CIBA FOUNDATION SYMPOSIUM ON THE

CHEMISTRY AND BIOLOGY OF

MUCOPOLY SACCHARIDES

Editors for the Ciba Foundation

G . E. W. WOLSTENHOLME, O.B.E., M.A., M.B., B.Ch.

and

MAEVE O'CONNOR, B.A.

With 48 Illustrations

LIITLE, BROWN AND COMPANY BOSTON

CHEMISTRY AND BIOLOGY OF MUCOPOLYSACCHARIDES

Ciba Foundation Symposia

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Mammalian Germ Cells - - - -

Preservation and Transplantation of Normal Tissues - - - - - -

LeukaemiaResearch - - - -

Chemistry and Biology of Pteridines -

Porphyrin Biosynthesis and Metabolism

Histamine - - - - - -

Extra Sensory Perception - - -

Bone Structure and Metabolism - -

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A leaJlet giving fuller details of these volumes, also of the Ciba Foundation Colloquia on Endocrinology and Colloquia

on Ageing, i s available from the Publishers,

CIBA FOUNDATION SYMPOSIUM ON THE

CHEMISTRY AND BIOLOGY OF

MUCOPOLY SACCHARIDES

Editors for the Ciba Foundation

G . E. W. WOLSTENHOLME, O.B.E., M.A., M.B., B.Ch.

and

MAEVE O'CONNOR, B.A.

With 48 Illustrations

LIITLE, BROWN AND COMPANY BOSTON

Library of Congress Catalog No. 58-7676

THE CIBA FOUNDATION

fo* the Promotion of International Co-operation in Medical and Chemical Research 41 PORTLAND PLACE, LONDON, W.1.

Trustees : THE RIGHT HON. LORD ADRIAN, O.M., F.R.Y.

THE RIGHT HON. LORD BEVERIDGE, K.C.B., F.R.4. SIR RUSSELL BRAIN, BT.

THE HON. SIR GEORGE LLOYD-JACOB SIR RAYMOND NEEDHAM, Q.C.

Executive Council : SIR RAYMOND NEEDHAM, Chairman LORD BEVERIDGE MR. PHILIP MAIR PROFESSOR A. HADDOW

PROFESSOR DR. R. MEIEB

PROFESSOR F. G. YOUNG, F.R.S.

Director, and Secretary to the Executive Council: DR. G. E. W. WOLSTENHOLME, O.B.E.

Deputy Director : DR. H . N. H. GENESE

Assistant Secretary : MISS N . BLAND

Librarian: Editorial Assistants : MISS JOAN ETHERINGTON MISS CECILIA M. O'CONNOR, BSc.

MISS MAEVE O'CONNOR, B.A.

ALL RIGHTS RESERVED

This book may not be reproduced by any means, in whole or in part, with- out the permission of the Publishers

Published in London by J . & A. Churchill Ltd.

104 Gloucesier Place, W.l

First Published 1958

Printed in Great Britain

PREFACE

SUBJECTS for conferences a t the Ciba Foundation are pro- posed by many individuals in various parts of the world. The multitudinous affairs in which polysaccharides are being found to play a part made their consideration of particular interest to an institution where workers separated in tradi- tional disciplines and their appropriate societies are en- couraged to pool their facts and opinions. The Director was, therefore, happy to accept valuable suggestions within this field, coming in the first place from Professor Z. Dische, Professor Paul Gyorgy, and Professor R. Meier, although to find a subject within the scope and scale of a Ciba Founda- tion symposium, discussion was eventually restricted to the " Chemistry and Biology of Mucopolysaccharides ".

Professor W. T. J. Morgan was persuaded to be Chairman of the symposium. He most kindly gave much time and care to its preparation and directed its course with friendly courtesy and skill.

There was some skirmishing beforehand on the definition of the word " mucopolysaccharides ", but it was decided for the conference and this volume to go ahead on the broad understanding that the term denoted carbohydrate-amino acid complexes which cannot yet be described in agreed exact terms.

A wide range of papers were accepted and are here repro- duced with the discussions they aroused. The group present was a small one, for the purpose of thorough discussion, and it is hoped that this volume will prove useful and enjoyable to those workers in this field who could not be asked to par- ticipate in person on this occasion, as well as to others not so directly engaged in such research.

To some readers this book may form an introduction to the work of the Ciba Foundation, and it may be helpful to add a few words about its interests.

V

vi PREFACE

Under its eminent Trustees, the Foundation is engaged in a number of activities with the purpose of improving co- operation in medical and chemical research between workers in different countries and different disciplines. At its house in London the Foundation provides accommodation for scientists, organizes conferences, conducts a medical post- graduate exchange scheme between Great Britain and France, arranges a variety of informal discussions, awards two annual lectureships, and is building up a library service in special fields. The Foundation assists international congresses and scientific institutions, and it is hoped that in its hospitality, its meetings, and in such a volume as this, it is also proving of value to the individual scientist.

C O N T E N T S PAGE

Chairman's opening remarks W. T. J. MORGAN . 1

General chemistry of the mucopolysaccharides by M. STACEY . 4

Discussion : CONSDEN, C~TP, DAVIES, DISCIIE, DORFMAN FOSTER, KABAT, KAHNT, KLENK, MORGAN, NEUBERGER, OGSTON, SPRINGER, STACEY, WESTPHAL, ZILLIKEN . . 16

Physicochemical studies on hyaluronic acids by B. S. BLUMBERG and A. G. OGSTON . . 22

fiscussion : BETTELHEIM, BLIX, CONSDEN, DISCHE, KABAT, KAIINT, MEIER, MORGAN, NEUBERGER, OGSTON, PARTRIDGE, STACEY, WESTPHAL, WINZLER . 37

Immunochemical approaches to polysaccharide and mucopolysaccharide structure

by E. A. KABAT . 4.2

DisCUSsiOn : KABAT, MORGAN, OGSTON, SPRINGER, STACEY, WATKINS . . 60

Biosynthesis of mucopolysaccharides : the uridine nucleo- tides of Group A streptococci

by A. DORFMAN and J. A. CIFONELLI . . 6 4 Discussion : DAVIES, DISCHE, DORFMAN, JEL~NLOZ, KLENK,

MORGAN, NEUBERGER, STACEY, ZILLIKEN . 81

Sulphated galactosamine-containing mucopolysaccharides by R. W. .JEANLOZ, P. J. STOFFYN, and MONIQUE TREMEQE 85

f i S C U S s i O l t : DISCIIE, D O R F M A S , I'OSTEli, .JI.24NI.OZ, KABAT. STACE\,, WINZLER, %II.I.IKEN . . . 90

The presence in cartilage of a complex containing chon- droitin sulphate combined with a non-collagenous protein

by S. M. PARTRIDGE and H. F. DAVIS . . 93 Discussion : BETTELHEIM, CONSDEN, C 6 ~ f , DISCHE, DORFMAN,

JEANLOZ, MEIER, MORGAN, NEUBERGER, OGSTON, PARTRIDGE, STACEY, WESTPHAL . . 110

vii

... Vl l l CONTENTS

PAGE:

The neutral heteropolysaccharides in connective tissue by Z. DISCHE, A. DANILCZENKO, and G. ZELMENIS .

Discussion: DISCHE, JEANLOZ, KEYSER, MEIER, MORGAN, STACEY, WINZLER .

@P.GYORGY . N-containing saccharides in human milk

DiSCUSsiOn : BETTELHEIM, RLIX, DISCIIE, DORFMAN, GYORGY, KAUAT, KLENK, MACLAGAN, MORGAN, NEUBERGER, SPRINGER, WESTPHAL, ZILLIKEN .

The pharmacological effects of polysaccharides by R. MEIER .

Discussion: DAVIES, DISCHE, KAHNT, SCHAR, WESTPHAL .

Mucopolysaccharides of Gram-negative bacteria: newer chemical and biological aspects

by 0. WESTPIIAL, 0. LUDERITZ, E. EICIIENBERGER, and E. NETER .

Discussion : DAVIES, DISCHE, GOTTSCIIALK, KAUAT, KLENK,

Mucopolysaccharides associated with blood group speci-

MORGAN, STACEY, WESTPHAL .

ficity by W. T . J. MORGAN .

Discussion ; DAVIES, DISCHE, KABAT, MORGAN, NEUBERGER, STACEY, WATKINS, WESTPHAL, ZILLIKEN .

Blood group active substances of plant origin by G. F. SPRINGER .

Discussion : C 6 ~ 6 , DAVIES, DISCHE, GYORGY, JEANLOL, KABAT, MORGAN, SPRINGER, STACEY, WATICINS, WESTPHAL .

Mucopolysaccharides of epithelial mucus

Glycoproteins of plasma

by L. ODIN .

by R. J. WINZLER . Discussion: C b ~ f , DAVIES, DISCHE, JEANLOZ, KABAT, KEYSER,

MACLAGAN, NEUBERGER, ODIN, SVENNERIIOLM, WESTPHAL, WINZLER .

,Colloidal properties of urinary mucopolysaccharides by N. F. MACLAGAN and A. J. ANDERSON .

Discussion : CONSDEN, DISCHE, DORFMAN, GYORGY, KEYSER, KLENIC, MACLAGAN, MORGAN, NEUBERGER, ODIN, OGSTON, SPRINGER, STACEY, WESTPHAL, WINZLER .

116

136

140

1.54

157

182

187

196

200

21 1

216

230

234

243

263

268

282

CONTENTS ix

PAGE The prosthetic group of some mucoproteins and its re- lationship to influenza virus

Neuraminic acid

by A. GOTTSCHALK . . 287

byE. KLENK . . 296 Discussion : BLIX, C ~ T E , DORFMAN, GOTTSCHALK, JEANLOZ.

KLENK, MORGAN, NEUBERGER, WINZLER, ZILLIKEN . . 302

General Discussion BETTELHEIM, BLIX, C ~ T E , DAVIES, DISCJIE, DORF~MAN, FOSTER, GOTTSCHALK, KLENK, MORGAN, SPRINGER, WINZLER . . 306

Chairman’s closing remarks W. T. J. MORGAN . . 312

List of those participating in or attending the Symposium on “ Chemistry and Biology of Mucopolysaccharides ”,

23rd-25th April, 1957

F. R. BETTELEEIM F. G. BLIX. . R. CONSDEN .

R. C6Th . D. A. L. DAVIES . 2. DISCHE .

A.DORFMAN . A. B. FOSTER . A. GoTTsCnALK .

P. G Y ~ R G Y . .

R. W. JEANLOZ . E.A.KABAT .

P. W. KAHNT J. W. KEYSER

N. F. MACLAGAN .

R. MEIER . W. T. J. MORGAN A.NEUBERGER .

L. ODIN .

A. G. OGSTON ,

Dept. of Biochemistry, University of Cambridge Dept. of Medical Chemistry, University of

Uppsala Special Unit for Juvenile Rheumatism,

Canadian Red Cross Memorial Hospital, Taplow, Bucks.

Lister Inst. of Preventive Medicine, London; and Canada

Microbiological Research Establishment, Por- ton, Wilts.

Dept. of Biochemistry, Columbia University College of Physicians and Surgeons, New York

Dept. of Pediatrics, University of Chicago Dept. of Chemistry, University of Birmingham Walter and Eliza Hall Inst. of Medical Re-

Dept. of Pediatrics, University of Pennsyl-

Massachusetts General Hospital, Boston Dept. of Microbiology, Columbia-Presbyterian

CIBA Ltd., Bade Dept. of Pathology and Bacteriology, The

Dept. of Physiological Chemistry, University

Dept. of Chemical Pathology, Westminster

CIBA Ltd., Bade Lister Inst. of Preventive Medicine, London Dept. of Chemical Pathology, St. Mary’s

Dept. of Clinical Chemistry, University

Dept. of Biochemistry, University of Oxford

search, Melbourne

vania, Philadelphia

Medical Center, New York

Royal Infirmary, Cardiff

of Cologne

Medical School, London

Hospital, London

Hospital, Uppsala

xi

xii LIST S. M. PARTRIDGE .

BERTHA SCHAR G. F. SPRINGER

M. STACEY . L. SVENNERHOLM .

WINIFRED M. ~VATKINY . 0. WESTPHAL . R. J. WINZLEI~ .

F. ZILLIKEN .

OF PARTICIPANTS University of Cambridge, and D.S.I.R. Low

Temperature Research Station, Cambridge CIBA Ltd., Bade Dept. of Immunology, University of Penn-

1)ept. of Chemistry, University of Birmingham Dept. of Medical Chemistry, University of

Lister Inst. of Preventive Medicine, London Dr. A. Wander Forschungsinstitut, Freibug Dept. of Biological Chemistry, University of

Dept. of Biochemistry, University of Penn-

sylvania, Philadelphia

Gothenburg

Illinois College of Medicine, Chicago

sylvania, Philadelphia

CHAIRMAN’S OPENING REMARKS

W. T. J. MORGAN

THE title of the main theme to be discussed a t this Ciba Foundation Symposium, “ The Chemistry and Biology of Mucopolysaccharides ”, might suggest that mucopolysacchar- ides were a well-defined group of substances. A glance at the abstracts of the communications shows, however, how widely those attending this Symposium interpret the term “ muco- polysaccharide”. It must be admitted that there is no generally accepted definition of a mucopolysaccharide and for that reason, a t this stage in the development of the subject, we must be prepared to include a wide range of carbohydrate- containing complexes in our discussions. In reaching an understanding of the nature of the biologically important parts of these complex macromolecules, however, the results of the study of relatively simple molecules, such as detached carbohydrate units, will make a valuable contribution to our discussions and for that reason must be included. We have much ground to cover in our allotted span of three days and I hope it will be possible to spend most of our time considering materials which contain carbohydrate and amino acids as integral parts of a complex macromolecule. In some instances, it will obviously be more convenient to discuss the carbo- hydrate moiety alone, where this moiety is readily released from its combination with protein in the native tissue, tissue fluid or secretion. Nevertheless the nature of the carbohydrate and its linkage to protein in the native complex should remain ever in our thoughts and be discussed where possible.

It is my impression that the groups of specialists attending former Ciba Foundation Symposia were more acquainted with or aware of all the problems and aspects under discussion than we shall be during the next three days. If this impression is

MUCO.-l 1

2 W. T. J. MORGAN

right we can be sure it is because of the very wide field covered under the heading " mucopolysaccharide ", and the large range of investigations covered will emphasize the difi - culty any one person will have in understanding and appre- ciating fully all approaches to the subject. For this reason it seems probable that some speakers will wish to spend more than usual of their allotted time describing briefly the broader aspects of their particular problem. I believe this will be welcomed by those present.

It would be idle to pretend that we have very much exact knowledge as yet in the field of mucopolysaccharide chemistry. It is true that the properties and behaviour of certain materials are in many instances fairly fully recorded, but even here after critical examination of the analytical figures and properties, it frequently seems probable that the materials are still not homogeneous in the sense that molecules similar in type which coexist with them in the native tissues and secretions still remain present to some extent. Those of us who are working with mucopolysaccharides which possess a readily detectable immunological specificity are in a much stronger position to detect contamination with materials chemically similar but which nevertheless possess a different specificity. By this means we can appreciate more easily the quality of our isolated materials and those who work with such serologically specific materials can have something of a shock when the results of tests for quite other specificities are recorded. The use of serologically specific tests makes us realise more fully the very real possibility of heterogeneity in what is frequently considered to be a homogeneous material.

We are fortunate indeed in being in a position to hear the very latest evidence concerning the constitution of sialic acid and its function in rnucoprotein complexes. This is obviously a most important material. When Dr. Wolstenholme and I considered subjects which might be suitably included in this Symposium, we agreed that sialic acid was of outstanding importance and presented for discussion many interesting problems in both chemistry and biology. It is now more than

CHAIRMAN’S OPENING REMARKS 3

twenty years since Prof. Blix made his important discovery and introduced to the scientific world a material which became known later as sialic acid. The full significance of this finding was not at first appreciated, but as the widespread occurrence of sialic acid was established and more efficient procedures for its isolation were developed, work on its structure was inten- sified and a t the point a t which the constitution of sialic acid and its derivatives seemed to be nearing completion, there has now come its synthesis, thus bringing to a close the important chemical problem of the exact structure of this elusive material. However, much remains to be done to establish the full biological significance of sialic acid and I believe this Symposium gives a unique opportunity for discussing for the first time the whole field of sialic acid chemistry and biology.

GENERAL CHEMISTRY OF THE MUCOPOLY SACCHARIDES

M. STACEY chemisty Department, The University, Birmingham

A DISCUSSION of this interesting group of complex macro- molecules is appropriate a t the present time since new prob- lems concerning their biology and chemistry are attracting attention almost every day. The papers to be presented during this meeting will serve to show the variety and significance of the approaches to the many topics under active investiga- tion in various parts of the world. The chemistry of the group is not yet very far advanced and presents us with an important challenge since there is no doubt that the structural secrets are not readily yielded up to the methods of orthodox chemistry. However the problems are being approached from a variety of angles. The various techniques used in my laboratories in the past to elucidate the structures of the carbohydrate compounds will be briefly commented upon and new lines of work being undertaken largely by groups under my colleagues Drs. Foster and Barker will be indicated.

Isolation In most tissues carbohydrate residues appear to be in firm

chemical combination with proteins or fats (or both), and quite drastic methods were often used to split the complexes. Decalcification where necessary, tissue mincing, defatting with various solvents, extraction with water, buffers, acids and alkalis, deproteinization by various means and fractionation by salt or solvent precipitation have all been valuable. Purity has been checked by analysis, optical activity and the usual physical techniques such as electrophoresis, ultracentrifu- gation, etc. However one can perhaps claim the preparation of

4

GENERAL CHEMISTRY OF MUCOPOLYSACCHARIDES 5

entities only when sensitive biological tests, such as blood group activity, can be applied. The lability of parts of some of these molecules to both acidic and alkaline hydrolysis may be very great indeed and can give rise to a variety of degradation products. We might all agree perhaps to explain our own artifacts! Our own early work was always carried out on what were clearly stated to be alkali-stable constituents.

Monosaccharide components Discussions on nomenclature of mucopolysaccharides and

mucoproteins lead nowhere at present but we might now agree perhaps that we will consider only those macromolecules which contain amino sugars. Glucosamine (2-amino-2-deoxy- glucose) and galactosamine (2-amino-2-deoxygalactose) are commonly found though many other amino sugar derivatives are now being identified by various investigators.

The amino sugars usually contain N-acyl residues such as N- acetyl while others may contain N-methyl groups and others sulphamido residues. Among the hexoses, galactose and man- nose are the most common with glucose sometimes occasionally being present.

Our original discovery of L-fucose (6-deoxy-~-galactose) (Bray, Henry and Stacey, 1946) as a constituent of the blood group A factor of pepsin has led to a lot of work on this interesting sugar. Rhamnose (6-deoxymannose) is often found in bacterial polysaccharides while the dideoxy sugars of Westphal found in Salmonellae are likely to prove of high importance in future. Acidic mucopolysaccharides contain units of glucuronic acid, and possibly iduronic acid and unsaturated hexuronic acids. Acidity in some compounds such as chondroitin sulphate and heparin is due partly to the presence of sulphate residues.

Methylation technique This technique, so valuable for normal polysaccharides, has

not so far yielded results of great value. Simultaneous de- proteinization and methylation were achieved in the case of

6 M. STACEY

ovomucoid (Stacey and Woolley, 1940, 1942) and a tangible methyl ether of an oligosaccharide was obtained, though not in high yield. Similar results can be obtained with seromucoids. Some results were obtained with a degraded chondroitin (Bray, Gregory and Stacey, 1944) though work with heparin and hyaluronic acid has not been very exciting. There is room for a new approach to methylation studies.

There is, however, much that can be done if we apply more recent advances in carbohydrate chemistry. Thus we can use for identification purposes, paper chromatography and paper ionophoresis with new staining techniques ; we have resin exchange and carbon columns; and we can use Cetavlon com- plexes of various kinds for purification. We can study deamin- ated compounds, we can make good use of infrared now that we have a ' fingerprint ' region for glycosides and we can supply the valuable technique of linkage analysis. The latter tech- nique consists of partial acidic hydrolysis, and separation and identification of the di- and oligosaccharides produced.

Acidic hydrolysis constitutes one of the first experiments to be performed in studying mucopolysaccharide structure. In many cases it permits the identification of the component monosaccharides by qualitative chromatographic and iono- phoretic analysis of the hydrolysates. Whilst most nitrogen- free neutral saccharides behave normally on acidic hydrolysis, yielding a near-theoretical yield of the free sugars, amino sugar derivatives do not, since the two pathways of hydrolysis shown in Fig. 1 may be followed simultaneously (Foster, Horton and Stacey, 1957). Thus, if cleavage of the glycosidic substituent Y precedes that of the N-substituent X, then pathway A is followed and the free amino sugar (111) is rapidly released. On the other hand if hydrolysis of X occurs first, then pathway B is followed. The first product (IV) of hydrolysis is strongly resistant to further attack by hydrions because of the electro-

static shielding effect of the -NH, group so that cleavage of the glycosidic group Y in (IV) will occur slowly under normal conditions of acidic hydrolysis.

The extent to which pathways A and B are followed depends

@

GENERAL CHEMISTRY OF MUCOPOLYSACCHARIDES 7

on the nature of X and Y. In most mucopolysaccharides Y is part of an interglycosidic linkage and X is an acetyl group. In a series of model compounds X and Y have been varied and also configuration at the glycosidic centre. In all cases (see Table I) where a glycosidic substituent was present, hydro- lysis by pathway B occurred to an appreciable extent (14- 37 per cent). In these hydrolyses the free amino sugars were determined by a colorimetric procedure (Elson and Morgan,

Pathway A --

7 NH-X\

.-.I, m

v - Y

@ NH, 1v

FIG. 1. Schematic representation of the acidic hydrolysis of 2-amino-2-deoxy-~-glucose (D- glucosamine) derivatives. A similar scheme

operates for the p-anomers.

1933; Belcher, Nutten and Sambrook, 1954); compounds of the type (IV) in Fig. 1 gave no colour in the test.

The existence of the two pathways of hydrolysis shown in Fig. 1 was recognized by Moggridge and Neuberger (1938) but the implications do not seem to have been realized. The re- actions in Fig. 1 undoubtedly operate during the acidic hydro- lysis of mucopolysaccharides under normal conditions, thereby preventing a complete release of the amino sugar moieties. Thus, low values for the hexosamine content of mucopoly- saccharides are likely to be obtained if the free amino sugar in

8 M. STACEY

their acid hydrolysates is determined by a colorimetric pro- cedure or is isolated by means of ion-exchange resins (Gardell, 1953). Providing that the mucopolysaccharides are protein- free then a more reliable method for the determination of the hexosamine content of acid hydrolysates involves deamination, for example under alkaline conditions (Tracey, 1952).

The effect of the -NH, group in compounds of the type (IV) in Fig. 1 is shown by the large times of half hydrolysis of methyl 2-amino-2-deoxy-a- and P-D-glucopyranosides (Table I).

Heparin, the physiological blood anticoagulant, provides an interesting example where acidic hydrolysis proceeds mainly by pathway B (Fig. 1). This is due to the fact that the muco- polysaccharide contains the acid-labile sulphamic acid group- ing NH.SO,H (Fig. 1, X = SO,H) and in this respect it is unique within the class of mucopolysaccharides (Stacey, 1946).

The sequence of events when heparin is subjected to acidic hydrolysis is shown in Fig. 2 (Foster and Huggard, 1955). Cleavage of the sulphamic acid groups occurs first to give

+-heparin (VI) in which the -NH, groups electrostatically shield the adjacent glucosaminidic linkage (Fig. 2, b ) from hydrions and direct further hydrolytic cleavage to the glucuronidic linkages (Fig. 2, a). Ultimately a resistant disaccharide (VII) is obtained.

In seeking other methods for the degradation of heparin we have studied the deamination of certain amino sugar deriva- tives. Treatment of the D-glucosamine derivatives (IX) with

e3

8

CH,OH CH,OH

H6 &JR H O @!". +R-oH

*", M X

nitrous acid results in a rapid reaction which involves a ring contraction and liberation of the glycosidic substituent (Foster, Martlew and Stacey, 1953). The reaction is clear-cut (Bera,

2- Am

ino

-

hydrolysis D

-glueose

2-deoxy-

(%I

2-Acetamid0-2-deoxy-~-g~ucose A

c H

4-43

100 2-Benzyloxycarbonylamino-2-deoxy-

Ph

. CH

, .O. CO

H

4-6

100

Methyl 2-acetam

ido-2-deoxy-P-

Ac

OM

e 4-8

82

Tim

e of half

(min.)

Derivative

x

Y

release

D-glU

CO

Se

illethyl 2-acetan

do

-2-d

eox

y-a-

Ac

OM

e 36

78 D

-glucopyranoside

D-glucopyranoside

OM

e 42

G3

M

ethyl 2-benzyloxycarbonylamine-

Methyl 2-benzyloxycarbonylam

ino- P

h.C

H,.O

. CO

O

Me

21 8

6

Eth

yl 2-benzyloxycarbonylarnin0-2-deox>~

Ph

.CH

,.O.C

O

OE

t 27

70

Ph

. CH

,. 0. CO

2-deoxy-cc-~-g1ucopyranoside

2-P-~

-glucopyranoside

cc-D-glucopyranoside

side hydrochloride M

ethyl 2-amino-2-deoxy-cc-~-glucopyranO- €I,€IC

I O

Me

8.5

X lo3

2

Dlethyl 2-amino-2-deoxy-~-~-g~ucopyrano- H

,HC

l O

Me

2.8X103

6

side hydrochloride

W

0

0

k B 5 s rn cc 0

kl

z 4 d R 0

'd 0 r E 8 k!

10 31. ST-4CE.l.

Foster and Stacey, 1956) and, further, the rate of deamination is dependent on the configuration a t the glycosidic centre. Deamination occurs most rapidly when the configuration is p. Application of this deamination reaction to +-heparin (VI, Fig. 2 ) , which may be isolated after mild acidic treatment of heparin, fragmented the polysaccharide a t a rate consistent with the presence of an a-glucosaminidic linkage (Table 11), to

b 6 b

-0

a v, Heparin d a

w MIC

FIG. 2. Schematic representation, in partial formulae, of the acidic and deaminative degradation of heparin.

yield mainly disaccharides of the type (VIII) in Fig. 2. In this process, the linkages which are most resistant in normal acidic hydrolysis of heparin are selectively cleaved. The structure of the degradation products is still under study.

The development of mild methods for selectively de-N-acetyl- ating the amino sugar moieties of other mucopolysaccharides would permit application of the deaminative degradation to yield fragments of potential value for sequence determination of the component monosaccharides.

GENERAL CHEMISTRY OF MUCOPOLYSACCHARIDES 11

Some progress has been made in the application of selective precipitants in the isolation of mucopolysaccharides and in the separation of mixtures. Thus 4-amino-4’-chlorodiphenyl (CAD) yields water-insoluble salts with highly sulphated poly- saccharides (Foster and Martlew, unpublished). A critical sul- phate content exists since $-heparin (de-N-sulphated heparin) and chondroitin hydrogen sulphate do not yield water- insoluble salts. The reagent is of obvious potential value for separating heparin-type polysaccharides from other, less highly sulphated, mucopolysaccharides.

Table I1

D-GLUCOSE (D-GLUCOSAMINE) DERIVATIVES RATES O F DEAXINATIVE DEGRADATION O F VARIOUS 2-AMINO-2-DEOXY-

(FOSTER, AIARTLEW AND STACEY, 1953)

Derivative

Methyl 2-amino-2-deoxy- a-D-ghcopyranoside

Methyl 2-amino-2-deoxy- P-D-ghcopyranoside

2-Amino-2-deoxy-u-gluco- pyranose

+-Heparin Chitosan (de-N-acetylated

chitin)

Conjiguration of Time of half linkage cleaved reaction (min.)

CL

P

5 . 0

1.0

a 4 ..5

a 5

5 . 5

1.0

a-Compounds, reaction essentially complete in 7 minutes P-Compounds, in 30 minutes

A more versatile precipitant is Cetavlon (cetyltrimethyl- ammonium bromide) ; originally introduced for the fractiona- tion of nucleic acids (Dutta, Jones and Stacey, 1953), its use has now been extended to the polysaccharide field. Cetavlon may be used (a) to separate acidic from neutral polysacch- arides, (b) to fractionate mixtures of acidic polysaccharides, (c) to fractionate mixtures of neutral polysaccharides, (d) in the isolation of mucopolysaccharides from natural sources. Examples of these uses are cited in the sequel.

Cetavlon yields water-insoluble salts with many acidic poly- saccharides, in which the acid functions are carboxyl groups

12 M. STACEY

and/or sulphate groups, and even with simple sugar sulphates but not with simple uronic acids. Neutral polysaccharides such as glycogen, blood group substances and dextran are not precipitated by the detergent from aqueous solutions at pH 7. Barker and Stacey (1956) made use of this fact in studying the polysaccharide content of the liver from a gargoylism case. The water-extracted polysaccharides were separated by Cet- avlon into neutral and acidic fractions. The neutral fraction contained, in addition to other polysaccharides, an amount of glycogen (< 0 . 3 per cent of the initial water-soluble extract) much less than that (8 per cent) found in a normal liver used for comparison purposes. On the other hand a larger amount (33-50 per cent) of acidic polysaccharide was found in the gargoylism liver than in the normal liver (20 per cent).

By using suboptimal controlled amounts of Cetavlon the acidic polysaccharide fraction from the gargoylism liver was further separated into fractions of high and low sulphate content which showed significant differences in certain physical properties.

The use of infrared absorption spectra was invoked in this investigation to demonstrate that there were marked differ- ences between the acidic polysaccharides present in the normal and gargoylism livers. The relevant portion of the spectra to be compared is that in the range 720-1000 cm.3, the so-called ' fingerprint ' region for carbohydrates.

The above use of Cetavlon simplified the fractionation procedure and enabled the demonstration of profound meta- bolic disorder in the liver of the gargoylism case.

Although neutral polysaccharides are not precipitated from aqueous solution at pH 7 by Cetavlon, the negatively charged complexes formed by certain polysaccharides in the presence of borate ions (see Foster, Newton-Hearn and Stacey, 1956) may be precipitated by Cetavlon. Several factors may determine whether a polysaccharide is precipitated under these conditions and these include (Table 111) the affinity of the polysaccharide for borate ions and the pH of the solution (Barker, Stacey and Zweifel, 1957).

GENERAL CHEMISTRY OF MUCOPOLYS-4CCHARIDES 13

Table I11 shows the behaviour of a series of neutral poly- saccharides when Cetavlon is added to their solutions in borate buffer and alkali. Thus, (1) a mixture of mannan and glycogen may be completely separated by first precipitating the mannan borate complex with Cetavlon from borate buffer pH 8 * 5 and then the glycogen borate complex a t pH 10, (2) a mixture of glycogen and inulin may be separated by precipitating the former as its borate complex with Cetavlon a t pH 10 and recovering the latter from the supernatant. The polysacch- arides may be recovered easily from their borate complexes.

Table I11 QUALITATIVE CETAVLON PRECIPITATIONS OF VARIOUS POLYSACCHARIDES

Polysaccharide

Yeast mannan Carob gum Laminarin Glycogen Inulin Dextran Blood group A substance Blood group B substance

[El; Borate buffer p R 0.1 iw-NaOH v 8 . 5 10.0

+ ++ +78' t ++ ++ + +4Qo - 1 6 O 4- + - I - ++ + + + - 3 2 O *

- + ++ i tT i

- -

+193" - -

+402" - - - - - - -

- L

The signs -, &, +, or ++ indicate the degree of precipitation observed when equal volumes of polysaccharide solution (1 yo) and Cetavlon solution (5%) were mixed.

The results in Table I11 indicate the method to be of wide potential application.

Cetavlon has also been used to simplify the procedure for the isolation of chondroitin hydrogen sulphate suitable for degradation studies, from bovine trachea (Bera, Foster and Stacey, 1955).

The need for study of simple compounds and model systems is emphasized by recent reports that iduronic acid (Hoffman, Linker and Meyer, 1956) and talosamine (Rluir, 1957) have been recognized in hydrolysates of chondroitin hydrogen sul- phate. The former is the C(,,-epimer of glucuronic acid and the latter the C(,,-epimer of galactosamine. To what extent epimeri- zation has occurred during the isolation and degradation of the

14 M. STACEY

chondroitin hydrogen sulphates from which these substances were isolated is not known.

The isolation of chitin from Crustacea cuticles normally involves drastic acid and alkaline treatments. It has been found (Foster and Hackman, 1957) that the powdered cuticles are rapidly decalcified under very mild conditions by ethylene- diaminetetra-acetic acid at pH 8-9 to leave chitin which can have undergone little or no degradation. It was concluded that the small amount (< 5 per cent) of protein present in the chitin was bound chemically to the polysaccharide. Fractional precipitation of the chitin from solution in anhydrous formic acid or lithium thiocyanate yielded fractions which had apparently the same protein content and no protein-free chitin fraction was obtained. Protein analysis of these fractions presented a problem and the only satisfactory method found involved acidic hydrolysis followed by zone electrophoresis of the hydrolysate in acetate buffer (pH 5). The amino acids were separated into acidic, neutral and basic zones which could be determined with ninhydrin.

Several prolonged treatments with hot alkali served to reduce the protein content but did not eliminate it. The conclusion drawn from these results is that the chitin in the cuticle is free of pendant groups other than a small amount of protein.

Controlled degradation of chitin is complicated by the insolubility of the polysaccharide. Chitin is however soluble in strong mineral acids and is slowly depolymerized. Com- plications arise, for example, in sulphuric acid where extensive sulphation occurs, and in hydrochloric acid where the difficulty of removal of acid makes the method impractical. Acetolysis (acetic anhydride - sulphuric acid) can be used to effect con- trolled degradation of chitin but the acetylated saccharides pro- duced cannot be deacetylated by alkaline treatment without the formation of decomposition products. It was ultimately found (Barker et al., 1957) that controlled fragmentation of the mucopolysaccharide could best be effected by subjecting the de-N-acetylated polysaccharide to acidic hydrolysis,

GENERAL CHEMISTRY O F MUCOPOLYSACCHARIDES 15

selectively M-acetylating the saccharides in the hydrolysate and then fractionating the mixture on a charcoal celite column. In this way a polymer-homologous series of oligosaccharides which contained N-acetyl-D-glucosamine was obtained. Data on these saccharides are shown in Table IV.

Table IV DATA ON THE OLIGOSACCHARIDES ISOLATED FROM CHITIN

I'ield Saccharide (g. )

(%)b

Monoa 0.475 (24.5)

Diu 0.262 (13-5)

Tria 0.256 (13.2)

Tetra 0.184

Pentae 0.138 (9.5)

(7.1)

01 10

Aqueous ethanol

elution c for

1-3

9-11.5

16-20

21-23

23.5-25.6

Molecular weigh@ ial

(eqf'il') Calc. Pound RF

+40.76' 221 208 0.43

+17.2' 424 409 0.34

+2*0.5" 627 652 0.24 432

-3.8' 830 871 0.16

-8.99" 1033 1049 0.10

a Isolated crystalline: monosaccharide, 1u.p. 20&7", [a]o+70.7" ( 5 nlin.) +40:7" (equp.); disaccharide, m.p. 260-2" (decomp.), [a]. +25.2" (3 min.) +17.2" (eqml.); trisaccharide m.p. 304-6" (decomp.). lalo +3.78 (13 min.) +2.18" (equil.). All the saccharides except the( trisaccharide were homogeneous on paper chromatography, the trisaccharide was fcirther purifled on charcoal celite. Percentage based on the weight of saccharide mixture introduced on to the column. From 3 8. of chitosan hydrochloride 1.94 g. of water soluble N-acetylated saccharides were obtained.

c Separation performed on a charcoal celite column about 29 x 3cm. Elution commenced with water (500 rnl.) and was continued with aqueous ethanol. The alcohol concentration in the eluent increased by 3.396 per li tre for 3 litres and then by 6.67!! per litre.

d Determined by hypoiodite oxidation. e Pyridine-amyl alcohol-water system. Discrete spots of hexa- and heptasaccharide were also

obtained.

The infrared absorption spectra of the oligosaccharides from chitin showed that as the series was ascended the spectra became more nearly identical, indicating a close structural similarity. Further; the spectrum of chitin itself was closely similar to that of the higher saccharides in Table IV, suggest- ing that the polysaccharide is essentially an extension of the structure present in the smaller fragments.

With the discovery and application of new techniques in the group of mucopolysaccharides we can be assured of useful discoveries in the days before us.

16 M. STACEY

Acknowledgements The author thanks Dr. A. B. Foster for his valuable help with this

communication. The expenses for recent work in this field have been met by a grant from the Nuffield Foundation.

REFERENCES

BARKER, S. A., FOSTER, A. B., STACEY, M., and WEBBER, J. M. (1957).

BARKER, S. A., and STACEY, M. (1956). J. din. Path., 9, 314. BARKER, S. A., STACEY, M., and ZWEIFEL, G. (1957). Chem. & Ind.

BELCHER, R., NUTTEN, A. J., and SAMBROOK, C. M. (1954). Analyst,

BERA, B. C., FOSTER, A. B., and STACEY, M. (1955). J. chem. SOC., 3788. BERA, B. C., FOSTER, A. B., and STACEY, M. (1956). J. chem. SOC., 4531. BRAY, H. G., GREGORY, H., and STACEY, M. (1944). Biochem. J . , 38,142. BRAY, H. G., HENRY, H., and STACEY, M. (1946). Biochem. J., 40,124,

DUTTA, S. K., JONES, A. S., and STACEY, M. (1953). Biochim. biophys.

ELSON, L. A., and MORGAN, W. T. J. (1933). Biochem. J., 27,1824. FOSTER, A. B., and HACKMAN, R. H. (1957). Nature, Lond., 180, 41. FOSTER, A. B., HORTON, D., and STACEY, M. (1957). J. chem. SOC., 81. FOSTER, A. B., and HUGGARD, A. J. (1955). Advanc. Carbohyd. Chem.,

FOSTER, A. B., MARTLEW, E. F., and STACEY, M. (1953). Chem. & Ind.

FOSTER, A. B., NEWTON-HEARN, P. A., and STACEY, M. (1956). J. chem.

GARDELL, S. (1953). Acta chem. scand., 7 , 201. HOFFMAN, P., LINKER, A., and MEYER, K. (1956). Science, 124, 1252. MOGGBIDGE, R. C. G., and NEUBERGER, A. (1938). J. chem. SOC., 745. MUIR, H. (1957). Biochem. J., 65, 33P. STACEY, M. (1940). Advanc. Carbohydr. Chem., 2, 161. STACEY, M., and WOOLLEY, J. M. (1940). J. chem. SOC., 184. STACEY, M., and WOOLLEY, J. M. (1942). J. chem. SOC., 550. TRACEY, M. V. (1952). Biochem. J., 52, 265.

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DISCUSSION

Dische: Is there any additional information about hexuronic acid in heparin? I think it is a rather controversial question.

Foster: The evidence all points to the hexuronic acid being glucuronic acid.

Dische: Colour reactions are not very conclusive as means of identi- fication, but there is some truth in them. Heparin reacts in a very