn. product, 123 Arabinonic acid, 3-0-8-~-galactopyrarloSyl-D-, 184 Arabinopyranoside, o-nitrophenyl Q-I,-, hydrolysis of, 269 Arahinopyranosides, 8-L-,hydrolysis of, 292 Arabinopyranosyl bromide, tri-0-acetyl-fl-D-, 88

380

SUBJECT INDEX, VOLUME

Arabinose, L-, 284 D-, from D-glUCitOl irradiation, 48 D-, from D-glucose irradiation, 35 . D-, from glycolipids, 209 D-, from D-rnannitol irradiation, 47 D-, from D-mannose irradiation, 41-45 in polysaccharides from pathogenic bacteria, 352 of soil, 338, 342 D-, from starch irradiation, 35 D-, from sucrose irradiation, 51, 52 D-, from wax D, 219, 220 -, 3-0-8-~-galactopyrano~yl-~-, 162, 183, 195 hexaacetate, 200 Arubinoside, ethyl a-r.-, ensyrnic synthesis of, 257 -, methyl p-u(and @-L)-,oxidn. products, 118 -, o-nitrophenyl a-L-,hydrolysis of, 268-272 Arabinosides, a+-, 280 o-L-, hydrolysis of, by 8-D-galaCtOsidases, 256 Arabinosyl group, transfer of, by enzymes, 262, 263 a+, transfer of, 8-galactosidase in, 256 L-, 7-ray effect on, 30 radiation effect on, 46 Ascorbic acids, 52, 53

16

1-Butanol, 313 complex with amylose, 304, 306 in starch fractionation, 300 -, 2-methyl-, complex with ttinylosc, 306,317 in starch fraotionation, 311 2-Butanol, amethyl-, in starch fractionation, 326 Butyl alcohol. See 1-Butanol. Butyric acid, 3-hydroxy-, 28 C

Calcium hydroxide, complex wit,h stnrrh, 327 Cane sugar. See Sucrose. Carbohydrates, complexes of, in #oil, 352, 353 decornp. of, in soil, 349-351 effect on chemical processes in soil, 354 on microbial activity in Roil, 354 in emulsins, 254 esters, 209 formation by soil micro-organisms, 351 8-galactosidase sepn. from, 247-248 in glycolipids of acid-fast bacteria, 208, 209 labeled with C14,self-decompn. of, 54 oxidn. products, properties of, 108 oxygen effect on irradiation of, 38 radiation effect on, 30, 37 B in sedimentary rocks, 335 in soil, 337, 338, 347-348 Bacillua Pnegatherium, polysaccharides degradation of, 350 from, 352 detn. of, 344 Barium hydroxide, complex with starch, effect on plant nutrition, 354 327 source of, 348 Barry degradation, 139 Bensirnidazole, 1-(2-deoxy-~-“galacto- spectra of irradiated solutions of, 53 Carbon, isotope of mass 14, self-desyl”)-, oxidn. product, 130 compn. of compds. labeled with, Benzoic acid, p-hydroxy-, polyester, 77 54 Benzophenone, 4-methoxy-, 83 Cellobiose, as acceptor in transgalacBifidus factor, 165 tosylation, 262 “Blue value,” 308 oxidn. of, 184 2,3-Butanediol, from ethanol irradiaradiation effect on, 50 tion, 25, 26 reaction with trifluoroacetic acid, 62 Butanol, complex with amylose, struc- -, octa-0-acetyl-, reaction with HF, 86 ture of, 303 Cellobioside, methyl 8-, 136 in starch fractionation, 327 oxidized, alkali effect on, 154

SUBJECT INDEX, VOLUME

38 1

16

Cellobiosyl fluoride, hepta-0-acetyl-, 86 Cellulose, 138 acetates, 67 benzoatcs, 68 decompn. of labeled, in Roil, 351 hydroxyinethyl ether, 08 radiution effect 011, 33, 34, 58 reaction with trifliioacetic acid, 60,62 with trifluoroacetic anhydride, CAOi in soil, 349, 350 Chitin, in soil, 350, 352 Chloral hydrate, complex with amyloar, 307 Citric acid, 28 CIuyH, mononncchr~ride adsorption by, 338 Conformation, of glucose in lactose, 183 of hemialdala, 116, 117 of methyl cr-L-rhamnopyranoside oxidn. product, 110 Conformational analysis, of oxidized Me a-D-glucoside, 12(1 Cord factor, 207, 209 degradation of, 212, 213 effect on leucocytes, 231 enzyme inhibition by, 231 as hapten, 234 homologs (lower) of, 216217 isolation of, 210 occurrence of, 210 structure and activity of, 232 structure of, 210-212, 218 synthesis of, 212-216 toxicity of, 232-233 Critical concentration, the term, 302 Cyclohexanol, in starch fractionation, 301 Cytidine, oxidn. product, 128 picrnte, oxidn. product, 128 Cytosine, 3-8-n-glucopyranosyl-, picrate, oxidn. product, 128

Dextrins, from amylose irradiation, 34 from starch irradiation, 34, 35 Dialdehyde methanolate, the term, 107 Difuco-di-(lacto-N-tetraose), 171 Difuco-tri-(lscto-N-tetraoRe),171 Di- (Iacto-N-tctrttose), 171 p-Dioxane, 3,5-dihydroxy-2-~"-meth oxy-G-n"-methyl-, 107 m - Dioxano[6,4 -e][l,4]dioxepan, dihy droxy -0a- methoxy - 2 - phenyltrane-, 107 ni.-Dioxin-G-carboxaldehyde, 2-phenyl-, 155 Disaccharides, 91 structure of, 137 Dosimeters, 21

D

B

Dambonitol, oxidn. product,, 120 Delta rays, the term, 10 Dextrans, 7-ray effect on, 52 radiation effect 011, 35, 36 sulfate, labeled with C", 57

E Emulsins, carbohydrate8 in, 254 8-galactosidase in, 240 11-Epicorticosterone, 66 trifluoroacetylation of, 62 Epilactose. See Mannose, 4-0-8-n-galactopyranosyl-n -. Erythritol, 145, 149 from methanol-C14, 24 -, 1,2-di-O-rnethyl-, 122 -, 1-0-methyl-, 115 Erythronic acid, D-, 150 -, 2-O-8-D-galaCtOpyrarlOSyl-n-, 162 Erythrose, D-, 115, 142, 154 from D-glucose irradiation, 36 from hexose irradiation, 41 from n-mannose irradiation, 42, 43 phenylosazone, 142 -, 2-O-j3-~-galactopyranosyl-~-, 162, 163 -, 2-O-methyl-, 115 Ethanol, irradiation of, 23, 25 Ethyl alcohol. See Ethanol. Ethylene glycol, from methanol-Cl4, 24 from methanol irradiation, 24 5-Etiocholenic acid, 3@-hydroxy-,methyl ester, 66

Friictopyranose, 3-O-acetyl -l , 2 - 0 -is0 propylidene-n-, oxidn. product, 131 -, penta-0-acetyl-B-n-, reaction with HF, 86

382

SUBJECT INDEX, VOLUME

16

Fructopyranoside, methyl I,-0-methyl- -, methyl 6-O-rnethyl-a-~-, oxidn. product, 122 (I-D-, 93, 94 -, methyl 3,4,5-tri-0-acetyl-l-O-meth--, o-nitrophenyl (I-D-, 292, 293 yl-a-D-, 93, 94 -, o-nitrophenyl D-D-, in detn. of p-gaFructopyranosyl fluoride, 1-0-methyllactosidase activity, 241, 242 a-D(and &D)-, reaction with -, o(and p)-nitrophenyl P-D-, 260 NaOMe, 93, 94 -, nitrophenyl a-D-,29 -, 3,4,5-tri-O-acetyl-p-~-, 86 -, o-nitrophenyl I-thio-p-D-, effect on 8-galactosidase, 250 Fructose, D-, 294 D-, from dextran irradiation, 52 -, o(and p)-nitrophenyl I-thio-p-D-, 2CiO D-, effect on pectin irradiation, 34 -, phenyl a-D-,297 polysaccharides contg., formation by a-galactosidase standardization by, 292 bacteria, 351, 352 hydrolysis of, 293, 295, 298 I)-, radiation effect on, 38, 46, 47, 50, 54 of soil, 338, 343 -, propyl P-D-, enzymic synthesis of, 257 D-, structure of, moiety i n sucrose, 93 -, O-tOlyl (I-D-, 293 I)-, from sucrose irradiation, 51, 52 Galactopyranosides, P-D-, enzyme effect on, 260, 261 -, 3,0-anhydro-u-, osotriazole oxidn. 0-D-, hydrolysis of, 293, 294 product, 119 -, alkyl (Y-D-, 293 -, 3-0-8-D-galaCtOsyl-D-, 2G1 -, aryl (I-D-, 293 Fucose, D-, 262 from hydrolyzed bacterial cultures, Galactopyranosyl bromide, tetra-0-ace351, 352 tyl-a-D-, 165 of soil, 342, 347, 351, 352 Galactopyranosyl fluoride, 8-D-,90 Galactosamine. See Galactose, 2-amino-, 2-0-methyl-, 224 Fucosides, p-D-, 260 2-deoxy-. Fucosyl group, transfer of, by enzymes, Galactosan, oxidn. product, 118 Galactose, D-, 160, 161 262 Fulvic acids, of soil, 337 D-, a-galactosidase inhibition by, 298 2-Furfuraldehyde, 340 D-, p-galactosidase inhibition by, 280, G

284

-D-glucose interconversion, 179 Galactaric acid, 181 D-, incubation with melibiase, 297 Galactitol, from lactose, 187 D-, from isolactal, 200 Galactobiose, 297 D-, metabolism by yeasts, 179, 180 Galactofuranoside, ethyl B-D-,260 D-, mycolates, 212 -, o-nitrophenyl 6-D-,260 in polysaccharides from pathogenic Galactometasaccharinic acid, “(I”-D-. bacteria, 352 See Hexonic acid, 3 - d e o x y - ~ - of soil, 337, 338, 342 xylo-. D-, from wax D, 219, 220 Galactonic acid, D-, 182 -, 2-amino-2-deoxy-~-,of soil, 343, 3-47, D-, 1,4-lactone, 298 352 L-, 1,4-lactone, radiation effect on, 52 from wax D, 220 Galactopyranose, (I-D-, radiation effect -, 1,6-anhydro-3,4-0-isopropylideneon, 38 8-D-, 199 -, 1,2:3,4-di-O-isopropylidene-~-, 198 -, 6-deoxy-. See Fucose. Galactopyranoside, ethyl (I-D-, 293 -, 6-O-@-lactosyl-~-,198 -, ethyl 0-D-,enzymic synthesis of, 257 -, 2(and 6)-O-mycoloyl-~-,232 -, methyl WD-, 293 -, 2,3,4, 6-tetra-0-methyl-D-, 161 hydrolysis of, 295 Galactosidases, (I-, 239, 290 -, methyl P-D-, 161 p-, 239 D-,

SUBJECT INDEX, VOLUME a-,acceptor specificity in presence of,

294 8-, amino acids from, 253 8-, in animals, 240 8-, in a-L-arabinosyl group transfer, 256 8-, binding sites of, 281 p - , from caIf intestine, 246-248 8-, carbohydrate sepn. from, 247-248 &, chem. composition of, 252, 253 8-, detn. of activity of, 241-242 8-, from Escherichia coli, 242-246 8-, in galactosyl group transfer, 255, 263 a-,hydrolysis by, 293, 294 P-D-, hydrolysis of a-L-arabinosides by, 256 8-, hydrolysis of glycosides by, 258,259 8-, imidazolium group of, 266 a-,inactivation of, 298 8-, inhibition of, 273-280, 283, 284, 297 B-, kinetics of, 262, 264, 282 8-, manganese ion in activation of, 242 8-, mechanism of action, 285-289 o-, metals in, 252, 277 8-, in micro-organisms, 240 p - , mol. wt. of, 249 a-,occurrence of, 291 & D - , occurrence of, 178 a-,pH effect on, 297 8-, pH effect on, 254, 265 b-, in plants, 240 a-,properties of, 292 8-, properties of, from different sources, 248-249 &, specificity of, 260 8-, sulfhydryl group of, 273 D-D-, synthesis of, 179 a-,synthesis (induced) of, 298 8-, synthesis (induced) of, 290 8-, temp. effect on, 254 8-, ultraviolet spectrum of, 251 8-, units of enzyme activity of, 242 Galactoside, ethyl P-D-, in 8-galactosidase detn., 241 -, methyl a-D-,oxidn. product, 123 -, methyl 8-u-, oxidn. product, 123 -, methyl 4,G-o-benzylidene-8-~-, oxidn. product, 115 -, o-nitrophenyl 8 - ~ -257, , 275, 280, 284 hydrolysis of, 268-272, 277, 287

16

383

hydrolysis with 8-galactosidase, kinetics of, 262, 264, 283 p H effect on, 264-266 -, p-nitrophenyl P-D-, hydrolysis of, 268-272 -, o-nitrophenyl I-thio-@-D-,280, 281, 282,284 -, p-nitrophenyl I-thio-fl-D-, 282 -, phenethyl l-thio-p-D-, 271, 281 --, phenyl &D-, 280 in 8-galactosidase detn., 241 hydrolysis of, 290 -, phenyl I-thio-D-D-, 280 Galactosides, P-D-, 261 @- D- , 8-galactosidase inhibition by, 280 -, I-thio-D-D-, hydrolysis of, 285 Galactosyl group, transfer by enzymes, 255, 262, 263, 294 Galactosyl phosphate, D-, 176 --, a-D-, 177, 179 Galactotriaose, 297 Galactowaldenase, 177 Gamma rays, effect on acid solns., 19 effect on L-ascorbic acid, 30 Gentianose, 137 Gentiobiose, 88 Glucans, a- and b-, fractionation, 311 Glucaric acid, D-, 181 of soil, 343 Glucitol, D-, acetals, 73 D-, derivs., 65, 66 D-, 1,3:2,4-diacetals, 74 D-, hexanitrate ester, 69 D-, isopropylidene acetals, 77 D- , from lactose, 187, 188 D-, radiation effect on, 38, 45, 47, 48-50 -, 3-0-(acetoxymethyl)-5-0-acetyl-l,6di-O-benzoyl-2,4-0-methylene D-, 70, 73 -, 5-0-acetyl-6-O-benzoyl-l ,3: 2,4-di-Omethylene-n-, 70 -, 5-O-acetyl-l , 6-di-0 -benzoyl-2,4-0methylene-D-, 70 -, 5-O-acetyl-l , 3: 2,4-di -0-ethylideneD-, 66 -, 6-O-acetyl-l , 3: 2,4-di-O-ethylidene5-O-methyl-~-,66 -, 5-O-acetyl-l , 3: 2,4-di-O-ethylidene6-O-trifluoroacetyl-D-, BG -, 3-O-acetyl-l,5,6-tri-O-benzoyl-2,40-methylene-D-, 72

384

SUBJECT INDEX, VOLUME

-, 5-O-acetyl-l , 3,6-tri -0-benaoyl-2 ,4-

16

D-,from D-glucose irradiation, 35, 45 D-,lactone, 54 0-methylene-D-, 71, 72 -, 2,5-anhydro-1,6-di-O-benzoyl-~-, D-,1,4-lactoneJ radiation effect on, 52 D-, from starch irradiation, 35 oxidn. product, 122 -, I ,5-anhydro-4-0-@-~-galactopyrano- D-, from sucrose irradiation, 51, 52 -, 2-keto-. See Hexulosonic acid, DSyl-0-, 199 arabino-. -, 2,4-0-benzylidene-o-, 77 -, tetra-O-methyl-D-, 1,4-lactjoue, 162 -, 1,6-di-O-acetyl-2,4:3,5-di-O-methylGlucopyranose, WD-, radiation effect ene-D-, 73 -, 5,6-di-O-acetyl-l,3:2,4-di-O-methylon, 38 -, 1,6-anhydro-8-~-,88, 96, 199 ene-D-, 73 -, l16-di-O-acetyl-2,4-O-methylene-~-, triacetate, reaction with H F , 87 -, 1,6-anhydr0-2-deoxy-2-p-toluene74, 77 sulfonamido-@-D-,90 -, 3,5-di-O-acetyl-2,4-0-met hylene-l,6-, 0-a-L-fucopyranosyl-(1-4) -0-B-D-gadi -0-propion yl -D-, 74 lactopyranosyl-(1+4)-~-,168 -, 1,6-di-O-benzoyl-2,4:3,5-di-O-nieth-, 0-a-L-fucopyranosyl - (1 -4)-0-@ - Dylene-n-, 70, 71, 72 galactopyranosyl- (1 +3) -0- (2-, 1 , 3:2,4-di-O-ethylidene-~-,77 acetamido -2-deoxy -@ - D-gluco derivs., 65 pyranoeyl)-(l-3)-0-@-~-galac-, 1,3: 2,4 - di - 0 - ethylidene8,6 - di -0topyranosyl- (1+4) -D-,169, 170 (trifluoroacetyl) -D-,66 -0-@-D-ga-, 1,3:2,4-di-O-ethylidene-5-0-methyl--, O-~-~-fucopyranosyl-(l-+2) lactopyranosyl- (1-4) -0- [a- L D-,66 f ucopyranosyl- (1+3) -1-D-, 169 -, 1 , 3:2,4-di-O-methylene-~-,77 -, 2,4:3,5-di-O-methyIene-l,6-di-O-pro-, 4-0-~-~-galactopyranosyl-n-. See pionyl-D-, 74 Lactose. -, 4-O-@-~-galactopyranosyl-~ -. See -, 0-@-D-galactopyranosyl-(1-3) -0-(2acetamido-2-deoxy-8-D-gluceLactitol. pyranosyl)-(l-+3)-0-B-~-galac-, hexa-0-acetyl-, D-, 76 topyranosyl-(l+4)-~-, 169 -, 2,4-0-methylene-~-,72, 74, 78 -, 2,4-0-methylene-l,6-di -0-propionyl- -, 0-~-~-galactopyranosyl-(1+3)-O-[~L-fucopyranosyl-(1-4))-0- (2D-,74 acetamido - 2 - deoxy -8 - D-gluco -, 1,3,5,6-tetra-O-acetyI-2,4-0-benzylipyranosyl)-(l-+3)-0-@-1~-gnlac dene-D-, 76 topyranosyl-(1+4)-~-,170 -, 1,3,5,6-tetra-O-acetyI-2,4-O-meth-, 1,2,3,4-tetra-O-acetyl-@-u-, 198 ylene-D-, 74 -, l15,6-tri-0-acetyl-2,4-O-niethylcnc - -, tri-0-acetyl-1,Z-anhydro-a-I>-,80 Glucopyranoside, a - D - glucopyrarionyl D-, 73 a-D-.See Trehalose. -, 1,5,6-tri-0-benzoyl-2,4-0-methyl-, methyl WD-, 295 ene-n-, 72 -, 1,3: 2,4: 5,6-tri-O-methylene-u-, 74, oxidn. product, 123-127 radiation effect on, 51 75, 77, 79 -, methyl @-D-,88 polyester from adipic acid and, 78 Glucofuranose, 3,5-0-benzylidene-l,2- -, methyl 3-0-beneoyl-4,6-0-benzylidene-a-D-, 63, 64 0-isopropylidene-6 -0- (methyl -, methyl 6-deoxy-6-fluoro-@-~-, 95 sulfonyl)-a-D-, 95 -, 3,5-O-benaylidene-l , 2-04 sopropyli- -, methyl 2-deoxy-2-(N-methyl-p-toluenesulfonamido)-wD-,93 dene-a-n-, 95 -, methyl 2-deoxy-2-p-toluenesulfonaGluconic acid, D-,161, 182 D-,from dextran irradiation, 36, 52 mido-@-D-,90 D-,from D-glucitol irradiation, 40 -, methyl 2-0-methyl-a+, 93

SUBJECT INDEX, VOLUME

-, methyl 6-O-trityl-a-~-,oxic'n. prod-

16

385

-n-galactose interconversion, 179 uct, 122 8-galactosidase inhibition by, 280, Glucopyranosylamine, 3,4,6-tri-O-ace284 tyl-2-amino-2-deoxy-p-~-, 96, 97 from D-glUCitOl irradiation, 48 Glucopyranosyl azide, &D-, 96 from glycolipids, 209 -, 2-amino-2-deoxy-p-n-, derivs., 96, as lactoee precursor, 174 103 from methyl a-u-glucopyranoside -, tetra-0-acetyl-pa-, 97 irradiation, 51 -, 3,4,6-tri-O-acetyl-2-amino-2-deoxy- mycolates, 212 B-D-, 97 oxidn. of, 182 Glucopyranosyl bromide, tetra-0-aceradiation effect on, 32, 35, 38, 39, tyl-ff-D-,66, 88, 96 42, 44, 45, 47, 54 -, 3,4 ,G-tri-O-acetyl-2-amino-2-deoxy- from starch irradiation, 35 ff-D-, 97 structure of, moiety in sucrose, 93 Gliicopyranosyl fluoride, CY-D-, 95 from sucrose irradiation, 51, 52 8-D-, 94 polysaccharides containing, formation CY-D-,reaction with NaOMe, 92 by soil bacteria, 351, 352 0-D-,reaction with NaOMe, 89 D-, oi soil, 337, 338, 342, 352 -, 2-amino-2-deoxy-a-~-,reaction with -, 2-acetamido-2-deoxy-o-, oligosacchaNaOMe, 92 rides contg., 168 -, 4,6-0-benzylidene-~-~-, 95 of soil, 343 -, 2-deoxy-2-(N-niethyl-p-toluenesul--, 2-ncetamido-2-deoxy-3(4, and 6 ) - 0 fonamido)-p-u-, reaction with @-u-galactoSyl-I>-, 261 NaOMe, 93 -, 2-amino-2-deoxy-, of soil, 343, 347, -, 2-deoxy-2-p-toluenesulfonamido-p352 D-, reaction with NaOMe, 90 -, 2-amino-2-deoxy-i)-,294 -, 2-O-methyl-n-, anomers, 94 mycolates, 212 -, 2-o-methyl-p-~-, reaction with from wax D, 220 NaOMe, 93 -, 3-amino-3-deoxy-~-,derivs., 114 -, tetra-0-acetyl-a-n-, 87 -, 2-amino-2-deoxy-2(and 6)-0-myco-, tetra-0-acetyl-p-D-, 87 lOyl-D-, 232 -, 2,3,4-tri -O-benzoyl-a-~-,88 -, 2,3,4-tri-0-benzoyl-6-0-(tetra-0-ace--, 1,2-anhydr0-4-0-p-~-galactopyrnnoSyl-D-, 199 acetyl-@-D-glucopyranosyl)-aD-, 88 -, 1,G-nnhydro-4-O-~-~-galactopyra1io-, G-O-trityl-cu-D(andp-D)-, 95 Syl-b-D-, 199 Glucosamine. See Glucose, 2-amino-2- -, 3(4, and 6)-0-a-~-arabi11osy1-D-, 256 deoxy-. -, 6-deoxy-6-fluoro-D-, 95 -, N-acetyl-. See Glncose, 2-acetamidoderivs., 95, 101 2-deoxy-. -, 2,3: 5,6-di-O-isopropylidene-~-, di Glucose, D-, 161 ethyl acetal, 165 D-, from amylose irradiation 34 -, 1,2-0-ethylene-4-0-p-~-galactopyD-, conformation in lactose, 183 ranosyl-D-, 198 D-, from cord factor, 211, 212 -, &O-&u-galactopyranosyl-D-, 168, 2GO decomp. of labeled, in soil, 351 -, 3-O-p-~-galactosyl-o-, 8-galactosidase D-, from dextran irradiation, 36, 52 in prepn. of, 257 D-, effect on pectin irradiation, 34 hydrolysis of, 255, 256 D-, esters, labeled with CI4 in lactose -, 3(and 4)-O-p-D-gttlaCtOSyl-D-, 261 biosynthesis, 174 -, 4(and 6)-O-p-D-galaCtOSyl-D-,phenylD-, ethyl acetoacetate condensation osazones, 261 product, oxidized deriv., 121

386

SUBJECT INDEX, VOLUME

-,

16

methyl 4,6-0-benzylidene-2-O-p-tolylBUlfOnyl-a-D-, 64 -, methyl 4,6-0-benzylidene-3-O-triBuoroacetyl-a-D-, 63 -, O-u-D-galaCtOSyl-(lj6)-~-a-D-galaCmethylation of, G3 tOSyI-(l+G)-D-, 294 -, 4-0-8-~-glucopyranosyl-~-. See Cello- -, methyl 4,6-O-o-bromobenzylidenea - ~ -oxidn. , product, 115 biose. -, 1,2- 0 -isopropylidene -3,5,6 - tri - 0 - -, methyl 4,6-0-o(and p)-chlorobenzylidene-a+-, oxidn. product, 115 (trifluoroacetyl) -D -, 61 -, methyl 4,6(and 5,G)-di-O-methyl-a-, 6-O-@-laCtoSyl-D-,198 D-, oxidn. product, 122 -, 2(and 6)-O-mycoloyl-o-, 232 -, 1,2,3,4,6 -penta - 0 - acetyl -CI (or 8) - -, methyl 4,6-0-ethylidene-a-~-,oxidn. product, 115 D-, reaction with HF, 87 -, 3,4,6-tri-O-acetyl-2-amino-2-deoxy--, methyl 4,6-0-ethylidene-B-~-,69 -, methyl tetra-0-acetyl-a-w, 67 D-, derivs., 80 -, 2,3,4-tri-O-methyl-n-, from cord -, methyl tetra-0-propionyl-a+, 67 -, methyl 2,3,4,G-tetra-O-(trifluorofactor, 212 acetyl)-a-D-, 61 -, 2,3,G-tri-o-methyl-~-,161 Glucose-C14, D-, self-decompn. of, 54, 55, Glucosone, D-, 161 from dextran irradiation, 52 56, 57 from D-fructose irradiation, 46 Glucose oxidase, in soil, 349 from D-glUCOSe irradiation, 46 GlucoBide, benzyl 2,3,4 ,0-tetra-0-(trifrom D-mannose irradiation, 43, 44 fluoroacetyl)-P-D-, 61 from sucrose irradiation, 51, 52 -, methyl WD-, oxidn. product, 123 Glucosyl bromide, tetra-0-acetyl-a+, -, methyl P - D - , 92, 294 reaction with silver fluoride, 87 oxidn. product, 123 Glucosyl phosphate, D-, 176, 177 oxidized, alkali reaction with, 154 a-D-,177 -, methyl 4-0- (1-acetoxyethyl)-6-0Glucuronic acid, D-, from dextran irradiacetyl-p-D-, 69 ation, 36 -, methyl 2(and 3)-0-acetyl-4,6-0D-, from D-glucose irradiation, 35 benzylidene-a-D-, 64 from starch irradiation, 35 -, methyl 3-amino-4,6-0-benzylidene- D-, D-,from sucrose irradiation, 51,52 ~-3-dcoxy-D-,114 -, tri - 0 - acetyl - 1- bromo - 1- deoxy - I), -, methyl 2-arnino-2-deoxy-8-1~-,92 methyl ester, 66 -, methyl 2-0-benzoyl-4,6-0-benzyli- Glyceritol, 295 dene-a-o-, 63, 64 from methanol-Cl4, 24 -, methyl 2-0-benzoyl-4,6-0-benzyli- 1 (and 2)-phosphates, x-ray effect on, dene-3-O-p-tolylsulfongl-a-~-, 28 64 -, 2-0-@-~-galactopyranosyl-, 164 -, methyl 4,6- 0 - benzylidene - CY - D - , -, 1-0-methyl-&, 122, 126, 127 oxidn. product, 107, 111-115, -, 1-0-methyl-L-, 122, 126 121 Glyceritol-l,S-Cs14, in lactose biosynoxidn. product, alkaline degradation thesis, 175 of, 155 Glycerol. See Glyceritol. -, methyl 4,6-O-benzylidene-@-~-,Glycerose, phenylosazone, 152 oxidn. product, 115 -, 3-O-~-arabinofuranosyl-, phenylosa-, methyl 4,6-0-benzylidene-3-deoxyzone, 139 3-phenylazo-a-~-,114 Glycine, N-lactosyl-, 194 -, methyl 4,6-0-benzylidene-2,3-di-Glycolic acid, radiation effect on, 30-31, 0-(trifluoroacetyl)-a-D-, 61, 63 32

-, 6-0-8-D-galaCtoSyl-D-, 8-galrtctosidase i n prepn. of, 257 hydrolysis of, 255, 256

SUBJECT INDEX, VOLUME

Glyc01ic-C~~ acid, calcium salt, 32 Glycolipids, of acid-faat bacteria, 209 biological activities of, 230 carbohydrates of, 208, 209 glycosidic, 223 peptido-, 218 Glycols, a-, reaction with periodates, 105, 106 Glycopeptides, 222 Glycosidases, 261 reactions of, 255 Glycosides, 207 6-deoxyB-fluoro-, reaction with almond emulsin, 95 enzymic synthesis of, 257 hydrolysis of, by &galactosidases, 258, 259 inositol, 207 phenolic, of soil, 339, 344 phosphatidyl-inositol, 228 prepn. of, 88 -, methyl 4,6-0-alkylidene-, oxidn. products, 111 Glycosiduronic acids, of soil, 346 Glycosylamines, rearrangements of, 194 Glycosylation, trans-, glycosidase catalyzed, 255 Glyco~ylazides, 102 Glyco~ylfluorides, properties of, 98 -, 2-amino-2-deoxy-~-,properties of, 100 Guanosine, oxidn. product, 128 Guloheptulosan, L-. See Heptulopyranose, 2,7-anhydro-p-t-gulo-. Gulonic acid, L-, from D-glucitol irradiation, 49 D- (and L)-, 1,4-lttctones, radiation effect on, 52 Gulosan, oxidn. product, 118 Gulose, L-, from D-glUCitOl irradiation, 48 -, 3-amino-l,6-anhydro-3-deoxy-~-, 118 Guloside, methyl WD-, oxidn. product, 123 -, methyl 4,6-0-benzylidene-p-~-, oxidn. product, 115 Gynolactose, 168 G value, detn. of, 18 the term, 18

H Hemiacetals, 108 Hemialdal group, 106, 110

16

387

Hemialdals, 108, 109 conformation of, 116, 117 Hemicelluloses, decomp. of labeled, in soil, 351 fractionation of, 311 in soil, 349 Heptonic acid, 4-O-~-~-galactopyranoSyl -D-glyCt?rO-D-gUb,195 Heptopyranose, 1,7-anhydro-~-glycero8-o-gulo-, oxidn. product, 129 Heptopyranosi de, phen yl D-glycero -a-D galacto-, 292 Heptoses, 0-methyl-, of soil, 342 Heptulopyranose, 2,7-anhydro-p-~altro-, oxidn. product, 129 -, 2,7-anhydro-&~-gulo-,oxidn. product, 129 -, 2,7-anhydro-p-~-ido-,oxidn. product, 129 1-Hexene, effect on EtOH irradiation, 23 Hexitols, cyclic acetals, 72, 74 derivs., 76 radiation effect on, 47 Hexokinase, 254 Hexonic acid, 3-deoxy-~-zylo-,189 -, 2-0x0-D-arabino-. See Hexulosonic acid, D-arabino-. Hexosamines, 66 Hexose, 3,6-anhydro-~-arabino-, phenylosazone, 191 -, 3,6-anhydro-~-ribo-,phenylosazone, 191 phenylosotriaxole, 192 -, 3,6-anhydro-4-0-~-~-galactopyrttnosyl-D-ribo-, phenylosazone, 192 phenylosotriazole, 192 -, D-arabino-, phenylosazone, 190, 191 -, 2-deoxy-~-lyxo-,262 -, 3-deoxy-~-xylo-,262 -, 4,5 -di -0- acetyl -3,G -anhydro - D arabino-, phenylosazone, 191 -, 3,6-dideoxy-o-zylo-, 262 -, D-lyzo, phenylosazone, 201 -, 2-oxo-~-arabino-.See Glucosone, D-. Hexoses, deoxy-0-methyl-, of glycolipids, 209, 223 G-deoxy-, of soils, 347 0-methyl-, of soils, 342 radiation effect on, 36 of soil, detn. of, 344 Hexosides, 8-deoxy-~-xylo-,2GO

388

SUBJECT INDEX, VOLUME

16

Hexosulose, D-arabino-. See Glucosone, by amylose, 301, 302 by starch, 301 D-. Hexulose, 1,6-anhydro-~-efylhro-,2,3complex with amylose, structure of, 303 phenylosazone, 201 -, 1,5-anhydro-4-0-8-~-galactopyrano-Ionization, by radiation, 14, 15 syl-D-arahino-, 201 Isoglucal, D-,and pentaacetate, 200 -, 1,5-anhydro-4-0-j3-~-galactopyranoIsolactal, 200 syl - D - erylhro - , 2,3 - phenyl- Isomaltose, from dextran irradiation, 36 Isomaltotriose, from dextran irradiation, osazone, 201 -, 5,6-dideoxy-~-threo-,131 36 -, 4-0-8-~-galactopyranosyl-~-arabino-. Isosaccharinic acid, “@’-D-, 189 iia , I -D-, calcium salt, 163 See Lactulose. -, D-lyzo-. See Tagatose, D-. ‘ W ’ - D -lactone, , 188, 189 Hexulosonic acid, D-arabino-, 354 K from D-fructose irradiation, 46 from D-mannOSe irradiation, 43 Ketone, isobutyl methyl. See 2-Pentafrom sucrose irradiation, 51, 52 none, 4-methyl-. -, 4-O-8-D-galaCtOpyranOSyl-D-aTabinO-,Ketones, alkyl aryl, 68 183 Ketoses, 1-amino-1-deoxy-, 194 5-Hexulosonic acid, D - ~ ~ / z o47- , Koenigs-Knorr reaction, 197, 198 Hibbert, Harold, obituary of, 1 1 Humic acids, 353 Lactal, 200 of soil, 337, 346 hydroxylation of, 200 Humus, 336, 337, 348 -, hexa-0-acetyl-, 165 Hyaluronic acid, radiation effect on, 33 ozonolysis of, 200 Hydrogen, formation in irradiation of prepn. of, 199 water, 16-19 reaction with alkali, 200 Hydrogen peroxide, formation in irradiarearrangement of, 200 tion of water, 17, 18 Hydrolysis, of 0-arabinosylglucoses, 256 Lactaminic acid. See Neuraminic acid, N -acetyl-. of glyco~idesby 8-galactosidases, 258, Lactase. See Galactosidme, P-D-. 259 Lactic acid, 28 of trifluoroacetates, 61, 63 Hydroxyl (radical), formation in irradia- Lactitol, 187, 188 IAaclobacil~u8 bijidus, 166, 107 tion of water, 16, 19 var. pennsylvanicus, 167 I Lactobionic acid, 161, 180, 182 I dohep t ulosan , u -.See Hcpt ulopy ranose , calcium salt, 183 oxidn. of, 183 2,7-anhydro-&-~-ido-. Idose, 3-amino-l,6-anhydro-3-deoxy-~-, 1,5-lactone, 162 118 -, 2-keto-. See Hexulosonic acid, 4-0-8Inositol, derivs., 228 D -galactopyranosyl -D -arabino-. glycosides, 207 -, octa-0-methyl-, 162 hexaacetate, 2% Lacto-N-difucohexaose, 168, 170 myo-, and phosphates, 227 Lactodif ucotetraose. See Glucopyranosc, and phosphates, of soil, 343 0-a-L-fucopyranosyl-(1-12) -0myo-, from phospholipids, 225 8-n-galactopyranosyl - (1+4) -0myu-, radiation effect on, 38 [a-~,-fucopyranosyl-(1+3)J-o-. Invertase, in soil, 349 Lacto-N-fucopentaose I. See GlucopyraIodine, adsorption, by amylopectin, 301, nose, 0-a-L-fucopyranosyl(1+2)-0-8-~-galactopyranosyl 303

SUBJECT INDEX, VOLUME

(1-+3)-0-(2-acetamido -2-deoxy 8 - D - glucopyranosyl) - (1-+3)-08-D-galactopyranosyl-(1-+4)-~-. Lacto-N-fucopentaose 11. See Glucopyranose, 0-8-~-galactopyranosyl(143) - 0 - [(Y- L - fucopyranosyl (1-+4)] - 0 -( 2 - a c e t a m i d o - 2 - d ~ oxy-8-u-glucopyranosyl) - (1-+3) O + - D -galactopyranosyl-(1-+4)D-.

Lacto-N-pentaose, 171 Lactopyranoside, ethyl a-,and derivs., 195 -, ethyl hepta-0-acetyl-a-, 195 -, ethyl hepta-0-methyl-a-, 195 Lactosamine. See Lactose, 2-amino-2deoxy-. Lactose, alkali degradation of, 188-189 alkyl carbonates, 190 a-, 260, 261 a - and B-, 203-204 a-, hydrate, 203 anhydro derivs., 198-199 8-, 261 biosynthesis of, 173-178 in blood, 173 compd. with pyridine, 193 in chromatography, 205 crystalline forms of, 201-202 derive., 8-D-galactosidase hydrolysis of, 179 detn. of, 182 diethyl dithioacetal, 195 dithioacetals, 195 esters, 195-197 with fatty acids, 196 in 8-galactosidase activity detn., 241 8-galactosidase inhibition by, 280 in prepn. of, 257 D-glucose conformation in, 183 hydrogenolysis of, 188 hydrolysis of, 161, 181, 255, 256, 268271, 283 labeled with C", 174-175 metabolism of, 178-180 mutarotation of, 201, 203 nitrogen heterocyclic compda. from, 193, 194 occurrence of, 165 octanitrate ester, 196 octa(phenylurethan), 196

16

389

"over-oxidn.", 186 oxidn. of, 164, 180, 181-187 oxime, 183 phenylhydrazone, hydrogenation of, 192 phenylhydrazones, 192 phenylosazone, lG4, 189-192 heptaacetate, 191 oxidn. of, 190 physical properties of, 201-206 in plants, 173 reaction with NHa and amino compds., 193 with glycine, 194 with hydrazines, 189 with proteins, 194 redn. of, 187-188 electrolytic, 188 reversion of, 181 skin absorption of, 173 solubility of, 204 spectrum of, 205 structure of, 160-165 thermodynamic properties of, 205 unsatd. derivs., 199-200 in urine, 172-173 -, N-acetyl-0-acetylneuramino-, 171 -, N-acetylneuramino-, 171, 172 -, 2-amino-2-deoxy-, 192 -, anhydro-, phenylosazone, and its pentaacetate, 191 -, 0-fucosyl-, 168 -, hepta-0-acetyl-, 196 -, octa-0-acetyl-, 19F (Y anomer, 195,196 8 anomer, 195, 196, 197 -, octa-0-[p-(p-nitropheny1azo)benzoyll-a (and 8)-, 196 -, 1-thio-, 195 Lactose4 -C", reaction with proteins, 194 Lactoseen, hepta-0-acetyl-, 201 Lactoside, benzyl hepta-0-acetyl-8-, 198 -, (2-chloroethyl) hepta-0-acetyl-8-, 198 -, (3-chloropropyl) hepta-0-acetyl-8-, 198 -, cholesteryl hepta-0-acetyl-8-, 198 -, deoxycorticosterone hepta-0-acetyl8-, 198 -, (2-hydroxyethyl) hepta-0-acetyl-8-, 198 -, l-menthyl hepta-0-acetyl-8-, 198

390

-,

SUBJECT INDEX, VOLUME

methyl j3-, 136, 197

16

from starch irradiation, 35 radiation effect on, 50 Maltotriose, from amylose irradiation, 34 Mannans, 139 Lactosides, 197-198 D-, 91 of alkaloids, 194 in hexokinme, 254 Lactosone, 161, 192 in soil, stability of, 350 oxidn. of, 183 “Manninositose,” 225 Lactosuria, 172 Mannitol, D-,49, 295 Lactosylamine, derivs., 193 D-, hexanitrate, 69 derivs. of sulfa drugs, 194 D-, isopropylidene acetals, 77 -, N-octadecyl-, 193 D-, radiation effect on, 47 -, N-p-tolyl-, 194 of soil, 343 Lactosyl bromide, hepta-0-acetyl-a-, -, 0-(acetoxymethy1)-0-acetyl-l,3: 2,5196, 197, 198 di -0-met hylene-D -,75 hydrogen bromide removal from, 201 -, di-0-(acetoxyme thyl) -di-0-acetylreaction products with alkaloids, 194 2,5-0-methyIene-n-, 75, 76 with pyridine, 194 -, 3,4-di-0-acetyl-l,2:5,6-di-0-i~oproredn. of, 199 pylidene-D-, 76 Lactosyl chloride, hepta-0-acetyl-a-, -, hexa-0-acetyl-n-, 75 196, 197 -, hexa-O-(trifluoroacetyl) - D - , 61 Lactosyl fluoride, a-,197 -, 1,3:2,5: 4,6-tri -0-benzylidene-D-,76 Lactosyl iodide, hepta-0-acetyl-a-, 197 -, 1,3: 2,5:4,6-tri -O-methylene-~-,75 Lactosyl phosphate, 178 Mannobioside, phosphatidylinositol D-, a- and j3-, 196 227-228, 237 Lncto-N-tetraose. See Glucopyranose, Mannonic acid, D-, from D-mannose ir 0-8-D-galactopyranosyl- (1-+3)radiation, 39, 42, 43, 44 0 - (2-acetamido-2-deoxy-j3-~-Mannopyranose, 1,6-anhydro-2,3-0-isoglucopyranosy1)- (1+3) -0-j3-Dpropylidene-D-, 165 galactopyranosyl- (1-14) - D - . -, 6-0-a-~-mannopyranosyl-a-~-, 228 Lactotriaose, 179, 261 -, penta-0-acetyl-&D-, 47 Lactulose, 189 Mannopyranoside, a-D(and b-D)-manno-, 1-amino-1-deoxy-, 192 pyranosyl W D - , 91, 92 Levans, formation by soil micro-organ- -, methyl a - D -90 , isms, 351 -, methyl 3-amino-3-deoxy-a-~-,hydroLevoglucosan, diacid from, 129 chloride, 126 oxidn. product, 118 , 91 Mannopyranosyl fluoride, a - D -90, Levomannosan, oxidn. product, 118 Mannose, D-, compds. from irradiation LYXOSe, D-, 189 of, 40 D-, from D-mannose irradiation, 42 D-,from glycolipids, 209 D-, from n-mannitol irradiation, 47, 49 M D-,polymerization in irradiation of, 44 Magnesium sulfate, in starch fractionapolysaccharides contg., formation by tion, 328 soil bacteria, 351, 352 Magnesium sulfite, in starch fractionaD-, radiation effect on, 39-45, 47 tion, 314 of soil, 342 Malic acid, 28, 29 D-,from wax D, 219, 220 Maltose, aa acceptor in transgalactosyl- -, 4-0-~-~-galactopyranosyl-~-, 165, 200 ation, 262 -, tetra-O-acetyl-4-0-(tetra-O-acetyl-~from amylose irradiation, 34 D -galac t opyranosyl) -a+-,165 oxidn. of, 184 -, 2,3,4, B-tetra-O-rnethyl-~-,228

-, 2-naphthyl 1-thio-B-, 199 -, phenyl 8-, 199 -, phenyl 1-thio-8-, 198

SUBJECT INDEX, VOLUME

16

391

-, 2,3,4-tri-O-methyl-~-,228 8-galactosidase inhibition by, 283 Mannose-04, D-, self-decompn. of, 57 oxidn. products, 108 Mannose-I-C", D-, radiation effect on, 41 of soil, 337 Mannoside, methyl a-D-,294 in soil, stability of, 350 oxidn. product, 123 -, 1,6-anhydro-, oxidn. products, 118 -, methyl B-D-, oxidn. product, 123 -, 3,6-anhydro-, osotriazoles, 119 -, methyl 4,6-0-benzylidene-a-~-, Mor, 336, 348 oxidn. product, 115 Mucic acid. See Galactaric acid. -, methyl 4-0-8-D-galaCtOpyranOSyl-a- Mull, 336, 348 D-,201 Muramic acid, 220 Mannosyl fluoride, 3,6-di-O-acety1-4- Mutarotation, of osazones, 190 0 - (2,3,4,6-tetra-0-acetyl-8 - D - Mycocerosic acids, 224, 230 glUCOSyl)-a-D-, 86 Mycolanoic acid, 211 Mannotriaoside, phosphatidylinositol D-, -, 3,x-dihydroxy-, 218 238 -, 3-hydroxy-, 211 Mannuronic acid, D-, from D-mannitol -, 3-hydroxy-x-methoxy-, 218 irradiation, 47 [I-Test], 211 D-, from D-mannOSe irradiation, 39, 42, -, 3-hydroxy-x-oxo-, [3-BCG], 211 43, 45 Mycolic acids, 209, 211 Melezitose, 137 biological activity of, 230, 235 Melibiase. See Galactosidases, a - . from cord factor, 211 Melibiose, hydrolysis of, 290, 293, 294 esters with monosaccharides, 212 Methanol, amylopectin precipn. by, 226, as haptens, 234 227 nomenclature of, 211 irradiation of, 24 Mycoside A, 209, 223, 224 reaction with trifluoroacetates, 61 Mycoside B, 207, 209,223, 224 reaction with methyl 4-0-methyl-2,3- Mycoside C, 207, 223, B4-225, 226 di - 0 - (trifluoroacetyl) - (Y - L - Mycosides, 209, 223 rhamnopyranoside, 64,65 Methanol-04, self-decompn. of, 24, 54 N Michaelis constant, of hydrolysis of Neolactose. See Altrose, 4-0-8-~-galactoglycosides with 8-galactosidases, pyranos yl -D-. 258 -, a-chloroacetyl-, 197 Milk sugar. See Lactose. Neuraminic acid, 171 Monofuco-di-(lacto-N-tetraose),171 Monofuco-lacto-N-tetraose I. See Gluco- -, N-acetyl-, 171 Nitrogen, in soil, 337, 347 pyranose, 0-a-L-f ucopyranosylNucleic acids, phosphate from, after (1-2) -0-p- D-galactopyranosyl irradiation, 27 (1-3) -0-(2-acetamido-2-deoxyradiation effect on, 26-28 &D - glucopyranosyl) - (1+3) -0Nucleosides, oxidn. products, 127 8-~-galactopyranosyl-(l+4)-D-. Nucleotides, 15G Monofuco-lacto-N-tetraose 11. See Glupurine, radiation effect on, 28 copyranose, 0-8-D -galactopyrapyrimidine, radiation effect on, 28 nosyl - (143)- 0-[a- L - fucopyra nosyl- (1-4)]- 0-(2-acetamido-20 deoxy - 8 - D - glucopyranosyl) (1-3) - 0 - j3 - D - galactopyra - Obituary, of Harold Hibbert, 1 nosyl-(l+4)-~-. 1-Octanol, in amylose fractionation, 304 Monofuco-tri- (lacto-N-tetraose) , 171 2-Octanol, in starch fractionation, 301, Monosaccharides, adsorption by clays, 302 338 Octyl alcohol. See 1-Octanol.

392

SUBJECT INDEX, VOLUME

Oligosaccharides, as acceptow in glycosy1 transfers, 262 2 -acetamid0 - 2 - deoxy - u - glucose contg., 168 from cow’s milk, 171 a-D-galactose-contg., 290 lactose-contg., of milk, 165-171 nitrogen-contg., 168 nonreducing, 91 prepn. of, 88, 198 sialic acid-contg., 168 in soil, stability of, 350 by transglycosylation, 179 Osazones, anhydro derivs., 191 formation of, mechanism of, 192 mutarotation of, 190 Oxalacetic acid, 29 -, hydroxy-, 30 Oxyalginic acid, 153 derivs., 158 Oxyamygdalin, 136 Oxyamylopectin, 143, 145 Oxycellulose, 108, 137, 140 acetates, 152 alkaline degradation of, 153, 154, 156 cuprammonium fluidity of, l r i nitrates, 151 oxidn. of, 150 physical properties of, 146-147 prepn. of, 146 reaction with a h . , 150, 151 with N compds., 147-148 redn. of, 149 sodium dichromate, 108 stabilization towurd alkuli, 156 uties of, 158 -, amino-, 148 Oxydextran, 153 Oxyinulin, 153 derivs, 158 Oxyraffinose, 137 Oxystarch, 139 aldehyde content of, 141 derivs. as antitubercular s u bs tances, 158 hydrogenation of, 145 oxidn. of, 145 physical properties of, 141 prepn. of, 140 reactions of, 142

16

reaction with N H 3 , 143 with MeOH, 142 with N compds., 144-145 water-soluble, 158 uses of, 157-158 Oxysucrose, 108, 134-135 pheoylhydrazine deriv., 140 Oxytrehalose, 135, 136 Oxyxylan, 152

P Pectins, radiation effect on, 33, 34 in soil, stability of, 350 a-pentanone, 4-methyl-, in starch fractionation, 302 Pentasol. See Amy1 alcohols. Pentonic acid, 3-deoxy-2-C-(hydroxymethyl)-, 167 Pentopyranosides, methyl a - ~ -osidn. , products, 109 Pentoses, of soils, detn. of, 345 Peptides, reaction of, with phenyl trifluoroacetate, 62 with trifluoroacetic anhydride, 62 from wax D, 220 Periodates, oxidn. of a-glycols by, 105, 106 Phenols, reaction with acyl trifluoroacetates, 81 Phosphates, from nucleic acids after irradiation, 27 Phosphoglycolipids, 209, 225 biological activity of, 231, 234 Phospholipids, 225 antigenic effect of, 233 nitrogen-contg., 233 nitrogen-free, 225 Phosphoric acid, alkyl entcrri, rutlirtlion effect on, 28 Phthienoic acids, 230 as haptens, 234 Phytase, in soil, 349 “Pmko,” antigenic properties of, 234 effect on leucocytes, 231 hypersensitivity induction by, 234 immunizing activity of, 237 Polygalacturonase, in soil, 349 Polygalitol, oxidn. product, 130 Polymerization, in D-glucose irradiution. 45

SUBJECT INDEX, VOLUME

16

393

on pyrimidine nucleotides, 28 in D-mannose irradiation, 44 on water, 1 6 1 7 in sugar irradiation, 32 excitation and ionization of gases by, Polysaccharides, from Bacillus mega15 therium, 351, 352 excitation of molecules by, 15 effect on soil structure, 337, 353 hydrogen and hydrogen peroxide foresters, 218 mation in, of water, 17, 18 extraction from soil, 338-340 hydrogen and hydroxyl radical formaformation by soil micro-organisms, 351 tion in, of water, 16, 19 of Mycobaclerium tubercitlosis, m , 222 ionization by, 14, 15 mycolates, 219 measurement of, 21-22 nonreducing, 91 Raffinose, 7-ray effect on, 52 oxidized, 137-140 hydrolysis of, 293 from pathogenic bacteria, 352 Ramalin, 325 phospholipo-, 233 Reaction kinetics, of hydrolysis of glyradiation effect on, 33 cosides with 8-galactosidases, of soil, 338, 341, 342 258 from soil, attempted fractionntion of, Reductone, from D-fructose irradiation, 341 47 purification of, 340, 341 Rhamnopyranoside, methyl WL-, oxidn. i n soil, degradation of, 350 product, 109, 110 in sucrase, 254 oxidized, alkali effect on, 153 2-Propano1, complex with tmylosr, 321 -, methyl 3-0-acetyI-2,4-di-O-methyl2-Propanone, 1,3-dihydroxy-, from I)CU-L-, 65 fructose irradiation, 47 -, methyl 3-0-acetyl-4-0-methyl-~~-~-, phosphate, 28 65 Pseudolactal, 200 -, methyl 2,3-0-isopropylidene-a-~-, 64 -, penta-0-acetyl-, prepn. of, 200 -, methyl .l-O-methyl-a-~-,64 reaction with alkali, 200 Psicose, 3,6-anhydro-~-, osotriazolr -, methyl 4-0-methyl-2,3-di-O-(trifluoroacety1)-a-L-, 64 oxidn. product, 119 reaction with MeOH, 64,65 Pyritline, trifluoroacetylation in pres-, methyl 4-0-methyl-3-0-trifluoroaceence of, 62 tyl-a-L-, 65 Pyridinium chloride, l-I)-glucopyrnnoRhamnose, L-, 294 Ryl-, 94 polysaccharides contg., formation by -, l-(2-0-methy~-~-g~ucopyranosyl)-, 94 soil bacteria, 351, 352 hepta-O-acetyl-@-lactosyl Pyridinium of soil, 337, 342, 347 sulfate, 1-(hepta-0-acetyl-p-lac-, 2,4-di-O-methyl-, 64-65, 224 tosyl)-, 194 -, 3,4-di-O-methyl-, 224 Pyruvaldehyde, in soil, 350 -, 3,4-di-O-methyl-~-,65 Pyruvic acid, 28 -, 2-O-methyl-, 224

R Radiation, chemical changes by, 13 effect on a h . , 22-26 on alkyl phosphates, 28 on carbohydrates, 30, 32 on glycolic acid, 30-31, 32 on nucleic acids, 2 6 2 8 on purine nucleotides, 28

, Rhamnoside, methyl a - ~ -107 Rhamnosides, of soil, 344 Ribonucleic acids, of soil, 344 -, deoxy-, of soil, 344 Ribose, D-, 294 of soil, 338, 342 from hydrolyzed bacterial cultures, 351, 352 -, 5-deoxy-5-fluoro-o-, derivs., 95, 101

394

SUBJECT INDEX, VOLUME

Riboside, methyl 3-amino-3-deoxy-BD(and &L)-, 118

s Saccharic acid. See Glucaric acid. Saccharides, acidic, of milk, 171 from irradiation of amylose and starch, 34 Saccharinic acids, from lactose, 188 Sedoheptulosan. See Heptulopyranose, 2,7-anhydro-&~-altro-. Sialic acid, 171 oligosaccharides contg., 168 Sodium hydroxide, starch leaching by, 306 Sodium sulfate, starch fractionation by, 328 Solanine, 137 Sorbitol. See Glucitol, D-. Sorbose, L-, 294 Spurs, the term, 16 Stachyose, 137 Starch, complexes, 303 complexes with alkaline-earth hydroxides, 326 degradation and solubilization of, 323 degradation of, decrease in, by magnesium sulfite, 314 temp. and, 314 dialdehyde. See Oxyatarch. dissolution of, 326 fractionation of, 312, 314, 324, 325, 327 acids in, 326 alcs. in, 320 alkalis in, 326 1-butanol in, 300 chloral hydrate in, 307 -contg. raw materials, 329 2-methyl-1-butanol in, 311 2-methyl-2-butanol in, 326 salt solutions in, 310 by sulfates in, 328 fractionation of, of low molecular weight, 329 gelatinization temp. of, magnesium sulfate effect on, 313 hydrolysis products, fractionrttion of, 311

16

iodine adsorption by, 300, 301 leaching, by sodium hydroxide, 306 by water, 306 properties of fractions from potato, 331 radiation effect on, 34, 35 salting-out of, 312, 316 in soil, stability of, 350 solubilization temp., 313 solutions of, 310 system: magnesium sulfate-wuter-, 317 Streptamine, N,N'-dibenzoyl-, oxidn. products, 109, 110 Strontium hydroxide, complex with starch, 327 Styracitol, oxidn. product, 130 Succinic acid, 0x0-. See Oxalacetic acid. Sucrase, 254 Sucrose, as acceptor in transgalactosylation, 262 effect on pectin irradiation, 34 8-galactosidase inhibition by, 280 octaacetate, 67 radiation effect on, 32, 60,51, 52 structure of D-frllCtOSe moiety in, 93 of D-glucose moiety in, 93 Sucrose-C~4,self-decompn. of, 55, 50 Sugars, amino, of soil, 338, 343 detn. of, 346 effect on plant growth, 354 esters with alanine, 207 irradiation of, polymerization i n , 32 0-methyl-, of soil, 342 radiation effect on, 36, 39 reducing, in soil, 346 Sulfone, 8-D-glucopyranosyl phenyl, oxidn. product, 130 Sulfones, 69 Superlose, 325

T Tagatose,

D-,

189

-, 3,6-anhydro-~(and L)-, osotrinzole oxidn. products, 119 Takadiastase, 240 Talose, 6-deoxy-, 224 -, G-deoxy-3-O-methyl-, 224 Tartaric acid, 30, 31 Tetralactose, 198

SUBJECT INDEX, VOLUME

16

395

W “Theta temperature,” 325 Trehalose, 295 Water, hydrogen and hydrogen peroxide a#-, 88 formation from, by radiation, 17, from cord factor, 212 18 esters, 217, 232 hydrogen and hydroxyl radical forwith 2-eicosyl-3-hydroxytetracosamation in irradiation of, 16, 19 noic acid, 217 radiation effect on, 15-17 with fatty acids, 210 Wax C, 210 and esters, 209 adjuvant action of, 236 from glycolipids, 209 cord factor in, 210 octaacetate, 67 hypersensitivity induction by, 234 -, di-0-acetyl-6,6’-di-O-mycoloyl-, 214 Wax D, 207, 209, 210 -, 6,6‘-di-0-corynomycoloyl-,217, 233 adjuvant action of, 235-237 -, 2,2’-di-0-(3-hydroxy-x-methoxymy- antigenic properties of, 233 colanoy1)-, 215 biological activity of, 230, 231, 234, 235 -, 2,2’-di-O-mycoloyl-, 232 composition of, 218-222 -, 6,6’-di-O-mycoloyl-, 214, 215, 216, 232 cord factor in, 210 aa structure for cord factor, 212 properties of, 219 -, 6,6’-di-O-p-tolylsulfonyl-, 215, 216 structure of, 221 -, 2,3,4,2‘, 3‘,4‘-hexa-O-acetyl-6,6’-dideoxy-6,6’-diiodo-, 216 X -, 2,3,4,2’,3’, 4’-hexa-O-acety1-6 ,G’-di- X-rays, diffraction of, by amylose, 330 0-mycoloyl-, 215, 216 diffraction of, by butanol-amylose, 303 -, 2,3,4,2’,3’,4’-hexa-O-aeetyl-6,6’-di- by iodine-amylose, 303 0-p-tolylsulfonyl-, 215 effect of glyceritol l(and 2)-phos-, 6-O-mycoloyl-, 214, 232 phates, 28 -, 2,6,6’-tri -0-mycoloyl-, 214,232 Xylan, in soil, stability of, 350 lH-1,2,3-Triazole, 4-phenyl-l-(tetra-O- Xylitol, 1,4-anhydro-, oxidn. product, acetyl-p-D-glucopyrmosyl)-, 97 130 Xylopyranoside, methyl WD-, oxdn. prodU uct, 118 Uridine, 5-(D-galactosyl pyrophosphate), Xylopyranosyl bromide, tri-0-acetyl-aD-,87 180 Xylose, D - and L-, 294 5-(~-glucosylpyrophosphate), 17ti L-, from D-glucitol irradiation, 48 oxidn. product, 128 D-,phenylosazone, 152 5-pyrophosphate, 177 polysaccharides contg., formation by 5-triphosphate, 177 bacteria, 351,352 Uronic acids, polysaccharides contg., in soil, 337, 338, 342, 352 formation by soil bacteria, 351, Xyloside, methyl a-D-,oxidn. product. 352 117 of soil, 342, 346 -, methyl 8-D-,oxidn. product, 115 detn. of, 345 -, methyl 3-amino-3-deoxy-p-~-,118

CUMULATIVE AUTHOR INDEX FOR VOLUMES 1-16 BOBBITT,J. M., I’eriodate Oxidntioii of A Carbohydrates, 11, 1-41 ADAMS,MILDREI). See Culdwell, Mury L. B~ESEKEN, J., The Use of Boric Acid for ANDERSON, ERNEST,and SANDS,LILA, the Determination of the ConfiguraA Discussion of Methods of Value in tion of Carbohydrates, 4, 189-210 Research on Plant Polyuronides, 1, BONNER,T. G., Applications of Tri329-344. fluoroacetic Anhydride in CarboANDERSON,LAURENB. See Angyal, S. J . hydrate Chemistry, 16, 59-84 ANGYAL,8.J., and ANDERSON, LAURENS, BONNER, WILLIAMA., Friedel-Crafts and The Cyclitols, 14, 135-212 Grignard Processes in the CarboASPINALL,G. O., The Methyl Ethers of hydrate Series, 6, 251-289 Hexuronic Acids, 9, 131-148 E. J., and PEAT,STANLEY, The ASPINALL, G. O., The Methyl Ethers of BOURNE, Methyl Ethers of D-Glucose, 5, 145D-Mannose, 8, 217-230 190 ASPINALL, G . O . , Struct,ural Chemistry BOURNE, E. J. See also, Barker, S. A. of the Hemicclluloses, 14, 429-468 BOUVENG,H. O., and LINDBERO,B., R Methods in Structural Polysaccharide Chemistry, 15, 53-89 BALLOU,CLINTONE., Alkali-Jensitive BRAY, G., D-Glucuronic Acid in Glycosides, 2, 59-95 Metabolism, 8, 251-275 BARKER, G . R., Nucleic Acids, 111 285BRAY,H. G,, A N D STAcEY,M., Blood

-

333 Group Polysaccharides, 4, 37-55 BARKER,8. A . . nnd BOURNE,E. J.. Acetals nnd’Ketds of the Tetritok; C Pentitols and Hexitols, 7, 137-207 BARRETT,ELLIOTTP., Trendn in the CAESAR,GEORGEV., Starch Nitrato, 13, Development of Granular Adsorb331-345 CALDWELL, MARYL. and AIMMS, MILents for Sugar Refining, 6,205-230 JOHN, DRED, Action of Certain Alptin BARRY,C. P., and HONEYMAN, Fructose anti its Derivatives, 7, Amylaaes, 5, 229-268 CANTOR,SIDNEYM. See Miller, I t o h - t 53-98 BAYNE,S., and FEWBTER, J. A,, The Ellsworth. Osones, 11, 43-96 W. G . , ConCAPON,B., and OVEREND, B E ~ L I KANDREW, , Kojic Acid, 11,145-183 stitution and Physicochemical l’ropBELL, D. J., The Methyl Ethers of Derties of Carbohydrates, 15, 11-51 CARR,C. JELLEFF, and KRANTZ, JOHN Galactose, 6, 11-25 BEMILLER,J. N. See Whistler, Roy L. C., JP.,Metabolism of the Sugar RINKLEY, W. W., Column ChromatograAlcohol8 and Their DerivativeN, 1, phy of Sugars and Their Derivatives, 175-192 CLAMP,JOHN R., HOUGH,L., HICKRON, 10, 55-94 BINKLEY, W. W., and WOLFROM, M. L., JOHNL., and WHISTLER,ROY I,., Composition of Cane Juice and Cane Lactose, 16, 159-206 Final Molasses, 8, 291-314 COMPTON,JACK, The Molecular ConBLAIR, MARYGRACE,The 2-Hydroxystitution of Cellulose, 3, 185-228 CONCHIE), J., LEVVY,G. A., and MARSH, glycals, 9, 97-129

396

CUMULATIVE AUTHOR INDEX FOR VOLS.

1-16

397

C. A., Methyl and Phenyl Glycosides of the Common Sugars, 12, 157-187 CRUM,JAMES D., The Four-carbon Saccharinic Acids, 13, 169-188

Desulfurization by Raney Nickel, 5, 1-28 FLETCHER, HEWITT G., JR. See also, Jeanloz, Roger W. FORDYCE, CHARLESR., Cellulose Esters of Organic Acids, 1, 309-327 D FOSTER, A. B., Zone Electrophoresis of DAVIEY,D. A. L., Polysaccharides of Carbohydrates, 12, 81-115 Gram-negative Bacteria, 15, 271-340 FOSTER, A. B., A N D HORTON, D., Aspects DEAN,G. R., and GOTTFRIED, J. B., The of the Chemistry of the Amino Commercial Production of CrystalSugars, 14, 213-281 line Dextrose, 5, 127-143 FOSTER, A. B., and HUQQARD, A. J., The DEITZ,VICTORR. See Liggett, R. W. Chemistry of Heparin, 10, 335-368 DEUEL,H. See Mehta, N. C. FOSTER, A. B., A N D STACEY, M., The DEUEL,HARRYJ., JR., and MOREHOUSE, Chemistry of the 2-Amino Sugars MARGARET G., The Interrelation of (2-Amino-2-deoxy-sugars),7,247-288 Carbohydrate and F a t Metabolism, FOSTER, A. B., and WEBBER, J. M., 2, 119-160 Chitin, 15, 371-393 DEULOFEU,VENANCIO,The Acylated Fox, J. J., and WEMPEN,I., Pyrimidine Nitriles of Aldonic Acids and Their Nucleosides, 14, 283-380 Degradation, 4, 119-151 FRENCH, DEXTER,The Raffinose Family DIMLER, R . J., 1 ,&Anhydrohexofuof Oligosaccharides, 9, 149-184 ranoses, A New Class of HexosanB, FRENCH,DEXTER, The Schardinger 7, 37-52 Dextrins, 12, 189-260 DOUI)OROFF, M. See Hassid, W. Z. G DUBACH, P. See Mehta, N. C.

E ELDERYIELD, ROBERTC., The Carbohydrate Components of the Cardiac Glycosides, 1, 147-173 ELLIS,G . P., The Maillard Reaction, 14, 63-134

GARC~A G O N Z ~ L E F., Z , Reactions of Monosaccharides with bela-Ketonic Esters and Related Substances, 11, 97-143

GOEIJP,RUDOLPHMAXIMILIAN, J R . See Lohmar, Rolland. GOODMAN, IRVING, Glycosyl Ureides, 13,

215-236 ELLIS, G. P., and HONEYMAN, JOHN, GOTTFRIED, J. B. See Dean, G. R . Glycosylamines, 10, 95-168 GOTTSCHALK, ALFRED,Principles UndcrEVANH,TAYLOR H., and HIBBERT, lying Enzyme Specificity in the HAROLD, Bacterid I’olysaccharides, Domain of Carbohydrates, 5, 49-78 2, 203-233 W., The Halogen OxidaEVANS,w. I,., REYNOLDB, D. n., and GREEN,JOHN tion of Simple Carbohydrates, ExTALLEY, E. A., The Synthesis of cluding the Action of Periodic Acid, Oligosaccharides, 6, 27-81 3,129-184

F

GREENWOOD,C. T., Aspects of the Physical Chemistry of Starch, 11

FEWBTER, J . A . See Bayne, 8. 335-385 FLETCHER, HEWITT,G., JR., The Chem- GREENWOOD, C. T., The Size and Shape istry and Configuration of the Cycliof Some Polysaccharide Molecules, tols, 3, 45-77 7, 289-332; 11, 385-393 FLETCHER, HEWITTG., JR., and RICHT- GURIN,SAMUEL, Isotopic Tracers in the MYER, NELSONK., Applications in Study of Carbohydrate Metabolism, the Carbohydrate Field of Reductive 3,229-250

398

CUMULATIVE AUTHOR INDEX FOR VOLS.

GUTHRIE,R. D., The “Dialdehydes” from the Periodate 0xidat.ion of Carbohydrates, 16, 106-168

H HARRIS,ELWINE., Wood Saccharification, 4, 163-188 HASKINS, JOSEPHF., Cellulose Ethers of Industrial Significance, 2, 279-294 HASSID,W. Z., and DOUDOROFF, M., Enzymatic Synthesis of Sucrose and Other Disaccharides, 5, 29-48 HAYNES, L. J., and NEWTH,F. H., The Glycosyl Halides and Their Derivatives, 10, 207-258 J., The SubstitutedHEHRE,EDWARD sucrose Structure of Melezitose, 8, 277-290 HELFERICH, BURCKHARDT, The Glycals, 7, 209-245 HELFERICH, BURCKHARDT, Trityl Ethers of Carbohydrates, 3, 79-1 11 See Evans, Taylor H. HIBBERT, HAROLD. HICKSON, JOHNL. See Clamp, John R. HINDERT,MARJORIE.See Karabinos,

J. V. HIRST,E. L., [Obituary of] James Colquhoun Irvine, 8, xi-xvii HIRST, E. L., [Obituary of] Walter Norman Haworth, 6, 1-9 HIRST, E . I,., and JONES, J. K. N., The Chemistry of Pectic Materials, 2, 236-251 HIRST,E . L., and Ross, A. G., [Obituary of] Edmund George Vincent Percival, 10, xiii-xx HODGE,JOHNE., The Amadori Rearrangement, 10, 169-206 HONEYMAN, JOHN,and MORGAN,J. W. W., Sugar Nitrates, 12, 117-136 JOHN. See also, Barry, C. P. HONEYMAN, HONEYMAN, JOHN. See also, Ellis, G. P. HORTON, D., Tables of Properties of 2Amino-2-deoxy Sugars and Their Derivatives, 15, 169-200 HORTON, 1).See also, Foster, A. B. J. K. N., The HOUGH,L., and JONES, Biosynthesis of the Monosaccharides, 11, 186-262 HOUGH,L., PRIDDLE, J. E., and THEOBALD, R. S., The Carbonates and

1-16

Thiocarbonates of Carbohydrates, 15, 91-168 HOUGH,L. See also, Clamp, John R. HUDSON, C. S., Apiose and the Glycosides of the Parsley Plant, 4,67-74 HUDSON, C. S., The Fischer Cyanohydrin Synthesis and the Configurations of Higher-carbon Sugars and Alcohols, 1, 1-36 HUDSON,C. S., Historical Aspects of Emil Fischer’s Fundamental Conventions for Writing Stereo-formulas in a Plane, 3, 1-22 HUDSON, C. S., Melezitose and Turanose, 2, 1-36 HUGGARD, A. J. See Foster, A. B.

J JEANLOZ, ROGERW., [Obituary of] Kurt Heinrich Meyer, 11, xiii-xviii ROGERW.,The Methyl Ethers of 2-Amino-2-deoxy Sugars, 13, 189214 JEANLOZ, ROGERW., and FLETCHER, HEWITTG., JR.,The Chemistry of Ribose, 6, 136-174 JONES, J. K. N., and SMITH,F., Plant Gums and Mucilages, 4, 243-291 JONES, J. K. N . Seealso, Hirst, E. L. JONES, J. K. N. See also, Hough, I,. JONSEN, J., and LALAND, S., Bacterial Nucleosides and Nucleotides, 15, 201-234 JEANLOZ,

K KARABINOS, J. V., Psicoae, Sorbose and Tagatose, 7, 99-136 MARKARABINOB, J. v., and HINDERT, JORIE, Carboxymethylcellulose, 9, 286-302 KENT,P. W. See Stacey, M. KERTESZ,2. I., and MCCOLLOCH, R. J., Enzymes Acting on Pectic Substances, 5, 79-102 KLEMER, ALMUTH.See Micheel, Fritz. KOWKABANY, GEORGEN., Paper Chromatography of Carbohydrates and Related Compounds, 9, 303-363 KRANTZ, JOHN C., JR.See Carr, C. Jelleff.

CUMULATIVE AUTHOR INDEX FOR VOLS.

L LAIDLAW, R . A., and PERCIVAL, E . G. V., The Methyl Ethers of the Aldopentoses and of Rhamnose and Fucose, 7, 1-36 LALAND, S. See Jonsen, J. LEDERER,E., Glycolipids of Acid-fast Bacteria, 16, 207-238 LEMIEUX,R . U., Some Implications in Carbohydrate Chemistry of Theories Relating to the Mechanisms of Replacement Reactions, 9, 1-57 LEMIEUX,R. U., and WOLFROM, M. L., The Chemistry of Streptomycin, 3, 337-384 LESPIEAU,R., Synthesis of Hexitols and Pentitols from Unsaturated Polyhydric Alcohols, 2, 107-118 LEVI, IRVING, and PURVES,CLIFFORD B., The Structure and Configuration of Sucrose (alpha-D-Glucopyranosyl beta-D-Fructofuranoside),4, 1-35 LEVVY, G. A., andMAmH, C. A., Preparation and Properties of 8-Glucuronidase, 14, 381-428 LEVVY,G. A. See also, Conchie, J. LIGQETT,R. W., and DEITZ,VICTORR., Color and Turbidity of Sugar Products, 9, 247-284 LINDBERQ, B. See Bouveng, H. 0. LOHMAR,ROLLAND,and GOEPP, R u DOLPH MAXIMILIAN, JR.,The Hexitols and Some of Their Derivatives, 4, 211-241

M MAHER,GEORGEG., The Methyl Ethers of the Aldopentoses and of Rhamnose and Fucose, 10, 257-272 MAHER,GEORGEG., The Methyl Ethers of &Galactose, 10, 273-282 MALHOTRA, OM PRAKASH. See Wallenfels, Kurt. MANNERS,D. J., The Molecular Structure of Glycogens, 12, 261-298 MARSH,C. A. See Conchie, J. MARSH,C. A. See Levvy, G. A. MCCLOSKEY, CHESTERM.,Benzyl Ethers of Sugars, 12, 137-156 MCCOLLOCH, R. J. See Kertesz, 2.I. MCDONALD, EMMAJ., The Polyfructo-

1-16

399

sans and Difructose Anhydrides, 2, 253-277 MEHLTRETTER,C. L., The Chemical Synthesis of D-Glucuronic Acid, 8, 231-249

MEHTA,N. C., DUBACH, P., and DEUEI,, H., Carbohydrates in the Soil, 16, 335-355 MESTER,L., The Formazan Reaction in Carbohydrate Research, 13, 105-167 MESTER,L., [Obituary of] G6za Zemplhn, 14, 1-8 MICHEEL,FRITZ, and KLEMER,ALMUTH, Glycosyl Fluorides and Azides, 16, 85-103 MILLER, ROBERT ELLSWORTH, and CANTOR, SIDNEYM., Aconitic Acid, a By-product in the Manufacture of Sugar, 6, 231-249 MILLS, J. A., The Stereochemistry of Cyclic Derivatives of Carbohydrates, 10, 1-53 MOREHOUSE, MARQARET G. See Deuel, Harry J., Jr. MORGAN, J. W. W. See Honeyman, John. MORI, T., Seaweed Polysaccharides, 8, 315-350 MUETQEERT,J., The Fractionation of Starch, 16, 299-333 MYRBACK,KARL, Products of the Enzymic Degradation of Starch and Glycogen, 3, 251-310

N NEELY,W. BROCK,Dextran: Structure and Synthesis, 15, 341-369 NEELY,W. BROCK,Infrared Spectra of Carbohydrates, 12, 13-33 NEUBERQ,CARL, Biochemical Reductions a t the Expense of Sugars, 4, 75-117 NEWTH,F. H., The Formation of Furan Compounds from Hexoses, 6, 83-106 NEWTH,F. H . See also, Haynes, L. J. NICKERSON, R . F., The Relative Crystallinity of Celluloses, 5, 103-126 NORD,F. F., [Obituary of] Carl Neuberg, 13, 1-7 0

OLSON,E. J. See Whistler, Roy L. OVEREND,W. G., and STACEY,M., The

400

CUMULATIVE AUTHOR INDEX FOR VOLS.

Chemistry of the 2-Desoxy-sugars, 8, 46-106 W. G . See also, Capon, B. OVEREND,

P PACSU, EUQENE,Carbohydrate Orthoesters, 1, 77-127 PEAT, STANLEY,The Chemistry of Anhydro Sugars, 2, 37-77 See also, Bourne, E. J. PEAT,STANLEY. PERCIVAL, E. G. V., The Structure and Reactivity of the Hydrasone and Osazone Derivatives of the Sugars, 3, 23-44

PERCIVAL, E. G . V. See also, Laidlaw, R. A. PERLIN,A. S., Action of Lead Tetraacetate on the Sugars, 14, 9-61 PHILLIPS, G . O., Radiation Chemistry of Carbohydrates, 16, 13-68 POLQLASE, W. J., Polysaccharides Associated with Wood Cellulose, 10, 283333 J . E. See Hough, L. PRIDDLE, I’URVES,CLIFFORD B. See Levi, Irving.

R RAYMOND, ALBERT L., Thio- and Selenosugars, 1, 129-146 REEVES,RICHARD E., CupranimoniumGlycoside Complexes, 6, 107-134 REYNOLDS, 1). D. See Evans, W. L. RICHTMYER, NELSONK., The Altrose Group of Substances, 1, 37-76 RICHTMYER, NELSONK., The 2-(aMoPolyhydrox yalkyl) benzimidazoles, 6, 176-203 RICHTMYER, NELSONK. See also, Fletcher, Hewitt G., Jr. Ross, A . G. See Hirst, E. L. S

SANDS,LILA.See Anderson, Ernest. SATTLER,LOUIS, Glutose and the Unfermentable Reducing Substances in Cane Molasses, 3, 113-128 SCAOCH, THOMAS JOHN, The Fractionation of Starch, l, 247-277 SHAFIZADEH, F., Branched-chitin Sugars of Natural Occurrence, 11, 263-283

1-16

SHAFIZADEH, F., Formation and Cleavage of the Oxygen Ring in Sugars, 13, 9-81 SMITH,F., Analogs of Ascorbic Acid, 2, 79-106 SMITH,F. See also, Jones, J. K. N. SOWDEN,JOHN C., The Nitromethane and 2-Nitroethanol Syntheses, 6, 291-318 SOWDEN,JOHNC., The Saccharinic Acids, 12, 36-79 SPECK,JOHN C., JR., The Lobry de Bruyn-Alberda van Ekenntein Transformation, 13, 63-103 D. B., The Biosynthesis of SI’RINSON, Aromatic Compounds from ii-Glucose, 15, 236-270 STACEY,M., The Chemistry of Mucopolysaccharides and Mucoproteinn, 2, 161-201 STACEY,M., and KENT, P. W., The Polysaccharides of Mycohncterirm tuberculosis, 3, 311-338 STACEY, M. See also, Bray, H. G. STACEY, M. See also, Foster, A. €3. STACEY, M. See also, Overend, W. G. STOLOBF, LEONARD, Polysaccharide Hydrocolloids of Commerce, 13,266-287 JAMES M., Relative ReacSU~IHARA, tivities of Hydroxyl Groups of Cnrbohydrates, 8, 1-44

T TALLEY, E. A. See Evans, W. L. TEAQUE, ROBERTS., The Conjugates of D-Glucuronic Acid of Animal Origin, 9,186-246 THEOBALD, R. S. See Hough, L. TIPSON,R. STUART,The Chemistry of the Nucleic Acids, 1, 193-246 TIPSON, R. STUART, [Obituary of] Harold Hibbert, 16, 1-11 TIPSON, R. Stuart, [Obituary of] Phoebus Aaron Theodor Levene, 12, 1-12 TIPSON, R. STUART,Sulfonic Esters of Carbohydrates, 8, 107-216

W WALLENFELS, KURT, and MALHOTRA, OM PRAKASH,Galactosidases, 16, 239-298

CUMULATIVEAUTHOR INDEX FOR VOLS.

WEBBER,J. M. See Foster, A. B. WEMPEN,I. See Fox, J. J. WHISTLER, ROY L., Preparation and Properties of Starch Esters, 1, 279307 WHISTLER,ROYL., Xylan, 5,269-290 WHISTLER, ROY L., and BEMILLER, J. N., Alkaline Degradation of Polysaccharides, 13, 289-329 WHISTLER,ROY L., and OLSON,E. J., The Biosynthesis of Hyaluronic Acid, 12, 299-319 WHISTLER, ROY L. See also, Clamp, John R . WHITEHOUSE,M. W. See Zilliken, F. WIQGINS, L. F., Anhydrides of the Pentitols and Hexitols, 5, 191-228

1-16

401

WIQQINS,L. F., The Utilization of Sucrose, 4, 293-336 WISE, LOUIS E., [Obituary of] Emil Heuser, 15, 1-9 WOLFROM, M. L., [Obituary of] Claude Silbert Hudson, 9, xiii-xviii WOLFROM, M. L., [Obituary of] Rudolph Maximilian Goepp, Jr., 3, xv-xxiii WOLFROM, M. L. Seealso, Binkley, W. W. Wolfrom, M. L. See also, Lemieux, R. U. Z

ZILLIKEN,F., and WHITEHOUSE, M . W., The Nonulosaminic Acids-Neuraminic Acids and Related Compounds (Sialic Acids), 13, 237-263

CUMULATIVE SUBJECT INDEX FOR VOLUMES 1-16 A

B

Acetals, of hexitols, pentitols, and tetritols, 7, 137-207 Acetic acid, trifluoro-, anhydride, applications of, in carbohydrate chemistry, 16, 59-84 Aconitic acid, 6, 231-249 Adsorbents, granular, for sugar refining, 6, 205-230 Alcohols, higher-carbon sugar, configurations of, 1, 1-36 unsaturated polyhydric, 2, 107-118 Aldonic acids, acylated nitriles of, 4, 119-151. Aldopentoses, methyl ethers of, 7, 1-36; 10, 257-272 Alkaline degradation, of polysaccharides, 13, 289-329 Altrose, group of compounds related to, 1,37-76 Amadori rearrangement, 10. 169-205 Amino sugars. See Sugars, 2-amino-2deoxy. Amylases, certain alpha, 6, 229-268 Anhydrides, difructose, 2, 253-277 of hexitols, 6, 191-228 of pentitols, 6, 191-228 Anhydro sugars. See Sugars, anhydro. Animals, conjugates of D-glucuronic acid originating in, B, 185-246 Apiose, 4, 57-74 Ascorbic acid, analogs of, 2, 79-106 Aromatic compounds, biosynthesis of, from D-glUCOSe, 16,

Bacteria, glycolipides of acid-fast, 16, 207-238 nucleosides and nucleotides of, 16,

235-270 402

201-234

polysaccharides from, 2, 203-233; 3, 311-336

polysaccharides of Gram-negative, 16. 271-340

Benzimidazoles, Z-(aZdo-polyhydroxyaIkyl)-,6. 175-203 Benzyl ethers, of sugars, 12, 137-156 Biochemical reductions, at the expense of sugars, 4, 75-117 Biosynthesis, of aromatic compounds from D - ~ I u cose, 16, 235-270 of hyaluronic acid, 12, 299-319 of the monosaccharides, 11, 185-262 Blood groups, polysaccharides of, 4, 37-55 Boric acid, for determining configuration of carbohydrates, 4, 189-210 Branched-chain sugars. See Sugars, branched-chain. C

Cane juice, composition of, 8, 291-314 Cane See Carbohydrates, applications of reductive desulfurization by Raney nickel, in the field of, 6, 1-28 applications of trifluoroacetic anhydride in chemistry of, 16, 59-84 aa components of cardiac glycosides, 1, 147-173

carbonates of, 16, 91-158 constitution of, 16, 11-51

CUMULATIVE SUBJECT INDEX FOR VOLS.

determination of configuration of, with boric acid, 4, 189-210 enzyme specificity in the domain of, 6, 49-78 formazan reaction, in research on, 13, 105-167 Friedel-Crafts and Grignard processes applied to, 6, 251-289 halogen oxidation of simple, 3, 129-184 infrared spectra of, 12, 13-33 mechanisms of replacement reactions in chemistry of, 9, 1-57 metabolism of, 2, 119-160; 3, 229-250 orthoesters of, 1, 77-127 periodate oxidation of, 11, 1 4 1 the “dialdehydes” from, 16, 105-158 physicochemical properties of, 16.11-51 radiation chemistry of, 16, 13-58 and related compounds, paper chromatography of, 9,303-353 relative reactivities of hydroxyl groups of, 8, 1-44 in the soil, 16, 335-355 stereochemistry of cyclic derivatives of, 10, 1-63 sulfonic esters of, 8, 107-215 thiocarbonates of, 16, 91-158 trityl ethers of, 3, 79-111 zone electrophoresis of, 12, 81-115 Carbonates, of carbohydrates, 16, 91-158 Carboxymet hyl ether, of cellulose, 9, ‘285-302 Cellulose, carboxymethyl-, 9, 285-302 eaters of, with organic acids, 1,309-327 ethers of, 2, 279-294 molecular constitution of, 3, 185-228 of wood, polysaccharides associated with, 10, 283-333 Celluloses, relative crystallinity of, 6, 103-126 Chemistry, of the amino sugars, 14, 213-281 of the 2-amino sugars, 7,247-288 of anhydro sugars, 2,37-77 of carbohydrates, applications of trifluoroacetic anhydride in, 16, 59-84 some implications of theories relat-

1-16

403

ing to the mechanisms of replacement reactions in, 9, 1-57 of the cyclitols, 3, 45-77 of the 2-deoxy sugars, 8. 45-105 of heparin, 10,335-368 of mucopolysaccharides and mucoproteins, 2, 161-201 of the nucleic acids, 1, 193-245 of pectic materials, 2, 235-251 of ribose, 6, 135-174 of streptomycin, 3, 337-384 physical, of carbohydrates, 16, 11-51 of starch, 11, 335-385 radiation, of carbohydrates, 16, 1358

stereo-, of cyclic derivatives of carliohydrates, 10. 1-53 structural, of the hemicelluloses, 14, 4w68 of polysaccharides, 16, 53-89 Chitin, 16, 371-393 Chromatography, column. See Column chromatography. paper. See Paper chromtttography. Color, of sugar products, 9. 247-284 Column chromatography, of sugars and their derivatives, 10, 65-94 Complexes, cuprammonium-glycoside, 6, 107-134 Configuration, of carbohydrates, determination of, 4, 189-210 of cyclitols, 3. 45-77 of higher-carbon sugar alcohols, 1,1-36 of sucrose, 4, 1-35 Conjugates, of D-glucuronic acid, 9, 185-246 Constitution, of carbohydrates, 16, 11-51 Crystallinity, relative, of celluloses, 6, 103-126 Cuprammonium-glycoside complexes, 6, 107-134 Cyanohydrin synthesis, Fischer, 1, 1-36 Cyclic derivatives, of carbohydrates, stereochemistry of, 10, 1-53

404

CUMULATIVE SUBJECT INDEX FOR VOLS. l-l(i

Cyclitols, 14, 135-212 chemistry and configuration of, 3, 45-77

D Degradation, of acylated nitriles of aldonic acids, 4, 119-151 enzymic, of glycogen and atarch, 3, 251-310 Deoxy sugars. flee Sugars, deoxy. Desulfurization, reductive, by Raney nickel, 6, 1-28 Dextran, structure and synthesis of, 16, 341-369 Dextrine, the Schardinger, 12, 189-260 Dextrom, commercial production of crystalline, 6, 127-143 ‘Dialdehydes,” from the periodate oxidation of carbohydrates, 16, 105-158 Difructose, anhydrides, 2, 253-277 Disacc harides, enzymic synthesis of, 6, 29-48

E Electrophoresis, zone, of carbohydrates, 1%81-115 Enzymes. See also, Amylases, Galactosidases, p-Glucuronidase. acting on pectic substances, 6, 79-102 degradition by, of starch and glycogen, 3, 261-310 specificity of, in the domain of carbohydrates, 6, 49-78 synthesis of sucrose and other disaccharides by, 6, 29-48 Esters, of cellulose, with organic acids, 1, 308-327 bela-ketonic (and related substances), reactions with monosaccharides, 11, 97-143 nitrate, of starch, 13, 331-345 of starch, preparation and properties of, 1, 279-307 sulfonic, of carbohydrates, 8, 107-215

Ethanol, 2-nitro-, syntheses with, 6, 291-318 Ethers, benzyl, of sugars, la, 137-156 carboxymethyl, of cellulose, 9, 285303 of cellulose, a, 27S29.1 methyl, of the aldopentoses, 7, 1-36; 10,257272 of 2-amino-2-deoxy sugars, 13, 189214 of fucose, 7, 1-36; 10, 257-272 of u-galactose, 6, 11-25; 10, 273-282 of D-glucose, 6, 145-190 of hexuronic acids, 9, 131-148 of D-mannose, 8, 217-230 of rhamnose, 7, 1-3G; 10, 257-274 t,rityl, of carbohydrates, 3, 79-111

F Fat, metabolism of, 2, 110-160 Formazltn reaction, in carbohydrate research, 13, 105-167 Formulas, stereo-, writing of, in a plane, 3, 1-22 Fractionation, of starch, 1, 247-277; 16, 299-333 Friedel-Crafts process, in the carbohydrate series, 6. 251-289 Fructans, 2, 253-277 Fructofuranoside, a-D-glucopyranosyl D-D-,4, 1-35 Fructosans, poly-. See Fructans. Fructose, and its derivatives, 7, 53-98 di-, anhydrides, 2, 253-277 Fucose, methyl ethers of, 7, 1-36; 10, 257-272 Furan compounds, formation from hexoses, 6, 83-10G G

Galactose, methyl ethers of D-, 6. 11-25; 10, 273232 Galactosidases, 16. 239-298

CUMULATIVE SUBJECT INDEX FOR VOLS.

Glucose. See also, Dextrose. biosynthesis of aromatic compounds from D-, 16. 235-270 methyl ethers of D-, 6, 145190 Clucuronic acid, D-, chemical synthesis of, 8,231-249 conjugates of, of animal origin, 9, 185-246 in metabolism, 8, 251-275 8-Clucuronidase, preparation and properties of, 14, 381428 Glutose, 3, 113-128 Glycals, 7, 209-245 -, 2-hydroxy-, 9, 97-129 Glycogens, enzymic degradation of, 3, 251-310 molecular structure of, 12, 261-298 Clycolipides, of acid-fast bacteria, 16, 207-238 Glycoside-cuprammonium complexes, 6, 107-134 Glycosides, alkali-sensitive, 9, 59-95 cardiac, 1, 147-173 methyl, of the common sugars, 12, 157-187 of the parsley plant, 4, 57-74 phenyl, of the common sugars, 12, 157187 Glycosiduronic acids, of animals, 9, 185-246 poly-, of plants, 1, 329-344 Glycosylamines, 10, 95-168 Glycosyl azides, 16, 85-103 Glycosyl fluorides, 16. 85-103 Glycosyl halides, and their derivatives, 10, 207-256 Goepp, Rudolph Maximilian , Jr., obituary of, 3, xv-xxiii Grignard process, in the carbohydrate series, 6. 251-289 Gums. See also, Hydrocolloids. commercial, 13, 265-287 of plants, 4, 243-291

H Halogen oxidation. See Oxidation, halogen . Haworth, Walter Norman, obituary of, 6, 1-9

1-16

405

Hemicelluloses, structural chemistry of, 14, 429468 Heparin, chemistry of, 10, 335-368 Heuser, Emil, obituary of, 16, 1-9 Hexitols, acetals of, 7, 137-207 nnhydrides of, 6, 191-228 and some of their derivatives, 4, 211241 synthesis of, 2, 107-114 Hexofuranoses, 1,6-anhydro-, 7, 37-52 Hexosans, 7, 37-52 Hexoses. See also, Hexofuranoses. formation of furan compounds from, 6, 83-106 Hexuronic acids, methyl ethers of, 9, 131-148 Hibbert, Harold, obituary of, 16, 1-11 Hudson, Claude Silbert, obituary of, 9, xiii-xviii Hyaluronic acid, biosynthesis of, 12, 299-319 Hydrazones, of sugars, 3, 23-44 Hydrocolloids, commercial, polysaccharidic, 13, 265287 Hydroxyl groups, relative reactivities of, 8, 1-44

I Infrared spectra, of carbohydrates, 12, 13-33 Irvine, James Colquhoun, obituary of, 8. xi-xvii Isotopic tracers. See Tracers, isotopic.

K Ketals. See Acetals. Kojic acid, 11, 145-183

L Lactose, 16, 159-206 Lead tetraacetate, action of, on the sugars, 14, 9-Gl Levene, Phoebus Aaron Theodor, obituary of, 12, 1-12

406

CUMULATIVE SUBJECT INDEX FOR VOLS.

Lobry de Bruyn-Alberda van Ekenstein transformation, 13, 63-103

M Maillard reaction, 14, 63-134 Mannose, methyl ethers of D-, 8. 217-230 Mechanism, of replacement reactions in carbohydrate chemistry, B, 1-57 Melezitose, 2, 1-36 structure of, 8, 277-290 Metabolism, of carbohydrates, 2, 119-160 use of isotopic tracers in studying, 3,229-250 of fat, 2, 119-160 of the sugar alcohols and their derivatives, 1, 175-192 D-glucuronic acid in, 8, 251-275 Methane, nitro-, syntheses with, 6, 291-318 Methods, in structural polysaccharide chemistry, 16, 63-89 Methyl ethers. See Ethers, methyl. Meyer, Kurt Heinrich, obituary of, 11. xiii-xviii Molasses, cane, 8, 113-128 cane final, composition of, 8, 291-314 Molecular structure, of glycogens, 12, 261-298 Monosaccharides, biosynthesis of, 11, 185-262 reactions of, with beta-ketonic esters and related substances, 11, 97-143 Mucilages. See also, Hydrocolloids. commercial, 13, 265-237 of plants, 4, 243-291 Mucopolysaccharides. See Polysaccharides, muco-. Mucoproteins. See Proteins, muco-. Mycobaclerium lubeTCUlO8i8, polysaccharides of, 3, 311-336

N Neuberg, Carl, obituary of, 13, 1-7

1-16

Neuraminic acids, and related compounds, 13, 237-263 Nickel, Raney. See Raney nickel. Nitrates, of starch, 13, 331-345 of sugars, 12, 117-135 Nitriles, acylated, of aldonic acids, 4, 119-151 Nonulosaminic acids, 13, 237-263 Nucleic acids, 1, 193-246; 11, 285-333 Nucleosides, bacterial, 16, 201-234 pyrimidine, 14, 283-380 Nucleotides, bacterial, 16, 201-234 0

Obituary, of Rudolph Maximilian Goepp, J r . , 3, xv-xxiii of Walter Norman Haworth, 6, 1-9 of Emil Heuser, 16, 1-9 of Harold Hibbert, 16, 1-11 of Claude Silbert Hudson, 9, xiii-xviii of James Colquhoun Irvine, 8, xi-xvii of Phoebus Aaron Theodor Levene, 12, 1-12 of Kurt Heinrich Meyer, 11, xiii-xviii of Carl Neuberg, 13, 1-7 of Edmund George Vincent Percivul, 10, xiii-xx of GBza ZemplBn, 14, 1-8 Oligosaccharides, the raffinose family of, 9, 149-184 synthesis of, 6, 27-81 Orthoesters, of carbohydrates, 1, 77-127 Osazones, of sugars, 3, 23-44 Oxones, 11, 43-96 Oxidations, halogen, of simple carbohydrates, 3, 129-148 lead tetraacetate, of sugars, 14, 9-61 periodate, of carbohydrates, 11, 1 4 1 the “dialdehydes” from, 16, 105-158 Oxygen ring, formation and cleavage of, in sugars, 13, 9-61

CUMULATIVE SUBJECT INDEX FOR VOLS.

P Paper chromatography, of carbohydrates and related compounds, B, 303-353 Parsley, glycosides of the plant, 4, 57-74 Pectic materials, chemistry of, 2, 235-251 enzymes acting on, 6, 79-102 Pentitols, acetals of, 7, 137-207 anhydrides of, 6, 191-228 synthesis of, 2, 107-118 Percival, Edmund George Vincent, obituary of, 10, xiii-xx Periodate oxidation. See Oxidation, periodate. Physical chemistry, of carbohydrates, 16, 11-51 of starch, 11, 335-385 Plants, glycosides of parsley, 4, 57-74 gums of, 4, 243-291 mucilages of, 4, 243-291 ' polyuronides of, 1, 329-344 Polyfru ctosans. See Fruc tans. Polyglycosiduronic acids. See Glycosiduronic acids, poly-. Polysaccharides. See also, Carbohydrates, Cellulose, Dextran, Dextrins, Fructans, Glycogen, Glycosiduronic acids (poly-), Pectic materials, Starch, and Xylan. alkaline degradation of, 13, 289-329 associated with wood cellulose, 10, 283-333 bacterial, 2, 203-233; 16, 271-340 blood group, 4, 37-55 hydrocolloidal, 13, 265-287 methods in structural chemistry of, 16, 53-89 muco-, chemistry of, 2, 161-201 of Gram-negative bacteria, 16,271-340 of Mycobacterium tuberculosis, 3, 311336 of seaweeds, 8. 315-350 shape and size of molecules of, 7, 289332;11, 385-393 Polyuronides, oi plants, i, 324-344

1-10

407

Preparation, of esters of starch, 1, 27!+307 of 8-glucuronidase, 14, 381-428 Properties, of 2-amino-2-deoxy sugars and their derivatives, 16, 159-200 of esters of starch, 1, 27!+307 of 8-glucuronidase, 14, 381428 physicochemical, of carbohydrates, 16, 11-51 Proteins, muco-, chemistry of, 2, 161-201 Psicose, 7, 99-136 Pyrimidines, nucleosides of, 14, 283-380

R Radiation, chemistry of carbohydrates, 16, 1358

Raffinose, family of oligosaccharides, B, 149-184 Raney nickel, reductive desulfurization by, 6. 1-28 Reaction, the formazan, in carbohydrate research, 13. 105-167 the Maillard, 14, 63-134 Reactivities, relative, of hydroxyl groups of carbohydrates, 8, 144 Rearrangement, the Amadori, 10, 169-205 Reductions, biochemical, at the expense of sugars, 4, 75-117 Replacement reactions, mechanisms of, in carbohydrate chemistry, 9, 1-57 Rhamnose, methyl ethers of, 7, 1-36; 10, 257-272 Ribose , chemistry of, 6, 135-174 S

Saccharification, of Wood, 4, 163-188 Saccharinic acids, 12, 3&79 four-carbon, 13. 169-188 Schardinger dextrins, 12, 189-260

408

CUMULATIVE SUDJECT INDEX FOR VOLS.

Seaweeds, polysaccharides of, 8, 315-350 Seleno sugars. See Sugars, seleno. Shape, of some polysaccharide molecules, 7, 289-332; 11, 385-393 Sialic acids, 13, 237-263 Size, of some polysaccharide molecules, 7, 289-332; 11, 385-393 Soil, carbohydrates in, 16, 335-355 Sorhose, 7, 99-136 Specificity, of enzymes, in the domain of carhohydrates, 6. 49-78 Spectra, infrared, of carbohydrates, 12, 13-33 Starch, enzymic degradation of, 3, 251-310 fractionat,ion of, 1, 247-277; 16, 299333 nitrates of, 13. 331-345 physical chemistry of, 11, 336-385 preparation and properties of esters of, 1, 279-307 Stereochemistry, of cyclic derivihves of carbohydrates, 10, 1-53 formulas, writing of, in a plane, 3, 1-22 Streptomycin , chemistry of, 3, 337-384 Structural chemistry, of the hemicelluloses, 14, 429-468 Structure, molecular, of dextran, 16, 341-369 of glycogens, 12, 261-298 of sucrose, 4, 1-35 Sucrose. See also, Sugar. enzymic synthesis of, 6, 2948 structure and configuration of, 4, 1-35 utilization of, 4, 293-336 Sugar, aconitic acid as by-product in manufacture of, 6, 231-249 Sugar alcohols, higher-carbon, configurations of, 1, 1-36 and their derivatives, metabolism of, 1. 175-192

1-16

Sugar products, color and turbidity of, 9, 247-284 Sugar refining, granular adsorbents for, 6, 205-230 Sugara, action of lead tetraacetate on, 14, 9-61 2-amino. See Sugars, 2-amino-2-deoxy. 2-amino-2-deoxyI 7, 247-288 aspects of the chemistry of, 14, 213281 methyl ethers of, 13, 189-214 properties of, 16, 159-200 anhydro, chemistry of, 2, 37-77 benzyl ethers of, 12, 137-150 biochemical reductions a t the expense of, 4, 75-117 branched-chain, of natural occurrcncc, 11, 263-283 2-deoxy, 8, 45-105 higher-carbon, configuration8 of, 1, 1-36 hydrazones of, 3, 23-44 methyl glycosides of the common, 12, 157-187 nitrates of, 12, 117-135 osazones of, 3, 23-44 oxygen ring in, formation and clenvage of, ia, 9-61 phenyl glycoaicles of the common, 12, 157-187 and their derivatives, column chronnrtography of, 10, 55-94 related to altrose, 1. 37-70 seleno, 1. 144-145 thio, 1, 129-144 Sulfonic esters, of carbohydrates, 8, 107-215 Syn t hesi 8, biochemical, of monosaccharides, 11, 185-202 chemical, of D-glucuronic acid, 8, 231249 of dextran, 16, 341-309 enzymic, of sucrose and other disaccharides, 6, 2948

T Tagatose, 7, 99-136 Tetritols, acatals of. 7. 137-207

CUMULATIVE SUBJECT INDEX FOR VOLS.

Thiocarbonates, of carbohydrates, 16, 91-158 Thio sugars. See Sugars, thio. Tracers, isotopic, 3, 229-250 Transformation, the Lobry de Bruyn-Alberda Ekenstein, 13, 63-103 Trityl ethers, of carbohydrates, 3, 79-111 Turanose, 2, 1-36 Turbidity, of sugar products, 9, 247-284

U Ureides, glycosyl, 13, 215-236

1-16

409

w Wood, polysaccharides associated with cellulose of, 10, 283-333 saccharification of, 4, 153-188 van

X Xylan, 6. 269-290 Z

ZemplBn, GBza, obituary of, 14, 1-8 Zone electrophoresis, of carbohydrates, 12, 81-115

ERRATA AND ADDENDA VOLUME12 read “l/d/Z.” Page 18, line 13 up. For ‘‘42” Page 29, lines 3, 7, and 0, and Reference 46. For “Coblenr” read “Coblentz.” Page 29, line 10. After “material,” insert “in saturated, aqueous solution.” Page 29, line 11. After “solutions,” insert “evaporated to sirups by gentle heating.’ Page 182, Table 111, entry 11, columns 2,3, and 5. Insert the following figures: 132133, -18.1, 142a; 131-133, -21, 142b; 130-131, -17.3, 142c. Page 182, line 3 up (of References). Insert (142a) H. G. Fletcher, Jr., and C. S. Hudson, J . Am. Chem. SOC.,72,4173 (1950). (142b) R. K. Ness and H. G. Fletcher, Jr., ibid., 78,4710 (1966). (142~)R. Bentley, ibid., 70, 1720 (1957).

VOLUME14 Page 227, lines 10 up, 13 up, and 14 up; and page 502. For “2-carboxy” read “l-carboxy.”

VOLUME15 Page 43, after Formula XXVII. ‘%H@”nieans “protonated solvent.” Page 113, line 2. For “carbohydrate” read “carbonate.” Page 160, column 3, entry 2 up. For ‘i-75.40” read “+75.4”.” Page 207, lines 2 up, 4 up, and 5 up; page 209, line 13; and page 294, line 14. For “2carboxyethyl” read “1-carboxyethyl,” Page 258, equations 11 and 12. For ‘IP”read “HP04B.” Page 262, equations. For “P” read “HPO,m,” Page 276, line 17. For ‘lane” read “and.” Page 420, line 5 under D. For “riductase” read “reductase.”

410